Citations

Abstract Description Glioblastoma (GBM), the most common and malignant primary brain tumor, exhibits profound immune suppression, hindering immunotherapy efficacy. GBM-derived extracellular vesicles (EVs) play a major role through the immunosuppressive polarization of myeloid cells. Inhibition of EV-myeloid cell interactions thus represents a novel target to mitigate GBM-mediated immune suppression. However, there are multiple mechanisms of EV-target interaction, and downstream pathways of GBM-EV signaling are poorly understood. We explored two pharmacological agents targeting different mechanisms of EV uptake: Methyl-B-cyclodextrin (MBCD), which disrupts lipid rafts, and cytochalasin D, which inhibits actin polymerization. Our results showed immunosuppressive myeloid cell (CD11b+/ CD15-/ HLA-DRlow/ CD14high) populations increased upon GBM-EV treatment. Following cytochalasin D treatment, fewer immunosuppressive myeloid cells were observed (7.40%±1.01 vs. untreated 9.66%±2.86), with rescuing T cell proliferation. Conversely, MBCD treatment increased immunosuppressive myeloid cell levels (10.42%±2.38 vs. untreated 8.87%±3.97) without impacting T cell proliferation. This study demonstrates the feasibility of pharmacologically targeting EV-myeloid cell interactions to rescue tumor-mediated immune suppression by inhibiting actin-dependent endocytosis with cytochalasin D. The converse results using MBCD treatment indicate differential importance of different EV uptake pathways in monocyte regulation. Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Description Although contact-dependent, perforin-mediated cytotoxicity is crucial for T cell-mediated immunity, it is an inefficient method of eliminating unwanted cells. This is because the directional release of cytotoxic granules across the immunological synapse allows only one target cell to be killed at a time. This serial killing approach is likely insufficient at controlling rapid infections or an influx of malignant cells. One explanation for the poor effectiveness of CAR-T cells at treating solid tumors may be that the low efficiency contact-dependent killing of target cells require high effector:target (E:T) ratio may not be attainable clinically. Thus, developing strategies that enable T cells to kill tumor targets at a higher efficiency (i.e., lower E:T ratio) is warranted. Using a murine CAR-T system, we observed a direct role for T cell-derived cytokines in killing tumor target cells. As these effector molecules are diffusible, we found that this killing did not require effector-to-target contact but rather killed in a “remote” manner. Furthermore, we found that remote killing can be substantially improved if we inhibit TNF signaling molecules in the target cells that are often disabled by pathogens. Thus, tumor targets that are deficient in TAK1, HOIP or TBK1/IKKε can be killed by CAR-T cells at low E:T ratios. This work suggests that concurrently inhibiting certain TNF signaling molecules may be a strategy to augment CAR-T cell therapy for improved clinical outcome. Funding Sources T32A170478 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Description Fungal species, such as Candida albicans, are commonly found as part of the normal microbiota in both the skin and intestine. However, these fungal species, including C. albicans, can also become opportunistic pathogens and have been associated with intestinal and allergic disorders. We hypothesized C. albicans colonization could occur in early life when the neonatal microbiota is permissive to pathogens and lacks colonization resistance compared to later times in life. To investigate this question, we sequenced stool of premature and term infants and frequently found C. albicans colonization, suggesting fungal colonization within the human GI tract. To investigate the impact of long-term C. albicans colonization to the immune system, we established colonization by oral administration of C. albicans to neonatal mice. Colonization of C. albicans was associated with increased diversity of bacterial species in the intestinal microbiota at weaning. Additionally, we observed significant increases of inflammatory cytokines IL-17 and G-CSF in the serum. Future research will expand on the impact of C. albicans colonization on the intestinal microbiota and immune systems. Funding Sources Center for Individualized Medicine Microbiome Program Research Team Science Award, Mayo Clinic NIH, NIDDK R01 DK134366 Topic Categories Mucosal and Regional Immunology (MUC)

Abstract Description Glioblastoma (GBM) is an aggressive brain cancer with poor survival. GBM patients have severe peripheral immunosuppression, including defective T cell responses. We showed that serum-derived factors were sufficient to inhibit T cell proliferation. This factor was nonsteroidal and larger than 100kDa. Mass-spectrometry and pathway analysis of suppressive serum implicated proteins involved in DNA response elements. We hypothesized that the suppressive factor is cell-free DNA (cfDNA). In support of this, serum levels of cfDNA are increased in glioma-bearing mice. Exposure to cfDNA from serum of glioma-bearing mice was sufficient to directly and potently inhibit T cell proliferation. Exposure to genomic DNA was not sufficient to induce proliferation defects in T cells, highlighting the unique features of cfDNA. Importantly, reducing cfDNA content using DNaseI was sufficient to partially restore proliferation capacity. We determined that the T cell proliferation defects were independent of AIM2 DNA sensing mechanisms, implicating a novel pathway of DNA sensing in T cells. Finally, to determine the origin of the cfDNA, we used methylation-seq and found that the majority of cfDNA originated from neutrophils, implicating NETosis. Together, we contend that cfDNA from neutrophils induces immunosuppressive effects in GBM. Our work highlights a potential role for targeting cfDNA, neutrophils, and/or NETs in order to reverse T cell dysfunction and improve patient outcomes in GBM. Funding Sources R00NS117799 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Hematopoiesis declines with age due to intrinsic and extrinsic dysregulation resulting in diminished lymphopoiesis. Hoxa9 is a pleiotropic hematopoietic regulator necessary for stem/progenitor cell (HSPC) maintenance and lymphoid differentiation. Interestingly, bone marrow (BM) HSPC and B lineage compartments of young hoxa9-/- mice mimic that which accompanies aging. Hoxa9 is a negative regulator of p16ink4a, a cyclin-dependent kinase inhibitor upregulated in hoxa9-/- HSPCs and a gatekeeper of the G1 to S cell cycle checkpoint. We hypothesized that increased expression of p16ink4a contributes to the HSPC and/or B progenitor alterations due to hoxa9-deficiency. Interestingly, BM B lymphopoiesis was normalized in hoxa9-/-p16ink4a-/- (DKO) mice, but alterations were less obvious in HSPCs. Flow analysis of the HSPC compartment proliferation showed contraction of unbiased but not myeloid-biased progenitors prior to lineage fate commitment. hoxa9-/- CFU-PreB were blunted then rescued by p16ink4a co-ablation. Primary BM transplant demonstrates DKO like hoxa9-/- has reduced reconstitution efficiency but differs with superior B lineage reconstitution similar to controls. Bioinformatic analysis of recent open access CUT&RUN data in HSPCs confirms Hoxa9 binds promoters in genes relevant to G1 to S cell cycle checkpoint, these findings currently being validated. Our work emphasizes HSPC regulatory factors as crucial targets in combating age-associated maladies of the immune system. Funding Sources Supported by NIH R01AG076163 Topic Categories Hematopoiesis and Immune System Development (HEM)

Abstract Description B7-H3 is a member of the B7 superfamily, which includes co-inhibitory receptors such as B7-H1/PD-L1 and co-stimulatory receptors such as B7-1/CD80 and B7-2/CD86. B7-H3 is upregulated by almost all types of tumors and is associated with worse prognosis. However, the exact mechanism by which B7-H3 suppresses anti-tumor immune response remains to be discovered as its ligand has yet to be identified. Sialic acid modifications are also increased in many cancers and are similarly associated with poor outcome. Here, we demonstrate for the first time that sialylated B7-H3 binds to the immune inhibitory receptor Siglec-9. Siglec-9 is expressed on many immune cells, including NK cells and T cells. We have shown that B7-H3 expression on target cells reduces killing by Siglec-9 expressing NK cells in vitro. We then hypothesized that antibodies that block B7-H3 binding to Siglec-9 will be an effective intervention to boost anti-tumor immunity. We have generated a high affinity anti-B7-H3 monoclonal antibody, F7, which blocks B7-H3 binding to Siglec-9. F7 functions to enhance NK cell killing of B7-H3 expressing target cells in vitro. F7 also dramatically enhances CART19 mediated killing of B7-H3 expressing Nalm6 cells in a relapse model in NSG mice in vivo. In summary, our work has demonstrated that B7-H3 binds Siglec-9, and blockade of such interaction can enhance anti-tumor immunity. Funding Sources • Mayo Clinic Center for Biomedical Discovery • NIH Glycobiology Common Fund supplement to R01CA243545-01A1 to VSS Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Abstract Description Early oral route antigen exposure decreases the incidence of food allergy while environmental exposures through non-oral routes, such as the skin or airways, increase the risk. The goal of this study was to investigate the immunologic mechanisms of how oral allergen consumption protects from sensitization to the same allergens through environmental exposure routes. Naive mice were orally administered ovalbumin (OVA) in drinking water. Mice were subsequently exposed intranasally (i.n.) or epicutaneously (e.c.) to OVA plus peanut flour, as an adjuvant. OVA-specific CD4+ T cells from draining lymph nodes (dLNs) were assessed by MHC-II tetramer staining and flow cytometry. Following i.n. or e.c. exposure to OVA plus peanut flour, mice developed OVA-IgE and OVA-IgG1 and showed symptoms of acute anaphylaxis upon OVA challenge. Oral OVA feeding prior to i.n. or e.c. antigen exposure dramatically reduced OVA-IgE titers and protected mice from anaphylaxis. Protection was OVA-specific, as OVA-fed mice still developed peanut-IgE and anaphylaxis upon i.p. peanut challenge. Total OVA-specific CD4+ T and CD4+Foxp3+ T regulatory (Treg) cell numbers in dLNs were similar between control and OVA-fed mice. However, the majority of OVA-specific CD4+ T cells in control dLNs were T follicular helper (Tfh) cells, which were nearly abolished in OVA-fed mice. Oral route antigen exposure suppresses development of antigen-specific Tfh cells in remote dLNs and prevents systemic allergic sensitization. Funding Sources Supported by NIH R37 AI71106 and Mayo Clinic Graduate School of Biomedical Sciences. Topic Categories Immediate Hypersensitivity, Asthma, and Allergic Responses (HYP)

