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-Chains and Closely Related 
-Chains1
Section of Immunobiology, Yale University School of Medicine and Howard Hughes Medical Institute, New Haven, CT 06510
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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- and
-chains contacting up to five residues of the peptide. We have used
two TCRs that recognize the same antigenic peptide and have identical
V8.2 chains, but differ in all three CDR of their related V2
chains, to examine the fine specificity of the TCR:peptide contacts
that lead to activation. By generating a peptide library containing all
20 aa residues in the five potential TCR contact sites, we were able to
demonstrate that the two similar TCRs responded differentially when
agonist, nonagonist, and antagonist peptide functions were examined.
Dual substituted peptides containing an agonist residue at the N
terminus, which interacts with CDR2, and an antagonist residue at
the C terminus, which interacts with the CDR3, were used to show
that the nature of the overall signal through the TCR is determined by
a combination of the type of signal received through both the TCR -
and -chains. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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-chain
complementarity-determining region 2 (CDR2) (5). The fifth
residue (I) was shown to interact with both the TCR - and the TCR
-chains via their CDR3 (5). In addition, the C-terminal
E in the eighth position of the CA peptide was shown to interact with
the TCR CDR3 (5). The crystal structure exhibiting
interactions between two different human TCRs and HLA-A2 was recently
determined and showed that the TCR binds the MHC class I molecule in a
diagonal pattern (6) consistent with the motif predicted
by Sant’Angelo and colleagues (5) for MHC class II
molecules. However, the crystal structure containing a complex of the
D10 TCR interacting with the MHC class II molecule
I-Ak bound with cognate Ag showed that the D10
TCR crossed the peptide in an orthogonal manner (7). The
D10 TCR:I-Ak/CA complex crystal
structure demonstrates interactions between the CA peptide residue 2
and the D10 TCR CDR1 and CDR3 domains, the CA peptide residue 5
with the D10 TCR CDR3 domain, and the CA peptide residue 8 with the
D10 TCR CDR3 and CDR3 domains, slightly modified from the
interaction we previously predicted (5, 7).
Single amino acid changes in the peptide TCR contacts have been shown
to alter the nature of the biochemical signal received through the TCR,
changing the response from an agonist to a nonagonist, partial agonist,
or antagonist T cell response (8, 9, 10). Each type of signal
has a unique biochemical signature pattern with agonist peptides
eliciting a signal characterized by TCR 
-chain tyrosine
phosphorylation to the p21 and p23 forms of equal intensity
(11, 12, 13, 14, 15). In addition, the tyrosine kinase Zap-70 is
recruited to the TCR complex and is activated following tyrosine
phosphorylation (16). Partial agonist peptides have been
described that alter the nature of the biochemical signal that leads to
T cell activation and cytokine production, but no progression through
the cell cycle (17, 18). Antagonist peptides lead to a
reduction in cellular responses associated with T cell activation and
are characterized biochemically by an alteration in the ratio of
phospho- p21:p23 and a lack of phosphorylation of the recruited
Zap-70 (8, 10, 11, 12, 19, 20) possibly by rapid recruitment
of the tyrosine phosphate Src homology protein 1 (SHP-1) to the
molecular complex involved in T cell Ag recognition (15).
Nonagonist peptides do not elicit any cellular responses or biochemical
signals (15).
In this study, we use two well-characterized T cell clones, D10 and
AK8, that were obtained in a similar fashion from an
H-2k mouse immunized with the Ag CA, and
recognizing the same I-Ak-binding peptide
CA134–146. The D10 and AK8 T cell clones have an identical V8.2
chain and two similar V2 chains, with amino acid differences in all
three CDRs. The sequence differences in the V2 chain alter how the
two TCRs interact with and respond to CA134–146
analogue peptides containing single amino acid changes in the TCR
contact residues. The D10 TCR is antagonized with peptides containing
substitutions of the E at position 8 (20), while AK8 is
antagonized by peptides with substitutions of the R at position 2
(21). Even though the TCR -chains are identical, AK8 is
not antagonized by position 8 analogue peptides, demonstrating a role
for both TCR chains in determining the nature of the TCR signal. We
also identified analogue peptides at positions 2, 5, and 8 that
resulted in differential agonist responses when D10 and AK8 were
compared. In addition, using a peptide with two different
substitutions, the first at position 2 that enhances D10 responsiveness
and the other at position 8 that mediates D10 antagonism, we show that
the overall nature of the D10 response is a combination of the two
responses. Thus, the TCR - and -chains contribute similarly and
function cooperatively in mediating T cell signaling leading to
cellular responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Mice were either purchased from The Jackson Laboratory (Bar Harbor, ME) or reared in our colony at Yale University (New Haven, CT). All mice were between 5 and 12 wk of age upon use.
