bluuebunny/crossref_metadata_2025 huggingface dataset:
Schistosoma Mansoni infection induces anti-atherogenic transcriptional changes in hepatic macrophages (MPF2P.758)
Authors: Fairfax Keke , Elvington Andrew , Pearce Edward , Randolph Gwendalyn
Published: January 2023

Hepatic macrophages play an essential role in the granulomatous response to infection with the parasitic helminth Schistosoma Mansoni, but the transcriptional changes that underlie this participation are poorly understood. To explore this, we sorted the two previously recognized hepatic macrophage populations (perivascular and Kupffer) from naïve and S. mansoni infected mice and performed microarray analysis as part of the Immunological Genome Project. Consistent with the pattern of great diversity identified in other organ macrophages, the two hepatic macrophage populations displayed signatures distinct from all other macrophages, with the two populations exhibiting remarkable differences between them. However, this diversity was greatly reduced following infection with S. mansoni, and in fact, many of the transcripts identified as uniquely perivascular or Kupffer cell specific were lost following infection, raising the possibility that both populations may be replenished by monocytes following infection. Our analysis showed a profound alteration in phospholipid and cholesterol metabolic pathways, including prostaglandin signaling, in addition to the expected M2 markers. These changes suggested a possible mechanism for the previously reported atheroprotective effects of S. mansoni infection. Indeed we find that ApoE null mice fed a high fat diet in combination with S. mansoni infection have reduced body mass in addition to reduced plaque area as compared to control mice.
Hepatic recruitment of the inflammatory cell subsets upon liver injury duo to Echinococcus multilocularis (MPF2P.761)
Authors: Zhang Chuanshan , Wen Hao , Lin Renyong
Published: January 2023

Alveolar echinococcosis (AE) is a chronic disease characterized by slowly expanding liver lesions.In addition to liver-resident Kuffer cells, infiltrating immune cells have recently been linked to the development of liver injury.The role of these inflammatory cell subsets and functional contributions of their differential recruitment into the injured liver are not defined.Here we studied in vivo the phenotype and function of infiltrating immune cells around the lesion in AE patients and mice model by means of immunohistochemistry and flow cytometry.AE patients with parasitic lesions displayed a significant increase of activation of predominantly CD163+ kuffer cells and CD14+ monocyte cells around lesion compared to the diatal liver tissue.Infected mice showed that inflammatory CD11bhi monocytes are massively recruited into the liver upon E.m-induced injury in CD11b++F4/80+ intrahepatic macrophages,and the proportion of resident macrophages and eosinophils increased after 6 weeks infection. In addition, CD8+ effector T cells displayed a less exhausted phenotype characterized by decreased expression of programmed cell death 1 (PD-1).These findings indicate that inflammatory monocytes recruited into the injured liver and the percentage of kuffer cells, eosinophils and CD8+PD-1lo T cells increased around lesion, promote the progression of liver injury. These data suggest that they could be related to resistance or sensitivity to infection by E. multilocularis in humans.
Role of IL-17 during Helicobacter pylori infection (MPF2P.750)
Authors: Contreras Diana , Patel Rachna , Algood Holly
Published: January 2023

Helicobacter pylori is a bacterium found in the gastric microbiota of a large percentage of the world’s population. H. pylori colonization results in mild inflammation in all patients, however some patients go on to develop peptic ulcers and gastric cancer. An effective adaptive immune response, which involves a mixed T helper (Th)1, Th17 and Treg response, is essential to minimize colonization but is also responsible for the increased inflammation. Th17 cells, which produce IL-17A, IL-17F, IL-21 and IL-22, are believed to be one of the driving immune cells in response to H. pylori infection. The purpose of our study was to investigate the differential roles of IL-17A, IL-17F, and IL-17A/F in controlling H. pylori colonization and H. pylori-induced gastritis. To this end, IL-17A, IL-17F, and IL-17A/F deficient mice were infected with H. pylori strain PMSS1. After 3 months of infection, the ability of wild-type mice and cytokine deficient mice to control colonization and activate T and B cell responses was assessed. Analyses included measurement of pro-inflammatory cytokines, antibody production, inflammation, and colonization. The pathology suggests that any deficiency of IL-17 leads to increased inflammation and increased cytokine production by other types T helper cells. Although inflammation is beneficial in keeping a low bacterial burden, increased inflammation during IL-17 deficiency may be a key factor in the adverse outcomes of some patients.
Local antigen and inflammation in the lung induce the preferential establishment and maintenance of CXCR6 and CD49a-expressing antigen-specific CD8 TRM cells in the lung parenchyma and airways (MUC1P.900)
Authors: McMaster Sean , Wilson Jarad , Wein Alex , Dunbar Paul , Matloubian Mehrdad , Kohlmeier Jacob
Published: January 2023

Acting as sentinels capable of quickly organizing a secondary immune response upon pathogen challenge, resident memory CD8 T (TRM) populations are initially primed by microenvironment cues and APC licensing following acute infection of peripheral tissues. Despite their capacity for heterologous protection against influenza virus (flu) challenge, scant knowledge exists as to factors necessary to establish and maintain airway and lung parenchymal (LP) CD8 TRM cells. In contrast to mechanisms described for other tissues, lung CD8 TRM cell establishment required cognate antigen recognition once cells were recruited to the lung by local inflammation. Furthermore, this combination of local antigen and inflammation formed long-lasting TRM populations in the LP and airways that highly express the chemokine receptor CXCR6 and adhesion molecule CD49a. During a primary intranasal (IN) flu infection, CXCR6 is preferentially expressed on virus-specific CD8 T cells in the airways and LP by day 7 post-infection. Selectively, LP CD8 TRM cells highly express CXCR6, while systemically circulating flu-specific cells achieve only an intermediate expression level. Finally, use of mixed bone marrow chimeras show that wild-type virus-specific memory CD8 T cells predominate over CXCR6-/- cells in the airway and LP after IN infection with Sendai or flu virus. Thus, our data show that local antigen and inflammation coupled with CXCR6 expression is key to establish CD8 TRM cells in the LP and airways.
Respiratory exposures to pneumococcus establish localized memory capable of heterotypic lung protection (MUC1P.914)
Authors: Stauffer Nicole , Coleman Fadie , Hilliard Kristie , Yamamoto Kazuko , Zabinski Mary , Jones Matthew , Quinton Lee , Mizgerd Joseph
Published: January 2023

