bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of high cytotoxic NK cell model for leukemia treatment (TUM10P.1041)
Authors: Twu Yuh-Ching
Published: January 2023

Natural killer (NK) cells play an important role in innate immune system to against virus-infected cells, transformed cells and tumor cells without further stimulation. The killing functions of NK cells regulated by the signals from a large set of activating receptors and inhibitor receptors. Moreover, either the directly interaction with other immune cells, including dendritic cells, macrophages and T cells, or the cytokines secreted from other cells also enhance the NK cell killing activity. The NK cell produces cytokines, such as IFN-γ and TNF-α, recruit immune cells to enhance the inflammatory response as well. Acute myeloid leukemia is characterized by the accumulation of immature cells in bone marrow and affects the hematopoiesis. By standard chemo- and radio-therapy, leukemia could be controlled on the remission status. However, the leukemia stem cells which are resistant to conventional treatments, cause the relapse leading to the death of AML patients. So far it is believed that hematopoietic stem cell transplantation is a curable treatment for AML, but it is limited by the patient age and the availability of HLA-matched donor. According to previous study, NK cells seem to have the potential to kill LSCs and lower the disease relapse. In this study, we try to develop a NK cell model with the ability to eliminate both leukemia cells and LSCs. If success, the highly cytotoxic NK model may apply to clinical AML treatment to prevent the relapse of AML.
HLA-G expressing NK cell subset with suppressive properties is expanded in breast cancer (TUM10P.1043)
Authors: Ostapchuk Yekaterina , Aktas Cetin Esin , Perfilyeva Yuliya , Yilmaz Abdullah , Belyaev Nikolai , Deniz Gunnur
Published: January 2023

NK cells were considered traditionally as cytotoxic effector cells rapidly killing infected and transformed cells. Recently, NK cells have been shown to act as potential regulatory cells interacting with various components of the immune system. HLA-G is an important immune-regulatory molecule.There is evidence that HLA-G expression contributes to escape of malignant cells from immune recognition and destruction by down-regulation of the immune response. Immunosuppressive T cells expressing HLA-G have been recently reported. In healthy donors, we have found a subset of circulating NK cells that constitutively express HLA-G (4.5±1.3, n=11), which occurs more frequently among CD57- than CD57+ NK cells. They are enhanced in response to PHA stimulation and enriched with IL-10- and TGF-β-producing NK cells. HLA-G+ NK do not show cytotoxic activity and strongly decrease both cytotoxicity and expression of perforin, granzyme B and CD107a of HLA-G- NK cells, but do not affect IFN-γ production. HLA-G neutralization antagonized the suppressive effects, leading to a 88% restoration of cytotoxic capacity of HLA-G- NK cells. In breast cancer patients, an increased number of circulating HLA-G+NK cells compared to healthy women (6.8±1.8, n=21; 4.0±1.1, n=6; p=0.001) has been detected. Obtained data suggest that increased number of suppressive HLA-G+ NK cells may be included into cohort of immunosuppressive cells mediating escape of NK-sensitive tumor cells from cytolysis.
SAHA prevents osteoblast-mediated protection of AML cells via upregulation of Nherf-1 (TUM10P.1050)
Authors: Hedin Karen , Kremer Kimberly , van Wijnen Andre , Westendorf Jennifer
Published: January 2023

Osteoblasts are known to protect acute myeloid leukemia (AML) cells in the bone marrow microenvironment, however, the identity of these protective signals is not known. Because disrupting this protection could help prevent the relapse of AML patients following chemotherapy, we characterized the molecular mechanisms. We previously showed that SDF-1 (CXCL12), a chemokine abundant in the bone marrow, induces apoptosis of AML cells in cocultures unless the leukemic cells are protected via proximity to differentiating osteoblasts. Treating differentiating osteoblasts with either suberoylanilide hydroxamic acid (SAHA) or LBH-589 histone deacetylase (HDAC) inhibitor drugs inhibited their protection of AML cells. RNAseq revealed that SAHA globally inhibits osteoblast differentiation while inducing expression of the scaffold protein, Nherf-1. Remarkably, expressing Nherf-1 in differentiating osteoblasts was both required and sufficient to block osteoblast-mediated protection of AML cells. Together, these results indicate that disrupting the differentiation of osteoprogenitors in the bone marrow inhibits their ability to protect AML cells from apoptosis, and that this can be achieved via either inhibiting HDACs or overexpressing Nherf-1 in osteoblast-lineage cells. HDAC inhibitors or treatments specifically designed to induce Nherf-1 expression either during or immediately prior to chemotherapy may therefore be a useful strategy for reducing the risk of disease relapse in AML patients.
Dissecting early mechanisms of melanoma cell resistance to cytotoxic T lymphocyte attack (TUM10P.1025)
Authors: khazen roxana , Puissegur Marie-pierre , Muller Sabin , Valitutti Salvatore
Published: January 2023

