THE WAKSMAN FOUNDATION OF JAPAN INC. 　

Report of Researchesin2011

Dr. Selman A.Waksman

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THE

WAKSMAN

FOUNDATION

OF

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INC.

Published by THE WAKSMAN FOUNDATION OF JAPAN INC.

30-8 Daikyo-cho, Shinjuku-ku, Tokyo, Japan

THE WAKSMAN FOUNDATIONOF JAPAN INC.

Report of Researches in 2011

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THE WAKSMAN FOUNDATIONOF JAPAN INC.

Report of Researches in 2011

THE WAKSMAN FOUNDATIONOF JAPAN INC.

Honorary Patron

H.I.H. Prince Akishino

Board of Directors

Chairman : Ichiro Kitasato, Adviser, The Kitasato Institute.Former Chairman of The Board, Meiji Seika Kaisha, Ltd.

Shogo Sasaki, Prof. Emeritus, Keio Univ.

Teruhiko Beppu, Prof. Emeritus, Tokyo Univ.

Takeshi Ishikawa, Vice PresidentRetirement Allowance Foundation of Private Colleges and Universities.

Tadakatsu Shimamura, Prof. Emeritus, Showa Univ.

Toshiro Sato, Adviser, The Kitasato Institute.

ManagingDirector : Takeji Nishikawa, Prof. Emeritus, Keio Univ.

Comp-troller : Yoshiharu Wakiyama, Senior Adviser, Kaken Pharmaceutical, Co., Ltd.

Shirow Enoki, Former President, Seikagaku Corporation.

Councilors

　Ryoichi Mori, Prof. Emeritus, Kyushu Univ.

　Keizo Takemi, Prof., Tokai Univ.

　Koichi Yamanishi, Director, General National Institute of Biomedical Innovation.

　Sachiko Goto, Prof. Emeritus, Toho Univ.

　Yoshihiro Miwa, President & CEO, Kowa Co., Ltd.

　Isao Uchida, Senior Adviser, Yokogawa Electric Corporation.

　Shigeo Koyasu, Prof., Keio Univ. Sch. Med.

　Takashi Shoda, Chairman of the Board, Representative Director, Daiichi Sankyo Co., Ltd.

　Yasuma Sugihara, Former Chairman & CEO, Mobil Sekiyu K.K.

Special Adviser

　Akira Uehara, Chairman & CEO, Taisho Pharmaceutical Holdings Co., Ltd.

　Masamichi Fujii, Prof. Emeritus, St. Marianna Univ.

　Osamu Sato, Prof. Emeritus, Tokai Univ.

　Osamu Nagayama, Chairman & CEO, Chugai Pharmaceutical Co., Ltd.

　Haruo Naito, President & CEO, Eisai Pharmaceutical Co., Ltd.

　Seiichi Sato, President & CEO, Sato Pharmaceutical Co., Ltd.

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Preface to the First Report (1962)

It is indeed a privilege to take this opportunity to write a few words of introduction to the first report of the Waksman Foundation of Japan Inc., covering five years of its activities and comprising the results of the work of the first two years of research carried out by various scholars in Japan in the fields of microbiology and medical science, supported by this Foundation.

In 1952, I accepted the invitation from Keio University and the Kitasato Institute, to deliver the centennial lecture in honor of the great Japanese bacteriologist, Shibasaburo Kitasato. Before departing for Japan, I proposed to the trustees of the Rutgers Research and Educational Foundation which owned the patents on streptomycin, to share the royalties under the patent in Japan, for the support of research in microbiology and allied fields in that country. The trustees heartily approved my recommendation that I make such announcement to that effect.

Soon upon my arrival in Japan (December 17, 1952), I invited a group of eminent microbiologists, biochemists, and clinical investigators to meet with me in order to discuss the plan. Everyone present was very enthusiastic about the proposal. It was decided that a proper committee be selected to work out the plan of a Foundation under which the royalties were to be received and distributed for the support of Japanese investigators working in different universities in Japan and elsewhere, in the fields of microbiology and medical research. The committee recommended that a Board of Directors be selected and the proposed Foundation be named THE WAKSMAN FOUNDATION OF JAPAN INCORPORATION.

The Rutgers Research and Educational Foundation approved at once the above recommendations and issued a statement, signed by Dr. Lewis Webster Jones, President of the Foundation, to the effect that

‘‘The Rutgers Research and Educational Foundation desires to emphasize that its principal concern is the advancement of scientific knowledge in the public interest and that it confidently expects that the Waksman Foundation for Microbiology and Medical Research in Japan will be similarly motivated, thereby serving the peoples of both countries.’’

This announcement was received with enthusiasm both by the scientific world and the popular press in Japan and in the United States. It took several years before the Waksman Foundation of Japan Inc. was properly organized, and before applications were received and approved. In 1958, I had the privilege of participating in the first official meetings of the Board of Directors of the Japanese Foundation and to greet personally the first group of scholars to whom grants had been made.

In summarizing these brief remarks in connection with the first cinqueannual report of the Waksman Foundation of Japan Inc., I would like to enphasize that this example of collaboration between universities and scientists of the United States and Japan may serve to encourage collaboration between scientific workers throughout the world towards a better understanding between men and women and towards a happier and healthier human race, so that all the nations on this earth can live in peace and that man may finally ‘‘break

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his swords and build out of them plowshares’’ for the betterment of mankind as a whole.

Selman A. Waksman

Professor EmeritusRutgers-State University N. J., U. S. A.

The ‘‘Waksman Foundation of Japan Inc.’’ was established in 1957 with the spirit of humanity by Dr. S.A. Waksman, Professor of Microbiology, Rutgers University, U.S.A. The Foundation’s operations are possible only because Dr. S.A. Waksman and the Rutgers Research and Educational Foundation donated patent royalties he received from the production in Japan of the discovery, Streptomycin.

Because of these royalties, each year many Japanese scholars and research workers in the fields of Microbiology and medical science are encouraged and find it possible to continue their work. Especially, in accordance with Dr. Waksman’s suggestion, the funds are distributed to scholars in local and economically hampered schools and laboratories and to those developing research workers who are endeavoring to expand in their fields. This thought of Dr. Waksman’s is most appreciated, as it matches our Oriental phylosophy, and results in the search for a jewel among ordinary stones, which is the highest work of the science-leader.

Some five years have now passed since the start of this Foundation, and many persons have received aid through this period.

The reports which are presented herein cover the first and second group of research workers who received financial assistance from the Foundation.

Toshio Katow, M. D.

Executive Director

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Contents

Yasuhiko Horiguchi:

Study on the mechanism causing paroxysmal cough in whooping cough ………………… 1

Hiroaki Takatori:

The Role of Lymphoid Tissue Inducer-like Cells in Chronic Antigen-induced Airway Inflammation …………………………………………………… 7

Norihiko Watanabe:

Immune mechanisms involved in the development of fatal autoimmune hepatitis in mice ……………………………………………………… 13

Haruhiko Suzuki:

Application of CD8+ regulatory T cells to Inflammatory Bowel Disease ………………… 21

Hidekazu Kuwayama:

Analysis of activation pathway of Huntington’s disease-causing gene in

Dictyostelium discoideum ………………………………………………………………… 31

Yukihiko Aramaki:

Induction of regulatory T cells following intranasal administration of liposomes composed of phosphatidylserine ……………………………………………… 41

── Report of Researches in 2011──

Study on the mechanism causing paroxysmal cough inwhooping cough

Yasuhiko Horiguchi

Department of Molecular Bacteriology, Research Institute for Microbial Diseases, Osaka University, Yamada-oka3-1, Suita Osaka 565-0871, Japan

Introduction

Whooping cough is a highly contagious, acuterespiratory disease caused by the pathogenic bacterium,Bordetella pertussis (1). Clinical manifestations of thedisease include paroxysmal coughing, pneumonia,apnea, and hypoxia. Severe cases can even result indeath. The disease is considered to occur in infants,but in fact, adolescents and adults can be infectedand a source of infection for infants. Although severaltypes of vaccines, whole-cell vaccines and acellularvaccines mainly comprising components derived frompertussis toxin and filamentous hemagglutinin, havebeen introduced, a resurgence of the disease hasbeen reported even in industrial countries with highvaccine coverage, and cases have increasingly occurredfor the last twenty years. According to the WorldHealth Statistics 2012 report by the World HealthOrganization, 129,265 whooping cough cases wererecorded in 2010. Thus, this disease is still a veryimportant medical problem in the world and a novelapproach to treatment is required. However, thepathogenesis of whooping cough is complex, and whilemany potential virulence factors have been recognized,the mechanism by which the bacteria establish thedisease is poorly understood. It is not known either howthe organisms cause the paroxysmal coughing.

A major obstacle to analyzing the course ofwhooping cough is a lack of suitable animal models.Notably, hardly any models that reproduce the parox-ysmal cough in whooping cough have been described.

Significant effort has been made to reproduce human-like pertussis in rabbits, guinea-pigs, puppies, monkeysand some primates, rats, and mice (2)(3). Mice, whichhave been most widely used, show some manifesta-tions resembling human whooping cough; e. g. highersusceptibility of suckling mice than adult mice toinfection, hypoglycemia, leukocytosis, and histaminesensitization. In addition, more recently, they haveoften been used to examine inflammation in responseto Bordetella infections. However, physiologically,mice are unable to cough and therefore do not displayparoxysmal coughing at any time during the infection.It is also problematic that a much larger number ofbacterial cells is required to establish the infection inmice, compared to natural infections.

In this study, we tried to find an animal model thatcan be readily utilized and reproduce the coughing inhuman whooping cough, by examining combinations ofvarious experimental animals and pathogenic bacteriabelonging to the genus Bordetella including B.pertussis and B. bronchiseptica. As a result, we foundrats infected with B. bronchiseptica to be the mostappropriate model. B. bronchiseptica, which is closelyrelated to B. pertussis, producing nearly identicalvirulence factors except pertussis toxin (4)(5), has ratsas its natural host whereas B. pertussis is highly adaptedto humans. B. bronchiseptica inoculated in amounts assmall as 10 cfu/rat, colonized the trachea and caused aparoxysmal cough indistinguishable from that observedin animals infected with a large amount of B. pertussis(108 cfu/rat). From these results, we consideredthat rats infected with B. bronchiseptica could be a

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model for human whooping cough. Moreover, whileanalyzing the B. bronchiseptica infection, we isolateda mutant strain that did not induce coughing in rats.To understand how Bordetella cause the paroxysmalcough, we have tried to identify the mutated gene andanalyze the pathogenesis of the mutant strain.

Materials and Methods

Bacterial strains and plasmids.

B. bronchiseptica strain RB50 was providedby P. A. Cotter, University of California, SantaBarbara, CA. B. bronchiseptica strain S798 (6), and B.pertussis strains Tohama and 18323 were maintainedin the laboratory. B. pertussis and B. bronchisepticawere grown on Bordet-Gengou (BG) agar plates(Gibco) containing 1% glycerol and 20% defibrinatedhorse blood. Avirulent phase III-type variants ofB. bronchiseptica were isolated from characteristicsmooth colonies on BG plates (1)(7). It was confirmedby Western blotting that the variants did not produceadenylate cyclase toxin, a virulence factor, indicatingthe phase III state. A calibration curve was preparedwith known concentrations of the bacterial cells andOD values at 650 nm and the correlation coefficientto estimate the number of cfu from the OD650nm wasobtained ; 1 OD650nm corresponds to 4.5 × 108 cfuof Bordetella. Escherichia coli used for all geneticexperiments was grown on Luria-Bertani (LB) agar orbroth. E. coli strains DH5α λ pir and HB101 harboringpRK2013 were used for the triparental conjugationtechnique.

An approximately 1-kbp fragment of DNApartly corresponding to a target gene was insertedinto pABB-CRS2-A1 (8) containing a gentamicin-resistance marker gene, which was used for thepreparation of insertion mutants. Clones of the insertionmutants were selected by growth on gentamicin-containing BG plates. For the preparation of deletionmutants, a chloramphenicol-resistance gene flanked byabout 1 kbp of DNA downstream and upstream ofthe target gene was inserted into pABB-CRS2-A1,which carries a sacB gene along with the gentamicin-resistance marker gene. The resultant plasmid was

used as a suicide vector, which was introduced intoB. bronchiseptica with the triparental conjugationtechnique. B. bronchiseptica clones in which targetgenes were deleted, were selected based on resistanceto gentamicin and subsequently to sucrose.

Animal experiments

The bacteria recovered from colonies on BG plateswere suspended in Stainer-Scholte (SS) medium tomake an OD650nm value of 0.2, and incubated at37◦C for an appropriate period with shaking to givea desired bacterial concentration. After incubation,the bacterial culture was diluted with SS mediumto 108 cfu/ml. Three-week old female Wistar rats(Japan SLC, Inc) were anesthetized with ether or amixture of medetomidine, midazolam, and butorphanolat final doses of 0.3, 2, and 5.0 mg/kg body weight,respectively, and inoculated intranasally with 103 cfuof test bacteria in 10 μ l, using a micropipet with aneedle-like tip. For the coughing analysis, each ratwas isolated in a separate cage, and coughing wasrecorded with a video camera (Canon HD ivisHF21,Canon) for 5 min every day for 15 days postinoculation.The recorded data were played on a computer screenwith the software Adobe Premier Pro CS5.5 (Adobe),which displays simultaneously moving images, sounds,and sound waveforms. By checking all the video data(moving images, sounds, and sound waveforms), anobserver enumerated the number of coughs.

On day 15 postinoculation, rats were euthanizedwith pentobarbital. The trachea was removed, andminced in Dulbecco’s PBS at 10 times the volume (ml)to trachea weight (mg) and strained with a Biomasher(Funakoshi, Tokyo). The resultant tissue extract wasserially diluted with Dulbecco’s PBS and plated onBG agar. After incubation at 37◦C for an appropriateperiod, the number of colonies on the plates wasenumerated.

Genome sequencing

DNA was purified with a QIAGEN Blood & Cellculture DNA kit (QIAGEN) from a spontaneous mu-tant strain (ΔC strain) that does not induce paroxysmal

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Fig. 1. Coughing of rats infected with B. bronchisepticaRB50. The data are for days 5 to 11 of infection.Values represent the mean ±SD (n = 8 for ratsinoculated with SS medium, n = 7 for those withRB50).

coughing in rats and the strain’s genome was sequencedin collaboration with Frontier Science Research Cen-ter, Miyazaki University. We compared the DNA se-quence with that of the parental strain RB50 (5) us-ing GenomeMacher (http://www.ige.tohoku.ac.jp/joho/gmProject/gmhome.html), nucmer (http://mummer.sourceforge.net/), and blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and identified the mutation sites, whichwere subsequently verified by direct sequencing.

