Journal: Molecular Medicine

Article Title: Binding of CLL Subset 4 B Cell Receptor Immunoglobulins to Viable Human Memory B Lymphocytes Requires a Distinctive IGKV Somatic Mutation

doi: 10.2119/molmed.2017.00003

Figure Lengend Snippet: CLL subset 4 IGs do not bind i or I carbohydrate antigens. (A) Glycan sequences of the lipid-linked oligosaccharides pLNH, pLNnH, LNnO and I-octa that were among 76 additional (i- and Fc10.2- antigen-negative probes) in the microarray. Abbreviations: Gal, galactose; GlcNAc, N-acetylglucosamine; Glc, glucose; DH, L-1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine; Cer, ceramide. (B), (C) Microarray analysis of CLL mAbs 141, 282, 183, 240, and 342, in either (B) native bivalent or (C) precomplexed condition. The human anti-i IgM PIA ELL, human anti-I Ma, and the murine IgM Fc10.2 were included as positive controls. The results are the means of fluorescence intensities of duplicate spots, printed at 5 fmol, with error bars representing half the difference between the two values.

Article Snippet: Cells were then incubated with CLL mAbs (5–50 μg/mL) for 1 h at 4°C and binding detected by FITC-conjugated F(ab’) 2 goat anti-human IgG (Southern Biotech), or by streptavidin PE-Cy7 (eBioscience) when biotin-conjugated mAbs (sulfo-NHS-LC-biotin, Thermo Scientific) were used.

Techniques: Glycoproteomics, Microarray, Fluorescence

Journal: Molecular Medicine

Article Title: Binding of CLL Subset 4 B Cell Receptor Immunoglobulins to Viable Human Memory B Lymphocytes Requires a Distinctive IGKV Somatic Mutation

doi: 10.2119/molmed.2017.00003

Figure Lengend Snippet: Subset 4 IGs bind to viable human B lymphocytes. Subset 4 mAbs (nos. 183, 240, 342) were incubated with Ramos or Jurkat cell line cells under nonpermeabilizing conditions. mAb141, which expresses IGHV4-34 but does not belong to subset 4, and mAb360, which is a member of subset 1 and does not express IGHV4-34, were used as comparators. Apoptosis was measured by annexin V staining. (A) Representative contour plots of CLL mAb binding to Ramos cells. Binding detected by FITC-labeled F(ab’)2 fragments of goat polyclonal antibodies (pAbs) specific for human IgG. (B) Average binding of live (annexin V–) and apoptotic/dead (annexin V+) Ramos and Jurkat cells by the following biotinylated CLL mAbs: subset 4 mAbs183, 240 and 342; non–subset 4 IGHV4-34–expressing CLL mAbs 141 and DO8; and subset 2 mAb282 (IGHV3-21/IGLV3-21). Binding was detected by streptavidin PE-Cy7. Data obtained from four independent experiments. Error bars represent SEM.

Article Snippet: Cells were then incubated with CLL mAbs (5–50 μg/mL) for 1 h at 4°C and binding detected by FITC-conjugated F(ab’) 2 goat anti-human IgG (Southern Biotech), or by streptavidin PE-Cy7 (eBioscience) when biotin-conjugated mAbs (sulfo-NHS-LC-biotin, Thermo Scientific) were used.

Techniques: Incubation, Staining, Binding Assay, Labeling, Expressing

Journal: Molecular Medicine

Article Title: Binding of CLL Subset 4 B Cell Receptor Immunoglobulins to Viable Human Memory B Lymphocytes Requires a Distinctive IGKV Somatic Mutation

doi: 10.2119/molmed.2017.00003

Figure Lengend Snippet: CLL IGs belonging to subset 4 bind with different intensities to memory and naïve B cells. Tonsil mononuclear cells were incubated with CLL subset 4 mAbs 183, 240 and 342, and with a panel of commercial antibodies reactive with membrane antigens that distinguish human B cell subsets. (A) Two subpopulations (dim and bright) of CD19+ cells can be identified based on CLL subset 4 mAb binding. mAb141, which uses IGHV4-34 but does not belong to subset 4, recognizes only one population. (B) After gating specifically on CD19+ cells, it is clear that the CD19+/CLL mAbbright fraction is predominantly CD27–/IgD+/CD38l°w (naïve B cells) and the CD19+/CLL mAbdim cells are mainly CD27+/IgD–/CD38l°w/high (memory B cells). In contrast, CD19+ cells bound by mAb141 are predominantly CD27–/IgD+/CD38l°w cells (naïve B cells).

Article Snippet: Cells were then incubated with CLL mAbs (5–50 μg/mL) for 1 h at 4°C and binding detected by FITC-conjugated F(ab’) 2 goat anti-human IgG (Southern Biotech), or by streptavidin PE-Cy7 (eBioscience) when biotin-conjugated mAbs (sulfo-NHS-LC-biotin, Thermo Scientific) were used.

