British Journal of Haematology, 2001, 112, 410±420
Myeloma-reactive allospecific cytotoxic T lymphocytes lyse target cells via the granule exocytosis pathway Maurizio Chiriva-Internati, 1 Jianhui Du, 1 Martin Cannon, 2 Bart Barlogie 1 and Qing Yi 1 1 Myeloma and Transplantation Research Center, Arkansas Cancer Research Center, and 2 Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA Received 26 June 2000; accepted for publication 13 September 2000
Summary. Accumulating evidence indicates that a graft-vs.myeloma effect (GVM) and its associated clinical remission of the disease can be induced by donor lymphocyte infusion in myeloma patients who have relapsed after allogeneic bone marrow transplantation. Although it is believed that GVM is induced by allospecific T cells, T-cell subsets and the mechanisms involved in the killing of myeloma cells by donor T cells have not been studied. In this study, we generated allospecific cytotoxic T lymphocyte (CTL) lines against three different myeloma cell lines, ARK, ARP-1 and U266, from unmatched healthy donors and examined their cytotoxicity against the target cells. Our results demonstrate that the allospecific CTLs efficiently lysed myeloma cells. The observed cytotoxicity was mediated mainly by CD81 T cells and inhibited by MHC class I-blocking antibody.
Furthermore, the CTLs lysed the target cells via the perforinmediated pathway, as concanamycin A, but not brefeldin A (the selective inhibitors for perforin- or Fas-mediated pathways respectively) or tumour necrosis factor-a (TNF-a)blocking antibody, abrogated the cytolytic activity of the cells. These CTLs expressed and produced predominantly TNF-a and interferon-g (IFN-g), indicating that they belong to the type 1 T-cell subsets. Taken together, these results indicate that CD81 allospecific T cells may be responsible for mediating GVM and that the granule-mediated lysis of target cells is the major pathway in the CD81 T-cell response against myeloma cells.
Multiple myeloma (MM) is a malignant plasma cell disorder accounting for approximately 1% of all cancers and 10% of haematological malignancies. Myeloma is still an incurable disease. With standard melphalan±prednisone or combination chemotherapy using additional cytotoxic drugs, complete remission rates have not exceeded 5% and the median survival has not been extended beyond 3 years (Alexanian & Dimopoulos, 1994; Barlogie, 1995). High-dose chemotherapy or chemo-radiotherapy with haematopoietic stem cell support increases the incidence of complete remission and extends event-free and overall survival (Attal et al, 1996; Barlogie et al, 1997). However, relapse of the underlying disease remains the primary cause of treatment failure (Tricot et al, 1995). Allogeneic bone marrow transplantation is a curative treatment for selected patients with haematological malignancies (Bortin et al, 1979; Gale & Champlin, 1984)
including MM (Gahrton et al, 1991). Its curative potential depends largely on an anti-tumour effect derived from the adoptive transfer of immunocompetent cells present in the donor graft (Bortin et al, 1979; Gale & Champlin, 1984). It was shown in chronic myeloid leukaemia relapsing after transplantation that donor lymphocyte infusion induced not only graft-vs.-host disease (GVHD), but also a graft-vs.leukaemia effect (GVL) that resulted in durable cytogenetic and molecular genetic remissions (Kolb et al, 1990; BaÈr et al, 1993; Drobyski et al, 1993). The anti-leukaemic effect of GVHD and donor lymphocyte infusion is now well documented in both acute leukaemia and chronic myeloid leukaemia (Sullivan et al, 1989; Kolb et al, 1990; BaÈr et al, 1993; Drobyski et al, 1993; Porter et al, 1994; Slavin et al, 1996). More recently, a graft-vs.-myeloma effect (GVM) has also been observed in the post-allograft relapse setting of MM, following infusion of donor lymphocytes (Tricot et al, 1996; Verdonck et al, 1996; Lokhorst et al, 1997). Taken together, these findings indicate the powerful anti-tumour effect mounted by allospecific donor T cells. There is no doubt that cytotoxic T lymphocytes (CTLs) are the major effector cells in protection against tumours
Correspondence: Qing Yi, M.D. Ph.D., Myeloma and Transplantation Research Center, University of Arkansas for Medical Sciences, 4301 West Markham Street, Slot 776, Little Rock, AR 72205, USA. E-mail:
[email protected]
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Keywords: alloantigen, CTL, perforin, multiple myeloma, tumours.
