Diminished TCR Signaling in Cutaneous T Cell Lymphoma Is

Diminished TCR signaling in cutaneous T cell lymphoma is associated with decreased activities of Zap70, Syk and membrane-associated Csk M-C Fargnoli1,2, RL Edelson1, CL Berger1, S Chimenti2, C Couture3, T Mustelin3 and R Halaban1

1Department of Dermatology, Yale University School of Medicine, New Haven, CT, USA; 2University of L’Aquila, L’Aquila, Italy; and 3Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

Malignant cells of patients with cutaneous T cell lymphoma + + specific antigen and have undergone antigen-driven acti- (CTCL) are of monoclonal origin and of the CD4 /CD45RO sub- vation, even though the nature of the antigen(s) remains to set. Since unlike their normal counterparts, triggering of their 13 TCR/CD3 in vitro elicits only a weak mitogenic response, we be elucidated. Antigen recognition by the TCR can lead to set out to determine which of the signal transduction molecules activation, proliferation, differentiation or cell death, 14 initiated by anti-CD3⑀ antibodies are affected in neoplastic depending on the maturation stage of the T cells. It is poss- cells. The results obtained from analysis of tumor cells from ible that CTCL cells develop a protective mechanism against four patients show a general reduction in basal and induced TCR-mediated apoptotic signals, since triggering of TCR/CD3 tyrosine phosphorylation of a wide range of signaling proteins. in CTCL cells in vitro produces only a weak proliferative Furthermore, the function of members from distinct families of protein tyrosine kinases was altered in neoplastic cells. The response (see for example Ref. 15). Because none of the pre- enzymatic activity of the membrane-bound fraction of Csk was vious studies determined the cause of this stunted response, suppressed, and its association with other cellular proteins we investigated whether immediate TCR signaling is impaired was altered. There was a decline in the amount and activity of in CTCL cells, and if so, we set out to identify the molecular Syk, and a slight decrease in the specific activity of Lck kin- basis for this deficiency. ases. Zap70 tyrosyl phosphorylation was reduced or undetect- TCR engagement rapidly induces tyrosine phosphorylation able and the kinase associated weakly, or not at all, with the TCR ␨ chain. We propose that dampened TCR-triggered of several cellular substrates in normal T cells (reviewed in responses in CTCL are caused by suppression of an array of Refs 16–19). At least five protein tyrosine kinases (PTKs) effector molecules required for coupling cell surface receptors belonging to three different families play active roles in to early and late signaling events. TCR/CD3-induced early signal transduction. They include Keywords: phosphoproteins; protein-tyrosine kinase; receptor– lck fyn zap + p56 (Lck) and p59 (Fyn) of the Src family, p70 (Zap70) CD3 complex; CD45RO ; protein tyrosine phosphatase and p72syk (Syk) of the Syk family, and p50csk (Csk) of the Csk family (reviewed in Refs 17, 18, 20 and 21). The tyrosine kinase activities of Lck and Fyn increase moderately within Introduction seconds after TCR cross-linking,22 and their activity is sub- sequently down-regulated perhaps by phosphorylation of their Cutaneous T cell lymphoma (CTCL, which includes mycosis C-terminal negative regulatory tyrosine residues by Csk fungoides and its leukemic variant, Se´zary syndrome) is a neo- (reviewed in Refs 18 and 21). This Csk repressive phosphoryl- plasm of clonal T cells that infiltrates the skin. The cells dis- ation can be reversed by the protein tyrosine phosphatase + + play a phenotype of CD4 helper/inducer, CD45RO T cells (PTPase) CD45 (reviewed in Ref 18). In addition, TCR engage- 1–5 characteristic of ‘memory’ T lymphocytes. A high percent- ment results in Zap70 activation after its binding to the tyrosyl- age of the malignant cells constitutively express BE2, a late phosphorylated immunoreceptor tyrosine-based activation activation marker of 78 kDa, that appears on cloned normal motifs (ITAMs) of the ␨ chains and the CD3 complex23,24 (for T cells only after stimulation with specific antigen, and on review see Ref. 25) and in rapid tyrosine phosphorylation and 6,7 freshly isolated normal T cells in response to mitogens, sug- activation of Syk.26 The signaling initiated by TCR activation gesting that CTCL cells have been activated by their surround- is transmitted from the cell membrane through the cytoplasm ing dermal environment. Since CTCL lymphocytes can pro- to the nucleus and induces quiescent T cells to undergo cell liferate in the cutaneous compartment but are extremely division and maturation, processes critical for clonal expan- difficult to cultivate in vitro, their neoplastic growth appears sion and initiation of the immune responses. to reflect in vivo stimulation, possibly through their TCR, or We selected for investigation several immediate key reac- antigen-independent pathways or cytokines. IL-7 might be one tions essential for T cell receptor-mediated signal transduc- of the required growth factors for CTCL cells in the skin since tion, proliferation and effector functions. Our results, based transgenic mice constitutively producing IL-7 developed on analysis of a limited sample of CTCL cells (from four extensive dermal infiltration followed by dermotropic lym- patients), show nevertheless, common characteristics, ie 8,9 phoma. However, although IL-7 is produced by kera- deficiencies in critical immediate functions associated with 10 tinocytes, it is a weak in vitro CTCL mitogen, and purified TCR activation. These include molecules participating in populations of CTCL cells were sustained in culture for only events proximal and distal to the TCR such as: (1) reduced a short period (such as a week) by mixtures of cytokines that levels of basal and stimulated tyrosyl-phosphorylated proteins 11,12 include IL-7 and IL-2. belonging to a wide range of signaling molecules; The preferential expression of CD45RO and the clonal TCR (2) suppressed specific activity of membrane-bound Csk and rearrangement suggest that CTCL clones have encountered a Lck; (3) failure to activate membrane-bound PTPase; (4) failure to activate Zap70 and to induce its association with the TCR␨ chain; and (5) reduced expression and activity of Syk. Limited Correspondence: R Halaban, Department of Dermatology, Yale Uni- + + versity School of Medicine, PO Box 20859, New Haven, CT 06520- comparative analysis with the CD4 /CD45RO subpopulation 8059, USA indicated that protein levels of several kinases in these cells Received 29 November 1996; accepted 18 April 1997 were intermediate between normal CD4+ T cells and CTCL Zap70, Syk and Csk in CTCL TCR signaling M-C Fargnoli et al 1339 cells. Our results imply that the reduced responsiveness of CTCL cells to TCR triggering is the result of impairment in a complex array of normal T cell functions. This may be relevant to the proliferation or survival of these neoplastic cells.

Material and methods

T cells and TCR stimulation

PBMCs were purified from six healthy adult volunteers and four selected patients with erythrodermic CTCL (designated BU, HA, RU and WO). These patients were chosen because they had marked clonal expansion of neoplastic cells rep- resenting 80–95% of the mononuclear cells (Table 1). Infor- Figure 1 CTCL cells display CD3 expression similar to that of nor- med consent was obtained for all specimens in accordance mal lymphocytes. Indirect immunofluorescence profiles generated by with the policy of Yale University Human Investigation flow cytometry of the staining of peripheral blood lymphocytes with Review Board. The CTCL cells were identified by their reac- ␣CD3 monoclonal antibody. The light line is the CD3 positive popu- tivity with mouse mAbs recognizing the variable region of the lation. The heavy line is the negative control demonstrated by the TCR ␣ or ␤ chains (T-Cell Diagnostic, Cambridge, MA, USA). binding of murine ascites fluid of the same isotype (IgG1) as the CD3 monoclonal antibody but of irrelevant specificity. There were 3500– Monthly phenotyping by flow cytometry indicated that the 5000 cells in each sample. See Table 1 for the expression of other CTCL cells maintained expression of both the CD3 molecule specific markers. and the T cell receptor (Table 1). As is characteristic in this group of patients, the marked expansion of the clonal malignant T cell population resulted in a reduced representation of well to therapy and at present has decreased numbers of cir- normal T and B cells. The B cell population, as identified by culating malignant cells. Therefore, it is not possible to ascribe the presence of CD19, ranged from 0–1.7%, compared to clinical significance to the relative reduction in CD3 10 ± 5% in normal healthy individuals (Table 1). The CD8+ expression found on one population of cells in this patient. subpopulation of cells was also reduced but the levels of CTCL cells from BU and HA were isolated at the time of diag- CD3⑀ and the ␣ and ␤ chains of the TCR were similar in the nosis (before treatment) as well as later when the patients were different populations of cells used in our studies, as determ- undergoing photopheresis.27 The other two patients (RU and ined by FACS analysis (Table 1), except for patient HA who WO) were receiving photopheresis throughout the study per- had two different populations of tumor cells with high and iod. CTCL cells were collected from patients under treatment low CD3 expression (Figure 1). The CTCL cells from this 1 month after a cycle of photopheresis and before the begin- patient also had comparatively low levels of CD45RO+ ning of the next one. Although we encountered some varia- (Table 1 and Figure 2f). Loss of membrane antigens in parti- bility in the expression of specific proteins in cells from the cular CD3 on the malignant T cells has been seen in some same patient between early and late collections (as discussed CTCL patients and may relate to subclone formation and more below), the general cellular phenotype remained the same. aggressive disease. However, this particular patient responded The normal and neoplastic cells isolated from the buffy coat by Ficoll–Histopaque 1077 density gradient centrifugation were washed twice in RPMI 1640 (GIBCO BRL, Grand Island, Table 1 Immunophenotype analysis of patients’ peripheral blood cells NY, USA) supplemented with antibiotics and 20% heat-inactivated FCS (GIBCO BRL). Only freshly isolated normal T cells Marker/Patient % Positive were used in the expreriments. In contrast, the CTCL cells, unless otherwise indicated, were immediately frozen in liquid Normal RU BU HA WO nitrogen until use. They retained 90% viability upon thawing as determined by trypan blue exclusion test, and remained T cells viable for at least a week in the presence of a mixture of cyto- CD3 72 (±7) 98.2 97.7 95.8 95.0 kines that included IL-2 and IL-7. However, we were unable CD4 45 (±10) 97.0 97.6 96.2 89.0 to maintain these cells in culture for longer than 10 days using CD8 28 (±8) 2.0 0 0 5.0 various combinations of cytokines, antibodies or other stimu- CD4/CD8 ratio 0.8–3.5 48.5/1 97.6/0 96.2/0 17.8/1 lants described in the literature (see for example Refs 11, 12). B cells ± Freshly isolated and frozen cells were incubated in RPMI/20% CD19 10 5 0.6 1.6 1.7 0 ° Others FCS medium overnight at 37 C before any further manipu- BE2 0 36.1 36.9 15.0 lations. Preliminary studies using freshly isolated and frozen CD45RO 15–40 98.0 79.9 20.5 88.0 cells from patients RU and BU indicated that storage in liquid CLA 8–23 1.1 0.3 0.4 2.0 nitrogen did not affect the cellular response (as described Monocyte below). population + + + MO-1 50.9 24.8 80.3 0 Normal CD4 and CD4 /CD45RO T cells from healthy T cell receptor donors were purified by negative selection as follows. Density + VB5 1–5 94.8 96.4 91.8 87.0 gradient purified PBMCs were depleted of CD8 T and B cells by incubation with magnetic beads precoated with mAb spe- Values for normal controls express range or mean ± s.d. cific for the CD8 and CD19 Ags (Dynal, Oslo, Norway), on a (standard deviation). rotator for 1 h at 4°C. After removal of the magnetic beads, Zap70, Syk and Csk in CTCL TCR signaling M-C Fargnoli et al 1340 the non-adhering mononuclear cells in the supernatants were washed twice in RPMI 1640/20% FCS (heat inactivated FCS, GIBCO BRL), and incubated overnight at 37°C. The CD4+/CD45RO+ subpopulation was enriched by removal of the CD45RA+ cells from the CD4+ population using anti- CD45RA mAb28 (anti-2H4; Coulter, Hialeah, FL, USA) followed by immunomagnetic depletion with sheep anti-mouse IgG1 (Fc) (Dynal). The percentage of CD4+ and CD4+/CD45RO+ T cells in the selected populations was determined by FACS analysis employing anti-CD4 and anti- CD45RO mAb (UCHL1, Dako, Carpinteria, CA, USA). Jurkat T leukemia cells were grown in RPMI-1640 supplemented with 5% heat inactivated FCS, L-glutamine and antibiotics. After overnight incubation in RPMI 1640/20% FCS, neoplastic T cells, normal CD4+ T cells and CD4+/CD45RO+ T cells were each washed twice with RPMI 1640 and resuspended at a concentration of 10 × 106 cells/ml. Half of the cells were stimulated with a mAb against the CD3⑀ chain of the TCR/CD3 complex (␣CD3⑀,20␮g/ml) at 37°C for 2 min. The ␣CD3⑀ antibody (CD3 (IgG1)) used was from Coulter (Miami, FL, USA), or was purified by us from the conditioned medium of the hybridoma producing cells (OKT3, ATCC CRL 8001). The normal and malignant cells were then processed further as described below.

