A two-step model of acute CD4 T-cell mediated cardiac allograft rejection

CD4 T cells are both necessary and sufficient to mediate acute cardiac allograft rejection in mice. This process requires "direct" engagement of donor MHC class II molecules. That is, acute rejection by CD4+ T cells requires target MHC class II expression by the donor and not by the host. However, it is unclear whether CD4+ T cell rejection requires MHC class II expression on donor hemopoietic cells, nonhemopoietic cells, or both. To address this issue, bone marrow transplantation in mice was used to generate chimeric heart donors in which MHC class II was expressed either on somatic or on hemopoietic cells. We report that direct recognition of hemopoietic and nonhemopoietic cells are individually rate limiting for CD4+ T cell-mediated rejection in vivo. Importantly, active immunization with MHC class II(+) APCs triggered acute rejection of hearts expressing MHC class II only on the somatic compartment. Thus, donor somatic cells, including endothelial cells, are not sufficient to initiate acute rejection; but they are necessary as targets of direct alloreactive CD4 T cells. Taken together, results support a two-stage model in which donor passenger leukocytes are required to activate the CD4 response while direct interaction with the somatic compartment is necessary for the efferent phase of acute graft rejection.     

Murine vascular endothelium activates and induces the generation of allogeneic CD4+25+Foxp3+ regulatory T cells

Unlike graft-resident donor-derived hemopoietic APCs, which decrease in number over time after transplantation, vascular endothelial cells are lifelong residents of a vascularized allograft. Endothelial cells are potent APCs for allogeneic CD8+ T lymphocytes but are unable to induce proliferation of allogeneic CD4+ T lymphocytes. Although the reason for this differential response has been poorly understood, here we report that alloantigen presentation by vascular endothelium to CD4+ T lymphocytes activates and induces CD4+25+Foxp3+ regulatory T cells, which can inhibit proliferation of alloreactive T cells both in vitro and in vivo. This process occurs independently of B7.1 costimulation but is dependent on programmed death ligand 1 (B7-H1). This finding may have important implications for tolerance induction in transplantation.     

Mouse vascular endothelium activates CD8+ T lymphocytes in a B7-dependent fashion

Despite several studies examining the contribution of allorecognition pathways to acute and chronic rejection of vascularized murine allografts, little data describing activation of alloreactive T cells by mouse vascular endothelium exist. We have used primary cultures of resting or IFN-gamma-activated C57BL/6 (H-2(b)) vascular endothelial cells as stimulators and CD8(+) T lymphocytes isolated from CBA/J (H-2(k)) mice as responders. Resting endothelium expressed low levels of MHC class I, which was markedly up-regulated after activation with IFN-gamma. It also expressed moderate levels of CD80 at a resting state and after activation. Both resting and activated endothelium were able to induce proliferation of unprimed CD8(+) T lymphocytes, with proliferation noted at earlier time points after coculture with activated endothelium. Activated endothelium was also able to induce proliferation of CD44(low) naive CD8(+) T lymphocytes. Activated CD8(+) T lymphocytes had the ability to produce IFN-gamma and IL-2, acquired an effector phenotype, and showed up-regulation of the antiapoptotic protein Bcl-x(L). Treatment with CTLA4-Ig led to marked reduction of T cell proliferation and a decrease in expression of Bcl-x(L). Moreover, we demonstrate that nonhemopoietic cells such as vascular endothelium induce proliferation of CD8(+) T lymphocytes in a B7-dependent fashion in vivo. These results suggest that vascular endothelium can act as an APC for CD8(+) direct allorecognition and may, therefore, play an important role in regulating immune processes of allograft rejection.     

