Allogeneic T lymphocytes as a source of peptide-dependent T cells specific for myeloma cells
Alexandrine Geffroy-Luseau,
Agnès Moreau-Aubry,
Régis Bataille and
Catherine Pellat-Deceunynck
Inserm UMR601, Institut de Biologie, 9 quai Moncousu, 44093 Nantes cedex 01, France
Correspondence to: C. Pellat-Deceunynck; E-mail: catherine.pellat-deceunynck{at}univ-nantes.fr
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Abstract
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We investigated the generation of myeloma-specific CTLs from normal donors HLA mismatched with the myeloma cell line SBN. The T-cell line obtained was cloned and each CTL was assessed against SBN and SBN-EBV (a B-EBV cell line obtained by EBV infection of B cells from SBN patient) simultaneously. Among >270 clones evaluated, 2 CTLs (Vbeta13.1 and Vbeta17) killed SBN but spared SBN-EBV cells. Antibodies against HLA-I, but not HLA-A2, molecules abrogated their recognition of SBN. Moreover, SBN recognition was abrogated by anti-HLA-Cw6 antiserum. Both clones recognized two other HLA-Cw*0602 myeloma cell lines. Neither of them recognized HLA-Cw*0602 B-EBV cell lines, the PBMCs of HLA-Cw*0602-unrelated donors or HLA-Cw*0602 melanoma cell lines. We showed that HLA-Cw6 molecules were more expressed at the cell surface of B-EBV cells as compared with myeloma cells, suggesting that the lack of reactivity against B-EBV cells was not related to a low level of HLA expression. Since CTL clones did not express any KIR or NKG2D, we excluded the fact that NK cell receptors could be involved in myeloma-specific recognition through KIRHLA-I or NKG2DMICA,B interactions. Cold target competition and acid elution experiments confirmed that myeloma cell recognition was peptide dependent.
Keywords: multiple myeloma, CTL
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Introduction
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Multiple myeloma (MM) is a fatal hematological malignancy characterized by an accumulation of monoclonal malignant plasma cells within the bone marrow (1). The most efficient treatment for patients under 65 years of age is intensive therapy (high-dose melphalan followed by autologous stem cell transplantation), which improves the remission rate, event-free survival and overall survival, but does not lead to a cure (2). Recent reports have shown that allogeneic stem cell transplantations provide effective therapy for patients with MM (37). Besides inducing the graft-versus-host disease (GVHD), the allogeneic transplantation demonstrated a graft-versus-myeloma (GVM) effect. These observations demonstrated that myeloma-specific CTLs could be induced in vivo, suggesting that myeloma-specific CTLs could recognize tumor-associated antigens (TAA) specifically expressed by myeloma cells. Indeed, others and we demonstrated that MM cells expressed TAA of the MAGE family as opposed to most hematological malignancies (810). Generation of myeloma-specific CTLs from patients with MM remains difficult, although possible, at least because available primary myeloma cells are few and do not proliferate in vitro (1113). In 1999, we reported the isolation of autologous myeloma-specific CTL clones in a patient in remission after the co-culture of myeloma cells with his PBMCs (14). Since this protocol was not applicable to numerous patients, we explored the possibility to obtain myeloma-specific CTLs from unrelated donors since it had been reported that alloreactivity could be a source of peptide-specific CTLs (1517). Indeed, Sadovnikova et al. reported the generation of tumor-reactive cytotoxic T cells against peptides, from cyclinD1 in humans or mdm-2 in mice, presented by non-self-MHC class I molecules (15, 16). Münz et al. (17) demonstrated that alloreactivity allowed them to isolate high-avidity CTLs that could not be obtained in autologous situation, probably since they had been deleted during thymic education. Moreover, existence of a functionally diverse allorestricted T-cell repertoire has been demonstrated (18). In the present study, we were interested in isolating myeloma-specific CTL clones from allogeneic ones by selecting them through their lack of reactivity against non-myeloma cells derived from the patient, i.e. B lymphocytes immortalized by EBV.
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Methods
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Samples and cell lines
Peripheral blood of normal donors was obtained in agreement with current French laws. PBMCs were isolated by classical FicollHypaque centrifugation. The human myeloma cell lines (HMCLs) were either derived in our laboratory from patients with MM (XG6, SBN, BCN) or purchased from the American Type Culture Collection (Rockville, MD, USA) or DSM (Braunschweig, Germany). All HMCLs were cultured in RPMI 1640 supplemented with 10% FCS and 2 x 105 M 2-mercaptoethanol with or without 3 ng ml1 IL-6 (19). BCN primary myeloma cells were obtained from blood of an MM patient (BCN) at the time of leukemic relapse and cryopreserved: a fraction of these cells gave rise to BCN HMCL after in vitro culture in RPMI 10% FCS with 3 ng ml1 IL-6. The B-EBV cell lines from patients (SBN-EBV and BCN-EBV) or normal donor (BARS-EBV) were obtained as described (14). Briefly, B cells from patients or normal donors were infected by EBV to obtain B-EBV cell lines that constitute a non-malignant B counterpart to myeloma cells. Melanoma cell lines M88 and M113 were obtained in the laboratory (F. Jotereau, U601, Nantes, France). HLA-I typing was performed at the Etablissement Français du Sang (EFS, Nantes, France).
Reagents
mAbs anti-CD4, anti-CD8, anti-TCRalpha/beta and anti-TCR-Vbeta were from Beckman Coulter (Villepinte, France). Hybridomas W6/32 (anti-HLA-I) and BB72 (anti-HLA-A2) were from the American Type Culture Collection, and B1.23.2 (anti-HLA-B/Cw) was a gift from F.A. Lemonnier, INSERM U152, Paris, France (20). Anti-HLA-Cw4/Cw6 and HLA-A32 antisera were from Pasteur Mérieux Connaught (Marcy l'Etoile, France). Anti-HLA-Cw6 single-chain antibody fragments (scFv) and scFv-B11 (irrelevant control scFv) were a kind gift from Matthias Marget (University of Kiel, Germany). mAbs anti-p58.1/CD158a EB6, anti-p58.2/CD158b GL183, anti-p140 DEC66 were a gift from E. Vivier (Centre d'Immunologie de Marseille-Luminy, Marseille, France), anti-p70 Z27.3.7, anti-NKG2A Z199 and anti-NKG2D ON72 were purchased from Beckman Coulter (Roissy, France).
Generation, expansion, selection and re-stimulation of SBN-specific T cells
Generation of SBN-specific T-cell line was performed by co-culturing fresh PBMCs with irradiated SBN as previously described (14). Cloning and monthly re-stimulation of clones were performed in 96-well U-bottom plates (0.3 cell per well for cloning and 10 000 cells per well for re-stimulation) in the presence of irradiated allogeneic PBMCs (107 cells per plate) and B-EBV cell lines (106 cells per plate) in 10% pooled human sera in the presence of 150 U ml1 IL-2 and 1 µg ml1 PHA (14).
Cytotoxicity and tumor necrosis factor release of CTLs
Cytotoxic assays were performed in 96-well V-bottom plates with 3000 51Cr-labeled target cells in 100 µl (4 h). The percentage of specific cytotoxicity was determined as follows: (experimental release spontaneous release)/(total release spontaneous release) x 100.
Tumor necrosis factor (TNF) release was determined by the Wehi 164 clone 13 bioassay after 6 h to overnight co-culture of CTLs with cellular targets as described previously (21). TNF release could not be determined in the co-cultures with B-EBV cells since these latter produce some TNF spontaneously.
Reverse transcription and PCRs
Total RNA was extracted with Trizol reagent method as recognized by the supplier (Invitrogen, Cergy-Pontoise, France). Reverse transcription (RT) of mRNA into cDNA was performed by incubating 4 µg of total RNA with 200 units of Moloney Murine Leukemia Virus Reverse Transcriptase (Invitrogen), 2 µM oligo(dT)1218 primer (Invitrogen), 10 mM dithiothreitol, 25 units of ribonuclease inhibitor (Promega, Madison, WI, USA) and 0.5 mM deoxyribonucleotide triphosphates in 20 µl. After 90 min at 42°C, the reaction was stopped by adding 80 µl of H2O on ice. cDNAs were stored at 20°C until used for PCR reactions.
HLA-Cw*0602 PCR reactions were performed using an Olerup SSPTMAB kit containing the specific primers (Bionobis, Monfort-L'Amaury, France). Samples were resolved in 2% agarose gels in TrisacetateEDTA buffer, stained with ethidium bromide and recorded under UV illumination. Amplicons were 240 bp and 110 bp for genomic and complementary HLA-Cw*0602 DNA, respectively. ß-Actin amplification reaction was performed under standard conditions with primers 5'-GGCATCGTGATGGACTCCG-3' and 5'-GCTGGAAGGTGGACAGCGA-3' (30 cycles). ß-Actin cDNA amplification product was 615 pb.
Staining and FACS analysis
HLA-Cw6 expression was detected by indirect staining (three steps). Cells were incubated 15 min at room temperature with c-myc-scFv directed against HLA-Cw6 (22), followed with mouse anti-c-myc mAb (Invitrogen, Carlsbad, CA, USA) and finally with PE-conjugated donkey anti-mouse (Jackson Immunoresearch, West Grove, PA, USA). BA-22.2 and BA-61.4 cells were incubated with mAbs directed against p58.1, p58.2, p70, p140, NKG2A, ILT2 and NKG2D followed with incubation with PE-conjugated donkey anti-mouse. Labeled cells were analyzed with FACSCalibur (Becton Dickinson).
Acid treatment of myeloma cells
One million SBN or XG6 cells were labeled with 51Cr for 1 h, and then incubated with 250 µl of 300 mM glycine, 1% BSA pH 2.5, for 3 min at room temperature. The acid was neutralized with 15 ml of RPMI medium and the cells were washed and treated with brefeldin A (5 µg ml1) for 1 h at room temperature. Acid-treated cells were then used as targets in the classical 51Cr release assay.
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Results
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Isolation of allogeneic CTLs recognizing SBN but not SBN-EBV
Our aim was to isolate myeloma-specific T cells either in the HLA-A2 context or in an HLA-B/Cw allogeneic context. For this purpose, we cultured blood cells of normal donors with the HMCL SBN. Normal donors and SBN were HLA matched only for HLA-A*0201 allele (see Table 1 for HLA-I typing). After 16 days of mixed PBMC/tumor culture between irradiated SBN and BA donor's PBMC, a T-cell line obtained showed significant cytotoxicity not only against SBN but also against SBN-EBV, a B-EBV cell line obtained after EBV infection of B cells from SBN patient (Fig. 1). We cloned the T-cell line by a limiting dilution culture and >270 CTLs were tested for their cytotoxicity against SBN and SBN-EBV simultaneously. We obtained 189 having both SBN and SBN-EBV reactivity (as BA-11), 79 having no reactivity (mainly CD4+, as BA-14) and 6 demonstrating SBN but not SBN-EBV cytotoxicity, as BA-22 (see Fig. 1A). These selected CTLs, all CD8+ alpha beta, were subcloned and we isolated a Vbeta13.1 clone (represented five times), BA-22.2, and a Vbeta17 clone (represented one time), BA-61.4. The same experiment was performed with another unrelated donor and we isolated one clone (from 160 CD8+ CTLs) demonstrating SBN but not SBN-EBV reactivity (data not shown).

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Fig. 1. Cytotoxic assays of BA T-cell line and derived CTLs. (A) Cytotoxicity assays were performed using a standard 51Cr-release assay (4 h) in triplicate wells (mean ± SD). CTLs BA-11, BA-14 and BA-22 were isolated from the BA T-cell line and cloned by limiting dilution as described in Methods. (B) Specific lysis of two clones, BA-22.2 and BA-61.4, against SBN, K562 and Raji. Assays were performed as described in (A). (C) HLA-I-blocking experiments. Cytotoxic assays were performed in the presence of decreasing amount of anti-HLA-I mAb, W632 (pan HLA-I), B1232 (pan HLA-B/Cw), BB72 (HLA-A2). W632 and B1232 were ascitis, BB72-purified mAb. Purified mAb or ascitis were incubated with SBN 30 min prior to CTL addition. Cytotoxic assays were performed as described in (A).
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As shown in Fig. 1(B), CTLs had no NK reactivity against SBN since they lysed neither K562 nor Raji cells. Moreover, BA-22.2 and BA-61.4 reactivity was blocked by anti-HLA-I (W632) and anti-HLA-B/Cw (B1232) but not by anti-HLA-A2 (BB72) mAbs (Fig. 1C).
