The strength of cell-mediated xenograft rejection in the mouse is due to the CD4+ indirect response

Abstract: Previous studies have shown that CD4+ T cells are responsible for the great strength of cell-mediated xenograft rejection in the mouse. In vitro studies have suggested that this CD4+ response is to xenogeneic antigens that are presented indirectly. The present studies were carried out in order to determine whether the strength of cell-mediated xenograft rejection in vivo is dependent on the CD4+ indirect response. We grafted pig skin onto mice that express class 11 MHC antigens only on their thymic epithelial cells (II-4+ mice). These mice have normal numbers of functional peripheral CD4+ T cells; however they lack class II MHC expression on their antigen presenting cells and are thus incapable of mounting a CD4+ T cell-mediated indirect response. Xenograft survival was prolonged on these mice. Furthermore, administration of cyclosporine and anti-CDS monoclonal antibodies to II-4+ recipients prolonged xenograft survival to at least the same extent as allograft survival, demonstrating that the strength of cell-mediated xenograft rejection resides in the CD4+ indirect response. Despite the increased survival time, xenograft rejection still occurred in the absence of the indirect pathway. Depletion of the II-4+ recipients of their CD4+ T cell population prolonged xenograft survival even further, suggesting the presence of a weaker CD4+ direct mechanism which was virtually undetectable in vitro.     

CD8+ T cells are capable of rejecting pancreatic islet xenografts

BACKGROUND: In this study, the capacity of CD8+ T cells to act as a potential effector mechanism in pancreatic xenograft rejection was examined. METHODS: The fate of pancreatic islet xenografts was studied in mice deficient in MHC class II molecules and CD4+ T cells. Fetal pig pancreas (FPP) or Wistar rat islets (RI) were transplanted into nondiabetic or streptozotocin-induced diabetic I-A knock-out (CII K/O) mice. RESULTS: CII K/O mice were capable of rejecting both RI and FPP grafts. RI graft survival was not prolonged compared with wild type C57BL/6 controls. However, FPP grafts did survive longer in CII K/O recipients than in C57BL/J6 mice. Both RI and FPP graft rejection were CD8+ T-cell phenomena in CII K/O mice, as anti-CD8 monoclonal antibody prolonged graft survival, there were increased CD8+ T cells in the grafts and spleens of CII K/O recipients, and cell-mediated cytotoxicity was a CD8+ T-cell phenomenon associated with activation of the perforin/granzyme B system. By contrast, RI and FPP graft rejection was a CD4+ T cell-dependent phenomenon in wild type C57BL/6 mice with graft survival prolonged by anti-CD4 monoclonal antibody. There were increased numbers of CD4+ T cells, and cell-mediated cytotoxicity was a CD4+ T-cell phenomenon associated with activation of the Fas/FasL lytic pathway. CONCLUSIONS: The results demonstrate that, in the absence of CD4+ T cells, CD8+ T cells were capable of rejecting both rat and pig pancreatic islet xenografts.     

Roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection

An essential role of murine CD4+ T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2b) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3+/-(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2k) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4+ T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8+ T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8+ T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8+ T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4+ T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8+ T cells also are capable of rejecting discordant human skin xenografts.     

Effect of GK1.5 monoclonal antibody dosage on survival of pig proislet xenografts in CD4+ T cell-depleted mice

Treatment of CBA/H mice with 5 injections of anti-CD4 (GK1.5 mAb) terminating on day 10 posttransplant resulted in long-term survival (greater than or equal to 6 weeks) of fetal pig proislet (pancreatic islet precursor) xenografts. The GK1.5 mAb dose determined the duration of CD4+ T cell depletion and the extent to which the survival of pig proislet xenografts was prolonged. Sustained depletion of CD4+ T cells (0%, 1%, and 9% of total T cells in peripheral lymph nodes at 2, 4, and 6 weeks, respectively) and survival of proislet xenografts at 6 weeks posttransplant was observed when transplant recipients were treated with 5.4 mg GK1.5 mAb/injection. Treatment of transplanted mice with a suboptimal dose of GK1.5 mAb (0.2 mg/injection) resulted in the same level of depletion at 2 weeks posttransplant but a more rapid recovery of CD4+ T cells in the periphery (24% of total T cells at 4 weeks) and only temporary prolongation in xenograft survival (less than or equal to 4 weeks). Control xenografts showed evidence of graft destruction by as early as 6-7 days posttransplant and were completely rejected by 2 weeks. The rejection reaction consisted predominantly of CD4+ T cells, eosinophils and F4/80-positive macrophages. Only small numbers of CD8+ T cells were identified. CD4+ T cells therefore represented the major T cell component of the cellular infiltrate. In contrast, surviving xenografts in GK1.5 mAb-treated recipient mice showed essentially an absence of CD4+ T cells but presence of CD8+ T cells. This finding may be attributable to the increase (1.7-3.1-fold) in the absolute size of the population of CD8+ T cells in the periphery following GK1.5 mAb treatment in vivo. Compared with isolated fetal pig proislets, which contained only a small population of insulin-producing cells in addition to glucagon- and somatostatin-positive cells, surviving pig proislet xenografts contained mainly insulin-positive beta cells with smaller populations of glucagon- and somatostatin-positive cells. Fetal pig proislets therefore differentiate into insulin-producing islet tissue posttransplant and thus show evidence of normal development of endocrine function.     

