Both innate and adaptive major histocompatibility complex class I-dependent immunity impair long-term islet xenograft survival

Natural killer (NK) cells have long been appreciated for their rapid, proinflammatory contribution to host defense. However, more recent studies show an unexpected regulatory role for host major histocompatibility complex (MHC) class I-dependent immunity and NK cells in promoting tolerance induction to islet allografts. It is unclear whether the potential tolerance induction to islet xenografts follows similar requirements to those found in allograft tolerance. In this study, we determined whether induced islet xenograft prolongation also showed a reliance on MHC class I-dependent immune pathways. In particular, we tested whether NK1.1+ cells and/or CD8 T cells were required for the long-term islet xenograft survival in a rat-to-mouse transplant model. Short-term host treatment with combined anti-CD154 plus anti-LFA-1 (CD11a) resulted in prolonged, but not indefinite, survival of WF rat islet xenografts in C57BI/6 mouse recipients. In stark contrast with similar islet allograft studies, adjunct treatment with anti-NK1.1 therapy combined wither anti-CD154/anti-LFA-1 treatment led to long-term (>100 days) survival of the majority of islet xenografts. In parallel studies, we determined whether CD8 T cells also contributed a barrier to xenograft survival. Similar to results found in anti-NK1.1-treated animals, CD8-deficient (knockout) recipients also demonstrated augmented xenograft prolongation after combined anti-CD154/anti-LFA-1 therapy. Taken together, NK1.1+ cells (NK/NKT cells) and CD8 T cells constitute differing MHC class I-dependent immune pathways forming a significant barrier to xenograft prolongation.     

Interferon-gamma is not a universal requirement for islet allograft survival

BACKGROUND: Although many transplantation studies have implicated a graft-destructive role for T helper (Th)1 cytokines and a graft-protective role for Th2 cytokines, more recent studies have challenged this paradigm by showing that long-term allograft survival can actually require the presence of Th1 cytokines, such as interleukin 2 and interferon (IFN)-gamma. The purpose of this study was to examine the requirement for IFN-gamma in the induction of islet allograft acceptance after monoclonal antibody therapy targeting conceptually distinct molecular pathways: the costimulatory molecule CD154, the CD4 coreceptor, or the beta2 integrin lymphocyte function-associated antigen (LFA)-1 (CD11a). METHODS: Diabetic C57Bl/6 (B6; H2b) mice were grafted with fully MHC mismatched BALB/c (H2d) islets, or reciprocally, diabetic BALB/c mice underwent transplantation with B6 islets and were treated with anti-CD154, anti-CD4, or anti-LFA-1. RESULTS: When IFN-gamma gene knockout mice were used as graft recipients, the requirement for IFN-gamma in allograft survival was found to be highly conditional, depending on both the host strain and the induction therapy used. In both strain combinations studied, anti-CD154 was effective in the presence or absence of IFN-gamma, whereas anti-CD4 lost therapeutic potential in the absence of this cytokine. Alternatively, the requirement for IFN-gamma for allograft prolongation by anti-LFA-1 therapy was noted only in B6 transplant recipients. CONCLUSIONS: IFN-gamma is not always requisite in islet allograft survival but rather varies according to the molecular target of induction therapy and the genetic background of the transplant recipient.     

The complement dependent cytotoxicity (CDC) immune effector mechanism contributes to anti-CD154 induced immunosuppression

BACKGROUND: In many situations, anti-CD154 (CD40 ligand) monoclonal antibody (mAb) treatment is very potent in producing allograft tolerance. In accordance to our previously reported results, combined donor specific transfusion (DST)3 plus anti-CD154 mAb (MR1) treatment enables the permanent engraftment of DBA/2 (H-2(d)) islets into B6AF1 (H-2(b/kd)) recipients in all cases. It has been widely assumed that the MR1 anti-154 is a noncytolytic neutralizing mAb, and it exerts immune suppressive effects by blockade of CD40/CD154 signal pathway. In this study, we sought to test the role of complement dependent cytotoxicity (CDC) immune effector mechanism in MR1 anti-CD154 induced immunosuppression. METHODS: We have evaluated the contributions of CDC in the context of the potent tolerizing effects of DST plus anti-CD154 mAb treatment regiment in recipients of islet allografts. We have used CD40 knockout (KO) mice and complement C5 deficient mice DBA/2 as islet allograft recipients as well as cobra venom factor (CVF), a complement blocker, treatment. RESULTS: The absence of direct and indirect CD40/CD154 pathway signals does not prevent islet allograft acute rejection. Interestingly, MR1 anti-CD154 induces islet allograft tolerance in the absence of CD40/CD154 pathway. In a wild-type major histocompatibility complex (MHC) mismatched strain combination, DST results in accelerated islet allograft rejection. Combination of DST and MR1 anti-CD154 treatment prevents presensitization and permits permanent engraftment. However, administration of CVF abolishes the tolerance induction. Moreover, DST plus MR1 anti-CD154 regiment, a potent tolerizing therapy, does not prevent acute islet allograft rejection when complement C5 deficient DBA/2 mice are used as recipients. Thus, the mechanisms of the tolerizing effects by MR1 anti-CD154 are not limited to blockade of CD40/CD154 signals. The CDC immune effector mechanism contributes to MR1 anti-CD154 induced immunosuppression.     

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.     

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.     

Indefinite survival of neonatal porcine islet xenografts by simultaneous targeting of LFA-1 and CD154 or CD45RB

A variety of transient therapies directed against molecules involved in T-cell activation and function result in long-term islet allograft survival. However, there are relatively few examples of durable islet xenograft survival using similar short-term approaches, especially regarding highly phylogenetically disparate xenograft donors. Previous studies demonstrate that combined anti-lymphocyte function-associated antigen-1 (LFA-1) plus anti-CD154 therapy results in a robust form of islet allograft tolerance not observed with either individual monotherapy. Thus, the aim of this study was to determine whether the perturbation of anti-LFA-1, either alone or in combination with targeting CD154 or CD45RB, would promote neonatal porcine islet (NPI) xenograft survival in mice. NPI xenografts are rapidly rejected in wild-type C57BL/6 mice but reproducibly mature and restore durable euglycemia in diabetic, immune-deficient C57BL/6 rag-1(-/-) recipients. A short course of individual anti-LFA-1, anti-CD154, or anti-CD45RB therapy resulted in long-term (>100 days) survival in a moderate proportion of C57BL/6 recipients. However, simultaneous treatment with anti-LFA-1 plus either anti-CD154 or anti-CD45RB therapy could achieve indefinite xenograft function in the majority of recipient animals. Importantly, prolongation of islet xenograft survival using combined anti-LFA-1/anti-CD154 therapy was associated with little mononuclear cell infiltration and greatly reduced anti-porcine antibody levels. Taken together, results indicate that therapies simultaneously targeting differing pathways impacting T-cell function can show marked efficacy for inducing long-term xenograft survival and produce a prolonged state of host hyporeactivity in vivo.     

Rat xenograft survival in mice treated with donor-specific transfusion and anti-CD154 antibody is enhanced by elimination of host CD4+ cells

BACKGROUND: Treatment with a donor-specific transfusion (DST) and a brief course of anti-mouse CD154 (anti-CD40-ligand) monoclonal antibody (mAb) prolongs the survival of both allografts and rat xenografts in mice. The mechanism by which allograft survival is prolonged is incompletely understood, but depends in part on the presence of CD4+ cells and the deletion of alloreactive CD8+ T cells. Less is known about the mechanism by which this protocol prolongs xenograft survival. METHODS: We measured rat islet and skin xenograft survival in euthymic and thymectomized mice treated with combinations of DST, anti-CD154 mAb, anti-CD4 mAb, and anti-CD8 mAb. Recipients included C57BL/6, C57BL/6-scid, C57BL/6-CD4null, and C57BL/6-CD8null mice. RESULTS: Pretreatment with a depleting anti-CD4 mAb markedly prolonged the survival of both skin and islet xenografts in mice given DST plus anti-CD154 mAb. Comparable prolongation of xenograft survival was obtained in C57BL/6-CD4null recipients treated with DST and anti-CD154 mAb. In contrast, anti-CD8 mAb did not prolong the survival of either islet or skin xenografts in mice treated with DST and anti-CD154 mAb. Thymectomy did not influence xenograft survival in any treatment group. Adoptive transfer of splenocytes from C57BL/6-CD4null recipients treated with DST and anti-CD154 mAb and bearing long-term skin xenografts revealed the presence of residual xenoreactive cells. CONCLUSIONS: These data suggest that treatment with DST and anti-CD154 mAb induces a state of "functional" transplantation tolerance. They also support the hypothesis that both the induction and maintenance of graft survival based on this protocol depend on different cellular mechanisms in allogeneic and xenogeneic model systems.     

