bluuebunny/crossref_metadata_2025 huggingface dataset:
Generation of knock-in mouse model expressing functional CD8ab without an active CD8aa (IRC5P.634)
Authors: Kang Yoon Joong
Published: January 2023

To understand functional significance of Recognition of CD8aa complexes in vivo, we generated transgenic mouse model and knock-in mouse model carrying a variant CD8ab (CD8am3b) capable of forming inactive CD8aa/pMHC1 complexes. These mice show a sub-optimal thymic differentiation with reduced populations of CD8 single-positive thymocytes. Transgenic CD8 T cells exhibit a compromised developmental capacity when competing with CD8 T cells exhibit normal effector function against viral infection. In addition, TCRab+ T cell populations expressing CD8ab+ or CD8aa reduced to about 50% in our TG mice compared to normal B6 mice, and none of these CD8aa+TCRab+ T cells were attained by TL tetramer. Furthermore, this mice show reduced inflammatory responses in DSS-induced colitis model. These findings obtained from our transgenic and knock-in animal models are going to be important to dissect the distinct function of CD8aa molecule.
Searching for natural Treg peptide-MHC ligands by engineering increased affinity/avidity Treg TCRs. (IRC5P.621)
Authors: Phillips Matt
Published: January 2023

Natural Tregs (nTregs) are produced in the thymus by selection to self peptide-MHC (spMHC) complexes. There is abundant data in the literature that suggests nTreg TCRs are higher affinity for self than conventional T cells and respond to spMHC in the periphery. To date, no nTreg ligands have been found, in part, possibly because nTregs may have TCR affinities that fall somewhere between the affinties required for positive and negative selection in the thymus for conventional T cells. This implies that although nTreg TCRs may be higher affinity for self, this affinity may still be lower than a conventional T cell TCR has for it's immunological antigen. To overcome this, we have developed a way to increase the affinity for some nTreg TCRs for pMHC while still mainting peptide specificity. Using these higher affinity nTreg TCRs, we can scan peptide-MHC libraries in order to find peptide mimitopes which may lead us to the actual self-peptides which are able stimulate specific nTregs.
New roles for IL-12: characterizing how IL-12 pretreatment alters human CD4 T cell responses (IRM5P.636)
Authors: Vacaflores Aldo , Chapman Nicole , Houtman Jon
Published: January 2023

Antigen experienced CD4 T cells are constantly exposed to IL-12 during infections and autoimmune disorders. When given concurrently with TCR induction, IL-12 enhances IFN-γ production. However, alterations of TCR-mediated antigen experienced CD4 T cell functions by prior IL-12 exposure remain unexplored. To address this knowledge gap, human antigen experienced CD4 T cells were exposed to IL-12, IL-12 was then removed, and the cells were stimulated with anti-TCR antibodies. We found that IL-12 treatment increased TCR-induced IFN-γ, TNF-α, IL-13, IL-4 and IL-10 production in comparison to untreated cells. This suggests that prior exposure to IL-12 primes the TCR-induced release of a range of cytokines. The potentiation of TCR-mediated cytokine release required at least 90 minutes of IL-12 treatment and only lasted for 6-12 hours after IL-12 removal. Interestingly, we found that IL-12 exposure selectively increased the transcription of IFN-γ and IL-10 without altering the transcript levels of the other cytokines. Furthermore, IL-12 treatment increased the phosphorylation of AKT, p38 and LCK following TCR stimulation without altering other TCR signaling molecules. Our data uncover a new function of IL-12; IL-12 primes antigen experienced CD4 T cells to enhance cytokine release by “rewiring” TCR signaling pathways. This novel function of IL-12 provides insight into potential methods to enhance or suppress CD4 T cell activity that could be harnessed as therapeutics.
Role of hydrogen sulfide in myocardial infarction via immue response of macrophages (IRM5P.645)
Authors: Zhu Yi Zhun
Published: January 2023

Macrophages are critical players in the inflammation, and are of central importance for healing after Myocardial infarction (MI). Hydrogen sulfide (H2S) has been demonstrated to possess cardioprotection in MI. However, it is unclear whether and how macrophage modulation by H2S can affect infarct repair. Thus, the present study was to evaluate the roles and mechanisms of H2S on macrophage post-MI. The cardiac function were improved in both WT mice and CSE KO mice, pretreated with different concentrations of NaHS (Exogenous sources of H2S). Meanwhile, the cardiac tissues that treated with NaHS exerted an increased immunohistochemical staining for macrophage marker (Galectin-3). Enhanced M2 macrophage polarization was observed in the infarcted heart treatment with NaHS following MI, along with increased production of M2 signature cytokines, such as interleukin-10 and CD163. In vitro, NaHS accelerated the migration of macrophage cell line RAW264.7. While, the inhibitors (refer to Pyk2, FAK, Src, and Rac1), not only significantly restored the migratory ability in response to the NaHS, but also blocked the activation of phospho-Src, -Pyk2, -FAK397, and -FAK925. Furthermore, the internalization of integrin β1 on the macrophage surface was induced by NaHS. Whereas, integrin β1 silencing inhibited the macrophage migration and the Src signaling activation. The results suggest that H2S may have the potential as an anti-infarct of MI by immune response of macrophages.
SOCS1 regulates IL-4-activated IRS-2 tyrosine phosphorylation in monocytes and macrophages via the proteasome (IRM5P.639)
Authors: McCormick Sarah , Gowda Nagaraj , Carrera Maria , Fang Jessie , Heller Nicola
Published: January 2023

