Generation of Potent Antitumor CTL from Patients with Multiple Myeloma Directed against HM1.24

Multiple myeloma is a B-cell malignancy characterized by the accumulation of terminally differentiated plasma cells in the bone marrow. Despite initial response to chemotherapy, most patients relapse with chemoresistant disease and there are few long-term survivors. The demonstration of autologous idiotype-specific T cells (1) and evidence of clinical response to allogeneic donor leucocyte infusions (2) indicate that antitumor responses can be generated. In addition, the disease course typically enters a plateau phase of minimal, or at least stable disease, following initial response to therapy, when immunotherapeutic strategies may have a role in eradicating remaining tumor cells. The generation of a protective antitumor response requires efficient presentation of the appropriate tumor antigen/s to stimulate an effective and robust T-cell response. The unique ability of dendritic cells to cross-present exogenous antigens (3), and hence to prime naive T cells, inducing both CD4 T helper as well as CD8 CTL responses, together with the development of protocols for the isolation, culture, and antigen loading of these cells has led to a great interest in their use in cancer immunotherapy. Work in animal models has shown that tumor antigen–pulsed dendritic cells are capable of inducing a protective and therapeutic antitumor response (4, 5), and several clinical trials have now been reported in a range of malignant diseases (reviewed ref. 6).

The idiotype determinant on the myeloma paraprotein is an obvious candidate tumor antigen, and several studies of vaccination with idiotype-pulsed dendritic cells have been reported (7–10). Although both cellular and humoral anti-idiotype immune responses have been seen, clinical responses have not been impressive. This may be because of the prevalence of the tumor antigen in the form of circulating paraprotein, or because the idiotype is not sufficiently immunogenic. There is thus a pressing need to identify alternative tumor antigens in multiple myeloma. Candidate tumor antigens include Muc-1 (11), telomerase (12), survivin (13), Sp-17 (14), PRAME, and members of the MAGE-A family (15). Members of the cancer testis group of antigens like Sp-17, and the epithelial antigen, Muc-1 are highly immunogenic, and CTLs have been generated against these antigens; however, these antigens are only expressed on a proportion of primary multiple myeloma cells (14, 16). HM1.24 is a recently described type II glycoprotein that is preferentially and highly expressed on malignant plasma cells (17). Expression was barely detectable on normal B cells and was not detected on other normal tissues, including bone marrow, liver, heart, kidney, and spleen (18). Immunotherapy with a monoclonal antibody (mAb) directed against HM1.24 dramatically reduced tumor size and improved survival in an animal model of myeloma (19), indicating that HM1.24 may be a potential tumor antigen. We have recently completed a phase I clinical trial of a humanized anti-HM1.24 mAb, in which there was very little toxicity (20).

The aim of this work was to evaluate the feasibility of generating HM1.24-specific antitumor CTL from patients with multiple myeloma using protein-pulsed dendritic cells and to evaluate the functional capabilities of these CTL.

Patients. Bone marrow and peripheral blood were obtained from patients with newly diagnosed or relapsed/refractory multiple myeloma after informed consent. Leucapheresis products were obtained at the time of peripheral blood stem cell harvest in preparation for high dose therapy. Approval for the study was obtained from the Joint UCL/UCLH Committees for the Ethics of Human Research.

Myeloma cell lines. RPMI 8226 cell line was cultured in RPMI 1640 (Invitrogen Co., Grand Island, NY) with 10% FCS (PAA Laboratories Gmbh, Linz, Austria). U266, MM1.R, MM1.S, KMS-BM, and KMS-PE cell lines were all cultured in RPMI 1640 with 10% fetal bovine serum (Perbio, Logan, UT). KPMM2 cells were cultured in RPMI 1640/20% fetal bovine serum supplemented with interleukin 6 (IL-6, 20 ng/mL). MOLP-5, which is a human bone marrow stroma–dependent cell line, was cultured in RPMI 1640/10% fetal bovine serum. RPMI 8226 and U266 lines were obtained from American Type Culture Collections (Rockville, MD). MM1.R and MM1.S (21) were a kind gift of Dr. S. Rosen (Northwestern University, Chicago, IL). KMS-BM and KMS-PE (22) were a kind gift of Dr. T. Ohtsuki (Kawasaki Medical School, Kurashiki, Japan) and KPMM2 (23) was supplied by Chugai Pharmaceutical (Tokyo, Japan). MOLP-5 was a kind gift of Dr. Y.Matsuo (Fujisaki Cell Center, Fujisaki, Okayama, Japan). Cell lines were passaged every 2 to 3 days and maintained at 1 to 5 × 10⁵/mL.

Primary malignant plasma cells. Fresh bone marrow aspirates from patients with newly diagnosed or relapsed multiple myeloma were collected in RPMI 1640 supplemented with preservative-free Heparin. Malignant plasma cells (MPC) were obtained from bone marrow mononuclear cells by selection with CD138 magnetic beads (LS separation columns, Miltenyi Biotech, Gladbach, Germany) according to manufacturers' instructions.

Production of soluble HM1.24 protein. A soluble HM1.24 antigen (Asn⁴⁹ to Ala¹⁶³) was designed to remove the putative cytoplasmic and transmembrane domains and 17 amino acid residues of the COOH terminus, and a HA-tag was added to its NH₂ terminus. A leader peptide from immunoglobulin was used to obtain efficient secretion into culture medium. The DNA sequence encoding this fusion protein was inserted into a pCHO1 (24) expression vector. Soluble HM1.24 antigen secreting Chinese hamster ovary cells were cultured in α MEM (Life Technologies, Grand Island, NY) medium containing 10% fetal bovine serum and 100 nmol/L 4-amino-10-methylpteroylglutamic acid (Sigma, St. Louis, MO). Soluble HM1.24 antigen protein was purified from culture supernatant by affinity chromatography using an anti-HM1.24 antibody conjugated column and reverse-phase chromatography (C4).

Generation of dendritic cells. Fresh leucapheresis fractions or peripheral blood from patients with multiple myeloma were centrifuged over Ficoll-Hypaque to obtain peripheral blood mononuclear cells (PBMC). An aliquot (15 × 10⁶ per well in a 6-well plate) was allowed to adhere for 2 hours in RPMI 1640/10% FCS at 37°C. Nonadherent cells were removed and the adherent cells were then cultured in X-VIVO 20 (Bio Whittaker, Walkersville, MD) with 10% autologous serum, IL-4 (500 units/mL), and granulocyte macrophage colony-stimulating factor (800 units/mL; both from Insight Biotechnologies, Middlesex, United Kingdom) for 7 days, with replenishment of cytokines on days 3 and 6. The remaining PBMC were cryopreserved at −80°C until required. On day 5, dendritic cell cultures were pulsed with 50 μg/mL HM1.24 protein, before maturation with CD40 ligand (300 ng/mL, Alexis Biochemicals, Nottingham, United Kingdom) on day 6. On day 7 of culture, dendritic cells were harvested, γ-irradiated, and set up in coculture with autologous PBMC. In selected experiments as indicated in Results, pooled human AB serum was used in place of autologous serum.

Generation of HM1.24-specific T lymphocytes. HM1.24 protein–loaded dendritic cells (1 × 10⁵/mL) were incubated with freshly isolated PBMC (1 × 10⁶/mL) in X-VIVO 20 with 3% autologous serum (or AB serum in some experiments, as indicated), IL-7 (20 ng/mL), and IL-12 (100 pg/mL) at 37°C and 5% CO₂. Cultures were replenished every 4 days with fresh cytokines and were restimulated with HM1.24-loaded dendritic cells every 7 days. At each restimulation, T cells were initially cultured for 48 hours without cytokines, after which IL-2 (20 IU/mL) and IL-7 (20 ng/mL) were added. IL-15 (10 ng/mL) was added for the final 7 days of culture. T cells were harvested after two rounds of restimulation and used in ELISPOT and cytotoxicity assays. All cytokines were obtained from Pepro Tech (Rocky Hill, NJ).

