bluuebunny/crossref_metadata_2025 huggingface dataset:
Th40 T cells are pathogenic in multiple sclerosis and EAE the mouse model of MS (P3121)
Authors: Wagner David , Waid Dan , Corboy John , Schreiner Teri
Published: January 2023

Multiple sclerosis (MS) is a neurodegenerative disease with established autoimmune components. We described a unique T cell subset expressing CD40, Th40 cells that achieve effector status in autoimmune diseases. Furthermore we discovered that Th40 cells are detected in human subjects and that during autoimmunity these T cells are significantly (p < 10(-7) expanded in number compared to normal controls. Th40 cells produce both IFNg and IL-17, suggesting an interesting option for pathogenicity development. The majority of patients carried the DR15 (DR2) haplotype, but a significant number do not. Th40 cells were predominantly CD45RO (memory phenotype) and unlike Th40 cells in T1D express both CCR5 and CXCR3. In many patients, an antigen response profile was determined; i.e. some patients respond to MOG while others respond to PLP etc. In the EAE mouse model of MS we discovered that prior to EAE onset levels of Th40 cells are systemically very low, disease induction increased Th40 cell percentages and numbers drastically. When Th40 cells are isolated from EAE induced mice , disease can be adoptively transferred to scid recipients. Histology reveals that Th40 cells infiltrate brain and spinal cord. Th40 cells isolated from the brains of stage 3 EAE mice are pro-inflammatory.
Impaired lymphocyte response in patients with chronic kidney disease (P3075)
Authors: Mansouri Ladan , Lundahl Joachim , Jacobson Stefan , Paulsson Josefin
Published: January 2023

In Chronic Kidney Disease (CKD), immune cells are affected due to uremic toxins. Given the central role of T lymphocytes in the progress of CKD, we analyzed lymphocyte proliferative response and immune modulators production to selected stimulators. We applied a method called Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) using whole blood and flow-cytometric analysis for detection of proliferation in addition to a multi-array method (Milliplex 26-plex) to analyze the profile of immune modulators. The absolute number of lymphoblasts, CD4+, CD8+ subpopulations in response to mitogens were equal comparing healthy controls, patients with eGFR <20 (ml/min/1.73 m2) and hemodialysis patients. However, levels of Interleukin (IL)-2, 10 and 15 in the Staphylococcus enterotoxin A -stimulated supernatant as well as levels of IL-15 and IL-5 in the Influenza A vaccine-stimulated supernatants of patients were lower compared to healthy controls. TNF-α, IL-10, IL-12, IL-15, IL-8, MCP-1, IP-10, IFN-α2, IL-1α and Eotaxin levels were higher in the patient plasma. Our results suggest that T-cells have no intrinsic defects in terms of proliferative response to mitogens but an inability to produce selected cytokines due to the uremic milieu or dialysis procedure. The reported aberrations may contribute to the increased frequency of infections in CKD patients and the systemic inflammatory state may be causative for atherosclerosis.
HMGB1, a major damage-associated molecule, is an early and critical mediator in an animal model of uveitis induced by uveitogenic T cells (P3161)
Authors: Shao Hui , Jiang Guomin , Sun Deming , Kaplan Henry
Published: January 2023

It is largely unknown how invading autoreactive T cells initiate the pathogenic process inside the diseased organ in organ-specific autoimmune disease. In this study, we used a chronic uveitis disease model in mice, experimental autoimmune uveitis (EAU), induced by adoptive transfer of uveitogenic, interphotoreceptor retinoid binding protein (IRBP)-specific T cells and showed that high mobility group box chromosomal protein 1 (HMGB1), an important endogenous molecule that serves as a danger signal, is rapidly released from retinal tissue cells into the extracellular matrix and intra-ocular fluid in response to IRBP-specific T cell transfer. HMGB1 levels in the intra-ocular fluid correlated with disease severity. The HMGB1-releasing tissue cells remained intact, showing that this molecule was not released by necrotic/apoptotic cells. HMGB1 release required direct cell-cell contact between retinal tissue cells and IRBP-specific T cells. Administration of HMGB1 antagonists markedly suppressed development of EAU via mechanisms including inhibition of migration of granulocytes/monocytes and Th1 cytokine production. Our study demonstrates that HMGB1 is an early and critical mediator of ocular inflammation initiated by autoreactive T cell invasion.
Role of calcium/calmodulin kinase IV on podocyte function in lupus nephritis (P3081)
Authors: Ichinose Kunihiro , Ushigusa Takeshi , Koga Tomohiro , Tsokos George , Kawakami Atsushi
Published: January 2023

[Objectives] Kidney podocytes and their slit diaphragms settle the integrity of renal basement membrane and prevent urinary protein loss. T cell from patients with systemic lupus erythematosus display increased expression of calcium/calmodulin kinase IV (CaMKIV). Here we evaluated the expression of CaMKIV in kidney biopsy specimens from patients with lupus nephritis (LN) and in a human podocyte cell line (AB8/13) after exposure to IgG from sera of patients with LN. [Methods] We examined the expression level of nephrin, a podocyte marker and CaMKIV in normal and LN kidney specimens by immunofluorescence staining. We cultured human podocytes with IgG purified from sera of normal individuals and LN patient for 24 hr and analyzed CaMKIV mRNA levels. We also performed microarray analysis to determine gene expression. [Results] Although the expression levels of nephrin were decreased, CaMKIV expression was found increased in podocytes of LN kidney biopsy specimens.Interestingly, culture of podocytes in the presence of IgG from LN sera led to 1.5-fold increase in the expression of CaMKIV mRNA.Gene array analysis revealed that the expression of genes related to the regulation of neural precursor cell proliferation decreased significantly (P<0.000441) in podocytes treated with IgG from LN patients. [Conclusion] We suspect that increased CaMKIV expression may contribute to the inability of podocytes to maintain the integrity of glomerular basement membrane in LN patients.
BANK1 controls IL-6 secretion and a Type I interferon signature via a p38 and MNK1/2- eIF4E translation initiation pathway (P4060)
Authors: Wu Ying-Yu , Kumar Ramesh , Dozmorov Mikhail , Haque Mohammed , Wren Jonathan , Castillejo-López Casimiro , Alarcón-Riquelme Marta
Published: January 2023

BANK1, an adaptor protein expressed in B-cells, is genetically associated with human SLE and plays a little understood role in B-cell signaling. Because BANK1 contains an N-terminal putative Toll-interleukin 1 receptor (TIR) domain, we used murine Bank1-/- splenic B cells to test if BANK1 affects signaling induced by the TLR9 agonist CpG. Following CpG stimulation BANK1 deficiency reduced p38 phosphorylation without affecting that of ERK or JNK and to reduced IL-6 secretion. Bank1-/- cells showed reduced phosphorylation of MNK1/2, confirming that BANK1 affects p38 signaling, and expression microarray analysis showed a disregulated type I interferon response. Microarray analysis and reduced phosphorylation of eIF4E indicated general repression of the eIF4F translation initiation complex in Bank1-/- cells. Together these data establish previously unrecognized roles for BANK1 in CpG-induced responses by splenic B cells. BANK1 may regulate type I IFN responses to TLR9 stimulation by influencing p38 signaling and the eIF4F complex, and thereby influence the course of viral infections or autoimmunity.
Blk haploinsufficiency promotes proinflammatory cytokine production and accelerates onset of kidney disease in B6.lpr mice (P4064)
Authors: Hayes Sandra , Samuelson Elizabeth , Laird Renee , Papillion Amber , Tatum Arthur , Princiotta Michael
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disorder whose etiology remains unknown. There is strong evidence for a genetic basis to this disease, and many genetic alleles that predispose an individual to SLE have been identified from studies in patients with SLE and in mouse models of lupus. With recent advances, however, such as the completion of the Human Genome Project and the International HapMap Project, it is possible to perform GWAS to identify additional susceptibility genes in humans. Indeed, several groups using this experimental approach have identified over twenty-five new susceptibility loci in SLE patients of different ethnicity and race. BLK is one of the newly identified susceptibility genes, and risk alleles that map to the BLK locus result in reduced expression. BLK encodes B lymphoid kinase, a Src family kinase whose expression was initially reported to be restricted to the B cell lineage, but we show that it is also expressed in plasmacytoid dendritic cells and IL-17-producing γδ and CD4-CD8- αβ T cells. To investigate the contribution of BLK risk alleles to disease development, we reduced Blk expression in autoimmune-prone B6.lpr mice. We report that Blk+/-.lpr mice exhibit enhanced proinflammatory cytokine production and accelerated onset of lymphoproliferation and kidney disease compared to B6.lpr mice. These data suggest that BLK risk alleles promote disease through the dysregulation of a proinflammatory cytokine network.
Differential regulation of immune responses in MyD88-deficient mice by cytokine-expressing Salmonella typhimurium (P4046)
Authors: al-Ramadi Basel , Issac Jincy , El-Ojali Samia , Mohamed Yassir , Al-Ketbi Mai , Fernandez-Cabezudo Maria
Published: January 2023

MyD88 is a critical component of most TLR signaling pathways. Earlier we demonstrated that MyD88-deficient (MyD88-/-) mice are hypersusceptible to infection by a vaccine strain (aroA-/aroD-) of Salmonella typhimurium (designated BRD509). Curiously, infected mice exhibited a state of hypergammaglobulinemia, with high levels of anti-Salmonella Abs of all IgG isotypes. The dysregulated Ab response also lead to production of anti-dsDNA and anti-thyroglobulin autoantibodies and immune complex deposition in kidneys. Increased susceptibility of MyD88-/- mice has been linked to poor type-1 cytokine responses. Therefore, we assessed the response of these mice to infection with a S. typhimurium engineered to express murine IFN-γ (GIDIFN). Infection studies using different doses of BRD509 and GIDIFN strains revealed that the latter strain was >100-fold less virulent in MyD88-/- mice than BRD509 and correlated with decreased bacterial loads in systemic organs and robust pathogen clearance starting at 7 days of infection. Although GIDIFN-infected mice exhibited high Ab titers, the degree of autoreactivity was significantly decreased compared to BRD509-infected animals. Our data suggest that TLR-MyD88 signaling pathway is critical for controlling Ab responses to bacterial antigens and for maintaining B cell self-tolerance and demonstrate that the two processes may be unlinked through the use of cytokine-expressing bacterial strains that modulate host immune responses.
A dominant CD4+ T cell response to Helicobacter pylori is associated with decreased risk of gastric diseases in human (P4328)
Authors: Zou Quanming
Published: January 2023