Allergic asthma is a chronic lung disease driven by Th2 cytokines IL-4/-13. In macrophages, IL-4/-13 bind IL-4 receptors, which differentially signal through Insulin Receptor Substrate (IRS)-2, inducing M2 macrophage differentiation. M2 macrophages correlate with disease severity and poor lung function. The mechanisms that regulate M2 polarization are poorly understood. Following IL-4 exposure, Suppressor Of Cytokine Signaling (SOCS)1 is induced in human monocytes. We found that siRNA knockdown of SOCS1 prolonged IRS-2 tyrosine phosphorylation and enhanced M2 differentiation, while knockdown of SOCS3 had no impact. We found human SOCS1 co-immunoprecipitated with IRS-2. Since SOCS1 is an E3 ubiquitin ligase, we examined IL-4-induced ubiquitination of IRS-2. siRNA knockdown of SOCS1 inhibited ubiquitination of IRS-2 while knockdown of SOCS3 did not change IRS-2 ubiquitination. IL-4 stimulation of monocytes from healthy and allergic individuals revealed that SOCS1 is induced in healthy monocytes but not allergic, whereas SOCS3 is induced in allergic monocytes. Healthy monocytes show greater ubiquitination of IRS-2 than allergic monocytes after IL-4 stimulation. Here we identify SOCS1 as critical negative regulator of IRS-2 phosphorylation, limiting IL-4 signaling and M2 differentiation. These findings provide novel insight into the immune bias in allergic monocytes that predispose them to M2 differentiation and present a novel therapeutic target for managing allergic disease.
Long-range chromatin interactions control trophoblast-restricted HLA-G expression during pregnancy (IRM6P.657)
Authors: Ferreira Leonardo , Meissner Torsten , Mikkelsen Tarjei , O'Donnell Charles , Sherwood Richard , Mallard William , Rinn John , Cowan Chad , Strominger Jack
Published: January 2023

Human pregnancy poses an immunological paradox: at the fetal-maternal interface, semi-allogeneic fetal extravillous trophoblasts (EVT) invade the uterine mucosa without being rejected by the maternal immune system. HLA-G is a nonclassical MHC class I molecule specifically expressed on the surface of EVT and is believed to be a key to fetus-induced immune tolerance. However, the EVT-specific expression of HLA-G is still poorly understood. A novel enhancer more than 10 kb upstream of HLA-G, Enhancer L, was discovered via dissection of the HLA-G locus using a Massively Parallel Reporter Assay (MPRA). DNase I Hypersensitive Site (DHS) mapping, Chromosome Conformation Capture (3C) and reporter gene assays established Enhancer L as a cell type-specific enhancer that loops into the HLA-G classical promoter. Strikingly, deletion of Enhancer L using a CRISPR/Cas9 dual guide approach results in complete ablation of HLA-G expression. Saturation mutagenesis of Enhancer L revealed the existence of motifs for transcription factors paramount in trophoblast development. Two of them, GATA2 and CEBPB, associate with Enhancer L and the HLA-G classical promoter, and are absolutely required for HLA-G expression. Interestingly, progesterone upregulates HLA-G, as well as GATA2, via a nonclassical membrane progesterone receptor complex in trophoblasts. These findings shed light on the mechanism by which HLA-G is specifically expressed in invasive trophoblasts during pregnancy.
Reconstitution of TCR/CD3 expression in B cell lymphoma to dissect the contribution of CD3 conformational change from downstream signaling events in the process of TCR/CD3 downregulation. (IRM7P.714)
Authors: Ferrer Alejandro , Reed Brendan , Schrum Adam , Gil Pages Diana
Published: January 2023

T cell receptor (TCR) stimulation induces conformational change in CD3 (CD3Δc), followed by phosphorylation of ITAMs that leads to receptor downregulation among other T cell responses. To begin to dissect the contribution of CD3Δc from downstream signal transduction mechanisms associated with these events, we have transduced the mouse B cell lymphoma X63.Ag8.653, which lacks expression of the src-family kinase Lck required for proper ITAM phosphorylation, with the OT-I TCR chains Vα2 and Vβ5, together with all CD3 subunits and the coreceptor CD8αβ, generating the cell line X63.OT1.CD8. Stimulation of the OT-I TCR with anti-CD3 antibody or the cognate antigen SIINFEKL-H2Kb induces CD3Δc in this cell line. As expected by the absence of Lck, CD3 ITAM phosphorylation was undetectable in our cell line and no calcium mobilization was found upon antibody stimulation. Unexpectedly, stimulation with either SIINFEKL-H2Kb or anti-CD3 did induce TCR/CD3 downregulation in our cells. These data suggest CD3ε PRS exposure via CD3Δc might be sufficient to transduce some signals that contribute to TCR downregulation in the absence of CD3 ITAM phosphorylation.
A systematic investigation on the mechanism of non-catalytic tyrosine phosphorylated receptor triggering (IRM7P.718)
Authors: Denham Eleanor , Goyette Jesse , van der Merwe P.
Published: January 2023

Non-catalytic tyrosine phosphorylated receptors (NTRs) are a large class of leukocyte receptors that bind to surface-associated ligands and include both activating and inhibitory members. They contain, or associate with adaptor molecules which contain, tyrosine residues within conserved cytoplasmic motifs that are phosphorylated and dephosphorylated by extrinsic kinases and phosphatases respectively. The mechanism by which NTR-ligand engagement leads to sustained phosphorylation of receptor tyrosine-based motifs and initiation of downstream signalling (‘receptor triggering’), is as yet unknown. Our hypothesis is that the NTRs signal using the kinetic segregation (KS) model. To systematically test whether predictions of the KS model hold for all the NTRs we have developed an artificial generic ligand system in which biophysical and biochemical properties of NTR-ligand interactions can be manipulated, including: NTR-ligand dimensions, ligand densities and NTR-ligand affinities. This system uses a Strep-tag II attached to the ectodomain of NTRs. We demonstrate that representative NTRs, both activating and inhibitory, can be triggered through ligation of this tag. In agreement with a key KS model prediction we further show that only ligation by surface-associated ligands leads to triggering, whilst ligation by soluble ligands does not. The flexibility of this system will allow us to further test other predictions of the model, in addition to other parameters of NTR signalling.
Identifying the precursor of natural IgM-producing plasma cells (LYM2P.736)
Authors: Reynolds Alexander , Kuraoka Masayuki , Kelsoe Garnett
Published: January 2023

