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

Abstract

20Effector-memory T-cells (TEM) are assumed to be short-lived cells that poorly proliferate upon 21 antigenic restimulation, thus depending on central-memory T-cells (TCM) to replenish their numbers 22 during homeostasis, largely depending on adoptive transfer evidence. Here we analyzed T cells in 23 their natural environment and observed robust long-term in vivo cycling within the TEM subset that 24 was stronger than the one in the TCM subset. Murine Cytomegalovirus (MCMV) induces inflationary 25 TEM responses that remain high during latency. We analyzed Ki67 expression during acute and latent 26 MCMV infection and found Ki67 hi Bcl2 lo TEM in latently infected mice, arguing for antigen-driven TEM 27 proliferation. TEM acquired deuterium more rapidly than TCM in an in vivo labeling experiment, and 28 were replenished more rapidly than TCM after memory depletion, suggesting that TEM cycle faster 29 than TCM. We depleted selectively the proliferating T-cells by Cre-overinduction, which resulted in a 30 selective loss of Ki67 hi Bcl2 lo effector T-cells, and an increase in the death of TEM in the spleen, while 31 it hardly affected the TCM subset, arguing for robust proliferation of TEM in the spleen. On the other 32 hand, TEM homing to the spleen upon adoptive transfer was substantially poorer than TCM, 33 explaining the previously reported expansions of TCM, but not TEM, upon transfer. In conclusion, our 34 data suggest that memory inflation is maintained by proliferation of antigen-specific TEM, rather 35 than by continued expansion and differentiation of TCM. 36 37 Running title: TEM proliferation during MCMV latency 3 38 Author Summary 39 The naïve T cell population consists of T cells that have the potential to recognize millions of 40 different pathogens. Upon infection, naïve T cells that recognize the pathogen expand, and 41 differentiate into effector T cells that eliminate infected cells. Once the infection is contained, the T 42 cell pool contracts and only a small population of central memory T cells remains that can expand 43 quickly upon re-infection. Cytomegaloviruses cause persistent infections that are not cleared from 44 the organism after the initial immune response. In infected individuals a pool of CMV-specific 45 effector memory T cells dominates the immune system in a phenomenon called memory inflation. 46 Previous research using the transfer of central memory or effector memory T cells from CMV-47 infected mice into mice with a matching infection, showed expansion of central memory T cells but 48 not effector memory T cells. Here we show that effector memory T cells have a reduced capacity to 49 home into lymphoid organs, where T cell activation takes place, compared to central memory T cells. 50 Using methods that do not interfere with T cell differentiation and homing, we show that effector 51 memory T cells are proliferating during the persistent phase of CMV infection, significantly 52 contributing to the upkeep of the inflationary population. 53 126 Ki67 hi CD8 T-cells was substantiall...