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Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, and
Cellular Immunology Section, Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains and a greater amount of phosphorylated ZAP-70
than less potent ligands, and the number of TCR down-modulated per
available ligand is higher with ligands showing this full agonist-like
pattern. The large number of receptors showing partial
phosphorylation following exposure to weak ligands indicates that the
true extent of TCR engagement and signaling, and thus the amount of
sequential engagement, is underestimated by measurement of TCR
down-modulation alone, which depends on full receptor activation. These
data provide new insight into T cell activation by revealing a clear
relationship among intrinsic ligand quality, signal amplification by
serial engagement, functional T cell responses, and observable TCR
clearance from the cell surface. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß complexes and their cognate ligands in vitro is generally in
the high micromolar range, with fast off-rates being characteristic of
these interactions 4, 5, 6, 7 . Thus, the engagement of a single TCR by a
single peptide:MHC molecule complex is not by itself able to evoke a
sustained intracellular signal. Prolonged signaling is also unlikely to
depend on sequential binding of a large number of TCR to a comparable
number of ligands because functional activation often requires only
tens to hundreds of specific peptide:MHC molecule complexes per
presenting cell 8, 9, 10, 11, 12, 13 . The problem posed by low affinity of TCR for
their ligands may in some degree be ameliorated by recruitment of the
CD4 or CD8 coreceptors 14 , which stabilize TCR-ligand binding 15, 16 and modify the biochemical characteristics of the signaling event
17, 18 . However, available data suggest that coreceptor function can
only extend the time of association of the TCR with its ligand a few
fold, failing to provide an adequate model of TCR signaling for
activation events requiring a substantially longer duration of
intracellular messenger generation. Consideration of these issues led Valitutti et al. 19 to explore the quantitative relationship between ligand density and TCR triggering and to provide evidence for an unexpected mechanism that contributes to sustained TCR signaling in the presence of small numbers of specific ligands on the APC membrane. These investigators used TCR down-modulation (internalization) to estimate the relationship between ligand number and TCR engagement. Their studies suggested that at low ligand densities (<100 per APC), each peptide:MHC molecule complex could serially trigger up to 200 separate TCR, amplifying and extending the duration of intracellular signaling. The required continued availability of peptide:MHC molecule ligands after disengagement from numerous TCR is consistent with the long lifespan of most such peptide:MHC molecule combinations reaching the cell surface 20 .
Although this model has great appeal, even investigators who have used it to explain their functional data on T cell activation 21 have not provided quantitative data confirming this hypothesis. Furthermore, despite progress in understanding some mechanistic aspects of receptor down-modulation following ligand engagement or cross-linking using Ab 14, 22, 23 , some more recent studies have questioned whether there is any direct relationship between TCR internalization itself and the elicitation of effector functions 24, 25 . These latter experiments have led several investigators to conclude that TCR loss from the cell surface is unrelated to effective T cell activation and that this parameter does not measure a physiologically important aspect of TCR signaling, raising questions about the validity or relevance of the serial engagement phenomenon.
In contrast, other reports suggest a direct correlation between levels of TCR down-modulation and T cell responses 26, 27 , though the techniques used do not permit one to argue directly for serial engagement as an underlying mechanism. Recent evidence for a hierarchical organization of effector response thresholds 27, 28, 29, 30 and the relationship among ligand quality, response, and TCR down-modulation 27 suggested to us a possible unifying explanation for these apparently divergent results of different laboratories. Therefore, we have reinvestigated the phenomenon of TCR down-modulation in a quantitative manner using a diverse set of mouse and human T cells, various ligands for individual TCR, and parallel biochemical signaling studies. Using highly potent ligands, our studies confirm for human clones the phenomenon of extensive serial TCR engagement reported by Valitutti et al. 19 . More importantly, we provide evidence for a clear relationship among the quality of biochemical signaling events involving an engaged TCR complex, the likelihood that this receptor complex will be removed from the cell surface, and the cell’s functional response. These data are discussed from the standpoint of how ligand density, the biochemistry of individual receptor activation, and the phenomenon of serial engagement combine to yield an effective stimulus for the T cell.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A.E7 and 3C6 are Th1 cell clones specific for pigeon cytochrome c (PCC)3 88–104 and I-Ek 31 . These clones were maintained as previously described 30 and used no earlier than 2 wk after restimulation and cytokine expansion. P13.9 is a supertransfected derivative of the DAP.3 fibroblast-derived transfectant DCEK Hi7 that expresses high levels of I-Ek, B7.1, and ICAM-1 32 . GDBP is a human T cell clone specific for myelin basic protein (MBP) 87–99 and HLA-DRB1*1302, grown and maintained as described 33 . GP-BC is an EBV-transformed B cell expressing DRB1*1302.