Techniques: Incubation, Membrane, Binding Assay

Journal: Molecular Medicine

Article Title: Binding of CLL Subset 4 B Cell Receptor Immunoglobulins to Viable Human Memory B Lymphocytes Requires a Distinctive IGKV Somatic Mutation

doi: 10.2119/molmed.2017.00003

Figure Lengend Snippet: Binding of subset 4 IGs to permeabilized and fixed HEp-2 cells and to DNA. (A) Reactivity of subset 4 IGs with permeabilized and fixed HEp-2 cells. Binding of Abs was detected using fluorochrome-labeled goat anti-human IgG pAbs. (B) Binding of native CLL IGs to DNA determined by ELISA. IGHV4-34+ subset 4 mAbs (nos. 183, 342 and 240), IGHV4-34+ non–subset 4 mAbs (nos. 141 and DO8) and subset 2 (IGHV3-21/IGLV3-21) mAb (no. 282) were tested for reactivity with dsDNA and ssDNA by enzyme immunoassay. Antibodies were used at a concentration of 25 ug/mL. Human sera containing antibodies reactive with dsDNA or with ssDNA were used as positive controls. Human serum without antibodies against dsDNA or ssDNA was used as negative control. Data are average ± SD of at least three independent experiments. Following the calculation and result interpretation information provided by the manufacturer, values ≥ 92.6 WHO units/mL for dsDNA binding (dashed blue line) and ≥ 68.6 U/mL for ssDNA binding (dashed red line) were registered as positive.

Article Snippet: Cells were then incubated with CLL mAbs (5–50 μg/mL) for 1 h at 4°C and binding detected by FITC-conjugated F(ab’) 2 goat anti-human IgG (Southern Biotech), or by streptavidin PE-Cy7 (eBioscience) when biotin-conjugated mAbs (sulfo-NHS-LC-biotin, Thermo Scientific) were used.

Techniques: Binding Assay, Labeling, Enzyme-linked Immunosorbent Assay, Concentration Assay, Negative Control

Journal: Molecular Medicine

Article Title: Binding of CLL Subset 4 B Cell Receptor Immunoglobulins to Viable Human Memory B Lymphocytes Requires a Distinctive IGKV Somatic Mutation

doi: 10.2119/molmed.2017.00003

Figure Lengend Snippet: Sequences of the IGHV-D-J and IGKV-J rearrangements of subset 4 IGs and of the various IGHV4-34 and IGKV2-30 point revertants. The (A) IGHV4-34- and (B) IGKV2-30–encoded parts of the IG H and L variable domains of CLL subset 4 mAbs (nos. 240, 342 and 183) are aligned with the corresponding germline counterpart. H and L chain revertants are indicated by arrows.

Article Snippet: Cells were then incubated with CLL mAbs (5–50 μg/mL) for 1 h at 4°C and binding detected by FITC-conjugated F(ab’) 2 goat anti-human IgG (Southern Biotech), or by streptavidin PE-Cy7 (eBioscience) when biotin-conjugated mAbs (sulfo-NHS-LC-biotin, Thermo Scientific) were used.

Techniques:

Journal: Molecular Medicine

Article Title: Binding of CLL Subset 4 B Cell Receptor Immunoglobulins to Viable Human Memory B Lymphocytes Requires a Distinctive IGKV Somatic Mutation

doi: 10.2119/molmed.2017.00003

Figure Lengend Snippet: Effect of individual IGHV4-34 reversions on the binding of subset 4 IG 240 to viable human B cells. (A) Effect of light chain reversion on binding to tonsil B lymphocytes. Tonsil mononuclear cells were incubated with biotinylated versions of CLL subset 4 mAb240 and the original 240 H chain paired with the L chain revertant rL-1, followed by incubation with streptavidin PE-Cy7. Note the significant drop in memory B cell binding and the increase in naïve B cell binding. (B) Effect of light chain reversion or light chain swapping on binding to Ramos B cells. Ramos B cell line cells were incubated with unmanipulated CLL subset 4 mAb240 or the original 240 H chain paired with the L chain revertant rL-1 (left panel) or with unmanipulated CLL subset 4 mAb183 or the original 183 H chain paired with the L chain rearrangement from CLL mAb 141, an IGHV4-34+ IG that is not a member of subset 4 (right panel). In both cases (left and right panels), each mAb was followed by incubation with streptavidin PE-Cy7. See Figure 5 for explanation of abbreviation for rL-1 revertant.

Article Snippet: Cells were then incubated with CLL mAbs (5–50 μg/mL) for 1 h at 4°C and binding detected by FITC-conjugated F(ab’) 2 goat anti-human IgG (Southern Biotech), or by streptavidin PE-Cy7 (eBioscience) when biotin-conjugated mAbs (sulfo-NHS-LC-biotin, Thermo Scientific) were used.

Techniques: Binding Assay, Incubation