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L Is a Partial Agonist for Outer Surface Protein A-Reactive T Cells1
*
Laboratory of Molecular Immunology, Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115;
Department of Pathology, and
Division of Rheumatology/Immunology, Tufts University School of Medicine, New England Medical Center, Boston, MA 02111
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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L at the T cell level and the inability to detect
Bb in the joint implicate an autoimmune mechanism. To
analyze the nature of response to OspA and LFA-1L, we
used OspA-specific T cell hybrids from DR4 transgenic mice, as well as
cloned human cells specific for OspA165–184, the
immunodominant epitope, from five DRB1*0401+ patients,
using OspA-MHC class II tetramers. Although OspA165–184
stimulated nearly all OspA-specific human T cell clones tested to
proliferate and secrete IFN-
and IL-13,
LFA-1L326–345 stimulated 
10% of these clones to
proliferate and a greater percentage to secrete IL-13. Assays with LFA-
or OspA-DR4 monomers revealed that higher concentrations of LFA-DR4
were needed to stimulate dual-reactive T cell hybrids. Our analysis at
the clonal level demonstrates that human LFA-1L326–345
behaves as a partial agonist, perhaps playing a role in perpetuating
symptoms of arthritis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have analyzed the Ag-specific CD4+ T cells
in treatment-resistant Lyme arthritis, using class II tetramers, and
have shown that arthritis-associated Borrelia burgdorferi
(Bb) sensu stricto outer surface protein A (OspA)-specific T
cells can be identified with this technology. We were able to visualize
and directly isolate these cells from the SF and PBMC of patients
suffering from treatment-resistant Lyme arthritis (7).
Infection of humans with Bb sensu stricto through the bite
of an infected ixodes tick results in arthritis in 50–60% of
untreated patients (8, 9). In most cases of Lyme
arthritis, joint inflammation resolves with antibiotic therapy
(8). However, in 10% of patients, the arthritis
persists despite multiple courses of antibiotics (10).
These individuals often develop high titers of OspA-specific Abs during
the treatment-resistant phase of disease (11, 12). There
is an association with the presence of RA-associated alleles, including
HLA-DRB1*0401 (13). We have recently reported that SF T
cells from these individuals respond in bulk functional assays to
bacterial OspA (14). Following analysis of overlapping
peptides of OspA, an important HLA*0401-binding OspA peptide, aa
165–173, was identified. A GenBank search of that amino acid
sequence revealed a homologous peptide within the
L-chain of human LFA-1, aa 332–340
(hLFA-1L (14)).
LFA-1L is expressed on T cells, macrophages,
and many other cells and is up-regulated at the site of inflammation.
Thus, sequence homology between bacterial and self antigenic epitopes
may be the basis for the molecular mimicry between host and bacteria
and may play an important role in the etiology of treatment-resistant
Lyme arthritis (14, 15). In both a Chlamydia
species infection model (16) and HSV-type 1 model
(17), T cells specific for the microbial Ag induced
tissue-specific destruction in the animal host.
In this study, we quantitatively measured the nature of the
OspA165–184/LFA-1L326–345
dual reactivity at the clonal level, examining whether a single T cell
is able to recognize and respond to both the bacterial and the self Ag
presented in the context of HLA-DRB1*0401. About 10% of OspA-reactive
T cell hybridomas (THy) generated from OspA-immunized DRB1*0401
transgenic mice secrete IL-2 in response to
hLFA-1L326–345. From a panel of human
OspA-specific T cell clones, derived by single cell cloning of
DRB1*0401/OspA tetramer-binding cells from patients with chronic Lyme
arthritis, 50% mount a response to
hLFA-1L. However, the quality and quantity of
the response to the two Ags are significantly different, as measured by
cytokine production and proliferation, respectively. Although OspA is
the full agonist that induces high proliferation and cytokine
production, LFA-1L behaves as a partial
agonist, reflecting the lack of high affinity
LFA-1L-specific T cells due to
self-tolerance.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients met the Centers for Disease Control and Prevention criteria for the diagnosis of Lyme disease (18). They had arthritis affecting one or both knees and a positive Ab response to Bb by ELISA and Western blot, interpreted according to the Centers for Disease Control and Prevention/Association of State and Territorial Public Health Laboratory Directors’ criteria (19). One patient was selected for extensive study. He was determined to be HLA DR*0401 homozygous. HLA analysis was performed by Lee Ann Baxter-Lowe (20). Treatment consisted of at least 2 mo of oral doxycycline or at least 1 mo of i.v. ceftriaxone, or both. After antibiotic treatment, repeated PCR tests (21) for Bb DNA in joint fluid were negative.