Natural antibody is constitutively present in the serum, and plays an important role in the clearance of cellular debris, the early control of viral and bacterial expansions, and the initiation of T-dependent humoral responses. However, despite widespread recognition of the critical role of natural IgM in the prevention of both infectious and autoimmune diseases, the source of natural antibody long remained obscure. We have found that most serum IgM in resting mice is produced by a discrete population of IgM plasma cells in the bone marrow, a substantial fraction of which are long-lived cells that occupy a survival niche distinct from that required by IgG plasma cells. Natural IgM plasma cells originate from precursors in the peritoneal cavity, as transfer of unfractionated peritoneal washout cells completely restores serum IgM and the specific compartment of bone marrow plasma cells in Rag1-deficient mice. Surprisingly, however, transfer of purified peritoneal B1a, B1b, or B2 cells did not efficiently reconstitute serum IgM, while a distinct peritoneal B-cell population reconstituted both natural IgM and peritoneal B1a cells in Rag1-deficient mice. We propose that natural IgM plasma cells represent an “innate B-cell” population descended from yolk sac progenitors, and that natural IgM plasma cells are not the progeny of B1a cells per se but instead share an earlier common ancestor also present in the peritoneal cavity.
NKAP is required for invariant natural killer T cell development and generation of ROR-γt expressing iNKT17 (LYM2P.738)
Authors: Thapa Puspa , Chen Meibo , McWilliams Doug , Belmonte Paul , Constans Megan , Hiebert Scott , Sant'Angelo Derek , Shapiro Virginia
Published: January 2023

iNKT cells differentiate into functional subsets defined as NKT1, NKT2 and NKT17, characterized by their expression of transcription factors, T-bet, GATA3 and ROR-γt and production of IFN-γ, IL-4 and IL-17, respectively. In our earlier studies, we showed that the transcriptional repressor NKAP is required for positive selection of DP thymocytes into the iNKT lineage, using CD4-cre NKAP conditional knockout (cKO) mice. To study the role of NKAP in subsequent stages of iNKT cell development and differentiation, we generated PLZF-cre NKAP cKO mice. In these mice, NKAP deletion occurs after entry into the iNKT lineage at stage 0, bypassing the requirement for NKAP in iNKT development at the DP stage. PLZF-cre NKAP cKO mice have a significant reduction in the absolute number of iNKT cells starting at Stage 1. This block in development is not due to defect in cell homeostasis or increased cell death, but is due to decreased rate of proliferation. PLZF-cre NKAP cKO mice have very few T-bet expressing NKT1 cells with similar amount of IFN-γ production to WT, whereas there was almost no ROR-γt expressing NKT17 cells with no IL-17 production. There is a decrease in the absolute number of NKT2 cells; however, IL-4 production by NKAP-deficient NKT2 cells is similar to WT. Thus, NKAP is required for proliferation of iNKT cells early in development, and is required for the differentiation of iNKT cells into the NKT17 lineage.
Reduced T follicular helper function in aged mice is correlated with increased numbers of T regulatory cells (LYM2P.725)
Authors: Masters April , Lefebvre Julie , Qiu Zhijuan , Hopkins Jake , Haynes Laura
Published: January 2023

T follicular helper (TFH) cells are critical for a germinal center response and a robust humoral response. Aging has a profound impact on the formation of a strong humoral/germinal center response, yet the impact of aging on TFH remains unclear. This study explores the effects of aging on TFH transcription factors, which could explain some of the age related germinal center defects. During influenza infection, young mice exhibit a very robust IgG response, which is significantly reduced in aged mice. Initial studies showed that there are no differences in the number of flu-specific TFH (CXCR5hi PD-1hi) during the course of the infection, as determined using MHC class II NP tetramer. To determine transcription factor expression and localization of TFH, we performed multicolor flow cytometry and confocal microscopy on spleens from these mice. There was no difference in BCL6 expression in young and aged flu-specific TFH. But, strikingly, flu-specific BCL6hi TFH from aged mice had increased co-expression of Foxp3, characteristic of T follicular regulatory cells, a critical controller of germinal center responses. We are currently exploring the contribution of whether the differences in TFH transcription factor expression are intrinsic to aged CD4+ T cells or are a result of the aged microenvironment. These results strongly suggest that aging has a dramatic impact on transcription factor expression in CD4 T cells, which can influence the establishment of a robust immune response.
Reduction of regulatory T cells during acute Toxoplasma gondii infection is caused by IFN-γ (MPF2P.759)
Authors: Olguin Jonadab , Fernández Jacquelina , Salinas Nohemí , Terrazas Luis , Saavedra Rafael
Published: January 2023

