Tuberculosis Treatment Response Monitoring by the Phenotypic Characterization of MTB-Specific CD4+ T-Cells in Relation to HIV Infection Status

Abstract

HIV infection causes systemic immune activation, impacts TB disease progression and hence may influence the diagnostic usability of Mycobacterium tuberculosis-specific T cell profiling. We investigated changes of activation and maturation markers on MTB-specific CD4+ T-cells after anti-tuberculosis treatment initiation in relation to HIV status and the severity of lung impairment. Thawed peripheral blood mononuclear cells from TB patients with (n = 27) and without HIV (n = 17) were analyzed using an intracellular IFN-γ assay and flow cytometry 2 and 6 months post-TB treatment initiation. H37Rv antigen was superior to the profile MTB-specific CD4+ T-cells phenotype when compared to PPD and ESAT6/CFP10. Regardless of HIV status and the severity of lung impairment, activation markers (CD38, HLA-DR and Ki67) on MTB-specific CD4+ T-cells declined after TB treatment initiation (p < 0.01), but the expression of the maturation marker CD27 did not change over the course of TB treatment. The MTB-specific T cell phenotype before, during and after treatment completion was similar between people living with and without HIV, as well as between subjects with severe and mild lung impairment. These data suggest that the assessment of activation and maturation markers on MTB-specific CD4+ T-cells can be useful for TB treatment monitoring, regardless of HIV status and the severity of lung disease.

Keywords: HIV; Mycobacterium tuberculosis (MTB)-specific; lung severity; tuberculosis.

Figure 1

Detection of dynamic changes in the expression of the activation and maturation markers on MTB-specific CD4+ T-cells upon TB treatment initiation. The frequency of T cells expressing the activation markers CD38, HLA-DR, Ki67 and CD27 in all subjects (n = 44, (A–D)) and per group (E–H): HIV/TB coinfected (n = 27) and TB monoinfected (n = 17) at baseline (n = 35), 2 months (n = 40) and 6 (n = 33) months after TB treatment. The red circles and blue squares represent HIV/TB coinfected and TB monoinfected subjects, respectively. The black dots in the HIV/TB coinfected plots represent the subjects ART naïve at baseline. MTB-specific CD4+ T-cells were characterized after H37Rv stimulation. Bars represent medians. Statistical analyses were performed using the Mann–Whitney test for unmatched samples and with the Wilcoxon signed rank test for paired samples. p-values are indicated.

Figure 2

The slopes (difference from baseline to month 2) of the expression of activation markers CD38 (A), HLA-DR (B) and Ki67 (C) on MTB-specific and total CD4+ T-cells were compared between and among the HIV/TB coinfected (n = 19) and TB monoinfected (n = 14) groups. The red circles and blue squares represent HIV/TB coinfected and TB monoinfected subjects, respectively. Bars represent medians. Statistical analyses were performed using the Mann–Whitney test. p-values are indicated.

Figure 3

The dynamic of the expression of the activation markers CD38 (A), HLA-DR (B) and Ki67 (C) as well as the maturation marker CD27 (D) on MTB-specific CD4+ T-cells from the beginning to the end of TB treatment (n = 28). The expression of these markers was analyzed among subjects with severe (n = 13) and mild (n = 15) lung function impairment (A–D) at baseline (n = 23), month 2 (n= 25) and month 6 (n = 22). The blue circles and red triangles represent subjects with severe and mild lung impairment, respectively. MTB-specific CD4+ T-cells were characterized after H37Rv stimulation. The mild group includes subjects with mild and moderate lung impairments. Bars represent medians. Statistical analyses were performed using the Mann–Whitney test for unmatched samples and the Wilcoxon signed rank test for paired samples. p-values are indicated.

Figure 4

STARD flow diagram of study subjects. Peripheral blood mononuclear cell samples from TB patients with or without HIV (n = 44) were selected at baseline, month 2 and month 6 after TB treatment initiation, in vitro stimulated with MTB antigens and analyzed by flow cytometry. The number of missing visits (n = 11) and those excluded due to CD4 IFN-γ count <20 (poor quality samples, n = 13) is indicated for each time point. All samples responded to positive control antigen, SEB.