Preparation of MHC II monomers and tetramers
Soluble MHC class II molecules were prepared using baculovirus,
as previously described (3, 5, 7, 22). Briefly, using a
two-promoter baculovirus transfer vector, the gene encoding the
extracellular region of the DRA1*0401 chain was cloned behind the p10
promoter, and the gene encoding the extracellular portion of the
DRB1*0401 chain was cloned behind the polyhedrin promoter. Sequence
encoding either the Bb OspA164–176
peptide, hLFA-1L332–343 (for plate-bound
assay), or the human cartilage glycoprotein,
gp-39263–275 peptide, and a 14-aa flexible
linker was inserted between the leader and the N terminus of the
DRB1*0401 domain. The C terminus of the DRB1*0401 chain carried a
peptide tag for biotinylation by BirA (Avidity, Denver, CO). DR4 was
purified from culture supernatant of infected insect cells by
immunoaffinity and size exclusion chromatography. The purified proteins
were biotinylated for use in plate-bound assays as monomers or
incorporated into multimeric complexes with PE-streptavidin (BioSource
International, Camarillo, CA), as previously described
(3).
Production of THy
Eight-week-old DR4 transgenic mice lacking mouse MHC class II
molecules (23) were immunized s.c. with 100 µg
OspA165–184 in CFA twice over the course of 3
mo. Lymph node cells were stimulated 48 h with 40 µg/ml
OspA165–184, washed extensively, mixed with
BW-TCR-/- cells (American Type Culture
Collection, Manassas, VA), and fused in 35% polyethylene glycol.
Washed fused cells were plated at 1 cell/well in
hypoxanthine/aminopterin/thymidine medium (Sigma, St. Louis, MO). Every
4 days, plates were fed with
hypoxanthine/aminopterin/thymidine-supplemented 10% FCS-RPMI
1640, until unfused BW-/- control cells
were dead. Subclones and lines were screened for Ag specificity by
incubating THy, irradiated syngeneic spleen cells plus
OspA165–184, control OspA peptide,
LFA-1L326–345, or plate-bound anti-CD3 or
medium alone for 48 h. Supernatants were harvested, and IL-2 was
measured by IL-2 ELISA.
Fluorescent cell sorting and T cell cloning
As previously reported, a frozen aliquot of patient cells was thawed and washed just before staining with tetramer (7). Staining reactions were set up at 300–400 µl final volume in complete RPMI 1640 containing 6–8 x 106 SF cells, 20 µg/ml OspA, or gp39 tetramer. Reactions were incubated at 37°C for 1.5–2.5 h. Anti-CD4 FITC and anti-CD64 CyChrome (BD PharMingen, San Diego, CA) were added to the reaction mixture, and the cells were incubated for an additional 30 min at room temperature. The cells were washed twice and resuspended in 5% human serum-RPMI 1640 plus additives. To exclude monocytes from analysis, only CD64- cells (monocyte-macrophage negative (24)) were included in the sort. OspA tetramer+ CD4+CD64- cells were single-cell sorted into 96-well round-bottom plates containing 100 µl 5% human serum-supplemented RPMI 1640 complete medium, 150,000 irradiated human PBMC (5000 rad), plus 2 µg/ml PHA, and incubated at 37°C. As control, OspA tetramer-negative, CD4+CD64- were also sorted. At 48 h, human rIL-2 (Teceleukin; National Cancer Institute, Frederick, MD) in complete medium was added for a final concentration of 20 U/ml in each well. Clones were fed or expanded every 3 days with 100 µl of rIL-2-containing medium. After 14 days, 168 OspA tetramer-positive and 8 OspA tetramer-negative clones were established; the cloning efficiency was 33%. A total of 2 x 104 resting cells from each clone was restimulated with 2 µg/ml PHA and 150,000 irradiated PBMC. At 48 h, rIL-2 was added to wells at a final concentration of 20 U/ml. After an additional 2 wk of feeding, the clones could be used for functional assays.
