Epitope-specific crosslinking of CD45 down-regulates membrane-associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

Abstract

CD45 engagement by monoclonal antibodies on human activated T cells triggers tumour necrosis factor-α (TNF-α) gene transcription in an epitope-specific manner. To dissect the early signalling events leading to TNF-α gene expression, we established that CD45 crosslinking resulted in tyrosine phosphorylation of p56lck, ZAP-70, CD3-ζ, LAT and Vav. This was accompanied by down-regulation of membrane-associated protein tyrosine phosphatase activity in the absence of demonstration of enhanced p56lck, p72syk and ZAP-70 kinase activity, which remained constitutive. These early events eventually triggered an intracellular Ca²⁺ rise and phosphoinositide turnover. We conclude that down-regulation of membrane-associated tyrosine phosphatase activity by CD45 extracytoplasmic domain multimerization led, in an epitope-specific fashion, to unopposed tyrosine kinase activity and to the activation of the T-cell receptor/CD3 complex signalling cascade, resulting in TNF-α gene expression. This model strongly suggests that CD45 extracytoplasmic tail multimerization may contribute to the modulation T-cell functions.

Introduction

CD45 belongs to a family of heavily glycosylated, high molecular weight transmembrane glycoproteins expressed on all cells of haematopoietic origin.^1,2 Structurally heterogeneous, CD45 consists of a family of eight isoforms ranging from 180 000 to 220 000 MW generated by alternative splicing of three exons differentially expressed on subpopulation of lymphocytes.² Isoform-specific expression was thought to distinguish between naive and memory T cells;^3–5 however, differences in isoform usage may instead be markers for cells differing in requirement for activation. The expression of various isoforms and extracytoplasmic domains suggests that so far unknown physiological ligands exist for the different members of the CD45 family.

CD45 protein tyrosine phosphatase (PTPase) activity plays a central role in T-cell activation.^6,7 Genetic studies in T cells deficient in CD45 expression demonstrated that T-cell clone proliferation was impaired,⁸ therefore resulting in uncoupling of signals transduced via the T-cell receptor (TCR) to the phosphatidylinositol pathway.^9,10 Transfection of the intracytoplasmic domain comprising PTPase activity restored TCR signalling capability, showing that the extracellular and transmembrane domains were dispensable for CD45 to interact with TCR/CD3 complex.^11–13 Other investigators have described a similar central role of CD45 in signals transmitted by the B-cell antigen receptor.¹⁴

While these experiments underline the key role of the PTPase domain of CD45, they do not exclude a modulating role for the extracytoplasmic domain. A regulatory role for the extracytoplasmic domain of CD45 has been suggested^15,16 and provides evidence that alternative CD45 isoforms are functionally distinct and control specific mechanisms regulating T-cell responsiveness. Jurkat cells expressing CD45 antisense RNA exhibited marked isoform-dependent differences in interleukin-2 (IL-2) production and Vav phosphorylation when reconstituted to express either the CD45RO or CD45ABC.¹⁵ A role for CD45 as a primary signalling molecule has been suggested in other models. While some anti-CD45 monoclonal antibodies (mAb) induced homotypic aggregation of T cells¹⁷ and thymocytes¹⁸ other anti-CD45 mAb directly down-regulated B-cell adhesion.¹⁹ In thymocytes, engagement of CD45 synergized with phorbol myristate acetate (PMA) to stimulate IL-2 and IL-2 receptor mRNA expression²⁰ and induction of homotypic aggregation via CD45 was dependent on the leucocyte function-associated antigen-1/intercellular adhesion molecule-3 pathway.¹⁸ Engagement of CD45 has finally been involved in the activation of cytokine genes in monocytes,²¹ natural killer (NK) cells,^22,23 neutrophils²⁴ and activated T cells,²⁵ as well as in T-cell programmed cell death.^26,27 Dramatic morphologic changes in murine T cells have been demonstrated upon CD45 crosslinking by immobilized antibodies.²⁸ Taken together, these data suggest that the extracellular domain of CD45 play a regulatory role in the fine tuning of signals transduced by the TCR. CD45 engagement by mAb 4B2 or 9.4 on human activated T cells led to tumour necrosis factor-α (TNF-α) gene transcriptional activation in an epitope-specific manner, whereas two other mAb (10G10 and UCHL1) showed no effect.^17,25 Using this panel of anti-CD45 mAb to target the CD45 extracytoplasmic domain, we herein examined the early signal transduction mechanisms induced by the epitope-specific engagement of CD45 and leading to TNF-α gene expression.

