bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage migration inhibitory factor upregulates expression of receptor activator of NF-kappa B ligand(RANKL) in rheumatoid arthritis synovial fibroblasts (98.16)
Authors: Lee Sang Heon , Kim Kyoung Woon , Kim Hae Rim , Cho Mi La , Min Jun Ki , Seo Youngil , Jung Young-Ok , Lee Chang-Keun
Published: January 2023

Rheumatoid arthritis(RA) is characterized by chronic progressive inflammatory destruction of bone and cartilage, which is mediated by inflammatory cytokines. Macrophage migration inhibitory factor (MIF) is abundantly expressed in the sera, synovial fluids and synovial tissues of RA patients, and its level correlates with disease activity. The aim of this study is to explore the role of MIF in regulating RANKL expression and osteoclastogenic activity in RA synovial fibroblasts (FLS). FLS were separated from the synovium of RA patients and stimulated with rMIF. RANKL expression was determined by real time PCR, western blot and immunostaining. Osteoclast precursor cells were cocultured with MIF-stimulated RA FLS and subsequently stained for tartrate-resistant acid phosphatase (TRAP) activity. MIF upregulated the expression of RANKL in RA-FLS. The increased expression of RANKL mRNA and protein was completely blocked by AP-1 inhibitor (curcurmin), p38 MAPK inhibitor (SB203580) or JAK II-STAT2(AG490). Increased number of TRAP positive cells were observed when osteoclast precursor cells were incubated with MIF-pretreated RA FLS. These results suggest that MIF could be involved in the inflammatory bone destruction by regulating RANKL expression in RA FLS. The interaction between MIF and RANKL could be a potential gateway leading to new therapeutic approach in treating bone destruction in RA.
Regulation and function of newly-recognized IL-1 family cytokines in human bronchial epithelial cells (98.18)
Authors: Kato Atsushi , Chustz Regina T , Schleimer Robert P
Published: January 2023

The IL-1 family of cytokines, which now includes 11 members, is well known to participate in inflammation. Although the latest IL-1 family cytokines (IL1F5-11) have been shown to be expressed in airway epithelial cells, the regulation of their expression has not been studied. We investigated the regulation of IL1F5-11 in normal human bronchial epithelial cells. Messenger RNAs for IL1F6 and IL1F9, but not IL1F5 or IL1F10, were significantly up-regulated by TNF, IL-1b, IL-17 and TLR3 ligand dsRNA. Messenger RNAs for IL1F7, IL1F8 and IL1F11 (IL-33) were weakly up-regulated by some of the cytokines tested. Notably, mRNAs for IL1F6 and IL1F9 were synergistically enhanced by the combination of TNF/IL-17 or dsRNA/IL-17. IL1F9 protein was detected in the supernatant following stimulation with dsRNA (1.6 ng/ml) or a combination of dsRNA and IL-17 (2.5 ng/ml). IL1F6 protein was detected by western blot. We screened the receptors for IL1F6 and IL1F9 and found that fibroblasts expressed these receptors. We found that IL1F9 activated the MAPKs and the transcription factors including NFkB in primary normal human lung fibroblasts. IL1F9 also stimulated the induction of CXCL8 and CCL20. These results suggest that epithelial activation by respiratory viral infection and exposure to cytokines from Th17 cells (IL-17) may amplify neutrophilic inflammation in the airway via induction of IL1F6 and IL1F9.
The protective effect of Vitamin C on cardiomyocytes damage and heart failure (98.22)
Authors: kim hyemin , Kang Jae Seung , Maeng Hyung Gun , Bae Se Yeon , Lee Na Eun , Kong Joo Myoung , Hwang Young Il , Lee Wang Jae
Published: January 2023

In the heart, reactive oxygen species play a pivotal role in the apoptosis of cardiomyocytes, which is one of the major factors for heart failure. Although there are several reports regarding the effect of vitamin C as an antioxidant scavenging free radicals on patients with cardiovascular disease, the specific mechanism is still being elucidated. In this study, the effects of vitamin C on the prevention and protection of heart failure are investigated under stressful conditions composed by cold water and restraint using Gulo(-/-) mice which could not synthesize vitamin C endogenously. Survival rates of mice checked under stressful conditions showed that Gulo(-/-) mice were more susceptible to stress than wild type or vitamin C-supplemented Gulo(-/-) mice. The levels of heart failure-related proinflammatory cytokines were examined, where the levels of TNF-alpha, IL-1 alpha, and IL-6 were increased in Gulo(-/-) mice without stress. In Gulo(-/-) mice under stressful conditions, not only the levels of IL-1 alpha, TNF-alpha and soluble Fas ligand but also lipid peroxidation were remarkably elevated. And, the extensive apoptosis of cardiomyocytes in cardiac tissues from dead Gulo(-/-) mice by stress was observed. Taken together, it is suggested that vitamin C could effectively prevent stress-induced heart failure through the regulation of heart failure-related cytokines and the inhibition of apoptosis of cardiomyocytes.
Identification of lipopolysaccharide binding site on High Mobility Group Box 1 Protein (98.23)
Authors: Youn Ju Ho , Shin Jeon-Soo
Published: January 2023

