regulation of γδ t cell survival by soluble hla-i: involvement of cd8 and activating killer...

Regulation of cd T cell survival by soluble HLA-I:Involvement of CD8 and activating killer Ig-like receptors

Alessandro Poggi1, Paola Contini2, Silvia Catellani3, Maurizio Setti4,Giuseppe Murdaca4 and Maria Raffaella Zocchi3

1 Laboratory of Experimental Oncology D, National Institute for Cancer Research,Genoa, Italy

2 Laboratory of Clinical Immunology, University of Genoa, Genoa, Italy3 Laboratory of Tumor Immunology, San Raffaele Institute, Milan, Italy4 Department of Internal Medicine, University of Genoa, Genoa, Italy

We show that human Vd1 or Vd2 T lymphocytes secrete FasL and undergo apoptosisupon incubationwith soluble HLA (sHLA)-I or after cross-linking of CD8, with a kineticsdifferent from that observed following ligation of TCR. sHLA-I-induced apoptosis wasblocked by anti-CD8 mAb; on the other hand, sHLA-I was not effective in CD8– clones,while an HLA-I mutated in the a3 domain, responsible for CD8 binding, was notfunctional on CD8+ clones. Purified sHLA-Cw3 or -Cw4 alleles, isolated from the Cw3-or Cw4-transfected 721.221 lymphoblastoid cell line, triggered cd T cell apoptosis,interacting with the specific receptors CD158j/KIR2DS2 or CD158 h/KIR2DS1,respectively, also known as activating isoforms of killer Ig-like receptors (KIR). Again,this effect was dependent on FasL secretion and it was blocked by specific mAb toKIR2DS2 or KIR2DS1. The engagement of CD8 or activating KIR also triggered theproduction of TNF-a. Noteworthy, sHLA-I molecules synergize with antigen-mediatedactivation of Vd2 T cells: Indeed, Vd2 T lymphocytes produced TNF-a when stimulatedwith isopentenyl pyrophosphate, and this effect was enhanced by sHLA-I. Thesefindings suggest that sHLA-I can regulate cd T cell survival and that activating KIR mayamplify antigen-specific Vd2 T cell responses.

Introduction

Human TCR cd T lymphocytes are though to beimportant effectors in the first-line defense againstinfections and tumors [1–5]. These cells share somephenotypic and functional characteristics typical of NKcells, reported to be the most potent cytolytic effector

cells in humans [5–7]. Indeed, TCRcd T cells can expressCD56 and CD16 cell surface receptors, which were firstlyconsidered as characteristic NK cell molecules [6–8],and are capable of MHC-independent killing oftransformed and infected cells [5, 8]. Actually, cdT cells can be subdivided into two subpopulations withdistinct functional behavior, i.e. circulating Vd2 T cells,representing the majority of peripheral blood cdT lymphocytes and mainly implied in the responseagainst viral or mycobacterial infections and hematolo-gical tumors, and Vd1 T cells, which are resident in themucosal-associated lymphoid tissue and are thought toplay a role in epithelial cancers [8–10]. Vd2 T cellsappear to recognize peculiar non-protein antigens suchas isopentenyl pyrophosphate (IPP) expressed bymycobacteria, while the nature of the antigens recog-nized by Vd1 lymphocytes remains elusive [1, 4, 8, 10].

Immunomodulation

Correspondence: Dr. Alessandro Poggi, Laboratory of Experi-mental Oncology D, National Institute for Cancer Research,Largo Rosanna Benzi 10, I-16132 Genoa, ItalyFax: +39-010-354282e-mail: [email protected]

Received 11/3/05Revised 8/7/05

Accepted 29/7/05

[DOI 10.1002/eji.200526177]

Key words:Apoptosis � soluble

HLA-I � cdT cells � CD8� Activating IRS

Abbreviations: CLIR: C-type lectin inhibitory receptor �FasL: Fas ligand � GAM: goat anti-mouse � IPP: isopentenylpyrophosphate � IRS: inhibitory-receptor superfamily �KIR: killer Ig-like inhibitory receptor � PI: propidium iodide �sHLA: soluble HLA

Alessandro Poggi et al. Eur. J. Immunol. 2005. 35: 2670–26782670

f 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji.de

Although cd T lymphocytes are thought to exert non-MHC-restricted antigen recognition, both cd T cellsubpopulations can bear the CD8 molecule that is ableto bind HLA-I [11, 12]. Recently, it has become evidentthat soluble HLA (sHLA)-I molecules, which can bereleased by transformed and virus-infected cells, caninduce the apoptosis of short-term mitogen-stimulatedpolyclonal ab T cell populations [13–15] and NK cellclones [16]. It has been reported that in alloreactive abCTL specific for HLA-A2 or HLA-B7, this effect is due toTCR engagement [13]; this was further confirmed usingsoluble MHC-peptide complexes with impaired CD8binding [17, 18]. However, it has been shown that in NKcells, which lack TCR, the interaction of sHLA-I withCD8 induces the synthesis and release of FasL, which inturn triggers apoptosis by engaging its receptor Fas atthe cell surface [14–16]. A similar mechanism has beenproposed for activated T lymphocytes; in these lym-phocytes, as well as in NK cells, sHLA-I-induced releaseof FasL and the consequent apoptosis are blocked byspecific anti-CD8 mAb [14–16].

