Restricted T-Cell antigen receptor repertoire in bronchoalveolar T cells from normal humans

ELSEVIER

Restricted T-Cell Antigen Receptor Repertoire in Bronchoalveolar T Cells from Normal Humans Vladimir V. Yurovsky, Barbara White

Eugene R. Bleecker,

ABSTRACT: The repertoire of variable ct (AV) and p (BV) TCR genes was compared in the peripheral blood and BAL fluid of five healthy individuals. Rearranged TCR transcripts were amplified by a reverse transcriptionpolymerase chain reaction, using oligonucleotide primers specific for 22 AV and 24 BV gene families. Nearly all AV and BV gene families were expressed in BAL T cells at levels similar to those in blood T cells. The diversity of AV and BV gene repertoire was examined further, testing the distribution of nucleotide lengths of TCR junctional regions. Most V gene families had a normal distribution of junctional region lengths in both blood and BAL T cells. Some gene families, particularly AV21 and BV9 in BAL samples, had a skewed banding pattern, with fewer bands or predominance of several bands. The limited diversity in TCR junctional region lengths was more prominent in CD8’ T cells from BAL fluids than from blood.

ABBREVIATIONS AC (Y constant gene segment AV (Y variable gene segment BAL bronchoalveolar lavage BC p constant gene segment base pairs bp BV /3 variable gene segment C constant CDR complementarity-determining

and

CD4’ T cells also contributed to the limited diversity in BAL T cells. The oligoclonal expansion of bronchoalveolar CD8’ T cells was confirmed by sequence analysis of AV2 1 -constant (Y(AC) and BVPBC junctional regions in the blood and BAL cells. The levels of V gene expression and the diversity of junctional region lengths were very similar in T cells obtained from three separate lobes of one donor. In general, skewed patterns of TCR junctional region lengths were not consistent over time in two donors, over periods of 3 and 17 months. Together, these data show that the T-cell repertoire is diverse within the lungs of normal humans, except for an oligoclonal predominance of a few V gene families in both CD4’ and CD8’ T cells. The T-cell repertoire in the lungs changes over time, which may reflect environmental exposures. Human

Immunology 50, 22-3

7 (1996)

diversity joining peripheral blood mononuclear reverse transcriptase-polymerase reaction T-cell antigen receptor variable

PBMC RT-PCR TCR V

cells chain

region

INTRODUCTION Lymphocytes tory

tract

that

localize

are important

to the surface in protective

inhaled

foreign

antigens

[l,

tective

immune

response

in the human

of the respira-

immunity

27. To understand lung,

against the pro-

more

needs

to be known capabilities Experimental vated

and

antigens From the Department of Medtcine, University of Maryland School of Medicine: and Medicine and Research Services, Veterans Administration Medical Center, Baltimore, Maryland, USA. Address rebrint reauests to Dr. V. V. Yuvovskv. Debartment of Medicine. University of ;2larylaLd School of Medicine. MS?F B’uilding, koom 8-23: Baltimore, MD 21201, USA. Received December 4, 1995; accepted April 24, 1996. Human Immunology 50, 22-37 (1996) 0 American Society for Histocompatibility

and Immunogenetics,

1996

about

memory

characteristics

of the lower

have

found shown

T cells

tissues

the

or have tract

functional

healthy

presence

lung

lung. of acti-

that

recognize

by the individual

T cells in the lung

151. T lymphocytes respiratory

and

in the

in the

encountered

whether

lymphoid

circulation in the profile

studies

previously

It is unclear local

the

of lymphocytes

been

{3,4].

are derived

recruited

on the epithelial exchange

slowly

from

from with

the

surface those

blood lb}. It is known that the surface marker of T cells obtained from the lung by bronchoal019%8859/96/$15.00 PI1 SO198-8859(96)00126,-7

23

TCR Repertoire in Bronchoalveolar Fluid

veolar lavage (BAL) differs from that in peripheral blood [7,8]. T cells in BAL fluid belong mainly to the memory (CD45RO’) subtype 191 and frequently express the very late antigens-l and -4 that are associated with a late activation stage of the cell [6, lo}. The level of expression of CD45RA, a marker of naive T cells, is significantly lower in BAL fluid than in peripheral blood 14, 111. Little is known about the T-cell repertoire in the healthy lung. It is likely that the repertoire can be modified by encounter with environmental antigens, but this has not been proven. A single study has suggested clonal expansion of nearly all variable (V) p gene families of T-cell antigen receptor (TCR) in BAL fluids from normal donors [12]. Expression of some TCR AV (VCX)and BV (VP) gene families has also been studied within the diseased lungs, mainly in patients with pulmonary sarcoidosis {13--151. Increased expression of BV8 and AV2S3 compared with the healthy lungs has been demonstrated 113, 141. Diversity of TCR arises from pairing of different (Yand P-chains [ 161, use of multiple variable, diversity (D), and joining (J) gene segments 1171, random nibbling of those segments, and random addition of non-germlineencoded N nucleotides between them during the gene rearrangement [18]. The V-D-J junctional region of TCR, corresponding to the third complementaritydetermining region (CDR3) of immunoglobulin, varies considerably in length and amino acid sequence. The junctional region determines antigen specificity of the TCR [19, 20). T-cell responses to antigens may lead to clonal expansion of T cells with identical junctional regions 12 I-231 and, thus, restricted nucleotide lengths of the junctional regions. An oligoclonal expression of some BV gene families within CDS’ T-cell subpopulation in normal human blood has been described [24-261, probably owing to the responses to infectious agents to which the individuals were exposed. We examined all the major TCR AV and BV gene families {27-29} in unfractionated and CD8’ T cells from peripheral blood and BAL fluid of five normal individuals. A reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used to amplify rearranged TCR transcripts across the junctional region, followed by two methods of analysis. First, we determined the relative expression of V genes, normalized to the expression of constant (C) region of the alternate strand. Second, we looked for evidence of restricted diversity among different V gene families, using sequencing gel electrophoresis followed by autoradiography. This method identifies TCR V gene families expressed with a limited diversity of nucleotide lengths of their junctional regions, which suggests a selected expansion of the corresponding T cells [30-331. DNA sequencing of the junctional regions was

used to confirm oligoclonality of TCR with limited versity of junctional region lengths.

MATERIALS

AND

di-

METHODS

Isolation of BAL and blood samples. BAL was performed on five healthy, nonsmoking male volunteers (age range 1831 years). No donor had a history of asthma, systemic illness, or recent respiratory illness. None had evidence of airway hyperresponsiveness by methacholine challenge. Each had ~20% reduction in their l-s forced expiratory volume after a cumulative dose of >223 U of methacholine. All had normal pulmonary function tests. None had positive intradermal skin tests to a panel of allergens that included house dust mite and pollens. Three had no family history of atopy; two had one parent with a history of hayfever. The right middle lobe and lingular segment of the left upper lobe were lavaged in donor 1. The same lobes and the right upper lobe were lavaged in donors 2-4. The right middle lobe, right upper lobe, and lingular segment were lavaged separately in donor 5. Donor 1 underwent two separate lavages 17 months apart and donor 2 was lavaged a second time 3 months apart. Each lobe was lavaged with six 25-ml aliquots of sterile saline at 37°C through a fiberoptic bronchoscope (Olympus BF type ITlO; Olympus, Lake Success, NY). The saline was recovered by gentle suction immediately after the infusion of each aliquot. Peripheral blood was obtained from the same donors. The protocol was approved by the institutional review board for human studies, and informed written consent was obtained from the subjects. Cell separation. Bronchoalveolar lymphocytes were isolated by centrifugation at 250 X g for 10 min at 4°C. The cell pellet was washed two times in RPMI-1640 medium (Mediatech Inc., Herndon, VA) and resuspended either in RPMI-1640 containing 5% heat-inactivated human serum (Sigma Chemical Co., St. Louis, MO) for positive selection of CD8’ cells or in Hanks’ balanced salt solution containing 5% horse serum and 0.1% sodium azide for immunofluorescent staining. Peripheral blood mononuclear cells (PBMC) were isolated from the blood by density gradient centrifugation using Histopaque-1077 (Sigma Chemical Co.), washed two times, and resuspended as above. Positive selection of CD8’ T cells was performed using Dynabeads M-450 CD8 (Dynal, Inc., Great Neck, NY), according to the manufacturer’s instructions. In the second lavage from donor 1, the positive selection of CD8’ T cells was followed by positive selection of CD4’ T cells, using Dynabeads M-450 CD4. Purity of CD8’ and CD4’ fractions was 97-99%, as assessed by two-color flow cytometry.

24

Flow cytometry. For two-color immunofluorescence analysis, 2-S x lo5 cells were stained with monoclonal antibdo y anti-Leu-4 (CD3) coupled to phycoerythrin and monoclonal antibody anti-Leu-3a+3b (CD4) or anti-leu2a (CDS) coupled to fluorescein isothiocyanate. All antibodies were obtained from Becton-Dickinson (Mountain View, CA). Immunofluorescent staining was performed by standard techniques 1341 and analyzed using a FACS flow cytometer (Becton-Dickinson).

RT-PCR amplification of TCR jmctional

region RNA. Total cellular RNA was isolated from unfractionated and CD8’ cells from PBMC and BAL by acid guanidinium thiocyanate-phenol-chloroform extraction 1351. The first strand cDNA was synthesized in a 60-pl reaction mixture containing 2 pg of total RNA, 50 mM Tris-HCl (pH 8.3), 75 mM KCI, 3 mM M&I,, 0.5 mM of each dNTP, 1 pM of random hexamer primers, and 400 U of Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, MD). The reaction mixture was incubated at 42°C for 60 min, heated at 95°C for 5 min, and diluted with H,O to a final volume of 250 1.11.Five-microliter aliquots of the cDNA products were amplified using one of a panel of 5’ oligonucleoide primers specific for TCR AV or BV gene families and a 3’ oligonucleoide primer specific for AC or BC, respectively. The AV, BV, AC, and BC primers were purchased from Clontech Laboratories, Inc. (Palo Alto, CA). The AV and BV primers were designed to anneal to areas of maximum V region sequence diversity, which are not in the same location for the different V genes. The size range of AV-AC amplification products was from 290 to 430 base pairs (bp), and for BV-BC products was from 180 to 370 bp. The AC and BC primers were end-labeled with 32P 1361. The PCR was performed in the manufacturer’s recommended buffer with the use of a CetusiPerkin Elmer thermal cycler for 35 cycles under the following conditions: denaturation at 95°C for 1 min, primer annealing at 55°C for 1 min, and primer extension at 72’C for 1 min. To obtain semiquantitative data about TCR V gene usage, simultaneous PCR amplification of C region of the alternate strand was performed in each V regionspecific PCR reaction tube. The AC-AC control primers were used with the primers for the BV family and, conversely, the BC-BC control primers were used with the primers for the AV family. One primer in each pair was end-labeled with ‘*P. The concentration of V regionspecific primers was 0.5 PM and that of control primers was 0.2 pM. The sizes of the internal standards were 604 and 146 bp for AC-AC and BC-BC PCR products, respectively. Analysis of RT-PCR prodmts. RT-PCR products were separated by electrophoresis on a 1.8% agarose gel. “P

V. V. Yurovsky

et al.

incorporation into AV-AC, BV-BC, AC-AC, and BC-BC amplification products was measured using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). To correct for differences due to pipetting errors and efficiency of amplification, the integrated intensity of each V region-specific band was normalized to the alternate Cregion band amplified in the same tube. The relative amount of each V gene family was expressed as a percentage of the total intensities of 22 AV or 25 BV bands after normalizing for BC or AC bands, respectively. To analyze the distribution of nucleotide lengths of the RT-PCR products, electrophoresis in a 6% Long Ranger (AT Biochem, Malvern, PA) sequencing gel was performed, using Ml 3mpl8 bacteriophage sequence as a size marker. M13mp18 single-stranded DNA (United States Biochemical, Cleveland, OH) was sequenced by the standard dideoxy-mediated chain termination method {37], using a j2P-labeled Ml3 (-20) primer (5 ‘-GTAAAACGACGGCCAGT-3 ‘) and Sequenase (United States Biochemical). The RT-PCR products and M13mp18 nucleotide sequence were detected by autoradiography using XAR-5 film (Eastman Kodak, Rochester, NY). The intensities of RT-PCR bands were measured by Computing Densitometer (Molecular Dynamics). Sequencing of AV21-AC and BV9-BC junctional regions. RT-PCR-amplified AV21-AC or BV9-BC cDNA were ligated into pCR II vector (Invitrogen Corp., San Diego, CA) and then used to transform INVaF’ competent cells, according to the manufacturer’s instructions. Plasmids containing the cDNA inserts were isolated by conventional techniques [38} and sequenced by the dideoxymediated chain termination method [37f, using the same AC or BC primer as for PCR amplification (Clontech Laboratories, Inc.). DNA sequences were analyzed by electrophoresis in a 7% Long Ranger gel.

RESULTS Flow cytometric analysis of PBMC and BAL cells. First, the percentages of CD4’ and CD8’ T cells were compared in the peripheral blood and BAL fluid of the five healthy, nonsmoking individuals. The percentages of CD4’ and CD8’ cells in CD3’ T cells were determined by direct immunofluorescence and flow cytometry (Table 1). Each pair of blood and BAL cells was tested, except the second set from donor 2. The CD4/CD8 ratios were similar in peripheral blood and BAL T cells for each individual, with ratios usually > 1. Relative expression of TCR A V and BV gene families in unfractionated PBMC and BAL cells. An RT-PCR procedure was carried out to amplify rearranged TCR transcripts for

TCR Repertoire in Bronchoalveolar Fluid

TABLE

1

25

CD4’ and CD8’ T cells in peripheral blood and BAL fluids” @ CD3’

cells

expressing marker BAL

Blood Donor

1

Lavage I Donor 1 Lavage II Donor 2 Donor

3

Donor

4

Donor

5

Combined

CD4 CD8

68 29

31

CD4

69

60

CD8

27

38

CD4

53

64

RUL

RML

Lingula

lar in the blood and the three lobes (Fig. 2). The few exceptions were AV3, AV8, AVl 1, AVI 5, and BV3 gene families, for which a twofold or greater difference was seen for at least one comparison. A twofold or greater difference in levels was seen for only 4.7% of all possible comparisons. Thus, the levels of AV and BV gene expression were similar in blood and BAL cells from separate lobes.

68

Limited junctional diversity of TCR AV und BV gene transcripts in BAL

CD8

36

33

CD4

64

62

CD8

32

36

CD4

52

44

CD8

41

47

CD4

50

51

53

52

CD8

40

28

28

30

” Unfractionated PBMC or BAL cells were double-stained by direct immunofluorescence with anti-CDS-phycwtythrin and anti-CD4 or -CD8 coupled to fluorescein isothiocyanate and were analyzed by flow cytometry. Mononuclear cells were gated on the basis of forward and orthogonal light scatter criteria. Results are given as percent of CD4’ or CDB’ cells in total CD3’ cells.

