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The diagnosis of pulmonary tuberculosis in HIV-infected individuals is particularly challenging as HIV-induced alterations of the immune system lead to reduced cavitation, limiting the sensitivity of sputum-based assays (1). Thus, alternate markers are needed to distinguish between latent tuberculosis infection (LTBI) and active tuberculosis (aTB) in this high-risk group. Several attributes of Mycobacterium tuberculosis (Mtb)-specific CD4 T cells have been shown to efficiently delineate LTBI and aTB in HIV-uninfected individuals, including their polyfunctional or memory profiles (2–4). Moreover, Adekambi and colleagues recently demonstrated that the activation profile of Mtb-specific CD4 T cells accurately discriminates between LTBI and aTB (5). As chronic HIV infection is characterized by persistent systemic immune activation (6), it is plausible that these blood-based markers may not be relevant for HIV-infected individuals. We, therefore, compared the potential of the activation and polyfunctional profiles of Mtb-specific CD4 T cells to distinguish between LTBI and aTB in HIV-uninfected and HIV-infected individuals. We analyzed 76 participants divided in four groups according to their TB and HIV status: LTBI/HIV (n = 17; median age, 22 yr; 47% female), aTB/HIV (n = 17; median age, 27 yr; 29% female), LTBI/HIV (n = 21; median age, 29 yr; 67% female; median CD4 count, 316 cells/mm; interquartile range [IQR], 231–543 cells/mm), and aTB/HIV (n = 21; median age, 35 yr; 57% female; CD4 count, 250 cells/mm; IQR, 155–295 cells/mm). LTBI was defined as tuberculin skin test positive, IFN-g release assay positive, sputum culture negative, and normal chest X-ray. aTB was diagnosed on the basis of symptoms suggestive of tuberculosis and Mtb-positive smear and/or sputum culture, as previously described (7). All HIV-infected participants were antiretroviral therapy–naive. The University of Cape Town ethics committee approved the study, and written consent was obtained from participants. Cryopreserved peripheral blood mononuclear cells were stimulated for 16 hours with early secretory antigenic target-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) peptide pool, and intracellular staining, using a live/dead marker and antibodies toward CD3, CD4, CD8, human leukocyte antigen-DR (HLA-DR), Ki67, CD38, IFN-g, tumor necrosis factor (TNF)-a, and IL-2, was performed. Positive ESAT-6/CFP-10 responses (defined as twice the background) were detectable in 16 subjects in the LTBI/HIV and LTBI/HIV groups, and in 15 and 18 individuals in the aTB/HIV and aTB/HIV groups, respectively. No significant differences were observed in the overall magnitude of IFN-g responses between the four groups (data not shown). We first compared the activation profile of IFN-g Mtbspecific CD4 T cells between the four groups (Figure 1A). As previously shown (5), in HIV-uninfected persons, HLA-DR, Ki67, and CD38 expression on IFN-g Mtb-specific CD4 T cells was significantly higher in aTB participants than in those with LTBI (Figure 1B). Interestingly, although HLA-DR expression on Mtb-specific CD4 T cells in the LTBI/HIV group (median, 41.7%; IQR, 25.7–54.6%) was significantly higher than in the LTBI/HIV group (median, 13.7%; IQR, 8.9–27.5%), HLA-DR expression on these cells was significantly further increased in HIV-infected individuals with aTB (median, 84%; IQR, 73.7–87.9%) (Figure 1B). Additional analyses showed that in LTBI/HIV individuals, HLA-DR expression on Mtb-specific CD4 T cells mirrors HLA-DR expression in the whole CD4 compartment (P = 0.02; r = 0.56), but this association was not apparent in aTB/HIV individuals (data not shown). Unlike HLA-DR, Ki67 and CD38 expression levels were comparable between HIV-uninfected and HIVinfected individuals with LTBI. In HIV-infected persons with aTB, Ki67 expression on IFN-g Mtb-specific CD4 T cells was significantly higher (P, 0.0001) than in LTBI, whereas the up-regulation of CD38 was more modest between these two groups (P = 0.03). Of note, in the aTB/HIV group, the expression of CD38 was significantly higher in individuals with a positive smear when compared with smear-negative participants (P = 0.01; data not shown), suggesting that CD38 expression could reflect bacterial load. To assess the accuracy of these markers to discriminate between LTBI and aTB status, receiver operating characteristic curves and crossover plots were performed. Figure 1C shows the data for HLA-DR; area under the curve (AUC) and P values reflect that HLA-DR expression on IFN-g Mtb-specific CD4 T cells distinguishes LTBI and aTB in both the HIV and HIV groups (AUC = 0.98 [P, .0001]; and AUC = 0.9 [P, 0.0001], respectively). However, the optimum cutoff values discriminating LTBI from aTB were distinct for HIV-uninfected (40%) and HIV-infected (70%) individuals. In our experimental setting, the expression of Ki67 and CD38 was less robust to differentiate TB status in HIV-uninfected (AUC = 0.896 [P = 0.00017], cutoff = 1.4%; AUC = 0.858 [P = 0.0007], cutoff = 4%, respectively) and HIV-infected (AUC = 0.89 [P = 0.0002], cutoff = 2.4%; AUC = 0.72 [P = 0.026], cutoff = 5%, respectively) individuals (data not shown). Our data were comparable to those of Adekambi and colleagues (5) despite disparity in the cutoff value for these markers, which could be explained by flow-cytometry technical differences. The polyfunctional profile of Mtb-specific CD4 T cells has also been shown to discriminate between LTBI and aTB in HIV-uninfected individuals (2, 3), but conflicting data exist for HIV-infected persons (8–10). Thus, we compared the profile of ESAT-6/CFP-10-specific CD4 T cells on the basis of their capacity to secrete IFN-g, TNF-a, and/or IL-2, between the four groups (Figure 2A). HIV-uninfected individuals with LTBI were C.R. is funded by the National Institutes of Health, Office of the Director (R21AI115977). K.A.W. is funded by the Medical Research Council UK. R.J.W. is supported by the Wellcome Trust (084323 and 104803), the Medical Research Council UK (U1175.02.002.00014.01), the European Union (FP7-Health-F3–2012–305578), South African National Research Foundation, and Medical Research Council South Africa Strategic Health Innovations Partnership.

Activation Profile of Mycobacterium tuberculosis–Specific CD4+ T Cells Reflects Disease Activity Irrespective of HIV Status

Activation Profile of Mycobacterium tuberculosis–Specific CD4⁺ T Cells Reflects Disease Activity Irrespective of HIV Status