BACKGROUND and AIMS: Immunodominance has been well-demonstrated as a key feature in antiviral, antitumoral and antibacterial cellular immune responses but much less so in the immune responses against Helicobacter pylori (H. pylori). Antigen-specific CD4+ T cells have been reported to play a pivotal role in protecting against H. pylori in murine systems. We investigated the immunodominant CD4+ T cell response to HpaA (also called neuraminyllactose-binding hemagglutinin), a conserved, H. pylori-specific colonization factor which has been considered as a promising vaccine candidate. METHODS: HpaA-specific CD4+ T cells were expanded with autologous peripheral blood mononuclear cells (PBMCs) loaded with recombinant HpaA and further characterized with overlapping synthetic peptides. The percentage of HpaA88-100-specific CD4+ T cells restricted to HLA-DRB1*1501 from 59 H. pylori-infected subjects with different diseases were compared. RESULTS: Several novel, immunodominant CD4+ T cell epitopes were identified and characterized. The immunodominant CD4+ T cell responses specific to HpaA88-100 were observed in most of the H. pylori-infected individuals who expressed HLA-DRB1*1501 and they were significantly more abundant in patients with less severe diseases (P<0.05). CONCLUSIONS: The HLA-DRB1*1501-restricted immunodominant CD4+ T cell response specific to HpaA88-100 is associated with decreased risk of severe gastric diseases. Further study of these and other similar immunodominant CD4+ T cell responses to H. pylori will help the field to gain critical insight into the mechanism of protective immunity against H. pylori in humans and the knowledge gained will improve potential vaccine design.
Tracking the CD4+ T cell response to BCR-ABL-induced leukemia (P4330)
Authors: Berquam-Vrieze Katherine , Farrar Michael
Published: January 2023

Patients with BCR-ABL+ acute lymphoblastic leukemia develop T cell responses to the BCR-ABL fusion peptide (referred to as BAp). However, these responses are not sustained and loss of the BAp-specific T cell response correlates with relapse. A major gap in our knowledge is why such epitopes do not elicit immune responses that eliminate BCR-ABL+ leukemia. This knowledge gap exists because of an inability to track the fates of endogenous BAp:I-Ab-specific T cells in vivo. To address this question we plan to generate peptide:MHCII tetramers that will allow us to track BAp:I-Ab-specific T cell responses. We have demonstrated that CD4+ T cells exist that recognize the BCR-ABL fusion junction in a mouse model of BCR-ABL+ leukemia. We are generating BAp:I-Ab tetramers that will allow us to track these cells in vivo. Using this reagent we will determine whether T cell responses are impaired due to deletion, immune deviation, Treg-mediated suppression or anergy. We will then assess whether enhancing antigen presentation or inhibiting anergy improves BAp:I-Ab-specific T cell responses. In addition, we will determine if altering the cytokine mileu can improve the CD4+ T cell response to BCR-ABL+ leukemia using both knock-out mouse mice and in vivo delivery of exogenous cytokines.
Use of mRNA for vaccination or gene therapy: delivery route determines the verdict (P4316)
Authors: Pollard Charlotte , Vandermeulen Gaëlle , Vanham Guido , Grooten Johan , Préat Véronique , De Koker Stefaan
Published: January 2023

The use of DNA and viral vector vaccines for the induction of cellular immune responses is increasingly gaining interest. However, concerns have been raised regarding the safety of these immunization strategies. Due to their lack of genome integration, mRNA-based vaccines have emerged as a promising alternative. In this study we evaluated the potency of antigen-encoding mRNA either delivered via complexation with the cationic lipid DOTAP/DOPE, or delivered via in vivo electroporation as a novel vaccination approach. By using luciferase-encoding mRNA we showed that in vivo electroporation of mRNA induces high levels of luciferase at the injection site, while protein expression in mice injected with DOTAP/DOPE complexed mRNA was barely above background levels. Despite the high levels of protein expression, in vivo electroporation of mRNA failed to elicit immune responses against the encoded antigen. In contrast, lipid-based delivery of mRNA induced antigen-specific, functional T cell responses resulting in specific killing of peptide-pulsed cells and the induction of humoral responses. In summary, in vivo electroporation of antigen-encoding mRNA induces high levels of protein expression but no immune responses, thus presenting an ideal tool for mRNA-based gene therapy, while lipid-based delivery offers advantages for vaccination purposes due to the induction of low protein levels but strong antigen-specific cellular immunity.
An effective cell-based approach for the generation and identification of functional monoclonal antibodies to cell surface targets (P4320)
Authors: Zhang Chonghui , Chan Nancy , Kikuchi Lena , Voegeli Karin
Published: January 2023

Monoclonal antibodies (mAbs) have long been remarkable tool reagents for discovery research, but have also recently emerged as a significant class of therapeutic biologics. The development of therapeutic mAbs, however, has been hindered by the challenges of purifying distinct antigens like cell membrane-bound proteins and by the lack of effective methods for identifying mAbs that recognize native epitopes of the antigen. Here we developed an effective cell-based approach for generating mAbs against cell surface targets. Targeted antigens of interest were molecularly cloned and stably expressed on the cell surface of a mouse pre-B cell line. The transfected cells were then used for immunization of a syngeneic strain of mice and for subsequent hybridoma selection by high-throughput flow cytometry. Panels of mAbs were raised against a large number of antigens ranging from cell surface proteins to viral targets. The mAbs generated by this approach were predominantly IgG isotypes and had minimal binding affinities of a nanomolar range. Most of the mAbs recognized antigens in their native conformation and were diversified in their Ig sequences. More importantly, many of these mAbs were found to have functional activity, such as receptor antagonism, protein kinase inhibition or neutralizing activity. This cell-based approach for immunization and mAb selection offers an effective means to generate high-quality mAbs for the needs of discovery research and therapeutic development.
Rewiring of the Bcr/Abl and PKCβII signal transduction pathways (P4322)
Authors: Hoekstra David , Carlson Louise , Lee Kelvin
Published: January 2023

