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. 2015 Sep 15;43(3):541-53.
doi: 10.1016/j.immuni.2015.08.007. Epub 2015 Aug 25.

Innate and Adaptive Humoral Responses Coat Distinct Commensal Bacteria With Immunoglobulin A

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Free PMC article

Innate and Adaptive Humoral Responses Coat Distinct Commensal Bacteria With Immunoglobulin A

Jeffrey J Bunker et al. Immunity. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1. IgA responses predominantly target commensal bacteria of the small intestine
(A) Representative staining of C57BL/6 feces and negative controls showing staining in the presence of excess purified IgA and of Rag2−/− Il2rg−/− mice lacking B cells or Aicda−/− mice lacking IgA. All bacterial flow cytometry plots were gated FSC+SSC+SYTO BC+DAPI. (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. (C) Staining and quantification of IgA+ bacteria in ileal or colonic aspirates from healthy humans. Lines connect samples from the same patient (n=6). (D) Representative pre- and post-MACS purity analysis of IgA+ and IgA fractions. (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. (F) Log10 relative abundance of each taxa in the IgA+ divided by relative abundance in IgA from panel (E). (G) Quantification of average % of colonic IgA+ or IgA taxa found at >1% relative abundance in the duodenum (black) or found at <1% relative abundance in the duodenum (white) in panel (E). See also Figure S1.
Figure 2
Figure 2. Colonic IgA+ and IgA bacteria differentially colonize the small intestine or colon
(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. (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.
Figure 3
Figure 3. T-independent and T-dependent IgAs coat distinct commensal bacteria
(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 TcrbCD3 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. (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. (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. (F) Relative enrichment of taxa in the colonic IgA+ fraction of controls (black) or knockouts (white). n=6 each genotype, representative of two independent experiments. See also Figure S2.
Figure 4
Figure 4. Germinal centers and somatic hypermutation are dispensable for commensal coating
(A) Absolute numbers of indicated populations in the mLN, PP, or small intestinal lamina propria of CD4-Cre Bcl-6 flfl mice or littermate controls. Data compiled from three independent experiments. (B) IgA bacterial coating, compiled from two independent experiments. (C) Fold enrichment of indicated taxa in colonic IgA+ fraction of Bcl-6ΔT mice or co-housed littermate controls. n=3 each genotype, representative of two independent experiments. (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. (E) IgM bacterial coating (Aicda−/− mice) or IgA bacterial coating (Aicda+/−) mice. Data compiled from two independent experiments. (F) Fold enrichment of indicated taxa in colonic IgM+ fraction of Aicda−/− mice or IgA+ fraction of Aicda+/− mice. n=5 each genotype. See also Figures S3 and S4.
Figure 5
Figure 5. Commensal-specific IgA+ plasma cells differentiate from B1b and B2 B cell precursors
(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. (C) IgA bacterial coating or free IgA in indicated recipient mice. (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. (E) Frequency of VH11 gene segments within the populations shown in (D) or number of sequences containing the indicated canonical B1a CDR3's. See also Figure S5.
Figure 6
Figure 6. B1b and B2 B cells coat diverse and overlapping commensal bacterial taxa
Fold enrichment of indicated taxa in colonic IgA+ fractions of Rag1−/− mice reconstituted with sorted B cell populations, as described in Figure 5. n=3 each group of mice, compiled from two independent experiments.
Figure 7
Figure 7. TI antibodies bind specifically to commensal bacteria
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 representative of two independent experiments.

Comment in

  • Independence Day for IgA.
    Macpherson AJ, McCoy KD. Macpherson AJ, et al. Immunity. 2015 Sep 15;43(3):416-8. doi: 10.1016/j.immuni.2015.08.024. Immunity. 2015. PMID: 26377894

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