bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterium tuberculosis infection decreases macrophage Tpl-2 protein levels over time which alters responses to TLR agonists (MPF4P.730)
Authors: Ni Bin , Torres Chelsea , Rajaram Murugesan , Schlesinger Larry
Published: January 2023

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis, is a host-adapted intracellular pathogen of macrophages. A complex, evolutionally adaptive relationship exists between M.tb and the host during which M.tb evades and subverts the immune response mounted by the macrophage. Understanding the interactions during primary infection between the macrophage and M.tb is important because it can impact the outcomes of infection and course of disease. We show that expression of the MAP3K Tpl-2, which was previously identified as a host defense molecule against M.tb in a murine knockout model, is down-regulated during M.tb infection of human primary macrophages. We observed downstream signaling effects of M.tb-induced suppression of Tpl-2 by studying ERK and MEK activation after stimulation with Toll-like receptor (TLR) ligands. M.tb lipomannan (ErdLM) stimulation of M.tb-infected macrophages showed decreased activation of ERK1/2 and MEK, congruent with decreased Tpl-2 levels after M.tb infection. However, ERK1/2 activation did not decrease significantly in M.tb-infected macrophages after treatment with the pure TLR agonists LPS, Pam3CysSK4, and CpG ODN, although decreased MEK phosphorylation was observed, suggesting that TLR ligands activate ERK1/2-mediated pathways that differ from those activated by ErdLM. These data indicate that M.tb has the capacity to suppress Tpl-2 over time and alter MAPK signaling to promote a survival advantage within the macrophage.
Dependency of TLR4 signaling for the anti-inflammatory effect of exopolysaccharide (EPS) of Bacillus subtilis (MPF4P.731)
Authors: Jones Sara , Paynich Mallory , Knight Katherine
Published: January 2023

Although probiotics offer a promising source of therapeutic compounds, few studies have identified the bacterial molecules or host signaling pathways required for the immunomodulatory effects. We previously demonstrated that exopolysaccharide (EPS) from probiotic Bacillus subtilis is the bacterial molecule that prevents disease induced by the murine pathogen Citrobacter rodentium, a pathogen that shares many similarities to the human pathogen enteropathogenic E. coli. To begin to elucidate how EPS functions, we determined which cells are required for protection and found that mice depleted of macrophages using clodronate loaded liposomes are not protected from the pathogen. Further, transfer of macrophage-rich peritoneal cells from wt, but not TLR4 KO, EPS-treated mice conferred protection from pathogen-induced colitis; these data suggest a role for TLR4 and macrophages. Using flow cytometry and microscopy, we find that EPS binds F4/80+CD11b+ macrophages and co-localizes with TLR4. We also find that EPS binds both wt and TLR4 KO macrophages, suggesting that EPS binds to a component of the TLR4 signaling complex instead of TLR4 directly. We are immunoprecipitating the EPS receptor, predicted to be a co-receptor of the TLR4 signaling complex, and determining if EPS is an agonist or antagonist of TLR4 signaling. Our studies should lead to a new class of bacteria-derived immunomodulatory molecules.
Luminal-GABA promotes inflammation in mouse models of colitis (MUC5P.756)
Authors: Dann Sara , Kelly Hannah , Suarez Diego , Hoppe Daniel , Banks MaChesha , Peniche Alex
Published: January 2023

Background: Inflammatory bowel disease (IBD) results from a dysregulated immune response towards commensal microbiota. Although the etiology of IBD is unclear, there is considerable interest in the role of commensals and their metabolites in regulating pathology. Gamma-aminobutyric acid (GABA) is one metabolite produced by several commensal species that has been reported to have anti-inflammatory effects which could impact severity of IBD. Objective: To determine the physiological role of GABA on intestinal inflammation. Methods & Results: C57BL/6 mice were administered GABA (2mg/ml) in their drinking water 3 days before induction of colitis (3% DSS or Clostridium difficile infection) and for another week thereafter. Intestinal tissue was collected and examined for histopathology, gene expression and cellular responses. In both models, administration of GABA exacerbated intestinal inflammation and damage, whereas inhibition of GABA with the receptor antagonist picrotoxin reduced disease severity. Damage was associated with increased expression of proinflammatory cytokines (IL-6 and IL-1β) and chemokines (CXCL1 and CXCL2) and alterations in macrophages and neutrophils. Conclusion: GABA increases host sensitivity to epithelial damage and inflammation. Identifying the effects of GABA on host immune defenses may reveal new approaches to treating and preventing IBD.
IL-1β secreted by macrophages in response to commensal bacteria acts together with bone morphogenetic protein signals to increase hepcidin expression in hepatocytes (MUC5P.758)
Authors: Cherayil Bobby , Shanmugam Nanda
Published: January 2023

Elevated expression of the iron regulating hormone hepcidin leads to abnormalities of iron metabolism in inflammatory diseases. The mechanisms involved in inflammation-associated hepcidin upregulation are not well understood. We have shown in mouse IBD models that the commensal microbiota significantly influences hepcidin expression. Here we describe experiments to elucidate how commensal bacteria upregulate hepcidin. Differentiated THP-1 macrophages were infected with the commensal Bifidobacterium infantis. Sterile supernatants of the infected, but not uninfected, THP-1 cells increased hepcidin expression in HuH7 human hepatocytes, whereas direct infection of HuH7 cells failed to induce hepcidin. The infected THP-1 cells secreted IL-1β, neutralization of which significantly inhibited induction of hepcidin expression in the HuH7 cells. Pretreatment of the HuH7 cells with inhibitors of the bone morphogenetic protein (BMP) signaling pathway also prevented the upregulation of hepcidin by the supernatant of B. infantis-infected THP-1 cells. Our findings demonstrate that IL-1β secreted by macrophages in response to commensal bacteria can induce increased expression of hepcidin in hepatocytes. An activator of the BMP signaling pathway is also involved but the identity and source of this molecule are yet to be determined. Furthermore, our results suggest that neutralization of IL-1β may be useful in preventing or treating the dysregulation of iron homeostasis associated with IBD.
Selective inhibition of Kv1.3 channels in memory T cells by targeted delivery with nanoparticles suppresses CD40 ligand expression in SLE (THER5P.908)
Authors: Chimote Ameet , Hajdu Peter , Kottyan Leah , Harley John , Conforti Laura
Published: January 2023

