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CD4-Dependent Potentiation of a High Molecular Weight-Melanoma-Associated Antigen-Specific CTL Response Elicited in HLA-A2/K^b Transgenic Mice¹

Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263

	Abstract

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The human high m.w.-melanoma-associated Ag (HMW-MAA) is an attractive target for the immunotherapy of melanoma, due to its relatively high expression in a high percentage of melanoma lesions and its restricted distribution in normal tissues. Active immunization with HMW-MAA mimics has been previously shown to induce a HMW-MAA-specific, T cell-dependent Ab response associated with an apparent clinically beneficial effect in advanced melanoma patients. Although T cells play an important role in controlling tumor growth, only limited information is available to date about the induction of HMW-MAA-specific CTL. In this report, we show that immunization of HLA-A2/K^b transgenic mice with HMW-MAA cDNA-transfected syngeneic dendritic cells elicited a CD8⁺ CTL response specific for HMW-MAA peptides with HLA-A2 Ag-binding motifs. The elicited CTL lysed HLA-A2⁺HMW-MAA⁺ melanoma cells in vitro, and mouse HLA-A2/K^b cells pulsed with HMW-MAA-derived peptides in vitro and in vivo. Although this CTL response could be generated in the absence of CD4⁺ T cell help, harnessing CD4⁺ T cell help in a noncognate Ag-specific manner with the polyclonal activator staphylococcal enterotoxin A augmented the CTL response. These results imply that dendritic cell-based immunization, in combination with CD4⁺ T cell help, represents an effective strategy to implement T cell-based immunotherapy targeting HMW-MAA in patients with HMW-MAA-bearing tumors.

	Introduction

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The human high m.w.-melanoma-associated Ag (HMW-MAA)⁴ is a membrane-bound proteoglycan that is overexpressed in a high percentage of melanoma lesions with low intra- and interlesional heterogeneity and has a restricted distribution in normal tissues (1, 2, 3). In a phase I/II clinical trial, we have shown that the anti-Id mAb MK2–23, which mimics the HMW-MAA determinant recognized by mAb 763.74 (4), elicited a T cell-dependent, HMW-MAA-specific Ab response that was associated with regression of metastatic lesions in a few patients (5) and with a significantly prolonged survival in patients with stage IV melanoma (6). Despite the demonstrated role of T cells in controlling tumor growth (7, 8), only limited information is available to date about the generation of HMW-MAA-specific CTL responses by active specific immunotherapy (9, 10). In this study, we used HLA-A2/K^b transgenic mice to assess whether immunization with the HMW-MAA cDNA-transfected syngeneic dendritic cell (DC) could induce HLA-A2-restricted, HMW-MAA-specific CTL responses. HLA-A2/K^b transgenic mice were selected for this purpose because their T cell repertoire bears extensive overlap with that of HLA-A*0201⁺ human subjects (11, 12). Therefore, the HLA-A2-restricted CTL response elicited in HLA-A2/K^b mice is expected to resemble the corresponding response in HLA-A*0201⁺ humans. Furthermore, a DC-based immunization strategy was selected because Ag-loaded DC-based immunization strategies can elicit robust immune responses to self-Ags (13, 14). Lastly, we have investigated the requirement for CD4⁺ T cells to generate a HMW-MAA-specific CTL response and their ability to potentiate this response, because CD4⁺ T cells play an important role in the maintenance of effective and durable CTL responses (15, 16, 17, 18, 19, 20), even though they may not be absolutely required to induce CD8⁺ CTL responses (21, 22, 23). The absence of CD4⁺ T cell help results in lethargy (17) or defective memory (18, 19, 20) of CD8⁺ T cells. In addition, although CD8⁺ T cells by themselves can elicit robust antitumor effects (24, 25), protective antitumor CTL immunity has been shown in certain instances to require CD4⁺ T cell help (15, 26, 27, 28). Because no information is currently available regarding the MHC class II Ag restriction of HMW-MAA in the HLA-A2/K^b mouse model, we have used the bacterial superantigen staphylococcal enterotoxin A (SEA) to recruit CD4⁺ T cell help in a noncognate Ag-specific manner. SEA interacts with MHC class II Ags and V chains of TCRs (29), leading to polyclonal CD4⁺ T cell activation and expansion across multiple V families (V1, 3, 10, 11, 12, and 17) (30, 31).

	Materials and Methods

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Mice

A breeder pair of HLA-A2/K^b transgenic mice (11) was kindly provided by Dr. L. Sherman (The Scripps Research Institute, La Jolla, CA). Mice were maintained and bred under specific pathogen-free conditions in the animal core facility at Roswell Park Cancer Institute (RPCI; Buffalo, NY). Six to 8-wk-old female mice were used for all the studies. Experiments were conducted under an animal protocol approved by the Animal Care and Use Committee at RPCI.

Cell lines

The human melanoma cell lines 1088 (HLA-A2⁺HMW-MAA⁺), M14#5 (HLA-A2⁺HMW-MAA^–), and Colo38 (HLA-A2^–HMW-MAA⁺) were maintained in RPMI 1640 medium (Tissue Culture Media Facility, RPCI) supplemented with 10% heat-inactivated FCS, 0.1 mM nonessential amino acids, 1 µM sodium pyruvate, 2 mM fresh L-glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, 50 µg/ml gentamicin, 0.5 µg/ml Fungizone (Invitrogen Life Technologies), and 50 µM 2-ME (Sigma-Aldrich) (complete medium). The murine EL-4 thymoma cell line transfected with the HLA-A2/K^b gene (EL-4/A2K^b; provided by Dr. L. Sherman, The Scripps Research Institute, La Jolla, CA) was maintained in complete medium containing 500 µg/ml of the selective antibiotic G418 (Invitrogen Life Technologies). All cell lines were maintained at 37°C in a humidified 5% CO₂ atmosphere.

Antibodies

The HLA-A2, A24, A28-specific mAb CR11-351 (32), the HMW-MAA-specific mAb 763.74 (33), the CD3-specific mAb 145-2C11 (34), the CD4-specific mAb GK1.5 (35), and the CD8-specific mAb 2.43 (36) have been previously described. mAbs were purified from ascitic fluid by sequential ammonium sulfate and caprylic acid precipitation (37). FITC, PE, or PE-Cy5-conjugated mAb to mouse CD3, CD4, CD8, CD11c, CD25, CD69, CD80, CD86, H-2 K^b, and H-2 I-A^b Ags; unconjugated mAb to mouse CD16/CD32; FITC, PE, or PE-Cy5-conjugated isotype-matched control Abs; and PE-labeled F(ab')₂ of goat anti-mouse Ig Abs were purchased from BD Biosciences.

