Induction Of Long-term Allograft Survival Research Articles

Editorial: Memory T Cells: Effectors, Regulators, and Implications for Transplant Tolerance.

Editorial: Memory T Cells: Effectors, Regulators, and Implications for Transplant Tolerance.

Suppression of Allograft Rejection By a Novel Protein ESAT-6.

Achieving long-term allograft survival or tolerance without continuously global immunosuppression is a highly desirable goal in transplantation. The objective of this study is to induce long-term allograft survival without long-term conventional immunosuppression via short-term treatments with a novel protein ESAT-6, which is originally identified as a T cell Ag in the short-term culture filtrate of M. tuberculosis. Previous studies have shown that ESAT-6 is a virulence factor that promotes the growth and pathogenesis of M. tuberculosis while briefly weakening an individual's immunity. In this study, C57BL/6 mice were transplanted with Balb/C islets and treated with ESTA-6 (0.05mg i.p. on days 0, 2, 4 and 6) and/or low doses of rapamycin (0.2 mg/kg/day for 14 days). One-way MLRs were set up to determine alloreactive T cell proliferation by 3H-TdR uptakes and Th cell differentiation by ELISA while Graft-infiltrating T cell proliferation was measured by the BrdU labeling. We found that ESAT-6 significantly extended islet allograft survival (MST = 32 vs. 14 days, P<0.05). Importantly, treatments with both rapamycin and ESAT-6 further prolonged islet allograft survival [MST = 91 versus 30 days (rapamycin alone) or 32 days (ESAT-6 alone)], with 50% of recipient mice achieving long-term allograft survival (>100 days). ESAT-6 also suppressed graft-infiltrating T cell proliferation one week after transplantation (BrdU+: 29±3 vs. 41±4 %, P<0.05) while ESAT-6 plus rapamycin further inhibited T cell proliferation (BrdU+: 15±2 vs. 41±4 %, P<0.05). Moreover, ESAT-6 reduced IFN-gamma (6.8±0.7 vs 12.5±0.1.4, ng/ml, P<0.05) but increased IL-4 (5.8±0.7 vs. 3.1±0.4, ng/ml, P<0.05) and IL-10 (5.4±0.6 vs. 2.8±0.3, ng/ml, P<0.05) levels in the supernatant of MLRs while inhibiting T cell proliferation in vitro (CPM x1000: 8±1 vs. 18±3, P<0.05). However, it did not significantly alter IL-17A level and FoxP3+ cell numbers. Thus, for the first time, our studies demonstrate that ESAT-6 suppresses alloreactive T cell proliferation and promotes the induction of long-term allograft survival by tipping the balance of Th1/Th2 cell differentiation. Therefore, ESAT-6 could be utilized as a novel agent to induce long-term allograft survival in clinic.

CD28 Aptamers as Powerful Immune Response Modulators

CD28 is one of the main costimulatory receptors responsible for the proper activation of T lymphocytes. We have isolated two aptamers that bind to the CD28 receptor. As a monomer, one of them interfered with the binding of CD28 to its ligand (B7), precluding the costimulatory signal, whereas the other one was inactive. However, dimerization of any of the anti-CD28 aptamers was sufficient to provide an artificial costimulatory signal. No antibody has featured a dual function (i.e., the ability to work as agonist and antagonist) to date. Two different agonistic structures were engineered for each anti-CD28 aptamer. One showed remarkably improved costimulatory properties, surpassing the agonistic effect of an anti-CD28 antibody. Moreover, we showed in vivo that the CD28 agonistic aptamer is capable of enhancing the cellular immune response against a lymphoma idiotype and of prolonging survival of mice which receive the aptamer together with an idiotype vaccine. The CD28 aptamers described in this work could be used to modulate the immune response either blocking the interaction with B7 or enhancing vaccine-induced immune responses in cancer immunotherapy.

