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Without CD4 Help, CD8 Rejection of Pig Xenografts Requires CD28 Costimulation But Not Perforin Killing¹

Walter and Eliza Hall Institute of Medical Research, Parkville, Australia

	Abstract

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Although CD4 cells are major mediators in cellular rejection of fetal pig pancreas (FPP) in the mouse, rejection still occurs in the absence of CD4 cells, albeit with delayed kinetics. CD4 cell-independent mechanisms of cellular rejection are poorly understood. To investigate the involvement of CD8 T cells in FPP rejection and their activation requirements, we used mice transgenic for anti-CD4 Ab; this is the most complete model of CD4 cell deficiency. We showed that in such mice FPP was infiltrated with CD8 cells starting from 2 wk posttransplantation and FPP was eventually rejected 8 wk posttransplantation. Ab depletion of CD8 cells greatly improved the survival of FPP and reduced cell infiltration at the graft site. This suggests that CD8 cells can mediate the rejection of porcine xenografts in the absence of CD4 cells. This CD8-mediated rejection of FPP is independent of their perforin-mediated lytic function, as graft survival was not affected in mice deficient in perforin. The production of IFN- and IL-5 by the graft infiltrates indicates that CD8 cells may act through cytokine-mediated mechanisms. Remarkably, in the absence of CD4 cells, lymphocyte infiltration at the graft site was absent in mice transgenic for CTLA4Ig such that the islet grafts flourished beyond 24 wk. In contrast, rejection was little affected by CD40 ligand deficiency. Therefore, we show that CD8 cells are activated to mediate FPP rejection independent of perforin and that this CD4-independent activation of CD8 cells critically depends on B7/CD28 costimulation.

	Introduction

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Replacement of insulin-producing cells represents a cure for type I diabetes. Shortage of human donors has led to the interest in xenotransplantation and most studies in xenotransplantation focus on pigs as the potential source. However, immunological rejection of xenografts remains one of the major hurdles to success (1, 2, 3). There are three types of xenorejection: preformed Ab-mediated hyperacute rejection, non-T cell-mediated delayed xenograft rejection, and T cell-mediated cellular rejection. T cell-mediated rejection can result in rapid graft loss in the absence of other rejection mechanisms. Studies in pig to mouse transplantation have established that CD4 lymphocytes are the major subset responsible for the strength of the cell-mediated response to xenografts (1, 4, 5, 6).

Despite the prominent role of CD4 cells in cellular rejection of xenografts, a number of studies have shown that porcine proislet xenografts were eventually rejected in the absence of CD4 cells (7, 8, 9, 10) and CD8 cells were prominent in the graft site in CD4-deficient mice (6, 8, 9). Some have argued that these CD8 lymphocytes in islet xenografts arrived there passively (6) and the combination of anti-CD4 and anti-CD8 Ab treatment is only sometimes better than anti-CD4 alone (11). In vivo depletion of murine CD8 cells alone did not influence xenograft outcome (i.e., rapid rejection) presumably due to the overriding strength of CD4 cell-mediated rejection. A recent study showed that the rejection of xenograft fetal pig pancreas (FPP)³ still occurred in CD4 cell-deficient MHC class II knockout (KO), and depletion of CD8 lymphocytes in these mice could further prolong the survival of FPP (9). Despite this observation, how CD8 cells are activated to reject xenografts remains to be elucidated. Moreover, the MHC class II KO model has a residual population of CD4 lymphocytes (12), which calls into question whether activation of CD8 cells in response to xenoantigens in studies using this model was truly independent of CD4 help.

Sustained T cell activation generally requires two signals: Ag/MHC and TCR interaction and costimulation between APC and T cells. B7-CD28 and CD40-CD40 ligand (L) represent two major costimulation pathways. Costimulation blockade has been shown to be a powerful way to induce transplant tolerance in at least some transplantation models (13). It has been reported that rejection of FPP xenografts was not inhibited by blocking B7-CD28 interaction with CTLA4Ig in wild-type mice (14), whereas the allospecific CD8 cell response was blocked by CTLA4Ig only when CD4 help was absent (15). This may reflect either that there are enough CD4 T cells that are not sensitive to such costimulation blockade to mediate rejection or that CD8 cells are absolutely dependent on this costimulation only when CD4 help is not available. As far as we know, we are the first to dissect the costimulation requirements for CD8 cell activation to FPP xenoantigens when CD4 cells are absent.

