Eur. J. Immunol. 1993. 23: 499-504 Clonal analysis of human intestinal yb cells 499

heterogeneity in y 6 T cell clones biopsies of patients with celiac

Gennaro De LiberoA, Maria Paola RocciO, Giulia CasoratiO., Claudia GiachinoO, Giuseppina Oderda., Kaveth Tavassoli. and Nicola MigoneO

Department of Research, University HospitalA, Basel, Basel Institute for Immunology., Basel, Dipartimento di GeneticaO, Biologia e Chimica Medica, University of ToMo and C.N.R. Immunogenetica e lstocompatibdita Torino and Pediatric Gastroenterologyw, University of ToMo, Torino

T cell receptor from intestinal disease*

In celiac disease large numbers of y6 T lymphocytes infiltrate the intestinal epithelia. We have iskated intestinal‘yG T celi clones from patients with celiac disease and have analyzed their T cell receptor repert0ire.T cell lines and clones were obtained from jejunal biopsies of 14 celiac patients and 12 individuals without celiac disease. These were analyzed by staining with monoclonal antibodies against CD3, ap and y6 T cell receptor, by Southern blot with y- and &specific probes and by polymerase chain reaction using VG-specific oligonu- cleotides. Intestinal y6 cells from patients with celiac disease differed from those of controls with normal jejunal histology in that V61+ cells and VGl-V62- cells were significantly increased. There was no evidence of the expansion of one or more clones expressing particular types of y6 T cell receptor.

1 Introduction

Celiac disease (CD) is characterized by lesions of the small-intestinal mucosa that appear after gluten intake and which result in malabsorption. The injury seems to be the result of a cell-mediated immune response, which origi- nates in the lamina propria. The lesions are infiltrated by a large number of lymphoid cells, many of which express the yG T cell receptor (TcR, for a recent review see [l]).

Although much is known about y and 6 genes and their products, the antigens recognized by y6 cells are still poorly defined and virtually nothing is known about the function of intestinal y6 cells. In the mouse, there is a striking compartmentalization of distinct y6 T cell subsets express- ing different y and/or 6 chains in different epithelial tissues [2,3]. Preliminary phenotypical analysis has indicated that in humans, the distribution of Vy and VG chains also differs in the various tissues, although not to the same extent as in the mouse [4-71.

The finding that many y6 cells are closely associated with epithelial cells in CD lesions [5, 71 suggests that these cells might be important in the pathogenesis of the disease. Studies on y6 cells from biopsies are very limited at present due to difficulties in isolating and expanding mucosal T cells in tissue culture. Here we report the isolation of y6 clones from human gut biopsies, and their phenotypic and genotypic characterization. The repertoire of y6 T cells

[I 106701 ~~

* This work was supported by grants from Swiss National Research Foundation (31-27971.89 to G.D.L.), and C.N.R. P.E Ingegneria Genetica and C.N.R.Target Project “Biotecnologie e Biostrumentazioni” (to N.M.)

Correspondence: Gennaro De Libero, Experimental Immunology, Department of Research, University Hospital, Hebelstr. 20, CH- 4031 Basel. Switzerland

Abbreviation: CD: Celiac disease

Key words: T cell receptor y6 / Intraepithelial lymphocytes / Celiac disease / T cell clones / V6 genes

from intestinal biopsies of CD patients differed from that of cells of normal or inflamed duodenum in that there was a preference for V61 and V63 usage as well as for Vy chains different from Vy4 and Vy9.

2 Materials and methods

2.1 Patients

Duodenal biopsies were taken from 26 children (mean age 8 years and 6 months), 14 with CD as diagnosed according to the criteria of the European Society of Pediatric Gastroenterology and Nutrition [8], and 12 with other gastrointestinal disorders (referred below as controls; for details see Table 1).

