A critical role for interleukin 4 in activating alloreactive CD4 T cells

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1 A criticl role for interleukin 4 in ctivting llorective CD4 T cells Jessmyn Bgley,Tokihiko Swd*,Yin Wu nd John Icomini To generte ntigen-specific responses, T cells nd ntigen presenting cells (APCs) must physiclly ssocite with ech other nd elorte solule fctors tht drive the full differentition of ech cell type. Immeditely fter T cell ctivtion, CD4 T cells cn produce oth interferon γ (IFN-γ) nd interleukin 4 (IL-4) efore polriztion into distinct T helper susets. Inhiition of IL-4 during mixed llogeneic lymphocyte culture resulted in defect in the ility of APCs to generte sufficient costimultory signls for ctivtion of llorective T cells. In vivo, deficiency in IL-4 production inhiited the ctivtion of llorective IL-2, IL-4 nd IFN-γ producing CD4 T cells in mice chllenged with llogeneic skin grfts, resulting in prolonged skin grft survivl.thus, production of IL-4 y CD4 T cells helps ctivte llorective T cells y ffecting APC function. T cells recognize llogeneic mjor histocomptiility (MHC) ntigens vi two distinct pthwys of ntigen presenttion 1. The direct pthwy involves the recognition of intct llogeneic MHC molecules on the surfce of donor cells y host T cells, wheres the indirect pthwy involves the recognition of donor MHC derived peptides tht re processed y host ntigen presenting cells (APCs) nd presented s peptides to T cells. In cses where host nd donor re mtched for MHC clss II, CD4 T cells cn lso recognize donor MHC-derived peptides in the context of donor MHC clss II, second form of indirect recognition. CD4 T cells tht recognize llogeneic ntigens through the indirect pthwy ply mjor role in llogrft rejection 2. However, the requirements for ctivting these CD4 T cell responses remin undefined. In ddition to MHC-restricted peptide recognition vi the T cell receptor (TCR), T cells must receive dditionl costimultory signls provided y APCs to ecome fully ctivted effector cells. Interction of CD28 on T cells with B7.1 (CD80) or B7.2 (CD86) on APCs provides sufficient costimultory signls to T cells to induce cytokine production nd cell prolifertion 3. APCs such s B cells nd dendritic cells express low B7.1 nd B7.2 in their resting stte, nd expression cn e up-regulted y mny inflmmtory stimuli including djuvnts, lipopolyscchride, IL-2, IL-4 nd signling through the cytoplsmic til of MHC clss II 4 6. B7.1 nd B7.2 costimulte T cells, nd the overexpression of these lignds increses the ility of APCs to ctivte T cells 7. Disrupting the interction of CD28 with B7.1 nd B7.2 leds to donor-specific tolernce in Figure 1. () Neutrliztion of IL-4 in mixed llogeneic cultures inhiits ctivtion of cytokine-producing CD4 T cells. CD8-depleted B10.AKM splenocytes were cultured with T cell depleted B10.MBR splenocytes in the presence of 100 µg/ml of either 11B11 (open rs) or control norml rt IgG (filled rs).after 5 dys, the frequency of IFN-γ or IL-4 producing cells ws determined y ELISPOT ssys. Results shown re n verge of two independent representtive experiments. () Asence of IL-4 in mixed llogeneic culture inhiits ctivtion of cytokine-producing CD4 T cells. CD8-depleted C57BL/6 splenocytes were cultured with T cell depleted B10.D2-H2 i7 splenocytes in the presence of 100 µg/ml of either 11B11 (open rs) or control norml rt IgG (filled rs). CD8-depleted IL-4 / splenocytes (htched rs) were cultured with T cell depleted B10.D2-H2 i7 splenocytes.after 3 dys, the frequency of IFN-γ, IL-2 or IL-4 producing cells ws determined y ELISPOT ssys. severl niml llogrft models 8, demonstrting the importnce of this interction to trnsplnt rejection. It hs een suggested tht immeditely fter T cells re stimulted, CD4 T cells cn produce oth interferon γ (IFN-γ) nd interleukin 4 (IL-4) 9. Additionl ntigenic stimultion polrizes CD4 T cells into either T helper type-1 (T H1) cells tht produce IL-2, tumor necrosis fctor α (TNF-α) nd IFN-γ, or T H2 cells tht produce IL-4, IL-5 nd