B-cell chronic lymphocytic leukemia (B-CLL) is a disease caused by the clonal expansion of a B lymphocyte expressing a unique monoclonal antibody (mAb) of a defined amino acid sequence. At least 27% of B-CLL patients share mAbs with very similar sequences, suggesting common antigen reactivity. A frequently occurring subset of B-CLL patients (at least 49 worldwide) carries an unmutated mAb with a nearly identical or stereotypic sequence that is characterized by a heavy (H) chain encoded by IGHV1-69, IGHD3-16, and IGHJ3 and a light (L) chain generally encoded by IGKV3-20. mAbs from this stereotypic subset (called subset 6) bind a characteristic cytoplasmic protein in human cells. We have recently shown that this protein is non-muscle myosin heavy chain IIA (MYHIIA).

Because MYHIIA is an intracellular protein, we hypothesized that MYHIIA becomes exposed on the cell surface during apoptosis, allowing the mAb of the B-CLL cell to interact with MYHIIA. In this report, we provide evidence that subset 6 mAbs recognize MYHIIA-containing apoptotic cell structures.

Apoptosis of the human T cell line (Jurkat), either spontaneous, or induced by treatment with camptothecin was assessed phenotypically by nuclear DNA rearrangement revealed by immunohistochemical staining with propidium iodide (PI). Costaining with rabbit anti-human MYHIIA followed by fluorescein isothiocyanate (FITC) labeled donkey anti-rabbit IgG revealed that MYHIIA becomes exposed during apoptosis in structures distinct from those containing fragmented nuclear DNA. PI costaining with recombinant subset 6 B-CLL IgG1 mAb and FITC labeled goat anti-human-IgG antibody shows that this B-CLL mAb binds similar apoptotic structures. Finally, Jurkat cells stained with subset 6 B-CLL mAb as before and rabbit anti-human MYHIIA followed by Rhodamine Red labeled donkey anti-rabbit IgG demonstrate that the B-CLL mAb colocalizes with MYHIIA containing structures.

To further study this observation, Jurkat cell apoptosis was assessed by flow cytometry after staining with 7-AAD and Annexin V-Phycoerytherin (AV-PE). Costaining with anti-MYHIIA shows that anti-MYHIIA is exposed in both early (7-AAD+ AV-PE) and late (7-AAD+ AV-PE+) apoptotic cells, but not in live (7-AAD, AV-PE) cells. Similarly, costaining with subset 6 B-CLL mAb demonstrates binding in both early and late apoptotic cells, but not live cells. Intriguingly, not all apoptotic Jurkat cells are MYHIIA+ or B-CLL mAb+.

In summary, subset 6 B-CLL mAbs react with early and late apoptotic cells exposing MYHIIA. Reactivity with a specific apoptotic cell structure(s) may result in the maintenance and expansion of the leukemic clone.

Disclosures: No relevant conflicts of interest to declare.

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