HMW-MAA cDNA and synthetic peptides

The plasmid construct containing full-length HMW-MAA cDNA was prepared as previously described (38). Briefly, HMW-MAA mRNA isolated from A375SM melanoma cells was reverse-transcribed and amplified by PCR, and the HMW-MAA cDNA product was inserted into the pcDNA3.1⁺ vector (Invitrogen Life Technologies). Synthetic peptides were purchased from the Molecular Genetics Instrumentation Facility, University of Georgia (Athens, GA).

Staphylococcal enterotoxin A

SEA was purchased from Sigma-Aldrich. It was reconstituted to a concentration of 10 µg/ml in sterile PBS before injection.

Flow cytometry

Flow cytometry analysis of cells was performed as described (39). Briefly, 5 x 10⁵ cells were incubated for 20 min at 4°C with 1 µg of unconjugated anti-mouse CD16/CD32 mAb to block surface FcRs, washed twice with 0.5% BSA in PBS (BSA-PBS), and then incubated for 20 min at 4°C with PE- or FITC-conjugated mAb, or for 20 min at 4°C with unconjugated mAb followed by 30 min at 4°C with PE-labeled goat anti-mouse Ig Abs. Cells were then washed twice, fixed in 2% paraformaldehyde, and analyzed with a FACScan flow cytometer (BD Biosciences) using CellQuest software.

Generation of DCs

DCs were generated in vitro from bone marrow precursors as previously described (40). Briefly, bone marrow cells were harvested from the tibias and femurs of 6- to 8-wk-old female HLA-A2/K^b mice and then cultured in complete medium supplemented with 10 ng/ml GM-CSF at 37°C in a 5% CO₂ atmosphere for 6 days. The culture medium was replenished every 2–3 days. On day 6 of culture, most of the nonadherent cells had acquired typical DC morphology, and these cells expressed moderate to high levels of MHC class I and class II Ags, CD11c, CD54, CD80, and CD86, as determined by flow cytometry (data not shown).

Transfection of DCs

Day 6 DCs were transfected with a plasmid containing the full-length HMW-MAA cDNA or with the empty vector pcDNA3.1 (Invitrogen Life Technologies), using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. Briefly, day 6 DCs (1 x 10⁶ cells/well) were incubated in 24-well plates (Costar 3524; Corning) with Lipofectamine-DNA complexes in Opti-MEM medium (Invitrogen Life Technologies) for 6 h. Then the medium was replaced with complete medium, and incubation was continued for an additional 18 h at 37°C.

Immunization with transfected DCs

After transfection and overnight culture at 37°C, DCs were washed three times in HBSS. DCs (5 x 10⁵/100 µl of HBSS) were injected s.c. bilaterally into the lower flanks of HLA-A2/K^b mice. Empty vector-transfected or untransfected DCs were used as immunization controls. Mice were immunized three times at 7-day intervals. Spleens were harvested 8 days after the final immunization.

In vivo cell depletion

CD4⁺ or CD8⁺ cells were depleted in vivo by injecting mice i.v. with CD4-specific mAb GK1.5 or CD8-specific mAb 2.43 (500 µg/mouse), respectively, 1 day before each immunization, as previously described (41). Flow cytometry analysis of splenocytes from mice injected with CD4-specific or CD8-specific mAb consistently revealed >95% depletion of the corresponding cell subset. The cell depletion was observed at 24 h after mAb injection and persisted for up to 7 days (data not shown).

Cytotoxicity assays

In vitro cytotoxicity assays were performed as described (42, 43). Pooled splenocytes (three mice per group) were either used directly as effector cells in a 10-h ⁵¹Cr-release assay (fresh splenocytes), or were stimulated in vitro for 5 days at 37°C with EL-4/A2K^b cells pulsed with three HMW-MAA-derived peptides and IL-2 (15 U/ml) before their use in a 6-h ⁵¹Cr-release assay (in vitro-stimulated splenocytes). Assays were performed in triplicates, and the percentage of specific cytotoxicity was calculated using the formula: percent cytotoxicity = (experimental cpm – spontaneous cpm)/(maximum cpm – spontaneous cpm) x 100. For all assays, the spontaneous ⁵¹Cr release was <25% of maximum ⁵¹Cr release.

The in vivo cytotoxicity assay was performed as described (44). Briefly, single-cell suspensions of HLA-A2/K^b splenocytes were split into two populations. One population was pulsed overnight with a panel of HMW-MAA-derived, HLA-A2-restricted peptides and labeled with 5 µM CFSE (Molecular Probes). The other population was left unpulsed and labeled with 0.5 µM CFSE. Equal numbers of cells from each population (1 x 10⁷ cells) were mixed together and adoptively transferred into HLA-A2/K^b mice. Four hours after adoptive transfer of CFSE-labeled splenocytes, mice were sacrificed and splenocytes were analyzed by flow cytometry. To calculate specific lysis, the following formulas were used: ratio = (percentage CFSE^low/percentage CFSE^high); percentage specific lysis = (1 – (ratio naive/ratio immunized) x 100).

Statistical analysis

Statistical analysis was performed using SigmaStat software (Jandel Scientific). Differences between groups were analyzed by the two-tailed, unpaired Student t test. A value of p < 0.05 was considered to be significant.

	Results

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CD69 up-regulation and IFN- production by CD8⁺ T cells from HLA-A2/K^b mice immunized with HMW-MAA-transfected DC

By flow cytometry analysis, empty vector- and HMW-MAA-transfected DC expressed higher levels of I-A^b and CD86 Ags than untransfected DC, suggesting that transfection with Lipofectamine 2000 enhances DC maturation (data not shown). However, the vector- and HMW-MAA-transfected DCs expressed similar levels of K^b, I-A^b, CD80, and CD86 Ags, suggesting that these DC populations are of a similar maturation state (Fig. 1). Approximately 27% of the HMW-MAA-transfected DCs were stained by HMW-MAA-specific mAb 763.74 (Fig. 1). In contrast, no staining of nontransfected or vector-transfected DCs by mAb 763.74 was detected (Fig. 1 and data not shown).

