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*
Howard Hughes Medical Institute and Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305;
Nuffield Department of Clinical Medicine, University of Oxford, Headington, United Kingdom; and
Third Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain of IE
molecules (5). A similar Ab recognizing an endogenously
generated complex of I-Ab bound to a fragment of
invariant chain has also been generated (6). Recently, Abs
reactive with peptides derived from an exogenous Ag (hen egg lysozyme)
bound to I-Ab or I-Ak have
been made using cells carrying a large number of such complexes as
immunogens (7, 8). In this study, we describe the production of three Abs that recognize a peptide representing the carboxyl-terminal nine amino acids (residues 95–103) of moth cytochrome c (MCC)3 (IAYLKQATK) bound to the class II MHC molecule IEk. The recognition of this complex by specific T cells has been the subject of many studies (9, 10, 11). The Abs described here show very little recognition of empty IEk molecules but react strongly when IEk is loaded with MCC (95–103). All of these mAbs are sensitive to some changes in the sequence of the helices of IEk and of the bound peptide, demonstrating that the epitope of these reagents is very similar to that recognized by TCRs interacting with these complexes. None of these mAbs shows reactivity with free peptide, and although their epitopes overlap they appear to be different. We also find that one of these reagents (G-35) specifically inhibits lymphokine release by two T cells reactive to IEk/MCC (88–103) but has no effect on a cell recognizing a different IEk-restricted peptide.
We have used the highest affinity of these Abs (D-4) to directly investigate by Scatchard analysis the relationship between the number of specific class II MHC/peptide complexes on an APC and the ability of that cell to stimulate T cells. We find that this relationship is very similar for B cells and a transfected fibroblast, suggesting that the number of complexes generated is the key event in T cell activation. We find that 200–400 complexes per APC are necessary and sufficient to induce a minimal response, whereas maximum stimulation requires >5000 complexes. Further study shows that although each APC in the population bears sufficient complexes to stimulate, activation of only a small fraction of the T cells present occurs. This indicates that T cell stimulation is stochastic when peptide is limiting, a finding that may help to explain the observations of antagonism and partial agonism in T cell responsiveness.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The T cell line 5C.C7 and hybridomas 2B4 and YO1 have been described previously (10, 12, 13). The IEk-bearing cell line (CHO-Ek) used to present peptides is a high expressing Chinese hamster ovary (CHO) transfectant (14). The indicator cell lines for measuring IL-3 and IL-2 concentrations, R6-X and HT-2, respectively, have also been described (15, 16). All cells were maintained in RPMI 1640 supplemented with 10% FBS, 50 mM ß-mercaptoethanol, 2 mM L-glutamate, 100 U/ml penicillin, and 100 U/ml streptomycin.
Synthetic peptides
Peptides were synthesized by the Stanford University PAN facility by standard F-moc chemistry. The peptides used had the following amino acid sequences: MCC (88–103), ANERADLIAYLKQATK; MCC (102S), ANERADLIAYLKQASK; Hb (64–76), GKKVITAFNEGLK.
Generation of IEk-peptide complexes
Soluble IEk was obtained as previously described by expression as a phosphoinositide-linked protein in CHO cells (17). Solubilized purified IEk was bound to appropriate peptide at pH 5 using conditions described previously (17). Free peptide was removed by ultrafiltration, and the buffer was exchanged to PBS. Approximately 70% of such molecules are occupied with offered peptide as assessed using ELISA (18).
Generation of mAbs
Mice were primed by i.p. injection using 10 µg purified IEk/MCC (88–103) complex emulsified in CFA (Calbiochem, La Jolla, CA). Mice were boosted by i.p. injection with 10 µg complex emulsified in IFA on days 15 and 42. Five weeks after the second boost, the mice were injected i.v. with 10 µg complex, and splenocytes were fused to the X63.Ag8.653 myeloma cell line at a ratio of 5. After selection in hypoxanthine-aminopterin-thymine, supernatants from wells containing fused hybridomas were screened for the appropriate reactivities by ELISA as described below. Hybridomas of interest were recloned at least twice to identify stably expressing cells.
