Th2-Biased Transcriptional Profile Predicts HIV Envelope-Specific Polyfunctional CD4+ T Cells That Correlated with Reduced Risk of Infection in RV144 Trial

Abstract

Ag-specific T cells play a critical role in responding to viral infections. In the RV144 HIV vaccine clinical trial, a rare subset of HIV-specific polyfunctional CD4⁺ T cells correlated with reduced risk of HIV-1 infection. Polyfunctional T cells are a subset of Ag-specific T cells that are able to simultaneously produce multiple effector cytokines. Little is known about what differentiates polyfunctional T cells from other vaccine-elicited T cells in humans. Therefore, we developed a novel live-cell multiplexed cytokine capture assay to identify, isolate, and transcriptionally profile vaccine-specific polyfunctional CD4⁺ T cells. We applied these methods to samples from subjects who received the RV144 vaccine regimen, as part of the HVTN 097 clinical trial. We identified two surface receptors (CD44 and CD82) upregulated on polyfunctional T cells and a Th2-biased transcriptional signature (IL-4, IL-5, and IL-13) that predicted the envelope-specific polyfunctional CD4⁺ T cell profiles that had 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 potential contributions of polyfunctional T cells to effective vaccine-elicited immunity.

Graphical abstract

FIGURE 1.

Comparison of env-specific CD4⁺ T cell responses postvaccination by multiplexed cytokine capture and ICS assays. (A) Example of multiplexed cytokine capture assay env and negative control stimulations of PBMCs from a HVTN 097 vaccine recipient. Data are gated on CD4⁺ T lymphocytes (live single, CD56⁻, CD19⁻, CD14⁻, CD3⁺, CD4⁺, and CD8⁻). The y-axis shows CD154 expression, and the x-axis shows IFN-γ (left), IL-2 (middle), and IL-4 (right). The percentage in the bottom right of each plot indicates the frequency of cytokine-positive events, and the percentage in the top right (in red) of each plot is the percentage of CD4 T cells that are coexpressing the indicated cytokine with CD154. (B) Background-subtracted percent of CD4 T cells expressing CD154, IFN-γ, IL-2, and IL-4 for each subject by multiplexed cytokine capture and ICS assays (n = 6). Significance was determined using a Wilcoxon rank test. *p < 0.05. (C) Pie charts show the median distribution of subsets among the env-specific CD4 T cells. Each pie slice indicates the proportion of env-specific CD4 T cells that express those cytokines (indicated in the legend) and is color-coded by the degree of polyfunctionality: monofunctional (blue), bifunctional (green), trifunctional (yellow/orange), and quad-functional (red). The pie arc colors allow visualization of the functions expressed by each slice. (D) Bar chart of the per subject counts of env-specific CD4⁺ T cell subsets by functional profile from ICS (out of a median of 556 total number of env-specific CD4⁺ T cells per subject) or multiplexed cytokine capture assay (out of a median of 1887 total number of env-specific CD4⁺ T cells per subject) and color-coded by the degree of polyfunctionality. The red boxes indicate the two subsets that correlated with reduced risk of HIV infection in the RV144 vaccine trial.

FIGURE 2.

Individual genes that are differentially expressed by polyfunctional env-specific CD4 T cells. (A) Violin plots of log counts (log₂-transformed normalized counts plus a pseudocount of one) of the genes that were significantly differentially expressed between monofunctional (producing only one cytokine/function) and polyfunctional (producing two or more cytokines/functions) env-specific CD4⁺ T cells. (B) Heatmap of Z scores (mean row-centered log₂ counts per million) of the same differentially expressed genes clustered by polyfunctionality profile.

FIGURE 3.

Individual genes that are differentially expressed by env-specific CD4 T cells expressing RV144 polyfunctional correlate profile compared with non-correlate env-specific CD4 T cells. (A) Violin plots of log counts of the scRNA sequences for the 14 significantly differentially expressed genes between the env-specific CD4⁺ T cells grouped by the cytokine protein expression profiles that correlated with reduced risk of HIV infection in RV144, labeled here as correlate (IL-4⁺, IL-2⁺, CD154⁺, and IFN-γ⁺ and IL-4⁺, IL-2⁺, and CD154⁺) or non-correlate. (B) Heatmap of Z scores of the differentially expressed genes clustered by T cells with correlate or non-correlate cytokine expression profile. Genes with FDR <0.05 and fold change of >1.25 or less than −1.25 were considered significantly differentially expressed between groups.

FIGURE 4.

Transcriptional signatures predicted rare env-specific polyfunctional CD4 T cell subsets that had correlated with reduced risk of HIV infection in the RV144 trial. XGBoost machine learning model was used to determine the genes that best predicted the two cytokine profiles associated with reduced risk of infection in Rv144 (correlate profile) versus other env-specific CD4⁺ T cell subsets (non-correlate profile). (A) Plot shows receiver operating characteristic (ROC) curves created from 5-fold cross-validation. The blue line depicts the mean across the five models. (B) Bar graph of the top 30 most important features based on mean feature importance across the five models. (C) SHAP values showing the contribution of each gene within each cell for the top 30 genes. Color indicates the level of gene expression in each cell for the corresponding gene.