Lipopolysaccharide (LPS), a bacterial endotoxin, triggers deleterious systemic inflammatory responses when released into blood circulation, causing severe sepsis, organ dysfunction and death. LPS binding components in serum play an important role in modifying LPS toxicity by facilitating its interaction with the LPS signaling receptor, which is expressed on LPS-responsive cells. We have previously shown that HMGB1 is able to bind LPS and this binding can increase LPS-induced TNF-α production in human peripheral blood monocytes. In this report, we identified several LPS binding regions within human HMGB1 sequence by pull-down experiments with synthetic biotinylated peptide derived from HMGB1. Using a conventional sequence-based comparison with several other LPS-binding proteins, we identified similar LPS-binding motifs in LPS-binding peptides from HMGB1, but inhibition assay demonstrated that the capacity of these peptides to interfere with LPS-HMGB1 interaction exhibits different characteristics We are now carrying out further experiments to evaluate which amino acid residues contribute to LPS binding and functional differences between different peptide in LPS-binding capacities. Our studies identified a defined region of HMGB1 involved in LPS binding, thereby implicating a physiological role of human HMGB1 as an LPS-binding protein. [This study was supported by a grant from the Korean Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A080694) and Brain Korea 21 project to Medical Science.]
The Effects of Leukocytes on Early Implantation in the Rhesus Monkey (98.24)
Authors: Rozner Ann E , Durning Maureen , Vielhuber Kathleen , Drenzek Jessica , Dambaeva Svetlana , Golos Thaddeus
Published: January 2023

At the time of implantation and throughout pregnancy, the primate uterus contains numerous leukocytes, consisting primarily of natural killer cells and macrophages. The cytokine balance at the maternal fetal interface and the expression of non-classical MHC class I molecules (HLA-G in humans and Mamu-AG in the rhesus monkey) on the trophoblast cells of the growing embryo are thought to be important immune modulators that allow the semi-allogenic embryo to implant and grow. However, study of the maternal fetal interface in human implantation is not feasible. We have established a co-culture system with rhesus monkey embryos to model implantation. Rhesus blastocysts stage embryos, derived by in vitro fertilization, were co-cultured with peripheral blood NK cells and/or monocytes obtained from the oocyte donor. Embryo growth was monitored and culture media were collected to determine cytokine and chorionic gonadotropin secretion. Trophoblast outgrowths from the embryo were noticeable after 6 days of co-culture. Embryos co-cultured with monocytes did not show signs of growth within the two weeks of co-culture, however embryos co-cultured with both monocytes and NK cells grew at similar rates to controls. Based on these results we conclude that the monocyte/NK cell balance and/or interaction is important and that using a rhesus model may lead to increased knowledge of the role of leukocytes during pregnancy.
Role of Protein Tyrosine Phosphatase Non-receptor 13 in Myofibroblast Apoptosis and Pulmonary Fibrosis (98.25)
Authors: Bamberg Alison B , Frankel Steve , Wynes Murry , Riches David W.H.
Published: January 2023

Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal, fibrotic lung disease of unknown origin. Fibrogenesis is mediated in part by the accumulation and persistence of myofibroblasts in fibroblastic foci wherein they secrete collagen and other matrix components. Impaired apoptosis is thought to contribute to myofibroblast accumulation in IPF. Human lung myofibroblasts basally express Fas but are resistant to Fas induced apoptosis; however, exposure to TNFα and IFNγ prior to Fas ligation overcomes basal resistance. In this study we address the hypothesis that exposure to TNFα and IFNγ down-regulates the expression of a Fas-associated inhibitory protein, Protein Tyrosine Phoshatase Non-receptor 13 (PTPN13), thereby enabling the formation of the death-inducing signaling complex (DISC). Exposure of lung myofibroblasts to TNFα and IFNγ decreased PTPN13 mRNA and protein expression and also increased assembly of the Fas DISC. In addition, specific knockdown of PTPN13 expression in human lung fibroblast cell lines sensitized cells to Fas induced apoptosis. Understanding the regulation of PTPN13 expression in lung tissues may reveal new approaches to treating this currently incurable disease.
IMMUNE RESPONSES IN AUTOIMMUNE HEPATITIS: EFFECT OF PREDNISONE AND AZATHIOPRINE TREATMENT (98.31)
Authors: Bluth Martin H , Kohlhoff Stephan , Nowowitz Kevin B , Silverberg Jonathan I , Chice Seto , Nowakowski Maja , Durkin Helen G , Smith-Norowitz Tamar A
Published: January 2023

Rationale: The role of the immune response in autoimmune hepatitis has not been studied before and after prednisone and azathioprine treatment.Methods: Distributions of blood lymphocytes (CD4+, CD8+, CD19+, CD23+, CD16/56+), levels of serum immunoglobulins (IgM, IgG, IgE, IgA) and cytokines (IFN-ã, IL-4, IL-12, TNFá ) were studied in a child (f/14 y/o) with autoimmune hepatitis before and after prednisone (20 mg/d) and azathioprine (50 mg/d) treatment (nephelometry, UniCAP Total IgE Fluoroenzymeimmunoassay, flow cytometry, cytokine ELISA). Patient was studied for 0-2.5 yrs; treatment was initiated 12 weeks post diagnosis.Results: Numbers of CD4+ T cells increased (50%), while CD19+ and CD23+ cells decreased (>50%) post treatment; other lymphocyte subsets were unaffected by treatment. Serum IgG and IgE levels decreased (>50%) after treatment; serum IgM and IgA were within normal range and were not affected by treatment High levels of IFN-ã (5-23 pg/ml) were initially detected in serum, which decreased after treatment (<0.1 pg/ml). In contrast, low levels of IL-4 (0.2 pg/mL) were detected before treatment, which were not detected after treatment (<0.1 pg/ml). Before treatment, IL-12 and TNFá were not detected in serum; however after treatment the levels of IL-12 and TNFá dramatically increased.Conclusions. Prednisone and azathioprine treatment decreased total serum IgG, IgE, IFN- â and IL-4 levels, and blood CD19+ and CD23+ cells; however serum IL-12, TNFá and blood CD4+ T cells increased with treatment. Understanding immunomodulation in autoimmune hepatitis will provide better insight and mechanisms of this disease and may tailor more effective therapeutic intervention.
Interphotoreceptor retinoid-binding protein (IRBP)-specific Th1 and Th17 cells in monophasic and recurrent experimental autoimmune uveitis (EAU) (99.10)
Authors: SHAO HUI , Bian Yang , Ke Yan , Wang Yali , Kaplan Henry J. , Sun Deming
Published: January 2023

