Differential Expression of Activation Markers by Mycobacterium tuberculosis-specific CD4+ T Cell Distinguishes Extrapulmonary From Pulmonary Tuberculosis and Latent Infection

Abstract

Background Diagnosis of active tuberculosis (ATB) currently relies on detection of M. tuberculosis (Mtb). Identifying patients with extrapulmonary TB (EPTB) remains challenging because microbiological confirmation is often not possible. Highly accurate blood-based tests could improve diagnosis of both EPTB and pulmonary TB (PTB), and timely initiation of anti-TB therapy. Methods A case-control study was performed using discriminant analyses to validate an approach using Mtb-specific CD4+T-cell activation markers in blood to discriminate PTB and EPTB from latent TB infection (LTBI) as well as EPTB from PTB in 270 Brazilian individuals. We further tested the effect of HIV co-infection on diagnostic performance. Frequencies of IFNγ+CD4+T-cells expressing CD38, HLADR, and/or Ki67 were assessed by flow cytometry. Results EPTB and PTB were associated with higher frequencies of CD4+T-cells expressing CD38, HLADR or Ki67 compared to LTBI (all p-values < .001). Moreover, frequencies of HLADR+ (p= .03) or Ki67+ (p< .001) cells accurately distinguished EPTB from PTB. HIV infection did not affect the capacity of these markers to distinguish ATB from LTBI or EPTB from PTB. Conclusion Cell activation markers in Mtb-specific CD4+T-cells distinguished ATB from LTBI, and EPTB from PTB, regardless of HIV infection status. These parameters provide an attractive approach for developing blood-based diagnostic tests for both active and latent TB.

Disease extent and anti‐tubercular treatment response correlates with Mycobacterium tuberculosis‐specific CD4 T‐cell phenotype regardless of HIV‐1 status

“…Translation of whole blood assay-based approaches into diagnostic and treatment monitoring tools requires use of simplified flow cytometry panels. Upon focusing our analyses solely on HLA-DR expression, our data concur with previous findings from our group and others that a 4-colour panel (including CD3, CD4, IFN-c and HLA-DR) could discriminate latent from aTB 15,[18][19][20] and could also be useful to monitor the response to treatment, as recently suggested by Ahmed et al 29 Here, we show for the first time that this approach: (1) could identify those at highest risk of poor treatment outcomes, (2) could gauge the extent of TB disease severity and (3) importantly, exhibit comparable performance in HIV-infected persons, who are at highest risk of TB disease. While flow cytometry may appear complex, its usage in clinical tests is becoming more common with the availability of compact flow cytometers.…”

“…14 Furthermore, several studies have highlighted the potential of blood-based assays as diagnostic tools; and while measuring the magnitude of Mtb-specific T-cell response does not offer much value for TB diagnostic 15,16 or treatment monitoring, 17 assessing the phenotypic profile of these cells showed promising results. Indeed, the activation, memory differentiation or functional profile of Mtb-specific CD4 T cells has been shown to relate to TB disease activity 15,[18][19][20][21][22][23][24][25][26][27] and evaluate treatment response. 18,19,28,29 However, very few of these studies have included HIV-infected participants.…”

Advancing new diagnostic tests for latent tuberculosis infection due to multidrug-resistant strains of Mycobacterium tuberculosis — End of the road?

“…Whole blood biomarkers that can better predict the risk of TB progression are being studied using RNA sequencing of blood from cohorts; these studies are identifying gene signatures for the risk of progression to active TB (Zak et al, 2016;Wang et al, 2019;Suliman et al, 2018;Warsinske et al, 2018;Deng et al, 2019;Gupta et al, 2020). Mtb-specific CD4+ T-cell activation markers in blood may discriminate pulmonary and extrapulmonary TB from LTBI (Silveira-Mattos et al, 2019). Recently, Mtb has been demonstrated in mesenchymal and hematopoietic stem cells (Mayito et al, 2019); thus whole genome sequencing of bone marrow specimens from LTBI patients may show the presence of Mtb with rpo gene mutations in stem cell CD34 populations.…”

Immune profiling of Mycobacterium tuberculosis-specific T cells in recent and remote infection

“…Expression of HLA-DR, a member of the MHC class II family typically expressed by antigen-presenting cells, is often used as a marker of T cell activation [15,16]. In the context of tuberculosis, studies have shown that disease, regardless of HIV status, is associated with significantly higher levels of T cell activation than M.tb infection, and that activation is reduced upon successful antimicrobial treatment [17][18][19][20][21][22]. These studies suggest that antigen-specific T cell activation could be used as a biomarker of microbial burden, and potentially infer in vivo antigen exposure during different stages of infection and disease.…”

“…TST and IGRAs have a low predictive value for tuberculosis [26]. Other functional assays that measure M.tbspecific T cell properties such as Th1 cytokine co-expression profiles [27,28], T cell differentiation (CD27, [29,30]) and T cell activation [17][18][19][20][21][22] have been proposed as new potential immunodiagnostic concepts that can distinguish between M.tb infection and disease. In line with this principle, potential biomarkers of recent M.tb infection based on proportions of TNF-only (IFN--IL-2-) TE (CD45RA-CCR7-CD127-) CD4 T cells or T cell proliferation, as well as a blood transcriptomic signature, have been described [31][32][33].…”