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*
Department of Pathology, Case Western Reserve University, Cleveland, OH 44106; and
Section of Endocrinology, University Hospital of Ulm, Ulm, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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release by a clonal
population of CD4 T cells was monitored on a clonal population of APC
while titrating the nominal peptide. The frequency of
cytokine-producing cells, the net per-cell output of cytokine, and the
onset of cytokine production were each found to be functions of the
signal strength. Sigmoidal dose-response curves were seen at the clonal
population level, but the activation thresholds for the individual T
cells followed a Gaussian distribution. Moreover, the overall
dose-response curve of the T cell clone revealed cyclic changes,
becoming increasingly shifted toward lower Ag concentrations with the
duration of time that elapsed since the last restimulation with Ag.
Therefore, responsiveness to Ag ("functional avidity") is not a
constant parameter of a T cell clone but a function of the T cell’s
history of last Ag encounter. The implications of such shifting
activation thresholds are discussed for autoimmune
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In general, is T cell affinity a constant feature of a clone, or one that can change dependent on the T cell’s state of activation? Therefore, while affinity is at the heart of understanding T cell function in general, and that of autoreactive T cells in particular, and while this term has been widely used to describe different signal strength-dependent reactions of T cells, it has remained challenging to conceptualize what affinity actually means in terms of T cell function.
As the original chemical term, affinity reflects the strength (free energy) of the binding between a monovalent receptor and a monovalent ligand at the state of equilibrium. In the context of Ag recognition by T cells, affinity could be defined as the strength of binding between a given (soluble) TCR with a given MHC-peptide ligand (1). The TCR-MHC-peptide complex being trimolecular, the peptide’s affinity for MHC codefines the overall strength of the TCR-MHC interaction (10). Therefore, even monovalent TCR-MHC-peptide binding reflects a higher-order reaction that, involving two equilibrium parameters, might be better described by the term "effective affinity." The effective affinity of the TCR-MHC-peptide binding can be measured, for instance, by using surface plasmon resonance. In such experiments, using TCR-peptide-MHC pairs with known thymic selection outcome, a correlation was shown between low-avidity binding and positive selection as well as between high-avidity binding and negative selection (11).
However, defining the effective affinity of the TCR-MHC-peptide binding does not suffice to describe TCR-triggered T cell functions. First, when the TCR-MHC-peptide ligations are considered during the T cell’s interaction with the APC, the actual dynamics of this process in the cell-cell contact area are fundamentally different from the same reaction in a solution (12). Accordingly, the multivalent binding of TCR-MHC-peptide pairs will define the overall avidity of the binding, being a function of both the densities of the TCRs on the T cell and of the MHC molecules bearing the specific peptide on the surface of the APC (13). Second, several additional cell surface molecule interactions contribute to the overall avidity of this interaction, including TCR dimerization (14), the lateral binding of the CD4/CD8 coreceptor molecules to the TCR-MHC-peptide complex (15), and attachments by general cell adhesion molecules constitutively expressed and induced during the cognate T cell-APC interaction. Third, as far as the functional consequences of these molecular interactions within the immunological synapse are concerned, the transduction of the T cell activating signal depends on serial triggering, whereby an optimal half-life of the TCR-MHC-peptide complex seems to be more important than the actual strength of the equilibrium binding (16, 17). A low off-rate associated with high avidity of TCR binding does not favor multiple TCR engagements and hence serial triggering by the same MHC-peptide ligand. Fourth, creating an additional level of complexity, the multiple coreceptor molecules involved in the T cell-APC interaction coaggregate with TCR molecules into supramolecular activation clusters (18) and, by contributing intracytoplasmic domains for the docking of kinases of tyrosine phosphorylation, they participate in the signaling events that lead to T cell activation (reviewed in Refs. 19 and 20). The cytoskeleton and plasma lipids actively contribute to the generation of the T cell-activating signal (21). Finally, the multiple levels of the signal transducing cascades are actively regulated. Because signal transduction events start immediately after cell-cell contact (22), neither of the above-mentioned processes actually reach equilibrium. Therefore, T cell avidity (describing the total binding energy generated by all the cell surface molecules involved in the T cell-APC interaction) cannot be directly translated into biochemical and biological reactions triggered in T cells; strictly speaking, the complexity of the signal transduction machinery and of the gene regulation processes involved preclude the description of T cell activation in terms of affinity/avidity.
