Comparative Proteomic Analysis of Bone Marrow Plasma Cells By Using Mass Spectrometry Based Bottom up Proteomic Strategies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5334-5334
Author(s):  
Ayhan Beycan ◽  
Duygu Fatma Ozel Demiralp ◽  
Klara Dalva ◽  
Meral Beksaç
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
B Cells ◽  
Molecular Mechanism ◽  
Plasma Cells ◽  
Hla Class I ◽  
Negative Control ◽  
Tumor Rejection ◽  
Class I ◽  
Apoptotic Process

Abstract Aim : As all other cancer types, multiple myeloma is a complex disease, yet its molecular mechanism that is not fully understood and needs to be furtherly investigated. Besides, the molecular mechanism responsible from the transition of healthy cells into malignant ones is still under investigation. In such comparative studies, human myeloma cell lines are preferred as positive control, while tonsillar plasma cells are used as negative control. The aim of this study is to perform a comparative proteomic analysis regarding the progression of Multiple Myeloma (MM) by using protein profiles of plasma cells obtained from MGUS, SMM, symptomatic myeloma patients, tonsil plasma cells obtained from healthy individuals and human myeloma cells of cell line origin to elucidate the underlying molecular mechanism involved in myeloma. Material and Methods: Five groups were adopted in the experiment. Of these groups, MM patients were classified mainly into three groups according to marrow plasma cell content (PCC) validated by flow cytometry: group1: 0-9, group 2: 10-20 and group 3: >20 %. The fourth group consisted of human myeloma cell line (HMCL) RPMI 8226 and was designated as positive control, while tonsil plasma cells were used as negative control and specified as the fifth group. Marrow samples were collected from 30 patients newly diagnosed with Multiple Myeloma ( n: 28 symptomatic and n: 1 smoldering (SMM)) and MGUS (n: 1)).Tonsil plasma cells were isolated from healthy tonsil tissue treated with trypsin enzyme and were then confirmed by using Selection Kit microbead specific for EasySep Human CD138 marker. Protein profile maps of groups were obtained via 2D gel electrophoresis and comparative analysis and detection of up/down regulated protein spots was performed by using PDQuest 8.01 software. Proteins from differently expressed protein spots were then identified using Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI) by Peptide Mass Fingerprinting (PMF) analysis Results: By using bottom up strategies, nine of the significantly expressed protein spots were identified with PMF analyses. The identified proteins are as follows:, Calreticulin (ERp60), Endoplasmin/ tumor rejection antigen (ERp99), MZB1(Marginal zone B and B1 cell specific protein/ pERp1), Actin cytoplasmic1 (ACTB), Thioredoxin domain-containing protein 5 (TXNDC5/ERp46), Protein disulfide isomerase (PDI) and HLA class I histocompatibility antigen (MHC class I antigen A*2). According to the results, calreticulin (which functions as chaperones and is responsible for Ca+2regulation and cell proliferation) showed a weak positive correlation with increase in patient PC content. The level was lowest in normal B cells. Endoplasmin/ tumor rejection antigen (which is assigned as chaperones and function in apoptotic process and Ca+2 regulation)declined by increase in PC content in patient samples. Level was lowest in HMCL. Marginal zone B and B1 cell specific protein (which functions as chaperones and is involved in apoptotic process, oxidoreductase activity, regulation of Ca+2, cell proliferation and activates B cell quantiity) correlated with PC percentage and was highest in HCML and lowest in normal B cells. Actin cytoplasmic1 protein (which functions in cell proliferation)synthesis showed a minimum decline in synthesis by increase in PC content .Level was highest in normal B cells. Thioredoxin domain-containing protein 5 which plays role in apoptotic process and shows oxireductase activity within MM/MGUS plasma cells also displayed a positive correlation but we were not able to detect a difference in HMCL and normal B cells which were synthesized less than the patient samples. Protein disulfide isomerase (which acts as chaperones and plays part in apoptotic process and shows oxireductase function) has a weak negative correlation with PC content and synthesis was lowest in HMCL. HLA class I histocompatibility antigen production did not correlate with PC content , it was higher in normal B cells compared to HMCL. Conclusion: In conclusion in this study we were able to confirm our previous findings which were presented at ASH 2013 by comparing the results with normal tonsil CD138+ plasma cells and a HMCL. Our study results suggest that the proteins which are involved in Calcium metabolism, chaperone binding and oxidative stress are also associated with low vs. high proliferation profile in myeloma. Disclosures No relevant conflicts of interest to declare.

