and CD3'q2 isoforms

Proc. Nati. Acad. Sci. USA Vol. 88, pp. 3842-3846, May 1991 Immunology

Differential signal transduction via T-cell receptor CD3'2, and CD3'q2 isoforms

CD3C-,v,

(Ca2+ mobilization/phosphatidylinositol turnover/protein tyrosine kinase/interleukin 2 production)

ANDREA BAUER*t, DAVID J. MCCONKEY*t, FRANK D. HOWARD*t, LINDA K. CLAYTON*t, DAVID NOVICK*, SHIGEO KOYASU*t, AND ELLIS L. REINHERZ*t *Laboratory of Immunobiology, Dana-Farber Cancer Institute, and Departments of tPathology and tMedicine, Harvard Medical School, Boston, MA 02115

Communicated by H. S. Lawrence, January 29, 1991 (received November 20, 1990)

It has been speculated that only TCRs containing a CD3C-21 heterodimer transmit signals inducing phosphatidylinositol (PI) turnover and that such signaling may be linked to activation-induced cell death (14, 15). To directly determine and compare the role of CD3C/CD3i1 structural isoforms in TCR signal transduction pathways, we herein characterized the signaling function of different types of TCR isoforms, derived from transfection of MA5.8, a CD3C-7- variant of the cytochrome c-specific I-Ek-restricted T-cell hybridoma 2B4.11 (16). The transfectants expressed CD3C2, CD3iq2, CD3C2/CD3C-i1, or CD3C2/CD3{-,q/CD3'q2 in association

The T-cell antigen receptor (TCR) consists of ABSTRACT an antigen-binding heterodimer, termed Ti, which is noncovalently associated with the invariant CD3 subunits (y, 8, £, C, and ii). The CD3C and -v, subunits form either homodimeric or heterodimeric structures in turn associated with the other components of the TCR complex. This feature increases the structural complexity of TCRs by creating "isoforms." Both CD3C and -p are thought to play an important role in signal transduction triggered by antigen/major histocompatibility complex. To compare signaling functions of TCR isoforms, MA5.8, a CD3{-,- variant of the cytochrome c-specific, I-Ek-restricted T-cell hybridoma 2B4.11, was stably transfected with cDNAs encoding CD3Wand/or CD3rp, and resulting clones were characterized. The findings indicate that signals inducing Ca21 mobilization, phosphatidylinositol turnover, and interleukin 2 production are each transmitted by the above TCR isoforms. In contrast, tyrosine phosphorylation of the CD3; subunit but not the CD3g subunit follows TCR stimulation. Given the general importance of tyrosine phosphorylation for receptor signaling, it is likely that this difference between TCR isoforms plays a regulatory role in T-lineage function by qualitatively or quantitatively altering signaling events.

with the pentamer Tia-f3CD3y3E TCR complex.

MATERIALS AND METHODS Cells and Antibodies. The mouse T-cell hybridoma 2B4.11, its variant MA5.8 (16), and the B-cell hybridoma LK35.2 were generous gifts of J. Ashwell (National Institutes of Health, Bethesda, MD). The cells were maintained in RPMI 1640 medium/10% fetal calf serum (FCS). The insert of pBS17 (mouse CD3iq) (10) was subcloned into pM2gpt, which drives insert expression with the adenovirus major late promoter and provides mycophenolic acid resistance (M. Concino, personal communication). MA'-vq301 and MA?201 were established by transfection of MAC15.4 (13) with pM2gpt-iq and pM2gptgrown in antisense-iq, respectively, and were continuously(Geneticin, RPMI 1640 medium/10%o FCS/G418 (500 ,g/ml) GIBCO)/mycophenolic acid (0.5 ,ug/ml) (Sigma)/hypoxanthine (15 ,ug/ml) (Sigma)/xanthine (250 ,ug/ml) (Sigma). The hamster-mouse somatic B-cell hybridoma 145.2C11 producing the monoclonal antibody (mAb) 2C11 against mouse CD3E (17) was generously provided by J. A. Bluestone (University of Chicago). mAb A2B4.2 specific for the Tia chain of the 2B4.11 TCR (18) was the generous gift of J. Ashwell. RNA Analysis. Total RNA was isolated from 2B4.11, MA5.8, and transfectants with guanidium isothiocyanate (19). RNA concentrations were measured spectrophotometrically and 10 or 20 ,ug of total RNA as indicated was electrophoresed and transferred to nitrocellulose. The RNA blots were hybridized separately to CD3;- and CD3-r-specific probes prepared by digesting pBS23 (13) with EcoNI and EcoRI and pBS17 with Ava II and EcoRI, respectively. Probes were labeled by the random-priming method (20) and hybridizations were performed as described (13). Blots were then exposed to Kodak X-Omat AR x-ray film at -70°C for

