B Cell Receptor Crosslinking Augments Germinal Center B Cell Selection when T Cell Help Is Limiting

Abstract

Antigen-dependent engagement of germinal center (GC) B cell receptors (BCRs) promotes antigen internalization and presentation for follicular helper T cells. However, whether BCR signaling is critical or synergistic with T cell help for GC B cell selection or differentiation is unclear. Here, by adapting an experimental approach that enables independent delivery of BCR-crosslinking antigen or T cell help to GC B cells in vivo, we showed that T cell help was sufficient to induce GC B cell expansion and plasmablast formation. However, although BCR crosslinking could not by itself promote GC B cell selection or differentiation, it could synergize with T cell help to enhance the GC and plasmablast responses when T cell help was limiting. These findings indicate that GC B cells can integrate variable inputs from T cell help and BCR signaling in vivo for an optimal process of selection and differentiation, critical for potent long-term humoral immunity.

Keywords: B cell receptor; B cell selection and differentiation; T cell help; follicular helper T cells; germinal centers; plasma cells.

Figure 1.. T Cell Help Is Sufficient to Rescue B Cell Participation in GC and PB Response

(A) Experimental outline for (B) and (C). Purified Hy10 Ig-transgenic (Tg) B cells were pulsed ex vivo for 5 min with 50 μg/mL DEL-OVA, washed, and 10⁶ were transferred to recipient B6 mice preinjected with splenocytes containing 5 × 10⁵ OTII Th cells and subcutaneously (s.c.) preimmunized with OVA in CFA. Four days after Ig-Tg transfer, recipient mice were s.c. reimmunized with mDEL, DEL-OVA, or PBS in IFA. (B and C) Accumulation of Ig-Tg GC (B) and PBs (C) per CD19⁺ cells in the inguinal lymph nodes (dLNs) of reimmunized recipient mice at 2 and 4 days post-reimmunization (6 and 8 days post-Ig-Tg B cell transfer). See also Figures S1A–S1E. (D) Experimental outline for (E) and (F). 10⁶ 50 μg/mL DEL-OVA-pulsed Ig-Tg B cells were recruited into GCs as in (A), and 4 d.p.t. recipient mice were s.c. reimmunized with PBS in IFA and injected with 10 μg of αDEC-OVAp or iso-OVAp. (E and F) Ig-Tg GC (E) and PB (F) accumulation in dLNs. See also Figure S1F. (G) Experimental outline for (I)–(N) (white bars). Recipient mice were preinjected with splenocytes containing 5 × 10⁴ OTII Th cells, immunized with OVA in CFA, and transferred with 10⁵ 0.5 μg/mL DEL-OVA-pulsed Ig-Tg B cells. At 4 d.p.t., mice were s.c. reimmunized with PBS in IFA and injected with the indicated amount of αDEC-OVAp or iso-OVAp. (H) Experimental outline for (I)–(K) (gray bars). Recipient mice were preinjected with splenocytes containing 5 × 10⁴ OTII Th cells, s.c. immunized with DEL-OVA in CFA, and transferred with 10⁵ 0.5 μg/mL DEL-OVA-pulsed Ig-Tg B cells. (I and L) Ig-Tg GC B cell accumulation in dLNs. (J and M) Fraction of Ig-Tg GC B cells in GCs. (K and N) Ig-Tg PB accumulation in dLNs. See also Figures S1G and S1H. (B and C) Data from 3–5 independent experiments, 3–6 mice percondition, shown as mean ± SEM. Kruskal-Wallis with Dunn’s post-test between PBS, mDEL, or DEL-OVA is shown. (E–N) Data from 2–4 independent experiments are shown. Each symbol represents one mouse. Mann-Whitney test (E and F) or Kruskal-Wallis with Dunn’s post-test between isotype and each αDEC-OVAp dose (I–N) is shown. *p < 0.05; **p < 0.01.