IFN-γ ELISPOT assays. Briefly, millititer HA plates (Millipore, Bedford, MA) were coated with anti-IFN-γ mAb (1-DK-1 clone; Mabtech, Stockholm, Sweden) at 2 μg/mL in HBSS (Life Technologies) overnight at 4°C. Plates were washed four times in PBS (without Ca²⁺ Mg²⁺; Life Technologies) and blocked with RPMI 1640/10% AB serum for 1 hour at 37°C in a humidified incubator. After a further round of washes with PBS, stimulator cells (1 × 10⁴ per well) and effector cells (1-5 × 10⁴ per well) were introduced as triplicates in 100 μL of RPMI 1640 and incubated at 37°C, 5% CO₂ for 20 hours. Stimulator cells included CD138 selected autologous and allogeneic MPC, HM1.24-loaded autologous PBMC (produced by incubating freshly isolated PBMC with 50 μg/mL HM1.24 protein overnight at 4°C) and unmanipulated autologous PBMC. After four washes with PBS, wells were incubated with a secondary biotinylated anti-IFN-γ mAb (0.2 μg/mL, Mabtech) at room temperature for 2 hours on a plate mixer. After further washing, wells were incubated with a streptavidin alkaline phosphatase conjugate (Caltag Laboratories, Burlingame, CA; 1:1,000 in PBS) for 1 hour on a plate mixer. An Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad Laboratories, Hercules, CA) containing an unmixed 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium solution and color development buffer was used to develop the spots. Plates were analyzed using an automatic plate reader (Elispot Reader System, AID, Strassberg, Germany).

Flow cytometric–based cytotoxicity assay. MPC were thawed and kept overnight at 4°C in RPMI 1640/10% FCS. The next day, cells were stained with PKH26 (4 μmol/L; Sigma-Aldrich, St. Louis, MO) for 2 minutes on ice. After addition of cold FCS, labeled cells were pelleted, washed once, and incubated with effector T cells at 37°C for 3 hours. Before analysis, TOPRO-3-Iodide (0.8 μmol/L, Sigma-Aldrich) was added to stain up dead cells. Percent cytotoxicity was analyzed by flow cytometry, gating on the PKH-positive population. Control target cells included autologous CD19-selected B cells, and allogeneic MPC. In some experiments, target cells were preincubated with antibodies to HLA class I (W6/32 clone, Serotec, Oxford, United Kingdom) or HLA class II (B1.12 clone, Immunotech, Marseille, France) at a concentration of 100 μg/mL for 30 minutes before the cytotoxicity assay. Antibodies were present throughout the assay. In experiments to test the mode of cytotoxicity, Concanamycin A (CMA, Sigma-Aldrich) or Brefeldin A (Sigma-Aldrich) were used as selective inhibitors of the perforin exocytosis and Fas secretion pathways, respectively. Effectors were pretreated with 100 nmol/L CMA or 10 μmol/L Brefeldin A for 2 hours at room temperature before cytotoxicity assays.

Use of autologous HM1.24-transfected dendritic cells as “cold competitors” in cytotoxicity assays. CDNA encoding HM1.24 was subcloned into the EcoRI and NotI sites of pCOS-1 vector. Plasmid DNA was isolated by Megaprep (Qiagen, Cawley, United Kingdom) and quantified by spectrophotometry. Dendritic cells were generated from autologous PBMC as above. After 5 days, dendritic cells were transfected with pCOS-1 vector containing HM1.24 cDNA. A transfection mixture comprising 2 μg cDNA with 6 μL of FUGENE (Roche Diagnostics, Mannheim, Germany) in a total of 100 μL of X-VIVO 10 (Bio Whittaker) was allowed to stand for 2 hours at room temperature before incubation with dendritic cells in serum-free medium in the presence of granulocyte macrophage colony-stimulating factor and IL-4. After 6 hours, serum was added to the dendritic cell cultures. After 48 hours, dendritic cells were harvested and expression of HM1.24 confirmed by staining with an FITC-conjugated anti-HM1.24 mAb. Transfected dendritic cells were then used in cytotoxicity assays as competitive targets (10:1 ratio with autologous plasma cells).

