International Immunology, Vol. 4, No. 8, pp. 931 -934

© 1992 Oxford University Press

T cell receptor usage by HLA-DQw8specific T cell clones Torbjern Hansen, Knut E. A. Lundin, Gunnar Markussen, and Erik Thorsby Institute of Transplantation Immunology, The National Hospital, N-0027 Oslo, Norway Key words: cellular immunity, HLA-DQ antigens, immunogenetics, insulin-dependent diabetes mellitus, T lymphocytes, TCR, transplantation immunology

Abstract

Introduction The TCR recognizes peptide fragments of antigen bound to HLA molecules (1). The TCR has a significant homology to the Fab fragment of immunoglobulins. Accordingly, it has been suggested that certain parts of the TCRa and TCR/S, namely the complementarity determining regions (CDR) 1 and 2 encoded by the V genes, interact with the a-helices of the HLA molecule, while CDR3, encoded by the V-(D) - J junction, interacts with the peptide in the groove (2 - 4). Thus, one might expect some homologies between TCR recognizing peptides bound by the same HLA molecule. The HLA- DQw8 heterodimer is associated with susceptibility to develop insulin-dependent diabetes mellitus (IDDM) (5,6). In IDDM, the insulin-producing pancreatic /S cells are lost, probably because of an autoimmune response by T lymphocytes. One explanation of the DQw8 - IDDM association is that DQw8 can present relevant peptides derived from the pancreatic 0 cells to autoimmune T lymphocytes. If this is a main pathogenic event, it becomes important to determine whether T cells recognizing peptides in the cleft of DQw8 demonstrate some structural homologies of their TCR. In this study we report the first TCR sequences of DQw8-specific T cells. Evidence of possible preferential usage of certain TCR structures will be presented. Methods T lymphocyte clones (TLC) The four alloreactive DQw8-specific TLC BL22, BL23, BL25, and BL33, derived from a DQw7-positive donor (BL), have been presented previously (7,8). Sequencing showed that the BL23

and BL25 had identical nucleotide sequences of both their TCRa and -/3 genes. Therefore, they are almost certainly sister clones and will be referred to jointly as BL25. The alloreactive DQw8specific TLC (0B44) was derived from another DQw7-positive donor (0B) in the same way as the BL TLC were generated (8). The Herpes simplex virus (HSV)-specific, DQw8-restricted TLC (BF37) was derived from donor BF, and has also been described in detail elsewhere (9). Apart from these five TLC, we know of only one other TLC with the same exquisite specificity for DQw8 (10). TCR gene sequencing TCRa was sequenced by a slight modification of the rapid amplification of the cDNA ends (RACE) technique (11). Cytoplasmic RNA from 0.5-2 x 108 TLC was purified by phenol extraction. Firstly, strand TCRa cDNA was synthesized for 1 h at 42°C using Moloney murine lymphoma reverse transcriptase (BRL) and the Ca primer 1: 5'-GCCACTTTCAGGAGGAG-3' [Ca sequence in (12)]. The cDNA was boiled for 2 min, and then subjected to gel filtration using Pharmacia Sephacryl S300 spun columns equilibrated with 0.15 M NaCI in TE buffer pH 8.0. The gel filtered material was precipitated in 0.3 M Na-acetate (pH 5.2) and ethanol, washed with 70% ethanol, and dissolved in 7 y\ H2O. To this was added 4 /J of 5 x tailing buffer (Boehringer Mannheim, Mannheim, Germany), 6 /d of 5 mM CoCI2, 2 /J of 2 mM dATP, and 1 /J terminal deoxynucleotidyle transferase (Boehringer Mannheim). Tailing was performed for 10 min at 37°C, and completed by incubation at 95°C for 5 min. The pdy(A) tailed first strand cDNA was

Correspondence to: T. Hansen Transmitting editor: H. Wigzell

Received 5 February 1992, accepted 8 May 1992

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To Investigate whether T cells recognizing the same HLA molecule may demonstrate homology in parts of their TCR, five different HLA - DQw8-specific T lymphocyte clones (TLC) were studied. The TCRa and -0 genes of four alloreactive, HLA - DQw8-spedfic and one antigen-specific TLC were sequenced. All TLC used different Va and Vff genes. However, four of the TLC shared a certain CDR10 motif and all five used either Jp2.3 or -2.5. In addition, two used the same Ja. The results Indicate a possible preferential usage of certain TCR structures by T cells specific for DQw8.

