The graft helps to define the character of the alloimmune response

Introduction: In mice, kidney and liver allografts may be spontaneously accepted, whereas cardiac and skin allografts in the same strain combinations are rapidly rejected. The reasons for this dichotomy in murine response outcomes remains to be determined. Methods and results: When DBA/2 (H-2d) cardiac allografts were placed in C57BL/6 (H-2b) recipients, they were rejected within 10 days, unless the allograft recipients were transiently treated with gallium nitrate (GN), at which time the allografts were accepted for > 150 days. The cardiac allograft rejector mice displayed DBA/2-reactive DTH responses, whereas the cardiac allograft acceptor mice displayed both TGFbeta- and IL10-mediated inhibition of DTH responses. In contrast, DBA/2 kidney allografts placed at the same location in C57BL/6 mice were spontaneously accepted without immunosuppression. These kidney allograft acceptor mice displayed TGFbeta-mediated, but not IL10-mediated inhibition of donor-reactive DTH responses. Conclusions: In the DBA/2-> C57B1/6 strain combination, cardiac allografts induce pro-inflammatory immunity and allograft rejection, while kidney allografts induce anti-inflammatory immunity and allograft acceptance despite the fact that both organs display the same strong MHC disparities and are implanted at the same location. Anti-inflammatory immunity and allograft acceptance are displayed by cardiac allograft recipients when they are transiently treated with select immunosuppressants. Thus, multiple immune response options are available to the organ allograft recipient, and the choice is determined, to some degree, by the allograft, itself.     

Transforming growth factor-beta and interleukin-10 subvert alloreactive delayed type hypersensitivity in cardiac allograft acceptor mice

We have previously reported that temporary treatment of cardiac allograft recipients with gallium nitrate (GN) results in indefinite graft survival, and the inability to mount donor-reactive delayed type hypersensitivity (DTH) responses. We report that antibodies to either transforming growth factor-beta (TGFbeta) or interleukin-10 (IL10) can uncover DTH responses to donor alloantigens in cardiac allograft acceptor mice. The DTH responses uncovered with TGFbeta-reactive antibodies can be blocked by exogenous IL10, and those uncovered with IL10-reactive antibodies can be blocked by exogenous TGFbeta. These data demonstrate that allograft acceptor mice are fully allosensitized, and poised to make donor-reactive cell-mediated immune responses. However, such responses are subverted by a donor alloantigen-dependent mechanism that involves TGFbeta and IL10, which in turn interfere with local cell-mediated immune responses.     

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.     

Allogeneic corneal tolerance in rodents with long-term graft survival

BACKGROUND: Healthy C57BL/6 orthotopic corneal allografts in place for more than 8 weeks in BALB/c mice (acceptor8w+) can survive indefinitely due to active suppression of the donor-specific delayed-type hypersensitivity (DTH) response. This suggests a state of tolerance in the acceptor mice, however, the mechanism(s) underlying this acceptance remains to be demonstrated. We investigated the relationship between tolerance-induction and the DTH response using murine re-grafting models to explore the possibility of promoting allogeneic corneal regraft acceptance in high-risk graft beds. METHODS: Acceptor8w+ BALB/c mice received C57BL/6- or C3H corneal regrafts onto the same eye. Re-grafting models were prepared by inducing corneal neovascularization in the graft beds of naive BALB/c mice 2 weeks before corneal allografting. These mice were intravenously (iv) injected with purified splenic T cells or T-cell-depleted splenocytes from acceptor8w+ mice at the time they received re-grafts of C57BL/6 corneas. We also iv injected acceptor8w+ splenocytes into mice bearing healthy primary corneal allografts for 4 weeks (acceptor4w) and assessed their DTH response to C57BL/6 alloantigen(s). In those experiments, acceptor4w mice received a C57BL/6 corneal regraft onto the same eye. RESULTS: In all acceptor8w+ mice there was indefinite survival of C57BL/6-, but not of C3H regrafts. The iv injection of T cells, but not of T-cell-depleted populations, from acceptor8w+ splenocytes promoted allograft survival. Acceptor4w mice iv injected with acceptor8w+ splenocytes manifested a reduced C57BL/6-specific DTH response and the survival rate of C57BL/6 regrafts was increased from 0% to 87.5%. CONCLUSION: As donor-specific T cells from acceptor8w+ mice induced prolonged regraft survival, we posit that the active suppression of DTH responses by T cells may have contributed to indefinite allogeneic regraft survival via the induction of corneal allograft tolerance.     