The pathogenesis of Systemic Lupus Erythematosus (SLE) is characterized by hyperactive memory T cells (TM). In SLE, the TM cells show a Ca2+-dependent increase in the costimulatory CD40 ligand (CD40L) which binds CD40 on B cells, resulting in B cell activation and autoantibody production. CD40L is widely recognized as a potential target for developing new therapies for SLE. Cytosolic Ca2+ levels, which increase during T cell activation and mediate CD40L expression, are regulated by Kv1.3 channels. We have developed lipid nanoparticles that can deliver Kv1.3 siRNAs (Kv1.3-NPs) selectively to TM cells and decrease the activation-induced Ca2+ influx (Hajdu et al., Biomaterials 2013). We studied whether these Kv1.3-NPs decrease the Ca2+-dependent overexpression of CD40L in SLE peripheral blood TM cells. We observed that nuclear translocation of NFAT, a transcription factor upstream to CD40L, in activated TEM cells was reduced by ~50% after treatment with Kv1.3-NPs. Furthermore, Kv1.3-NP treatment selectively reduced CD40L expression in activated TEM cells by ~40% in healthy donors and ~60% in SLE patients, while CD40L levels remained unchanged in cells that did not incorporate the nanoparticles. Interestingly, we observed that treatment with Kv1.3-NPs in SLE patients resulted in T cell phenotype switching from a predominantly TM phenotype to a naïve phenotype. Our findings open the possibility of using Kv1.3-NPs as potential targeted immune suppressive therapeutic agents in SLE.
Time-lapse imaging of contact dynamics between mesenchymal stem cells and T cells (THER5P.913)
Authors: Pyo Minji , Lee Hong Kyung , Uhm Jinbum , Ryu Hwa Sun , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Mesenchymal stem cells (MSC) ameliorate the pathogenesis of systemic lupus erythematosus through inhibiting T cell functions via soluble mediators and direct cell-cell contact. However, little is known about the contact dynamics at the single cell level. Using time-lapse imaging, we show that their contact is dependent on CCL2, but not CCL3, CCL4, CXCL10, and CXCL12, which are produced by MSCs. First, we analyzed contact dynamics of MSCs. Each WT MSC contacted 11 T cells and each contact lasted 107 min. In the same condition, each CCL2-/- MSC contacted 10 T cells and each contact continues 34 min. Subsequently, CCL2-/- MSCs did not inhibit T cell functions. Next, we analyzed migration dynamics of only T cells, since MSCs did not move well. When calculating the percentage of T cells contacting MSCs more than one time for 6 h, 68% T cells contacted MSCs and 59% T cells contacted CCL2-/- MSCs. T cells moved at average speeds of 7 um/min without contact and did not move well during the contact period, which was similar in cultures with either WT or CCL2-/- MSCs. Together, our data demonstrate that MSC-derived CCL2 might increase T cell migration, prolong contact duration with T cells, and finally inhibit T cell functions.
Antigen specific tolerance and the prevention of autoimmune type 1 diabetes occurs through indirect antigen presentation and requires programmed death ligand 1 (THER5P.918)
Authors: Spanier Justin , Heffernan James , Mitchell Jason , Pauken Kristen , Sahli Nathanael , Fife Brian
Published: January 2023

Antigen-coupled ethylene carbodiimide-fixed cells is an attractive therapeutic platform for treating human patients with type 1 diabetes since these cells can prevent and reverse diabetes in the non-obese diabetic mouse model of diabetes. However, the mechanisms contributing to the induction of T cell tolerance in vivo following treatment are unclear. Therefore, we used the BDC2.5 T cell transfer model to study how p31-coupled cells inhibit T cell function in vivo. Our results demonstrate that indirect antigen presentation was critical for tolerance induction in vivo. We further demonstrate that PD-L1 on the host antigen presenting cells is required for tolerance induction. Importantly, using two-photon microscopy we show that BDC2.5 T cells arrest on CD11c+ dendritic cells during the induction of tolerance, suggesting that these cells are responsible for presenting coupled cell antigen and tolerance induction. To increase the feasibility of translating antigen coupled cells to the clinic, we demonstrate that whole blood preparations and frozen cells are sufficient for tolerance induction, a significant concern for donor coupled cell sources when considering translation to human patients. These data further our understanding of how antigen-coupled cells induce tolerance and how this tolerance protocol could be altered to increase clinical feasibility.
MHC class II-disparate heart but not skin allografts accelerate recipient cardiovascular disease (TRAN1P.934)
Authors: Zhou Jing , yi tai , Qin Lingfeng , Ali Rahmat , Li Qingle , Jiao Yang , Li Guangxing , Zhang Jiasheng , Sadeghi Mehran , Pober Jordan , Tellides George
Published: January 2023

