B lymphocyte memory: role of stromal cell complement and FcgammaRIIB receptors

Abstract

To dissect the influence of CD21/CD35 and FcgammaRIIB in antigen retention and humoral memory, we used an adoptive transfer model in which antigen-primed B and T lymphocytes were given to sublethally irradiated wild-type mice or mice deficient in CD21/CD35 (Cr2(-/-)) or FcgammaRIIB receptors (FcgammaRIIB(-/-)). Cr2(-/-) chimeras showed impaired memory as characterized by a decrease in antibody titer, reduced frequency of antibody secreting cells, an absence of affinity maturation, and significantly reduced recall response. The impaired memory in Cr2(-/-) chimeras corresponded with the reduced frequency of antigen-specific memory B cells. Interestingly, FcgammaRIIB(-/-) chimeras showed a differential phenotype with impaired splenic but normal bone marrow responses. These data suggest that CD21/CD35 on stroma, including follicular dendritic cells, is critical to the maintenance of long-term B lymphocyte memory.

Figure 1.

Frequency of NP-specific ASCs in chimeric mice 16 wk after receiving memory B lymphocytes. Recipient BM (a and c) and spleen (b and d) before (a and b) or 1 wk after antigen challenge intravenously with 50 μg NP₅-KLH (c and d) were analyzed by ELISPOT for IgG-secreting plasma cells specific for NP. Data are represented as means ± SEM (a and b shows three independent experiments and c and d shows four independent experiments). n = number of mice in each group. *, statistically significant differences upon comparisons to WT chimeras (P < 0.035 BM and P < 0.02 spleen).

Figure 2.

Frequency of donor-derived, NP-specific memory B lymphocytes in chimeric mice 16 wk after adoptive transfer. (a) Representative scheme to identify antigen-specific, donor-derived memory B lymphocytes by FACS^®. Frequency of NP-binding CD45.1⁺Igλ⁺ B lymphocytes (CD19⁺) was determined from mouse BM (b and c) and spleen (d and e) before (b and d) or 1 wk after antigen challenge intravenously with 50 μg NP-KLH (c and e). Cells were also confirmed as CD3⁻, IgM⁻, CD138⁻, and CD11b⁻. Data are represented as means ± SEM of three independent experiments. n = number of mice in each group. *, statistically significant differences upon comparisons to WT chimeras (P < 0.01 BM and spleen prechallenge and P < 0.04 after challenge).

Figure 3.

Changes in relative serum affinity with time after antigen exposure. Sera collected from chimeric mice were titrated in ELISA for the amount of high affinity (NP₅-BSA) and the amount of total (NP₁₅-BSA) IgG. The ratio of NP₅:NP₁₅ titers is expressed as mean ratios ± SEM and are compiled from four independent experiments. ▪, WT chimeras (n = 34); ♦, Cr2^−/− chimeras (n = 31); •, FcγRIIB^−/− chimeras (n = 32); *, statistically significant differences. Statistical comparisons were made to the 2–3-wk time point for each group (P < 0.01).

Figure 4.

Memory responses after secondary transfer. Splenic B lymphocytes from original chimeric mice were adoptively transferred intravenously with KLH-primed T cells and NP-KLH into sublethally irradiated WT (a and c) and Cr2^−/− (b and d) recipients. 3 wk after secondary transfer into WT (a) and Cr2^−/− (b) mice, NP-binding serum IgG was measured by ELISA. Each symbol represents a single mouse. Horizontal bars represent mean antibody titers. (c and d) ELISPOTs were performed on recipient splenocytes to determine ASC frequency. Data are represented as means ± SEM from a single experiment. *, statistically significant differences. Statistical comparisons to WT chimeras were made (P < 0.01).

Figure 5.

Dependence on CD21/CD35⁺ stroma for localizing antigen in GCs. 16 wk after adoptive transfer of memory B lymphocytes, WT (a), FcgRIIB^−/− (b), and Cr2^−/− (c) chimeric mice were injected with biotinylated NP-BSA. (d) WT chimeras not receiving biotinylated antigen. 5-μM splenic cryosections were stained with anti-IgM (blue) and PNA (green) to delineate B lymphocyte zones and GCs, respectively. Arrows point to detectable antigen, as visualized using SA-TRITC (red). Colocalized IgM, PNA, and antigen are detected in white. Staining patterns shown are representative of multiple sections (>5) from five mice per chimeric group.