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Flow cytometry analysis of surface marker expression on HMW-MAA-transfected DCs. Bone marrow-derived DCs from HLA-A2/Kb mice were transfected with a plasmid containing the full-length HMW-MAA cDNA. The cells were stained with FITC- or PE-conjugated mAb to CD11c, Kb, I-Ab, CD80, or CD86, or sequentially with HMW-MAA-specific mAb 763.74 and PE-conjugated goat anti-mouse Ig Abs. The cells were fixed, analyzed by flow cytometry, and CD11c+ gated (G1) cells were assessed for surface expression of Kb, I-Ab, CD80, CD86, and HMW-MAA. The values indicate the percentages of marker-positive cells among gated CD11c+ cells. Bone marrow-derived DCs transfected with the empty vector were used as controls. The results shown are representative of three independent experiments.

View larger version (35K): [in this window] [in a new window]   FIGURE 2. Activation of T cells in HLA-A2/Kb mice immunized with HMW-MAA-transfected DC. A, Single-cell suspensions of splenocytes obtained from mice immunized with HMW-MAA-transfected DCs were analyzed by flow cytometry either immediately after harvest (direct ex vivo splenocytes), or after in vitro culture for 5 days in the presence of 15 U of IL-2 and EL-4/A2Kb cells pulsed with HMW-MAA peptides 769, 1063, and 2238 (in vitro-stimulated splenocytes). Cells were stained with PE-Cy5-conjugated mAb to CD8, in combination with PE-conjugated mAb to CD25 or CD69. Single-cell suspensions of splenocytes obtained from mice immunized with empty vector-transfected DCs were used as controls. The results shown are representative of three independent experiments. B, Splenocytes (2.0 x 106 cells/well) were cocultured in complete medium with EL-4/A2Kb cells pulsed for 24 h with HMW-MAA peptides 769, 1063, and 2238 (5.0 x 105 cells/well). After 24 h, the culture supernatants were collected and tested for IFN- production by ELISA. Splenocytes were cocultured with EL-4/A2Kb cells pulsed with HER-2/neu369–377 as a specificity control, and IFN- production was <50 pg/ml (data not shown). Single-cell suspensions of splenocytes obtained from mice immunized with empty vector-transfected DC were used as controls. The results shown are representative of three independent experiments, and the values are expressed as mean ± SD of triplicate wells. *, p < 0.05 for the indicated comparisons.

Requirement for multiple immunizations with HMW-MAA-transfected DC to induce detectable HMW-MAA-specific cytotoxicity

Splenocytes were harvested from HLA-A2/K^b mice 8 days after the first, second, and third immunization with HMW-MAA-transfected DCs, and were cultured at 37°C for 5 days in vitro in the presence of HMW-MAA peptide-pulsed EL-4/A2K^b cells and IL-2 (15 U). In vitro-stimulated splenocytes from mice immunized three times with HMW-MAA-transfected DC generated 40% specific lysis of HMW-MAA peptide-pulsed EL-4/A2K^b target cells (E:T ratio of 25:1; Fig. 3). In vitro-stimulated splenocytes from mice that had been immunized twice with HMW-MAA-transfected DC lysed target cells to a low extent, while in vitro-stimulated splenocytes from mice that had been either not immunized or immunized only once with HMW-MAA-transfected DC did not lyse HMW-MAA peptide-pulsed target cells.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Requirement for multiple immunizations with HMW-MAA-transfected DCs to induce detectable HMW-MAA-specific cytotoxicity. Splenocytes from HLA-A2/Kb mice immunized with HMW-MAA-transfected DCs were harvested 8 days after the first (), second (), or third () immunization. After in vitro stimulation, cytotoxic activity was measured using EL-4/A2Kb cells pulsed with HMW-MAA peptides 769, 1063, and 2238 as target cells in a standard 6-h 51Cr-release assay. Splenocytes harvested from naive HLA-A2/Kb mice (•) were used as a control. The results shown are representative of three independent experiments, and the values are expressed as mean ± SD of triplicate wells. *, p < 0.05 vs naive HLA-A2/Kb mice, or HLA-A2/Kb mice immunized once or twice.

Five-day in vitro-stimulated splenocytes from mice immunized with HMW-MAA-transfected DC lysed HMW-MAA peptide-pulsed EL-4/A2K^b cells (Fig. 4A), in an Ag-dependent manner (Fig. 4B). Moreover, the splenocytes lysed HLA-A2⁺HMW-MAA⁺ 1088 melanoma cells (Fig. 4C), but lysed neither HLA-A2⁺HMW-MAA^– M14#5 melanoma cells (Fig. 4D) nor HLA-A2^–HMW-MAA⁺ Colo38 melanoma cells (Fig. 4E).

View larger version (11K): [in this window] [in a new window]   FIGURE 4. Induction of HMW-MAA-specific CTL in HLA-A2/Kb mice by immunization with HMW-MAA-transfected DCs. Splenocytes harvested from mice immunized three times with HMW-MAA-transfected DC () were stimulated in vitro, and the cytotoxic activity of these splenocytes were tested by a standard 6-h 51Cr-release assay using as target cells: A, EL-4/A2Kb cells pulsed with the HMW-MAA peptides 769, 1063, and 2238; B, unpulsed EL-4/A2Kb cells; C, HLA-A2+HMW-MAA+ 1088 cells; D, HLA-A2+HMW-MAA– M14#5 cells; or E, HLA-A2–HMW-MAA+ Colo38 cells. Splenocytes harvested from mice immunized with empty vector-transfected DCs (•) or untransfected DCs () were used as controls. The results shown are representative of three independent experiments, and the values are expressed as mean ± SD of triplicate wells. *, p < 0.05 vs HLA-A2/Kb mice immunized with vector-transfected DCs or untransfected DCs.

View larger version (11K): [in this window] [in a new window]   FIGURE 5. CD8+ cell dependence and HLA-A2 restriction of HMW-MAA-specific CTL in HLA-A2/Kb mice immunized with HMW-MAA-transfected DCs. A, HLA-A2/Kb mice were injected with CD4-specific mAb GK1.5 (500 µg/mouse; ) or with CD8-specific mAb 2.43 (500 µg/mouse; ) 1 day before each immunization with HMW-MAA-transfected DC. Splenocytes were harvested 8 days after the third immunization and were stimulated in vitro. Cytotoxic activity was measured by a standard 6-h 51Cr-release assay using EL-4/A2Kb cells pulsed with the HMW-MAA peptides 769, 1063, and 2238 as target cells. Mice injected with an isotype-matched control Ab (500 µg/mouse; ) and untreated mice (•) were used as controls. The results shown are representative of three independent experiments, and the values are expressed as mean ± SD of triplicate wells. *, p < 0.05 vs all other groups. B, Splenocytes from mice immunized with HMW-MAA-transfected DC were harvested 8 days after the third immunization and were stimulated in vitro. Cytotoxic activity was measured by a standard 6-h 51Cr-release assay using EL-4/A2Kb cells pulsed with the HMW-MAA peptides 769, 1063, and 2238 as target cells in the presence of HMW-MAA-specific mAb 763.74 (), HLA-A2, A24, A28-specific mAb CR11-351 (), an isotype-matched control mAb () or no mAb (•). The results shown are representative of three independent experiments, and the values are expressed as mean ± SD of triplicate wells. *, p < 0.05 vs all other groups.

