Evidence for Protein Tyrosine Kinase Involvement in CD6-Induced T Cell Proliferation

CELLULAR IMMUNOLOGY

166, 44–52 (1995)

Evidence for Protein Tyrosine Kinase Involvement in CD6-Induced T Cell Proliferation1 LYDA M. OSORIO,* COSME ORDONEZ,* CARLOS A. GARCIA,* MIKAEL JONDAL,†

AND

SEK C. CHOW‡,2

*Departamento de Biologia, Instituto Nacional de Oncologia y Radiobiologica (INOR), 29 y E, 10400, Vedado, La Habana, Cuba; and Departments of †Immunology and ‡Toxicology, Karolinska Institutet, Box 60400, S-104 01, Stockholm, Sweden Received February 9, 1995; accepted June 6, 1995

action between the T cell and antigen that is expressed in conjunction with either MHC class I or class II molecules on an antigen-presenting cell. However, the signals induced through the TCR during antigen-specific T cell activation are insufficient to account for all the events observed during activation. It is now clear that interactions between a number of cell surface molecules (accessory molecules) are required to regulate the transition of a resting T cell to blast transformation, and subsequent proliferation and differentiation (1–3). The TCR is believed to deliver one set of signal and another receptor, delivering a second set of signal that is required for T cell activation. Some of these accessory molecules may function to replace or bypass the need for antigen-presenting cells. Various reports have suggested that the CD6 cell surface molecule can mediate this second signal, and that it synergized with TCRinduced signals to enhance T cell proliferation (4–6). The CD6 antigen is a membrane glycoprotein present on the surface of mature T cells and is weakly expressed in cortical thymocytes (7–9). It is also expressed in some malignant B cells as well as a subset of normal B cells (10, 11). Recent isolation and characterization of a cDNA clone encoding the human CD6 antigen revealed that the amino acid sequence of the mature protein is closely related to a large family of proteins, which includes the CD5 surface antigen and the type I macrophage scavenger receptor (12). CD6 is constitutively serine phosphorylated (105 kDa) in resting T cells and is hyperphosphorylated (130 kDa) in cells after protein kinase C (PKC)3 activation by 12O-tetradecanoylphorbol 13-acetate (TPA) or exposure to serum (13, 14). Although the cytoplasmic domain of the CD6 molecule has no kinase activity (15), CD6 is

Several studies have demonstrated that addition of soluble anti-CD6 mAbs to 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated naive T cells can induce cell proliferation. We showed in the present study that cell proliferation in TPA-treated T cell cultures can be enhanced several fold when the anti-CD6 mAbs are either immobilized or crosslinked with rabbit anti-mouse immunoglobulins (RAM Ig). Using a src family protein tyrosine kinase (PTK) inhibitor, herbimycin A, the cell proliferation induced by the anti-CD6 mAb, IOR-T1, in TPA-treated T cells were effectively abolished. Analysis of the cellular proteins in these cells after crosslinking the CD6 receptor with IOR-T1 (followed by RAM Ig) in the presence of TPA resulted in an increased level of tyrosine phosphorylation. Pretreatment of naive T cells with herbimycin A (0.5 and 1 mg/ml) for 18 hr completely inhibited the tyrosine phosphorylation on cellular substrates in T cell cultures stimulated with IOR-T1/RAM Ig and TPA. Similar concentrations of herbimycin A also inhibited the increase in IL-2 mRNA expression and cell proliferation in T cell cultures after IOR-T1/RAM Ig and TPA treatment. Furthermore, the increase in cytosolic free Ca2/ concentration in naive T cells after crosslinking of the CD6 receptor with IOR-T1/RAM Ig was also inhibited by herbimycin A. Taken together, our results suggest that CD6-mediated T cell proliferation is IL-2 dependent, and involves tyrosine kinase activity which is strictly dependent on protein kinase C activation. q 1995 Academic Press, Inc.

INTRODUCTION It is well established that antigen-specific T cell activation leading to T cell proliferation involves the inter-

3 Abbreviations used: RAM Ig, rabbit anti-mouse immunoglobulins; PLC, phospholipase C; DG, diacylglycerol; InsP3 , inositol 1,4,5triphosphate; PTK, protein tyrosine kinase; TPA, 12-O-tetradecanoylphorbol 13-acetate; AM, acetoxymethyl ester; [Ca2/]i , cytosolic free Ca2/ concentration; TCR, T cell receptor; FCS, fetal calf serum; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PBS, phosphate-buffered serum; RT/PCR, reversed transcription/polymerase chain reaction.

1

This study was supported by grants from the Swedish Agency, SAREC (to M.J.), the Swedish Medical Association, and Karolinska Institute (to S.C.C.). 2 To whom correspondence and reprint requests should be addressed at present address: Centre for Mechanisms of Human Toxicity, MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom. 44

