RESTRICTED CDR3 LENGTH OF THE HEAVY-CHAIN IS CHARACTERISTIC OF 6 RANDOMLY ISOLATED DISEASE-ASSOCIATED V-H J558(-PRONE MOTH-EATEN MICE() IGMAUTOANTIBODIES IN LUPUS)

To investigate the origin of disease-associated IgM autoantibodies (AA b), we compared the genetic and structural characteristics of IgM AAb from autoimmune prone motheaten (me(v)) mice with natural autoantibodi es (NAAb) from normal background C57/BL6 strain. Six hybridoma-derived IgM molecules each were obtained both from me(v) mice, at the termina l stage of systemic autoimmune disease, and from mitogen-stimulated C5 7/BL6 mice. These were randomly selected for V-H J558 gene expression (aberrantly expressed in me(v) mice). The variable regions of the IgM molecules, both from autoimmune and normal mice, were encoded by unmut ated germline V-H genes. Disease-associated AAb from me(v) mice were p redominantly encoded by the J558 subfamily 186.2, whereas five J558 su bfamilies were utilized in NAAb originating from normal mice. Junction al diversity as a result of N or P nucleotide insertions and D-D fusio ns was noted among IgMs originating from both me(v) (mostly B-1 lympho cytes) and C57BL/6 (mostly B-2 lymphocytes) mice. Interestingly, all s ix J558(+) IgMs from me(v) mice showed a restricted CDR3 length of 10 amino acids, with similar hydrophobicity indices. Four unique V-DJ rea rrangements were observed among these IgMs. None of the IgMs were poly reactive and three of the six were subsequently observed to express mo nospecific autoreactivity with synthetic peptides (residues 81-92 and 37-53) representing segments of the T cell CD4-accessory molecule. Thr ee IgM antibodies had hydrophilic arginine residues in their CDR3 heav y chain region. By contrast, all six J558(+) IgMs from C57/BL6 mice ha d variable CDR3 length, distinct VDJ rearrangements and a local negati ve charge in the CDR3 region. Four of these IgMs demonstrated polyreac tivity with multiple conserved autoantigens and, hence, were classifie d as NAAb. These findings provide evidence for either positive or impa ired negative selection of B-1 lymphocytes secreting disease-associate d IgM AAb in me(v) mice. This likely results from a reduced threshold of responsiveness to autoantigens due to PTP1C deficiency, which is ta rgeted at the CDR3 length of the variable region of the heavy chain. I n addition, characteristic differences in the size and hydrophobicity pattern of the CDR3 of the heavy chain allow structural distinction be tween monospecific disease-associated IgM AAb and the polyreactive IgM NAAb.