Results

Fig. 1 shows that rats infected with 103 cfu of B.bronchiseptica RB50 had a persistent cough. Coughingwas observed as early as day 5 postinfection and itsfrequency reached a peak at day 9. The total numberof coughs for 5 days from day 6 to 11 of infectionranged from 56 to 242/30 min (5 min × 6 days). Incontrast, rats given SS medium (Fig. 2) or the phaseIII variants of B. bronchiseptica hardly exhibited anycoughing (data not shown). Similar coughing was alsoobserved in rats infected with another strain of B.bronchiseptica, S798, or B. pertussis inoculated at108 cfu/rat (data not shown). During the course ofthese experiments, we unexpectedly isolated a mutantstrain of B. bronchiseptica, tentatively named ΔC,

Fig. 2. Coughing of rats infected with variants of B.bronchiseptica RB50. Shown is the total numberof coughs during 6 days (day 6 -day 11)of observation. Bars show the mean value ineach group. Numbers of rats used are given inparentheses.

which colonized the trachea but did not cause coughingparoxysms (Fig. 2).

To identify the mutated gene in ΔC, whichmight be responsible for the cough in infected rats,we sequenced the whole genome of the mutant andcompared it with that of the parental strain RB50.The first screening of mutation sites revealed single-base substitutions at 41 sites and deletion/insertionmutations at 6 sites. These regions of the ΔC andRB50 genomes were directly sequenced and eventually,we found a single-base insertion and a single-basesubstitution in geneA and geneB (tentative names) ofΔC, respectively. It was predicted that the formermutation should result in a translational frameshiftand the latter should cause an amino-acid substitution.When geneA but not geneB in RB50 was disrupted byinsertional mutagenesis, the resultant variant failed tocause coughing paroxysms (data not shown). On day 15postinoculation, the geneA-deficient RB50 strain wasrecovered from the trachea to a similar extent to thewild-type RB50, indicating that the strain colonized thetrachea but did not cause coughing. Thus, we consider

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geneA, directly or indirectly, to be involved in thecoughing in infected rats.

Discussion

This study is not the first to use rats as an animalmodel of Bordetella infections. In 1939, Hornibrookand Ashburn described that young rats infected with B.pertussis via intranasal instillation developed coughingparoxysms resembling those seen in patients withwhooping cough (9). Later, it was shown that ratsinfected with B. pertussis reproduce manifestations ofthe human infection including leukocytosis and thecough paroxysms (10)(11). However, the B. pertussisneeded to be encased in agar beads, which weredirectly introduced into the trachea surgically. Probablybecause of these cumbersome procedures, their workwas little reexamined by other groups. In contrast,we utilized a rat model focusing on coughingparoxysms in this study. For this purpose, we used B.bronchiseptica instead of B. pertussis as the pathogen.B. bronchiseptica shares with B. pertussis variousvirulence factors that are highly homologous in amino-acid sequence. Several animal species including pigsand dogs exhibit a persistent cough when infected withB. bronchiseptica (1)(12). B. pertussis, which is highlyadapted to humans, is not suitable for establishingan infection model in experimental animals. Thisprompted us to use B. bronchiseptica for the ratmodel to explore the pathogenesis of B. pertussis. Inaddition, we used video cameras to record the coughingand video-audio playback software to enumeratethe coughing by checking both sounds (and soundwaveforms) and images.

Three-week-old female Wistar rats were foundto respond to B. bronchiseptica infections with apersistent cough. A large amount of B. pertussis,although the infection did not persist, caused asimilar cough, which assured us that rats infectedwith B. bronchiseptica could be a model of humanwhooping cough, at least in terms of the coughing.The frequency of coughing of rats infected withwild-type B. bronchiseptica was 140 coughs/30 minon average (Fig. 2). We consider this coughing tobe paroxysmal and the rat model to be useful for

exploring the pathogenesis of Bordetellae. We alsoisolated ΔC, a spontaneous mutant strain, which did notcause coughing in rats. A comparison of the genomesof ΔC and the parental strain RB50 revealed DNAmutations in two distinct genes, tentatively designatedgeneA and geneB. Notably, the present results implythat geneA is the more probable candidate involvedin the coughing of infected rats. The coughing isa common manifestation of the diseases caused byBordetellae including B. pertussis, B. parapertussis,and B. bronchiseptica. In a future study, we hope toclarify the mechanism responsible for the coughingparoxysms associated with B. bronchiseptica infectionsand eventually those seen in human whooping coughby analyzing the functions of geneA-like genes in B.pertussis.

Conclusion

We identified geneA of B. bronchiseptica, whichis probably involved in the paroxysmal coughing seenin infected rats. Work is now in progress to clarify thegene’s function and to the cause of the coughing seenin human whooping cough.

References

(1) Mattoo, S. & Cherry, J. D. Molecular pathogene-sis, epidemiology, and clinical manifestations ofrespiratory infections due to Bordetella pertus-sis and other Bordetella subspecies. 18, 326–382(2005).

(2) Elahi, S., Holmstrom, J. & Gerdts, V. The benefitsof using diverse animal models for studyingpertussis. Trends Microbiol 15, 462–468 (2007).

(3) Warfel, J. M., Beren, J., Kelly, V. K., Lee, G.& Merkel, T. J. Nonhuman Primate Model ofPertussis. Infect Immun 80, 1530–1536 (2012).

(4) Hausman, S. Z., Cherry, J. D., Heininger,U., Wirsing von Konig, C. H. & Burns, D.L. Analysis of proteins encoded by the ptxand ptl genes of Bordetella bronchiseptica andBordetella parapertussis. Infect Immun 64, 4020–4026 (1996).

(5) Parkhill, J. et al. Comparative analysis of the

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genome sequences of Bordetella pertussis, Borde-tella parapertussis and Bordetella bronchiseptica.Nat Genet 35, 32–40 (2003).

(6) Horiguchi, Y., Sugimoto, N. & Matsuda, M.Stimulation of DNA synthesis in osteoblast-likeMC3T3-E1 cells by Bordetella bronchisepticadermonecrotic toxin. Infect Immun 61, 3611–3615(1993).

(7) Leslie, P. H. & Gardner, A. D. The Phasesof Haemophilus pertussis. J. Hyg. 31, 423–434(1931).

(8) Sekiya, K. Supermolecular structure of the en-teropathogenic Escherichia coli type III secretionsystem and its direct interaction with the EspA-

sheath-like structure. Proc. Natl. Acad. Sci. 98,

11638–11643 (2001).(9) Hornibrook, J. & Ashburn, L. A study of

experimental pertussis in the young rat. PublicHealth Rep 54, 439–444 (1939).

(10) Hall, E., Parton, R. & Wardlaw, A. C.Cough production, leucocytosis and serologyof rats infected intrabronchially with Bordetellapertussis. J Med Microbiol 40, 205–213 (1994).

(11) Woods, D. E. et al. Development of a rat modelfor respiratory infection with Bordetella pertussis.Infect Immun 57, 1018–1024 (1989).

(12) Goodnow, R. A. Biology of Bordetella bron-chiseptica. Microbiol Rev 44, 722–738 (1980).

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The Role of Lymphoid Tissue Inducer-like Cells inChronic Antigen-induced Airway Inflammation

Hiroaki Takatori

Department of Allergy and Clinical Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana,Chiba City, Chiba 260-8670, Japan

Introduction

It has been shown that CD4+CD3−CD11c−B220−

cells (lymphoid tissue inducer-like cells; LTi-like cells)produce large amounts of TNF-α , LT-α , and LT-β , and are involved in the proper formation of thesecondary lymphoid organ (SLO) such as lymph nodes,spleen, and gut-associated lymphoid tissue (GALT)in the intestine (1-3). The interaction among LTi-likecells, IL-21-producing CXCR5+PD-1+CD4+T cells(follicular helper T cell; TFH cells), and GL-7+Fas+Bcells (germinal center B cells; GC-B cells) has beenshown to be required for the development of SLO (4, 5).Recently, we have shown that not only Th17 cells butalso LTi-like cells are able to produce both IL-17Aand IL-22 in spleen (6). On the other hand, it has beenreported that ectopic lymphoid tissues spontaneouslydevelop in the site of inflammatory diseases suchas rheumatoid arthritis (RA), Sjogren syndrome, andmultiple sclerosis (MS) in humans (7-11), and murinedisease models such as collagen-induced arthritis(CIA) and experimental allergic encephalomyelitis(EAE) (10-13). These ectopic lymphoid tissues in thesite of inflammatory diseases have been named astertiary lymphoid organ (TLO). However, the role ofLTi-like cells, TFH cells, or GC-B cells in the formationof TLO and the chronicity of the inflammatory diseasesremains largely unknown.

In this study, we first established murine modelof chronic allergic airway inflammation (CAAI) byrepetitive inhalation of ovalbumin (OVA) on OVA-

sensitized mice. We found that the numbers of LTi-like cells, TFH cells, and GC-B cells were dramaticallyincreased in the lungs of CAAI-induced mice ascompared with those in control mice, and that TLO-like lymphoid tissues were observed in CAAI-inducedmice but not in control mice. Moreover, we found thatintranasal administration of LPS or zymosan enhancedthe accumulation of LTi-like cells but not of TFH cellsor GC-B cells in the lungs of CAAI-induced mice.Finally, we found that the recruitment of neutrophilsand eosinophils into the airways was significantly lowerin CAAI-induced RORγ t-deficient mice, which arecongenitally lacking LTi-like cells (14), than CAAI-induced WT mice. Our results suggest that LTi-likecells play a critical role in the amplification andchronicity of allergic airway inflammation by inducingTLO formation in the lung.

Materials and Methods

Mice

BALB/c mice were purchased from CLEA(Tokyo, Japan). RORγ t-/- mice were kindly providedby Dr. Iwakura (University of Tokyo, Japan) and thenthose mice were backcrossed over eight generationsonto BALB/c mice. All mice were housed inmicroisolator cages under specific pathogen-freeconditions. The Chiba University Animal Care and UseCommittee approved the animal procedures used in thisstudy.

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Reagents and flow cytometric analysis (FACS) ofimmune cells from lung

Monoclonal antibodies to CD3 (145-2C11), CD4(H129.19), B220 (RA3-6B2), CD11c (N418), CXCR5(2G8) were purchased from Biolegend (San Diego,CA). Monoclonal antibodies to GL7 and Fas (Jo2)were purchased from BD Pharmingen (San Diego, CA).Cells were stained with the antibodies and analyzed ona FACSCalibur (BD Biosciences). Fluorescent eventswere collected and analyzed with Flow Jo software(Tree Star Inc., Ashland, OR).

Chronic antigen-induced allergic inflammation in theairways (CAAI)

Mice (age 6-8 weeks) were immunized intraperi-toneally twice with 6μg OVA (Sigma Chemical Co, StLouis, Mo) in 4 mg aluminum hydroxide (alum) at 2-week intervals. Two weeks after the second immuniza-tion, the sensitized mice were given with aerosolizedOVA (50 mg/mL) dissolved in 0.9% saline by a DeVil-biss 646 nebulizer (DeVilbiss Corp, Somerset, Pa) for20 minutes 6 or 12 times at a 24–hour interval. Asa control, 0.9% saline alone was administered by thenebulizer. Twenty-four hours after the last inhalation,a sagittal block of left lung and trachea was excised,fixed in 10% buffered formalin, and embedded in paraf-fin. Sections of lung (3 mm thick) were stained withhematoxylin-eosin (HE) according to standard proto-cols. The number of eosinophils and neutrophils re-covered in the bronchoalveolar lavage fluid (BALF)was evaluated and a fraction of the cells was subjectedto a flow cytometric analysis for the lymphocyte sur-face phenotyping. In some experiments, LPS (20 mgper mouse) or zymosan (20 mg per mouse) (Sigma, St.Louis, MO) was administrated intranasally just beforeeach inhaled OVA challenge.

Isolation of immune cells from the lungs

Lungs of CAAI-induced mice were cut into smallfragments and digested with collagenase A solution(1 mg/ml, Roche, Indianapolis, IN) in Hanks’ balanced

salt solution containing 10% fetal calf serum for10 minutes at 37◦C with continuous agitation. Afterfiltering through 0.07 mm nylon mesh to removeaggregates, viable lymphocytes were collected fromthe cell suspension by using Lympholyte-M solution(Cedarlane laboratories, Ontario, Canada) according tothe manufacture’s instructions.

Data analysis

Data are summarized as means ± SDs. Thestatistical analysis of the results was performed bythe unpaired t test. P values < .05 were consideredsignificant.

Results

LTi-like cells, GC-B cells, and TFH cells are increasedin the lungs of CAAI-induced mice

To determine whether immune cells suchas CD4+CD3−CD11c−B220− LTi-like cells,B220+GL7+Fas+ GC-B cells, and CD4+CD3+PD-1+

TFH cells are involved in antigen-induced lung inflam-mation and TLO development, we first examined thenumbers of these cells in the lungs of CAAI-inducedmice. As shown in Figure 1, the frequency of LTi-likecells, GC-B cells, and TFH cells was dramaticallyincreased in the lungs of CAAI-induced mice (Fig. 1b,f, j) as compared with that in control mice (Fig. 1a, e,i). Because it has been shown that infectious agentsfacilitate exacerbation and chronicity of inflammatorydiseases, we next examined the effect of intranasaladministration of LPS and zymosan, which are derivedfrom cell wall of Gram-negative bacteria and yeast,respectively, on the induction of LTi-like cells, GC-Bcells, and TFH cells in the lungs of CAAI-induced mice.Interestingly, both LPS and zymosan up-regulated thefrequency of LTi-like cells (Fig. 1c, d). On the otherhand, neither LPS or zymosan did not up-regulate thefrequency of GC-B cells and TFH cells (Fig. 1). Inaddition, the absolute numbers of LTi-like cells, GC-Bcells, and TFH cells were significantly increased byadditional inhalation of OVA (Fig. 2). These resultsindicate that lymphoid tissue-organizing immune cells

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Fig. 1. The frequencies of LTi-like cells, GC-B cells, and TFH cells are increased in the lung ofCAAI-induced mice.OVA-sensitized BALB/c mice were challenged with OVA inhalation 6 times. Whereindicated, LPS or zymosan was administrated intranasally to mice just before each OVAinhalation. Immune cells were isolated from the lungs and analyzed by FACS. RepresentativeFACS profiles of CD4 versus CD3 staining on CD11c−B220− cells (upper panels), GL-7versus Fas staining on B220+ cells (middle panels), or PD-1 versus CXCR5 staining onCD4+T cells (bottom panels) are shown (n = 4 mice, each).

such as LTi-like cells, GC-B cells, and TFH cells canemerge not only in SLO but also in the inflammatorylesions and that these cells are significantly increasedin the lungs in accordance with numbers of antigeninhalation.

TLO-like tissues develops in the lungs of CAAI-inducedmice

We next investigated whether TLO-like tissuesdeveloped in the lung in CAAI-induced mice. Asexpected, histological analysis revealed that TLO-liketissues developed at the peribronchial and perivascularinflammatory sites in the lungs of CAAI-induced mice(Fig. 3). Consistent with the data shown in Fig. 2,the extent of TLO-like tissues became severer in

accordance with numbers of OVA inhalation (Fig. 3).Those results suggest that LTi-like cells together withGC-B cells and TFH cells may play an important role inthe development of TLO-like tissues in CAAI-inducedmice.