Toxoplasmosis is an infection disease caused by the protozoan parasite Toxoplasma gondii. A TH1 immune response is essential for protection against the parasite, IFN-γ being the major mediator of resistance. CD4+Foxp3+ Regulatory T cells (Treg) maintain immune homeostasis by actively suppressing pathological and physiological immune responses. Intraperitoneal infection of BALB/c mice with T. gondii cysts leads to high mortality rate and exacerbated IFN-γ levels in sera both of which parallel a decreased Treg number in blood and spleen. Since we observed an inverse correlation between high IFN-γ levels and low Treg cell number in infected mice, we analyzed if this cytokine was involved in Treg reduction. Since STAT-1 is involved during signaling by IFN-γ, we infected STAT-1-/- KO mice with T. gondii and found that the Treg cell number was unchanged in blood and increased in spleen, although all mice died; IFN-γ levels in sera in these mice were lower than those observed in infected WT mice, and CD4+ - and CD8+-producing IFN-γ cells were also reduced. These results strongly suggested that IFN-γ has a major role in the reduction of Treg cells. Thus we injected a neutralizing mAb against IFN-γ to infected Foxp3EGFP transgenic mice and unchanged Treg cell number was detected in blood. Our results thus demonstrate that exacerbated IFN-γ levels during acute toxoplasmosis are responsible of collapse of Treg cell number in this model.
Tumour necrosis factor-related apoptosis inducing ligand promotes the development of experimental chronic obstructive pulmonary disease (MUC1P.905)
Authors: Hansbro Philip , Haw Tatt , Nair Prema , Hanish Irwan , Nguyen Duc , Liu Gang , Inman Mark , Kim Richard , Collison Adam , Knight Darryl , Yagita Hideo , Mattes Joerg , Horvat Jay , Starkey Malcolm
Published: January 2023

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide and causes significant healthcare and economic burden. Cigarette smoking is a major risk factor. There is a lack of effective treatments for COPD due to the poor understanding of the underlining mechanisms. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is implicated in respiratory diseases such as asthma and pulmonary fibrosis. However, the role of TRAIL in the pathogenesis of COPD is unknown. In this study, TRAIL mRNA expression and/or protein levels in the lung (airway and parenchyma) and serum were increased in a mouse model of cigarette smoke-induced experimental COPD. Genetic deletion of TRAIL significantly reduced cigarette smoke-induced pulmonary inflammation, expression of key pro-inflammatory mediators, emphysema-like alveolar enlargement and improved lung function in experimental COPD. Interestingly, genetic deletion of TRAIL led to spontaneous small airway remodelling characterized by increased airway epithelial cell thickness and collagen deposition. Importantly, antibody-mediated neutralization of TRAIL reduced cigarette smoke-induced pulmonary inflammation, emphysema-like alveolar enlargement and small airway remodelling. Our study is the first to show that TRAIL plays an important role in the pathogenesis of COPD and provides further evidence for TRAIL being a pivotal inflammatory cytokine in respiratory diseases.
Effect of anti-high mobility group box 1 on innate lymphoid cells of dry eyes in mouse model of Sjögren syndrome (MUC2P.927)
Authors: Kim Mee , Jeong HyunJeong , Jin Suk Ryu , Kim Dong Hyun , Wee Won Ryang
Published: January 2023

How innate lymphoid cells (ILCs) secrete cytokines is not well understood. High mobility group box (HMGB) may be involved in secreting IL17 or IL22 in ILCs. Extracellular HMGB1 may be related with the pathogenesis of Sjögren syndrome. We here investigated that administration of anti-HMGB1 might attenuate dry eyes by modulating ILCs in mouse model of Sjögren syndrome. This study was conducted in conformance with the FASEB Statement of Principles for the use of animals in research and education. 10 weeks-old NOD. B10 or B6 mice had been subconjunctivally injected by 0.02 to 2 ug of anti-HMGB1 antibodies or PBS for 2 weeks. Injection of anti-HMGB1 attenuated corneal epithelial erosions significantly by fluorescein staining and increased tear secretion transiently by phenol-red thread test. After stimulation of the cells from Peyer’s patches or cervical drainage lymph nodes by IL1b and IL23, cells were negatively gated using anti-mouse CD3-PerCP-Cy5.5 and B220-FITC, and were positively gated using anti-mouse CD45-APC-Cy7, NKp46-APC and IL22-PE. The percentages and the numbers of NKp46+IL22 or NKp46-IL22 secreting cells were increased in cervical lymph nodes of anti-HMGB1 injected mice compared with those in PBS injected mice. While, those were not significantly changed in Peyer’s patches of anti-HMGB1 or PBS injected mice. Together, data demonstrate that administration of anti-HMGB1 attenuates dry eyes by increasing IL-22-secreting ILCs in mouse model of Sjögren syndrome.
Modeling and implications of antibody transcytosis by human genital epithelial cells during Chlamydia trachomatis infection and challenge (MUC2P.930)
Authors: Albritton Hannah , Buckner Lyndsey , Taylor Stephanie , Lillis Rebecca , Martin David , Kozlowski Pamela , Quayle Alison
Published: January 2023

Chlamydia trachomatis (CT) is a common sexually transmitted pathogen; there is no vaccine and infection can lead to infertility and increased susceptibility to HIV acquisition. An obligate intracellular bacterium, CT is tropic for genital columnar epithelial cells, and the endocervix is the primary site of infection in women. CT infections are usually asymptomatic, endure for months, and immunity is short-lived. However, recent epidemiology studies have identified a subgroup of women who spontaneously clear infection without treatment and are protected from re-infection. We hypothesize that genital secretions from these women will aid in the identification of site-specific immune correlates and inform vaccine design. Since animal and clinical studies suggest antibodies may play a role in protection and clearance of CT, we developed novel clinical sampling techniques and an ELISA that allows normalization and quantification of local CT-specific antibody isotypes from CT-infected women, and report significantly higher levels of CT-specific IgG/A in genital secretions compared to serum. Development of a novel in vitro endocervical epithelial model that can be infected with CT now allows us to (i) determine the protective efficacy of defined (clearer v non-clearer) patient samples against CT-challenge and (ii) explore recent observations that inflammation modulates endocervical immunoglobulin receptor expression, likely influencing antibody transport in the CT-infected cervix.
Intestinal epithelial cell mediated miscommunication in type 1 diabetes: a tipping point for disease? (MUC2P.933)
Authors: Graves Christina , Lapato Melissa , Wallet Shannon
Published: January 2023

