Translational imaging of TSPO reveals pronounced innate inflammation in human and murine CD8 T cell-mediated limbic encephalitis.

Autoimmune limbic encephalitis (ALE) presents with new-onset mesial temporal lobe seizures, progressive memory disturbance, and other behavioral and cognitive changes. CD8 T cells are considered to play a key role in those cases where autoantibodies (ABs) target intracellular antigens or no ABs were found. Assessment of such patients presents a clinical challenge, and novel noninvasive imaging biomarkers are urgently needed. Here, we demonstrate that visualization of the translocator protein (TSPO) with [18F]DPA-714-PET-MRI reveals pronounced microglia activation and reactive gliosis in the hippocampus and amygdala of patients suspected with CD8 T cell ALE, which correlates with FLAIR-MRI and EEG alterations. Back-translation into a preclinical mouse model of neuronal antigen-specific CD8 T cell-mediated ALE allowed us to corroborate our preliminary clinical findings. These translational data underline the potential of [18F]DPA-714-PET-MRI as a clinical molecular imaging method for the direct assessment of innate immunity in CD8 T cell-mediated ALE.

CD8+ T-Lymphocyte-Driven Limbic Encephalitis Results in Temporal Lobe Epilepsy.

OBJECTIVE: Limbic encephalitis (LE) comprises a spectrum of inflammatory changes in affected brain structures including the presence of autoantibodies and lymphoid cells. However, the potential of distinct lymphocyte subsets alone to elicit key clinicopathological sequelae of LE potentially inducing temporal lobe epilepsy (TLE) with chronic spontaneous seizures and hippocampal sclerosis (HS) is unresolved. METHODS: Here, we scrutinized pathogenic consequences emerging from CD8+ T cells targeting hippocampal neurons by recombinant adeno-associated virus-mediated expression of the model-autoantigen ovalbumin (OVA) in CA1 neurons of OT-I/RAG1-/- mice (termed "OVA-CD8+ LE model"). RESULTS: Viral-mediated antigen transfer caused dense CD8+ T cell infiltrates confined to the hippocampal formation starting on day 5 after virus transduction. Flow cytometry indicated priming of CD8+ T cells in brain-draining lymph nodes preceding hippocampal invasion. At the acute model stage, the inflammatory process was accompanied by frequent seizure activity and impairment of hippocampal memory skills. Magnetic resonance imaging scans at day 7 of the OVA-CD8+ LE model revealed hippocampal edema and blood-brain barrier disruption that converted into atrophy until day 40. CD8+ T cells specifically targeted OVA-expressing, SIINFEKL-H-2Kb -positive CA1 neurons and caused segmental apoptotic neurodegeneration, astrogliosis, and microglial activation. At the chronic model stage, mice exhibited spontaneous recurrent seizures and persisting memory deficits, and the sclerotic hippocampus was populated with CD8+ T cells escorted by NK cells. INTERPRETATION: These data indicate that a CD8+ T-cell-initiated attack of distinct hippocampal neurons is sufficient to induce LE converting into TLE-HS. Intriguingly, the role of CD8+ T cells exceeds neurotoxic effects and points to their major pathogenic role in TLE following LE. ANN NEUROL 2021;89:666-685.

Transient receptor potential melastatin subfamily member 2 cation channel regulates detrimental immune cell invasion in ischemic stroke.

BACKGROUND AND PURPOSE: Brain injury during stroke results in oxidative stress and the release of factors that include extracellular Ca(2+), hydrogen peroxide, adenosine diphosphate ribose, and nicotinic acid adenine dinucleotide phosphate. These alterations of the extracellular milieu change the activity of transient receptor potential melastatin subfamily member 2 (TRPM2), a nonselective cation channel expressed in the central nervous system and the immune system. Our goal was to evaluate the contribution of TRPM2 to the tissue damage after stroke. METHODS: In accordance with current quality guidelines, we independently characterized Trpm2 in a murine ischemic stroke model in 2 different laboratories. RESULTS: Gene deficiency of Trpm2 resulted in significantly improved neurological outcome and decreased infarct size. Besides an already known moderate neuroprotective effect of Trpm2 deficiency in vitro, ischemic brain invasion by neutrophils and macrophages was particularly reduced in Trpm2-deficient mice. Bone marrow chimeric mice revealed that Trpm2 deficiency in the peripheral immune system is responsible for the protective phenotype. Furthermore, experiments with mixed bone marrow chimeras demonstrated that Trpm2 is essential for the migration of neutrophils and, to a lesser extent, also of macrophages into ischemic hemispheres. Notably, the pharmacological TRPM2 inhibitor, N-(p-amylcinnamoyl)anthranilic acid, was equally protective in the stroke model. CONCLUSIONS: Although a neuroprotective effect of TRPM2 in vitro is well known, we can show for the first time that the detrimental role of TRPM2 in stroke primarily depends on its role in activating peripheral immune cells. Targeting TRPM2 systemically represents a promising therapeutic approach for ischemic stroke.