Peptides
The CA134–146 synthetic peptide (HRGAIEWEGIESG) coding for residues 134–146 of CA and the CA134–146 analogue peptides R2H, R2G, I5A, I5G, I5N, I5L, I5M, I5V, W7L, W7M, E8A, E8D, E8N, R2HE8A, and R2GE8A were synthesized and HPLC purified by the W. M. Keck Foundation Biotechnology Resource Laboratory at Yale University. CA134–146 synthetic peptides with substitutions of all 20 aa at positions R2, G3, I5, W7, and E8 were synthesized using the multipin cleavable peptide synthesis kit from Chiron Mimotopes Peptide Systems (San Diego, CA) using GAP linker pins, as previously described (20). The substituted peptides were screened for stimulatory activity and production of IL-4 by incubating 5 or 10 µl of the peptide mixture with D10 or AK8 cells and APC, as described below.
Cells
The D10.G4.1 (D10)- and AK8-cloned Th2 lines were developed in this laboratory and have been previously described (21, 22). D10 and AK8 were maintained by restimulation in Click’s Eagle’s-Hanks’ amino acids medium containing 5% FCS (Click’s 5%) and 100 µg/ml CA (Sigma, St. Louis, MO) in the presence of inactivated spleen cells every 2–4 wk.
Proliferation and antagonist assays
D10 and AK8 were stimulated as above and allowed to rest for at least 9 days before use. Cells were washed four times, and 1 x 104 cells were cocultured with B10.BR splenocytes (I-Ak) as APC in the presence of the CA134–146 Chiron peptide library diluted 1/10 or 1/20. For proliferation assays conducted with HPLC-purified peptides, the peptides were diluted 1/10 from 0.001 to 10 µM. For the antagonist assay, the spleen cells were prepulsed in Click’s 0% containing various doses of CA134–146 for 2 h at 37°C. The APC were washed four times, and 2 x 105 cells were added to each well. Chiron peptides were added at a 1/10 or 1/20 dilution, and the HPLC-purified peptides were diluted 1/3 from 1.2 to 100 µM. Cultures were pulsed after 48 h with 1 µCi [3H]TdR and harvested after 15–18 h.
Cytokine secretion
Secretion of IL-4 by D10 and AK8 was detected in culture supernatants collected 24 h following stimulation of D10 and AK8 with CA134–146 analogue peptides presented by B10.BR spleen cells and assayed by ELISA for the presence of IL-4 using anti-IL-4 mAb purchased from PharMingen (San Diego, CA), as previously described (20).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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We have previously described and extensively studied the cellular
responses of the CD4 Th2 D10 clone after TCR ligation
(22). The D10 clone bears a TCR consisting of AV2S5 and
BV8S2 restricted to I-Ak specific for a peptide
of CA encompassing residues 134–146 (CA134–146)
(HRGAIEWEGIESG) (23). In addition, we have previously
described the AK8 Th2 clone with the same MHC restriction and peptide
specificity as D10 (21). AK8 and D10 share an identical
V-chain, but differ in the V2 gene segment use that alters CDR1
and CDR2, as well as differing markedly in the CDR3, resulting in
several changes in specificity. D10 is alloreactive to a variety of MHC
class II molecules (22, 24, 25), but AK8 has no
alloreactivity (26). In addition, the position of the
single amino acid change rendering the CA134–146
wild-type (wt) peptide antagonist differs for the D10 and AK8 clones
(20, 21).
