Proc. Nati. Acad. Sci. USAVol. 87, pp. 2339-2343, March 1990Immunology

A signaling role for the cytoplasmic segment of the CD8 a chaindetected under limiting stimulatory conditions

(T-cell recognition/Ly-2,3 molecule/fluorescence photobleaching/p561d tyrosine kinase/transmembrane segment)

FRAN(OIS LETOURNEUR, JEAN GABERT, PIERRE COSSON, DOMINIQUE BLANC, JEAN DAVOUST,AND BERNARD MALISSENCentre d'Immunologie, Institut National de la Sante et de la Recherche Mddicale-Centre National de la Recherche Scientifique de Marseille Luminy, Case906, 13288 Marseille Cedex 9, France

Communicated by J. F. A. P. Miller, December 26, 1989 (receivedfor review September 11, 1989)

ABSTRACT To test for the functional importance of thecytoplasmic segment of the CD8 molecule, a mouse T-cellhybridoma expressing a T-cell receptor specific for the class Imajor histocompatibility complex product H-2Kb was trans-fected with a set of CD8 a-chain (Ly-2) and/or #-chain (Ly-3)genes encoding polypeptides with carboxyl-terminal trunca-tions or substitutions. When challenged with Kb-positive sple-nocytes, transfectants expressing Ly-2 homodimers that lackedcytoplasmic tails responded nearly as effectively as wild-typeLy-2 transfectants. However in marked contrast to the wild-type Ly-2 transfectants, tailless Ly-2 transfectants were greatlyimpaired in their ability to respond to Kb-transfected L cells.Coexpression of the Ly-3 gene did not restore this impairedresponse. The unique functional property of the Ly-2 a cyto-plasmic segment was further supported by the analysis of achimeric Ly-3 subunit in which the cytoplasmic segment wasreplaced by the one from the Ly-2 a subunit. When associatedwith a soluble Ly-2 subunit lacking a transmembrane segment,the chimeric Ly-3 was indeed sufficient to restore the responseto Kb-transfected L cells. Since the lateral mobility of thetailless Ly-2 molecules on the cell surface was nearly identicalto that ofthe wild-type Ly-2 molecules, their partially impairedfunction may indicate that they have lost their cis-actingsignaling properties but retained their ability to bind class Iproducts of the major histocompatibility complex.

The CD8 molecule is a disulfide-linked af3 heterodimerexpressed almost exclusively on mature T lymphocytes spe-cific for class I major histocompatibility complex (MHC)products. CD8 has been postulated to contribute to thestabilization of T cell-target cell interactions through therecognition of monomorphic sites expressed on target cellclass I MHC molecules. Strong support for this model hasbeen derived from the studies of Salter et al. (1), whichdemonstrated in a cell binding assay that the human CD8 achain can specifically bind to the a3 domain of class I MHCmolecules in the absence of T-cell antigen receptor (TCR).Moreover, the observation that T-cell clones vary in theirsusceptibility to inhibition by anti-CD8 antibodies has under-lined the fact that the CD8 function is a conditional one,required only when either antigen-MHC complexes becomelimiting on target cells or the affinity of a given TCR isinsufficiently strong to result in efficient triggering (2).More recently, it has been proposed that duringMHC class

I recognition, the CD8 molecule and the TCR-CD3 complexare brought into close contact through their respective abilityto recognize different sites of the same MHC class I mole-cule. As a consequence, they synergize so that signaling bythe resulting TCR-CD3-CD8 complexes is far more efficient

for T-cell activation than the signaling mediated by theTCR-CD3 complexes alone (3, 4). In further experiments, thepossibility of a role for CD8 in signal-transduction mecha-nisms has been reinforced by the demonstration of its asso-ciation with the intracellular p56Ick tyrosine protein kinase (5,6). For instance, once juxtaposed with the TCR-CD3 poly-peptides, the CD8-p56Ick complexes can phosphorylate cel-lular substrates (including the TCR-CD3 ; polypeptide) andthus positively modify, in an as yet undefined way, thesignaling properties of the TCR-CD3 complexes.To ask which structural features of the CD8 molecule

