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γδ T cell responses to activated T cells in multiple sclerosis patients induced by T cell vaccination
Journal of Neuroimmunology 87 Ž1998. 94–104
gd T cell responses to activated T cells in multiple sclerosis patients induced by T cell vaccination Piet Stinissen a
a,b,)
, Jingwu Zhang
a,b,1
, Caroline Vandevyver c , Guy Hermans
a,b
, Jef Raus
a,b,c
Multiple Sclerosis Research Unit, Dr. L. Willems-Instituut, UniÕersitaire Campus, B-3590 Diepenbeek, Belgium b Limburgs UniÕersitair Centrum (LUC), UniÕersitaire Campus, B-3590 Diepenbeek, Belgium c Biotechnology Unit, Dr. L. Willems-Instituut, UniÕersitaire Campus, B-3590 Diepenbeek, Belgium Received 29 December 1997; revised 10 February 1998; accepted 11 February 1998
Abstract To explore the hypothesis that gd T cells may regulate activated ab T cells, we studied gd T cell responses to ab T cell clones in Multiple Sclerosis ŽMS. patients who received attenuated autologous autoreactive T cells. We recently conducted a pilot study of T cell vaccination with myelin basic protein reactive T cells in MS. Since T cell vaccination upregulates the anti-vaccine T cell responses, we evaluated gd T cell reactivity towards the vaccine in the vaccinated patients. Lymphocytes were stimulated in vitro with irradiated vaccine cells and the responding lines were checked for the presence of gd T cells. Our data demonstrate that in the majority of vaccinated MS patients gd T cells expand upon stimulation with the vaccine cells. The responding gd T cells were predominantly Vd 1qrVg 1q, and represented diverse clonal origins. The gd T cells could not inhibit in vitro proliferation of the vaccine T cells and displayed low cytotoxic reactivity towards the vaccine clones. However, they produced high levels of IL2, TNFa and IL10. These results indicate that gd T cells can be stimulated by activated ab T cells, and that these gd T cell responses are upregulated after T cell vaccination. These findings suggest that gd T cells are involved in peripheral mechanisms to control activated autoreactive T cells. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Multiple sclerosis; gd T lymphocytes; Immunotherapy; T cell vaccination
1. Introduction Myelin basic protein ŽMBP. reactive T cells are considered to play an important role in the autoimmune processes of Multiple Sclerosis ŽMS. Žreviewed in Stinissen et al., 1997.. Although these T cells are part of the normal T cell repertoire, recent data indicate that MBP reactive T cells circulate in an activated state in patients with MS. For instance, activated MBP reactive T cells were shown to accumulate in the central nervous system ŽCNS. of patients with MS, and clonally expanded MBP reactive T cell populations persist for several years in the periphery of some patients ŽChou et al., 1992; Salvetti et al., 1993; Wucherpfennig et al., 1994; Zhang et al., 1994a,b; Vandevyver et al., 1995.. Wucherpfennig and Strominger Ž1995. provided experimental evidence that viral epitopes sharing )
Corresponding author. Tel.: q32 11 269211; fax: q32 11 269209; e-mail: [email protected] 1 Present address: Department of Neurology, Baylor College of Medicine, Houston, TX, USA. 0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 0 6 0 - 5
sequence homologies with MBP can stimulate MBP reactive T cells in vitro. In this scenario, the autoreactive T cells may become activated in the periphery, which allows them to migrate into the CNS and initiate a pathogenic cascade of immune mediated events leading to demyelination. The pathogenic potential of MBP reactive T cells has been further demonstrated in experimental autoimmune encephalomyelitis ŽEAE., an animal model of MS, where CD4q T cells specific for immunodominant MBP epitopes can induce encephalomyelitis ŽBen-Nun et al., 1981a; Vandenbark et al., 1985; Zamvil et al., 1985.. Based on the potential pathogenicity of MBP reactive T cells in MS, several immunotherapeutic strategies have been designed to specifically inactivate these T lymphocytes Žreviewed in Martin and McFarland, 1996.. One of these approaches, termed T cell vaccination, involves immunization with attenuated autoreactive T cells to elicit specific immunity to the pathogenic T cell populations. As shown in EAE, animals vaccinated with attenuated MBP reactive T cells are resistant to subsequent induction of the disease ŽBenNun et al., 1981b..
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In a recent pilot study, we have inoculated 13 MS patients with autologous MBP reactive T cell clones to assess the safety of and immunological responses to T cell vaccination ŽZhang et al., 1993; Medaer et al., 1995.. Cellular anti-vaccine responses were observed in all vaccinated patients, associated with a specific depletion of circulating MBP reactive T cells ŽZhang et al., 1993.. CD8q anticlonotypic T cells specifically recognizing the vaccine clones were isolated from the vaccinated patients and found to induce MHC class I restricted cytotoxicity of the T cell clones used as a vaccine, most likely by recognition of a T cell receptor ŽTCR. related sequence on the vaccine cells ŽZhang et al., 1993, 1995.. The treatment was safe and induced clinical stabilization or reduced progression of the disease in most patients, encouraging further clinical trials of T cell vaccination in MS ŽMedaer et al., 1995; Stinissen et al., 1996.. In this report, we further explored the cellular mechanism of T cell vaccination. In preliminary experiments we noted an unusual expansion of gd T cells when peripheral blood mononuclear cells ŽPBMC. of the vaccinated patients were stimulated with irradiated vaccine T cells. gd T cells represent only a minor fraction of the blood T cells. Although their role in the immune system is controversial, it has been speculated that gd T cells may play a role in the first line immune defense ŽChien et al., 1996.. gd T cells express a limited TCR variable gene diversity, and their activation may occur without processing of antigen by antigen presenting cells. gd T cells may also play a role in the pathogenesis of MS, most likely in the later stages of plaque formation. Recent data also indicated a pathogenic role for gd T cells in EAE ŽRajan et al., 1996.. We and others demonstrated the accumulation of gd T cells in CSF and brain lesions of MS patients, although the antigens responsible for the gd T cell expansion remain unknown ŽSelmaj et al., 1991; Wucherpfennig et al., 1992; Shimonkevitz et al., 1993; Hvas et al., 1993; Battistini et al., 1995; Nick et al., 1995; Stinissen et al., 1995a.. Here we demonstrate that T cell clones used for vaccination, but also PHA induced T cell blasts induce an expansion of gd T cells in primary PBMC cultures of the vaccinated patients. Because the gd T cell expansion is more frequently observed in vaccinated patients than in healthy subjects or unvaccinated MS patients, the gd T cell reactivity most likely is related to the T cell vaccinations. The responding gd T cells predominantly express the Vg 2y phenotype which is uncommon in peripheral blood. To evaluate whether the responding gd T cells play an active role in T cell vaccination, we studied the functional properties of these cells. Interestingly, the isolated gd T cell lines or clones did not inhibit in vitro proliferation of the vaccine T cells. Upon stimulation, these cells produce low levels of IFNg and IL4, but high levels of IL2, TNFa and IL10. The gd T cells lysed EBV transformed B cells, but expressed little cytotoxic reactivity towards the vaccine cells. Our data demonstrate that gd T
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cells can be stimulated by activated T cells in primary cultures and suggest a role for these T cells in regulatory T–T cell interactions.
2. Materials and methods 2.1. Reagents Human MBP was purified from human brain tissue by the method of Deibler et al. Ž1972.. The medium used for cell culture was RPMI 1640 supplemented with 10% heatinactivated FCS and L-glutamine, sodium pyruvate, nonessential amino acids, and 10 mM HEPES ŽLife Technologies, Gent, Belgium.. Recombinant human IL-2 ŽrIL-2. used for T cell culture was obtained from Boehringer Mannheim ŽMannheim, Germany.. 2.2. Patients and immunization protocol 13 MS patients with MS enrolled in a pilot study of T cell vaccination with MBP reactive T cell clones. The patients received three immunizations with irradiated autologous MBP-reactive T cells as previously described ŽZhang et al., 1993.. Briefly, MBP reactive T cell lines were generated from all patients by stimulation of PBMC with MBP under limiting dilution conditions ŽZhang et al., 1993.. Resulting lines were further cloned at single cell densities by stimulation with PHA.P in the presence of irradiated feeder cells. The clones were characterized for their epitope reactivity and TCR V–D–J rearrangements. The clones selected for vaccination were activated with MBP pulsed PBMC as a source of antigen presenting cells, washed, irradiated and injected s.c. in PBS at a dose of 10–15 million cells per clone. Three subsequent vaccinations at intervals of 2 to 4 months were performed. 2.3. Primary stimulation of PBMC with T cells and isolation of gd T cells PBMC were isolated from heparinized blood by Ficoll gradient centrifugation. To study the primary gd T cell responses induced by the vaccine T cells or other nonspecific T cells, PBMC were cultured in the presence of irradiated T cells. To this end, 40,000 PBMC were plated in each of 60 wells of a microtiterplate together with 40,000 irradiated stimulator T cells in a total volume of 200 m l culture medium. Culture medium was RPMI 1640 supplemented with 10% heat inactivated autologous serum. Stimulator cells were the vaccine cells, other MBP reactive T cells, PHA stimulated T cell blasts, or EBV transformed B-cells. The stimulating T cells were all used 5–7 days after activation with MBP pulsed PBMC or with PHA and feeder cells, and cultured in IL-2 containing medium Ž5 Urml.. After 7 days, the primary cultures were restimulated with 40,000 cells per well of irradiated stimulator
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cells. After 14 to 18 days of culture, gd T cell lines were identified by positive staining Ž) 10%. with a TCR gd specific antibody ŽTCR gd-1, Becton Dickinson, San Jose, CA.. gd T cell clones were obtained by plating out at 0.3 or 1 cell per well in culture medium in the presence of 10 5 irradiated feeder cells and stimulation with 2 m grml PHA.P. After 24 h rIL-2 was added at 5 Urml. The cultures were fed with fresh medium containing 5 Urml rIL-2 every 3 to 4 days. Growth positive wells were microscopically identified after 10–12 days of culture and were tested for their purity by antibody staining. 2.4. Flow-cytometry
gd T cell lines were identified and analyzed by staining with appropriate monoclonal antibodies as previously described ŽStinissen et al., 1995b.. Briefly, 2–10 = 10 4 cells were split in a V-bottom microtiterplate and washed with 250 ml FACS-buffer ŽPBS containing 5% vrv FCS and 0.01% grv sodium azide.. The plates were subsequently centrifugated for 5 min at 2500 rpm. For direct staining with conjugated antibodies, the cells were stained with antibodies Ž5% vrv. for 30 min on ice. After two final washes the cells were analyzed by flow cytometry using a FACScan ŽBecton Dickinson. with gates set to read the total lymphocyte population. The following antibodies were used in pairs for direct dual staining: phycoerythrin ŽPE. conjugated anti-TCR grd-1 ŽPAN TCR gd .rfluorescein ŽFITC. conjugated anti-TCR-1 arb and FITC-anti-IgG1rPE-anti-IgG2a Žsimultest control. to detect background staining. The antiTCR grd-1, anti-TCR-1 ab and simultest control antibodies were purchased from Becton Dickinson. To analyze the Vg surface expression, gd T cell clones were double stained with phycoerythrin ŽPE. conjugated anti-TCR grd1 and FITC-1B10. 1B10 is a Vg 2 specific monoclonal antibody that was previously developed in our laboratory ŽStinissen et al., 1995b..
