Proc. Nati. Acad. Sci. USAVol. 89, pp. 8706-8710, September 1992Immunology

Defective signal transduction by the CD2 molecule in immatureT-cell receptor/CD3- thymocytesLAURENCE A. TURKA*t, MARY C. FLETCHERt, NANCY CRAIGHEADf, CRAIG B. THOMPSON*§1,AND CARL H. JUNEtDepartments of *Medicine and §Microbiology/Immunology, and the lHoward Hughes Medical Institute, University of Michigan, Ann Arbor, MI 48109; andtlmmune Cell Biology Program, Naval Medical Research Institute, Bethesda, MD 20889

Communicated by J. Lawrence Oncley, May 26, 1992 (receivedfor review January 17, 1992)

ABSTRACT The CD2 accessory molecule mediates anactivation pathway in mature T cells, transducing sialssimilar to those observed following stimulation of the T-cellreceptor/CD3 (TCR/CD3) complex. CD2 is also one of theearliest cell surface markers to appear during thymic ontogenyand has been proposed to be a stimulatory pathway forimmature thymocytes that have not yet expressed TCRs ontheir surface (TCR/CD3-). To examine this hypothesis highlypurified TCR/CD3- human thymocytes were stimulated usingmitogenic combination of antl-CD2 monoclonal antibodies orindividual biotinylated anti-CD2 monoclonal antibodiescrosslinked with avidin. TCR/CD3+ thymocytes respondedreadily to either stimulus as determined by anti-phosphoty-rosine immunoblotting, and the pattern of tyrosine phosphor-ylated substrates was similar to that of mature T cells. Incontrast, TCR/CD3- thymocytes responded weakly and witha distinct substrate pattern. In addition, the altered signaltransduced by CD2 in TCR/CD3- thymocytes did not lead toa rise in intracellular calcium, failed to induce interleukin 2receptor expression, and did not serve as a comitogen withphorbol ester or interleukin 2, functions that were all intact inTCR/CD3+ thymocytes. Failure ofTCR/CD3- thymocytes torespond to CD2 stimulation was not due to an int c defectin these cells as they responded normally to phorbol ester pluscalcium ionophore. In TCR/CD3- thymocytes, CD2 stimula-tion also failed to affect steady-state mRNA levels of therecombination-activating genes RAG] and RAG2, whereas inTCR/CD3+ cells activation of the CD2 pathway terminatedtheir expression. Together, these data support the concept thatCD2 engagement does not deliver a stimulus to TCR/CD3-thymocytes and sugges that this molecule may not directlyparticipate in the earliest stages of thymic development.

Although antigen-specific activation of T cells requires en-gagement of the clonotypic T-cell receptor (TCR) het-erodimer (1), various accessory molecules present on thesurface ofT cells are capable of augmenting signals initiatedby the TCR/CD3 complex (reviewed in ref. 2). In general,stimulation of most accessory molecules is by itself insuffi-cient to induce T-cell activation; however, stimulation ofmature T cells through the CD2 pathway can transduce amitogenic signal even in the absence of TCR/CD3 engage-ment (3). This can be accomplished by stimulation of CD2with its natural ligand LFA-3 plus a single anti-CD2 mono-clonal antibody (mAb) (4) or by using combinations of mAbsdirected at distinct epitopes of the CD2 molecule (3).An important, but unresolved, question is whether the CD2

pathway is functional in' T cells that do not express theTCR/CD3 complex. CD2'TCR/CD3- T cells can be gener-ated by mutation ofT cell lines (4) or by using anti-CD3 mAbsto modulate TCR/CD3 from the cell surface (5). Most studies

of this type have reported that TCR/CD3- cells do notrespond to CD2 stimulation; however, the heterogeneity ofthese systems as well as the varying levels ofCD2 expressionhave led to conflicting results (6-8). An additional concern iswhether the means employed to generate TCR/CD3- cellsmight also alter the functional characteristics of these cells.The issue of whether CD2 functions in T cells that do not

express TCR/CD3- is an important one for developmentalimmunology, as the CD2 molecule has been proposed to havea role in T-cell ontogeny in the thymus (9-11). CD2, one ofthe first surface antigens expressed following colonization ofthe thymus by T-cell precursors, is detectable on the cellsurface prior to the TCR/CD3 complex (12). Despite theirlack of TCR/CD3 expression, at least a fraction ofCD2+CD3- thymocytes is proliferating, perhaps serving as agenerative pool (12). The forces driving the proliferation ofthese immature thymocytes are unknown but have beenproposed to include stimuli delivered by thymic stromal cellsin the form of cytokines and/or cell-cell contact. In partic-ular, LFA-3 is found on thymic stromal cells, and activationof the CD2 pathway by these cells has been postulated toinduce the proliferation of immature CD2'TCR/CD3- thy-mocytes prior to the expression ofCD3 on their surface (12).Here, we present evidence that CD2+TCR/CD3- thymo-

cytes differ dramatically from CD2+TCR/CD3+ thymocytesin their response to CD2 stimulation. Although both celltypes display altered patterns of protein tyrosine phosphor-ylation following CD2 stimulation, CD2 crosslinking ofhighlypurified human CD2+TCR/CD3- thymocytes fails to gener-ate a calcium flux, to induce interleukin 2 receptor (IL-2R)expression, or serve as a comitogen with phorbol ester orIL-2. Furthermore, although the expression of the recombi-nation-activating genes RAG) and RAG2 can be terminatedby CD2 stimulation of TCR/CD3+ thymocytes, RAG) andRAG2 expression is not affected by CD2 crosslinking ofTCR/CD3- thymocytes. Together, these results indicate thatCD3- thymocytes are refractory to activation by the CD2molecule and support the concept that this pathway does notparticipate in the earliest stages of T-cell maturation.

MATERIALS AND METHODSmAbs and Reagents. mAbs 9.6 (anti-CD2) and G19-4 (anti-

CD3) were provided by Jeffrey Ledbetter (Bristol-MyersSquibb, Seattle). mAb 9.1 (anti-CD2) was a gift ofBo Dupont(Sloan-Kettering, New York). mAbs T112 (anti-CD2) andT113 (anti-CD2) were a gift of David Fox (University ofMichigan). mAb CD2.1 (anti-CD2) was a generous gift ofDaniel Olive (Institut National de la Sante et de la Recherche

Abbreviations: TCR/CD3, T-cell receptor/CD3; PMA, phorbol 12-myristate 13-acetate; mAb, monoclonal antibody; IL-2R, interleukin2 receptor.tTo whom reprint requests should be addressed.

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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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Medicale, Marseille). Leu-6 (anti-CDla) was purchased fromBecton Dickinson.

Isolation of Thymocytes. Thymic tissue was obtained fromchildren under age 3 who underwent routine thymectomy atthe time of cardiothoracic surgery. TCR/CD3- thymocyteswere isolated by negative selection using anti-CD3 mAb andgoat anti-mouse immunoglobulin-coated magnetic beads asdescribed (13).

Cell Culture. Thymocytes were cultured at a density of0.5-1 x 106 per ml in RPMI-1640 medium supplemented with10% fetal calf serum. When specified, the medium wassupplemented with IL-2 (Calbiochem) at 64 half-maximalunits/ml. Phorbol 12-myristate 13-acetate (PMA) was used at10 ng/ml, and ionomycin was used at 250 ng/ml. Unlessotherwise indicated, mAbs were used at 1 Ag/ml.Northern Blot Analysis. Total cellular RNA was extracted

with guanidinium isothiocyanate (14). Equalized RNA sam-ples were separated on 1% agarose/formaldelyde gels andtransferred to nitrocellulose. The DNA probes used werelabeled by nick-translation and hybridized to the membranes(13), after which the membranes were exposed to x-ray filmat -70'C. The probes for IL-2R (15), actin (16), RAG] (17),and RAG2 (17) have been described.Measurement of Intracellular Calcium. Intracellular cal-

cium was measured with indo-1 (Molecular Probes) as de-scribed (18). Cells were stimulated either by incubation with10 pg of the indicated biotinylated monoclonal antibody perml for 5 min followed by crosslinking with 40 ug of avidin perml or, instead, by the addition of each of a stimulatory pairof anti-CD2 mAbs at 10 pug/ml.

