Antigen-specific memory in B-1a and its relationship to natural immunity

Abstract

In the companion article by Yang and colleagues [Yang Y, et al. (2012) Proc Natl Acad Sci USA, 109, 10.1073/pnas.1121631109], we have shown that priming with glycolipid (FtL) from Francisella tularensis live-vaccine strain (i) induces FtL-specific B-1a to produce robust primary responses (IgM >>IgG); (ii) establishes persistent long-term production of serum IgM and IgG anti-FtL at natural antibody levels; and (iii) elicits FtL-specific B-1a memory cells that arise in spleen but rapidly migrate to the peritoneal cavity, where they persist indefinitely but divide only rarely. Here, we show that FtL rechallenge alone induces these PerC B-1a memory cells to divide extensively and to express a unique activation signature. However, FtL rechallenge in the context of a Toll-like receptor 4 agonist-stimulated inflammatory response readily induces these memory cells to migrate to spleen and initiate production of dominant IgM anti-FtL secondary responses. Thus, studies here reveal unique mechanisms that govern B-1a memory development and expression, and introduce B-1a memory as an active participant in immune defenses. In addition, at a practical level, these studies suggest previously unexplored vaccination strategies for pathogen-associated antigens that target the B-1a repertoire.

Fig. 1.

FtL rechallenge induces anti-FtL memory B-1a in PerC to express a unique activation signature. Gene expression in sorted splenic or PerC anti-FtL B-1a subsets (groups A–E) from FtL primed or boosted C57BL6/J mice. The text Top Left defines the phenotype of the sorted subsets and the immune status of the donors. Each dot represents a single datapoint obtained for 100 sorted cells of the indicated subset (n = 6 data points per subset). Data are represented as fold-change relative to expression level (dashed line) of PerC B cells from nonimmunized mice (group F). Data for one of three experiments with similar results are shown.

Fig. 2.

FtL rechallenge does not induce activated anti-FtL memory B-1a in PerC to generate the secondary anti-FtL antibody responses. (A) Number of anti-FtL B-1a cells in spleen or PerC from BALB/c mice at indicated days after FtL priming or boost. (B) Serum IgM anti-FtL levels in C57BL6/J or TCRβ^−/−δ^−/− mice with indicated immune histories. Each dot shows data for an individual mouse (n = 4 per group). Values are expressed as microliter equivalents of a standard serum pool from 5-d FtL primed C57BL6/J mice.

Fig. 3.

PerC anti-FtL memory B-1a cells produce strong secondary anti-FtL antibody responses to FtL rechallenge when transferred intravenously to naïve allotype-congenic recipients, but not when transferred intraperitoneally either to naive recipients or intravenously to primed recipients. (A) PerC from FtL-primed Igh^b mice were transferred either intravenously or intraperitoneally to naïve allotype congenic Igh^a recipients that were immunized with FtL the next day. Anti-FtL responses in recipients or nontransfer mice were measured 5 d later after FtL immunization. (Top) Live splenic B (CD19⁺) cells were gated to reveal donor anti-FtL cells (circled population). (Middle) Live splenic anti-FtL plasma cells (CD138⁺) were gated to distinguish donor or recipient-derived anti-FtL plasma cells (boxed populations). (Bottom) Live PerC anti-FtL B-1a cells were gated to distinguish donor and recipient anti-FtL cells (boxed populations). (B) Donor IgM^b (Upper) or host-derived IgM^a (Lower) anti-FtL levels in recipient sera. n = 5–6 per group; each dot shows data for an individual mouse. Values are expressed as microliter equivalents of a standard serum pool from 5-d FtL primed nontransfer Igh^a or Igh^b mice.

Fig. 4.

FtL rechallenge in the context of MPL stimulation mobilizes antigen-activated anti-FtL memory cells to migrate from PerC to spleen, where they differentiate to plasma cell producing anti-FtL antibodies. (A) FACS analysis of spleen and PerC from C57BL6/J mice with indicated immunization history. Live splenic B cells (CD19⁺) were gated to reveal anti-FtL B-1a (circled population, Top) and further gated to reveal anti-FtL plasma cells (boxed population, Middle). Anti-FtL B-1a (circled population) in gated PerC B cells are shown (Bottom). (B) Serum IgM anti-FtL levels in C57BL6/J mice. n = 6–7, per group. Each dot shows data for an individual mouse. (C) FACS analysis of naïve recipients to which PerC from FtL immunized donors was transferred intraperitoneally. (Upper) Live splenic plasma cells (CD19⁺CD138⁺) from recipients gated to reveal donor-derived anti-FtL (FtL⁺) and other plasma cells (FtL^–) (circled and boxed populations). (Lower) Donor anti-FtL memory cells in recipients PerC (circled populations).