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Phenotypic detection of clonotypic B cells in multiple myeloma by specific immunoglobulin ligands reveals their rarity in multiple myeloma.

Trepel M, Martens V, Doll C, Rahlff J, Gösch B, Loges S, Binder M - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Hematology, BMT with section Pneumology, University Medical Center Hamburg-Eppendorf, Hubertus Wald Tumorzentrum/University Cancer Center Hamburg, Hamburg, Germany. m.trepel@uke.de

ABSTRACT
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.

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Related in: MedlinePlus

Selection of specific phage-displayed peptide ligands binding to myeloma Ig.A: 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: 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). C: 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. D: 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).
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pone-0031998-g002: Selection of specific phage-displayed peptide ligands binding to myeloma Ig.A: 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: 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). C: 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. D: 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).

Mentions: We used combinatorial phage display peptide library screenings selecting epitope-mimicking ligands which specifically bind to myeloma Ig. Peptides were selected on all myeloma Igs, irrespective of PCR-based evidence for clonotypic B cells. For the first phage library selections, myeloma Ig was recombinantly expressed as IgG-Fab in a prokaryotic system (selections on MM001, MM003, MM008 and MM020). As this recombinant approach is very labor-intensive, we also evaluated naturally occurring IgG or IgA paraproteins purified from serum as a source of target Ig. Paraproteins were purified by protein-A (IgG) or jacalin (IgA) chromatography and purity was assessed by coomassie staining of electrophoretically separated eluted Ig fractions (data not shown). Given that all patients had highly elevated Ig serum levels at the time of sample collection, contamination by non-paraprotein IgG or IgA was deemed negligible. Indeed, in most of the cases, binding phage could be enriched over three to four selection rounds on recombinantly expressed Fab as well as on purified paraprotein (exemplarily shown in Figure 2A) with at least one out of four random peptide phage libraries with different insert design (X7, X12, X18 and X4CX6CX4 = beta-sheet conformation [BS]). In only three out of 15 myeloma cases selections did not yield binding phage, potentially indicating that none of our peptide designs mimics the natural epitope of these antibodies. Table 4 lists the myeloma Igs and the peptide sequences selected thereon. All of the selected phage, but not random insert control phage, bound specifically to the myeloma Ig on which they were selected and not to control Igs, exemplarily shown in Figure 2B for MM021-selected phage FLNGCDKEDWMCWVTT binding to MM021 paraprotein. Phage FLNGCDKEDWMCWVTT, selected on the paraprotein of patient MM021, also bound to recombinantly expressed MM021 monoclonal antibody, providing proof of concept that selections on purified paraprotein yield phage clones selectively binding to the clonotypic Ig and not to potentially contaminating normal serum Ig (Figure 2B). The higher binding level of this phage to MM021 monoclonal antibody probably reflects the higher degree of purity as compared to MM021 paraprotein. Likewise, phage selected on recombinantly expressed antibodies bound specifically to the respective patient's native paraprotein verifying correct folding of the recombinant antibody used as target for phage selections (data not shown). To confirm that the selected phage displayed peptides bind the respective myeloma Ig independently of other phage components, we exemplarily performed competition assays with a GST-fusion protein containing the phage-derived peptide FLNGCDKEDWMCWVTT which inhibited binding of phage FLNGCDKEDWMCWVTT to MM021 paraprotein in a concentration-dependent manner (Figure 2C). To evaluate our phage clones for cross-reactivity with different myeloma Igs, we performed extensive binding assays as shown for three exemplary phage clones selected on MM021, MM034 and MM036 as well as respective control phage (Figure 2D). These assays suggest that phage clones selected on myeloma Ig bind their respective target in a highly specific manner and do not cross-react with other myeloma Igs or unrelated proteins.


Phenotypic detection of clonotypic B cells in multiple myeloma by specific immunoglobulin ligands reveals their rarity in multiple myeloma.

Trepel M, Martens V, Doll C, Rahlff J, Gösch B, Loges S, Binder M - PLoS ONE (2012)

Selection of specific phage-displayed peptide ligands binding to myeloma Ig.A: 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: 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). C: 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. D: 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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285203&req=5

pone-0031998-g002: Selection of specific phage-displayed peptide ligands binding to myeloma Ig.A: 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: 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). C: 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. D: 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).
Mentions: We used combinatorial phage display peptide library screenings selecting epitope-mimicking ligands which specifically bind to myeloma Ig. Peptides were selected on all myeloma Igs, irrespective of PCR-based evidence for clonotypic B cells. For the first phage library selections, myeloma Ig was recombinantly expressed as IgG-Fab in a prokaryotic system (selections on MM001, MM003, MM008 and MM020). As this recombinant approach is very labor-intensive, we also evaluated naturally occurring IgG or IgA paraproteins purified from serum as a source of target Ig. Paraproteins were purified by protein-A (IgG) or jacalin (IgA) chromatography and purity was assessed by coomassie staining of electrophoretically separated eluted Ig fractions (data not shown). Given that all patients had highly elevated Ig serum levels at the time of sample collection, contamination by non-paraprotein IgG or IgA was deemed negligible. Indeed, in most of the cases, binding phage could be enriched over three to four selection rounds on recombinantly expressed Fab as well as on purified paraprotein (exemplarily shown in Figure 2A) with at least one out of four random peptide phage libraries with different insert design (X7, X12, X18 and X4CX6CX4 = beta-sheet conformation [BS]). In only three out of 15 myeloma cases selections did not yield binding phage, potentially indicating that none of our peptide designs mimics the natural epitope of these antibodies. Table 4 lists the myeloma Igs and the peptide sequences selected thereon. All of the selected phage, but not random insert control phage, bound specifically to the myeloma Ig on which they were selected and not to control Igs, exemplarily shown in Figure 2B for MM021-selected phage FLNGCDKEDWMCWVTT binding to MM021 paraprotein. Phage FLNGCDKEDWMCWVTT, selected on the paraprotein of patient MM021, also bound to recombinantly expressed MM021 monoclonal antibody, providing proof of concept that selections on purified paraprotein yield phage clones selectively binding to the clonotypic Ig and not to potentially contaminating normal serum Ig (Figure 2B). The higher binding level of this phage to MM021 monoclonal antibody probably reflects the higher degree of purity as compared to MM021 paraprotein. Likewise, phage selected on recombinantly expressed antibodies bound specifically to the respective patient's native paraprotein verifying correct folding of the recombinant antibody used as target for phage selections (data not shown). To confirm that the selected phage displayed peptides bind the respective myeloma Ig independently of other phage components, we exemplarily performed competition assays with a GST-fusion protein containing the phage-derived peptide FLNGCDKEDWMCWVTT which inhibited binding of phage FLNGCDKEDWMCWVTT to MM021 paraprotein in a concentration-dependent manner (Figure 2C). To evaluate our phage clones for cross-reactivity with different myeloma Igs, we performed extensive binding assays as shown for three exemplary phage clones selected on MM021, MM034 and MM036 as well as respective control phage (Figure 2D). These assays suggest that phage clones selected on myeloma Ig bind their respective target in a highly specific manner and do not cross-react with other myeloma Igs or unrelated proteins.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Hematology, BMT with section Pneumology, University Medical Center Hamburg-Eppendorf, Hubertus Wald Tumorzentrum/University Cancer Center Hamburg, Hamburg, Germany. m.trepel@uke.de

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus