Ex-vivo characterization of regulatory T cells in pulmonary tuberculosis patients, latently infected persons, and healthy endemic controls

Abstract

Background: Regulatory T cells (Treg) are an essential arm of adaptive immunity not only in tolerance and autoimmunity but also in infectious diseases. In Tuberculosis (TB), it has been suggested that the frequency of Tregs is higher in the blood of TB patients when compared to healthy controls with subsequent decline after treatment. However, with the discovery that FOXP3, the hallmark marker of Tregs, is not exclusive to Tregs and the lack of specific markers for Tregs, it has been a challenge to fully understand the role of Tregs in TB.

Method: We isolated PBMC from smear positive TB patients (TB, N = 13) before and after treatment, latent TB infected participants (LTBI, N = 8), and healthy endemic controls (EC, N = 9) and evaluated the frequency of different populations of Tregs and expression of FOXP3 by flowcytometry using six markers.

Results: The findings in this study showed that the association of Treg frequency with TB disease depends on the phenotypic markers used. While the frequency of CD4(+)CD25(+/hi) T cells was higher in TB patients compared to LTBI individuals, there was no difference in the frequency of CD4(+)CD25(+)FOXP3(+)CD127(lo) Treg among TB, LTBI, or EC. However, delineation of Tregs into active and naïve subsets revealed a significant increase in FOXP3 expression in active primed Tregs (CD4(+)CD25(+)FOXP3(+)CD127(lo)CD45RO(+)Ki-67(+)) of TB patients compared to LTBI and EC; and a significantly higher frequency of resting primed (CD45RO(+)Ki-67(-)) Treg in QuantiFERON negative EC compared to TB patients. After treatment completion, there was a significant decline in the frequency of active primed Treg, median (IQR) from 12.4% (9.5-21.9) of Tregs to 9.3% (7.0-12.2); P = 0.003 Wilcoxon signed rank test. We conclude that Treg subsets may be differentially regulated and expressed in TB disease, cure, and infection.

Keywords: FOXP3; Regulatory T cells; Tuberculosis.

Figure 1

Gating strategy for regulatory T cells. After gating on CD4 T cells, CD25 is plotted against CD4 to identify CD4⁺CD25⁺ and CD4⁺CD25^hi T cells (A, left); CD25 is plotted against CD127 to identify CD4⁺CD25⁺CD127^lo T cells (A, middle); and CD25 is plotted against FOXP3 to identify CD4⁺CD25⁺FOXP3⁺ T cells (A, right). From the latter, a CD127 vs. FOXP3 plot is used to gate on CD127^lo population and thereby identify CD4⁺CD25⁺FOXP3⁺CD127^lo Treg (B, left); from which Ki-67 is plotted against CD45RO to identify the three subsets of Tregs; naïve Treg (CD45RO⁻Ki-67⁻), active primed Treg (CD45RO⁺Ki-67⁺), and resting primed Treg (CD45RO⁺Ki-67⁻) (B, right).

Figure 2

Comparison of Treg gates. Regulatory T cells were defined using five combinations of CD4, CD25, CD127, FOXP3, and CD127. The frequency of regulatory T cells as a percentage of CD4 T cells (A) and MFI of FOXP3 in the different phenotypes of Tregs (B) in TB patients (TB, N = 13), latently infected participants (LTBI, N = 8), and healthy endemic controls (EC, N = 9) are shown. Box-plots represent inter-quartile range (IQR) with line at the median and whiskers indicate Min to Max value. Significant differences among the study groups determined by Kruskale–Wallis test are indicated by dashed lines. Asterisks indicate P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).

Figure 3

Comparisons of Treg Subsets. Tregs defined as CD4⁺CD25⁺FOXP3⁺CD127^lo were further characterized based on their expression of Ki-67 and CD45RO. The frequency of Treg subsets; Active Primed (CD45RO⁺Ki-67⁺), Resting Primed (CD45RO⁺Ki-67⁻), or Naïve (CD45RO⁻Ki-67⁻) as a percentage of Tregs is shown (A, Line at Median). Expression of FOXP3 (MFI) in these Treg subsets (B) among TB patients (TB), latently infected participants (LTBI), and healthy endemic controls (EC) is shown in B. Box-plots represent IQR with line at the median and whiskers indicate Max to Min values. Results of one way ANOVA for comparison of MFI among study groups (dashed lines) and repeated measures ANOVA for comparison of MFI in the different Treg subsets within each study group (solid lines) followed by Tukey’s test for multiple comparison are indicated by P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****).

Figure 4

Treg subsets in TB patients before and after anti-TB treatment. Frequency of CD4⁺CD25⁺FOXP3⁺CD127^lo Treg (A) and primed Treg (B) in TB patients at diagnosis (TB) and after 24 weeks of MDT (TB Cured). The frequency (C) and FOXP3 expression (D) of Treg subsets are shown as Active Primed (Red), Resting Primed (Green), or Naïve Treg (Blue) in TB patients before and after treatment; box plots show median and inter-quartile range with whiskers at Min to Max values. P value of Wilcoxon signed rank test are indicated as P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****). Pie chart of distribution of subsets of Treg (mean) among TB patients, TB patients after 24 weeks of MDT (TB-cured), latently infected participants (LTBI) and healthy endemic controls (EC) are shown in E. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)