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1.
J Vis Exp ; (144)2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30882785

RESUMO

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Ativação Linfocitária/fisiologia , Humanos , Transfecção
2.
Sci Rep ; 7(1): 9923, 2017 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-28855662

RESUMO

Epstein-Barr virus (EBV) is a common gammaherpesvirus associated with various human malignancies. Antibodies with T cell receptor-like specificities (TCR-like mAbs) provide a means to target intracellular tumor- or virus-associated antigens by recognising their processed peptides presented on major histocompatibility complex (MHC) class I (pMHC) complexes. These antibodies are however thought to be relevant only for a single HLA allele. Here, we show that HLA-A*02:01-restricted EBV antigenic peptides EBNA1562-570, LMP1125-133 and LMP2A426-434 display binding degeneracy towards HLA-A*02 allelic microvariants, and that these pMHC complexes are recognised by anti-EBV TCR-like mAbs E1, L1 and L2 raised in the context of HLA-A*02:01. These antibodies bound endogenously derived pMHC targets on EBV-transformed human B lymphoblastoid cell lines expressing A*02:01, A*02:03, A*02:06 and A*02:07 alleles. More importantly, these TCR-like mAbs mediated both complement-dependent and antibody-dependent cellular cytotoxicity of these cell lines in vitro. This finding suggests the utility of TCR-like mAbs against target cells of closely related HLA subtypes, and the potential applicability of similar reagents within populations of diverse HLA-A*02 alleles.


Assuntos
Anticorpos Monoclonais/metabolismo , Antígeno HLA-A2/genética , Herpesvirus Humano 4/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Citotoxicidade Celular Dependente de Anticorpos , Linhagem Celular Tumoral , Variação Genética , Antígeno HLA-A2/química , Antígeno HLA-A2/imunologia , Infecções por Herpesviridae/imunologia , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo
3.
Blood ; 128(10): 1396-407, 2016 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-27338099

RESUMO

Epstein-Barr virus (EBV) is an oncovirus associated with several human malignancies including posttransplant lymphoproliferative disease in immunosuppressed patients. We show here that anti-EBV T-cell receptor-like monoclonal antibodies (TCR-like mAbs) E1, L1, and L2 bound to their respective HLA-A*0201-restricted EBV peptides EBNA1562-570, LMP1125-133, and LMP2A426-434 with high affinities and specificities. These mAbs recognized endogenously presented targets on EBV B lymphoblastoid cell lines (BLCLs), but not peripheral blood mononuclear cells, from which they were derived. Furthermore, these mAbs displayed similar binding activities on several BLCLs, despite inherent heterogeneity between different donor samples. A single weekly administration of the naked mAbs reduced splenomegaly, liver tumor spots, and tumor burden in BLCL-engrafted immunodeficient NOD-SCID/Il2rg(-/-) mice. In particular, mice that were treated with the E1 mAb displayed a delayed weight loss and significantly prolonged survival. In vitro, these TCR-like mAbs induced early apoptosis of BLCLs, thereby enhancing their Fc-dependent phagocytic uptake by macrophages. These data provide evidence for TCR-like mAbs as potential therapeutic modalities to target EBV-associated diseases.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos B/imunologia , Antígeno HLA-A2/imunologia , Herpesvirus Humano 4/imunologia , Neoplasias Hepáticas/prevenção & controle , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Apoptose , Proliferação de Células , Células Cultivadas , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/imunologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/virologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fagocitose/imunologia
4.
Front Biosci (Landmark Ed) ; 16(8): 3014-35, 2011 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-21622218

RESUMO

CD8-positive T cells respond to small antigenic peptide fragments presented on class I major histocompatibility complexes (MHCs). Those specific T cell epitopes capable of precipitating a cellular immune response are either derived from (altered) self (i.e. they are autoimmune- or cancer-associated) or come from foreign sources (i.e. they are pathogen-associated). Identification of T cell epitopes provides elementary information that can be employed in technologies that monitor and predict the likely outcome of an immune response, as well as in therapeutic and vaccine development efforts. The coexistence between host and pathogen has largely driven the diversification of both their systems of immune surveillance and their antigenic determinants, respectively. In this review, we discuss the multitude of factors that introduce diversity to the T cell response from both sides of the host-pathogen interaction. Furthermore, we provide an overview of a variety of commonly employed methods and tools to characterize class I MHC restricted antigen presentation and recent endeavors towards the harmonization of reporting data concerning T cell responses.


Assuntos
Epitopos de Linfócito T/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Variação Antigênica/genética , Bases de Dados de Proteínas , Epitopos de Linfócito T/genética , Antígenos HLA/genética , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Técnicas Imunológicas
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