Cell fractionation

Cells were suspended in 1 ml hypotonic lysis buffer (10 mM Tris-HCl pH 7.2, 10 mM NaCl, 10 mM ␤-glycerolphosphate,

2mM MgCl2,10mMKCl, 1 mM EDTA, 1 mM EGTA, 1 mM ␮ ␮ Na3VO4, 0.4 mM PMSF, 10 g/ml leupeptin, 10 g/ml aprotinin) on ice for 30 min. The suspensions were then slightly sonicated for 2 s on ice to ensure disruption (monitored by microscopic observations). Nuclei were Figure 2 CTCL cells display reduced tyrosyl-phosphorylated pro- removed by low-speed centrifugation and the supernatants teins and an altered pattern of kinase expression. (a, b, c and e) Anti- phosphotyrosine (␣PY) Western blots of whole cell lysates prepared were subjected to ultracentrifugation at 100 000 g for 1 h at + ° from normal CD4 T cells (NTC), CTCL cells (from patients HA, BU 4 C. The pellets (particulate fractions) were resuspended in or RU, as indicated), or CD4+/CD45RO+ selected normal T cells 100 ␮l hypotonic lysis buffer supplemented with 1% NP-40, (CD45RO). Cells were harvested without any further stimulation (−), sonicated on ice for 2 s at the lowest output and spun at ␣ ⑀ + ° or after stimulation with CD3 mAb ( ) for 2 min at 37 C. In (a), 10 000 r.p.m. for 10 min to remove insoluble material. The freshly prepared CTCL cells were used in the case of patients BU and high-speed supernatants (cytosolic fractions) were concen- RU, and frozen cells of patient HA. The numbers on the top of (b) indicate weeks of storage of CTCL cells in liquid nitrogen prior to trated 10-fold using Centricon 10 (Amicon, Beverly, MA, USA) stimulation. Normal T cells freshly isolated from four different healthy and supplemented with NP-40 to a final 1% concentration. donors are represented in (a–f). Spear-headed arrow points at the com- Protein concentration was determined with the Bio-Rad pro- plex of phosphorylated proteins with Mr 21 to 26 kDa that is likely tein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). to represent differentially phosphorylated forms of the ␨ and CD3 chains and arrowhead at the approximate position of Zap 70 as determined by immunoblotting with the respective antibody (not shown). (d) Successive probing of the membrane presented in (c) (with Western blotting, immunoprecipitation and antibodies repeated stripping except when indicated) with antibodies against CD45, PLC␥, STAT and (without stripping) Vav and MAPK; arrows For Western blotting of whole cell lysates, the stimulated and indicate the position of the signaling proteins as listed. (e) Normal unstimulated cells (3 × 106 cells/condition) were centrifuged CD4+T (CD4+) and CD4+/CD45RO+-selected (CD45RO) cells were ° ␣ at 15 000 r.p.m. for 2 min at 4 C, and resuspended directly purified from the same donor and proteins blotted with PY. into boiling sample buffer, kept for an additional 5 min at (f) Successive probing with antibodies against Csk, Fyn, Lck and ° CD45; numbers under each blot indicate arbitrary units of scanned 100 C, sonicated to disrupt DNA, and centrifuged. Super- bands. The panel designated %CD45RO+ indicates the percentage of natants were subjected to SDS-PAGE and Western blotting cells expressing CD45RO of the same population used for Western using the NOVEX gel apparatus, 10% polyacrylamide gels, blotting as measured by FACS. Equal protein loading in all lanes was PVDF (polyvinylidene difluoride) protein-sequencing mem- verified by staining the polyacrylamide gels after electrotransfer with branes (Bio-Rad Laboratories) following the manufacturer’s Coomassie brilliant blue. The equal levels of signaling proteins in (d) is instructions (NOVEX, San Diego, CA, USA). Antigen-antibody also an indication for equal protein loading. Positions of prestained high molecular size markers (Bio-Rad Laboratories) are given on the complexes were detected by enhanced chemiluminescence left in kDa according to the manufacturer’s size determinations. (ECL; Amersham, Arlington Heights, IL, USA), according to the manufacturer’s instructions. For successive probing, the membranes were stripped of the preceding antibodies with a sol- ution containing 7 M guanidine hydrochloride, 50 mM glycine Zap70, Syk and Csk in CTCL TCR signaling M-C Fargnoli et al 1341 (pH 10.8), 50 ␮M EDTA, 100 mM KCl, and 20 mM 2-ME for 10 muscle enolase (both from Sigma) showed that it was the most min at room temperature on a shaking platform. The mem- efficient target in our system. The A/G PLUS-agarose-bound branes were then rinsed, non-specific binding sites blocked antibody/antigen complexes were washed twice in lysis with 5% BSA for 1 h, and probed with the next antibody. The buffer, once in kinase buffer (20 mM Hepes pH 7.6, 20 mM ␤ efficiency of stripping was confirmed by the lack of signal after MgCl2,20mM MnCl2,20mM -glycerophosphate, 20 mM p- blotting the membranes with secondary antibody only. nitrophenylphosphate, 0.1 mM Na3V04) and then incubated For immunoprecipitation, stimulated and unstimulated cells with 10 ␮g Raytide in kinase buffer supplemented with 5 ␮M (1 × 108 cells/assay) were lysed in buffer containing 1% NP ATP and 5 ␮Ci [␥-32P]-ATP (3000 Ci/mmol, NEN Research 40, 10 mM Tris-HCl pH 7.2, 10 mM NaCl, 10 mM ␤-glycerol- Products, Boston, MA, USA), for 10 min at 30°C. Preliminary