IL To determine the function of IL-4 production we exmined the possile role of IL-4 production during T cell ctivtion. Our dt suggest tht IL-4 is criticl for the up-regultion of costimultory molecules on APCs, which in turn llows for ctivtion nd expnsion of llorective CD4 T cells. Thus, production of IL-4 fter ntigen encounter ensures efficient costimultion of ntigen rective T cells. Results IL-4 in llorective CD4 T cell ctivtion To determine the function of IL-4 in T cell ctivtion, the MHC clss I disprte congenic B10.AKM (H-2K k, I k, D q ) nd B10.MBR (H-2K, I k, D q ) mice were used to exmine the role of IL-4 in ctivting llorective CD4 T cells in vitro using mixed llogeneic cultures nd in vivo y skin grft rejection. CD8 T cell depleted splenocytes from B10.AKM mice primed in vivo y grfting with B10.MBR skin were stimulted in vitro with T cell depleted B10.MBR splenocytes in the presence of either the IL-4 neutrlizing ntiody 11B11 or norml rt IgG. After 5 dys the frequency of IL-4 nd IFN-γ producing cells in the cultures ws determined y enzyme-linked immunospot (ELISPOT) ssys (Fig. 1). The Trnsplnttion Biology Reserch Center, Msschusetts Generl Hospitl, MGH-Est, Building 149, 13th Street, Boston, MA 02129, USA. *Present Address: Kidney Center, Tokyo Women s Medicl College 8-1 Kwd-Cho Shinjuku,Tokyo 162 Jpn. Correspondence should e ddressed to J.I. (icomini@helix.mgh.hrvrd.edu). septemer 2000 volume 1 no 3 nture immunology 257

2 IFN-γ, IL-2 nd IL-4 producing cells oserved using T cells from IL-4 deficient mice ws sttisticlly the sme s the frequency oserved in cultures contining C57BL/6 T cells nd 11B11 (P>0.08 in ll cses). Collectively, these dt suggest tht IL-4 is involved in ctivtion of llorective CD4 T cells. Figure 2. () Neutrliztion of IL-4 in mixed llogeneic culture does not led to T cell nergy. CD8-depleted B10.AKM splenocytes were stimulted with T cell depleted B10.MBR splenocytes in the presence of 100 µg/ml of either 11B11 (open rs) or control norml rt IgG (filled rs) for 3 dys.the cells were then collected nd CD4 T cells purified y mgnetic cell sorting.the T cell preprtions were then stimulted with fresh T cell depleted splenocytes from B10.MBR mice in ELISPOT ssys. One representtive experiment of three is shown. () Neutrliztion of IL-4 inhiits the ility of APC to stimulte llorective T cells. CD8-depleted B10.AKM splenocytes were stimulted with T cell depleted B10.MBR splenocytes in the presence of 100 µg/ml of either 11B11 (open rs) or control norml rt IgG (filled rs) for 5 dys.the cells were then collected nd T cells depleted to purify APC. The T cell depleted APC preprtions were then used to stimulte fresh CD8 T cell depleted splenocytes from primed B10.AKM mice in ELISPOT ssys. One representtive experiment of three is shown. frequency of IL-4 producing cells ws significntly lower in 11B11-treted cultures (10±10 per 10 6 splenocytes) thn in norml rt IgG treted controls (280±70 per 10 6 splenocytes, P<0.01), consistent with the oservtion tht IL-4 supports differentition of T H2 cells 10. The frequency of IFN-γ producing cells ws lso significntly decresed in 11B11-treted cultures (30±20 per 10 6 splenocytes) when compred with controls (230±110 per 10 6 splenocytes, P<0.01). Similr results were oserved fter 3 nd 4 dys of stimultion with T cell depleted B10.MBR splenocytes (not shown). Detection of cytokine-producing cells required the presence of llontigen during the stimultion phse of the ssy nd, in control studies, T cell depletion of stimulted cells led to significnt decrese in the frequency of cytokine producing cells (not shown). To exmine whether the inhiition of llorective T cell ctivtion fter IL-4 neutrliztion ws strin specific, we lso exmined the response of primed CD8 T cell depleted splenocytes from C57BL/6 (H-2 ) mice to T cell depleted splenocytes from MHC clss I mismtched B10.D2-H2 i7 mice in the presence of 11B11 or norml rt IgG. C57BL/6J nd B10.D2- H2 i7 re not congenic strins, nd re mismtched t H-2D nd H-2L s well s multiple