bluuebunny/crossref_metadata_2025 huggingface dataset:
Choline acetyltransferase+ lymphocytes regulate endothelial nitric oxide synthase (MUC2P.939)
Authors: Olofsson Peder , Hanes William , Steinberg Benjamin , Oswald Michaela , Ahmed Mohamed , Hudson LaQueta , Valdes-Ferrer Sergio , Liu Shu , Pavlov Valentin , Chavan Sangeeta , Andersson Ulf , Diamond Betty , Miller Edmund , Gregersen Peter , Tracey Kevin
Published: January 2023

Mammalian blood vessels are innervated by adrenergic neurons that increase blood pressure. The neurotransmitter mechanism is dependent upon norepinephrine that mediates contraction of vascular smooth cells to increase arterial resistance. Vasodilation is mediated by acetylcholine, which lowers blood pressure through a mechanism dependent upon relaxation of vascular smooth muscle cells in response to endothelial release of nitric oxide. Endothelial cells are not innervated by cholinergic neurons, and it was unknown whether acetylcholine release by lymphocytes might modulate blood pressure. Here, we observed lymphocytes expressing choline acetyltransferase (ChAT), the enzyme that catalyzes biosynthesis of acetylcholine, in the blood. Co-culture of ChAThi lymphocytes with endothelial cells significantly increased calcium concentration and phosphorylation of eNOS in endothelial cells (p<0.05 vs. ChATlow lymphocytes). Passive transfer of engineered ChAThi Jurkat T cells resulted in an immediate significant decrease in blood pressure in recipient mice to 83±5 % of baseline, while blood pressure after transfer of ChATlow Jurkat T was 104±4 % of baseline (p=0.02). Pre-treatment with the NO synthesis inhibitor L-NG-monomethyl arginine citrate prevented the blood pressure reduction by ChAThi Jurkat T cells, and blood pressure remained at 98%±5% of baseline in these animals (p=0.21). These findings suggest a novel mechanism of lymphocyte-mediated blood pressure regulation.
Antimicrobial peptide gene expression and functionality in P-glycoprotein deficient models of colitis (MUC3P.943)
Authors: Lorenz Robin , Tanner Scott , Daft Joseph , Little Marguerite
Published: January 2023

Inflammatory bowel diseases (IBD), such as Crohn’s disease and ulcerative colitis, are chronic inflammatory conditions of the digestive tract. Antimicrobial peptides (AMPs) are produced in the intestines and function to control bacterial growth. AMP deficiency can result in unfavorable microbiota, a proposed IDB risk factor. FVB.mdr1a-/- mice, lacking the multidrug resistance gene (MDR) and its functional product, P-glycoprotein (P-gp), develop spontaneous colitis. We hypothesized that P-gp deficiency alters AMP expression, resulting in changes in the microbiota. To investigate this, intestinal AMP mRNA expression from control and FVB.mdr1a-/- were compared. This revealed altered expression of small intestinal and colonic AMPs. Next, FVB.mdr1a-/- mice were treated with antibiotics to determine if these changes are due to P-gp deficiency or secondary to microbial colonization. Intestinal AMP mRNA expression was analyzed and no changes were observed, suggesting that the absence of P-gp alters AMP expression, regardless of microbiota. Functionality of these AMPs were investigated, showing higher killing capabilities in the FVB.mdr1a-/- mice. For insight into human IBD, AMP mRNA expression were measured in the human colon adenocarcinoma cell line (Caco2-C) and a P-gp knockdown (Caco2-MR) after treatment with bacterial products. Here, we observed changes in AMP expression. These studies provide insight into the role of the MDR gene in the development of colitis.
A vital control of intracellular Chlamydia infection requires interleukin-17 receptor-mediated signaling (MUC3P.946)
Authors: Tram Cynthia , Kampen Rachel , Halperin Scott , Wang Jun
Published: January 2023

IL-17A is a pleiotropic proinflammatory cytokine that has a vital role in host resistance against extracellular bacterial and fungal infections. However, its role in controlling intracellular bacterial infection remains elusive. Here, we examined the IL-17/IL-17 receptor-mediated signaling in controlling intracellular Chlamydia muridarum infection in non-hematopoietic cells in vitro and in vivo. Remarkably, Chlamydia replication in mouse embryonic fibroblasts (MEF) was significantly increased in IL-17RC-knockout (IL-17RCKO) MEF and, to a lesser extent, in IL-17RAKO MEF compared to WT MEF at 6 and 12 hrs post-infection. To verify this finding in vivo, a respiratory Chlamydia infection model was established in bone marrow (BM) chimeric mice using WT BM donor cells transferred into γ-irradiated WT, IL-17RAKO and IL-17RCKO mice. Both IL-17RAKO and IL-17RCKO BM chimeric mice had significantly elevated bacterial burden in the lung compared to WT mice at days 5 and 11 post-infection. However, IL-17RCKO, but not IL-17RAKO, BM chimeric mice exhibited significant body weight loss compared to WT mice during early infection. While IL-17RAKO BM chimeric mice displayed an increased IL-17A response, IL-17RCKO mice showed an increased IFN-γ response in the lung. Our data demonstrates an indispensable role of the IL-17RC-mediated signaling in controlling Chlamydia infection and suggests differential roles of IL-17RA- and IL-17RC-mediated signaling in shaping Chlamydia-induced host responses.
Substance P and IL-33 synergistically stimulate mast cells to release IL-1β and TNF-α implicated in psoriasis; inhibition by the flavonoid methoxyluteolin (THER2P.960)
Authors: Taracanova Alexandra , Theoharides Theoharis
Published: January 2023

