Type I1 collagen-specific human T cell lines

Eur. J. Immunol. 1990. 20: 931-934

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Short paper Michael Lacour, Ulrike Rudolphi, Michael Schlesier and Hans-Hartmut Peter

Type I1 collagen-specific human T cell lines established from healthy donors*

Abteilung Rheumatologie und klinische Immunologie, Medizinische Universitatsklinik, Freiburg

Four type I1 collagen-specificTce11lines established from the peripheral blood of a healthy donor were studied in detail. These CD4+ Tcell lines with an a / P Tcell receptor proliferated in response to native and denatured human and chicken type 11collagen and human type I collagen, but not to human type IVcollagen or other potentially arthritogenic antigens. The Tcell response showed typical dose response characteristics, peaked between 30 and 48 h, was major histocompatibility complex class 11 restricted and not due to a mitogenic effect. ’Qpe I1 collagen-reactive T cells could hardly be detected in freshly isolated peripheral blood mononuclear cells from healthy donors, as revealed by limiting dilution analysis (frequency < l/lOOOOO). By prestimulation in bulk cultures for 10 days, type 11 collagen-reactive T cells could be approximately 1000-fold enriched. However, in these limiting dilution cultures, collagen-reactive Tcells could only be observed in a narrow window of cell concentrations, suggesting that type I1 collagen-reactiveT cells may be controlled by suppressive mechanisms in healthy persons.

1 Introduction Two animal models, the type 11collagen-induced arthritis [l]and the proteoglycan-reactive adjuvants arthritis [2], are thought to be relevant animal models in studying the pathogenesis of rheumatoid arthritis (RA; [3,4]). Transfer of arthritis by Tcells from arthritogenic animals [5, 61 and the demonstration of in vivo activated autoreactiveTcellsin patients with RA [7] provide evidence that T cells are involved in the progressive joint destruction seen in RA patients.

included. Mononuclear cells were obtained from defibrinated peripheral blood by Ficoll (Biochrom, Berlin, FRG) density gradient centrifugation. 2.2 Antigens

Lyophilized preparations of collagen type I and type IV from human placenta and collagen type I1 from chicken were kindly provided by Prof. K. Kuhn (Martinsried, FRG), human type 11collagen by Dr. B. Ohlrogge (Hannover, FRG). Collagens were dissolved at 2 mg/ml in 0.5% Recently,T cell clones isolated from the synovial tissue or acetic acid and sterilized under UV light (254nm) for fluid of RA patients have been shown to respond to type I1 15 min on ice. Aliquots were stored frozen at - 20°C. collagen [8, 91. However, to test the role of type I1 collagen Denatured collagens were prepared by heating the acid as a pathogenic autoantigen in RA, one ought to compare solution at 56°C for 30 min on the day of experiment. All frequencies of type I1 collagen-reactive T cells in healthy collagens were diluted in RPMI 1640 to a final concentrapersons and patients with RA. We therefore started to tion of 20 pg/ml unless otherwise stated. Control antigens identify type I1 collagen-specific T cells in healthy indi- were cartilage proteoglycan (20 pg/ml, Dr. B. Ohlrogge, viduals. Hannover, FRG), Mycobacterium tuberculosis H37 ( 5 kg/ml) and 65-kDa heat-shock protein of M . tuberculosis (0.5 pg/ml, both from Prof. S. H. E. Kaufmann, Ulm, FRG), small nuclear ribonucleoprotein particles (20 pg/ml, 2 Materials and methods Prof. R. Luhrmann, Marburg, FRG), PPD (20 pg/ml, Behring, Marburg, FRG) and EBV (laboratory standard 2.1 Blood donors and cell preparation titer). All experiments were performed with blood samples from two healthy individuals, MLR (male, 30 years old, HLA A2,32; B15,22; DRw6, x) and MSR (male, 37 years old, 2.3 Establishment of type II collagen-specificT cell lines HLA Al; B8,w60; DR3,S). Inone experiment (seenble 1, Sect. 3) two additional HLA-DR typed donors were Mononuclear cells from the peripheral blood were cultured at 1 x 106 celldml in RPMI 1640 supplemented with 10% autologous serum and 20 pg/ml native or denatured human [I 79861 collagen type 11. After 3 days of culture, 20 U/ml human rIL 2 was added. After an additional 7 days of culture, the * Supported by BMFTgrant No. 01VM860510. cells were collected and plated in U-shaped microtiter Correspondence: Michael Schlesier, Abt . Rheumatologie und plates at cell numbers ranging from 156 to 20000 celldwell klinische Immunologie, Medizinische Universitatsklinik, Hugstet- (24 replicates each) in RPMI 1640 supplemented with 10% terstr. 55, D-7800 Freiburg, FRG autologous serum. Autologous irradiated (30 Gy, @)Co) 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990

