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Department of Microbiology and Carter Immunology Center, University of Virginia, Charlottesville, VA 22908
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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accumulation, and cross-reactivity of the resulting T cell populations.
We found a direct correlation between the ability of a T cell
population to respond in vitro to low concentrations of the precise
peptide expressed on the tumor and its ability to delay the outgrowth
of B16 melanoma after adoptive transfer. Surprisingly, we found that
some T cells that exhibited high functional avidity and were effective
in controlling tumor outgrowth exhibited low structural avidity, as
judged by MHC-tetramer staining. Our results establish strategies for
the development and selection of CD8+ T cell populations
that persist despite peripheral tolerance, and that can control
melanoma outgrowth. Furthermore, they support the use of human MHC
class I-transgenic mice as a preclinical model for developing effective
immunotherapies that can be rapidly extended into therapeutic
settings. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two independent techniques have been described to isolate high-avidity CD8+ T cell populations in vitro. One approach is based on the original observation that the level of MHC-tetramer binding per T cell correlates with monovalent TCR affinity, as determined by surface plasmon resonance, and avidity (12, 13). Several studies have since used MHC-tetramer staining as a surrogate readout for TCR affinity, and have found that strong tetramer staining is correlated with high functional avidity, as measured by the dose of peptide required to stimulate an effector activity (14, 15, 16). Thus, MHC-tetramer staining has been used to isolate T cells from melanoma patients with enhanced tumor reactivity and sensitivity to lower concentrations of antigenic peptide pulsed onto target cells (16, 17). Alternatively, Alexander-Miller et al. (18) have demonstrated that HIV gp160-specific CD8+ T cells with higher functional avidity can be enriched by using lower concentrations of peptide during in vitro culture, and that such cells were more effective in limiting viral replication in vivo. This approach has been extended into tumor immunotherapy, in which the adoptive transfer of TRP-2-specific CD8+ T cells with higher functional avidity preferentially reduced the tumor burden in a B16 lung metastasis model (19).
We have previously studied the generation of CD8+
T cell responses to MDPs in transgenic mice expressing AAD, a chimeric
MHC class I molecule composed of the 
1 and 2 domains of
HLA-A*0201, and 3 domain of H2-Dd. Because the
murine and human MDP epitopes presented by HLA-A*0201/AAD are highly
homologous or identical, this model provides information that can be
usefully incorporated into the design of clinical trials using these
Ags. We previously demonstrated that AAD-transgenic mice are
unresponsive to HLA-A*0201-restricted epitopes derived from murine
tyrosinase (Tyr369, FMDGTMSQV) (5)
or gp100 (gp100209, ITDQVPFSV) (6).
Although CD8+ T cell populations that react to
these Ags could be generated by immunizing with single amino
acid-substituted variant peptides, it had not been determined whether
the cross-reactive T cells could be manipulated to recognize tumors. In
this study, we have extended this work by making use of cumulative in
vitro selection approaches, based on the manipulation of peptide
concentration and specificity, to engineer high-avidity, tumor-reactive
CD8+ T cell populations that are effective in
controlling the outgrowth of the aggressive B16 melanoma after adoptive
transfer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice expressing a chimeric MHC class I composed of
the 1 and 2 domains of HLA-A*0201 and the 3 domain of
H2-Dd (AAD) have been described previously
(20). Mice were maintained in specific pathogen-free
facilities at the University of Virginia.
Cell lines
C1R-AAD has been previously described (20). The C57BL/6-derived melanoma cell lines B16-F10 (CRL-6475) and B16-F1 (CRL-6323) were obtained from the American Type Culture Collection (Manassas, VA). The generation of AAD+ transfectants of B16-F10 and B16-F1 is described in detail elsewhere (47). Transfectants were cultured in RPMI 1640 containing 5% FBS supplemented with SerXtend (Irvine Scientific, Santa Ana, CA) and 300 µg/ml G418 (Life Technologies, Grand Island, NY), and screened regularly by flow cytometric analysis for AAD expression using the HLA-A2-binding Abs CR11-351 or BB7.2.
Peptides
Synthetic peptides were made by standard F-moc chemistry using a model AMS422 peptide synthesizer (Gilson, Middleton, WI) in the Biomolecular Core Facility at the University of Virginia. All peptides were purified to >98% purity by reverse-phase HPLC on a C-8 column (Vydac, Hesperia, CA). Purity and identity were confirmed using a triple quadrupole mass spectrometer (Finnigan, San Jose, CA).
