bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunization with E1A expressing tumor cell line enhances antigen specific tumor immunity (74.5)
Authors: Korrer Michael , Zhang Yuwen , Zhang Jian , Routes John
Published: January 2023

The stable expression of Adenovirus (Ad) E1A in tumor cells reduces tumorigenicity by 1,000 fold, an effect dependent on the elicitation of an NK cell and T cell mediated antitumor response. We generated B6 fibrosarcoma cell lines (MCA tumor cells) that stably expressed ovalbumin (MCA-OVA) or full-length E1A fused to ovalbumin (MCA-E1A-OVA) to test the hypotheses that E1A could function as a molecular adjuvant to induce a robust anti-tumor immune response to other weakly or non-immunogenic tumor antigens. MCA E1A-OVA was more susceptible to killing by NK in vitro and 1000 fold less tumorigenic than MCA or MCA-OVA in vivo. Immunization with 1X10^5 live MCA-OVA or MCA- E1A-OVA tumor cells elicited comparable numbers of IFNγ- and TNFα -producing OVA specific CD8 T cells that had similar cytolytic activity against OVA-expressing target cells in in vivo CTL killing assays. Immunization of MCA-OVA or MCA-E1A-OVA cells also increased the number of MCA-OVA cells required to form tumors by 1,000 fold in an anatomically distant site. However, primary tumors developed at the site of immunization with MCA-OVA but not MCA-E1A-OVA cells. These data demonstrate that immunization of live MCA-E1A or MCA-OVA tumor cells can elicit a robust, systemic anti-tumor immune response. However, primary tumor progression occurs at the site of inoculation following immunization with MCA-OVA cells despite the induction of an anti-tumor immune response.
Granulocytic and monocytic populations of tumor-infiltrating myeloid-derived suppressor cells (MDSC) suppress T cell proliferation through independent mechanisms (74.7)
Authors: Raber Patrick , Wyczechowska Dorota , Rodriguez Paulo
Published: January 2023

Granulocytic and monocytic myeloid-derived suppressor cells (G-MDSC and M-MDSC) are hallmarks of chronic inflammatory environments and major mediators of T cell suppression in cancer. We aimed to determine the mechanisms by which tumor-associated G-MDSC and M-MDSC suppress T cell proliferation. Our results suggest that MDSC isolated from lung carcinoma-bearing mice impaired the proliferation of activated T cells thorough an arrest in the G0-G1 phase of the cell cycle. This effect was not caused by a decreased T cell activation, as an increased expression of the early activation markers CD25 and CD69, and high production of IL-2 were detected in T cells co-cultured with MDSC. The anti-proliferative effect induced by MDSC was prevented after the addition of inhibitors of arginase and inducible nitric oxide synthase (iNOS). Interestingly, arginase I was exclusively expressed in G-MDSC, whereas iNOS was expressed mainly in M-MDSC. Additional results showed that G-MDSC produced higher levels of reactive oxygen species (ROS), whereas M-MDSC produced more peroxynitrites (PNT). Expression of gp91phox in G-MDSC regulated their production of ROS. In addition, M-MDSC-induced suppression was overcome using a PNT scavenger or an inhibitor for S-nitrosylation. These results suggest that sub-populations of MDSC suppress T cell proliferation through independent mechanisms. Targeting of these pathways may enable the design of new therapeutic approaches to reverse T cell tolerance in cancer.
Induction of differentiation of hematopoietic cells by the vaccine adjuvants (75.10)
Authors: Yang Ya-Wun , Chang Chih-Feng
Published: January 2023

Vaccine adjuvants have been used to enhance the immune responses against the co-administered antigens. The mechanisms of action by the adjuvants however remain poorly understood. We attempt to investigate in this study the effect of vaccine adjuvants on differentiation of the hematopoietic system. C57BL/6J mice were vaccinated with ovalbumin (OVA) in L121-adjuvant, an emulsion adjuvant consists of Pluronic L121 that previously shown to stimulate cytotoxic lymphocyte (CTL) effect. Cells were isolated from bone marrow and thymus after vaccination, followed by flow cytometric analysis. Our data showed that treatment of animals with L121-adjuvant skewed the differentiation of hematopoietic cells, resulting in a significant increase of the CD11b+ population at the expense of lineage (lin)+ cells, attributing mainly to a dramatic reduction of the B220+ population. A considerable increase of Lin-/Sca1+/c-Kit+ (LSK) hematopoietic stem/progenitor cells was observed in the bone marrow of immunized mice, suggesting an induction of enhanced cell expansion by the vaccine adjuvant. Treatment of animals with L121-adjuvant also affects the differentiation of cells in the thymus with a decrease of the CD4+/CD8+ double-positive (DP) cells, and a concurrent increase of the CD4+ and CD8+ single positive (SP) populations. These experimental results suggested the differentiation effect of vaccine adjuvants on the hematopoietic system, accounting for the enhanced immunity after immunization.
Human recombinant lactoferrin as a new adjuvant in specific DNA vaccination against ErbB2 (75.2)
Authors: Spadaro Michela , Casule Gabriele , Montone Monica , Merighi Irene , Pericle Federica , Cavallo Federica
Published: January 2023

