bluuebunny/crossref_metadata_2025 huggingface dataset:
Adiponectin deficiency suppresses lymphoma growth in mice by modulating NK cells, CD8 T cells and myeloid-derived suppressor cells (P2141)
Authors: Yang Young , Han Sora
Published: January 2023

We previously found that adiponectin (APN) suppresses IL-2-induced natural killer (NK) cell activation by down-regulating IFN-γ-inducible tumor necrosis factor-related apoptosis-inducing ligand and Fas ligand expression. Even though APN’s anti-tumor function has been reported in several solid tumors, with few controversial results, no lymphoma studies have been conducted. In this study, we assessed APN’s role in the function of immune cells, including NK cells, cytotoxic T-lymphocytes (CTL), and myeloid-derived suppressor cells (MDSC), using EL4 and B16F10-bearing C57BL/6J based APN-knockout (APNKO) mice. We observed attenuated EL4 growth in APNKO mice. Increased numbers of splenic NK cells were found under naive conditions and splenic CTLs under EL4-challenged conditions. Splenic NK cells in APNKO mice showed enhanced cytotoxicity, with and without IL-2 stimulation. In addition, decreased MDSC accumulation was found in EL4-bearing APNKO mice. Enforced MHC class I (MHC I) expression in B16F10 led to attenuated growth in APNKO mice. Thus, our results suggest that EL4 regression in APNKO mice is due to not only enhanced anti-tumor immune system but also to high level expression of MHC I.
Engagement of CD47 induces G1 arrest in EBV-transformed B cells by controlling CDK-cyclin-CDKi and activating p38 MAPK/JNK pathway (P2149)
Authors: Park Ga Bin , Choi Yunock , Kim Ja Young , Kim Hae Jin , Kim Seonghan , Kim Yeong-Seok , Lee Hyun-Kyung , Cho Dae-Ho , Lee Wang Jae , Bae Ki Won , Hur Dae Young
Published: January 2023

CD47 possesses a variety of anti-tumor, anti-angiogenic, and anti-metastatic properties in several tumors, but its roles on EBV-transformed B cells are still not fully understood. Here we first observed that EBV infection induced CD47 expression on B cells surface, and CD47 stimulation of EBV-transformed B cells with anti-CD47 mAb (B6H12) reduced cell proliferation and induced the cell cycle arrest at G1-phase. We showed that CD47-induced G1-phase arrest was mediated through increased expression of CDKi (p16INK4a, p21WAF1, and p53), a simultaneous decreased expression in CDK2, 4, 6 and cyclin D1, D2 and E and enhanced binding of CDKi-CDK. Among the relevant pathways, CD47 mAb induced outstanding activation of p38 MAPK and JNK, whereas phosphorylation of Akt and ERK1/2 were both reduced. In particular, p38 inhibitor SB203580 and JNK inhibitor SP600125 powerfully blocked the decreased expression of CDK and the increased expression of CDKi and dramatically suppressed binding of phospho-p38-CDKi. Accordingly, these data demonstrate that the p38 MAPK and JNK pathway participates in CDKi induction, subsequently leading to a decrease in the levels of cyclin D1-CDK4/6 and cyclin E-CDK2 complexes and CD47-dependent EBV-transformed B cell growth inhibition. In conclusion, these findings concerning the molecular mechanisms of CD47 signaling in EBV-transformed B cells provides a theoretical basis for clinical approaches for the use of therapeutic agents in treating EBV-associated tumors.
Colorectal tumor-specific regulatory and effector CD4+ T cell responses: translating laboratory results to the clinic (P2159)
Authors: Scurr Martin , Gallimore Awen , Godkin Andrew
Published: January 2023

The adaptive immune response to colorectal cancer (CRC) has a crucial role in prolonging host survival. Increased tumor infiltrates of CD3+ T cells can improve patient outcome, yet other T cell subsets exist that are capable of suppressing anti-tumor responses. Our lab has found that suppression of tumor-specific Th1 responses is associated with progression of CRC. Here, the phenotype and function of CD4+ T cells derived from PBMC, colon and tumor samples were analysed for suppressive markers by FACS, and anti-tumor responses by IFN-γ ELISPOT. CRC patients with more advanced tumors responded to fewer epitopes and generated a significantly weaker epitope-specific T cell response to the oncofetal antigen, 5T4 than healthy donors (p=0.0006). The mechanism of loss of T cell response is independent of HLA-DR subtype or patient age, but human depletion experiments both in vitro and in vivo indicates suppression by Foxp3+ regulatory CD4+ T cells. These cells were found in abundance amongst tumor-infiltrating lymphocytes; however, another equally prominent population of IL-10 and TGF-β-producing CD4+Foxp3- T cells were found to be >100-fold more suppressive. Thus, a major caveat to cancer immunotherapy is the suppressive tumor microenvironment, which contributes to the selective decline of measurable anti-tumor CD4+ T cell responses as tumors progress. These responses are enhanced in CRC patients by depleting regulatory T cells.
HIF-1/2α mediates PAR-2-induced TGF-α expression, but not VEGF production, in human pancreatic cancer cells (P2163)
Authors: Pan Shiow-Lin , Chang Li-Hsun , Teng Che-Ming
Published: January 2023

