Proc. Nati. Acad. Sci. USA Vol. 89, pp. 1492-1496, February 1992

Immunology

CD3C dependence of the CD2 pathway of activation in T

lymphocytes and natural killer cells

(sial transduction/acceory moecul/inereukln 2 productlon/Ca2+ mobilization)

PHILIPPE MOINGEON*tt, JEANNE L. LUCICH*, DAVID J. MCCONKEY*t, FRAN4oIS LETOURNEUR§, BERNARD MALISSEN§, JAREMA KOCHANI, HSIU-CHING CHANG*t, HANS-REIMER RODEWALD*t, AND ELLIS L. REINHERZ*11 *Laboratory of Immunobiology, Dana-Farber Cancer Institute and Departments of VPathology and 'Medicine, Harvard Medical School, Boston, MA 02115;

*Centre d'Immunologie, Institut National de la Sante et de la Recherche Medicale-Centre National de la Recherche Scientifique, de Marseille-Luminy, 13288 Marseille Cedex 9, France; and IDepartment of Molecular Genetics, Hoffman-LaRoche, Nutley, NJ 07110

Communicated by Dr. Stephen C. Harrison, November 12, 1991 (received for review August 6, 1991)

ABSTRACT In T lymphocytes, signal transduction through the CD2 adhesion molecule requires surface expression of the CD3-Ti T-cell receptor (TCR) complex. In contrast, in natural killer (NK) cells, CD2 functions in the absence of a TCR. Because the TCR on T lymphocytes and the CD16 low-affinity IgG Fc receptor (Fcy receptor type III) complex on NK cells share a common CD3Csubunit, we reasoned that CD3 may be critical for CD2 sialing in T lymphocytes and NK cels. Here we show that tnsfection of a cDNA encoding a tAnmembrane form ofCD16 into TCR- variants of the Jurkat T-cell line results in CD16 expression in aiaton with endogenous CD3C homodimers and restores CD2 sin function. To test dhdfty the role of CD3Cin CD2 triggering, we then trnected human CD2 into each of two murine T-T hybrdomas that express TCRs containing either a fufl-length CD3 subunit or a truncated CD34 subunit incapable of transducing signals. Despite expression of equivalent surface levels of TCR, CD2-nedlated signaing is seen only in the T cells onin wild-type CD3C. These findings show that (i) CD16 on NK ceis is fuc lly equivalent to the TCR on T lymphocytes for cig CD2 to sgling pathways and (u) CD2 sial t ducin depends upon the CD3 subunit of the TCR complex and, by inference, the CD34 subunit of the CD16 complex.

(14, 15), we also directly analyzed the involvement of CD3C in CD2 triggering. Our findings support an important role for CD3C in CD2 signaling function in both cell types.

MATERIALS AND METHODS Cell Lines and Transfection Experiments. The CD16 cDNA (16-18) consisting of a 5' EcoRI-Pst I fragment derived from clone PBL14 ligated to the 3' Pst I-EcoRI fiagment of clone HL12 (16) was subcloned into the pPink2 vector (19), which contains a neomycin-resistance gene, to create a plasmid termed CD16pPink2. Linearized CD16pPink2 was transfected into 31-13 cells by electroporation (using a cell porator system, BRL with a capacitance set at 330 1F). Transfectants were selected in culture medium (RPMI 1640 medium/10%o fetal calf serum/1 mM sodium pyruvate/2 mM L-glutamine/1% penicillin-streptomycin) supplemented with G418 at 1.5 mg/ml. Neomycin-resistant clones were further selected for CD16 expression by indirect immunofluorescence analysis (4). Production and characterization of murine T-T hybridomas expressing either the wild-type (DC262) or partially deleted CD3C (DC264 series) molecule will be reported in detail elsewhere (F.L. and B.M., unpublished work). Briefly, CD3C- mutants ofthe DOll-10 murine T-T hybridomas were transfected with either a cDNA encoding the wild-type murine CD3C (DC262) or a truncated molecule in which residues 44-131 have been deleted and residues 132-136 have been mutated from Tyr-Asp-Ala-Leu-His to Gln-Ala-CysLys-Leu (DC264). These structural alterations remove the six tyrosine residues as well as a putative nucleotide-binding site present in the cytoplasmic domain (20). For transfection of DC262 and DC264 cells, the human CD2 cDNA was subcloned into the BamHI site ofthe pPink2 vector (19). This construct was transfected into DC262 or DC264 cells by electroporation, and transfectants were selected as above, except that neomycin at 1.0 mg/ml was used. Neomycinresistant clones were further selected for CD2 expression by indirect immunofluorescence analysis. Analysis of Ca2+ mobilization and determination of IL-2 production have been described elsewhere (21, 22). Immunoprecipitation and Immunobot Analysis. Immunoprecipitations were done from cell lysates obtained in 1% Triton X-100 as described (21) using Sepharose beads coupled with an irrelevant (rabbit anti-mouse) antibody and then with Sepharose beads coated with specific antibody [either anti-CD8 (21Thy2D3), anti-CD3 (clone Leu4), or anti-CD16 (clone 3G8)]. For immunoblotting experiments, cells were

