Augmentation of Recipient Adaptive Alloimmunity by Donor Passenger Lymphocytes within the Transplant.

Chronic rejection of solid organ allografts remains the major cause of transplant failure. Donor-derived tissue-resident lymphocytes are transferred to the recipient during transplantation, but their impact on alloimmunity is unknown. Using mouse cardiac transplant models, we show that graft-versus-host recognition by passenger donor CD4 T cells markedly augments recipient cellular and humoral alloimmunity, resulting in more severe allograft vasculopathy and early graft failure. This augmentation is enhanced when donors were pre-sensitized to the recipient, is dependent upon avoidance of host NK cell recognition, and is partly due to provision of cognate help for allo-specific B cells from donor CD4 T cells recognizing B cell MHC class II in a peptide-degenerate manner. Passenger donor lymphocytes may therefore influence recipient alloimmune responses and represent a therapeutic target in solid organ transplantation.

Diversity of the CD4 T Cell Alloresponse: The Short and the Long of It.

MHC alloantigen is recognized by two pathways: "directly," intact on donor cells, or "indirectly," as self-restricted allopeptide. The duration of each pathway, and its relative contribution to allograft vasculopathy, remain unclear. Using a murine model of chronic allograft rejection, we report that direct-pathway CD4 T cell alloresponses, as well as indirect-pathway responses against MHC class II alloantigen, are curtailed by rapid elimination of donor hematopoietic antigen-presenting cells. In contrast, persistent presentation of epitope resulted in continual division and less-profound contraction of the class I allopeptide-specific CD4 T cell population, with approximately 10,000-fold more cells persisting than following acute allograft rejection. This expanded population nevertheless displayed sub-optimal anamnestic responses and was unable to provide co-stimulation-independent help for generating alloantibody. Indirect-pathway CD4 T cell responses are heterogeneous. Appreciation that responses against particular alloantigens dominate at late time points will likely inform development of strategies aimed at improving transplant outcomes.

Copresentation of intact and processed MHC alloantigen by recipient dendritic cells enables delivery of linked help to alloreactive CD8 T cells by indirect-pathway CD4 T cells.

In transplantation, direct-pathway CD8 T cells that recognize alloantigen on donor cells require CD4 help for activation and cytolytic function. The ability of indirect-pathway CD4 T cells to provide this help remains unexplained, because a fundamental requirement for epitope linkage is seemingly broken. The simultaneous presentation, by host dendritic cells (DCs), of both intact MHC class I alloantigen and processed alloantigen would deliver linked help, but has not been demonstrated definitively. In this study, we report that following in vitro coculture with BALB/c DCs, small numbers (~1.5%) of C57BL/6 (B6) DCs presented acquired H-2(d) alloantigen both as processed allopeptide and as unprocessed Ag. This represented class I alloantigen provides a conformational epitope for direct-pathway allorecognition, because B6 DCs isolated from cocultures and transferred to naive B6 mice provoked cytotoxic CD8 T cell alloimmunity. Crucially, this response was dependent upon simultaneous presentation of class II-restricted allopeptide, because despite acquiring similar amounts of H-2(d) alloantigen upon coculture, MHC class II-deficient B6 DCs failed to elicit cytotoxic alloimmunity. The relevance of this pathway to solid-organ transplantation was then confirmed by the demonstration that CD8 T cell cytotoxicity was provoked in secondary recipients by transfer of DCs purified from wild-type, but not from MHC class II-deficient, C57BL/6 recipients of BALB/c heart transplants. These experiments demonstrate that representation of conformationally intact MHC alloantigen by recipient APC can induce cytotoxic alloimmunity, but simultaneous copresentation of processed allopeptide is essential, presumably because this facilitates linked recognition by indirect-pathway CD4 Th cells.

Germinal center alloantibody responses are mediated exclusively by indirect-pathway CD4 T follicular helper cells.

