bluuebunny/crossref_metadata_2025 huggingface dataset:
A strategy to improve current MSC-based therapies (TECH1P.833)
Authors: Li Yan , Lin Feng
Published: January 2023

Mesenchymal stem cell (MSC) hold promise in the therapy of many inflammatory and degenerative disease. However, result from recent clinical trials using these cells are disappointing, suggesting that current MSC-based therapies need to be improved. We previously demonstrated that, immediately after their contact with serum following infusion, MSCs active complement of the innate immunity and are injured by the activated complement despite the presence of native cell surface complement regulators. In this work, we found that systemic administration of heparin, a widely used drug which is also a potent complement inhibitor, protects MSCs from serum-mediated damage after i.v. infusion. To minimize the side effects of systemic heparin administration, we also developed a method to paint heparin onto the surface of MSCs, We found that chemically modified heparin can be painted onto MSCs, and that these heparin-painted MSCs are better protected from serum (complement)-medicated attack and retain better immunosuppressive activity than regular MSCs. These data suggest that the cell surface engineering of MSC with heparin would be a simple and effective approach to improve the outcomes of current MSC-based clinical trials.
An efficient way to analyze function of immune cells (TECH1P.843)
Authors: Wang Zhong
Published: January 2023

Cellular assays for suspension cells, especially immune cells, provide investigators an opportunity to interrogate the biology of cellular function of immune cells or immunotherapeutic drugs. Despite significant technical advances made during the last few decades, working with suspension cells, such as primary blood cells, PBMCs (peripheral blood mononuclear cells), leukemia, myeloma, or lymphoma cells, current cellular assay approaches still suffer from limitations. For example, significant cell loss can occur during assay process. This loss can be further exacerbated when multi-step staining procedures are required. We applied DropArray plates, which employ surface tension to retain cells on the plate surface, for our suspension cellular assays. For a variety of suspension cells, including PBMCs, AML cells, or B cells, efficient retention (50% to 80%) is achieved during multiple washes. Reproducibility and quality of the experimental results are improved with DropArray plates. Furthermore, specific cytotoxic action of therapeutic antibodies on tumor cells can be also observed in real time. In summary, this system presented an efficient approach for phenotypic study of suspension cells, especially rare population of cells.
Profiling secreted cytokines from human Th2 and Th17 cells using antibody arrays (TECH1P.845)
Authors: Finkel David , James Amy , Brumbaugh Kathy , Dhawan Sanjeev , Goetz Christine , Bonnevier Jody , Wegner Greta
Published: January 2023

Naive T Helper cells can differentiate into Th1, Th2, and Th17 lineages which each secrete a characteristic signature of molecules. In this study, we demonstrate the utility of antibody arrays to monitor T cell differentiation processes. Naive T cells were isolated from PBMC preparations obtained from different donors. Following activation, the generation of each T cell subtype was confirmed by flow cytometry. Secreted proteins present in the cell culture supernatants were analyzed with a Human XL Cytokine Array containing 102 analytes. More than 30 proteins were identified, including cytokines specific to each Th subtype. ELISA data were used to quantitatively confirm the increases and decreases of multiple Th-specific cytokine levels observed using the array. These data demonstrate that antibody arrays provide an efficient tool to simultaneously study multiple parameters in complex biological processes where alternate differentiation protocols are being tested along with evaluating the impact of donor-to-donor variability.
Cohort-wide automated identification of morbid alleles with the human gene connectome (TECH1P.835)
Authors: Itan Yuval , Zhang Shen-Ying , Abel Laurent , Casanova Jean-Laurent
Published: January 2023

To determine the disease-causing allele(s) underlying primary human inborn errors, high-throughput genomic methods are applied and provide thousands of gene variants per patient. We recently reported a novel approach, the “human gene connectome” (HGC) - the set of all in silico-predicted biologically plausible routes and distances between all pairs of human genes. We demonstrated that the HGC is the most powerful approach for prioritizing high-throughput genetic variants in Mendelian disease studies. However, there is currently no available method for automating the selection of candidate disease-causing mutant alleles in the absence of a known morbid gene in at least one patient with the disease of interest, posing a major bottleneck in the field in high-throughput clinical genomics. We hypothesized that within a cohort of patients with the same Mendelian (or nearly Mendelian) disease, the cluster that contains the key disease-causing gene for each patient is the HGC-predicted biologically smallest cluster. We then developed and applied a Mendelian clustering algorithm, which estimates the biologically smallest HGC-predicted cluster that contains one allele per patient. By that we estimated and statistically validated a set of disease-causing alleles in a whole exome sequencing cohort herpes simplex encephalitis patients. This new unbiased approach should facilitate the discovery of morbid alleles in patients with primary inborn errors that lack a genetic etiology.
Th22 cells shape colorectal cancer stemness via epigenetic regulation (TUM2P.877)
Authors: Kryczek Ilona , Lin Yanwei , Nagarsheth Nisha , Peng Dongjun , Zhao Lili , Zhao Ende , Vatan Linda , Szeliga Wojciech , Dou Yali , Zgodzinski Witold , Majewski Marek , Wallner Grzegorz , Fang Jingyuan , Huang Emina , Zou Weiping
Published: January 2023