The effector mechanisms used by murine CD8 cells to damage xenografts are less clear. Mouse CD8 cells after activation can kill xenogeneic human tumor cells via a perforin-dependent mechanism (16), although the induction of CTL in the system was totally dependent on CD4 cell help (17). Alternatively, CD8 cells could produce cytokines that in turn activate other effector cells such as macrophages. Activated macrophages have been shown to be capable of rejecting pig xenografts (18, 19, 20). We now wish to investigate whether CD8 cells can be activated in the absence of CD4 cells to produce such cytokines.

We recently derived a new type of mouse deficient in CD4 cells, the GK mouse (21). These mice lack peripheral CD4 cells as a consequence of persistent endogenous transgenic anti-CD4 Ab. In the current study, we used GK mice and xenogeneic FPP as the source of xenoantigens. We aim to examine 1) whether CD8 cells mediate FPP xenograft rejection in the absence of CD4 cells; 2) whether perforin-mediated effector mechanisms are used by CD8 cells to destroy pig xenografts; and 3) whether the induction of CD8 lymphocyte response against xenoantigens can be inhibited by costimulation blockade.

	Materials and Methods

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Mice

C57BL/6 mice with mutations in MHC class I:C.H-2^bm1 (termed bm1) were used as wild-type mice. Transgenic mice were produced on a bm1 background. The production of GK-transgenic (Tg) mice (GK) and CTLA4Ig2c Tg mice (CTLA4Ig Tg) has been described previously (15, 22). GK mice were crossed to CTLA4Ig Tg to produce double Tg mice (GK/CTLA4Ig Tg) to CD154 KO mice (23) (provided by Dr. B. Heath in this Institute) to produce GK/CD154 KO mice to perforin KO mice (24) (provided by Dr. J. Trapani/M. Smyth, Peter McCallum Institute, Melbourne, Australia) to produce GK/perforin KO mice. These animals were bred and housed in our Institute.

Antibodies

The mAb YTS169 (anti-CD8) used for in vivo experiments were purified in our laboratory. The conjugated Abs 53-6.7 (anti-CD8) and H129 (anti-CD4) were purchased from Sigma-Aldrich (St. Louis, MO) and Caltag Laboratories (Burlingame, CA), respectively. Abs used for cytokine ELISA were purchased from BD PharMingen (San Diego, CA). Anti-CD40L Ab MR1 were prepared from Hybridoma Laboratory in the Institute.

Preparation of FPP fragments and FPP lysates

Pancreata were harvested from fetal pigs at 85–100 days of gestation. The pancreas tissue was cut into 4-mm³ pieces. The pancreatic segments were cultured at 37°C in 10% CO₂ on 0.45-µm Millipore filters on a gelfoam (Upjohn, Kalamazoo, MI) support for 4–7 days. The medium was DMEM supplemented with 10% (v/v) FCS and was changed twice weekly. FPP lysates were prepared by salt extraction. FPP were incubated overnight in 3 M KCl (a FPP in 5 ml) at 4°C with continuous rotation. After removing insoluble material by centrifugation at 164,000 x g for 1 h at 4°C, the supernatant was concentrated and dialyzed against PBS. The protein concentration was determined by the Lowry assay.

Transplantation

For FPP transplantation, mice were transplanted with four pieces of FPP under the kidney capsule. At various time points posttransplantation, graft-bearing kidneys were harvested. Paraffin sections of grafts were either stained with H&E for morphological evaluation or for insulin to monitor graft survival. Grafts were scored blindly on a scale 0–4 as previously described (8): 0, fibrous scar with no insulin-staining graft tissue; 1, scattered residual insulin-staining cells; 2, insulin-staining cell cluster with intragraft infiltrate; 3, well-differentiated insulin-staining clusters with perigraft infiltrate; and 4, well-differentiated insulin-staining clusters, no infiltrate. Inflammation was scored as follows: 0, no inflammatory cell infiltrate present; 1, a few perigraft inflammatory cells; 2, numerous perigraft inflammatory cells; 3, intragraft inflammatory cells with residual graft tissue; and 4, massive intragraft inflammatory cell infiltrate with no graft tissue. Statistical differences were assessed using a one-tailed Mann-Whitney U test (GraphPad Prism version 2.0a; GraphPad, San Diego, CA).

Flow cytometry of lymphoid cells

Leukocyte suspensions of spleen were analyzed by flow cytometry. For quantifying lymphocytes, lymphoid cells were stained with directly fluorochrome-conjugated Abs. Cells were analyzed on a FACScan (BD Biosciences, Mountain View, CA) with CellQuest software.