Peptides
Peptides were synthesized and purified by HPLC in the National Institute of Allergy and Infectious Diseases, Biological Resources Branch, Laboratory of Molecular Structure (Rockville, MD). The sequences of PCC88–104, moth cytochrome c (MCC) 88–103, MBP87–99, and MBP87–99 (F88 K94 K95 F98) were KERADLIAYLKQATAK, ANRADLIAYLKQATK, VHFFKNIVTPRTP, and VFFFKNIKKPRFP, respectively.
APC preparation and quantitation of peptide:MHC class II complexes
To create a surface pool of peptide/MHC class II complexes on APCs, P13.9 was cultured overnight with the indicated peptide, or GP-BC was cultured with MBP87–99 for 2 h. In quantitative analyses, N-terminal biotinylated peptides were used rather than unsubstituted peptides. The APC were then washed three times with PBS and used for incubation with T cells (see below) or for quantitation of surface peptide/MHC class II molecule complexes. For the latter, after an additional wash with PBS containing 1% BSA and 0.1% NaN3, cells were stained with streptavidin-phycoerythrin (PE; PharMingen, San Diego, CA) or PE avidin D (Vector Laboratories, Burlingame, CA) at a saturating concentration. Dead cells were gated out based on staining with propidium iodide. Cells (1 x 104) were analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The number of peptides was calculated from FL2 mean intensity, the PE:protein ratio of the streptavidin reagents and standard curves obtained using beads with known PE content (Flow Cytometry Standards, San Juan, PR), as follows: number of peptides = [(mean intensity of APC with peptides) - (mean intensity of APC without peptide)] x (slope element from standard curve)/(PE:protein ratio).
Coincubation method for inducing TCR down-modulation
Mouse T cells were mixed with control or peptide-pulsed APC at a
ratio of 1 T cell:2 APC in 5-ml polypropylene tubes (FALCON 2058;
Becton Dickinson, Franklin Lakes, NJ), pelleted, and incubated at
37°C for the indicated time. In human cell experiments, T cells and B
cells were cultured in 96-well U-bottom plates for 5 h. PBS
containing 0.5 mM EDTA was added after incubation to dissociate
conjugates. To stain mouse T cells for the TCR complex, 145-2C11
(anti-CD3
34) or KJ25 35 Ab-containing supernatant was added
at 4°C and incubated for 30 min. The cells were then washed with PBS
containing 1% BSA and 0.1% NaN3, and FITC
anti-hamster IgG Ab (Caltag Laboratories, South San Francisco, CA)
was added for another 30 min in the cold. After washing, TCR staining
was analyzed by excluding the L cell APC using foward scatter/side
scatter parameters and dead cells using propidium iodide.
For quantitative analysis of TCR complex expression, mouse T cells from cultures performed as above were stained directly with FITC-conjugated anti-Vß3 or anti-TCR Cß Ab (H57-597; PharMingen), whereas human T cells were stained with FITC-conjugated anti-human CD3 (PharMingen), all at saturating concentrations. Cells (10,000) were then analyzed and the number of TCR on each cell was calculated from the FL1 mean intensity, the fluorescein:protein (F:P) ratio of the Abs, and standard curves derived using beads with known FITC content (Flow Cytometry Standards) as follows: number of down-regulated TCR = [(mean intensity with APC without peptide) - (mean intensity with APC with peptide)] x (slope element from standard curve)/(F:P ratio).
T cell functional assays
P13.9 cells were cultured with peptide overnight at 37°C, then treated with mitomycin c (20 µg/ml) at 37°C for 1 h and washed four times with PBS. T cell clones (2.5 x 104/well) were cultured with APC (5 x 104/well) in 96-well flat-bottom plates. For use with human T cells, GP-BC was cultured with peptides for 2 h, and unbound peptides were washed away after 30 Gy irradiation. GDBP (1 x 104/well) was cultured with peptide-pulsed GP-BC (5 x 104/well) in 96-well U-bottom plates. After 48 h of culture, 1 µCi of [3H]thymidine was added, and the cells were harvested 16 h later to measure proliferation based on [3H]thymidine incorporation into high m.w. DNA.