The degree of phylogenetic disparity of islet grafts dictates the reliance on indirect CD4 T-cell antigen recognition for rejection

Cellular xenograft rejection involves a pronounced contribution of CD4 T-cells recognizing antigens in association with recipient MHC class II molecules. However, the requirement for such "indirect" antigen recognition for acute islet xenograft is not clear, especially as a function of the phylogenetic disparity between the donor and recipient species. In vitro studies show that C57BL/6 (B6) mouse T-cells respond directly to either allogeneic BALB/c or phylogenetically related xenogeneic WF rat stimulator cells while having undetectable responses to phylogenetically disparate porcine stimulator cells. Although all types of grafts rejected acutely in wild-type mice, this response demonstrated markedly differing dependence on host MHC class II antigen presentation, depending on the donor species. While BALB/c islet allografts were acutely rejected in B6 MHC class II-deficient (C2D) recipients, WF rat xenografts demonstrated marked prolongation in C2D hosts relative to wild-type recipients. Interestingly, neonatal porcine islet (NPI) xenografts uniformly survived long term (>100 days) in untreated C2D hosts despite transfer of wild-type CD4 T-cells, demonstrating that survival in C2D recipients was not secondary to a lack of CD4 T-cells seen in such mice. Taken together, these results show a marked hierarchy in the requirement for host MHC class II-restricted indirect pathway in the rejection of pancreatic islet grafts. Thus, while cellular rejection of porcine xenografts is generally quite vigorous, this pathway is relatively finite, displaying a major reliance on host MHC class II-dependent antigen presentation for acute rejection.     

Patterns of immune responses evoked by allogeneic hepatocytes: evidence for independent co-dominant roles for CD4+ and CD8+ T-cell responses in acute rejection.

INTRODUCTION: This is the first in a series of reports that characterizes immune responses evoked by allogeneic hepatocytes using a functional model of hepatocyte transplantation in mice. METHODS: "Donor" hepatocytes expressing the transgene human alpha-1-antitrypsin (hA1AT-FVB/N, H2q) were transplanted into C57BL/6 (H2b) or MHC II knockout (H2b) hosts treated with anti-CD4, anti-CD8, or a combination of anti-CD4 and anti-CD8 monoclonal antibodies (mAbs). Hepatocyte rejection was determined as a loss of circulating ELISA-detectable transgene product (hA1AT). In addition, some C57BL/6 mice underwent transplantation with FVB/N heterotopic cardiac allografts and were treated with anti-CD4 mAb. Cardiac allograft rejection was determined by palpation. Graft recipients were tested for donor-reactive alloantibodies and donor-reactive delayed-type hypersensitivity (DTH) responses. RESULTS: The median survival time (MST) of allogeneic hepatocytes in normal C57BL/6 mice was 10 days (no treatment), 10 days (anti-CD4 mAb), 14 days (anti-CD8 mAb), and 35 days (anti-CD4 and anti-CD8 mAbs). The MST of hepatocytes in B6 MHC class II knockout mice was 10 days (no treatment) and 21 days (anti-CD8 mAb). The MST of cardiac allografts was 11 days (no treatment) and >100 days (anti-CD4 mAb). Donor-reactive DTH responses were readily detected in both untreated and mAb-treated recipients. Donor-reactive alloantibody was barely detectable in untreated hosts. CONCLUSIONS: These studies demonstrate that allogeneic hepatocytes are highly immunogenic and stimulate strong cell-mediated immune responses by both CD4+ and CD8+ T cells, even when treated with agents that can cause acceptance of cardiac allografts. Indeed, CD4+ or CD8+ T cells seem to independently cause hepatocellular allograft rejection. Allogeneic hepatocytes evoked strong donor-reactive DTH responses but were poor stimuli for donor-reactive antibody production. This is an unusual pattern of immune reactivity in allograft recipients.     

