RESUMO
NK cell alloreactivity is governed largely through failure to detect self-HLA class I ligands by the clonally distributed inhibitory killer Ig-like receptors (KIR) expressed on the NK cell surface. In this study, we investigated the extent to which HLA class I-KIR interactions influence human NK cell proliferation in the allogeneic setting. NK cells were cultured with feeder cells either matched or mismatched for inhibitory KIR ligands, the latter lacking one or more ligands present in the NK cell donor. In postculture cytotoxicity assays, the ability of polyclonal NK cells to kill KIR ligand-mismatched targets was enhanced by exposure to appropriately mismatched feeder cells in prior culture. This corresponded with an increased frequency of postculture donor NK cells expressing a given inhibitory KIR if the allogeneic feeder cells used in the culture lacked its ligand. Similar skewing of KIR distribution was seen in clonally expanded NK cells. Finally, a flow cytometry-based proliferation assay was used to show KIR-specific NK cell division in response to missing self. The findings demonstrate that KIR distribution among a population of alloresponding peripheral blood NK cells is shaped by the HLA class I environment.
Assuntos
Proliferação de Células , Citotoxicidade Imunológica , Teste de Histocompatibilidade , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Receptores KIR/metabolismo , Células Cultivadas , Células Clonais , Técnicas de Cocultura , Citotoxicidade Imunológica/genética , Regulação da Expressão Gênica/imunologia , Antígenos HLA-B/metabolismo , Antígenos HLA-B/fisiologia , Antígenos HLA-C/metabolismo , Antígenos HLA-C/fisiologia , Humanos , Células Matadoras Naturais/metabolismo , Ligantes , Receptores KIR/biossíntese , Receptores KIR/deficiência , Receptores KIR/genética , Receptores KIR/fisiologia , Receptores KIR3DS1/deficiência , Receptores KIR3DS1/metabolismo , Receptores KIR3DS1/fisiologiaRESUMO
Human cytomegalovirus (HCMV) remains one of the most common opportunistic infections causing disease following stem cell transplantation, despite the availability of anti-viral therapies. Adoptive immunotherapy has the potential to further aid in counteracting chronic viral reactivation and subsequent disease by restoring viral immunity through the transfer of virus-specific T cells from transplant donors to their recipients. Our study refines the production and purification of a recombinant HCMV protein containing two of the most immunodominant antigens (IE1 and pp65) for the generation of polyclonal HCMV-specific T cells. In doing so, a 6x His-tagged IE1-pp65 protein was generated using a serum-free baculovirus/insect cell expression system and soluble IE1-pp65 protein was subsequently purified using Ni-NTA affinity chromatography under stringent conditions to obtain a highly pure product. The ability of the recombinant IE1-pp65 protein to elicit a functional T cell mediated immune response was demonstrated by the vigorous reactivation and expansion of HLA-A2-restricted pp65(495-503)-specific CD8+ T cells. This recombinant IE1-pp65 protein can potentially generate a multitude of HLA-restricted HCMV-specific T cells, providing a better alternative to using costly overlapping peptides or HCMV lysates for expansion of T cells for use in adoptive immunotherapy strategies.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/isolamento & purificação , Epitopos Imunodominantes/imunologia , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/isolamento & purificação , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas Imediatamente Precoces/química , Proteínas Imediatamente Precoces/imunologia , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/imunologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Spodoptera , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/imunologiaRESUMO
The minor histocompatibility antigen HA-1H is a potential immunotherapeutic molecule. It can be used as a target for graft versus leukaemia reactions to eliminate residual HA-1H expressing leukaemic cells in patients following haemopoietic stem cell transplantation, whereby HA-1H primed donor cells can be transferred into a patient via adoptive immunotherapy. However, thus far only synthetic peptides corresponding to a HLA-A *0201 restricted HA-1H epitope have been used to generate HA-1H specific T cells. We are the first laboratory to clone, express and purify a region of HA-1H using an Escherichia coli expression system. The recombinant HA-1H protein was purified under denaturing conditions and the affinity purification tag removed using thrombin to remove non-specific amino acids. The 92 amino acid recombinant protein was characterised by mass spectrometry. Our rationale is that by using a recombinant HA-1H protein rather than peptide, HA-1H specific T cells may be generated from presentation of multiple HA-1H epitopes complexed in different HLA molecules. A multi-epitope approach may have wider applicability and maybe more effective at leukaemia control. The recombinant HA-1H protein may also be used as a research tool to identify novel CD4(+) helper T cell and CD8(+) cytotoxic T cell epitopes.