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1.
Nat Methods ; 21(5): 846-856, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38658646

RESUMO

CD4+ T cells recognize peptide antigens presented on class II major histocompatibility complex (MHC-II) molecules to carry out their function. The remarkable diversity of T cell receptor sequences and lack of antigen discovery approaches for MHC-II make profiling the specificities of CD4+ T cells challenging. We have expanded our platform of signaling and antigen-presenting bifunctional receptors to encode MHC-II molecules presenting covalently linked peptides (SABR-IIs) for CD4+ T cell antigen discovery. SABR-IIs can present epitopes to CD4+ T cells and induce signaling upon their recognition, allowing a readable output. Furthermore, the SABR-II design is modular in signaling and deployment to T cells and B cells. Here, we demonstrate that SABR-IIs libraries presenting endogenous and non-contiguous epitopes can be used for antigen discovery in the context of type 1 diabetes. SABR-II libraries provide a rapid, flexible, scalable and versatile approach for de novo identification of CD4+ T cell ligands from single-cell RNA sequencing data using experimental and computational approaches.


Assuntos
Linfócitos T CD4-Positivos , Epitopos de Linfócito T , Antígenos de Histocompatibilidade Classe II , Linfócitos T CD4-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Animais , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/química , Camundongos , Humanos , Diabetes Mellitus Tipo 1/imunologia , Peptídeos/imunologia , Peptídeos/química , Apresentação de Antígeno/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Camundongos Endogâmicos NOD , Análise de Célula Única/métodos
2.
Cytotherapy ; 26(3): 266-275, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38231165

RESUMO

T cell receptor engineered T cell (TCR T) therapies have shown recent efficacy against certain types of solid metastatic cancers. However, to extend TCR T therapies to treat more patients across additional cancer types, new TCRs recognizing cancer-specific antigen targets are needed. Driver mutations in AKT1, ESR1, PIK3CA, and TP53 are common in patients with metastatic breast cancer (MBC) and if immunogenic could serve as ideal tumor-specific targets for TCR T therapy to treat this disease. Through IFN-γ ELISpot screening of in vitro expanded neopeptide-stimulated T cell lines from healthy donors and MBC patients, we identified reactivity towards 11 of 13 of the mutations. To identify neopeptide-specific TCRs, we then performed single-cell RNA sequencing of one of the T cell lines following neopeptide stimulation. Here, we identified an ESR1 Y537S specific T cell clone, clonotype 16, and an ESR1 Y537S/D538G dual-specific T cell clone, clonotype 21, which were HLA-B*40:02 and HLA-C*01:02 restricted, respectively. TCR Ts expressing these TCRs recognized and killed target cells pulsed with ESR1 neopeptides with minimal activity against ESR1 WT peptide. However, these TCRs failed to recognize target cells expressing endogenous mutant ESR1. To investigate the basis of this lack of recognition we performed immunopeptidomics analysis of a mutant-overexpressing lymphoblastoid cell line and found that the ESR1 Y537S neopeptide was not endogenously processed, despite binding to HLA-B*40:02 when exogenously pulsed onto the target cell. These results indicate that stimulation of T cells that likely derive from the naïve repertoire with pulsed minimal peptides may lead to the expansion of clones that recognize non-processed peptides, and highlights the importance of using methods that selectively expand T cells with specificity for antigens that are efficiently processed and presented.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Apresentação de Antígeno , Receptores de Antígenos de Linfócitos T , Mutação , Peptídeos , Antígenos HLA-B/genética
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