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Department of Microbiology and Immunology, Wake Forest University School of Medicine, Winston-Salem, NC 27157
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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vs CD8
and the ability of CTLs to colocalize the TCR and CD8 in the membrane.
Colocalization of these molecules was mediated by lipid rafts and
importantly, raft disruption resulted in the conversion of high avidity
CTL into a lower functional avidity phenotype. These novel findings
provide insights into the control of functional avidity in response to
viral infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The ability of CTLs to respond functionally to Ags displayed on the surface of APCs depends on a number of molecular interactions. Tetramer binding studies have shown that in some cases the affinity of the TCR for peptide/MHC complexes correlates with functional avidity (2, 3, 4). However, it should be noted that this correlation does not hold true in all cases. In addition, the levels of CD8 and LFA-1 have been shown to influence the amount of peptide/MHC required for activation (5, 6, 7, 8, 9).
Using a lymphocytic choriomeningitis virus (LCMV)3 TCR transgenic (P14) mouse crossed onto a recombinase-activating gene-2-/- background, we demonstrated that CTLs of high and low functional avidity can be generated independently of TCR affinity (10). Low avidity CTLs, although expressing a TCR identical with high avidity CTLs, required more TCR engagement events than high avidity CTLs to become activated. This appeared to be the result of TCR engagement events that do not produce a productive signal.
Furthermore, we made two new important observations concerning
the role of CD8 and functional avidity. First, although the surface
expression of CD8 was similar between high and low avidity CTLs,
CD8 expression was significantly increased in high avidity CTLs.
These data are consistent with the notion that high avidity CTLs
express a greater proportion of CD8 heterodimers relative to low
avidity CTLs. Next, we demonstrated that low avidity CTLs were less
efficient than high avidity CTLs at cointernalizing the CD8 molecule
with the TCR. Because the cointernalization of CD8 with TCR following
CD3 cross-linking occurred in the absence of engagement of CD8 with MHC
class I molecules, we concluded that the TCRs on the surface of high
avidity CTLs are more highly associated with the CD8 molecule than on
low avidity CTLs. The increased internalization of CD8 with TCR
demonstrated by high avidity CTLs and the ability to respond
functionally to fewer TCR engagement events suggested that the higher
functional avidity apparent in these CTLs may be a direct result of the
organization of these molecules within membrane microdomains.
Studies by Zhang et al. (18) have shown that TCR-mediated
signaling is enhanced upon oligomerization (11). This
aggregation of TCR resulting in increased signaling efficiency may be
influenced by the recruitment of these receptors into lipid rafts upon
engagement with peptide/MHC molecules on APCs (12). In
addition to TCR, a number of signaling molecules have been shown to
reside in lipid rafts, including p59fyn, CD8,
the linker of activation of T cells, and p56lck
(LCK) (12, 13, 14, 15, 16, 17, 18). The kinase p56lck
serves to phosphorylate ITAMS of the CD3
chains. Once
phosphorylated, these tyrosine residues serve as "docking sites"
for molecules containing src homology domain 2 binding motifs, such as
ZAP-70 and p59fyn (19, 20), further
amplifying signal transduction cascades. Additionally, Lck has
src homology domain 2 domain, and can bind to phosphorylated ZAP-70
associated with the CD3 chain, effectively coupling the coreceptor
to the TCR (21). Indeed, the cointernalization of TCR and
CD8 results from the association of coreceptor-associated Lck to
ZAP-70/CD3 of triggered TCR (22). Given our previous
findings that high avidity CTLs are more sensitive to TCR engagement
and are better able to cointernalize CD8 with the TCR
(10), we hypothesized that high avidity CTLs would
demonstrate a greater degree of colocalization of TCR with CD8, and
that this colocalization would be mediated in part by organization of
these molecules into lipid rafts.
