Impact of both donor and recipient strains on cardiac allograft survival after blockade of the CD40-CD154 costimulatory pathway

BACKGROUND: The effectiveness of anti-CD154 monoclonal antibodies in prolonging the survival of mouse allografts is dependent on the strain combination. In this report, we examined the impact of the donor and the recipient strains on the success of CD40-CD154 blockade. MATERIALS AND METHODS: Cardiac allograft survival was monitored in different donor/recipient strain combinations. Morphometric analyses on the allograft coronary arteries allowed quantification of vessel intimal thickening. RESULTS: Prolonged cardiac allograft survival after the administration of an anti-CD154 monoclonal antibody was found to be dependent on the donor and the recipient strains. The influence of the donor and the recipient strains lay in the ability of CD8 T cells to cause graft rejection despite CD40-CD154 blockade. Elimination of CD8 T cells before transplantation resulted in similar graft prolongation irrespective of the genotype of the donor or the recipient strain. CONCLUSION: These data show that both donor and recipient strains contribute to CD40-CD154-independent CD8 T-cell-mediated rejection.     

CD154 on the surface of CD4+CD25+ regulatory T cells contributes to skin transplant tolerance

BACKGROUND: It is known that the infusion of whole blood from donors (donor-specific transfusion) into recipients combined with anti-CD154 therapy can prolong allograft survival. It has generally been agreed that the effectiveness of anti-CD154 therapy is caused by the inactivation of alloreactive CD4+ and CD8+ effector T cells. The recent literature has implicated CD4+CD25+ regulatory T cells in the suppression of autoimmunity and graft rejection, and we therefore examined whether CD154 blockade is effective because of its blockade of inflammatory T-cell activation or because of a direct impact on the regulatory T cells. METHODS: RAG(-/-) mice were adoptively transfused with CD4+ T cells or a subset of the population (CD4+CD25+ or CD4+CD25- T cells) alone or in combination with donor-specific transfusion and anti-CD154 and given an allo-skin transplant. The longevity of the transplant was determined over time. CD154(-/-)CD4+ T cells were used to assess the importance of CD154 in graft rejection and acceptance. RESULTS: CD154 blockade (or loss of CD154) on CD4+CD25+ regulatory T cells enhanced their immunosuppressive activities and was a contributing factor to anti-CD154-induced immune suppression in vivo. In a model of allograft tolerance, suppression was elicited by antigen and anti-CD154 or antigen alone if the CD4+CD25+ regulatory T cells were deficient in CD154 expression. CONCLUSIONS: Neutralizing the function of CD154 on regulatory T cells upon antigen exposure induces heightened levels of suppressive activities and is likely a contributing factor to the long-lived therapeutic effects of anti-CD154 treatment.     

A novel CD154 monoclonal antibody in acute and chronic rat vascularized cardiac allograft rejection

BACKGROUND: The CD40-CD154 interaction is critically important in the cell-mediated immune responses. Blockade of this costimulatory pathway has been shown to prevent acute allograft rejection in murine, as well as nonhuman primate models. However, the role of the CD40-CD154 pathway in the development of chronic rejection and the effects of CD154 targeting on progression of chronic rejection have not been evaluated. METHODS: We examined the effect of AH.F5, a new hamster anti-rat CD154 monoclonal antibody, in a fully allogeneic acute(u) into Lewis [LEW] (RT11) and chronic [WF.1L (RT1l) into LEW (RT1l)] vascularized cardiac allograft rejection model. In the chronic model, the antibody was evaluated for prevention (starting day of transplant) and interruption of progression (starting day 30 or 60 after transplant) of chronic vasculopathy. Graft survival, morphology, and immunohistology were evaluated. RESULTS: In the acute rejection model, anti-CD154 therapy alone prevented acute allograft rejection and resulted in 50% long-term allograft survival (>200 days) and donor-specific tolerance. In recipients treated with anti-CD154 monoclonal antibody in combination with a short course of cyclosporine, 100% of allografts survived long-term and all recipients achieved donor-specific tolerance. In the chronic rejection model, allografts from animals treated with the anti-CD154 antibody had a statistically significant lower score of graft arteriosclerosis and fibrosis in both the prevention and 30-day interruption groups when compared with control allografts. In addition, immunohistochemistry showed a decrease in intragraft mononuclear cell infiltration and activation. CONCLUSION: A new anti-CD154 antibody not only prevents acute allograft rejection, but also inhibits and interrupts the development of chronic rejection. In the acute rejection model cyclosporine acts synergistically with anti-CD154 therapy to prolong allograft survival and induce tolerance. In the chronic rejection model relatively early initiation of therapy is essential to prevent progression of chronic allograft vasculopathy and fibrosis.     

