Effective Deletion of Anti-Donor Host Memory Effector Cells by Anti-3rd Party Veto CTLs: Implications to Tolerance Induction in Presensitized Bone Marrow Recipients.
Authors: Reich-Zeliger Shlomit , Bachar-Lustig Esther , Reisner Yair 
Published: October 2019

Abstract
Recently we demonstrated that veto CTLs enhance engraftment of mismatched T cell depleted BM in recipient mice following reduced intensity conditioning. This desirable tolerance induction can be further enhanced by combining veto CTLs with CD4+CD25+ cells and Rapamycin. While these results are encouraging, they were largely based on models in which the resistant effector T cells mediating the allorejection are naive CTLp. However, considering that many patients undergoing BMT are presensitized by transfusions of different blood products, memory T cells could play an important role in graft rejection and, therefore, their sensitivity to veto cells could be critical to the implementation of the latter cells in BMT. Clearly, memory T cells respond under less stringent conditions to foreign antigens, compared to their naïve counterparts. In particular, they are programmed to be activated promptly, with a reduced requirement for costimulatory signals and therefore they might be more resistant to veto cells. To address this question we used the 2C mouse model, the CD8 T cells of which express a transgenic TCR against H-2d. The CD8 T cells bearing the TCR transgene can be followed by FACS using staining with a clontypic antibody (1B2) against the transgene. In this model, addition of veto CTLs was shown to inhibit expansion of CD8+1B2+ effector cells by induction of apoptosis which can be monitored by annexin V staining. Thus, in a total of 10 experiments the addition of 5% veto cells to 3 day MLR culture of naive 2C effector cells in the presence of H-2d stimulator cells, led to 76%±9% inhibition of expansion. In order to compare the sensitivity of memory cells in the same model, memory cells were established by immunizing 2C transgenic mice with 1x106 irradiated splenocytes from Balb/c donors (H-2d origin). Six weeks later, splenocytes were harvested and after Ficoll separation were shown to be enriched with memory CD8 T cells(CD44+high CD45Rb+ CD62L+, average in 16 different experiments was 73%±11). Upon addition of 5% veto cells to MLR culture of memory 2C spleen cells in the presence of stimulator cells, 78%±7% inhibition of 2C expansion was found. This veto activity was associated with increased apoptosis of allospecific memory CD8 T cells. Thus, in the absence of veto cells the CD8+1B2+ memory cells exhibited a low level of Annexin V (6%±3%) while in the presence of 5% veto cells, a high level of Annexin V (25%±9%) was detected. The deletion of the 2C memory effectors, as previously shown for naive 2C cells, is largely dependent on the presence of Fas-FasL interaction, as indicated by using memory cells from 2C- lpr mice that lack Fas receptor on the cell surface. Upon addition of veto cells to MLR culture with 2C memory spleen cells from lpr mice, only a minor reduction of expansion (5.5%±6% in the presence of 10% veto CTLs) was detected. In conclusion, these results suggest that veto cells can delete memory effector cells as efficiently as exhibited on naive effector cells and by a similar Fas-FasL dependent mechanism. This finding might have significant implications not only for BMT, but also for the treatment of autoimmune diseases in which memory T cells play a major role.