Little is known about the direct impact of immune elements on human colorectal cancer invasiveness and stemness. We investigated the interaction between IL-22+ cells and cancer (stem) cells in colorectal cancer patients. We have demonstrated that Th22-derived IL-22 targets disruptor of telomeric silencing 1-like (DOT1L). DOT1L methylates histone 3 lysine 79 (H3K79) and, in turn, leads to core stem cell gene activation, resulting in increased colon cancer stemness and tumorigenic potential. In addition, increased levels of DOT1L and H3K79me2 in colorectal cancer predict poor patient survival. Collectively, this work identifies that Th22 cells control colon cancer stemness via epigenetic regulation that affects patient outcome. Efforts to target this network might be a strategy in treating patients with colorectal cancer.
Very low avidity endogenous memory CD8+ T cells control tumor growth (TUM2P.893)
Authors: Stagliano Katherine , Zhu Ziqiang , Hurwitz Arthur
Published: January 2023

In this study, we describe mice bearing a very low avidity TCR transgene (TCRvlo) specific for a melanoma antigen. Remarkably, a majority of peripheral TCRvlo CD8+ T cells display a phenotype consistent with the expression pattern for endogenous memory (EM) T cells: CD44hi, CXCR3hi, Gr1hi, GITRhi, CD69lo, CD25lo, CD49dlo. This phenotype was not observed among thymic CD8+CD4- TCRvlo T cells. EM-T cells are proposed to arise from naïve T cells in the absence of activation by foreign antigen and are a very rare population of cells which may contribute to primary immune responses, but have not been studied in cancer models. We hypothesize that EM-T cells may provide a unique reservoir of cells that can control tumor growth. Our previous observations indicate that T cells bearing a high avidity TCR with identical antigenic specificity initially control tumor growth but are prone to tumor-induced tolerance. In our preliminary studies, compared to wildtype animals, TCRvlo transgenic mice displayed delayed subcutaneous B16 melanoma growth. In an adoptive transfer setting, TCRvlo T cells also delayed B16 tumor growth compared to untreated animals. Ongoing studies are focused on characterizing the functional responses of the TCRvlo CD8+ T cells to antigen and cytokines as well as determining whether TCRvlo T cells resist tolerization in the tumor microenvironment. Results from these studies may enable us to use EM-T cells as a resource for generating more durable tumor immunity.
Cancer immunotherapy: treatment of established breast cancer or prevention of tumor relapse and metastasis? (TUM2P.903)
Authors: Payne Kyle , Graham Laura , Bear Harry , Manjili Masoud
Published: January 2023

Cancer vaccines and adoptive immunotherapies (AITs) are being tested both in preventive and therapeutic settings. In order to determine the most effective immunotherapeutic strategy, we investigated the efficacy of AIT against established advanced carcinoma and minimal residual disease. We used Decitabine to improve immunogenicity of the tumor cells by inducing the expression of cancer testis antigens (CTA), in vivo. Tumor-sensitized immune cells were activated and reprogrammed, ex vivo, using bryostatin 1 and ionomycin, along with IL-2, IL-7, and IL-15; this produced a central memory phenotype in 73% of T cells, and an activated phenotype in 27% of NK cells and 94% of NKT cells. FVBN202 transgenic mice were protected from developing lung metastases following AIT, but failed to cure established lung metastases. The reprogrammed immune cells also demonstrated immunological memory that protected animals from recall tumor challenge. In vitro studies showed that reprogrammed immune cells were able to kill 82% of viable Adriamycin-treated quiescent tumor cells. Our results suggest that AIT could be efficacious against chemotherapy-resistant dormant tumor cells in a setting of low tumor burden and prevent distant metastasis of breast cancer. These findings are significant because breast cancer mortality results from metastatic disease that develops from residual tumor cells remaining dormant, sometimes for prolonged periods following conventional therapies.
Identification of follicular helper T cells as a novel cell population potentially involved in the pathogenesis of Rheumatoid Arthritis (HUM3P.251)
Authors: Zhang Xin , Singla Shikha , Pound Jerry , Zakem Jerald , Collin Kismet , Webb-Detiege Tamika , Davis William , Quinet Robert
Published: January 2023

Background. RA is an autoimmune disease characterized by chronic inflammation of the synovium, causing progressive joint destruction. The treatment for RA is largely based on the clinic manifestations, which are remote from the fundamental pathophysiologic defects. Identification of cellular subsets or biomarkers is a necessary first step toward more accurate prognosis and better therapeutic decisions. Here, we identified a novel T cell subset, follicular helper T cells (Tfh) in RA patients, and examined the hypothesis that Tfh cells may account for RA pathogenesis by providing a fostering environment for self-reactive B cells, resulting in autoantibody production. Methods. Peripheral blood was collected from 26 RA patients and healthy donors. Synovial fluid from actively inflamed joints of RA patients was also collected. Clinical disease activity (DAS-28) and serum laboratory results were obtained. Tfh cells were defined by their signature surface markers via flow cytometry. IL-21 expression and plasmablasts were measured by intracellular staining. Results. A novel T cell subset with Tfh cell signature molecule and cytokine was expanded in the peripheral blood of active RA patients. The percentage of Tfh cells correlated positively with the level of pathogenic auto-antibody (anti-CCP) and disease activity. Conclusions. Circulating Tfh cells may serve as a biomarker of pathogenesis in RA patients. Targeting RA-Tfh cells may provide earlier and precise treatment strategies.
Antigen-specific regulatory T cells from pluripotent stem cells ameliorates arthritis in mice (HUM3P.252)
Authors: Haque Mohammad , Fino Kristin , Song Jianxun (Jim)
Published: January 2023