The host-environment dialogue at the intestinal interface has been strongly implicated in the development of inflammatory diseases. We are interested in understanding the role of this dialogue in autoinflammatory and autoimmune diseases such as type 1 diabetes (T1D). Although the gastrointestinal tract, a dynamic and highly regulated immunologic organ, has been demonstrated to be impaired in T1D, the mechanisms of any (mis)communications taking place remain unknown. Here we describe the global intestinal inflammatory environment and the phenotype of intestinal lymphocytes in human T1D. In addition, we evaluated the intestinal epithelial cell (IEC) innate immune responses using primary cell culture. Our data indicate a whole organ reduction in tolerogenic mediators concomitant with elevated IEC-induced pro-inflammatory mediators. Significant alterations in intestinal leukocyte populations were observed, whereby increased frequencies of pathogenic T cells and alterations in tolerogenic DCs were noted. Finally, T1D-derived primary IEC cultures demonstrated a lack of tolerogenic responses concomitant with induction of inflammatory response to microbial ligand stimulation as compared to non-T1D derived cultures. Together these data suggest a loss of gastrointestinal homeostasis in T1D potentially as a result of a dysfunctional IEC-mediated host-environment dialogue, though whether this is a cause of consequence of disease remains to be defined.
Effect of 17-allylaminogeldanamycin on antimicrobial peptide and cytokine expression in HaCaT cells after IL-17 and heat stimuli (MUC2P.941)
Authors: Kim Bo-Kyung , Park Minhwa , Kim Ji-Yon , Lee Kyung Ho , WOO SOYOUN
Published: January 2023

The pathogenesis of inflammatory skin disease involves interactions between Th17-mediated immune response and keratinocytes. Immersion in pools of thermal mineral water is often considered to have chemical, thermal, mechanical, and immune modulatory benefits to inflammatory skin disease. Therefore, we questioned the effect of thermal treatment on IL-17-mediated immune response in keratinocytes. We evaluated the effect of HSP90 inhibitor on IL-17-mediated cytokine and antimicrobial peptide expression in keratinocytes after heat treatment including IL-1β. To examine the effect of IL-17 receptor-mediated expression of antimicrobial peptides or inflammatory chemokines in HaCaT cells, Act1 siRNA-transfected HaCaT cells were heat stimulated at 42oC and 1 uM of 17-allylaminogeldanamycin (17-AAG) was added. At 37oC, Act1 knockdown decreased BD-1, LL-37, S100A8, S100A9, CXCL1, CXCL5, and CXCL8 expression; however, expression of these antimicrobial peptides and chemokines increased after incubation at 42oC. This thermal-mediated effect was suppressed upon treatment with 17-AAG. We also found that IL-1β expression decreased by 17-AAG after heat stimulation. Although HSP90 enhanced the expression of antimicrobial peptides or chemokines in keratinocytes after heat treatment, HSP90 inhibitors reversed these effects. Therefore, HSP90 inhibitors after heat stimulation (e.g., thermal baths) may be a beneficial therapeutic approach for IL-17-mediated inflammatory skin disease.
Candida albicans morphology and DC subsets determines T helper differentiation (MUC2P.920)
Authors: Igyarto Botond , Kashem Sakeen , Gerami-Nejad Maryam , Kumamoto Yosuke , Mohammed Javed , Jarrett Elizabeth , Drummond Rebecca , Zurawski Sandra , Zurawski Gerard , Berman Judith , Iwasaki Akiko , Brown Gordon , Kaplan Daniel
Published: January 2023

Candida albicans is a dimorphic fungus responsible for chronic mucocutaneous and often fatal systemic infections. Mucocutaneous immunity to C. albicans requires Th17 differentiation that is thought to depend on recognition of filamentous C. albicans while systemic resistance is considered T cell independent. Using a murine skin infection model, we compared in vivo T helper responses to yeast and filamentous C. albicans. Unexpectedly, we found that only yeast induced Th17 responses through a mechanism that required Dectin-1 mediated expression of IL-6 by Langerhans cells. Filamentous forms induced Th1 without Th17 due to the absence of Dectin-1 ligation. Notably, Th17 responses provided selective protection against secondary cutaneous infection while Th1 provided protection against systemic infection. Thus, C. albicans morphology drives distinct Th responses that provide tissue specific protection. These findings provide novel insight into compartmentalization of Th responses, C. albicans pathogenesis and have critical implications for vaccine strategies.
Trogocytosis and endocytosis of HLA-G from extravillous trophoblasts by decidual NK cells and its functional consequence (MUC2P.922)
Authors: Tilburgs Tamara , Evans James , Crespo Angela , Strominger Jack
Published: January 2023

The interaction of non-cytotoxic decidual NK cells (dNK) and extravillous trophoblasts (EVT) at the fetal-maternal interface has been studied. Confocal microscopy revealed that many dNK can interact with a single large EVT, leading to the formation of synapses. Filamentous projections from EVT enriched in HLA-G were shown to contact dNK and may represent the initial stage of synapse formation. A small fraction of dNK as isolated (~3%) contain surface HLA-G. However, HLA-G negative dNK had internalized HLA-G. Activation of dNK by culture in IL-15 resulted in disappearance of the internalized HLA-G in parallel with restoration of cytotoxicity. Surface HLA-G could be reacquired by incubation with EVT. However, under the conditions of the current experiments, re-acquisition of surface HLA-G by IL-15 activated dNK did not suppress their cytotoxicity. These data indicate a cycle in which HLA-G is acquired from EVT, endocytosed, degraded and, finally, reacquired from EVT. The HLA-G cycle is related to the loss and reacquisition of dNK cytotoxicity. The fetal-maternal interface offers an opportunity to study how evolution has met the challenge of generating a localized and specific tolerized immune compartment that does not reject an allogeneic fetus while maintaining the ability to respond to infections at this site.
The effect of sympathetic innervation on murine corneal epithelial wound healing (MUC3P.944)
Authors: Li Zhijie , Xue Yunxia , Smith Wayne
Published: January 2023

