STRUCTURAL-ANALYSIS OF V(H)4-21 ENCODED HUMAN-IGM ALLOANTIBODIES AND AUTOANTIBODIES AGAINST RED-BLOOD-CELLS

We have sequenced the variable heavy chain regions of a number of V(H)4-21 encoded monoclonal IgM anti-Rh(D) antibodies produced in response to deliberate immunization. These were compared with the sequences of similarly encoded IgM anti-I cold agglutinins (CA) derived from patients with lymphoproliferative diseases. The anti-Rh(D) antibodies show evidence of clonal expansion and somatic diversification. Even though they are produced in response to an antigenic stimulus, they demonstrate limited hypermutation in the variable heavy chain (V-H) segments and there is no evidence of selective pressure acting on the complementarity determining regions (CDRs). The CA demonstrate a higher rate of mutation and yet this results in a lower ratio of replacement to silent mutations (R:S) in the CDRs than seen in the anti-Rh(D) antibodies. It is not clear whether the different pattern of mutations seen in the CA is related to their auto-reactivity or their tumour origin. In both groups of antibodies the region encoded by the V(H)4-21 segment can be found in germline configuration at the amino-acid level indicating that other V-gene structures, i.e. light chains or CDR(H)3s, are crucial to the generation of either specificity. A role of the CDR(H)3 is indicated by the identification of a motif shared by four CAs and one Rh(D) antibody which also demonstrates CA activity independent of its anti-Rh(D) specificity. Amongst the anti-Rh(D) antibodies there seems to be an obligatory combination with V-L having closest homology to the DPL16 germline segment indicating this as particularly important in generation anti-Rh(D) specificity.