Citations

Abstract Description In the tumor microenvironment of glioblastoma, myeloid cells act as a double-edged sword—they are a major cellular component suppressing the immune response while serving as a promising therapeutic target. Our study sheds light on the voltage-gated proton channel Hv1, predominantly expressed in myeloid cells, as a pivotal regulator of their physiological functions. Elevated Hv1 expression correlates strongly with poor prognosis in glioblastoma patients, underscoring its clinical significance. Using a glioma mouse model, we demonstrated that Hv1 depletion markedly extended survival. To overcome the limitations of existing tools, we engineered novel transgenic mouse lines optimized for Hv1 research. Our findings revealed tumor-induced Hv1 upregulation, with glioma-associated macrophages identified as the principal contributors. Crucially, we identified that Hv1 in infiltrating macrophages as the major driver of survival phenotype differences. Through a multi-faceted approach combining in vivo two-photon imaging, spectral flow cytometry, and spatial transcriptomic sequencing, we discovered that Hv1 depletion limits macrophage infiltration and enhances antigen presentation, ultimately fostering a stronger adaptive immune response. These findings establish the Hv1 channel as a crucial new immune regulator within the brain tumor milieu, offering a promising target for reprogramming macrophage function to combat glioblastoma. Funding Sources Supported by NIH R35NS132326 (LJW); Mayo Clinic Cancer Center and Mayo Clinic Center for Biomedical Discovery. Topic Categories Neuroimmunology (NEUR)

Abstract Description Measles is a highly contagious virus that presents as fever, cough, and a rash that spreads from the face to the rest of the body. Other serious complications, such as pneumonia, deafness, and encephalitis, can occur in immunocompromised and unvaccinated populations. Although there is an effective vaccine, there are currently no FDA-approved antivirals for measles. Moreover, there has been an increased incidence of measles outbreaks in the United States. Two main targets for measles virus antiviral therapies include its surface glycoproteins, hemagglutinin (H) and fusion (F), which mediate attachment and fusion of the viral and cellular membranes, respectively. After binding of the H-protein to SLAM or nectin-4 on the host cell surface, the F-protein undergoes a conformational change to initiate a fusion event. Using a hybridoma-based approach, we isolated a panel of human monoclonal antibodies from the peripheral blood B cells of vaccinated or naturally infected measles donors that bind either the H or F proteins. These antibodies potently neutralize against a pseudotyped vesicular stomatitis virus expressing measles H and F proteins in vitro by inhibiting viral attachment or fusion. Structural studies will identify critical contact residues between these antibodies and their respective antigens and inform mutational escape assays for likely sites for immune evasion. The protective efficacy of these antibodies will also be assessed using a cotton rat in vivo model. Topic Categories Vaccines and Immunotherapy (VAC)

Abstract Description Follicular Lymphoma (FL) is a germinal center-derived malignancy representing approximately 20% of all non-Hodgkin’s lymphomas. Despite a relatively indolent disease course, FL is a biologically and clinically heterogeneous disease which poses significant clinical challenges due to its propensity for relapse and transformation into aggressive and untreatable forms of lymphoma. Here, we have leveraged computational techniques with WES, RNA-sequencing, and immune profiling data from FL tumors to gain further insight into the biologic basis for FL heterogeneity. We performed computational reconstruction of the B cell receptor repertoire on B cell sorted RNA-sequencing data from normal lymph nodes (n = 5) and FL tumors (n = 89). To gain a better understanding of FL B cell biology at the single cell level, we further investigated the clonal architecture of dominant malignant clonotypes in FL tumors using computational analysis of paired single cell V(D)J and 5’ GEX sequencing data from FL tumors (n = 21) and non-malignant lymph nodes (n = 3), allowing us to characterize the germinal center programs in malignant versus non-malignant B cells. Our data suggest that a subset of FL tumors consist of complex polyclonal B cell populations, some of which represent a pre-malignant FL precursor. Our data provide a deepened understanding of FL biology, including precursor cells, and contributes a refined transcriptomic characterization of malignant and non-malignant B cells in Follicular Lymphoma. Funding Sources This work was supported by the National Institutes of Health (R01 CA212162-01A1 to A.J.N and J.R.C., and P50 CA097274 to J.R.C., and A.J.N) Topic Categories Computational and Systems Immunology (COMP)

