Fady K. Baddoura

Recall and propagation of allospecific memory T cells independent of secondary lymphoid organs

The allospecifc T cell population responding to a transplanted organ consists of both naïve and memory lymphocytes. Although it is established that naive T cells are activated by antigen within the organized structures of secondary lymphoid organs (the spleen, lymph nodes, and mucosal lympoid tissues), it is not clear whether memory T cell activation and propagation depend on homing to these organs. To answer this question, we investigated whether allospecific naïve or memory T cells can mediate acute cardiac allograft rejection in mutant mice that lack all of their secondary lymphoid tissues. The results of our experiments demonstrated that antigen-experienced memory T cells have two advantages over naïve T cells: (i) memory T cells mount a vigorous immune response that leads to allograft rejection independent of secondary lymphoid organs; and (ii) memory T cells generate more memory T cells without homing to secondary lymphoid organs. These unique properties of memory T cells were further confirmed by showing that memory-like T cells that arise from the homeostatic proliferation of naive T cells in the absence of antigenic stimulation are suboptimal at rejecting allografts and do not generate memory T cells in mice devoid of secondary lymphoid tissues.

Technical and immunological complications in pediatric liver transplantation: A multivariate analysis in 100 living-related and 135 post-mortem donor grafts.

Lymphoid neogenesis is the process by which ectopic lymphoid accumulations that resemble lymph nodes arise in nonlymphoid tissues. Such lymphoid accumulations, known as tertiary lymphoid organs (TLO), are observed in chronic autoimmunity and they propagate immune pathology by setting up local antigen presenting sites. Whether lymphoid neogenesis occurs in transplanted organs and contributes to rejection is not well understood. To begin to address this question, we retrospectively analyzed 319 murine cardiac allografts for microscopic evidence of lymph‐node‐like structures. We found 78 allografts that had either classical TLO, characterized by discrete T‐ and B‐cell zones and high endothelial venules (HEV) expressing peripheral node addressin (PNAd) (n = 34), or PNAd+ HEV without organized lymphoid accumulations (n = 44). These changes were present in both short‐ and long‐lived allografts and were invariably associated with rejection. Importantly, they occurred in 78% of allografts undergoing chronic rejection (n = 85) but in only 7% of allografts undergoing primarily acute rejection (n = 184). These findings indicate that, like autoimmunity, alloimmunity is associated with lymphoid neogenesis in the target organ and suggest a role for local T‐cell activation in chronic allograft rejection.

Acute rejection of vascularized heart allografts in the absence of IFNgamma.

It is generally assumed that IFNgamma plays a central role in acute allograft rejection. To test this hypothesis, we transplanted fully allogeneic (MHC class I and II incompatible) C3H/HeJ (H2k) murine hearts to IFNgamma-/- (IFNgamma gene-knockout) and IFNgamma+/+ BALB/c (H2d) mice. The phenotype of IFNgamma-/- mice was confirmed by demonstrating absent IFNgamma protein production by Con A stimulated IFNgamma-/- splenocytes. Both IFNgamma-/- and IFNgamma+/+ strains rejected transplanted hearts acutely: graft survival (mean +/- SD) was 5.2+/-0.4 and 6.0+/-0.0 days, respectively. Histologic examination revealed similar patterns of acute cellular rejection in both mouse groups. IFNgamma mRNA was present in hearts rejected by IFNgamma+/+ mice but was absent in those rejected by IFNgamma-/- mice. IL-2, IL-4, IL-10, and TNFalpha mRNA expression, on the other hand, was similar in grafts rejected by either strain. We also observed that hapten-induced delayed-type hypersensitivity (DTH) response was significantly reduced but not absent in IFNgamma-/- mice. Our results demonstrate that IFNgamma is not required for acute cellular rejection of fully allogeneic murine hearts. We propose that non-DTH mechanisms of allograft destruction could be enhanced in the absence of IFNgamma and thus lead to robust acute rejection.

Tertiary Lymphoid Tissues Generate Effector and Memory T Cells That Lead to Allograft Rejection

Tertiary lymphoid tissues are lymph node‐like cell aggregates that arise at sites of chronic inflammation. They have been observed in transplanted organs undergoing chronic rejection, but it is not known whether they contribute to the rejection process by supporting local activation of naïve lymphocytes. To answer this question, we established a murine transplantation model in which the donor skin contains tertiary lymphoid tissues due to transgenic expression of lymphotoxin‐α(RIP‐LTα), whereas the recipient lacks all secondary lymphoid organs and does not mount primary alloimmune responses. We demonstrate in this model that RIP‐LTα allografts that harbor tertiary lymphoid tissues are rejected, while wild‐type allografts that lack tertiary lymphoid tissues are accepted. Wild‐type allografts transplanted at the same time as RIP‐LTα skin or 60 days later were also rejected, suggesting that tertiary lymphoid tissues, similar to secondary lymphoid organs, generate both effector and memory immune responses. Consistent with this observation, naive T cells transferred to RIP‐LTα skin allograft but not syngeneic graft recipients proliferated and differentiated into effector and memory T cells. These findings provide direct evidence that tertiary lymphoid structures perpetuate the rejection process by supporting naïve T‐cell activation.