The effector function of naturally occurring Cytotoxic T cells (CTL) or of CTL induced by immunetherapy protocols is often insufficient to achieve clinical remission of melanoma. Consistent with these, we have recently shown that cytotoxic T cells are equally activated upon encountering melanoma cells as compared to conventional targets; yet the apoptotic process seems to be defective or “delayed” in melanoma cells. We are presently interested to investigate mechanisms of melanoma resistance to CTL attack during cell/cell interaction at the immunological synapse, in aiming to improve the effectiveness of melanoma therapies. Our results show that melanoma cell resistance is not mainly related to a deficient intrinsic apoptotic process. Conversely, it seems that melanoma cells have evolved a resistance mechanism which causes a reduced entry and activity of granzyme B, which we showed to be related to a deficiency of perforin binding and polymerization on melanoma cells surface. In addition, melanoma cells exhibited a constitutive lysosome exposure, which increased upon specific contact with CTL, resulting in an intense exocytic activity towards the lytic synapse that contributes to their escape mechanism. Moreover, silencing several key molecules of secretory pathway in melanoma cell increased their susceptibility to cytotoxic attack. Together, our results reveal a new pathway of melanoma cell resistance that might become a potential target of new therapeutic strategies.
Synergistic COX2 induction by IFNγ and TNFα self-limits type-1 immunity in cancer tissues (TUM10P.1027)
Authors: Obermajer Natasa , Wong Jeffrey , Odunsi Kunle , Edwards Robert , Kalinski Pawel
Published: January 2023

Maintenance of CTL-, Th1-, and NK cell-mediated type-1 immunity is essential for effective anti-tumor responses. Unexpectedly, we observed that IFNγ and TNFα, the critical soluble mediators produced by type-1 immune effector cells, synergize in the induction of cyclooxygenase 2 (COX2), the key enzyme in prostaglandin (PG)E2 synthesis, and the hyper-activation of myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment (TME) of ovarian cancer patients. This MDSC hyper-activation by type-1 immune cells and the subsequent over-expression of indoleamine 2,3-dioxygenase (IDO), inducible nitric oxide synthase (iNOS/NOS2), IL-10, and additional COX2 result in strong feedback suppression of type-1 immune responses. Importantly, this self-limiting feedback could be eliminated by COX2 blockade, allowing amplification of type-1 immunity and prolonged survival of tumor bearing mice. Our data demonstrate a new mechanism underlying the self-limiting nature of type-1 immunity in the human TME and provide rationale for targeting the COX2-PGE2 axis to enhance the effectiveness of cancer immunotherapies.
Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein (VAC11P.1106)
Authors: Sun Lina , Wei Jingshuang , Qu Jing , Li Dexin , Liang Mifang
Published: January 2023

Currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test (RFFIT) in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site Ⅱ of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.
Bacterial lysates activates B cell differentiation and IL-10 production via Toll-Like Receptor-2 recognition (VAC11P.1064)
Authors: Jimenez-Martinez Maria , Salazar Alberto , Galicia-Carreon Jorge , Castillo Axel , Andaluz Maria , Nieto Jane
Published: January 2023

Bacterial lysates (BL) are used to improve immune response in patients with respiratory infections. BL are constituted by a mixture of bacterial antigens derived from different bacterial species; species included in these lysates are 14 different bacteria: S. pneumoniae, K. pneumoniae, B. catarrahalis, S. aureus, H. influenza, S. alpha, and beta, S. faecalis, S. epidermidis, B. pertussis, Proteus sp., Pseudomonas sp., E. coli, and C. pseudodiphteriae (BL-14) It has been suggested that BL activate innate response through TLR, however if BL-14 are able to induce B cell activation is not known and this was the aim of our study. Peripheral mononuclear blood cells (PBMC) were obtained from healthy donors and cultured with/without BL-14 (IPI ASAC Pharma) at different concentrations, after stimulation with BL-14, supernatant were collected and IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A (BD Biosciences), were measured with cytometric bead arrays. PBMC were harvested at 24, 48 and 72 h, labelled against anti- CD19, CD38, CD69 or TLR2 (BD Biosciences), and analysed by flow cytometry. We observed dose-dependent increased activation of CD19+. After 72 h we observed a diminished frequency of B cells precursors and expansion of transitional B cells, these cells were TLR2+ cells. A significant increased concentration of IL-10 and IL-6 was observed at 24h, and 48 h after BL-14 stimulation. BL induces expansion of transitional B cells and IL-10 production through TLR2 recognition
Different alpha fetoprotein epitope-specific CD8 T cells possess distinct antitumor function (VAC13P.1130)
Authors: He Yukai , Wu Sha , Peng Yibing , Wang Lan , Hong Yuan , Celis Esteban , Wu Juan
Published: January 2023

Using lentivector (lv) immunization and epitope-optimization, we have recently identified three CD8 epitopes in the hepatocellular carcinoma (HCC) associated antigen of murine alpha fetoprotein (AFP) that could effectively activate CD8 T cells in mice, resulting in potent antitumor effect that can completely prevent mice from tumor challenge and prevent carcinogen-induce autochthonous HCC. In this report, we studied whether the epitope-specific CD8 responses can be further enhanced by peptide boost and investigate if the newly identified epitope specific T cells can kill AFP+ tumor cells and have equal antitumor activity. We found that the lv-primed AFP-specific CD8 responses can be boosted by AFP peptides even at the peak of primary responses. As much as 20 folds increase of AFP-specific CD8 responses can be achieved by peptide boost. However, the kinetics and the antitumor effect of different epitope-specific CD8 responses are drastically different. While the epitope AFP499-specific CD8 T cells possess potent CTL activity against AFP+ tumor cells and generate strong antitumor effect in vivo, the AFP212-specific CD8 T cells have a very limited CTL activity and antitumor effect though they can produce high level of IFNg. The mechanisms underpinning the functional differences will be reported. This finding demonstrates that cancer vaccines must be able to elicit tumor-killing immune effector cells in order to generate antitumor effect.
Neuronal nitric oxide synthase: a possible inflammatory mediator responsible for host-induced susceptibility to herpes simplex encephalitis (VIR9P.1153)
Authors: Ferraioli Adrianna , Lundberg Patric
Published: January 2023