The T-cell receptor (TCR) is a molecular complex composed of at least seven individual subunits (reviewed in refs. 1-4). The five CD3 subunits are involved in signal transduction functions and, unlike the Ti a and f3 antigen/major histocompatibility complex (MHC) binding subunits, are invariant in their primary sequence. CD3y, CD38, and CD3e are structurally related molecules encoded by a set of linked genes on mouse chromosome 9 (5-8). CD3' and CD3i1 represent alternatively spliced products of a single locus on mouse chromosome 1 (9, 10, 33). CD3; is found primarily as a disulfide-linked homodimer within the CD3-Ti complexes, whereas 410% of the CD3; chains are expressed as heterodimers with CD37. Thus, the possibility exists that there are different functional TCR classes, even in the same cell (11, 12). We recently reported the primary structure of murine CD3'q as deduced from protein microsequencing and cDNA cloning (10). CD3vq consists of a 9-amino acid extracellular segment, a 21-amino acid transmembrane segment, and a 155-amino acid cytoplasmic tail. The first 122 amino acids of CD3? and CD3,q were found to be identical, whereas the COOH-terminal regions diverge. Of note, CD3ij lacks a tyrosine residue at position 132 and a putative nucleotide binding site present in CD3C. The potential for CD3,q-iq homodimer formation as part of the TCR was also demonstrated (13).

the indicated times.

Immunoprecipitation and Western Blotting Analysis. Cells were solubilized at 2.5 x 107 cells per ml in TBS (0.8% NaCl/0.02% KCI/25 mM Tris-HCl, pH 7.5) containing 1% Abbreviations: TCR, T-cell receptor; MHC, major histocompatibility complex; mAb, monoclonal antibody; IP, inositol phosphate; PI, phosphatidylinositol; IL-2, interleukin 2; anti-P-Tyr, antiphosphotyrosine mAb; FCS, fetal calf serum; PMSF, phenylmethylsulfonyl fluoride; PTK, protein-tyrosine kinase.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3842

Immunology:

Bauer et al.

Proc. Nati. Acad. Sci. USA 88 (1991)

digitonin (Sigma), 10 mM iodoacetamide, leupeptin (10 ,ug/ ml), antipain (50 gg/ml), 1 mM phenylmethylsulfonyl fluoride (PMSF), soybean trypsin inhibitor (10 gg/ml), aprotinin (10,ug/ml), and pepstatin (1 gg/ml) (all from Sigma). Postnuclear supernatants were incubated with either 50 ,l of 2C11-coupled Sepharose CL-4B (4 mg/ml) or 25 ,l of rabbit anti-CD3;/,q antibody 387 (kindly provided by R. D. Klausner, National Institutes of Health) in the presence of 50 pl of protein A-Sepharose CL-4B. Antigen-antibody complexes were extensively washed and solubilized in 50 ,l of nonreducing Laemmli's sample buffer at 100°C for 3 min (21). Proteins were resolved by one-dimensional nonreducing SDS/PAGE or two-dimensional nonreducing/reducing SDS/PAGE (12.5% acrylamide) and transferred to nitrocellulose (Bio-Rad). The blots were incubated with antibody 387 followed by '25I-labeled protein A (New England Nuclear) as described (12). After extensive washing, blots were exposed for 24 hr at -70°C to Kodak X-Omat AR x-ray film. Calcium Mobilization in the Transfectants. Cytosolic Ca2l concentrations were determined as described (22). Cells (2 x 106) were pelleted and resuspended in 200 ,I of RPMI 1640 medium containing 10o FCS and 1 ,uM acetoxymethyl ester of indo-1 (Molecular Probes). The cells were incubated for 30 min at 37°C and then diluted 1:10 with RPMI 1640 medium containing lo FCS. Analysis was performed with an Epics V cell sorter. Each arbitrary unit represents =100 nM cytosolic Ca2l concentration. Measurement of PI Hydrolysis. [3H]Inositol phosphates (IPs) were separated from the specified cell populations and quantified as described (23, 24). Incorporation of [3H]inositol into phospholipid was achieved by incubating the T-cell hybridomas (107 cells per ml) with [3Hjinositol (40 ,uCi/ml) (37 MBq/ml; New England Nuclear). LK35.2 cells were washed and resuspended at 1 x 107 cells per ml in RPMI 1640 medium containing 10% FCS with 2C11 (50 ,ug/ml) or 100 ,uM cytochrome c peptide (KKANDLIAYLKQATK; ref. 13). After preincubation for 10 min, the labeled hybridomas were added and incubated at 37°C until the indicated time points. The cells were lysed and total IPs (IP1, IP2, and IP3) were separated as described (23, 24). In~mmnoprecipitation of Phosphorylated Proteins. Cells were stimulated by crosslinking the TCR with either anti-CD3E or anti-Ti mAb and species-specific anti-IgG antibodies for 30 min, harvested, and suspended in 0.1 ml of a buffer solution

