bluuebunny/crossref_metadata_2025 huggingface dataset:
KMT2D histone methyltransferase modulates chromatin accessibility during regulatory T cell development (LYM6P.714)
Authors: Placek Katarzyna , Cui Kairong , Hu Gangqing , Lee Ji-Eun , Wang Chaochen , Konkel Joanne , Zhang Dunfang , Chen WanJun , Ge Kai , Zhao Keji
Published: January 2023

Gene regulatory elements are associated with histone H3 lysine 4 (H3K4) methylation and H3 acetylation and DNase I hypersensitivity. Whilst much is known about changes in histone modification patterns and chromatin accessibility during T cell differentiation, little is known about the role of histone modifying enzymes in this process. KMT2D is a histone methyltransferase that shapes enhancer landscape by regulating H3K4me and H3K27ac. To determine whether KMT2D plays a role in T cell development, we deleted KMT2D in T cells. Our data show that KMT2D deficiency results in reduced number of T cells in periphery. Moreover in the absence of KMT2D, differentiation of regulatory T (Treg) cells is compromised. KMT2D co-localizes with active chromatin marks. We showed that DNaseI hypersensitive sites (HS) bound by KMT2D tend to decrease in accessibility upon KMT2D deletion in CD4+ T cells that leads to reduced expression of nearby genes. The decreased HS are linked to genes involved in Treg differentiation and are enriched in transcription factor (TF) binding motif important for CD4+ T cell development. We found that KMT2D co-localizes and interacts with TF in CD4+ T cells. KMT2D binds to Treg master regulator: Foxp3 upstream enhancer and imposes H3K4me1 and H3K4me2 at Foxp3 regulatory elements. Our data suggest that KMT2D modulates CD4+ T cell development by maintaining chromatin accessibility and thus enabling TFs binding at genes involved in Treg cell differentiation.
Survival advantage of E. coli in the inflamed gut is mechanistically linked to enterobactin-mediated inhibition of myeloperoxidase and regulation by lipocalin 2 (MPF4P.736)
Authors: Singh Vishal , Yeoh Beng San , Bachman Michael , Borregaard Niels , Joe Bina , Vijay-Kumar Matam
Published: January 2023

During an inflammatory response in the gut, several commensal bacteria such as E.coli not only survive but also thrive and consequently contribute to the disease pathogenesis. The mechanisms by which such opportunistic pathogens bloom despite the hostile pro-inflammatory milieu of the inflamed gut remain largely unknown. We demonstrated that enterobactin (Ent), a siderophore released by E. coli, is a potent inhibitor of a key bactericidal enzyme of the host innate immune system, myeloperoxidase (MPO). The inhibitory activity is specific as glycosylated Ent (salmochelin) and non-catecholate siderophores such as yersiniabactin and ferrichrome failed to inhibit MPO activity. An E. coli ferrienterobactin permease mutant (ΔfepA) overexpressing Ent, but not 3-dehydroquinate synthase (ΔaroB)/ΔfepA double mutant inhibited MPO activity and exhibited enhanced survival in inflamed guts. This survival advantage was counter-regulated by the host siderophore binding protein, lipocalin 2, which rescued MPO from Ent-mediated inhibition. Spectral analysis revealed that Ent interferes with compound I [oxoiron, Fe(IV)=O] and reverts the enzyme back to its native ferric [Fe(III)] state. These findings define a fundamental mechanism by which E. coli surpasses the host innate immune responses during inflammatory gut diseases and gains a distinct survival advantage.
The yype I IFN pathway in C. difficile infection: impact on morbidity and mortality in mice (MPF4P.737)
Authors: Snyder Deann , Hedges Jodi , Robison Amanda , Broadaway Susan , Walk Seth , Jutila Mark
Published: January 2023

Infections with C. difficile are increasingly prevalent in individuals in health care settings and are becoming a concern in food animals. C. difficile is a gram positive, anaerobic bacteria known for its ability to form spores that are exceedingly difficult to kill. Disruption of the normal gut microbiota allows C. difficile spores to germinate, divide and produce toxins that compromise the intestinal barrier resulting in extreme inflammation. Currently, little is known about the innate immune response in Clostridium difficile infection (CDI). We hypothesized that type I IFNs may play a crucial role in innate protection from CDI. We found that IFNAR-deficient mice succumbed to CDI more rapidly compared to wild type mice. In addition, wild type mice treated with type I IFN-inducing dsRNA mimetic, polyIC, had delayed onset of symptoms compared to mice treated with saline. Since type I IFNs can block damaging inflammasome activity, this potential mechanism was explored. Wild type and IFNAR-deficient mice were treated with DMSO or the inflammasome-blocking agent, Glibenclamide. Glibenclamide-treated IFNAR-deficient mice faired better than all groups. This finding suggests that type I IFN signaling may protect from CDI in part by blocking the inflammasome. Considering that recombinant type I IFN is already used clinically, investigation of this pathway may present new treatment options for this emerging infection.
Inflammasomes recruit natural killer cells to prevent infection by a ubiquitous environmental bacterium (MPF4P.726)
Authors: Maltez Vivien , Tubbs Alan , Aachoui Youssef , Cook Kevin , Whitmire Jason , Miao Edward
Published: January 2023