Emerging evidence supports the notion that the IL-17 producing T cells (Th17 cells) play an important role in the inflammatory response of autoimmune diseases. We wished to determine whether eye-infiltrating autoreactive T cells differ between monophasic and relapsing uveitis, and whether Th1 and Th17 autoreactive T cells play distinctive roles in monophasic versus recurrent uveitis. Analysis of the cellular components of eye-infiltrating cells revealed that within the inflamed eye of m-EAU, Th17 cells were dominant in the earlier stage, and declined gradually with disease resolution. In contrast; the number of Th1 cells was lower than Th17 cells in the early stages of disease but gradually increased during the peak and remission of eye inflammation. During r-EAU, abundant IFN-r single positive cells and IFN-rIL-17 double positive T cells were found in the early stage of disease; the double positive cells gradually declined, while the IFN-r T cells persisted in the entire disease process. Only a few IL-17 single positive T cells were found in the inflamed eye during the entire disease episode. Our studies indicate that a change in the ratio of autoreactive T cell subsets between Th17 and Th1 appears to correlate with disease recovery of m-EAU. The appearance of IFN-rIL-17 double positive autoreactive T cells may correlate with the acute attack of eye inflammation in r-EAU. The pathogenic and immunoregulatory function of single positive and double positive autoreactive T cells remain to be further investigated.
Defective programmed death ligand-1 (PD-L1) expression on monocytes from systemic lupus erythematosus (SLE) patients can be corrected by a soluble T lymphocyte factor (99.15)
Authors: Ou Jing-Ni , Mozaffarian Neelufar , Wiedeman Alice , Stevens Anne
Published: January 2023

PD-L1 is an inhibitory protein expressed on monocytes (Mo) to maintain peripheral T cell tolerance. We previously reported deficient expression of PD-L1 on Mo from patients with active SLE. To investigate the mechanisms for dysregulated PD-L1 expression in SLE we isolated peripheral blood mononuclear cells (PBMC) from juvenile SLE patients and age-matched controls. PD-L1 expression was measured by flow cytometry and realtime quantitative RT-PCR. Cytokines were analyzed by Luminex.

We found that PD-L1 expression on SLE Mo was significantly induced by PBMC or CD4+ T lymphocytes from healthy subjects, and also by supernatant from healthy PBMC. Conversely, PD-L1 expression was decreased in healthy donor Mo by addition of SLE PBMC or supernatant. SLE cells had an increased rate of apoptosis, and inhibition of caspase activity led to decreased apoptosis and upregulation of PD-L1. PD-L1 mRNA expression did not correlate with protein expression. Among 15 cytokines tested, only IL-2 and IL-10 were elevated in plasma of SLE patients with active disease.

These results suggest that expression of PD-L1 in juvenile SLE is controlled by caspases at the protein level via soluble factors normally produced by T lymphocytes. Abnormal T lymphocyte activity in SLE may prevent induction of PD-L1, leading to loss of peripheral tolerance.

Supported by the Lupus Foundation and the Lupus Research Institute
Altered PD-1-PD-L1 Pathway in CD8+ T cells of (New Zealand Black x New Zealand White) BWF1 Mice with Anti-PD-1 antibody (99.29)
Authors: Wong Maida , Hahn Bevra , La Cava Antonio , Singh Ram P
Published: January 2023

PD1-PD-L1 is a costimulatory pathway that regulates self-tolerance by providing negative signals to T cells, resulting in anergy and decreased cytotoxicity. We have shown that induction of suppressive CD8+ T cells (CD8+Ti) reduces PD-1 expression on suppressor cells, which links to increased Foxp3 and TGF-£] expression. These induced CD8+Ti suppress proliferation of CD4+CD25- T cells; however, suppression is abrogated by silencing of PD-1. In untreated mice, the number and percentage of CD8+Ti in PBMC decreased over time, with increased PD-1. Blocking PD-1 increased TGF-ƒÒ expression and decreased anti-DNA and total IgG production Untreated mice developed >2+ proteinuria at week 30, and began to expire at week 39. Yet, mice treated with anti-PD-1 Ab had <2+ proteinuria until 45 weeks old, and survival was 100% until 50 weeks old.

PD-1 is likely one of the core participants in inherent autoreactivity of CD8+Ti and in the mechanisms of immune tolerance in our model. Blocking PD-1 in untreated mice delays autoantibody production and nephritis for weeks. TGF£] up-regulation suggests that blocking PD-1 may affect on CD8+ T cell suppression by TGF£] secretion. However, PD-1 abrogation 1 prevents the suppressive capacity of tolerized CD8+Ti. Downregulation may be needed to release the inhibitory capacity of the cells to permit full generation of inhibitory and cytotoxic CD8+ T cells. PD-1 expression has to be finely tuned to enable CD8+Ti to control autoimmunity.
Analysis of gender-based disparity in Treg populations in young lupus-prone mice (99.30)
Authors: Tucker Colleen F , Parnell Sarah A , Alard Pascale , Kosiewicz Michele M
Published: January 2023

