Sclerosis Individuals and Patients with Multiple TCR Signaling in T ...

The Journal of Immunology

The 77C3 G Mutation in the Human CD45 (PTPRC) Gene Leads to Increased Intensity of TCR Signaling in T Cell Lines from Healthy Individuals and Patients with Multiple Sclerosis1 Hue-Tran Do,* Wiebke Baars,* Katja Borns,* Anja Windhagen,† and Reinhard Schwinzer2* The 77C3 G mutation in exon A of the human CD45 gene occurs with low frequency in healthy individuals. An enhanced frequency of 77C3 G individuals has been reported in cohorts of patients suffering from multiple sclerosis, systemic sclerosis, autoimmune hepatitis, and HIV-1. To investigate the mechanisms by which the variant allele may contribute to disease susceptibility, we compared T cell reactivity in heterozygous carriers of the mutation (healthy individuals and multiple sclerosis patients) and wild-type controls. In vitro-generated T cell lines and freshly isolated CD4ⴙCD45R0ⴙ primed/memory T cells from 77C3 G individuals aberrantly expressed CD45RA isoforms and showed enhanced proliferation and IL-2 production when stimulated with anti-TCR/CD3 mAb or Ag. Mutant T cell lines contained a more active pool of p56lck tyrosine kinase and responded with increased phosphorylation of Zap70 and TCR-␨ and an enhanced Ca2ⴙ flux to TCR/CD3 stimulation. These data suggest that 77C3 G may act as a risk factor for certain diseases by increasing the intensity of TCR signaling. The Journal of Immunology, 2006, 176: 931–938.

T

he hemopoietic cell-specific protein tyrosine phosphatase, CD45 is essential for Ag receptor-mediated signaling in T and B cells (1). The CD45 protein is encoded by a single gene (protein tyrosine phosphatase, receptor-type C (PTPRC)), and different isoforms can be generated by alternative splicing of three variable exons A, B, and C (2, 3). Expression of the different CD45 isoforms is tightly regulated and depends upon cell type and state of activation (4, 5). In humans, naive/unprimed T cells express high-molecular-mass isoforms containing exon A, which can be detected by CD45RA mAb (CD45RA⫹ cells). During activation of naive cells, CD45RA isoforms are down-regulated and the low-molecular-mass CD45R0 isoform is expressed in which exons A, B, and C are spliced out. Therefore, CD45R0⫹ T cells are regarded as representative of the primed/memory T cell subset. A variant pattern of CD45 isoforms has been described by our group in humans which is characterized by continuous expression of CD45RA isoforms also on primed/memory and activated T cells (6, 7). The molecular basis for the aberrant expression of CD45RA is a point mutation at position 77 in exon A (77C3 G) (8, 9). The mutation does not change the encoded amino acids but disrupts an exon-splicing silencer that normally represses the use of the 5⬘ splice site of exon A (10). Thus, disturbed splicing of exon A results in overexpression of CD45RA isoforms on all cell types. So far, only heterozygous carriers of the 77G allele have been found. Among the European population, the frequency of 77C3 G indi-

viduals is low, ranging from ⬃1 to 2%. However, there may be significant differences between different ethnic groups because the mutation seems to be absent in the African population, whereas a high proportion of variant individuals (13%) has been found in a population living in the Pamir mountains of Central Asia (11). The observation that the 77G allele cosegregated with the disease in three multiple sclerosis (MS)3 nuclear families and was enhanced in three independent cohorts of MS patients (12) was the first indication that 77C3 G could be associated with autoimmune diseases. Because an association with MS has been confirmed in some studies (13, 14) but not in others (15, 16), the role of 77C3 G in MS is not yet clear. Nevertheless, recent reports on an excess of 77C3 G individuals in cohorts of patients suffering from systemic sclerosis (17), autoimmune hepatitis (18), and HIV-1 infection (19) strongly suggested that the mutation and aberrant CD45 splicing may alter the immune responses of affected individuals. With the aim of defining possible mechanisms by which 77C3 G may contribute to disease susceptibility, we searched for functional consequences of the polymorphism. By comparing the in vitro responses of T cells obtained from a group of six 77C3 G individuals (three healthy, three MS patients) with cells from eight individuals with wild-type CD45 (five healthy, three MS patients), we could show that 77C3 G is associated with T cell hyperreactivity.

Materials and Methods Blood donors

*Transplantationslabor, Klinik fu¨r Viszeral-und Transplantationschirurgie and †Neurologie, Medizinische Hochschule, Hannover, Germany Received for publication May 11, 2005. Accepted for publication October 26, 2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from the Deutsche Forschungsgemeinschaft (Schw437/2) and the Hochschulinterne Leistungsfo¨rderung Program of the Medizinische Hochschule Hannover. 2 Address correspondence and reprint requests to Dr. Reinhard Schwinzer, Transplantationslabor, Klinik fu¨r Viszeral-und Transplantationschirurgie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, 30623 Hannover, Germany. E-mail address: [email protected]

Copyright © 2006 by The American Association of Immunologists, Inc.