0008-8749/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

/ m3715$7961

09-22-95 09:30:55

cia

AP: Cell Immuno

CD6-INDUCED T CELL PROLIFERATION

phosphorylated on tyrosine (Tyr) residues after T cell activation via the T cell receptor/CD3 (TCR) complex (16). The relationship between the phosphorylation state of CD6 and the biochemical events involved in CD6-induced enhancement of T cell proliferation remains unclear. Some of the earliest events observed during MHCrestricted antigen-specific T cell activation through the TCR is the rapid activation of a number of tyrosine kinases, including ZAP-70, p56lck, and p59fyn, as well as the CD45 phosphotyrosine phosphatase (17, 18). This result in the tyrosine phosphorylation of the phosphoinositide-specific phospholipase C g-isoform (PLC-g), which in turn induce the hydrolysis of phosphatidylinositol 4,5-bisphosphate to produce two putative second messengers, inositol 3,4,5-trisphosphate (InsP3) and diacylglycerol (DG). InsP3 promotes the release of Ca2/ from the intracellular Ca2/ stores resulting in an increase in cytosolic free Ca2/ concentration ([Ca2/]i), whereas DG binds to PKC and in the presence of a [Ca2/]i increase translocates from the cytosol to the plasma membrane and subsequently activation of PKC (19, 20). In contrast to this well-established signal transduction pathway mediated through the TCR/CD3 complex, costimulatory signals induced by accessory molecules which are necessary for promoting T cell proliferation and effector functions may be different, for instance, the signaling events through CD28 stimulation (21). Most of the biochemical signals delivered by these accessory molecules are still unknown, although an emerging theme of signal transduction by some of these molecules appears to involve the nonreceptor protein tyrosine kinase (PTK). In the present study, crosslinking of the CD6 cell surface molecule on resting T cells with the mAb, IORT1, in the presence of TPA consistently resulted in the tyrosine phosphorylation of various cellular substrates and the induction of IL-2 mRNA expression. These events are completely blocked by pretreatment of the cells with herbimycin A, suggesting that the costimulatory signals transduced through CD6 is coupled to a PTK pathway. MATERIALS AND METHODS Materials. Rabbit anti-mouse immunoglobulins (RAM Ig) was obtained from Dakopatts (Denmark). Herbimycin A, fetal calf serum (FCS), and RPMI 1640 were purchased from GIBCO BRL (Gaithersburg, MD). TPA, ionomycin, and fura-2/AM were obtained from Sigma Chemical Co. (St. Louis, MO). Ficoll–Hypaque was from Pharmacia (Uppsala, Sweden). Dynabeads coupled with anti-CD4 or anti-CD8 mAbs and Detachabeads were obtained from Dynal A.S. (Oslo, Norway). mAbs. The anti-CD6 mAb, IOR-T1 (IgG2a, clone F5/43/27/F6), was produced and purified as previously

/ m3715$7961

09-22-95 09:30:55

cia

45

described (22, 23). Other anti-CD6 mAbs used were anti-T12 (IgM, clone 3pT12B8, Coulter Clone), DakoCD6 (IgG2a, clone ST23, Dakopatts, Denmark), anti2H1 (IgG1) and anti-6D3 (IgG2a) (the latter two mAbs were provided as ascitic fluid by Dr. S. F. Schlossman, Harvard Medical School, Boston, MA). OKT3 mAb (CD3, IgG2a), was purified from hybridoma supernatant (ATCC, Rockville, MD). Phycoerythrin (PE)-conjugated CD25 mAb (anti-human IL-2R) was from Beckton Dickinson. Isolation of purified T cells. Human PBMC were isolated from venous blood samples from healthy adult volunteers by Ficoll–Hypaque density gradient centrifugation. To obtain purified CD4/ and CD8/ T cells, PBMC were incubated with Dynabeads coated with anti-CD4 or anti-CD8 mAbs for 60 min at 47C. The bead-coated cells were isolated using a magnetic particle concentrator and resuspended in ice-cold PBS. The procedure was repeated once and the bead-coated cells were pooled and then incubated with Detachabead for 45 min at room temperature to allow detachment of cells from the beads. The detached cells were recovered after removing the beads with the magnetic particle concentrator. This isolation procedure routinely yielded a population of T cells that was 99% CD3/ as assessed by flow cytometry. Proliferation assay. Purified T cells (1 1 105 cells/ well) in 200 ml of RPMI 1640 supplemented with 10% AB/ human serum, 2 mM L-glutamine, and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) were cultured in flat-bottomed 96-well plates (Costar, Cambridge, MA) for 72 hr at 377C under an atmosphere of 5% CO2 in air. Cells were activated with CD6 mAbs in the presence or absence of TPA (4 ng/ml) and ionomycin (100 ng/ml) either alone or in combination. To crosslink the CD6 mAbs, culture plates were preincubated overnight at 47C with 100 ml of RAM Ig, diluted in 0.05 M bicarbonate buffer (pH 9.6) to a final concentration of 10 mg/ml. Unbound antibodies were removed by washing the wells with PBS followed by PBS containing 1% human serum. Antibodies and agents of interest were added to the wells of the culture plates at concentrations where indicated before addition of cells. In experiments where herbimycin A were used, the drug (0.5 and 1 mg/ml) was added to the cells 18 hr prior to the addition of stimuli. The cells were pulsed for the last 18 hr with 1 mCi/well of [3H]thymidine and then harvested onto glass fiber filters (Skartron Inc., Sterling, VA). The filters were dried and radioactivity determined using liquid scintillation counting. Flow cytometry analysis. Cells were cultured in 24well plates (Falcon, Becton Dickinson) and stimulated with TPA and ionomycin either alone or in combination, in the presence or absence of IOR-T1. To crosslink anti-CD6 mAb, IOR-T1, the plates were precoated with

AP: Cell Immuno

46

OSORIO ET AL.