Organ transplantation is the most effective therapy for patients with end-stage organ failure but is limited by premature deaths from cardiovascular disease (CVD). The underlying mechanism is poorly understood. Using the ApoE-/- mouse model of atherosclerosis we investigated the effect of allograft rejection on recipient CVD by transplanting MHC class II-disparate cardiac or skin allografts from BM12 donor mice into C57BL/6 (B6) ApoE-/- recipient mice. Isografts from B6 donor mice were used as controls. Mice were observed for 12 weeks post transplantation. Allograft rejection of the BM12 hearts occurred around 60 days whereas the skin allografts were rejected within 15 days. About half of the mice with cardiac allografts died before 12 weeks although all mice with the skin grafts or heart isografts survived. The mice of heart allografts exhibited impaired cardiac function (decreased fractional shortening and enlarged left ventricles) and larger atherosclerotic lesions compared with those of cardiac isografts or skin grafts. CD4 T cells from blood and atherosclerotic lesions expressed higher levels of the early activation marker CD69 in hosts of heart allografts but not in those of isografts or skin allografts. Only the mice with cardiac allografts showed elevated mRNA levels for IFN-γ and IL-12a in their native hearts. Our data indicate that chronic allorecognition by alloreactive CD4 T cells elicits sustained inflammation and plays a key role in promoting recipient CVD.
Reduced and skewed TCR Vβ repertoire in CD8 compared to Foxp3+ regulatory T cells in acute gastrointestinal graft versus host disease (TRAN1P.947)
Authors: Tran Dat , Maher Ossama , Rivas Charlotte , Moree Shannon
Published: January 2023

It is unclear if skewing of TCR repertoire in CD4, CD8 and Foxp3+Tregs plays a role in acute gastrointestinal (GI) GVHD. This prospective clinical study examined the TCR Vβ diversity among CD4, CD8 and Foxp3+Tregs from 18 adult patients (median age 53) post allogeneic stem cell transplant (SCT) at day 80 with acute GI GVHD (giGVHD, >grade 1, n=9) vs. nonGVHD (n=9). Fresh PBMC were analyzed by FACS using IOTest Beta Mark 24 TCR Vβ and Foxp3 (236A/E7)/Helios eBioscience kits. Mann Whitney test was used. Mean %CD4 was lower in giGVHD vs. nonGVHD (44vs72%, p=0.02) while no difference in %CD8 (51vs55%), %Tregs (16vs16%) and %Helios+Tregs (72vs76%). %Total Vβ were similar for CD4 (60), CD8 (60) and Tregs (64) in nonGVHD. In giGVHD, %CD8 (40) was lower than %Tregs (69), p<0.001 and %CD4 (56), p=0.08. Between giGVHD vs. nonGVHD, there was a difference for %TCR in CD8 (p=0.02) but none for CD4 or Tregs. In nonGVHD, there was a direct correlation between %CD8 TCR vs. %CD8 and %CD4 TCR vs. %CD4 but no correlation for %Treg TCR vs. %Tregs. In giGVHD, there was an inverse correlation between %CD8 TCR vs. %CD8 and %CD4 TCR vs. %CD4 but no correlation for %Treg TCR vs. %Tregs. These results indicate a restricted, reduced and skewed TCR repertoire within the CD8 as compared to Tregs and CD4 in giGVHD. These CD8 might be the pathogenic cells escaping Treg suppression. In conclusion, a reduced and skewed TCR diversity in CD8 at day 80 might be predictive for the development of chronic GVHD.
Effect of chemotherapy on the myeloid-derived suppressor cells percentages in the peripheral blood of advanced non-small cell lung cancer patients (TUM6P.965)
Authors: Vetsika E. , Gioulmpasani Marianthi , Skalidaki Eirini , Katsarou Afroditi , Koinis Filippos , Aggouraki Despoina , Koutoulaki Anna , Mavroudis Dimitris , Georgoulias Vassilis , Kotsakis Athanasios
Published: January 2023

In our previous study, high levels of one granulocytic (G-MDSC) and two monocytic (M-MDSCs) subpopulations of immunosuppressive MDSCs were found and their overexpression was correlated with worse prognosis in NSCLC patients. Using flow cytometry, the impact of chemotherapy on the percentages and functionality of M-MDSC (CD14+CD15-CD11b+CD33+HLA-DR-Lin- and CD14+CD15+CD11b+ CD33+HLA-DR-Lin-) and G-MDSC (CD14-CD15+CD11b+CD33+HLA-DR-Lin-) was evaluated in the peripheral blood of patients (n=141) prior to chemotherapy and after the 3rd and 6th cycle of treatment. The M-MDSC (CD14+CD15-CD11b+CD33+HLA-DR-Lin-) percentages were found significantly decreased post chemotherapy compared to baseline, whereas chemotherapy had no effect on CD14+CD15+CD11b+CD33+HLA-DR-Lin- M-MDSCs. In contrast, the production of iNOS by M-MDSCs was significantly increased compared to the baseline. Both G-MDSC levels and their ROS expression were not affected by chemotherapy. In addition, both subtypes of MDSCs, when co-cultured with CD3+ T cells, were able to significantly suppress IFN-γ secretion by T cells. Finally, the M-MDSC (CD14+CD15-CD11b+CD33+HLA-DR-Lin-) percentages were found significantly decreased in patients that did not develop progressive disease during or after treatment completion, compared to patients with disease progression. Chemotherapy seems to have an impact on MDSC expression and functionality, hence targeting these cells may lead to a better clinical outcome in NSCLC patients.
A translational study of monocyte receptor expression and chemotaxis between dogs and humans with and without osteosarcoma (TUM6P.975)
Authors: Fogle Jonathan
Published: January 2023