phosphate, 2 mM MgCl2,10mM KCl, 1 mM EDTA, 1 mM experiments indicated that the kinase reaction was linear up to ␮ EGTA, 1 mM Na3VO4, 0.4 mM PMSF, 10 g/ml leupeptin, and 20 min. The assays were terminated by spinning the reaction 10 ␮g/ml aprotinin (lysis buffer) and kept on ice for 30 min. mixtures for 30 s and blotting 10 ␮l of the supernatants on to Lysates were slightly sonicated and centrifuged at 15 000 1 × 2 cm phosphocellulose strips (Whatman P81; Maidstone, r.p.m. for 20 min at 4°C, to remove insoluble material. Lysates UK). The strips were then air dried, rinsed four times in 0.5% (200 ␮g protein) were incubated with the indicated antibodies phosphoric acid, and the radioactivity measured in a liquid for 1 h and then for an additional hour with protein A/G PLUS scintillation counter. Immunoprecipitation with rabbit IgG agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, was included for each cell type (negative control). The phos- USA) on ice, on a shaking platform. To detect Zap70 and its photransferase values of the negative controls were sub- associated proteins, the immobilized anti-Zap70 precipitates stracted from the experimental values for each condition. (rabbit polyclonal antibodies26) were washed extensively with PTPase activity was assayed by measuring the release of 32P high salt lysis buffer (20 mM Tris-HCl, pH 7.5, 650 mM NaCl, from the tyrosine-phosphorylated Raytide. Raytide was phos- 5mM EDTA, 1% NP-40 containing the protease inhibitors), phorylated in vitro by v-abl tyrosine kinase (Oncogene and the eluted proteins were fractionated on a 10% SDS- Science) and [␥-32P]-ATP in a kinase reaction mixture as PAGE gel, transferred to nitrocellulose, and immunoblotted as described.32 Cells were preincubated with 37 ␮M phenylar- described below. sine oxide (PhAsO; Sigma) for 8 min at 37°C, followed by To affinity purify Csk binding proteins, particulate and cyto- stimulation of half of the population with ␣CD3⑀ (2 min). For solic fractions (200 ␮g proteins each) were incubated with membrane preparation, cells were lysed in hypotonic buffer 10 ␮g of GST-SH2/SH3-Csk expressed as a bacterial fusion with 0.1% 2-ME and processed as above (cell fractionation). protein29 in 1 ml of lysis buffer for 2 h on ice, followed by The particulate fraction was resuspended in phosphatase incubation with preswollen glutathione beads (50 ␮l) (Sigma, buffer (20 mM Hepes pH 7.4, 150 mM KCl, 340 mM Sucrose, St Louis, MO, USA) for 1 h on ice, on a shaking platform. The 0.4 mM PMSF, 10 ␮g/ml leupeptin and 10 ␮g/ml aprotinin). beads were then washed three times in lysis buffer and pro- For immune complex phosphatase assays, cells were dis- teins were eluted with 50 ␮l hot sample buffer and subjected solved in lysis buffer supplemented with 0.1% 2-ME, incu- to Western blotting as described above. bated with ␣CD45 mAb GAP 8.3, antigen–antibody com- The following antibodies were used: 4G10 mAb against plexes bound to A/G PLUS-agarose beads washed twice in phosphotyrosine (␣PY; Upstate Biotechnology, (UBI), Lake lysis buffer without 2-ME and once in phosphatase buffer. Placid, NY, USA); anti-Fyn mAb (fyn(15) Santa Cruz Membrane fractions (10 ␮g) or A/G PLUS-agarose beads Biotechnology), or rabbit polyclonal antibodies (FYN3; Santa bound CD45 were incubated with the radiolabeled Raytide Cruz), or rabbit anti-p59fyn (UBI); anti-Lck mouse mAb (3A5; (°20 000 c.p.m./assay) for 2 min at 30°C and the reactions Santa Cruz) or rabbit polyclonal antibodies (No. 2102; Santa were terminated by adding 750 ␮l of 10% activated charcoal ␣ 30 Cruz); anti-Csk rabbit antiserum ( CSKC), or mAb in 0.9 M HCl, 90 mM Na4P2O7,2mMNaH2PO4. Aliquots of (Transduction Laboratories, Lexington, KY, USA), or rabbit the supernatant were counted in a scintillation counter. polyclonal antibodies (Santa Cruz); anti-CD45 mAb (Biosource International, Camarillo, CA, USA), or the mAb GAP 8.3 made from the supernatant medium of the Results hybridoma clone (a gift from Dr D Rothstein, Yale University, New Haven, CT, USA), recognizing all the isoforms of the CTCL cells display reduced levels of tyrosyl- leukocyte common antigen; anti-MAPK rabbit polyclonal Ab phosphorylated proteins and altered kinase expression 691;31 anti-PLC␥ mAb (Transduction Laboratories); anti-Vav mAb (UBI); anti-Syk rabbit antiserum26 or affinity-purified rab- The earliest response to TCR cross-linking is an increase in bit polyclonal Abs (Santa Cruz); anti-Zap rabbit polyclonal tyrosine phosphorylation of multiple proteins, several of Abs26 or mAb raised against GST-fusion protein corresponding which have been identified. Therefore, we first analyzed the to the two tandem SH2 domains of human Zap70 (UBI); anti- pattern of tyrosine phosphorylation on signaling proteins to GAP