 / T-Cell Lines Derived from Airway Biopsies
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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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/
T cells have been postulated to play an important role in
the immune response at epithelial boundaries, but have not
been well described in human lung tissue. We have identified
and characterized / T-cell lines from human airway biopsies
and compared them with T-cell lines from paired peripheral
blood samples. Airway-derived T-cell lines stimulated with tetanus toxoid (TT) contained a greater proportion of / T cells
compared with T-cell lines stimulated with mitogens, other
antigens, or without antigen. TT-stimulated airway T cells expressed different T-cell receptors (TCRs) than did blood-
derived T cells, and used predominately variable region (V)I
family genes rather than VII family genes. Airway-derived /
T cells produced high levels of interferon- and were associated with T helper 1-like cytokine profiles. This study describes the presence and antigen-dependent proliferation of
/ T cells from human airway tissue, and demonstrates differences in lung-derived / TCRs compared with / T cells derived from peripheral blood. The data suggest that / T cells
may be functionally enriched in human airways relative to peripheral blood.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The role of / T cells in human health and disease remains unclear. Emerging data suggest a potential immmunoregulatory role for / T cells in diseases such as allergy
and autoimmunity (1), as well as important roles in resistance to bacterial, viral, and parasitic diseases (8, 9). Much
of our understanding of human / T cells has been deduced from studies in mice, where / T cells comprise a
substantial proportion (~ 30%) of T cells found in the mucosal layers of the lung as well as in the skin and gut (10).
In mice, restrictions in the T-cell receptor (TCR) repertoire of / T cells have been well documented and distinct differences in TCR gene usage are related to anatomical
location (i.e., lung, skin, blood) and developmental stage
(9).
Information on / T cells in humans is much more limited. Using conventional immunocytochemistry techniques
/ T cells are undetectable or represent < 1% of T cells in
both fetal and normal adult lung tissue (12). / T cells
have been identified in adenocarcinomas in a subset of lung
cancer patients (15, 16). Limited studies that have attempted to characterize T-cell lines derived from human airway biopsies have identified primarily CD4+ or CD8+ T cells,
presumably of the 
/
type (15, 17). However, / T
cells are readily detectable in human bronchoalveolar lavage (BAL) fluid (BALF), and are significantly increased
in several lung diseases, including sarcoidosis, hypersensitivity pneumonitis, and asthma (6, 20, 21). / T cells in
BALF (normally ~ 2% of CD3+ cells) may reach as high
as 30% in some patients (20, 21). These BALF / T cells
express a restricted range of antigen receptors, suggesting
that they might be directed against an immunodominant epitope encountered locally or represent a developmentally distinct subpopulation (22, 23). The role of / T cells
in the pathogenesis of these human lung diseases, as well
as in normal lung mucosal immunity, remains unclear.
One clinically important antigen to which human / T
cells have previously been shown to respond is tetanus toxoid (TT), a crucial target for protective immunity to the
toxic effects of Clostridium tetani infection. Major histocompatibility complex (MHC) class II restricted human /
T cells that recognize TT have been cloned and TT has
been shown to induce proliferation of human / T cells
from peripheral blood in vitro (24, 25). TT vaccination has
also been shown to increase circulating levels of / T cells
in vivo in certain patients suffering from immunoglobulin (Ig) A nephropathy, and / T cells have been linked to
TT-induced IgA responses in mice (26, 27).
The present report describes the identification and initial characterization of functionally responsive / T cells
from human airway endobronchial biopsies and paired peripheral blood samples from four human subjects. / T
cells proliferated in response to TT in the presence of autologous antigen-presenting cells (APCs) and interleukin
(IL)-2, and were found in higher percentages in T-cell lines
derived from airway biopsies compared with paired peripheral blood. This is the first report we are aware of describing the outgrowth of / T cells from human airway
biopsies. The limited previous reports of human airway-
derived T-cell lines (15, 17) have described only / T
cells but have not probed directly for / T cells. In the present study human airway and blood / T cells were identified in TT-driven T-cell lines, and were characterized by flow
cytometry, reverse transcriptase/polymerase chain reaction (RT-PCR), and TCR sequence analysis. Comparative analysis of the / T cells from these distinct anatomical locations and the potential functional role of human / T cells
from the lung are discussed.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Patients and Tissue Samples
Four human subjects participated in these studies: two atopic (subjects 1 and 4) and two nonatopic adult males (subjects 2 and 3). The study was approved by the Human Investigations Committee at Yale University, and written informed consent was obtained from each subject. Subject 1, age 26, was skin test-positive for dust, cat and horse allergens. Subjects 2 and 3 were 44- and 40-yr-old nonatopic individuals, respectively. Subject 4, age 27, was skin test-positive for cats, dust, pollen, and mold. Airway biopsy samples were obtained during bronchoscopy as previously described (28) and peripheral blood samples were obtained immediately before bronchoscopy.