The following study uses high and low avidity lines generated
from TCR transgenic mice to examine the role of membrane microdomain
organization of CD8 and TCR in determining the functional avidity of
CTL. We report the novel finding that high and low avidity CTLs differ
dramatically in the spatial arrangement of CD8 and TCR on their
surface. Specifically, high avidity CTLs colocalize substantially more
TCR with CD8 compared with low avidity CTLs, which fail to associate a
significant portion of TCR following CD8 capping. The efficiency with
which high avidity CTLs cocap TCR with CD8 is abolished following
treatment with methyl--cyclodextrin (MBCD), indicating that the
arrangement is mediated by lipid rafts. Importantly, the ability of
high avidity CTLs to respond functionally to fewer TCR engagement
events than low avidity CTLs is directly related to the integrity of
lipid rafts on their surface, as treatment with MBCD decreases the
apparent functional avidity of CTL. These data support a mechanism
whereby high functional avidity is determined by the optimal membrane
localization of TCR and CD8, which is mediated by compartmentalization
of these molecules into lipid rafts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 mice were purchased from Frederick Cancer Research and Development Center (Frederick, MD). The TCR LCMV P14/recombinase-activating gene-2 mice were obtained from Taconic Farms (Germantown, NY). The LCMV P14 peptide (KAVYNATM) encompasses residues 33–41 of the gp33 protein and was synthesized at the Comprehensive Cancer Center Protein Analysis Core Laboratory at Wake Forest University School of Medicine (Winston-Salem, NC).
Generation of CTL lines
For CTL lines generated from transgenic mice, 2 x 106 spleen cells were cocultured with 3.5 x 106 C57BL/6 splenocytes (2000 rad irradiated) previously pulsed with either high (10-5 M) or low (10-10 M) concentrations of LCMV (P14) peptide. Before coculture, stimulators were washed to remove unbound peptide. Cultures were maintained in 24-well plates containing 2 ml of RPMI 1640 medium supplemented with 2 mM L-glutamine, 0.1 mM sodium pyruvate, nonessential amino acids, 100 U/ml penicillin, 100 µg/ml streptomycin, 2-ME (0.05 mM), 10% FBS, and 10% T-stim (Collaborative Biomedical Products, Bedford, MA) as an IL-2 source. CTL lines were established from primary cultures and maintained by weekly restimulation of 3–5 x 105 cells/well in the presence of 5 x 106 irradiated (2000 rad) C57BL/6 spleen cells pulsed with the appropriate concentration of peptide.
Flow cytometry and confocal microscopy
For intracellular staining, cells were fixed in 3%
paraformaldehyde solution for 20 min at room temperature and
permeabilized with a solution of 1% saponin, 10% FBS, and 0.1%
NaN3 in PBS on ice for 30 min. Before adding
primary Ab, cells were blocked with 5% normal goat serum for 20 min on
ice to prevent nonspecific binding of Abs. Cells were stained with
anti-Lck Ab (BD Transduction Laboratories, San Diego, CA) for 20
min on ice. Cells were washed and stained with the appropriate
FITC-conjugated secondary Abs (Caltag Laboratories, Burlingame, CA).
Lipid raft ganglioside GM1 was labeled using FITC-conjugated
cholera toxin B subunit (CT-X; Sigma-Aldrich, St. Louis, MO).
Expression of CD8 and CD8 were measure by flow cytometry using an
anti-CD8 (clone CT-CD8a) or anti-CD8 (clone CT-CD8b)
FITC-conjugated Ab (Caltag Laboratories). For CD8/CD8
cocapping studies, cells were labeled with FITC-conjugated
anti-CD8 Ab (clone CT-CD8b; Caltag Laboratories) for 30 min on
ice followed by washing and cross-linking with anti-rat IgG for 30
min at 37°C to induce capping. Cells were washed in ice-cold PBS
containing sodium azide stained with Cy5-conjugated anti-CD8
(clone 53-6.7; BD PharMingen, San Diego, CA), and fixed for analysis by
confocal microscopy. In Fig. 2
, the CD8 is depicted in green and the
CD8 in red. These changes in representation were made to maintain
consistency with the subsequent figures in which CD8 is depicted in
green. For TCR/CD8 cocapping studies, cells were incubated for 30 min
on ice with PE-conjugated anti-TCR Ab (clone H57-597; Caltag
Laboratories) and FITC-conjugated rat anti-mouse CD8 (clone
CT-CD8a; Caltag Laboratories). Cells were washed in cold PBS
supplemented with 2% FBS. CD8 cross-linking was done on ice by
adding goat anti-rat IgG, followed by washing and further
cross-linking with rabbit anti-goat IgG on ice. Cells were then
resuspended in 37°C cell culture media and incubated in a 5%
CO2 incubator for 20 min to induce capping. Cells
were pelleted and resuspended in ice-cold PBS containing 0.2% sodium
azide. Cells were fixed to poly(L) lysine-coated coverslips and
fluorescence measured by confocal microscopy. The goat anti-rat
secondary, used to cap the CD8 Ab, was tested to ensure that it was
not capable of binding the anti-TCR Ab. For cocapping studies
following raft disruption, CTLs were treated with 5 mM MBCD as
described below and subjected to the same capping method as above.