Growth and purification of mAbs
mAbs were purified from the supernatants of large scale hybridoma cultures grown in roller bottles or a hollow fiber bioreactor using affinity chromatography with a 1:1 mixture of protein A- and protein G-linked Sepharose (Sigma, St. Louis, MO). The purity of Ab was assessed by 12% SDS-PAGE (19).
ELISA
Reactivity of supernatants and purified Abs was assessed by coating plates (Immulon IV, Nunc, Naperville, IL) overnight at 4°C with 1 µg/ml purified IEk-peptide complexes in PBS. After blocking with PBS containing 2% BSA, Ab at the appropriate concentration, containing competitors where appropriate, was incubated for 1 h at 37°C. After washing, the amount of bound Ab was detected by an incubation with alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma) and color development with Sigma 104 phosphatase substrate. OD was monitored at 405 nm with a microplate reader (Molecular Devices, Palo Alto, CA).
FACS analysis
Adherent cells were released by trypsinization and pulsed with peptide in solution where necessary. Cells were stained with 5 µg/ml of the appropriate Ab in PBS containing 10% FBS, and the amount of bound Ab was detected by subsequent incubation with FITC-labeled goat anti-mouse IgG (Sigma). FACS analysis was performed on a FACScan, and cell sorting was performed on a FACSvantage.
Scatchard analysis
Fab' fragments of D-4 were prepared by papain digestion (20), purified by size exclusion on a TSK 3000 SW column, and labeled with 125I (21) to a specific activity of 1 x 1013 cpm/mol. Preliminary study showed that Fab' binding to cells reached equilibrium within 1 h. Cells at a concentration of 106/ml were pulsed with different concentrations of MCC (88–103) for 6 h and were washed three times. Binding assays for Scatchard analysis were performed essentially as described (22). Briefly, serial dilutions of labeled Fab' were incubated with cells for 60 min at room temperature in a final volume of 15 µl RPMI 1640 with 5% FBS. Cell-bound radioactivity was separated from free Fab' by centrifugation through an FBS cushion. Nonspecific binding was defined as the cpm bound in the presence of a 500-fold excess of unlabeled Fab'. The data were analyzed as described (23).
T cell assays
Assays were performed essentially as described (24). Briefly, CHO-Ek cells that had been prepulsed with different concentrations of peptide were incubated with T cells at the appropriate ratio for 24 h in a volume of 200 ml at 37°C.
Lymphokine activity assays
The supernatants from activation wells were analyzed for IL-3 (5CC7) or IL-2 (hybridomas) levels using indicator cell lines R6-X and HT-2, respectively, as described (17). Briefly, 5 x 103 indicator cells were cultured in six serial dilutions of each supernatant in a volume of 100 µl for either 20 (HT-2) or 40 (R6-X) h. Cells were then pulsed with 0.5 µCi [3H]thymidine for 4 h and harvested onto glass microfiber filters. The radioactivity incorporated was determined using a Packard Matrix 96 direct beta counter, and 1 U/ml IL-2 or IL-3 was defined as the concentration of lymphokine at which half-maximal incorporation occurred. The IL-2 and IL-3 activities were calculated by simultaneously fitting the data from a standard and the test supernatants with a four-parameter logistic curve using the ARCUS RADIMM program.
BIAcore analysis
Abs were immobilized on a BIAcore flow cell by amine-targeted chemistry (25) as described previously (26). Binding of IEk-peptide complexes to the immobilized Ab was performed at room temperature in PBS using a flow rate of 15 µl/min and 15-µl injections of protein at various concentrations with a BIAcore 2000 (Pharmacia, Piscataway, NJ).