Spontaneous canine osteosarcoma (OSA) is an excellent translational model for human OSA. Published reports of both canine and human OSA indicate that monocyte activation is important for controlling primary and metastatic disease. The purpose of this study was to identify phenotypic and chemotactic differences between monocytes from dogs with OSA, healthy dogs and to determine if the same findings occur in human OSA. Canine peripheral blood mononuclear cells (PBMCs) were incubated with antibodies against CD14, CD32, CD62L, CD16, CD11c, CCR2, CCR7, CD43, CX3CR1, and CXCR2 and analyzed by flow cytometry. In OSA dogs (n=18), CD16 surface expression was increased, and CD62L, CCR2, CCR7, CD43, CX3CR1 and CXCR2 expression was decreased, compared to controls (n=13, p<0.01, MWU). Additionally, decreased chemokine receptor expression was associated with increased peripheral blood monocyte counts. Preliminary data from human OSA patients suggests the monocyte receptor expression profile is similar to that in canine OSA. Based upon these results, we hypothesize that monocytes exhibit altered chemotaxis in OSA patients. Assessment of in vitro monocyte chemotaxis in healthy patients compared with OSA patients, in both dogs and humans, is currently underway.
Colorectal tumour associated macrophages are more pro-inflammatory than adjacent control bowel tissue macrophage populations (TUM6P.1003)
Authors: Kemp Roslyn , Norton Samuel , Taylor Edward , Dunn Elliott , Munro Fran , Black Michael , McCall John
Published: January 2023

Infiltration of macrophages in colorectal cancer is associated with patient prognosis. Using multiparameter flow cytometry, multiple distinct macrophage populations were detected in tumour tissue recovered from colorectal cancer patients, highlighting the heterogeneity of myeloid populations within complex tissues. Population frequencies of five key macrophage populations were assessed in tumour and matched non-transformed bowel tissue to reveal specific changes caused by the direct presence of the tumour. The frequency of conventional (pro-inflammatory) macrophages (CD45+CD11b+CD33-CD64+CD14-CD206+CD163-) was significantly increased in tumour tissue compared to healthy bowel tissue. This population was analogous to pro-inflammatory IL-12-producing macrophages derived in vitro from blood. A large population of gut resident macrophages was also observed in both tumour and control bowel tissue. “Homeostatic” gut resident macrophages (CD45+CD33+CD14-CD11b-CD64-) and “inflammatory” gut resident macrophages (CD45+CD33+CD14+CD11b+CD64+) were decreased and increased, respectively, in tumour tissue compared with healthy bowel tissue of the same patients. This suggests that macrophages provide a pro-inflammatory environment within the tumour tissue and may explain why these cells are often associated with improved patient outcome in colorectal cancer. Data gained from this study could be used for future investigation of macrophages as both prognostic and therapeutic targets.
Use of [11C]PBR28 to localize and quantify tumor associated macrophages in pancreas cancer (TUM6P.1008)
Authors: Perusina Lanfranca Mirna , Pasca Di Magliano Marina , Piert Morand , Zou Weiping , Frankel Timothy
Published: January 2023

Tumor associated macrophages (TAM) are an important part of the immune microenvironment in pancreas cancer (PC) and are associated with poor prognosis. We investigated the use of a novel radiolabeled small molecule ([11C]PBR28), which binds to the macrophage translocator protein (TSPO), to track TAMs in-vivo. A murine PC line was generated from a KRAS/P53 mutant transgenic mouse. Immunoblotting confirmed lack of endogenous TSPO expression. After subcutaneous implantation, mice were administered [11C]PBR28. After micro-PET imaging, organs and tumors were harvested for autoradiography. Immunoflourescent co-staining of tumors confirmed TAM infiltration with co-expression of F4/80 and TSPO. Following [11C]PBR28 injection, micro-PET successfully localized tumors and autoradiography demonstrated a 3-fold increase in tumor radioactivity compared to background (muscle). To confirm the observed activity was TAM specific, the experiment was repeated in CD11b+ diphtheria toxin receptor (DTR) transgenic mice. After tumor inoculation, diphtheria toxin (DT) was administered and macrophage depletion confirmed by immunofluorescence and flow cytometry. Autoradiography demonstrated equal tumor uptake relative to background and decreased levels compared to tumors from wild-type mice. We demonstrated a non-invasive method to quantify pancreatic cancer TAM infiltration in-vivo using a novel radiotracer. This could be used prognostically and to determine response to TAM based immunotherapy.
Unusual immunogenicity of live cancer cells (TUM6P.957)
Authors: Das Mohapatra Alok , Tirrell Isaac , Srivastava Pramod
Published: January 2023

The ability of irradiated tumor cells to elicit tumor immunity has been widely established. Using two murine tumor models, we observe that injection of about 20,000 live tumor cells fails to develop tumors in recipient mice. (About ten times that dose leads to a successful tumor implantation.) However, mice injected with this sub-tumorigenic dose of cells become resistant to a subsequent lethal challenge. Remarkably, as many as 20 million (M) irradiated tumor cells (i.e. 1,000 times the sub-tumorigenic dose) are required to elicit resistance to tumor challenge. We assumed that the live cells in the sub-tumorigenic dose multiply in vivo and generate a large immunizing dose, before they are rejected. Indeed, imaging in vivo of the mice challenged with the sub-tumorigenic dose revealed that the tumor cells multiply at the site of challenge ~100 fold to ~2M cells over a week followed by a rapid decline. Thus the total antigen load in a sub-tumorigenic live inoculum (2M cells) is still only about 10% of the antigenic load in an irradiated cell inoculum (20M cells). While we explore the mechanisms of the unusual immunogenicity of live cells, we presently conclude (i) that higher immunogenicity is not a direct function of antigen load, and (ii) live cells cross-prime the naïve T cell response in a way that is distinctly different from, and more efficient than irradiated cells.
Myeloid dendritic cell-specific mTORC2 deficiency enhances Th1 and Th17 cell responses and inhibits B16 melanoma growth (TUM6P.959)
Authors: Raich-Regue Dalia , Rosborough Brian , Fabian Kellsye , Turnquist Heth , Storkus Walter , Thomson Angus
Published: January 2023