Intrathymic delivery of plasmid-encoding endoplasmic reticulum signal-sequence-deleted MHC class I alloantigen can induce long-term allograft survival

Abstract Intrathymic (IT) delivery of donor alloantigen is a potent strategy to induce operational tolerance. In this study we determined whether this effect was dependent on direct aliorecognition of the tolerogen. Ten microgrammes of plasmid, encoding either the wildtype major histocompatibility complex (MHC) class I molecule Kb or a truncated form in which the signal sequence for translocation into the endoplasmic reticulum was deleted, preventing cell surface expression and direct allorecognition of the tolerogen, was administered intrathymically to CBA.Ca (H2k) recipients. In addition, recipients were treated with anti-CD4 antibody (YTA3.1) at the time of IT injection and underwent transplantation 28 days later with a fully mismatched C57BL/10 (H2b) cardiac allograft. Wildtype, as well as truncated Kb genes, were able to induce long-term survival of the cardiac allografts, in contrast to empty control plasmid. Reversetranscriptase PCR showed expression of the Kb genes for up to 28 days in thymus and spleen of pretreated recipients. These data show that direct allorecognition of the tolerogen was not required for the induction of long-term allograft survival following the introduction of plasmid-encoded MHC alloantigen into the thymus.

Intrathymic delivery of plasmid-encoding endoplasmic reticulum signal-sequence-deleted MHC class I alloantigen can induce long-term allograft survival.

Intrathymic (IT) delivery of donor alloantigen is a potent strategy to induce operational tolerance. In this study we determined whether this effect was dependent on direct allorecognition of the tolerogen. Ten microgrammes of plasmid, encoding either the wildtype major histocompatibility complex (MHC) class I molecule K(b) or a truncated form in which the signal sequence for translocation into the endoplasmic reticulum was deleted, preventing cell surface expression and direct allorecognition of the tolerogen, was administered intrathymically to CBA.Ca (H2(k)) recipients. In addition, recipients were treated with anti-CD4 antibody (YTA3.1) at the time of IT injection and underwent transplantation 28 days later with a fully mismatched C57BL/10 (H2(b)) cardiac allograft. Wildtype, as well as truncated K(b) genes, were able to induce long-term survival of the cardiac allografts, in contrast to empty control plasmid. Reverse-transcriptase PCR showed expression of the K(b) genes for up to 28 days in thymus and spleen of pretreated recipients. These data show that direct allorecognition of the tolerogen was not required for the induction of long-term allograft survival following the introduction of plasmid-encoded MHC alloantigen into the thymus.

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

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. 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. 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. 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.

Active suppression of allogeneic proliferative responses by dendritic cells after induction of long-term allograft survival by CTLA4Ig

AbstractCostimulatory blockade using cytotoxic T lymphocyte–associated antigen 4 immunoglobulin (CTLA4Ig) efficiently down-regulates immune responses in animal models and is currently used in autoimmune and transplantation clinical trials, but the precise cellular and molecular mechanisms involved remain unclear. Rats that received allogeneic heart transplants and were treated with adenoviruses coding for CTLA4Ig show long-term allograft survival. The immune mechanisms regulating induction of long-term allograft acceptance were analyzed in splenocytes using mixed leukocyte reactions (MLRs). MLRs of splenocytes but not purified T cells from CTLA4Ig-treated rats showed higher than 75% inhibition compared with controls. Splenocytes from CTLA4Ig-treated rats inhibited proliferation of naive and allogeneically primed splenocytes or T cells. MLR suppression was dependent on soluble secreted product(s). Production of soluble inhibitory product(s) was triggered by a donor antigen-specific stimulation and inhibited proliferation in an antigen-nonspecific manner. CTLA4Ig levels in the culture supernatant were undetectable and neither interleukin-10 (IL-10), transforming growth factor β1 (TGFβ1), IL-4, nor IL-13 were responsible for suppression of MLRs. Inhibition of nitrous oxide (NO) production or addition of IL-2 could not restore proliferation independently, but the combined treatment synergistically induced proliferation comparable with controls. Stimulation of APCs using tumor necrosis factor (TNF)–related activation-induced cytokine (TRANCE) or CD40L and addition of IL-2 normalized MLRs of CTLA4Ig-treated splenocytes. Finally, dendritic cells (DCs), but not T cells, from CTLA4Ig-treated rats inhibited naive MLRs. Altogether, these results provide evidence that after in vivo CTLA4Ig treatment, splenocytes, and in particular DCs, can inhibit alloantigen-induced proliferative responses through secretion of inhibitory products, thus promoting alloantigen-specific tolerance in vivo.

