immunodominance correlates with t-cell receptor (ap) gene usage

8
Immunology 1994 82 343-350 Immunodominance correlates with T-cell receptor (ap) gene usage in the class TI-restricted response to influenza haemagglutinin C. A. SMITH, C. M. GRAHAM & D. B. THOMAS National Institutefor Medical Research, London SUMMARY Class II-restricted T-cell clones elicited by natural infection with influenza A virus (H3N2 subtype) exhibit extensive diversity in their recognition specificity for the envelope glycoprotein, haemagglutinin, and focus on hypervariable regions of the HAl subunit that feature in antigenic drift. However, T-cell clones established from the same individual focus on a single antigenic site with differing fine specificity for mutant viruses. We wished to determine whether such diversity of the haplotype and contrasting immunodominance of the individual's repertoire was mirrored in T- cell receptor (TcR) gene usage. A structural analysis was undertaken of the a and f chains of TcR from a panel of CD4+ T-cell memory clones established in vitro after natural infection with X31 virus and specific for eight distinct antigenic sites of the HAl subunit: p48-67 (Ak), p58-73 (Ad), p120-139 (Ak), pl77-199 (Ad), pl86-200 (Ad), p226-245 (Ek), p246-265 (Ek) and p269-288 (Ak). Direct sequencing of the a and P chains, using the polymerase chain reaction, revealed that T-cell clones derived from the same donor used identical Vp Dp Jf, and Vc, Ja, elements. Moreover there was extensive diversity in usage of Vp (VpI or V64 or Vp68) genes between individual mice, in association with diverse J# and Va, Ja, elements for the recognition of a common antigenic peptide. We conclude that the CD4+ T-cell memory repertoire of the individual, following primary exposure to infectious virus, is oligoclonal and recruited from a limited number of precursor cells. INTRODUCTION A distinguishing feature of the class II-restricted T-cell repertoire for influenza haemagglutinin (HA) of the H3 subtype elicited by natural infection with X31 virus is the diversity of antigenic peptides that can be recognized in association with a single Ta restriction element.'-5 The Ad_ and Ak-restricted T-cell clones, so far characterized, recognize a majority of surface accessible regions on the membrane distal ectodomain of the HAl subunit, amounting to five antigenic peptides for a single haplotype. The amino acid sequences of the T-cell epitopes correspond, in the three-dimensional structure of the HA molecule to hypervariable regions that feature in antigenic drift.6'7 Moreover T-cell clones are sensitive to drift substitutions that have occurred in these sites and fail to recognize mutant viruses. What is particularly noteworthy, however, is that individual mice, albeit of the same inbred strain and infected with X31 virus under identical conditions of dose and route, recognize different antigenic sites. Even so, within an individual's repertoire, T-cell clones that focus on a common antigenic site do exhibit some differences in their fine specificity for mutant HA. The purpose of this study, therefore, was to Received 16 December 1993; accepted 8 March 1994. Correspondence: Dr D. B. Thomas, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. determine whether diversity of the haplotype, and contrasting immunodominance of the individual's T-cell repertoire, was mirrored in T-cell receptor (TcR) a4 gene usage. Particular attention was paid to T-cell clones from the same individual, in an attempt to correlate TcR structure with fine specificity for antigen. MATERIALS AND METHODS cDNA synthesis Total cellular RNA was extracted from individual T-cell clones 6-8 days post-viral stimulation using the single-step acid guanidinium thiocyanate-phenol-chloroform method.8 Single-stranded cDNA synthesis was carried out using 10 pg total RNA with AMV reverse transcriptase and an antisense oligonucleotide specific for the constant region of either the a chain or the P chain genes. For the a chain, 13 nmol of the primer C a9 were reacted in 50mM Tris pH 8-3, 10mM MgCl2, 70 mm KCl, 80 mm 2-mercaptoethanol, 0 5 mm deoxy- nucleotide triphosphates (dNTP), 24 U reverse transcriptase in a 50 Mp volume. For the ,B chain, 12 7 nmol of the primer COA (TGATGGCTCAAACAAGGAGAC) were reacted in 50mM Tris pH 8-3, 10 mM MgCl2, 140 mm KCl, 200 mm dithiothreitol, 1 mM dNTP, 24 U reverse transcriptase in a 50 pl volume. The cDNA product was extracted with phenol/chloroform, ethanol precipitated, and resuspended in 100 p1 (a cDNA) or 20p (I cDNA) of distilled water. 343

Upload: vuongcong

Post on 11-Feb-2017

217 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Immunodominance correlates with T-cell receptor (ap) gene usage

Immunology 1994 82 343-350

Immunodominance correlates with T-cell receptor (ap) gene usage in theclass TI-restricted response to influenza haemagglutinin

C. A. SMITH, C. M. GRAHAM & D. B. THOMAS National Institutefor Medical Research, London

SUMMARY

Class II-restricted T-cell clones elicited by natural infection with influenza A virus (H3N2 subtype)exhibit extensive diversity in their recognition specificity for the envelope glycoprotein,haemagglutinin, and focus on hypervariable regions of the HAl subunit that feature in antigenicdrift. However, T-cell clones established from the same individual focus on a single antigenic sitewith differing fine specificity for mutant viruses. We wished to determine whether such diversity ofthe haplotype and contrasting immunodominance of the individual's repertoire was mirrored in T-cell receptor (TcR) gene usage. A structural analysis was undertaken of the a and f chains of TcRfrom a panel of CD4+ T-cell memory clones established in vitro after natural infection with X31virus and specific for eight distinct antigenic sites of the HAl subunit: p48-67 (Ak), p58-73 (Ad),p120-139 (Ak), pl77-199 (Ad), pl86-200 (Ad), p226-245 (Ek), p246-265 (Ek) and p269-288 (Ak).Direct sequencing of the a and P chains, using the polymerase chain reaction, revealed that T-cellclones derived from the same donor used identical Vp Dp Jf, and Vc, Ja, elements. Moreover therewas extensive diversity in usage of Vp (VpI or V64 or Vp68) genes between individual mice, inassociation with diverse J# and Va, Ja, elements for the recognition of a common antigenic peptide.We conclude that the CD4+ T-cell memory repertoire of the individual, following primaryexposure to infectious virus, is oligoclonal and recruited from a limited number of precursor cells.

INTRODUCTION

A distinguishing feature of the class II-restricted T-cellrepertoire for influenza haemagglutinin (HA) of the H3subtype elicited by natural infection with X31 virus is thediversity of antigenic peptides that can be recognized inassociation with a single Ta restriction element.'-5 The Ad_and Ak-restricted T-cell clones, so far characterized, recognize amajority of surface accessible regions on the membrane distalectodomain of the HAl subunit, amounting to five antigenicpeptides for a single haplotype. The amino acid sequences ofthe T-cell epitopes correspond, in the three-dimensionalstructure of the HA molecule to hypervariable regions thatfeature in antigenic drift.6'7 Moreover T-cell clones are sensitiveto drift substitutions that have occurred in these sites and fail torecognize mutant viruses.

What is particularly noteworthy, however, is that individualmice, albeit of the same inbred strain and infected with X31virus under identical conditions of dose and route, recognizedifferent antigenic sites. Even so, within an individual'srepertoire, T-cell clones that focus on a common antigenicsite do exhibit some differences in their fine specificity formutant HA. The purpose of this study, therefore, was to

Received 16 December 1993; accepted 8 March 1994.

Correspondence: Dr D. B. Thomas, National Institute for MedicalResearch, The Ridgeway, Mill Hill, London NW7 1AA, U.K.

determine whether diversity of the haplotype, and contrastingimmunodominance of the individual's T-cell repertoire, wasmirrored in T-cell receptor (TcR) a4 gene usage. Particularattention was paid to T-cell clones from the same individual, inan attempt to correlate TcR structure with fine specificity forantigen.

MATERIALS AND METHODS

cDNA synthesisTotal cellular RNA was extracted from individual T-cell clones6-8 days post-viral stimulation using the single-step acidguanidinium thiocyanate-phenol-chloroform method.8

Single-stranded cDNA synthesis was carried out using10 pg total RNA with AMV reverse transcriptase and anantisense oligonucleotide specific for the constant region ofeither the a chain or the P chain genes. For the a chain, 13 nmolof the primer C a9 were reacted in 50mM Tris pH 8-3, 10mMMgCl2, 70mm KCl, 80mm 2-mercaptoethanol, 0 5mm deoxy-nucleotide triphosphates (dNTP), 24 U reverse transcriptase ina 50 Mp volume. For the ,B chain, 12 7 nmol of the primer COA(TGATGGCTCAAACAAGGAGAC) were reacted in 50mMTris pH 8-3, 10 mM MgCl2, 140mm KCl, 200mm dithiothreitol,1 mM dNTP, 24 U reverse transcriptase in a 50 pl volume. ThecDNA product was extracted with phenol/chloroform, ethanolprecipitated, and resuspended in 100 p1 (a cDNA) or 20p (IcDNA) of distilled water.

343

Page 2: Immunodominance correlates with T-cell receptor (ap) gene usage

C. A. Smith, C. M. Graham & D. B. Thomas

Polymerase chain reaction (PCR)Oligonucleotide primers were synthesized (Applied Biosystems380 DNA synthesizer; Cheshire, U.K.) to correspondto previously published sequences of specific VP regions10-14as follows: V _1-4ACTCAGTCACCAAGATAT, Vp2-GTG-ACTTTGCTGGAGCAAAAC, Vp3-4GTCATTCAGACTC-CAAGATAT, Vp4-1'CTGGTGGCAGTCACAGGGAGC,VP5. 1-3GGGGTTGTCCAGTCTCCAAGA, V66-'-GGTGG-CATCATTACTCAGACA, Vp7-'GACATGAAAGTAACC-CAGATG, Vp8.1-'AGGCTGCAGTCACCCAAAGTC, Vp9-'GATACTACGGTTAAGCAGAAC, V1 1_2GCTGGTGTC-ATCCAAACACCT, V#14-'GCTCAGACTATCCATCAA-TGG, Vp15-'GGAGCACTCGTCTATCAATAT, Vpl6-'GGACCCAAAGTCTTACAGATC.