An increase in the survival of murine H-2-mismatched cultured fetal pancreas allografts using depleting or nondepleting anti-CD4 monoclonal antibodies, and a further increase with the addition of cyclosporine

Depletion of CD4+ T lymphocytes with monoclonal antibodies (mAbs) has been shown to prolong allograft survival in mice. In this study, two rat anti-CD4 mAbs, H129.19 and GK1.5, were administered either alone or in combination with cyclosporine (CsA) to recipients of MHC-mismatched (H-2k to H-2d) cultured fetal pancreas allografts to determine their effect on graft survival. When compared with control mice, splenic CD4+ cells of GK1.5-treated mice were depleted by greater than 95%, but in H129.19-treated mice no depletion of CD4+ cells occurred. Instead, rat Ig was present on the surface of CD4+ cells in H129.19-treated mice. Anti-CD4 therapy with either H129.19 or GK1.5 prolonged fetal pancreas allograft survival to a similar extent, but did not lead to indefinite survival. Blockade of the CD4 antigen by the mAb H129.19 was as effective as the depletion of CD4+ cells by GK1.5 in prolonging allograft survival. Rejection of grafts by day 28 posttransplantation occurred in the absence of CD4+ cells, as determined by both flow cytometric examination of spleen cells and immunoperoxidase staining of the graft site. CsA alone did not prolong graft survival, but its addition to either H129.19 or GK1.5 mAb treatment significantly increased the survival rate of grafts at 28 days compared with mAb treatment alone. These results suggest that CD4+ cell depletion is not essential for effective anti-CD4 mAb therapy--and, further, that CsA may have a direct inhibitory effect on CD8+ cells during allograft rejection.     

Antibody-induced rejection of pig proislet xenografts in CD4+ T cell-depleted diabetic mice

Reversal of diabetes in mice was achieved following in vivo depletion of host CD4+ T cells and transplantation of xenogeneic fetal pig proislets (pancreatic islet precursors). These procedures resulted in xenograft tolerance since established pig proislet xenografts were not rejected by antipig antibodies produced in the host, and rejection was not induced following the administration of donor major histocompatibility complex--specific pig lymphocytes. Proislet xenografts were rejected following the administration of donor MHC-specific hyper-immune antipig PBL serum raised in normal mice. Although established proislet xenografts in anti-CD4-treated mice are sensitive to antibody-mediated destruction, such hosts are unable to produce an antibody response that leads to graft rejection. The study indicates that the mechanism of preventing xenograft rejection by anti-CD4 treatment in vivo involves not only initial CD4+ T cell depletion but also quantitative and/or qualitative modulation of a CD4+ T cell-dependent antibody response. As a consequence, an apparent state of xenograft tolerance is produced.     

Transplantation of organ cultured fetal pig pancreas in non-obese diabetic (NOD) mice and primates (Macaca fascicularis)

Abstract: Xenografts of organ cultured fetal pig pancreas in prediabetic NOD mice can survive for prolonged periods (>20 weeks) in recipients treated with anti-T cell monoclonal antibodies (MAb) directed against host CD4 and CD3 cell surface molecules. Anti-CD4 MAb treatment alone is only partly effective and xenograft rejection occurs over a period of many weeks. In diabetic recipients, by contrast, recurrence of autoimmune disease in isografts is rapid (<28 days) despite similar depletion of CD4+ve T cells. In spontaneously diabetic NOD mice immunosuppressed with anti-CD3 and anti-CD4 MAbs xenograft function occurs and the recipient's blood glucose levels fall into the pig range. Organ cultured fetal pig pancreas transplanted into cynomolgus monkeys is rapidly but not hyperacutely rejected when azathioprine-cyclosporin A-prednisolone immunosuppression is used. Anti-T-cell MAb treatment is now being studied in this primate model.     

