Michel Y Braun

A role for eosinophils in transplant rejection

Eosinophils release inflammatory mediators and cationic proteins that are instrumental in the pathogenesis of allergic diseases such as bronchial asthma. Here, we review experimental observations indicating that eosinophils are also involved in the rejection of allografts. We propose that their role as effectors of transplant damage becomes crucial when classical pathways of rejection are inhibited and T helper 2 (Th2) cells dominate the alloimmune response.

Mediation of acute but not chronic rejection of MHC-incompatible rat kidney grafts by alloreactive CD4 T cells activated by the direct pathway of sensitization

It has been previously postulated that there are two pathways of sensitization of MHC-incompatible kidney allografts: a direct pathway in which the host responder T cells are activated by MHC-incompatible passenger dendritic cells of the graft, and an indirect pathway, in which graft alloantigens are processed like “nominal” T cell antigens by host accessory cells, and presented as self-MHC restricted moieties. We show here that a rat AS anti-August alloreactive CD4+ T cell line, and a presumptive clone, activated through the direct pathway are capable in an adoptive transfer model of initiating rejection of normal August kidney grafts. However, neither the T cell line nor the presumptive clone initiates rejection of passenger cell-depleted August kidneys. The results support the hypothesis that direct pathway-sensitized T cells play a dominant role in early acute rejection, but not in chronic rejection.

Critical influence of natural regulatory CD25+ T cells on the fate of allografts in the absence of immunosuppression.

Background. Allografts are occasionally accepted in the absence of immunosuppression. Because naturally occurring CD4+CD25+ regulatory T cells (natural CD25+ Treg cells) have been shown to inhibit allograft rejection, we investigated their influence on the outcome of allografts in nonimmunosuppressed mouse recipients. Methods. We compared survival times of male CBA/Ca skin grafts in female CBA/Ca recipients expressing a transgenic anti-HY T-cell receptor on a RAG-1+/+ (A1[M]RAG+) or a RAG-1−/− (A1[M]RAG−) background. Depletion of natural CD25+ Treg cells in A1[M]RAG+ mice was achieved by in vivo administration of the PC61 monoclonal antibody. The influence of natural CD25+ Treg cells on the fate of major histocompatibility complex class II-mismatched (C57BL/6× bm12)F1 skin or bm12 heart transplants in C57BL/6 recipients was also assessed. Finally, we investigated the impact of natural CD25+ Treg cells on the production of T-helper (Th)1 and Th2 cytokines in mixed lymphocyte cultures between C57BL/6 CD4+ CD25− T cells as responders and bm12 or (C57BL/6× bm12)F1 antigen-presenting cells as stimulators. Results. Male allografts were spontaneously accepted by female A1(M)RAG+ mice but readily rejected by female A1(M)RAG+ mice depleted of natural CD25+ Treg cells by pretreatment with the PC61 monoclonal antibody. Depletion of CD25+ Treg cells also enhanced eosinophil-determined rejection of (C57BL/6× bm12)F1 skin grafts or bm12 cardiac grafts in C57BL/6 recipients. Finally, natural CD25+ Treg cells inhibited the production of interleukin (IL)-2, interferon-γ, IL-5, and IL-13 in mixed lymphocyte culture in a dose-dependent manner. Conclusion. Natural CD25+ Treg cells control Th1- and Th2-type allohelper T-cell responses and thereby influence the fate of allografts in nonimmunosuppressed recipients.

Acute Rejection in the Absence of Cognate Recognition of Allograft by T Cells

We studied the effects of the indirect pathway of allograft recognition using T cells from TCR transgenic Marilyn mice, which recognize the male Ag H-Y in an I-Ab-restricted fashion. The T cells are not alloreactive to the H-2k haplotype, because they are not activated when adoptively transferred into recombinase-activating gene-2−/− common γ-chain−/− double-mutant H-2k male or female mice. However, skin from H-2k males, but not from H-2k females, is acutely rejected by recombinase-activating gene-2−/− transgenic female recipients. In vitro, Marylin spleen cells primed by H-2k skin grafting proliferated and secreted both IL-4 and IFN-γ in response to H-2k male stimulators. However, the removal of H-2b APC from the responding population abolished the response. Taken together, these results show that the indirect recognition that triggers rejection in this model is due to the recognition of H-Y Ag shed from H-2k male allograft and presented by the recipient’s own I-Ab APC to transgenic T cells. This study demonstrates unequivocally the capacity of naive CD4+ T cells to promote the rejection of allografts through mechanisms that involve indirect destruction of grafted tissues.