contribute to its putative trans-acting (binding) and cis-acting(signaling) functions, we have devised a gene-transfer systemin which the TCR a and 1 genes isolated from a CD8+cytotoxic clone denoted KB5-C20 and specific for the classIMHC molecule H-2Kb are introduced into and expressed bythe DO-11.10 T-cell hybridoma. In this system, transfectionof the KB5-C20 TCR genes alone failed to confer anyreactivity for Kb even though the DO-11.10 transfectantsexpressed the appropriate TCR and could respond to stim-ulation by either of two anti-clonotypic antibodies (Desire-1and B20.2) specific for the KB5-C20 TCR. However, oncesupertransfected with the CD8 a (Ly-2) gene, the primarytransfectants acquired the ability to respond to Kb (7).Comparison of the mouse CD8 a protein sequence with thehomologous rat and human sequences indicates that thetransmembrane and cytoplasmic segments are the mosthighly conserved between species (8) and suggests that theymay exert a cis-acting signaling role. To test for the functionalimportance of these conserved segments, DO-11.10 cellsexpressing the KB5-C20 TCR have been supertransfectedwith chimeric CD8 a and/or P genes that encode polypep-tides with truncation or substitution of their carboxyl-terminal ends.

MATERIALS AND METHODSPlasmid Constructions. Plasmids Ly-2/Ad, Ly-2/Kk, and

Ly-2ATM (Fig. LA) were obtained by exon-shuffling, usingthe Ad (9), Ly-2.2 (7, 10), and Kk (11) genes. A plasmidcontaining the entire Ly-3.2 gene was constructed by insert-ing a 15.8-kilobase (kb) BamHI fragment from cosmid 18.W7(13) into the single BamHI site of plasmid pSV2-his (14).Finally, plasmid Ly-3/Ly-2CY was constructed from theBamHI/Sal I-opened plasmid pSV2-his(EcoRI)SalI by inser-tion of a 13.2-kb BamHI-Xho I fragment isolated from theLy-3.2 gene (13) together with the 3.8-kb Xba I-Sal I frag-ment containing exons 4 and 5 of the Ly-2.2 gene (10).

Transfection by Protoplast Fusion. Transfections of hybrid-omas with protoplasts were as described (7).

Abbreviations: MHC, major histocompatibility complex; TCR, T-cell antigen receptor; IL-2, interleukin 2.

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

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A150 160 170 180 190 200 210 220

TRANSFECTANT MP TM CYX M i

DC236 Ly-2 a DCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHRSRKIVCPRPLVRQEGKPRPSEXIV( Ly-2 ' ----- SR

DC237 Ly-2 /Apd - -----RLL

TM CY

DC238 Ly-2/Kk ----EPPPSTVST'I IAVLVVLGAAIVTGAVVAFVMKNRRTGKGGDYALAPGSQTSDLSLPDCiADC253 Ly-2aT1M -----G --

B 1 2 3a b a b a b

Ucx-

C1 2

-39

-39

FIG. 1. CD8 a constructs. (A) Predicted structures of the Ly-2 polypeptides with carboxyl-terminal deletions and/or substitutions. Thesequence of the membrane-proximal region (MP), transmembrane region (TM), and cytoplasmic tail (CY) of the Ly-2 a polypeptide is shownin the single-letter amino acid code. The sequences of the Ly-2 a' polypeptide (12) and of the various modified Ly-2 constructs are indicatedunder the Ly-2 a sequence. Amino acid identities with the latter are indicated with dashes. The Ly-2 residues are numbered according to ref.10. The predicted amino acid sequence encoded by exon 6 of A$ is from ref. 9, whereas the one encoded by exons 5-8 of Kk is from ref. 11.The names of the cell lines transfected with each plasmid are also indicated. (B) SDS/PAGE analysis of cell surface Ly-2 molecules. 125I-labeledDC179.1 cells transfected with the wild-type Ly-2 gene (DC236.8; lanes 1), the Ly-2/Kk gene (DC238.1; lanes 2), or the Ly-2/A$ gene (DC237.16;lanes 3) were immunoprecipitated with an anti-mouse IgG3 (lanes a) or an anti-Ly-2 (lanes b) antibody. (C) SDS/PAGE analysis of secretedLy-2 molecules. Secreted proteins from [35S]methionine-labeled DC253.2 (Ly-2ATM) cells were immunoprecipitated with an anti-mouse IgG3(lane 1) or an anti-Ly-2 (lane 2) antibody. Position of a molecular weight standard (Mr 39,000) is indicated, as are the bands corresponding tothe wild-type Ly-2 a and a' polypeptides.