Psoriasis is an autoimmune skin disease characterized by keratinocyte hyperproliferation and inflammation. The pathogenesis of psoriasis appears to involve interactions among keratinocytes, mast cells (MC) and cytokines. MC are immune cells that secrete various cytokines and chemokines including interleukin (IL)-1 family of cytokines (IL-1β, 18, 33), which can stimulate keratinocyte activation. IL-33 also augments activation of MC with further release of several mediators such as TNF-α, IL-6, and prostaglandin D2. Also, IL-33 has synergistic effect with a neuropeptide substance P (SP) on VEGF release in MC. Both IL-33 and SP are upregulated in psoriatic skin. Moreover, SP stimulates keratinocytes to secrete IL-1β. Our findings show that SP and IL-33 synergistically stimulate MC to produce and release IL-1β and TNF-α, pro-inflammatory mediators involved in psoriasis pathogenesis. The combination of SP and IL-33 also significantly up-regulates IL-1β and TNF-α gene expression. The natural flavonoid methoxyluteolin (Metlut) is a potent inhibitor of both keratinocyte and MC activation. Here we report that Metlut significantly inhibits IL-1β and TNF-α gene expression, production and release triggered by SP and IL-33 combination. Therefore, Metlut-based formulations could be a novel treatment for psoriasis.
High-throughput functional assay to screen anti-BDCA2 candidates for systemic lupus erythematosus therapeutics (THER3P.973)
Authors: Li Jenny , Ladi Ena , Pappu Rajita , Chan Pamela
Published: January 2023

Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease characterized by a large number of aberrations in the immune system. Studies with SLE patients have shown that heightened levels of the cytokine interferon-α (IFNα) can interfere with immune regulation and effect lupus pathogenesis. The plasmacytoid dendritic cells (pDCs) are the main producer of type I IFNs and have the capacity to synthesize up to 109 IFNα molecules per cell after activation. Crosslinking BDCA2, a typeII C-type lectin specific to pDCs, results in the inhibition of IFNα production in pDCs stimulated with TLR7/9. To develop an agonistic therapeutic antibody to BDCA2, a large number of antibody candidates have to be screened in a functional assay that detects blocking of IFNα production in pDCs isolated from peripheral blood mononuclear cells (PBMC). pDCs constitute less than 0.4% of PBMCs making pDC purification inefficient and challenging. Here we report an automated high-throughput BDCA2-agonistic functional assay using fraction 5 (F5) cells from an elutriated leukopak as substitute for purified pDCs coupled with a robust INFα detection method. The Integrated automation program in 384well format has provided a powerful tool for antibody screening and can be applied to other projects requiring a similar screening strategy.
Molecular profiling of CD8 T cells from autochthonous melanoma identifies Maf as driver of T cell exhaustion (TUM2P.1004)
Authors: Verdeil Gregory , Giordano Marilyn , Henin Coralie , Maurizio Julien , Bourdely Pierre , Buferne Michel , Auphan-Anezin Nathalie , Speiser Daniel , Schmitt-Verhulst A
Published: January 2023

T-cells infiltrating neoplasms express surface molecules typical of chronically virus-stimulated T-cells, often termed “exhausted” T-cells. We analyzed the transcriptome of “exhausted” CD8 T-cells infiltrating autochthonous melanomas compared to those of naïve and acutely stimulated CD8 T-cells. In spite of strong similarities between transcriptional signatures of tumor- and virally-induced exhausted CD8 T-cells, notable differences appeared. Among transcriptional regulators, Nr4a2 was expressed in both exhaustions, whereas Maf was highly over-expressed only in tumor-exhausted T-cells. We confirmed Maf and nr4a2 up-regulation in Melan-A/MART-1 specific CD8 T cells from tumor infiltrated LN from melanoma patients. Anti-tumor CD8 T-cells transduced to express Maf showed dampened anti-tumor activity upon adoptive transfer, whereas Nr2a4 over-expression was without effect. Maf-expressing CD8 T-cells showed unaltered homeostasis but failed to accumulate in tumor-bearing hosts and developed defective anti-tumor secondary responses. We also found that Maf expression in CD8 T cells induced part of the transcriptional program associated with tumor-induced exhaustion. We further identified TGFβ as a main contributor to Maf expression in CD8 T-cells. Therefore the melanoma microenvironment contributes to skewing of CD8 T-cell differentiation programs, in part by TGFβ-mediated induction of Maf.
Longitudinal analyses of CD3ζ-chain expression in the correlation of the disease status in a cohort of head and neck cancer patients (TUM2P.1033)
Authors: Upreti Deepak , Zhang Man-li , Bykova Elena , Pathak Alok , Kung Sam
Published: January 2023

Purpose: Despite advances in multimodality treatment, the 5-year survival rate of Head and neck squamous cancer (HNSCC) patients has unimproved over the last 4 decades. CD3ζ has emerged as a clinically important immunological molecule in HNSCC. Its relevance as a prognostic biomarker of HNSCC, however, has not been formally addressed in a longitudinal study. Methods: Peripheral blood mononuclear cells (PBMC) were collected from 46 patients and 53 controls at the time of diagnosis and post treatments (upto 2 year period). Expression of CD3ζ in the T cells of the PBMC samples were analyzed in flow cytometry. Results: We standardized a method for longitudinal analyses of intracellular CD3z expressions in the PBMC samples. We considered a <10% baseline increase in the normalized MFI of CD3ζ expression as a predictor of disease status in evaluating the follow-up samples of the HNSCC patients. Correlation analysis showed that 27/29 HNSCC patients who showed an increase in the CD3ζ expression relative to their baselines were disease free (negative predictive value. 93.1%). 10/17 HNSCC patients who showed a reduced/no change in the CD3ζ expression died or had recurrent disease (positive predictive value, 58.8 %). Overall accuracy of the assay was 80.43%. The sensitivity and specificity were respectively 83.3% and 79.41%. Conclusion: Our longitudinal analyses supported that a >10% increase in the CD3ζ expression against baseline could be a good prognostic biomarker in HNSCC patients.
Effector CD8+ T cells abrogate fibroblasts-mediated chemoresistance in ovarian cancer (TUM2P.1007)
Authors: Wang Weimin , Kryczek Ilona , Tan Lijun , Zhao Lili , He Gong , Vatan Linda , Szeliga Wojciech , Zou Weiping , Liu Rebecca
Published: January 2023