Tyrosine phosphorylation analysis
Peptide-prepulsed APC were prepared as described above. Two million A.E7 and 4 x 106 P13.9 were mixed and cultured for 5 min at 37°C. In human experiments, GDBP (1 x 106) and GP-BC (2 x 106) were cultured for the indicated time at 37°C. Thereafter, cells were lysed with 1% Nonidet P-40, 140 mM NaCl, 10 mM Tris-HCl (pH 7.2), 2 mM EDTA, 5 mM iodoacetamide, 1 mM Na3VO4, and proteinase inhibitor mixture (Boehringer Mannheim, Indianapolis, IN) on ice for 25 min. After centrifugation, the supernatant was incubated with anti-ZAP-70 Ab 36 for 4 h and precipitated with Staphylococcus aureus protein A (Pansorbin; Calbiochem, La Jolla, CA). Precipitated proteins were analyzed using 10% SDS-PAGE with reducing (mouse) or nonreducing (human) conditions and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Phosphorylated proteins were detected with 4G10 (Upstate Biotechnology, Lake Placid, NY) and horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad Laboratories, Richmond, CA). Horseradish peroxidase activity was detected with SuperSignal ULTRA chemiluminescence substrate (Pierce, Rockford, IL).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To study TCR down-modulation in a quantitative manner, we established a model system in which measurement of ligand density without cell disruption is possible and in which the APC does not undergo substantial changes in its adhesion or costimulatory molecule repertoire during interaction with the T cell. We first examined numerous T cell clones and hybridomas for TCR down-modulation upon Ag exposure. Even using the same APC displaying a given amount of ligand, the extent of TCR loss from the surface varies for different T cells, including a mouse T hybridoma and a human transfectant expressing the identical TCR. In general, immortalized T cells (T hybridomas, Jurkat) show low to intermediate levels of TCR down-modulation, whereas some, though not all nontransformed T cell clones show loss of between 70% and 90% of the total surface pool of TCR at high Ag concentrations (data not shown). Because of the limited down-modulation in the former cells, whose responses to Ag are also attenuated compared with nontransformed lymphocytes, we focused our studies on T cell clones that show extensive receptor loss and that are physiologically similar to those studied previously in mouse and human model systems of TCR down-modulation.
To establish the kinetics of TCR clearance, the T cell clone 3C6 was
incubated with APC bearing the agonist ligand
PCC88–104/I-Ek for various lengths of time, then stained
for surface TCR complexes (Fig. 1
A). A clear reduction in TCR
levels is seen at 15 min. Although the rate of down-modulation
decreases after 
1 h of T cell:APC contact, it takes nearly 5 h
to reach a plateau level of TCR loss at 10 µM peptide. Therefore,
cells were incubated for 5 h in subsequent experiments. T cell
clones A.E7 (Fig. 1B) and 3C6 (Fig. 1C) were then
cultured for 5 h with APC bearing various numbers of
PCC88–104/I-Ek complexes. A total of 70–90% of A.E7 TCRs
are down-modulated, but only 50–60% of 3C6 TCRs are lost at 10 µM
of PCC88–104. In all cases, the down-modulation appears to involve the
entire pool of cells in the population, even though these cloned cells
show functional heterogeneity 30 .
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A), and in
accord with these functional data, MCC88–103 also induces TCR
down-regulation slightly more effectively than PCC88–104 (Fig. 2B). On the other hand, the 3C6 clone does not show a
heteroclitic functional response to the MCC peptide (Fig. 2A), and in agreement with these results, PCC88–104 induces
more TCR down-modulation than MCC88–103 (Fig. 2B). Because
the same MCC- and PCC-peptide-bearing APC are used in a single
experiment for stimulating the two T cell clones, the inverse nature of
these effects cannot be attributed to variations in ligand density, but
rather clearly reflects the relative efficiency of the two ligands
(PCC/I-Ek and MCC/I-Ek) for triggering both
down-modulation and effective intracellular signaling by the slightly
different TCR of these cells.
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A variety of techniques have been used to quantitate the number of
specific peptide/MHC molecule complexes on APC. Among those permitting
the direct analysis of peptide-associated surface molecules on live
APC, the most widely used involves biotinylated peptides detected with
fluorescent streptavidin conjugates 37 . We chose this approach
because, compared with methods involving MHC molecule purification, it
is less susceptible to underestimating true ligand number due to
dissociation of peptide 38 . I-Ek-bearing L cells were
incubated with biotin-PCC88–104 and washed, and PE-conjugated
streptavidin was added at a saturating concentration to promote
univalent binding. The number of PE molecules was determined using
standard curves generated by analysis of beads conjugated with known
numbers of PE fluorochromes. At 100 µM and 10 µM peptide pulsing
concentrations, the mean number of PCC88–104 peptides bound is
9.7 x 104 and 3.9 x 104,
respectively (Fig. 3A). The
background binding to DAP.3 cells lacking expression of class II
molecules is negligible. The lowest number of peptides that can be
measured directly by this assay is 500 at 1 nM offered peptide.
However, a log-log plot of offered peptide concentration vs bound
ligand number follows a straight line with an r > 0.99
for values in the detectable range, and it is thus possible to
extrapolate with some confidence to substantially lower absolute ligand
values (Fig. 3A). This is critical, as the extent of serial
engagement is claimed to rise rapidly as ligand density decreases,
especially below a few hundred complexes per APC 19 .
