analysis of splenic gr-1int immature myeloid cells in tumor-bearing mice

Microbiol Immunol 2008; 52: 47–53doi:10.1111/j.1348-0421.2008.00009.x

EDITOR COMMUNICATED PAPER

Analysis of splenic Gr-1int immature myeloid cells intumor-bearing miceYoshiko Yamamoto1,2, Hirohito Ishigaki1, Hideaki Ishida1, Yasushi Itoh1, Yoichi Noda2 and KazumasaOgasawara1

Departments of 1Pathology and2Gynecology, Shiga University of Medical Science, Ohtsu, Japan

CorrespondenceKazumasa Ogasawara, Department ofPathology, Shiga University of Medical Science,Seta Tsukinowa-cho, Ohtsu 520-2192, Japan.Tel: +81 77 548 2172; fax: +81775482423;email: [email protected]

Received 25 November 2007; accepted 29November 2007.

List of Abbreviations: CTL, cytotoxic Tlymphocytes; GM-CSF,granulocyte/macrophage colony-stimulatingfactor; ImC, immature myeloid cells; IFN-γ,γ-interferon; IL-10, interleukin-10; MHC, majorhistocompatibility complex; NO, nitricmonoxide; OVA, ovalbumin; PD-1,programmed death-1; PDL1, programmeddeath ligand 1; ROS, reactive oxygen species;TCR, T-cell antigen receptor; TGF-β,transforming growth factor-β; Th2, T helper 2.

Key wordsGr-1int ImC, immunosuppression, tumor.

ABSTRACTIt is knownthatthenumber of ImC, expressing myeloidmarkers, CD11b andGr-1, increase with tumor growth and ImC play a role in the escape of tumorcells from immunosurveillance in tumor-bearing mice and cancer patients.However, the mechanisms by which ImC suppress immune responses intumor-bearing mice have not been completely elucidated. In the presentstudy, we investigated the function of splenic ImC freshly isolated fromtumor-bearing mice and splenic ImC differentiated in vitro by GM-CSF.Freshly isolated splenic ImC were divided into two groups depending onGr-1 expression, Gr-1 high (Gr-1hi) and intermediate (Gr-1int). Freshly iso-lated splenic Gr-1int ImC, but not Gr-1hi ImC, from tumor-bearing micereduced production of IFN-γ in CD8+ T cells, but neither splenic Gr-1int

ImC nor Gr-1hi ImC isolated from naive mice did. Both Gr-1int and Gr-1hi

ImC differentiated in vitro by GM-CSF inhibited production of IFN-γ inboth CD8+ and CD4+ T cells. In addition, the differentiated Gr-1int ImC,one-third of which were CD11c+F4/80+ cells, and their culture supernatantssuppressed proliferative responses of T cells stimulated by CD3 ligation, butthe differentiated Gr-1hi ImC and their culture supernatants did not. Theseresults suggest that Gr-1int ImC are altered to immune-suppressive cells intumor circumstances and that they are differentiated by GM-CSF progres-sively into CD11c+F4/80+ cells with further suppressive activity againstT cells.

T-cell tolerance induced by tumors is one of the ma-jor mechanisms for escape from immunosurveillance. Toachieve effective immunotherapy, it is important to an-alyze these mechanisms in cancer patients and tumor-bearing animals. One possible mechanism is an effectof tumor-related factors suppressing immune responses,such as TGF-β and IL-10 (1–3). Another possible mech-anism is immune tolerance induced by CD4+CD25+

regulatory T cells that accumulate in tumors (4–6). Fur-thermore, it has been shown that the proportion of ImCsuppressing T-cell responses, especially the function ofCD8+ T cells, in the spleen and blood is related to ex-pansion of tumors in tumor-bearing mice and cancerpatients, suggesting that ImC are involved in the non-

responsiveness of T cells in tumor-bearing individuals(7–9).