Embryonic stem cells have the ability to grow indefinitely while maintaining pluripotency. Under the right circumstance, pluripotent stem cells (PSC) can produce almost all of the cells in the body including regulatory T cell (Tregs). Tregs are essential for normal immune surveillance systems, and their dysfunction leads to the development of diseases, such as autoimmune disorders. However, the right circumstances for the development of antigen (Ag)-specific Tregs from PSC (i.e., PSC-Tregs) has not been well defined. So the purpose of this study is to develop antigen-specific Tregs from pluripotent stem cells for treatment of Ag-induced arthritis (AIA) in mice. In this study mouse iPS cells were transduced with FoxP3 and ovalbumin-specific TCR (OTII). iPS cells were driven toward T lymphocyte lineage by co-culture on OP9 cells expressing delta-like (DL1), a Notch ligand. The combination of Notch signaling, FoxP3, and TCR drove iPS cell to differentiation into Ag-specific Tregs. In vitro generated Tregs were adoptively transferred into AIA mice. Our results showed that TCR transduced iPS cells differentiated into Tregs and express CD3, TCRβ, CD4, CD8, CD25, CTLA4 in vitro. Adoptive transfer of such Tregs dramatically suppressed autoimmunity in a well-established AIA model, including the inflammation, joint destruction, cartilage prostaglandin depletion, osteoclast activity, and Th17 production.
A commensal bacteria-derived lipid, significantly decreased in the serum of multiple sclerosis patients, causes attenuation of experimental autoimmune encephalomyelitis (HUM3P.269)
Authors: Anstadt Emily , Fujiwara Mai , Nemati Reza , Yao Xudong , Nichols Frank , Clark Robert
Published: January 2023

The role of the microbiome in multiple sclerosis (MS) remains unknown. We have identified a novel commensal bacteria-produced lipodipeptide, Lipid 654 (L654), which functions as a toll-like receptor 2 agonist. We reported that L654 can be recovered in healthy human serum and that levels are significantly lower in patients with MS. The purpose of this study was to understand the functional relevance of the decreased serum L654 level in MS by determining: 1) if L654 attenuates clinical disease in a transfer model of EAE, and 2) the mechanism by which L654 mediates this attenuation. L654 administration significantly attenuated clinical disease severity in an adoptive transfer model of EAE in SJL/J mice (p < 0.0001). Cellular analysis of mononuclear cells in the spinal cord at day 18 showed a significant decrease in total cell number (p = 0.02) as well as a decrease in percentage of IL-17 producing, IFNγ producing and IL-17/IFNγ double-producing CD4+ T cells in mice administered L654. At day 11, we found a decrease in total mononuclear cell number and an increase in percentage of CD39+ Tregs and IL-10 producing CD11b+ cells in mice administered L654. These results suggest that L654, normally present in human serum but at significantly lower levels in MS serum, has the ability to mediate an immunoregulatory and disease inhibitory effect on CNS autoimmunity. L654 may thus have a role both in the abnormal immunoregulation in MS and as a potential therapeutic in this disease.
CD160 expression defines a functional CD8 T cell subset during HIV-1 infection (HUM4P.270)
Authors: Zhang Anli
Published: January 2023

T cell exhaustion, indicated by elevated expression of inhibitory receptors such as PD-1 and Tim-3 and impaired T cell function, is a hallmark of HIV-1 chronic infection. Glycosylphosphatidylinositol-anchored molecule CD160 expression on CD8+T cells is significantly increased in HIV-1 infected individuals. We show here, CD160 expression is higher in slow progressors than in typical progressors. Levels of CD160 expression on CD8+T cells correlated positively with CD4 T cell count and inversely with HIV-1 plasma viral load. CD160 co-expressed with HLA-DR but not CD38. Functionally, CD160 expressing T cells produced more IFN-γ and proliferate more vigorously in response to HIV-1 specific peptides stimulation. Together, our current study suggests CD160 defines a functional instead of a typical exhausted CD8 T cell subset, targeting CD160 pathway might help in controlling HIV-1 infection.
Immunodeficiency in a hypomorphic mouse model of propianic acidemia (HUM4P.300)
Authors: Castillejo Becerra Clara , Guenzel Adam , Barry Michael
Published: January 2023

Propionic Acidemia (PA) is a recessive disorder resulting from mutations in the PCCA or PCCB genes encoding the two subunits of propionyl-CoA carboxylase. Patients with PA generate propionate and potentially toxic metabolites after consumption of certain amino acids and odd-chain fatty acids leading to systemic health complications. In addition, PA patients are exceptionally sensitive to infections due to a compromised immune system. We developed a hypomorph mouse model of PA (Pcca-/-(A138T) that recapitulates many phenotypes associated with the human disease. In this study, we tested A138T mouse immune function and their susceptibility to infections. A138T mice had an overall reduction in white blood cells, lymphocytes, monocytes, granulocytes, and immunoglobulins when compared to control mice. When wild type and A138T mice were infected i.v. with adenovirus 5 (Ad5) expressing GFP-Luciferase, we observed higher luciferase expression in the livers of the Pcca-/-(A138T) suggesting that the mutant mice were more susceptible to the virus due to defects in the reticuloendothelial system. The A138T mice also generated lower levels of anti-GFP antibodies than control mice 7 weeks after injection suggesting weaker adaptive immune responses. Overall, our results show that the Pcca-/-(A138T) mouse model recapitulates the immunodefiency seen in humans and can be used to further explore their susceptibility to infections as well as the benefits or dangers of vaccination to PA patients.
Common and differential roles of inducible NO synthase and poly (ADP-ribose)polymerase in allergen-induced inflammation and airway hyperresponsiveness: a potential connection to NO levels (HYP5P.320)
Authors: Ibba Salome' , Ghonim Mohamed , Pyakurel Kusma , Naura Amarjit , Mishra Anil , Boulares Hamid
Published: January 2023

