Author Correction: A multi-cohort study of the immune factors associated with M. tuberculosis infection outcomes.

The spelling of author Qianting Yang was corrected; the affiliation of author Stephanus T. Malherbe was corrected; and graphs in Fig. 4b and c were corrected owing to reanalysis of the data into the correct timed intervals.

A multi-cohort study of the immune factors associated with M. tuberculosis infection outcomes.

Most infections with Mycobacterium tuberculosis (Mtb) manifest as a clinically asymptomatic, contained state, known as latent tuberculosis infection, that affects approximately one-quarter of the global population1. Although fewer than one in ten individuals eventually progress to active disease2, tuberculosis is a leading cause of death from infectious disease worldwide3. Despite intense efforts, immune factors that influence the infection outcomes remain poorly defined. Here we used integrated analyses of multiple cohorts to identify stage-specific host responses to Mtb infection. First, using high-dimensional mass cytometry analyses and functional assays of a cohort of South African adolescents, we show that latent tuberculosis is associated with enhanced cytotoxic responses, which are mostly mediated by CD16 (also known as FcγRIIIa) and natural killer cells, and continuous inflammation coupled with immune deviations in both T and B cell compartments. Next, using cell-type deconvolution of transcriptomic data from several cohorts of different ages, genetic backgrounds, geographical locations and infection stages, we show that although deviations in peripheral B and T cell compartments generally start at latency, they are heterogeneous across cohorts. However, an increase in the abundance of circulating natural killer cells in tuberculosis latency, with a corresponding decrease during active disease and a return to baseline levels upon clinical cure are features that are common to all cohorts. Furthermore, by analysing three longitudinal cohorts, we find that changes in peripheral levels of natural killer cells can inform disease progression and treatment responses, and inversely correlate with the inflammatory state of the lungs of patients with active tuberculosis. Together, our findings offer crucial insights into the underlying pathophysiology of tuberculosis latency, and identify factors that may influence infection outcomes.

Multidimensional analysis of immune responses identified biomarkers of recent Mycobacterium tuberculosis infection.

The risk of tuberculosis (TB) disease is higher in individuals with recent Mycobacterium tuberculosis (M.tb) infection compared to individuals with more remote, established infection. We aimed to define blood-based biomarkers to distinguish between recent and remote infection, which would allow targeting of recently infected individuals for preventive TB treatment. We hypothesized that integration of multiple immune measurements would outperform the diagnostic performance of a single biomarker. Analysis was performed on different components of the immune system, including adaptive and innate responses to mycobacteria, measured on recently and remotely M.tb infected adolescents. The datasets were standardized using variance stabilizing scaling and missing values were imputed using a multiple factor analysis-based approach. For data integration, we compared the performance of a Multiple Tuning Parameter Elastic Net (MTP-EN) to a standard EN model, which was built to the individual adaptive and innate datasets. Biomarkers with non-zero coefficients from the optimal single data EN models were then isolated to build logistic regression models. A decision tree and random forest model were used for statistical confirmation. We found no difference in the predictive performances of the optimal MTP-EN model and the EN model [average area under the receiver operating curve (AUROC) = 0.93]. EN models built to the integrated dataset and the adaptive dataset yielded identically high AUROC values (average AUROC = 0.91), while the innate data EN model performed poorly (average AUROC = 0.62). Results also indicated that integration of adaptive and innate biomarkers did not outperform the adaptive biomarkers alone (Likelihood Ratio Test χ2 = 6.09, p = 0.808). From a total of 193 variables, the level of HLA-DR on ESAT6/CFP10-specific Th1 cytokine-expressing CD4 cells was the strongest biomarker for recent M.tb infection. The discriminatory ability of this variable was confirmed in both tree-based models. A single biomarker measuring M.tb-specific T cell activation yielded excellent diagnostic potential to distinguish between recent and remote M.tb infection.

Antigen-Specific T-Cell Activation Distinguishes between Recent and Remote Tuberculosis Infection.