This study investigated the influence of murine corneal sympathetic nerves on epithelial wound healing. The wound model involved mechanical removal of a 2mm diameter area of the central corneal epithelial layer. We evaluated re-epithelialization, leukocyte extravasation, and platelet accumulation in the limbus and cornea at different time intervals after injury. The potential influence of sympathetic nerves in healing and inflammation was assessed by chemical sympathectomy, as well as topically applied adrenergic agonists and antagonists. Sympathectomy and the β2 receptor antagonist, timolol, each significantly accelerated the corneal epithelial healing process, including re-epithelialization and epithelial cell division. The agonist isoproterenol significantly inhibited re-epithelialization and epithelial cell division. Neutrophil migration into the wounded cornea was significantly inhibited by either sympathectomy or topical timolol, while isoproterenol restored neutrophil migration in the sympathectomized corneas. Platelet accumulation was inconsistently influenced by sympathectomy and timolol, but markedly inhibited by isoproterenol. These results are consistent with the interpretation that corneal sympathetic nerves can modulate corneal epithelial wound closure and platelet accumulation, and augment neutrophil influx.
Development of anti-CXCL13 monoclonal antibody for the treatment of autoimmune and immuno-oncological disorders (THER2P.950)
Authors: Klimatcheva Ekaterina , Mallow Crystal , Reilly Christine , Torno Sebold , Bussler Holm , Howell Alan , Scrivens Maria , Wang Wei , Balch Leslie , Murante Frank , Foster Cathrine , Kirk Renee , Giralico Susan , Leonard John , Paris Mark , Fisher Terrence , Evans Elizabeth , Smith Ernest , Zauderer Maurice
Published: January 2023

The chemokine CXCL13 is expressed in secondary lymphoid organs by follicular dendritic cells and macrophages and is also expressed by TH17 cells. It is the only known ligand for the CXCR5 receptor expressed on mature B cells, multiple subsets of T cells, macrophages and endothelial cells. Aberrant expression of CXCL13 within ectopic germinal centers has been linked to the development of autoimmune and cancer-promoting inflammatory disorders. Over-expression of CXCR5 receptor in certain cancers has been reported to induce CXCL13-dependent cell proliferation and metastasis. We developed a human IgG1 monoclonal antibody that specifically binds to human, rodent and primate CXCL13 and is capable of neutralizing CXCL13 function of these various species in in vitro functional assays. For preclinical in vivo studies we have engineered a chimeric antibody that contains the same human heavy and light chain variable genes as the human antibody along with mouse constant regions. This antibody has demonstrated efficacy in murine models of autoimmunity (Experimental Autoimmune Encephalomyelitis, Collagen-Induced Arthritis) and immuno-oncology (MALT lymphoma). In adoptive transfer and immunization studies, treatment with this antibody reduced B cell trafficking to the spleen and lymph nodes and interfered with the formation and expansion of germinal centers. The human antibody is currently undergoing pre-clinical development to support a future FDA Investigational New Drug application.
Nicotine exerts protective effect on ulcerative colitis via miR-124/STAT3 pathway (THER2P.962)
Authors: Liu Xia
Published: January 2023

A large body of epidemiological studies showed that smoking has a protective effect in patients with ulcerative colitis (UC). However, the underlying mechanism remains unknown. We previously reported nicotine could inhibit inflammation via miR-124-STAT3 pathway. Whether this pathway is involved in the protective effect of nicotine, accounting for the beneficial effect of smoking on UC, should be further explored. We found nicotine treatment could hamper weigh loss, improve disease activity index, decrease HE score, and increase miR-124 expression in the colon tissue of UC mice. Colon biopsies from patients with UC also showed higher miR-124 expression when compared with normal colons. Furthermore, miR-124 knockdown could markedly impair the beneficial role of nicotine in UC mice, and attenuat the inhibitory role of nicotine in the expression of STAT3 /p-STAT3 in colon tissues. In vitro, nicotine elevated miR-124 expression in colon epithelial cell HT-29, and miR-124 agomir or antagomir could decrease or increase STAT3/p-STAT3 expression and TNF-a production in these cells. Our data provide the first evidence that miR-124-STAT3 pathway is essential for the nicotine therapeutic effect on UC, and miR-124 is a potential therapeutic target for the treatment of UC.
Immune responses to chimeric human-FIX-mouse-Fc fusion protein in a hemophilia B mouse model (THER2P.963)
Authors: Golding Basil , Jiang Haiyan , Sauna Zuben , Levin Ditza
Published: January 2023

The fusion of a protein with the Fc-region of an antibody is used to increase the circulating half-life of therapeutic proteins. Using a mouse hemophilia B (HB) model we compared the immune responses to infusions of recombinant human FIX (rFIX) and human FIX-mouse Fc (hFIX-mFc) fusion protein. HB mice received weekly IV injection of rFIX or hFIX-mFc for 5 weeks. Anti-FIX IgG, IgE and cytokines were measured. Splenic cells were used to determine FIX-specific T and B cell functional and memory responses. Our results showed that, following IV treatment with rFIX or hFIX-mFc, there are no statistically significant differences in the titers of anti-FIX IgG Ab and numbers of long term memory B cells. However, a significantly larger proportion of mice treated with rFIX developed elevated titers of total IgE which correlated with a Th2 bias following rFIX. In contrast, hFIX-mFc induced a Th1-like response and no detectable IgE. Our study suggests, that in mice, hFIX-mFc fusion protein promotes a Th1-like response and is less likely than rFIX to induce an IgE response, which may lead to anaphylaxis, a rare severe adverse event associated with the treatment of HB patients.
IL-10 is critical in the regulatory function of CD1dhiCD5+ B cells in Experimental Autoimmune Myasthenia Gravis (THER2P.964)
Authors: Sheng JianRong , Soliven Betty
Published: January 2023

