GM-CSF and IL-12 production by malignant plasma cells promotes cell-mediated immune responses against monoclonal Ig determinants in a light chain myeloma model

Abstract

Immune responses towards malignant plasma cells have clearly been demonstrated in the course of monoclonal B cell dyscrasias and shown to be mostly specific for idiotypic determinants of the monoclonal immunoglobulin (Ig). These responses are specifically efficient against lymphoma cells expressing a membrane form of the monoclonal Ig. In myeloma, such immune responses are often weak and a number of strategies are currently assayed in order to boost the cell-mediated responses against the secreted monoclonal Ig. The use of cytokines promoting Th1 responses could be helpful for the induction of anti-tumour immunity and the control of residual disease in patients treated with myeloablative therapy, and such strategies need to be evaluated.

In a light chain myeloma model where the monoclonal Ig can only be secreted, we tried to induce protective immune responses through immunization of animals with transfected malignant plasma cells. An expression plasmid encoding GM-CSF and IL-12 proved to be highly efficient for the induction of both cytotoxic and proliferative responses after immunization of animals with transfected and irradiated tumour cells. Anti-tumour immunization according to this protocol was successful in protecting 93·4% of the animals against a subsequent tumour challenge.

INTRODUCTION

Myeloma cells generally secrete an Ig which displays antigenic determinants (termed idiotype, Id) within the variable regions of both heavy and light chains. Since this malignancy is monoclonal, all of the cells of each individual tumour produce the same Ig which may be regarded as a tumour specific antigen [1] which distinguishes neoplastic from normal plasma cells. Malignant B cells can potentially be recognized by T helper cells and lysed by cytotoxic T lymphocytes (CTL) because the intracellularly processed Ig generates Id-derived peptides which are expressed on the cell surface in the context of the major histocompatibility complex (MHC) class I molecules [2]. Furthermore, anti-Id antibodies can neutralize the circulating Ig molecules.

Standard therapies like radiation and chemotherapy, largely utilized in medical practice, can both induce remissions, but the majority of patients relapse and cannot be cured. Therefore, the recent understanding of T-cell importance in eliminating tumour cells suggests new ways to control the expansion of the malignant clone in established tumours or in residual disease. A major strategy to promote Id-specific T-cell responses is to render Id determinants more immunogenic. A large number of murine experimental models showed that the immunization with purified monoclonal component protected the animals from a lethal tumour challenge in an anti-Id, T-cell dependent manner [3,4]. More recent clinical trials in humans, based on the immunization with the purified Id [5] or Id-pulsed dendritic cells (DC) [6] plead for the participation of the cellular arm of the immune response in the control of malignant clone expansion.

The S67 human κ LC-producing malignant plasma cell line was previously shown to induce solid extramedullary tumours when injected to BALB/c mice [7]. Immunization of mice with mitomycin C-treated S67 cells resulted in a protective immune response against the graft of viable cells. This immunity was demonstrated to be specific for the CHEB antigen, but despite immunization some mice still developed tumours [7].

Interleukin-12 (IL-12) and granulocyte macrophage-colony stimulating factor (GM-CSF) are known to play important roles in enhancing the cytolytic activity of CTLs and a local recruitment of professional antigen-presenting cells (APC), respectively. Following stimulation with IL-12, helper T cells secrete IFN-γ which is one of the key cytokines able to promote Th1 immune responses [8]. GM-CSF was previously shown to actively contribute in the rejection of tumour cells in numerous experimental models [9,10]. The combined activities of these cytokines have never been tested in the case of myeloma anti-tumoral immunity.

In an attempt to an efficient immunization protocol against myeloma cells producing the CHEB κ LC, we constructed the plasmid pCK1 encoding GM-CSF and IL-12. The S67 plasmacytoma clone was then stably transfected with the pCK1 vector to generate a GM-CSF and IL-12-secreting clone, S67CK. In the present study we evaluate the tumorigenicity of the S67CK clone and the effectiveness of vaccination with such cytokine and Id-producing cells for the induction of a protective immunity against a further lethal tumour challenge with myeloma cells.