Blood samples were obtained from three individuals carrying 77C3 G identified among healthy voluntary blood donors recruited at the Department of Transfusion Medicine (Hannover Medical School). Samples from a group of five healthy individuals with wild-type CD45 were used as controls. Nine carriers of 77C3 G were identified among MS patients recruited at the Department of Neurology (Hannover Medical School). All of them presented with clinically definite and laboratory supported MS. Detailed analyses were performed with cells from three of the mutant MS individuals who had not been treated with immunosuppressive or immunomodulatory drugs or had received steroids within the past 6 mo. Samples from three untreated MS patients with wild-type CD45 served as controls. 3 Abbreviations used in this paper: MS, multiple sclerosis; PTK, protein tyrosine kinase; PVDF, polyvinylidene difluoride.

0022-1767/06/$02.00

932 The ages of all subjects in this study were between 29 and 61 years. Informed consent was obtained from patients and the local ethics committee approved the study.

Isolation of T cell subsets PBMC were isolated from heparinized venous blood by Ficoll gradient centrifugation and were cryopreserved until further use. T cell subsets were negatively isolated by MACS (Miltenyi Biotec). To isolate the CD4⫹CD45R0⫹ T cell subset, PBMC were incubated with an Ab mixture consisting of the mouse mAbs anti-CD14 (clone CD14; American Type Culture Collection (ATCC)), anti-HLA-DR (ATCC), anti-CD8 (AICD8.1, a gift of B. Schraven, University of Magdeburg, Magdeburg, Germany), anti-CD56 (T199, a gift of T. Pietsch, University of Bonn, Bonn, Germany), and anti-CD45RC (MT2; IQ Products). Labeled cells were washed twice and incubated with magnetic goat anti-mouse IgG MicroBeads. The cells were washed again, resuspended in MACS buffer (PBS (pH 7.2) supplemented with 0.5% BSA and 2 mM EDTA), applied to separation columns and subjected to a magnetic field according to manufacturer’s instructions. Unbound cells were eluted from the columns and used for further analyses. Enrichment of CD4⫹CD45RA⫹ cells was achieved by using the same mixture but with anti-CD45R0 (UCHL-1; a gift of P. C. Beverley, Jenner Institute, Compton, U.K.) instead of anti-CD45RC. Purity of CD4⫹CD45R0⫹ and CD4⫹CD45RA⫹ subsets was checked by flow cytometry and was usually ⬎95%.

Flow cytometry For the detection of cell surface molecules, the following directly labeled mAbs were used: anti-CD4-FITC (BD Biosciences/BD Pharmingen), antiCD45RA-FITC (MEM-56; a gift of V. Horejsi, Institute of Molecular Genetics, Prague, Czech Republic), and anti-CD45R0-PE (BD Biosciences/BD Pharmingen). Cells were stained with saturating concentrations of the mAbs and analyzed on a FACSCalibur flow cytometer (BD Biosciences). Data were processed by using CellQuest software.

Generation of T cell lines To establish alloreactive T cell lines, CD4⫹ T cells were isolated from wild-type and mutant blood donors (HLA-DR 4, 7-negative) and cultured with irradiated allogeneic EBV-transformed B cells (line Laz 509; HLA-DR 4, 7-positive) (20) plus IL-2 (20 U/ml; Roche) in RPMI 1640 medium supplemented with 10% FCS, 50 U/ml penicillin/50 ␮g/ml streptomycin, and 4 mM L-glutamine. The cells were kept in culture for several weeks and were restimulated every 7–10 days with Laz 509 cells. Mitogenreactive T cell lines were established by stimulating PBMC for 24 h with PHA (2.5 ␮g/ml). The cells were then washed and expanded by the addition of exogenous IL-2 (20 U/ml).

Analysis of T cell functions Proliferative responses of freshly isolated CD4⫹CD45R0⫹ or CD4⫹CD45RA⫹ T cells were determined by culturing 5 ⫻ 105 cells/ml (1 ⫻ 105/well) in RPMI 1640 medium in round-bottom microtiter plates precoated with increasing concentrations of mAb BMA 031 (anti-TCR␣␤; a gift of R. Kurrle, Aventis Pharma, Frankfurt, Germany) plus anti-CD28 mAb (L293; BD Biosciences). After 3 days, cultures were pulsed with 1 ␮Ci of [3H]TdR and harvested after an additional 16 h incubation period. Incorporation of [3H]TdR was determined by using a Microbeta scintillation counter (Wallac). The amount of cytokines induced by TCR/CD28mediated activation of T cell subsets was determined by using the human Th1/Th2 Cytometric Bead Array (BD Biosciences). Alloreactive T cell lines were usually cultured for 4 –5 wk and then rested for at least 10 days (culture without Laz 509 cells and IL-2) before being used in functional assays. Alloantigen-induced proliferation was determined by stimulating cells from T cell lines (5.0 ⫻ 104/well) with irradiated (50 Gy) Laz 509 cells (2.5 ⫻ 104/well) in round-bottom microtiter plates.