Pneumonia is a persistent and pervasive public health burden, often caused by diverse serotypes of Streptococcus pneumoniae. We hypothesize that repeated respiratory infections establish capsule-independent adaptive immunity that protects against subsequent unrelated pneumococcal pneumonia. We have established a mouse model in which mice are infected with diverse pneumococci in the respiratory tract, given time to recover, and then challenged with a mismatched virulent serotype 3 (Sp3) in the lung, which they cannot typically eliminate. Prior exposures to unrelated pneumococci proved protective, with multi-log reductions in lung CFU in mice previously exposed to unrelated pneumococci, compared to vehicle controls. RNAseq analysis of lungs 24h after Sp3 infection revealed an enrichment of lymphocyte-related pathways due to prior pneumococcal exposures, including immunoglobulin and other B cell-related genes (suggesting a local production of antibodies) as well as T helper cell signatures (particularly relating to Th17 cells). Prior to final infection, there were no differences in numbers of B cells or T cells in the lung, and no inflammation in H&E sections. However, ex vivo stimulation of CD4+ T cells revealed such lungs to contain more IL-17A producers, suggesting resident memory. We conclude that serial respiratory infections establish lung resident memory Th17 and B cells that cooperate to protect against pneumonia.
Direct effects of alcohol on the mucosal barrier and the implications on HIV transmission (MUC2P.935)
Authors: Nichols Whitney , Amedee Angela
Published: January 2023

Alcohol abuse is associated with an increased risk of HIV sexual transmission; however, the biological changes in the genital tract are unknown. The first line of defense is provided by the cervicovaginal epithelium acting as a physical barrier to infection. The epithelial cell innate properties are affected by various factors within the genital milieu. We hypothesize that alcohol abuse compromises cervicovaginal epithelial barrier function resulting in enhanced penetration of HIV to the underlying submucosa. Using a vaginal epithelial cell line grown at an air-liquid interface in a transwell, we modeled chronic alcohol exposure by supplementing the basolateral media with physiologically relevant levels of ethanol (50 mM). Epithelium barrier disruption was evident in the alcohol-treated cells by a significant decrease in TEER (p=0.001) and an increased permeability to 10kDa FITC-dextran (p=0.01) relative to control. Cell viability was unaffected by chronic alcohol exposure. Epithelial cells of the female genital tract exhibit significant changes in physical barrier integrity after exposure to intoxicating levels of alcohol. These changes in epithelial cell innate properties provide evidence for a biological mechanism of enhanced HIV penetration and infection in women who abuse alcohol. Future studies in alcohol-exposed cell culture models of both the upper and lower genital mucosa will identify specific mechanisms of HIV transmission.
Mesenchymal stem cells: new directions in treating asthma (THER2P.955)
Authors: Bonfield Tracey , Sutton Morgan , Lennon Donald , Caplan Arnold
Published: January 2023

INTRODUCTION: Mesenchymal stem cells (MSC) therapy has been investigated for inflammatory diseases. We studied MSCs efficacy in the murine model of chronic asthma hypothesizing that MSCs have the capacity to resolve inflammation and remodeling. METHODS: MSCs were administered to the ovalbumin murine model of chronic asthma after sensitization and 3 weeks of challenge. Animals were euthanized 7 days post-MSCs followed by bronchoalveolar lavage (BAL) for total cell count, differentials and cytokines. A separate set of animals were used to evaluate histopathology and markers of remodeling using ImagePro program and insoluble and soluble collagen levels. In vitro cultures of fibroblasts were used to study how MSCs impact remodeling. RESULTS: MSCs decreased weight loss, clinical score, epithelial cell hyperplasia, recruitment of BAL eosinophils, mucins, cytokines and systemic levels of IgE (P<0.05 for each). MSCs decreased soluble (28±6 to 13±9 mg collagen/lung) and insobuble (31±11 to 24±3 mg collagen/lung) collagen levels consistent with our ImagePro quantification. MSCs decreased type I and III collagen expression by 54±11% (n=3, p=0.04) and 30±8% (n=3, p=0.02) respectively. CONCLUSIONS: MSCs decreased airway inflammation and remodeling in the chronic asthma model emphasizing their unique therapeutic potential. ACKNOWLEDGEMENTS: Funded by the George and Virginia Baldwin Fund and American Lung Association: RG-169572.
Aberrant IL-17 production, mediated by SOCS1 deficiency, promotes inflammatory skin pathologies and lymphadenopathy in an IFN gamma independent manner. (THER2P.957)
Authors: Lam Brandon , Armbruster Cristina , Cain Antia , Collins Teresa , Larkin Joseph
Published: January 2023

