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Division of Hematology-Oncology, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02114
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Melanoma has been studied as a model disease for immunotherapy because of the identification of melanoma-associated Ags and their corresponding CTL epitopes (4, 5, 6, 7, 8, 9, 10). Most of the identified epitopes derived from melanoma-associated Ags, including MAGE-1, MAGE-3, gp100, MART-1, and tyrosinase, have been shown to effectively induce CTL reactivity in vitro (6, 7, 8, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). However, these peptides are weakly immunogenic in generating CD8+ T cells in comparison with peptides derived from viral Ags, probably because they bind to the HLA-A2 restriction elements with low affinities and have a rapid dissociation rate (21, 22, 23). It has been shown that a majority of high affinity natural peptides bound to HLA-A2 have a restricted size of 9–10 residues and contain 2 dominant anchor residues within the sequence: leucine (L) or methionine (M) at position 2 and valine (V) at position 9 (24, 25). Substitution of anchor residues at position 2 or 9 (residues not in contact with TCR) may be chosen to increase the binding affinities of HLA-A2 for the modified peptide and thereby to increase immunogenicity.
For example, gp100 parental peptide (aa 209–217; G209)2 is a 9-aa peptide derived from human gp100 beginning at position 209. G209-2M is derived from G209 by substituting methionine for valine at position 2 of G209 peptide. G209-2M has been shown in vitro to enhance the generation of CTL recognizing native G209 as well as melanoma (26), and has also been tested in a clinical trial as immunotherapy for melanoma (27). In most patients treated with G209-2M peptide, vaccination led to an increase in Ag-specific CTL precursors that recognize both modified and native G209 peptides. However, no patients demonstrated clinical responses (cancer regression) after vaccination with G209-2M peptide alone, although most patients (10 of 11) showed immune responses, and objective clinic responses were observed only in patients also receiving IL-2 (27). Our studies (unpublished data) also showed that many peptide-specific CTL generated by in vitro peptide stimulation failed to kill melanomas. This discrepancy between apparent successful immunization and lack of clinical effects prompted us to characterize CTL responses to stimulation with G209-2M in vitro.
In the present study, we chose G209-2M as a model CTL epitope and characterized the CTL generated from normal donors when stimulated with autologous dendritic cells (DC) pulsed with G209-2M. We show that CTL generated against G209-2M peptide can be classified into three categories: G209-2M-specific CTL which are cytotoxic only to G209-2M-pulsed targets; peptide-specific CTL which recognize both native G209- and G209-2M-pulsed targets but not HLA-A2+gp100+ melanomas; and melanoma-reactive CTL which recognize peptide-pulsed targets as well as HLA-A2+gp100+ melanomas. Furthermore, the ability of CTL to lyse HLA-A2+gp100+ melanomas is correlated to their TCR avidities and MHC/peptide density on targets in both an in vitro assay and a human melanoma model in nude mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human melanoma lines DM6 (HLA-A2, -B12, -Cw1, 2, gp100+), DM13 (HLA-A2, -A31, -B13, 18, gp100+), DM14 (HLA-A11, -A28; -B5, 8, -Cw2, 4, gp100-) were a gift from Dr. T. L. Darrow and Dr. H. F. Seigler (Duke University Medical Center, Durham, NC) (28), and melanoma A375 (HLA-A2+gp100-) was purchased from American Type Culture Collection (ATCC; Manassas, VA). Tumor cells were cultured in DMEM supplemented with 5% FCS (Life Technologies, Gaithersburg, MD), antibiotics (penicillin 100 U/ml, streptomycin 100 µg/ml, amphotericin B 0.25 µg/ml, gentamicin 50 µg/ml), L-glutamine 450 µg/ml, and sodium bicarbonate 2.5 mg/ml in 75-cm2 T flasks (Costar, Cambridge, MA). The melanoma tumor cell lines were grown as monolayer cultures and were passaged at confluence by trypsinization (0.25% trypsin with EDTA). Before cytotoxicity assays, the cells were harvested without using trypsin and EDTA to avoid alteration and loss of MHC and tumor-associated Ag (TAA). Instead, the cells were washed once with PBS and then incubated in PBS for 5–10 min at room temperature to detach them from the flask. Cells were then labeled with 51Cr.