The immunobiology of pig‐to‐nonhuman primate islet xenotransplantation: insights,innovation, and impact

Previous studies of pig‐to‐non‐human primate (NHP) islet xenotransplantation have provided important insights into the immune recognition and effector pathways operative in this relevant preclinical model. The specifics of the xenograft product, microenvironment at the implantation site, and the immunosuppressive regimen significantly influence the mechanisms underlying the rejection of xenogeneic islets. Our current understanding of the immunological barriers to survival of pig islets in NHPs is largely based on studies on intraportal islet xenografts and on comparisons with islet allografts. The demonstration of cell‐mediated rejection of intraportal porcine islet xenografts at about 1 month posttransplant in monkeys immunosuppressed with the same protocols that prevent monkey islet allograft rejection indicates that islet xenograft rejection involves cellular mechanisms that are not present in acute islet allograft rejection. While these mechanisms remain poorly defined the demonstration of long‐term diabetes reversal after intraportal islet xenotransplantation in non‐human primates immunosuppressed with anti‐CD40L but not with anti‐CD40 antibody‐based protocols suggests that the therapeutic efficacy of anti‐CD40L in this transplantation setting likely involves the depletion of donor‐reactive, activated T cells besides CD40:CD40L costimulation blockade. Rejection of intraportal islet xenografts in NHPs immunosuppressed with CTLA4‐Ig and rapamycin was mediated largely by IL‐15‐primed, CXCR3+CD8+ memory T cells recruited by IP‐10 (CXCL10) positive pig islets and macrophages that showed staining for IL‐12 and iNOS. Adding basiliximab induction and tacrolimus maintenance therapy to this protocol prevented rejection in 24 of 26 recipients followed for up to 275 days. Comparison of both groups suggests, though by no means conclusive, that prolongation of graft survival in this large cohort was associated with reduced direct T cell responses to xenoantigens, reduced proportion of intrahepatic (intragraft) B cells and IFN‐γ+ and IL‐17+ CD4 and CD8 T cells, and increased local production of immunoregulatory molecules linked with Tregs, including TGF‐β, Foxp3, HO‐1, and IL‐10. Anti‐pig non‐Gal IgG antibody elicitation was suppressed in both groups. We are currently exploring the concept of negative vaccination to markedly minimize the need for immunosuppression in islet xenotransplantation. Peritransplant administration of donor apoptotic cells extended pig‐to‐mouse islet xenograft survival to >250 days when combined with peritransplant B cell‐depletion and rapamycin. This costimulation blockade‐sparing, antigen‐specific immunotherapy is expected to cause rapid pretransplant clonal deletion of indirect and anergy of direct xenospecific T cells while inducing regulatory T cells. As anti‐CD40L antibodies, B cell depleting antibodies are expected to interfere with indirect antigen presentation, costimulation, and cytokine production required for optimal T cell proliferation, memory formation, and intragraft CD8+ effector function. It is conceivable that additional strategies must be employed in NHPs and eventually in diabetic patients to achieve – as previously with anti‐CD40L antibodies – more complete, yet selective depletion of donor‐reactive, activated T‐cells for the purpose of stable xenograft acceptance.     

CD4+ T cells initiate pancreatic islet xenograft rejection via an interferon-gamma-dependent recruitment of macrophages and natural killer cells

BACKGROUND: In this study, the mechanisms by which CD4+ T cells interact with the innate immune system in xenograft rejection were investigated. METHODS: Fetal pig pancreas (FPP) grafts were transplanted into female SCID mice. The FPP recipient SCID mice were reconstituted with exogenous leukocytes obtained from male BALB/c mice. RESULTS: Although nonreconstituted SCID recipients or recipients reconstituted with CD4+ T cell-depleted leukocytes showed indefinite FPP graft survival with very few macrophages infiltrating their grafts, reconstitution of SCID recipients with as few as 2x10(5) CD4+ T cells was sufficient to induce rapid xenograft rejection. CD4+ T cells secreted interferon-gamma but not interleukin-4 and initiated the activation and accumulation of macrophages and natural killer cells, that were responsible for the rapid graft destruction. Suppression of interferon-gamma prolonged graft survival and suppressed the macrophages and natural killer cell accumulation and activation. CONCLUSIONS: These results demonstrate that CD4+ T cell-dependent cellular xenograft rejection was a result of macrophage and natural killer cell accumulation and activation, but was not mediated by eosinophils. Consistent with this was the finding that interferon-gamma but not interleukin-4 was in part responsible for mediating this effect.     