We have previously described an orientation of the D10 TCR
to the peptide/MHC complex demonstrating critical
molecular contacts for TCR function between the TCR CDR
and domains and amino acid residues at positions R2, I5, and E8 of
the CA134–146 peptide (5). This
identification of the important TCR contact residues in
CA134–146 led us to further investigate the
cellular responses invoked by CA134–146 analogue
peptides containing amino acid substitutions in the TCR contacts to
more accurately define the TCR:peptide:MHC interaction. Specifically,
we were interested in identifying and comparing TCR contact residues
that were critical for mediating cellular responses leading to
cell proliferation and cytokine secretion in D10 and AK8. To
accomplish this goal, we generated a peptide library containing
CA134–146 analogue peptides with substitutions
at the TCR contact residues: R at position 2 (R2), G at position 3
(G3), I at position 5 (I5), W at position 7 (W7), and E at position 8
(E8) with all 20 aa (Fig. 1
). We then
used D10 and AK8 to screen our peptide library for the ability of
individual analogue peptides to induce cell proliferation and IL-4
secretion (Fig. 1). When positions R2 and G3 were substituted with all
20 aa, the resulting 40 peptides retained the ability to stimulate D10
(Fig. 1, A and B). Similarly, when the D10
culture supernatants were examined 24 h after stimulation with R2-
and G3-substituted peptides for the production of IL-4 by ELISA,
measurable amounts of IL-4 were detected with all 40 peptides (Fig. 1, F and G). Although the level of cell
proliferation was similar for all 40 R2- and G3-substituted peptides,
the level of IL-4 production varied a great deal. Thus, these data
indicate that the production of IL-4 by D10 is a more sensitive means
to identify alterations in biological responses invoked by a
stimulating peptide than is cell proliferation. These data are in
accordance with our previous finding and with a recent study
demonstrating the importance of position R2 contacts with the TCR for
TCR functionality (5, 7), and although differences in the
level of IL-4 secretion were detected in the supernatants of D10
stimulated with R2 and G3 analogue peptides, these two residues do not
appear to be critical for cellular proliferation mediated through the
D10 TCR. The disparity between proliferation and IL-4 production,
however, suggests that some peptides deliver a greater or lesser signal
via the TCR (R2G, G3Y; Fig. 1, F and G).
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, K
and P). Like D10, substitutions at G3 had little effect on
AK8 with only two substitutions, L and E, resulting in decreased
proliferation and IL-4 secretion and P resulting in a loss of effector
function (Fig. 1, L and Q). Thus, these data show
that AK8 cellular responses are dependent upon contacts between the TCR
and position R2 of CA134–146, with G3 contacts
with either TCR being of minimal importance. The lack of effect with
G3-substituted peptides is consistent with the D10
TCR:CA/I-Ak complex crystal structure that shows
the G3 residue buried with no exposure to the TCR or MHC
(7).
Substitutions at position I5 with A, S, T, Q, K, M, and P led to a
complete loss of proliferation by D10 (>98%), while substitutions
with G, E, and F led to partial loss in the proliferation of D10
(90–98%) (Fig. 1C). Similarly, residues G, E, Q, K, F, and
P led to a complete loss in IL-4 production (>98%), while residues S,
T, H, R, M, and Y led to a partial loss of IL-4 production (90–98%)
(Fig. 1H). However, there was not a direct correlation in
the loss of cell proliferation and reduction in IL-4 production. For
example, the residues R, L, and C resulted in >86% reduction in
secreted IL-4, while maintaining cell proliferation at the same level
as the wt peptide I5I (Fig. 1, C and H). This
indicates that proliferation of the D10 T cell clone is very sensitive
to IL-4. AK8 cellular responses were very sensitive to I5 substitutions
with all substitutions except V, L, T, H, M, F, and Y, reducing cell
proliferation by >90% (Fig. 1M). Likewise, only
substitutions V, L, and M led to IL-4 secretion that was >90% of that
to the control I5I peptide (Fig. 1R). The loss of both cell
proliferation and IL-4 production by I5 substitutions confirms the
major role of this amino acid in mediating both D10 and AK8 cellular
responses (5). Because the interaction of the D10 TCR with
the CA/I-Ak complex is thought to center on
position I5 (7), our findings demonstrate both common and
disparate effects of I5 substitutions. Strong responses of D10 to the
I5L and I5N substitutions are mimicked by AK8 only in the strong
response to I5L, but not I5N, while AK8 responds strongly to I5V and
I5M, which do not stimulate D10 (Fig. 1, H and
R). This is most likely due to differences in CDR3
residues GSFNKLTFGAG in D10 and PNTDVVT in AK8.
The substitution of amino acids at position W7 had a more dramatic
effect on responsiveness of D10 than the I5 substitutions. All
substitutions except V, S, F, and Y decreased D10 cell proliferation by
more than 98% (Fig. 1D). When the loss of IL-4 production
was examined, all substitutions except S, F, and Y resulted in >80%
reduction in the production of measurable IL-4 by D10 (Fig. 1I). Similarly, when AK8 cell proliferation and IL-4
production were measured, all substitutions except S and Y resulted in
a >90% reduction (Fig. 1, N and S). A study by
Reinherz et al. (7) showed the W7 residue, which has a
bulky indole ring, partially exposed to the TCR-binding surface and
interacting with the TCR CDR3 domain residue
Phe101, which also contacts the I5 residue. Thus,
it is conceivable that amino acid substitutions in the W7 residue could
alter TCR CDR3 interactions with residue I5, altering recognition
due to the pivotal role of I5 in TCR recognition (7).