ImC are immature cells of myeloid lineage expressingGr-1 and CD11b, and ImC isolated from tumor-bearingmice have been shown to suppress T-cell proliferation byCD3 or CD3/CD28 ligation, antigen-driven T-cell prolif-erative responses, and activity of CTL (8). It has also beenshown that ImC in tumor-bearing mice differentiate intoresting dendritic cells to induce anergy but not to inducethe differentiation of regulatory T cells (10), whereas it hasalso been shown that tumor cells converted ImC to cellssecreting TGF-β to induce regulatory T cells (11). ImCproduce immunosuppressive reagents, including NO,IL-10, prostaglandin E2 and TGF-β, which directly affect

c© 2008 The Societies and Blackwell Publishing Asia Pty Ltd 47

Y. Yamamoto et al.

effector cells (12–15). It was also demonstrated that ROSproduced by ImC derived from tumor-bearing mice inhib-ited ζ-chain expression in T cells through cell-to-cell con-tact and inhibited antigen-induced cell proliferation (16).Therefore, elimination of ImC in tumor-bearing mice im-proved CD8+ T-cell responses and allowed eradication oftumors (15). Furthermore, administration of GM-CSF totumor-bearing mice enhanced differentiation of ImC todendritic cells and eliminated tumors (17).

The mechanism of immune suppression by ImC is stillnot clear. We speculated that the function of ImC wasincomprehensible because of their heterogeneity; there-fore, we divided ImC into two groups depending on Gr-1expression, Gr-1 high (Gr-1hi) and intermediate (Gr-1int)and then analyzed differentiation and function of thesecells. Freshly isolated splenic Gr-1int ImC, but not Gr-1hi

ImC, from tumor-bearing mice reduced the productionof IFN-γ in CD8+ T cells, but neither Gr-1int ImC norGr-1hi ImC isolated from naive mice did. In addition,Gr-1int ImC, but not Gr-1hi ImC, isolated from tumor-bearing mice differentiated partly into CD11c+F4/80+

cells in vitro in the presence of GM-CSF, and the differen-tiated Gr-1int ImC inhibited T-cell proliferative responses.The suppression was conducted by soluble factors but notby cell-to-cell contact dependent on PDL1. These findingssuggest that Gr-1int ImC are immune suppressors in thetumor circumstances.

MATERIALS AND METHODS

Mice and tumor cell lines

Female BALB/c and C57BL/6 (B6) mice (6–12 weeks ofage) were obtained from Japan SLC (Hamamatsu, Japan).Mice were housed under specific-pathogen-free condi-tions in the Research Center for Animal Life Science,Shiga University of Medical Science. All experiments wereperformed with the approval of the Shiga Universityof Medical Science Animal Experiment Committee, andthree mice were usually used in each group of experi-ments to reduce the number of experimental animals.B6 mice were inoculated s.c. with OVA-transfected EL-4,E.G7-OVA (E.G7) or B16-B6 melanoma; and BALB/cmice were inoculated subcutaneously with CC26 coloncarcinoma, CC36 colon carcinoma, or Meth A sarcoma(1 × 106 cells/mouse). After tumors had grown to 10 mmin diameter, the animals were used as tumor-bearing micein the experiments.

Flow cytometry

The expression of cell-surface antigens on ImC was an-alyzed by staining with the following monoclonal an-

tibodies after treatment with anti-FcγRII/III (2.4G2):fluoresceine isothiocyanate (FITC)-anti-CD11c (NA18),FITC-anti-Gr-1 (RB6-8C5), FITC-anti-CD11b (M1/70),FITC-anti-CD4 (GK1.5), FITC-anti-CD8 (53-6.7), phy-coerythrin (PE)-anti-CD11b, PE-anti-CD11c, PE-anti-mouse Gr-1, PE-anti-MHC class II (NIMR-4),PE-anti-PDL1 (MIH5), allophycocyanin (APC)-anti-F4/80 (BM8), APC-anti-mouse CD11c, APC-anti-IFN-γ(XMG1.2) and APC-anti-IL-4 (11B11). The antibodieswere purchased from eBioscience (San Diego, CA, USA).Flow cytometric analysis was performed using a FACSCal-ibur cytometer (Becton Dickinson, San Jose, CA, USA).

Cell preparation

Splenic CD11b+ cells were obtained by sequential pu-rification using anti-CD11b microbeads (Miltenyi Biotec,Auburn, CA, USA) through a MACS column, accord-ing to the instructions of the manufacturer (MiltenyiBiotec), followed by sorting of Gr-1 highly expressedcells and Gr-1 intermediately expressed cells by FACSAria(Becton Dickinson) after staining with PE-anti-Gr-1 an-tibody. The purity of each population (>96%) wasconfirmed by flow cytometry analysis before experi-ments. In some experiments, sorted cells were culturedin RPMI-1640 containing fetal calf serum (10%) and 2-mercaptoethanol (50 μM) with murine recombinant GM-CSF (4 ng/mL; PeproTech, Rock Hill, NJ, USA).

