(A) Histological staining of spleen sections for peanut agglutinin (PNA) (red) and DWEYS-tetramer (green, left 2 panels) or RAG2 (green, right panel). Original magnification: ×100 (left and right panels); ×400 (middle panel). (B) Histological staining of spleen sections for B220 (red), tetramer binding (green), and RAG2 (blue). RAG2 is coexpressed in extrafollicular tetramer-binding cells. Original magnification: ×100 (top row); ×400 (middle and bottom rows). Three to five mice were used in each group. Four to six pictures were taken for each spleen section. (C) Bivariate plots showing the gates for sorting the specified B cell subsets. (D) qPCR analysis of RAG1 and RAG2 expression in antigen-reactive and nonreactive B cells. BM immature B cells (B220⁺AA4.1^hiIgM⁺IgD^–) were used as a positive control. Mice were immunized with DWEYS-MAP in CFA on day 0 and boosted with DWEYS-MAP in incomplete Freund adjuvant on day 7. On day 16, specified B cell subsets were sorted by flow cytometry for preparation of RNA. RNA polymerase 2a (Polr2a) was used as internal control gene. Data (mean ± SEM) are representative of at least 4 independent experiments. Relative level is defined as the relative expression of mRNA normalized to that of Polr2a.

(A) Flow cytometry analysis of IL-7R expression on Tet⁺B220^lo (solid line), Tet⁺B220^hi (dotted line), and Tet^–B220^hi (line over shaded area) subsets. Splenic cells were prepared on day 16 from DWEYS-MAP–immunized mice. The experiment was repeated twice with 5 mice at each time. The Tet⁺B220^lo cells display the highest increase in the level of IL-7R protein. (B) qPCR of Il7r on the specified B cell subsets. Cells from DWEYS-MAP–immunized mice were sorted by flow cytometry on day 16. Data (mean ± SEM) represent 1 of 3 independent experiments. Tet⁺B220^lo cells show the greatest increase in Il7r mRNA. (C) Histological staining of spleen sections for B220 (red) and RAG2 (green). Mice were immunized with DWEYS-MAP on day 0, boosted on day 7, and treated with anti–IL-7R–blocking antibody or with isotype control on days 8, 11, and 14. Spleens were harvested on day 16. Three mice were included in each group. A minimum of 6 pictures were taken for each spleen. The experiment was repeated twice. Spleens from mice treated with anti–IL-7R antibody showed decreased RAG2 expression (green). Follicles are identified by B220 (red). Original magnification: ×50. (D) qPCR analysis of Rag2 in Tet⁺B220^lo cells from DWEYS-MAP–immunized mice treated with anti–IL-7R Ab or with isotype control. The procedure for immunization and administration of antibodies was described in C. Cell sorting was done on day 16, following immunization. Data (mean ± SEM) are representative of 3 independent experiments. Rag2 mRNA is undetectable in mice treated with anti-IL7R antibody. For all these experiments, the mice were immunized with DWEYS-MAP in CFA on day 0 and boosted with DWEYS-MAP in incomplete Freund adjuvant on day 7.

BALB/c mice were immunized with DWEYS-MAP peptide and administered anti–IL-7R antibody or isotype control as described in Figure 3. Eight weeks after the primary immunization, the mice were boosted one more time with DWEYS-MAP in incomplete Freund adjuvant. Anti-dsDNA (A) and antipeptide (B) IgG serum antibodies were measured by ELISA after the boost. Data are the mean ± SEM of 5 mice in each group, representative of 2 independent experiments. The P value was determined by 2-way ANOVA.

BALB/c mice were immunized and treated with BSA or 10-2–BSA as described in Figure 6. Mice treated with 10-2–BSA were divided into 2 groups. On days 12 and 15, one group was administered with 1 mg of anti–IL-7R antibody, the other group with isotype control antibody. Mice were bled weekly for serum collection. Anti–10-2 IgM (A) and IgG (B) serum antibodies were measured by ELISA. Data are the mean ± SEM of 5 mice in each group, representative of 2 independent experiments. **P < 0.01, *P < 0.05, 2-tailed Student’s t test. The P values show the comparison of the anti–IL-7R–treated group to the group treated with isotype control antibody.

(A) qPCR analysis of transcripts of μ and γ1 chains in antigen-reactive and nonreactive B cells. (B) Sequence (Seq) analysis of VH and Vκ genes expressed by antigen-reactive B cells. Filled circles and triangles represent the number of point mutations in individual VH genes (n = 41) and Vκ genes (n = 26) expressed by Tet⁺B220^lo cells. (C) qPCR analysis of Aid, Blimp1, and Xbp1 in antigen-reactive and nonreactive B cells. Data (mean ± SEM) are representative of 3 independent experiments (A and C). ND, not detected. Mice were immunized with DWEYS-MAP in CFA on day 0 and boosted with DWEYS-MAP in incomplete Freund adjuvant on day 7. Cells were sorted on day 16 (A–C). (D) Flow cytometry analysis of surface CD80 and CD95 on Tet⁺B220^lo (solid line), Tet⁺B220^hi (dotted line), and Tet^–B220^hi (line over shaded area) subsets. Mice were immunized with DWEYS-MAP in CFA on day 0 and boosted with DWEYS-MAP in incomplete Freund adjuvant on days 7 and 56. Spleen and BM cells were prepared 4 days after the second boost for analysis. Representative histogram from 4 to 5 mice was shown.

(A) qPCR for l chain expression (Igl-V1) on Tet⁺B220^lo cells. Mice were immunized with DWEYS-MAP and treated with anti–IL-7R antibody or isotype control as described in Figure 3, and spleen cells were sorted on day 16. The experiment was performed twice. (B and C) ELISA of serum levels of l chain associated with IgM (B) or IgG (C) from immunized mice. Arrows indicate the time of injection of anti–IL-7R antibody or isotype control on days 8, 11, and 14. Data are the mean ± SEM of 5 mice in each group. The P value was determined by 2-way ANOVA.

(A) Expression of RAG in DWEYS tetramer-binding cells requires the presence of circulating dsDNA in the plasma. BALB/c naive splenocytes were adoptively transferred to Rag2^–/– mice. Three weeks later, the recipient mice were immunized with DWEYS-MAP as described in Figure 3. From day 9 through day 15 following immunization, 500 μg active or heat-inactivated bovine DNase I were administered to the mice. Tetramer-reactive cells were sorted on day 16, and Rag1 and Rag2 were analyzed by qPCR. (B) Soluble 10-2–BSA induces RAG in the spleen of mice immunized with 10-2–KLH. (C and D) Receptor revision is induced in antigen-reactive B cells by soluble 10-2–BSA. BALB/c mice were immunized with 10-2–KLH in CFA on day 0 and boosted with 10-2–KLH in incomplete Freund adjuvant on day 7. On days 13, 14, and 15, 1 mg of 10-2–BSA or BSA were administered. qPCR analysis for the mRNA of l chain (Igl-V1) was done on 10-2–tetramer reactive cells, and nonreactive naive B cells were sorted on day 16 (C). Data (mean ± SEM) are representative of 2 independent experiments. Serum levels of Igλ were measured by ELISA (D). Five mice were included in each group. The experiment was repeated twice. **P < 0.01, 2-tailed Student’s t test.