Functional and Activation Profiles of Mucosal-Associated Invariant T Cells in Patients With Tuberculosis and HIV in a High Endemic Setting

Abstract

Background: MAIT cells are non-classically restricted T lymphocytes that recognize and rapidly respond to microbial metabolites or cytokines and have the capacity to kill bacteria-infected cells. Circulating MAIT cell numbers generally decrease in patients with active TB and HIV infection, but findings regarding functional changes differ. Methods: We conducted a cross-sectional study on the effect of HIV, TB, and HIV-associated TB (HIV-TB) on MAIT cell frequencies, activation and functional profile in a high TB endemic setting in South Africa. Blood was collected from (i) healthy controls (HC, n = 26), 24 of whom had LTBI, (ii) individuals with active TB (aTB, n = 36), (iii) individuals with HIV infection (HIV, n = 50), 37 of whom had LTBI, and (iv) individuals with HIV-associated TB (HIV-TB, n = 26). All TB participants were newly diagnosed and sampled before treatment, additional samples were also collected from 18 participants in the aTB group after 10 weeks of TB treatment. Peripheral blood mononuclear cells (PBMC) stimulated with BCG-expressing GFP (BCG-GFP) and heat-killed (HK) Mycobacterium tuberculosis (M.tb) were analyzed using flow cytometry. MAIT cells were defined as CD3⁺ CD161⁺ Vα7.2⁺ T cells. Results: Circulating MAIT cell frequencies were depleted in individuals with HIV infection (p = 0.009). MAIT cells showed reduced CD107a expression in aTB (p = 0.006), and reduced IFNγ expression in aTB (p < 0.001) and in HIV-TB (p < 0.001) in response to BCG-GFP stimulation. This functional impairment was coupled with a significant increase in activation (defined by HLA-DR expression) in resting MAIT cells from HIV (p < 0.001), aTB (p = 0.019), and HIV-TB (p = 0.005) patients, and higher HLA-DR expression in MAIT cells expressing IFNγ in aTB (p = 0.009) and HIV-TB (p = 0.002) after stimulation with BCG-GFP and HK-M.tb. After 10 weeks of TB treatment, there was reversion in the observed functional impairment in total MAIT cells, with increases in CD107a (p = 0.020) and IFNγ (p = 0.010) expression. Conclusions: Frequencies and functional profile of MAIT cells in response to mycobacterial stimulation are significantly decreased in HIV infected persons, active TB and HIV-associated TB, with a concomitant increase in MAIT cell activation. These alterations may reduce the capacity of MAIT cells to play a protective role in the immune response to these two pathogens.

Keywords: MAIT activation; MAIT cell; MAIT cell function; human immunodeficiency virus; tuberculosis.

Figure 1

Frequencies of MAIT cells in patients with HIV infection, active TB, and HIV-associated TB. (A) Gating strategy used to identify MAIT cells. First, doublets and dead cells were excluded, then leukocytes selected, followed by CD3⁺ T cells. From the CD3⁺ T cells, MAIT cells were identified. (B) Frequencies of MAIT cells (%CD3 T cells) in HC, HIV, aTB, and HIV-TB. (C) MAIT cell counts (number of events in MAIT cell gate) in HC, HIV, aTB, and HIV-TB. (D) Relationship between MAIT cell frequencies and CD4 counts of HIV and HIV-TB group participants was investigated using Spearman rank correlation. (E) Effect of CD4 counts; and (F) ART on MAIT cell frequencies. P-values reported from Kruskal-Wallis test with a Dunn's post-hoc test for multiple comparisons and p < 0.05 reported as statistically significant. HC, Healthy controls group; HIV, HIV only group; aTB, active TB only group; HIV-TB, HIV-associated TB group.

Figure 2

The frequencies of MAIT cell subsets during HIV infection, active TB, and HIV-associated TB. (A) Representative plots showing MAIT subsets gated from total MAIT cells. (B) Frequencies of CD4, CD8, and DN MAIT cell subsets in HC, HIV, aTB, and HIV-TB. (C) t-SNE analysis of MAIT cell subsets in the different study groups, CD4 MAIT- Blue, CD8 MAIT- Green, DN- Red. P-values reported from Kruskal-Wallis test with a Dunn's post-hoc test for multiple comparisons of groups and HC and p < 0.05 reported as statistically significant. HC, Healthy controls group; HIV, HIV only group; aTB, active TB only group; HIV-TB, HIV-associated TB group.

Figure 3

MAIT cell responses after BCG and HK-M.tb stimulation. (A) Representative plots showing IFNγ and CD107a expression after stimulation with BCG 1, heat-killed M.tb (HK-M.tb), and phytohemagglutinin (PHA). (B,C) Expression of CD107a and IFNγ after BCG stimulation and HK-M.tb stimulation, respectively. (D) Contour plots for t-SNE analysis of MAIT cell functions between the different groups. red-cytokine-MAIT cells; dark-blue-CD107a+ MAIT cells; green-IFNg+ MAIT cells; light blue-HLA-DR+ MAIT cells. (E) CD4 counts of HIV and HIV-TB group participants and frequencies of MAIT cells expressing CD107a and IFNγ, assessed using Spearman correlation. (F) Effect of ART on MAIT cells expressing CD107a and IFNγ. (G) Effect of CD4 counts on MAIT cells expressing CD107a and IFNγ. P-values reported from Kruskal-Wallis test with a Dunn's post-hoc test for multiple comparisons and p < 0.05 reported as statistically significant. HC, Healthy controls group; HIV, HIV only group; aTB, active TB only group; HIV-TB, HIV-associated TB group.

Figure 4

Activation status of MAIT cells in HIV, aTB, and HIV-associated TB represented by the MFI of HLA-DR. Activation status of resting (unstimulated) MAIT cells in HC, HIV, aTB group, and HIV-TB group (at the top) and activation status of BCG stimulated IFNγ+ MAIT cells and MAIT cell subsets (at the bottom). (A) Bulk MAIT cells. (B) CD4 MAIT subsets. (C) CD8 MAIT cells. (D) DN MAIT cells. P-values reported from Kruskal-Wallis test with a Dunn's post-hoc test for multiple comparisons and p < 0.05 reported as statistically significant. HC, Healthy controls group; HIV, HIV only group; aTB, active TB only group; HIV-TB, HIV-associated TB group.

Figure 5

Effect of TB treatment on MAIT cell frequencies and functions. (A) Frequencies of MAIT cells and MAIT cell subsets at start of treatment (Week 0) and at 10 weeks of TB treatment (Week 10). (B,C) MAIT cell and MAIT cell subset responses (CD107a expression and IFNγ expression) at start of treatment (Week 0) and after 10 weeks of TB treatment (Week 10). P-values reported from Wilcoxon-ranked test between paired samples and p < 0.05 reported as statistically significant.