Clin. exp. Immunol. (1992) 89, 83-88

T lymphocyte anergy during acute infectious mononucleosis is restrictedto the clonotypic receptor activation pathway

M. PEREZ-BLAS*, J.R. REGUEIRO*t, J. RUIZ-CONTRERASt, & A. ARNAIZ-VILLENA*Departments of *Immunology and tPediatrics, Universidad Complutense, Hospital 12 de Octubre, Madrid, and

tDepartment of Pediatrics, Universidad de Valladolid, Valladolid, Spain

(Acceptedfor publication 19 March 1992)

SUMMARY

The transient T cell anergy associated with acute infectious mononucleosis (IM) caused by theEpstein-Barr virus has been analysed in a sample of 14 IM children. Peripheral blood mononuclearcells (PBMC) obtained from IM patients showed a significant specific impairment in theirproliferative response to both phytohaemagglutinin (PHA; P< 005) and to an anti-CD3 MoAb(P< 0-00 1), although both responses reached normal control levels by addition of a submitogenicdose of either phorbol myristate acetate (PMA) or recombinant IL-2 (rIL-2). In contrast, activationsignals delivered through other surface molecules (CD2, CD28) or other transmembrane pathways(PMA plus a calcium ionophore) elicited normal or high proliferative responses in most IM PBMC.In a group of five patients tested, the synthesis of IL-2 by IM PBMC in the presence of PMA wasimpaired when PHA or anti-CD3 was used as stimulus, but it reached normal levels with anti-CD2 orionophore. Lastly, PHA failed to induce IL-2a receptor (IL-2Ra) expression in IM PBMC from fourtested patients, but the presence of PMA completely corrected this defect. Taken together, theseresults strongly suggest that the T cell anergy associated with acute IM is due to a T cell receptor(TCR)-specific impairment in the induction of genes involved in T cell proliferation (including thosecoding for IL-2 and IL-2Rox) upon membrane signalling to otherwise normal T lymphocytes, sinceCD2, CD28 and certain transmembrane activation pathways are uncoupled from CD3 in theseparticular pathological conditions (and perhaps in most in vivo situations). This and other similarexperimental approaches to transient secondary immunodeficiencies may help to unravel thephysiopathological role of different surface molecules in T cell activation.

Keywords T lymphocyte infectious mononucleosis anergy T cell receptor activationdisease

INTRODUCTION antigens [4]. It is possible, however, to activate T lymphocytes in

Acute infectious mononucleosis (IM) is a self-limiting lympho- a polyclonal fashion by using lectins (phytohaemagglutininproliferative disease caused by the Epstein-Barr virus (EBV) [1]. (PHA)), antibodies directed against different monomorphicProfound immunoregulatory cellular dynamics follow the pri- surface molecules (CD2, CD3, CD28) or reagents that bypassmary infection, including the activation and expansion of CD8 membrane signals (like phorbol esters and calcium ionophores)lymphocytes which is thought to result from intense stimulation [5]. Since CD3 is the invariant component of the TCR,by EBV-infected B lymphocytes [2]. In addition, a transient activation of T cells through this structure is considered toimpairment ofnormal T cell responses (i.e. anergy) is associated mimic physiological stimuli. Ligands for both CD2 and CD28with the acute phase of IM, as assessed by both in vivo (skin (CD58 and B7, respectively) have been identified on the surfacetests) and in vitro (response to antigens and mitogens) assays [3]. ofseveral antigen-presenting cells [6], making their participationHowever, the precise mechanism underlying this EBV-induced in the adhesion step of the T cell response very likely. But theT cell anergy is mostly unknown. physiological role of the signal delivered through CD2 and/or

Resting T lymphocytes are activated and acquire specific CD28 to the T cell remains a matter of speculation.effector (cytotoxic/helper) functions only after engagement of In the present work, an attempt has been made to character-their clonally distributed T cell receptor (TCR) by appropriate ize the EBV-induced T cell anergy associated to TIM by analysing

both surface (PHA, CD2, CD3, CD28) and transmembraneCorrespondence: Antonio Arnaiz Villena, Inmunologia Hospital 12 (phorbol myristate acetate (PMA); Tonomycin) activation path-

de Octubre, 28041 Madrid, Spain. ways in TM patients. Our results indicate that the impairment of83

84 M. PNrez-Blas et al.

T lymphocyte function associated with IM is mostly restricted La Roche) 100 U/ml, pokeweed mitogen (PWM) 1% v/v, fromto the T cell receptor/CD3 activation pathway. Difco.

