Citations

Abstract Description The PD-L1/PD-1 receptor pathway allows cancer cells to evade immune surveillance, yet only a small percentage of patients benefit from PD-1/PD-L1 inhibitors. This limited efficacy is likely due to gaps in our understanding of PD-1/PD-L1 biology, particularly the lesser-known intracellular roles of PD-L1 compared to its extracellular interactions. Our research found that intracellular PD-L1 functions as an RNA-binding protein, interacting with NBS1 and BRCA1 mRNAs to protect them from degradation, thereby increasing cellular resistance to DNA damage. Given that current PD-1/PD-L1 inhibitors have little effect on intracellular PD-L1, we aimed to develop new drugs targeting this pathway. We created a novel PD-L1 antibody that disrupts the binding of PD-L1 to CMTM6, promoting PD-L1 degradation via the lysosome instead of blocking the PD-1/PD-L1 interaction. This mechanism enables the antibody to inhibit intracellular PD-L1 functions, something conventional immunotherapies cannot achieve. In vivo studies demonstrated that our antibody significantly sensitized tumors to radiation and chemotherapy in patient-derived xenograft models. In a humanized immune-competent triple-negative breast cancer PDX model, it outperformed existing PD-L1 inhibitors, suggesting it enhances anti-tumor immunity through PD-L1 degradation. This novel antibody offers a promising dual approach by targeting both intracellular and extracellular PD-L1, providing a compelling new strategy for cancer therapy. Funding Sources This work was supported in part by the American Society for Radiation Oncology, the National Cancer Institute of the National Institutes of Health under Award Number CA203561, P50CA116201, K12 HD065987, HALT Cancer at X, NCI R21 CA197878 (HD), R01-AI095239 and NIAID R01 AI095239. Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

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)

Abstract Description Common variable immunodeficiency (CVID) is a common inborn error of immunity, but a monogenic cause is rare. Biallelic loss-of-function pathogenic variants in the gene encoding BAFF-Receptor (TNFRSF13C) is one such cause. A common missense variant in BAFF-R, p.Pro21Arg, was proposed as contributing to the pathogenesis of non-monogenic CVID by Peiper et al. 2014 with an allelic frequency of 6.7% in controls versus 10.2% in CVID patients (OR 1.57, p = 0.0026) and Block et al. 2023 with an 11.36% allele frequency in CVID when compared to the Non-Finnish European population in the gnomAD database (OR = 1.316, p = 0.0145) and to an internal cohort of controls (OR = 1.316, p = 0.0458). Exact population composition matching is critical, as the frequency of this variant is highly variable even in Europe. We found the allelic frequency of BAFF-R p.Arg21Pro to be 10% from 90 non-familial CVID patients from the University of Utah Health Sciences Center, similar to Pieper et al. (10.2%) and Block et al. (11.36%), but comparing our Utah CVID patients to the 1000 Genomes project Utah Residents (CEU) with Northern and Western European Ancestry, which matches our CVID cohort (9.6% allelic frequency); we obtained an OR of 1.0468 (95% CI: 0.5309 to 2.0638; p = 0.8950). For further studies on BAFF-R p.Pro21Arg, we recommend closely matching the reference populations, and not using a broad European or other control populations. Topic Categories Translational and Interventional Immunology (TI)

