Citations

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 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 &lt; 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)

AbstractObjectiveTherapeutic strategies for patients with febrile infection‐related epilepsy syndrome (FIRES) are limited, ad hoc, and frequently ineffective. Based on evidence that inflammation drives pathogenesis in FIRES, we used ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) to characterize the monocytic response profile before and after therapy in a child successfully treated with dexamethasone delivered intrathecally six times between hospital Day 23 and 40 at 0.25 mg/kg/dose.MethodsPBMCs were isolated from serial blood draws acquired during refractory status epilepticus (RSE) and following resolution associated with intrathecal dexamethasone therapy in a previously healthy 9‐year‐old male that presented with seizures following Streptococcal pharyngitis. Cells were stimulated with bacterial or viral ligands and cytokine release was measured and compared to responses in age‐matched healthy control PBMCs. Levels of inflammatory factors in the blood and CSF were also measured and compared to pediatric healthy control ranges.ResultsDuring RSE, serum levels of IL6, CXCL8, HMGB1, S100A8/A9, and CRP were significantly elevated. IL6 was elevated in CSF. Ex vivo stimulation of PBMCs collected during RSE revealed hyperinflammatory release of IL6 and CXCL8 in response to bacterial stimulation. Following intrathecal dexamethasone, RSE resolved, inflammatory levels normalized in serum and CSF, and the PBMC hyperinflammatory response renormalized.SignificanceFIRES may be associated with a hyperinflammatory monocytic response to normally banal bacterial pathogens. This hyperinflammatory response may induce a profound neutrophil burden and the consequent release of factors that further exacerbate inflammation and drive neuroinflammation. Intrathecal dexamethasone may resolve RSE by resetting this inflammatory feedback loop.

Abstract Same day processing of biospecimens such as blood is not always feasible, which presents a challenge for research programs seeking to study a broad population or to characterize patients with rare diseases. Recruiting sites may not be equipped to process blood samples and variability in timing and technique employed to isolate peripheral blood mononuclear cells (PBMCs) at local sites may compromise reproducibility across patients. One solution is to send whole blood collected by routine phlebotomy via overnight courier to the testing site under ambient conditions. Determining the impact of shipping on subsequent leukocyte responses is a necessary prerequisite to any experimental analysis derived from transported samples. To this end, whole blood was collected from healthy control subjects and processed fresh or at 6, 24 and 48 hours after collection and handling under modeled shipping conditions. At endpoint, whole blood was assessed via a complete blood count with differential and immunophenotyped using a standardized panel of antibodies [HLADR, CD66b, CD3, CD14, CD16]. PBMCs and neutrophils were isolated from whole blood and subjected to ex vivo stimulation with lipopolysaccharide and heat-killed Staphylococcus aureus. Stimulated release of cytokines and chemokines was assessed by cytometric bead array. RNA was also isolated from PBMCs to analyze transcriptional changes induced by shipping. The complete blood count with differential revealed that most parameters were maintained in shipped blood held for 24 hours at ambient temperature. Immunophenotyping indicated preservation of cellular profiles at 24 hours, although with broadening of some populations and a decrease in CD16 intensity on classical monocytes. At the transcriptional level, RNAseq analysis identified upregulation of a transcription factor module associated with inflammation in unstimulated PBMCs derived from whole blood shipped overnight. However, these changes were limited in both scale and number of impacted genes. Ex vivo stimulation of PBMCs further revealed preservation of functional responses in cells isolated from shipped blood held for 24 hours at ambient temperature. However, neutrophil responses were largely abrogated by this time. By 48 hours neither cell population responded within normal parameters. These findings indicate that robust immunophenotyping and PBMC stimulated response profiles are maintained in whole blood shipped overnight and processed within 24 hours of collection, yielding results that are representative of those obtained from the sample immediately following venipuncture. This methodology is feasible for many patient recruitment sites to implement and allows for sophisticated immunological analysis of patient populations derived from large geographic areas. With regard to rare disease research, this meets a universal need to enroll patients in sufficient numbers for immunoprofiling and discovery of underlying pathogenic mechanisms.

The immune system consists of two broad branches: innate and adaptive. In the innate branch of the immune system, one will find natural killer (NK) cells, which are responsible for recognizing and killing virally infected or cancerous cells within the human body. Numerous immunodeficiency syndromes arise from mutations in genes that encode proteins essential to NK cells including those involved in NK cell development and effector functions. Among the most important NK cell effector functions is cell‐mediated cytotoxicity. Our lab has been interested in understanding the molecular machinery that regulates this process, in particular those regulated by the small GTP‐binding proteins of the Rho family. With this in mind, the ArhGAP12 protein, which functions as a GTPase activating protein for members of the Rho family, was tested in NK cells to see if it had an effect on cytotoxicity. Significantly, knockdown of ArhGAP12 in NK cells resulted in diminished NK cell‐mediated cytotoxicity. In addition, we found that ArhGAP12 is tyrosine phosphorylated following stimulation through the NKG2D+2B4 activating receptors, indicating that it is linked to this important signaling pathway. As ArhGAP12 is a multi‐modular signaling protein containing a src‐homology 3 (SH3), WW, pleckstrin homology (PH), and GAP domains, we performed proteomics to identify potential interacting partners using the SH3 domain as bait. We found that ArhGAP12 interacts strongly with multiple regulators of F‐actin dynamics including WAVE2, WASP, Evl and VASP. Taken together, these results suggest that ArhGAP12 is linked to activating receptors on NK cells, associates with F‐actin regulators, and is involved in NK cell‐mediated cytotoxicity. Support or Funding Information Mayo Clinic Graduate School of Biomedical Sciences SURF Program and the R01 grant AI120949 to D.D.B. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .