Mycobacterium tuberculosis Immune Response in Patients With Immune-Mediated Inflammatory Disease

Abstract

Subjects with immune-mediated inflammatory diseases (IMID), such as rheumatoid arthritis (RA), have an intrinsic higher probability to develop active-tuberculosis (TB) compared to the general population. The risk ranges from 2.0 to 8.9 in RA patients not receiving therapies. According to the WHO, the RA prevalence varies between 0.3% and 1% and is more common in women and in developed countries. Therefore, the identification and treatment of TB infection (TBI) in this fragile population is important to propose the TB preventive therapy. We aimed to study the M. tuberculosis (Mtb) specific T-cell response to find immune biomarkers of Mtb burden or Mtb clearance in patients with different TB status and different risk to develop active-TB disease. We enrolled TBI subjects as example of Mtb-containment, the active-TB as example of a replicating Mtb status, and the TBI-IMID as fragile population. To study the Mtb-specific response in a condition of possible Mtb sterilization, we longitudinally enrolled TBI subjects and active-TB patients before and after TB therapy. Peripheral blood mononuclear cells were stimulated overnight with Mtb peptides contained in TB1- and TB2-tubes of the Quantiferon-Plus kit. Then, we characterized by cytometry the Mtb-specific CD4 and CD8 T cells. In TBI-IMID, the TB therapy did not affect the ability of CD4 T cells to produce interferon-γ, tumor necrosis factor-α, and interleukin-2, their functional status, and their phenotype. The TB therapy determined a contraction of the triple functional CD4 T cells of the TBI subjects and active-TB patients. The CD45RA^- CD27⁺ T cells stood out as a main subset of the Mtb-specific response in all groups. Before the TB-preventive therapy, the TBI subjects had higher proportion of Mtb-specific CD45RA^-CD27⁺CD4⁺ T cells and the active-TB subjects had higher proportion of Mtb-specific CD45RA^-CD27^-CD4⁺ T cells compared to other groups. The TBI-IMID patients showed a phenotype similar to TBI, suggesting that the type of IMID and the IMID therapy did not affect the activation status of Mtb-specific CD4 T cells. Future studies on a larger and better-stratified TBI-IMID population will help to understand the change of the Mtb-specific immune response over time and to identify possible immune biomarkers of Mtb-containment or active replication.

Keywords: CD27; IFN-γ; M. tuberculosis; immune-mediated inflammatory disease; tuberculosis.

Figure 1

Gating strategy and representative panel of Mtb-specific CD4 and CD8 T-cell response in one patient with TBI-IMID. Briefly, lymphocytes were gated according to FSC and SSC parameters, doublets were excluded (FSC-A/FSC-H), and CD4 and CD8 T cells were gated inside the CD3-T cells subset. Frequency of IFN-γ, IL-2, and TNF-α producing T cells was evaluated inside CD4 and CD8 subsets. CD45RA and CD27 proportion on Mtb-specific T cells was evaluated inside the total cytokine response. The CD45RA and CD27 gating position was decided on bulk CD4 and CD8 T-cell subsets. (A) TB2-specific CD4 and CD8 T cells; (B) Cytokine panels of CD8 T cells stimulated with NIL and MITOGEN; (C) Cytokine panels of CD4 T cells stimulated with NIL and MITOGEN. FSC, forward scatter; SSC, side scatter; TB2, peptides of TB2 tube of QFT-plus kit; MITOGEN, positive control stimulation of QFT-plus kit; NIL, unstimulated tube of QFT-plus kit.

Figure 2

CD4 T cells producing IFN-γ, IL-2, and TNF-α in response to Mtb antigen stimulation before and after TB therapy completion. PBMC of patients enrolled before (T0) and after TB therapy (T1) were stimulated overnight with TB1 and TB2 peptides. (A, B) TBI-IMID; (C, D) TBI subjects; (E, F) Active-TB patients. Number of responders over total enrolled patients is reported below each panel. Horizontal lines indicate the median. Statistical analysis was performed using the Wilcoxon matched-pairs signed rank test and the p value was considered significant if ≤0.05. TB, tuberculosis; TBI, tuberculosis infection; IMID, immune mediated inflammatory disease; TB1, peptides of TB1 tube of QFT-plus kit; TB2, peptides of TB2 tube of QFT-plus kit.

Figure 3

CD8 T cells producing IFN-γ, IL-2, and TNF-α in response to Mtb antigen stimulation. PBMC of patients enrolled before (T0) and after TB therapy (T1) were stimulated overnight with TB1 and TB2 peptides. (A, B) TBI-IMID; (C, D) TBI patients; (E, F) Active-TB patients. Number of responders over total enrolled patients is reported below each panel. Horizontal lines indicate the median. Statistical analysis was performed using the Wilcoxon matched-pairs signed rank test and the p value was considered significant if ≤0.05. TB, tuberculosis; TBI, tuberculosis infection; IMID, immune mediated inflammatory disease; TB1, peptides of TB1 tube of QFT-plus kit; TB2, peptides of TB2 tube of QFT-plus kit.

