*Purpose: HLA-E is an MHC class-1b molecule that is under-studied in the transplant setting. Recent clinical studies have revealed a robust association between allograft outcome and HLA-E polymorphism. Based on previous studies in autoimmune disease, we hypothesized that Qa-1, a mice homolog of HLA-E, and T cell receptors (TCR) of regulatory CD8 cells (CD8 Treg) binding plays key role in development of antibody mediated allograft rejection. To test this hypothesis, we used Qa-1 mutant mice (D227K) that harbor a single mutation that abrogates binding of Qa-1 to the CD8 TCR.

*Methods: Fully mismatched BALB/c hearts were heterotopically transplanted to C57BL/6 mice (WT) or D227K mice. Recipients received either high dose CTLA4-Ig [500υg on post-operative day (POD) 0 followed by 250υg on POD 2,4,6 and 8] or low dose CTLA4-Ig [250υg on POD 2 only]. Recipients treated with low dose CTLA4-Ig were sacrificed on POD 28 for mechanistic analysis. Grafts, spleens and lymph nodes were harvested for histology and immunophenotyping. Serum was collected for DSA measurement.

*Results: Preliminary data showed that D227K but not WT recipients were resistant to tolerance induction with high dose CTLA4-Ig. Similarly, D227K recipients of BALB/c hearts exhibited a shorter allograft survival compared to WT when treated with low dose CTLA4-Ig (MST 20 vs. 33, P<0.05, n=3-4/group). Harvested allografts revealed cardiomegaly with gross cellular infiltrations in D227K compared to WT. In parallel, we observed increased germinal center (GC) formation and size of spleens from D227K when compared to WT. Flow cytometry data from spleens (n=6/group) revealed significant fold change increase of effector CD4 (1.5, P<0.001), effector CD8 cells (2.2, P<0.01), germinal center follicular helper T cells (1.9, P<0.01), as well as class-switched B cells (2.4, P<0.05) and plasma cells (2.0, P<0.01) in D227K compared to WT. Similar trend was observed on draining lymph nodes analysis. We also found an 8-fold increase in allo-antibodies in the serum of D227K mice compared to WT mice (P<0.001).

*Conclusions: Our study shows that disrupting the binding of Qa-1-peptide complex and TCR on Qa-1-restricted CD8 Treg results in unchecked GC formation and follicular helper T cell proliferation leading to robust antibody mediated rejection. Where previous studies have focused on classical CD4 regulatory T cells, the significance of Qa-1/HLA-E suggests unmet needs in investigating the role of CD8 Treg in solid organ transplantation.

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