bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of costimulation in obesity-induced adipose inflammation and insulin resistance: pro- or anti-inflammatory? (P3105)
Authors: Zhong Jixin , Rao Xiaoquan , Braunstein Zachary , Sun Qinghua , Rajagopalan Sanjay
Published: January 2023

Activation of T cells is thought to play an increasingly important role in the pathogenesis of inflammation and insulin resistance in obesity and Type 2 diabetes. Costimulation is essential for amplification of the T cell response, with the co-stimulatory molecules CD80 and CD86 playing an important role in the pathogenesis of a variety of inflammatory diseases. In this study, we used CD80/CD86 double knockout (B7 KO) mice to investigate the involvement of these key molecules in diet-induced obesity (DIO) and insulin resistance. Wild-type (WT) and B7 KO mice were fed either high fat diet (HFD) or normal chow. After 12 weeks of HFD feeding, WT and B7 KO mice had similar weight gain. B7 KO mice had less epidydymal fat (1.31±0.04 g vs. 0.92±0.06 g for WT vs. B7 KO) which was confirmed by MRI measures. Despite less systemic T cell activation, insulin resistance was significantly worse in the B7 KO mice. B7 KO mice had decreased Treg content which was a result of both impaired development and proliferation. A defect in Treg cells conferred enhanced classical activation of macrophages in the adipose tissue. Adoptive transfer of Treg into B7 KO mice restored insulin sensitivity in B7 KO mice on HFD. Taken together, lack of B7-mediated costimulation results in impaired development and proliferation of Treg and enhanced adipose inflammation and insulin resistance. Our results suggest an essential role for B7 in maintaining Treg numbers in experimental DIO.
CXCR6/CXCL16 in liver disease (P3145)
Authors: Parker Richard , Weston Chris , Adams David
Published: January 2023

Chemokines primarily mediate lymphocyte migration. Steatohepatitis caused by metabolic syndrome or alcohol is a common cause of liver disease. We investigated the role of CXCL16/CXCR6 in the development of steatohepatitis. Expression of CXCL16 in liver and isolated cells was analysed with rt-PCR and immunohistochemistry. Serum and supernatant concentrations of CXCL16 were measured with ELISA. Expression of CXCR6 was investigated with flow cytometry. Lymphocyte adhesion was assessed using freshly isolated lymphocytes from liver or peripheral blood flowed over a confluent layer of isolated human hepatic sinusoidal endothelium (HSEC). Whole liver expression of CXCL16 was increased in disease with increasing expression with increasing severity of disease. Isolated HSEC, biliary epithelial cells (BEC) and hepatoma cell lines increased expression of CXCL16 and released soluble CXCR6 in response to proinflammatory cytokines, particularly the combination of TNFα and IFNγ. Liver infiltrating lymphocytes and monocytes were enriched for CXCR6 expression compared to peripheral blood. CD3+CD56+ NKT cells and CD56+ NK cells most commonly expressed CXCR6. Inhibition of CXCR6 or CXCL16 inhibited transmigration of lymphocytes across HSEC. Transmigration was increased by incubating lymphocytes with soluble CXCL16. CXCL16 is expressed in diseased liver where it plays a role in the transmigration of lymphocytes across endothelium. This may represent a new therapeutic target in liver disease.
The stenotic kidney of Rag1 mice subjected to 2K1C hypertension is protected from atrophy (P3078)
Authors: Hartono Stella , Grande Joseph
Published: January 2023

Renal artery stenosis is a common finding in patients with cardiovascular disease. Recent studies have provided evidence that innate immunity plays a critical role in the development of chronic renal disease in renovascular hypertension. We have previously demonstrated that interstitial infiltration of lymphocytes is an early event in the development of renal atrophy in the 2K1C model of renovascular hypertension. We therefore sought to test the hypothesis that lymphocytes play a role in the development of progressive atrophy/fibrosis in this model. We subjected mice to renal artery stenosis through placement of a polytetrafluoroethylene cuff on the right renal artery. In accordance with our previous observations, wild type mice subjected to RAS developed renal atrophy within 2 weeks following placement of the cuff (70.5 +/- 11.9%). Chemokine expression in renal cortex of the stenotic kidney reflected predominantly a Th1 response (STAT1, STAT4, TBX21); CCL2, CCL17, CCL12, and TNF mRNA expression were also upregulated, but not IL-6 and IL-10. In striking contrast to wild type mice, Rag1 mice developed significantly less atrophy (23.1 +/- 10.4, p<0.0037 vs wild type). Unlike wild type mice, histologic evaluation of renal cortex of Rag1 mice showed no significant interstitial infiltration with CD3+ T cells or F4/80+ macrophages. We propose that T cell-mediated inflammation plays a critical role in the development of renal atrophy in 2K1C mice.
HMGB1 impairs erythropoiesis during sepsis (P3163)
Authors: Valdes-Ferrer Sergio , Blanc Lionel , Li Jian Hua , Sangeeta Chavan , Tracey Kevin
Published: January 2023

