Immunoglobulin A (IgA) is prominently secreted at mucosal surfaces and coats a fraction of the intestinal microbiota. However, the commensal bacteria bound by IgA are poorly characterized and the type of humoral immunity they elicit remains elusive. We used bacterial flow cytometry coupled with 16S rRNA gene sequencing (IgA-Seq) in murine models of immunodeficiency to identify IgA-bound bacteria and elucidate mechanisms of commensal IgA targeting. We found that residence in the small intestine, rather than bacterial identity, dictated induction of specific IgA. Most commensals elicited strong T-independent (TI) responses that originated from the orphan B1b lineage and from B2 cells, but excluded natural antibacterial B1a specificities. Atypical commensals including segmented filamentous bacteria and Mucispirillum evaded TI responses but elicited T-dependent IgA. These data demonstrate exquisite targeting of distinct commensal bacteria by multiple layers of humoral immunity and reveal a specialized function of the B1b lineage in TI mucosal IgA responses.

To characterize the commensal bacterial targets of IgA, we utilized bacterial flow cytometry coupled with 16S rRNA gene sequencing (IgA-Seq) (). We found that IgA coated many but not all commensals in the homeostatic state and that dramatic differences were associated with bacterial localization along the gastrointestinal tract. Using murine genetic models of immunodeficiency, we found that most IgA-bound taxa were specifically targeted by TI IgA. We further demonstrated that natural antibacterial B1a specificities did not contribute to IgA coating. In contrast, innate B1b—a phenotypically related but poorly understood, “orphan” lineage—and adaptive B2 B cells each contributed diverse commensal-reactive specificities. Finally, we identified an atypical subset of commensals that evaded TI responses but elicited TD IgA. Together, these data indicate that multiple layers of humoral immunity are elicited by distinct commensal bacteria in the small intestine and reveal a novel specialization for the B1b lineage in mucosal TI responses.

While TI antigens can stimulate circulating follicular B2 B cells, they can also activate innate B1 B cells that reside primarily in the peritoneal cavity (). In contrast, TD responses are thought to predominantly involve B2 B cells. Both B1 and B2 B cells can differentiate into intestinal IgAplasma cells, although the relative contributions of these lineages remain controversial (). Two subsets of B1 B cells, B1a and B1b, are present in the peritoneal cavity. Although limited data suggest differential capacity of B1a and B1b to undergo IgA class-switch recombination (), it is not known whether both subsets coat commensal bacteria in vivo. Peritoneal B1a secrete “natural” antibodies that react with conserved microbial antigens and have been hypothesized to contribute to control of the microbiota (). In contrast, very little is known about the role of B1b except that they can generate protective TI responses against Borrelia hermsii and Salmonella typhimurium outer membrane proteins and Streptococcus pneumoniae capsular polysaccharides after systemic infection ().

B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae.

Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity.

An intrinsic propensity of murine peritoneal B1b cells to switch to IgA in presence of TGF-β and retinoic acid.

Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity.

Mucosal IgAplasma cells can be generated by both T-dependent (TD) and T-independent (TI) mechanisms. However, the relative contributions of each pathway remain unclear. TD responses are typically directed against protein antigens and occur in gut-associated lymphoid tissues including Peyer’s patches (PPs) and mesenteric lymph nodes (mLNs), where germinal centers (GCs) are constitutively active. TD responses require signals from CD4T follicular helper (Tfh) cells that direct the selection and differentiation of high affinity GC B cells into long-lived plasma cells. In contrast, TI responses may occur both in organized lymphoid tissues and in non-lymphoid tissues (). In both TD and TI pathways, factors in the intestinal microenvironment such as transforming growth factor β (TGF-β), interleukin-10 (IL-10), and retinoic acid direct class switch recombination to the IgA isotype (). TI IgA responses may produce primarily “natural,” polyreactive specificities with low affinity for commensal bacteria (), but have been demonstrated against a limited number of commensal model antigens (). Thus, although protective immune responses to many enteric pathogens are TD (), it is unclear whether IgA coating of commensal bacteria is more dependent on TD or TI responses.

Host-commensal symbiosis is mediated at mucosal surfaces by secreted host-derived factors including mucus, antimicrobial peptides, and immunoglobulin A (IgA) (). Mammals invest significant resources into IgA production: more than 80% of all human plasma cells secrete IgA and reside in the intestinal lamina propria. IgA can mediate protective immunity to enteric pathogens including viruses, bacteria, and toxins (). However, IgA also contributes to intestinal homeostasis. Mice and humans with defective IgA secretion show increased susceptibility to inflammatory bowel disease, celiac disease, and allergy (). IgA may regulate commensal community composition, gene expression, and motility, which in turn influence host epithelial physiology and innate immunity (). Notably, IgA coating of commensal bacteria can be detected by flow cytometric and microscopic analysis of fecal samples from healthy mice and humans (). However, the commensal bacteria bound by IgA are poorly characterized and the mechanisms by which they induce specific IgA are unclear.

The amount of secreted IgA may not determine the secretory IgA coating ratio of gastrointestinal bacteria.

Development of a method for the identification of S-IgA-coated bacterial composition in mouse and human feces.

