Expression of plasma cell alloantigen 1 defines layered development of B-1a B-cell subsets with distinct innate-like functions

Abstract

Innate-like B-1a cells contribute significantly to circulating natural antibodies and mucosal immunity as well as to immunoregulation. Here we show that these classic functions of B-1a cells segregate between two unique subsets defined by expression of plasma cell alloantigen 1 (PC1), also known as ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1). These subsets, designated B-1a.PC1(lo) and B-1a.PC1(hi), differ significantly in IgH chain utilization. Adoptively transferred PC1(lo) cells secreted significantly more circulating natural IgM and intestinal IgA than PC1(hi) cells. In contrast, PC1(hi) cells produced more IL-10 than PC1(lo) cells when stimulated with LPS and phorbol 12-myristate 13-acetate (PMA). PC1(hi) cells were also more efficient than PC1(lo) cells in regulating Th1 cell differentiation, even though both B-1a subsets were comparably active in stimulating T-cell proliferation. Furthermore, PC1(lo) cells generated antigen-specific IgM responses to pneumococcal polysaccharide antigens, whereas PC1(hi) cells do not. We found that PC1(lo) cells develop from an early wave of B-1a progenitors in fetal life, whereas PC1(hi) cells are generated from a later wave after birth. We conclude that identification of B-1a.PC1(lo) and B-1a.PC1(hi) cells extends the concept of a layered immune system with important implications for developing effective vaccines and promoting the generation of immunoregulatory B cells.

Fig. 1.

Expression of PC1 distinguishes peritoneal B-cell subsets. Peritoneal cells were stained with indicated antibodies and were analyzed by FACS. The numbers are percentages of cells falling in each gate. FMO, fluorescence minus one control.

Fig. 2.

B-1a subset ontogeny in fetal and adult life. (A) Fetal liver (day 18) and peritoneal cells of 3- and 8-wk-old CB17 mice were analyzed by FACS. Cells were gated on CD19⁺CD5⁺ B-1a cells. The numbers are percentages of cells falling in each gate. (B) Combined data for six to eight mice per group.

Fig. 3.

Persistence and migration of B-1a subsets in vivo. Equal numbers (3 × 10⁵) of sort-purified B-1a subsets were injected i.p. to Rag1^−/− mice (8 wk old). Peritoneal cells and spleens of recipients were analyzed by FACS at 2 wk and 2 mo following transfer. Cells were gated on lymphocytes. Data represent multiple mice from at least three independent experiments. PerC, peritoneal cavity.

Fig. 4.

B-1a subsets express different IgH chain repertoires. Sort-purified B-1a subsets from three mice were cloned and sequenced for IgH VDJ regions as indicated. Data were pooled from two independent experiments. Totals of 222 and 213 sequences were analyzed for PC1^lo and PC1^hi cells, respectively. Frequencies of V_H, D, and J_H sequences (A) and N nucleotide additions (B) are depicted for PC1^lo and PC1^hi subsets. N⁻, no N nucleotide; N⁺, with more than one N nucleotide.

Fig. 5.

B-1a subsets show different capacities to secret IgM and IgA. (A) Rag1^−/− mice were reconstituted i.p. with equal numbers of sort-purified B-1a subsets for 2 wk. Serum IgM levels were measured by ELISA. Data are pooled from three independent experiments. Each symbol represents a mouse. Nonreconst, Rag1^−/− mouse serum control. (B) Rag1^−/− mice were reconstituted i.p. with B-1a subsets for 2 or 6 mo. The IgA levels in intestinal lavage were measured by ELISA. Each symbol represents a mouse. Small-intestinal tissue was examined by immunofluorescence microscopy (Lower). IgA-secreting cells in lamina propria appear as green. Data represent two independent experiments with similar results. (C) Rag1^−/− mice were reconstituted i.p. with equal numbers of sort-purified B-1a subsets for 2 wk before they were immunized i.p. with PPS-3. The serum levels of total IgM before immunization were measured by ELISA to confirm efficient reconstitution. Antigen-specific IgM following immunization was quantified by ELISA. Data are means ± SEM (n = 7–8 mice per group) and represent two independent experiments with similar results.

Fig. 6.

Antigen presentation and differential IL-10 secretion by B-1a subsets. (A) Sort-purified peritoneal B-cell subsets were cultured for 3 d with OVA_323–396 peptide, CD4⁺ T cells purified from OT-II transgenic mice and TGFβ ± anti-IL-10 Ab. The cells were stained for CD4 and intracellular IFN-γ and analyzed by FACS. Representative data of three independent experiments is shown. (B) Sort-purified peritoneal B-cell subsets were cultured with and without PMA plus LPS overnight. IL-10–secreting cells were visualized by ELISPOT. Data represent two independent experiments with similar results.