Severe sepsis is one of the leading causes of mortality and disability. Anemia is common in acute sepsis and related to decreased quality of life and increased mortality in sepsis survivors. Serum levels of HMGB1, a critical mediator of sepsis pathophysiology, remain elevated for weeks after onset of sepsis. We used a murine model of severe abdominal sepsis to study effect of HMGB1 on erythropoesis. Mice subjected to sepsis have significant decrease in hemoglobin (p=0.03 by day 3) and hematocrit (p=0.05 by day 7). Reticulocytosis, an indicator of bone marrow proliferation, was only transiently found on day 10. Administration of neutralizing anti-HMGB1 antibody to survivors beginning 9 days post- sepsis significantly improved hemoglobin and hematocrit levels. Exposure of erythropoetic stem cells, generated by culturing cord blood CD34+ cells with erythropoietin, stem cell factor and IL-3, to recombinant HMGB1 (rHMGB1) induced a decrease in hemoglobinization, and expression of GPA (CD235a) and transferrin receptor (CD71), indicating suppression of erythroid differentiation. rHMGB1 induced suppression of erythropoesis when added before day 8 and not later indicating that HMGB1 inhibits initial phases of differentiation. Together, these findings indicate that HMGB1 impairs erythropoiesis by altering differentiation of erythropoetic stem cells, and suggest that it may be possible to prevent or reverse anemia in sepsis survivors by administration of anti-HMGB1 antibodies.
Immune dysregulation in a major hematologic disease (P3167)
Authors: Joshi Sunil , Carey David , Chavez-Suarez Maria , Lang Mark , Broyles Robert , White Gary
Published: January 2023

Sickle cell disease is the most prevalent genetic disease on the planet, and is a major cause of morbidity and death. Much of the morbidity in sickle cell disease is due to frequent, severe infections due to immune dysregulation associated with progression of disease. Both innate and adaptive immunity are impaired. It is well established that T and B cell responses are required for host defense. The CD4+ Th cells express TCR that recognize antigen bound to MHC II molecules expressed on APCs such as DCs, B cells and macrophages. Thus the cognate interaction between receptor-ligand clusters on Th and APCs led to the activation of naïve CD4+ Th cells causes them to release cytokines, which influence the activity of many cell types, including the NKT, CD8+ CTLs and the APCs that activated them. Using a “humanized” sickle mouse model of sickle cell disease, we recently found that the transition from SPF to ‘dirty’ (conventional) housing significantly dysregulated the immune-system. These changes are characterized by a hyper-inflammatory state with the activation of innate-immune response (upregulation of TLR4) and loss of antigen-presentation machinery (loss of MHC class II expression) as the disease progresses. Specifically, our results indicate the loss of MHC II expression on APCs from various tissue of sickle mouse with the disease progression.
Resveratrol blunts unwanted platelet activation that occurs during platelet storage and as a consequence of red blood cell transfusion (P3171)
Authors: Lannan Katie , Spinelli Sherry , Casey Ann , Blumberg Neil , Phipps Richard
Published: January 2023

Blood transfusions are a common medical treatment dating back to the early 19th century and are now widely accepted as a life saving practice. However, red blood cell and platelet transfusions are associated with alterations in hemostasis and can lead to platelet-mediated adverse outcomes, such as thrombosis, immune dysregulation and inflammation. Platelets are intimately involved in hemostasis and inflammation, and can cause severe problems when excessively or insufficiently activated. A major unmet medical need in the field of transfusion medicine is to develop new agents that safely prevent unwanted platelet activation in transfused patients and in patients with underlying cardiovascular complications. Here, we investigate the potential of naturally occurring compounds to be used as novel anti-platelet agents, which could be used to mitigate transfusion complications. Naturally occuring compounds, such as resveratrol, have been suggested to lower the risk of cardiovascular complications. We demonstrate the ability of resveratrol to dampen unwanted platelet activation, which can occur after RBC transfusion. Additionally, we demonstrate that resveratrol can be used to effectively dampen platelet activation during the storage of platelet concentrates for transfusion. Resveratrol, as well as other natural compounds, could be used to mitigate platelet-mediated adverse transfusion reactions and could be used to improve the safety and efficacy of stored platelet concentrates.
Moyamoya syndrome is associated with production of autoantibodies to a broad spectrum of self-antigens and correlated with disease phenotype (P3175)
Authors: Li Quanzhen , Wang Ling , Zhang Zhengshan , Duan Lian
Published: January 2023

Moyamoya syndrome (MMS) is a chronic cerebrovascular disorder characterized by a slowly progressive occlusion of the internal carotid artery. Previous study suggested that immune disorder and autoantibody production may involve in the pathogenesis of this disease. In order to elucidate the correlation of the autoAbs with the disease phenotype and to identify possible biomarkers, we utilized an autoantigen microarray bearing 83 specificities to characterize IgG and IgM autoantibodies expressed in the sera of MMS patients (n=56) and healthy controls (HC, n=31). 13 IgG autoantibodies and 35 IgM autoantibodies were significantly increased in MMS patients compared with HC (p<0.05). The increased expression of anti-MPO, anti-MBP and anti-PR3 in MMS sera were further confirmed by ELISA. Cluster analysis identified 4 autoAb patterns including chromosome related (anti-centromere and histone), ECM related (anti-matrigel, laminin), RNA related (anti-Ro60 and SSA/SSB) and thyroid related (anti-thyroglobulin and TPO) antibodies which were increased in MMS and correlated with different clinical manifestations including course of disease, number of involved brain vessels and thyroid activity, etc. Furthermore, MMS sera exhibited positive reaction with the patient brain vessels by IHC staining. The above findings indicate that IgG and IgM autoAbs may play an important role in the pathogenesis of MMS and could be useful biomarkers for the prediction of the disease
The role of syndecan-4 in the development of rheumatoid arthritis (P4052)
Authors: Endo Tsutomu , Morimoto Junko , Ito Koyu , Iwasaki Norimasa , Uede Toshimitsu
Published: January 2023

