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To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B- cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline- encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti- I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens.

SummaryTo study the association of autoimmunity and human B cell neoplasia, we have established a model of a B cell lymphoma which expresses a pathogenic autoantibody of defined specificity. The Ig V gene expressed in this neoplasm was analyzed longitudinally using clinical specimens taken from the splenic lymphoma (S) at diagnosis and from lymph node relapses 3 and 4 yr later (N3 and N4). Southern analysis and oligonucleotide hybridization experiments demonstrated that clonally related predominant and minor tumor cell populations were present in S at diagnosis, and that the minor population became the predominant population in the relapse specimens, N3 and N4. Although the Ig specificity and idiotype were the same at diagnosis and at both relapses, analysis of the expressed V gene sequences showed 14 base changes between S and N3, and 2 further changes at N4 . Little sequence heterogeneity was observed at each sampling time, indicating that the ongoing mutation frequency was low. The relevant germline precursor V gene was determined from autologous germline DNA and compared to the expressed genes. Based on the pattern of shared and unshared mutations, we were able to establish the genealogic relationship of the germline V gene and the expressed clonotypes of S, N3 and N4. Taken together, the findings from Southern blotting, oligonucleotide hybridization, and sequence analysis permit us to describe a molecular aspect of tumor progression, "clonotypec shift", wherein subpopulations of the malignant clone, marked by different V gene clonotypes, emerge and predominate at different time points in the evolution of the lymphoma. Furthermore, the sequential and nonrandom pattern of the V mutations, correlated with the observed conservation of autospecificity and idiotype, implies that clonal selection may have influenced the pathogenesis of the lymphoma . utoimmune phenomena can occur in association with seviA eral human clonal B cell disorders, such as idiopathic cold agglutinin disease (1), chronic lymphocytic leukemia (2), nonHodgkin's and Hodgkin's lymphomas (3, 4), and the clonal B cell expansions seen in HIV infection (5-7) . The reasons for the association of autoimmunity and B cell neoplasia are poorly understood, and it is unlikely that a single mechanism will explain every example. In the normal humoral immune response, antigen plays a central role in provoking clonal expansion of B cells; theoretically, antigen could play an analogous role in some B cell clonal diseases. Disease states in which autoantibodies are found in association with B cell clonal disorders offer the opportunity to investigate the role of antigen, specifically autoantigen, in the pathogenesis of the clonal disease.The Ig gene can serve as a marker in clonal analysis because unique combinations of V and J or V, D, and J segments are formed in the preimmune repertoire and are generally conserved within a clone thereafter. The nucleotide sequence of the CDR3, with its N sequences, is generated by these rearrangements and is unique to a par...

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