Recruitment of neutrophils and eosinophils into theairways is attenuated in CAAI-induced RORγ t-deficientmice

To investigate whether LTi-like cells are requiredfor the induction of CAAI, we finally examined sever-ity of airway inflammation in CAAI-induced RORγ t-deficient mice which are congenitally lacking LTi-likecells. As shown in Fig. 4, antigen-induced neutrophilrecruitment into the airways was significantly attenu-

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Fig. 2. Absolute numbers of LTi-like cells, GC-B cells, and TFH cells are significantly increased inthe lungs of CAAI-induced mice.OVA-sensitized BALB/c mice were challenged with OVA inhalation once, 6 times, or 12times. Immune cells were harvested from lungs at 24 hours after the last OVA inhalationand analyzed by FACS. The numbers of CD4+CD3−CD11c−B220− cells (LTi-like cells),GL7+FAS+B cells (GC-B cells), and CXCR5+PD-1+ CD4+T cells (TFH cells) were shown.Data are means ±SD; n = 4 mice in each group; ∗P < 0.05, ∗∗P < 0.01.

Fig. 3. TLO-like lymphoid tissues develop in the lungs of CAAI-induced mice. OVA-sensitizedBALB/c mice were challenged with OVA inhalation once, 6 times, or 12 times.Representative photomicrographs of lung sections (HE staining) are shown (n = 4).

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Fig. 4. Neutrophil and eosinophil recruitment into the airways is down-regulated in CAAI-inducedRORγ t-/- mice. OVA-sensitized RORγ t-/- mice and WT mice on a BALB/c background werechallenged with the inhalation of OVA 6 times. The numbers of neutrophils and eosinophilsin the BALF were evaluated at 18 hours (upper panels) and 48 hours (bottom panels) afterthe last OVA inhalation. Data are means ±SD (n = 4). ∗∗P < 0.01. ND, not detectable.

ated in CAAI-induced RORγ t-deficient mice as com-pared with that in CAAI-induced WT mice at neu-trophilic phase (Fig. 4, upper panels). Antigen-inducedeosinophil recruitment into the airways was also signif-icantly attenuated in CAAI-induced RORγ t-deficientmice as compared with that in CAAI-induced WT miceat eosinophilic phase (Fig. 4, lower panels). These re-sults suggest that LTi-like cells may be essential for thedevelopment of CAAI in mice.

Discussion

In the current study, we show that the numbersof LTi-like cells, GC-B cells, and TFH cells, whichhas been shown to function as the organizer of SLOformation (1-5), are markedly increased in the lungsof CAAI-induced mice (Fig. 1 and 2). Moreover, wefound that repetitive inhalation of OVA (6 and 12

times) enhanced the formation of TLO-like tissuesin the peripronchial and perivascular sites of lungs(Fig. 3). Regarding the role of LTi-like cells in vivo,we have previously reported that the administrationof zymosan induces the production of IL-22 fromsplenic LTi-like cells (6). Recently, it has also beenshown that IL-22-producing LTi-like cells are requiredfor the clearance of bacterial infection in the murineintestine (15). However, physiological relevance of LTi-like cells to inflammatory diseases is still largelyunknown. Interestingly, we show here that LTi-likecells also emerge in the inflamed lung tissue in CAAI-induced mice. In addition we found that the deficiencyof LTi-like cells resulted in the attenuation of neutrophiland eosinophil recruitment into the airways in CAAI-induced mice. Our results suggest that LTi-like cells,in concert with GC-B cells and TFH cells, may beinvolved in the persistence of allergic inflammation by

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forming TLO-like tissues and raise the possibility thatthose cells could be novel therapeutic targets in allergicdiseases such as bronchial asthma.

Conclusion

LTi-like cells may induce TLO-like tissues inconcert with GC-B cells and TFH cells and may playa critical role in the amplification and chronicity ofallergic airway inflammation.

References

(1) Mebius, R. E. 2003. Organogenesis of lymphoidtissues. Nat Rev Immunol 3: 292-303.

(2) Ruddle, N. H., and E. M. Akirav. 2009. Secondarylymphoid organs: responding to genetic andenvironmental cues in ontogeny and the immuneresponse. J Immunol 183: 2205-2212.

(3) van de Pavert, S. A., and R. E. Mebius. Newinsights into the development of lymphoid tissues.Nat Rev Immunol 10: 664-674.

(4) McHeyzer-Williams, L. J., N. Pelletier, L. Mark,N. Fazilleau, and M. G. McHeyzer-Williams.2009. Follicular helper T cells as cognateregulators of B cell immunity. Curr Opin Immunol21: 266-273.

(5) Linterman, M. A., and C. G. Vinuesa. Signals thatinfluence T follicular helper cell differentiationand function. Semin Immunopathol 32: 183-196.

(6) Takatori, H., Y. Kanno, W. T. Watford, C. M.Tato, G. Weiss, Ivanov, II, D. R. Littman, and J. J.O’Shea. 2009. Lymphoid tissue inducer-like cellsare an innate source of IL-17 and IL-22. J Exp Med206: 35-41.

(7) Weyand, C. M., and J. J. Goronzy. 2003.Ectopic germinal center formation in rheumatoidsynovitis. Ann N Y Acad Sci 987: 140-149.

(8) Stott, D. I., F. Hiepe, M. Hummel, G. Steinhauser,and C. Berek. 1998. Antigen-driven clonalproliferation of B cells within the target tissue ofan autoimmune disease. The salivary glands ofpatients with Sjogren’s syndrome. J Clin Invest102: 938-946.

(9) Prineas, J. W. 1979. Multiple sclerosis: presenceof lymphatic capillaries and lymphoid tissue in thebrain and spinal cord. Science 203: 1123-1125.

(10) Drayton, D. L., S. Liao, R. H. Mounzer, and N.H. Ruddle. 2006. Lymphoid organ development:from ontogeny to neogenesis. Nat Immunol 7:344-353.

(11) Aloisi, F., and R. Pujol-Borrell. 2006. Lymphoidneogenesis in chronic inflammatory diseases. NatRev Immunol 6: 205-217.

(12) Han, S., S. Cao, R. Bheekha-Escura, and B.Zheng. 2001. Germinal center reaction in thejoints of mice with collagen-induced arthritis:an animal model of lymphocyte activation anddifferentiation in arthritis joints. Arthritis Rheum44: 1438-1443.

(13) Magliozzi, R., S. Columba-Cabezas, B. Serafini,and F. Aloisi. 2004. Intracerebral expressionof CXCL13 and BAFF is accompanied byformation of lymphoid follicle-like structures inthe meninges of mice with relapsing experimentalautoimmune encephalomyelitis. J Neuroimmunol148: 11-23.

(14) Eberl, G., S. Marmon, M. J. Sunshine, P. D.Rennert, Y. Choi, and D. R. Littman. 2004.An essential function for the nuclear receptorRORgamma(t) in the generation of fetal lymphoidtissue inducer cells. Nat Immunol 5: 64-73.

(15) Sonnenberg, G. F., L. A. Monticelli, M. M. Elloso,L. A. Fouser, and D. Artis. CD4(+) lymphoidtissue-inducer cells promote innate immunity inthe gut. Immunity 34: 122-134.

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Immune mechanisms involved in the development offatal autoimmune hepatitis in mice

Norihiko Watanabe

Center for Innovation in Immunoregulative Technology and Therapeutics, and Department of Gastroenterology andHepatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

Introduction

Fulminant hepatitis, potentially fatal liver disease,is a rare but develops globally in children and adultsof both sexes in diverse ethnic groups. Fulminanthepatitis rapidly develops and often progresses to life-threatening acute hepatic failure. Liver transplantationis considered an established life-saving procedurefor fulminant hepatitis, whereas the patients treatedwithout liver transplantation have very high mortality.Immune dysregulation in the liver appears to beinvolved in the development of fulminant hepatitiscaused by various etiologies, such as autoimmunity,virus infection and drug injury. However, the lowincidence of fulminant hepatitis makes it difficultto clarify mechanisms of the development. To findany clues to overcoming therapeutic insufficiency forfulminant hepatitis, preclinical animal models for detailexamination are needed.

Although the liver exhibits a distinctive form ofimmune privilege called liver tolerance, autoimmuneliver diseases including autoimmune hepatitis (AIH)develop. AIH shows varied clinical manifestationsranging from non-symptomatic mild chronic hepatitisto fulminant hepatic failure. The histological findingsof AIH are characterized by a mononuclear-cellinfiltration invading the parenchyma, ranging frompiecemeal necrosis to submassive lobular necrosis. Theserologic hallmark of AIH is the production of a varietyof characteristic circulating autoantibodies, includinganti-nuclear Abs (ANAs). Although AIH appears to bea T-cell mediated autoimmune disease, it is unclear

which type of effector T cells are involved and howthe dysregulated T cells trigger the development of fatalAIH.

Recently, we developed the first mouse modelof spontaneous fatal AIH resembling acute-onset AIHpresenting fulminant hepatic failure in humans. Neitherprogrammed cell death 1-deficient mice (PD-1-/- mice)nor BALB/c mice thymectomized three days after birth(NTx mice), which severely reduces the number ofnaturally arising Foxp3+ regulatory T cells (Tregs)in periphery, developed any inflammation of theliver. However, PD-1-/- BALB/c mice with neonatalthymectomy (NTx–PD-1-/- mice) developed fatal AIH,suggesting that immune dysregulation by a concurrentloss of naturally arising Tregs and PD-1–mediatedsignaling can induce the development of fatal AIH.Because of the massive destruction of the parenchymaof the liver, these mice started to die as early as twoweeks of age, with most dying by four weeks. FatalAIH in NTx–PD-1-/- mice was characterized by CD4+

and CD8+ T-cell infiltration with massive lobularnecrosis and huge ANA production. We showed thatthe infiltrated CD4+ and CD8+ T cells in the severelydamaged liver produced large amounts of inflammatorycytokines, such as IFN-γ and TNF-α .

In this study, using the new mouse model of AIHmanifesting fulminant hepatic failure in humans, weexamined immune mechanisms in the development offulminant hepatitis.

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Materials and Methods

Mice

BALB/c mice were purchased from JapanSLC (Shizuoka, Japan), and PD-1-/- mice on aBALB/c background were generated as describedpreviously (1-3). These mice were bred and housedunder specific pathogen-free conditions. Thymectomyand splenectomy of the mice three days after birthwas performed as described previously (1-3). Allmouse protocols were approved by the Institute ofLaboratory Animals, Graduate School of Medicine,Kyoto University.

Administration of Abs in vivo

NTx–PD-1-/- mice at one day after thymectomywere intraperitoneally injected every week with 100 μgof indicated Abs (1-3). All isotypes were fromeBioscience or R&D Systems. After four injections,mice at four weeks of age were sacrificed, and theirspleens and livers were harvested.

Histological and immunohistological analysis

Organs were fixed in neutral buffered formalin,embedded in paraffin wax, and cut into sections 4 μmthick. These sections were stained with hematoxylinand eosin for histopathology. Fluorescence immunohis-tology was performed on frozen sections using FITC-conjugated anti-CD4 or anti-CD8α (eBioscience, SanDiego, CA), peanut agglutinin (PNA, Vector Labora-tories, Burlingame, CA), biotin-labeled anti-B220 (BDBiosciences, San Jose, CA) followed by Texas red-conjugated avidin (Vector Laboratories) as describedpreviously (1-3).

Depletion of CD4+ T cells and Treg cell isolation

Single cells were isolated from the spleen of3-week-old NTx–PD-1-/- mice. CD4+ T cells weredepleted as described (1-3). Purity was assessed byflow cytometry, and CD4− splenocytes reached > 99%

purity. For CD4+CD25+ Treg cell isolation, single cellswere isolated from the spleen of adult BALB/c or PD-1deficient mice. Treg cells were isolated with a CD4+Tcell isolation kit followed by a CD25 microbead kit(Miltenyi Biotec) to reach > 90% purity.

Adoptive transfer

CD4+ T-cell depleted, splenocytes were preparedfrom NTx–PD-1-/- mice as described above. Spleno-cytes (1×106) were intravenously injected into RAG2-deficient recipient mice on BALB/c background at 4-6week of age. For Treg cell transfer, 1×106 of Treg cellsprepared from adult BALB/c or PD-1 deficient micewere intraperitoneally injected into NTx–PD-1-/- mice.

Real-time quantitative reverse-transcription poly-merase chain reactions (RT-PCR)

Real-time quantitative RT-PCR was performed asdescribed (1-3). Spleen and liver tissues or isolatedcells from the spleen and liver were frozen inRNAlater. RNA was prepared with an RNeasymini kit (Qiagen, Hilden, Germany), and single-strand complementary DNA was synthesized withSuperScriptTM II reverse transcriptase (Invitrogen).Real-time quantitative RT-PCR was performed usingSYBR Green I Master (Roche Applied Science, Basel,Switzerland) and the primers as described (1-3). Thereal-time quantitative reactions were performed using aLight Cycler 480 (Roche Applied Science) according tothe manufacturer’s instructions. Values are expressed asarbitrary units relative to glyceraldehyde-3-phosphatedehydrogenase (GAPDH).

Flow cytometry analysis

Single cells from the livers and spleens were pre-pared, and flow cytometric analysis was performedas described (1-3). Stained cells were analyzed withFACSCantoTM II (BD Biosciences). Data were ana-lyzed using Cell Quest ProTM (BD Biosciences). Deadcells were excluded based on side- and forward-scattercharacteristics. The number of viable indicated cellswas calculated as follows: (the percentage of cells in

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the cell type) x (the number of viable cells).

Enzyme-linked immunosorbent assay (ELISA)

Serum concentrations of cytokines were measuredby using ELISA sets according to the manufacturer’sprotocols. To detect serum ANAs, microtiter plates(Nunc, Roskilde, Denmark) were incubated with10 μg/ml antigens, and the nuclear fraction wasprepared from normal liver. Ab sets for detection ofmouse IgG1, IgG2a, IgG2b, and IgG3, from BDBiosciences (San Jose, CA) and anti-mouse IgM fromAbD Serotec (Oxford, UK) were used.

Statistical analysis

The data are presented as the mean values ±SD.Statistical analysis was performed by the Student’s t-test for unpaired data to compare the values betweentwo groups; variance was analyzed with the Tukey-Kramer test for multiple comparisons. Survival rateswere estimated by the Kaplan-Meier method andcompared with the log-rank test. P-values below .05were considered significant.

Results

Dysregulated generation of follicular helper T cells inthe spleen triggers fatal AIH.

In humans, liver tissue injury in AIH is mediatednot only by CD4+ but also by CD8+ T cells. Especiallyin acute-onset human AIH, activated CD8+ T cellsare thought to play a crucial role in the pathogenesis.To examine which T cells are indispensable for thedevelopment of fatal AIH, NTx–PD-1-/- mice wereinjected intraperitoneally at one day after NTx andthen once a week with either anti-CD4 or anti-CD8mAbs in vivo. After four injections of anti-CD4 or anti-CD8, the number of CD4+ or CD8+ T cells in theperiphery was greatly reduced, respectively, and fatalAIH was suppressed by these treatments. Importantly,depletion of CD4+ T cells inhibited the infiltrationof CD8+ T cells in the liver, whereas depletion ofCD8+ T cells allowed CD4+ T cells to infiltrate in

the liver. These data suggest that both CD4+ andCD8+ T cells are indispensable for the developmentof fatal AIH. In addition, in the development of fatalAIH, the recruitment of CD8+ T cells in the liver isregulated by CD4+ T cells, whereas activated CD8+ Tcells are mainly involved in progression to fatal hepaticdamage. We had previously demonstrated that transferof total but not CD4+ T-cell depleted splenocytesfrom NTx–PD-1-/- mice into RAG2-/- mice inducedthe development of severe hepatitis. Interestingly,neonatal splenectomy in NTx–PD-1-/- mice suppressedmononuclear infiltration as well as destruction of organstructure in the liver, showing a significantly highersurvival rate. Taken together, these data suggest thatsplenic CD4+ T cells are responsible for induction offatal AIH.