Autoimmune Myasthenia Gravis (MG) is a T cell-dependent, B cell-mediated autoimmune disorder in which autoantibodies is produced targeting the skeletal muscle acetylcholine receptor (AChR). IL-10-competent subset within CD1dhiCD5+ B cells, also known as B10 cells, has been shown to regulate autoimmune diseases. In our previous study, adoptive transfer of CD1dhiCD5+ B cells expanded in vivo by GM-CSF prevented and suppressed Experimental Autoimmune Myasthenia Gravis (EAMG), which was associated with downregulation of mature dendritic cell marker and expansion of regulatory T cell resulting in the suppression of acetylcholine receptor (AChR)-specific T cell and B cell response. To further examine the role of IL-10 in the regulatory function of B10 cells in EAMG, we induced EAMG in both wild type and IL-10 knockout mice, and also performed the adoptive transfer IL-10 deficient CD1dhiCD5+ cells to EAMG mice. We found that IL-10 knockout mice showed more severe EAMG disease with more Th1 cytokine production and less Foxp3 expression compared with wild type mice. Adoptive transfer of CD1dhiCD5+ B cells from IL-10 knockout mice to EAMG mice could not prevent and suppress disease. Our observations further confirmed that IL-10 derived from B10 cells played a critical role in maintaining and restoring immune homeostasis in EAMG mice. Administration IL-10 competent CD1dhiCD5+ B cells may represent an effective strategy in the treatment of human myasthenia gravis.
Autophagy reduces the accumulation of misfolded HLA-B27 in a rat model of spondyloarthritis (THER2P.951)
Authors: Navid Fatemeh , Layh-Schmitt Gerlinde , Colbert Robert
Published: January 2023

The MHC class I allele HLA-B27 is strongly associated with the development of spondyloarthritis (SpA) in humans, and when expressed in rats causes a SpA-like inflammatory disease. HLA-B27 has the tendency to misfold, and when up regulated by IFNγ and TNFα can cause ER stress leading to an unfolded protein response (UPR), which promotes pro-inflammatory cytokine production and may be critical for disease development. We examined whether alteration of autophagy affects HLA-B27 misfolding and UPR activation. Bone marrow-derived macrophages were obtained from HLA-B27-transgenic (TG), HLA-B7-TG and Lewis control (WT) rats and autophagy was induced with rapamycin. Western blot (WB) and immunofluorescence analysis of LC3B-II expression revealed a normal autophagic response in HLA-B27 expressing cells. Activation of autophagy with rapamycin reduced the accumulation of misfolded HLA-B27 induced by TNFα and IFNγ by 50%, while properly folded HLA-B27 heavy chains were minimally affected. Inhibition of autophagic flux with bafilomycin resulted in an exacerbation of the UPR upon HLA-B27 upregulation as measured by XBP1 splicing and expression of UPR target genes. These results demonstrate for the first time that induction of autophagy can reduce the accumulation of misfolded HLA-B27, and blocking autophagic flux exacerbates the UPR in HLA-B27 expressing cells. Alteration of the autophagic pathway may be a promising therapeutic tool in the treatement of SpA.
Characterization of the function and co-expression patterns of human FOXP3 isoforms and their role in IPEX syndrome development (THER2P.952)
Authors: Darrasse-Jeze Guillaume
Published: January 2023

The transcription factor FOXP3 plays a critical role in the development and function of regulatory T cells (Tregs) that maintain self-tolerance. Contrary to mice, humans possess three isoforms of FOXP3 with unknown function, and the expression of these isoforms is not restricted to Tregs. We determined the levels of expression and distribution of the isoforms of FOXP3 as well as other transcription factors in normal human T-cell subsets and in T cell subsets of patients with autoimmunity at the single-cell level by multiplex RT-qPCR and flow cytometry. We observed that the expression of these isoforms is not restricted to Tregs, but can also be detected in naïve and activated/memory helper (Th) and in activated/memory CD8+ T cells. Distinct patterns of co-expression of the FOXP3 isoforms are modulated in response to inflammatory or autoimmune stimuli. Moreover, we observed that normal FOXP3 isoforms could induce bona fide regulatory function in Th cells. Our work supports the view that lack of FOXP3 expression in the effector T-cell populations in IPEX patients may contribute to the pathogenesis of the disease, and our preliminary data suggest that FOXP3 deficiency can be corrected by gene transfer of the appropriate FOXP3 isoforms into conventional T-cells, which has important implication for the treatment of IPEX patients.
The potent, selective RORγt blocker, VTP-43742, suppresses Th17 production in vivo and provides greater benefit than IL-17 blockade in the EAE model of autoimmunity (THER3P.968)
Authors: Zhao Yi , Meng Shi , Noto Paul , Bukhtiyarov Yuri , Stadanlick Jason , Zhuang Linghang , Dillard Lawrence , Claremon David , Gregg Richard , McGeehan Gerard , Lala Deepak
Published: January 2023