rabbit antiserum (UBI); and anti-FAK mAb (Transduction evaluate responsiveness of CTCL cells to TCR stimulation. In Laboratories). The intensities of the bands were determined by order to examine three to four different tumor samples in par- scanning the ECL-X-ray films with the Computing Densi- allel, we had to use frozen material for most of our experi- tometer. ments since patients with high tumor load were only available at monthly intervals and we were unable to maintain the cells in culture for more than a week. We therefore determined first Immune complex kinase and phosphatase assays whether storage in liquid nitrogen affected cellular response. Western blotting with ␣PY antibody showed that basal as well All kinase assays were performed on immunoprecipitates in as induced levels of tyrosyl-phosphorylated proteins were azide-free conditions employing the synthetic peptide Raytide markedly reduced in CTCL cells compared with normal T cells (Oncogene Science, Manhasset, NY, USA) as a substrate since (Figure 2a, b and c). In some cases, as in Figure 2a, the tumor preliminary experiments with poly Glu-Tyr 1:1 and rabbit cells did not respond at all to ␣CD3 stimulation whether they Zap70, Syk and Csk in CTCL TCR signaling M-C Fargnoli et al 1342 were used immediately after isolation or after a freezing period (Figure 2a, compare responses of stored cells from patient HA to freshly used cells from patients BU and RU). The degree of unresponsiveness might be related to disease state or to the particular treatment the patient received prior to isolation. In addition, the limited responsiveness displayed by CTCL cells from patient BU was not affected by storage in liquid nitrogen for up to 26 weeks (Figure 2b and c). Furthermore, the reduced responsiveness was not due to diminished levels of CD3⑀, as indicated before (Figure 1), or low expression of several known signaling proteins as shown by the presence of Figure 3 Differences in Csk activity between normal and neoplas- ␥ tic cells. Histograms represent in vitro Csk phosphotransferase activity equal levels of CD45, PLC , Vav, STAT and MAPK (Figure 2d in immune complex kinase assays precipitated from whole cell lysates as indicated). It thus appears that CTCL suffer from severe (a) , or cell fractions (b and c). Cell type and treatments are as impairments in immediate signal transduction which affected described for Figure 2, and as marked in the Figure. The level of Csk a wide spectrum of molecules, including, in addition to those in each fraction prior to immunoprecipitation is shown by the Csk mentioned above, reduced phosphorylation of Zap, CD3 and Western blots in (b) and (c). These blots indicate that, except for ␨ chains of the TCR complex (Figure 2a, b as indicated by the patient HA, Csk levels were higher in the particulate fractions of the arrowhead and speared arrow, respectively). neoplastic compared to normal cells but yet exhibited lower total kin- + + ase activity, implicating reduced specific activity compared to normal Since CTCL cells are of memory CD4 /CD45RO subtype, control T cells. we went on to determine if the stunted responsiveness is a general characteristic of this population of memory T cells. As shown in Figure 2e, ␣CD3 stimulation triggered a similar increase in the intensity of tyrosyl-phosphorylated proteins in previous report34 ␣CD3⑀ caused redistribution of Csk as CD4+ and CD4+/CD45RO+ selected subtype of normal T cells shown by a loss in kinase activity in the cytosolic fraction and purified from the same donor. This is in spite of the fact that a gain in the membranous fraction (Figure 2b,c, normal T cells the population of selected memory T cells had profiles of and the CTCL of patient BU). The overall activity of Csk in CD45 expression, as well as expression of several critical kin- whole lysates of the neoplastic cells was greatly suppressed ases known to mediate T cell receptor engagement similar to in comparison to that of the cytosolic fractions and resembled that of CTCL cells isolated from two patients (Figure 2f as that of the membrane-associated fractions (Figure 3, compare marked). Probing of membranes with antibodies to three kin- a to b), suggesting inhibition of Csk by a tightly binding factor ases showed, that with some variation in the case of patient released by the detergent from the membrane. The normal Csk HA, Csk was consistently more abundant whereas Fyn and activity in cytosols of CTCL cells from patient BU is a good Lck were expressed at variable levels (see also below), in the indication that the use of frozen cells did not compromise Csk selected CD4+/CD45RO+ memory T and CTCL cells compared kinase activity in our studies (Figure 3c, compare BU to to unselected CD4+ T cells. NTC/CD4+).