In vitro-stimulated splenocytes from mice immunized with HMW-MAA-transfected DC generated 45–55% lysis of EL-4/A2K^b target cells pulsed with a combination of HMW-MAA peptides 769, 1063, and 2238 (E:T ratio of 25:1; Figs. 3, 4, and 6A). These effector cells did not lyse EL-4/A2K^b target cells that were pulsed with the irrelevant HLA-A2-binding peptide HER-2/neu_369–377 (45) (Fig. 6B). Interestingly, the same effector cells generated 25–35% lysis of EL-4/A2K^b target cells pulsed with HMW-MAA peptides 769, 1063, or 2238 separately (E:T ratio of 25:1; Fig. 6, C–E), indicating that the effector cell population consists of subsets that specifically recognize each of these HLA-A2-binding peptides.

View larger version (10K): [in this window] [in a new window]   FIGURE 6. Fine specificity of HMW-MAA-specific CTL in HLA-A2/Kb mice immunized with HMW-MAA-transfected DCs. Splenocytes were harvested from mice immunized three times with HMW-MAA-transfected DCs. These splenocytes were stimulated for 5 days in vitro, and the cytotoxic activity of the splenocytes were tested by a standard 6-h 51Cr-release assay using EL-4/A2Kb cells pulsed for 24 h with a mixture of HMW-MAA peptides 769, 1063, and 2238 (A, •), or EL-4/A2Kb cells pulsed with only peptide 769 (C, •), 1063 (D, •), or 2238 (E, •) as target cells. EL-4/A2Kb cells pulsed with the irrelevant HLA-A2-binding peptide HER-2/neu369–377 (B, •) or unpulsed EL-4/A2Kb cells (A–E, ) were used as controls in cytotoxicity assays. The results shown are representative of three independent experiments, and the values are expressed as mean ± SD of triplicate wells. *, p < 0.05

To rule out the contribution of in vitro stimulation on the HMW-MAA-specific CTL responses, we performed an in vivo CTL assay in HLA-A2/K^b mice immunized with HMW-MAA-transfected DCs. CFSE-labeled HLA-A2/K^b splenocytes that were pulsed with a combination of HMW-MAA peptides 769, 1063, and 2238 were used as target cells. In contrast to naive HLA-A2/K^b mice (Fig. 7A) or HLA-A2/K^b mice immunized with untransfected DCs (Fig. 7B), the 46% loss of CFSE^high splenocytes in HLA-A2/K^b mice immunized with HMW-MAA-transfected DCs (Fig. 7C) demonstrates that robust HMW-MAA-specific CTL activity can be detected in vivo in mice immunized with HMW-MAA-transfected DCs.

View larger version (20K): [in this window] [in a new window]   FIGURE 7. In vivo HMW-MAA-specific CTL in HLA-A2/Kb mice immunized with HMW-MAA-transfected DCs. Splenocytes from naive HLA-A2/Kb mice were either pulsed with a panel of HLA-A2-restricted, HMW-MAA-derived peptides (769, 1063, and 2238) and labeled with 5 µM CFSE (CFSEhigh), or were left unpulsed and labeled with 0.5 µM CFSE (CFSElow). An equal number of cells from each population (1 x 107 cells) were mixed together and adoptively transferred into naive HLA-A2/Kb mice (A), or HLA-A2/Kb mice on day 8 after the third immunization with untransfected (B) or HMW-MAA-transfected (C) DCs. As a control, a separate group of HLA-A2/Kb mice were adoptively transferred with 5 days in vitro-cultured HMW-MAA-specific CTL (2.0 x 107 cells/mouse) 30 min before the in vivo cytotoxicity assay (D). Four hours after adoptive transfer of CFSE-labeled splenocytes, mice were sacrificed and total splenocytes were analyzed by flow cytometry, with gating on CFSE-positive cells. The values indicate the percentages of target cells killed, compared with the same target cells in naive HLA-A2/Kb mice. The results shown are representative of three independent experiments.

View larger version (11K): [in this window] [in a new window]   FIGURE 8. Ex vivo HMW-MAA-specific CTL in HLA-A2/Kb mice immunized with HMW-MAA-transfected DCs. A, Splenocytes were harvested from mice immunized three times with HMW-MAA-transfected DCs (open symbols). They were either used directly in a 10-h 51Cr-release cytotoxicity assay (circles), or were stimulated for 5 days in vitro, as described in Fig. 2, followed by a 6-h 51Cr-release assay (triangles). EL-4/A2Kb cells pulsed with HMW-MAA peptides 769, 1063, and 2238 were used as target cells. Splenocytes harvested from mice immunized three times with untransfected DCs (filled symbols) were used as a control. The results shown are representative of three independent experiments, and the values are expressed as mean ± SD of triplicate wells. *, p < 0.05 vs the corresponding untransfected DC group. B, HLA-A2/Kb mice were injected with CD4-specific mAb GK1.5 (500 µg/mouse; ) or with CD8-specific mAb 2.43 (500 µg/mouse; ) 1 day before each immunization with HMW-MAA-transfected DC, as described in Fig. 5. Splenocytes were harvested 8 days after the third immunization and were used directly in a 10-h 51Cr-release cytotoxicity assay using EL-4/A2Kb cells pulsed with a mixture of HMW-MAA peptides 769, 1063, and 2238 as target cells. Mice injected with an isotype-matched control Ab (500 µg/mouse; ) or untreated mice (•) were used as controls. The results shown are representative of three independent experiments, and the values are expressed as mean ± SD of triplicate wells. *, p < 0.05 vs all other groups.