Abstract Description Combination of radiotherapy (RT) and immunotherapy has gained popularity to turn up the heat on cold tumors. However, intrinsic resistance is prevalent, hence better therapeutic strategies are needed to improve the outcome of patients. We recently demonstrated in metastatic prostate cancer that circulating levels of tumor-derived extracellular vesicles (tdEVs) increased shortly after RT and was associated with poor survival. TdEVs are mediators of antitumor immunity but their role in response to RT remains unknown. Our study elucidates the impact of RT on immunomodulatory roles of tdEVs. Mouse tumors models expressing a bioluminescent EV reporter were treated with clinically used doses of RT. TdEV organotropism was measured by ex-vivo imaging at different timepoints. TdEVs were detected in blood, spleen, lymph nodes and immune cells. OT-1 transgenic mice and in vitro co-culture assays showed a reduction in CD8 T-cell cytotoxic activity in presence of tdEVs from irradiated cells. To identify key players of tdEV-mediated immunosuppression in response to RT, proteomic analysis revealed the immune checkpoint molecule B7-H3 was overexpressed in tdEVs after irradiation. Genetic deletion of B7-H3 inhibited the suppressive activity of tdEVs from irradiated cells resulting in enhanced T-cell cytotoxicity. This study describes tdEVs as a mediator of systemic immunosuppression in response to radiotherapy and B7-H3 as a potential therapeutic target to boost antitumor immunity. Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description Therapies targeting the PD-1/PD-L1 axis are among the leading treatments of cancer, but only a minority of patients benefit. Many therapies focus on preventing PD-L1 from binding to its receptor, PD-1, but do not address PD-L1’s ability to be recycled to the cell surface and any intrinsic functions it may possess. Our lab has developed an antibody (H1A) that binds to PD-L1 and prevents its association with a stabilizing protein, CMTM6, therefore targeting it for lysosomal degradation. Consequently, H1A limits any PD-L1 intrinsic function while also decreasing interactions with PD-1. Here, we use human-derived immune cells to test H1A’s effect on immune responses through flow cytometry, multiplex screening, and cytotoxicity assays. Additionally, we have developed a humanized PD-1/PD-L1 mouse model that allows us to test H1A’s effect on tumor growth inhibition in an immunocompetent host. Our results show that H1A specifically targets human myeloid cells, resulting in increased co-stimulation, antigen presentation, and cytokine secretion. H1A demonstrated enhanced T cell cytotoxicity in a whole PBMC culture. Finally, H1A resulted in greater tumor growth inhibition and survival in our in vivo tumor models. This work highlights the importance of targeting beyond PD-1/PD-L1 ligation and that myeloid cells are instrumental to enhancing the immune response against cancer. H1A is emerging as a new immunotherapy tool to target PD-L1 and CMTM6 in myeloid cells for treatment of cancers. Funding Sources NIH R01 92389022; NIH T32 AI170478; Mayo Clinic Graduate School of Biomedical Sciences Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description RNA- and DNA-binding proteins are crucial for regulating gene expression by affecting DNA accessibility, transcription, and repair, as well as RNA splicing, transport, and stability. Dysfunction, mislocalization, aggregation, and liquid-liquid phase separation of these proteins are common in neurodegenerative and neuroinflammatory diseases. TDP43 mislocalization is notably implicated in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. In 97% of ALS cases, the loss of nuclear TDP43 and the formation of cytoplasmic aggregates (TDP43 proteinopathy) in motor neurons are observed. This loss or mislocalization of TDP43 disrupts nucleic acid sequestration and may activate cytosolic DNA and RNA sensors such as RIG1 and cGas-STING pathways, leading to the expression of interferon-stimulated genes (ISGs). Evidence suggests that the ubiquitin-like protein ISG15 contributes to neuron loss. Using primary murine cortical neurons, human IPSC-derived motor neurons, and mixed neural cultures, we report the impact of TDP43 knockdown and mislocalization on neuronal interferon signaling and ISG15 expression. We also use ISG15-deficient neurons to measure ISG15-dependent impacts on neuron viability. Our findings show ISG15-dependent effects on cGas-STING signaling, astrogliosis, and neuron loss in an AAV-C9orf72 mouse model of ALS using global and conditional ISG15 KO mice. Funding Sources Supported by Dominium Foundation Career Development Award (Clarkson). Topic Categories Neuroimmunology (NEUR)