The mechanistic/mammalian target of rapamycin (mTOR) is a key integrative kinase that functions in at least two independent complexes: mTOR complex (C)1 and mTORC2. While mTORC2 in T cells promotes Th2 differentiation, its immunologic function in dendritic cells (DC) is largely unknown. We defined the role of rictor, a key mTORC2 component, in myeloid DC and their ability to polarize allogeneic T cells. When stimulated with TLR ligands, bone marrow-derived DC generated from conditional Rictor knockouts displayed lower co-inhibitory B7-H1 (CD274) molecule expression, enhanced IL-12p70, IL-23, IL-6 and TNFα production and augmented allogeneic T cell stimulatory ability. Analysis of antigen-specific CD4+ T cells after activation by Rictor-/- DC in vivo revealed significant expansion of IFN-γ+ and IL-17+, but not IL-10+ or CD4+Foxp3+ T cells. Consistent with this pro-inflammatory effect, intratumoral injection of Rictor-/- DC markedly slowed B16 melanoma growth in B6 WT mice compared to control DC (p<0.05), promoting enhancement of CD4+IL-17+ and CD8+INFγ+ T cells in the spleens of Rictor-/- DC treated animals compared with controls. These data indicate that DC lacking mTORC2 activity exhibit an enhanced pro-inflammatory profile with ability to promote Th1/Th17 responses and slowed melanoma progression versus control DC. Therefore, targeting mTORC2 provides new insight into molecular regulation of DC function with therapeutic implications for cancer.
Dectin-1 activation subverts the suppression of myeloid-derived suppressor cells by inducing PMN-MDSC apoptosis and monocytic MDSC differentiation to potent antigen-presenting cells in cancer (TUM6P.961)
Authors: Albeituni Sabrin , Ding Chuanlin , Liu Min , Hu Xiaoling , Luo Fengling , Yan Jun
Published: January 2023

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells that promote tumor progression. Herein, we demonstrate that activation of a C-type lectin receptor, dectin-1, in MDSC differentially modulates the function of different MDSC subsets. Yeast-derived particulate β-glucan (WGP), a ligand to engage dectin-1, oral treatment in vivo significantly decreases tumor weight and splenomegaly in tumor-bearing mice by reducing the accumulation of PMN-MDSC but not M-MDSC in spleens and tumors, and decreases PMN-MDSC suppression in vitro through the induction of respiratory burst and apoptosis. On a different axis, WGP-treated M-MDSC differentiate into F4/80+CD11c+ cells that can uptake and present OVA antigen to OVA-specific CD4+ T cells and cross-present antigen to OVA-specific CD8+ T cells in a dectin-1 dependent manner. In addition, ERK1/2 phosphorylation is required for the acquisition of APC properties in M-MDSC, since M-MDSC treated with ERK inhibitor and WGP do not differentiate into APC. Moreover, WGP-treated M-MDSC do not promote tumor growth in vivo when inoculated subcutaneously with LLC cells. Interestingly, patients with non-small cell lung cancer (NSCLC) that received WGP treatment prior to any treatment for two weeks had a decreased frequency of CD14-HLA-DR-CD11b+CD33+ in the peripheral blood. Overall, these data indicate that WGP may be a potent immune modulator of MDSC suppressive function and differentiation in cancer.
Carcinoembryonic antigen signaling suppresses the activation of human T cells by a SHP-independent mechanism : a potential mechanism for tumor-mediated suppression of T cell immunity. (TUM7P.1025)
Authors: Bae Eun-ah , Lee Kyoo A , Song You Chan , Kang Chang-Yuil
Published: January 2023

Carcinoembryonic antigen (CEA) is a well-known tumor antigen that is found in the serum of patients with various cancers and is correlated with an increased risk of cancer recurrence and metastasis. To understand the tumor environment and to develop anti-tumor therapies, carcinoembryonic antigen has been studied as an antigen to activate/tolerate specific T cells. In this study, we show that carcinoembryonic antigen can function as a co-inhibitory molecule and can inhibit the activation of human PBMC-derived T cells. Addition of CEA-overexpressing tumor cells or immobilized CEA dampened both cell proliferation and the expression of IL-2 and CD69 expression in T cells after TCR stimulation. The phosphorylation of ERK and translocation of NFAT were hampered in these cells, while the phosphorylation of proximal TCR signaling molecules such as ZAP70 and PLCγ was not affected by immobilized CEA. To determine the relevance of CEACAM1 and SHP molecules to carcinoembryonic antigen-mediated suppression, we tested the effect of the SHP inhibitor, NSC-87877 on carcinoembryonic antigen-mediated suppression of T cells, but it did not reverse the effect of carcinoembryonic antigen. Collectively, these results indicate that carcinoembryonic antigen can function as a modulator of T cell responses suggesting a novel mechanism of tumor evasion.
Immunometabolic editing: a new type of immunoediting in tumorigenesis (TUM7P.1027)
Authors: Tyson Danielle , Ho Ping-Chih , Tsui Yao-Chen , Kaech Susan
Published: January 2023