Calcium release
Cells were peptide loaded as in the other assays, and T cells were loaded with the calcium sensitive dye fura-2. The intracellular calcium concentration during the interaction of the cells was followed by fluorescence microscopy as described (27, 28). Briefly, fura-2-loaded T cells were added onto a patch of confluent CHO cells on the microscope stage. Intracellular calcium in T cells was determined using C-Imaging-1280 System hardware and the Simca Quantitative Fluorescence Analysis Software Package; both from Compix Imaging Systems (Mars, PA). The imaging system was coupled to a Nikon Diaphot 300 inverted microscope which was equipped with the epifluorescence attachment and a 75-W xenon arc lamp (Nikon, Melville, NY). Alternate excitation of fura-2-loaded T cells at 340 and 380 nm was achieved using a Ludl high speed dual filter wheel (Ludl Electronic Products, Hawthorne, NY) controlled by the Simca software. Images were collected with a charge-coupled device camera (Dage-MTI CCD72) in combination with a SuperGenII intensifier (Dage-MTI, Michigan City, IN) to amplify fluorescence. Analysis of intracellular calcium and the generation of figures were achieved with the Simca software package. Per run, 30–40 cells were picked at random and analyzed. Data of two sets of experiments were pooled.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IEk heterodimers produced as
glycophosphatidylinositol-linked chimeras (piIEk)
are functionally "empty" of endogenous ligand, and >60% of these
molecules can be loaded with a given peptide in vitro (18, 29). This prompted us to try to generate Abs specific for a
complex of IEk and a particular peptide, using
the carboxyl-terminal residues of MCC(95–103) as a model. Either
syngeneic (H-2k) or allogeneic
(H-2d) mice were immunized and boosted with
piIEk/MCC(88–103) complexes using a standard
protocol. ELISA of serum samples showed that both strains made a
polyclonal Ab response that reacted with both the immunogen and with
piIEk that was not prebound to any peptide.
Approximately 200 hybridomas were recovered from both strains, and
supernatants from these were screened by ELISA for reactivity to
piIEk either unbound or associated with
MCC(88–103). Twenty-six hybridomas had measurable reproducible
reactivity, of which the majority (23) were BALB/c
derived. Most of these hybridomas, exemplified by G-23, produce
IEk-reactive Abs with peptide-independent
specificity as assessed by titration of supernatant activity for
IEk ± MCC (Fig. 1
A). Three independently
derived clones from the fusion of BALB/c spleen cells (D-4, G-32, and
G-35) produced Abs that preferentially recognize
IEk associated with MCC as assessed by ELISA
(Fig. 1A).
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B). In contrast, the G-32 Ab is able to recognize the
IEk molecules on these cells even if they had not
been treated with peptide, although the signal does increase slightly
after pulsing (Fig. 1B). These results are compared with
those of a fourth Ab obtained in this series (G-23), which shows no
indication of peptide sensitivity in the ELISA or in the FACS assay
(Fig. 1B). Sensitivity of Abs to peptide and MHC substitutions
To map the epitopes recognized by these Abs, we examined the
effects of substitutions of either the peptide or
IEk sequence on reactivity. Fig. 2 shows the sensitivity of recognition to
selected monosubstituted analogues of MCC(88–103) peptide bound to
soluble IEk as assessed by ELISA. Separate
peptides substituted at residues 97, 99, and 102 were used because
these three positions have been shown to interact with certain TCRs
(11, 30). Two analogues at each position were tested. At
positions 97 and 102, the original amino acid was replaced with either
a positive (lysine) or a negative (glutamate) charge to maximize
potential disruption. At position 99 where the original residue was
positively charged (lysine), a glutamine or glutamate was introduced.
All six substituted peptides bind to IEk as
effectively as the wild-type peptide (11) and are capable
of stimulating specific T cell responses (31).
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To analyze the effect of MHC sequence on Ab recognition, we used the 13
mutants of IEk described by Ehrich et al.
(32), where each cell line expresses a molecule specifying
a single substitution of an amino acid predicted to reside on the
helix. These cell lines were pulsed with wild-type MCC(88–103)
peptide, stained with Ab, and analyzed by cytofluorography. Fig. 3 shows a summary of the results of this
analysis. Only 2 of the 13 mutants show significant alterations in
staining intensity with these Abs. Cell line ß64 shows diminished
staining with all three peptide-dependent Abs but is recognized
effectively by G-23 and other IE-specific Abs (32). In
addition, mutant ß59 affects staining by G-35 and G-32 but not by D-4
or G-23. These results demonstrate that the Abs D-4, G-32, and G-35
have similar but unique specificities, the reactivities of which are
affected by changes in either component of their bipartite ligand.
