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and IL-10 Secretion in Patients with Chronic Borrelia burgdorferi Infection1
*
Laboratory of Molecular Immunology, Center for Neurologic Diseases, Department of Neurology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115;
Division of Rheumatology, Department of Medicine, Tufts University School of Medicine, Tupper Research Institute, New England Medical Center, Boston, MA 02111
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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- and IL-10-secreting human T
cells that differentiate in the presence of exogenous IL-12 in vitro
has recently been described. Whether this T cell population exists in
vivo is unknown. Borrelia burgdorferi, the etiologic agent
of Lyme disease, can induce a chronic infection in the presence of a
vigorous humoral immune response. We established T cell lines specific
for B. burgdorferi and tetanus toxoid from subjects with
chronic B. burgdorferi infection and healthy controls in
limiting dilution experiments and assessed proliferation and cytokine
secretion. As expected, higher frequencies of B.
burgdorferi-specific precursor T cells were observed in Lyme
patients compared with controls. In both groups of subjects, T cell
lines specific for B. burgdorferi secreted high amounts of
IFN-. However, in patients with Lyme disease, 27% of T cell lines
secreted not only IFN- but also IL-10, which was only observed in
0.6% of B. burgdorferi-reactive T cell lines generated
from controls and in none of the tetanus toxoid-reactive T cell lines
generated from either Lyme patients and controls. Single cell PHA
cloning confirmed that both cytokines were secreted from one clonally
expanded precursor cell. Whole mononuclear cells from B.
burgdorferi-infected individuals, but not from controls, secreted
IL-12. Moreover, neutralizing anti-IL-12 mAbs inhibited the
generation of the IFN-/IL-10 population. These data demonstrate that
this novel population of IL-12-induced IFN-/IL-10-secreting T cells
is generated in response to chronic B. burgdorferi
infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF-ß. IL-4 induces Th2 T cells that exhibit an
anti-inflammatory effect by secreting IL-4, IL-5, IL-10, and IL-13,
thus providing B cell help and inhibiting IFN- (3). In particular,
allergens can induce clear Th2 subsets, while viral and bacterial
infections are associated with a Th1 type of response. While the
Th1/Th2 paradigm of various infectious diseases is well defined in
animal models, the differentiation of T cells in humans is not as
clearly understood (4).
We and others have observed recently a novel population of human T
cells that differentiate in the presence of IL-12 into an
IFN-/IL-10-secreting subset (5, 6). The identification of this T
cell subset has not been observed without adding exogenous IL-12 to the
culture. Whether this T cell population exists in vivo or is associated
with inflammatory human disease is unknown. IL-12 is secreted by
monocytes and B cells in response to bacteria and intracellular
parasites, and stimulates T cell IFN- secretion (7, 8, 9, 10). A role for
IL-12 in the immune defense against infectious agents has been reported
in Mycobacterium tuberculosis and Leishmania
donovani infections (11, 12). In HIV-infected individuals,
impaired IL-12 production has been related to a defective immune
response to bacteria and parasites (13, 14). Thus, we postulated that
in chronic infectious disease, IL-12 secreted by APC may induce a major
population of T cells secreting both IFN- and IL-10 in
vivo.
Lyme disease is a multisystemic infectious disorder caused by Borrelia burgdorferi, a tick-borne spirochete. Acute B. burgdorferi infection is characterized by an expanding skin lesion called erythema migrans. Untreated or cases with delayed treatment may show clinical progression to systemic phases of the disease characterized by arthritis, carditis, and neurologic manifestations (15, 16, 17). There is evidence that spirochetes establish a chronically active infection in advanced stages of Lyme disease, although they become more difficult to recover as the disease progresses. The occasional identification of B. burgdorferi in diverse organs of patients with late Lyme disease (18, 19), the detection of B. burgdorferi by PCR in joint or cerebral spinal fluid samples from patients with Lyme arthritis or neuroborreliosis (20, 21), as well as the expanding Ag specificity of B. burgdorferi-directed Abs in untreated patients with advanced stages of disease (22) are indicative of the persistence of active spirochetal infection even in later stages of the disease. Thus, untreated patients with Lyme arthritis or neuroborreliosis are thought to have a chronically active infection that causes persistent activation of the immune system.
