RESUMO
NK cell responses are regulated by a balance of inhibitory and activating signals, reflecting the net effect of interactions between receptors and ligands on target and effector cell surfaces. The identification of ligands for orphan NK cell receptors is key to enhancing our understanding of NK cell biology. Here we describe a strategy (protocol) for the identification of ligands for orphan NK cell receptors using signaling reporter cells in combination with a virus rescue system.
Assuntos
Receptores de Células Matadoras Naturais/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , Ligantes , Camundongos , Ligação Proteica , Receptores de Células Matadoras Naturais/genéticaRESUMO
NK cells use surface NK receptors to discriminate self from non-self. The NK receptor ligand-binding domain (NKD) has been considered the sole regulator of ligand binding. Using a prototypic murine NK receptor, Ly49A, we show that the membrane proximal nonligand binding ecto-domain (the stalk region) is critical to ligand binding and signaling. The stalk region is required for receptor binding to ligand on target cells (trans interaction), but is dispensable for receptor binding to ligand on the same cell (cis interaction). Also, signaling in a trans manner depends on the stalk region mediating the formation of the immunological synapse. Thus, our data modeling receptor function at the cellular level reveal an essential role for the stalk region as a specific mediator of receptor signal integration, by which NKD-ligand interactions at the interface initiate and deliver information to the spatially separated cytoplasmic domain.
Assuntos
Sinapses Imunológicas/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/química , Subfamília A de Receptores Semelhantes a Lectina de Células NK/imunologia , Transdução de Sinais/imunologia , Sequência de Aminoácidos , Animais , Deleção de Genes , Genes Reporter , Ligantes , Camundongos , Modelos Imunológicos , Dados de Sequência Molecular , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , SolubilidadeRESUMO
CMV infection is one of the most common complications in immunocompromised individuals, such as organ and bone marrow transplant patients. Both innate and adaptive immune responses are required for defense against CMV infection. In murine CMV (MCMV) infection, strains harboring the MCMV-specific NK cell activation receptor, Ly49H (Klra8), are resistant. In contrast, MCMV infection of mice lacking Ly49H gene causes early mortality due to uncontrolled viral replication. In this study, we report the successful protection of mice from lethal MCMV infection with gene-transferred polyclonal CD8 T cells. CD8 T cells expressing a chimeric receptor comprising Ly49H extracellular and CD3zeta cytoplasmic domains are capable of killing target cells expressing the MCMV protein, m157. CD8 T cells expressing the chimeric receptor protect mice in vivo from lethality in the acute phase of MCMV infection, leading to the establishment of long-term protection. These data provide proof-of-principle evidence that a novel strategy for harnessing CD8 cytolytic function through TCR-independent yet pathogen-specific receptor can result in effective protection of hosts from pathogens.