Plate-bound MHC monomer assay
ELISA plates were coated overnight with 50 µl of a 100 µg/ml solution of extrAvidin (Sigma) in 50 mM Tris, 100 mM NaCl, pH 7.4. After blocking with 2% FBS/HBSS for 2 h, followed by four washes with the same solution, biotinylated soluble monomeric class II molecules (OspA-DR4, LFA-DR4, and gp39-DR4) were added in decreasing concentrations, beginning with 20 µg/ml, and plates were incubated for 2 h. Following four washes, 1 x 105 THy cells were added to wells and incubated overnight at 36°C in 5% CO2. Serial 2-fold dilutions of duplicate culture supernatants were analyzed for IL-2 secretion in the murine IL-2 ELISA, as described below.
Proliferation assays
The human T cell clones were tested for their Ag specificity in
a split well assay. Autologous EBV-transformed B cells (lymphoblastoid
cell lines) were treated with mitomycin C in <0.5% human serum for
1 h at 37°C for use as APC. Following three washes, 2 x
104 APC were added to each well of round-bottom
96-well plates. Ag was added to one-half of the wells to a final
concentration of 10 µg/ml OspA165–184 or
LFA-1L326–345. A total of 2 x
104 cells of each T cell clone was added to all
wells. At 48 h, one-half of the medium was removed for later
cytokine analysis, and plates were pulsed with 0.5 µCi
[3H]thymidine for 18 h. Plates were
harvested and counted on a beta-plate scintillation counter (EG&G
Wallac, Gaithersburg, MD). Data are expressed as 
-cpm, which is
equal to the average counts obtained after culture with Ag minus
average counts with medium alone.
Cytokine ELISA
Wells were coated with purified Abs to human IL-4, IL-5, IL-13
(BD PharMingen), or IFN- (Endogen, Woburn, MA) in 0.1 M
NaHC03 buffer overnight. Plates were blocked with
1% BSA-PBS in 200 µl for 2 h at room temperature. Serial 2-fold
dilutions of human recombinant standards were made in 5% human RPMI
1640 complete medium starting at 2000 pg/ml (rIL-4 and rIL-5,
Boehringer Mannheim, Indianapolis, IN; rIL-13, R&D Systems,
Minneapolis, MN; IFN-, Life Technologies, Gaithersburg, MD). After
washing plates, 50-µl samples (diluted 1/1 in medium) and 50 µl
appropriate standards were added. Following a 2-h incubation, plates
were washed before adding biotinylated secondary Abs (b-IL-4,
b-IL-5, b-IL-13; BD PharMingen), diluted 1/1000 in 1% BSA-PBS.
Plates were incubated for 1 h at room temperature.