Skin tests for the evaluation of T cell function in vivo wereperformed in some patients (n = 9) using a commercial intrader-

PATIENTS AND METHODS mal multi-antigen applicator (Multitest IMC, Inst. Merieux,

Patients Lyon, France) as previously described [11].A total of 14 acute IM children (5 + 3 years old) were examined. IL-2 synthesis was assayed on PMA-stimulated PBMC as

Clinical diagnosis was based on fever, lymphocytosis, spleno-megaly and atypical lymphocytes in the peripheral blood [7]. All Statisti InIyithe patients were heterophile antibody positive and had specific calpansis, All comparisons between acute IM patients and normal controlsIgM antibodies for the viral capsid. Twenty-two age-matched were done using Student's t-tests. The standard error (s.e.) of the(6+3 years old) normal controls were used for phenotypic mean was calculated as s.d/In. For variables in which theanalyses. However, for proliferative assays the control popu-

n

lation consisted of 36 normal healthy volunteers between 5 and number ofsampes avaiable formanlyisuwa les anwthe30 years of age, because no differences could be found in these tota numbe ofpatienssassays between normal children and adults. The control samples used in the comparisons.were obtained at the same time as the acute IM samples (within1 week of diagnosis) and were handled identically. RESULTS

Quantification of absolute and relative numbers of lymphocyteCytofluorographic analysis subsetsWhole blood samples were stained by direct immunofluores- The phenotypic characterization of lymphocyte subsets in ourcence with different MoAb, their erythrocytes lysed and the sample of acute IM revealed expected and previously describedremaining cells fixed for flow cytofluorometry analysis by alterations [3]: increased percentages ofCD8 cells and decreasedstandard techniques (Q-Prep, Coulter, Hialeah, FL) as de- percentages of CD4, CD57 and CD21 lymphocytes; in absolutescribed [8]. The results were recorded as: (i) the percentage of terms, the main finding is a significant increase in total andpositive cells for each MoAb (those displaying fluorescence mature T cells (CD2, CD3) mostly due to the CD8 subset (Tableintensities above the upper limit ofa negative control) within the 1). CD28 lymphocytes were normally represented in purifiedlymphocyte population (electronically gated by size/complexity acute IM PBMC (not shown).criteria); and (ii) the absolute number of positive cells for eachMoAb per microlitre of whole blood calculated as a product of Stimulation with lectins and skin teststhe previous percentage and the absolute total lymphocyte Upon stimulation of IM PBMC with PHA, the previouslycounts determined in parallel venous blood samples by cyto- described IM-associated T cell unresponsiveness was observedmetry (Coulter). OK-series MoAb were purchased from Ortho (Fig. 1 a). Similar results were obtained with Con A (data notPharmaceuticals (Raritan, NJ) and Leu-7 from Becton Dickin- shown) but not with PWM (Table 2). In vivo analysis of IMson (Sunnyvale, CA). Anti-CD25 (IL-22 receptor (IL-2Ra)) T cell function by delayed hypersensitivity skin tests essentiallyMoAb was purchased from Coulter and anti-CD28 (Kolt-2) confirmed the existence of a partial T cell anergy, although thewas a generous gift from K. Sagawa (Tokyo, Japan). difference from controls did not reach statistical significance