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Seven days after routine stimulation, CTLs were washed twice in
PBS and resuspended in serum-free OPTI-MEM (Life Technologies,
Rockville, MD) containing MBCD (Sigma-Aldrich, St. Louis, MO) at a
concentration of 5 mM. Following a 30-min incubation at 37°C, cells
were washed three times in PBS and resuspended in OPTI-MEM for use in
the IFN-
ELISA or CD8/TCR cocapping studies.
IFN- ELISA
Seven days following routine stimulation, CTLs (5 x
104/well) were either treated with MBCD or left
untreated and incubated in 96-well flat-bottom plates that had
previously been incubated overnight at 4°C with various
concentrations of anti-CD3 Ab (clone 2C11; BD PharMingen) followed
by PBS washing. CTLs were cultured for 18 h at 37°C in a 5%
CO2 incubator serum-free OPTI-MEM. Supernatant
was harvested and assayed for the presence of IFN- by ELISA (OptEIA
Mouse IFN- set; BD PharMingen).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The activation of a CTL depends on the phosphorylation of ITAMs in
the CD3 chains by src family kinases. The 56-kDa kinase Lck plays a
significant role in these phosphorylation events, and signal
transduction resulting in the internalization of the TCR is dependent
on p56lck activity (23). Our
previous studies have shown that high avidity CTLs down-regulate
surface TCR at lower concentrations of anti-CD3 Ab compared with
low avidity CTLs (10). Presumably, an increase in the
expression of Lck could translate into an increased ability to
phosphorylate the ITAMs of the TCR following engagement, which could in
turn result in an increased efficiency of TCR down-regulation following
engagement with anti-CD3 Ab. Thus, we tested the hypothesis that
TCR signal transduction is more efficient in high avidity CTL due to
increased Lck expression. Intracellular levels of Lck were measured by
flow cytometry, and Fig. 1 shows that the
expression of Lck between high and low avidity CTL was nearly
identical. These findings were reproduced in an additional pair of high
and low avidity CTL lines. Therefore, differential expression of Lck
does not explain the increased sensitivity to CD3 engagement in high
avidity CTL.
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The above data demonstrate that the overall level of expression of
Lck does not explain the difference in sensitivity to TCR engagement
between high and low avidity CTLs, but did not preclude the possibility
that the localization of Lck might influence the efficiency of ITAM
phosphorylation. One mechanism by which Lck is localized to the TCR
complex is by associating with the coreceptor CD8, which binds the 3
domain of the MHC class I molecule. The coordinate binding of both CD8
and TCR with the same MHC class I molecule allows for efficient
localization of CD8-associated Lck with the TCR. In addition,
TCR/coreceptor colocalization brings p56lck into
proximity of TCR-associated kinases such as fyn and Zap-70,
which facilitate kinase-dependent signal transduction cascades
(20, 24).
In an earlier study, we found that the increased efficiency of
anti-CD3-triggered cointernalization of CD8 with TCR was due at
least in part to the increased expression of CD8 heterodimers vs
homodimers by high avidity CTL, as CD8 molecules expressing the
-chain were selectively cointernalized with the TCR
(10). This increased coreceptor internalization in the
absence of ligand binding provides strong functional evidence for an
increased association of CD8 and TCR in high avidity CTL relative to
low avidity CTL. As previously demonstrated (10), the high
and low avidity CTL lines generated from TCR transgenic mice express
similar levels of CD8, but differed dramatically in the expression
of CD8. In Fig. 2
A, low
avidity CTLs express 
40% less CD8 than high avidity CTLs, yet
express similar levels of CD8. Although expression levels of CD8
and CD8 by flow cytometry were consistent with the notion that there
were more CD8 homodimers on low avidity CTL relative to high
avidity CTL, this technique was insufficient to provide direct evidence
of a differential expression of CD8 and CD8 isoforms.