Lactoferrin triggers several innate immunity mechanisms. Repeated oral administrations of a recombinant human lactoferrin (Talactoferrin, TLF, Agennix, Houston, TX) hamper the growth of both transplantable tumors and autochthonous mammary carcinomas arising in BALB/c mice transgenic for the rat ErbB2 oncogene (BALB-neuT mice). To evaluate the mechanisms that delay the tumor growth and enhance the immune response elicited by a DNA vaccine against rat ErbB2 oncogene, BALB-neuT mice received by gavage PBS or TLF (1 g/kg) once daily, 5 days per week, 3 weeks on and 1 week rest for seven cycles. Following oral TLF we observed: decrease of serum IL4 and IL5; increase of serum IL12 and IFNγ; decrease of immature myeloid cells in tumor draining lymph nodes. When BALB-neuT mice receiving oral TLF were vaccinated with 50 µg of DNA plasmid coding for the extracellular and transmembrane domain of rat ErbB2 we found: extended tumor-free survivals; increased overall survivals; decrease in the number of tumors/mouse; higher titers of vaccine-induced anti-ErbB2 antibodies; similar cytotoxicity activities against ErbB2+ tumors. These findings suggest that TLF is an interesting new adjuvant in DNA vaccination against ErbB2.
Exosomes loaded with α-galactosylceramide amplify antitumor immunity via iNKT cells (75.5)
Authors: Gabrielsson Susanne , Gehrmann Ulf , Hiltbrunner Stefanie , Karlsson Mikael , Näslund Tanja
Published: January 2023

Exosomes are nanosized membrane vesicles with endosomal origin that can be isolated from a variety of cell types and serve as means of communication between cells. Dendritic cell (DC) derived exosomes can induce adaptive, as well as innate immune responses and have been tested as therapeutic agents in cancer. Here, we investigated whether exosomes can be loaded with the iNKT cell activating ligand α-galactosylceramide (αGC) and whether exosomes loaded with both αGC and the protein antigen ovalbumin (OVA) could amplify both innate and adaptive immune responses. We show that DC-derived exosomes express CD1d and that αGC-loaded exosomes (Exo-αGC) activate iNKT cells in vitro in a CD1d-dependent manner. In vivo, αGC- and OVA-loaded exosomes (Exo(αGC-OVA)) induced NK cell and γδ T cell responses secondary to iNKT cell activation and increased anti-OVA T and B cell responses. Moreover, treatment of tumor-bearing mice with Exo(αGC-OVA) slowed down tumor growth and led to immune cell infiltration into tumor tissue in a OVA-expressing mouse melanoma model. Our results show that activation of iNKT cells by exosomes amplifies both innate and adaptive immune responses in a way that might be beneficial for cancer immunotherapy.
Scavenger Receptor, SRECI activates dsRNA-mediated TLR3 innate immune responses (76.1)
Authors: Murshid Ayesha , Gong Jianlin , Calderwood Stuart
Published: January 2023

SRECI is a scavenger receptor expressed by immune cells and has a role in CD8+ T cell immunity. SRECI can also modulate the function of Toll like receptors which have essential roles in innate immunity. Here we show that SRECI enhances double stranded RNA (dsRNA)-mediated Toll like receptor-3 (TLR3) activation. Viral double stranded RNA (dsRNA) is a pathogen associated molecular pattern (PAMP) indicating viral infection. We found that human monocyte/macrophage THP1 cells as well as bone marrow derived macrophages from mice exhibited increased responses to polyinosine-polycytidylic acid (PolyI:C) and enhanced production of inflammatory cytokines with overexpressed SRECI. Our data also show that intracellular/endocytic TLR3 can directly interact with SRECI in the presence of Poly I:C. Internalized ligand along with TLR3 co-localize in the endosomes of macrophages and TLR3-expressing Hek293 cells. In the absence of Poly I:C there was no such co-localization between these two receptors. We also found that SRECI can stimulate dsRNA mediated TLR3 activation of downstream signaling via NFkB, MAPK and IRF3 molecules which lead to the activation of inflammatory cytokines. We also hypothesize that SRECI is a cell surface receptor capable of internalizing Poly I:C as well as activating TLR3 mediated downstream cytokine production in macrophages.
B cells in primitive vertebrate act as pivotal antigen presenting cells in priming adaptive immunity (P5035)
Authors: Zhu Lv-yun , Nie Li , Shao Tong , Dong Wei-ren , Xiang Li-xin , Shao Jian-zhong
Published: January 2023

The long-held paradigm that B cells are unable to phagocyte large particles has been broken by a new discovery that teleost B cells have potent phagocytic and microbicidal abilities. This finding provides preliminary clues that B cells in primitive vertebrates might act as pivotal antigen presenting cells (APCs) in priming adaptive immunity. In this study, we provide evidence for this suggestion in zebrafish model. By in vitro antigen presentation and in vivo adoptive transfer assays, we found that zebrafish B cells have potent capacity to present both soluble and particulate antigens to prime naive CD4+ T cells. The CD86 and CD83 co-stimulatory molecules were identified to be essential for this process. Besides, IL-4 was found to be able to dramatically promote the B cell-mediated CD4+ T cell priming by up-regulating the expression of CD86, CD83 and MHC II molecules on B cells. Our results suggest that B cells in primitive vertebrates were important professional APCs for the initiation of adaptive immunity, whose functional characterization resembles the dendritic cells (DCs). The CD86 and CD83 were two ancient co-stimulatory molecules originating from teleost fish, whose regulatory functions were conserved from fish to mammals throughout vertebrate evolution. These results benefit to the better understanding of the evolutionary history of B subsets such as the origin of B1 cells, as well as the origin of immune-regulatory signaling networks in adaptive immunity.
Enhancement of antigen presenting ability in the leukemic plasmacytoid dendritic cell line (PMDC05) by lentiviral vector-mediated transduction of CD80 gene (P5059)
Authors: Takahashi Masuhiro , Yamahira Akie , Iwabuchi Minami , Satoh Naoya , Uchiyama Takayoshi , Hashimoto Shigeo , Bogan Brooke , Faure Emmanuelle , Kasahara Noriyuki , Narita Miwako
Published: January 2023