HLA Ligandome Analysis in Multiple Myeloma Reveals Novel Naturally Presented Myeloma-Associated Antigens Showing Stable Expression Under Proteasome Inhibitor Therapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3438-3438
Author(s):  
Simon D. Walz ◽  
Daniel J. Kowalewski ◽  
Heiko Schuster ◽  
Katja Weisel ◽  
Helmut R. Salih ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cell ◽  
Proteasome Inhibitor ◽  
Plasma Cells ◽  
Hla Class I ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Fold Change ◽  
Class I ◽  
Myeloma Cells

Abstract Results of allogeneic transplantation and reports that multiple myeloma (MM) cells can act as antigen presenting cells, but also the favorable immune effector-to-target cell ratio in the minimal residual disease (MRD) setting suggest that MM, which previously was considered to be weakly immunogenic, may be targeted effectively by T cell based immunotherapy. Following conventional therapy, peptide-based immunotherapy might offer a low side-effect opportunity to extend time to disease progression and possibly eradicate MRD. Therefore we aim to develop a multi-peptide vaccine for the immunotherapy of MM patients based on naturally processed and presented HLA ligands. In a first step we analyzed HLA surface expression on MM cells, which constitutes a prerequisite for effective T cell recognition. In MM patients (n=20), HLA class I expression was found to be heterogeneous with a mean of 415,000 ± 55,000 molecules on malignant cells. This expression was significantly (p<0.005, unpaired t-test) higher than that on autologous healthy B cells (200,000 ± 20,000), T cells (165,000 ± 15,000) and hematopoietic progenitor cells (205,000 ± 30,000). In addition, HLA class I expression on primary MM cells was also found to be significantly higher than that on plasma cells of healthy volunteers (HV, n=15) (290,000 ± 25,000, p<0.05). Mean HLA-DR expression on myeloma cells (25,000 ± 7,000) was significantly lower compared to HLA class I expression (p<0.0001) without significant difference compared to expression levels on autologous bone marrow cells as well as normal plasma cells of HVs (40,000 ± 5,000). Using the approach of direct isolation and identification of naturally presented HLA peptides by affinity chromatography and mass spectrometry, we identified more than 15,000 different HLA class I ligands from 8 primary MM samples, 2 plasma cell leukemia samples and 5 myeloma cell lines (MCLs). Comparative mapping of MM HLA ligandomes and the ligandomes of 40 HVs (30 blood and 10 bone marrow samples) revealed 15 ligandome-derived tumor-associated antigens (LiTAAs) represented by 36 corresponding HLA peptides (LiTAPs), showing exclusive representation in >30% of myeloma samples. Among them we identified 3 novel HLA peptides derived from Multiple Myeloma SET domain(MMSET), which is an established driver of myeloma cell proliferation, stimulating the expression of c-myc. We further studied the effect of the second-generation proteasome inhibitor carfilzomib on HLA surface expression and HLA ligandome composition. Notably, a slight albeit not significant increase in mean HLA class I expression was observed after 24h (t24) and 48h (t48) of carfilzomib treatment in MCL (n=5, fold change t24=1.4, pt24=0.37, fold change t48=1.6, pt48=0.25) as well as in primary myeloma cells in vitro (n=7, fold change t24=1.2, pt24=0.21, fold change t48=1.1, pt48=0.46). No significant loss or down regulation of HLA class I expression on MM cells was observed in ex vivo analysis of primary samples obtained from 2 myeloma patients before therapy and after 4 weeks of carfilzomib treatment. In order to analyze the impact of proteasome inhibition on the composition of the HLA class I ligandome we incubated MCLs (U266, MM.1S) with carfilzomib and performed quantitative mass spectrometry at baseline as well as at t24 and t48. On U266, 4.6% (4.0%) and 4.4% (8.6%) of total identified peptides (n=2444) were significantly up- or down regulated after 24h/48h of treatment with carfilzomib, respectively, whereas on MM.1S (n=1693) 1.6% (12.4%) and 10.8% (18.0%) were significantly regulated after 24h/48h. Focusing on the LiTAPs presented on these MCLs, 76.5% (88.2%) on U266 and 83.3% (66.7%) on MM.1S remained stably expressed after incubation with carfilzomib. Further analyses evaluating overall changes in peptide motifs and the occurrence of novel, cryptic peptides after carfilzomib treatment are ongoing. Taken together our data demonstrate that myeloma cells are valid targets for T cell based immunotherapy and provide a panel of novel, naturally presented myeloma-associated antigens, which exhibit stable presentation under proteasome inhibitor therapy. Disclosures No relevant conflicts of interest to declare.