MATERIALS AND METHODS

Animals

Female BALB/c mice, aged 6–8 week, were purchased from Iffa-Credo (L'Arbresle, France). Mice were housed under conventional conditions in our animal facilities, following principles of laboratory animal care (NIH publication 85–23, revised 1985). Animals with important tumour progression were killed with carbon dioxide, according to recommendations of the local ethical committee.

Isolation of coding sequences

All the cytokine coding sequences used in the immunization construct were isolated through reverse-transcriptase (RT) PCR amplification. The GM-CSF and IL-12 p35 sequences were amplified from mRNA isolated from Ficoll-separated mouse lymphocytes. The IL-12 p40 sequence was amplified from 2 × 10⁶ lymphocytes stimulated for 24h with 25μg/ml LPS (Sigma, L'Isle d'Abeau Chesnes, France). Total RNA was isolated using the Extract-All purification kit (Eurobio, Les Ulis, France) following the manufacturer's instructions. Isolated RNA was used as template for synthesizing first-strand cDNA, by extending an oligo(dT) primer (Amersham Pharmacia Biotech, Orsay, France) with Super Script II RT (Life Technologies, Cergy Pontoise, France). The sequences of the PCR amplimers were as follows: 5′GM-CSF (AGG ATG TGG CTG CAG AAT TTA CTT TTC), 3′GM-CSF (TCA TTT TTG GAC TGG TTT TTT GCA TTC), 5′IL-12 p35 (AGC ATG TGT CAA TCA CGC TAC), 3′IL-12 p35 (TCA GGC GGA GCT CAG ATA GCC), 5′IL-12 p40 (AAG ATG TGT CCT CAG AAG CTA), and 3′IL-12 p40 (CTA GGA TCG GAC CCT GCA GGG). PCR amplification for GM-CSF (426bp) and IL-12 p35 (646bp) was performed with the Taq DNA polymerase (Amersham Pharmacia Biotech, Orsay, France), and for the longer IL-12 p40 cDNA (1008 bp) using the Pfu DNA polymerase (Promega, Charbonnieres, France), followed by a single cycle of 30min at 72°C in the presence of the Taq DNA polymerase.

Construction of the immunization vector

The resulting PCR products were cloned into the pCRII-TOPO plasmid (Invitrogen, Groningen, the Netherlands), then sequenced by the dideoxy method using Big-dye terminators and a capillary electrophoresis system (Applied Biosystems, Foster City, CA, USA). Sequencing allowed us to select clones without any mutation and to determine the orientation of the cDNA inserts in the corresponding plasmids. The GM-CSF and IL-12 p35 genes were excised from the pCRII-TOPO plasmid by a Kpn I/Apa I digestion, then religated into the pcDNA3·1-Hygro(+) (Invitrogen, Groningen, the Netherlands) vector previously cut out by the same enzymes, to obtain the pGM-CSF and pIL-12-p35 vectors, respectively. Similarly, the IL-12 p40 gene was excised from the pCRII-TOPO vector through Spe I/Not I digestion, and inserted into Xba I/Not I sites of pcDNA3·1-Hygro(+) to obtain the pIL-12-p40 plasmid. All the genes cloned into the pcDNA3·1-Hygro(+) expression vector are preceded by the CMV promoter and followed by the same bovine growth hormone (BGH) polyadenylation (poly(A)) signal. Expression cassettes containing the IL-12 p35 and p40 genes, flanked by the CMV promoter and the BGH poly(A) signal, were excised from their corresponding plasmids by Mfe I/Pvu II and Mfe I/Sph I digestions, respectively. The two cassettes were treated with the T4 DNA polymerase to generate blunt-ended fragments, and were then sequentially inserted into the pGM-CSF plasmid, using the unique Mfe I and Ssp I sites, respectively. The expression plasmid pCK1 (Fig. 1), encoding the 2 subunits p35 and p40 of IL-12, and the GM-CSF, was thus finally generated.

Fig. 1.