Our lab has shown that inflammasome-triggered pyroptosis ejects microbes from replication niches in macrophages, exposing them to neutrophil killing. Because bona fide intracellular pathogens must evade pyroptosis in vivo, the role of pyroptosis in defense has primarily been observed in pathogens deprived of evasion strategies. To identify naturally occurring bacteria that are cleared via pyroptosis, we considered pathogens that are specific to chronic granulomatous disease (CGD), a neutrophil killing deficiency. We hypothesize that a subset of these microbes may be detected by inflammasomes and cleared via pyroptosis. In a survey of CGD pathogens, we discovered a ubiquitous environmental microbe, Chromobacterium violaceum, that is avirulent in WT mice (which resist a 1,000,000 CFU challenge), but is extremely virulent in inflammasome deficient mice (succumb to 100 CFU). This is the most penetrant inflammasome phenotype against any infection: bacterial, viral, fungal, or parasitic. Further, our studies with C. violaceum reveal a unique link between inflammasome driven IL-18 and NK cell cytotoxicity that functions in parallel to pyroptosis to eliminate intracellular replication niches. We posit that a subset of environmental microbes can be developed as powerful in vivo tools to study innate immune responses in the context of an appropriate and fully functional inflammasome response.
Simulated microgravity disrupts intestinal homeostasis and increases colitis susceptibility (MUC5P.763)
Authors: Ge Qing , Li Pingping , Shi Junxiu
Published: January 2023

The innate and adaptive immune systems can be altered by spaceflight in many aspects. But microgravity-related mucosal immune changes and its clinical significance has not been well studied. To investigate whether simulated microgravity influences the intestinal homeostasis and increases the susceptibility to colon inflammation, a hindlimb unloading (HU) microgravity mouse model was combined with dextran sulfate sodium (DSS)-induced colitis model. Compared to ground control mice, the HU ones revealed an impaired intestinal homeostasis and increased susceptibility to DSS-induced colitis. This includes an early change in intestinal microbiota, a decrease in regulatory T cell (Treg) numbers and IL-10 production, an increase in colonic IL-1β expression, an increase in circulating neutrophils and colonic neutrophil infiltration. The application of antibiotics ameliorated Treg and IL-10 reductions but did not significantly dampen neutrophilia and elevated expression of colonic IL-1β. These results indicate that the intestinal microflora and innate immune system both respond to simulated microgravity and together contribute to the proinflammatory shift in the gut microenvironment. The data provide useful insights in the changes of interactions between the hosts and microbiota under microgravity. It also emphasizes the necessity for evaluating the susceptibility of inflammatory bowel diseases in distant space travels.
Defining the immune regulatory function of LIGHT specific LTβR interactions in mucosal tissue. (MUC5P.747)
Authors: Zahner Sonja , Krause Petra , Kim Gisen , Morris Venetia , Tumanov Alexei , Fu Yang , Kronenberg Mitchell
Published: January 2023

Inflammatory bowel disease is characterized by an exacerbated inflammation of the intestine. While blocking tumor necrosis factor α (TNFα) helps many patients, it also causes significant side effects including a hampered host defense. TNFSF14/LIGHT, another member of the TNF superfamily, can bind to two receptors, HVEM and lymphotoxin β receptor (LTβR). Though LIGHT expression by T cells has been described to be inflammatory, we have recently shown that LIGHT interaction with LTβR is protective in murine colitis, indicating therapeutic promise for LIGHT. In absence of LIGHT-LTβR interactions, the innate immune system prolongs and exacerbates intestinal inflammation, with increased IL-1β and oncostatin M expression leading to increased IL-6. To define the cell-cell interactions leading to the excessive inflammation, we have analyzed mice with a conditional deletion of the LTβR. A dendritic cell-specific lack of the LTβR caused aggravated colitis compared to wild type but less severe colitis than in LIGHT-deficient mice. Thus, other LTβR-expressing cell types may be involved. To identify the cell types involved in the protective LIGHT-LTβR interaction in mouse colitis, we generated conditional mutants of LIGHT and are currently analyzing these mice. Intriguingly, mice lacking lymphotoxin β (LTβ), a subunit of the other ligand for the LTβR, were protected from severe disease. These data suggest that there are antagonistic effects of LIGHT and LTβ engagement of the LTβR.
Parenteral immunization with double-mutant heat labile toxin engages CD103+ dermal dendritic cells and redirects T cell immunity to the gut mucosa. (MUC5P.751)
Authors: Frederick Daniel , Cunningham David , Freytag Lucy , McLachlan James
Published: January 2023

The majority of infectious agents enter via mucosal compartments. Despite this, most current vaccines are administered by parenteral injection, which induce limited mucosal T cell responses. It is known that certain adjuvants can induce mucosal antibody responses regardless of route; however, there is minimal evidence that these injections direct antigen-specific effector T cells to the mucosa, where they are likely needed for relevant protection. In the current study we examine the potential of dmLT (double-mutant heat-labile toxin), a detoxified adjuvant derived from the mucosal human pathogen enterotoxigenic Escherichia coli, to drive antigen-specific T cells to the intestinal mucosa after parenteral immunization. By using a combination of antibodies and tetramers specific for a known peptide:MHCII, we can track intradermal delivery of dmLT plus antigen, with regards to antigen-presentation and the antigen-specific CD4 T cell response. We show this immunization results in T cell upregulation of the gut mucosal homing marker, α4β7 in local draining lymph nodes, mucosal lymph nodes, colon, and small intestine when compared to immunization with CpG plus the same antigen. We also observed an increased frequency of CD103+ dendritic cells in response to dmLT administration that were presenting the injected antigen. Lastly, we determined that dmLT imparts a balanced Th1/Th17 cytokine response and is capable of providing protection against oral Salmonella typhimurium challenge.
GMSCs suppress osteoclastogenesis and bone erosion in collagen-induced arthritis through curbing NF-KB and RANKL signals (THER5P.900)
Authors: Zheng Song , Gu Jian , Wang Julie , Olsen Nancy
Published: January 2023

Osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA) and gingiva-derived mesenchymal stem cells (GMSCs) can inhibit experimental collagen-induced-arthritis (CIA) model. This study aims to determine if GMSCs also suppress osteolastogenesis and bone erosion in CIA. Osteoclasts were induced from bone-marrow CD11b+ cells with RANKL and macrophage colony-stimulating factor (M-CSF), and assessed with tartrate-resistant acid phosphatase (TRAP) staining. Human osteoclasts were induced from human CD14+ cells. GMSCs were generated and added to cultures with different ratios with CD11b+ or CD14+ cells. NF-κB activation was determined by western blot. 2×106 GMSCs or fibroblast cells were adoptively transferred to DBA1/J mice on day 14 after immunization with CII/CFA. CIA onset and severity were monitored and bone erosion was examined by HE staining. We showed that GMSCs but not fibroblast cells completely suppressed osteoclastogenesis in vitro for human and mice cells. GMSCs injected after immunization and before of onset of CIA significantly suppressed disease development. Treatment with GMSCs dramatically decreased the levels of NF-κB p65/p50 in osteoclasts in vitro and P65/50 and RANKL expression by synovial tissues in vivo. These results suggest that the manipulation of GMSCs may have therapeutic effects on rheumatoid arthritis and other bone erosion related diseases.
Computational modeling of transplant rejection: a systems view of the dynamic roles of T cells, antigen presenting cells, and cytokines during rejection (TRAN1P.941)
Authors: Raimondi Giorgio , Maturo Andrew , Brandacher Gerald , Arciero Julia
Published: January 2023

The quality of life of organ transplant recipients is compromised by complications that arise from ensuing life-long immunosuppression and by limited long-term graft survival due to absence of tolerance induction. There is then an urgent need to better understand the complex interactions of the immune system leading to transplant rejection, so to identify innovative strategies to promote transplant acceptance while preserving immune competence. In this study we have pioneered a systems biology approach in which an experimentally-based mathematical model is used to predict how alterations in the immune response influence the rejection of mouse heart transplants. The model correctly predicts that in the absence of CD4 T cells the transplant survives indefinitely, while in the absence of CD8 T cells it is quickly rejected, providing also dynamics of innate cells that fit recently published data. As expected, decreasing the migration of effectors to the graft delays transplant rejection. This latter relation is not monotonic as increasing the translocation of CD8 T cells has a beneficial effect. This unexpected result derives from the interconnections with the relative concentrations of pro- and anti-inflammatory cytokines and with the ratio between Treg and T cells. This suggests possible key points of intervention for a testable novel strategy of intervention that highlights the usefulness of this innovative multidisciplinary approach.
Aquaporin 4 blockade is a novel strategy targeting ischemia/reperfusion injury in heart transplantation (TRAN1P.942)
Authors: Ayasoufi Katayoun , Kohei Naoki , Farr George , Pelletier Marc , McGuirk Paul , Fairchild Robert , Valujskikh Anna
Published: January 2023

Aquaporin 4 (AQP4) is the major water channel in the brain and its absence reduces brain edema in murine models of stroke.Recently, human and mouse cardiomyocytes were shown to express AQP4. Our goal was to test whether therapeutic inhibition of AQP4 reduces IRI and improves cardiac allografts survival. B6 mice were transplanted with BALB/c heart allografts subjected to 8 h of cold ischemia. Donors received AQP4 inhibitor 0.5h before harvest; hearts were perfused with and stored for 8 h on ice with the inhibitor. Recipients were given AQP4 inhibitor every 6h for 5 days. In the absence of additional immunosuppression, treatment with AQP4 inhibitor significantly prolonged heart allograft survival (MST 2d, n=24 vs. 6 d, n=5), decreased early T cell infiltration and the intragraft expression of IFNγ, granzyme B, perforin, FASL, CXCR3, ICOS, and CXCL10 in comparison to untreated controls. The frequencies of donor-reactive spleen cells secreting IFNγ in recipients treated with AQP4 inhibitor were also four fold lower than those in untreated controls. Blocking AQP4 during allograft storage and early after transplantation prolongs the survival of fully MHC-mismatched heart allografts subjected to 8 h CIS. The graft prolongation is associated with the reduced early memory T cell infiltration into the graft and decreased anti-donor responses. Our data identify AQP4 as a promising therapeutic target to diminish the detrimental effects of prolonged CIS on transplant outcome.
Effector T cell migration to pancreatic islet allografts is dependent on donor antigen recognition and not Gai chemokine signaling. (TRAN1P.945)
Authors: Zhang Qianqian , Dai Hehua , Zeng Qiang , Williams Amanda , Oberbarnscheidt Martin , Lakkis Fadi
Published: January 2023

Host effector T cell graft infiltration is a key step in the pathogenesis of both acute and chronic rejection. Recent data indicate effector T cell migration into primary vascularized organ transplants such as heart and kidney is driven by donor antigen recognition, not via Gai-coupled chemokine receptor signaling. Whether it is true for neovascularized pancreatic islet allografts is unknown. We co-transferred pertussis toxin (Ptx)-treated and untreated TCR-tg (OT-1) effector T cells into B6 recipients 7-10 days after transplanting B6-Act-OVA or B6 islet grafts under the kidney capsule. Ptx irreversibly blocks Gai-dependent signaling. Grafts were imaged 20-24 hours after T cell transfer by two-photon intravital microscopy. We observed B6-Act-OVA grafts were heavily infiltrated with OT-1 effector T cells irrespective of pre-treatment with Ptx or not (453.7+/-170.0 vs. 632.8+/-221.1 per imaging volume, n = 10, p = 0.0522 ), while B6 grafts, which lack the cognate antigen recognized by OT-1 T cells, were free of infiltrate (<5 cells, n = 7). These results indicate that, similar to heart and kidney transplants, islet allograft infiltration with host effector T cells is driven by donor antigen, not by Gai signaling. The paradigm of antigen-driven effector T cell migration is therefore generalizable to both primary vascularized grafts, in which donor antigens are abundantly expressed on the endothelium, and neovascularized cellular grafts whereby the endothelium is of host origin.
Allergic pulmonary inflammation accelerates breast cancer metastasis via increase of myeloid cells in the lung microenvironment (TUM6P.1005)
Authors: Libreros Stephania , Garcia-Areas Ramon , Gazaniga Nathalia , Robinson Philip , Keating Patricia , Iragavarapu-Charyulu Vijaya
Published: January 2023

Inflammation is known to contribute to tumor initiation and metastasis. Since it is not known the effect of pre-existing inflammation on metastasis, we combined a model of allergic pulmonary inflammation with a breast cancer model. Using this model, we found that allergen sensitized mice implanted with mammary tumors had a 5-fold increase in metastatic foci. More importantly allergic tumor bearing mice showed 3X faster tumor cell infiltration in their lungs with accelerated tumor growth and shorter survival. Since macrophages and MDSCs play a key role in the immunosuppressive processes associated with both inflammation and metastasis, we hypothesized that allergic pulmonary inflammatory microenvironment attracts myeloid cells that support incoming tumors cells and accelerates metastasis. We found an increase in the alveolar MO (CD11b-CD11C+CD68+F4/80+Ly6G-), interstitial MO (CD11b+CD11C-CD68+F4/80+Ly6G-), monocytic MDSC (CD11b+Ly6ChighLy6G-F4/80-), and granulocytic MDSC (CD11b+Ly6ClowLy6G+F4/80-) subpopulations in mice with allergic pulmonary inflammation prior to tumor inoculation. These populations were further increased after tumor cell inoculation with the exception the granulocytic MDSCs. Depletion of macrophages and MDSCs prior to tumor cell inoculation in allergen mice decreased tumor growth and metastasis in allergic tumor bearing mice. Targeting specific immune populations could lead to safer and more efficient strategies against breast cancer metastasis.
Impact of MDSC emergence during ovarian cancer in cytokine-deficient mice. (TUM6P.1011)
Authors: Riggs James , DePierri Kelley
Published: January 2023

We have been studying the emergence of myeloid-derived suppressor cells (MDSCs) following i.p. transfer of ID8 cells (mouse ovarian surface epithelial cell carcinoma) into intact, syngeneic, C57BL/6J (wild type, WT) mice. Our current work studies the impact of the cytokine (CK) environment on ID8 expansion and MDSC emergence in the peritoneal cavity (PerC). We wanted to determine if particular pro- (IFNg) or anti- (IL4, IL10) inflammatory CKs were drivers of disease in this model. We found that, relative to WT mice, the development of hemorrhagic ascites was accelerated in IL10-/-, IL4-/-, and IFNgR-/- mice. The greatest percentage and number of MDSCs was observed in IL10-/- mice, which also had the greatest expansion of ID8 cells. ID8 triggered a marked increase in PerC Mfs, particularly in the IL10-/- mice, and lymphocytes, particularly in the IFNgR-/- and IL4-/- mice. However, for all mice, the percent representation of B cells in the total PerC cell pool declined markedly, particularly in the IL10-/- mice (> 70%). Interestingly, beyond the peritoneal locus of disease, both B and T lymphocyte percentages dropped in the spleens of all mice. This was seen without a concomitant increase in the number of MDSCs or appearance of ID8 in the spleen. These observations reinforce that inflammation drives MDSC emergence and suggest that changes in lymphocyte populations could serve as biomarkers for assessing ovarian cancer.
HMGB1 promotes MDSC survival through autophagy (TUM6P.956)
Authors: Parker Katherine , Ostrand-Rosenberg Suzanne
Published: January 2023

Myeloid-derived suppressor cells (MDSC) are immune suppressive cells that are elevated in most individuals with cancer where their accumulation and suppressive activity are driven by inflammation. Because MDSC globally inhibit anti-tumor immunity, we are determining how their turn-over is regulated. We have established that the damage-associated molecular pattern molecule (DAMP) High Mobility Group Box protein 1(HMGB1) is critical for MDSC accumulation and function, and is ubiquitously present in the tumor microenvironment. Studies by others demonstrated that HMGB1 is induced by reactive oxygen species (ROS) and facilitates tumor cell survival by inducing autophagy. These findings have led us to hypothesize that HMGB1 regulates MDSC turn-over by propagating a pro-autophagic state that is self-sustained by the high levels of ROS produced by MDSC. Since autophagy involves translocation of HMGB1 from the nucleus to the cytosol, we have tested this hypothesis by starving MDSC and inhibiting HMGB1 translocation into the cytosol, and by inhibiting MDSC production of ROS. Inhibition of HMGB1 translocation limited MDSC autophagy and decreased MDSC survival, while inhibition of ROS reduced autophagy in MDSC. These results suggest that HMGB1 prolongs MDSC survival by inducing a pro-autophagic state in MDSC.
Inhibiting fatty acid oxidation blocks the immunosuppressive functions of myeloid-derived suppressor cells and enhances cancer therapies (TUM6P.960)
Authors: Hossain Fokhrul , Al-Khami Amir , Wyczechowska Dorota , Hernandez Claudia , Zheng Liqin , Reiss Krzystoff , Del Valle Luis , Trillo-Tinoco Jimena , Rodriguez Paulo , Ochoa Augusto
Published: January 2023

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T cell immunity through various mechanisms including the depletion of arginine and the production of nitric oxide and peroxinitrites. Blocking of any single mechanism has only moderate therapeutic effects because MDSC show high adaptability and upregulate the remaining suppressive pathways. We studied whether disrupting the energy metabolic pathway(s) of MDSC would modulate their immunosuppressive functions and enhance antitumor effects. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by increased mitochondrial mass and function, key FAO enzymes, and oxygen consumption rate. These metabolic changes paralleled with the induction of immunosuppressive mechanisms. Inhibition of FAO blocked immunosuppressive mechanisms and functions in T-MDSC and decreased their production of immunosuppressive cytokines. Interestingly, it also diminished the number of regulatory T cells and resulted in a T cell-dependent decrease in tumor growth. FAO inhibition also significantly increased antitumor effects with low-dose chemotherapy or cellular immunotherapy. Human MDSC demonstrated similar characteristics. These data support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. *F. Hossain and A.A. Al-Khami contributed equally to this work.
Genetic depletion of cyclin dependent Kinase-5 results in enhanced tumor rejection via a CD4+ T cell dependent mechanism (TUM7P.1023)
Authors: Dorand Rodney , Petrositue Agne , Nthale Joseph , Scrimieri Francesca , Pareek Tej , Myers Jay , Barkauskas Deborah , Letterio John , Stearns Duncan , Huang Alex
Published: January 2023

CDK5 is a non-canonical member of the CDK family that does not require cell cycling for activity but relies on binding to its obligate activators, p35 and p39 which are mostly expressed in post mitotic neurons. CDK5 is required for normal Central Nervous System (CNS) development. Medulloblastoma (MB), derived from primitive cerebellar granule neuron precursors, is the most common pediatric CNS malignancy. Utilizing a short hairpin RNA approach to silence Cdk5 expression in a C57BL/6 (B6) syngenic MB line derived from p53+/- PTCH-/- (MM1), we found that 19 days after subcutaneous inoculation, mice with silenced Cdk5 (MM1-CDK5-KD) revealed a 78% tumor free survival (N=36) compared to 4% (N=24) of mice inoculated with wild type MB (MM1-WT). Furthermore, when injected into Nu/Nu mice, MM1-WT and MM1-CDK5-KD had similar subcutaneous growth kinetics. When CD4+ cells were depleted in B6 mice inoculated with MM1-CDK5-KD tumor free survival decreased to 28% (N=15). We sought to interrogate the role of the immune system in enhanced killing of MM1-CDK5-KD tumors. When exposed to IFN-γ stimulation in vitro, genetic profiling of MM1-CDK5-KD revealed a decrease in PD-L1 expression, a known immune checkpoint surface molecule, compared to MM1-WT. These results indicate a functional role for Cdk5 in MB immune evasion which, when circumvented via Cdk5 inhibition, leads to increased susceptibility to anti-tumor surveillance.
Diversity index of mucosal resident T lymphocyte repertoire predicts clinical prognosis in gastric cancer (TUM7P.1028)
Authors: Wan Ying , Jia Qingzhu , Zhou Junfeng , Chen Gang , Shi Yan , Yu Haili , Lin Regina , Jiang Ning , Yu Peiwu , Li Qi-Jing
Published: January 2023

A characteristic immunopathology of human cancers is the induction of tumor antigen-specific T lymphocyte responses within solid tumor tissues. Current strategies for immune monitoring focus on the quantification of the density and differentiation status of tumor-infiltrating T lymphocytes; however, properties of the TCR repertoire ‒ including antigen specificity, clonality, as well as its prognostic significance ‒ remain elusive. In this study, we enrolled 28 gastric cancer patients and collected tumor tissues, adjacent normal mucosal tissues, and peripheral blood samples to study the landscape and compartmentalization of these patients’ TCR beta repertoire by deep sequencing analyses. Our results illustrated antigen-driven expansion within the tumor compartment and the contracted size of shared clonotypes in mucosa and peripheral blood. Most importantly, the diversity of mucosal T lymphocytes could independently predict prognosis, which strongly underscores critical roles of resident mucosal T-cells in executing post-surgery immunosurveillance against tumor relapse.
Imaging in cancer immunology: phenotyping of multiple immune cell subsets in-situ in FFPE tissue sections (TUM7P.1029)
Authors: Mansfield James , Hoyt Clifford , Stack Ed , Feldman Michael , Bifulco Carlo , Fox Bernard
Published: January 2023

There has been a rapid grown in the field of tumor immunobiology in recent years as a result of recent successes in cancer immunotherapies, and it is clear that immune cells play many sometimes conflicting roles in the tumor microenvironment. However, obtaining phenotypic information about the various immune cells in and around the tumor has been a challenge. Existing methods can deliver phenotypic information on homogenous samples (e.g., flow cytometry or PCR) or morphologic information in single stain IHC. We present here a methodology for delivering quantitative per-cell marker expression and phenotyping, analogous to that obtained from flow cytometry, but from cells imaged in situ in FFPE tissue sections. This methodology combines: the sequential multi-marker labeling of up to 6 antigens using antibodies all of the same species; automated multispectral imaging to remove problematic FFPE tissue autofluorescence and correct cross-talk between fluorescent channels; and an automated analysis that can quantitate the per-cell marker expression, determine the cellular phenotype, count these cells separately in the tumor compartment and in the stroma and provide high-resolution images of their distributions. We will show a 6-plex assay in breast cancer showing the application of the multiplexed staining, per-cell quantitation and cellular phenotyping in FFPE tissue sections, as well as methods to explore the spatial distributions of the phenotyped cells in and around the tumor.
STING protects against colitis-associated colorectal tumorigenesis by modulating intestinal inflammation (TUM7P.1018)
Authors: Zhu Qifan , Kanneganti Thirumala-Devi
Published: January 2023