The potential role of iNOS as a viable therapeutic target for the treatment of asthma was severely hampered by the negative clinical trial results showing that a selective iNOS inhibitor did not affect airway hyperreactivity (AHR) or airway inflammation after allergen challenge in human subjects with asthma. Our laboratory has shown that iNOS inhibition is protective against airway inflammation upon acute, but not chronic, exposures to ovalbumin. More importantly, iNOS inhibition protected against lung fibrosis suggesting that iNOS inhibition may be protective against some aspects of asthma. Here, we show that iNOS inhibition, pharmacologically by L-Nil or by gene knockout, provided an excellent protection against AHR upon an acute, but not chronic, exposure to ovalbumin. Our laboratory also established a reciprocal relationship between iNOS and PARP-1. However, PARP inhibition, protected against inflammation and AHR upon acute and chronic exposure to ovalbumin. It is interesting that PARP-1 inhibition does not completely abrogate expression of iNOS leaving the possibility that the protective effect of PARP inhibition against inflammation and AHR may be associated with reduction but not the complete inhibition of iNOS and associated production of moderate levels of NO. Studies are being conducted to verify this hypothesis and clarify the intricate roles of iNOS in asthma pathogenesis allowing the possibility of being a viable target for the treatment of the disease.
Chimeric antigen receptors based on low affinity mutants of FcεRI re-direct T cell specificity to cells expressing membrane IgE (HYP5P.321)
Authors: Ma Zhengyu , Ward Dana , Han Huihui
Published: January 2023

IgE is the key mediator of allergic immune responses. Targeting the source of IgE, the IgE-expressing B cells, represents an attractive therapeutic approach for atopic diseases. An adoptive T cell therapy (ACT) approach that redirects T cell killing to IgE-expressing B cells is becoming increasingly feasible due to the recent advance of ACT in efficacy and safety. Here, we designed chimeric antigen receptors (CARs) based on FcεRIα and tested their abilities to recognize cells expressing the transmembrane form of IgE (mIgE), a marker for IgE-expressing B cells. The ectodomains of the wild type (WT) and six low affinity mutants of FcεRIα were fused with CD3ζ to form WT and mutant CARs. When expressed on Jurkat cells, four of the six mutant CARs mediated robust T cell responses to U266 myeloma cells expressing mIgE, and the WT CAR mediated lower T cell responses. The presence of free IgE significantly inhibited the activity of WT CAR but did not affect mutant CARs. CAR+ Jurkat cells did not respond to cells with IgE bound through FcεRI or FcεRII (CD23). CAR+ Jurkat cells did not lead to degranulation of LAD2 mast cells with IgE bound through FcεRI. Taken together, CARs based on low affinity FcεRIα mediated robust and specific T cell responses to mIgE+ cells, even in the presence of free IgE, but not to cells with IgE bound through FcεRII or FcεRI. These CARs therefore have the potency and specificity required for targeting IgE-expressing B cells in ACT for atopic diseases.
Mitigation of bacterial translocation and subsequent sepsis in γ-irradiated mice treated with CCL1 antisense ODN (INC4P.331)
Authors: Kobayashi Makiko , Ito Ichiaki , Nishiguchi Tomoki , Suzuki Sumihiro , Loucas Bradford , Suzuki Fujio
Published: January 2023

Gut bacteria-associated sepsis is a serious concern in persons who have radiation-induced injuries in the gastrointestinal system. Such infection does not normally occur in healthy hosts with M1Mϕ. However, M1Mϕ are not generated in γ-irradiated mice (WBI-mice), even though Mϕ are still present. WBI-mice are carriers of M2bMϕ, which inhibit Mϕ conversion from resident Mϕ to M1Mϕ. We have previously reported that sepsis stemming from Enterococcus faecalis oral infection is markedly mitigated in 5 Gy WBI-mice after M2bMϕ polarization using CCL1 antisense ODN. In this study, we examined the effect of CCL1 antisense ODN on spontaneous bacterial translocation and subsequent sepsis in 8 Gy WBI-mice. The ODN was administered (s.c., twice daily) to 8 Gy WBI-mice for 1 week starting 3 days after γ-irradiation. The efficacy of the treatment was evaluated by an increase in the percentage of survival in test groups, as compared with that of control groups. All control mice died within 22 days of γ-irradiation. However, 70% of the WBI-mice treated with 25 to 125 μg/mouse of the ODN survived more than 30 days after γ-irradiation. Severe bacteremia and bacterial growth in MLNs were not seen in WBI-mice treated with the ODN. M1Mϕ were isolated from MLNs of these mice, while M2bMϕ were not. These results indicate that the Mϕ polarization therapy using CCL1 antisense ODN is effective to mitigate spontaneous bacterial translocation and subsequent sepsis in mice exposed to 8 Gy of γ-irradiation.
Possible roles of LC3-independent autophagy in elimination of Shigella in human colon cells (INC4P.343)
Authors: Ra Eun , Lee Sungwook , Park Boyoun
Published: January 2023

Autophagy is essential for the defense against bacterial invasion. The intestinal tract is quite specialized in order to co-exist with extracellular bacteria, and thus, this system efficiently exploits autophagy to regulate the number of bacteria and to control the immune response to preventing chronic inflammation. Bacterial elimination is mediated by microtubule-associated protein 1A/1B-light chain 3 (LC3), which is critical for the activation of the autophagic canonical pathway in mammals. Although both human colon HT-29 cell line and the human cervical HeLa cell line display normal LC3 expression, HeLa cells are very susceptible to Shigella infection, whereas HT-29 cells efficiently restrict the number of intracellular Shigella. Our results suggest that colon cells may utilize LC3-independent noncanonical autophagy to regulate bacterial growth and survival.
Caspase-11 regulates HMGB1 release from liver during polymicrobial sepsis (INM2P.348)
Authors: Deng Meihong , Scott Melanie , Billiar Timothy
Published: January 2023