Role of CD4+ and CD8+ T cells in allorecognition: lessons from corneal transplantation

Corneal transplantation represents an interesting model to investigate the contribution of direct vs indirect Ag recognition pathways to the alloresponse. Corneal allografts are naturally devoid of MHC class II+ APCs. In addition, minor Ag-mismatched corneal grafts are more readily rejected than their MHC-mismatched counterparts. Accordingly, it has been hypothesized that these transplants do not trigger direct T cell alloresponse, but that donor Ags are presented by host APCs, i.e., in an indirect fashion. Here, we have determined the Ag specificity, frequency, and phenotype of T cells activated through direct and indirect pathways in BALB/c mice transplanted orthotopically with fully allogeneic C57BL/6 corneas. In this combination, only 60% of the corneas are rejected, while the remainder enjoy indefinite graft survival. In rejecting mice the T cell response was mediated by two T cell subsets: 1) CD4+ T cells that recognize alloantigens exclusively through indirect pathway and secrete IL-2, and 2) IFN-gamma-producing CD8+ T cells recognizing donor MHC in a direct fashion. Surprisingly, CD8+ T cells activated directly were not required for graft rejection. In nonrejecting mice, no T cell responses were detected. Strikingly, peripheral sensitization to allogeneic MHC molecules in these mice induced acute rejection of corneal grafts. We conclude that only CD4+ T cells activated via indirect allorecognition have the ability to reject allogeneic corneal grafts. Although alloreactive CD8+ T cells are activated via the direct pathway, they are not fully competent and cannot contribute to the rejection unless they receive an additional signal provided by professional APCs in the periphery.     

Split tolerance induced by chick embryo thymic epithelium allografted to embryonic recipients

To test the capacity of the epithelial component of the chick embryo thymus to induce tolerance to major histocompatibility complex (MHC) antigens, pre-colonized thymic rudiments were grafted into chick embryonic recipients. Semi-allogeneic or allogeneic transplantations were done between two lines of chickens histocompatible at the MHC locus. Approximately 10% of these thymic chimeras hatched and were studied 3 mo after hatching. Thymic grafts were not rejected by the allogeneic host. The tolerance of chimeric chickens to thymus donor MHC antigens was tested by using a skin graft rejection test and a graft-vs-host (GvH) assay. Chimeric chickens that received an MHC-incompatible thymic graft during the embryonic life tolerated skin graft with the MHC haplotype of the thymus donor. Nevertheless, the lymphocytes within the thymic graft, the host thymus, and the blood were tolerant to the host MHC antigens but were alloreactive in GvH reaction for the MHC antigens of the thymic graft type. These results suggest that the epithelial component of the thymus when taken before the starting of the colonization by hemopoietic precursors and grafted into an early chick embryonic host can induce a tolerance for the MHC determinants involved in allograft rejection but not in the GvH reaction.     

Lymphocyte subsets differentially induce class II human leukocyte antigens on allogeneic microvascular endothelial cells

Increased expression of major histocompatibility complex class II (Ia) antigens on vascular endothelium is a common observation in allografts undergoing acute rejection. This phenomenon is generally ascribed to the host immune response directed against graft alloantigens, but its cellular and molecular basis are incompletely understood. In the present study we show that constitutively Ia-negative human microvascular endothelial cells (EC) can be induced to express surface class II human leukocyte antigens shortly after exposure to allogeneic lymphocytes in vitro. CD16+ (natural killer) and CD8+ (cytotoxic/suppressor) lymphocytes were efficient in triggering Ia antigen expression by EC, whereas CD4+ (helper/inducer) lymphocytes induced EC Ia expression only if cultured in the presence of autologous monocytes. Binding of lymphocytes to EC was shown to be essential for the subsequent induction of EC Ia, and anti-CD18 (LFA-1) antibody, which blocks lymphocyte-EC adhesion, was the only antibody of a panel of antilymphocyte antibodies that completely blocked the induction of EC Ia. Antibodies to interferon-gamma, which is a potent inducer of EC Ia, and to the CD3 T cell-surface antigen partly inhibited the induction of EC Ia by T cells, but neither antibody had any effect on Ia induction mediated by CD16+ cells, suggesting that T cells and natural killer cells utilize different mechanisms to induce Ia on EC. When combined with data from other laboratories indicating that Ia+ but not Ia- EC stimulate allogeneic T cell proliferation and cytotoxicity, our results suggest that the binding of EC by lymphocyte subpopulations followed by the induction of Ia antigen may represent the initial stage of incompatible allograft rejection.     