Immune evasion and deregulated metabolic transformation are hallmarks of cancer that contribute to tumor aggressiveness and metastasis through different mechanisms. However, it has not been examined whether there is a connection between these two hallmarks. In this study, we approach this question by both melanoma engraftment and a genetically modified melanoma mouse model. First we investigated whether the presence of CD4 or CD8 T cells during tumorigenesis has any effect on a tumors metabolic profile by depleting these cells post-tumor induction. We found that T cell depletion in the early phase of tumorigenesis led to down regulation of hexokinase 2 and pyruvate kinase isozyme M2. Then we investigated whether the effector functions of T cells, especially CD40L and interferon gamma (IFNγ), promote tumor cells to acquire deregulated metabolic activity. It appears the IFNγ may be a major player in the metabolic selection process. Finally, we aim to examine whether the metabolic activity of tumor cells has an effect on the immune activity. In the pilot test, we identified three different melanoma cell lines derived from genetically modified melanomas that have distinct glycolytic activity and oxidative phosphorylation. These tools lay a foundation for future analysis to investigate whether the metabolic features of melanoma cells can play a regulatory role in controlling anti-tumor immunity and the formation of an immunosuppressive microenvironment.
Regulation of CTL trafficking into tumors and anti-tumor immunity by chemoattractant receptors BLT1 and CXCR3 (TUM7P.1015)
Authors: Chheda Zinal , Sharma Rajesh , Jala Venkatakrishna , Bodduluri Haribabu
Published: January 2023

A major hurdle limiting the success of tumor immunotherapies in the clinic is poor recruitment of CD8+ T cells (Cytotoxic T Lymphocytes-CTL) into tumors despite efficient CTL responses in the periphery. However, little is known about the mechanisms that regulate their infiltration into tumors. We investigated the role of leukotriene B4 receptor, BLT1 and CXCR3, the receptor for CXCL9 and CXCL10 in anti-tumor immunity using a syngeneic B16 melanoma mice model. Significant acceleration in tumor growth and reduced survival was observed in both BLT1-/- and CXCR3-/- mice as compared to WT mice. Tumor immune infiltration studies revealed significant reduction of CTLs in BLT1-/- and CXCR3-/- tumors as compared to WT tumors, their frequencies being similar in the periphery. Adoptive transfer of WT but not BLT1-/- or CXCR3-/- CTLs reduced tumor growth in Rag2-/- animals due to enhanced WT CTL infiltration to tumors. Furthermore, adoptive transfer of BLT1-/-, CXCR3-/- or BLT1-/-CXCR3-/- CTLs together with WT CTLs showed that initial migration of BLT1+/+ CTLs to tumors facilitates additional CTL recruitment in a CXCR3 dependent manner. An anti-PD-1 antibody based vaccine completely lost its efficacy in both BLT1-/- and CXCR3-/- mice suggesting a requirement for these receptors in generating sustained anti-tumor immune response. These results demonstrate a critical role for BLT1 and CXCR3 in CTL migration to tumors and thus can be targeted to elicit effective anti-tumor responses.
Rescuing CTLs from undergoing TCR restimulation induced necroptosis improves tumor control (VAC7P.1034)
Authors: Kesarwani Pravin , Gudi Radhika , Chatterjee Shilpak , Scurti Gina , Nishimura Michael , Mehrotra Shikhar
Published: January 2023

Engineering T cells with tumor epitope reactive T cell receptors (TCR) for adoptive cell transfer (ACT) therapy requires rapid proliferation and expansion, resulting in replicative senescent phenotype and increased susceptibility to cell death. Cell death may occur as apoptosis (programmed) or necrosis (un-programmed), or programmed necrosis (termed necroptosis). Our data shows that human T cells engineered with tumor epitope tyrosinase specific TIL1383I TCR upon restimulation with cognate antigen undergo caspase independent and reactive oxygen species mediated cell death cell death that involves JNK. Further, electron microscopic studies revealed that primarily effector-memory phenotype bearing T cells underwent antigen stimulated cell death resembling necroptosis, which was averted by pretreating with necroptosis inhibitor NecroX2 that inhibits RIPK1. Further, in vivo studies done to treat subcutaneously established B16-F10 tumors in C57BL/6 mice showed better tumor control when adoptively transferred tumor epitope gp100 reactive T cells were pretreated with NecroX2. Long-term tracking studies showed that NecroX2 pretreated T cells persisted better than the untreated T cells alone, and could account for differences in tumor growth. Our data highlights that understanding the morphology/type of cell death that a tumor reactive T cell may undergo upon TCR restimulation is important to effectively rescue anti-tumor T cells for increasing persistence and improving ACT.
Functional deficiencies in chimeric antigen receptor T cells from geriatric donors (VAC7P.1035)
Authors: Guha Prajna , Cunetta Marissa , Thorn Mitchell , Point Gary , Junghans Richard , Katz Steven
Published: January 2023

Age-related immune dysfunction poses unique and poorly defined challenges to the use of cancer immunotherapy in geriatric patients. We sought to test our hypothesis that CAR-T from geriatric patients (gCAR-T, n=3, >65 years) are functionally impaired relative to CAR-T from younger patients (yCAR-T, n=3, 18-45 years). Donor T cells were genetically modified to express CAR specific for carcino-embryonic antigen (CEA), presently in use in our phase I trials. Untransduced and CAR-T cells were expanded ex vivo with IL2 (3000IU/ml) or IL15 (5ng/ml) for 20 days. A trend towards higher transduction efficiencies (35.2% yCAR-T vs 24.9% gCAR-T, p=0.09) and cell expansions of 2.5 fold, (p=0.01, two-tailed t-test) were observed in yCAR-T cells for both treatments as compared to gCAR-T. yCAR-T contained higher proportions of CD4 effector cells (EC) (64.3+7.6%,p=0.003), CD4 effector memory (EM) cells (43.2+6.8%,p=0.002) and CD8 effector memory cells (EM) (16.5+3.6%,p=0.002). In accordance with higher numbers of CD4 and CD8 TEM, yCAR-T demonstrated higher levels of (1.3-fold over gCAR-T, p=0.03) CEA specific cytotoxicity compared to gCAR-T, with maximum cytotoxicity observed in IL15 treated yCAR-T cells. Enhanced tumor killing by yCAR-T correlated with increased levels of perforin (1.3+0.1 ng/ml,p=0.01) and granzymeB (0.4+0.03 ng/ml, p=0.02). Our pilot study suggests gCAR-T demonstrate age-related deficiencies that may impact production and anti-tumor efficacy.
Evaluating use of the probiotic yeast Saccharomyces boulardii for the synthesis and delivery of oral therapeutics (VAC8P.1061)
Authors: Hudson Lauren , McDermott Courtney , Fasken Milo , Kuiper Emily , Guiliano David , McBride Shonna , Corbett Anita , Lamb Tracey
Published: January 2023