Determination of Protein Tyrosine Phosphorylation. Cellswere suspended in 1 ml in Microfuge tubes. At time = -5min, each biotinylated mAb was added at 10 pg/ml, and themAbs were crosslinked at time = 0 min by the addition of 40,ug of avidin per ml. When combinations of nonbiotinylatedanti-CD2 mAbs were used instead, the two mAbs weresimultaneously added at 10 ,ug/ml at time = 0 min. At time= 5 min the cells were pelleted and lysed with 150 txl of SDSsample buffer containing 25 mM dithiothreitol and 50 mMsodium orthovanadate, and the lysates were subjected toSDS/PAGE on 10% gels and transferred to membranes asdescribed (19). The membranes were probed first with puri-fied rabbit anti-phosphotyrosine antiserum and then with125I-labeled staphylococcal protein A (ICN), followed byexposure to x-ray film. Bands were quantified by densitom-etry of the membrane using a PhosphorImager (MolecularDynamics, Sunnyvale, CA).

RESULTSIsolation of TCR/CD3- Thymocytes. As previously re-

ported, unfractionated thymocytes exhibit a trimodal patternof distribution when stained for CD3 (Fig. 1 Upper and ref.20), and these three fractions have been termed CD3-,

Log Fluorescence

CD3+(din), and CD3+(bighlt). TCR/CD3- thymocytes wereisolated by negative selection using anti-CD3 mAb and goatanti-mouse immunoglobulin-coated magnetic beads. Theyield was 10-15% of the starting number of cells, and cellscontained in these preparations were >98% CD3- (Fig. 1Upper). Unfractionated and CD3- thymocytes were >98%CD2+ and the density of CD2 surface expression was similarin both sets of cells (Fig. 1 Lower).

Protein Tyrosine Phosphorylation in Response to CD2 Stim-ulation. Stimulation of mature T cells by the CD2 or CD3 cellsurface molecules activates phosphatidylinositol-specificphospholipase C (21). This may be the result of proteintyrosine kinase activation, as induction of tyrosine kinaseactivity is the earliest signal seen following stimulationthrough these pathways, occurring prior to a rise in intracel-lular calcium (19). To examine this signaling pathway, un-fractionated thymocytes and TCR/CD3- thymocytes wereincubated in medium or stimulated by crosslinking CD2 (Fig.2). Consistent with our previous work, a number of proteinswere constitutively tyrosine phosphorylated in unfraction-ated thymocytes (22). A similar pattern was seen in TCR/CD3 cells. Stimulation of unfractionated thymocytes byCD2 crosslinking up-regulated the phosphorylation of exist-ing substrates as well as induced new ones. Particularlyprominent bands were seen at 110, 85, and 40 kDa. A differentpattern in response to CD2 crosslinking was observed inpurified TCR/CD3- thymocytes. Here, a much smaller in-crease in tyrosine phosphorylation of the 40-kDa substrate(7-fold in TCR/CD3- cells vs. 25-fold in unfractionated cells)was observed. In contrast, TCR/CD3- thymocytes exhibiteda 3-fold increase in phosphorylation of a 70-kDa substrate,whereas no change was seen in unfractionated cells. Thesedifferences were reproducibly observed in four independentexperiments.

In the experiment shown in Fig. 2, we examined 107unseparated thymocytes and CD3 thymocytes. To controlfor the small amount of residual CD3+ thymocytes contam-inating the CD3 preparation, we also analyzed the responseof 3.5 x 105 unseparated thymocytes. No signal was detect-able with this number of cells. Thus, the response seen in theCD3- lane with 107 cells is wholly attributable to CD3thymocytes.Calcium Flux in Unfractionated Thymocytes and CD3-

Thymocytes. Although CD2 stimulation of TCR/CD3- cells

CELLS UNSEP CD3

CELL # i 3XIO 10C\1 C~1 C\2

STIM

*^ %3t5 200

0 97

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FIG. 1. Expression of CD3 on

unfractionated and purified TCR/CD3- thymocytes. Unfraction-ated thymocytes (dashed line) andTCR/CD3- thymocytes (solidline) were stained for CD3 usinganti-CD3 mAb G19-4 followed byfluorescein isothiocyanate-conju-gated goat anti-mouse antibody.