Females have a higher incidence of autoimmune disease than males. There is a consistently greater disparity in Foxp3+ Treg% between young females and males in autoimmune- vs non-autoimmune prone strains of mice, i.e., lupus-prone BWF1 female mice had lower Treg% than males. This disparity was not due to differences between female and male BWF1 mice in thymic production or homeostatic proliferation of Tregs in vivo, or the ability to convert Tregs in response to TGFβ, but could be due to the significantly higher rates of non-Treg CD4+ cell proliferation in vivo in female mice. These data also raised the interesting possibility that female BWF1 Tregs are less effective at controlling CD4 cell proliferation in vivo than males. However, Tregs from female and male BWF1 mice exhibited no differences in regulatory function in vitro. Likewise, no differences in the sensitivity of female and male CD4+ responders to suppression by Tregs or in the ability of splenic APC from female and male mice to activate regulatory cell function in vitro were detected. These data suggest that the failure to control CD4 cell activation in female BWF1 mice very likely contributes to disease development; this failure is due not to defects inherent in either the cells that regulate or are regulated, but rather to environmental factors that actively and transiently affect either or both cellular components of the response. Supported by LRI
Immune stress-induced generation of tumor variants deficient in CXCL9/Mig expression (100.11)
Authors: Gorbachev Anton , Petro Marianne , Fairchild Robert
Published: January 2023

Mechanisms promoting expansion of non-melanoma skin cancers remain poorly understood. Studies using a murine model of cutaneous fibrosarcoma demonstrated that although primary tumor growth is suppressed by IFN-g-dependent host immunity, the tumor eventually escapes this immunosurveillance. To investigate mechanisms of this process we used ELISPOT assays to demonstrate that the ability of tumor cells to produce the IFN-g-inducible chemokine CXCL9/Mig decreased during progression of cutaneous fibrosarcomas in immunocompetent wild type mice, but not in immunodeficient RAG1-/- mice. Tumor cell suspensions obtained from tumors excised at different time points were subcloned by limiting dilution and clones tested for IFN-g induced Mig. The frequencies of Mig-deficient tumor cells were higher in tumors growing in wild type mice vs. from RAG1-/- mice. Mig-deficient variants were highly tumorigenic in comparison to Mig-producing tumor clones when implanted into wild type mice. Furthermore, Mig-deficient tumor variants appeared within tumors induced by implantation of Mig-producing tumor clones into wild type mice, but not within tumors implanted into RAG1-/- mice. The results suggest that Mig-deficient variants arise from Mig-producing tumor cells and expand as a result of T cell-mediated immune stress that eliminates Mig-producing tumor cells faster than Mig-deficient tumor variants
Calcineurin Aβ signalling is required for Treg-cell generation and prevention of T-cell activation and B-cell lymphomagenesis (100.13)
Authors: Bommireddy Ramireddy , Chen Dora , Gard Connie , Molkentin Jefferey , Rimsza Lisa , Doetschman Tom
Published: January 2023

Introduction: Calcineurin (CN), a protein phosphatase activated by Ca2+-calmodulin, is involved in activation of NFAT. CN inhibitors such as cyclosporine A (CsA) and FK506 are potent inhibitors of T-cell responses and are commonly used to prevent graft rejection. Consistently, CNAβ-deficient mice exhibit defective thymocyte maturation in young mice. However, T cells undergo spontaneous activation in older Cnab-/- mice, and succumb to B-cell lymphomas. Methods: Splenocytes are cultured in the presence of anti-CD3/CD28 beads, TGFβ1 and IL-2 for 3 days. Cytokines in culture supernatants are determined by ELISA, CBA and MSD methods. Cells are stained with CD4, CD25, CD62L and intracellular FOXP3. Results: We observed that FOXP3+ Treg cells are decreased while mature activated T cells (CD62L-CD44+) are increased in KO mice compared to control mice. While TGFβ1 induced the expression of FOXP3 in WT cultures, it failed to induce FOXP3 in KO cultures. Cytokine profiles revealed that CNAβ is required to inhibit IL-4, IL-6, IL-10, IL-17 and IFNγ by TGFβ1. IHC analyses of lymphomas revealed that the lymphomas are of B cell origin and they do not express FOXP1, a marker for activated B cells (ABCs). Discussion: These data show that CNAβ is important for the development and homeostasis of mature T cells and Treg cells. The data also suggest that in the absence of CNAβ, a defective T-cell regulation results in activation of T cells, production of inflammatory cytokines and cause cancer.
Myeloid-derived suppressor cells express the death receptor Fas and apoptose in response to T cell-expressed FasL (100.18)
Authors: Sinha Pratima , Chornoguz Olesya , Clements Virginia , Artemenko Konstantin , Zubarev Roman , Ostrand-Rosenberg Suzanne
Published: January 2023

Myeloid-derived suppressor cells (MDSC) accumulate to high levels in individuals with a variety of conditions including cancer, chronic inflammation, and stress, where they are potent inhibitors of adaptive and innate immunity. Some of the factors that induce their accumulation are known; however, physiological conditions that regulate their turn-over have not been identified. Mass spectrometry analysis revealed prominent expression of apoptosis pathway proteins by MDSC and led us to hypothesize that Fas/FasL-mediated apoptosis may be a key mechanism that regulates MDSC turn-over. This hypothesis was confirmed by the findings that MDSC levels significantly regress after removal of primary mammary tumors from STAT6-deficient or CD-1-deficient mice, but do not regress in mice doubly deficient for FasL and STAT6 or CD-1. Flow cytometry and confocal microscopy studies examining activated caspase 3 expression and cytochrome C localization revealed that MDSC apoptosis is triggered via the extrinsic, and not the intrinsic, pathway. Surprisingly, activated FasL+ T cells mediated MDSC apoptosis in vivo, demonstrating a retaliatory relationship between these two cell populations, and suggesting that Fas-FasL interactions could be exploited as a strategy to reduce in vivo levels of MDSC.
Administration of cannabinoids enhances growth and metastasis of 4T1 murine breast cancer through the induction of myeloid derived suppressor cells. (100.2)
Authors: Jackson Austin , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