TCRcd T lymphocytes resemble NK cells also in theexpression of members of the inhibiting-receptor super-family (IRS) that interact with discrete HLA-I alleles [19,20]. Two structural types of IRS can be distinguished:(i) One consists of immunoglobulin (Ig) superfamilyreceptors, including killer Ig-like receptors (KIR); and(ii) the C-type lectin inhibitory receptors (CLIR), likethe NKG2/CD94 complex [11, 12]. Engagement of IRSmembers with a long cytoplasmic tail such as KIR2DL1or KIR2DL2 (CD158a and CD158b) by their naturalligands, the HLA-Cw4 and HLA-Cw3 alleles, leads to therecruitment of tyrosine phosphatases and to theblockade of early signals in the activation cascade[12, 20]. On the other hand, members of IRSwith a shortcytoplasmic domain (such as KIR2DS1 and KIR2DS2,also called CD158 h and CD158j) exert activating ratherthan inhibiting functions; these receptors can bind thesame HLA-I alleles recognized by their inhibitingcounterparts [20–22]. Recently, we have demonstratedthat the engagement of activating IRS with theappropriate sHLA-I allele can trigger NK cell apoptosisand IFN-c production [23, 24].

It has been shown that the majority of CD8-positivecdT cells, in particular the Vc9/Vd2 subset, express CLIRand KIR, or both, and high levels of CCR5, the receptorthat enables HIV-1 entry [25–27]. Interestingly, KIRexpressed by Vc9/Vd2 T lymphocytes have been re-ported to down-regulate TCR-mediated signaling inresponse to HLA-I-expressing B cell lymphomas [28].

At present, it has not been clarified whether sHLA-Iengagement of CD8 or KIR2DS onTCRcd T lymphocytescan induce apoptosis and/or cytokine production.Moreover, it is still to be defined whether theengagement of these receptors for HLA-I can amplify

the TCR-mediated immune response of cd T cells. In thispaper, we show that both CD8 and KIR2DS can induce inTCRcd T cells the release of FasL, which by interactingwith Fas triggers lymphocyte apoptosis. This effect isaccompanied by the production of immunoregulatorycytokines, such as TNF-a. More importantly, sHLA-Ialleles have a synergistic effect with phosphate antigensin activating cd T cells, suggesting that sHLA-I may bothamplify and regulate TCRcd T cell immune responses.

Results

sHLA-I induces FasL-dependent cd T cellapoptosis with kinetics different from TCR

It has been reported that activated CD8+ TCRab T cellscan undergo apoptosis upon interaction with sHLA-I[13–15]. To determine whether on cd T cells sHLA-I canregulate cell survival, we first selected from fourdifferent healthy donors 80 Vd1 (20 from each donor)and 95 Vd2 T cell clones (20 clones from two donors,30 clones from the third donor and 25 clones from thefourth donor) for the expression of CD8 antigen, and wefound that 25/80 Vd1+ and 45/95 Vd2+ T cell cloneswere CD8+ (not shown). To induce T cell apoptosis,sHLA-I was isolated from sera of healthy donors (whichrepresents a mixture of HLA-A, -B and -C alleles).Noteworthy, treatment with sHLA-I led to the apoptosisof Vd2+CD8+ (Fig. 1A) and Vd1+CD8+ (Fig. 1B) T cellclones; a similar effect was observed by CD8 oligomer-ization using the specific mAb (Fig. 1A, B). T cell death,detectable at 24 h (20% of apoptotic cells), was maximal(60–70%) 48 h after CD8 cross-linking or incubationwith sHLA-I (Fig. 1A, B). Interestingly, the apoptosisinduced on cd T cells by the engagement of TCRdisplayed different kinetics compared to that inducedby anti-CD8 mAb or sHLA-I, as it was maximal (>90% ofapoptotic cells) at 12 h for Vd2 and at 24 h for Vd1 T cellclones (Fig. 1A, B).