Although it has been previously reported that mice deficient in SOCS1 develop lupus-like pathologies including skin lesions, the contributing pathogenic mechanisms have not been fully elucidated. We have observed that SOCS1+/- IFNγ-/- mice, but not IFNγ-/-, SOCS1+/-, or wild-type mice develop significant spontaneous skin pathology on the back and ears. The hyperplasia was characterized by significant accumulations of CD3+ T cells, F4/80+ macrophages, and neutrophils. Moreover, SOCS1+/- IFNγ-/- mice presented with lymphadenopathy and splenomegaly. Notably, SOCS1+/- IFNγ-/- leukocytes stimulated with αCD3/αCD28 in vitro were hyper proliferative and produced significant amounts of IL17 and IL-6 compared to IFNy-/- controls. To assess the contribution of excess IL17 production in this overt pathology, SOCS1+/- IFNy-/- mice were treated with IL17 neutralizing antibodies or an IgG control. Strikingly, administration of αIL17 mediated a significant reduction in lymphadenopathy, comparable to IFNγ-/- mice. Moreover, αIL17 treatment significantly reduced dermal leukocyte infiltration and skin lesions compared to SOCS1+/- IFNγ-/- mice receiving IgG control or no treatment. Together, these results present a novel role of SOCS1 in the regulation of IL17 production and subsequent onset of spontaneous skin pathology. Furthermore, these data suggest that amelioration of SOCS1 deficiency using SOCS1 mimetic peptides may have implications in the regulation of inflammatory skin pathologies.
Anthocyanin extracted from black soybean seed coats prevents autoimmune arthritis by suppressing the development of Th17 cells and synthesis of proinflammatory cytokines by such cells, via inhibition of NF-κB (THER3P.972)
Authors: Kim Sung-Min , Min Hong-Ki , Baek Seung-Ye , Woo Jung-Won , Park Jin-Sil , Cho Mi-La , Lee Jennifer , Kwok Seung-Ki , Park Sung-Hwan
Published: January 2023

We investigated the therapeutic effects of anthocyanin extracted from black soybean seed coats (AEBS) in a murine model of collagen-induced arthritis (CIA) and human peripheral blood mononuclear cells (PBMCs), and explored possible mechanisms by which AEBS might exert anti-arthritic effects. A CIA model was induced in DBA/1J mice.. Joints were assessed in terms of arthritis incidence, clinical arthritis scores, and histological features. The extent of oxidative stress in affected joints was determined by measuring the levels of nitrotyrosine and inducible nitric oxide synthase. NF-κB activity was assayed via Western blotting. Osteoclast formation was assessed via TRAP staining. In the CIA model, AEBS decreased the incidence of arthritis, histological inflammation, cartilage scores, and oxidative stress. AEBS reduced the levels of proinflammatory cytokines in affected joints of CIA mice and suppressed NF-κB signaling. AEBS repressed differentiation of Th17 cells and expression of Th17- associated genes in vitro, in both splenocytes of CIA mice and human PBMCs. Tartrate-resistant acid phosphatase+ (TRAP) multinucleated cells fell, in a dose-dependent manner, upon addition of AEBS. The anti-arthritic effects of AEBS were associated with decreases in Th17 cell numbers, and the levels of proinflammatory cytokines synthesized by such cells, mediated via suppression of NF-κB signaling. Additionally, AEBS suppressed osteoclastogenesis and reduced oxidative stress levels.
Blockade of surface bound TGF-β on regulatory T cells abrogates suppression of effector T cell function within the tumor microenvironment (TUM2P.1015)
Authors: Budhu Sadna , Schaer David , Li Yongbiao , Houghton Alan , Silverstein Samuel , Merghoub Taha , Wolchok Jedd
Published: January 2023

Regulatory T cells (Treg) play a role in suppression of anti-melanoma immunity; however, the exact mechanism is poorly understood. Through intravital two photon microcopy, we found that tumor-specific Pmel-1 effectors engage in cell-cell interactions with tumor resident Tregs. To determine if contact between Tregs and Teff hinders killing of tumor cells in vivo, we utilized ex-vivo three-dimensional collagen-fibrin gel cultures of B16 melanoma cells. Collagen-fibrin gel cultures recapitulated the in vivo suppression, rendering the dissociated tumor resistant to killing by in vitro activated antigen specific T cells. In vivo depletion of Tregs in Foxp3-DTR mice prior to tumor excision reversed the suppression. In vivo modulation of Tregs by GITR ligation also reduced the number of intra-tumor Tregs leading to ex-vivo tumor killing. Using neutralizing antibodies, we found that blocking TGF-β reversed the suppression. In addition, soluble factors from collagen-fibrin gel tumors do not inhibit killing suggesting that suppression is contact or proximity dependent. The CD8 T cells recovered from these gels exhibit a decrease in Granzyme B expression and an increase in expression of T cell exhaustion marker PD-1. These findings support the conclusion that intra-tumor contact with Tregs during the effector phase of the immune response is responsible for inhibiting anti-melanoma immunity in a TGF-β dependent manner shedding light into novel ways to inhibit intratumoral Tregs.
A novel monoclonal antibody to the HLA of cisplatin resistant ovarian cancer cells (TUM2P.1018)
Authors: Kaabinejadian Saghar , Patterson Andrea , Bardet Wilfried , Jackson kenneth , Mcmurtrey Curtis , Wichner Timea , Hawkins Oriana , Weidanz Jon , Hildebrand William
Published: January 2023

