Phenotypic Detection of Clonotypic B Cells in Multiple Myeloma by Specific Immunoglobulin Ligands Reveals their Rarity in Multiple Myeloma

In multiple myeloma, circulating “clonotypic” B cells, that express the immunoglobulin rearrangement of the malignant plasma cell clone, can be indirectly detected by PCR. Their role as potential “feeder” cells for the malignant plasma cell pool remains controversial. Here we established for the first time an approach that allows direct tracking of such clonotypic cells by labeling with patient-specific immunoglobulin ligands in 15 patients with myeloma. Fifty percent of patients showed evidence of clonotypic B cells in blood or bone marrow by PCR. Epitope-mimicking peptides from random libraries were selected on each patient's individual immunoglobulin and used as ligands to trace cells expressing the idiotypic immunoglobulin on their surface. We established a flow cytometry and immunofluorescence protocol to track clonotypic B cells and validated it in two independent monoclonal B cell systems. Using this method, we found clonotypic B cells in only one out of 15 myeloma patients. In view of the assay's validated sensitivity level of 10−3, this surprising data suggests that the abundance of such cells has been vastly overestimated in the past and that they apparently represent a very rare population in myeloma. Our novel tracing approach may open perspectives to isolate and analyze clonotypic B cells and determine their role in myeloma pathobiology

Detection of surface Ig-positive clonotypic B cells by flow cytometry using patient-individual ligands.

<p><b>A and B:</b> Ig-selected phage bind to surface Ig-positive parental B cells as detected by flow cytometry. As proof of principal a flow cytometry staining protocol was set up using two different monoclonal B cell systems as positive controls as in the immunofluorescence setting illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031998#pone-0031998-g002" target="_blank">Figure 2A and B</a>. Phage-CA46 selected on the Ig of the Burkitt lymphoma cell line CA46 (panel A, second plot) as well as control phage (panel A, first plot) were used to label CA46 cells. Phage-CLL024 selected on the Ig of CLL patient 024 (panel B, second plot) as well as control phage (panel B, first plot) were used to label primary CLL024 cells. Cell-bound phage were stained with fluoresceinisothiocyanate-conjugated secondary detection (denominated “phage-FITC”), B cells were stained with a CD19 allophycocyanin-conjugated antibody (denominated “CD19-APC”). To determine the sensitivity level of the flow cytometry protocol, control phage-stained CA46 cells were spiked with CA46 cells stained with phage-CA46 in a 5%, 2.5%, 1%, 0.5%, 0.1%, 0.05% and 0.005% ratio (panel A, plots 3–7). The same spiking experiment was performed with primary CLL024 cells and the specifically binding phage-CLL024 (panel B, plots 3–7). All measurements recorded 50 000 events on a FACSCalibur flow cytometer (BD) and were analyzed with the FlowJo software (Treestar). <b>C:</b> Staining of PBMCs of patients MM031, MM034 and MM021 with phage selected on the respective patients' myeloma Ig (phage AGHPKDSGNEWA, phage ADSSKRCNFNQC and phage FLNGCDKEDWMCWVTT) as well as with control phage to detect potential surface Ig-positive clonotypic B cells. Flow cytometry measurements were performed as in A and B. PBMC = peripheral blood mononuclear cell.</p

Phage displayed peptide sequences binding to myeloma immunoglobulins.^*

<p>*Libraries with 7-mer linear (X7), 12-mer linear (X12), 18-mer linear (X18), and 14-mer constrained beta sheet (BS) inserts were selected on myeloma Ig of patients MM001–MM036. The table lists the peptide insert sequences (single letter amino acid code) of phage clones for which specific binding to the respective Fab fragment and/or paraprotein was validated in single clone binding assays.</p>†<p>phage selection on monoclonal Fab fragment.</p>‡<p>phage selection on paraprotein.</p

Selection of specific phage-displayed peptide ligands binding to myeloma Ig.

