Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which leads to an estimated 1. 5 million deaths worldwide each year. Although the immune correlates of protection against Mtb infection and TB disease have not been well-defined, natural killer (NK) cells are increasingly recognized as a key component of the innate immune response to Mtb and as a link between innate and adaptive immunity. In this study, we evaluated NK cell phenotypic and functional profiles in QuantiFERON-TB (QFT) + and QFT − adults in a TB endemic setting in Kisumu, Kenya, and compared their NK cell responses to those of Mtb-naïve healthy adult controls in the U.S. We used flow cytometry to define the phenotypic profile of NK cells and identified distinct CD56 dim NK cell phenotypes that differentiated the Kenyan and U.S. groups. Additionally, among Kenyan participants, NK cells from QFT + individuals with latent Mtb infection (LTBI) were characterized by significant downregulation of the natural cytotoxicity receptor NKp46 and the inhibitory receptor TIGIT, compared with QFT − individuals. Moreover, the distinct CD56 dim phenotypic profiles in Kenyan individuals correlated with dampened NK cell responses to tumor cells and diminished activation, degranulation, and cytokine production following stimulation with Mtb antigens, compared with Mtb-naïve U.S. healthy adult controls. Taken together, these data provide evidence that the phenotypic and functional profiles of NK cells are modified in TB endemic settings and will inform future studies aimed at defining NK cell-mediated immune correlates that may be protective against acquisition of Mtb infection and progression to TB disease.

Mycobacterium tuberculosis (Mtb) is a serious public health concern, infecting a quarter of the world and leading to 10 million cases of tuberculosis (TB) disease and 1. 5 million deaths annually. An effective type 1 CD4 T cell (TH1) immune response is necessary to control Mtb infection and defining factors that modulate Mtb-specific TH1 immunity is important to better define immune correlates of protection in Mtb infection. Helminths stimulate type 2 (TH2) immune responses, which antagonize TH1 cells. As such, we sought to evaluate whether co-infection with the parasitic helminth Schistosoma mansoni (SM) modifies CD4 T cell lineage profiles in a cohort of HIV-uninfected adults in Kisumu, Kenya. Individuals were categorized into six groups by Mtb and SM infection status: healthy controls (HC), latent Mtb infection (LTBI) and active tuberculosis (TB), with or without concomitant SM infection. We utilized flow cytometry to evaluate the TH1/TH2 functional and phenotypic lineage state of total CD4 T cells, as well as CD4 T cells specific for the Mtb antigens CFP-10 and ESAT-6. Total CD4 T cell lineage profiles were similar between SM + and SM − individuals in all Mtb infection groups. Furthermore, in both LTBI and TB groups, SM infection did not impair Mtb-specific TH1 cytokine production. In fact, SM + LTBI individuals had higher frequencies of IFNγ + Mtb-specific CD4 T cells than SM − LTBI individuals. Mtb-specific CD4 T cells were characterized by expression of both classical TH1 markers, CXCR3 and T-bet, and TH2 markers, CCR4, and GATA3. The expression of these markers was similar between SM + and SM − individuals with LTBI. However, SM + individuals with active TB had significantly higher frequencies of GATA3 + CCR4 + TH1 cytokine + Mtb-specific CD4 T cells, compared with SM − TB individuals. Together, these data indicate that Mtb-specific TH1 cytokine production capacity is maintained in SM-infected individuals, and that Mtb-specific TH1 cytokine + CD4 T cells can express both TH1 and TH2 markers. In high pathogen burden settings where co-infection is common and reoccurring, plasticity of antigen-specific CD4 T cell responses may be important in preserving Mtb-specific TH1 responses.

Antigen-specific CD4 and CD8 T cells are important components of the immune response to , yet little information is currently known regarding how the breadth, specificity, phenotype, and function of-specific T cells correlate with infection outcome in humans. To facilitate evaluation of human-specific T cell responses targeting multiple different Ags, we sought to develop a high throughput and reproducible T cell response spectrum assay requiring low blood sample volumes. We describe here the optimization and standardization of a microtiter plate-based, diluted whole blood stimulation assay utilizing overlapping peptide pools corresponding to a functionally diverse panel of 60 Ags. Using IFN-γ production as a readout of Ag specificity, the assay can be conducted using 50 μl of blood per test condition and can be expanded to accommodate additional Ags. We evaluated the intra- and interassay variability, and implemented testing of the assay in diverse cohorts of-unexposed healthy adults, foreign-born adults with latent infection residing in the United States, and tuberculosis household contacts with latent infection in a tuberculosis-endemic setting in Kenya. The -specific T cell response spectrum assay further enhances the immunological toolkit available for evaluating-specific T cell responses across different states of infection, and can be readily implemented in resource-limited settings. Moreover, application of the assay to longitudinal cohorts will facilitate evaluation of treatment- or vaccine-induced changes in the breadth and specificity of Ag-specific T cell responses, as well as identification of-specific T cell responses associated with infection outcomes.

HIV infection is a significant risk factor for reactivation of latent Mycobacterium tuberculosis infection (LTBI) and progression to active tuberculosis disease, yet the mechanisms whereby HIV impairs T cell immunity to M. tuberculosis have not been fully defined. Evaluation of M. tuberculosis –specific CD4 T cells is commonly based on IFN-γ production, yet increasing evidence indicates the immune response to M. tuberculosis is heterogeneous and encompasses IFN-γ–independent responses. We hypothesized that upregulation of surface activation-induced markers (AIM) would facilitate detection of human M. tuberculosis –specific CD4 T cells in a cytokine-independent manner in HIV-infected and HIV-uninfected individuals with LTBI. PBMCs from HIV-infected and HIV-uninfected adults in Kenya were stimulated with CFP-10 and ESAT-6 peptides and evaluated by flow cytometry for upregulation of the activation markers CD25, OX40, CD69, and CD40L. Although M. tuberculosis –specific IFN-γ and IL-2 production was dampened in HIV-infected individuals, M. tuberculosis– specific CD25 + OX40 + and CD69 + CD40L + CD4 T cells were detectable in the AIM assay in both HIV-uninfected and HIV-infected individuals with LTBI. Importantly, the frequency of M. tuberculosis– specific AIM + CD4 T cells was not directly impacted by HIV viral load or CD4 count, thus demonstrating the feasibility of AIM assays for analysis of M. tuberculosis– specific CD4 T cells across a spectrum of HIV infection states. These data indicate that AIM assays enable identification of M. tuberculosis– specific CD4 T cells in a cytokine-independent manner in HIV-uninfected and HIV-infected individuals with LTBI in a high-tuberculosis burden setting, thus facilitating studies to define novel T cell correlates of protection to M. tuberculosis and elucidate mechanisms of HIV-associated dysregulation of antimycobacterial immunity.

Background: Helminth infections can modulate immunity to Mycobacterium tuberculosis (Mtb). However, the effect of helminths, including Schistosoma mansoni (SM), on Mtb infection outcomes is less clear. Furthermore, HIV is a known risk factor for tuberculosis (TB) disease and has been implicated in SM pathogenesis. Therefore, it is important to evaluate whether HIV modifies the association between SM and Mtb infection. Setting: HIV-infected and HIV-uninfected adults were enrolled in Kisumu County, Kenya, between 2014 and 2017 and categorized into 3 groups based on Mtb infection status: Mtb-uninfected healthy controls, latent TB infection (LTBI), and active TB disease. Participants were subsequently evaluated for infection with SM. Methods: We used targeted minimum loss estimation and super learning to estimate a covariate-adjusted association between SM and Mtb infection outcomes, defined as the probability of being Mtb-uninfected healthy controls, LTBI, or TB. HIV status was evaluated as an effect modifier of this association. Results: SM was not associated with differences in baseline demographic or clinical features of participants in this study, nor with additional parasitic infections. Covariate-adjusted analyses indicated that infection with SM was associated with a 4% higher estimated proportion of active TB cases in HIV-uninfected individuals and a 14% higher estimated proportion of active TB cases in HIV-infected individuals. There were no differences in estimated proportions of LTBI cases. Conclusions: We provide evidence that SM infection is associated with a higher probability of active TB disease, particularly in HIV-infected individuals.