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). The
peptide-independent Ab G-23 inhibits its own reactivity but does not
affect recognition by D-4, G-32, or G-35. In contrast, recognition with
biotinylated G-35 is inhibited in a dose-dependent manner by G-35 and
more effectively by G-32 and D-4. The reactivities of biotinylated G-32
and D-4 were both inhibited in a similar dose-dependent way by
unmodified G-32 and D-4 but were only marginally affected by G-35.
These results suggest that D-4, G-32, and G-35 have overlapping
epitopes on the IE-MCC complex but that G-35 has a lower affinity.
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Affinity of Abs
To further examine the specificity of the Abs with regard to
peptide occupancy, we directly assessed their affinity for
IEk molecules bound to different peptides using
surface plasmon resonance. Abs were immobilized on separate sections of
a BIAcore (Pharmacia Sensor) chip, and increasing concentrations of
IEk preloaded with either MCC(88–103) or
Hb(64–76) were passed over the surface. Fig. 5 shows representative binding curves for
Abs D-4, G-35, and the anti-IEk mAb 14.4.4S
(33). Both complexes bound to immobilized 14.4.4S, and the
amount associated increased at higher concentrations. In contrast,
whereas binding of IEk complexed with MCC to D-4
and G-35 was clearly evident, no binding of IEk
preloaded with the Hb peptide was detectable. Table I indicates the calculated association
and dissociation rate constants and the inferred dissociation
equilibrium constant for these interactions. The
Kd of 14.4.4S for interaction with
IEk complexed with either MCC or Hb peptide is
100 nM. This compares with values for D-4 and G-35 interacting with
IEk bound to MCC of 10 and 700 nM, respectively.
Both of these Abs have on rates at least as fast as 14.4.4S, but their
off rates differ considerably: D-4 dissociation is 10-fold slower,
whereas G-35 is 10-fold faster. The inability to measure association of
D-4 and G-35 with IEk/Hb complexes using this
technique indicates that their affinities must be <100 µM, because
we can measure such values for interaction of the same
IEk-peptide complexes with soluble TCR
(26).
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To test whether the
IEk-MCC(88–103)-specific Abs generated
represented "surrogate" TCRs, we next examined their ability to
specifically block T cell recognition (Fig. 6). The T cell clone 5C.C7 and the T cell
hybridoma 2B4 both recognize MCC(88–103) restricted by
IEk, and their specificities have been
extensively studied (11), whereas the hybridoma YO1
recognizes Hb(64–76) in conjunction with IEk
(13). Although the response of all three T cells is
inhibited by Ab G-32, those to MCC are abrogated at 0.1 µg/ml,
whereas equivalent inhibition of YO1 requires >10 µg/ml Ab.
Recognition by all three T cells is also inhibited by the
peptide-independent G-23 Ab at concentrations >1 µg/ml. In contrast
to these results, although Ab G-35 inhibits the response of both 5C.C7
and 2B4 at >1 µg/ml, it has no effect on the responsiveness of YO1
at concentrations up to 200 µg/ml. Surprisingly, the Ab D-4, the
epitope of which overlaps that of G-32 and G-35 (Figs. 2 and 3), and
which has the highest affinity (Table I), is very poor at inhibiting
responses by the MCC(88–103)-specific T cells. Additional experiments
showed that the concentration of G-35 necessary to inhibit specific
recognition is dependent on the concentration of peptide used to pulse
the APCs (data not shown). Additionally, high levels of D4 partially
inhibit T cell responses specifically when APCs are pulsed with very
low peptide concentrations (data not shown).