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B), whereas cells pulsed with 0.3 nM peptide
show a loss of 700 TCRs. Fig. 3C shows the number of
down-regulated TCR per peptide:MHC complex derived from the data in
Fig. 3, A and B, while Fig. 3D
converts the data into the ratio "TCR down-modulated/ligand"
plotted against available ligand density. At high ligand density (equal
to or greater than the number of TCR on the T cell), the ratio of TCR
down-regulated to the number of available ligands is <1 (Fig. 3D). At 1 nM, which generates ligands at the limit of direct
detection (500 complexes per APC), the ratio rises slightly to 2
for both A.E7 and 3C6. At 10 pM, when extrapolation indicates each APC
would have 50 ligands, the ratios rise further to 7 for both
cells. Measurement of serial TCR engagement using human T cell clones and modified TCR ligands
Because the triggered TCR:ligand ratio of mouse T cell clones was
substantially lower than expected based on data obtained using human T
cell clones, we examined various parameters that differed between the
two sets of experiments to search for an explanation. Attempts to use
iodinated peptides for quantitation of ligand in the manner of
Valitutti et al. 19 were unsuccessful with a large background of
precipitated counts even from pulsed cells lacking MHC class II
expression (data not shown). However, we have been able to confirm the
accuracy of our ligand-counting methodology using Scatchard analysis
with Fab fragments of an Ab to a peptide-MHC class II combination
(Y-Ae) 39 in comparison to measurements using biotinylated peptides
in the same system (data not shown). Another potential difference lies
in the species origin of the T cell and APC. Therefore, we applied our
method to the combination of a transformed human B cell and a human
CD4+ T cell clone, which is specific for the self ligand
MBP87–99 bound to DRB1*1302 (Fig. 4A). Experiments were done
with both the natural ligand and a modified peptide we had previously
identified as "superagonist" for this clone (MBP87–99 (F88 K94 K95
F98)) 40 , which in functional assays is up to 1000 times more potent
than the wild-type peptide. This comparison was considered important
because several studies have indicated a relationship between the
overall extent of TCR down-modulation and ligand potency 19, 26, 27 .
Furthermore, we have found that many peptide:MHC molecule combinations
used to propagate human T cell clones are not optimal agonists, as
judged by either functional or biochemical signaling criteria 27 .
Numerous modified peptide ligands are more potent in stimulating these
clones than the original immunogen and, as shown in Fig. 2, heteroclitic ligands show an enhanced capacity to promote TCR
down-modulation.
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B), even though the latter generated more complexes
with DRB1*1302 at equal peptide concentrations (Fig. 5A). At 30 µM offered
peptide, about 6 x 104 MBP87–99 containing complexes
are detected per APC, with the lowest detectable number being 900 at 30
nM (Fig. 5A). As with the mouse system, there is a linear
log-log relationship between offered peptide and the number of
complexes formed, so that we can extrapolate to ligand densities
approaching 50 per APC. Using MBP87–99 at 30 µM and 0.3 nM, 1.4
x 104 and 1.1 x 103 complexes (which is
the smallest difference we could measure compared with the starting
number of receptors on resting T cells) were down-modulated,
respectively. MBP87–99 (F88 K94 K95 F98) induced a greater amount of
TCR loss at any given input peptide concentration, and down-modulation
could be seen under conditions in which peptide binding could not be
directly measured by our methods (Fig. 5, A and
B). Thus, at high ligand densities, both peptides form
complexes that lead to <1 TCR down-modulation event per available
bound peptide (Fig. 5C). With 900 MBP87–99 complexes on
GP-BC, the ratio rises to 3.5, and at an estimated 50 ligands per APC,
to 20. This latter ratio is three times greater than that seen with
mouse T cell clones (Fig. 5D), but still substantially lower
than that claimed for human clones 19 . However, using MBP87–99 (F88
K94 K95 F98)-containing complexes at low density, the predicted number
of TCR engagements per ligands rises to 100, a number close to that
reported by Valitutti et al. 19 .
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The above data all support the previous results reported by us
27 and by Valitutti et al. 19 that a functionally more potent
ligand induces a greater extent of TCR down-modulation than an
equal density of less potent ligand. In our prior study of this
phenomenon, we demonstrated that TCR loss from the cell surface
parallels the proportion of receptors showing biochemical evidence of
optimal signaling events (full TCR phosphorylation/ZAP-70
activation). Furthermore, equal levels of TCR down-modulation and of
ZAP-70 phosphorylation produced at different densities of these related
ligands result in equivalent functional responses of the T cells. These
data indicate that TCR that engage ligand but show a partially
phosphorylated status of associated -chains do not undergo efficient
internalization, and they do not appear to contribute to elicitation of
most effector responses. In the context of the present results, these
prior findings suggest that calculation of the number of ligand-TCR
engagement events based only on loss of TCR from the cell surface may
substantially underestimate the true number of relevant receptor-ligand
interactions, in that many such engagements would lead to measurable
but incomplete TCR signaling, a failure to internalize and hence, a
failure to be counted using flow cytometric approaches.
