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2V
2 T Cells Produce IFN- and TNF-
with an On/Off/On Cycling Pattern in Response to Live Bacterial Products1
Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells is rapidly regulated
by exposure to peptide Ag, the mechanisms regulating cytokine
production by 
T cells are unknown. In this study, we demonstrate
that human V2V2 T cells produce IFN- and TNF- as early as
2 h after Ag exposure, and that they produce these cytokines in a
dose- and time- dependent manner in response to stimulation with a live
bacterial product, iso-butylamine (IBA), but not to dead
bacteria or LPS. T cells began, ceased, and then resumed IFN-
and TNF- generation in an on/off/on cycling pattern, both in vitro
and in vivo, depending on the presence or absence of IBA. IFN- and
TNF-, whose optimum production was dependent on IBA-stimulated
T cells, were critical for monocyte-mediated killing of
Escherichia coli. By limiting cytokine production to
periods of direct contact with live bacteria, T cells focus
their resources at the site of infection, while limiting systemic
immunopathology. Thus, human T cells may mediate innate
resistance to extracellular bacteria via tightly regulated cytokine
production without necessarily expanding in
number. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2V2 T cells recognize nonpeptide
organophosphate and alkylamine Ags secreted by bacteria in a
TCR-dependent, and MHC- and CD1-unrestricted manner
(1, 2, 3). The human pathogens Morganella morganii
(1), Salmonella typhimurium (4, 5), and Listeria monocytogenes (6, 7)
produce the antigenic alkylamines iso-butylamine
(IBA),3
iso-amylamine (IAA), and n-butylamine,
respectively, and these bacterial infections cause in vivo T
cell expansions in humans. Other bacteria such as Yersinia
enterocolitica and Escherichia coli, which produce the
antigenic alkylamines IBA and IAA (4), cause the expansion
of human T cells in vitro (8, 9). Clostridium
perfringens and Bacteroides fragilis secrete millimolar
concentrations of n-butylamine and n-propylamine
(10), which are antigenic alkylamines capable of expanding
T cells in vitro (1). The parasite Trichinella
pseudospiralis, which causes regressive and regenerative changes
in muscle tissue, produces large amounts of n-butylamine
(11).
T cells expand 3–7 days after microbial infection (5, 9, 12, 13). However, the antibacterial effect of human T
cells in vivo is evident in as few as 17 h postinfection,
indicating that expansion of T cells is not required for an
antibacterial response (36). Several lines of evidence
suggest that T cells participate in the immune response to
microbial pathogens by producing factors such as IFN- and TNF-,
by direct cell-to-cell contact leading to cytotoxicity
(14, 15, 16), or by producing granulysin and perforin
(17). In animal models, one of dominant mechanisms for
elimination of bacteria is dependent on monocyte-mediated killing,
which is up-regulated by T cell-derived IFN- (14, 15, 18).
T cells comprise only 2–5% of CD3+ cells
in human peripheral blood, but are much more efficient cytokine
producers on a per cell basis than T cells (19).
Release of IFN- and TNF- can be harmful and sometimes lethal to
the host (20, 21, 22, 23, 24). To maintain and mediate an effective
immune response while minimizing systemic immunopathology, cytokine
production should be precisely regulated. Production of IFN- and
TNF- in T cells is rapidly regulated by exposure to peptide
Ag (25). It is not known how human T cells are
regulated to produce those cytokines. Ideally, human T cells
should produce IFN- and TNF- at a very early time in
response to live bacterial products such as IBA or IAA, but not to dead
bacteria or LPS. Once bacteria are killed, T cells should cease
cytokine generation to reduce and avoid unnecessary immunopathological
reaction. Further, T cells should quickly resume their cytokine
production when bacterial infection occurs again. Polyclonal expansion
of T cells might be unnecessary in most circumstances such as in
subclinical bacterial infections.
In this study, we demonstrate that human T cells
produce IFN- and TNF- as early as 2 h after exposure to the
live bacterial product IBA, a nonpeptide alkylamine Ag, but not to dead
bacteria or LPS, in a dose- and time-dependent manner. T cells
began, ceased, and resumed IFN- and TNF- generation in an
on/off/on cycling pattern dependent on the presence or absence of IBA.
