T helper 2 transcriptional profile predicts single-cell HIV envelope-specific polyfunctional CD4+ T cells correlated with reduced risk of infection in RV144 trial

Despite the critical role antigen-specific T cells play in responding to viral infections, their aggregate frequencies in peripheral blood have not correlated with clinical protection during HIV infection. However, a subset of HIV-specific CD4+ T cells, termed polyfunctional T cells, can produce multiple effector cytokines simultaneously. In the RV144 HIV vaccine trial, polyfunctional T cells correlated with reduced risk of HIV infection. Little is known about what differentiates polyfunctional T cells from other vaccine-elicited T cells. Therefore, we developed a novel live-cell multiplexed cytokine capture assay, to identify and transcriptionally profile vaccine-specific polyfunctional CD4+ T cells. We applied these methods to samples from the HVTN 097 clinical trial of the same vaccine regimen as RV144. We discovered two surface receptors that were enriched among polyfunctional CD4+ T cells and a Th2-biased signature (IL-4, IL-5, and IL-13) that specifically predicted the envelope-specific polyfunctional CD4+ T cells that were correlated with reduced risk of HIV infection in RV144. By linking single-cell transcriptional and functional profiles, we may be able to further define the role of vaccine-elicited polyfunctional T cells in contributing to effective immunity. Key PointsO_LINovel ex vivo multiplexed cytokine capture assay to enumerate and single-cell sort polyfunctional T cells for downstream analyses C_LIO_LIPolyfunctional T cells were specifically detected among the HIV envelope-stimulated CD4+ T cells C_LIO_LISingle-cell RNA sequencing identified novel surface markers enriched among vaccine-specific polyfunctional CD4+ T cells C_LIO_LITh2 transcriptional signature predicted polyfunctional CD4+ T cell profile that had correlated with reduced risk of HIV infection in the RV144 HIV efficacy trial C_LI