The CD2 molecule mediates adhesion and signal transduction function in T lymphocytes, thymocytes, and natural killer (NK) cells (1-3). Previous studies using variants of the Jurkat human T-cell line showed that CD2 is nonfunctional in CD3-Ti- mutants (4-6). CD2 signaling function could be restored by gene transfection that replaced the defective T-cell receptor (TCR) subunit with a functional subunit, thereby reconstituting TCR surface expression (4). The data established a requirement for surface TCR expression to facilitate CD2-mediated signal transduction in those T cells. In contrast, CD2 can transduce activation signals that result in cytosolic Ca2+ rise, initiation ofthe cytotoxic program, and expression of the p55 chain of the interleukin 2 (IL-2) receptor in NK cells, even though NK cells lack CD3-Ti on their surface (7-9). To address the apparent paradox concerning TCR dependency of CD2 function in T cells as opposed to NK cells, we determined whether CD16, the low-affinity IgG Fc receptor known to mediate signaling events in CD2+CD3-Ti- NK cells (10), could replace the TCR expression requirement for CD2-mediated signaling events. In addition, given that CD16 associates with either CD3C and/or Fcv receptor type Iy (FceRly) proteins on the cell membrane (11-13) and that human NK cells and T cells share an identical CD3C subunit

Abbreviations: mAb, monoclonal antibody; NK, natural killer; TCR, T-cell receptor; IL-2, interleukin 2; FceRIy, Fce receptor type Iy; [Ca2+]j, intracellular Ca2' concentration. tTo whom reprint requests should be addressed.

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. 1492

Immunology: Moingeon et al.

Proc. Natl. Acad. Sci. USA 89 (1992)

present study are 5'-CTCGCCTGCATCCTCCACGTGCGG-3' [sense, base pairs (bp) 97-120] and 5'-AGCGAGGGGCCAGGGTCTGC-3' (antisense, bp 565-546) for CD3C (24); the same sense amplimer and 5'-GAAGAATTCGAACTGCACTGCTTGGAAGTGG-3' (antisense, bp 595574) for CD3r1 (25); and 5'-TGATCGCCAGCTCCCAGCGC-3' (sense, bp 1-20) and 5'-GTGAGACTCGAGGATCAGGG-3' (antisense, bp 530-511) for FceRIy (26).

