Introduction: Adoptive Treg therapy has shown to be effective in a variety of immune diseases in preclinical studies (Bézie et al., Front Immunol.2017, Blood Adv.2019) and is currently moving to phase I and II to demonstrate its effectiveness. However, autologous Tregs are expensive and slow to produce, which is why we are considering using ready-to-use allogeneic Tregs for future cell therapy.

Method: CD8⁺CD4^-CD45RC^low/-CD56^- T cells were sorted by FACS Aria II, stimulated with anti-CD3 and CD28 mAbs for 2 days, nucleofected with CRIPR/Cas9 to Knock Out MHC molecules, and cultured for 19 additional days in medium supplemented with rapamycin, IL-2 and IL-15, and stimulated with anti-CD3 and CD28 mAbs every week. Suppression capacity was assessed in vitro on allogeneic CD4⁺T cells proliferation in response to third party APCs and in vivo on 1.5 Gy-irradiated NSG mice co-injected with allogeneic human PBMCs. Allogenicity of KO Tregs was assessed on allogeneic CFSE-labeled effector T cells.

Results: First, to explore the feasibility of off-the-shelf CD8⁺Treg-cell based therapy, we investigated the capacity of CD8⁺Tregs to control allogeneic immune responses. Interestingly, CD8⁺Treg could inhibit third-party APC-induced allogeneic effector T cell proliferation in vitro as efficiently as syngeneic cells. In addition, CD8⁺Tregs still control the development of GVHD induced by PBMCs from of foreign donor in vivo in humanized NSG mice. However, as CD8⁺Tregs express high levels of MHC-I and -II after ex vivo expansion, we evaluated their immunogenicity against allogeneic T cells. As expected, CD8⁺Tregs activated and initiated the proliferation of allogeneic CD8⁺ and CD4⁺ effector T cells. Thus, we developed a method to invalidate HLA molecules using CRISPR/Cas9 technology. By targeting B2M and CIITA genes encoding the proteins required for the expression of MHC-I and -II respectively, we succeeded in generating MHC-I and MHC-II deficient CD8⁺Tregs with preserved suppressive function, and which, unlike MHC⁺CD8⁺Tregs, did not activate effector T cell proliferation in vitro.

Conclusion: We have shown the therapeutic potential of MHC-deficient CD8⁺Tregs for off-the-shelf cell therapy. Experiments are underway to explore the impact of MHC-I deficiency of CD8⁺Tregs on their susceptibility to NK cell lysis.