Syndecan-4 (Syn4), a cell-surface heparan sulfate proteoglycan (HSPGs) expressed by several types of cells, has been implicated as a critical mediator for inflammatory responses. Recent report suggested that HSPGs including Syn4 expressed by B lymphocytes regulate B cell development and survival. Here, we have investigated the role of Syn4 in the development of murine models of Rheumatoid Arthritis. In type-II collagen-induced arthritis (CIA) model, which is both T and B cell-dependent model, we found that Syn4-deficiency showed attenuated clinical severity and pathological features, although Syn4-deficient (Syn4-KO) mice generated unimpaired numbers of Th1 and Th17 cells in the draining inguinal LN (dLN). Because Syn4 expression was highly detectable on B cells, we analyzed type-II collagen-specific antibody (Ab) production, and found that Syn4-KO mice generated comparable levels of IgM, but significantly reduced levels of total IgG, IgG2a and IgG2b compared with WT mice. Reduction of collagen-specific IgG Abs in Syn4-KO mice was also evident by defective formation of germinal center (GC) in the dLN. In addition, we found that Syn4-KO mice showed reduced numbers of B cell subsets (total B cells, follicular B cells and GC B cells) in the dLN following immunization, although the numbers of follicular helper T cells were comparable between both groups. These results suggest that Syn4 modulates CIA development through enhancing the generation of GC formation and Ab production.
Role of the TAM receptor Axl in differentiation and activation of B cells (P4058)
Authors: Nundel Kerstin , Rothlin Carla , Marshak-Rothstein Ann
Published: January 2023

In dendritic cells (DCs), TLR activation upregulates TAM receptor expression and these TAM receptors initiated pathways subsequently shut down cytokine production. Moreover, TAM receptor deficient mice develop a severe SLE-like disease. TLR9 and TLR7 play a key role in the detection of autoantigens by both DCs and B cells in the context of systemic autoimmunity. To better understand the direct effects of TAM receptors on B cells, we have used BCR Tg mice (AM14) that recognize autologous IgG2a. AM14 B cells are activated by immune complexes (ICs) that incorporate either DNA or RNA through pathways dependent on TLR9 and TLR7, respectively. Contrary to DCs, expression of the TAM receptor Axl is not induced by TLR ligands but is dependent on crosslinking of the BCR per se. However, BCR/TLR9, but not BCR/TLR7, co-engagement inhibits Axl upregulation, pointing to a role for TLR9 in the regulation of Axl expression. Activation of AM14 B cells by RNA-containing ICs induces TLR7-dependent differentiation into plasma cells and these plasma cells express high levels of Axl. In contrast, BCR/TLR9-dependent activation of AM14 B cells by DNA-containing ICs blocks Axl expression and does not lead to plasma cell differentiation. These data identify unique outcomes of BCR/TLR7 and BCR/TLR9 co-engagement on B cell differentiation and Axl expression. Furthermore, these B cell intrinsic responses may account for the opposing roles of TLR7 and TLR9 in the regulation of systemic autoimmunity.
FLEx-autoAb transgenic mice, a novel mouse model for studying peripheral tolerance to B cells that acquire self-reactivity following somatic hypermutation. (P4034)
Authors: Koh Yi , Nemazee David , Rickert Robert , Radic Marko , Cascalho Marilia , Casellas Rafael , Theofilopoulos Argyrios , Kono Dwight
Published: January 2023

Uptake of nucleic acid-containing antigens by antigen receptors (BCRs) on B cells leading to endosomal TLR activation is thought to be critical for antinuclear autoantibody (ANA) production in lupus. However, it has been difficult to study this in peripheral B cells that acquire ANA specificity because current ANA BCR models are subjected to central tolerance mechanisms. To overcome this, we generated a FLEx-autoantibody (autoAb) transgenic model in which VH specificity can be changed from a non-self-reactive anti-nitrophenyl (NP) to anti-chromatin (3H9-56R) reactivity following Cre recombinase expression induced by tamoxifen (all B cells) or the activation induced cytidine deaminase promoter (during class-switch recombination/somatic hypermutation). In nonautoimmune C57BL/6 mice, B cells that acquire self-reactivity to chromatin were completely deleted after several days at all stages of development, although numbers of plasma cells were too low to analyze. Faslpr mutation enabled the development of only low levels of anti-id positive (56R+) plasma cells but did not rescue B cells at any other stages of development after tamoxifen. The provision of excess T cell help, through induction of chronic graft-versus-host disease in B6-Faslpr mice resulted in a marked increase in 56R+ plasma cells. These findings suggest that ANA-specificity and engagement of endogenous TLR is not sufficient to rescue autoreactive B cells even in mice with Fas deficiency.
IgG immune complexes promote systemic lupus erythematosus by elevating BAFF and inducing T cell infiltration into the glomeruli (P4036)
Authors: Kang SunAh , Fedoriw George , Jiang Chuancang , Diaz Marilyn , Vilen Barbara J.
Published: January 2023

In systemic lupus erythematosus, the presence of autoantibodies, and the formation of immune complexes are closely related to disease pathogenesis. Although the deposition of immune complexes in the kidneys is a characteristic of SLE, the impact of immune complexes during the onset of disease remains unclear. Here, we show that FcγR/IgG-bound immune complexes containing nuclear self-antigens are displayed on DCs and MFs from C57BL/6 mice at low levels (3-fold), while increased in MRL/lpr mice (10-15 fold). To investigate the biological function of accumulated IgG-immune complexes, we developed a passive antibody transfer system using AID-/-MRL/lpr mice. When treated with anti-nucleosome antibody (IgG), AID-/-MRL/lpr mice accumulated IgG-immune complexes on DCs and MFs, coincident with a dramatic increase in the numbers of splenic B cells, BAFF-secreting DCs, and serum autoantibody titers. The mice also developed lupus nephritis reminiscent of MRL/lpr mice, and showed infiltration of T cells into the glomeruli. These effects were not observed in MRL/lpr mice lacking FcγRI. Our data suggest a mechanistic link between IgG-immune complexes, BAFF secretion, and glomerular infiltration of immune cells. To investigate the role of BAFF in autoimmunity and T cell infiltration into the glomeruli, we will partially neutralize BAFF to restore serum BAFF levels to that of non-diseased mice. This will allow us to define the role of BAFF in autoimmunity, and lupus nephritis.
Cre-based deletion of COX-2 within B cells in vitro: steps toward specific nanoparticle-driven COX-2 inactivation in vivo. (P4044)
Authors: Haque Shabirul , Li Jianhua , de Marco Ario , Ishikawa Tomo-o , Herschman Harvey , Spretz Ruben , Noriega Sandra , Nunez Luis , Mongini Patricia
Published: January 2023