Herpes Simplex Virus Type-1 (HSV-1) infection can result in a severe disease of the central nervous system termed Herpes Simplex Encephalitis (HSE). In both animals and humans, the immune response is known to play a role in HSE progression. Using resistant and susceptible HSE mouse models, we have characterized late-stage (>day 7 post-infection (p.i.)) inflammation by which susceptible mice succumb to HSE. Previous data have indicated that immune dysregulation between the two strains is evident at day 6 p.i. Thus, we propose that activation of specific pathways before day 6 p.i. result in an inflammatory cascade that contributes to HSE. To demonstrate this, resistant C57BL/6 and susceptible 129S6 male mice were infected with HSV-1 and brainstems were collected days 1-4 p.i. Microarrays, Ingenuity Pathway Analysis (IPA), and QRT-PCR were performed on RNA, and protein levels were determined via westerns. IPA predicated several inflammatory functions, such as migration of immune cells, to be 129S6-biased (p<0.05). Among the dysregulated transcripts identified by the microarray, neuronal nitric oxide synthase (nNOS) was shown to be significantly upregulated in susceptible 129S6 mice on day 3 p.i at both the RNA (p<0.01) and protein level (p<0.001). Our results implicate nNOS as a regulator of HSE pathogenesis and we are currently performing animal studies to better define the role of nNOS in HSE progression.
The application of clustered regularly interspaced short palindromic repeats genome engineering to the study of host antiviral factors (VIR9P.1160)
Authors: Omokehinde Tolu , Soll Steven , Bieniasz Paul
Published: January 2023

Human immunodeficiency virus (HIV), is a retrovirus that infects cells of the human immune system, including CD4­+ T cells, macrophages and dendritic cells. Previously, two of the candidate genes identified showed a capacity to protect cells from HIV-1 infection were Tripartite motif containing 56 (TRIM56) and 2’, 3’-cyclic-nucleotide 3’-phosophodiesterase (CNP). Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (CAS) protein 9 is a nuclease system involved in defense against invading phages and plasmids in bacteria. This system can be programmed to induce DNA double stranded breaks (DSBs), which when targeting coding regions of genes can create frame shift insertion/deletion (indel) mutations that result in gene knock-out cell lines. Using the CRISPR/Cas9 system, we were able to clone guide sequences targeting TRIM56 and CNP into a LentiCRISPR and LentiGuide plasmid. The cloned plasmids were then used to generate viruses that would deliver the necessary machinery to target and cleave TRIM56 and CNP genes. To test the efficiency of the CRISPR/Cas9 system, guide sequence targeting GFP were cloned. GFP deletion was observed in a 293T-GFP stable cell line and knockout efficiency was determined by analysis. Knockout results of TRIM56 and CNP are still pending, but we expect that TRIM56 and CNP deleted cell lines will demonstrate the contribution of each of these innate immune factors to the interferon response to HIV-1.
HIV infection in humanized mice: role of type I interferon on disease progression (VIR9P.1139)
Authors: Abraham Sojan , Choi Jang Gi , Ye Chunting , Schreiber Gideon , Shankar Premlata , Swamy Manjunath
Published: January 2023

HIV and SIV disease progression is associated with chronic expression of type I IFN and IFN-stimulated genes. A recent study showed that a high-dose injection of IFN-1 early during primary SIV infection in the rhesus macaque model was protective, while neutralization of endogenous IFN-1 was deleterious. In contrast, in the same animal model, prolonged IFN-1 administration accelerated disease development in the chronic stage of the infection. To better understand the role of IFN-1 during HIV infection, we used humanized-PBL mouse model for this study. The advantage of this model is that there is no endogenous interferon production upon HIV infection, allowing analysis of IFN-1 therapy. Since continuous presence of IFN-1 is detrimental in the SIV model, we hypothesized that intermittent treatment with IFN-1 may be protective against HIV infection. To test this hypothesis, we carried out intermittent pegasys (peg-interferon alpha) treatment after HIV infection in human PBL infused NOD-scid IL2rγcnull (hu-PBL) mice. This treatment with pegasys diminished HIV replication and delayed CD4 depletion compared to control mice. To further test the role of IFN, we are currently using humanized BLT mouse model of HIV infection. HIV infection in BLT mice leads to continuous production of high levels of type-I IFNs; and thus, we are studying the effects of continuous or intermittent IFN blockade using type I IFN antagonist. The results will be discussed.
Mechanism of HIV-1 capsid-nucleocapsid polyprotein in genome recognition (VIR9P.1143)
Authors: McCowin Sayo , Keane Sarah , Summers Michael
Published: January 2023