However, T cells clearly have Ag dose-response characteristics that define their reactions and fates. Therefore, it might be more appropriate to define "T cell avidity" in functional terms, by measuring the Ag dose-dependent activation of effector function such as the induction of proliferation or cytokine production, and to refer to it as "functional avidity" (23). In such dose-response experiments, naive and memory cells of the same specificity have different sensitivity for Ag stimulation (24), and memory cells were shown to display variable "tunable" activation thresholds dependent on different costimulatory molecules expressed on different types of APC (25, 26, 27, 28). Moreover, evidence emerged that different responses of a T cell—such as killing, proliferation, and the production of different cytokines—is each achieved at different activation thresholds (7, 8). Therefore, different functions of the same T cell may have different functional avidities.
Using ELISPOT analysis, we provide here the first detailed single-cell
analysis of a T cell clone’s secretory IFN- response, including its
kinetics and the quantification of the cytokine produced per cell at
various signal strengths and at various stages of the clone’s Ag
history. As opposed to intracytoplasmic cytokine staining that requires
the pharmacologic treatment of the cells with brefeldin A or monensin
to disrupt the Golgi apparatus and that measures the synthesized
cytokine inside the cell, the ELISPOT assay directly visualizes the
cytokine actually secreted by pharmacologically untreated cells. In the
ELISPOT assay, the released cytokine is being continuously captured on
a membrane around the secreting cell over the assay’s duration.
Therefore, the size of spots reflects an integrated amount of cytokine
produced over the assay time period, allowing quantification of the
per-cell cytokine output for the individual cells (29).
New image analysis capabilities permitted us to measure the per-cell
cytokine production in thousands of individual cells as a function of
time and of signal strength. By obtaining high-resolution data on these
parameters of the cytokine response of a T cell clone, we attempted to
gain insight into T cell activation thresholds and functional
avidity.
For this study, we selected a previously well-defined human
CD4+ T cell clone that is specific for
hemagglutinin (HA)4
peptide 307–319. This T cell clone was found to undergo cyclic changes
(30). With increasing time since the last restimulation,
the clone’s proliferative responsiveness increased. In the same
report, such cyclic behavior of proliferative responsiveness was seen
for all four randomly chosen clones studied, suggesting that this
response pattern might be generalizable. Studies of the expression of
24 different cell surface molecules showed consistently increased
expression of CD26, LFA, and very late Ag-1 on the "late memory
cells" (21 days after the last Ag restimulation) vs "early memory
cells" (5 days after Ag stimulation), while the expression of other
molecules including TCR and CD4 were unchanged at these time points. We
were interested in learning whether such cyclic changes in the
proliferative potential of the T cell also reflect changes in the
clone’s activation threshold for IFN--production. If the activation
threshold/functional avidity for engaging an effector function is not a
constant feature of a T cell clone, but one that undergoes cyclic
changes, then this finding might need to be accounted for in models of
positive and negative selection and might contribute to a better
understanding of mechanisms underlying T cell-mediated autoimmune
diseases.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HA peptide 307–319 (PKYVKQNTLKLAT) was synthesized by the
solid-phase method and was purified by reverse-phase HPLC. NBHAC25 is a
human CD4+ T cell clone that specifically
recognizes this HA peptide on DRB1*0101 as the restricting class II
molecule; NBHAC25 expresses V
3 and an undefined V
(30). NBHAC25 T cell clone was maintained in complete RPMI
1640 medium (94% RPMI 1640 + 5% human serum AB + 1%
L-glutamine) that was supplemented with 10 ng/ml
recombinant human IL-2. The medium was changed every 5 days. The T cell
assays were done on the respective 5th day, before feeding with IL-2
medium. For the periodic restimulation with Ag (monthly for routine
propagation of the clone), NBHAC25 cells (3 x
105/ml) were cultured with HA peptide (1 µg/ml)
and irradiated (10,000 rad) LG2 cells (EBV-transformed,
DRB1*0101-positive B cell clone, at 6 x
105/ml) in recombinant human IL-2 (2
ng/ml)-containing medium. LG2 cells were grown in complete RPMI
1640 medium, splitting the cultures every 5 days. Both the NBHAC25 and
the LG2 clone, as well as the HA peptide, were a gift from Dr. Z. A
Nagy (Hoffmann-La Roche, Nutley, NJ).