release–spontaneous releasermaximum release–spontaneous release. = 100%. 2.6. Analysis of T cell receptor Vg and Vd usage by PCR The TCR V gene usage of gd T cell clones was determined as described elsewhere ŽStinissen et al., 1995a.. Briefly, total RNA was extracted using the RNeasy method ŽQiagen. and 0.5–5 mg RNA was reverse transcribed into first-strand cDNA using the Reverse Transcriptase system of Promega ŽLeiden, the Netherlands.. The cDNA was then added to a standard amplification mixture containing 2.5 U Taq polymerase ŽAmpliTaq, Perkin-Elmer, Norwalk, CT., and 30 pmol of TCR DC primer or GC primer and 30 pmol of a TCRDV1, DV2, DV3, DV4, DV5 or TCRGV1, GV2, GV3 or GV4 specific primer. After 35 cycles of amplification of 1 min at 958C, 1 min at 558C and 2 min at 728C, the PCR products were Southern-blotted and hybridized with digoxigenin ŽDIG. end-labeled TCRDC or GC probes. The primer and probe sequences were published previously ŽStinissen et al., 1995a; nomenclature according to Strauss et al., 1987.. Detection of amplification products was performed with a DIG luminescent detection kit ŽBoehringer Mannheim, Mannheim, Germany., and exposure to a Hyperfilm MP ŽAmersham.. 2.7. Analysis of cytokine production of gd T cells
gd T cell clones Ž10,000 cells per well. were stimulated with PHA.P Ž2 m grml. and irradiated feeder cells Ž100,000 cells per well., or irradiated feeder cells alone Žcontrol.. The feeder cells were irradiated at 8000 rad in a 137Cs source. Cell supernatants were harvested after 72 h of culture, and the production of IL-2, IL-4, IL-10, IFNg and TNFa was determined in the supernatants using commercially available ELISA kits according to the manufacturer’s instructions ŽBiosource, Camarillo, CA..
3. Results 2.5. Cytotoxicity assays T cells ŽMBP reactive T cell clones or PHA stimulated T cell blasts., K562 cells or EBV transformed autologous B cells were used as target cells in a standard chromium release assay. The target cells were labeled with 200 m Ci 51 Cr ŽAmersham, Buckinghamshire, UK. for 1 h at 378C. The labeled target cells were washed four times to remove the free radioactivity, and incubated with the gd T cell clones Žeffectors. at an effector to target ratio between 5 and 20. After 5 h of incubation the supernatants were harvested and the released radioactivity was measured in a gamma counter ŽPackard, Meriden, CT.. The maximum and spontaneous chromium release were determined in wells containing H 2 SO4 or medium alone. Percentage of specific cytolysis was calculated as Žexperimental
3.1. Primary gd T cell responses are detected in PBMC cultures from Õaccinated MS patients stimulated with actiÕated Õaccine T cells To study the cellular immune responses induced by T cell vaccination, we stimulated post-vaccination PBMC samples with irradiated vaccine clones. The PB samples were taken 2 to 8 weeks after the third inoculation. The vaccine clones were all TCR abq and CD4q. The PBMC were plated at 40,000 cells per well in 60 wells in the presence of 40,000 cells per well of irradiated vaccine T cells. The cultures were restimulated with irradiated vaccine T cells after one week, and further cultured for an additional week in the presence of a low dose of exogenous IL-2 Ž5 Urml.. When evaluating the phenotypic
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Fig. 1. Primary gd T cell responses induced by autologous T cell stimuli in vaccinated patients. Post-vaccination PBMC samples from four MS patients were cultured at 40,000 cells per well in 60 wells of a microtiter plate and stimulated with 40,000 cells per well of an irradiated T cell stimulus. The T cell stimuli were the MBP reactive T cell clones used for vaccination Žvaccine clone 1 or 2., unrelated MBP reactive T cell clones with different TCR gene expression Žnon-vaccine clone 1 or 2., or PHA stimulated T cell blasts ŽPHA blasts.. In patients TV4 and TV5 we used two different MBP reactive T cell clones as a stimulus which were both incorporated in the T cell vaccine. After one week, all cultures were restimulated with their respective irradiated stimulator T cells Ž40,000 cells per well., and at day 14 the cultures were microscopically scored for cell growth. Growth positive cultures were stained with a PAN TCR gd monoclonal antibody and evaluated by flow-cytometry. Cultures containing more than 10% gd T cells were scored as TCR gd positive.
Fig. 2. Primary gd T cell responses induced by different autologous and allogeneic cellular stimuli. Post-vaccination PBMC of one patient ŽTV3. were stimulated with various stimuli, and restimulated with the respective stimuli after one week. At day 14 the responding T cell lines were scored for growth and presence of gd T cells Žat least 10%. as described in the legend of Fig. 1. In panel A the stimuli used were an irradiated autologous MBP reactive clone used for vaccination Žvaccine clone., irradiated autologous PHA stimulated T cell blasts ŽPHA blasts. and irradiated autologous EBV transformed B cells ŽEBV-B cells.. In panel B the stimuli used were irradiated autologous PHA stimulated T cell blasts and irradiated allogeneic PHA stimulated T cell blasts from two different donors. The experiment in panel B was performed with PBMC of patient TV3 obtained 1 month after the experiment illustrated in panel A.
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profile of the responding cultures at day 14, we noted that a substantial fraction of these cultures contained high amounts of gd T cells. For example, in some subjects more than 20% of the primary cultures contained 10% gd T cells or more. The percentage of gd T cells in these cultures ranged from 10 to 80%, with a mean percentage between 20 and 30% in all patients. This expansion was not observed in untreated blood lymphocytes of these patients as determined by flow-cytometry. Indeed, the frequency of T cells stained with anti-TCR gd antibody was between 2 and 8% in these patients, which is comparable to the frequencies observed in control subjects Ždata not shown.. To examine if this response was specific for the vaccine clones we stimulated PBMC of 4 vaccinated patients with different autologous T cell stimuli. As seen in Fig. 1, gd T cell enriched primary cultures Ž) 10% gdq. were not only observed with the vaccine clones as a stimulus, but also with other unrelated T cell stimuli: MBP reactive T cells which were not used for vaccination and PHA stimulated T cell blasts ŽPHA blasts.. These T cell stimuli express different TCR variable regions Žnot shown., indicating that the gd T cell response is not induced by a particular variable TCR segment. In one patient ŽTV3. we also included autologous EBV transformed B cells as a stimulus. Fig. 2A illustrates that no gd T cell containing cultures were obtained when EBV transformed B cells were used as a stimulus, indicating that only T cells, but not B cells induce the gd T cell expansion in the primary cultures. These data suggest that the gd T cell expansion is induced by a T cell derived growth factor or a nonspecific T cell surface marker. Finally, we tested the stimulating capacity of PHA blasts from different donors to study the MHC restriction pattern of the responding gd T cells. To this end, PBMC were isolated from a blood sample of patient TV3 taken 1 month after the experiment illustrated in Fig. 2A. The PBMC were stimulated with autologous and allogeneic PHA blasts. As illustrated in Fig. 2B, at this second sampling time again a high fraction of gd T cell positive cultures was observed when using autologous PHA blasts as stimulus. In addition, although the fraction of gd T cell positive cultures varied after stimulation with the different PHA T blasts, the data indicate that primary gd T cell expansion can be induced with activated T cells from different donors suggesting that this response is not restricted by classical MHC molecules. 3.2. Primary gd T cell responses induced by PHA stimulated T cell blasts in a group of Õaccinated patients and control subjects To examine whether the gd T cell responses are related to T cell vaccination, we tested a group of 9 vaccinated MS patients, 9 non-vaccinated MS patients and 6 healthy subjects for their primary gd T cell response to autologous PHA stimulated T cell blasts. PHA blasts were used as a
stimulus since the previous experiments illustrated similar stimulatory efficiencies of PHA blasts and MBP reactive T cell clones to generate gd T cell enriched cultures. In addition, the use of PHA blasts avoided the need to isolate MBP reactive T cell clones from all subjects. The blood samples from the vaccinated MS patients were drawn 2 to 10 weeks after the final inoculation. As seen in Fig. 3, gd T cell containing cultures were obtained in 6 out of 9 vaccinated patients, but only in 2 out of 9 unvaccinated patients and 1 out of 6 healthy subjects. The mean fraction of gd T cell positive cultures Žmore than 10% gd T cells. was 31.5% in 6 responding vaccinated MS patients, 5.9% in 2 responding unvaccinated MS patients, and 3.3% in the one responding healthy subject. These data suggest that the gd T cell sensitization might be related to T cell vaccination since significantly more gd T cells positive cultures were obtained in the vaccinated patients as compared to the control groups Žunvaccinated MS patients and healthy subjects.. 3.3. The gd T cells induced by actiÕated T cells are predominantly Vg 2 y In contrast to ab T cells, gd T cells express only a limited number of TCR V gene elements, and the V gene expression seems to correlate with the site where the gd T cell subsets are localized. gd T cells in the peripheral blood predominantly express Vd 2 and Vg 2 elements, while most of the gd T cells present in thymus and some epidermal tissues are Vd 1qrVg 1q. We studied the phenotype of the vaccine-responsive gd T cell lines by flow-cytometry using a Vg 2-specific monoclonal antibody Ž1B10. which was previously developed in our laboratory ŽStinissen et al., 1995b.. A total number of 215 primary gd T cell lines from 5 vaccinated patients were stained with 1B10 and a PAN-TCR gd antibody. Three different patterns were obtained: Vg 2y lines ŽTCRgdqr1B10y ., Vg 2q lines ŽTCRgdqr1B10q ., and lines which contained b o th a Vg 2 q a n d a Vg 2 y p o p u la tio n ŽTCRgdqr1B10qr1B10y .. Interestingly, in two patients the responding gd T cells almost exclusively expressed the Vg 2y phenotype, while in two other patients T cells expressing the Vg 2y phenotype represented the major gd subset ŽFig. 4.. In total, 59.0% of the tested lines were Vg 2y, 28.4% of the lines were Vg 2q, and 12.6% of the lines contained a Vg 2y and a Vg 2q population. Taken together, the data indicate that the activated T cells predominantly, though not exclusively, stimulate the minor Vg 2y subset of gd T cells in peripheral blood. In this regard, the gd T cell subset that is sensitive to stimulation with activated T cells seems to differ from the PB gd subset that is stimulated with PHA and IL-2. Indeed, as observed in our previous studies, gd T cell lines from the PB of MS patients and healthy subjects obtained by stimulation of PBMC with PHA and IL-2 were predominantly Vd 2qrVg 2q ŽStinissen et al., 1995a..
