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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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T cells play a pivotal role in initiating and orchestrating allergic responses in asthma. The goal of this work
was to learn whether ragweed challenge in the lungs alters the T-cell repertoire expressed in the blood and
lungs of atopic asthmatics. Analyses of cell numbers, differentials, and T-cell subsets in bronchoalveolar lavage (BAL) fluids showed that ragweed challenge was associated with preferential recruitment of CD4+
T cells into the lungs. A reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify
T-cell receptor (TCR) gene transcripts from unfractionated, CD4+, and CD8+ T cells in blood and BAL
fluids. As judged by RT-PCR, the usage of TCR V
and V
gene families in BAL fluids was similar to
that in blood. Ragweed challenge did not change the levels of expression of these V gene families. The
clonality of T cells was estimated by analyzing the diversity of TCR V-(D)-J junctional region nucleotide lengths associated with each V and V gene family, using sequencing gel electrophoresis. Most V gene
families in blood and BAL fluids were associated with multiple junctional region lengths before and after
ragweed challenge, indicating polyclonal expression. Some V gene families were expressed in an oligoclonal manner in unfractionated, CD4+, and CD8+ T cells in BAL fluids before ragweed challenge, as indicated by a few predominant junctional region lengths. The majority of these V gene families became
polyclonal after challenge, compatible with polyclonal T-cell influx during inflammation immediately after ragweed challenge. However, some V gene families became oligoclonal or developed a new oligoclonal pattern of junctional region lengths in BAL T cells after ragweed challenge. Surprisingly, this occurred in both CD4+ and CD8+ T cells. In one of these instances, DNA sequencing of V21 junctional regions in CD8+ T cells confirmed a change from polyclonal to oligoclonal expression after ragweed challenge. These findings show that ragweed challenge is associated with polyclonal influx and oligoclonal activation of both CD4+ and CD8+ T cells in the lungs.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Activated T cells contribute to the pathophysiology of
atopic asthma through the production of cytokines that
cause inflammation and promote IgE production (1, 2).
Allergen-specific activation of T cells occurs when T-cell
antigen receptors (TCR) bind peptide fragments of the allergen bound to major histocompatibility complex molecules and the T cell receives a co-stimulatory signal. Most TCRs consist of an chain encoded by rearranged variable (V), joining (J), and constant (C) region genes and a
chain encoded by V, diversity (D), J, and C genes (3, 4).
The V-(D)-J junctional region sequence determines antigen specificity of the TCR (5, 6). T-cell responses to allergens and other conventional protein antigens may be oligoclonal, as characterized by use of a limited set of V or
V gene families with conservation of junctional region
sequences (7).
Most previous studies of the T cells in the lungs of asthmatic patients have tested numbers of lymphocytes, percentages of CD4+ and CD8+ T cells, and markers of T-cell activation. T cells, especially CD4+ T cells, are increased in bronchial mucosal biopsies from asthmatics and express more CD25 (interleukin-2 receptor) and HLA-DR (11- 13). Numbers of T cells are similar in bronchoalveolar lavage (BAL) fluids from stable asthmatics and control subjects in many (14), but not all (17, 18), reports. After allergen challenge, numbers of T cells in BAL fluids may remain unchanged (19) or increase (22). In studies in which T-cell numbers increased, both CD4+ and CD8+ T cells have been reported to increase in BAL fluids after allergen challenge (22).
In contrast to many studies that test effects of allergen
exposure on numbers and types of lymphocytes in the
lungs, there are limited data that address the effect of allergen exposure on the T-cell repertoire expressed in the
lungs. One previous report used heteroduplex analysis of
TCR to test the effects of house dust mite, parietaria, and
cat allergens on the TCR diversity in the lungs (25).
Clonally expanded T cells were found in the lower respiratory tract of asthmatic patients after allergen inhalation. In
this study, we test the effect of ragweed challenge on levels of V or V gene expression and TCR clonality of blood
and BAL T cells in atopic asthmatics, including fractionated CD4+ and CD8+ populations.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Subjects
Six nonsmoking atopic asthmatics entered the study. Each
met American Thoracic Society criteria for the clinical diagnosis of asthma (26). None had an asthma exacerbation
for 2 mo before the study or status asthmaticus within the
past 2 yr. All subjects had a 3+ or 4+ positive skin test to
ragweed, as judged by area of intradermal induration 15 min after scratch test with short ragweed antigen in a glycerine base (Greer Laboratories Inc., Lenoir, NC). All subjects had a fall of 20% or more in forced expiratory volume
in 1 s (FEV1) in response to a provocative methacholine dose less than 25 mg, confirming airway hyperresponsiveness (27). Short-acting 2 agonists were withheld for 8 h
and theophylline preparations for 48 h before the methacholine challenge. FEV1 was measured with a wet spirometer (Sensomatics Corp., Anaheim, CA) and was required
to be greater than 50% of predicted value before methacholine challenge. One normal nonatopic volunteer entered
the study as a control. Subject characteristics are given in
Table 1.
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All subjects gave their written informed consent. The protocol of the study was approved by the Institutional Review Board of the University of Maryland School of Medicine.
Ragweed Challenge
Asthma therapy was withheld from all patients before the
study. Inhaled corticosteroids were withheld for 4 wk, cromolyn sodium for 2 wk, and 2 agonists for 24 h. The protocol consisted of two BAL procedures. The FEV1 before
the first BAL was always greater than 70% of predicted
value. Fentanyl (Janssen Pharmaceutica, Titusville, NJ)
and midazolam-HCl (Hoffmann-LaRoche Inc., Nutley, NJ) were administered intravenously for light sedation
and amnesia. Topical anesthesia was given by inhalation of
5 ml of 4% lidocaine with a DeVilbiss nebulizer #646
(DeVilbiss, Somerset, PA). Additional local anesthesia
was provided directly into the airways as needed with 2%
lidocaine through the bronchoscope (Model BF10; Olympus, Lake Success, NY). The right middle lobe and lingula
of each asthmatic patient were each lavaged with 120 to
140 ml (six or seven 20-ml aliquots) of sterile saline prewarmed to 37°C. The lavage fluids were recovered by gentle suction. Purified ragweed antigen (Greer Laboratories
Inc.), 480 protein nitrogen units in 5 ml saline, was then instilled in the right upper lobe. The subject was observed after bronchoscopy until sedation resolved and pulmonary
function was stable. Subjects withheld 2 agonists unless a
greater than 20% decrease in peak flow measurements occurred. Twenty-four hours later, a second BAL was done
of the ragweed-challenged right upper lobe of each asthmatic patient, with 120 to 140 ml (six or seven 20-ml aliquots) of sterile saline prewarmed to 37°C. The subject was again observed after bronchoscopy until stable.
Eighteen months after the initial study, Patients 2 and 4 were challenged with saline in an identical manner to serve as placebo control subjects.
The normal donor was challenged with ragweed in a similar manner, except the right middle lobe, right upper lobe, and lingula were each lavaged with six 20-ml aliquots of prewarmed sterile saline before the challenge. Purified ragweed antigen, 480 protein nitrogen units in 5 ml saline per lobe, was then instilled into the right middle lobe and the lingula. Twenty-four hours later, the two ragweed-challenged lobes were each lavaged with six 20-ml aliquots of prewarmed sterile saline. It was necessary to lavage three lobes at baseline and two lobes after ragweed challenge to obtain enough cells for analyses. It was shown previously that pooling cells from different lobes does not bias the comparison of the TCR repertoires in the lung (28).
Thirty milliliters of blood was drawn before each bronchoscopy. Blood and BAL fluids were kept on ice until processed.