Hypoxia-inducible factor (HIF) can influence tumor associated immune cells toward assisting in tumor progression and control of immune protection. In this study, we demonstrated that activated protease-activated receptor-2 (PAR-2) upregulated both HIF-1α and HIF-2α protein level, resulting in enhanced TGF-α. Downregulation of HIFs-α by siRNA or YC-1, a HIF inhibitor, resulted in depleted levels of TGF-α protein. Further, PAR-2 through integrin-linked kinase (ILK) signaling, including the p-AKT, promoted HIFs protein expression. Diminishing of ILK by siRNA decreased PAR-2-induced p-AKT, HIFs-α and TGF-α protein. Our results suggest that ILK is involved in the PAR-2-mediated TGF-α via a HIF-α-dependent pathway. Furthermore, the culture medium from PAR-2-treated pancreatic cancer cells enhanced HUVEC proliferation and tube formation, which was blocked by the MEK inhibitor PD98059. We also found that activated PAR-2 enhanced tumor angiogenesis through the release of vascular endothelial growth factor-A (VEGF-A) from cancer cells, independent of the ILK/HIFs-α pathways. Consistent with microarray analysis, activated PAR-2 induced TGF-α and VEGF-A gene expression. In conclusion, the activation of PAR-2 signaling induced human pancreatic cancer progression through the induction of TGF-α expression by ILK/HIFs-α, as well as through MEK/VEGF-A-mediated angiogenesis, and it plays a role in the interaction between cancer progression and cancer-related inflammation.
Modeling leukemia immunoediting in mouse-human chimeras (P2169)
Authors: Oliveira Giacomo , Toffalori Cristina , Garcia-Manteiga Jose Manuel , Camisa Barbara , Crucitti Lara , Lazarevic Dejan , Peccatori Jacopo , Bernardi Massimo , Bordignon Claudio , Bondanza Attilio , Stupka Elia , Ciceri Fabio , Fleischhauer Katharina , Bonini Chiara , Vago Luca
Published: January 2023

Acute Myeloid Leukemia (AML) can be recognized and eliminated by the immune system, as demonstrated by the clinical efficacy of allogeneic hematopoietic stem cell transplantation. Still, immune-resistant variants of AML can outgrow upon the selective pressure of the transplanted immune system and determine clinical relapse, in a process called “leukemia immunoediting”, the biological bases of which remain largely unknown. To unravel novel mechanisms of immunoediting, we engrafted primary human AML in immunocompromised NOD/SCID γ-chainnull mice and modeled immune pressure by serial infusions of human T cells, either autologous or allogeneic to the leukemic cells. HLA-mismatched allogeneic T cells eradicated AML from 6/6 treated mice, whereas HLA-identical T cells granted only temporary control in 3/3 mice and autologous T cells were completely inefficacious in 3/3 mice. To fine-tune immune pressure from HLA-mismatched T cells, and thus model a phase of equilibrium before leukemia immune escape, we genetically modified allogeneic T cells to express the HSV-Tk suicide gene. The activation of the suicidal machinery abated circulating T cell counts in 3/3 mice, halted the ongoing antileukemic response and resulted in leukemia outgrowth. Gene expression profiling of the AML blasts purified from the mice upon escape from immune pressure demonstrated the selective and significant deregulation of genes involved in immune processes, including antigen processing and presentation.
Systemic 4-1BB activation induces a novel T-cell phenotype driven by high expression of Eomesodermin (P2197)
Authors: Curran Michael , Geiger Theresa , Montalvo Welby , Kim Myoungjoo , Reiner Steven , Al-Shamkhani Aymen , Sun Joseph , Allison James
Published: January 2023

Following treatment with 4-1BB agonist antibody, a novel population of KLRG1+ T-cells infiltrate the tumors of mice. Compared to their KLRG1- counterparts, these T-cells express high levels of cytotoxicity associated genes in both the CD4 and CD8 lineages and also demonstrate enhanced tumor-specific killing in vitro. The phenotype of these KLRG1+ cells is dependent on high expression of the T-box transcription factor Eomesodermin (Eomes). The unique ability of α4-1BB to generate this phenotype stems from the expression of 4-1BB by antigen presenting cells (APC) which respond to its activation by producing cytokines which then drive the development of these Eomes+KLRG1+ T-cells. By analyzing changes in APC cytokine production in vivo, as well as by using a series of gene knockout mice we have begun to identify the factors necessary to generate this novel T-cell lineage. Among these factors, IL-27, IL-15, and IL-10 appear to be paramount. These T-cells represent a novel polarity we have termed ThEO (CD4) and TcEO (CD8) which resolves multiple questions associated with 4-1BB activation including how 4-1BB enhances tumor-specific cytotoxicity, and how 4-1BB can promote tumor immunity while repressing autoimmunity. Understanding the nature of this novel lineage of highly tumoricidal T-cells in both tumor and pathogen-specific immunity may provide critical information for converting sub-optimal anti-tumor responses to therapeutically successful ones.
Macrophage phenotypes influence lung cancer migration (P2221)
Authors: Rohrbach Timothy , Deshane Jessy , Steele Chad , Willey Christopher
Published: January 2023

Background: Lung cancer is the leading cause of cancer mortality. Studies have demonstrated that high macrophage density correlated with high microvessicle density and poor patient prognosis. Tumor associated macrophage are believed to predominantly possess an alternatively activated phenotype (M2). Hallmarks of this population include pro-angiogenesis, wound repair and extracellular matrix degradation. We investigated the role macrophage play in lung cancer migration. Methods: Human lung cancer lines A549, H1299, H1792, H1975 & H1650 were used. Murine bone marrow differentiated macrophages were stimulated with IFN-γ or IL-4 to achieve classically activated (M1) and M2 populations. Conditioned media (24 h) was collected and used in migration assays. Migration was assessed using BD 24 well FluoroBlok cell culture inserts. Results: qRT-PCR showed that stimulated M1 had a 344 fold increase (p<0.01) of iNos and stimulated M2 had a 39 fold increase (p<0.01) in Arg-1. Migration increased 20% (p<0.01) in A549 cells when cultured in M2 conditioned media. However, when macrophage and lung cancer cells were co-culture, migration of H1299 and H1650 increased by 82% (p<0.01) and 184% (p<0.01) respectively. Conclusions: Our studies suggest that macrophage produce cytokines that influence the migratory abilities of lung cancer cell lines.
Control of T cell fates and immune tolerance by p38α signaling in mucosal dendritic cells (P3177)
Authors: Huang Gonghua , Wang Yanyan , Chi Hongbo
Published: January 2023