To demonstrate the presence of CD8 homodimers in low avidity CTL
and to determine whether CD8 heterodimers vs CD8 homodimers
could be differentiated within the intact membrane, CD8 molecules
expressing the -chain were fluorescently labeled and capped, and the
distribution of CD8 and CD8 were visualized by confocal
fluorescence microscopy. In Fig. 2, B and C, the
staining patterns of CD8 molecules (green) and CD8 molecules
(red) are shown for low (Fig. 2B) and high (Fig. 2C) avidity CTLs. Under these conditions, CD8
heterodimers are identified by colocalization of CD8 and CD8
(yellow). CD8 is found in low avidity CTL in both capped and
uncapped regions. In contrast, all of the CD8 in high avidity CTL is
found colocalized with CD8. These data provide direct evidence that
high avidity CTLs express CD8 on their surface predominantly in the
CD8 heterodimeric form, whereas a substantial portion of surface
CD8 molecules on low avidity CTLs are in CD8 homodimeric
form.
Because our previous findings showed that high avidity CTLs were better
able to cointernalize CD8 molecules with TCR in the absence of MHC
engagement (10), we hypothesized that CD8 was more highly
associated with the TCR in high avidity CTLs. The ability of molecules
to cocap has been used previously as a measure of their association in
the membrane. Studies by Kwan Lim et al. (25) have shown
that capping with Abs to the CD8 heterodimer resulted in the
efficient cocapping of TCR. Thus, we tested the ability of TCR and CD8
to cocap in both high and low avidity CTLs. Importantly, the
colocalization of CD8 and TCR was independent of its engagement with
peptide/MHC. In Fig. 3, the CD8 molecule
is shown in green and the TCR in red for both low (Fig. 3A)
and high (Fig. 3B) avidity CTLs. Two important findings are
readily apparent. First, the morphology of the cocapped regions differs
between high and low avidity CTLs. The low avidity CTLs appear to have
incomplete capping (a pattern designated as patching by others), while
the high avidity CTLs show a more concentrated or uniform CD8/TCR
cocap. These data suggest that the cytoskeletal-dependent organization
of membrane receptors may be suboptimal in the low avidity CTL. Second,
the ability of the TCR molecule to cocap with CD8 is reduced in low
avidity CTL. Although a large portion of the TCR complexes are
colocalized with CD8 (yellow), a significant amount of TCR does not
colocalize with the capped CD8 molecules, suggesting that engagement of
these TCR would not lead to efficient signal transduction. In contrast,
the high avidity CTLs demonstrate an increased ability to cocap the TCR
with the CD8 molecule, with nearly all of the TCR colocalizing with
CD8. One would predict that this close association of TCR and CD8
independent of TCR/CD8 interaction with class I MHC would be very
efficient for signal transduction.
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. Approximately 2% of the confocal
images generated from the low avidity CTL line were scored as
colocalized and appeared similar to those in Fig. 3A.
Conversely, 91% of the images taken of the high avidity CTL line were
scored as colocalized, appearing similar to those in Fig. 3B. These data demonstrate a different spatial arrangement
of TCR and CD8 within the plasma membrane between high and low
avidity CTLs.
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The importance of lipid rafts in facilitating the optimal
orientation of signaling molecules is well-documented (16, 26, 27, 28, 29). The reduced cocapping of CD8 and TCR observed in low
avidity CTL might be explained by a decrease in the expression of lipid
rafts on the surface of these cells. To test this hypothesis, the
expression level of the ganglioside GM1 was measured using FITC-labeled
CT-X. GM1 is preferentially associated with lipid rafts and is
considered to be a raft marker (30). As shown in Fig. 4, high and low avidity CTLs bind CT-X
with similar efficiency, suggesting that a difference in the expression
of lipid rafts is not responsible for the differences in capping.