BAL Fluid Cell Count and Differential
The volume of BAL fluid was measured. BAL fluid was filtered through loose sterile gauze to remove mucus and then centrifuged at 450 × g, 4°C, for 10 min. The cell pellet was washed twice and resuspended in 10 ml of phosphate-buffered saline (PBS). A cell count was done with a hemocytometer. Cells per milliliter and total cells were determined. Cytocentrifuge slides were made from 5 × 105 BAL cells. Slides were stained with a modified Giemsa stain (Baxter Scientific Products, Glendale, CA). Cell differential was done on 400 cells.
Peripheral Blood Mononuclear Cells
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) and washed with PBS.
CD4+ and CD8+ Cell Separation
Positive selection of CD8+ and CD4+ cells from PBMC and BAL cells was done with sequential use of Dynabeads M-450 CD8 and CD4 (Dynal Inc., Great Neck, NY), according to the manufacturer's directions. Our previous flow cytometric analyses of cells isolated in an identical manner revealed that > 95% of T cells expressed the selected marker.
Flow Cytometric Analyses of Cell Surface Markers
The PBMC and BAL cells were resuspended in Hanks'
balanced salt solution containing 5% horse serum and
0.1% sodium azide. For two-color immunofluorescence
analyses, 3 × 105 to 5 × 105 cells were stained with the desired pair of monoclonal antibodies (mAbs), using standard techniques. Antibodies were directly coupled to fluorescein isothiocyanate (FITC) or phycoerythrin (PE). To determine percentages of CD3 cells that coexpressed
CD4, CD8, or 
/
TCR, cells were stained with anti-CD3
mAb (anti-Leu-4-PE) and anti-CD4 (anti-Leu-3a+3b-FITC), anti-CD8 (anti-Leu-2a-FITC), or anti-/ TCR
(TCR1-FITC) mAb. Percentages of CD3+, CD4+, and
CD8+ T cells that expressed CD25, HLA-DR, and the
memory marker CD29 were also determined. Cells were
stained with anti-Leu-4-FITC, anti-Leu-3a+3b-FITC, or
anti-Leu-2a-FITC mAb and anti-CD25-PE, anti-HLA-DR-PE, or anti-CD29-PE mAb. The mAb TCR1 was
from T Cell Sciences (Boston, MA). All other mAbs were
from Becton-Dickson (Mountain View, CA). Cellular fluorescence was measured with a FACScan flow cytometer
(Becton-Dickson), with data gathered in the list mode. Viable lymphocytes were selected for analysis based upon
forward and 90° side light scatter characteristics.
RT-PCR Amplification of TCR Junctional Region Transcripts
Reverse transcriptase-polymerase chain reaction (RT-PCR) of V and V TCR junctional region RNA was performed as previously described (29). Total cellular RNA
was isolated from unfractionated, CD4+, and CD8+ T cells
from PBMC and BAL by acid guanidinium thiocyanate-phenol-chloroform extraction, using RNA STAT-60TM
(Tel-test "B," Inc., Friendswood, TX). First strand cDNA
was synthesized in a 60-µl reaction mixture containing 2 µg
of total RNA, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.5 mM of each dNTP, 0.4 µM of C 3'
primer (5'-ATCATAAATTCGGGTAGGATCC-3') and
C 3' primer (5'-CATTCACCCACCAGCTCAGCT-3'),
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 then diluted with
water to a final volume of 250 µl. Five microliters of cDNA were amplified using one of a panel of 5' oligonucleotide
primers specific for TCR 22 V or 25 V gene families and
a 3' oligonucleotide primer specific for C or C, respectively (Clonetech Laboratories, Palo Alto, CA). The C region primers were end-labeled with 32P. The PCR was
done in the manufacturer's recommended buffer, using a
Cetus/Perkin 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. The V and V primers were designed to anneal to areas of maximum V region sequence diversity,
which were different for each V gene. The size range of
the different V-C amplification products was 290 to 430 bp. The size range of V-C amplification products was
180 to 370 bp.