Human Immunodeficiency Virus type-1 (HIV-1) is a retrovirus that is responsible for a widespread epidemic affecting nearly 36 million individuals worldwide. During the late phase of the viral life cycle, the Gag polyprotein specifically binds two copies of the unspliced viral genomic RNA. Gag consists of three structured domains: matrix (MA), capsid (CA), and nucleocapsid (NC). We have shown that mature NC binds to both the monomeric and dimeric 5’-untranslated region (5’-UTR) RNAs, but fails to explain the observation that the dimeric genome is preferentially packaged. Additionally, mature NC does not exist in the cytoplasm, where genome recognition occurs. Thus, this has led to the belief that other domains of Gag may also be involved in the selection of dimeric genomes. We hypothesize that the CA domain contributes to diploid RNA genome selection. In order to test this hypothesis, the polyprotein Capsid-Nucleocapsid (CA-NC) will be purified and titrated into 5’-UTR viral RNA. Previous work has shown that this CA-NC polyprotein is unstable and aggregates in the presence of RNA due to hexameric interactions mediated at the C-terminal domain (CTD). We will utilize the strategy of Gamble et al., by introducing two critical mutations, W184A and M185A, in the CTD domain of CA. These mutations have been shown to block CTD dimerization resulting in the monomeric form of the CA protein, while preserving necessary characteristics of the wild-type CA.
Virulence determinants enhance the CD8+ T cell response to a recombinant live virus vaccine (VIR10P.1168)
Authors: Pavelko Kevin , Bell Michael
Published: January 2023

Safe and effective vaccine vectors that drive T-cell responses are critical for the development of immunotherapies that treat cancer and infectious disease. We have developed Theiler’s murine encephalomyelitis virus (TMEV) as a vector for promoting CD8 T-cell responses to epitopes embedded within its genome. Previously, we demonstrated that the site of epitope integration within the viral genome can alter virulence and promote viral clearance. However, its inability to drive complete elimination of tumors suggests that further development is necessary. The TMEV species is comprised of two subspecies, the persisting group Daniel’s (DA) and a non-persistent virulent group GDVII. We introduced a genomic fragment of GDVII into the DA strain that contains the capsid and flanking regions. Virus generated from this vector demonstrates increased virulence and replicative capacity. To determine whether this vector could drive enhanced CD8 T-cell responses to vaccine epitopes we engineered the CD8 epitope SIINFEKL into the leader protein of the GDVII chimeric virus. Infection of B6 mice with this vaccine enhances the number of SIINFEKL specific CD8 T-cells when compared to wild-type vector. Studies to identify the precise determinants that are necessary for this enhancement and how effectively this vaccine can promote anti-tumor immunity will be important for future vector development.
CD44 controls T cell exhaustion and viral persistence during chronic viral infection (VIR10P.1170)
Authors: Carrette Florent , Tinoco Roberto , Barraza Monique , Bradley Linda
Published: January 2023

During a chronic viral infection, inhibitory receptors play a crucial role in controlling viral persistence and T cell exhaustion. However, the role of homing molecules in this process has been poorly investigated. Using the chronic LCMV virus model, Clone 13, we found that expression of CD44, a cell surface glycoprotein broadly used to identify activated T cells, dampens antigen specific T cell responses. In CD44-deficient hosts, we observed a significant increase in antigen specific CD4 and CD8 T cells functions with decreased PD-1 expression and a striking increase in multiple cytokine production. T cell accumulation was not due to increased proliferation based on BrdU incorporation, and the increased CD8 T cell response required CD4 T cell help because CD8 T cell exhaustion was maintained in CD4 depleted CD44-deficient mice. Using a bone marrow chimera approach, we found that restricting the CD44 deficiency in the non-hematopoietic compartment was sufficient to reproduce the observations made in the complete CD44 deficient hosts. Finally CD44-deficiency resulted in viral clearance by d15pi. Importantly, treatment of WT mice with a CD44-blocking antibody increased antigen specific CD4 and CD8 T cell recovery and some aspects of T cell function as early as d9pi. Taken together, these results indicate that CD44 is a novel inhibitory receptor that can be targeted to improve T cell response during chronic viral infections.
Deficiency in the receptor tyrosine kinase EphB2 attenuates experimentally-induced liver fibrosis in mice (CAM1P.156)
Authors: Mimche Patrice , Brady Lauren , Bray Christian , Mimche Sylvie , Thapa Manoj , King Thayer , Quicke Kendra , Lee Choon , Grakoui Arash , Morgan Edward , Lamb Tracey
Published: January 2023

Beyond the well-defined role of Eph receptor tyrosine kinases in biological processes, cell migration, adhesion, nothing is known about their implication in liver pathologies. During blood-stage rodent malaria infection, EphB2 mRNA and proteins are upregulated in the liver, a result likely driven by elevated surface expression on macrophages. This is significant for malaria pathogenesis because EphB2-/- mice are protected from malaria-induced liver fibrosis despite having a similar liver parasite burden compared with littermate mice. This protection is correlated with a defect in inflammatory potential of hepatocytes from EphB2-/- mice resulting in a reduction in adhesion molecules, chemokine/chemokine receptors RNA levels and infiltration of leukocytes including Kupffer cells which mediate liver fibrosis during malaria. These observations are recapitulated in the well-established carbon tetrachloride (CCL4) model of liver fibrosis in which EphB2-/- CCL4-treated mice showed a significant reduction of liver fibrosis compared to CCL4-treated littermate mice. EphB2 is predominantly expressed by Kupffer cells and depletion of macrophages abrogates liver EphB2 mRNA and protein increase in malaria infected mice, as well as fibrosis. Altogether these results reinforce the critical role played by EphB2 in promoting liver inflammation and fibrosis. To our knowledge, this work is the first to reveal the potential profibrotic nature of EphB receptor tyrosine kinases in liver injury.
The middle tyrosine residue of tyrosine-based inhibitory signalling motifs is a key molecule in inhibitory functions of the leukocyte immunoglobulin-like receptor B4 in FcγRI mediated cytokine production and bacterial phagocytosis. (CCR3P.211)
Authors: Park Mijeong , Tedla Nicodemus
Published: January 2023

The leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory cell surface receptor, primarily expressed on mono-myeloid cells. LILRB4 is a potent inhibitor of FcγRI-mediated pro-inflammatory cytokine production by monocytes and induces tolerogenic dendritic cells in vitro. It is believed that LILRB4 regulates monocyte/macrophage activation through its three intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) by dephosphorylation of non-receptor tyrosine kinases via recruitment of Src homology phosphatase-1 (SHP-1). However, the relative contribution of these ITIMs to LILRB4-mediated inhibition of monocyte/macrophage functions is poorly characterised. To address this, the three functional tyrosine residues of LILRB4 ITIMs at positions 337, 389 and 419 were single, double or triple mutated to non-functional phenylalanine and stably transfected into THP-1 cells. The effects of these mutants on FcγRI-mediated monocyte functions were then assessed. We show that the middle tyrosine residue (Y389) alone maximally inhibited but the proximal tyrosine residue (Y337) enhanced FcγRI-mediated TNF-α production and Fc-dependent bacterial phagocytosis. These results for the first time show that LILRB4 may have variable functions in vitro dependent on the position of the tyrosine residues in its ITIMs. The underlying mechanisms and in vivo implications of these adaptable functions require further investigations.
Signal transduction via CD103 and BAFF-receptor cooperatively play a role in the abnormal activation of T cells. (CCR3P.216)
Authors: Yoshimoto Keiko , Kurasawa Takahiko , Suzuki Katusya , Takeuchi Tsutomu
Published: January 2023

Background and Purpose: It was reported that the expression of CD103 (αEβ7) was elevated in infiltrated T cells in lachrymal glands of patients with Sjögren’s syndrome (SS), suggesting that abnormal expression of CD103 in T cells is involved in the pathogenesis of SS. Our previous study suggests that BAFF plays a role in the development of SS. In this study, we investigated a possible interaction between CD103 and BAFF in T cells. Materials and Methods: Jurkat, a human T cell leukemia cell line, was transfected with both human αE and β7 genes. Stable transfectants were stimulated with anti-CD3 to induce production of IL-2. The cells were further stimulated with anti-CD103, recombinant human soluble BAFF (rhsBAFF) or both. Results and Discussion: It may be noteworthy that the IL-2 production by T cells upon stimulation with ant-CD3 was elevated by the overexpression of CD103. The data suggest that CD103 plays a role in the activation of T cells. Anti-CD103 and rhsBAFF significantly enhanced the effect of anti-CD3 possibly in an additive manner. FACS and quantitative RT-PCR revealed that the expression level of a BAFF receptor (BR3) was increased in the transfectants, suggesting that the effect of CD103 on the response of the cells to rhsBAFF is mediated through the enhancement of BR3 expression. The data collectively suggest that abnormal expression of CD103 in T cells is involved in the development of SS by affecting BAFF signaling pathway.
Runx1 is required for T cell maturation (HEM2P.246)
Authors: Dash Barsha , Hsu Fan-Chi , Belmonte Paul , Constans Megan , Chen Meibo , McWilliams Douglas , Shapiro Virginia
Published: January 2023

T cell development depends on coordinated gene expression changes in order to progress through developmental checkpoints. The loss of transcription factor Runx1 in CD4-cre Runx1 conditional knockout (cKO) mice leads to decreased numbers of CD4 single positive (SP) thymocytes, CD4 peripheral T cells and lower expression of IL-7Rα, a key player of T cell homeostasis, by CD4+ Runx1 deficient T cells . Low IL-7Rα is thought to cause decreased CD4 T cell survival. However, we show that constitutive expression of an IL-7Rα transgene in CD4-cre Runx1 cKO mice does not restore thymic or peripheral CD4 cellularity. We also see similar expression of Bcl-2 and Bcl-xL in WT and Runx1-deficient CD4 T cells, indicating that a Runx1-mediated IL-7Rα-independent mechanism might be responsible. As IL-7Rα expression increases over the course of T cell maturation, we asked if Runx1 is needed for maturation. During thymic maturation, post-positive selection CD4 SP thymocytes migrate from cortex to medulla, changing from CCR7lowCD24high to CCR7highCD24high and finally CCR7highCD24low before thymic egress. We saw a block at the CCR7lowCD24high stage in CD4-cre Runx1 cKO CD4 SP thymocytes. Additionally, other maturation markers including CD55 and Qa2 were also downregulated. Thus, Runx1 is required for thymic maturation of CD4 SP thymocytes.
Retention and tolerance of autoreactive CD4+ recent thymic emigrants in the liver (HEM2P.249)
Authors: Ge Qing , Xu Xi , Zhang Shusong
Published: January 2023

Mechanisms of peripheral tolerance play a critical role in preventing T cells that escape from negative selection in the thymus from initiating autoimmune reactions. To investigate the site of peripheral tolerance induction, we examined migration and activation of recent thymic emigrants (RTEs) in liver, spleen, lymph node and peripheral blood. We show that a fraction of RTE precursors were retained in the liver independent of the secondary lymphoid organs. Compared to RTEs from the lymph nodes, RTEs from the liver proliferated more and many exhibited an activated phenotype with the capability of producing IL-10 upon activation. Liver RTEs also responded poorly to interleukin (IL)-7 and were more prone to apoptosis. Following transfer into RAG-/- recipients, liver RTEs induced more severe inflammation and T cell infiltration in the lung and colon. The extrathymic expression of MHC and Aire is required for the acquisition of tolerogenic phenotype of newly generated thymic emigrants in the liver. These results suggest that the liver is the first checkpoint in the periphery to filter, retain, and enforce tolerance to autoreactive CD4+ thymic emigrants that escape from negative selection.
Interleukin-15 receptor α signaling constrains the development of IL-17-producing γδ T cells (HEM2P.232)
Authors: Colpitts Sara , Puddington Lynn , Lefrançois Leo
Published: January 2023