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We next decided to use Scatchard analysis with D-4 to directly
examine the relationship between number of MHC-peptide complexes on an
APC and its ability to stimulate T cells. Fab' fragments were prepared,
and their affinity after iodination for IEk-MCC
on peptide pulsed cells was found to be 2–5 nM (data not shown), in
general agreement with the value measured using a biosensor. We next
determined the number of D-4-reactive sites generated on
CHO-Ek cells that had been prepulsed with
different concentrations of MCC(88–103). After removal of excess
peptide, the ability of these cells to stimulate different T cells at
varying T:APC ratios was also established, and the same cells were
analyzed by FACS after staining with D-4. Fig. 7A shows the response of the
cell line 5C.C7 to these cells using standard assay conditions. This
shows that these APCs can cause this T cell to release lymphokine only
after being pulsed with >30 nM peptide. Similar results were observed
for the hybridoma 2B4 and for T cells obtained from a mouse transgenic
for the TCR expressed by 5C.C7 (34). Fig. 7A
also shows the result of the Scatchard analysis for these cells, and
Fig. 7B shows the relationship between number of complexes
generated and stimulation capacity. The data presented in Fig. 7C indicates that there are 200 endogenous
IEk-peptide complexes on the
CHO-Ek cells that can interact with D-4,
representing 10-4 of total
IEk molecules expressed. At least a further
200–400 specific complexes per cell, corresponding to a peptide pulse
of 10–30 nM, are needed for the cells to be active in T cell
stimulation, whereas maximum stimulation is attained at 5000
complexes per cell. Fig. 7D shows that the staining
intensity of D-4 by FACS mirrors the increase in the number of
complexes. In a separate experiment, we also determined the number of
complexes generated after pulsing with a partial agonist for this cell
line, MCC(102S), and found these to be almost identical (Fig. 7, A and D). In addition, we examined the relative
ability of a B cell line (CH-27) compared with
CHO-Ek cells to stimulate the 5C.C7 cell line
(Fig. 7C). Although these cells load peptide more poorly,
the relationship between the number of complexes formed and T cell
stimulation capacity was almost identical.
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A,
restricting the number of T cells present imposes a limit on the
maximum IL-3 that can be released, representing maximal activation of
each cell. Thus, 820 T cells produce a maximum of 2 U/ml IL-3,
indicating that each cell can release 0.0025 U/ml (0.0005 U/cell). In
addition, Fig. 8B shows that a fixed number of APCs with
different numbers of complexes have only a limited capacity to
stimulate. Thus, cells pulsed with the limiting concentration of
peptide (30 nM) can elicit only 3 U/ml of IL-3 irrespective of the
number of T cells added. This corresponds to a maximal stimulation of
1200 T cells.
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C, this had no effect
on T cell stimulation. Additionally, fitting the Scatchard data to a
binomial equation, assuming that peptide loading is noncooperative,
illustrates that the SD for the number of complexes per cell after a 30
nM pulse would only be ±18 (Dr. Ron Kantor, unpublished observation).
This implies that none of the cells in this population would express
more than 500 IEk-MCC complexes. Taken together,
these findings indicate that all of the cells in the APC population
have approximately equivalent stimulatory capacity.
We next examined whether the limited activation observed represented
partial activation of all added T cells, or full activation of a
limited subset. Previous studies in a different system using a reporter
gene suggested that the latter is the case (35). In our
studies, we decided to examine the Ca2+ release
after stimulation with cells carrying different numbers of complexes.
Accordingly, cells were pulsed with peptide as above and were incubated
with T cells. Ca2+ release was assessed by
fluorescence microscopy, and the results are shown in Fig. 8D. This indicates that when peptide is limiting, only a
small number of T cells are being stimulated, even though the analysis
above shows that all of the APCs have equivalent number of complexes.
