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T Cells by TNF-
1
*
National Jewish Medical and Research Center, Denver, CO 80206;
Division of Biostatistics and
Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80262;
§
Department of Microbiology, Oregon State University, Corvallis, OR 97331; and
¶
Littman-Hart Information Division, Englewood, CO 80111
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells influence
both early innate and Ag-specific adaptive host responses, it has
remained unclear what triggers T cell reactivity. Investigating
very early T cell activation in mouse and human models of bacterial
infection, we measured CD69 expression as an indicator of early
cellular activation. Both murine 
ß and T cells responded
polyclonally to systemic bacterial infections, and to LPS. However,
T cells responded more strongly to the bacteria and to LPS. In
vitro LPS-stimulated human T cells showed a similar differential
response pattern. We identified TNF- as mediator of the early
differential T cell activation, and of differential proliferative
responses. The stronger response of T cells to TNF- was
correlated with higher inducible expression levels of TNF-Rp75.
Among unstimulated splenocytes, more T cells than ß T cells
expressed CD44 at high levels. The data suggest that TNF-Rp75
determines the differential T cell reactivity, and that most T
cells in the normal spleen are present in a presensitized state. As
TNF- stimulates activated T cells, it may early preferentially
connect T cell functions with those of cells that produce this
cytokine, including activated innate effector cells and Ag-stimulated T
lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells
have been
observed to proliferate and produce cytokines in many diseases (1). In
addition, studies in animal models suggest that these cells contribute
to host resistance
against infections (2), and that they influence inflammation (3),
epithelial regeneration (4), and mucosal tolerance to Ags (5, 6).
However, it has remained unclear what triggers T cell
reactivity, and to what extent T cell-activating stimuli differ
from those of ß T cells and B lymphocytes. For example, although
T cells respond during bacterial and viral infections, they have
not been firmly linked to Ag-specific adaptive immunity, and the
natural ligands for TCRs are still a matter of conjecture (7).
On the other hand, although T cell reactivity tends to be
associated with inflammation, it is not limited to early stages of the
host response to infection or injury. For instance, a study of murine
influenza A infection suggested that T cell functions in this
disease are focused not on the elimination of the virus or
virus-infected cells, but rather on the regulation of the host
response, because certain T cells in the lung expanded only
after the virus had been cleared completely (8). This response depended
upon a preceding response by ß T cells, and was concomitant with
the resolution of pulmonary inflammation (9). Additionally, in two
mouse models of infection with the facultative intracellular bacterium
Listeria monocytogenes, depletion of T cells resulted
in prolonged and exacerbated inflammation of the target organs, which
underwent extensive tissue destruction (3, 10, 11). Depletion of ß
T cells did not have the same consequences, despite comparable or
increased bacterial loads. Similar findings were also recently reported
in a mouse model of lung infection with Mycobacterium
tuberculosis (12). Whether T cell reactivity in these
infections is directly dependent on Ag recognition by these cells, or
instead is merely driven by the innate and adaptive host responses to
the bacteria, or both, has not been resolved. Although stimulation of
T cells by bacterial components has been well documented (1),
the exacerbating effects of T cell depletion in a mouse model of
collagen-induced arthritis (13), and during infection-induced
autoimmune orchitis (11), suggest that regulatory T cell
responses can also be triggered during inflammation in the absence of
pathogen-derived Ags.
Particularly strong T cell responses have been noted after
infection of mice with certain Gram-negative bacteria, including
Escherichia coli and Salmonella strains (14, 15, 16).
Whether T cells contribute to host protection against these
pathogens is presently controversial and probably depends on the
particular model studied (17). However, since T cells can also
be stimulated by LPS (18, 19, 20), it seemed likely that they would
contribute to host responses during Gram-negative sepsis. We have
therefore examined T cell responses during bacterial sepsis or
after inoculation of LPS. To define unique requirements for T
cell activation, we have compared the concurrent and ß T
cell responses under these conditions, using induction of the C-type
lectin receptor family member CD69 as an early activation marker (21),
a decrease of TNF-R expression as indicator of a reaction with TNF-
(22), CD44 expression as an indicator of prior sensitization, and cell
proliferation as a late activation marker. We found that T cells
responded more strongly to two types of systemic bacterial infection
and to LPS than did ß T cells. After stimulation with LPS in vivo
or in vitro, early activation of T cells was largely dependent
on TNF-, whereas unstimulated ß T cells showed little response
to this cytokine. Among T cells, early activation was more
prominent within the CD44high subset. TNF- also
preferentially costimulated TCR-dependent proliferative responses of
T cells. The differential T cell sensitivity to LPS and TNF-
was strictly dependent on TNF-R expression, and correlated with higher
levels of TNF-Rp75 expressed by T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/10, C57BL/6, C3H/HeN, and C3H/HeJ mice were purchased from
The Jackson Laboratory (Bar Harbor, ME) and cared for in the animal
facility at National Jewish Medical and Research Center (Denver, CO).