Materials and methods

Hybridomas, antibodies and reagents

Hybridomas secreting mAb 4B2 and 9.4 (immunoglobulin G2a; IgG2a) were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA). Together with hybridoma 10G10 (IgG1)¹⁷ they produce IgG-recognizing non-overlapping determinants common to all CD45 isoforms.¹⁷ Hybridoma UCHL1 (IgG2a), specific for CD45RO,²⁹ was a kind gift from Dr V. von Fliedner (Ludwig Institute, Lausanne, Switzerland). Anti-CD3 hybridoma OKT3 (IgG2a) was from ATCC. Anti-major histocompatibility complex (MHC) class II DR/DP/DQ hybridoma 3B12 (IgG1)³⁰ and murine myeloma protein UPC10 (IgG2a; ICN Biomedicals, Eschwege, Germany) were used as controls. Non-commercial mAb were purified from hybridoma supernatants by affinity chromatography on Protein A-Sepharose columns (Amersham Biosciences, Uppsala, Sweden). For immunoprecipitation, rabbit polyclonal anti-p56lck antiserum was previously described.³¹ Rabbit polyclonal anti-ZAP-70 and anti-Vav antisera were purchased from UBI (Lake Placid, NY). Rabbit polyclonal anti-p72syk and anti-LAT antisera were from Santa Cruz Biotechnology (Santa Cruz, CA). For immunoblotting, anti-ZAP-70 mAb was obtained from UBI, and anti-p72syk and anti-CD3-ζ mAb was from Santa Cruz Biotechnology. Protein tyrosine kinase (PTK) inhibitors genistein, tyrphostin and herbimycin A, and protein kinase C (PKC) inhibitor calphostin C were purchased from Life Technologies (Gaithersburg, MD). PTPase inhibitors phenylarsine oxide (PAO) and sodium pervanadate (Na₃VO₄/H₂O₂), and rabbit enolase came from Sigma (St Louis, MO). The Src family PTK inhibitor PP2, PMA and ionomycin were from Calbiochem (La Jolla, CA).

Cell culture and stimulation assays

Alloreactive human T cells were obtained from peripheral blood mononuclear cells as described.³⁰ Briefly, T cells were stimulated with irradiated (7500 rad) Epstein–Barr virus-transformed B cells every 10–15 days and expanded in rhuIL-2 (20 U/ml; BioSource, Camarillo, CA). CD45⁺ Jurkat T cells (line H₃₃HJ-JA1) and CD45^– Jurkat T cells were obtained from ATCC. All cells were grown in RPMI-1640 (Life Technologies) supplemented with 10% fetal calf serum and 2 mm glutamine (Life Technologies). For TNF-α gene expression assay, a solid phase T cell stimulation assay was performed. T cells (3 × 10⁶) cultured for 8–10 days together with feeder cells were incubated for 2 hr in 24-well cell culture dishes (Becton-Dickinson, Franklin Lakes, NJ) previously coated with anti-CD45 or control mAb (20 µg/ml). Alternatively, for immunoprecipitation experiments, a soluble phase T-cell stimulation protocol was applied. Primary activated human T cells or Jurkat T cells (1 × 10⁷) were first preincubated with soluble anti-CD45 mAb (20 µg/ml) for 3 min, washed in culture medium, and subsequently crosslinked with rabbit or goat affinity-purified anti-mouse IgG (20 µg/ml; Cappel, Durham, UK).

Quantitative reverse transcription–polymerase chain reaction (RT–PCR) assay

Quantitative RT–PCR assay was carried out as described previously.³² Total RNA was isolated according to Chomczinski and Sacchi.³³ RT reaction mixtures containing (0·25 µg of oligo-dT, 1 µl AMV buffer (500 mm Tris-HCl pH 8·3, 500 mm KCl, 50 mm MgCl₂), 1 µl 25 mm dithiothreitol, 1 µl 10 mm dNTP, 5 U RNasin, and 0·2 U AMV reverse transcriptase), 1 µl of total cellular RNA, 1 µl of internal standard (GeneAmplimer pAW109 RNA; Perkin Elmer, Branchburg, NJ) and water up to 10 µl were incubated at 42° for 60 min, heated at 95° for 5 min and then quickly chilled on ice. PCR amplification was carried out in a 30 µl final volume, by mixing 10 µl of RT mixture with 20 µl PCR buffer (50 mm Tris-HCl pH 8·3, 50 mm KCl), containing 50 pmol of each 5′ and 3′ primers, 0·25 µCi α-[³²P]dCTP, 0·5 U Taq DNA polymerase (Perkin Elmer). After 3 min of denaturation at 94°, samples were submitted to 22 amplification cycles. The PCR conditions comprised denaturation at 95° for 1 min, annealing at 60° for 30 s and extension at 72° for 1 min. PCR products from pAW109 RNA amplified with TNF-α primers were 301 bp long and were designed to be shorter than PCR products from target mRNA (325 bp). PCR products were denatured and separated on a urea sequencing gel in Tris-borate 0·45 m, EDTA 0·01 m, pH 8·0 (TBE). mRNA derived signals were quantified on an Instant Imager^® (Packard Instruments, Meriden, CT). Sample data were reported to the internal standard, corrected for sample concentration and expressed as fold induction over control sample.

Immunoprecipitation

After stimulation, T cells were rapidly pelleted and lysed in 40–100 µl of lysis buffer (LB) containing TBS (10 mm Tris-HCl pH 8·0, 150 mm NaCl), 20 mm HEPES (Life Technologies), 1% (v/v) Nonidet-P40, 1% (v/v) glycerol, 5 mm Na₃VO₄, 1 mm NaF (Fluka, Buchs, Switzerland), 1 mm benzamidine (Sigma), 1 mm phenylmethylsulphonyl fluoride (PMSF), leupeptin, chymostatin, antipain and pepstatin (each 1 µg/ml; Sigma), 10 µg/ml aprotinin (Sigma) and 1 mm ethyleneglycoltetraacetic acid (Fluka). After 5 min on ice, cells were centrifuged at 0° for 2 min at 10 000 g. Supernatants were precleared for 1 hr at 4° with 20 µl protein G-Sepharose (Amersham Biosciences) and normal rabbit serum (5 µl). Then, supernatants were incubated for 2 hr at 4° with antibodies specific for intracellular proteins, followed by precipitation with Protein G–Sepharose at 4° for 2–14 hr. Beads were washed 4 times in LB, and twice in LB/0·5 m NaCl, resuspended in LB, boiled at 100° for 3 min and finally loaded onto a denaturing 6·5% to 10% gradient polyacrylamide gel.