Structure of the cytokine-expressing vector pCK1. Genes encoding GM-CSF and the two subunits, p35 and p40, of IL-12 are both preceded by the P_CMV promoter, and followed by the BGH pA signal.

Cell lines

The SP2/0 cell line is a murine plasmacytoma (ATCC; CRL-1581) which expresses H-2d molecules and does not synthesize any murine Ig component. S67 cell line was obtained by a stable transfection of SP2/0 with a plasmid encoding CHEB human κ light chain (LC) [11,12]. The resulting clone secretes high levels (30μg/ml/106 cells per 24h) of the CHEB κ LC. BALB/c mice injected with S67 cells develop rapidly growing solid extramedullary plasmacytoid tumours.

S67 cells were stably transfected by electroporation (250V, 960μF using a Gene Pulser apparatus, Bio-Rad, Ivry sur Seine, France) with the plasmid pCK1 to obtain the S67CK clone.

All cultured cells were routinely maintained in RPMI 1640 culture medium supplemented with 10% heat-inactivated FCS, 2mm L-Gln, 50U/ml penicillin, and 50μg/ml streptomycin. For the experiments, the S67CK clone was either used as viable cells to estimate their immunogenicity or were irradiated (20000 rad) using a source of ⁶⁰Co, in order to immunize mice.

ELISAs

To confirm the expression of the IL-12 and GM-CSF genes, COS-7 cells (ATCC; CRL-1651) were transiently transfected with the plasmid pCK1 using the Superfect transfection kit (Qiagen, Hilden, Germany) and following the supplier's instructions. Culture supernatants, harvested from 2 × 10⁵ cells 24h post-transfection, were tested for the production of IL-12 and GM-CSF, using the OptEIA™ Mouse IL-12 (p70) and GM-CSF sets (Pharmingen, San Diego, CA, USA), respectively. Transfected COS-7 cells produced up to 9pg/ml IL-12 and 1·4pg/ml GM-CSF. Similarly, supernatants from viable or irradiated S67CK cells were assayed for the production of both cytokines.

A sandwich ELISA was used to detect the production of the CHEB human κ LC. Briefly, plates were coated overnight at 4°C with a goat antiserum to human κ chains (Kallestad, Chaska, MN, USA) diluted at 1/1000 in 50mm bicarbonate buffer pH 9·0. After washing and blocking, cell supernatants were added to plates and incubated 2h at room temperature. A goat anti-human κ LC (bound and free) alkaline phosphatase-conjugated (Sigma, L'Isle d'Abeau Chesnes, France) was used for detection. The substrate was p-nitrophenyl phosphate (Sigma, L'Isle d'Abeau Chesnes, France). A standard curve was obtained using a purified human κ LC.

Anti-κ LC antibodies were assessed by an antigen-capture ELISA as described [7]. Titres were estimated by comparison with a standard curve obtained with a murine monoclonal anti-κ LC antibody (HP6053, a kind gift of Dr P. Aucouturier). The 1/625 dilution of this antibody was fixed as a 1 arbitrary unit (A.U.) reference.

Inoculation of mice

Two groups of 30 mice were intraperitoneally (i.p.) injected with either 5 × 10⁶ viable S67 cells or 5 × 10⁶ viable S67CK cells. A third 30 mice group was first immunized i.p. with 2 × 10⁶ irradiated S67CK cells; one and two weeks later, the animals were boosted with the same number of irradiated S67CK cells. After a one week interval, the immunized mice received i.p. 5 × 10⁶ viable S67 cells, i.e. twice the number required for 50% tumour take in BALB/c mice, as previously determined [7]. Another group of 46 control mice were immunized according to the same protocol but with mitomycin C-treated S67 cells instead of S67CK, before receiving i.p. viable S67 cells.

Tumour development

Tumour growth, expressed in millimeters, was measured with a calliper every 2 days and calculated as the mean of the narrowest and longest surface length. Animals surviving tumour challenge were followed for 2 months and were defined as ‘tumour-free’. Mice that developed tumours exceeding 20mm were sacrificed.