Mice genetically deficient for B cells, µMT-/-
(23), IFN- (24), and the -chain and ß-chains of the TCR (25, 26) were cared for according to guidelines for immunodeficient animals.
Mice genetically deficient for TNF-Rp75 (27) and TNF-Rp55 (28) as well
as for both TNF-Rp75/p55 were a gift from David Lynch (Immunex,
Seattle, WA).
Bacteria and reagents
E. coli strain 1677, a uropathogenic clinical isolate
with serotype O6 that expresses type 1 and p fimbriae and produces
hemolysin (kindly provided by Jessica Jones-Carson, National Jewish
Medical and Research Center, Denver, CO), was used for Gram-negative
infection (29). Before i.v. inoculation into mice, a fresh culture of
E. coli was grown from frozen stocks in thiamine (T-4625;
Sigma, St. Louis, MO)-enriched tryptose broth (Difco, Detroit, MI) for
48 h, followed by a second 24-h incubation in fresh tryptose
broth. E. coli was recovered from the broth culture by
centrifugation, washed in sterile PBS, and resuspended at a
concentration of 2 x 108 CFU/10 ml of sterile PBS.
For Gram-positive infection, L. monocytogenes strain EGD
(kindly provided by Priscilla Campbell, National Jewish Medical and
Research Center) was maintained by serial passages in BALB/c mice, and
expanded in vitro before i.v. injection. In both bacterial infection
models, 1 x 108 CFU (live bacteria) was injected. LPS
from E. coli strain 055:055 (L-9023; Sigma),
Salmonella minnesota (L-2137; Sigma), lipid A diphosphoryl
from S. minnesota RE-595 (L-0774; Sigma), and detoxified LPS
of S. minnesota (L-1523; Sigma) were purchased. LPS from
E. coli strain 124 was a gift from Klaus Jann (MPI-Freiburg,
Freiburg, Germany). Endotoxin-free 0.9% NaCl was purchased from Abbott
Laboratories (Chicago, IL). Murine rTNF-
(rmTNF-)3 was purchased
from R&D (410-MT; Minneapolis, MN), and rat IgG (I-4131) from
Sigma.
Antibodies
Anti-murine TCR-ß mAb H57-597 has been described previously
(30). Anti-murine TCR- mAb (GL3) was a gift from Leo Lefrancois
(University of Connecticut Health Center, Farmington, CT) (31).
Anti-murine TCR-V1 mAb (2.11) was a gift from Pablo Pereira and
provided by Roger Sciammas (Ben May Institute for Cancer Research,
University of Chicago, Chicago, IL) (32). Anti-murine CD3
mAb (KT3)
was a gift from Kyuhei Tomonari (Transplantation Biology Section,
Middlesex, U.K.) (33), and the anti-murine CD69 mAb (H1.2F3) was a
gift from Ethan Shevach (National Institutes of Health, Bethesda, MD)
(34). Anti-murine TNF- mAb (MP6XT22) was a gift from DNAX (Palo
Alto, CA), and anti-murine FcR mAb 2.4G2 was purchased from
American Type Culture Collection (ATCC HB 197; Rockville, MD). mAbs
were prepared from Ab-secreting hybridoma cell lines by purification
from either cell culture supernatants or ascites, and quantified.
FITC-conjugated anti-murine TCR-V4 mAb (UC3-10A6);
FITC-conjugated anti-murine CD69 mAb (H1.2F3); and mAbs with
specificities for CD4 (mAb GK1.5), CD8 (mAb 54-6.7), and CD44 (mAb IM7)
were purchased from PharMingen (San Diego, CA). Biotin-conjugated
anti-murine TNF-Rp55 mAb (HM104) and anti-murine TNF-Rp75 mAb
(HM102) were a gift from Bob Johnson (Caltag Laboratories,
Burlingame, CA).
T cell purification
Spleens were carefully teased apart, RBC lysed with Gey’s solution, and lysis-resistant splenocytes passed through nylon wool columns to yield an enriched T cell preparation containing >70% CD3+ cells (35). After purification, cells were either analyzed directly or placed in tissue culture for in vitro stimulation.
For in vivo stimulated cells, indicated periods of stimulation refer to the time span between injection of the stimulus and the isolation of the tissue. When cells were nylon wool purified before Ab staining, CD69 expression continued to increase ex vivo, so that purified cells overall expressed CD69 at somewhat higher levels.