22 major AV families and 24 major BV families in unfractionated PBMC and BAL cells. For semiquantitative analysis of V gene usage, simultaneous PCR amplification of an internal standard, the C region of the alternate TCR strand, was performed in each reaction tube. All TCR AV and BV gene families were detected in blood and BAL fluids (Fig. 1). There was no difference in the mean level of expression of most AV and BV gene families in blood and BAL fluids, with p > 0.05 for all comparisons except BV2 and BV4, using two-tailed paired t test. The difference was significant for BV2 at p = 0.03, which is not significant if a lower p value is required for multiple (47) comparisons. The difference in expression of the BV4 family in blood and BAL fluids was minimal, ~1%~ but reached statistical significance at p < 0.001 because of very small variability. In general, BVl to BV3 and BV6 to BV9 gene segments were expressed in both blood and BAL fluid at higher level than the remaining BV segments (p < 0.0001, unpaired two-tailed t test). An occasional gene family was expressed more than twofold higher in BAL fluid than in blood in an individual donor, including AV19 in donors 1 and 4, AV2 1 and BV6 in donor 2, BVll, BV15, BV17, and BV18 in donor 3, and BV21, BV22, BV23, BV24 in donor 4. To address whether BAL cells from one lobe are representative of the entire lung, T cells from the blood, right upper lobe, right middle lobe, and lingula of donor 5 were tested separately. The percentages of CD4’ and CD8’ T cells were nearly identical in each lobe (Table 1). The level of TCR AV and BV gene expression was simi-

cells. The diversity of AV and BV gene repertoire was further analyzed by the distribution of nucleotide lengths of TCR junctional regions, using polyacrylamide sequencing gel electrophoresis with an M I3mpl8 sequence as a size marker. The amplified TCR transcripts and Ml3mpl8 nucleotide sequence were detected by autoradiography. Figures 3 and 4 show analyses of several AV and BV gene families in unfractionated and CD8’ T cells from the peripheral blood and BAL fluid of the four donors. The amplified products within each band represent TCR (Y or p rearrangements of identical nucleotide length. Bands were 3 or 6 bp apart compared with the M13mp18 sequencing ladder, which would be expected for an open reading frame. The degree of 32P incorporation reflected the number of amplified cDNA molecules, corresponding to the level of mRNA expression of rearranged TCR cx or p genes. The majority of V gene families had multiple banding pattern of their junctional region lengths both in the blood and BAL fluid, indicating diversity of the TCR. Frequently, there was a normal distribution of nucleotide lengths of the junctional regions, especially in the blood (e.g., AV8, AVI5, AV18, AV21, BV3, BV17, and BV19 gene families in the blood of donor 1) (Fig. 3). Occasionally, multiple junctional region lengths were observed, but with skewing away from a normal distribution. This was seen more frequently in BAL samples (e.g., AV15, AV21, and BV19 gene families in BAL fluid from donor 1) (Fig. 3). In contrast, a few V gene families possessed limited diversity in junctional region lengths, with predominance of one or two bands. The relative intensities of RT-PCR bands were measured with a Computing Densitometer. If the intensities of one or two bands constituted >50% in a given sample, this was designated as oligoclonal V gene family expression for purposes of description. Such a pattern of expression was observed for BV9 gene family in donor 1 (Fig. 3) and for all V gene families presented in Figure 4. In each of these cases, oligoclonality was present in BAL T cells, but not in peripheral blood T cells. Some V gene families had a banding pattern in BAL fluid different from that in the blood, but identical in unfractionated and CDS’ cells from BAL fluid [e.g.,

26

V. V. Yurovsky

16-

A

0

15

16

16

17

17

18

19

20

et al.

Blood BAL

21

22

AV Segments

l6

B

1

2

3

4

5.1

6

8

9

10

11

12

13

14

L5

18

19

20

21

22

23

24

BV Segments FIGURE 1 Relative amounts of TCR AV (Panel A) and BV (Panel B) gene segments expressed in unfractionated T cells from blood and BAL fluids from four individuals. 32P-labeled RT-PCR-amplified products were separated on 1.8% agarose gel and quantitated using PhosphorImager. The value of each V gene family was normalized to the corresponding internal standard and presented as a percent of the total 22 AV or 25 BV values, respectively. The mean f SEM of results in the four donors are presented. and A)].

BAL T cells. Of note, the AV2 1 gene family in BAL fluid from donor 2 and the AV19 family in BAL fluid from

These results show that CDS’ T cells contribute to differences in junctional region lengths between blood and

donor 4 (Fig. 4) showed a different distribution of bands in unfractionated and CDB’ cells, and both differed from

BV9 BV9

in donor in donor

1 (Fig. 3), AV19 in donor 2, AV7 3, BV9 and BV20 in donor 4 (Fig.

TCR

Repertoire

in Bronchoalveolar

27

Fluid

0

Blood

I

Lingula

rnRML RUL

1

15

3

2

4

5

6

7

8

9

11

12

AV

Segments

10

13

14

15

16

17

18

19 20

21

22

B 1

1

2

3

4 5.1 5.2 6

7

8

9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 BV

FIGURE lingula,

Segments

Relative expression of TCR AV (Panel A) and BV (Panel B) segments in unfractionated PBMC 2 right middle lobe (RML), and right upper lobe (RUL) from donor 5. See Figure 1 for explanation.

and BAL cells from

28

V. V. Yurovsky et al.

the blood. AV21 family in donor 4 was not expressed in CDS’ cells from either the blood or BAL, but was expressed in both unfractionated samples, although with different patterns (Fig. 3). These last examples suggest that CD4’ T cells also contribute to differences in TCR junctional region diversity between blood and BAL T cells. To further test the possible contribution of CD4’ T cells to the limited junctional region diversity in BAL T cells, positive selection of both CD4’ and CD8’ T cells was performed from a second set of blood and BAL samples from donor 1 (Fig. 5). Although multiple junctional region lengths were seen, some patterns were skewed away from a normal distribution within the CD4’ T cell subset (Fig. 5, AV8 and BV17, lanes e). The consistency over time of limited diversity of V gene junctional region lengths was tested by comparing

FIGURE

3

Analysis of junctional

region lengths of TCR AV and BV gene families in unfractionated

the blood and BAL fluid of donor 1. Lanes: a: unfractionated cells. M13mp18 bacteriophage sequence was electrophoresed

a

AV15 a b c d

AV8

results from the first and second sets of blood and BAL samples from donors 1 and 2 (Fig. 6A and B, respectively). For both donors, most V gene families continued to be expressed in a polyclonal manner, with similar patterns of multiple junctional region lengths in both blood and BAL T cells from both sets of samples (for example, AV18). When skewing of TCR junctional region lengths was seen in a particular V gene family in one set of samples, the pattern was usually different in the second set. As examples, compare AV8, AV17, AV2 1, and BV17 in CD8’ T cells from donor 1 and BV17 in CD8’ T cells from donor 2 (Fig. 6). However, two V gene families had a similar skewed distribution of TCR junctional lengths in each set (BV9 and BV21 in BAL T cells). In BV9 gene family, the similarity was more prominent in unfractionated than in CD8’ BAL cells.

b c

d

a

a

BV3 b c

d

AV17 b c d

a

a

BV9 bc d

PBMC; 6: CDS’ PBMC; c: unfractionated as a size marker.