Abstract Description Sequencing antigen-specific T cell receptors (TCR) remains a major technologic challenge, requiring labor- and cost-intensive single-cell approaches. We have developed a rapid and economical method for sequencing TCR mRNA from antigen stimulated peripheral blood mononuclear cells using targeted long-read nanopore sequencing with Oxford Nanopore Technologies’ (ONT) Minion sequencer. Targeted PCR amplification of TCR alpha and beta cDNA was performed with a template switching oligo (TSO). Dual unique molecular identifiers (UMI) were used for single cell, oligoclonal, or bulk cell sample specific sequence identification followed by nanopore sequencing. The reads were demultiplexed by UMIs and aligned to germline V,D, and J gene sequences from the IMGT database for identification. Error corrected sequences were then used to determine the non-templated complementarity determining region 3 (CDR3) sequence for the TCRs. From bulk PBMCs, TCR Beta Chain had 796 unique TRBV-TRBJ combination and TCR Alpha Chain had 2053 unique TRAV-TRAJ combinations. Human PBMC samples were spiked with 10% Jurkat cells and sequenced using this method for 12 hours resulting in 200K reads. After read demultiplexing, filtering (Q > 25), and alignment, it was found that 8.8% of TCR beta and 7.0% TCR alpha reads matched the Jurkat TCR sequence in the 10% spiked sample. This method provides a fast and cost-effective approach to generating antigen specific TCR sequences for downstream functional studies. Funding Sources Supported by NIH/NCI 5U01CA281660 Topic Categories Technological Innovations in Immunology (TECH)

Abstract Description Background Primary sclerosing cholangitis (PSC) is an immune-mediated liver disease characterized by chronic inflammation and fibrosis of the bile ducts. The primary cells impacted in PSC are bile duct epithelial cells called cholangiocytes. Mucosal associated invariant T (MAIT) cells are enriched in human liver and associated with fibrosis. We hypothesize that cholangiocyte-activated MAIT cells drive fibrosis in PSC. Results Flow cytometry of PSC patient bile duct brushings show that 30% of T cells are MAIT cells, suggesting their co-existence with cholangiocytes within the peri-biliary environment. Re-analysis of single cell RNA sequencing data of PSC patient cholangiocyte organoids reveals upregulation of MR1 gene, indicating their enhanced ability to activate MAIT cells. Proteomics of human MAIT cells identify the expression of multiple profibrotic factors including secreted protein acidic and rich in cysteine (SPARC). Flow cytometry of TCR-activated MAIT cells show a significant and dose-dependent increase in a SPARClow MAIT population, indicating release of SPARC. Re-analysis of Olink proteomics on supernatant of PSC cholangiocyte organoids show IL-17C secretion. IL-17C treatment of TCR-activated MAIT cells result in significant upregulation of SPARC gene. Conclusion MAIT cells secrete profibrogenic mediators and MAIT cell profibrogenicity is enhanced by IL-17C, a novel cytokine released by cholangiocytes in PSC. Funding Sources C-SIG P30DK084567 Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description Immune responses to allergens vary depending on the route of sensitization, but the mechanisms behind these differences are not clear. Damage signals from the microenvironment dictate the immune response magnitude and may regulate route of sensitization differences. Extracellular ATP (eATP) is a danger signal released by inflamed tissues and sensed by immune cells via P2RX7. The role of P2RX7 in CD4+ Th2 cell responses to allergen is unclear, with past reports suggesting opposite effects of P2RX7 knockout (KO). Here, we tracked the role of P2RX7 on CD4+ T cell-mediated lung allergy in response to different routes of sensitization. Comparing Papain + Ovalbumin (OVA) intranasal versus subcutaneous sensitization, we found increased lung type 2 inflammation and lung CD4+CD69+GATA3+ T cells in response to the subcutaneous route, compared to intranasal route. P2RX7-KO led to decreased lung CD4+ Th2 cell responses and reduced inflammation in mice exposed to subcutaneous, but not intranasal sensitization. Subcutaneous OVA + Papain led to a stronger eosinophilic response, while both eosinophils and neutrophils were elicited by intranasal route. Our results indicate that sensing of eATP through P2RX7 exacerbate the intensity of subcutaneous allergen-induced Th2 responses. We are currently comparing the Th2 responses induced by subcutaneous versus intranasal allergen sensitization, and how P2RX7 oppositely regulate such responses. Funding Sources NIH K99/R00A139381 NIH R01A170649 Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Abstract Description Infants are vulnerable to infectious diseases due to the immaturity of their immune system, including a delay in producing pathogen-targeting antibodies. Maternal immunization strategies have increased in use to protect infants from disease by vertical transfer of protective pathogen-specific antibodies. Late-onset sepsis (LOS) is a substantial contributor to neonatal mortality that results from a bloodstream infection at least 72 hours after birth. The implicated pathogen is often a member of the intestinal microbiome before systemic infection, suggesting translocation from the intestine as the path to the bloodstream. We hypothesized that maternal immunization of animals prior to pregnancy could protect nursing pups in a murine model of LOS. Following maternal immunization against Enterobacteriaceae taxa implicated in sepsis, we observed significantly decreased pup mortality following an oral challenge with Escherichia coli. This protection was associated with increases in antigen-specific IgG and IgA in both the mammary tissue of vaccinated dams and stomach contents of nursing pups. Interestingly, we did not observe differences in the infant microbiota following maternal vaccination, suggesting increased pathogen-specific IgG and IgA did not perturb the developing microbiota. This work suggests maternal immunization may be useful to protect offspring from enteric pathogens, and further supports the role of human milk and human milk factors in protection from LOS. Funding Sources Combating Antibiotic Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) Topic Categories Vaccines and Immunotherapy (VAC)