Dendritic cells (DCs) bridge innate and adaptive immunity. Our previous studies have shown that DCs employ the intracellular p38 and MKP-1 signaling axis to instruct T cell lineage choices and T cell-dependent autoimmune and infectious diseases (Huang et al. Immunity 2011; Huang et al. Nature Immunology 2012). Aside from launching protective adaptive immunity, DCs also play a crucial role in maintaining immune tolerance. To further explore the intracellular signaling pathways that program DCs to promote tolerogenic responses, we used a combination of approaches including mouse genetics, cellular immunology and biochemistry, as well as models of immune tolerance. We found that p38α signaling in mucosal DCs reciprocally regulated the differentiation of anti-inflammatory induced regulatory T (iTreg) cells and proinflammatory Th1 cells from naïve precursors, and promoted mucosal tolerance. Deficiency of p38α in mucosal DCs inhibited the generation of iTreg cells while promoting Th1 cell development in a TGF-β-dependent manner. Consequently, loss of p38α in DCs prevented induction of mucosal tolerance in vivo. Moreover, p38 signaling was constitutively activated in mucosal DCs and p38α activity was required for the expression of retinoid acid-metabolizing enzymes and imprinting gut homing receptors on responding T cells. Our studies identify a key mechanism of DC-mediated coupling of T cell differentiation and trafficking that orchestrates mucosal immune tolerance.
CD4+Foxp3+CD25+ regulatory T cells differentiate into IL-17-producing cells and promote immunopathology during Chlamydia genital tract infection (P3211)
Authors: Wang Jun , Moore-Conners Jessica , Halperin Scott
Published: January 2023

CD4+FoxP3+CD25+ regulatory T cells (Tregs) have been shown to promote de novo Th17 differentiation and to themselves convert into IL-17-producing cells in certain inflammatory settings. However, whether Tregs can potentiate IL-17/Th17 responses to intracellular bacterial infection has not been established. Using a murine model of Chlamydia, we found that Tregs produced IL-17 upon Chlamydia infection and promoted IL-17 production by conventional CD4+ T cells in vitro. Correspondingly, in vivo depletion of Tregs by anti-CD25 antibody dramatically reduced the induction of IL-17-producing CD4+ T cells during Chlamydia genital tract infection. Consistent with reduced IL-17 responses, Treg-depleted mice displayed striking amelioration of neutrophil infiltration and pathology in genital tract tissue relative to control mice, despite comparable bacterial burden throughout the course of infection. Collectively, our data demonstrate an important role for Tregs in promoting IL-17/Th17 responses during intracellular bacterial infection.
Goblet cells deliver luminal retinoids and imprint CD103+ lamina propria dendritic cells (P3223)
Authors: McDonald Keely , Wheeler Leroy , Knoop Kathryn , Miller Mark , Newberry Rodney
Published: January 2023

The intestinal lamina propria (LP) underlies the villous epithelia and contains a large population of CD103+ myeloid DCs. CD103+ LP-DCs generate the vitamin A metabolite all-trans retinoic acid (ATRA) and promote intestinal type responses including generating Foxp3+ T regulatory cells, imprinting gut homing expression on lymphocytes, and facilitating IgA production. In vivo studies demonstrated that luminal retinoids from the diet or bile are required to condition intestinal LP-DCs to generate ATRA. How luminal retinoids confer this capacity is not known. Goblet cell associated antigen passages (GAPs) provides a portal for CD103+ LP-DCs to acquire soluble luminal substances and goblet cell (GCs) proteins, suggesting a role for GCs and GAPs in imprinting CD103+ LP-DCs. Using an in vivo two-photon (2P) imaging approach, we found that GAPs delivered luminal retinoids and the enzyme required to metabolize pro-vitamin A to LP-DCs. In the absence of GCs, LP-DCs are not imprinted with ALDH activity. We observed that LP-DCs express CCR6, which was required for LP-DCs to associate with the small intestine epithelium. CCR6 deficient LP-DCs do not acquire GC proteins or luminal antigens, do not become imprinted with ALDH activity, and are impaired at inducing ATRA dependent events. These findings demonstrate that GAPs deliver luminal retinoids and the enzymes required to metabolize these retinoids to CD103+ LP-DCs, and accordingly play a critical role in imprinting CD103+ LP-DCs.
Modulation of intestinal macrophages for control of Type 1 diabetes (P3183)
Authors: Acharya Nandini , Basu Sreyashi
Published: January 2023

We have previously reported that oral administration of Capsaicin (CP), the pungent component of chili peppers inhibits proliferation and activation of auto-reactive T cells and protects mice from development of Type-1 diabetes (T1D) in a dose dependent manner. Here, we demonstrate that orally administered CP leads to induction of CX3CR1+ macrophages specifically in lamina propria and in the pancreatic lymph nodes. CX3CR1+ macrophages also known as regulatory macrophages (Mregs) play a critical role in maintenance of immune homeostasis in the gut. Inflammation hampers the differentiation of monocytes into CX3CR1+ macrophages in the gut. However, the factors responsible for facilitating the differentiation into Mregs remain elusive. Using mice deficient in Vanilloid Receptor 1 (VR1), the receptor for CP, we report that VR1 is important for the differentiation of macrophages into Mregs, which subsequently play a key role in tolerance induction via the induction of immune-suppressive CD4+IL10+FoxP3- Tr1 cells. This process culminates in providing protection from T1D in NOD mice. This study elucidates how a common food additive, CP, can act as a potent immune-modulatory agent and ameliorate autoimmune disease like T1D.
Critical role for PGI2 in the regulation of IL-22 production by intestinal innate immune cells (P3191)
Authors: Simons Bryan , Ferrini Maria , Jaffar Zeina , Roberts Kevan
Published: January 2023