Stimulating PD-1-negative signals concurrent with blocking CD154 co-stimulation induces long-term islet allograft survival

BACKGROUND: A balanced network of positive and negative T-cell co-stimulatory signals is important in regulating T-cell activation. Blocking CD28, CD154 (CD40L), or both co-stimulatory molecules has been efficacious in preventing acute allograft rejection in certain but not all transplantation models. In the present study, the authors tested the hypothesis that stimulating programmed death 1 (PD-1)-triggered negative signals concurrent with blocking CD154 co-stimulatory signals would facilitate islet allograft tolerance. METHODS: The authors used a dimeric PD-L1 immunoglobulin (Ig) fusion protein to stimulate the inhibitory receptor PD-1, and a monoclonal antibody to block CD154. The effects of PD-1 engagement and CD154 blockade on lymphocyte activation were determined by cell proliferation, flow cytometry, and a model of islet transplantation. RESULTS: PD-L1Ig inhibited the proliferation of both CD4+ and CD8+ T cells stimulated by anti-CD3. The inhibitory effect of PD-L1Ig was enhanced by concurrent blockade of CD154 co-stimulatory signals, as demonstrated by T-cell proliferation and expression of cell surface activation markers. PD-L1Ig and anti-CD154 also synergistically blocked the activation and maturation of antigen-presenting cells. In an islet transplantation model, treatment of recipient C57BL/6 (H-2b) mice with PD-L1Ig and anti-CD154 induced long-term survival of DBA/2 (H-2d) islet allografts, whereas treatment with each reagent alone failed to prevent islet allograft rejection. CONCLUSIONS: These results suggest that engaging the negative receptor PD-1 exhibits critical immunoregulatory effects in the allograft response, and blocking positive co-stimulatory molecules with active delivery of inhibitory signals may represent a novel therapeutic strategy in transplantation.     

Regulation of skin and islet allograft survival in mice treated with costimulation blockade is mediated by different CD4+ cell subsets and different mechanisms

BACKGROUND: Donor-specific transfusion (DST) and a brief course of anti-CD154 monoclonal antibody (mAb) induces permanent islet and prolonged skin allograft survival in mice. Induction of skin allograft survival requires the presence of CD4 cells and deletion of alloreactive CD8 cells. The specific roles of CD4 and CD4CD25 cells and the mechanism(s) by which they act are not fully understood. METHODS: We used skin and islet allografts, a CD8 T cell receptor (TCR) transgenic model system, and in vivo depleting antibodies to analyze the role of CD4 cell subsets in regulating allograft survival in mice treated with DST and anti-CD154 mAb. RESULTS: Deletion of CD4 or CD25 cells during costimulation blockade induced rapid rejection of skin but only minimally shortened islet allograft survival. Deletion of CD4 or CD25 cells had no effect upon survival of healed-in islet allografts, and CD25 cell deletion had no effect upon healed-in skin allograft survival. In the TCR transgenic model, DST plus anti-CD154 mAb treatment deleted alloreactive CD8 T cells, and anti-CD4 mAb treatment prevented that deletion. In contrast, injection of anti-CD25 mAb did not prevent alloreactive CD8 T cell deletion. CONCLUSIONS: These data document that (1) both CD4CD25 and CD4CD25 cells are required for induction of skin allograft survival, (2) CD4CD25 T cells are not required for alloreactive CD8 T cell deletion, and (3) CD4CD25 regulatory cells are not critical for islet allograft tolerance. It appears that skin and islet transplantation tolerance are mediated by different CD4 cell subsets and different mechanisms.     