10 mg/ml RAM Ig as described above. After 24 hr the cells were collected and stained with PE-conjugated anti-CD25 mAb (IL-2 receptor) for 30 min at 47C, before flow cytometry analysis using a FACScan analyzer equipped with an argon laser (Beckton Dickinson). Forward and side scatter gatings were set to include lymphocytes and lymphoblasts. A total of 1 1 104 cells of the gated population were analyzed. Antiphosphotyrosine immunoblotting. Purified T cells (10 1 106/ml) were stimulated by crosslinking the anti-CD6 mAb, IOR-T1 (10 mg/ml) with RAM Ig (40 mg/ ml) in the absence or presence of TPA (4 ng/ml). At the indicated time the cells were rapidly washed in ice-cold PBS containing 400 mM Na3VO4 , 5 mM EDTA, and 10 mM NaF. The cells were then lysed on ice with a lysis buffer containing 50 mM Tris, 0.5% Triton X-100, 300 mM NaCl, 1 mM Na3VO4 , 5 mM EDTA, 5 mg/ml aprotinin, and 2 mM PMSF at pH 7.6 for 45 min. The cell lysates were centrifuged at 20,000g to remove nuclei and cell debris. The supernatants were then mixed with an equal volume of 21 SDS sample buffer, and samples equivalent to 5 1 106 cells were separated on 8% SDS–PAGE and subsequently electroblotted to nitrocellulose paper. The blots were stained with ponceau S (Sigma) to ensure that equivalent amount of proteins were transferred from each lane. After washing off the ponceau S stain with PBS, nonspecific binding sites were blocked by incubating the nitrocellulose membrane with PBS containing 5% skim milk. The immunoblots were then incubated overnight with the antiphosphotyrosine mAb, 4G10 (Upstate Biotechnology, Lake Placid, NY) at 47C. The blots were washed twice before incubating with horseradish peroxidase-conjugated monoclonal anti-mouse IgG (Amersham). After washing the blots were developed by the enhanced chemiluminescence method according to the manufacturer’s specifications (ECL Western blotting detection, Amersham, UK). Measurement of cytosolic free Ca2/ concentration. Changes in [Ca2/]i were determined as previously described (24). Briefly, cells at 2 1 107/ml in RPMI 1640 medium were loaded with fura-2/AM (5 mM) for 30 min at room temperature. Fura-2-loaded cells (5 1 106) were resuspended in 2 ml modified Krebs–Henseleit buffer (1.3 mM CaCl2 , supplemented with 5.5 mM glucose and 10 mM Hepes, pH 7.2) in a quartz cuvette maintained at 307C in a Sigma ZFP 22 dual-wavelength spectrofluorometer (Sigma Instruments, Berlin West, FRG) with excitation wavelengths 334 and 366 nm (emission was measured using a 500-nm cutoff filter). To minimize dye leakage all experiments were performed at 307C instead of 377C (25). Changes in [Ca2/]i were monitored by recording changes in the fura-2 fluorescence ratio signal and calibrated in terms of [Ca2/]i as previously described (26).

/ m3715$7961

09-22-95 09:30:55

cia

RNA extraction and PCR amplification of mRNA. Total RNA were extracted from 10 1 106 cells as previously described (27). The mRNA levels of IL-2 were analyzed using the reversed transcription/polymerase chain reaction (RT/PCR) described elsewhere (28). In brief, total RNA was denatured at 907C for 5 min and quickly chilled on ice. First-strand cDNA was generated using random hexanucleotides (Pharmacia-LKB, Uppsala, Sweden) and reverse transcriptase (BRL, Life Technologies, Inc., MD) in a reaction volume of 40 ml. PCR amplification of the resultant cDNA was carried out in a final volume of 50 ml containing 5 ml of 101 PCR buffer (100 mM Tris–HCl, 500 mM KCl, 0.1% w/ v gelatine, pH 8.3), 1 ml dNTP (5 mM each), 5 ml of 5 mM IL-2 or b-actin specific primer (control gene), 0.25 ml (5 U/ml) of Taq polymerase (Boeringher Mannheim), 10 ml of the first-strand cDNA, and water. PCR conditions were 1 min at 947C for denaturation, 1 min at 557C for annealing, 1 min at 727C for primer extension, and amplified for 30 cycles. The PCR products were electrophoresed on 2% agarose gel and stained with ethidium bromide before visualization using uv illumination (FMC BioProducts, Rockland, MD). The oligonucleotide primers used were: IL-2, 5*-TGTACAGGATGCAACTCCTG (sense), 5*-CAATGGTTGCTGTCTCATCAG (antisense), and b-actin, 5*-AGCGGGAAATCGTGCGTC (sense), 5*-CAGGGTACATGGTGGTGCC (antisense). The resultant PCR products were 400 bp for IL-2 and 339 bp for b-actin. RESULTS Effect of anti-CD6 mAbs on T cell proliferation in the presence of TPA or ionomycin. Previous reports have shown that the ligation of the CD6 surface antigens with anti-CD6 mAbs is comitogenic for T lymphocyte cultures in the presence of TPA (5, 15, 29). Using the anti-CD6 mAb, IOR-T1, which have been shown recently to induce greater responsiveness in TCR/CD3mediated T cell proliferation (6), we examined its effect on cell proliferation in the presence of TPA or ionomycin. As illustrated in Fig. 1A, soluble IOR-T1 have little effect on cell proliferation in the presence of either TPA (4 ng/ml) or ionomycin (0.1 mg/ml) alone or in combination. However, crosslinking IOR-T1 with RAM Ig stimulated a substantial incorporation of [3H]thymidine into T cell cultures incubated in the presence of TPA but not ionomycin (Fig. 1B). As expected, IORT1 (soluble or crosslinked with RAM Ig), TPA, or ionomycin when added alone induced negligible cell proliferation in T cell cultures as shown by the low level of [3H]thymidine uptake. Other anti-CD6 mAbs, such as T12, 2H1, Dako-CD6, and 6D3, have effects similar to IOR-T1 on cell proliferation in TPA- or ionomycintreated T cell cultures when added in soluble form or crosslinked with RAM Ig (results not shown). All subsequent experiments were performed using RAM Ig to

