Citations

ABSTRACT Interferon-γ (IFNγ) signaling is prominent in inflammatory CNS microenvironments across many neurological disorders, but the neuronal peptides presented on HLA class I under these conditions and their functional consequences for CD8 + T cells remain incompletely defined. Here we combine human iPSC-derived human neural aggregates (HNAs), HLA class I immunoprecipitation coupled to LC-MS/MS immunopeptidomics, and microfluidic co-culture assays to map IFNγ-induced neuronal antigen presentation and test antigen-specific cytotoxicity. IFNγ stimulation induced robust HLA class I expression in HNAs and enabled recovery of a canonical 8-12-mer class I ligandome enriched for 9-mers. Neuron-restricted expression of a synapsin-driven polyepitope cassette yielded presentation of defined neoantigen 9-mer peptides on donor HLA class I molecules and, in the presence of IFNγ, elicited activation of autologous antigen-specific CD8 + T cells and consequent antigen-dependent neurite injury. Across four donors, comparative immunopeptidomics identified large IFNγ-unique neural peptide repertoires distinct from matched fibroblasts and revealed a consistent enrichment of predicted high-affinity binders on HLA-B allotypes. Finally, β2-microglobulin deletion ablated peptide recovery, and neuron-restricted β2-microglobulin reconstitution enabled identification of neuron-derived peptides, including peptides derived from neurofilament light (NEFL) that were shared across donors and presented on multiple HLA allotypes. Together, these data provide an integrated platform for neuronal autoantigen discovery and functional validation and support a model in which IFNγ-driven neuronal HLA class I presentation creates an HLA-B-weighted epitope landscape that can be recognized by autoreactive cytotoxic CD8 + T cells.

Abstract Description The pathophysiology of autoimmune diseases involves human leukocyte antigen (HLA)-dependent interactions between effector T and antigen presenting cells. In multiple sclerosis (MS) major histocompatibility complex (MHC) class I-restricted CD8+ T cells predominate in active lesions and are necessary and sufficient to cause axonal injury and loss of function in demyelination models. Preliminary work showed that demyelination drives presentation of neuron-specific neoantigens on axonal MHC class I and that neuroantigen-specific CD8+ T cells infiltrate the central nervous system and injure such antigen-presenting axons, supporting a pathogenic role for autoreactive anti-neuronal CD8+ T cells in MS progression. The current study investigates T cell-mediated neurotoxicity in MS patients using a coculture assay designed to address critical gaps in understanding immune-neuronal interactions. Live cell imaging showed neurite injury in cocultures of patient-dervied CD8+ T cells and autologous iPSC-derived neurons, while HLA blockade partially prevented neuronal death. To expand the utility of this assay, “universal” HLA-ABC knockout neural stem cells were reconstituted with patient-specific HLA molecules, enabling scale up of the coculture system without the need to derive neurons from each patient. Future directions include live cell reporters for granzyme B and NFAT activity, interferon signaling, and calcium transients to detect T cell activation concomitant with neuronal injury. Funding Sources NIH R01N5115126 (to Charles L. Howe) Topic Categories Neuroimmunology (NEUR)

Abstract Description Background Glutamic acid decarboxylase 65 (GAD65) antibodies are found in patients with stiff-person syndrome (SPS), cerebellar ataxia (CA), epilepsy and type-I diabetes (T1D). Given the intracellular localization of the antigenic target, a direct pathogenic role for GAD65 antibody (GAD65Ab) is unlikely. Methods PBMC-derived dendritic cells (DCs) from GAD65Ab+ patients with SPS (n = 6), epilepsy (n = 4), CA (n = 5), or T1D (n = 4) were pulsed with full-length GAD65 proteins or with 15-mer peptides from GAD65. T cell activation, exhaustion, and cytotoxicity were measured via cytometric bead array, FluoroSpot, flow cytometry, and live cell imaging. Results CD8+ T cells from patients with GAD65Ab-associated neurological disorders (AD) were activated by exposure to DCs pulsed with full-length GAD65 (p < 0.01 vs unstimulated, Mann-Whitney test; n = 16). We found 3 peptides of interest that caused a 10-30% increase in activated CD8+ T cells in GAD65AbAD patients, as assessed by surface CD69+ labeling. Using NetMHCpan4.2, we identified 8 different 9-mer peptides with high predicted affinity for HLA-C*03:03/04 and HLA-C*07:01/02. Two of these peptides showed high binding to HLA-C and subsequently demonstrated tetramer labeling of CD8+ T cells in GAD65AbAD patients. Conclusions We demonstrated that both full-length GAD65 protein and specific GAD peptides activated CD8+ T cells derived from patients with GAD65AbAD, indicating a potential pathogenic role for these effectors in these disorders. Funding Sources Support from Aditxt Immune Health Project (EU CT number: 2023-504798-19-00) and the Center for MS and Autoimmune Neurology in Mayo Clinic. Topic Categories Neuroimmunology (NEUR)

Multiple sclerosis is characterized by CNS infiltration of autoreactive immune cells that drive both acute inflammatory demyelination and chronic progressive axonal and neuronal injury. Expanding evidence implicates CD8 + antineural T cells in the neurodegeneration that underlies irreversible clinical progression in multiple sclerosis, yet therapies specifically targeting this cell population are limited. CD8 + T cells from patients with MS exhibit increased engagement of the pentose phosphate pathway. Pharmacologic inhibition of the pentose phosphate pathway reduced glycolysis, glucose uptake, NADPH production, ATP production, proliferation, and proinflammatory cytokine secretion in CD8 + T cells activated by ligation of CD3 and CD28. Pentose phosphate pathway inhibition also prevented CD8 + T cell–mediated antigen-specific neuronal injury in vitro and in both an adoptive transfer–based cuprizone model of demyelination and in mice with experimental autoimmune encephalomyelitis. Notably, transcriptional profiling of CNS-infiltrating CD8 + T cells in patients with MS indicated increased pentose phosphate pathway engagement, suggesting that this pathway is involved in CD8 + T cell–mediated injury of axons and neurons in the demyelinated CNS. Inhibiting the pentose phosphate pathway disrupts CD8 + T cell metabolic reprogramming and effector functions, suggesting that such inhibition may serve as a therapeutic strategy to prevent neurodegeneration in patients with progressive MS.