Epidermal Resident Memory T Cell Fitness Requires Antigen Encounter in the Skin

Reviewer #1 (Public review):

Summary:

Weiss et. al. seek to delineate the mechanisms by which antigen-specific CD8+ T cells outcompete bystanders in the epidermis when active TGF-b is limiting, resulting in selective retention of these cells and more complete differentiation into the TRM phenotype.

Strengths:

They begin by demonstrating that at tissue sites where cognate antigen was expressed, CD8+ T cells adopt a more mature TRM transcriptome than cells at tissue sites where cognate antigen was never expressed. By integrating their scRNA-Seq data on TRM with the much more comprehensive ImmGenT atlas, the authors provide a very useful resource for future studies in the field. Furthermore, they conclusively show that these "local antigen-experienced" TRM have increased proliferative capacity and that TCR avidity during TRM formation positively correlates with their future fitness. Finally, using an elegant experimental strategy, they establish that TCR signaling in CD8+ T cells in epidermis induces TGFBRIII expression, which likely contributes to endowing them with a competitive advantage over antigen-inexperienced TRM.

Weaknesses:

The main weakness in this paper lies in the authors' reliance on a single model to derive conclusions on the role of local antigen during the acute phase of the response by comparing T cells in model antigen-vaccinia virus (VV-OVA) exposed skin to T cells in contralateral skin exposed to DNFB 5 days after the VV-OVA exposure. In this setting, antigen-independent factors may contribute to the difference in CD8+ T cell number and phenotype at the two sites. For example, it was recently shown that very early memory precursors (formed 2 days after exposure) are more efficient at seeding the epithelial TRM compartment than those recruited to skin at later times (Silva et al, Sci Immunol, 2023). DNFB-treated skin may therefore recruit precursors with reduced TRM potential. In addition, TRM-skewed circulating memory precursors have been identified (Kok et al, JEM, 2020), and perhaps VV-OVA exposed skin more readily recruits this subset compared to DNFB-exposed skin. Therefore, when the DNFB challenge is performed 5 days after vaccinia virus, the DNFB site may already be at a disadvantage in the recruitment of CD8+ T cells that can efficiently form TRM. In addition, CD8+ T cell-extrinsic mechanisms may be at play, such as differences in myeloid cell recruitment and differentiation or local cytokine and chemokine levels in VV-infected and DNFB-treated skin that could account for differences seen in TRM phenotype and function between these two sites. Although the authors do show that providing exogenous peptide antigen at the DNFB-site rescues their phenotype in relation to the VV-OVA site, the potential antigen-independent factors distinguishing these two sites remain unaddressed. In addition, there is a possibility that peptide treatment of DNFB-treated initiates a second phase of priming of new circulatory effectors in the local-draining lymph nodes that are then recruited to form TRM at the DFNB-site, and that the effect does not solely rely on TRM precursors at the DNFB-treated skin site at the time of peptide treatment.

Secondly, although the authors conclusively demonstrate that TGFBRIII is induced by TCR signals and required for conferring increased fitness to local-antigen-experienced CD8+ TRM compared to local antigen-inexperienced cells, this is done in only one experiment, albeit repeated 3 times. The data suggest that antigen encounter during TRM formation induces sustained TGFBRIII expression that persists during the antigen-independent memory phase. It remains unclear why only the antigen encounter in skin, but not already in the draining lymph nodes, induces sustained TGFBRIII expression. Further characterizing the dynamics of TGFBRIII expression on CD8+ T cells during priming in draining lymph nodes and over the course of TRM formation and persistence may shed more light on this question. Probing the role of this mechanism at other sites of TRM formation would also further strengthen their conclusions and enhance the significance of this finding.

