Antigen-specific antibody responses in B-1a and their relationship to natural immunity

Abstract

B-1a cells are primarily thought of as natural antibody-producing cells. However, we now show that appropriate antigenic stimulation induces IgM and IgG B-1a antibody responses and long-lived T-independent antigen-specific B-1a memory that differs markedly from canonical B-2 humoral immunity. Thus, we show here that in the absence of inflammation, priming with glycolipid (FtL) from Francisella tularensis live vaccine strain induces splenic FtL-specific B-1a to mount dominant IgM and activation-induced cytidine deaminase-dependent IgG anti-FtL responses that occur within 3-5 d of FtL priming and fade within 1 wk to natural antibody levels that persist indefinitely in the absence of secondary FtL immunization. Equally surprising, FtL priming elicits long-term FtL-specific B-1a memory cells (IgM>>IgG) that migrate rapidly to the peritoneal cavity and persist there indefinitely, ready to respond to appropriately administrated secondary antigenic stimulation. Unlike B-2 responses, primary FtL-specific B-1a responses and establishment of persistent FtL-specific B-1a memory occur readily in the absence of adjuvants, IL-7, T cells, or germinal center support. However, in another marked departure from the mechanisms controlling B-2 memory responses, rechallenge with FtL in an inflammatory context is required to induce B-1a secondary antibody responses. These findings introduce previously unexplored vaccination strategies for pathogens that target the B-1a repertoire.

Fig. 1.

FtL priming establishes persistent long-term production of serum IgM and IgG anti-FtL at natural antibody levels. IgM and IgG anti-FtL levels measured in sera from WT (BALB/c) and syngenic mutant (Rag1^−/− and AID^−/−) mice primed with FtL or injected with PBS, as indicated. Each dot represents a single mouse; n = 5 per group. Horizontal lines in “quartile box plots” indicate the 25th, median, and 75th percentiles. The dashed line shows background levels (Rag^−/− and AID^−/− sera). Values are expressed as microliter equivalents of a standard serum pool from 5-d FtL primed BALB/c mice (Fig. S1 shows FACS data).

Fig. 2.

FtL-induced isotype switching in anti-FtL B-1a requires AID but occurs without T-cell help or GC support. (A) Live FtL-binding Igκ⁺ cells from spleen of C57BL6/J, TCRβ^−/−δ^−/−, and AID^−/− syngenic mice 5 d after FtL immunization were gated to reveal CD138 and IgG expression. Boxes show IgG⁺ CD138⁻ and IgG⁺CD138⁺ plasma cells. One of four experiments is shown. (B) AID and BCL6 expression in sorted anti-FtL B-1a (CD138^–) and anti-FtL plasma cells (CD138⁺) from spleen of day 5 FtL-primed C57BL6/J mice. Data for each gene is shown as fold-change relative to expression level (dashed line) in PerC B cells from nonimmunized mice. Each dot represents data for 100 sorted cells of each subset, n = 6. One of three experiments is shown.

Fig. 3.

FtL immunization induces splenic anti-FtL B-1a responses in IL-7^−/− mice. Live splenic CD19⁺ B cells from IL-7^−/− mice injected with PBS or immunized with FtL for 3–4 d were gated to reveal FtL-binding Igκ⁺ B cells (“diagonal” in circled population, Upper), and further gated to distinguish anti-FtL plasma cells (CD138⁺CD5^–) (boxed population, Lower) from CD138^– cells, >95% of which are CD5⁺. (For serum responses, see Fig. S3C).

Fig. 4.

Naïve spleen, but not naïve PerC, produce anti-FtL primary responses to FtL challenge when transferred intravenously to naïve recipients. (Top) 10⁷ PerC (from one to two naïve mice) or indicated number of spleen cells from naïve Igh^a mice were transferred intravenously to naïve congenic Igh^b recipients immunized with FtL the next day. Donor and recipient anti-FtL responses were measured 5 d later. (Middle) Live B cells from indicated recipient spleen were gated to identify donor anti-FtL B-1a (IgM^a+) (circled population, Top), which are then further gated to show donor anti-FtL plasma cells (CD267^hiCD138⁺, boxed population, Rightmost Middle). CD267, transmembrane activator, and calcium signal-modulating cyclophilin ligand interactor (TACI) is a marker induced during plasma cell differentiation. The boundary for FtL binding is determined from the fluorescence-minus-one (FMO) (40) staining control in which fluorochrome-labeled FtL was omitted from the stain-set (Rightmost Top). Live PerC anti-FtL B-1a were gated to distinguish donor or recipient-derived cells (boxed populations, Middle). (Bottom) Numbers of donor (IgM^a) anti-FtL B-1a in recipient spleen (Left); donor-derived (IgM^a) anti-FtL in sera of recipients (Right). Each point represents data from an individual mouse (n = 5, per group). Values are expressed as microliter equivalents of a standard serum pool from 5-d FtL-primed Igha mice.

Fig. 5.

FtL priming induces the development of anti-FtL B-1a (IgM>>IgG) populations that persist long-term in the PerC. (A) Anti-FtL B-1a (tight diagonal, Upper) in gated live BALB/c PerC B at indicated days after FtL immunization. IgM or isotype-switched (IgM^–) anti-FtL B-1a are shown in boxes (Lower). (B) Anti-FtL B-1a (circled population, Upper) gated from PerC B cells from FtL-immunized BALB/c for indicated days were further gated to reveal anti-FtL subsets expressing IgG1 or IgG3 (gray line boxed populations, Lower). The remaining cells (IgG1^–IgG3^–, dashed line boxed population) express IgM anti-FtL. (C) Numbers of PerC anti-FtL B-1a from BALB/c mice at indicated days after FtL immunization. Each point represents data from an individual mouse (n = 5, per group). Mean value for each group is connected by dashed line.

Fig. 6.

MPL administered simultaneously with FtL priming dampens anti-FtL primary responses and inhibits development of FtL-specific B-1a memory in PerC. Absolute number of anti-FtL B-1a in spleen (Top) or in the PerC (Middle), and serum IgM anti-FtL antibodies levels (Bottom) in C57BL6/J mice at d 5 after PBS, MPL, FtL, or FtL plus MPL injection. Each dot represents data from an individual mouse (n = 4–5, per group). Values are expressed as microliter equivalents of a standard serum pool from 5-d FtL primed C57BL6/J mice.