Abstract Description Late-onset neonatal sepsis (LOS) is often the result of systemic bacteremia developing between 3-90 days of age, predominantly in prematurely born populations. An initial spike of IL-6 serum levels is a hallmark diagnostic tool and has been implicated as a source for the resulting cytokine storm. Yet, other than antibiotics, there is a lack of effective therapeutics targeting the overwhelming inflammatory response. We performed single-organism infections of pathogenic and commensal clinical E. coli isolates to compare the acute cytokine response in our LOS model in neonatal (postnatal day 5-7) and periweaning (postnatal day 17-20) pups. As expected, neonatal pups had increased bacterial burden and mortality during pathogenic infection compared to periweaning-age and commensal infection. An in-depth, 32-cytokine analysis of serum, liver, and spleen revealed overall elevated and tissue-specific inflammation in the neonates with a positive correlation of IL-6 serum levels to many cytokines (MCP-1, M-CSF, MIP-1β, etc.). Isolate-specific responses were assessed in an in vitro co-culture model with RAW-blue macrophages. While both isolates induced inflammatory NFκB/AP-1 activation, commensal E. coli caused elevated IL-1β while pathogenic E. coli led to significantly elevated IL-6. Both age- and pathogenic-specific inflammatory activation suggest unique signaling from the E. coli infection and the neonatal response, calling for neonatal-specific treatment options. Funding Sources Mayo Clinic Graduate School of Biomedical Sciences R01 DK134366 (Knoop) T32 DK124190-04 (Muske) Topic Categories Microbial, Parasitic, and Fungal Immunology (MPF)

Abstract Description Neutrophils heavily infiltrate the brain following acute and chronic brain insults. Neutrophils infiltrating brain tumors display a prolonged lifespan compared to blood neutrophils. We investigated presence and potential function of neutrophils in the brain following infection with Theiler’s Murine Encephalomyelitis (TMEV) and kainic acid-induced seizures. Both total myeloid cells and neutrophils started infiltrating the brain as early as day 1, peaked at day 3, and remained elevated at least 7 days post-infection. Further characterization of the neutrophils revealed upregulation of MHC-II, indicating a potential role in antigen presentation. We detected similar accumulation of total myeloid cells and neutrophils in the brain of mice following seizures. MHC-II expression was upregulated on infiltrating neutrophils. Given the sustained and unusual accumulation of neutrophils in the brain past innate time points, we evaluated the bone marrow niche for evidence of myelopoiesis. Frequencies of Lin-Sca-1+c-Kit + (LSK) were significantly higher in all bone marrow compartments in mice infected intracranially with TMEV. This increase in LSK cells concurred with an increase in frequencies of total myeloid cells. These changes were not observed when mice were infected with TMEV systemically. Our data highlights dysregulation of bone marrow hematopoiesis and a potential novel role of neutrophils in response to neuroinflammation following intracranial pathologies. Topic Categories Neuroimmunology (NEUR)

Abstract Description Broad spectrum antibiotics are being increasingly used in obstetric patients without long term follow up on neonatal risks. Previous data have suggested antibiotics for preterm premature rupture of membranes increases neonatal late onset sepsis and nectrozing enterocolitis (NEC), but the mechanism is unknown. We analyzed samples from the Mechanisms of Maternal Immunoglobulin A control over the neonatal microbiota and the development of NEC (MAMI) trial. MAMI enrolled infants born under 32 weeks and collected maternal-infant biospecimens. We performed a detailed chart review for maternal antibiotic exposure and analyzed breast milk samples collected 10-45 days post delivery by ELISA. Data were compared by t test and one way ANOVA. Breast milk IgA was higher than IgG and IgM (p < 0.0001, p < 0.005). When compared by antibiotic exposure, IgA and IgM were decreased with any antibiotic exposure (p = 0.02, p = 0.01). IgA was decreased by length of antibiotic exposure (p = 0.02). We stratified by the antibiotic spectrum of activity into narrow (penicillin, ampicillin) and broad (2nd and 3rd generation cephalosporin, gentamicin, etc) and found that the spectrum of antimicrobial activity was associated with a decrease in maternal IgA with broad spectrum antibiotics with the least amount of IgA (p < 0.004). Our data demonstrate mammary IgA and IgM are decreased with antenatal antimicrobial exposure. These data suggest a mechanism by which antenatal antibiotics may lead to neonatal sepsis and NEC. Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description The two key determinants of successful vaccines are the ability to induce a protective immune response and a long-lived immune memory. The mechanism determining durability and its contribution to the lower vaccine efficacy in older adults is elusive. We use varicella zoster virus (VZV) vaccination as a model that relies on cellular, not humoral immunity. We compared long-lived antigen-specific memory T cells in adults who were vaccinated at young (<20 years) or older age (>50 years) with a life-attenuated vaccine that confers sustained protection only in young age, or who received an adjuvanted subunit vaccine that is protective at older age. We stimulated PBMCs of vaccine recipients years after vaccination and collected VZV-reactive T cells for spectral flow cytometry and single-cell sequencing. While CD4 T cells were resilient to aging, VZV-specific CD8 T cells lost diversity and lacked stem-like features while gaining end-differentiated, NK-like signatures. These age-associated changes in CD8 T cells were not reversed with the effective subunit vaccine. Instead, this vaccine induced pro-inflammatory VZV-specific TH17 cells. TH17 cells in the less efficient vaccine response, albeit present, were skewed towards a FOXP3+ regulatory phenotype and deficient in TH1 polyfunctionality – a phenotypic shift that is in part controlled by lipid metabolic pathways. Our study uncovers traits of T cell dysfunction with age and informs vaccination efforts tailored towards healthy aging. Funding Sources NIH U19AI057266, R01AG045779, R01AI108891, R01AI129191, T32AG049672, Glenn Foundation for Medical Research Postdoctoral Fellowship, Robert and Arlene Kogod Center on Aging Career Development Award. Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)

