LETTER doi:10.1038/nature10698 Acquisition of a multifunctional IgA 1 plasma cell phenotype in the gut Jo ¨rg H. Fritz 1 {*, Olga Lucia Rojas 1 *, Nathalie Simard 1,2 , Douglas D. McCarthy 1 , Siegfried Hapfelmeier 3 {, Stephen Rubino 4 , Susan J. Robertson 1 , Mani Larijani 1 {, Jean Gosselin 5 , Ivaylo I. Ivanov 6 , Alberto Martin 1 , Rafael Casellas 7 , Dana J. Philpott 1 , Stephen E. Girardin 4 , Kathy D. McCoy 3 , Andrew J. Macpherson 3 , Christopher J. Paige 1,2 & Jennifer L. Gommerman 1 The largest mucosal surface in the body is in the gastrointestinal tract, a location that is heavily colonized by microbes that are normally harmless. A key mechanism required for maintaining a homeostatic balance between this microbial burden and the lymphocytes that densely populate the gastrointestinal tract is the production and transepithelial transport of poly-reactive IgA (ref. 1). Within the mucosal tissues, B cells respond to cytokines, sometimes in the absence of T-cell help, undergo class switch recombination of their immunoglobulin receptor to IgA, and dif- ferentiate to become plasma cells 2 . However, IgA-secreting plasma cells probably have additional attributes that are needed for coping with the tremendous bacterial load in the gastrointestinal tract. Here we report that mouse IgA 1 plasma cells also produce the antimicrobial mediators tumour-necrosis factor-a (TNF-a) and inducible nitric oxide synthase (iNOS), and express many molecules that are commonly associated with monocyte/granulocytic cell types. The development of iNOS-producing IgA 1 plasma cells can be recapitulated in vitro in the presence of gut stroma, and the acquisition of this multifunctional phenotype in vivo and in vitro relies on microbial co-stimulation. Deletion of TNF-a and iNOS in B-lineage cells resulted in a reduction in IgA production, altered diversification of the gut microbiota and poor clearance of a gut-tropic pathogen. These findings reveal a novel adaptation to maintaining homeostasis in the gut, and extend the repertoire of protective responses exhibited by some B-lineage cells. Most class switch recombination (CSR) to IgA takes place in the Peyer’s patches and requires encounters between B cells and cytokine- secreting T cells within germinal centres. However, IgA CSR can also take place outside of Peyer’s patches within isolated lymphoid follicles of the lamina propria 2 . Local production of nitric oxide (NO) via the inducible nitric oxide synthase (NOS2 or iNOS) has been shown to be a critical mediator of CSR to IgA within the small intestinal lamina propria of mice 3,4 . Because lymphotoxin-deficient mice have a signifi- cant, unexplained IgA defect 5 , we hypothesized that this could be due to a lack of iNOS in the gut. Indeed, we identified a population of CD11c lo iNOS 1 cells by flow cytometric analysis of intestinal lamina propria cell preparations, and these CD11c lo iNOS 1 cells were decreased in both lymphotoxin-b-deficient and lymphotoxin-b-receptor-deficient mice (Ltb 2/2 and Ltbr 2/2 , respectively; Fig. 1a and Supplementary Table 1), confirming a relationship between iNOS and the generation/ maintenance of IgA 1 plasma cells 4 . The absence of iNOS-expressing cells in lymphotoxin-deficient mice prompted us to ask whether B-lineage cells could influence the expres- sion of iNOS within the gut, as lymphotoxin-deficient mice lack some B-cell subsets in the small intestinal lamina propria 5 . Accordingly, we examined B-cell-deficient mice for evidence of iNOS expression in the gut and found that CD11c lo iNOS 1 cells were absent in JH 2/2 mice, Rag2 2/2 mice and strongly reduced in mMt mice (Fig. 1b, Supplementary Table 1 and Supplementary Fig. 1a). Because lymphotoxin-deficient mice also lack IgA 1 plasma cells, and iNOS-expressing cells are strongly reduced in B-cell-deficient