TI IgA responses might give rise to polyreactive specificities (). Further, IgA might interact with bacteria nonspecifically via the IgA fragment crystallizable (Fc) region or secretory component (). We generated recombinant monoclonal antibodies from single Tcrbsmall intestinal IgAplasma cells and engineered them to express the human IgG1 Fc instead of mouse IgA Fc. Five out of five antibodies recognized Rag1small intestinal or colonic commensal bacteria ( Figure 7 ). Each antibody bound to a discrete bacterial subset and their mixture stained an even greater fraction of bacteria, revealing distinct specificities. Recognition was specific, as none of the antibodies reacted with the colonic commensal Bacteroides fragilis ( Figure 7 ).

Staining of total small intestinal or colonic bacteria from Rag1 −/− mice or pure cultures of Bacteroides fragilis with indicated recombinant monoclonal antibodies derived from single Tcrb −/− d −/− small intestinal IgA + plasma cells and engineered to express human IgG1 Fc instead of mouse IgA Fc Staining was performed using supernatants from transduced HEK293T cells expressing indicated antibody constructs. Untransfected supernatant was used as a negative control. Data are representative of two independent experiments.

To identify commensal bacteria bound by B1b or B2-derived IgA, we performed IgA-Seq on colonic samples from Rag1mice reconstituted with B1 or B2 B cells. This analysis revealed that TI B1b and B2 B cells were each sufficient to coat a diverse array of commensal bacteria ( Figure 6 ). B1b and B2 coated overlapping bacterial taxa and there were no apparent differences in efficacy of coating certain taxa by either subset ( Figure 6 ). This is consistent with the observation that both B1b and B2 B cells possess broad and diverse immunoglobulin repertoires ( Figure 5 D). Addition of T cells did not alter the taxa targeted by B1b or B2 B cells, further supporting the conclusion that most commensal bacteria primarily induce TI IgA ( Figure 6 ).

Fold enrichment of indicated taxa in colonic IgAfractions of Rag1mice reconstituted with sorted B cell populations, as described in Figure 5 . n = 3 each group of mice, compiled from two independent experiments. Error bars indicate SE.

We next assessed the ability of peritoneal B1a and B1b subsets to differentiate into IgAplasma cells by i.p. transfer of sorted B1a or B1b into Rag1recipients. While B1b B cells gave rise to a substantial population of IgAplasma cells and coated commensal bacteria, B1a B cells did not differentiate into IgAplasma cells ( Figures 5 A–5C). We verified that both B1a and B1b B cells stably reconstituted the peritoneal cavity of recipient mice ( Figure S5 ). To further assess whether B1a B cells contribute to the IgAplasma cell pool, we performed immunoglobulin repertoire sequencing of peritoneal B1a and B1b B cells, splenic B2 B cells, WT IgAplasma cells, and TcrbIgAplasma cells. The B1a B cell repertoire is enriched in canonical rearrangements of the V11 gene family that encode specificities toward conserved microbial antigens such as phosphorylcholine (). Indeed, we found that the B1a repertoire was partially restricted and that approximately 8% of sequences were of the V11 gene family ( Figures 5 D and 5E). The B1a repertoire also contained prominent clonal populations with conserved CDR3 sequences representing the V11, V1-55, and V1-9 gene families ( Figures 5 D and 5E). In contrast, V11 gene segments made up a negligible fraction of the repertoires of splenic B2, peritoneal B1b, WT IgAplasma cells, or TcrbIgAplasma cells and canonical B1a CDR3s were completely absent ( Figures 5 D and 5E). The peritoneal B1b repertoire was broad and of comparable diversity to that of splenic B2 B cells with little evidence of clonal expansion or conserved immunoglobulin rearrangements. Together, these data suggest that B1a B cells do not contribute to the IgAplasma cell repertoire or IgA coating of commensals. We conclude that commensal-specific IgA is derived from B1b and B2 B cell precursor populations, although the precise contributions of each subset remain unclear due to limitations in cell transfer studies and a lack of genetic tools to dissect these responses in intact mice.

Analysis of B1 B cells is complicated by a lack of genetic tools, such as fate-mapping models, to study these cells in vivo. Moreover, genetic alterations that disrupt B1 lineage development involve BCR signaling-related molecules that also disrupt the activation and function of B2 B cells (). Therefore, to assess whether B1 or B2 B cells were sufficient to generate commensal-specific IgA, we reconstituted immunodeficient Rag1mice with exclusively B1 or B2 B cells by transferring pure sorted populations. We initially established groups of Rag1recipients reconstituted by: (1) intraperitoneal (i.p.) transfer of B1 B cells, (2) i.p. B1 B cells + splenic CD4and CD8T cells (B1+T), (3) intravenous (i.v.) transfer of B2 B cells, and (4) i.v. B2 B cells + splenic CD4and CD8T cells (B2+T). We readily detected IgAplasma cells and bacterial IgA coating in mice reconstituted with either B1 or B2 B cells ( Figures 5 A–5C). Addition of T cells did not affect B1 differentiation into IgAplasma cells but did induce free IgA, albeit at low titers ( Figures 5 A–5C). In contrast, T cells markedly increased the number of B2 B cell-derived IgAplasma cells and supported generation of high titers of free IgA ( Figures 5 A–5C). Thus, TI B1 B cells and TI and TD B2 B cells can coat commensal bacteria while free IgA is predominantly derived from TD B2 B cells.