Several lines of evidence indicate that COX-2 and downstream prostaglandin (PG) positively influence germinal center development and Ig heavy chain class switching. Although activated B cells upregulate COX-2 and downstream effector molecules, it is unclear whether autocrine production of PG is a major driver of B cell autoimmunity. One approach to investigate this is through Cre-mediated, B cell lineage-specific COX-2flox recombination in mice prone to autoimmunity. Because Cre insertion into B lineage genes, CD19 and mb-1, compromises expression of molecules needed for BCR-triggered autoimmunity, we are seeking to achieve B cell-specific Cre-Lox recombination through an alternative route: B cell-specific NP bearing Cre as a payload. We here report that purified recombinant Cre protein rapidly achieves specific in vitro recombination within PCR amplicons of COX-2 with inserted pLox sites. Furthermore, one hour exposure of viable splenocytes from NOD.B10 COX-2flox mice to Cre protein at 37oC results in ~ 90% recombination. Cells treated with active Cre and subsequently stimulated with BCR-L, IL-4 and BAFF, display significantly less COX-2 protein upon reculture than cells similarly treated with heat-inactivated Cre. We additionally report the preparation of CD22-specific nanoparticles composed of PEG and PLA. In future studies, similar NP will be employed to target Cre specifically to B cells in Sjogren’s Syndrome-susceptible NOD.B10 COX-2flox mice.
The development of genetically modified K562 cells as artificial antigen presenting cells for NKT cells (P4310)
Authors: Tian Gengwen , Jena Bipulendu , Liu Daofeng , Heczey Andras , Torikai Hiroki , Lee Dean , Cooper Laurence , Metelitsa Leonid
Published: January 2023

CD1d-restricted type-I Natural Killer T cells (NKTs) have been shown to mediate anti-tumor immune responses in mouse models and are associated with improved outcome in several types of cancer. However, the therapeutic application of NKTs has been limited by low numbers and functional defects of these cells in patients with cancer. To provide a means for effective ex-vivo NKT-cell expansion for cell and gene therapy applications we explored native and engineered properties of K562 cells to function as artificial antigen presenting cells (aAPC). K562 cells express an activating allele of HLA-C and propagate NK cells which compete with NKTs in the same culture. Therefore, we used zinc finger nucleases to delete the endogenous HLA-C gene in K562 cells and transduced parental and HLA-Cnull K562 cells with CD1d cDNA followed by single cell sorting and clonal expansion. The clones exhibited a range of CD1d expression. They were pulsed with a NKT ligand, αGalactosylceramide and tested as aAPC for NKTs using CFSE proliferation assay. We found that aAPC clones with an intermediate level of CD1d expression induced the highest rate of NKT-cell proliferation. No difference was observed between K562/CD1d and HLA-CnullK562/CD1d clones when purified NKTs were used as responders. In contrast to K562/CD1d, HLA-CnullK562/CD1d clones did not expand NK cells when added to primary PBMC. Therefore, these HLAnullCD1d+ aAPC can be used to selectively expand primary NKTs for adoptive cell therapy.
Development of a fluorescent dual-cytokine cellular immune response assay: preliminary results from sipuleucel-T treated subjects (P4312)
Authors: Wei Jessica , Perdue Nikole , Sheikh Nadeem , Dela Rosa Corazon , Trager James
Published: January 2023

In the Phase III IMPACT study evaluating sipuleucel-T, an immunotherapy for asymptomatic or minimally symptomatic metastatic castrate resistant prostate cancer, post-treatment antigen-specific cellular responses were detected using an IFNγ ELISPOT assay. While this assay is a robust readout of memory responses, it only detects one cytokine, providing minimal information of the responding cells. As clinical trial samples are limited, simultaneous detection of multiple cytokines is a pragmatic alternative that provides additional information without using more cells. The UV-fluorescent FluoroSpot assay detects multiple cytokines, providing an understanding of the phenotype of the immune response. FluoroSpot results were concordant to the ELISPOT data; antigen-specific IFNγ responses were seen after treatment in sipuleucel-T subjects, indicating treatment produced immunological memory. However, we saw that IL-2 secreting cells were maintained throughout treatment, whereas IL-5 secreting (Th2) cells decreased after the first infusion. Most importantly, the FluoroSpot assay showed the generation of polyfunctional T cells (IFNγ/IL-2 and IFNγ/IL-5) after treatment. The presence of the distinct dual cytokine functional T cell signatures supports the supposition that sipuleucel-T induces proliferative antigen-specific T cell immunological responses. The multi-analyte FluoroSpot assay may provide insight into the roles of other different cell populations after sipuleucel-T treatment.
IL-33 precursor regulates inflammation in vivo in a T1/ST2-independent, Th2-independent fashion (P6270)
Authors: Atamas Sergei , Lockatell Virginia , Kopach Pavel , Todd Nevins , Luzina Irina
Published: January 2023

