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The Journal of Immunology, 2002, 169: 531-539.
Copyright © 2002 by The American Association of Immunologists

Antimelanoma Activity of CTL Generated from Peripheral Blood Mononuclear Cells After Stimulation with Autologous Dendritic Cells Pulsed with Melanoma gp100 Peptide G209-2M Is Correlated to TCR Avidity

Sixun Yang, Gerald P. Linette, Simonne Longerich and Frank G. Haluska1

Division of Hematology-Oncology, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02114


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Anchor residue-modified peptides derived from tumor-associated Ag have demonstrated success in engendering immune responses in clinical studies. However, tumor regression does not always correlate with immune responses. One hypothesis to explain this is that CTL resulting from such immunization approaches are variable in antitumor potency. In the present study, we evaluated this hypothesis by characterizing the activity of tumor-associated Ag-specific CTL. We chose an anchor residue-modified peptide from gp100, G209-2M, and used peptide-pulsed dendritic cells to generate CTL from PBMC of HLA-A2+ normal donors. The specificities and avidities of the resulting CTL were evaluated. The results demonstrate that CTL generated by G209-2M 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 G209 and G209-2M peptides but not melanomas; and melanoma-reactive CTL which recognize peptide-pulsed targets as well as HLA-A2+gp100+ melanomas. CTL that kill only peptide-pulsed targets require a higher peptide concentration to mediate target lysis, whereas CTL that lyse melanomas need a lower peptide concentration. Increasing peptide density on melanomas by loading exogenous G209 peptide enhances their sensitivity to peptide-specific CTL. High avidity CTL clones also demonstrate potent antimelanoma activity in melanoma model in nude mice. Injection of G209 peptide around transplanted tumors significantly enhances the antitumor activity of low avidity CTL. These results suggest that peptide stimulation causes expansion of T cell populations with a range of avidities. Successful immunotherapy may require selective expansion of the higher-avidity CTL and intratumor injection of the peptide may enhance the effect of peptide vaccines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cytotoxic T lymphocytes recognize antigenic peptides of 8–12 residues, bound by MHC class I molecules and presented on the surface of APCs and tumor cells (1). Recent efforts toward the development of anticancer vaccines have focused on the generation of tumor-specific CTL responses (2, 3).

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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor cell lines

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 {beta}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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CTL generated from PBMC by autologous DC pulsed with G209-2M are heterogeneous

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 IGo). 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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  		Table I. Summary of specificity of in vitro CTL response from normal individuals to modified anchor amino acid peptide G209-2M derived from melanoma-associated Ag gp1001

 
We then further investigated whether the resultant G209-2M-reactive CTL were able to recognize native peptide presented on T2 targets and endogenously processed peptide on HLA-A2+ melanoma cells expressing gp100. Among the 62 G209-2M-reactive CTL lines from the six donors, 24% (15 of 62) only lyse T2 targets pulsed with G209-2M peptide (named G209-2M-specific CTL), and 76% (47 of 62) recognize both the native G209 peptide and G209-2M peptide presented on T2 targets (named peptide-specific CTL). However, only 52% (32 of 62) lyse melanoma cells expressing both HLA-A2 and gp100 that presumably present endogenously processed peptide on their surface (named melanoma-reactive CTL). In addition, none of G209-2M-specific CTL lyse HLA-A2+gp100+ melanomas (Table IGo). 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. 1Go, 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. 1Go, 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. 1Go, A and B). As expected, the CTL did not lyse DM14 transduced with Ad2/hgp100 or A375 transduced with Ad2/MART-1 (Fig. 1GoA).



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  		FIGURE 1. Ag specificity and CTL responses to anchor residue-modified gp100 peptide G209-2M. CD8+ T lymphocytes from a normal donor were stimulated in vitro with G209-2M loaded onto autologous DC. The resultant CTL lines C1 (A) and E7 (B) were tested for their specificity against various melanoma targets. A, Cytolytic activity of CTL C1 against different targets at an E:T ratio of 10:1. B, Cytotoxicity of CTL E7 against melanoma targets at E:T ratios of 2:1 to 20:1. DM14/gp, A375/gp, and A375/Mart are DM14, A375 melanomas transduced with adenoviral vector containing the human gp100 gene and MART-1 gene, respectively. The transduced cells were labeled with 51Cr and used as targets 24–30 h after transduction. C, Cold target blocking of peptide-specific CTL. Peptide-specific CTL C1 and E7 were incubated with unlabeled cold targets DM13 or DM14 for 1 h at 37°C and then incubated with 51Cr-labeled hot target DM6 for an additional 4 h. The effector-hot target ratio is 10:1. Med, Medium.