Proliferation assay

Spleen cells (1 × 105/well) from B6 mice were stimu-lated with anti-CD3ε antibody (500A2, 0.1 μg/mL, eBio-science) as responder T cells in the presence of splenicImC (1 × 105/well) in 96-well plates (13). After 2 days ofculture, cell proliferation was assessed by incorporation of[3H]-thymidine.

Intracellular cytokine staining

Spleen cells (1 × 105) were stimulated for 72 hr withanti-CD3ε antibody with or without 5 × 104 freshly iso-lated splenic ImC or previously cultured splenic ImCfor 1 week in the presence of GM-CSF (4 ng/mL).Activated cells were harvested and washed once with ice-cold phosphate-buffered saline (PBS). They were restimu-lated with phorbol myristrate acetate (PMA) (0.1 μg/mL)and ionomycin (1 μg/mL) in the presence of monensin(2 μM) for 4 hr. After restimulation, cells were harvestedinto conical tubes and washed twice with ice-cold PBS.The cells were then suspended in ice-cold PBS (50 μL foreach test = 1–2 × 105 cells) and stained with FITC-anti-CD4, FITC-anti-CD8, and ethidium monoazide bromide(Molecular Probes, Eugene, OR, USA). After washing oncewith ice-cold PBS, the cells were fixed with 500 μL of 4%

48 c© 2008 The Societies and Blackwell Publishing Asia Pty Ltd

Splenic immature myeloid cells

paraformaldehyde. After washing with PBS containing0.1% bovine serum albumin (BSA) and saponin buffer(prepared freshly in PBS with 0.1% saponin, 1% heat-inactivated fetal calf serum, and 0.1% w/v sodium azide),the cells were stained with APC-anti-IFN-γ or APC-anti-IL-4 on ice for 30 min followed by washing twice withsaponin buffer and ice-cold PBS containing BSA.

Statistical analysis

The statistical significance between values was determinedby Student’s t-test. Probability values < 0.05 were consid-ered as significant differences.

RESULTS

ImC were increased in the tumor-bearingmice

The proportion of Gr-1+CD11b+ ImC in spleens of naı̈veB6 mice and mice bearing E.G7 thymoma were deter-mined. The proportion of splenic ImC was higher intumor-bearing mice than in naı̈ve mice (3.6% vs 28.2%).The ratio of ImC was also increased in BALB/c micebearing Meth A fibrosarcoma (4.4% vs 11.3%). A simi-lar tendency was observed in B6 mice carrying B16-B6melanoma and in BALB/c mice carrying CC26 and CC36colon carcinomas (Table 1). We found that ImC could bedivided into two groups depending on Gr-1 expression,Gr-1 high (Gr-1hi) and intermediate (Gr-1int). The ratioof Gr-1hi ImC was increased in the spleens of mice bearingE.G7 (14.5%) compared with that in mice bearing no tu-mor (0.7%) (Fig. 1). Similarly, the ratio of Gr-1int ImC wasincreased in the spleens of tumor-bearing mice (1.9% vs27.5%). Therefore, increase in the ratio of Gr-1+CD11b+

ImC in spleens of tumor-bearing mice was thought to beindependent of mouse strain and tumor type.

Table 1 The proportion of ImC in the spleen of mice bearing tumor

Mouse Tumor ImC in spleen (%)

C57/BL6 None 3.6E.G7 28.2B16-B6 8.7

BALB/c None 4.4CC26 15.3CC36 11.5Meth A 11.3

Percentage of Gr-1+CD11b+ ImC in spleens of naı̈ve and tumor bearingmice. The percentage of splenic ImC was increased in E.G7 thymoma-or B16-B6 melanoma-bearing mice compared with that in C57BL/6naı̈ve mice. Similarly, the percentage of ImC was increased in Meth Afibrosarcoma or CC26 or CC36 colon carcinoma-bearing BALB/c micecompared with that in naı̈ve BALB/c mice.

Fig. 1. Increase of ImC in tumor-bearing mice. E.G7 cells (1 × 106)were inoculated subcutaneously in B6 mice. When tumor diameter hadreached 10 mm, spleen cells were stained with anti-CD11b and Gr-1 andanalyzed by a flow cytometer. The numbers indicate the percentages ofGr-1hi and Gr-1int ImC in whole spleen cells of a normal mouse (a) anda tumor-bearing mouse (b). Results are representative of those for threemice.

