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CD4⁺ T-Cell Recognition of Mutated B-RAF in Melanoma Patients Harboring the V599E Mutation

Surgery Branch, National Cancer Institute, Center for Cancer Research, NIH, Bethesda, Maryland

	ABSTRACT

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The potential of antigen-directed cancer immunotherapy has not been fully realized, perhaps because many commonly targeted tumor associated proteins are not essential to maintaining the malignant cell phenotype. A constitutively activating mutation in the signaling molecule BRAF is expressed frequently in melanomas and may play an important role in the biology of this disease. A 29-mer B-Raf peptide incorporating the V599E mutation was used for in vitro stimulation of lymphocytes derived from melanoma patients, generating MHC class II-restricted CD4⁺ T cells specific for this peptide as well as for melanoma cells expressing B-Raf V599E. Mutated B-Raf exemplifies targets that may be ideal for immunotherapy.

	Introduction

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Investigators have long sought to use the power of the immune system to combat established or incipient cancers. The promise of antigen (Ag)-directed immunotherapy as an anticancer modality has not yet been fully realized, perhaps in part due to the choice of target Ag for vaccine development and adoptive T-cell transfer. Most of the current information on the nature of cancer Ag recognized by the immune system has been derived from studies of human melanoma, and, hence, the majority of clinical efforts have focused on this disease. Using commonly expressed nonmutated Ag such as tyrosinase, gp100, and MART-1/MelanA as targets for immunotherapy has the advantage of accessing a significant portion of the patient population, and yet loss of expression of these nonessential molecules provides an escape route for tumors refractory to treatment (1) . Identification and targeting of tumor Ag vital to maintaining the malignant phenotype may be critical to realizing the full potential of cancer immunotherapy.

A constitutively activating somatic mutation in the signaling molecule B-Raf is associated with >60% of malignant melanomas (2) . B-Raf is a member of the mitogen-activated protein (MAP) kinase cascade that transduces extracellular mitogenic signals to the cell nucleus, controlling cell growth, differentiation, and survival. Complete activation of B-Raf requires the phosphorylation of two residues, Thr598 and Ser601, with Thr598 playing the dominant role (3) . The BRAF point mutation T1796A commonly expressed in melanomas results in the missense V599E conversion, presumably mimicking phosphorylation of Thr598 and conferring activation. Of note, the B-Raf V599E mutation is associated not only with melanomas but also with 82% of benign nevi (4) , suggesting a role in carcinogenesis. In addition, its importance for maintaining the malignant cell phenotype has been revealed recently with RNA interference techniques (5) . Pharmacological inhibition of B-Raf has thus been proposed as a treatment for melanoma (6) . Considering mutated B-Raf as a potential target for immunotherapy, we sought in the current study to assess the capacity of CD4⁺ T cells from melanoma patients harboring the V599E mutation to specifically recognize this molecule.

	Materials and Methods

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Cell Lines.
Melanoma cultures were initiated from enzymatic digests of metastatic lesions. EBV-transformed B-cell lines were generated according to standard methods. All of the cell lines were maintained in RPMI 1640 + 10% FCS and were free of Mycoplasma contamination.

BRAF DNA Sequencing.
Genomic DNA was isolated from fresh cryopreserved peripheral blood mononuclear cells (PBMCs), cultured melanomas, and EBV-B cells, and fresh cryopreserved enzymatically digested single cell suspensions of metastatic melanoma lesions using the Easy-DNA kit (Invitrogen, Carlsbad, CA). BRAF exon 15 was amplified with 35 cycles of PCR, and the products were gel-purified and directly sequenced. Previously published intronic primers were used for the PCR and for genomic DNA sequencing (2) . Because melanin in the heavily pigmented melanomas 553-mel, 1011-mel, 1088-mel, and 1350-mel interfered with the PCR, it was necessary to use reverse-transcribed cDNA rather than genomic DNA as the template for PCR and sequencing of these cell lines. The following primers (based on GenBank sequence accession no. M95712) were used for reverse transcription-PCR with an annealing temperature of 50°C to produce and sequence an amplicon of 380 bp: forward, 5'- GACAGACTGCACAGGGCATG-3'; reverse, 5'-GTTACTCCGTACCTTACTGA-3'.