Dendritic cells (DC)
DC were generated as described (21), with modifications (6). Immature DC were isolated on a StemSep column after incubation with a mixture of Abs that enrich for DC, then incubated at a 2:1 ratio overnight with irradiated CD40 ligand (CD40L)-transfected NIH-3T3 fibroblasts (a kind gift from R. Lapointe, National Cancer Institute, Bethesda, MD). These activated DC expressed high levels of MHC class I, MHC class II, CD80, CD86, and CD40, and demonstrated intracellular accumulation of IL-12 after a 5-h incubation in the presence of 10 µg/ml brefeldin A (Sigma-Aldrich, St. Louis, MO).
Immunization
DC were pulsed with the indicated concentration of peptide for
4 h at 37°C in HBSS containing 5% FBS and 5 µg/ml human
2-microglobulin (Calbiochem, La Jolla, CA),
washed twice, and resuspended in HBSS. Mice were injected in tail veins
with 105 DC.
Generation of peptide-specific HLA-A*0201-restricted CD8+ T cells
Spleens from primed mice were harvested at least 3 wk after immunization. A total of 1.5 x 107 responder cells was incubated in upright 25-cm2 tissue culture flasks (Costar, Cambridge, MA) with 7 x 106 autologous irradiated (2500 rad) spleen cells that had been pulsed with the indicated concentration of peptide for 3 h at 37°C. After culture for 6–7 days, cytotoxic activity and cytokine production were assessed, as described above. T cell lines were established from the initial cultures and maintained by weekly restimulations, as described above, except that 5 x 105 T cells were incubated with 5 x 106 peptide-pulsed stimulators in 12-well plates (Costar) after week 2, and cultures were supplemented with 10 Cetus U/ml IL-2 on the day of restimulation and then 3 days later.
Intracellular cytokine staining
Cytokine expression was examined either ex vivo in freshly isolated spleen cells or in long-term T cell lines maintained in vitro. For ex vivo analysis, CD8+ T cells were isolated from spleens of immunized mice after incubation with a mixture of Abs to enrich for CD8 cells, followed by passage over StemSep column (StemCell Technologies, Vancouver, BC, Canada). Preparations were consistently 85–95% CD8+, as assessed by flow cytometry. Enriched CD8+ T cells were then directly assessed for cytokine production after 5-h incubation with C1R-AAD stimulator cells that had been pulsed overnight with the indicated concentration of peptide.
To measure the production of intracellular cytokines, peptide-pulsed
stimulator cells were incubated with T cells for 5 h at a ratio of
1:1 in medium supplemented with 50 U/ml IL-2 and 10 µg/ml brefeldin
A. Stimulated cells were counterstained with a 1/1000 dilution of
either PE- or APC-conjugated anti-CD8 (BD PharMingen, San Diego,
CA), washed, then fixed and permeabilized in PermWash/Fix (BD
PharMingen), followed by staining with a 1/50 dilution of
FITC-conjugated anti-IFN- (BD PharMingen). Flow cytometry was
conducted on a FACScan using CellQuest software (BD Biosciences, San
Jose, CA). Normalized staining values were calculated by using the
formula ((experimental value - background value for unpulsed
stimulators)/(maximal value (using 100 µg/ml peptide-pulsed
stimulators) - background value)) x 100.
Cytotoxicity assay
Lytic activity of T cell populations was assessed using standard
chromium release assays. Briefly, target cells were labeled overnight
with 51Cr (Amersham, Boston, MA) at a
concentration of 100 µCi/106 cells. The target
cells were washed and counted, and 2000 cells were aliquoted into wells
containing the indicated concentration of peptide that had been diluted
in medium containing 5 µg/ml
2-microglobulin. After 2 h at 37°C, T
cells were added at a ratio that had been predetermined to produce 65%
lysis of target cells pulsed with 10 µg/ml peptide.
Tetramer staining
HLA-A*0201 tetramers that had been folded around ITDQVPFSV (gp100209) or IMDQVPFSV (gp100209(M)) were produced by the National Institutes of Health Tetramer Facility at Emory University (Atlanta, GA). T cells were coincubated for 45 min at room temperature with the indicated concentration of tetramer and a 1/1000 dilution of anti-CD8, washed twice, and fixed in 2% paraformaldehyde. Staining was quantitated on a FACScan using CellQuest software. Tetramer-staining values on a CD8+ T cell with irrelevant specificity were subtracted.