Evidence that a similar range of alloimmune responses can develop in murine and human allograft recipients

Based on discrepancies between various laboratory and clinical observations, skepticism has developed regarding the pre-clinical value of rodent immunologic studies. In this article, we review the progression of our murine and human studies over the last several years, which have demonstrated that humans and mice can make very similar types of immune responses in vivo to allografts. Early studies by ourselves and others, demonstrated that mice can make either pro-inflammatory (rejection) or anti-inflammatory (acceptance) immune responses to graft alloantigens. We demonstrated that donor-reactive DTH assays could be used to monitor which type of alloimmune response had been selected by the allograft recipient. To help determine if similar immune response options are available to humans and detectable by DTH assays, we first developed the transvivo DTH assay. In this system, mice are used as a receptacle in which DTH responses made by human PBMC can be induced and measured. These transvivo DTH studies revealed that human allograft recipients, like mice, commonly make either pro-inflammatory or anti-inflammatory immune responses to graft alloantigens. In transplant patients, this rarely correlates with the development of donor-reactive alloantibodies during the post-transplant period.     

MHC-identical heart and hepatocyte allografts evoke opposite immune responses within the same host

BACKGROUND: Purified allogeneic hepatocytes are highly antigenic and elicit immune responses that are not easily controlled. However, it is not clear whether hepatocytes are not capable of protective immune mechanisms or whether they are not to protection by immune mechanisms that permit long-term survival of other allografts. The purpose of the current study was to determine whether donor-matched allogeneic hepatocytes are protected from rejection in mice that have been induced to accept heart allografts. METHODS: Transient treatment with anti-CD4 monoclonal antibody (mAb) or gallium nitrate (GN) was used to induce acceptance of heterotopic FVB/N (H-2(q)) heart allografts by C57BL/6 (H-2(b)) mice. Transgenic hA1AT-FVB/N hepatocytes were sequentially transplanted into C57BL/6 mice that had accepted FVB/N heart allografts more than 60 days (heart acceptor mice), CD8 depleted C57BL/6 heart acceptor mice, or B-cell knockout (BCKO, H-2(b)) heart acceptor mice. Hepatocyte survival was determined by the detection of secreted transgenic product hA1AT by enzyme-linked immunosorbent assay (ELISA). RESULTS: FVB/N hepatocytes were rejected by day 10-14 posttransplant, while FVB/N heart allografts continued to function in C57BL/6, BCKO, and CD8 depleted heart acceptor mice. When FVB/N hepatocytes and heart allografts were transplanted into C57BL/6 or BCKO mice under short-term cover of anti-CD4 mAb or GN, hepatocyte rejection occurred by day 10 posttransplant, while most heart allografts survived for more than 60 days. CONCLUSIONS: Hepatocyte rejection does not appear to interfere with the of mechanisms that permit heart allograft acceptance. However, immune responses to allogeneic hepatocytes are not to regulation by mechanisms induced in heart acceptor mice. The simultaneous rejection of FVB/N allogeneic hepatocytes and continued acceptance of FVB/N-matched heart allografts is independent of host CD8+ T cells and humoral immunity.     

Peripheral nerve transplantation: the role of chemical acellularization in eliminating allograft antigenicity

This study tests the hypothesis that a chemically acellularized peripheral nerve allograft is as immunologically inactive as a peripheral nerve isograft. Cellular and acellular sciatic nerves were transplanted from BALB/c into C57BL/6 mice. C57BL/6 sciatic nerves were also transplanted into C57BL/6 recipients as isograft controls. Fourteen days post-transplantation, recipient splenocytes were isolated, stimulated with donor alloantigens, and IL-2, IL-4, IL-5, and gamma-IFN production was quantified using the ELISPOT technique. Cellular peripheral nerve allografts stimulated robust Th1 and Th2 systemic immune responses, whereas acellular peripheral nerve allografts elicited a response that is comparable to or lower than that quantified following peripheral nerve isograft transplantation. Chemical acellularization of peripheral nerve allografts dramatically reduces the cellular and humoral immunologic responses. These data indicate that chemically acellularized peripheral nerve constructs are relatively non-antigenic and may be a readily available source of nerve for peripheral nerve reconstruction.     