AV18 b c d

BV17 abed

AV21 a b c d

BV19 abed

and CD8’ T cells from BAL cells; d: CD8’

M131npl8 GATC

M13mp18 GATC

by

bp -250

-220

BAL

TCR Repertoire

in Bronchoalveolar

Fluid

29

Donor 3

Donor 2 AV19 a h c- du_mzz.e_.=c

a

AV21 b c d

AVl b c

a

BV9 b c

a

d

d

: > _ i .‘

- 340 -360

-

330

310

.

Donor 4

AV19 a b c d

a

BV9 ;1 b c

AV21 b c d

a

d

BV20 b c

d

-340

- 240

FIGURE 4 TCR AV and BV gene families which showed oligoclonal banding pattern in BAL samples from donors 2-4. Lanes are marked as in Figure 3. The size range of the RT-PCR products (in bp) is given and is dependent upon the particular V-C primer pair.

Junctional region diversity of TCR AV and BV gene families

determine

in differeent lobes. The diversity

region

resents

that

middle

region

lengths

lengths

lobe,

in the

and right

blood,

upper

lingular

in TCR

junctional

segment,

lobe in donor

right

5 was tested

to

whether

the T-cell

of the entire

were strikingly

lung

repertoire (Fig.

for AV and BV gene

similar.

This

in one lobe rep7). The

families

similarity

junctional in each lobe

held true even

FIGURE 5 TCR AV and BV gene families in unfractionated, CD4’, and CDS’ T cells from the second isolation of blood and BAL fluid from donor 1. Lanes: u: unfractionated PBMC; b: CD4’ PBMC; u CD8’ PBMC; d: unfractionated BAL cells; e: CD4’ BAL cells; fi CDS’ BAL cells. The size range of the RT-PCR products (in bp) is given. AVX

AVII

AVIX

UVO

AV?I a b c d e c -360

UVl7

30

V. V. Yurovsky

A a

b

c

II abed

d

AV18

AVl7

AV8

I

I

a

b

c

abed

d

et al.

I

11

a

b

c

d

a bl’c

a

b

c

d

abed

d

- 360

AV2l

1

II abed ,_

abed

II abed

I

a

bc

d

-360

BV21

BV17 a

I abed

11

I b _c

d

a

b

c

d

a

11 b c

I abed

d

- 250

II a

b

c

d

-260

- 205

- 230

- 193

- 240

FIGURE 6 TCR AV and BV gene families in the first (I) and second (II) isolations of blood and BAL fluid from donors 1 (Panel A) and 2 (Panel B). Lanes: a: unfractionated PBMC; b: CD8’ PBMC; c: unfractionated BAL cells; d: CD8’ BAL cells.

when the pattern cells

differed

of junctional

from

that

regions

in peripheral

lengths

in BAL T

T cells.

As

ex-

in the AV15, BV7, and BV17 gene families, a similar polyclonal distribution of the junctional region lengths was observed in T cells from blood and each lobe. In the AV19, AV21, BV9, and BV13 gene families, a similar pattern of junctional region lengths was seen in all three lobes that was different from that in the blood. The AVll, AVI6, and BV21 gene families showed an oligoclonal pattern of junctional region lengths in each of the three lobes, as determined with a Computing Densitometer. Together, these data demonstrate altered diversity in junctional region lengths of some TCR V gene families in BAL fluid of normal humans. Table 2 summarizes the data of AV and BV gene family expression in these five donors, as presented in Figures 3-7. amples,

Sequence analysis of junctional regions in CD8’ PBMC and BAL T cells. The presence of TCR rearrangements of predominant lengths is consistent with the oligoclonal expansion of T cells expressing the given V segment. To support this conclusion, AV2 l-AC PCR amplification products from the blood and lung CD8’ T cells of donor 2 and BV9-BC PCR products from donors 3 and 4 were subcloned into a bacterial vector and sequenced across the junctional region. Nine clones were isolated from AV21-AC PCR products from blood CDS’ T cells; each was isolated just once (Fig. 8). The nucleotide lengths of their junctional region sequences corresponded to the 33%35O-bp size range of the PCR amplification products shown in Figure 4 (Donor 2, AV21, lane b). In contrast, four of nine sequences isolated from the bronchoalveolar CDS’ cells were identical (BAL-3 in Fig. 8), with a 39-bp length of CDR3 region, corresponding to

TCR Repertoire

in Bronchoalveolar

AVll ab c d

AV15

Fluid

31

AV21 AV19 abcdabcdGATC

AV16

M13mp18 -360

- 280

320

BV13

BV9

BV7

BV 17

BV21

FIGURE 7 TCR AV and BV gene families in unfractionated PBMC and BAL cells from three lobes from donor 5. Lanes: a: PBMC; 6: lingular segment; c: right middle lobe; d: right upper lobe. The size range of the RT-PCR products (in bp) is given.

the upper major band of 344 bp in lane d (Fig. 4). One more sequence, BAL-4, had the same nucleotide length. Two other sequences, BAL-5 and BAL-6, were of a 33-bp CDR3 length, corresponding to the lower major band of 338 bp in lane d. Thus, the diversity of TCR AV21-AC junctional region sequences confirmed the oligoclonal character of the expansion of bronchoalveolar CDS’ T cells versus polyclonal expansion of blood CDS’ T cells. It is noteworthy that some identical sequences were isolated from both blood and BAL samples (Blood-5 and BAL-3, Blood-9 and BAL-6).

TABLE 2

Expression of TCR AV and BV gene families

Sequence analysis of BV9-BC junctional region (Fig. 9) resulted in isolation of 19 distinct sequences from the blood CD8’ T cells of donor 3, with nucleotide lengths corresponding to a 187-2O5-bp size range of the PCR amplification products shown in Figure 4 (Donor 3, BV9, lane b). One of these sequences was isolated twice. Six distinct BV9-BC junctional sequences were isolated from the blood CD8’ T cells of donor 4; all were found just once (Fig. 9). From the BAL CDS’ T cells of donor 3, 14 distinct BV9-BC junctional sequences were isolated. One of them (BAL-2), corresponding to the upper in peripheral

