bluuebunny/crossref_metadata_2025 huggingface dataset:
Tyrosine phosphatase PTP1B functions to limit cytokine responses and leukocyte trafficking during allergic inflammation (P6057)
Authors: Berdnikovs Sergejs , Abdala Valencia Hiam , Cook-Mills Joan
Published: January 2023

Allergic inflammation is orchestrated by an intricate cellular communication network operated by cytokines. Members of protein tyrosine phosphatase family are potentially critical in balancing cytokine-triggered cellular activation. However, virtually nothing is known about function of specific tyrosine phosphatases in immune-mediated diseases. We interrogated role of protein tyrosine phosphatase PTP1B during allergic inflammation using ovalbumin asthma models in PTP1B KO and WT mice. Flow cytometry and intravital microscopy were used to characterize trafficking of leukocytes to the lung. In this study, we observed exaggerated allergen-driven eosinophilopoiesis and early-induced alterations in leukocyte recruitment to the lungs of KO mice. Intravital microscopy revealed profound differences in kinetics of leukocyte binding to endothelium between challenged WT (in hours) and KO mice (in minutes). In concert with stimulated eosinophilopoiesis and enhanced leukocyte trafficking, there was an increase in eotaxins and a number of Th2 and tissue-derived cytokines in KO mice compared to WT mice. Eosinophils and mast cells cultured from bone marrow of PTP1B KO mice exhibited cytokine-induced hyperproliferation, hyperactivation, as well as an increased chemotaxis and upregulation of markers for migration. In summary, phosphatase PTP1B is a critical endogenous negative regulator of inflammation and a possible target for cytokine-oriented therapies in asthma.
IKKalpha is critical for the maintenance of a normal pool of regulatory T cells and for the expansion of both regulatory and effector T cells (P1095)
Authors: Chen Xin , Willette-Brown Jami , Wu Xueqiang , Howard O.M. Zack , Hu Yinling , Oppenheim Joost
Published: January 2023

Our previous studies show that TNF activates and expands Tregs through TNFR2. It was reported that TNF-TNFR2 interaction results in the activation of alternative NFκB pathway through p100 processing, in IKKα-dependent manner. We therefore hypothesize that IKKα may play an important role in determining the size of Treg pool. To this end, we generated a mouse strain with conditional KO of IKKα in CD4 cells (CD4-IKKα-/-) which showed a marked reduction in the number of Tregs and in the proportion of Tregs in CD4 cells, indicating that IKKα KO had more profound impact on Tregs than Teffs. In vitro, naïve CD4 cells deficient in IKKα could be differentiated as usual into Foxp3-expressing cells by TGFβ, suggesting that reduction of Tregs in CD4-IKKα-/- mice was likely due to a defect in the maintenance of the Treg pool, rather than a malfunction in the induction of Foxp3 expression. The function of Tregs deficient in IKKα was examined by co-transfer to Rag1-/- mice together with naïve CD4 cells. Although WT Tregs was able to inhibit colitis induced by transfer of WT naïve CD4 cells, IKKα-deficient Tregs failed to inhibit colitis which was associated with their incapacity to reconstitute in Rag1-/- mice. Nevertheless, naïve CD4 cells deficient in IKKα also failed to reconstitute Rag1-/- mice. Therefore, our study indicate that IKKα is important for the maintenance of normal Treg pool and is critical for the expansion of Treg cells as well as Teff cells.
A non-coding interspecies conserved motif of the Immune System Released Activating Agent induced potential immunoregularory effects (P1071)
Authors: Bakhiet Moiz , Taha Safa
Published: January 2023

We have recently discovered ISRAA as a novel nervous system-induced factor inducing immune responses in mouse spleen. ISRAA gene was cloned and its sequence was mapped to chromosome 14. Herein, in silico simulation approaches were used to identify the human ISRAA. The data showed that ISRAA gene constitutes a sequence within human chromosome 10 in Zmiz1 gene intron with no similarity between coding sequences. Even though, this mouse protein showed potential biological activities on human cells. Titration of mouse ISRAA on human cells depicted dose-dependent effects since 5μg exhibited positive apoptosis while 50pg revealed significant proliferation (p<0.0005) and increased IFN-γ production. Cells from immunosupressed patients did not proliferate after PHA stimulation, but demonstrated significant proliferation to 50 pg ISRAA (p<0.005). A concentration of 500ng ISRAA showed cytotoxic effects on two sets of tumor cell lines, but not on healthy cells. Furthermore, a predicted active site of ISRAA gene was constructed and a mutant ISRAA lacking this motif was produced and tested. This motif was found to have 72% similarity with the interspecies conserved signal peptide motif of TNFR1. Mutant ISRAA showed no biological activity compared to its wild type. Thus, functional interspecies non-coding conserved domains exist, and ISRAA effects on immunocompromised cells and tumor cell lines can be explored in future therapies to treat immunosuppressive and cancer disorders.
Development of a vaccine to attenuate viral exacerbation of asthma (P4276)
Authors: White Maria , Debley Jason , Lindell Dennis
Published: January 2023