HLA Typing.
The major histocompatibility complex (MHC) class II genotypes of melanoma patients under study were determined from fresh PBMCs by the NIH W.G. Magnuson Clinical Center HLA Laboratory (Bethesda, MD) using sequence-specific PCR techniques. The HLA type of patient 2069 was DRB1*0404, 14; DQB1*03, 0503; DRB3*02; DRB4*01; and DPB1*0201, 0401. In addition, melanoma cell cultures and EBV-B cells used in this study were subjected to HLA genotyping.

Peptides.
Peptides used in this study were synthesized with Fmoc chemistry, and their purity was confirmed with mass spectrometry. A 29-mer B-Raf peptide containing the V599E mutation, B-Raf 585–613_mut, had the sequence EDLTVKIGDFGLATEKSRWSGSHQFEQLS. The artificial "universal" pan-DR epitope designated PADRE had the sequence AKFVAAWTLKAAA (7) . The mutated triosephosphate isomerase (TPI) peptide TPI 26–38 T28I, specifically recognized by CD4⁺ tumor infiltrating lymphocyte (TIL) 1558 reactive against autologous melanoma cells, had the sequence IGILNAAKVPADT (8) . The influenza hemagglutinin (HA) peptide HA 307–319 had the sequence PKYVKQNTLKLAT. The tetanus toxoid 830–843 peptide had the sequence QYIKANSKFIGITE.

T-Cell Cultures.
CD4⁺ T-cell microcultures were initiated by peptide stimulation of fresh cryopreserved PBMCs from three melanoma patients whose metastatic tumors harbored the T1796A (V599E) BRAF mutation. PBMCs were cultured in flat-bottomed 96-well plates at 3 x 10⁵ cells/well in RPMI 1640 +10% heat-inactivated human AB serum. Granulocyte macrophage colony-stimulating factor (GM-CSF; 200 units/ml) and interleukin 4 (100 units/ml; PeproTech Inc., Rocky Hill, NJ) were added to cultures only on day 0 to generate dendritic cells as antigen presenting cells (APCs), along with 50 µM of the mutated B-Raf peptide or a positive control peptide pool consisting of 10 µM each of the peptides tetanus toxoid 830–843, HA 307–319, and PADRE. Recombinant interleukin 2 (150 IU/ml) was added to T-cell cultures on day 6 and replenished every 4–7 days. Thereafter, T-cell microcultures were restimulated every 10–14 days with irradiated autologous PBMCs or EBV-B cells pulsed with the appropriate peptide(s) at 1 x 10⁵ feeder cells/well. Long-term CD4⁺ T-cell cultures were maintained in 300 IU/ml interleukin 2 and 20% conditioned medium from lymphokine activated killer cell cultures. As a control in some experiments, CD4⁺ TILs from patient number 1558, recognizing the HLA-DR1-restricted mutated epitope TPI 26–38 T28I, were used (8) .

T-Cell Recognition Assays.
To assess specific peptide recognition by T cells, 0.2–1 x 10⁵ T cells/well were stimulated overnight in flat-bottomed 96-well plates with 1 x 10⁵ autologous PBMCs or EBV-B cells that had been prepulsed for 16–24 h with 50 µM of peptide, either B-Raf 585–613_mut or an irrelevant peptide (TPI 26–38 T28I or HA 307–319). Culture supernatants were then harvested, and GM-CSF or IFN secretion by T cells was measured using commercially available ELISA kits (R&D Systems, Minneapolis, MN). When allogeneic EBV-B cells were used as APCs to determine the MHC restriction of peptide-specific T cells, care was taken to wash excess peptide off APC before combining them with T cells. Whole tumor cell recognition was tested by incubating T cells (1 x 10⁵/well) in microtiter plates with melanoma cells (1 x 10⁵/well) for 20 h. To up-regulate the expression of MHC class II molecules on melanoma cells, melanomas were cultured in the presence of IFN (500 units/ml) for 3 days before T-cell recognition assays, and cell surface expression of MHC molecules was assessed by flow cytometry on the day of the assay. In some assays, monoclonal antibodies (mAbs) directed against MHC molecules were used to inhibit T-cell reactivity, including W6/32 (IgG2a, anti-MHC class I), IVA12 (IgG1, anti-HLA-DR, -DP, ?-DQ), L243 (IgG2a, anti-HLA-DR), IVD12 (IgG1, anti-HLA-DQ3), Genox 3.53 (IgG1, anti-HLA-DQ1), G2b.2 (IgG2a, anti-HLA-DQ1; all purified hybridoma supernatants, American Type Culture Collection, Manassas, VA), and B7/21 (IgG3, anti-HLA-DP; BD Biosciences, San Diego, CA). Final concentrations of mAb in blocking assays were 5 µg/ml for B7/21 and 20 µg/ml for the others. The mAb NFLD.D1 specific for HLA-DR4 (Accurate Chemical, Westbury, NY) was used for flow cytometry.