Tumor infiltrating effector CD8+ T cells have been described to play a major role in antitumoral activity and to be a favorable prognostic factor in ovarian cancer. The strategies of immunotherapy to improve effector T cells response have also shown potential clinical benefit in subset of ovarian cancer patients. Currently platinum-based chemotherapy is still the most active regimen for ovarian cancer treatment. However, the therapeutic resistance is a major challenge to limit the therapeutic efficiency. Here we found primary fibroblasts isolated from ovarian tumor speciman could induce resistance against cisplatin in cancer cells when the two cell types were co-cultured in vitro or co-inoculated in vivo. Activated CD8+ T cells abolished fibroblasts-mediated cisplatin resistance through IFNγ secretion. Next, we found fibroblasts induced cisplatin resistance through increasing intracellular Glutathione (GSH) level and reducing the cisplatin accumulation in cancer cells. We also identified both intact GSH and Cysteine released by fibroblasts contributed to cisplatin resistance in cancer cells. IFNγ producted by activated CD8+ T cells promoted fibroblasts extracellular GSH degradation through up-regulation of Gamma-glutamyl transpeptidase, and also diminished Cysteine generation by fibroblasts through down-regulation of System xc⁻ cystine/glutamate antiporter.These date implicate effector T cells immunotherapy as a powerful regimen to overcome the resistance to chemotherapy.
Optimizing the efficacy of dual-costimulation cancer therapy by engaging tumor-unrelated CD4 T cell help (TUM2P.1009)
Authors: Mittal Payal , Wang Xi , Uthaya Dinesh , St. Rose Marie-Clare , Vella Anthony , Adler Adam
Published: January 2023

Tumor escape following immune therapy can be caused by loss of specifically targeted MHC class I-restricted epitopes. It would thus be beneficial to develop therapies that engage multipronged anti-tumor responses. Agonist mAbs to the TNF/TNFR family costimulatory receptors CD134 plus CD137 (dual costimulation) not only elicits CD8+ CTL, but also cytotoxic CD4 Th1 cells that can directly target MHC-II+ tumors. Further, dual costimulated cytotoxic CD4 Th1 cells not only provide linked help to CD8 T cells responding to cross-presented antigen, but can also provide non-linked help that programs Th1/Tc1 functions in antigen-non-responding (bystander) T cells. Here we tested whether engagement of tumor-unrelated CD4 help augments dual costimulation-mediated control of established B16 melanoma that can be induced to express both MHC class I and II. Notably, engagement of tumor-unrelated CD4 T cells dramatically enhanced the ability of dual costimulation to control B16 tumor growth, this effect depended upon both CD8 and CD4 cells, and therapeutic efficacy correlated with increased tumor infiltration of GzmB-expressing effector CD8 and CD4 T cells with a concomitant decrease in Foxp3+CD4+ cells. Interestingly, the dual costimulated tumor-unrelated CD4 helper T cells themselves appear to require help from a CD134-responsive but presumably antigen-non-responding cell to attain their maximal function.
Role of natural killer cells in ovarian cancer chemoresistance (TUM3P.1050)
Authors: Scholler Nathalie , Repellin Claire
Published: January 2023

Novel immunotherapeutic approaches revolutionize cancer prognosis but the role of innate immunity in chemoresistance remains poorly understood. For example, toll-like receptor (TLR) ligands in combination with anticancer treatments yielded unpredictable effects, either beneficial or deleterious. We hypothesized that the function of tumor-infiltrating immune cells depends on constitutive TLR variants. We characterized healthy donor T cells, B cells, monocytes and natural killer (NK) for functional polymorphism after transwell co-culture with chemosensitive (OVCAR5) vs. chemoresistant (SKOV3TR) ovarian tumor cells +/- (1) medium only; (2) LPS; (3) PamCys3; (4) Paclitaxel (PTX); (5) OVCAR5; (6) OVCAR5+PTX; (7) SKOV3TR; (8) SKOV3TR+PTX. CD8+ T cell activation was measured by perforin staining, monocyte and B cell maturations by cytokine expression array, and NK-mediated cytotoxicity by PI staining of GFP-transfected K562 after 4 hour-incubation. We observed minor functional variations between donor T cells, B cells and monocytes. However, while all NK cells killed GFP-K562 cells as expected, in some donors NK cytotoxicity was specifically inhibited by SKOV3RT co-culture. NK genotyping and phenotyping are ongoing including tumor-dependent NKG2D expression, and PTX-dependent MICA release by tumor cells. These results support the hypothesis that chemoresistant tumors release soluble factors that modulate NK function though specific interactions with innate immune receptor variants.
A novel class of small molecule agonists with preference for human over mouse TLR4 activation (VAC3P.1056)
Authors: Maynard Sean , Marshall Jason , Heeke Darren , Rao Eileen , Hornigold David , McCrae Chris , Fraser Neil , Williams Nicola , King Sarah , Cooper Martin , Frantz Chris , Hajjar Adeline , Woo Jennifer
Published: January 2023

The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.
Therapeutic immunization with a mixture of herpes simplex virus type 1 Glycoprotein D derived “asymptomatic” human CD8+ T cell epitopes decreases spontaneous ocular shedding in latently infected HLA transgenic rabbits: association with low frequency of local PD-1+TIM-3+CD8+ exhausted T cells (VAC4P.1114)
Authors: Benmohamed Lbachir , Srivastava Ruchi , Khan Arif , Nesburn Anthony
Published: January 2023