Va primers were synthesized according to previouslypublished sequences.9 These Va, and VP primers were used inindividual PCR reactions'5 together with an appropriateconstant region antisense oligonucleotide; Cab9 to determineVa usage and CpB (AGTCACATTTCTCAGATCCTC) todetermine VP usage. For the a chain, 0' 5yl cDNA was

amplified in the presence of 0-3 yM each of Cab and one Vc,primer, 12SMm dNTP, 20mM, Tris pH8-2, 10mM KCl, 2mMMgC12, 6mM (NH4)2 SO4, 0-1% Triton X, lOg/ml bovineserum albumin (BSA), 5% glycerol, and 1 25 U DNApolymerase (Stratagene) in a 50 MI reaction volume. Theamplification cycle was 1 x (950/3 min, 570/1 min, 72°/1 min),35 x (95°/1 min, 570/1 min, 72°/1 min), 1 x (72°/l0min). Forthe chain, Ipl cDNA was amplified in the presence of 0 1 Mm

each of C#B and one Vl primer, 200Mm dNTP, 10 mm TrispH 8-3, 50mM KCI, l 5mM MgCl2, 001% (w/v) carageenan,and 2-5 U Taq DNA polymerase (Perkin-Elmer Cetus, Nor-walk, CT) in a 100 MI reaction volume. The amplification cyclewas x (95°/3 min, 50/1 min, 70/1-5 min), 40 x (95/1 min,50/l min, 700/15 min), I x (70/5 min).

PCR product sequence analysisPCR products were separated on a 2% agarose gel and theband of interest cut out. The cleaned product was directlysequenced by a modified chain-termination method'6 toconfirm VP and V, usage and determine the Dp JO andJa, genes used. Primers used were as for PCR, and withan additional Vpl-specific primer 226ATATCTGCCGTG-GATCCAGAA.

CD4+ T-cell memory clonesThe derivations of the HA-specific T-cell clones used in thisstudy have been described elsewhere. 2'4 CBA/Ca or BALB/cmice were infected intranasally with X31 virus (H3N2 subtype;5 HAU) and CD4+ T-cell lines were established > 6 weeksafter recovery from primary infection, and followingrechallenge in vivo with purified, inactivated virus (500 HAU)or HA (5 g). T-cell clones were obtained by limiting dilutionand maintained in vitro by restimulation with X31 virus(100 HAU/ml) and irradiated (3000 rads) syngeneic spleencells as antigen-presenting cells every 10-12 days with theaddition of interleukin-2 (IL-2)-containing culture supernatant

3 days after antigen.

FACS analysisT-cell clones were analysed on the FACS for Vp expression

using the following mAb-specific reagents: V62 (B20.6.517), V,3

(KJ25'8) V.4 (KT4.1019), Vp6 (44.2.120), Vp7 (TR31021) Vp8(F23.122) and a fluorescein isothiocyanate (FITC)-conjugatedanti-mouse IgG antibody (Sigma Chemical Co., Poole, U.K.).

Accessory molecule expression was determined by FACSanalysis using FITC-conjugated anti-mouse CD1 la (Pharmin-gen, San Diego, CA), or phycoerythrin-conjugated anti-mouseCD4 (Becton Dickinson, Mountain View, CA).

RESULTS

Iad_ and Iak-restricted recognition of HA

Figure 1 shows the location of Ad, or Ak, or Ek-restricted T-cell

epitopes, and their donor origin, on the three-dimensionalstructure of the HA monomer. Assignments were made on thebasis of proliferative responses of CD4+ T-cell clones,established from individual mice, to a panel of syntheticpeptides corresponding to the primary sequence of X31 virus.2There was no obvious homology between different peptidespresented by the same Ta molecule, and the minimum peptidelength for stimulation was between 15 and 20 amino acidresidues.

Clonal diversity for immunodominant sites

Although clones from the same donor recognized a common

antigenic site, in several instances they could be distinguished in

Ia dla

Figure 1. Location of the Ad_, Ak- and Ek-restricted T-cell recognition

sites on the three-dimensional structure of the HA monomer (H3

subtype; refs 6,7). The amino acid sequences of individual peptides are

presented in refs 2 (Ak or Ek) and 4 (Ad).

344

Page 3: Immunodominance correlates with T-cell receptor (ap) gene usage

Influenza haemagglutinin-specific TcR 345

Table 1. Recognition specificity of T-cell clones established from donor BA5 (BALB/c; H-2d) or CB12 (CBA/Ca; H-2k) for natural variant viruses ofthe H3 subtype (see refs 2, 3 and 7 for amino acid sequence data) or laboratory mutants ofX31 virus, isolated at this Institute, differing from wild typevirus by single amino acid substitutions in the HAl subunit at the positions indicated: (U) proliferative responses to virus (50-200 HAU/ml)

equivalent to (80-150%) those for immunizing X31 virus; (El) proliferative responses < 5% of X31-specific responses

Natural variants Laboratory mutants

135 193 193 193 198 199

Donor Clone Specificity X31 ENG69 HK71 ENG72 PC73 HAN73 SC074 VIC75 TEX77 BK79 CN84 G/R S/N S/R S/I A/E S/P

BA5 T6 (Ad, p186-200) * * U* U* O C C E*E C C*BA5 Tll1(Ad, pl86-200) * CO * * CO * C CO C E*JEBA5 T16 (Ad, p186-200) U C C U U * U * U C C U U U U U U