However, these results did not preclude the possibility that the
organization of lipid rafts or the arrangement of molecules within
lipid rafts might differ between high and low avidity CTLs, resulting
in the differential display of TCR and CD8 on the cell surface.
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. Following cholesterol depletion with MBCD, both
high and low avidity CTLs display a nearly uniform distribution of TCR
and CD8 on their cell surface (Fig. 5).
These data show that the ability to cocap TCR with CD8 has been
completely abrogated, and thus support the hypothesis that the
colocalization of TCR following capping of CD8 is mediated by lipid
rafts.
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, it seemed likely that this increased
colocalization could be responsible for the increased sensitivity of
high avidity CTL to TCR engagement. Optimal localization of Lck and TCR
would allow for improved efficiency of phosphorylation of TCR ITAMs,
translating into increased sensitivity to TCR engagement. Therefore, we
hypothesized that disruption of lipid rafts (and thus colocalization of
CD8 with TCR) in high avidity CTLs would induce a lower avidity
functional phenotype. The effect on low avidity CTLs should be less
significant, as they already demonstrated a reduced ability to
colocalize CD8 with TCR. To test this hypothesis, we measured the
effect of MBCD treatment on the ability of high and low avidity CTLs to
produce IFN- in response to anti-CD3 stimulation. Treatment of
high avidity CTLs with MBCD shifted the dose-response curve toward that
of the low avidity CTLs (Fig. 6). High
avidity CTLs now required 8-fold more anti-CD3 Ab to induce
half-maximal IFN- production (average of three experiments).
Treatment of low avidity CTLs with MBCD had only a minimal effect,
shifting the dose-response curve by only 1.5-fold (average of three
experiments). The viability of both lines was 95% in the presence of
MBCD. Therefore, the differential effect on the dose-response curves
was not the result of a difference in the toxicity of the drug in high
vs low avidity lines. Similar shifts in dose response were seen
following treatment with nystatin, which also disrupts lipid raft
integrity (data not shown). These data demonstrate that high avidity
CTLs are much more sensitive to raft disruption compared with low
avidity CTLs. These results support a mechanism in which high avidity
CTLs display TCR and CD8 on their surface in an array that is favorable
for optimal signaling following TCR engagement. Disruption of lipid
raft integrity abrogates the optimal positioning of TCR and CD8,
resulting in a decreased efficiency of signaling through the TCR, which
translates into a lower functional avidity phenotype.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous work demonstrated that high and low avidity CTLs differed
in the expression levels of CD8 but not CD8 expression. These
data were consistent with the notion that high avidity CTLs express CD8
predominantly as CD8 heterodimers, whereas low avidity CTLs
express a significant population of CD8 as CD8 homodimers. In the
present study, using confocal microscopy we formally demonstrate that
high avidity CTLs display CD8 on their surface predominantly in the
CD8 heterodimeric form, whereas the low avidity CTLs express a
significant portion of CD8 in the CD8 homodimeric form. The
expression of CD8 homodimers on the surface of low avidity CTLs
has a direct effect on the localization of Lck, which binds to the
cytoplasmic tail of the CD8-chain. Whereas both CD8 and
CD8 molecules are able to bind Lck, only CD8 molecules are
localized into lipid rafts by virtue of the palmitoylation site found
in the cytoplasmic tail of CD8 (13). Therefore, the
form in which CD8 is expressed (CD8 or CD8) can have a
dramatic impact on the localization of CD8-associated Lck relative to
raft-associated TCR.
We hypothesize that the preferential expression of CD8 as
heterodimers allows for efficient localization of CD8-associated Lck
with raft-resident TCR resulting in a high functional avidity
phenotype. This hypothesis is supported by our finding that treatment
with MBCD, which completely abrogated the cocapping of TCR and CD8 for
both high and low avidity CTLs, resulted in an 8- to 10-fold increase
in the amount of anti-CD3 Ab required for 50% maximal IFN-
production in high avidity CTLs, while shifting the dose-response curve
for low avidity CTLs only minimally (1- to 2-fold more anti-CD3
Ab). These studies provide functional evidence that the optimal
localization of TCR and CD8 can enhance CTL functional avidity by
improving the efficiency of signaling. This localization is highly
dependent on the organization of these molecules within lipid rafts.