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Granule-mediated CTL Lysis of Myeloma Cells (Takesue et al, 1990; Greenberg, 1991). As GVHD is caused by recognition of host histocompatibility antigens by donor lymphocytes (Billingham, 1967; Antin & Ferrara, 1992), it is thus conceivable that the GVM is also mediated by allospecific T cells. However, despite the clinical evidence, no study has been published to examine whether and how donor lymphocytes kill myeloma cells and which subsets of T cells, e.g. CD41 or CD81 cells, are involved in this process. In this study, we generated myeloma-reactive CTLs from MHC-unmatched healthy donors and examined their cytotoxicity against the target cells. Our results show that the CD81 CTLs play a major role in the lysis of myeloma tumour cells via the granule exocytosis pathway. PATIENTS AND METHODS Myeloma cell lines and healthy donors. The U266 (A2, B49,56, Cw1,w7, DR1,4, DQ5,3) human myeloma-derived cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). The ARP-1 (A1, B15,27, Cw2,w3, DR11,14, DQ5,7) and ARK (A66,68, B41,44, Cw5,w17, DR4,13, DQ7) cell lines were established at the Arkansas Cancer Research Center from bone marrow aspirates of patients with MM (Harding et al, 1994) and were provided by Dr J. Epstein. Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Grand Island, NY, USA) containing 10% heat-inactivated (568C, 30 min) fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin and 4 mmol l-glutamine (Gibco). Blood was obtained from three healthy blood donors and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Hypaque density centrifugation. The cells were frozen at 2808C until use. The MHC type of the donors was A1, B44,27, Cw2, DR1,11, DQ5,7 (donor no. 1), A24,11, B7,11, Cw7, DR15,13, DQ6 (donor no. 2) and A1,2, B44,13, Cw5,w6, DR4,7, DQ2,7 (donor no. 3). Monoclonal antibodies. Fluoroscein isothiocyanate (FITC)and phycoerythrin (PE)-labelled antibodies against human CD3, CD4, CD8, CD14, CD19, CD38, CD138 and CD56 were purchased from Immunotech (Marseille, France). FITClabelled anti-Fas and a purified antibody against human FasL were from PharMingen (San Diego, CA, USA). To study MHC restriction of T-cell responses, monoclonal antibodies against human MHC class II molecules HLA-DR (IgG2b; Immunotech), class I molecules HLA-ABC (IgG2b; Chemicon International, Temecula, CA, USA) and an isotypic control IgG2b (Immunotech) were used. For intracellular staining of cytokine and perforin expression, PE-conjugated monoclonal antibodies against human interferon-g (IFN-g), interleukin-2 (IL-2), IL-4 and tumour necrosis factor-a (TNF-a) were from R & D Systems (Minneapolis, MN, USA). Perforin and isotypic control IgG were from PharMingen. Flow cytometric analysis. The expression of various membrane-bound molecules was determined using direct immunofluorescence with FITC- and PE-conjugated antibodies or indirect immunofluorescence using a purified antiFasL antibody (IgG1, NOK-1, PharMingen), followed by PE-conjugated rat anti-mouse IgG1 (Becton Dickinson Immunochemistry System, San Jose, CA, USA). Cells were
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analysed using a FACScan flow cytometer (Becton Dickinson) as described previously (Yi et al, 1992). For intracellular cytokine or perforin staining, a Cytofix/ CytoPerm Plus (with GolgiPlug) kit (PharMingen) was used. Briefly, cells were fixed and permeabilized in Cytofix/ Cytoperm solution for 20 min and then washed once with Perm/Wash solution. PE- and FITC-conjugated antibodies or isotypic control IgG were added to the cells and incubated on ice for 20 min. After incubation, cells were washed twice with the Perm/Wash solution and resuspended in phosphate-buffered saline (PBS) for analysis. Generation of myeloma-reactive allospecific CTL lines. Myeloma-reactive allospecific CTL lines were prepared by stimulating donor lymphocytes with myeloma cell lines. Donor PBMCs were plated in growth medium in flatbottomed 24-well plates (Becton Dickinson Labware, Franklin Lakes, NJ, USA) at 1 106 cells per well and stimulated with 2 105 irradiated (50 Gy) cells of each individual myeloma cell line. After 7 d of co-culture, the cells were harvested and live cells were isolated by Ficoll-Hypaque density centrifugation and subcultured in flat-bottomed 24well plates at 5 105 per well, and restimulated with 1 105 per well of irradiated myeloma cells. On d 3, the cultures were treated with human IL-2 (Boehringer Mannheim, GMbH, Germany) at 5 U/ml and recombinant human IL-7 (R & D Systems) at 2´5 ng/ml. Four days after the addition of the cytokines, cells were harvested and restimulated with the target cells at a ratio of 5:1. IL-2 (10 U/ml), rIL-7 (5 ng/ml) and 1 106 irradiated autologous PBMCs (as the feeder cells) were added on d 3. Thereafter, the cells were restimulated