Despite an increasing understanding of the pathogenesis of Lyme disease
(23), basic aspects of the immune response to B. burgdorferi
are not well understood. The cellular immune response to B.
burgdorferi infections has been studied primarily in the mouse
model. In murine Lyme disease, T cells play a critical role in
modulating the severity of acute arthritis and determining the outcome
of B. burgdorferi infections. CD4 T cells control
spirochetal growth, since abrogation of this subset by treatment with
anti-CD4 mAb increases the severity of arthritis and the bacterial
burden. CD8 T cells promote B. burgdorferi infection in
susceptible mice during the later stages of disease, as depletion of
this subset leads to a reduced severity of arthritis and a decreased
recovery rate of B. burgdorferi spirochetes (24, 25, 26). In
disease-susceptible mice, B. burgdorferi infection elicits a
Th1 cytokine response since disease progression has been demonstrated
to be associated with IFN- production. In contrast, mice resistant
to B. burgdorferi arthritis secrete predominantly IL-4, thus
exhibiting a Th2 cytokine response. Furthermore, adoptive transfer of
B. burgdorferi-specific Th2 clones provided resistance to
B. burgdorferi infection in susceptible mice, stressing the
protective role of Th2 cytokines (27, 28).
In this study, we investigated the cellular immune response to B.
burgdorferi and tetanus toxoid
(TT)4 in patients with Lyme
arthritis and neuroborreliosis. Short-term B.
burgdorferi-specific and TT-specific T cell lines were generated,
and their cytokine profile was assessed. We found that human Lyme
disease is associated with a strong response to B.
burgdorferi that is characterized by a novel subset of T cells
secreting both IFN- and IL-10. IL-12 is critical for the
differentiation of these cells, as endogenous IL-12 secretion was
observed in patients with Lyme disease, but not in control subjects,
and T cell secretion of IFN- with IL-10 was suppressed in the
presence of neutralizing Abs to IL-12. The presence of IFN-/IL-10
secretion was only observed in B. burgdorferi-, but not
TT-specific T cell lines from infected subjects. These data demonstrate
that B. burgdorferi infection induces IL-12 secretion that
induces a population of T cells characterized by secretion of both
IFN- and IL-10, a novel functional T cell subset in humans.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Twelve patients diagnosed with chronic neuroborreliosis and five patients with Lyme arthritis were enrolled in this study. All 17 patients were seropositive for B. burgdorferi by ELISA and Western blot using the CDCcriteria (29). The control group consisted of five healthy individuals with negative Lyme serologic test results. Human subject approval and informed consent were obtained before blood drawing.
Reagents
Whole sonificated B. burgdorferi Ags were prepared as previously described (30). TT was kindly provided by Cyanamid (Pearl River, NY). IL-12 and neutralizing anti-IL-12 Ab were purchased from R&D Systems (Minneapolis, MN). T-stimulans containing IL-2 and other unspecified growth factors was obtained from Collaborative Biomedical Products (Bedford, MA), and rIL-4 was purchased by Boehringer Mannheim Corp. (Indianapolis, IN). Ab directed against CD3 and CD28 was obtained from American Type Culture Collection (Rockville, MD).
Generation of Ag-specific T cell lines
B. burgdorferi- and TT-specific short-term T cell lines were established from PBMC of seven patients with Lyme disease and five healthy control subjects aged 25 to 54 yr following modified, previously published methods (31, 32). In brief, PBMC were isolated from heparinized venous blood by Ficoll-Hypaque density-gradient centrifugation (Pharmacia, Piscataway, NJ) and resuspended in RPMI 1640, 10% autologous serum, 4 mM L-glutamine, 25 mM HEPES buffer, 50 U/ml penicillin, and 50 µg/ml streptomycin (Whittaker Products, Walkersville, MD). An equal number of wells with 5 x 104 and 1 x 105 PBMC from patients with Lyme disease and controls, respectively, were plated in 96-well round-bottom plates for the appropriate conditions. Ag concentration was evaluated with titration experiments to achieve optimal T cell stimulation; PBMC were incubated at 107/ml with B. burgdorferi (100 µg/ml) or at 5 x 106/ml with TT (62.5 Lf/ml) for 1 h and then diluted to 106/ml in cell culture medium to a final concentration of B. burgdorferi of 10 µg/ml and TT of 12.5 Lf/ml. On day 7, medium was changed and T cell lines were restimulated with 105 autologous, irradiated PBMC/well that were pulsed with either B. burgdorferi or TT at the same concentrations as before. After 3 further days of culture, 5% T-stimulans and rIL-4 at a final concentration of 5 U/ml were added. Between days 14 and 19, T cell lines were split in case of overgrowth with renewal of medium containing T-stimulans and rIL-4. T cell lines were maintained until day 21 to 24 without further interventions to allow highly activated lines to return to a resting state before repeated activation with Ag. After 21 to 24 days, T cell lines were washed twice with RPMI to remove T-stimulans and rIL-4, resuspended in medium without additional cytokines, and split into four aliquots to test Ag specificity and cytokine secretion.