Splenic ImC from tumor-bearing miceinhibited IFN-γ production of CD8+ T cells

In order to analyze the function of ImC, we first investi-gated whether Gr-1int or Gr-1hi ImC inhibited IFN-γ pro-duction of CD8+ T cells stimulated by anti-CD3. Neitherfreshly isolated Gr-1int nor Gr-1hi ImC from non-tumor-bearing mice reduced IFN-γ production of CD8+ T cells(Fig. 2a vs 2b). In contrast, freshly isolated Gr-1int ImCfrom tumor-bearing mice reduced IFN-γ production ofCD8+ T cells, but Gr-1hi ImC did not (Fig. 2a vs 2c). Thesefindings suggest that splenic Gr-1int ImC, but not Gr-1hi

ImC, acquire the capacity to inhibit IFN-γ production ofCD8+ T cells in the presence of tumor.

Splenic Gr-1int ImC from tumor-bearing micedifferentiated into CD11c+F4/80+ cells in thepresence of GM-CSF

It has been demonstrated that GM-CSF is involved in fur-ther differentiation of ImC (17). Therefore, Gr-1int andGr-1hi ImC sorted from spleens of tumor-bearing micewere cultured with GM-CSF, and surface molecules wereanalyzed after 7 days. The cultured Gr-1int ImC had re-duced Gr-1 expression, and one-half of Gr-1int ImC be-came F4/80+ cells or one-third of Gr-1int ImC changedto CD11c+ cells after being cultured with GM-CSF. As adouble staining with anti-F4/80 and anti-CD11c showedthat 77% of F4/80+ cells expressed CD11c on their surface(data not shown), one-third of Gr-1int ImC differentiatedinto CD11c+F4/80+ cells in the presence of GM-CSF. Incontrast, after culture with GM-CSF, the Gr-1 expressionlevel in Gr-1hi ImC was maintained and there were onlya few CD11c+F4/80+ cells (Fig. 3a). CD11c+F4/80+ cells


Y. Yamamoto et al.

Fig. 2. Inhibition of IFN-γ production of T cells by ImC freshly isolatedfrom tumor-bearing mice. Splenic Gr-1int and Gr-1 hi ImC were freshlyisolated from naı̈ve B6 mice and B6 mice bearing E.G7. B6 spleen cellswere stimulated with anti-CD3 for 72 hr in the absence of ImC (a) orin the presence of ImC isolated from mice not bearing a tumor (b) ortumor-bearing mice (c). Thereafter, T cells were stimulated with phorbolmyristrate acetate (PMA) and ionomycin for 4 hr. IFN-γ production wasanalyzed in CD8+ T cells. Numbers indicate the percentages of cytokine-positive cells in CD8+ T cells. Results are representative of those for threemice.

expressed MHC class II molecules at high levels and CD86at low levels (Fig. 3b). These results suggest that Gr-1int

ImC could differentiate into other cells but that Gr-1hi

ImC might be terminally differentiated cells, like granu-locytes.

Differentiated Gr-1int ImC and Gr-1hi ImC inthe presence of GM-CSF inhibited IFN-γproduction of T cells

We investigated whether the differentiated Gr-1int andGr-1hi ImC inhibited IFN-γ production of T cells stimu-lated by anti-CD3. Both Gr-1int and Gr-1hi ImC reducedIFN-γ production by CD8+ T cells (Fig. 4a), and bothGr-1int and Gr-1hi ImC suppressed IFN-γ production byCD4+ T cells (Fig. 4b). However, differentiated Gr-1int

ImC did not polarize CD4+ T cells to Th2 cells withIL-4 production, whereas differentiated Gr-1hi ImC in-duced slightly enhanced IL-4 production by CD4+ T cells(Fig. 4c). These results suggest that differentiated Gr-1int

ImC in the presence of GM-CSF in tumor-bearing mice

Fig. 3. Analysis of surface molecules of Gr-1int and Gr-1hi ImC afterculture with GM-CSF. The Gr-1int and Gr-1hi ImC were separated by acell sorter from E.G7-bearing mice and cultured with GM-CSF for 7 days.Thereafter, surface molecules of the differentiated ImC were stained withanti-Gr-1 and anti-F4/80 or anti-Gr-1 and anti-CD11c (a). Similarly, thedifferentiated ImC were stained with anti-MHC class II and anti-F4/80,anti-MHC class II and anti-CD11c, anti-CD86 and anti-F4/80, or anti-CD86 and anti-CD11c (b). Results are representative of those for threemice.

acquired the capability of inhibiting IFN-γ production byT cells without Th2 polarization.

