Precursor-like T cells maintain tissue resident memory T cell heterogeneity and restrain their effector differentiation during reactivation

Tissue resident memory T (TRM) cells are non-recirculating lymphocytes that provide localized immunity against intracellular pathogens and cancer. Upon antigen reencounter, TRM cells can differentiate into effector cells and give rise to another generation of memory cells. While considerable heterogeneity of TRM cells has been described, it is unclear how distinct populations contribute to their maintenance or recall capacity. Here we show that TRM cells that express the transcriptional regulators HOBIT, TCF1 and ID3 exhibited precursor-like properties, contributing to the maintenance of the TRM cell pool over time and the reformation of CD103+ TRM cells after recall. TGF-beta (TGF-b) and retinoic acid signalling were required for inducing and maintaining TCF1 and ID3 expression and restrained secondary effector differentiation during reinfection. Finally, we show that TRM cells could differentiate into tissue confined and recirculating CX3CR1+ effector cells thereby contributing to protective immunity during recall. Thus, distinct precursor-like TRM cells contribute to the maintenance of tissue memory and anamnestic secondary memory responses. Overall design: Adoptively co-transferred CD45.1+/CD45.2+ HobitTomCre P14 and CD45.2+ Tcf7flox/floxHobitTomCre P14 cells from the SI-IEL and SI-LP were harvested at day 35 post LCMV Armstrong infection. Additional parallel cohorts of LCMV-immune recipient mice were treated with anti-Gr1 (NIMP-R14) to deplete recirculating antigen specific memory cells one week prior to intravenous secondary rechallenge with LM-gp33. To minimize P2RX7 mediated cell death, all recipient mice received 50ug of Artc2.2 nanobody (BioLegend) intravenously 30min prior to euthanasia. Single cell suspensions from mouse tissues were prepared as mentioned above. Viable CD45.1+/CD45.2+ and CD45.2+ TCRb+CD44+CD8+Va2+ cells were pooled from recipient mice and sorted for single cell RNA sequencing.