Journal of Autoimmunity (1991) 4,795~806
Peptide Competition at the Level of MHC-binding Sites Using T Cell Clones from a Rheumatoid Arthritis Patient
Patricia Mkndez-Samperio Department0
and Luis Jimknez-Zamudio
de Inmunologia, Escuela National de Ciencias Bioldgicas, I.P.N. y Plan de Ayala, Mbxico, D.F. 11340, Me’xico
Carpio
(Received 14 March 1991 and accepted 12 June 1991) The inhibition of antigen presentation in rheumatoid arthritis by blocking peptide binding to MHC at the antigen presenting cell (Al%) level was investigated using various synthetic peptides derived from the 65 kDa mycobacterial protein. Human T cell clones from tuberculosis and rheumatoid arthritis patients were stimulated with peptides in the presence of irradiated APCs (autologous or DR homozygous EBV-B cell lines). Two peptides (residues 65-85 and 412-426) were found to be able to bind to the HLA-DRl protein. Cross-competition was observed between these peptides when APCs were cultured with a suboptimal concentration of stimulator peptide in the presence of various concentrations of competitor peptides and T cell clones from tuberculosis patients as responder cells. These T cell clones responded not only to the peptides but also to the native protein. In other experiments, we used T cell clones from a rheumatoid arthritis patient to demonstrate the blocking of MI-X-binding sites by adding the ~412-426 in the recognition of DRl restricted T cell clone specific to ~65-85; MHC binding was not observed using a control peptide (residues 198-217). This approach has permitted the identification of MIX-specific blockers. Further experimentation is required to determine the particular amino acids involved in MI-XCbinding. Our data support the idea that modulation of antigen presentation by peptide competition could be a useful tool for immunotherapy in autoimmune diseases.
Introduction
It is known that peptides associated with MHC class I or II molecules are recognized by T cells [ 11, and that it is possible to modulate antigen presentation by interfering Correspondence to: Patricia Mbndez-Samperio, Departamento de Imnunologia, Escuela National de Ciencias Biolbgicas, I.P.N, Carpio y Plan de Ayala, Mexico, D.F. 11340, M&xico. 795 0896-841 l/91/050795
+ 12 $03.00/O
0 1991 Academic Press Limited
796 P. Mkndez-Samperio and L. Jimbnez-Zamudio with the binding of peptides to MHC molecules [2]. So far, peptide competition for antigen presentation has been used in different applications: for example, to assess the immunological relevance of peptide binding to purified MHC class II molecules [3]; to demonstrate that alloreactivity results from the recognition of peptides presented by an allogenic MHC molecule [4]; to select candidate T cell epitopes for incorporation into a vaccine [5], to inhibit the T cell response to insulin [6] by blocking the peptide binding at the antigen-presenting cell (AK) level; or to prevent autoimmune diseases by peptide competition in experimental autoimmune encephalomyelitis [7]. Therefore, competition for antigen presentation by blocking MHC binding sites could be used to prevent the presentation of antigens involved in the participation of human autoimmune diseases. It is widely believed that autoimmune diseases result from the activation of self-reactive T cells by autoantigens, and one characteristic of these diseases is the increased frequency of certain HLA class II alleles in affected individuals. These disease-associated HLA class II molecules have the capacity to bind the autoantigen and present it to T cells, resulting in induction and maintenance of autoimmune disease. Inhibition of antigen presentation by disease-associated HLA molecules could be possible by blocking the peptide-MHC class II interactions with a synthetic peptide. This is seen as an alternative to immunotherapy with antibodies to T cell antigens or MHC molecules. Recent studies have revealed that T cells taken from patients with rheumatoid arthritis [8] and cloned T cells capable of inducing adjuvant arthritis in the rat [9] are reactive with the mycobacterial65 kDa protein. These results imply that peptides of this protein may be recognized by T cells activated in these autoimmune diseases. In this paper we have investigated the inhibition of antigen presentation at the level of MHC-binding by using synthetic peptides