weekly with irradiated target cells and expanded with the addition of cytokines and feeder cells. After 4±6 weeks of antigen stimulation, the cells were expanded by culturing with fourfold irradiated (30 Gy) autologous PBMCs, 20% of irradiated (80 Gy) individual myeloma cell lines and 20 U/ml IL-2 added on d 2. The specificity and function of the cultured T cells were monitored weekly by examining their cytotoxic and proliferative activity. Allogeneic mixed lymphocyte reaction (MLR). The proliferative activity of the T cells was examined by allogeneic MLR. PBMCs and T cells were cultured at 1 105 per well in U-bottomed 96-well plates (Becton Dickinson Labware) with various numbers of irradiated myeloma cells for 6 d. Six hours before harvest,37 kBq/well of [3H]-thymidine (Dupont-NEN Life Science Products, Boston, MA, USA) was added. The cells were collected using a Skatron combi cell harvester (Skatron A/S, Lier, Norway) and radioactivity was measured in a liquid scintillation analyser (Packard, Meriden, CT, USA). The results are expressed as mean counts per minute (c.p.m.) of triplicates. Cytotoxicity assay. Target cells were labelled with 1´85 MBq of 51Cr (Amersham, Buckinghamshire, UK) for 1 h and washed three times in RPMI-1640 medium with 10% FBS. Labelled cells (104) were added to triplicate wells of U-bottomed 96-well plates. Effector cells suspended at various E:T ratios in 100 ml were added to target cells. Plates were centrifuged at 800 r.p.m. for 2 min and then incubated at 378C for 4 h. After incubation, the plates were
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centrifuged again at 1000 r.p.m. for 3 min and supernatants (100 ml) were collected and chromium release was measured in a gamma counter (Packard). The results are expressed as the mean c.p.m. of the triplicates. The percentage of specific cytotoxicity was calculated as followed: (experimental c.p.m. 2 c.p.m. spontaneous release)/(c.p.m. maximal release 2 c.p.m. spontaneous release) 100. Spontaneous release was below 20% of the maximum 51Cr uptake. To determine whether cytolysis was restricted by MHC class I or class II molecules, target cells were preincubated for 30 min with the antibodies against HLA-ABC or HLADR and an isotypic control mouse IgG at 50 mg/ml. In some cases, the antibodies or control IgG were added to the cultures at a concentration of 20 mg/ml. Inhibition of perforin-, Fas- and TNF-a -mediated pathways of cytolysis. Concanamycin A (CMA; Sigma, St Louis, MO, USA), an inhibitor of vacuolar type [H1]-adenosine triphosphate (ATPase), or brefeldin A (Sigma) were used as selective inhibitors of perforin-mediated and Fas-mediated cytotoxicity respectively. Effector cells were pretreated with 100 nmol/l CMA or 10 mmol/l brefeldin A for 2 h and assayed for cytotoxicity in the presence of the drugs, as described previously (Kataoka et al, 1996). To examine the effect of TNF-a on cytotoxicity, a monoclonal antibody against human TNF-a (MAB210, R & D) at a final concentration of 50 mg/ml was used to pretreat effector cells for 1 h before the assay was performed. This procedure has been shown to inhibit membrane-bound TNF-amediated cytotoxicity (Aversa et al, 1993). Each cytotoxicity assay was repeated at least three times. Enzyme-linked immunospot (ELISPOT) assay. The detailed methods of the ELISPOT assay for the enumeration of antigen-specific cytokine-secreting cells have been described previously (Yi et al, 1993, 1995). Briefly, plates (MillititerHAM; Millipore, Bedford, MA, USA) were coated with mouse monoclonal anti-human IFN-g (1 mg/ml), IL-4 (2´5 mg/ml) or TNF-a (2´5 mg/ml) (R & D Systems). Cultured T cells (104 cells/well) were added and incubated with the same amount of irradiated myeloma cells for 24 h at 378C. Cells incubated without myeloma cells or with phytohaemagglutinin (PHA) (10 mg/ml; Sigma) were used as controls. After incubation, cells were detached from the plates by washing and polyclonal anti-human IFN-g (250 ng/ml), IL-4 (1 mg/ml) (R & D System) or TNF-a (4 mg/ml; Serotec, Oxford, UK) were added and incubated for 2 h. The spots were developed by sequential incubation with biotinylated anti-goat (1:500; IFN-g and IL-4) or anti-rabbit (1:1000; TNF-a) antibodies (Vector Labs, Burlingame, CA, USA), followed by peroxidase staining using the substrate 3-amino-9-ethyl-carbazol (Sigma). Spots corresponding to the cytokine-secreting cells were enumerated under a dissection microscope (Stemi SV6, Zeiss, Jena, Germany). All samples were run in duplicate. Data are expressed as the mean number of cytokine-secreting cells/104 T cells. To determine whether cytokine secretion was restricted by MHC class I or class II molecules, antibodies against HLA-ABC or HLA-DR and an isotypic control mouse IgG at 10 mg/ml were added to the cultures at the initiation of the culture.
RESULTS Generation of myeloma-reactive allospecific CTLs CTLs against three different myeloma cell lines (ARK, ARP-1 and U266) were generated from three MHC unmatched healthy donors by co-culturing donor PBMCs with each individual myeloma cell for 4±6 weeks and subsequently expanding T cells using autologous PBMCs, myeloma cell lines and IL-2. During the process, a sample of cultured T cells was taken out and their activity and specificity against the target cells were examined in allogeneic MLR and [51Cr]release assays. Figure 1 depicts representative results of the experiments monitoring the T-cell proliferative response during repeated, weekly stimulation with the myeloma cells. Myeloma cell lines ARK and U266 induced a higher T-cell proliferation in the primary cultures and the response increased as T cells were further stimulated and selected by the myeloma cells on a weekly basis. However, ARP-1 was less efficient at stimulating donor T cells, despite their MHC disparity. The specificity of the T-cell response to the alloantigen was confirmed as the other two myeloma cell
Fig 1. Allogeneic MLR showing T-cell proliferation (c.p.m.) with (B) or without (A) alloantigen stimulation by individual myeloma cell lines ARK (A), ARP-1 (B) and U266 (C) in primary (week 0) and secondary (week 1) cultures and after three consecutive weekly cultures (weeks 2, 3 and 4). PBMCs were from donor no. 1. Mean ^ SD were determined from triplicate values of three experiments. In these tests, T-cell response against the other two myeloma cell lines (control cells) were also examined and c.p.m. obtained in such cultures were low (# 8000).
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Granule-mediated CTL Lysis of Myeloma Cells lines, i.e. the cell lines that were not used to stimulate and expand the T cells, induced much weaker T-cell proliferation (c.p.m. generally less than 8000). Similar to the results depicted in Fig 1, ARK and U266, but not ARP-1, induced a strong T-cell proliferation with PBMCs from the other two donors (data not shown). T cells recovered from week-2 cultures and thereafter were also examined for their cytotoxic activity against myeloma cells in a standard 4-h [51Cr]-release assay. As exemplified by the experiments in Fig 2 monitoring the cytotoxicity of T cells during the culture period, U266- and ARK-stimulated T cells recovered after 2 weeks of antigen stimulation efficiently lysed the target cells, and the percentages of cytotoxicity increased by repeated, weekly stimulation with alloantigen and reached almost 90% at week 5 at an effector:target ratio of 40:1. T cells derived in culture with ARP-1 showed less cytotoxicity (about 30%) against their target cells. These results were reproduced in other experiments using the same or other donor cells (Fig 3). After a 4-week antigen stimulation and selection, the percentage of cytotoxicity against the target cells of CTLs derived from the donors ranged between 65% and 90% for ARK- and U266-stimulated cells, and between 10% and 35% for ARP-1-stimulated cells respectively. Less than 20% of cell lysis was observed with control target cells including other myeloma cell lines and autologous PBMCs. Thus, it appears that myeloma cell lines ARK and U266 were efficient allo-antigen-presenting cells (APCs); they were able to activate CTLs and could be lysed by the T cells, while ARP-1 was less efficient at generating CTL response. The cytotoxicity was not contributed by natural killer (NK) activity as no lysis was observed with an NK-sensitive target K562 (data not shown). These data indicate that myelomareactive allospecific T-cells with strong cytotoxicity/CTLs had been generated. Phenotype of CTL lines. After 4 weeks of antigen stimulation, the T cells were pooled and expanded to cell lines by coculturing with irradiated autologous PBMCs, myeloma cell lines and IL-2. We had obtained CTL lines against the myeloma cell lines from all the blood donors and examined their phenotype and function. First, flow cytometric analysis was applied to examine the subsets of CTL lines and the
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representative results are shown in Fig 4. While both CD41 and CD81 T cells were detected, it was clear that CD81 T cells were the in majority in ARK- and U266-reactive CTL lines. In contrast, ARP-1-reactive T-cell lines contained predominantly CD41 T cells. In these T-cell lines, CD561 NK cells were absent or at very low percentages. Similar results were obtained with T-cell lines from other donors (ARK-CTLs; CD41: 19±49%, CD81: 50±78%, NK: 0±3%; ARP-1-CTLs; CD41: 66±90%, CD81: 9±30%, NK: 1±5%; U266-CTLs; CD41: 20±40%, CD81: 58±78%, NK: 1±8% respectively). Thus, a predominant CD81 CTL response was generated. In the following experiments examining MHC restriction and cytolytic mechanisms of CTLs, we focused on ARK- and U266-reactive CTL lines from different donors, as these two cell line-reactive CTLs had strong cytotoxic activity against their target cells. MHC restriction To investigate whether CTL-mediated