AP: Cell Immuno

CD6-INDUCED T CELL PROLIFERATION

47

FIG. 1. Effect of the anti-CD6 mAb, IOR-T1, on T cell proliferation in the presence of TPA and/or ionomycin. Purified T cells were cultured in flat-bottomed 96-microwell plates with soluble (A) or crosslinked (B) IOR-T1 (1 mg/ml) in the presence of TPA (4 ng/ml) or ionomycin (0.1 mg/ml). To crosslinked IOR-T1, the plates were precoated with 10 mg/ml RAM (RAM Ig) overnight at 47C before used. [3H]Thymidine incorporation was determined after 3 days and the results were expressed as means (SD õ15%) of triplicate cultures from one representative experiment out of three.

crosslink IOR-T1, either by immobilizing the RAM Ig onto culture plates or used as secondary antibodies in soluble form. Effects of herbimycin A on anti-CD6-induced T cell proliferation in the presence of TPA. Although PKC activation is necessary for T cell proliferation stimulated through the CD6 antigen, little is known about the nature of the biochemical signal delivered through the CD6 receptor during the ligation of CD6. Using a selective inhibitor of the src family PTK, herbimycin A (30, 31), the possible involvement of tyrosine kinases during CD6-induced T cell proliferation was examined. Purified T cells were pretreated with herbimycin A (0.5 and 1 mg/ml) for 18 hr before stimulated by crosslinking the various anti-CD6 mAbs with RAM Ig alone or in the presence of TPA. The results in Table 1, showed that herbimycin A pretreatment markedly inhibited the incorporation of [3H]thymidine to near control levels in T cells stimulated with the crosslinking of the anti-CD6 mAbs in the presence of TPA. The decrease in cell proliferation after herbimycin A treatment is not due to downregulation of the CD6 antigens since flow cytometry analysis indicates that the cell surface expression of CD6 on the T cells remain unaffected (results not shown), and the concentration of the drug used is not toxic over the course of the experiments. To ensure that herbimycin A did not affect cellular responses due to TPA, the cells were examined for their expression of CD25 (IL-2 receptor) after TPA treatment (32). Flow cytometry analysis indicated that TPA-induced expression of CD25 was unaffected by the presence of herbimycin A at the concentrations used (results not shown). Furthermore, herbimycin A had neg-

/ m3715$7961

09-22-95 09:30:55

cia

ligible effect on T cell proliferation induced by the combination of TPA and ionomycin (results not shown). These results are consistent with previous reports showing that herbimycin A does not inhibit the serine/ threonine protein kinases, c-raf, and PKC (31). The present results strongly suggest that the signals induced through the ligation of the CD6 receptor, which is comitogenic in TPA-treated T cells is herbimycin A sensitive. Crosslinking of the CD6 receptor with IOR-T1 induces herbimycin A-sensitive protein tyrosine phosphorylation in TPA-treated T cells. The inhibition of T cell proliferation induced by crosslinking IOR-T1 in the presence of TPA after herbimycin A pretreatment (Table 1) suggest that protein tyrosine phosphorylation may be involved in the signaling mechanism through the CD6 molecule. Using immunoblot analysis, cell lysates from CD6-stimulated T cells in the presence or absence of TPA were examined for tyrosine phosphorylated substrates. As illustrated in Fig. 2, few changes in tyrosine phosphorylation pattern of cellular proteins were detected in T cells stimulated with TPA or soluble IOR-T1 (lanes 2 and 3, respectively). A slight increase in tyrosine phosphorylation of two proteins (approximately 75 and 118 kDa) was observed in cell lysates from T cells stimulated with the crosslinking of IORT1 alone with RAM Ig (lane 4). However, when IORT1 was crosslinked with RAM Ig in the presence of TPA, several proteins with approximate molecular mass of 75, 94, 102, and 118 kDa were markedly tyrosine phosphorylated (lane 5). Time course studies indicate that tyrosine phosphorylation of the 75-kDa substrate reaches a maximum level after 5 min and then

AP: Cell Immuno

48

OSORIO ET AL.

TABLE 1 Effect of Herbimycin A on CD6/TPA-Induced T Cell Proliferation [3H]Thymidine incorporation (cpm)

mAb

Medium

TPA (4 ng/ml)

TPA (4 ng/ml) / herbimycin A (0.5 mg/ml)

TPA (4 ng/ml) / herbimycin A (1 mg/ml)

Experiment 1 Medium IOR-T1 Anti-T12 Dako-CD6 Anti-2H1

1,132 2,505 1,565 1,807 1,919

12,040 90,110 79,012 61,695 67,861

1,353 2,944 6,438 3,291 444

544 906 466 838 271

3,597 11,981 7,381 7,784 10,105 7,302

879 1,903 440 921 588 1,717

Experiment 2 Medium IOR-T1 Anti-T12 Dako-CD6 Anti-2H1 Anti-6D3

1,276 1,938 929 1,302 914 318

27,904 46,048 50,095 100,220 57,303 34,712

Note. Purified T cells from normal donors were cultured in triplicate using 96-well plates precoated with RAM Ig. The anti-CD6 mAbs, IOR-T1, anti-T12, and Dako-CD6 were used at final concentrations of 1 mg/ml, whereas anti-2H1 and anti-6D3 were used at a 1:5000 final dilution of the ascitic fluid. Herbimycin A was added 18 hr before the addition of the mAbs and/or TPA. [3H]Thymidine incorporation was determined after 3 days of culture. Results are expressed as the means of triplicate cultures with SD õ15%.

declined slightly by 10 min (lanes 6 and 7, respectively). Tyrosine phosphorylation of all other proteins (94, 102, and 118 kDa) remained unchanged over the 10-min (lanes 5–7). In contrast, pretreatment of the T cells with herbimycin A (0.5 mg/ml) prior to stimulation with IOR-T1 (followed by RAM Ig) in the presence of TPA, completely inhibited the tyrosine phosphorylation of cellular substrates in T cells (lanes 11–14). We then compared the protein tyrosine phosphorylation pattern in T cells induced by crosslinking CD6 (in the presence of TPA) with that observed after ligation of the CD3 complex with OKT3 in the presence or absence of TPA. As illustrated in Fig. 3 the tyrosine phosphorylation pattern of proteins in T cells after CD6 and CD3 stimulation were virtually identical. Although CD6 have previously been shown to be tyrosine phosphorylated during TCR/CD3 complex stimulation, our immunoblot analysis showed that none of the tyrosine phosphorylated protein bands (102 and 118 kDa) were part of the CD6 antigen (results not shown). Herbimycin A inhibits [Ca2/]i increase induced by multivalent crosslinking of the CD6 receptor. One of the early signaling events involved in T cell activation is a rapid increase in [Ca2/]i (20). We therefore examined whether IOR-T1 can induce changes in [Ca2/]i in T cells. As shown in Fig. 4A,, soluble IOR-T1 has little effect on [Ca2/]i in fura 2-loaded T cells (trace b), but subsequent crosslinking of the IOR-T1 mAb with RAM Ig resulted in a rapid increase in [Ca2/]i . This increase in [Ca2/]i is, however, much lower in magnitude com-

/ m3715$7961

09-22-95 09:30:55

cia

pared to that induced by crosslinking of OKT3 (antiCD3 mAb) with RAM Ig (trace a). The presence of 4 ng/ml TPA (10 or 60 min pretreatment) did not alter the rate or magnitude of the [Ca2/]i increase induced by the crosslinking of IOR-T1 with RAM Ig (trace c and d). Pretreatment of T cells with 0.5 mg/ml herbimycin A for 18 hr completely abolished the increase in [Ca2/]i induced by IOR-T1/RAM Ig (Fig. 4B, trace b), whereas the rise in [Ca2/]i induced by ionomycin was unaffected (results not shown). Control T cells incubated for 18 hr without any treatment responded the same way as freshly purified T cells (trace a). CD6-induced IL-2 mRNA expression in TPA-treated T cells. Since tyrosine phosphorylation is an early event in TCR-mediated signaling, we next examined whether CD6/RAM Ig stimulation in the presence of TPA can induce the expression of IL-2 mRNA, a distal event typically observed during T cell activation. Using IL-2-specific primers, first-strand cDNA synthesized from RNA isolated from treated T cells was amplified using PCR. As illustrated in Fig. 5A, IL-2 mRNA was expressed in T cells after crosslinking IOR-T1 with RAM Ig in the presence of TPA (lane 5), whereas crosslinking of IOR-T1 in T cells in the absence of TPA resulted in no detectable level of IL-2 mRNA (lane 4). These results correlate well with the incorporation of [3H]thymidine in T cells under similar conditions (Table 1 and Fig. 1). As expected an increased in IL-2 mRNA was observed in T cells after crosslinking OKT3 with RAM Ig in the presence of TPA (lane 7), but not

AP: Cell Immuno

49

CD6-INDUCED T CELL PROLIFERATION

DISCUSSION

FIG. 2. Crosslinking of the CD6 receptor by the CD6 mAb, IORT1, induces tyrosine phosphorylation of cellular substrates in TPAtreated cells that is inhibited by herbimycin A. Purified T cells were left unstimulated (lane 1); stimulated for 15 min with TPA (4 ng/ml) (lane 2); stimulated for 15 min with IOR-T1 (10 mg/ml) (lane 4); stimulated for 15 min with IOR-T1 followed by crosslinking with 40 mg/ml RAM (RAM Ig) for 2 min (lane 4); stimulated for 15 min with IOR-T1 and TPA followed by crosslinking with RAM for 2, 5, and 10 min (lanes 5, 6, and 7 respectively). In some cultures, herbimycin A (0.5 mg/ml) was added 18 hr before stimulation (lanes 8–14). Cell lysates were prepared and separated by SDS–PAGE, transferred on to nitrocellulose membrane and immunoblotted with anti-pTyr mAb as described under Materials and Methods. Molecular weight markers (kDa) are shown on the left.