Rationale: Current diagnostic tests fail to identify individuals at higher risk of progression to tuberculosis disease, such as those with recent Mycobacterium tuberculosis infection, who should be prioritized for targeted preventive treatment. Objectives: To define a blood-based biomarker, measured with a simple flow cytometry assay, that can stratify different stages of tuberculosis infection to infer risk of disease. Methods: South African adolescents were serially tested with QuantiFERON-TB Gold to define recent (QuantiFERON-TB conversion <6 mo) and persistent (QuantiFERON-TB+ for >1 yr) infection. We defined the ΔHLA-DR median fluorescence intensity biomarker as the difference in HLA-DR expression between IFN-γ+ TNF+Mycobacterium tuberculosis-specific T cells and total CD3+ T cells. Biomarker performance was assessed by blinded prediction in untouched test cohorts with recent versus persistent infection or tuberculosis disease and by unblinded analysis of asymptomatic adolescents with tuberculosis infection who remained healthy (nonprogressors) or who progressed to microbiologically confirmed disease (progressors). Measurements and Main Results: In the test cohorts, frequencies of Mycobacterium tuberculosis-specific T cells differentiated between QuantiFERON-TB- (n = 25) and QuantiFERON-TB+ (n = 47) individuals (area under the receiver operating characteristic curve, 0.94; 95% confidence interval, 0.87-1.00). ΔHLA-DR significantly discriminated between recent (n = 20) and persistent (n = 22) QuantiFERON-TB+ (0.91; 0.83-1.00); persistent QuantiFERON-TB+ and newly diagnosed tuberculosis (n = 19; 0.99; 0.96-1.00); and tuberculosis progressors (n = 22) and nonprogressors (n = 34; 0.75; 0.63-0.87). However, ΔHLA-DR median fluorescent intensity could not discriminate between recent QuantiFERON-TB+ and tuberculosis (0.67; 0.50-0.84). Conclusions: The ΔHLA-DR biomarker can identify individuals with recent QuantiFERON-TB conversion and those with disease progression, allowing targeted provision of preventive treatment to those at highest risk of tuberculosis. Further validation studies of this novel immune biomarker in various settings and populations at risk are warranted.

Prevention of M. tuberculosis Infection with H4:IC31 Vaccine or BCG Revaccination.

BACKGROUND: Recent Mycobacterium tuberculosis infection confers a predisposition to the development of tuberculosis disease, the leading killer among global infectious diseases. H4:IC31, a candidate subunit vaccine, has shown protection against tuberculosis disease in preclinical models, and observational studies have indicated that primary bacille Calmette-Guérin (BCG) vaccination may offer partial protection against infection. METHODS: In this phase 2 trial, we randomly assigned 990 adolescents in a high-risk setting who had undergone neonatal BCG vaccination to receive the H4:IC31 vaccine, BCG revaccination, or placebo. All the participants had negative results on testing for M. tuberculosis infection on the QuantiFERON-TB Gold In-tube assay (QFT) and for the human immunodeficiency virus. The primary outcomes were safety and acquisition of M. tuberculosis infection, as defined by initial conversion on QFT that was performed every 6 months during a 2-year period. Secondary outcomes were immunogenicity and sustained QFT conversion to a positive test without reversion to negative status at 3 months and 6 months after conversion. Estimates of vaccine efficacy are based on hazard ratios from Cox regression models and compare each vaccine with placebo. RESULTS: Both the BCG and H4:IC31 vaccines were immunogenic. QFT conversion occurred in 44 of 308 participants (14.3%) in the H4:IC31 group and in 41 of 312 participants (13.1%) in the BCG group, as compared with 49 of 310 participants (15.8%) in the placebo group; the rate of sustained conversion was 8.1% in the H4:IC31 group and 6.7% in the BCG group, as compared with 11.6% in the placebo group. Neither the H4:IC31 vaccine nor the BCG vaccine prevented initial QFT conversion, with efficacy point estimates of 9.4% (P=0.63) and 20.1% (P=0.29), respectively. However, the BCG vaccine reduced the rate of sustained QFT conversion, with an efficacy of 45.4% (P=0.03); the efficacy of the H4:IC31 vaccine was 30.5% (P=0.16). There were no clinically significant between-group differences in the rates of serious adverse events, although mild-to-moderate injection-site reactions were more common with BCG revaccination. CONCLUSIONS: In this trial, the rate of sustained QFT conversion, which may reflect sustained M. tuberculosis infection, was reduced by vaccination in a high-transmission setting. This finding may inform clinical development of new vaccine candidates. (Funded by Aeras and others; C-040-404 ClinicalTrials.gov number, NCT02075203 .).