blood and BAL fluids Number of V gene families detected” AV

Donor Donor Donor Donor Donor

1 2 3 4 S

Oligoclonal V gene families detected

BV

AV

BV

Blood

BAL

Blood

BAL

Blood

22 21 21 21 22

21 21 21 21 22

23 23 24 24 24

24 24 24 24 24

0 0

0 AV19,

0 0

AV7 AV19,

0

Awl,

G From 22 AV and 24 BV gene families tested

BAL

Blood

BAL

21

0 0

BV9 BV9,

21 16

0 0 0

BV9 BV9, 20 BV21

17, 21

32

V. V. Yurovsky et al.

Source

Junctional AV21

Blood 1 TGTGCAGCAA

BAL

sequence N

AJ ACCXXCAGGAGAGC~lTAClTllGGG

GCGGGGCGCGGGAC

2 TGTGCAGCAA

GCGAlTCAGGA

3 TGTGCAGCAAC

GCTGGACCATAGAAGG

4 TGTGCAGCM

GCCCCCA

5 TGTGCAGCAA

GCGCGAGGACT

6 TGTGCAGCAA

GGAGGGATA

7 TGTGCAGCAA

as

8 TGTGCAGCAA

GCGTAGG

9 TGTGCAGCAA

GTCCT

GGAGGTGCTGACGGACTCACC-ITEGC AATGCCAGACTCATGT-ITGGA

AAA-ITCCGGGTATGCACTCAACTTCGGC

AJ Segment

CDR3 length

Number of isolates

16.6

45

1

9.7

42

1

3.2

42

1

9.10

39

1

GGGGCAAACAACCTClTCTTTGGG

17.5

39

1

ACTATGGTCAGAATTTTGTCTTTGGT

13.2

39

1

AAAGCAGCGGAGACAAGCTGACT’ITI-GGG

FFill.l

36

1

CTATGGTCAGAATTTTGTCTTTGGT

13.2

36

1

ACCGGTAACCAGlTCTATTlTGGG

17.3

33

1

17.8

42

1

4.1

42

1

GGGGCAAACAACCTClTCTlTGGG

17.5

39

4

4 TGTGCAGCAACC T

TAGGAGGAGGTGCTGACGGACTCACCTTTGGC

9.7

39

1

5T

TCCGG

ACTCAGGGCGGATCTGAAAAGCTGGTCmGGA

9.16

33

1

6 TGTGCAGCAA

GTCCT

33

1

1 TGTGCAGCAACC CGAT

CTAACTTTGGAAATGAGAAATTAACCTTTGGG

2 TGTGCAGC

GCCCCGAGGG

3TGTGCAGCAA

GCGCGAGGACT

GAAACCAGTGGCTCTAGGTTGACUTTGGG

ACCGGTAACCAGTTCTATTTTGGG

17.3

amplified from the blood and lung CD8’ T cells from donor 2. FIGURE 8 Nucleotide sequences of AV21-AJ rearrangements RT-PCR products were subcloned into a bacterial vector and the plasmid inserts were sequenced using the same AC primer as for PCR. Assignment of AJ segments was made according to nomenclature [39], except the Blood-7 sequence, which AJ segment corresponds to a germline transcript reported in reference [40). CDR3 length was calculated as the number of nucleotides from, but not including, the J region+ncoded GXG triplet (where G is glycine and X is any amino acid) to the nearest preceding V region-encoded cysteine [41) or phenylalanine in the BAL-5 sequence.

major

band of 205 bp in lane d (Fig. 4), was found

times;

and three

length

(BAL-9,

lower major

sequences BAL-10,

of identical BAL-11)

junctional

corresponded

three region to the

band of 193 bp. From the BAL CDS’ T cells

of donor 4, nine distinct BV9-BC junctional sequences were isolated (Fig. 9). Sequences corresponding to the lower major band of 193 bp in Figure 4 (Donor 4, BV9, lane d) were isolated 13 times (BAL-6, BAL-7, and BALS), further confirming the oligoclonal character of the expansion of bronchoalveolar CDS’ T cells.

DISCUSSION The T-cell repertoire was analyzed in the blood and lungs of healthy individuals. Nearly all AV and BV gene families were expressed at both sites in each individual. Interindividual variability in the level of expression of many AV gene families was several fold. As examples, AV2 was expressed by 2-l 1% and AV14 was expressed by 5-19% of peripheral blood TCR transcripts. These variations are in agreement with the data on AV expression determined by DNA sequencing 1391. Analysis of the TCR BV repertoire revealed that BVl to BV3 and BV6 to BV9 segments were generally detected at higher level than other BV segments in both blood and BAL fluid. Similar observations have been made in the blood of normal subjects {24] and in BAL fluid from patients with pulmonary sarcoidosis 1151.

BAL fluids are used as the source of T cells for several reasons. The lavage can be obtained easily and repeatedly, with low risk to the patient. Millions of T cells with excellent viability can be recovered from BAL fluid, which facilitates isolation of subpopulations. T cells in the airways are likely to represent those in adjacent mucosal tissue. The phenotype of BAL T cells correlates well with those obtained from tissue in both normal and diseased lungs 1431. The major exception is interstitial lung disease, in which T cells are underrepresented in BAL cells {44, 451. Compared with testing BAL T cells, analysis of the T-cell repertoire in a transbronchial, thoracoscopic, or open lung biopsy is associated with a greater risk to the patient from obtaining the sample. Moreover, bias in results may be introduced by biopsy site and size, with analyses of fewer T cells. We looked for evidence that superantigens or conventional peptide antigens skewed the T-cell repertoire in BAL fluids from normal humans. T-cell activation by superantigens can cause a polyclonal increase 146-491 or decrease [SO, 5 l] in expression of certain BV gene families, without conservation of junctional region sequences. In contrast, T-cell activation by nominal antigens can cause oligoclonal expansion of T cells that conserve junctional regions in one or both TCR chains 121-23, 5254]. Limited data suggest the possibility that superantigens may influence the T-cell repertoire in healthy donors. In each individual, a few BV gene families were

TCR Repertoire

in Bronchoalveolar

Junctional BV9

Source

Donor 3 Blood 1 AGCCMG 2 AGCCA 3AGC 4 AGCCMG 5 AGCCAAG G&CC 7 AGCCAAG

Fluid

33

BJ

2.1 1.2

51 40

AATGAAAAACTGWGGC CACAGATACGCAGTATmGGC AAGAGACCCAGTACTTCGGG ATCAGCCCCAGCATTTTGGT

1.4 2.3 2.5 1.5

40 48 48 45

2.1

45

2.2

45

1

CCTACGAGCAGTACTTCGGG

2.7

45

1

AACACCGGGGAGCTG”GGA GAGACCCAGTACITCGGG CAAGAGACCCAGTAClTCGGG

2.2 2.5

42

1

42

AGAGACCCAGTACTTCGGG CACCGGGGAGCTGTTJJ-I’TGGA

2.5 2.5 2.2

39 39 39

1 1 1 1

TACAACGTGGUXGACAGGAG GGGTATCTCCCAGGGTWGG cx.MAA-m AACCCAGCGGGGTACGCT

11 AGCCAAG GAACAGGGVTAGG 12 AGCCAAG CGGCTCAC 13 AGCCAAG ATGAAGTGGG 14AGC ITAGMCGGGC

ATGAGCAGTKXTCGGG AACACCGGGGAGCTGlTll-fTGGA

15NXC TGTGGACAGCCG 16 AGCCAAG ATAGG

BAL

1 AGC

TAGGATG TATGAUXGAG TTCGGCGGCA lTMGGCCTCllCAGGGCGCATCGAAGATG

GAATTAGGGGGACAGGGGGTTCTA 2AGcc JAGCC CCCCGGGTCAGGGCATCGAGAA 4 AGCCAAG GTCCGGGACGGACTC 5 AGCCAAG ATCCGGGCCCACG 6&X-C CACCGGGTAGT 7 AGCCAAG ATCGTGTCAGG 8A ACCTACAGGGGTCA QAGC TCGACAGGGG lOAGCGAAGGGllCEXXXX= 11 AGCCAAG ATCGCGGCCCCTT 12MCC CT13 AGCCAAG CCTTGATGGC 14KCC CTTCCTTCC