New insights into the interactions between T-cell costimulatory blockade and conventional immunosuppressive drugs

OBJECTIVE: To determine the precise in vivo interaction between T-cell costimulatory blockade and conventional immunosuppression in transplantation. SUMMARY BACKGROUND DATA: Blocking B7 or CD154 T-cell costimulatory activation pathways prevents allograft rejection in small and large animal transplant models and is considered a promising strategy for clinical organ transplantation. METHODS: A fully MHC-mismatched vascularized mouse cardiac allograft model was used to test the interactions between anti-CD154 or CTLA4Ig monotherapy and conventional immunosuppressive drugs in promoting long-term graft acceptance. The frequency of alloreactive T cell was measured by ELISPOT. Chronic rejection was examined by histology. RESULTS: Cyclosporine, tacrolimus, and anti-IL-2R monoclonal antibody therapy abrogated the effect of a single-dose protocol of anti-CD154 therapy. In contrast, rapamycin acted synergistically with anti-CD154 therapy in promoting long-term allograft survival. The addition of calcineurin inhibitors did not abolish this synergistic effect. Intense CD154-CD40 blockade by a multiple-dose schedule of anti-CD154 resulted in long-term graft survival and profound alloreactive T-cell unresponsiveness and overcame the opposite effects of calcineurin inhibitors. CTLA4Ig induced long-term graft survival, and the effect was not affected by the concomitant use of any immunosuppressive drugs. CONCLUSIONS: The widespread view that calcineurin inhibitors abrogate the effects of T-cell costimulatory blockade should be revisited. Sufficient costimulatory blockade and synergy induced by CD154 blockade and rapamycin promote allograft tolerance and prevent chronic rejection.     

Neutralizing IL-7 Promotes Long-Term Allograft Survival Induced by CD40/CD40L Costimulatory Blockade

Memory T cells are somewhat resistant to immunosuppresion. They therefore pose a threat to inducing long‐term allograft survival. IL‐7 is essential for memory T‐cell generation. Here, we investigated whether neutralizing IL‐7 promotes allograft survival. We found that neutralizing IL‐7 alone did not significantly prolong allograft survival. However, blocking both IL‐7 and CD154 signaling synergistically prolonged allograft survival. In contrast, neutralizing IL‐2 failed to further prolong allograft survival induced by CD40/CD154 costimulatory blockade. Allospecific memory CD8+ T‐cell generation was severely impaired under the treatment of anti‐IL‐7 plus anti‐CD154 Ab while administering recombinant IL‐7 enhanced CD8+ memory generation even under donor‐specific transfusion plus anti‐CD154 Ab treatment. Neutralizing IL‐7, but not IL‐2, together with blocking CD154 synergistically suppressed the proliferation of naïve/effector CD8+ T cells infiltrating grafts. Nevertheless, neutralizing IL‐7 did not alter regulatory T‐cell generation while neutralizing IL‐2 suppressed their generation. Hence, targeting IL‐7 represents a new strategy to prolong allograft survival by acting on both naïve and memory T cells. Long‐term allograft survival may be achieved by neutralizing IL‐7 plus CD40/CD154 blockade, since CD40/CD154 costimulatory blockade prevents acute rejection while neutralizing IL‐7 suppresses the generation of memory T cells that persist and mediate late or chronic rejection.     

CD8 T cell-mediated rejection of intestinal allografts is resistant to inhibition of the CD40/CD154 costimulatory pathway

BACKGROUND: Disruption of the CD40/CD154 pathway inhibits rejection in numerous models. The importance of this pathway on intestinal allograft rejection was examined in this study. METHODS: Intestinal grafts from B6C3F1 mice transplanted into C57BL/6 recipients were assessed histologically for rejection. RESULTS: The monoclonal antibody to CD154, MR1, failed to inhibit rejection in wild-type mice. Similarly, CD154-/- recipient mice rejected intestinal allografts. MR1 did inhibit early rejection in CD8-/- mice, but had no effect in CD4-/- recipients. All MR1-treated CD8-/- recipients eventually developed rejection. No benefit was observed when blockade of the CD40/CD154 pathway by MR1 was combined with blockade of the CD28/B7 pathway by mCTLA4Ig. CONCLUSIONS: These data suggest that CD4+ T cells mediating intestinal allograft rejection may be more dependent upon the CD40/CD154 pathway than CD8+ T cells. This finding highlights the importance of identifying agents that suppress CD8+ T cell-mediated rejection.     