IFN- and TNF-, whose production was dependent on IBA-stimulated
T cells, were critical for monocyte-mediated killing of
extracellular bacteria. Thus, by limiting cytokine production to
periods of direct contact with live bacteria, T cells focus
their resources at the site of infection, while limiting systemic
immunopathology.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mAb ascites against T cell Ags used were as follows:
control mAb (P3), pan- TCR (anti-TCR1), V1 (A13),
V1/J1 (TCS1), V2 (BB3), V2 (7A5), and CD3 (OKT3). The
specificity of these mAbs is reviewed in Porcelli et al.
(26). Other reagents were purchased as follows:
FITC-conjugated F(ab')2 goat anti-mouse IgG
(catalog number AMI4708; BioSource International, Camarillo, CA); IBA
(catalog number I-3634; Sigma-Aldrich, St. Louis, MO); mouse
anti-human IFN- (catalog number 554698; BD PharMingen, San
Diego, CA); mouse anti-human TNF- (catalog number 18630D; BD
PharMingen); human TNF- and IFN- (recDNA; National Cancer
Institute, Bethesda, MD); and LPS (catalog L2880 from E.
coli serotype 055:B5; Sigma-Aldrich).
PBMC
Human PBMC obtained from random healthy donor leukopacks
(Dana-Farber Cancer Institute, Boston, MA) were isolated by
Ficoll-Hypaque centrifugation (Pharmacia, Peapack, NJ). PBMC were
screened for reactivity to V2V2 T cell Ags by culturing them at
one million cells per well in 24-well flat-bottom plates in RPMI 1640
medium containing 10% FBS, 2 mM glutamine, 1 nM 2-ME, and 100 IU of
penicillin and streptomycin in the presence or absence of IBA (0.4 mM)
at 37°C. On day 3, IL-2 was added to a final concentration of 0.5
nM and on days 10–12, the cells were counted and analyzed by
flow cytometry using TCR V gene-specific mAbs. Before expansion in
culture, the percentage of V2 T cells ranged from 1–5% of T cells.
PBMC were considered positive responders to IBA if they showed a 5- to
40-fold expansion of V2V2 T cells (around 95% of screened
donors) or produced intracellular IFN- and TNF- (100% of
screened donors). The screened PBMC were cryopreserved in FBS
containing 10% DMSO at -196°C until use.
Depletion of V2V2 T cells
Depletion of V2V2 T cells was performed using mouse
anti-human V2 Ab (BB3), or P3, an isotype-matched mock control,
and goat anti-mouse IgG Dynabeads M-450 (catalog number 110.06;
Dynal Biotech, Oslo, Norway) according to the manufacturer’s
instructions. For most depletions, P3, an isotype-matched control mAb,
was substituted for the anti-V2 mAb. Over 95% of V2 T cells,
confirmed by surface marker staining and analysis of flow cytometry,
were depleted. We screened several donors by two-color fluorescence and
found that 100% of V2-bearing T cells also expressed V2. Though
the V2 TCR chain paired with V1 or V3 have been described,
they are extremely rare, and there is no evidence that they respond to
nonpeptide Ags. Thus, the likelihood that we are studying a population
other than V2V2+ T cells is very
remote.
Stimulation of PBMC with IBA, dead bacteria, or LPS in vitro
Fresh or cryopreserved PBMC were cultured in RPMI 1640 medium in
the presence or absence of either 2 mM IBA, dead E. coli
(25922; American Type Culture Collection, Manassas, VA; inactivated at
56°C for 2 h, and diluted to final concentration of 5 x
105 CFU/ml), or LPS (final concentration of 1
µg/ml). The culture supernatant at the indicated time points was
collected for analysis of IFN- and TNF- levels by ELISA, and PBMC
were analyzed by three-color flow cytometry to quantitate percentage of
V2V2 T cells producing IFN- and TNF-.
