Purpose and Experimental Design: Twenty-eight patients with immunoglobulin G myeloma stages I to II were immunized i.d. over 110 weeks with autologous M protein combined with interleukin-12 (IL-12; n = 15) or with IL-12 and granulocyte macrophage colony-stimulating factor (GM-CSF; n = 13). Idiotype-specific T-cell responses were assessed by [ 3 H]thymidine incorporation, enzyme-linked immunospot assay, and delayed-type hypersensitivity reaction. Results: Based on these three assays, idiotype-specific immune responses were noted in 5 of 15 (33%) patients in the IL-12 group and in 11 of 13 (85%) patients in the GM-CSF/IL-12 group (P < 0.01). Immune response was seen only in patients with M-component concentration of <50 g/L. Three of 16 (19%) responders showed a gradually increasing idiotype-specific T-cell response, whereas 11 of 16 (69%) patients showed initial response, which then disappeared rapidly; the latter pattern was frequently associated with subsequent progressive disease. Immune nonresponse was associated with an increase in the numbers of CD4 + /CD25 + cells (regulatory T cells), which was absent in responding patients. Median time to progression for immune responders (n = 16) was 108 weeks compared with 26 weeks for nonresponders (n = 12; P = 0.03). Conclusions: These results indicate that idiotype immunization of myeloma patients with GM-CSF and IL-12 may induce specific T-cell response more frequently than with IL-12 alone and that immune response may correlate with time to progression and nonresponse with increased numbers of regulatory T cells.

Purpose: Erythropoietin (EPO) and EPO receptor (EPO-R) expression have been reported in solid tumors and are claimed to regulate tumor growth; however, no data have been published on this issue in B-cell malignancies or normal lymphoid cells. This report describes genomic/protein EPO-R expression and in vitro effects of recombinant human EPO (epoetin) in B-cell chronic lymphocytic leukemia (B-CLL), mantle-cell lymphoma (MCL), and multiple myeloma (MM). Experimental Design: Blood samples were obtained from patients with B-CLL, MCL, and healthy volunteers, and bone marrow was obtained from MM patients. EPO-R mRNA was detected by reverse transcription-PCR. EPO-R surface expression was investigated by flow cytometry using digoxigenin-labeled epoetin and polyclonal rabbit anti^EPO-R antibody for intracellular receptor. Tumor cell stimulation was determined in vitro using [ 3 H]thymidine incorporation and CD69 expression after exposure to epoetin a or h or darbepoetin a. Results: EPO-R mRNA was detected in mononuclear cells from 32 of 41 (78%) B-CLL and 5 of 7 (71%) MCL patients, and 21of 21 (100%) MM samples. Expression was also detected in highly purifiedTcells from six of eight B-CLL patients, four of four MM patients, and normal donor B and T cells. Surface EPO-R protein was not detected. Intracellular EPO-R staining with anti^EPO-R antibodies was unspecific. No tumor-stimulatory effect was observed with high epoetin concentrations. Conclusions: EPO-R gene is frequently expressed in lymphoid malignancies and normal B and Tcells. However, there was no surface protein expression and no epoetin-induced in vitro stimulation of tumor B cells, indicating that epoetin therapy in vivo is likely to be safe in patients with lymphoid malignancies.Erythropoietin (EPO), the principal regulator of erythropoiesis, is a glycoprotein hormone produced by the kidney and fetal liver (1). EPO prevents apoptosis, stimulates growth, and promotes the differentiation of RBC progenitors by interacting with a specific transmembrane receptor (EPO-R) expressed on the cell surface (2). EPO-R is a member of a cytokine receptor family that lacks the tyrosine kinase domain (2). Upon activation, EPO-R homodimers undergo conformational changes and initiate a Janus kinase signal transducer, which, in turn, activates the transcription factor STAT5 that regulates cell proliferation and differentiation (2 -4). EPO and EPO-R expression have recently been shown in several nonhematopoietic tissue types, including embryonic, nervous system, uterine, ovarian, and endothelial cells, suggesting a broader biological role for EPO signaling (5 -8). The EPO -EPO-R pathway might be involved in a variety of cellular functions, including cellular growth, survival, and proliferation as well as in the promotion of angiogenesis and the prevention of ischemic and toxic-stress tissue injuries (5 -14).Great concern and debate have recently emerged about the potential role of EPO and recombinant human EPO in promoting tumorigenesis not only in vitro (2 -4) but als...

The time kinetics of five cytokines [interleukin-2 (IL-2), IL-5, interferon-g (IFN-g), granulocyte macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor-a (TNF-a)] and one cytotoxic effector protein (granzyme B) was analysed by real-time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)].