Relative Frequencies of Alloantigen-Specific Helper CD4 T Cells and B Cells Determine Mode of Antibody-Mediated Allograft Rejection.

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient (Tcrbd-/-) C57BL/6 recipients against donor H-2Kd MHC class I alloantigen was provided by adoptively transferred "TCR75" CD4 T cells that recognize processed H-2Kd allopeptide via the indirect-pathway. Transfer of large numbers (5 × 105) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2Kd humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient Rag2-/- recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) Tcrbd-/- recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to Rag2-/- recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of Sh2d1a-/- TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in Tcrbd-/- recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (103), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity. This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019.

Germinal Center Alloantibody Responses Mediate Progression of Chronic Allograft Injury.

Different profiles of alloantibody responses are observed in the clinic, with those that persist, often despite targeted treatment, associated with poorer long-term transplant outcomes. Although such responses would suggest an underlying germinal center (GC) response, the relationship to cellular events within the allospecific B cell population is unclear. Here we examine the contribution of germinal center (GC) humoral alloimmunity to chronic antibody mediated rejection (AMR). A murine model of chronic AMR was developed in which T cell deficient (Tcrbd-/-) C57BL/6 recipients were challenged with MHC-mismatched BALB/c heart allografts and T cell help provided by reconstituting with 103 "TCR75" CD4 T cells that recognize self-restricted allopeptide derived from the H-2Kd MHC class I alloantigen. Reconstituted recipients developed Ig-switched anti-Kd alloantibody responses that were slow to develop, but long-lived, with confocal immunofluorescence and flow cytometric characterization of responding H-2Kd-allospecific B cells confirming persistent splenic GC activity. This was associated with T follicular helper (TFH) cell differentiation of the transferred TCR75 CD4 T cells. Heart grafts developed progressive allograft vasculopathy, and were rejected chronically (MST 50 days), with explanted allografts displaying features of humoral vascular rejection. Critically, late alloantibody responses were abolished, and heart grafts survived indefinitely, in recipients reconstituted with Sh2d1a-/- TCR75 CD4 T cells that were genetically incapable of providing TFH cell function. The GC response was associated with affinity maturation of the anti-Kd alloantibody response, and its contribution to progression of allograft vasculopathy related principally to secretion of alloantibody, rather than to enhanced alloreactive T cell priming, because grafts survived long-term when B cells could present alloantigen, but not secrete alloantibody. Similarly, sera sampled at late time points from chronically-rejecting recipients induced more vigorous donor endothelial responses in vitro than sera sampled earlier after transplantation. In summary, our results suggest that chronic AMR and progression of allograft vasculopathy is dependent upon allospecific GC activity, with critical help provided by TFH cells. Clinical strategies that target the TFH cell subset may hold therapeutic potential. This work is composed of two parts, of which this is Part II. Please read also Part I: Alsughayyir et al., 2019.

CD8 T-cell recognition of acquired alloantigen promotes acute allograft rejection.

Adaptive CD8 T-cell immunity is the principal arm of the cellular alloimmune response, but its development requires help. This can be provided by CD4 T cells that recognize alloantigen "indirectly," as self-restricted allopeptide, but this process remains unexplained, because the target epitopes for CD4 and CD8 T-cell recognition are "unlinked" on different cells (recipient and donor antigen presenting cells (APCs), respectively). Here, we test the hypothesis that the presentation of intact and processed MHC class I alloantigen by recipient dendritic cells (DCs) (the "semidirect" pathway) allows linked help to be delivered by indirect-pathway CD4 T cells for generating destructive cytotoxic CD8 T-cell alloresponses. We show that CD8 T-cell-mediated rejection of murine heart allografts that lack hematopoietic APCs requires host secondary lymphoid tissue (SLT). SLT is necessary because within it, recipient dendritic cells can acquire MHC from graft parenchymal cells and simultaneously present it as intact protein to alloreactive CD8 T cells and as processed peptide alloantigen for recognition by indirect-pathway CD4 T cells. This enables delivery of essential help for generating cytotoxic CD8 T-cell responses that cause rapid allograft rejection. In demonstrating the functional relevance of the semidirect pathway to transplant rejection, our findings provide a solution to a long-standing conundrum as to why SLT is required for CD8 T-cell allorecognition of graft parenchymal cells and suggest a mechanism by which indirect-pathway CD4 T cells provide help for generating effector cytotoxic CD8 T-cell alloresponses at late time points after transplantation.

Operative salvage of radiocephalic arteriovenous fistulas by formation of a proximal neoanastomosis.

OBJECTIVE: We examined the outcomes of radiocephalic arteriovenous fistulas salvaged by formation of a neoanastomosis in the proximal cephalic vein segment. METHODS: Patients with a radiocephalic arteriovenous fistula revised by formation of a neoanastomosis in the proximal cephalic vein segment were identified from a prospectively maintained database and outcomes retrospectively analyzed. RESULTS: Eighty patients had 81 radiocephalic arteriovenous fistulas revised by formation of a neoanastomosis in the proximal cephalic vein segment. Failure to mature was the indication for revision in 39 (48.1%), 17 (21.0%) were revised for poor flows during dialysis, and 25 (30.9%) were performed for thrombosis. Primary patency of the 81 neoanastomoses at 12, 24, and 36 months was 78.5%, 68.9%, and 54.9%, respectively. Compared with neoanastomoses that were performed on 50 immature radiocephalic arteriovenous fistulas, those performed on the 31 mature fistulas exhibited improved patency rates (P = .04). There was no difference in the primary patency of the neoanastomosis between those performed for 25 failed fistulas and 56 failing (but patent) fistulas (P = .15). There was one case (1.2%) each of bleeding, infection, and steal after neoanastomosis. Four patients (4.9%) required further interventions on their neoanastomoses. CONCLUSIONS: Operative salvage of radiocephalic arteriovenous fistulas by formation of a neoanastomosis in the proximal cephalic vein segment demonstrates good patency and low complication rates and can be performed with reasonably good results in patients with failed or failing (but patent) radiocephalic arteriovenous fistulas. These patients should not automatically proceed to elbow fistula formation; rather, proximal neoanastomosis should be considered.

Treatment of dialysis access-associated steal syndrome with the "revision using distal inflow" technique.

PURPOSE: Dialysis access-associated steal syndrome (DASS) is a common, serious complication of antecubital fossa (ACF) arteriovenous fistulas (AVFs). We describe our experience of the "revision using distal inflow" (RUDI) technique for the treatment of DASS and review the literature. METHODS: Patients underwent fistula ligation at the anastomosis with re-establishment of inflow via the proximal radial or ulnar arteries using a venous interposition graft or venous collateral. A retrospective analysis of outcomes of all patients undergoing this procedure at our center was carried out. RESULTS: Seven patients with autogenous ACF AVFs underwent the RUDI procedure, four under local anesthesia. Interposition vein grafts were used in five patients, and inflow was achieved through the proximal radial artery in four cases. The median post-operative rise in digital systolic blood pressure was 65.5 mmHg. Follow-up at 7-36 months found that three fistulas had failed (one at 8 months, two within days), two patients had died with patent fistulas, one patient was transplanted with a functional AVF, and the remaining patient continues to dialyze through the fistula. No patients developed DASS post-operatively and no further interventions were required to maintain patency. CONCLUSIONS: Although RUDI was successful at treating DASS, a high rate of AVF failure was seen. With technical modifications and further experience, RUDI may become a valuable tool in the surgical armamentarium.