Study of the effect of indoleamine 2,3-dioxygenase on murine mixed lymphocyte reactions and skin allograft rejection

ACUTE REJECTION of allografts is mediated by the coordinated infiltration and effector functions of alloantigen-specific T cells. After infiltration of transplanted allografts, host T cells are activated to produce proinflammatory cytokines and to express cytolytic activities that mediate destruction of the graft tissue. It is known that interferon(INF) is often produced and detected in allografts during rejection. Although INFup-regulates MHC molecule expression and enhances alloantigen presentation in vitro, it is not required for acute rejection of fully allogeneic (MHC class I–disparate and MHC class II–disparate) grafts. Instead, INFhas an essential immunoregulatory function in vivo. INFcan facilitate the induction of long-term allograft survival and tolerance to protein antigens partly by limiting the proliferation of activated T lymphocytes. Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme in the L-tryptophan-kynurenine pathway that converts the essential amino acid, Ltryptophan (L-TRP), to N-formylkynurenine in mammalian extrahepatic tissues. It recently was proposed that IDO-mediated immunosuppression might prevent rejection of the fetus by the maternal T-cell response. Although several studies have circumstantially linked IDO induction to certain anti-proliferative and antimicrobial effects of immune stimuli, different and potentially detrimental consequences of IDO induction also have been proposed. Although the mechanism of IDO induction in vivo is unclear, it is widely accepted that IDO is up-regulated by INF– dependent and INF– independent mechanisms, and down-regulated by interleukin (IL)-4 and TGF. It has not yet been established, however, whether the physiological role of IDO regulation by these cytokines is beneficial or detrimental, nor whether this enzyme has an effect on allograft rejection. Several studies suggest that INFinduction of IDO exerts anti-microbial and anti-proliferative effects, possibly by depleting L-TRP. By treating skin allograft mice with 1-methyl-DL-tryptophan, which competes with L-TRP for IDO, we tested whether IDO might play an important role in the skin allograft rejection as a mechanism for regulating T-cell responses after producing INF. MATERIALS AND METHODS Mice BALB/c (H-2), C57BL/6 (H-2), B6.C-H2bm1, and B6.C-H2bm12 female mice, aged 6 to 8 weeks, were purchased from the Animal Production Area, NCI-Frederick (Frederick, Md, USA).

Regulation of alloantigen-mediated T-cell proliferation by endogenous interferon-gamma: implications for long-term allograft acceptance.

Recent data suggest that interferon (IFN)-gamma is not an essential mediator of acute rejection but, instead, is critical for the induction of long-term allograft acceptance. The in vivo mechanisms by which endogenous IFN-gamma regulates the alloimmune response and thus facilitates the induction of long-term allograft survival are not known. We examined long-term cardiac and skin allograft survival, alloantigen-induced T-cell proliferation, and alloantigen-induced T-cell apoptosis in wild-type (IFN-gamma+/+) and IFN-gamma gene-knockout (IFN-gamma-/-) mice treated with either B7-CD28 T-cell costimulation blockade alone or B7-CD28 T-cell costimulation blockade combined with donor splenocyte transfusion. We found that IFN-gamma is essential for long-term allograft survival induced by treating mice with either B7-CD28 T-cell costimulation blockade alone or B7-CD28 T-cell costimulation blockade combined with donor splenocyte transfusion. Alloantigen-induced T-cell proliferation in vivo was significantly greater in IFN-gamma-/- mice than in IFN-gamma+/+ mice, and T-cell costimulation blockade abrogated alloantigen-induced T-cell proliferation in wild-type mice but failed to do so in mice that lack IFN-gamma. In contrast, alloantigen-induced T lymphocyte apoptosis in vivo did not differ between IFN-gamma+/+ and IFN-gamma-/- mice, and T-cell costimulation blockade enhanced alloantigen-induced T-cell apoptosis in both mouse strains. These data suggest that endogenous IFN-gamma facilitates the induction of long-term allograft survival by limiting the proliferation of alloactivated T lymphocytes. The data also suggest that B7-CD28 T-cell costimulation blockade exerts immunosuppressive actions by inhibiting the proliferation of activated T lymphocytes and by promoting their apoptosis.