This was reevaluated here both as possible explanation for the
difference between the mouse and human clone studies and as the basis
for the failure of some investigators to see a clear relationship
between functional T cell activation and TCR down-modulation. The A.E7
mouse clone and the GDBP human clone were stimulated with either of two
ligands differing in their capacity to induce down-modulation when
offered at equal density on the APC and thus, showing a difference in
the extent of serial engagement estimated using this approach. As seen
in Fig. 6A, for A.E7, the
heteroclitic ligand MCC induced a higher proportion of the fully
phosphorylated p23 form of TCR and a greater amount of
phosphorylated ZAP-70 as compared with the less active PCC peptide when
offered at an equivalent concentration. The amount of phosphorylated
ZAP-70 was similar, however, at concentrations of MCC (1 µM) and PCC
(10 µM) that led to a similar decrease in TCR expression and to equal
functional responses, consistent with down-modulation reflecting the
number of individual TCR complexes undergoing full activation upon TCR
engagement. Likewise, for GDBP, the superagonist peptide induced a
higher ratio of fully phosphorylated -chain (p38 dimer =
p23 monomer analyzed in the mouse system 27) and more
phosphorylated ZAP-70 than the less potent wild-type MBP87–99 peptide
(Fig. 6, B and C). At concentrations of the
modified peptide (3 µM) that gave TCR down-modulation close to that
seen with 30 µM MBP87–99, the total level of phosphorylated ZAP-70
was comparable for the two ligands. Thus, TCR engagements resulting in
incomplete receptor activation as assessed by the phosphorylation
status of and ZAP-70 do not appear to contribute to TCR
down-modulation, leading to a substantial underestimate of the extent
of actual TCR-ligand interactions nevertheless able to initiate
signaling events.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain phosphorylation and
ZAP-70 activation, even as a much larger number of TCR in the same
cells shows evidence of ligand engagement in the form of partially
phosphorylated -chains and recruited but not activated ZAP-70 27 .
Our findings relating ligand quality, signaling biochemistry, and TCR
down-modulation agree with functional studies of others using altered
TCR ligands to induce TCR internalization 26 . These recent observations appeared to us to have clear relevance to the model of serial engagement of TCR by peptide:MHC ligands, which was postulated to permit substantial amplification of signals in the face of low ligand densities on APC 19 . This striking observation has not been directly confirmed in a quantitative manner in published work by other laboratories, even though some investigators ascribe their functional observations to this phenomenon 11, 21 . Others have failed to observe any relationship between TCR down-modulation and certain measures of T cell activation, leading them to question the physiological relevance of this measurement or the derived serial engagement model 25 .
Our own analysis of numerous mouse T cell clones and hybridomas showed
that some but not all cells responded to ligand with extensive loss of
surface TCR expression. The loss of TCR was similar when measured using
anti-CD3, anti-TCR Cß, anti-TCR Vß, and anti-TCR
V reagents, removing one source of possible discrepancy based on the
reagents employed for analysis 47 . Nevertheless, hybridomas bearing
the same TCR as nontransformed clones showed a different extent of TCR
down-modulation upon exposure to the same APC bearing the same ligand.
This argues that down-modulation is not controlled simply by the
strength of the TCR-ligand binding event, but also depends on
properties of the cell expressing the TCR. For many T hybridomas, the
pattern of TCR signaling upon ligand exposure is more like that seen
with partial agonists than full agonists, as compared with T cell
clones expressing the same TCR (unpublished observations), a phenomenon
that may in part relate to the lower level of Lck and/or coreceptor
found in these hybridomas as compared with normal T cells 18 . These
observations fit the data obtained here using mouse and human clones,
which show that TCR down-modulation closely tracks the extent of full
phosphorylation and/or ZAP-70 activation. They are also consistent
with the work of La Face et al. 48 , who observed a lack of TCR
down-modulation with mouse clones exposed to antagonist ligands capable
of inducing only partial chain phosphorylation. Thus, among a large
number of tested cells, the extent of TCR engagement is substantially
underestimated by examining down-modulation, as interactions leading to
partial signaling events are not translated into internalization
events.
This dichotomy between engagement events leading to at least limited TCR-associated phosphorylation and those resulting in measurable TCR down-modulation can potentially explain the problems faced by many investigators attempting to replicate the results of Valitutti et al. 19 . For many human and a smaller number of mouse T cell clones we have studied, the immunizing ligand used to maintain the cells and activate them to effector function are partial, not full agonists, as defined by the pattern of TCR-associated phosphorylation (Ref. 27 and Stefanova et al., unpublished observations). Use of such partial agonist ligands to explore the phenomenon of serial engagement will lead to a low calculated ratio of engaged TCR to ligand, as only a small number of all interactions leading to detectable phosphorylation changes results in full activation of the TCR and internalization.
