Final Report Summary - NEUROKINE (Initial Training Network for Neurogical disorders orchestrated by cytoKines)
During the duration of NeuroKine, the UZH team focused on the interaction of CNS-resident and immune cells under inflammatory conditions. They were able to delineate the capacity of IL-23 to impart a pro-inflammatory/Encephalitogenic signature onto T cells thereby enabling them to invade the CNS. Among the signature cytokines, GM-CSF emerged as the main communication conduit between T cells and myeloid cells to drive tissue inflammation and immunopathology. How IL-23 but also another cytokines, IL-1 affect the development of GM-CSF producing T cells was also a focus in the project of the partner UMC. They found that IL-1 is not needed for the development of pathogenic T cells, but only for their propagation. Partner UMC also investigated the role of IL-10 produced by T cells in CNS inflammation. Surprisingly, they found that if all T cells lack this cytokine, autoimmunity is not enhanced, as expected but halted. The reason for that is that IL-10 is indeed a suppressive cytokine, but its also important for proper stimulation of T cells. In its absence, T cells are triggered too strongly and die, resulting in reduced autoimmunity.
Also the partner Apitope investigated IL-10, and demonstrated that antigen-specific peptide immunotherapy led to an increase in the number of regulatory T cells and IL-10-secreting CD4+ T cells in organs throughout the whole body. In addition to an increase in the number of CD4+ T cells with regulatory properties, effector T cells were prevented from entering the CNS. In addition, Apitope demonstrated the important principle that tolerised CD4+ T cells can mediate regulation of not only T cells specific for the treatment peptide, but also CD4+ T cells that recognise immunodominant peptides from related proteins. They found that tolerised T cells were able to exert linked bystander suppression of other T cells in vivo, most successfully so when both peptides where presented by the same antigen-presenting cell by coupling the two antigens with a linker sequence.
Also the research from Rotterdam focused on cytokines, and has shed light on a possible role of EBV in MS and analyzed disruption of cytokines signaling pathways in primate models of MS (IL-7 and IL-23). Furthermore, they performed a comprehensive analysis of radical mediated tissue damage and mitochondrial injury in primate EAE, paralleling work previously done in MS, and observed that primate models can be used for testing novel therapeutics directed at alleviating oxidative tissue injury.
The first objective of the partner from Charite was to exchange resident microglia, which are the CNS macrophages with peripheral myeloid cells (PDMCs) in the Alzheimer’s disease (AD) context. This exchange happens when microglia are ablated using the partner’s previously published CD11b-SHVTK mouse model. To assess the morphological and functional differences between the microglia and PDMCs they implanted a cranial window on the transgenic mice. They found that the process activity of both microglia and PDMCs reduced in the proximity of plaques, while myeloid cells further away from plaques but in the AD environment were equally functional as cells in a wild-type environment. This leads to the hypothesis that the PDMCs are primed in the periphery by the soluble Aβ levels in the blood as well as inflammatory markers associated with AD.
Paraneoplastic neurological disorders (PND) are rare immune-mediated diseases that develop in the setting of malignancy and offer a unique prospect to analyze the interplay between tumour immunity and autoimmunity. The Toulouse group generated new mouse models that allow expression of a self-antigen in the Purkinje neurons of the cerebellum and in tumor cells. They found that mice developed severe cerebellar inflammation upon anti-CTLA-4 treatment, with subsequent loss of Purkinje neurons, but the tumour was rejected. Therefore, the enhanced tumour control was obtained at the expense of autoimmune paraneoplastic cerebellar degeneration. Consequently, they showed that the immune checkpoint therapy in our mouse model elicits T cell migration into the cerebellum and local destructive autoimmunity.
The partner Wien attempted to define the nature of the inflammatory response in MS lesions in relation to that in other inflammatory and non-inflammatory diseases and age matched controls in humans. Using quantitative immunocytochemical techniques they revealed the presence of T-lymphocytes (especially CD8+) and of CD20 positive B-cells in MS brains. Within the CD8 population a low and variable fraction expressed granzyme B as a marker for cytotoxic activation. This study support a very prominent role of B-lymphocytes in the inflammatory process of MS, which is also supported by the recently reported success of anti-CD20 treatment in MS patients.