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Fig. 3. Primary gd T cell responses induced by autologous PHA stimulated T cells blasts in T cell vaccinated MS patients, in control MS patients Žunvaccinated., and in healthy subjects. PBMC of 9 MS patients vaccinated with MBP reactive T cells, 9 MS patients Žnot vaccinated. and 6 healthy subjects ŽNS. were stimulated with irradiated autologous PHA stimulated T cell blasts. The cultures were restimulated after one week, and scored for growth and the presence of gd T cells at day 14 as described in the legend of Fig. 1. In three vaccinated MS patients, 4 control MS patients and 4 healthy subjects no growth positive cultures were obtained at day 14.
In one vaccinated patient ŽTV3. we further studied the TCR V gene usage by RT-PCR. As seen in Table 1, all 15 gd T cell isolates studied expressed the Vg 1 gene. Some of the isolates co-expressed Vg 3 mRNA, however, these Vg 3 gene elements are possibly not functionally rear-
ranged due to a reported splicing defect in these gene segments ŽZhang et al., 1994a,b.. With respect to Vd expression, RT-PCR revealed preferential expression of Vd 1 Ž11r13., while only 2 isolates expressed Vd 3, and one isolate was Vd 2q ŽTable 1..
Fig. 4. Phenotypic characterization of gd T cells responding to stimulation with autologous T cells. gd T cell isolates were dual stained with conjugated PAN TCR gd antibody and conjugated 1B10 antibody and analyzed by flow-cytometry. 1B10 specifically stains the Vg 2q T cell subset. The gd T cells were scored as Vg 2q, Vg 2y and Vg 2qrVg 2y and the fraction of these subtypes for each patient is represented in the figure. A total number of 215 primary gd T cell lines derived by stimulation with autologous T cells Žvaccine T cell clones or PHA stimulated T cell blasts. from 5 vaccinated patients were studied.
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Table 1 TCR V gene usage and TCRDV–D–J amino acid sequences of gd T cells responding to the vaccine T cell clones in patient TV3 Isolate
V gene usage
DV amino acid sequence
DV
GV
DV–DJ
V
N–D–N
J
C
18.31 18.32 P.4 P.15 P.24 P.5 P.7 P.10 P.25 P.12
1 1 1 1 1 1 1 1 1 1, 3
1 1 1, 3 1, 3 1, 3 1, 3 1, 2, 3 1, 3 1, 3 1, 3
5.1 P.13 P.28 30.20 30.21
3 1 2 NT NT
1, 3 1 1, 3 1, 3 1, 3
V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V3–J1 V3–J1 NT NT NT NT
YFCAL YFCAL YFCAL YFCAL YFCAL YFCAL YFCAL YFCAL YFCAL YFCAL YYCAF YYCA
GELLTGLPTRGLTGGS GESTFLPSILVTGSY AEPNINWGIN GEKKTLTYWGIRLG GEPNLNWGID GGKRLYWDT GGKRLYWDT GGRILPPPLGRRK GGRILPPPLGRRK GGRILPPPLGRRK SLRPSYPF STCGGDP
DKLIFGKGTRVTVEP KLIFGKGTRVTVEP KLIFGKGTRVTVEP TDKLIFGKGTRVTVEP KLIFGKGTRVTVEP KLIFGKGTRVTVEP KLIFGKGTRVTVEP TDKLIFGKGTRVTVEP TDKLIFGKGTRVTVEP TDKLIFGKGTRVTVEP DKLIFGKGTRVTVEP TDKLIFGKGTRVTVEP
RSQP RSQP RSQP RSQP RSQP RSQP RSQP RSQP RSQP RSQP RSQP RSQP
3.4. TCR d chain V–D–J sequences of gd T cells Since the clonal diversity of the responding gd T cells may provide information about the nature of the antigenic stimuli, we determined the TCR DV chain sequences of a panel of 11 independent gd T isolates obtained from one patient ŽTV3.. As shown in Table 1, all isolates expressed the DJ1 gene element. The majority of lines Ž9. rearranged a DV1–DJ1 chain, while one line ŽP5.1. expressed a DV3–DJ1 transcript, and one line ŽP.12. expressed both a
DV1–DJ1 and a DV3–DJ1 chain. Interestingly, among the 10 DV1–DJ1 we observed two clonally expanded populations sharing identical DV-chains ŽTable 1.. However, the remaining clones represented unrelated clonal origins. 3.5. Functional properties of gd T cells: cytotoxic and inhibitory reactiÕity towards the T cell Õaccine clones and cytokine profile To study the functional properties of the gd T cell isolates we tested a panel of 38 gd T cell lines isolated
Fig. 5. Cytotoxic reactivity of gd T cells responding to stimulation with autologous T cells. 10 gd T cell lines derived from one vaccinated patient by stimulating PBMC with autologous PHA stimulated T cell blasts were tested for their cytotoxic reactivity towards autologous PHA stimulated T cell blasts, autologous EBV transformed B cells and K562 cells in a standard 51 Cr-release assay. The effector to target ratio was 20. The percentage of gd T cells is indicated for each line.
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from 3 vaccinated MS patients for their cytotoxic reactivity towards the T cells that were originally used as T cell stimuli. The gd T cell isolates tested induced only low specific killing Ž- 10% lysis. of their respective activated T cell targets at an effector to target ratio of 10–20 Ždata not shown.. In contrast, CD8q anticlonotypic ab T cells typically induce 40 to 90% cytolysis of the target vaccine cells ŽZhang et al., 1993.. A panel of 10 gd enriched T cell lines was further studied for their cytotoxic effect on autologous PHA stimulated T blasts, autologous EBV transformed B cells and natural killer sensitive K562 cells. As shown in Fig. 5, almost all lines tested induced high specific cytolysis of the EBV transformed B cells and K562 cells, but they induced no or very low cytolysis of the autologous PHA stimulated T blasts. Thus, the data indicate that the majority of the derived gd T cells induce little or no cytolysis of the activated T cells that were used as the original stimulus, despite their in vitro cytotoxic potential towards other target cell populations. Next, we tested whether the gd T cells could inhibit the antigen induced proliferation of the vaccine T cells. To this end, we cocultured MBP reactive T cells with MBP pulsed antigen presenting cells in the presence and absence of irradiated gd T cells. A panel of 13 gd T cell lines derived from 3 vaccinated MS patients was studied for their inhibitory reactivity. None of the tested gd T cell lines inhibited significantly Ž) 5%. the antigen induced proliferation of the antigen reactive T cell clones. In a similar assay, CD8q anticlonotypic T cells generally induced more than 70% inhibition of their respective indicator T cell clones ŽZhang et al., 1993.. As a final possible functional mechanism involved in the regulation of activated T cells we studied the cytokine profile of the isolated gd T cells. Therefore, two gd T cell clones responding to stimulation with the vaccine T cells, were stimulated with PHA and irradiated feeders, and the production of IFNg , IL4, IL2, TNFa and IL10 was quantified in the cell supernatants after 72 h. using commercial
Table 2 Cytokine production of gd T cell clones Clone
2G4-8
2G4-7
a
Cytokine production Žpgrml. a
control stimulation net production control stimulation net production
IFNg
IL4
IL2
TNFa
IL10
0 12 12 0b 0 0
0 11 11 0 0 0
236 479 243 506 1098 592
64 312 248 61 274 213
16 96 80 22 173 151
10,000 gd T cells were cultured in the presence of 10 5 irradiator feeder cells Žcontrol., or in the presence of 10 5 irradiator feeder cells q2 m grml PHA.P Žstimulation.. Cell supernatants were harvested after 72 h of culture, and cytokine production was determined by ELISA. The cytokine production of PHA stimulated irradiated feeder cells was below the detection limit. b Production below detection limit Ž5 pgrml.