To assess whether the HMW-MAA-specific CTL response could be augmented in a CD4⁺ T cell-dependent manner, we evaluated whether the bacterial superantigen SEA, a polyclonal CD4⁺ T cell activator (29, 30, 31), could enhance the HMW-MAA-specific CTL response. We chose an injection schedule consisting of multiple administrations across a time frame spanning each DC immunization, because the efficacy of SEA-mediated enhancement of CD4⁺ T cell responses has been shown to depend on either multiple or continuous administration (46). Injection of HLA-A2/K^b mice with SEA (10 µg/mouse i.p. twice a day) for 4 days markedly expanded the population of CD4⁺ T cells, but not that of CD8⁺ T cells (Fig. 9A). Based on these initial findings, HLA-A2/K^b mice were injected with the same route, dose, and schedule of SEA, and were immunized with HMW-MAA-transfected DCs on day 3. This immunization regimen was repeated a total of three times; spleens were harvested 8 days after the third immunization. In the presence of SEA treatment, both fresh and in vitro-stimulated splenocytes were more effective in lysing HMW-MAA peptide-pulsed EL-4/A2K^b cells in vitro (Fig. 9, B and C). In addition, the level of in vivo cytotoxicity to HMW-MAA peptide-pulsed splenocytes was higher in HLA-A2/K^b mice immunized with HMW-MAA-transfected DCs in combination with SEA injection than in mice immunized with HMW-MAA-transfected DCs only (Fig. 10, B and C). The immunostimulatory effect of SEA is due to the selective expansion of the CD4⁺ T cell population, because mAb depletion of CD4⁺ cells before SEA injection and DCs immunization resulted in a loss of augmentation of the HMW-MAA-specific CTL activity (Fig. 10D).

View larger version (44K): [in this window] [in a new window]   FIGURE 9. Augmentation of in vitro HMW-MAA-specific CTL activity by enhancement of CD4+ T cell activity with SEA. HLA-A2/Kb mice were injected i.p. with SEA (10 µg/injection/mouse, twice a day) for 1, 2, 3, or 4 days. Spleens were harvested 10 days after final treatment. Single-cell suspensions were stained with FITC-conjugated mAb to CD3, in combination with PE-conjugated mAb to CD4 or CD8, and were analyzed by flow cytometry (A). HLA-A2/Kb mice were treated with SEA for 4 days (as described above, ), and were immunized with HMW-MAA-transfected DCs on day 3. This immunization regimen was repeated a total of three times. Spleens were harvested 8 days after third immunization. HLA-A2/Kb mice immunized with HMW-MAA-transfected () or untransfected (•) DCs in the absence of SEA treatment were used as controls. Splenocytes were either used directly in a 10-h 51Cr-release cytotoxicity assay (B), or were stimulated for 5 days in vitro, as described in Fig. 2, followed by a 6-h 51Cr-release assay (C). EL-4/A2Kb cells pulsed with HMW-MAA peptides 769, 1063, and 2238 were used as target cells. EL-4/A2Kb cells pulsed with HER-2/neu369–377 were used as controls in cytotoxicity assays; specific lysis was <5% at an E:T ratio of 25:1 (data not shown). The results shown are representative of three independent experiments, and the values are expressed as mean ± SD of triplicate wells. *, p < 0.05 vs HMW-MAA-transfected DC immunization in the absence of SEA treatment.

View larger version (19K): [in this window] [in a new window]   FIGURE 10. Augmentation of in vivo HMW-MAA-specific CTL activity by enhancement of CD4+ T cell activity with SEA. HLA-A2/Kb mice were injected with SEA for 4 days and were immunized with HMW-MAA-transfected DC on day 3, as described in Fig. 9. This immunization regimen was repeated a total of three times. As target cells, splenocytes from naive HLA-A2/Kb mice were either pulsed with a panel of HLA-A2-restricted, HMW-MAA-derived peptides (769, 1063, and 2238) and labeled with 5 µM CFSE (CFSEhigh), or were left unpulsed and labeled with 0.5 µM CFSE (CFSElow). An equal number of cells from each population (1 x 107 cells) were mixed together and adoptively transferred into HLA-A2/Kb mice on day 8 after the third immunization (C). As controls, the same target cells were also transferred into naive HLA-A2/Kb mice (A), HLA-A2/Kb mice immunized three times with HMW-MAA-transfected DC in the absence of SEA treatment (B), or HLA-A2/Kb mice immunized with HMW-MAA-transfected DC in the presence of SEA, but depleted of CD4+ cells with CD4-specific mAb GK1.5 (500 µg/mouse) 1 day before the start of each immunization cycle (D). Four hours after adoptive transfer of CFSE-labeled splenocytes, mice were sacrificed and total splenocytes were analyzed by flow cytometry, with gating on CFSE-positive cells. The values indicate the percentages of target cells killed, compared with the same target cells in naive HLA-A2/Kb mice. The results shown are representative of three independent experiments.

	Discussion

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Our findings complement two previous reports of HLA-A2 Ag-restricted, HMW-MAA-specific CTL in the peripheral blood lymphocytes obtained from melanoma patients immunized with MEL- IMMUNE, which consists of the murine anti-Id mAb MEL-2 and MF11-30. These two anti-Id mAb mimic distinct Ab-defined epitopes of HMW-MAA (9, 10). Analysis of the fine specificity of the HMW-MAA-specific CTL response identified the HLA-A2 Ag-restricted CTL epitope HMW-MAA_76–84, on the basis of its homology to a linear amino acid sequence in the V_H region of mAb MF11-30 (10). Although the previous finding demonstrated that HMW-MAA mimics such as anti-Id mAb generated a HMW-MAA-specific CTL response, we have established in this report that the nominal Ag can also generate a specific CTL response, when presented in an immunogenic context. Moreover, we have identified three HLA-A2 Ag-restricted CTL epitopes from HMW-MAA, which can be recognized by CTL elicited by immunization with HMW-MAA-transfected DC. These CTL epitopes are located in regions of HMW-MAA that are distinct from the previously identified epitope. Two (HMW-MAA_769–777 and HMW-MAA_1063–1071) are located in membrane-proximal regions of the extracellular domain, while the other (HMW-MAA_2238–2246) is located in the transmembrane domain.