(mean scores 8 + 6 mm in IM patients versus 13 + 6 mm in age-Isolation of lymphocytes matched controls). This partial anergy could not be due to aPeripheral blood mononuclear cells (PBMC) were obtained by general depression in the effector capacity ofIM T cells, becausedensity gradient-centrifugation using Ficoll-Hypaque (Lym- addition of both protein kinase C (PKC) activators (phorbolphoprep, Nyegaard, Norway) as described [9]. ester PMA) and calcium ionophore (Ionomycin) restored their

full proliferation potential, which turned out to be significantlyProliferative andfunctional assays higher than control cells (Table 2).Cells (1 x 105) were placed in round-bottomed microtitre wells(Nunc, Roskilde, Denmark) in 0.2 ml of final culture medium: A normal response to PHA is restored with both exogenousRPMT 1640 (Flow Laboratories, UK) supplemented with 10% rIL-2 or PMAfetal calf serum (FCS, Flow), 1% Penicillin-Streptomycin An attempt was then made to correct IM-associated anergy to(Difco, UK) and 1% L-glutamine (200 mM) (Flow). After 3 days PHA by addition of exogenous rIL-2 or PMA to the cultures.of in vitro culture, wells were individually pulsed with 1 jiCi of The results, shown in Table 2, demonstrate that both reagents3H-TdR for 15-18 h and washed using a Titertek cell harvester restore normal proliferative responses to PHA in IM PBMC.(Flow). 3H-TdR uptake was measured in a liquid scintillation Thus, it is deduced that: (i) the observed T cell anergy may becounter [10] (Beckman, Brea, CA) The following stimuli or due to an impairment in the induction of IL-2 or IL-2R by PHA;their combination were used: anti-CD3 (OKT3) 12 5 ng/ml, (ii) accessory cell signals are normal in IM; and (iii) insufficientanti-CD2 (CD2.1 and CD2.9 from D. Olive (Villejuif, France)) signals are being delivered through membrane receptors for1 pg/ml, anti-CD28 (9.3 from P. Martin, Seattle, WA) 1:10000 PHA to PKC moieties in IM PBMC.dilution of ascites fluid, concanavalin A (Con A; Calbiochem,La Jolla, CA) 10 ig/ml, PHA (Difco, Detroit, MT) 10 ,ug/ml, Stimulation with MoAb against CD2, CD3 and CD28PMA (Sigma, St. Louis, MO) 1-10 ng/ml, Tonomycin (Calbio- PHA stimulation offers a crude estimate of T cell function,chem, La Jolla, CA) l tIM, rTL-2 (kindly supplied by Hoffman because it may use both antigen-specific (i.e. TCR/CD3-

T cell anergy in infectious mononucleosis 85

Table 1. Lymphocyte marker analysis of acute infectious mononucleosis (IM) patients

IM patients (n = 14) Controls (n = 22)

Absolute AbsoluteCD Per cent of lymphocyte Per cent of lymphocyte

Monoclonal antibody (subset) equivalent lymphocytes (no. per PI) total lymphocyte (no. per pI)

OKTl 1 (total T cells) CD2 83+4 4677+ 896t 78+1 3499+ 59OKT3 (mature T cells) CD3 67+4 4681 + 839t 64 + 2 2983 + 70OKT4 (helper T cells) CD4 23+3* 1706+377 42+1 1910+67OKT8 (cytotoxic/supressor T cells) CD8 39+4t 2696+553* 28+ 1 1262+ 52Leu-7 (NK cells) CD57 4+ 1 395 +65 6+1 263 +49OKB7(Bcells) CD21 11+2t 853+201 16+1 715+42

Results are expressed as mean + s.e.* P value <0001 compared with normal controls.1 P value < 0.05 compared with normal controls.NK, Natural killer.