Culture media
Powdered Iscove’s modified Dulbecco’s medium (IMDM) was dissolved in sterile water according to manufacturer’s instructions (I-7633; Sigma). A mixture of additional growth factors was added to IMDM. The mixture was made up in 1 L of IMDM containing 7.5 g dextrose (G-7021; Sigma), 75 ml of a 50x essential amino acids solution (320-1130AH; Life Technologies, Gaithersburg, MD), 140 ml of a 100x nonessential amino acids solution (320-1140G; Life Technologies), 100 ml of a 100x sodium pyruvate solution (320-1360AG; Life Technologies), 8.5 g of sodium bicarbonate (S-5761; Sigma), 500 mg of gentamicin (G-3632; Sigma), 600 mg of penicillin G (860-1830 MJ; Life Technologies), 1 g of streptomycin sulfate (S-9137; Sigma), and 34 µl of 2-ME (4196; Kodak, Rochester, NY). The standard medium (culture medium of IMDM plus 10% FBS (ICTM)) was made up fresh with 9% (v/v) of the mixture, 10% FCS, and 81% IMDM.
Flow microfluorimetry (FMF)
For FMF, mAbs were conjugated with
N-hydroxysuccinimido-biotin (H-1759; Sigma) or FITC isomer I
on Celite (F-1628; Sigma). Then, 1 to 2 x 106 cells
in 96-well plates (Falcon; Becton Dickinson, Franklin Lakes, NJ) were
stained by using one- or two-color techniques and analyzed
cytofluorographically on XL (Coulter, Miami, FL) counting 150,000
events per gated region (forward and side scatter gated to exclude dead
cells, debris, and cellular aggregates) for freshly prepared cells.
Additional gates were included to identify or
TCR-ß-positive cells. For in vitro cultured T cells, 50,000 events
per gated region were collected. For each of the gated populations, the
mean fluorescence intensity (MFI) of CD69+ or
TNF-R+ cells was determined as a response parameter.
TNF-R-expressing mouse fibrosarcomas WEHI 164 (ATCC 1751-CRL) and L929
were used to establish optimal staining dilution for FMF analysis for
biotin-conjugated mAbs against murine TNF-Rs. Briefly, we used
streptavidin-phycoerythrin (diluted at 1/100 per 1 x
106 cells, SNN1007; Tago Immunologics Biosource, Camarillo,
CA) for detection of the biotin-conjugated Abs, including appropriate
controls (as suggested in 73 .
In vitro culture
Nylon wool-purified T cells from the spleen were seeded at a
concentration of 5 x 106 cells/ml of standard
medium in 24-well plate (Falcon). To the appropriate well, the
following stimuli were added, as indicated in the figure legends: 100
ng rmTNF- (410-MT; R&D), rmTNF- plus 25 µg anti-TNF
(MP6XT22), as an isotype control 25 µg/ml rat IgG (I-4131; Sigma), 5
µg/ml Con A from Canavalia ensiformis type IV-S (C-5275;
Sigma), and plate-bound anti-CD3 (precoated at 10 µg/ml).
After the indicated incubation periods, cells were washed and stained
for CD69 and TNF-R expression. Statistical analysis of CD69 expression
in cultured cells was based on two-way analysis of variance of multiple
experiments with individual linear contrast. For two paired groups,
Student’s t or Sign tests were used. Correlations between
CD69 induction and TNF-Rp75 reduction were analyzed using Pearson’s
r correlation coefficient.
Proliferation assay
Nylon-wool purified T cells isolated from mouse spleen were
seeded at a concentration of 1 x 105 cells/well
in 100 µl of ICTM in triplicate using flat-bottom 96-well plates
(Falcon) at 37°C for 48 h in a 10% CO2 atmosphere.
During the last 16 h, cells were pulsed with 0.5 µCi/well of
[3H]thymidine (NET027; NEN/Life Science, Boston, MA), and
the incorporated radioactivity was measured using a MicroBeta 1450
counter (Wallac Oy, Turku, Finland), following the manufacturer’s
instructions. Proliferation was measured either in the absence or in
the presence of the following stimuli: plate-bound anti-CD3 Ab
(10 µg/ml), plate-bound anti-CD69 Ab (10 µg/ml), combination of
plate-bound anti-CD3 and anti-CD69 in a ratio of 1:1,
rmTNF- (100 ng), rmTNF- in combination with plate-bound mAb
anti-CD3 and/or anti-CD69. Statistical analyses were
performed on all three independent experiments using two-way analysis
of variance with individual linear contrast.