CB12 T4 (Ak, pl2O139) * * U U U C C C C C C U* U* UCR12 T5 (Ak, pl20-139) * C U U U* U C C C C UE***CB12 TiI (Ak, p120-139) * * O * * * * C C C U U U U U

their responses to natural and/or laboratory mutant viruses ofthe H3 subtype. Such clonal diversity is illustrated in Table 1

for 'families' of T-cell clones established from donor BA5 (Ad_restricted for p186-200) or CB12 (Ak-restricted for p120-139).Representative dose-response curves (Fig. 2) illustrate thediffering effects of single amino acid substitutions in laboratoryvariants (HA1 193 S-.R; 198 A-.E) on the responses of clonesBA5-T6 or BA5-Ti and have been extensively documentedelsewhere.2-5

Consider donor BA5: all of the T-cell clones studied fromthis individual could be classified into three distinct specificitygroups according to their responses to mutant viruses contain-ing known amino acid substitutions within the T-cell epitope,HAl 186-200. These included either natural variant viruses or

laboratory mutants of X31 virus differing from wild type virusby single substitutions at HAl 193 (S-.N, S-.R, S-I) or HAl198 (A-.E) or HAl 199 (HAl S-+P). T-cell clones establishedfrom donor CB12 also differed in their proliferative responses

30-

20 -

10-

0 1 10 100 500

Virus concentration (HAU/ml)

Figure 2. The proliferative responses of T-cell clones BA5-T6 (-AN)or BA5-Tll (OQA ) to X31 virus (0), or laboratory variantsthereof containing single amino acid substitutions in the HAl subunit(AA 193 S R; E[1 198 A-.E). Stimulation index (SI) is the ratio ofmean values for [3H]thymidine incorporation in the presence or absenceof virus, and irradiated (3000 rads) syngeneic spleen cells as antigen-presenting cells. Background values in the absence of antigen were

between 150 and 2000c.p.m.

to natural variant viruses of the H3 subtype containing driftsubstitutions within the primary sequence HAl 120-139 (seerefs 2, 4 and 7 for sequence data of variant viruses).

FACS analysis for Vp expression

Our initial approach in defining TcR usage was to screen T-cellclones using a panel of currently available Vp-specific mAb(VO2, 3, 4, 6, 7, 8). In most instances, clear-cut assignments werepossible with mAb specific for Vp4 or V,#6 or V#8. The results ofsuch a screen are summarized in Table 2, and representativeFACS profiles are shown in Fig. 2.

It was not possible however to assign Vp8 usage to any of theT-cell clones established from donor BA5, and specific forp186-200, as baseline values were obtained in the FACS witheach of the mAb used herein.

PCR sequence analysis of (VDJ)p usage

The aims of the PCR analysis were twofold. First, to determineVp chain usage for T-cell clones that were negative by FACSanalysis (donor BA5); and secondly to ascertain whether thefine specificity differences seen for T-cell clones from the sameindividual, in their recognition of a common antigenic site(Table 1), were the result of junctional region variation. Forthis purpose, we synthesized a series of oligonucleotide primers

Table 2. Vp expression by T-cell clones, as determined by FACSanalysis

mAb reactivity T-cell clone*

Vp4+ (13.10) (4.4) (BA5E-6/69)Vp6+ (BA6E-1/69)Vp8+ (CB12-T5) (CB12-TI 1) (1.14) (1.99) (3F.10)

(ML7P-67)

*Antigen specificity and class II restriction, and donor origin of T-cell clones are given in Fig. 1 (and Table 6) with the exception of clone4.4 (Ak; HAI 120-139) and clone BA5E-6/69 (Ad; HAl 58-73).Representative FACS profiles are shown in Fig. 2.

Page 4: Immunodominance correlates with T-cell receptor (ap) gene usage

C. A. Smith, C. M. Graham & D. B. Thomas

c

0

0

Channel no.

-ILI IIdF A..~~~~.. ...L F TV...".._...~~~~~~

Figure 3. FACS analysis of representative T-cell clones after indirectimmunofluorescent staining with Vp-specific mAb: (a) (b) clone CB12-T5 with V.8-specific mAb (F23.1); (c) (d) clone BA6E-1/69 with Vp6-specific mAb (44.2.1); (e) (f) clone BA5E-6/69 with Vp 4-specific mAb(KT4.10). Control samples (a, c, e) were incubated with FITC-conjugated anti-mouse Ig antibody alone. See the Materials andMethods for Vp-specific mAb reagents used for FACS analysis. Verticalline = 256.

specific for the different Vp elements (see the Materials andMethods), and two constant region Cp primers that were usedfor either amplification or direct sequencing of cDNA.

Donor BA5: productive Vpl Dp1 Jpl.1 rearrangement

All of the T-cell clones established from donor BA5 generated a

PCR product of the correct size with the Vpl-specificoligonucleotide primer. Productive rearrangement of thechain was confirmed by complete sequencing of the PCR

products and shown to be derived from genomic V#1 D.81 Jpl.1(Table 3). Moreover, each of the T-cell clones had identicaljunctional region sequences.

Donor CB12: productive Vp8.2 Dpi Jp2.1 rearrangement

T-cell clones from donor CB12 had identical junctional regionsequences for Vp8.2 rearranged to Jp2.1 (Table 3) despiteapparent differences in recognition specificity for variantviruses (Table 1).