Stimulator of IFN genes (STING) is an innate immune sensor for cytosolic cyclic dinucleotides and also serves as an adaptor molecule for a number of intracellular DNA sensors. While STING has been shown to be critically involved in modulating immune responses during infections and autoimmune diseases, its physiological role in cancer remains largely unknown. In this study, we demonstrated that STING-deficient mice are highly susceptible to colitis-associated colorectal cancer with overt intestinal proliferation during early stages of tumorigenesis. Hyperactive NF-kB and STAT3 signaling was responsible for increased levels of proinflammatory cytokines observed in STING-deficient mice. Furthermore, intestinal tissues deficient in STING exhibited elevated level of DNA damage maker, γH2AX and a defect in caspase-1-mediated colonic IL-18 release, highlighting its fundamental functions in maintaining genomic integrity and activating inflammasome. Therefore, our results identify an unexpected and important role for STING in mediating protection against colorectal tumorigenesis.
Epigenetic silencing of Th1-type chemokines dictates effector T cell tumor trafficking and predicts patient outcome (TUM7P.1019)
Authors: Peng Dongjun , Kryczek Ilona , Nagarsheth Nisha , Zhao Lili , Wei Shuang , Zhao Ende , Vatan Linda , Szeliga Wojciech , Kotarski Jan , Tarkowski Rafal , Dou Yali , Cho Kathleen , Liu Rebecca , Zou Weiping
Published: January 2023

Epigenetic silencing including histone modifications and DNA methylation is an important tumorigenic mechanism in cancer. However, its role in cancer immunopathology and immunotherapy is poorly understood. In this study, we focused on the infiltration of effector T lymphocytes into ovarian tumor and established the histone H3 lysine 27 trimethylation (H3K27me3) and DNA hypermethylation as epigenetic silencing mechanisms regulating Th1-type chemokines CXCL9 and CXCL10 production. We found that a polycomb group (PcG) protein, enhancer of zeste 2 homolog (EZH2)-based histone H3 lysine 27 trimethylation (H3K27me3) alone or in collaboration with DNA methyltransferases (DNMTs) selectively control the tumor production of CXCL9 and CXCL10, and subsequently determine effector T cell trafficking to the tumor microenvironment. Treatment with epigenetic modulators removes the repression and increases effector T cell tumor infiltration, slows down tumor progression, and improves therapeutic efficacy of PDL1(B7-H1) checkpoint blockade and adoptive T cell transfusion. Moreover, tumor EZH2 and DNMT1 are negatively associated with tumor infiltrating CD8+ T cells and patient outcome. Our results indicate that epigenetic silencing of Th1-type chemokine is a novel tumor immune evasion mechanism. Selective epigenetic reprogramming alters T cell landscape in cancer and enhances clinical efficacy of cancer therapy.
IL-2 complex rescues CD8+ T cell tolerance for cancer immunotherapy (VAC7P.1041)
Authors: Swier Lauryn , Berrien Melissa , Teague Ryan
Published: January 2023

IL-2 is a well-characterized T cell growth factor that promotes proliferation and effector function of CD8+ T cells. For this reason, IL-2 has been used to boost immune responses in patients with melanoma. However, IL-2 treatment requires large potentially toxic doses of IL-2, and often only limited therapeutic efficacy is acheived. Efforts to enhance the efficacy and safety of IL-2 have recently focused on the use of cytokine/antibody complexes. These complexes demonstrate greater bioactivity in vivo relative to IL-2 alone, making them an attractive approach for improved immunotherapy. One of the major impediments to anti-tumor immunity is CD8+ T cell tolerance, where tumor-reactive T cells are either eliminated by apoptosis or rendered dysfunctional/anergic. The influence of IL-2 complex (IL-2c) on tolerant CD8+ T cells has not been explored. We utilized a mouse model of T cell tolerance to examine how IL-2c treatment impacts tolerant tumor-reactive T cells during immunotherapy for leukemia. Specifically, IL-2c was administered daily starting 3 days after T cell transfer into tumor-bearing mice. The function and persistence of transferred tumor-reactive T cells was assessed 5 days later. Treatment with IL-2c prevented apoptotic deletion of transferred CD8+ T cells and improved effector function. These results suggest that IL-2c treatment is sufficient to overcome peripheral T cell tolerance, and support further exploration of IL-2c as an immunotherapeutic for cancer.
Immunogenicity of mucosally-administered modified vaccinia Ankara expressing HIV-1 Env protein gp120 in rabbits (VAC8P.1047)
Authors: Jones Dorothy , Pickup David , Staats Herman
Published: January 2023

Modified vaccinia Ankara (MVA) used as a vaccine vector expressing heterologous antigens has been demonstrated to be immunogenic when delivered parenterally while its immunogenicity after mucosal delivery is less defined. To determine the immunogenicity of MVA when administered via different mucosal routes, NZW rabbits were vaccinated on day 0 and 28 with 3 x 108 PFU MVA expressing HIV-1 gp120 by nasal, rectal, or gastric routes. Intramuscular immunization with 1 x 108 PFU of the MVA vaccine was used as the positive control. Serum was collected on days 21 and 42 and antigen-specific antibodies were determined by ELISA. While intramuscular vaccination was the only route that induced a significantly elevated anti-gp120 serum IgG titer, nasal vaccination induced detectable anti-gp120 IgG response on day 42. Immunization by rectal or gastric routes did not induce measurable anti-gp120 IgG responses. Intramuscular and intranasal vaccination, but not gastric or rectal vaccination with MVA, induced significantly elevated serum IgG responses to pox proteins A33R and B5R. We conclude that, under the conditions tested, MVA vaccine vectors are immunogenic when delivered intramuscularly and intranasally but not gastrically or rectally. Insert specific responses after nasal vaccination were not comparable to those achieved by intramuscular vaccination, indicating that use of MVA as a nasal vaccine vector will need to be optimized.
Sendai virus is a safe and immunogenic Jennerian vaccine for human parainfluenza virus-type 1 in 3-6 year old children (VAC8P.1053)
Authors: Penkert Rhiannon , Adderson Elisabeth , Branum Kristen , Sealy Robert , Jones Bart , Surman Sherri , Freiden Pamela , Slobod Karen , Gaur Aditya , Hayden Randall , Allison Kim , Howlett Nanna , Utech Jill , Allay Jim , Knight James , Sleep Susan , Meagher Michael , Russell Charles , Portner Allen , Hurwitz Julia
Published: January 2023

