B-CLL Antibodies Encoded by Stereotypic V_H1-69, D3-16, and J_H3 Rearrangements Immunoprecipitate Non-Muscle Myosin Heavy Chain IIA.

B-cell chronic lymphocytic leukemia (B-CLL) patient clinical outcomes can be segregated based on the degree of mutation of immunoglobulin (Ig) variable region genes. This observation suggests a functional role for antigen binding by the Ig B-cell receptor in B-CLL. Furthermore, over 20% of B-CLL patients share antibodies with very similar Ig heavy chain (H) variable region CDR3 sequences, suggesting common antigen reactivity. For example, sixteen unrelated B-CLL patients have been reported to express an unmutated Ig heavy chain encoded by V_H1-69, D3-16, and J_H3 with nearly identical HCDR3 sequences. The probability that these patients developed B-CLL with identical IgH variable regions by random chance is extremely small. This stereotypic set of antibodies strongly bind cytoplasmic structures in HEp-2 cells as observed by immunofluorescence. Therefore, HEp-2 cell extracts were used as the source of material to isolate the cellular antigen recognized by these antibodies. These B-CLL antibodies were recombinantly overexpressed and purified as human IgG1 molecules. Recombinant antibody was bound to protein G agarose beads and incubated with cell extracts overnight. Bound cellular antigen was immunoprecipitated, washed, eluted, run on SDS-PAGE, and stained with Coomassie Blue. B-CLL antibodies from this stereotypic set immunoprecipitated protein bands at approximately 45 and 225 kDa in size. These bands were isolated and identified by mass spectrometry analysis of trypsin-digested peptides. The 45 kDa and 225 kDa proteins are cytoplasmic actin and non-muscle myosin heavy chain IIA (MYHIIA), respectively. The V_H1-69, D3-16, and J_H3 encoded stereotypic set of antibodies has been reported to be polyreactive against multiple autoantigens, such as DNA, Ig, and actin. However, MYHIIA has not been previously reported as an antigen recognized by this stereotypic set of antibodies. Therefore, we confirmed that MYHIIA was recognized by this stereotypic antibody set by probing B-CLL antibody immunoprecipitates by Western blot with anti-MYHIIA antibody. Furthermore, anti-MYHIIA antibody immunofluorescent staining of HEp-2 cells co-localized with this B-CLL stereotypic antibody immunofluorescent staining. Thus, stereotypic V_H1-69, D3-16, and J_H3 encoded B-CLL antibodies are polyreactive with common cellular proteins, including the newly identified autoantigen MYHIIA. The cellular antigens recognized by these stereotypic antibodies may be important in the pathogenesis, diagnosis, and treatment of B-CLL.