Caspase-11 was recently identified as the cytosolic LPS receptor, although little is known about its role in a polymicrobial sepsis. We subjected WT and caspase-11-/-(casp-11-/-) mice to polymicrobial sepsis in the form of cecal ligation and puncture(CLP). By 18h caspase-11 was upregulated and activated in multiple tissues, including liver. Casp-11-/- mice had significantly lower bacterial levels in peritoneum (3.9E+5±2.4E+5vs 8.3E+6±4.8E+6CFU/mL;p<0.05), lower systemic cytokine levels (IL1b;IL6) and reduced peritoneal neutrophil death (3.1±0.3vs6.7±0.5%;p<0.05) vs WT. HMGB1 has been associated with late mortality during sepsis and hepatocytes (HC) are major producers of HMGB1 after CLP, as shown by significantly decreased plasma HMGB1 in HC-specific HMGB1-/- mice at 18h after CLP vs WT(11±3vs53±9ng/mL;p<0.05). Similarly, plasma HMGB1 levels in casp-11-/- mice were significantly lower after CLP vs WT(14±2vs39±8ng/mL;p<0.05). To confirm a role for caspase-11 in HMGB1 release, we stimulated primary isolated HC with LPS(100ng/mL) for 24h in vitro. Media HMGB1 level increased significantly in WT HC after LPS stimulation, but not in casp-11-/- HC. Furthermore, HC cell death was not different between WT and casp-11-/- HC +/- LPS suggesting the release of HC-HMGB1 regulated by caspase-11 is not passive via induction cell death. Our data suggest novel roles for caspase-11 in regulation of immune responses during sepsis including the regulation of HMGB1 release by epithelial cells.
The STING of inflammation (INM2P.356)
Authors: Packard Thomas , Mohning Michael , Barthel Lea , Henson Peter , Janssen William , Cambier John
Published: January 2023

Stimulator of Interferon Genes (STING, a.k.a. MPYS, TMEM173) is a critical intracellular transducer of innate immune signals induced by cytosolic DNA. Activation of STING drives both apoptosis and production of type 1 interferon (IFN1). Thus STING is a vital defensive player on the front line of innate immunity to infectious agents. Recent studies have suggested that STING may also be activated by host-derived DNA, particularly in the context of reduced nuclease activity, and could play a role in autoimmune processes. We hypothesize that during inflammation STING can be activated by host-derived DNA acting as a damage-associated molecular pattern (DAMP). To begin to address this role of STING, we utilized multiple models of in vivo sterile inflammation. Our studies demonstrate that STING-deficient animals have reduced inflammatory responses and more rapid resolution of sterile acute lung injury (ALI) and peritonitis. Further, using in vitro and in vivo models we found that STING plays a role in responses to infiltrating neutrophils and neutrophil-derived debris. Studies to be presented support a model wherein STING is activated by host-derived cellular components, such as DNA, and drives production of type 1 interferon and cell death. Ultimately this exacerbates tissue damage, driving a vicious cycle serving to amplify and perpetuate inflammation.
Dendritic cells metabolize and activate retinol into all-trans retinoic acid which stimulates antimicrobial activity in M. tuberculosis-infected cells via paracrine interaction (INM3P.406)
Authors: Liu Philip , De Leon Avelino , Kim Elliot , Radu Roxana , Montoya Dennis
Published: January 2023

Understanding the innate immune response to the Mycobacterium tuberculosis, is a key step to development of effective immunotherapies for tuberculosis (TB). Epidemiological evidence correlates low serum vitamin A (retinol) levels to active TB; however, retinol is biologically inactive. Previously, we demonstrated that stimulation of M. b-infected macrophages with the active vitamin A hormone, all-trans retinoic acid (ATRA), triggers a NPC2-dependent antimicrobial activity. Therefore, understanding the mechanism by which the immune system metabolizes and activates retinol at the site of infection will solve the mystery of how retinol, despite being biologically inactive, can influence the immune response to infection. Through gene expression and functional analysis of human macrophage subsets and dendritic cells (DCs) we identified that GM-CSF-derived DCs express enzymes capable of the two step conversion of retinol into ATRA including, ALDH1A2 and DHRS9, which is subsequently released from the cell. Conditioned medium, from DCs cultured with retinol, stimulated expression of NPC2 from monocytes which was inhibited by a retinoic acid receptor inhibitor (RARi). Inclusion of DEAB, an ALDH1A2 inhibitor, during DCs and retinol incubation also abrogated this response. Expression levels of vitamin A metabolic enzymes are lower in TB lung tissues compared with normal lung. These results help us incorporate vitamin A into clinical care and therapeutic modalities for TB.
Cigarette smoke exposure augments the ability ofStaphylococcus aureus to evade host innate immune responses. (INM3P.407)
Authors: Kulkarni Ritwij , Singh Sanjay , Baral Pankaj , Jeyaseelan Samithamby
Published: January 2023

