Long-term effects of idiotype vaccination on the specific T-cell response in peripheral blood and bone marrow of multiple myeloma patients.

OBJECTIVES: To elucidate long-term effects of idiotype (Id) vaccination on Id-specific T cells of multiple myeloma (MM) patients and compare Id-specific T-cell responses of peripheral blood with those of bone marrow (BM). MATERIALS AND METHODS: Id-specific T-cell responses of peripheral blood mononuclear cells (PBMC) were compared with those of BM mononuclear cells (BMMC) in 10 MM patients vaccinated with the Id protein at a median time of 41 months since the last immunization. The PBMC responses at late follow-up were also compared with those during active immunization. The responses were assessed by a proliferation assay, enzyme-linked immunospot (ELISPOT) (gamma-interferon), cytometric bead array (CBA) for secreted cytokines and quantitative real-time polymerase chain reaction (QRT-PCR) for cytokine gene expression. RESULTS: At the late testing time, an Id-specific response was detected in PBMC of five patients (ELISPOT, CBA, QRT-PCR) and in BMMC of four patients (CBA, QRT-PCR). A response in both compartments was noted only in three patients. The cytokines gene profile was consistent with a predominance of Th(2) cells [interleukin (IL)-4, IL-5, IL-10]. Comparison of the Id-specific responses of PBMC during active immunization with those at the late follow-up showed that the frequency and magnitude of the responses had decreased significantly by time (proliferation/ELISPOT) (P < 0.02) and shifted at the gene level from a Th(1) to a Th(2) profile (P < 0.05). CONCLUSION: Id-specific T-cells may decline overtime and shift toward a Th(2) response and may be found at a similar frequency of patients in blood and BM.

Expression of erythropoietin receptor and in vitro functional effects of epoetins in B-cell malignancies.

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 alpha or beta or darbepoetin alpha. RESULTS: EPO-R mRNA was detected in mononuclear cells from 32 of 41 (78%) B-CLL and 5 of 7 (71%) MCL patients, and 21 of 21 (100%) MM samples. Expression was also detected in highly purified T cells 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 T cells. 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.

Long-term idiotype vaccination combined with interleukin-12 (IL-12), or IL-12 and granulocyte macrophage colony-stimulating factor, in early-stage multiple myeloma patients.

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.

Idiotype protein vaccination in combination with adjuvant cytokines in patients with multiple myeloma--evaluation of T-cell responses by different read-out systems.

Anti-idiotypic T cells were analyzed in myeloma patients (n=18) vaccinated with idiotypic protein together with the adjuvant cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interleukin-12 (IL-12). In the group given IL-12/GM-CSF, 78% developed idiotype specific T cells as compared to 22% in the group given only IL-12 (proliferation/ELISPOT assays) (p<0.05). The percentage of immune-responding patients increased when quantitative real time polymerase chain reaction assays for cytokines were included. A predominance of a Th1 (IFN-gamma/TNF-alpha) immune response was noted in the IL-12 group while a Th2 (IL-5) response prevailed in the IL-12/GM-CSF group (p=0.053). Application of multiple read-out systems improved the characterization of the immune response.