[Aim] PMDC05, a leukemic plasmacytoid dendritic cell (pDC) line which was established from CD4+CD56+ leukemia cells in our laboratory, showed a capacity of generating antigen-specific cytotoxic T lymphocytes (CTLs). In order to enhance an antigen presenting ability of PMDC05, PMDC05 was transduced with CD80 gene by lentiviral vector, which was named as PMDC11. We investigated whether PMDC11 could enhance allogeneic T cell response. [Materials and Methods] Surface antigen presentation-associated molecules and antigen presenting ability were analyzed in PMDC05 and PMDC11 with or without stimulation by phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore (CI) by flow cytometry or mixed leukocyte culture (MLC). [Results] CD80 gene transduction induced a definite up-regulation of CD1a and HLA-DR in addition to CD80. In original PMDC05 cells, which had antigen presenting ability without any stimulation, PMA/CI could enhance the ability significantly. PMDC11 cells without stimulation had an antigen presenting ability equivalent to PMA/CI-stimulated PMDC05. Measurement of IFN-γ in supernatant of MLC demonstrated that PMDC11 stimulated allogeneic lymphocyte to produce much more IFN-γ compared with PMDC05. [Conclusion] Our data suggested PMDC11 cells, which were established by transducing CD80 gene into PMDC05 cells, could be efficiently applied as antigen presenting cells in adoptive cellular immunotherapy for tumors and severe infections.
Metformin reduces gene expression of adipogenesis and inflammatory cytokines through the modulation of T cell priming in obese-induced Type II diabetic mice (P5063)
Authors: Shin Seulmee , Hyun Bobae , Lee Aeri , Lee Sungwon , Kong Hyunseok , Han Shinha , Song Youngcheon , Kim Kyungjae
Published: January 2023

Inflammation is increasingly regarded as a key process underlying metabolic diseases in obese individuals where obese adipose tissue shows features characteristic of active local inflammation. Metformin is a biguanide derivative used in the treatment of T2D and one of the world's most widely prescribed drugs and have been suggested that metformin has anti-inflammatory properties. Here we show that metformin efficiently attenuates the metabolic disorder in DIO mice and suppresses MHC-restricted antigen presentation in dendritic cells. This study was to investigate the therapeutic effect of metformin on diabetic symptoms in DIO mice. Metformin decreased aipogenesis related gene, FAS, SCD-1, GPAT, SREBP1a, but also suppressed gene expression of innate cytokines than control mice. Metformin decreased both class I- and class II-restricted presentation of exogenous OVA in dendritic cells. Metformin also decreased expression of the co-stimulatory molecules, ICAM-1 and B7-1/-2, and MHC class I and II molecules but not phagocytic activity of DCs. Metformin decreased MHC class II-restricted exogenous antigen presentation in peritoneal macrophages in vivo. These results show that metformin attenuates immune responses followed by decreases MHC-restricted presentation of exogenous antigen in DCs via intracellular processing events. Theses finding suggest that modulation of the antigen presentation pathway could provide a novel means for regulating T cell responses in metabolic diseases.
Analysis of HLA-DQ protein and peptide interaction in association to auto-immune mechanism of narcolepsy. (P5069)
Authors: Sophia Hsuan-Jung Chen , Hiroko Miyadera , Katsushi Tokunaga
Published: January 2023

Human narcolepsy is a sleep disorder characterized by excessive daytime sleepiness (EDS), cataplexy, and abnormalities of rapid eye movement (REM). Strong HLA class II association was determined where the narcoleptic individuals of diverse ethnic backgrounds share a specific HLA class II haplotype, DQA1*01:02-DQB1*06:02. It has been also reported that hypocretin in the CSF of narcoleptic patients are significantly decreased and that hypocretin producing cells are disrupted in patients’ brain. We hypothesized that hypocretin or other proteins specifically expressed in hypocretin producing cells in the hypothalamus, are presented by HLA-DQA1*01:02-DQB1*06:02, and induce activation of self-reactive T cells. To analyze the peptide-binding reperitore of HLA-DQA1*01:02-DQB1*06:02, we transduced murine fibroblast cells with HLA-DQA1*01:02-DQB1*06:02 (suscceptible), DQA1*01:03-DQB1*06:01 (resistant), DQA1*01:03-DQB1*06:03 (resistant), and DQA1*01:02-DQB1*06:04 (neutral) alleles by retrovirus-vectors and confirmed stable DQ proteins expression. Using these cell lysates we analyzed binding affinity of synthetic peptides designed for hypocretin-A, -B, and other proteins expressed in hypocretin producing cells.
Analysis of HLA-DR protein and peptide interaction in association to type 1 diabetes in Japanese. (P5071)
Authors: Chen Cindy Chia-Jung , Miyadera Hiroko , Tokunaga Katsushi
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disorder that mediates destruction of insulin-producing pancreatic cells. Previous genetic association studies reported HLA II to be the major factor for increased susceptibility to T1D. Yet, knowledge remained limited on disease mechanism in Japanese. The purpose of current study is to elucidate immunological mechanism of risk and protective alleles and to determine the peptide binding repertoire of T1D susceptible DR alleles in the Japanese for Zinc Transporter 8 (ZnT8), one of the established auto-antigenic peptides. We expressed DR9 (DRA*01:01-DRB1*09:01), DR4(DRA*01:01-DRB1*04:05), DR8 (DRA*01:01-DRB1*08:02) contributing to susceptibility to T1D in the Japanese and DR15 (DRA1*01:01-DRB1*15:01) that are associated with protection in mammalian cells. A plate based peptide binding assay system was established using nickel coated microtiter plates to measure the binding of synthetic peptides to recombinant DR proteins. Established DR9 binding motif was searched in ZnT8 sequences. Binding characteristics of risk and protective DR allele products including DR9 are examined in utilization of a 20mer ZnT8 peptide library.
Lysosomal transporter SLC15A4 regulates Toll like receptor 7/9-mediated antibody production (P4195)
Authors: Kobayashi Toshihiko , Toyama-Sorimachi Noriko
Published: January 2023