	Results and Discussion

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DNA Sequencing for the T1796A (V599E) BRAF Mutation.
To identify melanoma patients harboring the V599E mutation who would be suitable for immunological studies, genomic DNA isolated from six fresh metastatic melanoma specimens was used to amplify and sequence BRAF exon 15 (Table 1) . Four of six fresh melanomas contained codon 599 mutations, encoding V599E (three samples) or V599K (one sample). In addition, we sequenced DNA or cDNA from 18 long-term melanoma cell lines generated from metastatic lesions that were later used as targets for T-cell recognition. Twelve of these contained codon 599 mutations (11 V599E and 1 V599K). Overall, 67% of melanomas contained codon 599 mutations, 88% of which encoded V599E. In contrast, autologous nonmelanoma samples from 10 patients contained only the wild-type BRAF sequence, as did PBMCs derived from 5 normal donors. These results are consistent with those of others demonstrating the prevalence and tumor specificity of somatic codon 599 BRAF mutations in melanomas (2 , 9, 10, 11, 12) .

CD4⁺ T Cells from Patient 2069 Use 3 Different HLA Alleles for Recognizing the Mutated B-Raf Peptide.
Three individual T-cell microcultures from patient 2069, designated A4, B5, and F2, were studied to identify the MHC allele(s) restricting recognition of the B-Raf_mut peptide. Cultures A4 and F2 were pure CD4⁺ T-cell populations as assessed by flow cytometry, whereas B5 was 83% CD4 positive. To determine the MHC restriction of these T cells, allogeneic EBV-B cell lines sharing various HLA-DR, -DQ, and/or -DP alleles with patient 2069 were used as APC for exogenously pulsed B-Raf_mut peptide. Peptide-pulsed K562 erythroleukemia cells devoid of cell surface MHC molecules were used as a negative control. As shown in Table 2 , CD4⁺ A4 T cells specifically recognized the B-Raf_mut peptide pulsed onto autologous EBV-B cells or onto allogeneic APCs sharing HLA-DQB1*05 and did not recognize the irrelevant HA 307–319 peptide. In contrast, B5 T cells required allogeneic APCs that expressed HLA-DPB1*0401 for B-Raf_mut peptide recognition. Finally, F2 T cells recognized the B-Raf_mut peptide pulsed onto 583-EBV sharing only HLA-DRB1*0404 with patient 2069. B-Raf_mut could be recognized at limiting concentrations of 0.5–5 µM (data not shown). Additional HLA typing of the DRB3, DRB4, and DRB5 loci did not reveal any patterns among APCs that were consistent with the profiles of T-cell activity observed (data not shown). Thus, it appeared that CD4⁺ T-cell cultures generated from 1 melanoma patient could use three different MHC class II alleles to recognize a single 29-mer B-Raf peptide containing the V599E mutation.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. MHC class II restriction of CD4+ T cells specific for the B-Rafmut peptide. Monoclonal antibodies as described in "Materials and Methods" were used to inhibit T-cell recognition of peptide-pulsed allogeneic antigen presenting cells expressing the deduced restriction elements (HLA-DPB1*0401 for B5 T cells, or HLA-DRB1*0404 for F2 T cells). Although both B5 and F2 T cells are inhibited by the pan-class II monoclonal antibody IVA12, B5 is also inhibited by B7/21 specific for HLA-DP, whereas F2 is blocked by L243 specific for HLA-DR molecules, consistent with the deduced restriction elements. T cells, 3 x 104/well; antigen presenting cells, 1 x 105/well; B-Raf peptide, 50 µM.