Flow cytometry. Isolated primary MPC and myeloma cell lines were analyzed for expression of HM1.24 using an FITC-conjugated HM1.24 mAb (14). Phenotype analysis of T cells was done using the following mAbs: CD3 FITC, CD4 allophycoerythrin, CD8 peridinin chlorophyll protein, CD45RO phycoerythrin, and CD45RA FITC. These mAbs were purchased from Becton Dickinson (UK). Cells were incubated with 10 μL mAb for 30 minutes on ice, washed in 2 mL PBS/0.5% bovine serum albumin, and fixed in 2% paraformaldehyde before fluorescence-activated cell sorting analysis. Fluorescence analysis was done on a flow cytometer (EPICS Elite, Beckman-Coulter).

Statistics. Data were compared using the Mann-Whitney U test for nonparametric data. P < 0.05 was considered significant.

HM1.24 is highly expressed on primary malignant plasma cells and myeloma cell lines. Bone marrow MPC from all patients with multiple myeloma examined displayed significant levels of HM1.24 expression, the majority of samples contained >80% cells positive for HM1.24 (median, 84.4% positive; range, 48.7-99.3%; n = 17). Flow histograms are illustrated for eight representative patients in Fig. 1 (1-8). All myeloma cell lines tested also showed high levels of HM1.24 expression (97.2 ± 3.6% positive; n = 8; Fig. 1, 9-16).

HM1.24-primed T cells secrete IFN-γ in response to HM1.24 expressing autologous malignant plasma cells but not allogeneic malignant plasma cells. The function of HM1.24-primed T cells was initially assessed in an ELISPOT assay for IFN-γ secretion. HM1.24-primed T cells were set up against a panel of stimulator cells including autologous HM1.24-loaded PBMC and autologous MPC. As illustrated in Fig. 2A, HM1.24-primed T cells showed a significantly greater cytokine response to HM1.24-loaded autologous PBMC (median, 342; range, 198-495 IFN-γ–producing units/10⁵) compared with the response to unloaded PBMC (median, 98; range, 7-137; P < 0.05; n = 5). Primed T cells also showed an enhanced response to autologous MPC (median, 227; range, 153-335) but not to allogeneic MPC (median, 57; range, 22-158; P < 0.05; n = 5). The response to the negative controls was not significantly greater than the baseline responses of T cells in the absence of stimulator cells (median, 30; range, 6-74; n = 5). The antigen specificity of our T-cell culture system was assessed by testing the responses of control T cells that had been cultured and restimulated with unloaded dendritic cells. In contrast to the responses of HM1.24-primed T cells, control T cells showed minimal responses to autologous or allogeneic HM1.24-expressing stimulator cells (Fig. 2B). We also tested freshly isolated T cells from our patients in the ELISPOT assay, to investigate whether HM1.24-specific T cells are present in the circulation. We found that freshly isolated T cells, unlike cultured and HM1.24-primed T cells had no cytokine response to HM1.24-expressing stimulators (data not shown).

HM1.24-primed CTL lyse autologous tumor cells in a HLA class I–dependent fashion. A total of 13 patients were investigated for their ability to generate HM1.24-directed CTL capable of lysing autologous MPC. In one patient, T cells failed to expand after the first restimulation step with protein loaded dendritic cells. T cells from the other 12 patients were tested in cytotoxicity assays against autologous, and in some cases, allogeneic MPC. All MPC from patients were found to express HM1.24 (>70% positive in 11 patients and 49% in the case of patient 02). Baseline target cell death in the assays was low, 7.5% (median; range, 2.8-24.3) for autologous MPC and 10.5% (range, 2.9-39.0) for allogeneic MPC. T cells from all 12 patients tested exhibited cytotoxic activity against autologous MPC (at 20:1 E/T ratio: median, 48% specific killing; range, 23-88%, at 10:1 E/T ratio: median, 43%; range 15-80%). Results for the first six individual patients (patients 01-06) are shown in Fig. 3A, which also illustrates that killing of allogeneic MPC was either absent or at very low levels. This would suggest that the CTL activity is MHC restricted.