932

TCR usage by HLA - D0w8-specific TLC

TCR& was amplified by synthesizing first strand cDNA and performing PCR using a degenerated primer specific for the leader sequence in conjunction with a C0 primer as described earlier (13), or by RACE. For RACE, we used the following primers. Ce primer 1 5'-CACTGTGCACCTCCTTC-3' (reverse transcription), Cp primer 2: 5'-CCATTCACCCACCGCTCAGCTCCA-3' (RACE-PCR), and C0 primer 3- 5'-GGAGAgCTCaaGCTTCTGATGGCTCA-3' (nested PCR) (14). For both TCRa and TCR0, at least three M13 plaques from each TLC were sequenced.

Table 1. TLC 0B44 HLA-DQw8 + cells only

is specifically

restimulated

Spont.

DR DQ DQA1 DQB1 0B44

Stimulator

None 16 w5 9015 WT24 9018 LO081785 3 w2 0501 4 w8 0301 9024 KT17 4 w8 0301 9092 BM92 4 w8 0301 9098 MT14B 9034 SAVC 4 w8 0302 4 w8 0301 9031 BOLETH 4 w7 0302 BL 7 w2 0201 9049 IBW9 9 w9 0302 9075 DKB

0201 0302 0302 0302 0302 0302 0301 201 0303

1624 1550 2256 9668 16572 22306 9812 18320 1886 2550 2378

by

± ± ± ± ± ± ± ± ± ± ±

a

196 332 329 256 533 675 291 b 879 084 120 078

360 346 724 728 484 446 876 708 236 502

± ± ± ± ± ± ± ± ± ±

24 31 44

08 17 83 24 37 32 25

"Mean c.p.m. ± SD of triplicates. b This restimulation with SAVC has not been reproducible.

Table 2. Summary of the TLC specificities TLC

BL22 (allo) BL25 (allo) BL33 (allo) 0B44 (allo) BF37 (HSV)

Stimulating DQw7 o1*0302 01*0301

cells D0w8 a1"0301 01"0302

_ -

+ + + + +

DGw8 a1*0302 0TO3O2

DQw9 a 1*0302 01*0303

Others1

+ + _

_ — -

_ _ -

nd 2

+

'Non-DOw8. Not determined.

2

Table 3. V and J gene usage by the TLC Results

TLC

Specificity of TLC Because detailed data concerning the TLC BL22, BL25, BL33, and BF37 have been published elsewhere (7-9), only relevant data concerning TLC 0B44 are provided (Table 1). TLC 0B44 was restimulated only by cells expressing DQw8, and this response was specifically inhibited using mAbs specific for DQ molecules (data not shown). The specificities of all TLC used are summarized in Table 2. The TLC reacted only when DQw8 was present in the stimulating/antigen presenting cells. In addition, BL33 discriminated between two variants of DQw8 with different a-chains. TCR sequences The Va, Ja, Vg, and Je usage of these TLC is displayed in Table 3, and the deduced aa sequences are shown in Fig. 1. BL22 had two productively rearranged TCRa genes, but we could only find one productively rearranged TCR0 gene. In addition, by sequencing 12 M13 clones from RACE-amplified BL22 TCRp, we identified a TCR0 'minigene' consisting of Je2.3 coupled to C02, but with germline DNA upstream of Jp2.3 (not shown). 0B44 had rearranged Va2.1 out of frame and BF37 had rearranged Va4 out of frame (not shown).

Ja

2.1

15

4.1a 3.1 19.0 21.1a 20.1

W 03 03 09

BL22BL25 BF37 BL33 0B44

1/0 71

2.3

5.1 5.3 82 6.7c

2.5 2.5 2.3 2.3

T w o in frame TCRa transcripts were found for BL22.

BL25 and BF37 both used members of the Vf5 family. All other TLC used Vas and V^s from different families ( < 7 5 % nucleotide sequence homology). Because it is assumed that only certain parts of the TCR, namely CDR1 and CDR2, bind to the HLA molecules, these regions were studied in detail. We defined CDR1 a as encompassing aa 2 5 - 3 1 , CDR1,, as aa 2 6 - 3 1 , CDR2a as aa 4 8 - 5 6 , and CDR2j, as aa 4 7 - 5 7 , according to Chothia et al. (3). As can be seen (Fig. 2), homology in the CDR1g was observed because all TLC, except BL22, used the motif ISGH. In the other CDRs there was little homology, although perhaps it may be significant that aa 53 and 55 of all TCRa, except for BL22, were basic.