minor ntigens. Thus, oth the precursor frequency of llorective T cells nd the speed of skin llogrft rejection in this strin comintion should e greter thn in the congenic B10.AKM nd B10.MBR strins tht re mismtched t only the H-2K locus. The frequency of C57BL/6 llorective IL-4 (25±5 versus 240±75 per 10 6 splenocytes, P<0.01), IFN-γ (25±35 versus 515±30 per 10 6 splenocytes, P<0.01) nd IL-2 (90±20 versus 350±75 per 10 6 splenocytes, P<0.02) producing cells ws significntly reduced in 11B11-treted cultures when compred with control cultures contining norml rt IgG (Fig. 1), suggesting tht the requirement for IL-4 in ctivting llorective T cells is not strin specific. To exmine whether the effect of IL-4 neutrliztion ws due to nonspecific inhiitory effect of the neutrlizing ntiody 11B11, we next exmined the response of CD8 T cell depleted splenocytes from primed C57BL/6 IL-4 knockout mice 11 when stimulted with MHC clss I mismtched B10.D2-H2 i7 T cell depleted splenocytes. The frequency of llorective CD4 T cells from IL-4 deficient mice tht produced IFN-γ (140±45 versus 515±30, P<0.02) nd IL-2 (180±30 versus 350±75, P<0.02) were significntly reduced when compred with the frequency oserved for C57BL/6 controls (Fig. 1). The frequency of llorective IL-4 nd APC-stimultion of llorective CD4 T cells To further exmine the role of IL-4 in the ctivtion of llorective CD4 T cells, CD8 T cell depleted splenocytes from primed B10.AKM mice were stimulted in vitro with T cell depleted splenocytes from B10.MBR mice for 5 dys in the presence of either 11B11 or norml rt IgG. CD4 T cells were then purified from the cultures y mgnetic cell sorting, nd cytokine ELISPOT ssys were done fter stimultion with freshly isolted T cell depleted B10.MBR splenocytes. CD4 T cells collected from 11B11-treted cultures were redily le to produce IFN-γ (400±11 per 10 6 splenocytes ), IL-2 (350±50 per 10 6 splenocytes) nd IL-4 (440±70 per 10 6 splenocytes) when stimulted for 24 h with T cell depleted llogeneic stimultors (Fig. 2), indicting tht IL-4 neutrliztion did not result in T cell nergy or deth. There ws no sttisticlly significnt difference in the frequency of cytokine-producing CD4 T cells collected from 11B11 or norml rt IgG treted cultures (Fig. 2). To determine whether the effect of IL-4 neutrliztion on T cell ctivtion ws due to chnges in APC function, we exmined the ility of APCs collected from similr cultures to stimulte freshly isolted CD4 T cells from primed B10.AKM mice. When nlyzed y flow cytometry, no significnt difference in the numer of CD45R + (B220) B cells or CD11c + dendritic cells ws oserved in 11B11-treted cultures when compred with norml rt IgG treted cultures (not shown). APCs collected from norml rt IgG treted cultures could stimulte oth llorective IFN-γ (270±120 per 10 6 splenocytes) nd IL-4 producing CD4 T cells (190±70 per 10 6 splenocytes) (Fig. 2). In contrst, APCs collected from 11B11- treted cultures could not stimulte llorective IFN-γ (40±60 per 10 6 Figure 3. Neutrliztion of IL-4 in vitro prevents up-regultion of B7.1 nd B7.2 surfce expression on APC. Splenocytes were collected from primed B10.AKM mice, CD8 T cell depleted nd then cultured for 7 dys with either T cell depleted B10.MBR splenocytes (T depleted) or sonicted B10.MBR splenocytes (sonicted). The cells were cultured in medi lone (dshed line), medi contining 100 µg/ml 11B11 (solid line) or medi contining 100 µg/ml 11B11 nd 5 ng/ml recominnt murine IL-4 (dotted line).to nlyze B7.1 nd B7.2 expression y flow cytometry, the cells were gted on either B220 + or CD11c + cells. One representtive experiment of four is shown. 258 nture immunology volume 1 no 3 septemer