Antibodies targeting IL-23 or IL-17 are highly effective in the treatment of psoriasis and psoriatic arthritis, validating the RORγt /Th17 pathway in human disease. VTP-43742 is a potent, selective and orally active inhibitor of RORgt that is being pursued as a treatment of autoimmune disorders. Herein, we present data showing the effects of VTP-43742 in two separate mouse studies using the MOG35-55/CFA immunized EAE model. In a subchronic study, prophylactic dosing of VTP-43742 for 11 days gave a dose-dependent suppression of both CD4+IL-17+IFNγ- and CD4+IL-17+IFNγ+ T cells in spleen and in draining lymph nodes. Splenocytes from these mice restimulated in vitro with the MOG35-55 peptide showed specific suppression of Th17 derived cytokines. In a chronic disease study, the maximal pharmacological effect of VTP-43742 was compared to maximal IL-17 blockade by mouse monoclonal antibody MM17F3 (eBioscience) in a prophylactic setting. High dose VTP-43742 completely suppressed the EAE clinical score, significantly beyond the reduction seen with MM17F3 (p<0.001). Analysis of spinal cord RNA showed significant suppression of Th17-associated markers by VTP-43742 with no significant reduction by MM17F3. Additionally, spinal cord histology showed qualitatively greater suppression of demyelination and inflammatory cell infiltration with VTP-43742 treatment versus MM17F3, suggesting that RORγt blockade may provide treatment benefit beyond anti-IL-17 targeted strategies in autoimmunity.
Metformin ameliorates inflammatory bowel disease by suppression of STAT3 signaling pathway and regulation of the Th17/Treg balance (THER3P.1001)
Authors: Lee Seon-Young , Lee Seung Hoon , Kim Eun-Kyung , Kim Jae-Kyung , Shin Dong-Yun , Cho Mi-La
Published: January 2023

Metformin is an effective pharmacological treatment for type 2 diabetes. The current study attempts to determine whether metformin reduces inflammation regulating p-STAT3 expression and Th17 cell proliferation in an experimental inflammatory bowel disease (IBD) mice model. IBD mice were intrarectally injected with either metformin once a day for 16 days. Histological analyses of the colons were performed via immunohistochemistry. AMPK, mTOR, p-STAT3 and p-STAT5 of the spleen and lymph node tissue were detected by confocal microscopy staining. Gene expression was calculated by real-time PCR and protein levels were measured using western blot enzyme-linked immunosorbent assay (ELISA). MTT assay was used to evaluate human HT-29 cell proliferation. Metformin reduced DAI score and inhibited weight loss. Metformin also decreased the colonic histological score and inflammatory mediators with increased colon length. Expression of IL-17, p-STAT3 and p-mTOR was inhibited with metformin treatment. On the contrary, metformin treatment increased the expression of p-AMPK and Foxp3. Additionally, inflammatory cytokines expression was reduced dose dependently in inflamed human HT-29 cells cultured in various concentrations of metformin treatment. Metformin attenuates the severity of IBD and declines inflammation through the inhibition of p-STAT3 and IL-17. These results support targeting therapeutic substances for IBD and to increase the understanding of IBD.
Immune cell responses to combined treatment of murine melanoma with ALT-803 and Sunitinib in normal and chronic alcohol consuming mice (TUM2P.1032)
Authors: Little Alexander , Gaither Kari , McBride Alisha , Castillo Savanna , Brar Kiran , Platt Amity , Zhu Zhaohui , Zhang Faya , Luong Dung , Zhang Hui , Meadows Gary
Published: January 2023

The immune enhancing IL-15 super agonist ALT-803 and Sunitinib, a tyrosine kinase inhibitor that inhibits immunosuppressive myeloid derived suppressor cells (MDSC) and T regulatory (Treg) cells, were examined for their single and combined effects on immune cell phenotype in normal and chronic alcohol consuming (CAC) mice inoculated with s.c. B16BL6 melanoma. ALT-803 alone and in combination with Sunitinib initially increased CD8+CD44hi and CD8+CD44hiCD62Llo memory phenotype T cells in the spleen and PBL. IFN-γ producing CD8+CD44hi T cells increased in the spleen and the increase was more robust in CAC versus normal mice. These responses decayed during the later stages of tumor growth, and declined more rapidly in the CAC mice. Sunitinib alone did not affect CD8+ T cells or their IFN-γ production. Sunitinib alone and in combination with ALT-803 prevented the increase in CD11b+Gr-1int MDSC in the spleen associated with no treatment or ALT-803 treatment in normal mice; however, the effect was less pronounced in CAC mice where MDSC did not increase in the untreated group over time. MDSC in the PBL increased over time in untreated and ALT-803-treated normal mice. Sunitinib prevented this increase until the late stages of tumor progression. Sunitinib alone and in combination suppressed CD4+CD25+FoxP3+ Treg cells in the spleen during tumor progression.
Oral vaccine by tumor cell-derived microparticles leads to effective anti-tumor immune responses (TUM2P.1041)
Authors: Huang Bo
Published: January 2023

In response to stimuli, tumor cells release size of 100-1,000 nm vesicles, called microparticles (MP) to the extracellular space. Our previous studies have demonstrated that tumor cell-derived microparticles (T-MP) can act as cell-free tumor vaccines. T-MPs present both tumor antigens and immunostimulatory signals to dendritic cells, leading to tumor-specific T cell responses. Here, we further examine whether the T-MPs are capable of inducing anti-tumor immune responses when administrated via oral route. We find that treatment with oral B16 melanoma-derived MPs effectively inhibits tumor growth and improves survival of B16 melanoma-bearing mice, compared with tumor lysate or whole tumor cell control group. However, these MPs do not induce the anti-tumor effect in nude mice. In addition, depletion of CD4 or CD8 T cells impairs T-MP-mediated anti-tumor immunity in immune-competent mice. Importantly, mice orally provided with MPs derived from OVA-B16 cells display significantly increased frequencies of OVA-positive CD8+ T cells in the blood and spleen. Also, the levels of IFN-γ are enhanced in mice immunized with OVA-B16-MPs. Interestingly, we find that epithelial cells transport T-MPs to dendritic cells, resulting in T cell responses. We use DC/EC coculture system and demonstrate that the transepithelial transport of T-MPs is mediated by the epithelial transcytosis. Collectively, our data suggest that oral delivery T-MPs elicit an effective anti-tumor immune responses.