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Nat Commun ; 9(1): 4297, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30327458


Functional CD8+ T cells in human tumors play a clear role in clinical prognosis and response to immunotherapeutic interventions. PD-1 expression in T cells involved in chronic infections and tumors such as melanoma often correlates with a state of T-cell exhaustion. Here we interrogate CD8+ tumor-infiltrating lymphocytes (TILs) from human breast and melanoma tumors to explore their functional state. Despite expression of exhaustion hallmarks, such as PD-1 expression, human breast tumor CD8+ TILs retain robust capacity for production of effector cytokines and degranulation capacity. In contrast, melanoma CD8+ TILs display dramatic reduction of cytokine production and degranulation capacity. We show that CD8+ TILs from human breast tumors can potently kill cancer cells via bi-specific antibodies. Our data demonstrate that CD8+ TILs in human breast tumors retain polyfunctionality, despite PD-1 expression, and suggest that they may be harnessed for effective immunotherapies.

Neoplasias da Mama/patologia , Linfócitos T CD8-Positivos/patologia , Linfócitos do Interstício Tumoral/patologia , Receptor de Morte Celular Programada 1/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Testes Imunológicos de Citotoxicidade , Feminino , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/metabolismo
J Proteomics ; 176: 13-23, 2018 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-29331515


To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cells. We determined the sequence of 3196 MHC class I ligands representing 1921 proteins from a panel of 20 breast cancer cell lines. After removing duplicate peptides, i.e., the same peptide eluted from more than one cell line, the total number of unique peptides was 2740. Of the unique peptides eluted, more than 1750 had been previously identified, and of these, sixteen have been shown to be immunogenic. Importantly, half of these immunogenic peptides were shared between different breast cancer cell lines. MHC class I binding probability was used to plot the distribution of the eluted peptides in accordance with the binding score for each breast cancer cell line. We also determined that the tested breast cancer cells presented 89 mutation-containing peptides and peptides derived from aberrantly translated genes, 7 of which were shared between four or two different cell lines. Overall, the high throughput identification of MHC class I-loaded peptides is an effective strategy for systematic characterization of cancer peptides, and could be employed for design of multi-peptide anticancer vaccines. SIGNIFICANCE: By employing proteomic analyses of eluted peptides from breast cancer cells, the current study has built an initial HLA-I-typed antigen collection for breast cancer research. It was also determined that immunogenic epitopes can be identified using established cell lines and that shared immunogenic peptides can be found in different cancer types such as breast cancer and leukemia. Importantly, out of 3196 eluted peptides that included duplicate peptides in different cells 89 peptides either contained mutation in their sequence or were derived from aberrant translation suggesting that mutation-containing epitopes are on the order of 2-3% in breast cancer cells. Finally, our results suggest that interfering with MHC class I function is one of the mechanisms of how tumor cells escape immune system attack.

Neoplasias da Mama/imunologia , Antígenos de Histocompatibilidade Classe I/análise , Sequência de Aminoácidos , Apresentação do Antígeno , Antígenos de Neoplasias , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Epitopos/genética , Antígenos HLA , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Mutação , Proteômica/métodos
Proc Natl Acad Sci U S A ; 113(29): 8272-7, 2016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27307436


Infiltration of T cells in breast tumors correlates with improved survival of patients with breast cancer, despite relatively few mutations in these tumors. To determine if T-cell specificity can be harnessed to augment immunotherapies of breast cancer, we sought to identify the alpha-beta paired T-cell receptors (TCRs) of tumor-infiltrating lymphocytes shared between multiple patients. Because TCRs function as heterodimeric proteins, we used an emulsion-based RT-PCR assay to link and amplify TCR pairs. Using this assay on engineered T-cell hybridomas, we observed ∼85% accurate pairing fidelity, although TCR recovery frequency varied. When we applied this technique to patient samples, we found that for any given TCR pair, the dominant alpha- or beta-binding partner comprised ∼90% of the total binding partners. Analysis of TCR sequences from primary tumors showed about fourfold more overlap in tumor-involved relative to tumor-free sentinel lymph nodes. Additionally, comparison of sequences from both tumors of a patient with bilateral breast cancer showed 10% overlap. Finally, we identified a panel of unique TCRs shared between patients' tumors and peripheral blood that were not found in the peripheral blood of controls. These TCRs encoded a range of V, J, and complementarity determining region 3 (CDR3) sequences on the alpha-chain, and displayed restricted V-beta use. The nucleotides encoding these shared TCR CDR3s varied, suggesting immune selection of this response. Harnessing these T cells may provide practical strategies to improve the shared antigen-specific response to breast cancer.

Neoplasias da Mama/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/metabolismo , Sequência de Bases , Linhagem Celular , Emulsões , Feminino , Humanos , Reação em Cadeia da Polimerase/métodos