Immunoblotting

After sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), samples were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). After extensive washing in phosphate-buffered saline (PBS)−0·05% (v/v) Tween 20 (PBS-T), membranes were blocked in 4% (w/v) powder milk/PBS or dried for 15 min at 37°, then incubated overnight at 4° with biotinylated-antiphosphotyrosine mAb 4G10 (0·25 µg/ml; UBI) in PBS-T/1% (w/v) powder milk (PBS-T-M). After 3 washes in PBS-T-M for 5 min, blots were incubated with streptavidin–horseradish peroxidase (HRP) (1 : 3000 dilution) in PBS-T-M. After 1 hr at room temperature, membranes were washed in PBS-T and twice in PBS. Phosphorylated proteins were revealed by enhanced chemiluminescence (Amersham Biosciences) on X-OMAT Kodak film. For quantitative control of immunoprecipitated proteins, membranes were stripped as described by the manufacturer and reincubated with specific polyclonal antisera or mAb for 14 hr at 4°, with HRP-labelled goat anti-rabbit IgG (Pharmingen, San Diego, CA) or goat anti-mouse IgG (Pierce) for 2 hr, and finally revealed using enhanced chemiluminescence.

PTPase assay

T cells (10⁶ primary activated T cells, CD45⁺ Jurkat T cells, or CD45^– Jurkat T cells) stimulated with soluble or crosslinked anti-CD45 mAb were lysed in 200 µl hypotonic buffer containing 25 mm Tris-HCl pH 7·5, 25 mm sucrose, 0·1 mm ethylenediaminetetraacetic acid (EDTA), 5 mm MgCl₂, 1 mm PMSF, and protease inhibitors (10 µg/ml each of leupeptin, antipain, chymopain and pepstatin)³⁴ and subjected to three freeze–thaw rounds followed by sonication for two cycles of 3 s using a Bandelin Sonoplus sonicator (Berlin, Germany). Lysates were centrifuged twice for 3 min at 300 g at 0° to eliminate nuclei. Supernatants were then centrifuged at 100 000 g in an Optima TLX Beckman ultracentrifuge (Palo Alto, CA) for 60 min at 0°. Pellets (membrane-cytoskeletal fractions) were resuspended in 100 µl of suspension buffer containing 50 mm HEPES pH 7·3, 150 mm NaCl, 0·2% (v/v) Triton-X-100, 2 mm EDTA, 1 mm PMSF and 10 µg/ml aprotinin prior to transfer to a 96-well microtitre plate. one hundred and fifty µl of 1·9 mg/ml para-nitrophenylphosphate (pNPP) substrate (Roche Molecular Diagnostics, Basel, Switzerland) in 50 mm HEPES pH 7·3, 10 mm dithiothreitol and 25 mm EDTA were added to each well.³⁵ After 30 min, the reaction was stopped by addition of 0·2 m NaOH. Free phosphate was quantified photometrically by measuring optical density at 405 nm. In these conditions, background PTPase activity of CD45^– Jurkat T cells used as control represented less than 20% of CD45⁺ Jurkat T cell whole PTPase activity.

Immune complex kinase assay

T cells were stimulated with anti-CD45 antibodies for the indicated period of time and lysed in LB as described for immunoprecipitation. Supernatants from 10⁷ cells were precleared with normal rabbit serum and protein G-Sepharose (Amersham Biosciences), then subjected to specific immunoprecipitation for 2 hr at 4° with antibodies preadsorbed to protein G–Sepharose. Beads were washed in LB³⁴ and immune complex kinase assay was carried out in 30 µl of kinase reaction buffer [0·5% (v/v) Nonidet P-40, 100 mm NaCl, 10 mm MnCl₂, 10 mm MgCl₂, 20 mm HEPES pH 7·4, 5 µm ATP, 2·5 µCi γ-[³²P]ATP (Hartmann Analytic)] at 30° using 2·5 µg/sample of rabbit muscle enolase (Sigma) as exogenous substrate.³⁶ Reactions were terminated by adding SDS–PAGE loading buffer. Immunoprecipitates were analysed by gradient SDS–PAGE, transferred to PVDF membranes, and exposed on X-OMAT Kodak film. Quantitative analysis of PVDF membranes was done on an Instant Imager™.

Phosphoinositide turnover assay

Inositolphosphates generation was quantitated as previously described.³⁰ Jurkat T cells (20 × 10⁶) in 199 Medium (Life Technologies) were loaded with myo-2-[³H]-inositol (Amersham Biosciences; 1·5 µCi/10⁶ cells) for 14 hr at 37°, washed and incubated for 20 min in the presence of 5 mm LiCl. T cells were stimulated with anti-CD45 mAb or control antibodies, and the reaction was terminated by mixing with a solution containing methanol : chloroform : concentrated HCl (200 : 100 : 1). After centrifugation at 2000 g for 30 min at 4°, the aqueous phase was sucked off and applied to 0·5 ml Dowex AG-1 × 8 columns (formate form; Bio-Rad, Hercules, CA). Columns were sequentially washed with H₂O, followed by 5 mm di-sodium tetraborate and 30 mm sodium formate solutions. Inositol phosphates were eluted with 0·1 m formic acid and 1·0 m ammonium formate.