Generation of T-Cell Lines
T-cell lines from airway biopsies and peripheral blood were established under the conditions described by Upham and colleagues (19). Briefly, five to eight airway biopsies were washed with CO2-independent media (GIBCO BRL, Grand Island, NY), pooled, and stimulated overnight in six-well plates with 10 ng/ml recombinant IL-2 (R&D Systems, Minneapolis, MN) to extract T cells. The next day the extracted cells were replated in 48-well plates with 5 × 105 autologous mitomycin C (Sigma, St. Louis, MO)- treated peripheral blood mononuclear cells (PBMCs) as APCs and antigen or mitogen. TT (Massachusetts Biological Laboratories, Jamaica Plain, MA) and recombinant dust mite antigen (DerP2, a generous gift of Drs. Mark Chapman [University of Virginia School of Medicine] and Philip Askenase [Yale School of Medicine]) were used at 50 and 10 µg/ml, respectively. Phytohemagglutinin A and conconavalin A (Sigma) were used at 1 and 5 µg/ml, respectively. On Day 3 of culture, 2 ng/ml of IL-2 was added; and after 1 wk, fresh antigen or mitogen, APCs, and IL-2, were added. Cultures were then propagated with fresh autologous APCs, IL-2, and antigen or mitogen weekly. Cultures were subject to Ficoll-Hypaque density gradient centrifugation every other week to remove dead APCs. T-cell lines from paired peripheral blood samples were generated under identical conditions starting with 3 to 5 × 106 PBMCs. Autologous patient serum (10%) was used as a media supplement throughout. The cell lines were analyzed by flow cytometry starting at Week 3, the earliest time at which enough cells could be harvested for flow cytometry analysis. All flow cytometry and cytokine studies were conducted between Weeks 3 and 5 of culture, during which time the cell-line phenotypes were stable.
Phenotypic Characterization of T-Cell Lines
T-cell lines were analyzed by three-color flow cytometry analysis using combinations of the following monoclonal antibodies (mAbs)
from Becton Dickinson (San Jose, CA): fluorescein isothiocyanate (FITC)-conjugated anti-CD3; FITC-conjugated anti-/; phycoerythrin (PE)-conjugated anti-CD4; PEcy5-conjugated anti-CD8;
and PEcy5-conjugated anti-CD3. The following reagents were also
used from PharMingen (San Diego, CA): PE-conjugated anti-V9;
PE-conjugated anti-/; and FITC-conjugated anti-2. Data were
acquired on a FACscan and analyzed using Cell Quest software
(Becton Dickinson).
Cytokine Analysis
Culture supernatants of T-cell lines were analyzed for cytokine
levels after 3 wk of stimulation with TT, APCs, and IL-2. Cytokine levels were quantitated in triplicate cultures 48 h after restimulation with antigen, APC, and IL-2, using Opti EIA kits (PharMingen) according to the manufacturer's protocol. Briefly, culture supernatants from paired T-cell lines were incubated for 2 h at room temperature in duplicate in enzyme-linked immunosorbent assay plates coated with anticytokine mAbs. Captured
cytokines were detected with horseradish peroxidase-labeled
secondary antibodies and concentrations were based on a standard curve generated using known concentrations of recombinant cytokine. Paired Student's t tests were performed to determine statistical significance of the differences between cytokine
production by different T-cell lines. Intracellular cytokine staining was performed by three-color flow cytometry using the CytoStain kit from Pharmingen. Briefly, 2 µM monensin was added
to cell cultures for 8 h before analysis. Cells were stained for surface markers CD3 and / TCR, fixed with 1% paraformaldehyde, and permeabilized before staining with PE-conjugated
anti-interferon (IFN)- (PharMingen).