To quantitate V gene expression, PCR amplification of
the C region of the alternate TCR strand was done in the
same reaction tube used to amplify the V region. A pair of
C-C control primers was used with V-C primer pairs,
and, conversely, a pair of C-C control primers was used
with V-C primer pairs. One primer in each pair was
end-labeled with 32P. The concentration of V region primers was 0.5 µM and that of the control primers was 0.1 µM.
Sizes of the internal standards were 604 bp and 146 bp for
C-C and C-C internal standards, respectively. The
RT-PCR products were separated by electrophoresis on a
1.8% agarose gel. 32P incorporation into V-C, V-C,
C-C, and C-C cDNA was measured using PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The integrated intensity of each V-C band was normalized to the
opposite C-C 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 V-C or 25 V-C
bands, after normalizing for C-C or C-C bands, respectively.
Analysis of TCR Junctional Region Lengths
Analysis of the distribution of nucleotide lengths of amplified TCR cDNAs was done as previously described (29).
The RT-PCR products were subjected to electrophoresis
in a 6% Long RangerTM (AT Biochem, Malvern, PA) sequencing gel. M13mp18 bacteriophage sequence was used
as the size marker. M13mp18 single-stranded DNA (United
States Biochemical, Cleveland, OH) was sequenced by the
standard dideoxy-mediated chain termination method, using a 32P-labeled M13 (
20) primer (5'-GTAAAACGACGGCCAGT-3') and SequenaseTM (United States Biochemical). The RT-PCR products and M13mp18 nucleotide sequence were detected by autoradiography using XAR-5
film (Eastman Kodak, Rochester, NY).
DNA Sequencing
RT-PCR-amplified V21-C cDNA was ligated into
pCRTMII vector (Invitrogen Corp., San Diego, CA) and
then used to transform INVF' competent cells, according
to the manufacturer's instructions. Plasmids containing the
cDNA inserts were isolated by conventional techniques
and sequenced by the dideoxy-mediated chain termination method, using the same C primer as for PCR amplification (Clontech Laboratories). DNA sequences were analyzed by electrophoresis in a 7% Long RangerTM gel.
Statistical Analyses
Statistical analyses of the effects of ragweed challenge on cell numbers and percentages, T-cell subsets, and V gene expression were done using a two-tailed paired t test, to compare data from the same donor before and after challenge. Because multiple comparisons of V gene expression were made, differences were not considered significant unless P < 0.01. A chi-square analysis was used to compare the frequencies of skewing of different patterns of TCR junctional length distribution in asthmatic patients after ragweed or saline challenge and in control donor.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cells in BAL Fluids
Cell counts and differentials were done on BAL fluids
from asthmatics before and after ragweed challenge (Table 2). Because different numbers of lobes were lavaged
before and after ragweed, the number of cells recovered
was corrected per 120 ml PBS infused. This was the volume of PBS usually used to lavage one lobe. Ragweed challenge caused a 3-fold increase in total cells recovered.
Total numbers of macrophages doubled after ragweed
challenge, but the percentage of macrophages decreased.
Total numbers of lymphocytes increased in concert with
the increase in total cells after ragweed challenge, leaving
percentage of lymphocytes unchanged. Ragweed challenge was associated with an increase in both numbers and percentages of neutrophils and eosinophils. However, the
increase in number of eosinophils was not statistically significant, because of wide individual variation. Compared
with the asthmatics, 10-fold fewer cells per 120 ml PBS
infused were recovered from the control donor before
and after ragweed challenge (695,000 cells before and
2,350,000 cells after). Ragweed challenge did not cause a change in percent macrophages (92% 
94%), lymphocytes (6% 6%), neutrophils (0.5% 0.5%), or eosinophils (0% 0%) in BAL fluid from the control donor.
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Flow cytometric analyses were done to measure T-cell subsets in blood and BAL fluids in asthmatic patients before and after ragweed challenge (Figure 1). First, comparisons were done to test whether T-cell subsets in BAL fluids were different from those in paired blood samples. More CD3+, CD4+, and CD8+ T cells in BAL fluids expressed HLA-DR, CD25, and CD29. This was true before and after ragweed challenge.