Donor 4 Blood 1AGcc

AATACCTAGGAACTGG 2 AGCCAAG GAGCAGGAGGACTGG 3ECC CGGGACAGG 4AG TCCAGGGGWCT 5 AGCCAAG ATGCG 6 AGCCAAG GCTC

BAL

1 AG

TCTCACTGACGGGCTAGGGG

2 AGCCAAG GCATCAGGGAC 3AGC TTAGAGGATGAGGGGG 4AGCC CCTTAGGGACGACACAG

1 1 1 1

TCCTACAATGAGCAGTKTTCGGG TATGGCTACACCTTCGGT

AGGGACCGGGTGGTAGC GGGAmGGACAGGGGGCTGGA

TTCAGGGGGGGAG 8 AGCCA Q AGCCAAG AGTGGGCAGCTCCCA TGATGGCTCCG IOAGCC

17 AGCCA 18AGC 1QAGC

BJ CDR3 Number Segment length of isolates

sequence N-BD-N

1 1 1

CCTACGAGCAGTACTTCGGG

2.7

39

1

AGCACAGATACGCAGTAT-I-ITGGC AATGAGCAGTTCTKGGG

2.3

39

2.1

1 2

TACGCAGTAmGGC ACGAGCAGTACTTCGGG

2.3 2.7

33 33 33

1 1

AACACTGAAGCTITCJ-I-I’GGA

1 .l

57

1

AAGAGACCCAGTACI-KGGG AGATACGCAGTATTI-TGGC ACAATGAGCAGTTCTTCGGG AGAGACCCAGTACTTCGGG

2.5

51

3

2.3 2.1 2.5 2.3 1 .l

46 45

1 1

42 42

1 1

42

2.1 2.2

42 39

1 1

TACGAGCAGTACTTCGGG

2.7

39

1

CGAGCAGTACTTCGGG

2.7 1.4

39 36 36 33

1 1 1 1

2.1

45

1

1.2 2.6 1.2

42 42

1 1

39

1

2.1 1.2

36

1

33

1

CCTACAATGAGCAG-I-KXTCGGG

2.1

48

1

TCCTACAATGAGCAGTTCTTCGGG ACTATGGCTACACCTTCGGT

45 42 42

1 1 4

AGCACAGATACGCAGTATTTTGGC AACACTGAAGCTTTCTTTGGA TCCTACAATGAGCAGTTCTTCGGG ACACCGGGGAGCTGTl-ETTGGA

AAAACTGTTTTTTGGC TGAGCAGTTCTTCGGG ACGAGCAGTACTTCGGG

CTACAATGAGCAGTTCTTCGGG ATGGCTACACCTTCGGT CTGGGGCCAACGTCCTGACmCGGG AACTATGGCTACACCTTCGGT TACAATGAGCAGTTCTTCGGG CTATGGCTACACCTTCGGT

2.1 2.7

1

GAAAAACTGTTTTTTGGC

2.1 1.2 1.4

CCTACGAGCAGTACTTCGGG

2.7

42

1

6MCC

CCCGGGCAGGAACA CGGCGGGGACCG

ACACTGAAGCllTCT-lTGGA

1.1

39

10

7AGCC

CAGGGCTCATG

AACACTGAAGCT-ITCTTTGGA

1.1

39

2

CCTACGAGCAGTACTTCGGG

2.7

39

1

2.7

36

1

5 AGCCA

8 AGCCAAG ATGGCCTAG 9AGc

FIGURE 9

TCAGGGGTAGAGGGCGG

GCAGTACTKGGG

Nucleotide sequences of BV9-BD-BJ rearrangements amplified from the blood and lung CD8” T cells from donors 3 and 4. The same BC primer was used for PCR and sequencing. Assignment of BJ segments was made according to nomenclature [42). CDR3 length was calculated as in Figure 8.

34

expressed in twofold or greater amounts in BAL fluids than in blood or vice versa. Many of these BV gene families (Vp2, VB3, Vp6, VBl2, V/314, Vpl5, Vpl7, and VBlS) are involved in T-cell responses to superantigens 147, 551. Complementary studies of TCR junctional region lengths showed that all but one of these BV gene families were expressed in a polyclonal manner. Thus, the polyclonal increase in T cells bearing these particular BV genes suggests that superantigens may influence the T-cell repertoire in the blood and lungs of normal adults. However, it remains possible that the minor increase in expression is related to chance, vagaries of quantification by RT-PCR technique, or preferential sequestration of particular T cells in the lung. We found evidence that conventional protein antigens may skew the T-cell repertoire in the lungs of healthy donors. T-cell activation by conventional antigen can be associated with conservation of junctional regions in either or both (X and p TCR chains 121-23, 52-541. Conservation of TCR junctional region sequences will reduce variability in nucleotide lengths of the junctional regions. In each donor, we identified AV or BV gene families with a restricted distribution of TCR junctional region lengths in BAL fluid. Other AV and BV gene families that were expressed at similar levels in the same samples displayed a normal distribution of junctional region lengths. Thus, it is unlikely that the restriction in TCR junctional region lengths was due to a small number of T cells being tested or limitations of RT-PCR technique. We interpret the finding of reduced diversity of TCR junctional region lengths as evidence that the corresponding T cells have undergone selection by conventional protein antigens. This interpretation is supported by DNA sequence analyses in this and other articles 125, 56, 571. In this study, evidence of antigen selection of T cells in normal subjects was found in CDS’ T cells. This is consistent with the reports of others that CDS’ T cells undergo selected expansion in the blood of normal 124, 251 and aged donors [58-601. Of note, in this study, skewing of the CD8’ T-cell repertoire was more pronounced in BAL fluids than in blood. Restricted diversity in junctional region lengths of V gene families expressed on CD8’ T cells was not always apparent when unfractionated T cells were tested. Thus, the sensitivity of using junctional region length analysis to detect skewing of the T-cell repertoire increases when CD8’ and CD4’ T cells are tested separately. Our results suggest that CD4’ T cells may also contribute to TCR junctional region diversity in BAL fluid. Using RT-PCR and single-strand conformation polymorphism analysis, Dohi et al. also found evidence of oligoclonality of T cells within the lungs of normal subjects [12]. They found that most of TCR BV gene fami-

V. V. Yurovsky et al.

lies tested had undergone clonal expansion in BAL fluids from five normal donors. Remarkably, in one donor, all BV gene families were involved. In contrast, our data suggest that only a few BV and AV gene families have undergone oligoclonal selection in a given donor. Our data suggest that the skewing of the CD8’ T-cell repertoire in normals does not persist over time in all instances. Variability in the TCR repertoire over time was examined in two individuals, with testing 3 and 17 months apart. Those AV or BV gene families that had skewed junctional region lengths in one set of samples usually had a different pattern in the other set. Encounter with different environmental antigens may be responsible for this variability in the TCR repertoire over time. Viral infection might lead to oligoclonal selection of CD8’ T cells. Nuclear or cytoplasmic self-antigens can also be presented in the context of MHC class I molecules 1611. Exposure to these self-antigens might explain the oligoclonal CD8’ T cells. The antigen specificities of these CD8’ T cells in normal humans remains to be tested. The skewing of a few V gene families expressed by CD4’ T cells may reflect antigen-specific responses of these cells to exogenous pathogens such as bacteria. One unexpected observation was the selective use of certain junctional region lengths in association with the BV9 gene family, without any selection for DNA sequences. The pattern of BV9 TCR junctional region lengths was similar in BAL T cells from four donors, with conservation of junctional regions of 193- and 205bp lengths. The reason for this conservation is unclear. It may be related to antigen specificity of the T cells expressing BV9. Antigen challenge can lead to homogeneity in the CDR3 length before selection of DNA sequences 1621. Alternatively, the preferential use of certain junctional region lengths with the BV9 gene family may be unrelated to antigen and may merely reflect more favorable structural characteristics imposed by those lengths. The persistence of similar patterns of BV9 junctional region lengths over time and in multiple individuals would be consistent with this latter possibility. An increased level of expression of a particular AV or BV gene family in tissue compared with blood has been used to identify T cells likely to be involved in disease processes 114, 63, 641. Our data suggest that this strategy will preferentially detect T cells that have undergone polyclonal expansion by superantigens and may overlook T cells that have undergone oligoclonal selection by conventional protein antigens. Most V gene families expressed in an oligoclonal manner in BAL were not present as an increased percentage of total TCR transcripts. This result emphasizes the importance of using limited heterogeneity of TCR junctional regions, rather than level of V gene expression, to screen for T cells likely to have been selected by conventional protein antigens.