Solute Carrier family (SLC) 15A4 is known to be a proton-coupled transporter expressed by the immune and nervous systems, specifically carrying free histidine and certain di- or tri-peptides. Previous studies reported that SLC15A4 gene is associated with the autoimmune disorders such as inflammatory bowel diseases and systemic lupus erythematosus. However, the precise mechanism of how this transporter contributes to pathogenesis of these diseases remains unclear. We have investigated the functional involvement of SLC15A4 in the innate immune system, as this transporter is highly expressed in innate immune cells, and is known to reside on the lysosomal membranes. We previously reported that SLC15A4 plays crucial roles in TLR9- and NOD1-mediated cytokine production in dendritic cells (DCs). Here we show that SLC15A4 is also important for TLR7-triggered immune responses not only in DCs but also in B cells. We found that SLC15A4 is also expressed in B cells, and activation of naïve SLC15A4-deficient B cells was attenuated when triggered by TLR7 or TLR9 ligand, showing impaired IFN-beta or IL-6 production. Moreover, SLC15A4-deficient B cells failed to produce IgG2c antibody in vitro and in vivo. Although further investigation is necessary to understand the precise mechanism, our findings suggest that TLR7 or 9 activity, such as antibody production could be regulated in lysosomes by SLC15A4.
Interferon regulatory factor 8 deficiency causes phenotypic and functional changes in myeloid cells inside and outside the CNS (P4145)
Authors: Campbell Iain , Terry Rachael , Xie Rui Dan , Almolda Beatriz , Villacampa Nàdia , Minten Carsten , King Nicholas , Gonzalez Berta , Castellano Bernardo
Published: January 2023

We identified IRF8 as a constitutive and IFN-γ-stimulated nuclear factor in microglia of the CNS. IRF8 is a transcription factor with a key role in the cellular response to IFN-γ but is also pivotal in myeloid cell differentiation. Here we employed IRF8 KO mice to determine the role of IRF8 in microglial development and function in the healthy CNS and in the microglial response to injury (facial nerve axotomy [FNA]) or infection (West Nile Virus [WNV] encephalitis). Microglia were present in the CNS of IRF8 KO mice but showed altered morphology and significantly reduced surface area and had alterations in a number of key markers including reduced levels of Iba1, CCR2 and CCR5 but increased levels tomato lectin binding. Following FNA the microglial response was markedly delayed and attenuated in IRF8 KO mice and the wrapping of motor neuron cell bodies by microglia involved in synaptic stripping and phagocytosis was incomplete. Finally, IRF8 KO mice had increased susceptibility to WNV accompanied by a marked reduction in both microglial cell activation and leukocyte accumulation in the brain. In the periphery, ly6Cint monocytes were increased in number but exhibited defective trafficking to the infected CNS while ly6Chi monocytes were almost completely absent. In conclusion: (1) IRF8 is a major intrinsic determinant of the microglial phenotype in the healthy and the diseased CNS, and (2) IRF8 has a crucial role in the development and trafficking of inflammatory monocytes.
The regulation of plasma cell function through soluble mediators of the CD28/B7 pathway (P1005)
Authors: Holthausen David , Njau Modesta , Jacob Joshy
Published: January 2023

CD28 is the secondary co-stimulatory molecule that is crucial for optimal activation of naïve T cells and their differentiation into effector T cells. Activation of T cells without co-stimulation, renders the cells anergic. The influence of CD28 is not limited solely to T cells. CD28 as well as its ligand B7.1/2 are found on short- and long-lived plasma cells. We have demonstrated that signaling via CD28 acts as a negative regulator of plasma cell function. The removal of the CD28 signal leads to a hyperactive plasma cell state, with enhanced longevity, and increased antibody production in individual plasma cells. Interestingly, soluble forms of CD28, B7 and CTLA-4, are seen in the sera of patients with chronic human diseases, including autoimmunity, allergic asthma, and chronic hepatitis. We hypothesize that soluble CD28/B7 mediators will transiently block the CD28/B7 interaction on plasma cells and enhance autoantibody production. The research presented will compare the appearance and time course of the soluble mediators in mouse models of chronic and acute disease.
A lamprey monoclonal VLR antibody recognizes a novel plasma cell antigen (P3020)
Authors: Yu Cuiling , Herrin Brantley , Jaye David , Cooper Max , Ehrhardt Götz
Published: January 2023

Variable lymphocyte receptor B (VLRB) antibodies of the jawless sea lamprey are composed of leucine-rich repeat (LRR) units instead of the Ig-based units of conventional mammalian antibodies. Structural analyses of three monoclonal VLRB antibodies paired with their cognate antigens indicate that antigens bind to variable residues in the beta-sheets located on the inner concave surface of the solenoid VLRB protein and to a flexible loop structure protruding from the C-terminal LRR. We recently demonstrated that VLRB antibodies can be used in standard laboratory techniques such as flow immunocytometry, western blotting, immunoprecipitation and mass spectrometry-based protein identification. In an effort to identify novel biomarkers on human plasma cells, we screened a VLRB library generated from lamprey larvae immunized with human bone marrow of multiple myeloma patient and isolated a monoclonal VLRB antibody (MM3) that selectively recognized plasma cells from myeloma patients, normal individuals and two non-human primates, Rhesus macaque and Sooty mangabey monkeys. Surprisingly, screening of a diverse panel of human cell lines indicated expression of the MM3 antigen by two of nine B cell lines, but not by plasmacytoma cell lines. Trypsin and periodate sensitivity of the MM3 epitope suggested the glycoprotein nature of the cell membrane antigen. We conclude that the recombinant VLR MM3 antibody recognizes a novel plasma cell marker.
Polymorphisms of the transforming growth factor-beta 1 gene in relation to idiopathic recurrent miscarriage (P6248)
Authors: Almawi Wassim , Finan Ramzi , Al-Zaman Intissar , Mustafa Fekria
Published: January 2023

Transforming growth factor, β1 (TGFB1) plays a significant role in the pregnancy outcome. We investigated the association of TGFB1 exon 1 (rs1800471, rs1800470) and promoter region (rs1800469, rs1800468) polymorphisms with recurrent pregnancy loss (RPL) in 675 Tunisian women: 304 women with a history of three consecutive pregnancy loss of unknown etiology with the same partner, and 371 age-matched multiparous control women. TGFB1 genotyping was done by TaqMan assays. Higher minor allele frequency (MAF) for rs1800471 (i.e. +915G/C or R25P) (P < 0.001), but not for rs1800470, rs1800469, or rs1800468 was found in cases compared to controls. Significant difference in the distribution of rs1800471 genotypes was seen between RPL cases and control women, irrespective of the genetic model used. Increased RPL risk was seen with rs1800471 allele C in both heterozygous and even more so in the homozygous state, thus establishing a dose-dependent effect. Haploview analysis revealed differential linkage disequilibrium between TGFB1 SNPs analyzed. TGFB1 haplotypes analysis identified 8 common haplotypes (rs1800471/rs1800470/rs1800469/rs1800468) with three (GTTG, Pc = 0.014; CCTG, Pc = 0.003, and CTCG, Pc = 0.01) being positively associated with RPL, while one (GCCG, Pc = 0.009) was negatively associated with RPL. This study provides the first evidence that TGFB1 genotype is possibly associated with RPL.
ATF4 is induced by ROS and regulates Th1 and Th17 cells (P6219)
Authors: Yang Xi , Gao Xin , Zhu Yibei , Li Gang , Zhang Xueguang , Xiao Guozhi , Cao Huiling , Chen Xinchun , Yang Qianting , Lu Binfeng
Published: January 2023

Reactive oxygen species (ROS) play a major role in the pathogenesis of chronic inflammatory and autoimmune diseases. It is well established that ROS modulate T cell-mediated immune responses. However, little is known about underlying molecular mechanisms of how ROS regulate T cell immune responses and how T cells adapt to high levels of ROS. We demonstrated that ROS induced ATF4 in activated T cells. ATF4 is a basic leucine-zipper (bZip) transcription factor, which regulates cellular redox state and amino acid metabolism. We found that, in the high ROS condition, ATF4-/- Th1 cells failed to produce IFN-γ whereas WT Th1 cells still made large amounts of IFN-γ. Inhibition of ROS by addition of antioxidants recovered the IFN-γ production by ATF4-/- Th1 cells, suggesting ATF4 is critically required for the function of Th1 cells in the presence of high levels of ROS. ATF4-/- antigen presenting cells (APC) promoted IL-17 production, suggesting ATF4 plays an important role in APCs in suppressing Th17-driving cytokines. In addition, we demonstrated that experimental allergic encephalomyelitis (EAE) was exacerbated in ATF4 deficient mice compared with wild type (WT) mice. The number of autoreactive Th1 cells was decreased whereas that of autoreactive Th17 cells was elevated in ATF4-/- mice compared with WT mice. Our study establishes that ATF4 promotes Th1 and suppresses Th17 immune responses and likely plays an important role in autoimmunity and cancer immunology.
TRPM2 links oxidative stress to the NLRP3 inflammasome activation (P1268)
Authors: Zhong Zhenyu , Zhai Yougang , Liang Shuang , Mori Yasuo , Han Renzhi , Sutterwala Fayyaz , Qiao Liang
Published: January 2023

Exposure to particulate crystals, either environmental irritants or endogenous metabolites, can induce oxidative stress in phagocytes, which in turn triggers NLRP3-inflammasome mediated interleukin 1β (IL-1β) secretion to initiate undesired inflammatory responses associated with many autoinflammatory and metabolic diseases. Although mitochondrial reactive oxygen species (ROS), accumulated under oxidative stress, has been shown essential for NLRP3 inflammasome activation, little is known how ROS signals the assembly of the NLRP3 inflammasome. Here, we first identified liposomes as a novel group of particulate activators for NLRP3 inflammasome, and further demonstrated that this immune recognition process also requires mitochondrial ROS. Moreover, we found that stimulation of particulate substances (liposomes and crystals) induced a ROS-dependent calcium influx via the TRPM2 ion channel that directs the assembly of NLRP3 inflammasome. Macrophages deficient in TRPM2 displayed drastically impaired NLRP3 inflammasome activation and IL-1β secretion in response to liposomes/crystals. Consistently, Trpm2−/− mice were resistant to crystals/liposomes-induced, IL-1β-mediated peritonitis in vivo. Taken together, these results identify TRPM2 as a novel player that links particulates-induced oxidative stress to the NLRP3 inflammasome activation. Thus, targeting TRPM2 may be beneficial for inflammatory disorders associated with deranged NLRP3 inflammasome activation.
Kinetics of NLRP3 inflammasome independent and dependent mitochondrial damage during inflammasome activation (P1276)
Authors: Heid Michelle , Watkins Simon , Salter Russell
Published: January 2023

The NLRP3 inflammasome is a protein complex that plays a central role in innate immunity and is involved with caspase-1 activation. In murine bone marrow derived macrophages (BMDM), NLRP3 inflammasome activation can lead to the processing and secretion of IL-1 beta, as well as pyroptotic cell death. Pore forming toxins, such as tetanolysin O (TLO), as well as the potassium ionophore nigericin, stimulate the NLRP3 inflammasome. In response to treatment with nigericin, we have observed that mitochondrial depolarization occurs independent of the presence of the NLRP3 inflammasome, however the loss of mitochondrial membrane integrity is NLRP3 dependent. This loss of mitochondrial integrity is preceded by NLRP3 dependent lysosomal membrane permeability. Hence, we have shown kinetically that NLRP3 inflammasome independent damage to the mitochondria precedes NLRP3 inflammasome dependent organelle damage and eventual cell death. The kinetic information provided via these data suggests a multi-step process involving both NLRP3 dependent and independent events during inflammasome activation.
Bacterial RNA:DNA hybrids: novel activators of the NLRP3 inflammasome (P1250)
Authors: Kailasan Vanaja Sivapriya , Rathinam Vijay , Kalantari Parisa , Fitzgerald Katherine , Leong John
Published: January 2023