The ability to differentiate leukemia cells directly into antigen presenting cells (APCs) in vivo has long been sought after as a method of cancer immunotherapy. While also being less proliferative, these newly differentiated APCs may present new antigens to the immune system and could potentiate a robust immune response. We have previously shown that canonical activation of protein kinase C isoform βII (PKCβII) drives dendritic cell differentiation and apoptosis in leukemic cells. With this as our pro-drug, we have rewired its signaling to be activated by the oncogenic kinase Bcr/Abl, which is essential for chronic myeloid leukemia cell survival. To do this, the abl kinase target motif was inserted into a specific regulatory sequence in PKCβII based off of molecular modeling techniques. Using ImageStream technology, we have found that the rewired PKCβII constructs are activated in a Bcr/Abl+ leukemia cell line, but not in Bcr/Abl- cells; introduction of Bcr/Abl into non-expressing cells allows for activation of the rewired PKCβII. Additionally, this activation was blocked by the Bcr/Abl inhibitor imatinib in leukemia cells sensitive to imatinib but persisted in resistant cells. Activation of PKCβII by Bcr/Abl led to a decrease in viability and an increase in apoptosis in Bcr/Abl+ leukemic cells. This novel strategy of activating differentiation machinery in leukemic cells in vivo provides a new method of treating patients who do not respond to traditional chemotherapy.
mTORC1 inhibition blocks the gut-homing phenotype induced by retinoic acid in naturally-occurring Treg but not in conventional CD4+ T cells (P6260)
Authors: Chen Leo , Rosborough Brian , Thomson Angus , Raimondi Giorgio
Published: January 2023

The vitamin A metabolite all-trans retinoic acid (ATRA) is known to induce a gut-homing phenotype in T cells by upregulating the integrin α4β7 and CCR9. We report that, unlike conventional CD4+ T cells, only about half of naturally-occurring Treg (nTreg) upregulate CCR9 in response to ATRA. The bimodal distribution of CCR9+ versus CCR9- Treg subsets was not due to differences in the extent of proliferation, intensity level of FoxP3, or expression of the nTreg/iTreg discriminator Helios. Furthermore, we found that addition of the mammalian target of rapamycin (mTOR) inhibitor rapamycin prevented the appearance of the CCR9+ subset of nTreg, but did not prevent CCR9 upregulation by T cells, suggesting nTreg-specific involvement of mTOR in regulation of the expression of CCR9. mTOR involvement was independent of Akt activity, as inhibition of Akt or its downstream target GSK-3β did not prevent CCR9 expression. Additionally, Rictor-/- Treg (which lack mTORC2) showed no diminution of the CCR9+ subset compared to wild-type controls, demonstrating selective participation of mTORC1. These results suggest that nTreg are a heterogeneous population with two subsets: one that does not upregulate CCR9 in response to ATRA, and one that does upregulate CCR9 in an mTORC1-dependent fashion. These findings reveal a novel difference between nTreg and conventional T cells, and provide a framework for which the migratory behavior of nTreg versus T cells may be differentially influenced.
Effects of osteopontin on B cells (P6285)
Authors: Dsilva Sehba , Rothstein Thomas
Published: January 2023

Osteopontin (OPN) is a protein responsible for diverse processes. OPN expression is linked to autoimmunity and cancer. Both innate and adaptive immune cells like macrophages, dendritic cells,T and B cells express OPN. OPN has many effects on non-B immune cells, the source of these effects is either the OPN produced by the cells themselves or OPN made by other cells. With regard to B cells, it’s been suggested that high levels of OPN contribute to chronic and sustained polyclonal B cell activation. However, the specific effects of OPN on B cell survival, migration and stimulated proliferation are unknown. Our objective is to identify these specific effects of OPN on B cells and determine the contribution to disease activity. For survival studies, purified B cells from WT mice-untreated and treated with exogenous OPN were used along with B cells from OPN transgenic mice. Increased survival was seen in both OPN treated B cells and B cells from transgenic mice. Thymidine incorporation and CFSE assays were used to examine proliferation of B cells from WT and OPN transgenic mice in response to various stimuli. The results indicate enhanced proliferation in B cells from the transgenic mice compared to those from WT mice. Using transwell migration assay, preliminary experiments also indicate that OPN induces migration of B cells. These developments suggest that OPN affects B cell survival, proliferation and migration, which could in turn contribute to propagation of disease activity.
The RNA-binding protein HuR coordinately regulates GATA-3 and Th2 cytokine gene expression in dose dependent manner (P6262)
Authors: Atasoy Ulus , Gubin Matthew , Calaluce Robert , Magee Joseph , Martindale Jennifer , Gorospe Myriam , Casolaro Vincenzo , Stellato Cristiana
Published: January 2023

Posttranscriptional control by RNA binding proteins (RBPs) is poorly understood. Th2 mediated diseases such as asthma, are driven by GATA-3, IL-4 and IL-13. The RBP, HuR, regulates IL-4 and IL-13 by increasing mRNA stability. We identified GATA-3, IL-4 and IL-13 as HuR targets and hypothesized HuR may be coordinately regulating Th2 differentiation by controlling mRNA stability and translatability. We made a HuR CD4+T transgenic mouse, and a HuR conditional knock out mouse (HuRfl/fl) to ablate HuR in T cells. HuR over-expression in transgenic CD4+ T cells stabilized GATA-3, IL-4 and IL-13 mRNAs, and increased mRNA, protein and Th2 polarization. HuR knock-down resulted in decreases in GATA-3, IL-4 and IL-13 expression. Findings were confirmed in human naïve and memory T cells. We defined HuR binding sites in GATA-3. Th2 cells with low HuR levels (26%) from HuRfl/+ mice, had decreases in IL-4, IL-13 and GATA-3 mRNA but not protein. Surprisingly, CD4+ T cells from HuRfl/fl mice (93% knockdown) showed increased IL-2, IL-4, IL-13 mRNA and protein. IL-4 transcription increased but mRNA stability remained unchanged. IL-2 and IL-13 transcription were unaltered but there were increases in mRNA stability. Polysomal gradient analysis revealed similar translation. We analyzed gene expression in KO vs. control CD4+ T cells to determine HuR targets. Ova challenge model of asthma corroborated ex vivo results. These data suggest HuR levels regulate Th2 cytokines and Th2 differentiation.
The TL1A-IL13 axis: a novel pathway for Type 2 immunity mediated by innate lymphocytes independent of helminth infection (P6264)
Authors: Meylan Francoise , Hawley Eric , Barron Luke , Barlow Jillian , Penumetcha Pallavi , Richard Arianne , Chen Xi , Paul William , Wynn Thomas , McKenzie Andrew , Siegel Richard
Published: January 2023