V. J. rearrangements for donor BA5 or CB12

As T-cell clones from the same individual could be distin-guished in their fine specificity for mutant HA, but neverthelessexpressed identical chains, it was imperative to determine thecorresponding a chain sequences. Table 4 shows that thejunctional region sequences for T-cell clones from donorBA5 (VA4) or donor CB12 (V 5) are identical. However, fordonor CB12 we have failed to obtain full-length sequencesacross the J regions and this raises the (remote) possibility thatdifferences in antigenic specificity can be attributed to a chainusage.

We propose that the sequence data presented here are notinconsistent with recruitment of a T-memory population,specific for HA, from a single progenitor cell during thecourse of a natural virus infection of limited duration (4-6days).

Frequent Vp4 or Vp8 usage with different Jp elements

There was no correlation between TcR V# J# usage and eitherclass II restriction or peptide specificity. Not unexpectedly therewas frequent representation of Vp4 or V.8. 1 or V68.2 by T-cellclones that were specific for six distinct antigenic peptides(Table 5). In each instance, the TcR chain repertoire ofindividual donors has been represented by a single clonotype as

sibling clones could not be distinguished in their recognition ofvariant viruses. For instance, all of the T-cell clones establishedfrom donor BA5E or BA6E exhibit similar specificity in theirfailure to recognize variant viruses containing the driftsubstitution HAl 63 Asp-+ Asn that introduces a novel N-

23glycosylation site.

Table 3. Nucleotide and deduced amino acid sequence comparison of chain junctional regions of CB12 and BA5 families

Donor

+-Vp Jm-+ Vp Dp Jp95 104

A S G D E R G R D A ECB12* GCC AGC GGT GAT GAG CGG GGG CGC GAT ... ... GCT GAG 8-2 1 2-1

-Vpl Jp-a95 103

A S S Q D W Q G N T EBA5* GCC AGC AGC CAA GAT TGG CAG GGG ... ... AAC ACA GAA 1 1 1.1

*All clones studied from the donor had the same sequence.Underlined text represents D regions.

c

A.I

I El u I

e

,I, I

346

Page 5: Immunodominance correlates with T-cell receptor (ap) gene usage

Influenza haemagglutinin-specific TcR

Table 4. Nucleotide and deduced amino acid sequence comparisons of a chain junctional regions of CB12 and BA5 families

347

Donor

4Vct Vc Jct80 93V Q E S D S A V Y Y C V L G L D R

BA5* GTG CAG GAG TCA GAC TCT GCT GTG TAC TAC TGT GTT CTG GGC CTC GAT AGAJar-.96G S A L G R . L H F G A G T QGGT TCA GCC TTA GGG AGG ... ... CTG CAT TFITT GGA GCT GGG ACT CAG 4 FN1

80 93A H P G D S A G Y Y C A V S

CB12* GCC CAT CCT GGG GAC TCA GCC GGG TAC TTC TGC GCA GTC AGT 5 ND

*All clones studied from the donor had the same sequence.

T-cell clones that recognized a common antigenic site but Conservation of aspartic acid at codon 97had been established from different donors, employed different A notable feature of the f chain junctional region sequences forV6 J, elements for their receptors as seen for donor BA5E (VA four donor mice, representative of both haplotypes (Table 6),Jpl.2) or donor BA6E (V86 J.82.3) that are Ad-restricted in theirrecognition of p58-73. This was also evident in the Ak_ is the presence of a conserved aspartic acid residue (rather than

glycine) at codon 97 which does not correlate with peptiderestricted recognition of pl20-139 by T-cell clones from donor specificity.4 (V$#4 Jp2.3) or donor CB12 (V,68.2 J,62.1). The junctionalregion sequences for this panel of T-cell clones are shown in DISTable 5. It should be emphasized that in this study a minimum CUSSIONof 10 T-cell clones have been characterized from each donor for This study has been concerned with an analysis of TcR usageboth antigen specificity and TcR usage. and junctional region sequences of HA-specific, CD4+ T-cell

Table 5. Nucleotide and deduced amino acid sequence comparison of (B chain junctional region sequences

T-cell -VP J Vfl Dfl Jclone

90 95 103F C A S S A T Q D T

3F10 TXT TTC TGT GCC AGC AGC GXG GCT ACC ... ... ... ... CAA GAC ACC 8.1 NA 2.5

90 95 103Y F C A S S Q D G G G A E T

4.4 TAC TTC TGT GCC AGC AGC CAA GAT GGG GGG GGT ... ... GCA GAA ACG 4 2 2.3

90 95 105S F C A S G E G K V Q A

1.14 TCC TTC TGT GCC AGC GGT GAG GGG AGG GTC CAG ... ... ... ... GCT 8.1 1 1.5

90 95 104Y F C A S S Q G W G G A G . D T

13.10 TAC TTC TGT GCC AGC AGC CAA GGG TGG GGG GGC GCA GGA ... GAC ACC 4 2 2.5

90 95 104Y F C A S S Q D L Q N S D

BASE- TAC TTC TGT GCC AGC AGC CAA GAT CTA CAG AAC ... ... TCC GAC 4 NA 1.26/9

90 95 103F L C A S S T G T G V A E T

BA6E- TTT CTC TGT GCC AGC AGT ACC GGG ACA GGC GTT ... ... GCA GAA ACG 6 1 2.31/69

91 95 102F N A S R L G G G . F G R L

ML7P- TTC TGG GCC AGC AGA CTG GGG GGG GGG ... ... CCC GGG CGG CTC 8.3 2 2.267

Page 6: Immunodominance correlates with T-cell receptor (ap) gene usage

C. A. Smith, C. M. Graham & D. B. Thomas

Table 6. Summary of the frequent usage of glycine (G) or aspartic acid(D) at amino acid residue 97 of the TcR ,B chain. The corresponding

