Characterization of the Primary Structure of T Cell Receptor # Chains in CellsInfiltrating the Salivary Gland in the Sicca Syndrome of HIV-1 InfectionEvidence of Antigen-driven Clonal Selection Suggested by Restricted Combinationsof Vft Jft Gene Segment Usage and Shared Somatically Encoded Amino Acid Residues

Edward Dwyer, Silviu Itescu, and Robert WinchesterDepartment of Pediatrics, Columbia University, College of Physicians and Surgeons, New York 10032

Abstract

Infection with HIV-1 occasionally results in a sicca syndrome,termed the diffuse infiltrative lymphocytosis syndrome, char-acterized by infiltration of the salivary glands with a predomi-nance of CD8T cells. This response is strongly associated withcertain MHCclass I and class II alleles. To define the salivarygland T cell receptor (TCR) repertoire, the primary structureof the TCR#-chains was determined using in situ cDNAsynthe-sis followed by the "anchored" polymerase chain reaction. Thesequences of 59 #-chains from five individuals with diffuse in-filtrative lymphocytosis syndrome shared structural featuressuggesting antigenic clonal selection. Certain combinations ofV13 JO3 gene segments were selectively overrepresented in therepertoire sample, demonstrating a commonrestricted usage ofcertain Vft and J(3 gene segments. The ,8-chains derived fromthese overrepresented Vft J(3 combinations revealed a prefer-ence for specific amino acids at position 97 in the third comple-mentarity-determining region, a residue postulated to contactpeptide antigen. Moreover, the nucleotides encoding this posi-tion were not germline in origin. TCR(-chains in nonoverrepre-sented V,@J(B combinations did not exhibit preferential usage ofselected somatically encoded residues. The pattern of TCR(3-chains expressed in the salivary gland of a control person withprimary Sjogren's syndrome was considerably more heteroge-neous and different from that found in diffuse infiltrative lym-phocytosis syndrome. (J. Clin. Invest. 1993.92:495-502.) Keywords: T cell repertoire * Sjogren's syndrome - diffuse infiltra-tive lymphocytosis syndrome - immunoglobulin * autoimmunity

Introduction

An unusual form of sicca syndrome termed the diffuse infiltra-tive lymphocytosis syndrome (DILS),' is a distinctive but in-frequent host response to infection with HIV. DILS is charac-terized by infiltration of predominantly CD8T cells into lacri-mal and salivary glands and is frequently accompanied by a

Address correspondence to Edward Dwyer, M.D., Department of Pedi-atrics, Columbia University, College of Physicians and Surgeons, 630W. 168th Street, NewYork, NY 10032.

Received for publication 12 August 1992 and in revised form 12January 1993.

1. Abbreviations used in this paper: CDR3, third complementarity-de-termining region; DILS, diffuse infiltrative lymphocytes; OR, odds ra-tio; TCR, T cell receptor.

significant peripheral CD8 T cell lymphocytosis as well as amaintenence of relatively normal levels of circulating CD4cells (1). The majority of peripheral CD8 and CD4 T cellsexhibit a mature memory phenotype characterized by theabundant expression of (31 and (2 integrins, without a detect-able increase in cells bearing markers of recent activation suchas HLA-DR molecules or IL-2 receptors (2). HIV- 1 antigensare identifiable only in monocyte lineage cells at sites of tissueinfiltration and the virus is culturable from blood monocytes(3). Susceptibility to developing DILS is strongly associatedwith the DRB1* 1102 and DRB1*1301 alleles of HLA-DR5and DR6, respectively, while HLA-B35 is significantly de-creased in frequency (4). These features contrast with the morecommon idiopathic autoimmune disorder of primarySjogren's syndrome which is characterized by infiltration of thesalivary glands with a predominance of CD4T cells and suscep-tibility is associated with HLA-DR2 or DR3 (5).

The ability of T cells to respond to foreign antigens such asHIV- I is determined by the clonally distributed a(3 T cell re-ceptor (TCR) which is a membrane-bound 88-kD heterodimerthat recognizes foreign peptides presented by self MHCmole-cules (6). The unique recognition structure of each clonal TCRis determined by the combinatorial pairing of distinct V, D,and J gene segments which together constitute each individualmolecule. In the case of the TCR# chain, the potential reper-toire is composed of 24 V: families, 2 D(3 segments, and 13 Jo3segments (7). Although several studies of the (3-chain TCRrepertoire in humans have demonstrated a nonrandom distri-bution of Vf3 family expression in both normals and variousdisease states (8-14), a recent investigation specifically focus-ing on the primary structure of the entire (-chain variable re-gion revealed a nonbiased pairing of individual V(3 familieswith specific J(3 segments ( 15). In addition to the use of thesemultiple germline elements, structural diversity of the (3 chainis greatly enhanced by exonucleic "nibbling" and N region di-versification during junctional pairing of individual V, D, andJ segments which results in random deletion or addition ofindividual nucleotides in generating nongermline encodedamino acids (6); this region of the molecule which exclusivelyemphasizes somatic diversity is referred to as the third comple-mentarity-determining region (CDR3), and it is this region ofthe TCR(-chain which is postulated to directly interact withforeign peptides presented by self MHCmolecules ( 16, 17). Insupport of this model of molecular interaction are several stud-ies which have demonstrated in vitro that murine T cell clonesresponsive to a cytochrome c-derived peptide express TCR(3chains that show not only restricted usage of specific V(3J(3combinations, but perhaps more importantly, also exhibit apreferential expression of particular nongermline somaticallyencoded amino acids in the CDR3 region, most notably atamino acid position 97 (18-20) which is five residues carboxyl

Selective Usage of T Cell Receptor ,8-Chain Genes in HIV-1 Infection 495

J. Clin. Invest.© The American Society for Clinical Investigation, Inc.0021-9738/93/00/0000/00 $2.00Volume 92, July 1993, 000-000

to the invariant cysteine at position 92. Further emphasizingthe importance of determining complete primary structuredata in obtaining evidence of antigen-specific immune respon-siveness is the recent finding that single nonconservative sub-stitutions at specific amino acid positions in the cytochrome cpeptide are capable of transferring responsiveness to alterna-tive clones which express TCR 13 chains featuring differentVflJfl combinations as well as different non-conservativeamino acid substitutions at position 97 of the CDR3 re-gion ( 18).

This study analyzes the structure of the TCR f1-chain togain insight into the biologic basis of the infiltration into tissuesby T cells in the DILS response to HIV-1 infection. Severalalternative possibilities exist for mechanisms underlying theaccumulation of lymphocytes, each of which have differentpredictions in terms of the character of the T cell repertoire asdetermined by the structure of the TCR13 chains. In view of theincreased frequency of B cell lymphoma in DILS (4), the lym-phocytic accumulation in tissues could represent a parallelpreneoplastic expansion of one or a few T cell clones whichwould be recognized by marked TCR13 chain homogeneity atthe level of nucleotide sequence. In contrast, a process drivenby nominal antigen should exhibit some evidence of selectionat the level of primary protein structure by demonstrating pref-erential usage of V13 and J13 elements, and especially, character-istic features of the CDR3region. In the instance of superanti-gen drive, which has been suggested by two reports of HIV +patients ( 12, 21), one would expect preferential deletion orenhancement of certain V13 families while allowing randomstructural features in the CDR3 region and the lack of anypreferential usage of specific Jo elements. Since CD4 lympho-cytes and monocytes which function as reservoirs of HIV- 1 aresystemically disseminated, this antigen or superantigen drivewould be expected in both the involved tissue and elsewhere inthe body. Finally, the process of tissue accumulation could beunrelated to any antigen drive or neoplastic event, perhapsreflecting secondary tissue recruitment of blood lymphocytesthat have undergone activation as a result of locally secretedcytokines, and in this case, no commonality of primary struc-ture would be evident in a comparison of TCR 13 chains fromdifferent individuals with DILS.

Current techniques assessing TCR structural features useeither a limited set of monoclonal antibodies to measure V13frequencies, or use potentially more informative molecular bio-logic techniques which may be divided into two general catego-ries. In one, illustrated by construction of a formal cDNA li-brary, the repertoire is determined by sequencing individual 1chain clones to define CDR3organization and obtain a fre-quency distribution of V13 and J13 usage by enumerating theiroccurrence. In the other, primers specific for each of the V1segments are used in quantitative PCRmethods which is rela-tively simple and has the important advantage that all knownV1 gene segments can be measured (22). However, indetermi-nate variabilities in the efficiency of particular primers intro-duces uncertainties into the quantitation process and themethod is presently incapable of determining V13J13 combina-torial frequencies or evaluating CDR3structure. This reportuses a novel approach which entails an initial stage of in situcDNAsynthesis performed on a frozen section, thereby elimi-nating any bias introduced by in vitro manipulation or differ-ential priming, and is followed by amplification of cDNA bythe anchored polymerase chain reaction (23) which has been

demonstrated to nonartifactually amplify all expressed V13 seg-ments ( 15, 24); the primary structure is obtained through nu-cleotide sequencing. This approach addresses the role of anti-gen specificity by infiltrating T cells in salivary gland tissue byconcentrating on the presence of preferential V13J1 combina-tions as well as the amino acid structure of the third comple-mentarity-determining region in TCR 13-chains isolated di-rectly from tissue.

Methods

Patients. Each individual with DILS met the diagnostic criteria of atleast 6 mo of salivary gland enlargement, HIV- I positivity confirmedby isolation of the virus and demonstration of specific antibodies byELISA and Western blotting, elevated CD8 Tcell levels in the periph-eral blood, and at least two foci of lymphocytic infiltration with a pre-dominance of CD8 lineage T cells demonstrated in lip biopsy speci-mens. The individual with primary Sjogren's syndrome had symptom-atic xerostomia, more than two lymphocytic foci on minor salivarygland biopsy, and anti-Ro/ SS-A autoantibodies. All tissue analyzed inthis report was residual diagnostic lip biopsy material obtained withinformed consent and under an approved protocol.

Methods. The method of in situ cDNAsynthesis was according to amodification of the method of Tecott et al. (25). Briefly, all specimenswere snap frozen with liquid nitrogen and stored until cut into 10-'qmsections. Each tissue section was hybridized for 12-16 h at room tem-perature with 50 ,l of a solution containing 50% formamide, 4X SSC,and a 36-bp primer (80 ng/ml) complementary to a sequence in theconstant region of the TCR d-chain (CCCATTCACCCACCCAGC-TCAGCTCCACGTGGTCGGG).After hybridization, sections werewashed in 2x SSCat room temperature for 10 min for three washes,with a final wash in .5x SSCat 40'C for 2 h. cDNAwas synthesized byadding to each tissue section a volume of 50 Al which contained 10 UAMVreverse transcriptase (Promega Corp., Madison, WI; buffer sup-plied) and 1 mMdNTP's, and incubated at 420C for 1 h. Each sectionwas washed three times in 2x SSC at room temperature for 10 min,followed by a final wash at 40'C for 2 h. For each sample, at least fivesections from each specimen were processed simultaneously accordingto this protocol. The cDNA was eluted from each tissue section with0.15 MNaOH. .25 volume of I MTris-HCl (pH = 6.0) was added toeach specimen followed by ethanol precipitation. The cDNAwas sub-sequently amplified by a modification of the anchored PCRprocedureof Loh et al. (23). Specifically, the first strand cDNA product wasG-tailed with terminal deoxynucleotidyl transferase (Promega Corp.;buffer supplied) using 1 mMdGTP for 30 min at 37°C, and thenamplified using a .1 -MM34-mer C-tailed primer which also has a restric-tion site for NotI (TTGACGCGGCCGCTACTAGCCCCCCCCCCC-CCCC), a .8-MuM primer containing only the NotI site (TTGACGCGG-CCGCTACTAGC),and a 1-AM third primer complementary to theconstant region, 5' of the original 36-mer (CCTGTGGCCAGGCAC-ACCAG). The PCRprotocol using a Hybaid (Hook and Tucker In-strumants, United Kingdom) thermal cycler consisted of denaturationat 94°C for 1 min, annealing at 53°C for 2 min, and synthesis at 72°Cfor 3 min. The product was size-selected using a low melting agarose geland 200-400-bp fragments were reamplified for 20 cycles (94°C for 1min, 55°C for 2 min, and 72°C for 3 min) using a constant regionprimer 5' of the previously used constant region primer (GTGTGG-CCTTTTGGGTGTGG)while the other primer was the 20-mer NotIprimer. The product was again size-selected on an agarose gel, digestedwith NotI, and ligated into Bluescript (Stratagene Inc., La Jolla, CA)which had been digested with NotI and SmaI. The f3-galactosidase sys-tem was employed to select white colonies which were then screened byrestriction digestion with SstI and Bgl II, which selectively cut a BglIIrestriction site present in the ,B-chain constant region just 5' of thelast-used primer. All clones with inserts revealed by BglII and SstIdigestion were sequenced using a DNAsequencer (373A; Applied Bio-systems Inc., Foster City, CA) using the Taq dye deoxy terminator

496 E. Dwyer, S. Itescu, and R. Winchester

sequencing kit (Applied Biosystems Inc.) in accordance with the manu-facturer's protocol.

Statistics. Statistical analysis used Chi-square analysis or Fisher'sexact test for which an odds ratio is given for each calculation. In eachinstance the null hypothesis for V(3 or J(3 frequency calculations wasthat there was an equal likelihood of the presence of each gene element.

Nomenclature. The V(3 family designation is according to Wilson etal. (26) and Robinson (27); the (-chain amino acid position number isaccording to Kabat et al. (28). The length of the CDR3 regions isarbitrarily defined as the number of amino acid residues between theinvariant cysteine at position 92 and the invariant phenylalanine atposition 108 from which is subtracted the length of the shortest CDR3region to give the absolute number of varying residues.

Results

The nucleotide sequence and inferred primary amino acid se-quence of 62 TCR 3-chains were obtained from lip biopsy speci-mens of five patients with HIV-associated DILS. Their lengthranged from 100 to 250 bp and reflected extension from theconstant region primer to well into the V(3 segment. Each (3chain had a different G-tailed terminus, indicating that theyarose from separate priming events. The number of A-chainsfrom each individual range from 10 to 17. 59 of the Vf3 seg-ments sequenced could be unambiguously assigned to one ofthe 24 known V: families and, with few exceptions, have se-quence identity with known V: alleles; the remaining 3 V(3sequences were too short to unequivocally assign to a specificV: family, but were included in the analysis of J(3 usage andCDR3structure. The patterns of V(3 and J(3 segment usage aswell as the organization of the structure of the CDR3 regionwas determined by enumeration of the (3 chain sequences. Forreference purposes and to control for the possibility of intrinsicsalivary gland specificity, 55 TCR(-chains derived from the lipbiopsy of an individual with primary Sjcgren's syndrome arealso included in the analysis.

V(3 Frequency distribution. Fig. 1 shows the frequency ofV( segment usage in the TCRof T cells infiltrating salivarytissue of each of the five samples from DILS as determinedfrom the sequence obtained for each isolate. 15 of the 24known V(3 families are collectively expressed in this sample.Although relatively heterogeneous, the frequency distributionof V(3 segments for the group shows a distribution biased to-wards the use of certain V(3 families and is non-Gaussian (x2= 63.65, P = 0.00001). Six V(3 families, V(32, 5, 6, 8, 12, and13, are expressed at a level greater than one standard deviationabove the median (median = 2.0, SD = 2.6) and collectivelyconstitute 64% of the total V(3 families expressed. With theexception of patient 3 where V(35 was used in four of the 10(3-chains isolated from that individual, there is not a dominantusage of one specific V(3 family by the entire group or by any ofthe other four patients. 8 of the 24 known V(3 families were notencountered in any of the (3 chain sequences from the fivesamples.

Fig. 2 compares the frequency of V(3 usage in the 59 (3chains of the DILS group with 53 TCR(-chains obtained fromthe same anatomic site from one individual with primarySjogren's syndrome. That 20 of the 24 V(3 families are repre-sented in the Sjogren's syndrome sample indicates the V(3 reper-toire diversity in this sinigle person exceeds that found in theentire DILS group. Further emphasizing differences in V(usage of these two diseases involving accumulation of lympho-cytes in the same organ is the observation that there are seven

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Figure 1. Frequency of TCRV(3 families in DILS. 15 V(3 families areexpressed in 59 TCR(-chains expressed in the salivary glands of fiveindividuals with DILS (median = 2.0, SD = 2.6). The distribution isnonrandom (x2 = 63.65, P = 0.00001 ) and exhibits six V(3 families(V(32, 5, 6, 8, 12, and 13) which are expressed at a level greater thanone standard deviation above the median which collectively accountfor 64% of the total V(3 distribution.

V(3 families, V(34, 9, 10, 11, 15, 16, and 23, expressed in theSjogren's syndrome sample that are not found in the sample ofDILS tissue. The TCRrepertoire observed in the Sjogren's syn-drome sample contains only three V(3 families, V(35, 6, 13,expressed at a level greater than one standard deviation abovethe median (median = 2.0, SD = 2.4), and these constitute45% of the total V( segment expression.

J( Frequency distribution. The frequency of J(3 segmentexpression for persons with DILS is shown in Fig. 3. All J(3

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Figure 2. Comparative distribution of TCRV(3 families in the salivaryglands of DILS (n = 59) and Sjbgren's syndrome (n = 53). One in-dividual with Sjogren's syndrome expresses 20 V(3 families whichrepresents greater diversity than the collective V(3 repertoire of fiveindividuals with DILS; this includes seven V(3 families (V(34, 9, 10,11, 1 5, 16, and 23 ) not present in the V(3 repertoire of any of the DILSpatients. There are three V(3 families (V(35, 6, and 13) in Sjogren'ssyndrome which are expressed at a level greater than one standarddeviation greater than the median (median = 2.0, SD = 2.4) whichcollectively account for 45% of the total V(3 distribution.

Selective Usage of T Cell Receptor ,(-Chain Genes in HIV-J Infection 497

segments analyzed are identical to previously reported J13 seg-ment sequences. Of the 13 known J# segments, 10 are repre-sented in the 1-chain repertoire of the T cells in the salivaryglands in DILS. The overall distribution is moderately heteroge-neous (median = 4.0, SD = 4.5) in that there is not a singulardominance of one J13 segment; however, there is a preferentialskewing of Jfl segment usage, with four of the total of 13 J13segments, J3 1. l, 2.1, 2.5, and 2.7, found to be present at a levelgreater than one standard deviation above the median and col-lectively account for 70% of the expressed J# segments in glan-dular tissue (X2 = 48.2, P = 0.000003). Three J13 segments,J#1.3, J132.4, and J#2.6, are not represented in any of the 62TCR 3-chains analyzed.

Fig. 4 shows the frequency distribution of J13 segment ex-pression from the individual with Sjogren's syndrome as com-pared to the that among the five persons with DILS. There isconsiderable overlap between the two groups with regard to thepreferential use of certain J13 segments, e.g., J 1. 1, J132. 1, andJ132.7; however, selective differences are evident as shown bythe significantly increased frequency of J#2.5 usage in DILS (P= 0.017, odds ratio = 9.72) and a trend of enhanced usage ofJ12.3 in Sjogren's syndrome which approaches statistical signif-icance (P = 0.086).

D13 Frequency distribution. Analysis of D13 segment usagereveals that there is not a significant difference in the usage ofeither of the two D13 segments, with D13l 1. found in 321-chainsand D12.1 in 30 1-chains. One 13-chain incorporated bothD13 1.1 and D12. 1 and 5 13-chain transcripts did not exhibit anidentifiable D13 segment. In addition, within each D13 segmentgroup, there was not a demonstrable preference for the use ofone particular translational reading frame for either D13 1. 1 orD12.1 (data not shown).

Inferred length of the CDR3 region. Fig. 5 illustrates thelength of the CDR3regions for the 1-chains of DILS as well asprimary Sjogren's syndrome, arbitrarily defined as the number

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Figure 3. Frequency of TCRJ(3 segments in DILS. 10 of the 13 J#segments are expressed in 62 TCR(-chains in the salivary glands offive individuals with DILS (median = 4.0, SD = 4.5) The distributionis nonrandom (X2 = 48.2, P = 0.000003) with four J/3 segments(Jj l.l, 2.1, 2.5, and 2.7) expressed at a frequency greater than onestandard deviation above the median which collectively account for70% of the total distribution.

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Figure 4. Comparative distribution of TCRJ13 segment expression inthe salivary glands of DILS (n = 62) and Sjogren's syndrome (n= 55). There is considerable similarity in the skewness of J# segmentusage in DILS and Sjogren's syndrome, with the only significant dif-ference being the preferential usage of J#2.5 by the DILS patients (P= 0.017).

of amino acid residues between the invariant cysteine at posi-tion 92 and the invariant phenylalanine at position 108 fromwhich is subtracted the length of the shortest CDR3region, 9residues, to derive the number of inserted residues. The totalnumber of amino acids in the CDR3 region of the DILS 1-chains is significantly longer (mean = 12.4, SD = 1.72) andshows less variability as compared to the Sjogren's patient(mean = 11.7, SD = 1.93, P = 0.04). The CDR3region of theDILS 1-chains show more size restriction in that 73% are 11,12, or 13 amino acids in length, or 2, 3, or 4 inserted residues,whereas the CDR3regions of the individual with Sjogren's syn-drome display more variability in size with a random distribu-tion from 9 to 14 amino acids in length, or from zero to fiveinserted residues.

