Abstract Streptococcus pneumoniae serotype 19F (SP19F) infection induces lung-resident memory lymphocytes that confer immunity against mismatched pneumococcal serotypes. We previously observed that CD40L blockade when extravascular (EV) lymphocytes peak in the infected lung prevents the subsequent pool of resident memory B cells (BRM) that produce local heterotypic antibodies protective against infection. We aimed to determine how CD40L impacts lung BRM cells. Deficiency of CD40 decreased lung BRM accumulation, implicating this specific receptor. We observed that CD40 was expressed on many different cell-types in the infected lung. To identify which CD40+ cells were being activated by CD40L in the infected lung, we used Labelling Immune Partnerships by SorTagging Intercellular Contacts (LIPSTIC) mice. We instilled Sp19F into the left lung lobe of LIPSTIC mice at days 0 and 7. We delivered labeling substrate into the left lung for 2 hours at day 9 and performed flow cytometry to identify the CD40+ cells that had been activated then and there by CD40L. Significant proportions of EV lung B cells and dendritic cells (DC) were labeled for CD40-CD40L interaction in the lung at day 9, with means of 6.1% and 3.4% respectively. In contrast, circulating (intravascular) B cells as well as EV neutrophils and interstitial macrophages in the infected lung were not labeled. CD40L blockade in LIPSTIC mice resulted in complete loss of signal, indicating that this approach accurately labeled CD40-CD40L interactions. These data reveal that B cells and DCs undergo CD40-CD40L interaction in the lung during pneumococcus infection. To determine whether CD40 signaling in B cells specifically is driving BRM development, we employed CD19cre+ CD40fl/fl (B40) mic. At 35 dpi, the “experienced” B40 mice showed a significant reduction of lung BRM cells compared to experienced Cre- littermate controls. Spectral flow cytometry revealed a significant decrease in some sub-populations of lung BRM cells including IgM B1, IgM B2, and class-switched B2 cells. The experienced B40 mice showed a reduction in heterotypic IgG in the blood and heterotypic IgG and IgA in the BAL. Upon 4 days of infection with mismatched serotype of pneumococcus (SP3), experienced B40 mice had significantly greater weight loss and multi-log increases in lung and blood bacteria, compared to experienced Cre- littermates. We interpret these results to suggest that CD40L activation of B cell CD40 in the lungs helps drive the development of lung BRM cell populations that prevent severe pneumonia from heterotypic infections. This abstract is funded by: PI R01
Vannini et al. (Fri,) studied this question.