The durable alloantibody responses that develop in organ transplant patients indicate long-lived plasma cell output from T-dependent germinal centers (GCs), but which of the two pathways of CD4 T cell allorecognition is responsible for generating allospecific T follicular helper cells remains unclear. This was addressed by reconstituting T cell-deficient mice with monoclonal populations of TCR-transgenic CD4 T cells that recognized alloantigen only as conformationally intact protein (direct pathway) or only as self-restricted allopeptide (indirect pathway) and then assessing the alloantibody response to a heart graft. Recipients reconstituted with indirect-pathway CD4 T cells developed long-lasting IgG alloantibody responses, with splenic GCs and allospecific bone marrow plasma cells readily detectable 50 d after heart transplantation. Differentiation of the transferred CD4 T cells into T follicular helper cells was confirmed by follicular localization and by acquisition of signature phenotype. In contrast, IgG alloantibody was not detectable in recipient mice reconstituted with direct-pathway CD4 T cells. Neither prolongation of the response by preventing NK cell killing of donor dendritic cells nor prior immunization to develop CD4 T cell memory altered the inability of the direct pathway to provide allospecific B cell help. CD4 T cell help for GC alloantibody responses is provided exclusively via the indirect-allorecognition pathway.

Donor CD4 T cells contribute to cardiac allograft vasculopathy by providing help for autoantibody production.

BACKGROUND: The development of autoantibody after heart transplantation is increasingly associated with poor graft outcome, but what triggers its development and whether it has a direct causative role in graft rejection is not clear. Here, we study the development of antinuclear autoantibody in an established mouse model of heart allograft vasculopathy. METHODS AND RESULTS: Humoral vascular changes, including endothelial complement staining, were present in bm12 heart grafts, explanted 50 days after transplantation. Alloantibody was not detectable, but long-lasting autoantibody responses developed in C57BL/6 recipients from the third week after transplantation. No autoantibody was generated if donor CD4 T cells were depleted before heart graft retrieval or in recipients that lacked B-cell major histocompatibility complex class II expression, indicating that humoral autoimmunity is a consequence of donor CD4 T-cell allorecognition of the major histocompatibility complex class II complex on recipient autoreactive B cells. An effector role for autoantibody in graft rejection was confirmed by abrogation of humoral vascular rejection, and attenuation of vasculopathy, in B-cell deficient recipients and by development of vascular obliteration and accelerated rejection in recipients primed for autoantibody before transplantation. CONCLUSIONS: Passenger CD4 T cells within heart transplants can contribute to allograft vasculopathy by providing help to recipient B cells for autoantibody generation.

Abrogation of antibody-mediated allograft rejection by regulatory CD4 T cells with indirect allospecificity.

Alloantibody is an important effector mechanism for allograft rejection. In this study, we tested the hypothesis that regulatory T cells with indirect allospecificity can prevent humoral rejection by using a rat transplant model in which acute rejection of MHC class I-disparate PVG.R8 heart grafts by PVG.RT1(u) recipients is mediated by alloantibody and is dependent upon help from CD4 T cells that can recognize the disparate MHC alloantigen only via the indirect pathway. Pretransplant treatment of PVG.RT1(u) recipients with anti-CD4 mAb plus donor-specific transfusion abrogated alloantibody production and prolonged PVG.R8 graft survival indefinitely. Naive syngeneic splenocytes injected into tolerant animals did not effect heart graft rejection, suggesting the presence of regulatory mechanisms. Adoptive transfer experiments into CD4 T cell-reconstituted, congenitally athymic recipients confirmed that regulation was mediated by CD4 T cells and was alloantigen-specific. CD4 T cell regulation could be broken in tolerant animals either by immunizing with an immunodominant linear allopeptide or by depleting tolerant CD4 T cells, but surprisingly this resulted in neither alloantibody generation nor graft rejection. These findings demonstrate that anti-CD4 plus donor-specific transfusion treatment results in the development of CD4 regulatory T cells that recognize alloantigens via the indirect pathway and act in an Ag-specific manner to prevent alloantibody-mediated rejection. Their development is associated with intrinsic tolerance within the alloantigen-specific B cell compartment that persists after T cell help is made available.

Dendritic cells internalise and re-present conformationally intact soluble MHC class I alloantigen for generation of alloantibody.