Assay for CTL

An independent group of 10 mice was immunized 3 times with irradiated S67 CK cells as described above. Cytolytic activity of splenocytes from these mice was assessed by measuring lactate dehydrogenase release using the Cytotox 96 Assay kit (Promega, Charbonnieres, France) as previously described [7], with the only modification consisting in the irradiation of the S67 stimulator cells before the coculture with splenocytes. For the assay, different splenocytes (effector, E) to target T (S67 or SP2/0) ratios (E:T ratios) were tested, and percent lysis was calculated according to the manufacturer's instructions using the formula:

Lymphocyte proliferation assay

Suspensions of splenocytes used for the detection of the CTL activity were also utilized in a lymphocyte proliferation assay. As stimulators, 2 × 10⁴ irradiated S67 and SP2/0 cells in 100μl RPMI 1640 containing 10% FCS, antibiotics and 50μm 2-ME were plated in sixplicate into flat-bottomed 96-well plates, and 100μl of splenocytes suspension were added to each well to give 37·5:1 and 50·0:1 splenocytes: stimulator cells ratios. Cells were cocultured for 72h and [³H]-thymidine (1μCi/ml, Amersham Pharmacia Biotech, Orsay, France) was added 18h before harvesting.

RESULTS

Obtaining cytokine-secreting malignant plasma cells

In order to obtain malignant plasma cells secreting GM-CSF and IL-12, the human CHEB κ LC-producing S67 clone was stably transfected with the plasmid pCK1 described above, and the resulting clones were screened in the presence of hygromycin at 600μg/ml. Five positive clones were then tested by ELISA for the secretion of GM-CSF and IL-12, and the best cytokine producer, S67CK, was retained for the experiments. This clone secretes about 1·7ng GM-CSF/ml/10⁶ cells/24h and 52pg IL-12/ml/10⁶ cells/24h.

The tumorigenicity of malignant plasma cells producing cytokines is dramatically decreased

To compare the ability of S67 and S67CK clones to induce tumours in BALB/c mice, 2 groups of 30 animals were injected i.p. with 5 × 10⁶ of these cells. The mice were observed for at least 2 months for the appearance of tumours and when it occurred, the tumour size was regularly measured. Among the mice inoculated with the S67 clone, 3 out of 30 (i.e. 10%) spontaneously rejected the tumours, comparing with 21 out of 30 (i.e. 70%) in the case of mice injected with the clone S67CK (Fig. 2a). This difference was highly significant (χ² test, P <0·0001). Moreover, when tumours occurred, their size was reduced in the case of mice inoculated with the S67CK comparatively to the animals receiving the S67 clone, and this was particularly true during the first 2 weeks of tumour growth. After 3 weeks, the tumour size finally reached similar sizes in both cases (Fig. 2b).

Fig. 2.

Tumour take in mice inoculated with viable S67 or S67CK cells. (a) Comparison of the tumorigenic potential of S67 and S67CK cells. (b) Differential growth of tumours in mice inoculated with S67 (•) or S67CK (▪) cells.

Irradiated S67CK cells still produces CHEB κ LC, GM-CSF and IL-12

Since we wished to immunize mice with irradiated S67CK cells, we checked that this treatment did not alter the production of the CHEB κ LC, GM-CSF and IL-12. Specific ELISA assays were used to assess the synthesis of these molecules in culture supernatants obtained 24 and 48 h after the irradiation of 1 × 10⁶ S67CK cells. The mean values from different assays are presented in Fig. 3. From these data it is clear that all three molecules are produced by the irradiated cells at levels comparing those in nonirradiated cells.

Fig. 3.

Secretion of the CHEB κ LC (a), GM-CSF (b) and IL-12 (c) by the irradiated S67CK cells.