Viral exacerbation of asthma exacerbation is a major cause of emergency department visits and hospitalization. Thus, vaccination against causative viruses would be an effective method for the attenuation of viral exacerbation for asthmatics. Because live/attenuated vaccines are not safe for asthmatics, inactivated viral vaccines need to be developed. We hypothesize that the innate immune response produces pro-inflammatory signals that determine the outcome of the adaptive immune response. We are evaluating the potential of an intranasal inactivated respiratory syncytial virus vaccine consisting of inactivated virus in conjunction with liposome (delivery agent) and TLR agonists (immune stimulatory agent) to attenuate viral asthma exacerbation. In vitro, bone marrow dendritic cells stimulated with inactivated RSV and TLR7/9 agonists induced more CXCL10, IFNβ, and DLL4 compared to inactivated RSV alone. In vivo, mice vaccinated with liposome-RSV and TLR 7/9 agonists enhanced viral clearance, without inducing an IL-33 or IL-17a response. Using a cockroach allergen RSV-exacerbated asthma mouse model, vaccinated mice had reduced viral load and increased activated lymphocytes and RSV specific antibodies. However, mucus secretion was also increased, indicating that further refining of the vaccine will be necessary. However, our data indicate successful vaccination against a virus clinically associated with asthma exacerbation in a well-defined animal model of asthma.
Long-term immune responses to AS03-adjuvanted, low antigen dose influenza vaccines in BALB/c mice (P4299)
Authors: Yam Karen , Brewer Angela , Gupta Jyotsana , Allen Elizabeth , Ward Brian
Published: January 2023

During the 2009 pandemic influenza outbreak, a vaccine formulated with the oil-in-water adjuvant AS03 and 25% of the usual antigen dose was selected for administration to Canadians. Since the long-term (LT) immune response to this vaccine remains unclear, our objective is to study AS03-adjuvanted low antigen dose influenza vaccines in mice with a focus on LT immunity. We hypothesize that LT memory following unadjuvanted full-dose vaccine will be superior to adjuvanted low-dose vaccination. Mice received 2 IM injections of influenza A/Uruguay H3N2 split vaccine formulated with 3µg antigen only or low antigen dose with AS03 adjuvant. At 3 weeks post-boost, serum hemagglutination inhibition (HAI) titers were: 276 (3µg unadjuvanted); 1365 (0.03µg+AS03); and 1681 (0.003µg+AS03). Lower antigen doses (0.00003-0.0003µg) with AS03 failed to consistently produce detectable antibodies. LT studies with these same formulations (3µg, 0.03µg+AS03, 0.003µg+AS03) are ongoing for up to 18 weeks post-boost; we are examining spleen and bone marrow cells (eg: ELISpots, cytokine profiles, B cell subsets by flow cytometry). At 1, 3 and 6 weeks post-boost, splenocytes of mice immunized with antigen alone produced higher levels of IL-4 and IL-10 than mice immunized with adjuvanted low-dose vaccines. Conversely, splenocytes from the adjuvanted low-dose groups produced higher levels of IL-2 than those from mice immunized with antigen alone. These differences in LT immunity may affect vaccine efficacy.
Systems biology assessment of human immune responses after seasonal trivalent inactivated influenza vaccine (P4307)
Authors: Hoek Kristen , Howard Leigh , Allos Tara , Samir Parimal , Niu Xinnan , Creech Buddy , Edwards Kathryn , Link Andrew
Published: January 2023

Systems biology represents a novel approach to comprehensively study the human immune response to vaccines at the global transcriptional and proteomic level. However, most systems vaccinology approaches utilize total PBMCs in their analyses. In this context, responses of underrepresented immune cell types in the blood are potentially obscured by the predominant cells in the PBMC fraction, and the contribution of PMNs is completely ignored. To investigate the contribution of individual cell types in the immune response following vaccination, we developed a rapid and efficient method for purifying large numbers of T cells, B cells, monocytes, NK cells, myeloid DCs and neutrophils from fresh venous human blood for systems vaccinology studies. This optimized protocol was applied to adult volunteers vaccinated with 2011-12 seasonal TIV. 100mL blood was obtained prior to and on days 1, 3, and 7 post-vaccination. Whole blood, PBMC and PMN fractions were subjected to phenotypic analysis by flow cytometry. Immune cells were fractionated and processed for RNA and protein extraction in a single day. RNA-Seq and quantitative proteomics were performed on purified cells in order to determine individual expression profiles. Our results show significant variation in the phenotypes and expression profiles of immune cells at each time point. This innovative systems approach is currently being utilized to evaluate vaccine safety and efficacy in an adjuvanted influenza clinical trial.
Involvement of the Aim2 inflammasome pathway in generating antibody responses elicited by DNA vaccination. (P4313)
Authors: Suschak John , Wang Shixia , Remington Karen , Fitzgerald Katherine , Lu Shan
Published: January 2023

Recent human study data suggests that DNA priming immunization can substantially enhance the protective antibody responses elicited by conventional influenza vaccines, but their mechanisms remain completely unknown. The current study explored the roles of innate immunity in regulating acquired immunity elicited by influenza vaccines. Innate pattern recognition receptors (PRRs), and their downstream signaling molecules, were investigated for their effect on DNA vaccination. Of particular interest is the absent in melanoma 2 (AIM2) protein pathway and its effects on the antigen-specific antibody response. AIM2 is a cytosolic DNA sensor important in both the maturation of the pro-inflammatory cytokines, IL-1b, IL-18, and an inflammatory form of cell death known as pyroptosis. Aim2 knockout mice and wild type controls were immunized with a DNA vaccine expressing the hemagglutinin antigen (HA) of H1 subtype influenza virus. Mouse serum anti-H1HA antibodies and B cell responses revealed that deletion of the Aim2 pathway led to a significant reduction of the anti-HA antibody response. Interestingly, non-antigen specific cytokine profile analysis did not demonstrate a dramatic change in cytokine signaling between wild type and knockout mice. Furthermore, deletion of IL-1 signaling did not significantly affect the anti-HA antibody responses. These results suggested that the Aim2 pathway plays an important role in shaping the antigen specific antibody response to DNA vaccines.
Therapeutic immunization with EBNA-1 elicits responses to novel T cell epitopes in latently Lymphocryptovirus infected rhesus macaques (P4323)
Authors: Silveira Eduardo , Fogg Mark , Leskowitz Rachel , Ertl Hildegund , Wiseman Roger , O'Connor David , Lieberman Paul , Wang Fred , Villinger Francois
Published: January 2023