3843

consisting of 150 mM NaCl, 1 mM orthovanadate, 1 mM PMSF, 10 mM Tris HCl (pH 8.0). Subsequently, cells were lysed in 0.4 ml of 0.5% Triton X-100/10 mM EDTA/10 mM EGTA/1 mM NaF/25 mM Tris HCl, pH 8.0, for 1 hr at 4°C. Immunoprecipitation was performed with 2C11-coupled Sepharose CL-4B (4-5 mg/ml) or anti-phosphotyrosine monoclonal antibody (anti-P-Tyr) 4G10 (25) (kindly provided by B. Druker and T. Roberts, Dana-Farber Cancer Institute, Boston) in the presence of protein A-Sepharose CL-4B. After washing with the lysis buffer solution supplemented with 1 mM orthovanadate and 1 mM PMSF, proteins were eluted by boiling in Laemmli's nonreducing sample buffer solution. Proteins were then resolved by one-dimensional nonreducing SDS/PAGE or two-dimensional nonreducing/reducing SDS/ PAGE (12.5% acrylamide), transferred to nitrocellulose, and blotted with the antibodies indicated. Immunoreactive proteins were visualized by using alkaline phosphatase-coupled second-step reagents (Bio-Rad). Determination of Interleukin 2 (IL-2) Activity. For antigenic stimulation, 3 x 105 cells were incubated in 1 ml of RPMI 1640 medium containing 10% FCS, 50 ,uM 2-mercaptoethanol, and the indicated concentration of cytochrome c peptide in the presence of either I-Ek-expressing B-lymphoma LK35.2 cells or B10.BR splenocytes. For CD3 crosslinking in experiment 2, 2 x 104 cells were stimulated in an Immulon 96-well ELISA plate (Dynatech Laboratories) coated with the indicated amounts of 2C11. After a 24-h incubation, supernatants were collected and IL-2 activities were assayed by the induction of DNA synthesis in an indicator cell line, CTLL-15G, as described (26) (kindly provided by K. A. Smith, Dartmouth Medical School, Dartmouth, NH).

RESULTS AND DISCUSSION MA;15.4 (expressing CD3;2), MA77i61.9 (expressing CD3172), and MA{-7752.9 (expressing CD3?2/CD32-77) were generated by transfection of MA5.8 cells with the expression vector pPink-2 containing the CD3; cDNA, CD377 cDNA, or both constructs, respectively (13). MA;201 (CD3;2) and MA2-77301 (CD3{2/CD3{-,q/CD3?72) were generated by transfection of MA;15.4 cells with the pM2gpt vector containing either the antisense (MA;201) or sense (MA{-Y7301) orientation of the CD3f7 cDNA. As shown in Fig. 1A, MA'-Y7301 and MA201 express levels of TCR 3- to 4-fold greater than 2B4.11. This level is comparable to that of the parental MA;15.4 cells (data