THE Fas-FasL PATHWAY OF ACTIVATION-INDUCED T CELL DEATH IS NOT CRITICAL FOR THE INDUCTION OF LONG-TERM ALLOGRAFT SURVIVAL

457 Fas belongs to a family of membrane receptors that mediate T cell apoptosis. Activation of T lymphocytes upregulates the number of Fas molecules and induces de novo Fas ligand (FasL) expression. Fas-FasL interactions then lead to the apoptosis of activated T cells. Studies in Fas-deficient or FasL-mutant (gld) mice provide strong evidence that the Fas-FasL pathway of activation-induced cell death (AICD) is critical for the clonal deletion of autoreactive T lymphocytes. In this study, we tested whether the Fas-FasL pathway is necessary for the induction of long-term allograft survival or for the activation-induced apoptosis of alloreactive T cells. We therefore analyzed the survival of C57BL/6 (H-2b) vascularized cardiac allografts transplanted to C3H/He (H-2k) wild-type (w.t.) or FasL-mutant (gld) mice. Recipients were either left untreated or received 200μg CTLA4Ig i.p. two days post-transplantation. Rejection was defined as cessation of heart beat and was confirmed histologically. To study alloantigen-mediated AICD, we measured the apoptosis (by flow analysis, TUNEL) of w.t. and gld lymph node T cells following repeated in vivo stimulation with fully allogeneic spleen cells. Results: Allograft survival was significantly longer in untreated gld recipients than untreated w.t. recipients. In contrast, CTLA4Ig induced long-term allograft acceptance in both w.t. and gld mice: (Table)TableAlloantigen-induced CD4+ and CD8+ T cell apoptosis (AICD) was greater in w.t. than in gld mice; however, AICD in the gld group was still significantly greater than background T cell apoptosis (syngeneic stimulation): (Table)TableConclusions: Fas-FasL interactions promote acute allograft rejection but are not critical for the induction of long-term allograft survival. Mechanisms other than the Fas-FasL pathway contribute to the activation-induced apoptosis of alloreactive T lymphocytes.

The Role of CD80, CD86, and CTLA4 in Alloimmune Responses and the Induction of Long-Term Allograft Survival

Blocking the interaction of the CD28 costimulatory receptor with its ligands, CD80 and CD86, inhibits in vivo immune responses, such as allograft rejection, and in some instances induces tolerance. Previously, we found that CTLA4Ig, which blocks the CD28/CTLA-4 (CD152) ligands CD80 and CD86, can be used to induce transplantation tolerance to vascularized allografts. Recent data suggest that an intact CD152-negative signaling pathway is essential for induction of tolerance to nominal Ags. Here, we show that blockade of CD152 using an anti-CD152 mAb at the time of transplantation prevents the induction of long-term allograft survival by agents that target CD80 and CD86. In contrast, CD152 signals are not required for the maintenance of established graft survival. We also report for the first time that blockade of CD86 alone can induce long-term graft survival. This requires that anti-CD86 mAb is given on the day of transplantation and also depends upon an intact CD152 pathway. This result, plus experiments using CD80-deficient mice, suggests a dominant role for CD80 molecules on donor cells as the relevant ligand for CD152. We additionally find that blockade of CD152 at the time of transplantation does not interfere with the effectiveness of anti-CD154 mAbs, suggesting distinct mechanisms for inhibition of graft rejection by blocking the CD28 vs CD154 pathways.