Association of V: with J1. Fig. 6 depicts the association thatwas found in a given 13 chain between the presence of a specificV13 family and the occurrence of a particular J13 segment ineach of the 59 TCR1-chains from DILS tissue. In addition tothe apparently background random combinatorial pairing ofsome V1 families with individual J13 segments, there is evi-dence for a non-random selective usage of particular V13J13 com-binations. For example, the combination of V135J132.1 is en-countered in four separate 1-chains derived from three differ-ent individuals (P = 0.009, OR= 13.14), accounting for 7%ofall 1-chains as against an expected frequency of 1.8%. TheV16J12.7 combination is represented in the repertoire of fourdifferent persons (P = 0.026, OR= 7.33), and the combina-tion of V#1 3J3 1.1 is expressed in three separate 13-chains (P= 0.022, OR= 12.00). For each of the V13 segments expressedin these three particular V13J13 combinations, the associated J13constitutes greater than 50%of the total J13 segments associatedwith that particular V1 family; V1 1 7J12. 1 also fulfills this crite-rion but only approaches statistical significance for a V13J13 com-bination (P = 0.08). The TCR 1-chains containing theV1J1 combinations V135J12. 1, V136J132.7, V:13 3J1 1. 1, andV13 1 7J132. 1, are designated "overrepresented" 13-chains.

With the exception of two 13-chains which represent exam-ples of V135J12. 1 combinations, the repertoire of Sjogren's syn-

498 E. Dwyer, S. Itescu, and R. Winchester

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AMINOACID LENGTHFigure 5. Amino acid length of the CDR3region in individual TCRf-chains in DILS (n = 62) and Sjogren's syndrome (n = 55). Thetotal length of the CDR3region is defined as the number of aminoacids between the invariant cysteine at position 92 and the invariantphenylalanine at position 108; the number of inserted amino acids,shown in parentheses, is determined by subtracting the number ofamino acids in the shortest CDR3region (e.g., 9 residues) from thetotal number of amino acids in each CDR3region. The total numberof amino acids in the CDR3region of the TCRd chains of the pa-tients with DILS (mean = 12.4, SD= 1.72) is significantly greater andshows less variability than that of Sjogren's syndrome (mean = 1 1.7,SD = 1.93, P = 0.04). 73% of CDR3regions in DILS exhibit two tofour inserted residues while the CDR3regions of the Sjogren's syn-drome patient demonstrate random variation between 0 and 5 in-serted residues.

drome does not include any examples of expressed chainswhich encode the overrepresented VfJf combinations presentin DILS (data not shown). Rather it demonstrates entirelydifferent overrepresented VflJf combinations which includeVf5Jf2.7 (P = 0.042, OR= 5.7), Vf6Jf1.I (P = 0.011, OR= 10.75), and VflI5Jf2.3 (P = 0.04, OR= 18.8).

Association of particular VflJf combinations with CDR3structure. In view of the observation that fl-chains with particu-lar VflJf combinations were found in salivary glands of pa-

tients with DILS at levels greater than expected by chance, theCDR3regions of fl-chains composed of these overrepresentedcombinations were compared to those where no preferentialVflJf combinations were observed. The primary structures ofthe CDR3regions of selected f-chains present in these overrep-

resented VfJfl combinations are shown in Table I. Specificallywith regard to amino acid position 97, it can be seen that thereis a strong relationship between the use of specific VflJfl combi-nations and the identity of the amino acid at position 97. Forexample, in the combination Vf 1 3J 1.1 fl-chains (Table I A),serine is present at position 97 in two of the three isolates (P =

0.01, OR= 54) and, as indicated by Fig. 7 which shows thetotal frequency of individual amino acids at position 97, thisaccounts for two of the four serine residues used at this positionin the entire sample of 62 f-chains. Similarly, glutamine ac-

counts for two of the four residues at position 97 in d chains

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Figure 6. Coexpression of VA families with specific Jfl segments inindividual TCRa chains in the salivary glands of DILS. There is apreferential association of certain Vfl families with specific J.: seg-ments as demonstrated by the pairings of Vfl5J,32.1 (P = 0.009, OR= 13.14), Vfl6Jfl2.7 (P = 0.026, OR = 7.33), and V,313J1l.l (P= 0.022, OR= 12.00).

with the combination Vf5JO2.1 (Table I B, P = 0.01, OR= 54), and in the case of the Vf6Jf2.7 combination (Table IC), aspartic acid is present at position 97 in two of the four:-chains (P = 0.01, OR= 54), again accounting for two of thefour occurrences of this amino acid at this position. A furtherexample of this association is found in the Vl 17 family wheretwo of the three isolates are paired with J02. 1. Table I showsthat both of these Vfl 17Jf2. 1 chains are encoded with a glu-tamic acid at position 97 (P = 0.006). In addition, emphasiz-ing the importance of similarity in residues found at position97 is the the presence of identical amino acids adjacent to posi-tion 97 in two of the four V#Jf3 categories, e.g., serine residuesat position 98 in the f chain with the Vf6Jf32.7 combinationand tyrosine residues at position 96 in the V: 1 3JDO 1. 1 combina-tion. Moreover, it should be noted that with the exception ofVl I 7Jf2. 1, where both of the f-chains are derived from thesame individual, all the other VOfJfl pairings are contributed bydifferent individuals.

