SummaryT helper-17 (Th17) cells are associated with inflammatory disorders and cancer. We report that environmental conditions resulting in cellular stress, such as low oxygen, glucose, and isotonic stress, particularly enhance the generation of Th17 cells. Pharmacological inhibition of cell stress reduces Th17 cell differentiation while stress inducers enhance the development of Th17 cells. The cellular stress response results in Th17 cell development via sustained cytoplasmic calcium levels and, in part, XBP1 activity. Furthermore, in an inflammatory environment, conditions resulting in cell stress can bring about de novo Th17 cell differentiation, even in the absence of transforming growth factor β (TGF-β) signaling. In vivo, cell stress inhibition enhances resistance to Th17-mediated autoimmunity while stress-exposed T cells enhance disease severity. Adverse metabolic environments during inflammation provide a link between adaptive immunity and inflammation and may represent a risk factor for the development of chronic inflammatory conditions by facilitating Th17 cell differentiation.

CD4 + Th cells play a key role in orchestrating immune responses, but the identity of the CD4 + Th cells involved in the antitumor immune response remains to be defined. We analyzed the immune cell infiltrates of head and neck squamous cell carcinoma and colorectal cancers and identified a subset of CD4 + Th cells distinct from FOXP3 + Tregs that coexpressed programmed cell death 1 (PD-1) and ICOS. These tumor-infiltrating lymphocyte CD4 + Th cells (CD4 + Th TILs) had a tissue-resident memory phenotype, were present in MHC class II–rich areas, and proliferated in the tumor, suggesting local antigen recognition. The T cell receptor repertoire of the PD-1 + ICOS + CD4 + Th TILs was oligoclonal, with T cell clones expanded in the tumor, but present at low frequencies in the periphery. Finally, these PD-1 + ICOS + CD4 + Th TILs were shown to recognize both tumor-associated antigens and tumor-specific neoantigens. Our findings provide an approach for isolating tumor-reactive CD4 + Th TILs directly ex vivo that will help define their role in the antitumor immune response and potentially improve future adoptive T cell therapy approaches.

Murine peripheral lymph node TCR gd T cells have been divided into type 1 and type 17 functional categories based on phenotypic and functional markers. Localized in the gut epithelial barrier, intestinal intraepithelial lymphocytes (iIEL) gd T cells constitute a peculiar subset of T lymphocytes involved in intestinal homeostasis. However, whether iIEL gd T cells obey the type 1/type 17 dichotomy is unclear. Using both global transcriptional signatures and expression of cell surface markers, we reveal that murine iIEL gd T cells compose a distinct population, expressing ~1000 specific genes, in particular genes that are responsible for cytotoxicity and regulatory functions. The expression of the transcription factor Helios is a feature of iIEL gd T cells, distinguishing them from the other TCR gd T subsets, including those present in the epithelia of other tissues. The marked expression of Helios is also shared by the other iIELs, TCRabCD8aa lymphocytes present within the intestinal epithelium. Finally, we show that Helios expression depends in part on TGF-b signaling but not on the microbiota. Thus, our study proposes iIEL gd T cells as a distinct subset and identifies novel markers to differentiate them from their peripheral counterparts. ImmunoHorizons, 2022, 6: 515-527.

No abstract

Both identity and plasticity of CD4 T helper (Th) cells are regulated in part by epigenetic mechanisms. However, a method that reliably and readily profiles DNA base modifications is still needed to finely study Th cell differentiation. Cytosine methylation (5mC) and cytosine hydroxymethylation (5hmC) are DNA modifications that identify stable cell phenotypes but their potential to characterize intermediate cell transitions has not yet been evaluated. To assess transition states in Th cells, we developed a new method to profile Th cell identity using cas9-targeted single molecule nanopore sequencing and found that 5mC and 5hmC can be used as markers of cellular identity. Targeting as few as 10 selected genomic loci, we were able to distinguish major differentiated T cell subtypes as well as intermediate phenotypes by their native DNA 5mC/5hmC patterns. Moreover, by using off-target sequences we were able to infer transcription factor activities relevant to each cell subtype. Our analysis demonstrates the importance of epigenetic regulation by 5mC and 5hmC modifications in the establishment of Th cell identity. Furthermore, our data highlight the potential to exploit this immune profiling application to elucidate the pathogenic role of Th transition states in autoimmune diseases.