Figure 2.. T Cell Help Is Sufficient for GC B Cell Selection and Differentiation into GCPB

(A) Experimental outline for (B)–(I). Mice were treated as in Figure 1G and, at 4 d.p.t., reimmunized with PBS in IFA and s.c. injected with 0.5 μg αDEC-OVAp, iso-OVAp, or unconjugated αDEC-205 for analysis 3 days later (7 days after Ig-Tg cell transfer). (B–D) Representative examples of Ig-Tg and endogenous LZ (CXCR4^lo CD86^high) and DZ (CXCR4^high CD86^lo) GC B cell gating after administration of iso-OVAp (B), unconjugated αDEC-205 (C), and αDEC-OVAp (D). (E) Ig-Tg GC DZ to LZ ratio. See also Figure S2B. (F) Ig-Tg DZ to LZ ratio normalized to the endogenous GC DZ to LZ cell ratio. See also Figure S2C. (G) Ig-Tg GC B cell accumulation in dLNs. See also Figure S2A. (H and I) Ig-Tg PB (H) and GCPB (I) accumulation in dLNs. See also Figures S2D–S2H. (E–I) Data from 3 independent experiments. Each symbol represents one mouse. *p < 0.05; Kruskal-Wallis with Dunn’s post-test.

Figure 3.. BCR Crosslinking Promotes GC and PB Response when T Cell Help Is Subsaturating

(A) Experimental outline. 0.5 μg/mLDEL-OVA-pulsed Ig-Tg B cells were recruited into GC responses as in Figure 1G. 4 d.p.t. recipient mice were s.c. reimmunized with 50 μg mDEL or PBS in IFA and injected with 0.5 μg iso-OVAp or with 0.5 μg or 10 μg αDEC-OVAp. (B–M) Representative examples of GC B cell gating analysis of Ig⁺ CD3~ lymphocytes in dLNs at 3 days post-reimmunization (7 days post Ig-Tg cell transfer; in B, F, and J) and accumulation of Ig-Tg GC B cells (C, G, and K), PB (D, H, and L), and GCPB (E, I, and M) in dLNs. Data from 2–4 independent experiments. Each symbol represents one mouse. *p < 0.05; Mann-Whitney test. See also Figure S3.

Figure 4.. BCR Crosslinking Enhances Early GCPB Differentiation when T Cell Help Is Subsaturating

(A) Gating strategy for Ig-Tg early and late PBs and GCPBs. 10⁵ Blimp1^yfpor CD45.1^pos Ig-Tg B cells were transferred to B6 recipient mice, which were immunized s.c. with 50 μg DEL-OVA in CFA. dLNs were analyzed 10 d.p.i. (B) Representative histographs of Blimp1, CD86, CD138, surface IgG₁, and CD45.1 in B220⁺ (red) and B220^lo (blue) GCPBs and non-GC Ig-Tg B cells (gray). For IgG₁ staining, non-GC B cells were gated on IgG₁^pos cells. Data representative of 2 or 3 independent experiments with 13 mice are shown. See also Figure S4A. (C) Experimental outline for (D)-(L). 0.5 μg/mL DEL-OVA-pulsed Ig-Tg B cells were recruited into GC responses as in Figure 1G. 4 d.p.t. recipient mice were s.c. reimmunized with 50 μg mDEL, sphDEL, or PBS in IFA and injected with 0.5 μg iso-OVAp or αDEC-OVAp. (D) Representative gating example for Ig-Tg and endogenous B220⁺ early GCPBs and B220^lo late GCPBs. (E–L) Accumulation of B220⁺ (E, F, I, and J) and B220^lo (G and K) GCPBs and GC B cells (H and L) in dLNs 1 day and 2 days post-reimmunization. Data from 4 independent experiments. Each symbol represents one mouse. *p < 0.05; Kruskal-Wallis with Dunn’s post-test between PBS and mDEL or sphDEL (left column) or Mann-Whitney test. See also Figures S4B–S4K.