(E) Frequency of V11 gene segments within the populations shown in (D) or number of sequences containing the indicated canonical B1a CDR3′s. See also Figure S5

(D) Immunoglobulin heavy chain repertoire sequencing of indicated populations. Tree plots are shown—each shape indicates a unique IgH CDR3 and size is scaled to relative clonal abundance.

(A) Representative staining and (B) absolute numbers of small intestinal or colonic IgA + plasma cells recovered from Rag1 −/− mice that received the indicated sorted populations. B1 populations were transferred i.p. (500,000 B1, 1,000,000 CD4/CD8 T cells, or 250,000 B1a or B1b) and B2 populations were transferred i.v. (1,000,000 B2, 1,000,000 CD4/CD8 T cells). Mice were analyzed 5 weeks after transfer. Data compiled from six independent experiments and two separate sorts for each transferred population.

IgAplasma cells can derive from both B1 and B2 B cell precursors, although the relative contributions of these lineages remain controversial (). Limited evidence based on mixed bone marrow-peritoneal cell chimeric mice suggest that many IgAplasma cells in Tcrbmice might be of B1 origin (). However, it is unclear whether both B1 and B2 B cells give rise to commensal-specific IgA and whether these lineages contribute differentially to IgA coating of certain commensal taxa.

Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity.

As a complementary approach, we analyzed IgA coating in a model of Tfh hyper-sufficiency. Our laboratory recently identified the E3 ubiquitin ligase Cullin-3 (CUL3) as a co-repressor that complexes with BCL-6 and limits Tfh differentiation (). Conditional deletion of CUL3 in T cells (CD4-Cre Cul3; Cul3) results in mLN hyperplasia driven by spontaneous, cell-intrinsic, antigen-specific expansion of Tfh and T follicular regulatory (Tfr) cells in the absence of observable pathology (). This expansion drove a 10- to 15-fold increase in mLN GC B cells and B220IgAB cells ( Figure S4 A) and a corresponding increase in B220IgAplasma cells in the proximal small intestinal lamina propria ( Figure S4 B). Cul3mice had an increased frequency of IgAbacteria compared to co-housed littermate controls ( Figure S4 C). Increased IgA coating was clearly driven by exaggerated TD responses against a single taxon, S24-7 ( Figure S4 D). Cul3mice also showed a notable absence of Mycoplasmataceae, which were abundant in littermate controls ( Figure S4 D). S24-7 did not require T cells for IgA targeting, as its relative enrichment in the IgAfraction was not altered in Tcrbmice ( Figures 3 F and S2 B). These data suggest that S24-7 can induce TD responses but, unlike SFB and Mucispirillum, does not require TD specificities to become IgA

Together, these data indicate that GCs and SHM are dispensable for IgA coating of commensal bacteria and support the hypothesis that commensal-specific IgA is primarily TI.

To identify bacteria coated in the absence of SHM, we performed IgM-Seq on Aicdamice and IgA-Seq on co-housed Aicdalittermate controls ( Figure S3 B). IgMbacteria in Aicdamice were identical to IgAbacteria in controls and all taxa were equally enriched in the IgMfraction of Aicdamice and the IgAfraction of controls ( Figure 4 F). SFB and Mucispirillum were both strongly IgMin Aicdamice, further suggesting that coating of these taxa is TD but independent of SHM.

As a second approach, we examined bacterial coating in mice lacking activation-induced cytidine deaminase (AID; encoded by Aicda). AID is required for somatic hypermutation (SHM) and class-switch recombination and thus Aicdamice produce unmutated antibodies of the IgM isotype (). We observed Tfh and GCs in Aicdamice ( Figure 4 D), and Aicdamice had B220IgMbut not B220IgAplasma cells in their small intestinal lamina propria ( Figure 4 D), as reported previously (). As expected, we found no IgAbacteria in Aicdamice ( Figure 1 A). Instead, we readily detected IgMbacteria in Aicdamice, but not in Aicdaor Rag1controls ( Figure S3 A). The frequency of IgMbacteria in Aicdamice was identical to the frequency of IgAbacteria in Aicdalittermate controls ( Figure 4 E).

IgA-Seq revealed that all IgAcommensal bacteria induced potent GC-independent IgA responses ( Figure 4 C) and that IgAbacteria in littermate controls were equally enriched in the IgAfraction of co-housed Bcl-6mice. We found that SFB and Mucispirillum were IgAin Bcl-6mice, suggesting that coating these bacteria is TD but GC-independent. These data support the conclusion that IgAcommensal bacteria prominently induce TI responses.

We first examined IgA responses in CD4-Cre Bcl-6(Bcl-6) mice, in which conditional deletion of the transcription factor BCL-6 in T cells prevents Tfh differentiation and GC formation (). Bcl-6mice lacked GCs and Tfh but had normal numbers of B220IgAB cells in mLNs and PPs ( Figure 4 A). Bcl-6small intestinal IgAplasma cell numbers were reduced 3-fold compared to controls ( Figure 4 A). Similar to Tcrbmice, bacterial IgA coating in Bcl-6mice was identical to controls and no differences in staining intensity were apparent ( Figure 4 B).