That cd T cell apoptosis induced by sHLA-I ismediated by the engagement of the CD8 receptor wasstrongly suggested by the finding that this phenomenonwas blocked by the covering of CD8 with anti-CD8 mAb,either on Vd2 clones (Fig.1C) or on Vd1 clones (Fig. 1D).More importantly, the pentameric MHC-peptide com-plex HLA-A1101, mutated in the a3 domain and unableto bind to CD8, was not able to deliver an apoptoticsignal, at variance with the pentameric wild-type HLA-A1101 (Fig. 1C). In turn, neither CD8 cross-linking noraddition of sHLA-I were effective when CD8 was notexpressed at the cell surface (Fig. 1D). It is of note thatthe representative T cell clones shown in Fig.1C and Ddid not express at the cell surface receptors for sHLA-Iother than CD8 (i.e. they were KIR and CLIR negative;

Eur. J. Immunol. 2005. 35: 2670–2678 Immunomodulation 2671


not shown). Both sHLA-I- and CD8-induced apoptosis ofVd2 or Vd1 T cell clones was dependent on theinteraction of FasL with Fas, as it was inhibited by theaddition of anti-Fas and/or anti-FasL mAb (Fig. 2A); thiswas confirmed by the analysis of DNA laddering (notshown). Moreover, sFasL was detected in the super-natant of such clones upon CD8 cross-linking or sHLA-Istimulation (Fig. 2B), suggesting that an autocrinepathway is responsible for cd T cell apoptosis.

Discrete sHLA-I alleles induce apoptosis of cdT cells through the engagement of activating KIR:Involvement of Fas/FasL interaction

To determine whether also KIR2DS receptors expressedby cd T cells could deliver an apoptotic signal, someexperiments were performed using discrete sHLA-Ialleles isolated from the allele-transfected 721.221lymphoblastoid cell line. cd T cell clones expressingKIR2DS1 and/or KIR2DS2 molecules were selected onthe basis of (i) their reactivity with anti-KIR2D mAb(GL183 mAb recognizing KIR2DL2 and KIR2DS2; EB6mAb recognizing KIR2DL1 and KIR2DS1) and (ii) theability of these mAb to trigger the cytolytic activity of agiven clone in a redirected killing assay, as described foridentifying NK cell clones bearing activating isoforms of

Fig. 1. Kinetics of apoptosis in cd T cells induced by sHLA-I orTCR engagement. (A, B) cd T cell clones [(A) Vd2 clones panel,(B) Vd1 clones] were incubated with either sHLA-I or anti-CD8or anti-TCR mAb, followed by GAM Ab-coated beads (-XL), andapoptotic cells were analyzed by annexin-V staining at theindicated time points. Means � SD from experiments with tenVd2 clones or eight Vd1 clones. (C, D) cd T cell clones [(C) Vd2clones, (D) Vd1 clones] were incubated with either sHLA-I oranti-CD8 or anti-TCR mAb, alone or followed by GAM Ab-coated beads (-XL), and apoptotic cells were analyzed byannexin-V staining at 48 h. In some experiments, soluble anti-CD8 mAb was added. In parallel samples, the MHC-peptidecomplex HLA-A1101/YVNVNMGLK, wild type (a3wt) or mu-tated in the a3 domain (a3 m) and unable to bind to CD8, wasused at 4 lg/mL (C). Anti-CD8 Ab cross-linking was alsoperformed on CD8– clones as a control (D). Means � SD fromexperiments with ten Vd2 clones or eight Vd1 clones and sixVd1 CD8– clones. GAM in (A–D): GAM Ab-coated beads alone.

Fig. 2. Apoptosis of cd T cells induced by sHLA-I is mediated byFasL/Fas interaction. (A) Vd1 and Vd2 clones were incubatedwith sHLA-I or anti-CD8 mAb followed by cross-linking withGAM Ab-coated beads (-XL) either alone or in the presence ofanti-Fas and/or anti-FasL mAb. Apoptotic cells were analyzedby annexin-V staining at 48 h of incubation. Results areexpressed as % of apoptotic cells (annexin-V+ PI– cells). Means� SD fromexperimentswith tenVd2 clones or eight Vd1 clones.GAM: GAMAb-coated beads alone. (B) Vd1 and Vd2 cloneswereincubatedwith sHLA-I or anti-CD8mAb or anti-TCRmAb (anti-Vd1 or anti-Vd2) followed by cross-linking with GAMAb-coatedbeads (-XL). Soluble FasL was measured by ELISA in clonesupernatant recovered after 24 h and referred to a standardcurve obtained with recombinant FasL. Means � SD fromexperiments with ten Vd2 clones or eight Vd1 clones. GAM:GAM Ab-coated beads alone.



