Study of CD27, CD38, HLA-DR and Ki-67 immune profiles for the characterization of active tuberculosis, latent infection and end of treatment

Abstract

Background: Current blood-based diagnostic tools for TB are insufficient to properly characterize the distinct stages of TB, from the latent infection (LTBI) to its active form (aTB); nor can they assess treatment efficacy. Several immune cell biomarkers have been proposed as potential candidates for the development of improved diagnostic tools.

Objective: To compare the capacity of CD27, HLA-DR, CD38 and Ki-67 markers to characterize LTBI, active TB and patients who ended treatment and resolved TB.

Methods: Blood was collected from 45 patients defined according to clinical and microbiological criteria as: LTBI, aTB with less than 1 month of treatment and aTB after completing treatment. Peripheral blood mononuclear cells were stimulated with ESAT-6/CFP-10 or PPD antigens and acquired for flow cytometry after labelling with conjugated antibodies against CD3, CD4, CD8, CD27, IFN-γ, TNF-α, CD38, HLA-DR, and Ki-67. Conventional and multiparametric analyses were done with FlowJo and OMIQ, respectively.

Results: The expression of CD27, CD38, HLA-DR and Ki-67 markers was analyzed in CD4⁺ T-cells producing IFN-γ and/or TNF-α cytokines after ESAT-6/CFP-10 or PPD stimulation. Within antigen-responsive CD4⁺ T-cells, CD27^- and CD38⁺ (ESAT-6/CFP-10-specific), and HLA-DR⁺ and Ki-67⁺ (PPD- and ESAT-6/CFP-10-specific) populations were significantly increased in aTB compared to LTBI. Ki-67 demonstrated the best discriminative performance as evaluated by ROC analyses (AUC > 0.9 after PPD stimulation). Data also points to a significant change in the expression of CD38 (ESAT-6/CFP-10-specific) and Ki-67 (PPD- and ESAT-6/CFP-10-specific) after ending the anti-TB treatment regimen. Furthermore, ratio based on the CD27 median fluorescence intensity in CD4⁺ T-cells over Mtb-specific CD4⁺ T-cells showed a positive association with aTB over LTBI (ESAT-6/CFP-10-specific). Additionally, multiparametric FlowSOM analyses revealed an increase in CD27 cell clusters and a decrease in HLA-DR cell clusters within Mtb-specific populations after the end of treatment.

Conclusion: Our study independently confirms that CD27^-, CD38⁺, HLA-DR⁺ and Ki-67⁺ populations on Mtb-specific CD4⁺ T-cells are increased during active TB disease. Multiparametric analyses unbiasedly identify clusters based on CD27 or HLA-DR whose abundance can be related to treatment efficacy. Further studies are necessary to pinpoint the convergence between conventional and multiparametric approaches.

Keywords: Mycobacterium tuberculosis; T-cells; activation markers; cluster; flow cytometry; immune response; multiparametric analysis; treatment.

Figure 1

CD27⁻, CD38⁺, HLA-DR⁺, and Ki67⁺ phenotype from Mtb-specific CD4⁺ T-cells of each patient group. Percentage of CD27⁻(A), CD38⁺ (B), HLA-DR⁺ (C), and Ki67⁺ (D) within TNF-α⁺ and/or IFN-γ⁺ CD4⁺ T-cells after stimulation with ESAT-6/CFP-10 or PPD (left and right half of graph, respectively) in patients with active TB in the beginning and end of treatment, as well as LTBI individuals. Data plotted with median and interquartile range. Differences between aTB and LTBI conditions were calculated using the two-tailed Mann–Whitney U-test. Differences between aTB and eTrt groups were calculated using a mixed statistical model controlling repeated measures on logit-transformed data. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. No indication of p value implies non significance. aTB, active TB; LTBI, latent tuberculosis infection; eTrt, aTB patients after anti-TB treatment.

Figure 2

CD27 MFI ratio is increased in aTB patients over LTBI individuals (ESAT-6/CFP-10-specific) and patients after anti-TB treatment (PPD-specific). A ratio of CD27 MFI was calculated as suggested by Portevin et al. The numbers result from the division of the MFI of CD27 in CD4⁺ T-cells over MFI of CD27 in TNF-α⁺ and/or IFN-γ⁺ CD4⁺ T-cells. (A) CD27 MFI ratio after ESAT-6/CFP-10 or (B) PPD stimulation in patients with active TB in the beginning and end of treatment, as well as LTBI individuals. Data plotted with median and interquartile range. Differences between aTB and LTBI conditions were calculated using the two-tailed Mann–Whitney U-test. Differences between aTB and eTrt groups were calculated using a mixed statistical model controlling repeated measures on logit-transformed data. ^*p < 0.05. aTB, active TB; LTBI, latent tuberculosis infection; eTrt, aTB patients after anti-TB treatment.

Figure 3

Results from multiparametric analyses of the samples. (A) UMAP based on the expression of CD4, CD8, TNF-α, IFN-γ, CD27, CD38, HLA-DR and Ki-67 markers in CD3⁺ cells from our dataset containing LTBI individuals and aTB patients before and after treatment, after stimulation with ESAT-6/CFP-10 or PPD. Colored clusters indicate populations with difference in abundance depending on disease status, obtained with FlowSOM and analyzed within TNF-α⁺ and/or IFN-γ⁺ subsets. In background, all 50 cell clusters defined. (B,D) Box and dot plots showing percentage (Y axis) of each cluster of interest in each respective group [(B) for samples after PPD stimulation, (D) for samples after ESAT-6/CFP-10 stimulation. Data plotted with median and interquartile range. (C,E) Volcano plot displaying logarithm scale of fold-change of percentage ratio of sample from aTB patients over LTBI individuals (left) and patients who completed treatment (right; C) for samples after PPD stimulation, (E) for samples after ESAT-6/CFP-10 stimulation]. Positive values (logFC>1) show clusters whose proportion is increased in aTB, while negative values (logFC<−1) show clusters whose proportion is decreased in aTB. Significant samples are represented above the threshold (q < 0.05). Volcano plots were automatically produced by the OMIQ software. aTB, active TB; LTBI, latent tuberculosis infection; eTrt, aTB patients after anti-TB treatment, UMAP: uniform manifold approximation; FDR: false discovery rate.