Cisplatin is widely used as a chemotherapeutic drug in the treatment of ovarian cancer. Resistance to cisplatin occurs in about one-third of women during the primary course of treatment. We hypothesized that the class I HLA of cisplatin-resistant ovarian cancer cells present peptides distinct to these cells as compared to sensitive cells and that HLA/peptide complexes unique to cisplatin-resistant cells would be valuable targets for immunotherapeutic intervention. To identify the peptides that are uniquely presented by cisplatin-resistant ovarian cancer cells, the intrinsic cisplatin-resistant cells (SKOV3) and sensitive cells (A2780, OV90, FHIOSE) were characterized by comparative mass spectrometry. Peptide sequences distinct to cisplatin-resistant cells include a peptide (VMF11) derived from thioredoxin interacting protein (TXNIP) that was in high abundance in SKOV3. Next a T cell receptor mimic monoclonal antibody (RL41A) against A*02:01/VMF11 complex was generated. The specificity and affinity of RL41A toward VMF11/A*02:01 complex was shown by staining peptide-pulsed T2 cells and surface plasmon resonance respectively. Staining of ovarian cancer cells by flow cytometry also demonstrates that RL41A stains cisplatin-resistant cells but not drug sensitive cells. We therefore report the successful development of a monoclonal antibody that represents an attractive candidate for further validation using cisplatin-resistant and sensitive primary ovary tissues.
Induction of robust antitumor immunity by exogenous interleukin-33 (TUM2P.1005)
Authors: Dominguez Donye , Ye Cong , Geng Zhe , chen siqi , Qin Lei , Fan Jie , long alan , Zhang Bin
Published: January 2023

IL-33 is associated with the induction of Th2-type immunity, however it has recently gained attention for its role in boosting Th1-type immunity and CD8+ T cell function. We have discovered that IL-33 can induce robust antitumor effect through a CD8+ T cell dependent mechanism. Systemic administration of recombinant IL-33 (rIL-33) alone was sufficient to inhibit growth of established tumors in multiple mouse tumor models. Notably, rIL-33 treatment promoted antitumor CD8+ T cell expansion and effector function. Mechanistically, rIL-33 therapy primarily and directly targets dendritic cells in tumor bearing mice, thereby restores CTL activity by increasing antigen cross-presentation within the tumor microenvironment. Furthermore, agonist activation of CD40, which is induced by rIL-33 treatment, stabilized and induced regression of established tumors. Our study has elucidated the potential use of rIL-33 as a novel immunotherapy option for the treatment of established cancers.
Devising a glycoantigen specific DC immunotherapy to develop an efficient anti-tumor response within the hypoxic environment of glioblastoma (TUM2P.1026)
Authors: Ginwala Rashida , Sagar Divya , Huang Xiaofang , Foss Catherine , Karakashev Sergey , Khan Zafar , Philip Ramila , Pomper Martin , Reginato Mauricio , Jain Pooja
Published: January 2023

Glioblastoma (GBM) is the most malignant type of brain tumor, its therapy hindered by the hypoxic microenvironment that promotes tumor resistance and inhibits immune cell function. We propose combining dendritic cell (DC) immunotherapy with hypoxia reversal to eradicate GBM. HIF-1α, a transcription factor that stimulates genes promoting angiogenesis and tumor growth, was expressed in both GBM cells and DCs under hypoxia. Hypoxia also downregulated expression of HLA-DR, CD86 and HLA-ABC on DCs affecting their antigen processing and presentation capability in addition to reducing the generation of pro-inflammatory cytokines such as TNF- α and IL-6. In vivo imaging revealed fluorescently labeled DCs migrating away from site of injection into spleen and mediastinal lymph nodes of mice with GBM indicating chemoattraction of DCs to areas of inflammation post hypoxia reversal using using antisense HIF-1α plasmid. In order to identify tumor antigens we used glycan analysis wherein GlcNAc was overexpressed in hypoxic GBM cells. We then enriched GlcNAc expressing glycoantigens and identified 46 glycopeptides derived from 33 glycoproteins. Amongst them Lamb1, SerpinH1, CD63 and others are associated with tumor survival, progression, invasion, immune evasion and therapy resistance. Lectins expressed on DCs complimentary to these glycans will be identified to devise a DC based vaccine, which in conjunction with hypoxia reversal will augment the efficacy of existing therapies against GBM.
A Toll-like receptor (TLR) 5 agonist entolimod mediates anti-metastatic activity via NK cell-dependent T cell activation (TUM2P.1029)
Authors: Brackett Craig , Kojouharov Bojidar , Veith Jean , Greene Kellee , Gollnick Sandra , Burdelya Lyudmila , Abrams Scott , Gudkov Andrei
Published: January 2023

Directing innate immunity against cancer is a promising but poorly developed approach to immunotherapy due to the risk of acute inflammation induced by innate immunity activators. In this regard, TLR5 is a unique innate immune receptor with a distinct tissue specificity of expression and induction of a specific cytokine profile upon activation by its agonist bacterial flagellin that is significantly safer than that induced by other TLR agonists. Entolimod is an optimized Salmonella flagellin derivative with demonstrated safety and efficacy in protecting normal cells from stresses such as radiation or chemotherapy, while not protecting or stimulating tumors. We show here that systemic administration of entolimod has potent antitumor effects in mouse syngeneic colon (CT26) and breast (4T1) cancer models, including suppression of liver metastases and development of CD8 T cell memory. In these models, entolimod treatment leads to blood-borne homing of NK cells to the liver followed by activation of CD4 and CD8 T cells. Antibody-mediated depletion and adoptive transfer experiments showed that all three of these cell types are essential for entolimod’s antitumor effects and demonstrate a pivotal role for NK cell-mediated CD8 and CD4 T cell activation and formation of CD8 T cell memory. Therefore, entolimod has strong promise as a safe, effective and broadly applicable immunotherapeutic agent against liver metastases, which are currently a major cause of cancer-associated mortality.
Un-masking the latent immunogenicity of neoepitopes by checkpoint blockade (TUM2P.1031)
Authors: Hodio Jennifer , Srivastava Pramod
Published: January 2023