from the 65 kDaprotein, recognized by T cell clones from tuberculosis and rheumatoid arthritis patients, and have demonstrated the ability to modulate antigen presentation by peptide competition. Materials and methods Antigens M. tuberculosis H37Rv was grown for 8 weeks as a surface pellicle on Sauton’s medium. A soluble extract (MTSE) was prepared by disruption of cobalt-irradiated organisms using a cell disintegrator at 4,000 rpm for 2 min at 5-10°C. Bacterial debris were removed by centrifugation at 30,000 x g for 1 h. The supernatant was filtered through a 0.45 lt filter and the protein concentration in the soluble extract was determined by the method of Lowry et al. [22] using bovine serum albumin as a standard. Mycobacterial 65 kilodalton (kDa) antigen, kindly supplied by Dr J. van Embden (RIVM, Bilthoven, The Netherlands), was isolated from a recombinant E. coli strain [lo, 111. Synthetic peptides corresponding to regions on the mycobacterial65 kDa protein (residues 65-85, 198-217 and 412-426) were prepared by solid phase synthesis as described previously [12]. The particular regions of the proteins that were synthesized were selected on the basis of the presence of a pattern of either four or five amino acids starting with a charged or glycine residue followed by two or three hydrophobic residues and then a polar amino acid [ 131, and were kindly provided by Dr J. Ivanyi (Hammersmith Hospital, London, UK).
Inhibition of antigen presentation at the APC level Preparation
of 65 kDa fractionated
797
antigen bound to nitrocellulose particles
This protein was obtained as previously described [ 141. Briefly, MTSE (100 pg per gel) were applied, using a blank comb with a single reference well for (molecular weight) (MW) markers (Sigma Chemical Co.). Protein bands were separated using SDS-PAGE (12% w/v acrylamide in the running gel and 4.8% in the stacking gel). The proteins were electrophoretically transferred to nitrocellulose paper in a Hoeffer Scientific Instrument using 0.02 M Tris-glycine buffer (pH 8.3). Nitrocellulose membranes were stained with amido black and the corresponding 65 kDa MW was assigned to fractions using markers (Sigma Chemical Co.). These fractions were cut, washed three times, and solubilized with 500 ~1 DMSO. Nitrocellulose particles were precipitated with 500 ~10.05 M carbonate-bicarbonate buffer and resuspended in 0.5 ml of RPM1 1640 (Sigma Chemical Co.) for use in the proliferation assay. Isolation of T cell clones
Peripheral blood mononuclear cells (PBMCs) from two tuberculosis patients [A.G. (DRl,DQWl) and J.M. (DR1.3)] were stimulated in vitro with peptides (~65-85 or p4 12-426) ( 10 pg/ml), expanded in interleukin 2-containing medium, and cloned by limiting dilution. Before use in proliferation assays, the T cell clones (clone I of patient A.G. and clone II of patient J.M.) were rested for 6 to 8 days after the last addition of APCs and antigen. We used ~65-85 to stimulate in vitro PBMCs from a patient with rheumatoid arthritis (typed as HLA DR1.5). From the in vitro primed PBMCs of this donor we obtained, by limiting dilution, a T cell clone (RA) which was stimulated with peptide 65-85, MTSE or recombinant 65 kDa mycobacterial protein (R.65). Isolation of EB V-B cell lines
Epstein-Bar virus-transformed B cells (EBV-B cell lines) were prepared from PBMCs (107/ml) of different donors with 1 ml of EBV. After incubation for 60 min at 37”C, the cells were washed three times and resuspended in complete RPM1 medium in the presence of 10 pg/ml cyclosporin (Sandimmun Cyclosporin, Sandoz-Pharmacy). One ml complete RPM1 medium was added after 1 week and subsequently changed twice weekly. Lymphocyte
proliferation
assays
T cells (5 x 104/ml) were stimulated with antigens in the presence of irradiated (5,000
rads) APCs (autologous or DR homozygous EBV-B cell lines) in 0.2 ml of complete medium (RPM1 1640 supplemented with 2 mM glutamine (In vitro S.A., Mexico City, Mtxico), 100 IU/ml penicillin and streptomycin (Sigma Chemical Co.) and 10% heat inactivated fetal calf serum (Flow Laboratories) with or without antigen. After 3 days’ incubation, an 8 h pulse of 1 VCi of [3H]-thymidine was added to each well and the cultures were harvested onto glass fibre filters. Proliferation, as correlated with [3H]-TdR incorporation, was measured by liquid scintillation spectroscopy.