Adoptive transfer of CD8+ T cells
Subcutaneous tumors were established in AAD mice by injection of 4 x 105 B16-F1-AAD or B16-F10-AAD in 200 µl of saline. Tumor cells were 100% viable by trypan blue exclusion and >98% AAD+ by flow cytometric analysis on the day of injection. All animals developed palpable tumors by day 10, at which point 1 x 107 cells of the indicated CD8+ T cell line were injected directly into the tumor. A total of 500 Cetus units of IL-2 was coadministered with the T cells, and additional injections of 500 Cetus units of IL-2 were delivered i.p. on the subsequent 3 days. Tumor growth was measured every 48–72 h using a Vernier caliper, and was recorded as the product of two orthogonal diameters (a x b). The first diameter was identified as the longest surface length (a), with the second diameter being the subsequent orthogonal width (b). Repeated observations with the Vernier caliper resulted in <5% SD.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of CD8+ T cell populations
that can be used for adoptive transfer requires the in vitro expansion
of Ag-experienced T cells. We previously showed that after immunization
with recombinant vaccinia viruses or peptide-pulsed mature DC,
responses to the self-Ags Tyr369 and
gp100209 were extremely weak (5, 6).
Since it has been reported that CD40L is a potent inducer of DC
immunogenicity, we first asked whether populations of MDP-reactive
memory cells that would be appropriate for further in vitro
manipulation could be generated in AAD mice immunized with DC that had
been activated in this manner. AAD mice were injected i.v. with
CD40L-activated DC that had been incubated with either
Tyr369 or gp100209
peptides, and CD8+ T cells were isolated 7 days
later at the peak of the primary response. These cells were incubated
with peptide-pulsed stimulator cells for 5 h and then analyzed by
flow cytometry for the accumulation of IFN-. No measurable number of
gp100209-specific T cells was detected in any of
seven mice immunized with gp100209, and only two
of six mice showed a discernible response to Tyr369
(Table I
). In contrast, consistently
strong responses were seen in AAD mice immunized using DC pulsed with
single amino acid-substituted variant peptides
Tyr369(Y) or gp100209(M) (Table I). Tyr369(Y) is a variant of Tyr369
carrying a conservative F to Y substitution at the P1 position, while
gp100209(M) is a variant of
gp100209 with enhanced binding to HLA-A*0201 due
to the substitution of M for T at P2 (22). Importantly,
40% of the CD8+ T cells activated by
immunization with Tyr369(Y) or
gp100209(M) also produced IFN- when stimulated
with Tyr369 and gp100209,
respectively (Fig. 1 and Table I). These
data confirm earlier work showing that T cells in AAD mice are largely
unresponsive to immunization with gp100209 and
Tyr369 self peptides, and demonstrate that
CD40L-activated DC are not effective in altering this. However,
immunization with closely related peptide variants enables the
expansion of T cell populations that show appreciable cross-reactivity
on the parental self peptides at the effector level, and provides a
basis for the further development of MDP-reactive
CD8+ T cell populations for adoptive
transfer.
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Although the preceding results were encouraging, it was of concern
that over several independent immunizations, on average over half of
the CD8+ T cells activated with epitope variants,
were not cross-reactive on the epitope itself (Table I
and data not
shown). Thus, further in vitro culture in the presence of the epitope
variants might lead to loss of cross-reactive T cells altogether
(6). On the other hand, the inability to demonstrate
significant primary responses upon immunization with
Tyr369 or gp100209 suggested
that these peptides might be unable to drive in vitro proliferation of
T cells. To evaluate these concerns directly, mice were immunized using
DC pulsed with either 0.1 or 10 µg/ml
gp100209(M). T cell lines were established from
mice primed with the lower concentration of peptide by in vitro culture
with 0.1 µg/ml of either gp100209(M) or
gp100209, and corresponding lines were
established from mice primed with the higher concentration by culture
with 10 µg/ml of either peptide. CD8+ T cell
lines were successfully generated in each case, demonstrating that
gp100209 can stimulate proliferation of primed
cells in vitro, despite its inability to stimulate naive T cells in
vivo (Fig. 2 and data not shown).