2.2 Establishment of T cell lines and clones

The biopsies were washed twice in RPMI 1640 (Flow Laboratories, Ltd., Herts, GB) containing 100 U/ml peni- cillin and 100 pg/ml streptomycin (Flow), finely minced and washed. The pellet was resuspended in 3 ml of the same medium supplemented with 10% FCS and incubated overnight at 37°C with collagenase type IV (200 U/ml) (Sigma, St. Luis, MO). Cells were immediately cloned and maintained as previously described [9]. PBMC were obtained through a Lymphoprep density gradient (Ny- corned AS, Oslo, Norway) and stored frozen until use. T cell lines and clones were established and maintained as described [9].

2.3 Monoclonal antibodies used in these studies

The following mAb were used: TR66 (anti-CD3), BMA 032 (anti-ap TcR), 61 (anti-CG), y61, (anti-Cy), GTCS1 (anti-VGl/J1-2), BB3 (anti-V62), TiyA (anti-Vy9), 4Al l (anti-Vy4) [9]. The mAb A13 (anti-VG1) was kindly provided by Dr. S. Ferrini [lo]. Immunofluorescence studies with single and double staining were performed as described [9].

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1993 0014-2980/93/0202-0499$3.50 + .25/0

500 G. De Libero, M. F! Rocci, G. Casorati et al. Eur. J. Immunol. 1993. 23: 499-504

2.4 Southern blot analysis of y and 6 genes

The DNA configuration of the y and 6 loci was determined in T cell clones by Southern blot analysis with at least four restriction enzymes (BamHI, EcoFU, HindIII and BglII) and the following TcR6 probes J1, J3,V61, and V62, and TcR y probes J1, JP1, JP and JP2, as previously described [4]. In addition, theV63 rearrangements were confirmed by means of a 850-bp, V63-containing probe, and the partial rearrangements (DDJ) by a D61 probe, i.e. 1.7-kb EcoRI- HindIII segment from the E2.6 clone, kindly donated by Dr. T. Mak [l l] .

2.5 Polymerase chain reaction (PCR) analysis

Total RNA was extracted from 15 x lo6 cells as described [4], the first cDNA strand was synthesized from 1-5 pg total RNA using an oligo(dT) primer and murine Moloney leukemia virus reverse transcriptase (BFU, Gaithersburg, MD). A fraction of each cDNA sample (l/20) was amplified using V6- and C6-specific primers. The sequences of the primers are the following: V61: 5'-AGCAACTTCCCAG- CAAAGAG-3'; V62: 5'-AGGAAGACCCAAGGTAAC- ACAA-3'; V63: 5'-CACTGTATATTCAAATCCAGA-3'; V64: 5'-TGACACCAGTGATCCAAGTTA-3'; V65: 5'- CTGTGACTATACTAACAGCATGT-3' ; V66: 5'-TATC- ATGGATTCCCAGCC-3' ; V67: 5'-CTTGATAACTGA- AGGATGATG-3' ; V68: 5'-AGTCTCAACCAGAGATG- TCTG-3'; C6: exon 1,5'-CTTCACCAGACAAGCGAC- AT-3'.V67 and V68 primers are derived from the sequence of twoV genes recently found rearranged to C6 (N. Migone et al., manuscript in preparation). The amplification pro- files were either 25 or 35 cycles (94 "C, 45 s; 50 "C, 45 s and 72 "C, 45 s). PCR products were loaded on a 1.5 YO agarose gel and transferred to a Zeta probe filter (Bio-Rad). Filters were hybridized using the following 32P-labeled oligonu- cleotides as probes: J61: 5'-ACTTGGTTCCACAGTCAC- AC-3'; 562: 5'-GTTCCACGATGAG'ITGTGTTC-3' ; 563 5'-AAACATCTGTCGGGTGTCCCA-3'.