Splenic Gr-1int ImC derived fromtumor-bearing mice suppressed T-cellproliferative responses after differentiationin the presence of GM-CSF

Next, we examined whether ImC differentiated in the pres-ence of GM-CSF suppress T-cell proliferative responses.Differentiated Gr-1int ImC or Gr-1hi ImC were mixedwith spleen cells at a 1:1 ratio then cultured for 48 hrin the presence of stimulatory anti-CD3 antibody. Dif-ferentiated splenic Gr-1int ImC from tumor-bearing micesuppressed proliferation of splenic T cells (P = 0.01), butdifferentiated Gr-1hi ImC from tumor-bearing mice didnot (Fig. 5a).

In previous studies, it was demonstrated that ImC sup-pressed T cell responses by soluble factors such as NO andTGF-β (12, 14, 15). It has also been shown that ImC neededcell-to-cell contact through the CD3ζ chain to inhibitT-cell responses (16). In order to examine how Gr-1int

ImC derived from tumor-bearing mice that had differen-tiated in culture with GM-CSF suppressed T-cell prolif-eration in our system, supernatants were collected afterculture of the differentiated ImC. The supernatant fromthe differentiated Gr-1int ImC suppressed T-cell prolifera-tive responses (P < 0.001) but that from the differentiatedGr-1hi ImC did not (Fig. 5b).



Fig. 4. Inhibition of IFN-γ production of T cells by Gr-1int and Gr-1hi

ImC differentiated by GM-CSF. Gr-1int and Gr-1hi ImC were prepared asdescribed in the legend to Fig. 3. The differentiated ImC were culturedwith spleen cells in the presence of anti-CD3 antibody for 72 hr as de-scribed in Materials and Methods. Then, T cells were stimulated withphorbol myristrate acetate (PMA) and ionomycin for 4 hr. IntracellularIFN-γ was analyzed in CD8+ T cells (a) and CD4+ T cells (b). IntracellularIL-4 was analyzed in CD4+ T cells (c). Results are representative of thosefor experiments with similar results. Numbers indicate percentages ofcytokine-positive cells in CD8+ or CD4+ cells.

Fig. 5. Inhibition of T-cell proliferative responses by ImC. Gr-1hi and Gr-1int ImC were sorted from spleen cells derived from B6 mice bearing E.G7and cultured as described in the legend to Fig. 3. Culture supernatantswere collected 1 day later. Spleen cells from B6 mice were stimulated withCD3 ligation. The cultured ImC (a) and supernatants from the culturedImC (b) were mixed with the spleen cells at a 1:1 ratio. Cells werecultured for 48 hr. Proliferative responses are indicated as incorporationof [3H]-thymidine. Probability values are shown as results from triplicateculture.

PDL1 on the differentiated Gr-1int ImC fromtumor-bearing mice was not involved insuppression of T-cell proliferation

In a previous study, it was shown that signals throughPD-1 induced apoptosis to suppress T-cell function (18).

Fig. 6. Expression of PDL1 on differentiated Gr-1int and Gr-1hi ImCfrom tumor-bearing mice and contribution of PDL1 to T-cell proliferativeresponses suppressed by Gr-1int ImC. Freshly isolated Gr-1hi ImC and Gr-1int ImC were sorted from spleens of B6 mice bearing E.G7. Cells werecultured as described in the legend to Fig. 3. The expression of PDL1 wasexamined after culture for 7 days with GM-CSF (a). Figures show rep-resentative results from three mice. The differentiated Gr-1int ImC weremixed with spleen cells at a 1:1 ratio in the presence of anti-CD3 anti-body (0.1 μg/mL) with or without anti-PDL1 antibody (10 μg/mL). Afterculture for 48 h, proliferative responses were assessed as incorporationof [3H]-thymidine. Probability values are shown as results from triplicateculture.

Therefore, we examined the expression and function ofPDL1 on the Gr-1int ImC from tumor-bearing mice thathad differentiated in culture with GM-CSF. Differenti-ated Gr-1int ImC upregulated the expression of PDL1,but upregulation on differentiated Gr-1hi ImC was weak(Fig. 6a). Next, we tested whether PDL1 on differenti-ated Gr-1int ImC contributed to the suppression of T-cellproliferation. Even in the presence of a blocking antibodyagainst PDL1, differentiated Gr-1int ImC still inhibitedT-cell proliferative responses. Therefore, PDL1 expressedon the differentiated Gr-1int ImC was not involved in thesuppression of T-cell proliferation (Fig. 6b).

