Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load

Abstract

High Ag load in chronic viral infections has been associated with impairment of Ag-specific T cell responses; however, the relationship between Ag load in chronic Mycobacterium tuberculosis infection and functional capacity of M. tuberculosis-specific T cells in humans is not clear. We compared M. tuberculosis-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads-that is, persons with latent M. tuberculosis infection (LTBI), with smear-negative pulmonary tuberculosis (TB), and smear-positive TB. Patients with smear-positive TB had decreased polyfunctional IFN-γ(+)IL-2(+)TNF-α(+) and IL-2-producing specific CD4 T cells and increased TNF-α single-positive cells, when compared with smear-negative TB and LTBI. TB patients also had increased frequencies of M. tuberculosis-specific CD8 T cells, compared with LTBI. M. tuberculosis-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear-positive TB, and correlated positively with ex vivo IFN-γ(+)IL-2(+)TNF-α(+) CD4 T cells, and inversely with TNF-α single-positive CD4 T cells. During 6 mo of anti-TB treatment, specific IFN-γ(+)IL-2(+)TNF-α(+) CD4 and CD8 T cells increased, whereas TNF-α and IFN-γ single-positive T cells decreased. These results suggest progressive impairment of M. tuberculosis-specific T cell responses with increasing mycobacterial load and recovery of responses during therapy. Furthermore, these data provide a link between specific cytokine-producing subsets and functional capacity of M. tuberculosis-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. These data have potentially significant applications for the diagnosis of TB and for the identification of T cell correlates of TB disease progression.

Figure 1. Reduced polyfunctional and IL-2-producing Mtb-specific CD4 T cells in individuals with smear+ TB, compared with smear− TB and LTBI

Whole blood from individuals with LTBI, smear− TB and smear+ TB was stimulated for 8 hours with Mtb peptide pools, PPD, or SEB, and analyzed by flow cytometry for intracellular expression of IFN-γ, IL-2, and TNF-α in CD4 T cells. (A) Representative whole blood ICS assay data from an LTBI donor (left), a smear− TB donor (middle), and a smear+ TB donor (right). The plots shown were gated on CD3⁺CD4⁺ T cells; cells expressing all 3 cytokines (3+; red), any 2 cytokines (2+; green), or any one cytokine only (1+; blue) are shown on each plot. (B) Qualitative analysis of ESAT-6 and PPD-specific CD4 T cell responses. The contribution of each cytokine-producing subset to the total ESAT-6 or PPD-specific CD4 T cell response was assessed in donors with positive responses in the ICS assay to ESAT-6 (n=23 LTBI, n=12 smear− TB, n=23 smear+ TB) and PPD (n=30 LTBI, n=16 smear− TB, n=38 smear+ TB). (C) Contribution of IL-2-producing CD4 T cells to the total ESAT-6, PPD, and SEB-specific CD4 T cell response. The total proportion of IL-2-producing cells to the total CD4 response was determined in each donor shown in panel B. All donors had a positive CD4 response to SEB, and were thus included in the contribution of IL-2 to the total SEB response. For the data in panels B and C, differences between the three groups were assessed by the Kruskal-Wallis test, and if significance was found (p<0.05), the Mann-Whitney test was used for comparison of two groups. For box and whiskers plots, the horizontal line represents the median, the boxes the interquartile range, and the whiskers the 10^th and 90^th percentiles.