Peptide-Specific F2 CD4⁺ T Cells Recognize Whole Melanoma Cells Containing a Mutated BRAF Allele.
T cells sensitized by repetitive in vitro stimulation with synthetic peptides may fail to recognize the naturally processed parent protein. This can occur because a hypothetical epitope is not actually generated by intracellular processing, because the synthetic peptide used for in vitro stimulation contains chemical impurities or sequence irregularities provoking T-cell reactivity, or because the conformation of an exogenously pulsed peptide complexed to MHC fails to reproduce the conformation of a naturally processed epitope loaded intracellularly onto MHC molecules (14) . Thus, we next sought to determine whether CD4⁺ T cells raised against and specific for the B-Raf_mut peptide could also specifically recognize whole melanoma cells expressing both mutated BRAF and the appropriate class II allele. F2 T cells were tested for recognition of a panel of cultured melanomas that had been characterized with BRAF exon 15 DNA sequencing (Table 1) as well as MHC class II genotyping. Because not all of the melanoma targets constitutively expressed significant amounts of cell surface class II molecules, they were cultured in the presence of IFN for 72 h before the assay, and the induction or enhancement of HLA-DR expression was confirmed with flow cytometry. As shown in Table 3 , F2 T cells secreted GM-CSF specifically in response to melanomas expressing both mutated B-Raf and HLA-DRB1*0404. Expression of either molecule alone was not sufficient for T-cell recognition. Thus, 1558-mel, expressing mutated B-Raf but not the appropriate restricting allele, was not recognized by F2 T cells but could be recognized by HLA-DR1-restricted CD4⁺ TIL 1558 specific for mutated TPI (8) . Similarly, 1011-mel, expressing the appropriate restriction element but not the V599E B-Raf mutation, was not recognized by F2 T cells. Whereas 397-mel was well recognized based on its coexpression of mutated B-Raf and HLA-DR4, there was no recognition of T lymphoblasts from patient 397 (397 TIL) that nevertheless demonstrated strong cell surface expression of DR molecules (Table 3) and specifically of HLA-DR4 (data not shown). Of note, 1898-mel was not recognized by F2 T cells despite containing the V599E mutation and DRB1*0404 as assessed on a genetic level. Unexpectedly, whereas flow cytometric analysis of 1898-mel with the mAb L243 showed adequate cell surface expression of HLA-DR (Table 3) , subsequent analysis with an HLA-DR4-specific mAb failed to detect expression of this allele, consistent with the failure of F2 recognition. In two separate experiments (data not shown), F2 T cells recognizing IFN-treated 397-mel failed to react against IFN-treated cultured fibroblasts from patient 2069 expressing HLA-DR, nor did they recognize additional allogeneic melanomas that contained mutated B-Raf but not HLA-DRB1*0404 (586-mel, 624-mel, 938-mel, and 1087-mel). Fresh 2069 melanoma cells expressed HLA-DR poorly by immunostaining and were not recognized. Taken together, these results demonstrate the Ag specificity and MHC class II restriction of CD4⁺ F2 T cells. Moreover, they provide evidence that endogenously expressed mutant B-Raf protein can be processed within melanoma cells and presented in association with MHC molecules, becoming a target for immune recognition.

In summary, our findings indicate that the B-Raf V599E mutation commonly expressed in melanomas can be displayed by tumor cells for CD4⁺ T-cell recognition. A single 29-mer peptide incorporating the V599E point mutation has the capacity to stimulate MHC class II-restricted responses through multiple alleles and, thus, has potential clinical utility. Because this mutation is somatically acquired, immunotherapies directed at B-Raf V599E may circumvent issues of immunological tolerance encountered with the clinical application of nonmutated self-Ag. Furthermore, molecules such as mutant B-Raf, of which the expression appears to be important for maintaining the malignant phenotype, may provide ideal targets for cancer immunotherapy.

	ACKNOWLEDGMENTS

 
We thank John Riley for peptide synthesis, Yong Li for DNA sequencing, Arnold Mixon and Shawn Farid for flow cytometry, John Wunderlich for providing melanoma cell lines, Sharon Adams and staff for HLA genotyping, and Richard Marais for providing a plasmid containing the mutant B-Raf sequence. We also thank Steven A. Rosenberg for advice and support, and to Douglas Lowy and Drew M. Pardoll for helpful discussions.

	FOOTNOTES

 
Grant support: Howard Hughes Medical Institute-NIH Research Scholar (S. Patel).

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.

Notes: M. S. Sharkey and G. Lizée contributed equally to this work.

Requests for reprints: Suzanne Topalian, Surgery Branch, National Cancer Institute, NIH 10/2B47, Bethesda, MD 20892-1502. Phone: (301) 496-4269; Fax: (301) 402-0922; E-mail: Suzanne_Topalian{at}nih.gov

Received 10/14/03. Revised 1/ 9/04. Accepted 1/15/04.

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