Derivation of T cell clones
PBMC were isolated from 40 ml of freshly collected peripheral
blood by Ficoll-Hypaque centrifugation. One million PBMC were
stimulated with alkylamine Ags in a 24-well plate using RPMI 1640 with
10% FBS. Fresh media containing 1 nM of rIL-2 was added on days 3 and
7. Flow cytometry performed on day 10 showed that T cells
expanded to 70–80% of CD3+ cells. Culture was
continued for another 2 wk. The cultured PBMC (1 x
107 cells) were reacted with the mAbs BMA031 and
OKT4 to remove T cells and CD4+ T cells.
respectively, by magnetic bead selection (27). The cells
were cloned by limiting dilution in round-bottom 96-well plates in RPMI
1640 with 10% FBS, 1 nM rIL-2. and 1/2000 PHA-P. Irradiated (5 Gy)
allogeneic PBMC (1 x 105) and
EBV-transformed B cells (DG.EBV and CP.EBV each 2.5 x
104) were added to each well as feeder cells. The
clones were maintained by periodic restimulation with PHA. T cells
(1–2 x 105/per well) were cultured with
irradiated (5 Gy) allogeneic PBMC (5 x 105)
and EBV-transformed B cells (DG.EBV and CP.EBV each 2.5 x
105) as feeders and PHA-P (1/4000) in RPMI 1640
supplemented with rIL-2 (1–2 nM) in 24-well plates.
Cytokine release assays
Stimulation of the T cell clones was performed in a
96-well flat-bottom plate with 2 x 104
responder cells per well in 0.2 ml. Mitomycin C-treated C1R cells
(5 x 104/per well) were used as accessory
cells. Half log dilutions of IBA, IAA, or the calcium ionophore,
ionomycin (at 1 µg/ml), plus 10 ng/ml PMA (as a positive control)
were used (2). After a 24-h incubation, the supernatant
was harvested and used at a final dilution of 1/8 to stimulate the
growth of the IL-2-dependent HT-2 cell line. IL-2 release assays were
performed in triplicate using 5 x 103 HT-2
cells per well in 96-well flat-bottom plates. After 18 h of
culture, the HT-2 cells were pulsed with
[3H]thymidine (1 µCi/well) for 24 h, and
were then harvested and counted by liquid scintillation on a Betaplate
system. The standard deviation of the triplicate determination was less
then 10% of the mean. IFN- release was analyzed by ELISA (described
below) after 24 h of antigenic stimulation.
Detection of IFN- in tissue culture supernatant
Human IFN- ELISA was performed according to procedures
recommended by the manufacturer (catalog number 2613KI; BD PharMingen).
The detection limit of the assay was 4.7 pg/ml.
Intracellular cytokine staining
PBMC were cultured in RPMI 1640 medium in presence or absence of
IBA (1–2 mM), heat-killed E. coli, M. morganii,
or S. typhii (1 x 105–1 x
107 CFU), or LPS (10 ng to 1 µg). Four hours
before staining intracellular cytokine, monensin (catalog number
2092KZ; BD PharMingen) was added and cells were cultured at 37°C,
which enhanced intracellular cytokine accumulation. Cells were washed
with PBS and stained with surface marker AlexaFluor-conjugated 488 IgG
control Ab or pan-TCR1 and CD3+ (the Abs were
purified and conjugated by our laboratory). After two washes, cells
were fixed with 2% formaldehyde in PBS and permeabilized with 0.5%
(w/v) saponin (catalog number 554715; BD PharMingen). Intracellular
IFN- and TNF- were stained with PE-conjugated Ab (catalog numbers
18905A and 18645A; BD PharMingen) in saponin buffer. After two washes,
cells were resuspended in PBS and analyzed by use of FACS flow
cytometer collecting 200,000 events per sample (BD Biosciences). The
data were analyzed by Flowjo software (Tree Star, San Carlos,
CA).
SCID mice
All animal experimental protocols were approved by the Internal Review Board of Brigham and Women’s Hospital and Harvard Medical School. Homozygous C.B-17 scid/scid (SCID) male mice, 5–6 wk old, were purchased from Taconic Farms (Germantown, NY) and maintained in microisolator cages. Animals were fed autoclaved food and water. All manipulations were performed under laminar flow. One day before PBMC inoculation, SCID mice were injected (i.p.) with rabbit anti-asialo GM1 Ab (25 µl/mouse; WAKO, Richmond, VA), which depletes murine NK cells (28). SCID mice were irradiated (3 Gy gamma irradiation, 137Cs source) before PBMC engraftment, which allows a high level of functional engraftment of human PBMC (29).
