Fate-specification signals and long-term maintenance mechanisms of the terminally differentiated effector-memory (terminal-$T_{EM}$) CD8 T cell population

Memory CD8 T cells were classically divided into central-memory ($T_{CM}$) and effector-memory ($T_{EM}$) subsets, but Milner et al. (2020) showed that the conventional CD62L-low $T_{EM}$ gate conflates two populations: a CD127-high $T_{EM}$ population and a CD127-low CD62L-low terminally differentiated effector-memory population (terminal-$T_{EM}$) that persists for months, up-regulates the survival and pro-memory molecules Bcl2 and Tcf1 relative to acute terminal effectors, and confers strong per-cell protection against bacterial rechallenge. What governs this population remains unresolved: the signals that direct a differentiating effector CD8 T cell into the terminal-$T_{EM}$ versus the $T_{EM}$ pathway during the effector-to-memory transition have not been identified; the molecular mechanisms (and cytokine requirements) that sustain terminal-$T_{EM}$ survival are unknown, with the predicted regulators Zeb2 and Rictor/mTORC2 never tested under the refined subset definitions; and the human CD45RO+ CD127-low CD62L-low counterpart's longitudinal stability and relationship to established human memory-marker panels are uncharacterized. Follow-up work on the closely related KLRG1+ long-lived effector population (IL-15 independence, flexibility to become tissue-resident memory) addresses adjacent but not identical questions, leaving the core open.

Background

After an acute intracellular infection, antigen-specific CD8 T cells expand and then contract, leaving long-lived memory cells. Two classical circulating memory lineages were defined by Sallusto et al. (1999) using the lymph-node homing receptors CCR7 and CD62L: central-memory cells ($T_{CM}$, CD62L/CCR7 high), which recirculate through lymphoid tissue, produce IL-2, and proliferate strongly on rechallenge, and effector-memory cells ($T_{EM}$, CD62L/CCR7 low), which are more cytolytic and traffic to inflamed peripheral tissues. During the effector phase, CD8 T cells bifurcate into short-lived terminal effector (TE) cells, phenotypically KLRG1-high and CD127-low (CD127 is the IL-7 receptor alpha chain, used as a memory-precursor marker), and memory-precursor cells (KLRG1-low, CD127-high); the transcription factors T-bet, Zeb2, Blimp1, and Id2 promote the TE/effector program, while Eomes, Bcl6, Tcf1 (encoded by Tcf7), Foxo1, and Id3 support memory. The homeostatic cytokines IL-7 and IL-15 maintain memory CD8 T cells, and IL-15 in particular supports KLRG1+ effector-like cells in some settings.

Milner, Nguyen, Omilusik et al. (2020, PNAS 117:25667-25678) revisited the circulating memory compartment in mice after acute LCMV (lymphocytic choriomeningitis virus) infection. They found that the conventional CD62L-low gate hides a persisting CD127-low CD62L-low population — terminal-$T_{EM}$ — which is terminally fated and cytolytic yet is a genuine memory population: it survives for months (though less well than $T_{EM}$ or $T_{CM}$), up-regulates Bcl2, Tcf1, and Bcl6 relative to day-7 terminal effectors, and confers the most robust per-cell protection against Listeria monocytogenes rechallenge. Redefining $T_{EM}$ as CD127-high CD62L-low overturned textbook attributes of the $T_{EM}$ subset (which turns out to be enriched in lymphoid tissue, IL-2 production, recall proliferation, and homeostatic fitness). Perturbation experiments showed terminal-$T_{EM}$ formation depends on T-bet, Bcl6, and Foxo1, whereas the redefined $T_{EM}$ needs Bcl6 and Foxo1 but not high T-bet or Blimp1. Ingenuity Pathway Analysis (a computational method ranking transcription factors expected to activate a gene-expression signature) predicted Rictor (the defining component of the mTORC2 kinase complex, a growth-factor-sensing pathway) and Zeb2, alongside T-bet, as top regulators of the terminal-$T_{EM}$ fate, but only T-bet was experimentally tested. In humans, single-cell RNA sequencing and flow cytometry of healthy-donor blood identified a transcriptionally analogous CD45RO+ CD127-low CD62L-low population in cross-sectional samples. A closely related population, KLRG1+ CD127-low long-lived effector cells (LLECs), was delineated concurrently by Renkema et al. (2020); its gate (KLRG1-based) overlaps but is not formally identical to the CD62L-based terminal-$T_{EM}$ definition. What the 2020 study did not establish — and what no subsequent work has settled — is which signals assign effector cells to the terminal-$T_{EM}$ versus the $T_{EM}$ fate, which molecular pathways sustain terminal-$T_{EM}$ survival (whether Bcl2/Tcf1 up-regulation suffices, which cytokines are required), whether Zeb2 and Rictor/mTORC2 actually control terminal-$T_{EM}$ under the refined definitions, and whether the human population is stable over time and how it maps onto established human memory subsets defined by markers such as CD57 and CD28 or chemokine receptors.

Problem Statement

Complete the mechanistic delineation of the terminally differentiated effector-memory CD8 T cell population (terminal-$T_{EM}$, mouse CD127-low CD62L-low; human CD45RO+ CD127-low CD62L-low) that Milner et al. (2020) separated from conventional $T_{EM}$. Under the refined CD127/CD62L-based subset definitions, the task is to resolve all three of the following open questions, each of which is mandatory: (1) what extrinsic signals and cell-intrinsic molecular pathways assign a differentiating effector CD8 T cell to the terminal-$T_{EM}$ fate rather than the CD127-high CD62L-low $T_{EM}$ fate during the effector-to-memory transition — the Ingenuity Pathway Analysis-predicted regulators Zeb2 and Rictor/mTORC2 remain experimentally untested in this setting, in which the source study perturbed only T-bet, Blimp1, Bcl6, and Foxo1; (2) what is the mechanism of terminal-$T_{EM}$ long-term survival — in particular whether the up-regulation of Bcl2 and Tcf7 relative to acute terminal effectors is sufficient for persistence, and which cytokine or other homeostatic pathways are required; and (3) whether the human peripheral-blood terminal-$T_{EM}$ population is stable over time and how it relates to human memory subsets defined by established marker panels. Completeness is judged against exactly these three questions; a solution addressing only a subset of them, or substituting additional questions for them, is not complete.

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