IL‐5 and eosinophils mediate the rejection of fully histoincompatible vascularized cardiac allografts: regulatory role of alloreactive CD8+ T lymphocytes and IFN‐γ

CD8+ T lymphocytes are known to inhibit the development of eosinophilia and IL‐5 synthesis in models of experimental lung disease. In transplantation, the rejection of fully mismatched cardiac allografts by recipients depleted of CD8+ T cells is characterized by the recruitment of eosinophils in the rejected organs. We show here that this intragraft eosinophilia is dependent on the production of IL‐5 since hearts transplanted into IL‐5‐deficient recipients depleted of CD8+ cells did not contain eosinophils. More importantly, allograft survival was significantly extended in these animals. In mixed lymphocyte cultures (MLC), the presence of CD8+ T cells in the responding cell population inhibited the secretion of IL‐5. This inhibition was IFN‐γ dependent since adding neutralizing anti‐IFN‐γ antibodies induced the production of IL‐5. Furthermore, spleen cells isolated from IFN‐γ receptor (IFN‐γR)‐deficient mice secreted IL‐5 upon allogeneic stimulation in primary MLC. In vivo, eosinophilia was observed in allografts rejected by IFN‐γR‐deficient recipients. On the contrary, grafts rejected by IFN‐γR‐deficient mice treated with neutralizing anti‐IL‐5 antibodies did not exhibit eosinophilic infiltration. Our study reveals the capacity of IL‐5‐secreting CD4+ T cells and eosinophils to promote the rejection of heart allograft and demonstrates the importance of CD8+ T cells and IFN‐γ in regulating this pathway of rejection.

NK Cell Regulation of CD4 T Cell-Mediated Graft-versus-Host Disease

CD3-negative NK cells are granular lymphocytes capable of producing inflammatory cytokines and killing malignant, infected, or stressed cells. We have recently observed a new role for NK cells in the control of the proliferation of CD4 T cells under persistent antigenic stimulation. Monoclonal anti-male CD4 T cells transferred into Rag2−/− male recipients did not expand or were rapidly eliminated. Remarkably, T cells transferred into NK cell-deficient Rag2−/− Il-2Rγc−/− male hosts expanded extensively and mediated tissue lesions usually observed in chronic graft-versus-host disease (GVHD). T cell failure to proliferate and to induce chronic GVHD was the result of NK cell activity, because depletion of the recipient’s NK1.1+ cells by Ab treatment induced T cell expansion and chronic GVHD. T cells under chronic Ag stimulation upregulated ligands of the activating receptor NKG2D, and regulatory activity of NK cells was inhibited by the injection of Abs directed to NKG2D. On the contrary, blocking NKG2A inhibitory receptors did not increase NK cell regulatory activity. Finally, we show that NK regulation of T cell expansion did not involve perforin-mediated lytic activity of NK cells, but depended on T cell surface expression of a functional Fas molecule. These results highlight the potential role played by NK cells in controlling the Ag-specific CD4+ T cells responsible for chronic GVHD.

CD4+CD25+ and CD4+CD25− T Cells Act Respectively as Inducer and Effector T Suppressor Cells in Superantigen-Induced Tolerance

The repeated injection of low doses of bacterial superantigens (SAg) is known to induce specific T cell unresponsiveness. We show in this study that the spleen of BALB/c mice receiving chronically, staphylococcal enterotoxin B (SEB) contains SEB-specific CD4+ TCRBV8+ T cells exerting an immune regulatory function on SEB-specific primary T cell responses. Suppression affects IL-2 and IFN-γ secretion as well as proliferation of T cells. However, the suppressor cells differ from the natural CD4+ T regulatory cells, described recently in human and mouse, because they do not express cell surface CD25. They are CD152 (CTLA-4)-negative and their regulatory activity is not associated with expression of the NF Foxp3. By contrast, after repeated SEB injection, CD4+CD25+ splenocytes were heterogenous and contained both effector as well as regulatory cells. In vivo, CD4+CD25− T regulatory cells prevented SEB-induced death independently of CD4+CD25+ T cells. Nevertheless, SEB-induced tolerance could not be achieved in thymectomized CD25+ cell-depleted mice because repeated injection of SEB did not avert lethal toxic shock in these animals. Collectively, these data demonstrate that, whereas CD4+CD25+ T regulatory cells are required for the induction of SAg-induced tolerance, CD4+CD25− T cells exert their regulatory activity at the maintenance stage of SAg-specific unresponsiveness.

