Figure 1. Alloreactive B cells are not deleted in tolerant recipients.

(A) B/c (H-2^d) or B/c.2W-OVA heart allografts were transplanted into C57BL/6 (B6, H-2^b) recipients that were untreated (acutely rejecting, AR) or treated with anti-CD154 (αCD154) on day 0 (D0), D7, and D14 plus DSCs (D0) to induce allograft tolerance. (B) Survival of allograft in AR or tolerant (Tol) mice. n = 10–40/group; P < 0.0001 by log-rank test. (C) Donor-specific antibodies–IgG (DSA-IgG) from Tol mice on postoperative day (POD) 0, 14, 45, 60, and 90 after heart transplant (HTx) and AR D14. n = 9–12/group. Representative flow plots of H-2K^d–binding B cells in naive B6 mice were identified using (D) double-positive (DP) donor MHC class I (K^d) tetramer conjugated to PE or APhC fluorochromes, and (E) decoy K^b (recipient MHC) tetramer conjugated to PE and AF647 in combination with K^d-PE tetramers. (F–H) Splenocytes and inguinal, axillary, and branchial lymph node cells were pooled and the total number of (F) K^d, (G) L^d, and (H) I-E^d tetramer–binding B cells from naive, Tol, or naive MD4 (anti-HEL BCR-Tg) mice were analyzed. n = 4–12/group. mse, mouse. (I–K) Normalized mean fluorescence intensity (MFI) of (I) K^d, (J) L^d, and (K) I-E^d tetramer–specific B cells from naive and Tol mice. n = 6–10/group. MFIs were normalized to DP or decoy tetramer–binding B cells of naive B6 mice. Data were pooled from 2 or more independent experiments and are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 2-way ANOVA with Tukey’s post hoc test for multiple comparisons (F–H) or 1-way ANOVA with Bonferroni’s post hoc test (C).