Calcium mobilization

To measure intracellular calcium (Ca²⁺), primary human T cells were loaded with Indo-1 AM (Sigma) as described.³⁷ Intracellular Ca²⁺ rise was analysed by flow on a FACStar cytofluorometer (Becton Dickinson, Mountain View, CA).

Results

PTK activity is required for TNF-α gene expression

To examine the role of PTK in cytokine gene expression induced by anti-CD45 mAb 4B2 or 9.4, human activated T cells were incubated with PTK inhibitors genistein, tyrphostin and herbimycin A (Fig. 1a–c) or with the Src family specific inhibitor PP2 (Fig. 1d). TNF-α mRNA transcript induction was inhibited in a dose-dependent manner down to background values. This inhibition appeared to be directed to PTKs upstream from PKC, since induction of TNF-α transcripts could be bypassed by PMA/ionomycin even in the presence of PTK inhibitors (Fig. 1f as a representative example). The signal cascade required preserved PKC activity, since gene expression was abrogated in a dose-dependent manner by T-cell incubation with calphostin C (Fig. 1e).

Figure 1.

TNF-α gene expression requires PTK and PKC activity. Primary activated T cells were preincubated for 30 min with PTK inhibitors genistein (a), tyrphostin (b), herbimycin (c), or PP2 (d), or with the PKC inhibitor calphostin C (e), then simulated for 2 hr with anti-CD45 mAb 4B2 crosslinked on plastic. Similar results were obtained with cells stimulated with mAb 9.4. Alternatively, T cells preincubated with tyrphostin were stimulated with PMA (20 ng/ml) and Ionomycin (1 µm) (f). Results are expressed as TNF-α mRNA fold induction over unstimulated T cells (representative sample in (a)). [³²P]-labelled PCR products quantified using an Instant Imager™ are presented in boxes (TNF-α transcripts: upper signal, internal standard transcripts: lower band).

Upstream PTKs p56lck, ZAP-70 and p72syk, adaptor protein LAT and exchange factor Vav are phosphorylated upon CD45 engagement

Because CD45 regulates PTKs including p56lck^38–40 or ZAP-70⁴¹ we examined whether PTKs from the src or Syk family exhibited enhanced tyrosine phosphorylation, as suggested from whole T cell lysate analysis (data not shown). Western blot analysis of anti-p56lck immunoprecipitates with antiphosphotyrosine mAb 4G10 revealed a transient raise in tyrosine phosphorylation peaking at 15 min as observed by the appearance of p60lck(Fig. 2a); p60lck is a post-translational product of p56lck known to be associated with an increase in serine/threonine phosphorylation.⁴² As expected, this was accompanied by an increase in the relative abundance of p60lck (Fig. 2c). p56lck/p60lck modulation was strictly dependent on crosslinking with mAb 9.4 (Fig. 2) or 4B2 (data not shown). Consistently, anti-CD45 mAb UCHL1, which was unable to induce TNF-α gene expression¹⁷ did not enhance tyrosine phosphorylation, similarly to isotype control mAb UPC10 (data not shown) or goat anti-mouse antibodies alone (Fig. 2), excluding a role for Fc receptor-mediated signalling. A similar modulation of p56lck/p60lck was also detected in primary activated T cells (data not shown).

Figure 2.

Upstream PTK p56lck, ZAP-70 and p72syk, adaptor protein LAT and exchange factor Vav are phosphorylated upon CD45 engagement. Jurkat T cells were stimulated with soluble (S) or crosslinked (XL) anti-CD45 mAbs 9.4 (or 4B2, data not shown) or UCHL1 (20 µg/ml) (or 10G10, data not shown), or left unstimulated for increasing periods of time. Whole cell lysates were immunoprecipitated with anti-p56lck, anti-ZAP-70, anti-p72syk, anti-LAT and anti-Vav antibodies. (a, b) Following separation on 6·5–10% polyacrylamide gradient gels in SDS and transfer to PVDF membranes, blots were incubated with antiphosphotyrosine mAb and revealed by chemiluminescence. (c, d) Blots were stripped prior to incubation with specific anti-p56lck, anti-ZAP-70, antip72syk, anti-CD3ζ, anti-LAT and anti-Vav antibodies.

Tyrosine phosphorylation of ZAP-70 and p72syk exhibited an identical kinetics, with no changes observed in control lanes (Fig. 2a). Crosslinking of activator mAb was essential to guarantee phosphorylation. In addition, hyperphosphorylated CD3-ζ (p23) was clearly identified in ZAP-70 immunoprecipitates.

A substrate for ZAP-70/p72syk, the 36 000–38 000 MW phosphorylated protein LAT triggers the recruitment of multiple signalling molecules. In agreement with the data described above, its kinetics of tyrosine phosphorylation following CD45 crosslinking did not differ from upstream PTKs. We finally examined the exchange factor Vav, which binds to phosphorylated LAT and links early PTK activation (ZAP-70, p72syk) with downstream induction of transcription factors, involving Ras- and Rho-related small GTPases.^43–45 We indeed observed an early hyperphosphorylation of Vav after 2–5 min of stimulation with crosslinked, but not soluble mAb 9.4.