3 Results

3.1 TcR expression by intestinal T lymphocytes from CD patients and controls

PHA lines were isolated from the biopsies of 14 CD patients and 12 age-matched controls (10 with histologically normal jejunal mucosa and 2 with duodenitis) (Table l ) , and were characterized by staining with mAb specific for TcR a@ and y6. The lines isolated from controls showed significantly lower levels of y6 cells compared to CD patients (medians: 2.9 YO vs. 19.6 YO, respectively, p = 0.047; the statistical analyses were based on Fisher' exact test on the medians). A single control line contained an exceptionally high proportion of y6 cells (63 %).The y6 cell fraction in CD lines were 22-53 YO in 7,8-15 YO in 6 and 3.8% in 1 patient. These findings are in agreement with previous immunohistochemical studies [5 , 71.

Analysis of the lines with mAb specific for the y and 6 variable (V) chains revealed a considerable variability. y6 Cells from five controls resembled the major y6 Tcell

subset in the blood [4], i.e. the expression of Vy9 and V62 was very high. The lines from five other controls predomi- nantly expressed V61, while those from two other controls prevalently expressed V6 chains other than V61 and V62. Vy4 was the second most frequently used y chain, after Vy9, in six control lines.

Most strikingly, in 11 out of 14 CD lines, V61 was the predominant 6 chain. The 6TCS1+ fraction of the CD3+ cells from control and CD patients differed significantly (p = 0.005). Vy different from Vy9 and Vy4 (defined as Vyx), and V6 different fromV61 and V62 (V6x) were more abundant in CD than in control lines (medians: Vyx, 25 % vs. 7.4%, p = not significant; V ~ X , 18% vs. 0.1 YO, p = 0.047). Interestingly, this different pattern of y and 6 chain expression was found both in lines from patients with villous atrophy and in patients whose intestines exhibited a normal histology after intake of a gluten-free diet (data not shown).

3.2 TcR gene usage of intestinal y6 clones from CD patients and controls

To study V6 and Vy chains in single cells, we established T cell clones from gut biopsies at day 0. From a total of 350 clones derived from 3 CD patients and 1 control, 91 were y6+. These clones were further analyzed with Vy- and V6-specific mAb (Table 2). The findings can be summa- rized as follows: (i) 59 % of the clones express V61, 15 % V62 and 25 % express neither V61 nor V62. These results are in agreement with the data of the CD lines described above, (ii) V62 chain usually pairs withVy9, (iii) a relevant fraction (25 YO) of the CD clones express a V6 different fromV61 and V62 (V6x) and (iv) in CD clonesV61 and V6x chains pair with different Vy.

To identify theV6x expanded in CD patients, we studied by Southern blot the genomic DNA of nine 61+ T cell clones from patients 10 and 13 which did not express V61-J1/2 (recognized by 6TCS1 mAb) or V62 (recognized by BB3 mAb). As shown in Fig. 1,V63 was found rearranged and expressed in five out of the six clones tested from patient 10; the same V6 gene was expressed in two out of the three clones analyzed from patient 13.The two remain- ing clones showed rearrangement of V65 and of a new V gene hitherto not found rearranged at the 6 locus (here called V68, N. Migone et al., manuscript in preparation). Southern blot analysis of theVy locus indicated that at least four different Vy genes (Vy3, Vy4, Vy8, Vy5) were rear- ranged and expressed together with V63 in these clones (Fig. 2), suggesting that V63 can pair with any Vy chain.

TheV6 gene repertoire within the V6x subset was analyzed more extensively by PCR.Two bulk cell lines, derived from biopsies of patients 10 and 13, were sorted with FACS as 61+, 6TCS1-, BB3-, and then cDNA amplified using primersspecificforV61,2,3,4,5,6,7,8andCG.Toanalyze the contribution of the different J6 elements to the V6 repertoire, the PCR products were electrophoresed, blot- ted and hybridized with J61-, 562- and J63-specific probes. Fig. 3 shows the typical V-J6 usage in a control population of y6+ cells derived from the PBL of a non-celiac donor and that of the two V6x+ sorted intestinal CD cell lines.