FIG. 2. Tyrosine phosphorylation induced through CD2 stimu-lation (STIM). Unfractionated thymocytes (UNSEP) and TCR/CD3- (CD3-) thymocytes were cultured in Microfuge tubes. At time= -5 min, 10 ,ug of biotinylated anti-CD2 (a-CD2) mAb (9.6) per mlwas added to the medium. The mAb was crosslinked at time = 0 minby the addition of40 Aig of avidin per ml. Cells were lysed after 5 minand subjected to SDS/PAGE. Following transfer, the filters were

incubated with anti-phosphotyrosine antibody followed by 1251-labeled protein A. Molecular masses are indicated in kDa.

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effectively induced protein tyrosine phosphorylation, thepattern seen differed from that observed in TCR/CD31 cells,and thus it was uncertain whether this altered pattern wasassociated with additional events characteristic of cell acti-vation. Therefore, unfractionated cells and TCR/CD3- cellswere loaded with indo-1 for determination of intracellularcalcium. Consistent with previous reports (23, 24), unfrac-tionated cells responded to stimulation by the CD2 or CD3molecules (Fig. 3). However, TCR/CD3- thymocytes failedto respond to either of these stimuli or to engagement of theCD1 antigen with anti-CDla mAb (data not shown). This didnot appear to be due to an intrinsic defect in the ability ofthese cells to mobilize calcium, as the direct G proteinactivator aluminum fluoride induced a calcium flux in 70% ofTCR/CD3- cells, at a magnitude equivalent to the responseseen in unfractionated cells (71% responding). Failure torespond to CD2 stimuli also did not appear to be the result ofthe specific mAb used, as cells were unresponsive tocrosslinking CD2 using mAb 9.6 (Fig. 3) or T112 (data notshown) or to the combination of 9.1 plus 9.6 or T112 plus T113(10 ug/ml of each, data not shown).

Induction ofIL-2R Expression Through CD2 Stimulation. Inmature T cells, stimulation of the CD2 or CD3 pathwayinduces the IL-2R p55 chain (3). This effect can also beelicited through direct activation of protein kinase C withphorbol esters. We therefore next investigated the effect ofCD2 stimulation on IL-2R expression (Fig. 4). Unfraction-ated thymocytes and purified TCR/CD3- thymocytes werestimulated with the phorbol esterPMA or with a combinationof anti-CD2 mAbs that is mitogenic for mature T cells (9.1plus 9.6). Cells were lysed after 18 hr and subjected toNorthern blot analysis. Transcripts for the p55 chain of the

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0 150 300 450 600 750Time (Seconds)

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FIG. 3. Calcium mobilization in unfractionated thymocytes andTCR/CD3- thymocytes. Unfractionated thymocytes (dashed line)and TCR/CD3- thymocytes (solid line) were loaded with indo-1 andwashed, and basal calcium measurements were obtained. [Ca2+]i,intracellular calcium. The cells were then stimulated by incubationwith 10 pg of biotinylated mAb to CD3 per ml (G19-4, Top) or CD2(9.6, Middle) for 5 min followed by crosslinking with 40 ,ug of avidinper ml (indicated by break in tracing). TCR/CD3- thymocytes werealso stimulated with aluminum fluoride by the addition of50mM NaFand 10 gM AICl3 to the medium (Bottom).

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FIG. 4. Induction of IL-2R expression by CD2 stimulation.Unfractionated thymocytes (UNSEP) and TCR/CD3- (CD3-) thy-mocytes were cultured with medium alone (MED), PMA (10 ng/ml),anti-CD2 mAbs (9.1 plus 9.6, each at 1 gg/ml), orPMA plus anti-CD2mAbs. Cells were harvested after 18 hr and RNA was isolated andequalized (Top). Northern blots were prepared and the filters werehybridized sequentially with cDNA probes specific for IL-2R (Mid-dle) and actin (Bottom). Lanes 1-8 on the left contain 4 Zg ofRNAper lane. To increase the sensitivity, 20 gg of RNA from CD2-stimulated cells (unfractionated and TCR/CD3-) was also analyzedon the same filter (far right two lanes).