Human parainfluenza viruses are responsible for tens of thousands of hospitalizations and hundreds of thousands of emergency room visits each year in the United States. Included in this group of viruses is human parainfluenza virus-type 1 (hPIV-1), the main causative agent of laryngotracheobronchitis (croup) in young children, for which there is currently no licensed vaccine. In response to this need, we have developed a Jennerian vaccine that employs the use of murine PIV-1 (Sendai virus, SeV) to produce cross-reactive antibodies towards hPIV-1. The vaccine was administered as a single intranasal dose to healthy, hPIV-1 seropositive, 3-6 year old children in a dose escalation study (5 x 105, 5 x 106 or 5 x 107 EID50). Importantly, the vaccine was well tolerated by all participants. No replication of the viral vaccine was detected in the airway of any participant. Within four weeks of vaccination, most participants showed an increase in binding and neutralizing antibodies towards hPIV-1. For several participants, antibody responses remained elevated at six months post-vaccination. The results of this study suggest that hPIV-1 vaccination may be beneficial to boost immunity in seropositive children, and encourage testing of the vaccine in young, seronegative children who are most at risk for respiratory virus disease.
Immunogenicity of inactivated polio vaccine, given with or without mucosal adjuvants dmLT and TCTA1T in rat model (VAC9P.1102)
Authors: park julien , Rho Semi , Kim Jae-Ouk , Song Man Ki , Dey Ayan
Published: January 2023

To eradicate polio, the use of oral polio vaccine (OPV) should be stopped to avoid circulation of vaccine-derived polioviruses (VDPVs). After OPV cessation, transition to inactivated polio vaccine (IPV) is recommended. This can only be accomplished when IPV induce better mucosal protection. This can be achieved by adding mucosal adjuvant to the IPV formulation. In the present study we used wistar rats to elucidate the adjuvant effect of double mutant heat-labile toxin (dmLT) of E. coli and recombinant cholera toxin (TCTA1T) on IPV. Rats were administered by intramuscular injection of 1.0-0.6-1.0 D-antigen units (DU) of Inactivated Polio type 1, 2 and 3 /rat. The E. coli dmLT and TCTA1T was used as adjuvant and was administered subcutaneously along with IPV. The control groups received IPV alone or PBS. Rats were immunized on days 0, 14 and 28 days. After last vaccination mice were sacrificed and spleen cells and blood were collected. IgA secreting cells specific for Polio Type1, 2 and 3 were measured by ELISPOT. Intramuscular immunization results in robust mucosal response in inactivated polio antigens + dmLT group compared to other groups. Further measurement of antibodies in serum doesn’t show significant difference among IPV and adjuvanted IPV groups. Thus the present data suggest that vaccination with IPV and dmLT results in specific induction of mucosal immunity, which will provide barrier to infection from oral fecal route.
Identification of MHC bound peptides from murine bone marrow derived dendritic cells infected with Salmonella (VAC9P.1108)
Authors: Karunakaran Karuna , Yu Hong , Jiang Xiaozhou , Chan Queenie , Goldberg Michael , Foster Leonard , Jenkins Marc , Brunham Robert
Published: January 2023

Salmonella infections cause a spectrum of diseases ranging from self-limiting diarrhea to life-threatening systemic disease. Diseases caused by Salmonella are treatable with antibiotics but successful antibiotic treatment has become increasingly difficult due to antimicrobial resistance. An effective vaccine is therefore the best strategy to reduce these diseases. A clear understanding of host immunity to Salmonella is essential to aid in the development of a vaccine. Protective immunity against Salmonella depends on a wide range of innate and adaptive immune mechanisms and T cell-mediated immune responses are important in the host control of intracellular Salmonella infection. Our laboratory used an immunoproteomics approach to identify Chlamydia T cell antigens that exhibited significant protection against Chlamydia infection in mice when used as vaccines. These results demonstrate that T cell antigens identified by immunoproteomics can be successfully exploited as T cell vaccines against an intracellular pathogen. Recently we generated dendritic cells from bone marrow of C57BL/6 mice and infected with Salmonella enterica SL1344 followed by elution of MHC class I and class II-bound peptides. The sequences of the purified peptides were then identified using tandem mass spectrometry. We identified 87 MHC class II and 23 MHC class I Salmonella derived peptides. These antigens are of use in Salmonella immunobiology research and as potential Salmonella vaccine candidates.
Effect of anticoagulants on the measurement of cholera vaccine-induced humoral immune responses (VAC9P.1101)
Authors: Yang Jae Seung , kang Seok Seong , Yun Cheol-Heui , Han Seung Hyun
Published: January 2023