Eur. J. Immunol. 1993. 23: 499-SO4 Clonal analysis of human intestinal y6 cells SO1

Table 1. Study groups

Patient Sex Age Histology Duration of disease (months)

5 p e of diet

Celiacs 10 11 12 13 14 15

22 24 23 32 35 37 39 43

F F F M F F

F M F F F M M M

10 m 12 y, 10 m

3Y 9Y

3 p l m 6 y , l m

1 y, 10 m 11 m 14 Y

15 y, 9 m 3 y , 5 m

22 Y 11 m 6Y

Non-celiac controlsb) 2 M 11 y, l m

3 F 9y, 10m

5 M 8Y

6 M 8 y , 7 m

7 M 14y,4m

9 F 9 y 9 7 m

33 F 13 y, 6 m

38 F 7y,11m

41 F 9 y , 1 1 m 42 M 6y,11m

36 M 15 Y

40 F 11 Y

Villous atrophy Villous atrophy

Villous subatrophy Villous subatrophy Normal duodenum Normal duodenum

SCG HP+a) Villous subatrophy

Villous atrophy Villous subatrophy SCG HP+ Villous subatrophy SCG HP- Villous subatrophy SCG HP-

Normal duodenum Villous atrophy SCG HP-

Villous subatrophy SCG HP-

Normal duodenum SCG HP- Duodenitis SCG HP+

Normal duodenum Peptic oesophagitis

Duodenitis SCG HP+

Normal duodenum SCG HP+

Normal duodenum Peptic oesophagitis

Normal duodenum SCG HP- Normal duodenum SCG HP- Normal duodenum SCG HP- Normal duodenum SCG HP- Normal duodenum SCG HP- Normal duodenum SCG HP+

6 72 24 96 24 60

14 5

120 150 36

240 1

68

24

36

60

48

24

48

24 18 6

12 15 18

Gluten containing Gluten containing

Gluten challenge (1 m) Gluten challenge (1 m)

Gluten free (13 m) Gluten free (6 m)

Gluten challenge (1 m) Gluten containing Gluten containing

Gluten free (since weaning) Gluten challenge (1 m)

Gluten free (6 m) Gluten containing (4 m)

Gluten containing (since weaning)

Indication for endoscopy and final diagnosis Abdominal pain Antral gastritis Abdominal pain

H. pylori gastritis Recurrent vomiting Reflux oesophagitis

Abdominal pain Duodenal ulcer Abdominal pain

H. pylori gastritis Recurrent vomiting Reflux oesophagitis

History of H. pylori gastritis History of H . pylori gastritis History of H. pylori gastritis History of H . pylori gastritis History of H. pylori gastritis History of H. pylori gastritis

a) SCG HP+: Superficial chronic gastritis associated with H. pylori infection. b) All non-celiac controls were on a normal (gluten-containing) diet since weaning.

Table 2. V6 and Vy gene usage by clones derived from gut biopsies

mAb Reactivity y6 Pair Donold) Total clones 81 6TCS1 BB3 TiyA 4All 7 10 12 13

+ + - - vyx V61 + + - + - vy9 V61 + + - - + vy4 V61 Total V61

+ + - vy9 V62 - - vyx V8X

- - + vy4 V6X

-

- + + +

- - -

Total V6x Total clones

9b 1 2

12(44 %) lO(37 %) 2 3 5(19%)

27

8 3 2

13(57 %) 3(13 %) 5 2 7(30 %)

23

6 1 6

13(68 %)

l(5 %) 5 0 5(26 %)

19

10 33 1 6 5 15

16(73 %)

0 14 3 15 3 8 6(27 %)

22 91

a) Donor 7 is a non-celiac subject. b) Number of clones.