ELISPOT assays
These assays were performed as described previously
(31). Briefly, ImmunoSpot plates (Cellular Technology,
Cleveland, OH) were coated overnight at 4°C with the IFN--specific
capture Ab (M700A; Endogen, Woburn, MA) at 2 µg/ml in PBS. Two types
of ELISPOT plates were used. One plate had the regular format of 96
wells, 200 µl per well (ImmunoSpot M200). The membrane in the other
plate is downsized 1:4 (ImmunoSpot P50), requiring 1/4 of the reagents.
The plates were then blocked with BSA (10 g/L in PBS) for 1 h and
washed three times with PBS. Cells were plated in complete RPMI 1640
medium. Irradiated LG2 cells (10,000 rad) were plated at 5 x
104 cells per well in the ImmunoSpot M200 plates
and at 1.25 x 104 per well in the
ImmunoSpot P50 plates. HA peptide was added as specified in the
figures. After 30 min incubation (to permit equilibrium binding of the
peptide to DRB1*0101; Ref. 30), the NBHAC25 cells were
added at 300 cells per well in the ImmunoSpot M200 plates or at 100
cells per well in the ImmunoSpot P50 plates (the exception being Fig. 1
B, where the T cells were serially diluted). After 24
h, or as specified, the plates were washed and the biotinylated
anti-IFN--detection Ab (M701; Endogen; biotinylated in our
laboratory) was added at 2 µg/ml in PBS/BSA/Tween (10 g/L BSA with
0.5% Tween). After an overnight incubation at 4°C, and washing three
times with PBS/Tween, streptavidin-HRP (1/2000 dilution; Dako,
Roskilde, Denmark) in PBS/BSA/Tween was added for 2 h at room
temperature. After washing with PBS/Tween, followed by PBS, the spots
were developed using a 3-amino-9-ethylcarbazole solution
(Pierce, Rockford, IL). The AEC stock solution was prepared by
dissolving 10 mg AEC in 1 ml N,N-dimethyl
formamide (Fischer Scientific, Fair Lawn, NJ). For the actual
development, 1 ml of this AEC stock solution was freshly diluted into
30 ml of 0.1 M sodium-acetate buffer (pH 5.0), filtered (0.45 µm),
and mixed with 15 µl
H2O2, 200 µl (ImmunoSpot
M200), or 100 µl (ImmunoSpot P50) of which was plated per well. The
plates were developed for 15 min, after which the reaction was stopped
by rinsing with tap water. This enzymatic reaction yields 1- to 3-fold
interassay variations in spot size generated; therefore, spot sizes
were internally controlled for each experiment, and data points to be
compared were generated in the same experiment. The plates were
air-dried overnight before subjecting them to image analysis.
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We used a Series 1 ImmunoSpot Image Analyzer (Cellular Technology) specifically designed for the ELISPOT assay. Digitized images were analyzed for the presence of areas in which color density exceeds background by an amount set on the basis of the comparison of experimental wells (containing T cells, APC, and Ag) and control wells (containing T cells and APC only). After background and noise subtraction, custom software is used to analyze spot morphology for circularity and density distribution to identify and separate touching and overlaying spots. Objects that meet these criteria are recognized as spots and counted. The measurement of spot size distribution is also a built-in function of the software; it is based on the array of spot sizes in a given well sorted according to distinct size categories.
Intracellular cytokine staining
Staining for flow cytometry analysis was performed as described
(32). T cell clone NBHAC25 (5 x
105/ml) was cultured with 10,000 rad irradiated
LG2 cells (1 x 106/ml) with or without HA
peptide (0, 4 µg/ml) in complete RPMI 1640 medium. After 2 h
cell culture, brefeldin A (BD PharMingen, San Diego, CA; 10
µg/106 cells) was added, and another 2 h
later the cells were fixed, permeabilized with 0.5% saponin in
PBS/BSA, and stained with PE-labeled anti-IFN- (BD Biosciences,
San Jose, CA) and PerCP labeled anti-CD3 Ab (BD Biosciences).
Two-color flow cytometry was performed on FACScan (BD Biosciences). The
isotype-matched control mAbs were obtained from BD Biosciences.
ELISA
In general, this assay was performed as described previously
(34). In particular, NBHAC25 and LG2 were cultured with HA
peptide in 96-well regular round-bottom tissue culture plates (Nunc
Immunoplate; Fisher Scientific, Pittsburgh, PA) in parallel to the
ELISPOT assays. Supernatants were collected after 24 h.