The killing seen in our cytotoxicity assays was unlikely to be mediated by natural killer cells, as >95% of the effector cells typed as CD3⁺ (see below), and in two patients tested, there was no killing of K562 cells (data not shown). Cytotoxicity of autologous MPC was inhibited by an anti-MHC class I mAb (median, 89% inhibition; range, 81.5-98%; n = 4), whereas anti-MHC class II mAb was without effect in three patients (median, 5.8% inhibition; range, 0-11.7%) and produced partial inhibition in a fourth (patient 01, 31.6% inhibition; Fig. 3B). The CTL activity of HM1.24-primed T cells was reproducible, as shown by repeat CTL experiments using the same targets 24 or 48 hours afterwards (data not shown).

To test the effector specificity of the T cell culture system, we generated control T cells by culture and restimulation with unloaded dendritic cells. In contrast to HM1.24-primed T cells, control T cells displayed minimal cytotoxicity against both autologous MPC (10:1 E/T ratio: median, 7.2% killing; range, 3.2-20.4%; n = 4) and allogeneic MPC (10:1 E/T ratio: median, 4.9%; range, 2.4-11.4%; n = 3; Fig. 3C). In addition, there is a clear dose relationship between E/T ratio and level of cytotoxicity displayed by HM1.24-primed T cells (Fig. 3D; patients 0.08 and 09).

HM1.24-specific CTL lyse autologous malignant plasma cells through the perforin-mediated pathway. To determine the mechanism by which CTL lyse MPC, effector cells were preincubated with either CMA or Brefeldin A as detailed in Materials and Methods. CMA treatment of HM1.24-primed CTL led to a significant reduction in target cell lysis (72.8% and 90.8% inhibition for patients 01 and 03, respectively), whereas Brefeldin A was without effect (Fig. 4). These results indicate that CTL activity against plasma cells is mediated by the granule exocytosis pathway. There was no effect of either inhibitor on the viability of CTL as assessed by TOPRO-3-Iodide staining in the cytotoxicity assay (78.4 ± 6.0% viable with CMA, 75.1 ± 4.3% viable with Brefeldin A, compared with 75.8 ± 4.3% viability of control cells).

Target specificity of CTL activity. To test the target specificity of the CTL generated in our system, we used autologous CD19-selected B cells as control targets. In two experiments (patients 011 and 012), we showed that whereas HM1.24-primed CTL exhibited killing of autologous MPC (46.4%, 23.8% cytotoxicity at E/T ratio of 10:1, respectively), there was only low level or no killing of autologous B cells (10% and 0% cytotoxicity at E/T ratio of 10:1, respectively). As dendritic cells were cultured in autologous serum that contained M-proteins, it is possible that the culture system also stimulated idiotype-specific T cells that may have contributed to the lysis of autologous MPC. To exclude this possibility, we carried out experiments using human AB serum in place of autologous serum in our culture system. We tested two patients (patients 07 and 09) in this manner and showed CTL activity against autologous MPC (Fig. 5). Additionally, when T cells from patient 09 were tested in the ELISPOT assay, HM1.24-primed T cells showed cytokine responses to HM1.24-loaded PBMC (148 IFN-γ–producing units/10⁵) but not to unloaded PBMC (48 IFN-γ–producing units/10⁵).

We also carried out one experiment in a patient with nonsecretory multiple myeloma (patient 010) and were able to show, using autologous serum in our cultures, that patient CTL effectively lysed autologous MPC (70% cytotoxicity at E/T ratio 20:1 and 61% at E/T ratio 10:1).