A comparison of antigen-specific T cell responses induced by six novel tuberculosis vaccine candidates.

Eradication of tuberculosis (TB), the world's leading cause of death due to infectious disease, requires a highly efficacious TB vaccine. Many TB vaccine candidates are in pre-clinical and clinical development but only a few can be advanced to large-scale efficacy trials due to limited global resources. We aimed to perform a statistically rigorous comparison of the antigen-specific T cell responses induced by six novel TB vaccine candidates and the only licensed TB vaccine, Bacillus Calmette-Guérin (BCG). We propose that the antigen-specific immune response induced by such vaccines provides an objective, data-driven basis for prioritisation of vaccine candidates for efficacy testing. We analyzed frequencies of antigen-specific CD4 and CD8 T cells expressing IFNγ, IL-2, TNF and/or IL-17 from adolescents or adults, with or without Mycobacterium tuberculosis (M.tb) infection, who received MVA85A, AERAS-402, H1:IC31, H56:IC31, M72/AS01E, ID93+GLA-SE or BCG. Two key response characteristics were analyzed, namely response magnitude and cytokine co-expression profile of the memory T cell response that persisted above the pre-vaccination response to the final study visit in each trial. All vaccines preferentially induced antigen-specific CD4 T cell responses expressing Th1 cytokines; levels of IL-17-expressing cells were low or not detected. In M.tb-uninfected and -infected individuals, M72/AS01E induced higher memory Th1 cytokine-expressing CD4 T cell responses than other novel vaccine candidates. Cytokine co-expression profiles of memory CD4 T cells induced by different novel vaccine candidates were alike. Our study suggests that the T cell response feature which most differentiated between the TB vaccine candidates was response magnitude, whilst functional profiles suggested a lack of response diversity. Since M72/AS01E induced the highest memory CD4 T cell response it demonstrated the best vaccine take. In the absence of immunological correlates of protection, the likelihood of finding a protective vaccine by empirical testing of candidates may be increased by the addition of candidates that induce distinct immune characteristics.

Diagnostic Accuracy of Early Secretory Antigenic Target-6-Free Interferon-gamma Release Assay Compared to QuantiFERON-TB Gold In-tube.

BACKGROUND: Early secretory antigenic target-6 (ESAT-6) is an immunodominant Mycobacterium tuberculosis (M.tb) antigen included in novel vaccines against tuberculosis (TB) and in interferon-gamma (IFN-γ) release assays (IGRAs). Therefore, the availability of an ESAT-6-free IGRA is essential to determine M.tb infection status following vaccination with ESAT-6-containing vaccines. We aimed to qualify a recently developed ESAT-6-free IGRA and to assess its diagnostic performance in comparison to QuantiFERON-TB Gold In-tube (QFT). METHODS: Participants with different levels of M.tb exposure and TB disease were enrolled to determine the ESAT-6-free IGRA cutoff, test assay performance in independent cohorts compared to standard QFT, and perform a technical qualification of antigen-coated blood collection tubes. RESULTS: ESAT-6-free IGRA antigen recognition was evaluated in QFT-positive and QFT-negative South African adolescents. The ESAT-6-free IGRA cutoff was established at 0.61 IU/mL, based on receiver operating characteristic analysis in M.tb-unexposed controls and microbiologically confirmed pulmonary TB patients. In an independent cohort of healthy adolescents, levels of IFN-γ released in QFT and ESAT-6-free IGRA were highly correlated (P < .0001, r = 0.83) and yielded comparable positivity rates, 41.5% and 43.5%, respectively, with 91% concordance between the tests (kappa = 0.82; 95% confidence interval, 0.74-0.90; McNemar test P = .48). ESAT-6-free IGRA blood collection tubes had acceptable lot-to-lot variability, precision, and stability. CONCLUSIONS: The novel ESAT-6-free IGRA had diagnostic accuracy comparable to QFT and is suitable for use in clinical trials to assess efficacy of candidate TB vaccines to prevent established M.tb infection.

A Quantitative Analysis of Complexity of Human Pathogen-Specific CD4 T Cell Responses in Healthy M. tuberculosis Infected South Africans.