Recombinant human IFN- (BD PharMingen) was serially diluted as the
standard. ELISA were done using mAb M700A (at 2 µg/ml) as the capture
Ab and the biotinylated mAb M701 (1 µg/ml) as the detection Ab.
Streptavidin-alkaline phosphatase (1/2000 dilution; Dako) followed by
p-nitrophenylphosphate (1.6 mg/ml; Research Organics,
Cleveland, OH) was used for the colorimetric reaction. The reaction
product was measured at 405 nm.
Proliferation assays
Irradiated (104 rad) LG2-B cells (4 x 104/well) were preincubated with HA peptide (in different concentrations as specified in Results) in microtiter plates (U-bottom shape; Nunc) for 30 min. Subsequently 2 x 104 NBHAC25 cells were added per well. After 72 h of culture, the proliferation rate was measured as [3H]thymidine incorporation for 12 h.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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secretion by clone NBHAC25 is measured in ELISPOT assay at
a single-cell resolution
The CD4 T cell clone that we used is specific for HA
peptide 307–319. The clone recognizes this peptide in an
HLA-DRB1*0101-restricted manner: HA307–319 binds
to the HLA-DRB1*0101 molecule with an IC50 of
0.33 µM. A cloned HLA-DRB1*0101-positive EBV-transformed B
cell line was used to provide a uniform population of APC.
A monolayer of such clonal APC was used in a 100-fold excess over the T
cells to assure the synchronized and immediate Ag contact by the T
cell. Because the peptide used does not require processing, it can be
directly loaded onto the APC’s surface on the HLA molecule
(30). Therefore, the amount of peptide added is
proportional to the number of peptide-MHC complexes formed. By
performing IFN- ELISPOT assays with this setup, the cytokine
response of individual T cells was studied under highly defined
conditions, with the MHC-peptide ligand density being the primary assay
variable.
In the first set of experiments, we tested whether our IFN- ELISPOT
assay—within the experimental setup used—would permit the direct
visualization of cytokine production by NBHAC25 cells at the
single-cell level, whether there were variations in the amount of
cytokine produced by individual cells within the clone, and where the
lower detection limit is for the per-cell-produced cytokine. We tested
T cell clone NBHAC25 by intracytoplasmic IFN- staining, and by
ELISPOT analysis, in parallel. Fig. 1
A shows that by
intracytoplasmic staining and flow cytometry, 
50% of the individual
T cells were induced to produce IFN- at 0.1 µg/ml HA peptide. When
the T cell clone was plated in serial dilutions with a fixed number of
the same APC (clonal EBV-transformed B cells) and the peptide was kept
at the same concentration (0.1 µg/ml), the number of spots detected
in the ELISPOT assay was 59 ± 8% of the T cells plated (Fig. 1B); this frequency was seen independent of the number of T
cells plated per well, resulting in a linear function between T
cells/well and peptide-induced spots/well that passes through the
origin (r = 0.98). Therefore, at the peptide
concentration tested, the number of T cells that were found to be
IFN- positive by intracytoplasmic staining were also detected by
ELISPOT, and the lower detection limit for IFN- expressed per cell
was comparable for the two assays, moderately in favor of the ELISPOT
approach.
Next, we kept constant both the number of T cells (300 cells/well) and
the number of the EBV-B cells (5 x 104
cells/well) and titrated the HA peptide (in this experiment, the
T cells were tested 20 days after their last stimulation with Ag) (Fig. 2A). At peptide concentrations
exceeding 0.01 µg/ml, 300 spots were counted, closely matching the
number of T cells plated per well (Fig. 2A). Therefore,
above this peptide concentration, all T cells plated were detected as
IFN--secreting cells. Jointly, these data show that the ELISPOT
measurements are made at single-cell resolution.
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by ELISPOT at
different peptide concentrations as a measure of functional avidity
Functional avidity can be defined as the concentration of peptide
that leads to activation of the 50% maximal number of T cells
(22, 23). Fig. 2A shows such a dose-response
curve, with the frequency of IFN--producing NBHAC25 cells vs the
corresponding peptide concentration. The curve obtained was closely
approximated by a sigmoidal function in the log scale (Fig. 2A) or a hyperbolic function on a linear peptide scale (data
not shown), asymptotically reaching a plateau at high concentrations of
the peptide. Because the minimally activating and maximally activating
peptide concentration cannot be accurately defined in such curves, we
used the 5 and 95% activation points to evaluate the respective
Ag dose.
The kinetics of the induction of cytokine secretion is a function
of the signal strength; frequencies of IFN--spot-forming cells at
various Ag doses reach plateau values within 24 h
We studied the kinetics of IFN- spot formation at increasing Ag
doses to test whether the low frequency of cytokine-producing cells
detected by 24 h at low peptide concentrations reflects delayed
activation of T cells or whether only a fraction of cells within the
clonal population becomes activated at such conditions. At maximally
stimulating peptide doses, spot formation started within 2 h after
the Ag challenge, and the number of spots gradually increased until all
of the plated T cells became activated, which occurred by 10 h after
the initial Ag contact (Fig. 2B). In the presence of
submaximally stimulating peptide doses, spot formation started with a
considerable delay (after up to 6 h), then increased to a plateau
at 18 h, reaching numbers that corresponded to only a fraction of
the T cells plated. The data suggest that, while the kinetics of the
per-cell IFN- production is Ag dose dependent, only a fraction of
the T cells within a clone become activated at a submaximally
stimulating peptide dose.
The net amount and kinetics of cytokine production by individual cells is a function of the signal strength; spot sizes at submaximal doses reach plateau values within 24–48 h
In all experiments, a high level of heterogeneity of IFN-
ELISPOT spot sizes was observed, closely mirroring the
heterogeneity seen when this T cell clone was studied by
intracytoplasmic staining by us (Fig. 1A) or when different
clones were studied by others (36). Fig. 3 provides an example for the range of
spot sizes seen in a 24-h assay at the maximally stimulating HA peptide
dose of 0.4 µg/ml vs the medium control well. The size of ELISPOTs is
proportional to the total amount of protein produced by the cell during
the assay’s duration (36). Therefore, the spot morphology
should reflect the kinetics of IFN- production. Using
computer-assisted image analysis, we studied the spot size distribution
and found that on a logarithmic scale this size distribution was
closely approximated by a normal distribution function. Similar to the
mean channel number in FACS analysis, we used the mean spot size (the
hatched line) to characterize this integrated parameter of the
secretory response for the entire T cell population.
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A), it was found to increase
with the peptide concentration and to reach a plateau (however, the
spot size distribution was Gaussian for all peptide concentrations
similar to Fig. 3, only the mean spot size differed, data not shown),
confirming the notion that cytokine production is indeed Ag dose
dependent; also, the IFN- concentration in the culture supernatants
was found to be peptide dose dependent at all time points tested (data
not shown). Thus, the net amount of cytokine produced by individual
cells within 24 h was proportional to the signal strength.
Accordingly, at 24 h, 0.4 µg/ml peptide not only activated all T
cells to secrete cytokine, but also induced the maximal production
rate. Spot size measurements at different time points (addressing the
kinetics of cytokine production) vs the peptide concentration showed
(Fig. 4B) that at the highly stimulatory peptide
concentrations (
0.025 µg/ml), the T cells started to produce
IFN- within 2 h, while following weak stimulation (0.0016
µg/ml), spots became detectable only after 6 h. At maximally
stimulating doses, the T cells continued to produce at a constant rate
(up to 72 h) that also appeared to be the maximal rate (in Fig. 4B the linear function between mean spot size and time for
doses was 0.4 µg/ml). However, below 0.4 µg/ml, the average spot
sizes increased more slowly (suggesting a lower production rate) and
reached a plateau within 24 h. Thus, at weakly stimulating peptide
doses, cytokine production was not only slowed down, but also started
with a delay and ceased earlier, involving both a lower production rate
and a shorter production period.
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production in these cultures.