Our findings suggest that although CD4⁺ cells are not absolutely required to elicit a HMW-MAA-specific CTL response by immunization with HMW-MAA-transfected DCs, the polyclonal expansion of noncognate T cells by SEA can enhance the HMW-MAA-specific CTL response in a CD4⁺ cell-dependent manner. These results are consistent with those of other investigators, who have noted that the presence of CD4⁺ T cells may potentiate the CD8⁺ T cell response (17), although CD4⁺ T cell help is not absolutely required for priming a CD8⁺ T cell response (21, 22, 23). In addition, these and other investigators have demonstrated that CD8⁺ T cells generated in the absence of CD4⁺ T cell help do not show a sustained response and memory during antigenic rechallenge (16, 18, 19, 20). In our study, it is likely that polyclonal expansion and activation of the CD4⁺ T cell population in a non-HMW-MAA-specific manner is sufficient to provide T cell help for the potentiation of a HMW-MAA-specific CD8⁺ T cell population. Several possibilities that are not mutually exclusive may account for the CD4⁺ T cell independence of the HMW-MAA-specific CTL response elicited by our DC immunization strategy, as well as the potentiation of this CTL response in the presence of CD4⁺ T cell help from SEA. First, the DC population that we have used is mature and activated in vitro, and therefore may be sufficient to elicit a HMW-MAA-specific CTL response independently of the requirement for CD4⁺ T cell help (47). However, the in vivo effects of DC immunization may be transient, and therefore the availability of CD4⁺ T cell help from SEA may prolong the HMW-MAA-specific CTL response (48). Second, the enhancement of the HMW-MAA-specific CTL response by CD4⁺ T cells may involve the production of Th1 cytokines (49), which may impact on the CTL directly and thus bypasses a direct interaction between the CD4⁺ T cells and DCs (50). Third, SEA may exert a direct stimulatory effect on DCs, which may enhance their ability to elicit a robust HMW-MAA-specific CTL response independently of CD4⁺ T cells. Direct APC activation by pathogens has been described to replace the CD4⁺ T cell-mediated helper effect (51). In this scenario, the role of the CD4⁺ T cells may be distinct from their role in the activation of DCs.

We expect the findings obtained in HLA-A2/K^b transgenic mice to be clinically relevant and a similar strategy to be translatable to the clinical setting, because there is 70% concordance between the TCR repertoire of HLA-A2/K^b mice and HLA-A2⁺ human subjects (12). Therefore, a similar immunization strategy as that which we have described may be able to generate HMW-MAA-specific CTL responses that may control HMW-MAA-bearing tumors. In addition, the identification of three HLA-A2 Ag-restricted HMW-MAA peptides that are recognized by HMW-MAA-specific CTL in HLA-A2/K^b mice suggests several CTL epitopes that may be useful for monitoring the HMW-MAA-specific cellular immune responses.

Lastly, at variance with most MAA, which have been shown to serve as targets for either Ab-based (52, 53) or T cell-based (7, 8) immunotherapy but not both, the findings that we have presented here, in conjunction with our previous results (5, 6, 54, 55), demonstrate that HMW-MAA can be recognized and targeted by both Abs and T cells. This feature of HMW-MAA offers the opportunity to enhance the apparent clinical benefits associated with the induction of HMW-MAA-specific Abs (5, 6), by the addition of T cell immunity. The validity of this combinatorial therapeutic modality has been demonstrated by the increased antitumor effects achieved by combining Ab and cellular immunity for the immunotherapy of a HER-2/neu-expressing tumor (56). Therefore, our findings are expected to broaden the strategies available for the immunotherapy of melanoma and other HMW-MAA-bearing tumors.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by Public Health Service Grants P01 CA89480 and R01 CA105500 (to S.F.) and R01 CA104645 (to P.A.S.) awarded by the National Cancer Institute, Department of Health and Human Services, and by a grant from the Harry J. Lloyd Charitable Trust (to S.F.).

² L.P. and E.K. contributed equally to this manuscript.

³ Address correspondence and reprint requests to Dr. Soldano Ferrone, Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. E-mail address: soldano.ferrone{at}roswellpark.org

⁴ Abbreviations used in this paper: HMW-MAA, high m.w.-melanoma-associated Ag; DC, dendritic cell; SEA, staphylococcal enterotoxin A.