Our previous studies have shown that prostacyclin (PGI2) plays an important role in regulating mucosal inflammatory responses and the development of IL-17-producing innate T cells. Using mice lacking the PGI2 receptor, IP, we have investigated the role of this prostanoid in innate immune responses in the intestine. IL-22 belongs to the IL-10 family of cytokines and targets innate immune pathways due to the restricted expression of IL-22 receptors on innate cells critical to maintaining barrier function, such as epithelial cells. Our results revealed that IP receptor deficient mice (IP-/- mice) had a marked increase in intestinal IL-22 production by innate lymphoid cells, but a clear defect in IgA production. Importantly, the number of CD3-NKp46+ lymphoid cells was dramatically increased in the Peyer’s patch of IP-/- mice compared to wild-type animals and the cells produced high levels of IL-22 in response to IL-23 exposure. Concomitant with the enhanced IL-22 production, altered intestinal mucus production and reduced number of inter-follicular cells in the Peyer’s patch was observed in IP-/- mice compared to wild-type mice. Collectively, these results demonstrate that PGI2 plays a critical role in regulating IL-22 production and limiting NKp46+ lymphoid cell responses in the intestine. Since expression of IL-22 is induced in several human gut inflammatory conditions including Crohn’s disease, these findings may have implications in inflammatory bowel disease.
Enhanced apoptosis of NOD.H2h4 thyrocytes by low concentrations of iodide: an intrinsic susceptibility factor in iodide-accelerated thyroiditis? (P4148)
Authors: Kolypetri Panayota , Carayanniotis George
Published: January 2023

Enhanced iodide intake in NOD.H2h4 mice accelerates the onset and severity of spontaneous autoimmune thyroiditis (ISAT) via an unknown mechanism. In this study, we have tested two hypotheses: 1) that NOD.H2h4 thyrocytes exhibit increased susceptibility to iodide-mediated apoptosis vs that of thyrocytes from other strains of mice; and 2) that progression of ISAT is associated with a reduction in the number of Treg cells in lymphoid tissues. We report that NOD.H2h4 thyrocytes cultured in vitro for 24 hr with 1-16 μM of NaI exhibited total numbers of apoptotic cells (ranging from 7.2 -32.9% above background) which were significantly higher than similarly treated CBA/J thyrocytes (ranging from 1.5 - 6.3% above background) upon staining with fluorescent caspase inhibitors. In addition, we report that flow cytometry of Treg cells in thyroid-draining lymph nodes (7.6-9.9% of CD4+ cells) or spleens (18.5 -19% of CD4+ cells) of NOD.H2h4 Foxp3-gfp mice did not reveal a significant alteration in their frequency, 7, 11 and 15 days following the initiation of enhanced iodide-intake (0.05 % NaI in drinking water). These results indicate that ISAT is not associated with a decrease of Treg frequency in lymphoid organs during the initial stages of the disease but support the view that an intrinsic high susceptibility of NOD.H2h4 thyrocytes to iodide-mediated apoptosis may be one of the triggering factors in ISAT.
Increased type I interferon disrupts marginal zone barrier integrity via dissociation of lymphotoxin-expressing B cells and marginal zone macrophages in systemic autoimmunity (P4160)
Authors: Li Hao , Wu Qi , Yang PingAr , Li Jun , Fu YangXin , Mountz John , Hsu Hui-Chen
Published: January 2023

In the spleen, membrane expressed Lymphotoxin (LT) on B cells maintains the integrity of marginal zone macrophages (MZM) which acts as the final barrier to prevent follicular entry of autoantigens derived from apoptotic debris. In this study, we found that there was an age-dependent decrease in the number of MZMs and clearance of apoptotic debris in the spleens of autoimmune BXD2 mice. Selective depletion of MZMs by repeated injection of recombinant LTR fusion protein to BXD2 mice accelerated autoimmune phenotypes. Interestingly, in BXD2 spleen, the major LT producing MZ and MZ precursor B cells are primarily localized in the follicles due to type I IFN-induced upregulation of CD69. Adoptive transfer of B cells from BXD2-Ifnar-/- but not BXD2 to BXD2-Rag2-/- mice resulted in retention of B cells in the MZ, leading to repopulation of MZMs. Consistent with this, there was no age-related loss of MZMs in the spleens of BXD2-Ifnar-/- mice. Reconstitution of BXD2-Rag2-/- with a mixed bone marrow of BXD2-GFP+Ifnar+ and BXD2-GFP-Ifnar- (1:1) resulted in a 50:50 repopulation of donor MZMs in recipient mice, thereby excluding the direct effect of type I IFNs on MZM maintenance. Our present study suggests that one novel mechanism associated with type I IFN-induced autoimmunity is related to the effects on promoting follicular migration of LT expressing B cells. The dissociation of LT+ B cells and MZMs disrupts MZ barrier and enables follicular entry of apoptotic antigens.
Differential efficacy of human mesenchymal stem cells on disease in murine lupus based on source of origin. (P4162)
Authors: Collins Erin , Gu Fei , Naga Osama , Gilkeson Gary
Published: January 2023