Analysis of human cells
Peripheral blood was collected from 11 different healthy
subjects. Whole blood was cultured for 3 h under the following
conditions: no stimulation, 1 µg LPS from E. coli 026:B6
(L-3755; Sigma) per 1 x 106 cells, or 10 ng human
rTNF- (210-TA-010; R&D) per 1 x 106 cells. After
the culture, whole blood was stained with Abs following the procedures
outlined by Becton Dickinson Sourcebook for Monoclonal
Antibodies ("Direct immunofluorescence staining of cell surface
antigens in unseparated blood," 1989; San Jose, CA). Stained cells
were fixed with FACS lysing (349202; Becton Dickinson). Staining
reagents included phycoerythrin-conjugated anti-human CD69 (clone
L78, 347823; Becton Dickinson), PerCP-conjugated anti-human CD45
(clone 2D1, 347464; Becton Dickinson), FITC-conjugated anti-human
TCR-ß (clone BW242/412; T Cell Sciences, Inc., Cambridge, MA), and
FITC-conjugated anti-human TCR- (clone 5A6.E9; T Cell
Sciences, Inc.). To examine TNF-R expression, lymphocytes were isolated
from peripheral blood samples by centrifugation on Ficoll-Hypaque
(Pharmacia, Piscataway, NJ) density gradients. Lymphocytes were then
cultured either in the presence of 10 ng human rTNF- (210-TA-010;
R&D) per 1 x 106 cells or in its absence for 3
h. Cells were then stained with biotin-conjugated anti-human TNF-R
Ab specific for either TNF-Rp75 (clone 4D1B10 (MR2-1); Caltag
Laboratories) or TNF-Rp55 (clone 2H10 (MR1-2); Caltag Laboratories).
Both Abs were a generous gift by Bob Johnson (Caltag Laboratories).
Cells were cytofluorographically analyzed using a FACSCalibur flow
cytometer (Becton Dickinson). When whole blood samples were examined,
10,000 gated events were counted, essentially as described for the
murine cells. To assess TNF-R expression, 30,000 gated events were
analyzed. MFI was used as a parameter to assess shifts in CD69 and
TNF-R expression in both T cell subsets, using three-color
analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß T cells in systemic
bacterial infections
To compare responses of murine and ß T cell
populations after systemic infection with large numbers of bacteria, we
monitored the CD69 cell surface molecule, a C-type lectin known to be
expressed very early in T cell activation (36, 37). L.
monocytogenes, a Gram-positive bacterium eliciting both ß and
T cell responses (2, 38, 39, 40), caused moderate increases in CD69
expression on splenic T cells 2 h after i.v. injections of
108 CFU. T cells expressed CD69 at approximately
twofold higher levels than ß T cells (Fig. 1
, A and B).
Injection of the same number of E. coli induced a far
stronger CD69 response in both types of T cells (Fig. 1, C
and D). Nevertheless, T cells still expressed
infection-induced CD69 at approximately 2.5-fold higher levels than
ß T cells. This differential expression of CD69 was early and
transient, since at later time points (e.g., 6 h),
infection-induced CD69 expression of both T cell subsets continued to
increase, with the difference between and ß T cells
diminishing (not shown). Note: Unless indicated otherwise, staining
experiments were conducted with nylon wool-purified cells. During this
procedure, CD69 expression somewhat increased ex vivo, accelerating the
kinetics shown by 30 to 45 min.
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and ß T cells by LPS
Since Gram-negative E. coli induced stronger responses
than Gram-positive L. monocytogenes, we next examined
whether LPS, a cell wall component of Gram-negative bacteria, also
induced CD69 expression. In this experiment, we have followed
LPS-induced CD69 expression for up to 48 h. For most of this time,
and ß T cells expressed CD69 at similar levels (data not
shown). However, very shortly after LPS inoculation, we found
differential CD69 expression in the two T cell populations (Fig. 2). We compared response kinetics of CD69
expression on both T cell types using a fixed high dose of LPS (10
µg/mouse), and their dose-response relationships at a fixed time
point (1 h). T cells responded more rapidly and strongly to the
fixed amount of 10 µg of LPS, and perhaps more importantly, also
exhibited greater sensitivity to low doses of LPS.
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and ß T cells in
induced CD69 expression was also evident in additional inbred mouse
strains tested (including C57BL/6, BALB/c, and C3H/HeN), as well as in
mutant mice lacking B lymphocytes (µMT-/-),
either one of the T cell subsets (TCR-ß-/- or
TCR--/- mice), or IFN-
(IFN--/- mice) (not shown). It was also evident with
LPS derived from different bacterial strains, including E.
coli strains 124 and 055:055, and S. minnesota, as well
as with lipid A from S. minnesota (not shown). Neither T
cell subset responded to detoxified polysaccharide from S.
minnesota. Finally, we examined possible effects of the route of
LPS administration (not shown). Intraperitoneal inoculation delayed
overall response kinetics in the spleen by approximately 2 h as
compared with i.v. inoculation but did not alter the differential
reactivity of the two T cell subsets.