Labeling, Immunoprecipitation, and SDS/PAGE. Analysisof CD8 proteins was performed as described (15).Monoclonal Antibodies and Cytofluorometric Analysis. The

various monoclonal antibodies used in this study have beendescribed (7, 13).H-2KbInduced Activation of Transfectants. In brief, 0.2-ml

microcultures were prepared containing 105 responding cellsand either 105 Kb-transfected L cells or 106 Kb-positiveB10.MBR splenocytes. These two different ratios of re-sponder to stimulator cells have been defined as the onesresulting in optimal stimulatory conditions (data not shown).Cultures were incubated for 24 hr, at which time the super-natants were harvested and assayed for interleukin 2 (IL-2)(16).

Determination of the Lateral Mobility of CD8 Molecules onSurfaces of Transfected T Cells. T hybridomas were culturedon glass coverslips and subjected to photobleaching as de-scribed (17).

RESULTSExpression, Function, and Lateral Diffusion of Ly-2 Poly-

peptides with Carboxyl-Terminal Modifications. Two chi-meric constructs derived from the Ly-2 gene were obtainedby exon-shuffling. As shown in Fig. 1A, the construct de-noted Ly-2/Ad should direct the synthesis of a "tailless"polypeptide possessing the extracellular and transmembranedomains of Ly-2 followed by the last four amino acids fromthe cytoplasmic domain of the Ad class II MHC product. TheLy-2/Ag mutant is intended to mimic the naturally occurringtailless Ly-2 a' form (see Fig. LA). On the other hand, theconstruct denoted Ly-2/Kk is expected to direct the synthesisof a polypeptide in which the extracellular domain of Ly-2 isfollowed by the transmembrane and cytoplasmic regions

derived from the Kk class I MHC molecule (Fig. IA). Thewild-type Ly-2 a gene (denoted Ly-2 a,a') and the twochimeric constructs were individually transfected into theDC179.1 T-cell line. DC179.1 is a derivative of the mouse Thybridoma DO-11.10 that has been transfected with plasmidpCA195 (7) and consequently expresses the KB5-C20 TCR(Fig. 2A). To determine whether the two Ly-2 chimericconstructs could be expressed at the cell surface, the corre-sponding transfectants were analyzed by cytofluorometryusing an anti-Ly-2 antibody. Both Ly-2/Ap (DC237.16) andLy-2/Kk (DC238.1) transfectants displayed levels of stainingsimilar to that observed with the wild-type Ly-2 transfectant(DC236.8). Furthermore, that the two chimeric constructsreacted intensely with three additional anti-Ly-2 antibodies(19/178, H35-27.9, and H59-101.7; data not shown) suggestedthat the overall conformation of the extracellular domain hadnot been grossly affected by the carboxyl-terminal modifi-cations. To establish whether the apparent molecular weightsof the mutated Ly-2 molecules corresponded to the onespredicted by the sequences shown in Fig. LA, surface-labeledLy-2 molecules were analyzed by SDS/PAGE under reduc-ing conditions. Analysis ofthe wild-type Ly-2 molecules (Fig.1B, lane lb) revealed both the a and the a' form of the Ly-2polypeptide. As expected, immunoprecipitates from trans-fectants that received the Ly-2/Ad gene displayed a singleband, which migrated at a position identical to that of theLy-2 a' form (lane 3b). Analysis of the Ly-2/Kk gene prod-ucts (lane 2b) revealed a single band, which migrated at aposition slightly above the Ly-2 a form observed in thewild-type Ly-2 transfectant. Both wild-type and modifiedLy-2 molecules were expressed as disulfide-bonded ho-modimers (data not shown). Further support for the predictedcarboxyl-terminal structures shown in Fig. 1A has been