(F) Fold enrichment of indicated taxa in colonic IgMfraction of Aicdamice or IgAfraction of Aicdamice. n = 5 each genotype. Error bars indicate SE. See also Figures S3 and S4

(D) Absolute numbers of indicated populations in the mLN, PP, or small intestinal lamina propria of Aicda −/− mice or Aicda +/− littermate controls. Data compiled from two independent experiments.

(C) Fold enrichment of indicated taxa in colonic IgA + fraction of Bcl-6 ΔT mice or co-housed littermate controls. n = 3 each genotype, representative of two independent experiments. Error bars indicate SE.

(A) Absolute numbers of indicated populations in the mLN, PP, or small intestinal lamina propria of CD4-Cre Bcl-6 fl/fl mice or littermate controls. Data compiled from three independent experiments.

Insights into the role of Bcl6 in follicular Th cells using a new conditional mutant mouse model.

We found prominent TI IgA coating in Tcrb −/− d −/− mice, but it was possible that these mice had defects related to the absence of T cells but unrelated to TD IgA. Therefore, we sought to validate these observations by examining bacterial coating in two additional models.

Together, these data suggest that TD and TI responses might coat non-overlapping commensal bacterial taxa. While most IgA + bacteria induce strong TI responses, atypical commensals such as SFB and Mucispirillum exclusively elicit TD responses.

We identified two taxa, segmented filamentous bacteria (SFB) and Mucispirillum, that were absent from the IgAfraction of Tcrbmice but enriched in the IgAfraction of Tcrblittermate controls ( Figures 3 F and S2 B). This pattern was observed in both colonic and jejunual samples ( Figures 3 F and S2 B). Thus, TD specificities appear necessary for IgA coating of these taxa. Notably, both SFB and Mucispirillum interact closely with the intestinal epithelium in the terminal ileum (). We hypothesize that SFB and Mucispirillum might possess atypical cell wall structures that poorly stimulate TI responses and might therefore come into close contact with the mucosa, allowing sampling by antigen-presenting cells and priming of TD IgA responses.

To identify commensal bacteria targeted by TI IgA, we performed IgA-Seq on colonic and jejunal samples from Tcrbmice and co-housed littermate controls. Co-housed Tcrbmice and Tcrblittermates displayed largely overlapping small intestinal and colonic microbial communities ( Figure S2 B). Most IgAbacteria found in controls were equally enriched in the IgAfraction of Tcrbmice ( Figures 3 F and S2 C). This trend was apparent in both the colon and jejunum ( Figures 3 F and S2 C). These data suggest that most IgAbacteria induce robust TI responses.

While commensal-specific IgA appeared largely intact in Tcrbmice, free IgA was significantly reduced ( Figure 3 E) (). Because this compartment was dramatically affected by the loss of T cells, we considered that it might contain TD specificities against non-microbial antigens and therefore assessed whether free IgA could bind commensal bacteria. We cohoused WT C57BL/6 mice with Rag1mice for 3 weeks, which equilibrated microbial communities ( Figure S2 A). We then isolated ileal or colonic free IgA from WT mice and used it to stain Rag1ileal or colonic bacteria, respectively. Although some free IgA reacted with Rag1bacteria, this staining was faint and insufficient to restore IgA coating to WT frequencies, even at high staining concentrations >100 μg/mL ( Figure 3 E). Thus, commensal-reactive specificities appear to be dilute in the free IgA and this compartment might contain primarily TD specificities against other luminal antigens.

To determine whether TI IgA is sufficient to coat commensal bacteria, we examined IgA responses in Tcrbmice and Tcrblittermate controls. Tcrbmice lack all αβ and γδ T cells and thus cannot mount TD antibody responses (). Tcrbmice did not form GCs in mLNs and PPs but had normal numbers of B220IgAclass-switched B cells ( Figures 3 A and 3B ), as previously reported (). Despite this, small intestinal and colonic lamina propria B220IgAplasma cells were reduced 10-fold in Tcrbmice ( Figure 3 C), consistent with previous reports (). TcrbIgAsmall intestinal plasma cells displayed a mixed surface IgAand IgAphenotype while WT cells were largely IgA; colonic IgAplasma cells were IgAin both Tcrbmice and controls ( Figure 3 C). Surprisingly, we observed substantial commensal IgA coating in Tcrbmice ( Figure 3 D). Indeed, IgAbacteria were found at identical frequencies to co-housed littermate controls and no appreciable differences in IgA staining intensity were apparent ( Figure 3 D). These observations suggest that TI IgA might account for most commensal bacterial coating.

(F) Relative enrichment of taxa in the colonic IgAfraction of controls (black) or knockouts (white). n = 6 each genotype, representative of two independent experiments. Error bars indicate SE. See also Figure S2

(E) (left panel) Free IgA in Tcrb −/− d −/− mice (n = 8) and littermate controls (n = 9) or (right panels) endogenous IgA coating in the ileum or colon of co-housed B6 and Rag1 −/− mice and staining of Rag1 −/− bacteria with B6 free IgA, as indicated. Error bars indicate SE.

(D) Representative staining and quantification of IgA + bacteria in Tcrb −/− d −/− mice (n = 8) and littermate controls (n = 9). Data compiled from four independent experiments. Error bars indicate SE.