The significance of the relationship between Vj3Jfl andamino acid residue at position 97 is further enhanced by theobservation that, in most of the ,8-chains, the amino acidsfound at and around position 97 are encoded by somaticallyadded nucleotides, rather than the individual V and J segmentgermline elements used elsewhere in the chain. The somati-cally added nucleotides are depicted in lower case letters. Ascan be seen in Table I different codons encode the same aminoacid at these positions, suggesting that factors operating at thelevel of the expressed protein rather than at the level of DNAare involved in this nonrandom representation of amino acidresidues. The 53 f-chains expressed in the salivary tissue of theone individual with primary Sjogren's syndrome do not exhibita significant association between any VflJf combination and aparticular amino acid residue at position 97, or at any adjacentposition in the CDR3region (data not shown).

Selective Usage of T Cell Receptor A-Chain Genes in HIV-J Infection 499

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AMINOACIDFigure 7. Distribution of amino acid residues used at position 97 inindividual TCRf-chains in DILS. *Amino acids represented in pref-erential V#Jf3 pairings. (A, Ala, C, Cys, D, Asp, E, Glu, F, Phe, G,Gly, H, His, I, Ile, K, Lys, L, Leu, M, Met, N, Asn, P, Pro, Q, Gln,R, Arg, S. Ser, T, Thr, V, Val, W, Trp, Y, Tyr).

DiscussionThe central finding of this study, using direct enumeration ofthe primary structural elements of each isolated TCRf3 chain,is that a subpopulation of T cells infiltrating the minor salivaryglands of HIV + individuals with DILS express a restricted fi-chain TCRrepertoire. This repertoire exhibits structural char-acteristics consistent with what would be predicted if the clonalselection of these T cells occurred through an antigen drivenmechanism. The findings that support an antigen driven selec-tion event include: the selective overrepresentation of particu-lar VflJf3 combinations, the presence of identical amino acidresidues at position 97 in fi chains included in these same VflJ,3combinations even if the chains are derived from different indi-viduals, and that the amino acids at this position are specifiedby different codons which are not specified by the germline. Asa counterpoise to these nonrandom events is the observationthat there is no evidence of bias in the use of the Dfl elementswhich adds further support to the considerable evidence in fa-vor of selective usage of the other TCRgene elements. Thatthese fl-chain structural features are specific to the immuneresponse of DILS is supported by the finding that three of thefour overrepresented VflJfl combinations of the DILS reper-toire are not represented in any of the :-chains of the personwith Sjogren's syndrome. More generally, a recent study of 250TCRf-chains from five healthy individuals, including a pair ofidentical twins, did not demonstrate a preferential associationbetween any particular Vfl family and Jf gene segment in indi-vidual TCR f chains ( 15 ). These observations argue againstthe presence of biasing structural features, either of the genomeor of the mature protein product, as being responsible for theselective VflJfl combinations present in DILS. Taken togetherwith the fact that DILS is a distinctive host response to HIV- Iinfection which occurs in the setting of particular MHCclass Iand II alleles, the present observations suggest the possibilitythat the unique features of the response of DILS are mediatedby the recognition properties of these TCRfi chains for certainHIV-encoded peptides.

The identification of a subset of fl-chains characterized byoverrepresented combinations of VflJfl segments allowed an

approach to the problem of identifying TCR's from clones thatmight have specifically responded to an antigen as opposed toTCR's from other clones that reflect nonspecific recruitmentmechanisms. The fact that analysis of the CDR3regions ofthese two catagories of fl-chains revealed a highly nonrandomoccurrence of particular amino acids at position 97 supportsthe rationale of this approach. For example, each of the V3Jfchains from the overrepresented groups in DILS (e.g.,Vf5Jfl2. 1, V06Jf2.7, V# I3Jl1. 1, and V13I7Jl2. I ) demon-strates a selectivity for specific amino acids at position 97 of theCDR3region, which is the same structural residue implicatedin direct interaction with the antigenic peptide in several invitro and transgenic investigations ( 18-20). The significanceof this association is enhanced by the observation that in eachof the four preferred VfJf classes the commonamino acid isencoded by nongermline nucleotides. The existence of severalstructurally distinct overrepresented VfJ# categories suggeststhe possible existence of several antigenic peptides responsiblefor eliciting the immune response in the salivary glands ofDILS.