3 splenocytes, P<0.01) nd IL-4 producing CD4 T cells (20±30 per 10 6 splenocytes, P<0.01). Thus, neutrliztion of IL-4 cused defects in the ility of APCs to stimulte llorective CD4 T cells. To determine the role of IL-4 in APC function, CD8 T cell depleted splenocytes from primed B10.AKM mice were stimulted in vitro with T cell depleted splenocytes from MHC clss I disprte B10.MBR mice in the presence of 11B11, nd cells were then exmined for B7.1 nd B7.2 expression y flow cytometry. B7.1 nd B7.2 were up-regulted on the surfce of B cells nd dendritic cells in mixed lymphocyte cultures contining CD4 T cells from primed B10.AKM mice nd T cell depleted B10.MBR splenocyte stimultors. In contrst, neutrliztion of IL-4 in culture with 11B11 prevented upregultion of B7.1 nd B7.2 on oth B cells nd dendritic cells, consistent with the oservtion tht IL-4 ffects expression of B7.1 nd B7.2 on APCs 12 (Fig. 3). The effect of 11B11 on B7.1 nd B7.2 expression ws concentrtion-dependent, nd required llogeneic stimultors (not shown). Addition of rt IgG to control cultures hd no effect on B7.1 nd B7.2 up-regultion (not shown). The effect of IL-4 neutrliztion ws pprent on oth syngeneic B10.AKM nd llogeneic B10.MBR APCs (not shown). Addition of exogenous IL-4 to 11B11-treted cultures restored B7.1 nd B7.2 up-regultion in culture, suggesting tht the inhiitory effects of 11B11 on B7.1 nd B7.2 surfce expression were specific to the ction of IL-4. Becuse llorective CD4 T cells cn recognize processed llontigen presented y MHC clss II on self-apcs, we lso exmined the effect of IL-4 neutrliztion on B7.1 nd B7.2 expression fter stimultion of CD8 T cell depleted splenocytes from primed B10.AKM mice with sonicted B10.MBR splenocytes. Neutrliztion of IL-4 with 11B11 in these cultures prevented the up-regultion of B7.1 nd B7.2 to similr degree s tht oserved using intct B10.MBR stimultors (Fig. 3). IL-4 expression in llogrfts undergoing rejection To determine whether IL-4 hs role in ctivting llorective CD4 T cells in vivo, we first exmined cytokine gene expression in llogeneic skin grfts undergoing CD4 T cell medited rejection. To this end, thymectomized nd CD8 T cell depleted (Thx-CD8 - mice) or untreted B10.AKM mice were grfted with oth B10.MBR nd syngeneic til skin. Grfts were collected for RNA isoltion 12 dys lter nd intrgrft expression of cytokine genes ws nlyzed y reverse trnscriptse-polymerse chin rection (RT-PCR). IL-2, IL-10, TNF-α nd IFN-γ trnscripts were expressed in oth llogeneic nd syngeneic grfts collected from oth Thx-CD8 - nd norml B10.AKM mice, suggesting tht expression of these cytokines ws not specific to llogrft rejection. Although IL-4 trnscripts were detected in 62% of rejected B10.MBR llogrfts, we were unle to detect expression of IL-4 in syngeneic B10.AKM grfts collected from the sme mice (Tle 1, n=8, P=0.02). IL-4 trnscripts were lso detected in 50% of llogeneic B10.MBR grfts collected from norml B10.AKM mice ut not in the syngeneic grfts (Tle 1, n=4, P>0.05). Expression of IL-4 in rejecting grfts ws still pprent t lter timepoints in oth Thx-CD8 - nd norml B10.AKM mice (not Tle 1. Cytokine gene expression in llogenic nd syngeneic skin grfts from Thx-CD8 B10.AKM nd untreted B10.AKM mice Thx-CD8 - B10.AKM Untreted B10.AKM Skin grft Skin grft Cytokine B10.MBR B10.AKM B10.MBR B10.AKM TNF-α 6/8 75% 8/8 100% 3/4 75% 4/4 100% IL-2 5/8 62% 5/8 62% 3/4 75% 3/4 75% IL-10 8/8 100% 8/8 100% 4/4 100% 4/4 100% IFNγ 6/8 75% 6/8 62% 2/4 50% 3/4 75% IL-4 5/8 62% 0/8 0% 2/4 50% 0/4 0% Numer of grfts expressing cytokine mrna. Percentge of grfts expressing cytokine mrna. shown). Thus, IL-4 is produced during the specific rejection response nd not simply produced s result of nonspecific inflmmtion. IL-4 in CD4 T cell medited llogrft rejection We next exmined whether neutrliztion of IL-4 in vivo could ffect llogrft survivl. Thx-CD8 - B10.AKM mice were injected intrperitonelly with either 11B11 or norml rt IgG 1 dy efore skin grfting nd then every other dy until the grfts were rejected. Survivl of clss I disprte grfts from B10.MBR congenic mice on B10.AKM mice treted with 11B11 (Fig. 4) ws significntly prolonged medin survivl time (MST)=30 dys when compred to survivl of B10.MBR grfts on mice treted with norml rt IgG (MST=17 dys, P=0.001). Survivl of B10.D2-H2 i7 skin grfts ws lso significntly prolonged on Thx-CD8 - C57BL/6 IL-4 knockout mice (MST=19 dys) when compred with survivl on Thx-CD8 - C57BL/6 controls (MST=11 dys, P=0.0001) (Fig. 4), suggesting tht the effect of 11B11 tretment ws neither strinspecific nor the result of nonspecific inhiitory effect of 11B11. After rejection of the B10.MBR skin grfts, the percentge of peripherl lood CD4 T cells in Thx-CD8 - B10.AKM mice treted with sline (8.0±0.42%) or norml rt IgG (7.2±0.43%) ws incresed when compred with the percentge oserved in the lood of Thx-CD8 - mice tht never received skin grft (3.4±0.19%, P<0.001) (Fig. 5). These dt suggest tht CD4 T cells undergo significnt expnsion in response to llontigen. In contrst, the percentge of CD4 T cells in 11B11-treted Thx-CD8 - B10.AKM mice tht rejected B10.MBR skin grft ws significntly lower (4.4±0.45%, P=0.004) thn the percentge of CD4 T cells in mice receiving norml rt IgG, nd did not differ significntly from the percentge of CD4 T cells in the lood of nïve Thx-CD8 - mice (P=0.09). Similrly, fter rejection of the B10.D2-H2 i7 skin grfts, the percentge of CD4 T cells in the lood of Thx-CD8 - C57BL/6 mice (15.7±1.7%) ws incresed when compred to the percentge oserved efore skin grfting (12.5±1.2%, P=0.02) (Fig. 5). In contrst, fter rejection of the B10.D2-H2 i7 skin grfts, the percentge of CD4 T cells in the lood of Thx-CD8 - C57BL/6 IL-4 knockout mice ws unchnged (P=0.8) when Figure 4. Neutrliztion or sence of IL-4 in vivo results in prolonged skin llogrft survivl.() Survivl of B10.MBR skin grfts on Thx-CD8 - B10.AKM mice treted with 11B11 (, n=18) or control norml rt IgG (, n=11). One representtive experiment of three is shown. () Survivl of B10.D2-H2 i7 skin grfts on Thx-CD8 - IL-4 / mice (, n=7) or C57BL/6 mice (, n=9). septemer 2000 volume 1 no 3 nture immunology 259

4 Figure 5. () Neutrliztion of IL-4 in vivo prevents expnsion of CD4 T cells. Blood ws collected from Thx-CD8 - B10.AKM mice 35 dys fter skin grfting, nd stined with sturting concentrtions of nti-mouse CD4. Shown is the percentge of CD4 T cells in the lood of control Thx-CD8 - mice tht were not treted, tht is they did not receive skin grft or ntiody tretment (, n=9),thx-cd8 - mice treted with HBSS nd grfted with B10.MBR skin (, n=4),thx-cd8 - mice treted with norml rt IgG nd grfted with B10.MBR skin (, n=9), nd Thx-CD8 - mice treted with 11B11 nd grfted with B10.MBR skin (, n=11).vlues for individul mice re plotted.the horizontl lines show the men vlues for ech group. () Asence of IL-4 in vivo prevents expnsion of CD4 T cells. The percentge of CD4 T cells in the lood of control Thx-CD8 - C57BL/6 mice efore receiving skin grft (, n=9), Thx-CD8 - C57BL/6 mice grfted with B10.D2-H2 i7 skin (, n=9),thx-cd8 - IL-4 / mice efore receiving skin grft ( n=7), nd Thx-CD8 - IL-4 / mice grfted with B10.D2-H2 i7 skin (, n=7) is shown.vlues for individul mice re plotted.the horizontl lines show the men vlues for ech group. compred to the percentge efore skin grfting. There ws no sttisticlly significnt difference in the numer of totl leukocytes per ml of lood etween ny groups (not shown). Together, these dt suggest tht IL-4 is directly involved in CD4 T cell medited llogrft rejection, nd tht neutrliztion of IL-4 in vivo prevents expnsion of CD4 T cells. To determine whether the overll decrese in the percentge of CD4 T cells in 11B11-treted mice reflected specific decrese in llorective cells, Thx-CD8 - B10.AKM mice were llowed to reject B10.MBR grfts while receiving 11B11 or norml rt IgG. Four weeks fter grft rejection, the mice were killed nd cytokine ELISPOT ssys were done to determine the frequency of IL-2, IFN-γ or IL-4 producing llorective T cells fter stimultion for 24 h in vitro with either T cell depleted syngeneic splenocytes or T cell depleted llogeneic B10.MBR splenocytes. The frequency of cytokine-producing cells ws expressed s the rtio of cytokine producing cells detected fter stimultion with llogeneic versus syngeneic splenocytes to normlize for potentil differences in the cell composition of the spleens of the 11B11- nd norml