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in a 5-h assay (SC50).
gp100209(M) was chosen to measure functional
avidity for all T cell lines because it was the peptide used to prime
all T cells in vivo. The T cell lines maintained with 0.1 µg/ml of
either gp100209 or
gp100209(M) showed similar and relatively high
functional avidities (SC50 of 3.7 x
10-9 and 2.9 x 10-9
g/ml, respectively; Fig. 2, A and B, and Table II) compared with those of the T cell
lines maintained with 10 µg/ml of either peptide
(SC50 of 4.9 x 10-7
and 9.9 x 10-7 g/ml, respectively; Fig. 2, C and D, and Table II). These relative
differences in functional avidity for gp100209(M)
among the lines were observed consistently in three independent
experiments, and were in concordance with previously work demonstrating
that the functional avidity of a T cell population inversely correlates
with the concentration of Ag used to generate the T cells
(18).
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40-fold
better than did the line maintained with 0.1 µg/ml
gp100209(M), despite their similar avidities for
gp100209(M) (Fig. 2, compare A and
B, and see Table II). Similarly, the T cell line maintained
with 10 µg/ml gp100209 recognized this peptide
6-fold better than did the line maintained with 10 µg/ml
gp100209(M) (Fig. 2, compare C and
D, and see Table II). Similar results were observed by
comparison of several other T cell lines developed from
gp100209(M)-immunized mice (data not shown). Two
important conclusions can be reached from these data. First, they
demonstrate that there are conformational differences between
gp100209 and gp100209(M)
that are detectable by T cells, despite the fact that the residue
altered between these two peptides is in a binding pocket and not
directly accessible at the cell surface. Second, in vitro culture with
gp100209 can select for T cells with enhanced
reactivity for gp100209. This can occur without
altering the functional avidity for the original stimulating Ag
(gp100209(M)), and despite the inability of
gp100209 to initiate T cell responses in
vivo.
We next evaluated whether a similar process could be used to select T
cells from AAD mice primed with Tyr369(Y). However, in
contrast to what was observed above, very little
CD8+ T cell expansion was observed after 1 wk of
in vitro culture with Tyr369 compared with culture
with Tyr369(Y), and we were unable to establish
CD8+ T cell lines in this manner (T. A.
Colella, T. N. J. Bullock, D. W. Mullins, and V. H. Engelhard,
manuscript in preparation). We therefore asked whether
CD8+ T cells with greater functional avidity and
cross-reactive recognition of Tyr369 could be
generated by manipulating the concentration of
Tyr369(Y) used for in vitro culture. It was found that
there was again an inverse correlation between the concentration of
Tyr369(Y) used to establish and maintain T cell lines
in vitro and the functional avidity of the lines, as measured by either
IFN- accumulation or cytotoxic activity (Fig. 3, A and B, and
Table II). Importantly, we also found that as functional avidity for
Tyr369(Y) increased, there was a comparable increase
in both the fraction of cells that recognized Tyr369
at the highest peptide dose, as well as the cross-reactivity
measured as the ratio of SC50 values for
Tyr369 and Tyr369(Y) (Fig. 3C and Table II). Indeed, the T cell line maintained with
the lowest concentration of Tyr369(Y) did not
substantially discriminate between this peptide and
Tyr369 (Fig. 3, B and C).
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The results obtained with the
Tyr369(Y)-stimulated T cells were in keeping with our
expectations, based on studies with Abs, that receptors with a higher
affinity for Ag would also show greater cross-reactivity on closely
related structures (23, 24). In contrast, the T cell lines
maintained on 0.1 µg/ml of either gp100209 or
gp100209(M) showed less cross-reactivity than T
cell lines maintained on 10 µg/ml of either peptide (Table II). To
gain further insight into the reasons for this, we analyzed the
interactions of TCR expressed on the four
gp100209-reactive T cell lines with tetramers
composed of HLA-A*0201 folded around either
gp100209 or gp100209(M).
Because of the multivalent nature of these interactions, we refer to
this parameter as structural avidity. Unfortunately, <2% of the T
cell lines maintained in the presence of 10 µg/ml of either
gp100209 or gp100209(M)
bound to either tetramer (data not shown). Thus, it was not possible to
evaluate whether the higher cross-reactivity of these lines was
reflected in the structural avidity of their TCR.
