Analysis of Mycobacterium tuberculosis-specific CD8 T-cells in patients with active tuberculosis and in individuals with latent infection

Abstract

CD8 T-cells contribute to control of Mycobacterium tuberculosis infection, but little is known about the quality of the CD8 T-cell response in subjects with latent infection and in patients with active tuberculosis disease. CD8 T-cells recognizing epitopes from 6 different proteins of Mycobacterium tuberculosis were detected by tetramer staining. Intracellular cytokines staining for specific production of IFN-gamma and IL-2 was performed, complemented by phenotyping of memory markers on antigen-specific CD8 T-cells. The ex-vivo frequencies of tetramer-specific CD8 T-cells in tuberculous patients before therapy were lower than in subjects with latent infection, but increased at four months after therapy to comparable percentages detected in subjects with latent infection. The majority of CD8 T-cells from subjects with latent infection expressed a terminally-differentiated phenotype (CD45RA+CCR7(-)). In contrast, tuberculous patients had only 35% of antigen-specific CD8 T-cells expressing this phenotype, while containing higher proportions of cells with an effector memory- and a central memory-like phenotype, and which did not change significantly after therapy. CD8 T-cells from subjects with latent infection showed a codominance of IL-2+/IFN-gamma+ and IL-2(-)/IFN-gamma+ T-cell populations; interestingly, only the IL-2+/IFN-gamma+ population was reduced or absent in tuberculous patients, highly suggestive of a restricted functional profile of Mycobacterium tuberculosis-specific CD8 T-cells during active disease. These results suggest distinct Mycobacterium tuberculosis specific CD8 T-cell phenotypic and functional signatures between subjects which control infection (subjects with latent infection) and those who do not (patients with active disease).

Figure 1. Comparison of the frequencies of tetramer⁺ CD8 T-cells in peripheral blood from LTBI subjects and TB patients with active disease before therapy (T0) and after four months of therapy (T4).

(A) In each group tested, LTBI subjects, TB patients at T0 and TB patients at T4, the median proportion of tetramer⁺ CD8 T-cells was estimated as 100% and the relative percentages of individual tetramer⁺ CD8 T-cells calculated accordingly. (B) Dot plot analysis of tetramer⁺ CD8⁺ T-cell populations of one representative LTBI subject, one TB patient at T0 and one TB patient at T4.

Figure 2. Phenotypic analysis of tetramer⁺ Mtb-specific CD8 T-cells.

Peripheral blood mononuclear cells (PBMC) were stained with individual tetramers, and anti-CD8, -CD45RA and -CCR7 mAbs to separate functionally distinct subpopulations. After gating on tetramers⁺ CD8⁺ cells, the percentage of cells expressing CD45RA and CCR7 was determined. (A) Representative phenotyping data for one subject with LTBI (LTBI), one TB patient before (T0) and one TB patient 4 months after therapy (T4). Numbers in the corners indicate the percentage of positive cells in each quadrant. (B) Summary cumulative data of the phenotype of tetramer⁺ Mtb-specific CD8 T-cells. Data are presented with box plot reporting the median values and the interquartile range. Black columns = LTBI; grey columns = T0; white columns = T4.

Figure 3. Polyfunctional cytokine production analysis of tetramer⁺ Mtb-specific CD8 T-cells.

Peripheral blood mononuclear cells (PBMC) were stimulated with the same individual peptides as those present in tetramers and were stained with mAbs to CD8, IFN-γ and IL-2, or with isotype-control mAbs. After gating on CD8⁺ cells, the percentage of cells expressing IFN-γ and IL-2 was determined. (A) Representative intracellular cytokine staining data in one subject with LTBI, one TB patient before therapy and one PPD⁻ healthy donor. Numbers in the corners indicate the percentage of CD8⁺ cytokine-positive cells in each quadrant. (B) Summary cumulative data of the IFN-γ and IL-2 secretion capability of tetramer⁺ Mtb-specific CD8 T-cells in LTBI subjects (white bars) and TB patients with active disease before therapy (black bars). The data are expressed as the percentage of CD8⁺ T-cells that are IFN-γ⁺/IL-2⁻ or IFN-γ⁺/IL-2⁺. The values reported are the mean percentage of the different subset analysed for each group tested ± standard deviations (SD). *p<0.001 and **p<0.01 when compared to values in LTBI subjects.

Figure 4. Peptides-specific CD8 T cell responses in LTBI subjects, in TB patients at T0 and T4.

Peptides-specific CD8 T cell responses are shown as a pie chart. Each portion of a pie chart indicates the percentage of peptides-specific T cells that responded with one or two functions, i.e. producing IFN-γ alone or the combination of IFN-γ and IL-2 (see legend).