Abstract Description The dura mater of the meninges recruits immune cells for CNS surveillance and response to infections, serving as a critical interface between the brain and the peripheral immune system. Theiler’s murine encephalomyelitis virus (TMEV) infection is a model neurotropic virus for which MHC class I molecules are critical for sterilizing immunity. Using spectral flow cytometry, we profiled the immune response in the dura mater in healthy and TMEV-infected wildtype mice. Acute TMEV infection resulted in the infiltration of diverse immune cell types into the dura mater, including increased cDC1, pDCs, monocytes, NK cells, CD4 T cells, and CD8 T cells. We next examined the importance of MHC class I molecules on diverse immune cell populations in the dura mater during acute TMEV infection. MHC class I deficiency resulted in increased numbers of CD4 T cells with high expression of PD1 in MHC class I-/- (H-2Kb-/-, H-2Db-/-) mice compared to WT mice 7 days post TMEV infection. This demonstrated the importance of MHC class I and CD8 T cells in modulating dura immune cell populations and implies a compensatory shift toward a CD4 T cell response during acute infection. Subsequent studies employing in vivo cell-specific ablation of MHC class I molecules in discrete cell types will further define the impact of antigen presentation on immune regulation and inflammatory outcomes within the dura mater. Topic Categories Neuroimmunology (NEUR)

Abstract Description Chronic infection-associated inflammatory arthritis is a poorly understood phenomenon and varies by the associated infection. Infections such as human immunodeficiency virus (HIV), hepatitis B and C, lyme disease, cytomegalovirus, and numerous others can not only cause patients to develop arthralgias, but synovial inflammation with inflammatory infiltrate. Based on our prior studies looking at HIV-infected humanized mouse models, there is significant inflammation that develops in the synovial tissue with elevated levels of GM-CSF, TNF, and IL-17. Our current studies are now focused on studying a diverse group of patients with chronic infections that developed inflammatory arthritis and look for patterns of immune cell distribution and cytokine profiles between the various infections. Patients were compared with healthy controls as well as patients with rheumatoid arthritis who tested negative for these infections. Increased inflammatory cells were seen in patients with chronic infections in the serum which was similar to the rheumatoid arthritis patients. Uniquely, elevated levels of GM-CSF were seen in the patients with HIV and hepatitis B and C that correspond with the prior mouse model data. Our data will help provide additional insight into not only the immunology and pathology associated with various infections, but insights into the development of various forms of inflammatory arthritis. Funding Sources Mayo Clinic Benefactor-funded Early Career Development Award in Rheumatology Topic Categories Immune Mechanisms of Human Disease (HUM)

Abstract Description Antigen receptor (CAR) T cells can be generated in vivo within the natural lymph node (LN) environment as the immune response to a virus infection unfolds. This in situ approach entails priming LNs with intradermal inoculations of replication defective adenoviruses encoding MHC class I and class II allo/xeno-antigens. Universal transplant antigens were devised for LN priming by introducing tryptophan into amino acid position 162 of the MHC HLA-A2:01 class I heavy chain and by using the mouse MHC class II beta chain to form chimeric class II antigens with endogenous class II alleles. The virus encoded mismatched MHC antigen presenting molecules are expressed functionally in the LN following intradermal inoculation provoking polyclonal T cells responses. Three days later CAR T cells are generated by directly injecting retroviruses encoding CARs of interest into the responding LN. Mouse CD19-specifc CAR T cells created in this manner in B6-DQ8 and DQ8/A2 mice effectively target endogenous B cells and are therapeutic in male and female Emu-myc lymphomas demonstrating the potential for the generating functional CAR T cells directly in vivo. While in situ CAR T cells are readily produced in B6, B6-IA0/DQ8, B6-IA0/DQ8/A2, and B6-IA0/DQ6 LNs, functional differences among the strains emerged in the periphery suggesting that properties of the autoimmune-associated HLA DQ8 class II allele is particularly reactive during the production of CAR T cells in vivo. Funding Sources MustangBio, Inc Mayo Clinic Center of Biomedical Research Mayo Clinic Department of Immunology Mayo Clinic Graduate School of Biomedical Science Fraternal Order of Eagles Mayo Clinic Ventures NIH R21 AI 097337 NIH R01 CA 178804 NIH T32 AI 07425 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)