300 -6(a) 60 (b) W(c)

250 - 50

* | 100 _' 200 740 * * *i

10 0 0i

t3 Nom. SMNoma SMNra_____ C)27 @0=14 n34 )b) (=

E 150 030____

00100 20

50 0 --00 ~~~~~~~~~00

IM Normal IM Normal IM Normalpatients controls patients controls patients controlsln =14) (n=27) (n =14) (n=34) (n=10) (n=l13)

Fig. 1. Proliferative response of peripheral blood mononuclear cells (PBMC) from infectious mononucleosis (IM) patients and normal control tophytohaemagglutinin (PHA) (a), anti-CD3 (b) and anti-CD2 + anti-CD28 (c) stimulation. Mean values oftriplicate samples are indicated by dots. Thegroup means + s.e. are also depicted. The observed differences are statistically significant in (a) and (b), but not (c) (P < 0 05, P < 0-001 and P > 0-05,respectively; see also Tables 2 and 3).

mediated) and non-antigen-specific (i.e. CD2-mediated) acti- Low IL-2 and IL-2RM synthesis upon stimulation with PHAvation pathways. Therefore, various T cell activation pathways To assess this point directly, the induction of both IL-2 and(CD2, CD3, CD28) were explored to try to map IM-associated IL-2Rc (CD25) was tested on stimulated IM PBMC from aT cell anergy to one or another. As shown in Figs I b, I c and group of five patients. The results, shown in Table 4 and Fig. 2,Table 3, only the CD3-mediated response is significantly indicate an acute IM-specific impairment in the induction ofimpaired in IM PBMC, and the difference from controls is more both IL-2 and IL-2Rc by PHA as compared with normalclear-cut than with PHA (compare Figs. la and b). controls. Indeed, a modulation ofpre-existing bright IL-2Roa on

IM-activated cells seems to take place after addition ofPHA toThe impaired response to anti-CD3 is corrected with both the cultures (25+4% with PHA versus 44+3% without it,exogenous rIL-2 or PMA Fig. 2). CD3 stimulation roughly confirmed these findings,As shown previously for PHA, the IM-associated proliferative although IL-2 induction on control cells was always lower thananergy to anti-CD3 can be restored to normal levels by addition that of PHA (Table 4). The induction of IL-2Ro by anti-CD3ofexogenous submitogenic rIL-2 orPMA to the cultures (Table was negligible in control cells in our culture conditions and3). In summary, the T cell anergy associated with acute TM maps therefore it was not tested. In contrast, measurable amounts ofto the TCR/CD3 activation pathway, apparently because IL-2 were secreted by TM PBMC upon stimulation with anti-insufficient IL-2 synthesis is induced upon membrane signalling CD2 or ionomycin (Table 4). Also, the addition of submitogenicto PKC moieties. doses of PMA to PHA-stimulated cultures revealed a normal

86 M. Prez-Blas et al.

Table 2. Stimulation of infectious mononucleosis (IM) lymphocytes Table 4. IL-2 synthesis by infectious mononucleosiswith lectins (IM) lymphocytes (%)

3H-TdR incorporation IM patients Normal controls(ct/min x 10-3) Stimulus (n = 5) (%) (n =4) (%)

IM patients Normal controls Medium 7 + 2 01 + 01Stimulus (n= 14) (n=36) PHA 18+7 77+25

Anti-CD3 6+3 28+ 16PHA 79+62* 119+55 Anti-CD2 81+10 107+5PHA+rIL-2 163 +62 164+70 lonomycin 100+0 100+0PHA+PMA (10 ng/ml) 170 + 78 175 + 73PWM 33+29 31+15PMA (1 ng/ml) + onomycin 1543+ 39 10+58 Peripheral blood mononuclear cells (PBMC) wererIL-2 14 + 14 9+5 cultured in the presence of the indicated stimuli andPMA (1-10 ng/ml) + 5+3 phorbol myristate acetate (PMA) (10 ng/ml). CulturePonomycinI1+1 2+12+ 1 supernatants were assayed at day 3 for IL-2 activity byMedium 2+ 1 3+ 1 standard techniques [11]. Due to the variability amongMedium 2+ 1 3 + I

distinct experiments, the results are shown as meanrelative response indexes + s.e. The response index was

Peripheral blood mononuclear cells (PBMC) were cultured in the calculated as (ct/min test-ct/min medium)/(ct/minpresence of the indicated stimuli and pulsed for 18 h with 1 pCi/well 3H- max-ct/min medium). Maximal proliferation wasTdR. The mean ct/min of triplicate samples was determined by liquid observed in most cases with ionomycin (equivalent to ascintillation on day 3. Group means + s.d. ct/min are shown for each range of 5-50 U/ml of IL-2). The statistical significancestimulus. of the observed differences between IM patients and

* P value < 0.05 compared with normal controls. normal controls could not be determined due to thePHA, Phytohaemagglutinin; PMA, phorbol myristate acetate; small sample size.

