Final Report Summary - CD2 AND CD244 (Regulation of immune responses by CD2, CD244 and related receptors)
The generation of immune responses to infections require not only the recognition of the foreign invader, but also communication among different cell types of the immune system. In many cases, this communication is mediated by proteins on the cell surface. The objectives of our study were to understand how the family of cell surface proteins, known as the CD2/SLAM family receptors, contribute to the development of immune responses. When the extracellular portions of the receptors bind to their ligands on nearby cells, they cluster together and initiate a signal through the intracellular regions of the proteins. Natural killer (NK) cells are a type of cell that can recognise and kill virally infected cells as well as some tumour cells. They express many of the SLAM family receptors, and our aim was to determine how these proteins signal the NK cell to kill its target.
The intracellular regions of all of the SLAM-family receptors contain short sequences that can bind to small adapter proteins called SAP and EAT2. A previous study in our laboratory had found that the enzyme Phospholipase C-gamma 1 (PLCγ1) could bind to a different region of EAT2. In this way, the cell surface proteins of the SLAM family can be coupled to enzymes inside the cell such as PLCγ1 that can activate NK cell cytotoxic functions. During the course of our study, we found that EAT2 might also recruit a second, related enzyme called PLCγ2.
We used a human NK cell line called NK92 and a gene-delivery system known as lentiviral transduction to determine how these signalling proteins influence the activation of NK cell responses through CD2 and SLAM family receptors such as CD244. By transducing short hairpin RNA sequences (shRNA) into the NK92 cell line, we were able to disrupt the expression of specific genes within NK cells, including small adapter proteins SAP and EAT2, and intracellular enzymes Phospholipase C-gamma 1 and Phospholipase C-gamma 2 (PLCγ1 and PLCγ2). We then measured the ability of different cell surface proteins to induce NK cell killing. We found that the SLAM family receptors could readily trigger cytotoxicity, but are completely dependent on both PLCγ1 and PLCγ2 for initiating cytotoxic responses. Reduction in expression of the adapter protein EAT2 had only mild effects on cytotoxicity, while reducing SAP expression in NK cells dramatically limited the NK cytotoxic responses through SLAM-family proteins CD244, NTBA and CRACC. Therefore, we think it is likely that SLAM family receptors use both of these adapter proteins to cause PLCγ1 and PLCγ2 activation.
A second critical function of NK cells is the production of small messenger proteins called cytokines. One such cytokine is interferon gamma (IFNγ) which can enhance the anti-tumour responses of NK cells through the activation of inflammatory macrophages. This cytokine can be produced by NK cells upon activating receptor stimulation, or after NK cells detect the presence of other cytokines such as IL-2 or IL-12. The disruption of PLCγ2 expression in NK cells resulted in decreased production of IFNγ after receptor stimulation, showing the overall importance of PLCγ2 in activating human NK cell function. Conversely, the loss of PLCγ1, SAP or EAT2 resulted in greater production of IFNγ in response to IL-2 stimulation. This finding indicates that proteins which are responsible for activating cytotoxic responses may also suppress intercellular communication through other pathways.
These findings are important for other medical researchers trying to harness the immune system to eradicate tumours, especially leukemias and lymphomas.
The intracellular regions of all of the SLAM-family receptors contain short sequences that can bind to small adapter proteins called SAP and EAT2. A previous study in our laboratory had found that the enzyme Phospholipase C-gamma 1 (PLCγ1) could bind to a different region of EAT2. In this way, the cell surface proteins of the SLAM family can be coupled to enzymes inside the cell such as PLCγ1 that can activate NK cell cytotoxic functions. During the course of our study, we found that EAT2 might also recruit a second, related enzyme called PLCγ2.
We used a human NK cell line called NK92 and a gene-delivery system known as lentiviral transduction to determine how these signalling proteins influence the activation of NK cell responses through CD2 and SLAM family receptors such as CD244. By transducing short hairpin RNA sequences (shRNA) into the NK92 cell line, we were able to disrupt the expression of specific genes within NK cells, including small adapter proteins SAP and EAT2, and intracellular enzymes Phospholipase C-gamma 1 and Phospholipase C-gamma 2 (PLCγ1 and PLCγ2). We then measured the ability of different cell surface proteins to induce NK cell killing. We found that the SLAM family receptors could readily trigger cytotoxicity, but are completely dependent on both PLCγ1 and PLCγ2 for initiating cytotoxic responses. Reduction in expression of the adapter protein EAT2 had only mild effects on cytotoxicity, while reducing SAP expression in NK cells dramatically limited the NK cytotoxic responses through SLAM-family proteins CD244, NTBA and CRACC. Therefore, we think it is likely that SLAM family receptors use both of these adapter proteins to cause PLCγ1 and PLCγ2 activation.
A second critical function of NK cells is the production of small messenger proteins called cytokines. One such cytokine is interferon gamma (IFNγ) which can enhance the anti-tumour responses of NK cells through the activation of inflammatory macrophages. This cytokine can be produced by NK cells upon activating receptor stimulation, or after NK cells detect the presence of other cytokines such as IL-2 or IL-12. The disruption of PLCγ2 expression in NK cells resulted in decreased production of IFNγ after receptor stimulation, showing the overall importance of PLCγ2 in activating human NK cell function. Conversely, the loss of PLCγ1, SAP or EAT2 resulted in greater production of IFNγ in response to IL-2 stimulation. This finding indicates that proteins which are responsible for activating cytotoxic responses may also suppress intercellular communication through other pathways.
These findings are important for other medical researchers trying to harness the immune system to eradicate tumours, especially leukemias and lymphomas.