502 G. De Libero, M. I? Rocci, G. Casorati et al. Eur. J. Immunol. 1993. 23: 499-504

Figure 1 . Southern blot hybridization of nine intestinal lympho- cyte y6 clones with four different TcR6 probes, and three different restriction enzymes. Clones 1-6 are from patient 10, clones 7-9 from patient 13. C, unrearranged control DNA. From the top: J61 and HindIII, 563 and BglII, D61 and EcoRI,V63 and HindIILThe size in kb of the rearranged fragments and the gene assignment (based on the hybridizations with these probes, plusV65 and V68, not shown), are indicated on the right. The germ-line size of the fragments containing the probes are indicated on the left. V63 is rearranged to J61 in lanes 2-6 and 9, to 563 in lane 8.The HindIII site between D61 and D62 is never deleted following the V63 rearrangement (V63 rearranges by inversion), thus indicating that in all seven cases D62 is used, D61 being germ line. The unproductive chromosome is partially rearranged in all cases, except in lane 9 where this typing remained unclear. D2-(D3)-J1 was found in cases 1-3 and 5-9, D2-(D3)-J3 in case 4.

In the PBL y6 line we could detect only the V61,V62 and V63 transcripts (the last one only after 35 cycles of PCR amplification), thus confirming that these are the V6 genes most frequently expressed in the peripheral blood [4]. As expected, in PBL J61 was the segment mostly involved in the rearrangements (Fig. 3, panel B). In the V61-J1/2-, V62- CD lines we noted a particular pattern of theV6 gene usage. Transcripts containing V63,V65 and V68 sequences were amplified in both samples, and in patient 10 a V64 band could also be detected.V61-J1/2 and V62 transcripts were also found and are probably derived from the unproductive allele (Fig. 3, sample 1-10 and 1-13, lanes 1 and 2).V61-J3 products were also detected (Fig. 3, sam- ple 1-10 and sample 1-13, lane 1). This finding is not surprising, since GTCS1 mAb does not detect V61-J3 gene products [4,12]. To confirm these data, we analyzed by FACS the same VGx-sorted bulk lines using A13 mAb, which detects all V61+ cells [lo], and found positive cells in both cell lines (data not shown).

In conclusion, both phenotypic and genotypic studies indicate that the y6 population which expanded in the gut of CD patients is heterogeneous. Indeed, V61 chain is rearranged either to J1, 52, or J3, and is associated with Vy9, Vy4 or other Vys without apparent preference. The

Figure 2. Southern blot hybridization of the same clones shown in Fig. 1 using the Jyl probe. EcoRI and BamHI digestions are shown in the top and in the bottom panel, respectively. The size in kb of the rearranged fragments and the Vy gene assignment (based on the size of the fragments that hybridize with the Jyl probe) [4] are indicated on the left.The EcoRI 1.55- and 3.2-kb bands represent the germ-line Jyl and Jy2 gene segments, respectively.The 2.75-kb band in lane 4 could not be assigned to any known rearranged or germ-line polymorphic segment.

Figure 3. PCR analysis of the TcR 6 gene expression in a PBL y6+ cell line derived from a non-celiac donor, and in two 61+ 6TCS1- BB3- intestinal lymphocyte bulk cell lines from patients 10 and 13. Panel A shows ethidium bromide-stained gels after 35 cycles of PCR.The DNA size marker is a lambda phage DNA digested with HindIII and a a x 1 7 4 phage RF DNA digested with HaeIII. The PCR-amplified fragments are between 350 and 500 bp. The numbers from 1 to 8 indicate theV6-specific primers used for PCR. The amplified products were transferred to filters and hybridized with 32P-labeled J61-, 562- and J63-specific probes (panel B, C and D, respectively). In panel C, the 562 probe cross-hybridizes with aberrant PCR products of smaller size whose nature is under investigation.

Eur. J. Immunol. 1993. 23: 499-504 Clonal analysis of human intestinal yS cells 503

V61-V62- population contains a variety of V6 elements, some of which, e.g.V63,V64,V65, and V68, seem to be preferentially selected in individual patients, and may associate with at least four different Vy genes.