junctional region nucleotide sequences are shown in Table 5

Peptide Amino acidT-cell clone Ia specificity at codon 97

CB12-T5 Ak p120-139 EBA5-TlO Ad p186-200 D3F10 Ek p226-245 A4.4 Ak p120-139 D1.14 Ak p269-288 G13.10 Ak p48-67 GBA5E-6/69 Ad pS6-76 DBA6E-1/69 Ad p56-76 GML7P-67 Ad p177-199 G

clones in an attempt to resolve an apparent paradox: extensivediversity of the T-cell repertoire for influenza HA (H3 subtype)seen within a haplotype (lad or lak) and contrasting dominanceof a single antigenic site in the individual's response to virusinfection. We find that T-cell clones, established from a

common donor, have identical productive rearrangements oftheir a and chains, despite apparent differences in finespecificity for mutant viruses (Table 1; Fig. 2). How then dowe relate our findings to determinant selection of the class II-restricted T-cell repertoire?

First, we do not consider that our findings identity of aand P TcR for the individual are an artefact of the PCRamplification and sequencing procedures used herein. Therewas concordance between FACS analysis for assignment of Pchain usage (Fig. 3) and sequencing data for PCR products(Tables 3 and 5). In each instance, productively rearrangedsequences confirmed Vi assignment. Moreover, PCR sequen-

cing of (VDJ)p junctional regions, for sibling clones from thesame donor, were conducted at different time intervals (weeksto months) and by different investigators (C.A.S. or C.M.G.)thereby excluding technical artefacts of cross-contamination.For the a chains of T-cell clones from donor CB12 (Table 4),however, we have not been able to obtain full-length sequences

for the J regions and it may be argued that antigen-specificitydifferences reside in the a chain.

Secondly, our initial classification of T-cell clones from thesame donor, according to proliferative responses to mutantviruses (Table 1; Fig. 2) might be flawed. We have establishedtheir fine specificity for variant viruses over a wide range ofantigen concentrations and by a variety of lymphocyteactivation assays including proliferation, IL-3 release, andcytotoxic T-lymphocyte (CTL) activity.-5'23 Moreover, Ak_restricted T-cell clones from the same individual (donor CB 12)differ in fine specificity, and show differing sensitivity to site-specific mutations in the a chain of Ak transfectants,24'25thereby indicating differences in contact sites for TcR-peptide-Ia interaction.

Thirdly, the observed bias in TcR usage for a specificantigenic peptide might be considered an artefact of in vitroselection of T cells with a high cloning efficiency. Such criticismcan be levelled at all clonal analyses of the immune repertoireand is not readily answered. It has not been possible toinvestigate TcR usage in 'bulk cultures' or T-cell lines freshly

established from previously infected donors because such celllines contain CD4+ T cells reactive to all major viral proteins(nucleoprotein > HA > matrix protein; D. B. Thomas, unpub-lished results) and it is necessary to first obtain clonalpopulations of HA-specific T cells by limiting dilution beforeTcR analysis. Our extensive analysis of T-cell clones (> 100)from different individual donors (H-2k or H-2d) would argue

against such bias in in vitro selection. Although oligoclonalrepresentation is seen in the memory CD4 + response

established in culture several months after the primaryinfection, we have no information on the potential 'breadth'of the T-cell repertoire. Given the above caveats, the observedimmunodominance may be the consequence of (a) clonalcompetition, (b) restricted number of T-cell precursors in theindividual's repertoire, (c) a consequence of the finite periodavailable for the recruitment of effector T cells during thecourse (3-6 days) of virus infection. There is no evidence ofvirus persistence in the respiratory tract, beyond day 6, or ofsystemic infection. Accordingly, there might be a stochasticelement in recruitment of CD4+ T cells from a limited numberof progenitor cells.

It is reasonable to assume that, despite apparent diversity intheir recognition specificity for variant viruses, all of the T-cellclones established from a common donor are recruited from a

single progenitor cell, and that during memory maturation (orin vitro selection) their TcR exhibit differences in recognition ofmutant viruses. This could be due, for instance, to differences inthe level of expression of accessory molecules. However, we

have not observed significant differences in CD4 or CDl la

expression (C. A. Smith, C. M. Graham and D. B. Thomas,unpublished data). Even so, we consider that differences inrecognition fine specificity for variant virus HA provide an

index of diversity within the individual's repertoire, which may

be due to differences in TcR affinity. Indeed, our previouslyreported findings24'25 that T-cell clones from a common donorare sensitive to different site-specific mutations in the AKa chainsupport our contention that there is indeed clonal diversity inthe T-cell memory pool, albeit derived from a common

progenitor cell.Previously reported examples of Vp dominance have been

attributed to peptide specificity of the TcR response rather thanto class I or class II restriction elements. For instance, Vpl andVp3 in association with Jp1.2 predominate in the responses ofT-cell clones from different donors (Ek_ or Es-restricted) topigeon cytochrome c, peptide 81-104.26 Similar findings havebeen reported for class II-restricted responses to myoglobin27or the class I-restricted responses to lymphocytic chorio-meningitis virus glycoprotein.28,29 In each instance, TcRspecific for a single antigenic peptide have been investigatedand shown to exhibit preference (not exclusively however) for a

certain Vp element.There is evidence to suggest that the putative CD3 loop of

the chain contributes to peptide specificity30 while the CDland CD2 loops of both a and chains associate with criticalmajor histocompatibility complex (MHC) residues. The usageof a particular Vp and/or Va element in the recognition of a

single antigenic peptide-MHC complex might not be unex-

pected, therefore. We have frequently found that for influenzaHA, T-cell clones that recognize a common antigenic peptide,and are derived from different donor mice, use different Vi J#elements. This is illustrated in Ad-restricted recognition of HAl