cytotoxicity against the target cells was MHC restricted, we studied the effect of blocking antibodies against MHC class I and class II molecules HLA-ABC and HLA-DR on the cytotoxicity of the CTLs. As shown in Fig 5A, MHC class I-blocking antibody significantly inhibited (60±90%) the cytotoxicity, whereas the class II-blocking antibody had less effect (20±30% of inhibition). The isotypic control IgG did not affect the cytotoxicity. Similar results were obtained with the CTL lines from the other two donors (55±90% inhibition by class I antibody and 10±40% by class II antibody respectively). Thus, the CTL-mediated cytotoxicity appeared to be predominantly MHC class I restricted. The perforin pathway predominates in the lysis of myeloma cells by allospecific CTL lines. Treatment of effector cells with brefeldin A or CMA selectively blocks the Fas- or granulemediated pathways respectively. Brefeldin A selectively inhibits Fas-based cytotoxicity by inhibition of intracellular glycoprotein transport and CMA is a selective inhibitor blocking only perforin-based cytotoxicity mostly as a result of accelerated degradation of perforin by an increase in the pH of lytic granules (Kataoka et al, 1996). The representative results shown in Fig 5B demonstrated that, while treatment of the CTLs with brefeldin A (10 mmol/l)
Fig 2. [51Cr]-release assay showing specific lysis of myeloma cell lines ARK (A), ARP-1 (B) and U266 (C) by their respective CTLs from donor no. 1 obtained after 2 (B), 3 (O) and 4 (X) weeks of culture with the target cells. Also shown are the lysis of control target cells, i.e. the other two irrelevant myeloma cell lines (A and K) and autologous PBMCs (W), by the CTLs obtained after 4 weeks of culture with the target cells. The data shown are representative of three separate experiments. q 2001 Blackwell Science Ltd, British Journal of Haematology 112: 410±420
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Fig 3. [51Cr]-release assay showing specific lysis of myeloma cell lines ARK (B), U266 (O) and ARP-1 (X) by their respective CTLs from donor no. 3 obtained after 4 weeks of culture with the target cells. Also shown are the lysis of control target cells, i.e. one of the other two irrelevant myeloma cell lines ARP-1 (A), ARK (K) or U266 (W), by the CTLs. The data shown are representative of four separate experiments.
produced some inhibition, CMA (100 nmol/l) almost totally abrogated the cytotoxicity of ARK- and U266-stimulated CTL lines against their target cells. Indeed, CMA treatment induced more than 80% inhibition of the cytotoxicity of all CTL lines, including ARP-1-stimulated T-cell lines, from the donors. However, the CTL lines could also lyse Fas-sensitive target cells. As exemplified by the experiments in Fig 5C, when Jurkat cells were used, a significant cytotoxicity was observed. Treatment of CTLs with CMA had no effect, while addition of a FasL-blocking antibody (NOK-1, PharMingen) to the culture at a final concentration of 20 mg/ml abrogated the cytotoxicity. We next examined the expression of surface FasL and intracellular perforin of the CTL lines, and representative
Fig 4. Flow cytometric analysis showing the percentages (mean ^ SD from three experiments) of CD41 T cells (B), CD81 T cells (B) and CD561 NK cells (A) in myeloma cell-reactive CTL lines from one donor.
Fig 5. Inhibition of CTL-mediated cytotoxicity by (A) antibodies against MHC class I (HLA-ABC), class II (HLA-DR) or an isotypic control IgG; and by (B) concanamycin A (CMA), brefeldin A and an anti-TNF-a-blocking antibody. Shown are the representative results from two CTL lines, ARK-reactive CTLs (B) and U266-reactive CTLs (A). Mean ^ SD were determined from three experiments. (C) shows cytotoxicity of ARK-reactive CTLs against Jurkat without (B) or with the treatment of the effector cells with CMA (A) or addition of a FasL-blocking antibody to the culture (X).
histograms of FasL and perforin expression by CTLs and a control cell, PHA-stimulated PBMCs, are shown in Fig 6. It is evident that a significant portion of the allospecific CTLs expressed perforin, while FasL was expressed only on a small population of the cells. Both perforin and FasL were expressed by PHA-stimulated PBMCs. Figure 7 depicts the representative results obtained with ARK- and U266stimulated CTL lines from a donor. In line with the blocking experiments, FasL was expressed only by a very small percentage of the T cells and the expression was not upregulated by alloantigen stimulation. On the contrary, intracellular expression of perforin was detected in both CTL lines and the percentages of perforin-expressing T cells were
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Fig 6. Flow cytometric analysis of FasL and perforin expression of myeloma-reactive CTLs. Shown are the results of U266reactive CTLs and control cells (PHAstimulated PBMCs from a healthy donor) staining with PE-labelled anti-FasL or perforin antibodies (histogram with bold line). The histogram with the thin line depicts staining with an isotype-matched control antibody.