The development and homeostasis of γδ T cells is highly dependent on distinct cytokine networks. Here we examine the role of interleukin (IL)-15 and its unique receptor, IL-15Rα, in the development of IL-17-producing γδ (γδ-17) T cells. Phenotypic analysis showed that CD44high γδ-17 cells expressed IL-15Rα and the common gamma chain (CD132) yet lacked the IL-2/15Rβ chain (CD122). Surprisingly, we found an enlarged population of γδ-17 cells in the peripheral and mesenteric lymph nodes of adult IL-15Rα KO mice but not IL-15 KO mice. γδ-17 cells were also increased in the peripheral lymph nodes of transgenic knock-in mice where the IL-15Rα intracellular signaling domain was replaced with the intracellular portion of the IL-2Rα chain (that lacks signaling capacity). The generation of mixed chimeras from neonatal thymocytes indicated that cell-intrinsic IL-15Rα expression was required to limit IL-17 production by γδ T cells. Finally, an analysis of neonatal thymi revealed that the CD44low precursors of γδ-17 cells were increased in newborn mice lacking IL-15Rα. Thus, these findings demonstrate that signaling through IL-15Rα regulates the development of γδ-17 cells early in ontogeny with long-term effects on their peripheral homeostasis in the adult.
Themis and the control of SHP1 phosphatase activity in TCR signal strength during thymocyte positive selection (HEM2P.234)
Authors: Gascoigne Nicholas , Brzostek Joanna , Mehta Monika , Zhao Xiang , Fu Guo , Paster Wolfgang , Acuto Oreste , Rybakin Vasily
Published: January 2023

Themis is a T cell-specific protein important for positive selection in the thymus. It associates, through the adaptor GRB2, with the LAT signalosome after stimulation. Recent data show that it acts as a negative regulator of TCR signal strength for positive selecting ligands during thymocyte development, through its interaction with the phosphatase SHP1. A Themis-GRB2-SHP1 complex is recruited to LAT, tuning down the TCR signaling cascade, so that intermediate-strength positive selecting ligands are not recognized as agonists. In Themis-knockout thymocytes, signaling in response to positive selecting ligands is strong, resulting in increased negative selection. In contrast, responses to strong, negative-selecting, ligands are not affected by the absence of Themis. SHP1 is under-phosphorylated in Themis-deficient thymocytes, and its phosphorylation is not significantly increased after TCR stimulation, in contrast to results from Themis-sufficient thymocytes. In concert with phosphorylation, phosphatase activity of SHP1 is increased after TCR stimulation, but this is much reduced in the absence of Themis. We have recently found that phosphatase activity can be coprecipitated with Themis from thymocytes, and this activity increases immediately after TCR stimulation. The amount of phosphatase activity bound to Themis varies with signal strength. These results will be discussed in the context of dynamic control of positive selection signaling during thymocyte development.
An intricate link between autophagy and microRNAs in cystic fibrosis (HUM1P.262)
Authors: Tazi Mia , Dakhlallah Duaa , Khalil Hany , Caution Kyle , Marsh Clay
Published: January 2023

Each year 1,000 children and adults are diagnosed with Cystic Fibrosis (CF), a fatal genetic disorder that critically affects the lungs. Autophagy, a highly-regulated biological process, normally functions to clear dysfunctional CFTR (CF transmembrane conductance regulator) proteins that aggregate within macrophages. However, this process is defective in CF patients and CF mice, as their macrophages express limited autophagy activity thus exacerbating inflammation. Present therapies to improve autophagy are ineffective. MicroRNAs (miRNAs, miRs) are non-coding RNAs that post-transcriptionally regulate targeted mRNA expression. The objective for this study is to elucidate the role of miRNAs in CF macrophages in an effort to restore autophagy. We hypothesize CF macrophages exhibit elevated cluster expression that downregulate autophagy targets thus contributing to autophagy dysfunction. We find that, CF macrophages exhibit decreased autophagy protein expression and elevated cluster expression compared to WT. When cluster expression is absent, autophagy protein expression is restored, suggesting the canonical inverse relationship between miRNA and protein expression. Predicted autophagy targets of specific miRs comprising the cluster were validated. In vivo downregulation of specific miRs comprising the cluster increases autophagy expression. Thus, this data demonstrates, microRNA cluster expression correlates to autophagy expression which modulates the pathophysiology of CF.
Myeloid dendritic cells with elevated CD1c expression in non-infectious uveitis patients exhibited mature phenotype through the regulation of TNFa-p38 MAPK pathway (HUM1P.273)
Authors: Chen Ping , Denniston Alastair , Hannes Susan , tucker william , Wei Lai , Liu Baoying , Hirani Sima , Li Zhiyu , Lee Richard , Sen H. , Nussenblatt Robert
Published: January 2023

