CD27low NK Cells Prolong Allograft Survival By Inhibiting Alloreactive CD8+ T Cell Responses.

Abstract

While NK cells are emerging as key effector cells in alloimmune responses, the underlying mechanisms by which distinct NK cell subsets promote either allograft rejection or tolerance remain unclear. Therefore, the purpose of this study was to investigate the role of distinctively mature NK cell subsets in alloimmunity in the context of their regulation by T-box-transcription factors. Since T-bet controls the maturation of NK cells from immature CD27high to terminally-differentiated CD27low NK cells, we created BALB/c Rag-KO mice on a T-bet-KO background to study the different roles of CD27low vs CD27high NK cells in a model of T cell-mediated allograft rejection. For this, Rag-T-bet double-KO (DKO) and Rag-KO recipients of fully mismatched B6 skin allografts (STx) were adoptively transferred with purified T cells from Foxp3-gfp-knockin BALB/c mice. Groups of recipient mice were treated with CTLA4Ig and anti-CD154 (MR-1) on day 0, 2, 4 and 6 after reconstitution. Alloimmune responses were analyzed by histology, ELISA, RT-PCR and flow cytometry. First, we found by flow cytometry that Rag-T-bet DKO, but not Rag-KO, mice fully lack mature CD27lowNKp46+ NK cells in the periphery, confirming that our model allows us to study different NK cell subsets in alloresponses. Importantly, T cell-reconstituted Rag-KO recipients (with CD27low NK cells) showed significantly prolonged allograft survival upon costimulatory blockade (MST 35±3.4d) when compared to DKO mice lacking CD27low NK cells (28±1.2d), indicating that CD27low NK cells promote allograft survival in Rag-KO mice. Critically, in the DKO recipients memory CD8+ T cells showed a strikingly increased proliferation response (CFSE assay) and higher IFN-γ production (3.7±0.5 vs 1.7±0.5 × 106 IFN-γ+ cells). Therefore, we hypothesized that CD27low NK cells regulate alloreactive CD8+ T cell responses under costimulatory blockade conditions. To test this, we transferred CD27low NK cells into DKO STx recipients and found that reconstitution with CD27low NK cells restores prolonged allograft survival (40.4±5.0d vs 29.6±0.6d) and reduces the proliferation of alloreactive IFN-γ+CD8+ T cells. We conclude that mature CD27low NK cells promote allograft survival under costimulatory blockade conditions by inhibiting alloreactive CD8+ T cell responses.