Impaired Mycobacterium tuberculosis-specific T-cell memory phenotypes and functional profiles among adults with type 2 diabetes mellitus in Uganda

Abstract

Background: Efforts to eradicate tuberculosis (TB) are threatened by diabetes mellitus (DM), which confers a 3-fold increase in the risk of TB disease. The changes in the memory phenotypes and functional profiles of Mycobacterium tuberculosis (Mtb)-specific T cells in latent TB infection (LTBI)-DM participants remain poorly characterised. We, therefore, assessed the effect of DM on T-cell phenotype and function in LTBI and DM clinical groups.

Methods: We compared the memory phenotypes and function profiles of Mtb-specific CD4⁺ and CD8⁺ T cells among participants with LTBI-DM (n=21), LTBI-only (n=17) and DM-only (n=16). Peripheral blood mononuclear cells (PBMCs) were stimulated with early secretory antigenic 6 kDa (ESAT-6) and culture filtrate protein 10 (CFP-10) peptide pools or phytohemagglutinin (PHA). The memory phenotypes (CCR7/CD45RA), and functional profiles (HLA-DR, PD-1, CD107a, IFN-γ, IL-2, TNF, IL-13, IL-17A) of Mtb-specific CD4⁺ and CD8⁺ T cells were characterised by flow cytometry.

Results: Naïve CD4⁺ T cells were significantly decreased in the LTBI-DM compared to the LTBI-only participants [0.47 (0.34-0.69) vs 0.91 (0.59-1.05); (p<0.001)]. Similarly, CD8⁺ HLA-DR expression was significantly decreased in LTBI-DM compared to LTBI-only participants [0.26 (0.19-0.33) vs 0.52 (0.40-0.64); (p<0.0001)], whereas CD4⁺ and CD8⁺ PD-1 expression was significantly upregulated in the LTBI-DM compared to the LTBI-only participants [0.61 (0.53-0.77) vs 0.19 (0.10-0.28); (p<0.0001) and 0.41 (0.37-0.56) vs 0.29 (0.17-0.42); (p=0.007)] respectively. CD4⁺ and CD8⁺ IFN-γ production was significantly decreased in the LTBI-DM compared to the LTBI-only participants [0.28 (0.19-0.38) vs 0.39 (0.25-0.53); (p=0.030) and 0.36 (0.27-0.49) vs 0.55 (0.41-0.88); (p=0.016)] respectively. CD4⁺ TNF and CD8⁺ IL-17A production were significantly decreased in participants with LTBI-DM compared to those with LTBI-only [0.38 (0.33-0.50) vs 0.62 (0.46-0.87); (p=0.004) and 0.29 (0.16-0.42) vs 0.47 (0.29-0.52); (0.017)] respectively. LTBI-DM participants had significantly lower dual-functional (IFN-γ⁺IL-2⁺ and IL-2⁺TNF⁺) and mono-functional (IFN-γ⁺ and TNF⁺) CD4⁺ responses than LTBI-only participants. LTBI-DM participants had significantly decreased dual-functional (IFN-γ⁺IL-2⁺, IFN-γ⁺ TNF⁺ and IL-2⁺TNF⁺) and mono-functional (IFN-γ⁺, IL-2⁺ and TNF⁺) central and effector memory CD4⁺ responses compared to LTBI-only participants.

Conclusion: Type 2 DM impairs the memory phenotypes and functional profiles of Mtb-specific CD4⁺ and CD8⁺ T cells, potentially indicating underlying immunopathology towards increased active TB disease risk.

Keywords: T cells; diabetes mellitus; functional profiles; latent tuberculosis infection; memory phenotypes.

