E. Harrington
Johns Hopkins NeurologyAuthor Of 1 Presentation
YI01.04 - Generation of MHC class I and MHC class II reporter mice for investigation of antigen presentation by oligodendroglia
Abstract
Background
Emerging evidence from both human multiple sclerosis (MS) tissue and rodent experimental autoimmune encephalomyelitis (EAE) models has demonstrated that subsets of oligodendrocyte lineage cells are capable of expressing antigen presenting and processing molecules, including MHC class I and MHC class II molecules, in an inflammatory environment. Antigen presentation by inflammatory oligodendroglia (iOPCs/iOLs) to CD4 and CD8 T cells may result in cytotoxic death of oligodendroglia and perpetuation of the inflammatory response. However, the dynamics of MHC class I and MHC class II expression by oligodendrocyte lineage cells in an inflammatory environment are unknown.
Objectives
To better define these dynamic changes in phenotype, we developed two new mouse reporter lines by targeting Beta-2-microglobulin (B2m) and CD74 invariant chain (Ii) genes, which are upregulated by oligodendroglia in human MS and mouse EAE. Beta-2-microglobulin (B2m) is a component of both classical and non-classical MHC class I molecules and CD74 invariant chain (Ii) is involved in occupancy of the peptide binding groove of MHC class II molecules in the endoplasmic reticulum and formation of stable MHC class II-antigen complexes.
Methods
We generated reporter lines by Crispr/Cas9-mediated insertion of a P2A-TdTomato-WPRE-pA sequence, replacing the stop codon of B2m (for class I reporter) and CD74 (for class II reporter).
Results
Both B2m-TdT and CD74-TdT mice demonstrate robust reporter expression in cultured OPCs upon exposure to interferon gamma. In naïve CD74-TdT reporter mice, TdT reporter expression was not detected in the brain or spinal cord parenchyma, but strong expression was observed in peripheral immune cells in the meninges and choroid plexus. B2m-TdT reporter mice exhibited TdT reporter expression in microglia throughout the brain and spinal cord, and some expression in spinal cord white matter oligodendroglia near the meningeal surface. No TdT reporter expression was detected in brain oligodendroglia at baseline. Cortical stab injury in B2m-TdT reporter mice resulted in TdT reporter co-localization with Iba1+ microglia and Mac2+ macrophages within the lesion core and Olig2+CC1+ oligodendrocytes in the corpus callosum ipsilateral and contralateral to the stab injury.
Conclusions
Further investigation of MHC class I and MHC class II expression in oligodendroglia in the setting of inflammatory demyelinating models will help reveal the spatial and temporal dynamics of this phenotypic change in oligodendroglia.
Presenter Of 1 Presentation
YI01.04 - Generation of MHC class I and MHC class II reporter mice for investigation of antigen presentation by oligodendroglia
Abstract
Background
Emerging evidence from both human multiple sclerosis (MS) tissue and rodent experimental autoimmune encephalomyelitis (EAE) models has demonstrated that subsets of oligodendrocyte lineage cells are capable of expressing antigen presenting and processing molecules, including MHC class I and MHC class II molecules, in an inflammatory environment. Antigen presentation by inflammatory oligodendroglia (iOPCs/iOLs) to CD4 and CD8 T cells may result in cytotoxic death of oligodendroglia and perpetuation of the inflammatory response. However, the dynamics of MHC class I and MHC class II expression by oligodendrocyte lineage cells in an inflammatory environment are unknown.
Objectives
To better define these dynamic changes in phenotype, we developed two new mouse reporter lines by targeting Beta-2-microglobulin (B2m) and CD74 invariant chain (Ii) genes, which are upregulated by oligodendroglia in human MS and mouse EAE. Beta-2-microglobulin (B2m) is a component of both classical and non-classical MHC class I molecules and CD74 invariant chain (Ii) is involved in occupancy of the peptide binding groove of MHC class II molecules in the endoplasmic reticulum and formation of stable MHC class II-antigen complexes.
Methods
We generated reporter lines by Crispr/Cas9-mediated insertion of a P2A-TdTomato-WPRE-pA sequence, replacing the stop codon of B2m (for class I reporter) and CD74 (for class II reporter).
Results
Both B2m-TdT and CD74-TdT mice demonstrate robust reporter expression in cultured OPCs upon exposure to interferon gamma. In naïve CD74-TdT reporter mice, TdT reporter expression was not detected in the brain or spinal cord parenchyma, but strong expression was observed in peripheral immune cells in the meninges and choroid plexus. B2m-TdT reporter mice exhibited TdT reporter expression in microglia throughout the brain and spinal cord, and some expression in spinal cord white matter oligodendroglia near the meningeal surface. No TdT reporter expression was detected in brain oligodendroglia at baseline. Cortical stab injury in B2m-TdT reporter mice resulted in TdT reporter co-localization with Iba1+ microglia and Mac2+ macrophages within the lesion core and Olig2+CC1+ oligodendrocytes in the corpus callosum ipsilateral and contralateral to the stab injury.
Conclusions
Further investigation of MHC class I and MHC class II expression in oligodendroglia in the setting of inflammatory demyelinating models will help reveal the spatial and temporal dynamics of this phenotypic change in oligodendroglia.