Epstein-Barr virus (EBV) is a gamma-Herpesvirus associated with lymphoma or nasopharyngeal carcinoma in immunocompromised patients. Lymphocryptovirus (LCV) infection of rhesus macaques replicates several characteristics of the human EBV infection. EBNA-1 is a nuclear virus protein expressed in all phases of EBV/LCV infections, essential for survival, malignant transformation, tumor progression and escape from host immune response. In spite of its importance for viral replication and survival, EBNA-1 is poorly immunogenic during lytic or latent infection. However, since protection against select experimental infections was achieved after enhancement of antigen immunogenicity through immunization, we hypothesized that eliciting EBNA-1-specific responses during chronic latent infection could increase control of EBV/LCV in vivo. We therefore attempted to enhance and broaden LCV EBNA-1 responses via immunization with an AdC68-LCV-EBNA-1 vector in groups of rhesus macaques that were latently infected with LCV but exhibiting low baseline responses. Following immunization, several CD4+ and CD8+ T cell epitopes were identified through IFN-g ELISPOT assays either as novel or markedly expanded responses. One such novel expanded CD8+ T cell epitope was mapped to a 9-mer peptide restricted by Mamu B*029, and a tetramer was developed for staining of specific CD8+ T cells. The study provides proof of concept that EBNA-1 may be a suitable vaccine target.
Vaccination with recombinant PIV5 expressing influenza neuraminidase provides protection against homologous and heterologous influenza virus infection (P4291)
Authors: Tompkins Stephen , Mooney Alaina , Li Zhou , Gabbard Jon , Dlugolenski Daniel , Johnson Scott , Tripp Ralph , He Biao
Published: January 2023

Vaccination is considered the most effective measure for controlling influenza virus infection, however current vaccination methods are inadequate. Virus-vectored vaccines offer an appealing alternative. Parainfluenza virus 5 (PIV5), a non-segmented, negative-stranded RNA virus (NNSV) in the family Paramyxoviridae is an appealing vector candidate as it has a stable genome without a DNA phase in its life cycle, is readily grown to high titers in approved vaccine cell lines, and infects many mammals without causing disease. Neuraminidase (NA), a glycoprotein found on the surface of influenza A virus as well as virus-infected cells is more conserved than the hemagglutinin surface glycoprotein, the primary antigen in current influenza vaccines. This increases the likelihood of achieving broad protection if used as a vaccine antigen. We show that vaccination with PIV5 expressing the NA from H5N1 highly pathogenic avian influenza (HPAI; rPIV5-N1 (H5N1)) or pandemic H1N1 (rPIV5-N1 (H1N1)) primes robust NA-specific antibody and T cell responses and confers complete protection against homologous (NA-matched) influenza virus challenge. Moreover, immunization also confers significant cross-protection against challenge with heterologous influenza virus within the same NA type. These results suggest that NA can be an effective vaccine antigen and protect against even a stringent challenge, such as with H5N1 HPAI virus, making it an appealing candidate pandemic influenza vaccine.
Dual functions of PD-1 in CD4+ T cell responses by sublingual mucosa-mediated antigen application (P3210)
Authors: Ohno Tatsukuni , Zhang Chenyang , Yagita Hideo , Azuma Miyuki
Published: January 2023

[Objective] PD-1 is a co-inhibitory T cell receptor and binds two ligands, B7-H1 and B7-DC. Although the B7-H1:PD-1 pathway is involved in the induction of peripheral tolerance, immunostimulatory effects of B7-H1 or B7-DC have been demonstrated. The sublingual mucosa (slm) has been used as a route for sublingual immunotherapy to induce tolerance against allergens. On the other hand, the slm is also used as vaccination sites to induce immune responses against pathogens. The mechanisms by which slm-mediated immune responses are controlled are unclear. In this study, we investigated the involvement of PD-1 in OVA-specific CD4+ T cell responses by slm-mediated antigen application with and without mucosal adjuvant. [Results and Discussion] CFSE- labeled DO11.10 T cells were transferred into immunodeficient mice and OVA was painted onto slm with or without cholera toxin (CT). In the presence of CT, blockade of PD-1 by mAb or deficiency of PD-1 inhibited Ag-specific CD4+ T cell proliferative responses, whereas without CT, similar PD-1 blockade enhanced the proliferation. PD-1 is comparably induced on Ag-specific T cells with and without CT. Blockade of B7-H1 did not clearly affect T cell responses even in the absence of CT. CT markedly inhibited the induction of B7-H1 in slm DCs. Our results suggest the existence of PD-1-mediated immunostimulatory pathway in the presence of CT. CT may affect the availability of B7-H1 and B7-DC on slm DCs and regulate the dual function of PD-1.
NALP3 regulates NALT B220+ cell-responses to ricin toxoid and induction of secretory IgA Abs (P3194)
Authors: Jee Junbae , Bonnegarde-Bernard Astrid , Remy Lauriane , Steiner Haley , Fial Michael , Boyaka Prosper
Published: January 2023