BA-22.2 and BA-61.4 CTLs were specific of HLA-Cw*0602 myeloma
To further identify the HLA molecules involved, we performed blocking experiments with antisera directed against HLA-Cw4/Cw6 and HLA-A32. TNF secretion of clones was blocked by anti-HLA-B/Cw mAb and by anti-HLA-Cw6 antiserum but not by anti-HLA-A32 (Fig. 2A). We assessed CTL reactivity against a panel of seven HMCLs. Only two of them that are HLA-Cw*0602, i.e. BCN and XG6, were recognized by both CTLs (Fig. 2B). The lack of SBN-EBV recognition was not restricted to this cell line since two other HLA-Cw*0602 B-EBV cell lines, BCN-EBV (derived from BCN patient) and BARS-EBV (derived from HLA-Cw*0602 donor), were not recognized either (Fig. 2C). Furthermore, normal HLA-Cw*0602 primary cells, i.e. T cells from SBN patient and PBMCs from normal donors (FB, MC), respectively, were not recognized either (Fig. 2B and C). Since cell lines are known to over-express or under-express genes as compared with primary tumor cells, we evaluated the reactivity of both clones against primary myeloma cells of patient BCN that had been cryopreserved prior to immortalization. As shown in Fig. 2(A), primary myeloma cells of patient BCN were also recognized by BA-22.2 and BA-61.4 CTLs. We wondered whether CTLs could recognize tumor cell types other than myeloma cells. We measured the reactivity of BA-22.2 and BA-61.4 CTLs against two HLA-Cw*0602 melanoma cell lines, namely M88 and M113, since melanoma cells are known to express most of the identified TAA and are good antigen-presenting cells. As shown in Fig. 2(B and C), no melanoma cell line was lysed nor induced TNF release by CTLs. All these data showed that BA-22.2 and BA-61.4 recognized HLA-Cw*0602 myeloma cells only.

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Fig. 2. (A) TNF release was determined by a bioassay after overnight co-culture of CTLs with target cells (20 000 targets : 10 000 effector cells) in 100 µl. For blocking experiments, mAb or antiserum (dilutions were 80 and 4 for anti-HLA-B/Cw mAb and anti-HLA-Cw4/Cw6 antiserum, respectively) was added to SBN or BCN 30 min prior to CTL addition. (B) TNF release was determined by a bioassay after overnight co-culture of CTLs with target cells (10 000 targets : 2500 effector cells) in 100 µl. (C) Cytotoxicity assays of CTLs against the indicated HLA-Cw*0602 target cells (HMCL, B-EBV, normal cells and melanoma cells) were performed as described in the Fig. 1. One experiment out of three.
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FACS analysis of HLA-Cw6 and NKCR expression
We evaluated HLA-Cw6 expression in all cells in order to exclude a possible link between HLA expression level and reactivity. By RTPCR, we observed that myeloma cells (SBN, BCN), as well as their B-EBV counterpart, expressed HLA-Cw*0602 mRNA (Fig. 3A). This result was further confirmed at the protein level with an scFv specific for HLA-Cw*0602 (22). As illustrated in Fig. 3(A), HLA-Cw6 was weakly expressed by all HLA-Cw*0602 cells, irrespective of their normal or malignant status. However, non-malignant cells expressed more HLA-Cw6 than myeloma cell lines. This observation was not restricted to HLA-Cw molecules since myeloma cells seem to express less HLA-I molecules than B-EBV cells (data not shown). This prompted us to evaluate NKCR expression in BA-22.2 and BA-61.4. Indeed, because malignant cells are known to express less HLA molecules than normal cells, T cells expressing KIR should lyse tumor cells only (23). On the other hand, MICA was recently reported to be expressed by myeloma cells (24). Interaction between MICA and its receptor NKG2D could decrease the threshold of the reactivity of T cells, allowing expression of cytotoxicity against MICA+ cells only. Therefore, we also evaluated NKG2D in BA-22.2 and BA-61.4. As shown in Fig. 3(B), neither BA-22.2 nor BA-61.4 (data not shown) expressed KIR or NKG2D. Together, these findings excluded that myeloma-specific recognition could be related to NKCR involvement. Taken together, our data suggested that myeloma recognition by both BA-22.2 and BA-61.4 ought to be peptide specific.