Abstract Description Evidence consistent with immunoediting in human cancer has been identified through associative studies. To provide direct evidence that differences in the neoantigen profile are immune-mediated, we compared the neoantigen profiles of cutaneous squamous cell carcinoma (cSCC) from immunocompetent and immunosuppressed patients. Despite consistency in the overall mutational burden and signature, immunocompetent patients have a lower clonal mutational burden compared to immunosuppressed patients. Furthermore, clonal mutations in cSCC from immunocompetent patients have a lower proportion of neoantigens predicted to bind MHC class I, supporting the role of the immune system in shaping the neoantigen profile. In single-cell RNA sequencing of intratumoral CD8+ T cells from a separate cohort of cSCC from immunocompetent and immunosuppressed patients, we observe an increase in exhausted CD8+ T cells in tumors from immunocompetent patients, suggesting an explanation for the persistence of binding neoantigens subclonally. Finally, we compare clonal and subclonal neoantigens as an unbiased screen for characteristics associated with neoantigen immunogenicity. Neoantigens with a large increase in predicted MHC class I binding relative to the normal peptide are enriched in subclonal neoantigens in immunocompetent patients. Overall, this work supports the immune system’s role in sculpting the neoantigen profile of cSCC. Funding Sources Supported by American Cancer Society - Simone Charitable Foundation - Discovery Boost Grant, AZ-DBG-23-1151311-01-IBCD; NIH F30CA281056; R35GM149235 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Cellular immune responses are critical for protection from environmental and genetic insults, including pathogens and cancer. However, immunity can also drive pathogenic responses that damage organs and drive pathogenesis. Immune response quality is influenced by several factors including genetics, age, sex and the environment. Environmental influences including everyday activities and lifestyle have been shown to modulate the immune system but are often difficult to monitor. We have developed a system of serial monitoring of peripheral blood mononuclear cells that coincided with wearable device activity monitoring to further understand how biometric data correlates with the composition of blood derived immune cells. Using a 31-marker CyTOF phenotyping panel we monitored blood derived immune cell composition over a 14-week period in an individual volunteer while biometric data was collected via wearable technology. To determine whether interventions can be introduced that change peripheral blood immune cell phenotypes our volunteer introduced lifestyle changes at specific intervals in this period of monitoring. These included intermittent fasting and the introduction of regular exercise. In addition, the individual was immunized against COVID-19 during this time to provide an immune challenge that would perturb immune homeostasis. In this study, we find that lifestyle changes can promote alterations in immune cell phenotypes identified using high parameter mass cytometry. Topic Categories Translational and Interventional Immunology (TI)