Secretory IgA (SIgA) Abs play an important role in the protection of mucosal surfaces including the respiratory tract. Several bacterial toxin and derivatives were shown to act as adjuvants and promote SIgA responses to co-administered antigens, but little is known about potential of plant toxins and derivatives to induce SIgA Abs. We found that chemically inactivated ricin toxoid (RT) increases the frequencies of CD11b+phospho-p65 NF-κB+ cells in nasopharyngeal-associated lymphoid tissues (NALT), an effect associated with increased expression of CD40 molecules on CD11b+ macrophages and enhanced pro-inflammatory cytokine responses. Three hours after intranasal administration of RT in vivo, NALT tissues showed a significant increase in the percentage of B220+ cells, which appeared to undergo rapid proliferation. When nasally co-administered with an unrelated antigen, RT induced broad antigen-specific T helper cell-cytokine responses and promoted antigen-specific SIgA Abs. This adjuvant effect of RT was altered in NALP3 KO mice, which exhibited lower number of B cells in NALT after intranasal administration of RT. Furthermore, antigen-specific T cell and B cell responses, as well as antigen-specific SIgA Ab responses were diminished significantly in NALP3 KO mice. Our results show that the plant toxin derivative RT is a mucosal adjuvant, which promotes SIgA via B cell-recruitment and activation in NALT tissues, and that NALP3 is a key regulator of this activity.
Genital chlamydia trachomatis infection associated immune responses alter host microRNAs expression (P3196)
Authors: Gupta Rishein , Arkatkar Tanvi , Yu JiehJuen , Wali Shradha , Chambers James , Guentzel Neal , Arulanandam Bernard
Published: January 2023

Chlamydia trachomatis (CT) is the leading cause of human bacterial sexually transmitted infections and associated reproductive damage. Given the involvement of microRNAs (miRs) as immune regulators and that TLR2 and CD4+ T cells are associated with host defenses and clearance of CT, we analyzed the modulation of miRs in genital tracts (GT) following CT challenge of wild type (WT), TLR2 deficient (TLR2-/-) and CD4+ deficient (CD4-/-) C57BL/6 mice. CT elementary bodies (5X104 IFU) were inoculated intravaginally for analysis at days 6 (early infection) and 12 (established infection). GT were removed and divided into lower (LGT; vagina & cervix), and upper (UGT) regions for RNA extraction and immunopathology associated miR PCR analyses. Within the WT LGT at day 6, miRs significantly down-regulated were mir-125b-5p (-17.34 fold change), miR-16(-8.87), mir-214(-4.30), mir-182(-19.29), and miR-30c(-19.15) while miR-135a(+13.89) was significantly upregulated, profiles not exhibited by day 12 in LGT and UGT. Moreover, we found significant differences in regulation of miR-214(-3.45) and miR-125b-5p(+1.85) in TLR2-/- GT and miR-214(-2.20) and miR-135a(-2.46) in CD4-/- GT compared to WT mice. Of importance, in vitro knockdown analyses with specific inhibitors of these miRs resulted in 30-45% increase in CT infectivity. Overall, this first report provides for further investigation on contribution of host miRs in CT ascension, associated inflammation and reproductive pathology.
Recognition of soluble yeast β-1,3/1,6 glucan by complement receptor 1 is essential for it to bind complement receptor 3 on human neutrophils and monocytes, and complement receptor 2 on B cells (P3048)
Authors: Bose Nandita , Chan Anissa , Bykowski Jonas Adria , Qiu Xiaohong , Grossman William
Published: January 2023

β-glucans are considered as fungal pathogen-associated molecular patterns that are recognized by innate human cells. Our previous studies demonstrated that Imprime PGG®, an analytically well-characterized form of yeast-derived soluble β−glucan, bound to human neutrophils and monocytes via complement receptor 3 (CR3) and B-cells via CR2, and that receptor binding was complement-dependent. The objective of this study was to further our understanding of the mechanism underlying the interaction of soluble β−glucan with immune cells, specifically, to investigate a role for complement receptor type 1 (CR1; CD35; C3b/C4b receptor) in the binding of Imprime PGG. Blocking of CR1 by anti-CR1 monoclonal antibody significantly abrogated binding of Imprime PGG on all three cell types. Cell-surface CR1, as opposed to soluble CR1, was shown to be more important in mediating Imprime PGG binding. The role of CR1 was further confirmed by demonstrating enhanced binding of Imprime PGG to HEK 293T cells transfected with a combination of CR1 and CR2 genes in comparison to either gene alone. These results suggest that CR1 receptor works in concert with CR3 on neutrophils and monocytes, and with CR2 on B cells in recognizing Imprime PGG. Modulation of Imprime PGG-mediated immune functions after blocking CR1 receptor will also be presented. These data demonstrate for the first time the interplay of several complement receptors in recognizing soluble β−glucan by human immune cells.
Inhibition of Cryptococcus neoformans-mediated phagolysosome damage by induced lysosomal renitence constrains fungal growth. (P3106)
Authors: Olszewski Michal , Davis Michael , Yafeng Qiu , Chapman Rebekah , Malachowski Antoni , Vedula Priya , Swanson Joel
Published: January 2023

Cryptococcus neoformans (C.neo) is a pathogenic yeast which primarily infects the lungs. The mechanisms which enable intracellular replication in unactivated macrophages by C.neo and the strategies employed by classically activated macrophages to combat this replication are incompletely understood. In this work we explore lysosomal damage as an important intracellular survival strategy of C.neo. We measured lysosome damage in C.neo infected murine bone marrow-derived macrophages using ratiometric-epifluorescence microscopy. C.neo growth inhibition data were confirmed by in vitro growth assay. C.neo-mediated lysosome damage was observed in a significant portion of the infected macrophages. The damage increased in frequency and average magnitude with time and replication of the intracellular yeast. Lysosome damage was significantly reduced in macrophages which had phagocytosed heat killed C.neo. Increased lysosome damage correlated with increased C.neo replication and reduced yeast killing. Finally, macrophage activation with IFN-γ but not IL-4 protected macrophages from lysosome damage and promoted increased killing of C.neo. Thus, we conclude that Lysosome damage may be important to the C.neo survival strategy in the host and that classically-activated host macrophages counter this strategy by inducing lysosome renitence. Thus, therapeutic strategies which decrease the ability of C.neo to cause lysosomal damage may be valuable in treating these infections.
Immunologically active cryogels for breast cancer therapy (P4329)
Authors: Bencherif Sidi , Draganov Dobrin , Lewin Sarah , Li Aileen , Sands Roger , Verbeke Catia , Dranoff Glenn , Mooney David
Published: January 2023