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Fig. 3. (A) RTPCR analysis of HLA-Cw*0602 expression. HLA-Cw*0602 and ß-actin cDNA amplicons were obtained as described in Methods. FACS analysis of HLA-Cw6 expression in HMCL, B-EBV and normal cells. All cells were typed HLA-Cw*0602 positive except LP1 and DAB-EBV that were used as control (see Table 1). Staining was performed as described in Methods. Each overlay histogram represents the control staining (thin line) and the HLA-Cw6 staining (thick line) of indicated cells. (B) FACS analysis of NKCR in BA-22.2 CTLs. Staining was performed as described in Methods. Each overlay histogram represents the control staining (thin line) and the indicated staining (thick line) of BA-22.2 CTLs.
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Cold target competition and acid elution assays
To further demonstrate that both clones did not recognize B-EBV cell lines, we performed cold target competition assays. XG6 cells were labeled with 51Cr and cytotoxic assays were performed in the presence of an increasing number of SBN or SBN-EBV cells. As shown in Fig. 4A, XG6 lysis by BA-22.2 or BA-61.4 was inhibited by cold SBN (>60%), but not by cold SBN-EBV. Cold non-HLA-Cw*0602 myeloma and B-EBV cells, i.e. LP1 and DAB-EBV, did not inhibit XG6 lysis. To further demonstrate the peptide dependency, we treated myeloma cells with acid to elute peptides bound to HLA (25). As illustrated in Fig. 4B, acid elution highly inhibited SBN lysis (as well as XG6, data not shown) by both BA-22.2 and BA-61.4 (>60% inhibition) as well as by CT6, an autologous HLA-A*0201 SBN-specific CTL that we derived from an SBN patient, as described previously (14).

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Fig. 4. (A) Cold target inhibition assay. Increasing numbers of SBN, SBN-EBV, LP1 or DAB-EBV (from 7500 up to 60 000) cells were added to 3000 51Cr-XG6 cells in 96-well plates prior to addition of 3000 CTLs. 51Cr release was measured in triplicate wells (mean ± SD) after 4 h incubation. One experiment out of three. (B) Acid elution treatment of SBN. SBN cells were first labeled with 51Cr, acid stripped 3 min at room temperature, incubated 1 h with brefeldin A and used as target in a 3-h 51Cr release assay (mean ± SD of triplicate wells). One representative experiment out of five.
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Discussion
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In this paper, we were interested in defining if myeloma-specific CTLs could be derived from normal unrelated donors. This question was addressed since it remains very difficult to isolate autologous CTLs in MM. Indeed in most patient samples, the number of available tumor cells is too low and immortalization of myeloma cells in vitro is a very rare event. Immortalization essentially occurs in patients with terminal disease, i.e. in leukemic phase. In these patients, T-cell collection is not possible anymore but immortalization of non-malignant cells of the same lineage, i.e. B cells, by EBV infection remains accessible. With these couples of autologous malignant/non-malignant cell lines, we were able to perform the present study. The first conclusion is that we did not isolate any CTL recognizing a tumor peptide within the HLA-A2 context. A possible explanation is that such CTLs, if present, were too rare and probably hidden by the most frequent alloreactive CTLs. The second conclusion is that we isolated CTLs that were selectively reactive against myeloma cells only. For one donor (BA), we further characterized two independent clones, named 22.2 and 61.4. We showed that they both recognized all the HMCL HLA-Cw*0602 cell lines so far tested, namely XG6 and BCN. On the other hand, they were reactive against neither non-malignant HLA-Cw*0602 cells lines, B-EBV, normal HLA-Cw*0602 leukocytes, nor HLA-Cw*0602 melanoma cell lines. We wondered whether the lack of recognition of normal cells could be related either to a too weak HLA-Cw expression or to the expression of NKCR on CTLs. We thus evaluated HLA-Cw6 expression since (i) HLA-Cw molecules are known to be regulated independently of HLA-A or -B molecules and (ii) HLA-Cw molecules are often weakly expressed (26). We found that HLA-Cw6 molecules were weakly expressed and more particularly so by myeloma cells than by B-EBV cells. On the other hand, we did not find any expression of NK-cell inhibitor receptors (including NKG2D) in CTLs. We thus excluded that lack of recognition of non-malignant cells was related to HLA-Cw6 expression level or to HLANKCR and MICANKG2D interactions. Assays of cold target inhibition as well as acid stripping indicated that both CTLs were peptide dependent. Alloreactive T cells are known to be heterogeneous, some recognizing non-self-MHC molecules independently of peptides and others recognizing MHC molecule peptide complexes (1518, 27). In the latter case, usually, T-cell clones recognized individual peptides. Our data highly suggest that we obtained such clones with specificity for myeloma. Since they were also able to recognize primary myeloma cells, their specificity was not restricted to myeloma cell lines.
