Type 2 diabetes mellitus is associated with altered CD8(+) T and natural killer cell function in pulmonary tuberculosis

Abstract

Type 2 diabetes mellitus (DM) is associated with expanded frequencies of mycobacterial antigen-specific CD4(+) T helper type 1 (Th1) and Th17 cells in individuals with active pulmonary tuberculosis (TB). No data are available on the role of CD8(+) T and natural killer (NK) cells in TB with coincident DM. To identify the role of CD8(+) T and NK cells in pulmonary TB with diabetes, we examined mycobacteria-specific immune responses in the whole blood of individuals with TB and DM (TB-DM) and compared them with those without DM (TB-NDM). We found that TB-DM is characterized by elevated frequencies of mycobacterial antigen-stimulated CD8(+) T cells expressing type 1 [interferon-γ and interleukin-2 (IL-2)] and type 17 (IL-17F) cytokines. We also found that TB-DM is characterized by expanded frequencies of TB antigen-stimulated NK cells expressing type 1 (tumour necrosis factor-α) and type 17 (IL-17A and IL-17F) cytokines. In contrast, CD8(+) T cells were associated with significantly diminished expression of the cytotoxic markers perforin, granzyme B and CD107a both at baseline and following antigen or anti-CD3 stimulation, while NK cells were associated with significantly decreased antigen-stimulated expression of CD107a only. This was not associated with alterations in CD8(+) T-cell or NK cell numbers or subset distribution. Therefore, our data suggest that pulmonary TB complicated with type 2 DM is associated with an altered repertoire of cytokine-producing and cytotoxic molecule-expressing CD8(+) T and NK cells, possibly contributing to increased pathology.

Trial registration: ClinicalTrials.gov NCT01154959.

Keywords: CD8+ T cells; diabetes; natural killer cells; tuberculosis.

Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Figures

Figure 1

Elevated antigen-stimulated frequencies of type 1 and type 17 cytokine secreting CD8+ T cells in patients with tuberculosis and diabetes mellitus (TB-DM). (a) A representative whole-blood intracellular cytokine assay flow data from a TB-DM individual showing expression of interferon-γ (IFN-γ), interleukin-2 (IL-2), tumour necrosis factor-α (TNF-α), IL-17A, IL-17F and IL-22. The plots shown are gated on CD3+, CD8+ T cells. (b) The baseline frequency of CD8+ T cells expressing type 1 (IFN-γ, IL-2, TNF-α) or type 17 (IL-17A, IL-17F and IL-22) cytokines is shown as scatter plots with the line representing the geometric mean of the frequency of CD8+ T cells expressing the respective cytokine(s) in TB-DM (n = 22) and TB–no diabetes mellitus (TB-NDM; n = 22) individuals. (c, d) The net frequency of CD8+ T cells expressing type 1 (IFN-γ, IL-2, TNF-α) or type 17 (IL-17A, IL-17F and IL-22) cytokines in response to culture filtrate protein-10 (CFP-10) (c) and early secretory antigen 6 (ESAT-6) (d) are shown in TB-DM and TB-NDM individuals. (e) The net frequency of CD8+ T cells expressing the different cytokines in response to anti-CD3 stimulation is shown in TB-DM and TB-NDM individuals. Net frequencies were calculated by subtracting baseline frequencies from antigen-stimulated or anti-CD3-stimulated frequencies. P-values were calculated using the Mann–Whitney test.

Figure 2

Elevated antigen-stimulated frequencies of type 1 and type 17 cytokine-secreting natural killer (NK) cells in tuberculosis–diabetes mellitus (TB-DM). (a) A representative whole-blood intracellular cytokine assay flow data from a TB-DM individual showing expression of interferon-γ (IFN-γ), interleukin-2 (IL-2), tumour necrosis factor-α (TNF-α), IL-17A, IL-17F and IL-22. The plots shown are gated on CD56+, CD56+ CD3− cells. (b) The baseline frequency of CD8+ T cells expressing type 1 (IFN-γ, IL-2, TNF-α) or type 17 (IL-17A, IL-17F and IL-22) cytokines is shown as scatter plots with the line representing the geometric mean of the frequency of NK cells expressing the respective cytokine(s) in TB-DM (n = 22) and tuberculosis–no diabetes mellitus (TB-NDM) (n = 22) individuals. (c, d) The net frequency of NK cells expressing type 1 (IFN-γ, IL-2, TNF-α) or type 17 (IL-17A, IL-17F and IL-22) cytokines in response to culture filtrate protein 10 (CFP-10) (c) and early secretory antigen 6 (ESAT-6) (d) is shown in TB-DM and TB-NDM individuals. Net frequencies were calculated by subtracting baseline frequencies from antigen-stimulated frequencies. P-values were calculated using the Mann–Whitney test.

Figure 3

Diminished frequencies of cytotoxic marker expressing CD8+ T cells in patients with tuberculosis and diabetes mellitus (TB-DM). (a) A representative whole-blood intracellular cytotoxic marker flow data from a TB-DM individual showing CD8+ T-cell expression of perforin, granzyme B and CD107a. (b) The baseline or antigen or anti-CD3 stimulated net frequencies of CD8+ T cells expressing the cytotoxic markers perforin, granzyme B and CD107a are shown as scatter plots with the line representing the geometric mean of the frequency of CD8+ T cells expressing the respective marker in TB-DM (n = 22) and tuberculosis–no diabetes mellitus (TB-NDM) (n = 22) individuals. Net frequencies were calculated by subtracting baseline frequencies from antigen-stimulated or anti-CD3-stimulated frequencies. P-values were calculated using the Mann–Whitney test.

Figure 4

Diminished frequencies of cytotoxic marker expressing natural killer (NK) cells in tuberculosis–diabetes mellitus (TB-DM). (a) A representative whole-blood intracellular cytotoxic marker flow data from a TB-DM individual showing NK cell expression of perforin, granzyme B and CD107a. (b) The baseline or antigen-stimulated net frequencies of NK cells expressing the cytotoxic markers perforin, granzyme B and CD107a are shown as scatter plots with the line representing the geometric mean of the frequency of NK cells expressing the respective marker in TB-DM (n = 22) and tuberculosis–no diabetes mellitus (TB-NDM) (n = 22) individuals. Net frequencies were calculated by subtracting baseline frequencies from antigen-stimulated frequencies. P-values were calculated using the Mann–Whitney test.

Figure 5

Tuberculosis–diabetes mellitus (TB-DM) is associated with increased frequencies of naive but not central or effector memory CD8+ T cells in TB-DM. (a) The absolute numbers of CD8+ T cells and natural killer (NK) cells in TB-DM and tuberculosis–no diabetes mellitus (TB-NDM) individuals. (b) Percentages of naive (defined as CD45RA+ CCR7+), central memory (defined as CD45RA– CCR7+), and effector memory (defined as CD45RA– CCR7–) CD8+ T cells in TB-DM and TB-NDM individuals. The results are shown as scatter plots with each circle representing a single individual. P-values were calculated using the Mann–Whitney test.