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ELISAs. To avoid potential interference with cytokines produced by contaminating ab T cells, only ‘pure’ gd T cell clones were used in this experiment. As summarized in Table 2, clone 2G4-8 produced low levels of IFNg and IL4, but high levels of IL2, TNFa and IL10. The second clone Ž2G4-7. produced no detectable IFNg and IL4, but again high levels of IL2, TNFa and IL10. The cytokine production of PHA stimulated irradiated feeder cells was below the detection limit. Thus, the gd clones do not fit in typical Th1 and Th2 phenotypes, but represent a mixed or Th0-like phenotype. Interestingly, these clones produce low levels of the proinflammatory cytokine IFNg but high levels of IL10, a cytokine known to suppress inflammatory responses ŽFiorentino et al., 1989..
4. Discussion We have previously demonstrated that vaccination with attenuated myelin-reactive T cell clones boosts anti-vaccine T cell responses which are potentially involved in the regulation of myelin-reactive T cells in MS ŽZhang et al., 1993.. In addition to the clonotype specific T cell responses, we also noted less selective T cell responses directed at other cellular antigens which are expressed on PHA induced T cell blasts ŽZhang et al., 1993.. These responses are potentially triggered by markers commonly expressed on the surface of activated T cells, and hence termed anti-ergotypic responses. Anti-ergotypic responses were previously reported to mediate protection to EAE in T cell vaccinated rats ŽLohse et al., 1989.. Recently, cytokine receptors were identified as candidate targets of anti-ergotypic T–T interactions ŽMor et al., 1996.. Here, we demonstrate that the cellular immune responses against the T cell vaccine also involve gd T cells. Our data are in agreement with those of Burns et al. Ž1995. who reported gd T cell responses to activated MBP reactive T cells in a few patients with MS. Similar gd T cell responses can be obtained when using proteolipid protein ŽPLP. specific T cells as stimulators as shown by Weiner et al. Ž1997.. Because the gd T cell responses are also observed when we used other activated T cells as stimulus, it can be speculated that the vaccine reactive gd T cells represent part of the anti-ergotypic response. Interestingly, gd T cell responses to activated T cells were less pronounced in unvaccinated MS patients and control subjects, indicating that this gd T cell response is upregulated by T cell vaccination. Since the upregulated gd T cell response in the vaccinated patients did not correlate with an increased percentage of gd T cells as determined by flow-cytometric analysis of blood lymphocytes, the data suggest that the vaccination does not induce a significant expansion of the total gd population in blood, but merely leads to the expansion of a subset of gd T cells reactive to activated T cells, which is not detected by FACS-analysis of total blood lymphocytes.
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Which molecules on the vaccine T cells induce the gd T cell proliferation? Our data suggest that gd T cells are triggered by ligands which are expressed on activated T cells but not on EBV transformed B cells. Alternatively, the gd T cell expansion could also be induced by unknown soluble factors such as cytokines released by the activated stimulatory T cell populations. To obtain further information on the possible gd T cell ligands, we studied the V gene expression of the gd T cells, since there seems to be a relationship between V gene expression and the reactivity pattern of gd T cells. For instance, Vd 2rVg 2 T cells frequently respond to mycobacterium tuberculosis derived antigens ŽKabelitz et al., 1991; Haregewoin et al., 1989.. In the present study, the responding gd T cells predominantly expressed the Vg 2y phenotype, which is uncommon in peripheral blood. Further PCR analysis revealed that most of these cells express DV1rDJ1 genes. Based on the preferential V gene expression pattern of these gd T cells, it is unlikely that the gd T cell expansion is induced by heat shock proteins potentially expressed on the surface of the activated T cells or by the recently identified non-peptidic phosphorylated molecules, as these ligands were demonstrated to specifically stimulate the Vd 2rVg 2 subset ŽConstant et al., 1994; Tanaka et al., 1995.. So far, only few ligands were identified that can stimulate Vd 1rVg 1 T cells. Vd 1q T cell clones were found to lyse certain tumor cells and EBV transformed B-cells ŽOrsini et al., 1994; Choudhary et al., 1995.. The gd T cells tested in our study also expressed cytotoxic but no proliferative reactivity to EBV transformed B cells. It remains to be studied whether these gd T cells express different functional characteristics with respect to proliferative and cytotoxic reactivity as previously described ŽHaecker and Wagner, 1994.. The rather heterogeneous TCR hypervariable regions expressed by the responding gd T cells could also reflect a superantigen-like stimulation ŽRust et al., 1990; Stinissen et al., 1995b.. In contrast to a ligand specific expansion, it can be argued that the observed gd T cell proliferation is a nonspecific ‘byproduct’ of the overall T cell reactivity towards the vaccine clones in the vaccinated patients. The lack of gd T cell responses in the unvaccinated MS patients and normal subjects would then be in line with the reduced proliferative responses towards the activated T cells in these subjects. However, if the gd T cell responses would be part of a nonspecific T cell proliferative response which is upregulated in the vaccinated patients one would expect to find a proliferation of the more common Vg 2q subset and not the Vg 2y subset identified in this report. Therefore, together with the data of Burns et al. Ž1995., our findings suggest that gd T cell expansion is induced by a ligand expressed on or released by the activated T cells and that the recognition is not restricted by classical MHC molecules. Further studies are necessary to identify the actual ligands which induce this gd T cell expansion. Another aspect of this study relates to the potential role
of gd T cells in the protective mechanisms of T cell vaccination. TCR ab anticlonotypic T cells induce class I restricted lysis of the vaccine clones, and inhibit the proliferation of the vaccine cells ŽZhang et al., 1993.. Therefore, we examined whether the vaccine reactive gd T cells exhibit a similar reactivity pattern. With respect to their cytotoxic reactivity, the gd T cells could efficiently lyse K562 and B cells, but they exhibit low cytotoxic reactivity to the activated T cells. Interestingly, Weiner and coworkers isolated gd T cells reactive to PLP peptide specific T cells which were able to lyse autologous T cells irrespective of their antigen specificity. The gd T cell induced cytotoxicity was not inhibited by anti-MHC class I antibodies ŽWeiner et al., 1997.. Since no detailed technical information was provided in their abstract, it is difficult to examine whether experimental differences may account for the low cytotoxic reactivity observed with our gd T cell lines. None of the gd clones tested was able to specifically inhibit the in vitro proliferation of the vaccine clones. However, upon stimulation, the gd clones produced low levels of the proinflammatory cytokine IFNg and relatively high levels of TNFa and IL10. IL10 has been shown to have immunosuppressive properties ŽFiorentino et al., 1989.. It is not clear whether the IL10 levels produced by the gd T cells are insufficient to suppress the in vitro proliferation of the vaccine T cells. Further studies need to resolve whether the IL10 production is a common characteristic of these gd T cells, and whether bystander suppression mediated by in vivo IL10 release contributes to a potential protective role of gd T cells in T cell vaccination. In summary, our data indicate that gd T cells are involved in T–T cell interactions with activated T cells, and suggest that this interaction is boosted by T cell vaccination. Our findings provide further support to a potential role of gd T cells in a cellular regulatory mechanism. Such a protective mechanism of gd T cells was recently suggested by Harrison et al. Ž1996., who demonstrated that gd T cells may suppress murine insulin dependent diabetes. The elucidation of the AgŽs. recognized by these gd T cells and their functional characteristics will provide further insights into the potential role of these cells in the regulation of autoreactive T cells.
Acknowledgements We thank C. Bocken, E. Smeyers, K. Engelen, J. Bleus, M. Steukers and L. Philippaerts for excellent technical help, Dr. R. Medaer for patient blood samples, Dr. L. Celis for helpful discussions, and Dr. M.-P. Jacobs for critical reading of the manuscript. This work was supported by grants from the Belgian ‘Nationaal Fonds voor Wetenschappelijk Onderzoek ŽNFWO.’, ‘NFWO-Levenslijn’, the foundation for ‘Wetenschappelijk Onderzoek in Multiple Sclerose ŽWOMS.’, the Belgian Charcot Foundation, the
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‘Limburgs Universitair Centrum ŽLUC.’, the ‘Nationale Loterij’, and the ‘Fonds ter Bevordering van het Wetenschapplijk Onderzoek in het Dr. L. Willems-Instituut ŽFWI.’. G.H. holds a fellowship from the Flemish ‘Instituut voor Wetenschappelijk en Technologisch Onderzoek in de Industrie ŽIWT.’.
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nating multiple sclerosis lesions. Proc. Natl. Acad. Sci. U.S.A. 89, 4588–4592. Wucherpfennig, K.W., Zhang, J., Witek, C., Matsui, M., Modabber, Y., Ota, K., Hafler, D.A., 1994. Clonal expansion and persistence of human T cells specific for an immunodominant myelin basic protein peptide. J. Immunol. 152, 5581–5592. Wucherpfennig, K.W., Strominger, J.L., 1995. Molecular mimicry in T cell mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 80, 695–705. Zamvil, S., Nelson, P., Trotter, J., Mitchell, D., Knobler, R., Fritz, R., Steinman, L., 1985. T cell clones specific for myelin basic protein induce chronic relapsing paralysis and demeylination. Nature 317, 353–358. Zhang, J.W., Medaer, R., Stinissen, P., Hafler, D.A., Raus, J., 1993.
