The B cell receptor itself can activate complement to provide the complement receptor 1/2 ligand required to enhance B cell immune responses in vivo

Abstract

B cells express complement receptors (CRs) that bind activated fragments of C3 and C4. Immunized CR knockout (KO) mice have lower antibody titers and smaller germinal centers (GCs), demonstrating the importance of CR signals for the humoral immune response. CR ligands were thought to be generated via complement fixation mediated by preexisting "natural" IgM or early Ab from inefficiently activated B cells. This concept was recently challenged by a transgenic (Tg) mouse model that lacks circulating antibody but still retains membrane IgM (mIgM) and mounts normal immune responses. To test whether CR ligands could be generated by the B cell receptor (BCR) itself, we generated similar mice carrying a mutated mIgM that was defective in C1q binding. We found that B cells from such mutant mice do not deposit C3 on B cells upon BCR ligation, in contrast to B cells from mIgM mice. This has implications for the immune response: the mutant mice have smaller GCs than mIgM mice, and they are particularly deficient in the maintenance of the GC response. These results demonstrate a new BCR-dependent pathway that is sufficient and perhaps necessary to provide a CR1/2 ligand that promotes efficient B cell activation.

Figure 1.

C3 is deposited on λ⁺ mIgM but not mIgM(P436S) splenocytes. Splenocytes of mIgM (A) and mIgM(P436) (B) mice were prepared. After lysis of red blood cells they were loaded with NP-BSA (fine line) or incubated in BSA alone (dark line). After washing off unbound NP-BSA, cells were injected into B cell–deficient mice and after 30 min blood was collected into Heparin/EDTA. Lymphocytes were purified by gradient centrifugation and stained for B220, λ, and C3. Histograms represent C3 staining gated on live, λ⁺ B cells. Histograms show a representative result out of 6 experiments with 1–4 animals per group. mIgM(P436) λ cells (B) display no difference in C3 staining of cells incubated with NP-BSA or BSA control. On mIgM cells (A) incubated with NP-BSA (fine line) elevated anti-C3 staining was detected to BSA controls (dark line).

Figure 2.

FACS^® analysis of immune responses. Red cell–depleted splenocytes were stained as indicated and 10⁵ events were collected per sample on a FACSCalibur™. Antigen-specific B cell expansion was measured by gating on λ⁺/NIP⁺, live splenocytes. Representative data of day 12 after immunization with NP₂₂-CGG are shown here and summarized in Fig. 3 and Table I.

Figure 3.

mIgM(P436S) mice show diminished responses to immunization with different Ag carrier and haptenation ratios. Both mouse strains were immunized with NP₂₂-CGG (A), NP₃-CGG (B), and NP₁₅-HSA (C) and the percentage of λ⁺/NIP⁺ splenocytes determined by FACS^® analysis on day 12. Error bars indicate SEM of 3–4 mice per group in each of 2–3 separate experiments (i.e., at least 6 mice per group total). Mutant mice (solid bars) showed lower Ag-specific B cell expansion than mIgM mice (open bars) after all immunizations (P < 0.005 for NP₂₂-CGG, NP₃-CGG, and 50 μg of NP₁₅-HSA) but responded to all of them except for 5 μg of NP₃-CGG, in which case the response is not substantially different from the alum control.

Figure 4.

mIgM(P436S) mice generate smaller and fewer GCs throughout most of the primary immune response. Spleens were frozen from each experiment and 5 μm frozen sections were cut and stained with anti λ-AP (blue) and PNA-bi/SA-HRP (red). mIgM spleens consistently show more GCs of larger size than mIgM(P436S) spleens.

Figure 5.

Time course analysis of the primary immune response to NP₂₂-CGG. Splenocytes from mIgM (solid symbols) and mIgM(P436S) (open symbols) were analyzed by FACS^® at several time points after immunization for the percentage of Ag specific B cells. Mice were given 5 μg (dotted lines, triangles) or 50 μg (solid lines, squares) of antigen. Error bars indicate SEM of 3–4 mice per group per experiment. Day 8 was done in two experiments, day 12 in three and the other time points are from 3–4 mice in one experiment (P < 0.005 at days 8, 12, and 20. At day 4 P < 0.05 for 5 μg and P < 0.01 for 50 μg). Diminished Ag-specific B cell expansion in mIgM(P436S) mice was seen throughout the primary immune response. Shown are the percentages of λ⁺/NIP⁺ live splenocytes minus the average percentage of λ⁺/NIP⁺ cells in the relevant preimmune Tg mouse.

Figure 6.

Quantitation of the GC response. Spleens obtained from mice immunized with 50 μg (A) and 5 μg NP₂₂-CGG (B) were frozen at the several time points during the primary immune response and cryosections stained for GCs (PNA) and the λ light chain. The area of GCs was measured as described in Materials and Methods. Bars show the total area of λ⁺ GCs in square mm per 40× field. Error bars indicate standard error of 5 to 8 pictures per mouse and 3 mice per group (P < 0.005 for days 4, 12, and 20. On day 8, P > 0.05). Solid bars represent mIgM(P436S) mice and open bars mIgM mice.

Figure 7.

Specific antiserum rescues the primary response in mIgM(P436S) mice. Serum was obtained from cardiac puncture of JhD and BALB/c (naive and 2 wk after immunization with CGG) mice. Tail vein injection of 200 μl serum into mIgM(P436S) mice was followed by i.p. immunization with 5 μg of NP₂₂-CGG three hours later. At day 12 the percentage of λ⁺/NIP⁺ (A) and λ⁺/PNA⁺ (B) splenocytes were determined by FACS^® analysis. Administration of immune BALB/c serum (black) led to reconstitution of the primary immune response in comparison to mIgM(P436S) mice that received no serum (white, P < 0.005), JhD serum (horizontal stripes, P < 0.005), naive BALB/c serum (diagonal stripes, P < 0.005), or immune BALB/c serum (black, P < 0.005). The influence of NP specific IgM on the response of mIgM(P436S) mice (gray) was compared with the response in mIgM (black) and mIgM(P436S) (white) mice at 5 μg and 50 μg NP22-CGG (C) (P < 0.01 for 5 μg and P < 0.05 for 50 μg between mIgM or mIgM(P436S)+Ab and mIgM(P436S)).