To validate the assumption that the spot size reflects the amount of
cytokine produced per cell, and to reproduce these observations by an
independent read out, we also measured IFN- by ELISA in the culture
supernatants. A close to linear correlation was seen between the net
amount of IFN- measured in the supernatants at different peptide
concentrations and the mean spot sizes (Fig. 5), suggesting that the spot size and the
total amount of cytokine produced by individual cells were directly
proportional. Based on these data, we conclude that the net per-cell
output of cytokine and the onset and the duration of IFN- production
are each functions of the signal strength. The observed heterogeneity
of spot sizes, even at maximally stimulating peptide doses, appears to
reflect both different productivity of the individual T cells
(resulting from their different activation thresholds) and the
asynchronous onset of cytokine production. Therefore, considerable
biologic complexity is concealed behind the close to perfect sigmoidal
dose-response curve of a clonal population of T cells. Although such
dose-response curves apparently do not fully characterize the clone’s
affinity (as genetically defined by the TCR specificity expressed),
they are suited to characterize the overall responsiveness of the cell
population. In vivo, T cell populations, not individual cells, mediate
effector functions, and the frequency of cytokine-producing cells along
with the per-cell cytokine production rate will define the magnitude of
effect. This overall Ag responsiveness seems to be well reflected in
the ELISPOT dose-response curve. Is functional avidity a constant
feature of a memory cell?
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In Fig. 6A, the
dose-response curve is shown for NBHAC25 cells 5 days after Ag
stimulation. Ninety-five percent of the 300 T cells plated produced
IFN- spots at HA peptide concentrations of 0.22 µg/ml, reaching
100% at higher peptide doses. The 5% activation threshold was at
0.0032 µg/ml. Fifty percent maximal activation was calculated to be
at 0.040 µg/ml peptide. When the T cell clone was kept in culture for
an additional 20 days (without restimulating with Ag), and was retested
on day 25, 100% of the T cells were eventually induced to produce
cytokine, but the dose-response curve was shifted to the left. The 5,
50, and 95% activating peptide doses were calculated to be at 0.0001,
0.0028, and 0.016 µg/ml, respectively, which are 20-fold lower
than when the clone was tested at the 5-day time point. The
dose-response curves measured at the 10- and 15-day time points gave
intermediate results with an incremental shift to the left with time
(Fig. 6A). To ascertain that these changes in peptide
responsiveness are intrinsic to the T cells, we restimulated the
NBHAC25 clone with Ag after the 25-day time point, and retested it at
days 5 and 10. The dose-response curves seen overlapped with the
measurements initially made at day 5 and 10, respectively; this
time-dependent shift was reproduced in two additional experiments (data
not shown). Therefore, the time-dependent changes in dose-response
curves seem to be cyclic changes in the activation thresholds for
IFN- production that are intrinsic to the clone. Parallel to these
ELISPOT measurements, we also performed proliferation assays.
Reproducing the previously published data on the proliferative
potential of the clone (30), we found that higher peptide
doses were required to induce a proliferative response at the 5-day
time point than at the 25-day time point (open symbols in Fig. 6A). At both time points shown (and at all the other time
points tested), the induction of IFN- production required
approximately one log lower concentration of peptide, suggesting
different activation thresholds for the two T cell functions.
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The significant increase in functional avidity of cloned T cells
with time might suggest that T cells require time before they can reset
their cytokine synthesis machinery. If this were the case, as opposed
to a general decrease in the T cell’s signaling functions, one might
expect decreased per cell cytokine production rates in the early memory
cells. Aided by the ability to study independently the frequencies and
per-cell cytokine output of T cells vs the signal strength, we
addressed this question. Fig. 6B shows the dose-response
graphs of average spot sizes vs the peptide concentration for the
NBHAC25 clone tested at 5, 15, and 25 days after the last stimulation
with Ag; the testing of the T cells with the different Ag history was
done in parallel. These data show that the day 5 cells require a
stronger signal to reach the same average spot size than cells of day
25, and cells from day 15 gave intermediate results. While there was a
parallel shift to the left on the peptide concentration axis, the cells
reach the same plateau level, but at different peptide concentrations.
Therefore, independent of the Ag history, the T cells had comparable
maximal IFN- productivity. These data suggest that the cyclic
changes in Ag responsiveness operate at the level of the signaling
machinery and do not result from a decreased ability of these cells to
perform these functions
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A). The
sigmoidal shape of the dose-response curves suggests that the
activation thresholds for the individual cells within the clone show
normal distribution (the Gaussian distribution being an integral of the
sigmoidal curve). Despite this considerable interindividual variation
in activation thresholds within a single clone, different T cell clones
can have fundamentally different dose-response characteristics, with
their own Gaussian spectrum of interindividual variations. Thus, in our
previous work we found that myelin basic protein (MBP)-specific T cell
clones isolated from MBP-expressing wild-type mice required a 10,000
times higher peptide dose for becoming stimulated than did clones
isolated from MBP-/- congenic mice
(37). With the same immunodominant MBP peptide and the
same APC involved, this fundamentally different Ag dose responsiveness
could only be attributed to different affinities of the clones, i.e.,
the deletion of high-affinity clones in the MBP-expressing host.