The nose is the most common reservoir for Staphylococcus aureus, which is present 30-40% of normal adults. Its presence in the nasopharynx varies with age, but greater in children than adults. The oxidant chemicals in cigarette smoke (CS) induce S. aureus biofilm formation by suppressing agr (accessory gene regulator) regulon. In addition to quorum sensing, agr regulon regulates virulence gene expression of S. aureus. Therefore, we hypothesized that CS exposure augments virulence of S. aureus by evading host immunity. Using RNAseq, S. aureus transcriptome analysis revealed upregulation of gene expression which includes the induction of effectors involved in the nasal colonization (clfB), survival inside macrophages (spa) and the escape from neutrophil extracellular traps (NETs; nuc). In accordance with the RNAseq data, CS-exposed S. aureus exhibited significantly reduced phagocytosis, improved intracellular survival, increased evasion NET-mediated killing in the in vitro assays using human and murine macrophages and neutrophils. Importantly, the mouse model of acute pneumonia by intranasal inoculation resulted in significantly increased pulmonary bacterial burden in mice infected with CS-exposed S. aureus as compared to those infected with control bacteria. These results, which are clinically relevant, support a novel paradigm that the normal microflora becomes hypervirulent following exposure to environmental stressors such as CS, leading to devastating infections in humans.
Existence of LysRS-MITF/Hint1 pathway in dendritic cells (IRM12P.642)
Authors: SHU SHIN LA , Yang Chris , Razin Ehud , Kemeny David
Published: January 2023

Using murine bone marrow derived dendritic cell line JAWS II, we investigated the existence of LysRS-MITF/Hint1 pathway in dendritic cells (DC). Based on florescence microscopy, unstimulated cells expressed LysRS predominantly in the cytoplasm. LysRS localization was increased in the nuclei 2 h after stimulation with LPS, RANKL, GM-CSF (LRG) and was sustained over 6 h. To examine its functional role, doxycycline (Dox)-controlled plasmid overexpressing a single point mutant of LysRS (Ser207 to Asp) that mimics constitutive phosphorylation (LysRSS207D) was stably transfected into the JAWS II cell line. Dox-treated LysRSS207D cells were more dynamic, showed increased cell spreading and motility compared to wild-type cells and to untreated LysRSS207D cells. Moreover, Dox-treated LysRSS207D cells exhibited up-regulation of MHCII, but not CD11c, CD80, CD86 and CD40. Primary cultures of bone marrow derived dendritic cells (BMDCs) from C57BL/6 mice were also examined. Bone marrow cells transfected with NUDT2 siRNA inhibited development into BMDCs compared to scrambled siRNA control. Taken together, LysRS-MITF/Hint1 pathway exists in DC and appears to play an important role in DC morphology, maturation and development. Next steps include measurement of cytokine levels, primary allogeneic T cell activation, and adoptive transfer in our asthma and flu models.
Investigating the factors that regulate the rate of intracellular IL-1β degradation (IRM12P.652)
Authors: Ainscough joseph , Gerberick G , Brough David , Kimber Ian , Dearman Rebecca
Published: January 2023

Interleukin (IL)-1β is a key player in the initiation and orchestration of inflammation. The secretion of IL-1β requires 2 independent processes; an initial signal to induce the upregulation of the inactive precursor (pro-IL-1β) and, a second signal to drive cleavage and subsequent secretion. In previous investigations using murine dendritic cells, we have demonstrated that in lipopolysaccharide-stimulated cells, pro-IL-1β is polyubiquitinated and degraded by the proteasome, resulting in a rapid reduction in the amount of intracellular IL-1β available for secretion. The current study has employed an immortalized bone marrow derived murine macrophage cell line that stably expresses fluorescent IL-1β to track the rate of degradation. Here, proteasome inhibition using MG132 led to an increase in cell fluorescence compared with baseline expression, as measured by flow cytometry. This was coupled with a corresponding increase in IL-1β protein levels, as measured by ELISA, and an increase in polyubiquitinated IL-1β, as detected by Western blot. These data indicate that polyubiquitination and subsequent proteasomal degradation of IL-1β are also features of macrophages and independent of cell activation. Furthermore, fluorescence is a reliable readout for measuring degradation in this cell line and thus, represents a useful tool to measure factors that regulate the rate of IL-1β degradation and potentially, the potency of the inflammatory response.
The actin capping protein alpha-adducin is required for CD28 costimulation (IRM12P.658)
Authors: Butte Manish , Thauland Tim
Published: January 2023

Alpha-adducin (Add1) is a critical component of the actin-spectrin network, acting to cap the barbed ends of actin filaments, and recruiting spectrin to these junctions. Add1 is expressed at high levels in naïve T cells and is down-modulated upon TCR stimulation, but the role that Add1 plays in the cytoskeletal rearrangements that occur in motile T cells and during immunological synapse formation have not been investigated. Using CD4 T cells from knockout mice, we show that Add1 is necessary for complete activation of T cells in response to low levels of antigen, as measured by proliferation and cytokine production. Interestingly, the defective proliferation we observed in Add1-/- T cells was due to an inability to respond to respond to CD28 costimulatory signals. Additionally, we demonstrate that naïve and recently activated T cells lacking Add1 have a defect in motility. Using CD4 T cells transduced with GFP-tagged Add1, we found that Add1 is located cortically in non-motile cells, but rapidly moves to the nascent uropod in T cells initiating a crawling phenotype. While the bulk of GFP-Add1 is located in the uropod of crawling cells, brief accumulations are seen at the leading edge. Real-time imaging of interactions between transduced T cells and peptide-loaded antigen presenting cells revealed that Add1 becomes concentrated at the distal pole complex. Together, these results demonstrate that Add1 plays an unexpected and central role in T cell activation and migration.
LPS up-regulates FasL expression in Breg population in mice (IRM12P.643)
Authors: Li Xiaojuan , Wang Keng , Zeng Xiangzhou , Tao Lei , Zhang Yueyang
Published: January 2023