Impaired Alloantigen-Mediated T Cell Apoptosis and Failure To Induce Long-Term Allograft Survival in IL-2-Deficient Mice

We examined whether IL-2 regulates alloimmune responses by studying allograft survival in wild-type (IL-2+/+) and IL-2 gene-knockout (IL-2-/-) mice. The acute rejection of vascularized, cardiac allografts and the generation of allospecific CTLs were not impaired in the absence of IL-2. In contrast, blocking the B7-CD28 T cell costimulation pathway with CTLA4Ig induced long-term allograft survival (> 100 days) in IL-2+/+ recipients but failed to do so in IL-2-/- mice or in wild-type mice that had been treated with IL-2-neutralizing Ab around the time of transplantation. Allografts rejected by IL-2-/- recipients exhibited extensive mononuclear cell infiltrates despite CTLA4Ig administration. In vivo allostimulation in the absence of IL-2 led to exaggerated T lymphocyte proliferation and impaired apoptosis of activated T cells in untreated and CTLA4Ig-treated mice. These findings indicate that endogenous IL-2 is required for the induction of long-term allograft survival, and that IL-2 regulates alloimmune responses by preparing activated T lymphocytes for alloantigen-induced apoptosis.

Synergism of the malononitrilamides 279 and 715 with cyclosporine A in the induction of long-term cardiac allograft survival.

We tested here the effects of malononitrilamide (MNA) 279 and MNA 715 (derivatives of A771726, the active metabolite of leflunomide) in monotherapy and in combination with cyclosporine A (CSA) on heterotopically transplanted rat cardiac allografts (BN) [Brown Norway Lewis]. Both MNAs (5-20 mg/kg) displayed a dose-dependent increase of efficacy. The combination of CSA (5 mg/kg) with the MNAs (5 and 10 mg/kg) showed a synergistic effect in the prolongation of allograft survival and in the induction of long-term allograft survival. To investigate the immunological mechanism responsible for long-term allograft survival, we transplanted second set (BN) and third party (Dark Agouti) skin allografts on the tail of long-term surviving rats. The cause for long-term allograft survival turned out to be a donor-specific tolerance. We formulate a hypothesis for the mechanism of the synergism of the combination MNA + CSA in the induction of tolerance.

Synergism of the malononitrilamides 279 and 715 with cyclosporine A in the induction of long-term cardiac allograft survival

Abstract We tested here the effects of malononitrilamide (MNA) 279 and MNA 715 (derivatives of A771726, the active metabolite of leflunomide) in monotherapy and in combination with cyclosporine A (CSA) on heterotopically transplanted rat cardiac allografts (BN) [Brown Norway Lewis]. Both MNAs (5–20 mg/kg) displayed a dose-dependent increase of efficacy. The combination of CSA (5 mg/kg) with the MNAs (5 and 10 mg/kg) showed a synergistic effect in the prolongation of allograft survival and in the induction of long-term allograft survival. To investigate the immunological mechanism responsible for long-term allograft survival, we transplanted second set (BN) and third party (Dark Agouti) skin allografts on the tail of long-term surviving rats. The cause for long-term allograft survival turned out to be a donor-specific tolerance. We formulate a hypothesis for the mechanism of the synergism of the combination MNA + CSA in the induction of tolerance.