Figure 4

Comparison of CD4 T cells producing IFN-γ, IL-2, and TNF-α in response to Mtb antigen stimulation at each time point. PBMC of patients enrolled before (T0) and after TB therapy (T1) were stimulated overnight with TB1 and TB2 peptides. (A, B) TB1 response at T0 and T1; (C, D) TB2 response at T0 and T1. Horizontal lines indicate the median. Statistical analysis was performed using the Mann–Whitney unmatched test, Bonferroni correction was applied, and the p value was considered significant if ≤0.017. TB, tuberculosis; TBI, tuberculosis infection; IMID, immune mediated inflammatory disease; TB1, peptides of TB1 tube of QFT-plus kit; TB2, peptides of TB2 tube of QFT-plus kit.

Figure 5

Functional profile of Mtb-specific CD4 T cells before and after TB therapy completion. PBMC of patients enrolled before (T0) and after TB therapy (T1) were stimulated overnight with TB1 and TB2 peptides. (A, B) TBI-IMID patients; (C, D) TBI subjects; (E, F) Active-TB patients. Cytokine profile was evaluated only on responders using Boolean gate combination. The number of CD4 and CD8 responders to TB1 and TB2 stimulation is reported in detail in Table 3. Horizontal lines indicate the median. Statistical analysis was performed using the Wilcoxon matched-pairs signed rank test, and the p value was considered significant if ≤0.05. TB, tuberculosis; TBI, tuberculosis infection; IMID, immune mediated inflammatory disease; TB1, peptides of TB1 tube of QFT-plus kit; TB2, peptides of TB2 tube of QFT-plus kit.

Figure 6

Comparison of functional profile of TB1 specific CD4 T cells at each time point. PBMC of patients enrolled before (T0) and after TB therapy (T1) were stimulated overnight with TB1 peptides. (A) T0; (B) T1. Cytokine profile was evaluated only on responders using Boolean gate combination. The number of CD4 and CD8 responders to TB1 and TB2 stimulation is reported in detail in Table 3. Horizontal lines indicate the median. Statistical analysis was performed using the Mann–Whitney unmatched test, Bonferroni correction was applied, and the p value was considered significant if ≤0.017. TB, tuberculosis; TBI, tuberculosis infection; IMID, immune mediated inflammatory disease; TB1, peptides of TB1 tube of QFT-plus kit; TB2, peptides of TB2 tube of QFT-plus kit.

Figure 7

Comparison of functional profile of TB2 specific CD4 T cells at each time point. PBMC of patients enrolled before (T0) and after TB therapy (T1) were stimulated overnight with TB2 peptides. (A) T0; (B) T1. Cytokine profile was evaluated only on responders using Boolean gate combination. The number of CD4 and CD8 responders to TB1 and TB2 stimulation is reported in detail in Table 3. Horizontal lines indicate the median. Statistical analysis was performed using the Mann–Whitney unmatched test, Bonferroni correction was applied, and the p value was considered significant if ≤0.017. TB, tuberculosis; TBI, tuberculosis infection; IMID, immune mediated inflammatory disease; TB1, peptides of TB1 tube of QFT-plus kit; TB2, peptides of TB2 tube of QFT-plus kit.

Figure 8

Phenotype of Mtb-specific CD4 T cells before and after TB therapy completion. PBMC of patients enrolled before (T0) and after TB therapy (T1) were stimulated overnight with TB1 and TB2 peptides, and CD45RA and CD27 proportions were evaluated on total cytokine response and T cells producing IFN-γ, IL-2, and TNF-α. (A, B) TBI-IMID patients; (C, D) TBI subjects; (E, F) Active-TB patients. Phenotype was evaluated only on responders. The number of CD4 and CD8 responders to TB1 and TB2 stimulation is reported in detail in Table 3. Horizontal lines indicate the median. Statistical analysis was performed using the Wilcoxon matched-pairs signed rank test, and the p value was considered significant if ≤0.05. TB, tuberculosis; TBI, tuberculosis infection; IMID, immune mediated inflammatory disease; TB1, peptides of TB1 tube of QFT-plus kit; TB2, peptides of TB2 tube of QFT-plus kit.

Figure 9

Comparison of phenotype of Mtb-specific CD4 T cells at each time point. PBMC of patients enrolled before (T0) and after TB therapy (T1) were stimulated overnight with TB1 and TB2 peptides, and CD45RA and CD27 proportions were evaluated on total cytokine response and T cells producing IFN-γ, IL-2, and TNF-α. (A, B) TB1 stimulation at T0 and T1; (C, D) TB2 stimulation at T0 and T1. Phenotype was evaluated only on responders. The number of CD4 and CD8 responders to TB1 and TB2 stimulation is reported in detail in Table 3. Horizontal lines indicate the median. Statistical analysis was performed using the Mann–Whitney unmatched test, Bonferroni correction was applied, and the p value was considered significant if ≤0.017. TB, tuberculosis; TBI, tuberculosis infection; IMID, immune mediated inflammatory disease; TB1, peptides of TB1 tube of QFT-plus kit; TB2, peptides of TB2 tube of QFT-plus kit.