Overcoming NK Cell-Mediated Allograft Rejection By Anti-3rd Party Central Memory Veto CD8 T Cells through Down-Regulation of the Activating Receptor DNAM-1 on Alloreactive NK Cells

• Published in: Blood 2024-11, Vol.144 (Supplement 1), p.3401-3401
• Main Authors: Liu, Wei-Hsin, Singh, Aloukick, Yadav, Sandeep, Bachar Lustig, Esther, Reisner, Yair
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2024-203601

Allograft rejection is a major challenge for allogeneic chimeric antigen receptor (CAR) cell therapy, particularly due to host T and NK cell responses. Previous studies demonstrated that anti-third-party central memory CD8 veto T cells (Tcm) can overcome graft rejection under mild conditioning without causing significant GvHD, suggesting these cells as an ideal platform for allogeneic CAR T cell therapy. We demonstrated that veto Tcm can effectively delete anti-donor T cell clones through a Fas-FasL mechanism, whereas the mechanism by which they neutralize alloreactive NK cells remains unknown. To address this question, we established a model for NK-mediated rejection, in the absence of T cell activity. GFP+ T depleted bone marrow (TDBM) cells from B6 donors (H2Kb) were transplanted into BALB/c nude mice (H2Kd) pre-conditioned with 3Gy TBI, in the presence or absence of anti-asialo GM1 antibody pretreatment that specifically eliminates NK cells. FACS chimerism analysis of the blood and spleen at 30 days post-transplant revealed significant differences between anti-asialo GM1 treated or untreated mice (unpaired t-test, blood p=0.0035 and spleen p=0.0018, n=5). Notably, a similar ability to overcome NK-mediated rejection was observed upon transplantation of TDBM plus B6-Tcm veto cells, leading to marked enhancement of chimerism at 30 days post-transplant (average GFP+ cells in blood 4.26%, and spleen 3.18%), compared to mice treated with TDBM alone (average GFP+ in blood 0.22%, and spleen 0.18%) (unpaired t-test, blood p=0.0035, and spleen p=0.0018, n=5 ). However, in contrast to the ablation of NK cells using anti-asialo GM1 antibody, FACS analysis revealed that administration of veto cells was not associated with significant reduction of host NK cell levels. Based on this observation, we hypothesized that the mechanism by which Tcm veto cells neutralize NK cell-mediated rejection might involve suppression of host NK cytotoxicity, as opposed to NK cell deletion. To address this possibility, we first interrogated the impact of Tcm veto cells on NK cell proliferation in mixed lymphocyte reaction (MLR). CFSE-labeled BALB/c splenocytes were co-cultured with irradiated (20 Gy) C57BL/6 splenocytes, in the presence or absence of B6-derived Tcm veto cells. While in the absence of Tcm veto cells, NK cells exhibited marked proliferation starting at 96 hours of culture, the addition of B6 Tcm veto cells led to a substantial arrest of NK cell proliferation (unpaired t-