This analysis did reveal unexpected complexity in the tetramer staining
of T cell lines maintained with 0.1 µg/ml of either peptide. More
than 75% of both lines expressed TCRs that bound to tetramers at the
highest concentrations tested (Fig. 4, A and B). Interestingly, however, the T cell line
maintained with 0.1 µg/ml gp100209 bound to
tetramers made with gp100209(M) only 5-fold
better than to tetramers made with gp100209 (Fig. 4B), although there was a 25-fold difference in its
functional avidity (Fig. 2B). Strikingly, the T cell line
maintained with 0.1 µg/ml gp100209(M) bound
both of the tetramers to a similar degree (Fig. 4A),
although there was a 1000-fold difference in the respective functional
avidities (Fig. 2A). Thus, for both T cell lines, the
difference in functional avidity for gp100209 and
gp100209(M) is always much larger than the
difference in structural avidity for these two peptides. Most
surprisingly, the structural avidities of the T cell line maintained
with 0.1 µg/ml gp100209 were always much lower
than those of the T cell line maintained with 0.1 µg/ml
gp100209(M), despite the fact that its functional
avidities for both peptides were equivalent or higher (cf Fig. 4, A and B, with Fig. 2, A and
B). Taken together, these results indicate that the
functional avidities of these T cell lines do not correlate with their
structural avidities, and that significant differences in effector
activity can occur upon interaction with Ags for which the TCR has
similar nominal structural avidity.
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Our ultimate goal was to develop CD8+ T
cells that could be used for adoptive immunotherapy of tumors. We
therefore screened the T cell lines described above for in vitro and in
vivo reactivity with B16-F1 or B16-F10 melanoma cells that had been
transfected to express AAD. Although 50% of cells in the line
maintained with 0.01 µg/ml Tyr369(Y) accumulated
IFN- when stimulated with B16-F1-AAD, only 2% of the line
maintained with 0.1 µg/ml Tyr369(Y) did so, and no
stimulation by B16-F1-AAD was detected in the lines maintained with 1
or 10 µg/ml of this peptide (Fig. 5A). Interestingly, none of
these lines recognized B16-F10-AAD (data not shown), suggesting that it
expresses a lower amount of this epitope than B16-F1-AAD. Among the T
cell lines initiated with gp100209(M), the line
maintained with 0.1 µg/ml gp100209 was
stimulated to the greatest extent by B16-F10-AAD, while the lines
cultured with either 10 µg/ml gp100209 or 0.1
µg/ml gp100209(M) were stimulated to a lower
and similar extent, and the line cultured with 10 µg/ml
gp100209(M) was not stimulated at all (Fig. 5B). In the case of the
gp100209-reactive lines, recognition of the
B16-F10-AAD tumor does not correlate with the functional avidity for
the gp100209(M) peptide nor with structural
avidity as measured with MHC-tetramers based on either peptide. Thus,
the ability of these T cell populations to produce IFN- upon
recognition of this tumor in vitro correlates most closely with their
functional avidities for Tyr369 or
gp100209.
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), in keeping with
their ability to recognize these tumors in vitro. Surprisingly, T cell
lines maintained with 1 µg/ml Tyr369(Y) or 10
µg/ml gp100209(M), which showed no recognition
of tumor in vitro, slowed tumor outgrowth to a limited degree. Neither
CD8+ T cells specific for an irrelevant epitope,
which did not recognize B16-AAD cells in vitro (data not shown), nor
administration of IL-2 alone had an effect on tumor outgrowth. Thus,
the ability of the T cell lines maintained with 1 µg/ml
Tyr369(Y) or 10 µg/ml
gp100209(M) to control tumor in vivo was Ag
specific, and demonstrated that the in vitro measurements of tumor
reactivity underestimated their therapeutic potential. Nevertheless,
the ability of these Tyr369- and
gp100209-reactive T cell populations to control
tumor outgrowth in vivo correlated most closely with their functional
avidity, and not with their structural avidity, for these two
Ags.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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accumulation. As expected, T cell lines with higher functional
avidities for Tyr369(Y) also showed greater
cross-reactivity on the parental Tyr369 peptide.
However, this correlation was inverted for T cell lines from mice
primed with gp100209(M). Surprisingly, we found
that T cell lines with low cross-reactivity on
gp100209, as determined using functional assays,
exhibited similar structural avidities, as defined with MHC tetramers,
for both peptides. However, the structural avidity of T cells
maintained with gp100209 was substantially lower
than the structural avidity of T cells maintained with
gp100209(M). These results demonstrate that
functional avidity, as measured by effector function, need not be
directly correlated with structural avidity. Finally, we found that the
ability of these T cells to control the outgrowth of a murine melanoma
correlated with their functional avidity for the original parent
peptides. Effective adoptive immunotherapies against tumors are dependent upon the generation of large numbers of highly tumor-reactive CD8+ T cells. The data presented in this work and in previous studies (5, 6) demonstrate that the HLA-A*0201-restricted responses to Tyr369 and to several gp100-derived epitopes are usually undetectable in AAD mice, as is the response to gp10025–33 restricted by H-2Db (25). In contrast, responses to TRP-2181–188 restricted by H-2Kb were observed after single in vivo immunization (19, 26, 27). Collectively, these studies indicate that the immunogenicity of different class I MHC-associated MDP epitopes can vary significantly. Although intermediate to high MHC-binding affinity of foreign peptides has generally correlated with their ability to induce immune responses (28), many poorly immunogenic MDP-derived peptides have affinities in this range (5, 6). This suggests that self-tolerance also plays an important role in determining whether different MDP-derived peptides are effective immunogens. Successful expansion of highly tumor-reactive T cells will therefore depend on level of peptide presentation and the mechanism by which self-tolerance prevents responses to MDPs.