Treatment of PBMC with V2V2 specific Ags, engraftment of SCID
mice, bacterial infection, and collection of engrafted PBMC
PBMC were cultivated in RPMI 1640 medium in the presence or absence of IBA (1 mM) for 18 h at 37°C, and all cells were collected and washed twice with RPMI 1640 medium. Groups of five SCID mice were injected with 0.5 ml (i.p.) of RPMI 1640 medium containing 3 x 107 human PBMC under aseptic conditions. E. coli (American Type Culture Collection 25922) was grown in Luria-Bertani (LB) broth at 37°C until the culture reached early stationary phase. E. coli was aliquoted (1 ml/vial) and stored in LB broth containing 10% glycerol at -80°C until use. Before infection, E. coli were washed once with 30 ml of PBS and plated on LB agar to determine CFU. Each SCID mouse was inoculated 0.5 ml (i.p.) PBS containing 5 x 106 CFU of live E. coli. Eighteen hours postinfection, PBMC were collected from the peritoneum with a 3-ml PBS lavage. The PBMC were washed three times with RPMI 1640 containing 100 IU/ml of penicillin and streptomycin, and 5 µg/ml of gentamicin. The intracellular cytokine staining was performed as described above.
Monocyte bactericidal assay (30)
After depletion or mock depletion of V2V2 T cells, human
PBMC (1 x 106 cells) in 1 ml of RPMI 1640
containing 1 mM IBA were cultivated in 24-well tissue culture
plates for 4 days in the presence or absence of anti-IFN- (1
µg/ml), anti-TNF- (1 µg/ml), IFN- (100 IU/ml), or TNF-
(100 IU/ml). The cells were washed three times with
phosphate-buffered balanced salt solution (BSS) and resuspended in 0.65
ml of BSS. Fifty microliters of ice-cold normal human serum and 0.3 ml
of E. coli (7.5 x 106 CFU/ml,
middle stationary phase) were added to each well. After 20 min of
cultivation at 37°C, cells were washed four to six times with 2 ml of
ice-cold BSS (Sorvall RT 6000D, 1000 rpm, 8 min). One plate was taken
as time zero and Triton X-100 (1.5% in PBS) was added to release
bacteria from the monocytes. Another plate was taken as time 90 min.
Nine hundred-fifty µl of BSS and 50 µl of normal human serum was
added to each well and was further incubated for 90 min at 37°C to
allow for monocyte killing of bacteria before adding Triton X-100.
Bacteria were diluted serially with water and was plated on LB agar and
cultivated overnight at 37°C. The number of monocytes in wells
treated with different conditions was the same, and the same number of
bacteria was added for each condition (30). The range of
bacterial counts after Triton X-100 treatment at the two time points
was 
800 CFU at time 0, and 20–400 at time 90. The percentage
of killing = [(test group T0 CFU - T90 CFU)/(mock-depletion
T0 CFU - T90 CFU)] x 100.
Statistics
Values are expressed as means ± SEM of the respective test or control group. Statistical significance between control and test groups was calculated by the Student’s t test (two-tailed) and among groups by analysis of variation. Data were representative of three to four experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells produced IFN- and TNF- in response to
the live bacterial product, IBA, but not to dead bacteria and LPS
To determine which bacterial component was responsible for
inducing cytokine production, we stimulated human T cells with
either IBA, heat-killed E. coli, heat-killed M.
morganii, or LPS for 18 h, and we then analyzed intracellular
IFN- and TNF- production in T cells using three-color flow
cytometry. Over 1,000 V2V2 T cells in each sample, collected from
200,000 events of gated lymphocytes, were analyzed. The experiments
were performed three times. None of the T cells produced IFN-
and TNF- in response to dead bacteria and LPS, whereas 15.5% of
T cells generated IFN- and 9% generated TNF- in response
to 2 mM IBA, a live bacterial product (Fig. 1
). IBA did not directly stimulate human
T cells, monocytes, NK cells, and NK T cells to produce IFN-
and TNF-, but LPS and dead bacteria did (data not shown). The dose
of IBA used in this experiment is below the 3.4 mM of concentration
detected in crude bacterial supernatant (1), suggesting
that similar stimulation of T cells might occur in vivo during
infection. T cells that responded to IBA stimulation and
produced IFN- and TNF- all expressed a TCR with markedly
restricted germline gene segment usage (V2V2) in this study (data
not shown).