Our longitudinal data has shown that the level of CD1c+ myeloid dendritic cells 1 (mDC1) from peripheral blood reflects disease activity in autoimmune non-infectious uveitis patients. However, the role of CD1c+ mDC1 is not clear in autoimmune uveitis patients. Here, we demonstrate that the mDC1 in patients with non-infectious uveitis (n=74) was increased compared to age and gender-matched healthy controls (n=96). The increased CD1c+ mDC1 exhibited high HLADR expression and less antigen uptake. CD1c+ mDC1 were further divided into two subpopulations based on the level of CD1c expression. CD1chi mDC1 subpopulation showed less antigen uptake and higher HLADR expression compared to CD1clo mDC1 subpopulation. Importantly, the CD1chi mDC1 subpopulation was significantly increased in uveitis patients. By utilizing monocyte-derived dendritic cells (MoDCs) for in vitro studies, MoDCs with less antigen uptake and higher levels of CD1c induced more CD4+ T helper cell activation and proliferation. TNFa, increased in uveitis patient sera, reduced the antigen uptake in a dose-dependent manner and increased the phosphorylated p38 expression on MoDCs. A p38 inhibitor significantly inhibited the expression of CD1c during dendritic cell differentiation from monocytes. In conclusion, CD1c is a maturation marker of mDC1, increased in uveitis patients and correlated with disease activity. The high CD1c expression and low antigen uptake are mediated by the TNFa-activated p38 MAPK pathway.
Healthy human pregnancy: inherently inflammatory or anti-inflammatory? (HUM1P.275)
Authors: CHILDStudyTeam . , Graham Caroline , Chooniedass Rishma , Becker Allan , Study Team CHILD
Published: January 2023

Although much is known of immunity at the maternal-fetal interface, the innate immune status of women during healthy pregnancy remains unclear. Whether it exhibits a bias towards inflammatory phenotypes (transiently enhancing host defense) or anti-inflammatory phenotypes (reducing potential responses to the fetus) is controversial. Here, in a longitudinal study of >100 healthy women giving birth to full term healthy infants, systemic innate immune status was examined during the 2nd/3rd trimester and again, one or three years postpartum. Following REB approval and informed consent, pro and anti-inflammatory plasma biomarkers were quantified. Constitutive CCL2, CXCL10 and TNFa levels were sharply reduced (p<0.003 to 0.0001) in pregnancy. Several anti-inflammatory biomarkers were elevated (sTNFRI, sTNFRII, IL-1Ra p<0.0001). Plasma IL-10, expressed in >85% of this population, was not altered during/post pregnancy. Kinetic studies revealed that pro-inflammatory biomarker expression (CXCL10, CCL2, CXCL8, IL-18, TNFa) was independent of gestational age. Conversely, the intensity of anti-inflammatory cytokine antagonist production increased with gestational age (Spearman p<0.0003). In summary, among women experiencing healthy full term pregnancies, basal systemic immunity is characterized by an increasingly intense bias towards an anti-inflammatory innate immune phenotype that is resolved by one year postpartum.
Assessment of the Fc receptor CD16A of NK cells of CFS patients for potential to mediate ADCC activity (HUM1P.303)
Authors: Silvestre Isabel , Peterson Daniel , Redelman Douglas , Anderson Stephen , Hudig Dorothy
Published: January 2023

Natural killer (NK) cytotoxicity of chronic fatigue syndrome (CFS) patients is decreased, as reported by several laboratories. This observation led us to query whether the NK cells also lack antibody-dependent cell-mediated cytotoxicity (ADCC). NK cells have the IgG Fc-receptor CD16A that mediates ADCC. To assess potential ADCC effectors, we examined the peripheral blood of 11 CFS patients from a Lake Tahoe cohort that met the Fukuda standards for CFS and had low SF36 scores. Healthy controls were age- and gender-matched. We stained peripheral blood cells for CD16A with mAb clone 3G8 and for CD3, CD56, and perforin, then assessed the cells by flow cytometry. We found that the percentages of NK cells expressing CD16A were slightly lower for CFS patients 86.0+/-11.5% vs. controls 93.0+/-6.6% (P=0.08). The median fluorescent indices (MFIs) of CD16A were lower,72% of the values for the CFS patients, 9342+/-3233 vs. control 12929+/-4425, though not statistically significant (P<0.17). Intracellular staining for perforin in the CD16Apos cells was similar for patients and controls. Our data indicate that a larger sampling of CFS patients vs. controls is required to determine if the fractions of CD16Apos NK cells and the levels of CD16A on these cells differs. These two observations are both in the direction of compromising ADCC activity of patients. In addition, a bias in F over V allelic variants of CD16A might add to impairment, since the F allele confers reduced ADCC.
Influence of human TLR and NLR genetic variants on colorectal cancer - involvement of commensal bacterial detection, gut immunity or microbiome diversity? (HUM1P.311)
Authors: Weber Alexander , Huhn Stefanie , Klimosch Sascha , da Silva Filho Miguel Inacio , Sanmuganantham Tharmila , Pichulik Tica , Padini Barbara , Naccarati Alessio , Schafmayer Clemens , Vodickova Ludmilla , Vodicka Pavel , Hemminki Kari , Försti Asta
Published: January 2023