Systemic lupus erythematosus (SLE) is a devastating autoimmune disease that targets multiple organ systems. A population of non-hematopoetic stem cells, mesenchymal stem cells (MSC), are of increasing interest as a therapeutic option for autoimmune diseases such as SLE. MSCs can be derived from various sources such as bone marrow or umbilical cords. In this study we examine the efficacy of human MSCs with varying origin on disease in murine based lupus. Human MSCs were harvested from umbilical cords, healthy donor bone marrow, and lupus patient bone marrow. We showed that in MRL/lpr mice, MSCs from all sources improved survival, increased body weight, and decreased proteinuria. Glomerular IgG, but not C3, deposition is significantly decreased in mice receiving umbilical cord and healthy donor MSCs. However, MSC from lupus patients caused a significant increase in glomerular C3 and IgG. Although no differences were seen in the percentage of CD4+ or CD8+ T, mice receiving lupus MSCs experienced a 2-fold increase in the percentage of Foxp3+ cells in the spleen. These mice also experienced this percentage increase in TCRβ+, B220+, and CD138+ cells in the bone marrow. Human MSC from various origins all made an impact on the disease severity of lupus prone mice. However, these results suggest that MSCs from healthy donors or umbilical cords appear to be more effective in murine lupus than the MSCs derived from lupus patients.
Dysfunction of hepatitis B virus-specific CD8 T cells depends on viral load and is partially regulated by PD-1:PD-L1 interaction in a mouse model of persistent infection (P6123)
Authors: Chen Chih-Yeh , Chou Ho-Yuan , Huang Ya-Hui , Tao Mi-Hua
Published: January 2023

The levels of functional impairment of CD8 T cells in chronic hepatitis B patients are correlated with viral loads, where the mechanisms underlying this dysfunction remain unclear. Taking advantage of a mouse model of chronic HBV infection mediated by adeno-associated viral vectors carrying HBV genome (AAV/HBV), we demonstrated that the function of HBV-specific CD8 T cells lost gradually during persistent infection with high viral load of AAV/HBV and was irreversible with therapeutic vaccination, while infection with low viral load resulted in a less extent of functional impairment, which was restored after vaccination. Consistently, CD8 T cells expressed increasing levels of inhibitory receptors in a viral load-dependent manner, especially programmed death 1 (PD-1). Furthermore, in PD-L1 KO mice infected with AAV/HBV, the levels of viral antigen decreased, concomitant with more potent function of specific CD8 T cells. However, the reduction maintained only in KO mice with low viral load, whereas the antigen levels rebounded in those infected with high load, suggesting the roles of other factors beyond PD-1:PD-L1 signaling pathway in regulating immune tolerance in chronic hepatitis B infection. Collectively, our data indicate that the viral load-dependent dysfunction of CD8 T cells induced in chronic HBV infection is partially regulated by inhibitory signal via PD-1, and blocking PD-1:PD-L1 alone may not be sufficient to rescue the immunity against persistent HBV infection.
Splenic natural killer cells act early to select viral escape mutants during murine cytomegalovirus infection (P6135)
Authors: Parikh Bijal , Han Patricia , Rivenbark Joshua , Yokoyama Wayne
Published: January 2023

The large double-stranded DNA genome of murine cytomegalovirus (MCMV) is stable both in vitro and in vivo in the absence of immune pressure. However, one month after inoculation of plaque-purified MCMV into T cell-deficient mice, viral clones emerge with mutations in the viral MHC class I-like molecule, m157, due to selection by natural killer (NK) cells. While the emergence of escape mutants is dependent on the cognate interaction of m157 with the Ly49H NK cell activation receptor, the mechanism for mutant virus selection has not been fully dissected. Surprisingly, we show here that diverse mutations were already present in the salivary glands of C57BL/6 mice 14 days post-infection, much earlier than previously reported. Upon re-inoculation, MCMV was then subjected to purifying selection with one dominant clone emerging. To address the role of NK cells and the specific organs involved, we designed a single nucleotide polymorphism genotyping assay to quantify selection for a mutant virus with a single point mutation in m157 during a mixed infection. Remarkably, our results suggest that the escape mutant was initially selected in the spleen, but not the liver, as early as 2 days post-infection. Together, these findings suggest that the selective pressure exerted by NK cells on MCMV occurs primarily in the spleen during the initial rounds of viral replication, indicating that the host innate immune system exerts pressure on the virus almost immediately upon infection.
Commensal bacteria and T cells control the generation of memory NK cells (P6103)
Authors: Kamimura Yosuke , Lanier Lewis
Published: January 2023

Previously, only T and B cells were thought able to generate long-lived memory cells; however, recent studies demonstrated that natural killer (NK) cells expressing the activating Ly49H receptor undergo clonal expansion, contraction, and generate long-lived memory cells after infection with mouse cytomegalovirus (MCMV). It is still unclear which subsets of NK cells preferentially give rise to memory NK cells. In this study, we show that expression of killer cell lectin-like receptor G1 (KLRG1) defines short-lived effector cells and precursor for long-lived memory cells. We found that sorted KLRG1-, but not KLRG1+ NK cells can generate memory cells. Interestingly, the percentage of KLRG1+ NK cells is significantly increased in Rag1-/- mice and Rag1-/- NK cells are largely impaired in the generation of memory cells. This phenotype was rescued by mixed bone marrow chimeric (wild-type:Rag1-/-) mice. Notably, the same results were also demonstrated in Tcra-/- or T cell-depleted mice. Meanwhile, the percentage of KLRG1+ NK cells was decreased in germ-free mice and antibiotic treatment enhanced memory generation. We further determined that steady-state cytokines induced by nucleotide-binding, oligomerization domain-containing protein (Nod) 1 and Nod2, but not Myd88 and Trif are responsible for the induction of KLRG1+ NK cells. Taken together, our findings demonstrate that commensal bacteria and T cells reciprocally regulate the generation of long-lived memory NK cells.
ZBTB20 is an adjuvant-specific regulator of long-term antibody responses (P1439)
Authors: Wang Yinan , Bhattacharya Deepta
Published: January 2023