Molecular Analysis of TCR Genes
RNA was extracted from Ficoll-Hypaque-purified T-cell lines using RNAeasy columns from Qiagen (Valencia, CA). The complementary DNA (cDNA) was prepared using a Superscript kit from GIBCO BRL. PCR was performed by adding 2 µl of cDNA to Platinum PCR supermix (GIBCO BRL), along with 20 pmol of each of the appropriate primers. PCR reactions were denatured for 5 min at 94°C; 30 cycles of 94°C, 55°C, and 72°C for 30 s each; and 5 min final extension at 72°C, using a Perkin-Elmer DNA Thermocycler (Perkin-Elmer, Gaithersburg, MD). The following primers were used on the basis of sequence data from the literature (22) (name, direction, and sequence 5' to 3'):
Variable region (V)I family, forward, GTC TAC ATC CAC
TGG TAC CT.
VII family, forward, GCA ACA TCT GTA TAT TGG TA.
VIII family, forward, CAC TGG TAC (C/T)GG CAG
AAA CCA.
TCR constant, reverse, GAA GGA AGA AAA ATA GTG
GGC TTG GGG GA.
PCR products were resolved on 2% agarose gels and the expected ~ 330-base pair (bp) bands were subsequently cloned from 1.2% low melting-point gels into the pTOPO-TA vector from Invitrogen (Carlsbad, CA). A total of 26 individual RT-PCR clones were sequenced, 20 from an airway biopsy-derived T-cell line and six from the paired blood-derived T-cell line, half each from two separate PCR reactions. Sequence analysis was performed by the Yale University DNA Sequencing Center (New Haven, CT).
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Generation and Characterization of Human T-Cell Lines from Airway Biopsies and Paired Peripheral Blood Samples
Human T-cell lines from airway biopsies and blood were
generated by stimulation with antigen or mitogen in the
presence of IL-2, autologous APC, and serum as described
in MATERIALS AND METHODS. The T-cell lines were initially
characterized by three-color flow cytometry for expression
of CD3, CD4, and CD8. In cultures stimulated with TT,
high percentages of CD4
CD8 T cells were consistently
noted in airway biopsy-derived T-cell lines from all four
patients. The increase in CD4CD8 T cells was paralleled by a relative decrease primarily in CD4+ T cells (Figure 1). In comparison, airway biopsy-derived T-cell lines
stimulated with mitogens or the allergen DerP2, or without antigen, did not contain high percentages of CD4
CD8 T cells. T-cell lines derived from paired peripheral
blood samples under identical antigenic stimuli also did not
contain the high percentages of CD4CD8 T cells found
in the airway biopsy-derived T-cell lines. The CD4CD8
T cells observed in the TT-driven airway biopsy-derived
T-cell lines were determined to be / T cells on the basis of
direct flow cytometry staining for the / receptor (Figure
2) and back gating. In one T-cell line, two populations of /
T cells, with different densities of / TCR and CD3 expression, were noted (Figures 2 and 3A). Similar dual populations of / T cells have been noted in murine studies (29),
but their significance remains unclear. The / T cells present
in TT-stimulated cultures were overwhelmingly CD4CD8,
except for one subject that did express a range of CD8+ /
T cells, although never to the high level expressed by CD8+
/ T cells in the same cultures (Figure 3B).
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Analysis of TCR Expression by Human / T Cells from
Airway and Blood
Significant differences in TCR expression by the TT-driven
T cells derived from airway biopsies and peripheral blood
were observed using a combination of analytical methods.
By flow cytometry, we documented that the / T cells in
TT-driven cultures from peripheral blood predominately
expressed the V92 TCR rearrangement most commonly
found in peripheral blood (8) (Figure 4). In contrast, airway-derived, TT-induced T cells were predominately negative for V92 expression by flow cytometry (Figure 4),
and expressed VI family genes as detected by RT-PCR
(Figure 5).
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Sequence analysis of the TCR VI family genes (Table
1) from the T-cell lines demonstrated preferential usage of
V8JP2 rearrangement by the airway-derived T cells. Restricted clonality, with 87% (seven of eight) of the functional
RT-PCR clones sequenced displaying identical CDR3s, was
also documented. In contrast, the few VI family expressing T cells from the blood predominately used V8J2 rearrangements and displayed clonality distinct from that expressed by the airway T cells, but similarly restricted. All
functionally rearranged VI family genes sequenced exhibited substantial N-terminal additions and deletions. This
finding, along with the restricted CDR3 clonality may suggest antigen-mediated or developmentally regulated selection (30).