Modulation of tissue-specific immune response to cardiac myosin can prolong survival of allogeneic heart transplants

The role of immune response to tissue-specific Ags in transplant rejection is poorly defined. We have previously reported that transplantation of cardiac allografts triggers a CD4(+) Th1 cell response to cardiac myosin (CM), a major contractile protein of the heart, and that pretransplant activation of proinflammatory CM-specific T cells accelerates rejection. In this study, we show that administration of CM together with IFA (CM/IFA) can prevent acute rejection of an allogeneic heart transplant. Prolongation of cardiac graft survival is associated with activation of CM- and allo-specific T cells secreting type 2 cytokines (IL-4, IL-5) and reduction of the frequency of proinflammatory IFN-gamma-secreting (type 1) alloreactive T cells. Blocking of IL-4 cytokine with Abs abrogates the prolongation. CM/IFA treatment prevents acute rejection of MHC class I-mismatched, but not fully mismatched grafts. However, if donor heart is devoid of MHC class II expression, CM-IFA administration delays rejection of fully allogeneic cardiac transplants. This finding suggests that the effect of CM modulation depends on the type (direct vs indirect) and strength of recipient's CD4(+) T cell alloresponse. Our results underscore the important role of host immunity to tissue-specific Ags in the rejection of an allograft. This study demonstrates that modulation of the immune response to a tissue-specific Ag can significantly prolong cardiac allograft survival, an observation that may have important implications for the development of novel selective immune therapies in transplantation.     

Differential effects of B and T lymphocyte attenuator and programmed death-1 on acceptance of partially versus fully MHC-mismatched cardiac allografts

Although fully MHC-mismatched murine cardiac allografts are rapidly rejected, allografts mismatched at only MHC class I or class II alleles survive long term; the immunologic basis for the long-term survival of MHC class I- or II-mismatched allografts is unknown. We examined the roles of two recently described inhibitory receptors, B and T lymphocyte attenuator (BTLA) and programmed death-1 (PD-1), in the survival of partially or fully MHC-mismatched allografts using gene-deficient recipients as well as through use of blocking mAbs in wild-type hosts. Partially MHC-mismatched allografts showed strong induction of BTLA, but not PD-1 mRNA and survived long term in wild-type recipients, whereas targeting of BTLA or its ligand, herpesvirus entry mediator, but not PD-1, prompted their rapid rejection. By contrast, fully MHC-mismatched cardiac allografts were acutely rejected in wild-type recipients despite the induction of both BTLA and PD-1. Targeting of PD-1 in several fully MHC-mismatched models accelerated rejection, whereas targeting of BTLA unexpectedly enhanced PD-1 induction by alloreactive CD4 and CD8 T cells and prolonged allograft survival. In vitro studies using allogeneic dendritic cells and T cells showed that at low levels of T cell activation, BTLA expression was primarily induced, but that with increasing degrees of T cell activation, the expression of PD-1 was strongly up-regulated. These data suggest that BTLA and PD-1 exert distinct inhibitory actions in vivo, with the BTLA/herpesvirus entry mediator pathway appearing to dominate in regulating responses against a restricted degree of allogeneic mismatch.     

Host dendritic cells alone are sufficient to initiate acute graft-versus-host disease

Alloantigen expression on host APCs is essential to initiate graft-vs-host disease (GVHD); however, critical APC subset remains to be elucidated. We compared the ability of dendritic cells (DCs) and B cells to initiate acute GVHD by an add-back study of MHC class II-expressing APCs (II(+/+)) into MHC class II-deficient (II(-/-)) mice that were resistant to CD4-dependent GVHD. Injection of host-derived, but not donor-derived, II(+/+) DCs or host-derived II(+/+) B cells, was sufficient to break GVHD resistance of II(-/-) mice and induced lethal acute GVHD. By contrast, host-derived II(+/+) B cells, both naive and LPS stimulated, failed to induce activation or tolerance of donor CD4(+) T cells. Similarly, in a model of CD8-dependent GVHD across MHC class I mismatch injection of allogeneic DCs, but not B cells, induced robust proliferation of donor CD8(+) T cells and broke GVHD resistance of chimeric recipients in which APCs were syngeneic to donors. These results demonstrate that host-derived DCs are critical in priming donor CD4(+) and CD8(+) T cells to cause GVHD, and selective targeting of host DCs may be a promising strategy to prevent GVHD.     