Immunization of mice with irradiated S67CK cells elicits a cellular immune response

A group of 10 mice was immunized by repeated i.p. injection (3 times) of irradiated 2 × 10⁶ S67CK cells. To estimate the capacity of these cells to induce a cellular immune response against the original S67 clone, and more specifically against the CHEB antigen, we first compared the cytolytic activity of splenocytes from the immunized mice versus either S67 or SP2/0 cells as targets. A specific lysis of S67 cells was obtained for all splenocytes versus target cells ratios, whereas only a minimal lysis was obtained when the SP2/0 cells were used as targets (Fig. 4a). As previously reported, no CTL activity could be demonstrated against SP2/0 cells transfected with an empty vector and producing only the neomycin resistance enzyme 7, and data not shown]. Moreover, we also tested the induction of a T-cell proliferation in response to the CHEB antigen. A [³H]-thymidine incorporation assay showed that S67 cells, but not SP2/0 cells, dramatically stimulate splenocyte proliferation (Fig. 4b).

Fig. 4.

Induction of T-cell responses in mice immunized with irradiated S67CK cells. (a) CTLs generated by immunizing mice specifically kill S67 cells (•), but not SP2/0 cells (▪), in a LDH-releasing assay. (b) Stimulation of splenocytes from immunized mice by either S67 (□) or SP2/0 () cells was assessed by incorporation of [³H]-thymidine in a 3-day coculture experiment.

Tumour challenge and immunization efficacy

To estimate the effectiveness of an immunization with irradiated S67CK cells in protecting mice against a tumorigenic dose of the original S67 clone, we initiated a statistical study. Thirty mice were immunized as described above, then the animals received i.p. 5 × 10⁶ viable S67 cells. The mice were observed for at least 2 months for the appearance of tumours, and when it occurred, the tumour development was recorded every two days. One week after the tumour graft, 4 out of 30 mice carried tumours. Among the 4 tumour-bearing mice, 2 had a rapid tumour development (mice A and B), while the 2 others showed a progressive tumour regression (mice C and D) by about 4 weeks after the tumour graft (Fig. 5a). Altogether, the percentage of protection against tumour progression was 93·4% among S67CK-immunized mice. The frequency of progressive tumours among S67CK-immunized mice (6·6%) was thus significantly lower than in control mice immunized with S67 (which developed tumours in 22·7% cases, P <0·01) and much lower than in nonimmunized mice (which developed tumours in 90% of cases, P <0·0001).

Fig. 5.

Several mice developed tumours despite immunization. (a) Fate of tumour growth in 4 out of 30 immunized mice which have developed tumours. • A; ▪ B; ○ C; □ D. (b) A CTL response could be detected in mice with progressive tumour development. • S67 cells; ▪ SP2/0 cells. (c) High titres of anti-human κ LC antibodies can be detected in immunized mice whether or not they carry tumours.

Following the immunization protocol, all the S67CK-immunized animals developed a cell-mediated immunity, which was specifically directed against the S67 cells. We next tried to understand if the tumour development observed in two animals despite immunization was caused by a loss of the cytolytic activity of T cells or if the proliferation rate of the malignant clone was rather responsible for the tumour take. A CTL assay revealed that splenocytes from the two mice with continuous tumour development are still able to lyse S67 cells, but not SP2/0 cells (Fig. 5b). Moreover, we measured the occurrence of a humoral immune response in both tumour-free and tumour-bearing immunized mice. An ELISA assay showed that both groups of mice produce comparable high levels of anti-human κ LC antibodies (Fig. 5c).

DISCUSSION

There is increasing evidence that the immune system, and particularly CTLs, can target malignant plasma cells [13,14]. The monoclonal Ig produced by these cells constitutes a tumour antigen that is specific for each individual malignant clone. Promising immunological and clinical responses were previously obtained by using this immunogen in patients with B-cell lymphomas [15]. In multiple myeloma, immunity is less easily obtained because of the lack of membrane receptors. However, several recent clinical trials showed that immunization with the monoclonal Ig produced by the recipient's neoplastic clone elicits detectable anti-Id cellular responses [16]. Österborg et al. [17] reported that immunization of 5 patients with their own purified monoclonal component, in combination with GM-CSF, improved the rate of response to the vaccine, because one patient showed a decrease in the serum concentration of the monoclonal Ig. Other clinical trials were based on the utilization of Id-pulsed DC [18,19], and there is hope that new immunotherapeutic approaches may promote better responses.