We looked for tumor peptides identified within the HLA-Cw*0602 since myeloma cells are known to express cancer-testis genes (8, 9). Two peptides have been reported derived from either GAGE-1,2,8 or NY-ESO-1, i.e. YRPRPRRY and ARGPESRLL, respectively (28, 29). We loaded SBN-EBV cells with both peptides, but we did not detect any lysis of loaded cells excluding these two candidates (data not shown). This result was in good agreement with the lack of melanoma recognition since most melanoma cell lines express both GAGE and NY-ESO-1 genes. To date, no myeloma-restricted antigen has been reported.
Myeloma cells are poorly immunogeneic and it remains very difficult to obtain specific autologous CTLs. Indeed, co-culture of PBMCs with autologous dendritic cells pulsed with apoptotic or necrotic myeloma cells did not allow us to isolate CTLs specific for myeloma (L. Garderet, C. Pellat-Deceunynck, unpublished results). Only very few articles reported successful obtention of myeloma-specific CTLs. Dhodapkar et al. (30) reported the obtention of myeloma-specific CTL lines after co-culture of PBMCs with dendritic cells loaded with myeloma cells coated with anti-CD138 mAb. On the other hand, Milazzo et al. (31) showed that DC transfected with RNA extracted from myeloma cell lines induced myeloma-specific CTL lines. In both reports, (i) myeloma cells were not primary cells but cell lines and (ii) CTL clone was neither isolated nor characterized. To date, disregarding our own autologous and allogeneic CTL clones, only one CTL specific of myeloma cells has been reported by Orsini et al. (7). This CTL was associated with GVM in a patient receiving DLI after relapse following allogeneic BMT from a HLA-identical sibling donor. This Vbeta13.1 CTL recognized myeloma cells only.
Taking all data together, we can conclude that allogeneic approach seems to be of particular interest to obtain myeloma-specific CTLs since (i) autologous approach is not easy and not very successful and (ii) allogeneic approach ought to allow the isolation of myeloma-restricted CTLs that recognize peptides encoded by normal over-expressed genes.
On the other hand, allogeneic peptide-dependent CTLs could be useful in vivo in patients receiving non-HLA-identical allograft. It is noteworthy that, in spite of inducing GVHD, transplantation of hematopoietic stem cells in patients with leukemia or myeloma may be associated with a beneficial anti-tumor effect (32). In HLA-identical allograft, beyond the recognition of minor histocompatibility antigens, the anti-tumor effect is supposed to be mediated by tumor-specific CTLs that could recognize TAA (7, 32, 33). In unrelated HLA-matched transplantation, HLA-Cw alleles may be mismatched since matching criteria do not involve them. Thus, in a HLA-Cw mismatch graft, our data indicate that allogeneic HLA-Cw-specific CTLs could account for a selective reactivity against tumor cells. Although transplantation of leukemic patients with HLA-Cw (but not HLA-A or -B) mismatch hematological graft enhanced GVHD, it tended to reduce tumor relapse (34, 35).
In conclusion, our results show that the allogeneic context is of interest to isolate myeloma-restricted CTLs that could permit the identification of myeloma-specific antigens. Such CTLs could have some impact in vivo on patients with MM receiving unrelated HLA-matched transplant. Selected allogeneic CTLs should be of interest for future adoptive immunotherapy of patients with MM.
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Acknowledgements
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We thank M. Marget for providing us with HLA-Cw6-specific scFv, M. Bonneville, E. Chalmeau, N. Gervois, H. Vié and T. Guillaume for helpful discussions, advices and critical reading of the manuscript and F. Bonneville for performing HLA-Cw*0602 PCR. This work was supported by the Ligue Contre Le Cancer (Labellisation 2004).
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Abbreviations
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GVHD | graft-versus-host disease |
GVM | graft-versus-myeloma |
HMCL | human myeloma cell line |
MM | multiple myeloma |
RT | reverse transcription |
scFv | single-chain antibody fragments |
TAA | tumor-associated antigen |
TNF | tumor necrosis factor |
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Notes
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Transmitting editor: L. Moretta
Received 23 March 2005,
accepted 13 June 2005.
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