(A) Representative staining and absolute numbers of indicated populations in the mLN, (B) PP, or (C) small intestinal and colonic lamina propria of Tcrb −/− d −/− mice or Tcrb +/− d +/− littermate controls. CD95 by Gl7 plots were gated CD19 + . B220 by IgA plots were gated Tcrb − CD3 − in the mLN and PP and Lin − (CD3, Tcrb, CD4, CD11c, NK1.1, F4/80) in the intestinal lamina propria. Data compiled from three independent experiments.

Commensal-specific IgAs have been posited to be largely TD (). However, the specificity of TD IgA remains poorly understood and it is not clear whether GC reactions target all IgAcommensals or only a subset. Although mucosal GCs depend in part on signals from the microbiota (), we detected GC B cells, Tfh cells, and IgA in germ-free mice, suggesting that non-microbial antigens such as dietary antigens might also stimulate TD responses (data not shown). Seminal work by Macpherson and colleagues demonstrated that TI IgA could react with model antigens expressed by E. coli or with lysates from the model culturable commensal Enterobacter cloacae (). However, it is unclear whether most commensal bacteria elicit TI responses in vivo and the commensals targeted by TI specificities have not been characterized.

In summary, we conclude that IgA predominantly targets small intestinal commensals and that most small intestinal bacteria elicit specific IgA. In contrast, bacteria found primarily in the colon are not major targets of IgA.

To assess whether colonic IgAbacteria could establish residence in the small intestine, we colonized germ-free mice with either IgAor IgAcolonic fractions and analyzed small intestinal and colonic communities 28 days later. Consistent with origins in the small intestine, colonic IgAbacteria stably colonized the jejunum of germ-free mice and gave rise to a community that closely resembled the input community ( Figure 2 A). In contrast, the colonic community of these mice did not resemble the input community, likely representing outgrowth of minor contaminants in the input fraction ( Figure 1 D). Beta diversity-based analysis of bacterial communities further verified that small intestinal communities of recipient mice were more similar to the IgAinput than colonic communities ( Figure 2 B). Mice colonized with a colonic IgAinoculum showed an opposite pattern: IgAbacteria stably colonized the colon and gave rise to a colonic community that resembled the IgAinoculum ( Figure 2 A). In contrast, the small intestinal communities of these mice did not resemble the inoculum. Beta diversity-based analysis verified that colonic communities were more similar to the IgAinoculum than small intestinal communities ( Figure 2 B). These data support the hypothesis that colonic IgAbacteria also reside in the small intestine, whereas colonic IgAbacteria are indigenous to the colon.

(B) Beta diversity analysis comparing intestinal microbial communities of mice colonized with IgA + colonic bacteria or IgA − colonic bacteria indicate similarity between samples shown in (A). Branch length is scaled to the weighted UniFrac distance.

(A) Average relative abundance of indicated taxa in the jejunum or colon of germ-free mice colonized with IgA + colonic bacteria (n = 4) or mice colonized with IgA − colonic bacteria (n = 3). Input fractions used to colonize recipient germ-free mice were from WT B6 mice. Recipients of IgA + or IgA − inocula were housed in separate gnotobiotic isolators and mice were analyzed 28 days after colonization.

We reasoned that segregation of colonic bacteria into IgAand IgAtaxa could be explained if (1) most bacteria indigenous to the colon were not targeted by IgA, and (2) colonic IgAbacteria also reside in the small intestine. In support of this, we found that the colonic IgAfraction closely resembled the duodenal community, whereas the colonic IgAfraction was mostly composed of taxa indigenous to the colon ( Figure 1 E). In total, more than 90% of colonic IgAbacteria were present at >1% relative abundance in the duodenum ( Figure 1 G). A member of Bacteroidetes, S24-7, was the only taxon found appreciably in the colonic IgAfraction and at >1% relative abundance in the duodenum, but this taxon was also found enriched in the IgAfraction in both locations ( Figure 1 E–1G). Thus, nearly all colonic IgAtaxa were also abundant in the small intestine, whereas most IgAtaxa were abundant only in the colon.

To identify commensal bacteria targeted by IgA, we fractionated samples into highly pure IgAand IgAfractions by stringent magnetic purification with an autoMACS separator ( Figure 1 D) and classified bacteria present in each fraction by IgA-Seq. We found that colonic bacteria markedly segregated into IgAand IgAtaxa ( Figures 1 E and 1F), as recently reported (). Numerous taxa were heavily enriched in the colonic IgAfraction, suggesting specific targeting by IgA ( Figure 1 F). Conversely, numerous colonic taxa were not targeted by IgA and were instead enriched in the IgAfraction ( Figures 1 E and 1F). These trends were also apparent in human colonic samples ( Figure S1 ). In stark contrast to the colon, duodenal bacteria did not segregate into IgAand IgAtaxa ( Figures 1 E and 1F). Instead, most duodenal taxa were found equally represented in both fractions. These data, as well as the high frequency of IgAbacteria in the duodenum ( Figure 1 B), suggest that most duodenal commensals elicit specific IgA, whereas many colonic commensals do not.