PWM, pokeweed mitogen. PHA, Phytohaemagglutinin.

Table 3. Stimulation of infectious mononucleosis (IM) lymphocytes pM Normalpatient controlwith MoAb against CD2, CD3, and CD28 - __--- - _--- - -

E|1 ~~~~~~48| ||8E3H-TdR incorporation

(ct/min x 10- 3) sjil

IM patients Normal controlsStimulus (n= 14) (n=36) |

Anti-CD2 1+ 1 2+ 1Anti-CD2 + IL-2 31 +21 45+21Anti-CD2+PMA (10 ng/ml) 181 +93 177 +68Anti-CD2 + Anti-CD28 27 + 18 42 + 22Anti-CD3 10+10* 24+ 12Anti-CD3 + rIL-2 68 + 24 82 + 30 62 56Anti-CD3 +PMA (10 ng/ml) 120+74 142+61 2Anti-CD28 1+ 1 2+1 +Anti-CD28+PMA (10 ng/ml) 88+64 102+44 IrIL-2 14+ 14 9+5PMA (10ng/ml) 5+5 5+3Medium 2 + 1 3 + 1 Fig. 2. The induction of IL-2Ra expression with different stimuli wasMedium2+1 3 + I

measured by direct immunofluorescence on infectious mononucleosis(IM) patients and normal controls after 72 h. Depicted graphs are from

Peripheral blood mononuclear cells (PBMC) were cultured in the a representative experiment out of four. The mean expression per-presence of the indicated stimuli and pulsed for 18 h with 1 uCi/well 3H- centages + s.e. were the following (IM patients versus normal controls):TdR. The mean ct/min of triplicate samples was determined by liquid medium (44 + 3 versus 6 + 1), phytohaemagglutinin (PHA) (25 +4 versusscintillation on day 3. Group means+s.d. ct/mmn are shown for each 35±2) and PHA+phorbol myristate acetate (PMA) (70±8 versusstimulus. 66 ± 2). The statistical significance of the observed differences between

*P value <0001 compared with normal controls. the two donor groups could not be determined due to the small samplePMA, Phorbol myristate acetate. size.

T cell anergy in infectious mononucleosis 87

induction of IL-2Rcx on IM lymphocytes (70 + 8% in IM and on IL-2 synthesis by CD4/helper T cells [20]. In addition, the use66 + 2% in controls; 25 +4% and 35 + 2%, respectively, are the ofmixed populations for the analysis ofT cell activation in vitrovalues without PMA, Fig. 2). may be more informative than that of purified subsets, because

T cell responses are the result of collaborative cellular interac-tions in vivo.

DISCUSSION An IgG serum blocking factor specific forT lymphocytes hasbeen previously described in IM [21]. This antibody suppressed

Secondary immunodeficiencies (SID) are a heterogeneous the T cell response of normal donors to both antigens andgroup of disorders that occur frequently together with other mitogens and apparently bound to CD2, because it significantlypathological conditions [13]. However, the molecular basis of inhibited sheep erythrocyte rosette formation. Although it hassuch SID are mostly unknown due to the transient nature of the not been formally ruled out, the existence of a CD2 blockingalterations, although they are by far the most common in factor in our IM sample is unlikely, because CD2 expressionclinical practice. In this work, an attempt has been made to levels are normal (Table 1), and activation of IM T cells throughcharacterize a very common herpesvirus-induced SID (IM) CD2 is also within normal limits (Table 3). Also, the TCR-because it offers the chance to study a homogeneous SID with specific T cell anergy associated with IM that has been detectedvery well defined cellular parameters. is probably not caused by a serum factor, because the cells were

