Figure 5. Donor-specific B cell tolerance is rapidly induced and prevented by agonistic anti-CD40 antibody.

(A) Experimental designs for various treatments and AdTr models for B–F. (B) Immunization with third-party antigen C3H DSCs and analysis of anti-C3H IgG on day 14 (D14) (normalized MFI). n = 5–6/group. (C) Agonistic anti-CD40 (αCD40; 100 μg, i.v.) was administered at the time of HTx and treatment with αCD154 plus DSCs. On D14, enriched B cells from naive (N-B) or αCD40-treated tolerant (Tol-B) mice were AdTr into MD4 hosts and challenged with 2 × 10⁷ B/c DSCs. αB/c IgG (MFI) was measured on D14 (open symbols) and D28 (filled symbols) after AdTr. (D) B6 mice transplanted with B/c heart graft were treated with CTLA-4Ig (250 μg/mse, i.p.) on D0, D2, D4, and D7 after HTx. On D14, B cells were isolated and AdTr into MD4 recipients and immunized with 2 × 10⁷ B/c DSCs. αB/c IgG (MFI) measured on D14 (open symbols) and D40 (filled symbols) after AdTr. n = 5–10/group. (E) N-B or Tol-B cells were isolated from D7, D14, D35, and >D60 post-HTx recipients receiving αCD154 plus DSCs and AdTr to MD4 hosts. Filled and open symbols indicate αB/c IgG measured on D14 and D28 after AdTr, respectively. n = 6–16/group. (F) Purified B cells (2 × 10⁷) from naive B6 mice on D35 posttreated with αCD154 plus DSCs without HTx or tolerant mice D35 after HTx, were AdTr into MD4 hosts, followed by immunization with 2 × 10⁷ B/c DSCs. αB/c IgG (MFI) measured on day 28. n = 5/group. MFIs were normalized to MD4 that received N-B cells without B/c DSCs. Data were pooled from 2 or more independent experiments. Data are presented as the mean ± SEM. *P < 0.05; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Bonferroni’s post hoc test.