Passive immunotherapy has become an effective adjunct for the treatment of HER2/neu-overexpressing breast cancers, as patients can respond well to monoclonal antibodies such as trastuzumab (anti-HER-2/neu antibody therapy). However, patients with late-stage disease, who often become immunosuppressed are unlikely to respond, motivating the development of new prophylactic vaccines. To this end, we have developed an injectable, polymer-based cryogel vaccine containing living, attenuated HER-2/neu-overexpressing breast cancer cells. The cryogel-based vaccine mimics key aspects of bacterial infection and directly controls immune-cell trafficking and activation in the body. This system provides a sustained release of GM-CSF to recruit host dendritic cells (DCs) to the porous material, and subsequently presents breast cancer antigens and TLR9 ligand CpG oligonucleotides to activate the resident DCs. Subcutaneous injection of the cryogel vaccines provide potent prophylactic protection against mammary cancer in mice, consistent with significantly higher titers of Her-2/neu-specific antibodies compared to the humoral responses induced by bolus injection of irradiated GM-CSF-secreting breast tumor cells. The cryogel-based vaccines induced a 70-fold increase in antibody production compared to untreated mice and 100% survival was achieved in vaccinated mice. The cryogel-based vaccines represent a promising tool for the development of novel active immunotherapeutic approaches to cancer.
Synergistic induction of anti-tumour immunity by co-delivery of protein and α-galactosylceramide on exosomes (P4457)
Authors: Gehrmann Ulf , Hiltbrunner Stefanie , Georgoudaki Anna-Maria , Karlsson Mikael C. , Näslund Tanja I. , Gabrielsson Susanne
Published: January 2023

Exosomes, nanosized endogenous vesicles, can induce adaptive immune responses and have been tested in cancer treatment. Similarly, the iNKT cell ligand α-galactosylceramide (αGC) presented on the MHC class I-like molecule CD1d has been used for therapeutic purposes in cancer trials. Here, we investigated if exosomes can be loaded with αGC to activate iNKT cells and potentiate a cancer-specific adaptive immune response. We show that αGC loaded exosomes readily activate iNKT cells both in vitro and in vivo. In addition, exosomes loaded with αGC and the model antigen ovalbumin (OVA) induced a potent NK and γδ T cell innate immune response, followed by synergistically amplified T and B cell responses. Interestingly, αGC/OVA-loaded exosomes did not induce iNKT cell anergy, in contrast to soluble αGC, and were more potent than soluble αGC and OVA in inducing adaptive immunity. Finally, tumor-bearing mice treated with αGC/OVA-loaded exosomes displayed decreased tumor growth and increased T cell tumor infiltration in comparison to mice immunized with OVA-loaded exosomes in an OVA-specific mouse melanoma model. These results show that exosomes loaded with protein antigen and the glycolipid αGC effectively activate both innate and adaptive immunity, important for future cancer immunotherapy design.
Both plasma kininogen and bradykinin receptors are required for collagen antibody-induced arthritis (P4079)
Authors: Wu Yi , Colman Robert
Published: January 2023

Although experimental and clinical studies have suggested that plasma kallikrein-kinin system (KKS) is involved in arthritis, their role in the disease pathogenesis has never been studied using gene deficient mouse model. High molecular weight kininogen (HK) is a major component of KKS, the activation of which induces HK cleavage to generate proinflammatory bradykinin (BK). In mice, kininogen-1 (Kng1) gene in liver drives production of HK in plasma. We generated a Kng1-/- mouse strain whose liver Kng1 mRNA and plasma HK protein were absent. To induce collagen antibody-induced arthritis (CAIA), three groups of mice (WT, Kng1-/-, and B2RB1R-/- that lack double BK receptors) were injected I.P. with anti-type II collagen antibody cocktail (3 mg) on day 0 and LPS (50 μg) on day 3. WT mice started to develop joint swelling and erythema on day 5, and the increase in ankle joint diameter became marked on day 6 (1.32±0.13 mm) and peaked on day 8 (1.41±0.17 mm). As detected by ELISA, plasma levels of IL-1β were increased on day 8 than day 0 (p<0.01). Real time PCR indicated that IL-1β mRNA in monocytes of WT mice was increased by 3 fold. In contrast, either Kng1-/- or B2RB1R-/- mice did not develop marked joint swelling, their plasma IL-1β and monocytic IL-1β mRNA levels were not significantly increased. Our results demonstrate an essential role of HK in the pathogenesis of arthritis, BK serving as a key mediator. Blockade of HK cleavage has a potential for anti-arthritic treatment.
Gut CD103+ dendritic cells from female lupus-prone (NZBxNZW) F1 (BWF1) mice are much less effective than male BWF1 mice at inducing iTregs (P4087)
Authors: Chhabra Anita , Alard Pascale , Kosiewicz Michele
Published: January 2023