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0.05 or 
P 
P Next, comparisons were done to test whether ragweed challenge changed T-cell subsets in blood or BAL fluids. Ragweed challenge in the lungs caused no change in T-cell subsets in the blood. However, ragweed challenge was associated with a change in T-cell subsets in BAL fluids. The percentage of T cells expressing CD4 increased, with a reciprocal decrease in the percentage of T cells expressing CD8 (Figure 1A). Thus, the increase in total lymphocytes in BAL fluids after ragweed challenge (Table ) was associated with preferential homing of CD4+ T cells. Expression of HLA-DR, CD25, and CD29 was not increased on BAL T cells 24 h after ragweed challenge (Figures 1B through 1D). If anything, expression of HLA-DR and CD25 tended to decrease on BAL T cells after challenge, which may reflect influx of T cells from the periphery.
TCR V and V Gene Usage in Unfractionated
PBMC and BAL Cells
The levels of expression of 22 V and 25 V gene families
were measured in unfractionated PBMC and BAL cells
before and after ragweed challenge (Figure 2). Being
aware of limitations of quantitative PCR, we involved the
coamplification of a reference template in the same reaction tube. The number of PCR cycles was carefully monitored to ensure that amplified product was proportional to the amount of starting template (29). The quantity of the
amplified reference template was similar in all samples in
a given experiment, so relative quantity of TCR V gene
transcripts could be inferred.
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All V gene families were expressed in BAL fluids
(Figure 2B) at levels similar to those in blood drawn at the
same time (Figure 2A). Ragweed challenge had no significant effect on the level of expression of any V gene family in blood or BAL fluids (Figures 2A and 2B). The same
findings were true for V gene families (Figures 2C and
2D). The expression of each V gene family was similar in
blood and BAL fluids at baseline and did not change significantly after ragweed challenge.
Increased Diversity of TCR Junctional Region Lengths in Unfractionated BAL T Cells from Asthmatics after Ragweed Challenge
The diversity of T-cell repertoire was analyzed by testing
the distribution of nucleotide lengths of TCR junctional
regions associated with the different V and V gene families. Polyclonal expression of T cells bearing any particular V gene will be associated with multiple TCR junctional
region lengths. Clonal T-cell populations have identical
TCR junctional region lengths.
Most V and V gene families in each asthmatic patient were expressed in a polyclonal manner with multiple
junctional region lengths in both blood and BAL fluids,
with no difference before and after ragweed challenge. For
example, Figure 3 shows multiple junctional region lengths
of TCR expressing V16, V21, V12, and V22 gene
families in blood and BAL fluid from Patient 1, before and
after ragweed challenge. The multiple lengths were frequently normally distributed.
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In contrast to this common polyclonal pattern, some V
gene families were expressed with a skewed pattern of
TCR junctional region lengths or a few predominant
lengths in unfractionated T cells in baseline BAL fluids
(Figure 3, compare BAL lane a to blood lane a). When
one or two bands predominated and comprised > 50% of
PCR amplification products by densitometry, this pattern was designated oligoclonal for purposes of description. A
number of V and V gene families were skewed or oligoclonal in baseline BAL but became more polyclonal after
ragweed challenge, with a less restricted pattern of TCR
junctional region lengths. Examples of such changes include the V9 and V20 gene families in Patient 4, the
V17 and V20 gene families in Patient 5, and the V20 gene family in Patient 6 (Figure 3, compare BAL lanes a
and b). This change from a skewed or oligoclonal pattern
to a more polyclonal pattern of TCR junctional region
lengths is compatible with polyclonal T-cell influx during
the inflammatory response.
In the control, the V8, V9, and V12 gene families
were expressed with skewed TCR junctional region
lengths in baseline BAL cells. In contrast to the findings
with the asthmatics, these patterns changed little after ragweed challenge (Figure 3, compare BAL lanes a and b in
the control). This difference from asthmatics is compatible
with a milder inflammatory response to ragweed challenge in the control donor. As an additional control, no changes
in TCR junctional region length distribution were seen
when Patient 4 was challenged with saline rather than ragweed (Figure 4, compare BAL lanes a and b).