Regulatory B cell plays important role in negative immune regulation, it can exert the inhibitory functions partly by the secretion of IL-10. FasL is also considered to be associated with the immune regulatory function of B cells. Here, we first observed the expression of FasL in CD5+CD1dhi Breg population as well as other B cell subsets after LPS stimulation.In this study, splenocytes were isolated from C57/BL6 wild-type mice and cultured with LPS for 24-48 hours to activate Breg. Results showed that the expression of FasL in CD5+CD1dhi B cell subset is much higher than CD19+CD5+CD1dlow and CD19+CD5neg B cell subset. The lowest FasL expression was seen in CD19+CD5neg B cell subset. This is consistent with the expression of IL-10 in corresponding B cell subsets. In addition, studies showed that the expression of CD25 molecules in CD19+CD5+CD1dhi Breg was significantly higher than other two B cell subsets after stimulating by LPS for 24 hours. CD86 molecule was highly expressed in all these three B cell subsets, and it is higher in CD19+CD5+CD1dhi Breg compared with other B subsets. In contact hypersensitivity model, the expression of FasL in CD19+CD5+CD1dhi Breg after LPS activation showed significant difference in comparison with control group.These results suggested that FasL might also contribute to the inhibitory function of CD5+CD1dhi Breg population after LPS stimulation and thus participate in the regulation of some inflammatory immune diseases.
Nuclear export of NF-κB inhibitor, IκBα, is crucial for T cell function (IRM12P.644)
Authors: Lisiero Dominique , Tejera Melba Marie , Davis Shelly , Neldner Brandon , Suresh Marulasiddappa , Miyamoto Shigeki
Published: January 2023

Numerous cell types, including T cells, respond to inflammatory stimuli by engaging the NF-κB signaling pathway. Classical studies with knockout mouse models demonstrated that T cell receptor (TCR) triggering of NF-κB is crucial for T cell function and proper response to pathogens. While the ability of IκBα, a main inhibitor of NF-κB, to dynamically localize between the nucleus and cytoplasm is a key regulatory mechanism in NF-κB signaling, the physiological significance of this nuclear shuttling in T cell physiology remains undefined. Our group has previously generated a knock-in mouse model, NfkbiaNES/NES, with a germline mutation in the nuclear export sequence (NES) of IκBα. T cell development in NfkbiaNES/NES mice is relatively unaffected as evidenced by normal thymus architecture and thymocyte development. However, mutant CD8+ and CD4+ T cells have severe defects in activation, function, and clonal expansion following infection by LCMV Armstrong. In particular, NfkbiaNES/NES CD8+ T cells failed to produce memory precursor effector cells. Even under competitive conditions in bone marrow chimeric mice, defective T cell expansion and memory formation were observed with NfkbiaNES/NES cells in response to LCMV infection. These findings reveal surprisingly widespread roles of nuclear export of IκBα in the regulation of activation induced NF-κB function in mature T cells in an infectious setting.
Macrophage tolerance to MyD88-dependent TLR agonists is mediated by LPS-/R848-induced miR-146a (IRM12P.649)
Authors: Nahid M. , Benso Lia , Shin John , Mehmet Huseyin , Hicks Alexandra , Ramadas Ravisankar
Published: January 2023

Toll like receptors (TLRs) TLRs facilitate the recognition of pathogens by immune cells and the initiation of the immune response, leading to the production of proinflammatory mediators. Production of proinflammatory mediators by innate immune cells such as macrophages is tightly regulated to facilitate pathogen clearance while limiting adverse impact on host tissue. Exposure to TLR ligands induces a state of temporary refractoriness to a subsequent exposure of a TLR ligand, a phenomenon referred to as ‘tolerance’. This study sought to evaluate the mechanistic regulation of TLR4 and TLR7/8 ligand induced tolerance to other TLRs by miR-146a. Using THP-1 macrophages as well as human M1 and M2 macrophages, we demonstrate that priming with a TLR4 agonist (LPS) or a TLR7/8 agonist (R848) induce tolerance to a panel of TLR ligands in macrophages, leading to the impaired production of a variety of cytokines and chemokines. We also demonstrate that overexpression of miR-146a is sufficient to mimic LPS- or R848-induced hyporesponsiveness. Conversely, the knockdown of miR-146a leads to LPS- or R848-induced TLR hyperresponsiveness. Furthermore, we demonstrate that while miR-146a dampens cytokine production following a primary stimulus with MyD88-dependent, but not MyD88- independent TLR pathways. Collectively, these data provide comprehensive evidence of the central role of miR-146a in TLR signaling tolerance to plasma membrane as well as endosomal TLR ligands in human macrophages.
Modification of self-reactivity on naïve T cell provides long lasting immune surveillance. (LYM6P.721)
Authors: Lee JunYoung , Kim Juhee , Surh Charles
Published: January 2023

It is reported that Ly6c+ CD4 T cell including Tregs and non Tregs exhibits relatively weaker self-reactivity to compare Ly6c- counterpart. Because mature T cells found in the thymus do not express Ly6c, it is possible that the weaker self-reactivity of Ly6c expressing CD4 T cells is obtained features in periphery rather than reflecting their natural bone TCR sensitivity. Furthermore, there are controversial reports for Ly6c inducing factor such as type 1 IFNs, developmental programming on T cell in thymus, and the influence of self-TCR signaling. Therefore, evaluating the true factors inducing Ly6c expression and meaning of Ly6c expression on T cell is required. By transferring Ly6c expressing Tregs into lymphopenic host, we found that self-reactivity of Ly6c+ Treg is almost absence rather than weaker to compare Ly6c- Treg. Ly6c+ Treg proportion in total Foxp3+ is lowest in neonatal period and highest in young adult mice but the ratio is declining with aging and the ratio is inversely correlates with effector Treg ratio. Since activation of Ly6c+ Treg cell readily down-regulate the expression of Ly6c, the decreased ratio of Ly6c+ Tregs may indicate activation of Ly6c+ Treg. Indeed, Ly6c+ Tregs can undergo self-ag induced proliferation in various conditions where there is accumulation of effector non Treg CD4 T cells. Our data suggest that the weaker self-reactivity of Ly6c+ CD4 T cell is not inherited from thymus but acquired phenotype.
Fungal recognition by Dectin-1 on dendritic cells induces IL-10 producing CD4+ T cells by GATA3 / C/EBP-β pathway (MPF4P.733)
Authors: Tajima Masaki , Fuss Ivan , Kitani Atsushi , Strober Warren
Published: January 2023