KIR [21, 23]. About 10% Vd1 (70 clones analyzed) orVd2 (90 clones analyzed) clones were either KIR2DS1+

or KIR2DS2+, and 2%were double positive (not shown).The Vd2 clone B25.15, which is CD8–CD158j+/KIR2DS2+ (Fig. 3A), the Vd1 clone A50.4, which isCD8–CD158h+/KIR2DS1+ (Fig. 3B), and the Vd1 cloneA50.12, which is CD8–CD158h+CD158j+ (Fig. 3C), were

incubated with discrete alleles of sHLA-I (Cw3 as ligandof CD158j or Cw4 as ligand of CD158 h) or with sHLA-A2 (as a negative control) for 48 h; then, cells wereevaluated for apoptosis. As shown in Fig. 3, sHLA-Cw3(A, C) and sHLA-Cw4 (B, C) induced cell death ofTCRcd T cells, in keeping with the expression of the Cw3receptor CD158j or the Cw4 receptor CD158 h. Inparticular, either sHLA-Cw3 or sHLA-Cw4 was effectiveon the Vd1 clone A50.12 that bears at the cell surfaceboth CD158 h and CD158j (Fig. 3C). Accordingly, sHLA-Cw3- (Fig. 3A, C) or sHLA-Cw4-induced apoptosis(Fig. 4B, C) was inhibited by blocking the specificreceptors with anti-KIR mAb, but not by the use of anti-TCR mAb; on the other hand, cross-linking of either KIR

Fig. 3. sHLA-I alleles induce cd T cell apoptosis through theengagement of activating KIR. The Vd2 clone B25.15,CD8–CD158j+/KIR2DS2+ (A), the Vd1 clone A50.4,CD8–CD158h+/KIR2DS1+ (B), and the Vd1 clone A50.12,CD8–CD158h+CD158j+ (C), were incubated with discrete allelesof sHLA-I (Cw3 as ligand of CD158j/KIR2DS2 or Cw4 as ligand ofCD158 h/KIR2DS1) or with sHLA-A2 (as indicated) for 48 h;then, cells were evaluated for apoptosis. sHLA-Cw3 (A, B) andsHLA-Cw4 (B, C) were used alone or in combination withblocking anti-KIR or anti-TCR mAb. In turn, anti-KIR or anti-TCR mAb were cross-linked with GAM Ab-coated beads (-XL).Apoptotic cells were analyzed by annexin-V staining at 48 h ofincubation. Results are expressed as % of apoptotic cells(annexin-V+ PI– cells). Means � SD from three independentexperiments with each clone. GAM: GAM Ab-coated beadsalone.

Fig. 4.Apoptosis of cd T cells induced by discrete sHLA-I allelesthrough the engagement of activating KIR is mediated by FasL/Fas interaction. cd T cell clones [(A) Vd1 clone A50.3CD8–CD158j+/KIR2DS2+, (B) Vd2 clone B50.8 CD8–CD158h+/KIR2DS1+] were incubated with discrete alleles of sHLA-I(Cw3 as ligand of CD158j/KIR2DS2 or Cw4 as ligand of CD158 h/KIR2DS1) or anti-KIR mAb, alone or followed by cross-linkingwith GAM Ab-coated beads (-XL), for 48 h; then, cells werestained with annexin-V and PI. Results are expressed as % ofapoptotic cells (annexin-V+ PI– cells). In some samples, anti-Fasand/or anti-FasL mAb were added, as indicated. Data are themeans � SD of three independent experiments with eachclone. GAM: GAMAb-coated beads alone. (C, D) cd T cell clones(Vd1 clone A50.3 and Vd2 clone B50.8) were incubated withsHLA-I alleles (Cw3, Cw4 or A2) or anti-KIR mAb followed bycross-linking with GAM Ab-coated beads (-XL). In someexperiments, the anti-pan-KIR2D mAb (anti-KIR) was usedas a soluble form to block the interaction of sHLA-I alleles withtheir counterreceptors. Soluble FasL was measured by ELISA inclone supernatants recovered after 24 h and referred to astandard curve obtained with recombinant FasL. Means � SDfrom three independent experiments with each clone. GAM:GAM Ab-coated beads alone.



or TCR was capable of inducing cd T cell apoptosis(Fig. 3). These data suggest that cd T cell apoptosisinduced through the engagement of activating KIR is notdependent on the interaction of sHLA-I alleles with TCR.

Interestingly, apoptosis of cd T cells induced bydiscrete sHLA-I alleles through the engagement ofactivating isoforms of KIR molecules is mediated byFas/FasL interaction. Indeed, as shown in Fig. 4, theapoptosis induced in the Vd1 clone A50.3, and the Vd2clone B50.8, by sHLA-Cw3 (A) and sHLA-Cw4 (B),according to the expression of the Cw3 receptor CD158jor the Cw4 receptor CD158 h, was blocked by anti-Fasand/or anti-FasL mAb (Fig. 4A, B). Along this line, anti-Fas and/or anti-FasL mAb inhibited also the apoptosiselicited upon KIR2DS1 or KIR2DS2 cross-linking at thesurface of either cd T cell clone (Fig. 4A, B). Finally,soluble FasL was detected in the supernatant of cloneA50.3 Vd1 (Fig. 4C) or clone B50.8 Vd2 (Fig. 4D) uponengagement of KIR2DS1 or KIR2DS2 by incubation withsHLA-Cw3 (Fig. 4C) or sHLA-Cw4 (Fig. 4D), accordingto the expression of their specific receptors. Accordingly,FasL secretion in these clones was abolished by theaddition of anti-pan-KIR2D mAb together with sHLA-Cw3 (Fig. 4C) or sHLA-Cw4 (Fig. 4D) alleles.

