Evaluation of Mycobacterium tuberculosis specific antigen-stimulated CD27-CD38+IFN-γ+CD4+ T cells for discrimination of active tuberculosis

Abstract

Background: Active tuberculosis (ATB) originates from primary Mycobacterium tuberculosis (MTB) infection or reactivation of latent tuberculosis. Besides bacteriological examination, MTB-reactive immunocytes detection can be an alternative testing for discrimination of active tuberculosis. The purpose of this study is to investigate the accuracy of peripheral blood CD27^-CD38⁺IFN-γ⁺CD4⁺T cells in ATB diagnosis.

Methods: This prospective diagnostic accuracy study was conducted at Shanghai Pulmonary Hospital between January 2019 and December 2021. Patients with ATB, non-tuberculosis mycobacterium infection (NTM), latent tuberculosis infection (LTBI), other respiratory diseases (OD), and healthy individuals (HC) were enrolled. The accuracy of CD27^-CD38⁺IFN-γ⁺CD4⁺/CD4⁺ and other phenotypic markers for ATB diagnosis was assessed.

Results: A total of 376 patients (237 ATB, 38 LTBI, 8 NTM, 50 OD, and 43 HC) were enrolled. The ratios of CD4⁺IFN-γ⁺CD27^- and CD4⁺IFN-γ⁺CD27^-CD38⁺ profiles in CD4⁺IFN^-γ⁺ cells and the ratios of CD4⁺IFN-γ⁺CD38⁺, CD4⁺IFN-γ⁺CD27^-, and CD4⁺IFN-γ⁺CD38⁺CD27^- profiles in CD4⁺ cells in the ATB group were significantly higher than in the other groups. The area under the curve (AUC) of CD27^-CD38⁺IFN-γ⁺CD4⁺/CD4⁺ for the diagnosis of ATB was the highest, with a value of 0.890. With the optimal cutoff value of 1.34 × 10^-4, the sensitivity and specificity of CD27^-CD38⁺IFN-γ⁺CD4⁺/CD4⁺ for ATB diagnosis was 0.869 and 0.849, respectively.

Conclusion: CD27^-CD38⁺IFN-γ⁺CD4⁺/CD4⁺ might be a potential biomarker for active tuberculosis diagnosis.

Keywords: Biomarker; CD27; CD38; Diagnosis; Flow cytometry; IFN−γ; Tuberculosis.

Fig. 1

Participant enrollment flowchart. TB tuberculosis, ATB active tuberculosis, LTBI latent tuberculosis infection, NTM non-tuberculous mycobacterium pulmonary disease, OD other respiratory diseases, HC healthy control, PTB pulmonary tuberculosis, EPTB extra-pulmonary tuberculosis, IGRA interferon-γ release assay

Fig. 2

Flow cytometry gating strategy for CD4⁺IFN-γ⁺CD38⁺CD27⁻ T cells from the peripheral blood lymphocyte of the participants. A Peripheral blood lymphocyte without stimulation by ESAT-6/CFP-10 peptides (negative control) and with 16-h stimulation by ESAT-6/CFP-10 peptides (E6C10). B The comparison of CD27 or CD38 expression in ESAT-6/CFP-10 peptides stimulated CD4⁺IFN-γ⁺ T cells to the unstimulated cells

Fig. 3

The frequency of CD27 and CD38-expressing populations in CD4⁺ IFN-γ⁺ T cells and in CD4⁺ T cells for the various groups. A CD4⁺IFN-γ⁺CD27⁺ and CD4⁺IFN⁻γ⁺CD27⁻, B CD4⁺IFN⁻γ⁺CD38⁺ and CD4⁺IFN-γ⁺CD38⁻, C CD4⁺IFN⁻γ⁺CD38⁺CD27⁻ and CD4⁺IFN-γ⁺CD27⁺CD38⁻, D CD4⁺IFN⁻γ⁺CD27⁻, E CD4⁺IFN⁻γ⁺CD38⁺, and F CD4⁺IFN⁻γ⁺CD38⁺CD27⁻ in CD4⁺ IFN-γ⁺ T cell in peripheral blood stimulated with ESAT 6/CFP10 were compared in ATB and other groups. The phenotypic markers G CD4⁺IFN⁻γ⁺CD27⁻, H CD4⁺IFN⁻γ⁺CD38⁺, and I CD4⁺IFN⁻γ⁺CD38⁺CD27⁻ on CD4⁺ T cell in peripheral blood stimulated with ESAT 6/CFP10 in each group was compared. ATB active tuberculosis, LTBI latent tuberculosis infection, NTM non-tuberculous mycobacterium pulmonary disease, OD other respiratory diseases, HC healthy control

Fig. 4

Receiver operating characteristics (ROC) curves and accuracy for the diagnosis of active tuberculosis (ATB). A ROC curves of different indicators for the diagnosis of ATB. B Sensitivity and specificity corresponding to the cutoff values. The dotted line indicates 80% sensitivity and 80% specificity. C The dotted line represents the cutoff value of CD4⁺IFN-γ⁺CD38⁺CD27⁻/CD4⁺ as 1.34 × 10^–4. ATB active tuberculosis, LTBI latent tuberculosis infection, NTM non-tuberculous mycobacterium pulmonary disease, OD other respiratory diseases, HC healthy control

Fig. 5

The phenotypic marker CD4⁺IFN⁻γ⁺CD38⁺CD27⁻ on CD4⁺ cells in different subgroups. A The phenotypic marker CD4⁺IFN-γ⁺CD38⁺CD27⁻ on CD4⁺ cells with negative TB culture stimulated with ESAT 6/CFP10. B The phenotypic marker CD4⁺IFN-γ⁺CD38⁺CD27⁻ on CD4⁺ cells with EPTB stimulated with ESAT 6/CFP10. C⁺: TB culture⁺; C⁻: TB culture⁻; PTB: pulmonary tuberculosis; EPTB: extrapulmonary tuberculosis; LTBI: latent tuberculosis infection; NTM: non-tuberculous mycobacterium pulmonary disease; OD: other respiratory diseases; HC: healthy control