Most blinding ocular herpetic disease is due to reactivation of herpes simplex virus type 1 (HSV-1) from latency rather than to primary acute infection. No herpes simplex vaccine is currently available for use in humans. In this study, we used the HLA-A*02:01 transgenic (HLA Tg) rabbit model of ocular herpes to assess the therapeutic efficacy of a therapeutic vaccine based on HSV-1 gD epitopes that are mainly recognized by CD8+ T cells from “naturally” protected HLA-A*02:01 positive, HSV-1 seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease). Three ASYMP CD8+ T cell epitopes (gD53-61, gD70-78 and gD278-286) were linked with a promiscuous CD4+ T-cell epitope (gD287-317) to create 3 separate pairs of CD4-CD8 peptides, which were then each covalently coupled to an Ne-palmitoyl-lysine moiety, a toll like receptor 2 (TLR-2) ligand. This resulted in the construction of 3 CD4-CD8 lipopeptide vaccines. Latently infected HLA-Tg rabbits were immunized with a mixture of these 3 ASYMP lipopeptide vaccines, delivered as eye drops in sterile PBS. The ASYMP therapeutic vaccination: (i) significantly reduced HSV-1 shedding detected in tears; (ii) boosted the number and function of local HSV-1-specific CD8+ T cells; and (iii) was associated with fewer exhausted HSV-1 gD-specific PD-1+TIM-3+CD8+ T-cells. The results underscore the potential of an ASYMP CD8+ T cell epitope-based therapeutic vaccine strategy against recurrent ocular herpes.
Reversal of peanut anaphylaxis by retinoic acid-differentiated dendritic cell immunotherapy. IL-27-dependent induction of Foxp3- non-Tr1 regulatory T cells (VAC4P.1100)
Authors: Gordon John , Li Chunyan , Churchman Laura , Dawicki Wojciech
Published: January 2023

Background. Semi-mature IL-10-induced DC (DC10) induce human or mouse effector Th2 cells (Teff) to differentiate into Foxp3+ Treg and thereby induce asthma tolerance. Retinoic acid (RA) and TGFβ-induced gut DC similarly induce tolerance to gut contents via Foxp3+ Treg, while immature hDC-10 induce IL-10-dependent Foxp3- Tr1 cells. Methods. We assessed the abilities of mature RA-induced DC to reverse anaphylaxis sensitivity, characterizing LPS-matured DC-RA and their tolerogenic activities in mouse models of OVA and peanut systemic anaphylaxis. Results. Mature DC-RA strongly expressed CD103 (αE integrin), MHCII, co-stimulatory markers, PDL1 & -L2, ICOSL, TGFβ, IL-27 and the RA-metabolizing enzyme Aldh1A2. They induced Th2 cells to convert into CD25+LAG3+CD49b-IL-10-Foxp3- Treg in an IL-10-independent, IL-27-dependent fashion, although IL-27 was dispensable for Th2 suppression by DC-RA. Wild-type, but not IL-27-/- DC-RA therapy reversed systemic anaphylaxis, including clinical scores, diarrhea, mast cell activation and Th2 responses, and lowered serum allergen-specific IgE and IgG1 levels. Conclusions. Allergen-presenting DC-RA induce food allergen tolerance by activating CD25+Foxp3-, non-Tr1 responses in an IL-27-dependent fashion. This data indicates that, like DC10, DC-RA can also be used for allergic disease immunotherapy, but that these two populations of DCreg induce distinct Treg responses, and this potentially provides us with important options DCreg immunotherapy
Forskolin increases propagation of influenza virus in Madin-Darby canine kidney cells (VAC5P.1118)
Authors: Lee Sang-Yeon , Sohn Sung-Hwa , Park Hye-Lim , Nam Jae-Hwan
Published: January 2023

Influenza virus is a pathogenic negative-sense RNA virus belonging to the Orthomyxoviridae, which causes severe respiratory illness worldwide. A vaccine against the influenza virus is the best way to control the spread of influenza. Recently, a cell-based influenza vaccine was developed to overcome the disadvantages of egg-based vaccines. Here, we investigated whether forskolin, an adenylyl cyclase activator, could increase the output of the cell-based influenza vaccine. We found that forskolin increased the propagation of three influenza virus subtypes, A/H1N1/California/4/09, A/H3N2/Mississippi/1/85, and B/Shandong/7/97, in MDCK cells. We performed a microarray analysis to identify the mechanism whereby forskolin enhances influenza virus replication. We compared a forskolin-treated group with untreated group after infection with influenza virus. The expression of importin α5 and Erk1/2, which can regulate the import and export of viral ribonucleoprotein complex (vRNP), is upregulated in the forskolin-treated group. By contrast, the expression of innate immunity factors and antiviral factors such as TLR3, RIG-I/MDA5, IRF3, MxA and viperin, is downregulated. Taken together, we suggest the reduction of the antiviral response and the increase of translocation of vRNP by forskolin enhance the propagation of influenza virus. Therefore, the addition of forskolin to cultures may be helpful to increase the production efficiency of cell-based vaccines for influenza virus.
SP17 as new immunotherapy target and biomarker for triple-negative breast cancer (VAC5P.1121)
Authors: Chiriva-Internati Maurizio , Mirandola Leonardo , Cobos Everardo , Dahlbeck Scott , Nguyen Diane , Figueroa Jose , Layeequr Rahman Rakhshanda
Published: January 2023