The duration of antibody production following immunization can vary greatly depending on the vaccine, but the molecular basis for these differences is unknown. Here, we demonstrated that ZBTB20 is an adjuvant-specific transcription factor critical for the durable antibody production by long-lived plasma cells. ZBTB20 was highly expressed in germinal center and memory B cells and in short and long-lived plasma cells, but not in naïve B cells.Zbtb20 deficiency due to a gene trap mutation in the Zbtb20 locus specifically led to defective accumulation of long-lived plasma cells following immunization, while short-lived plasmablasts and germinal center B cells were unaffected. Competitive fetal liver reconstitution experiments demonstrated a B cell-intrinsic requirement for ZBTB20 in long-term antibody production following alum-adjuvanted immunization. Strikingly, inclusion of Toll-like receptor ligands in the adjuvant completely rescued long-term antibody production in Zbtb20-deficient fetal liver chimeras. Thus, different immunization conditions induce long-lived plasma cells with qualitatively distinct transcriptional programs to regulate their turn-over.
Children diagnosed with common variable immune deficiency show distinct T cell maturation phenotypes when compared with adults (P3333)
Authors: Issa-El-Khoury Karine , McCusker Christine , Mazer Bruce , Alizadehfar Reza , Ben-Shoshan Moshe
Published: January 2023

INTRODUCTION: Common variable immune deficiency (CVID) is considered primarily a B cell disorder characterized by low immunoglobulin levels, defective specific antibody production and susceptibility to sinopulmonary infections. Recent studies have implicated T cells in the pathogenesis of CVID. OBJECTIVES: To compare T cell maturation in children and adults with CVID. METHODS: Through PRIMES (Canadian Primary IMmunodeficiency Evaluation Study) registry, we recruited eight children and ten adults with CVID. T cell phenotype was assessed by flow cytometry. Patients were compared to healthy controls (thirteen children and ten adults). Wilcoxon Rank Sum test was used to assess differences in T cell subsets. RESULTS: Total lymphocyte count, CD3, CD4 and CD8 lymphocytes were decreased in children with CVID compared to controls (p=0.0006, p=0.0003, p=0.0003, p=0.03). Percentage and absolute numbers of naïve (p=0.012, p=0.002) and absolute numbers of memory cells (p=0.001) were decreased in children with CVID. However, there were no statistical differences between adults with CVID and healthy controls. DISCUSSION: Our data shows decreased T cell subsets in childhood CVID while levels in adults were normal. Studies assessing temporal trends in T cell subsets are required to determine if these differences in children are due to age-related compensatory mechanisms or if different mechanisms play a role in the pathogenesis of childhood versus adult onset CVID.
Shared epitope impacts arthritis by influencing CD4 T cell differentiation (P3321)
Authors: Luckey David , Behrens Marshall , Luthra Harvinder , David Chella , Taneja Veena
Published: January 2023

HLA-DRB1*0401 has been associated with predisposition to develop rheumatoid arthritis (RA) while *0402 has been linked with resistance to develop RA. Using transgenic mice, we showed that *0401 mice are susceptible to develop collagen-induced arthritis (CIA) that mimics human disease in histopathology, production of autoantibodies and sex-bias while *0402 mice are not susceptible. In this study, we determined if *0401 mice have CD4 T cell repertoire that is predetermined to produce proinflammatory cytokines. The data shown here supports that both *0401 and *0402 mice can produce Th1/TH17 cytokines although conditions required for production of Th17 cytokines differ between the 2 strains. In context of CIA, *0402 mice generate a Th2 response that may explain its resistance to develop CIA. Further, our data supports that a significant subset of naïve CD4 T cells when activated in polarizing conditions can generate T regulatory cells in *0402 but not *0401 mice. We hypothesize that *0401 has been evolutionarily selected due to its ability to clear infection. Our data suggests that *0401 generates a storm of cytokines in response to bacterial products and may be more efficient in clearing infection than *0402. Autoimmunity is a bystander effect of the cytokine storm ensuing immune dysregulation along with the presence of lower number of T regulatory cells in *0401 mice.
Engaging students in immunology via easy, low risk, active learning exercises (P4525)
Authors: Lukin Kara
Published: January 2023

Apathy toward science in high school and college students has reached alarming proportions. The lack of scientific understanding and basic knowledge of biology is creating both health and economic crises in the United States. Traditionally, college science courses have been completely lecture-based. However, lecture-free teaching and active learning strategies engage students more successfully in the content and help students solidify content knowledge more effectively. Students also find these educational formats more enjoyable than lectures. Here, several low risk activities which force students to prepare content prior to its explanation in class are presented. In particular, the use of immediate-feedback assessment tool cards and student-led lectures in teaching immunology are detailed.
Discovering optimal targets for adoptive T-cell immunotherapy of leukemia (P4346)
Authors: Vincent Krystel , Hadj-Mimoune Sarah , Hardy Marie-Pierre , Perreault Claude
Published: January 2023

