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

Abstract

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. Murine Cytomegalovirus (MCMV) induces inflationary TEM responses that remain high during latency. We analyzed Ki67 expression during acute and latent MCMV infection and found Ki67hiBcl2lo TEM in latently infected mice, arguing for antigen-driven TEM proliferation. TEM acquired deuterium more rapidly than TCM in an in vivo labeling experiment, and were replenished more rapidly than TCM after memory depletion, suggesting that TEM cycle faster than TCM. We depleted selectively the proliferating T-cells by Cre-overinduction, which resulted in a selective loss of Ki67hiBCl2lo effector T-cells, and an increase in the death of TEM in the spleen, while it hardly affected the TCM subset, arguing for robust proliferation of TEM in the spleen. On the other hand, TEM homing to the spleen upon adoptive transfer was substantially poorer than TCM, explaining the previously reported expansions of TCM, but not TEM, upon transfer. In conclusion, our data suggest that memory inflation is maintained by proliferation of antigen-specific TEM, rather than by continued expansion and differentiation of TCM.\n\nAuthor SummaryThe naive T cell population consists of T cells that have the potential to recognize millions of different pathogens. Upon infection, naive T cells that recognize the pathogen expand, and differentiate into effector T cells that eliminate infected cells. Once the infection is contained, the T cell pool contracts and only a small population of central memory T cells remains that can expand quickly upon re-infection. Cytomegaloviruses cause persistent infections that are not cleared from the organism after the initial immune response. In infected individuals a pool of CMV-specific effector memory T cells dominates the immune system in a phenomenon called memory inflation. Previous research using the transfer of central memory or effector memory T cells from CMV-infected mice into mice with a matching infection, showed expansion of central memory T cells but not effector memory T cells. Here we show that effector memory T cells have a reduced capacity to home into lymphoid organs, where T cell activation takes place, compared to central memory T cells. Using methods that do not interfere with T cell differentiation and homing, we show that effector memory T cells are proliferating during the persistent phase of CMV infection, significantly contributing to the upkeep of the inflationary population.