Robust engraftment of human gammadelta T cells in humanized NSG TM -SGM3-IL-15 × MHC I/II double knockout mice for the evaluation of gammadelta T cell-based therapeutics

Background Gamma delta (gammadelta) T cells represent a minor subset of T lymphocytes capable of recognizing a wide range of universally conserved antigens expressed by cancer cells. Unlike conventional T cells, their recognition is independent of MHC, making them strong candidates for next-generation universal cancer therapeutics targeting various malignancies. Our preliminary donor screening results demonstrated that engrafting human peripheral blood mononuclear cells (PBMCs) into irradiated NSG TM -SGM3-IL-15 × MHC I/II DKO (SDKO) mice could lead to the expansion of circulating gammadelta T cells to a concentration comparable to those in human and robust gammadelta T cell recovery from tissues. To demonstrate our PBMC-SDKO mice platform for in vivo evaluation of gammadelta T cell related therapeutics, we conducted gammadelta T cell engraftment experiments using irradiated SDKO mice and PBMC from 9 donors. We also assessed the anti-cancer functionality of these isolated gammadelta T cells from PBMC engrafted mice using in vitro co-culture experiments with human lymphoma or triple-negative breast cancer cell lines. Methods Irradiated SDKO mice were engrafted with PBMCs from 9 different human donors. Leukocyte engraftment in the periphery was monitored weekly post PBMC injection using flow cytometry to enumerate the frequency and numbers of alphabeta T cells, gammadelta T cells and their subpopulations Vdelta1, and Vdelta2. gammadelta T cells engrafted in spleen and lungs were isolated using antibody conjugated magnetic beads and then co-cultured in vitro with Raji or MDA-MB-231 cells to assess their anti-tumor functionality. Results We found that circulating gammadelta T cells expanded greatly in engrafted SDKO mice. In some of the donors, the concentration of gammadelta T cells reached up to 4×10 2 cells/µL - a level comparable to that of circulating human B cells, which is a major population in peripheral blood. Among the gammadelta T cell subsets, the Vdelta2 subset exhibited the most robust and consistent expansion, while other gammadelta T cell subsets such as the Vdelta1 were donor dependent. In addition to peripheral blood, we also observed robust engraftment and recovery of gammadelta T cells from the lungs and spleens of engrafted SDKO mice. Our in vitro co-culture experiments demonstrated that the gammadelta T cells isolated from the spleens and lungs of SDKO mice showed elevated cell surface expression of CD69, granzyme B secretion in response to cancer cells and reduced the number of Raji or MDA-MB-231 cell numbers at effector to target ratios of 1:1 and 3:1. Our results suggest that gammadelta T cells can be robustly expanded and recovered from the SDKO mice and that the recovered cells possess potent cytolytic activity against tumor cells.