To understand the biology of CD4+ cytotoxic T lymphocytes (CD4-CTLs), Patil et al. used single cell RNASeq on the CD4-TEMRA subset (effector memory T cells expressing CD45RA), which is known to be enriched for CD4-CTL. They observed significant expression of cytotoxic and survival/co-stimulatory genes and substantial but variable clonal expansion. Clustering within the CD4-TEMRA subset differentiated cytotoxic cells and their memory precursor cells, with the latter marked by high expression of IL-7R, consistent with a memory phenotype.

CD4(+) cytotoxic T lymphocytes (CD4-CTLs) have been reported to play a protective role in several viral infections. However, little is known in humans about the biology of CD4-CTL generation, their functional properties, and heterogeneity, especially in relation to other well-described CD4(+) memory T cell subsets. We performed single-cell RNA sequencing in more than 9000 cells to unravel CD4-CTL heterogeneity, transcriptional profile, and clonality in humans. Single-cell differential gene expression analysis revealed a spectrum of known transcripts, including several linked to cytotoxic and costimulatory function that are expressed at higher levels in the TEMRA (effector memory T cells expressing CD45RA) subset, which is highly enriched for CD4-CTLs, compared with CD4(+) T cells in the central memory (TCM) and effector memory (TEM) subsets. Simultaneous T cell antigen receptor (TCR) analysis in single cells and bulk subsets revealed that CD4-TEMRA cells show marked clonal expansion compared with TCM and TEM cells and that most of CD4-TEMRA were dengue virus (DENV)-specific in donors with previous DENV infection. The profile of CD4-TEMRA was highly heterogeneous across donors, with four distinct clusters identified by the single-cell analysis. We identified distinct clusters of CD4-CTL effector and precursor cells in the TEMRA subset; the precursor cells shared TCR clonotypes with CD4-CTL effectors and were distinguished by high expression of the interleukin-7 receptor. Our identification of a CD4-CTL precursor population may allow further investigation of how CD4-CTLs arise in humans and, thus, could provide insights into the mechanisms that may be used to generate durable and effective CD4-CTL immunity.

Author Info: (1) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. (2) Division of Vaccine Discovery, La Jolla Institute

Author Info: (1) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. (2) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. (3) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. (4) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Southampton, UK. (5) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. (6) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. Genetech Research Institute, Colombo, Sri Lanka. (7) Division of Infectious Diseases and Vaccinology, School of Public Health, University of California, Berkeley, Berkeley, CA 94720, USA. (8) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. Department of Medicine, University of California San Diego, 9500 Gilman Drive #0656, La Jolla, CA 92093, USA. (9) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. (10) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. (11) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. Department of Medicine, University of California San Diego, 9500 Gilman Drive #0656, La Jolla, CA 92093, USA. (12) Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA. vijay@lji.org. Department of Medicine, University of California San Diego, 9500 Gilman Drive #0656, La Jolla, CA 92093, USA. Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, Faculty of Medicine University of Southampton, Southampton, UK.

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