Regulatory CD4+Foxp3+ cells (Tregs) either develop in the thymus (nTregs) or are converted from non-Tregs in the periphery (iTregs). Lupus is much more prevalent in females than males, and although we have found that female BWF1 mice have lower Treg% than males and Treg% are increased in androgen-treated females and decreased in castrated males, it is unclear whether it is the nTregs or iTregs that are affected. In this study, we compared the ability of female and male BWF1 cells to mediate conversion. We found no differences in the ability of female and male BWF1 in vitro generated (bone marrow-derived) dendritic cells (DC) treated with TGFβ to induce Foxp3+ iTregs in vitro. Next, we analyzed CD103+DC from mesenteric lymph nodes (MLN) that can spontaneously induce iTregs. CD103+DC from female BWF1 mice exhibited a dramatically decreased ability to induce iTregs in vitro. CD103+DC from castrated male mice also had a similarly reduced ability to induce Tregs compared to sham-castrated males. Differences in CD103+DC function correlated with Treg% in MLN which were lower in females than males. There were no differences in CD103+DC% in MLN between female and male BWF1 mice. Interestingly, treatment in vivo with a blocking anti-CD103 antibody induced/accelerated disease and increased mortality in male BWF1 mice. Our data suggest 1) androgens may increase tolerogenic function of CD103+DC and 2) CD103+DC may induce a population of iTregs that are important for controlling lupus.
Mechanisms of AIM2 inflammasome regulation (P4093)
Authors: Prasad Amit , Ramirez-Ortiz Zaida , Means Terry
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune syndrome typified by autoantibodies against DNA, chromatin, and RNA-associated proteins. Experimental evidence from our group and others suggests that RNA- and DNA-containing immune complexes isolated from SLE patients activates dendritic cells (DCs) through the nucleic acid sensors TLR7 and TLR9, respectively. Using a highly parallel shRNA pooled screening strategy; we identified several additional genes that positively regulate innate responses to stimulatory RNA and DNA complexes that contribute to SLE pathology. Here we show that triggering receptor expressed on myeloid cells like 4 (Treml4) regulates innate sensing of dsDNA and ssRNA in macrophages and DCs. To validate these findings we generated TREML4-deficient mice. We found that dsDNA or ssRNA stimulation of DCs isolated from TREML4-deficient mice produced significantly less IL-1 beta compared to wild-type DCs. We also observed lower caspase-1 and pro-inflammatory cytokines in Treml4 deficient DCs. In addition, AIM2 expression and function are significantly impaired in TREML4-deficient cells compared to wild-type cells. Our results suggest further study of this new signaling intermediate will give better insight into autoimmune diseases, such as SLE and may serve as a potential therapeutic target.
Modulation of dendritic cell function by neuroantigen-specific CD8+ T cells (P4065)
Authors: Kashi Venkatesh , Ortega Sterling , Tyler Andrew , Karandikar Nitin
Published: January 2023

Experimental autoimmune encephalomyelitis (EAE) studies have established that myelin reactive T cells contribute significantly to the pathology of multiple sclerosis (MS). While auto-reactive CD4+ T cells have long been considered to be pathogenic, the role of CD8+ T cells is relatively unknown. We previously showed that myelin oligodendrocyte glycoprotein-specific CD8+ T cells (MOG-CD8+) suppress EAE; however, the mechanism of disease suppression is not completely understood. Here we show that CD11c+ dendritic cells (DC) from MOG-CD8+ recipient mice are less efficient in priming naïve and in activating recall CD4+ T cell response as compared to control ovalbumin-CD8+ recipients. In response to lipopolysaccharide stimulation, DC from MOG-CD8+ recipients demonstrated an anti-inflammatory cytokine profile with significantly lower IL-12 but higher IL-10 secretion. This was also confirmed in CD8-/- mice where DC from MOG-immunized CD8-/- mice secreted higher levels of IL-12 as compared to wild-type mice. MOG-CD8+ transfer to naïve mice did not affect DC function indicating that DC modulation requires cognate antigen presentation. Kinetic studies revealed that DC were modulated prior to disease onset at day 7 and persisted until day 30 post CD8+ transfer. Overall, our data suggest that transfer of CNS-antigen specific CD8+ T cells modulates DC function and can be an important mechanism of maintaining peripheral tolerance.
Autophagy: a dichotomous player in Toll-like receptor 7-mediated autoimmunity. (P4073)
Authors: Weindel Chi , Richey Lauren , Bolland Silvia , Huber Brigitte
Published: January 2023

The endosomal Toll-like receptor, TLR7, acts as critical line of defense against viral pathogens; however, overexpression of TLR7 has been shown to lead to spontaneous lupus-like disease (SLE). Thus, tight regulation of this pattern recognition receptor is crucial in preventing a hyperactive cellular state. We hypothesized that autophagy plays a role in TLR7 activation by delivering ligand to endosomes and by controlling subsequent inflammatory responses. To test this model, we crossed TLR7 transgenic (tg) mice that succumb to SLE with mice where ATG5, a critical autophagy gene, was deleted in dendritic cells (DCs) or B cells or both. These mice were compared to TLR7 tg mice with intact autophagy for morbidity and the inflammatory response. We observed that loss of ATG5 in either DCs or B cells significantly increased the survival of TLR7 tg mice, while ATG5 deletion in both cell types only modestly improved survival. On the other hand, loss of autophagy led to an elevated inflammatory response compared to TLR7 tg mice with intact autophagy, as evidenced by vastly increased lymphoid organ mass and elevated numbers of granulocytes and macrophages. The inflammatory mediators involved in this process are currently under investigation. These data suggest that autophagy plays an important and dichotomous role in TLR7-mediated autoimmunity, both enabling and ameliorating symptoms of SLE. It is likely that autophagy is affecting multiple TLR7-dependent signaling processes.
TCR signal strength via IRF4 influences CD8 T cell responses to acute LCMV infections (P6096)
Authors: Nayar Ribhu , Prince Amanda , Enos Megan , Daniels Keith , Brehm Michael , Welsh Raymond , Berg Leslie
Published: January 2023