Synthetic peptides
HLA-A*0201-restricted peptides G209 (ITDQVPFSV) and its anchor residue-modified counterpart G209-2M (IMDQVPFSV) were synthesized at the Massachusetts General Hospital Biopolymer Core Facility (Charlestown, MA). Peptides were purified to >90% by reverse phase HPLC as confirmed by mass spectrometry. Peptides were dissolved in DMSO and then diluted with PBS at 2 mg/ml (the final concentration of DMSO in stock solution is <5%, v/v), filtered through a 0.2-µm pore size membrane, and stored at -80°C.
Melanoma cell transduction with recombinant adenoviral vectors containing the gp100 or MART-1 genes
Cells were harvested, washed twice in serum-free medium, resuspended in Ex-Vivo-15 (BioWhittaker, Walkersville, MD) at 10 x 106/ml, and equilibrated to 37°C in a water bath before transduction. Adenovirus constructs were provided by Genzyme (Framingham, MA) and have been described in detail previously (29, 30). Adenovirus stocks were thawed on ice and added to cell suspension at a multiplicity of infection of 300. Cells were gently mixed by agitation and placed immediately back in a 37°C water bath. After a 20-min incubation, warm Ex-Vivo 15 medium was added to dilute the cells to a final concentration of 1 x 106/ml. Transduced cells were transferred to flasks, maintained at 37°C under 5% CO2 overnight, and used the next day as targets for 51Cr release assays.
Generation of DC from PBMC
DC were generated from PBMC as described by Romani et al. (31) with some modifications (30). Briefly, PBMC isolated from normal donors’ buffy coat or leukapheresis products were cultured for 2 h in RPMI 1640 (Life Technologies) supplemented with 1% human AB serum at 5 x 106 cells/ml in triple flasks. Nonadherent cells were then gently washed off and saved as responders for CTL generation. The remaining adherent cells were cultured with 100 ng/ml GM-CSF (Immunex, Seattle, WA) and 20 ng/ml IL-4 (PeproTech, Rocky Hill, NJ) in RPMI 1640 containing 1% human male AB+ serum. On day 6, the cells were harvested, washed and replated in ultralow adherence six-well culture trays (Costar; Ultralow No. 3471) and matured with CD40 ligand-trimeric (CD40LT, 1 µg/ml; Immunex) for 24 h.
Purification of PBMC and CD8+ cells
PBMC from HLA-A2+ normal donors were purified by centrifugation in Ficoll-Paque (Pharmacia, Peapack, NJ) from buffy coat or leukapheresis products. CD8+ T cells were isolated by negative selection of PBMC or nonadherent PBMC using a panel of mAb and magnetic beads as follows. PBMC were incubated with anti-CD4 (OKT4; ATCC), anti-HLA-DR (L243; ATCC), anti-CD20 (1F5; ATCC), anti-CD14 (3C10; ATCC), and anti-CD56 (B159; BD PharMingen, San Diego, CA) at saturating concentrations for 60 min at 4°C. Cells were washed twice and then incubated with magnetic particles coated with goat anti-mouse IgG (PerSeptive Biosystems, Framingham, MA) for 60 min with rocking at 4°C. Cell separation was performed with a strong magnet. Cells were collected, washed twice, and resuspended in RPMI 1640 containing 10% human male AB+ serum. Purified CD8+ T cells were >85% positive for expression of CD3CD8 and <10% positive for CD3CD4.
CTL generation
DC were pulsed with 20 µg/ml peptide at a cell concentration
of 1–2 x 106/ml in the presence of 3
µg/ml 
2-microglobulin for 3 h at 37°C
and were irradiated (25 Gy) before use. Purified
CD8+ T cells were mixed with peptide-pulsed
autologous DC at a ratio of 20:1 in the presence of IL-7 (10 ng/ml;
PeproTech). Cultures were set up in 48-well tissue culture trays
(Costar) by distributing 0.5 ml of the mixture (containing 1 x
104 DC and 2 x 105
CD8+ T cells) to each well. CTL in each culture
well were restimulated individually every 7–10 days with irradiated
autologous DC pulsed with peptide in the presence of IL-2 (100 U/ml;
Chiron, Emeryville, CA). CTL activity was tested 7 days after
stimulation, following three to six cycles of stimulation.