It is apparent from this study and previous work (5, 6) that peptide variants of the poorly immunogenic Tyr369 and gp100209 can provide an effective means to activate MDP-specific responses in AAD mice, and indicates that self-tolerance does not delete or inactivate all T cells reactive with these epitopes. The mechanism of action of these variant peptides remains to be elucidated. The relative weak HLA-A*0201-binding affinity exhibited by some MDP-derived peptides provided the rationale for the initial design of variant peptides (22), and enhanced responses were attributed to more stable MHC-peptide complexes, allowing more productive TCR engagement (6, 7, 22, 29, 30). However, the immunogenicity of Tyr369(Y) cannot be explained in this manner, as it binds to HLA-A*0201 with nearly identical affinity as Tyr369, and both peptides are equally immunogenic in AAD+ mice that do not express tyrosinase (5). An alternative explanation is that the Tyr369-specific T cells that persist in tyrosinase+ mice perceive this epitope as a partial or weak agonist (31, 32) that is capable of activating effector functions such as perforin and cytokine release, but not proliferation. However, these T cells would perceive Tyr369(Y) as a full agonist, which elicits the full range of effector activities and proliferation.
The substitution of Met for Thr at the P2 anchor position of
gp100209 to produce
gp100209(M) was originally shown to stabilize its
binding to HLA-A*0201 while preserving its ability to be recognized by
gp100209-reactive T cells (22).
Indeed, some of the data reported in this work and elsewhere (6, 7) are consistent with this explanation of its improved
immunogenicity. However, while the binding affinity of
gp100209 for HLA-A*0201 is 10-fold lower than
that of gp100209(M) (Table I), the
cross-reactivity of different T cell lines was up to 1000-fold lower.
These results demonstrate that these two peptides are antigenically
distinguishable by at least some T cells, and that for many of those
cells gp100209 is perceived as a weak agonist.
Interestingly, while some studies have demonstrated that stronger
agonist epitopes, as measured by functional assays, exhibited
relatively higher affinities for TCR (33, 34), others have
shown that this correlation does not always hold (35, 36, 37).
In the present work, the observation that a T cell line responded
functionally to gp100209(M) up to 1000-fold
better than gp100209, yet showed very similar
structural avidities for these two peptides, is consistent with these
latter observations. Furthermore, the T cell line maintained on
gp100209 responded functionally to
gp100209(M) and gp100209
equivalently and 100-fold better, respectively, than the line
maintained on gp100209(M). However, the
structural avidities of the former line were up to 1000-fold lower than
those of the latter. These data demonstrate a significant disparity
between functional and structural avidities. This suggests that
differences in the efficiency with which signals are transduced by
these receptors in response to each of these epitopes play an important
role in shaping T cell functional avidity, and that this may also be
shaped by in vitro culture conditions.
It remains to be determined how functional avidity can be regulated independently of structural avidity, and particularly how a T cell line with low structural avidity can show high functional avidity. The expression of CD8 was similar between the two cell lines (data not shown), and CD8 involvement in tetramer binding was prevented by Abs to CD8 (38, 39). Thus, differences in structural avidities between these cell lines cannot be accounted for by the differential involvement of CD8. In addition, when CD8 involvement was minimized in the functional assay, there was no change in the relative avidities for gp100209 and gp100209(M) (data not shown). Together, these data suggest that the differences in structural and functional avidities are independent of CD8 engagement and/or p56lck recruitment (40). It is interesting that both structural avidity and T cell functional activity have been shown to be augmented by the organization of TCR into membrane lipid rafts (41, 42, 43, 44). Thus, it is possible that the differences in these parameters for the lines maintained in gp100209 and gp100209(M) reflect differences in both the true affinity of their TCRs and the extent to which these TCR are distributed into lipid rafts.