Although substitutions at position 7 were not tested with D10 single
chain transgenic mice, it appears from these data that the shared
-chains may account for this similarity in response of D10 and AK8
to the set of position 7 substitutions.
Finally, the E8 substitutions G, A, V, S, T, K, C, M, F, and Y resulted
in a >98% loss in D10 proliferation, and the I substitution resulted
in a >95% loss (Fig. 1E). When IL-4 secretion was
examined, all substitutions except L, D, N, and H resulted in a >90%
reduction in the level of IL-4 detected (Fig. 1J). AK8 cell
proliferation was reduced by >95% with the G, A, V, L, I, S, T, H, K,
C, M, F, Y, W, and P E8 substitutions (Fig. 1O), and all E8
substitutions resulted in a >95% loss in IL-4 secretion (Fig. 1T).
Because the multipin cleavable peptide synthesis kit from Chiron
Mimotopes Peptide Systems generated crude preparations of peptides that
did not allow for control of peptide quality or concentration, a direct
comparison of the level of stimulatory activity and peptide
concentration was not possible for any given peptide or between
peptides. Thus, the peptides were titrated over a 100-fold range to
confirm their potency (data not shown). The data shown in Fig. 1 are
representative of the titrations performed.
The lack of D10 cell proliferation and subsequent IL-4 production
induced by peptides with substitutions at positions I5, W7, and E8, all
of which contact the CDR3 in the D10 -chain (7),
demonstrates that critical contacts leading to D10 activation are
mediated through the -chain contacting the C-terminal half of the
CA134–146 peptide. In addition, contacts between residue E8 and the
CDR3 of the D10 -chain were found to be equally important for
effector functions, demonstrating a critical role for the TCR -chain
in D10 TCR signaling as well. The importance of the C-terminal residues
was further reinforced by the effects of substitutions at position R2,
which had little impact on D10 cellular responses. In comparison, like
D10, positions I5, W7, and E8 were important for AK8 cellular
responses. One major difference between D10 and AK8 was the importance
of position R2 in AK8 responsiveness. As previously discussed, the AK8
- and D10 -chains have amino acid variability in all three CDRs.
These differences render the contacts between the CDR domains with
the CA134–146 peptide essential for AK8 TCR
signaling.
Differential recognition of position 2, 5, and 8 CA134–146-substituted peptides by D10 and AK8
To further confirm the unique specificities of the D10 and AK8
TCR, we chose those peptides examined in Fig. 1 that exhibited opposing
cellular responses in the two clones for further study, and had them
resynthesized and HPLC purified. This allowed for a direct comparison
of the stimulatory activity of the individual peptide analogues based
on known protein concentrations. Using IL-4 as the more sensitive
readout of T cell activation, we chose to examine the peptides R2H,
R2G, E8D, E8N, and I5N for the ability to stimulate D10, but not AK8
activation, and peptides I5L, I5M, and I5V for the ability to stimulate
AK8, but not D10 activation. Shown in Fig. 2A is a comparison of the
proliferative response of D10 and AK8 to the
CA134–146 wt peptide, with both T cell clones
responding to 0.1 µM peptide with a similar stimulation profile.
Interestingly, while the D10 antagonist E8A (20) is a
nonagonist for AK8 (Fig. 1O), the AK8 antagonist peptide R2H
(21) induced a proliferative response in D10 similar to
the CA134–146 peptide (Fig. 2B).
Thus, the sequence differences in the CDR domains of AK8 and D10
determine how the N-terminal residues of the peptide are recognized.
Further confirmation of this is the proliferative response to the R2G
peptide. AK8 is nonresponsive to R2G, while this peptide is a
superagonist for the D10 TCR. D10 responds to the R2G peptide at a
concentration of 0.01 µM, 10-fold less peptide than required for the
response observed with the CA134–146 peptide
(Fig. 2, A and C). In addition to position
2-substituted peptides, differential recognition of peptides by D10,
but not AK8, is also observed with the position 8-substituted peptides
E8D (Fig. 2D) and E8N (Fig. 2E) and the position
5-substituted peptide I5N (Fig. 2F). By comparison, the only
peptide we could identify that resulted in AK8 responsiveness, but not
D10 responsiveness, was the I5M peptide (Fig. 2H). In
addition, we identified the I5L and I5V peptides, which are weak D10
agonists requiring a peptide dose of 1–10 µM to observe a
proliferative response, while proliferation of AK8 was observed at 0.1
µM (Fig. 2, G and I). Further analysis of the
I5V peptide showed that the peptide is a partial agonist based on
tyrosine-phosphorylation patterns following engagement of the D10 TCR
with I5V (our unpublished observations). We confirmed this by
performing semiquantitative RT-PCR to identify IL-4 transcripts
following engagement with I5V (our unpublished observations). Thus, the
high level of D10 proliferation observed in Fig. 1C, not
observed in Fig. 2I, is most likely due to a low level of
IL-4 production induced by a high level of peptide concentration that
could not be measured due to the nature of the crude peptide
preparation used in Fig. 1.
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To determine which TCR contact residue(s) of
CA134–146 could mediate TCR antagonism of D10
and AK8, we screened our peptide library for the ability to inhibit D10
and AK8 cell proliferation. We have previously demonstrated antagonism
of AK8 cell proliferation with the position 2-substituted peptide R2H
(21). Thus, to confirm the specificity of our peptide
library, we screened the R2-substituted peptides for the ability to
inhibit AK8 cell proliferation in an antagonist assay. As shown in Fig. 3E, the R2 substitutions G, A,
S, Q, and H inhibited AK8 cell proliferation to prepulsed APC by 80,
85, 75, 80, and 78%, respectively. In comparison, D10 was not
antagonized by any of the R2-substituted peptides (Fig. 3A).
The suppression observed with the G and H substitutions in Fig. 3A most likely is not due to antagonism, as indicated by the
robust IL-4 production shown in Fig. 1F, but rather may be
explained by the presence of peptide in greater concentration than the
concentration required for a maximal proliferative response, which is
known to be inhibitory. No antagonism was observed for either D10 or
AK8, with G3-substituted peptides as predicted from the proliferative
responses to these peptides shown in Fig. 1 (data not shown). The
examination of I5 substitutions showed no D10 antagonism (Fig. 3B) and only peptide I5A-antagonized AK8, inhibiting
proliferation by 77% (Fig. 3F). Similar results were
observed with the W7 substitutions with no antagonism of D10 (Fig. 3C) and antagonism of AK8 with only the W7L peptide leading
to a 75% inhibition of proliferation (Fig. 3G). We have
previously reported that the D10 TCR is antagonized with E8-substituted
peptides (20), and this result is confirmed in Fig. 3D, showing antagonism with the G, A, V, S, and T
substitutions leading to inhibition of proliferation by 92, 93, 95, 92,
and 89%, respectively. Because D10 and AK8 share an identical TCR
-chain, we reasoned that AK8 would also be antagonized by
substitutions at position E8. However, the data shown in Fig. 3H show no antagonism of AK8 with the E8-substituted
peptides and demonstrate that the TCR -chains of D10 and AK8 must
interact differently, as the identical peptide:MHC molecule leads to
distinct cellular responses.
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were resynthesized, HPLC purified, and screened for their ability to
inhibit proliferation of D10 and AK8. As shown for D10 in Fig. 4A, only the position 8
substitutions resulted in antagonism of D10, with E8A resulting in
complete antagonism. The E8-substituted peptides E8T, E8S, E8V, and E8G
are also antagonist for D10 (20 and data not shown). Upon
further examination, the I5G and I5M peptides were found to be weak
agonists of D10 proliferation, thus not inhibiting proliferation in an
antagonist assay (Fig. 4A and data not shown). The W7L and
W7M peptides were found to be nonagonist peptides failing to induce D10
proliferation or IL-4 production (Ref. 15 ; Fig. 2, D and
I), and having no inhibitory effect in an antagonist assay
(Fig. 4A). Using R2H to confirm antagonist activity on AK8
(Fig. 4B) (21), we found that the I5G, W7M, and
E8A peptides, although nonagonist (Fig. 1, M, N,
and O), did not demonstrate any antagonist activity (Fig. 4B). In contrast, the peptides R2G, I5A, and W7L, which
exhibited antagonism in Fig. 2, resulted in a dose-dependent inhibition
of AK8 proliferation (Fig. 4B). These data confirm our
previously published findings describing antagonist peptides for D10
and AK8 and clearly demonstrate differences in recognition of peptides
by D10 and AK8. Of particular interest is the antagonism of AK8 with
position 2-, 5-, and 7-substituted peptides, while D10 is only
antagonized with position 8 analogue peptides. Thus, the nature of the
cellular response stimulated is influenced by interactions of both the
TCR - and -chains with the peptide:MHC complex, as the presence
of an identical TCR -chain in the two T cell clones is not
sufficient to render the signal delivered through the TCR identical.