TCR Repertoire

in Bronchoalveolar

ACKNOWLEDGMENTS

The authors thank Jung Choi and Mary Ann Johns for excellent technical assistance. This work was supported by a Career Investigator Award (B.W.) and a Merit Award from the Veterans Administration (B.W.). V. V. Yurovsky is a fellow of the Arthritis Foundation.

REFERENCES 1. Agostini

C, Chilosi M, Zambello R, Trentin L, Semenzato G: Pulmonary immune cells in health and disease: lymphocytes. Eur Resp J 61378, 1993.

2. Zambello R, Feruglio C, Cipriani A, Cadrobbi P, Semenzato G: B-ly-7, a monoclonal antibody labeling of activated lung lymphocytes. Blood 77:1855, 1991. S, Bouchonnet F, Smiejan JM, Hance AJZ: 3. Dominique Expression of surface antigens distinguishing “naive” and previously activated lymphocytes in bronchoalveolar lavage fluid. Thorax 45:391, 1990. of nor4. Becker S, Harris DT, Koren HS: Characterization mal human lung lymphocytes and interleukin-2-induced lung T cell lines. Am J Respir Cell Mol Biol 3:441, 1990. MA, Rose A, Ford J, 5. Holt PG, Kees UR, Shon-Hegrad Bilyk N, Bowman R, Robinson BWS: Limiting dilution analysis of T cells extracted from solid human lung tissue: comparison of precursor frequencies for proliferative responses and lymphokine production between lung and blood T cells from individual donors. Immunology 64: 649, 1988. 6. Saltini C, Hemler ME, partmentalized on the spiratory tract express complex VLA- 1. Clin 1988.

Crystal RG: T-lymphocytes comepithelial surface of the lower rethe very late activation antigen Immunol Immunopathol 46:22 1,

7. Davidson BL, Faust J, Pessano S, Daniele RP, Rovera G: Differentiation and activation phenotypes of lung Tlymphocytes differ from those of circulating Tlymphocytes. J Clin Invest 76:60, 1985. 8. Yamada M, Tamura N, Shirai T, Kira S: Flow cytometric analysis of lymphocyte subsets in the broncho-alveolar lavage fluid and peripheral blood of healthy volunteers. Stand J Immunol 24:559, 1986.

9 Saltini C, Kirby M, Trapnell

BC, Tamura N, Crystal RG: Biased accumulation of T-lymphocytes with “memory”type CD45 leukocyte common antigen expression on the epithelial surface of the human lung. J Exp Med 17 1: 1123, 1990.

10

35

Fluid

Ho1 BEA, Krouwels FH, Bruinier B, Reijneke RMR, Mengelers HJJ, Koenderman L, Jansen HM, Out TA: Cloning of T lymphocytes from bronchoalveolar lavage fluid. Am J Respir Cell Mol Biol 7:523, 1992.

11. Garlepp MJ, Rose AH, Bowman RV, Mavaddat N, Dench J, Holt BJ, Baron-Hay M, Holt PG, Robinson BW: A clonal analysis of lung T cells derived by bronchoalveolar lavage of healthy individuals. Immunology 77:31, 1992.

12. Dohi M, Yamamoto K, Masuko K, Ikeda Y, Matsuzaki G, Sugiyama H, Suko M, Okidaira H, Mizushima Y, Nishioka K, Ito K: Accumulation of multiple T cell clonotypes in lungs of healthy individuals and patients with pulmonary sarcoidosis. J Immunol 152:1983, 1994. 13. Moller DR, Konishi K, Kirby M, Balbi B, Crystal RG: Bias toward use of a specific T cell receptor B-chain variable region in a subgroup of individuals with sarcoidosis. J Clin Invest 82:1183, 1988. 14. Grunewald J, Janson CH, Eklund A, Ohrn M, Olerup 0, Persson U, Wigzell H: Restricted Vo2.3 gene usage by CD4 + T lymphocytes in bronchoalveolar lavage fluid from sarcoidosis patients correlates with HLA-DR3. Eur J Immunol 22:129, 1992. 15. Forrester JM, Wang Y, Ricalton N, Fitzgerald JE, Loveless J, Newman LS, King TE, Kotzin BL: TCR expression of activated T cell clones in the lungs of patients with pulmonary sarcoidosis. J Immunol 153:4291, 1994. 16. Davis MM, Bjorkman and T-cell recognition.

PJ: T cell antigen receptor Nature 334:395, 1988.

genes

17.

Davis MM: T cell receptor Ann Rev Biochem 59:475,

18.

Wilson RK, Lai E, Concannon P, Barth RK, Hood LE: Structure, organization and polymorphism of murine and human T-cell receptor (Y and B chain gene families. Immunol Rev 101:149, 1988.

I?.

Chothia C, Boswell DR, Lesk AM: The outline structure of the T cell alpha/beta receptor. EMBO J 7:3745, 1988.

gene diversity 1990.

and selection.

20. Jorgensen JL, Reay PA, Ehrich EW, Davis MM: Molecular components of T-cell recognition. Ann Rev Immunol 10835, 1992. 21. Aebischer T, Oehen S, Hengartner H: Preferential usage of Va4 and VP10 T cell receptor genes by choriomeningitis virus glycoprotein-specific H-2Db-restricted cytotoxic T cells. Eur J Immunol 20:523, 1990. 22. Moss PAH, Moots RJ, Rosenberg WMC, Rowland-Jones SJ, Bodmer HC, McMichael AJ, Bell JI: Extensive conservation of (Y and B chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc Nat1 Acad Sci USA 88:8987, 1991. 23. Deckhut AM, Allan W, McMickle A, Eichelberger M, Blackman MA, Doherty PC, Woodland DL: Prominent usage of VB8.3 T cells in the H-2Db-restricted responses to an influenza A virus nucleoprotein epitope. J Immunol 151:2658, 1993. 24. Akolkar PN, Gulwani-Akolkar B, Pergolizzi R, Bigler RD, Silver J: Influence of HLA genes on T cell receptor V segment frequencies and expression levels in peripheral blood lymphocytes. J Immunol 150:2761, 1993. 25. Hingorani R, Choi I-H, Akolkar P, Gulwani-Akolkar B, Pergolizzi R, Silver J, Gregersen PK: Clonal predominance of T cell receptors within the CD8’ CD45RO’ subset in normal human subjects. J Immunol 151~5762, 1993. 26. Posnett

DN, Sinha R, Kabak S, Russo C: Clonal popula-

36

V. V. Yurovsky

tions of T cells in normal elderly humans: the T cell equivalent to “benign monoclonal gammapathy.” J Exp Med 179:609, 1994. 27. Kimura N, Toyonaga B, Yoshikai Y, Du R-P, Mak TW: Sequences and repertoire of the human T cell receptor (Y and p chain variable region genes in thymocytes. Eur J Immunol 17:375, 1987. 28.

