Immune Phenotype and Functionality of Mtb- Specific T-Cells in HIV/TB Co-Infected Patients on Antiretroviral Treatment

Abstract

The performance of host blood-based biomarkers for tuberculosis (TB) in HIV-infected patients on antiretroviral therapy (ART) has not been fully assessed. We evaluated the immune phenotype and functionality of antigen-specific T-cell responses in HIV positive (+) participants with TB (n = 12) compared to HIV negative (-) participants with either TB (n = 9) or latent TB infection (LTBI) (n = 9). We show that the cytokine profile of Mtb-specific CD4+ T-cells in participants with TB, regardless of HIV status, was predominantly single IFN-γ or dual IFN-γ/ TNFα. Whilst ESAT-6/CFP-10 responding T-cells were predominantly of an effector memory (CD27-CD45RA-CCR7-) profile, HIV-specific T-cells were mainly of a central (CD27+CD45RA-CCR7+) and transitional memory (CD27+CD45RA+/-CCR7-) phenotype on both CD4+ and CD8+ T-cells. Using receiving operating characteristic (ROC) curve analysis, co-expression of CD38 and HLA-DR on ESAT-6/CFP-10 responding total cytokine-producing CD4+ T-cells had a high sensitivity for discriminating HIV+TB (100%, 95% CI 70-100) and HIV-TB (100%, 95% CI 70-100) from latent TB with high specificity (100%, 95% CI 68-100 for HIV-TB) at a cut-off value of 5% and 13%, respectively. TB treatment reduced the proportion of Mtb-specific total cytokine+CD38+HLA-DR+ CD4+ T-cells only in HIV-TB (p = 0.001). Our results suggest that co-expression of CD38 and HLA-DR on Mtb-specific CD4+ T-cells could serve as a TB diagnosis tool regardless of HIV status.

Keywords: CD38; HLA-DR; immune activation; treatment response.

Figure 1

Study flow diagram. Samples were excluded for the following reasons: no follow-up sample available (n = 7), poor sample viability (n = 6), or technical errors (n = 8).

Figure 2

Comparison of the polyfunctional capacity of ESAT-6/CFP-10 CD4+ T-cells between HIV+TB, HIV−TB, and LTBI. (A) Representative plot of the expression of IL-2, IFN-γ, and TNFα in an HIV+TB participant. NS = unstimulated, ESCF = ESAT-6/CFP-10, MtbLy = Mtb-Lysate, PHA = Phytohemaglutinin. (B) Frequencies of CD4+ T-cells producing any of the possible combinations of IL-2, IFN-γ, and TNFα in response to ESAT-6/CFP-10 stimulation. Horizontal bars show the median and first and third quartile (Q1, Q3). Only statistically significant differences by the Kruskal–Wallis test with Dunn’s correction for multiple comparisons are shown on the graph.

Figure 3

Polyfunctionality analysis of ESAT-6/CFP-10 specific CD4+ T-cell responses (A) Heatmap of combinatorial polyfunctionality analysis of antigen-specific T-cells (COMPASS) posterior probabilities of the distribution of responses in ESAT-6/CFP-10 stimulated CD4+ T-cells. Columns correspond to the different cell subsets modeled using the COMPASS package in R [19], color coded by the cytokines they express (white = “off”, shaded = “on”, grouped by color = “degree of polyfunctionality”) ordered by degree of functionality from one function on the left to all three functions on the right (shaded from light blue to green on the bottom). Rows correspond to participants; one on each line, the color of each cell represents the probability (range 0–1) that the cell subset exhibits an antigen-specific response. Each cell of the heatmap shows the probability that a given cell-subset (column) has an antigen-specific response in the corresponding participant (the groups are coded on the right of the heatmap; pink = HIV+TB, blue = HIV−TB, and green = LTBI). Plots of polyfunctional scores (PFS) stratified by patient group (B) and timepoint for HIV+TB (C) and HIV−TB (D). Red dots represent HIV+TB, blue dots HIV−TB, and black dots LTBI. Differences between groups were compared using the Kruskal–Wallis test with Dunn’s correction for multiple comparisons are shown on the graph. We assessed differences between timepoints using the Wilcoxon signed-rank test.