Inhibition of cellular immune responses to encapsulated porcine islet xenografts by simultaneous blockade of two different costimulatory pathways

BACKGROUND: Transplantation of human islets has been successful clinically. Since human islets are scarce, we are studying microencapsulated porcine islet xenografts in nonobese diabetic (NOD) mice. We have evaluated the cellular immune response in NOD mice with and without dual costimulatory blockade. METHODS: Alginate-poly-L-lysine-encapsulated adult porcine islets were transplanted i.p. in untreated diabetic NODs and NODs treated with CTLA4-Ig to block CD28/B7 and with anti-CD154 mAb to inhibit CD40/CD40-ligand interactions. Groups of mice were sacrificed on subsequent days; microcapsules were evaluated by histology; peritoneal cells were analyzed by FACS; and peritoneal cytokines were quantified by ELISA. Controls included immunoincompetent NOD-Scids and diabetic NODs given sham surgery or empty microcapsules. RESULTS: Within 20 days, encapsulated porcine islets induced accumulation of large numbers of macrophages, eosinophils, and significant numbers of CD4 and CD8 T cells at the graft site, and all grafts were rejected. During rejection, IFNgamma, IL-12 and IL-5 were significantly elevated over sham-operated controls, whereas IL-2, TNFalpha, IL-4, IL-6, IL-10, IL-1beta and TGFbeta were unchanged. Treatment with CTLA4-Ig and anti-CD154 prevented graft destruction in all animals during the 26 days of the experiment, dramatically inhibited recruitment of host inflammatory cells, and inhibited peritoneal IFNgamma and IL-5 concentrations while delaying IL-12 production. CONCLUSIONS: When two different pathways of T cell costimulation were blocked, T cell-dependent inflammatory responses were inhibited, and survival of encapsulated islet xenografts was significantly prolonged. These findings suggest synergy between encapsulation of donor islets and simultaneous blockade of two host costimulatory pathways in prolonging xenoislet transplant survival.     

Rejection of porcine islet xenografts mediated by CD4+ T cells activated through the indirect antigen recognition pathway

We have previously demonstrated that human T cells responding to porcine islets are primarily CD4+ and recognized porcine major histocompatibility complex class I molecules through the indirect pathway of antigen presentation. To determine whether this mechanism is responsible for rejection of adult porcine islets xenografts, porcine islets from adult pigs were transplanted under the kidney capsule of streptozotocin-treated CD4-knockout (KO), CD8-KO, Ig-KO and normal C57BL/6 mice. Islet xenografts were acutely rejected with similar kinetics when transplanted into normal C57BL/6 (MST=17.6 +/- 3.5 days) and Ig-KO (MST=19.0 +/- 1.7 days) mice. Interestingly, islet xenografts were rejected significantly earlier when transplanted into CD8-KO mice as compared with normal C57BL/6 (MST=7.0 +/- 0.01 days, P=2 x 10-4). Histopathological analysis revealed classical acute cellular rejection with severe diffuse interstitial cellular infiltrates in all rejected islet xenografts. In contrast, islet xenografts were not rejected when transplanted into CD4-KO mice (MST >/= 100 days, P=1 x 10-9). Histopathological analysis revealed no cellular infiltrates and intact islet xenografts. CD4+ T cells from both normal C57BL/6 and CD8-KO xenograft recipients showed detectable proliferative responses to porcine islets in the presence but not in the absence of syngeneic antigen-presenting cells. In addition, the anti-islet proliferative responses observed in normal C57BL/6 mice were significantly lower than those observed in CD8-KO mice. IgG anti-porcine antibodies were readily detected in C57BL/6 and CD8-KO xenograft recipients but not in Ig-KO or CD4-KO recipients. These results indicate that indirectly activated CD4+ T cells mediate acute rejection of adult porcine islet xenografts and that xenoreactive CD8+ T cells and antibodies are not necessary in this process.     