Testing for Ag specificity
To assess Ag reactivity, T cell lines were split and duplicate
wells were incubated with 105 autologous Ag-pulsed and
irradiated PBMC or plate-bound anti-CD3/anti-CD28 (1 µg/ml
each); another two wells received nonpulsed irradiated PBMC. After
44 h of incubation, 180 µl of cell culture supernatant was
collected from each well for measurement of cytokines and stored at
-80°C until used. Supernatant was replaced with medium, and cell
proliferation was measured by incorporation of
[3H]thymidine after another 18 h. Lines with a
stimulation index (mean cpm + Ag/mean cpm, no Ag) of >3 and 
cpm >2000 were considered positive.
Limiting dilution analysis
To determine the precursor frequency of Ag-specific T cells, limiting dilution analysis (LDA) was performed following previously published methods with modifications (30, 33). T cell lines (837 B. Burgdorferi-specific and 570 TT-specific) were established in parallel at varying densities in four patients with Lyme disease and five healthy controls. PBMC were plated at 5 x 104, 5 x 103, and 5 x 102 in patients with Lyme disease, and 1 x 105, 1 x 104, and 1 x 103 in control subjects. After 21 days, B. burgdorferi- and TT-specific T cell lines were assayed for their Ag reactivity by assessment of proliferation, as described above.
IL-12 and anti-IL-12 inhibition experiments
B. burgdorferi-specific T cell lines from two
patients with neuroborreliosis were established in the presence and
absence of anti-IL-12 Abs at a final concentration of 2 µg/ml
(according to the manufacturer’s suggestions) and from two patients in
the presence of IL-12 at a final concentration of 10 ng/ml. It has been
shown previously by our laboratory that at this concentration, IL-12
induces significant IFN- production and anti-IL-12 induces
significant inhibition of IFN- by a CD4+ T cell clone
(6). In the IL-12 condition, no rIL-4 was added during the culture
period. T cell lines were cultured for 21 days and then tested for Ag
reactivity by B. burgdorferi-pulsed autologous PBMC or
plate-bound anti-CD3/anti-CD28 Abs (1 µg/ml each).
Preliminary experiments have shown insufficient activation of T cell
lines with anti-CD3 alone that are 21 days or older, but optimal
stimulation of anti-CD3 in combination with anti-CD28 Abs (data
not shown).
Measurement of cytokine secretion
Supernatants (pooled from two duplicate wells) of all lines
established from 5 x 104 and 1 x
105 PBMC/well in patients with Lyme disease and controls,
respectively, were assayed by ELISA for cytokine secretion of IL-4,
IL-10, and IFN- regardless of their proliferation status. Lines
established in LDA experiments from 5 x 102 and
1 x 103 PBMC were tested only for cytokine secretion,
when the proliferation assay was positive. For the detection of IL-4
and IL-10, 1 µg/ml of capture mouse anti-human IL-4 mAb and rat
anti-human IL-10 mAb and 0.5 µg/ml of biotinylated rat
anti-human IL-4 and rat anti-human IL-10 were used (purchased
from PharMingen, San Diego, CA) following the manufacturer’s
suggestions. For IFN-, 1 µg/ml monoclonal mouse anti-human
IFN- and 1 µg/ml polyclonal rat anti-human IFN- (obtained
from Endogen, Cambridge, MA) and biotinylated anti-rat IgG (Sigma
Chemical Co., St. Louis, MO) were used. Assays were done in duplicate.
The sensitivities of the assays were 15 pg/ml for IL-4 and IL-10 and 30
pg/ml for IFN-. A net cytokine secretion (concentration of cytokine
in +Ag condition minus concentration of cytokine in non-Ag condition)
of 40 pg/ml IL-4, 50 pg/ml IL-10, and 50 pg/ml IFN- was considered
positive. Only results with an SD between the duplicates of 15% or
less were used for analysis.