The NLRP3 inflammasome is essential for IL-1β production in response to Enterohemorrhagic Escherichia coli (EHEC). To determine the mechanism of NLRP3 inflammasome activation by EHEC, we screened EHEC mutants lacking various virulence genes and found that none of them are essential for inflammasome activation. However, lysosomal acidification following infection was found to be essential for IL-1β production implying that bacterial lysis and its products such as nucleic acids play a role. Consistent with this, pretreating macrophages with RNase A specifically reduced EHEC-induced IL-1β response. Also, EHEC RNA was found to be co-localizing with NLRP3 in the cytosol, revealing a potential mechanism for NLRP3 activation. Interestingly, treating cells with RNase H, which degrades RNA of RNA:DNA hybrids, also markedly reduced IL-1β activation indicating that RNA:DNA hybrids formed as replication and transcription intermediates in bacteria also play a role in NLRP3 activation. Supporting this idea, synthetic EHEC RNA:DNA hybrids potently induced IL-1β production in a NLRP3-dependent manner. Importantly, an E. coli rnhA mutant incapable of producing RNase H induced greater than wild type levels of IL-1β and complementing this mutant with rnhA reduced IL-1β to wild type levels. Altogether these findings identify bacterial RNA:DNA hybrids as novel NLRP3 activators and demonstrate that multiple nucleic acid species such as RNA and RNA:DNA hybrid activate NLRP3 in bacterial infections.
The timing of stimulation of primary memory CD8 T cells impacts the rate of secondary memory generation and differentiation (P1402)
Authors: Khan Shaniya , Martin Matthew , Zhou Xinyuan , Xue Hai Hui , Badovinac Vladimir
Published: January 2023

Memory CD8 T cell responses are essential in establishing long-lasting immunity, as these responses confer protection from repeated infections. Various factors have shown to influence the generation of memory CD8 T cells, suggesting that the process of memory differentiation is not fixed and can be manipulated. Here, we asked whether the timing of stimulation (or recruitment) of primary memory CD8 T cells impacts the generation and differentiation of secondary memory CD8 T cell responses after bacterial and viral infections. We show that despite decreased secondary expansion, the differentiation of primary memory CD8 T cells into populations with the long-term secondary memory phenotype (ex. CD62Lhi, CD27hi) and function (ex. tissue distribution, IL-2 production, antigen-driven proliferative expansion) is facilitated by late recruitment into the immune response. Additionally, we show that the expression of survival factors and transcription factors known to be involved in memory CD8 T cell differentiation (ex. Bcl2, Eomes, Tcf1) is increased in late-stimulated secondary effector CD8 T cells. Thus, these data suggest that the timing of antigen-stimulation modulates the program of secondary memory CD8 T cell differentiation, which may have relevance for the design of future prime-boost vaccine protocols.
Requirements for tissue resident T cell migration in the salivary gland following LCMV infection (P1428)
Authors: Stolp Bettina , Pieczyk Markus , Kreutzfeldt Mario , Merkler Doron , Fukui Yoshinori , Stein Jens
Published: January 2023

Following clearance of viral infection memory T cells are retained to protect the host from reinfection. Central memory (CM) T cells efficiently migrate within the loose network of fibroblastic reticular cells inside the lymph node (LN) parenchyma and monitor antigen presenting cells for cognate antigen. Tissue-resident effector memory (EM) T cells cope with the denser environment within peripheral organs, migrating between endo- and epithelial cells and dense extracellular matrix. Little is known about migration behavior and mechanisms required for EM T cell tissue surveillance. In this study we compare the migratory behavior of CM and EM T cells within the same mouse by consecutive multiphoton intravital imaging of the popliteal LN and the salivary gland (SG), respectively. We establish intravital imaging of the SG as a model for a peripheral organ harboring tissue-resident effector memory cells in between the tightly packaged endo- and epithelial cells. Comparison of the migratory behavior of cognate T cells during the acute and the memory phase of a LCMV infection revealed efficient migration of effector T cells within the SG at all timepoints, although with reduced velocity compared to the LN parenchyma. During the memory phase of infection EM T cells show a probing phenotype, projecting dendrites in between the epithelial cells. Using the ROCK-inhibitor Y27632 and DOCK8-deficient T cells we refine the picture of EM T cell migration within peripheral organs.
Characterization of IL-15 independent memory CD8 T cell subsets (P1438)
Authors: Fraser Kathryn , Schenkel Jason , Vezys Viava , Masopust David
Published: January 2023

IL-15 is required for the maintenance and homeostatic proliferation of conventionally characterized memory CD8 T cells, and thus sets a carrying capacity for the size of the memory CD8 T cell compartment. However, we recently demonstrated that heterologous prime boost (HPB) vaccine regimens that establish very abundant memory CD8 T cell populations result in an enlargement of the memory CD8 compartment, which is almost entirely due to the addition of vaccine-specific memory T cells that lack CD62L expression. Here, we test the hypothesis that memory CD8 T cells resulting from HPB are less dependent on IL-15. We demonstrate that boosted memory cells express decreased levels of IL15Rβ and undergo little homeostatic proliferation. When co-transferring primary and boosted memory CD8 T cells into IL15KOs, boosted memory CD8 T cells that lacked CD62L expression were better able to persist compared to primary memory CD8 T cells (regardless of CD62L expression). We extended these studies by demonstrating that primary memory CD8 T cells that are resident in several peripheral tissues were also maintained in the absence of IL-15, and in some cases, exhibited IL-15 independent homeostatic proliferation. These observations point to a distinct lineage, not wholly defined by lack of CD62L expression, that harbors less dependence on IL-15 for maintenance. This has ramifications for regulation of memory CD8 T cell quantity following certain vaccine modalities and at front line tissues.
TLR7/9 stimulation by viral RNA is not a major driver of immune activation in pathogenic SIV infection (P6191)
Authors: Barratt-Boyes Simon , Kader Muhamuda , Smith Amanda , Watkins Simon , Guiducci Cristiana , Barrat Franck
Published: January 2023