MHC restricted clonotypic depletion of human myelin basic proteinreactive T cells by T cell vaccination. Science 261, 1451–1454. Zhang, J.W., Markovic, S., Lacet, B., Raus, J., Weiner, H.L., Hafler, D.A., 1994a. Increased frequency of interleukin 2-responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. J. Exp. Med. 179, 973–984. Zhang, X.-M., Tonelle, C., Lefranc, M.-P., Huck, S., 1994b. T cell receptor g cDNA in human fetal liver and thymus: Variable regions of g chains are restricted to V g I or V9, due to the absence of splicing of the V10 and V11 intron. Eur. J. Immunol. 24, 571. Zhang, J.W., Vandevyver, C., Stinissen, P., Raus, J., 1995. In vivo clonotypic regulation of human myelin basic protein reactive T cells by T cell vaccination. J. Immunol. 155, 5868–5877.
gd T cell responses to activated T cells in multiple sclerosis patients induced by T cell vaccination Piet Stinissen a
a,b,)
, Jingwu Zhang
a,b,1
, Caroline Vandevyver c , Guy Hermans
a,b
, Jef Raus
a,b,c
Multiple Sclerosis Research Unit, Dr. L. Willems-Instituut, UniÕersitaire Campus, B-3590 Diepenbeek, Belgium b Limburgs UniÕersitair Centrum (LUC), UniÕersitaire Campus, B-3590 Diepenbeek, Belgium c Biotechnology Unit, Dr. L. Willems-Instituut, UniÕersitaire Campus, B-3590 Diepenbeek, Belgium Received 29 December 1997; revised 10 February 1998; accepted 11 February 1998
Abstract To explore the hypothesis that gd T cells may regulate activated ab T cells, we studied gd T cell responses to ab T cell clones in Multiple Sclerosis ŽMS. patients who received attenuated autologous autoreactive T cells. We recently conducted a pilot study of T cell vaccination with myelin basic protein reactive T cells in MS. Since T cell vaccination upregulates the anti-vaccine T cell responses, we evaluated gd T cell reactivity towards the vaccine in the vaccinated patients. Lymphocytes were stimulated in vitro with irradiated vaccine cells and the responding lines were checked for the presence of gd T cells. Our data demonstrate that in the majority of vaccinated MS patients gd T cells expand upon stimulation with the vaccine cells. The responding gd T cells were predominantly Vd 1qrVg 1q, and represented diverse clonal origins. The gd T cells could not inhibit in vitro proliferation of the vaccine T cells and displayed low cytotoxic reactivity towards the vaccine clones. However, they produced high levels of IL2, TNFa and IL10. These results indicate that gd T cells can be stimulated by activated ab T cells, and that these gd T cell responses are upregulated after T cell vaccination. These findings suggest that gd T cells are involved in peripheral mechanisms to control activated autoreactive T cells. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Multiple sclerosis; gd T lymphocytes; Immunotherapy; T cell vaccination
1. Introduction Myelin basic protein ŽMBP. reactive T cells are considered to play an important role in the autoimmune processes of Multiple Sclerosis ŽMS. Žreviewed in Stinissen et al., 1997.. Although these T cells are part of the normal T cell repertoire, recent data indicate that MBP reactive T cells circulate in an activated state in patients with MS. For instance, activated MBP reactive T cells were shown to accumulate in the central nervous system ŽCNS. of patients with MS, and clonally expanded MBP reactive T cell populations persist for several years in the periphery of some patients ŽChou et al., 1992; Salvetti et al., 1993; Wucherpfennig et al., 1994; Zhang et al., 1994a,b; Vandevyver et al., 1995.. Wucherpfennig and Strominger Ž1995. provided experimental evidence that viral epitopes sharing )
Corresponding author. Tel.: q32 11 269211; fax: q32 11 269209; e-mail: [email protected] 1 Present address: Department of Neurology, Baylor College of Medicine, Houston, TX, USA. 0165-5728r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 5 7 2 8 Ž 9 8 . 0 0 0 6 0 - 5
sequence homologies with MBP can stimulate MBP reactive T cells in vitro. In this scenario, the autoreactive T cells may become activated in the periphery, which allows them to migrate into the CNS and initiate a pathogenic cascade of immune mediated events leading to demyelination. The pathogenic potential of MBP reactive T cells has been further demonstrated in experimental autoimmune encephalomyelitis ŽEAE., an animal model of MS, where CD4q T cells specific for immunodominant MBP epitopes can induce encephalomyelitis ŽBen-Nun et al., 1981a; Vandenbark et al., 1985; Zamvil et al., 1985.. Based on the potential pathogenicity of MBP reactive T cells in MS, several immunotherapeutic strategies have been designed to specifically inactivate these T lymphocytes Žreviewed in Martin and McFarland, 1996.. One of these approaches, termed T cell vaccination, involves immunization with attenuated autoreactive T cells to elicit specific immunity to the pathogenic T cell populations. As shown in EAE, animals vaccinated with attenuated MBP reactive T cells are resistant to subsequent induction of the disease ŽBenNun et al., 1981b..
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In a recent pilot study, we have inoculated 13 MS patients with autologous MBP reactive T cell clones to assess the safety of and immunological responses to T cell vaccination ŽZhang et al., 1993; Medaer et al., 1995.. Cellular anti-vaccine responses were observed in all vaccinated patients, associated with a specific depletion of circulating MBP reactive T cells ŽZhang et al., 1993.. CD8q anticlonotypic T cells specifically recognizing the vaccine clones were isolated from the vaccinated patients and found to induce MHC class I restricted cytotoxicity of the T cell clones used as a vaccine, most likely by recognition of a T cell receptor ŽTCR. related sequence on the vaccine cells ŽZhang et al., 1993, 1995.. The treatment was safe and induced clinical stabilization or reduced progression of the disease in most patients, encouraging further clinical trials of T cell vaccination in MS ŽMedaer et al., 1995; Stinissen et al., 1996.. In this report, we further explored the cellular mechanism of T cell vaccination. In preliminary experiments we noted an unusual expansion of gd T cells when peripheral blood mononuclear cells ŽPBMC. of the vaccinated patients were stimulated with irradiated vaccine T cells. gd T cells represent only a minor fraction of the blood T cells. Although their role in the immune system is controversial, it has been speculated that gd T cells may play a role in the first line immune defense ŽChien et al., 1996.. gd T cells express a limited TCR variable gene diversity, and their activation may occur without processing of antigen by antigen presenting cells. gd T cells may also play a role in the pathogenesis of MS, most likely in the later stages of plaque formation. Recent data also indicated a pathogenic role for gd T cells in EAE ŽRajan et al., 1996.. We and others demonstrated the accumulation of gd T cells in CSF and brain lesions of MS patients, although the antigens responsible for the gd T cell expansion remain unknown ŽSelmaj et al., 1991; Wucherpfennig et al., 1992; Shimonkevitz et al., 1993; Hvas et al., 1993; Battistini et al., 1995; Nick et al., 1995; Stinissen et al., 1995a.. Here we demonstrate that T cell clones used for vaccination, but also PHA induced T cell blasts induce an expansion of gd T cells in primary PBMC cultures of the vaccinated patients. Because the gd T cell expansion is more frequently observed in vaccinated patients than in healthy subjects or unvaccinated MS patients, the gd T cell reactivity most likely is related to the T cell vaccinations. The responding gd T cells predominantly express the Vg 2y phenotype which is uncommon in peripheral blood. To evaluate whether the responding gd T cells play an active role in T cell vaccination, we studied the functional properties of these cells. Interestingly, the isolated gd T cell lines or clones did not inhibit in vitro proliferation of the vaccine T cells. Upon stimulation, these cells produce low levels of IFNg and IL4, but high levels of IL2, TNFa and IL10. The gd T cells lysed EBV transformed B cells, but expressed little cytotoxic reactivity towards the vaccine cells. Our data demonstrate that gd T
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cells can be stimulated by activated T cells in primary cultures and suggest a role for these T cells in regulatory T–T cell interactions.