Breast cancer is the most prevalent type of cancer afflicting women with more than 190,000 new cases diagnosed per year and over 40,000 deaths. The active ingredient in marijuana (Cannabis sativa), delta9-tetrahydrocannabinol (THC) as well as endogenous cannabinoids, such as anandamide, have been shown to exert effects on immune cells that express the cannabinoid(CB) receptors. We have previously shown that when mice are inoculated with 4t1 murine mammary carcinoma and treated with THC, the tumor grows and metastasizes more rapidly when compared to vehicle controls, resulting from immunosuppression. However, the mechanism for the loss of immune surveillance following THC treatment is not clear. Upon treatment of tumor-bearing mice, there was a significant increase in primary tumor size following THC treatment when compared to controls. Histopathological analysis demonstrated more tumor nodules in the lungs in groups exposed to THC. The splenocytes and pulmonary lymphocytes were assessed for the presence of MDSCs. There was an increase in MDSC in the lungs and spleens in cannabinoid-treated mice when compared to controls. Our studies therefore demonstrated that 4T1 mammary tumor-bearing mice elicited the development of MDSC in the spleens and lungs and the frequency of these cells was enhanced following administration of THC. Thus, we suggest a role for MDSCs in the cannabinoid-induced immune suppression in breast cancer patients that may result in enhanced tumor growth.
Defective antigen-presenting cell function of MDSCs from late stage tumor-bearing mice can be recovered by ATRA treatment. (100.3)
Authors: Lee Jung-Mi , Kim Eun-Kyung , Kim Yeon-Jeong , Kim Yun-Sun , Kang Chang-Yuil
Published: January 2023

Myeloid-derived suppressor cells (MDSCs), which accumulate during tumor progression, have recently been shown to function as the main suppressor cells. In our previous study, we showed that poorly immunogenic MDSCs could function as immunogenic antigen presenting cells (APCs) with the help of activated natural killer T (NKT) cells. However, depending on the tumor stage, we found that MDSCs had variable capacity as APCs. NKT ligand-loaded MDSCs from late stage tumor-bearing mice (late MDSCs) were less immunogenic as APCs compared to those from early stage tumor-bearing mice. As tumor growth progresses, surface molecule expression required for APC function on MDSCs was largely down-regulated. NKT ligand-loaded late MDSCs were also poorly differentiated and induced decreased NKT activation and antigen-specific cytotoxic T lymphocyte (CTL) response. However, these functional defects were recovered by the treatment of all-trans retinoic acid (ATRA). NKT ligand-loaded, ATRA-treated late MDSCs were able to be transformed into immunogenic APCs to induce incremental immune responses. These effects were accompanied by the recovered activation and differentiation of late MDSCs. Collectively, we concluded that although the functional defect of MDSCs as APCs was found with tumor progression, it can be recovered by treatment with ATRA. Therefore, these findings suggest that late MDSCs can function as effective APCs.
A population of lymphoid progenitor cells inversely correlated with outcome in canine non-Hodgkin B-cell lymphoma (100.34)
Authors: Modiano Jaime , Ito Daisuke , O’Brien Timothy
Published: January 2023

Disease progression and response to treatment in people and in pet dogs with non-Hodgkin B-cell lymphoma (NHL) can be unpredictable, even for patients with the same subtype of disease, reflecting the heterogeneous nature of these tumors. Tumor angiogenesis has been correlated with stage and progression of lymphoid tumors in humans and in dogs, but the prognostic value of mature endothelial cells (ECs) and endothelial progenitor cells (EPCs) as surrogate markers for angiogenesis in NHL is unclear. Furthermore, the significance of lymphoid progenitor cells (LPCs) in this disease has not been addressed. Here, we evaluated the predictive value of ECs, EPCs and LPCs in dogs with naturally occurring NHL. We used flow cytometry to identify prospectively cells that co-expressed CD34, c-Kit, and/or CD133 with αvβ3-integrin (EPCs); or with CD45 and lymphoid lineage markers (LPCs), as well as cells that expressed CD146 (ECs), in blood and lymph nodes from dogs with NHL treated using standard of care multi-agent chemotherapy. The normalized frequency of ECs, EPCs and LPCs was compared with patient outcomes. ECs, EPCs, and LPCs were routinely present in all lymph node samples. No correlation was observed between ECs and EPCs and patient outcomes; conversely, there was a significant (p<0.05) inverse correlation between LPCs and overall survival. Our results suggest that a population of cells with lymphoid progenitor phenotypes is predictive for survival outcomes in spontaneous canine NHL.
Expression of CD39 and CD73 as a means of evading anti-tumor immune reponses in lung cancer (100.7)
Authors: Meyer Christian , Rosinsky Carolyn , Alme Angela , Powell Jonathan
Published: January 2023

A major hurdle for cancer immunotherapy is the ability of tumors to inhibit anti-tumor immune responses. CD39 and CD73 are ectonucleotidases that promote the generation of extracellular adenosine from ATP. In light of the ability of adenosine to inhibit T cell responses by activating the adenosine A2a receptor (A2aR) we wanted to explore the expression of these two molecules on the surface of cancer cells. We investigated CD39 and CD73 expression and function utilizing murine and human tissues. In vitro studies revealed that Murine Lewis Lung Carcinoma cells did not express ectonucleotidases. However, upregulation of CD39 was found in vivo at multiple time points after injection into C57BL/6 mice. This expression was not T or B cell mediated as injection into RAG knockout mice produced the same expression patterns. Interestingly, characterization of multiple human lung cancer cells lines in vitro revealed near universal expression of CD73 and no CD39 expression. To determine if the upregulation of adenosine could have consequences in vivo, we injected Lewis lung carcinoma cells into A2aR knock out mice or wild-type mice treated with an adenosine receptor antagonist. In both cases improvements in tumor free and overall survival were observed. Overall our data suggests that CD39 and CD73 expression on the surface of lung cancer cells may contribute to their resistance to immunotherapy and that successful immunotherapy in lung cancer might include A2aR antagonists.
Enhanced Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1) and Soluble TREM-1 Levels in the Myeloid Cells of Tumor-Bearing Mice and Patients with Renal Cell Carcinoma (100.8)
Authors: Ford Jill , Howard O.M. Zack , Subleski Jeff , Yang Youfeng , Rayman Pat , Weiss Jon , Linehan W. , Sayers Tom , Finke James , McVicar Daniel
Published: January 2023

Myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) contribute to cancer-related inflammation and tumor progression. TREM-1 amplifies the innate immune response and is thus emerging as an important player in inflammation and disease. Soluble TREM-1 (sTREM-1) is found in plasma of those with various inflammatory conditions and is associated with disease progression. Because TREM-1 is expressed on myeloid cells in healthy donors, we predicted TREM-1 would be expressed on MDSC and TAM within tumors and sTREM-1 would be elevated in the periphery. In this study, we examined TREM-1 and sTREM-1 levels in mice and patients with cancer. Using the 4T1 tumor model, we found that TREM-1 was present on blood and splenic MDSC in tumor-bearing mice and that levels increased over time after tumor challenge. TREM-1 was expressed on MDSC and TAM in the tumor. Ligation of TREM-1 on MDSC enhanced TNF release, indicating that the TREM-1 is functional. sTREM-1 was elevated in plasma of tumor-bearing mice and correlated with tumor volume. In renal cell carcinoma patients, TREM-1 was expressed on both tumor-infiltrating monocytes and neutrophils and plasma sTREM-1 levels increased with cancer stage. Taken together, these data show that TREM-1/sTREM-1 is regulated during tumor development, define TREM-1 as a novel MDSC marker, and demonstrate that TREM-1 on MDSC is functional. Future studies are needed to determine if TREM-1 can modulate tumor-associated inflammation.
Inhibition of cervical cancer cell growth by IL-24 (101.22)
Authors: Lee Ki-Mo , Kang Hyung-Sik
Published: January 2023

HeLa cervical cancer cells stably overexpressing human papilomavirus (HPV)16 E6, HE6F cells, were established to investigate the growth inhibitory effect by interleukin-24 (IL-24). The expression of angiogenesis-associated genes was upregulated in HE6F cells compared to that in mock-transfection control cells, which led to the suppression of apoptosis and consequent the augmentation of their growth. However, when HE6F cells were treated with recombinant human IL-24 (rhIL-24), the growth was inhibited by downregulation of angiogenesis-associated gene expression and induction of apoptosis. In our previous results, overexpression of Axl receptor tyrosine kinase was induced by HPV16 E6 in cervical cancer cells. To investigate whether growth inhibition of HE6F cells induced by rhIL-24 is accomplished through the regulation of Axl expression, Axl promoter assay and western blot analysis were performed. We found that rhIL-24 inhibited the growth of HE6F cells via induction of downregulated Axl expression in both transcriptional and translational levels.
A critical role for mast cells in TLR2-mediated inhibition of tumor growth in vivo (101.24)
Authors: Oldford Sharon , Haidl Ian , Howatt Mackenzie , Leiva Carlos , Johnston Brent , Marshall Jean
Published: January 2023

Mast cells are abundant surrounding solid tumors where they typically promote angiogenesis and enhance tumor growth and metastasis. In the context of infection, mast cells respond to pathogens via innate immune receptors, including TLR, to produce mediators which aid host defense and cell recruitment. Some TLR agonists are effective in tumor immunotherapy but the role of mast cells in their mechanism of action is unclear. Using a melanoma model in wild-type C57BL/6 and mast cell deficient KitW-sh/W-sh mice, mast cells were shown to be crucial for TLR2-agonist (Pam3CSK4) induced tumor growth inhibition. Activation of TLR2 on mast cells reversed their established pro-tumorigenic role. The tumor inhibitory effects of Pam3CSK4 were restored in KitW-sh/W-sh mice by local reconstitution with wild-type, but not TLR2-deficient mast cells. Tumor growth inhibition occurred independent of mast cell derived TNF and tumor cytotoxicity but was associated with angiogenesis suppression and NK and T cell recruitment. The mast cell mediated, tumor inhibitory effects of Pam3CSK4 were also observed in an LLC1 lung cancer model. This study reveals a novel mechanism of action for TLR2 agonists in vivo and has important implications for the design of immunotherapeutic strategies harnessing the innate immune functions of mast cells.
Conditional expression of tissue-specific antigen regulates CD8 T cell tolerance induction (101.3)
Authors: Huang Lan , Lacuanan Aimee , Eisenlohr Laurence
Published: January 2023

We have developed a carefully-designed transgenic model to learn how expression of an autoantigen influences the degree and nature of tolerance. The surrogate antigen, directed to thyroid by a tissue-specific promoter, consists of a cell-surface glycoprotein (human interleukin-2 receptor α chain, Tac) to which well-defined B cell, CD8+ T cell, and CD4+ T cell epitopes have been appended in order to assess all three lymphocyte subset responses. A LoxP-flanked stop cassette was inserted between the tissue-specific promoter and the transgene open reading frame (ORF) to allow for conditional expression. To avoid positional and copy number effects in traditional transgenesis, the transgene was targeted to HPRT locus with a single-copy insertion. Here we show that embryonic but not adult expression of Tac transgene results in a profound epitope-specific CD8 T cell tolerance. However, prolonged expression of postnatal antigen is able to facilitate tolerance induction. Moreover, the ability to induce Tac-specific immune tolerance correlates with the loss of susceptibility to rejection of Tac-expressing tumor cells. These findings provide direct evidence of dynamic tolerance induction to normal or tumor antigens.
The effect of alpha2M levels on the immunogenicity of HSP-peptide complexes (101.32)
Authors: Zhou Yu , Binder Robert
Published: January 2023