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T cells to express the
activation markers CD25 and CD69 (16, 31), it is possible
that they work synergistically with IBA in cytokine production.
Interestingly, neither dead bacteria nor LPS augmented IBA-dependent
cytokine secretion (Fig. 1
), further demonstrating that T
cell-mediated production of IFN- and TNF- was strictly regulated
by the live bacterial product, IBA (Fig. 1).
Human V2V2 T cells produced IFN- and TNF- as early as
2 h after Ag exposure, showing a time- and dose-dependent pattern
The prognosis of infection is dependent on the speed of immune
system reaction and pathogen proliferation. Thus, early response to
live bacterial infection is critical for the immune system to eliminate
pathogens. To determine how quickly human T cells could generate
IFN- and TNF- after exposure to the live bacterial product, IBA,
we assessed intracellular cytokine production at different times after
stimulation with 2 mM IBA. About 1% of T cells began to produce
IFN- and TNF- as early as 2 h after Ag exposure, and this
increased to 6% at 18 h, showing a time-dependent (Fig. 2, a and b) and
dose-dependent response (Fig. 2c). A V2V2 T cell clone
derived by stimulation with IAA showed a similar dose-dependent
response curve to IBA and IAA in production of IL-2 and IFN- (Fig. 2, d and e). In contrast to recently stimulated
V2V2 T cells, most V2V2 T cell clones tested in this
study lost their capacity to secrete TNF- (data not shown),
suggesting that data interpretation should be cautious when
comparing the response of different T cell preparations.
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T cells produced detectable IFN- and
TNF- as early as 2 h after Ag exposure suggests that T
cells participate in the early innate immune response. The time- and
dose-dependent patterns of T cells in response to the live
bacterial products, IBA and IAA, suggest that amount of IFN- and
TNF- produced by T cells correlates with numbers of bacteria
and infective time. If exposed to IBA for 1 day, T cells did not
expand (Fig. 3), but they did produce
cytokines (Fig. 1). Continued stimulation for 10 days with IBA, but not
with dead bacteria (such as heat-killed E. coli, M.
morganii, or S. typhii) and LPS, caused T cell
expansion (Fig. 3). This expansion started at day 6 and maximized at
days 10–13 (data not shown). In addition, continuous IBA exposure
caused T cells to continually produce cytokines, with production
peaking at days 3 and 4 (data not shown). These data demonstrate that
both cytokine production and cellular expansion of T cells were
regulated by the live bacterial product, IBA, and that the immune
function of T cells correlates with the dose and duration of Ag
exposure.
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T cells show on/off/on cycling in generation of
IFN- and TNF-
Virus-specific CD8+ T cells show a
pattern of rapid on/off cycling in cytokine production that is
dependent on peptide MHC complexes (25). To determine
whether T cells could cease and resume production of IFN- and
TNF- dependent on the absence or presence of the live bacterial
product, IBA, we washed PBMC after 18 h of IBA stimulation,
cultured these IBA-primed PBMC in medium, and analyzed them by
intracellular cytokine staining. The number of T cells producing
cytokine declined by up to 70% after 2 h and became undetectable
by 6 h in the absence of IBA (Fig. 4a). When IBA stimulation was
restored, T cells resumed IFN- and TNF- production (Fig. 4b). V2V2 T cell viability after IBA re-exposure did
not change significantly. Because production of IFN- and TNF- was
abolished by the addition of actinomycin D (data not shown), T
cell-mediated production of these cytokines, as in T cells
(25), required de novo RNA synthesis. This regulation was
specific for T cells because IBA did not directly stimulate
T cells, NK cells, NK T cells, or monocytes to produce
cytokines. Thus, during a mild bacterial infection, V2V2 T cells
do not expand, but they do produce IFN- and TNF-, showing
"on/off/on" cycling of cytokine production.