In vitro tyrosine kinase assay Protein tyrosine kinase (PTK) activity was assayed by using a tyrosine kinase assay kit (Chemicon International). The assay bases on the phosphorylation of the synthetic biotinylated substrate poly(Glu:Tyr), 4:1, which contains multiple tyrosine residues and can be phosphorylated by a wide range of PTK. Cells were lysed in lysis buffer and postnuclear fractions were precipitated with anti-p56lck Ab (3a5; Santa Cruz Biotechnology). After extensive washing, the immunoprecipitate (10 ␮l) was incubated for 30 min at 30°C with a reaction mixture containing 25 mM Tris (pH 7.2), 50 mM MgCl2, 5 mM ATP, and the peptide substrate. Reactions were stopped by adding EDTA. Fifty microliters of the reaction mix was

HYPERREACTIVITY OF 77C3 G T CELLS transferred to streptavidin-coated microwells, washed, and incubated for 1 h with HRP-conjugated anti-phosphotyrosine mAb PY20. After washing and 10 min of incubation with tetramethylbenzidine substrate, the enzyme reaction was stopped and adsorbance at 450 nm of each microwell was determined using a microplate reader. Kinase activity was calculated by comparing the OD values with a phosphopeptide standard according to the manufacturer’s instructions.

Immunoprecipitation and immunoblotting A total of 10 ⫻ 106 cells were loaded with 3 ␮g/ml anti-TCR/CD3 mAb (OKT3; ATCC) and cross-linked for 1 min with 10 ␮g/ml goat anti-mouse Ig (Dianova). The cells were lysed in lysis buffer (1% Nonidet P-40 in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 10 mM NaF, 10 mM iodoacetamide) and lysates were separated from insoluble material by centrifugation for 10 min at 8000 ⫻ g. Samples were precleared twice before TCR-␨ and Zap70 proteins were precipitated with anti-TCR-␨ (mouse mAb TIA-2; Coulter) or anti-Zap70 (mouse mAb 2F3.2; Upstate Biotechnology) coupled to protein G-Sepharose (Amersham Biosciences). The immunoprecipitates were washed four times with 50 mM Tris-HCl (pH 7.4) containing 0.5% Nonidet P-40, boiled in sample loading buffer (100 mM Tris-HCl (pH 6.8), 20% glycerol, 2% SDS, 0.2 mg/ml bromphenol blue, 0.4 M DTT), and subjected to SDSPAGE (15% gels). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) by semidry electroblotting, and phosphotyrosine proteins were detected by incubating the membrane with the 4G10 mouse mAb (Upstate Biotechnology) followed by biotinylated goat antimouse and HRP-conjugated streptavidin and then exposure to ECL reagents (Pierce). Membranes to be reprobed were rehydrated in 100% methanol and washed in PBS/0.05% Tween 20 (Sigma-Aldrich). The membranes were then stripped in 100 mM 2-ME, 62.5 mM Tris (pH 6.8), 2% (w/v) SDS at 50°C for 30 min and washed with PBS/0.05% Tween 20, and restained with anti-TCR-␨ or anti-Zap70. Western blot analyses of p56lck precipitates were performed by using the anti-p56lck mAb 3a5 (Santa Cruz Biotechnology) or the rabbit polyclonal Ab lck-pY505, which is specific for p56lck phosphorylated at Tyr505 (BioSource International). CD45 molecules were precipitated by protein G-Sepharose-coupled mAb AICD45.2 (a gift of B. Schraven), which is directed to an epitope present on all CD45 isoforms. Precipitates were electrophoresed through 8% SDSpolyacrylamide gels and transferred to PVDF membranes. CD45 isoforms were detected after incubation of the membrane with mAb AICD45.2. To study anti-TCR/CD3-induced changes of the tyrosine phosphorylation patterns in whole-cell lysates, cells (5 ⫻ 106/ml) were suspended in RPMI 1640 without FCS, prewarmed at 37°C for 10 min, and then loaded with anti-TCR/CD3 mAb (OKT3; ATCC). Ab-loaded cells were washed and cell-bound mAbs were cross-linked by the addition of 10 ␮g/ml goat anti-mouse Ig. Reactions were stopped at suitable intervals (0 s, 30 s, 1 min, 2 min, and 3 min) by the addition of lysis buffer. Samples were boiled for 10 min before loading the equivalent of 5 ⫻ 105 cells/track onto 12.5% SDS-PAGE. Proteins were transferred to the PVDF membrane by semidry electroblotting. Membranes were blocked with blocking buffer containing PBS (pH 7.2), 1% casein, and 0.05% Tween 20 to prevent nonspecific binding of detecting reagents. Phosphotyrosine proteins were detected using mAb 4G10 as described above.

Measurement of intracellular Ca2⫹ concentration levels The concentration of cytoplasmic calcium was determined using the fluo-3 method and flow cytometry (21). T cells (5 ⫻ 106/ml) were loaded with 4 ␮M Fluo-3 AM and 8 ␮M fura-red AM (Molecular Probes) in 25 mM HEPES-buffered RPMI 1640 without FCS at 37°C in the dark. After 20 min, the cell concentration was diluted to 1 ⫻ 106/ml and the incubation time prolonged for 40 min. After washing, cells were suspended in Ca2⫹ buffer (25 mM HEPES (pH 7.2), 140 mM NaCl, 1.8 mM CaCl2 ⫻ 2 H2O, 1 mM MgCl2 ⫻ 6 H2O, 3 mM KCl, 10 mM D-glucose) and stored at room temperature in the dark. At a final dilution of 2 ⫻ 105/ml, cells were analyzed on a FACSCalibur flow cytometer. TCR ligation was achieved by treatment with goat anti-mouse IgG (20 ␮g/ml) for 1 min followed by the anti-TCR/CD3 mAb OKT3 (10 ␮g/ml). Mean ratio of Fluo-3/fura-red fluorescence was measured during the acquisition time course and expressed graphically to indicate Ca2⫹ flux.