Following organ transplantation soluble MHC class I is released from the graft and may contribute to alloimmunity. We determined in a well-established rat model whether DC are able to internalise soluble MHC class I alloantigen and then re-present intact alloantigen to B cells and T cells for generation of an alloantibody or CD8 T cell response. PVG.RT1(u) BM-derived DC internalised (via an active process) and retained intact a recombinant soluble form of RT1-A(a) (sRT1-A(a)). When PVG.RT1(u) rats were immunised with sRT1-A(a)-pulsed syngeneic DC, they developed a strong anti-sRT1-A(a) alloantibody response and showed accelerated rejection of RT1-A(a)-disparate PVG.R8 heart grafts. Alloantibody production and accelerated heart graft rejection were both dependent on immunisation with viable sRT1-A(a)-pulsed DC. The alloantibody response to sRT1-A(a)-pulsed DC was directed exclusively against conformational epitopes expressed by sRT1-A(a) and not epitopes expressed, for example, by non-conformational sRT1-A(a) heavy chain. Immunisation with sRT1-A(a)-pulsed syngeneic DC did not stimulate a CD8 T cell response. Our findings suggest a novel alloantigen recognition pathway whereby soluble MHC class I alloantigen released from an allograft may be taken up by recipient DC and presented in an intact unprocessed form to B cells for the generation of an alloantibody response.

Evidence of macrophage receptors capable of direct recognition of xenogeneic epitopes without opsonization.

BACKGROUND: We have previously demonstrated that porcine livers perfused with human blood remove most of the erythrocytes from three units of human blood over the course of a 72-h extracorporeal perfusion. Red blood cell loss did not appear to involve classical complement pathway-mediated hemolysis, but instead resulted from porcine Kupffer cell phagocytosis. METHODS: We developed a method incorporating collagenase digestion and metrizamide separation to isolate and maintain porcine Kupffer cells in primary culture. An in vitro rosetting assay was used to assess the binding of human and porcine erythrocytes to porcine Kupffer cells. Immunohistochemistry was used to confirm the presence of porcine macrophages. The rosetting assay was quantified using 51Cr-labeling of erythrocytes to assay for both rosette formation and phagocytosis. RESULTS: Porcine Kupffer cells were successfully isolated and maintained in primary culture. The presence of porcine macrophages was confirmed using the monoclonal antibody 74-22-15A. Human, but not porcine, erythrocytes were bound in an in vitro rosetting assay as confirmed by immunohistochemistry, electron microscopy and 51Cr-quantitation. Porcine Kupffer cells bound human erythrocytes regardless of the presence of opsonizing antibody. Approximately 70% of the isolated porcine Kupffer cells demonstrated the capacity to bind non-opsonized human erythrocytes. Phagocytosis was not observed. CONCLUSIONS: Using primary porcine Kupffer cell cultures, we have demonstrated that a subpopulation of porcine macrophages has the ability to recognize specifically xenogeneic human erythrocyte epitopes without the need for prior opsonization. The possibility is discussed that lectin-mediated carbohydrate binding plays a role in the cellular and humoral recognition and rejection of xenografts.

Classical pathway complement destruction is not responsible for the loss of human erythrocytes during porcine liver perfusion.

BACKGROUND: Porcine livers perfused with human blood destroy 85% of human erythrocytes (red blood cells [RBC]) during prolonged extracorporeal perfusion, raising the possibility of a complement-mediated graft-versus-host effect. METHODS: Isolated porcine livers were perfused with fresh human blood. Plasma samples were analyzed for complement production by reverse CH50 analysis and porcine immunoglobulin class and specificity by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Anti-CD59 and anti-decay accelerating factor (DAF) monoclonal antibody were used to investigate whether human complement regulatory proteins inhibit porcine complement. RESULTS: After 64 hr of perfusion of porcine livers with human blood, mean complement activity in the perfusate was 95% of the starting value and increasing, whereas perfusion in the absence of a liver showed a falling complement activity of 28.7%. ELISA demonstrated porcine immunoglobulin (Ig) G and IgM in the xenoperfused human plasma. Whereas in a previous study flow cytometry demonstrated porcine antibodies specific for antigens on human T lymphocytes, in this study, anti-human RBC antibodies were not found. Xenoperfused human plasma did not lyse fresh human RBC. Human complement was consistently more efficient at lysing porcine RBC than was porcine complement at lysing human RBC, and human plasma inhibited the ability of porcine plasma to lyse human RBC, raising the possibility of cross-species complement regulation. Complement regulatory proteins on human RBC were blocked using mouse monoclonal anti-human CD59 and DAF. Blocking CD59, but not DAF, augmented lysis of human RBC by porcine complement. CONCLUSIONS: Human CD59 inhibits porcine complement. The production of porcine complement from xenoperfused porcine livers is unlikely to result in clinically significant injury mediated through the classical pathway of complement activation.