Non-hematopoietic allograft cells directly activate CD8+ T cells and trigger acute rejection: an alternative mechanism of allorecognition

Despite evidence that human non-hematopoietic cells, such as vascular endothelium, can activate allogeneic T lymphocytes in vitro, the prevailing view has been that hematopoietic antigen-presenting cells are required to trigger alloimmune responses in vivo. Here we report that mouse non-hematopoietic cells activate alloreactive CD8+ T lymphocytes in vitro and in vivo. We also show that vascularized cardiac allografts are acutely rejected via CD8+ direct allorecognition even if the alloantigen is not presented by hematopoietic professional antigen-presenting cells. Because activation of alloreactive CD8+ T cells by donor-type non-hematopoietic cells can continue for the life of the allograft, these findings present a new clinically relevant mechanism of allorecognition and should be taken into consideration when developing strategies to prevent allograft vasculopathy or to induce tolerance.     

EBV-specific CD4+ T cell clones exhibit vigorous allogeneic responses

Alloreactive T cells play a key role in mediating graft-vs-host disease and allograft rejection, and recent data suggest that most T cell alloreactivity resides within the CD4 T cell subset. Particularly, T cell responses to herpesvirus can shape the alloreactive repertoire and influence transplantation outcomes. In this study, we describe six distinct EBV-specific CD4(+) T cell clones that cross-reacted with EBV-transformed lymphoblastoid cell lines (LCLs), dendritic cells, and endothelial cells expressing MHC class II alleles commonly found in the population. Allorecognition showed exquisite MHC specificity. These CD4(+) T cell clones efficiently killed dendritic cells or LCLs expressing the cross-reactive allogeneic MHC class II molecules, whereas they did not kill autologous LCLs. Endothelial cells expressing the proper allogeneic MHC molecules were poorly killed, but they induced high-level TNF-alpha production by the EBV-specific CD4(+) T cell clones. As already proposed, the strong alloreactivity toward LCLs suggest that these cells could be used for selective depletion of alloreactive T cells.     

Dynamics of monocytes/macrophages and T lymphocytes in acutely rejecting rat renal allografts

We examined the infiltration of acutely rejecting renal allografts (DA→LEW) by ED1⁺ and ED2⁺ macrophages and T lymphocytes at intervals of 24 h after transplantation. Donor and recipient macrophages were differentiated by MHC class II antigen expression in double-staining experiments with ED1. Proliferation was assayed after pulse-labelling with BrdU. We subdivided allograft infiltration into three consecutive phases: 1) During phase I on days 1 to 2 after allogeneic kidney transplantation, perivascular infiltrates developed that contained numerous donor and recipient macrophages. Allograft rejection could already be diagnosed 24?h after transplantation by perivascular infiltration of T lymphocytes, whereas T cells were rarely found in isografts. 2) Phase II of allograft rejection from day 3 to 4 was characterized by massive propagation of the infiltrate. About equal numbers of interstitial donor and recipient macrophages were counted. Both macrophages and T lymphocytes proliferated in situ and macrophages outnumbered T cells until complete rejection. 3) During phase III the allograft was destroyed. Large intravascular monocytes surprisingly expressed the ED2 antigen. In the interstitium of viable graft regions, the population of recipient macrophages grew, whereas the population of donor macrophages and of T lymphocytes decreased.     

Embryonic thymic epithelium naturally devoid of APCs is acutely rejected in the absence of indirect recognition.

