Virtual memory T cells are foreign antigen‐inexperienced T cells that have acquired memory‐like phenotype and constitute 10–20% of all peripheral CD8+ T cells in mice. Their origin, biological roles, and relationship to naïve and foreign antigen‐experienced memory T cells are incompletely understood. By analyzing T‐cell receptor repertoires and using retrogenic monoclonal T‐cell populations, we demonstrate that the virtual memory T‐cell formation is a so far unappreciated cell fate decision checkpoint. We describe two molecular mechanisms driving the formation of virtual memory T cells. First, virtual memory T cells originate exclusively from strongly self‐reactive T cells. Second, the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T‐cell compartment via modulating the self‐reactivity of individual T cells. Although virtual memory T cells descend from the highly self‐reactive clones and acquire a partial memory program, they are not more potent in inducing experimental autoimmune diabetes than naïve T cells. These data underline the importance of the variable level of self‐reactivity in polyclonal T cells for the generation of functional T‐cell diversity.

The animal facility of the IMG is a part of the Czech Centre for Phenogenomics and the work there was supported in part by following grants: LM2015040, LM2018126, OP RDI CZ.1.05/2.1.00/19.0395, OP RDI BIOCEV CZ.1.05/1.1.00/02.0109 provided by the Czech Ministry of Education, Youth and Sports and the European Regional Development Fund. AM performed most of the experiments. VN, LS, MP, AD, RS, TM, AN, KK, PS, OS performed experiments. AM, VN, and OS analyzed data and finalized figures. JKu and JM analyzed the transcriptomic data. JN analyzed the TCR profiling data. DC and JKr analyzed the S16 sequencing data. RS and PS provided feral mice. TH and HK provided germ-free mice. MK, HK, JKr, PS, and OS supervised the work. OS conceived the study.

Virtual memory T cells are foreign antigen-inexperienced T cells that have acquired memory-like phenotype and constitute for 10-20% of all peripheral CD8 + T cells in mice. Their origin, biological roles, and relationship to naïve and foreign antigen-experienced memory T cells are incompletely understood. By analyzing TCR repertoires and using retrogenic monoclonal T-cell populations, we show that virtual memory T cells originate exclusively from strongly self-reactive T cells. Moreover, we show that the stoichiometry of the CD8 interaction with Lck regulates the size of the virtual memory T-cell compartment via modulating the self-reactivity of individual T-cell clones. We propose a so far unappreciated peripheral T-cell fate decision checkpoint that eventually leads to the differentiation of highly self-reactive T cells into virtual memory T cells. This underlines the importance of the variable level of self-reactivity in polyclonal T cells for the generation of functional T-cell diversity. Although virtual memory T cells descend from the highly self-reactive clones and acquire a partial memory program, they do not show higher capacity to induce autoimmune diabetes than naïve T cells. Thus, virtual memory T cells are not generally more responsive than naïve T cells, because their activity highly depends on the immunological context. SummaryWe conclude that virtual memory T cells are formed from self-reactive CD8 + T cells in a process regulated by CD8-Lck stoichiometry. Despite their self-reactivity and partial memory differentiation program, virtual memory T cells did not show a strong autoimmune potential.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.