Hepatic damage from AIH in NTx–PD-1-/- micewas apparent at two to three weeks of age. Whenwe looked in situ at the spleen of two-week-oldNTx–PD-1-/- mice, most of the CD4+ T cells werepreferentially localized within B220+ B-cell folliclesthat autonomously developed PNA+ germinal centers(GCs), whereas CD8+ T cells were mainly localizedoutside the follicles. The rapid accumulation of CD4+

T cells in the follicles with GC formation dependedon concurrent loss of naturally arising Tregs and PD-1 mediated signaling, because neither PD-1-/- mice norNTx-BALB/c mice at same age showed any of thesephenotypes. In addition, NTx-BALB/c mice injectedwith blocking mAbs to both PD-L1 and PD-L2 showedan accumulation of CD4+ T cells in the follicles anddevelopment of GCs in the spleen. Moreover, Foxp3expression of CD4+ T cells in the spleen of NTx–PD-1-/- mice was severely reduced and transfer of Tregsfrom either normal BALB/c or PD-1-/- mice into NTx–PD-1-/- mice suppressed GC formations in the spleenand the development of fatal AIH.

We next examined whether accumulated CD4+ Tcells in the follicles of the spleen display the molecularsignature of follicular helper T (TFH) cells. TFH cellsprovide a helper function to B cells and are responsiblefor GC formation in the B cell follicles. TFH cells arisefrom activated T cells that express the transcriptionfactor Bcl-6. Differentiated TFH cells express IL-21, IL-21R, inducible costimulator (ICOS), CXCR5, and PD-

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1. Both IL-21 and ICOS are indispensable for TFH-cellgeneration and helper function to B cells. Chemokinereceptor CXCR5 promotes the colocalization of T andB cells in GCs. Indeed, CD4+ T cells in the spleenof one-week-old NTx–PD-1-/- mice showed increasedIL-21 mRNA expression. In addition, CD4+ T cellsisolated from the spleen of 2.5 to 3-week-old NTx–PD-1-/- mice expressed Bcl-6, IL-21, ICOS and CXCR5,indicating some key features of TFH cells.

To examine the link between generation ofTFH cells and the development of fatal AIH, weadministrated blocking mAb to ICOS or IL-21 in vivo.After four injections of anti-ICOS in vivo, NTx–PD-1-/-

mice at four weeks of age greatly reduced accumulationof CD4+ T cells in the follicles, the development ofGC, hyper-gammaglobulinemia and ANA production,including class-switched Abs. Although four-week-oldmice injected with isotype control developed severeAIH, anti-ICOS injection completely suppressed thedevelopment of fatal AIH. In addition, after fourinjections of antitIL-21 blocking antibodies in vivo,NTx–PD-1-/- mice at four weeks of age showedmarkedly suppressed generation of TFH cells and GCformation in the spleen as well as the development offatal AIH. These data suggest a link between generationof TFH cells and development of fatal AIH.

CCR6-CCL20 axis-dependent migration plays a cru-cial role for dysregulated TFH cells to induce fatal AIH

Tissue infiltration of activated T cells expressedspecific chemokine receptors depends on expressionof the its ligands in the inflamed tissues. We foundthat CD4+ T cells in the spleen and liver expressedCCR6 in NTx–PD-1-/- mice and the CCR6+ cells,including CXCR5+CCR6+ cells, were increased insplenic and hepatic CD4+ T cells in two-week-oldmice. In addition, gene expression of CCR6 ligandCCL20 was elevated in the liver of 1.5 and 2-week-old NTx–PD-1-/- mice. To evaluate whetherCCR6 expressing CD4+ T cells migrate into CCL20expressing liver and trigger the development of fatalAIH, we administrated blocking mAb to CCL20 invivo. After four injections of anti-CCL20, diffuseaccumulation of TFH cells in the GC+follicles were

observed, whereas the development of fatal AIH wassuppressed. These data suggest that CCR6-CCL20axis-dependent migration plays a crucial role fordysregulated TFH cells to induce fatal AIH (Figure 1).

Inflammatory Th1 responses are dominant in theprogression process of fatal AIH.

Next, we examined mechanisms in the progressionprocess to identify key molecules triggering the fatalprogression of AIH. Inflamed liver tissues of NTx–PD-1-/- mice at three weeks of age expressed Th1 lineage-specific transcription factor T-bet together with IFN-γ and TNF-α , showing an inflammatory Th1 profile.Indeed, in the progression phase of AIH in three-week-old NTx–PD-1-/- mice, infiltrated CD4+ andCD8+ T cells in the liver produced large amounts ofinflammatory cytokines, such as IFN-γ and TNF-α . Inaddition, we examined serum levels of IFN-γ and TNF-α at one to three weeks of age and found that the serumlevels of both IFN-γ and TNF-α were elevated fromone to two weeks of age through the development offatal AIH.

Using neutralizing mAb to mouse IFN-γ , we eval-uated whether IFN-γ is essential in the development offatal AIH. NTx–PD-1-/- mice were injected intraperi-toneally with 100 μg of anti- IFN-γ . After four injec-tions, mice at four weeks of age showed that neutraliza-tion of IFN-γ did not suppress, but rather exacerbatedinflammation of the liver in AIH. Four-week-old NTx–PD-1-/- mice with injection of the isotype control de-veloped severe mononuclear cell infiltration in the liverand a massive degeneration of hepatocytes, whereas in-jection of anti-IFN-γ showed more severe mononuclearcell infiltration in the liver with a massive degenerationof hepatocytes. In addition, injection of anti-IFN-γ re-vealed significantly increased serum concentrations ofAST at four weeks of age. These data suggest that neu-tralization of IFN-γ worsens inflammation of the liverin AIH, implying that IFN-γ acts as negative regulatorfor the development of AIH in NTx–PD-1-/- mice.

Next, we examined whether neutralization ofTNF-α can suppress the development of fatal AIHat four weeks of age. NTx–PD-1-/- mice at one dayafter thymectomy were injected intraperitoneally every

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Fig. 1. Splenic CD4+ T cells in NTx–PD-1 KO mice are localized within GC-bearing B-cell folliclesand display the molecular signature of TFH (T Follicular Helper) cells. In the induction ofAIH, migration of splenic T cells into the liver is regulated by CCR6-CCL20 axis.

week with 100 μg of neutralizing mAb to mouseTNF-α or the isotype control. After four injections,neutralization of TNF-α suppressed mononuclearcell infiltration in the liver and degeneration ofhepatocytes and showed significantly decreased serumconcentrations of aspartate aminotransferase andalanine aminotransferase, and total bilirubin as well assignificantly increased survival rate at four weeks ofage. These data indicate that neutralization of TNF-αsuppressed the development of fatal AIH in NTx–PD-1-/- mice, suggesting an essential role for TNF-α in thedevelopment of AIH. Next, we investigated whetherneutralization of TNF-α could suppress progressionof fatal AIH even after the development of AIH.In NTx–PD-1-/- mice from two to three weeks ofage, mononuclear cell infiltrations in the liver rapidlyprogressed and were followed by massive destructionof the parenchyma of the liver. NTx–PD-1-/- mice at 14days after thymectomy were injected with anti–TNF-α; after the second injection, mice at four weeks ofage were sacrificed. The neutralizing TNF-α did notsignificantly increase the survival rate, and it did notreduce a massive degeneration of hepatocytes or severe

CD4+and CD8+ cell infiltration in the liver at fourweeks of age. Thus, neutralization of TNF-α after thedevelopment of hepatitis did not significantly suppressprogression of fatal AIH.

Since production of ANA in IgG2a subclassincreased in NTx–PD-1-/- mice, we examined whetherneutralization of IL-12 (which play decisive roles inthe development of Th1 cells) affect ANA productionand the development of fatal AIH. After four injectionsof neutralizing anti-IL-12p40 in vivo, production ofANA in the IgG2a subclass was significantly reduced.However, these neutralizations did not suppress thedevelopment of fatal AIH, suggesting that IL-12 is notexclusively involved in differentiation into T cells withfeatures of Th1 cells in the progression of fatal AIH.

IL-18–mediated signaling is critical for the develop-ment of fatal AIH in NTx–PD-1-/- mice.

Next, we performed global quantitative mRNAscreening of Th1-related cytokine receptors in isolatedsplenic and hepatic CD8+ and CD4+ T cells fromthree-week-old NTx–PD-1-/- mice and found that gene

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expression of IL-18Rα were elevated in 3-week-oldNTx–PD-1-/- mice. When we looked at serum levels ofIL-18 at one to three weeks of age, the serum levels ofIL-18 were elevated from two to three weeks throughthe progression of AIH.

IL-18 receptor complex (IL-18R) is the het-erodimer IL-18Rα and IL-18Rβ subunits. The IL-18Rβ subunit is nonbinding but confers high affinitybinding for the ligand and is responsible for biologi-cal signals. Next, to examine the roles of IL-18 in thedevelopment of fatal AIH, NTx–PD-1-/- mice were in-jected with neutralizing IL-18Rβ mAb. We found thatadministering anti-IL-18Rβ suppressed fatal progres-sion of AIH suggesting that IL-18–mediated signalingis critical for the development of fatal AIH in NTx–PD-1-/- mice.

Discussion

Although immune dysregulation in the liverappears to be involved in the development of fulminanthepatitis, it has been unclear immune mechanisms ofthe development. We found that immune dysregulationby a concurrent loss of naturally arising Tregs and PD-1–mediated signaling can induce the development offulminant hepatitis.

Treg cells are critical to maintain immunologicself-tolerance and to regulate a variety of pathologicaland physiological immune responses. Treg cellsspecifically express the transcription factor Foxp3that is required for their development and regulatoryfunction. In patients with AIH, Treg cells are reducednumerically and functionally, and Foxp3 expression ofTreg cells is lower than in normal subjects. However,loss-of-function mutations in the gene encoding Foxp3in humans result in the development of organ-specificautoimmune diseases without fulminant hepatitis.Therefore, it had been unclear whether dysfunction ofTreg cells is primarily involved in the development ofAIH resembling fulminant hepatitis.

On the other hands, PD-1 provides negative co-stimulation to antigen stimulation. The expression ofPD-1 can be induced not only in T cells but alsoin B cells and myeloid cells. The ligands for PD-1 are PD-L1 and PD-L2. PD-L1 is constitutively

expressed in T cells, B cells, macrophages, anddendritic cells, and upregulated following activationof these cells. The expression of PD-L1 is alsodetected on non-lymphoid cells. By contrast, PD-L2 expression is detected on activated macrophagesand DCs. In the human liver, numbers of PD-1–expressing lymphocytes are increased in chronic liverdiseases including viral hepatitis and AIH, and PD-1 ligand expression is upregulated on Kupper cells,DCs, sinusoidal endothelial cells, and hepatocytes.In mice, PD-L1 is constitutively expressed in theliver. Mice deficient in PD-L1 displayed increasedconstitutive accumulation of activated CD8+ T cells inthe liver, suggesting that PD-L1 might be involved inthe elimination of CD8+ T cells. While the PD-L1–PD-1 pathway contribute to the regulation of immunereaction in the liver, it had been unclear whetherdysfunction of PD-1 pathway is primarily involved inthe development of fulminant hepatitis.

In this study, we identified key moleculestriggering the progression of fatal AIH. In NTx–PD-1-/- mice, the dysregulated generation of TFH cellsinitially occurred in the spleen and these TFH cellsin the spleen directly migrated into the liver via theCCR6-CCL20 axis, triggering the induction of fatalAIH. Following the induction, fatal progression of AIHis mediated by IL-18. IL-18 stimulates Th1-mediatedimmune responses and activates Th1 cells, whichhighly express functional IL-18 receptor, producinglarge amounts of IFN-γ . Indeed, in our model, CD4+

and CD8+ T cells in the spleen and liver increased IL-18Rα mRNA expression. IL-18 may directly and/orindirectly modulate further differentiation of T cells infatal progression of AIH.

The chemokine receptor CXCR3 and its ligands—CXCL9, CXCL10, and CXCL11— constitutean inflammatory chemokine system coordinatingmigration of Th1 type CD4+ and effector CD8+ T cellsinto the inflamed sites in various immunoinflammatorysettings. In the fatal progression phase in these mice,CD8+ T cells, and to a lesser extent CD4+ T cells,were extensively increased in the liver. Interestingly,we found that splenic and hepatic CD4+ and CD8+ Tcells mainly expressed CXCR3. In addition, NTx–PD-1-/- mice showed markedly elevated gene expression of

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the CXCR3 ligand CXCL9 but not of CXCL10 andCXCL11. Indeed, anti-CXCL9 injections suppressedfatal progression of AIH, suggesting that in theprogression phase of fatal AIH, the CXCR3-CXCL9axis is crucial for migration of effector T cells into theliver to lead Th1 responses. Notabely, blocking of IL-18signal reduced the number of CXCR3+ T cells. Thus,IL-18 may directly and/or indirectly modulate furtherdifferentiation of TFH cell into Th1-like cells in fatalprogression of AIH.

IL-18 is known to be produced by various typesof cells, including activated macrophages, Kupffercells, DCs, B cells, T cells and epithelial cellsand is involved in the crosstalk between the innateimmunity and adaptive immunity. In our model, wefound that DCs in the spleen and liver highly producedIL-18. Innate immunity including mucosal immunityinduced by commensal bacteria can modulate DCactivation status. Commensal bacteria may directlyand/or indirectly modulate IL-18 production of DCsand final differentiation of T cells in the fatalprogression of AIH.

It is at present unknown whether IL-18 isinvolved in the fatal progression of human fulminanthepatitis. However, in several inflammatory diseases,including autoimmune diseases, IL-18 has been shownto be involved in disease processes associated withexcessive Th1 responses in humans. Patients withfulminant hepatitis show elevated serum levels of IL-18. Therefore, blocking IL-18R signaling may bea therapeutic option to prevent fatal progression offulminant hepatitis.

In conclusion, we have identified the pivotal roleof the IL-18 in fatal progression of AIH, implying thatblocking IL-18 signaling may have clinical potential

for protecting against fatal progression of fulminanthepatitis.

Acknowledgements

We thank Dr. Dovie Wylie for assistance inpreparation of the manuscript. This work is supportedby Grants-in-Aid for Scientific Research by TheWaksman Foundation of Japan. Center for Innovationin Immunoregulative Technology and Therapeutics issupported in part by the Special Coordination Fundsfor Promoting Science and Technology of the JapaneseGovernment and in part by Astellas Pharma Inc. inthe Formation of Innovation Center for Fusion ofAdvanced Technologies Program.

