Lung microenvironment effects on T cells following Francisella tularensis vaccination

Pulmonary infections often fail to produce long-lived immune memory and the underlying mechanism(s) remain unclear. We undertook a multiomics approach to compare adaptive immunity in two bacterial infections: Bordetella pertussis and Francisella tularensis. Both infections require CD4+ T cells, but B. pertussis results in long-lived memory responses whereas F. tularensis does not. Single cell RNA sequencing and flow cytometry revealed CD4+ T cells favored proliferation immediately after secondary B. pertussis infection but stagnated during F. tularensis challenge. Instead, rapid IFN-? production by F. tularensis-elicited T cells induced IDO1 expression in endothelial cells, depleting pulmonary tryptophan and increasing kynurenine. This metabolic shift correlated with poor T cell proliferation and retention. Additionally, IDO1 knock-out resulted in larger T cell pools and improved survival during F. tularensis infection. Imaging confirmed the spatial association of immune cell centers and IDO1-expressing macrovascular endothelial cells underscoring the microenvironment's impact on T cell function. Our study highlights the utility of multiomics in understanding cellular interactions within complex tissues. Overall design: To generate immune animals, mice were intranasally inoculated with 1x105 CFU Bp or 150 CFU of LVS in 25 µL PBS delivered to a single nare after anesthetizing with ketamine/xylazine. Where indicated, mice were intranasally challenged with 1x106 CFU Bp or 25 CFU of Ftt in 25 µL PBS to a single nare after being anesthetized with ketamine/xylazine. Single cell suspensions were generated by mincing and then digested with Liberase TM (Sigma-Aldrich) for 45 minutes at 37°C. Red blood cells were lysed with ACK lysis buffer (Life Technologies). A TC20 automated cell counter (Bio-Rad) was used to determine the total number of viable cells using trypan blue exclusion. Samples were processed through the 10X Genomics V3.1 processing and prep of single-cell libraries