lysed in a buffer containing 1% digitonin, 150 mM NaCi, 10 mM EDTA, 10 mM EGTA, 1 mM NaF, and protease inhibitors at pH 7.2, and CD3 or CD16 was immunoprecipitated as above. Proteins were transferred to Immobilon membranes (Millipore) as described elsewhere (23) before incubation with the anti-CD3C rabbit heteroantiserum 387 (20). Immunoreactive proteins were then visualized by using an alkaline phosphatase-coupled second-step goat anti-rabbit reagent (Bio-Rad). For metabolic labeling of DC264 cells, 6 x 107 cells were incubated for 3 hr with 1.25 mCi (1 Ci = 37 GBq) of [35S]methionine plus [35S]cysteine (Tran35S label, ICN) in 6 ml of methionine and cysteine-free Dulbecco's modified Eagle's medium (GIBCO), washed, and incubated an additional 3 hr with RPMI 1640/10%o fetal calf serum. To immunoprecipitate surface TCRs, cells were incubated for 30 min at 40C with purified 145-2C11 monoclonal antibody (mAb) (20 ,ug/ml, final concentration), washed twice, and lysed before adding protein A-Sepharose beads. Immunoprecipitates were then processed as above. After a 20-min incubation in Enlightning (NEN), the gel was dried and exposed with intensifying screens for up to 2 weeks. PCR Experiments. Total RNA was extracted from 5 x 106 cells using the vanadyl ribonucleoside complex method. One microgram of RNA was used to synthesize single-strand cDNAs with an oligo(dT) synthetic primer and 200 units of Moloney murine leukemia virus reverse transcriptase (BRL). With this material as a template, 35 cycles of amplification were done using a GeneAmp kit (Perkin-Elmer/Cetus) under the following conditions: denaturation at 94°C for 1 min, annealing at either 68°C (CD3C), 65°C (CD3i1), or 60°C (FcERIy) for 1 min, and extension at 72°C for 0.5 min for a total of 35 cycles of amplification. "Amplimers" used in the

RESULTS AND DISCUSSION A cDNA encoding a transmembrane form of CD16 (16) was transfected into the 31-13 TCR- variant of Jurkat cell, which has a defect in Ti f gene expression (4). A TCR+ 31-13 derivative termed WT6 was previously reconstituted with a Ti 13 cDNA by transfection and used as a control. As shown in indirect immunofluorescence assays, transfection of 31-13 cells with the CD16 cDNA construct resulted in the isolation of 10 independent G418-resistant clones expressing CD16. Three of these clones, termed CD16R1, CD16R2, and CD16R3, were extensively characterized (Fig. 1A). As expected, the anti-CD16 mAb was unreactive with 31-13 and WT6 clones. Both anti-CD3 and anti-clonotype mAbs (antiTi; REX9H5) were reactive with WT6 but not with 31-13, CD16R1, CD16R2, or CD16R3. All cell lines expressed comparable levels of the CD2 molecule. To characterize the CD16 protein expressed on the surface of the above cells, immunoprecipitation experiments were conducted by using lysates from surface-iodinated cells (Fig. 1B). SDS/PAGE analysis of anti-CD3 immunoprecipitates reveals the characteristic human 26-kDa CD3y and 20-kDa CD38 and CD3e proteins in lysates from WT6. In contrast, no surface CD3 proteins were detected from 31-13, CD16R1, or CD16R2, confirming the fluorescence-activated cell sorter D

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FIG. 1. Expression of the transmembrane form of CD16 in CD2+CD3-Ti- Jurkat variants restores CD2 signaling. (A) Immunofluorescence of Jurkat mutants transfected with a CD16 cDNA. 31-13 is a CD2+CD3-Ti- mutant of the Jurkat human T-cell line. TCR surface expression has been restored in WT6 cells after transfection of 31-13 with a Ti P cDNA (4). CD16R1, CD16R2, and CD16R3 cells were derived from 31-13 cells by transfection with the cDNA encoding the transmembrane form of CD16. Histograms represent reactivity with different antihuman mAbs (thick line) compared with an equivalent amount of an irrelevant antibody (1HT4 4E5, thin line). First-step antibodies are as follows: anti-CD16 (clone 3G8), anti-CD3 (clone OKT3), anti-Ti (clone REX9H5), and anti-CD2 (clone 3T4-8B5). (B) Immunoprecipitation of CD3 and CD16 molecules from surface-iodinated cells. Molecular mass numbers are as follows: 97 kDa, phosphorylase B; 69 kDa, bovine serum albumin; 46 kDa, ovalbumin; 30 kDa, carbonic anhydrase. Immunoprecipitates were resolved on a 10%o SDS/polyacrylamide gel under reduced conditions. (C) Coprecipitation of CD16 and CD3C. Anti-CD16 or anti-CD3 mAb immunoprecipitates from the indicated cells were size-fractionated on a 10%o SDS/polyacrylamide gel under nonreducing conditions, blotted onto an Immobilon membrane, and probed with anti-human CD3C heteroantisera. (D) Variation of intracellular calcium after CD16, CD3, or CD2 triggering. Human cells were labeled with indo-1 and incubated with either anti-CD16 (clone 3G8), anti-CD3 (clone 2Ad2A2, IgM), or anti-T112 plus anti-T113 (open triangle) using ascites at 1:200. The arrow and the closed triangle correspond, respectively, to the addition of either rabbit anti-mouse (RAM) second-step heteroantisera (10 ,ug/ml, final concentration) or Ca2+ ionophore (1 ,ug/ml). Samples were monitored continuously for up to 12 min on an Epics V cell sorter. Results are expressed as 410/480 nm fluorescence ratio in arbitrary units (ordinate) vs. time in min (abscissa). One arbitrary unit represents 200 nM of Ca2+ increase (as evaluated in quantitative parallel experiments using the quin-2 fluorescent dye) (4).

1494

Proc. Natl. Acad. Sci. USA 89 (1992)

Immunology: Moingeon et al.

(FACS) analysis. Using the anti-CD16 antibody 3G8, a 55kDa band corresponding to the transmembrane form ofCD16 is detected in lysates from a human polyclonal NK cell line (15) and from CD16R1 and CD16R2 cells (and CD16R3 cells, data not shown) but is absent from 31-13. This 55-kDa species is specific for CD16 because it is not present in control immunoprecipitates using the irrelevant anti-CD8 mAb. Although it is known that CD16 must associate either with the CD3C subunit or with the FcERIy subunit to be expressed on the cell surface (11-13), the presence of the latter was excluded in Jurkat cells and related variants by PCR analysis (data not shown). Therefore, we determined whether the transfected human CD16 product associates with the endogenous human CD3C to form a complex on the surface of CD16R1 T-cell transfectants by lysing cells in digitonin and immunoprecipitating with either anti-CD3 or anti-CD16 mAbs followed by nonreducing SDS/PAGE and immunoblotting using a rabbit anti-CD3C heteroantisera. Fig. 1C shows a 32-kDa protein coprecipitating specifically with anti-CD3 but not with anti-CD16 in the CD3+CD16- WT6 cells. A band of identical molecular mass is found in anti-CD16 but not antiCD3 immunoprecipitates in CD3-CD16+ CD16R1 cells, whereas neither of the two mAbs coprecipitate CD3C in CD3-CD16- 31-13 cells. Two-dimensional nonreducing/ reducing gel analysis confirmed that this 32-kDa protein runs as a 16-kDa off-diagonal spot under reducing conditions and is, therefore, a C4 homodimer (data not shown). Having demonstrated that CD16 is surface expressed in association with endogenous CD3C in our transfectants, we then analyzed whether the signaling function of CD2 is restored in these cells. To this end, cells were loaded with the Ca2+ sensitive dye indo-1, and changes in intracellular free Ca2+ ([Ca2+]I) were monitored using an Epics V cytofluorometer after stimulation with mAbs specific for CD3 (2Ad2), CD2 (anti-T112 plus anti-T113 mAbs), or CD16 (3G8). As shown in Fig. 1D, none of these antibodies activated 31-13 cells, consistent with our earlier observation that the CD2 pathway of activation is not functional in the absence of TCR surface expression (4). However, a marked increase in cytosolic free Ca2+ is seen after addition of the Ca2' ionophore A23187, establishing that 31-13 cells were loaded with indo-1. The CD3-Ti+ WT6 cells can be stimulated via CD3-Ti or CD2 to increase [Ca2+]1, whereas an anti-CD16 mAb with or without addition of a second-step rabbit anti-mouse antibody as a crosslinking agent is without effect. In contrast, incubation of CD16R1, CD16R2, or CD16R3 cells with an antiCD16 mAb reproducibly induced a low, but detectable, Ca2+ increase in -10-20o of the cells, whereas further crosslinking of CD16 molecules results in a large Ca2+ flux in virtually all cells of each CD16 transfectant population. This finding suggests that the protein encoded by the transfected CD16 cDNA couples to the signal-transduction machinery in these cells. As expected, anti-CD3 mAb has no effect on [Ca2+]i in the CD3-CD16+ cells. More importantly, in the presence of the stimulatory combination of CD2-specific mAbs, a readily detectable Ca2+ flux is induced in virtually all cells of the CD16R1, CD16R2, and CD16R3 clones. These results show that the CD2 pathway of activation is functional in these transfectants with respect to regulated changes in [Ca2lji. Although not shown, we were unable to obtain a rise in [Ca2W+i with F(ab')2 fragments of anti-T112 plus anti-T113. These results are consistent with other studies of NK cells in which engagement of both CD16 and CD2 is required to produce a detectable rise in [Ca2+]j (8). The presence of CD16 in TCR- Jurkat variants also restores the ability of the above anti-CD2 mAbs to trigger IL-2 production (Table 1). We note that F(ab')2 fragments of the anti-CD2 mAbs clearly stimulate IL-2 production, indicating that with regard to lymphokine activation, CD2 signaling is independent, at least in part, of the ectodomain of CD16. It