Received for publication July 29, 2005. Accepted for publication November 23, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Natali, P. G., K. Imai, B. S. Wilson, A. Bigotti, R. Cavaliere, M. A. Pellegrino, S. Ferrone. 1981. Structural properties and tissue distribution of the antigen recognized by the monoclonal antibody 653.40S to human melanoma cells. J. Natl. Cancer Inst. 67: 591-601. [Medline]
2. Wilson, B. S., K. Imai, P. G. Natali, S. Ferrone. 1981. Distribution and molecular characterization of a cell-surface and a cytoplasmic antigen detectable in human melanoma cells with monoclonal antibodies. Int. J. Cancer 15: 293-300.
3. Ferrone, S., P. Giacomini, P. G. Natali, D. Ruiter, G. Buraggi, L. Callegaro, U. A. Rosa. 1983. A human high molecular weight-melanoma associated antigen (HMW-MAA) defined by monoclonal antibodies: a useful marker to radioimage tumor lesions in patients with melanoma. Proceedings of the 1st International Symposium on Neutron Capture Therapy 174-183. Brookhaven National Laboratory, Associated Universities, Upton, NY.
4. Kusama, M., T. Kageshita, Z. J. Chen, S. Ferrone. 1989. Characterization of syngeneic antiidiotypic monoclonal antibodies to murine anti-human high molecular weight melanoma-associated antigen monoclonal antibodies. J. Immunol. 143: 3844-3852. [Abstract]
5. Mittelman, A., Z. J. Chen, C. C. Liu, S. Hirai, S. Ferrone. 1994. Kinetics of the immune response and regression of metastatic lesions following development of humoral anti-high molecular weight-melanoma associated antigen immunity in three patients with advanced malignant melanoma immunized with mouse antiidiotypic monoclonal antibody MK2–23. Cancer Res. 54: 415-421. [Abstract/Free Full Text]
6. Mittelman, A., Z. J. Chen, H. Yang, G. Y. Wong, S. Ferrone. 1992. Human high molecular weight melanoma-associated antigen (HMW-MAA) mimicry by mouse anti-idiotypic monoclonal antibody MK2–23: induction of humoral anti-HMW-MAA immunity and prolongation of survival in patients with stage IV melanoma. Proc. Natl. Acad. Sci. USA 89: 466-470. [Abstract/Free Full Text]
7. Sutmuller, R. P., L. R. Schurmans, L. M. van Duivenvoorde, J. A. Tine, E. I. van Der Voort, R. E. Toes, C. J. Melief, M. J. Jager, R. Offringa. 2000. Adoptive T cell immunotherapy of human uveal melanoma targeting gp100. J. Immunol. 165: 7308-7315. [Abstract/Free Full Text]
8. Saleh, F. H., K. A. Crotty, P. Hersey, S. W. Menzies. 2001. Primary melanoma tumour regression associated with an immune response to the tumour-associated antigen melan-A/MART-1. Int. J. Cancer 94: 551-557. [Medline]
9. Pride, M. W., S. Shuey, A. Grillo-Lopez, G. Braslawsky, M. Ross, S. S. Legha, O. Eton, A. Buzaid, C. Ioannides, J. L. Murray. 1998. Enhancement of cell-mediated immunity in melanoma patients immunized with murine anti-idiotypic monoclonal antibodies (MELIMMUNE) that mimic the high molecular weight proteoglycan antigen. Clin. Cancer Res. 4: 2363-2370. [Abstract/Free Full Text]
10. Murray, J. L., M. Gillogly, K. Kawano, C. L. Efferson, J. E. Lee, M. Ross, X. Wang, S. Ferrone, C. G. Ioannides. 2004. Fine specificity of high molecular weight-melanoma-associated antigen-specific cytotoxic T lymphocytes elicited by anti-idiotypic monoclonal antibodies in patients with melanoma. Cancer Res. 64: 5481-5488. [Abstract/Free Full Text]
11. Vitiello, A., D. Marchesini, J. Furze, L. A. Sherman, R. W. Chesnut. 1991. Analysis of the HLA-restricted influenza-specific cytotoxic T lymphocyte response in transgenic mice carrying a chimeric human-mouse class I major histocompatibility complex. J. Exp. Med. 173: 1007-1015. [Abstract/Free Full Text]
12. Wentworth, P. A., A. Vitiello, J. Sidney, E. Keogh, R. W. Chesnut, H. Grey, A. Sette. 1996. Differences and similarities in the A2.1-restricted cytotoxic T cell repertoire in humans and human leukocyte antigen-transgenic mice. Eur. J. Immunol. 26: 97-101. [Medline]
13. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245-252. [Medline]
14. Steinman, R. M., M. Dhodapkar. 2001. Active immunization against cancer with dendritic cells: the near future. Int. J. Cancer 4: 459-473.
15. Toes, R. E., F. Ossendorp, R. Offringa, C. J. Melief. 1999. CD4 T cells and their role in antitumor immune responses. J. Exp. Med. 189: 753-756. [Free Full Text]
16. Bourgeois, C., B. Rocha, C. Tanchot. 2002. A role for CD40 expression on CD8⁺ T cells in the generation of CD8⁺ T cell memory. Science 297: 2060-2063. [Abstract/Free Full Text]
17. Bourgeois, C., H. Veiga-Fernandes, A. M. Joret, B. Rocha, C. Tanchot. 2002. CD8 lethargy in the absence of CD4 help. Eur. J. Immunol. 32: 2199-2207. [Medline]
18. Janssen, E. M., E. E. Lemmens, T. Wolfe, U. Christen, M. G. von Herrath, S. P. Schoenberger. 2003. CD4⁺ T cells are required for secondary expansion and memory in CD8⁺ T lymphocytes. Nature 421: 852-856. [Medline]
19. Shedlock, D. J., H. Shen. 2003. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300: 337-339. [Abstract/Free Full Text]
20. Sun, J. C., M. J. Bevan. 2003. Defective CD8 T cell memory following acute infection without CD4 T cell help. Science 300: 339-342. [Abstract/Free Full Text]
21. Rahemtulla, A., W. P. Fung-Leung, M. W. Schilham, T. M. Kundig, S. R. Sambhara, A. Narendran, A. Arabian, A. Wakeham, C. J. Paige, R. M. Zinkernagel. 1991. Normal development and function of CD8⁺ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353: 180-184. [Medline]
22. Bodmer, H., G. Obert, S. Chan, C. Benoist, D. Mathis. 1993. Environmental modulation of the autonomy of cytotoxic T lymphocytes. Eur. J. Immunol. 23: 1649-1654. [Medline]
23. Hou, S., X. Y. Mo, L. Hyland, P. C. Doherty. 1995. Host response to Sendai virus in mice lacking class II major histocompatibility complex glycoproteins. J. Virol. 69: 1429-1434. [Abstract]
24. Melief, C. J.. 1992. Tumor eradication by adoptive transfer of cytotoxic T lymphocytes. Adv. Cancer Res. 58: 143-175. [Medline]
25. Peng, L., J. Kjaergaard, G. E. Plautz, D. E. Weng, S. Shu, P. A. Cohen. 2000. Helper-independent, L-selectin^low CD8⁺ T cells with broad anti-tumor efficacy are naturally sensitized during tumor progression. J. Immunol. 165: 5738-5749. [Abstract/Free Full Text]
26. Hung, K., R. Hayashi, A. Lafond-Walker, C. Lowenstein, D. Pardoll, H. Levitsky. 1998. The central role of CD4⁺ cells in the antitumor immune response. J. Exp. Med. 188: 2357-2368. [Abstract/Free Full Text]
27. Ossendorp, F., E. Mengede, M. Camps, R. Filius, C. J. Melief. 1998. Specific T helper cell requirement for optimal induction of cytotoxic T lymphocytes against major histocompatibility complex class II negative tumors. J. Exp. Med. 187: 693-702. [Abstract/Free Full Text]