A MA5.8

2B4.11

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FIG. 1. (A) Fluorescence-activated cell sorter analysis of transfectants and parental cell lines. Cells were analyzed for surface TCR expression using the anti-Tia mAb A2B4.2 (18), followed by a fluorescein isothiocyanate conjugated goat anti-mouse antibody. MA;15.4 (13) was transfected with pM2gpt-iq or pM2gpt-antisense-,q to generate MAC-71301 and MA;201, respectively. (B) Expression of CD3C and CD3tq mRNAs in 2B4. 11, MA5.8, and MAC-,q301. Either 10;lg (for CD3,q) or 20 ,g (for CD3J of total RNA isolated from the indicated cells was size fractionated on a 1% agarose gel containing formaldehyde and transferred to nitrocellulose. The filter was hybridized to CD3fl- or CD33-specific probes as shown and exposed for 2 weeks (CD3i1) or 2.5 days (CD33) at -70°C with an intensifying screen. Positions of ribosomal 28S and 18S RNAs are indicated. (C and D) Association of CD3C/CD3,q isoforms with the TCR. Digitonin lysates of the indicated cell lines were immunoprecipitated with either anti-CD3e mAb 2C11 (17) or rabbit anti-CD3C/fq peptide antibody 387 (12) and analyzed by SDS/PAGE followed by immunoblotting with antibody 387. (C) MAC201 and MAC-i13O1 lysates immunoprecipitated with 2C11 and resolved by SDS/PAGE under nonreducing conditions prior to Western blotting. Each lane contains material from 2.5 x 107 cells. (D) MAC-77301 lysate immunoprecipitated by antibody 387 and resolved by two-dimensional nonreducing/reducing SDS/PAGE prior to Western blotting with antibody 387. The gel contains material from 2.5 x 107 cells. Migration positions of prestained molecular weight markers (BRL) are indicated.

Immunology: Bauer et al.

3844

Proc. Natl. Acad. Sci. USA 88 (1991)

not shown). As previously reported (16), 2B4.11 has a 5-fold higher surface TCR copy number than MA5.8. MAC-i1301 expresses CD3i1 as well as CD3C mRNA; the sizes of the transcripts derived from the expression vector are larger than the endogenous 2.0-kilobase (kb) CD3C and 1.7-kb CD3fl transcripts in 2B4.11 (Fig. 1B). As expected, no transcripts for CD3C or CD3iq are present in MA5.8. To analyze TCR-associated CD3C and CD3?q proteins in these transfectants, the TCR complex was immunoprecipitated from digitonin lysates of MAC201 and MAC-v1301 with anti-CD3e mAb (2C11), resolved on nonreducing SDS/ PAGE and analyzed by Western blotting with rabbit antiCD3C/fq antibody 387, which recognizes both CD3C and CD3i1 (12) (Fig. 1C). As shown, three types of dimers were observed in MAC-q301: a CD3C2 homodimer at 32 kDa, a CD3C-,q heterodimer at 37 kDa, and a CD3q2 homodimer at 42 kDa. These results unequivocally demonstrate that three different types of CD3;/CD3iq dimers are associated with the TCR of MAC-q1301 cells. In contrast, MAC201 expresses only CD3C2. The dimeric nature of the three proteins in MAC-v301 is shown by two-dimensional diagonal nonreducing/reducing SDS/PAGE analysis of MAC-q1301 lysates immunoprecipitated and immunoblotted with antibody 387 (Fig. 1D). The amount of CD3iq2 detected in the TCR complex (Fig. 1C) is lower than that present in the cells as defined by antibody 387 (Fig. 1D), suggesting that the association of CD3q2 to the TCR is weaker than that of CD3C2 or CD3C-iq. As reported previously, MA?-iq52.9 cells express CD3C2 and CD3C-,q but, unlike MAC-iq301, do not express CD3q2 (13). The low level of CD3T, mRNA in MAC-i152.9 relative to MAC-v1301 cells seems to account for this difference (data not shown). Thus, the preference for subunit assembly in a stable TCR complex is likely to be CD3;2 > CD3C-i > CD3i72. Of note, transfection of CD3,q into MA015.4 cells, which already express CD3C, does not affect the TCR surface level observed in the resultant transfectant MAC-q301 (Fig. 1A). Signal transduction through TCR stimulation was assessed in transfectants as well as 2B4.11 and MA5.8. Crosslinking of the TCR with anti-Ti mAb A2B4.2 results in Ca2' mobilization in all the transfectants and in the parental 2B4.11 cells. In contrast, no Ca2' mobilization is detected in MA 5.8 (Fig. 2). Similar results were obtained when cells were triggered with the anti-CD3e mAb (data not shown). These results indicate that CD3C and CD3q either alone or in combination are capable of transmitting signals responsible for Ca2' mobilization. While the level of Ca2' flux in MAq61.9 is reproducibly lower than that in the other transfectants, this is likely a consequence of either clonal variability or the lower copy number of surface TCR relative to the other transfec6 2B4'11