Figure 2. Increased frequency of Mtb-specific CD8 T cell responses in individuals with pulmonary TB, compared with LTBI

Whole blood from individuals with LTBI, smear− TB and smear+ TB was stimulated for 8 hours with Mtb peptide pools, PPD, or SEB, and analyzed by flow cytometry for intracellular expression of IFN-γ, IL-2, and TNF-α in CD8 T cells. (A) Representative whole blood ICS assay data from an LTBI donor (left), a smear− TB donor (middle), and a smear+ TB donor (right). The plots shown were gated on CD3⁺CD8⁺ T cells; cells expressing all 3 cytokines (3+; red), any 2 cytokines (2+; green), or any one cytokine only (1+; blue) are shown on each plot. (B) Comparison of the frequency of each cytokine-producing CD8 T cell subset specific for CFP-10 (left) or ESAT-6 (right) in LTBI (n=30), smear− TB (n=16), and smear+ TB donors (n=38). Background cytokine production in the negative control sample has been subtracted. Differences between the groups were assessed by Kruskal-Wallis test; if significance was found (p<0.05), the Mann-Whitney test was used for comparison between two groups. (C) Qualitative analysis of cytokine-producing CFP-10 and ESAT-6-specific CD8 T cell responses. The contribution of each cytokine-producing subset to the total CD8 T cell response was assessed in donors with positive responses to either CFP-10 or ESAT-6 (n=6 LTBI, n=6 smear− TB, n=23 smear+ TB). (D) Frequency of Mtb-specific CD8 T cell responders. The percentage of LTBI, smear− TB, and smear+ TB donors who had a positive CD8 T cell response in the ICS assay to either CFP-10 and/or ESAT-6 is shown. Differences in the frequency of recognition of these antigens were determined by the Fisher’s exact test. For all graphs, white bars represent individuals with LTBI, light grey bars smear− TB, and dark grey bars smear+ TB.

Figure 3. Polyfunctional Mtb-specific CD4 T cell responses increase following reduction in mycobacterial load by initiation of anti-TB treatment

Whole blood ICS assays were performed on a subset of individuals with pulmonary TB (n=4 smear− TB, n=9 smear+ TB) prior to or within 7 days of anti-TB treatment (pre-TB tx), and again at 2 and 6 months following initiation of TB treatment. (A) Representative ICS data from PPD-stimulated whole blood of an individual with smear+ TB pre-treatment, and 2 and 6 months on TB treatment. Plots are gated on CD3⁺CD4⁺ T cells; cells expressing all 3 cytokines (3+; red), any 2 cytokines (2+; green), or any one cytokine only (1+; blue) are shown on each plot. The percentages shown are after subtraction of background cytokine production by CD4 T cells in the negative control at each time point. (B) Longitudinal analysis of the total frequency of IFN-γ, TNF-α, and IL-2-producing CD4 T cells following 8-hour stimulation of whole blood with CFP-10 and ESAT-6 peptide pools, and PPD. Each symbol represents a different donor, with the same symbols used for each donor in all three graphs. Differences in the total frequency of Mtb-specific CD4 T cells over the three time points were determined by the Freidman test; if significance was found (p<0.05), the Wilcoxon matched pairs test was used for comparison between two time points. (C) Contribution of each cytokine-producing subset to the total PPD-specific CD4 T cell response pre-treatment, and 2 and 6 months on TB treatment. All 13 individuals followed longitudinally maintained a positive PPD-specific response throughout treatment, and were therefore included in this analysis. Differences in the contribution of each subset between the three time points were determined by the Freidman test; if significance was found (p<0.05), the Wilcoxon matched pairs test was used for comparisons between two time points. (D) Longitudinal analysis of the proportion of cytokine-producing CD4 T cells to the total PPD-specific response. The four different cytokine-producing subsets of PPD-specific CD4 T cells that showed a significant change on TB treatment (panel C) were assessed on an individual donor level at each time point. Each symbol represents a different donor, with the same symbols used for each donor in all 4 graphs. The same symbols were used for each donor in panels B and D.