in T cells stimulated with TPA, OKT3, or IOR-T1 alone (lanes 2, 4, and 6, respectively). In the presence of 0.5 mg/ml herbimycin A, the expression of IL-2 mRNA observed in TPA-treated T cells after crosslinking of IORT1 was completely abolished (Fig. 5B, lane 6) but the expression of CD25 was unaffected (results not shown). The concentrations of herbimycin A required to block the expression of IL-2 mRNA in T cells were identical to those used in preventing the incorporation of [3H]thymidine (Table 1) and the inhibition of tyrosine phosphorylation of cellular proteins (Fig. 2). In addition, the crosslinking of IOR-T1 with RAM Ig in TPA-treated cells further upregulates the expression of CD25 (96% positive cells) compared to 65% positive cells after TPA treatment alone (Fig. 6). This enhanced expression of CD25 was equivalent to that obtained in cells treated with the combination of optimal concentrations of TPA and ionomycin. Crosslinking of CD6 with IOR-T1, followed by RAM Ig in T cell cultures treated with TPA/ ionomycin did not further upregulate the expression of IL-2R. The T cell cultures stimulated with IOR-T1/ RAM Ig did not express IL-2R above control levels (Fig. 6)

/ m3715$7961

09-22-95 09:30:55

cia

Multivalent crosslinking of the CD6 molecule with anti-CD6 mAbs in TPA-treated naive T cells have previously been shown to induce cell proliferation (5, 15, 29). In the present study, we have confirmed these observations and further demonstrate that an essential requirement for the induction of T cell proliferation via the ligation of the CD6 molecule is the immobilization of the anti-CD6 mAbs. This is further supported by the low level of cell proliferation observed in TPA-treated T cell cultures when the anti-CD6 mAbs were present in soluble form. To investigate the role of PTK in CD6-induced T cell proliferation, a src family tyrosine kinase inhibitor, herbimycin A, was used. When added to T cell cultures 18 hr prior to CD6 mAbs/TPA treatment, a concentration-dependent inhibition of cell proliferation was observed. Since the internal domain of the CD6 molecule does not possess any intrinsic tyrosine kinase activity (15), these results suggest that CD6-induced T cell proliferation may be coupled to a nonreceptor tyrosine kinase. The selectivity of herbimycin A for src family PTK has been reported previously (30, 31) and is further supported by our observations that this drug has no effect on the upregulation of CD25 or IL-2 receptor expression (data not shown), one of several cellular responses induced through the activation of PKC by TPA (32). The involvement of PTK during the ligation of the CD6 receptor in TPA-treated T cells was further corroborated using an immunoblot assay, where several detergent soluble cellular substrates were found to be

FIG. 3. Comparison of the protein tyrosine phosphorylation induced by the CD6 and CD3 antigen in T cells. Purified T cells were stimulated with TPA (4 ng/ml) alone for 15 min (lane 1), stimulated with IOR-T1 (10 mg/ml) and TPA for 15 min followed by crosslinking with RAM (RAM Ig) for 5 min (lane 2), or stimulated for 15 min with OKT3 (10 mg/ml) in the absence (lane 3) or presence of TPA (lane 4) followed by RAM. Cells lysates were prepared and analyzed as outlined in legend to Fig. 2. Molecular weight markers (kDa) are shown on the left.

AP: Cell Immuno

50

OSORIO ET AL.

FIG. 4. Herbimycin A inhibits the increase in T cell [Ca2/]i induced by crosslinking of the CD6 receptor with IOR-T1. Changes in cytosolic free Ca2/ concentration were monitored using the Ca2/ fluorescence dye, fura-2 as described under Materials and Methods. The anti-CD3 mAb, OKT3 (10 ng/ml), anti-CD6 mAb, IOR-T1 (100 ng/ml), and TPA (4 ng/ml) were added where indicated followed by RAM (RAM Ig) which was added at 40 mg/ml.

tyrosine phosphorylated upon crosslinking of the CD6 receptor by the anti-CD6 mAb, IOR-T1, followed by RAM Ig. The CD6-induced tyrosine phosphorylation was observed within 2 min, indicating that the coupling

FIG. 5. Crosslinking of the CD6 receptor by the anti-CD6 mAb, IOR-T1, induces IL-2 mRNA expression in TPA-treated T cells that is inhibited by herbimycin A. (A) Purified T cells (10 1 106 cells/well) were incubated for 18 hr in RAM (RAM Ig)-coated 24-well plates. Cells were stimulated as follows: Control, lane 1; TPA (4 ng/ml), lane 2; IOR-T1 mAb (10 mg/ml), lane 4; TPA plus IOR-T1 mAb, lane 5; OKT3 mAb (10 mg/ml), lane 6; TPA plus OKT3 mAb, lane 7. (B) Purified T cells were pretreated with (lanes 5–7) and without (lanes 1, 3, and 4) herbimycin A (0.5 mg/ml) for 18 hr prior to activation in RAM-coated 24-well plates with Control (lanes 1 and 5), TPA plus IOR-T1 mAb (lanes 3 and 6), or OKT3 mAb plus TPA (lanes 4 and 7). The total RNA was isolated, reverse transcribed into cDNA, and amplified by PCR as described under Materials and Methods. Molecular weight markers are in base pairs of 50–1000.