To study the commensal bacteria targeted by IgA under homeostatic conditions, we established a flow cytometric assay to visualize IgA-bound (IgA) bacteria in murine feces. We found that approximately 20% of bacteria were IgAin the feces of wild-type (WT) C57BL/6 mice and verified that this staining was specific and absent from Rag2and Aicdafeces ( Figure 1 A), as reported previously (). While the frequency of IgAbacteria in the colon was relatively constant, we found substantial differences along the gastrointestinal tract. IgA coated a significantly greater fraction of bacteria in the small intestine than the colon (40%–80% IgAversus 10%–30% IgA Figure 1 B), as reported previously (). This correlated with significantly higher titers of luminal free IgA in the small intestine ( Figure 1 B) and 10–15 fold more IgAplasma cells in the small intestinal lamina propria relative to the colonic lamina propria ( Figure 1 B). Similar trends were apparent in WT BALB/c and C3H mice (data not shown). We also observed a higher frequency of IgAbacteria in small intestinal aspirates of healthy humans relative to colonic aspirates ( Figure 1 C). These data suggest that IgA responses against commensal bacteria are most prominent in the small intestine.

(G) Quantification of average % of colonic IgAor IgAtaxa found at >1% relative abundance in the duodenum (black) or found at <1% relative abundance in the duodenum (white) in (E). See also Figure S1

(F) Log 10 relative abundance of each taxa in the IgA + divided by relative abundance in IgA − from (E). Error bars indicate SE.

(E) Average relative abundance of taxa in indicated fractions as assessed by 16S sequencing. Duodenal and colonic samples were taken from the same mice, n = 3.

(C) Staining and quantification of IgA + bacteria in ileal or colonic aspirates from healthy humans. Lines connect samples from the same patient (n = 6).

(B) Representative staining and quantification of IgA + bacteria measured by flow cytometry (n = 12) or free IgA measured by ELISA (n = 19) or absolute numbers of IgA + plasma cells. Data compiled from five independent experiments. Error bars indicate SE.

(A) Representative staining of C57BL/6 feces and negative controls showing staining in the presence of excess purified IgA and of Rag2 −/− Ιl2rg −/− mice lacking B cells or Aicda −/− mice lacking IgA. All bacterial flow cytometry plots were gated FSC + SSC + SYTO BC + DAPI − .

The amount of secreted IgA may not determine the secretory IgA coating ratio of gastrointestinal bacteria.

The amount of secreted IgA may not determine the secretory IgA coating ratio of gastrointestinal bacteria.

Development of a method for the identification of S-IgA-coated bacterial composition in mouse and human feces.

Distinct Regulation of IgA Synthesis in the Small Intestine and Colon of Mice and Humans

Discussion

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et al. Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease. + and induced specific IgA. Although a fraction of colonic bacteria were IgA+, colonic IgA+ taxa were also abundant in the small intestine and homed to the small intestine upon transfer into germ free mice. It is possible that colonic IgA+ bacteria represent small intestinal contaminants rather than indigenous colonic flora, and distinct physiological properties might contribute to the differential ability of colonic IgA+ and IgA− commensals to colonize the small intestine and colon. However, it seems unlikely that small intestinal bacteria are more immunogenic than colonic bacteria. Instead, this anatomical regulation is likely due to extensive priming of commensal-specific IgA+ plasma cells in secondary lymphoid tissues accompanying the small intestine. PPs and isolated lymphoid follicles are predominantly associated with the small intestine and possess a specialized epithelium that allows sampling of luminal antigens ( Tsuji et al., 2008 Tsuji M.

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Fagarasan S. Requirement for lymphoid tissue-inducer cells in isolated follicle formation and T cell-independent immunoglobulin A generation in the gut. The commensal bacteria targeted by IgA have remained enigmatic. Recent studies suggest that IgA targets particularly immunogenic or invasive bacteria (). In contrast, we found that anatomical location was the primary factor that determined whether a particular taxon elicited an IgA response. Under homeostatic conditions, most small intestinal bacteria were IgAand induced specific IgA. Although a fraction of colonic bacteria were IgA, colonic IgAtaxa were also abundant in the small intestine and homed to the small intestine upon transfer into germ free mice. It is possible that colonic IgAbacteria represent small intestinal contaminants rather than indigenous colonic flora, and distinct physiological properties might contribute to the differential ability of colonic IgAand IgAcommensals to colonize the small intestine and colon. However, it seems unlikely that small intestinal bacteria are more immunogenic than colonic bacteria. Instead, this anatomical regulation is likely due to extensive priming of commensal-specific IgAplasma cells in secondary lymphoid tissues accompanying the small intestine. PPs and isolated lymphoid follicles are predominantly associated with the small intestine and possess a specialized epithelium that allows sampling of luminal antigens (). Our data suggest that these tissues prime IgA against nearly all bacteria present in the small intestinal lumen.