Comparison of transcriptome sequences of the BALB/c mouse fibrosarcoma Meth A with the normal BALB/c genome led to identification of 1528 heterozygous tumor-specific point mutations, which, using the NetMHC algorithm, generated 203 Kd-restricted potential neoepitopes1. BALB/c mice were immunized with each of 11 candidate-neoepitopes which had a high binding affinity to Kd (IC50 <500 nM), and were challenged with live Meth A cells. Not a single candidate-neoepitope elicited tumor rejection. Mice were then immunized with the same candidate-neoepitopes in combination with 9D9, an antagonistic antibody to CTLA-4 (mouse anti-mouse IgG2b). In preliminary results, a small number of candidate-neoepitopes that did not elicit tumor rejection on their own appear to be active in tumor rejection, when combined with checkpoint blockade of CTLA-4. These results as well as a mechanistic dissection of this un-covering of latent immunogenicity by checkpoint blockade shall be presented. 1Duan F, Duitama J, Al Seesi S, Ayres CM, Corcelli SA, Pawashe AP, Blanchard T, McMahon D, Sidney J, Sette A, Baker BM, Mandoiu II, Srivastava PK. Genomic and Bioinformatic Profiling of Mutational Neoepitopes Reveals New Rules to Predict Anticancer Immunogenicity." J Exp Med. 211 (2014): 2231-248. PMID: 25245761
Effects of anti-VISTA mAb and its combination therapy in antitumor immunity (TUM2P.1035)
Authors: Kondo Yuta , Ohno Tatsukuni , Azuma Miyuki
Published: January 2023

[Purpose] V-domain Ig suppressor of T-cell activation (VISTA) is a novel immune checkpoint molecule that regulate T-cell activation. VISTA is expressed on both T cells and antigen-presenting cells. In this study, we generated a new mAb against VISTA (MIH63) and examined effects of monotherapy and combined therapy with either CTLA-4 or PD-1 blockade in murine SCCVII and CT26 tumor models. [Results] In a SCCVII tumor model, VISTA was expressed on all TIL and regional lymph node (rLN) T cells, whereas CTLA-4 was selectively induced in all CD8⁺ TIL and LN cells and PD-1 was induced on some T cells. Monotherapy with MIH63 did not clearly affect the growth of SCCVII and CT26, but significantly elevated CD8+ T cell activation assessed by CD25, IFN-g and TNF-a expression. MIH63 significantly reduced the number of CD8+ T cells. Monotherapy of CTLA-4 blockade efficiently reduced the tumor growth and a combination therapy with MIH63 further decreased the tumor growth. Consistently, MIH63 in the combination therapies enhanced CD8+ T cell activation, but decreased the ratio of CD8+ T cells. Blockade of FcγR by 2.4G2 restored the depletion of CD8+ T cells by MIH63, but not the CD8+ T cell activation. [Discussion] Our results suggest that MIH63 mainly acts as an antagonistic mAb to interfere regulatory function of VISTA, but in some part, induce CD8+ T cell-depletion via FcγR-mediated ADCC. The use of non-depleting anti-VISTA mAb may develop more effective results in the VISTA blockade.
Rescuing cancer immunity through formins and the actin cytoskeleton (VAC3P.1063)
Authors: Howard Andrew , Alberts Arthur
Published: January 2023

Immunotherapy strives to replace radiation, chemo and surgery in the fight against cancer by reversing cancer’s suppression of the immune system. CTLA-4 and PDL1 checkpoint blockade inhibitors are emerging therapies that release anti-tumor T cells to kill the tumor. However, cancer also alters myeloid activation to systemically suppress the immune system, which may explain the range of responses to checkpoint blockade inhibitors. We strive to better understand myeloid activation in cancer and develop therapies to modulate these altered responses. Through in vitro myeloid activation assays, we investigated the mechanism of action for two established immunomodulatory compounds, lenalidomide and tipifarnib, as well as tested novel small molecule Intramimics, developed by our lab, for immunomodulatory function. We found that lenalidomide and tipifarnib modulate immune responses through their activation of Rho GTPases and their downstream effectors to promote F-actin assembly. Interestingly, Intramimics prove to be better at modulating immune responses as they directly activate the diaphanous related formin effectors of Rho GTPases. We propose that modulation occurs mechanistically through the MADS box transcription factor SRF, which senses actin dynamics within the cell. We intend to confirm the dependency of SRF in immunomodulation and test these therapeutics in combination with checkpoint blockade inhibitors in preclinical cancer models in the future.
Genetically modified live attenuated L. donovani parasite induces classical activation of macrophages (M1 phenotype) leading to generation of Th1 response in BALB/c mice (VAC4P.1102)
Authors: Bhattacharya Parna , Dey Ranadhir , Dagur Pradeep , kruhlak Michael , Debrabant Alain , Okeeffe Nevien Ismail , McCoy John , Nakhasi Hira
Published: January 2023