Our data demonstrate that T cells with a high functional avidity may express TCR with a low structural avidity. Such dissociation of structural and functional avidity has also been noted in two other recent studies, suggesting that this phenomenon may be more prevalent than previously appreciated (45, 46). The implication of these observations is that the selection of T cell populations based solely on high structural avidity may exclude T cells that could have high functional avidity and be therapeutically useful. Thus, while tetramers are an important tool for monitoring the expansion of T cells in an immune response, functional studies are required to assess therapeutic potential of the activated populations.
The principal goal of this study was to use the AAD-transgenic mouse to examine strategies for the generation of CD8+ T cells specific for peptide Ags that are relevant to human melanoma. We have demonstrated a direct correlation between impact on tumor outgrowth and the T cell’s functional avidity for the exact peptide expressed on the tumor. Although these results are similar to those observed by others using either viral peptides or strongly immunogenic tumor-associated peptides (18, 19), we have extended this paradigm to include situations in which the tumor-associated peptides are weakly immunogenic at best. The challenge in this situation was to enrich for T cells that are capable of recognizing the tumor-associated Ag. We found that in one case, T cells with augmented cross-reactive recognition of the tumor-associated Ag could be selected simply by using very low concentrations of a closely related peptide variant. In the second case, however, selection of T cells with this specificity was augmented by sequential use of a relatively immunogenic peptide variant and the tumor-associated peptide, both in low concentration. It will be interesting to examine the utility of these alternative strategies to derive similar tyrosinase- or gp100-reactive CD8+ T cells for use in melanoma patients.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Department of Microbiology and Carter Immunology Center, Room 4072C, MR4 Building, University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail address: vhe{at}virginia.edu
3 Abbreviations used in this paper: MDP, melanocyte differentiation protein; DC, dendritic cell; TRP, tyrosinase-related protein; CD40L, CD40 ligand.
Received for publication March 14, 2001. Accepted for publication September 10, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 and 3 domains in the recognition of self and non-self MHC molecules. J. Immunol. 156:2473.[Abstract]
domains in CD8 coreceptor function: importance for MHC I binding, signaling, and positive selection of CD8+ T cells in the thymus. Immunity 12:409.[Medline]
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  M. J. Smithey, S. Brandt, N. E. Freitag, D. E. Higgins, and H. G. A. Bouwer Stimulation of Enhanced CD8 T Cell Responses Following Immunization with a Hyper-Antigen Secreting Intracytosolic Bacterial Pathogen J. Immunol., March 1, 2008; 180(5): 3406 - 3416. [Abstract] [Full Text] [PDF]
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R.-H. Voss, R. A. Willemsen, J. Kuball, M. Grabowski, R. Engel, R. S. Intan, P. Guillaume, P. Romero, C. Huber, and M. Theobald Molecular Design of the C{alpha} Interface Favors Specific Pairing of Introduced TCR{alpha} in Human T Cells J. Immunol., January 1, 2008; 180(1): 391 - 401. [Abstract] [Full Text] [PDF]
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 R. M. Wong, R. R. Scotland, R. L. Lau, C. Wang, A. J. Korman, W. M. Kast, and J. S. Weber Programmed death-1 blockade enhances expansion and functional capacity of human melanoma antigen-specific CTLs Int. Immunol., October 1, 2007; 19(10): 1223 - 1234. [Abstract] [Full Text] [PDF]
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 D.-W. Liu, Y.-C. Yang, H.-F. Lin, M.-F. Lin, Y.-W. Cheng, C.-C. Chu, Y.-P. Tsao, and S.-L. Chen Cytotoxic T-Lymphocyte Responses to Human Papillomavirus Type 16 E5 and E7 Proteins and HLA-A*0201-Restricted T-Cell Peptides in Cervical Cancer Patients J. Virol., March 15, 2007; 81(6): 2869 - 2879. [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. W. Mullins and V. H. Engelhard Limited infiltration of exogenous dendritic cells and naive T cells restricts immune responses in peripheral lymph nodes. J. Immunol., April 15, 2006; 176(8): 4535 - 4542. [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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 R. Salerno-Goncalves, R. Wahid, and M. B. Sztein Immunization of Volunteers with Salmonella enterica Serovar Typhi Strain Ty21a Elicits the Oligoclonal Expansion of CD8+ T Cells with Predominant V{beta} Repertoires Infect. Immun., June 1, 2005; 73(6): 3521 - 3530. [Abstract] [Full Text] [PDF]