rt IgG treted mice. As expected, the rtio of IL-4 producing T cells cple of responding to llogeneic versus syngeneic splenocytes ws significntly reduced (1.3±0.15) in 11B11-treted mice when compred to those in norml rt IgG treted controls (3.0±0.7, P 0.01) (Fig. 6). The rtio of cells producing IL-2 or IFN-γ ws lso reduced in 11B11-treted mice. The rtio of IL-2 producing cells ws 1.2±0.07 in 11B11-treted mice compred with 2.6±0.7 (P=0.01) in norml rt IgG treted controls, nd the rtio of Figure 6. () Neutrliztion of IL-4 in vivo inhiits the ctivtion of llorective CD4 T cells. Thx-CD8 - B10.AKM mice were grfted with B10.MBR skin nd treted with either 11B11 (open rs) or norml rt IgG (filled rs). Five weeks fter skin grft rejection, spleens were collected nd ELISPOT ssys done on splenocytes to mesure IL-4, IFN-γ nd IL-2 production fter stimultion with either T cell depleted B10.MBR or B10.AKM splenocytes. Control mice (htched rs) were thymectomized nd CD8 T cell depleted s descried, ut did not receive B10.MBR skin grfts. Results re expressed s the rtio of llorective to utorective cells. A rtio of 1 indictes no difference over ckground oserved using syngeneic stimultors.vlues shown represent the verge of six mice in ech group. () Asence of IL-4 in vivo inhiits the ctivtion of llorective CD4 T cells. Thx-CD8 - IL-4 / (open rs, n=7) or Thx-CD8 - C57BL/6 (filled rs, n=5) mice were grfted with B10.D2-H2 i7. Four weeks fter skin grft rejection, spleens were collected nd ELISPOT ssys done on splenocytes to mesure IL-4, IFN-γ nd IL-2 production fter stimultion with either T cell depleted C57BL/6 splenocytes or B10.D2-H2 i7 splenocytes.vlues shown represent n verge of two independent experiments. cells producing IFN-γ ws 1.1±0.07 in 11B11-treted mice nd 4.0±0.96 (P 0.01) in norml rt IgG treted mice. Similrly, four weeks fter rejection of B10.D2- H2 i7 skin grfts the rtio of cytokine-producing T cells cple of responding to llogeneic versus syngeneic splenocytes ws significntly reduced in Thx-CD8 - C57BL/6 IL-4 knockout mice when compred with Thx- CD8 - C57BL/6 controls (Figure 6). Thus, s suggested y our in vitro experiments, neutrliztion of IL-4 impirs formtion of llorective CD4 T cells. Discussion Our dt suggest tht IL-4 plys criticl role in ctivting llorective CD4 T cells. Neutrliztion of IL-4 in vitro led to defect in the ility of APCs to stimulte cytokine-producing CD4 T cells. Although T cells collected from cultures in which IL-4 hd een neutrlized responded to fresh APCs in the presence of IL-4, APCs collected from these cultures were unle to stimulte fresh llorective CD4 T cells. Our nlysis of APCs collected from these cultures showed tht the up-regultion of t lest two key costimultory molecules, B7.1 nd B7.2, ws inhiited fter IL-4 neutrliztion. In vivo, in the sence of IL-4, the ility to ctivte llorective CD4 cells ws inhiited, resulting in prolonged skin llogrft survivl. Although IL-4 knockout mice cn reject llogrfts 13 nd generte T H1 responses under pproprite conditions, we suggest tht rejection in these cses my e medited y CD8 T cells tht cn reject skin grfts in the sence of CD4 T cells 14,15. Although neutrliztion of IL-4 does not result in permnent grft survivl in the models we tested, APC mturtion cn e induced y mny stimuli other thn IL-4. We suggest tht stimuli, such s the shedding of necrotic or poptotic cells from the grft, re likely to occur in vivo nd could overcome the requirement for IL-4 16,17. Nevertheless the ility to prolong grft survivl, rigorous test of T cell function, y inhiiting IL-4 shows tht this cytokine plys mjor role in mediting rejection response. Although role for IL-4 in ctivting IL-2 or IFN-γ producing CD4 T cells my seem contrry to the current findings in IL-4 knockout mice, IL-4 knockout mice do not generte norml CD4 T cell responses under ll conditions 18,19. Experiments in IL-4 knockout mice hve suggested tht IL-4 my e required for the genertion of tumor-specific T H1 cells 20. Elimintion of IL-4 producing cells fter stimultion in vitro prevents production of IFN-γ producing T H1 cells nd T H2 cells 21. Together, these dt support the hypothesis tht IL-4 is criticlly involved in priming of T H1 nd T H2 responses. Our results suggest tht inhiition of IL-4 leves T cells in stte of clonl ignornce, consistent with the oservtion tht preventing the interction with B7 molecules leves T cells unctivted ut functionl 22. In some systems, it hs een suggested tht peripherl T cell tolernce to llogrfts my e IL-4 dependent 23. Becuse the induction of T cell 260 nture immunology volume 1 no 3 septemer

5 nergy requires costimultion through B7 molecules 22, our findings suggest tht IL-4 medited up-regultion of B7.1 nd B7.2 my ply role in promoting T cell nergy in some systems through its ction on APCs. Becuse in our model llorective CD4 T cells must recognize processed llogeneic peptides rther thn intct cell surfce llogeneic MHC clss I, ntigen recognition in this system is similr to tht oserved in norml T cell responses to protein ntigens. The requirement for IL-4 in CD4 T cell responses to nontrnsplnttion ntigens is not yet known. Bsed on our dt, we suggest tht IL-4 helps ensure productive T cell APC interction in some systems. Methods Mice. 6 to 12-week-old femle B10.AKM/SnJ, B10.MBR/Sx, C57BL/6J, C57BL/6J-IL-4 tm/cgn (IL-4 knockouts) nd mle or femle B10.D2-H2 i7 mice were purchsed from The Jckson Lortory (Br Hror, ME) nd mintined under microisoltor conditions on utoclved feed nd wter. C57BL/6J-IL-4 tm/cgn mice were ckcrossed to C57BL/6 mice for 12 genertions. All niml procedures were pproved y the institutionl review ord t Msschusetts Generl Hospitl. Mixed llogeneic culture. To increse the frequency of llorective T cells, B10.AKM mice were primed y grfting with B10.MBR skin. Two weeks fter grft rejection, the mice were killed. Splenocytes were then collected nd depleted of CD8 T cells using the monoclonl ntiody to CD8, 2.43 (rt IgG2) 24, nd got nti-rt microeds (Miltenyi Biotech., Sunnyvle, CA) in conjunction with the MACS mgnetic cell sorting system ccording to the mnufcturer s specifictions (Miltenyi Biotech., Auurn, CA). Splenocytes were collected from nïve B10.MBR mice nd T cell depleted using 2.43 nd the monoclonl ntiody to CD4, GK1.5 (rt IgG2) 25, in conjunction with MACS cell sorting s ove. Similrly, C57BL/6 nd IL-4 knockout mice were primed y grfting with B10.D2-H2 i7 skin. All mgneticlly purified splenocytes were checked for complete depletion y FACS using ntiodies not locked y those used for depletion ( nti-cd8, RM4-5 nti-cd4, Phrmingen). CD8 T cell depleted B10.AKM nd T cell depleted B10.MBR splenocytes were plted t 1:1 rtio, t concentrtion of cells/ml in DMEM medi contining 15% fetl clf serum nd either 100 µg/ml 11B11 (rt nti-mouse IL-4) 26 or 100 µg/ml norml rt IgG (Jckson ImmunoReserch, West Grove, PA). All norml rt IgG preprtions were dsored on B10.AKM mouse thymocytes to remove ntiodies rective ginst unknown mouse epitopes. Purified CD4 T cells were prepred y collecting splenocytes from mixed llogeneic cultures contining CD8-depleted B10.AKM nd T-depleted B10.MBR splenocytes fter 3 dys s descried ove. CD4 T cells were purified using GK1.5 in conjunction with mgnetic cell sorting ccording to the mnufcturers specifictions (Miltenyi Biotech.). Similrly, APCs were prepred y collecting splenocytes from mixed llogeneic cultures contining CD8-depleted B10.AKM nd T-depleted B10.MBR splenocytes fter 3 dys s descried ove. Splenocytes were then further T cell depleted y using GK1.5 nd 2.43 in conjunction with mgnetic cell sorting ccording to the mnufcturers specifictions (Miltenyi Biotech.). ELISPOT. ELISPOT ssys were done with Millipore HA pltes (Millipore, Bedford, MA) ccording to the mnufcturer s specifictions. Monoclonl ntiodies BVD-1D11 (nti IL- 4), JES6-1A12 (nti IL-2) nd R4-6A2 (nti IFN-γ) (Cltg Lortories, Burlingme, CA) were used t concentrtions of 10 µg/ml to cot pltes T cell depleted llogeneic or syngeneic stimultor cells were dded to ech well. Responder cells were ssyed t 10 