Title : Antigen-specific CD4+T cell activation, proliferation and functional markers of adult pulmonary tuberculosis patients in Addis Ababa, Ethiopia.
The poor diagnostic and prognostic potential of existing tools potentially hinder the tuberculosis (TB) prevention and control program. Currently, antigen-specific blood-based biomarkers have become alternatives to improve the diagnostic and prognostic challenges related to tuberculosis (TB) diagnosis and treatment. In this study, we evaluated antigen-specific CD4+T cell activation (HLA-DR, CD-38, CD40L), proliferation (Ki-67), and functional (IFN-γ, TNF- α) markers as alternative tools to diagnose smear positive and negative PTB and monitor the effectiveness of anti-TB treatment for adult pulmonary TB using polychromatic flow cytometry. Alongitudinal cohort study was conducted from August 2020 to July 2021. Newly diagnosed HIV-negative, smear positive (n= 34), and smear-negative pulmonary TB (n=29) adults were prospectively recruited and followed at baseline, second, and sixth months of anti-TB treatment from selected TB clinics in Addis Ababa, Ethiopia. In addition, patients with confirmed non-TB respiratory illness (n= 33) and apparently healthy QFT positive (n=30) and negative (n=23) were included in the study as comparators. M. tuberculosis identification and confirmation from sputum were done by direct microscopy, LJ/MGIT culture, and RD9 PCR. Fresh peripheral mononuclear cells and plasma were used for different immunological analyses (IGRA, surface, and intracellular staining for flow cytometry). Flow cytometry data were analyzed with FlowJo analytical software (version 9.9.6). GraphPad Prism (Version 6.0) was used to analyze data and for graphic display. Comparison between groups was performed using the non-parametric Mann-Whitney for baseline data and Wilcoxon matched-paired rank test for follow-up data. A p-value less than 0.05 was considered statistically significant. Our baseline study showed that the percentage expression of activation markers from PPD-specific IFN-γ+CD4+Tcells or TNF-α+ CD4+T cells was substantially higher among smear positive and negative pulmonary TB patients compared to apparently healthy controls (p-value = 0.0004 and p-value = 0.0056, respectively) and to a lesser extent among patients with non-TB respiratory illnesses (p-value =0.034). However, for the majority of the cases, the percentage of PPD-specific CD4+T cells expressing activation and proliferation markers did not significantly change from baseline data during the second month of anti-TB treatment follow-up. Furthermore, after six months of standard anti-TB therapy, the percentage of PPD-specific CD4+T cells expressing activation markers (HLA-DR, CD-38, and CD40L) was significantly lower in smear-positive PTB patients following treatment (p-value, = 0.0177,0.0163, and 0.0061, respectively) compared to the baseline. However, a consistent decline in these markers was less apparent among smear-negative PTB patients both in the second and sixth months compared with the baseline. The ex vivo expression pattern of activation (HLA-DR, CD38, CD40L) and proliferation (Ki-67) markers among the total unstimulated CD4+T cells was higher at the baseline and decreased significantly after therapy, particularly at month 6 (p-value = 0.003, < 0.0001, 0.002, and 0.01, respectively). Our study indicated that that PPD-specific cytokine-producing CD4+T cells co-expressing activation markers (HLA-DR, CD-38, CD40L) in a multicolor flow cytometry assay could distinguish smear positive and negative PTB from non-TB respiratory illness and healthy controls. Moreover, during the second month of TB treatment follow-up, we observed a limited prognostic role of activation, proliferation, and cytokine markers from antigen-specific CD4+T cells in smear-positive and smear-negative PTB patients. After six months of standard anti-TB therapy, a considerable percentage reduction of PPD-specific CD4+T cells that produce cytokines as well as percentage expression of those cells' co-expressing activation markers (HLA-DR, CD-38, and CD40L) may indicate a prognostic role in smear positive PTB patients. On the other hand, a persistent percentage of activation markers from PPD-specific CD4+T cells among smear-negative PTB patients may suggest that these markers have little value in monitoring and predicting anti-TB treatment responses. Also, the considerable percentage reduction in ex-vivo activation marker expression during anti-TB therapy follow-up may suggest the prognostic value of those markers in smear-positive and negative PTB patients.
Keywords: Pulmonary tuberculosis, smear microscopy, diagnostic and prognostic biomarkers, PPD-specific CD4+T cells.