 
Cold target blocking experiments also showed that lysis of DM6 (HLA-A2+gp100+) by melanoma-reactive CTL C1 and E7 (derived from the same normal donor) was competitively blocked by another HLA-A2+gp100+ melanoma DM13, but not by DM14 (HLA-A2-gp100-) (Fig. 1GoC). 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 IIGo). 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. 2Go 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. 3Go, 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 IIGo, 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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  		Table II. Summary of MxLD50 of the peptide-specific CTL tested and their activity against melanoma1

 


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  		FIGURE 2. Peptide-specific CTL with high avidity but not low avidity are cytolytic toward HLA-A2+ melanoma expressing human gp100. CD8+ T cells were stimulated with autologous DC pulsed with G209-2M three times at 7- to 10-day intervals and then cloned as described in Materials and Methods. The resultant CTL were tested for their avidities by using various concentrations of G209 peptide-loaded T2 targets. Simultaneously, melanoma DM6 and DM14 were also used to detect ability of the CTL to recognize endogenously processed peptide. E:T 10:1. A, Titration of MxLD50 of peptide-specific CTL. B, Activity of peptide-specific CTL toward melanomas DM6 (HLA-A2+gp100+) and DM14 (HLA-A2-gp100-). CTL 32 C1, 41 D7 and 41 E7 exhibit high avidity and antimelanoma activity.

 


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  		FIGURE 3. Regression analysis of relationship between CTL avidity and tumor reactivity. CTL avidity is expressed as MxLD50 as defined in the legend to Table IIGo. Net lytic activity toward HLA-A2+gp100+ melanomas for each CTL (y-axis) vs corresponding MxLD50 (x-axis) is plotted. The trend line for the CTL is shown, with a regression coefficient of -0.96 (% lysis vs log MxLD50). Regression analysis demonstrates that the p value for a two-sided test is <0.00001 and the 95% confidence interval on the correlation of percent lysis and logMxLD50 is (-0.90, -0.98)

 
Because the activity of a CTL is correlated to its MxLD50, we designated melanoma-reactive CTL as high avidity and melanoma-nonreactive peptide-specific CTL as low avidity in this study. Previous work by Alexander-Miller et al. (32) showed that in vitro stimulation with very low concentration of virus-derived peptide selectively expanded high avidity CTL. We thus tested whether low concentration of G209-2M peptide stimulation expanded high avidity CTL in human settings. The activity of CTL generated by autologous DC pulsed with various concentrations of G209-2M peptide from three donors was tested toward melanomas and summarized in Table IIIGo. 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 IIIGo).


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  		Table III. Activity of CTL generated from PBMC by autologous DC pulsed with various concentrations of G209-2M peptide1

 
Loading with exogenous G209 peptide increases the sensitivity of melanoma cells to low avidity G209-specific CTL

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. 4Go, 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. 4Go). 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. 4Go). 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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  		FIGURE 4. Loading melanoma cells with exogenous G209 peptide increases their sensitivity to peptide-specific CTL. Peptide-specific CTL clones (2B1, 2B4, 2D1, 1E5, 1F1 and 1F2) were generated from a normal donor 1119227 as described in Fig. 2Go. CTL activity was detected simultaneously against various concentration of G209 (0–1000 nM)-pulsed T2; T2 pulsed with G209-2M (1 µM); and DM13, DM14, and DM13 pulsed with G209 peptide (100 nM). E:T 10:1. A, Titration of MxLD50 of peptide-specific CTL. 2M, T2 pulsed with G209-2M (1 µM). B, Activity of peptide-specific CTL toward melanomas DM13 (HLA-A2+gp100+), DM13 pulsed with G209 (100 nM), and DM14 (HLA-A2-gp100-).