References

(1) M. Kido, N. Watanabe, T. Okazaki, T. Akamatsu,J. Tanaka, K. Saga, A. Nishio, T. Honjo, T. Chiba,Fatal autoimmune hepatitis induced by concurrentloss of naturally arising regulatory T cells andPD-1-mediated signaling, Gastroenterology 135(2008) 1333-1343.

(2) N. Aoki, M. Kido, S. Iwamoto, H. Nishiura, R.Maruoka, J. Tanaka, T. Watanabe, Y. Tanaka, T.Okazaki, T. Chiba, N. Watanabe, DysregulatedGeneration of Follicular Helper T cells in thespleen Triggers Fatal Autoimmune Hepatitis inMice, Gastroenterology 140 (2011) 1322-1333.

(3) S. Iwamoto, M. Kido, N. Aoki, H. Nishiura, R.Maruoka, A. Ikeda, T. Chiba, N. Watanabe. IFN-γis reciprocally involved in the concurrent develop-ment of organ-specific autoimmunity in the liverand stomach. Autoimmunity 45 (2012) 186-198.

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Application of CD8+ regulatory T cells to InflammatoryBowel Disease

Haruhiko Suzuki

Department of Immunology, Nagoya University Graduate School of Medicine 65 Tsurumai-cho, Showa-ku, Nagoya466-8550, Japan

Introduction

Conventional mature T cells expressing αβ -typeT cell receptor (TCR) are largely divided into CD4+

cells and CD8+ cells. It is somehow believed that CD4+

cells are the “helper” T cells that secret cytokines andhelp the function of many kinds of immune-relatedcells including CD4+ T cells themselves, CD8+ Tcells, B cells, natural killer (NK) cells, NKT cells,macrophages, granulocytes, dendritic cells (DC) andso on, and that CD8+ cells are the “cytotoxic” or“killer” T cells that kill virus-infected cells, allogeneictransplanted cells and possibly tumor cells. In additionto the “helper” and “cytotoxic/killer” T cells, thefunctional classification of T cells requires the thirdpopulation of T cells that has suppressive activity forimmune responses.

More than 30 years ago, T cells that suppress theaction of other T cells were first identified and werenamed as “suppressor” T cells. The study of suppressorT cells rapidly expanded but suddenly shrank by somereason. The weakest point of suppressor T cells wasthat such suppressor T cells could not be distinguishedfrom other conventional helper or cytotoxic T cells bysome markers. Investigators working on suppressor Tcells tried to induce suppressor T cells and succeededin enriching suppressor cells. However, no one couldanswer how many percentages of cells were realsuppressor cells in such a mixture of different typesof cells even if the suppressor cells were enriched,and no one could indicate which individual cell wasthe suppressor cell because there were no reliable

markers that indicated suppressor cells at the single celllevel. Other investigators tried to establish suppressorT cell clones or suppressor T cell hybridomas andsucceeded in establishing them. However, such clonesand hybridomas lost their suppressive function asthey continued the in vitro culture and, even worse,they could not be maintained in vivo. In those daysof suppressor T cells, clones and hybridomas wereCD8+-dominant. This phenomenon does not seem toreflect the real dominance of suppressor T cells inthe natural condition in vivo because CD8+ cellsgenerally grow faster than CD4+ cells if only sufficientlevel of interleukin (IL)-2 is supplied in vitro culture.Therefore, it is likely that CD8+ clones grew fasterand made visible colonies after the grow from a singlecell such as limiting dilution earlier than CD4+ clonesdid, and then were picked up and expanded morepredominantly than the natural existence ratio in vivo.

Although we could not judge whether it wasa coincidence or not, the revival of T cells withsuppressive function started and grew rapidly when thestudies of “suppressor” T cells shrank. In this secondtime expansion, there were several key points that ledto the success of the study of these new cells.

First, the cells were newly named as “regulatory”T cells that gave the impression of different cells fromthe old “suppressor” cells to the investigators althoughthe real difference was unclear at that time and maybeit is rather impossible to say the relation between themnow.

Second, the regulatory T cells could be markedwith the cell surface molecules of CD4 and CD25. Itdid not mean that all the CD4+CD25+ cells were the

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regulatory T cells but did mean that CD4+CD25− cellsdid not contain the regulatory T cells. For purifying theregulatory T cells (Treg), we had to find other markermolecules.

Third, the term “naturally occurring” or “naturallyarising” was frequently used to explain the newlyidentified Treg. This means that the fate of T cellsis already determined at some point of T celldifferentiation whether the cell is going to be aconventional effector T cell or a regulatory T cell. Thisstatement is still partially true now because immatureT cells that have been determined to become Treg anddevelop to Treg in thymus really exist, but was partiallyfound to be wrong because Treg was also induced frommature non-Treg cells by the stimulation of TGF-β .

Fourth, the method to evaluate the activity of theTreg is very easy because Treg cells themselves do notproliferate in vitro culture but survive and suppress theproliferation of other T cells. Thus, a simple assay of3H-thymidine uptake reflects the proliferation of thetarget cells, which well corresponds to the regulatoryactivity of the co-cultured Treg.

Fifth, the master gene of Treg, Foxp3, wasidentified though the discovery of it was about 8 yearslater than that of Treg itself. “Master gene” means thekey gene that determines the characterization of thecell. In other words, when the master gene is expressed,the cell become to change or keep the characterizationin accordance with the order of the master geneproduct. For example, when Foxp3 gene is introducedand expressed in conventional effector CD4+ T cells,the cells become regulatory T cells. Furthermore, theexpression of Foxp3 is tightly correlated with theregulatory function. When a cell expresses Foxp3, wecan say the cell is a regulatory cell with almost 100%confidence. The only one weak point of Foxp3 may bethe intra-nuclear localization of Foxp3 protein, whichrequires killing cells to stain Foxp3 with an antibody.

Treg have been attracting much attention of notonly basic immunologists but also clinicians who worksfor the patients suffering from immune-based diseases.In most cases when the immunologists or the cliniciansuse the tem “Treg” without some specific explanation, itseems to mean CD4+CD25+Foxp3+ regulatory T cells.The studies regarding Treg that have been published in

international journals listed by PubMed have reachedmore than 5,000, most of which are related to the CD4+

Treg. The suppressive power of CD4+ Treg has beenwell proved in various different experimental systemsincluding both in vitro and in vivo that also containmany animal models of autoimmune diseases.

When CD4+ Treg was attracting more and moreattention, how was the progress of the study of CD8+

regulatory T cells? As it was expected from theold studies of the “suppressor” T cells, investigatorssearched CD8+ regulatory T cells with some regulatoryT cell-specific markers. In contrast to the unificationto CD25+Foxp3+ phenotype in CD4+ T cell popu-lation, regulatory T cells in CD8+ population is inconfusion. There have been tens of reports describingCD8+ regulatory T cells with different markers.When some examples of such regulatory T cellsare listed up, they include CD28−, CD28−Foxp3+,CD25+Foxp3+, CD25+CTLA-4+Foxp3+GITR+,CD122+, LAP+ (naturally occurring type up tohere), CCR7+CD45RO+, TGFβ +CD25−Foxp3-, IL-10+CD25−Foxp3−, CD28−, CD103+, CD28−CD56+,CD103highLAPhighFoxp3+, CD25+Foxp3+LAG3+,CD25+CD69+CTLA-4+Foxp3+, IL-10+, CTLA-4+Foxp3+GITR+, CD25+CD28+Foxp3+,CD25+CTLA-4+Foxp3+, IL-16+, Foxp3+PD-1+,CD28−Foxp3+, CD11c+, CD62L+TGFβ +GITR+,NKG2A+CD90+, CD45RO+CD101+CD103+ (in-duced type up to here). Some of the markers suchas Foxp3, CD25, CTLA-4, and GITR, are just mim-icking that of CD4+ Treg, exposing the lack of theiroriginality.

While the research of CD8+ regulatory T cells waschaotic, my study group identified the CD8+CD122+

regulatory T cells (CD122+ Treg). After the patientand hard work of searching regulatory T cells thatimprove the condition of CD122 (IL-2 receptor β -chain)-knockout mice, we found that CD8+ cells inwild-type mice with higher expression level of CD122than other T cells had an activity to normalize thederegulated activation of T cells in CD122-deficientmice. Furthermore, we proved that these CD122 high-expressing (CD122+) cells also had an activity toregulate CD8+CD122− cells derived from wild-typemice. Since we published the initial article describing

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the discovery of CD122+ Treg in J. Exp. Med. 2004,we have published 6 original articles and 1 reviewregarding CD122 Treg. This number is far moresuperior to those of all the other regulatory T cellsraised in the former paragraph, most of which have onlyone article.

In this report, I describe the effect of CD122+

Treg on inflammatory bowel disease (IBD). The formerhalf is about the experimental study using the animalmodel of IBD and the latter half is about the humanstudy using the peripheral blood samples obtained frompatients of ulcerative colitis (UC) and Crohn’s disease(CD). Although the study of human samples turned outto be negative results, it may give some information tothe related field of both scientific and clinical medicine.

Materials and Methods

Mice

C57BL/6j mice were purchased from Japan SLC,Inc. (Hamamatsu, Japan). Recombination activatinggene (RAG)-2-gene-targeted mice with C57BL/6genetic background were originally purchased fromTaconic Farms (Hudson, NY) and maintained in ouranimal facility. IL-10-deficient mice with C57BL/6genetic background were originally purchased from theJackson Laboratory (Bar Harbor, ME) and maintainedin our animal facility.

Antibodies

Fluorescent isothiocyanate (FITC)-conjugatedanti-mouse CD8a, FITC or Phycoerythrin (PE)-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD45RB, biotin-conjugated anti-mouse CD122antibodies and streptavidin-PE-Cy5 conjugate werepurchased from eBioscience (San Diego, CA). Cy-chrome-conjugated anti-human CD4, Cy-5-conjugatedanti-human CD25, PE-conjugated anti-human CTLA-4/CD152 antibodies were purchased from BDPharmingen (San Diego, CA), PE-conjugated anti-human mouse IgG2a antibody was purchased fromMiltenyi Biotech (Auburn, CA), anti-human mouseIgG2a negative control antibody was purchased from

DAKO (Glostrup, Denmark), and PE-conjugatedanti-human GITR antibody was purchased from R&D(Mineapolis, MN).

Induction of IBD in mice

4 × 105 CD4+CD45RBhigh cells were intra-venously injected into RAG-2 deficient mice. Progres-sion of IBD was chased by examining the fecal blood,body weight, and serum amyloid-α (SAA) levels, andat 12wks after T cell transfer, mice were sacrificed andthe grade of colitis was judged by the histological ex-amination of descending colon.

Patients

Total of 23 IBD patients (11 UC and 12 CD) and6 healthy volunteers participated in this study. Beforestarting the study, all the patients were informed andagreed to join the study. Patients with CD active index(CDAI) of higher than 150 were defined as active CD,and those with CDAI of lower than 150 were defined asinactive CD. The activity of UC was determined by UCdisease active index (UCDAI) that reflected the totalhealth condition including the number of defecation ina day, fecal blood, and the observations in endoscopicexamination. Patients with UCDAI score of higher than8 were defined as active UC and those with UCDAIscore of lower than 8 were defined as inactive UC.

Isolation of human peripheral blood lymphocytes

Peripheral blood was obtained from patientsof UC, CD, and normal healthy volunteers. Bloodsamples were diluted twice with PBS, layeredonto Ficoll-Paque (GE Healthcare Bioscience), andcentrifuged at 1750rpm 4◦C for 30 minutes. Afterthe centrifugation, the boundary layer was recovered,washed with PBS twice and suspended into completecell culture medium. Cells were cultured underthe stimulation with anti-CD3 and anti-CD28-coatedmicro-beads (DynaBeads, Dynal) in IL-2-containingmedium (1 ng/ml, Peplotech) for 12 h. Then, micro-beads were removed and the cells were transferred intofresh medium in a well of 96-well plate, and the culture

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Table 1. Fecal occult blood of mouse IBD induced by T cell transfer

Weeks after T Cell TransferTransferred Cells 3 4 5 6 7 8 9 10 11 12

CD4+CD45RBhigh 0/4 1/4 3/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4

CD4+CD45RBhigh

+CD4+CD45RBlow 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4

CD4+CD45RBhigh

+CD8+CD122+ 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4

CD4+CD45RBhigh

+CD8+CD122−0/4 0/4 1/4 3/4 4/4 4/4 4/4 4/4 4/4 4/4

was continued for 12 h. During the last 5 h, cells werefurther stimulated with PMA (50 ng/ml) + ionomycin(1 μg/ml) in GolgiPlug (BD Bioscience)- containingcell culture medium.

Staining intracellular IL-10 and flow cytometricanalysis

Harvested cells were washed with PBS, treatedwith Cytofix/Cytoperm (BD Bioscience), incubatedwith FITC-conjugated anti-human CD8a and PE-conjugated anti-human IL-10 for 20 min on ice, andanalyzed using FACSCalibur (BD Bioscience).

Results

In vivo experiments using mice

IBD was induced in RAG-2 deficient miceby transferring CD4+CD45RBhigh cells. When onlyCD4+CD45RBhigh cells were transferred into RAG-deficient mice, those mice started to lose their bodyweight around 4-6 weeks after the T cell transfer(pink square marks, Fig. 1A). SAA level was alreadyhigh at 2 weeks after the T cell transfer and furtherincreased as time passed (pink square marks, Fig.1B). Histological examination of the colon revealeda massive infiltration of inflammatory cells under theepithelium and thickening of the colon wall (Fig. 1C).Histological score showed significant elevation (Fig.1D). Fecal blood was 100% (4/4) positive later than

6 weeks after the transfer of CD4+CD45RBhigh cellsalone (Table 1). In contrast, when CD8+CD122+ cellswere co-transferred with CD4+CD45RBhigh cells, thebody weight loss was not observed (yellow trianglemarks, Fig. 1A) and SAA level was not increased(yellow triangle marks, Fig. 1B). Histology of thecolon was normal (Fig. 1C). Histological score wasnot significantly higher than the non-T cell-transferredcontrol group (Fig. 1D). Fecal blood was not detected inall the mice (0/4) throughout the experiment (Table 1).These results indicated that CD8+CD122+ regulatoryT cells effectively prevented IBD.

Examination of human CD8+ Treg in IBD

In our former study, we found that there wereno human CD8+ T cells that expressed high levelof CD122 the level of which was compatible withthat of murine CD122. Instead of CD122, we foundthat the expression of CXCR3 could be a markerof CD8+ regulatory T cells in human peripheralblood lymphocytes (PBL). However, because I knewthat even murine CD8+CD122+ cells containedremarkable percentage of non-regulatory T cells andCD8+CXCR3+ cells involved rather more cells thanCD8+CD122+ cells, I thought that the examinationof simple number or percentage of CD8+CXCR3+

cells was of little meaning. Therefore, I chose theexamination of IL-10 that was thought to be producedfrom CD8+CXCR3+ regulatory T cells and work as asuppressive effector molecule.