Cytotoxicity assays
Cultured CTL were tested for cytotoxicity in a standard 4-h 51Cr release assay. Melanoma cells (1–2 x 106/ml) were labeled with 100 µCi sodium [51C]chromate for 1 h at 37°C. Peptide-pulsed targets (1 x 106/ml in the presence of different concentrations of peptide in 1 ml of medium) were labeled with 100 µCi sodium [51C]chromate for 2 h at 37°C. Target cells (5000 targets/well) were added to wells containing effector CTL. The percent specific 51Cr release was calculated as: [(mean experimental cpm - mean spontaneous cpm)/(mean maximum cpm - mean spontaneous cpm)] x 100%, in which spontaneous release represents cpm in supernatants from wells containing target cells with medium only and maximum release represents cpm in supernatants from wells containing target cells in medium with 2% Triton X-100. Spontaneous release was always <20% of maximum release. The SD of duplicate wells was <10%.
Cold target blocking assay
CTL (1 x 105) were incubated with 15 x 104, 5 x 104, or 1.5 x 104 unlabeled "cold" targets for 1 h at 37°C, before the addition of 5 x 103 51Cr-labeled "hot" targets. The final effector-hot target ratio was 10:1, and the final cold-hot target ratios are as indicated in the figures. After an additional 4-h incubation, the supernatants were harvested, and specific chromium release was calculated as described above.
Cloning and expansion of peptide-specific CTL
After testing for peptide recognition (day 21 of culture), CTL
cultures were plated at 0.5 cell/well in 96-well round-bottom plates
with 5 x 104 irradiated (25 Gy) autologous
PBMC and 1 x 104 irradiated (100 Gy)
EBV-transformed B cells in 200 µl of medium supplemented with 30
ng/ml OKT3. After 24 h and every 3 days thereafter, 300 U/ml IL-2
was added for 
20 days. Wells positive for clonal growth were
identified 15–20 days after plating and were tested for peptide
reactivity. Peptide-specific clones were transferred to 24-well plates
(2 ml/well) and restimulated with OKT3, and irradiated autologous PBMC
(2 x 106/well) and EBV-transformed B cells
(5 x 105/well) were added as feeder cells.
The expanded clones were tested for their avidities, cytolytic activity
to melanoma, and in vivo antimelanoma activity in nude mice.
Adoptive immunotherapy of human melanoma in nude mice by peptide-specific CTL
Nude mice (nu/nu, BALB/c; Charles River Breeding Laboratories, Wilmington, MA) were inoculated s.c. with the human melanoma cell line DM6 (1 x 107/mouse). Seven days after inoculation, when the diameter of the transplanted tumor reached 2–6 mm, the mice were labeled with ear tags and randomized into six groups with five mice in each group. Mice were treated as follows: the PBS group received PBS by i.v. injection (0.5 ml/mouse); the HLA-A2-restricted, influenza virus matrix peptide (Flu M1) group, CTL (from the same donor as 2B4 and 1F1 clones) specific for influenza virus matrix peptide M1 (i.v., 1 x 107 cells/mouse); the 2B4 group, CTL clone 2B4 (i.v., 1 x 107 cells/mouse); the 2B4/G209 group, i.v. 2B4 plus s.c. injection of G209 peptide (200 µg/mouse in 0.1 ml) around tumor; the 2B4/G280 group, i.v. 2B4 plus s.c. G280 peptide (200 µg/mouse); and the 1F1 group, i.v. CTL clone 1F1 (1 x 107 cells/mouse). Mice were treated twice as described above at 3-day intervals. Tumor size was measured every 5 days for 30 days.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To favor the expansion in vitro of peptide-specific CTL, we used
reverse purified CD8+ T cell populations (purity
>85% CD8+ T cells) that were stimulated with
autologous DC pulsed with G209-2M. IL-7 was included in the cultures in
the first 7 days. The cultured cells were restimulated every 7–10 days
with autologous DC pulsed with peptide in the presence of IL-2.
Cytolytic activity was first measured 7 days after the third
stimulation (days 21–30) by using T2 and T2 pulsed with G209-2M.
Peptide-specific CTL were generated from six of eight donors with
variable response rates (Table I
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However, we failed to generate any G209- or G209-2M-specific CTL from
the other two donors even after six cycles of stimulation with
autologous DC pulsed with G209-2M.