This biochemical insight into the serial engagement problem does not, however, fully account for the differences we have observed using mouse vs human clones. The mouse clones employed and the ligands for their TCR give patterns of signaling comparable to the best of the ligands tested using GDBP, despite the 10- to 20-fold difference in calculated engagement ratios. Evidence that the mouse ligands fall into the same potent agonist class as those used with human cells also comes from examination of the concentrations of peptide required to generate comparable ligand density (a measure of the efficiency of peptide:MHC class II binding) and the concentration of peptide needed to achieve similar biological responses. The MCC peptide employed with A.E7 achieves half-maximal proliferation at <10 pM, an even lower concentration than required by the superagonist ligand of GDBP. One is thus left with either concluding that murine peptide:MHC ligands and TCR cannot undergo the type of serial triggering seen with their human counterparts, or as we think is more likely, that down-modulation itself is less efficient in mouse cells than human cells, even given comparable phosphorylation signatures at the individual TCR level. Because down-modulation measurements can only place a lower limit on the extent of actual engagement of TCR, it would seem imprudent to conclude that mouse T cells do not use this mechanism as extensively as human cells. We clearly see evidence for some serial triggering with mouse cells, and it is likely that this number is an underestimate even with potent ligands, providing an additional explanation for difficulties in some laboratories in appreciating the relationship between serial TCR:ligand binding and response.
Finally, on the broader issue of the relationship between TCR down-modulation and effector responses, the apparent lack of a direct association between these two parameters in some studies 25 can be explained by combining our observations on TCR signaling and down-modulation with data on the hierarchical organization of thresholds for elicitation of different TCR responses 26, 27, 28, 29, 30, 49 . Some activation thresholds are reached with very low levels of overall TCR signaling, which can be provided by a very low fraction of all engaged TCR generating the type of full intracellular signal that would lead to internalization. Because signaling in excess of this very low required level does not increase response above the maximum, exposure to potent ligands can give much more down-modulation than weaker ligands for the same functional outcome. Other responses require a much greater number of full signaling events, and their pattern of elicitation by related ligands should and does largely track TCR down-modulation because one is not examining effects at a plateau level of signaling 27 . Serial engagement does not improve the quality of signaling at the individual TCR complex level, because the nature of the phosphorylation events show only a change in amount and not pattern as ligand density varies over a range markedly altering the extent of serial triggering 2, 46 . What this process does do is reduce the quantitative difference in overall signaling engendered by high and low ligand densities on the APC, as compared with what would be the case if each ligand only activated a single TCR complex. Thus, serial engagement helps to provide a higher absolute level of signal, sustained over a longer time, allowing these response thresholds to be exceeded with only a few ligands present on the APC membrane.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ronald N. Germain, Lymphocyte Biology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10, Room 11N311, 10 Center Drive, MSC-1892, Bethesda, MD 20892-1892. E-mail address:
3 Abbreviations used in this paper: PCC, pigeon cytochrome c ; MBP, myelin basic protein; MCC, moth cytochrome c; PE, phycoerythrin.
Received for publication September 28, 1998. Accepted for publication November 10, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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I. Munitic, P. E. Ryan, and J. D. Ashwell T Cells in G1 Provide a Memory-Like Response to Secondary Stimulation J. Immunol., April 1, 2005; 174(7): 4010 - 4018. [Abstract] [Full Text] [PDF]
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 P. A. Gonzalez, L. J. Carreno, D. Coombs, J. E. Mora, E. Palmieri, B. Goldstein, S. G. Nathenson, and A. M. Kalergis T cell receptor binding kinetics required for T cell activation depend on the density of cognate ligand on the antigen-presenting cell PNAS, March 29, 2005; 102(13): 4824 - 4829. [Abstract] [Full Text] [PDF]
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M. von Essen, C. M. Bonefeld, V. Siersma, A. B. Rasmussen, J. P. H. Lauritsen, B. L. Nielsen, and C. Geisler Constitutive and Ligand-Induced TCR Degradation J. Immunol., July 1, 2004; 173(1): 384 - 393. [Abstract] [Full Text] [PDF]
