Models.

Allotype analysis of IgG2a anti-dsDNA Abs in 3H9.KI and 3H9/56R.KI mice after cGVH. The use of the sd-tg (a) allotype (left hand graphs) or the nonsd-tg (b) allotype (right hand graphs) in IgG2a anti-dsDNA Abs was determined by ELISA in sera. (A) 3H9(+), □, and bm12→3H9(+), gray diamond, mice. (B) 56R(+), □, and bm12→56R(+), ♦, mice.

Genealogical trees generated from hybridoma sequences, demonstrating ongoing somatic VH region diversification (Hybridomas are those analyzed in Figs. 3 and 4). The individual clones sequenced were assigned to a particular genealogical tree on the basis of shared and unique VH mutations. Although we interpret most shared mutations as representing single events, we did observe independent parallel mutations (presumed hot spots). Multiple hybridomas with identical nucleotide sequences share a line. The height of each line shows the number of mutations recorded. For clarity, the hybridomas are identified by number within the ovals. Note the high number of mutations carried by each of the sequences analyzed. All hybrids except 169, 177, 2, and 107 bind dsDNA.

Nucleotide sequences of the recombinational junctions of hybridoma H chains from the bm12→56R(+) fusion. Hybridomas in this figure are the same ones depicted in Fig. 3, and are presented in the same order. The top line of each group represents the presumed V, D, and J germline (GL) genes used. Sequence identities with the germline VH or JH are denoted by dashes. The DH segments are indicated on the right. DH sequences are determined by identifying at least 4 nucleotide identities to a known DH segment. These, as well as the original DH sequences, are indicated in parentheses. The non-DH, non-VH sequences are assumed to be N sequences. As in Fig. 3, the VH sequences are assigned to the known VH germline from the GenBank or Haines et al. (reference 30). Nucleotides that differ from germline are underlined.

Anti-DNA Abs in 3H9/56R.KI mice after cGVH. Groups tested: n = 4–5 mice per group. Experimental GVH group: bm12 spleen cells injected into 3H9/56R.KI recipients (bm12→56R(+), ♦). Positive GVH control: bm12 spleen cells injected into 3H9/56R.KI negative littermates (bm12→56R(−), gray triangle). Negative controls: syngeneic B6 spleen cells injected into 3H9/56R.KI positive mice (B6→56R(+), □); B6 spleen cells injected into 3H9/56R.KI negative littermates (B6→56R(−), ○). Mice were bled at 4-wk intervals. The presence of anti-DNA Abs in sera was tested by ELISA. Results represent means ± SEM. (A) anti-ssDNA IgG. (B) anti-dsDNA IgG. Titers of anti-dsDNA in bm12→56R(+) differed from the positive control bm12→56R(−) at all weeks (P ≤ 0.05), while for anti-ssDNA, the difference was only at the prebleed. Titers of both anti-ssDNA and anti-dsDNA from B6→56R(+) mice differed significantly from those of B6→56R(−) mice at all time points. Anti-dsDNA titers from bm12→56R(+) were significantly higher than those from 3H9(+) at 4 and 8 wk after cGVH (P ≤ 0.05) (unpublished data). MRL/lpr sera tested at the same concentration gave an OD of 0.9 for anti-dsDNA. * represents statistical difference (P ≤ 0.05) from the appropriate negative control.

Nucleotide and deduced amino acid sequences of hybridoma H chains from the bm12→56R(+) mouse after the induction of cGVH. The panel shows nucleotide sequences of the H chains used by 56Rtg⁻ hybridomas derived from the bm12→56R(+) mouse. The sequences for each hybrid were determined by using the LD-JHCH PCR, as described in Materials and Methods. For each hybridoma, sequences were analyzed for homology to published VH gene segments using both GenBank/EMBL/DDBJ databases and B6 VH gene segments determined by Haines et al. (reference 30). Established germline codons and their corresponding single letter amino acids are shown in the top rows. Sequences from hybridomas using the same H chain are grouped together below these codons. Hybridomas 153, 223, 216, 228, 135, and 155 represent potential members of the same clone. Hybridomas 127, 214, and 107 represent potential members of a second clone. Clone assignments were made as described in Results. Dashes represent bases identical to those of the germline. Upper case letters indicate replacement mutations; silent mutations are in lower case. The J558.B3, J558.B4, and J558.B18 sequences described in this paper are available from GenBank/EMBL/DDBJ under the following accession nos.: AF296429, AF296421, AF296435, respectively. CDR, complementarity-determining regions.