increased after overnight exposure to the target cells. However, incubation of CTLs with control cells, such as other myeloma cell lines or autologous PBMCs, did not alter the percentage of perforin-expressing T cells (data not shown). Collectively, perforin expression was detected in 10±40% of CTLs obtained from the donors. Expression of membrane-bound TNF-a TNF-a exists in both membrane-bound and secretory forms, and the membrane-bound form is known to be expressed on some CD81 and CD41 CTLs and exerts cytotoxicity against appropriate target cells (Kriegler et al, 1988; Yasukawa et al, 1993). Flow cytometric analysis showed that membranebound TNF-a was expressed on about 60% of our allospecific CD81 T cells, but only on about 3% of the CD41 T cells. To evaluate the role of membrane-bound TNF-a in lysis of the myeloma cells, we examined the effect of an anti-TNF-a antibody on the cytotoxicity of ARK- and U266-stimulated CTL lines. This antibody has been shown to efficiently block membrane-bound TNF-amediated cytotoxicity (Aversa et al, 1993). As shown in Fig 5B, no inhibition of cytotoxicity was induced by the pretreatment of effector cells with the antibody, suggesting that membrane-bound TNF-a was not involved. Cytokine production Cytokine expression and secretion by the CTL lines were examined using intracellular staining and the ELISPOT assay. Figure 8 shows histograms of intracellular cytokine staining of CTLs. The representative results shown in Fig 9A were obtained by the detection of intracellular cytokines of ARK- and U266-reactive CTL lines and demonstrate that the majority of the CTLs expressed TNF-a. IFN-g was expressed in 25±40% and IL-4 in less than 5% of the cells. Antigen stimulation did not increase the percentages of
positive cells. The ELISPOT assay revealed that the unstimulated cells contained a relatively higher number of cells secreting TNF-a and IFN-g (40±200 per 104 T cells) than those secreting IL-4 (7±10 per 104 T cells) (Fig 9B). After exposure to the target cells, the number of TNF-a- and IFN-g-secreting cells increased 10- to 20-fold, while the number of IL-4-secreting cells increased only onefold. Incubation with control cells, such as the other irrelevant myeloma cell lines, did not affect the percentage of cytokineexpressing or cytokine-secreting T cells (data not shown). The predominance of TNF-a and IFN-g production also was observed in ARP-1-stimulated T-cell lines (data not shown). The increase in the TNF-a- and IFN-g-secreting cells induced by co-culture with the target cells was inhibited by MHC class I-blocking antibody (60±80% of inhibition), while class II-blocking antibody had less effect (20±30%
Fig 7. Flow cytometric analysis showing the percentages (mean ^ SD from four experiments) of ARK-(B) and U266-reactive (A) CTL lines (CD31 T cells) expressing FasL and perforin. Shown are the results of unstimulated cells and cells stimulated with the target cells overnight.
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Fig 9. Cytokine expression and production of ARK- and U266reactive CTL lines evaluated by intracellular staining (A) and ELISPOT assay (B). Cytokines examined are TNF-a (B), IFN-g (B) and IL-4 (A) and the results are shown as the percentage of CD31 cells expressing the cytokines (A) and the number of cytokinesecreting cells per 104 T cells (B). Mean ^ SD were calculated from three experiments.
Fig 8. Flow cytometric analysis of cytokine expression of myelomareactive CTLs. Shown are the results of U266-reactive CTLs staining with PE-labelled anti-TNF-a, IFN-g and IL-4 antibodies (histogram with bold line). The histogram with the thin line depicts staining with an isotype-matched control antibody.
inhibition). These results were reproduced from the other donors (data not shown) and indicate that CD81 T cells were the main producers of the cytokines. DISCUSSION It is becoming increasingly clear that a GVM effect, although associated with GVHD, can be evoked after donor lymphocyte infusion (Tricot et al, 1996; Verdonck et al, 1996; Lokhorst et al, 1997). It is believed that GVHD and, probably, GVM are attributed to donor T cells recognizing antigens encoded by the minor or major, if not completely matched, histocompatibility complex genes of the host (Billingham, 1967; Antin & Ferrara, 1992). As CTLs are the major effector cells in tumour setting, it is conceivable that allospecific donor CTLs may be responsible for mediating the GVM. The primary goal of this study was to generate allospecific myeloma-reactive CTLs from MHCunmatched healthy donors and to examine their cytotoxicity against the target cells. In addition, we investigated the
cytolytic pathways, e.g. FasL-, perforin- or TNF-a-mediated cytotoxicity, MHC restriction and patterns of cytokine secretion, in order to identify T-cell subset(s) that are responsible for GVM. To generate myeloma-reactive allospecific CTLs from donors, we used myeloma cell lines as the target cells. We obtained several CTL lines against ARK, ARP-1 and U266 and examined their cytolytic activity against the target cells. Our results demonstrated that the allospecific CTLs efficiently lysed myeloma cells. Although both CD41 and CD81 T cells were present and the percentages of these cells varied among our CTL lines, the cytolytic activity appeared to be correlated with the proportion of CD81 T cells. Indeed, a strong cytolytic activity was observed in U266- and ARKspecific CTLs, in which the majority of the cells were CD81 T cells. Moreover, we have examined purified CD41 and CD81 T cells and found that the percentages of cytotoxicity mediated by these cells were 20±30% and 80±90% respectively. Further support came from analysis of MHC restriction; class I-blocking antibody induced 60±90% inhibition, whereas class II antibody resulted in 20±30% inhibition of the cytotoxicity. Taken together, these results indicate that allospecific CD81 CTLs are the major effector cells in the lysis of myeloma cells. This observation is in line with several clinical and experimental studies suggesting that CD81 T cells play a critical role in the pathogenesis of GVHD (Champlin et al, 1990; Truitt & Atasoylu, 1991; Soiffer et al, 1993; Nimer et al, 1994; Fowler et al, 1996).