The -chain sequence of the two distinct T cell clones makes a
difference both in response to N-terminal substituted peptides that
interact with the TCR -chain and to C-terminal substituted peptides
that interact with the TCR -chain.
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- and -chains
To examine the contribution that the - and -chains make to
the overall TCR signal received in D10, we generated peptides with two
amino acid substitutions at positions 2 and 8. As previously shown in
Fig. 2, the R2G peptide is a superagonist peptide requiring
10-fold less peptide to induce the same response as the
CA134–146 wt peptide (Fig. 5) (23). In contrast, the
R2H peptide requires 
10-fold more peptide than
CA134–146 to generate a similar response (Fig. 5). Because the E8A antagonist peptide does not induce cell
proliferation, we generated peptides with the E8A substitution and the
R2G or R2H substitution to determine whether the nonstimulatory E8A
residue would affect the level of D10 activation observed with R2G or
R2H alone. The dual substituted peptide R2GE8A resulted in a
proliferative response that required 100-fold more peptide than the R2G
peptide to induce the same level of proliferation (Fig. 5). Similarly,
the R2H residue in combination with E8A rendered the peptide a
nonagonist, leading to no D10 proliferation (Fig. 5). These data
demonstrate that the overall strength of the signal through the D10 TCR
is contributed to by contacts between peptide:MHC complexes with both
the TCR -chain CDRs and the TCR -chain CDR3 domain. It also shows
that the nature of the signal received through both chains determines
the overall quality of signal leading to agonist, nonagonist,
superagonist, or antagonist responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Bonnie N. Dittel, Blood Research Institute, The Blood Center of Southwest Wisconsin, P.O. Box 2178, Milwaukee, WI 53201-2178.
3 Abbreviations used in this paper: CA, hen egg conalbumin; CDR, complementarity-determining region; EAE, experimental autoimmune encephalomyelitis; wt, wild type.
Received for publication May 25, 2000. Accepted for publication August 30, 2000.
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References |
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and lack of Zap70 recruitment in APL-induced T cell anergy. Cell 79:913.[Medline]
phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science 267:515.
tefanová, Jr C. A. Janeway. 1999. Cross-antagonism of a T cell clone expressing two distinct T cell receptors. Immunity 11:289.[Medline]
chain. Cell 71:649.[Medline]
in T helper 1, T helper 2, and T helper 0 clones bearing the same TCR. J. Immunol. 158:4065.[Abstract]
chain variable domain. Cell 69:999.[Medline]
and tumor necrosis factor production. J. Exp. Med. 180:2227.This article has been cited by other articles:
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  J. S. Rossman, N. G. Stoicheva, F. D. Langel, G. H. Patterson, J. Lippincott-Schwartz, and B. C. Schaefer POLKADOTS Are Foci of Functional Interactions in T-Cell Receptor-mediated Signaling to NF-{kappa}B Mol. Biol. Cell, May 1, 2006; 17(5): 2166 - 2176. [Abstract] [Full Text] [PDF]
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 E. I. Buzas, A. Hanyecz, Y. Murad, F. Hudecz, E. Rajnavolgyi, K. Mikecz, and T. T. Glant Differential Recognition of Altered Peptide Ligands Distinguishes Two Functionally Discordant (Arthritogenic and Nonarthritogenic) Autoreactive T Cell Hybridoma Clones J. Immunol., September 15, 2003; 171(6): 3025 - 3033. [Abstract] [Full Text] [PDF]
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) was cocultured with B10.BR
splenocytes in the presence of 1/10 dilutions of CA134–146
(A), R2H (B), R2G (C), E8D
(D), E8N (E), I5N (F), I5L
(G), I5M (H), or I5V (I)
from 0.001 to 10 µM. Proliferation was detected as described for Fig. 1
), W7M (
), or E8A (
) from 1.2 to
100 µM. For AK8, the 5 µg/ml CA134–146 was used for
prepulsing, and peptides R2G (•), R2H (
), and E8A (+) were used. Proliferation was detected
as described for Fig. 1