The main barrier in allogeneic hematopoietic cell transplantation is the risk of developing graft-versus-host disease. This can be prevented by the injection of CD8 T cells targeted to leukemia associated antigens (LAAs) or minor histocompatibility antigens (MiHAs). Several studies in humans have established the value of LAAs and MiHAs as target for immunotherapy on solid tumors and leukemia, respectively. Importantly, their therapeutic efficacy has never been assessed on a per antigen basis. Thus, our goal is to directly compare the therapeutic efficacy of T-cells targeted to LAAs or MiHAs expressed on EL4 cells. We selected 4 MiHAs and 4 LAAs and confirmed their immunogenicity by in vitro cytotoxicity assays. We evaluated the anti-leukemic activity of antigen-specific CD8 T cells in vivo. Notably, mice immunized against MiHAs showed enhanced survival compared to mice immunized against LAAs. We found that decreased survival of EL4 bearing mice cannot be explained by weaker MHC I/Peptide interactions. Interestingly, we showed that T cells specific for 4 LAAs and 1 MiHA are undetectable by flow cytometry and that the abundance of tetramer positive cells correlates strongly with survival of EL4 bearing mice. We propose that CD8 T cells, mainly targeted to LAAs, bind weakly to their MHC I/Peptide complexes, leading to decreased immunogenicity. We believe that the insights gained from our studies will serve as guide for selecting the best antigens for future clinical trials.
Immunomodulation through CD40 and TLR-4: an approach to control tuberculosis (P4516)
Authors: Khan Nargis , Agrewala Javed
Published: January 2023

The immune system have nature's one of the most fascinating mechanisms which continuously protects us against billions of pathogens. It operates through a very well organized network of cells that constantly rove, communicate and coordinate to protect the host. The cells of the immune system regulate through sequence of events involving myriad of molecules expressed on their surface (costimulatory molecules, toll-like receptors, etc.) and also by soluble mediators (cytokines, chemokines, defensins, complement components, etc.). Moreover, the intricate interaction of these molecules among different immune cells like T cells, B cells, dendritic cells (DCs), macrophages, etc., elicit their activation or inhibition, thereby conferring resistance against various pathogens and maintain homeostasis of the immune system. Signaling of these molecules that are expressed on the surface of immune cells can further enhance their efficacy in elimination of pathogens. The study conducted by us showed that concomitant signaling through CD40 and Toll-Like Receptor-4 augmented the production of nitric oxide and proinflammatory cytokines like IL-6, TNFa and IL-12 in Mycobacterium tuberculosis (Mtb) infected DCs. Further, activated DCs showed enhanced microbicidal function and significantly reduced the intracellular survival of Mtb. This study indicates that concomitant signaling through CD40 and TLR-4 can be employed as a therapeutic strategy to curb the intracellular growth of pathogens.
Highly immunostimulatory RNA derived from a SeV defective interfering viral genome. (P4522)
Authors: Lopez Carolina , Mercado Xiomara
Published: January 2023

Defective interfering viral genomes (DIVGs) are generated during RNA viral replication. They are strong inducers of dendritic cell maturation and cellular immune responses. Virus stocks rich in DIVGs induce the expression of high levels of IFN-β, IL-12, IL-6 and other cytokines in cell cultures. Previous studies suggest that DIVGs induce cytokine expression through the action of cell pattern recognition receptors, specifically the members of the RIG-I like receptors. Whether DIVG recognition by these viral sensors and the resulting high cytokine expression depends on the DIVG sequence, structure, or length has not been addressed. In vitro transcribed RNA of Sendai virus DIVGs of different lengths and compositions were generated and tested for their stimulatory ability. Our results suggest that DIVGs specific structures and sequences are essential for their ability to trigger high cytokine expression thought RIG-I like receptor recognition. Moreover, we have generated a shorter DIVG with the identified minimal stimulatory motif that induce higher levels of IFN-β expression in vitro. In order to determine its immunostimulatory potential in vivo, we injected mice with DIVG RNA. Higher expression of pro-inflammatory cytokines was detected in the footpad tissue as early as 6 hours post-injection. This SeV-derived oligonucleotide with strong immunostimulatory activity may be then harnessed as a potent adjuvant for vaccination.
Temporal requirement for B cells in CD4 T cell responses (P4523)
Authors: Mollo Sarah , Harrington Laurie
Published: January 2023

B cell depletion therapy is used to treat patients with non-Hodgkin’s lymphoma and autoimmune diseases, yet how a reduction in the B cell population effects the development and maintenance of memory CD4 T cells is not fully understood. Infection models using B cell-deficient mice suggest B cells play a critical role in CD4 memory T cell development; however, it has been difficult to ascertain whether this effect is due to disrupted lymphoid architecture or a direct requirement for B cells during infection. Initial experiments demonstrate that MHCII on B cells is required for optimal CD4 T cell responses to Listeria monocytogenes (LM) infection, suggesting that B cells are essential for antigen-presentation to CD4 T cells. In order to determine the temporal requirements for B cells in the development and maintenance of memory CD4 T cells, wild-type mice were treated with anti-CD20 at different stages during LM infection. B cells are not required during the contraction and/or maintenance phases for LM-specific CD4 T cell memory responses; however, when B cells are depleted prior to CD4 T cell priming there is a reduced frequency and number of LM-specific effector and memory CD4 T cells capable of producing IFNγ and IL-2. Additionally, B cells are required for recall responses after the formation of memory CD4 T cells. These studies suggest an indispensable role for B cells in the generation of memory CD4 T cells and the initiation of robust secondary responses during infection.
A novel programmed death 1 isoform-based fusion DNA vaccine enhances functional antigen-specific CD8+ T cell immunity (P4499)
Authors: Cheung Allen , Zhou Jingying , Liu Henggui , Tan Zhiwu , Chen Zhiwei
Published: January 2023