We performed a quantitative analysis of the HLA restriction, antigen and epitope specificity of human pathogen specific responses in healthy individuals infected with M. tuberculosis (Mtb), in a South African cohort as a test case. The results estimate the breadth of T cell responses for the first time in the context of an infection and human population setting. We determined the epitope repertoire of eleven representative Mtb antigens and a large panel of previously defined Mtb epitopes. We estimated that our analytic methods detected 50-75% of the total response in a cohort of 63 individuals. As expected, responses were highly heterogeneous, with responses to a total of 125 epitopes detected. The 66 top epitopes provided 80% coverage of the responses identified in our study. Using a panel of 48 HLA class II-transfected antigen-presenting cells, we determined HLA class II restrictions for 278 epitope/donor recognition events (36% of the total). The majority of epitopes were restricted by multiple HLA alleles, and 380 different epitope/HLA combinations comprised less than 30% of the estimated Mtb-specific response. Our results underline the complexity of human T cell responses at a population level. Efforts to capture and characterize this broad and highly HLA promiscuous Mtb-specific T cell epitope repertoire will require significant peptide multiplexing efforts. We show that a comprehensive "megapool" of Mtb peptides captured a large fraction of the Mtb-specific T cells and can be used to characterize this response.

Differential Recognition of Mycobacterium tuberculosis-Specific Epitopes as a Function of Tuberculosis Disease History.

RATIONALE: Individuals with a history of tuberculosis (TB) disease are at elevated risk of disease recurrence. The underlying cause is not known, but one explanation is that previous disease results in less-effective immunity against Mycobacterium tuberculosis (Mtb). OBJECTIVES: We hypothesized that the repertoire of Mtb-derived epitopes recognized by T cells from individuals with latent Mtb infection differs as a function of previous diagnosis of active TB disease. METHODS: T-cell responses to peptide pools in samples collected from an adult screening and an adolescent validation cohort were measured by IFN-γ enzyme-linked immunospot assay or intracellular cytokine staining. MEASUREMENTS AND MAIN RESULTS: We identified a set of "type 2" T-cell epitopes that were recognized at 10-fold-lower levels in Mtb-infected individuals with a history of TB disease less than 6 years ago than in those without previous TB. By contrast, "type 1" epitopes were recognized equally well in individuals with or without previous TB. The differential epitope recognition was not due to differences in HLA class II binding, memory phenotypes, or gene expression in the responding T cells. Instead, "TB disease history-sensitive" type 2 epitopes were significantly (P < 0.0001) more homologous to sequences from bacteria found in the human microbiome than type 1 epitopes. CONCLUSIONS: Preferential loss of T-cell reactivity to Mtb epitopes that are homologous to bacteria in the microbiome in persons with previous TB disease may reflect long-term effects of antibiotic TB treatment on the microbiome.

Optimization and Interpretation of Serial QuantiFERON Testing to Measure Acquisition of Mycobacterium tuberculosis Infection.

RATIONALE: Conversion from a negative to positive QuantiFERON-TB test is indicative of Mycobacterium tuberculosis (Mtb) infection, which predisposes individuals to tuberculosis disease. Interpretation of serial tests is confounded by immunological and technical variability. OBJECTIVES: To improve the consistency of serial QuantiFERON-TB testing algorithms and provide a data-driven definition of conversion. METHODS: Sources of QuantiFERON-TB variability were assessed, and optimal procedures were identified. Distributions of IFN-γ response levels were analyzed in healthy adolescents, Mtb-unexposed control subjects, and patients with pulmonary tuberculosis. MEASUREMENTS AND MAIN RESULTS: Individuals with no known Mtb exposure had IFN-γ values less than 0.2 IU/ml. Among individuals with IFN-γ values less than 0.2 IU/ml, 0.2-0.34 IU/ml, 0.35-0.7 IU/ml, and greater than 0.7 IU/ml, tuberculin skin test positivity results were 15%, 53%, 66%, and 91% (P < 0.005), respectively. Together, these findings suggest that values less than 0.2 IU/ml were true negatives. In short-term serial testing, "uncertain" conversions, with at least one value within the uncertainty zone (0.2-0.7 IU/ml), were partly explained by technical assay variability. Individuals who had a change in QuantiFERON-TB IFN-γ values from less than 0.2 to greater than 0.7 IU/ml had 10-fold higher tuberculosis incidence rates than those who maintained values less than 0.2 IU/ml over 2 years (P = 0.0003). By contrast, "uncertain" converters were not at higher risk than nonconverters (P = 0.229). Eighty-seven percent of patients with active tuberculosis had IFN-γ values greater than 0.7 IU/ml, suggesting that these values are consistent with established Mtb infection. CONCLUSIONS: Implementation of optimized procedures and a more rigorous QuantiFERON-TB conversion definition (an increase from IFN-γ <0.2 to >0.7 IU/ml) would allow more definitive detection of recent Mtb infection and potentially improve identification of those more likely to develop disease.

