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Antigen interaction with B cells in two proliferative disorders: CLL and MGUS
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Immunology and Transfusion Medicine.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of the work presented in this thesis was to elucidate B cell interaction with antigen in the two B cell proliferative disorders chronic lymphocytic leukemia (CLL) and monoclonal gammopathy of undetermined significance (MGUS). In the first part we investigated the antigen specificity of CLL cells and characterized Epstein-Barr virus (EBV)-transformed CLL cell lines with regard to phenotype and genotype. The second part consists of studies on the antigen presenting capacity of myelin protein zero (P0) specific MGUS B cells and their relation to T cells and development of polyneuropathy.

The aim of the work presented in this thesis was to elucidate B cell interaction with antigen in the two B cell proliferative disorders chronic lymphocytic leukemia (CLL) and monoclonal gammopathy of undetermined significance (MGUS). In the first part we investigated the antigen specificity of CLL cells and characterized Epstein-Barr virus (EBV)-transformed CLL cell lines with regard to phenotype and genotype. The second part consists of studies on the antigen presenting capacity of myelin protein zero (P0) specific MGUS B cells and their relation to T cells and development of polyneuropathy.

CLL cells are considered antigen experienced and different patient-derived CLL cells expressing B cell receptors (BCR) with highly homologous antigen binding sites are believed to have been selected by a common antigen at some point during the leukemogenesis. In paper I we investigated the antigen specificity of CLL-cell derived antibodies (Abs) with various IGHV gene usage and stereotyped BCR subset belonging. Identified CLL antigens included vimentin, filamin B, cofilin-1, proline rich acidic protein-1, cardiolipin, oxidized low density lipoprotein and Streptococcus pneumoniae capsular polysaccharides. Many of the CLL Abs studied displayed an oligo- or polyreactive antigen binding pattern and the identified antigens were either associated with apoptotic cells or microbial infection. This is similar to what has been described for innate natural antibodies, possibly indicating that CLL cells are derived from a natural-antibody- producing B cell population. Further characterization of CLL homology subset-2 antigen specificity showed binding to glands in human gastric mucosa corpus tissue sections for a CLL homology subset-2 Ab with HCDR3 motif-1, suggesting that this CLL subset recognize an autoantigen much like the CLL Abs tested in Paper I.

Characterization of EBV-transformed CLL and normal lymphoblastoid cell lines (LCLs) in paper II showed that eight of the CLL cell lines were verified to be of authentic neoplastic origin. Indication for a biclonal CLL was found in two of the cell lines and two of the presumably normal LCLs turned out to represent the malignant CLL clone. For three cell lines no conclusive evidence for CLL origin could be found emphasizing the importance of verifying the identity of cell lines used in research.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2010. , 59 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1158
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-53281ISBN: 978-91-7393-475-6 (print)OAI: oai:DiVA.org:liu-53281DiVA: diva2:288139
Public defence
2010-01-15, Aulan, Katastrofmedicinskt Centrum, Campus US, Linköpings universitet, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2010-01-20 Created: 2010-01-20 Last updated: 2017-09-22Bibliographically approved
List of papers
1. A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies
Open this publication in new window or tab >>A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies
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2008 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 111, no 7, 3838-3848 p.Article in journal (Refereed) Published
Abstract [en]

The restricted immunoglobulin (Ig) repertoire found in B-cell chronic lymphocytic leukemia (CLL) implies a role for antigen(s) in the leukemogenesis. The nature of the antigens has, however, not been characterized, although examples of autoantigens have been demonstrated. We have analyzed a panel of 28 CLL cell lines and primary cultures, producing monoclonal Ig with different Ig heavy-chain variable region gene usage and mutational status, including several complementarity determining region 3 homology subset members. Using mass-spectrometry, immunoassays, or protein macroarrays, we have discovered novel antigens binding to CLL Igs. These antigens included cytoskeletal proteins vimentin, filamin B, and cofilin-1, but also phosphorylcholine-containing antigens (eg, Streptococcus pneumoniae polysaccharides and oxidized low-density lipoprotein [oxLDL]). Additional new antigens identified were cardiolipin and proline-rich acidic protein-1. Remarkably, these antigens represent molecular motifs exposed on apoptotic cells/blebs and bacteria, and several CLL Igs bound to apoptotic Jurkat cells. In conclusion, these intriguing data, showing a limited target structure recognition, indicate that CD5+ CLL B cells are derived from a cell compartment that produces "natural antibodies," which may be instrumental in elimination and scavenging of apoptotic cells and pathogenic bacteria.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16339 (URN)10.1182/blood-2007-11-125450 (DOI)
Available from: 2009-01-16 Created: 2009-01-16 Last updated: 2017-12-14Bibliographically approved
2. Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia
Open this publication in new window or tab >>Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia
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2013 (English)In: Leukemia and Lymphoma, ISSN 1042-8194, E-ISSN 1029-2403, Vol. 54, no 8, 1769-1779 p.Article in journal (Refereed) Published
Abstract [en]

Chronic lymphocytic leukemia (CLL) B-cells resemble self-renewing CD5 + B-cells carrying auto/xeno-antigen-reactive B-cell receptors (BCRs) and multiple innate pattern-recognition receptors, such as Toll-like receptors and scavenger receptors. Integration of signals from BCRs with multiple surface membrane receptors determines whether the cells will be proliferating, anergic or apoptotic. To better understand the role of antigen in leukemogenesis, CLL cell lines producing monoclonal antibodies (mAbs) will facilitate structural analysis of antigens and supply DNA for genetic studies. We present here a comprehensive genotypic and phenotypic characterization of available CLL and normal B-cell-derived lymphoblastoid cell lines (LCLs) from the same individuals (n = 17). Authenticity and verification studies of CLL-patient origin were done by IGHV sequencing, fluorescence in situ hybridization (FISH) and DNA/short tandem repeat (STR) fingerprinting. Innate B-cell features, i.e. natural Ab production and CD5 receptors, were present in most CLL cell lines, but in none of the normal LCLs. This panel of immortalized CLL-derived cell lines is a valuable reference representing a renewable source of authentic Abs and DNA.