The TNF-family cytokine TL1A costimulates T cells through its receptor DR3 and is required for diverse autoimmune disease models, but its role in innate lymphocyte biology has not been explored. Transgenic mice chronically expressing TL1A have increased activated T cells and develop small intestinal pathology characterized by high levels of IL-13, muscular and goblet cell hyperplasia, and infiltration with immune cells, all of which depend on DR3. Genetically eliminating T cells reduces T cell hyperactivation and gut infiltration with lymphocytes but not IL-13 production or mucosal hyperplasia. These data suggest that TL1A can induce another cell type to produce IL-13 and induce gastrointestinal pathology. Using a DsRED reporter mouse for IL-13, we find that the major IL-13 producing cells in TL1A transgenic mice lack T and B cell lineage markers and are phenotypically similar to ’type 2’ innate lymphocytes (ILC2) which expand and produce IL-13 in response to IL-25, IL-33 or parasitic infection. TL1A directly promotes IL-13 production by ILC2 ex-vivo. However, DR3 deficient mice mount a vigorous response to the intestinal nematode Nippostrongylus brasiliensis, and IL-13 producing innate lymphocytes can be expanded by IL-25 in the absence of DR3. Thus, TL1A can directly induce IL-13 production by innate lymphocytes through mechanisms distinct from parasitic infection. TL1A may be an attractive target for immunotherapy of diverse diseases associated with this cytokine.
Interleukin 32 promotes angiogenesis: a new role for a versatile cytokine. (P6266)
Authors: Rudloff Ina , Zepp Jarod , Dinkel Hannah , Palmer Brent , Farkas Laszlo , Cool Carlyne , Taraseviciene-Stewart Laima , Kim Soo-Hyun , Dinarello Charles , Voelkel Norbert , Nold Claudia , Nold Marcel
Published: January 2023

IL-32 is a multi-faceted cytokine with roles in infection, autoimmunity and cancer. It aggravates inflammation, inhibits virus propagation and acts as a critical regulator of endothelial cell (EC) function. We now discovered that IL-32 also possesses angiogenic properties. The hyperproliferative EC of pulmonary hypertension and glioblastoma multiforme exhibited a markedly increased abundance of IL-32, and the cytokine colocalized with integrin αVβ3. In vitro, VEGF receptor blockade resulted in hyperproliferation of EC and a 3.4-fold increase in IL-32. When IL-32 was silenced by siRNA, the proliferation of adult and fetal EC was reduced (change in cell number +58% vs -15%). Mechanistic studies revealed that siIL-32-transfected EC produced 3-fold more activin A and angiopoietin 2, but 61% less NO, whereas neither apoptosis nor the abundance of VEGF or TGF-β were affected. Interestingly, pre-treatment with IFNγ was required to render EC responsive to exogenous IL-32γ. To obtain in vivo evidence for these angiogenic effects, we injected mice with matrigel plugs loaded with IL-32γ, VEGF or vehicle, and performed CD31- and H&E-staining after 14d. As anticipated, 25 ng/ml of VEGF increased neocapillarization 8-fold, but unexpectedly the effect of 100 ng/ml IL-32γ was even stronger (8.5-fold). In summary, we add VEGF-independent angiogenic properties to the portfolio of IL-32, implicating a role in pulmonary hypertension and neoplastic diseases that is possibly exerted via αVβ3.
Dendritic cell derived exosomes need to activate both T and B cells to induce antitumor immunity (P4250)
Authors: Näslund Tanja , Gehrmann Ulf , Qazi Khaleda , Karlsson Mikael , Gabrielsson Susanne
Published: January 2023

Exosomes are secreted membrane nanovesicles of endosomal origin and are considered as potential cancer vaccine vectors. Phase I clinical trials have been successfully conducted with tumor peptide-loaded exosomes derived from dendritic cells (dexosomes) and a phase II clinical trial is currently ongoing. However, much is still unknown regarding the in vivo role of dexosomes and if the immunogenicity of dexosomes can be enhanced. We have previously reported that dexosomes induce CD4+ T cell responses in a B cell dependent manner, suggesting that immunizing with dexosomes only carrying T cell peptides induce suboptimal immune responses. Here we show that CD8+ T cell responses were induced in vivo when mice were immunized with protein, but not peptide, loaded dexosomes. We could also show that the cytotoxic T cell response was totally dependent on CD4+ T cells and, interestingly, also on B cells. Mice deficient in complement activation and antigen shuttling by B cells have lower responses to protein-loaded dexosomes showing involvement of these B cell mediated mechanisms. Finally, protein-loaded dexosomes were superior in protecting against tumor growth. In conclusion, proper activation of CD4+ T and B cells need to be considered when designing cancer vaccines to ensure full potential of the treatment.
Novel mechanisms of anti-tumor action for therapeutic T-bet gene-modified dendritic cells (P4228)
Authors: Chen Lu , Taylor Jennifer , Storkus Walter
Published: January 2023

Clinical implementation of Type-1 polarized dendritic cell (DC) in vaccine formulations holds significant promise in the cancer therapy setting. We have recently demonstrated that DC engineered to express the T cell Type-1 transactivator protein T-bet (TBX21) promote robust Type-1 T cell-mediated immunity via a unique mechanism that is largely interleukin (IL)-12p70-independent. In the current study, we investigated the anti-tumor mechanisms of action associated with intratumoral administration of DC.Tbet developed from syngenic IL-12p35 -/-, IL-12p40 -/-, IFNγ -/- or β2m -/- mice. We observed that Type-1 anti-tumor immunity associated with slowed tumor growth was effectively developed in animals receiving DC.Tbet deficient in their ability to produce IL-12 family cytokines (i.e. IL-12, IL-23, IL-35) or IFN-γ. Surprisingly, we observed that the ability of DC.Tbet to present MHC class I/peptide complexes (i.e. lost in DC developed from β2m -/- mice) was not important for early priming of anti-tumor CD8+ T cells, but was essential for the sustained therapeutic efficacy of DC.Tbet (and treatment-induced anti-tumor CD8+ T cells) within the tumor microenvironment (TME). In order to best translate this cancer treatment modality into the clinic, we are currently evaluating how DC.Tbet conditions the TME for improved function of Ag-primed Type-1 T effector cells and how these APC collaborate with host DC to diversify the therapeutic T cell repertoire.
Adjuvant effect of a natural TLR4 ligand on dendritic cells (P4240)
Authors: Kim Hyung Sook , Ryu Hwa Sun , Lee Hong Kyung , Kim Yeon Jin , Kim Ji Sung , Park Yunsoo , Park Mi-jeong , Hong Jin Tae , Kim Youngsoo , Han Sang-Bae
Published: January 2023

Defect of dendritic cell (DC) maturation in tumor microenvironments is an important immunological problem limiting the success of cancer immunotherapy. In this study, we investigated the immunostimulatory effect of a Mori fructus polysaccharide (MFP) on DCs, a type of potent antigen presenting cells. MFP induced phenotypic maturation of DCs, as proven by the elevated expression of CD40, CD80/86, and MHC-I/II molecules. MFP induced functional maturation of DCs, in that MFP increased the expression of IL-12, IL-1β, TNF-α, and IFN-β, decreased antigen capture capacity, and enhanced allogenic T cell stimulation. MFP efficiently induced maturation of normal toll-like receptor4 (TLR4) DCs from C57BL/6 and C3H/HeN mice, but not mutated TLR4 DCs from C3H/HeJ mice, suggesting that TLR4 might be one of the membrane receptors of MFP. We showed that MFP increased phosphorylation of mitogen-activated protein kinase (MAPKs), and nuclear translocation of NF-κB p65 subunit, which are signaling molecules downstream from TLR4. These results indicate that MFP induced DC maturation through TLR4 signaling.
Effect of low level laser and Mesenchymal Stem Cells therapies on cigarette-induced lung inflammation in mice. (P4200)
Authors: Ligeiro de Oliveira Ana Paula , Silva Vanessa , Neves Vinicius , Vieira Rodolfo , Jazedje Tatiana , Pellati Mayra , Czeresnia Carlos , Perin Paulo , Maluf Mariângela , Nisenbaum Marcelo , Evangelista Lucila , Halpern Silvio , Zatz Mayana , Peron Jean Pierre
Published: January 2023

Chronic Obstructive Pulmonary Disease (COPD) has a high prevalence and economic and social cost. In this context, new therapeutic approaches are of great interest, and the use of Mesenchymal Stem Cells (MSCs) or low level laser therapy (LLL) may be innovative and promising approaches. Thus we investigated the effect of LLL (670 nm) and htMSCs (human tubal-derived Mesenchymal Stem Cells) in the treatment of cigarette-induced pulmonary inflammation in mice. C57Bl/6 mice were submitted to cigarette smoke for 75 days (2 times/day). Next, animals were treated with LLL (COPD+LLL group) or htMSCs (COPD+htMSCs group) 7 and 15 days before the experiment, on day 76th. Animals were sacrified for morphologic and functional analysis of the lung. Bronchoalveolar lavage (BAL) analysis showed that COPD+LLL and COPD+htMSCs groups had a significant decrease in neutrophils and lymphocytes when compared to COPD group. This was associated with reduced mucous secretion and collagen deposition. Moreover, LLL significantly reduced IL-6 and TNF-alpha, which was not observed aftetr htMSCs. In vitro experiments showed reduction in IL-6 secretion by bronchial epithelial cells (BEC) and TNF-alpha by pneumocytes type II (PII) exposed to cigarette extract at 2,5% and further irradiated with LLL. These results indicate that LLL and htMSCs therapies have anti-inflammatory effect on cigarette-induced lung inflammation reducing local inflammation and may be considered interesting therapeutic approaches.
Oral Salmonella expressing Colonization Factor Antigen I stimulates the rapid induction of Galectin-1 on induced Treg cells (P4204)
Authors: Jun Sangmu , Hoffman Carol , Hoyt Teri , Pascual David
Published: January 2023

Galectin-1 (Gal-1) is one of the 15 Galectin family members that bind to β-galactoside. Gal-1 binding to various cell surface glycoproteins leads to deletion of T effector functions. Gal-1 engagement to CD43 on APCs converts these cells to tolerogenic supporting the generation of Treg cells. Our previous work have shown that oral Salmonella-CFA/I strain H696 (expresses CFA/I fimbriae from enterotoxigenic E. coli) induces a highly suppressive Foxp3+ CD25+CD4+ Treg cells capable of suppressing the development of experimental autoimmune encephalomyelitis (EAE). Gal-1 expression was rapidly induced within 3 days by both Foxp3+ Treg cells obtained from Salmonella-CFA/I-immunized mice unlike mice vaccinated with isogenic Salmonella vaccine (strain H647) control. To assess the function of Gal-1, isolated Treg cells from H696- and H647-vaccinated mice were assessed for their ability to suppress OT-2 OVA-specific effector T cell proliferation following r estimulation with OVA peptide. Upon neutralization of Gal-1, Salmonella-CFA/I-induced Treg cells were unable to suppress OT-2 cell proliferation; similar neutralization of Salmonella vector-derived Treg cells had no effect. Kinetic analysis of Gal-1 expression by dendritic cells (DCs) following H696 imminization also showed early induction of Gal-1 suggesting the importance of Gal-1 to induce tolerogenic T cells and DCs. These results suggest Gal-1 is a major effector molecule for the Salmonella-CFA/I-induced Treg cells.
CD8 T cell exhaustion is influenced by genetic background (P6076)
Authors: Christiaansen Allison , Boggiatto Paola , Hartwig Stacey , Varga Steven
Published: January 2023

Lymphocyte choriomeningitis virus (LCMV) induces a robust CD8 T cell response allowing for viral clearance and limited disease following an acute LCMV Armstrong (Arm) infection in both C57BL/6 (B6) and BALB/c mice. In contrast, infection of B6 mice with LCMV Clone-13 (Cl-13) results in CD8 T cell exhaustion leading to the establishment of a chronic infection. We examined Cl-13 infection of BALB/c mice and observed a does-dependent increase in disease following infection. BALB/c mice infected with either Arm or a low dose of Cl-13 induced a robust NP118-126-specific CD8 T cell response whereas infection with a high dose of Cl-13 significantly blunted the induction of the NP118-126-specific CD8 T cell response. In addition, BALB/c mice infected with a high dose of Cl-13 failed to control the infection with prolonged virus replication in multiple peripheral organs. The more focused immunodominant CD8 T cell response elicited by LCMV in BALB/c versus B6 mice does not appear to account for the increased disease following high dose Cl-13 infection. Cl-13 infected B10.D2 mice exhibit an increased NP118-126-specific CD8 T cell response as compared to BALB/c infected mice. Furthermore, BALB.B mice exhibit a reduced magnitude of CD8 T cells specific to the three immunodominant epitopes as compared to B6 mice. Our results indicate that non-MHC genes influence the rate of CD8 T cell exhaustion and subsequent disease following a chronic Cl-13 infection.
SHP-1 deficiency does not restore the CD8+ T cell response to an LCMV viral variant (P6079)
Authors: Shorter Shayla , Evavold Brian
Published: January 2023

Viruses employ various mechanisms to evade recognition by host immune effectors and establish persistence in the host. We have previously shown that CD8+ T cells responding to a viral variant of lymphocytic choriomeningitis virus (LCMV) have high SHP-1 activity. Src homology region 2 domain-containing phosphatase-1 (SHP-1) is a non-receptor phosphatase found in hematopoietic cells and is thought to play a primarily negative regulatory role in many signaling pathways. SHP-1 exerts important negative regulation on proximal TCR signaling by blocking early T cell activation. Because of its role in dampening TCR signaling, we hypothesized that removal of SHP-1 in T cells would restore the response to the viral variant. Using CD4 Cre Fl SHP-1 mice, we evaluated the generation, activation and functionality of CD8+ T cells generated in response to the viral escape mutant. Loss of SHP-1 did not greatly affect response of the P14 TCR Tg cells line assessed in vitro. In vivo analysis of the response to the viral escape mutant did not demonstrate increased T cell numbers or functionality (IFNg secretion) in response to the viral escape mutant or the control chronic viral LCMV infection. Polyclonal T cell responses to the viral challenges were unaffected by SHP-1 deficient. These observations suggest that removal of a negative regulator may not have the expected therapeutic benefit of an enhanced T cell response.
Selective requirement of Ikaros zinc fingers in Treg and Th17 fate decision. (P1137)
Authors: Heller Jennifer , Schjerven Hilde , Qiu Ju , Lee Aileen , Smale Stephen , Zhou Liang
Published: January 2023

TGF-β is a common factor important for the differentiation of pro-inflammatory Th17 and anti-inflammatory inducible Treg cells. However, the precise molecular mechanisms underlying the fate decision of differentiating CD4+ T cells in the presence of TGF-β is poorly understood. Here, we show that distinctive N-terminal DNA-binding zinc fingers of Ikaros play essential roles in Treg and Th17 fate decision. Ikaros has a highly conserved DNA-binding domain near the N-terminus with four tandem zinc fingers. Zinc fingers 2 and 3 are required for stable binding to DNA, whereas fingers 1 and 4 appear to be important for differentially modulating binding properties to specific sites at target genes. Our data show that T cells lacking Ikaros zinc finger 4 but not 1 failed to differentiate into Foxp3+ Tregs upon TGF-β stimulation. Instead, TGF-β-skewed Ikaros zinc finger 4 mutant cells displayed aberrant upregulation of Th17-associated cytokines IL-17 and IL-22. IL-17 but not IL-22 upregulation is dependent on transcription factor RORγt. Aryl hydrocarbon receptor, an essential transcription factor required for IL-22 expression, was unexpectedly decreased. Together, our data uncover a novel selective requirement for Ikaros zinc fingers in the differentiation of Treg and Th17 cells and an intricate interplay among various transcription factors in programming Th17/Treg lineages. We are currently examining the role of Ikaros zinc finger 4 in infection and autoimmunity.
B-lymphocyte induced maturation protein 1 (Blimp-1) is required to limit the number of IL17A-producing CD4+ T cells in vivo. (P1139)
Authors: Bankoti Rashmi , Salehi Soofia , Benevides Luciana , Couse Michael , Silva Joao , Dhall Deepti , Meffre Eric , Targan Stephan , Martins Gislaine
Published: January 2023