Requirement of CD4 cells for induction and maintenance of unresponsiveness in islet xenografted mice

Abstract: Long-term survival of islet xenografts in the hamster to mouse model can be induced by a short-course treatment with a nondepleting anti-CD4 mAb but not with a depleting anti-CD4 mAb (Lu et al. Xenotransplantation 1998; 5:154–1631. Although CD4 cells are known to play a key role in the rejection of islet xenografts, it remains unclear whether CD4 cells are also required for the induction and/or maintenance of specific unresponsiveness to xenografts. To investigate this problem, islets were isolated from golden hamsters and transplanted into streptozotocin-induced diabetic CBA/J mice. Nondepleting mAb YTS 177.9 was used to block CD4 cells for the induction of islet xenograft unresponsiveness and subsequently depleting mAb GK1.5 to deplete CD4 cells in the unresponsive recipients.
First, we now confirm that second donor-strain xenografts were permanently accepted in recipients that had been unresponsive to the first grafts, whereas Lewis rat islet xenografts, used as third-party grafts, were rejected like a primary graft within 7–8 days. Second, we depleted CD4 cells in recipient mice, which had been treated perioperatively with the nondepleting mAb YTS 177.9 and became unresponsive to their primary hamster islet graft, by using a depleting anti-CD4 mAb at different time points post-transplant. Depletion of CD4 cells in the unresponsive recipients by the depleting anti-CD4 mAb GK1.5 did abrogate this unresponsive state, since the grafts were always rejected within an average of 25.5 days after the mAb GK1.5 injections. Therefore, our results strongly suggest that CD4 positive cells play an active suppressive role and that their presence in the recipients appears essential for both induction and maintenance of long-term islet xenograft survival or specific unresponsiveness.     

CTLA-4Ig in Combination with Anti-CD40L Prolongs Xenograft Survival and Inhibits Anti-Gal Ab Production in GT-Ko Mice

The generation of GT-Ko mice has provided unique opportunities to study allograft and xenograft rejection in the context of anti-alpha1,3-Gal antibody (anti-Gal Ab) responses. In this study we used the allotransplantation model of C3H hearts into galactosyltransferase-deficient (GT-Ko) mice and the xenotransplantation model of baby Lewis rat hearts into GT-Ko mice to investigate the ability of CTLA-41g in combination with anti-CD40L mAb to control graft rejection and anti-Gal Ab production. Murine CTLA-41g or anti-CD40L monotherapy prolonged allograft survival, and the combination of these reagents was most immunosuppressive. However short-term treatment with murine cytotoxic T lymphocyte associated antigen-4 (muCTLA-41g) and/or CD40 ligand (CD154) monoclonal antibodies (anti-CD40L mAbs) was unable to induce indefinite allograft survival. CTLA-4-immunoglobulin fusion protein (CTLA-41g) or anti-CD40L monotherapy only marginally prolonged xenograft survival; the combination of human CTLA-41g and anti-CD40L significantly prolonged xenograft survival (74days), while the combination of murine CTLA-41g and anti-CD40L resulted in graft survival of >120days. CTLA-41g or anti-CD40L monotherapy or the combination of these agents inhibited the production of alloAbs, including anti-Gal Abs. CTLA-41g or anti-CD40L monotherapy partially controlled xenoAb and anti-Gal Ab production, while the combination was more effective. These observations corroborate our previous observations that humoral, including anti-Gal Ab, responses and rejection following allograft or concordant xenograft transplantation in GT-Ko mice are T-cell dependent and can be controlled by costimulation blockade.     

Combination of anti-CD4 with anti-LFA-1 and anti-CD154 monoclonal antibodies promotes long-term survival and function of neonatal porcine islet xenografts in spontaneously diabetic NOD mice