The present set of experiments demonstrates that the T cell thoroughly washed before culture and thereafter cultured foranergy that takes place during acute IM in children is due to a 3 days at 370C before harvesting. Any factor binding to T cellsselective impairment of the TCR/CD3 complex activation should be detached in 1 h in such conditions.pathway in otherwise normal T lymphocytes. A lack of The normal response of most IM PBMC to PWM, but notcorrelation was observed between proliferative responses and PHA or Con A, is a striking finding. However, normal responsesskin test results. However, it should be noted that skin tests in to PWM have been reported previously in the absence of achildren (< 15 years old) are quantitatively lower and therefore normal response to other lectins in some ID with T cellless reliable than in adults [11]. membrane defects [9, 22]. Also, PWM differs markedly from

An obvious possibility to explain IM-associated T cell PHA and Con A in the spectrum of cell surface receptors it cananergy could be that the strong activation and expansion of bind [23], and therefore its mitogenic mechanism may beCD8 T cells as a consequence ofEBV infection [14] leads to TCR different too. Since PWM is a T cell-dependent B cell mitogen,modulation and, therefore, to T cell unresponsiveness. How- our data support that sufficient T cell 'help' is available in IMever, two lines of evidence stand against this idea: first, CD3 PBMC for a normal PWM response, although IM T cells fail toexpression levels were normal in all tested patients (Table 1 and synthesise normal levels of IL-2 and IL-2RM in response tounpublished fluorescence intensity data); second, TCR modula- TCR-dependent stimuli.tion leads to a transient unresponsiveness to both CD2- and Our results strongly suggest thatIM-associatedT cell anergyCD28-mediated activation signals [15], and this does not is associated with an impaired synthesis of IL-2 and IL-2Rcxhappen in our IM patients sample (Table 3). In fact, it is ofsome upon TCR crosslinking. The fact that exogenous rIL-2 restoresinterest that T lymphocytes from most tested IM patients may both PHA and OKT3-induced proliferation to normal levels inbe unresponsive to anti-CD3 MoAb (10 of 14 (71 %) were below IM may be due to an rIL-2-dependent induction of IL-2Rx onthe lower 70% confidence limit (around 1 s.d.) of the control) IM PBMC, although this has not been directly assessed. In anybut not to anti-CD2 and/or anti-CD28 MoAb (only three of 10 event, this would be consistent with the fact that IL-2 gene(30%)). Our data support that CD2 and CD28 activation induction is strictly dependent on signals from the TCR [24],pathways may be uncoupled from CD3 in certain pathophysio- whereas IL-2Ra may be induced by TCR-independent signalslogical conditions, as suggested by others' in vitro studies [16, 17] (tumour necrosis factor (TNF), IL-1, IL-2, PMA) [25]. Thus,

The fact thatIM-associated T cell anergy can be more clearly accessory signals for the normal induction of IL-2Rcx in IMdemonstrated using anti-CD3 MoAb rather than PHA (com- T cells may be available in our system, further supporting thatpare Fig. la and b) further supports that PHA stimulates T cells the observed T cell anergy is a TCR-specific phenomenon.through non-antigen-specific pathways (i.e. CD2) [18] and also Alternatively, TCR/CD3 engagement may enhance apoptosis,that CD2 is functional in acute IM T lymphocytes. It could be rather than proliferation, in IM T lymphocytes, causing theargued, however, that a general surface impairment for the observed anergy in vitro [26]. Certain signals (through CD2,induction of IL-2 synthesis (non-TCR-specific) is the basis of CD28 or CD25/IL-2R) would rescue IM T cells from theIM-associated T cell anergy, because transmembrane PKC apoptotic pathway, as shown previously for IL-2 [27].activators (PMA) were used to test both CD2 and CD28 The reduction in IL-2 synthesis observed in IM patientspathways. However, this is very unlikely because a combination using PHA+PMA and anti-CD3 +PMA (Table 4), contrastsof anti-CD2 and anti-CD28 MoAb (in the absence of PMA), with the normal proliferation shown by the same cells to thosewhich is known to drive T cell proliferation through an stimuli (Tables 2 and 3). This may reflect that IM T lymphocytesautocrine IL-2/IL-2R pathway [19], induced normal mitogenic receive some (proliferation) but not all (IL-2 synthesis) acti-responses in most tested IM PBMC (seven of 10, Table 3 and vation signals, as shown previously in certain mutant T cellsFig. Ic). [28]. Indeed, the TCR/CD3 complex has recently been shown to