DISCUSSION

Immature myeloid cells or myeloid suppressor cells havebeen defined as the cell population expressing CD11band Gr-1. In general, ImC are thought to have the ca-pacity to suppress host immunity. However, it is diffi-cult to analyze the suppressive function of ImC againstimmune responses due to their heterogeneity. Therefore,we analyzed their suppressive function against T-cell re-sponses after separating splenic ImC into two subpopu-lations depending on Gr-1 expression, Gr-1hi and Gr-1int

ImC. Freshly isolated Gr-1int ImC, but not Gr-1hi ImC,from tumor-bearing mice reduced IFN-γ production ofCD8+ T cells. Neither Gr-1hi ImC nor Gr-1int ImC frommice not bearing a tumor inhibited IFN-γ production.


Y. Yamamoto et al.

However, freshly isolated Gr-1hi ImC and Gr-1int ImCfrom both tumor-bearing mice and mice not bearing atumor did not suppress T-cell proliferative responses, butGr-1int ImC from tumor-bearing mice differentiated invitro by GM-CSF diminished the T-cell responses. Thesuppression was attributed mainly to soluble factors de-rived from the differentiated Gr-1int ImC and not to cell-to-cell contact. These findings suggest that Gr-1int ImCacquire the capability to inhibit responses of T cells ac-cording to differentiation/maturation stages in tumor cir-cumstance.

It has been suggested that GM-CSF induced differen-tiation of ImC to mature antigen-presenting cells (16,17). Therefore, we examined how Gr-1hi and Gr-1int

ImC differentiated in vitro in the presence of GM-CSF.GM-CSF induced differentiation of Gr-1int ImC fromtumor-bearing mice into CD11c+F4/80+ cells. How-ever, two-thirds of the Gr-1int ImC differentiated intoGr-1−CD11c−F4/80− cells, indicating the possibility thatGr-1int ImC are still heterogeneous. In contrast, Gr-1hi ImC from tumor-bearing mice were not altered byGM-CSF, indicating that Gr-1hi ImC might be termi-nally differentiated cells, like granulocytes. In addition,the differentiated Gr-1int ImC expressed low levels of CD86and high levels of MHC class II. This expression patternof surface molecules suggests that these cells might beinvolved in anergy induction. In the near future we willexamine this possibility.

Both Gr-1hi ImC and Gr-1int ImC from tumor-bearingmice that had been differentiated by GM-CSF suppressedthe differentiation of CD4+ and CD8+ T cells into IFN-γ-producing cells, although freshly isolated Gr-1int ImC, butnot Gr-1hi ImC, from tumor-bearing mice reduced IFN-γproduction of CD8+ T cells. After culture with GM-CSF,Gr-1int ImC expressed F4/80 and CD11c molecules andshowed suppressive activity against T-cell proliferative re-sponses. Therefore, we speculate that splenic Gr-1int ImCmigrate into tumor sites and are differentiated by GM-CSFinto cells with upgraded suppressive activity against hostT-cell immunity. However, it is unclear whether GM-CSFis always involved in ImC differentiation in the tumorcircumstances. Indeed, our previous study showed thatF4/80+CD11c+ ImC isolated from tumor sites did notinhibit T-cell proliferative responses (13). Thus, hereinwe merely mention that splenic Gr-1int ImC become up-graded suppressor cells in the presence of GM-CSF in vitro.

We have demonstrated that suppression of T-cell pro-liferation is due to soluble factors derived from differen-tiated Gr-1int ImC. IL-10, TGF-β or NO, however, wasnot involved in the suppression (data not shown). Othercandidates produced from ImC are ROS and peroxyni-trite, which might induce modification of TCR and CD8molecules, resulting in loss of the ability of CD8+ T cells

to bind to the peptide-MHC complex (19). However, aswe used non-specific stimulation through CD3 to activateCD8+ T cells, this mechanism is not likely to explain thelow responsiveness of CD8+ T cells in our system.

