Whole-Blood Flow-Cytometric Analysis of Antigen-Specific CD4 T-Cell Cytokine Profiles Distinguishes Active Tuberculosis from Non-Active States

Abstract

T-cell based IFN-γ release assays do not permit distinction of active tuberculosis (TB) from successfully treated disease or latent M. tuberculosis infection. We postulated that IFN-γ and IL-2 cytokine profiles of antigen-specific T cells measured by flow-cytometry ex vivo might correlate with TB disease activity in vivo. Tuberculin (PPD), ESAT-6 and CFP-10 were used as stimuli to determine antigen-specific cytokine profiles in CD4 T cells from 24 patients with active TB and 28 patients with successfully treated TB using flow-cytometry. Moreover, 25 individuals with immunity consistent with latent M. tuberculosis infection and BCG-vaccination, respectively, were recruited. Although the frequency of cytokine secreting PPD reactive CD4 T cells was higher in patients with active TB compared to patients with treated TB (median 0.81% vs. 0.39% of CD4 T cells, p = 0.02), the overlap in frequencies precluded distinction between the groups on an individual basis. When assessing cytokine profiles, PPD specific CD4 T cells secreting both IFN-γ and IL-2 predominated in treated TB, latent infection and BCG-vaccination, whilst in active TB the cytokine profile was shifted towards cells secreting IFN-γ only (p<0.0001). Cytokine profiles of ESAT-6 or CFP-10 reactive CD4 T cells did not differ between the groups. Receiver operator characteristics (ROC) analysis revealed that frequencies of PPD specific IFN-γ/IL-2 dual-positive T cells below 56% were an accurate marker for active TB (specificity 100%, sensitivity 70%) enabling effective discrimination from non-active states. In conclusion, a frequency lower than 56% IFN-γ/IL-2 dual positive PPD-specific circulating CD4 T-cells is strongly indicative of active TB.

The risk of tuberculosis in transplant candidates and recipients: a TBNET consensus statement

“…The role of a T-helper (Th)1 response, especially of CD4 and CD8 interferon (IFN)-c producing T-cells, is considered essential. Polyfunctional memory CD4 T-cells that simultaneously produce IFN-c, tumour necrosis factor (TNF)-a and interleukin (IL)-2 appear to be mediators of long lasting protection against TB reactivation [9][10][11][12][13].…”

“…More recently, flow cytometry has been used to quantify specifically activated T-cells based on the intracellular accumulation of IFN-c or other cytokines. Although this technique has so far largely been applied in experimental settings, its potential to perform multi-parameter analyses may hold promise as an immune-based technique to distinguish LTBI from active disease [9,[11][12][13].…”

“…New diagnostic approaches are needed that allow for a more targeted identification of patients at risk to develop TB. This may involve modifications of in vitro immunodiagnostic assays such as the use of novel stimulatory antigens [15,164,165], alternative biomarkers other than IFN-c [166][167][168][169][170][171][172], variations in incubation time [173,174], the readout system [9,12,13,175,176] or the clinical specimen instead of blood [177][178][179]. In addition, both for the detection of LTBI with a risk of reactivation and for suspected active TB, a novel approach based on a whole blood transcriptional signature could provide a biomarker system with high discriminative potential [180].…”

Superior Sensitivity of Ex Vivo IFN-γ Release Assays as Compared to Skin Testing in Immunocompromised Patients

“…These studies have stimulated further interest in exploring cytokine profiling for other clinical applications such as improved identification of patients at risk for developing tuberculosis or for monitoring treatment success (30). Most studies have used flow-cytometric analysis of stimulation-induced cytokines after intracellular accumulation (31)(32)(33). Of note, given that the detection of PPD-specific T cells by the FACS approach was least affected by immunosuppressive drug therapy, cytokineprofiling may also serve as a valuable tool to distinguish active from latent infection in immunocompromised patients such as transplant recipients.…”

Association of Genetic Polymorphism of IL-2 Receptor Subunit and Tuberculosis Case

“…따라서 혈중 IL-2Rα의 농도는 T cell 활성의 지표가 되기도 한다 (Rubin et al, 1985;Wang et al, 2012). 또한 결핵균이 잠복 감염된 환자에서 IL-2와 IFN-γ 를 모두 발현하는 T cell의 비율이 현성 감염 환자에 비하 여 높은 것으로 나타났다는 결과를 보고한 연구도 진행되 었다 (Casey et al, 2010;Sester et al, 2011). (Table 3). )…”