Howe, Charles L.

Assessing balance is crucial in neurological rehabilitation, yet while wearable sensors enable real-world monitoring, identifying reliable digital biomarkers remains challenging. This study utilized a high-fidelity instrumented walkway to determine which gait parameters best predict balance impairment, providing robust targets for future wearable applications. We analyzed 49 steady-state gait metrics from 140 individuals with diverse neurological conditions. Using statistical analysis and machine learning, we evaluated these parameters against objective force plate sway scores and clinical fall-history labels. Group analysis identified 16 parameters significantly distinguishing fallers from non-fallers, and a neural network classified fallers with an area under the curve of 0.75. Across all analytical approaches, overall gait variability, e.g., Stride Width S.D. and the Gait Variability Index, emerged as a universal predictor of balance impairment and fall risk. Furthermore, while traditional linear models emphasized spatial postural control, machine learning classification uniquely identified inter-limb asymmetry as a premier driver of fall prediction. These findings indicate that instrumented gait analysis effectively identifies digital biomarkers for balance deficits. Isolating these specific metrics provides a clear blueprint for meaningful metrics required for continuous objective monitoring and future development of personalized, adaptive rehabilitation strategies.

Neurons upregulate major histocompatibility complex class I (MHC I) during demyelination, enabling the presentation of self-antigens to cytotoxic CD8 + T cells. While this phenomenon is well described in multiple sclerosis lesions, its functional significance for disease progression remains poorly understood. Here, we test the hypothesis that neuronal MHC I expression promotes CD8 + T cell-mediated neurodegeneration in demyelinating disease. Using genetic and viral approaches to selectively ablate β ₂-microglobulin (β₂M) in neurons, we demonstrate that the loss of neuronal MHC I limits antigen presentation, attenuates the activation of neuron-antigen–specific CD8 + T cells, and reduces the recruitment of these cells to the demyelinated central nervous system (CNS). In myelin oligodendrocyte glycoprotein (MOG₃₅–₅₅)-induced experimental autoimmune encephalomyelitis, CD8 + T cell ablation during active disease limited neuronal injury and improved clinical recovery. Similarly, neuronal β ₂M ablation decreased clinical disease burden without affecting CNS T cell infiltration. Likewise, in the context of cuprizone intoxication, neuronal β ₂M deletion reduced peripheral activation and CNS recruitment of CD8 + T cells recognizing a neuron-restricted neoantigen. It protected neoantigen-expressing neurons from cognate CD8 + T cell-mediated cytotoxicity. Collectively, these findings identify neuronal MHC I–dependent antigen presentation as a driver of both the immune surveillance of neuronal antigens and neuronal injury during demyelination.

