Final Report Summary - NKG2D-IMMUNO (The role of NKG2D in CD4+ T cell function and autoimmunity)
The immune system is a highly developed and complex system that serves the body with a simple task: to find and kill the invaders. However, more than 5 million people in Europe live with chronic inflammatory diseases partially mediated by inappropriate T cell response. Although current medicine and medical science puts great effort into understanding the basis of chronicity and finding the proper treatment, the mechanisms driving improper T cell activation are largely unknown and deserve further investigation. Particularly, how pro-inflammatory cells adapt to function and survive in chronic inflammation, what are the markers defining and regulating their function and differentiation during pathogenesis and whether they can be modulated to prevent pathology would be of major interest.
CD4+ T helper cells are a major source of pro-inflammatory cytokines, such as IFN-gamma, TNF and IL-17, during viral infections and inflammation. The effector functions of these cells are not solely dependent on the presence of the antigen (i.e. TCR stimulation) but their functions can also be modulated by cytokines and/or sensing danger signals via surface expressed receptors. One of the best-described danger-recognizing receptors in lymphocyte biology is NKG2D. NKG2D is a potent C-type lectin like receptor, initially described as an activating receptor of NK cells. It was shown to be important in recognizing virus-infected and transformed cells and it mediated the cytotoxicity of NK cells towards these targets. It is now also established that the expression of this receptor is more promiscuous, being found on effector and memory CD8+ T cells, NKT cells and gamma/delta T cells, as well as in CD4+ T cells under pathological conditions. Interestingly, while NKG2D can clearly function as a co-stimulatory molecule in CD8 T cells, its role in CD4+ T cells is still rather unclear.
The aim of this project was to identify whether NKG2D is a marker and a critical regulator of CD4+ T cell function and differentiation. In addition, we wanted to contribute to better understanding of pro-inflammatory cytokine profile of pathogenic NKG2D-expressing CD4+ T cells and dissect the molecular mechanisms of a potential regulatory role of NKG2D signaling in cytokine expression.
By establishing the multicolour flow cytometry profiling experiments, the fellow could show that an innate activating receptor NKG2D was expressed on a proportion of CD4+ Th1 cells in various ex vivo tested lymphoid and non-lymphoid organs. In addition, NKG2D could be induced de novo on naïve CD4+ T cells polarized towards Th1 lineage and most surprisingly, NKG2D+ Th1 cells were associated with higher expression of immunoregulatory cytokine IL-10.
To identify the molecular basis of NKG2D-mediated cytokine expression by Th1 cells the fellow performed global transcriptome analysis on in vitro generated Th1 cells and identified the transcription factor Blimp-1 as a differentially regulated candidate gene between NKG2D+ Th1 and NKG2D-/- Th1 cells. This indicates that Blimp-1 might be involved in NKG2D-mediated regulation of IL-10 in Th1 cells.
To address the functionality of NKG2D on CD4+ T cells the fellow established streptavidin-mediated crosslinking of Th1 cells, previously labeled with biotinylated anti-CD3, alone or in combination with anti-NKG2D, upon which the phosphorylation of molecules was assessed by flow cytometry. By this approach, it could be shown that the co-stimulation of cells with anti-NKG2D in combination with low dose anti-CD3 resulted in phosphorylation of Erk1/2, a signaling molecule in the PI3 kinase pathway, which is known to be important for IL-10 production in Th1 cells and Blimp-1 expression in plasma cells, as shown by others.
To elucidate the role of NKG2D+ Th1 cells in vivo, the fellow established various mouse models of inflammation, and could show that NKG2D+ Th1 cells were present and enriched also under inflammatory condition, and were particularly enriched in IL10/IFN-gamma co-producing cells in the model of anti−CD3 induced tolerance, a model in which tolerance is mediated by IL-10 producing Th1 cells.
Altogether, our findings reveal the unexpected role of NKG2D in modulating cytokine expression in Th1 cells as well as its possible role in inducing regulatory features in Th1 cells. This finding might have important application in therapies that aim at modulating pathogenic profile of CD4+ T cell in chronic inflammations as well as autoimmune diseases.
Apart from scientific achievements, we have aimed at providing the best possible training opportunities for the researcher in charge. We believe that the fellow has expanded her technical and technological knowledge by having access to state-of-the art flow cytometry and sorting facility. The fellow has supervised two Master Students, held numerous scientific presentations on regular in-house meetings as well as scientific conferences, thereby being questioned and challenged by experts in the field, which helped the fellow in reaching scientific maturity and independent thinking. The fellow, however, brought research qualities and both scientific and technical knowledge in mouse NK cell receptor biology which in return contributed to the host institution. Further, fellow has actively participated in the developing of research projects within the host group, writing manuscript and grant proposals as well as in establishing new collaborations.