CD8+ T cells undergo rapid proliferation, differentiation and contraction following acute infections. One of the key factors influencing these CD8+ T cell responses is the strength of TCR-ligand interactions. We have previously shown that the transcription factor Interferon Regulatory Factor 4 (IRF4) is important in the differentiation of CD8 T cells. IRF4 is not expressed in naïve CD8 T cells, but is rapidly up-regulated upon T cell activation. Further, this up-regulation is directly regulated by the strength of TCR signaling, such that stronger signaling leads to higher levels of IRF4 expression. To test the function of IRF4 in vivo, we have utilized the acute LCMV infection model together with mice expressing graded levels of IRF4. These studies indicated that CD8 T cell proliferation and differentiation into effector and memory populations is regulated by IRF4. Further, we found that IRF4 was impacting the expression of major downstream transcription factors, Eomes, TBet, and TCF1, that are critical for effector responses and memory differentiation. Together these data establish a transcription factor network that is highly responsive to the strength of TCR-ligand interactions during infection.
Inflammasome adaptor protein ASC is critical for the immune response and survival in West Nile virus encephalitis (P6110)
Authors: Verma Saguna , Roe Kelsey , Kumar Mukesh , Orillo Beverly , Muruve Daniel , Nerurkar Vivek , Gale, Jr. Micheal
Published: January 2023

Innate immune responses are essential for WNV clearance, dissemination and neurovirulence. Pattern recognition receptors and other danger signals activate assembly of inflammasome complex, which modulates antiviral defense responses. ASC is an important adaptor molecule, which mediates downstream signaling pathways of NLRP3 inflammasomes. In this study we defined the role of ASC in WNV immunity. Infection of cultured BMDCs showed that ASC was essential for the activation of caspase-1, a key component of inflammasome assembly. ASC-/- mice were highly susceptible to WNV infection marked by increased mortality, enhanced viremia and virus replication in the CNS. As compared to wild type mice, key cytokines including IL-1β and IFN-γ were significantly reduced in the sera of ASC mice. Additionally, antiviral IFN-α levels were also decreased in the sera of ASC-/- mice. Increased virus titers in the brain of ASC-/- mice correlated with increased inflammation and neuronal apoptosis. However, intracranial inoculation of WNV did not result in significant differences in the virus titers in both groups suggesting that impaired immune response coupled with failure to clear virus in the periphery leads to increased neuropathology and mortality in ASC-/- mice. Collectively, our data provides new insights into the role of ASC as an essential modulator of inflammasome-dependent and -independent immune response to effectively control WNV infection.
Regulation of CXCR3 chemokine production and CD8+ T cell infiltration in the metastatic melanoma microenvironment (P5091)
Authors: Clancy-Thompson Eleanor , Mullins David
Published: January 2023

Vaccine-based immune therapy has shown minimal efficacy against established tumors. However, vaccination in the adjuvant setting prolongs disease-free survival. We previously demonstrated that CXCR3 expression by tumor antigen-specific T cells correlates with this. We hypothesized that melanoma engraftment may induce spatially- and temporally-restricted expression of CXCR3 chemokines in the tumor microenvironment, leading to transient infiltration of CXCR3+ T cells into tumor. We used a model of murine metastatic-like melanoma to evaluate tyrosinase expression as a measure of tumor burden. We observed a positive correlation between tyrosinase and CXCL9 (r2 = 0.73) and CXCL10 (r2 = 0.86) expression over time, demonstrating that melanoma growth in the lung is sufficient to induce CXCR3-cognate chemokines. In a time-course study, we observed that CXCL9 and CXCL10 expression decrease after day 15; we hypothesize that diminished chemokine production is due to reduced interferon signaling. We are investigating this possibility. Our preliminary flow cytometry analyses suggest a positive correlation between CD8+ T cell infiltration and the expression of CXCL9/10, suggesting that metastatic melanomas are transiently receptive to T cell infiltration. Therefore, successful immunotherapy of metastatic melanoma may require adjuvant therapy to induce or extend the chemotactic window for T cell infiltration.
IL-12 inhibits memory CD8+ T cells localization to the respiratory tract and reduces protection against Influenza A virus. (P5093)
Authors: Slutter Bram , Harty John
Published: January 2023

Memory CD8 T-cells specific for conserved antigens from Influenza A virus (IAV), like nucleoprotein (NP), may provide an important line of defense when neutralizing antibodies are absent (e.g. IAV pandemic). Here we show, through local CD8 T-cell depletions, that NP specific memory CD8 T-cells have superior protective capacity against IAV when they are localized inside the respiratory tract (BAL fluid). Expression of the chemokine receptor CXCR3 on memory CD8 T-cells was essential for the maintenance of a local memory population in the respiratory tract. Interestingly, the inflammatory milieu during immunization seems critical for the ability of memory CD8 T-cells to express CXCR3. Exposure to interleukin 12 (IL-12), but not other signal 3 cytokines, led to reduced expression of CXCR3 on memory CD8 T-cells, through a STAT4 and T-bet dependant pathway. Neutralization of IL-12 during priming or selection of adjuvants that induce little IL-12, significantly increases CXCR3 expression on memory CD8 T-cells, enhances their numbers in the respiratory tract and provides superior protection against IAV challenge. Thus, CXCR3+ memory CD8 T-cells have a superior protective capacity against IAV, due to their capacity to populate the respiratory tract and IL-12 is a key regulator for the establishment of CD8 T-cell memory inside the respiratory tract.
EBI2 guides osteoclast precursors to bone niches and regulates osteoclastogenesis and bone mass homeostasis. (P5099)
Authors: Nevius Erin , Horowitz Mark , Insogna Karl , Ishii Masaru , Pereira João
Published: January 2023