Therefore, the sigmoidal graphs of dose-dependent frequency
measurements of cytokine-producing cells permit us to characterize
clonal (or bulk) T cell populations with respect to average functional
avidity and activation threshold distribution.
So far, it was not possible to directly visualize the signal
strength-dependent production of cytokine by individual cells.
Therefore, the very nature of the T cell activation threshold has
remained unresolved. It has been suggested that individual T cells
"count" the number of TCR ligations by MHC-peptide complexes on the
APC, and that the T cell becomes activated as soon as a critical number
of TCR ligations have occurred (a number that has been approximated in
the order of several thousands) (16, 39). Accordingly, at
low peptide concentrations (MHC-peptide ligand density), the individual
T cell may simply require more time for "counting" before the
threshold number of TCR ligations occurs. Therefore, is there a defined
minimum number of MHC-peptide complexes on the APC above which TCR
ligation does result in induction of IFN-, or do weak signals merely
lead to delayed cytokine production?
Studying the spot size distributions vs peptide concentration by
ELISPOT, we showed that per-cell cytokine output is a function of the
signal strength, confirming previous observations (32, 35). However, our detailed analysis of the kinetics of spot
formation suggests that not only the rate of production was affected,
but also the time required for the beginning of secretion and duration
of the production period itself. The plots for the average spot size as
the function of the peptide concentration showed a similar sigmoidal
curve, as did the dose-response frequency plots (Fig. 2A vs
4A) with a Gaussian distribution of spot sizes at each
concentration (Fig. 3), comparable to the activation threshold curve
derived from the dose-response frequency plots. Most likely, this
outcome reflects the normal distribution of "avidities" for
individual T cells within the clone (if cell "A" within the clone
has a lower avidity than cell "B," "B" will produce more
cytokine and a bigger spot at a given peptide concentration). However,
we cannot rule out that the potency to produce (a certain maximal
amount of) cytokine is also normally distributed (if cell "A"
produces half the amount of cytokine at concentration "x" than does
cell "B," it might still produce half the amount of cytokine at
concentration "y", presuming both cells have the same avidity),
also contributing to the Gaussian shape of the curves. In fact, this is
likely, as at the maximally stimulating doses we still see a normal
distribution with a similar range, though shifted to higher values.
This parallel shift of close to normal distribution toward higher cytokine output implies that the dose responses for the individual cells should follow (sigmoidal) curves similar to the average spot size graphs. Indeed, our analysis of spot size distributions clearly demonstrates that activation of individual T cells is not a quantum process with a simple on/off threshold, but follows a sigmoidal/asymptotic behavior. Therefore, it is impossible to determine an absolute cutoff representing the activation threshold for individual T cells. However, an arbitrary cutoff can be defined at a certain spot size, similar to the 5% frequency threshold.
Although the ELISPOT system can detect per-cell cytokine production
over a wide range (>2 logs of spot sizes and densities, Fig. 3), we
found no evidence for even minimal cytokine production below a certain
range of Ag doses during an up to 72 h incubation period (Figs. 2 and 4). Therefore, although a discrete lower threshold for the T cell
activation may not exist, it seems that when the T cell "counts"
MHC-peptide ligations, these TCR engagements have to occur at a minimal
frequency rate, to generate a stimulatory signal, consistent with the
kinetic proofreading model (39). Confirming this notion,
subthreshold triggering seems to lead to the formation of inactive
phosphorylation intermediates of the signal-transducing TCR-
-chain
(17).
It was striking that the Ag responsiveness of a T cell clone (at the
population and single-cell level) was not a constant. T cells 5 days
after the last Ag stimulation required 20-fold higher peptide
concentrations to start secreting IFN- and to reach 50% cell
activation than they did 25 days after the last Ag contact (Fig. 6).
Although it has been previously described that the activation threshold
for a given T cell can vary when it interacts with a different type of
APC (that expresses different levels of MHC and costimulatory
molecules), in our experimental setting the APCs were clonal and
constant. This T cell clone’s different activation threshold and
functional avidity for IFN- production was found to be cyclic (a
function of the time that elapsed since the last restimulation by Ag).
