M. Negus

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.

Pathways of helper CD4 T cell allorecognition in generating alloantibody and CD8 T cell alloimmunity.

Background. The relative contributions of the “direct” and “indirect” pathways of CD4 T cell allorecognition in providing help for generating effective humoral and CD8 T cell alloimmunity remain unclear. Here, the generation of alloantibody and cytotoxic CD8 T cell responses to a vascularized allograft were examined in a murine adoptive-transfer model in which help could only be provided by transferred CD4 T cells recognizing alloantigen exclusively through the direct pathway. Methods. Rejection kinetics and the development of alloantibody and cytotoxic CD8 T cell responses to MHC-mismatched H-2d heart grafts were compared when CD4 T cell help was present (wild-type H-2b recipients), or absent (CD4 T cell deficient, MHC class II−/− H-2b recipients [B6CII−/−]), or available only through the direct pathway (B6CII−/− mice reconstituted with wild-type CD4 T cells). Results. BALB/c allografts were rejected by B6 mice rapidly (median survival time [MST] 7 days) with strong CD8 T cell effector and alloantibody responses, but were rejected by B6CII−/− mice more slowly (MST 23 days), with markedly reduced CD8 T cell responses and no detectable alloantibody. CD4 T cell reconstitution of B6CII−/− recipients accelerated heart graft rejection to near that of wild-type recipients (MST 13 days), with complete restoration of cytotoxic CD8 T cell responses but without detectable IgM or IgG alloantibody. Conclusions. Different pathways of helper T cell allorecognition are responsible for generating humoral and CD8 T cell alloimmunity. CD4 T cell help provided exclusively through the direct pathway generates strong cytotoxic CD8 T cell responses that effect rapid heart graft rejection.

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.RT1u 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.RT1u 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.

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-2d 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-2d 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.

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

Summary 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.

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.RT1u BM‐derived DC internalised (via an active process) and retained intact a recombinant soluble form of RT1‐Aa (sRT1‐Aa). When PVG.RT1u rats were immunised with sRT1‐Aa‐pulsed syngeneic DC, they developed a strong anti‐sRT1‐Aa alloantibody response and showed accelerated rejection of RT1‐Aa‐disparate PVG.R8 heart grafts. Alloantibody production and accelerated heart graft rejection were both dependent on immunisation with viable sRT1‐Aa‐pulsed DC. The alloantibody response to sRT1‐Aa‐pulsed DC was directed exclusively against conformational epitopes expressed by sRT1‐Aa and not epitopes expressed, for example, by non‐conformational sRT1‐Aa heavy chain. Immunisation with sRT1‐Aa‐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.

Diversity of the CD4 T cell alloresponse: the short and the long of it

Summary 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.

ACQUISITION OF INTACT ALLOANTIGEN BY RECIPIENT DCS PROVOKES CYTOTOXIC CD8 T CELL AND ALLOANTIBODY RESPONSES.: 3054

S. Sivaganesh1, C.J. Callaghan2, T. Conlon3, R. Motallebzadeh4, K. Saeb-Parsy3, M. Negus3, E.M. Bolton3, J.A. Bradley3, G.J. Pettigrew3 1Surgery, University of Cambridge, Cambridge/UNITED KINGDOM, 2Department Of Surgery, Addenbrooke’s Hospital, University of Cambridge, Cambridge/UNITED KINGDOM, 3Department Of Surgery, University of Cambridge, Cambridge/UNITED KINGDOM, 4Surgery, Cambridge University, Cambridge/UNITED KINGDOM

CYTOTOXIC CD8 T CELLS RECEIVE HELP FROM INDIRECT PATHWAY CD4 T CELLS BY PRESENTING PROCESSED ALLOANTIGEN ON ACQUIRED MHCII: 1729

Introduction: Indirect CD4 T cell allorecognition is fundamental to allograft rejection, but how this primes development of effector mechanisms for graft destruction is not clear. We investigate how indirect-pathway CD4 T cells that recognise processed alloantigen presented by recipient APCs provide ‘unlinked’ help for direct pathway cytotoxic CD8 T cells recognising intact allo-MHCI on donor cells. Methods: Female Mar (B6 RAG2-/-) mice, whose monoclonal CD4 T cells recognise self-restricted male H-Y peptide, were reconstituted with effector female B6 CD8 T cells and received BALB/c or B6xBALB/c heart transplants. CD8 and CD4 T cell activation was analysed using IFN-γ ELISPOT and division of CFSE-labelled Mar cells. Results: Mar recipients rejected male BALB/c heart grafts acutely (Fig 1A), but only if reconstituted with WT CD8 T cells. Female BALB/c grafts survived >50d, confirming that CD8 T cellmediated rejection was dependent on indirect help from Mar CD4 T cells. Surprisingly, although the epitopes for Mar CD4 and CD8 T cell recognition are on different APCs (recipient vs donor), CD8 T cell-reconstituted Mar recipients challenged with male B6 APCs (to activate Mar CD4 T cells) mounted minimal cytotoxic CD8 T cell responses and did not reject female BALB/c grafts. Effective help was thus only generated when H-Y and MHCI alloantigens were co-expressed on graft cells. We hypothesised that, analogous to the provision of cognate T cell help to B cells, this requirement for co-expression reflects acquisition of H-Y antigen from graft cells by allospecific CD8 T cells, with subsequent processing and presentation in the context of MHCII for Mar CD4 T cell recognition. In support, Mar recipients reconstituted with MHCII-/CD8 T cells rejected male BALB/c grafts more slowly and reconstitution with CD8 T cells from H-2DMa mice (which are unable to process antigen) resulted in indefinite graft survival. In contrast, male B6xBALB/c grafts (which enable ‘linked’ help via direct allorecognition of both Mar CD4 and CD8 T cell epitopes on donor APCs) were rejected rapidly when reconstituted with WT, MHCII-/or H-2DMa CD8 T cells (fig 1A), indicating that antigen processing and MHCII expression by CD8 T cells is necessary only for indirect-pathway CD4 T cell help. Finally, mouse CD8 T cells, which do not normally express MHCII, acquired MHCII upon culture with WT APCs (fig 1B) and also when adoptively transferred to MHCII+/+ TCR-/mice which lack T cells (fig 1C). Conclusion: Indirect pathway CD4 T cells provide help to allospecific CD8 T cells through recognition of alloantigen that is internalised by CD8 T cells via the TCR and presented as processed allopeptide on acquired MHC II.