348

Page 7: Immunodominance correlates with T-cell receptor (ap) gene usage

Influenza haemagglutinin-specific TcR 349

56-76 by donor BA5E (V64 Jfl.2) or donor BA6E (VP6 Jp2.3);and Ak-restricted recognition ofHAl 120-139 by donor 4 (V,64Jp2.3) or donor CB12 (V#8.2 Jl2.1). TcR usage does notcorrelate with antigen specificity.

Similarly, Taylor et al.31 have reported highly diverse A4 TcRusage in Ed-restricted recognition ofthe HAl 110-120 peptide ofHi subtype influenza virus, although no information waspresented on the donor origin of T-cell hybridomas; and thesewere generated by a variety of immunization protocols (notincluding natural infection). Nevertheless, it is particularlyrelevant to the peptide specificity of TcR that a comparison ofjunctional region sequences indicates frequent occurrence ofaspartic acid (in addition to glycine) at codon 97 of the fi chain(Table 6) for Ad- and Ak-restricted T-cell clones specific forthree distinct antigenic regions of the HAl subunit. This is not acommon feature for previously reported TcR fi chain sequences.

In summary, our findings emphasize the importance ofimmune repertoire studies at the level of the individual donorbecause, during influenza virus infection, different individualswould appear to focus on different antigenic sites of the HAmolecule. This is consistent with recruitment of a T-cellmemory population from a single progenitor cell, contrastswith the diversity of Ad gene usage within a given haplotype,and suggests that there is a stochastic element in TcR repertoireselection during virus infection.

REFERENCES

1. MILLS K.H.G., SKEHEL J.J. & THOMAS D.B. (1986) Extensivediversity in the recognition of influenza virus hemagglutinin bymurine T helper clones. J. exp. Med. 163, 1477.

2. BURT D.S., MILLS K.H.G., SKEHEL J.J. & THOMAS D.B. (1989)Diversity of the Class II (I-Ak/I-Ek) restricted T cell repertoire forinfluenza haemagglutinin and antigenic drift: six non-overlappingepitopes on the HAl subunit are defined by synthetic peptides. J.exp. Med. 170, 383.

3. THOMAS D.B., BURT, D.S., BARNETr B.C., GRAHAM C.M. & SKEHELJ.J. (1989) B- and T-cell recognition of influenza hemagglutinin.Cold Spring Harbor Sym. Quant. Biol. LIV, 487.

4. BARNETT B.C., GRAHAM C.M., BURT D.S., WARREN A.P., SKEHELJ.J. & THOMAS D.B. (1989) I-Ad restricted T cell recognition ofinfluenza hemagglutinin: synthetic peptides identify multipleepitopes corresponding to antibody-binding regions of the HAIsubunit. J. Immunol. 143, 2663.

5. GRAHAM C.M., BARNETT B.C., HARTLMAYR I., BURT D.S., FAULKESR., SKEHEL J.J. & THOMAS D.B. (1989) The structural requirementsfor Class II (I-Ad) restricted T cell recognition of influenzahemagglutinin: B cell epitopes define T cell epitopes. Eur. J.Immunol. 19, 523.

6. WILSON I.A., SKEHEL J.J. & WILEY D.C. (1981) Structure of thehemagglutinin membrane glycoprotein of influenza virus at 3Aresolution. Nature, 289, 366.

7. WILEY D.C., WILSON I.A. & SKEHEL J.J. (1981) Structuralidentification of the antibody binding sites of Hong Konginfluenza hemagglutinin and their involvement in antigenicvariation. Nature, 289, 373.

8. CHOMZYSKI P. & SACCHI N. (1987) Single step method of RNAisolation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal. Biochem. 162, 156.

9. CASANOVA J.-L., ROMERO P., WIDMANN C., KOURILSKY P. &MARYANSKI J.L. (1991) T cell receptor genes in a series of Class IMajor Histocompatibility complex-restricted cytotoxic T lympho-cyte clones specific for a Plasmodium berghei nonapeptide:

implications for T cell allelic exclusion and antigen specificrepertoire. J. exp. Med. 174, 1371.

10. HEDRICK S.M., NIELSEN E.A., KAVALER J., COHEN D.I. & DAVIS

M.M. (1984) Sequence relationships between putative T cellreceptor polypeptides and inimunoglobulins. Nature, 308, 153.

11. CHOU H.S., ANDERSON S.J., LOUIE M.C., GODAMBE S.A., PozziM.R., BEHLKE M.A., Huppi K. & LOH D.Y. (1987) Tandem linkageand unusual RNA splicing of the T cell receptor fi chain variableregion gene. Proc. natl. Acad. Sci. U.S.A. 84, 1992.