In summary, we believe that the scientific and socio-economic output of this Marie Curie project is outstanding, giving an example of a fruitful collaboration between the fellow and her host institution, reaching most of its objectives and providing a great opportunity for scientific maturation of a promising young European intellectual.
CD4+ T helper cells are a major source of pro-inflammatory cytokines, such as IFN-gamma, TNF and IL-17, during viral infections and inflammation. The effector functions of these cells are not solely dependent on the presence of the antigen (i.e. TCR stimulation) but their functions can also be modulated by cytokines and/or sensing danger signals via surface expressed receptors. One of the best-described danger-recognizing receptors in lymphocyte biology is NKG2D. NKG2D is a potent C-type lectin like receptor, initially described as an activating receptor of NK cells. It was shown to be important in recognizing virus-infected and transformed cells and it mediated the cytotoxicity of NK cells towards these targets. It is now also established that the expression of this receptor is more promiscuous, being found on effector and memory CD8+ T cells, NKT cells and gamma/delta T cells, as well as in CD4+ T cells under pathological conditions. Interestingly, while NKG2D can clearly function as a co-stimulatory molecule in CD8 T cells, its role in CD4+ T cells is still rather unclear.
The aim of this project was to identify whether NKG2D is a marker and a critical regulator of CD4+ T cell function and differentiation. In addition, we wanted to contribute to better understanding of pro-inflammatory cytokine profile of pathogenic NKG2D-expressing CD4+ T cells and dissect the molecular mechanisms of a potential regulatory role of NKG2D signaling in cytokine expression.
By establishing the multicolour flow cytometry profiling experiments, the fellow could show that an innate activating receptor NKG2D was expressed on a proportion of CD4+ Th1 cells in various ex vivo tested lymphoid and non-lymphoid organs. In addition, NKG2D could be induced de novo on naïve CD4+ T cells polarized towards Th1 lineage and most surprisingly, NKG2D+ Th1 cells were associated with higher expression of immunoregulatory cytokine IL-10.
To identify the molecular basis of NKG2D-mediated cytokine expression by Th1 cells the fellow performed global transcriptome analysis on in vitro generated Th1 cells and identified the transcription factor Blimp-1 as a differentially regulated candidate gene between NKG2D+ Th1 and NKG2D-/- Th1 cells. This indicates that Blimp-1 might be involved in NKG2D-mediated regulation of IL-10 in Th1 cells.
To address the functionality of NKG2D on CD4+ T cells the fellow established streptavidin-mediated crosslinking of Th1 cells, previously labeled with biotinylated anti-CD3, alone or in combination with anti-NKG2D, upon which the phosphorylation of molecules was assessed by flow cytometry. By this approach, it could be shown that the co-stimulation of cells with anti-NKG2D in combination with low dose anti-CD3 resulted in phosphorylation of Erk1/2, a signaling molecule in the PI3 kinase pathway, which is known to be important for IL-10 production in Th1 cells and Blimp-1 expression in plasma cells, as shown by others.
To elucidate the role of NKG2D+ Th1 cells in vivo, the fellow established various mouse models of inflammation, and could show that NKG2D+ Th1 cells were present and enriched also under inflammatory condition, and were particularly enriched in IL10/IFN-gamma co-producing cells in the model of anti−CD3 induced tolerance, a model in which tolerance is mediated by IL-10 producing Th1 cells.
Altogether, our findings reveal the unexpected role of NKG2D in modulating cytokine expression in Th1 cells as well as its possible role in inducing regulatory features in Th1 cells. This finding might have important application in therapies that aim at modulating pathogenic profile of CD4+ T cell in chronic inflammations as well as autoimmune diseases.
Apart from scientific achievements, we have aimed at providing the best possible training opportunities for the researcher in charge. We believe that the fellow has expanded her technical and technological knowledge by having access to state-of-the art flow cytometry and sorting facility. The fellow has supervised two Master Students, held numerous scientific presentations on regular in-house meetings as well as scientific conferences, thereby being questioned and challenged by experts in the field, which helped the fellow in reaching scientific maturity and independent thinking. The fellow, however, brought research qualities and both scientific and technical knowledge in mouse NK cell receptor biology which in return contributed to the host institution. Further, fellow has actively participated in the developing of research projects within the host group, writing manuscript and grant proposals as well as in establishing new collaborations.
In summary, we believe that the scientific and socio-economic output of this Marie Curie project is outstanding, giving an example of a fruitful collaboration between the fellow and her host institution, reaching most of its objectives and providing a great opportunity for scientific maturation of a promising young European intellectual.