Ex-Vivo Acquisition of Central Memory Phenotype Is Critical for Tolerance Induction by Donor Anti-3rd party CD8 T Cells in Allogeneic Bone Marrow Transplantation.
Authors: Edelshtein Yaki , Ophir Eran , Bachar-Lustig Esther , Afik Ran , Reisner Yair 
Published: April 2020

Abstract
The robust immunity remaining after reduced intensity conditioning represents a major barrier to durable engraftment of MHC disparate T cell depleted BM transplants (TDBMT). One approach to address this challenge could be afforded by using donor CD8 CTLs, endowed with potent veto activity (the ability of a cell to specifically suppress CTL precursors directed against its antigens, while sparing other CTLs capable of affording protection against infections and malignancies). However, their use is limited due to their marked GVH reactivity. This problem can be addressed effectively by stimulation of donor CD8 T cells against 3rd party stimulators under exogenous IL2 deprivation. The anti-3rd party veto CTLs support engraftment of TDBMT without causing GVHD. Nevertheless, although these cells displayed highly efficient veto activity in vitro, they were less efficient in vivo, requiring administration in conjunction with Rapamycin. In our study we found, using in vivo imaging, that veto CTLs display poor homing to the host lymph nodes (LNs). Therefore, we hypothesized that the relatively low efficiency of veto CTLs in vivo could be attributed to their failure to co-localize with the host T cells (HTCs) at the LNs, during the critical time at which the HTCs are triggered against donor antigens. To enhance LNs homing we generated anti-3rd party CD8 T cells under culture conditions favoring the acquisition of central memory (Tcm) phenotype (CD44highCD62Lhigh). In order to monitor CTLs or Tcm cells adoptively transferred into irradiated recipients of BM allografts, we used (Host x Donor)F1 cells co-expressing host and donor MHC that can be distinguished by FACS. When CD62Lhigh Tcm cells, expanded in the presence of IL15, were administered, the number of cells located at the host LNs two days post transplant was 40-fold higher compared to that found for veto CTLs expanded in the presence of IL2, which exhibit a CD62Llow effector phenotype (p&lt;0.05). Moreover, the total number of Tcm cells harvested from various organs was increased by 9 folds between days 2 to 6 post BMT (p&lt;0.05) in sharp contrast to the CTLs which displayed a non significant proliferation, indicating that Tcm cells possess superior proliferative capabilities. In addition, the Tcm cells exhibited marked durable persistence in-vivo when tested 100 days post transplant, comprising 17%±9% of the total CD8 T cell compartment following infusion of 1x106 cells. When evaluated in a stringent mouse model for T cell mediated BM allograft rejection, Tcm cells displayed marked tolerizing activity. In this model, 3x106 Balb-Nude BM is transplanted into lethally irradiated C3H hosts that were previously inoculated with 1.25x104 HTC (a number sufficient to induce BM rejection and lethality). Thus, administration of 5x106 anti-3rd party Tcm cells derived from (C3H x BALB)F1 mice, with no further immunosuppression, led to overall survival of 83% (19/23) at 100 days post BMT, while administration of 107 anti-3rd party CTLs (bearing an effector phenotype) could only afford survival of 19% (3/16) (p&lt;0.05) in the absence of Rapamycin. To evaluate the risk of GVHD, allogeneic Balb/c derived anti-3rd party Tcm cells or naive CD8 T cells were administered in conjunction with 5x106 Balb/c-Nude BM cells into lethally irradiated C3H hosts. As expected, naive CD8 cells caused lethal GVHD and only 6% (1/16) of the mice which received 2x106 naive cells survived 100 days post BMT. In contrast, the mice receiving Tcm cells displayed 83% survival (10/12 or 5/6 for 2x106 or 5x106 Tcm cells, respectively) with no signs of GVHD. Furthermore, when evaluated in the graft rejection model, 5x106 fully allogeneic donor Tcm cells induced survival, accompanied with complete donor chimerism at 100 days post transplant in 73% (11/15) of the recipients. The similar efficacy of overcoming rejection displayed by both fully allogeneic and F1 derived Tcm cells is in accordance with a veto mechanism, as opposed to potential ablation of host resistance by donor alloreactive clones. Collectively, the ability of anti-3rd party CD8 Tcm cells to expand and persist in vivo without GVHD, their marked LN homing and their capacity to overcome rejection of TDBMT in the absence of further immunosuppression, strongly suggest that these cells hold marked potential for tolerance induction in allogeneic hematopoietic stem cell transplantation.