12. BEHLKE M.A., SPINELLA D.G., CHOU H.S., SHA W., HART D.L. &LOH D.Y. (1985) T cell receptor f chain expression: dependence on

relatively few variable region genes. Science, 229, 566.13. LOUIE M.C., NELSON C.A. & LOH D.Y. (1989) Identification and

characterization of new murine T cell receptor fi chain variableregion VP genes. J. exp. Med. 170, 1987.

14. BARTH R.K., KIM B.S., LAN N.C., HUNKAPILLER T., SOBIECK N.,WINOTO A., GERSHENFELD, H., OKADA C., HANSBURG D., WEISSMANI.L. & HOOD L. (1985) The murine T cell receptor employs a limitedrepertoire of expressed Vp chain segments. Nature, 316, 517.

15. MULLIS K.B. & FALOONA F.A. (1987) Specific synthesis ofDNA invitro via a polymerase catalysed chain reaction. Meth. Enzymol.155, 335.

16. WINSHIP P.R. (1989) An improved method for directly sequencingPCR amplified material using dimethyl sulphoxide. Nucl. Acid.Res. 17, 1266.

17. FINKEL T.H., CAMBIER J.C., KuBo R.T., BORN W.K., MARRACK P.& KAPPLER J.W. (1989) The thymus has two functionally distinctpopulations of immature a/ + T cells: one population is deleted byligation of a/P TcR. Cell, 58, 1047.

18. PULLEN A.M., MARRACK P. & KAPPLER J. (1988) The T cellrepertoire is heavily influenced by tolerance to polymorphic selfantigens. Nature, 335, 796.

19. TOMONARI K., LOVERING E. & SPENCER S. (1990) Correlationbetween Vp4+ CD8+ T cell population and the H-2d haplotype.Immunogenetics, 31, 333.

20. MACDONALD H.R., SCHNEIDER R., LEES R.K., HowE R.C., ACHA-ORBEA H., FESTENSTEIN H., ZINKERNAGEL R. & HANGARTNER H.

(1988) T cell receptor Vp use predicts reactivity and tolerance toMlsa encoded antigens. Nature, 332, 40.

21. OKADA C.Y., HOLTZMAN B., GuIDos C., PALMER E. & WEISSMANI.L. (1990) Characterization of a rat monoclonal antibody specificfor a determinant encoded by the Vp7 gene segment. Depletion ofVy7+ T cells in mice with Mls-la haplotype. J. Immunol. 144, 3473.

22. STAERZ U.D., RAMMENSEE H., BENEDETTO J.D. & BEVAN M.J.(1985) Characterization of a murine monoclonal antibody specificfor an allotypic determinant on T cell antigen receptor. J. Immunol.134, 3994.

23. THOMAS D.B., HODGSON J., RISKA P.F. & GRAHAM C.M. (1990) Therole of the endoplasmic reticulum in antigen processing: N-glycosylation of influenza hemagglutinin abrogates CD4+ cyto-toxic T cell recognition of endogenously processed antigen. J.Immunol. 144, 2789.

24. WARREN A.P., PASCHEDAG I., BENOIST C., PECCOUD J., MATHIS D. &THOMAS D.B. (1990) Defects in antigen presentation of mutantinfluenza hemagglutinins are reversed by mutations in the MHCClass II molecule. EMBO. J. 9, 3849.

25. GRAHAM C.M., WARREN A.P. & THOMAS D.B. (1992) Do antigenicdrift residues in influenza hemagglutinins of the H3 subtype qualifyas contact sites for MHC Class II interaction? Int. Immunol. 4, 917.

26. HEDRICK S.M., ENGEL I., MCELLIGOTT D.L., FINK P.J., Hsu M.-L.,HANSBURG D. & MATIS L.A. (1988) Selection ofamino acid sequences

in the P chain of the T cell antigen receptor. Science, 239, 1541.27. DANSKA J.S., LIVINGSTONE A.M., PARAGAS V., ISHIHARA T. &

FATHMAN C.G. (1990) The presumptive CDR3 regions of both Tcell receptor a and ,B chains determine T cell specificity formyoglobin peptides. J. exp. Med. 172, 27.

28. YANAGI Y., MAEKAWA R., COOK T., KANAGAWA 0. & OLDSTONE

Page 8: Immunodominance correlates with T-cell receptor (ap) gene usage

350 C. A. Smith, C. M. Graham & D. B. Thomas

M.B.A. (1990) Restricted V segment usage in T cell receptors fromcytotoxic T lymphocytes specific for a major epitope oflymphocytic choriomeningitis virus. J. Virol. 64, 5919.

29. AEBISCHER T., OEHEN S. & HENGARTNER H. (1990) Preferentialusage of Vx4 and V.10 T cell receptor genes by lymphocyticchoriomeningitis virus glycoprotein-specific H-2Db restrictedcytotoxic T cells. Eur. J. Immunol. 20, 523.

30. ENGEL I. & HENDRICK S.M. (1988) Site directed mutations in theVDJ junctional regions of a T cell receptor P chain cause changes inantigenic peptide recognition. Cell, 54, 473.

31. TAYLOR A.H., HABERMAN A.M., GERHARD W. & CATON A.J. (1990)Structure-function relationships among highly diverse T cells thatrecognize a determinant from influenza virus haemagglutinin. J.exp. Med. 172,1643.