A previous study showed that signals through PD-1 induced apoptosis to suppress T-cell function (18).PDL1 was expressed weakly on differentiated Gr-1hi ImCbut highly on CD11c+F4/80+ differentiated Gr-1int ImC.However, a blocking antibody to PDL1 did not resultin recovery of T-cell proliferation from suppression bythe differentiated Gr-1int ImC. In addition, a previousstudy showed that deletion of L-arginine by arginase 1induced by IL-4 in ImC resulted in T-cell unresponsive-ness (20). However, Gr-1int ImC did not elicit Th2 po-larization in the presence of GM-CSF. Similarly, freshlyisolated Gr-1int ImC from tumor-bearing mice did not in-duce IL-4 production of T cells (data not shown). Takentogether, the results suggest that soluble factors mainlycontribute to the immunosuppression induced by Gr-1int

ImC without Th2 polarization, although further investiga-tion is needed to identify surface markers of differentiatedImC.

It is well known that ImC comprise a heterogeneouspopulation and an exact definition of ImC is still unclear.Recently, it has been reported that immature myeloidsuppressor cells (Gr-1+CD115+F4/80+) induced in vivodevelopment of Foxp3+ T regulatory cells, which wereanergic and suppressive (21). ImC, including immaturemyeloid suppressor cells, have mainly been isolated fromspleens of tumor-bearing mice. However, a new type ofImC (CD34+CD11b+) has been shown to be recruitedfrom bone marrow to the tumor invasion front. Thesecells express matrix metalloproteinase MMP9 and MMP2and are thought to facilitate tumor invasion (22). Thesecells have not been shown to possess immune suppressivecapacity. Thus, further studies using ImC in tumor sitesare required to determine the true function of ImC andprovide a new strategy for cancer therapy.

REFERENCES

1. Arteaga C.L., Hurd S.D., Winnier A.R., Johnson M.D., Fendly B.M.,Forbes J.T. (1993) Anti-transforming growth factor (TGF)-betaantibodies inhibit breast cancer cell tumorigenicity and increasemouse spleen natural killer cell activity. Implications for a possiblerole of tumor cell/host TGF-beta interactions in human breastcancer progression. J Clin Invest 92: 2569–76.

2. Jarnicki A.G., Lysaght J., Todryk S., Mills K.H. (2006) Suppressionof antitumor immunity by IL-10 and TGF-beta-producing T cellsinfiltrating the growing tumor: influence of tumor environment onthe induction of CD4+ and CD8++ regulatory T cells. J Immunol177: 896–904.

3. Won J., Kim H., Park E.J., Hong Y., Kim S.J., Yun Y. (1999)Tumorigenicity of mouse thymoma is suppressed by soluble type IItransforming growth factor beta receptor therapy. Cancer Res 59:1273–7.



4. Bronte V., Serafini P., De Santo C., Marigo I., Tosello V., MazzoniA., Segal D.M., Staib C., Lowel M., Sutter G., Colombo M.P.,Zanovello P. (2003) IL-4-induced arginase 1 suppresses alloreactiveT cells in tumor-bearing mice. J Immunol 170: 270–8.

5. Onizuka S., Tawara I., Shimizu J., Sakaguchi S., Fujita T., NakayamaE. (1999) Tumor rejection by in vivo administration of anti-CD25(interleukin-2 receptor alpha) monoclonal antibody. Cancer Res 59:3128–33.

6. Sutmuller R.P., van Duivenvoorde L.M., van Elsas A., SchumacherT.N., Wildenberg M.E., Allison J.P., Toes R.E., Offringa R., MeliefC.J. (2001) Synergism of cytotoxic T lymphocyte-associated antigen4 blockade and depletion of CD25(+) regulatory T cells inantitumor therapy reveals alternative pathways for suppression ofautoreactive cytotoxic T lymphocyte responses. J Exp Med 194:823–32.

7. Almand B., Clark J.I., Nikitina E., van Beynen J., English N.R.,Knight S.C., Carbone D.P., Gabrilovich D.I. (2001) Increasedproduction of immature myeloid cells in cancer patients: amechanism of immunosuppression in cancer. J Immunol 166:678–89.

8. Kusmartsev S., Nagaraj S., Gabrilovich D.I. (2005)Tumor-associated CD8+ T cell tolerance induced by bonemarrow-derived immature myeloid cells. J Immunol 175: 4583–92.

9. Melani C., Chiodoni C., Forni G., Colombo M.P. (2003) Myeloidcell expansion elicited by the progression of spontaneousmammary carcinomas in c-erbB-2 transgenic BALB/c micesuppresses immune reactivity. Blood 102: 2138–45.