Combined use of Mycobacterium tuberculosis-specific CD4 and CD8 T-cell responses is a powerful diagnostic tool of active tuberculosis.

Immune-based assays are promising tools to help to formulate diagnosis of active tuberculosis. A multiparameter flow cytometry assay assessing T-cell responses specific to Mycobacterium tuberculosis and the combination of both CD4 and CD8 T-cell responses accurately discriminated between active tuberculosis and latent infection.

Rapid perturbation in viremia levels drives increases in functional avidity of HIV-specific CD8 T cells.

The factors determining the functional avidity and its relationship with the broad heterogeneity of antiviral T cell responses remain partially understood. We investigated HIV-specific CD8 T cell responses in 85 patients with primary HIV infection (PHI) or chronic (progressive and non-progressive) infection. The functional avidity of HIV-specific CD8 T cells was not different between patients with progressive and non-progressive chronic infection. However, it was significantly lower in PHI patients at the time of diagnosis of acute infection and after control of virus replication following one year of successful antiretroviral therapy. High-avidity HIV-specific CD8 T cells expressed lower levels of CD27 and CD28 and were enriched in cells with an exhausted phenotype, i.e. co-expressing PD-1/2B4/CD160. Of note, a significant increase in the functional avidity of HIV-specific CD8 T cells occurred in early-treated PHI patients experiencing a virus rebound after spontaneous treatment interruption. This increase in functional avidity was associated with the accumulation of PD-1/2B4/CD160 positive cells, loss of polyfunctionality and increased TCR renewal. The increased TCR renewal may provide the mechanistic basis for the generation of high-avidity HIV-specific CD8 T cells. These results provide insights on the relationships between functional avidity, viremia, T-cell exhaustion and TCR renewal of antiviral CD8 T cell responses.

Mycobacterium tuberculosis-specific CD8+ T cells are functionally and phenotypically different between latent infection and active disease.

Protective immunity to Mycobacterium tuberculosis (Mtb) remains poorly understood and the role of Mtb-specific CD8(+) T cells is controversial. Here we performed a broad phenotypic and functional characterization of Mtb-specific CD8(+) T cells in 326 subjects with latent Mtb infection (LTBI) or active TB disease (TB). Mtb-specific CD8(+) T cells were detected in most (60%) TB patients and few (15%) LTBI subjects but were of similar magnitude. Mtb-specific CD8(+) T cells in LTBI subjects were mostly T EMRA cells (CD45RA(+) CCR7(-)), coexpressing 2B4 and CD160, and in TB patients were mostly TEM cells (CD45RA(-) CCR7(-)), expressing 2B4 but lacking PD-1 and CD160. The cytokine profile was not significantly different in both groups. Furthermore, Mtb-specific CD8(+) T cells expressed low levels of perforin and granulysin but contained granzymes A and B. However, in vitro-expanded Mtb-specific CD8(+) T cells expressed perforin and granulysin. Finally, Mtb-specific CD8(+) T-cell responses were less frequently detected in extrapulmonary TB compared with pulmonary TB patients. Mtb-specific CD8(+) T-cell proliferation was also greater in patients with extrapulmonary compared with pulmonary TB. Thus, the activity of Mtb infection and clinical presentation are associated with distinct profiles of Mtb-specific CD8(+) T-cell responses. These results provide new insights in the interaction between Mtb and the host immune response.

Lack of Mycobacterium tuberculosis-specific interleukin-17A-producing CD4+ T cells in active disease.