4 Discussion

In this report we describe the phenotype and the V gene repertoire of y8 T cell lines and clones derived from the small intestine of 14 celiac patients and 12 control subjects who had suffered from other gastrointestinal disturbances but had a normal villous architecture of the jejunum at the time of biopsy. The data presented here show that in the Tcell lines from 13 out of 14 CD patients the y6 cell fraction was significantly increased, and confirm that the V61-positive subset was mainly responsible for this phe- nomenon [5,7, 131. In addition, compared to the control group, the Val-, V62- y6 cells appeared significantly expanded in CD (this fraction was above 15% of the y6 cells in 8/14 patients but only in 2/12 controls). Expansion of the V6x phenotype in CD was previously suggested on the basis of immunohistochemical studies [5, 71, and more recently by Rust et al. [13]. However, no systematic study on the molecular heterogeneity of this cell subset has so far been reported. Intrigued by the possible role of this cell population in CD, we have characterized in detail the TcR V chains used by these cells through the analysis of the y and 6 V and J segments expressed by 6TCS1- and BB3- T cell lines and clones. Furthermore, our screening included the six V6 elements previously described [14], plus two additional V genes,V67 and V68. It is worth noting that like most V6 genes,V67 and V68 have been previously characterized asVa members (Va28 and Va14, respective- ly), and we have recently found them expressed in y6 clones from thymus and PBL (N.M., manuscript submitted). Interestingly, V88 represents the human homolog of the murine V64, which is known to be abundantly expressed in intraepithelial lymphocytes of strains with particular MHC haplotypes [ 151.

We demonstrated that the V6x population uses at least four different V6 genes, including the newly described V68, variably joined to any J6 element. The Val-, V62- clones showed a high frequency of V63 usage. Overall, our findings are consistent with a polyclonal expansion, since all the V63+ clones tested carried different rearrangements at the y locus.

From the y6 cells selected for being 6TCS1- and BB3-, Val-positive cDNA were also obtained. The molecular analysis demonstrated that they were mostly due to V61- 563 products which are not detectable by 6TCS1 mAb [4, 121. Although we cannot exclude that a fraction of the V61-J63 cDNA may partly derive from the unproductive chromosome, it is interesting to note that such products are infrequent or barely detectable in peripheral blood. If this tendency is confirmed on paired samples from the same individuals it might suggest a higher clonal heterogeneity among intestinal V61+ T cells.

The a@ oligoclonality recently observed in intestinal muco- sa of bona fide normal subjects [ 16, 171 contrasts with the heterogeneity of y6 TcR in our CD lines. It is unclear at present whether these differences are related to present or

previous pathological conditions, to peculiar in vivo distri- bution of the two cell types within the intraepithelial and lamina propria lymphocytes, or to the distinct functions exerted by y6 and a@ cells.

The finding of heterogeneous y6 populations in the intes- tinal mucosa of CD patients might suggest that they were expanded in response to a variety of antigens to which celiac patients may be particularly reactive, such as differ- ent gliadin fractions, or adenovirus antigens.

It is not known whether y6 cells participate in the generation of local tissue damage. We and other authors have found increased numbers of y6 intestinal lymphocytes also in patients on a gluten-free diet with normal jejunal villous architecture [5, 7, 13, 18l.These results may suggest that the y6 cells expanded in the intestinal mucosa are not autoaggressive, and their presence in the lesion could be an epiphenomenon not associated with the pathogenesis of CD [l]. Thus, y6 cells could have been expanded after the inflammation was initiated by other cells.

In the mouse, a major population of gut lymphocytes, which lack the Thy-1 marker and express the CD8 aa homodimer, it locally generated [19]. Human intestine might also promote T cell maturation, since human intes- tinal lymphocytes with a phenotype resembling that of immature thymocytes and expressing CD8 aa homodimers have been described [6,20]. In CD, the presence of an altered microenvironment and/or genetic factors might favor the extrathymic differentiation of precursor cells into y6 T lymphocytes.

We thank Dr. W Haas for invaluable discussions and critical reading of the manuscript: Drs. S. Abrignani, J. Baumann, K . Karjalainen, A . Lanzavecchia and L. Mori for reading the manuscript; Drs. M . Brenner, J. Borst, Z Hercend, L. Moretta, and S. Ferrini for providing mAb.

Received May 19, 1992; in final revised form October 22, 1992.

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