Most studies examining either tissue-specific or peripheralVf repertoires have demonstrated elements of nonrandomnessand while the Vfl repertoire of DILS identifies six of 24 Vflfamilies (Vf2, 5, 6, 8, 12, and 13) as accounting for 64% of thetotal Vf segments isolated, the principal finding here is that theVB repertoire of each individual with DILS is more similar toother individuals with DILS than it is to the individual withSjogren's syndrome. For example, the one individual withSjogren's syndrome exhibits greater diversity in expressing 20of 24 Vfl families than the collective repertoire of five individ-uals with DILS which only express 15 Vfl families. Moreover,seven of the Vf families expressed in the Sjogren's syndromepatient's repertoire are absent from any of the DILS reper-toires. Additionally, it should be noted that the data from theindividual with Sjogren's syndrome are in general agreementwith a recent study which found a preferential usage of V,32and Vol 3 from lip biopsies in primary Sjogren's syndrome(29). In the present study Vf 13 is the most frequently used Vflfamily, accounting for 17% of the Vfl repertoire; however, Vfl2comprises only 3.7% of the total repertoire. Both DILS andSjogren's syndrome exhibit considerable overlap in expressingskewed Jf segment usage where both diseases emphasize only 4of 13 Jol segments in accounting for 70% of the repertoire.

While a superantigen mechanism has been postulated as anexplanation for distortions in the frequency of Vf family repre-sentation in AIDS ( 12), it is unlikely that the present findingsof skewed Vfl expression in DILS are a consequence of expo-sure to an HIV-related superantigen; such a mechanism fails toprovide an explanation for the selectivity of the Jo segmentsand the similarities in other features of CDR3structure thatwere found in the present study. Another possible explanationfor Vfl gene segment restriction could be selective traffickinginto the salivary gland of T cells expressing particular VOfami-lies. Comparative analysis with primary Sjdgren's syndrome,used here principally as a methodologic and biologic control,suggests that the Vf3 bias in DILS is not simply the result ofsalivary gland tropism of a select subset of T cells since the twodiseases display Vfl repertoires that are only partially overlap-ping.

The inclusion of only 8 of 59 or 13% of overrepresentedTCR f chains as representative of an antigen-reactive group isconsistent with previous analyses investigating the frequency of

Selective Usage of T Cell Receptor ,8-Chain Genes in HIV-J Infection 501

antigen-specific T cells in pathologic lesions. For example,Bloom and colleagues demonstrated that only 2% of infiltrat-ing T cells in cutaneous tuberculoid leprosy lesions respondedin an antigen-specific manner to Mycobacterium leprae (30),and the relevance of these data to the current study is enhancedby the recent finding of a subset of peptide-specific human Tcell clones responsive to M. leprae that also use TCRf-chainswithin a single Vfl family and also encode identical amino acidsat position 97 of the CDR3region (31 ). Similarly, in studyingthe antigen-induced animal model of autoimmunity, experi-mental autoimmune encephalomyelitis, Cohen and colleaguesrecently reported that only 5% of T cells derived from patho-logic lesions were reactive to the stimulating antigen myelinbasic protein (32). The estimation of an even higher frequencyof antigen-specific T cells in DILS salivary tissue may be par-tially explained by the findings of several recent studies whichhave consistently shown that HIV + individuals demonstratemarkedly elevated numbers of HIV-reactive T cells even in theperipheral blood (33-35); presumably this is partly a conse-quence of the systemic distribution of HIV- 1 coupled with theanatomical localization of the infection to the immune systemitself. In fact, despite elements of organ specificity such as thatexhibited in DILS, the disseminated nature of the infectionpredicts that many of the same structurally selective TCR'scould be isolated from the peripheral blood as those isolatedfrom the salivary gland, especially in consideration of theuniquely prominent peripheral CD8 lymphocytosis in thisHIV + group.

Acknowledgments

Wethank Ms. Modrie Payne for excellent assistance in preparation ofthe manuscript.

This work was funded in part by a grant from the NewYork chapterof the Arthritis Foundation (E. Dwyer), a Postdoctoral Fellowshipfrom the Arthritis Foundation (S. Itescu), National Institutes ofHealth grants Al 1941 1 and AR39626, and American Foundation forAIDS Research grant 500255-12-PG.

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