IL-33 precursor (IL33PR) released from damaged cells is known to bind to the receptor T1/ST2 and activate cells in culture. However, we found that in patients with interstitial lung diseases (ILD) and in the bleomycin mouse model of lung injury, pulmonary expression of IL33PR is elevated intracellularly and intranuclearly. Recombinant adenoviral gene delivery of IL33PR to mouse lungs in vivo did not lead to IL-33 maturation and produced a phenotype that was strikingly different from that induced by mature IL33 (MIL33). IL33PR induced pulmonary lymphocytosis and neutrophilia, whereas MIL33 induced eosinophilia, goblet cell hyperplasia, and increases in Th2 cytokines. The effects of MIL33 were, but the effects of IL33PR were not, abrogated by gene deficiency of T1/ST2. When combined with bleomycin injury, IL33PR potentiated lymphocytosis, collagen accumulation, and the expression of proinflammatory and profibrotic cytokines TGF-β, IL-6, MCP-1, and MIP-1α in the lungs. Gene expression profiling in cultured fibroblasts and epithelial cells overexpressing IL33PR revealed 10 - 100 fold increases in HSP70 mRNA, further confirmed by RT-Q-PCR. Previous reports have associated HSP70 with ILD. Immunohistochemical analyses showed strong HSP70 protein increases in mice challenged with bleomycin, IL33PR gene delivery, and particularly in mice challenged with these factors combined. Thus, IL33PR promotes ILD by inducing proinflammatory and profibrotic cytokines and HSP70.
Lymphopenia modulates levels of STAT1 expression leading to enhanced CD4 T cell responsiveness to Type-I IFN (P6272)
Authors: Le Saout Cecile , Hasley Rebecca , Imamichi Hiromi , Tcheung Lueng , Luckey Megan , Smith Mindy , Hu Zonghui , Sneller Michael , Rehm Catherine , Lane H. , Catalfamo Marta
Published: January 2023

To maintain the size of T cell pools throughout life, homeostatic mechanisms regulate both survival and proliferation through IL-7 and TCR signaling. IL-7 (through lymphopenia-induced proliferation/LIP) can also mediate expansion of autoreactive clones leading to the onset of autoimmune disease or enhance effector function against tumors and viruses. In the present study, we hypothesized that in a setting of chronic inflammatory environment, such as HIV infection, LIP could contribute to the immune activation leading to CD4 T cell depletion and CD8 T cell expansion. Type-I IFN is an essential cytokine in host defense during viral infection. In HIV infected patients, the genes associated with γc cytokines and Type-I IFN signaling were differentially expressed in CD4 and CD8 T cells. Using a lymphopenic murine model, we present evidence that, in vivo, IL-7 differentially regulated the expression of the total-Signal Transducer and Activator of Transcription 1 (t-STAT1) in CD4 and CD8 T cells undergoing LIP and in so doing enhanced CD4 T cell responsiveness to Type-I IFN. In this setting, chronic treatment with IFN-α led to decreased CD4 T cell counts and CD8 T cell expansion. This interplay between IL-7 and Type-I IFN might be advantageous in immunity against pathogens, however chronic stimulation of this pathway could be deleterious for CD4 T cell homeostasis and may contribute to the aberrant immune activation and eventual CD4 T cell depletion observed during HIV infection.
Blocking of PD-L1 on therapeutic dendritic cells modulate in vitro T cells proliferation and differentiation to Th1 and Th17 subpopulations (P4230)
Authors: Lopez Mercedes , Behrens Carolina , Falcon-Beas Cristian , Tempio Fabian , Salazar-Onfray Flavio
Published: January 2023

DCs constitute a promising treatment against melanoma. Recently, we showed the effectiveness of an allogeneic melanoma lysate DC-based immunotherapy for improving long-term survival in melanoma patients. Moreover, an elevated proportion of regulatory T lymphocytes (Tregs) was observed in non-responder patients. PD-L1 is an important co-inhibitory molecule expressed on DCs binding to PD-1 on T cells. This interaction promotes the induction, conversion and maintenance of Tregs, suggesting a role in immunosuppression. Here, we studied if the blockade of PD-1-PD-L1 interaction reverses Tregs differentiation and increases T helper populations. Monocyte-DCs were generated in the presence of pro and anti-inflammatory factors, and the PD-L1/PD-1 signaling was blocked by anti PD-L1 mAbs. Allo-stimulatory capacity of DCs was impaired, and inversely correlated with PD-L1/CD86 ratio. However, blocking PD-L1 on DCs increased of Th1 and Th17 proliferation, concomitant with a Tregs reduction, independently of the DCs phenotype and functional characteristics. We demonstrated that the PD-L1/CD86 ratio play a key role in the functional capability of DCs. Therefore, blocking this molecule on DCs becomes an attractive strategy to improve the efficacy of DCs-based vaccines in melanoma patients.
Recognition and neutralization of bovine herpesvirus-1 by bovine antibody variable heavy- and light-chain domains (P4182)
Authors: Pasman Yfke , Nagy Eva , Kasuhik Azad
Published: January 2023

Mono- and multimeric single chain variable fragments (scFvs), expressed in Pichia pastoris, are capable of neutralizing Bovine Herpesvirus type-1 (BoHV-1) in vitro [Koti et al. 2011. Vaccine 29; Pasman et al., 2012. Clin Vaccine Immunol 19]. The cDNA encoding these scFvs originated from a mouse x cattle hybridoma that secreted bovine IgG1 capable of neutralizing BoHV-1. The molecular modelling of heavy (VH) and light (VL) chain domains predicted that the VL might only have a supportive role in antigen recognition. VH and VL were expressed as single domains (Fd), in P. pastoris with an objective to assess their role in viral recognition and neutralization. The ability to recognize and neutralize BoHV-1 of the monomeric scFv3-18L, VH and VL linked via 18 amino acid linker, was compared to the ability of the individual FdVH and FdVL domains, in an ELISA and in vitro plaque reduction assays. These experiments demonstrated that while FdVH, but not FdVL, recognized BoHV-1 in an ELISA, FdVH did not neutralize BoHV-1. The complexities associated with such antibody functions will be discussed. [Supported by NSERC Canada]
Leptin antagonist reduces STAT1/3 activation, lessens Smad7 expression, restores TGF-β1 signaling and induces regulatory T cells (Tregs) to ameliorate chronic colitis (P4208)
Authors: Singh Udai , Singh Narendra , Busbee Brandon , Guan Hongbing , Price Robert , Taub Dennis , Mishra Manoj , Nagarkatti Mitzi , Nagarkatti Prakash
Published: January 2023

