Multi-functional flow cytometry analysis of CD4+ T cells as an immune biomarker for latent tuberculosis status in patients treated with tumour necrosis factor (TNF) antagonists

Abstract

Although monitoring tuberculosis (TB) infection during long-term treatment with tumour necrosis factor (TNF) antagonists is of great importance, no monitoring strategy has yet proved successful. Indeed, even the newly proposed interferon-gamma release assays (IGRAs) are known to produce dynamic changes in IFN-γ plasma levels, making them unreliable indicators of patients' pathological/clinical status. We used intracellular cytokine flow cytometry (ICCFC) to investigate the performance of multi-functional CD4(+) T cells producing IFN-γ, interleukin (IL)-2 and/or TNF in response to Mycobacterium tuberculosis-specific antigens in subjects treated with TNF antagonists. Patients were classified into three groups based on their TB status before commencement of treatment and on IFN-γ level fluctuations evaluated by IGRA during a 36-month follow-up period. The cytokine profile of M. tuberculosis-specific CD4(+) T cells showed that latent tuberculosis infection (LTBI) subjects had a higher frequency of double-positive IFN-γ(+) IL-2(+) CD4(+) T cells and triple-positive IFN-γ(+) IL-2(+) TNF(+) CD4(+) T cells compared to those without LTBI, who showed IFN-γ-level fluctuations over time. In contrast, this latter group of patients showed similar proportions of cells producing IFN-γ alone, IL-2 alone and IL-2 in combination with TNF in response to M. tuberculosis-specific antigens. It therefore appears that patients with and without LTBI infection are characterized by different intracellular cytokine profiles. This is the first study evaluating ICCFC in patients treated with TNF antagonists, and suggests that multi-functional analysis of CD4(+) T cells could be useful for ruling out TB infection in patients classified at screening as LTBI-negative but who show IGRA fluctuations under long-term TNF antagonist treatment.

Characterization of CD4/CD8+ αβ and Vγ2Vδ2+ T cells in HIV-negative individuals with different Mycobacterium tuberculosis infection statuses

“…However, the differences in antigen-specific T cells in subjects with different M.tb infection statuses 1 in humans are not clear. Most studies have focused on differences between active 2 tuberculosis and healthy individuals [23][24][25]. Studies on the distribution of T cell 3 subsets in populations with different M.tb infection statuses were limited [26][27][28].…”

Flow cytometric immune profiling in infliximab-associated tuberculosis

“…5 The FC-based assays of T-cell markers could potentially provide an additional diagnostic tool to identify patients on TNFA with latent and active TB. [9][10][11] Combinatorial interferon gamma release assays and FC assays assessing TB antigen-induced T-cell CD25 (interleukin-2 receptor α chain) and CD134 (TNF-α receptor superfamily member) co-expression were recently described as a method to risk stratify patients with LTBI. 7 Furthermore, this strategy has been used to identify patients with LTBI with HIV co-infection.…”

Treatment of latent tuberculosis infection induces changes in multifunctional Mycobacterium tuberculosis-specific CD4+ T cells

“…The TB antigens are pools of overlapping peptides and pooled together as a single stimulation condition. Whole blood was co-stimulated with anti-CD28 plus anti-CD49d (5 μl/ml, BD Bioscience, Pharmingen, Italy) as indicated by several authors [11,12,24], and Brefeldin A (10 μg/ml, Sigma-Aldrich) was immediately added to each tube, as previously described [16,20]. In brief, after 18 h of incubation, the cell surface staining was performed with the following markers: anti-CD45-VioBlue, anti-CD4 PE-Vio770, and anti-CD8 PerCP (Miltenyi Biotec, Germany) and the red cells were lysed with 1 ml FACS Lysing Solution (BD Bioscience).…”

“…First and foremost, we analysed a relatively small number of patients within each clinical group, even if the number of subjects enrolled is comparable to those described in similar reports [10,16,20,29]. Secondly, this is a cross-sectional study, and determining the clinical utility of cytokine profiles of Mtb-specific T cells for treatment monitoring will require a longitudinal study.…”

“…No significant differences between untreated and treated LTBI subjects in terms of Mtb-specific CD8 + T cell cytokine profiles (p > 0.05) were identified. The significant changes in the cytokine profiles of Mtb-specific T cells after INH therapy suggest that analysis IGRA to detect TB infection [17][18][19][20]. Indeed, ICCFC has been reported to improve discrimination between active TB and LTBI [10][11][12][13][14][15][16] and to enable more reliable monitoring of responses to TB treatment [11-13, 21, 22].…”