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Importance of microenvironment and antigen in the regulation of growth and survival of CLL cells
Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Arts and Sciences.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Chronic lymphocytic leukemia (CLL) cells rapidly die when put in culture implying that microenvironmental signals delivered by accessory cells confer CLL cells with a growth advantage. Recent findings show that CLL cells are antigen experienced and antigen binding play a critical role in the pathogenesis of the disease. The overall aim of this thesis was to study the influence of the microenvironment and antigen binding in CLL.

In paper I, we studied the influence of the small redox-regulatory molecule thioredoxin (Trx) on CLL cell survival and proliferation. We found Trx to be highly expressed in CLL lymph nodes (LNs), secreted from stromal cells surrounding proliferating CLL cells in proliferation centers, indicating growth promoting properties. Secreted Trx was also shown to protect CLL cells from apoptosis.

In paper II, oxidized LDL was added to subset #1 CLL cells. However, in contrast to our hypothesis, we could not observe activation and proliferation of CLL cells. Instead subset #1 CLL cells were unresponsive/anergic through the B cell receptor (BcR). This anergic state could however be overcome by “wash out” of bound antigen or addition of toll-like receptor 9 stimulation in some patients.

Gene expression profiles differ between groups of CLL patients and in peripheral blood (PB) and LN compartment, due to different microenvironments. However, it is not known whether these differences also apply for DNA methylation. In paper III, we identified various genes that were alternatively methylated between IGHV mutated (M) and unmutated (UM) groups. For example prognostic genes, CLLU1 and LPL, genes involved in B cell signaling, IBTK, as well as numerous TGF-β and NF-κB/TNF pathway genes.

The intensity and duration of BcR signals are fine-tuned by enhancing or inhibitory coreceptors. SHP-1 inhibits BcR-signals by dephosphorylation. In paper IV, we compared the expression and activity of SHP-1 in CLL cells from LN with matched PB samples. However, in contrast to our hypothesis, SHP-1 activity/phosphorylation status in PB and LN, did not differ significantly.

This thesis, add another piece to the puzzle, on how the microenvironment and antigens influence CLL pathogenesis. Since great variations among individuals are seen, further studies in different groups of patients are necessary to elucidate the importance of antigen for the development of CLL.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2016. , 83 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1501
National Category
Cell and Molecular Biology Clinical Medicine
Identifiers
URN: urn:nbn:se:liu:diva-124576DOI: 10.3384/diss.diva-124576ISBN: 978-91-7685-867-7 (print)OAI: oai:DiVA.org:liu-124576DiVA: diva2:900455
Public defence
2016-02-26, Berzeliussalen, Campus US, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2016-02-04 Created: 2016-02-04 Last updated: 2017-09-22Bibliographically approved
List of papers
1. Thioredoxin, produced by stromal cells retrieved from the lymph node microenvironment, rescues chronic lymphocytic leukemia cells from apoptosis in vitro
Open this publication in new window or tab >>Thioredoxin, produced by stromal cells retrieved from the lymph node microenvironment, rescues chronic lymphocytic leukemia cells from apoptosis in vitro
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2007 (English)In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 92, no 11, 1495-1504 p.Article in journal (Refereed) Published
Abstract [en]