Inducing an effective CD8+ T cell immunity is important in the protection and elimination of infectious pathogens. Programmed death-1 (PD1) up-regulation in in chronic infections (e.g. HIV-1 and TB) results in “exhausted” function of CD8+ T cells, but is restored by blockade of the PD1/PD-L pathway with antibodies or a soluble form of (s)PD1. Apart from sPD1, the other three spliced variants previously identified currently have no known function. In this study, we identified a new isoform of human PD1 that contains a 42-nucleotide in-frame deletion located at exon 2 near its IgV domain found expressed in PBMCs, named Δ42PD1. We found that Δ42PD1 is distinct from PD1 as it does not engage PD-L1 or PD-L2 and capable of inducing the production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β). Next, we used Δ42PD1 as an intramolecular adjuvant to construct a DNA fusion vaccine with HIV-1 Gag p24 antigen. Following immunization in Balb/c mice, a significantly enhanced level of anti-p24 antibody titer and p24-specific CD8+ T cell responses were elicited that persisted for at least 7.5 months. Furthermore, vaccinated mice were protected against pathogenic vaccinia-Gag virus challenge, likely due to the improved proliferative and cytotoxic functions of the elicited CD8+ T cells. Therefore, our study demonstrates that a novel Δ42PD1 variant that amplifies the generation of antigen-specific CD8+ T cell immunity when used in a DNA vaccine.
Two HLA-A*0201-restricted epitopes identified from herpes glycoprotein B are recognized exclusively by CD8+ T cells from asymptomatic individuals and protect against ocular herpes (P4517)
Authors: BenMohamed Lbachir , Dervillez Xavier , Qureshi Huma , Chentoufi Aziz , Diaz Oscar , Sidney John , Villacres Maria , Wechsler Steven , Sette Alessandro , Nesburn Anthony
Published: January 2023

Identification of human “asymptomatic” HSV-1 epitopes that induce CD8+ T cells would help in designing an immunotherapeutic vaccine against herpes. Here, the HSV-1 gB sequence was screened for potential HLA-A*0201 CD8+ T cell epitopes using a variety of algorithms. The ability of these epitopes to induce multi-functional CD8+ T cell responses was compared in 10 “symptomatic” vs. 10 “asymptomatic” individuals. Ten potential epitope peptides were tested for affinity to HLA-A*0201. gB441-449 and gB561-569 exhibited high affinity to, and significantly stabilized, HLA-A*0201 molecules on the surface of T2 cells. Peptide-specific CD8+ T cell proliferation (CFSE dilution) and CD107a/b cytotoxic degranulation against these two epitopes was found in all 10 “asymptomatic” individuals. Recognition by “asymptomatic” CD8+ T cells was validated for the two epitopes using HLA/peptide tetramers. Staining for IFN-γ, IL-2, and TNF-α revealed dual- and triple-positive CD8+ T cells, indicating that these gB peptide-specific CD8+ T cells were (poly)functional. The CD8+ T cell responses to these two epitopes were absent in “symptomatic” individuals. A lipopeptide/rAdv5 prime/boost mucosal vaccine using these asymptomatic CD8+ epitopes induced strong, long lasting CD8+ T-cell protective immunity against ocular herpes in susceptible HLA-A*0201 “humanized” transgenic mice. The HSV-gB-epitope repertoire for human CD8+ T cells is more restricted than previously suspected.
Proinflammatory responses of neuroglia cells after radiofrequency fields exposure (P5036)
Authors: Lu Yonghui , Zhong Min , Xu Shangcheng , Zhang Lei , He Mindi , Chen Chunhai , Yu Zhengping , Zhou Zhou
Published: January 2023

Worldwide use of mobile phones has provoked intense concern regarding the potential biological effects of radiofrequency (RF) fields on the brain. Given the important roles of inflammation and immunization in neural physiopathologic processes, we investigated the proinflammatory responses of neuroglia cells after RF exposure. In the present study, we exposed cultured mouse microglia cell line (N9) and mouse astrocyte cell line (CRL-2541) to 1800 MHz RF modulated by 217 Hz for 1, 3, 6, 12 and 24 hours with a special absorption rate (SAR) of 2 W/kg and a duration of 5 min on/10 min off. The RF exposure significantly increased the mRNA levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) in N9 and CRL-2541 cells. Concomitant with this finding, the TNF-α and IL-1β releases were elevated by RF exposure in both neuroglia cell culture mediums. In addition, the RF exposure enhanced the gene and protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX 2) in both cell lines. The releases of prostaglandin E2 (PGE2) and nitric oxide (NO), which are the productions of iNOS and COX 2 respectively, were also increased by the RF exposure in the two cell lines. Our results indicate that exposure to 1800 MHz RF (SAR=2 W/kg) may evoke proinflammatory responses in mouse microglia and astrocyte.
NR4A1 (Nur77) dependent monocytes patrol the lung vasculature and inhibit tumor cell invasion (P5068)
Authors: Hanna Richard , Cekic Cagler , Chittezhath Manesh , Herrley Erica , Chodaczek Grzegorz , Biswas Subhra , Hedrick Catherine
Published: January 2023

The nuclear receptor NR4A1 (Nur77) is expressed in monocytes and macrophages, yet its function in regulating inflammation associated with cancer is uncertain. Our previous studies identified that Nur77 regulates the development of patrolling Ly6C- monocytes and is involved in the resolution of inflammation. In the current study, we sought to determine how absence of Nur77 in hematopoietic cells impacts tumor growth and metastasis. Mice deficient in Nur77 showed a significant increase in growth and lung metastasis of syngeneic B16F10 melanoma. Increased tumor seeding of the lung was visible after only 4 days post IV tumor transfer, and appears to be specific to the lung. In vivo imaging reveals that Nur77-defficient mice have a drastic reduction in cells patrolling the lung vasculature, and that these patrolling monocytes establish early immune interactions with tumor cells and granulocytes in the lung. Furthermore, over-expression of Nur77 in human monocytes inhibited angiogenesis and invasion of human tumor cells in co-culture assays. We conclude that the absence of Nur77-regulated patrolling monocytes in combination with polarization of myeloid cells towards a pro-inflammatory and pro-angiogenic phenotype contributes to early tumor cell invasion of the vasculature and growth in the lung. These studies indicate that Nur77 is a novel target for modulating the inflammatory phenotype of monocytes and macrophages in inflammatory diseases, including cancers.