Induction of transplantation tolerance by intraportal injection of allogeneic bone marrow cells

Intraportal inoculation of C57BL/6 marrow cells into sublethally (400 rad) irradiated BALB/c recipients resulted in durable chimerism and the permanent acceptance of C57BL/6 skin allografts. Sublethally irradiated recipients of a similar number of marrow cells inoculated systemically did not develop chimerism or any significant prolongation of the survival of C57BL/6 skin allografts. Consequently, lethal graft-versus-host disease developed only in recipients of intraportal marrow allografts (80%). The intraportal injection of allogeneic C57BL/6 marrow cells into nonirradiated recipients resulted in significant, although not permanent, prolongation of skin allograft survival without durable chimerism, suggesting that the introduction of alloantigens intraportally may favor the induction of nonresponsiveness to alloantigens even across strong major histocompatibility barriers. The relevance of these findings is discussed regarding the intraportal inoculation of allogeneic bone marrow cells for the treatment of genetic disorders in utero through the induction of neonatal tolerance.     

Induction of transplantation tolerance by intraportal injection of allogeneic bone marrow cells: Possible implications for intrauterine bone marrow transplantation across major histocompatibility barriers

Abstract. Intraportal inoculation of C57BL/6 marrow cells into sublethally (400 rad) irradiated BALB/c recipients resulted in durable chimerism and the permanent acceptance of C57BL/6 skin allografts. Sublethally irradiated recipients of a similar number of marrow cells inoculated systemically did not develop chimerism or any significant prolongation of the survival of C57BL/6 skin allografts. Consequently, lethal graft-versus-host disease developed only in recipients of intraportal marrow allografts (80%). The intraportal injection of allogeneic C57BL/6 marrow cells into nonirradiated recipients resulted in significant, although not permanent, prolongation of skin allograft survival without durable chimerism, suggesting that the introduction of alloantigens intraportally may favor the induction of nonresponsiveness to alloantigens even across strong major histocompatibility barriers. The relevance of these findings is discussed regarding the intraportal inoculation of allogeneic bone marrow cells for the treatment of genetic disorders in utero through the induction of neonatal tolerance.     

Real-time polymerase chain reaction analysis reveals an evolution of cytokine mRNA production in allograft acceptor mice

BACKGROUND: The relative contribution of pro-inflammatory and anti-inflammatory cytokines in promoting the rejection or acceptance of experimental cardiac allografts remains controversial. We hypothesized that the posttransplantation induction of a new immune response to graft alloantigens at a distant delayed-type hypersensitivity (DTH) site would force the immune system to reveal its current disposition toward graft alloantigen as it initiates the new immune response. Thus, we should be able to monitor the evolution of the immunologic relationship between allograft recipients and their grafts at any time posttransplantation by challenging the recipient for DTH responses to donor alloantigen and evaluating the cytokine profiles displayed at the DTH site. METHODS: We have used the sensitive and quantitative technique of real-time polymerase chain reaction to evaluate the patterns of donor alloantigen-induced cytokine mRNA production for interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-10, and transforming growth factor (TGF)-beta. We evaluated cytokine mRNA expression in cardiac allografts and in donor alloantigen-challenged DTH sites in mice that have either accepted or rejected cardiac allografts. RESULTS: We observed the following. (1) Normal hearts and pinnae exhibited detectable baseline production of cytokine mRNAs: TGF-beta>IFN-gamma=IL-10>IL2->IL-4. (2) Both the accepted and rejecting cardiac allografts produced increased amounts of all cytokine mRNAs tested and displayed few quantitative differences in cytokine mRNA production. Notably, accepted allografts displayed enhanced IL-10 mRNA production on day 7 posttransplantation, but not on day 60 posttransplantation and reduced IFN-gamma mRNA production on day 60, but not day 7. (3) There was a high degree of variability in production levels among the various cytokine mRNAs, both for background levels and for allograft-stimulated levels. (4) Donor-reactive DTH sites of allograft rejector mice displayed a broad array of cytokine mRNAs, whereas the DTH sites of allograft acceptor mice displayed only IL-4 mRNA production. (5) Provision of exogenous TGF-beta or IL-10 at a DTH challenge site of allograft rejector mice caused a shift in the cytokine mRNA profile that minimized IFN-gamma and IL-2 mRNA production but spared IL-4, IL-10, and TGF-beta mRNA production. CONCLUSIONS: A broad array of cytokine mRNAs may be stockpiled for future use in cardiac allografts, regardless of whether the grafts will be accepted or rejected. This stockpile is continuously replenished for as long as the graft survives, thereby obscuring any changes in immune disposition of the graft recipient toward graft alloantigens. However, such changes can be revealed by challenge with donor alloantigens at a distant site (DTH challenge). In allograft acceptor mice, this disposition evolves from pro-inflammatory to anti-inflammatory.     