Membrane-bound Csk activity is markedly reduced in Differential association of target proteins to the CTCL cells SH2/SH3 domain of Csk in vitro

The stunted responsiveness of CTCL cells presented in Csk may be a part of a multiprotein complex that could regu- Figure 2 could be due to diminished PTK activity and/or an late its activity since the SH2 (Src homology 2) domain of Csk increase in protein tyrosine phosphatase (PTPase) activity. The associates with several tyrosyl-phosphorylated proteins in Jur- kinase activity of Csk was tested first since this PTK occupies kat and normal T cells independently of TCR/CD3 acti- a central role in signal transduction. It may down-regulate the vation.29,35–38 We therefore looked for differences in GST- T cell receptor (TCR) signaling by phosphorylating the SH2/SH3-Csk-associated proteins in cell fractions from normal (inhibitory) C-terminal tyrosine of Lck and Fyn and potentially and neoplastic cells. The ␣PY Western blots of the affinity pur- of the CD45 PTPase. The results show that Csk basal and ified proteins revealed qualitative differences between the two induced kinase activity was three-to four-fold lower in CTCL cell types. Normal T cells preferentially displayed phosphoryl- cells than normal T cells (Figure 3a). Because Csk is found in ated SH3/SH2-Csk binding proteins of 140 kDa and 50 kDa in both cytoplasm and membrane,30,33 the immune complex the membrane fraction, and 50 kDa and several other higher assay was repeated using soluble and particulate fractions. molecular weight species in the cytosolic fraction This set of experiments showed that Csk activity was drasti- (Figure 4a,b, arrows). However, a major protein of about cally diminished in the particulate fraction of CTCL cells, with 120 kDa was apparent only in the membranes prepared from no or very small differences in the cytosol when compared to CTCL cells but not from normal T cells (Figure 4a, solid normal T cells (Figure 3b,c). This suppressed activity could not arrowhead). Reprobing the membrane with antibodies against be attributed to low levels of Csk protein because, except for p120FAK or p120GAP, tyrosyl-phosphorylated signaling proteins patient HA in the experiment described here, Csk was more failed to detect any association (data not shown). The 60 kDa, abundant in CTCL compared to normal CD4+ T cells (Figure 3 and 110 kDa Csk-SH2-associated proteins described blots in b and c, see also Figure 2f). The levels of Csk varied before29,36 were present at almost equal levels in both normal in HA cells, probably due to changes in the cell population and malignant cells (Figure 4a,b, marked by heavy arrows), (as seen in Figure 1). In addition, reduced kinase activity could whereas the 90 kDa was less abundant in the malignant cells not be caused by a Csk-associated PTPase, since none was (Figure 4a, marked by empty arrow). These results reinforced detected in immune complex PTPase assays performed on the notion that Csk activity could be regulated by a mem- anti-Csk precipitates (data not shown). In agreement with a brane-resident protein. Zap70, Syk and Csk in CTCL TCR signaling M-C Fargnoli et al 1343

Figure 4 Differences in Csk binding proteins between normal and neoplastic cells. ␣PY Western blots of afﬁnity-puriﬁed proteins eluted from control GST, or GST-SH2/SH3-Csk fusion protein bound to glutathione beads. Precipitations were done with membrane (a) or cytosolic (b) fractions. Small arrows point at GST-SH2/SH3 Csk binding proteins in normal T cells, not detected in CTCL cells; arrowhead in (a) (right-hand side) indicates the °120 kDa ␣PY reactive GST- SH2/SH3 Csk binding protein present in neoplastic but not in normal T cells. Heavy and empty arrows indicate the position of proteins reported to bind Csk-SH2 in other cellular systems.

