The durable alloantibody responses that develop in organ transplant patients indicate long-lived plasma cell output from T-dependent germinal centres (GCs), but which of the two pathways of CD4 T cell allorecognition are responsible for generating allospecific T follicular helper (TFH) cells remains unclear. This was addressed by reconstituting T-cell deficient mice with monoclonal populations of TCR-transgenic CD4 T cells that recognised 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 days after heart transplantation. Differentiation of the transferred CD4 T cells into TFH cells was confirmed by follicular localisation 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 immunisation 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.

Tertiary lymphoid organs (TLOs) may develop within allografts, but their contribution to graft rejection remains unclear. Here, we study a mouse model of autoantibody-mediated cardiac allograft vasculopathy to clarify the alloimmune responses mediated by intragraft TLOs and whether blocking lymphotoxin-β-receptor (LTβR) signaling, a pathway essential for lymphoid organogenesis, abrogates TLO development. TLOs (defined as discrete lymphoid aggregates associated with high endothelial venules) were detectable in 9 of 13 heart allografts studied and were predominantly B cell in composition, harboring germinal-center activity. These are most likely manifestations of the humoral autoimmunity triggered in this model after transplantation; TLOs did not develop if autoantibody production was prevented. Treatment with inhibitory LTβR-Ig fusion protein virtually abolished allograft TLO formation (mean TLOs/heart: 0.2 vs. 2.2 in control recipients; P=0.02), with marked attenuation of the autoantibody response. Recipients primed for autoantibody before transplantation rejected grafts rapidly, but this accelerated rejection was prevented by postoperative administration of LTβR-Ig (median survival time: 18 vs. >50 d, respectively, P=0.003). Our results provide the first demonstration that TLOs develop within chronically rejecting heart allografts, are predominantly B cell in origin, and can be targeted pharmacologically to inhibit effector humoral responses.

Background-The development of autoantibody after heart transplantation is increasingly associated with poor graftoutcome, 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. (Circ Heart Fail. 2009;2:361-369.)

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Introduction We have recently demonstrated that a tolerant state among B cells responding to blood group-A antigens develops following blood group A-to-O pediatric liver transplantation. Blood group antigen-reactive B cells might be tolerized through their interaction with the liver sinusoidal endothelial cells (LSECs), which exclusively express blood group antigens together with Fas-L and PDL1. Methods In order to address this possibility, we have used α1,3-galactosyltransferase-deficient (GalT -/-) mice, since the α-Gal epitope is very similar in structure for blood groups A and B. Immune fluorescence staining of the wild-type mouse livers reveals that Gal epitopes predominantly express on the LSECs, resembling blood group antigens in human livers. The α-Gal-expressing LSECs isolated from wild-type GalT +/+ B6 mice were adoptively transferred via the portal vein into the congeneic GalT -/mice (4 × 10 6 cells/mouse); they were intraperitoneally injected 2 days before the transplantation with the pyrrolizidine alkaloid monocrotaline (3-400 mg/kg), a genotoxin, which impaired host LSECs, conferring proliferative advantage to the transplanted LSECs. Three to four weeks after the adoptive transfer of LSECs, the recipient mice underwent myeloablative radiation and reconstitution with bone marrow cells (BMCs) with/ without splenocytes from GalT -/mice. Four weeks after the immune reconstruction, the recipient mice were immunized with α-Galexpressing rabbit erythrocytes. Results After the immunization, high levels of anti-Gal Abs were detected in the sera of the GalT -/mice that had not received LSECs but were repopulated with BMCs from GalT -/mice. In contrast, anti-Gal Abs were persistently undetectable in the sera of the GalT -/mice that had received LSECs from GalT +/+ mice and were consequently repopulated with BMCs from GalT -/mice. To rule out the possibility that anti-Gal Abs were merely absorbed by the Gal epitope expressed on the engrafted GalT +/+ LSECs, the presence of anti-Gal-producing cells was assessed by ELISPOT assay. Cells producing anti-Gal Abs were undetectable in the bone marrow of those GalT -/mice that had received LSECs from GalT +/+ mice. In contrast, in the GalT -/mice repopulated with both BMCs and splenocytes from GalT -/mice, anti-Gal Abs were highly detectable, despite LSEC engraftment from GalT +/+ mice. In addition, when the LSECs from GalT +/+ B6 mice were adoptively transferred into the splenectomized congeneic GalT -/mice, these GalT -/mice lost their ability to produce anti-αGal Abs. Even when the LSECs from FasL-deficient (c-gld) GalT +/+ B6 mice were adoptively transferred into the splenectomized congeneic GalT -/mice, the GalT -/recipient mice still lost their ability to produce anti-αGal Abs. Conclusion These findings suggest that after ABO-incompatible liver transplantation, the LSECs tolerize immature B cells but do not tolerize mature B cells specific for blood group carbohydrate antigens. For inducing such immune-regulatory effects of the LSECs on B cells, Fas/FasL pathway m...

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