/ m3715$7961

09-22-95 09:30:55

cia

of the kinase through the activation of CD6 is an early event. This kinase appears to be similar to that being activated during CD3-induced T cell activation, as the phosphorylation patterns of the proteins induced by both CD6 and CD3 were virtually identical. The presence of TPA, presumably through the activation of PKC was found to be absolutely necessary for the tyrosine kinase activity as shown by the lack of tyrosine-phosphorylated substrates in T cells stimulated by crosslinking the CD6 receptor with IOR-T1 alone. Under these conditions, there was a corresponding decrease in the uptake of [3H]thymidine, suggesting that the CD6-induced tyrosine kinase activity is crucial for the T cell proliferation induced by CD6/RAM Ig in the presence of TPA. Indeed, the pretreatment of T cells with herbimycin A completely abolished the increased in tyrosine phosphorylation of cellular proteins and inhibited cell proliferation in these cells upon crosslinking of the CD6 receptor in the presence of TPA. Similar to antigen-specific T cell activation, the CD6/RAM Iginduced cell proliferation in TPA-treated naive T cells appears to proceed through an IL-2-dependent mechanism as shown by the expression of the p55 chain of the IL-2R on the cell surface, and the expression of IL2 mRNA. Pretreatment of T cell cultures with concentrations of herbimycin A that inhibit CD6/TPA-stimulated tyrosine phosphorylation and cell proliferation also inhibits the expression of IL-2 mRNA, further supporting the role of tyrosine phosphorylation in CD6/ TPA-induced T cell proliferation. Taken together, these

AP: Cell Immuno

51

CD6-INDUCED T CELL PROLIFERATION

FIG. 6. Crosslinking of the CD6 receptor with IOR-T1 potentiated the TPA-induced IL-2R expression. Purified T cells (1 1 106 cells/well) were incubated in RAM (RAM Ig)-coated 24-well plates with TPA (4 ng/ml) and/or ionomycin (0.5 mg/ml) in the presence or absence of IOR-T1 (1 mg/ml). After 24 hr, the cells were stained with PE-conjugated anti-CD25 (IL-2R) and analyzed by flow cytometry. The x axis represents the log fluorescence intensity and the y axis represents cell number. The percentage of positive cells is shown in the upper right corner.

results suggest that one of the earliest biochemical signals observed through the ligation of the CD6 receptor leading to cell proliferation in T cell cultures involves protein tyrosine phosphorylation, which is strictly dependent on the activation of PKC. It is known that a number of proteins are phosphorylated when PKC is activated by TPA or through the physiologically released DG. The latter is an important early step during T cell activation through the TCR/ CD3 complex (18). How PKC activation primes naive T cells to become responsive to CD6 stimulation is at present unclear, although several possibilities exist. One possibility is that the coupling of CD6 to the PTK pathway involves the phosphorylation of the CD6 receptor by PKC, as shown by the hyperphosphorylation of serine residues on the CD6 molecule after TPA treatment (13, 14). Alternatively, the association of the CD6 receptor to PTK could involve the phosphorylation of the kinase(s) by PKC, which become active only after interacting with the internal domain of the CD6 recep-

/ m3715$7961

09-22-95 09:30:55

cia

tor. Recently, it was shown that activation of naive T cells via the TCR/CD3 complex leads to the phosphorylation of both tyrosine and serine residues of the CD6 internal domain, presumably through the activation of PTK and PKC (16). When mAbs to CD6 are cocrosslinked with mAbs to the TCR/CD3 complex, severalfold increases in cell proliferation above that obtained with anti-CD3 mAbs alone were observed (4, 6). Based on the present observations with TPA, the enhanced cell proliferation observed in T cell cultures stimulated through the CD6/TCR complex is most likely due to the activation of PKC. Since CD6 is also phosphorylated on tyrosine residues during TCR/CD3 complex activation, a role for PTK in priming naive T cells into becoming responsive to CD6 stimulation cannot be ruled out. Further studies are needed to determine the relationship between the phosphorylation state of CD6 and its activation potential. Crosslinking of the CD6 receptor with anti-CD6 mAbs also induces an increase in [Ca2/]i albeit lower in magnitude compared to that induced by the ligation of the TCR/CD3 complex with anti-CD3 mAbs. It is well established that InsP3 , which mediates the release of stored Ca2/ from intracellular stores, is generated through the hydrolysis of phosphatidylinositol 4,5-biphosphate by PLC-g during T cell activation (20). Since PLC-g is regulated by PTK (33), the low PTK activity observed during CD6 crosslinking in the absence of TPA may be sufficient for mediating some PLC-g activity. It would be of interest to know if the increased in [Ca2/]i after CD6 mAbs treatment involves PLC-g and the generation of InsP3 . Incidently, the increase in [Ca2/]i after crosslinking mAbs to CD6 remained unchanged in the presence of TPA, but was completely abolished in herbimycin A-treated cells. This suggests that CD6 may be associated with at least two T-cellspecific src-like family PTK. One appears to be involved in the regulation of [Ca2/]i increase after CD6 crosslinking, while the other which requires PKC activation and plays a major role in T cell activation. Taken together, the present results demonstrate that one of the earliest biochemical events in CD6/TPA-induced T cell proliferation involves the activation of src family PTK(s), and cell proliferation activated through this pathway proceeds through an IL-2-dependent pathway, which is similar to antigen-specific T cell activation. REFERENCES 1. Jenkins, M. K., and Johnson, J. G., Curr. Opin. Immunol. 5, 361, 1993. 2. Allison, J. P., Curr. Opin. Immunol. 6, 414, 1994. 3. Collins, T. L., Kassner, P. D., Bierer, B. F., and Burakoff, S. T., Curr. Opin. Immunol. 6, 385, 1994. 4. Gangemi, R. M. R., Swack, J. A. Gaviria, D. M., and Romain, P. L., J. Immunol. 143, 2439, 1989.