HM1.24-transfected but not untransfected autologous dendritic cells inhibit CTL killing of autologous plasma cells. To confirm that killing of autologous MPC was HM1.24 specific, we carried out “cold competition” assays, using HM1.24-expressing autologous dendritic cells as competitive targets. Autologous dendritic cells were transfected with plasmid containing HM1.24 cDNA on day 4 of culture and showed high antigen expression 48 hours later, as assessed by staining with FITC-conjugated anti-HM1.24 mAb and flow cytometry (Fig. 6A). HM1.24-transfected dendritic cells were used as “cold competitors” at 10:1 ratio with PKH-labeled autologous plasma cells in cytotoxicity assay. As seen in Fig. 6B, the presence of HM1.24-transfected dendritic cells almost completely blocked cytolysis of autologous MPC (89.2% and 97.2% inhibition for patients 05 and 06, respectively; Fig. 6B). In contrast, unmanipulated dendritic cells were ineffective in blocking the CTL mediated killing of target cells. In one further experiment, autologous dendritic cells transfected with a control plasmid were used as control competitors; these dendritic cells were unable to block killing of autologous MPC, whereas HM1.24-transfected dendritic cells produced 80% inhibition of killing. This indicates that the cytolytic activity of HM1.24-primed CTL is specific for HM1.24 epitopes on target MPC.

HM1.24-primed CTL comprise both CD4⁺ and CD8⁺ cells and exhibit an activated CD45RO⁺ phenotype. CTL harvested for cytotoxicity assays were analyzed for surface phenotype. Greater than 95% of CTL generated in our coculture system were CD3⁺ T cells and these comprised varying proportions of CD4⁺ and CD8⁺ cells (Fig. 7A). Both CD4⁺ and CD8⁺ T cell populations displayed mainly an activated CD45RO⁺ phenotype (Fig. 7B) and a distinct cell population displaying a central memory phenotype (CCR7⁺ and CD62L⁺) could be identified in two of three patients tested (Fig. 7C).

The successful generation of an effective immune response against tumor cells is a prerequisite for any immunotherapeutic strategy for cancer. Here we show, for the first time, the ex vivo generation of HM1.24 antigen–specific CTL from the blood of patients with multiple myeloma, which display high levels of lytic activity against primary autologous MPC. CTL activity was induced following stimulation with HM1.24 protein–pulsed dendritic cells, was MHC restricted and mediated by the perforin exocytosis pathway. The antigen specificity of the CTL activity was confirmed by the inhibition seen when autologous HM1.24-transfected dendritic cells were used as competitors in the CTL assay. T cells produced by our culture system comprise both CD8⁺ and CD4⁺ cells, with a predominantly activated/memory phenotype. These results indicate that HM1.24 is not only immunogenic but is also a suitable CTL target for immunotherapy in the treatment of multiple myeloma.

Several tumor antigens have been tested in multiple myeloma, among them Muc-1, telomerase, survivin, Sp-17 and of course, idiotype. Although many studies have shown CTL activity against the putative tumor antigen (11–14), most of these studies have used normal donors and cell lines as targets. An exception is the report by Chiriva-Internati et al. on generation of CTL from three patients with multiple myeloma, which were capable of killing Sp-17–positive autologous tumor cells (14). Although clearly immunogenic, Sp-17 is only expressed on a fraction of malignant plasma cells. In contrast, HM1.24 is expressed on all primary myeloma cells, as well as multiple myeloma cell lines. Our study is therefore unique in demonstrating potent autologous antitumor CTL activity (at levels approaching 60% using E/T ratios of 10:1) directed against a tumor antigen expressed on all myeloma cells. The HM1.24 antigen has also recently been tested in a system employing AAV-mediated gene transfer into dendritic cells to generate CTL from healthy donors (25). In contrast to our work, those authors used only healthy donors whereas the responses that we show here are all autologous antitumor responses in patients with multiple myeloma.

An alternative approach to the use of a particular tumor antigen is to employ whole myeloma cells/apoptotic bodies or tumor lysates thereof because of the potential to use all myeloma antigens (24, 26). Yet another approach is to use tumor-derived RNA to load dendritic cells (27). Notably, both tumor lysates and apoptotic multiple myeloma cells when loaded onto dendritic cells were able to stimulate the production of CTLs, which lysed autologous plasma cells (26, 28). These approaches, however, suffer from the drawback that they require large numbers of tumor cells at the outset and may generate immune responses to some self antigens. The advantages of our culture system are the relative ease of generating antigen-specific CTL without requiring large numbers of tumor cells. Whereas peptide-based approaches are restricted by patient HLA type, the strength of our system that uses whole tumor protein is that immune responses are generated against multiple epitopes; hence, it is applicable to patients irrespective of HLA type. As we are able to generate significant CTL responses using whole protein, there seems little to be gained by exploring peptide based strategies for HM1.24. Any such strategies would have to employ a cocktail of peptides, each tailored to the individual patient HLA type, so as to induce both CD8⁺ and CD4⁺ responses. Our work not only provides important confirmatory data on this tumor antigen but forms the basis for clinical application of this antigen. Our culture system is able to generate large numbers of antigen-specific CTL and is readily adaptable to GMP standards, unlike gene therapy–based protocols.