A second explanation for IM-induced T cell anergy is a T cell contain at least two signal-transducing modules [29], perhapssubset imbalance (i.e. relative lack of helper cells) as evidenced one of which may be impaired in TM T lymphocytes (the oneby phenotype analysis (Table 1). However, this seems also signalling for paracrine IL-2 synthesis). The IL-2 synthesis mayunlikely in view of the normal responses recorded for some T cell thus be sufficient for autocrine, but not paracrine, purposes instimuli (anti-CD2 and/or anti-CD28), which are also dependent TM patients.

88 M. Perez-Blas et al.

Lastly, IM PBMC showed a significantly higher prolifera-tive response to PMA plus lonomycin than control cells (Table2), together with high IL-2R levels (Fig. 2). Although alternativeinterpretations are not excluded, these two observations may berelated, perhaps reflecting a pre-activation status on IM T cellsthat allows for a stronger proliferation upon appropriate(transmembrane) triggering.

Further work is required to elucidate the molecular basis ofIM-associated T cell anergy, because the steps that couple TCRto PKC activation are ill defined at the present time. Inparticular, G protein defects may be involved in this and otherID [4].

ACKNOWLEDGMENTS

Continued supply of reagents by A. Bernard, P. Martin, D. Olive, K.Sagawa, Hoffman-La Roche and Eurocetus is gratefully acknowledged.We thank A. Blanco, J. F. Guisasola, E. Barbosa, P. Iglesias-Casarrubios and A. Garcia for their help. The contribution ofM. P&rez-Blas and J. R. Regueiro to this work is equal, and the order ofauthorship is arbitrary. This work is supported in part by an FIS grant.

REFERENCES

1 Okano M, Thiele GM, Davis JR et al. Epstein-Barr virus andhuman diseases: recent advances in diagnosis. Clin Microbiol Rev1988; 1:300-9.

2 Tosato G, Magrath I, Koski I et al. Activation of suppressor T cellsduring Epstein-Barr-virus-induced infectious mononucleosis. NewEng J Med 1979; 301:1133-7.

3 Tomkinson BE, Wagner DK, Nelson DL et al. Activated lympho-cytes during acute Epstein-Barr virus infection. J Immunol 1987;139:3802-7.

4 Chatila T, Wong R, Young M et al. An immunodeficiencycharacterized by a defective signal transduction in T lymphocytes.New Eng J Med 1989; 320:696-702.

5 Moretta A, Ciccone E, Pantaleo G et al. Surface molecules involvedin the activation and regulation ofT or natural killer lymphocytes inhumans. Immunol Rev 1989; 111: 145-75.

6 De Franco AL. Between B cells and T cells. Nature 1991; 351:603.7 Sumaya CV, Ench Y. Epstein-Barr virus infectious mononucleosis

in children: clinical and general laboratory findings. Pediatrics 1985;75:1003-19.

8 Caldwell CW, Taylor HM. A rapid, no-wash method for immuno-phenotypic analysis by flow cytometry. Am J Clin Path 1986;86:600-7.

9 Regueiro JR, Lopez-Botet M, Landazuri MO et al. An in vivofunctional immune system lacking polyclonal T-cell surface ex-

pression of the CD3/Ti (WT31) complex. Scand J Immunol 1987;26:699-707.