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Oligoclonal T-Cell Responses to Ragweed Challenge in Unfractionated BAL Cells from Asthmatics
Ragweed challenge was associated with a second change
in the T-cell repertoire in the BAL fluids of the asthmatic
patients. The TCR junctional region lengths associated
with some V gene families displayed a new skewed or oligoclonal pattern after ragweed challenge. The development of a new pattern of restricted junctional region
lengths occurred in BAL T cells in Patient 2 in the V21, V9, V10, and V17 gene families, in Patient 4 in the
V21 gene family, in Patient 5 in the V21 gene family,
and in Patient 6 in the V8 gene family (Figure 5, compare
BAL lanes a and b). The most striking example was the
V10 family, in which one predominant junctional region
length was seen 24 h after ragweed exposure (Figure 5).
These findings are compatible with ragweed-associated recruitment of T cells bearing these V genes. In the control
donor challenged with ragweed and in two asthmatic patients challenged with saline, no V or V gene family in
blood or BAL T cells developed a more restricted pattern
of TCR junctional region lengths after challenge. These
results show that the induction of restricted diversity of
the TCR was specific for the asthmatic phenotype and for
allergen challenge.
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Changes in the CD4+ and CD8+ T-Cell Repertoire in BAL Fluids in Asthmatics after Ragweed Challenge
In Subjects 2, 5, and 6 and in the control donor, CD4+ and
CD8+ T-cell subpopulations were isolated from blood and
BAL cells. The TCR junctional region lengths expressed
by these subpopulations were determined. Findings with
the CD4+ and CD8+ cells confirmed the previous results
with unfractionated cells. The most common finding was a
polyclonal pattern of TCR junctional region lengths in
both blood and BAL cells, both before and after ragweed
challenge (data not shown). Similarly, as seen with unfractionated cells, some V gene families were expressed with a
skewed or oligoclonal pattern of junctional region lengths
in baseline BAL cells. These patterns became less restricted after ragweed challenge, compatible with the polyclonal influx of both CD4+ and CD8+ T cells. In CD4+
T cells, this was seen in Patient 2 in the V5.1 and V21
gene families, in Patient 5 in the V21 and V22 gene families, and in Patient 6 in the V22 gene family (Figure 6,
panel A, compare lanes a and b). This was also seen in
CD8+ T cells, in Patient 2 in the V21 gene family, in Patient 5 in the V17 and V22 gene families, and in Patient
6 in the V17 gene family (Figure 6, panel B, compare
lanes a and b). In contrast to the findings with the asthmatics, ragweed challenge of the nonatopic control donor did
not cause any change in patterns of TCR junctional region
lengths in CD4+ and CD8+ T cells from blood or BAL
fluid (data not shown). Similarly, no changes in patterns
of TCR junctional region lengths in CD4+ or CD8+ BAL
T cells were seen when Patient 2 was challenged with saline as a placebo control 18 mo after the ragweed study
(Figure 6, compare lanes c and d). Of note, the changes in
baseline expression of the V21 gene family in CD8+ BAL
T cells over time (Figure 6, panel B, V21, compare lanes a and c) may reflect the T-cell exposure to different environmental antigens.
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A few V gene families were expressed with a more restricted or different pattern of TCR junctional region
lengths following ragweed challenge. This was demonstrated in the V15 gene family in CD4+ T cells from Patient 2 (Figure 7, panel A, compare lanes a and b). Surprisingly, this was also seen in CD8+ T cells, in Patient 2 in the
V17 and V20 gene families, in Patient 5 in the V20 and
V21 gene families, and in Patient 6 in the V8, V9, and
V20 gene families (Figure 7, panel B, compare lanes a
and b). Thus, the V20 gene family became expressed
with a more restricted or different pattern of TCR junctional region lengths in CD8+ T cells from these three donors. Such changes in patterns of TCR junctional region
lengths in CD4+ or CD8+ BAL T cells were not seen when
Patient 2 was challenged with saline (Figure 7, compare
lanes c and d) or when normal donor was challenged with
ragweed (data not shown).