Type 1 diabetes mellitus (T1DM) is caused by the autoimmune destruction of pancreatic islet beta-cells, which are required for the production of insulin. Islet transplantation has been shown to be an effective treatment option for TIDM; however, the current shortage of human islet donors limits the application of this treatment to patients with brittle T1DM. Xenotransplantation of pig islets is a potential solution to the shortage of human donor islets provided xenograft rejection is prevented. We demonstrated that a short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) was highly effective in preventing rejection of neonatal porcine islet (NPI) xenografts in non-autoimmune-prone B6 mice. However, the efficacy of this therapy in preventing rejection of NPI xenografts in autoimmune-prone nonobese diabetic (NOD) mice is not known. Given that the current application of islet transplantation is for the treatment of T1DM, we set out to determine whether a combination of anti-LFA-1 and anti-CD154 mAbs could promote long-term survival of NPI xenografts in NOD mice. Short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs, which we found highly effective in preventing rejection of NPI xenografts in B6 mice, failed to promote long-term survival of NPI xenografts in NOD mice. However, addition of anti-CD4 mAb to short-term treatment of a combination of anti-LFA-1 and anti-CD154 mAbs resulted in xenograft function in 9/12 animals and long-term graft (>100 days) survival in 2/12 mice. Immunohistochemical analysis of islet grafts from these mice identified numerous insulin-producing beta-cells. Moreover, the anti-porcine antibody as well as autoreactive antibody responses in these mice was reduced similar to those observed in naive nontransplanted mice. These data demonstrate that simultaneous targeting of LFA-1, CD154, and CD4 molecules can be effective in inducing long-term islet xenograft survival and function in autoimmune-prone NOD mice.     

Co-stimulatory molecules in islet xenotransplantation: CTLA4Ig treatment in CD40 ligand-deficient mice

Previous work has demonstrated that short-term systemic administration of cytotoxic T lymphocyte antigen-4 (CTLA-4) Ig blocks human pancreatic islet xenograft rejection in mice and induces long-term, donor-specific tolerance, whereas studies on pig pancreatic islet rejection in mice have failed to demonstrate a role for CTLA4Ig in preventing rejection. Treatment with anti-CD40 ligand (L) monoclonal antibodies alone is somewhat effective in prolonging the survival of islet xenografts, but ineffective when applied to skin xenografts. However, simultaneous blockade of the CD28 and CD40 co-stimulatory pathways prolongs the survival of pig skin on recipient mice. To evaluate the role of CD28 and CD40 co-stimulatory pathways in pig islet-like cell cluster (ICC) xenograft rejection in mice, CD40L-deficient mice transplanted with fetal porcine ICCs were given posttransplant treatment with human (h) CTLA4Ig or a human IgG1 chimeric mAb (hL6). Xenografts were evaluated 6 or 12 days after transplantation. Fetal porcine ICC xenografts were protected from rejection in hCTLA4Ig-treated CD40L-deficient mice, whereas xenograft rejection persisted in untreated CD40L-deficient mice. Simultaneous blockade of the CD28 and CD40 co-stimulatory pathways is mandatory to inhibit ICC xenograft rejection in the pig-to-mouse model, because the CD28 and CD40 co-stimulatory pathways seem capable of efficiently substituting for one another.     

Human leukocyte antigen-DR matching improved skin graft survival from transgenic pigs to accommodate SCID mice reconstituted with human peripheral blood mononuclear cells

The shortage of human organs has encouraged scientists to develop genetically modified pigs for xenotransplantation, such as CD55 or CD46, and CD59 transgenesis as well as alpha-galactosyl transferase gene knockouts. In allotransplantation, the match of human leukocyte antigen class II (HLA-II) may improve graft survival although the role of HLA-II in xenotransplantation is unknown. HLA-II transgenic pigs, including DP, DQ, and DR, have been successfully generated and HLA-DR15+ transgenic pig skin pieces grafted onto severe congenital immunodeficiency (SCID) mice reconstituted intraperitoneally with HLA-DR15+ or HLA-DR15(-) human peripheral blood mononuclear cells (hPBMCs). This study sought to develop an animal model to evaluate the effects of HLA-DR matching on xenograft survival. Human CD4+ and CD8+ were detected from days 7 to 29 after hPBMC reconstitution in SCID mice. Both CD4+ and CD8+ cells of HLA-DR15(-) reconstituted SCID mice were significantly higher at day 29 postgrafting compared with HLA-DR15+ reconstituted SCID mice. An HLA-DR15+ transgenic pig dermal graft survived and integrated into SCID mice reconstituted with hPBMCs/HLA-DR15+ as proven by the histopathological finding that the collagen layer remained intact with little lymphocytic response. In contrast, the transgenic pig dermal graft showed more collagen disruption as well as mild to moderate lymphocytic infiltration when reconstituted in an hPBMC/HLA-DR15(-) SCID mouse. The results suggested that HLA-DR matching eased xenograft rejection; however, it was not yet clear that the response was mediated by T cells.     

CD4+ T cell recognition of a single discordant HLA-A2-transgenic molecule through the indirect antigen presentation pathway induces acute rejection of murine cardiac allografts.