Visceral Leishmaniasis (VL) causes significant mortality and there is no effective vaccine. We have tested live attenuated L. donovani parasites (Ldp27-/-and LdCen-/-) (LAPs) as vaccine candidates and demonstrated the protection through induction of an adaptive immune response. However the immunological basis of LAP mediated protection in terms of host innate response is poorly understood. In this study we showed, LAPs induced classical activation in bone marrow derived macrophages (BMDMs) as observed by significantly higher production of proinflammatory cytokines and nitric oxide and attenuated arginase-1 expression compared to LdWT parasites. Unlike LdWT infection, LAP infection did not alter the membrane fluidity of BMDMs, showed no impairment of antigen presentation and induced robust CD4+ T cell activation in vitro. Similarly, parasitized splenic macrophages from LAP infected mice showed up regulation of genes encoding classical activation markers such as IL-1β, TNF- α , IL-12 and iNOS2 and down regulation of genes associated with alternative activation like IL-10, YM1, Arg-1 and MRC-1 compared to LdWT infection. Furthermore, BMDM from LAP infected mice induced more IFN-γ and less IL-10 production by Leishmania antigen specific T cells than WT infected mice, thereby leading to proliferation of Th1 cells suggesting that LAP infection promotes a state of classical activation in macrophages in vitro and in vivo, leading to the generation of host protective Th1 response.
MicroRNA-155 plays a critical role in the establishment of latency in murine γ-herpesvirus infection (VIR1P.1148)
Authors: Crepeau Rebecca , Usherwood Edward
Published: January 2023

Gammaherpesvirus infection in immunosuppressed populations is associated with severe disease. MicroRNA-155 (miR155) has been shown to play significant roles in the immune response, including in the formation of germinal centers (GC) and the development and maturation of T follicular helper cells (Tfh). We sought to determine the relative contribution of miR-155 in the T and B cell compartments to latent murine gammaherpesvirus infection. Mice deficient in miR-155 had a 100-fold decrease in latent viral load, measured by qPCR, and exhibited decreases in both follicular helper cells and germinal center B cells. However, infection of mixed bone marrow chimeric mice showed a similar latent viral burden in both WT and miR-155 deficient B cells. Therefore the role of miR-155 may not be B cell intrinsic, but is likely mediated in part due to the regulation that miR155 exerts on the T follicular helper cell population. Studies using a YFP marker virus also address roles for miR-155 in virus reactivation and maintenance of latency. Further elucidating the relative role of miR155 on these cell populations will provide key insights into the development of latency in this model system.
Regulation of EBI2 expression by the EBV BRRF1 gene in B lymphocytes (VIR1P.1152)
Authors: Poole Brian , Cornaby Caleb , Birrell Cameron
Published: January 2023

Background: Epstein Barr Virus Induced gene 2 (EBI2), also known as GPR183, plays a role in migration of B cells. Expression of EBI2 is activated by Epstein Barr Virus (EBV). We investigated the mechanism of EBV activation of EBI2. By regulating EBI2 expression EBV may be able to manipulate B cell migration and interactions between B and T cells. Materials and Methods: EBI2 and EBV gene expression was measured using quantitative-PCR. The effects of the EBV gene BRRF1 were investigated using an EBV strain deficient for the Na protein and lentiviral transfection of the Na gene (BRRF1). Results: B cell lines and ex vivo B cells up-regulate EBI2 after EBV infection (p < .005). Treatment with inactivated virus did not affect EBI2 expression. EBI2 expression is higher in primary B cells than cell lines containing EBV (LCL and Ramos cells; LCL p =0.012, Ramos p=0.027, N=3). EBV BRRF1 expression correlated with EBI2, and BRRF1 deficient EBV did not affect EBI2 expression. Overexpression of BRRF1 using a lentiviral vector resulted in elevated expression of EBI2. Conclusions: EBI2 expression is directly influenced by EBV infection, rather than general cellular activation. BRRF1 is the viral gene responsible for EBI2 upregulation during infection of B cells with EBV. Since BRRF1 is expressed early in infection and during lytic infection, there may be an advantage to EBV in disrupting B cell migration at these times.
TCR-independent suppression of IFNγ production in vivo (VIR1P.1156)
Authors: Hosking Martin , Flynn Claudia , Whitton J Lindsay
Published: January 2023

After encountering cognate antigen in vivo, CD8+ rapidly secrete the pleiotropic anti-viral cytokine IFNγ. Interestingly, after producing a transient burst of IFNγ immediately following secondary viral infection, memory CD8+ T cells fail to sustain IFNγ production in vivo, even though ample viral antigen remains (Hosking et al. JI 2013). We did not know whether CD8+ T cells were (i) refractory to further TCR stimulation or (ii) actively suppressed and thus incapable of IFNγ production. We have, therefore, attempted to model discrete encounters with cognate antigen in vivo by injecting LCMV-immune mice with a cocktail of peptides corresponding to a subset of immunodominant CD8+ T cell epitopes. Virus-specific CD8+ memory T cells receiving TCR stimulation transiently produced a burst of IFNγ in vivo and phenotypically resembled antigen-specific CD8+ T cells responding to a secondary viral infection. On the other hand, bystander virus-specific CD8+ T cells that did not receive peptide stimulation did not produce IFNγ. To determine whether prior TCR stimulation limits subsequent cytokine production, peptide stimulated LCMV-immune mice were immediately rechallenged with LCMV, and CD8+ T cell cytokine production was assessed in vivo. Both prior-peptide stimulated and bystander virus-specific CD8+ memory T cells failed to produce IFNγ following infection. TCR-independent suppression of cytokine production was also observed within CD4+ T cells and appears to be mediated by IFNγ.
Intra-spike crosslinking overcomes antibody evasion by HIV-1 (VIR1P.1131)
Authors: Galimidi Rachel , Klein Joshua , Politzer Maria , Bai Shiyu , Seaman Michael , Nussenzweig Michel , West Anthony , Bjorkman Pamela
Published: January 2023

Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. We postulated that anti-HIV-1 antibodies primarily bind monovalently because HIV’s low spike density impedes bivalent binding through inter-spike crosslinking, and the spike structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a “molecular ruler” to measure intra-epitope distances on virion-bound spikes and construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders-of-magnitude increased potency (>100-fold average increases across virus panels) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection.
Study of binding characteristics of human monoclonal antibodies to differentially matured dengue virus particles (VIR2P.1172)
Authors: Tsai Wen-Yang , Lloyd Yukie , Wu Yi-Chieh , Lin Hong-En , Wang Wei-Kung
Published: January 2023

Dengue virus (DENV) is the leading cause of arboviral disease. The ectodomain of envelope protein, containing three domains, DI, DII and DIII, is the major target of DENV vaccine. Studies on monoclonal antibodies (mAbs) showed that DIII mAbs are more potent neutralizing (NT) than those mAbs binding DII fusion-loop (FL). Due to the inefficiency of furin protease activity, produced DENV is a mixture of mature, immature and partially immature virions. It remains unknown regarding the epitope accessibility, avidity and NT potency of different mAbs on different maturation status of DENV. Immature and mature virions were generated from DENV1 infected BHK cells with treatment of NH4Cl and transfected with furin, respectively. Capture ELISA were performed to examine human anti-FL and anti-DIII mAbs (kindly provided by Dr. J. Mongkolsapaya and Dr. F. Sallusto). Anti-FL mAbs had higher maximum binding (Bmax) to immature than mature virions (P=0.0015) and no difference in dissociation constant (Kd) to both virions, suggesting that they have more accessible epitopes on immature virions and bind both with similar avidity. Anti-DIII mAbs showed no difference in Bmax or Kd to both virions but had lower Kd to mature virions than Anti-FL mAbs (P=0.04). Considering the greater infectivity of mature than immature virions, our findings of higher avidity of Anti-DIII mAbs to mature virions compared with Anti-FL mAbs provide a mechanistically explanation for stronger NT potency of Anti-DIII mAbs.
Increased susceptibility to influenza A in a mouse model of MeCP2 duplication syndrome (VIR2P.1163)
Authors: Cronk James , Kim Taeg , Moser Emily , Sharma Ashish , Braciale Thomas , Kipnis Jonathan
Published: January 2023

MeCP2 duplication syndrome is a devastating neurodevelopmental disorder caused by duplication of the Xq28 region of the X chromosome, primarily affecting males. Methyl-CpG-binding protein 2 (MeCP2) ubiquitously binds methylated CpG sites in the genome, and regulates gene expression by recruitment of transcriptional mediators. MeCP2 is expressed highly in both neurons and immune cells, thus mutations or over-expression of MeCP2 are associated with neurologic pathology (including seizures, motor dysfunction, and cognitive impairment), and immunologic pathology. Many patients die by the age of 25, and recurrent respiratory infections are a common cause of death. We are studying the pathogenesis of influenza A virus in a mouse model of MeCP2 overexpression, the MeCP2Tg3 mouse, which expresses MeCP2 at ~3 fold normal levels. MeCP2Tg3 mice display significant mortality approximately one week post infection, with T and B cell deficiencies in both number and activation, further characterized by high viral titer and low virus-specific antibody levels in the infected lung. Preliminary evidence suggests that vascular and/or lymphatic impairment may underlie both immunologic defects and the increased mortality observed in MeCP2Tg3 mice during influenza A virus infection. In sum, these findings uncover MeCP2 as an important regulator of host response to respiratory virus infection and may inform treatment of human patients with MeCP2 duplication syndrome.
Location, abundance, and phenotype of follicular SIV-specific CTL during chronic infection (VIR2P.1169)
Authors: Skinner Pamela
Published: January 2023

The inability of HIV- and SIV-specific CTL to fully suppress virus replication may be due to a deficiency of CTL in B cell follicles. HIV-1 and SIV replication is concentrated within B cell follicles during chronic disease; whereas HIV and SIV-specific CTL fail to accumulate in large numbers in follicles. It is not known whether virus-specific CTL migrate throughout the entire follicular area including germinal centers, nor whether virus specific-CTL within B cell follicles are functional. We evaluated these questions using in situ tetramer staining and IHC in tissues from chronically SIV infected macaques. We found that SIV-specific CTL migrated into germinal centers at similar levels detected in other follicular areas. In addition, subsets of follicular SIV-specific CTL expressed the activation and proliferation marker Ki67, and subsets expressed the effector molecules perforin and granzyme B. However, we also found some in contact with Foxp3+ Tregs. Thus, the inability of SIV-specific CTL to clear follicular SIV producing cells is not due to a complete inability of these cells to migrate into germinal centers. In addition, although subsets of follicular SIV-specific CTL express Ki67 and effector molecules, their relative low abundance and contact with Tregs likely inhibits their ability to clear follicular SIV+ cells. These findings suggested that a functional cure for HIV might include strategies that augment functional virus-specific CTL within B cell follicles.
Heterologous protective immunity can lead to impaired activation, memory differentiation and inflation of new naive responses (VIR2P.1170)
Authors: Agarwal Priti , Kraft Anke , Watkin Levi , Kenney Laurie , Daniels Keith , Chen alex , Brehm Michael , Selin Liisa
Published: January 2023