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 Background High-titer glutamic acid decarboxylase 65 (GAD65) antibodies are found in patients with GAD65 antibody-associated neurological disorders, including stiff-person syndrome (SPS), GAD65 cerebellar ataxia (CA), and GAD65 epilepsy. Given the intracellular localization of the antigenic target, a direct pathogenic role for GAD65 autoantibodies is unlikely. Instead, the autoantibody may be a biomarker for the existence of pathogenic anti-GAD65 autoreactive CD8 + T cells. Methods PBMC-derived dendritic cells (DCs) from 20 GAD65 antibody-positive patients with neurological manifestations (SPS, n=10; CA, n=7; epilepsy, n=3) and 15 healthy controls were pulsed with full-length GAD65 protein, full-length GAD67 protein, overlapping 15-mer peptide pools spanning GAD65 and GAD67, individual GAD65 15-mer peptides, or GAD65 9-mer peptides selected from predicted class I binding hotspots. T cell activation was quantified by flow cytometry-based activation-induced marker (AIM) assays using CD69 upregulation. Class I and class II HLA haplotypes were determined by high resolution typing. NetMHCpan v4.2c was used to generate residue-level peptide:HLA binding density maps across GAD65, and candidate 9-mers were validated for HLA binding by peptide:MHC monomer affinity testing. GAD65-peptide-HLA-restricted CD8 + T cells were identified by co-staining with two separately assembled tetramers carrying the same peptide:HLA complex on different fluorophores (APC and BV421), with double-positive events scored as antigen-specific. HLA-restricted cytotoxicity was measured by coculture of patient CD8 + T cells with GAD65-expressing HEK-293T cells reconstituted with defined HLA class I alleles using AAV-delivered Cre-dependent HLA-2A-eGFP cassettes. Results CD8 + T cells from GAD65 antibody-positive patients showed increased activation in response to DCs pulsed with full-length GAD65 relative to healthy controls (P=0.0157, Welch’s t-test), whereas responses to GAD67 did not differ significantly between groups. CD4 + T cells responded to both GAD65 (P=0.0004) and GAD67 (P=0.0051). Peptide pool screening of GAD65 identified discrete CD8 + and CD4 + immunogenic regions, with refinement using individual 15-mers localizing CD8 + activity to multiple subdomains within GAD65 (205-300) , GAD65 (316-435) , and GAD65 (447-520) . HLA class I haplotyping in 16 non-Hispanic White GAD65 + patients revealed enrichment of HLA-B*08:01 (3.0-fold) and HLA-B*40:01 (4.1-fold) relative to USA NMDP European Caucasian reference frequencies (both BH q < 0.05), with suggestive enrichment of HLA-C*03:04 (2.9-fold; q = 0.09). Additionally, 44% of patients carried the HLA-A*01:01, HLA-B*08:01, and HLA-C*07:01 8.1 ancestral haplotype, approximately four-fold higher than the expected population frequency. Dual-fluorophore tetramer staining identified CD8 + T cells in GAD65 + subjects that bound a subset of HLA-A*11:01- and HLA-B*08:01-restricted GAD65 9-mers, with the clearest disease-skewed signals localized to GAD65 (213-221) , GAD65 (257-265) , and GAD65 (529-537) . In an HLA-reconstituted target-cell killing assay, CD8 + T cells from an HLA-B*08:01-positive GAD65 + patient mediated antigen- and HLA-restricted depletion of GAD65-expressing HEK-293T cells, with HLA-restricted target loss also observed in single-donor experiments across additional HLA-A, -B, and -C contexts. Conclusions Patients with GAD65 antibody-associated neurological disorders harbor circulating CD8 + T cells that recognize discrete HLA class I-restricted GAD65 peptides and that are capable of cytotoxicity against GAD65-expressing HLA-matched target cells. We characterize the immunogenetic and cellular features of class I-restricted CD8 + T cell responses in GAD65 autoimmunity, including overrepresentation of the 8.1 ancestral haplotype, complementing the historical focus on antibodies and CD4 + T cell help, and we provide a panel of validated GAD65 peptide:HLA tetramers for prospective isolation, clonotypic analysis, and longitudinal monitoring of candidate pathogenic CD8 + T cell populations across the GAD65 antibody-associated neurological disease spectrum.