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Proc. Natl. Acad. Sci. USA 87 (1990) 2341

I1gG3 TCR IgG3

Ly-3 Ly-2 Ly3Ly-2

E DC259 F DC253.2

IgG3 Ig03TCR Ly-2 TCR

Ly3 L y-3

G DC257.1 H DC277.6

EgG3 Ly-3 IgG3 Ly-2

TCR Ly-2 Ly-3

Relative fluorescence intensity (log scale)

FIG. 2. Flow cytometry analysis of the DC179.1 cells transfectedwith various CD8 a (Ly-2) and/or (Ly-3) gene constructs afterstaining with an anti-Ly-2 antibody (H58-55.3), an anti-Ly-3 antibody(H35-17.2), or an anticlonotypic antibody specific for the TCRexpressed on KB5-C20 cells (B20.2). The nature of the CD8 gene(s)expressed by each transfectant is as indicated in Table 1. Eachfluorescence histogram is compared with a negative control histo-gram obtained after staining with an anti-mouse IgG3 antibody(H139-69.2. 1).

obtained from the comparison oftheir phosphorylation status(18).To determine the functional effects of the carboxyl-

terminal modifications, clones originating from the Ly-2wild-type (DC236), Ly-2/Ad (DC237), and Ly-2/Kk (DC238)transfections were analyzed in parallel for the relativeamount of Ly-2 expressed at their surface as well as for theirability to respond to stimulation with Kb molecules expressedeither on splenocytes or on transfected L cells. As previouslyexemplified (7), transfectants expressing wild-type Ly-2 mol-ecules readily responded to Kb-positive cells (Fig. 3 A and B).Furthermore, each of them reacted more vigorously to Kb_positive splenocytes than to Kb-transfected L cells. In con-trast, the transfectants expressing tailless Ly-2 moleculesshowed a marked dichotomy in their ability to respond to Kbexpressed on either splenocytes or L cells. For instance,when compared to wild-type Ly-2 transfectants, the Ly-2/A,transfectants were not markedly diminished in their ability torespond to Kb-bearing splenocytes (compare A and C in Fig.3). On the other hand, in spite of the expression of a range ofLy-2 levels similar to that observed on the wild-type Ly-2transfectants, the bulk of the tailless Ly-2/Ad transfectantsdid not respond to Kb-transfected L cells (Fig. 3D). In fact,the few Ly-2/A -expressing clones able to give a weak butsignificant IL-2 response were the ones bearing the highestlevels of Ly-2/Ad homodimers. Finally, the Ly-2/Kk con-structs showed an even lower ability to synergize with theKB5-C20 TCR. For instance, when expressed at nearlyequivalent levels and analyzed against Kb splenocytes, theLy-2/Kk transfectants appeared to be 400 times less effec-tive than wild-type Ly-2 transfectants (Fig. 3 A and E). It

104.

102

10

1

'U-

- 103

0 102

1-

Kb splenocytes

A 19

Is~ 13a

415 0 °o1 16

a 0 5

l ,12 °6

11

104 XE103

1020

X2 9o a

1 6- I

0 100 200Ly-2,

Kb fibroblasts

B

5 13

Is9 * is I

a0 a224 %

1

D

14 1I: 16

F

.I

300 400 0 100 200mean fluorescence intensity

DC-236

DC-237

DC-238

300 400

FIG. 3. IL-2 production ofDC179.1 cells transfected with variousCD8 a (Ly-2) gene constructs in response to stimulation by Kb-positive cells. Fourteen clones expressing Ly-2 a and a' polypeptides(DC236), 17 clones expressing Ly-2/Apd polypeptides (DC237), and6 clones expressing Ly-2/Kk polypeptides (DC238) were individuallyexamined for the production of IL-2 in response to stimulation withKb expressed at the surface of either B10.MBR splenocytes (r) orKb-transfected L cells (w). The corresponding results are expressedas units of IL-2 per ml of undiluted supernatant. On the same day,the various transfectants were analyzed by flow cytometry to de-termine the relative amount of Ly-2 expressed at their surface.Cytofluorometric data are presented as mean fluorescence intensityat equivalent linear amplifier gain. In each panel, the amount of IL-2produced by individual transfectants is plotted as a function of thecorresponding level ofcell surface Ly-2 molecules. The identificationnumber of each cloned transfectant is given next to the correspond-ing point. Data shown are representative of three experiments.

should be noted that all the above transfectants expressednearly equivalent levels of KB5-C20 TCR and LFA-1 mole-cules (Table 1 and data not shown), and showed comparablelevels of IL-2 production in response to the anti-clonotypicantibody Desire-1 (data not shown).An important step in T-cell recognition/activation events

involves the redistribution of accessory molecules (includingCD8) to the areas where T cells specifically contact theantigen-bearing cells (19). Therefore, to analyze whether theimpaired function of the Ly-2 carboxyl-terminal mutantscorresponded to modification or restriction in their lateralmobility, the various Ly-2 transfectants were subjected tophotobleaching analysis. Both the Ly-2/Ad and the Ly-2/Kkmolecules displayed a lateral mobility nearly identical to thatof the wild-type Ly-2 molecules (Table 2).The Impaired Function of the Modified Ly-2 Molecules Is

Not Rescued by Ly-3 Expression. As shown in the aboveexperiments, Ly-3 is not required for the functional expres-sion of Ly-2. However, the Ly-2 homodimers may presentfunctional differences as compared to the Ly-2,3 het-erodimers. To investigate this point, we analyzed whether thepresence ofLy-3 would enhance the function ofthe wild-typeLy-2 subunit or complement the impaired function of theLy-2/Ad and Ly-2/Kk polypeptides. Each of the resultingdouble transfectants expressed high levels of Ly-2,3 het-erodimers. When challenged with Kb-splenocytes, they allproduced 2-3 times more IL-2 than their matched Ly-

C1 3

5 19 0 0 146 0° na 010 0 12 016

g7 0°oo 0 13 0 0

20

6o1

Immunology: Letourneur et al.

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Table 1. Antigen-specific IL-2 production and surface phenotype of DC179.1 cells transfected with various CD8 a and/orf3 gene constructs

IL-2 secreted in responseTransfected responder cells to Kb cells, units/ml Relative fluorescence intensity

Parental Transfected B10.MBR Kb L Mouse KB5-C20Exp. Name cells CD8 gene splenocytes cells IgG3 TCR Ly-2 Ly-3

1 DC179.1* None <1 <1 3 65 4 3DC236.8 DC179.1 Ly-2 a,a' 76 17 3 23 153 2DC237.16 DC179.1 Ly-2/Ag 19 2 3 29 155 3DC238.1 DC179.1 Ly-2/Kk 2 <1 3 47 149 3DC257.1 DC236.8 Ly-3 208 89 4 62 300 342DC266.11 DC237.16 Ly-3 49 3 3 22 441 525DC267.1 DC238.1 Ly-3 5 <1 3 37 220 224

2 DC259 DC179.1 Ly-3 <1 <1 3 27 3 4DC253.2 DC179.1 Ly-2ATM <1 <1 3 27 3 4DC277.6 DC253.2 Ly-3 25 <1 2.5 7 131 212DC278.7 DC253.2 Ly-3/Ly-2CY 52 8 3 8 118 222

Transfected cells were assayed for IL-2 production in response to Kb-positive cells. On the same day, the various transfectants were analyzedby flow cytometry after staining with antibodies specific for mouse IgG3 (H139-69.2.1), the KB5-C20 TCR (B20.2), Ly-2 (H58-55.3), or Ly-3(H35-17.2). All cells showed similar bright staining with the anti-LFA-1 antibody H35-89.9 (data not shown).*A derivative of mouse hybridoma DO-11.10 that expresses the transfected KB5-C20 TCR genes.

2-expressing parents (Table 1). However, these increasedresponses may not be solely due to the presence of Ly-3,since the levels of Ly-2 polypeptides were correlativelyaugmented in the double transfectants. Regardless of thatlimitation, in no instance was the presence of Ly-3 able torestore the ability ofthe Ly-2/Ad or Ly-2/Kk transfectants torespond to Kbtransfected L cells (Table 1).The Ly-2 a Cytoplasmic Tail Is Endowed with Unique

Functional Properties. To further substantiate the putativesignaling role of the Ly-2 a cytoplasmic tail, an experimentwas set up based on the following grounds. First, deletion ofexon 3 of the Ly-2 gene directed the synthesis of a secretedLy-2 polypeptide that lacked the transmembrane region(Ly-2ATM; Fig. 1 A and C) and that appeared functionallysilent (DC253.2; Table 1). Second, transfection of the Ly-3gene alone did not result in serologically (DC259; Fig. 2E) orfunctionally (Table 1) detectable surface expression of Ly-3polypeptides. Third, the concomitant expression of the Ly-2ATM and Ly-3 genes rescued the expression of both prod-ucts at the cell surface and resulted in high levels offunctionalLy-2ATM-Ly-3 heterodimers (DC277.6; Table 1 and Fig.2H). Fourth, coexpression ofLy-2ATM with a chimeric Ly-3gene deiqoted Ly-3/Ly-2CY, in which the exons coding forthe cytoplasmic tail were replaced by the corresponding onesfrom the Ly-2 a gene, resulted in the surface expression ofhigh levels of functional Ly-2ATM-Ly-3/Ly-2CY het-erodimers (DC278.7; Table 1). Both heterodimers were dis-ulfide-linked (data not shown). Given these findings, wedetermined whether the ectopic localization of the Ly-2 acytoplasmic segment in the Ly-2ATM-Ly-3/Ly-2CY mole-

Table 2. Lateral mobility of CD8 on surfaces of transfectedT cells

Diffusion Mobilecoefficient x 1011, fraction,

Cells cm2/sec %DC236.16 (Ly-2 wild-type) 6.88 ± 0.55 83 ± 4DC237.16 (Ly-2/AA) 6.82 ± 0.09 80 ± 3DC238.1 (Ly-2/Kk) 8.55 ± 0.83 82 ± 4DC257.1 (Ly-2,3) 6.76 ± 0.22 83 ± 4

Transfected cells plated on coverslips were labeled for 30 min at4°C with an F(ab')2 fragment of a rhodamine-conjugated anti-Ly-2monoclonal antibody and subjected to fringe-pattern fluorescencephotobleaching at 200C as described (17). Data are expressed as themean ± SD. From 9 to 12 individual measurements were made foreach transfectant.

cules resulted in a functional increment relative to the Ly-2ATM-Ly-3 heterodimers. To this end, clones originatingfrom the DC277 (Ly-2ATM-Ly-3) and DC278 (Ly-2ATM-Ly-3/Ly-2CY) transfections were analyzed in parallel for thelevels ofLy-2,3 molecules they expressed and for their abilityto respond to Kb. Each of the DC277 and DC278 clonesreacted to Kb-splenocytes (Fig. 4 A and C) in spite of thepresence of an identical low level of KB5-C20 TCR on all ofthe analyzed clones (Table 1). However, when matched forequivalent levels of CD8, cells expressing the Ly-2ATM-Ly-3/Ly-2CY molecules responded much better.Such a functional increment was particularly apparent whenKb-transfected L cells were used as stimulators. In the latterinstance, the Ly-2ATM-Ly-3/Ly-2CY heterodimers werethe only ones capable of restoring a significant response (Fig.4 B and D).

DISCUSSIONTransfection of the wild-type Ly-2 gene resulted in theexpression of both a and a' forms and restored the ability ofDC179.1 cells to respond to Kb-positive cells. That such aresponse was not due to a cooperation occurring between thea and a' forms and essentially mediated by the former hasbeen substantiated by the fact that DC179.1 cells expressinga full-length Ly-2 a cDNA displayed a pattern of responsive-ness similar to the one observed with the Ly-2 a,a' trans-fectants (ref. 12 and unpublished data). By contrast, themajority of the a'-like, Ly-2/AP transfectants no longerresponded to Kb-transfected L cells. However, when chal-lenged with Kb splenocytes, the Ly-2/Ad transfectants werefound to respond nearly as well as wild-type Ly-2 transfec-tants. Therefore, the conditions of stimulation reached withKb-transfected L cells appear operationally more limiting,perhaps because the LFA-1/ICAM-1 adhesion pathway isnot implicated in T cell-L cell interactions (20). Our data arereminiscent of those obtained with a deletion mutant of thehuman CD4 molecule that lacked a cytoplasmic tail (21). Thetruncated CD4 molecule was found to be as efficient as theintact CD4 molecule at enhancing the responsiveness toantigen-bearing cells. However, transfectants expressing themutant CD4 molecule were slightly less efficient than thewild-type CD4 transfectants when challenged with antigenincorporated into liposomes. In both instances, the functionalimpairment resulting from the truncation of the cytoplasmictail is not an absolute one, since it may be partially relievedby either expressing high levels of Ly-2/Al homodimers (see

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1000

100'

10

0 <1._

C*; 1000

100,

10 a

Kb splenocytes

)

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DC277

80 100 120 140 60 80 100 120 140

80 100

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Ly-2, mean fluorescence intensity100 120

DC278

140

FIG. 4. IL-2 production of DC253.2 cells transfected with CD8 P (Ly-3) gene constructs in response to stimulation by Kb expressed at thesurface of either B10.MBR splenocytes (c) or Kb-transfected L cells (m). Ten clones expressing Ly-2ATM-Ly-3 heterodimers (DC277) and 10clones expressing Ly-2ATM-Ly-3/Ly-2CY heterodimers (DC278) were individually analyzed as described in the legend of Fig. 3.

clones 12, 14, and 16 in Fig. 3D) or increasing the surfacelevels of the complemented TCR (21).

Since the Ly-2 a cytoplasmic segment is the only CD8structural component capable ofbinding the p561ck kinase (12)and no measurable change in lateral mobility was observedfor the Ly-2/Ad homodimers, the partially impaired functionof the latter molecules may indicate that they have lost theirp56lck-mediated signaling properties but retained their MHCclass I binding function. However, it is presently impossibleto exclude a converse interpretation in which the CD8 a

cytoplasmic tail constitutes a unique target for intracellularsecond messengers resulting from TCR engagement andleading to an increase in CD8 avidity (see ref. 22).

In contrast to the situation observed with the Ly-2/Addimers, expression of the Ly-2/Kk molecules resulted in anearly complete lack of responsiveness against Kb spleno-cytes. Although it does not appear to modify the lateralmobility of the Ly-2/Kk dimers, the presence of the Kktransmembrane segment may negatively affect the ability ofCD8 to contact the TCR-CD3 complex and subsequentlyrecognize the same class I MHC molecule as the TCR.

Finally, our data bring into question the exact role of theLy-3 chain, since the coexpression of both Ly-2 and Ly-3subunits did not result in a dramatic increase of the respon-siveness to Kb.

We thank P. Golstein and R. Zamoyska for discussions. This workwas supported by grants from the Centre National de la RechercheScientifique, the Institut National de la Sante et de la RechercheMedicale, and the Association pour le Developpement de la Recher-che sur le Cancer.

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