798
P. Mhdez-Samperio
and L. Jimbnez-Zamudio Competition
of antigen presentation
This competition was performed with fixed EBV-B cell lines as APCs. Cells were fixed with 0.1 Y0 formaldehyde in PBS. The reaction was stopped by 0.2 M lysine in PBS after 30 s, and the cells were resuspended in complete medium. Fixed APCs (1 x lo5 cells per well) were incubated with various concentrations of the competitor peptide (l-100 pM) and suboptimal concentrations of the stimulator peptide. After incubation for 20 h at 37”C, the cells were washed three times and cultured with 5 x IO4 cloned T cells. The cultures were pulsed with 1 uCi of [3H]-thymidine at day 3 and were harvested onto glass fibre filters 8 to 16 h later. Results Interaction
of p6585
andp412-426
with DRl
molecules
To investigate the interaction of p65-85 and ~412-426 with HLA class II molecules, we established two human T cell clones restricted to DRl, each specific for a different peptide (Figure 1). The T cells were stimulated with antigens in the presence of irradiated APCs (autologous or DR homozygous EBV-B cell lines). The results show the binding of both peptides (~65-85 and ~412-426) to the MHC class II HLA-DRl molecules. Inhibition of antigen presentation
by competition assay
Inhibition of antigen presentation was determined by incubating fixed EBV-B cells (lo5 cells per well) with 1 ug/ml peptide 65-85 (clone I) or peptide 412-426 (clone II) and l-100 ug/ml competitor peptide for 18 h. APCs were then washed and T cells from clones I or II (5 x lo4 cells per well) were added. After 3 days, the cultures were pulsed with [3H]-thymidine (Figure 2). Responses (mean of triplicates) in the presence or absence of stimulator peptide were 38, 612 and 862 cpm, respectively, for clone I and 37,721 and 632 cpm, respectively, for clone II. Competition was noted when ~412-426 prevented ~65-85 from interaction with HLA class II-DRl molecules (clone I), and when ~65-85 competed with ~412-426 to interact with HLA class II-DRl molecules (clone II). ~198-217 (control peptide) failed to inhibit the response. No inhibition of antigen presentation was observed for any clone when HLA-DR4 homozygous EBV-B cells (negative control) were used as APCs in this system (data not shown). In vitro induced anti-peptide
T cell clones recognized
the whole protein
The T cell clones I and II were assessed for their ability to recognize the soluble extract of M. tuberculosis as well as a recombinant 65 kDa protein containing the ~65-85 and ~412-426. As shown in Figure 3, both the native and the recombinant proteins were clearly recognized at different concentrations. The response to the native as well as the recombinant was lower in magnitude than that to the peptide when compared by weight. However, when compared on a molar basis (antigens were tested at equimolar concentrations, 3 pM) the proliferative responses were comparable (Table 1).