Blimp-1 is a transcription factor required for embryonic development and immunity. Mice with T-cell-specific deletion of Blimp-1 (Blimp-1CKO) develop severe intestinal inflammation apparently mediated by CD4+Th cells, but little is known about Blimp-1’s role in regulating Th cell differentiation in vivo. Here we show that Blimp-1 restrains Th cell production of IL17 and IFNγ in vivo. Blimp-1CKO mice accumulate IL17 and IFNγ-producing TCRβ+CD4+cells in lymphoid organs and intestinal mucosa. Increased numbers of IL17-producing Th cells are also observed in WT and Blimp-1CKO-mixed BM chimeric mice containing WT regulatory T (treg) cells, suggesting a Treg-independent intrinsic role for Blimp-1 in constraining IL17production. Blimp-1-/-naïve CD4+Tcells are more prone to differentiate into IL17+IFNγ+ cells and cause severe colitis when transferred to Rag1-/- mice. In vitro-stimulated Blimp-1-/- CD4+T cells show increased expression of il17a and other Th17-related genes, suggesting that Blimp-1 could inhibit transcription of Th17 genes. Using ChIP assays we identified at least one site at the il17a gene that can be bound by Blimp-1 in Th cells, suggesting that Blimp-1 could function as a direct repressor of il17a. Single cell analysis reveals that Blimp-1 expression is selectively regulated during Th differentiation and directly correlates with suppression of Th17 traits in Th1cells. Collectively, these results establish a new role for Blimp-1 in regulating IL17 production in vivo
Effects of intracellular communication between T helper cells on Th2 differantiation (P1149)
Authors: Reich-Zeliger Shlomit , Antebi Yaron , Rimer Jacob , Hoberman-Arieli Tamar , Friedman Nir
Published: January 2023

Cytokines provide a main regulatory mechanism of immune responses, which allows for intercellular communication and enables cells to make collective decisions. Existing evidence for stochastiticity in expression of cytokines and their receptors raises the question: how does cytokine communication performs reliably in the face of noise? We study the effect of heterogeneity in IL4R expression level during Th2 differentiation as a model system. Naïve CD4 T cells were separated into different groups according to their IL4R expression levels and the different groups were cultured separately, under different IL4 concentrations driving their differentiation. Expression levels of Th2 markers, IL4 and GATA3, were measured at the single cell level. Our results reveal differences in the differentiation abilities of the different groups - higher initial receptor levels facilitate faster Th2 differentiation, at lower levels of IL4 as an input. Interestingly, we found that these differences in the differentiation abilities vanish when cells with high and low initial IL4R levels are co-cultured. These results suggest that intercellular interactions can decrease the potential heterogeneity in the population that results from noise in gene expression. These experiments provide an opportunity to evaluate the importance and roles played by intercellular communication in Th cell differentiation, and to assess the effects of stochasticity in protein levels on the differentiation process.
Analysis of factors that induce memory T cells. (P1157)
Authors: Soh Tetsuyoshi , Oki Yutaro , Uchiumi Fumiaki , Kishimoto Hidehiro
Published: January 2023

Naïve T cells recirculate between blood, lymph ducts and secondary lymphoid organs. Consequently, it should be different time that required for Ag-specific T cells, which are located in the various tissues, to populate and enter the draining lymph nodes in late time of the immune response. Late coming T cells were also primed in different environments and differentiated into memory T cells. We have investigated the kinetics of both OVA specific TCR Tg (OT-1 and OT-2) T cells stimulated earlier (early-stimulated T cells) and later in an immune response (late-stimulated T cells). To ascertain above studies, naïve T cells isolated from lymph nodes of OT-1/CD45.2+ mice and OT-2/CD45.2+ mice were injected i.v. into CD45.1+/CD45.2+ B6 host mice. For immunization of mice, ex-vivo expanded DCs that were induced from murine bone marrow cells by culturing with GM-CSF were incubated with OVA protein and LPS, and were injected s.c. into host mice. The early-stimulated T cells were developed by adoptive-transfer of Tg T cells into host mice before Ag-pulsed activated DCs were injected and the late-stimulated T cells were developed by adoptive-transfer of Tg T cells into mice 5-7 days after injection of Ag-pulsed activated DCs. The late-stimulated T cells divide slower and proliferate less than the early-stimulated T cells. These results suggest that the timing of recruitment of T cells into the draining lymph nodes affects T cells-fate that are influenced by different environments.
BCAP, a TLR/IL1R signaling adapter, intrinsically regulates differentiation of T helper cells (P1159)
Authors: Troutman Ty , Brewer Travis , Pasare Chandrashekhar
Published: January 2023

T helper cells utilize IL18 and IL1 signals provided by myeloid cells to achieve optimal Th1 and Th17 effector capacities. The IL1 and IL18 receptors belong to a family of receptors including Toll-like receptors (TLRs). The TLR/IL1R family share signaling components and (with the exception of TLR3) depend on the adapter MyD88 for engagement of downstream pathways. Signals from the receptor to the adapter are transmitted through homotypic interaction of TIR (Toll-Interleukin-1 receptor) homology domains found in all TLR/IL1R family members and their adapters. We have previously shown that BCAP (B-cell adapter for PI3K) contains a functional TIR domain enabling its participation in the TLR signaling pathway. Importantly, BCAP plays an obligate role in linking TLRs to activation of phosphoinositide 3-kinase (PI3K). Because the TLR/IL1R family share signaling components, we hypothesized that BCAP may regulate T cell differentiation through IL1R family signaling. Indeed, we found that BCAP intrinsically regulated differentiation of naïve T cells towards Th1 and Th17 effector lineages by participation in the IL1R family signaling pathways. Further, BCAP intrinsically regulated T cell proliferation and survival. Our continued efforts will further explore the molecular and transcriptional mechanisms by which BCAP regulates T cell activation. Significantly, this work will increase the understanding of key signaling pathways involved in disease and inflammation.