Chronic immune activation is a fundamental component of AIDS pathogenesis but the mechanism responsible for this process is not understood. Emphasis has been placed on plasmacytoid dendritic cells (pDC), given their capacity to produce interferon (IFN)-α in virus infections and the link between persistent IFN-stimulated gene (ISG) expression and immune activation. We used a dual antagonist of TLR7 and 9 to selectively inhibit pDC responses in SIV infection. Administration of TLR7/9 antagonist blocked blood pDC production of IFN-α and TNF-α following SIVmac251 infection of rhesus macaques, but infection alone induced pDC hyporesponsiveness, whereas blood mDC gained the capacity to secrete IFN-α. In lymph nodes pDC were major but not exclusive producers of IFN-α, which was transient, and inhibited by TLR7/9 blockade. In contrast, TNF-α production in SIV-infected lymph nodes derived from macrophages and T cells, was sustained and was not impacted by TLR7/9 blockade. TLR7/9 blockade had no impact on plasma IFN-α production or ISG expression and did not diminish activation and proliferation of CD4+ and CD8+ memory T cells in either blood or lymph node. These experiments reveal that virus-mediated TLR7/9 activation is not a major contributor to immune activation and suggest that other pathways such as persistent TNF-α production may be significant factors in this process.
Impact of IFNγ on HIV-specific CD4 T cell and antigen presenting cell function (P6163)
Authors: Porichis Filippos , Hart Meghan , Barblu Lucie , Zupkosky Jennifer , Kavanagh Daniel , Kaufmann Daniel
Published: January 2023

One of the major impediments of HIV infection is the abnormally high levels of immune activation that contributes to immune dysregulation and impairs viral clearance. While IFNγ secretion is used as a marker of the functional state of T lymphocytes and NK cells, its role in regulating immune responses is poorly understood. We isolated CD8-depleted PBMCs from HIV infected individuals (n=13). We measured proliferation and cytokine production by HIV-specific CD4 T cells stimulated with HIV Gag in the presence of isotype control, anti-IFNγ and/or anti-PD-L1. We performed apoptosis measurements using Annexing-V binding assays. We also determined the modulatory impact of IFNγ on APCs by measuring expression of PD-1 ligands, HLA-DR, HLA-I and IL-12 secretion. Neutralization of IFNγ produced by Gag stimulated CD4 T cells enhanced proliferation (p=0.0161) and reduced apoptosis of HIV-specific CD4 T cells. IFNγ blockade enhanced IL-13 secretion (p=0.0039) but had no effect on IL-2, IL-10 and TNFα levels. IFNγ induced strong up-regulation of PD-L1, HLA-DR and HLA-I on monocytes but not on B, T and NK cells. IFNγ also induced IL-12 secretion by APCs that acted in a positive feedback loop to further enhance IFNγ secretion. Concurrent blockade of IFNγ and PD-L1 led to a more prominent increase in proliferation of HIV-specific CD4 T cells than blockade of individual pathways. These data provide mechanistic insight on the causal role of IFNγ in T-helper dysregulation in HIV infection.
Rapid Galectin-9 release occurs following HIV-1 acquisition in acute HIV-1 infection (P6165)
Authors: Tandon Ravi , Fujita Tsuyoshi , Chew Glen , Byron Mary Margaret , Niki Toshiro , Hirashima Mitsuomi , Barbour Jason , Ostrowski Mario , Clayton Kiera , Lanteri Marion , Ishii Naoto , Borrow Persephone , Norris Philip , Hecht Frederick , Ndhlovu Lishomwa
Published: January 2023

Despite successes with antiretroviral therapy, HIV infection continues to be a significant health burden with patients remaining at risk of non-AIDS defining events. The earliest immunological responses induced during HIV infection are key events in the control of HIV disease progression. Soluble biomarkers may aid in understanding HIV-immunopathogenesis, inflammation and prophylactic vaccine design. Galectin-9 (Gal-9) is a bidirectional immune regulator that promotes tissue inflammation and cell death but induces immune tolerance in part through engagement with the Tim-3 receptor. We measured circulating levels of Gal-9 in the plasma of 10 acute HIV-infected subjects at multiple time points over a 42-day period, before and after detectable HIV RNA. Plasma HIV RNA levels increased rapidly in all subjects. We found that Gal-9 levels rose with the first median elevation time of 5 days after detectable viremia and a median peak of 57.4 pg/ml. Overall Gal-9 levels generally tracked with viremia. In vitro, recombinant Gal-9 significantly increased IFN-γ production by CD8+ T cells compared to untreated cells (0.02% vs 1.2%) in cultures from early HIV-infected subjects and this was reversed by α-lactose blockade. Our findings reveal that Gal-9 is a novel plasma factor that is rapidly elevated during the first wave of the cytokine storm after HIV emergence and may have important roles in CD4+ T cell loss or the response to tissue damage.
Peroxisome proliferator-activated receptor-gamma in CD11c+ lung dendritic cells programs peripheral CD4+ T cells for Foxp3 induction during airway tolerance. (P6001)
Authors: Khare Anupriya , Krishnamoorthy Nandini , Raundhal Mahesh , Morse Christina , Ray Prabir , Ray Anuradha
Published: January 2023