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Zhang W.

et al. Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease. −/−d−/− small intestinal IgA+ plasma cells recognized commensal bacteria. Although we did not directly measure their affinity, these TI antibodies were clearly sufficient to brightly stain commensal bacteria. TI responses generate short-lived plasma cells, and active turnover of commensal-specific plasma cells might facilitate rapid, dynamic IgA responses upon exposure to novel commensal antigens. TI responses might also prevent pathological activation of commensal-specific T cells by sequestering bacteria away from antigen-presenting cells in the intestinal epithelium. Previous work has suggested that TI IgA responses generate low affinity antibodies that react poorly with commensal bacteria (). Further, recent studies of dysbiotic mice suggested that many commensals might elicit TD responses (). In contrast, we found strong commensal Ig coating in mice lacking T cells, GCs, or SHM. These data indicate that most commensals elicit strong TI responses and that TI IgA is completely sufficient to coat most commensal bacteria at frequencies and staining intensities found in WT mice. This conclusion was also strongly supported by our finding that five out of five antibodies generated from Tcrbsmall intestinal IgAplasma cells recognized commensal bacteria. Although we did not directly measure their affinity, these TI antibodies were clearly sufficient to brightly stain commensal bacteria. TI responses generate short-lived plasma cells, and active turnover of commensal-specific plasma cells might facilitate rapid, dynamic IgA responses upon exposure to novel commensal antigens. TI responses might also prevent pathological activation of commensal-specific T cells by sequestering bacteria away from antigen-presenting cells in the intestinal epithelium.

Mestecky, 2005 Mestecky J. Mucosal Immunology. Pabst, 2012 Pabst O. New concepts in the generation and functions of IgA. Mathias and Corthésy, 2011 Mathias A.

Corthésy B. Recognition of gram-positive intestinal bacteria by hybridoma- and colostrum-derived secretory immunoglobulin A is mediated by carbohydrates. −/−d−/− IgA+ plasma cells engineered to express the human IgG1 Fc region instead of mouse IgA Fc, we demonstrated Fab-dependent binding to discrete subsets of commensal bacteria, but not to cultured B. fragilis. Ongoing work in our laboratory is focused on characterizing the commensal bacteria and specific antigens recognized by these antibodies. TI responses might promote the generation of polyreactive specificities (), and IgA might bind nonspecifically to bacteria via the Fc region or secretory component (). However, by generating recombinant monoclonal antibodies from single TcrbIgAplasma cells engineered to express the human IgG1 Fc region instead of mouse IgA Fc, we demonstrated Fab-dependent binding to discrete subsets of commensal bacteria, but not to cultured B. fragilis. Ongoing work in our laboratory is focused on characterizing the commensal bacteria and specific antigens recognized by these antibodies.

Alugupalli et al., 2004 Alugupalli K.R.

Leong J.M.

Woodland R.T.

Muramatsu M.

Honjo T.

Gerstein R.M. B1b lymphocytes confer T cell-independent long-lasting immunity. Gil-Cruz et al., 2009 Gil-Cruz C.

Bobat S.

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et al. The porin OmpD from nontyphoidal Salmonella is a key target for a protective B1b cell antibody response. Haas et al., 2005 Haas K.M.

Poe J.C.

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Tedder T.F. B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae. B1a cells constitute the most abundant peritoneal B cell lineage and have been extensively investigated and shown to produce natural IgM antibodies with antimicrobial and self reactivities. However, the minor “sister” B1b lineage has remained largely elusive and has only been reported to participate in humoral responses against B. hermsii, S. typhimurium, and S. pneumoniae (). Our results extend these early reports and reveal a specialization of the B1b lineage in TI IgA responses against intestinal commensal bacteria. Future work should address the development of B1b cells and the sites at which they encounter intestinal antigens and undergo class-switch recombination in vivo.

Baumgarth, 2011 Baumgarth N. The double life of a B-1 cell: self-reactivity selects for protective effector functions. Mestecky, 2005 Mestecky J. Mucosal Immunology. Pabst, 2012 Pabst O. New concepts in the generation and functions of IgA. Slack et al., 2012 Slack E.

Balmer M.L.

Fritz J.H.

Hapfelmeier S. Functional flexibility of intestinal IgA - broadening the fine line. + plasma cell immunoglobulin repertoire did not include canonical “natural” B1a specificities and that B1a B cells did not differentiate into IgA+ plasma cells, consistent with a previous study ( Roy et al., 2013 Roy B.

Brennecke A.M.

Agarwal S.

Krey M.

Düber S.

Weiss S. An intrinsic propensity of murine peritoneal B1b cells to switch to IgA in presence of TGF-β and retinoic acid. Previous work has suggested that TI B1a B cells may give rise to “natural” IgA in the form of intestinal free IgA, which resembles “natural” low-affinity IgM found in circulation in the absence of immunization (). However, we found that the IgAplasma cell immunoglobulin repertoire did not include canonical “natural” B1a specificities and that B1a B cells did not differentiate into IgAplasma cells, consistent with a previous study (). IgA plasma cells contributing to the free IgA compartment appeared to be predominantly derived from TD B2 B cells. Thus, although many potential antigens might stimulate free IgA, this compartment does not represent “natural” IgA and appeared largely specific for non-microbial antigens.

Lécuyer et al., 2014 Lécuyer E.

Rakotobe S.

Lengliné-Garnier H.

Lebreton C.

Picard M.

Juste C.

Fritzen R.

Eberl G.

McCoy K.D.

Macpherson A.J.

et al. Segmented filamentous bacterium uses secondary and tertiary lymphoid tissues to induce gut IgA and specific T helper 17 cell responses. Lécuyer et al., 2014 Lécuyer E.

Rakotobe S.

Lengliné-Garnier H.

Lebreton C.

Picard M.

Juste C.

Fritzen R.

Eberl G.

McCoy K.D.

Macpherson A.J.

et al. Segmented filamentous bacterium uses secondary and tertiary lymphoid tissues to induce gut IgA and specific T helper 17 cell responses. Lécuyer et al., 2014 Lécuyer E.