Limiting dilution cloning
T cell clones were generated from T cell lines secreting both
IFN- and IL-10 by PHA cloning methods. Quantities amounting to 1 and
0.3 T cells/well were plated on 96-well V-bottom plates containing
tissue culture medium and 106/ml irradiated,
heterologous mononuclear feeder cells with 1 µg/ml PHA-Purified
(Murex, Norcross, GA). At 48 h, 100 µl medium/well was removed
and replaced by new medium containing 20% T-stimulans. One hundred
microliters of medium from each well were replaced every 3 to 4 days
with fresh medium containing 10% T-stimulans. After 14 days, cells
from growth-positive wells were transferred to 96-well U-bottom plates
to a final concentration of 5000 cells/well and restimulated with
PHA-Purified and feeder cells. After further 14 days, Ag reactivity to
B. burgdorferi was assessed with autologous, Ag-, and
non-Ag-pulsed PBMC, as described above. Cytokine secretion was tested
in clones with a positive proliferation ( cpm >3000). The cloning
efficiency was 50% in the assays starting with 1 cell/well, and 15%
starting with 0.3 cells/well.
Measurement of endogenous IL-12 secretion
PBMC from patients with Lyme disease and healthy controls were cultured for 2 days at 1 x 106 cells/ml in polypropylene culture tubes (Fisher Scientific, Pittsburgh, PA) in RPMI 1640 supplemented with 10% FBS, 4 mM L-glutamine, 25 mM HEPES buffer, 50 U/ml penicillin, and 50 µg/ml streptomycin (all from Whittaker Products). After 2 days, PBMC were washed twice and then activated with 1 µg/ml of anti-CD3 mAb, and culture supernatants were collected 24 h later. Supernatants were stored at -80°C until used. IL-12 was determined using an ELISA kit purchased from R&D Systems. The assay’s sensitivity for measurement of IL-12 was 2 pg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The precursor frequency of B. burgdorferi- and
TT-specific T cells in four patients with Lyme disease in comparison
with five healthy controls was measured by LDA (Fig. 1
). The calculated frequency for B.
burgdorferi-specific T cells was 1:500 in patients with Lyme
disease and 1:100,000 in normal individuals. LDA was performed
similarly using the recall Ag TT. Although all individuals had this
vaccination between 5 and 10 yr before blood drawing, the precursor
frequency of TT-specific T cells was markedly higher in patients with
Lyme disease (1:1,000) as compared with control subjects
(1:30,000).
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and IL-10
As the frequency of B. burgdorferi-specific T cells was
low in normal individuals, T cell lines were established from wells
plated with 1 x 105 PBMC in four controls as compared
with 5 x 104 PBMC/well in another series of four
patients with Lyme disease. Each well was examined for cytokine
secretion and [3H]thymidine incorporation after B.
burgdorferi-specific stimulation by split well assay. In patients
with Lyme disease, the predominant cytokine was IFN- secreted by
147/240 (61%) of the lines tested. However, an additional 66/240
(27.5%) lines secreted both IFN- and IL-10. This was observed in
all four patients with Lyme disease. In contrast, B.
burgdorferi-specific T cell lines generated from seronegative,
healthy controls secreted IFN- alone in 55% (98/177), but only
1/177 (0.6%) lines secreted both IFN- and IL-10. Interestingly,
there was no IL-4 secretion in B. burgdorferi-specific T
cell lines in patients with Lyme disease (0/240) and little (1/177) in
the control subjects (Fig. 2).
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with IL-10
represented single clones or multiple lines. Therefore, we generated T
cell lines at a frequency of 5 x 102 PBMC/well and
examined them for cytokine secretion. At this density, LDA indicates
that the majority of wells contain a single clonally expanded
Ag-specific T cell line. As shown in Figure 3, we continued to observe T cell lines
secreting both IFN- with IL-10 at the same frequency (21/65 =
32%) observed in higher cell numbers per well.
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T cell lines were established after primary stimulation with TT
from 5 x 104 PBMC/well in two patients with Lyme
disease, and 1 x 105 PBMC/well in two control
subjects, and examined for cytokine secretion. In contrast with
B. burgdorferi-specific T cell lines, TT-specific T cell
lines secreted either a Th1 or Th0 profile. Forty-seven percent (28/60)
and fifty percent (21/42) of TT-specific lines secreted IFN- alone
in patients with Lyme disease and controls, respectively. IFN-
together with IL-4 was secreted in 47% (28/60) and 17% (7/42) in
patients with Lyme disease and controls, respectively. Simultaneous
secretion of IFN- and IL-10 in TT-specific T cell lines was not
observed in patients with Lyme disease and controls. In Figure 2
, the
cytokine pattern of TT-specific T cell lines is contrasted to that of
B. burgdorferi-specific T cell lines. Whereas the cytokine
pattern of TT-specific T cell lines did not differ between patients
with Lyme disease and controls, the concentrations of IFN- and IL-4
secreted by T cells were significantly higher in patients with Lyme
disease than in control subjects (Fig. 4).
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by T cells
To prove that individual T cells were secreting both IFN- and
IL-10, we performed single cell PHA cloning from a series of B.
burgdorferi-specific T cell lines secreting both cytokines and
generated stable T cell clones. T cell clones were stimulated with
autologous Ag and non-Ag-pulsed PBMC, and the
[3H]thymidine uptake (proliferation) and cytokine
secretion were assessed. The characteristics of three representative
clones secreting IFN- with IL-10, in contrast to two clones
secreting IFN- alone, are shown in Table I. While IFN- is
secreted only by T cells, IL-10 can be secreted by both macrophages and
T cells. Thus, it was important to demonstrate that the secretion of
IL-10 was by T cells and not secondary to activated T cells inducing
macrophage secretion of the cytokine. Therefore, B.
burgdorferi-reactive T cell clones were expanded in the absence of
feeder cells, and thus did not contain viable macrophages. Stimulation
of these clones by a combination of anti-CD3 and anti-CD28 mAbs
cross-linked on plastic induced proliferating T cells that secreted
both IFN- and IL-10 (Table I). Generation of B.
burgdorferi-reactive T cells in the presence of IL-12 further
increased the secretion of IL-10 (Tables I and II).
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We recently observed a high frequency of T cell lines secreting
both IFN- and IL-10 after in vitro Ag stimulation of T cells from
normal individuals with either myelin basic protein or TT in the
presence of IL-12 (6). Thus, it was of obvious interest to determine
whether there was increased secretion of IL-12 in PBMC from patients
with Lyme disease, possibly inducing the population of
IFN-/IL-10-secreting T cells we observed in these subjects. As shown
in Figure 5, PBMC from 9 of 12 patients
with Lyme disease secreted significant amounts of IL-12 after
anti-CD3 stimulation, whereas in healthy controls there was no
IL-12 secretion. No IL-12 secretion was noted after stimulation by
whole B. burgdorferi Ag (data not shown).
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with IL-10 is blocked
by anti-IL-12-neutralizing Abs
To determine whether IL-12 secretion by PBMC in patients with Lyme
disease induced the differentiation of T cells secreting IFN- and
IL-10, B. burgdorferi-specific T cell lines were generated
in the absence and presence of anti-IL-12-neutralizing Abs in two
patients. T cell lines were tested for Ag-specific proliferation and
cytokine secretion after stimulation with B. burgdorferi as
in previous assays. The frequency of Ag-specific T cell lines, as
measured by [3H]thymidine incorporation, was less in the
presence of anti-IL-12-neutralizing Abs; 118/120 lines (98%) in
the absence, and 35/66 (53%) in the presence of anti-IL-12 Abs
proliferated upon Ag-specific stimulation. As T cell lines can secrete
cytokines in the absence of proliferation, cytokine secretion was
examined regardless of [3H]thymidine incorporation.
Although Ag-reactive T cells were still present, the presence of
IFN-/IL-10-secreting T cell lines was abrogated completely by the
anti-IL-12-neutralizing Abs (Fig. 6).