The etiology of Crohn’s disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease (IBD) are still unknown. However, there is evidence that chronic intestinal inflammation caused by excessive immune response to mucosal antigens affects millions of people around the world. Studies clearly support the role for TGF-β1 in the initiation and maintenance of tolerance by induction of Tregs to control gut inflammation. Leptin is a satiety hormone produced primarily by adipose tissue, which increases during colitis progression. In the present study, we investigated the mechanism of how pegylated leptin antagonist (PG-MLA) mediates the inhibition of experimental colitis. The PG-MLA significantly attenuates the clinical score and reverses pathogenesis including a decrease in body weight associated with colitis. PG-MLA reduces systemic and mucosal inflammatory cytokines. Further, PG-MLA increases the percentage of systemic and mucosal Tregs and TGF-β1 expressing CD4+ T cells. In addition STAT1/3 activation in immune cells, and expression of Smad7 in colon also lessens after PG-MLA treatment. These findings clearly suggest that PG-MLA diminishes Smad7 expression, reduces STAT1/3 activation and restores TGF-β1 signaling that leads to the induction of Tregs resulting in amelioration of chronic colitis. These results support a link between inflammation and nutritional status in controlling immune self-tolerance by inducing Treg responsiveness and function.
Abnormally short serum half-lives of chimeric and human IgGs in NOD-SCID mice (P4184)
Authors: Pop Laurentiu , Liu Xiao-yun , Pop Iliodora , Vitetta Ellen
Published: January 2023

The immunodeficient mice are the standard animal model in antibody research, which include the inbred NOD-SCID strain. We report here the aberrant pharmacokinetics of chimeric and human IgGs with a human IgG1 Fc domain in NOD-SCID mice. These IgGs displayed very short in vivo half-lives (25.0±0.4 hrs and 27.6±1.3 hrs, respectively) when injected at a dose of 10 μg. At a dose of 500 μg the half-lives were extended (131.1±3.1 hrs and 220.3±14.3 hrs, respectively), but still shorter than those observed in other strains of mice. The half-lives of these IgGs in NOD MrkTac, BALB/c, Swiss Webster and another immunodeficient strain of mice, C.B-17 SC-R, were similar to previous reports (300-400 hrs). In contrast, no differences in half-lives were observed for murine, rat, bovine and goat IgGs in any mouse strain studied here. RT-PCR for FcRn cDNA showed no difference in FcRn gene sequence and the binding affinity of chimeric and human IgGs to the FcRn-positive intestinal brush border of suckling mice was similar for all mice. However, chimeric and human IgGs showed higher in vitro binding affinities for Fc gamma receptors on macrophages from NOD-SCID mice vs. other strains of mice studied, suggesting that at low doses, these antibodies might bind preferentially to Fc gamma receptors-expressing macrophages, impairing their pharmacokinetics. Taken together, the NOD-SCID mice and their derivatives may not be suitable models for studying the in vivo activity of chimeric and human IgGs.
Therapeutic clearance of the virally infected nervous system is mediated by noncytopathic T cell interactions with resident myeloid cells (P4220)
Authors: Herz Jasmin , McGavern Dorian
Published: January 2023

Several viruses can infect the mammalian nervous system, some with devastating consequences, and others with little or no overt pathology. Adoptive immunotherapy is an approach that has shown promise in clinical studies for the treatment of CMV, EBV and adenovirus infections. Our laboratory models immunotherapy by transferring memory T cells into mice persistently infected from birth with lymphocytic choriomeningitis virus. Here, we demonstrate that memory T cells can completely purge the brain of persistently infected mice without causing blood brain barrier breakdown or severe tissue damage. This is accomplished by a tailored release of chemoattractants that recruit adaptive immune cells, but few pathogenic innate immune cells such as neutrophils and inflammatory monocytes. Interestingly, memory T cells enlist the support of nearly all brain resident myeloid cells (microglia) by converting them into CD11c-expressing antigen-presenting cells (APC). Two-photon imaging studies revealed that anti-viral CD8 T cells are more likely to decelerate and form stable interactions with brain-resident APC than CD4 T cells. Importantly, microglia do not undergo cell death following T cell engagement and appear to protect themselves by upregulating serine protease inhibitors like Spi-6. We propose that non-cytopathic viral clearance from the brain by therapeutic memory T cells results from tailored chemoattractant production and interactions with resident myeloid cells protected by Spi-6.
NB4 cells are superior to HL-60 cells as a basis for the Activated Transfected Killer (ATAK) cell platform (P4224)
Authors: Lin Lin , Tan Brandon , Pantapalangkoor Paul , Spellberg Brad
Published: January 2023

We are developing a cellular immunotherapy to enable neutrophil transfusions to treat neutropenic infections. We have found that HL-60 cells (AML M2 cells) can be transfected with a suicide trap and activated to differentiate into neutrophil-like cells. However, we now report that NB4 (AML M3 cells) result in far superior neutrophil differentiation by morphology, superoxide production, and CD11b expression. NB4-based ATAK cells possess superior microbicidal characteristics against Staphylococcus aureus, Acinetobacter baumannii, and Candida albicans than HL-60-based ATAK cells. NB4 cell microbicidal activity and morphology are similar to freshly harvested neutrophils.. Our current results confirm marked improvement in performance of the technology platform based on use of an NB4 cell line.
Dynamics of natural killer cell responses to hepatitis delta virus infection during the course of interferon alpha treatment (P6070)
Authors: Lunemann Sebastian , Malone David , Manns Michael , Sandberg Johan , Cornberg Markus , Ljunggren Hans-Gustaf , Wedemeyer Heiner , Björkström Niklas
Published: January 2023

Infection with the hepatitis delta virus (HDV) causes the most severe form of viral hepatitis. 15-20 million people are chronically infected with HDV. Hepatitis delta is believed to be an immune-mediated disease however adaptive immune responses against HDV are hardly detectable. This suggests a possible role of natural killer (NK) cells in the pathogenesis of HDV infection. To study this, we collected blood samples from 34 patients with hepatitis delta, 16 out of which were sampled during antiviral treatment with pegylated interferon alpha (peg-IFNalpha). Expression levels of activation and inhibitory NK cell receptors and multi-functional NK cell responses were assessed using 15-color flow cytometry. HDV infected patients exhibited higher baseline frequencies of NK cells. Peg-IFNalpha treatment was associated with a redistribution of NK cell subsets with increased numbers of CD56bright NK cells whereas total and CD56dim NK cells decreased. During treatment the most mature CD56dim NK cell subsets disappeared from the periphery and the frequency of immature CD56dim NK cells increased. Furthermore, the treatment resulted in decreased multi-functional NK cell responses against K562 cells, with and without additional IFNα stimulation. Taken together, we show that HDV infection increases the amount of NK cells. Furthermore our data suggest a reshaping of the NK pool by IFNα during treatment resulting in a loss of mature cells and decreased functional responses.
Reduced immune cells infiltration and increased neuroinflammation in type 2 diabetic mice infected with West Nile virus (P6038)
Authors: Kumar Mukesh , Roe Kelsey , Nerurkar Pratibha , Orillo Beverly , Verma Saguna , Nerurkar Vivek
Published: January 2023

Diabetes is a significant risk factor for developing West Nile virus (WNV)-associated encephalitis (WNVE) in humans. Using a diabetic mouse model, db/db, we recently demonstrated that diabetes enhanced WNV replication and susceptibility of mice to WNVE. Herein, we have examined immunological and pathological events in the brain of wild-type (WT) and db/db mice after WNV infection. C57BL/6 WT and db/db mice were infected with WNV and leukocyte infiltration, expression of cell adhesion molecules (CAM), neuroinflammatory responses and neuronal death were analyzed. We demonstrate that infiltration of CD45+ leukocytes and CD8+ T cells was significantly reduced in the brains of db/db mice, which was correlated with attenuated expression of CAM such as E-selectin and ICAM-1. WNV infection in db/db mice was associated with enhanced inflammatory response in the brain characterized by significantly high levels of multiple cytokines and chemokines. Elevated levels of cytokines correlated with increased astrocytes activation and neuronal damage in the brain of db/db mice. These data suggest that reduced leukocytes recruitment in the brain, in part, due to lower levels of CAM, results in failure to clear WNV infection leading to increased neuroinflammation, which mediates increased neuronal death and mortality in db/db mice. This is the first study to elucidate neuropathogenesis of WNV infection in a diabetic mouse model and can be used to develop therapies to manage WNVE in diabetics.
Role for Blimp-1 in the generation and function of cytotoxic CD4 effector T cells identified by NKG2A/C/E expression (P6046)
Authors: Marshall Nikki , Brauner Matthew , Kuang Yi , Berg Leslie , Swain Susan
Published: January 2023

CD4 T cells are best known for helper roles in immunity, but they can also acquire cytotoxic activity (ThCTL) and kill MHC class II+ targets presenting cognate antigen. We have identified a subset of CD4 effectors in the lung of influenza A/PR8 (IAV)-infected mice that peak at 8 days post-infection. They mediate potent perforin-dependent, Fas-independent antigen-specific cytotoxic activity and express the NK cell-associated receptors NKG2A/C/E. CD4 T cells from lung, spleen and draining lymph nodes express mRNA for the NKG2A inhibitory receptor, while mRNA for the activating receptors, NKG2C and NKG2E, are only detected in the lung. Interestingly, blocking NKG2A/C/E receptors or their ligand, non-classical MHC class I molecule Qa-1, significantly inhibits ThCTL-mediated specific killing of target cells. The NKG2A/C/E+ ThCTL subset co-expresses PD-1, CD27, PSGL-1 and Ly6c, but not CD103, as well as transcription factors Blimp-1 and Eomes. We analyzed the role of transcriptional repressor Blimp-1 in the CD4 response and found that although the conditional KO of Blimp-1 in T cells did not affect total numbers of CD4 effectors in IAV-infected lung, it significantly reduced the number of ThCTL, their expression of the aforementioned phenotype and their cytotolytic function. NKG2A/C/E is a valuable marker for identifying ThCTL that are generated during an acute anti-viral response through a unique program that involves Blimp-1.
Inflammatory cytokines extend the division of activated CD8 T cells in vivo by maintaining high affinity IL-2 signaling (P6050)
Authors: Starbeck-Miller Gabriel , Xue Hai-Hui , Harty John
Published: January 2023

Activation of naïve CD8 T cells requires several signals including TCR ligation and costimulation. Although these signals induce division, accumulation of activated CD8 T cells is dependent on a third signal provided by inflammatory cytokines. In vitro studies suggest that “signal 3”specifically enhances survival; however, the mechanisms for optimal accumulation of activated CD8 T cells in vivo is unknown. To explore these mechanisms, we transfer ~600 OT-I tg-CD8 T cells into congenic hosts and immunize hosts with OVA257-coated dendritic cells in the absence (DC) or presence of inflammation (CpG; DC+CpG). OT-I numbers were similar in DC and DC+CpG recipients at day 5 p.i. However, the presence of inflammation during immunization increased accumulation and extended division of OT-I at day 6-7 p.i. while not altering survival. Extended division of DC+CpG OT-I depended on the maintenance of IL-2/PI3K signaling that was regulated by early/direct IL-12 signaling. These findings indicate that "signal 3" enhances accumulation by maintaining division (not survival) of activated CD8 T cells in a manner dependent on a temporally orchestrated sequence of cytokine signals.
IRF-1 is a key transcriptional regulator of Tr1 differentiation (P1135)
Authors: Karwacz Katarzyna , Yosef Nir , Kuchroo Vijay
Published: January 2023

IL-10 producing Type 1 regulatory T cells (Tr1) are crucial for maintenance of peripheral tolerance and prevention of autoimmune inflammation. Using IL-27 as the differentiation factor, we undertook a detailed whole genome transcriptional network analysis of differentiating Tr1 cells. We identified IRF-1 as a key transcriptional regulator of Tr1 differentiation. Our studies demonstrate a critical requirement for IRF1 in the generation of Tr1 cells both in vitro and in vivo. IRF1-/- mice immunized for the development of Experimental autoimmune encephalomyelitis (EAE) develop very severe clinical disease and fail to recover. Furthermore, CD4+ T cells in IRF1-deficient mice with active disease showed increased number of IL-17 secreting cells in the CNS. In fact, over-expression of IRF1 during Th17 differentiation suppresses induction of both IL17 and IL23 receptor, the role opposite to the one played by another IRF family member - IRF4. Interestingly, loss of IRF-1 enhances IRF4 binding to the key DNA-binding sites involved in Th17 differentiation and functional assays further support that IRF1 can actively repress IRF4-mediated transactivation of IL17A, pointing to a cross-competition of IRF1 and IRF4 for the same DNA-binding sites in CD4+ T cells. Collectively these data demonstrate a critical role of IRF-1 in the generation of Tr1 cells, but at the same time, the induction of IRF-1 represses Th17 differentiation by actively inhibiting IRF-4 mediated Th17 differentiation.
Cross-talk between T cell receptor and cytokine signaling is mediated by Itk: implications for the balance between Th17 and regulatory T cells (P1141)
Authors: Gomez-Rodriguez Julio , Wohlfert Elizabeth , Handon Robin , Wu Julie , Belkaid Yasmine , Schwartzberg Pamela
Published: January 2023

A proper balance between Th17 and T regulatory cells (Tregs) is critical for the generation of immune protective immune responses while minimizing autoimmunity. We show that the Tec family kinase Itk influences the balance between Th17 and T regulatory cells by regulating crosstalk between T Cell Receptor (TCR) and cytokine signaling. Under both Th17 and Tregs differentiation conditions, Itk-/- CD4 T cells develop higher percentages of functional Foxp3+ cells, associated with increased responsiveness to IL-2. Itk-deficient CD4 cells show reduced TCR-induced phosphorylation of mTOR targets, including ribosomal S6 and AKT, accompanied by changes in metabolic signatures that are affected by mTOR pathways. Surprisingly, iTreg Itk-/- cells also show reduced IL-2-induced mTOR pathways, despite increased STAT5 phosphorylation. We find that TCR stimulation in WT CD4+ T cells leads to a dose-dependent repression of expression of Pten; however, at low TCR stimulation or in the absence of Itk, Pten is not effectively repressed, thereby uncoupling STAT5 phosphorylation and PI3K/mTOR pathways and increasing Foxp3 expression. Functional studies show that naïve Itk-/- CD4 T cells also preferentially develop into Tregs in an in vivo colitis model. Furthermore Itk-deficient Tregs are capable to inhibit the development of colitis demonstrating that Treg function is independent of Itk. Our results suggest that Itk is a potential target for altering the balance between Th17 and Treg cells.
Immune memory in the CNS (P1177)
Authors: Garcia Samantha , Portocarrero Carla , Kean Rhonda , Schutsky Keith , Hooper Douglas
Published: January 2023

Due to specialized neurovasculature, referred to as the blood-brain barrier (BBB), CNS tissues are immunologically privileged and only infiltrated by lymphocytes under certain circumstances. One of these is the clearance of attenuated rabies virus (RABV) from the CNS which is dependent upon CD4 T and B cell entry into CNS tissues and local production of virus neutralizing antibody (VNA). Despite spread to the CNS, these processes do not occur during wild-type RABV infection. Differences in the spread and induction of immunity to different RABV in mice allow us to determine if immune effector accumulation in CNS tissues results in the establishment of immune memory in this location. The attenuated recombinant RABV TriGAS has a severely curtailed ability to spread and does not reach the CNS when administered to the gastrocnemius. Mice infected with TriGAS via this route are fully protected against a peripheral challenge with wild-type RABV but not against intra-nasal infection with the same virus. Protection against a CNS challenge with wild-type RABV is only established by vaccination regimens that deliver RABV-specific immune effectors to the CNS. Lymphocytes remain in the CNS for extended periods of time following clearance of attenuated virus and appear to rapidly divide upon local challenge with wild-type RABV. This suggests that these cells may represent CNS resident immune memory cells capable of reactivating to produce VNA upon local challenge.