Gene expression and cytokine profile correlate with mycobacterial growth in a human BCG challenge model.

BACKGROUND: Bacillus Calmette-Guerin (BCG) vaccine is the most widely administered vaccine in the world, yet its mechanism of action remains unclear. We hypothesize that certain immune pathways are associated with reduced mycobacterial growth following BCG challenge in human volunteers. METHODS: We used samples from a mycobacterial challenge in which previously BCG-vaccinated or BCG-naive adults in the United Kingdom were challenged intradermally with a standard dose of BCG. Any remaining BCG was quantified in a skin biopsy specimen obtained 2 weeks after challenge and used as a measure of BCG growth and functional antimycobacterial immunity. We measured the immune response over the 2-week challenge, using DNA microarrays and flow cytometry, and correlated this with mycobacterial growth. RESULTS: The magnitude of the immune response to BCG is greater in previously vaccinated volunteers, and this correlates with reduced mycobacterial growth but increased scarring at the vaccination site. In particular, the interferon γ and interleukin 17 pathways are strongly induced in previously vaccinated volunteers and correlate with reduced mycobacterial growth in this population. CONCLUSION: This study identifies pathways associated with control of mycobacterial growth in vivo in human volunteers and supports the use of BCG challenge as a tool for evaluating vaccine efficacy and identifying mechanisms of antimycobacterial immunity

Tools for Assessing the Protective Efficacy of TB Vaccines in Humans: in vitro Mycobacterial Growth Inhibition Predicts Outcome of in vivo Mycobacterial Infection.

Tuberculosis (TB) remains a leading global cause of morbidity and mortality and an effective new vaccine is urgently needed. A major barrier to the rational development of novel TB vaccines is the lack of a validated immune correlate or biomarker of protection. Mycobacterial Growth Inhibition Assays (MGIAs) provide an unbiased measure of ability to control mycobacterial growth in vitro, and may represent a functional correlate of protection. However, the biological relevance of any potential correlate can only be assessed by determining the association with in vivo protection from either a controlled mycobacterial infection or natural development of TB disease. Our data demonstrate that the direct MGIA using peripheral blood mononuclear cells (PBMC) is measuring a biologically relevant response that correlates with protection from in vivo human BCG infection across two independent cohorts. This is the first report of an MGIA correlating with in vivo protection in the species-of-interest, humans, and furthermore on a per-individual as well as per-group basis. Control of mycobacterial growth in the MGIA is associated with a range of immune parameters measured post-BCG infection in vivo including the IFN-γ ELISpot response, frequency of PPD-specific IFN-γ or TNF-α producing CD4+ T cells and frequency of specific sub-populations of polyfunctional CD4+ T cells. Distinct transcriptomic profiles are associated with good vs. poor mycobacterial control in the MGIA, with good controllers showing enrichment for gene sets associated with antigen processing/presentation and the IL-23 pathway, and poor controllers showing enrichment for hypoxia-related pathways. This study represents an important step toward biologically validating the direct PBMC MGIA for use in TB vaccine development and furthermore demonstrates the utility of this assay in determining relevant immune mechanisms and pathways of protection

Process of assay selection and optimization for the study of case and control samples from a phase IIb efficacy trial of a candidate tuberculosis vaccine, MVA85A.

The first phase IIb safety and efficacy trial of a new tuberculosis vaccine since that for BCG was completed in October 2012. BCG-vaccinated South African infants were randomized to receive modified vaccinia virus Ankara, expressing the Mycobacterium tuberculosis antigen 85A (MVA85A), or placebo. MVA85A did not significantly boost the protective effect of BCG. Cryopreserved samples provide a unique opportunity for investigating the correlates of the risk of tuberculosis disease in this population. Due to the limited amount of sample available from each infant, preliminary work was necessary to determine which assays and conditions give the most useful information. Peripheral blood mononuclear cells (PBMC) were stimulated with antigen 85A (Ag85A) and purified protein derivative from M. tuberculosis in an ex vivo gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) and a Ki67 proliferation assay. The effects of a 2-h or overnight rest of thawed PBMC on ELISpot responses and cell populations were determined. Both the ELISpot and Ki67 assays detected differences between the MVA85A and placebo groups, and the results correlated well. The cell numbers and ELISpot responses decreased significantly after an overnight rest, and surface flow cytometry showed a significant loss of CD4(+) and CD8(+) T cells. Of the infants tested, 50% had a positive ELISpot response to a single pool of flu, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) (FEC) peptides. This pilot work has been essential in determining the assays and conditions to be used in the correlate study. Moving forward, PBMC will be rested for 2 h before assay setup. The ELISpot assay, performed in duplicate, will be selected over the Ki67 assay, and further work is needed to evaluate the effect of high FEC responses on vaccine-induced immunity and susceptibility to tuberculosis disease

Single-Cell Transcriptomics of Regulatory T Cells Reveals Trajectories of Tissue Adaptation.

Non-lymphoid tissues (NLTs) harbor a pool of adaptive immune cells with largely unexplored phenotype and development. We used single-cell RNA-seq to characterize 35,000 CD4+ regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, their respective draining lymph nodes (LNs) and spleen. In these tissues, we identified Treg cell subpopulations with distinct degrees of NLT phenotype. Subpopulation pseudotime ordering and gene kinetics were consistent in recruitment to skin and colon, yet the initial NLT-priming in LNs and the final stages of NLT functional adaptation reflected tissue-specific differences. Predicted kinetics were recapitulated using an in vivo melanoma-induction model, validating key regulators and receptors. Finally, we profiled human blood and NLT Treg and Tmem cells, and identified cross-mammalian conserved tissue signatures. In summary, we describe the relationship between Treg cell heterogeneity and recruitment to NLTs through the combined use of computational prediction and in vivo validation

Cytomegalovirus infection is a risk factor for tuberculosis disease in infants.

Immune activation is associated with increased risk of tuberculosis (TB) disease in infants. We performed a case-control analysis to identify drivers of immune activation and disease risk. Among 49 infants who developed TB disease over the first 2 years of life, and 129 healthy matched controls, we found the cytomegalovirus-stimulated (CMV-stimulated) IFN-γ response to be associated with CD8+ T cell activation (Spearman's rho, P = 6 × 10-8). A CMV-specific IFN-γ response was also associated with increased risk of developing TB disease (conditional logistic regression; P = 0.043; OR, 2.2; 95% CI, 1.02-4.83) and shorter time to TB diagnosis (Log Rank Mantel-Cox, P = 0.037). CMV+ infants who developed TB disease had lower expression of NK cell-associated gene signatures and a lower frequency of CD3-CD4-CD8- lymphocytes. We identified transcriptional signatures predictive of TB disease risk among CMV ELISpot-positive (area under the receiver operating characteristic [AUROC], 0.98, accuracy, 92.57%) and -negative (AUROC, 0.9; accuracy, 79.3%) infants; the CMV- signature was validated in an independent infant study (AUROC, 0.71; accuracy, 63.9%). A 16-gene signature that previously identified adolescents at risk of developing TB disease did not accurately classify case and control infants in this study. Understanding the microbial drivers of T cell activation, such as CMV, could guide new strategies for prevention of TB disease in infants

The role of interleukin 33 in intestinal homeostasis

IL-33 is a pleiotropic cytokine that orchestrates both innate and adaptive immunity. It is commonly associated with type 2 immune responses but recently expression of the IL-33 receptor, ST2, was reported on Treg cells found preferentially in non-lymphoid tissues, such as the visceral adipose tissue, muscle or colon. A crucial role of Tregs in maintaining intestinal homeostasis has been well described. However, little is known about the functional relevance of the ST2-expressing Treg population in the colon. Phenotypic and functional characterisation of Tregs in the gut revealed the presence of two distinct populations: ST2+/Gata3+ and Ror&gamma;t+ Tregs. Thymic-derived ST2+/Gata3+ Tregs showed a more activated phenotype and produced IL-10 under homeostatic conditions. Upon microbial challenge and colitis, ST2+/Gata3+ Tregs were decreased, while Ror&gamma;t+ Tregs expanded. Furthermore, in vitro experiments demonstrated that IL-33 directly induced activation of the Gata3 pathway in Tregs, which enhanced expression of Foxp3 and ST2. Additionally, amphiregulin was also induced in Tregs upon stimulation with IL-33. However, in vivo, IL-33 was dispensable for both the maintenance of Treg cells under homeostatic conditions and Treg function in Helicobacter hepaticusâdriven colitis. Investigation of the negative regulators of IL-33 showed that IL-23 inhibited IL-33-mediated effects on Tregs. We also observed increased production of soluble ST2 by stromal cells during intestinal inflammation, which likely contributed to the reduction of IL-33 bioavailability. Finally, a systematic analysis of the cellular source of IL-33 revealed that PDGRF&alpha;+ stromal cells located in the T cell zone of secondary and tertiary lymphoid tissues were a major IL-33-producing cell population in the gut. Collectively, our findings suggest that signals received by the stromal compartment upon cell injury may trigger a specific phenotype of Tregs with a repair capacity, and thus, promote intestinal homeostasis. These findings improve our understanding of tissue-resident Tregs and open an exciting avenue to explore heterocellular signalling between stromal cells and Tregs.</p