Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA

Abstract

Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

Figure 1

CLL mAb with stereotypic IGHV1-69, IGHD3-16, and IGHJ3 rearrangements immunoprecipitate observable proteins. A total of 6% SDS-PAGE of immunoprecipitate from HEp-2 cell extracts treated with mAb having stereotypic IGHV1-69, IGHD3-16, and IGHJ3 rearrangements (CLL068, 2 different recombinant mAb preparations) or not (CLL412, 2 different recombinant mAb preparations, or IgG) is shown with protein size (kilodalton) markers. Another mAb having stereotypic IGHV1-69, IGHD3-16, and IGHJ3 rearrangements (CLL258) showed similar results (Figure S1). mAb heavy chain (IgH) and light chain (IgL) as well as 225- and 45-kDa protein bands are indicated.

Figure 2

LC-MS/MS identification of 225- and 45-kDa proteins. After LC-MS/MS analysis, 225-kDa protein band peptide sequence matches to MYHIIA (A) and 45-kDa protein band peptide sequence matches to cytoplasmic beta-actin (B) are shown in bold and are underlined.

Figure 3

CLL mAb with stereotypic IGHV1-69, IGHD3-16, and IGHJ3 rearrangements immunoprecipitate MYHIIA. Antibodies from CLL068 (2 different recombinant mAb preparations) or human IgG were used to immunoprecipitate HEp-2 cell extracts. Immunoprecipitate (IP) and supernatant (Sup) samples were electrophoresed in 6% SDS-PAGE as in Figure 1. After blotting to nitrocellulose, the membrane was probed with rabbit antihuman anti-MYHIIA as shown.

Figure 4

Binding of CLL mAb with stereotypic IGHV1-69, IGHD3-16, and IGHJ3 rearrangements colocalizes with MYHIIA. (A) HEp-2 cells stained with CLL068 antibody (green) and anti-MYHIIA antibody (red) were visualized by confocal microscopy separately and merged together (overlap in yellowish orange). Untreated or blebbistatin-treated SNB19 cells were stained with CLL068 antibody (green) and (B) anti-MYHIIA antibody (red) or (C) phalloidin-stained F-actin (red). Cells were visualized by confocal microscopy separately and merged together.

Figure 5

MYHIIA siRNA treatment removes most cellular staining by CLL mAb with stereotypic IGHV1-69, IGHD3-16, and IGHJ3 rearrangements. (A) Immunoblot of SNB19 cell extracts either untreated or after transfection with MYHIIA siRNA or Lipofectamine 2000 alone. Top part of membrane containing high MW proteins (> 75 kDa) was probed with anti-MYHIIA, and the lower part with low MW proteins was probed with anti-alpha-tubulin. MYHIIA siRNA reduced MYHIIA protein levels by 99.6% relative to Lipofectamine 2000 alone. Lipofectamine 2000 alone (Control) or siRNA-transfected SNB19 cells were stained with CLL068 antibody (green) and (B) anti-MYHIIA antibody (red) or (C) phalloidin stained F-actin (red). Cells were visualized by confocal microscopy separately and merged together.

Figure 6

Model of cycle of CLL autoantigen stimulation. CLL B cell indicated by circle labeled with 3 Y-shaped antibodies and the number 5 (for CD5). Activated CLL B cells are striped. Boxes represent 2 cellular compartments: solid tissue, lymphoid organs such as bone marrow or lymph nodes, or blood. Other types of cells, possibly stromal cells as suggested in this illustration, present antigen represented by different shades and shapes. Sizes of pathway arrows indicate amount of cell traffic within and between compartments.