Breast cancer (BC) is a main health concern for women worldwide. Immunotherapies directed against specific tumor-associated antigens enhancing cytotoxic T lymphocyte (CTL)-mediated antitumor responses, have not been equally effective against all BCs: targeted, specific anti-hormonal therapies are now available against the estrogen (ER) and progesterone (PR)-positive BC, as well as HER-2/neu receptor. Triple negative breast cancer (TNBC) displays a negative immunohistologic pattern for ER, PR, and HER-2/neu. As neither of the hormone receptors nor the HER-2 are expressed in TNBC, the hormonal and molecular therapies used in other subtypes are not effective. Current efforts are focused on identifying molecular targets for TNBC. The CTA, SP17, is aberrantly expressed in an array of neoplasms. In this study, we investigated SP17 expression and immunogenicity in triple-negative breast cancer cell lines and tissues, and we tested the efficacy SP17 to stimulate a specific CTL-mediated antitumor response. We showed, for the first time, that SP17 was expressed in TNBC cell lines and primary breast tumor samples. Furthermore, we identified specific anti-SP17 antibodies in TNBC patients’ sera and we were able to generate SP17-specific, HLA class I-restricted, cytotoxic T lymphocytes capable of efficiently killing TNBC cells in vitro. Hence, SP17 may function as a novel biomarker, as well as vaccine target in this disease.
Simultaneous monitoring of in vivo humoral and cellular immune responses in the avian model (VAC5P.1123)
Authors: Erf Gisela , Alaamri Olfat , Jang Hyeonmin , Falcon Daniel , Byrne Kristen , Aguilar Zoraida , Dienglewicz Robert
Published: January 2023

Using the dermis (pulp) of growing feathers (GF) as a test-tissue together with blood sampling, we monitored cellular and humoral immune responses in chickens immunized with 3 different formulations of mouse IgG test-antigen (T-Ag). Test-antigen (26 μg) was injected i.m. in 2 groups of chickens either alone, mixed with Alum adjuvant or conjugated to iron-oxide nanoparticles (10 nm). Group 1 was immunized at 7 weeks and Group 2 at 7 and 11 weeks (n = 18 chickens/group). Antibody production (IgM, IgG) to T-Ag was monitored for 4 weeks following both the primary and secondary i.m. immunizations. To examine the local effector response to T-Ag, T-Ag was injected into 20 GF per bird (10 μL/GF) on Day 10 (Group 1) or Day 5 (Group 2) post-primary or secondary immunization, respectively, and GF collected 0.25, 1, 2, 3, 4, 5, & 7 days post-injection. Analysis of GF pulp cell suspensions by flow cytometry revealed temporal, qualitative and quantitative differences (P < 0.05) in leukocyte-infiltration between immunization treatments and primary/memory effector-responses. Cytokine expression analysis (qRT-PCR) is in progress. ELISA also revealed temporal, qualitative, and quantitative differences (P < 0.05) in the humoral responses to T-Ag following the different primary/secondary i.m. immunizations. Minimally invasive, non-terminal procedures such as sampling of injected GF and blood provides unique insight into cellular and humoral immune activities in the same individual over time.
The inhibition of CD1d-mediated antigen presentation upon an HSV-1 infection is VP22-dependent (VIR1P.1134)
Authors: Liu Jianyun , Gallo Richard , Duffy Carol , Brutkiewicz Randy
Published: January 2023

CD1d-restricted T (NKT) cells are important for controlling a herpes simplex virus (HSV) infection. One of the mechanisms of immune evasion by HSV is to down-regulate CD1d-mediated activation of NKT cells. VP22 is an HSV-1-encoded protein responsible for reorganizing the host cell’s cytoskeletal network and viral spreading. We have shown that modification of the cytoskeleton can alter CD1d-mediated antigen presentation. In this study, we found an HSV-1 lacking VP22 (ΔUL49) was impaired in its ability to inhibit CD1d-mediated antigen presentation compared to wild-type (WT) virus; this was reversed by a repair virus (UL49R) in CD1d-expressing cells. Of importance, the effects of VP22 were shown to be exerted on the T322/S323 dyad, critical residues for the virus to reduced antigen presentation. We further demonstrated that CD1d recycling was inhibited by infection with WT and UL49R, but not ΔUL49 virus. Rather than inhibiting antigen presentation by CD1d, ectopic expression of VP22 in CD1d-expressing cells complemented the VP22-deficient virus in inhibiting antigen presentation. Therefore, VP22 is required (but not sufficient) for the inhibition of CD1d-mediated antigen presentation by an HSV-1 infection.
Viral cyclin control of infection and associated disease (VIR1P.1135)
Authors: Niemeyer Brian , Oko Lauren , Van Dyk Linda
Published: January 2023

Gammaherpesviruses (gHV), including Epstein-Barr virus and Kaposi’s sarcoma associated herpesvirus, are lymphotropic viruses, distinctive in their ability to exploit the lymphocytes as their lifelong reservoir. gHV infection is ubiquitous, and can result multiple pathologies in immune-compromised individuals. Murine gHV68 is homologous to the human viruses, and serves as a small animal model. Previously, our lab demonstrated that a conserved virally encoded homolog of host cyclins, (v-cyclin), is critical for specific aspects of gHV68 infection. A virus lacking the v-cyclin is is dramatically defective in reactivation from latency, viral persistence, and progression of disease. Using an enzymatically marked virus, we now identify infected cells during latent infection with v-cyclin sufficient or deficient viruses. We found that cells infected with or without the v-cyclin are not significantly different in infected B cell subset distribution. However, we found the number of cells with detectable levels of gene expression was significantly depressed in the absence of the v-cyclin. These data suggest that the v-cyclin supports active gene expression during latency, likely corresponding to increased reactivation capacity. This system parallels the complex gene expression programs of Epstein Barr virus, and may permit us to distinguish the specific features of infected cells poised for reactivation and virus propagation, along with their relative roles in virus induced pathology.
BNIP3-mediated mitophagy promotes the generation of natural killer cell memory (VIR1P.1139)
Authors: O'Sullivan Timothy , Sun Joseph
Published: January 2023

Natural killer (NK) cells are innate lymphocytes that possess traits of adaptive immunity such as clonal expansion, contraction, and generation of long-lived “memory” cells, processes poorly understood at the molecular level. Here we find that as proliferating NK cells accumulate dysfunctional mitochondria during viral infection, a protective mitophagy pathway is induced during the contraction phase to promote their survival in a reactive oxygen species (ROS)-dependent manner. Inhibition of mammalian target of rapamycin (mTOR) or activation of AMPK during the contraction to memory phase of the antiviral response increased autophagic activity and enhanced memory NK cell numbers through an Atg3-dependent mechanism. Furthermore, we demonstrate a temporally regulated role for mitophagy-inducing proteins BNIP3 and BNIP3L in the generation of robust NK cell memory. Thus, our study reveals the functional importance of mitophagy during the dynamic response of these cytolytic innate lymphocytes against infection.
Dysregulation of the B cell compartment correlates with development of pulmonary arterial hypertension in a non-human primate model of HIV-infection (VIR1P.1142)
Authors: Kling Heather , Tarantelli Rebecca , Norris Karen
Published: January 2023

HIV-infected persons are at increased risk for co-morbidities associated with advanced age, such as pulmonary arterial hypertension (PAH), though underlying mechanisms are not understood. HIV infection is also associated with B cell dysregulation, which may contribute to tissue damage and premature onset of disease. We have previously shown that simian immunodeficiency virus (SIV) infection of macaques results in increased pulmonary arterial pressures and pulmonary vascular remodeling. In the current study, we examine whether B cell activation/exhaustion is associated with PAH in SIV infection. Rhesus macaques (n=10) were mucosally infected with SIVΔB670. Peripheral blood (PB) and bronchoalveolar lavage fluid (BALF) cells were collected prior to and monthly following SIV infection, stained and analyzed by flow cytometry. Right ventricular systolic pressures (RVSP) were assessed by right-heart catheterization at 3-4 month intervals. Percent of CD95+ B cells was increased in PB and BALF (p<0.0001) following SIV infection and correlated with RVSP (PB, p=0.0317; BALF, p=0.0007). Additionally, elevated frequency of exhausted B cells (CD27-CD10-CD21lo) correlated with RVSP (p=0.043). CD95 expression was strongly correlated with age-associated B cells (ABC; CD21-CD11c+) (p<0.0001), the frequency of which also correlated with RVSP (p=0.017). Collectively, these results support the hypothesis that chronic B cell activation in HIV infection is associated with PAH development.
Heart rate variability in humans infected with the cytomegalovirus (VIR1P.1145)
Authors: Koenig Julian , Jarczok Marc , Rector Jerrald , Fischer Joachim , Sternberg Esther , Thayer Julian
Published: January 2023

Humans infected with the cytomegalovirus (CMV+) have been shown to have a higher risk of cardio-vascular-disease (CVD) and mortality. Previous research supports a neuroimmunomodulatory reflex via vagally mediated t-cells in the splenic nerve end. Vagally-mediated heart rate variability (vmHRV) is a independent predictor of CVD mortality and associated with inflammatory processes. Existing studies investigated the association of CMV infection and HRV only in small samples of CMV infected infants or CMV-infected fetuses. The aim of the present study was to explore potential differences on vmHRV in individuals infected with CMV compared to their non-infected counterparts. 24-hour recordings of HRV were obtained by ECG holter monitoring and the high-frequency (HF) power was derived as index of vagal activity. CMV activity was determined by an enzyme-linked immunosorbent assay on plasma from fasted blood samples. Data from a total of 485 participants (10% females; mean age 46±10 years,) was available for analysis. Participants diagnosed as CMV+ (37%, n = 181) showed significantly (t(1.98) = 0.024, p = .024) lower vmHRV (HF 236±16.5 ms2) compared to individuals without CMV (CMV-, n = 304, HF 284±15.9 ms2).
The role of CD40 in LP-BM5 infection of mixed glial cells (VIR1P.1158)
Authors: Grlickova-Duzevik Eliza , Malon Jennifer , Cao Ling
Published: January 2023

LP-BM5 retrovirus infection is an established murine model of HIV infection in humans due to that susceptible strains of mice (such as B6) develop immunodeficiency syndrome. Previously, we have shown that infected B6 mice developed peripheral neuropathy and CD40 knockout (KO) mice displayed less mechanical hypersensitivity (behavioral sign of peripheral neuropathy) post-LP-BM5 infection. The spinal cord viral RNA levels were significantly lower in CD40 KO mice compared to wild type (WT) B6 mice, which is correlated with decreased lumbar spinal cord cytokines, IL-1-beta, IL-6 and IFN-gamma. It is known that LP-BM5 can infect glial cells directly. We hypothesized that glial cells in CD40 KO mice were less susceptible to LP-BM5 infection. To test this hypothesis, primary spinal cord mixed glial cells from both WT and CD40 KO mice were infected with various doses of LP-BM5. Consistent with our in vivo findings, levels of viral RNA are significantly lower in mixed glial cells from CD40 KO mice than that from WT mice. Further, levels of cytokines, IL-1-beta, IL-6, TNF-alpha, IFN-gamma, and IL-12p40 were measured in the supernatants. LP-BM5 only induced minimal increase of these cytokines and there no significant differences between WT and CD40 KO mice were observed. Our data indicate that glial cells lacking CD40 are more resistant to LP-BM5 infection and this resistance does not seem to be due to the decreased production of proinflammatory cytokines by glial cells.
Temporal transcription analysis of innate immune response to respiratory syncytial virus infection (VIR2P.1164)
Authors: Gao Yajing , Song Ran , Dozmorov Igor , Viswanathan Kasthuribai , Levitz Ruth , Kahn Jeffrey , Wakeland Edward
Published: January 2023

Respiratory syncytial virus (RSV) is a leading cause of severe respiratory disease in infants. Little is known about the cell-intrinsic innate immune responses of RSV infected epithelial cells or responding immune cells. We studied two clinical-isolates from the RSV-B subgroup: NH1125 that induces high cytokine responses (measured by CCL5 and IL-6); and NH1067, which induces low cytokine responses. We used RNA sequencing technologies to profile the transcriptome of non-immune and immune-lineage cells following infection with these two strains. The innate immune response of A549, a human epithelial cell line, was distinct from that of monocyte-derived macrophages and dendritic cells at 2, 4, 8, and 24 hours following infection. These responses were compared at a high multiplicity of infection (MOI=5) for a homogeneous response and low MOI (MOI=0.3), which more closely resembles in vivo conditions, to determine the extent of communication between infected and un-infected cells. Our analyses revealed 6 classes of genes marking the early- and late-stage cellular responses. Pathway analyses identified miRNA let-7f, EIF2 signaling and mitochondrial dysfunction signaling as early activated pathways and pattern-recognition receptor and Interferon pathway at later time points. A cross-comparison also reveals that RSV gene expression and viral replication is greater in A549 cells than macrophages, consistent with a more potent cell-intrinsic innate defense in myeloid lineage cells.
Extra-cellular Matrix protein 1 (ECM1) Ameliorates Experimental Autoimmune Encephalomyelitis (EAE) through Inhibition of MMP9 Activity
Authors: Sun Bing , Su Pan
Published: January 2023

Multiple sclerosis (MS) is the most common inflammatory demyelinating disease of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE), an animal model representing human multiple sclerosis (MS), is mainly mediated by Th1 and Th17 cells. Matrix metalloproteinase-9 (MMP9), a zinc bound protease, plays an important role in the invasion or infiltration of pathogenic Th cells into CNS during MS/EAE. In current study, we find that ECM1 could inhibit MMP9 activity through protein/protein interaction in vitro and ameliorate MOG-induced EAE via blocking MMP9-mediated invasion/infiltration of pathogenic Th cells into CNS in vivo. Giving ECM1 protein treatment on the late periods of pathogenic T cell migration in EAE model, the EAE disease is significantly attenuated. This treatment functions by reduction of infiltrating pathogenic Th cells into CNS, but had no impact on Th1/Th17 cell development in the peripheral lymphoid tissues. Our results suggest that ECM1 is potential an inhibitor to treat EAE disease.
The innate immune receptor NOD2 is protective against autoimmune-induced arthritis in SKG mice
Authors: Napier Ruth J , Vance Emily , Snow Paige , Lee Ellen J , Sakaguchi Shimon , Rosenzweig Holly L
Published: January 2023

Rheumatoid arthritis remains a major public health concern, estimated to affect 1.5 million adults in the U.S. However, the etiology and underlying immunological mechanisms of this disease remain largely speculative. SKG mice harbor a spontaneous mutation in ZAP-70 and develop arthritogenic T cells, leading to chronic, self-sustained joint destruction. Arthritis in SKG mice replicates a current theoretical paradigm that environmental factors, including microbes, activate preexisting autoreactive T cells in genetically predisposed individuals to cause disease. Currently, the mechanisms by which innate immune receptors augment T cell activation as well as disease onset and progression are not understood. Here we sought to characterize the role of the intracellular pattern recognition molecule, NOD2, in disease pathology. Using NIR-spectroscopy and clinical scoring methods we determined that SKG mice lacking NOD2 develop markedly worse arthritis when triggered by fungal associated molecules, including curdlan and zymosan, as compared to SKG/NOD2+/+ mice. Intracellular cytokine staining showed no difference between Th17 and Th1 cytokine production by CD4+ and CD8+ T cell splenocytes between genotypes. Importantly, neutralization of IL-17A reduced disease severity in SKG/NOD2−/− mice to levels of disease exhibited by SKG/NOD2+/+, suggesting IL-17A as the prominent worsening factor in SKG/NOD2−/− mice. Our data suggest that NOD2 serves a protective role against autoimmune arthritis, in part due to regulation of IL-17A. Thus, understanding how microbial recognition pathways like NOD2 factor into the induction and pathology of arthritis is key to the development of effective a therapy.
IL-23 compensates for the absence of IL-1 signaling on CD4 T cells in EAE model
Authors: Mufazalov Ilgiz , Schelmbauer Carsten , Regen Tommy , Kuschmann Janina , Wanke Florian , Gabriel Laureen , Wolk Michaela , Hauptmann Judith , Muller Werner , Pinteaux Emmanuel , Kurschus Florian , Waisman Ari
Published: January 2023

Interleukin-1 (IL-1) is implicated in numerous pathologies, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE), which is believed to be a T cell-mediated disease. However, the lack of specific tools so far limited our understanding of the precise mechanisms of IL-1 action. We therefore generated a mouse strain allowing the conditional deletion of the only known signaling receptor for IL-1, IL-1 receptor type 1 (IL-1R1). Crossing of these mice to CMV-Cre or CD4-Cre transgenic mice resulted in IL-1R1 deletion globally or specifically in αβ T cells, respectively. When we induced EAE in these mice using a standard protocol, we found that Pertussis toxin administration leads to IL-1R1 dependent recruitment of myeloid cells to the draining lymph nodes and subsequent IL-1β production. This myeloid-derived IL-1β did not vitally contribute to the generation and plasticity of Th17 cells in general, but rather promoted expansion of a GM-CSF+ Th17 cell subset, thereby enhancing its encephalitogenic potential. Mice lacking IL-1R1 specifically in T cells, in contrast to complete IL-1R1 deficient animals, were not resistant to active EAE induction but rather developed a mild form of paralysis. CNS-infiltrating CD4 T cells included GM-CSF+ Th17 cells yet in reduced frequencies and numbers compared to wild type control animals. Moreover, the pathogenicity of IL-1R1 deficient T cells was fully restored by IL-23 polarization in vitro prior to transferring of sufficient numbers of these IL-17A+ cells into Rag1−/− recipient mice. Therefore, our data emphasize an important but dispensable function of IL-1 signaling in T cell mediated neuropathology.
Mercury modulates splenic immune cell proportion in mice
Authors: Jin Guang-Bi
Published: January 2023