Activation of crossreactive memory CD8 T cells leads to heterologous protective immunity. Here we question whether early activation of crossreactive memory CD8 T cells alters the activation of new naïve CD8 responses to the second virus. Pichinde virus (PICV) and lymphocytic choriomeningitis virus (LCMV) contain a crossreactive usually subdominant epitope, NP205, which differs in 6 of 8 amino acids. During PICV infection of LCMV-immune mice the NP205 memory response become immunodominant and mediates protective immunity. PICV is cleared early and does not persist. The usually dominant non-crossreactive new naïve PICV NP38 CD8 T cell response appears to be subdominant day 7 post infection. However, surprisingly over the next 100 days post infection the NP38 response continues to gradually expand and then stabilizes. These NP38-specific CD8 T cells appear to arrest in an intermediate effector T cell phenotype (KLRG1low, CD62Llow IL-7Rhi), with altered functionality (decreased TNFα production). They appear to have altered expression of apoptotic genes on gene array. This NP38 inflationary response did not occur when mice were immunized with an LCMV mutant that did not present the crossreactive NP205 epitope. Our data shows that the strong crossreactive response kills DC’s expressing NP205 early (day 2-3) in PICV infection. This may result in an early termination of antigen-presentation leading to inadequate activation of the naïve NP38 response and inflation.
Engineering MHC class I fine structure for activation of self-tolerant T cells
Authors: Parks Christopher A , Hansen Michael J , Ju Josephine M , Reed Brendan K , Kranz David M , Bordner Andrew J , Pease Larry R
Published: January 2023

Central tolerance imposed during thymic development and mechanisms of peripheral tolerance result in a mature T cell repertoire capable of cross-reacting weakly on self, but usually incapable of activation. We have observed mutations in class I heavy chains capable of activating normally tolerant T cells which can then cross-react effectively on self. We hypothesized that mutation of the class I heavy chain, based on our understanding of MHC fine structure, could be used to generate engineered MHC molecules capable of activating T cells normally incapable of activation due to self-tolerance that could then cross-react effectively on self, but without altering the presentation of bound peptides. Known ternary complex (pMHC:TCR) crystal structures were modeled to predict amino acid substitutions within the class I heavy chain resulting in increased stable contacts between pMHC and TCR without altering the structure of bound peptides. Four substitutions within the class I heavy chain meeting these criteria were predicted by the structure based modeling. Cell lines were established which demonstrated stable surface expression of the engineered class I molecules. In a direct test of binding, a single chain TCR was detected with 20 fold greater intensity on the surface of cells expressing engineered MHC molecules compared with wild-type MHC, while competitive inhibition of binding a labeled peptide demonstrated unaltered peptide presentation. Stimulation of T cells with a weak antigen presented by the engineered MHC resulted in enhanced activation and proliferation compared with wild-type MHC. In an in vivo transplant model, protective immunity was induced against a native tumor after challenge with tumor expressing engineered MHC.
An internalizing antibody induces the production of pro-inflammatory cytokines via Fc-dependent pathway in THP-1 cell.
Authors: Park Hyun Joon , Kwon Myung-Hee
Published: January 2023

Conventionally, the extracellular effector functions of antibodies are mediated by the heavy chain constant regions of Ig molecules, but recently, intracellular effector functions of antibodies have been reported. After antibodies binds to the virus and the antibody-virus complex enters the cytosol, the complex can be detected by TRIM21 that is an intracellular Fc receptor, leading to the degradation of the virus and activation of transcription factors related to immune response. In this process, the minimum requirement is the presence of antibody in the cytosol for interaction with TRIM21. We previously have shown that 3D8 IgG originated from an autoimmune-prone MRL-lpr/lpr mouse is an internalizing antibody that can spontaneously enter the cytosol. Here, we investigated whether 3D8 IgG regulates the immune response in diverse cells without viral infection. Treatment of 3D8 IgG wild type induces pro-inflammatory cytokines (IL-8 and TNF-α) in human monocyte cell line THP-1. In contrast, treatment of 3D8 IgG mutant which does not bind to TRIM21 up-regulates production of the cytokines, compared to 3D8 IgG wild type. Moreover, 3D8 scFv (single chain fragment variable) which is devoid of Fc region did not induce the production of cytokines. Our results demonstrate that the production pathway(s) of cytokines are TRIM21-independent and Fc-dependent manner. We suppose that Fc-interacting unknown molecules would induce the secretion of pro-inflammatory cytokines. Our study would provide a new insight to the pathogenesis of autoimmune disease.
Characterizing surface costimulatory molecule expression on plasmacytoid dendritic cells in male and female B6.Nba2 lupus-prone mice.
Authors: Gubbels Bupp Melanie R , Thoner Timothy , Jorgensen Trine N
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disorder that primarily affects women and is characterized by the production of autoreactive antinuclear autoantibodies and elevated levels of serum interferon-alpha (IFN-α). Female B6 mice congenic for the Nba2 lupus susceptibility interval develop a lupus-like disease including antinuclear autoantibodies, glomerulonephritis, and elevated serum IFN-α. Plasmacytoid dendritic cells (pDC) are capable of secreting high amounts of IFN-α and also present antigen. Ablation of Siglec H+ plasmacytoid dendritic cells in female B6.Nba2 mice is sufficient to reduce serum IFN-α and prevent the production of antinuclear autoantibodies as well as the development of glomerulonephritis. We hypothesized that genetic and/or sex-specific differences in pDC activation states may contribute to differential susceptibility to lupus. Therefore, we compared the expression levels of specific costimulatory molecules on pDCs in male and female control (B6) and lupus-prone (B6.Nba2) mice. Plasmacytoid DCs were categorized into 3 subsets: Siglec H+ CD19−, CD19+ Siglec H−, or double negative. Using flow cytometry, the expression of CD80, CD86, CD40, CD40L, ICOS, B7RP1, and MHC Class II were quantified in each subset. This study may provide insights into the genetic and sex-specific mechanisms by which pDCs contribute to SLE pathogenesis.