INTERFERON-GAMMA (IFN??) IS CRITICAL FOR REGULATING THE PROLIFERATION OF ALLOANTIGEN-ACTIVATED T CELLS

25 We have observed that B7-CD28 T cell costimulation blockade with CTLA4Ig, administered two days after transplantation, induces longterm cardiac allograft survival in wildtype (IFNγ+/+) mice but fails to do so in IFNγ gene-knockout (IFNγ-/-) recipients or in IFNγ+/+ mice treated with IFNγ-neutralizing antibody[mean survival time = 51±15 (n=8; 2/8 grafts survived >100 days), 16±2 (n=7), and 10±1 (n=3) days, respectively]. In this study, we investigated the mechanisms by which endogenous IFNγ facilities induction of longterm allograft survival by analysing thein vivo proliferation and apoptosis of alloantigen-activated T cells in IFNγ+/+ and IFNγ-/- mice. IFNγ+/+ or IFNγ-/- BALB/c (H-2d) mice were injected in the footpads with 1 × 107 allogeneic C3H/He(H-2k) splenocytes on days 0 and 4. Mice were either left untreated or received 200µg CTLA4Ig i.p. on day 2. Lymph node T cells were isolated on day 6 and analysed for DNA replication (BrdU uptake) and for apoptosis (TUNEL) by flow cytometry. Control mice were injected with syngeneic splenocytes. Results: Alloantigen-induced in vivo T cell proliferation was markedly increased in IFNγ-/- mice. Moreover, CTLA4Ig failed to completely inhibit T cell proliferation in the absence of IFNγ: Table Similarly, exaggerated in vitro proliferation of IFNγ-/- splenocytes was observed in primary MLRs. Superantigen-induced Vβ8+ T cell expansion was also significantly increased in IFNγ-/- mice. On the other hand, alloantigen-inducedin vivo T cell apoptosis was neither impaired nor increased in the absence of IFNγ: Table Results shown are representative of 3 to 4 independent experiments. Exaggerated proliferation in the absence of IFNγ was observed in both the CD4 and CD8 T lymphocyte compartments.Conclusions: (1) IFNγ is critical for limiting in vivo expansion of alloreactive T lymphocytes, possibly by regulating their proliferation. This essential function of IFNγ may account for failure to induce longterm allograft survival in IFNγ-deficient mice.(2) IFNγ is not required for activation-induced apoptosis of alloreactive T lymphocytes.

INTERFERON-GAMMA (IFNγ) IS REQUIRED FOR LONGTERM ALLOGRAFT SURVIVAL INDUCED BY COMBINED ADMINISTRATION OF DONOR-SPECIFIC SPLENOCYTE TRANSFUSION(DSST) AND CTLA4Ig.

433 Administration of donor splenocytes or bone marrow enhances the salutary effect of B7-CD28 costimulation blockade on allograft survival in rodents. Because IFNγ has important immunoregulatory functions, we addressed whether this cytokine plays a role in DSST+CTLA4Ig-mediated immunosuppression by comparing the survival of fully allogeneic cardiac or skin grafts in wildtype (IFNγ+/+) recipients to that in IFNγ gene-knockout(IFNγ-/-) mice. DSST (1 × 107 donor splenocytes) was administered i.v. at the time of transplantation. CTLA4Ig (200μg) was administered i.p. in a single dose two days after transplantation. Cardiac rejection was defined as cessation of palpable heart beat and was confirmed histologically. Skin rejection was defined as >90% necrosis of the graft(trunk skin). Results: DSST+CTLA4Ig-mediated prolongation of cardiac allograft survival (C3H/He → BALB/c) was significantly greater in IFNγ+/+ than IFNγ-/- recipients:TableThese data are consistent with our previous findings that endogenous IFNγ facilitates induction of longterm cardiac allograft survival in mice treated with CTLA4Ig alone. Similarly, DSST+CTLA4Ig-mediated prolongation of skin allograft survival(BALB/c → C57BL/6), although modest, was significantly greater in IFNγ+/+ than IFNγ-/- recipients:TableConclusions: (1) Endogenous IFNγ does not hinder but rather facilitates induction of longterm allograft survival in mice treated with donor-specific splenocyte transfusion and CTLA4Ig. (2) These preliminary data suggest that IFNγ plays an essential regulatory role in alloimmunity. Because cyclosporine inhibits IFNγ production, cyclosporine could abrogate the potential clinical utility of donor blood, bone marrow, or splenocyte administration to recipients of solid organ allografts.

INTERFERON-GAMMA (IFNγ) IS CRITICAL FOR REGULATING THE PROLIFERATION OF ALLOANTIGEN-ACTIVATED T CELLS.