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932

M. Lacour, U. Rudolphi, M. Schlesier and H.-H. Peter

mononuclear cells were added as APC (5 x lo4 cells/well) together with 20 pg/ml native or denatured human collagen type II. After 3 days, the cultures were supplied with 20 U/ml rIL 2 and subsequently split and supplied with fresh IL 2-containing medium if neccessary. After 14 days, the T cell lines were washed, split and tested for their proliferative response to autologous APC alone or in the presence of 20 pg/ml type I1 collagen. Cell lines derived from cultures that received 625 cells/well or less and showed a proliferative reponse to collagen were expanded and supplied every 14 days with autologous APC, native or denatured human collagen type I1 and rIL 2. Phenotyping of the cell lines was performed by indirect immunofluorescence using the following monoclonal antibodies: anti-CD4 (OKT4), anti-CDS (OKT8; Ortho, Heidelberg, FRG); anti-CD3 (BMA), anti-TcR a / p (BMA031; Behring); antiCD29 (4B4), anti-CD45R (2H4; Coulter, Krefeld, FRG).

2.4 Assay for proliferative responses

T cell lines (1 x 104cells/well) were cultured in triplicates in U-shaped plates in RPMI 1640 supplemented with 10% autologous serum or human pooled serum. Autologous or allogeneic APC were added at 5 x 104 cells/well together with different antigen preparations. For the analysis of MHC restriction, mAb specific for MHC class I (W6/32; culture SN, MOO), MHC class I1 (Clonab DR/DP, Tu35, 1:2000; Biotest, Dreieich, FRG) or HLA-DR (Clonab DR, Tu34, 1/200 or 1: 2000, Biotest) were added. After 48 h, unless otherwise stated, [3H]dThd (18.5 kBq, Amersham, Braunschweig, FRG) was added for a further 24 h period. Cultures were harvested (Multimash, Dynatech, Alexandria, VA) and incorporated radioactivity (in cpm) was measured by means of a liquid scintillation counter (Rackbeta 1219,Pharmacia-LKB, Freiburg, FRG). Results given represent the mean of triplicate values. SD ranged from 5% to 20%.

2.5 LD analysis LD cultures were set up either with freshly isolated mononuclear cells or mononuclear cells prestimulated with collagen type I1 in bulk culture for 10 days.The cell numbers ranged from 156to 20 OOO cells/well with 24 replicates each. Autologous APC, collagen and I L 2 were added as described in Sect. 2.3. After 14 days, the cultures were washed, split and tested for their proliferative response ([3H]dThd incorporation after 48 h) to autologous APC (autoreactive response) or autologous APC plus collagen type I1 in the absence of exogenous IL 2. The autoreactive response always increased with increasing cell concentrations. The response to collagen was considered to be positive, if it was at least twice as high as the autologous control and exceeded the response of the internal negative control of autologous Con A blasts [7]. The fraction of negative cultures was plotted against the cell concentration (nonresponder fraction plot) and frequencies were calculated from the descending part of the resulting curves according to the zero term of the Poisson distribution [lo] by x2 minimization [ll].