CD91 is an endocytic receptor on Antigen Presenting Cells for the immunogenic Heat Shock Proteins (HSPs) gp96, hsp70, hsp90 and calreticulin. Binding of HSPs to CD91 initiates immune responses through cross presentation of the HSP-chaperoned peptides and elaboration of co-stimulatory signals. In vitro, various CD91 ligands compete with HSPs for receptor binding; the serum protein alpha-2-macroglobulin (a2M) is one such ligand. In light of this observation, we hypothesize that modulation of a2M levels in animals will affect the immunogenicity of HSP-vaccines. Elevated levels of a2M are achieved by initiating an acute inflammatory response and monitored by ELISA. Immune responses following HSP vaccination are measured by in vivo CTL assays. The results from these experiments will impact on-going and future clinical trials in cancer patients vaccinated with HSP-peptide complexes.
Secretory Leukocyte Protease Inhibitor (SLPI) delays development of murine squamous cell carcinoma and inhibits metastasis. W Jin, J Wen, MF Starost, G McGrady, SD Shapiro and SM Wahl, OIIB, NIDCR, NIH, and DVR, NIH, Bethesda, MD and Univ Pittsburg, Pittsburg, PA. (101.33)
Authors: Jin Wenwen , Wen Jie , Starost Matthew , McGrady George , Shapiro Steven , Wahl Sharon
Published: January 2023

Development of squamous cell carcinomas (SCC) represents a multistage process of transformation, invasion and metastasis dependent on genetic aberrancies and contributions of surrounding stroma. Dissemination to distant sites depends on proteolytic enzymes to enable migration and entry into vessels, underscored by a protease-protease inhibitor imbalance. Deletion of SLPI, a serine protease inhibitor expressed in most epithelia, which potently inhibits elastase, is linked to altered tumor progression. In rodents, SLPI is also generated by inflammatory cells, and has antimicrobial and anti-inflammatory activities, in addition to inhibition of proteases. To establish which of these functions are critical in regulating tumorigenesis, we chemically induced SCC in SLPI null and wildtype mice and monitored skin tumor progression, invasion and lung metastasis. In the absence of SLPI, increased inflammatory infiltrates in pre-malignant skin tissues along with more evident protease accumulation was associated with earlier papilloma development, but malignant transformation from the papilloma, once formed, to SCC was similar in null and wildtype mice. Subsequent tumor cell invasion and metastasis to the lungs was significantly enhanced in the absence of SLPI. Resetting the balance of protease-protease inhibitor through deletion of elastase (elastase null) abrogated tumorigenesis, suggesting that elastase promotes tumor progression if unopposed by its endogenous inhibitor, SLPI.
Peroxiredoxin 1 stimulates secretion of proinflammatory cytokines by interacting with Toll-Like Receptor 4 (101.34)
Authors: Riddell Jonah , Minderman Hans , Wong Xiang , Gollnick Sandra
Published: January 2023

Elevated Peroxiredoxin 1 (Prx1) expression is a poor prognostic factor in various cancers. Prx1 is an intracellular antioxidant and molecular chaperone protein that is non-classically secreted from transformed or stressed cells. Prx1 was found to increase the maturation state and secretion of pro-inflammatory cytokines from macrophages and dendritic cells in a Toll-Like Receptor (TLR4) dependent manner. Prx1 bound macrophages in a saturable, competitive, and TLR4 dependent manner. Prx1 immunoprecipitated and colocalized with TLR4. Extracellular Prx1 increased nuclear factor kappa B (NF-kB) translocation and DNA binding activity in a TLR4 dependent manner. In vivo, Prx1 injected intravenously in mice increased systemic interleukin 6 (IL-6) levels in a TLR4 dependent manner. Prx1 promotes innate immunity through the interaction in TLR4; however, the role of extracellular Prx1 in cancer remains undefined. Prx1 delayed tumor growth in prostate cancer models and additionally affected the numbers of CD11b+ Gr1HI F4/80+ tumor associated macrophages that have been identified as a myeloid derived suppressor cell population. This is the first time that extracellular Prx1 is demonstrated to interact with TLR4 on immune cells and that the interaction has consequential effects on macrophage populations and tumor progression.
Cytotoxic chemotherapy rescues tumor-driven aberrant CD4+ T cell differentiation and restores an activated polyfunctional helper phenotype (101.9)
Authors: Zhou Gang , Ding Zhi-Chun , Blazar Bruce , Mellor Andrew , Munn David
Published: January 2023

The functional development of tumor-specific CD4+ T cells critically impacts the outcome of anti-tumor immune responses. Adoptive immunotherapy involving tumor-specific CD4+ T cells has shown encouraging clinical benefits in some cancer patients. In order to mount an effective anti-tumor immunity, it is desirable to elicit activated type 1 T helper cells (Th1). In this study, we report that Th1-like effector cells that arose in tumor-bearing hosts progressively expressed Programmed Death 1 (PD-1) during tumor growth. The PD-1hi effector cells displayed a dysfunctional phenotype, characterized by heightened apoptosis and poor anti-tumor efficacy. This tumor-driven aberrant T cell response could be prevented by a single dose of the widely-used chemotherapy agent cyclophosphamide. We show that chemotherapy conditioned the host environment, creating a transient window for optimal effector differentiation for adoptively transferred CD4+ T cells. This robust effector differentiation, which was antigen-driven and mechanistically dependent on an intact host response to type I interferon, gave rise to activated polyfunctional T helper cells with potent anti-tumor activity against established B cell lymphomas. We hypothesize that prevention of tumor-induced effector cell dysfunction is a major mechanism contributing to the efficacy of combined chemo-immunotherapy.
Computational studies of B cell receptor diffusion can elucidate the mechanism of B cell synapse formation (130.1)
Authors: Raychaudhuri Subhadip , Tsourkas Philippos
Published: January 2023