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). The activity of melanoma-reactive CTL toward melanomas is
lower than that toward peptide-pulsed T2 targets. That is, specific
lysis against T2 pulsed with optimal concentration of peptide was
generally 50–80%, whereas the specific lysis toward melanoma was
20–40% at the same E:T ratio of 10:1. G209-specific CTL that lyse melanomas are HLA-A2 restricted and human gp100 specific
Two of the melanoma-reactive CTL lines elicited with G209-2M were
further studied by testing their capacity to recognize various HLA-A2
melanoma cells expressing gp100, and by determining the ability of
HLA-A2+gp100+ cold
(nonradiolabeled) targets to inhibit the lysis of another
HLA-A2+gp100+ melanoma
line. The results presented in Fig. 1, A and B, show that the melanoma-reactive CTL are
capable of killing HLA-A2 matched, gp100+
melanomas DM6 and DM13, but not
HLA-A2+gp1000- and
HLA-A2-gp100- melanomas
A375 and DM14, respectively (Fig. 1, A and B).
Furthermore, the CTL were also cytolytic to
HLA-A2+gp100- melanoma
cell line A375 transduced with adenovirus containing the human gp100
gene (Ad2/hgp100; Fig. 1, A and B). As expected,
the CTL did not lyse DM14 transduced with Ad2/hgp100 or A375 transduced
with Ad2/MART-1 (Fig. 1A).
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C). Taken together, these results indicate that the
melanoma-reactive CTL are HLA-A2-restricted and human
gp100-specific. TCR avidities of peptide-specific CTL correlate with melanoma reactivity
To determine the relationship between TCR avidities and melanoma
reactivity of peptide-specific CTL, avidities of 19 CTL clones
generated from 3 donors were tested for their activity toward T2 cells
pulsed with various concentrations of G209 peptide and against
melanomas. These 19 CTL were isolated by limited dilution (0.5
cell/well in 96-well plates) from 576 wells (Table II). The avidity, expressed as the
concentration of peptide required for half-maximal lysis of T2 loaded
with optimal concentration of peptide in cytotoxicity assay (MxLD50),
was calculated using the MS Excel program. Optimal concentration of
peptide was defined as the peptide concentration loaded on T2 cells
that gives the highest lytic activity. For most CTL clones, optimal
concentration of peptide is 1 µM; for a few clones, optimal
concentration is 10 µM. Fig. 2 shows
titration of MxLD50 of CTL with various TCR avidities and their
capacity for lysis of melanomas. To determine whether CTL avidity is
correlated to melanoma reactivity, MxLD50 is plotted against net lytic
activity against melanomas. As shown in Fig. 3, MxLD50 correlates well with melanoma
reactivity with regression coefficient -0.80. In the present study, we
arbitrarily define that a CTL with >10% net cytolytic activity toward
HLA-A2+gp100+ melanomas at
E:T ratio of 10:1 as melanoma reactive. As summarized in Table II, the
mean MxLD50 for the 9 melanoma-reactive CTL is <1 nM (mean ± SD,
0.47 ± 0.52 nM), and the MxLD50 for the 10 melanoma-nonreactive
CTL is 23.02 ± 13.93 nM.
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. No significant differences in
melanoma recognition were observed between the 10 µM and 0.1 µM
groups. However, reduction of peptide concentration to 0.001 µM
failed to engender either peptide or melanoma reactive CTL (Table III).
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A variety of studies have shown that CTL generated by synthetic
peptides often fail to recognize the same peptides endogenously
processed and presented by tumor cells due to the low determinant
density on tumor cells (15, 33, 34). To test whether
failure to recognize melanoma by low avidity CTL was due to the low
peptide density on the cell surface, we pulsed melanoma cells with
exogenous G209 and detected their sensitivity to high and low avidity
CTL. As seen from Fig. 4, high avidity
CTL 2B1, 1E5, and 1F1, which are melanoma-reactive CTL per se,
demonstrate increased cytolytic activity in variable degree to DM13
pulsed with exogenous G209 peptide. However, low avidity CTL 2B4 and
1F2, which are melanoma nonreactive, are rendered highly cytotoxic to
melanoma DM13 when the melanoma cells are pulsed with exogenous G209
peptide (Fig. 4). CTL 2D1, which is G209-2M specific and does not
recognize native G209 peptide presented on T2 cell, does not recognize
either parental melanoma cells or melanoma cells exogenously pulsed
with G209 peptide (Fig. 4). These results suggest that the naturally
processed epitope density on melanoma cells may too low to be
recognized by peptide-specific low avidity CTL.
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To investigate the in vivo antitumor efficacy of low and high
avidity CTL, peptide-specific CTL clones 2B4 (low avidity) and 1F1
(high avidity) were used to treat a human melanoma model in nude mice.