10. Kusmartsev S., Cheng F., Yu B., Nefedova Y., Sotomayor E., Lush R.,Gabrilovich D. (2004) All-trans-retinoic acid eliminates immaturemyeloid cells from tumor-bearing mice and improves the effect ofvaccination. Cancer Res 63: 4441–9.

11. Ghiringhelli F., Puig P.E., Roux S., Parcellier A., Schmitt E., SolaryE., Kroemer G., Martin F., Chauffert B., Zitvogel L. (2005) Tumorcells convert immature myeloid dendritic cells intoTGF-beta-secreting cells inducing CD4+CD25+ regulatory T cellproliferation. J Exp Med 202: 919–29.

12. Gabrilovich D.I., Velders M.P., Sotomayor E.M., Kast W.M. (2001)Mechanism of immune dysfunction in cancer mediated byimmature Gr-1+ myeloid cells. J Immunol 166: 5398–406.

13. Ishigaki H., Yamamoto Y., Ishida H., Kajino K., Itoh Y., Fujiyama Y.,Ogasawara K. (2006) Preparation and functional analysis oftumor-infiltrating stroma cells using bone marrow chimera mice.Microbiol Immunol 50: 655–62.

14. Pelaez B., Campillo J.A., Lopez-Asenjo J.A., Subiza J.L. (2001)Cyclophosphamide induces the development of early myeloid cellssuppressing tumor cell growth by a nitric oxide-dependentmechanism. J Immunol 166: 6608–15.

15. Terabe M., Matsui S., Park J.M., Mamura M., Noben-Trauth N.,Donaldson D.D., Chen W., Wahl S.M., Ledbetter S., Pratt B.,Letterio J.J., Paul W.E., Berzofsky J.A. (2003) Transforming growthfactor-beta production and myeloid cells are an effector mechanismthrough which CD1d-restricted T cells block cytotoxic Tlymphocyte-mediated tumor immunosurveillance: abrogationprevents tumor recurrence. J Exp Med 198: 1741–52.

16. Kusmartsev S., Nefedova Y., Yoder D., Gabrilovich D.I. (2004)Antigen-specific inhibition of CD8+ T cell response by immaturemyeloid cells in cancer is mediated by reactive oxygen species. JImmunol 172: 989–99.

17. Li Q., Pan P.Y., Gu P., Xu D., Chen S.H. (2004) Role of immaturemyeloid Gr-1+ cells in the development of antitumor immunity.Cancer Res 64: 1130–9.

18. Probst H.C., McCoy K., Okazaki T., Honjo T., van den Broek M.(2005) Resting dendritic cells induce peripheral CD8+ T celltolerance through PD-1 and CTLA-4. Nature Immunol 6:280–6.

19. Nagaraj S., Gupta K., Pisarev V., Kinarsky L., Sherman S., Kang L.,Herber D.L., Schneck J., Gabrilovich D.I. (2007) Alteredrecognition of antigen is a mechanism of CD8+ T cell tolerance incancer. Nat Med 13: 828–35.

20. Curiel T.J., Coukos G., Zou L., Alvarez X., Cheng P., Mottram P.,Evdemon-Hogan M., Conejo-Garcia J.R., Zhang L., Burow M., ZhuY., Wei S., Kryczek I., Daniel B., Gordon A., Myers L., Lackner A.,Disis M.L., Knutson K.L., Chen L., Zou W. (2004) Specificrecruitment of regulatory T cells in ovarian carcinoma fostersimmune privilege and predicts reduced survival. Nat Med 10:942–9.

21. Huang B., Pan P.Y., Li Q., Sato A.I., Levy D.E., Bromberg J., DivinoC.M., Chen S.H. (2006) Gr-1+CD115+ immature myeloidsuppressor cells mediate the development of tumor-induced Tregulatory cells and T-cell anergy in tumor-bearing hosts. CancerRes 66: 1123–31.

22. Kitamura T., Kometani K., Hashida H., Matsunaga A., Miyoshi H.,Hosogi H., Aoki M., Oshima M., Hattori M., Takabayashi A.,Minato N., Taketo M.M. (2007) SMAD4-deficient intestinal tumorsrecruit CCR1+ myeloid cells that promote invasion. Nat Genet 39:467–75.


analysis of splenic gr-1int immature myeloid cells in tumor-bearing mice

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