sHLA-I triggers TNF-a production by cdT lymphocytes and synergizes with TCR

We further investigated whether the engagement of CD8or activating KIR members also induced the productionand release of pro-inflammatory cytokines. To this aim,the Vd2 T cell clones B75.4 (CD158j+CD8–) and B32.8(CD8+CD158–), or the Vd1 T cell clones A25.2(CD158h+CD8–) and A13.3 (CD8+CD158–), weretriggered with the sHLA-Cw3 or sHLA-Cw4 forms, orwith antibodies directed to KIR2DS1 or KIR2DS2 or CD8molecules followed by goat anti-mouse (GAM) Ab-coated beads; supernatants were harvested at 24 h andanalyzed for the presence of TNF-a. As shown in Fig. 5A,oligomerization of KIR or CD8 triggered TNF-a produc-tion; moreover, secretion of TNF-awas elicited by eithersHLA-Cw3 or sHLA-Cw4 on cd T lymphocytes, depend-ing on the expression of either CD158j/KIR2DS2 orCD158 h/KIR2DS1, respectively (Fig. 5A). Conversely,no IFN-c production was observed in either Vd1 or Vd2T cell clones following stimulation with sHLA-I alleles(not shown).

It is noteworthy that the engagement of KIR2DS withsHLA-I synergizes with antigen-mediated activation ofcd T cells. Indeed, Vd2+ T lymphocytes (two represen-tative Vd2 CD158j+CD8– clones out of six tested aredepicted in Fig. 5B) produced TNF-a when challengedwith IPP, which is known to specifically stimulate cellsbearing the Vd2 TCR [10] (Fig. 5B). Moreover, a strongincrease (fourfold) of TNF-a secretion was observed

when Vd2+ T cells were incubated with IPP and sHLA-I-Cw3 (counterligand for CD158j/KIR2DS2) compared toeither IPP or sHLA-I alone, as shown in Fig. 5B.Altogether, these findings suggest that sHLA-I, interact-ing with activating KIR, may amplify the antigen-specificcd T cell response. This might be relevant whenpathogens producing phosphate antigens and/or releas-ing sHLA-I molecules are involved, as occurs duringmicobacterial or HIV-1 infection.

Discussion

In this paper, we provide evidence that occupancy ofCD8 with sHLA-I, or of activating KIR with specific

Fig. 5. The engagement of the activating KIR with discretesHLA-I alleles triggers cytokine production by cdT lymphocytes and synergizes with TCR. (A) The Vd2 B75.4and B32.8 or the Vd1 A25.2 and A13.3 T cell clones weretriggeredwith sHLA-I alleles, or with antibodies directed to theindicated surfacemolecules followed by GAMAb-coated beads(-XL). Cross-linking of CD54 molecules was used as a negativecontrol. Supernatants were harvested at 24 h and analyzed forthe presence of TNF-a by ELISA. Results are expressed as pg/mLand are the means � SD from three independent experimentswith each clone. (B) Two representative Vd2 T cell clones(CD158j+/KIR2DS2+CD8–) out of six were stimulated for 24 hwith either sHLA-Cw3 or IPP or with sHLA-Cw3 + IPP. Super-natants were analyzed for the presence of TNF-a by ELISA.Results are expressed as pg/mL and are the means � SD fromthree independent experiments with each clone. None: Cellsincubated with culture medium alone.



sHLA-I alleles, can induce in cd T cells the release ofFasL, which by interacting with Fas triggers lymphocyteapoptosis. This effect is accompanied by the productionof immunoregulatory cytokines, such as TNF-a. Moreimportantly, sHLA-I has a synergistic effect withphosphate antigens in activating cd T cells, suggestingthat sHLA-I may regulate and amplify the function ofthis lymphocyte population, besides controlling theirsurvival.

It is still under debate whether sHLA-I can triggerprogrammed cell death (PCD) due to its direct bindingto CD8. Indeed, in alloreactive ab CTL, HLA-I-inducedapoptosis is conceivably due to TCR rather than CD8engagement [13], as it can be obtained using solubleMHC-peptide complexes with impaired CD8 binding[17, 18]. However, in our experiments, sHLA-I did notelicit apoptosis in cd T cell clones lacking CD8; more-over, in CD8+ clones, sHLA-I-induced release of FasLand the consequent apoptosis were blocked by specificanti-CD8 mAb. More importantly, an MHC-peptidecomplex mutated in the a3 domain and unable to bindto CD8 was not able to deliver an apoptotic signal,suggesting that sHLA-I-CD8 interaction can be respon-sible for cd PCD.