Viral infection abrogates CD8(+) T-cell deletion induced by costimulation blockade

BACKGROUND: Treatment with a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (mAb) prolongs skin allograft survival in mice. It is known that prolongation of allograft survival by this method depends in part on deletion of alloreactive CD8(+) T cells at the time of tolerance induction. Recent data suggest that infection with lymphocytic choriomeningitis virus (LCMV) abrogates the ability of this protocol to prolong graft survival. METHODS: To study the mechanism by which viral infection abrogates allograft survival, we determined (1) the fate of tracer populations of alloreactive transgenic CD8(+) T cells and (2) the duration of skin allograft survival following treatment with DST and anti-CD154 mAb in the presence or absence of LCMV infection. RESULTS: We confirmed that treatment of uninfected mice with DST and anti-CD154 mAb leads to the deletion of alloreactive CD8(+) T cells and is associated with prolongation of skin allograft survival. In contrast, treatment with DST and anti-CD154 mAb in the presence of intercurrent LCMV infection was associated with the failure to delete alloreactive CD8(+) T cells and with the rapid rejection of skin allografts. The number of alloreactive CD8(+) cells actually increased significantly, and the cells acquired an activated phenotype. CONCLUSIONS: Interference with the deletion of alloreactive CD8(+) T cells mediated by DST and anti-CD154 mAb may in part be the mechanism by which viral infection abrogates transplantation tolerance induction.     

Combined treatment with CD40 costimulation blockade, T-cell depletion, low-dose irradiation, and donor bone marrow transfusion in limb allograft survival

To determine the efficacy of a regimen based on CD40 costimulation blockade and donor bone marrow in the limb allograft model, C57Bl/6 mice received limb allografts from Balb/c mice and either no treatment or a combination of MR1 (anti-CD40 ligand monoclonal antibody), CD4+ and CD8+ T-cell-depleting antibodies, low-dose irradiation, and bone marrow transfusion from Balb/c donors for 1 or 2 weeks. Recipients treated for 1 week showed rejection at 38.2 +/- 5.4 (mean +/- SEM) days, while those treated for 2 weeks had allograft survival of 56.5 +/- 9.9, with a range up to 91 days. Histology demonstrated rejection which was less cell-mediated and suggestive of transplant vasculopathy. Differential rejection of skin occurred first. Thus, a combined regimen based on CD40 costimulatory blockade and donor marrow significantly prolonged allograft survival. However, tolerance was not achieved, and histology suggests chronic rejection as a possible cause of allograft loss.     

Development of a combined cardiac and aortic transplant model to investigate the development of transplant arteriosclerosis in the mouse.

BACKGROUND: The degree of transplant arteriosclerosis in murine cardiac allografts is difficult to assess. Aortic allografts represent an alternative model for evaluating the impact of novel transplant strategies on transplant arteriosclerosis in which the vascular changes can be quantified easily. However, it remains controversial as to whether vascular lesions seen in this model are equivalent to those that develop in solid-organ transplants. The aim of this study was to develop a model of combined cardiac and aortic transplantation to allow more precise quantification of transplant arteriosclerosis and to establish a correlation between the lesions that develop in the 2 types of graft. METHODS: CBA (H2(k)) recipients received a C57BL/10 (H2(b)) cervical cardiac allograft on Day 0 and a C57BL/10 (H2(b)) abdominal aortic allograft on Day 1. Recipients were treated with anti-CD154 mAb (MR1) on Days 0, 2, and 4. We performed histology and morphometric measurements for both grafts 30 days after transplantation. RESULTS: We observed significant intimal proliferation in both the cervical cardiac and abdominal aortic allografts from recipients treated with anti-CD154 mAb (heart, 64% +/- 9%; aorta, 67% +/- 8%; n = 5). Abdominal aortic grafts transplanted alone into anti-CD154-treated recipients developed a degree of transplant arteriosclerosis equivalent to that seen in the aortic grafts of the combined group (aorta alone, 68% +/- 9%, vs aorta + heart, 67% +/- 8%; n = 5). CONCLUSIONS: This combined cardiac and aortic transplant model permitted quantitative assessment of transplant arteriosclerosis while monitoring graft survival by cardiac palpation. Furthermore, development of transplant arteriosclerosis was equivalent in abdominal aortic allografts either in the presence or absence of an additional solid- organ transplant.     