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Fig. 1. Prevention of colitis by CD8+CD122+ regulatory T cells.A, CD4+CD45RBhigh cells (4 × 105) in combination with either 2 × 105 CD4+CD45RBlow cells, 2 × 105

CD8+CD122+ cells, or 2×105 CD8+CD122− cells were transferred into RAG-2-/- mice. Change of body weightwas monitored until 12 weeks after T cell transfer. Data were obtained from three independent experiments (N=3),each of which contained 4 mice (n = 4 each and total n = 12, error bar=SD). Data of transfer of CD4+CD45RBlow

cells, CD8+CD122+ cells or CD8+CD122− cells mixed with CD4+CD45RBhigh cells were compared with that oftransfer of CD4+CD45RBhigh cells alone, and data with statistically significant difference are marked with asterisks(*) (p < 0.005, Student’s t test and Bonferroni correction) and data without significant difference are marked withsharps (#) (p > 0.005, Student’s t test and Bonferroni correction).B, Serum amyloid α (SAA) was monitored (n = 4). Data of transfer of CD4+CD45RBlow cells, CD8+CD122+

cells or CD8+CD122− cells mixed with CD4+CD45RBhigh cells were compared with that of transfer ofCD4+CD45RBhigh cells alone, and data with statistically significant difference are marked with asterisks (*)(p < 0.005, Student’s t test and Bonferroni correction) and data without significant difference are marked withsharps (#) (p > 0.005, Student’s t test and Bonferroni correction).C, Mice were sacrificed at 12 weeks after T cell transfer and dissected colon was analyzed by HE staining.Representative pictures of each group are shown.D, Histological scores of individual mice are shown as circles (first experiment, n = 4), triangles (secondexperiment, n = 4), and squares (third experiment, n = 4) and the averages of each group are shown as horizontalbars (total n = 12). Statistical analysis was performed and data with significant difference between two groupsare marked with asterisks (*) (p < 0.005, Student’s t test and Bonferroni correction) and data without significantdifference is marked with a sharp (#) (p > 0.005, Student’s t test and Bonferroni correction).

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Table 2. Profile of IBD patients and healthy volunteers

UC or CD active (a) or IL-10+ inage sex CAI CDAI

or NC remission (r) CD8+ (%)UC r 62 F 4 - 0.61UC r 46 F 3 - 0.21UC r 48 F 3 - 0.99UC r 36 F 5 - 1.13UC r 41 F 5 - 7.58UC a 35 F 10 - 1.97UC a 42 F 13 - 2.98UC a 59 M 11 - 2.63UC a 55 F 6 - 1.19UC a 33 M 9 - 0.98UC r 56 M 3 - 0.18CD a 69 M - 332 0.39CD a 39 M - 285 0.63CD r 43 M - 139 2.07CD r 45 F - 154 2.99CD r 46 M - 26 1.91CD a 55 F - 240 1.35CD r 42 M - 170 0.42CD a 25 M - 314 1.51CD a 22 M - 281 1.49CD a 31 M - 292 4.29CD r 32 M - 183 0.6CD r 28 M - 177 0.67NC - 35 M - - 1.4NC - 32 M - - 1.6NC - 36 M - - 1.1NC - 48 M - - 4.23NC - F - - 1.6NC - 52 M - - 2.67

PBL were collected from patients or controlvolunteers and the IL-10 production from CD8+ cellswas measured as described in Materials and Methods.The profiles and the experimental results of eachpatient and healthy volunteer are shown in Table 2.Summarized graph demonstrating the production of IL-10 from CD8+ cells of UC patients, CD patients, andnormal control (NC) is shown in Fig. 2. UC patients andCD patients are further divided into two phases withmilder symptoms of remission phase (UCr and CDr)and with severer symptoms of active phase (UCa andCDa). Graphs and statistic analysis are shown as the

patients separated by remission phase and active phase.The result of statistic analysis is summarized in Table3 (UC vs. NC) and Table 4 (CD vs. NC). The overallresults showed that there are no statistically significantdifferences in the percentage of IL-10-producing cellsamong total CD8+ cells between UC patients andnormal control, and between CD and normal control.

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Fig. 2. Rate of IL-10 producing cells in CD8+ cells of human peripheral blood. Lymphocytes were prepared fromperipheral blood of patients of ulcerative colitis (UC), Crohn’s disease (CD), and control normal volunteers (NC) asdescribed in Materials and Methods. Cells were cultured under stimulation with CD3 plus CD28-coated microbeadsand then stained with anti-human CD8a, then intra cellular IL-10 was stained and measured by flow cytometricanalysis. Patients were further divided into in remission group (UCr and CDr) and active group (UCa and CDa).Data are presented as mean value obtained from 5-6 samples in each group with error bars of standard deviation.No significant difference was observed in any combination of comparison between two groups.

Table 3. Statistic analysis of IL-10 producing cells in CD8+ cells of UC patients

t-TestUC NC UCr NC

Average 1.859091 2.1 0.46 2.1Variance 4.419829 1.37036 0.2109 1.37036t −0.2573 −2.27527P value (two-sided) 0.80044 0.057033

UCa NC UCr UCaAverage 2.816667 2.1 0.46 2.816667Variance 6.236387 1.37036 0.2109 6.236387t 0.636493 −1.56853P value (two-sided) 0.538751 0.160745

Discussion

In vivo experiments using mice

The suppressive effect of CD8+CD122+ regula-tory T cells was clearly observed in this animal modelof IBD. Although the mechanism of suppression is notdemonstrated in this report, my research group fur-ther investigated on this point and obtained some ex-perimental results showing that the production of IL-10 from the cells in colon tissue could be a key for

the anti-inflammatory reaction for the development ofIBD. IL-10-deficient mice suffer from severe colitisthat looks more serious than other colitis-developingmouse models such as T cell-deficient mice transferredwith CD4+ Treg-lacking CD4+ T cells (described in theformer part of this report), IL-2-related gene-targetedmice including IL-2-deficient mice, CD25 (IL-2 re-ceptor α chain)-deficient mice, and CD122 (IL-2/IL-15 receptor β chain)-deficient mice. A striking abnor-mality that is observed at almost 100% rate in IL-10-deficient mice is prolapse of rectum, which is rare inthe other colitis-developing mouse models mentioned

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Table 4. Statistic analysis of IL-10 producing cells in CD8+ cells of CD patients

t-TestCD NC CDr NC

Average 1.705 2.1 1.99 2.1Variance 1.472783 1.37036 0.0128 1.37036t −0.63827 −0.12595P value (two-sided) 0.5336 0.903884

CDa NC CDr UCaAverage 1.63375 2.1 1.99 2.816667Variance 1.862741 1.37036 0.0128 6.236387t −0.67056 0.352794P value (two-sided) 0.515199 0.733357

above. It is proposed that the spontaneous movementof gastro-intestinal tract is produced by the activity ofinterstitial cells of Cajal (ICC) that are also called asthe pace maker cells of gastro-intestinal (GI) tract. Oneof my collaborators who investigates the movement ofGI tract found that the receptor for IL-10 is expressedin the ICC cells and possibly conducting negative sig-nal into the ICC cells (my personal communication).Therefore, it is reasonable that frequency of the move-ment of GI tract in IL-10-deficient mice is augmented,which may be the cause of inevitable prolapse of rec-tum in IL-10-deficient mice.

When we performed the experiments of transfer-ring Treg-lacking CD4+ cells ± CD8+CD122+ regu-latory T cells into RAG-deficient mice, we believedthat CD8+CD122+ regulatory T cells produced IL-10and such IL-10 was the main effector molecule thatprevent and cure (data not shown in this report) col-itis caused by Treg-lacking CD4+ T cells. However,after publishing the effect of CD8+CD122+ regula-tory T cells on CD4+ cell-induced colitis, we foundthat CD8+CD122+ cells could be divided into two ormore subpopulations by the expression level of CD49d(VLA-4, integrin α4 chain), whereas CD8+CD122−

cells were monotonously stained with anti-CD49d an-tibody at intermediate expression level. When we ex-amined the production of IL-10 from the CD49dhigh+

(CD49d+) subpopulation and the CD49d− subpopula-tion, CD49d+ cells produced detectable level of IL-10but CD49d− cells did not. This experimental result putan idea into my mind. The idea was that CD49d+ cells

were regulatory T cells and CD49d− cells were not reg-ulatory T cells, possibly another type of cells like mem-ory T cells.

After observing the IL-10 production from theCD8+CD122+CD49d+ T cells, we concentrated onthe work of resolving regulatory mechanism ofCD8+CD122+CD49d+ T cells. However, a shocksuddenly appeared in front of us. That was theco-culture of CD8+CD122+CD49d+ T cells andCD8+CD122− T cells and we expected that IFN-γ production from CD8+CD122− T cells should besuppressed by the activity of IL-10 produced fromCD8+CD122+CD49d+ T cells. As a negative control,we prepared the co-culture of CD8+CD122+CD49d−

T cells and CD8+CD122− T cells. After 48hof co-culture, an amazing result came up. Thenumber of CD8+CD122− T cells co-cultured withCD8+CD122+CD49d− T cells dramatically decreasedwhereas that co-cultured with CD8+CD122+CD49d+

T cells was unchanged. Repeated experiments gavealmost same results, forcing me to think thatCD8+CD122+CD49d− T cells killed CD8+CD122− Tcells.

For finally judging which cells are real regulatoryT cells, the in vivo experiment was inevitable.CD8+CD122− T cells alone (n=18), CD8+CD122−

T cells plus CD8+CD122+CD49d− T cells (n=17),and CD8+CD122− T cells plus CD8+CD122+CD49d+

T cells (n=20) were transferred into RAG-2-deficienthost mice. The result was striking. All the hostmice that had received CD8+CD122− T cells alone

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died by 16 weeks after the T cell transfer and themice that had received CD8+CD122− T cells plusCD8+CD122+CD49d+ T cells died almost equally asmice that had received CD8+CD122− T cells alone.In contrast, mice that had received CD8+CD122−

T cells plus CD8+CD122+CD49d− T cells survivedsignificantly longer than mice of 2 other groups andmore than half of them survived longer than 20 weeksafter the T cell transfer. According to the result of invivo experiment, we conclude that the regulatory Tcells in the CD8+ population are CD122+CD49d− cellsand the mechanism of suppression is cytotoxicity to thetarget cells, not IL-10 production.

Examination of human CD8+ Treg in IBD

In the human study, we measured IL-10 produc-tion from CD8+ cells. As it is described above, how-ever, IL-10 production is not reflecting the regulatoryactivity of CD8+ T cells in the case of murine exper-

iments. It is reasonable that we could not get positiveresults by the analysis of IL-10 production in humansin this study because the function of IL-10 should notbe much different between humans and mice. We needto establish an assay system to measure cytotoxic activ-ity of human CD8+ T cells as soon as possible.

Conclusion

In this study, we first proved the preventive effectof CD8+ regulatory T cells for CD4+ T cell-inducedIBD model of mice. Next strategy is to refine theeffective regulatory T cell population. In the studyusing human PBL samples, we could not obtainpositive result but now we surely know the reasonof failure. It means that the chance to get a positiveresult by the improved trial is reasonably high. Ihope that the application of the regulatory T cells weare investigating is going to be close to the concretesuccess.

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Analysis of activation pathway of Huntington’s disease-causing gene in Dictyostelium discoideum.

Hidekazu Kuwayama

Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Tennodai, 1-1-1, Ibaraki 305-8572,Japan

Introduction

Huntington’s disease is a neurodegenerativegenetic disorder that affects muscle coordination andleads to cognitive decline and psychiatric problemsand there exists no effective method which can curethis serious disease. This typically occurs at mid-adult life and even the mechanism how this diseasecan occur is not completely understood, yet. Thecasual gene, named Huntingtin, was identified in1993 by an international consortium and the geneticbasis of Huntingtin was clarified. The Huntingtin geneprovides the genetic information for the protein whichcontains expansion of a CAG triplet repeat stretchat around N-terminal region results in a different(mutant) form of the protein, which gradually damagescells in the brain, although mechanisms that are notcompletely understood. The researches on how themutated Huntingtin protein causes the disease havebeen extensively on going, but little is known aboutthe normal cellular function of Huntingtin and howthis might be disturbed in Huntington disease. Invitro studies identified a wide range of Htt-interactingproteins and suggest that wild-type Huntingtin maybe involved in such diverse biological processes suchas intracellular trafficking, cytoskeleton, postsynapticsignalling, transcriptional regulation and anti-apoptoticfunctions.

A model organism is a very powerful toolto identify the gene function. So far, the fruitfly Drosophila melanogaster serves genetic and

experimental advantages for Huntingtin research.Drosophila shares many essential features with higher-order organisms. It was previously shown showedthat the human Htt-Q75 and -Q120 transgenes inducedegeneration of fly photoreceptor neurons. And it wasidentified later a Drosophila homolog of huntingtin.However, the normal function of Drosophila huntingtinremains elusive. By reverse transcription-PCR andin situ hybridization, it was shown that huntingtinwas expressed ubiquitously during all stages of flydevelopment. Also, with an anti-huntingtin antibody,it was confirmed that, like its mammalian homolog,Drosophila huntingtin was a cytoplasmic protein.Furthermore, to explore the normal function ofhuntingtin, they created a huntingtin knockout (KO) flyusing flipase–FRT-mediated recombination. Previousmouse models that are null for the murine Huntingtinhomolog have been difficult to study since they dievery early in embryogenesis. Unlike mouse huntingtinhomozygous knockout mice, the KO flies survivedinto adulthood. Further, in contrast to a previousreport that used RNAi, knockout flies developed ata similar rate to their wild-type counterparts, withembryonic and larval CNS, muscles, eyes and otherimaginal discs appearing morphologically normal.It was also observed normal axonal pathfinding,muscle innervation, overall synapse structure, axonaltransport and typical crawling behavior during larvalstages. Immunostaining revealed normal pre- and post-synaptic structures, and proper synaptic componentdelivery (including synapsin, FasII and Dlg). However,the KO flies were not completely normal; they

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displayed phenotypes in mushroom bodies (MBs),particularly a decrease in FasII staining, as well asreduced numbers of varicosities and branches in theaxonal termini of giant fiber neurons (Zhang et al.).The mature dhtt-ko flies also demonstrated late-onsetdefects in mobility and viability, and phototransductiondeficits during temperature-induced stress.

It was surprising that KO fly displayed no devel-opmental defects, in contrast to the early embryoniclethality observed in huntingtin knockout mice. It stillremains possible that a redundant pathway might com-pensate for the loss of fly huntingtin. There may also besubtle defects in KO flies that escaped detection undernormal conditions that might intensify with stress. Itwould be interesting to further investigate whetherthe mild abnormalities in MBs and axonal termini areresponsible for the reduced adult mobility and viability,although it would result in exhausting efforts in vain.Although it was argue that fly Huntingtin may havedistinct functions from human Huntingtin, no solid con-clusion can be made until it is shown whether humanHuntingtin can rescue the phenotypes of the KO flies.