Semiquantitative RT-PCR A total of 1.5 ⫻ 106 cells were stimulated with 10 ␮g/ml immobilized anti-TCR/CD3 mAb (OKT3) for 0, 1, 2, and 4 h. Total RNA was isolated from nonstimulated and stimulated cells using peqGold Trifast FL (peqlab). A total of 0.5 ␮g of total RNA was reverse-transcribed in the presence of 0.6 mM dNTPs, 20 pM random hexamers (Amersham Biosciences), 1.5 ␮l of 10-fold reverse transcription buffer (Stratagene), and 20


933

U M-MuLV reverse transcriptase (Stratagene) in a final reaction volume of 15 ␮l. Reactions were conducted at 37°C for 1 h, followed by a 10-min step at 95°C to the inactivate enzyme. PCR was performed in a final volume of 20 ␮l containing 3 ␮l of reverse-transcription transcript, 2 ␮l of 10-fold PCR buffer (Qiagen), 0.25 ␮M of each primer, and 1.5 U TaqDNA polymerase (Qiagen). The following primers were used: ␤-actin sense, 5⬘-GAA ACT ACC TTC AAC TCC ATC-3⬘, antisense, 5⬘-GTA GAA GCA TTT GCG GTG GAC-3⬘; IL-2 sense, 5⬘-ACT CAC CAG GAT GCT CAC AT-3⬘; IL-2 antisense, 5⬘-AGG TAA TCC ATC TGC TGT TCA GA-3⬘. Samples were amplified using 25 cycles for ␤-actin and 30 cycles for IL-2. PCR products (10 ␮l) were electrophoresed on ethidium bromide-stained agarose gels (2%) and visualized under UV light. Fragment sizes were 300 bp for ␤-actin and 222 bp for IL-2. mRNA expression of ␤-actin and IL-2 was determined using a video densitometry system (Eagle eye II; Amersham Biosciences). Subsequently, the IL-2:␤-actin ratio in densitometric units was calculated in the analytical process.

Statistical analysis Proliferative responses and cytokine production of mutant and wild-type cells were compared using the Student t test.

Results

Hyperreactivity of mutant CD4⫹CD45R0⫹“memory” T cells to stimulation with anti-TCR/CD3 mAb The 77C3 G mutation prevents splicing of exon A and downregulation of CD45RA isoforms. Thus, CD4⫹CD45R0⫹ primed/ memory T cells usually do not express CD45RA isoforms but remain CD45RA-positive in heterozygous carriers of 77C3 G (Fig. 1a). In CD4⫹CD45RA⫹ (naive/unprimed) T cells, however, 77C3 G is not associated with a strong phenotype (Fig. 1a). Therefore, we assumed that functional consequences of 77C3 G should be most pronounced in the CD4⫹CD45R0⫹ T cell subset. To test this hypothesis, the proliferative responses of CD4⫹CD45R0⫹ T cells from three healthy individuals carrying the variant CD45 allele and five wild-type controls were compared after stimulation with anti-TCR/CD3 plus anti-CD28 mAb. The Abs induced a dose-dependent response in T cells from both groups (Fig. 1b). However, at all Ab concentrations, mutant T cells proliferated significantly stronger than T cells from control individuals. To confirm that T cell hyperreactivity indeed results from overexpression of CD45RA isoforms on variant CD4⫹CD45R0⫹ cells, we also examined CD4⫹CD45RA⫹CD45R0⫺ T cells expressing the same CD45 isoforms in 77C3 G carriers and controls. In these cells, no differences in the strength of Ab-induced proliferation were observed (Fig. 1b). To study the influence of variant CD45RA expression on cytokine production, we stimulated CD4⫹CD45R0⫹ T cells with antiTCR/CD3 plus anti-CD28 mAb and determined the amount of various cytokines in the cell culture supernatant. These experiments revealed significantly enhanced production of IL-2, IL-4, and IL-10 by 77C3 G T cells (Fig. 2). There was also a tendency of 77C3 G T cells to produce more IL-5 than cells from wild-type controls. This difference, however, was statistically not significant because of great interindividual variability. In addition, we did not find clear-cut differences in the amount of TNF-␣ and IFN-␥ produced by activated wild-type and mutant T cells. Studies on the underlying mechanisms of T cell hyperreactivity were hampered by the fact that the number of CD4⫹CD45R0⫹ cells isolated from peripheral blood usually was too small to perform detailed biochemical studies on TCR signal transduction. Because in vitro-activated T cells of variant and control individuals express the same CD45RA/CD45R0 pattern as the CD4⫹CD45R0⫹ primed/memory subset (variant: CD45RA⫹CD45R0⫹, control: CD45RA⫺CD45R0⫹) (8), we used in vitro-generated alloreactive or mitogen-reactive T cell lines for additional experiments. Detailed studies on the isoform combination by immunoprecipitation experiments revealed that CD45R(0), CD45R(B), and CD45R(AB) iso-

