Low-Density Granulocytes Affect T-SPOT.TB Assay by Inhibiting the Production of Interferon-γ in T Cells via PD-L1/PD-1 Pathway

Abstract

BackgroundT-SPOT TB (T-SPOT) assay is widely used for detection of Mycobacterium tuberculosis infection that is based on the detection of M. tuberculosis-specific interferon-γ-secreting T cells (ISCs) in peripheral blood mononuclear cells (PBMCs). Recently, high frequencies of low-density granulocytes (LDGs) were found in the PBMCs of tuberculosis patients. Whether these LDGs affect the detection of T-SPOT has not been investigated. The impact of LDGs on T-SPOT assay and related mechanism were investigated in this study.MethodsThe correlations between the frequencies of LDGs and the results of T-SPOT were analyzed. T-SPOT with LDG-removed PBMCs and PBMCs with exogenous addition of LDGs were performed. The possible mechanism was explored by detecting the levels of negative immune regulatory molecules on LDGs. The impact of programmed death ligand 1 (PD-L1) on T-SPOT was evaluated and confirmed by function blocking with neutralizing antibody.ResultsThe positive rates of T-SPOT and ISCs in tuberculosis patients with low LDGs frequency (n = 22) were significantly higher than those with high LDGs frequency (n = 39). Removal or exogenous addition of LDGs significantly increased or decreased the ISCs and the positive rate of T-SPOT. The frequencies of interferon-γ-producing T cells were negatively correlated with the frequencies of LDGs. The expression of PD-L1 was significantly elevated on LDGs. Pretreatment of LDGs with anti-PD-L1 antibody significantly counteracted the impact of LDGs on T-SPOT. Treatment of PBMCs with anti-PD-L1 antibody resulted in comparable ISCs with that of LDG removal.ConclusionLDGs can inhibit the production of interferon-γ in T cells and decrease the positive rated of T-SPOT assay via highly expressed PD-L1.

Rapid and Visual Differentiation of Mycobacterium tuberculosis From the Mycobacterium tuberculosis Complex Using Multiplex Loop-Mediated Isothermal Amplification Coupled With a Nanoparticle-Based Lateral Flow Biosensor

“…However, traditional detection methods (i.e., AFB smear, L-J culture, and biochemical tests) usually cannot meet the requirements necessary for rapid detection in terms of time and sensitivity. PCR and PCR-based assays (e.g., real-time quantitative PCR and GeneXpert techniques) and immunologic tests (e.g., T-SOPT TB and enzyme-linked immunosorbent assays) can achieve rapid detection for MTB, but the special instrument requirements and/or expensive reagents hinder their application in the field and in resource-poor areas ( Gallo et al, 2016 ; Lekhak et al, 2016 ; Rao et al, 2021 ). Among a wide variety of rapid examination methods, LAMP, as a low-cost and valuable technique, has been widely used in the detection of various pathogens, including viruses, bacteria, and fungi ( Han et al, 2007 ; Hsu et al, 2017 ).…”

“…Currently, conventional methods, including acid-fast bacilli (AFB) smears, solid cultures (Lowenstein–Jensen medium, L-J), biochemical tests, and immunological tests (e.g., T-SOPT TB and enzyme-linked immunosorbent assays), are mainly used in clinical examinations to detect MTBC strains ( Otu et al, 2008 ; Rodriguez-Morales and Castañeda-Hernández, 2014 ; Agrawal et al, 2016 ; Lyashchenko et al, 2020 ). In particular, the T-SPOT TB assay, which is based on the detection of MTB-specific interferon-γ-secreting T cells in peripheral blood mononuclear cells, is widely used to detect MTB infections ( Rao et al, 2021 ). Nevertheless, it is difficult to meet the sensitivity and time requirements necessary for rapid examination using the abovementioned methods ( Otu et al, 2008 ; Wadhwa et al, 2014 ; Agrawal et al, 2016 ; Lyashchenko et al, 2020 ).…”

Exploring the Role of Low-Density Neutrophils During Mycobacterium tuberculosis Infection

“…Although in vitro work showed that LDNs upregulated IL-10 expression, addition of an anti-IL-10 antibody did not change the differences in the T cell proliferation observed in the presence of LDNs versus NDNs ( La Manna et al., 2019 ), indicating that the effect of LDNs on T cell proliferation in this assay is IL-10-independent. The association between LDNs and T cell responses during Mtb infection has also been analyzed in active TB patients using the T-SPOT.TB test to assess IFN-γ secretion by T cells in response to the Mtb antigens ESAT-6 and CFP-10 ( Rao et al., 2021 ). Active TB patients with high LDN frequencies had lower numbers of IFN-ү-secreting cells than patients with low LDN frequencies ( Figure 2 ) ( Rao et al., 2021 ).…”

Lipoarabinomannan as a Point-of-Care Assay for Diagnosis of Tuberculosis: How Far Are We to Use It?

“…Although the sensitivity and specificity of urine LAM test are currently improving, studies have shown that the clinical profile of a TB patient interferes with the diagnostic assays, as also happens in the interferon-gamma release assays (Rao et al, 2021 (Bulterys et al, 2019). Given the current situation, we consider that focusing our efforts on improving the diagnostic tools based on the identification and quantification of LAM in urine or plasma is imperative, as it could have several characteristics to become a gold-standard diagnostic test.…”