In this report we show in a murine experimental model that the immunization with autologous malignant plasma cells secreting GM-CSF and IL-12 could elicit cellular immune responses which are able to efficiently protect mice against a lethal tumour challenge.

The previously described S67 plasmacytoma cell line [7] was stably transfected with the plasmid pCK1 encoding the GM-CSF and the two subunits of IL-12, to obtain the S67CK clone. When the ability of both S67 and S67CK cells to induce tumours was compared, we obtained 10% of spontaneously tumour rejection in the first case, compared to 70% in the latter. When tumours occurred, their size was significantly reduced in mice inoculated with S67CK cells, by comparison with mice receiving the parental clone. In this case, a likely explanation for the strongly decreased tumour take is the induction of an anti-tumoral immune response due to the production of GM-CSF and IL-12 at the site of the tumour cell inoculation. This immunity is more effective at the beginning of the tumour development. Since S67CK cells like S67 cells are actively proliferating, the tumour size may in some instances reach a threshold after which it exceeds the capacity of tumour rejection by the immune system. Our data indicate that transfection of the original clone S67 with the GM-CSF and IL-12 coding sequences contributes significantly to an increase of spontaneous tumour rejection, although it does not reach a hundred percent efficiency in this matter.

We next show that irradiated S67CK cells were still able to produce CHEB κ LC, GM-CSF and IL-12, and could be efficiently used for immunization. Mice immunized with irradiated S67CK cells developed an efficient T-cell-mediated immune response that could be functionally detected in cytotoxic and proliferation assays. Splenocytes from immunized mice lysed the CHEB κ LC-secreting S67 cells, but not SP2/0 cells. When S67 and SP2/0 cells were used to stimulate [³H]-thymidine incorporation by the splenocytes, only the first one was a potent stimulator. These results suggest the ability of irradiated S67CK cells to elicit cytotoxic and proliferative immune responses mainly directed against the S67 cells but not against the SP2/0 cells. Then, the immune response seems to be specifically induced by the CHEB antigen, expressed only by the S67 cells, rather than by other potential plasma cell antigens.

We further evaluated the efficacy of a vaccination based on the inoculation of irradiated malignant plasma cells producing the monoclonal Ig and cytokines, against a tumorigenic dose of the original clone producing only the monoclonal Ig. From 30 mice immunized with irradiated S67CK cells, 26 survived and remained tumour-free (i.e. 86·7%), and among the 4 tumour-bearing mice, 2 had a rapid tumour development and were sacrificed, while the 2 remaining showed tumour regression until no macroscopic tumours were visible. Taken together, the percentage of protection reached 93·4%. This response was significantly stronger than in mice immunized with S67 cells which were not transfected with GM-CSF/IL-12 expression vector (77·3% protection).

Again, the appearance of tumours in a few animals was related to the high proliferation rate of malignant plasma cells. Indeed, a CTL assay revealed that splenocytes from tumour-bearing mice were still able to efficiently lyse S67 cells. Moreover, the CTL activation was accompanied by a significant humoral response: both tumour-free and tumour-bearing immunized mice showed similar high levels of anti-human κ LC antibodies in their sera. We can thus conclude that the immunization of mice with irradiated S67CK cells can effectively stimulate both arms of the immune system and protect the animals against the inoculation of a tumorigenic dose of S67 cells. However, in a few number of cases the rapidity of malignant cell division seems to be critical for the tumour take in immunized mice.

In conclusion, using a murine experimental model, we provide evidence that the secreted monoclonal component in LC myeloma may represent the main target of specific immune responses. Although efficient anti-tumoral immunity is readily obtained in such a murine model where the secreted human monoclonal LC is strongly immunogenic, anti-tumour responses restricted to idiotypic determinants of the LC are likely to be much weaker in patients. As a strategy to enhance immune responses, it is thus promising that autologous malignant plasma cells engineered to produce haematopoietic growth factors (GM-CSF) and IL-12 proved to be more immunogenic in mice and to induce an enhanced protective immunity in an anti-tumoral vaccination protocol.