Protective immunity to Mycobacterium tuberculosis (Mtb) is commonly ascribed to a Th1 profile; however, the involvement of Th17 cells remains to be clarified. Here, we characterized Mtb-specific CD4(+) T cells in blood and bronchoalveolar lavages (BALs) from untreated subjects with either active tuberculosis disease (TB) or latent Mtb infection (LTBI), considered as prototypic models of uncontrolled or controlled infection, respectively. The production of IL-17A, IFN-γ, TNF-α, and IL-2 by Mtb-specific CD4(+) T cells was assessed both directly ex vivo and following in vitro antigen-specific T-cell expansion. Unlike for extracellular bacteria, Mtb-specific CD4(+) T-cell responses lacked immediate ex vivo IL-17A effector function in both LTBI and TB individuals. Furthermore, Mtb-specific Th17 cells were absent in BALs, while extracellular bacteria-specific Th17 cells were identified in gut biopsies of healthy individuals. Interestingly, only Mtb-specific CD4(+) T cells from 50% of LTBI but not from TB subjects acquired the ability to produce IL-17A following Mtb-specific T-cell expansion. Finally, IL-17A acquisition by Mtb-specific CD4(+) T cells correlated with the coexpression of CXCR3 and CCR6, currently associated to Th1 or Th17 profiles, respectively. Our data demonstrate that Mtb-specific Th17 cells are selectively undetectable in peripheral blood and BALs from TB patients.

NYVAC immunization induces polyfunctional HIV-specific T-cell responses in chronically-infected, ART-treated HIV patients.

We report the results of the Theravac-01 phase I trial, which was conducted to evaluate the safety and immunogenicity of a poxvirus-based vector, NYVAC, expressing Gag, Pol, Nef, and Env from an HIV clade B isolate. NYVAC-B vaccine was injected intra-muscularly into ten HIV-infected patients successfully treated with antiretroviral therapy, twice on day 0 and again at week 4. Safety and immunogenicity were monitored for 48 weeks. HIV-specific T-cell responses following immunization were quantitatively analyzed using an IFN-γ ELISPOT assay and qualitatively characterized for their functional profile (including multiple cytokines secretion plus cytotoxic and proliferation capacity) by polychromatic flow cytometry. Our results indicate that the NYVAC-B vaccine is safe and highly immunogenic, as indicated by increased HIV-specific T-cell responses in virtually all vaccinees. Interestingly, both an expansion of preexisting T-cell responses, and the appearance of newly detected HIV-specific CD4(+) and CD8(+) T-cell responses were observed. Furthermore, immunization mostly induced an increase in Gag-specific T-cell responses. In conclusion, NYVAC-B immunization induces broad, vigorous, and polyfunctional HIV-specific T-cell responses, suggesting that poxvirus-based vaccine regimens may be instrumental in the therapeutic HIV vaccine field.

Expression of specific HLA class II alleles is associated with an increased risk for active tuberculosis and a distinct gene expression profile.

Several HLA allelic variants have been associated with protection from or susceptibility to infectious and autoimmune diseases. Here, we examined whether specific HLA alleles would be associated with different Mycobacterium tuberculosis (Mtb) infection outcomes. The HLA alleles present at the -A, -B, -C, -DPA1, -DPB1, -DQA1, -DQB1, -DRB1, and -DRB3/4/5 loci were determined in a cohort of 636 individuals with known Mtb infection outcomes from South Africa and the United States. Among these individuals, 203 were QuantiFERON (QFT) negative, and 433 were QFT positive, indicating Mtb exposure. Of these, 99 QFT positive participants either had active tuberculosis (TB) upon enrollment or were diagnosed in the past. We found that DQA1*03:01, DPB1*04:02, and DRB4*01:01 were significantly more frequent in individuals with active TB (susceptibility alleles), as judged by Odds Ratios and associated p-values, while DPB1*105:01 was associated with protection from active TB. Peripheral blood mononuclear cells (PMBCs) from a subset of individuals were stimulated with Mtb antigens, revealing individuals who express any of the three susceptibility alleles were associated with lower magnitude of responses. Furthermore, we defined a gene signature associated with individuals expressing the susceptibility alleles that was characterized by lower expression of APC-related genes. In summary, we have identified specific HLA alleles associated with susceptibility to active TB and found that the expression of these alleles was associated with a decreased Mtb-specific T cell response and a specific gene expression signature. These results will help understand individual risk factors in progressing to active TB.

T cell receptor repertoires associated with control and disease progression following Mycobacterium tuberculosis infection.

