Induction of tolerance in arthritogenic B cells with receptors of differing affinity for self-antigen -- Huang et al. 103 (10): 3734 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Huang et al. 10.1073/pnas.0600214103.

Supporting Information

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Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Text

Supporting Figure 6

Fig. 6. Targeted insertion of anti-GPI H and L chains. (A and C) Partial restriction maps of the targeting vector, the wild-type locus, the targeted allele in ES cell and in mice for the H and L chains, respectively. The beginning and end of homologous regions in targeting vector were marked by dashed lines. The PCR primers used to screen for homologous recombination and the probe used for confirmation by Southern blot analysis are shown. E, EcoR I; S, SacI. (B and D) Southern blot analysis of ES clones for the heavy and light chain knock-in, respectively. Genomic DNA isolated from PCR-positive clones was digested with SacI and hybridized to the probe shown in A and C. Lane 1 is control, lanes 2-4 are PCR-positive clones.

Supporting Figure 7

Fig. 7. Heavy chain usage and inclusion. (A) Expression of the 121 knock-in heavy chain (IgM^a allotype) vs. the endogenous IgM^b allotype in B220⁺ splenocytes. (B) Few IgM^a/b dual expressors among anti-GPI B cells, limited to the GPI-dull population (Middle Right). The IgM^a+GPI^dull cells included many of the dual light chain cells. Representative of three mice.

Supporting Figure 8

Fig. 8. Assessment of population kinetics by BrdUrd labeling. H^121/+L^121/h mice were pulsed with injected BrdUrd (A) or given long-term BrdUrd in drinking water (B-D) and B220⁺ splenocytes analyzed for GPI-binding activity and light-chain allelic inclusion. (A) Short-term labeling showing the absence of proliferation in either of the populations confirming that they were not cycling very actively. (B) Representative profile for mice given BrdUrd for 7.5 days. (C) Kinetic profiles of BrdUrd labeling for populations G, D, and E from H^121/+L^121/h mice were compared to immature (B6 AA4⁺) and mature (B6 AA4^-) splenocytes from B6 mice. The unedited B cells of population "G" became labeled with a much steeper slope than did the cells of populations D and E, indicating a faster turnover rate. The G curve appeared to be biphasic, suggesting that population G may contain two subsets: one with the fast turnover rate characteristic of immature B cells, and the other more long-lived. (D) Similar labeling of B220⁺ splenocytes from H^121/+L^121/+Rag^-/- and TCRa-deficient (Ca^-/-) mice, where Ca^-/- mice were used to provide an equivalent T-deficient control. Two to six mice were used for each time point of labeling.

Supporting Figure 9

Fig. 9. In vitro proliferation of anti-GPI B cells in response to different stimuli. Splenocytes from H^121/+L^121/+Rag^-/- and control (Ca^-/-) mice were cultured in vitro for 72 h with or without various stimuli. Stimulation by F(ab’)₂ anti-IgM (A), LPS (B), anti-CD40 (C), or anti-CD40 in the presence of a fixed concentration of IL-4 (50 units/ml) (D). Representative of five experiments.