Study objective: To characterize physiologic autonomic dysfunction in humans after chronic spinal cord injury (SCI) relative to uninjured controls and to identify autonomic correlates to neuroimmune deficits. Hypothesis: Individuals with greater autonomic dysfunction after SCI will have more pronounced immune dysfunction, which will be further influenced by sympathetically mediated increases in catecholamine levels. Methodology: Both uninjured controls and individuals with SCI completed a battery of autonomic physiology tests aimed at characterizing preservation of supralesional/sublesional sympathetic activation and global sympathetic inhibition. The autonomic testing battery included 3-minute hand and foot cold pressor tests, 3 Valsalva maneuvers with goal expiratory pressure of 30 mmHg, a novel bladder pressor test (5 minutes of sustained bladder pressure at 35 cmH20 with room temperature sterile water infusion), paced breathing at 0.25 Hz for heart rate/blood pressure variability, and successive doses of bolus intravenous phenylephrine (Oxford technique). A subset of individuals in both cohorts had baseline and cold pressor stressed levels of catecholamines/cortisol drawn and immunologic assessments. Peripheral blood mononuclear cells were extracted from this baseline sample and assessed with flow cytometry for cellular exhaustion markers (CD69, PD-1, TIGIT, TIM-3). Antigen recall response, with/without primed exposure of catecholamines was also completed to identify how sympathetic activation variably impacted functional immune performance with exposure to viral peptides. Data: n=35 uninjured controls; n=29 individuals with SCI Summary of Results: When compared to uninjured controls, individuals with SCI demonstrated significant autonomic dysfunction in all three domains, with decreased supralesional sympathetic activation (Valsalva pressure recovery time, p< 0.0001), abnormal sublesional sympathetic activation (increased bradycardia, p< 0.0001), and impaired sympathetic inhibition (larger dose/body-weight adjusted increase in mean arterial pressure, p=0.0006). As expected, autonomic dysfunction in those with SCI existed on a continuum, with some having largely normal autonomic regulation and others exhibiting profound deficits. Principal component analysis confirmed these three domains as the primary drivers of variability. Cellular exhaustion markers were increased in those with SCI, with positive correlation (R2=0.42) between degree of global autonomic dysfunction (Z-score over 2.0) and CD3+ CD8+ T-cell TIM-3 immunologic exhaustion markers. Antigen recall response was impaired in those with SCI and autonomic dysfunction on physiologic testing, with pronounced decrement in response when catecholamines were added (mean 57% of response lost in CD69 T-cells to Epstein-Barr virus antigen exposure, 53% lost to Flu antigen). Conclusions: In humans with SCI, early evidence supports that individuals have greater autonomic dysfunction and that this is correlated to increased immunologic deficits. Funding Sources: NIH K23HD102663, R01NS115126 This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Description CD8+ T cell responses to neuronal antigens are believed to contribute to CNS autoimmune diseases and neurodegenerative disease pathophysiology. However, the specific endogenous human neuron antigens and their MHC restrictions remain unknown. We characterized the immunopeptidome of human iPSC-derived neurons and verified their ability to elicit cytopathic responses in autologous T cells. After maturation, &gt;100M neurons were treated with IFNg to induce MHC I expression. MHC class I molecules were immunoprecipitated, and associated peptides were analyzed using mass spectrometry. For comparison we also analyzed identically treated human adult dermal fibroblasts and neural ectoderm cells, and neurons differentiated from B2M KO NSCs. Immunopeptidomes were analyzed my Gibbs clustering, netMHC affinity and immunogenicity prediction for patient allotypes. We identified peptides uniquely presented by IFNg-treated neurons and analyzed these for brain enrichment. Among neural enriched proteins detected across multiple subjects, proteins for which CNS autoimmunity has been described were found to be enriched. We also identified putative immunodominant peptides from 29 potential MS patient autoantibody targets (including CRYAB) presented by MHC I on human neurons. Finally, human neurons transduced to express immunodominant peptides from CMV, EBV, and influenza (CEF) showed detectable MHC I peptide presentation and could be injured by autologous CEF-stimulated CD8+ T cells by live imaging. Funding Sources Don and Fran Herdrich NIH grant NS064571 (to CLH) Mayo Clinic Eugene and Marcia Applebaum fellowship award (to BDSC) The Center for Multiple Sclerosis and Autoimmune Neurology at Mayo Clinic (to CLH). Topic Categories Neuroimmunology (NEUR)

Abstract Description Neurons upregulate MHC Class I during infection and neuroinflammation associated with demyelinating diseases, allowing them to present both self and foreign antigens to CD8+ T cells. We hypothesized that ablation of neuronal MHC I expression reduces neural antigen-specific CD8+ T cell recruitment to demyelinated CNS and reduces neuron loss. We have previously shown that demyelination induces expression of MHC Class I on neurons and axons, allowing neuron-specific neoantigens to be presented to CD8+ T cells, leading to neuroinflammatory damage. Elevated MHC-I and β2-microglobulin expression in white and grey matter tissue from MS patients further supported the role of CD8+ T cells in neuronal injury. Here we demonstrate that neuron specific β2-microglobulin ablation in the B6 MOG35-55 EAE model of multiple sclerosis reduces disease progression and severity via clinical scoring. Our preliminary findings suggest that MHC-I ablation may reduce peripheral activation and CNS recruitment of neural antigen-specific CD8+ T cells as well as CD8+ T cell-mediated neurodegeneration in in the context of demyelinating disease. Future studies are aimed at generating a humanized mouse model reconstituted with patient immune cells to understand the role of MHC I expression in neurons, astrocytes, and oligodendrocytes. Specifically, using this model, we will characterize brain infiltrating T cells from patients with CNS autoimmune disease and suspected T cell involvement. Funding Sources Generous donation from Don and Fran Herdrich. Topic Categories Neuroimmunology (NEUR)