Inhibition of antigen presentation at the APC level
799
APC (0) Autoiogous
DRI
DR5 I 20
I IO
0
I 40
I 30
,5(
APC (b)
Autologous
:q 0
,
,
5
IO cpm
,
,
,
15
20
25
(thousands)
Figure 1. Antigen specificity and MHC restriction of two human T cell clones used in a competition assay. T cells (5 x lo4 cells per well) were cultured in triplicate wells with irradiated autologous or DR homozygous EBV-B cells ( lo5 cells per well) in the presence of 10 ug of (a) ~65-85 (clone I) or(b) ~412-426 (clone II). Background counts were
Peptide Competition at the Level of MHC-binding Sites Using T Cell Clones from a Rheumatoid Arthritis Patient
Patricia Mkndez-Samperio Department0
and Luis Jimknez-Zamudio
de Inmunologia, Escuela National de Ciencias Bioldgicas, I.P.N. y Plan de Ayala, Mbxico, D.F. 11340, Me’xico
Carpio
(Received 14 March 1991 and accepted 12 June 1991) The inhibition of antigen presentation in rheumatoid arthritis by blocking peptide binding to MHC at the antigen presenting cell (Al%) level was investigated using various synthetic peptides derived from the 65 kDa mycobacterial protein. Human T cell clones from tuberculosis and rheumatoid arthritis patients were stimulated with peptides in the presence of irradiated APCs (autologous or DR homozygous EBV-B cell lines). Two peptides (residues 65-85 and 412-426) were found to be able to bind to the HLA-DRl protein. Cross-competition was observed between these peptides when APCs were cultured with a suboptimal concentration of stimulator peptide in the presence of various concentrations of competitor peptides and T cell clones from tuberculosis patients as responder cells. These T cell clones responded not only to the peptides but also to the native protein. In other experiments, we used T cell clones from a rheumatoid arthritis patient to demonstrate the blocking of MI-X-binding sites by adding the ~412-426 in the recognition of DRl restricted T cell clone specific to ~65-85; MHC binding was not observed using a control peptide (residues 198-217). This approach has permitted the identification of MIX-specific blockers. Further experimentation is required to determine the particular amino acids involved in MI-XCbinding. Our data support the idea that modulation of antigen presentation by peptide competition could be a useful tool for immunotherapy in autoimmune diseases.
Introduction
It is known that peptides associated with MHC class I or II molecules are recognized by T cells [ 11, and that it is possible to modulate antigen presentation by interfering Correspondence to: Patricia Mbndez-Samperio, Departamento de Imnunologia, Escuela National de Ciencias Biolbgicas, I.P.N, Carpio y Plan de Ayala, Mexico, D.F. 11340, M&xico. 795 0896-841 l/91/050795
+ 12 $03.00/O
0 1991 Academic Press Limited
796 P. Mkndez-Samperio and L. Jimbnez-Zamudio with the binding of peptides to MHC molecules [2]. So far, peptide competition for antigen presentation has been used in different applications: for example, to assess the immunological relevance of peptide binding to purified MHC class II molecules [3]; to demonstrate that alloreactivity results from the recognition of peptides presented by an allogenic MHC molecule [4]; to select candidate T cell epitopes for incorporation into a vaccine [5], to inhibit the T cell response to insulin [6] by blocking the peptide binding at the antigen-presenting cell (AK) level; or to prevent autoimmune diseases by peptide competition in experimental autoimmune encephalomyelitis [7]. Therefore, competition for antigen presentation by blocking MHC binding sites could be used to prevent the presentation of antigens involved in the participation of human autoimmune diseases. It is widely believed that autoimmune diseases result from the activation of self-reactive T cells by autoantigens, and one characteristic of these diseases is the increased frequency of certain HLA class II alleles in affected individuals. These disease-associated HLA class II molecules have the capacity to bind the autoantigen and present it to T cells, resulting in induction and maintenance of autoimmune disease. Inhibition of antigen presentation by disease-associated HLA molecules could be possible by blocking the peptide-MHC class II interactions with a synthetic peptide. This is seen as an alternative to immunotherapy with antibodies to T cell antigens or MHC molecules. Recent studies have revealed that T cells taken from patients with rheumatoid arthritis [8] and cloned T cells capable of inducing adjuvant arthritis in the rat [9] are reactive with the mycobacterial65 kDa protein. These results imply that peptides of this protein may be recognized by T cells activated in these autoimmune diseases. In this paper we have investigated the inhibition of antigen presentation at the level of MHC-binding by using synthetic peptides from the 65 kDaprotein, recognized by T cell clones from tuberculosis and rheumatoid arthritis patients, and have demonstrated the ability to modulate antigen presentation by peptide competition. Materials and methods Antigens M. tuberculosis H37Rv was grown for 8 weeks as a surface pellicle on Sauton’s medium. A soluble extract (MTSE) was prepared by disruption of cobalt-irradiated organisms using a cell disintegrator at 4,000 rpm for 2 min at 5-10°C. Bacterial debris were removed by centrifugation at 30,000 x g for 1 h. The supernatant was filtered through a 0.45 lt filter and the protein concentration in the soluble extract was determined by the method of Lowry et al. [22] using bovine serum albumin as a standard. Mycobacterial 65 kilodalton (kDa) antigen, kindly supplied by Dr J. van Embden (RIVM, Bilthoven, The Netherlands), was isolated from a recombinant E. coli strain [lo, 111. Synthetic peptides corresponding to regions on the mycobacterial65 kDa protein (residues 65-85, 198-217 and 412-426) were prepared by solid phase synthesis as described previously [12]. The particular regions of the proteins that were synthesized were selected on the basis of the presence of a pattern of either four or five amino acids starting with a charged or glycine residue followed by two or three hydrophobic residues and then a polar amino acid [ 131, and were kindly provided by Dr J. Ivanyi (Hammersmith Hospital, London, UK).
Inhibition of antigen presentation at the APC level Preparation
of 65 kDa fractionated
797
antigen bound to nitrocellulose particles
This protein was obtained as previously described [ 141. Briefly, MTSE (100 pg per gel) were applied, using a blank comb with a single reference well for (molecular weight) (MW) markers (Sigma Chemical Co.). Protein bands were separated using SDS-PAGE (12% w/v acrylamide in the running gel and 4.8% in the stacking gel). The proteins were electrophoretically transferred to nitrocellulose paper in a Hoeffer Scientific Instrument using 0.02 M Tris-glycine buffer (pH 8.3). Nitrocellulose membranes were stained with amido black and the corresponding 65 kDa MW was assigned to fractions using markers (Sigma Chemical Co.). These fractions were cut, washed three times, and solubilized with 500 ~1 DMSO. Nitrocellulose particles were precipitated with 500 ~10.05 M carbonate-bicarbonate buffer and resuspended in 0.5 ml of RPM1 1640 (Sigma Chemical Co.) for use in the proliferation assay. Isolation of T cell clones
Peripheral blood mononuclear cells (PBMCs) from two tuberculosis patients [A.G. (DRl,DQWl) and J.M. (DR1.3)] were stimulated in vitro with peptides (~65-85 or p4 12-426) ( 10 pg/ml), expanded in interleukin 2-containing medium, and cloned by limiting dilution. Before use in proliferation assays, the T cell clones (clone I of patient A.G. and clone II of patient J.M.) were rested for 6 to 8 days after the last addition of APCs and antigen. We used ~65-85 to stimulate in vitro PBMCs from a patient with rheumatoid arthritis (typed as HLA DR1.5). From the in vitro primed PBMCs of this donor we obtained, by limiting dilution, a T cell clone (RA) which was stimulated with peptide 65-85, MTSE or recombinant 65 kDa mycobacterial protein (R.65). Isolation of EB V-B cell lines
Epstein-Bar virus-transformed B cells (EBV-B cell lines) were prepared from PBMCs (107/ml) of different donors with 1 ml of EBV. After incubation for 60 min at 37”C, the cells were washed three times and resuspended in complete RPM1 medium in the presence of 10 pg/ml cyclosporin (Sandimmun Cyclosporin, Sandoz-Pharmacy). One ml complete RPM1 medium was added after 1 week and subsequently changed twice weekly. Lymphocyte
proliferation
assays
T cells (5 x 104/ml) were stimulated with antigens in the presence of irradiated (5,000
rads) APCs (autologous or DR homozygous EBV-B cell lines) in 0.2 ml of complete medium (RPM1 1640 supplemented with 2 mM glutamine (In vitro S.A., Mexico City, Mtxico), 100 IU/ml penicillin and streptomycin (Sigma Chemical Co.) and 10% heat inactivated fetal calf serum (Flow Laboratories) with or without antigen. After 3 days’ incubation, an 8 h pulse of 1 VCi of [3H]-thymidine was added to each well and the cultures were harvested onto glass fibre filters. Proliferation, as correlated with [3H]-TdR incorporation, was measured by liquid scintillation spectroscopy.