The avidities and cytolytic activity of the 2 CTL clones were described
above (Fig. 4). A Flu M1-specific CTL derived from the same donor as
2B4 and 1F1 was also used as a control. As shown in Fig. 5, all mice in the PBS control group
(treated with PBS; Fig. 5A), the Flu M1 control group
(treated with Flu M1-specific CTL, Fig. 5B) and the 2B4 group (treated
with low avidity CTL 2B4; Fig. 5C) demonstrated aggressive
tumor growth. In contrast, tumors in mice treated with high avidity CTL
1F1 were eradicated from three of five mice, and the three responding
mice remained tumor free 30 days after treatment. The growth of
melanoma in the remaining two tumor-bearing mice in this group was
significantly inhibited (Fig. 5D). We next sought to test
whether addition of peptide to in situ melanoma would sensitize
macroscopic tumor to lysis by low avidity CTL, in parallel to our in
vitro observations. Although treatment with low avidity CTL 2B4 alone
had no antitumor effect in vivo (Fig. 5C), four of five mice
that were coinjected with G209 peptide around the melanoma had
significantly smaller tumors and melanoma in another mouse was
eliminated 10 days after the first treatment and remained tumor free
thereafter (Fig. 5E). To control for the possibility that
such peptide inoculation actually was immunizing, we used cotreatment
with peptide G280, which is also derived from gp100 and is HLA-A2
restricted; it had no synergetic or additive effect on impairing tumor
growth with 2B4 (Fig. 5F). The data are consistent with our
in vitro experiments that show increased sensitivity of melanomas to
low avidity CTL when the melanomas are pulsed with exogenous
peptide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nearly one-fourth of CTL lines generated in this study are G209-2M specific and thus would not be expected to lyse the melanomas because they do not recognize the native G209 peptide. Anchor residues most probably are not involved in TCR contact. Altering the anchor residue at position 2 of HLA-A*0201 binding peptide G209 from threonine to methionine should not affect the recognition of peptide by a TCR. However, our results suggest that an HLA-A2 molecule bound with G209-2M peptide may have a different conformation than an HLA-A2 bound with native G209 peptide. Apparently, some CTL are able to discern the difference. Therefore, conformational alterations due to substitutions in anchor residue could influence the interaction between the TCR and MHC-peptide complex. This observation is consistent with other studies using T cell clones and a panel of peptides with different anchor residue alterations. Chen et al. (42) reported that four CD8+ T cell hybridomas, specific for the naturally processed OVA peptide restricted by H2-Kb, showed different responses to peptides with single amino acid changes at the MHC anchor residues. Recently, Hsu et al. (43) showed that a single anchor residue change in mouse hemoglobin peptide Hb64–76 (I-Ek restricted) influenced recognition by some, but not all Hb64–76-specific T cell clones. Most recently, using PBMC from G209-2M-immunized melanoma patients, Clay et al. (44) demonstrated that 25% (5 of 20) of PBMC-derived peptide-specific CTL recognized only G209-2M, which was very similar to our observation. Although G209-2M-specific CTL clones could not recognize parental peptide G209, some of them also recognized other modified peptide such as G209-2I and G209-2L (44). We suggest that some peptide-specific T cell clones are able to detect a conformational difference between MHC molecules bound with peptides differing at a single anchor residue and thereby demonstrate different responses to the native vs the modified peptide. In contrast, other peptide-specific T cells ignore the conformational difference and respond to both the native and modified peptides.
Regression analysis shows that melanoma reactivity is well correlated
to CTL avidity. Among the G209-reactive CTL lines that recognize both
native and anchor-modified peptides, only one-half recognize
endogenously processed gp100 peptide on melanoma cells. According to
the criteria described in Table II, these represent high avidity CTL
(with MxLD50 1 nM). Failure of low avidity CTL to recognize the
melanomas could be due to a lower level of MHC expression and/or
presence of NK-inhibitory receptors on melanoma cells as shown by Ikeda
et al. (45). However, all the low avidity G209-reactive
CTL that initially failed to lyse
HLA-A2+gp100+ melanomas
demonstrated increased cytolytic activity against the melanomas loaded
with exogenous native G209 peptide (Fig. 4 and data not shown). This
suggests that low avidity CTL need a higher determinant density on
targets for efficient TCR and MHC-peptide interaction. Failure of low
avidity CTL to react to melanomas is probably not due to the low level
of HLA-A2 or the presence of NK-inhibitory receptors on melanomas but
likely due to the low epitope density on tumor cells.