B cell receptor (BCR) clustering in the form of an immune synapse (membrane-bound antigens) or a cap structure (soluble antigens) is a central event in adaptive immunity because such BCR clustering takes part in BCR recognition of non-self antigens. We approached the problem of B cell synapse formation using a Monte Carlo computational study of B cell receptor diffusion at a single molecule level. Recent experiments (Immunol. Rev. 221:64-76 2008), based on single molecule tracking, have elucidated that BCR molecules diffuse at a much slower rate in the regions of antigen binding. The mean-square displacement of BCR molecules, as obtained from our computational studies, shows an affinity dependent sub-diffusive behavior indicating an affinity dependent mechanism of B cell receptor clustering in immunological synapses. Parallel theoretical analysis in a simplified model system clarified how binding of receptor-ligand pairs at a curved cell-bilayer junction could lead to affinity-dependent sub-diffusive behavior. Our recent efforts in this direction are focused on exploring various possible mechanisms of biased transport of BCR molecules. Initial results indicate biased diffusion can also generate sub-diffusion, albeit in an affinity-independent manner, and such biased transport is necessary for BCR clustering in immunological synapses.
A Thr/Ser-based signal in the cytoplasmic tail regulates the functional expression of human CD1d following an HSV infection (130.21)
Authors: Liu Jianyun , Glosson Nicole , Du Wenjun , Gervay-Hague Jacquelyn , Brutkiewicz Randy
Published: January 2023

CD1d molecules are MHC class I-like molecules that present lipids to a unique subpopulation of T cells called natural killer T (NKT) cells. We have previously shown that the Thr322 residue in the cytoplasmic tail of human CD1d is important for its intracellular trafficking and functional expression. We proposed that the phosphorylation of T322 is a signal for CD1d lysosomal targeting and degradation. In the current study, we generated dual mutants by substituting the T322 and S323 residues with Ala (non-phosphorylatable) or Asp (mimicking phosphorylation) and expressed them in human 293 cells. We found that the surface expression level of the CD1d mutants was in this order: T322AS323A>WT>T322A>S323A>S323D>T322D>T322DS323D. Our results suggest that the phosphorylation of both T322 and S323 has a cumulative negative effect on the functional expression of CD1d. Interestingly, substitution of the T322 and S323 residues with Ala inhibited CD1d degradation and prevented a decrease in CD1d-mediated antigen presentation following an HSV infection. This suggests that the T322/S323 dyad may be phosphorylated and target CD1d for lysosomal degradation during a viral infection as a means of immune evasion. Thus, our findings strongly suggest that T322 and S323 form a dual residue motif that can regulate the functional expression of CD1d.
CD99 differentially modulate transcriptional regulation of classical MHC class I and CD1a molecules (130.23)
Authors: Mahiddine Karim , Bernard Ghislaine , Larbret Frederic , Mallavialle Aude , Bzioueche Hanene , Bernard Alain
Published: January 2023

We have previously established that CD99, a ubiquitous surface molecule expressed as two isoforms, could regulate classical MHC class I (class I) expression. Fine tuning of class I during differentiation and activation of professional APC should be crucial event which deserve to be carefully examinated. Surprisingly, we found that expression of CD1a was inversely regulated to class I expression by a mechanism involving CD99. Using the Jurkat cell line, we have demonstrated that Class I and CD1a transcriptions levels are dictated by CD99 long isoform (CD99LF). CD99LF enhances Class I transcription and therefore its expression, while down regulating CD1a. We also showed that this regulation is counteracted in the presence of CD99 short form (CD99SF). Therefore, expression of both CD99 isoforms allows expression of class I and non classical MHC CD1a molecules at plasma membrane without requiring β2M modulation. To confirm our results, we used CD14+ monocytes and their derived dendritic cells. Performing qRT-PCR, flow cytometry and CD99 silencing experiments, we have demonstrated that generation of CD1a positive dendritic cells (mDC1) depends on a mechanism involving an expression switch from CD99LF to both CD99 isoforms. We hypothesize that lack of CD99 switches leads to CD1a negative (mDC2) generation. This regulatory mechanism appears to be therefore crucial for mDC1 or mDC2 orientations, which are distinguished by their different capacities to direct Th cell differentiation.
Immune cross recognition of a conserved Flavivirus epitope (130.28)
Authors: McMurtrey Curtis , Piazza Paolo , Meeks Janet , Bardet Wilfried , Buchli Rico , Imrie Allison , Rinaldo Charles , Hildebrand William
Published: January 2023

We previously characterized an immunodomiant, HLA-A*0201 restricted, West Nile virus (WNV) peptide determinant. This dominant WNV peptide epitope, termed SVG9, lies within a highly conserved region of the envelope glycoprotein and demonstrates 100% homology among contemporary WNV strains. This region of the virus envelope is also highly conserved among other Flaviviruses such as Japanese encephalitis virus and Dengue virus (DV). Since immune cross reactivity has been reported within the Dengue virus serogroup, we hypothesized that T cells specific for this A*0201 restricted WNV stretch of envelope would cross-recognize different flaviviruses due to epitope conservation. As an initial test of this hypothesis we synthesized the corresponding envelope peptide for WNV (SVG9) and DV type 1 (SIG9). We utilized a competitive peptide binding assay to demonstrate that SVG9 and SIG9 have comparable and high A*0201 binding affinities. We then utilized PBMC from WNV and DV1 seropositive donors to assess T cell cross recognition of SVG9 and SIG9. T cells specific for the WNV SVG9 were cross-reactive for their DV1 Flaviviral counterpart SIG 9 and vice versa. These data demonstrate that cellular immunity is cross-reactive among different Flaviviruses. This observation has implications for vaccine development and for unraveling the immunopathology associated with infection.