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M. R. Betts, D. A. Price, J. M. Brenchley, K. Lore, F. J. Guenaga, A. Smed-Sorensen, D. R. Ambrozak, S. A. Migueles, M. Connors, M. Roederer, et al. The Functional Profile of Primary Human Antiviral CD8+ T Cell Effector Activity Is Dictated by Cognate Peptide Concentration J. Immunol., May 15, 2004; 172(10): 6407 - 6417. [Abstract] [Full Text] [PDF]
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N. L. La Gruta, H. Liu, S. Dilioglou, M. Rhodes, D. L. Wiest, and D. A. A. Vignali Architectural Changes in the TCR:CD3 Complex Induced by MHC:Peptide Ligation J. Immunol., March 15, 2004; 172(6): 3662 - 3669. [Abstract] [Full Text] [PDF]
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 K.-H. Lee, A. R. Dinner, C. Tu, G. Campi, S. Raychaudhuri, R. Varma, T. N. Sims, W. R. Burack, H. Wu, J. Wang, et al. The Immunological Synapse Balances T Cell Receptor Signaling and Degradation Science, November 14, 2003; 302(5648): 1218 - 1222. [Abstract] [Full Text] [PDF]
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A. Margalit, S. Fishman, D. Berko, J. Engberg, and G. Gross Chimeric {beta}2 microglobulin/CD3{zeta} polypeptides expressed in T cells convert MHC class I peptide ligands into T cell activation receptors: a potential tool for specific targeting of pathogenic CD8+ T cells Int. Immunol., November 1, 2003; 15(11): 1379 - 1387. [Abstract] [Full Text] [PDF]
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A. K. Stanic, R. Shashidharamurthy, J. S. Bezbradica, N. Matsuki, Y. Yoshimura, S. Miyake, E. Y. Choi, T. D. Schell, L. Van Kaer, S. S. Tevethia, et al. Another View of T Cell Antigen Recognition: Cooperative Engagement of Glycolipid Antigens by Va14Ja18 Natural TCR J. Immunol., November 1, 2003; 171(9): 4539 - 4551. [Abstract] [Full Text] [PDF]
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 V. Hurez, A. Saparov, A. Tousson, M. J. Fuller, T. Kubo, J. Oliver, B. T. Weaver, and C. T. Weaver Restricted Clonal Expression of IL-2 By Naive T Cells Reflects Differential Dynamic Interactions with Dendritic Cells J. Exp. Med., July 7, 2003; 198(1): 123 - 132. [Abstract] [Full Text] [PDF]
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A. E. Annenkov, G. M. Daly, T. Brocker, and Y. Chernajovsky Clustering of immunoreceptor tyrosine-based activation motif-containing signalling subunits in CD4+ T cells is an optimal signal for IFN-{gamma} production, but not for the production of IL-4 Int. Immunol., May 1, 2003; 15(5): 665 - 677. [Abstract] [Full Text] [PDF]
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S.-J. E. Lee, Y. Hori, and A. K. Chakraborty Low T cell receptor expression and thermal fluctuations contribute to formation of dynamic multifocal synapses in thymocytes PNAS, April 15, 2003; 100(8): 4383 - 4388. [Abstract] [Full Text] [PDF]
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 Y. R. Carrasco, M. N. Navarro, and M. L. Toribio A Role for the Cytoplasmic Tail of the Pre-T Cell Receptor (TCR) alpha Chain in Promoting Constitutive Internalization and Degradation of the Pre-TCR J. Biol. Chem., April 11, 2003; 278(16): 14507 - 14513. [Abstract] [Full Text] [PDF]
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B. M. Badran, S. M. Wolinsky, A. Burny, and K. E. Willard-Gallo Identification of Three NFAT Binding Motifs in the 5'-Upstream Region of the Human CD3gamma Gene That Differentially Bind NFATc1, NFATc2, and NF-kappa B p50 J. Biol. Chem., November 27, 2002; 277(49): 47136 - 47148. [Abstract] [Full Text] [PDF]
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L. V. Overtvelt, M. Andrieu, V. Verhasselt, F. Connan, J. Choppin, V. Vercruysse, M. Goldman, A. Hosmalin, and B. Vray Trypanosoma cruzi down-regulates lipopolysaccharide-induced MHC class I on human dendritic cells and impairs antigen presentation to specific CD8+ T lymphocytes Int. Immunol., October 1, 2002; 14(10): 1135 - 1144. [Abstract] [Full Text] [PDF]
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A. G. Schrum and L. A. Turka The Proliferative Capacity of Individual Naive CD4+T Cells Is Amplified by Prolonged T Cell Antigen Receptor Triggering J. Exp. Med., September 16, 2002; 196(6): 793 - 803. [Abstract] [Full Text] [PDF]
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C. Dumont, N. Blanchard, V. Di Bartolo, N. Lezot, E. Dufour, S. Jauliac, and C. Hivroz TCR/CD3 Down-Modulation and {zeta} Degradation Are Regulated by ZAP-70 J. Immunol., August 15, 2002; 169(4): 1705 - 1712. [Abstract] [Full Text] [PDF]
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Z. Borovsky, G. Mishan-Eisenberg, E. Yaniv, and J. Rachmilewitz Serial Triggering of T Cell Receptors Results in Incremental Accumulation of Signaling Intermediates J. Biol. Chem., June 7, 2002; 277(24): 21529 - 21536. [Abstract] [Full Text] [PDF]