Background and Objectives: The redox-regulatory protein thioredoxin has several functions including transcriptional regulation, and antioxidant, cytokine, and chemokine activities. We have previously shown that extracellular thioredoxin protects B-cell chronic lymphocytic leukemia (CLL) cells from apoptosis in vitro. In this study we were interested to determine whether thioredoxin is produced by cells surrounding the CLL cells in the in vivo microenvironment and whether this cell-derived thioredoxin has any leukemia growth-promoting effect in vitro. Design and Methods: Lymph nodes from CLL patients (n=25) were analyzed for thioredoxin expression by immunohistology. Stromal cells purified from the lymph nodes were analyzed for thioredoxin secretion at the single cell level using an ELIspot assay. The survival effect of the stromal-derived thioredoxin was tested by co-culturing stromal- and CLL cells with and without Fab-fragments of an anti-thioredoxin antibody. Results: The results indicated that the thioredoxin production correlated with the amount of proliferating cells and was mainly localized to the proliferation centers (pseudofollicles) in the CLL lymph nodes. The leukemia cells per se showed minimal thioredoxin levels, in contrast, stromal cells strongly expressed thioredoxin. Purified primary stromal cells, which secreted extracellular thioredoxin, significantly protected the CLL cells from undergoing apoptosis in 72 h co-cultures. Interestingly, this anti-apoptotic effect could be abrogated by addition of Fab-fragments of an anti- thioredoxin antibody. Interpretation and Conclusions: In conclusion, we have shown that stromal cells in the lymph node microenvironment produce thioredoxin and that the thioredoxin production is localized to the proliferation centers of the CLL lymph nodes. In addition, thioredoxin produced by purified stromal cells rescued CLL cells from apoptosis in vitro. ©2007 Ferrata Storti Foundation.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-40728 (URN)10.3324/haematol.11448 (DOI)54003 (Local ID)54003 (Archive number)54003 (OAI)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2017-12-13
2. Silenced B-cell receptor response to autoantigen in a poor-prognostic subset of chronic lymphocytic leukemia
Open this publication in new window or tab >>Silenced B-cell receptor response to autoantigen in a poor-prognostic subset of chronic lymphocytic leukemia
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2014 (English)In: Haematologica, ISSN 0390-6078, E-ISSN 1592-8721, Vol. 99, no 11, 1722-1730 p.Article in journal (Refereed) Published
Abstract [en]

Chronic lymphocytic leukemia B-cells express auto/xeno-antigen-reactive antibodies that bind to self-epitopes and resemble natural IgM antibodies in their repertoire. One of the antigenic structures recognized is oxidation-induced malonedialdehyde present on low-density lipoprotein, apoptotic blebs, and on certain microbes. The poor-prognostic stereotyped subset #1 (Clan I IGHV genes-IGKV1(D)-39) express IgM B-cell receptors that bind oxidized low-density lipoprotein. In this study, we have used for the first time this authentic cognate antigen, since it is more faithful to B-cell physiology than anti-IgM, for analysis of downstream B-cell receptor-signal transduction events. Multivalent oxidized low-density lipoprotein showed specific binding to subset #1 IgM/IgD B-cell receptors, whereas native low-density lipoprotein did not. The antigen-binding induced prompt receptor-clustering, followed by internalization. However, the receptor-signal transduction was silenced, revealing no Ca2+ mobilization or cell-cycle entry, while phosphorylated extracellular-regulated kinase1/2 basal levels were high and could not be elevated further by oxidized low-density lipoprotein. Interestingly, B-cell receptor responsiveness was recovered after 48 hours culture in the absence of antigen in half of the cases. Toll-like receptor 9-ligand was found to breach the B-cell receptor-signaling incompetence in 5 of 12 cases pointing to intra-subset heterogeneity. Altogether, this study supports B-cell receptor-unresponsiveness to cognate self-antigen on its own in poor-prognostic subset #1 chronic lymphocytic leukemia indicating that these cells proliferate by other mechanisms that may override B-cell receptor-silencing brought about in a context of self-tolerance/anergy. These novel findings have implications for the understanding of chronic lymphocytic leukemia pathobiology and therapy.