These findings are in agreement with a recent study by Arcaro et al.
(32), which demonstrated that in T cell hybridomas, CD8
couples the TCR to raft-associated CD8/p56lck
complexes. Specifically, the portion of the CD8 molecule containing
the palmitoylation site that allows for CD8 to associate with lipid
rafts was shown to be critical for efficient TCR/CD8 association. These
findings are also consistent with our CD8 cointernalization studies
(10), which demonstrated that CD8 molecules expressing the
-chain were selectively internalized with TCRs that had transduced a
signal. The present study extends our understanding of the functional
consequences of the increased association of CD8 and TCR by
establishing a correlation between the raft-dependent localization of
these two molecules and the sensitivity to TCR engagement (functional
avidity).
Importantly, the morphology of cocapped TCR and CD8 was also found to differ between high and low avidity CTLs. High avidity CTLs displayed very discrete, concentrated caps that were highly polarized. The capping of CD8 and TCR on low avidity CTLs appears much less polarized, with patches of colocalization appearing in a comparatively random distribution on the cell surface. The clustering of lipid rafts on the surface of T cells is known to be dependent on actin cytoskeleton reorganization (33, 34). Therefore, the differences in the morphology of capped CD8/TCR indicate that high avidity CTLs demonstrate an enhanced cytoskeletal rearrangement of these raft-resident molecules into more defined polarized regions relative to low avidity CTLs, resulting in more efficient propagation of signals. Another possible outcome of an increased ability to concentrate TCR and coreceptor into highly polarized caps is the increased phosphorylation of the ITAMs on unengaged TCR. Such "bystander" phosphorylation events may serve to amplify signaling through the TCR when TCR ligand is limiting.
Because the preferential expression of CD8 as an heterodimer in
high avidity CTLs correlated with an increased ability to associate
with the TCR, we predicted that disruption of lipid raft integrity
would decrease the ability of TCR to cocap with CD8 and induce a lower
avidity phenotype. We found that treatment with MBCD completely
abrogated the cocapping of TCR and CD8 for both high and low avidity
CTLs. Importantly, these results demonstrated that the ability to
efficiently concentrate TCR and CD8 into distinct caps is dependent
upon the organization of these molecules into lipid rafts. Furthermore,
by disrupting the higher order organization of these molecules through
cholesterol depletion, the functional response of high avidity CTLs was
dramatically attenuated. High avidity CTLs now display a lower
functional avidity, requiring significantly more TCR engagement events
following treatment with MBCD to exert effector function (8-fold more
anti-CD3 Ab). These studies provide functional evidence that the
optimal localization of TCR and CD8 can enhance CTL functional avidity
by improving the efficiency of signaling. This localization is highly
dependent on the organization of these molecules within lipid
rafts.
The ability to regulate functional avidity by the organization of
molecules in membrane microdomains or the expression of CD8 vs
CD8 opens the possibility that functional avidity may be an
inducible phenomenon. Currently, it is unknown whether the functional
avidity of a CTL is induced in response to environmental signals during
the initial activation of a naive precursor in vivo or whether our
ability to generate CTL lines of distinct avidity is a reflection of
the preferential expansion of a subpopulation of CTL in vitro. Under
conditions in which peptide Ag is abundant, CD8 expression may occur
at basal levels. However, when the amount of Ag is very high or when
signaling through the TCR is very strong, CD8 expression could be
decreased, effectively inducing a lower avidity functional phenotype.
Another possibility is that the initial encounter with an APC can
influence the organization of molecules within membrane microdomains.
Lipid rafts have been shown to play an important role in the efficient
presentation of peptide/MHC class II by APCs (26). Thus,
it is tempting to speculate that the organization of peptide/MHC on the
APC is influencing the initial activation of a CTL such that a defined
organization of membrane receptors is established that influences the
subsequent response of that CTL.