Although T cells are scattered throughout the spleen, many are
found adjacent to sinusoids (41). Because it seemed possible that the
two T cell subsets might therefore be exposed differently to the
injected LPS, we examined the responses of dissociated, in vitro
stimulated splenocytes (Fig. 3A). Again, T
cells expressed LPS-induced CD69 at nearly twofold higher levels than
did ß T cells, indicating that the different responses of
and ß T cells did not depend on their tissue location. Since
differential activation could be based on direct or indirect
stimulation with LPS, we also tested whether purified T cells could
respond to this stimulus (Fig. 3B). However, in the
absence of non-T splenocytes, LPS induction of CD69 expression was
virtually absent in both types of T cells, emphasizing the dependence
of this early T cell response on non-T accessory cells.
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and ß T cells to TNF-
The dependence of the differential T cell response to LPS on
accessory cells suggested an indirect mechanism of T cell stimulation,
perhaps mediated through cytokines. Because LPS is a particularly
strong stimulator of TNF- production (42), we tested whether a
TNF--neutralizing Ab (MP6XT22) could inhibit in vitro LPS-induced
CD69 expression (Fig. 3
). This Ab partially inhibited LPS-induced CD69
expression in T cells, whereas isotype-matched nonrelevant Abs
had no significant effect. In ß T cells, LPS-induced CD69
expression was too weak at this time point to ascertain Ab inhibition.
We therefore performed two alternative experiments. First, we examined
the effect of adding rmTNF- to purified splenic T cells in vitro to
test its potential for inducing differential CD69 expression (Fig. 4A and Table I). Treatment with rmTNF- increased
CD69 expression in T cells 1.5- to 2-fold within 3 h of in
vitro stimulation, but caused much smaller increases in ß T cells.
Maximal induction of CD69 expression was reached at cytokine
concentrations of 10 to 100 ng/ml. The differential response was
abolished in the presence of the TNF--neutralizing mAb, but not with
the isotype-matched nonrelevant Ab. These data showed that TNF-
indeed could provide a differential stimulus. To address the question
of whether TNF- was a mediator of the differential LPS stimulation
in vivo, we injected mice genetically deficient in receptors for
TNF- (TNF-Rp55-/-, TNF-Rp75-/-, and
TNF-Rp55-/-/p75-/- (double knockout) mice)
with LPS and measured the induction of CD69 expression. One hour after
LPS inoculation (Fig. 5A), LPS-induced CD69
expression of T cells was comparatively reduced in all of the
TNF-R-deficient mice tested. This effect was most prominent in mice
genetically deficient for both TNF-Rp75 and TNF-Rp55, but was also
detectable with mice lacking only one TNF-R. This confirmed TNF- as
an early mediator of the differential T cell response after LPS
stimulation in vivo. At 1.5 h after LPS inoculation, CD69 levels
began to rise also in the TNF-R-deficient mice, suggestive of a
secondary mechanism of LPS stimulation (Fig. 5B).
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as an early mediator of differential CD69
induction in T cells, these experiments did not address whether the
larger increase of CD69 expression in T cells was due to greater
sensitivity for TNF-, or to a different regulation of CD69
expression, i.e., an inherent ability of T cells to express CD69
at higher levels than ß T cells. We thought the latter to be
unlikely because at later time points (>12 h) after LPS stimulation,
both types of T cells reach equally high surface levels of CD69 (not
shown). In addition, we examined the mitogen Con A for its ability to
induce CD69 expression in the two types of T cells (Fig. 6). As before, stimulation with TNF-
resulted in a twofold increase of CD69 expression in T cells,
whereas ß T cells showed little effect. In contrast, the mitogen
induced CD69 expression in both types of T cells, with larger increases
of CD69 in ß T cells. This result favors differential sensitivity
to TNF- as the reason for the differential CD69 induction in T
cells.
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and ß T cells
directly after their isolation from normal or LPS-stimulated mice, and
after in vitro culture. Cells freshly isolated from 11 normal C57BL/10
mice exhibited variable levels of TNF-R, although with one exception,
T cells always expressed TNF-Rp75 at higher levels than did
ß T cells. The average MFI (mean value +/- SD) of unstimulated
T cells was 1.39 +/- 0.65, and of unstimulated ß T cells,
1.02 +/- 0.33. Pairwise comparison of ß and T cells in
individual animals showed that the higher expression of TNF-Rp75 in
T cells was a significant difference (Sign test,
p < 0.05). This was also evident in LPS-stimulated
mice (p < 0.01), although the levels of
TNF-Rp75 on the cells from these mice did not change significantly.