MA5.8

MAZ15 4.

tants (13). Furthermore, given that inositol 1,4,5-trisphosphate, a product of PI turnover, is implicated as being responsible for an increase in intracellular free calcium (27), the findings suggested that PI turnover can be stimulated by TCR crosslinking in all transfectants. To directly test this possibility, we measured PI turnover in the same cell populations. As shown in Fig. 3A, upon stimulation with antiCD3e mAb, we readily detected PI turnover in 2B4.11, MAC- j301, MAC201, MAC15.4, and MAq161.9 as measured by an increase in total IPs. In contrast, only a low level of PI turnover was observed in these transfectants when the cells were stimulated with 100 gM cytochrome c peptide fragment in the presence of LK35.2 cells as antigen-presenting cells (Fig. 3B). Analysis of 14 CD3C, 6 CD3fl, and 9 CD3C-iq independent experiments showed no statistically significant difference in the level of PI turnover among individual cell types. As expected, little, if any, PI turnover was detected in MA5.8. The basis of the disparity between antibody and antigen stimulation is unclear. However, PI hydrolysis presumably occurs in the transfectants only after substantial TCR aggregation; such aggregation is achieved by CD3e crosslinking but not by antigen/MHC stimulation. The suggestion of Ashwell and colleagues (14) that PI hydrolysis requires the CD3C-iq heterodimer was based on the observation that CD3C transfectants lacking CD3C!-- heterodimers underwent little, if any, PI turnover upon antigen/ MHC stimulation. However, parallel examination of CD3C2, CD3q2, and CD3C-2q transfectants as shown above reveals low but detectable levels of PI turnover in response to antigen/MHC stimulation in each case. We conclude that there is no significant difference in the capacity of CD3O and CD3vq proteins to transmit signals for PI turnover in MA5.8 transfectants. To next characterize the ability of various cell types to produce IL-2, 2B4.11, MA5.8, and the transfectants were stimulated with cytochrome c peptide and LK35.2 cells or BlO.BR splenocytes as antigen-presenting cells; IL-2 secretion was then quantitated by measuring proliferation of an IL-2-dependent cell line. The data in Table 1 demonstrate that 2B4.11 and all the transfectants produce IL-2 in an

0

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FIG. 2. Calcium mobilization in the transfectants. Cells were stimulated with 20 g1 of anti-Ti mAb A2B4.2 culture supernatant (v) and 40 ,ug of rabbit anti-mouse (v). As a control, 10 t1. of calcium ionophore A23187 (2 mg/ml; Molecular Probes) was added to MA5.8

(G).

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Time (min) FIG. 3. PI turnover upon stimulation in transfectants. PI turnover induced by anti-CD3e mAb crosslinking (A) or antigenic stimulation (B) was measured in various transfectants. The amounts of total labeled IPs at indicated time points are presented as percentage increase over unstimulated control.

Immunology.-

Bauer et A

Proc. Natl. Acad. Sci. USA 88 (1991)

3845

Table 1. IL-2 production of CD3; and CD3i1 transfectants IL-2 production, units per ml per 3 x 105 cells Treatment 2B4.11 MA5.8 MA?15.4 MA{43.2 MAT-,q52.7 MA{-i752.9 MAi161.3 MAq61.9 MA;201 MA;-iy301 Exp. 1 Antigen (AM) O