28. Marzo, A. L., B. F. Kinnear, R. A. Lake, J. J. Frelinger, E. J. Collins, B. W. Robinson, B. Scott. 2000. Tumor-specific CD4⁺ T cells have a major "post-licensing" role in CTL mediated anti-tumor immunity. J. Immunol. 165: 6047-6055. [Abstract/Free Full Text]
29. Marrack, P., J. Kappler. 1990. The staphylococcal enterotoxins and their relatives. Science 248: 705-711. [Abstract/Free Full Text]
30. Callahan, J. E., A. Herman, J. W. Kappler, P. Marrack. 1990. Stimulation of B10.BR T cells with superantigenic staphylococcal toxins. J. Immunol. 144: 2473-2479. [Abstract]
31. Takimoto, H., Y. Yoshikai, K. Kishihara, G. Matsuzaki, H. Kuga, T. Otani, K. Nomoto. 1990. Stimulation of all T cells bearing V1, V3, V11 and V12 by staphylococcal enterotoxin A. Eur. J. Immunol. 20: 617-621. [Medline]
32. Russo, C., A. K. Ng, M. A. Pellegrino, S. Ferrone. 1983. The monoclonal antibody CR11–351 discriminates HLA-A2 variants identified by T cells. Immunogenetics 18: 23-35. [Medline]
33. Giacomini, P., A. K. Ng, P. R. Kantor, P. G. Natali, S. Ferrone. 1983. Double determinant immunoassay to measure a human high-molecular-weight melanoma-associated antigen. Cancer Res. 43: 3586-3590. [Abstract/Free Full Text]
34. Leo, O., M. Foo, D. H. Sachs, L. E. Samelson, J. A. Bluestone. 1987. Identification of a monoclonal antibody specific for a murine T3 polypeptide. Proc. Natl. Acad. Sci. USA 84: 1374-1378. [Abstract/Free Full Text]
35. Wilde, D. B., P. Marrack, J. Kappler, D. P. Dialynas, F. W. Fitch. 1983. Evidence implicating L3T4 in class II MHC antigen reactivity; monoclonal antibody GK1.5 (anti-L3T4a) blocks class II MHC antigen-specific proliferation, release of lymphokines, and binding by cloned murine helper T lymphocyte lines. J. Immunol. 131: 2178-2183. [Abstract]
36. Sarmiento, M., A. L. Glasebrook, F. W. Fitch. 1980. IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt 2 antigen block T cell-mediated cytolysis in the absence of complement. J. Immunol. 125: 2665-2672. [Abstract]
37. Temponi, M., T. Kageshita, F. Perosa, R. Ono, H. Okada, S. Ferrone. 1989. Purification of murine IgG monoclonal antibodies by precipitation with caprylic acid: comparison with other methods of purification. Hybridoma 8: 85-95. [Medline]
38. Yang, J., M. A. Price, C. L. Neudauer, C. Wilson, S. Ferrone, H. Xia, J. Iida, M. A. Simpson, J. B. McCarthy. 2004. Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms. J. Cell Biol. 165: 881-891. [Abstract/Free Full Text]
39. Holmes, K., B. J. Fowlkes, I. Schmid, J. V. Giorgi. 2002. Immunofluorescence staining of single-cell suspensions for detection of surface antigens. J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and W. Strober, eds. Current Protocols in Immunology 5.3.1-5.3.3. Wiley, New York.
40. Inaba, K., M. Inaba, N. Romani, H. Aya, M. Dequchi, S. Ikehara, S. Muramatsu, R. M. Steinman. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176: 1693-1702. [Abstract/Free Full Text]
41. Shu, S., T. Chou, S. A. Rosenberg. 1987. In vitro differentiation of T cells capable of mediating the regression of established syngeneic tumors in mice. Cancer Res. 47: 1354-1360. [Abstract/Free Full Text]
42. Mizobata, S., K. Tompkins, J. F. Simpson, Y. Shyr, F. J. Primus. 2000. Induction of cytotoxic T cells and their antitumor activity in mice transgenic for carcinoembryonic antigen. Cancer Immunol. Immunother. 49: 285-295. [Medline]
43. Xiang, R., F. J. Primus, J. M. Ruehlmann, A. G. Niethammer, S. Silletti, H. N. Lode, C. S. Dolman, S. D. Gillies, R. A. Reisfeld. 2001. A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice. J. Immunol. 167: 4560-4565. [Abstract/Free Full Text]
44. Coles, R. M., S. N. Mueller, W. R. Heath, F. R. Carbone, A. G. Brooks. 2002. Progression of armed CTL from draining lymph node to spleen shortly after localized infection with HSV-1. J. Immunol. 168: 834-838. [Abstract/Free Full Text]
45. Fisk, B., T. L. Blevins, J. T. Wharton, C. G. Ioannides. 1995. Identification of an immunodominant peptide of HER-2/neu protooncogene recognized by ovarian tumor-specific cytotoxic T lymphocyte lines. J. Exp. Med. 181: 2109-2117. [Abstract/Free Full Text]
46. Chen, L., M. Koyanagi, K. Fukada, K. Imanishi, J. Yagi, H. Kato, T. Miyoshi-Akiyama, R. Zhang, K. Miwa, T. Uchiyama. 2002. Continuous exposure of mice to superantigenic toxins induces a high-level protracted expansion and an immunological memory in the toxin-reactive CD4⁺ T cells. J. Immunol. 168: 3817-3824. [Abstract/Free Full Text]
47. Lanzavecchia, A.. 1998. Immunology: licence to kill. Nature 393: 413-414. [Medline]
48. Bourgeois, C., C. Tanchot. 2003. CD4 T cells are required for CD8 T cell memory generation. Eur. J. Immunol. 33: 3225-3231. [Medline]
49. Mosmann, T. R., S. Sad. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today 17: 138-146. [Medline]
50. Lu, Z., L. Yuan, X. Zhou, E. Sotomayor, H. I. Levitsky, D. M. Pardoll. 2000. CD40-independent pathways of T cell help for priming of CD8⁺ cytotoxic T lymphocytes. J. Exp. Med. 191: 541-550. [Abstract/Free Full Text]
51. Medzhitov, R., C. A. Janeway, Jr. 2002. Decoding the patterns of self and nonself by the innate immune system. Science 296: 298-300. [Abstract/Free Full Text]
52. Houghton, A. N., D. Mintzer, C. Cordon-Cardo, S. Welt, B. Fliegel, S. Vadhan, E. Carswell, M. R. Melamed, H. F. Oettgen, L. J. Old. 1985. Mouse monoclonal IgG3 antibody detecting GD3 ganglioside: a phase I trial in patients with malignant melanoma. Proc. Natl. Acad. Sci. USA 82: 1242-126. [Abstract/Free Full Text]
53. Vadhan-Raj, S., C. Cordon-Cardo, E. Carswell, D. Mintzer, L. Dantis, C. Duteau, M. A. Templeton, H. F. Oettgen, L. J. Old, A. N. Houghton. 1988. Phase I trial of a mouse monoclonal antibody against GD3 ganglioside in patients with melanoma: induction of inflammatory responses at tumor sites. J. Clin. Oncol. 6: 1636-1648. [Abstract/Free Full Text]
54. Chen, Z. J., H. Yang, C. C. Liu, S. Hirai, S. Ferrone. 1993. Modulation by adjuvants and carriers of the immunogenicity in xenogeneic hosts of mouse anti-idiotypic monoclonal antibody MK2–23, an internal image of human high molecular weight-melanoma associated antigen. Cancer Res. 53: 112-119. [Abstract/Free Full Text]
55. Luo, W., J. C. Hsu, C. Y. Tsao, E. Ko, X. Wang, S. Ferrone. 2005. Differential immunogenicity of two peptides isolated by high molecular weight-melanoma associated antigen (HMW-MAA)-specific monoclonal antibodies with different affinities. J. Immunol. 174: 7104-7110. [Abstract/Free Full Text]
56. Reilly, R. T., J. H. Machiels, L. A. Emens, A. M. Ercolini, F. I. Okoye, R. Y. Lei, D. Weintraub, E. M. Jaffee. 2001. The collaboration of both humoral and cellular HER-2/neu-targeted immune responses is required for the complete eradication of HER-2/neu-expressing tumors. Cancer Res. 61: 880-883. [Abstract/Free Full Text]

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