CD45RB-targeting strategies for promoting long-term allograft survival and preventingchronic allograft vasculopathy

BACKGROUND: CD45RB is a potent immunomodulatory target to achieve long-term allograft survival. We evaluated the in vivo effect of anti-CD45RB monoclonal antibody (mAb) treatment in combination with conventional immunosuppression or costimulatory blockade strategies as a therapeutic modality for future clinical application. METHODS: A fully MHC-mismatched vascularized mouse cardiac allograft model was used to test the interactions between anti-CD45RB mAb and conventional immunosuppressive drugs or costimulatory blockade of the CD40/CD154 or B7/CD28 pathway. Chronic rejection was examined histologically for development of chronic allograft vasculopathy. RESULTS: Cyclosporine significantly abrogated the effect of anti-CD45RB therapy. In contrast, rapamycin acted synergistically with anti-CD45RB mAb in promoting long-term allograft survival. CD154 blockade further enhanced the tolerogenic efficacy of anti-CD45RB mAb. These synergistic effects of combination treatments also prevented the development of chronic allograft vasculopathy. CONCLUSION: CD45RB-targeting strategy in combination with the use of rapamycin or costimulatory blockade promotes allograft tolerance and prevents chronic rejection.     

Critical Role for CD8+ T Cells in Allograft Acceptance Induced by DST and CD40/CD154 Costimulatory Blockade

Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8(+) T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4(+) and CD8(+) T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.     

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.     

Increased expression of transforming growth factor-beta and eosinophil infiltration is associated with the development of transplant arteriosclerosis in long-term surviving cardiac allografts

BACKGROUND: Transplant arteriosclerosis is a major limiting factor for long-term function of allografts in clinical transplantation. This study investigated the impact of three different protocols capable of inducing long-term allograft survival on the development of transplant arteriosclerosis and immune response in cardiac allografts. METHODS: CBA.Ca (H2k) recipients of fully allogeneic C57/BL10 (H2b) heart grafts received a short-term course of anti-CD154 antibody or were pretreated with anti-CD4 antibody in combination with donor alloantigen in the form of CBK (H2k+Kb) bone marrow or C57BL/10 donor-specific transfusion (DST). Grafts were analyzed on day 40 or 100 after transplantation for transplant arteriosclerosis and expression of interferon-gamma, interleukin (IL)-2, IL-4, IL-10, IL-12p40, inducible nitric oxide synthase, and transforming growth factor (TGF)-beta1 mRNA. Serum was analyzed for the presence of alloantibodies. RESULTS: Intimal proliferation was 62%+/-11% on day 40 in the anti-CD154 group, progressed from 31%+/-10% on day 40 to 68%+/-8% on day 100 in the CBK-bone marrow group, but remained stable at 39%+/-4% in the DST group. Increased transplant arteriosclerosis on day 100 was associated with high intragraft TGF-beta1 mRNA production and eosinophil infiltration, but not alloantibody production. Progressing transplant arteriosclerosis was associated with increased IL-4 expression. CONCLUSION: Treatment protocols for the induction of long-term allograft survival can differ substantially in the extent and kinetics of transplant arteriosclerosis. IL-4 and TGF-beta1 may be two potential therapeutic targets to attenuate the development of transplant arteriosclerosis in the long term.     

Aggressive skin allograft rejection in CD28-/- mice independent of the CD40/CD40L costimulatory pathway.

CD28-/- mice have been utilized to study the role of B7/CD28 and B7-CTLA4 interactions. There is evidence that CTLA4 ligation may be critical for tolerance induction. The aim of the current study is to further investigate rejection responses of CD28-/- mice and to define the role of B7-CTLA4 interactions in the absence of the CD40 and CD28 pathways. Balb/c skin allografts were transplanted onto C57BL/6 (B6) wild type or CD28-/- mice treated with anti-CD40L, CTLA4-Ig, or combination blockade. To investigate the cellular mechanism of rejection in CD28-/- recipients, mice were treated with anti-CD4 or anti-CD8 antibodies prior to treatment with costimulation blockade. The fluoroscein dye CFSE was utilized to study T cell expansion in vivo. Surprisingly, treatment of B6 CD28-/- mice with CTLA4-Ig alone (MST 12d), anti-CD40L alone (MST 13d), or combined blockade (MST 13d) had no effect on allograft survival compared to untreated B6 CD28 mice (MST 11d). CD28-/- recipients depleted of CD4+ cells and treated with CTLA4-Ig, anti-CD40L, or combination blockade also did not have prolonged survival compared with untreated mice (MST 10d). In contrast, CD28-/- recipients depleted of CD8+ cells had markedly prolonged allograft survival when treated with either anti-CD40L alone (MST 49d) or with combination blockade (MST 57d). Studies utilizing CFSE demonstrated that CD28-/- CD8+ T cells are not defective in in vivo proliferation responses compared with wild type CD8 cells. Thus, CD28-/- CD8+ T cells are responsible for aggressive rejection responses of CD28-/- mice independent of the CD40 pathway. In addition, CD40L blockade does not result in CD4+ T cell tolerance in CD28 recipients, despite an intact B7-CTLA4 pathway.     