IL-2R were readily detected in unfractionated thymocytesstimulated with eitherPMA or anti-CD2 mAbs. Furthermore,the combination of the two stimuli acted synergistically ininducing IL-2R. In contrast, although PMA effectively in-duced IL-2R gene expression in TCR/CD3- thymocytes,stimulation of the CD2 pathway had no effect and was unableto synergize with PMA in augmenting IL-2R expression. Toverify that IL-2R transcripts were not induced through CD2stimulation, Northern analysis was done using a 5-foldgreater amount of RNA (20 pg per lane) for CD2-stimulatedcells (Fig. 4, far right two lanes). Anti-CD2-induced IL-2Rtranscripts were again detectable only in unfractionatedthymocytes.

Induction of Proliferation. We also investigated the abilityof CD2 stimulation to provide a comitogenic signal forTCR/CD3- thymocytes. Mature T cells can proliferate inresponse to anti-CD3 stimulation (if the mAb is immobilized)or to combinations of anti-CD2 mAbs (3), as these stimuliinduce IL-2R expression and the production of IL-2. Thy-mocytes do not produce IL-2 in response to these stimuli butcan respond if exogenous IL-2 is provided (13, 24). Inaddition, IL-2 production in response to anti-CD3 or anti-CD2 treatment can be induced if phorbol ester is present inthe culture medium (25). Therefore, unfractionated thymo-cytes and TCR/CD3- thymocytes were stimulated withanti-CD2 mAbs (9.1 plus 9.6), PMA, or IL-2 either alone orin combination (Table 1). Unfractionated thymocytes andTCR/CD3- thymocytes proliferated in response to PMA plusIL-2 or to PMA plus the calcium ionophore ionomycin.However, only unfractionated thymocytes responded toPMA plus anti-CD2 mAbs or IL-2 plus anti-CD2 mAbs.Identical results were seen when the anti-CD2 mAbs T112plus T113 or CD2.1 plus 9.6 were used in place of 9.1 plus 9.6.

Effects of CD2 Stimulation on Expression of the Recombina-tion-Activating Genes RAG) and RAG2. The recombination-activating genesRAG] and RAG2 are constitutively expressedin TCR/CD3- thymocytes and TCR/CD3'CD4+CD8+ thy-mocytes (17). Expression of these genes is terminated uponcell activation with the combination ofPMA plus ionomycin orwith anti-CD3 mAb (in the case of TCR/CD31 cells). Theintracellular signals required for cessation ofRAG expression

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Table 1. CD2 does not transduce a proliferative signal inTCR/CD3- thymocytes

cpm

Cell fraction Stimulus Exp. 1 Exp. 2Unseparated None 191 ± 106 380 ± 85

PMA 142 ± 28 339 ± 125CD2 100 38 245 ± 58IL-2 545 ± 155 2,131 ± 2208PMA + CD2 17,305 ± 557 74,936 ± 5586CD2 + IL-2 7,934 ± 688 27,371 ± 3057PMA + IL-2 10,054 + 1306 22,788 ± 2471PMA + ionomycin 9,404 ± 868 20,398 ± 1873

TCR/CD3- None 162 + 35 285 ± 101PMA 280 + 89 634 ± 122CD2 135 ± 78 314 ± 20IL-2 1,049 ± 281 498 ± 68PMA + CD2 212 ± 17 238 ± 52CD2 + IL-2 1,320 ± 60 564 ± 132PMA + IL-2 19,854 ± 913 10,509 ± 515PMA + ionomycin 10,482 ± 1970 6,224 ± 162

Cells were cultured for 4 days in 96-well microtiter plates. PMAwas used at 10 ng/ml, ionomycin was at 250 ng/ml, and IL-2 was at64 half-maximal units/ml. All cultures were pulsed with 1 ,ICi (1 Ci= 37 GBq) of [3H]thymidine 18 hr prior to harvesting. CD2 stimu-lation consisted of mAbs 9.1 and 9.6, each at 1 pg/ml. Data arerepresentative of five separate experiments; cpm are expressed asmean SD.

have not been characterized, and therefore it was of interestto determine the effects of CD2 stimulation on RAG expres-sion (Fig. 5). RAG) and RAG2 transcripts were readily de-tected in unfractionated cells and TCR/CD3- cells. Stimula-tion with anti-CD2 mAbs strongly down-regulated RAG) andRAG2 steady-state mRNA levels in unfractionated thymo-cytes. In contrast, no significant effect of CD2 stimulation onRAG expression was observed in isolated TCR/CD3- cells.We have noted that the level of RAG expression in unfrac-tionated thymocytes is frequently higher than that in TCR/CD3 thymocytes (data not shown), a finding likely to beattributable to the higher level of expression in TCR/CD3+CD4+CD8+ cells vs. TCR/CD3- cells. The strongdown-regulation of RAG) and RAG2 in CD2-stimulated un-fractionated thymocytes occurs because the CD2-responsiveTCR/CD3+CD4+CD8+ cells account for most of the signalobserved in unfractionated cells. With longer exposures, a

UNSEP CD3-

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FIG. 5. Effects of CD2 stimulation onRAG expression. Unfractionated thymo-cytes (UNSEP) and TCR/CD3- (CD3-)thymocytes were cultured with mediumalone (MED) or anti-CD2 mAbs (9.1 plus9.6, each at 1 pg/ml). Cells were har-vested after 6 hr and RNA was isolatedand equalized. Northern blots were pre-pared and the filters were hybridizedsequentially with cDNA probes specificfor RAG) (second panel), RAG2 (thirdpanel), and actin (lower panel).

small amount ofRAG) and RAG2 message was seen followingCD2 stimulation of unfractionated cells (data not shown),consistent with continued RAG expression by the TCR/CD3-cells. As previously reported (17), RAG expression in TCR/CD3- cells was terminated following treatment with PMA plusionomycin (data not shown).

DISCUSSIONCD2 appears very early in thymocyte development, prior tothe appearance of surface TCR/CD3, and hence a subpop-ulation of CD2+TCR/CD3- thymocytes can be identified(12). The observation that some of these cells are cycling hasled to the concept that proliferation of these cells may bedriven through CD2 stimulation (12), a hypothesis that issupported by the fact that the CD2 ligand LFA-3 is expressedon the surface of thymic epithelial cells (26). The workpresented here fails to support this hypothesis and argues,instead, that the ability of the CD2 signaling pathway toactivate thymocytes develops coincidentally with the cellsurface expression of TCR/CD3. Our studies demonstratethat highly purified TCR/CD3- thymocytes do not respondto CD2 signals by generating a calcium flux. This appears tobe due to defective CD2 signal transduction rather than to anintrinsic defect of immature cells, as the direct G proteinactivator aluminum fluoride is able to induce a rise inintracellular calcium. Similarly, although CD2 stimulationwas sufficient to induce IL-2R expression in TCR/CD3+thymocytes, it was unable to do so in TCR/CD3- cells or toserve as a comitogen for these cells in combination witheither phorbol ester or IL-2. Finally, CD2 stimulation termi-nates steady-state RAG expression in TCR/CD3+ thymo-cytes but not in TCR/CD3- cells. Though direct assays ofrecombinatorial ability in this cell system are not available,this is in agreement with our current concept of thymicontogeny, as continued expression of the variable(diversi-ty)joining recombination machinery would be desired in cellsthat have not yet productively rearranged their TCR genes.CD2 stimulation ofTCR/CD3- cells reproducibly induced