We should note that the concentrations of sHLA-I(*3 lg/mL; 50 nM), able to induce apoptosis of cdT cells through CD8aa interaction, are more than 1000-fold lower than the Kd affinity (*200 lM) of sHLA-I forCD8aa. A possible explanation of the observed effect, atthese very low concentrations, is that multimericaggregates of sHLA-I are present in our preparations.Alternatively, the affinity of sHLA-I for CD8aa might bedifferent when CD8 is cell membrane associatedcompared to the soluble form used for Kd determination[29].

A similar molecular mechanism has been proposedfor NK cells, which lack TCR, and for activatedT lymphocytes where synthesis and release of FasLand triggering of apoptosis by sHLA-G1 are inhibited byspecific anti-CD8 mAb [14–16]. Nevertheless, we cannotexclude that other ligands, such as a human analog ofthe murine thymic leukemia antigen [30], can bind toCD8aa, the homodimer expressed by NK and cd T cells,and are responsible for the sHLA-I biological effects invivo.

We have previously shown that sHLA-I can induceapoptosis of NK cells through the engagement of theactivating isoforms of KIR [23, 24], and here we showthat this may also occur in human Vd1+ or Vd2+

T lymphocytes. Indeed, sHLA-Cw3 selectively leads toapoptosis of cd T cells expressing its counterligandCD158j/KIR2DS2, while the sHLA-Cw4 allele exerts thesame effect only on cd T lymphocytes bearing itsreceptor CD158 h/KIR2DS1; in both cases, FasL releaseand apoptosis are inhibited by blocking the sHLA-I allele

binding to KIR2DS2 or KIR2DS1 with the specific mAb.Moreover, secretion of TNF-a was elicited by eithersHLA-Cw3 or sHLA-Cw4 on cd T lymphocytes, depend-ing on the expression of either CD158j/KIR2DS2 orCD158 h/KIR2DS1, respectively. These data suggestthat sHLA-I molecules, through the engagement ofactivating KIR, can amplify the immune response byinducing the production of pro-inflammatory cytokines,but also provide a feedback control of NK and cd T cellactivation by inducing at the same time the release of apro-apoptotic factor such as FasL.

During the immune response to several pathogens,the innate arm of the immune system is activated: NKand T cells bearing the cd TCR can be recruited to thesite of inflammation, where they may play a role inarming an effective response [2, 3, 7]. However, severalinflammatory cytokines and effector cells can lead to thedeath of epithelial or mucosal cells and the release ofsHLA-I molecules [31, 32]. Recently, it has becomeevident that sHLA-I molecules can also be released bytransformed and virus-infected cells, thus possiblycontributing to the depletion of CD8+ effector lympho-cytes [31, 32]. In two different studies, CD8+ cd T cellshave been found to express genes controlling T cellproliferation and inducing apoptosis, while CD8– cdT lymphocytes were activated and apoptosis resistant[33, 34]. A high proportion of pre-apoptotic cells wasalso found among circulating CD8+ cd T lymphocytes inpatients suffering from Plasmodium falciparum malaria;these cells were also the primary IFN-c producers [35].Along this line, extensive Fas/FasL-dependent death ofcd T cells following infection with Lysteria monocyto-genes has been described in mice [36]. Again, these dataprovide evidence for an activation-induced suicide,mediated by Fas/FasL interaction, during chronicinfections leading to a cd T cell response. Moreover,mycobacterial and phospho-antigens have been de-scribed to induce T cell apoptosis through Fas/FasLinteraction [36, 37]. Interestingly, we found that sHLA-I,besides inducing FasL release, in the same clonessynergizes with IPP in inducing TNF-a production.

Altogether, these data and our findings suggest thatsHLA-I, released by damaged cells, can regulate on oneside cd T cell survival and on the other side may amplifyan antigen-specific cd T cell response, both through CD8and activating KIR. This might be relevant whenpathogens producing phosphate antigens and/or releas-ing sHLA-I molecules are involved, as occurs duringmycobacterial or HIV-1 infection. We have previouslyreported in HIV-1-infected patients a reduction ofcirculating Vd2 T lymphocytes compared to healthydonors [38]. Interestingly, in the sera of these patients,high amounts of sHLA-I (1.4–6.4 lg/mL vs. 0.1–0.8 lg/mL of healthy donors) were found, together withsignificant levels of sFasL (0.7–1.2 ng/mL vs.



0.0–0.2 ng/mL of healthy donors) and high HIV-1 RNAcopy numbers. Conversely, in patients showing a normalor increased percentage of circulating Vd2T lymphocytes, low or undetectable levels of eithersHLA-I (0.2–1.0 lg/mL) or soluble FasL (0.2–0.6 ng/mL) and <80 HIV-1 RNA copies were found.

Accordingly, peripheral Vd2 T cell deletion andanergy to non-peptidic mycobacterial antigens has beenreported in HIV-1-infected individuals, as the conse-quence of a chronic antigenic stimulation similar to thatleading to activation-induced cell death [39]. Finally,serum levels of anti-Fas autoantibodies and soluble Fashave been shown to concur in abT cell depletion in HIV-1 infection [40].

