Receptor Revision Diminishes the Autoreactive B Cell Response after Antigen. PNA Tet. Day 8. Day 16

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1 Receptor Revision Diminishes the Autoreactive Cell Response after Antigen Activation Ying-Hua Wang and etty Diamond Supplemental data: PNA Tet Supplemental Figure 1: Kinetic analysis of tetramer-binding cells in the spleens from DWEYS-MAP immunized mice. Histological staining of spleen sections for PNA (red), tetramer binding (green). Few tetramer-binding cells were detected on day 5 following immunization. Tetramer-binding cells first appeared within GCs on day 8, and then were detected in extrafollicular areas on day 11. Original magnification: x 1. Three mice were used in each group. Four to six pictures were taken for each spleen section.

2 Anti-DWEYS IgG (μg/ml) Tet + Tet - Tet + Tet - Anti-DNA IgG (μg/ml) Tet + Tet - Tet + Tet - Supplemental Figure 2: The tetramer binding population contains memory cells. Splenocytes (3 x 1 6 ) with (Tet + ) or without (Tet - ) tetramer-binding subsets were adoptively transferred from DWEYS-MAP immunized AL/c mice on day 35 to μmt mice (3 mice in each group). CD138 + cells were gated out during cell sorting. The recipient mice were boosted once after transfer. Serum was collected 2 weeks after boost. Anti-peptide (A) and anti-dna () IgG were measured by ELISA.

3 RAG1 RAG Relative level Relative level Tet Tet - 22 hi Tet + 22 hi Tet + 22 lo. Tet Tet - 22 hi Tet + 22 hi Tet + 22 lo Subsets Subsets Supplemental Figure 3: RAG is de novo expressed in mature cells by antigen challenge. AA4.1 - /HSA lo splenic lymphocytes (2x1 6 ) were isolated from naïve AL/c mice of 1-12 weeks and adoptively transferred to RAG2 -/- mice. After 3 weeks, the recipient mice were immunized with DWEYS-MAP. Antigen-binding and nonbinding cells were sorted by flow cytometry on day 16 following immunization. The mrna levels of RAG1 and RAG2 were measured with qpcr analysis. Data (mean ± SEM) are representative of two independent experiments.

4 1 % of Max IL-7R Relative level IL-7R C Mutations/sequence Heavy chain Supplemental Figure 4: The Tet + 22 lo population expresses IL-7R and mutated IgH V genes, even in mice treated with anti-il-7r blocking antibody. (A) Flow cytometry analysis of IL-7R expression on Tet + 22 lo and Tet - 22 hi cells from DWEYS-MAP immunized mice, treated with anit-il-7r or isotype control antibody. Immunization and administration of antibodies were performed as described in the text. Spleen cells were analyzed on day 16 post immunization. Shaded line, Tet - 22 hi subset from mice treated with isotype control; solid line, Tet - 22 hi cells from mice treated with anti-il-7r; dashed line, Tet + 22 lo subset from mice treated with isotype control; dotted line, Tet + 22 lo subset from anti-il-7r treated mice. A representative mouse was shown for each group, which includes three to five mice. () qpcr of IL-7R on Tet + 22 lo cells from DWEYS-MAP immunized mice, treated with anti-il-7r or isotype control antibody. Data (mean ± SEM) are representative of three independent samples. (C) Sequence analysis of IgH Vgenes expressed by Tet + 22 lo cells from DWEYS-MAP immunized mice, treated with anti-il-7r. Dots represent the number of point mutations in individual VH genes (n=27). Procedures for immunization, antibody treatment and cell sorting were described in previous figures.

5 1 3 Total IgM (μg/ml) μg/ml) Total IgG ( Supplemental Figure 5: antibody does not change total serum Ig levels in mice immunized with DWEYS-MAP. AL/c mice were immunized with DWEYS- MAP peptide and administered anti-il-7r antibody or isotype control as described. The mice were bled on the specified days for serum collection. Total serum IgM (A) and IgG () were measured by ELISA. Data are the mean ± SEM of five mice in each group.

6 Anti-DNA IgM (μg/ml) C Anti-DNA Ig G (μg/ml) * * Anti-peptide IgM (μg/ml) D Anti-pep tide IgG (μg/m l) Supplemental Figure 6: antibody does not affect IgM anti-dna titers, but increase IgG anti-dna titers in the primary response to DWEYS-MAP. AL/c mice were immunized with DWEYS-MAP peptide and administered anti-il-7r antibody or isotype control as described above. The mice were bled on specified days thereafter. IgM anti-dsdna (A), IgM anti-peptide (), IgG anti-dna (C) and IgG anti-peptide (D) serum antibodies were measured by ELISA. Data are the mean ± SEM of five mice in each group. *, P<.5, Student s t test.

7 nti-klh IgG (μg/ml) SA 1-2-SA/ 1-2-SA/ Supplemental Figure 7: Soluble hapten and receptor revision does not change IgG antibody response to the carrier protein, KLH. AL/c mice were immunized with 1-2- KLH. Treatment with soluble antigen and antibody was performed as described in Figures 7 and 8. Anti-KLH IgG in the serum collected on D21 after immunization was measured by ELISA.