798
P. Mhdez-Samperio
and L. Jimbnez-Zamudio Competition
of antigen presentation
This competition was performed with fixed EBV-B cell lines as APCs. Cells were fixed with 0.1 Y0 formaldehyde in PBS. The reaction was stopped by 0.2 M lysine in PBS after 30 s, and the cells were resuspended in complete medium. Fixed APCs (1 x lo5 cells per well) were incubated with various concentrations of the competitor peptide (l-100 pM) and suboptimal concentrations of the stimulator peptide. After incubation for 20 h at 37”C, the cells were washed three times and cultured with 5 x IO4 cloned T cells. The cultures were pulsed with 1 uCi of [3H]-thymidine at day 3 and were harvested onto glass fibre filters 8 to 16 h later. Results Interaction
of p6585
andp412-426
with DRl
molecules
To investigate the interaction of p65-85 and ~412-426 with HLA class II molecules, we established two human T cell clones restricted to DRl, each specific for a different peptide (Figure 1). The T cells were stimulated with antigens in the presence of irradiated APCs (autologous or DR homozygous EBV-B cell lines). The results show the binding of both peptides (~65-85 and ~412-426) to the MHC class II HLA-DRl molecules. Inhibition of antigen presentation
by competition assay
Inhibition of antigen presentation was determined by incubating fixed EBV-B cells (lo5 cells per well) with 1 ug/ml peptide 65-85 (clone I) or peptide 412-426 (clone II) and l-100 ug/ml competitor peptide for 18 h. APCs were then washed and T cells from clones I or II (5 x lo4 cells per well) were added. After 3 days, the cultures were pulsed with [3H]-thymidine (Figure 2). Responses (mean of triplicates) in the presence or absence of stimulator peptide were 38, 612 and 862 cpm, respectively, for clone I and 37,721 and 632 cpm, respectively, for clone II. Competition was noted when ~412-426 prevented ~65-85 from interaction with HLA class II-DRl molecules (clone I), and when ~65-85 competed with ~412-426 to interact with HLA class II-DRl molecules (clone II). ~198-217 (control peptide) failed to inhibit the response. No inhibition of antigen presentation was observed for any clone when HLA-DR4 homozygous EBV-B cells (negative control) were used as APCs in this system (data not shown). In vitro induced anti-peptide
T cell clones recognized
the whole protein
The T cell clones I and II were assessed for their ability to recognize the soluble extract of M. tuberculosis as well as a recombinant 65 kDa protein containing the ~65-85 and ~412-426. As shown in Figure 3, both the native and the recombinant proteins were clearly recognized at different concentrations. The response to the native as well as the recombinant was lower in magnitude than that to the peptide when compared by weight. However, when compared on a molar basis (antigens were tested at equimolar concentrations, 3 pM) the proliferative responses were comparable (Table 1).
Inhibition of antigen presentation at the APC level
799
APC (0) Autoiogous
DRI
DR5 I 20
I IO
0
I 40
I 30
,5(
APC (b)
Autologous
:q 0
,
,
5
IO cpm
,
,
,
15
20
25
(thousands)
Figure 1. Antigen specificity and MHC restriction of two human T cell clones used in a competition assay. T cells (5 x lo4 cells per well) were cultured in triplicate wells with irradiated autologous or DR homozygous EBV-B cells ( lo5 cells per well) in the presence of 10 ug of (a) ~65-85 (clone I) or(b) ~412-426 (clone II). Background counts were