Excitotoxic neuronal cell death during an oligodendrocyte-directed CD8+ T cell attack in the CNS gray matter.

BACKGROUND: Neural-antigen reactive cytotoxic CD8+ T cells contribute to neuronal dysfunction and degeneration in a variety of inflammatory CNS disorders. Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from 'leaky' immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes. METHODS: Wild-type and T cell receptor transgenic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations. RESULTS: Following T cell receptor stimulation, CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter. CONCLUSION: Glutamate release may represent a crucial effector pathway of neural-antigen reactive CD8+ T cells, contributing to excitotoxicity in CNS inflammation.

TRPM2 cation channels modulate T cell effector functions and contribute to autoimmune CNS inflammation.

TRPM2, a highly Ca(2+)-permeable member of the transient receptor potential melastatin-related (TRPM) family of cation channels, is expressed in cells of the immune system. We demonstrate firstly that TRPM2 cation channels on T cells critically influence T cell proliferation and proinflammatory cytokine secretion following polyclonal T cell receptor stimulation. Consistently, trpm2-deficient mice exhibited an attenuated clincal phenotype of experimental autoimmune encephalomyelitis (EAE) with reduced inflammatory and demyelinating spinal cord lesions. Importantly, trmp2-deficient T cells were as susceptible as wildtype T cells to oxidative stress-induced cell death as it occurs in inflammatory CNS lesions. This supports the notion that the attenuated EAE phenotype is mainly due to reduced T cell effector functions but unaffected by potential modulation of T cell survival at the site of inflammation. Our findings suggest TRPM2 cation channels as a potential target for treating autoimmune CNS inflammation.

Residues at the tip of the pore loop of NR3B-containing NMDA receptors determine Ca2+ permeability and Mg2+ block.

BACKGROUND: Members of the complex N-methyl-D-aspartate receptor (NMDAR) subfamily of ionotropic glutamate receptors (iGluRs) conventionally assemble from NR1 and NR2 subunits, the composition of which determines receptor properties. Hallmark features of conventional NMDARs include the requirement for a coagonist, voltage-dependent block by Mg2+, and high permeability for Ca2+. Both Mg2+ sensitivity and Ca2+ permeability are critically dependent on the amino acids at the N and N+1 positions of NR1 and NR2. The recently discovered NR3 subunits feature an unprecedented glycine-arginine combination at those critical sites within the pore. Diheteromers assembled from NR1 and NR3 are not blocked by Mg2+ and are not permeable for Ca2+. RESULTS: Employing site-directed mutagenesis of receptor subunits, electrophysiological characterization of mutants in a heterologous expression system, and molecular modeling of the NMDAR pore region, we have investigated the contribution of the unusual NR3 N and N+1 site residues to the unique functional characteristics of receptors containing these subunits. Contrary to previous studies, we provide evidence that both the NR3 N and N+1 site amino acids are critically involved in mediating the unique pore properties. Ca2+ permeability could be rescued by mutating the NR3 N site glycine to the NR1-like asparagine. Voltage-dependent Mg2+ block could be established by providing an Mg2+ coordination site at either the NR3 N or N+1 positions. Conversely, "conventional" receptors assembled from NR1 and NR2 could be made Mg2+ insensitive and Ca2+ impermeable by equipping either subunit with the NR3-like glycine at their N positions, with a stronger contribution of the NR1 subunit. CONCLUSIONS: This study sheds light on the structure-function relationship of the least characterized member of the NMDAR subfamily. Contrary to previous reports, we provide evidence for a critical functional involvement of the NR3 N and N+1 site amino acids, and propose them to be the essential determinants for the unique pore properties mediated by this subunit.