FIGURE 1. CD4⫹CD45R0⫹ T cells from 77C3 G individuals express CD45RA isoforms and are hypersensitive to anti-TCR/CD3-induced activation. a, Flow cytometry analysis of isolated cells. CD4⫹CD45R0⫹ and CD4⫹CD45RA⫹ T cells of a donor with wild-type CD45 or the variant 77C3 G allele were negatively isolated from peripheral blood T cells. Expression of CD45RA and CD45R0 isoforms was detected after staining the cells with the mAb combination CD45RA-FITC/CD45R0-PE. b, Proliferative responses of T cell subsets. CD4⫹CD45R0⫹ and CD4⫹CD45RA⫹ T cells (1 ⫻ 105/well) were cultured in microtiter-plates precoated with increasing concentrations of anti-TCR/CD3 plus anti-CD28 mAb. Proliferation was determined after 3 days. Results represent the proliferative responses (mean cpm ⫾ SD) obtained with T cells from blood donors with variant (n ⫽ 3) and wild-type CD45 (n ⫽ 5). ⴱ, p ⫽ 0.014; ⴱⴱ, p ⫽ 0.022; ⴱⴱⴱ, p ⫽ 0.010.

forms are expressed by 77C3 G cells whereas only CD45R(0) and CD45R(B) molecules are present in controls (Fig. 3). Enhanced responsiveness of 77C3 G T cells to stimulation with Ag To address the question of whether hyperreactivity of variant T cells can also be demonstrated after stimulation with Ag instead of agonistic Abs, we stimulated alloreactive CD4⫹ T cell lines from 77C3 G carriers and wild-type controls with specific alloantigen. In accordance with the data obtained by Ab stimulation, the proliferative responses of variant cells were stronger than those of wild-type cells (Fig. 4). The differences were more pronounced at later time points, also suggesting a prolonged response, compared with controls. In each experiment, control and variant cell lines were compared side-by-side to exclude day-to-day variability as a reason for differential responses. Under these conditions, we observed stronger proliferative responses in all cell lines generated from 77C3 G carriers (Table I). Enhanced alloreactivity of 77C3 G T cells was also suggested by our preliminary data, revealing a high frequency of IL-2-producing cells in 77C3 G T cell lines stimulated with alloantigen (22).

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HYPERREACTIVITY OF 77C3 G T CELLS

FIGURE 3. Variant T cell lines aberrantly express the CD45R(AB) isoform. T cells from blood donors with wild-type and variant CD45 were cultured for 4 wk with the allogeneic EBV-transformed B cell line Laz 509 plus IL-2. Cells were lysed and postnuclear lysates were immunoprecipitated with mAb AICD45.1 which is directed to all CD45 isoforms. Samples were resolved by SDS-PAGE and immunoblotted with AICD45.1.

FIGURE 2. TCR/CD3-induced cytokine production by 77C3 G and wild-type T cells. CD4⫹CD45R0⫹ T cells (1 ⫻ 105/well) were cultured in microtiter plates precoated with anti-TCR/CD3 plus CD28 mAb. After 24 h, supernatants were harvested and the concentration of cytokines was determined by using the Cytometric Bead Array. The bars represent the cytokine concentrations (mean ⫹ SD) detected in three independent experiments. In each experiment, cells from wild-type and 77C3 G individuals were analyzed side-by-side.

77C3 G cells contain a more active pool of p56lck kinase molecules The crucial role of CD45 in TCR signaling results from the capacity to activate the kinase p56lck by dephosphorylating its downregulatory tyrosyl residues (Tyr505) and enabling the kinase to achieve an active conformation (23). We assumed that additional expression of the CD45R(AB) isoform on activated T cells from 77C3 G carriers (Fig. 3) might have an influence on p56lck activity. To assess this, we precipitated p56lck and compared the amount of catalytically inactive molecules between variant and control cell lines by using an Ab specific for lck phosphorylated at Tyr505 (pTyr505). There was no difference in the total amount of p56lck between wild-type and 77C3 G cell lines (Fig. 5a). However, in cell lines from variant individuals, we observed a selective reduction of Tyr505-phosphorylated lck. To evaluate whether the reduced levels of pTyr505 indeed reflects a particularly active pool of p56lck in variant cell lines, we then performed in vitro kinase assays. The kinase activity determined in p56lck precipitates from variant cells was, on average, 2-fold higher than the activity calculated for precipitates from control cells (Fig. 5b). Increased intensity of TCR-mediated signaling in 77C3 G T cells To examine whether enhanced activity of p56lck might alter TCRmediated signaling, we next compared the phosphorylation pattern

following activation of variant and control cells with cross-linked anti-TCR/CD3 mAb. Despite the enhanced kinase activity in variant cells, no differences were observed in the basal tyrosine phosphorylation levels between variant and control cells (Fig. 6a). After TCR stimulation, however, differences in the phosphorylation pattern and kinetics were detected. Whereas in controls only proteins at ⬃30 kDa were stronger phosphorylated than in unstimulated cells, a more complex pattern was observed in variant cells showing increased phosphorylation of proteins at ⬃30 kDa and proteins between 40 and 54 kDa. Furthermore, the time course for the induction of tyrosine phosphorylation was accelerated in variant T cells reaching a maximum already at 30 s. In controls, the maximum intensity was found between 1 and 2 min. The overall greater increase in tyrosine phosphorylation and the accelerated kinetics observed in variant T cells suggested that TCR signal transduction might be more effective than in controls. To follow this hypothesis, we compared the phosphorylation specifically of TCR-␨ by immunoprecipitation and immunoblotting for phosphotyrosine. The basal level of phosphorylated TCR-␨ did not differ between cells from variant individuals and controls. However, upon TCR-directed stimulation, TCR-␨ appeared to be more heavily phosphorylated in variant cells (Fig. 6b). To confirm enhanced phosphorylation of TCR-␨ as a consequence of variant CD45RA expression, we repeated these experiments using cells from two additional carriers of 77C3 G and wild-type controls. Quantification of the immunoblots by densitometry revealed that the intensity of TCR-␨ phosphorylation was enhanced by 25– 45%