A major role for host MHC class I antigen presentation for promoting islet allograft survival

The purpose of this study was to determine the role for CD8 T cells versus generalized MHC class I-restricted antigen presentation in islet allograft rejection and tolerance. Diabetic C57BI/6 (B6, H-2(b)) controls, C57BI/6 CD8-deficient (CD8 KO), or MHC class I-deficient C57BI/6 (beta 2m KO) recipients were grafted with allogeneic BALB/c (H-2(d)) islets. Islet allografts were acutely rejected in untreated B6, CD8 KO, and in beta 2m KO mice, indicating that neither CD8 T cells nor host MHC class I is required for allograft rejection. We then determined the efficacy of costimulation blockade in these same strains. Costimulation blockade with anti-CD154 therapy facilitated long-term islet allograft survival in both B6 and in CD8 KO recipients. However, anti-CD154 treated beta 2m KO recipients were completely refractory to anti-CD154 therapy; all treated animals acutely rejected islet allografts with or without therapy. Also, anti-NK1.1 treatment of wild-type B6 mice abrogated graft prolongation following anti-CD154 therapy. Taken together, results show a dramatic distinction between two forms of MHC class I-restricted pathways in allograft prolongation. Although anti-CD154-induced allograft survival was CD8 T-cell independent, an intact host MHC class I-restricted (beta 2m-dependent) pathway is nevertheless necessary for allograft survival. This pathway required NK1.1+ cells, implicating NK and/or NKT cells in promoting allograft prolongation in vivo.     

Cytokine regulation of chronic cardiac allograft rejection: evidence against a role for Th1 in the disease process.

BACKGROUND: Transient depletion of CD4+ T cells in cardiac allograft recipients prolongs allograft survival; however, grafts exhibit signs of chronic rejection characterized by collagen deposition and neointima development. Although it is believed that Th1 cells promote acute graft rejection, the role of these cells in chronic rejection remains unclear. Hence, our study evaluated whether Th1 cells are associated with the development of chronic cardiac allograft rejection. METHODS: Splenocytes obtained from C57BL/6 recipients bearing BALB/c hearts with signs of chronic rejection were adoptively transferred into C57BL/6 SCID cardiac allograft recipients. As a measure of Th1 function, interferon-y production was determined after restimulation of recipient splenocytes with donor alloantigens. RESULTS: Transfer of splenocytes in SCID allograft recipients resulted in accelerated chronic rejection in the majority of mice. Characterization of these cells before transfer revealed hyporesponsive Th1 function. However, donor-specific proliferative responses and precursor interleukin-2 producing helper and cytotoxic T lymphocyte frequencies were comparable to that of naive splenocytes. Further, splenocytes obtained from SCID recipients with advanced signs of chronic rejection remained deficient in Th1 function, suggesting that Th1 are not involved in this disease process. This possibility was further supported by the development of chronic rejection in IL-12 knockout recipients. Finally, when splenocytes used for adoptive transfer retained Th1 function, transfer of these cells into SCID recipients resulted in acute allograft rejection. CONCLUSIONS: We have established a model in which the mediators of chronic rejection may be further explored. In this system, the absence rather than the presence of donor-reactive Th1 is associated with chronic rejection. These data indicate that Th1-independent effector mechanisms are responsible for chronic rejection in this model.     

Intrathymic alloantigen-mediated, tolerant, completely major histocompatibility complex-mismatched mouse hearts are specifically rejected by adoptively transferred anti-class I L(d+)-specific 2C cells

BACKGROUND: Tolerance to cardiac allografts can be induced in mice and rats by the injection of donor alloantigen into the thymus in combination with a CD4 T-cell-depleting antibody. CD8(+) cells in these animals are hyporesponsive to graft-specific alloantigens. Most of the CD8(+) T cells in the transgenic 2C mouse express a T-cell receptor specific for the class I major histocompatibility complex L(d+) locus. This study was designed to determine whether the adoptive transfer of these 2C T cells could precipitate rejection of a tolerant, completely major histocompatibility complex-mismatched L(d+) or L(d-) heart. METHODS: C57BL/6 mice (L(d-)) were given 10 x 10(6) cells of BALB/c (L(d+)) or dm2 (BALB/c background lacking L(d) [L(d-)]) splenocytes intrathymically and GK1. 5 (10 mg/kg) intraperitoneally. Twenty-one days later, BALB/c or dm2 hearts were transplanted. On the day of transplantation or after long-term allograft acceptance, recipients received naive 2C cells or 2C cells sensitized by in vitro mixed lymphocyte culture with BALB/c (L(d+)). RESULTS: Mean survival time of BALB/c cardiac allografts in untreated C57BL/6 mice was 7.3 days, although 73% of the mice that were pretreated with BALB/c splenocytes IT plus GK1.5 accepted the donor antigen-specific heart allografts indefinitely. All recipients that were pretreated with the intrathymic plus GK1.5 and that were injected with naive 2C cells at the time of heart transplantation experienced rejection of the BALB/c (L(d+)), but not the dm2 (L(d-)) hearts. In contrast, naive 2C cells could not reject tolerant (>30 days acceptance) BALB/c (L(d+)) hearts. 2C cells sensitized in vitro against L(d) were able to reject established BALB/c hearts but could not reject the L(d-) dm2 hearts. CONCLUSIONS: L(d)-specific 2C T-cell receptor transgenic T cells that are adoptively transferred to recipients will precipitate the rejection of accepted hearts that express class I L(d+) in mice rendered tolerant by an intrathymic injection of alloantigen plus anti-CD4 monoclonal antibodies.     

Cardiac allograft rejection in the absence of macrophage migration inhibitory factor

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a secreted proinflammatory lymphokine essential for elicitation of delayed-type hypersensitivity (DTH) reactions in vivo. We tested whether MIF blockade-absence affected acute or chronic murine cardiac allograft rejection. METHODS: Wild-type (WT) C57BL/6 (B6) mice underwent transplantation with BALB/c hearts with or without blocking anti-MIF antibody, and MIF knockout (KO) B6 mice underwent transplantation with MIF KO BALB/c hearts. Chronic immune injury was induced in WT and KO recipients using donor-specific transfusion and anti-CD40L antibody. RESULTS: Unexpectedly, the blockade or genetic absence of MIF did not prolong graft survival even if recipient T-cell cytotoxicity was additionally impaired. The histologic manifestations of acute and chronic immune injury to the allograft were similar between groups. CONCLUSIONS: MIF is not required for acute or chronic allograft rejection in mice. The findings raise questions about the role of DTH as an important mediator of cardiac allograft injury.     

ICAM-1 deficiency suppresses host allosensitization and rejection of MHC-disparate corneal transplants

BACKGROUND: We used a murine model of orthotopic corneal transplantation to determine whether host deficiency in ICAM-1 promotes survival of corneal grafts with different degrees of allodisparity. METHODS: ICAM-1-/- and wild-type C57BL/6 (ICAM-1+/+) received corneal grafts from the following strains of mice: BALB/c (fully mismatched), BALB.b (mismatched at multiple minor H only), or B10.D2 [including major histocompatibility complex (MHC) mismatch]. Graft rejection, induction of allospecific delayed-type hypersensitivity (DTH) responses, and leukocytic infiltration of grafts were measured. RESULTS: There were no differences in long-term survival of allografts that were either fully mismatched or had only minor H disparity in ICAM-1+/+ vs. ICAM-1-/-hosts. However, whereas B10.D2 grafts were accepted in only 58% of the ICAM-1+/+ hosts, graft survival in ICAM-1-/- recipients was 100% (P=0.006). Moreover, none of the ICAM-1-/- mice receiving B10.D2 grafts developed allospecific DTH. CONCLUSIONS: Prolonged survival seen in MHC-mismatched grafts in ICAM-1-/- mice, along with a suppressed DTH response to donor alloantigens after transplantation, suggest that ICAM-1 is associated with recipient sensitization to MHC alloantigens.     

"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.     