remains to be determined whether dfflerent activation requirements and/or different detection sensitivities of the two assays can account for the CD16 dependence of CD2mediated Ca2' mobilization, as opposed to the CD16 independence of CD2-mediated IL-2 production. Given that Fcy receptor type III and TCR complexes share the CD3C subunit (14, 15), CD2 signaling function might be linked to CD3C. To test this possibility, CD3C- variants ofthe DOll-10 murine T-T hybridoma have been serially transfected with cDNAs encoding either the wild-type murine CD3C molecule or a truncated version of the CD3C molecule and subsequently human CD2. The truncated as well as full-length CD3C associates with the Ti a-. CD3y8E complex and reconstitutes TCR surface expression (see Fig. 2C). Although the truncation of CD3C (lacking cytoplasmic amino acid residues 44-131) was recently shown to eliminate the transduction of signals generated by antigenic stimulation, it does not affect signals elicited by crosslinking of the TCR using the 145-2C11 anti-murine CD3e mAb (20). Initially, the CD3F- variant of DO11-10 was transfected with full-length or truncated CD3C to create DC262 and DC264 cells, respectively. To characterize the CD3C components associated with the TCRs expressed on the surface of these transfectants, cells were surface-iodinated and lysed in 1% digitonin; lysates were then immunoprecipitated with mAb 145-2C11 coupled to Sepharose beads. The proteins in immunoprecipitates were resolved in two-dimensional nonreducing/reducing 15% SDS/polyacrylamide gels. Fig. 2A shows that a 16-kDa off-diagonal spot with the characteristic mobility of CD3C (24) is found in immunoprecipitates obtained from DC262 but not from DC264 cells. The truncated CD3C protein was not visualized in DC264 after lactoperoxidase catalyzed iodination despite intentional overexposure of the autoradiogram. However, SDS/PAGE analysis of surface TCR complexes metabolically labeled with [35S]cysteine plus [35S]methionine revealed the presence of an off-diagonal spot of -8 kDa in DC264 cells (Fig. 2A Inset) consistent with it being the receptor-associated truncated CD3C protein. Although the other TCR components are not Table 1. CD16 transfection restores CD2-triggered IL-2 production in TCR- T-cell variants IL-2 production, units/ml

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