Reviewer #2 (Public review):

Summary:

The authors set out to dissect the mechanistic basis of their previously published finding that encountering cutaneous antigen augments the persistence of CD8+ memory T cells that enter skin (TRM) (Hirai et al., 2021, Immunity). Here they use the same murine model to study the fate of CD8+ T cells after antigen-priming in the lymph nodes, (1) those that re-encounter antigen in the skin via vaccinia virus (VV) versus (2) those that do not encounter antigen in skin but rather are recruited via topical dinitrofluorobenzene (DNFB) (so-called "bystander TRM"). The authors' previous publication establishes that this first group of CD8+ TRM has a persistence advantage over bystander TRM under TGFb-limiting conditions. The current paper advances this finding by elucidating the role of TGFBR3 in regulating CD8+ TRM skin persistence upon topical antigen exposure. Key novelty of the work lies in the generation and use of the CD8+ T cell-specific TGFBR3 knockout model, which allows them to demonstrate the role of TGFBR3 in fine-tuning the degree of CD8+ T cell skin persistence and that TGFBR3 expression is promoted by CD8+ TRM encountering their cognate antigen upon initial skin entry. Future work directly measuring active TGFb in the skin under different conditions would help identify physiologic scenarios that yield active TGFb-limiting conditions, thus establishing physiologic relevance.

Strengths:

Technical strengths of the paper include (1) complementary imaging and flow cytometry analyses, (2) integration of their scRNA-seq data with the existing CD8+ TRM literature via pathway analysis, and (3) use of orthogonal models where possible. Using a vaccina virus (VV) model, with and without ovalbumin (OVA), the authors investigate how topical antigen exposure and TCR strength regulate CD8+ TRM skin recruitment and retention. The authors use both FTY720 and a Thy1.1 depleting antibody to demonstrate that skin CD8+ TRM expand locally following both a primary and secondary recall response to topical OVA application.

A conceptual strength of the paper is the authors' observation that TCR signal strength upon initial TRM tissue entry helps regulate the extent of their local re-expansion on subsequent antigen re-exposure. They achieved this by applying peptides of varying affinity for the OT-I TCR on the DNFB-exposed flank in tandem with initial VV-OVA + DNFB treatment. They then measured TRM expansion after OVA peptide rechallenge, revealing that encountering a higher-affinity peptide upon skin entry leads to greater subsequent re-expansion. Additionally, by generating an OT-I Thy1.1+ E8i-creERT2 huNGFR Tgfbr3fl/fl (Tgfbr3∆CD8) mouse, the authors were able to elucidate a unique role for TGFBR3 in CD8+TRM persistence when active TGFb in skin is limited.

Weaknesses:

Overall, the authors' conclusions are well supported, although there are some instances where additional controls, experiments, or clarifications would add rigor. The conclusions regarding skin-localized TCR signaling leading to increased skin CD8+ TRM proliferation in-situ and increased TGFBR3 expression would be strengthened by assessing skin CD8+ TRM proliferation and TGFBR3 expression in models of high versus low avidity topical OVA-peptide exposure. The authors could further increase the novelty of the paper by exploring whether TGFBR3 is regulated at the RNA or protein level. To this end, they could perform analysis of their single-cell RNA sequencing data (Figure 1), comparing Tgfbr3 mRNA in DNFB versus VV-treated skin.

For clarity, when discussing antigen exposure throughout the paper, it would be helpful for the authors to be more precise that they are referring to the antigen in the skin rather than in the draining lymph node. A more explicit summary of some of the lab's previous work focused on CD8+ TRM and the role of TGFb would also help readers better contextualize this work within the existing literature on which it builds.

For rigor, it would be helpful where possible to pair flow cytometry quantification with the existing imaging data. Additional controls, namely enumerating TRM in the opposite, untreated flank skin of VV-only-treated mice and the treated flank skin of DNFB-only treated mice, would help contextualize the results seen in dually-treated mice in Figure 1. In figure legends, we suggest clearly reporting unpaired T tests comparing relevant metrics within VV or DNFB-treated groups (for example, VV-OVA PBS vs VV-OVA FTY720 in Figure 3F). Finally, quantifying right and left skin draining lymph node CD8+ T cell numbers would clarify the skin specificity and cell trafficking dynamics of the authors' model.