 
Immunotherapeutic effects of low and high avidity G209-specific CTL on human melanoma in nude mice

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. 4Go). A Flu M1-specific CTL derived from the same donor as 2B4 and 1F1 was also used as a control. As shown in Fig. 5Go, all mice in the PBS control group (treated with PBS; Fig. 5GoA), the Flu M1 control group (treated with Flu M1-specific CTL, Fig. 5BGo) and the 2B4 group (treated with low avidity CTL 2B4; Fig. 5GoC) 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. 5GoD). 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. 5GoC), 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. 5GoE). 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. 5GoF). 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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  		FIGURE 5. Comparison of antimelanoma activity of low and high avidity peptide-specific CTL in vivo. High and low avidity CTL 1F1 and 2B4 as described in Fig. 4Go were chosen in adoptive immunotherapy of human melanoma in nude mice. BALB/c nude mice were inoculated with 1 x 107 DM6 melanoma (HLA-A2+gp100+) s.c. Mice were treated 7 days later, when tumor diameter reaches 2–6 mm, with i.v. CTL (1 x 107 cells/mouse in 0.5 ml) with or without coinjection of peptide (200 µg/mouse in 0.2 ml) around tumor. Each panel illustrates serial tumor measurements for the five individual mice in the experimental group. A, PBS control; B, Flu M1-specific CTL control; C, CTL 2B4; D, CTL 1F1; E, 2B4 plus G209; F, 2B4 plus G280. Tumor size was measured every 5 days for 30 days. n = 5.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The use of synthetic peptides derived from TAA, either emulsified in adjuvant or loaded on DC, is a promising immunotherapy approach for the treatment of cancer (27, 35, 36, 37, 38, 39, 40). However, in a published trial, immunization with G209-2M alone in patients with melanomas rarely led to melanoma regression, although increased CTL responses to both parental and modified peptides were observed in most cases (27). The reasons for the failure of expanded CTL to eliminate tumor are not clear, but analysis of fine specificity and features of CTL generated with peptides may provide some explanation. In the present study, we have demonstrated that CTL generated by G209-2M can be classified into three groups: G209-2M-specific CTL which lyse only G209-2M-bearing targets; peptide-specific CTL which lyse both native and modified peptide-bearing targets but not melanomas; and melanoma-reactive CTL which lyse melanoma as well as peptide (both native and modified)-pulsed targets. None of G209-2M-specific CTL lyse melanomas and none of the melanoma-reactive CTL fail to recognize peptide (both G209 and G209-2M) presented on T2 cell targets in our system. Furthermore, among the G209-specific CTL, melanoma reactivity is correlated to TCR avidity; i.e., only CTL with high avidity can recognize endogenously processed peptide on melanomas. In addition, the G209-specific, melanoma-nonreactive CTL are able to kill melanoma cells if antigenic determinant density on the melanoma cell surface increased by loading with exogenous peptide. Furthermore, consistent with our in vitro observation, only high avidity CTL showed antimelanoma behavior in our human melanoma model in nude mice as observed in our in vivo study. The data are also consistent with study by Sherman’s group (41). Using a hemagglutinin (HA)-transgenic mouse model, they found that only high avidity HA-specific CTL induced diabetes in HA-transgenic mice after adoptive transfer of HA-specific CTL (41). Taken together, the studies suggest that high, but not low, avidity Ag-specific CTL contribute to autoimmune responses and effective antitumor immunity.

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 IIGo, 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. 4Go 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. 2Go and 4Go). This observation differs from that reported by Dudley et al. (38). In their study, in which IFN-{gamma} 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. 2Go and 4Go 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.


   Acknowledgments
 
We thank Drs. Timothy L. Darrow and H. F. Seigler (Duke University Medical Center, Durham, NC) for kindly providing melanoma and A. Khatri (Massachusetts General Hospital) for peptide synthesis. We thank Mark Vangel for statistical assistance.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Frank G. Haluska, Jackson Building, GRJ 1021, Hematology-Oncology Unit, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114. E-mail address: haluska.frank{at}mgh.harvard.edu Back

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. Back

Received for publication January 7, 2002. Accepted for publication April 18, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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