C106-12 Characterising Lung Mucosal and Systemic Immune Response in Human Following Inhalation of Attenuated Mycobacterium Bovis BCG Using Single Cell Sequencing

Abstract Rationale The development of tuberculosis (TB) vaccines is hampered by a limited understanding of the human host-pathogen interactions, the uncertain predictive value of animal models, and the absence of validated immune correlates of protection. The aerosol BCG human challenge model enables the investigation of immune responses to mycobacterial infection at defined timepoints. Methods Healthy, BCG-naïve UK adults received a 1x10⁷ CFU BCG Danish 1331 or saline aerosol infectious challenge. Bronchoalveolar lavage (BAL) samples were collected from volunteers in five groups on Days 2, 7, 14, 28, and 56, respectively. Peripheral blood mononuclear cells (PBMC) were collected at various times from the same volunteers. Samples underwent single-cell RNA sequencing and T-cell receptor (TCR) sequencing. TCR repertoires in PBMC were also profiled using bulk TCR sequencing. The trial was registered at ClinicalTrials. gov, NCT04777721. Results The activation of myeloid cells in PBMC post aerosol BCG challenge was more transient compared to myeloid cell activation in the lung mucosa. BCG-expanded T cells were identified through comparing the frequency of a TCR in PBMCs on D7 post-aerosol BCG challenge and that on D0. The frequency of BCG-expanded T cells was highly correlated with the magnitude of IFNg+ T cell in PBMCs measured by Ex vivo enzyme-linked immunospot (ELISpot) assay, suggesting they were highly likely to be BCG-specific. Most Purified protein derivative (PPD) -reactive T cells detected on D7 in PBMCs were not BCG-expanded and did not enrich in the lung mucosa. Most BCG-expanded T cells in both the lung mucosa and PBMCs can be detected before aerosol BCG challenge and thus were pre-existing. These pre-existing BCG-expanded T cells were more likely to be activated CD4+ T cells and enriched in the lung mucosa compared to BCG-expanded T cells detected exclusively post BCG challenge. BCG-expanded activated CD4+ T cells in PBMCs were more likely to enrich in the lung mucosa compared with BCG-expanded regulatory CD4+ T cells. BCG-expanded TCR clusters, which contains highly similar BCG-expanded TCRs, were identified. TCRs from clusters that included TCRs from different volunteers—one of which contained TCRs from over half of the BCG-challenged participants—were shown to be reactive to PPD. This was demonstrated using monocytes-differentiated dendritic cells from the volunteers who had the TCRs and reporter T cell lines that expressed these TCRs exogenously. Discussion Human infection model provides unique insights into the dynamics of human mucosal and systemic immunity following mycobacterial infection, informing the design of more effective TB vaccines. This abstract is funded by: Wellcome Trust; National Institute for Health and Care Research (NIHR) Oxford Biomedical Research Centre (BRC)