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Multiple Myeloma |
From the Immune and Gene Therapy Laboratory, Cancer Centre Karolinska, Stockholm, Sweden (AOA, LH, IE, HR, HM, AO); Departments of Hematology and Oncology, Karolinska University Hospital Solna (LH, EDR, HM, AO); Department of Oncology/Pathology, Karolinska Institutet, Solna, Stockholm, Sweden (AOA, LH, IE, BN-G, HR, HM, AO)
Correspondence: Håkan Mellstedt, MD, Ph.D., Professor, Department of Oncology, Karolinska University Hospital Solna, SE-171 76, Stockholm, Sweden. E-mail: hakan.mellstedt{at}karolinska.se
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ABSTRACT |
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 TOP ABSTRACT Design and MethodsResults and DiscussionReferences |
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/TNF-
) 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.
Key words: idiotype, vaccination, myeloma, T-cell response.
The idiotype (Id) is a B-cell tumor antigen, and a unique target for immunotherapy of multiple myeloma (MM). A detailed characterization of a vaccine-induced T-cell response is crucial for the evaluation of the efficacy of a vaccine and the assessment of even a low frequency of antigen-specific T cells might be of value. A T-cell response against tumor antigens, which are often weak self-antigens, such as the Id, might be enhanced by adjuvant cytokines.1 Granulocyte-macrophage colony-stimulating factor (GM-CSF) augments the activity of antigen presenting cells and interleukin-12 (IL-12) favors a Th1 polarized immune response,2 which is of importance for an anti-tumor effect. The main objectives of this study were to characterize in detail the anti-Id T-cell response in MM patients vaccinated with idiotypic protein in combination with GM-CSF and/or IL-12 and to evaluate the potential of different read-out systems for the detection of Id-specific T cells.
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Design and Methods |
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TOPABSTRACTDesign and MethodsResults and DiscussionReferences |
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Immunization protocol and immune testing
The Id vaccine was prepared as previously described.4 Patients were allocated to receive 0.5 mg of the Id vaccine i.c. on day 1 in the left arm, and 2 µg IL-12 (Genetics Institute, Inc., Wyeth-Ayerst Research, Cambridge, MA, USA) s.c. in the contralateral arm (IL-12 group) (n=9) or the Id vaccine and IL-12 as described above plus 75 µg of GM-CSF (Schering-Plough, Kenilworth, NJ, USA) i.c. at the vaccine site daily, days 1 to 4 (IL-12/GM-CSF group) (n=9). The vaccination was repeated at weeks 2, 4, 6, 8, 14 and 30. Patients were tested for cellular immune responses before vaccination and at weeks 4, 8, 10, 14, 30, and 32.
Immune monitoring
Id-specific T cells were evaluated by several read-out systems. Peripheral blood mononuclear cells were isolated from blood and stimulated with F(ab)2 fragments of the autologous or isotype matched monoclonal IgG (1 pg/mL to 100 µg/mL).5 Proliferation assay, ELISPOT for interferon [IFN] polymerase chain reaction, quantitative real time PCR (QRT-PCR) for cytokines (IFN-, tumor necrosis factor- [TNF-], IL-5) and cytotoxic proteins (granzyme B, perforin) and cytokine flow cytometry for intracellular IFN- were performed as described elsewhere. 6,7 The CBA (cytometric bead array) Human Th1/Th2 Cytokine kit (Becton-Dickenson, Mountain View, CA, USA) was used for analysis of secreted cytokines (IFN-, TNF-, IL-5).
Criteria for a vaccine-induced Id-specific T-cell response, Th1/Th2 profiling, and clinical response
A vaccine-induced Id-specific T-cell response was considered to be present when (i) the autologous Id-specific response was greater than the isotype-matched one; (ii) above the corresponding cut-off level; (iii) more than twice the pre-vaccination value; (iv) and detected at two or more separate times. A cellular response was defined as Th1 when only Th1 cytokine genes (IFN- and/or TNF-) were expressed and as Th2 when only the IL-5 gene was expressed. When all three cytokine genes were expressed the response was considered as mixed Th1/Th2 with a Th1 predominance when the IFN- and/or TNF- gene fold increase ratio was higher or expressed at more testing times than IL-5 and as a mixed Th1/Th2 with a Th2 predominance when the IL-5 gene fold increase ratio was higher than that of IFN- and/or TNF-. A clinical response was defined according to the Myeloma Subcommitte of the European Group for Blood and Marrow Transplantation (EBMT).8
Statistical methods
The 
2 test was used to compare differences between groups and Spearman’s rank correlation test to estimate correlations between assays.
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Results and Discussion |
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TOPABSTRACTDesign and MethodsResults and DiscussionReferences |
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), QRT-PCR for cytokine genes] used to evaluate all 18 patients for Id-specific T cells (Tables 1, 2). Nine of the patients (50%) developed an Id-specific T-cell response in the proliferation and/or ELISPOT assays. Seven of the responding patients were in the IL-12/GM-CSF group (78%) while only two were in the IL-12 group (22%) (p<0.05). Vaccine-induced immunity was noted more frequently in patients with a pre-vaccination Id-specific immunity (7/9) than in those without (2/9) (proliferation/ELISPOT) (p<0.02). A vaccine-induced response at the gene level (QRT-PCR) was noted in five patients in the IL-12 group and six in the IL-12/GM-CSF group (Table 2). There was a strong trend towards a Th1 polarized response in the Il-12 group (4/5) whereas this type of response was less common in the IL-12/GM-CSF group (2/8) in which Th2 immunity prevailed (p=0.053) (Table 1). By adding determination of the Id-specific response at the gene level, the percentage of overall Id-specific responses increased from 50% (9/18) to 78% (14/18) (Table 1).