Mercury is one of the widely spread environmental pollutants, inducing autoimmunity characterized by a T cell-dependent polyclonal activation of B cells. However, its mechanisms remain unclear. In this study, we investigated the role of mercury on the immune cell proportion. Eight-week old BALB/c mice were injected (s.c.) three times a week with HgCl2 (1 mg/kg body weight) throughout the duration of the experiment. Control mice received a sterile saline in the same manner. At 2, 3, 4, 5, 10 and 12 weeks after first HgCl2 injection, single splenic cells were stained with fluorochrome-conjugated mAbs for flow cytometer analysis. Our results showed that mercury treatment decreased the percentage of Treg (CD4+CD25+FoxP3+) in the spleen at time-points of 3–10 weeks, while increase in the percentage of B cells (CD19+CD3−) at 4–12 weeks. Mercury treatment did not affect the proportion of dendritic cells (DCs) in the spleen, including myeloid DCs (CD11chighMHCII+B220−) and plasmacytoid DCs (CD11clowmPDCA1+B220+). The dose and frequency of our HgCl2 injection did not affect body weight in the BALB/c mice. However, mercury transiently increased spleen weight at 2 and 3 weeks. Our findings suggest that mercury reduces Treg in the spleen, which may contribute to mercury-mediated autoimmunity.

Funding: Supported by the Grant from National Natural Science Foundation of China (81460503), and Research Funds from Jilin Provincial Science & Technology Department (20150101207ZC), Education Department of Jilin Province (JIJIAOKEHEZI2015No.3), Health and Family Planning Commission of Jilin Province (2014Z089).
The Ena/VASP family of cytoskeletal proteins selectively regulates activated T cell trafficking by promoting the diapedesis step of transendothelial migration.
Authors: Jacobelli Jordan , Estin Miriam L. , Traxinger Brianna , Fisher Marlie H. , Friedman Rachel S.
Published: January 2023

Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins that have been implicated in T cell actin cytoskeleton remodeling in response to T cell receptor signaling. However, the role of these proteins in T cell motility, transendothelial migration, and in vivo trafficking remained unknown. We investigated the role of EVL and VASP in T cell trafficking and found that these cytoskeletal effectors are selectively required for activated but not naïve T cell trafficking to the lymph nodes and spleen. Moreover, in a model of Multiple Sclerosis, EVL/VASP deficiency severely impaired activated T cell trafficking to the inflamed central nervous system (CNS) of mice with experimental autoimmune encephalomyelitis (EAE). Deletion of EVL and VASP resulted in impaired Very Late Antigen-4 (VLA-4) expression and function, but unexpectedly did not cause alterations in shear-resistant adhesion and motility of T cells on primary brain microvascular endothelial cells in vitro. Instead, deletion of EVL and VASP caused a significant impairment in the ability of T cells to initiate and complete the diapedesis step of transendothelial migration. Furthermore, upon blockade of VLA-4, T cell diapedesis became equivalent between control and EVL/VASP-deficient T cells. Together, these data suggest that that EVL and VASP selectively regulate activated T cell trafficking by specifically mediating the diapedesis step of transendothelial migration in a VLA-4-dependent manner.
NET formation induced by gout-associated monosodium urate crystals requires IL-8-mediated neutrophil migration and is inhibited by the P2Y6 receptor antagonist MRS2578.
Authors: Sil Payel , Hayes Craig , Reaves Barbara , Sokolove Jeremy , Rada Balazs
Published: January 2023

Human neutrophils generate inflammatory responses within the joints of gout patients upon encountering Monosodium Urate (MSU) crystals. Studies (including ours) show that neutrophil extracellular traps (NETs) are found abundantly in the synovial fluid (SF) of gout patients and are crucial for the onset of the autoinflammatory cascade. Our goal is to shed light on possible roles of purinergic signaling and neutrophil migration in mediating in vitro NET formation induced by MSU crystals.

Our data show that gout SF supernatants have significantly higher levels of the neutrophil granule markers, myeloperoxidase (MPO) and human neutrophil elastase (HNE), and NETs than SF specimens from MSU-negative osteoarthritis patients. MSU crystal-triggered NETs in human neutrophils contain active DNA-bound MPO and HNE. MRS2578, a purinergic P2Y6 receptor antagonist, reduces the release of extracellular DNA, MPO and HNE in MSU crystal-stimulated neutrophils. Releases of MPO, HNE and NETs were measured by commercial ELISA and our proprietary NET-DNA ELISA assays. We found that MRS2578 reduces MSU crystal-induced IL-8 release, neutrophil chemotaxis and characteristic formation of DNA aggregates. The store operated calcium entry (SOCE) channel inhibitor (SK96365) blocked MSU crystal-induced NET release. Our results indicating that the P2Y6-SOCE-IL-8 axis is involved in MSU crystal-induced NET formation provide a novel mechanism potentially relevant in gout inflammation. Our work on the role of the P2Y6-SOCE-IL-8 axis in MSU crystal-triggered NET formation could potentially lead to a better understanding of the mechanism of gout joint inflammation and, thereby, help in improving the care of gout patients.