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. 2012 Apr 30;109(20):7811–7816. doi: 10.1073/pnas.1204509109

Fig. 2.

Fig. 2.

Excess CD40L perturbs self-tolerance of anti-Sm/RNP Vκ38C+ B cells in 56R mice. (A–C) Hybridomas were generated from LPS-stimulated spleen cells obtained from 56R and CD40L/56R mice at 8 and 33 wk of age. 56R H chain+ hybridomas were analyzed for expression of Ig κ and λ L chain and reactivity to dsDNA by ELISA and for Vκ gene segment use by PCR (A). Total numbers of 56R H chain+ hybridomas are indicated by the numeral in the center of each circle diagram. Frequencies of hybridomas using Vκ21D and Vκ38C are indicated by the inner circle shading. Frequencies of anti-DNA hybridomas are indicated by the outer circle shading. Detailed results of VL gene use are shown in Table S1. Antibodies (3 μg/mL) from hybridoma supernatants using the indicated Vκ and Vλ gene segments obtained from 8-wk-old mice were assayed by ELISA for reactivity to dsDNA, ssDNA, histone, and Sm/RNP (B). Dots represent individual hybridomas. Numbers of hybridomas that were analyzed are indicated in parentheses. Out of 52 Vκ21D+ hybridomas, 44 hybridomas that do not react to any of these antigens are not shown. R462 is shown by red dots. Supernatant from the Vκ38C+ hybridoma D627 derived from CD40L/56R mice was examined for ANA staining pattern (C). (D) Anti-Sm/RNP antibody production was examined in sera from 16- to 24-wk-old WT (C57BL/6 × BALB/c) F1, 56R, CD40L, and CD40L/56R mice by ELISA. The antibody titer was determined relative to a standard curve generated from pooled MRL/lpr sera. The numbers in parentheses indicate the numbers of mice that were analyzed.