Transplants of tissues depleted of passenger leukocytes are upon in vitro culture usually accepted in allogeneic recipients. Accordingly, fully allogeneic embryonic thymic epithelium was suggested to be poorly immunogenic. However, this tissue is capable of inducing donor-specific tolerance to peripheral tissues, when restoring T cell development in nude mice, through the production of regulatory cells. In the present work, adult immunocompetent allogeneic recipients were grafted with embryonic tissues isolated at stages before hemopoietic colonization or even before the establishment of circulation. Allogeneic thymic epithelium of day 10 embryos and heart primordium of day 8 embryonic donors were always rejected. Acute rejection of the thymic anlagen takes place in less than 12 days, with maximal CD4(+) and CD8(+) T cell infiltrates at 10 days post-transplant. In addition, a significant infiltrate of NK1.1(+) cells is observed, although without any essential role in this process. Furthermore, recipients lacking the indirect pathway of Ag presentation to CD4(+) T cells do not reveal any significant delay in rejection, even when CD8(+) T cells are also eliminated. Thus, our experimental approach reveals acute allograft rejection in the absence of all known pathways of naive T cell activation and therefore unveils a novel graft rejection mechanism that should be mediated by direct recognition of parenchymal cells. Given the importance of dendritic cells in naive T cell activation, it is likely that cross-reactive memory T cells may also drive rejection.     

Tolerance to antigen-presenting cell-depleted islet allografts is CD4 T cell dependent

Pretreatment of pancreatic islets in 95% oxygen culture depletes graft-associated APCs and leads to indefinite allograft acceptance in immunocompetent recipients. As such, the APC-depleted allograft represents a model of peripheral alloantigen presentation in the absence of donor-derived costimulation. Over time, a state of donor-specific tolerance develops in which recipients are resistant to donor APC-induced graft rejection. Thus, persistence of the graft is sufficient to induce tolerance independent of other immune interventions. Donor-specific tolerance could be adoptively transferred to immune-deficient SCID recipient mice transplanted with fresh immunogenic islet allografts, indicating that the original recipient was not simply "ignorant" of donor antigens. Interestingly, despite the fact that the original islet allograft presented only MHC class I alloantigens, CD8+ T cells obtained from tolerant animals readily collaborated with naive CD4+ T cells to reject donor-type islet grafts. Conversely, tolerant CD4+ T cells failed to collaborate effectively with naive CD8+ T cells for the rejection of donor-type grafts. In conclusion, the MHC class I+, II- islet allograft paradoxically leads to a change in the donor-reactive CD4 T cell subset and not in the CD8 subset. We hypothesize that the tolerant state is not due to direct class I alloantigen presentation to CD8 T cells but, rather, occurs via the indirect pathway of donor Ag presentation to CD4 T cells in the context of host MHC class II molecules.     

Donor MHC class II antigen is essential for induction of transplantation tolerance by bone marrow cells

Posttransplant infusion of donor bone marrow cells (BMC) induces tolerance to allografts in adult mice, dogs, nonhuman primates, and probably humans. Here we used a mouse skin allograft model and an allogeneic radiation chimera model to examine the role of MHC Ags in tolerance induction. Infusion of MHC class II Ag-deficient (CIID) BMC failed to prolong C57BL/6 (B6) skin grafts in ALS- and rapamycin-treated B10.A mice, whereas wild-type B6 or MHC class I Ag-deficient BMC induced prolongation. Removal of class II Ag-bearing cells from donor BMC markedly reduced the tolerogenic effect compared with untreated BMC, although graft survival was significantly longer in mice given depleted BMC than that in control mice given no BMC. Infusion of CIID BMC into irradiated syngeneic B6 or allogeneic B10.A mice produced normal lymphoid cell reconstitution including CD4+ T cells except for the absence of class II Ag-positive cells. However, irradiated B10.A mice reconstituted with CIID BMC rejected all B6 and a majority of CIID skin grafts despite continued maintenance of high degree chimerism. B10.A mice reconstituted with B6 BMC maintained chimerism and accepted both B6 and CIID skin grafts. Thus, expression of MHC class II Ag on BMC is essential for allograft tolerance induction and peripheral chimerism with cells deficient in class II Ag does not guarantee allograft acceptance.     