FIGURE 4. Variant T cells are hyperreactive to stimulation with alloantigen. CD4⫹ T cell lines from HLA-DR 4/7-negative variant and control donors were established by culturing with the EBV-transformed B cell line Laz 509 (HLA-DR 4/7). The cell lines were cultured for 4 wk, rested for 10 days (culture without Laz 509 and IL-2), and then restimulated with irradiated Laz 509 cells. Proliferation was determined after the indicated period of time. Results are expressed as mean cpm ⫾ SD of triplicate cultures. Data represent one of five independent experiments comparing the reactivity of cells from three variant with five control individuals.


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Table I. Enhanced proliferation of mutant T cell lines to activation by alloantigena Proliferation to Stimulation with Expt.

Cells

Medium

Alloantigen

1

Co1 Va1 Co2 Va2 Co3 Va1 Co4 Va3 Co5 Va2

415 ⫾ 25b 434 ⫾ 14 498 ⫾ 28 418 ⫾ 61 470 ⫾ 29 208 ⫾ 42 144 ⫾ 8 208 ⫾ 30 366 ⫾ 36 353 ⫾ 39

23,780 ⫾ 2,333 39,373 ⫾ 4,128 28,609 ⫾ 1,867 47,122 ⫾ 2,776 29,979 ⫾ 1,570 48,630 ⫾ 1,451 15,245 ⫾ 2,045 28,134 ⫾ 483 28,751 ⫾ 1,640 42,813 ⫾ 2,190

2 3 4 5

a T cell lines from three variant donors (Va1-Va3) and five individuals with wildtype CD45 (Co1-Co5) were generated by repetitive stimulation with the EBV-transformed B cell line Laz 509. The cells were cultured for 4 wk, rested for 10 days, and restimulated with irradiated Laz 509 cells. Proliferation was determined after 3 days by [3H] TdR incorporation. Variants and controls differed in HLA-DR from Laz 509 cells (HLA-DR 4, 7). b Results are expressed as mean cpm ⫾ SD of triplicate cultures.

in T cells from individuals carrying the variant allele (data not shown). There was also increased phosphorylation of Zap70 in 77C3 G T cells (Fig. 6c) and an enhanced Ca2⫹ flux (Fig. 6d) further substantiating the assumption of a greater signaling response as consequence of variant expression of CD45RA isoforms.

with wild-type CD45. In each experiment, cells from one variant individual were compared side-by-side with cells from one or two wild-type control individuals. Because we reproducibly observed stronger responses of 77C3 G T cells (Table I) it seems unlikely that hyperreactivity of variant cells may be due to experimental variability. Second, in variant T cells, there was a clear-cut correlation between the strength of phenotypic alterations induced by 77C3 G and the intensity of functional consequences. Thus, the CD4⫹CD45R0⫹ T cell subset and in vitro-activated T cells, which expressed two CD45 isoforms (CD45RB, CD45R0) in controls but three (CD45RB, CD45R0, CD45RA) in variants (Fig. 3), were hyperreactive. CD4⫹CD45RA⫹ T cells, however, expressing a slightly enhanced concentration of CD45RA molecules in variants but an otherwise “normal” combination of isoforms (CD45RA, CD45RB) responded as strongly as control cells. If reasons other than variant expression of CD45RA would be responsible for the enhanced T cell responses, one should find hyperreactivity not only in cell types showing the most pronounced phenotypic differences between 77C3 G and wild-type individuals. The fact that only some cell types are significantly affected by the 77C3 G mutation might also be the reason for our failure to detect functional consequences in previous studies when we compared the in vitro reactivity of the entire lymphocyte population of variants and controls (6). By which mechanisms may aberrant expression of CD45RA isoforms enhance T cell reactivity? The crucial role of CD45 in

T cell reactivity is enhanced also in MS patients carrying 77C3 G The 77C3 G mutation has been described as occurring with enhanced frequency in certain autoimmune diseases. To examine the functional consequences of 77C3 G in one autoimmune disease, we compared the reactivity patterns of cells from MS patients carrying 77C3 G (n ⫽ 3) and patients with wild-type CD45 (n ⫽ 3). In accordance with the data obtained with T cell lines from healthy carriers of 77C3 G, we also found increased enzymatic activity of p56lck, enhanced proliferation to stimulation with anti-TCR/CD3 mAb, and an increased Ca2⫹ flux (data not shown). Hyperreactivity of T cells from variant MS patients could also be demonstrated by semiquantitative PCR analyses. These experiments revealed an increased amount of IL-2 mRNA in variant cells after anti-TCR/ CD3 stimulation (Fig. 7).