Figure 4. Polyfunctional Mtb-specific CD8 T cell responses increase following reduction in mycobacterial load by initiation of anti-TB treatment

Whole blood ICS assays were performed on a subset of individuals with pulmonary TB as described in Figure 3. (A) Representative whole blood ICS data from ESAT-6 peptide pool-stimulated whole blood of an individual with smear+ TB pre-treatment, and 2 and 6 months on TB treatment. Plots are gated on CD3⁺CD8⁺ lymphocytes; cells expressing all 3 cytokines (3+; red), any 2 cytokines (2+; green), or any one cytokine only (1+; blue) are shown on each plot. The percentages shown are after subtraction of background cytokine production by CD8 T cells in the negative control at each time point. (B) Longitudinal analysis of the total frequency of IFN-γ, TNF-α, and IL-2-producing CD8 T cells following 8-hour stimulation of whole blood with CFP-10 and ESAT-6 peptide pools. Each symbol represents a different donor, with the same symbols used for each donor in both graphs. No differences in the total frequency of CFP-10 or ESAT-6-specific CD8 T cells over the three time points were found (Friedman test). (C) Contribution of each cytokine-producing population to the total CFP-10 and ESAT-6-specific CD8 T cell response pre-treatment, and 2 and 6 months on TB treatment. Seven individuals followed longitudinally maintained a positive CFP-10 or ESAT-6-specific CD8 T cell response at each time point, and were therefore included in this analysis. Differences in the contribution of each subset between the three time points were determined by the Freidman test; if significance was found (p<0.05), the Wilcoxon matched pairs test was used for comparisons between two time points. (D) Longitudinal analysis of the proportion of IFN-γ⁺TNF-α⁺IL-2⁺ and IFN-γ-single positive CD8 T cells contributing to the total CFP-10 or ESAT-6-specific response. These two cytokine-producing subsets of CFP-10 and ESAT-6-specific CD8 T cells showed a significant change on TB treatment (panel C), and were assessed on an individual donor level at each time point. Each symbol represents a different donor; the same symbols were used for each donor in panels B and D.

Figure 5. Impaired proliferative capacity of Mtb-specific T cells in smear+ TB, compared with LTBI

Freshly isolated PBMC from individuals with LTBI (n=12) and smear+ TB (n=14) were labeled with Oregon Green (OG) and stimulated with CFP-10 and ESAT-6 peptide pools, PPD, or SEB for 6 days. (A) Representative CD4 T cell proliferation assay data from an individual with LTBI (top row) and smear+ TB (bottom row); plots are gated on Vivid^lowCD3⁺CD4⁺ T cells. The percentages indicate the frequency of proliferating (OG^low) cells in the gated population. (B) Representative CD8 T cell proliferation assay data from an individual with LTBI (top row) and smear+ TB (bottom row); plots are gated on Vivid^lowCD3⁺CD8⁺ T cells. The percentages indicate the frequency of proliferating (OG^low) cells in the gated population. Summary data of CD4 and CD8 T cell proliferative capacity in individuals with LTBI (closed symbols) and smear+ TB (open symbols) are shown in (C) and (D), respectively. Background proliferation in the negative control has been subtracted. Horizontal lines represent the median; differences were assessed by the Mann-Whitney test.

Figure 6. Mtb-specific CD4 T cell proliferative capacity correlates with the quantity and quality of ex vivo cytokine-producing CD4 T cells

Correlations between the frequency of proliferating T cells with the total frequency of cytokine-producing T cells in the ex vivo whole blood ICS assay are shown for PPD-specific CD4 T cells (A), CFP-10/ESAT-6-specific CD4 T cells (B), and CFP-10/ESAT-6-specific CD8 T cells (C). Correlations between the frequency of proliferating CD4 T cells and the proportion of IFN-γ⁺IL-2⁺TNF-α⁺ T cells contributing to the total ex vivo ICS response are shown for PPD-specific CD4 T cells (D) and CFP-10/ESAT-6 specific CD4 T cells (E). (F) Correlation between the frequency of CFP-10/ESAT-6-specific proliferating CD4 T cells with the proportion of CFP-10/ESAT-6-specific TNF-α-single positive CD4 T cells ex vivo. Black circles represent LTBI donors and white circles represent smear+ TB donors. Correlations were assessed by the Spearman rank test.