BCG induces CX3CR1hi effector memory T cells to provide cross-protection via IFN-y mediated trained immunity

After a century of the Bacillus Calmette-Guérin (BCG) vaccine, our understanding of its protection against homologous (Mycobacterium tuberculosis) or heterologous (e.g. influenza virus) infections is still limited. Here we show that systemic (intravenous) BCG vaccination (BCG-iv) provides significant protection against subsequent influenza A virus (IAV) infection in mice. We further demonstrate that the BCG-mediated cross-protection against IAV is largely due to the enrichment of conventional CD4+ αβ effector memory T cells that express high levels of CX3CR1hi in circulation trafficking into the lung parenchyma. Importantly, pulmonary CX3CR1hi T cells limit early viral infection in an antigen-independent manner via potent IFNγ production, which subsequently enhances long-term antimicrobial activity of the innate immune system like alveolar macrophages. Similarly, we uncover a prominent IFNγ signature in which its increased basal production was associated with enhanced BCG-mediated heterologous innate memory responses in BCG-vaccinated humans. These results offer insight into the unknown mechanism by which BCG has persistently displayed broad protection against non-tuberculous infections via a crosstalk between adaptive and innate memory responses. BCG vaccination administered intravenously generated a significant enrichment of effector memory T cells chracterized by their CX3CR1hi surface marker expression. Our data provides evidence that the expansion of these cells is key in the subsequent cross-protection between BCG vaccination and influenza infection. The increase in numbers of these cells in BCG-iv mice is evident, however asssessing the transcriptional profile of these cells can reveal any functional signature of the induction of memory T cells by BCG. We thus vaccinated mice with BCG-iv in paralell with PBS controls and after 28 days, purified CX3CR1lo and CX3CR1hi splenic T cells. We subjected the cells to Bulk-RNAseq and assessed the transcriptional landscape.