Background: Tumefactive demyelination (TD) is a rare variant of multiple sclerosis (MS) characterized by tumor-like lesions that often require aggressive management. Genome-wide association studies (GWAS) identified variants associated with MS; similar analyses in TD are lacking. Objective: A GWAS was performed to identify variants associated with TD. Methods: The case–control study included 142 TD cases and 293 controls. TD patients were required to have a demyelinating event and magnetic resonance imaging (MRI) showing one or more lesions. Controls were patients without a neurologic or systemic inflammatory disease or cancer. Logistic regression was used to compare cases versus controls for each variant; age, sex, and principal components were included as covariates. A p -value threshold of 5 × 10 −8 was GWAS significant and 5 × 10 −6 nominally significant. A polygenic risk score (PRS) was compared across TD and controls. Results: Variants on chromosome 14 (rs117797734, p = 2.06 × 10 −11 , odds ratio (OR) = 13.14) and chromosome 6 (most significant rs6936540, p = 5.5 × 10 −7 , OR = 2.61) near DCBLD1 were significant. Seven non-MHC and two MHC variants associated with MS were associated with TD. The PRS was significantly higher in TD versus controls. Conclusion: We identified novel regions associated with TD, demonstrating the importance of performing GWAS in homogeneous subtypes of MS. Further validation and functional experiments are necessary.

ABSTRACT Objectives To investigate the value of cytokine, chemokine, and neurofilament light chain (NfL) concentrations in predicting relapse risk, chronic epilepsy, and functional impairment in LGI1 autoimmune encephalitis (AE). Methods Cytokines/chemokines (IL‐1‐beta, IL‐2, IL‐4, IL‐5, IL‐6, IL‐8/CXCL8, IL‐10, IL‐12p70, IL‐13, IL‐17A, GM‐CSF, TNF‐alpha, IFN‐gamma, CXCL9, CXCL10, CXCL13, BAFF) and NfL concentrations were measured in CSF and paired serum from LGI1‐AE patients evaluated at Mayo Clinic (01/2015–02/2024), using a multiplex immunoassay system (ELLA, Bio‐Techne) and correlated with clinical outcomes. A laboratory‐based cohort of LGI1‐IgG‐positive patients and control cohorts, including patients with mixed non‐inflammatory disorders (MNID), Alzheimer's disease (AD), and temporal lobe epilepsy (TLE) were analyzed. Results Forty‐four patients with LGI1‐AE were included; 29 (66%) were male, with a median age of 68.5 years (range, 8–85). Median time from symptom onset to CSF sampling was eight months (IQR, 3–17); 19/42 (45%) experienced a clinical relapse and 27% developed chronic epilepsy. Serum IL‐6, serum and CSF IL‐8/CXCL8, and IL‐17A were higher in LGI1‐IgG positive patients than MNID ( p < 0.05). TLE cytokine/chemokine profiles were similar to LGI1 AE; AD patients had lower serum IL‐6 and CSF IL‐8/CXCL8 ( p = 0.04; p = 0.01), and higher serum IL‐17A and GM‐CSF ( p = 0.004; p = 0.01) than LGI1‐AE. Higher CSF IL‐6 and IL‐8/CXCL8 in LGI1‐AE associated with clinical relapse ( p < 0.05) and higher CSF NfL associated with chronic epilepsy ( p = 0.01). Conclusion Elevations in IL‐6, IL‐8/CXCL8, and IL‐17A were identified in this LGI1‐AE cohort. CSF IL‐6, IL‐8/CXCL8, and NfL levels are potential prognostic biomarkers for risk of relapse and chronic epilepsy in LGI1‐AE.