Osteoclasts (OCs) are bone-lining cells derived from the fusion of monocytic precursors. Their exclusive positioning in contact with bone enables OC differentiation and the remodeling of bone outgrowth. However, the guidance cues that osteoclast precursors (OCP) follow for positioning at the bone surface remain poorly characterized. We demonstrate that EBI2 (Gpr183) was abundantly expressed in OCPs, and that autocrine EBI2 signaling was necessary for differentiation into mature osteoclasts in vitro. The expression of EBI2 and its ligands suggested that EBI2 signaling regulates OCP motility. Time lapse microscopy of OCP differentiation revealed reduced velocity and displacement of EBI2-deficient OCPs and mature OCs in vitro. Mice deficient in EBI2 or EBI2-ligands were osteopetrotic under homeostasis, and were protected from osteoporosis induced by estrogen deficiency. As osteoblasts (OBs) control osteoclast differentiation we hypothesized that they also recruit OCPs to bone niches. Indeed, OBs expressed EBI2-ligand synthesis enzymes, Ch25h and Cyp7b1, and secreted EBI2 ligands in vitro. We further demonstrate that EBI2-deficient OCPs were unable to efficiently migrate towards bone, and differentiated into smaller sized osteoclasts in vivo. Remarkably, overexpression of EBI2 was sufficient for directing OCPs to bone surfaces. Combined, these studies revealed a novel EBI2 controlled bone marrow niche, and identified a new candidate for the treatment of bone disorders.
Distribution, morphology and phenotypical characterization of lipid-laden multilocular cells in the aging thymus. (P5107)
Authors: de Mello Coelho Valeria , Andrade Leonardo , Taub Dennis , Borojevic Radovan , Langhi Larissa
Published: January 2023

Age-related thymic involution is characterized by decreased thymopoiesis and increased white adipose tissue in the perivascular space. Previously, we described the presence of intrathymic lipid-laden multilocular cells (LLMC) expressing factors known to promote thymic atrophy. In this study, we aimed to characterize the distribution, morphology and phenotype of LLMC. Our results show that LLMC are distributed in thymic capsule, parenchyma and surrounding blood vessels and increase with aging. Based on their shape and/or location, LLMC present globular, fibroblastoid or pericyte-like morphology. Our ultrastructural analyses showed that LLMC contain lipid droplets with distinct electrondensities and autolipophagossome-like structures in their cytoplasm. In addition, we found LLMC interacting directly with lymphocytes, endothelial cells and mast cells in the thymic parenchyma. Furthermore, mast cells were quantified and increase in thymus with age. By immunohistochemistry, we analyzed the phenotype of LLMC and we found cells positively stained with specific markers for pericytes (α-SMA, NG2), macrophages (CD11b, F4/80), pre-adipocytes (PPARγ2) and brown adipocytes (UCP1) in the aging thymus. In conclusion, LLMC are not simply differentiating adipocytes, but correspond to distinct cellular populations, including macrophages and mesenchymal-derived cells that assume a lipid-laden morphology and are able of interacting with thymocytes and distinct thymic microenvironmental cells.
Coxsackievirus B3 regulates T cell infiltration by LFA-1 modification through Rap1 activation (P5075)
Authors: Shim Seung-Hyun , Kim Dae-Sun , Nam Jae-Hwan
Published: January 2023

Coxsackievirus B3 (CVB3) infection has the potential to induce myocarditis. The following may be the mechanisms of CVB3-induced pathogenesis: viral infection directly causes injuries to cardiomyocytes through viral cytopathic effect and virus-activated T cell infiltration indirectly causes injuries to cardiomyocytes through inflammation. However, the detailed mechanism of T cell infiltration remains unknown. The present study observed that infiltrated T cells could be detected in the hearts of CVB3-infected mice. Meanwhile, attenuated CVB3 (YYFF) did not elicit T cell infiltration in infected mouse hearts. Rap-1, Ras family of small GTPases, is a critical regulator of T cell activation. CVB3 infection was found to activate Rap1 through cAMP induction. The downstream molecules of Rap1, ERK and p-CREB, which are involved in T cell activation and differentiation, were also activated after CVB3 infection. However, this result did not occur in the case of YYFF. This Rap-1 activation leads to an active form of LFA-1 on T cells that can be bound to intracellular adhesion molecule-1 (ICAM-1) on myocytes for T cell infiltration after CVB3 infection. This mechanism of CVB3-induced T cell infiltration may contribute to be a potential therapeutic agent for virus-induced myocarditis. Keywords: Coxsackievirus B3, YYFF, LFA-1
Non-invariant NKT cells cause diet-induced obesity by tissue inflammation (P5111)
Authors: Satoh Masashi , Eshima Koji , Takeuchi Emiko , Tamauchi Hidekazu , Iwabuchi Kazuya
Published: January 2023

The progression of obesity accompanies a chronic inflammatory process that involves both innate and acquired immunity. Mϕ, T cells and other immune cells have been shown to affect the development of obesity by modulating the chronic inflammation in visceral adipose tissue. NKT cells recognize lipid antigens and are also distributed in visceral adipose tissue. To examine the involvement of NKT cells in the development of obesity, WT and two NKT-cell-deficient strains, Jα18-/- and CD1d-/-, were fed a high fat diet. We reported that CD1d-/- but not Jα18-/- mice fed a high fat diet showed the least gain of body weight, and glucose intolerance and insulin resistance were not aggravated because of a reduced tissue inflammation. Furthermore NKT cells (non- invariant NKT) in visceral adipose tissue were accumulated in WT and Jα18-/- mice fed a high fat diet. In the present study we further examined stromal vascular fraction obtained from visceral adipose tissue. First, TCR-Vβ usage of NKT cells (TCBβ+NK1.1+) were analyzed. Notably, Vβ7+, Vβ8.1+ NKT cells were major subsets in spleen and liver, whereas Vβ5+, Vβ11+, Vβ12+ cells were mainly observed in stromal vascular fraction in WT mice fed a high fat diet. Vβ5+ subsets were also observed in Jα18-/- mice. Further characterization of NKT subsets and analyses of interactions of NKT cells and other SVF components may elucidate the development of obesity.