Supporting Text
Targeting Constructs.
The Ig heavy and light chain cDNAs encoding the anti-GPI mAb 6.121 were amplified and sequenced from total RNA purified from the 6.121 hybridoma as described (1), using primers specific for the IgG1 constant region. Primers complementary to the start codon ATG and 3' sequence of the J segment were used to amplify the corresponding genomic DNA fragments (6.121VDJ and 6.121VJ). These primers also included restriction enzyme sites suitable for cloning purposes. 6.121VDJ was amplified by primers 5'- CTGAGCGTCGACACCATGAGATCACTGTTCTCTC-3' and 5'-CAGAGCATCGATGATTGTCACTGTTCCACAGGC-3'. 6.121VJ was amplified by primers 5'-CGAGAGACTAGTACCATGAAGTTGCCTGTTAGGTTG-3' and 5'-GGCAGCGTCGACCCTTAACACCTGATCTGAGAATGG-3'. For the Ig heavy chain knock-in construct, the D23 promoter (gift of Klaus Rajewsky; amplified by primers 5'-GAACTGCTCGAGAGTACCATGTCCCTAAGTGGC-3' and 5'-GCAGTCGTCGACTCTGATGGAGGCTCTATGAG-3'), 6.121VDJ and a short arm of homology sequence from BALB/c mice (corresponding to the J-C intron and amplified by primers 5'-GACTACATCGATACTGTCTAGGGATCTCAGAGCC-3' and 5'-GATTACGCGGCCGCTGCAGGTGTTC-3') were joined and cloned into the unique SalI/NotI sites of the pIVhL2neo vector (2). For the Ig light chain knock-in construct, the 3-83 promoter (amplified from the plasmid pRVk3-83-2neo by primers 5'-CGAGTCTAGAGCGGCCGCACAC-3' and 5'-GCATCGACTAGTTTTTCCTTTGAATTTTGGTCCC-3') and the 6.121VJ fragments were joined and cloned into the SalI/NotI sites of the pRVK3-83-2neo vector (3). Embryonic stem (ES) Cells and Mice.
The resulting targeting constructs were transfected into the 129/Sv-derived ES cell line PC3 (4). Ig heavy chain homologous recombinant ES clones were screened by PCR using the primers 5'-GCCTGTGGAACAGTGACAATC-3' and 5'-AGCCTTGCCACATGACCTGC-3'. Light chain homologous recombinant clones were screened by PCR using the primers 5'-TCTGCAAATGTCTGATGAGT-3' and 5'-CTCGTGCTTTACGGTATCGCC-3'. PCR-positive clones were verified by Southern blot analysis. Two verified clones for each targeting construct were injected into C57BL/6 (B6)-derived blastocysts, and the resulting chimeric mice were bred to B6 mice for germline transmission. The Neo^r gene, used to select homologous recombinant ES cells, was deleted during spermatogenesis in male chimera mice due to the protamine-promoter-driven Cre transgene in PC3 ES cells from 129/SV (4). The deletion of Neo^r in progeny was confirmed by PCR analysis. Experimental mice are on a mixed B6/129 background or partially backcrossed to B6. All animals were housed and bred under specific pathogen-free conditions at the Harvard Medical School Center for Animal Research and Comparative Medicine, and experiments were conducted in compliance with federal and institutional guidelines and with the approval of the Institutional Animal Care and Use Committee at Harvard University. Immunohistochemistry.
Cryosections of spleen were fixed in acetone, blocked with 10% horse serum and stained with various fluorochrome-conjugated Abs. For detection of GPI-specific B cells, we stained the sections with FITC-labeled GPI and the signal was amplified sequentially with an Alexa Fluor-488 rabbit anti-fluorescein and an Alexa Fluor-488 goat anti-rabbit IgG (Molecular Probes/Invitrogen). Staining of sections by MOMA-1 (specific for mouse splenic metallophilic macrophages) and the acquisition of photomicrographs was done as described (5). Cell Preparation and Flow Cytometric Analyses.
Suspensions of bone-marrow and spleen cells were prepared with Dulbecco’s Modified Eagles Medium (DMEM) (without phenol red) containing 3% heat-inactivated horse serum and 0.05% sodium azide. Stainings of cells were carried out in 96-well plates on ice in a final volume of 50 ml. Biotinylated Abs were revealed by incubating with fluorochrome-conjugated streptavidin after twice washing the cells. Anti-mouse kappa (mCk) Ab was applied first, and then the cells were washed and blocked with normal mouse serum (10%) before adding other Abs.

The AA4.1 hybridoma was a gift from David Allman. Purified AA4.1 mAb was conjugated with phycoerythrin (PE) or allophycocyanin (APC) following standard protocols. IgM^a (RS3.1)and IgM^b (MB86) (hybridomas were gifts from Klaus Rajewsky) were labeled with FITC and PE following standard protocols. Commercially obtained mAbs used in these studies included: FITC-anti-IgM, PE-anti-IgM, FITC-anti-IgD, PE-anti-IgD, PE-anti-CD23, PE-anti-CD43, PE-Cy7-anti-B220, FITC-anti-mCk, PE-anti-mCk (all from BD PharMingen), FITC-anti-hCk and biotin-anti-hCk (both from Biosource). Recombinant mouse GPI was labeled with NHS-biotin (Vector). Biotin-labeled GPI was used at 5-10 mg/ml to stain anti-GPI B cells, and was revealed by incubating with fluorochrome-conjugated streptavidin.

Analyses were conducted on a Coulter Elite cytometer (Beckman) or a MoFlo cell sorter (Cytomation). Cytometry data were analyzed by the Expo32 (Beckman) or Summit (Cytomation) programs.

BrdUrd Labeling and Detection.
Adult mice were given 0.8 mg/ml BrdUrd (Sigma) in the drinking water for the specified amount of time. The BrdUrd water-bottle was wrapped in aluminum foil and changed every other day. For a short pulse 100 ml of 5 mg/ml BrdUrd was injected i.p. in three-hour intervals. Cells were harvested and stained for surface expression of various markers, and detection of BrdUrd by FITC-anti-BrdUrd was carried out following the manufacturer’s protocol (BD PharMingen).

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