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T
cells in an in vivo model. Human PBMC pretreated with 1 mM IBA for
18 h resulted in 8% of T cells producing IFN- and
TNF-. We then washed the PBMC, immediately reconstituted SCID mice
with these PBMC, and simultaneously infected the mice with 5 x
106 CFU of live E. coli. Eighteen
hours later, human PBMC were collected from the peritoneal lavage of
SCID mice for intracellular cytokine staining. The recovery rate of
human V2V2 T cells from hu-SCID mice, similar to whole PBMC, was
30–50%. Two-color analysis shows that 6.27 and 10.9% of human
T cells from SCID mice infected with the live E. coli
expressed intracellular IFN- and TNF-, respectively, whereas only
0.61 and 1.43% of T cells from mock-infected mice expressed
intracellular IFN- and TNF-, respectively (Fig. 5). These data suggest that T
cells cease cytokine production in the absence of stimulation in
vivo.
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and TNF- produced by T cells are critical for
monocyte-mediated killing of extracellular bacteria
Fresh PBMC containing an average of 20,000 V2V2 T cells that
were stimulated with nonpeptide alkylamine Ags secreted up to 700
µg/ml of IFN- or TNF-. IFN- and TNF- are critical for
monocyte-mediated killing of extracellular bacteria (14, 15, 18). To test the ability of human T cells to influence
monocyte-mediated killing of E. coli in vitro, mock-depleted
PBMC or those depleted of V2 T cells were cultured in medium
containing the V2V2 T cell-specific Ag, IBA, for 4 days.
Monocytes from these cultures were then analyzed for their ability to
kill E. coli. Monocytes from PBMC cultures that were mock
depleted of V2 T cells killed up to 3-fold more bacteria than
monocytes from the cultures depleted of V2 T cells. Inclusion of
neutralizing mAbs to either IFN- or TNF- during the 4-day culture
abrogated the V2 T cell-dependent monocyte-mediated killing of
E. coli, whereas adding back either IFN- or TNF- to
the cultures completely restored bacterial killing activity (Fig. 6). These data strongly suggest that
IFN- and TNF- produced by human T cells in response to
stimulation by the live bacterial product, IBA, are critical for
monocyte-mediated killing of extracellular bacteria.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2V2 T cells
continually secrete cytokines and expand later. However, cytokine
production ceases upon disappearance of the live bacterial product,
IBA, and resumes when antigenic contact is restored (Fig. 4), showing
on/off/on cycling. V2V2 T cells do not expand, but they do
produce IFN- and TNF- in response to less than 24 h of
stimulation with >125 µM IBA (
Figs. 1–3). These data indicate that
there exists a time and dose threshold for stimulation of V2V2 T
cells by live bacterial products. The significance of this finding has
not been appreciated, nor has the underlying mechanism been described.
We speculate that V2V2 T cells produce cytokines in response to a
mild bacterial infection and cease cytokine generation when bacteria
are killed; if the infection lasts longer and is more severe,
V2V2 T cells continue cytokine production and subsequently
expand. These expanded V2V2 T cells may then play an important
role in killing infiltrated macrophages to control inflammation
(32, 33).
It is well known that IFN- and TNF- are required for
monocyte-mediated bactericide, in particular, IFN-. These two
cytokines work synergistically in host resistance to bacterial
infection (34). Because many immune cells produce IFN-
and TNF-, depletion of V2 T cells substantially reduces but does
not completely deplete the total levels of IFN- or TNF-. In our
experiment (Fig. 6), the addition of either exogenous IFN- or
TNF- completely restored monocyte-mediated bactericide, reflecting
the synergistic effects of exogenous and endogenous TNF- and
IFN-.
