RESUMO
Adaptive immune responses begin with cognate antigen presentation-dependent specific interaction between T cells and antigen-presenting cells. However, there have been limited reports on the isolation and analysis of these cellular complexes of T cell-antigen-presenting cell (T/APC). In this study, we successfully isolated intact antigen-specific cellular complexes of CD8+ T/APC by utilizing a microfluidics cell sorter. Using ovalbumin (OVA) model antigen and OT-I-derived OVA-specific CD8+ T cells, we analyzed the formation of antigen-specific and antigen-non-specific T/APC cellular complexes and revealed that the antigen-specific T/APC cellular complex was highly stable than the non-specific one, and that the intact antigen-specific T/APC complex can be retrieved as well as enriched using a microfluidics sorter, but not a conventional cell sorter. The single T/APC cellular complex obtained can be further analyzed for the sequences of T cell receptor Vα and Vß genes as well as cognate antigen information simultaneously. These results suggested that this approach can be applied for other antigen and CD8+ T cells of mice and possibly those of humans. We believe that this microfluidics sorting method of the T/APC complex will provide useful information for future T cell immunology research.
Assuntos
Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Separação Celular/métodos , Microfluídica/métodos , Animais , Linfócitos T CD8-Positivos/metabolismo , Comunicação Celular/imunologia , Linhagem Celular Tumoral , Citometria de Fluxo/métodos , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Reprodutibilidade dos TestesRESUMO
Human herpesvirus 6 (HHV-6) infects over 90% of people. The HHV-6 subtype, HHV-6B in particular, is often associated with exanthem subitum in early childhood. Exanthem subitum is usually self-limiting and good prognosis disease; however, some infants primarily infected with HHV-6B develop encephalitis/encephalopathy, and half of the patients developed encephalopathy reported to have neurological sequelae. Furthermore, after primary infection, HHV-6B remains in a latent state and sometimes reactivated in immunosuppressed patients, causing life-threatening severe encephalopathy. However, effective immunotherapies or vaccines for controlling HHV-6B infection and reactivation have not yet been established. Recently, we have found that the HHV-6B tetrameric glycoprotein (g) complex, gH/gL/gQ1/gQ2 is a promising vaccine candidate, and currently under preclinical development. To confirm our vaccine candidate protein complex induce detectable T-cell responses, in this study, we comprehensively screened CD4+ and CD8+ T-cell epitopes in the gH/gL/gQ1/gQ2 tetrameric complex protein in mice immunisation model. Both BALB/c and C57BL/6 mice were immunised with the tetrameric complex protein or plasmid DNA encoding gH, gL, gQ1, and gQ2, and then restimulated with 162 20-mer peptides covering the whole gH/gL/gQ1/gQ2 sequences; multiple CD4+ and CD8+ T-cell-stimulating peptides were identified in both BALB/c and C57BL/6 mice. Our study demonstrates that gH/gL/gQ1/gQ2 tetramer-targeted vaccination has potential to induce T-cell responses in two different strains of mice and supports the future development and application of T-cell-inducing vaccine and immunotherapies against HHV-6B.
Assuntos
Epitopos de Linfócito T/imunologia , Glicoproteínas/imunologia , Herpesvirus Humano 6/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
The identification of Human herpesvirus 6B (HHV-6B) epitopes that are recognized by T-cells could contribute to the development of potential vaccines and immunotherapies. Here, we identified CD4+ and H-2Kd-restricted CD8+ T-cell epitopes on the glycoprotein Q1 of HHV-6B (BgQ1), which is a unique glycoprotein and essential for HHV-6B viral entry, by using in vivo electroporation with a plasmid DNA encoding BgQ1, overlapping peptides spanning the BgQ1 sequence, ELISPOT assay for quantification of gamma interferon (IFN-γ), and computer-based T-cell epitope prediction programs. The CD4+ and CD8+ T-cell epitopes identified in BALB/c mice in this study could be a good animal model system for use in the development of T-cell responses, inducing HHV-6B vaccines or immunotherapies.