Off-target effects of Cre recombinase reveal limits of adoptive T cell transfers and persistent proliferation of effector CD8 T-cells

Effector-memory T-cells (TEM) are assumed to be short-lived cells that poorly proliferate upon antigenic restimulation, thus depending on central-memory T-cells (TCM) to replenish their numbers during homeostasis, largely depending on adoptive transfer evidence. Here we analyzed T cells in their natural environment and observed robust long-term in vivo cycling within the TEM subset that was stronger than the one in the TCM subset. We compared the non-persistent vaccinia virus and the persisting murine Cytomegalovirus (MCMV), which induces inflationary TEM responses that remain high during viral latency. We analyzed Ki67 expression during acute, resolved and latent infection and found Ki67hiBcl2lo TEM in acutely or latently infected mice, arguing for antigen-driven TEM proliferation. In vivo labeling with deuterium showed that TEM acquired deuterium more rapidly than TCM, and were rapidly lost during chase. Similarly, antibody-mediated depletion of primed CD8 T cells in latenly infected mice revealed that TEM replenished more rapidly than TCM, suggesting that TEM cycle faster than TCM. Finally, we utilized the ability of Tamoxifen-induced Cre-ERT2 recombinase to induce chromosomal translocations when large amounts of Tamoxifen are administered for an extended time, which resulted in a selective depletion of proliferating Ki67hi cells that hardly affected the TCM subset, but drove a selective loss of Ki67hiBcl2lo effector T-cells, and an increase in the death of TEM in the spleen, arguing that TEM preferentially proliferate in the spleen. Since our results contradicted previous evidence from adoptive transfer experiments, we tested T cell homing to the spleen upon adoptive transfer. TEM homing was substantially poorer than the one of TCM, likely explaining the previously reported expansions of TCM, but not TEM, upon transfer into latently infected mice. In conclusion, our data suggest that memory inflation is largely maintained by splenic proliferation of antigen-specific TEM, rather than by continued expansion and differentiation of TCM.