Prolonged allograft survival in anti-CD4 antibody transgenic mice: lack of residual helper T cells compared with other CD4-deficient mice

BACKGROUND: Investigations of the role of CD4 T lymphocytes in allograft rejection and tolerance have relied on the use of mouse models with a deficiency in CD4 cells. However, in mice treated with depleting monoclonal antibody (mAb) and in MHC class II knockout (KO) mice, there are residual populations of CD4 cells. CD4 KO mice had increased CD4- CD8-TCRalphabeta+ helper T cells, and both strains of KO mice could reject skin allografts at the normal rate. In this study, transgenic mice with no peripheral CD4 cells were the recipients of skin and heart allografts. Results were compared with allograft survival in CD4 and MHC class II KO mice. METHODS: GK5 (C57BL/6 bml mice transgenic for a chimeric anti-CD4 antibody) had no peripheral CD4 cells. These mice, and CD4 and class II KO mice, received BALB/c or CBA skin or cardiac allografts. Some GK5 mice were treated with anti-CD8 mAb to investigate the role of CD8 cells in rejection. CD4 and CD8 cells were assessed by FACS and immunohistochemistry. RESULTS: BALB/c skin on GK5 mice had a mean survival time +/- SD of 24+/-6 days, compared with 9+/-2 days in wild-type mice. Anti-CD8 mAb prolonged this to 66+/-7 days. BALB/c skin survived 10+/-2 days on class II KO and 14+/-2 days on CD4 KO, both significantly less than the survival seen on GK5 recipients (P<0.001). BALB/c hearts survived >100 days in GK5 recipients and in wild-type recipients treated with anti-CD4 mAb at the time of grafting, in contrast to a mean survival time of 10+/-2 days in untreated wild-type mice. Immunohistochemistry revealed that long-term surviving heart allografts from the GK5 recipients had CD8 but no CD4 cellular infiltrate. These hearts showed evidence of transplant vasculopathy. CONCLUSIONS: The GK5 mice, with a complete absence of peripheral CD4 cells, provide the cleanest available model for investigating the role of CD4 lymphocytes in allograft rejection. Prolonged skin allograft survival in these mice compared with CD4 and MHC class II KO recipients was clearly the result of improved CD4 depletion. Nevertheless, skin allograft rejection, heart allograft infiltration, and vascular disease, mediated by CD8 cells, developed in the absence of peripheral CD4 T cells.     

ICOS-Dependent and -Independent Functions of Memory CD4 T Cells in Allograft Rejection

Donor-reactive memory T cells undermine the survival of transplanted organs through multiple pathways. We have previously reported that memory CD4 T cells resist treatment with anti-CD154 antibody and donor-specific transfusion (DST/MR1) and promote cardiac allograft rejection via generation of effector CD4 T cells and alloantibody. We hypothesized that the helper functions of memory CD4 T cells are independent of T-cell costimulation through CD154 but instead are regulated by alternative costimulatory pathways. This study investigated how blocking ICOS/B7RP-1 interactions affects functions of donor-reactive memory CD4 T cells. Treatment with blocking anti-ICOS mAb synergized with DST/MR1 and prolonged mouse cardiac allograft survival despite the presence of donor-reactive memory CD4 T cells. While blocking ICOS did not diminish the expansion of preexisting memory CD4 T cells or the induction of allospecific effector T cells, it did inhibit recruitment of the activated memory and effector T cells into the graft. In addition, anti-ICOS mAb treatment in combination with DST/MR1 prevented help provided by memory CD4 T cells for production of donor-specific IgG antibody. These results demonstrate the potential efficacy of ICOS blockade in sensitized transplant patients and provide the foundation for rational use of ICOS blockade in combination with other graft-prolonging strategies.