The focus of the labs at the WIS and USR is to understand the cross talk between the immune system and the brain, and design new therapies for autoimmune of the CNS. The lab of Michal Schwartz discovered that immune cells can enter the CNS through the blood cerebrospinal fluid barrier (BCSFB), and that activity of this barrier to promote trafficking of leukocytes is dependent on the availability of IFN-γ. They also found that in neurodegenerative diseases, in analogy to cancer, systemic immunosuppressive cells and pathways, such as regulatory T cells and inhibitory immune checkpoint pathways, respectively, might curtail the immune response needed to fight this pathology.
USR focused more on the treatment of CNS autoimmunity, and specifically on trying to treat the CNS via the stimulation of neural precursor cells (NPCs). They found that NPCs secrete TGFβ2 that inhibits monocyte-derived dendritic cells differentiation and maturation, favouring the switch towards an anti-inflammatory phenotype. USR further dissected the role of NPC residing in the subventricular zone of the brain taking advantage of in house generated transgenic mouse line where NPCs can be selectively ablated in this region. Using these novel mice, they found that endogenous NPCs play a protective role in acute but not in chronic inflammatory disorders of the CNS.
Two industrial partners developed new technology that will be to the benefit of the whole community. The partner Biontech focused on setting up a pipeline enabling high-throughput sequencing of αβTCR genes from single murine T cells (scTCRseq). ScTCRseq allows the identification and reconstitution of functional αβTCR pairs, even when material is limited, as often the case with mouse models (of CNS disorders). They could show general feasibility of this method using two sets of TCR α and β chain-encoding in vitro transcribed mRNA libraries with known sequences. Their platform established is now ready to use for the unbiased determination of the individual αβTCRs from up to 9216 (96x96) single murine T cells in parallel.
The hypothesis of the project of the partner Miltenyi was that different subpopulations of reactive astrocytes (ASCA-2+) exist in the inflamed CNS with distinct cytokine/chemokine secretion profile and perform different roles in disease development and recovery. Using methods that allow to isolate astrocytes from neonates and adult mice, they Miltenyi group found 856 genes were expressed at a significantly higher level in neonatal ACSA-2-positive cells relative to adult cells. In contrast, 102 genes were expressed at a significantly higher level in adult ACSA-2- positive cells relative to neonatal cells. As determined by annotation enrichment analysis, the terms translation, nucleotide metabolism, intracellular import/export, and others were enriched with high significance among the genes with higher expression levels in neonatal cells. In summary, single-cell transcriptome analyses revealed a highly diverse expression profile of neonatal and adult astrocytes.
Further information please find on our website: http://www.unimedizin-mainz.de/neurokine/uebersicht.html
Also the partner Apitope investigated IL-10, and demonstrated that antigen-specific peptide immunotherapy led to an increase in the number of regulatory T cells and IL-10-secreting CD4+ T cells in organs throughout the whole body. In addition to an increase in the number of CD4+ T cells with regulatory properties, effector T cells were prevented from entering the CNS. In addition, Apitope demonstrated the important principle that tolerised CD4+ T cells can mediate regulation of not only T cells specific for the treatment peptide, but also CD4+ T cells that recognise immunodominant peptides from related proteins. They found that tolerised T cells were able to exert linked bystander suppression of other T cells in vivo, most successfully so when both peptides where presented by the same antigen-presenting cell by coupling the two antigens with a linker sequence.
Also the research from Rotterdam focused on cytokines, and has shed light on a possible role of EBV in MS and analyzed disruption of cytokines signaling pathways in primate models of MS (IL-7 and IL-23). Furthermore, they performed a comprehensive analysis of radical mediated tissue damage and mitochondrial injury in primate EAE, paralleling work previously done in MS, and observed that primate models can be used for testing novel therapeutics directed at alleviating oxidative tissue injury.
The first objective of the partner from Charite was to exchange resident microglia, which are the CNS macrophages with peripheral myeloid cells (PDMCs) in the Alzheimer’s disease (AD) context. This exchange happens when microglia are ablated using the partner’s previously published CD11b-SHVTK mouse model. To assess the morphological and functional differences between the microglia and PDMCs they implanted a cranial window on the transgenic mice. They found that the process activity of both microglia and PDMCs reduced in the proximity of plaques, while myeloid cells further away from plaques but in the AD environment were equally functional as cells in a wild-type environment. This leads to the hypothesis that the PDMCs are primed in the periphery by the soluble Aβ levels in the blood as well as inflammatory markers associated with AD.