Antigen-specific, MHC-restricted αß T cells are necessary for protective immunity against Mycobacterium tuberculosis, but the ability to broadly study these responses has been limited. In the present study, we used single-cell and bulk T cell receptor (TCR) sequencing and the GLIPH2 algorithm to analyze M. tuberculosis-specific sequences in two longitudinal cohorts, comprising 166 individuals with M. tuberculosis infection who progressed to either tuberculosis (n = 48) or controlled infection (n = 118). We found 24 T cell groups with similar TCR-ß sequences, predicted by GLIPH2 to have common TCR specificities, which were associated with control of infection (n = 17), and others that were associated with progression to disease (n = 7). Using a genome-wide M. tuberculosis antigen screen, we identified peptides targeted by T cell similarity groups enriched either in controllers or in progressors. We propose that antigens recognized by T cell similarity groups associated with control of infection can be considered as high-priority targets for future vaccine development.

Proliferation capacity and cytotoxic activity are mediated by functionally and phenotypically distinct virus-specific CD8 T cells defined by interleukin-7R{alpha} (CD127) and perforin expression.

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127(+) perforin(-)/CD127(-) perforin(+), and CD127(-)/perforin(-) CD8 T cells, respectively. CD127(-)/perforin(-) and CD127(-)/perforin(+) cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin(+)/IL-2(-) or perforin(-)/IL-2(+) cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P < 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127(+)/IL-2-secreting) and cytotoxic (perforin(+)) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.

Meeting report: Virtual Global Forum on Tuberculosis Vaccines, 20-22 April 2021.

The Global Forum on Tuberculosis (TB) Vaccines was held virtually from 20 to 22 April 2021, marking its 20th anniversary. The Global Forum on TB Vaccines is the world's largest gathering of stakeholders striving to develop new vaccines to prevent TB. The program included more than 60 speakers in 11 scientific sessions, panel discussions, and workshops. It provided an overview of the state of the field, and an opportunity to share the latest research findings, as well as new and innovative approaches to TB vaccine research and development (R&D). This year, it was held against the backdrop of the COVID-19 pandemic and convened researchers, developers, funders, and other stakeholders remotely to discuss opportunities and challenges for TB vaccine R&D in these unprecedented times.

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

BACKGROUND: Recent Mycobacterium tuberculosis (M.tb) infection is associated with a higher risk of progression to tuberculosis disease, compared to persistent infection after remote exposure. However, current immunodiagnostic tools fail to distinguish between recent and remote infection. We aimed to characterise the immunobiology associated with acquisition of M.tb infection and identify a biomarker that can distinguish recent from remote infection. METHODS: Healthy South African adolescents were serially tested with QuantiFERON-TB Gold to define recent (QuantiFERON-TB conversion <6 months) and persistent (QuantiFERON-TB+ for >1.5 year) infection. We characterised M.tb-specific CD4 T cell functional (IFN-γ, TNF, IL-2, CD107, CD154), memory (CD45RA, CCR7, CD27, KLRG-1) and activation (HLA-DR) profiles by flow cytometry after CFP-10/ESAT-6 peptide pool or M.tb lysate stimulation. We then assessed the diagnostic performance of immune profiles that were differentially expressed between individuals with recent or persistent QuantiFERON-TB+. FINDINGS: CFP-10/ESAT-6-specific CD4 T cell activation but not functional or memory phenotypes distinguished between individuals with recent and persistent QuantiFERON-TB+. In response to M.tb lysate, recent QuantiFERON-TB+ individuals had lower proportions of highly differentiated IFN-γ+TNF+ CD4 T cells expressing a KLRG-1+ effector phenotype and higher proportions of early differentiated IFN-γ-TNF+IL-2+ and activated CD4 T cells compared to persistent QuantiFERON-TB+ individuals. Among all differentially expressed T cell features CFP-10/ESAT-6-specific CD4 T cell activation was the best performing diagnostic biomarker of recent infection. INTERPRETATION: Recent M.tb infection is associated with highly activated and moderately differentiated functional M.tb-specific T cell subsets, that can be used as biomarkers to distinguish between recent and remote infection. FUNDING: US National Institutes of Health (NIH), Bill and Melinda Gates Foundation, South African National Research Foundation, South African Medical Research Council, and Aeras.