Studies on TLR and NLR knock out mice have suggested these pattern recognition receptors (PRRs) drastically affect gut mucosal immunity and composition of the gut microbiome, which may give rise to diseases such as colorectal cancer. We have identified several coding SNPs in TLR5 and different NLR (including the so far enigmatic group of development-associated NLRs) to be associated with different parameters of colorectal cancer, and for TLR5 have observed a functional effect in human blood immune cells. With a view to further elucidating the underlying mechanisms of these SNPs in colorectal cancer, we have begun to investigate the role of these PRR variants on gut immune activation levels and microbiome diversity in humans. Our hypothesis is that functional TLR and NLR variants are associated with different immune potential in the gut, shaping or being shaped by the host microbiome. We have built-up, genotyped and started to assay a cohort of 125 healthy volunteers and established stool assays for sIgA and cytokine levels by ELISA, cellular contents by flow cytometry analysis and PRR stimulating potential. Additionally, a functional characterization of coding NLR SNPs in cellular model systems is being carried out. Our studies dissect the functional impact and relationship of host genetic factors and the microbiome in humans, which may reveal novel targets in the diagnosis or treatment of colorectal cancer or other gut-related diseases.
Clinically relevant positive controls validate the use of serial FACS analysis, and video endoscopy in two murine adoptive transfer models of colitis (HUM1P.315)
Authors: Beal Dominic , Graham Sean , Lyng Gregory , Sonis Stephen
Published: January 2023

The adoptive transfer of naïve TH cells into immunodeficient mice is one of the best characterized immunological models of chronic colitis (CC). Traditional disease severity endpoints of weight loss and histopathology are often misleading since colitis may occur in the absence of weight loss, mice may not engraft, and weight loss and colitis may respond independently to established or experimental treatment. To overcome these limitations, we analyzed peripheral blood to track engraftment, and performed serial endoscopies to assess onset and severity of CC in two murine models of adoptive transfer-induced colitis: naive T cells from C57Bl/6J males sorted as either CD4+, CD45RBHIGH (FACS), or CD4+, CD44-, CD62L+ (negative magnetic separation) were injected IP into RAG2-/- males. Onset of colitis corresponded with the onset of weight loss, and day 14 peripheral blood CD4+ counts correlated well with both final weight loss (R2=0.74) and final endoscopy score (R2=0.79), indicating that day 14 CD4+ count was a reliable predictor of disease severity. Treatment with anti-TNFα had little effect on terminal weight loss, but significantly affected endoscopy score (2.0±0.15 vs. 1.1±0.14 at day 49 on a scale of 0-3). Together these observations suggest that endoscopy is a useful addition to weight change as a complete readout for assessing in-life disease severity in these models.
C/EBP homologous protein expression in alveolar macrophages promotes airway inflammation in an ovalbumin-based mouse model of asthma (HYP2P.329)
Authors: Thevenot Paul , Ghonim Mohamed , Boulares Hamid , Rodriguez Paulo
Published: January 2023

Lungs from asthmatic subjects are in a state of chronic Th2-driven inflammation with substantial shifts in myeloid cell function. We have recently shown that C/EBP homologous protein (Chop), a transcription cofactor linked with Integrated Stress Responses, is highly expressed in myeloid cells under chronic inflammatory processes. Therefore, we hypothesized that Chop plays a central role in the polarization of Th2 inflammation in a murine aerosol sensitization and challenge model. Increased expression of Chop was observed in alveolar macrophages (AMΦ) from ovalbumin (OVA)-challenged mice, compared to naïve controls. Chop expression appears to be mediated by Th2 cytokines as treatment of naïve AMΦ by a combination of IL-4 and IL-13 promoted a substantial increase in Chop expression. Interestingly, Chop deletion impaired AMΦ M2 polarization upon exposure to IL-4 and IL-13, suggesting an upstream role for Chop in promoting M2 responses. Using an OVA-based mouse model of asthma, we show that systemic Chop deletion blocked airway hyperresponsiveness, eosinophilia, and ovalbumin-specific IgE levels in mice following sensitization and challenge. Moreover, OVA-challenged Chop deficient mice exhibited decreased influx of F4/80+ AMΦ into the lungs accompanied by reduced accumulation of CD4+ T cells and increased numbers of CD8+ T cells. These results indicate a potential role for Chop in AMΦ in promoting Th2 inflammation in asthma.
Vasculotide can attenuate the immune inflammatory response and airway remodeling in chronic experimentally-induced airway inflammation in mice. (HYP2P.343)
Authors: Bourdeau Annie , Van Slyke Paul , Trop Sebastien , Dumont Daniel
Published: January 2023

Inflammation and airway remodeling are features associated with chronic asthmatic lungs. Previous reports have shown the benefits of the angiopoietin-1/Tie2 signaling axis on acute experimentally-induced asthma in mice. However, the more chronic effects as well as the interaction between the inflammatory and vascular components is poorly. Thus, we evaluated the impact of vasculotide, a synthetic Tie2 agonist in a chronic model of asthma. During the chronic allergic response in mice, we show that in addition to endothelial Tie2 receptor expression, there is an increased proportion of dendritic cells and monocytes expressing Tie2. Treatment of experimentally-induced chronic airway inflammation in mice with vasculotide correlated with a skewed CD4+ TH subsets and cytokine profile in the bronchoalveolar lavage of animals. This modified cytokine milieu promoted an altered endothelial cell response characterized by reduced expression of CD62E, CD54 and CD106 on endothelial cells, decreased cellular migration of inflammatory cells to the lung as well as reduced mucus accumulation. Analysis of lung tissues of animals treated with vasculotide revealed a decrease in collagen deposition and thinning of the smooth muscle cell layer in the airways of mice with chronic allergic airway inflammation. Our results demonstrate that vasculotide can impact the inflammatory and remodeling components in chronic asthma through its action on both cells of the immune response and on endothelial cells.