2. Materials and methods 2.1. Reagents Human MBP was purified from human brain tissue by the method of Deibler et al. Ž1972.. The medium used for cell culture was RPMI 1640 supplemented with 10% heatinactivated FCS and L-glutamine, sodium pyruvate, nonessential amino acids, and 10 mM HEPES ŽLife Technologies, Gent, Belgium.. Recombinant human IL-2 ŽrIL-2. used for T cell culture was obtained from Boehringer Mannheim ŽMannheim, Germany.. 2.2. Patients and immunization protocol 13 MS patients with MS enrolled in a pilot study of T cell vaccination with MBP reactive T cell clones. The patients received three immunizations with irradiated autologous MBP-reactive T cells as previously described ŽZhang et al., 1993.. Briefly, MBP reactive T cell lines were generated from all patients by stimulation of PBMC with MBP under limiting dilution conditions ŽZhang et al., 1993.. Resulting lines were further cloned at single cell densities by stimulation with PHA.P in the presence of irradiated feeder cells. The clones were characterized for their epitope reactivity and TCR V–D–J rearrangements. The clones selected for vaccination were activated with MBP pulsed PBMC as a source of antigen presenting cells, washed, irradiated and injected s.c. in PBS at a dose of 10–15 million cells per clone. Three subsequent vaccinations at intervals of 2 to 4 months were performed. 2.3. Primary stimulation of PBMC with T cells and isolation of gd T cells PBMC were isolated from heparinized blood by Ficoll gradient centrifugation. To study the primary gd T cell responses induced by the vaccine T cells or other nonspecific T cells, PBMC were cultured in the presence of irradiated T cells. To this end, 40,000 PBMC were plated in each of 60 wells of a microtiterplate together with 40,000 irradiated stimulator T cells in a total volume of 200 m l culture medium. Culture medium was RPMI 1640 supplemented with 10% heat inactivated autologous serum. Stimulator cells were the vaccine cells, other MBP reactive T cells, PHA stimulated T cell blasts, or EBV transformed B-cells. The stimulating T cells were all used 5–7 days after activation with MBP pulsed PBMC or with PHA and feeder cells, and cultured in IL-2 containing medium Ž5 Urml.. After 7 days, the primary cultures were restimulated with 40,000 cells per well of irradiated stimulator
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cells. After 14 to 18 days of culture, gd T cell lines were identified by positive staining Ž) 10%. with a TCR gd specific antibody ŽTCR gd-1, Becton Dickinson, San Jose, CA.. gd T cell clones were obtained by plating out at 0.3 or 1 cell per well in culture medium in the presence of 10 5 irradiated feeder cells and stimulation with 2 m grml PHA.P. After 24 h rIL-2 was added at 5 Urml. The cultures were fed with fresh medium containing 5 Urml rIL-2 every 3 to 4 days. Growth positive wells were microscopically identified after 10–12 days of culture and were tested for their purity by antibody staining. 2.4. Flow-cytometry
gd T cell lines were identified and analyzed by staining with appropriate monoclonal antibodies as previously described ŽStinissen et al., 1995b.. Briefly, 2–10 = 10 4 cells were split in a V-bottom microtiterplate and washed with 250 ml FACS-buffer ŽPBS containing 5% vrv FCS and 0.01% grv sodium azide.. The plates were subsequently centrifugated for 5 min at 2500 rpm. For direct staining with conjugated antibodies, the cells were stained with antibodies Ž5% vrv. for 30 min on ice. After two final washes the cells were analyzed by flow cytometry using a FACScan ŽBecton Dickinson. with gates set to read the total lymphocyte population. The following antibodies were used in pairs for direct dual staining: phycoerythrin ŽPE. conjugated anti-TCR grd-1 ŽPAN TCR gd .rfluorescein ŽFITC. conjugated anti-TCR-1 arb and FITC-anti-IgG1rPE-anti-IgG2a Žsimultest control. to detect background staining. The antiTCR grd-1, anti-TCR-1 ab and simultest control antibodies were purchased from Becton Dickinson. To analyze the Vg surface expression, gd T cell clones were double stained with phycoerythrin ŽPE. conjugated anti-TCR grd1 and FITC-1B10. 1B10 is a Vg 2 specific monoclonal antibody that was previously developed in our laboratory ŽStinissen et al., 1995b..
release–spontaneous releasermaximum release–spontaneous release. = 100%. 2.6. Analysis of T cell receptor Vg and Vd usage by PCR The TCR V gene usage of gd T cell clones was determined as described elsewhere ŽStinissen et al., 1995a.. Briefly, total RNA was extracted using the RNeasy method ŽQiagen. and 0.5–5 mg RNA was reverse transcribed into first-strand cDNA using the Reverse Transcriptase system of Promega ŽLeiden, the Netherlands.. The cDNA was then added to a standard amplification mixture containing 2.5 U Taq polymerase ŽAmpliTaq, Perkin-Elmer, Norwalk, CT., and 30 pmol of TCR DC primer or GC primer and 30 pmol of a TCRDV1, DV2, DV3, DV4, DV5 or TCRGV1, GV2, GV3 or GV4 specific primer. After 35 cycles of amplification of 1 min at 958C, 1 min at 558C and 2 min at 728C, the PCR products were Southern-blotted and hybridized with digoxigenin ŽDIG. end-labeled TCRDC or GC probes. The primer and probe sequences were published previously ŽStinissen et al., 1995a; nomenclature according to Strauss et al., 1987.. Detection of amplification products was performed with a DIG luminescent detection kit ŽBoehringer Mannheim, Mannheim, Germany., and exposure to a Hyperfilm MP ŽAmersham.. 2.7. Analysis of cytokine production of gd T cells
gd T cell clones Ž10,000 cells per well. were stimulated with PHA.P Ž2 m grml. and irradiated feeder cells Ž100,000 cells per well., or irradiated feeder cells alone Žcontrol.. The feeder cells were irradiated at 8000 rad in a 137Cs source. Cell supernatants were harvested after 72 h of culture, and the production of IL-2, IL-4, IL-10, IFNg and TNFa was determined in the supernatants using commercially available ELISA kits according to the manufacturer’s instructions ŽBiosource, Camarillo, CA..
3. Results 2.5. Cytotoxicity assays T cells ŽMBP reactive T cell clones or PHA stimulated T cell blasts., K562 cells or EBV transformed autologous B cells were used as target cells in a standard chromium release assay. The target cells were labeled with 200 m Ci 51 Cr ŽAmersham, Buckinghamshire, UK. for 1 h at 378C. The labeled target cells were washed four times to remove the free radioactivity, and incubated with the gd T cell clones Žeffectors. at an effector to target ratio between 5 and 20. After 5 h of incubation the supernatants were harvested and the released radioactivity was measured in a gamma counter ŽPackard, Meriden, CT.. The maximum and spontaneous chromium release were determined in wells containing H 2 SO4 or medium alone. Percentage of specific cytolysis was calculated as Žexperimental
3.1. Primary gd T cell responses are detected in PBMC cultures from Õaccinated MS patients stimulated with actiÕated Õaccine T cells To study the cellular immune responses induced by T cell vaccination, we stimulated post-vaccination PBMC samples with irradiated vaccine clones. The PB samples were taken 2 to 8 weeks after the third inoculation. The vaccine clones were all TCR abq and CD4q. The PBMC were plated at 40,000 cells per well in 60 wells in the presence of 40,000 cells per well of irradiated vaccine T cells. The cultures were restimulated with irradiated vaccine T cells after one week, and further cultured for an additional week in the presence of a low dose of exogenous IL-2 Ž5 Urml.. When evaluating the phenotypic
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Fig. 1. Primary gd T cell responses induced by autologous T cell stimuli in vaccinated patients. Post-vaccination PBMC samples from four MS patients were cultured at 40,000 cells per well in 60 wells of a microtiter plate and stimulated with 40,000 cells per well of an irradiated T cell stimulus. The T cell stimuli were the MBP reactive T cell clones used for vaccination Žvaccine clone 1 or 2., unrelated MBP reactive T cell clones with different TCR gene expression Žnon-vaccine clone 1 or 2., or PHA stimulated T cell blasts ŽPHA blasts.. In patients TV4 and TV5 we used two different MBP reactive T cell clones as a stimulus which were both incorporated in the T cell vaccine. After one week, all cultures were restimulated with their respective irradiated stimulator T cells Ž40,000 cells per well., and at day 14 the cultures were microscopically scored for cell growth. Growth positive cultures were stained with a PAN TCR gd monoclonal antibody and evaluated by flow-cytometry. Cultures containing more than 10% gd T cells were scored as TCR gd positive.
Fig. 2. Primary gd T cell responses induced by different autologous and allogeneic cellular stimuli. Post-vaccination PBMC of one patient ŽTV3. were stimulated with various stimuli, and restimulated with the respective stimuli after one week. At day 14 the responding T cell lines were scored for growth and presence of gd T cells Žat least 10%. as described in the legend of Fig. 1. In panel A the stimuli used were an irradiated autologous MBP reactive clone used for vaccination Žvaccine clone., irradiated autologous PHA stimulated T cell blasts ŽPHA blasts. and irradiated autologous EBV transformed B cells ŽEBV-B cells.. In panel B the stimuli used were irradiated autologous PHA stimulated T cell blasts and irradiated allogeneic PHA stimulated T cell blasts from two different donors. The experiment in panel B was performed with PBMC of patient TV3 obtained 1 month after the experiment illustrated in panel A.