CD4+ T-cell-independent rejection of corneal allografts

BACKGROUND: Several studies suggest that a significant number of corneal allografts undergo rejection in the absence of CD4 T cells. This study examined the role of CD4 T cell-independent mechanisms of corneal allograft rejection. METHODS: BALB/c corneal allografts were transplanted to C57BL/6 beige nude mice that received either CD8 or CD8 T cells from C57BL/6 CD4 knockout (KO) mice that had rejected BALB/c corneal allografts. Immune effector functions of CD8 or CD8 T cells from C57BL/6 CD4 KO mice were assessed using delayed-type hypersensitivity assays and Annexin V apoptosis assays respectively. RESULTS.: Both CD8 and CD8 T cells from CD4 KO corneal allograft rejector mice mediated corneal allograft rejection following adoptive transfer to nude mice. CD8 T cells, but not CD8 T cells, from CD4 KO mice adoptively transferred donor-specific DTH and induced apoptosis of BALB/c corneal endothelial cells in vitro. Apoptosis of BALB/c corneal endothelial cells was mediated by double negative (DN) T cells, as treatment of CD8 cells from CD4 KO mice with anti-Thy 1.2 plus complement abolished their effector function. CONCLUSION: The results support the proposition that CD4 T cell-independent rejection of corneal allografts can be mediated by either CD8 or CD8 T cells. The CD8 T cells represent a unique DN T cell population that might mediate rejection by either direct cytolysis or by inducing apoptosis of the donor corneal endothelium.     

Immunopathogenesis of accelerated allograft rejection in sensitized recipients: humoral and nonhumoral mechanisms

INTRODUCTION: Accelerated rejection (AccR) in sensitized recipients (second-set rejection) is considered a classic humorally mediated form of allograft rejection, although additional effector mechanisms may be involved. METHODS: We developed a model of AccR in which C57BL6 mice are sensitized by BALB/c skin grafts, followed 10 days later by transplantation of BALB/c hearts. We undertook analysis of various humoral and cellular components in this model using knockout or monoclonal antibody-treated allograft recipients. RESULTS: Sensitized mice rejected cardiac allografts in 34+/-7 hr. AccR was accompanied by endothelial deposition of immunoglobulins, complement, and fibrin, but also by dense expression of multiple chemokines and a mixed polymorphonuclear and mononuclear cellular infiltrate. Whereas neutrophil or complement depletion had no significant effect on the tempo of AccR, surprisingly B cell-deficient recipients still underwent AccR (41+/-7 hr) in conjunction with T cell and macrophage recruitment. In contrast, T cell-deficient (nude) mice maintained functioning cardiac allografts for >720 hr despite prior skin engraftment. CONCLUSIONS: AccR in sensitized experimental recipients involves multiple effector pathways. Although most previous studies have emphasized the key role of humoral pathways in mediating AccR, our data indicate that T cell-dependent mechanisms can also promote AccR, alone or in conjunction with humoral responses.     

CXCL9 Antagonism Further Extends Prolonged Cardiac Allograft Survival in CCL19/CCL21-Deficient Mice

CCL19/MIP-3beta and CCL21/SLC are essential for chemotactic recruitment of mature dendritic cells (DC) to T-cell areas of secondary lymphoid tissue. Paucity of lymph node T-cells (plt/plt) mice lack CCL21-serine (ser) and CCL19 expression. We tested plt/plt and wild type (wt) BALB/c (H2d) mice as recipients of heart or skin allografts from C57BL/10J (H2b) donors. Donor DC trafficking to secondary lymphoid tissue was markedly reduced in plt heart but not skin allograft recipients. Heart, but not skin grafts survived significantly longer in plt recipients. Accordingly, T cells from plt heart transplant recipients demonstrated poor anti-donor responses in ex vivo MLR, compared to wt heart or wt and plt skin recipients. Moreover, donor-reactive T cells from plt heart recipients exhibited Th2-skewing in comparison to T cells from wt heart or skin graft recipients. Anti-CXCL9/Mig was administered for 2 weeks post-transplant to determine whether impairment of activated T-cell migration could further prolong cardiac allograft survival in plt recipients. CXCL9-antagonism extended graft survival significantly only in plt mice, likely due, in part, to retention of alloactivated T cells in secondary lymphoid tissue/reduction of graft-infiltrating T cells. Thus, targeting DC and activated T-cell migration concomitantly has additive effects in prolonging heart graft survival with potential for therapeutic application.