Rakotobe S.

Lengliné-Garnier H.

Lebreton C.

Picard M.

Juste C.

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McCoy K.D.

Macpherson A.J.

et al. Segmented filamentous bacterium uses secondary and tertiary lymphoid tissues to induce gut IgA and specific T helper 17 cell responses. Robertson et al., 2005 Robertson B.R.

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On S.L.

Fox J.G.

Lee A. Mucispirillum schaedleri gen. nov., sp. nov., a spiral-shaped bacterium colonizing the mucus layer of the gastrointestinal tract of laboratory rodents. Interestingly, SFB and Mucispirillum evaded TI IgA and instead elicited TD specificities to become IgA coated. Yet, IgA coating of these taxa was independent of GCs and SHM and thus these organisms might induce IgA by atypical mechanisms that are dependent on T cells or T cell-derived factors. SFB is known to be particularly immunogenic and can induce formation of GCs and tertiary lymphoid structures, as well as effector T cell differentiation (). SFB also induces large quantities of free IgA that is not SFB-specific (). In contrast to SFB, the immunogenicity of Mucispirillum remains uncharacterized. Ongoing work in our laboratory is focused on culturing and characterizing the properties of this organism. Notably, SFB and Mucispirillum both closely associate with the intestinal epithelium in the terminal ileum (). Thus, these organisms may inhabit similar niches and possess atypical immunogenic properties, allowing them to evade TI responses and come into close contact with the mucosa where they can be sampled by antigen-presenting cells and elicit TD responses.

+; however, we found no instances in which 100% were IgA+. As small intestinal IgA− bacteria appeared taxonomically similar to IgA+ bacteria, a fraction of bacteria may escape coating. This might result from phase variation of surface capsular polysaccharide antigens ( Peterson et al., 2007 Peterson D.A.

McNulty N.P.

Guruge J.L.

Gordon J.I. IgA response to symbiotic bacteria as a mediator of gut homeostasis. Moon et al., 2015 Moon C.

Baldridge M.T.

Wallace M.A.

Burnham C.A.

Virgin H.W.

Stappenbeck T.S. Vertically transmitted faecal IgA levels determine extra-chromosomal phenotypic variation. We consistently observed that most small intestinal commensal bacteria were IgA; however, we found no instances in which 100% were IgA. As small intestinal IgAbacteria appeared taxonomically similar to IgAbacteria, a fraction of bacteria may escape coating. This might result from phase variation of surface capsular polysaccharide antigens (). Alternatively, commensal-specific IgA may be limiting or may be actively degraded by bacterial IgA proteases (). Understanding the specificity of individual IgA antibodies will shed light on this important issue.

+, similar to reports by Kroese et al. (1996) Kroese F.G.

de Waard R.

Bos N.A. B-1 cells and their reactivity with the murine intestinal microflora. Tsuruta et al. (2009) Tsuruta T.

Inoue R.

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Hara H.

Yajima T. The amount of secreted IgA may not determine the secretory IgA coating ratio of gastrointestinal bacteria. Palm et al. (2014) Palm N.W.

de Zoete M.R.

Cullen T.W.

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Stefanowski J.

Hao L.

Degnan P.H.

Hu J.

Peter I.

Zhang W.

et al. Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease. Palm et al. (2014) Palm N.W.

de Zoete M.R.

Cullen T.W.

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Hao L.

Degnan P.H.

Hu J.

Peter I.

Zhang W.

et al. Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease. Palm et al. (2014) Palm N.W.

de Zoete M.R.

Cullen T.W.

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Hao L.

Degnan P.H.

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et al. Immunoglobulin A coating identifies colitogenic bacteria in inflammatory bowel disease. hi bacteria that was enriched in commensals with pathogenic properties and largely targeted by TD IgA. This observation might be limited to a dysbiotic flora because we did not observe an IgAhi population in healthy mouse microbiota. We found that ∼20% of colonic bacteria were typically IgA, similar to reports byand, but higher than the ∼8% reported by. These differences may be technical, as we used a polyclonal anti-IgA antibody, whereasused a monoclonal antibody.also described a population of IgAbacteria that was enriched in commensals with pathogenic properties and largely targeted by TD IgA. This observation might be limited to a dysbiotic flora because we did not observe an IgApopulation in healthy mouse microbiota.

In summary, our data reveal the prominent role of the enigmatic B1b lineage in control of the microbiota and suggest a model whereby multiple layers of humoral immunity contribute to homeostatic IgA coating of microbiota in the small intestine. Most commensals induce TI responses from B1b and B2 B cells, and these responses sequester bacteria away from the intestinal epithelium, preventing T cell activation. However, atypical commensals including SFB and Mucispirillum evade TI responses and penetrate the mucus layer, where they interact with antigen-presenting cells and prime T cell responses. Humoral regulation of commensal bacteria might have represented a substantial evolutionary force promoting the diversification and maintenance of peripheral B cell lineages, including the elusive B1b lineage. While these data clarify the homeostatic regulation of commensal-specific IgA, a further understanding of the antigens targeted by IgA might shed light on the regulation of IgA responses and allow opportunities for therapeutic intervention in microbiota-associated pathologies such as inflammatory bowel disease, obesity, diabetes, and celiac disease.