Tyrosine phosphorylation of PTKs ZAP-70 and p72syk, adaptor protein LAT and p23-ζ upon CD45 engagement is dependent on p56lck kinase activity

To assess the specificity of this enhanced tyrosine phosphorylation, Jurkat T cells were preincubated for 30 min with the Src family kinase inhibitor PP2^46,47 prior to stimulation with anti-CD45 mAb 9.4 for 15 min. Using equivalent amounts of cell lysate (Fig. 3a), we demonstrated that tyrosine phosphorylation of p72syk, ZAP-70, p56lck, LAT and p23-ζ was markedly reduced or abrogated upon PP2 treatment in a dose-dependent manner (Fig. 3b). The inhibition of p56lck autophosphorylation (as shown by the absence of tyrosine phosphorylation of its p60 post-translational product) with 10 µm PP2 strongly suggests that p56lck played a crucial role as an upstream PTK in the phosphorylation cascade triggered by CD45 crosslinking that led to ZAP-70, p72syk, p23-ζ and LAT tyrosine phosphorylation.

Figure 3.

Tyrosine phosphorylation of PTK ZAP-70 and p72syk, adaptor protein LAT and p23-ζ induced by CD45 engagement is dependent on p56lck kinase activity. Jurkat T cells were preincubated or not with PP2 (10 or 100 µm) for 30 min, and were then stimulated with soluble (S) or crosslinked (XL) anti-CD45 mAbs 9.4 (20 µg/ml) (or 4B2, data not shown) for 15 min, or left unstimulated. Whole cell lysates were immunoprecipitated with specific anti-p72syk, anti-ZAP-70, anti-p56lck, anti-LAT and anti-CD3-ζ antibodies. (a) Following separation on 6·5–10% polyacrylamide gradient gels in SDS and transfer to PVDF membranes, blots were incubated with antiphosphotyrosine mAb and revealed by chemiluminescence. (b) Blots were stripped prior to incubation with antip72syk, anti-ZAP-70, anti-p56lck, anti-LAT and anti-CD3-ζ antibodies.

Membrane-associated PTPase activity is down-regulated by CD45 crosslinking in an epitope-dependent manner

Since enhanced tyrosine phosphorylation of cellular substrates may be the result of a decrease in cell PTPase activity, we examined whether cell-membrane associated PTPase activity was down-regulated by anti-CD45 mAb crosslinking. To directly evaluate this hypothesis, primary activated T cells or Jurkat T cells were incubated with soluble or crosslinked anti-CD45 mAb and lysed in hypo-osmotic buffer (Fig. 4). Membrane preparations of equal protein and CD45 content (as demonstrated by immunoblotting, data not shown) were then incubated with the phosphatase synthetic substrate pNPP. CD45 crosslinking by mAb 9 4 or 4B2 resulted in a sharp decline in membrane PTPase activity, both in primary activated T cells (mean percentage inhibition: 48% and 53%, respectively, Fig. 4a) and in Jurkat cells (mean percent inhibition: 50% and 47%, respectively, Fig. 4b). In contrast, membrane PTPase activity was unaffected by CD45 crosslinking with mAb 10G10 or UCHL1, or by incubation with control anti-MHC class II mAb 3B12 or myeloma protein UPC10. This indicated that CD45 engagement by mAb crosslinking led to an epitope-dependent decrease in membrane-associated PTPase activity, which in turn resulted in enhanced tyrosine phosphorylation of specific cellular substrates because of unopposed kinase activity. The dominant representation of CD45 among membrane PTPases strongly suggests that inhibition of CD45 activity is instrumental to the mechanism of activation. In Jurkat T cells lacking CD45, background membrane-associated PTPase activity represented less than 20% of the total membrane-associated PTPase activity of wild type Jurkat T cells (data not shown). This does not however, exclude a role for other membrane-associated PTPases, which might be indirectly affected by CD45 crosslinking. The specific involvement of CD45 reflected by partial down-regulation of membrane-associated PTPase activity in crosslinking experiments was further underscored by the total abrogation of TNF-α gene expression when T cells were preincubated with broad spectrum PTPase inhibitors such as Na₃VO₄/H₂O₂ or PAO (Fig. 5a, b). Both approaches identified thus two different pathways involving each the down-regulation of cell PTPase activity, but leading in one case (partial down-regulation via CD45 crosslinking) to T-cell activation and TNF-α expression, and in the other (broad PTPase inhibition) to cell substrate hyperphosphorylation in the absence of gene induction.⁴⁸ In addition, Fig. 5(c) demonstrates that TNF-α gene expression induced by CD45 crosslinking was further enhanced by T-cell pretreatment with the serine/threonine phosphatase inhibitor okadaic acid in a dose-dependent manner.

Figure 4.

Membrane-associated PTPase activity is down-regulated by CD45 crosslinking in an epitope-dependent manner. T cells (10⁶; a, primary activated T cells; b, Jurkat T cells) stimulated for 30 min with soluble or crosslinked anti-CD45 mAbs 9.4, 4B2, 10G10 or UCHL1, or with control anti-MHC class II mAb 3B12 (each at 20 µg/ml) were disrupted in hypotonic buffer, and the membrane cytoskelettal fraction isolated by ultracentrifugation. PTPase activity was measured using a colorimetric assay with pNPP as substrate. Activity from samples treated with crosslinked mAb was expressed as percent inhibition of that obtained from samples treated with soluble mAb. Data represents the mean ± SD from three independent experiments.