MHC-mismatched islet allografts are vulnerable to autoimmune recognition in vivo

When transplanted into type 1a diabetic recipients, islet allografts are subject both to conventional allograft immunity and, presumably, to recurrent autoimmune (islet-specific) pathogenesis. Importantly, CD4 T cells play a central role both in islet allograft rejection and in autoimmune disease recurrence leading to the destruction of syngeneic islet transplants in diabetic NOD mice. However, it is unclear how NOD host MHC class II (I-A(g7))-restricted, autoreactive CD4 T cells may also contribute to the recognition of allogeneic islet grafts that express disparate MHC class II molecules. We hypothesized that islet-specific CD4 T cells can target MHC-mismatched islet allografts for destruction via the "indirect" (host APC-dependent) pathway of Ag recognition. To test this hypothesis, we determined whether NOD-derived, islet-specific CD4 T cells (BDC-2.5 TCR transgenic cells) could damage MHC-mismatched islets in vivo independent of conventional allograft immunity. Results demonstrate that BDC-2.5 CD4 T cells can vigorously destroy MHC class II-disparate islet allografts established in NOD.scid recipients. Tissue injury is tissue-specific in that BDC-2.5 T cells destroy donor-type islet, but not thyroid allografts established in the same NOD.scid recipient. Furthermore, BDC-2.5 CD4 T cells acutely destroy MHC class II-deficient islet allografts in vivo, indicating that autoimmune pathogenesis can be completely independent of donor MHC class II expression. Taken together, these findings indicate that MHC-mismatched islet allografts can be vulnerable to autoimmune pathogenesis triggered by autoreactive CD4 T cells, presumably through indirect autoantigen recognition in vivo.     

Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients

4-1BB is expressed on activated CD4(+) and CD8(+) T cells; its ligand, 4-1BB ligand is expressed on APCs. Despite expression on both T cell subpopulations, 4-1BB has been reported to predominantly affect CD8(+) T cell responses. By quantifying graft-vs-host disease alloresponses in vivo, we demonstrate that both CD4(+) and CD8(+) T cell-mediated alloresponses are regulated by 4-1BB/4-1BB ligand interactions to approximately the same extent. 4-1BB receptor-facilitated CD4(+) T cell-mediated alloresponses were partly CD28 independent. In two distinct marrow graft rejection systems, host CD8(+) and CD4(+) T cells each separately contributed to host anti-donor T cell-mediated allograft rejection. alpha 4-1BB mAb increased the graft-vs-leukemia effect of a suboptimal number of donor splenocytes given later post bone marrow transplantation by bolstering allogeneic responses resulting in leukemia elimination. In summary, 4-1BB ligation is a potent regulator of CD4(+) and CD8(+) T cell-mediated allogeneic responses in vivo. Modifying the ligation of 4-1BB represents a new approach to altering the graft-vs-host disease and graft-vs-leukemia effects of allogeneic T cells post bone marrow transplantation.     

Comprehensive analysis of MHC-II expression in healthy human skin

A number of antigen-presenting cells (APCs) expressing major histocompatibility complex class II (MHC-II) have been identified in healthy human skin including the Langerhans cells of the epidermis and the three recently defined dermal APC subsets. It is well documented that in other tissues HLA-DR expression is not exclusive to APCs. Following a comprehensive analysis of the cells in human skin using flow cytometry and fluorescence immunohistochemistry, we have identified additional cell subsets that express HLA-DR. Using markers exclusive for blood and lymphatic endothelium, we demonstrated that both of these cell populations have the capacity to express HLA-DR. In addition, a small subset of dermal T lymphocytes was found to express low-level HLA-DR suggesting an activated phenotype. Dermal T lymphocytes were often in intimate contact with either CD1a(+) CD207(-) dermal APCs or CD1a(+) CD207(+) dermal Langerhans cells, possibly explaining the activated phenotype of a subset of dermal T lymphocytes.