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profile of the responding cultures at day 14, we noted that a substantial fraction of these cultures contained high amounts of gd T cells. For example, in some subjects more than 20% of the primary cultures contained 10% gd T cells or more. The percentage of gd T cells in these cultures ranged from 10 to 80%, with a mean percentage between 20 and 30% in all patients. This expansion was not observed in untreated blood lymphocytes of these patients as determined by flow-cytometry. Indeed, the frequency of T cells stained with anti-TCR gd antibody was between 2 and 8% in these patients, which is comparable to the frequencies observed in control subjects Ždata not shown.. To examine if this response was specific for the vaccine clones we stimulated PBMC of 4 vaccinated patients with different autologous T cell stimuli. As seen in Fig. 1, gd T cell enriched primary cultures Ž) 10% gdq. were not only observed with the vaccine clones as a stimulus, but also with other unrelated T cell stimuli: MBP reactive T cells which were not used for vaccination and PHA stimulated T cell blasts ŽPHA blasts.. These T cell stimuli express different TCR variable regions Žnot shown., indicating that the gd T cell response is not induced by a particular variable TCR segment. In one patient ŽTV3. we also included autologous EBV transformed B cells as a stimulus. Fig. 2A illustrates that no gd T cell containing cultures were obtained when EBV transformed B cells were used as a stimulus, indicating that only T cells, but not B cells induce the gd T cell expansion in the primary cultures. These data suggest that the gd T cell expansion is induced by a T cell derived growth factor or a nonspecific T cell surface marker. Finally, we tested the stimulating capacity of PHA blasts from different donors to study the MHC restriction pattern of the responding gd T cells. To this end, PBMC were isolated from a blood sample of patient TV3 taken 1 month after the experiment illustrated in Fig. 2A. The PBMC were stimulated with autologous and allogeneic PHA blasts. As illustrated in Fig. 2B, at this second sampling time again a high fraction of gd T cell positive cultures was observed when using autologous PHA blasts as stimulus. In addition, although the fraction of gd T cell positive cultures varied after stimulation with the different PHA T blasts, the data indicate that primary gd T cell expansion can be induced with activated T cells from different donors suggesting that this response is not restricted by classical MHC molecules. 3.2. Primary gd T cell responses induced by PHA stimulated T cell blasts in a group of Õaccinated patients and control subjects To examine whether the gd T cell responses are related to T cell vaccination, we tested a group of 9 vaccinated MS patients, 9 non-vaccinated MS patients and 6 healthy subjects for their primary gd T cell response to autologous PHA stimulated T cell blasts. PHA blasts were used as a
stimulus since the previous experiments illustrated similar stimulatory efficiencies of PHA blasts and MBP reactive T cell clones to generate gd T cell enriched cultures. In addition, the use of PHA blasts avoided the need to isolate MBP reactive T cell clones from all subjects. The blood samples from the vaccinated MS patients were drawn 2 to 10 weeks after the final inoculation. As seen in Fig. 3, gd T cell containing cultures were obtained in 6 out of 9 vaccinated patients, but only in 2 out of 9 unvaccinated patients and 1 out of 6 healthy subjects. The mean fraction of gd T cell positive cultures Žmore than 10% gd T cells. was 31.5% in 6 responding vaccinated MS patients, 5.9% in 2 responding unvaccinated MS patients, and 3.3% in the one responding healthy subject. These data suggest that the gd T cell sensitization might be related to T cell vaccination since significantly more gd T cells positive cultures were obtained in the vaccinated patients as compared to the control groups Žunvaccinated MS patients and healthy subjects.. 3.3. The gd T cells induced by actiÕated T cells are predominantly Vg 2 y In contrast to ab T cells, gd T cells express only a limited number of TCR V gene elements, and the V gene expression seems to correlate with the site where the gd T cell subsets are localized. gd T cells in the peripheral blood predominantly express Vd 2 and Vg 2 elements, while most of the gd T cells present in thymus and some epidermal tissues are Vd 1qrVg 1q. We studied the phenotype of the vaccine-responsive gd T cell lines by flow-cytometry using a Vg 2-specific monoclonal antibody Ž1B10. which was previously developed in our laboratory ŽStinissen et al., 1995b.. A total number of 215 primary gd T cell lines from 5 vaccinated patients were stained with 1B10 and a PAN-TCR gd antibody. Three different patterns were obtained: Vg 2y lines ŽTCRgdqr1B10y ., Vg 2q lines ŽTCRgdqr1B10q ., and lines which contained b o th a Vg 2 q a n d a Vg 2 y p o p u la tio n ŽTCRgdqr1B10qr1B10y .. Interestingly, in two patients the responding gd T cells almost exclusively expressed the Vg 2y phenotype, while in two other patients T cells expressing the Vg 2y phenotype represented the major gd subset ŽFig. 4.. In total, 59.0% of the tested lines were Vg 2y, 28.4% of the lines were Vg 2q, and 12.6% of the lines contained a Vg 2y and a Vg 2q population. Taken together, the data indicate that the activated T cells predominantly, though not exclusively, stimulate the minor Vg 2y subset of gd T cells in peripheral blood. In this regard, the gd T cell subset that is sensitive to stimulation with activated T cells seems to differ from the PB gd subset that is stimulated with PHA and IL-2. Indeed, as observed in our previous studies, gd T cell lines from the PB of MS patients and healthy subjects obtained by stimulation of PBMC with PHA and IL-2 were predominantly Vd 2qrVg 2q ŽStinissen et al., 1995a..
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Fig. 3. Primary gd T cell responses induced by autologous PHA stimulated T cells blasts in T cell vaccinated MS patients, in control MS patients Žunvaccinated., and in healthy subjects. PBMC of 9 MS patients vaccinated with MBP reactive T cells, 9 MS patients Žnot vaccinated. and 6 healthy subjects ŽNS. were stimulated with irradiated autologous PHA stimulated T cell blasts. The cultures were restimulated after one week, and scored for growth and the presence of gd T cells at day 14 as described in the legend of Fig. 1. In three vaccinated MS patients, 4 control MS patients and 4 healthy subjects no growth positive cultures were obtained at day 14.
In one vaccinated patient ŽTV3. we further studied the TCR V gene usage by RT-PCR. As seen in Table 1, all 15 gd T cell isolates studied expressed the Vg 1 gene. Some of the isolates co-expressed Vg 3 mRNA, however, these Vg 3 gene elements are possibly not functionally rear-
ranged due to a reported splicing defect in these gene segments ŽZhang et al., 1994a,b.. With respect to Vd expression, RT-PCR revealed preferential expression of Vd 1 Ž11r13., while only 2 isolates expressed Vd 3, and one isolate was Vd 2q ŽTable 1..
Fig. 4. Phenotypic characterization of gd T cells responding to stimulation with autologous T cells. gd T cell isolates were dual stained with conjugated PAN TCR gd antibody and conjugated 1B10 antibody and analyzed by flow-cytometry. 1B10 specifically stains the Vg 2q T cell subset. The gd T cells were scored as Vg 2q, Vg 2y and Vg 2qrVg 2y and the fraction of these subtypes for each patient is represented in the figure. A total number of 215 primary gd T cell lines derived by stimulation with autologous T cells Žvaccine T cell clones or PHA stimulated T cell blasts. from 5 vaccinated patients were studied.
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Table 1 TCR V gene usage and TCRDV–D–J amino acid sequences of gd T cells responding to the vaccine T cell clones in patient TV3 Isolate
V gene usage
DV amino acid sequence
DV
GV
DV–DJ
V
N–D–N
J
C
18.31 18.32 P.4 P.15 P.24 P.5 P.7 P.10 P.25 P.12
1 1 1 1 1 1 1 1 1 1, 3
1 1 1, 3 1, 3 1, 3 1, 3 1, 2, 3 1, 3 1, 3 1, 3
5.1 P.13 P.28 30.20 30.21
3 1 2 NT NT
1, 3 1 1, 3 1, 3 1, 3
V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V1–J1 V3–J1 V3–J1 NT NT NT NT
YFCAL YFCAL YFCAL YFCAL YFCAL YFCAL YFCAL YFCAL YFCAL YFCAL YYCAF YYCA
GELLTGLPTRGLTGGS GESTFLPSILVTGSY AEPNINWGIN GEKKTLTYWGIRLG GEPNLNWGID GGKRLYWDT GGKRLYWDT GGRILPPPLGRRK GGRILPPPLGRRK GGRILPPPLGRRK SLRPSYPF STCGGDP
DKLIFGKGTRVTVEP KLIFGKGTRVTVEP KLIFGKGTRVTVEP TDKLIFGKGTRVTVEP KLIFGKGTRVTVEP KLIFGKGTRVTVEP KLIFGKGTRVTVEP TDKLIFGKGTRVTVEP TDKLIFGKGTRVTVEP TDKLIFGKGTRVTVEP DKLIFGKGTRVTVEP TDKLIFGKGTRVTVEP
RSQP RSQP RSQP RSQP RSQP RSQP RSQP RSQP RSQP RSQP RSQP RSQP
3.4. TCR d chain V–D–J sequences of gd T cells Since the clonal diversity of the responding gd T cells may provide information about the nature of the antigenic stimuli, we determined the TCR DV chain sequences of a panel of 11 independent gd T isolates obtained from one patient ŽTV3.. As shown in Table 1, all isolates expressed the DJ1 gene element. The majority of lines Ž9. rearranged a DV1–DJ1 chain, while one line ŽP5.1. expressed a DV3–DJ1 transcript, and one line ŽP.12. expressed both a
DV1–DJ1 and a DV3–DJ1 chain. Interestingly, among the 10 DV1–DJ1 we observed two clonally expanded populations sharing identical DV-chains ŽTable 1.. However, the remaining clones represented unrelated clonal origins. 3.5. Functional properties of gd T cells: cytotoxic and inhibitory reactiÕity towards the T cell Õaccine clones and cytokine profile To study the functional properties of the gd T cell isolates we tested a panel of 38 gd T cell lines isolated
Fig. 5. Cytotoxic reactivity of gd T cells responding to stimulation with autologous T cells. 10 gd T cell lines derived from one vaccinated patient by stimulating PBMC with autologous PHA stimulated T cell blasts were tested for their cytotoxic reactivity towards autologous PHA stimulated T cell blasts, autologous EBV transformed B cells and K562 cells in a standard 51 Cr-release assay. The effector to target ratio was 20. The percentage of gd T cells is indicated for each line.