The cyclic Ag responsiveness of this clone, and of three others
studied, was previously noted using proliferation assays
(30), which we reproduced here (Fig. 6A). It is
unlikely that these changes in activation thresholds reflect different
maturation stages of the clone. Rather, the changes are likely to
result from activation-state-dependent variations in cell surface
molecule expression. Thus, in a previous report (30), the
proliferative behavior of this clone (and three others) was correlated
with the cell surface expression of 24 molecules. The level of TCR and
CD4 expression was comparable early and late after restimulation (which
we confirmed, data not shown). Therefore, changes in the avidity of
TCR-MHC-peptide binding are unlikely to account for the cyclic changes
of the activation threshold. A consistent increase in the expression of
CD26, LFA-1, and very late Ag-1 was noted on late time points after
restimulation (30). Being molecules involved in
costimulation, the up-regulation of these molecules might explain the
lowered activation thresholds. Additional molecular correlates seem
possible, and even likely. Because these changes did not affect the T
cells’ cytokine expression machinery (the maximal per-cell cytokine
production was unimpaired, Fig. 6B), it is likely that this
tuning of activation threshold occurs at the level of the signal
transduction apparatus. It was suggested that the increased
proliferative potential of the late vs early memory cell helps in
maintaining the memory T cell pool when the Ag becomes limiting in the
body (30). Although such changes in the proliferative
responsiveness of foreign Ag-reactive T cells can be well reconciled
with the regulation of clonal sizes and with the maintenance of an
effective immune system, such tunable activation thresholds for T cell
effector functions are more difficult to accommodate in the theoretical
framework of T cell biology, particularly with respect to self/nonself
discrimination.
It is at present thought that all the T cells that are positively
selected in the thymus must have a minimal affinity for self-MHC and an
associated self-peptide, and that this low level of TCR stimulation (by
the positively selecting MHC-self-peptide complex) has to be maintained
also in the immune periphery for the naive T cell to survive (3, 4). Therefore, all naive T cells in the immune periphery are
weakly autoreactive. When naive T cells become activated, they are
thought to become more responsive to Ag (24); that is,
they seem to decrease their activation threshold. The reason for this
might be in part that the memory T cell up-regulates accessory molecule
expression. Indeed, it requires either immunization with an autoantigen
or an infection with a cross-reactive microorganism to convert
"ignorant" naive autoreactive T cells into autoaggressive effector
cells; after becoming primed, these autoreactive memory cells start to
become stimulated by the same APC whose constitutive level of
autoantigen presentation was ignored by the naive T cell
(9). The data presented here suggest that this lowering of
the T cell activation threshold from that of the naive T cell to that
of effector/memory T cell is not a discrete quantum leap, but one that
continues after the priming event. Accordingly, many low-affinity
autoreactive T cell clones that are engaged by a peripheral
immunization or cross-reactive infection will further lower their
activation threshold, while they make the transition to the late memory
cell stage. Only after these 2 wk will they be stimulated by the
level of autoantigen generally presented (Fig. 7).
|
). The
time course of relapses, up to weeks apart, are consistent with the
cyclic regulation of the T cell activation threshold, as seen in
Fig. 6. In summary, we show here that while dose-response curves do not fully characterize a T cell clone’s affinity as genetically defined by the TCR specificity expressed, they are suited to characterize the overall Ag responsiveness of the cell population. Also this Ag responsiveness is not solely defined by the TCR specificity expressed, but codefined by the clone’s Ag history. Cyclic changes in T cells’ Ag responsiveness might contribute to the maintenance of immune memory and might explain the intermittent course of T cell-mediated autoimmune diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul V. Lehmann, Institute of Pathology, School of Medicine, Case Western Reserve University, BRB 929, 10900 Euclid Avenue, Cleveland, OH 44116. E-mail address: pvl2{at}po.cwru.edu
3 P.V.L. and M.T.-L. contributed equally to this study.
4 Abbreviations used in this paper. HA, hemagglutinin; MBP, myelin basic protein.
Received for publication January 11, 2001. Accepted for publication June 1, 2001.
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, with a total number of 411 spots assessed).
The mean spot size is indicated with the hatched line. A total of four
spots were counted in the four medium control wells (
). Details of
image analysis are provided in Materials and Methods.
The results are representative for all experiments reported in this
paper.