Kinase activity of Csk-substrates

As mentioned above, Csk substrates include the C-terminal Figure 5 Src-family kinases and PTPase activity are not markedly inhibitory tyrosine residues of the Src-family kinases, and altered in CTCL cells. (a and b) Histogram shows phosphotransferase possibly Tyr-1193 of CD45 phosphatase (see reviews, Refs activity in Fyn and Lck immune complex kinase assays. Blot on the 17–20). A decrease in Csk activity might be expected to result bottom shows the levels of the respective kinases in the whole cell lysates used in these experiments. (c) PTPase activity in membrane in increased specific kinase activity of Lck, Fyn and possibly, fractions. (d) Immune complex CD45 PTPase activity. Unstimulated a decrease in CD45 PTPase activity in CTCL cells compared (−) and ␣CD3⑀ stimulated (+) normal CD4+ (NTC) and neoplastic cells + to normal CD4 T lymphocytes. However, repeated immune from patients (as indicated) were used in these experiments. complex kinase assays showed, that the specific activity of each of the Src-family kinases in the malignant cells was not increased, but rather, in some cases, was reduced by 50– membranes with ␣Zap70 mAb showed that, in one experi- 80% (Figure 5a,b). ment equal amounts of Zap70 was precipitated from the nor- Likewise, very little impact of suppressed membrane-bound mal and neoplastic HA, and in the second experiment, from Csk was found on PTPase activity. Basal PTPase activity in neoplastic RU and Jurkat T cells (Figure 6, right panels, the intact membrane fraction or in CD45 immune precipitates ␣Zap70). In the experiment presented in Figure 6a, for derived from CTCL cells was similar (or slightly increased) unknown reasons very little Zap70 was precipitated from RU compared to that of normal T lymphocytes (Figure 5c and d). CTCL cell lysate (Figure 6a, right panel). Furthermore, As expected, there was a two-fold increase in the membrane- additional experiments also showed that Zap70 protein is bound phosphatase activity in normal T cells but little or expressed at normal levels in the neoplastic cells of HA, RU no enhancement in the neoplastic T cells after ␣CD3⑀ and BU, but does not become tyrosyl-phosphorylated in stimulation (Figure 5c). response to stimulation with ␣CD3⑀ (Figure 7, compare pattern of tyrosyl phosphorylation with position of Zap70 protein band in cell lysates). These results indicate that activation of Dysfunctional Syk family kinases Zap70 is deficient in CTCL cells. Since Syk can compensate for lack of Zap70,20 we determ- The status of Syk and Zap family of kinases was also examined its expression and activity in the different cell types. Syk ined, since reduced phosphorylation of Zap was suggested in PTK positively correlated with its protein levels and was our ␣PY immunoblotting experiments (Figure 2). Differential directly proportional to the levels of tyrosyl-phosphorylated activation of Zap70 with ␣CD3⑀ was clearly demonstrated in proteins (Figure 7a,b,c). Reprobing the same membrane with the ␣Zap70 immunoprecipitation/immunoblotting experi- antibodies against Csk Fyn, and Lck showed, as before, that ments. Induction of Zap70 tyrosyl-phosphorylation and these enzymes were expressed at higher levels in CTCL cells, association with the phosphorylated TCR ␨ chains was with no correlation to the tyrosyl-phosphorylation pattern detected in normal and Jurkat T cells (Figure 6a,b, left panels, (data not shown). Thus, Syk could not substitute for Zap70 in speared shaped and empty arrows, respectively). In contrast, CTCL cells. the weakly tyrosyl-phosphorylated Zap70 precipitated from the neoplastic cells of one patient (HA) was not associated with the phosphorylated ␨ chain (Figure 6a, left panel, HA). Discussion In cells from another patient (RU), Zap70 did not become tyrosyl-phosphorylated, and only a faint band of ␨ chain was Our studies examined, for the first time, signals transduced visible in one experiment (Figure 6a, left panel, RU), but not in through the stimulation of the TCR in highly purified clonal the second one (Figure 6b, left panel, RU). Probing the same populations of CTCL cells isolated directly from the patients Zap70, Syk and Csk in CTCL TCR signaling M-C Fargnoli et al 1344 without any adaptation to culture conditions. The information derived from these studies is particularly valuable since these cells cannot be established in culture as immortal cell lines. CTCL cells, from four erythrodermic patients possessing high percentages of circulating neoplastic T cells, did not initiate, or did so to a much lesser degree, the activation cascade characteristic to ␣CD3 stimulation in normal CD4+ T cells. The stunted response was manifested as reduced levels of tyrosyl-phosphorylated proteins and was not due to changes in the level of expression of components of the TCR. Likewise, this phenotype could not be the result of selective expression of CD45RO in these cells, since in our hands the CD4/CD45RO+ subpopulation of normal T cells responded to TCR ligation in a manner similar to that of unselected CD4+ T cells, and extensive studies by others failed to detect consist- ent differences in TCR signaling between cells expressing different isoforms of CD45 (see for example review, Refs 39 and 40). In fact, memory T cells proliferate at a faster rate than naive cells when stimulated with anti-CD3 monoclonal antibody (see for example Ref. 41). The block in CTCL cells involved key members of at least two families of kinases, the membrane-bound Csk, Zap70 and Syk. Csk, Zap70 and Syk occupy central roles in the activation of immediate and proximal signals. Suppression of membrane- bound Csk may explain the failure to activate membrane- bound PTPase (presumably CD45, the major phosphatase in membranes of T cells, see review, Ref 42), since Csk controls not only the Src-like kinases but possibly also CD45.43 On the other hand, reduction in Zap70 function in CTCL can, by Figure 6 Triggering of Zap70 is aborted in CTCL cells. Proteins of + itself, be the cause for the unresponsiveness of CTCL cells. normal T cells (CD4 ), CTCL (HA and RU), or Jurkat T cells were + precipitated (ip) with ␣Zap70 rabbit polyclonal antibodies (a and b, Peripheral CD4 T cells in patients carrying inactivating Zap70), or with normal rabbit serum (b, NRS) and subjected to West- mutations in Zap70 are