AP: Cell Immuno

52

OSORIO ET AL.

5. Bott, C. M., Doshi, J. B., Morimoto, C., Romain, P. L., and Fox, D. A. Int. Immunol. 5, 783, 1993. 6. Osorio, L. M., Garcia, C. A., Jondal, M., and Chow, S. C. Cell Immunol. 154, 123, 1994. 7. Reinherz, E. L., Meuer, S., Fitzgerald, K. A., Hussey, R. E., Levine, H., and Schlossman, S. F. Cell 30, 735, 1982. 8. Reinherz, E. L., Geha, R., Rappeport, J. M., Wilson, M., Penta, A. C., Hussey, R. E., Fitzgerald, K. A., Daley, J. F., Levine, H., Rosen, F. S., and Schlossman, S. F., Proc. Natl. Acad. Sci. USA 79, 6047, 1983. 9. Reiter, C., in Leukocyte Typing, IV. White Cell Differentiation Antigens (Knapp, W., Dorken, B., Gilks, W. R., Reiber, E. P., Schmidt, R. E., Stein, H., and Kr von dem Borne, A. E. G., Eds.), p. 339. Oxford Univ. Press, New York, 1989. 10. Kamoun, M., Kadin, M. E., Martin, P. J., Nettleton, J., and Hansen, J. A., J. Immunol. 127, 987, 1981. 11. Endres, N., Riethmuller, G., and Rieber, E. P., in Leukocyte Typing, IV. White Cell Differentiation Antigens (Knapp, W., Dorken, B., Gilks, W. R., Rieber, E. P., Schmidt, R. E., Stein, H., and Kr. von dem Borne, A. E. G, Eds.), p. 340. Oxford Univ. Press, New York, 1989. 12. Aruffo, A., Melnick, M. B., Linsley, P. S., and Seed, B., J. Exp. Med. 174, 949, 1991. 13. Cardenas, L., Carrera, A. C., Yague, E., Pulido, R., SanchezMadrid, F., and de Landazuri, M. O., J. Immunol. 145, 1450, 1990. 14. Swack, J. A., Mier, J. W., Romain, P. L., Hull, S. R., and Rudd, C. E., J. Biol. Chem. 266, 7137, 1991. 15. Swack, J. A., Gangemi, R. M. R., Rudd, C. E., Morimoto, C., Schlossman, S. F., and Romain, P. L., Mol. Immunol. 26, 1037, 1989.

/ m3715$7961

09-22-95 09:30:55

cia

16. Wee, S. F., Shieven, G. L., Kirihara, J. M., Tsu, T. T., Ledbetter, J. A., and Aruffo, A., J. Exp. Med. 177, 219, 1993. 17. Iwashima, M., Irving, B. A., ran Oers, N. S. C., Chan, A. C., and Weiss, A., Science 263, 1136, 1994. 18. Weiss, A., and Littman, D. R., Cell 76, 263, 1994. 19. Rao, A., Crit. Rev. Immunol. 10, 495, 1991. 20. Premack, B. A., and Gardner, P., Am. J. Physiol. 263, (32), C1119, 1992. 21. Rudd, C. E., Janssen, O., Cai, Y. C., da Silva, A. J., Raab, M., and Prasad, K. V. S., Immunol. Today 15, 225, 1994. 22. Garcia, C. A., Interferon Biotecnol. 1, 28, 1984. 23. Garcia, C. A., Barral, A., and Torres, K., Biotec. Aplic. 2, 70, 1991. 24. Chow, S. C., and Jondal, M., J. Biol. Chem. 265, 902, 1990. 25. Treves, S., Di Virgilio, F., Cerundolo, V., Zanovello, P., Collavo, D., and Pozzan, T., J. Exp. Med. 166, 33, 1987. 26. Grynkiewicz, G., Poenie, M., and Tsien, R. Y., J. Biol. Chem. 260, 3440, 1985. 27. Chomczynski, P., and Sacchi, N., Anal. Biochem. 162, 156, 1987. 28. Pisa, E. K., Pisa, P., Hansson, M., and Wigzell, H., Scand. J. Immunol. 36, 745, 1992. 29. Morimoto, C., Rudd, C. E., Letvin, N. L., Hagan, M., and Schlossman, S. F., J. Immunol. 140, 2165, 1988. 30. Uehara, Y., Fukazawa, H., Murakami, Y., and Mizuno, S., Biochem. Biophys. Res. Commun. 163, 803, 1989. 31. June, C. H., Fletcher, M. C., Ledbetter, J. A., Schieven, G. L., Siegel, J. N., Phillips, A. F., and Samelson, L. E., Proc. Natl. Acad. Sci. USA 87, 7722, 1990. 32. Isakov, U., Mally, M. I., Scholz, W., and Altman, A., Immunol. Rev. 95, 89, 1987. 33. Weiss, A., Koretzky, G., Schatzman, R. C., and Kadlecek, T., Proc. Natl. Acad. Sci. USA 88, 5484, 1991.

AP: Cell Immuno