Eur. J. Immunol. 1990.20: 931-934

3 Results and discussion Sixty type I1 collagen-reactive T cell lines were established from two healthy donors by the LD technique following type I1 collagen prestimulation in bulk cultures for 10 days. Four T cell lines from one donor were arbitrarily selected and studied in detail. The phenotype of these cell lines was C D ~ + T C R ~ P + C D ~ + C D ~ - C D ~A~strong + C Dpro~~R-. liferative response was observed to native and denatured human type I1collagen, chicken type I1collagen and human type I collagen (all fibril-forming collagens), but not to human type IV collagen (network-forming collagen) as shown for the representative Tcell line MSR-N1 in Fig. 1. The same antigen specificity of theTcell lines was obtained irrespective of prestimulation with either native (MSR-N1 or MSR-N2) or denatured (MSR-D1 or MSR-D2) collagen type I1 (Table 1). This reaction pattern, which was maintained by Tcell clones derived from cell line MSR-N1 (not shown), presumably reflects a cross-reactivity between human type I and type I1and chicken type I1collagen,which share a 60% to 95% sequence homology [12]. The T cell lines did not respond to control antigens like cartilage proteoglycan, heat-killed Mycobacterium tuberculosis strain H37, PPD, the 65-kDa heat shock protein of Mycobacterium tuberculosis, small nuclear ribonucleoprotein or EBV (Fig. 1). The T cell response to native and denatured human and chicken type I1 as well as to human type I collagen preparations was dose dependent and peaked between 30 to 48 h (shown for native human type I1 collagen in Fig. 2).The fact that dose response and kinetics were similar for all collagen preparations again argues in favor of a cross-reactive recognition by the Tcell lines. The stronger response to chicken as compared to human type 11 collagen may be explained by a more efficient uptake and processing of chicken collagen by APC and/or a higher 25000r

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Figure 1. Specificity of collagen-specific T cell line MSR-N1.The proliferative response (cpm) of MSR-N1 was tested in medium (Med), in the presence of autologous APC (Aut), and in the presence of autologous APC supplemented with native ( a n ) or denatured (C2d) human type I1 collagen, chicken type I1 collagen (c2n, c2d), human type I collagen (Cln, Cld) and human type IV collagen (C4n, C4d). All collagens were used at 20 pglml. Denaturation was performed by heating for 30 min at 56°C immediately before the test. Control antigens tested in the presence of autologous APC were cartilage proteoglycan (PG, 20 pg/ml), heat-killed Mycobacterium tuberculosis H37 (5 pglml) , PPD (20 pglml), 65-kDa heat-shock protein of M . tuberculosis (65K, 0.5 pglml), small nuclear ribonucleoprotein particles (RNP, 20 pg/ml) and EBV

v p e I1 collagen-specific human T cell lines

Eur. J. Immunol. 1990.20: 931-934

933

Table 1. MHC restriction of collagen-specificT cell lines

Collagen type added

APC Autologous (HLA-DIUS)

Allogeneic (HLA-DR3.7)

Allogeneic (HLA-DR4,7)

Allogeneic (HLA-DR w ~ , x )

None

None Human C2n Chicken c2n Human Cln Human C4n None Human C2n Chicken c2n Human Cln Human C4n None Human C2n Chicken c2n Human Cln Human C4n None Human C2n Chicken c2n Human Cln Human C4n None Human C2n Chicken c2n Human Cln Human C4n

In)

25000-

Collagen-specificT cell lined MSR-N2 MSR-D1 MSR-D2

MSR-N1 1931 13403 23 354 12502 1410 1521 7402 19 176 10211 2 140 167 81

156 18420 31316 11021 125 170 3444 10567 3977 108

288 196 123 519 137 370 105 126 274 113 236 147 135 290 260

97 282 84

128 84 129 221 70 62 78 57 157 48

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362 168 272 566 139 647 114 169 253 221 1% 376 337

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728

149 154

57

68

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62 153 65

126 262

115

a) The proliferative response (cpm) of collagen-specificTcell lines to native collagen preparations (20 pg/ml, abbreviations as in Fig. 1) was tested in the absence of APC or in the presence of autologous or allogeneic APC. Denatured collagens were also tested and gave comparable results.

affinity of MHC or TcR for the relevant collagen peptide. Similar observations have been reported in the mouse system [13]. Anti-MHC class I1 and anti-HLA-DR, but not anti-MHC class I mAb inhibited the collagen-specificTcell response (Fig. 3). No Tcell response was observed in the absence of autologous, or in the presence of HLADR-incompatible allogeneic APC. However, HLA-DR

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Figure 2. Dose-response characteristics (A) and time kinetics (B) of the proliferative response of Tcell lines MSR-N1 (0-O), MSR-Dl (W-B) and MSR-D2 ( 0 .. .O) to native human type I1 collagen. (A) T cell lines (1 X 104 celldml) were cultured in the presence of autologous APC (5 x 104celldwell) plus human native collagen type I1 at concentrations indicated. [3H]dThduptake was measured after 48 h. (B) T cell lines were co-cultured with autologous APC plus human native collagen type I1 (20 pg/ml). [3H]dThd incorporation was measured by a 24-h pulse after time intervals indicated. Each data point represents the mean of triplicates (A) or the mean of triplicates reduced by the cpm values of cultures without collagen (B). The SD was