Avidin-peroxidase conjugate (Sigma) at 1/5,000 to 1/10,000 dilution was
added to all wells for 45 min. In the IFN assay, the anti-IFN-
secondary Ab (Endogen) was not biotinylated. Following a 1-h incubation
with 1/1000 dilution of anti-IFN- and washes, a 1/10,000
dilution of peroxidase goat anti-rabbit IgG (BioSource) was added
for 1 h. All plates were washed extensively, and
3,3',5,5'-tetrametylbenxidine (Kirkegaard & Perry Laboratories,
Gaithersburg, MD) substrate was added. Substrate development was
stopped using 0.1 M H3PO4,
and plates were read at 450 nm. Values were determined by comparing
OD450 values to the standard curve generated for
each plate. For measurement of murine IL-2, anti-mouse IL-2 and
biotinylated anti-mouse IL-2 (BD PharMingen) and mouse rIL-2 (R&D
Systems) were used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L in THy from
DR4 transgenic mice
We prepared a panel of THy from the lymph node cells of DR4
transgenic mice (23) that had been immunized and boosted
with OspA in vivo. Because these mice do not express murine class II
molecules, all their CD4+ T cells are restricted
to HLA-DRB1*0401. As expected from previous results obtained with bulk
T cell cultures (14, 25), the majority of these THy were
specific for OspA165–184. An initial screen
revealed that 10% of these OspA-reactive THy also secreted IL-2 in
response to the LFA-1L cross- reactive
peptide, LFA-1L326–345 (results not shown).
When these dual-reactive THy were tested for their ability to bind
tetramers, nearly the whole clonal population stained with
OspA165–184-DR4 tetramer (7), but
were negative for staining with the
LFA-1L326–345-DR4 tetramer (results not
shown). Because these clones had responded to
LFA-1L326–345 peptide in vitro, we analyzed
the reactivity pattern of these cloned T cells in more detail. THy
cells were cultured in the presence of increasing concentrations of
monomeric OspA-, LFA-1-, or control gp39-DR4 molecules (Fig. 1
, A, B, or
C, respectively), bound to wells via streptavidin, and
measured secretion of IL-2. Two representative THy lines, 4–41 and
4–38, secreted large amounts of IL-2 in response to plate-bound OspA.
Plate-bound LFA monomers were also able to stimulate these clones,
although significantly higher concentrations of reagent were required.
In contrast, control gp39-DR4 monomer did not stimulate any IL-2
production in these THy. These results indicate that the LFA tetramer
is able to bind TCR and stimulate OspA-specific cloned THy.
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L peptides in
patient samples
SF and/or PBMC from eight individuals with treatment-resistant
Lyme arthritis (six patients were DRB1*0401+, two
patients were DRB1*0401-) and two non-Lyme
disease controls (DRB1*0401+) were stained with
OspA165–184-DR4-PE tetramer (as reported in Ref.
7). OspA-responsive PBMC are not common in
treatment-responsive patients. In Lyme arthritis patients, 0.01 to
3.05% of SF cells bound the OspA tetramer, while lower percentages,
<0.005 to 0.11%, were identified in peripheral blood. To analyze the
cross-reactivity, T cell clones were generated from SF and/or PBMC from
three treatment-resistant Lyme arthritis patients by stimulating
OspA-DR4 tetramer-sorted cells with PHA in the presence of irradiated
allogeneic feeder cells and feeding with IL-2-containing medium, as
described previously (7). No Ag-specific clones could be
generated from the controls. Although dual-reactive clones were
identified in two patients (from SF and/or PBMC), in one patient who
was DRB1*0401 homozygous, about one-half of the OspA-reactive T cell
clones also respond to LFA-1L326–345. In this
patient, the vast majority (93%) of clones generated by sorting OspA
tetramer+ SF cells had strong responses to OspA,
as measured by proliferation, while the strength of their responses to
LFA-1L326–345 varied greatly (Fig. 2). CD4+ OspA
tetramer- cells were also sorted into single
wells and expanded. Although 1 of 20 sort-negative clones responded
minimally to OspA in vitro (1500 cpm), further analysis proved that the
response was not significant (results not shown).
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L peptides
To begin identifying characteristics of
OspA/LFA-1L-reactive vs OspA-only-reactive T
cell clones, we measured the amount of cytokines secreted in response
to stimulating Ag. Cloned T cells plus autologous EBV-transformed
lymphoblastoid cell lines were incubated with peptide Ags. Supernatants
were removed from cultures for cytokine ELISA at 48 h. Stimulation
with OspA165–184 induced strong proliferation
and led to the secretion of large amounts of IFN- and IL-13 in
nearly all clones tested and lesser amounts of IL-4 and IL-5 in a
subset of clones (Fig. 3A,
). In contrast, when the stimulating Ag was
LFA-1L326–345, proportionally more IL-13 was
made than IFN- at 10 µg/ml peptide tested (Fig. 3A,
•). The amount of IFN- produced seemed to reflect the level of
proliferation, with OspA-stimulated clones secreting more IFN- than
LFA-1L-stimulated clones (Fig. 3A).
OspA-stimulated clones secreted more of each cytokine tested than
LFA-1L-stimulated clones. When cytokine
secretion patterns were compared with each other, the OspA-stimulated
clones could be divided in two groups, reflecting higher and lower
levels of IL-13, IL-4, and IL-5 secretion (Fig. 3B).
Additionally, IFN-, a Th1 cytokine, was not secreted to the
exclusion of Th2 cytokines such as IL-4 and IL-5. OspA-responsive
clones were very sensitive to OspA stimulation in dose-response
experiments. They proliferated and secreted high amounts of IFN- and
somewhat less IL-13 in response to as little as 0.1 ng/ml OspA. When
the clones were stimulated with OspA164–185, 0.1
ng/ml was enough to induce half-maximal proliferation or cytokine
secretion, while clones required 10 µg/ml
LFA-1L326–345 to reach half-maximal response.
Interestingly, when LFA-1L326–345
concentration was increased to 40 µg/ml, it also induced a low, but
measurable IFN- production (data not shown). Neither OspA nor
LFA-1L responses were measurable in
sort-negative control clones.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L326–345.
These results led to the hypothesis that inflammation at the initial
site of Bb-induced arthritis is perpetuated via molecular
mimicry between a bacterial Ag and a self-determinant (14, 15, 17). The observed HLA-DRB1 restriction of these patients to
*0401 and related alleles (13) led us to postulate that a
self-response is initiated in the chronic phase of the disease. Thus,
it is vital that we carefully evaluate the nature of the Bb
OspA immune response in individuals with treatment-resistant Lyme
arthritis. Chronic treatment-resistant Lyme arthritis is not prevalent
in Europe. This may be due to the fact that B. garinii and
B. afzelii, which are primarily responsible for the
infections there, have different sequences in the cross-reactive
position of OspA than Bb sensu stricto strains, which cause
the infection in the U.S.
To directly test the molecular mimicry hypothesis, we analyzed whether
the cross-reaction between OspA and LFA-1L
could be seen at the single cell level. Two approaches were used: 1)
generation of THy from OspA-immunized DRB1*0401 transgenic mice, and 2)
single cell cloning of OspA-specific T cells from chronic
treatment-resistant Lyme arthritis patients, using
DRB1*0401/OspA165–184 tetramers
(7). A subpopulation of
OspA165–184-specific human T cells or mouse THy
responded to the LFA-1L mimic peptide,
although we identified proportionally more dual-responsive human clones
with methods used. In preliminary TCR V
sequence analysis, multiple
clones reactive with both OspA and LFA-1L used
single unique TCR -chains (data not shown). This indicates that the
same TCR can respond to the two related peptides in the context of
DRB1*0401.
In these dual-reactive clones, the OspA response differs quantitatively
and qualitatively from the LFA-1L response, as
measured by proliferation and cytokine production, respectively.
Typically, the human T cell clones proliferated vigorously in response
to OspA and produced large amounts of IFN-, IL-4, IL-5, and IL-13,
even at low concentrations of Ag. In contrast, dual-reactive clones
proliferated less vigorously in response to higher concentrations of
LFA-1L and secreted less IFN-, yet still
produced significant amounts of IL-13, but not other Th2 cytokines,
IL-4 or IL-5. When clones were tested with high doses, 40–200 µg/ml,
of LFA-1L peptide, IFN- was secreted,
although at much lower amounts than following OspA stimulation. All
dual-reactive THy produced significantly less IL-2 in response to
LFA-1L compared with OspA, as tested over a
wide dose range. These results indicate that the nature of the peptide
determines the quality and quantity of the signal delivered through the
TCR. Perhaps IL-13 is a cytokine that characterizes weak TCR/Ag
interactions. The Hammer algorithm predicts that both the
OspA165–184 and
LFA-1L326–345 peptides bind to the
HLA-DRB1*0401 molecule with similar affinities (27, 28),
suggesting that the two peptides are presented to the T cell with equal
efficiency. Despite this similarity in Ag presentation, the
three-dimensional interaction of the peptide/MHC complex with the TCR
may differ. The OspA peptide is strongly agonistic and induces a
vigorous response, even at low concentrations of Ag (data not shown),
indicative of high affinity TCR recognition. In contrast, the
LFA-1L peptide induces a less efficient
interaction, reminiscent of a partial agonist. Stimulation of the TCR
with a partial agonist induces a different set of downstream effects
that may result in minimal proliferation and, in this case, a different
pattern of cytokine secretion at a given concentration of peptide.
Furthermore, given more efficient endogenous Ag presentation in situ,
it is possible that it is not necessary to achieve
LFA-1L concentrations equal with those needed
in vitro to maintain chronic stimulation in the joint.
Nearly all OspA-reactive T cells tested produce both Th1-type
inflammatory and Th2-type B cell-stimulatory cytokines, IFN-, IL-4,
IL-5, and IL-13, respectively. This lack of clearly defined Th1/Th2
response seems to be a common feature of human Ag-specific T cell lines
and contrasts with the pattern seen in murine T cell lines
(29). Of special interest is that stimulation with
LFA-1L results predominantly in IL-13
production, with little IFN- secretion only. The role of IL-13 has
not been fully established yet (30). IL-13 is strongly
associated with allergic reactions, but it has also been shown to play
a role in fibrosis following Schistosome infection (31).
Thus, IL-13 from LFA-1L326–345-stimulated T
cells could be contributing to the persistent joint inflammation in
chronic treatment-resistant Lyme patients.
The degeneracy of TCR specificity was not fully appreciated until the
advent of combinatorial peptide libraries, which allow the probing of
the full spectrum of a TCR’s binding requirements
(32, 33, 34). These studies have revealed that the TCR is
flexible and can recognize a surprisingly wide array of amino acid
sequences. Hemmer et al. (28) applied a positional
scanning peptide combinatorial library to delineate peptide recognition
of a T cell clone generated from the cerebrospinal fluid of a patient
with chronic neuroborreliosis, another form of chronic Lyme disease.
This clone preferentially recognizes peptides of Bb
bacterial origin, but also responds to CNS-derived human peptides from
myelin-associated oligodendrocyte basic protein and somatostatin
receptor, as well as peptides from human TGF-3. Similar to our
observation, they found that higher concentrations of human self Ags
than Bb Ags were necessary to obtain optimal proliferative
responses in this T cell clone. Using peptide spot synthesis analysis,
Maier et al. (35) determined a TCR-binding motif of
peptide recognition of THy generated from a Bb
OspA-immunized DR4 transgenic mouse. Database searches revealed a large
number of both bacterial and human peptides with this binding motif,
including hLFA-1L. Not all peptides identified
were functional, as tested on a small panel of these DR4-restricted
THy. Curiously, the LFA-1L326–345 peptide,
which stimulates a significant number of our human
HLA-DRB1*0401-restricted and DR4 transgenic mouse-derived OspA-specific
T cells, was not recognized by their hybridomas. This discrepancy may
be explained by significant differences between the two DR4 transgenic
mouse lines used by them (35) and us (23).
Additionally, given that we see LFA-1L
cross-reactivity in 1/10 OspA-specific THy, they may have missed
this reactivity in their small panel of OspA-specific hybridomas
tested.
Possible mechanism of treatment-resistant Lyme arthritis
It is expected that high affinity
LFA-1L-reactive T cells are eliminated in the
thymus during negative selection. However, lower affinity
LFA-1L-reactive cells may remain. Some of
these same T cells have a higher affinity for and are fully responsive
to the homologous peptide of OspA. In the absence of OspA, the
activation threshold of these self-responsive T cells can only be
overcome by a high local level of the self Ag,
LFA-1L, as would be expected in an
inflammatory environment such as an affected joint. During the course
of Lyme arthritis, macrophages in the synovium present Bb
Ags to immune cells, including those of OspA. In genetically
susceptible individuals, this may include
OspA165–184 (Fig. 4A). As the immune response
proceeds, OspA-specific T cells become stimulated and cytokines such as
IFN- are secreted. An increased level of IFN- enhances the
general state of activation in the synovium, while local levels of
LFA-1L expressed on T cells, macrophages, and
dendritic cells increase. Although Bb organisms are thought
to be cleared eventually during antibiotic treatment, presentation of
the abundantly expressed self Ag LFA-1L
continues. Ultimately, LFA-1L peptides from
endogenous production and scavenged cells are most likely presented by
MHC class II expressed on macrophages and dendritic cells at the site
(Fig. 4B). Then, OspA-expanded T cells can cross-react with
MHC-presented self LFA-1L peptide, a partial
agonist for the same TCR, inducing a slightly different cytokine
milieu, including more IL-13. This unintentional but likely
presentation of LFA-1L and the continued
stimulation of T cells at the site of chronic inflammation could
explain why the LFA-1L response in
treatment-resistant Lyme arthritis patients is apparently limited to
the site of intense inflammation. Although the role of IL-13 in the
joint is unknown, it is possible that
LFA-1L-induced IL-13 may contribute to
persistent joint inflammation, and if the amount of
LFA-1L is high enough, secretion of IFN-
supports the inflammatory environment.
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L.
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Acknowledgments |
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Footnotes |
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2 C.T. and A.L.M are co-first authors.
3 Address correspondence and reprint requests to Dr. Brigitte T. Huber, Department of Pathology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111.
4 Abbreviations used in this paper: SF, synovial fluid; Bb, Borrelia burgdorferi; hLFA, human LFA; OspA, outer surface protein A; THy, T cell hybridoma.
Received for publication November 14, 2000. Accepted for publication February 8, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 T. A. Willett, A. L. Meyer, E. L. Brown, and B. T. Huber An effective second-generation outer surface protein A-derived Lyme vaccine that eliminates a potentially autoreactive T cell epitope PNAS, February 3, 2004; 101(5): 1303 - 1308. [Abstract] [Full Text] [PDF]
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 F. Goulhen, D. Grenier, and D. Mayrand ORAL MICROBIAL HEAT-SHOCK PROTEINS AND THEIR POTENTIAL CONTRIBUTIONS TO INFECTIONS Critical Reviews in Oral Biology & Medicine, November 1, 2003; 14(6): 399 - 412. [Abstract] [Full Text] [PDF]
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D. S. Pikas, E. L. Brown, S. Gurusiddappa, L. Y. Lee, Y. Xu, and M. Hook Decorin-binding Sites in the Adhesin DbpA from Borrelia burgdorferi: A SYNTHETIC PEPTIDE APPROACH J. Biol. Chem., August 15, 2003; 278(33): 30920 - 30926. [Abstract] [Full Text] [PDF]
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 A. C. Steere Lyme Disease N. Engl. J. Med., July 12, 2001; 345(2): 115 - 125. [Full Text] [PDF]
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) and 4-38 (•) were generated from lymph
node cells of OspA-immunized DR4 transgenic mice and were screened for
their ability to secrete IL-2 in response to OspA165–184.
Dual reactivity was tested by culturing the THy (105) for
24 h with increasing concentrations of OspA-DR4
(A), LFA-DR4 (B), or gp39-DR4
(C) monomeric molecules, bound via extrAvidin to ELISA
plates. Supernatants were harvested and analyzed for IL-2 content by
ELISA. Values were determined by comparison with standard curve.
), are expressed in 
