Innate response activator (IRA) B cells are GM-CSF-producing B cells that increase in number during inflammation. (A) Quantification of GM-CSF-producing cells retrieved from tissues in the steady state and in response to 4 daily i.p. injections of LPS (means ± SEM, n = 3–5). *P < 0.05. (B) Identification of GM-CSF-producing cells in the spleen. Representative plots show percentage of B cells and their production of GM-CSF retrieved from spleens during inflammation. Data represent at least ten independent experiments. (C) Western blot for GM-CSF conducted on sorted cells. One of three independent experiments is shown. (D) Co-localization of representative GM-CSF-producing cells with IgM. (E) Red pulp sections with markers against CD11b (green) and GM-CSF (red) (left panel) and B220 (green) and GM-CSF (red) (right panel). Co-localization of green and red cells is yellow and the scale bar is shown in white. (F) Quantification of GM-CSF⁺ B cells and other cells on histological sections of the spleen in the red pulp and white pulp in the steady state and after LPS (means ± SEM, n = 3–4). *P < 0.05. (G) Splenic GM-CSF expression detected by RT-PCR and conducted on sorted cells and on unprocessed spleen tissue taken from wild type and B cell knockout (μMT) mice (means ± SEM, n = 3–4). *P < 0.05.

IRA-B cells are a distinct subset with a unique phenotypic signature. (A) Flow cytometric analysis of the phenotype of IRA-B cells. Plots show B cell phenotypes retrieved from spleens during steady state and inflammation. Representative from n > 10 is shown. (B) Plots show the phenotype of GM-CSF-producing cells in the spleens during inflammation. IRA-B cells are IgM^high, CD23^low CD43⁺ CD93⁺. (C) Plots show the phenotype of IRA-B cells with respect to VLA4 and CD138 expression as determined by flow cytometry. Representative from n > 5 is shown. (D) Hierarchical clustering dendrogram based on whole-genome microarray data of sorted samples of B cell subsets retrieved from LPS-treated animals and steady-state B1a. (E) Principal Component Analysis (PCA) of the different cell subsets shown in (D).

IRA-B cells develop from B1a B cells via TLR4/MyD88 and reside in tissue through LFA-1/VLA-4. (A) Flow cytometric analysis of the percent chimerism is shown in spleens of CD45.1⁺ mice that had been in parabiosis with CD45.2⁺ mice for 3 weeks prior to LPS injection. Mice were sacrificed 2 days after LPS injection. Representative plots from two independent experiments are shown. (B) Adoptive transfer of peritoneal B1a B cells yields IRA-B cells. Cells from steady state CD45.2⁺ mice were transferred to CD45.1⁺ mice that then received LPS for 3 days. Animals were analyzed 72 hours after transfer. Representative plots from flow cytometric analysis of n = 4–5 mice are shown. (C) Flow cytometric analysis of the development of IRA-B cells in Tlr4^−/−, Myd88^−/−, Ticam1^−/− (the gene the encodes TRIF), μMT, Tnfrsf13c^−/− (the gene that encodes BAFFR), and Cd19^−/− mice. Representative plots from n = 4 mice are shown. (D) Enumeration of IRA-B cells in steady state and inflammation in wt (C57BL/6) mice and in the mice shown in (D) (means ± SEM, n = 4–10). *P < 0.05. (E) Flow cytometric analysis of the adoptive transfer of CD45.1⁺ B1a cells into congenic Tlr4^−/− CD45.2⁺ mice injected with LPS. Representative from n = 3 mice is shown. (F) Flow cytometric analysis of the effect of blocking VLA-4/LFA-1 on IRA-B cell retention in the spleen. Representative from n = 3 mice is shown.

IRA-B cells protect against polymicrobial sepsis. (A) Generation of mixed chimeras (GM/μMT). (B) Kaplan-Meier curve showing survival of GM/μMT and control animals after cecal ligation and puncture (CLP). n = 10–20/group. (C) Enumeration of total leukocytes and neutrophils in the peritoneum of GM/μMT (dark red) and control (black bars) mice 20 h after CLP. (D) Serum levels and (E) peritoneal levels of inflammatory cytokines in GM/μMT (dark red) and control (black bars) mice 20 h after CLP. (F) Ex vivo phagocytosis assay showing capacity of neutrophils to phagocytose E. coli from GM/μMT (dark red) and control (black bars) mice 20 h after CLP. (G) serum levels of IgM and IgG 20 h after CLP in same groups as above. (H) Representative H&E stain of liver and lung sections 20 h after CLP in same groups as above. (I) Blood from GM/μMT and control mice 20 h after CLP was plated for 1 day. Representative plate shows bacterial colonies. (J) Enumeration of bacteremia in the peritoneum and blood of GM/μMT (dark red) and control (black bars) mice 20 h after CLP. *P < 0.05 [means ± SEM, n = 10–20/group for (C)–(G), (J). Four independent experiments were performed and data were grouped].