This in vitro antibacterial effect has been recapitulated in an in vivo
hu-SCID model. T cells recovered from E.
coli-infected, but not uninfected, hu-SCID mice produced IFN-
and TNF- (Fig. 5). Hu-SCID mice receiving PBMC that were mock
depleted of T cells had significantly higher resistance to
bacterial challenge compared with those mice receiving PBMC depleted of
T cells. Whereas in vivo expansion of human T cells in
reconstituted, infected SCID mice did not occur until 7 days
postinfection, the antibacterial effect of human T cells in our
studies was evident in as few as 17 h postinfection for S.
aureus and 27 h for E. coli, indicating that
expansion of T cells is not required for an antibacterial
response. SCID mice reconstituted with mock-depleted human PBMC had
higher levels of serum human IFN- compared with mice reconstituted
with human PBMC depleted of V2 T cells. Further, there was a
remarkable inverse correlation between blood IFN- levels and
E. coli CFU in hu-SCID mice (36). Therefore, it
is likely that early antibacterial effects result from a
cytokine-dependent mechanism.
In contrast, cytokine release can be harmful and sometimes lethal to
the host (21, 22, 23, 24, 35). Cytokines are responsible for many
of the symptoms of viral and bacterial infection. Several cytokines, in
particular TNF- and IFN-, have been shown to be responsible for
pathological reactions, which may lead to shock and death observed
during infection with gram-negative bacteria and in response to
endotoxins (20).
The ability to turn cytokines on and off without cell expansion shows
the versatility of the cellular immune response and provides a
mechanism for maintaining and mediating an effective immune response
while reducing immunopathology. As cytokines are rapidly secreted,
continued production in the absence of Ag would lead to their
inappropriate release in noninfected tissues as activated V2V2 T
cells migrated through various organs or the bloodstream.
By maintaining cytokine production only upon continued IBA contact,
V2V2 T cells minimize the immunopathology that would occur if
these cytokines were secreted in a continual manner. Further, by
limiting cytokine production to periods of direct contact with infected
targets, V2V2 T cells focus their resources at the site of
infection.
V2V2 T cells, as an intact functional unit within PBMC, produced
cytokines in an on/off/on pattern in response to the live bacterial
product, IBA. It is not clear whether the same T cell clones that
ceased cytokine production subsequently resumed production on
re-exposure to Ag, but regardless, this pattern of cytokine secretion
consequently results in an immune response that is tightly controlled
by the presence or absence of Ag. Effector memory type T cells can
produce cytokines rapidly in response to Ag stimulation, whereas naive
or central memory T cells need several signaling steps for activation.
It is possible that only certain subpopulations of memory V2V2 T
cells produce cytokines in response to IBA stimulation, and further
studies are necessary to define these subpopulations.
Time and dose thresholds of V2V2 T cells in response to
alkylamines existed not only at high concentrations, but also at
physiological concentrations of IBA. V2V2 T cells cultured in low
doses of IBA over 13–14 days had up to 2-fold higher cellular
expansion in response to a subsequently high dose of IBA stimulation as
compared with those cultured in absence of IBA (our unpublished data).
Physiologically low concentrations of alkylamine Ags, which may be
derived from either plant foodstuffs or from commensal gut bacteria,
are ubiquitously found in human body fluids (1). These low
concentrations of alkylamines may prime T cells to respond to
the higher concentrations of secreted alkylamine Ags associated with a
bacterial infection, thereby leading to enhanced bacterial
resistance.
Depending on conditions, stimulation of fresh human PBMC with IBA in
vitro induced only 2–15% of V2V2 T cells to produce IFN- and
TNF-, compared with 3–50% of peptide-specific T cells
(25). However, these cells are much more efficient
at cytokine production on a per cell basis than T cells
(19). Further, these V2V2 T cells, reactive to
nonpeptide Ags, are 5- to 50-fold more numerous than T cells
specific for any one peptide, and may thus have a major physiologic
impact.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jack F. Bukowski, Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Smith Building, Room 526D, One Jimmy Fund Way, Boston, MA 02115. E-mail address: jbukowski{at}rics.bwh.harvard.edu
3 Abbreviations used in this paper: IBA, iso-butylamine; IAA, iso-amylamine; LB, Luria-Bertani; BSS, balanced salt solution.
Received for publication June 26, 2001. Accepted for publication September 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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