Assessment of intracellular TAP‐1 and TAP‐2 in conjunction with surface MHC class I in plasma cells from patients with multiple myeloma

1997 ◽  
Vol 98 (2) ◽  
pp. 426-432 ◽  
Author(s):  
Brian E. Crucian ◽  
Lynn C. Moscinski ◽  
Matthew Androlewicz ◽  
Oscar F. Ballester ◽  
Raymond H. Widen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mhc Class I ◽  
Plasma Cells ◽  
Class I

scholarly journals "Intermediate" Cell Types and Mixed Cell Proliferation in Multiple Myeloma: Electron Microscopic Observations

Blood ◽  
1966 ◽  
Vol 27 (2) ◽  
pp. 212-226 ◽  
Author(s):  
JORGE E. MALDONADO ◽  
ROBERT A. KYLE ◽  
ARNOLD L. BROWN ◽  
EDWIN D. BAYRD
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Plasma Cells ◽  
Cell Transformation ◽  
Cell Types ◽  
Lymphoid Cells ◽  
Intermediate Cell ◽  
Electron Microscopic ◽  
Mixed Cell ◽  
Reticulum Cells

Abstract Bone marrow studies of multiple myeloma revealed, in some cases, a conspicuous proliferation of "lymphoid" cells, virtually indistinguishable by light microscopy from those seen in lympho-proliferative disorders. Electron microscopy demonstrated a variety of cells ranging from typical lymphocytes to cells with plasmocytoid features. Between these two types of elements there were cells with intermediate characteristics. In addition, in several cases of myeloma the presence of fixed reticuloendothelial cells and "reticular" plasma cells (or reticulum cells with plasmocytic features) was frequently noted. The presence of reticulum cells and lymphocytes and of cells apparently "intermediate" between these cellular elements and plasma cells, as judged from electron microscopic observations, is suggestive morphologic evidence of a phenomenon of cell transformation and evidence of a mixed cell proliferation in certain cases of multiple myeloma.

scholarly journals HLA Class I Antibody-Mediated Endothelial Cell Proliferation via the mTOR Pathway

2008 ◽  
Vol 180 (4) ◽  
pp. 2357-2366 ◽  
Author(s):  
Peter T. Jindra ◽  
Yi-Ping Jin ◽  
Enquire Rozengurt ◽  
Elaine F. Reed
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cell ◽  
Hla Class I ◽  
Mtor Pathway ◽  
Class I ◽  
Endothelial Cell Proliferation

scholarly journals T-cell malignancies with mature phenotypes: altered cell cycle regulation by HLA class I molecules

Blood ◽  
1991 ◽  
Vol 78 (8) ◽  
pp. 2045-2052 ◽  
Author(s):  
MC Turco ◽  
F Alfinito ◽  
M De Felice ◽  
A Lamberti ◽  
S Ferrone ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Dna Synthesis ◽  
Hla Class I ◽  
Class I ◽  
Mitogenic Effect ◽  
Prolymphocytic Leukemia ◽  
T Lymphocyte Proliferation ◽  
Hla Class I Molecules