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from 3 vaccinated MS patients for their cytotoxic reactivity towards the T cells that were originally used as T cell stimuli. The gd T cell isolates tested induced only low specific killing Ž- 10% lysis. of their respective activated T cell targets at an effector to target ratio of 10–20 Ždata not shown.. In contrast, CD8q anticlonotypic ab T cells typically induce 40 to 90% cytolysis of the target vaccine cells ŽZhang et al., 1993.. A panel of 10 gd enriched T cell lines was further studied for their cytotoxic effect on autologous PHA stimulated T blasts, autologous EBV transformed B cells and natural killer sensitive K562 cells. As shown in Fig. 5, almost all lines tested induced high specific cytolysis of the EBV transformed B cells and K562 cells, but they induced no or very low cytolysis of the autologous PHA stimulated T blasts. Thus, the data indicate that the majority of the derived gd T cells induce little or no cytolysis of the activated T cells that were used as the original stimulus, despite their in vitro cytotoxic potential towards other target cell populations. Next, we tested whether the gd T cells could inhibit the antigen induced proliferation of the vaccine T cells. To this end, we cocultured MBP reactive T cells with MBP pulsed antigen presenting cells in the presence and absence of irradiated gd T cells. A panel of 13 gd T cell lines derived from 3 vaccinated MS patients was studied for their inhibitory reactivity. None of the tested gd T cell lines inhibited significantly Ž) 5%. the antigen induced proliferation of the antigen reactive T cell clones. In a similar assay, CD8q anticlonotypic T cells generally induced more than 70% inhibition of their respective indicator T cell clones ŽZhang et al., 1993.. As a final possible functional mechanism involved in the regulation of activated T cells we studied the cytokine profile of the isolated gd T cells. Therefore, two gd T cell clones responding to stimulation with the vaccine T cells, were stimulated with PHA and irradiated feeders, and the production of IFNg , IL4, IL2, TNFa and IL10 was quantified in the cell supernatants after 72 h. using commercial
Table 2 Cytokine production of gd T cell clones Clone
2G4-8
2G4-7
a
Cytokine production Žpgrml. a
control stimulation net production control stimulation net production
IFNg
IL4
IL2
TNFa
IL10
0 12 12 0b 0 0
0 11 11 0 0 0
236 479 243 506 1098 592
64 312 248 61 274 213
16 96 80 22 173 151
10,000 gd T cells were cultured in the presence of 10 5 irradiator feeder cells Žcontrol., or in the presence of 10 5 irradiator feeder cells q2 m grml PHA.P Žstimulation.. Cell supernatants were harvested after 72 h of culture, and cytokine production was determined by ELISA. The cytokine production of PHA stimulated irradiated feeder cells was below the detection limit. b Production below detection limit Ž5 pgrml.
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ELISAs. To avoid potential interference with cytokines produced by contaminating ab T cells, only ‘pure’ gd T cell clones were used in this experiment. As summarized in Table 2, clone 2G4-8 produced low levels of IFNg and IL4, but high levels of IL2, TNFa and IL10. The second clone Ž2G4-7. produced no detectable IFNg and IL4, but again high levels of IL2, TNFa and IL10. The cytokine production of PHA stimulated irradiated feeder cells was below the detection limit. Thus, the gd clones do not fit in typical Th1 and Th2 phenotypes, but represent a mixed or Th0-like phenotype. Interestingly, these clones produce low levels of the proinflammatory cytokine IFNg but high levels of IL10, a cytokine known to suppress inflammatory responses ŽFiorentino et al., 1989..
4. Discussion We have previously demonstrated that vaccination with attenuated myelin-reactive T cell clones boosts anti-vaccine T cell responses which are potentially involved in the regulation of myelin-reactive T cells in MS ŽZhang et al., 1993.. In addition to the clonotype specific T cell responses, we also noted less selective T cell responses directed at other cellular antigens which are expressed on PHA induced T cell blasts ŽZhang et al., 1993.. These responses are potentially triggered by markers commonly expressed on the surface of activated T cells, and hence termed anti-ergotypic responses. Anti-ergotypic responses were previously reported to mediate protection to EAE in T cell vaccinated rats ŽLohse et al., 1989.. Recently, cytokine receptors were identified as candidate targets of anti-ergotypic T–T interactions ŽMor et al., 1996.. Here, we demonstrate that the cellular immune responses against the T cell vaccine also involve gd T cells. Our data are in agreement with those of Burns et al. Ž1995. who reported gd T cell responses to activated MBP reactive T cells in a few patients with MS. Similar gd T cell responses can be obtained when using proteolipid protein ŽPLP. specific T cells as stimulators as shown by Weiner et al. Ž1997.. Because the gd T cell responses are also observed when we used other activated T cells as stimulus, it can be speculated that the vaccine reactive gd T cells represent part of the anti-ergotypic response. Interestingly, gd T cell responses to activated T cells were less pronounced in unvaccinated MS patients and control subjects, indicating that this gd T cell response is upregulated by T cell vaccination. Since the upregulated gd T cell response in the vaccinated patients did not correlate with an increased percentage of gd T cells as determined by flow-cytometric analysis of blood lymphocytes, the data suggest that the vaccination does not induce a significant expansion of the total gd population in blood, but merely leads to the expansion of a subset of gd T cells reactive to activated T cells, which is not detected by FACS-analysis of total blood lymphocytes.
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Which molecules on the vaccine T cells induce the gd T cell proliferation? Our data suggest that gd T cells are triggered by ligands which are expressed on activated T cells but not on EBV transformed B cells. Alternatively, the gd T cell expansion could also be induced by unknown soluble factors such as cytokines released by the activated stimulatory T cell populations. To obtain further information on the possible gd T cell ligands, we studied the V gene expression of the gd T cells, since there seems to be a relationship between V gene expression and the reactivity pattern of gd T cells. For instance, Vd 2rVg 2 T cells frequently respond to mycobacterium tuberculosis derived antigens ŽKabelitz et al., 1991; Haregewoin et al., 1989.. In the present study, the responding gd T cells predominantly expressed the Vg 2y phenotype, which is uncommon in peripheral blood. Further PCR analysis revealed that most of these cells express DV1rDJ1 genes. Based on the preferential V gene expression pattern of these gd T cells, it is unlikely that the gd T cell expansion is induced by heat shock proteins potentially expressed on the surface of the activated T cells or by the recently identified non-peptidic phosphorylated molecules, as these ligands were demonstrated to specifically stimulate the Vd 2rVg 2 subset ŽConstant et al., 1994; Tanaka et al., 1995.. So far, only few ligands were identified that can stimulate Vd 1rVg 1 T cells. Vd 1q T cell clones were found to lyse certain tumor cells and EBV transformed B-cells ŽOrsini et al., 1994; Choudhary et al., 1995.. The gd T cells tested in our study also expressed cytotoxic but no proliferative reactivity to EBV transformed B cells. It remains to be studied whether these gd T cells express different functional characteristics with respect to proliferative and cytotoxic reactivity as previously described ŽHaecker and Wagner, 1994.. The rather heterogeneous TCR hypervariable regions expressed by the responding gd T cells could also reflect a superantigen-like stimulation ŽRust et al., 1990; Stinissen et al., 1995b.. In contrast to a ligand specific expansion, it can be argued that the observed gd T cell proliferation is a nonspecific ‘byproduct’ of the overall T cell reactivity towards the vaccine clones in the vaccinated patients. The lack of gd T cell responses in the unvaccinated MS patients and normal subjects would then be in line with the reduced proliferative responses towards the activated T cells in these subjects. However, if the gd T cell responses would be part of a nonspecific T cell proliferative response which is upregulated in the vaccinated patients one would expect to find a proliferation of the more common Vg 2q subset and not the Vg 2y subset identified in this report. Therefore, together with the data of Burns et al. Ž1995., our findings suggest that gd T cell expansion is induced by a ligand expressed on or released by the activated T cells and that the recognition is not restricted by classical MHC molecules. Further studies are necessary to identify the actual ligands which induce this gd T cell expansion. Another aspect of this study relates to the potential role
of gd T cells in the protective mechanisms of T cell vaccination. TCR ab anticlonotypic T cells induce class I restricted lysis of the vaccine clones, and inhibit the proliferation of the vaccine cells ŽZhang et al., 1993.. Therefore, we examined whether the vaccine reactive gd T cells exhibit a similar reactivity pattern. With respect to their cytotoxic reactivity, the gd T cells could efficiently lyse K562 and B cells, but they exhibit low cytotoxic reactivity to the activated T cells. Interestingly, Weiner and coworkers isolated gd T cells reactive to PLP peptide specific T cells which were able to lyse autologous T cells irrespective of their antigen specificity. The gd T cell induced cytotoxicity was not inhibited by anti-MHC class I antibodies ŽWeiner et al., 1997.. Since no detailed technical information was provided in their abstract, it is difficult to examine whether experimental differences may account for the low cytotoxic reactivity observed with our gd T cell lines. None of the gd clones tested was able to specifically inhibit the in vitro proliferation of the vaccine clones. However, upon stimulation, the gd clones produced low levels of the proinflammatory cytokine IFNg and relatively high levels of TNFa and IL10. IL10 has been shown to have immunosuppressive properties ŽFiorentino et al., 1989.. It is not clear whether the IL10 levels produced by the gd T cells are insufficient to suppress the in vitro proliferation of the vaccine T cells. Further studies need to resolve whether the IL10 production is a common characteristic of these gd T cells, and whether bystander suppression mediated by in vivo IL10 release contributes to a potential protective role of gd T cells in T cell vaccination. In summary, our data indicate that gd T cells are involved in T–T cell interactions with activated T cells, and suggest that this interaction is boosted by T cell vaccination. Our findings provide further support to a potential role of gd T cells in a cellular regulatory mechanism. Such a protective mechanism of gd T cells was recently suggested by Harrison et al. Ž1996., who demonstrated that gd T cells may suppress murine insulin dependent diabetes. The elucidation of the AgŽs. recognized by these gd T cells and their functional characteristics will provide further insights into the potential role of these cells in the regulation of autoreactive T cells.
Acknowledgements We thank C. Bocken, E. Smeyers, K. Engelen, J. Bleus, M. Steukers and L. Philippaerts for excellent technical help, Dr. R. Medaer for patient blood samples, Dr. L. Celis for helpful discussions, and Dr. M.-P. Jacobs for critical reading of the manuscript. This work was supported by grants from the Belgian ‘Nationaal Fonds voor Wetenschappelijk Onderzoek ŽNFWO.’, ‘NFWO-Levenslijn’, the foundation for ‘Wetenschappelijk Onderzoek in Multiple Sclerose ŽWOMS.’, the Belgian Charcot Foundation, the
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‘Limburgs Universitair Centrum ŽLUC.’, the ‘Nationale Loterij’, and the ‘Fonds ter Bevordering van het Wetenschapplijk Onderzoek in het Dr. L. Willems-Instituut ŽFWI.’. G.H. holds a fellowship from the Flemish ‘Instituut voor Wetenschappelijk en Technologisch Onderzoek in de Industrie ŽIWT.’.
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