To further define the role of indirect allorecognition, cardiac allografts from HLA-A2-transgenic (HLA-A2+) C57BL/6 mice were heterotopically transplanted into normal C57BL/6, CD4 T cell-knockout (KO) C57BL/6 mice, CD8 T cell-KO C57BL/6 mice, fully MHC-discordant BALB/c mice (allogeneic control), and HLA-A2+ C57BL/6 mice (syngeneic control). HLA-A2+ grafts were acutely rejected when transplanted into BALB/c mice (mean survival time: 10+/-0.8 days), normal C57BL/6 mice (mean survival time: 16.5+/-2.1 days) as well as CD8-KO mice (mean survival time: 12.8+/-1.3 days). Histopathological analysis revealed classical acute cellular rejection with moderate to severe diffuse interstitial CD4+ and CD8+ cellular infiltrates and significant intra-graft deposition of IgG and complement. In contrast, HLA-A2+ grafts were not rejected when transplanted into CD4-KO mice or HLA-A2+ mice. CD8-KO recipients treated with an anti-CD4 monoclonal antibody, but not with an anti-NK monoclonal antibody, failed to reject their allografts with prolonged administration of antibody (30 days). Spleen cells from mice rejecting HLA-A2+ allografts failed to lyse HLA-A2+ target cells indicating a lack of involvement of CD8+ T cells in the rejection process. In contrast, spleen cells from rejecting animals proliferated significantly to both HLA-A2+ cells and to a peptide derived from the HLA-A2 molecule. Development of anti-HLA-A2 antibodies was observed in all animals rejecting HLA-A2+ allografts. These results suggest that indirect allorecognition of donor MHC class I molecules leads to rejection of cardiac allografts and development of alloantibodies in this unique transplant model in which there is a single MHC discordance between donor and recipient.     

Requirement of MyD88 for macrophage-mediated islet xenograft rejection after adoptive transfer

BACKGROUND: Porcine antigen primed and CD4+ T-cell activated macrophages are able to migrate to and destroy porcine xenografts. However, the specific signaling mechanisms involved remain to be identified. METHODS: In this study macrophages which lack the universal toll-like receptor (TLR) adaptor MyD88 were used to investigate the role of TLR in the recognition and activation of macrophages in islet xenograft rejection. Macrophages were isolated from rejecting MyD88(-/-) and wild-type C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts, and were transferred to NPCC recipient NOD-SCID mice. RESULTS: Both wild-type C57BL/6 and MyD88(-/-) mice rejected NPCC xenografts 8 and 10 days, respectively after transplantation, and the grafts were heavily infiltrated with CD4+ T cells and macrophages. However, graft infiltrating macrophages from rejecting MyD88(-/-) recipients demonstrated impaired up-regulation of TLR expression and impaired activation phenotype, when compared to those from rejecting C57BL/6 recipients. Transfer of NOD-SCID recipients with macrophages from rejecting C57BL/6 mice resulted in NPCC xenograft rejection along with massively infiltrated macrophages 8 days after transfer, whereas NPCC xenografts in NOD-SCID mice transferred with macrophages from rejecting MyD88(-/-) mice remained intact until the end of this study, 90 days after transfer, with insulin-positive islets and no infiltration by macrophages. CONCLUSION: This study demonstrates that deletion of MyD88 causes impaired macrophage activation after pig islet xenotransplantation. However, graft survival is not prolonged and xenografts are rejected rapidly by alternate mechanisms.     

The critical role of mouse CD4+ cells in the rejection of highly disparate xenogeneic pig thymus grafts