Figure 5.

Broad spectrum PTPase inhibitors block TNF-α gene expression. Primary activated T cells (3 × 10⁶) were preincubated for 30 min (a) with PTPase inhibitors PAO, or (b) with sodium pervanadate (Na₃VO₄/H₂O₂), then stimulated for 2 hr with anti-CD45 mAb 4B2 (20 µg/ml)crosslinked on plastic. (c) Primary activated T cells were incubated (30 min) with the serine/threonine phosphatase inhibitor okadaic acid prior to stimulation with anti-CD45 mAb 4B2 as for (a) and (b), or left unstimulated (unstim.). Total RNA was isolated and analysed by RT-PCR using TNF-α-specific primers. Results are expressed as fold induction over unstimulated T cells. Similar results were obtained with cells stimulated with mAb 9.4.

CD45 crosslinking and tyrosine kinase activity

Because p56lck, ZAP-70 and p72syk showed enhanced tyrosine phosphorylation upon CD45 engagement in an epitope-specific manner, we examined whether this contributed to modify their intrinsic activity. As far as p56lck and ZAP-70 were concerned, both kinases were able to autophosphorylate as well as to phosphorylate rabbit enolase (Fig. 6a, b). Autophosphorylation of the p60 post-translational product of p56lck was readily apparent (Fig. 6a, b). However, no enhanced kinase activity in CD45 stimulated cells was detected as judged by a comparable capacity of both kinases to phosphorylate the exogenous rabbit enolase substrate under any conditions (Fig. 6b, d). A non-significant normalized fold induction of peak dot densities of 1·38 was measured after cell stimulation with control mAb UCHL-1 in Fig. 6(d) (lane 8 versus lane 9, ZAP-70 autophosphorylation and enolase phosphorylation), but was not observed in two other experiments. Although a trend in enhanced p72syk kinase activity upon CD45 crosslinking with mAb 9.4 was observed in some experiments, we were unable to demonstrate a consistent increase in several experimental replicates (data not shown). Overall, these data indicated the absence of a significant up-regulation of intrinsic PTK activity after CD45 engagement by mAb 9.4 or 4B2.

Figure 6.

PTK activity is not enhanced in response to CD45 engagement. Following stimulation of Jurkat T cells with soluble (S) or crosslinked (XL) anti-CD45 mAb 9.4, 4B2, 10G10 or UCHL1 for 15 min at 37°, p56lck, ZAP-70 or p72syk (data not shown) were immunoprecipitated with specific antibodies. (a) Equivalent amounts of p56lck (as evaluated by immunoblotting using an anti-p56lck mAb) were assayed (b) for kinase autophosphorylation and for phosphorylation of rabbit enolase (10 min) (incorporation of [³²P]-phosphate into rabbit enolase revealed by autoradiography). (c, d) Assessment of ZAP-70 kinase activity was conducted similarly.

Epitope-specific CD45 engagement leads to Ca²⁺ fluxes and phosphoinositide turnover

To determine whether the observed tyrosine hyperphosphorylation led to downstream signalling events, we examined the turnover of membrane phosphoinositides (PI) as well as the rise in intracellular Ca²⁺ upon CD45 engagement. Total phosphoinositide generation was markedly enhanced (2·2-fold) in a dose-dependent (Fig. 7a) and epitope-specific (Fig. 7b) manner in Jurkat T cells upon CD45 engagement by mAb 9.4 or 4B2 (data not shown). The level of PI turnover was even better than upon direct CD3 crosslinking used a positive experimental control. In primary activated T cells or Jurkat T cells loaded with indo-1, there was no rise in intracellular Ca²⁺ in the absence of mAb 4B2 or 9.4 crosslinking. In contrast, a significant Ca²⁺ flux was observed both in primary activated T cells (data not shown) and in Jurkat T cells (Fig. 7c) after mAb crosslinking. This was not the case with control anti-CD45 mAb UCHL1. Intracellular Ca²⁺ rise was markedly inhibited by pretreating T cells with the PTK inhibitor herbimycin A.

Figure 7.

Epitope-specific CD45 crosslinking induces phosphoinositide turnover and intracellular calcium fluxes. (a) Jurkat T cells were loaded with myo-2-[³H]-inositol and stimulated for 35 min with increasing concentrations of crosslinked mAb 9.4 (or mAb 4B2, data not shown), or left unstimulated. Total [³H]-phosphoinositide generation was expressed in counts per minute (mean of triplicates ± SD). (b), Jurkat T cells loaded with myo-2-[³H]-inositol were stimulated for 35 min with a panel of crosslinked anti-CD45 mAbs (10G10, UCHL1, 9.4: 20 µg/ml), or control antibodies (UPC10, 20 µg/ml; anti-CD3 mAb OKT3, 1 µg/ml). Total [³H]-phosphoinositide generation was expressed in counts per min (mean of triplicates ± SD). (c) Jurkat T cells loaded with Indo-1 were stimulated for 3 min with soluble (S) anti-CD45 mAb 4B2, 9.4 or UCHL1, then crosslinked (XL) with goat anti-mouse antibodies. Results are expressed as FL2 fluorescence ratios over time. Intracellular Ca²⁺ rise induced by mAb 9.4 crosslinking was inhibited by pretreatment with herbimycin A 10 µg/ml for 30 min.