Materials and methods

Monoclonal antibodies and reagents

The anti-CD8a (astra 102, IgG1) mAb, the anti-CD54(14D12D2, IgG1) mAb and the anti-pan-KIR2D mAb (NKVFS1,IgG1) recognizing both KIR2DL1, KIR2DL2 and a commonepitope of CD158 h/KIR2DS1 and CD158j/KIR2DS2 wereproduced as described [16]. The anti-FasL mAb (NOK-1, IgG1)was from PharMingen International (San Diego, CA). The anti-CD158 h/KIR2DS1 (EB6, IgG1) and the anti-CD158j/KIR2DS2(GL183, IgG1) mAb were from Serotec (Kidlington, Oxford).The blocking anti-Fas mAb ZB4 (IgG1) and the apoptosis-inducing anti-Fas mAb CH11 (IgM) were from MBL (Naka-kuNagoya, Japan); the anti-FasL mAb Alf-2.1a was from AncellCorporation (Bayport, MN). The anti-CD8a chain OKT8 mAbwas fromOrtho (Milan, Italy). ThemAbW6/32 and TP25.99 tothe HLA-I heavy-chain a3 domainwere a kind gift of S. Ferrone(Roswell Park Memorial, Buffalo, NY). The anti-Vd1 A13 mAband the anti-Vd2 BB3 mAb were produced as described [38].Annexin-V-FITC, propidium iodide (PI) and IPP (used at 1 lg/106 cells/mL) were from Sigma (Milan, Italy). The affinity-purified GAM isotype-specific antiserum was from SouthernBiotechnology (Birmingham, AL) and the immunomagneticbeads coated with GAM Ab were from Oxoid (Dynal, Oslo,Norway).

Isolation and culture of cd T cells

PBMC from healthy donors were isolated by Ficoll-Hypaque(Sigma, Milan, Italy) gradient. Highly purified CD3+ cd T cellswere obtained from PBMC, after depletion of adherent cells,following staining with anti-Vd1 and anti-Vd2 mAb and cellsorting using immunomagnetic nanobeads (StemCell Tech-nology, Vancouver, Canada). To obtain clones and lines, cellswere seeded at either 1 or 10 cells/well, respectively, in 96-well U-bottom microtiter plates (Greiner Labortechnik,Nurtingen, Germany) and cultured in RPMI 1640 mediumsupplemented with 200 mM L-glutamine, penicillin-strepto-mycin (Biochrom, Berlin, Germany), 5% FCS, 5% AB serum,1 lg/mL phytohemagglutinin (PHA) (Sigma), 25 U/mL rIL-2(Proleukin; Chiron Italia, Siena, Italy). Cells were thenexpanded with rIL-2 and restimulated every 3 wk with PHA

and irradiated feeder cells (3000 rad) according to standardprocedures [38]. Each T cell clone was analyzed for theexpression of TCRcd or KIR by specific mAb and immuno-fluorescence. To select cd T cell clones expressing KIR2DS1and/or KIR2DS2, a given clone was stained with anti-CD158 h/KIR2DS1 (EB6) or anti-CD158j/KIR2DS2 (GL183)mAb. As these mAb can also react with the inhibitory isoformsof KIR (KIR2DL1 and KIR2DL2), each clone was tested in aredirected killing assay using the FccR+ murine mastocytomaP815 cell line, as described [16, 23]. Indeed, in this assay, thecytolytic activity of cd T cell clones bearing KIR2DS1 orKIR2DS2 activating receptors was triggered in the presence ofthe anti-CD158 h or the anti-CD158j mAb (>40%P815 lysis vs.<5% in the absence of mAb at an E/T ratio of 10 : 1 (notshown).

Indirect immunofluorescence and cytofluorometricanalysis

Immunofluorescence staining of cultured cells was performedas described [38]. Briefly, aliquots of 105 cells were stainedwith the corresponding mAb followed by FITC- or PE-conjugated isotype-specific GAM serum (Southern Biotechnol-ogy). Control aliquots were stained with isotype-matchedirrelevant mAb followed by FITC- or PE-GAM Ab or with thefluorescent reagent alone. Double staining was performedwith FITC- and PE-conjugated mAb. Samples were analyzed ona flow cytometer (FACSort; Becton Dickinson) equipped withan argon ion laser exciting PE at 488 nm. Calibration wasassessed with CALIBRITE particles (Becton Dickinson) usingthe AutoCOMP computer program (version 2.1.2).