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Granule-mediated CTL Lysis of Myeloma Cells 1
Thus, considering that CD8 T cells are the major player in both GVHD and GVM, depletion of CD81 T cells from donor lymphocytes (Champlin et al, 1990; Nimer et al, 1994; Alyea et al, 1998) may reduce the incidence of GVHD, but might also compromise their anti-tumour effect. Cytokine expression by myeloma-reactive CTLs was analysed by intracellular staining and ELISPOT assay. Results from both assays show that the CTLs produced mainly TNF-a and IFN-g. IL-4-producing cells were very low. Interestingly, overnight incubation of CTLs with their target cells did not elevate the percentages of cytokineexpressing T cells determined by intracellular staining, but such a treatment resulted in 10- to 20-fold increases in the numbers of IFN-g- and TNF-a-secreting cells (Fig 7). These results may indicate that the T cells were in effector phase; further antigenic exposure had no effect on expansion of the cytokine-expressing T-cell population, but triggered the T cells to secrete effector molecules such as IFN-g and TNF-a. Thus, according to the current subclassification of T cells based on their cytokine profile (Cox & Liew, 1992; Mosmann & Sad, 1996), our CTL lines belong to the type 1 T-cell subsets. This observation is in agreement with a previous study of a murine leukaemia model showing that the type-1 cells generated GVL, while the type-2 cells abrogated the effect (Fowler et al, 1996). We have shown previously that myeloma cells can act as APCs, i.e. they could activate allospecific T cells and also present recall antigens to autologous T cells (Yi et al, 1997). The present study investigated further whether these cells were able to stimulate the generation of allospecific CTLs in vitro. The prerequisite for alloAPCs is to efficiently present alloantigen to T cells and induce T-cell activation, which requires the expression of MHC class I and II antigens, costimulatory molecules B7-1 and B7-2, and adhesion molecules on the cell surface (Unanue & Allen, 1987; Thompson, 1995). Myeloma cells, especially myeloma cell lines, express some, if not all, of the molecules, such as HLAABC, CD44, CD56, CD54 and VLA-4 (Dupperray et al, 1989; Leo et al, 1992). Expression of HLA-DR, CD40, CD28, CD80 and CD86 was also observed on a small portion of primary myeloma cells and cell lines (Pellat-Deceunynck et al, 1994; ElaÈsser et al, 1996; Yi et al, 1997). Thus, myeloma cells may belong to the category of nonprofessional APCs. Indeed, we demonstrated here that most of the myeloma cell lines tested were able to present alloantigens to donor T cells and activate CTLs. However, it is also worth mentioning that one cell line, ARP-1, was much less efficient at stimulation of allospecific T cells in primary culture and activation of CTLs, despite their MHC disparity on both class I and II antigens. Flow cytometric analysis revealed that all cell lines expressed HLA-ABC and small percentages of the cells also expressed HLA-DR (U266: 17%; ARK: 1%; and ARP-1: 2% respectively). Expression of normal levels of MHC class I, but low levels of class II, antigens has also been demonstrated on primary myeloma cells (ElsaÈsser et al, 1996; Yi et al, 1997). Thus, the disparity in MHC expression on the tumour cells may have contributed to the generation of a dominant CD81 CTL response in this study. CD40 and CD80 were detected on
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0´2±1% of the cells, while CD86 expression was present on 4% of U266, 3% of ARK and 1´8% of ARP-1 respectively. Whether the small difference in the expression of CD86 on ARP-1, compared with other cell lines, or other intrinsic factors accounted for the inability of this cell line to activate allospecific CTLs remains to be investigated. Moreover, our preliminary study with primary myeloma cells from two patients also demonstrates that the cells were inefficient for activation of CTLs, despite the observation that they were able to stimulate a modest proliferation of donor T cells in an allogeneic MLR (data not shown). In accordance, a previous study in pre-B acute lymphoblastic leukaemia showed that the primary tumour cells were inefficient alloAPCs despite the expression of alloantigens and adhesion molecules, and that the ability of the tumour cells to activate allospecific T cells correlated with their expression of B7 antigens (Cardoso et al, 1996). In view of the possibility of developing an adoptive immunotherapy using myeloma-specific CTLs, it is therefore important to identify means of conditioning primary myeloma cells so that they can serve as better APCs. Such methods may include the use of cytokines such as IFN-g (Yi et al, 1997) or CD40L (Cardoso et al, 1996; Schultze et al, 1997) to upregulate the expression of MHC and co-stimulatory molecules on myeloma cells. CTL recognition of target cells via their T-cell receptor activates two distinct mechanisms of cell lysis (Kagi