Paraneoplastic neurological disorders (PND) are rare immune-mediated diseases that develop in the setting of malignancy and offer a unique prospect to analyze the interplay between tumour immunity and autoimmunity. The Toulouse group generated new mouse models that allow expression of a self-antigen in the Purkinje neurons of the cerebellum and in tumor cells. They found that mice developed severe cerebellar inflammation upon anti-CTLA-4 treatment, with subsequent loss of Purkinje neurons, but the tumour was rejected. Therefore, the enhanced tumour control was obtained at the expense of autoimmune paraneoplastic cerebellar degeneration. Consequently, they showed that the immune checkpoint therapy in our mouse model elicits T cell migration into the cerebellum and local destructive autoimmunity.
The partner Wien attempted to define the nature of the inflammatory response in MS lesions in relation to that in other inflammatory and non-inflammatory diseases and age matched controls in humans. Using quantitative immunocytochemical techniques they revealed the presence of T-lymphocytes (especially CD8+) and of CD20 positive B-cells in MS brains. Within the CD8 population a low and variable fraction expressed granzyme B as a marker for cytotoxic activation. This study support a very prominent role of B-lymphocytes in the inflammatory process of MS, which is also supported by the recently reported success of anti-CD20 treatment in MS patients.
The focus of the labs at the WIS and USR is to understand the cross talk between the immune system and the brain, and design new therapies for autoimmune of the CNS. The lab of Michal Schwartz discovered that immune cells can enter the CNS through the blood cerebrospinal fluid barrier (BCSFB), and that activity of this barrier to promote trafficking of leukocytes is dependent on the availability of IFN-γ. They also found that in neurodegenerative diseases, in analogy to cancer, systemic immunosuppressive cells and pathways, such as regulatory T cells and inhibitory immune checkpoint pathways, respectively, might curtail the immune response needed to fight this pathology.
USR focused more on the treatment of CNS autoimmunity, and specifically on trying to treat the CNS via the stimulation of neural precursor cells (NPCs). They found that NPCs secrete TGFβ2 that inhibits monocyte-derived dendritic cells differentiation and maturation, favouring the switch towards an anti-inflammatory phenotype. USR further dissected the role of NPC residing in the subventricular zone of the brain taking advantage of in house generated transgenic mouse line where NPCs can be selectively ablated in this region. Using these novel mice, they found that endogenous NPCs play a protective role in acute but not in chronic inflammatory disorders of the CNS.
Two industrial partners developed new technology that will be to the benefit of the whole community. The partner Biontech focused on setting up a pipeline enabling high-throughput sequencing of αβTCR genes from single murine T cells (scTCRseq). ScTCRseq allows the identification and reconstitution of functional αβTCR pairs, even when material is limited, as often the case with mouse models (of CNS disorders). They could show general feasibility of this method using two sets of TCR α and β chain-encoding in vitro transcribed mRNA libraries with known sequences. Their platform established is now ready to use for the unbiased determination of the individual αβTCRs from up to 9216 (96x96) single murine T cells in parallel.
The hypothesis of the project of the partner Miltenyi was that different subpopulations of reactive astrocytes (ASCA-2+) exist in the inflamed CNS with distinct cytokine/chemokine secretion profile and perform different roles in disease development and recovery. Using methods that allow to isolate astrocytes from neonates and adult mice, they Miltenyi group found 856 genes were expressed at a significantly higher level in neonatal ACSA-2-positive cells relative to adult cells. In contrast, 102 genes were expressed at a significantly higher level in adult ACSA-2- positive cells relative to neonatal cells. As determined by annotation enrichment analysis, the terms translation, nucleotide metabolism, intracellular import/export, and others were enriched with high significance among the genes with higher expression levels in neonatal cells. In summary, single-cell transcriptome analyses revealed a highly diverse expression profile of neonatal and adult astrocytes.
Further information please find on our website: http://www.unimedizin-mainz.de/neurokine/uebersicht.html