NASAL KLEBSIELLA PNEUMONIAE IMMUNIZATION GENERATES HETEROGENEOUS POPULATIONS OF RESIDENT MEMORY B CELLS AND PLASMA CELLS IN THE LUNG

AMEZCUA VESELY MARIA CAROLINA; BERENSTEIN ARIEL; BRUNOTTO VALENTINA; ALMADA LAURA; GARECA JULIO; GAZZONI YAMILA; ACOSTA RODRIGUEZ EVA VIRGINIA; MONTES CAROLINA L; ADRIANA GRUPPI

Congreso; LXXII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI) & 3ER CONGRESO FRANCO-ARGENTINO DE INMUNOLOGÍA (FAIC); 2022

The development of a proper adaptive immune response against different microbes protects against future challenges through the generation of memory B and T cells. Memory immune responses can be induced locally or systemically after immunization or infection. Systemic immunizations generate circulatory memory B and T cells but local immunizations induce the generation of both circulatory and tissue resident memory T and B cells (TRM and BRM).The development of lung specific memory cells is crucial to quickly deal with respiratory tract infections. Previous results indicate that intranasal immunization with heat-killed Klebsiella pneumoniae (Kp) protects mice from infection through the generation and re-activation of CD4 TRM cells. Based on these results, our main goal was to characterize memory BRM after intranasal immunization. C57Bl/6 mice were intranasally immunized on day 0 and 7 with heat killed Kp. On day 30, we used an in-vivo labeling method by injecting intravenously (iv) anti-CD45-A700 antibody 5 minutes before mouse sacrifice and lung collection to exclude circulating immune cells from downstream methods. Then, we sorted lung CD45iv negative and CD4negative cells from naïve and immunized mice to perform single cell RNA-seq and SingleR to identify clusters of hematopoietic and non-hematopoietic cells. Among hematopoietic cells, we selected the B cell cluster using a single-cell-Gate tool to evaluate BRM heterogeneity. As expected, the number of lung infiltrating B cells was higher in immunized versus naïve mice. Immunized mice had heterogeneous populations of BRM based on cluster numbers (6 clusters immunized mice vs 3 clusters naïve mouse) and also an increase in IgA+ resident plasma cells compared with naïve mice. Preliminary evaluation using flow cytometry shows agreement with single cell RNA-seq data. In conclusion, we determined that intranasal Kp immunization generates different types of BRM whose specificity and function will be further evaluated.