10 Alarcon B, Regueiro JR, Arnaiz-Villena A et al. Familial defect inthe surface expression of the T-cell receptor-CD3 complex. NewEngl J Med 1988; 319:1203-8.

11 Forse RA, Christon NV, Meakins JL. Reliability of skin testing as a

measure of nutritional state. Arch Surg 1981; 116:1284-8.

12 Gillis S, Ferm MN, Ou W et al. T cell growth factor: parameters ofproduction and quantitative microassay for activity. J Immunol1978; 120:2027-32.

13 Amman AJ. Immunodeficiency diseases. In: Stites DP, Stobo JD,Wells JV, eds. Basic and clinical immunology. CT: Appleton andLange, 1987:347-53.

14 Tomkinson BE, Brown MC, Carrabis S et al. Soluble CD8 duringT cell activation. J Immunol 1989; 142:2230-6.

15 Clevers H, Alarcon B, Wileman T et al. The T cell receptor/CD3complex: a dynamic protein ensemble. Ann Rev Immunol 1988;6:629-62.

16 Pierres A, Lopez M, Cerdan C et al. Triggering CD28 moleculessynergize with CD2 (TI 1.1 and TI 1.2)-mediated T cell activation.Eur J Immunol 1988; 18:685-90.

17 Wakasugi H, Mahe Y, Huet S et al. Comparison of signals deliveredthrough CD3 and CD2 for T-cell activation: the role of calciuminflux and interleukin 1. Hum Immunol 1988; 23:163-78.

18 Leca G, Boumsell L, Fabbi MM et al. The sheep erythrocytereceptor and both alpha and beta chains of the human T lymphocyteantigen receptor bind the mitogenic lectin (Phytohaemagglutinin)from Phaseolus vulgaris. Scand J Immunol 1986; 23:535-44.

19 Van Lier RAW, Brouwer M, Aarden LA. Signals involved in T cellactivation. T cell proliferation induced through the synergisticaction of anti-CD28 and anti-CD2 monoclonal antibodies. Eur JImmunol 1988; 18:167-72.

20 June CH, Ledbetter JA, Lindstein T et al. Evidence for theinvolvement of three distinct signals in the induction of IL-2 geneexpression in human T lymphocytes. J Immunol 1990; 143:153-61.

21 Veltri RW, Kikta VA, WainwrightWH et al. Biologic and molecularcharacterization of the IgG serum blocking factor (SBF-IgG)isolated from sera of patients with EBV-induced infectious mono-nucleosis. J Immunol 1981; 127:320-8.

22 Gehrz RC, McAuliffe JJ, Linner KM et al. Defective membranefunction in a patient with severe combined immunodeficiency. ClinExp Immunol 1980; 39:344-8.

23 Chilson OP, Kelly-Chilson AE. Mitogenic lectins bind to the antigenreceptor on human lymphocytes. Eur J Immunol 1989; 19:389-96.

24 Crabtree GR. Contingent genetic regulatory events in T lymphocyteactivation. Science 1989; 243:355-61.

25 Toribio ML, Gutierrez-Ramos JC, Pezzi L et al. Interleukin-2-dependent autocrine proliferation in T-cell development. Nature1989; 342:82-5.

26 Moss DJ, Bishop CJ, Burrows SR et al. T lymphocytes in infectiousmononucleosis. I. T cell death in vitro. Clin Exp Immunol 1985;60:61-9.

27 Bishop CJ, Moss DJ, Ryan JM et al. T lymphocytes in infectiousmononucleosis. II. Response in vitro to interleukin-2 and establish-ment of T cell lines. Clin Exp Immunol 1985; 60:70-7.

28 Perez-Aciego P. Alarcon B, Arnaiz-Villena A et al. Expression andfunction of a variant T cell receptor complex lacking CD3y. J ExpMed 1991; 174:319-26.

29 Wegener AMK, Letourner F, Hoeveter A et al. The T cell receptor/CD3 complex is composed of at least two autonomous transductionmodules. Cell 1992; 68:83-95.