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Sequence Analysis of V21-C Junctional
Regions in CD8+ BAL T Cells
To confirm that the presence of TCR rearrangement of
predominant length is consistent with the oligoclonal expansion of T cells expressing the given V segment, V21-C PCR amplification products from CD8+ BAL T cells
from Patient 5 before and after ragweed challenge (see
Figure 7, V21, lanes a and b) were subcloned into a bacterial vector and sequenced across the junctional region.
Nine distinct clones were isolated before the challenge,
each of them just once (Figure 8). The nucleotide lengths
of their CDR3 sequences varied from 33 to 45 bp, which
corresponded to the multiple bands of PCR amplification
products seen in Figure 7 (V21, lane a). Twenty-five independent clones were isolated after the challenge, 22 of
them identical, with their CDR3 length corresponding
to the major band seen in Figure 7 (V21, lane b). Thus,
the diversity of TCR V21-C junctional sequences confirmed the oligoclonal character of expansion of CD8+
BAL T cells after the ragweed challenge. Of note, 27 of total 34 junctional sequences used J2.1 segment.
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Introduction
Materials & Methods
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Discussion
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This study tested whether segmental allergen exposure in the lung altered the T-cell repertoire expressed in the blood or lung during a late-phase allergic response. BAL fluids were 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 (32). Compared to 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.
All TCR V and V gene families were expressed in
blood and BAL fluids of atopic asthmatics. Levels of V gene
expression in BAL fluids were similar to those of the same
V gene in blood. Expression of V1 through V9 gene
families was generally higher than other V families. This
finding is not unexpected. Increased usage of V1 through
V9 has been reported previously in blood (33) and BAL
fluids (28) of normal donors and in BAL fluids from patients with sarcoidosis (34, 35) and systemic sclerosis (36).
At baseline and following ragweed challenge, most V gene families were expressed in a polyclonal manner, as assessed
by multiple TCR junctional region lengths associated with
each V gene. These findings indicate that the TCR repertoire is diverse in blood and BAL fluids of atopic asthmatics, before and after ragweed challenge.
Despite this diversity, some V and V gene families
were expressed in baseline BAL fluids with restricted
TCR junctional region lengths. The set of V gene families
that were expressed with restricted junctional region lengths
was different in each subject, although the V20 gene family showed restricted junctional region lengths in baseline
BAL cells from three donors. This baseline skewing of
TCR junctional region lengths was not due to small numbers of T cells tested nor to limitations of RT-PCR technique. V gene families that were oligoclonal in some donors were expressed in other donors at the same level, in a
polyclonal manner. The reproducibility of patterns of TCR
junctional region lengths in different lobes and over time
has been shown previously in normal donors (28).