Discussion In this article, we show that freshly isolated primed/memory T cells and in vitro-generated T cell lines from heterozygous carriers of the 77C3 G polymorphism aberrantly express CD45RA isoforms and are hyperreactive to TCR-mediated activation. In light of these functional data and various reports on an excess of 77C3 G individuals in some autoimmune diseases (12, 17, 18) and in a cohort of HIV-1 patients (19), the following issues concerning this polymorphism may be important: 1) is aberrant expression of CD45RA indeed the reason for T cell hyperreactivity or could there be other explanations, 2) how is the mechanism by which overexpression of CD45RA isoforms alter T cell reactivity, and 3) could the functional peculiarities of variant T cells have an impact on the susceptibility of 77C3 G carriers for certain diseases? The conclusion that 77C3 G and the resulting aberrant expression of CD45RA isoforms is the underlying reason for T cell hyperreactivity is supported by two different arguments. First, we found enhanced T cell reactivity in all six tested individuals carrying the 77G allele, compared with a group of eight individuals

FIGURE 5. 77C3 G cells contain a more active pool of p56lck kinase molecules. T cell lines from three variant and five control individuals were generated by cultivation with PHA plus IL-2 for 2 wk. a, Phosphorylation of Tyr505-p56lck. Cells were lysed, and postnuclear lysates were precipitated with anti-p56lck. Samples were resolved by SDS-PAGE and immunoblotted with anti-p56lck or anti-pTyr505-p56lck. Intensity of the bands was measured by densitometry. b, Analysis of kinase activity. p56lck was precipitated and assayed against the phosphorylated peptide. The amount of phosphopeptide was calculated by comparing the OD values with values obtained with a phosphopeptide standard curve. Horizontal lines indicate the mean phosphopeptide amount calculated for wild-type and 77C3 G cell lines, respectively. Inset, Immunoblotting of p56lck precipitated from a representative control (1) and variant (2) cell line.

936

HYPERREACTIVITY OF 77C3 G T CELLS

FIGURE 6. 77C3 G increases the intensity of TCR-mediated signal transduction. CD45 wild-type and mutant T cell lines were analyzed, which had been established either by in vitro stimulation with alloantigen (Laz 509 cells) or PHA plus IL-2. Cultured cells were rested for 3– 4 days (culture without Ag/PHA and IL-2) before TCR-mediated signaling was studied. a, Phosphorylation patterns after stimulation with anti-TCR/CD3 mAb. The cells were loaded with anti-TCR/CD3 mAb and cross-linked with goat anti-mouse IgG. At the indicated time points, the cells were lysed and proteins were separated by SDS-PAGE, transferred to PVDF membrane, and probed with anti-phosphotyrosine Ab. Protein loading was controlled by stripping the blots and reblotting with actin. Data represent one of three independent experiments comparing T cells from three variant and three control individuals. b, Phosphorylation of TCR-␨. The cells were left untreated (⫺) or stimulated for 1 min with cross-linked anti-TCR/CD3 mAb (⫹). Cells were lysed, and postnuclear lysates were immunoprecipitated with anti-TCR-␨. Samples were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine. Intensity of the bands was measured by densitometry. As a protein-loading control, the same blots were stripped and reblotted with TCR-␨. A representative result is shown. Similar patterns were observed when cells from two additional carriers of 77C3 G were compared with two more wild-type controls. c, Phosphorylation of Zap70. Unstimulated cells and cells that had been stimulated by cross-linked anti-TCR/CD3 mAb were lysed, and postnuclear lysates were immunoprecipitated with anti-Zap70. Samples were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine. The same blots were stripped and reblotted with anti-Zap70. d, Kinetics of [Ca2⫹]i changes. The cells were loaded with the fluorophores fluo-3 and Fura Red, stimulated by the addition of anti-TCR/CD3 mAb, and analyzed in a FACSCalibur flow cytometer. The traces shown were obtained in one experiment. Enhanced Ca2⫹ flux of 77C3 G cells was also observed in a second experiment in which cells from another variant individual were analyzed together with cells from two blood donors with wild-type CD45.

TCR signaling results from its capacity to activate Src family PTKs like p56lck by dephosphorylating their down-regulatory tyrosyl residues and enabling them to assume an active conformation (24). The enhanced activity of p56lck found in variant cells (Fig. 5) could therefore indicate an elevated CD45 phosphatase activity in these cells. Testing of this hypothesis, however, was difficult because of methodological reasons. The activity of CD45 is usually analyzed by measuring the phosphatase activity of CD45 molecules precipitated by anti-CD45 mAb. This, however, may introduce artificial dimer/multimer formation. Because several lines of evidence suggest that dimerization of CD45 down-regulates its functions (25, 26), it is unlikely that the phosphatase activity of CD45 precipitates reflects the in vivo situation. In fact, we found no differences in phosphatase activity between CD45 precipitates from variant and control cells (data not shown). Nevertheless, by studying the lectin-binding patterns of variant CD45RA molecules, we obtained some hints that the CD45 phosphatase activity could be enhanced in 77C3 G T cells. These experiments revealed high concentrations of ␣ 2,6-linked sialic acids on variant, but not on “normal”, CD45RA molecules (H.-T. Do et al., manuscript in preparation). Because dimerization is impeded by sialylation, it is

likely that a high proportion of the heavily glycosylated variant CD45RA molecules exists as monomers. This may shift the equilibrium between CD45 monomers and dimers toward monomers thereby enhancing the phosphatase activity of a cell. Therefore, it is tempting to speculate that additional expression of CD45RA isoforms enhances the phosphatase activity of variant cells thereby generating a more active pool of p56lck kinase molecules. However, alternative explanations for the enhancement of TCR signaling by variant CD45RA isoforms should also be considered in particular because recent crystal structure data show that the cytoplasmic CD45 domains probably cannot dimerize (27). The localization of molecules within lipid rafts is crucial for their effects on TCR signaling (28, 29). Thus, it will be of interest to determine the distribution of variant CD45RA molecules between raft and nonraft subdomains of the cell membrane. The findings presented here allow for the first time an estimation of 77C3 G as a risk factor for certain autoimmune diseases on the basis of functional data. It is well-established that T cells specific for myelin autoantigens are present in healthy individuals but usually remain innocuous throughout life (30). This phenomenon is


FIGURE 7. T cell reactivity is enhanced in MS patients carrying 77C3 G. T cell lines were generated by cultivation of purified T cells with PHA plus IL-2. After 2 wk, the cells were washed, rested for 3– 4 days (cultivation without PHA and IL-2), and restimulated with cross-linked anti-TCR/CD3 mAb. Total RNA was isolated, and RT-PCR was performed using primers to ␤-actin and IL-2. PCR products were electrophoresed, visualized under UV light, and quantified by densitometry. The relative amount of IL-2 mRNA was determined by calculating the IL-2:␤-actin ratio. In each experiment, cells from one variant and one wild-type individual were analyzed side-by-side. a and b, MS patients; c, healthy individuals.

explained by the existence of mechanisms which control the activity of autoaggressive cells at different levels. Regulatory T cells are thought to play an important role in this scenario (31). The autoaggressive potential may also be controlled at the level of TCR signaling sensitivity. Thus, a high activation threshold usually might prevent activation of T cells by autoantigens. Because a particularly sensitive TCR will lower the activation threshold, variant T cells may have a greater chance than wild-type cells of being activated by autoantigens. Although our data may help to develop models of the mechanisms by which 77C3 G contributes to disease susceptibility, one should keep in mind that the main observations were derived from in vitro-generated T cell lines which may not entirely reflect the situation pertaining to in vivo. According to the model that the host’s genes affect its susceptibility to autoimmunity at different levels (32), the variant CD45 allele would belong to the category of genes that affect the overall reactivity of the immune system and thus can predispose the individual to many different types of autoimmune diseases. Recent data on another CD45 polymorphism in the human (138A3 G in exon C) (33) supports a role of CD45 in disease susceptibility. However, the 138G allele seems to be protective because heterozygous carriers were reduced in cohorts of patients with autoimmune Graves’ disease and hepatitis B infection, and homozygotes were absent from a cohort of Hashimoto’s thyroiditis patients (34).

937 Although 77C3 G may enhance the susceptibility for certain diseases, this allele apparently is not a main risk factor because the great majority of patients suffering from MS, systemic sclerosis, and autoimmune hepatitis carry wild-type CD45. It is therefore likely that 77C3 G contributes to disease susceptibility only in the context of additional genetic and possibly also environmental factors. The mode of interaction of various susceptibility genes in determining a disease phenotype is not well-understood yet. The existence of 77C3 G in healthy individuals and the excess of heterozygous carriers in various diseases is compatible with the view of 77C3 G as a genetic modifier. Thus, T cell hyperreactivity alone does not necessarily result in disease but modifies susceptibility of the individual. If additional genetic components which further enhance susceptibility are also present, manifestation of the disease may occur. Polymorphisms in cytokine gene promoters influencing the level of transcription could represent one category of such genetic factors. Following this concept, the combination of 77C3 G with different additional risk factors could be the reason why the 77G allele occurs with enhanced frequency in different autoimmune diseases. Genetic modifiers may also influence the disease phenotype (35). Because T cells from variant MS patients were hyperreactive (Fig. 7), one could expect an association of 77C3 G with the severity of the disease. However, due to the great variability in the clinical course of MS, large numbers of 77C3 G patients would be required to define a modifying effect of the mutation on disease progression. In fact, the nine variant MS patients studied here did not exhibit marked differences in their clinical course, compared with patients with wild-type CD45. In summary, our data suggest that aberrant expression of CD45RA isoforms on CD45R0-expressing cells of healthy individuals and MS patients carrying 77C3 G increases the intensity of TCR signal transduction. This observation touches on several issues of human T cell biology. For example, our data imply that the combination of CD45 isoforms is more critical in determining the strength of a T cell response than the concentration of an individual isoform. These and other hypotheses are testable and should motivate additional experiments that will result in a better understanding of the role of individual CD45 isoforms in human T cell activation. Furthermore, we assume that 77C3 G and the resulting T cell hyperreactivity are part of a genetic background which may increase the risk of developing certain diseases. The detailed knowledge of the functional consequences of the 77C3 G polymorphism may provide the basis for identifying further genetic components of this background which act in combination with the 77G allele.

Acknowledgments We thank K. Wonigeit for helpful discussions and constructive comments on the manuscript.

Disclosures The authors have no financial conflict of interest.

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