Place, publisher, year, edition, pages
Ferrata Storti Foundation, 2014
Keyword
Anergy; B-cell Receptor Signaling; Chronic Lymphocytic Leukemia; Oxidized LDL; Stereotyped subsets
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-109020 (URN)10.3324/haematol.2014.106054 (DOI)000347016300013 ()25085355 (PubMedID)
Available from: 2014-07-28 Created: 2014-07-28 Last updated: 2017-12-05
3. 450K-array analysis of chronic lymphocytic leukemia cells reveals global DNA methylation to be relatively stable over time and similar in resting and proliferative compartments
Open this publication in new window or tab >>450K-array analysis of chronic lymphocytic leukemia cells reveals global DNA methylation to be relatively stable over time and similar in resting and proliferative compartments
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2013 (English)In: Leukemia, ISSN 0887-6924, E-ISSN 1476-5551, Vol. 27, no 1, 150-158 p.Article in journal (Refereed) Published
Abstract [en]

In chronic lymphocytic leukemia (CLL), the microenvironment influences gene expression patterns; however, knowledge is limited regarding the extent to which methylation changes with time and exposure to specific microenvironments. Using high-resolution 450K-arrays, we provide the most comprehensive DNA methylation study of CLL to date, analysing paired diagnostic/follow-up samples from IGHV-mutated/untreated and IGHV-unmutated/treated patients (n=36) and patient-matched peripheral blood and lymph node samples (n=20). On an unprecedented scale, we revealed 2239 differentially methylated CpG sites between IGHV-mutated and unmutated patients, with the majority of sites positioned outside annotated CpG islands. Intriguingly, CLL prognostic genes (e.g. CLLU1, LPL, ZAP70, NOTCH1), epigenetic regulator (e.g. HDAC9, HDAC4, DNMT3B), B-cell signaling (e.g. IBTK) and numerous TGF-ß and NF-κB/TNF pathway genes were alternatively methylated between subgroups. Contrary, DNA methylation over time was deemed rather stable with few recurrent changes noted within subgroups. Although a larger number of non-recurrent changes were identified among IGHV-unmutated relative to mutated cases over time, these equated to a low global change. Similarly, few changes were identified between compartment cases. Altogether, we reveal CLL subgroups to display unique methylation profiles and unveil methylation as relatively stable over time and similar within different CLL compartments, implying aberrant methylation as an early leukemogenic event.Leukemia accepted article preview online, 27 August 2012; doi:10.1038/leu.2012.245.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-80705 (URN)10.1038/leu.2012.245 (DOI)000313511400021 ()22922567 (PubMedID)
Available from: 2012-08-29 Created: 2012-08-29 Last updated: 2017-12-07
4. B cell receptor signaling suppressor SHP-1 is active in CLL lymph node and peripheral blood
Open this publication in new window or tab >>B cell receptor signaling suppressor SHP-1 is active in CLL lymph node and peripheral blood
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Protein tyrosine phosphatase SHP-1 expression and activity is downregulated or lost in several leukemias and lymphomas due to DNA promotor hypermethylation, catalytic site mutation or oxidation, or phosphorylation at inhibitory sites, implying a negative role of SHP-1 in development of leukemias/lymphomas. In chronic lymphocytic leukemia (CLL), B cell receptor (BcR) and microenvironment signal levels are important in the pathogenesis. Considering that SHP-1 is a BcR signaling suppressor, we hypothesized that SHP-1 would be down-regulated and/or inactivated in the proliferative center lymph node (LN) cells. We analyzed PTPN6 (SHP-1) gene expression, SHP-1 protein expression and phosphorylation status in matched CD5+/CD19+ peripheral blood (PB) and LN cells from 6 CLL patients, and in comparison, BcR (anti-IgM) in vitro triggered CLL PB cells from 10 patients. Gene expression of PTPN6 was significantly higher in PB compared to LN CLL cells in 50% of the cases. SHP-1 protein expression level and phosphorylation at SHP-1Y536 and SHP-1S591 were, however, equal in PB and LN samples. SHP-1 phosphorylation at Y536 and S591, in PB CLL cells cultured ex vivo was significantly reduced upon BcR engagement in all patient samples. These results indicate that in vivo BcR signaling in CLL is paralyzed.

Keyword
B cell, chronic lymphocytic leukemia, SHP-1, suppressor, tyrosine phosphorylation
National Category
Cell and Molecular Biology Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-124575 (URN)
Available from: 2016-02-04 Created: 2016-02-04 Last updated: 2017-09-22Bibliographically approved

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