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In the airway biopsy-derived, TT-driven T-cell line from
subject 4, significant expression of a clonal V2J2 rearrangement, with "in frame" J- and C-regions but a stop codon in
the CDR3, was also noted (39%, or seven of 18, of the total RT-PCR clones sequenced). It remains uncertain whether
the V2J2 rearrangement represents the allelic partner of
the functional V8JP2 rearrangement, given their similar
cloning frequencies. Alternatively, the nonfunctional V2J2
rearrangement could be present in the / T cells of the culture, as might be predicted by a sequential or concurrent TCR rearrangement model of T-cell development
(30, 31). Nonetheless, taken together, the data on TCR expression by paired samples of human airway and blood-derived T-cell lines demonstrate significant differences in
the TCR genes of T cells derived from these different anatomic locations under the same antigenic stimulation.
Analysis of Cytokine Secretion by Human / T Cells
To determine whether human airway / T cells manifest
prototypical T helper (Th)1-like or Th2-like phenotypes,
we analyzed cytokine production by an airway-derived,
TT-driven T-cell line that contained predominately (98%)
/ T cells. A marked Th1-like cytokine profile was measured in the culture supernatant of this cell line, which differed from the mixed Th1/Th2 profile observed for the
paired blood-derived TT-stimulated T-cell line from the same subject, that contained only 23% / T cells and 75%
/ T cells (Figure 6A). Culture supernatant from the airway-derived T-cell line (predominately /+) contained significantly higher levels of IFN- and tumor necrosis factor
(TNF)- and lower levels of IL-4, IL-5, and IL-13 (P < 0.001). Analysis of the TT-stimulated T-cell lines, by intracellular cytokine staining, further verified that these human airway / T cells secrete significant amounts of IFN-
(Figure 6B). Th1 skewed cytokine secretion profiles were
also observed in other airway biopsy-derived T-cell lines
that contained elevated percentages of / T cells (data not
shown).
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The present study generated and characterized human T-cell
lines from human airway biopsies and peripheral blood. We
documented the presence of functionally responsive / T
cells in T-cell lines from human airway biopsies, and demonstrated that these / T cells were preferentially expanded
in response to the human vaccine antigen TT in vitro. TT-driven T-cell lines from human airway biopsies contained
significantly elevated percentages of / T cells compared
with TT-driven T-cell lines from paired blood samples, as
well as airway and blood T-cell lines driven with other
stimuli. Further, airway biopsy-derived / T cells differed from paired blood sample-derived / T cells in their TCR
gene usage. Airway / T cells produced significant amounts
of IFN- and were associated with Th1-like cytokine profiles. The data suggest that / T cells may be functionally
enriched in human airways relative to peripheral blood,
and support the hypothesis that human lung / T cells may
be important contributors to normal immunity and diseases
of the airways in humans.
The present study demonstrates the feasibility of generating T-cell lines from human endobronchial biopsies, a
potentially powerful technique for investigating local airway mucosal immunity in humans, where larger surgical or
autopsy samples are rarely available. Previous studies characterizing human airway mucosal T cells have been limited, partly due to the difficulties in obtaining human samples and to technical difficulties in cloning T cells from
endobronchial biopsies that are typically < 1 mm3 in size.
The few prior studies that have generated and characterized T-cell lines derived from human airway tissue biopsies have identified CD4+ and/or CD8+ T cells, presumably expressing / TCR, and have not described / T cells.
The majority of these investigations used nonspecific stimuli, such as IL-2 alone and/or mitogens, to expand the T-cell lines studied, and / TCR usage was not directly assessed
(15, 17). The recall response of airway mucosal T cells to
human vaccine antigens, such as TT, has not been reported.
Human airway biopsy-derived / T cells induced by TT
in this study were characterized by analysis of their TCR expression and shown to differ from blood-derived / T cells
generated under identical conditions. / T cells from airway biopsies expressed predominately VI family genes
whereas blood-derived / T cells expressed predominately
VII family genes. Further differences in clonality and J-region usage of the airway- and blood-derived / T cells were
also documented, and are consistent with previous studies
describing discrete differences in TCR usage by / T cells
from different tissues (8, 9). In mice, it has been well documented that / T cells from the lung, peripheral blood, skin, and gut use different and distinct and TCR genes.
In humans, tissue-restricted usage of / TCR genes is less
defined. V9 and 2 are predominately used in peripheral
blood and lymphoid organs (8, 9) whereas V1 family genes
are predominately expressed in other tissues, such as intestinal epithelium, nasal mucosa, and synovial fluid (33).
Human V gene usage has not been as well studied but one
report has described a relative increase in V8 gene usage
among / T cells in the epithelium of the gut and inflamed
synovial tissue (36). VI family genes have also been shown
to be expressed by human BAL-derived / T-cells, however / TCR usage in human airway tissue-derived T cells
has not previously been described. This study documents
preferential VI family usage in antigen-driven / T cells
from human airway mucosa.
