W. Henry Boom

Immune and clinical outcomes to Mycobacterium tuberculosis (Mtb) infection vary greatly between individuals, yet the underlying genetic and cellular mechanisms driving this heterogeneity remain poorly understood. We performed a cellular genome-wide association study to identify genetic variants associated with Mtb-induced monocyte transcriptional expression of IL1B , IL6 , TNF , and IFNB1 via RNA-Seq in a Ugandan cohort. Significantly associated variants were assessed for transferability in an independent Seattle cohort, further validated in vitro, and assessed for clinical phenotype associations. We identified 77 loci suggestively associated with Mtb-induced cytokine expression in monocytes in Uganda. SNPs associated with Mtb-induced TNF were enriched within α-linolenic acid metabolism pathway genes, which was validated in vitro using PLA2 inhibitors. Four loci maintained significant associations in Seattle. We validated a cytokine effect with siRNA knockdown for two of these loci, which mapped to the genes SLIT3 and SLC1A1 . Furthermore, exogenous treatment of macrophages with SLIT3 enhanced Mtb intracellular replication. Finally, SLC1A1 and SLIT3 variants were associated with susceptibility to tuberculous meningitis and subsequent survival, respectively, in a Vietnamese cohort. In summary, we identified multiple variants and pathways associated with Mtb-induced cytokine transcriptional responses that were validated in vitro and were associated with clinical tuberculosis susceptibility.

T cells are required for protective immunity against Mycobacterium tuberculosis . We recently described a cohort of Ugandan household contacts of tuberculosis cases who appear to “resist” M . tuberculosis infection (resisters; RSTRs) and showed that these individuals harbor IFN-γ–independent T cell responses to M . tuberculosis –specific peptide antigens. However, T cells also recognize nonprotein antigens via antigen-presenting systems that are independent of genetic background, known as donor-unrestricted T cells (DURTs). We used tetramer staining and flow cytometry to characterize the association between DURTs and “resistance” to M . tuberculosis infection. Peripheral blood frequencies of most DURT subsets were comparable between RSTRs and latently infected controls (LTBIs). However, we observed a 1.65-fold increase in frequency of MR1-restricted T (MR1T) cells among RSTRs in comparison with LTBIs. Single-cell RNA sequencing of 18,251 MR1T cells sorted from 8 donors revealed 5,150 clonotypes that expressed a common transcriptional program, the majority of which were private. Sequencing of the T cell receptor α/T cell receptor δ (TCRα/δ) repertoire revealed several DURT clonotypes were expanded among RSTRs, including 2 MR1T clonotypes that recognized mycobacteria-infected cells in a TCR-dependent manner. Overall, our data reveal unexpected donor-specific diversity in the TCR repertoire of human MR1T cells as well as associations between mycobacteria-reactive MR1T clonotypes and resistance to M . tuberculosis infection.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a leading cause of pediatric morbidity and mortality. Young children are at high risk of TB following Mtb exposure, and this vulnerability is secondary to insufficient host immunity during early life. Our primary objective was to compare CD4+ and CD8+ T-cell production of proinflammatory cytokines IFN-gamma, IL-2, and TNF-alpha in response to six mycobacterial antigens and superantigen staphylococcal enterotoxin B (SEB) between Ugandan adults with confirmed TB (n = 41) and young Ugandan children with confirmed (n = 12) and unconfirmed TB (n = 41), as well as non-TB lower respiratory tract infection (n = 39). Flow cytometry was utilized to identify and quantify CD4+ and CD8+ T-cell cytokine production in response to each mycobacterial antigen and SEB. We found that the frequency of CD4+ and CD8+ T-cell production of cytokines in response to SEB was reduced in all pediatric cohorts when compared to adults. However, T-cell responses to Mtb-specific antigens ESAT6 and CFP10 were equivalent between children and adults with confirmed TB. In contrast, cytokine production in response to ESAT6 and CFP10 was limited in children with unconfirmed TB and absent in children with non-TB lower respiratory tract infection. Of the five additional mycobacterial antigens tested, PE3 and PPE15 were broadly recognized regardless of TB disease classification and age. Children with confirmed TB exhibited robust proinflammatory CD4+ and CD8+ T-cell responses to Mtb-specific antigens prior to the initiation of TB treatment. Our findings suggest that adaptive proinflammatory immune responses to Mtb, characterized by T-cell production of IFN-gamma, IL-2, and TNF-alpha, are not impaired during early life.

Tuberculosis (TB) remains a major public health threat globally, especially in sub-Saharan Africa. Both human and Mycobacterium tuberculosis (MTBC) genetic variation affect TB outcomes, but few studies have examined if and how the two genomes interact to affect disease. We hypothesize that long-term coexistence between human genomes and MTBC lineages modulates disease to affect its severity. We examined this hypothesis in our TB household contact study in Kampala, Uganda, in which we identified three MTBC lineages, of which one, L4.6-Uganda, is clearly derived and hence recent. We quantified TB severity using the Bandim TBscore and examined the interaction between MTBC lineage and human single-nucleotide polymorphisms (SNPs) genome-wide, in two independent cohorts of TB cases (n = 149 and n = 127). We found a significant interaction between an SNP in PPIAP2 and the Uganda lineage (combined p = 4 × 10⁻⁸). PPIAP2 is a pseudogene that is highly expressed in immune cells. Pathway and eQTL analyses indicated potential roles between coevolving SNPs and cellular replication and metabolism as well as platelet aggregation and coagulation. This finding provides further evidence that host–pathogen interactions affect clinical presentation differently than host and pathogen genetic variation independently, and that human–MTBC coevolution is likely to explain patterns of disease severity.

After extensive exposure to Mycobacterium tuberculosis (Mtb), most individuals acquire latent Mtb infection (LTBI) defined by a positive tuberculin skin test (TST) or interferon-γ release assay (IGRA). To identify mechanisms of resistance to Mtb infection, we compared transcriptional profiles from highly exposed contacts who resist TST/IGRA conversion (resisters, RSTRs) and controls with LTBI using RNAseq. Gene sets related to carbon metabolism and free fatty acid (FFA) transcriptional responses enriched across 2 independent cohorts suggesting RSTR and LTBI monocytes have distinct activation states. We compared intracellular Mtb replication in macrophages treated with FFAs and found that palmitic acid (PA), but not oleic acid (OA), enhanced Mtb intracellular growth. This PA activity correlated with its inhibition of proinflammatory cytokines in Mtb-infected cells. Mtb growth restriction in PA-treated macrophages was restored by activation of AMP kinase (AMPK), a central host metabolic regulator known to be inhibited by PA. Finally, we genotyped AMPK variants and found 7 SNPs in PRKAG2, which encodes the AMPK-γ subunit, that strongly associated with RSTR status. Taken together, RSTR and LTBI phenotypes are distinguished by FFA transcriptional programs and by genetic variation in a central metabolic regulator, which suggests immunometabolic pathways regulate TST/IGRA conversion.

Humans have been infected with Mycobacterium tuberculosis (Mtb) for thousands of years. While tuberculosis (TB), one of the deadliest infectious diseases, is caused by uncontrolled Mtb infection, over 90% of presumed infected individuals remain asymptomatic and contain Mtb in a latent TB infection (LTBI) without ever developing disease, and some may clear the infection. A small number of heavily Mtb-exposed individuals appear to resist developing traditional LTBI. Because Mtb has mechanisms for intracellular survival and immune evasion, successful control involves all of the arms of the immune system. Here, we focus on immune responses to Mtb in humans and nonhuman primates and discuss new concepts and outline major knowledge gaps in our understanding of LTBI, ranging from the earliest events of exposure and infection to success or failure of Mtb control.

Background: Tuberculosis (TB) is the most deadly infectious disease globally and highly prevalent in the developing world, especially sub-Saharan Africa. Even though a third of humans are exposed to Myocbacterium tuberculosis (Mtb), most infected immunocompetent individuals do not develop active TB. In contrast, for individuals infected with both TB and the human immunodeficiency virus (HIV), the risk of active disease is 10% or more per year. Previously, we identified in a genome-wide association study a region on chromosome 5 that was associated with resistance to TB. This region included epigenetic marks that could influence gene regulation so we hypothesized that HIV-infected individuals exposed to Mtb, who remain disease free, carry epigenetic changes that strongly protect them from active TB. To test this hypothesis, we conducted a methylome-wide study in HIV-infected, TB-exposed cohorts from Uganda and Tanzania. Results: In 221 HIV-infected adults from Uganda and Tanzania, we identified 3 regions of interest that included markers that were differentially methylated between TB cases and LTBI controls, that also included methylation QTLs and associated SNPs: chromosome 1 ( RNF220 , p=4×10 ⁻⁵ ), chromosome 2 (between COPS8 and COL6A3 genes, p=2.7×10 ⁻⁵ ), and chromosome 5 ( CEP72 , p=1.3×10 ⁻⁵ ). These methylation results colocalized with associated SNPs, methylation QTLs, and methylation × SNP interaction effects. These markers were in regions with regulatory markers for cells involved in TB immunity and/or lung. Conclusion: Epigenetic regulation is a potential biologic factor underlying resistance to TB in immunocompromised individuals that can act in conjunction with genetic variants.

Lymph node tuberculosis (LNTB) is the most frequent extrapulmonary form of tuberculosis (TB). Studies of human tuberculosis at sites of disease are limited. LNTB provides a unique opportunity to compare local in situ and peripheral blood immune response in active Mycobacterium tuberculosis (Mtb) disease. The present study analysed T regulatory cells (Treg) frequency and activation along with CD4+ T cell function in lymph nodes from LNTB patients. Lymph node mononuclear cells (LNMC) were compared to autologous peripheral blood mononuclear cells (PBMC). LNMC were enriched for CD4+ T cells with a late differentiated effector memory phenotype. No differences were noted in the frequency and mutifunctional profile of memory CD4+ T cells specific for Mtb. The proportion of activated CD4+ and Tregs in LNMC was increased compared to PBMC. The correlation between Tregs and activated CD4+ T cells was stronger in LNMC than PBMC. Tregs in LNMC showed a strong positive correlation with Th1 cytokine production (IL2, IFNγ and TNFα) as well as MIP-1α after Mtb antigen stimulation. A subset of Tregs in LNMC co-expressed HLA-DR and CD38, markers of activation. Further research will determine the functional relationship between Treg and activated CD4+ T cells at lymph node sites of Mtb infection.

Appraisal delay is the time a patient takes to consider a symptom as not only noticeable, but a sign of illness. The study’s objective was to determine the association between appraisal delay in seeking tuberculosis (TB) treatment and geographic distance measured by network travel (driving and pedestrian) time (in minutes) and distance (Euclidean and self-reported) (in kilometers) and to identify other risk factors from selected covariates and how they modify the core association between delay and distance. This was part of a longitudinal cohort study known as the Kawempe Community Health Study based in Kampala, Uganda. The study enrolled households from April 2002 to July 2012. Multivariable interval regression with multiplicative heteroscedasticity was used to assess the impact of time and distance on delay. The delay interval outcome was defined using a comprehensive set of 28 possible self-reported symptoms. The main independent variables were network travel time (in minutes) and Euclidean distance (in kilometers). Other covariates were organized according to the Andersen utilization conceptual framework. A total of 838 patients with both distance and delay data were included in the network analysis. Bivariate analyses did not reveal a significant association of any distance metric with the delay outcome. However, adjusting for patient characteristics and cavitary disease status, the multivariable model indicated that each minute of driving time to the clinic significantly (p = 0.02) and positively predicted 0.25 days’ delay. At the median distance value of 47 min, this represented an additional delay of about 12 (95% CI: [3, 21]) days to the mean of 40 days (95% CI: [25, 56]). Increasing Euclidean distance significantly predicted (p = 0.02) reduced variance in the delay outcome, thereby increasing precision of the mean delay estimate. At the median Euclidean distance of 2.8 km, the variance in the delay was reduced by more than 25%. Of the four geographic distance measures, network travel driving time was a better and more robust predictor of mean delay in this setting. Including network travel driving time with other risk factors may be important in identifying populations especially vulnerable to delay.

Mycobacterium tuberculosis (Mtb) cell wall glycolipid mannose‐capped lipoarabinomannan (ManLAM) inhibits CD4 ⁺ T‐cell activation by inhibiting proximal T‐cell receptor (TCR) signaling when activated by anti‐CD3. To understand the impact of ManLAM on CD4 ⁺ T‐cell function when both the TCR–CD3 complex and major costimulator CD28 are engaged, we performed label‐free quantitative MS and network analysis. Mixed‐effect model analysis of peptide intensity identified 149 unique peptides representing 131 proteins that were differentially regulated by ManLAM in anti‐CD3‐ and anti‐CD28‐activated CD4 ⁺ T cells. Crosstalker, a novel network analysis tool identified dysregulated translation, TCA cycle, and RNA metabolism network modules. PCNA, Akt, mTOR, and UBC were found to be bridge node proteins connecting these modules of dysregulated proteins. Altered PCNA expression and cell cycle analysis showed arrest at the G2M phase. Western blot confirmed that ManLAM inhibited Akt and mTOR phosphorylation, and decreased expression of deubiquitinating enzymes Usp9x and Otub1. Decreased NF‐κB phosphorylation suggested interference with CD28 signaling through inhibition of the Usp9x‐Akt‐mTOR pathway. Thus, ManLAM induced global changes in the CD4 ⁺ T‐cell proteome by affecting Akt‐mTOR signaling, resulting in broad functional impairment of CD4 ⁺ T‐cell activation beyond inhibition of proximal TCR–CD3 signaling.