Figure 4

Functional analysis of HIV+TB. The functional profile of CD4+ T-cells specific for different antigens was assessed for CD4+ T-cells from HIV+TB patients at the time of TB diagnosis. (A) Each slice of the pie corresponds to a distinct combination of cytokines shown in (B) and defined by Boolean analysis in FlowJo. A key to the colors used for the arcs in the pie charts is shown on the top right. Pie charts were compared using the SPICE permutation test with p < 0.05 considered significant. (B) Frequency of cells producing any possible combination of IFN-γ, IL-2, or TNFα in response to the different stimulations. Horizontal bars represent median and interquartile range (IQR). Statistical comparisons were made using Kruskal–Wallis with Dunn’s test for multiple comparisons. MtbLy = Mtb-Lysate; ESCF = ESAT-6/CFP-10.

Figure 5

Comparison of the activation profiles of Mtb-specific CD4+ T-cells between HIV+TB, HIV−TB, and LTBI participants. (A) Representative dot plots of the activation profile on CD4+ T-cells of an HIV+TB patient in response to ESAT-6/CFP-10 stimulation. The proportion of each subset on ESAT-6/CFP-10 (B) and Mtb-Lysate (C) stimulated total cytokine+CD4+ T-cells. Statistical comparisons were made using the Kruskal–Wallis with Dunn’s test for multiple comparisons. We used Boolean gates to derive all possible combinations of the co-expressed markers.

Figure 6

Receiver operating characteristics (ROC) curve analysis of the activation phenotype on total cytokine producing CD4+ T-cells for discriminating between TB and LTBI. Phenotypic analysis was only conducted in participants with a positive ESAT-6/CFP-10 response defined by a false discovery rate (FDR) < 0.05 using mixture models for single cell assays (MIMOSA) analysis: HIV+TB (n = 9); HIV−TB (n = 9); LTBI (n = 8). Comparison of the area under the curve for the phenotype CD38+HLA-DR+Ki67+ (top panel, A,B) and CD38+HLA-DR+Ki67- (bottom panel, C,D) in HIV+TB (red) and HIV−TB (blue). ROC curve analysis was done in Prism. The area under the curve (AUC) and p-values are shown. The dotted lines represent an AUC of 0.5.

Figure 7

Comparison of the memory phenotype of ESAT-6/CFP-10-stimulated CD4+ T-cells between active TB and LTBI participants. (A) Representative plot of the total cytokine production in the memory compartment in response to ESAT-6/CFP-10 stimulation at baseline and two months (fu) timepoints. (B) Comparison of the memory phenotype of ESAT-6/CFP-10 stimulated total cytokine+CD4+cells between HIV+TB and HIV−TB. Changes in the memory phenotype between baseline and two months of TB treatment in HIV+TB (red, (C)) and HIV−TB (blue, (D)). Statistical comparisons were made using Kruskal–Wallis with Dunn’s test for multiple comparisons. Differences between time points were assessed using the Wilcoxon signed rank test.

Figure 8

Sensitivity of the CD27MFI ratio on ESAT-6/CFP-10-specific CD4+ T-cells to detect active TB. (A) Comparison of the CD27MFI ratio between active TB and latent TB. CD27MFI ratio was calculated as the CD27MFI on bulk CD4 T-cells divided by CD27MFI on total cytokine+ CD4+ T-cells. Change in the CD27 MFI ratio of total cytokine+CD4+ T-cells between baseline and two months of TB treatment in (B) HIV+TB, (C) HIV−TB, and Gag-stimulated total cytokine+CD8+T-cells (D). Differences between time points were assessed using the Wilcoxon signed rank test. (E) and (F) show the receiver operating characteristics (ROC) curves of the CD27 MFI ratio in HIV−TB (blue) and HIV+TB (red), respectively, as assessed by the ROC curve analysis in Prism. The dotted lines represent an area under the curve (AUC) of 0.5.