Figure 1

Type 2 DM alters the memory phenotype of Mtb-specific CD4⁺ and CD8⁺ T cells. (A, B) Representative flow cytometry plots are shown for CD4⁺ and CD8⁺ CCR7/CD45RA-defined T-cell memory subsets, respectively. PBMCs were stimulated and cultured for 18 hours with ESAT-6 and CFP-10 peptide pools plus brefeldin A and stained for surface markers. (C, D) Percentage expression of memory phenotypes in CD4⁺ and CD8⁺T cells, respectively. (E, F) Heat maps for the percentage distribution of all memory phenotypes in the three CD4⁺ and CD8⁺ T cell participant groups. Size of participant groups: LTBI-DM (n = 21), LTBI (n = 17), DM (n = 16). Data represent medians and interquartile ranges. The non-parametric Kruskal-Wallis and Mann-Whitney U tests were used to determine the statistical significance between the medians. p<0.05 (*), p< 0.01 (**), p<0.001 (***). Non-significant p-values were not shown.

Figure 2

Type 2 DM impairs the HLA-DR, PD-1, and CD107A expression of Mtb-specific CD4+ and CD8+ T cells. The PBCMs were surface stained with HLA-DR and PD-1 antibodies after 18 hours of incubation with ESAT-6 and CFP-10 peptide pools and brefeldin A. (E, F) For degranulation analysis of CD4⁺ and CD8⁺ T cells, CD107a was added during stimulation. (A-D) Representative plots for HLA-DR and PD-1. Size of participant groups: LTBI-DM (n = 21), LTBI (n = 17), DM (n = 16). Data represent medians and interquartile ranges. The non-parametric Kruskal-Wallis and Mann-Whitney U tests were used to determine the statistical significance between the medians. p< 0.05 (*), p< 0.01 (**), p<0.001 (***) and p< 0.0001 (****). Non-significant p-values were not shown.

Figure 3

Type 2 DM impairs the production of Mtb-specific Th-1, Th-2 and Th-17 cytokines by CD4⁺ and CD8⁺ T cells. The PBMCs were cultured and stimulated for 18 hours with ESAT-6 and CFP-10 peptide pools, brefeldin A, and intracellularly stained for cytokines. (A-J) Representative plots for CD4⁺ and CD8⁺ T-cell producing TNF, IFN-γ, IL-2, IL-13, IL-17A cytokines. Size of participant groups: LTBI-DM (n = 21), LTBI (n = 17), DM (n = 16). Data represent medians and interquartile ranges. The non-parametric Kruskal-Wallis and Mann-Whitney U tests were used to determine the statistical significance between the medians. p< 0.05 (*), p< 0.01 (**). Non-significant p-values were not shown.

Figure 4

Type 2 DM impairs dual and mono-functional Mtb-specific CD4⁺ and CD8⁺ T-cell responses. (A) Polyfunctional Mtb-specific CD4⁺ T-cell responses. (B) Polyfunctional Mtb-specific CD8⁺ T-cell responses. The X-axis represents the frequencies of Mtb-specific CD4⁺ T cells producing all possible IFN-γ, IL-2 and TNF combinations. Data represent medians and interquartile ranges. The non-parametric Kruskal-Wallis and Mann-Whitney U tests were used to determine the statistical significance between the medians. p< 0.05 (*), p< 0.01 (**), p<0.001 (***) and p< 0.0001 (****).

Figure 5

Type 2 DM impairs triple, dual, mono-functional Mtb-specific central and effector memory CD4⁺ T-cell responses. (A) Polyfunctional Mtb-specific central memory CD4⁺ T-cell responses. (B) Polyfunctional Mtb-specific effector memory CD4⁺ T-cell responses. The X-axis represents the frequencies of Mtb-specific central and effector memory CD4⁺ T cells producing all possible combinations of IFN-γ, IL-2 and TNF. Data represent medians and interquartile ranges. Kruskal-Wallis and Mann-Whitney U tests were used to determine the statistical significance between the medians. p< 0.05 (*), p< 0.01 (**), p<0.001 (***) and p< 0.0001 (****). CM, Central memory; EM, Effector memory.