Nearly a quarter of the global population is infected with Mycobacterium tuberculosis (Mtb), with 10 million people developing active tuberculosis (TB) annually. Co-infection with human immunodeficiency virus (HIV) has long been recognized as a significant risk factor for progression to TB disease, yet the mechanisms whereby HIV impairs T cell-mediated control of Mtb infection remain poorly defined. We hypothesized that HIV infection may promote upregulation of inhibitory receptors on Mtb-specific CD4 T cells, a mechanism that has been associated with antigen-specific T cell dysfunction in chronic infections. Using cohorts of HIV-infected and HIV-uninfected individuals with latent Mtb infection (LTBI) and with active TB disease, we stimulated peripheral blood mononuclear cells (PBMC) for 6 hours with Mtb peptide pools and evaluated co-expression profiles of the inhibitory receptors BTLA, CTLA-4, and PD-1 on IFN-γ + /TNF-α + Mtb-specific CD4 T cells. Mtb-specific CD4 T cells in all participant groups expressed predominately either one or no inhibitory receptors, unlike cytomegalovirus- and HIV-specific CD4 T cells circulating in the same individuals, which were predominately CTLA-4 + PD-1 + . There were no significant differences in inhibitory receptor expression profiles of Mtb-specific CD4 T cells between HIV-uninfected and HIV-infected individuals with LTBI. Surprisingly, BTLA expression, both alone and in combination with CTLA-4 and PD-1, was markedly downregulated on Mtb-specific CD4 T cells in HIV-infected individuals with active TB. Together, these data provide novel evidence that the majority of Mtb-specific CD4 T cells do not co-express multiple inhibitory receptors, regardless of HIV infection status; moreover, they highlight a previously unrecognized role of BTLA expression on Mtb-specific CD4 T cells that could be further explored as a potential biomarker of Mtb infection status, particularly in people living with HIV, the population at greatest risk for development of active TB disease.

Background The spread of mono-resistant and multi-drug resistant tuberculosis (MDR-TB) has been enhanced by delays in the identification of resistant strains. However, resistance gene patterns and the extent and distribution of mono-resistant TB and MDR-TB is unknown, particularly for western Kenya where Human Immunodeficiency Virus (HIV) is common. As such, the overall objective of the current study was to identify cases of mono-resistant TB and MDR-TB among enrolled patients in health facilities in western Kenya. Methods Patients with a suspected TB history were referred by clinicians to the health facilities for TB and HIV diagnosis. HIV testing was done using the Unigold and Abbott Determine kits. Early morning sputum samples were collected and cultured on Mycobacteria growth indicator tubes (MGIT) and incubated at 37°C. Drug susceptibility testing (DST) using the SIRE® kit was done on ZN smear positive MGIT tubes and line probe assay (LPA) performed to identify specific mutations on the rpo B, kat G and inh A genes. Mutations on discordant samples were confirmed by the BigDye® Terminator v3.1 Cycle Sequencing Kit. Results The proportion of MDR-TB, RIF mono-resistant (RMR) TB and INH mono-resistant TB as estimated by LPA and DST, was as follows: MDR-TB: 1.38% / 1.26%; RMR-TB: 1.2% / 0.72%; INH mono-resistant TB: 2.1% / 2.4%, respectively. Our study showed that the H526Y rpo B and S315T1 kat G mutations were common in HIV-positive patients (8% and 18% respectively) and that the S3I5T1 and S531L was the most common mutation in MDR-TB strains in both HIV-positive and HIV-negative patients (5% and 8% respectively). Binary logistic regression, indicated that RMR-TB significantly predicted HIV status ( p=0.025). Conclusions Our findings show that RIF mono-resistant TB predicts HIV infection.

BackgroundIn 2015, 10.4 million people worldwide had tuberculosis (TB) and 1.4 million deaths occurred, 400 000 of whom were HIV-positive. Sub-Saharan Africa accounted for 81% of these cases. In western Kenya, current data on the distribution of rifampicin (RIF) and isoniazid (INH) mutations is not available. The association of gene mutations with HIVinfection and the treatment response of HIV-infected and -uninfected patients with TB are not known. This study „determined the proportion of drug-resistant Mycobacterium tuberculosis in sputum isolates and investigated the association of RIF and INH gene mutations with HIV status and monitored the treatment response of TB/HIV-co-infected patients.MethodsThe present study was longitudinal, and enrolment was done between 2012 and 2014 after the revision of the TB treatment regimen. Patients with confirmed drug-resistant TB were followed up for one year to establish the TB treatment response as confirmed by sputum smear microscopy.ResultsA total of 1381 new and 18 previously treated TB patients were enrolled. Sputum samples were cultured on Mycobacteria-growth indicator tubes; drug susceptibility tests and line probe assay were performed to identify drug resistance and specific mutations on the rpo B, kat G and inh A’genes. Discordant samples were sequenced. Conversion rate was calculated by finding the percentage of smear-negative and -positive patients at follow-up and initial visit, respectively. Regression analysis showed that RIF resistance was associated with HIV status (p=0.025). Mann-Whitney tests revealed that the conversion time of HIV-infected and -uninfected patients with TB drug mutations was comparable (p=0.180).ConclusionThe study showed that INH mono-resistance was common. Detection of INH mono-resistance in TB-endemic areas should be scaled-up as well as TB contact investigation studies to increase early detection of resistant strains.