University of Leicester
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In vitro Generation of Cytotoxic T Cells With Potential for Adoptive Tumor Immunotherapy of Multiple Myeloma

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journal contribution
posted on 2019-08-29, 14:07 authored by Wafaa S. Khalaf, Mamta Garg, Yehia S. Mohamed, Cordula M. Stover, Michael J. Browning
Multiple myeloma is a life-threatening hematological malignancy, which is rarely curable by conventional therapies. Immunotherapy, using tumor antigen-specific, cytotoxic T-lymphocytes, may represent an alternative or additional treatment for multiple myeloma. In this study, we used hybrid cell lines, generated by fusion of an EBV B-lymphoblastoid cell line (B-LCL) and myeloma cells, to stimulate in vitro peripheral blood lymphocytes (PBLs) from patients with multiple myeloma. We investigated induction of antigen-specific, cytotoxic T-lymphocytes to the well-defined tumor associated antigens (TAAs) hTERT, MUC1, MAGE-C1 and CS1, which have been shown to be expressed in a high proportion of cases of multiple myeloma. HLA-A2-peptide pentamer staining, interferon-γ and perforin ELISpot assays, as well as cytotoxicity assays were used. Following several rounds of in vitro stimulation, the hybrid cell lines induced antigen-specific, cytotoxic T-lymphocytes to four candidate TAAs in PBLs from HLA-A2+ multiple myeloma patients, using known HLA-A2 restricted peptide epitopes of the TAAs. In contrast, the HLA-A2+ myeloma cell line U266 failed to induce antigen-specific, cytotoxic T-lymphocytes in vitro. Our data indicate that B-LCL/myeloma hybrid cell lines induce antigen-specific, cytotoxic T-lymphocytes in PBLs isolated from multiple myeloma patients in vitro and may represent a novel strategy for use in adoptive immunotherapy of multiple myeloma.


WK was supported by a Ph.D. studentship award from the Government of Egypt.



Frontiers in Immunology, 2019, 10:1792.

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/Organisation/COLLEGE OF LIFE SCIENCES/School of Medicine/Department of Infection, Immunity and Inflammation


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The Supplementary Material for this article can be found online at: Supplementary Table 1. Patient characteristics from whom the starting material for CTL culture was obtained (time of study: 2015–2016; NBD, no paraprotein band detected in serum). Supplementary Table 2. Numbers of PBMCs after each stimulation in a representative example of one stimulation experiment. Supplementary Figure 1. Percentages of positive MUC1, h TERT, CS1, MAGE-C1 and WNF HLA-A*201 restricted, PE peptide pentamer-stained CTL clones. PBMCs isolated from four HLA A2- patients with MM were stimulated in vitro for 4 weeks using the hybrid cell lines HRC and HU266, or the parent cell lines HMY2 and U266 as stimulators. The cultures were stained with HLA-A2-peptide pentamers incorporating cognate peptide antigen form MUC 1, hTERT, CS1, MAGE-C1, or the irrelevant WNF peptide, followed by flowcytometric analysis. Responses of individual cell lines are shown on the X-axis. Percent of pentamer-positive cells is shown on the Y-axis. Results are presented as mean ± SEM. Supplementary Figure 2. IFNγ ELISpot assays. Responder cells (PBMCs, isolated from three different HLA-A2- MM patients and stimulated with HMY2, HU266, HRC or U266) were mixed with T2 stimulator cells after pulsing with MUC1, h TERT, CS1, MAGE-C1 or WNF derived peptides) in IFNγ ELISpot assays. Responder:stimulator cell ratios are shown on the X-axis. Number of elicited spots is shown on the Y-axis. Data are presented as mean value of the patients' responses ± SEM. Stimulated PBMCS in the long-term culture of the used cell lines without further stimulation were used as a negative control in each corresponding ELISpot assay of the same cell lines. (There were insufficient cells in the cultures to perform perforin Elispot assays). Supplementary Figure 3. IFNγ releasing ELISpot assay in one of HLA-A2 negative patient. There are observed increases of IFNγ releasing responses in the case of MUC1 (in all of the used ratios), h TERT (in 1:100 and 1:50), and CS1 only in 1:100 ratio (A). Flowcytometric histograms of HLA-A2 expression by the HLA-A2 negative patient (in the upper row), and one of the HLA-A2 positive patients in the lower row. The left hand histogram of each line represents the isotype control staining of this patient, and the right hand histogram represents cells stained with the HLA-A2 specific mAb BB7.2 (B). Flow cytometric analysis of the stability of HLA-A2 molecule of T2 cell line pulsed with the examined MM antigens, irrelevant WNF peptide antigen, or unpulsed T2 cell line (C). Stability of HLA-A2 pulsed with WNF, MUC1, h TERT, CS1 and MAGE-C1 using flow cytometric analysis (MFI of HLA-A2 expression on T2 pulsed peptide) and Syfpeithi score (D). Supplementary Figure 4. The cytotoxic activity of the long-term stimulated antigen specific T lymphocytes. PBMCs isolated from three different HLA-A2- MM patients, were stimulated in vitro as above, and directed against T2 cell line pulsed with the relevant HLA-A2 restricted TAA-derived peptides of MUC1, h TERT, CS1 and MAGE-C1, or an irrelevant antigen peptide (WNF virus). K562 cells were used as NK activity controls. Responder:stimulator cell ratios are shown on the X-axis. Percent specific cytolysis is shown on the Y-axis. Data are presented as mean ± SEM of % specific lysis. No statistically significant responses were seen in any of the assays compared with negative controls.