incapable of transducing normal TCR- ern blotting (ib) with mAbs to phosphotyrosine (PY), and, after strip- mediated signals manifested as severe combined immuno- ping, to Zap70. Protein phosphorylation and Zap70 expression in deficiency (reviewed in Refs 20, 44). Furthermore, altered immunoprecipitates was compared to that of whole cell lysates peptide ligands (APLs) that induce anergy, mediate their sig- (WCL). Spear-headed arrows indicate Zap70 and open arrows point ␨ ° ␣ nals through an alternative TCR pathway involving the altered at the co-precipitated phosphorylated chains. The 40 kDa PY ␨ reactive band in the stimulated sample of normal T cells in (a) is not phosphorylation pattern of TCR and failure to activate and yet identified. The °55 kDa band in (b) is OKT3 reacting with the recruit Zap70.45,46 The TCR signaling in CTCL cells could not secondary antibodies in the ECL detection system. Stimulation of cells be rescued by Syk, whose expression and activity was also was as described before. reduced. Membrane-bound Csk was clearly regulated at the activity level, since the protein was, on average, °5-fold more abundant in CTCL cells compared to normal T cells. Csk localiz- ation and catalytic activity is regulated by TCR cross-linking, but the exact mechanism of its activation is still unknown since post-translational modifications, such as phosphorylation on Tyr and/or Ser/Thr have not been found.29 The marked reduction in overall Csk activity in detergent-lysed CTCL cells but not in cytosolic fractions, suggests that the membrane fraction contains a Csk-binding inhibitor. Indeed, a °120 kDa tyrosine-phosphorylated protein associated with the GST-SH2/SH3-Csk from the membrane fraction of CTCL cells but not from normal CD4+ T cells. We ruled out the possibility that this protein is GAP or the focal adhesion kinase FAK. Association of the SH2 domain of Csk with a selective set of phosphotyrosine containing substrates, including a Figure 7 Aberrant expression and/or activity of Syk family of kin- 120 kDa protein was recently demonstrated in mouse T cell 38 ases. Syk expression and activity positively correlate with levels of hybridoma cells expressing constitutively active Lck. Further tyrosyl phosphorylated proteins. (a) ␣PY Western blot of whole cell identification of this protein and studies on its effect on Csk lysates as described above. Spear-headed arrows indicate the position activity will determine if indeed it is the putative Csk inhibitor. ␨ of phosphorylated forms of the and CD3 chains and an arrowhead Reduced activity of membrane-bound Csk did not cause a that of Syk. (b) Expression of Syk. Western blot of the stripped mem- steady-state activation of the Src-like kinases Lck and Fyn in brane in (a) with ␣Syk. The histogram shows arbitrary units of scanned bands of the Syk immunoblot (presented on the bottom). CTCL cells, as would be expected from the known physiologi- (c) Phosphotransferase activity in Syk immune complex kinase assays cal role of Csk. This is surprising since point mutations in the were performed on the same lysates as presented in (a). inhibitory C-terminal tyrosine residues phosphorylated by Csk Zap70, Syk and Csk in CTCL TCR signaling M-C Fargnoli et al 1345 induced hyper-reactivity and massive tyrosine phosphoryl- stimulation and growth of T cells. J Invest Dermatol 1990; 94: ation responses to TCR/CD3 stimulation.47 Furthermore, cell 452–455. lines derived from Csk knock-out mouse embryos displayed 8 Rich BE, Leder P. Transgenic expression of interleukin 7 restores T cell populations in nude mice. J Exp Med 1995; 181: 1223–1238. an order of magnitude increase in the activity of Src and Fyn 9 Rich BE, Campos-Torres J, Tepper RI, Moreadith RW, Leder P. 48 kinase. Therefore, the activity of the Src-like kinases in CTCL Cutaneous lymphoproliferation and lymphomas in interleukin 7 cells, might be mostly influenced by the cytosolic enzyme transgenic mice. J Exp Med 1993; 177: 305–316. whose activity is not altered in the neoplastic cells. In 10 Dalloul A, Laroche L, Bagot M, Mossalayi MD, Fourcade C, addition, Csk-related enzymes, such as Lsk or Ctk,49,50 may Thacker DJ, Hogge DE, Merle-Beral H, Debre P, Schmitt C. play a role. Interleukin-7 is a growth factor for Sezary lymphoma cells. J Clin ␣ ⑀ Invest 1992; 90: 1054–1060. The reduced responsiveness to CD3 stimulation in CTCL 11 Abrams JT, Lessin S, Ghosh SK, Ju W, Vonderheid EC, Nowell P, might be a result of their malignant phenotype and might con- Murphy G, Elfenbein B, DeFreitas E. A clonal CD4-positive T-cell fer a growth advantage. Stimulation of the TCR can produce line established from the blood of a patient with Sezary syndrome. a spectrum of functional responses that include not only J Invest Dermatol 1991; 96: 31–37. increases in the proliferation of reactive clones that mediate 12 Foss FM, Koc Y, Stetler-Stevenson MA, Nguyen DT, O’Brien MC, immunity and inflammation, but also cell death, in order to Turner R, Sausville EA. Costimulation of cutaneous T-cell lym- limit further inflammation. Whereas naive, resting T cells are phoma cells by interleukin-7 and interleukin-2: potential autocrine or paracrine effectors in the Sezary syndrome. J Clin resistant to receptor-stimulated suicide, activated T cells of all Oncol 1994; 12: 326–335. 51,52 subsets are sensitive. Recent experiments also suggested 13 Heald PW, Yan SL, Edelson RL, Tigelaar R, Picker LJ. Skin-selec- a requirement for PTK activity (possibly that of fyn) in super tive lymphocyte homing mechanisms in the pathogenesis of leu- antigen-induced programmed cell death of peripheral T cells kemic cutaneous T-cell lymphoma. J Invest Dermatol 1993; 101: in vivo.53 Therefore, the ability to avoid apoptotic signaling 222–226. may protect the neoplastic lymphocytes in CTCL and provide 14 Janeway C Jr, Bottomly K. Signals and signs for lymphocyte responses. (Review). Cell 1994; 76: 275–285. a selective growth advantage. 15 Hansen ER, Vejlsgaard GL, Cooper KD, Heidenheim M, Larsen JK, Ho VC, Ross CW, Fox DA, Thomsen K, Baadsgaard O. Leukemic T cells from patients with cutaneous T-cell lymphoma demonstrate Acknowledgements enhanced activation through CDw60, CD2, and CD28 relative to activation through the T-cell antigen receptor complex. J Invest Dermatol 1993; 100: 667–673. 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