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W. Ise, M. Totsuka, Y. Sogawa, A. Ametani, S. Hachimura, T. Sato, Y. Kumagai, S. Habu, and S. Kaminogawa Naive CD4+ T Cells Exhibit Distinct Expression Patterns of Cytokines and Cell Surface Molecules on Their Primary Responses to Varying Doses of Antigen J. Immunol., April 1, 2002; 168(7): 3242 - 3250. [Abstract] [Full Text] [PDF]
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K.-H. Lee, A. D. Holdorf, M. L. Dustin, A. C. Chan, P. M. Allen, and A. S. Shaw T Cell Receptor Signaling Precedes Immunological Synapse Formation Science, February 22, 2002; 295(5559): 1539 - 1542. [Abstract] [Full Text] [PDF]
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 K. Katagiri, M. Hattori, N. Minato, and T. Kinashi Rap1 Functions as a Key Regulator of T-Cell and Antigen-Presenting Cell Interactions and Modulates T-Cell Responses Mol. Cell. Biol., February 15, 2002; 22(4): 1001 - 1015. [Abstract] [Full Text] [PDF]
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 H. M. Diepolder, N. H. Gruener, J. T. Gerlach, M.-C. Jung, E. A. Wierenga, and G. R. Pape Different Levels of T-Cell Receptor Triggering Induce Distinct Functions in Hepatitis B and Hepatitis C Virus-Specific Human CD4+ T-Cell Clones J. Virol., September 1, 2001; 75(17): 7803 - 7810. [Abstract] [Full Text] [PDF]
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M. A. Derby, J. Wang, D. H. Margulies, and J. A. Berzofsky Two intermediate-avidity cytotoxic T lymphocyte clones with a disparity between functional avidity and MHC tetramer staining Int. Immunol., June 1, 2001; 13(6): 817 - 824. [Abstract] [Full Text] [PDF]
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C. Chan, A. J. T. George, and J. Stark Cooperative enhancement of specificity in a lattice of T cell receptors PNAS, May 8, 2001; 98(10): 5758 - 5763. [Abstract] [Full Text] [PDF]
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G. T. Belz, W. Xie, and P. C. Doherty Diversity of Epitope and Cytokine Profiles for Primary and Secondary Influenza A Virus-Specific CD8+ T Cell Responses J. Immunol., April 1, 2001; 166(7): 4627 - 4633. [Abstract] [Full Text] [PDF]
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T. O. Cameron, J. R. Cochran, B. Yassine-Diab, R.-P. Sekaly, and L. J. Stern Cutting Edge: Detection of Antigen-Specific CD4+ T Cells by HLA-DR1 Oligomers Is Dependent on the T Cell Activation State J. Immunol., January 15, 2001; 166(2): 741 - 745. [Abstract] [Full Text] [PDF]
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C. Forestier, F. Deleuil, N. Lapaque, E. Moreno, and J.-P. Gorvel Brucella abortus Lipopolysaccharide in Murine Peritoneal Macrophages Acts as a Down-Regulator of T Cell Activation J. Immunol., November 1, 2000; 165(9): 5202 - 5210. [Abstract] [Full Text] [PDF]
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A. L. Meyer, C. Trollmo, F. Crawford, P. Marrack, A. C. Steere, B. T. Huber, J. Kappler, and D. A. Hafler Direct enumeration of Borrelia-reactive CD4 T cells ex vivo by using MHC class II tetramers PNAS, September 22, 2000; (2000) 190335897. [Abstract] [Full Text]
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A. Qadri, C. G. Radu, J. Thatte, P. Cianga, B. T. Ober, R. J. Ober, and E. S. Ward A Role for the Region Encompassing the c'' Strand of a TCR V{alpha} Domain in T Cell Activation Events J. Immunol., July 15, 2000; 165(2): 820 - 829. [Abstract] [Full Text] [PDF]
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A. G. Schrum, A. D. Wells, and L. A. Turka Enhanced surface TCR replenishment mediated by CD28 leads to greater TCR engagement during primary stimulation Int. Immunol., June 1, 2000; 12(6): 833 - 842. [Abstract] [Full Text] [PDF]
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J. V. Spencer and T. J. Braciale Incomplete CD8+ T Lymphocyte Differentiation as a Mechanism for Subdominant Cytotoxic T Lymphocyte Responses to a Viral Antigen J. Exp. Med., May 15, 2000; 191(10): 1687 - 1698. [Abstract] [Full Text] [PDF]
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V. Lafont, J. Liautard, M. Sable-Teychene, Y. Sainte-Marie, and J. Favero Isopentenyl Pyrophosphate, a Mycobacterial Non-peptidic Antigen, Triggers Delayed and Highly Sustained Signaling in Human gamma delta T Lymphocytes without Inducing Down-modulation of T Cell Antigen Receptor J. Biol. Chem., May 4, 2001; 276(19): 15961 - 15967. [Abstract] [Full Text] [PDF]
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J. R. Cochran, T. O. Cameron, J. D. Stone, J. B. Lubetsky, and L. J. Stern Receptor Proximity, Not Intermolecular Orientation, Is Critical for Triggering T-cell Activation J. Biol. Chem., July 20, 2001; 276(30): 28068 - 28074. [Abstract] [Full Text] [PDF]
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A. L. Meyer, C. Trollmo, F. Crawford, P. Marrack, A. C. Steere, B. T. Huber, J. Kappler, and D. A. Hafler Direct enumeration of Borrelia-reactive CD4 T cells ex vivo by using MHC class II tetramers PNAS, October 10, 2000; 97(21): 11433 - 11438. [Abstract] [Full Text] [PDF]
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