With regard to TNF-Rp55 expression, no difference was detectable
between the two types of T cells in either normal or LPS-stimulated
mice. After 3 h in unstimulated cultures, TNF-Rp75 was expressed
at increased levels compared with freshly isolated cells (Fig. 4B and Table I), with a further increased difference between
and ß T cells (approximately threefold higher on T
cells). In contrast, TNF-Rp55 was expressed on both types of T cells at
approximately the same levels. Addition of rmTNF- to these cultures
led to a reduction of TNF-Rp75 expression in both T cell subsets, a
predicted effect shown by others to be correlated with TNF--mediated
stimulation (43, 44). TNF--mediated reduction of TNF-Rp75 levels was
reversed by the TNF--neutralizing mAb, but not by an isotype-matched
nonrelevant Ab. Increases in CD69 expression by T cells
correlated with decreases in TNF-Rp75 expression (Table I, Pearson’s
linear correlation coefficient r = -0.995;
p < 0.06). Since T cells initially expressed
TNF-Rp75 at higher levels than ß T cells, this correlation between
activation and TNF-R decrease suggested that the greater sensitivity of
T cells for TNF- is based on higher levels of TNF-Rp75.
Consistently, we found that T cells derived from
TNF-Rp75-/- mice failed to up-regulate CD69 after
stimulation with TNF- in vitro (not shown).
Differential effect of TNF- on TCR-dependent and ß T
cell responses
To test whether the differential activation of and ß T
cells with TNF- has consequences for TCR-dependent responses (Table II), we stimulated both types of T cells
with plate-bound anti-CD3 mAb, using T cell preparations from
the normal spleens of mice genetically deficient for TCR-ß or
TCR-. Anti-CD3 mAb as a substitute for cognate TCR ligands was
chosen with the assumption that the immediate consequences of CD3
cross-linking are similar for and ß T cells. Within 48
h, both types of T cells were induced to proliferate by this treatment,
although enriched ß T cells showed a comparatively stronger
proliferative response to CD3 cross-linking. When exposed to TNF- at
the beginning of the culture period in addition to plate-bound
anti-CD3 mAbs, both types of T cells showed an enhanced
proliferative response. However, the effect on T cells was
stronger than that on ß T cells. We next examined the functional
significance of CD69 expression in the two types of T cells, by
cross-linking surface-expressed CD69 with plate-bound anti-CD69 mAb
(21). In contrast to anti-CD3 mAb, plate-bound anti-CD69 mAb
alone did not induce proliferation in either type of T cell (Table II),
nor did the combination of anti-CD69 mAb and TNF- have any
effect. A combination of anti-CD69 and anti-CD3 mAbs resulted
in only a small increase in proliferation in both types of T cells.
Most clearly, however, T cells stimulated with anti-CD3 mAb,
or both anti-CD3 and anti-CD69 mAbs, showed large increases of
proliferation when also in the presence of TNF- (>sixfold increase
over anti-CD3-stimulated and >fivefold increase over anti-CD3
plus anti-CD69-stimulated responses), whereas only small increases
were seen with ß T cells (2.7- and 1.6-fold, respectively). Note
that fold increases in Table II are all calculated in reference to
anti-CD3-stimulated responses.
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and ß T cells with LPS
and TNF-
We also examined responses of T cells in whole human blood to LPS
and TNF- (Table III; note that
cytofluorometry with human cells was conducted with a different flow
cytometer, resulting in different MFIs). Unlike the murine splenic T
cells, unstimulated human blood T cells showed quite variable
background levels of CD69 expression. Similarly to mice, however,
unstimulated T cells of most normal healthy individuals tested
(9 of 11) already expressed somewhat higher CD69 levels than ß T
cells. Stimulation with LPS induced substantial increases of CD69
expression in both and ß T cells. After stimulation,
T cells expressed CD69 at higher levels than ß T cells in 10 of 11
individuals tested. Due to the higher background levels of CD69
expression in T cells, the fold increase among ß T cells
was sometimes larger, however. Human T cells were also tested after
stimulation with TNF-. For T cells, TNF- was a strong
stimulus, inducing CD69 to expression levels almost as high as with
LPS. In contrast, and like the murine cells, human ß T cells
responded only weakly to TNF-. In addition, we have
measured TNF-R expression on T cells of one individual (ML), after
isolating PBL on Ficoll gradients. As with murine T cells, TNF-Rp75 was
expressed predominantly on T cells (not shown). Thus, human and
mouse and ß T cell populations resembled each other in
their differential responses to LPS and TNF- as well as their
differential expression of TNF-Rp75.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells respond more readily to TNF- than do ß T cells. In
vitro, unstimulated T cells were induced by this cytokine to
express the very early activation Ag CD69. In contrast, unstimulated
ß T cells hardly responded at all. In vivo, genetic TNF-R
deficiencies had a strong impact on the ability to induce CD69
expression on T cells. Importantly, not only murine but also
human T cells in general exhibited stronger responses to
TNF- than did ß T cells.
