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Lineage-determining transcription factors (LD-TFs) drive the differentiation of progenitor cells into a specific lineage. In CD4+ T cells, T-bet dictates differentiation of the TH1 lineage, whereas GATA3 drives differentiation of the alternative TH2 lineage. However, LD-TFs, including T-bet and GATA3, are frequently co-expressed but how this affects LD-TF function is not known. By expressing T-bet and GATA3 separately or together in mouse T cells, we show that T-bet sequesters GATA3 at its target sites, thereby removing GATA3 from TH2 genes. This redistribution of GATA3 is independent of GATA3 DNA binding activity and is instead mediated by the T-bet DNA binding domain, which interacts with the GATA3 DNA binding domain and changes GATA3′s sequence binding preference. This mechanism allows T-bet to drive the TH1 gene expression program in the presence of GATA3. We propose that redistribution of one LD-TF by another may be a common mechanism that could explain how specific cell fate choices can be made even in the presence of other transcription factors driving alternative differentiation pathways.

T-bet is the lineage-specifying transcription factor for CD4+ T helper type 1 (TH 1) cells. T-bet has also been found in other CD4+ T cell subsets, including TH 17 cells and Treg, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells that have naïve cell surface markers and a naïve cell transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4+ T cells. Naïve-like T-bet-experienced cells are polarised to the TH 1 lineage, predisposed to produce IFNγ upon cell activation, and resist repolarisation to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can polarise T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T helper response. This article is protected by copyright. All rights reserved.

T-bet is the lineage-specifying transcription factor for CD4

T-bet is the lineage-specifying transcription factor for CD4+ T helper type 1 (TH1) cells. T-bet has also been found in other CD4+ T cell subsets, including TH17 cells and TREG, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells with naïve cell surface markers and that this novel cell population is phenotypically and functionally distinct from conventional naïve CD4+ T cells. These cells are also distinct from previously described populations of memory phenotype and stem cell-like T cells. Naïve-like T-bet-experienced cells are polarised to the TH1 lineage, predisposed to produce IFNγ upon cell activation, and resist repolarisation to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can function to polarise T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T helper response.

Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these relationships. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional states associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC.

Summary In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation., Graphical Abstract, Highlights • Tumor-infiltrating CD4+ T cells exhibit T helper and cytotoxic features • Treg cells limit IL-2 availability vital for the acquisition of cytotoxic features • T-bet is required for IFN-γ, but not GzmB, expression in CD4+ TILs • Blimp-1 is required for GzmB expression in response to IL-2 in CD4+ TILs, Śledzińska et al. show that upon depletion of regulatory T cells, a surplus of interleukin-2 in the tumor microenvironment supports acquisition of cytotoxic activity by T helper cells orchestrated by the transcription factor Blimp-1. These polyfunctional CD4+ T cells exhibit potent anti-tumor activity in an adoptive transfer setting with therapeutic implications.