The social amoebae, Dictyostelium discoideum,cells is a novel candidate for studying the normalfunction of huntingtin, since its genome has beenrevealed to possesses the huntingtin homologue. Thesolitary amoeboid cells grow by simple division innutrient. In removal of nutrient, the amoebae startto aggregate by chemotactic movement and behavecooperatively to construct multicellularlity. During thisprocess, single cells transform into a multicellularorganism, which results in a fruiting body consistingof a cellular stalk to suspend globular sori at its apicalend within 24 h. This life-cycle is very simple and hasbeen studied as a model system for cell differentiationand morphogenesis.

In this study, we found that disruption of agene encoding a huntingtin disease causing gene(huntingtin) homologue, Dd huntingtin, resulted inabnormal development in D. discoideum. The nullmutant cells exhibited extremely long developmentalcycle and reduced chemotacitc activity. And by YeastTwo-Hybrid system, the gene product was shown tointeract with activated form of a Galpha protein whichtransduces the extracellular chemotactic ligand signal

via a cell surface 7TM receptor. Furthermore, in vivointeraction was suggested by FRET analysis. Theseresults indicate that Dd Huntingtin may function asan intracellular signalling molecule which controlsthe orientation of cell motility, thus effects ondevelopmental time course in D. discoideum.

Materials and Methods

Dictyostelium Cell Culture and Development

Wild type Dictyostelium discoideum AX2 cellswere cultured at 21◦C in HL5 medium with 100 μg/mlstreptomycin sulfate and 100 units/ml benzylpenicillinpotassium. For culturing the transformed cells usedin this study, the HL5 medium was supplementedwith the selecting drugs, 10 μg/ml blasticidin S or20 μg/ml G418. Cells were collected by centrifugationand resuspended in PB (10 mM Na2HPO4 and 10 mMNaH2PO4 [pH 6.5]). For developmental studies, cellswere developed on 1.5 % agar PB plates or nitrocellu-lose filters atop the agar plates at a surface density ofstated in figure legends. For FRET experiment, cellswere suspended in PB at a density of 1× 107 cells/mland shaken at 125 rpm for 1hr and then added withrepeated pulses at 30 nM at every 6 min. Then, celldensity was adjusted at a density of 1×108 cells/ml.

Vector Construction and Transformation

Gene-targeting construct for generating Dd hunt-ingtin null mutant was prepared by fusing the blasti-cidin S resistance gene expression cassette (bsr) withgenomic DNA fragments in the coding region to eachend by fusion PCR technique. The construct was am-plified by PCR for transformation. Full length of Ddhuntingtin was amplified from cDNA mixture preparedfrom aggregation stage mRNA using oligo dT primerand verified by sequencing. For constructing the over-expression or fluorescent protein fusion vectors, the fulllength or truncated cDNA was cloned into the Dic-tyostelium discoideum expression vector, pHK12neoand pHK12bla or pHK12neo-N-Venus, which give riseto constitutive levels of expression. The cerulean genewas inserted into the vector for the expression of

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Fig. 1. Construction of Dd huntingtin KO cells.Gene-targeting construct for generating Dd huntingtin KO mutant was prepared by fusing the blasticidin Sresistance gene expression cassette (bsr) with genomic DNA fragments in the coding region to each end by fusionPCR technique. The construct was amplified by PCR for transformation.

Gα2-Cerulean was constructed by inserting cDNA ofcerulean into in the loop between the αA and the αBhelices, of the Gα2 cDNA as described previously.Transformation and selection of transformants wereperformed using 10 μg of DNA by electroporation asdescribed previosuly.

Nucleic Acid Anaylses

Total RNA was extracted by using RNAeaykit (Qiagen). Bloting and detection for northern andsouthern analyses were performed using DIG-easyhybri kit.

FRET Analysis

Differentiated cells starved for 6 h were suspendedat a density of 1× 108 cells/ml in PB. FRET analysiswas performed with a Spectrofluorophotometer RF-5300PC (Shimadzu Japan). Cell suspension was keptto be stirred during the measurements.

Results

Huntingtin KO shows slow growth, development

Our previous study revealed that D. discoideumhuntingtin (Ddhtt) can potentially interact with adominant active form of Galpha 2. In order toinvestigate the function of Ddhtt, we constructed DdhttKO vector and isolated the KO cells (Fig. 1).

The growth of the KO was investigated. Theparental wild type AX2, requires 8-10 h for doubleduring the exponential growth phase. On the otherhand, the KO cells are relatively slow in growth (Fig.2). This observation indicates that Ddhtt is related tocell growth.

Under starvation, wild type, AX2 cells start toaggregate at 6 h, form slug at 16 h and construct fruitingbody at 24 h after starvation (Fig. 3). On the otherhand, the KO cells do not show any symptom forcell aggregation for 10 hr. The weak aggregation isonly observed at 24 h, tight mound formation at 36 h,

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Fig. 2. Growth of huntingtin KO cellsCell growth of in AX2 (◆), Dd huntingtin KO (■) cells. Cells were cultured in HL5 liquid medium, at 21◦C, inthe with shaking at 125 rpm

slug formation at 48 h and fruiting body at 72 h afterstarvation (Fig. 3). These observations shows that theKO cells are extremely slow in development, especiallytake much longer time to initiate cell aggregation.

It has been known that, in D. dicsoideum,development is dependent on cell density. In AX2 cells,development was observed at a density of 1.25 × 106

cells/cm2 and even at the half density (Fig. 4). Onthe other hand, the KO cells require much higher celldensity to complete development. They develop only ata density of higher than 2.5 × 106 cells/cm2 (Fig. 4).This indicates that KO cells are defective in cell-cellinteraction or intercellular signalling as chemotaxis.

Aggregation and chemotactic activities of huntingtinKO cells

In order to investigate the aggregation activityin detail, cells were allowed to be observed underhigher magnification under submerged condition withphosphate buffer. In AX2, cells started to apparentlyaggregate at 8 h and form aggregation-mound at10 h after starvation (Fig. 5). On the other hand,

although the KO cells cell shape and motility werenot distinctly different from those of AX2 cells,cells started to form small aggregations but did notlast for long time (Fig. 5). Then cells repeatedaggregation and disaggregation several times (arrowsin Fig. 5). However, no distinct aggregation wasobserved until 42 h after starvation. These observationsindicate that KO cells can initiate cell aggregation andinteract with each other probably via cAMP-dependentchemotactic signalling, but cannot properly organizethe developmental process.

To further analyse the aggregation activity in KOcells, we performed chemotaxis assay by the agardrop method. In AX2 cells which starved for 6 h,chemotactic activity was observed in a wide rangeof cAMP concentration (Fig. 6). On the other hand,KO cells which starved for the same time, 6 h, nochemotactic activity was observed. Considering thatKO cells require much longer time, KO cells whichwere starved for 30 h were used for the chemotaxisassay. In the result, KO cells show very weakchemotaxis and require higher cAMP concentrationthan AX2 cells (Fig. 6). These observations suggest

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Fig. 3. Development of huntingtin KO cellsDevelopment of in the parental AX2 strain (the parental strain), Dd huntingtin KO cells were assessed on non-nutrient agar plates; cells were plated at a density of 1.25×106 cells/cm2. In AX2 cells, mounds formed at 12 hr ofdevelopmentafter plating;, slugs at were visible at 16 hr;, culmination at occurred by 20 h,r and fruiting bodies arewere observed at 24 hr. Scale bar, 1 mm.

that Huntingtin plays a regulatory role on intracellularchemotactic signalling.

Dd Huntingtin Directly Interact With Activated Form ofGα2

To study the kinetics of the interaction of DdHuntingtin with the activated form of Gα2 in livingcells upon ligand stimulus, monitoring fluorescenceresonance energy transfer (FRET) between fusionproteins of a yellow fluorescent protein, Venus to C-terminus of Dd Huntingtin and a cyan fluorescentprotein, Cerulean inserted into the first helical domainof Gα2 was adopted. Fusion of Venus to Dd Huntingtinretained the function of wild-type Dd Huntingtindue to being rescued the phenotypic character ofDd Huntingtin KO mutant by Venus-Dd Huntingtin(data not shown). FRET was measured in living anddifferentiated cells excited at 440 nm of light wave

and recorded the emission at 525 nm upon additionof cAMP stimulus. The cotransformed cells of DdHuntingtin-Venus and Gα2-Cerulean showed the rapidand significant increase in FRET efficiency followedby readily decrease (Fig. 7). Cell lines expressing thefusion proteins alone or mixtures of cells containingeither the fusion proteins alone did not display anyresponse (data not shown). These results showed thatGα2 directly interacts with Dd Huntingtin in a Gprotein coupled receptor dependent manner.

Discussion

Huntington’s disease is a hereditary neurodegener-ative disorder. This disease is typically a late onset typewhich shows various symptoms like disturbances inmovement, mood, cognition. At around the N-terminusof huntingtin, poly glutamate stretch is contained lessthan 37 repeats in health people, but more than 40 in

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Fig. 4. Density effect on Development of huntingtin KO cellsDevelopment of the parental AX2 strain (the parental strain), Dd huntingtin KO cells were assessed on non-nutrientagar plates; cells were plated at various densities. Scale bar, 1 mm.

the patients. So far, unfortunately it has not discoveredyet what kind of clinical treatments is effective on thepatient. In order to find an effective drug, it is criticallyimportant to unravel the molecular functions of normalhuntingtin which can be compared to that of mutatedone and thus, it can be possible to design a novel drugto act only on the mutated huntingtin in neuron. In thisstudy, we used a mode organism, Dictyostelium dis-

coideum and indicate that Dd Huntingtin plays a regula-tory role in intracellular chemotactic signalling. And itis shown that Dd huntingtin is interacted with a trimericG protein alpha subunit at the onset of the activation ofits interacting cell surface receptor, car1. These findingsmay lead to a breakthrough to unravel how huntingtinis activated in cell and what is a molecular function ofhuntingtin in neuron, since the intracellular signalling

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Fig. 5. Aggregation of huntingtin KO cellsAggregation of the parental AX2 strain (the parental strain), Dd huntingtin KO cells were assessed in the phosphatebuffer as a submerged condition. Red arrows indicate aggregation centers. Scale bar, 0.1 mm.

pathway of D. discoideum possesses almost identicalmolecular machinery and dynamics.

We are now investigating the human counterpartof G alpha subunits. At present, we finished cloninghuman huntingtin binding domain and 16 human G

alpha subunits which are mutated to dominant activeforms to examine the activity-dependent interactionwith human huntingtin. These investigations may leadto explore not only the function of normal huntingtinand lead to an effective clinical treatment but also a

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Fig. 6. Chemotaxis of huntingtin KO cellsA) Chemotaxis acitivity of the parental AX2 strain (the parental strain), Dd huntingtin KO cells were the agar dropassay. A droplet containging about 1,000 cells and a various concentration of cAMP were dropped side by side andthe attractions of cells to cAMP droplet were measured. Scale bar, 1 mm.B) Chemotaxis activitiy in the parental AX2 strain (the parental strain), Dd huntingtin KO cells.

biological importance of huntingtin molecules in cellsensing system and development.

Conclusion

In this study, we found that disruption of hunt-ingtin gene resulted in abnormal development inD. discoideum and the null mutant cells exhibitedextremely long developmental cycle and reduced

chemotacitc activity. By Yeast Two-Hybird system, thegene product was shown to interact with activated formof a Galpha protein which transduces the extracellularchemotactic ligand signal via a cell surface 7TM re-ceptor. Furthermore, in vivo interaction was suggestedby FRET analysis. These results indicate that Dd Hunt-ingtin may directly interact with the activated form ofthe G alpha subunit and controls the orientation of cellmotility, and duration of development in D. discoideum.

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Fig. 7. FRET analysis of the interaction of Dd Huntingtin with G alpha 2 proteinTemporary change of FRET efficiency in Dd huntingtin-C-Venus and Gα2-Cerulean co-expression cells weremeasured. The 8 h starved cells were resuspended at a density of 1× 108 cells/ml and excited at 440 nm, and theemission spectrum at 525 nm was monitored before and after adding 3 μM cAMP stimulation. cAMP was added attime 0 sec.

Acknowledgements

This work was supported by The WaksmanFoundation of Japan.

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Induction of regulatory T cells following intranasaladministration of liposomes composed of phos-phatidylserine

Yukihiko Aramaki

Department of Drug Delivery and Molecular Biophramaceutics, School of Pharmacy, Tokyo University of Phar-macy and Life Sciences 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, JAPAN

Introduction

It is well known that regulatory T cells (Treg)have a crucial role in protection against autoimmunityand regulation in immune responses by suppressingT cell proliferation. The absence or dysfunction ofTreg can lead to severe autoimmune diseases, whileincreased Treg numbers can prevent efficient immuneresponses against tumors or invading pathogens. Tregare divided into two types, naturally occurring Treg(nTreg) and inducible Treg (iTreg). nTreg are developedin the thymus, where they acquire expression on thetranscription factor Foxp3 in response to self-antigenpresentation. On the other hand, iTreg are developedfrom naıve CD4+ T cells in the lymphoid tissue inresponse to environmental antigens in association withtransforming growth factor β1 (TGF-β ).

TGF-β is a multifunctional cytokine that regulatesnumerous physiological processes, including cellgrowth, differentiation, apoptosis, adhesion, and thesynthesis of extracellular matrix proteins. Engulfmentof apoptotic cells is thought not only to remove themfrom the tissue but also to provide protection fromlocal damage resulting from the release or discharge ofproinflammatory contents, and the immune-suppressiveeffect was largely inhibited by TGF-β -neutralizingantibodies. Liposomes composed of phosphatidylserine(PS-liposomes) can mimic the release of cytokines toapoptotic cells, and indeed the release of TGF-β frommacrophages has been identified in vitro. Furthermore,

PS-liposomes inhibited immune responses in vivothrough a PS-specific receptor, and suggested thecontribution of TGF-β in this inhibition.

In our series of studies [1-3], PS-liposomesinhibited the production of nitric oxide (NO) andtumor necrosis factor (TNF)-α from thioglycollate-elicited mouse peritoneal macrophages stimulated withLPS, and TGF-β is one of the factors producedby PS-liposomes that suppresses the productionsof NO and TNF-α in macrophages. Furthermore,we demonstrated that the PI3K/Akt pathway andthe downstream extracellular signal-regulated kinase(ERK), a MAP kinase, signaling pathway via PS-specific receptors are intimately involved in theproduction of TGF-β by macrophages treated with PS-liposomes.

It is well known that TGF-β and IL-2 arecritical factors for developing Foxp3+ iTreg in vitro.In this study, we focused on the induction of Tregin mesenteric lymph nodes and nasal cavity followingintraperitoneal and intranasal administration of PS-liposomes in mice. Our data indicated that PS-liposomes promoted the release of TGF-β in the nasalcavity and induced Treg in the nasal passage but not innasopharynx-associated lymphoid tissue (NALT).

Materials and method

Materials

Phosphatidylserine (PS) from bovine brain and

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cholesterol (Chol) were obtained from Sigma Co., Ltd.(St. Louis, MO). Phosphatidylcholine (PC) from eggyolk was purchased from Nippon Oil and Fat Co.(Tokyo, Japan).

Mice

BALB/c mice were obtained from SLC Japan(Hamamatsu, Japan) and maintained in barrier-protected animal facilities under pathogen-free con-ditions. Animal use and relevant experimental proce-dures were approved by the Tokyo University of Phar-macy and Life Science Committee on the Care andUse of Laboratory Animals. Mice (n=5 per group) werenasally administered with liposomes or apoptotic cells(Jurkat cells) at indicated time.

Preparation of liposomes

Multilamellar liposomes composed ofPS:PC:Cholesterol = 2:1:1 (by molar ratio, PS-liposomes) and PC:Cholesterol = 3:1 (by molar ratio,PC-liposomes) were prepared by vortexing and passedthrough a membrane filter (0.45 μm; Corning Glass-works, Corning, NY) before use. Retinoic acid (RA)containing PS-liposomes were also prepared as thesame procedure. Liposomal size and Zeta potentialwere measured with a dynamic light-scattering spec-trophotometer (DLS-7000, Otsuka Electronics, Tokyo)and Nicomp 380 ZLS (PSS Nicomp Particle SizingSystem). The mean diameter and Zeta potential ofSA-liposomes were 380 nm and -25 mV, respectively.

Induction of apoptotic cells

Jurkat T cells purchased from Riken Cell Bank(Ibaraki, Japan) were exposed to UV irradiation at 254nm for 5 min or treated with 10 μM camptothecin for 6hr. The cells were cultured in RPMI 1640 with 10% fe-tal calf serum (Gemini Bio-Products) for 2 h at 37◦C in5% CO2. Cell apoptosis was evaluated by determiningthe externalization of phosphatidylserine (PS) by flowcytometry (FACS Calibur, Becton Dickinson, San Jose,CA) using TACSTM Annexin V-FITC apoptosis detec-tion kits according to the manufacturer’s instructions

(Biovision, Mountain View, CA), and the proportion ofcells expressing PS at the surface was generally 60%.

Isolation of mononuclear cells

Mononuclear cells from nasal passage, NALT, andmesenteric lymph nodes (MLN) were isolated by themechanical dissociation method using gentle teasingthrough stainless steel screens as reported previously.

Detection of TGF-β1

BALB/c mice were administered with PS-liposomes (400 nmol as lipid, 50 μg OVA), andmononuclear cells from nasal passage and NALT wereobtained at a specified time. TGF-β1 concentrations inthe culture supernatant of both cells were determinedby ELISA using pairs of purified capture andbiotinylated detection antibodies recognizing murineTGF-β1 according to the manufacturer’s directions(BD Biosciences).

FACS analysis for Foxp3+/CD4+ cells

BALB/c mice were nasally or intraperitoneallyadministered with PS-liposomes (400 nmol as lipid,50 mg OVA) or apoptotic Jurkat cells (1 × 106 cells),and mononuclear cells from nasal passage, NALT andMLN were obtained at a specified time. Each singlecell suspension (1 × 106 ) of pooled nasal passage,NALT, and MLN from six treated mice were treatedwith FcR Blocking Reagent (Militenyi Biotec) for10 min at 4◦C, and then stained with fluorochrome-labelled mAbs using Mouse Treg Flow kit (Biolegend)according to the instruction manual. The expression ofCD4 and Foxp3 on the cell surface was evaluated byflow cytometry (FACSCant, Becton-Dickinson).

Results and discussion

TGF-β induction following intranasal administrationof liposomes

Liposomes are artificially made into membranousvesicles composed essentially of naturally occurring

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phospholipids and have been found to serve as a carrierof drugs and an immunological adjuvant. After beingintravenously injected, they are quickly removed fromthe blood circulation and trapped by Kupffer cells ofthe liver and macrophages of spleen. However, thechanges liposomes exert in these cells with whichliposomes interact remain unresolved. In our recentstudies [1-3], PS-liposomes inhibited the production ofNO and TNF-α from mouse peritoneal macrophagesstimulated with LPS. Additionally, we demonstratedthat TGF-β is one of the factors produced by PS-liposomes that suppressed the macrophage functions,and the ERK, a MAP kinase, signaling pathway viaPS-specific receptors was intimately involved in theproduction of TGF-β in vitro. To clarify whether TGF-β is induced by PS-liposomes in vivo, PS-liposomeswere nasally administered in mice and the productionof TGF-β was evaluated by ELISA. As shown in Fig.1, the high production in TGF-β was observed in theculture supernatant obtained from nasal passage cells,but not in cells obtained from NALT. In mice treatedwith PC-liposomes, no increase in TGF-β productionin both culture supernatants of single cells from nasalpassage and NALT was observed. In spleen, TGF-β levels increased following iv. administration of PS-liposomes. These findings indicated that PS-liposomesinduce TGF-β not only in macrophages in vitro, butalso in cells of nasal passage and spleen following thenasal and iv. administration.

TGF-β induction by apoptotic cells

Apoptosis is a critical process in tissue homeosta-sis and results in the immediate removal of dying cellsby phagocytes such as macrophages and dendritic cells.This process should prevent inflammation and autoim-mune responses against the intracellular antigens thatcan be released from apoptotic cells. Although thereare a variety of mechanisms by which macrophagescould phagocytose apoptotic cells, the best knownmolecule involved is PS. The PS-specific receptor hasbeen cloned and found to contribute to the phagocyto-sis of apoptotic cells like PS-liposomes, and the con-tribution of TGF-β1 is suggested in the preventions ofinflammation and autoimmune responses by apoptotic

Fig. 1. Balb/c mice were nasally administered twice (day0 and 7) with liposomes (400 nmol as lipid/mouse)composed of phosphatidylcholine (PC-liposomes)or phosphatidylserine (PS-liposomes) in combina-tion with OVA (50 μg/mouse). Single cells fromnasal passage and NALT were obtained on day 8,and cultured for 24 hr. TGF-β1 concentrations inthe culture supernatant of both cells were deter-mined by ELISA.

cells. We thus investigate whether apoptotic cells in-duce TGF-β in macrophage–like cell line, RAW264.7cells. RAW264.7 cell were treated with apoptotic Ju-rkat T cells, which were obtained UV (254 nm) irra-diation for 5 min, and TGF-β production was evalu-ated by ELISA. As shown in Fig. 2, TGF-β produc-tion increased in proportion to the amount of apoptoticcells, and indicated that apoptotic cells induce TGF-βin macrophages in vitro.

Treg induction in MLN

TGF-β is a regulatory cytokine with multiplefunctions in control of T cell responses. TGF-β -deficient mice or mice with T cell-specific deletionof TGF-β receptor develop early fatal multifocalinflammatory diseases, indicating crucial role for TGF-β in T cell response. Activation of naıve CD4+ Tcells in the presence of TGF-β induces transcriptionalfactor Foxp3 expression and the differentiation ofinduced T reg (iTreg). iTreg are differentiated inthe lymphoid tissues, and they may control immunetolerance to environmental antigens such as commensalflora. iTreg differentiation by TGF-β is mediated by

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Fig. 2. Effects of PS-liposomes or apoptotic cells oninduction of TGF-β in mesenteric lymph nodes.Balb/c mice were intraperitoneally injected withPS- or PC-liposomes (in each case, liposomaldose is 1 mg phospholipid/mouse). Furthermore,mice were injected with UV-irradiated andcamptothecin-induced apoptotic Jurkat T cells(4×107 cells/mouse). MLN lysate were subjectedto Western blot analysis. The values are the mean± SD of triplicate culture from three independentexperiments. ∗∗; p < 0.05 compared with control.

the recruitment of its downstream transcription factorSmad3 to a Foxp3 enhancer element and consequentinduction of Foxp3 gene expression. Moreover, thevitamin A metabolite, retinoic acid (RA), promotesTGF-β -mediated Foxp3 expression during in vitrodifferentiation of iTreg and preferentially supportsFoxp3+ iTreg formation.

We first examined the TGF-β production inMLN following intraperitoneal injection of liposomesand apoptotic Jurkat cells. TGF-β production wasevaluated by Western blotting, and the band intensitywas evaluated by NIH image. In MLN, TGF-β levelsincreased in PS-liposome- and apoptotic cell-treatedmice, but not in PC-liposome- treated mice (Fig. 3).These data is interesting that PS-liposomes enhanceTGF-β production in MLN even in the peritonealinjection.

We thus examined the induction of iTreg in MLNfollowing intraperitoneal injection of liposomes. PS-liposomes, PS-liposomes+RA (0.1 μmol/mouse), or

Fig. 3. Effect of apoptotic Jurkat T cells on TGF-β1production from macrophage-like cell line,RAW264.7 cells. RAW264.7 cells (1 × 106

cells/well) were treated with apoptotic Jurkat Tcells at indicated cell numbers for 18 hr. Culturesupernatants were collected and evaluated TGF-β concentrations by ELISA. The values are themean ± SD of triplicate culture from threeindependent experiments. ∗∗; p < 0.005 comparedwith control.

PC-liposomes (in each case, liposomal lipid was 1 mgas phospholipid/mouse) were intraperitoneally injected5 times every 4 days, and the single cells of MLNwere collected. The cells were incubated with APC-labeled anto-CD4 mAb and FITC-Foxp3 mAb, andtwo color fluorescences was analyzed on FACSCant.Furthermore, the effect of apoptotic Jurkat T cells wasalso investigated. Fig. 4 shows that CD4+ and Foxp3+

cells are about 5-6%, and no difference was observedbetween control and PS-liposome-treated groups. It isreported that RA promotes TGF-β -mediated Foxp3expression during in vitro differentiation of iTreg.However, no increase in double positive cells wasobserved, even in PS-liposome+RA group.

Treg induction in nasal cavity

It is well established that mucosal (oral andnasal) administration of many antigens can induceperipheral tolerance, often referred to as oral tolerance,which is characterized by a decreased immuneresponse to systemic immunization with the sameantigen. Mucosally induced tolerance, which appears

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Fig. 4. Balb/c mice were intraperitoneally injected with PS-liposomes, PS-liposomes+RA (0.1 μmol/mouse), or PC-liposomes (in each case, liposomal lipid was 1 mg as phospholipid/mouse) were intraperitoneally injected 5 timesevery 4 days. Furthermore, mice were injected with apoptotic Jurkat T cells (apoJ: 4× 107 cells/mouse). Singlecells from MLN were collected, and stained with fluorochrome-labelled mAbs using Mouse Treg Flow kit. Theexpression of CD4 and Foxp3 on the cell surface was evaluated by flow cytometry (FACSCant, Becton-Dickinson).

to be a physiologically important way of avoidingdevelopment of allergic or other harmful immunologicreactions to ingested or inhaled food or environmentalantigens, has for a long time been recognized as apromising approach to prevent allergic or autoimmunedisorders. Recently, in allergic rhinitis and foodallergy, the disorder in T cell differentiation wassuggested, and an insufficient Treg development maybe associated with the allergic inflammations. Toclarify whether nasally administered liposomes orapoptotic cells alter the Treg development, changesin the number of CD4+ and Foxp3+ cells inNALT and nasal passage were evaluated by flowcytometry. PS-liposomes, PS-liposomes+RA (1 nmolRA/mouse), or PC-liposomes (in each case, liposomallipid was 400nmol phospholipid/mouse) were nasally

administered 4 times every 3 days, and the singlecells of NALT were collected and subjected to flowcytometry. Furthermore, the effect of apoptotic JurkatT cells was also investigated. Fig. 5 shows that CD4+

and Foxp3+ cells are about 2-3% in each group, and nochange was observed between PS- and PC-liposomes,and also liposomes and apoptotic cells. This result mayattribute to the finding that TGF-β a critical factorfor developing Foxp3+ iTreg, could not be inducedby PS-liposome in NALT (Fig. 1). RA promotesTGF-β -mediated Foxp3 expression during in vitrodifferentiation of iTreg. However, no increase in doublepositive cells was observed, even in PS-liposome+RAgroup. This may come from the difference between invitro and in vivo, and the distinction could be clarified.

Furthermore, the changes in the number of CD4+

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Fig. 5. Mice were nasally administered with PS-liposomes, PS-liposomes+RA (1 nmol/mouse), or PC-liposomes (in eachcase, liposomal lipid was 400 nmol as phospholipid/mouse) were intraperitoneally injected 4 times every 3 days.Furthermore, mice were also administered with apoptotic Jurkat T cells (apoJ: 2× 106 cells/mouse). NALT wasremoved and single cells were prepared according to the Method section. Single cells were stained with withfluorochrome-labelled mAbs using Mouse Treg Flow kit. The expression of CD4 and Foxp3 on the cell surface wasevaluated by flow cytometry (FACSCant, Becton-Dickinson).

and Foxp3+ cells in nasal passage were evaluated byflow cytometry. As shown in Fig. 6, the number ofdouble positive cell is low, but twice number of CD4+

and Foxp3+ cells were observed in PS-liposome andapoptotic Jurkat cell administered groups. No effect ofRA on the development of iTreg was observed even inthe nasal passage.

In this study, PS-liposomes composed of naturallyoccurring phospholipid have an ability to induce TGF-β in vivo, and suggested the induction of iTreg in thenasal passage. To clarify whether PS-liposomes havea possibility to induce iTreg, further investigations arerequired.

Conclision

I t is well known that negatively chargedliposomes composed of phosphatidylserine (PS) orphosphatidic acid (PA) are preferentially taken upby phagocytic cells such as macrophages. There arenumerous reports about liposomes having a potentialvalue as carriers of antigens to macrophages or immuneadjuvants. However, the considerable attention hasnot been paid to the effects of liposomes on themacrophage function. Our recent studies demonstratedthat PS-liposomes suppressed inflammatory substancesin response to LPS, and TGF-β was a factor produced

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Fig. 6. Mice were nasally administered with PS-liposomes, PS-liposomes+RA (1 nmol/mouse), or PC-liposomes (in eachcase, liposomal lipid was 400 nmol as phospholipid/mouse) were intraperitoneally injected 4 times every 3 days.Furthermore, mice were also administered with apoptotic Jurkat T cells (apoJ: 2×106 cells/mouse). Nasal passagewas collected and single cells were prepared according to the Method section. Single cells were stained with withfluorochrome-labelled mAbs using Mouse Treg Flow kit. The expression of CD4 and Foxp3 on the cell surface wasevaluated by flow cytometry (FACSCant, Becton-Dickinson).

by PS-liposomes that suppressed macrophage functions[1-3]. Treg have a crucial role in protection againstautoimmunity and regulation in immune responses bysuppressing T cell proliferation. Treg are divided intotwo classes, n-Treg and iTreg, and iTreg are developedfrom naıve CD4+ T cells in the lymphoid tissuein response to environmental antigens in associationwith TGF-β . In this study, we focused on theinduction of iTreg by PS-liposomes. Following intranaladministration of PS-liposomes in Balb/c mice, theincrease in TGF-β level was observed, and iTreginduction was found in the nasal passage. Data obtainedin this study were still preliminary, and further

investigation will be required to clarify the involvementof PS-liposomes in the induction of iTreg in vivo.

References

(1) R. Matsuno, Y. Aramaki, S. Tsuchiya, Biochem.Biphys. Res. Commun. 281, 614(2001).

(2) M. Otsuka, S. Tsuchiya, Y. Aramaki, Biochem.Biphys. Res. Commun. 324, 1400(2007).

(3) M. Otsuka, Y. Negishi, Y. Aramaki, FEBS-lett.581, 325(2007).

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