Recent studies have explored the possibility of using microorganisms to synthesize and deliver therapeutics for the treatment of gastrointestinal disorders. Probiotic organisms, particularly the eukaryotic probiotic yeast Saccharomyces boulardii, may be well suited for this task given their current clinical applications for the treatment of gastrointestinal infections and inflammatory bowel diseases. Saccharomyces is furthermore capable of performing eukaryotic post translational modifications and can thus be made to express a wide array of therapeutic proteins. In order to increase the ease with which S. boulardii can be transformed to express recombinant protein, we developed auxotrophic mutant strains of S. boulardii using UV mutagenesis. Unlike wild type S. boulardii, these mutant S. boulardii can be genetically manipulated without reliance on antibiotic selection. These mutants also show a similar profile of resistance to conditions found in the gut, such as low pH and bile acid salts, relative to wild type S. boulardii. Critically, we demonstrate through oral gavage experiments that viable mutant S. boulardii recovered from murine gastrointestinal immune tissues continue to express recombinant protein. These mutant S. boulardii will thus permit studies to evaluate the ability of eukaryotic probiotics to deliver oral recombinant therapeutics and provide adjunctive therapy for the treatment of gastrointestinal disorders.
Leukocyte immune-type receptors are markers of antiviral cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus (VET1P.1125)
Authors: Taylor Erin , Wilson Melanie , Bengten Gertrud
Published: January 2023

LITRs are a multigene family in teleosts that encode immunoglobulin superfamily receptors with activating or inhibitory signaling capability, but their functional role in the immune response is not well understood. These immunoregulatory receptors exhibit sequence homology to both Fc receptors and molecules encoded in the leukocyte receptor complex. By using a combination of immunoprecipitation and protein identification by LC-MS/MS, we determined that the monoclonal antibody CC41 binds to LITRs on clonal cytotoxic T lymphocytes (CTL). Consequently, we hypothesize that this antibody can be used to monitor the in vivo antiviral CTL response. To test this hypothesis, we injected gynogenetic catfish with a channel catfish virus (CCV)-infected MHC-matched clonal T cell line (G14D-CCV) and collected peripheral blood lymphocytes (PBL) at 3, 5, and 9 days after injection for analyses by flow cytometry. The percentage of CC41+ cells was elevated five days after primary immunization and three days after booster immunization. When the CC41+ cell population was magnetically isolated from the PBL, they were found to specifically kill CCV-infected targets, as shown by 51Cr-release assays, and this killing is partially inhibited by the addition of CC41. When subjected to flow cytometric mRNA analyses, the CC41+ cells were found to express message for CD3γδ, perforin, and eomesodermin (EOMES), indicating that these cells are in fact CTL.
Diminished innate, but not adaptive, immune responses in migrating adult Chinook salmon, Oncorhynchus tshawytscha (VET1P.1127)
Authors: Dolan Brian , Fisher Kathleen , Colvin Michael , Benda Susan , Danley Courtney , Peterson James , Kent Michael , Zwollo Patty , Schreck Carl
Published: January 2023

In wild systems, animals have to balance the energy required to mount an immune response with other requirements necessary to survive and procreate. An extreme example is migrating adult Pacific salmon species, which cease feeding at the start of their migration from salt water to fresh water. During the migration, salmon must swim against strong currents, compete for mates, evade predation, and contend with freshwater pathogens. We quantified the immune response in migrating adult Chinook salmon, Oncorhynchus tshawytscha, at different times and locations during the run using several different measures. We measured the levels of immunoglobulin heavy chain mu and heavy chain tau mRNA transcript in the anterior kidney and found that levels of both secreted and membrane bound forms of the heavy chain did not diminish during migration, and importantly, that they remained unchanged even after spawning. We also measured the innate anti-bacterial ability of plasma isolated from fish to prevent the growth of a lab strain of E. coli. Contrary to the humoral immune response, the innate anti-bacterial components of plasma diminished at later time points in the run leading up to spawning. Diminished immune response was not due to IL-10 secretion, as transcripts for this cytokine were similar at all time points that were examined. These data suggest that in the face of limited energy resources, innate immune responses diminish while adaptive responses remain unchanged.
Characterizing the murine immune response to Corynebacterium pseudotuberculosis: approaches to developing a C. pseudotuberculosis vaccine (VET1P.1119)
Authors: Hino Christopher , Crowley Daniel , Chisholm Gregory , Geiger Kristina , Sumida Megan , Molinder Karen , Pollock Roberta
Published: January 2023