Even the melanoma-reactive CTL generated by G209-2M demonstrate
reactivity to melanoma that is much lower than their reactivity toward
T2 pulsed with G209 peptide (1 nM to 10 µM; Figs. 2 and 4). This
observation differs from that reported by Dudley et al.
(38). In their study, in which IFN-
production from CTL
was measured as the study endpoint, melanomas were better than or equal
to T2-G209 as stimulators. In our study, we never observed that the
lytic activity to melanomas was higher than that to T2 pulsed with G209
peptide even at concentration as low as 0.1 nM (Figs. 2 and 4 and data
not shown). The difference between Dudley’s (38) and our
observations is probably due to the different readouts (cytokine
release vs cytotoxicity by CTL) used. In fact, in another study
reported by Dudley et al. (46), they found discordance
between lytic activity and cytokine production of peptide-specific CTL.
That is, some clones produced high levels of cytokines with less lytic
activity and vice versa.
Although about one-half of peptide-specific CTL elicited by G209-2M stimulation cannot lyse melanomas, G209-2M peptide has been shown to be a more potent immunogen than the native G209 peptide. In our own direct comparison study using both G209 and G209-2M, it is much more difficult to generate CTL response when G209 was used as immunogen. This situation makes it difficult to compare fine specificity of CTL elicited by G209 and G209-2M. Because of thymic education and/or peripheral tolerance, self-reactive T cells with highest affinities have been inactivated either by deletion or anergy, and only low to intermediate affinity T cells exist in peripheral circulation. Cells bearing low affinity TCRs for the G209/HLA-A2 ligand may be the majority of the T cells effectively stimulated by the G209-2M/HLA-A2 ligand. This may be an explanation for the observation that G209 peptide-specific CTL engendered after G209-2M vaccination fail to mediate tumor regression in vivo. We examined one strategy to overcome this failing. After immunization with G209-2M peptide, we augmented peptide presentation on melanoma cells by intratumoral injection of native peptide. This may enhance the cytolytic activity of low avidity CTL expanded by G209-2M, enabling tumor killing.
Because only high avidity CTL lyse tumors, the best strategy for immunotherapy may be to selectively engender specific CTL of high avidities in vivo. Using a murine model, Alexander-Miller et al. (32) demonstrated that use of a low concentration of virus-derived peptide selectively expanded high avidity CTL, which were much more effective in eliminating virus from the host than the low avidity CTL. Using the same approach as described by Alexander-Miller et al. (32), Zeh et al. (47) also successfully generated high and low avidity tumor-specific CTL from B16/GM-CSF melanoma immunized mice after in vitro stimulation with high and low concentrations of peptide, respectively. High avidity CTL also demonstrated superior antitumor activity compared with low avidity CTL. However, we failed to generate any CTL responses when the concentration of G209-2M was reduced to 0.001 µM. Failure to generate CTL by very low concentration peptide from TAA may occur because the frequency of autoreactive CTL with high avidity is very low compared with that of viral reactive CTL. Another explanation for our observation is also possible. In both studies cited above, the T cells used for in vitro stimulation were from either virus or GM-CSF gene-modified tumor-immunized mice, whereas our study used PBMC from unimmunized normal blood donors to induce primary in vitro responses. It is very likely that unprimed T cells require a higher dose of Ag to reach their activation thresholds. Nevertheless, high avidity CTL can be identified and expanded in vitro as described in the present study and the CTL can be used individually or in pools in adoptive immunotherapy protocols to treat cancer patients as described previously by Rosenberg et al. (48) and Walter et al. (49).
The present study clearly shows that MxLD50 of a CTL correlates well with its ability to tumor lysis, suggesting that MxLD50 may be a good parameter of functional avidity and antitumor capacity of Ag-specific CTL. Yee et al. (50) showed that magnitude of tetramer binding was correlated with T cell avidity and suggested that tetramer staining could be used to isolate or identify high avidity CTL. However, further study (51) demonstrated that the relative efficiency of staining of Ag-specific CTL with tetramer could considerably vary with staining conditions and did not necessarily correlate with functional avidity. Instead, it was found that there was a clear correlation between functional avidity and the stability of tetramer and interaction with TCR (51). The staining intensity of tetramer may well correlate with functional avidity only for very high and very low avidity CTL as suggested by Derby et al. (52). For the "intermediate avidity" CTL, functional avidity of a CTL could not be predicted by magnitude of tetramer staining (52).