6, 10 5 nd 10 4 cells per well. Cytokine production ws detected fter 24 h using concentrtions of 10 µg/ml of iotinylted monoclonl ntiodies BVD-24G2 (nti IL-4), JES6-5H4 (nti IL-2) nd XMG1.2 (nti IFN-γ). Flow cytometry. B10.AKM mice were primed y llogrfting s descried ove nd killed 5 weeks fter the grfts were rejected. Splenocytes were collected from primed B10.AKM nd nïve B10.MBR mice CD8 T cell depleted B10.AKM splenocytes were then plced in culture with either T cell depleted B10.MBR cells or n equivlent numer of sonicted B10.MBR splenocytes. B10.MBR splenocytes were sonicted for 5 mins using W-380 ultrsonic processor (Het Systems, Frmingdle, NY). No vile cells were oserved y Trypn lue exclusion. Cells were cultured in DMEM medi contining 15% fetl clf serum or medi contining either 100 µg/ml 11B11 or 100 µg/ml norml rt IgG with or without 5 ng/ml exogenous IL-4. The culture medi, including the ntiodies indicted, ws replced every 2 dys. After 5 7 dys in culture t 37 C, 5% CO2, the cells were collected nd exmined for B7.1 nd B7.2 expression y flow cytometry using nti-cd80 (RMMP-1) nd nti- CD86 (RMMP-2) purchsed from Cltg Lortories. All cell surfce stining nd flow cytometry ws performed s descried previously 14. In vivo neutrliztion of IL-4. B10.AKM mice were thymectomized t 4 to 6 weeks of ge. Two weeks fter thymectomy, the mice were injected intrperitonelly with 100 µg of purified 2.43 (nti-cd8) or 100 µl of 2.43 scities fluid t dys 7 nd 14 fter thymectomy to produce Thx-CD8 - mice 14. Before skin grfting, T cell depletion ws confirmed y flow cytometry. One dy efore skin grfting, mice were injected intrperitonelly with 100 µg purified 11B11 or 100 µg of purified norml rt serum IgG. The following dy, til skin grfts from 4 to 8-weekold B10.MBR mice were plced on the flnk of control nd experimentl mice. Skin grfting nd thymectomy were performed s descried B11 or control rt IgG ws dministered every other dy until the grfts were rejected. C57BL/6 nd C57BL/6J-IL-4 tm/cgn mice were thymectomized nd CD8-depleted s descried, nd grfted with til skin from 4 to 6-week-old B10.D2-H2 i7 mice. Grfts were monitored every dy until rejection Intrgrft cytokine PCR. Skin grfts were collected 12 dys fter grfting nd totl RNA ws isolted s descried µg of RNA ws then used to generte cdna using n oligo dt primer nd the SuperScript pre-mplifiction system (GIBCO BRL, Githersurg, MD) ccording to the mnufcturer s specifictions. PCR primers specific for ech cytokine were otined from Clontech (Plo Alto, CA). All cytokine primers were designed either to not mplify genomic DNA or llow mplifiction of cdna nd genomic DNA to e distinguished sed on the size of the PCR product otined. Amplifiction of β-ctin or glycerldehyde 3-phosphte dehydrogense trnscripts ws used to control for the qulity nd mount of cdnamplified in ech experiment. Sttistics. All sttisticl clcultions were performed using GrphPd Prism 2.01 softwre (GrphPd Softwre Inc., Sn Diego). Comprison of proportions ws performed y the Fisher exct test. The Kpln nd Meier method with 95% confidence intervl ws used for the clcultion of survivl curves. Comprison of survivl curves ws performed using the log rnk test. Comprisons of CD4 T cell levels efore nd fter grft rejection were performed using pired t-test. All other sttistics were clculted using two-tiled Student s t-test. Acknowledgments We thnk S. Pilli, J. J. Lfille nd H.Winn for criticl review of the mnuscript; N. Cretin for ssistnce with skin grfts; P. Heeger for dvice on performing ELISPOT ssys; nd D. H. Schs for monoclonl ntiodies 11B11, 2.43 nd GK1.5. Supported in prt y the Ntionl Institutes of Helth grnt RO1 AI43619 (to J.I.). Received 11 My 2000; ccepted 7 August Auchincloss, H. Jr & Sultn, H.Antigen processing nd presenttion in trnsplnttion. Curr. Opin. Immunol. 8, (1996). 2. Gould, D. & Auchincloss, H. Jr Direct nd indirect recognition: the role of MHC ntigens in grft rejection. Immunol.Tody 20, (1999). 3. Turk, L.A. et l. 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