Immunohistochemistry for detection of insulin and leukocytes

To detect insulin-producing cells, paraffin-embedded sections were incubated with guinea pig anti-pig insulin Ig (DAKO, Carpinteria, CA) and then with peroxidase-conjugated rabbit anti-guinea pig Ig. To detect infiltrated leukocytes, frozen sections were incubated with biotinylated rat anti-mouse CD8 mAb (53-6.7; BD PharMingen), B220 (RA3-6B2; BD PharMingen), or F4/80 (American Type Culture Collection, Manassas, VA) followed by streptavidin-biotinylated peroxidase complex (DAKO) and diaminobenzidine, and counterstained with hematoxylin.

Cell culture and cytokine detection

Splenocytes or FACS-sorted cells from FPP-transplanted mice and unprimed mice were cultured at 5 x 10⁶ cell/ml in a 2-ml volume in the presence of FPP lysates for 24 or 72 h. Cytokine levels in the supernatants were evaluated by capture ELISA. The cytokine standards, coating Ab and biotinylated Ab, were obtained from BD PharMingen.

To detect cytokine mRNA, grafts were snap-frozen in hexane over liquid nitrogen and RNA was extracted with RNAzol (Biotecx Laboratories, Houston, TX). RT-PCR was performed with cytokine-specific primers (25) with actin mRNA as control.

	Results

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CD8 cells were activated in FPP-transplanted CD4 cell-deficient mice

Extensive studies have shown that CD4 cell depletion dramatically delays rejection and reduces infiltration in pig to mouse islet xenotransplantation (6, 26). However, in most cases, FPP xenografts are eventually rejected, even in GK mice which are permanently deficient in CD4 cells (Tg expression results in 0.1 mg/ml circulating anti-CD4 Ab in these mice) (8). GK mice and their crosses used in this study all had <0.1% CD4 cells in the spleen and were used in this study to investigate CD4-independent rejection. The size of the splenic CD8 population was not significantly different among the types of CD4-deficient mice (data not shown). We has shown previously in GK mice that FPP were rejected within 2 wk in wild-type mice and were rejected around 8 wk in most GK mice and that CD8⁺ cells infiltrated rejecting FPP graft in GK mice (8).

FPP rejection in CD4-deficient mice is much slower than CD4-mediated rejection. Some workers have suggested that infiltrated cells at the graft site in the absence of CD4 cells were inactive (27). To investigate this premise, grafts were harvested and analyzed for their cytokine profile by RT-PCR. As expected, grafts from wild-type mice transcribed IFN-, IL-2, and IL-5 one wk after transplantation (Fig. 1A). In GK mice, some IFN- mRNA can be detected at the graft site between 1 and 2 wk posttransplantation. By 3 wk, IFN-, IL-2, and IL-5 mRNA were all detected from FPP grafts in GK mice. IFN- mRNA but not IL-5 mRNA remained detectable at 8 wk after FPP transplantation (data not shown). Thus, the cytokine profile in graft infiltrates in CD4-deficient mice would argue against the cells being functionally inactive.

View larger version (36K): [in this window] [in a new window]   FIGURE 1. Cytokine production by FPP-transplanted mice: A, Cytokine mRNA expression by graft infiltrates, Mice were transplanted with FPP under kidney capsules. At various times after transplantation, grafts were removed and RNA was extracted. Cytokine mRNA was detected by RT-PCR with specific primers. B, Cytokine production by splenocytes from FPP-transplanted mice. Splenocytes were harvested from bm1 and GK mice that were transplanted with FPP for 4 wk. Cells were cultured at 8 x 106 cells/2 ml wells in the presence of 100 µg/ml FPP lysates, and culture supernatants were harvested 24 h later for IL-2 and IL-4 detection and 72 h for IL-5 and IFN- detection. C, Cytokine production by splenic CD8 from FPP-transplanted mice. CD8 cells from transplanted animals were purified by FACS sorting. Cells were cultured at 2 x 105 cells/well in 0.2 ml in 96-well plates in the presence of 100 µg/ml FPP lysates, 100 pg/ml mouse IL-2, and 5 x 105 (20 Gy) irradiated bm1 splenocytes for 72 h. Culture supernatants were assayed for IL-5 and IFN- detection. Samples from control mice had levels of cytokine below detection limits. The RT-PCR experiments were performed three times and the cytokine measurements of culture supernatants were performed twice. The results of all five experiments concur that the CD8 cells in the absence of CD4 cells were activated by the xenograft. Mean and SE are shown.

CD8 T cells mediated FPP xenograft rejection in the absence of CD4 cells

To investigate whether CD8 cells are involved in xenograft rejection in CD4-deficient mice, GK mice were treated with a short course of depleting anti-CD8 Ab (1 mg of YTS 169 at -1, +7, and +14 days relative to FPP transplantation), and graft survival was evaluated at 3 and 10 wk after transplantation. The depletion regimen resulted in >90% of reduction of CD8 population in spleen and lymph nodes initially (day 3 after the last Ab injection); the CD8 population eventually recovered to half that of undepleted mice 3 wk after the last Ab injection. At 3 wk after transplantation, both anti-CD8 Ab-treated and untreated GK mice had intact grafts containing insulin-positive cells. However, the graft infiltration from anti-CD8 Ab-treated GK mice was much reduced compared with that from untreated GK mice. The paradoxical finding that grafts survived in untreated GK mice at 3 wk, despite moderate to heavy infiltration, suggests that with the severe CD4 help for deficiency in GK mice the CD8 cells are slow to become aggressive. At 10 wk posttransplantation, grafts from anti-CD8 Ab-treated mice still had abundant well-differentiated insulin-producing tissues. Moreover, mononuclear cell infiltration at the graft site was much reduced. In contrast, grafts in untreated FPP-transplanted GK mice contained no or a few residual insulin-positive cells with heavy infiltration by mononuclear cells (Fig. 2A). Scoring of graft survival and infiltration based on insulin staining and H&E staining from five mice in each group revealed that CD8 depletion significantly (p < 0.01) improved survival of insulin-producing FPP (Fig. 2B). Treatment with anti-CD8 Ab beginning at 4 wk posttransplantation, when infiltration had already developed in FPP-transplanted GK mice, was also found to delay FPP rejection in GK mice (data not shown). Amelioration of graft rejection by the anti-CD8 Ab in the GK mice indicated that the rejection of FPP xenografts in the absence of CD4 help was CD8 mediated.

View larger version (49K): [in this window] [in a new window]   FIGURE 2. FPP survival in anti-CD8 Ab-treated GK mice. GK mice were transplanted with FPP and half of them were given three doses of anti-CD8 Ab (YTS 169). Grafts were harvested at 3 and 10 wk after transplantation. Survival of insulin-producing grafts and cell infiltration was assessed from insulin-stained and H&E-stained sections. A, Photomicrograph of insulin-stained FPP grafts. Arrow, Insulin-positive cells; M, infiltrated mononuclear cells. Original magnification, x200. B, Score of graft survival and infiltrate. The graft survival and infiltrate levels were scored blindly at a range of 0–4 according to the criteria described in Materials and Methods. Data represent the mean and SD of five individual mice in each group. Experiments were performed three times. *, p < 0.05; **, p < 0.01 compared with untreated mice by Student’s t test.

CD8 cells can execute their effector functions through a perforin/granzyme-dependent lytic pathway as well as a cytokine-mediated indirect pathway. Murine CD8 cells have been shown previously to reject xenogeneic human tumor cells by a perforin-mediated cytotoxic mechanism (16). To test whether such a mechanism is used by CD8 cells to destroy FPP xenografts, GK mice were crossed to C57BL/6 perforin KO mice. GK/perforin KO mice were transplanted with FPP and the fate of xenografts in these mice was monitored and compared with that of GK mice. The kinetics of FPP graft survival and inflammatory infiltrate was not significantly different in the presence or the absence of perforin. Like GK mice, GK/perforin KO mice developed mononuclear cell infiltration and only residual insulin-producing grafts remained at 8 wk posttransplantation (Fig. 3A). We conclude that perforin is not important for CD8 cell-mediated FPP rejection in GK mice, and our results are consistent with a cytokine-mediated mechanism. Indeed, grafts from three GK/perforin-deficient mice all transcribed IFN- and IL-2 mRNA (Fig. 3B). There was no IL-5 message detected in either GK or GK/perforin KO mice at 8 wk posttransplantation (data not shown).

View larger version (47K): [in this window] [in a new window]   FIGURE 3. FPP survival in perforin-deficient GK mice. GK mice and GK/perforin KO mice were transplanted with FPP. Mice were sacrificed at 8 wk posttransplantation. A, Photomicrograph of FPP grafts from H&E-stained and insulin-stained sections. Arrow, Insulin-positive cells; M, infiltrated mononuclear cells. Original magnification, x200. B, Cytokine mRNA expression by graft infiltrate at 8 wk posttransplantation. RNA was extracted from graft tissue. Cytokine mRNA was detected by RT-PCR with specific primers.

B7-CD28 and CD40-CD40L are two major pathways of T cell costimulation. To test whether CD8-mediated rejection of FPP can be inhibited by CD28 costimulation blockade, GK/CTLA4Ig doubly Tg mice were produced. When such mice were used as recipients of FPP xenografts, grafts survived beyond 24 wk without any infiltration (Fig. 4A). It was also evident that FPP matured and differentiated in the absence of rejection. By 24 wk, the earlier patchy distribution of small clusters of insulin-producing cells had developed into well-differentiated islets of insulin-producing cells. The graft survival in GK/CTLA4Ig mice was significantly better than in that in control GK mice (Fig. 4B). Not surprisingly, cytokine mRNA (IFN-, IL-2, and IL-5) was not detected from grafts in GK/CTLA4Ig doubly Tg mice (Fig. 1A).

View larger version (62K): [in this window] [in a new window]   FIGURE 4. FPP survival in GK/CTLA4Ig doubly Tg mice: GK mice and GK/CTLA4Ig mice were transplanted with FPP. Grafts were harvested at various times after transplantation. Survival of insulin-producing grafts and cell infiltration were assessed from insulin-stained and H&E-stained sections. A, Photomicrograph of insulin-stained FPP grafts in GK/CTLA4Ig mice. B, Score of graft survival and infiltrate from 8-wk transplanted mice. Data represent the mean and SD of five individual mice in each group. Five similar experiments were performed. **, p < 0.01 Student’s t test, compared with FPP-transplanted GK mice. Original magnification, x200.

To test whether CD40-CD40L interaction plays a role in the induction of CD4-independent CD8 cell responses to FPP xenografts, FPP were transplanted into GK mice that were also deficient in CD40L. Although the insulin-producing cells were slightly more abundant in GK/CD40L KO mice, infiltration in GK/CD40L KO mice was similar to GK mice (Fig. 5). IFN- and IL-2 messages in the grafts were, however, relatively lower in GK/CD40L KO mice compared with that in GK mice (data not shown).

View larger version (43K): [in this window] [in a new window]   FIGURE 5. FPP survival in GK/CD40L KO mice. GK mice and GK/CD40L mice were transplanted with FPP. Grafts were harvested at various times after transplantation. Survival of insulin-producing grafts and cell infiltration were assessed from insulin-stained and H&E-stained sections. A, Photomicrograph of insulin-stained FPP grafts in GK/CD40L KO mice. B, Score of graft survival and infiltrate from 4- and 8-wk transplanted mice. Data represent the mean and SD of five individual mice in each group. Five similar experiments were performed. Original magnification, x200.

As a control for the experiments performed in the CD4-deficient mice, mice with an intact CD4 compartment were also used in this study to dissect the costimulation requirement and effector mechanisms. In the presence of CD4 cells, Tg CTLA4Ig was incapable of blocking graft rejection. By 2 wk posttransplantation, there were no insulin-producing cells left. Blocking CD40-CD40L interaction by anti-CD40L Ab only marginally improved graft survival. Only two of nine mice had insulin-producing grafts at 2 wk posttransplantation. On the other hand, grafts survived beyond 8 wk in CTLA4Ig Tg/CD40L KO mice. Interestingly, infiltration developed from 2 wk posttransplantation despite excellent graft survival. Not surprisingly, porcine islet xenografts were rejected in perforin KO mice by 2 wk posttransplantation (Fig. 6).

View larger version (98K): [in this window] [in a new window]   FIGURE 6. FPP rejection in the presence of CD4 cells. CTLA4Ig Tg mice, anti-CD40L Ab-treated mice (i.p. injection of 3 mg in 3 doses at days 0, 3, and 7 referring to transplantation time), CTLA4Ig Tg/CD40L KO mice, and perforin KO mice were transplanted with FPP. Grafts were harvested at various times after transplantation. Survival of insulin-producing grafts and cell infiltration were assessed from insulin-stained sections. Photomicrograph of insulin-stained FPP grafts are shown. Experiments were repeated at least twice for all strains of mice. Original magnification, x200.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

T cell activation requires the interaction of CD28 molecules on T cells and B7 (CD80/86) molecules on APC. Blocking CD28-mediated costimulation in allo- and xenotransplantation has been shown to prolong graft survival (29, 30). In the presence of CD4 cells, CTLA4Ig is not very effective in blocking the rejection of porcine islet xenografts in this and an earlier study (14). This is also the case for other allo- and xenografts (15, 31) and may reflect the involvement of other costimulation pathways. Nevertheless, in the absence of CD4 cells, costimulation blockade by Tg CTLA4Ig herein resulted in long-term survival of pig xenografts. Remarkably, there was no infiltration; this being reminiscent of what we find in SCID mice. The results highlight the critical role of CD28-B7 interaction in activation of CD8 cells in response to xenoantigens, when CD4 help is absent. Extensive studies have shown that CD28-B7 interaction is important in the initiation of activation of T cells by decreasing the threshold of activation (32, 33), in the expansion of activated T cells by inducing IL-2 production (34), and in T cell recruitment by inducing the production of chemokines (35). CTLA4Ig could potentially block at all these levels and thus prevent CD8 cell-mediated rejection.

Blockade of another costimulation pathway, CD40L-CD40, in the current study did not have a significant effect on the development of infiltration at the graft site in CD4 cell- deficient mice. The reduced dependency of CD40L-CD40 interaction for CD8 activation to xenoantigen in this study can be related to the absence of CD4 help in the system. Activation of CD8 cells to many cellular Ags required CD4 help (36) and CD4 cells can execute this help via a CD40L-CD40 interaction with APC in addition to providing IL-2 to CD8 cells (37). Considering that CD40-CD40L interaction was critical to sustain a CD8 response rather than prime a CD8 response (38), it is possible that the CD8 cell-mediated rejection of porcine islet xenograft progresses slowly in the absence of CD4 cells and therefore the demand for expansion of activated CD8 cells is not grave. The CD8 cell response in this setting may not require CD40L-CD40 interaction for such sustaining signals. The reduced dependency of CD40L-CD40 interaction for CD8 activation to xenoantigen in this study can also be related to the absence of CD4 help in the system. In the presence of CD4 cells, blockade of CD40-CD40L marginally prolonged the graft survival. This could be explained by some compensatory mechanism provided by another costimulation signal. Indeed, when both B7-CD28 and CD40-CD40L are blocked, graft survival was dramatically prolonged in the current study.

CD8 cells can execute their effector functions via perforin/granzyme B lytic activity as well as via cytokine production to activate other effector cells such as macrophages. It has been shown previously that mouse CD8 cells are activated in vivo in response to immunization of xenogeneic human tumor cell lines and activated CD8 cells can directly kill human tumor cells by perforin-mediated mechanisms (16). However, we show here that CD8 cell-mediated rejection of pig xenografts in the mouse is independent of perforin in vivo. Fas-FasL cytotoxicity has been shown to be used by human CD4 cells to lyse pig cells directly (39); thus indicating the compatibility of human FasL with pig Fas. It seems unlikely that the Fas-FasL pathway is important in CD8-mediated FPP rejection in the mouse, as it has been shown that very few CD8 cells in FPP-transplanted mice had FasL (9). Given the fact that the graft-infiltrating lymphocytes did produce cytokine locally, it is reasonable that activated CD8 cells might reject grafts through production of cytokines, primarily IFN-, to activate other effector cells like macrophages.

In conclusion, CD8 cells can be activated to mediate FPP rejection in the absence of CD4 help. Activation of CD8 cells in response to FPP xenografts critically depends on B7-CD28 costimulation when CD4 help is absent. Activated CD8 cells mediate rejection of FPP xenografts independent of a perforin-mediated lytic mechanism. The contribution of CD8 cells needs to be considered in strategies to overcome xenograft rejection.

	Acknowledgments

 
We thank Steven Mihajlovic, Ellen Tsui for assistance with histology, and Miriam Buttigieg and Michelle Latimer for assistance with mouse maintenance. We thank Dr. Bill Heath, Dr. Tom Mandel, Dr. Tom Kay, and Prof. Len Harrison for reading this manuscript.

	Footnotes

 
¹ This work was supported by grants from the Juvenile Diabetes Research Foundation, Ramaciotti Foundation, Diabetes Australia, and National Health and Medical Research Council of Australia.

² Address correspondence and reprint requests to Dr. Andrew M. Lew, Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Parkville 3050, Victoria, Australia. E-mail address: lew{at}wehi.edu.au

³ Abbreviations used in this paper: FPP: fetal pig pancreas; KO: knockout; L, ligand; Tg, transgenic.

Received for publication June 29, 2001. Accepted for publication October 2, 2001.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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