Pigeon Fever is a major infectious equine disease caused by the Gram-positive, intracellular bacterium Corynebacterium pseudotuberculosis. The disease manifests as external abscesses, internal infections, or ulcerative lymphangitis. Horses with external abscesses have a very low mortality rate, while horses with the less common internal infections have a high mortality rate of up to 40%. We have developed a mouse model for this disease to examine the roles of different T-cell and antibody responses to C. pseudotuberculosis infection. BALB/c and C57Bl/6 mice were challenged intradermally with varying doses of live bacteria following immunization with crude bacterial fractionations. Analysis of serum antibody levels of unvaccinated mice suggests that both mouse strains did not mount a significant humoral immune response against C. pseudotuberculosis infection. By contrast, mice mounted a high IgG response after being immunized with crude bacterial fractions, which providing partial protection against infection. We have generated monoclonal antibodies from immunized mice and used these antibodies to immunoprecipitate their respective antigens. Three proteins have been identified by mass spectrometry, and are being cloned so that the recombinant proteins can be tested for their effectiveness as vaccine components. These studies highlight the potential of a vaccine strategy based on the use of immunogenic bacterial proteins for use in a protective vaccine.
Th17 deviation under influence of Th1 response attenuates the inflammation in late phase of influenza virus infection (VIR5P.1138)
Authors: Huang Ching-Tai , Dutta Avijit , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia
Published: January 2023

Overwhelming inflammation with cytokine storm has been postulated as a major cause of severe influenza disease in human. In our influenza Ag- specific mouse experimental system, we discovered a Th17 deviation of CD4+ T cell response with resultant attenuation of inflammation in the late phase of influenza infection. Naïve hemagglutinin (HA)-specific CD4+ T cells respond to acute influenza virus infection with Th1 phenotype. In the presence of antigen specific Th1 CD4+ T cells, naïve CD4+ T cells adoptively transferred 4 days after the acute infection are activated with deviation to the Th17 phenotype. The second batch of naïve HA-specific CD4+ T cells is activated in an environment enriched with Th1 cytokines and active TGF-β. They proliferate, secrete IL-17 but not IFN-γ and express LAG-3 on the cell surface. They can suppress T cell activation in vitro and decrease lung inflammation in vivo. The 6.5 mice, with monotonous TCR repertoire responsive to influenza HA, reveal a Th17 deviated CD4+ T cell response and significant survival benefit in influenza virus infection. Consecutive waves of naïve CD4+ T cells from the thymus in 6.5 mice are probably activated as subsequent batches of naïve CD4+ T cells, with Th17 deviation which attenuates the inflammation in late phase of influenza virus infection.
Herpes simplex virus tegument protein VP11/12-derived epitopes preferentially recall polyfunctional effector memory CD8+ T cells from seropositive asymptomatic individuals and protect “humanized” HLA-A*02:01 transgenic mice against ocular herpes (VIR6P.1172)
Authors: Benmohamed Lbachir , Khan Arif , Srivastava Ruchi
Published: January 2023

In this study, we used multiple prediction computer-assisted algorithms to identify 10 potential HLA-A*02:01-restricted CD8+ T cell epitopes from the 716 amino acids sequence of Herpes Simplex Virus type 1 virion tegument phosphoprotein 11/12 (HSV-1 VP11/12). Three out of ten epitopes exhibited high to moderate binding affinity to HLA-A*02:01 molecules. In ten sequentially studied HLA-A*02:01 positive, HSV seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease), the most frequent, robust and polyfunctional effector CD8+ T-cell responses, as assessed by a combination of tetramer frequency, granzyme B, granzyme K, perforin, CD107a/b cytotoxic degranulation, IFN-g and multiplex cytokines assays, were predominantly directed against three epitopes: VP11/1266-74, VP11/12220-228 and VP11/12702-710. Interestingly, ASYMP individuals had significantly higher proportion of CD45RAlowCCR7lowCD44highCD62LlowCD27lowCD28lowCD8+ effector memory T cells (TEM) specific to the three epitopes, compared to symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpetic disease). Moreover, immunization of HLA-A*02:01 transgenic mice with the three ASYMP CD8+ TEM cell epitopes induced a strong protective immunity against ocular herpes infection and disease. Our findings outline the features of protective HSV-specific CD8+ T cells that should guide the development of an effective T-cell-based herpes vaccine.
Mouse polyomavirus tropism alters magnitude and functionality of virus-specific CD8 T cells (VIR6P.1173)
Authors: Qin Qingsong , Jin Ge , Lukacher Aron
Published: January 2023

Mouse polyomavirus (MPyV) is a ubiquitous persistent natural mouse pathogen. Human JC polyomavirus variants associated with progressive multifocal leukoencephalopathy have discrete amino acid mutations in VP1, the major capsid protein, that affects tropism for glial cells. A glutamic acid (E) to glycine (G) difference at position 92 of VP1, shifts the profile to tumors induced by MPyV from epithelial to mesenchymal cell origin. Here, we asked if this shift in tropism affects the MPyV-specific CD8 T cell response, which controls MPyV infection/tumorigenesis. We found that the A2 (92E-VP1) and RA (92G-VP1) MPyV strains differ sharply in MPyV-specific CD8 T cell responses. A2 virus generates a robust, high magnitude virus-specific CD8 T cell response, whereas RA virus elicits a markedly smaller anti-MPyV CD8 T cell response. Infection by an A2 92G-VP1 mutant virus recapitulates the antiviral CD8 T cell response seen with RA virus. Notably, the antiviral CD8 T cell recall response during persistent infection by this A2 mutant significantly exceeded that of A2 virus. Using immunization with peptide-loaded dendritic cells in the setting of bystander inflammation by A2 or the mutant virus, we found that A2 virus stimulates a higher magnitude MPyV-specific CD8 T cell response; this difference is negated in DC-deficient Flt3-/- mice. Together, these data indicate that viral tropism for DCs is a major determinant of the quantity and quality of the anti-MPyV CD8 T cell response.