IRF4 silencing inhibits Hodgkin lymphoma cell proliferation, survival and CCL5 secretion

Interferon regulatory factor 4 (IRF4) expression is detected in many lymphoid and myeloid malignancies, and may be a promising therapeutic target. IRF4 is strongly expressed in classical Hodgkin lymphoma (cHL) and its expression is up-regulated by CD40L and down-regulated by both anti-proliferative and pro-apoptotic stimuli. This study analysed the effects of IRF4 silencing in a panel of HL-derived cell lines. We demonstrated that IRF4 down-modulation determined a remarkable decrease of both cell number and clonogenic growth in L-1236, L-428, KM-H2 and HDLM-2 cells, but not in IRF4-negative L-540 cells. IRF4 silencing induced apoptosis, as evaluated by caspase-3 activation and Annexin-V staining and up-regulation of the pro-apoptotic molecule Bax. CD40 engagement by both soluble and membrane bound-CD40L almost totally reduced IRF4 down-modulation and growth inhibition by IRF4 silencing in both L-1236 and L-428 cells. Finally, IRF4 silencing decreased CCL5 secretion in all HL cell lines tested and CCL17 in KM-H2 cells. Taken together, our results demonstrated that IRF4 down-modulation by IRF4 silencing was reversed by CD40 engagement, inhibited HL cells proliferation, induced apoptosis and decreased CCL5 secretion, thus suggesting that IRF4 may be involved in HL pathobiology.     

Novel epitope evoking CD138 antigen-specific cytotoxic T lymphocytes targeting multiple myeloma and other plasma cell disorders

The development of an immunotherapeutic strategy targeting CD138 antigen could potentially represent a new treatment option for multiple myeloma (MM). This study evaluated the immune function of CD138 peptide-specific cytotoxic T lymphocytes (CTL), generated ex vivo using an HLA-A2-specific CD138 epitope against MM cells. A novel immunogenic HLA-A2-specific CD138(260-268) (GLVGLIFAV) peptide was identified from the full-length protein sequence of the CD138 antigen, which induced CTL specific to primary CD138(+) MM cells. The peptide-induced CD138-CTL contained a high percentage of CD8(+) activated/memory T cells with a low percentage of CD4(+) T cell and naive CD8(+) T cell subsets. The CTL displayed HLA-A2-restricted and CD138 antigen-specific cytotoxicity against MM cell lines. In addition, CD138-CTL demonstrated increased degranulation, proliferation and γ-interferon secretion to HLA-A2(+) /CD138(+) myeloma cells, but not HLA-A2(-) /CD138(+) or HLA-A2(+) /CD138(-) cells. The immune functional properties of the CD138-CTL were also demonstrated using primary HLA-A2(+) /CD138(+) cells isolated from myeloma patients. In conclusion, a novel immunogenic CD138(260-268) (GLVGLIFAV) peptide can induce antigen-specific CTL, which might be useful for the treatment of MM patients with peptide-based vaccine or cellular immunotherapy strategies.     

4q loss is potentially an important genetic event in MM tumorigenesis: identification of a tumor suppressor gene regulated by promoter methylation at 4q13.3, platelet factor 4

In this study, we have elucidated the chromosomal imbalances in the multistep pathogenesis and delineated several critical tumor suppressor gene (TSG) loci in multiple myeloma (MM). By using comparative genomic hybridization, allelotyping, and multicolor interphase fluorescence in situ hybridization, 5 MM cell lines and bone marrow CD138+ plasma cells from 88 Chinese patients with monoclonal gammopathy of undetermined significance (MGUS) and early and advanced stages of MM were investigated. In all MGUS and MM samples, chromosome copy number abnormalities were detected. A higher number of chromosomal imbalances and specific genetic alterations are involved in MGUS to MM transition (-6q, +3p, and +1p) and MM progression (+2p and +9q). In addition to -13q, we first found high frequencies (42% to 46%) of -4q involving high percentages (70% to 74%) of clonal plasma cells in both MGUS and MM, suggesting that inactivation of TSG in this region is also a potentially critical genetic event in MM tumorigenesis. By high-resolution allelotyping, we defined a common deletion region on 4q13.3 and found that a candidate TSG, platelet factor 4, was frequently silenced by promoter hypermethylation in MM (15 of 28) and MM cell lines (5 of 5). These data have opened up a new approach in the molecular targeting therapy and provide novel insights into MM tumorigenesis.     