This strongly suggests that there is a stochastic component to
T cell activation within this range. The finding that there is a
maximum degree of stimulation also indicates that those cells that are
not activated by such an encounter become refractory to further
stimulation within the time course of the assay.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The similarity between these reagents and TCRs is further suggested by
the observation that one of them (G-35) specifically and efficiently
inhibits the release of lymphokines by different T cells in response to
IEk-MCC. Interestingly, Ab D-4, which has a
higher affinity for the IEk-MCC complex (Table I), was extremely poor at inhibiting lymphokine release by these T
cells. This differential blockade of T cell activation in vitro by G-35
and D-4 is mirrored by their relative effectiveness at blocking
positive selection of developing thymocytes specific for
IEk-MCC in vivo (37). Although these
Abs interact with different kinetics and affinities, it seems unlikely
that this is an explanation for preferential inhibition by G-35 because
this has the lowest affinity and fastest off rate (Table I). The faster
on rate for G-35 is unlikely to be exerting an effect because the APCs
were preincubated with Ab in our experiments. Although partially
overlapping, the epitopes for these Abs are clearly different, and it
is possible that this influences efficiency of inhibition. Thus,
whereas D-4 is most sensitive to substitutions throughout the MCC
peptide sequence (Fig. 2), it may be unable to block access for TCR
engagement, although this seems unlikely because a soluble TCR inhibits
interaction of this Ab with IEk-MCC complexes on
cells (26). Alternatively, it is possible that G-35 is
more potent because it binds in a such a way that it also hinders
additional interactions of IEk. Recognition by
G-35 is sensitive to alterations in more of the ß helical residues of
IEk tested than D-4, suggesting that it may
interact more with the MHC molecule itself. The region of
IEk to which G-35 appears to bind (ß59-ß64,
Fig. 3) partially overlaps the site in HLA-DR1 (ß58–ß69) detected
by Ab KL-295, which undergoes a conformational change after association
with peptide (38). Such a
peptide-dependent alteration may play an important role in generating
arrangements of IEk-MCC complexes that are
stimulatory for T cells, perhaps by altering multimerization on the
surface of the APC. Supporting such a role is the observation that Ab
G-32, which has an epitope similar to that of G-35 but is less
sensitive to sequence of bound peptide, is particularly effective at
inhibiting responses of T cells (Fig. 6). This area may be less
occluded in class II MHC molecules than the corresponding region of
class I after TCR binding as a result of the more orthogonal mode of
interaction (39).
The generation of these mAbs has allowed us to directly quantitate the relationship between number of antigenic complexes per cell and lymphokine release by T cells. We find that 200–400 complexes per cell are necessary and sufficient to activate at least a small fraction of coincubated T cells. This is consistent with previous indirect studies that have estimated fractional class II MHC occupancy using either radiolabeled peptide or a functional readout (40, 41, 42). We find that the same number of limiting complexes is sufficient to stimulate two different T cells maintained in vitro as well as ex vivo T cells. The quantitation of loading of a B cell line (CH27) with MCC peptide assessed using D4 is almost identical with that previously reported by Kimachi et al. (42) using a radioiodinated peptide of related sequence. In contrast to this result, it has been reported that the number of complexes necessary to stimulate class I MHC-restricted T cells can vary greatly (43). Indeed, one report has suggested that a single complex may be sufficient (44). Although it is possible that the activation of class I MHC-restricted T cells is much more sensitive than class II MHC-restricted cells, such indirect measurements are susceptible to a number of errors (43). In particular, the need to use much higher concentrations of cells when binding radiolabeled peptide to achieve quantifiable signal compared with those used in pulsing for T cell lysis may underestimate loading necessary for stimulation because the effective concentration of receptors differs. Analysis similar to that described in this report using the recently described Abs specific for class I MHC-peptide complexes (36, 45) may resolve some of these discrepancies.
Previous quantitative studies have been unable to measure the number of complexes generated on per cell basis. We have therefore used these reagents to investigate what "minimal activation" represents. We find that a population of cells expressing minimal levels of antigenic complexes can only elicit a fractional lymphokine release, irrespective of the number of T cells added. This is not due to a skewed distribution of complexes within the population because removal of the top 20% of APCs with the highest D-4 expression had no effect on stimulation capacity. We also find that at this margin only a fraction of T cells are induced to release their intracellular stores of calcium. The direct implication of these results is that at limiting Ag dose, which is probably the level at which most real T cell responses occur, activation of T cells is stochastic even when the APC has sufficient complexes to activate. A recent report (46) indicated that very small numbers of class II MHC-peptide complexes (1, 2, 3, 4, 5, 6, 7, 8, 9, 10), although being unable to elicit lymphokine release, can induce an anergic effect on T cells. This may underpin the findings in this study, because at the margin the decision may be variable. The molecular reasons for this density-dependent effect are unclear but may be related to the rate at which complexes multimerize or otherwise interact to induce the TCR down-regulation that seems to occur (47, 48). These findings also suggest a possible explanation for the finding that the positive or negative selection of some developing class I MHC-restricted T cells can be mediated by different concentrations of the same peptide (49).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Philip A. Reay, Nuffield Department of Clinical Medicine, University of Oxford, John Radcliffe Hospital II Headington, OX3 9DU, U.K.