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G. Dorothee, I. Vergnon, F. El Hage, B. L. M. Chansac, V. Ferrand, Y. Lecluse, P. Opolon, S. Chouaib, G. Bismuth, and F. Mami-Chouaib In Situ Sensory Adaptation of Tumor-Infiltrating T Lymphocytes to Peptide-MHC Levels Elicits Strong Antitumor Reactivity J. Immunol., June 1, 2005; 174(11): 6888 - 6897. [Abstract] [Full Text] [PDF]
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J. W. Hodge, M. Chakraborty, C. Kudo-Saito, C. T. Garnett, and J. Schlom Multiple Costimulatory Modalities Enhance CTL Avidity J. Immunol., May 15, 2005; 174(10): 5994 - 6004. [Abstract] [Full Text] [PDF]
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M. A. Lyman, C. T. Nugent, K. L. Marquardt, J. A. Biggs, E. G. Pamer, and L. A. Sherman The Fate of Low Affinity Tumor-Specific CD8+ T Cells in Tumor-Bearing Mice J. Immunol., March 1, 2005; 174(5): 2563 - 2572. [Abstract] [Full Text] [PDF]
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 K. Narayanan, A. Jaramillo, N. D. Benshoff, L. G. Campbell, T. P. Fleming, J. R. Dietz, and T. Mohanakumar Response of Established Human Breast Tumors to Vaccination with Mammaglobin-A cDNA J Natl Cancer Inst, September 15, 2004; 96(18): 1388 - 1396. [Abstract] [Full Text] [PDF]
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E. M.-L. Choi, J.-L. Chen, L. Wooldridge, M. Salio, A. Lissina, N. Lissin, I. F. Hermans, J. D. Silk, F. Mirza, M. J. Palmowski, et al. High Avidity Antigen-Specific CTL Identified by CD8-Independent Tetramer Staining J. Immunol., November 15, 2003; 171(10): 5116 - 5123. [Abstract] [Full Text] [PDF]
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 D. W. Mullins, S. L. Sheasley, R. M. Ream, T. N.J. Bullock, Y.-X. Fu, and V. H. Engelhard Route of Immunization with Peptide-pulsed Dendritic Cells Controls the Distribution of Memory and Effector T Cells in Lymphoid Tissues and Determines the Pattern of Regional Tumor Control J. Exp. Med., October 6, 2003; 198(7): 1023 - 1034. [Abstract] [Full Text] [PDF]
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M. J. Pittet, V. Rubio-Godoy, G. Bioley, P. Guillaume, P. Batard, D. Speiser, I. Luescher, J.-C. Cerottini, P. Romero, and A. Zippelius {alpha}3 Domain Mutants of Peptide/MHC Class I Multimers Allow the Selective Isolation of High Avidity Tumor-Reactive CD8 T Cells J. Immunol., August 15, 2003; 171(4): 1844 - 1849. [Abstract] [Full Text] [PDF]
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T. N. J. Bullock, D. W. Mullins, and V. H. Engelhard Antigen Density Presented By Dendritic Cells In Vivo Differentially Affects the Number and Avidity of Primary, Memory, and Recall CD8+ T Cells J. Immunol., February 15, 2003; 170(4): 1822 - 1829. [Abstract] [Full Text] [PDF]
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M. K. Slifka, J. N. Blattman, D. J. D. Sourdive, F. Liu, D. L. Huffman, T. Wolfe, A. Hughes, M. B. A. Oldstone, R. Ahmed, and M. G. von Herrath Preferential Escape of Subdominant CD8+ T Cells During Negative Selection Results in an Altered Antiviral T Cell Hierarchy J. Immunol., February 1, 2003; 170(3): 1231 - 1239. [Abstract] [Full Text] [PDF]
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 H. Echchakir, G. Dorothee, I. Vergnon, J. Menez, S. Chouaib, and F. Mami-Chouaib Cytotoxic T lymphocytes directed against a tumor-specific mutated antigen display similar HLA tetramer binding but distinct functional avidity and tissue distribution PNAS, July 9, 2002; 99(14): 9358 - 9363. [Abstract] [Full Text] [PDF]
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)
or variant peptide (
), or left unpulsed (
). The results are the
average of two mice per group, and the tyrosinase and gp100 arms of the
experiment were performed independently. One experiment of three is
presented.
)
or nonspecific T cell activity (
). Error bars are hidden by the
symbols.