Moreover, the frequency of T cell lines secreting IFN- alone was
also diminished. These data provide definitive evidence that T cells
are capable of secreting both IL-10 and IFN-, and that this subset
is induced by endogenous IL-12 secretion.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-10, which was induced by IL-12 secreted
by PBMC. T cell secretion of IFN- and IL-10 was suppressed in the
presence of neutralizing Abs to IL-12. The presence of IFN-/IL-10
secretion was observed only in B. burgdorferi- and not in
TT-specific T cell lines. These data demonstrate that chronic B.
burgdorferi infection causes IL-12 secretion that induces a
population of T cells characterized by secretion of both IFN- and
IL-10. The significance of the cellular immune response to B. burgdorferi in Lyme disease has been investigated earlier. A strong T cell response to B. burgdorferi was detected early in the course of illness, often preceding the development of the humoral immune response (33). Despite a vigorous humoral as well as cellular immune response, untreated B. burgdorferi infection tends to become chronic with a low bacterial load. Since during the course of chronic illness the Ab specificity develops a broader antigenic spectrum and spirochetal Ag and genome can be detected, a chronically activated Ag-specific immune response to B. burgdorferi can be assumed (22). This persistently activated cellular immune response may play a pivotal role in perpetuating Lyme disease in chronic B. burgdorferi infection.
Proliferative responses of T cells to whole sonificated B. burgdorferi Ag have been investigated in 5-day bulk cultures in patients with Lyme disease. Significant differences in the mean proliferative responses to B. burgdorferi between Lyme patients and seronegative controls have been observed (34, 35). Similar results have also been observed in a murine model, in which an Ag dose-dependent specific T cell response of lymph node cells as well as splenic T cells to B. burgdorferi could only be observed in B. burgdorferi-infected, but not in uninfected mice after 4 days of culture (25).
We were interested in further investigating this phenomenon of
increased proliferative responses to B. burgdorferi by
establishing B. burgdorferi-specific T cell lines in LDA
experiments. While B. burgdorferi-specific precursor T cells
were present in every seronegative individual, the frequency of these T
cells was increased significantly in patients with Lyme disease (Fig. 1). In contrast to previously published data, in which proliferative
responses to B. burgdorferi were found only inconsistently
(34), in our experiments an increased frequency of B.
burgdorferi-specific T cells was found consistently in all
patients with Lyme disease. This may be due to different technologies,
as the generation of B. burgdorferi-specific T cell lines
from freshly collected blood samples is by far more sensitive than
5-day bulk cultures from frozen PBMC. LDA analysis has been performed
in one patient with Lyme disease earlier (33). In this singular case,
the frequency of B. burgdorferi-specific T cells has been
estimated to 1 in 3700. Our calculations, however, revealed clearly
higher frequencies (Fig. 1). This may be explained by different
experimental procedures: while in this earlier published experiment
frequencies were calculated from 6-day bulk culture assays, in our
study, LDA was performed from 21-day B. burgdorferi-specific
T cell lines.
As our LDA experiments revealed significantly increased frequencies of
B. burgdorferi-specific T cell lines in patients with Lyme
disease as compared with healthy controls, we were also interested in
comparing these findings with the frequency of T cells specific for TT,
a control Ag. Surprisingly, the frequency of TT-specific T cell lines
was also increased in Lyme patients compared with controls, although
there was no history of recent TT vaccinations in these patients. This
may be explained by the presence of a chronically activated immune
system. In this regard, it has been demonstrated that whole mononuclear
cells from naive individuals stimulated with B. burgdorferi
in vitro showed a secondly enhanced response to TT (36). Thus, patients
with chronic B. burgdorferi infection, with persistent
presentation of B. burgdorferi Ag, may exhibit enhanced
proliferative responses to unrelated Ags. This is underscored by the
observation that TT-specific T cells cloned from patients with Lyme
disease secrete higher amounts of cytokines compared with control
subjects (Fig. 4). The increased cytokine secretion was not the result
of more TT-reactive T cells/well and a higher precursor frequency of
Ag-reactive T cells in Lyme patients, as individual T cells in LDA also
secreted greater amounts of cytokines.
To further characterize the nature of the cellular immune response to
B. burgdorferi, we assessed the cytokine profile of B.
burgdorferi-specific T cells. By generating T cell lines from
normal individuals seronegative for B. burgdorferi, we
directly compared the presumably naive B.
burgdorferi-reactive T cells with the differentiated memory T
cells from patients with late Lyme disease. The B.
burgdorferi-reactive T cell lines from normal individuals secreted
IFN-, although qualitatively less than the B. burgdorferi
T cell lines from patients with Lyme disease. This finding, however, is
in contrast to the previously published observation that only T cells
from patients with Lyme disease, but not from healthy controls, are
capable of secreting IFN- on a single cell level in response to
B. burgdorferi Ag (37). However, in the latter study,
cytokine secretion was not tested from B.
burgdorferi-specific T cell lines or clones.
Whereas there was no difference in the IFN- detection of B.
burgdorferi-specific T cell lines between Lyme patients and
controls, the major difference in the characteristics of B.
burgdorferi-reactive T cells from patients with Lyme disease was
the secretion of IFN- and IL-10. IL-10 can be secreted by T cells, B
cells, and other mononuclear cell populations. As IL-10 may have been
secreted in response to B. burgdorferi by monocytes, it was
important to demonstrate that IL-10 was secreted by T cells. Thus, we
stimulated B. burgdorferi-specific T cell lines in duplicate
culture with B. burgdorferi-pulsed APCs or with
anti-CD3/anti-CD28 in the absence of any other cells. These
experiments confirmed that IL-10 and IFN- were secreted by the T
cells. To further exclude the possibility that IFN- and IL-10 were
secreted by two different populations of T cells, we performed LDA and
single cell cloning with PHA. B. burgdorferi-reactive T
cells were cloned at 0.3 cells/well with PHA and feeder cells. The
expanded T cell clones were then stimulated again with either B.
burgdorferi-pulsed APCs or a combination anti-CD3 and
anti-CD28 mAb in the absence of accessory cells, and again these
clones secreted IFN-/IL-10. Thus, these experiments provided
definitive evidence that B. burgdorferi-specific T cells
from patients with Lyme disease secrete both IFN- and IL-10. This
phenomenon was observed for B. burgdorferi-reactive T cells,
but not in TT-specific T cell lines (Fig. 3).
IL-12 has been described as a proinflammatory cytokine secreted by
macrophages and B cells in response to bacterial infections. IL-12
induces IFN- secretion from T cells and NK cells. An important role
of IL-12 in the human immune response to infectious agents, in
particular in AIDS, tuberculosis, and L. donovani
infections, has been shown (12, 13, 32). IL-12 and IL-12-induced
IFN- favor Th1 T cell differentiation and Ag-specific proliferation
(10). However, we and others have observed recently that Ag stimulation
of T cells in vitro in the presence of IL-12 induces a population of T
cells that secrete IFN- and IL-10 (5, 6). This led us to examine
IL-12 secretion by PBMC from subjects with Lyme disease. It was
reported previously that B. burgdorferi-derived lipoproteins
can induce macrophage-derived IL-12 (38, 39). In our assays using whole
B. burgdorferi bacteria as Ag and PBMC instead of macrophage
cultures, stimulation of PBMC with B. burgdorferi-pulsed
autologous APC did not induce detectable amounts of IL-12 secretion
(data not shown). However, we could observe significant secretion of
IL-12 from PBMC stimulated with anti-CD3 mAb that was not observed
in the control individuals (Fig. 5). Using the same assay in patients
with chronic autoimmune disease, IL-12 secretion was mediated by CD40
ligand expressed on T cells (40). Moreover, to further prove that IL-12
induces T cells secreting both IFN- and IL-10, neutralizing Abs
against IL-12 were able to totally abrogate the generation of this
subset (Fig. 6). These data provide the first direct evidence that the
newly described population of IFN-/IL-10-secreting T cells can be
associated with increased PBMC secretion of IL-12 induced in vivo by a
chronic systemic infection.
As strong IL-10 secretion in the presence of IFN- has been
demonstrated in T cell lines generated with IL-12 stimulation, we
investigated whether the amounts of IL-10 secreted by B.
burgdorferi-specific T cells could be increased with exogenous
IL-12. Thus, we established B. burgdorferi-reactive T cell
lines with IL-12 stimulation and measured the cytokine secretion after
anti-CD3/CD28 stimulation. Increased amounts of IL-10 with IFN-
were secreted (Table II), and these concentrations correspond with
those published by others (5, 6).
|
alone, thus
playing a critical role in perpetuating chronic Lyme disease. Since
IL-12 is capable of triggering the secretion of both IFN- and IL-10
from T cells, our data further suggest that this new T cell subset may
play a crucial role in the evolution and establishment of chronic Lyme
disease.
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Footnotes |
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2 Current address: Children’s Hospital, Ludwig-Maximilians-University, 80337 Munich, Germany.
3 Address correspondence and reprint requests to Dr. David A. Hafler, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address:
4 Abbreviations used in this paper: TT, tetanus toxoid; LDA, limiting dilution analysis.
Received for publication July 17, 1997. Accepted for publication October 27, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD3/