3 Abbreviations used in this paper: MCC, moth cytochrome c; CHO, Chinese hamster ovary.
Received for publication November 30, 1999. Accepted for publication March 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ßT cell receptors. Annu. Rev. Immunol. 16:523.[Medline]
T cells. Cell 76:29.[Medline]
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B. Laugel, J. M. Boulter, N. Lissin, A. Vuidepot, Y. Li, E. Gostick, L. E. Crotty, D. C. Douek, J. Hemelaar, D. A. Price, et al. Design of Soluble Recombinant T Cell Receptors for Antigen Targeting and T Cell Inhibition J. Biol. Chem., January 21, 2005; 280(3): 1882 - 1892. [Abstract] [Full Text] [PDF]
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T. Mareeva, T. Lebedeva, N. Anikeeva, T. Manser, and Y. Sykulev Antibody Specific for the Peptide{middle dot}Major Histocompatibility Complex: IS IT T CELL RECEPTOR-LIKE? J. Biol. Chem., October 22, 2004; 279(43): 44243 - 44249. [Abstract] [Full Text] [PDF]
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N. J. Poloso, A. Muntasell, and P. A. Roche MHC Class II Molecules Traffic into Lipid Rafts during Intracellular Transport J. Immunol., October 1, 2004; 173(7): 4539 - 4546. [Abstract] [Full Text] [PDF]
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F. Andre, N. Chaput, N. E. C. Schartz, C. Flament, N. Aubert, J. Bernard, F. Lemonnier, G. Raposo, B. Escudier, D.-H. Hsu, et al. Exosomes as Potent Cell-Free Peptide-Based Vaccine. I. Dendritic Cell-Derived Exosomes Transfer Functional MHC Class I/Peptide Complexes to Dendritic Cells J. Immunol., February 15, 2004; 172(4): 2126 - 2136. [Abstract] [Full Text] [PDF]
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T. J. Dillon, V. Karpitski, S. A. Wetzel, D. C. Parker, A. S. Shaw, and P. J. S. Stork Ectopic B-Raf Expression Enhances Extracellular Signal-regulated Kinase (ERK) Signaling in T Cells and Prevents Antigen-presenting Cell-induced Anergy J. Biol. Chem., September 19, 2003; 278(38): 35940 - 35949. [Abstract] [Full Text] [PDF]
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O. Acuto T Cell-Dendritic Cell Interaction in Vivo: Random Encounters Favor Development of Long-Lasting Ties Sci. Signal., July 22, 2003; 2003(192): pe28 - pe28. [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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E. M. Hiltbold, N. J. Poloso, and P. A. Roche MHC Class II-Peptide Complexes and APC Lipid Rafts Accumulate at the Immunological Synapse J. Immunol., February 1, 2003; 170(3): 1329 - 1338. [Abstract] [Full Text] [PDF]
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 Q. Ge, J. D. Stone, M. T. Thompson, J. R. Cochran, M. Rushe, H. N. Eisen, J. Chen, and L. J. Stern Soluble peptide-MHC monomers cause activation of CD8+ T cells through transfer of the peptide to T cell MHC molecules PNAS, October 15, 2002; 99(21): 13729 - 13734. [Abstract] [Full Text] [PDF]
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R. J. DiPaolo and E. R. Unanue Cutting Edge: Chemical Dominance Does Not Relate to Immunodominance: Studies of the CD4+ T Cell Response to a Model Antigen J. Immunol., July 1, 2002; 169(1): 1 - 4. [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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P. S. Andersen, C. Geisler, S. Buus, R. A. Mariuzza, and K. Karjalainen Role of the T Cell Receptor Ligand Affinity in T Cell Activation by Bacterial Superantigens J. Biol. Chem., August 31, 2001; 276(36): 33452 - 33457. [Abstract] [Full Text] [PDF]
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P. S. Andersen, C. Menne, R. A. Mariuzza, C. Geisler, and K. Karjalainen A Response Calculus for Immobilized T Cell Receptor Ligands J. Biol. Chem., December 21, 2001; 276(52): 49125 - 49132. [Abstract] [Full Text] [PDF]
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