Peroxisome proliferator-activated receptor-gamma (PPARγ) is a widely expressed molecular regulator with anti-inflammatory functions. However, whether it has an anti-inflammatory role in dendritic cells (DCs) that influence the Treg/Teffector balance has not been experimentally addressed. In this study, we investigated the role of PPARγ in DCs regulating the delicate balance between tolerance versus inflammation in the periphery using a model of airway induced tolerance by inhaled allergen. Using mice with selective deletion of PPARγ in CD11c+ cells, we observed that PPARγ expression in CD11chi lung DCs is essential for de novo Foxp3 expression in CD4+ T cells. We demonstrate that PPARγ plays a crucial role in enhancing aldh1a2 expression in lung CD103+ DCs under tolerogenic conditions with significantly less expression of the enzyme in these cells when mice are immunized for airway inflammation. When tolerized CD11ccre/+ PPARγfl/fl mice were antigen-challenged, a complete loss of immune tolerance with increased neutrophil-dominated airway inflammation was observed. Taken together, our study identifies PPARγ as a central regulator in DC-mediated programming of Foxp3 expression in naive CD4+ T cells dictating tolerance versus inflammation upon antigen provocation.
Novel grass pollen antigens are the major inducers of Th2 cytokine producing T cells in allergic individuals (P6039)
Authors: Schulten Veronique , Greenbaum Jason , Hauser Michael , Baker Denise , Sidney John , Kolla Ravi , Lindestam Arlehamn Cecilia , Oseroff Carla , Alam Rafeul , Broide David , Ferreira Fatima , Grey Howard , Sette Alessandro , Peters Bjoern
Published: January 2023

T cells play an important role in the pathogenesis of allergic diseases. However, potential immunogens of allergenic T cell responses have traditionally been limited to those that induce IgE responses. Timothy grass (TG) pollen is an inhaled allergen for which major IgE-reactive allergens have also been shown to trigger Th2 responses. Here we examined whether other TG pollen proteins are recognized by Th2 responses independently of IgE-reactivity. A TG pollen extract was analyzed by 2D-gel electrophoresis and IgE/IgG immunoblots using pooled sera from allergic donors. Mass spectrometry in combination with de-novo sequencing of the whole TG pollen transcriptome identified 93 novel proteins for further study, 64 of which were not targeted by IgE. Peptides from the novel TG proteins were screened for T cell reactivity in PBMCs from allergic donors. Strong IL-5 production was detected in response to peptides from several novel proteins, most of which were not targeted by IgE. Responses against the dominant novel epitopes were associated with memory T cells and could be detected directly ex vivo after Th2 cell enrichment. These findings demonstrate that a combined unbiased transcriptomic-, proteomic-, and immunomic approach identifies a greatly broadened repertoire of protein antigens targeted by T cells involved in allergy pathogenesis. The discovery of proteins that induce Th2 cells but are not IgE reactive may allow the development of safer immunotherapeutic strategies.
Effect of NADPH oxidase 2 deficiency on allergic lung inflammation and differentiation of helper T cells (P6045)
Authors: Kwon Bo In , Lee Seung-Hyo
Published: January 2023

Nox2 has been originally identified in phagocytes, which kills engulfed pathogens by producing reactive oxygen species (ROS). It has been also revealed that activated T cells produce ROS that is an important second messenger for the functions of effector T cells. Recent studies have shown that Nox2 dificient CD4 T cells contain increased IL-17 producing capability and Nox2 knock-out mice have a tendency an enhanced susceptibility to spontaneous arthritis with age. To investigate whether Nox2 or ROS deficiency in T cells affect differentiation and function of CD4 T cells in T cell intrinsic manner. Specifically we adopted allergic asthma model to clarify the function of Th2 cells without Nox2 or ROS. Asthma phenotypes were analyzed in fungal protease mixed with chicken egg ovalbumin (OVA) allergen (APO) induced experimental asthma. To understand mechanisms of exaggerated effector T cell functions, we investigated the degree of T cell activation and differentiation of Treg cells with Nox2 deficient and wild type CD4 T cells. All experimental asthma phenotypes including airway hyper-responsiveness, lung inflammation along with eosinophilia and mucus production were increased in Nox2 deficient mice. Nox2 deficient T cells also showed enhanced hyper-activation, reduced AICD and decreased Treg cell differentiation. Our results indicate that Nox2/ROS deficiency showed exaggerated experimental asthma, which was caused by enhanced function of Th2 cells.
Respiratory infection with rhinovirus interferes with the induction of tolerance to aeroallergens (P6007)
Authors: Mehta Amit , Duan Wei , Soroosh Pejman , Doerner Astrid , Zuraw Bruce , Croft Michael
Published: January 2023

Respiratory viral infections can influence the course of asthma at different time points. Rhinovirus (RV) infections during early age associate with a higher frequency of asthma in later childhood. Because airway exposure to innocuous antigen normally engenders T cell tolerance and prevents the development of airway inflammation, we examined the effect of RV infection on airway tolerance induced by exposure to intranasal OVA. We found that simultaneous infection with RV1B, a minor serotype, that binds LDL receptors in mice, abrogated tolerance induced by exposure to OVA, suppressing the generation of antigen-specific CD4+forkhead box protein 3 (FOXP3)+ regulatory T (Treg) cells while at the same time promoting IL-4-secreting effector CD4+ T cells. Rhinovirus infection in-vivo resulted in selective lung expression of the innate cytokines thymic stromal lymphopoietin (TSLP) and IL-33, and the T cell costimulatory TNF family molecule OX40L that is regulated by TSLP. Furthermore, RV infection subsequently resulted in full susceptibility to develop asthmatic disease, which was blocked by targeting TSLPR, IL-33R, or OX40L. These results suggest that RV infection of the respiratory tract can prevent the induction of tolerance to inhaled antigen through a mechanism involving TSLP, IL-33 and OX40L that results in susceptibility to developing acute allergic lung inflammation.