Preclinical development of hybrid cell vaccines for multiple myeloma

Immunotherapy may provide alternative or supplementary treatment of multiple myeloma (MM). We propose that hybrid cells, formed by fusing professional antigen-presenting cells with malignant plasma cells, would induce immune responses capable of mediating tumour regression. The human B-lymphoblastoid cell line, HMy2, was fused in vitro with CD138+ bead-separated myeloma plasma cells from five patients with MM. The hybrid cell lines generated in these studies grew stably in tissue culture, and maintained their phenotypic and functional characteristics, providing self-renewing cell lines with potential for therapeutic vaccination. The hybrid cells stimulated allogeneic and autologous T-cell proliferative responses in vitro to a considerably greater degree than their respective parent myeloma plasma cells, and directly activated both CD4+ and CD8+ T-cell responses. The enhanced T-cell stimulation correlated with expression of CD80 on the hybrid cells, and was inhibited by CTLA4-Ig fusion protein. The hybrid cell lines expressed several tumour-associated antigens known to be expressed in myeloma. These data show that self-replicating cell lines with enhanced immunostimulatory properties and potential for therapeutic vaccination can be generated by in vitro fusion of ex vivo myeloma cells and B-lymphoblastoid cell lines.     

Expression of MUM1/IRF4 selectively clusters with primary effusion lymphoma among lymphomatous effusions: implications for disease histogenesis and pathogenesis

Primary effusion lymphoma (PEL) is a peculiar B-cell lymphoma characterized by infection by human herpesvirus type-8/Kaposi sarcoma-associated herpesvirus (HHV-8/KSHV) and by preferential growth in the serous body cavities. Histogenetic studies have suggested that PEL originates from B cells at a late stage of differentiation. In this study, we have investigated PEL for the expression status of MUM1/IRF4 (multiple myeloma 1/interferon regulatory factor 4) protein, which is involved in physiological B-cell maturation and represents a histogenetic marker of late B-cell differentiation. Using multiple detection assays, all cases of PEL (n = 22) were found to express MUM1/IRF4 molecules. MUM1/IRF4 expression was a selective feature of PEL among lymphomas involving the serous body cavities as secondary lymphomatous effusions generally failed to express the protein. In reactive lymphoid tissues, MUM1/ IRF4 expression clustered with advanced stages of B-cell differentiation. Comparison of MUM1/IRF4 expression with that of other histogenetic markers defined two phenotypic variants of PEL, i.e. MUM1/IRF4+, CD138/syndecan-1+, B-cell antigen- (20 out of 22 cases) and MUM1/IRF4+, CD138/syndecan-1-, B-cell antigen+ (2 out of 22 cases), suggesting a certain degree of heterogeneity in the disease histogenesis. The implications of these data are threefold. First, MUM1/IRF4 expression corroborates the notion that PEL originates from post-germinal centre, preterminally differentiated B-cells. Second, MUM1/IRF4 may help in the differential diagnosis of PEL among other lymphomas involving the serous body cavities. Finally, MUM1/IRF4 may interact with HHV-8/KSHV-encoded interferon regulatory factors (IRFs) and thus contribute to PEL escape from interferon-mediated control of viral infection.     

Characterization of clonogenic multiple myeloma cells

The identity of the cells responsible for the initiation and maintenance of multiple myeloma (MM) remains unclear largely because of the difficulty growing MM cells in vitro and in vivo. MM cell lines and clinical specimens are characterized by malignant plasma cells that express the cell surface antigen syndecan-1 (CD138); however, CD138 expression is limited to terminally differentiated plasma cells during B-cell development. Moreover, circulating B cells that are clonally related to MM plasma cells have been reported in some patients with MM. We found that human MM cell lines contained small (< 5%) subpopulations that lacked CD138 expression and had greater clonogenic potential in vitro than corresponding CD138⁺ plasma cells. CD138^- cells from clinical MM samples were similarly clonogenic both in vitro and in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, whereas CD138⁺ cells were not. Furthermore, CD138^- cells from both cell lines and clinical samples phenotypically resembled postgerminal center B cells, and their clonogenic growth was inhibited by the anti-CD20 monoclonal antibody rituximab. These data suggest that MM "stem cells" are CD138^- B cells with the ability to replicate and subsequently differentiate into malignant CD138⁺ plasma cells.