25 We have observed that B7-CD28 T cell costimulation blockade with CTLA4Ig, administered two days after transplantation, induces longterm cardiac allograft survival in wildtype (IFNγ+/+) mice but fails to do so in IFNγ gene-knockout (IFNγ-/-) recipients or in IFNγ+/+ mice treated with IFNγ-neutralizing antibody [mean survival time = 51±15 (n=8; 2/8 grafts survived >100 days), 16±2 (n=7), and 10±1 (n=3) days, respectively]. In this study, we investigated the mechanisms by which endogenous IFNγ facilitates induction of longterm allograft survival by analysing the in vivo proliferation and apoptosis of alloantigen-activated T cells in IFNγ+/+ and IFNγ-/- mice. IFNγ+/+ or IFNγ-/- BALB/c (H-2d) mice were injected in the footpads with 1×107 allogeneic C3H/He (H-2k) splenocytes on days 0 and 4. Mice were either left untreated or received 200μg CTLA4Ig i.p. on day 2. Lymph node T cells were isolated on day 6 and analysed for DNA replication (BrdU uptake) and for apoptosis (TUNEL) by flow cytometry. Control mice were injected with syngeneic splenocytes. Results: Alloantigen-induced in vivo T cell proliferation was markedly increased in IFNγ-/- mice. Moreover, CTLA4Ig failed to completely inhibit T cell proliferation in the absence of IFNγ: TableSimilarly, exaggerated in vitro proliferation of IFNγ-/- splenocytes was observed in primary MLRs. Superantigen-induced Vβ8+ T cell expansion was also significantly increased in IFNγ-/- mice. On the other hand, alloantigen-inducedin vivo T cell apoptosis was neither impaired nor increased in the absence of IFNγ: TableResults shown are representative of 3 to 4 independent experiments. Exaggerated proliferation in the absence of IFNγ was observed in both the CD4 and CD8 T lymphocyte compartments. Conclusions: (1) IFNγ is critical for limiting in vivo expansion of alloreactive T lymphocytes, possibly by regulating their proliferation. This essential function of IFNγ may account for failure to induce longterm allograft survival in IFNγ-deficient mice. (2) IFNγ is not required for activation-induced apoptosis of alloreactive T lymphocytes.

IFN-γ Is Critical for Long-Term Allograft Survival Induced by Blocking the CD28 and CD40 Ligand T Cell Costimulation Pathways

AbstractIt is postulated that IFN-γ production hinders long-term acceptance of transplanted organs. To test this hypothesis, we compared survival of skin and heart allografts in wild-type (IFN-γ+/+) mice to that in IFN-γ gene knockout (IFN-γ−/−) mice. We found that perioperative blockade of the CD28 and/or CD40 ligand T cell costimulation pathways induces long-term skin and heart allograft survival in IFN-γ+/+ recipients but fails to do so in IFN-γ−/− mice or in wild-type mice treated with IFN-γ-neutralizing Ab at the time of transplantation. In vitro studies showed that endogenously produced IFN-γ down-regulates T cell proliferation and CTL generation in MLCs. These actions of IFN-γ were not mediated by TNF-α production or Fas-Fas ligand interactions. In vivo studies revealed exaggerated expansion and, subsequently, impaired deletion of superantigen-reactive T lymphocytes in IFN-γ−/− mice injected with staphylococcal enterotoxin B. Taken together, our findings indicate that IFN-γ does not hinder but instead facilitates induction of long-term allograft survival possibly by limiting expansion of activated T cells.

Prevention and therapy of allograft rejection reactions and induction of long-term allograft survival by MNA 279 and MNA 715

Prevention and therapy of allograft rejection reactions and induction of long-term allograft survival by MNA 279 and MNA 715

Prevention and therapy of allograft rejection reactions and induction of long-term allograft survival by MNA 279 and MNA 715

Prevention and therapy of allograft rejection reactions and induction of long-term allograft survival by MNA 279 and MNA 715