Bone marrow-derived immature dendritic cells prime in vivo alloreactive T cells for interleukin-4-dependent rejection of major histocompatibility complex class II antigen-disparate cardiac allograft

BACKGROUND Dendritic cells (DC) at the immature state express low levels of major histocompatibility complex and costimulatory molecules and are poor stimulators of primary T-cell response in vitro. Injection of immature bone marrow-derived DC, however, was shown to prime in vivo alloreactive CD4 T lymphocytes toward type 2 cytokine-producing cells in the absence of CD8 T-cell activation. METHODS We undertook the present study to determine whether Th2-immunization by immature DC could lead to allograft rejection. We first analyzed, in the major histocompatibility complex class II antigen-disparate B6-anti-bm12 combination, the capacity of immature DC to regulate the activity of alloreactive CD4 T cells. We then determined, in this model of weak antigenicity, whether injection of bm12 DC in B6 recipients before transplantation could modify the survival of vascularized bm12 cardiac allografts. RESULTS We confirmed that in vitro immature DC are poor stimulators of T-cell alloresponse. However, when given in vivo, immature bm12 DC primed anti-bm12 T cells for the production of interleukin (IL)-4. Moreover, they induced the acute rejection of bm12 cardiac allograft. The process of rejection was dependent on IL-4 because immunization of IL-4-deficient mice did not trigger rejection. CONCLUSIONS Allogeneic immature DC generated with granulocyte-macrophage colony-stimulating factor are potent stimulators of primary alloreactive response in vivo and prime for transplant rejection. Our results indicate that strategies based on immature DC for the induction of transplantation tolerance should be considered with caution.

Reviving Function in CD4+ T Cells Adapted to Persistent Systemic Antigen

In bone marrow-transplanted patients, chronic graft-versus-host disease is a complication that results from the persistent stimulation of recipient minor histocompatibility Ag (mHA)-specific T cells contained within the graft. In this study, we developed a mouse model where persistent stimulation of donor T cells by recipient’s mHA led to multiorgan T cell infiltration. Exposure to systemic mHA, however, deeply modified T cell function and chronically stimulated T cells developed a long-lasting state of unresponsiveness, or immune adaptation, characterized by their inability to mediate organ immune damages in vivo. However, analysis of the gene expression profile of adapted CD4+ T cells revealed the specific coexpression of genes known to promote differentiation and function of Th1 effector cells as well as genes coding for proteins that control T cell activity, such as cell surface-negative costimulatory molecules and regulatory cytokines. Strikingly, blockade of negative costimulation abolished T cell adaptation and stimulated strong IFN-γ production and severe multiorgan wasting disease. Negative costimulation was also shown to control lethal LPS-induced toxic shock in mice with adapted T cells, as well as the capacity of adapted T cells to reject skin graft. Our results demonstrate that negative costimulation is the molecular mechanism used by CD4+ T cells to adapt their activity in response to persistent antigenic stimulation. The effector function of CD4+ T cells that have adapted to chronic Ag presentation can be activated by stimuli strong enough to overcome regulatory signals delivered to the T cells by negative costimulation.

Foxp3+CD25+ T regulatory cells stimulate IFN-gamma-independent CD152-mediated activation of tryptophan catabolism that provides dendritic cells with immune regulatory activity in mice unresponsive to staphylococcal enterotoxin B.

Mice made unresponsive by repeated injection of staphylococcal enterotoxin B (SEB) contained SEB-specific CD25+CD4+TCRBV8+ T cells that were able to transfer their state of unresponsiveness to primary-stimulated T cells. About one-half of these cells stably up-regulated the expression of CD152. We undertook the present study to determine whether CD152high cells seen in this system were T regulatory cells responsible for suppression or whether they represented SEB-activated CD4+ T effector cells. Our results show that, among SEB-specific TCRBV8+ T cells isolated from unresponsive mice, all CD152highCD25+CD4+ T cells expressed Foxp3, the NF required for differentiation and function of natural T regulatory cells. Moreover, suppression by CD25+CD4+TCRBV8+ T cells was fully inhibited by anti-CD152 Abs. Following stimulation by soluble CD152-Ig, dendritic cells (DC) isolated from unresponsive mice strongly increased the expression and the function of indoleamine-2,3-dioxygenase (IDO), the enzyme responsible for the catabolism of tryptophan. This capacity to activate IDO was independent of IFN-γ production by DC because CD152-Ig stimulation of DC isolated from SEB-treated IFN-γ-deficient animals activated IDO expression and function. Finally, adding 1-methyl-tryptophan, an inhibitor of tryptophan catabolism, increased substantially the capacity of DC from unresponsive animals to stimulate primary T cell response toward SEB. Thus, we conclude that IFN-γ-independent CD152-mediated activation of tryptophan catabolism by Foxp3+CD25+ T regulatory cells provides DC with immune regulatory activity in mice unresponsive to SEB.