The Allograft Defines the Type of Rejection (Acute versus Chronic) in the Face of an Established Effector Immune Response

Transplanted organs fail due to either acute or chronic rejection. The prevailing view is that the nature or magnitude of the recipient’s immune response to donor Ags determines the type of rejection. In variance with this view, we show in this study that the status of the graft itself plays a dominant role in defining the type of rejection even in the face of an established alloimmune response. Using adoptive transfer mouse models in which the graft is exposed to a constant number of effector lymphocytes, we found that newly transplanted heart allografts are rejected acutely, while healed-in allografts undergo chronic rejection. Acute rejection of healed-in allografts was largely recapitulated by subjecting the grafts to ischemia-reperfusion injury similar to that present in newly transplanted organs. Ischemia-Reperfusion injury altered the outcome of rejection by enhancing the accumulation of effector T cells within the graft. The accumulation of effector T cells in the graft was dependent on the presence of both ischemia-reperfusion injury (inflammation) and alloantigens. These findings demonstrate that the graft plays a dominant role in shaping the outcome of rejection by controlling the trafficking of effector T cells.

Interferon-gamma is necessary for initiating the acute rejection of major histocompatibility complex class II-disparate skin allografts.

BACKGROUND Although interferon (IFN)gamma has immunostimulatory functions, it is not essential for the acute rejection of fully allogeneic grafts in mice. It is not known whether IFNgamma plays a critical role in the acute rejection of MHC class I- or MHC class II-disparate allografts. METHODS We studied the survival of skin allografts transplanted from fully allogeneic (BALB/c), MHC class I-disparate (bml), or MHC class II-disparate (bm12) donors to C57BL/6 wild-type (IFNgamma+/+) and IFNgamma gene-knockout (IFNgamma-/-) recipients. We also investigated the in vitro responses of IFNgamma+/+ and IFNgamma-/- T cells to MHC class II-disparate splenocytes. RESULTS We found that IFNgamma-/- recipients reject BALB/c and bml skin grafts at the same rate as IFNgamma+/+ mice but are not capable of rejecting bm12 skin. Despite the inability of IFNgamma-/- mice to reject bm12 skin grafts, IFNgamma-/- T cells displayed vigorous proliferation and cytotoxic responses when stimulated with bm12 splenocytes in vitro. Furthermore, priming IFNgamma-/- recipients with bm12 splenocytes enabled these mice to reject bm12 skin grafts at a normal rate and to mount a cutaneous delayed-type hypersensitivity response to the bm12 antigen. CONCLUSION The data demonstrate that IFNgamma is not necessary for generating effector mechanisms associated with acute transplant rejection but that it is required for initiating alloimmune responses to MHC class II-disparate skin grafts.

Direct Antigen Presentation by a Xenograft Induces Immunity Independently of Secondary Lymphoid Organs

The location of immune activation is controversial during acute allograft rejection and unknown in xenotransplantation. To determine where immune activation to a xenograft occurs, we examined whether splenectomized alymphoplastic mice that possess no secondary lymphoid organs can reject porcine skin xenografts. Our results show that these mice rejected their xenografts, in a T cell-dependent fashion, at the same tempo as wild-type recipients, demonstrating that xenograft rejection is not critically dependent on secondary lymphoid organs. Furthermore, we provide evidence that immune activation in the bone marrow did not take place during xenograft rejection. Importantly, immunity to xenoantigens was only induced after xenotransplantation and not by immunization with porcine spleen cells, as xenografted mutant mice developed an effector response, whereas mutant mice immunized by porcine spleen cells via i.p. injection failed to do so. Moreover, we provide evidence that antixenograft immunity occurred via direct and indirect Ag presentation, as recipient T cells could be stimulated by either donor spleen cells or recipient APCs. Thus, our data provide evidence that direct and indirect Ag presentation by a xenograft induces immunity in the absence of secondary lymphoid organs. These results have important implications for developing relevant xenotransplantation protocols.

Innate immunity alone is not sufficient for chronic rejection but predisposes healed allografts to T cell-mediated pathology.

BACKGROUND Acute allograft rejection is dependent on adaptive immunity, but it is unclear whether the same is true for chronic rejection. Here we asked whether innate immunity alone is sufficient for causing chronic rejection of mouse cardiac allografts. METHODS We transplanted primarily vascularized cardiac grafts to recombinase activating gene-knockout (RAG(-/-)) mice that lack T and B cells but have an intact innate immune system. Recipients were left unmanipulated, received adjuvants that stimulate innate immunity, or were reconstituted with B-1 lymphocytes to generate natural IgM antibodies. In a second model, we transplanted cardiac allografts to mice that lack secondary lymphoid tissues (splenectomized aly/aly recipients) and studied the effect of NK cell inactivation on T cell-mediated chronic rejection. RESULTS Acute cardiac allograft rejection was not observed in any of the recipients. Histological analysis of allografts harvested 50 to 90 days after transplantation to RAG(-/-) mice failed to identify chronic vascular or parenchymal changes beyond those observed in control syngeneic grafts. Chronic rejection of cardiac allografts parked in splenectomized aly/aly mice was observed only after the transfer of exogenously activated T cells. NK inactivation throughout the experiment, or during the parking period alone, reduced the severity of T cell-dependent chronic rejection. CONCLUSIONS The innate immune system alone is not sufficient for causing chronic rejection. NK cells predispose healed allografts to T cell-dependent chronic rejection and may contribute to chronic allograft pathology.