Transcriptional profile of tuberculosis antigen-specific T cells reveals novel multifunctional features

Abstract

In latent tuberculosis infection (LTBI) spread of the bacteria is contained by a persistent immune response, which includes CD4(+) T cells as important contributors. In this study we show that TB-specific CD4(+) T cells have a characteristic chemokine expression signature (CCR6(+)CXCR3(+)CCR4(-)), and that the overall number of these cells is significantly increased in LTBI donors compared with healthy subjects. We have comprehensively characterized the transcriptional signature of CCR6(+)CXCR3(+)CCR4(-) cells and found significant differences to conventional Th1, Th17, and Th2 cells, but no major changes between healthy and LTBI donors. CCR6(+)CXCR3(+)CCR4(-) cells display lineage-specific signatures of both Th1 and Th17 cells, but also have a unique gene expression program, including genes associated with susceptibility to TB, enhanced T cell activation, enhanced cell survival, and induction of a cytotoxic program akin to CTL cells. Overall, the gene expression signature of CCR6(+)CXCR3(+)CCR4(-) cells reveals characteristics important for controlling latent TB infections.

Figure 1. TB-specific memory CD4⁺ T cells are restricted to the CCR6⁺CXCR3⁺CCR4⁻ subset and produce IFNγ, TNFα and IL-2 but no IL-17

Representative dot plots from one donor. Plots are gated on total CD4⁺ memory T cells (grey background) or epitope-specific CD4⁺ memory T cells (red dots) (A). Percentage of tetramer⁺ T cells divided into 4 Th subsets; CCR6⁺CXCR3⁺CCR4⁻, CCR6⁻CXCR3⁺CCR4⁻ (Th1), CCR6⁺CXCR3⁻CCR4⁻ (Th17), and CCR6⁻CXCR3⁻CCR4⁺ (Th2) cells. Data represent median ± interquartile range from 5 donors (B). Epitope-specific IFNγ, TNFα, IL-2 and IL-17 production by CCR6⁺CXCR3⁺CCR4⁻ T cells measured after 6 h stimulation. Representative dot plots from one donor. Plots are gated on CCR6⁺CXCR3⁺CCR4⁻ T cells stimulated with media (control, top panel) or peptide pool (bottom panel) (C). Percentage of responding CCR6⁺CXCR3⁺CCR4⁻ expressing each of the fifteen possible combinations of IFNγ, TNFα, IL-2 and IL-17. Each dot represents one donor; mean ± SEM is indicated (D).

Figure 2. The CCR6⁺CXCR3⁺CCR4⁻ subset is increased in subjects with LTBI compared to HC

Contour plots for memory T cells from representative LTBI (left panel) and HC (right panel) gated on CCR6^+/− (top panel) and further gated on the 4 Th subsets; CCR6⁺CXCR3⁺CCR4⁻ (6⁺X3⁺4⁻), CCR6⁺CXCR3⁻CCR4⁻ (Th17) (middle panel), CCR6⁻CXCR3⁺CCR4⁻ (Th1), and CCR6⁻CXCR3⁻CCR4⁺ (Th2) (bottom panel). Numbers indicate percentage of subset in CD4⁺ T cells (A). Percentage of CD4⁺ T cells divided into 4 Th subsets as in (A) comparing LTBI (white bars) and HC (grey bars). Data represent median ± interquartile range from 12 LTBI and 12 HC donors. Mann Whitney test, **, p<0.01. Filled circles represent data from individual donors (B).

Figure 3. The transcriptional program of TB-specific cells are conserved in the CCR6⁺CXCR3⁺CCR4⁻ compartment

Mapping of short mRNA reads to genes encoding CXCR3, CCR4 and CCR6 in Th2 (red), Th1 (green), Th17 (purple) and 6⁺X3⁺4⁻ (orange) cells. Dot plots show expression for each individual sample tested. Data represents mean ± SEM (A). MA plots comparing gene expression between groups of samples. Geometric mean of expression between the samples (x-axis) is compared to fold change in expression between the samples (y-axis). Differentially expressed genes (red circles) were identified based on having an adjusted p-value < 0.05 according to the DESeq analysis and if they showed an at least 2-fold change in expression. Genes that did not meet these cutoffs were plotted in gray (B). Number of differentially expressed genes comparing groups of samples from Th subsets within LTBI and HC donor cohorts (C). Number of differentially expressed genes when comparing samples from the same subset between HC vs. LTBI donors and when comparing tetramer positive cells to the different Th subsets (D).

Figure 4. Expression pattern of genes differentially transcribed between sets of samples

Samples from different donors and cell types were clustered based on their similarity in gene expression in the complete set of 1,670 differentially expressed genes. Dendogram showing the sample clustering is shown on the upper right with samples from the same cell type making up the four main clusters. Disease state of the donor of each samples is indicated next to the dendogram (orange = LTBI, blue = HC). The heat map displays expression of each gene (column) as a fold change of the median expression in all samples with blue indicating lower and red indicating higher expression. Selected groups of genes are shown in the heat map: 1) CCR6⁺CXCR3⁺CCR4⁻ signature genes (6⁺X3⁺4⁻ from LTBI or HC donor being 2-fold higher or 2-fold lower than all other Th subsets from the same donor cohort). 2) Genes that are significantly different in Th1 vs. Th17 samples in either LTBI or HC donors (Th1 vs. Th17 signature), subdivided by into three groups that show either 6⁺X3⁺4⁻ expression similar to Th1, 6⁺X3⁺4⁻ expression similar to Th17 or 6⁺X3⁺4⁻ expression that is intermediate between the two (A). Mapping of short mRNA reads to genes differentially expressed in 6⁺X3⁺4⁻ (orange), Th1 (green), Th17 (purple), Th2 (red) and Tet⁺ (brown) cells. Dot plots show expression for each individual sample tested. Data represents mean ± SEM (B).

Figure 5. CCR6⁺CXCR3⁺CCR4⁻ cells produce a wide range of cytokines after mitogen stimulation

mRNA expression of cytokine genes after stimulation with mitogen compared to the resting state. The cytokines examined were previously described as being produced by human T cells in an epitope-specific manner in PMA/Ionomycin stimulated CCR6⁺CXCR3⁺CCR4⁻ cells. Shown are cytokines with at least three-fold induction ranked from highest to lowest. Data represents average ± standard deviation on a log scale for 3 LTBI donors.

Figure 6. The transcriptional profile of the CCR6⁺CXCR3⁺CCR4⁻ subset is reflected by a similar expression profile of proteins

CD4⁺ T cells were stained for CCR2 and KIT and expression was compared between different subsets. Top panel; CCR6⁺ vs. CCR6⁻, bottom panel; 6⁺X3⁺4⁻, Th1, Th17 and Th2. Each dot represents one donor, median ± interquartile range is indicated. Unpaired one-tailed t test, *, p<0.05, **, p<0.01, ***, p<0.001, ****, p<0.0001 (A). CCR2 (top) and KIT (bottom) expression in tet⁺ CCR6⁺CXCR3⁺CCR4⁻ cells (black dots) compared to CCR6⁺CXCR3⁺CCR4⁻ (grey dots) (B). Percentage of tet⁺ CCR6⁺CXCR3⁺CCR4⁻ cells expressing CCR2 and KIT. Data represent median ± interquartile range from 3 donors (C). Percentage increase in tetramer⁺ cells compared to CCR6⁺CXCR3⁺CCR4⁻ if CCR2⁺ and/or KIT⁻ is included in the staining panel. Data represent median ± interquartile range from 3 donors (D).