C. Albicans is known to dramatically transit its morphology between the yeast and hyphal form, depending on its environment. In order to show how these two types of C. Albicans may induce T cell responses, naïve T cells were stimulated with bone marrow-derived dendritic cells in the presence of either form of this organism. The cells stimulated with the hyphal form produced high amount of immunosuppressive IL-10, but not when stimulated with the yeast form. As C. Albicans expresses various innate receptor ligands, T cells were stimulated in presence of both Dectin-1- and/or TLR2-specific ligands. The T cells produced IL-10 when stimulated with Dectin-1 ligand alone, but not when stimulated with both Dectin-1 and TLR2 ligands. This suggests that the hyphal form predominantly express Dectin-1 ligands whereas the yeast form express both Dectin-1 and TLR2 ligands. In accompanying molecular studies, we found that Dectin-1 stimulation (but not Dectin-1 plus TLR2 stimulation) leads to IL-4 production and that Dectin-1-stimulated IL-4-, STAT6-, and GATA3-deficient CD4+ T cells produce greatly reduced amounts of IL-10. Furthermore, CCAAT/enhancer-binding protein beta (C/EBP-β) KO CD4+ T cells were also deficient in IL-10 production. Finally, we found that C/EBP-β interacts with GATA3 and influences its function. Thus, Dectin-1-induced IL-10 production stimulated by certain fungal forms is set in motion by a GATA3 / C/EBP-β mediated effect on IL-10 transcription.
Co-inhibitory and co-stimulatory crosstalk regulates helper T cell differentiation and humoral immunity during Plasmodium infection (MPF5P.742)
Authors: Zander Ryan , Obeng-Adjei Nyamekye , Guthmiller Jenna , Kulu Divine , Li Jun , Ongoiba Aissata , Traore Boubacar , Crompton Peter , Butler Noah
Published: January 2023

Pathogen-specific T cells are subject to co-stimulatory and co-inhibitory signals, the balance of which promotes pathogen control while limiting immunopathology. During blood-stage Plasmodium infection, parasite-specific CD4 T cells express PD-1 and LAG-3 co-inhibitory receptors that coordinately limit CD4 T cell function. Blockade of PD-1/LAG-3 co-inhibitory signaling during chronic Plasmodium infection enhances CD4 T cell function and parasite control. In contrast to co-inhibitory receptors, the role of T cell co-stimulatory receptors during established Plasmodium infection has not been investigated. Here we show that CD4 T cells exhibit sustained expression of the co-stimulatory receptor OX40 during both human and experimental malaria. We hypothesized that co-stimulatory signaling can functionally counteract co-inhibitory circuits to maintain protective Plasmodium-specific CD4 T responses. In support of this, therapeutically activating OX40 during established experimental malaria enhances CD4 T cell activity, humoral immunity and parasite clearance. Our data also show that the effects of OX40 signaling are modified following simultaneous PD-1 blockade, which markedly impairs parasite-specific T follicular helper cell responses in an IFN-g-dependent manner. Collectively, our results identify that OX40 can be targeted to limit malaria parasite replication and reveal a previously unrecognized role of IFN-g as a negative regulator of Plasmodium-specific humoral immunity.
A pivotal interplay between TNF alpha and the IL-23-axis cytokines in regulating primary human intestinal epithelial cell apoptosis (MUC5P.753)
Authors: Juncadella Ignacio , Wang Chao-ting , Caviness Gary , Nabozny Gerald
Published: January 2023

TNFα induces intestinal epithelial cell (IEC) cell death, likely responsible for barrier dysfunction and mucosal damage. However, studies addressing the role of TNFα on epithelial damage rely on Caco2 or HT29 cell lines and rarely primary IECs. Interleukin-23 (IL-23) pathway is genetically associated to IBD pathogenesis. IL-23-responsive cells produce the signature cytokines, interleukin-17 (IL-17) and interleukin-22 (IL-22). These cytokines are elevated in IBD patients, and may contribute to disease pathology. Here, we describe the synergistic interplay between TNFα and the IL-23 axis on IEC apoptosis. Using human IECs and organotypic cultures, we demonstrate that; TNFα treatment induces moderate to low proinflammatory gene expression compared to IL-17 and IL-22, TNFα and IL22 stimulation leads to upregulation of proinflammatory and proapoptotic genes and, IL22 but not IL17 enhances TNFα-induced cell death. Interestingly, the effect of IL22 was independent of IFNγ, contrary to previous findings. IL-22 stimulation resulted in TNFRI and TNFRII expression, likely making the epithelium more responsive to TNFα-induced cell death. In conclusion, this is the first demonstration between TNFa and the IL23-axis in driving IEC apoptosis and cell death. Also, while TNFα and the IL-23 axis contribute to IBD pathogenesis via different approaches; the interplay of these pathways may be linked to promote epithelial cell damage and barrier dysfunction resulting in disease progression.