Anticoagulated blood is widely used for the simultaneous evaluation of humoral and cellular immune responses after the vaccination. However, anticoagulants in the plasma may interfere with the quantitative and qualitative analyses of antibodies. In the present study, we examined the potential interference of the widely-used anticoagulants including heparin, acid citrate dextrose (ACD), and ethylenediaminetetraacetic acid (EDTA) on bactericidal activity against V. cholerae and its LPS-specific IgG, IgM, and IgA levels in sera and plasma samples from cholera patients or vaccinees. Serum vibriocidal activity was inhibited in the presence of EDTA and ACD whereas no change was found when heparin was used. Moreover, vibriocidal titer of plasma, obtained from vaccinees using tube containing heparin, was 100% identical with the titer of corresponding sera while 37.5%, 50% in the plasma containing EDTA and ACD were matched, respectively. Pearson correlation coefficient (r) for LPS-specific IgG, IgM, and IgA between serum and heparinized plasma were 0.997, 0.945, and 0.716, respectively. Collectively, heparin is suggested to be an appropriate anticoagulant for collection of blood for measuring vibriocidal activity and antibody in the plasma after cholera vaccination.
Genetic markers for resilience and susceptibility to Johne's disease In red deer (Cervus elaphus) (VET1P.1120)
Authors: Brennan Liam , O'Brien Rory , Griffin Frank
Published: January 2023

Mycobacterium avium subspecies paraturberculosis is the causative agent of Johne’s disease, an enteritis infection that affects ruminants.This disease has been studied in deer, but there is little information regarding immunity or disease immunopathology. There is evidence that host genetics contribute to resilience or susceptibility to the disease. This study sought to identify genes in the innate and adaptive immune systems as biomarkers for resilient (R) or susceptible (S) phenotypes in red deer. We have bred over 60 animals from sires with a confirmed R or S phenotype. Animals were bled routinely and monocyte derived macrophages (MDMs) or whole PBMCs were obtained and cultured in vitro. Gene expression was determined using qPCR. The 15 most informative gene targets were chosen from a panel of 60 using GenEx software MDMs showed higher levels of expression of immune genes such as IL1A, TNFA, IL23A and IL12A in S animals, as well as genes not typically associated with the immune system, such as PKLR that exhibit this pattern. Conversely, PBMC samples from these animals exhibit higher levels of expression of immune genes such as IFNY, IL17A and IL2 in R animals. These results have been reproduced in animals bred from different sires over several years. We intend to incorporate these gene markers into diagnostic tests to identify R and S animals at an early age. These genes may also provide deeper insights into immune parameters that determine disease outcome in an animal.
Resistin-like molecules reduce influenza A virus morbidity in mice (VIR5P.1158)
Authors: Samarasinghe Amali , Lin Yanyan , LeMessurier Kim , Woolard Stacie , McCullers Jonathan
Published: January 2023

Resistin-like molecules (RELMs) are recently discovered cysteine rich secreted proteins abundant in a variety of organs including the lungs. RELMα has an inhibitory role in the lungs by downregulating inflammation, while RELMβ promotes inflammation. We noted a markedly greater expression of RELMs in the lungs of mice with acute asthma and influenza, which were protected against severe influenza morbidity and hypothesized that RELMα and RELMβ proteins enhance anti-viral defenses. Recombinant RELMα and RELMβ proteins were administered intranasally to C57BL/6 mice starting three days prior to viral infection and continued on a daily basis following viral infection. Mice were infected with 1000 TCID50/ml of A/CA/04/2009 pandemic influenza A virus. Animals were sacrificed on days 3, 5, 7, and 9 following viral infection for sample acquisition. Animals not treated with exogenous RELMs served as controls. Administration of RELM proteins led to a reduction in airway resistance after influenza virus infection. Inflammation in the airways increased in RELM-treated mice with macrophages and neutrophils taking precedence and a prominent increase in CD8+ T cells at late timepoints. A significant reduction in viral burden occurred in the lungs of RELM treated mice. The administration of RELM proteins directly impacted the host immune response against influenza A virus. Future studies will investigate the mechanisms by which RELMs mediate protection against influenza.
Characterizing the T cell receptor clonotype repertoire of an atypical HLA class II-restricted CD8 T cell response in HIV-1 infection (VIR6P.1170)
Authors: Lamothe Pedro , Ranasinghe Srinika , Crawford Frances , White Janice , Clayton Gina , Power Karen , Garcia-Beltran Wilfredo , Allen Todd , Shalek Alex , Kappler John , Walker Bruce
Published: January 2023

CD8 T cells targeting peptides presented by human leukocyte antigen (HLA) class II are atypical. Little is known about how the CD8 T cell receptor (TCR) recognizes peptides on HLA class II. We analyzed the TCR repertoire of a class II-restricted CD8 T cells targeting a HIV peptide. We identified an atypical HIV-specific CD8 response to DV16 peptide on HLA-DRB1*11. We sequenced the TCR of the class II-tetramer sorted cells. To measure the binding kinetics of the TCR with the peptide-MHC we used surface plasmon resonance (SPR). Analysis of 68 sequences showed that TCR-beta repertoire has a single TRBV2*01 clonotype. We looked at 64 sequences of TCRalpha and found that the repertoire had two clonotypes: TRAV26-1*02 and TRAV6*02. SPR revealed that only the TRAV6 was able to bind to the peptide-MHC. We studied 27 sequences of the CD4 T cells targeting the same MHC class II-peptide. We observed a polyclonal response of 16 clonotypes. 13 clonotypes used the same TRBV2*01 gene but had different rearrangements. Interestingly, two of these sequences are exactly the same as the dominant clonotype from the CD8 response. We found that the TCR is shared between CD8 and CD4 T cells targeting the same class II HLA-peptide. These data suggest that atypical CD8 cells express two different TCR alpha, possibly due to inefficient allelic exclusion during development. The use of the same TRBV2*01 by different CD4 clonotypes may suggest an atypical docking of TCR in the binding with peptide-HLA.