<p><b>A:</b> Enrichment of selectively binding phage on MM021 paraprotein over three selection rounds. A X₁₈ random peptide phage display library was screened on immobilized MM021 paraprotein. Bound phage were recovered by K91 bacterial infection, amplified overnight, purified and subjected to the next selection round. Negative preselection of the library was performed in selection round two and three. The selected phage were tested for binding to MM021 paraprotein and the control human polyclonal IgG after each selection round. Bacteria transduced by recovered phage were grown on LB plates containing tetracycline to determine the number of transducing units (TU) by colony counting. Ig = immunoglobulin, MM = Multiple Myeloma. <b>B:</b> Phage selected on myeloma Ig specifically bind the antibody on which they were selected. Examplarily, binding of paraprotein MM021-selected phage FLNGCDKEDWMCWVTT and control phage fd tet to MM021 monoclonal antibody, MM021 paraprotein and control human IgG is shown. Bound phage were quantified by Enzyme-linked Immunosorbent Assay (ELISA). Data are shown as means from triplicate experiments (± SEM). <b>C:</b> The protein GST-FLNGCDKEDWMCWVTT blocks binding of phage FLNGCDKEDWMCWVTT to MM021 paraprotein. MM021-selected phage FLNGCDKEDWMCWVTT was incubated on MM021 paraprotein in the presence of increasing amounts of GST-FLNGCDKEDWMCWVTT or GST alone. Bound phage were quantified as in B. Data are shown as relative values compared to binding of a control phage (means from triplicate experiments ± SEM). GST = glutathione S-transferase. <b>D:</b> Phage selected on myeloma Ig specifically bind their target and do not cross-react with other Igs. Binding of phage clones selected on the myeloma Ig of patients MM021, MM034 and MM036 as well as random control phage YMTPPLSSQQKS and fd tet to different myeloma paraproteins and recombinantly expressed Fab fragments (MM001, MM003, MM008, MM020) as well as control polyclonal IgG and Bovine Serum Albumin is shown. Bound phage were quantified as numbers of transducing units (TU) based on bacterial infection (means from triplicate platings ± SEM).</p

Clinical characteristics of myeloma patients.^*

<p>*MM = multiple myeloma, PR = partial remission, SD = stable disease, PD = progressive disease.</p

Malignant plasma cell immunoglobulin rearrangements.^*

<p>*MM = multiple myeloma, VH = variable heavy chain, VL = variable light chain, HCDR3 = heavy chain complementarity determining region 3, LCDR3 = light chain complementarity determining region 3, Mutational Status = percentage of variable region germline sequence, M = mutated (>98% identity to variable region germline sequence), n.e. = not evaluated.</p

Detection of clonotypic Ig rearrangements by semi-nested PCR.

<p><b>A:</b> Illustration of semi-nested PCR approach for the detection of clonotypic Ig rearrangements. Ig genes were amplified using VH family-specific forward leader primers and constant region specific reverse primers (1^st PCR). A secondary PCR was used to specifically amplify the clonotypic Ig rearrangement with a patient-individual HCDR3-specific primer. PC = plasma cell, Ig = immunoglobulin, VH = heavy chain variable region, CH = heavy chain constant region, HCDR3 = heavy chain complementarity determining region 3. <b>B:</b> Detection of clonotypic Ig-rearrangements in myeloma PBMCs and BMMNCs of patients MM021, MM025 and MM036. Semi-nested PCRs were performed as described in A. PCR products were loaded on agarose gels stained with ethidium bromide. MM = Multiple Myeloma, PBMC = peripheral blood mononuclear cell, BMMNC = bone marrow mononuclear cell.</p

Detection of surface Ig-positive clonotypic B cells by immunofluorescence using patient-individual ligands.

<p><b>A and B:</b> Ig-selected phage bind to surface Ig-positive parental B cells as visualized by immunofluorescence. As proof of principal an immunofluorescence staining protocol was set up using two different monoclonal B cell systems as positive controls as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031998#s2" target="_blank">Methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031998#s3" target="_blank">Results</a> sections. Phage-CA46 selected on the Ig of the Burkitt lymphoma cell line CA46 (panel A, upper row) as well as control phage (panel A, bottom row) were used to stain CA46 cells. Phage-CLL024 selected on the Ig of CLL patient 024 (panel B, upper row) as well as control phage (panel B, bottom row) were used to stain primary CLL024 cells. Phage were visualized by immunofluorescence (FITC; green). Nuclei were stained with DAPI (blue). Images are shown at low (right panel) and high magnification (left panel). Images were obtained by confocal microscopy (Leika TCS SP2 AOBS; lens 63×) and analyzed using the Leika confocal software. CLL = chronic lymphocytic leukemia. <b>C:</b> Staining of MM031 PBMCs with MM031 Ig-selected phage AGHPKDSGNEWA and control phage to detect potential surface Ig-positive clonotypic B cells. Staining and imaging was performed as in A and B. PBMC = peripheral blood mononuclear cell. <b>D:</b> Staining of MM021 PBMCs with MM021 Ig-selected phage FLNGCDKEDWMCWVTT and control phage to detect potential surface Ig-positive clonotypic B cells. Staining and imaging was performed as in A and B. The white arrows point to single surface Ig-positive clonotypic B cells from patient MM021.</p