Airway biopsy-derived / T cells in the present study
were shown to produce significant amounts of IFN- and
were associated with Th1-like cytokine secretion profiles.
An airway-derived T-cell line that was > 98% / T cells
secreted high levels of IFN- and low levels of IL-4, IL-5,
and IL-13 compared with a paired blood-derived T-cell line
that was > 75% / T cells. Further proof that human airway / T cells produce significant levels of IFN- was obtained through intracellular cytokine staining. The Th1-like
profile of human airway / T cells observed in the present
study is consistent with previously reported studies of human / T cells derived from other tissues, i.e., blood, intestine, synovial fluid, and gingiva (37). Such a Th1-like
profile suggests that human airway / T cells could play a
protective role in bacterial and allergic airway diseases or
might directly participate in granulomatous lung diseases
such as sarcoidosis. The dependence of human airway /
T cell Th1 cytokine secretion on specific antigenic stimuli
remains unknown but warrants further investigation.
Although the outgrowth of / T cells from human airway
biopsy cultures stimulated with TT verifies their presence
in these tissues, it does not prove that these cell types respond directly to TT antigen. Blood-derived / T cells have
previously been shown to recognize TT in an MHC II-
restricted manner. However, TT may also stimulate / T
cell proliferation indirectly, possibly via factors produced
by / T cells and/or APCs (25). Sample size limitations of
the present study did not permit studies on the antigen specificity of the T cells expanded in vitro from human airways.
However, control cultures stimulated with mitogens or without antigen did not induce significant increases in / T cells,
suggesting an antigen dependence for / T-cell growth. In
the present studies, increased percentages of / T cells in airway biopsy-derived T-cell lines followed several weeks
of culture with continuous stimulation. The induction of /
T-cell proliferation, therefore, might be secondary to direct
antigenic stimulation of another cell population capable of
stimulating / T cells. Finally, all of the subjects in this study
had been previously immunized and boosted with TT, raising the possibility that / T cells might participate in the
immune memory response to recall antigens.
A major limitation of the present study was the small
quantities of human airway tissue available, which precluded direct characterization of the starting cell populations in these specimens. Thus, we could not determine
whether / cells had increased precursor frequencies in
airway biopsies compared with peripheral blood, or whether
other factors in the T-cell lines favored their expansion. Another limitation is the relatively small number of subjects studied, limiting any analysis related to the presence
of atopy or disease status. Further studies are needed to
better characterize the / T cells present in human airway
tissue from "normal" subjects as well as individuals with
lung diseases such as asthma.
In summary, we have documented the presence of / T
cells in human airway biopsies and their proliferation following TT stimulation. TT-stimulated T cells from airway
tissue expressed different / TCRs than did TT-stimulated T cells derived from paired blood samples, suggesting a difference in epitope specificity or the development
of different / T cells from these two different compartments. Airway biopsy-derived / T cells were also associated with Th1-like cytokine production. The data suggest
that / T cells may be functionally enriched in human airways relative to peripheral blood, and support the hypothesis that / T cells may play an important role in normal
human lung immunity and disease. Further studies in a larger
number of human subjects will help identify potential differences in airway / T cells in patients with airway diseases such as asthma compared with healthy individuals.
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Footnotes |
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Address correspondence to: Adam V. Wisnewski, Ph.D., Yale School of Medicine, 333 Cedar St., LCI-105, New Haven, CT 06520. E-mail: adam. wisnewski{at}yale.edu
(Received in original form August 16, 2000 and in revised form November 16, 2000).
Acknowledgments: The authors acknowledge Drs. Christina Herrick and Anuradha Ray for critical review of the manuscript, as well as Drs. Kim Bottomly and Brian Smith for helpful discussions. The authors also thank Dr. Jack Elias and the members of the Yale School of Medicine Asthma SCOR Program for their support and constructive criticisms. This work was supported by the American Lung Association and National Institutes of Health (1P01HL56389, 1R01HL62622, K08HL03129 and NIH/NCRR/GCRL, Program Grant RR00125).
Abbreviations antigen-presenting cell, APC; fluorescein isothiocyanate, FITC; interferon, IFN; interleukin, IL; monoclonal antibody, mAb; phycoerythrin, PE; reverse transcriptase/polymerase chain reaction, RT-PCR; T-cell receptor, TCR; T helper, Th; tetanus toxoid, TT; variable region, V.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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