SRP469438 PRJNA1034093 Marginal zone B cells protect against TB by shaping the cytokine pattern. Exploring the role of B cells in tuberculosis (TB) is important for developing new TB vaccines. To investigate the immune landscape of B cells in TB, BALB/c mice were infected with 100-200 colony-forming units of Mycobacterium tuberculosis (Mtb) through aerosol inhalation to serve as an animal model. Subsequently, immune landscapes of B cells in the lungs and spleen were analyzed using flow cytometry.Our results showed that marginal zone B (MZB) cells expand in the lungs and spleen in response to infection. Importantly, splenic MZB cells systemically protect against infection by shaping the cytokine pattern to create an anti-tuberculosis environment. Both pulmonary and splenic MZB cells produced multiple cytokines against Mtb directly. Our results highlight that, beyond the conventional focus on antibody production, targeting multiple-cytokine production of B cells can be a valuable strategy for developing new TB vaccines.In response to infection, pulmonary and splenic MZB cells displayed distinct characteristics from follicular B (FoB) cells. These MZB cells exhibited an activated and memory-like phenotype and produced multiple cytokines. To compare RNA signatures between MZB and FoB cells, we sorted MZB (live, CD45+CD93-B200+CD1dhiCD23-) and FoB (live, CD45+CD93-B200+CD1dmiCD23+) cells from the lungs and spleen of infected mice at 12-weeks post-infection. MZB and FoB cells from uninfected mice served as uninfected controls. Sorted B cells were preserved in TRIzol (Thermo Fisher Scientific). Total RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform method and cleaned up using RNeasy Micro kit (Qiagen). The extracted RNA quality was evaluated on the Bioanalyzer (Agilent) with RNA Integrity Number (RIN) ranging from 7.4 to 9.9. cDNA libraries were prepared from 2 ng of RNA using the Smart-seq2 protocol (S Picelli et al., 2014), modified by adding 20 micromolar Template Switching Oligo and utilizing 200 pg of cDNA with one-fifth of a reaction of the Illumina Nextera XT Kit (Illumina). The length distribution of the cDNA libraries was measured using DNA High Sensitivity Reagent Kit on the Labchip system (PerkinElmer). All samples underwent an indexed paired-end sequencing run of 2 x 151 cycles on the HiSeq 4000 system (Illumina), with each accommodating 24 samples. The results indicate that pulmonary and splenic MZB cells share a similar RNA signature but differ from their FoB counterparts.