Oligoclonal V gene familes were found among unfractionated, CD4+, and CD8+ T cells in baseline BAL fluids from asthmatic patients. Oligoclonal CD4+ and CD8+ T cells have been previously found in lungs of normal humans (28). Therefore, the presence of oligoclonal CD4+ and CD8+ T cells in baseline BAL fluids may or may not be related to atopic asthma in these subjects. Following ragweed challenge, several changes occurred in the T-cell repertoire in BAL fluids. Some V gene families that were expressed with restricted junctional region lengths in baseline BAL fluids developed more diverse junctional region lengths after ragweed exposure. This was seen in unfractionated, CD4+, and CD8+ T cells from BAL fluids. This finding is compatible with an influx of polyclonal T cells. This interpretation is supported by the increase in total CD4+ and CD8+ lymphocytes within BAL fluids after ragweed exposure, although there is preferential migration of CD4+ cells. Our finding of polyclonal changes in the T-cell repertoire in BAL fluids 24 h after ragweed challenge may reflect mechanisms governing lymphocyte recruitment and retention, the timing of the lavage, or the dose of antigen used. It takes lymphocytes 12 to 15 h to travel through the lungs (37). Given timing of the lavage 24 h after ragweed exposure, it is likely that many of the sampled T cells have entered the lung because of its inflamed state but may not be retained. Of note, in an animal model of secondary immune response in the lung, numbers of antigen-specific B cells peak on Day 4 and are back to baseline by Day 10 (38). Not all T cells are retained equally well in the lungs (39, 40). The presence of relevant antigen may (41) or may not (40) be an important determinant of retention. Future studies are needed to test later times after ragweed challenge to determine whether, once the polyclonal influx has resolved, an oligoclonal subset of T cells is retained within the lungs. Serial studies in individual patients are more difficult than analyses at a single time point as they require allergen challenge of several lobes at the same time and multiple subsequent lavages of the same patient. This design may provoke a more significant decline in pulmonary function than allergen challenge of a single lobe.
The observation that the T-cell repertoire becomes more polyclonal after segmental ragweed challenge may also be related to the high local concentration of antigen that occurs with segmental allergen challenge. This induces a pronounced inflammatory response that appears to cause nonspecific recruitment of lymphocytes. Exposure to smaller doses of antigen may allow for preferential activation and proliferation of ragweed-specific T cells within the lungs, without inducing the nonspecific inflammatory infiltrate. In dogs primed in the lungs with sheep red blood cells, rechallenge with small antigen doses leads to no inflammation or increase in vascular permeability but is associated with proliferation of antigen-specific B lymphocytes (42).
In addition to a polyclonal influx of T cells, ragweed
challenge was associated with a second change in the T-cell
repertoire. This change was the emergence of a new skewed
or oligoclonal pattern of TCR junctional region lengths for
some V gene families. This pattern was observed in unfractionated, CD4+, and CD8+ T cells. The finding that CD8+
T cells became oligoclonal after ragweed challenge was
unexpected, especially since this included the V20 gene
family in three donors. Given the typical processing pathway for soluble exogenous antigens (43), it is expected that
ragweed peptides would be recognized by CD4+, not CD8+,
T cells. The specificity and function of oligoclonal CD8+
T cells that arise in response to allergen challenge is unknown at this time. We speculate that they may provide a
beneficial downregulatory effect on the inflammatory response induced by CD4+ allergen-specific T cells. Indeed,
some reports suggest a beneficial effect of developing a
CD8+ T-cell response as part of atopy. CD8+ T cells are
increased in BAL fluids from patients who are protected from developing a late-phase asthmatic response to allergen challenge (23). Successful immunotherapy is associated with the development of suppressive CD8+ T cells
(44).
In summary, we have observed changes in the unfractionated, CD4+, and CD8+ T-cell repertoire in BAL fluids from atopic asthmatics 24 h after segmental allergen challenge. These changes suggest both a polyclonal influx of T cells and ragweed-associated activation of T cells within the lungs. Future studies are needed to correlate changes in the T-cell repertoire in BAL fluids with those in mucosal biopsies.
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Footnotes |
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Address correspondence to: Dr. Vladimir V. Yurovsky, Department of Medicine, University of Maryland, MSTF Room 8-23, 10 S. Pine St., Baltimore, MD 21201. E-mail: vyurovsk{at}umabnet.ab.umd.edu.
(Received in original form February 20, 1997 and in revised form July 16, 1997).
Acknowledgments: This work was supported by a Veterans Administration Career Investigator Award (to B.W.).
Abbreviations BAL, bronchoalveolar lavage; C, constant; CDR, complementarity-determining region; D, diversity; FEV1, forced expiratory volume in 1 s; FITC, fluorescein isothiocyanate; J, joining; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PE, phycoerythrin; RT-PCR, reverse transcriptase-polymerase chain reaction; TCR, T-cell antigen receptor(s); V, variable.
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References |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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