Role of CD40-CD154 pathway in the rejection of concordant and discordant xenogeneic islets

BACKGROUND: Costimulatory blockade has been shown to allow long-term survival of xenogeneic islets. The aim of the present study was to evaluate the role of recipient CD40 and CD154 in the rejection process of concordant and discordant islet xenotransplantation (Tx). METHODS: Diabetic C57BL/6 mice, CD40- or CD154 knockout (KO) mice were transplanted with either concordant rat or discordant human islets. Experimental design: group 1, control (ie, C57BL/6 mice received islet Tx without therapy); group 2, C57BL/6 mice received islet Tx with anti-CD154 monoclonal Ab (mAb) therapy; group 3, CD40 KO mice; and group 4, CD154 KO mice were used as recipients without therapy. Mouse anti-rat mixed lymphocyte reactions (MLR) were performed using mouse splenocytes obtained from animals transplanted with rat islets in groups 1 to 4. RESULTS: In group 2, short-term anti-CD154 mAb therapy significantly prolonged rat-to-mouse and human-to-mouse xenograft survival, compared to controls. In CD40-KO and CD154-KO recipients, survival of concordant or discordant islets was not prolonged significantly compared to control groups. Mouse anti-donor rat cellular responses were reduced approximately 50% in group 2 but remained unmodified in groups 3 and 4, when compared to group 1. CONCLUSIONS: Improved graft survival and reduced MLR responses against donor cells in vitro among the anti-CD154 mAb-treated mice could be explained by specific targeting of activated T cells with subsequent inactivation by anergy and/or elimination by apoptosis, or complement- or cellular-mediated mechanisms. Rejection of xenografts and strong MLR responses against donor cells in vitro in CD40 or CD154 KO animals is possible through efficient activation of alternate pathways of costimulation.     

Antibody‐Mediated Rejection of Single Class I MHC‐Disparate Cardiac Allografts

Murine CCR5^?/? recipients produce high titers of antibody to complete MHC‐mismatched heart and renal allografts. To study mechanisms of class I MHC antibody‐mediated allograft injury, we tested the rejection of heart allografts transgenically expressing a single class I MHC disparity in wild‐type C57BL/6 (H‐2^b) and B6.CCR5^?/? recipients. Donor‐specific antibody titers in CCR5^?/? recipients were 30‐fold higher than in wild‐type recipients. B6.K^d allografts survived longer than 60 days in wild‐type recipients whereas CCR5^?/? recipients rejected all allografts within 14 days. Rejection was accompanied by infiltration of CD8 T cells, neutrophils and macrophages, and C4d deposition in the graft capillaries. B6.K^d allografts were rejected by CD8^?/?/CCR5^?/?, but not μMT^?/?/CCR5^?/?, recipients indicating the need for antibody but not CD8 T cells. Grafts recovered at day 10 from CCR5^?/? and CD8^?/?/CCR5^?/? recipients and from RAG‐1^?/? allograft recipients injected with anti‐K^d antibodies expressed high levels of perforin, myeloperoxidase and CCL5 mRNA. These studies indicate that the continual production of antidonor class I MHC antibody can mediate allograft rejection, that donor‐reactive CD8 T cells synergize with the antibody to contribute to rejection, and that expression of three biomarkers during rejection can occur in the absence of this CD8 T cell activity.     

"Indirect" acute islet allograft destruction in nonobese diabetic mice is independent of donor major histocompatibility complex and requires host B lymphocytes

Islet allografts are destroyed rapidly in spontaneously diabetic nonobese diabetic (NOD) mice. However, whether this process is more similar to conventional allograft immunity, islet-specific autoimmune pathogenesis, or both remains controversial. In particular, we sought to determine whether C57BI/6 donor islet major histocompatibility complex (MHC) class I or class II expression was required for islet allograft destruction in autoimmune prone NOD mice versus non-autoimmune-prone BALB/c mice. Results show that islet allografts deficient in both MHC I and II are uniformly accepted in BALB/c mice. In sharp contrast, such MHC-deficient allografts were destroyed acutely in spontaneously diabetic NOD mice. Such donor MHC-independent rejection implicates "indirect" (host MHC-restricted) immunity as a pathway responsible for islet injury. To determine whether host NOD B lymphocytes could contribute to indirect graft recognition, wild-type and MHC I/II-deficient allografts were grafted into B-lymphocyte-deficient (microMT) NOD mice. Whereas wild-type NOD mice could reject MHC-I/II-deficient islet allografts, such grafts were all accepted in B-lymphocyte-deficient NOD mice. Taken together, these results indicate that NOD mice are capable of vigorous donor MHC-independent islet allograft rejection not found in non-autoimmune-prone recipients. Importantly, B lymphocytes may play a key role as antigen-presenting cells in this exuberant host 'indirect' response found in NOD mice.     

Prolonged survival of neonatal porcine islet xenografts in mice treated with a donor-specific transfusion and anti-CD154 antibody

BACKGROUND: Combined treatment with a single donor-specific transfusion (DST) and a brief course of anti-mouse CD154 monoclonal antibody (mAb) to induce co-stimulation blockade leads to long-term murine islet allograft survival. The authors hypothesized that this protocol could also induce long-term survival of neonatal porcine islet cell clusters (NPCC) in chemically diabetic immunocompetent mice and allow their differentiation into functional insulin-producing cells. METHODS: Pancreata from 1- to 3-day-old pigs were collagenase digested and cultured for 8 days. NPCC were recovered and transplanted into the renal subcapsular space. Recipients included chemically diabetic nonobese diabetic (NOD)-scid and C57BL/6 mice that were otherwise untreated, treated with anti-CD154 mAb alone, or treated with DST plus anti-CD154 mAb. Plasma glucose concentration and body weight were measured, and xenografts were examined histologically. RESULTS: NPCC fully differentiated and restored normoglycemia in four of five diabetic NOD-scid recipients but were uniformly rejected by diabetic C57BL/6 recipients. Anti-CD154 mAb monotherapy restored normoglycemia in 4 of 10 (40%) NPCC-engrafted, chemically diabetic C57BL/6 mice, but combined treatment with DST and anti-CD154 mAb restored normoglycemia in 12 of 13 (92%) recipients. Reversal of diabetes required 5 to 12 weeks. Surviving grafts were essentially free of inflammatory infiltrates 15 weeks after transplantation. CONCLUSIONS: Combination therapy with a single DST and a brief course of anti-mouse CD154 mAb without maintenance immunosuppression permits survival and differentiation of NPCC in diabetic C57BL/6 mice. Successful grafts were associated with durable restoration of normoglycemia and the absence of graft inflammation.     

Absence of host B7 expression is sufficient for long-term murine vascularized heart allograft survival

CD28 antagonists have been shown to promote long-term graft survival and induce donor-specific tolerance. In this study, the role of CD28/B7 costimulation and the relative importance of host versus donor B7 expression in allograft rejection was assessed in a murine abdominal vascularized heterotopic heart transplant model. Wild-type, CD28-deficient, or B7-1/B7-2-deficient C57BL/6 (B6) mice were grafted with allogeneic wild type or B7-1/B7-2-deficient hearts. The results demonstrate allogeneic heart grafts survive long-term in mCTLA4Ig-treated B6 and untreated B7-1/B7-2-deficient B6 recipients but not CD28KO B6 mice. B7-1/B7-2KO B6 recipients treated with anti-CD28 (PV-1) or recombinant human IL-2 rejected the heart transplants indicating that these mice are immunologically competent to reject grafts if costimulatory signals are supplied or bypassed. Finally, there was no difference in rejection between normal animals transplanted with wild-type versus B7-1/B7-2-deficient hearts. These results support a critical role for B7-expressing host antigen presenting cells in the rejection of heart allografts in mice and differences among B7KO and CD28KO animals.     

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.     

Induction of antigen-specific unresponsiveness to pancreatic islet allografts by antilymphocyte serum

The mechanism (or mechanisms) underlying the indefinite survival of DBA/2 islet allografts in strongly histoincompatible (C57BL/6xA)F1 (B6AF1) mice, induced either by the combined use of Ficoll-prepared crude islets and treatment of recipients with antilymphocyte serum (ALS), or by the use of handpicked, purified islets in nonimmunosuppressed recipients, was examined. B6AF1 mice bearing DBA/2 crude islet allografts for more than 100 days following ALS treatment accepted secondary DBA/2 crude islet allografts, but acutely rejected third-party A.SW crude islet allografts. This antigen-specific unresponsiveness to islet allografts can be successfully transferred into syngeneic B6AF1 mice by spleen cells. However, the state of unresponsiveness to donor antigen observed in these animals appears to be relatively weak, since transplantation of DBA/2 skin allografts or injection of DBA/2 spleen cells (5 x 10(7] caused acute rejection of long-term-accepted islet allografts. In contrast, B6AF1 mice bearing DBA/2 purified islet allografts over 100 days without immunosuppression rejected the secondary DBA/2 crude islet allografts acutely. Transfer of spleen cells obtained from these animals to syngeneic B6AF1 mice failed to induce prolongation of DBA/2 crude islet allografts. Thus, the mechanism (or mechanisms) involved in the long-term acceptance of islet allografts induced by the use of ALS and crude islets appears to be different from that involved in the long-term acceptance of purified islet allografts. The possible roles played by ALS in the induction of specific unresponsiveness to islet allografts are discussed.     

Role of CD4+ and CD8+ T cells in murine skin and heart allograft rejection across different antigenic desparities

The factors that influence the relative contribution of the T cell subsets to allograft rejection remain unclear. We compared skin and heart rejection in CD4 Knockout (KO), and CD8 KO mice across full-, minor-, and class II histocompatibility antigen (HA) mismatches. Skin allografts were rejected by either CD4+ or CD8+ T cells alone at any degree of antigenic mismatch. However, either the absence of CD4+ cells or a lesser degree of HA mismatch resulted in prolongation of graft survival. In contrast, fully allogeneic heart grafts were accepted in CD4 KO recipients, and minor HA mismatched heart grafts were accepted by both CD4 KO and CD8 KO mice. Thus, the T cell subsets required for allograft rejection are determined by the immunogenicity of the tissue transplanted. In the absence of CD8+ T cells, perforin and Fas ligand (FasL) but not granzyme B mRNA were detected in rejecting grafts. Thus, granzyme B is a CD8+ cytotoxic T lymphocyte (CTL)-specific effector molecule.     

Evidence for tissue-directed immune responses: analysis of CD4- and CD8-dependent alloimmunity

BACKGROUND: Transplant rejection has generally been considered a CD4 T-cell-dependent immune process. CD4-independent, CD8 T-cell rejection pathways have recently gained attention because of their relative resistance to immunosuppression. In the current study, the role of the allograft tissue in activation of these distinct pathways was examined by comparing host-immune responses with allogeneic pancreatic islets or hepatocytes transplanted across the same genetic disparity. METHODS: To compare activation of CD4-dependent versus CD8-dependent alloimmunity, islets or hepatocytes retrieved from FVB/N (H-2) mice were transplanted into CD8 or CD4 T-cell-reconstituted severe combined immunodeficiency mice, CD4 or CD8 knockout (KO) mice, and anti-CD4 monoclonal antibody (mAb) or anti-CD8 mAb treated C57BL/6 mice (all H-2). The ability to immunomodulate CD4-dependent allograft rejection (in CD8 KO mice) was examined in the context of several mechanistically distinct immunotherapeutic strategies, including anti-CD4 mAb, donor-specific transfusion and anti-CD154 mAb, and anti-lymphocyte function-associated antigen-1 mAb. RESULTS: The studies demonstrate that, whereas hepatocytes evoke alloreactive CD4-dependent and (CD4-independent) CD8 T-cell immune responses, allogeneic islets only activate CD4-dependent immune pathways. CD4-dependent host-immune responses initiated by pancreatic islet allografts were readily suppressed by a variety of short-term immunotherapies, whereas hepatocyte-initiated CD4-dependent alloimmune responses were not. CONCLUSIONS: These results demonstrate that immune characteristics of the specific allograft tissue uniquely influence the pattern of host immune responses such that the propensity to activate CD4- or CD8-dependent alloimmune responses can be distinguished. Furthermore, CD4-dependent immune responses activated by different tissues from the same donor strain are distinguished by their susceptibility to specific immunotherapy.     

Long-term survival and function of intrahepatic islet allografts in baboons treated with humanized anti-CD154.

Clinical islet cell transplantation has resulted in insulin independence in a limited number of cases. Rejection, recurrence of autoimmunity, and impairment of normal islet function by conventional immunosuppressive drugs, e.g., steroids, tacrolimus, and cyclosporin A, may all contribute to islet allograft loss. Furthermore, intraportal infusion of allogeneic islets results in the activation of intrahepatic macrophages and endothelial cells, followed by production of proinflammatory mediators that can contribute to islet primary nonfunction. We reasoned that the beneficial effects of anti-CD154 treatment on autoimmunity, alloreactivity, and proinflammatory events mediated by macrophages and endothelial cells made it an ideal agent for the prevention of islet allograft failure. In this study, a nonhuman primate model (Papio hamadryas) was used to assess the effect of humanized anti-CD154 (hu5c8) on allogeneic islet engraftment and function. Nonimmunosuppressed and tacrolimus-treated recipients were insulin independent posttransplant, but rejected their islet allografts in 8 days. Engraftment and insulin independence were achieved in seven of seven baboon recipients of anti-CD154 induction therapy administered on days -1, 3, and 10 relative to the islet transplant. Three of three baboons treated with 20 mg/kg anti-CD154 induction therapy experienced delayed rejection episodes, first detected by elevations in postprandial blood glucose levels, on postoperative day (POD) 31 for one and on POD 58 for the other two. Re-treatment with three doses of anti-CD154 resulted in reversal of rejection in all three animals and in a return to normoglycemia and insulin independence in two of three baboons. It was possible to reverse multiple episodes of rejection with this approach. A loss of functional islet mass, as detected by reduced first-phase insulin release in response to intravenous glucose tolerance testing, was observed after each episode of rejection. One of two baboons treated with 10 mg/kg induction therapy became insulin independent post-transplant but rejected the islet graft on POD 10; the other animal experienced a reversible rejection episode on POD 58 and remained insulin independent and normoglycemic until POD 264. Two additional baboon recipients of allogeneic islets and donor bone marrow (infused on PODs 5 and 11) were treated with induction therapy (PODs -1, 3, 10), followed by initiation of monthly maintenance therapy (for a period of 6 months) on POD 28. Rejection-free graft survival and insulin independence was maintained for 114 and 238 days, with preservation of functional islet mass observed in the absence of rejection. Prevention and reversal of rejection, in the absence of the deleterious effects associated with the use of conventional immunosuppressive drugs, make anti-CD154 a unique agent for further study in islet cell transplantation.     

The role of autoimmunity in islet allograft destruction: major histocompatibility complex class II matching is necessary for autoimmune destruction of allogeneic islet transplants after T-cell costimulatory blockade

Although it has often been assumed that transplanted allogeneic islets can be destroyed by recurrent autoimmunity in recipients with type 1 diabetes, definitive evidence is lacking and the settings in which this may occur have not been defined. To address these issues, we compared the survival of islet transplants (subject to tissue-specific autoimmunity) with cardiac transplants (not subject to tissue-specific autoimmunity) from various major histocompatibility complex (MHC)-matched and -mismatched donors transplanted into autoimmune NOD recipients. We found that when recipients were treated with combined B7 and CD154 T-cell costimulatory blockade, hearts survived best with better MHC matching, whereas islets survived worst when the donor and recipient shared MHC class II antigens. In the absence of full or MHC class II matching, there was no difference in the survival of islet and cardiac allografts. We also found that the tendency of NOD mice to resist tolerance induction by costimulation blockade is mediated by both CD4+ and CD8+ T-cells, not directly linked to the presence of autoimmunity, and conferred by non-MHC background genes. These findings have clinical importance because they suggest that under some circumstances, avoiding MHC class II sharing may provide better islet allograft survival in recipients with autoimmune diabetes, since mismatched allogeneic islets may be resistant to recurrent autoimmunity. Our results may have implications for the design of future clinical trials in islet transplantation.