Discussion

We have shown in this paper that the engagement of CD45 by mAb resulted, in an epitope-specific fashion, in a decrease in membrane-associated PTPase activity and in enhanced tyrosine phosphorylation of several key signalling elements such as PTKs p56lck, ZAP-70, and p72syk, adaptor protein LAT and exchange factor Vav. However, these phosphorylation events did not affect PTK activity, but did contribute to trigger membrane phospholipid turnover and Ca²⁺ fluxes. This suggests that the engagement of the CD45 extracytoplasmic domain induces interactions between PTKs and key phosphorylated substrates of the signalling cascade ultimately leading to distal events such as cytokine gene expression.

Anti-CD45 mAb may differentially induce either T cell homotypic adhesion¹⁷ or TNF-α gene transcription and TNF-α secretion.²⁵ These events are triggered in an epitope-specific manner, because mAb recognizing non-overlapping determinants common to all CD45 isoforms (mAb 10G10, mAb 4B2, mAb 9.4) or specific for the CD45RO isoform (mAb UCHL1) were able to selectively regulate either TNF-α transcription (mAb 4B2, mAb 9.4) or homotypic aggregation (mAb 10G10, mAb UCHL1). Taking advantage of these antibodies to dissect CD45 signalling pathway(s) leading to cytokine gene expression, we first examined the requirement for PTK activity. A dose-dependent inhibition of PTK activity by specific inhibitors strongly suggested that tyrosine kinase activity was crucial in inducing TNF-α gene expression (Fig. 1). Furthermore, crosslinking CD45 with mAb 9.4 or 4B2 resulted in tyrosine phosphorylation of multiple cellular substrates, including kinases or adaptor proteins such as p72syk, ZAP-70, p56lck, LAT, Vav and p23-ζ (Fig. 2). Tyrosine phosphorylation inhibition by the Src specific inhibitor PP2 (within its range of specificity of 10 µm) of not only p56lck, but also p72syk, ZAP-70, LAT and p23-ζ underlined the central role of p56lck in the signalling cascade triggered by CD45 engagement (Fig. 3). This observation was further supported by the marked or complete abrogation of TNF-α gene expression after PP2 pretreatment within a range of concentration as low as 0·2–5 µm (Fig. 1d). Like TNF-α gene expression, tyrosine phosphorylation was strictly dependent on the recognition of specific CD45 epitopes by mAb 9.4 or 4B2, and was not observed when cells were crosslinked with mAb 10G10 or UCHL1, or in the absence of crosslinking. Although phosphorylation of cellular substrates upon CD45 crosslinking was also previously observed in other models, including NK cells²³ murine T-cell clones²⁸ and neutrophils,²⁴ our results represent the first demonstration that the specific triggering of non-overlapping epitopes led to specific downstream signalling events.

Enhanced tyrosine phosphorylation may result either from down-regulation of PTPase activity or from unopposed tyrosine kinase activity. Using T-cell membrane preparations to optimally preserve the biochemical environment of CD45³⁴ we demonstrated that mAb 9.4 and 4B2, but not 10G10, UCHL1 nor unrelated control mAb markedly decreased membrane-associated PTPase activity without enhancing PTK activity (Figs 4 and 6). Although previous reports demonstrated that dimerization of chimeric transmembrane receptor PTPase led to functional inhibition of CD45 PTPase activity^49–51 our model significantly differs from the homodimer model. Indeed, whereas chimeric transmembrane receptor PTPase dimerization by its ligand (epidermal growth factor) blocked TCR-mediated signalling with decreased protein phosphorylation and abrogation of Ca²⁺ mobilization, we showed here that engagement of specific epitopes on CD45 extracytoplasmic domain, and subsequent multimerization of CD45, led in contrast to unopposed tyrosine phosphorylation of key PTKs, adaptor proteins and exchange factors of the TCR signalling cascade and finally to cytokine gene expression. These events were independent of direct TCR triggering. Overall, our data based on pinpointed interactions of a battery of mAbs suggest that CD45 extracellular domains may potentially be the targets of differential ligands or of cell-surface molecular interactions able to modulate membrane-associated PTPase activity and to affect subsequent downstream signalling pathway(s).

CD45 crosslinking mediated down-regulation of PTPase activity was specifically associated with the up-regulation of distal events such as TNF-α gene expression. In contrast, inhibition mediated by Na₃VO₄/H₂O₂ or PAO with a broad spectrum of action led to the abrogation of TNF-α gene expression (Fig. 5). This indicates that the hyperphosphorylation resulting from mAb crosslinking involved specific signalling elements or pathways. This also suggests that some residual PTPase activity including non-CD45 PTPase activity may be (indirectly) required for TNF-α gene induction. In opposition to the abrogation of TNF-α gene expression induced by inhibitors such as Na₃VO₄/H₂O₂ or PAO, pretreatment of cells stimulated by crosslinked anti-CD45 mAb with the serine/threonine phosphatase inhibitor okadaic acid led to further enhancement in TNF-α gene expression (Fig. 5c). As previously described, okadaic acid increases TNF-α RNA stability and induces AP1 and nuclear factor (NF)-κB in human T cells.^52,53 This apparently led to a synergy between CD45 engagement and serine/threonine phosphatase inhibition, compatible with a negative regulatory role of serine/threonine phosphatases on signalling events triggered by CD45.

In T cells, there is large evidence of physical and regulatory interactions between CD45 and PTK from the src or syk family, such as p56lck^38,39,54,55 and ZAP-70.⁴¹ CD45 acts by dephosphorylating p56lck on the carboxy terminal tyrosine Y505, thus coupling TCR occupancy with increased kinase activity and triggering of the phosphatidylinositol pathway.⁹ The phosphorylation events following CD45 crosslinking and their potential functional consequences on p56lck, ZAP-70 or p72syk were thus further examined. CD45 crosslinking with mAb 9.4 and 4B2 resulted in a marked post-translational modification of p56lck and in enhanced phosphorylation of ZAP-70 and p72syk. Post-translational alterations of p56lck have been associated with enhanced serine/threonine phosphorylation of the kinase upon signals transduced via CD2, CD3 or by phorbol esters.^42,56 This also indirectly suggests that epitope-specific engagement of CD45 may induce the activation of serine/threonine kinase(s). In immune complex kinase assay, p56lck was active, as reflected by its capacity to autophosphorylate both p56 and p60 species. However, although p56lck was hyperphosphorylated, there was no evidence that its kinase activity was reduced.⁵⁷ These data are reminiscent of observations made in CD45 negative T-cell lines where p56lck activity was not inhibited either.⁵⁸ The absence of significant effect on p56lck kinase activity is in agreement with data obtained with murine T-cell clones.²⁸ Our results are further supported by the observation that serine/threonine phosphorylation of p56lck upon CD3 crosslinking did not affect its kinase activity.⁴² In a different model, Marie-Cardine et al. showed a rise in p56lck activity, but had to use simultaneously two anti-CD45 mAb or an association of anti-CD45 and anti-CD2 mAb.⁵⁹ We were furthermore unable to demonstrate significant changes in ZAP-70 and p72syk kinase activity (Fig. 6), thus reinforcing the conclusion that the overall observed hyperphosphorylation of cellular substrates was mainly related to down-regulation of membrane-associated tyrosine phosphatase activity (Fig. 4).

Protein tyrosine phosphorylation plays a critical role in coupling TCR/CD3 stimulation to transcriptional activation.⁶⁰ The most proximal signals are initiated by the activation of Src family PTKs. Subsequently, CD3-ζ is tyrosine phosphorylated, most likely by activated p56lck. Analysis of ZAP-70 immunoprecipitates indicated that phosphorylated ZAP-70 was recruited by phosphorylated CD3-ζ (p23) (Fig. 2). These initial events were strongly suggesting that T-cell activation following CD45 crosslinking was proceeding via molecular interactions similar to those used by the TCR/CD3 pathway. The tyrosine phosphorylation of LAT and Vav is further supportive of this hypothesis (Fig. 2). LAT is a substrate for ZAP-70/Syk tyrosine kinases and is a docking site for critical molecules of the TCR/CD3 signalling pathway, such as Grb2, phospholipase Cγ, p85 subunit of phophoinositol-3-kinase or Vav.⁶¹ The 95 000 MW proto-oncogene product Vav, phosphorylated by the Src and Syk families of PTKs, is essential for T- and B-cell antigen receptor-mediated signal transduction.^44,62 Syk kinases and Vav co-operate to activate NF-AT synergistically, coupling immune recognition receptors to signalling pathways involved in lymphokine production, a late event of CD45 engagement.²⁵ Vav may thus in a similar way represent a sensitive step in the signal transduction pathway mediated by CD45.

Altogether, enhanced tyrosine phosphorylation induced by CD45 crosslinking was essentially dependent on the down-regulation of membrane-associated PTPase activity, as we were unable to demonstrate enhanced intrinsic tyrosine kinase activity. Hyperphosphorylation, generated in an epitope-dependent manner, appears to lead to functional signals, because it was followed by Ca²⁺ fluxes and phosphoinositides turnover (Fig. 7). The way signals proceed is strongly reminiscent of the cascade of events triggered by antigen receptor stimulation.⁶⁰ Phosphorylation and clustering of PTKs and adaptor molecules may further lead to the translocation of the phosphorylated molecules to detergent-insoluble membrane microdomains.^63,64 Recently, CD45 has been shown to negatively regulate cytokine receptor signalling by dephosphorylating JAKs.⁶⁵ Targeted disruption of the cd45 gene in mice led to hyperactivation of the JAK-STAT pathway, cell haematopoiesis and enhanced interferon-α-receptor regulated antiviral responses.⁶⁵ Furthermore mutation to arginine of a single critical glutamate in the inhibitory structural wedge of CD45 led in vivo to lymphoproliferative syndrome and severe autoimmune nephritis.⁶⁶ We provide herein novel evidence that engagement of CD45 extracellular domain may specifically modulate cell membrane-associated PTPase activity and up-regulate essential T-cell functions such as cytokine gene expression. The precise extracellular interactions in physiological situation and molecular mechanisms leading to these events still need to be defined.

Abbreviations

LB

lysis buffer

PTK

protein tyrosine kinase

PAO

phenylarsine oxide

PTPase

protein tyrosine phosphatase

PBS-T

PBS-Tween 0·05%

PBS-T-M

PBS-T/milk 1%