et al, 1994a; Kojima et al, 1994). The first is granule exocytosis mediated by the pore-forming perforin and granzyme A and B. The second involves interaction between the Fas ligand on effector cells and Fas molecules expressed on the target cells. A third pathway, which is mediated by membranebound TNF-a and lymphotoxin, has also been identified (Kriegler et al, 1988; Browning et al, 1991). Although our CTLs expressed membrane-bound TNF-a and secreted the cytokine as well, TNF-a-blocking antibody had no inhibitory effect in the 4-h cytotoxicity assay. However, one cannot exclude the fact that the cytokine may have an effect on the tumour cells, as cytotoxicity mediated by the cytokine needs a longer culture period to become apparent than that mediated by the perforin±granzyme and Fas±FasL systems (Lee et al, 1996). It has been shown that the granule exocytosis pathway is the major pathway used by CD81 CTLs, while CD41 CTLs lyse target cells via the Fas±FasLmediated pathway (Kagi et al, 1994a; Kagi et al, 1994b). In our study, flow cytometric analysis showed that perforin, but not FasL, was expressed in a large population of the CTLs that could be further upregulated by antigen stimulation. The low expression of FasL on antigen-specific CTLs and its upregulation by mitogen, but not by antigen, stimulation was reported previously (Shankar et al, 1999). Also, Fas antigen was weakly expressed on U266, but absent on ARP-1 and ARK cells (data not shown). Moreover, the cells appeared to lyse the target cells mainly via the perforinmediated pathway, as CMA, the selective blocking agent, induced 90% of inhibition of the cytotoxicity. Brefeldin A, a selective inhibitor of the Fas-mediated pathway, resulted in only 10±25% inhibition. These results are consistent with the findings mentioned above and provide further support that perforin-producing CD81 CTLs are the major effector
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cells in the lysis of the myeloma cells. Nevertheless, it should be recognized that CD41 CTLs within the cell lines also had some cytolytic activity against the target cells and may account for up to 20% of the cytotoxicity. This conclusion is built on the results of MHC class II antibody and brefeldin A inhibition experiments. Our findings that myeloma-reactive CTLs lysed the target cells via the perforin-mediated pathway is of special importance in view of published results on Fas expression on myeloma cells. It has been shown that Fas antigen point mutation was detected in 10% of patients' bone marrow samples. The mutations were located in the cytoplasmic region that is involved in transduction of an apoptotic signal and, thus, renders the cells resistant to Fas-induced apoptosis (Landowski et al, 1997a). Furthermore, they found that myeloma cells induced to be drug resistant also became resistant to Fas-mediated apoptosis (Landowski et al, 1997b). Thus, in view of the finding that even Fas1 myeloma cells may be resistant to Fas-mediated apoptosis induced by Fas agonistic antibodies (Shima et al, 1995; Tong et al, 2000), the use of CTLs that are cytotoxic via the Fasmediated pathway may be limited, but the pore-forming CTLs can be used for the treatment of drug-resistant myeloma. GVHD and GVL/GVM are mediated by donor T cells recognizing host histocompatibility antigens (Billingham, 1967; Antin & Ferrara, 1992). These allospecific T cells may recognize alloMHCs associated with specific peptides (Wang et al, 1998; Obst et al, 2000). In line with these results, alloMHC-restricted, antigen/peptide-specific T cells have been identified (Sadovnikova & Stauss, 1996). Thus, it is highly conceivable that different allospecific T cells are involved; GVL/GVM-mediating T cells may include cells recognizing allo-MHCs associated with tumour-derived peptides, whereas T cells mediating GVHD may recognize allo-MHCs associated with peptides derived from epithelial cells or other tissue cells. Therefore, CTLs generated against the myeloma cell lines in the present study may contain tumour-specific T cells and these T cells might be the same cells as those that mediate GVM in vivo. Further studies are warranted to examine the antigen specificity of such CTLs and their role in GVHD and GVM. In summary, our study of myeloma-reactive allospecific CTLs suggests that CD81 T cells may be responsible for mediating GVM and that perforin-mediated lysis of the target cells is the major pathway in the CD81 T-cell response against myeloma cells. Our study also suggests that, if using primary myeloma to activate CTLs, it may be necessary to condition these cells to be better APCs. ACKNOWLEDGMENT This work was supported in part by a grant from The Leukaemia & Lymphoma Society (Qing Yi is a Leukaemia & Lymphoma Society Translational Research Awardee). REFERENCES Alexanian, R. & Dimopoulos, M. (1994) The treatment of multiple myeloma. New England Journal of Medicine, 7, 484±489.
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