a strong increase in tyrosine phosphorylation of a 70-kDaprotein. Tyrosine phosphorylation ofa similarly sized proteinwas reported to occur in Jurkat (27) or normal T cellsfollowing CD3, but not CD2, stimulation. In our studies, thisprotein was strongly observed on phosphotyrosine immuno-blots of unfractionated thymocytes stimulated through CD3crosslinking (data not shown) and much more weakly appar-ent following CD2 crosslinking (Fig. 2). Since isolated TCR/CD3 thymocytes yielded a strong band for this proteinfollowing CD2 crosslinking, it seems likely that these cellsaccounted for most of the signal seen with CD2 stimulationof unfractionated thymocytes. The fact that pp7O is tyrosinephosphorylated on CD2-stimulated TCR/CD3- thymocytes,but weakly or not at all tyrosine phosphorylated on CD2-stimulated TCR/CD3+ thymocytes or mature T cells, furthersuggests that signals transduced by CD2 differ qualitatively inimmature lymphocytes. It is also noteworthy that tyrosinephosphorylation of a 40-kDa substrate was observed follow-ing CD2 stimulation of TCR/CD3- thymocytes and unfrac-tionated thymocytes, although the signal was much strongerin the latter population. This was the sole response to CD2stimulation clearly attributable to TCR/CD3+ thymocytesand TCR/CD3- thymocytes. The phosphorylation of pp4Oand pp7O in TCR/CD3- cells leaves open the possibility thatCD2-mediated signal transduction may play a role in thedevelopment of TCR/CD3- thymocytes.

In addition to our current work, the following lines ofevidence also argue against the ability of these cells totransduce stimulatory signals in TCR/CD3- thymocytes. (i)The distribution of CD2 has not been highly conserved duringmammalian evolution. Whereas in humans, CD2 is expressed

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at uniformly high levels on virtually all thymocytes andmature T cells, in mice and in sheep, CD2 appears relativelylater in thymic development and increases in cell surfacedensity as cells progressively mature (28-30). (ii) Treatmentof embryonic thymus in organ culture or in vivo injection ofnewborn mice with anti-CD2 mAb does not interfere withnormal thymocyte differentiation (29, 31). (iii) Only TCR/CD3+ cells have been shown to bind to thymic epithelial cellsthrough a CD2-LFA-3 interaction or to be stimulated as aresult of this interaction (32).Our work stands in contrast to three previous reports of

CD2-induced responses in TCR/CD3- thymocytes (11, 25,33); however, the reasons for the discrepancy are not imme-diately apparent. Nonetheless, using highly purified (>98%)TCR/CD3- thymocytes, we were unable to observe a re-sponse to CD2 stimulation as measured by several differentparameters. Though it is possible that differences in ourresults might be due to the mAbs used, we found no responseusing multiple stimuli with several different anti-CD2 mAbs.Ohno et al. (7) have recently reported that CD2 could mediatemature T-cell activation of TCR/CD3- mutants if the levelsof cell surface CD2 were sufficiently high. Although thisfinding may provide interesting insights into the nature of theCD2-CD3 interaction, it does not appear to influence theinterpretation of our results, as CD2 is expressed at similarlevels in TCR/CD3- thymocytes and TCR/CD3+ thymo-cytes as well as mature T cells (Fig. 1 and ref. 20).Although it is uncertain iftyrosine phosphorylation ofpp4O

and pp70 following CD2 stimulation of TCR/CD3- thymo-cytes is of functional significance, the CD2 molecule mayserve a role in the development of TCR/CD3- thymocytesindependent of intracellular signal transduction. A CD2-LFA-3 adhesive interaction may be important to maintaincell-cell contact during thymic ontogeny. In addition, stim-ulation ofLFA-3 has been shown to induce thymic epithelialcells to release interleukin 1 (IL-1) (10), and thus, if TCR/CD3- thymocytes do interact through CD2 with thymicepithelial cells, then induction of the release of IL-1 (andperhaps other cytokines) may serve to promote their matu-ration and/or proliferation.

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9. Fox, D. A., Hussey, R. E., Fitzgerald, K. A., Bensussan, A.,Daley, J. F., Schlossman, S. F. & Reinherz, E. L. (1985) J.Immunol. 134, 330-335.

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11. Dalloul, A. H., Mossalayi, M. D., Dellagi, K., Bertho, J. &Debre, P. (1989) Eur. J. Immunol. 19, 1985-1990.

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