and lymphotoxin-deficient mice, we examined the possibility that IgA 1 plasma cells may have the capacity to produce iNOS. Indeed, when we gated on iNOS 1 cells, we found that they expressed IgA (Fig. 1a) and low levels of B220 (not shown), indicating that IgA-producing cells may account for significant iNOS expression within the gut in the steady state. In agreement with this result, we found that purified CD11c lo iNOS 1 cells exhibited evidence of a rearranged V-D-JH4 product (Supplementary Fig. 1b). We next asked what proportion of IgA 1 cells express iNOS. Compared to knockout controls, a subset of IgA 1 plasma cells expresses iNOS and TNF-a, the expression of which has also been described in monocyte-derived cells 6 (see Fig. 1c and Supplementary Table 2 for relative frequencies of each population and Fig. 1e for immunofluorescence microscopy). We further confirmed these observations by performing cytospins on small intestinal lamina- propria-derived cells (Supplementary Figs 2 and 3). Collectively, these data indicate that gut-resident B-lineage cells contribute towards iNOS and TNF-a expression within the small intestinal lamina propria. To determine better which B-lineage cells were expressing TNF-a/ iNOS, we examined whether previous expression of activation induced cytidine deaminase (AID), an enzyme that is required for both CSR and somatic hypermutation in B cells 7 , correlated with iNOS and TNF- a expression. Using AID-Cre 3 YFP mice 8 , we observed three popula- tions of cells in the small intestinal lamina propria: (1) YFP 2 IgA 2 cells , that is, any lamina-propria-derived cells that have not undergone CSR nor somatic hypermutation, such as a dendritic cells or naive B cells; (2) YFP 1 IgA 2 cells, that is, B cells that have undertaken somatic hypermutation or have undergone CSR to a class of immunoglobulin other than IgA; and (3) YFP 1 IgA 1 cells, that is, B cells that have undergone CSR to IgA (Fig. 1d and Supplementary Table 3 for relative frequencies of each population). Confirming that YFP 1 IgA 1 cells probably represent IgA 1 plasma cells, YFP 1 IgA 1 cells also expressed low levels of the plasma cell marker syndecan-1 (CD138) and were CD19 low (compare YFP 1 IgA 1 (red trace) with YFP 1 IgA 2 B cells (blue trace), which are CD138 2 CD19 hi ; Fig. 1d). Using this approach, we found that some YFP 1 IgA 1 cells (red box) expressed iNOS as well as TNF-a (Fig. 1d and Supplementary Table 4 for the relative expres- sion of TNF-a/iNOS for each population). Furthermore, YFP 1 IgA 1 cells also expressed leukocyte surface markers commonly associated *These authors contributed equally to this work. 1 Department of Immunology, University of Toronto, Toronto M5S 1A8, Canada. 2 Ontario Cancer Institute, Princess Margaret Hospital, University Health Network, Toronto M5G 2M9, Canada. 3 University of Bern, Department Klinische Forschung (Gastroenterologie), Mutenstrasse 35, 3010 Bern, Switzerland. 4 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto M5S 1A8, Canada. 5 Department of Molecular Medicine, University of Laval, Quebec G1V 4G2, Canada. 6 Department of Microbiology and Immunology, Columbia University Medical Center, College of Physicians and Surgeons, New York, New York 10032, USA. 7 Genomics and Immunity, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. {Present addresses: Complex Traits Group, Department of Microbiology and Immunology, McGill University, Montreal H3G 0B1, Canada (J.H.F.); University of Bern, Institute of Infectious Disease, Friedbu ¨ hlstrasse 51, 3010 Bern, Switzerland (S.H.); Division of Biomedical Sciences, Faculty of Medicine, Memorial University of Newfoundland, St John’s A1B 3V6, Canada (M.L.). 12 JANUARY 2012 | VOL 481 | NATURE | 199 Macmillan Publishers Limited. 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