In summary, the data herein provide a novel mechanism to explain the
increased sensitivity to TCR engagement demonstrated by our high
avidity CTLs generated from TCR transgenic mice. By expressing CD8
predominantly in the heterodimeric form rather than the
homodimeric form, high avidity CTLs display CD8 and TCR with greater
colocalization relative to low avidity CTLs. This preferential
association of CD8 and TCR effectively brings CD8-associated Lck into
proximity of the TCR on high avidity CTLs. The enhanced colocalization
of CD8 and TCR on high avidity CTLs depends on the integrity of lipid
rafts; however, capping studies suggest that the cytoskeletal
reorganization of these two molecules differs as well, with high
avidity CTLs forming more polarized caps relative to low avidity CTLs.
These data extend our understanding of mechanisms that contribute to
the control of functional avidity by demonstrating that functional
avidity can be influenced by the form of CD8 ( homodimeric or
heterodimeric), the manner in which TCR and CD8 are organized on
the cell surface, and the efficiency of cytoskeletal rearrangement of
these two molecules. The advances in our understanding of avidity
provided by the results presented herein are of significant importance,
given the crucial role for functional avidity in determining the
outcome of viral infection.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Martha A. Alexander-Miller, Department of Microbiology and Immunology, Wake Forest University School of Medicine, Room 5108, Gray Building, Medical Center Boulevard, Winston-Salem, NC 27157. E-mail address: marthaam{at}wfubmc.edu
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; MBCD, methyl--cyclodextrin; CT-X, cholera toxin B subunit.
Received for publication May 22, 2002. Accepted for publication July 22, 2002.
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References |
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  E. B. Walker, D. Haley, U. Petrausch, K. Floyd, W. Miller, N. Sanjuan, G. Alvord, B. A. Fox, and W. J. Urba Phenotype and Functional Characterization of Long-term gp100-Specific Memory CD8+ T Cells in Disease-Free Melanoma Patients Before and After Boosting Immunization Clin. Cancer Res., August 15, 2008; 14(16): 5270 - 5283. [Abstract] [Full Text] [PDF]
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 C. J. Kroger, S. Amoah, and M. A. Alexander-Miller Cutting Edge: Dendritic Cells Prime a High Avidity CTL Response Independent of the Level of Presented Antigen J. Immunol., May 1, 2008; 180(9): 5784 - 5788. [Abstract] [Full Text] [PDF]
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C. J. Kroger and M. A. Alexander-Miller Cutting Edge: CD8+ T Cell Clones Possess the Potential to Differentiate into both High- and Low-Avidity Effector Cells J. Immunol., July 15, 2007; 179(2): 748 - 751. [Abstract] [Full Text] [PDF]
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L. A. Johnson, B. Heemskerk, D. J. Powell Jr., C. J. Cohen, R. A. Morgan, M. E. Dudley, P. F. Robbins, and S. A. Rosenberg Gene Transfer of Tumor-Reactive TCR Confers Both High Avidity and Tumor Reactivity to Nonreactive Peripheral Blood Mononuclear Cells and Tumor-Infiltrating Lymphocytes J. Immunol., November 1, 2006; 177(9): 6548 - 6559. [Abstract] [Full Text] [PDF]
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 D. A. Price, J. M. Brenchley, L. E. Ruff, M. R. Betts, B. J. Hill, M. Roederer, R. A. Koup, S. A. Migueles, E. Gostick, L. Wooldridge, et al. Avidity for antigen shapes clonal dominance in CD8+ T cell populations specific for persistent DNA viruses J. Exp. Med., November 21, 2005; 202(10): 1349 - 1361. [Abstract] [Full Text] [PDF]
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S. Yang, J. W. Hodge, D. W. Grosenbach, and J. Schlom Vaccines with Enhanced Costimulation Maintain High Avidity Memory CTL J. Immunol., September 15, 2005; 175(6): 3715 - 3723. [Abstract] [Full Text] [PDF]
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S. Yang, K.-Y. Tsang, and J. Schlom Induction of Higher-Avidity Human CTLs by Vector-Mediated Enhanced Costimulation of Antigen-Presenting Cells Clin. Cancer Res., August 1, 2005; 11(15): 5603 - 5615. [Abstract] [Full Text] [PDF]
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) for 30 min as in Fig. 5