For some time, TNF- has been known to enhance Ag- or mitogen-induced
T cell proliferation and cytokine production. However, only activated T
cells were found to be susceptible to TNF- costimulation (45, 46),
presumably because of an absence of TNF-R on resting ß T cells. As
in these earlier studies, we did not find significant effects of
TNF- alone on the proliferation of unstimulated T cells in vitro,
for either ß or T cells. Nevertheless, unstimulated
T cells, but not ß T cells, readily responded to TNF- by
up-regulating cell surface expression of the very early activation Ag
CD69. Measuring [3H]TdR incorporation as indicator
of proliferative responses, T cells were also more susceptible
to TNF- costimulation when simultaneously activated through the TCR
(by plate-bound anti-CD3 mAbs), or through the TCR and CD69
together (by additional cross-linking anti-CD69 mAbs). Whether the
later effects on proliferation are merely a reflection of the early
differential response or indicate, in addition, late differential
reactivity with TNF- remains to be seen. In either case, our data
justify the conclusion that T cells respond at early time points
more strongly to TNF-. In addition, they might indicate that
T cells, unlike ß T cells, do not require additional activation to
become susceptible to TNF-, or alternatively, that T cells
are already in a sensitized state. Consistently with this second
possibility, a higher fraction of T cells than of ß T cells
expressed CD44 at high levels, directly after their isolation from
normal mice (Fig. 7). High levels of CD44
have been associated with lymphocyte activation (47), and upon in vivo
stimulation with LPS, CD69 surface expression was preferentially
induced on CD44high ß T cells (48). We show in this
study that the same is true for T cells. Although the procedures
of isolation may have caused differential T cell activation, an
intriguing alternative is that T cells, unlike ß T cells,
normally exist in a state of activation, or are activated more
easily.
|
T cells is the
second novel finding of this study. but not ß T cells
markedly increased their expression of TNF-Rp75 when briefly cultured
in vitro. In their preferential expression of TNF-Rp75, T cells
resemble B-lymphoid and myeloid cells (49, 50). Whether the increased
expression of TNF-Rp75 in T cells is due to active stimulation
or merely derepression is still unclear. Others have reported that
prolonged culture (several days) of human T cells in the
presence of IL-12 results in increased TNF-Rp75 expression (51). In
contrast, we have observed highest levels of TNF-Rp75 within 3 h
after unstimulated in vitro culture. IL-12 is among the first cytokines
to be produced in inflammation (52). Whether this cytokine could cause
the very rapid increase in TNF-Rp75 expression seen with murine
T cells shortly after they have been placed into culture remains to be
addressed.
TNF-Rp75 exhibits greater specificity and higher affinity in its
interactions with TNF- than does TNF-Rp55 (53). It has been proposed
that at low concentrations of TNF, TNF-Rp75 functions as a catcher by
binding TNF and delivering it to TNF-Rp55 (54). Moreover, unlike
TNF-Rp55, TNF-Rp75 seems to function in transmitting activating
signals, but not signals for apoptosis (53, 54, 55, 56, 57). Although our data have
only provided a correlation between TNF-Rp75 expression and
responsiveness to TNF-, it thus is reasonable to assume that the
stronger response of T cells to this cytokine is related to
their higher initial levels of TNF-Rp75 expression. Conceivably, the
stronger T cell response as far as CD69 is concerned could also
be due to differential regulation of CD69 expression in these cells.
This possibility seems less likely for two reasons. First, not all
stimuli applied induced higher levels of CD69 expression in T
cells. Thus, we found that the mitogen Con A induced larger increases
in CD69 expression in ß T cells. Second, at later time points
after stimulation with LPS (i.e., >12 h), the two types of T cells
expressed CD69 at equally increased levels (not shown). Others have
demonstrated TNF--induced CD69 expression (58) and have found
TNF--responsive elements upstream of the CD69 promoter region. In
fact, TNF- appears to activate the promoter of CD69 via the nuclear
factor-
B/Rel family members c-Rel and RelA (59). A direct
relationship between differential TNF-R expression, responsiveness to
TNF-, and CD69 induction therefore seems to be the most probable
mechanism underlying the stronger T cell response.