In summary, this study clearly demonstrates that the activity of peptide-specific CTL is correlated with their TCR avidity and determinant density on tumor cells. The study suggests that monitoring of peptide-specific CTL changes in circulation after peptide vaccination will require functional characterization of resulting cellular responses. It will be necessary to determine avidities of resultant CTL using either cytotoxic assays or other functional studies. Such information may enhance our understanding the complex mechanisms underlying the success and failure of vaccination therapy for cancer.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: G209, gp100 parental peptide (aa 209–217); G209-2M, gp100 peptide G209 with methionine substitution at the second position; DC, dendritic cell; Flu M1, HLA-A2-restricted, influenza virus matrix peptide; MxLD50, the amount of peptide required to mediate half-maximal lysis of target cells pulsed with optimal concentration of peptide in cytotoxicity assay; TAA, tumor-associated Ag; HA, hemagglutinin.
Received for publication January 7, 2002. Accepted for publication April 18, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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G. M. Bendle, A. Holler, L.-K. Pang, S. Hsu, M. Krampera, E. Simpson, and H. J. Stauss Induction of Unresponsiveness Limits Tumor Protection by Adoptively Transferred MDM2-Specific Cytotoxic T Lymphocytes Cancer Res., November 1, 2004; 64(21): 8052 - 8056. [Abstract] [Full Text] [PDF]
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 B. Wang, H. Chen, X. Jiang, M. Zhang, T. Wan, N. Li, X. Zhou, Y. Wu, F. Yang, Y. Yu, et al. Identification of an HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of SARS-CoV spike protein Blood, July 1, 2004; 104(1): 200 - 206. [Abstract] [Full Text] [PDF]
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S. Yang and F. G. Haluska Treatment of Melanoma with 5-Fluorouracil or Dacarbazine In Vitro Sensitizes Cells to Antigen-Specific CTL Lysis through Perforin/Granzyme- and Fas-Mediated Pathways J. Immunol., April 1, 2004; 172(7): 4599 - 4608. [Abstract] [Full Text] [PDF]
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N. C. V. Verra, A. Jorritsma, K. Weijer, J. J. Ruizendaal, A. Voordouw, P. Weder, E. Hooijberg, T. N. M. Schumacher, J. B. A. G. Haanen, H. Spits, et al. Human Telomerase Reverse Transcriptase-Transduced Human Cytotoxic T Cells Suppress the Growth of Human Melanoma in Immunodeficient Mice Cancer Res., March 15, 2004; 64(6): 2153 - 2161. [Abstract] [Full Text] [PDF]
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T. W. Kim, C.-F. Hung, D. Boyd, J. Juang, L. He, J. W. Kim, J. M. Hardwick, and T.-C. Wu Enhancing DNA Vaccine Potency by Combining a Strategy to Prolong Dendritic Cell Life with Intracellular Targeting Strategies J. Immunol., September 15, 2003; 171(6): 2970 - 2976. [Abstract] [Full Text] [PDF]
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S. Xu, G. K. Koski, M. Faries, I. Bedrosian, R. Mick, M. Maeurer, M. A. Cheever, P. A. Cohen, and B. J. Czerniecki Rapid High Efficiency Sensitization of CD8+ T Cells to Tumor Antigens by Dendritic Cells Leads to Enhanced Functional Avidity and Direct Tumor Recognition Through an IL-12-Dependent Mechanism J. Immunol., September 1, 2003; 171(5): 2251 - 2261. [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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L. Novellino, N. Renkvist, F. Rini, A. Mazzocchi, L. Rivoltini, A. Greco, P. Deho, P. Squarcina, P. F. Robbins, G. Parmiani, et al. Identification of a Mutated Receptor-Like Protein Tyrosine Phosphatase {kappa} as a Novel, Class II HLA-Restricted Melanoma Antigen J. Immunol., June 15, 2003; 170(12): 6363 - 6370. [Abstract] [Full Text] [PDF]
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S. Oh, J. W. Hodge, J. D. Ahlers, D. S. Burke, J. Schlom, and J. A. Berzofsky Selective Induction of High Avidity CTL by Altering the Balance of Signals from APC J. Immunol., March 1, 2003; 170(5): 2523 - 2530. [Abstract] [Full Text] [PDF]
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