Autoreactive B-1 B cells: constraints on natural autoantibody B cell antigen receptors

Abstract

B-1 B-cells constitute a distinctive population of cells that are enriched for self-reactive B cell receptors (BCRs). These BCRs are encoded by a restricted set of heavy and light chains, including heavy chains that lack nontemplated nucleotide additions at the V-D and D-J joining regions. One prototype natural autoantibody produced by B-1 B cells binds to a cryptic determinant exposed on senescent red blood cells that includes the phosphatidylcholine (PtC) moiety. The V(H)11Vkappa9 BCR, which accounts for a large fraction of the anti-PtC specificity, is underrepresented in other B-cell populations, including newly formed B cells in bone marrow, and the transitional B cells, follicular B cells, and marginal zone B cells in spleen. Previous work has shown that V(H)11 heavy chains pair ineffectively with surrogate light chain (SLC) and so do not promote development in bone marrow, but instead allow fetal liver maturation because of a fetal preference for weaker pre-BCR signaling. Such inefficient SLC pairing constitutes one constraint on the maturation of B cells containing V(H)11 rearrangements that biases their generation to fetal development. Here, we examine another possible bottleneck to the B1 cell repertoire: light chain pairing with V(H)11 heavy chain, finding very significant preferences.

Figure 1

Single cell suspensions were stained with a combination of fluorescent reagents, FL-anti-V_H11id(3H7), PE-anti-CD21/35(7G6), Cy5PE-anti-Vκ9id(13B5), Cy55PE-anti-CD93(AA4.1), APC-anti-CD5(53-7), Cy55APC-anti-CD19(1D3), Cy7APC-anti-CD23(B3B4), CasB-anti-IgM(331.12), QDot605-anti-CD43(S7). Cells were stained with propidium iodide to allow elimination of dead cells (PI⁺). Forward light scatter height versus area gating was used to eliminate aggregates and forward versus side scatter gating was used as a “lymphoid cell” gate to eliminate small debris and large granular non-lymphoid cells. In spleen, Fo cells were gated CD19⁺IgM⁺CD93⁻CD23⁺; B1 cells were gated CD19⁺IgM⁺CD93⁻CD23⁻CD43⁺CD5⁺; MZ cells were gated CD19⁺IgM⁺⁺CD93⁻CD23⁻CD21/35⁺⁺. In peritoneal cavity (PerC), B-2 cells were gated CD19⁺IgM⁺CD43⁻CD5⁻; B-1 cells were gated CD19⁺IgM⁺CD43⁺CD5⁺. In bone marrow, newly-formed B cells (Fr. E) were gated CD19⁺IgM⁺CD93⁺.

Figure 2

B lineage cells in VH11 Transgenic (BR5 line) spleen from newborn and adult animals. Single cell suspensions were stained with fluorescent antibodies specific for CD45R/B220(6B2), V_H11id, and endogenous IgM (b allotype). Panels show B220⁺ gated cells.

Figure 3

Levels of TdT mRNA in transduced cell lines, assayed by quantitative PCR. Transduced cells were purified by sorting GFP⁺ cells, then RNA was prepared and analyzed as described in the Methods. Maximum levels, detected in the parental R1 line were arbitrarily set to 100%.

Figure 4

Cell surface IgM expression on pre-B cells transduced with V_H11 and several different light chains. Transduced cells were stained with APC-anti-IgM(331.12) and then gated for V_H11 (CFP⁺) and kappa light chain (GFP⁺) expression. Fluorescence intensity determined by flow cytometry, normalized for transduction level variation as described in the text. Level on Vκ9 cells arbitrarily set to 1.0.

Figure 5

Model for V_H11Vκ9 natural autoantibody B cell generation, showing the two bottlenecks discussed in the text and illustrating a fetal bias in production.