Klein MH, Concannon P, Everett M, Kim LDH, Hunkapiller T, Hood L: Diversity and structure of human T-cell receptor a-chain variable region genes. Proc Nat1 Acad Sci USA 84:6884, 1987.

29. Robinson MA: The human T-cell receptor beta chain complex contains at least 57 variable gene segments. J Immunol 146:4392, 1971. 30. Cachet M, Pannetier C, Regnault A, Darche S, Leclerc C, Kourilsky P: Molecular detection and in uivo analysis of the specific T cell response to a protein antigen. Eur J Immunol 22:2639, 1992. 31. Yurovsky VV, Schulze DH, White B: Analysis of diversity of T cell antigen receptor genes using polymerase chain reaction and sequencing gel electrophoresis. J Immunol Methods 175:227, 1994. 32. Gorski J, Yassai M, Zhu X, Kissella B, Keever C, Flomenberg N: Circulating T cell repertoire complexity in normal individuals and bone marrow recipients analysed by CDR3 size spectratyping. Correlation with immune status. J Immunol 152:5109, 1994. 33. Cotrrez F, Auriault C, Capron A, Groux H: Analysis of the VP specificity of superantigen activation with a rapid and sensitive method using RT PCR and an automatic DNA analyser. J Immunol Methods 17285, 1974. 34. Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W (eds): Current Protocols in Immunology. New York, John Wiley & Sons, 1991, p. 5.3.1. 35. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156, 1987. 36. Maxam AM, Gilbert W: Sequencing with base-specific chemical cleavages. 65:499, 1980. 37. Sanger F, Nicklen chain-terminating 7415463, 1777.

end-labeled DNA Methods Enzymol

S, Coulson AR: DNA sequencing with inhibitors. Proc Nat1 Acad Sci USA

38. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press, 1989. 39. Moss PAH, Rosenberg WMC, Zintzaras E, Bell JI: Characterization of the human T cell receptor a-chain repertoire and demonstration of a genetic influence on Vo usage. Eur J Immunol 23:1153, 1993. 40. Ricken G, Pluschke G, Krawinkel U: T cell receptor a-chain germ line transcripts from activated lymphocytes. Immunol Lett 32:97, 1992. 41. Rock EP, Sibbald PR, Davis MM, Chien Y: CDR3 length

in antigen-specific 323, 1994.

immune

receptors.

et al.

J Exp Med

179:

42. Toyonaga B, Yoshikai Y, Vadasz V, Chin B, Mak TW: Organization and sequences of the diversity, joining, and constant region genes of the human T-cell receptor p chain. Proc Nat1 Acad Sci USA 82:8624, 1985. 43. Berman JS, Beer DJ, Theodore AC, Kornfeld H, Bernard0 J, Center DM: Lymphocyte recruitment to the lung. Am Rev Respir Dis 142:238, 1990. 44. Hunninghake GW, Fulmer JD, Young RC, Gadek JE, Crystal RG: Localization of the immune response in sarcoidosis. Am Rev Respir Dis 120:49, 1979. 45. Kradin RL, Divertie MB, Colvin RB, Ramirez J, Ryu J, Carpenter HA, Bhan AK: Usual intersitial pneumonitis is a T-cell alveolitis. Clin Immunol Immunopathol 40:224, 1986. 46. MacDonald HR, Lees RK, Schneider R, Zinkernagel RM, Hengartner H: Positive selection of CD4’ thymocytes controlled by MHC class II gene products. Nature 336: 471, 1988. 47. Choi Y, Kotzin B, Herron L, Callahan J, Marrack P, Kappler J: Interaction of Staphylococcus az/rez1Jtoxin “superantigens” with human T cells. Proc Nat1 Acad Sci USA 86:8941, 1989. 48. Tomonari Immunol

K: Positive selection 4:1195, 1992.

of VB2’CD8’

T cells. Int

49. White J, Herman A, Pullen AM, Kubo R, Kappler JM, Marrack P: The VP-specific superantigen staphylococcal entertoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell 56:27, 1989. 50. Webb S, Morris C, Sprent J: Extrathymic tolerance mature T cells: clonal elimination as a consequence immunity. Cell 63:1249, 1970.

of of

5 1. Kawabe Y, Ochi A: Programmed cell death and extrathymic reduction of VpS+ CD4’ T cells in mice tolerant to Staphylorocczs aelFezs entertoxin B. Nature 349:245, 1991. 52. Sherman DH, Hochman PS, Dick R, Tizard R, Ramachandran KL, Flavell RA, Huber BT: Molecular analysis of antigen recognition by insulin-specific T-cell hybridomas from B6 wild-type and bmI2 mutant mice. Mol Cell Biol 7:1865, 1787. 53. Acha-Orbea H, Steinman L, McDevitt HO: T cell receptors in murine autoimmune diseases. Ann Rev Immunol 7:371, 1989. 54. Danska JS, Livingstone JM, Paragas V, Ishihara T, Fathman CG: The presumptive CDR3 regions of both T cell receptor alpha and beta chains determine T cell specificity for myoglobin peptides. J Exp Med 172:27, 1990. 55. Tomai MA, Aelion JA, Dockter ME, Majumdar G, Spinella DG, Kotb M: T cell receptor V gene usage by human T cells stimulated with superantigen streptococcal M protein. J Exp Med 174:285, 1991. 56. DerSimonian H, Sugita M, Glass DN, Maier AL, Weinblatt ME, Reme T, Brenner MB: Clonal Vc~12.1’ T cell

TCR Repertoire

in Bronchoalveolar

Fluid

expansion in the peripheral blood of rheumatoid patients. J Exp Med 177:1623, 1993.

37

arthritis

57. Yurovsky VV, Sutton PA, Schulze DH, Wigley FM, Wise RA, Howard RAF, White B: Expansion of selected V61’ y/6 T cells in systemic sclerosis patients. J Immunol 153: 881, 1994. 58. Clarke GR, Humphrey CA, Lancaster FC, Boylston AW: The human T cell antigen receptor repertoire: skewed use of V beta gene families by CDS’ T cells. Clin Exp Immunol 96:364, 1994. 59. Okumura M, Fujii Y, Takeuchi Y, Inada K, Nakahara K, Matsuda H: Age-related accumulation of LFA- 1h’gh cells in a CD8’CD45RAh’sh T cell population. Eur J Immunol 23:1057, 1993. 60. Gabriel H, Schmitt B, Kindermann W: Age-related increase of CD45RO’ lymphocytes in physically active adults. Eur J Immunol 23:2704, 1993.

61. Hunt DF, Henderson RA, Shabanowitz J, Sakaguchi K, Michel H, Sevilir N, Cox AL, Appella E, Engelhard VH: Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry. Science 255: 1261, 1992. 62. McHeyzer-Williams MG, Davis MM: Antigen-specific development of primary and memory T cells in vivo. Science 268:106, 1995. 63. Palliard X, West SG, Lafferty JA, Clements JR, Kappler JW, Marrack P, Kotzin BL: Evidence for the effects of a superantigen in rheumatoid arthritis. Science 253:325, 1991. 64. Uematsu Y, Lanchbury J, repertoire in toid arthritis 8534, 1991.

Wege H, Straus A, Ott M, Bannwarth W, Panayi G, Steinmetz M: The T-cell-receptor the synovial fluid of a patient with rheumais polyclonal. Proc Nat1 Acad Sci USA 88: