Article Figures & Data
Figures
- FIGURE 1.
Mean (±SEM) IgG anti-peptide and autoantibody responses in peptide-immunized and unmanipulated B/W mice. Thirteen-week-old B/W mice were immunized with MAP-DWEYSVWLSN (n = 14) or MAP-control peptide (n = 9) in CFA, and boosted with 100 μg of MAP-DWEYSVWLSN or MAP control in IFA after 3, 6, and 9 wk. A, IgG anti-peptide. B, IgG anti-cardiolipin. C, IgG anti-dsDNA. D, IgG anti-laminin. E, Comparison of IgG anti-peptide and autoantibody titers in 26-wk-old MAP-DWEYSVWLSN-immunized (n = 14), MAP-control-immunized (n = 9), and age- and sex-matched unmanipulated (n = 8) B/W mice. An ∗ appears over time points in which the difference between MAP-DWEYSVWLSN- and MAP-control-immunized mice is statistically significant; the # appears over time points in which the difference between MAP-DWEYSVWLSN-immunized and unmanipulated B/W mice is statistically significant (see text for details).
- FIGURE 2.
Mean (±SEM) IgG anti-dsDNA titers in peptide-immunized B/W mice. A, Sera from 26-wk-old MAP-DWEYSVWLSN-immunized (n = 14), MAP-control-immunized (n = 9), and unmanipulated (n = 8) B/W mice at an initial dilution of 1/200 were serially diluted on dsDNA-coated plates. B, The last serum dilution at which the OD was higher than the mean + 3 SDs of control sera (n = 5) was defined as 1/titer.
- FIGURE 3.
Isotypes of the anti-peptide and anti-dsDNA responses. A, Anti-DWEYSVWLSN, and B, anti-dsDNA responses in MAP-DWEYSVWLSN-immunized (n = 14), MAP-control-immunized (n = 9), and unmanipulated B/W mice (n = 8). Results are shown as mean serum concentration ± SEM. An ∗ appears over time points in which the difference between MAP-DWEYSVWLSN- and MAP-control-immunized mice is statistically significant; the # appears over time points in which the difference between MAP-DWEYSVWLSN-immunized and unmanipulated B/W mice is statistically significant (see text for details).
- FIGURE 4.
Peptide inhibition of A, sera from MAP-DWEYSVWLSN-immunized B/W mice (n = 14), and B, mAb binding to dsDNA. A, Serum at a dilution giving 50% maximal DNA binding was preincubated with 125 μmol of MAP-DWEYSVWLSN for 1 h, and transferred to dsDNA-coated plates. B, mAbs at 10 μg/ml were preincubated with serial dilutions of MAP-DWEYSVWLSN for 1 h, and transferred to dsDNA-coated plates. Percent inhibition is calculated by: ((OD without inhibitor) − (OD with inhibitor))/OD without inhibitor. Results are shown in A as mean ± SEM.
- FIGURE 5.
Kidney histology in peptide-immunized and unmanipulated B/W mice at 29 wk of age. Low (×200; A, C, E, and G) and high (×400; B, D, F, H, I, and J) power views of H&E (A–H)- and PAS-stained (I–J) kidney sections from peptide-immunized and control B/W mice demonstrating severe glomerular and tubular disease in two DWEYSVWLSN-immunized B/W mice (E and F and G and H, respectively), but not in a control-immunized (A and B) or an unmanipulated B/W mouse (C and D). MAP-DWEYSVWLSN-immunized B/W mice display severe proliferative glomerulonephritis with crescent formation, as well as marked tubulointerstitial disease with tubular dilation and protein casts. I and J, Glomerular PAS-positive staining consistent with Ig deposits in a MAP-DWEYSVWLSN-immunized B/W mouse.
Tables
Isotype dsDNA ssDNA Peptide Cardiolipin Histone Sm/RNP B/W1,19-2 IgM 3.5 3.5 3.5 3.5 2.86 3.5 B/W1,17-15 IgM 3.5 3.5 0.17 0.16 0.12 0.14 B/W2,18-2 IgM 0.1 0.12 0.25 3.5 0.13 0.17 B/W2,27-7 IgM 3.5 1.49 0.09 0.43 0.16 0.78 B/W1,23-37 IgM 0.41 0.4 1.51 2.46 0.4 0.71 B/W2,25-32 IgM 2.04 2.65 2.96 3.1 0.61 1.08 B/W3,12-5 IgM 3.5 3.5 1.45 2.01 0.14 0.24 B/W2,28-22 IgM 3.5 2.48 0.15 1.08 0.14 0.91 B/W2,7-13 IgM 3.24 2.86 0.29 0.24 0.32 0.57 B/W3,14-1 IgM 3.5 3.5 3.5 3.5 0.92 1.67 B/W1,21-28 IgM 0.44 0.87 1.33 0.77 1.14 1.86 B/W2,24-13 IgM 1.45 2.39 2.48 2.35 0.49 0.74 B/W3,11-6 IgM 3.5 3.5 1.08 1.06 0.15 0.22 B/W3,24-4 IgM 0.67 1.52 0.68 1.56 0.12 0.16 B/W2,19-19 IgG1 0.31 0.76 1.66 1.58 0.75 0.77 B/W2,3-4 IgG2a 3.5 3.5 0.12 0.12 0.14 0.15 B/W2,30-4 IgG2a 0.1 0.16 0.22 1.54 0.14 0.13 B/W3,29-2 IgG2b 0.36 1.0 2.04 1.79 1.05 0.89 B/W3,20-3 IgG2b 3.5 2.7 1.05 1.52 3.5 1.8 B/W3,9-18 IgG2b 0.24 0.42 1.58 0.93 0.46 0.83 B/W2,30-2 IgG3 0.41 0.63 0.76 0.56 0.31 0.55 -
a Normalized supernatants at 10 μg/ml were incubated on Ag-coated plates for 2 h, followed by the appropriate alkaline phosphatase-linked goat anti-mouse Ig and substrate. Numbers reflect OD readings at 405 nm. mAbs are defined as positive (in bold) for a given specificity if the OD value was greater than the average of at least two isotype-matched control Abs plus 2 SD.
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Isotype BSA Keyhole Limpet Hemocyanin Lysozyme Cytochrome c B/W1,19-2 IgM 2.33 3.44 2.48 2.36 B/W1,23-27 IgM 0.08 2.6 0.13 0.13 B/W2,25-32 IgM 0.15 0.83 0.19 0.19 B/W3,12-5 IgM 0.07 0.18 0.08 0.09 B/W2,7-13 IgM 0.09 0.1 0.08 0.09 B/W3,14-1 IgM 0.18 2.81 0.26 0.26 B/W1,21-28 IgM 0.11 0.37 0.38 0.21 B/W2,24-13 IgM 0.13 0.56 0.15 0.15 B/W3,11-6 IgM 0.07 0.15 0.08 0.08 B/W3,24-4 IgM 0.07 0.16 0.09 0.07 B/W2,19-19 IgG1 0.1 0.16 0.19 0.19 B/W3,29-2 IgG2b 0.11 0.22 0.19 0.2 B/W3,20-3 IgG2b 0.08 0.16 0.12 0.27 B/W3,9-18 IgG2b 0.1 0.24 0.21 0.14 -
a Normalized supernatants at 10 μg/ml were incubated on Ag-coated plates for 2 h, followed by alkaline phosphatase-linked goat anti-mouse Ig and substrate. Numbers reflect OD readings at 405 nm. mAbs are defined as positive (in bold) for a given specificity if the OD value was greater than the average of at least two isotype-matched control Abs plus 2 SD.
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dsDNA/Peptide VH DH JH Vk Jk B/W1,19-2 +/+ J558 DFL16.1 2 VOx-1 4 B/W1,17-15 +/− J558 DQ52 3 Vk1-b 1 B/W2,18-2 −/− J558 ? 2 Vk21 2 B/W2,27-7 +/− J558 DFL16.2c 3 Vk1-a 4 B/W1,23-37 +/+ VH10 ? 3 Vk21 2 B/W2,25-32 +/+ Q52 DFL16.2 2 Vk8 2 B/W3,12-5 +/+ J558 DQ52 2 Vk8 1 B/W2,28-22 +/− J558 DFL16.1 2 Vk1-a 4 B/W2,7-13 +/+ J558 DFL16.1c 2 Vk9 1 B/W3,14-1 +/+ J558 DFL16.1 1 Vk1-c 5 B/W1,21-28 +/+ Q52 DSP2.3 4 Vk21 1 B/W2,24-13 +/+ Q52 DFL16.2 2 Vk8 2 B/W3,11-6 +/+ J558 DQ52 2 Vk8 1 B/W3,24-4 +/+ J558 DFL16.1 2 Vk21 1 B/W2,19-19 +/+ J558 DSP2.6 4 Vk4 1 B/W2,3-4 +/− J558 DQ52 2 Vk1-c 2 B/W2,30-4 −/− J558 DFL16.2 3 Vk8 2 B/W3,29-2 +/+ J558 DFL16.1 3 Vk1-c 1 B/W3,20-3 +/+ J558 DSP2.9 4 Vk4 2 B/W3,9-18 +/+ J558 DSP2.6 3 Vk32 4 B/W2,30-2 +/− Q52 DFL16.2 4 Vk8 1 -
a Total RNA from 2 × 107 hybridoma cells was reversed transcribed with an oligo(dT) primer. PCR for the H and L chain genes was performed using gene-specific primers, and V region sequences were compared to murine Ig sequences in GenBank using the Advanced BLAST program from the National Center for Biotechnology Information. The column labeled dsDNA/Peptide details mAb specificity for dsDNA and/or peptide.
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