Hypoxia acts as an environmental cue for the human tissue-resident memory T cell differentiation program
Farah Hasan, Yulun Chiu, Rebecca M. Shaw, Junmei Wang, Cassian Yee
Farah Hasan, Yulun Chiu, Rebecca M. Shaw, Junmei Wang, Cassian Yee
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Research Article Immunology

Hypoxia acts as an environmental cue for the human tissue-resident memory T cell differentiation program

Abstract

Tissue-resident memory T cells (TRM) provide frontline defense against infectious diseases and contribute to antitumor immunity; however, aside from the necessity of TGF-β, knowledge regarding TRM-inductive cues remains incomplete, particularly for human cells. Oxygen tension is an environmental cue that distinguishes peripheral tissues from the circulation, and here, we demonstrate that differentiation of human CD8+ T cells in the presence of hypoxia and TGF-β1 led to the development of a TRM phenotype, characterized by a greater than 5-fold increase in CD69+CD103+ cells expressing human TRM hallmarks and enrichment for endogenous human TRM gene signatures, including increased adhesion molecule expression and decreased expression of genes involved in recirculation. Hypoxia and TGF-β1 synergized to produce a significantly larger population of TRM phenotype cells than either condition alone, and comparison of these cells from the individual and combination conditions revealed distinct phenotypic and transcriptional profiles, indicating a programming response to milieu rather than a mere expansion. Our findings identify a likely previously unreported cue for the TRM differentiation program and can enable facile generation of human TRM phenotype cells in vitro for basic studies and translational applications such as adoptive cellular therapy.

Authors

Farah Hasan, Yulun Chiu, Rebecca M. Shaw, Junmei Wang, Cassian Yee

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Figure 1

Hypoxia and TGF-β1 synergize to induce a CD69+CD103+ population that expresses human TRM-associated markers.

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Hypoxia and TGF-β1 synergize to induce a CD69+CD103+ population that exp...
Naive CD8+ T cells sorted from PBMCs were activated in 20% O2 (AtmosO2) or 2% O2 (hypoxia) for 4 days and then for an additional 2 days with the addition of recombinant human TGF-β1 (rhTGF-β1). Expression levels of TRM-associated genes were analyzed via quantitative real-time PCR. (A–C) Fold change of gene transcript levels in 2% O2 + TGF-β1 over 20% O2 + TGF-β1. (D) The frequency of the CD69+CD103+ TRM-like population and (E) expression of TRM-associated markers were assessed by flow cytometry. Representative results from 1 donor are shown (D and E). Blue histograms represent CD69+CD103+ cells from 20% O2 + TGF-β1, red histograms represent CD69+CD103+ cells from 2% O2 + TGF-β1, and gray histograms represent fluorescence minus one (FMO). (A–C) n = 7, 3 independent experiments; paired t test with Benjamini, Krieger, and Yekutieli correction for multiple comparisons; **q < 0.01, ***q < 0.001, ****q < 0.0001; FDR < 0.05, data are mean ± SEM. (D) n = 8, 3 independent experiments; ratio paired t test; ****P < 0.0001. Naive CD8+ T cells were activated in 20% O2 or 2% O2 for 4 days and then for an additional 2 days with or without rhTGF-β1. (F) Frequency of the CD69+CD103+ population and (G) expression of TRM-associated markers on CD69–CD103– cells (20% O2), CD69+CD103– cells (2% O2), and CD69+CD103+ cells (2% O2 + TGF-β1) were assessed by flow cytometry; representative pseudocolor plots shown for 1 donor, n = 8. Two-way ANOVA (F) or repeated measures 1-way ANOVA (G), *P adj < 0.05, **P adj < 0.01, ***P adj < 0.001, ****P adj < 0.0001; data are mean ± SEM.