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and cytokine bead array for secreted cytokines. The results are summarized in Figure 1. An Id-specific proliferative T-cell response was noted in two patients while Id-specific IFN- protein production was observed in three patients in ELISPOT and four by cytokine bead array. Id-specific IFN- gene expression (evaluated by QRT-PCR) was noted in three patients. Id-specific intracellular IFN- protein was detected by cytokine flow cytometry in four patients. The frequency of specific T cells detected by cytokine flow cytometry varied between 1.3% and 2.6% and the T cells were of both CD4 and CD8 phenotypes (data not shown). Assessment of the Id-specific T-cell response by cytokine bead array measuring two additional cytokines (TNF- and IL-5) showed that both Th1 (IFN- and/or TNF-) and Th2 (IL-5) cytokines were secreted (Figure 1). No correlation was noted between the expression of these cytokines (measured by cytokine bead array) and the corresponding genes (measured by QRT-PCR). A statistically significant correlation was only seen comparing the Id-specific IFN- gene and IFN- protein expression determined by cytokine flow cytometry (r=0.79, p<0.0001).
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In the present study IL-12 seemed to favor a Th1 polarized response, while the combination of IL-12 and GM-CSF favored Th2 immunity. The combination of the two cytokines has also been reported to down-regulate the magnitude of the immune response as compared to that produced by IL-12 alone.14 The impact of GM-CSF on a Th1/Th2 response is not clear. We have previously reported that GM-CSF may favor a Th1 response,5 while others have observed an unbiased mixed cellular response.15 The timing of GM-CSF administration might be of importance. The administration of GM-CSF 3 days before an HIV-DNA vaccine induced mainly a Th2 response whereas simultaneous administration evoked both Th1 and Th2 responses. When GM-CSF was injected 3 days after the vaccine, a Th1 response predominated.16 In this study, patients with spontaneous Id-specific immunity more frequently mounted Id-specific immunity compared to those with no pre-vaccination immunity. This indicates that the Id antigen has the potential to break tolerance and might be a good candidate vaccine. These results support those of previous studies showing that patients with a pre-existing T cell immunity are more prone to mount an immune response than are tumor antigen immune naïve patients.17
Antigen-specific T cells consist of a polyclonal population of cells producing various cytokines. Measuring only IFN-, as in ELISPOT, might underestimate the frequency of antigen-specific cells.18 By adding QRT-PCR to evaluate multiple cytokines, T-cell subsets producing different cytokines might be detected. Indeed, when this assay was included, the frequency of immune responders increased and functional capability could be predicted. The addition of granzyme B and perforin assays indicated that the majority of the patients had Id-specific T cells with cytolytic activity.
A significant correlation was noted between the Id-specific expression of the IFN- gene (measured by QRT-PCR) and the protein as detected by cytokine flow cytometry. A strong correlation has previously been reported between QRT-PCR and cytokine flow cytometry findings for IFN-.19 In our study there were, however, testing times when the IFN- protein was detected but not the corresponding gene transcript. The time point selected for analyzing cytokine gene expression after antigen activation might be critical. We previously showed that 18 h was an acceptable time of antigen activation for the detection of IFN- against a recall antigen, but there was a great inter-individual variation.6 The time kinetics of cytokine gene expression might vary for different antigens and gene expression might be missed, despite the presence of the corresponding protein, if evaluations are conducted at only a few activation times.
More than 20 Id vaccine trials have been performed in MM patients. Roughly 55% of the patients mounted an Id-specific response, but only 12% had a clinical response. Clinical responders were also noted among patients having no detectable Id-specific response.20 In the present study, during the first 32 weeks of follow-up, none of the patients had a clinical response regardless of whether they were immune responders (n=14) or not (n=4). A clinical benefit might, however, be noted late during follow-up and a longer observation period is needed. Based on the present limited material, firm recommendations on the choice of assays for immune monitoring cannot be made. Extended vaccine trials are needed. However, the application of multiple read-out assays seems to improve the detection and characterization of a weak immune response.
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Footnotes |
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AOA: performed all the real time PCR assays and analysis and wrote the manuscript. LH: analyzed proliferation and ELISPOT assays and defined vaccine induced responses by these assays. IE, BN-G: carried out the proliferation, ELISPOT, CBA, and flow cytometry assays and contributed to the analysis; EDR: put out the cut-off level for flow cytometry analysis; HR: contributed to the design of project and real time PCR probes and primers; HM: group leader and provided all the necessary supervision, study design and corrections; AO: senior co-supervisor and contributed to planning the project arranging material and correcting various versions of the manuscript.
The authors reported no potential conflicts of interest.
Received for publication April 3, 2006. Accepted for publication November 21, 2006.
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References |
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TOPABSTRACTDesign and MethodsResults and DiscussionReferences |
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This article has been cited by other articles:
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  A. O. Abdalla, P. Kokhaei, L. Hansson, H. Mellstedt, and A. Osterborg Idiotype vaccination in patients with myeloma reduced circulating myeloma cells (CMC) Ann. Onc., June 1, 2008; 19(6): 1172 - 1179. [Abstract] [Full Text] [PDF]
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