Abstract Soluble anti-HLA class I monoclonal antibodies (MoAbs) modulate normal T-lymphocyte proliferation induced via the CD3/Ti and the CD2 pathway, but do not induce proliferation of normal T lymphocytes in the absence of additional mitogenic stimuli. In this report, we show that anti-HLA class I MoAbs induce DNA synthesis in peripheral blood mononuclear cells from a patient with a CD4+CD8+T-prolymphocytic leukemia (T-PLL) and from a patient with a CD4-CD8+ T-chronic lymphocytic leukemia (T- CLL), in the absence of detectable additional mitogenic stimuli. Proliferation of leukemic T cells is induced by both whole Igs and Fab' fragments of anti-HLA class I MoAbs, arguing in favor of their direct interactions with the proliferating cells as the mechanism underlying the mitogenic effect. This interpretation is also supported by the ability of anti-HLA class I MoAbs to induce proliferation of leukemic T- cell preparations, depleted of accessory cells. DNA synthesis in T-CLL and T-PLL cells is preceded by expression of G1-specific messenger RNAs, ie. c-myc, 2F1, Tac, and interferon-gamma, in activated cells. Cell proliferation is inhibited by the protein kinase C inhibitor H7, indicating that activation of this enzyme is required for the mitogenic effect of anti-HLA class I MoAbs. The latter inhibit the proliferation of T-CLL cells as well as that of normal T cells stimulated with anti- CD3 MoAbs and enhance that of both types of cells stimulated with anti- CD2 MoAbs. In addition, anti-HLA class I MoAb Q6/64 in combination with anti-CD2 MoAb 9.6 or MoAb 9–1 induces proliferation of leukemic T cells to a greater extent than the individual MoAbs, but is not mitogenic for normal T cells. Anti-HLA class I MoAbs restore the cytolytic activity of T-CLL cells that is lost after 5 days of incubation of control medium, suggesting that HLA class I antigens may mediate a signal contributing to the activation state. The present results indicate that leukemic T-cell proliferation can be triggered via HLA class I molecules and suggest a potential role for these antigens in the in vivo growth of malignant clones.

scholarly journals Fas-Independent Apoptosis of Activated T Cells Induced by Antibodies to the HLA Class I α1 Domain

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3629-3639 ◽  
Author(s):  
Laurent Genestier ◽  
Romain Paillot ◽  
Nathalie Bonnefoy-Berard ◽  
Geneviéve Meffre ◽  
Monique Flacher ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Heavy Chain ◽  
Hla Class I ◽  
Class I ◽  
T Cell Proliferation ◽  
Activated T Cells ◽  
Hla Class I Molecules

Abstract In addition to their major function in antigen presentation and natural killer cell activity regulation, HLA class I molecules may modulate T-cell activation and proliferation. Monoclonal antibodies (MoAbs) that recognize distinct epitopes of HLA class I molecules were reported to interfere with T-cell proliferation. We show here that two MoAbs (mouse MoAb90 and rat YTH862) that bind to an epitope of the α1 domain of HLA class I heavy chain induce apoptotic cell death of activated, but not resting, peripheral T lymphocytes. Other reference anti-HLA class I antibodies specific for distinct epitopes of the α1 (B9.12.1), α2 (W6/32), or α3 (TP25.99) domains of the heavy chain decreased T-cell proliferation but had little or no apoptotic effect. Apoptosis shown by DNA fragmentation, phosphatidylserine externalization, and decrease of mitochondrial transmembrane potential was observed whatever the type of T-cell activator. Apoptosis did not result from Fas/Fas-L interaction and distinct though partly overlapping populations of activated T cells were susceptible to Fas– and HLA class I–mediated apoptosis, respectively. Induction of apoptosis did not require HLA class I cross-linking inasmuch as it could be observed with monovalent Fab′ fragments. The data indicate that MoAb90 and YTH862 directed against the α1 domain of HLA class I trigger apoptosis of activated T lymphocytes by a pathway which does not involve Fas-ligand.

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