TNF- is produced in response to numerous stimuli and by a variety of
cells. In endotoxemia, TNF- is one of the first cytokines to appear
in the bloodstream, followed by IL-1 and IL-6 (60, 61). Activated
macrophages stimulated by LPS are considered among the largest sources
(42). For this reason, we examined TNF- as a candidate mediator of
the differential T cell response found after LPS stimulation. The
arguments of this study in favor of TNF- as the major mediator of
LPS-induced early T cell activation can be summarized as
follows: first, both ß and T cells showed little, if any,
activation by LPS in vitro in the absence of accessory cells. With
regard to ß T cells, this result was predicted by earlier studies
(62, 63). Second, TNF-, like LPS, induced differential responses of
and ß T cells, as indicated by higher induced levels of
CD69 expression in T cells. Third, early after LPS injection,
induced CD69 expression was much diminished in genetically
TNF-R-deficient mice, and in fact was practically nonexistent when both
TNF-R were absent. These findings are most easily explained if TNF-
is a major mediator of the early differential T cell response to LPS.
As might be expected from the differential expression of TNF-Rp75 in
the two types of T cells, mice deficient in this receptor also
exhibited the largest loss of CD69 expression. However, the experiments
with genetically TNF-R-deficient mice suggested that TNF-Rp55 is also
required for full activation of T cells.
The role of TNF- as a major mediator of the differential T cell
response to LPS appears to be early and transient, however. By 90 min
following LPS inoculation, T cells from TNF-R-deficient mice also
showed signs of activation but there was no difference between
and ß T cells. This could indicate the existence of a slower,
non-differential and TNF- independent pathway of stimulation. More
likely perhaps, it reflects a mechanism of LPS stimulation independent
of TNF-, for example via IL-1 (64). It is also conceivable that
T cells are directly activated by LPS (18, 19, 20). The observed
dependence on accessory cells for the in vitro response to LPS does not
strictly rule out T cell stimulation through the LPS molecule
itself (65), and our difficulties to block the T cell response
to LPS in vitro with a mAb quite capable of blocking the response to
added rmTNF- could be due to such alternative pathways of
stimulation.
We chose to examine T cell responses to LPS because systemic
inoculation of Gram-negative E. coli bacteria induced the
strongest immediate T cell reactivity. However, differential T cell
responses, as indicated by increases of CD69 expression, were also
evident after inoculation of Gram-positive L. monocytogenes
bacteria. Infections with L. monocytogenes also elicit
strong innate host responses characterized by the release of large
amounts of inflammatory cytokines, among them TNF- (66). The fact
that T cell responses have been noted in many situations in
which TNF- is produced (2, 3, 8, 15, 67) is consistent with the
notion that this cytokine is one of the major signals triggering
T cell reactivity. The greater susceptibility of T cells for
TNF- stimulation could thus bias T cell responses in favor of
T cell reactivity whenever this cytokine is present, or might ensure
preferential T cell reactivity at limiting cytokine levels.
Since T cells themselves can produce TNF-, they might also be
autostimulatory under appropriate conditions. Moreover, other studies
have provided evidence for a role of T cells in up-regulating
TNF- production, suggestive of a feedback loop in which T
cells could perpetuate the generation of their own major stimulus
(68, 69).
The fact that substantial fractions of ß T cells up-regulate CD69
expression after bacterial infection or stimulation with LPS has led
others to suggest that this early activation step occurs independently
of conventional Ag recognition (48). We found that even larger
fractions of T cells respond to LPS stimulation by increasing
CD69 expression. The fact that the same is accomplished by their
exposure to TNF- suggests that their early activation is also
independent of ligand recognition via the TCR. T cell activation
independently of Ag recognition most likely is a common occurrence in
infectious and autoimmune inflammation. Our experiments with T
cells as well as those of others with ß T cells (48) suggest
interactions between T cells and effectors of the innate immune system
(70) as cause for this early activation. The functional significance of
early T cell activation, and the difference between and ß T
cell responses remain uncertain, however. Engagement of CD69 has been
found by others, and in this study, to enhance T cell reactivity (37, 71, 72). Thus, polyclonally expressed CD69 most likely increases the
probability of T cell responses following TCR-ligand interactions, a
state that might be described as heightened cellular alertness.
However, the ligand(s) for CD69 has not yet been found, and polyclonal
CD69-ligand interactions might alter the course of an immune response
in other, as yet unknown ways.
|
Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michael Lahn, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206.
3 Abbreviations used in this paper: rmTNF, recombinant murine tumor necrosis factor; FMF, flow microfluorimetry; IMDM, Iscove’s modified Dulbecco’s medium; MFI, mean fluorescence intensity; Per CP, pevidin-chlorophyll-protein.
Received for publication October 3, 1997. Accepted for publication January 28, 1998.
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