Long-term survival of fetal pig thymus (FP THY) grafts and efficient repopulation of mouse CD4+ T cells is achieved in thymectomized (ATX) B6 mice that receive T and NK cell depletion by injection of a cocktail of mAbs (GK1.5, 2.43, 30-H12, and PK136) and fetal pig thymus/liver (FP THY/LIV) grafts. The requirement for each mAb in this conditioning regimen in order to avoid the rejection of FP THY grafts has not yet been defined. In our present studies, CD4 cell-depleted ATX B6 mice and euthymic MHC class II-deficient (IIKO) mice were employed to investigate the role of mouse CD4+ cells in the rejection of FP THY grafts in vivo. After grafting FP THY/LIV to CD4+ cell-depleted ATX B6 mice, efficient repopulation of mouse CD4+ T cells was observed in the periphery. However, only two of four mice had remaining FP THY grafts by 17 weeks post-implantation, and these were of poor quality, whereas four of four T and NK cell-depleted ATX B6 mice had well-developed FP THY grafts. Furthermore, three of four FP THY/LIV-grafted, CD4+ cell-depleted ATX B6 mice rejected donor MHC-matched pig skin grafts. In contrast, three of three FP THY/LIV grafted, T and NK cell-depleted, ATX B6 mice accepted donor MHC-matched pig skin grafts, suggesting that optimal tolerance to xenogeneic pig antigens was not achieved in mice conditioned only with anti-CD4 mAb. ATX B6 mice treated with only anti-CD8 mAb rejected FP THY completely by 6 weeks post-grafting, a time when CD4+ cell-depleted ATX B6 mice had well-vascularized FP THY grafts. In addition, when euthymic IIKO mice were pre-treated with the standard conditioning regimen that includes four different mAbs, FP THY grafts survived and supported the repopulation of mouse CD4+ T cells in the periphery, while high levels of mouse CD8+ T cells developed in host thymi. These studies suggest that mouse CD4+ T cells play a critical role in the acute rejection of xenogeneic FP THY grafts. Without help from CD4+ cells, mouse CD8+ cells, NK, NK/T, and TCR(gamma/delta)+ T cells do not mediate acute rejection of FP THY grafts. Furthermore, our results suggest that other cell subsets besides CD4+ T cells play a role in the delayed rejection of highly disparate xenogeneic FP THY grafts.     

Non-depleting anti-CD4, but not anti-CD8, antibody induces long-term survival of xenogeneic and allogeneic hearts in alpha1,3-galactosyltransferase knockout (GT-Ko) mice

The anti-galactose-alpha1,3-galactose (Gal) antibody (Ab) response following pig-to-human transplantation is vigorous and largely resistant to currently available immunosuppression. The recent generation of GT-Ko mice provides a unique opportunity to study the immunological basis of xenograft-elicited anti-Gal Ab response in vivo, and to test the efficacy of various strategies at controlling this Ab response [1]. In this study, we compared the ability of non-depleting anti-CD4 and anti-CD8 to control rejection and antibody production in GT-Ko mice following xenograft and allograft transplantation. Hearts from baby Lewis rat or C3H mice were transplanted heterotopically into GT-Ko. Non-depleting anti-CD4 (YTS177) and anti-CD8 (YTS105) Abs were used at 1 mg/mouse, and given as four doses daily from day -2 to 1 then q.o.d. till day 21. Xenograft rejection occurred at 3 to 5 days post-transplantation in untreated GT-Ko recipients, and was histologically characterized as vascular rejection. Anti-CD4, but not anti-CD8, Ab treatment prolonged xenograft survival to 68 to 74 days and inhibited anti-Gal Ab as well as xeno-Ab production. In four of the five hearts from anti-CD4 mAbs-treated GT-Ko mice, we observed classic signs of chronic rejection, namely, thickened intima in the lumen of vessels, significant IgM deposition, fibrosis and modest mononuclear cell infiltrate of Mac-1+ macrophages and scattered T cells (CD8>CD4). Xenograft rejection in untreated, as well as anti-CD4- and anti-CD8-treated, recipients was associated with increased intragraft IL-6, IFN-gamma and IL-10 mRNA. C3H allografts were rejected in 7 to 9 days by untreated GT-Ko mice and were histologically characterized as cellular rejection. Treatment with anti-CD4 and anti-CD8 mAb resulted in graft survivals of >94.8 and 11.8 days, respectively. Anti-CD4 mAb treatment resulted in a transient inhibition of alloreactive and anti-Gal Ab production. The presence of circulating alloreactive and anti-Gal Abs at >50 days post-transplant was associated with significant IgM and IgG deposition in the graft. Yet, in the anti-CD4 mAb-treated group, the allografts showed no signs of rejection at the time of sacrifice (>100 days post-transplantation). All rejected allografts had elevated levels of intragraft IL-6, IFN-gamma and IL-10 mRNA, while the long-surviving anti-CD4-treated allografts had reduced mRNA levels of these cytokines. Collectively, our studies suggest that the elicited xeno-antibody production and anti-Gal Ab production in GT-Ko mice are CD4+ T-cell dependent. The majority of xenografts succumbed to chronic rejection, while allografts survived with minimal histological change, despite elevated levels of circulating alloAbs. Thus, immunosuppression with anti-CD4 mAb therapy induces long-term survival of allografts more effectively than to xenografts.