One problem facing vaccination strategies in cancer therapy is that in vivo immune pathways in these patients are likely to be ineffective. This may be because tumor antigens are only weakly immunogenic, or that specific factors in vivo impair either or both effector and antigen-presenting cell function. Peripheral blood T cells from patients with multiple myeloma are reported to contain fewer CD4⁺ cells and to have impaired mitogenic and CTL responses (29), while peripheral blood dendritic cells may be functionally impaired in responses to activation or maturational stimuli (30). In addition, the tumor microenvironment contains factors that may impair antitumor immune responses. Vascular endothelial growth factor, for example, which is up-regulated in the multiple myeloma bone marrow, has been reported to inhibit the maturation of dendritic cells, resulting in T-cell tolerance rather than activation (31). Bone marrow sera from patients with multiple myeloma have been reported to inhibit dendritic cell activation, an effect attributed to high concentrations of vascular endothelial growth factor and IL-6 (28). Thus, many mechanisms may cooperate in vivo to induce tolerance to tumor antigens. Hence, the lack of clinical responses seen after injecting idiotype-pulsed immature dendritic cells may be due to inhibitory cytokines preventing dendritic cell maturation. The use of matured dendritic cells may be a more promising approach, although evidence of clinical benefit is still awaited (10). Therefore, to generate an effective immune response in patients with multiple myeloma, it may be necessary to break anergy. We adapted a dendritic cell/T-cell coculture system already in use in the department for the generation of cytomegalovirus-specific CTL (32). Our studies show that, under appropriate in vitro culture conditions, dendritic cells from multiple myeloma patients can function as antigen presenting cells to prime a strong CTL response, and also that T cells from these patients are competent to develop into antitumor CTL. This suggests that antimyeloma T cells are not deleted, but rather, rendered anergic in vivo. The class I dependence in our system also indicates that antitumor cytotoxic activity is mediated largely by CD8⁺ cells primed to recognize HM1.24 epitopes displayed on MHC class I molecules. This in turn suggests that dendritic cells are taking up and processing exogenous antigen via the well-recognized cross-presentation pathway. On the other hand, the system also generates CD4⁺ T cells, as indicated by phenotyping, and the presence of these cells may be important, not only to provide T-cell help but may also contribute to the cytokine responses seen.

It is important that any clinical strategy should produce a response that is both detectable in vitro and protective in vivo. Given that the immune response pathways in vivo may be impaired, the use of ex vivo generation of antigen-specific CTL for reinfusion is an attractive option. To ensure a robust response the infused T cells must be able to expand in response to further antigen stimulation in vivo. This entails two prerequisites. First, infused T cells must contain, in addition to peripheral effector cells, sufficient numbers of central memory T cells which will expand in response to antigen stimulation in vivo, as has been shown by our previous experience in some patients receiving cytomegalovirus-specific adoptive T-cell therapy (33). In support of this, we have shown that HM1.24-primed T cells generated in our culture system include cells with a central memory phenotype. Second, antigen presentation mechanisms in vivo must be effective at driving the expansion of these memory T cells. Thus, a dual approach combining adoptive cellular therapy with ex vivo generated HM1.24-specific CTL with immunization using ex vivo antigen-loaded dendritic cells may be required.

In conclusion, we have shown, using protein loaded dendritic cells, the ex vivo generation of HM1.24-specific CTL from patients with multiple myeloma which have potent lytic activity against autologous MPC. We also describe a culture system that provides the platform for an immunotherapeutic strategy in multiple myeloma.