The cytokine/chemokine pattern in the bone marrow environment of multiple myeloma patients.

OBJECTIVE: The interaction of multiple myeloma (MM) with its bone marrow (BM) microenvironment is important for the homing pattern, survival, and proliferation of malignant plasma cells. We aimed at answering the question which cytokines, chemokines, and growth factors are typically found in the BM of untreated MM patients as well as in MM patients after allogeneic stem cell transplantation (alloSCT). MATERIALS AND METHODS: We determined the concentrations of 34 cytokines/chemokines in the supernatants of 10 myeloma cell lines, as well as in the plasma derived from BM and peripheral blood samples of 10 newly diagnosed MM patients, 20 MM patients who had received allogeneic stem cell transplantation (alloSCT), and 20 healthy donors. RESULTS: Besides cytokines/chemokines known to be secreted by myeloma cell lines, such as interleukin-1 receptor antagonist (IL-1RA), IL-8, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1ß, and MIP-3α, we also detected significant levels of epidermal growth factor (EGF), hepatocyte growth factor (HGF), IL2R, IL-12p40/p70, IL-22, interferon-γ (IFN-γ)-inducible protein 10 (IP-10), monokine induced by IFN-γ (MIG), and regulated on activation normally T-cell expressed and secreted (RANTES) in culture supernatants. The BM environment in MM patients evidenced elevated concentrations of HGF, IL-2R, IL-16, EGF, IL-1RA, IP-10, MCP-1, and monokine induced by IFN-γ. Additionally, in the BM of MM patients post alloSCT, we found selectively elevated concentration of IL-4, IL-6, IL-8, IL-12p40/p70, and eotaxin. Eotaxin levels were particularly high in patients with chronic graft-vs-host disease. CONCLUSIONS: Our study demonstrates characteristic cytokine/chemokine patterns in the BM environment of MM patients before and after alloSCT. Certain factors, such as MIP-1α, MCP-1, HGF, IL-16, IP-10, and eotaxin, might not only be developed into diagnostic instruments and/or predictive biomarkers, but are also potential targets for future myeloma- or graft-vs-host disease-specific therapies.

Expression, epigenetic regulation, and humoral immunogenicity of cancer-testis antigens in chronic myeloid leukemia.

OBJECTIVE: Cancer-testis (CT) antigens represent attractive targets for tumor immunotherapy based on their tumor-restricted expression and immunogenicity. However, a broad picture of the expression of CT antigens and associated humoral immune responses in chronic myeloid leukemia (CML) is still missing. METHODS: We screened CML cell lines and bone marrow (BM) samples from healthy donors by RT-PCR for the expression of 31 CT antigens before and after treatment with epigenetic agents. Expression of tumor-restricted antigens was further examined in 60 CML patients and humoral immune responses against 15 CT antigens were screened by ELISA. RESULTS: In untreated cell lines we detected the expression of 17 CT antigens that were absent from normal BM. Expression of most antigens increased following demethylating treatment with 5'-Aza-2'-Deoxycytidine. In these samples, only PRAME was repeatedly detected and expression correlated with several clinicopathological parameters and decreased overall survival. We further show that a lower frequency of PRAME-positive samples during imatinib treatment was not caused by gene-specific downregulation. Analyzing the patients' antibody responses we found that the vast majority of patients lacked spontaneous immunity against CT antigens including PRAME. CONCLUSIONS: CT antigen expression can be increased by the application of epigenetic agents and the expression of PRAME correlates with clinicopathological parameters and overall survival in patients with CML, but does not lead to humoral immune responses. PRAME-specific immunotherapy might represent a promising approach for the eradication of residual therapy-resistant leukemic cells due to its frequent expression and stability under imatinib treatment.

An optimized assay for the enumeration of antigen-specific memory B cells in different compartments of the human body.

OBJECT: In the framework of our current study we set out to develop an optimized assay for the quantification of antigen-specific B cells in different compartments of the human body. METHODS: Mononuclear cells (MNC) derived from the peripheral blood, bone marrow (BM), or human tonsils were incubated with different combinations of stimuli. The stimulated cells and culture supernatants were then applied to IgG-ELISPOT and ELISA read-out assays and tetanus toxoid (TT)-specific B cell responses were quantified. RESULTS: We found that a combination of CD40L, CpG, and IL21 was optimal for the induction of TT-specific IgG-producing cells from memory B cell (mBc) precursors. This cocktail of stimuli led to a proliferation-dependent induction of CD19(intermediate)CD38(high)CD138(high)IgD(negative) terminally differentiated plasma cells. Applying our optimized methodology we were also able to quantify mBc specific for cytomegalovirus and influenza virus A. Most importantly, the same method proved useful for the comparison of mBc frequencies between different compartments of the body and, accordingly, we were able to demonstrate that TT-specific mBc preferably reside within tonsillar tissue. CONCLUSION: Here, we optimized an assay for the quantification of antigen-specific B cells in different human tissues demonstrating, for example, that TT-specific mBc preferably reside in human tonsils but not in the BM or the peripheral blood. We suggest that our approach can be used for the enumeration of mBc specific for a wide variety of Ag (microbial, tumor-related, auto-antigens), which will lead to significant improvements regarding our knowledge about the biology of humoral immunity.

Cancer-testis antigens MAGE-C1/CT7 and MAGE-A3 promote the survival of multiple myeloma cells.

BACKGROUND: Multiple myeloma is a life-threatening disease and despite the introduction of stem cell transplantation and novel agents such as thalidomide, lenalidomide, and bortezomib most patients will relapse and develop chemoresistant disease. Therefore, alternative therapeutic modes for myeloma are needed and cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 have been suggested to represent a class of tumor-specific proteins particularly suited for targeted immunotherapies. Surprisingly, the biological role of cancer-testis genes in myeloma remains poorly understood. DESIGN AND METHODS: We performed the first investigation of the function of two cancer-testis antigens most commonly expressed in myeloma, MAGE-C1/CT7 and MAGE-A3, using an RNA interference-based gene silencing model in myeloma cell lines. Functional assays were used to determine changes in proliferation, cell adhesion, chemosensitivity, colony formation, and apoptosis resulting from gene-specific silencing. RESULTS: We show that the investigated genes are not involved in regulating cell proliferation or adhesion; however, they play an important role in promoting the survival of myeloma cells. Accordingly, knock-down of MAGE-C1/CT7 and MAGE-A3 led to the induction of apoptosis in the malignant plasma cells and, importantly, both genes were also essential for the survival of clonogenic myeloma precursors. Finally, silencing of cancer-testis genes further improved the response of myeloma cells to conventional therapies. CONCLUSIONS: Cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 play an important role in promoting the survival of myeloma cells and clonogenic precursors by reducing the rate of spontaneous and chemotherapy-induced apoptosis and might, therefore, represent attractive targets for novel myeloma-specific therapies.