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precipitated with Na-acetate and ethanol as detailed above, washed in 70% ethanol, and dissolved in TE buffer. A PCR was performed in 100 /J, using the tailed cDNA, 20 pmol of the Ca PCR primer 2: 5'-TCGGAACCCAATCACTGACAG-3', 5 pmol of the primer 5'-CCACTGCAGTCGACTCTAGAC 11 111 11 I 11 I I I I I I I-3' (anchor+ T), 20 pmol of the primer 5'-CCACTGCAGTCGACTCTAGAC-3' (anchor-T), and 2.5 U Taq polymerase (Perkin Elmer Cetus, Norwalk, CT, USA). The cycling conditions were 95°C for 1 min, 55°C for 5 min, and 72°C for 40 min, followed by 40 cycles of 95°C for 40 s, 55°C for 1 min 30 s, and 72°C for 1 mm 30 s. After the last cycle, the samples were incubated at 72°C for an additional 10 min. If necessary, a small fraction of the RACE product was subjected to nested PCR using the upstream Ca primer 3: 5'-GGATCTAGAGTCTCTgAGCTcGTACACGGC-3' and the anchor-T primer. The RACE products were size-selected by agarose gel electrophoresis and cloned in M13 using one of the restriction sites in the anchor primer, and either the natural Hin6\\\ site upstream of the Ca PCR primer 1, or the Sst I site introduced in the Ca PCR primer 2. The white M13 plaques were checked for the presence of TCRa insert by PCR using the M13 primer. 5'-CACAGGAAACAGCTATGACCATGA-3', and the Ca PCR primer 4: 5'-TACACGGCAGGGTCAGGGTTC-3'. Only plaques which gave rise to a PCR product of the expected size were sequenced.

TCR usage by HLA - DQw8-spedfic TLC 933

Vp BL22I KL25I TTJ**TJ/WTTyrftirJ \\J1Ffr*TwlrmlMlWM

(2J) CHIH1U7I

Fig 1. Deduced aa sequences of the TCR for (A) TCRa and (B) TCR0. Two in-frame TCRa transcnpts were found for BL22. The constant cysteines at aa positions 22 and 23 of Va and V/3, respectively, are indicated.

All TLC expressed either J? 2.3 or Je 2.5, and two TLC (BL25 and BF37) both expressed Ja 03.

CDRIa U25-31

CDtta U48-36

SDKCSQS

8I«Y(NQ3K

CDKlf)

1L221 IBOLTSSVM

BL25I

KT8lmL

TimurTP

1L331

TVSMFUI

SIS8UDIH

«44i

HHIATBD

IIQGTKTKV

BT37i

SVCISAL

LssaraacBG

Tl'flT

imonvpiDD

We sequenced the TCRa and TCRfi genes of tour alloreacbve, DQw8-specific, and one HSV-specific, DQw8-restricted TLC. We found that all TLC expressed different Va and Vp genes. However, there was some homology in the CDRIp and J 3 regions. We cannot establish which TCRa is expressed by the alloreactive TLC BL22 because two in frame TCRa transcripts were found. We consider it unlikely that TLC BL22 is a double clone because we could only find one TCRfi transcript using RACE, but this possibility cannot be excluded. There are several examples of TLC with two transcripts of either TCRa or -/3 (15,16). The Va were quite heterogeneous. However, two of the TLC (BL25 and BF37) both used Ja03. Because 61 different J a are known (T cell workshop, 11th International Histocompatibility Workshop, Yokohama 1991), this is probably not a coincidence. The V3 were more homogeneous. Two TLC (BL25 and BF37), used members of the Vfl 5 family. Furthermore, four of the TLC had the motif ISGH in the putative CDR1fl. In a study of DR4-restricted TLC, we found that nine of 35 TLC had this motif (Hansen etal., submitted). This difference may be statistically significant (P < 0.0313 by Fischer's exact test). The presence of such an homology in CDR1^ of TCR recognizing the same HLA molecule is in support of the current model of interaction of the TCR CDR1 and CDR2 with the a-helices of HLA. A more striking homology was found in the J^ region, because all five TLC used either J 3 2.3 or 6e 2.5, which are very similar. The sequence (D/E)TQYFGPGTRL(UT)VL is unique for these J