sHLA-Imolecule preparation and sHLA-Imeasurement

sHLA-I molecules were obtained from serum of healthysubjects by precipitation and low-medium pressure chromato-graphy, as described [16], and purified by affinity chromato-graphy on anti-HLA class I mAbW6/32 (10 lg/mL) coupled tocyanogen bromide-activated Sepharose 4B (Pharmacia). Thepurity of sHLA-I molecule preparations was analyzed by SDS-PAGE followed by silver staining or immunoblotting with anti-HLA class I mAb TP25.99 and ECLmethod [15, 16]. sHLA-A2, -Cw3 and -Cw4 were prepared from culture supernatants of721.221 cells transfected with the corresponding HLA-I allelesas described [15, 16]. sHLA-I was measured by ELISA in thesera obtained from 20 healthy donors and 20 HIV-1 patients,provided informed consent, at the Infectious Diseases Depart-ment (Department of Internal Medicine, University of Genoa,Italy). Patients were on stage A of the disease, definedaccording to the CDC (Center for Disease Control andPrevention, Atlanta, GA) criteria, and naive antiretroviraltreatment.

Detection of soluble FasL and TNF-a

Soluble FasL was measured by ELISA in clone supernatant,treated as above, or in the sera of healthy donors and HIV-1patients: Supernatants or sera, diluted 1 : 4 in PBS, wereadded to a microtiter plate coated with anti-FasL mAb NOK-2and incubated for 45 min at room temperature (RT). After



5 washes with PBS/0.2% Tween-20, biotinylated anti-FasLmAb NOK-1 was added for an additional 45 min at RT.Streptavidin-horse radish peroxidase (Pierce, Rockford, IL)was added for 1 h at RT, and the reaction was developed for15 min at RT in the darkwith o-phenylenediamine (40 lg/mL)in phosphate citrate buffer (pH 5.0) supplemented with 0.04%v/v H2O2. Optical density was read with a spectrophotometerat 490 nm against the blank (PBS/BSA 5%). A standard curvewas obtained using progressive dilution of recombinant FasLfrom Alexis Co. (Leufelfingen, Switzerland). Results wereexpressed as mean � SD of triplicate wells. Supernatantsharvested from cd T cells incubated under the above-mentioned conditions were analyzed for their ability to induceapoptosis of the human lymphoid T cell line Jurkat (Fas+) todetermine whether the FasL present was functional [15, 16].Culture supernatants derived from cd+ T cells incubated withthe stimuli indicated in the results section were collected afterdifferent periods of time (12, 24, 48 h) and analyzed for thepresence of TNF-a with the corresponding ELISA assay(Peprotec, London, UK).

Induction and detection of apoptosis

cd T cell clones (105 cells/mL) were cultured in 24-well flat-bottom plates (Becton Dickinson, Oxnard, CA) in the presenceof sHLA-I (0.5–8 lg/mL) for 12, 24, 48, 72 h at 37�C. Theoptimal sHLA-I concentration was 4 lg/mL. Cells wereincubated with either the anti-Vd1 or anti-Vd2 TCR mAb orwith the anti-CD8 mAb (astra 102) alone or in combinationwith the anti-FasL (Alf-2) mAb; in other samples, sHLA-Cw3 or-Cw4 were added, alone or in combination with the anti-pan-KIR2D mAb, for 30 min at 4�C; samples were then washed andincubated for different times with 4-per-cell of GAM Ab-coatedmagnetic beads (Dynal) to induce cross-linking of thecorresponding molecules [15, 16]. Beads were extensivelywashed before use in order to discard sodium azide; in controlsamples, GAM Ab-coated magnetic beads were added to thecells alone or after incubation with an anti-CD54mAb. In someexperiments, anti-CD8 or anti-pan-KIR2D mAb in soluble formwere added in order to inhibit the binding of sHLA-I or sHLA-Ialleles to their counterreceptors. In other experiments, thepentameric MHC-peptide complex HLA-A1101/YVNVNMGLK(ProImmune, Oxford, UK), wild type or mutated in thea3 domain and unable to bind to CD8, was used at 4 lg/mLconcentration. Early apoptotic events were evaluated by theannexin-V labeling method, to show the exposure ofphosphatidylserine at the external side of the plasmamembrane. Viable apoptotic cells were differentiated fromnecrotic cells by flow cytometry after PI staining of non-permeabilized cells. Apoptotic cells were identified as annexin-V+ PI– cells [15, 16]. FACSort calibration was assessed withCALIBRITE particles (Becton Dickinson) and the CellQuestcomputer program. At least 104 cells per samplewere analyzedand results plotted as percentage of annexin-V+ PI– cells. Insome experiments, 5 � 105 cells werewashed and analyzed ona Epics Elite flow cytometer (Coulter) after permeabilizationand PI staining (615 nm to analyze DNA staining and toidentify cells with <2n DNA as apoptotic cells). In parallelexperiments, apoptosis was also detected by DNA extractionand agarose gel electrophoresis [15, 16].

Acknowledgements: This work was supported bygrants from Istituto Superiore di Sanit� AIDS project(ISS 40F.84) and by Associazione Italiana per la Ricercasul Cancro (AIRC) and CIPE (IMM-03).

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