Place, publisher, year, edition, pages
Informa Healthcare, 2013
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-16340 (URN)10.3109/10428194.2013.764418 (DOI)000321763800032 ()
Available from: 2009-01-16 Created: 2009-01-16 Last updated: 2017-12-14Bibliographically approved
3. IGHV3-21 stereotyped subset-2 chronic lymphocytic leukemia cells make autoantibodies that bind to an 11.5kDa gastric mucosal antigen
Open this publication in new window or tab >>IGHV3-21 stereotyped subset-2 chronic lymphocytic leukemia cells make autoantibodies that bind to an 11.5kDa gastric mucosal antigen
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: The immunoglobulin heavy chain variable region (IGHV) gene mutational status is an important prognostic factor in chronic lymphocytic leukemia (CLL). Patients with mutated IGHV genes show significantly longer survival than unmutated cases. CLL patients with mutated IGHV3-21 genes, however, have a shorter survival compared to other mutated CLL cases. Recently, we showed that CLL cells react with oxidized epitopes exposed on apoptotic cells and bacteria. The gastric mucosal reactivity was of special interest to further characterize in detail.

Methods: We collected corpus biopsies from seven Helicobacter pylori (H.p.)+ study subjects withnon-atrophic gastritis, six subjects with H.p.- atrophic gastritis, eight subjects with H.p.+ atrophicgastric and from eight controls without gastritis. The binding pattern of CLL subset-2 IGHV3-21antibodies was analyzed by immunohistochemistry and the antigen was biochemically purified byaffinity chromatography.

Results: The subset-2 IGHV3-21 Abs bound to mucosal glands in 18 of 29 cases regardless of H.p.status or diagnosis. It also showed staining of connective tissue in all of the biopsies. The antigen waspurified and a protein of 11.5 kDa MW was isolated.

Conclusion: The results from this study indicate that IGHV3-21 subset-2 CLL Abs bind an 11.5kDaprotein present in gastric mucosal glands or connective tissue. This autoantigen has no associationwith H.p. since it is present in normal, non-atrophic H.p.+ and atrophic H.p.+/H.p.- gastric mucosafrom corpus. The exact nature of the 11.5 kDa protein is currently under detailed structure massspectrometry analysis in order to reveal whether bacterial mimicry is the mechanism behind theinduction of this particular autoantibody.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-53279 (URN)
Available from: 2010-01-20 Created: 2010-01-20 Last updated: 2010-01-20Bibliographically approved
4. Myelin protein zero is naturally processed in IgM MGUS B cells: Aberrant triggering of patient T cells
Open this publication in new window or tab >>Myelin protein zero is naturally processed in IgM MGUS B cells: Aberrant triggering of patient T cells
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2010 (English)In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 95, no 4, 627-636 p.Article in journal (Refereed) Published
Abstract [en]

Background and Objectives: Monoclonal gammopathy of undetermined significance (MGUS) of IgM isotype is a condition with clonally expanded B cells, recently suggested having an infectious origin. MGUS is frequently associated with polyneuropathy and antibodies against myelin protein zero (P0), whereas the role of the T cells remains largely unknown. Here we have analyzed P0-specific B cells, as antigen-presenting cells, and their capacity to activate T helper cells.

Design and Methods: We used a well-characterized MGUS-derived B cell line, TJ2, expressing anti-P0 IgM. The ability of TJ2 cells to bind, endocytose, process, and present P0 was investigated by receptor-clustering and immunofluorescence. The activation of P0-specific autologous T cells was studied by measuring IL2 and IFNγ with flow cytometry, immunobeads, and ELISPOT.

Results: Surface-receptor clustering and endocytosis of receptor-ligand (IgM/P0) complexes were pronounced after P0 exposure. Naturally processed or synthetic P0 peptide (194-208)-pulsed TJ2 cells significantly induced IL2 secretion from autologous T cells compared to control antigen pulsed cells (p<0 .001). The numbers of IFNγ producing T helper cells, including CD4+/CD8+ cells, were also significantly increased (p=0.0152). Affinity-isolated naturally processed myelin peptides were potent IFNγ stimulators for autologous PBMCs, but not for control PBMCs.

Interpretation and conclusions: We show for the first time that myelin P0 is naturally processed in IgM MGUS B cells, acting as aberrant antigen-presenting cells in activation of patients T helper cells. Our findings cast new light on the important role of autoreactive P0-specific B cells in he induction of the pathogenic T cell responses found in nerve lesions of MGUS patients with peripheral neuropathy.

Keyword
Monoclonal gammopathy of undetermined significance, myelin P0, peripheral neuropathy, CD5+B cells
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-53280 (URN)10.3324/haematol.2009.015123 (DOI)000277144800017 ()
Available from: 2010-01-20 Created: 2010-01-20 Last updated: 2017-12-12

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