bluuebunny/crossref_metadata_2025 huggingface dataset:
The immune system and cancer stemness cross-talk can predict therapeutic response and patient outcome (P3030)
Authors: Kryczek Ilona , Tanikawa Tkashi , Szeliga Wojciech , Vatan Linda , Zou Weiping
Published: January 2023

Myeloid-derived suppressor cells (MDSCs) inhibit anti-tumor immunity. However, their involvement in cancer stem cell (CSC) biology, and their molecular, clinical and therapeutic relevance of this involvement have not been studied. Here, we investigated MDSCs in human breast cancer. We show that MDSCs prompt breast cancer stemness through enhance CSC gene expression, sphere formation and cancer metastasis. The effects depend on the cross-talk between interleukin (IL)-6/signal transducer and activator of transcription 3 (STAT3) and nitric oxide (NO)/NOTCH signaling pathways. MDSCs initiate STAT3 phosphorylation through IL-6, and activate Notch and sustain prolonged STAT3 phosphorylation via NO in CSCs. Furthermore, MDSCs positively correlate with CSC numbers and signature genes in breast cancer, and serve as a negative predictor for patient survival and herceptin response. Collectively, our study has revealed novel extrinsic cellular and molecular mechanisms controlling cancer stemness, and suggests that targeting both MDSCs and cancer is essential for engendering potent therapeutic efficacy in cancer patients. Efforts to target this network might be a valuable strategy in anti-cancer therapy.
IL-4-dependent regulation of skin barrier function. (P3042)
Authors: Serezani Ana Paula , Riley Jon , Spandau Dan , Goenca Shreevrat , Kaplan Mark
Published: January 2023

Atopic dermatitis (AD), a common manifestation of allergic disease, can be an early indicator of subsequent atopic phenotypes. Mutations in the Filaggrin gene, a component of the Epidermal Differentiation Complex (EDC) has emerged as a predisposing factor for the development of AD. EDC genes are critical for barrier function in the skin, but the Th2-type cytokines IL-4 and IL-13 inhibit expression of EDC genes in keratinocytes. We hypothesized that pro-allergic cytokines alter skin barrier function and facilitate allergen exposure by altering keratinocyte differentiation. Thus, to define the mechanisms of IL-4-dependent changes in gene expression, the activation of STAT6 was investigated in normal human keratinocytes (HK). We observed that STAT6+ cells were induced by IL-4, resulting in a peak of positive cells after 30 minutes of stimulation. Moreover, IL-4 significantly inhibited EDC genes expression in HK differentiated in vitro. IL-4 also resulted in a significant increase in the expression of the chemokine CCL26 (Eotaxin3) and the matricellular protein periostin, which is involved in persistent allergic inflammation. Thus, IL-4-dependent changes in keratinocyte gene expression are likely a key component in AD pathogenesis.
IL-18-MyD88 signaling axis is important in the attainment of a robust virus-specific effector NK cell response but dispensable for memory formation (P1248)
Authors: Madera Sharline , Sun Joseph
Published: January 2023

Although natural killer (NK) cells are considered part of the innate immune system, studies have demonstrated the ability of NK cells to contribute to potent recall responses similar to T and B cells. The precise signals that promote the generation of a long-lived NK cell response are largely undefined. Using cytokine and cytokine receptor deficient mice we investigated the role of the pro-inflammatory cytokine IL-18 on NK homeostasis, activation, expansion, and memory cell formation in response to mouse cytomegalovirus (MCMV) infection. We demonstrate that IL-18 receptor-deficient (IL-18RKO) NK cells exhibit a defect in NK cell expansion following MCMV infection; however, the generation of IL-18RKO memory NK cells was unaffected, as IL-18RKO and wildtype NK cells were detected greater than 5 weeks following MCMV challenge. NK cells deficient in MyD88, the adapter protein which mediates signaling downstream of the IL-1 and IL-18 receptors, also showed a similar defect in expansion. No defect was observed in IL-1RKO NK cells. Interestingly, IL-18RKO NK cells exhibit comparable homeostatic proliferation as wildtype. Thus, these data together highlight the importance of an IL-18-MyD88 signaling axis in NK cell priming, and in the attainment of a robust virus-specific effector NK cell response. Understanding the full contribution of the signals necessary for a protective anti-viral NK cell response will be of interest in the development of vaccines and therapeutics.
The abundance of memory-like T cells depends on TCR/MHC interactions (P1442)
Authors: Kazansky Dmitry , Silaeva Yulia , Kalinina Anastasia , Vagida Murad , Khromykh Ludmila , Deikin Alexei , Ermolkevich Tatiana , Sadchikova Elena , Goldman Igor
Published: January 2023

To investigate impact of TCR transgenic chains in formation of activation surface phenotype of T lymphocytes, we have generated mouse strain expressing transgenic beta-chain of TCR 1D1 (belonging to Vbeta6 family) on genetical background B10.D2(R101). Genomic sequence encoding TCR 1D1 beta-chain was isolated from memory T-hybridoma specific to Kb molecule. In thymus of transgenic mice accumulation of CD4-CD8-CD3- T lymphocytes takes place on the rate exceeding by several times the number of these cells in wild type animals. In transgenic animals about 70-80% of peripheral T lymphocytes express Vbeta6, whereas the percent of these cells does not exceed 5-7% in wild type animals. The ratio of peripheral CD4 and CD8 T lymphocytes stayed unchanged in transgenic mice. The percent of T cells with naive phenotype CD44-CD62L+ was significantly increased, whereas the levels of effector memory CD44+CD62L- and central memory CD44+CD62L+ T lymphocytes were markedly decreased. T lymphocytes expressing endogenous beta-chains mostly had the phenotype of memory-like T cells. Expression of 1D1 beta-chain was associated with naïve phenotype and lowered MLR-responses. These results suggest that appearance of memory-like T cells depends on interactions between TCRs and endogenous MHC/peptide complexes, whereas abundances of naive and memory-like T cells are regulated by competition of naive T cells for restricted number of appropriate MHC/peptide complexes in lymphoid tissue.
Infant immune activation, HIV susceptibility and ochratoxin A exposure in Khayelitsha, South Africa (P6159)
Authors: Wood Lianna , Jaspan Heather , Sodora Donald
Published: January 2023

In Khayelitsha, South Africa, 25% of new mothers are HIV-infected, and despite HIV acquisition through breast milk, WHO guidelines recommend breastfeeding to reduce infant mortality. One unexplained observation is that mixed fed infants (breast milk with formula/cereals) have up to an 11 fold increased risk of HIV acquisition. We hypothesized that this increase in HIV susceptibility is due to higher immune activation in response to non-breast milk foods. In fact, using non-invasive oral mucosa sampling, we observed increased CD4+ cells in mixed fed infants by flow cytometry. One immunomodulatory factor that may induce this immune activation in mixed fed infants is the common food contaminant Ochratoxin A (OTA). We measured OTA levels in plasma samples from infants in Khayelitsha, and found that at 2 weeks of age all infants had low OTA levels. However, at 6 weeks of age, OTA levels increased by 2 fold only in non-breastfed infants. Additionally, OTA plasma levels positively correlated with plasma levels of CXCL10 and TNF-alpha and negatively with IFN-alpha. Together, these data show that: 1. mixed fed infants have increased mucosal HIV target cells, 2. infant foods in Khayelitsha are contaminated with OTA, and 3. OTA exposure correlates with increased inflammatory and decreased antiviral activity in infant plasma. These findings suggest a mechanism for increased HIV susceptibility in mixed-fed infants and a role for food contaminants in infant HIV susceptibility.
Role of MGL and galectin-1 cross-talk in the regulation of T cell apoptosis (P1288)
Authors: Ilarregui Juan , García-Vallejo Juan , Rabinovich Gabriel , van Kooyk Yvette
Published: January 2023

Recently we showed that the interaction of MGL, expressed by tolerogenic dendritic cells (DCs), with GalNAc-decorated CD45 on effector cells inhibits T cell function leading to T cell death. The LacNAc binding lectin Gal1 also binds CD45 inducing apoptosis of Th1 and Th17 effector T cells. However, whether these lectins may act in concert to control effector T cell functions remain to be explored. Firstly, we incubated both lectins with Jurkat T cells and measured apoptosis by annexin V. We found higher and faster apoptosis induction compared to each lectin alone. A mechanistic analysis of this result by ImageStream® technology revealed that MGL triggers Gal1 expression that later colocalizes with CD45 in the membrane inducing high levels of receptor clustering and, in this manner, leads to augmented apoptosis. This effect is CD45 mediated and T cell specific, since Jurkat T cells devoid of this receptor or CD45-expressing carcinoma or adenocarcinoma cell lines are protected from apoptosis when incubated with MGL. Next, we isolated and activated mouse CD4+ T cells and showed that MGL1 but not MGL2 homologue induces apoptosis. However, there is no additive effect in primary CD4+ T cells. Although more work is required to fully elucidate the observed effects, these results indicate that both MGL and Gal1 may modify effector T cell function, through the interaction with specific CD45 glycoforms, potentially contributing to the homeostasis of the immune response.
Dysregulated neutrophil function promotes chronic intestinal parasite infection (P3068)
Authors: Hughes Michael , Antignano Frann , Snyder Kim , Gold Matthew , Oudhoff Menno , Zaph Colby , McNagny Kelly
Published: January 2023

Clearance of intestinal helminth infection and acquired resistance is dependent on an appropriate type-2 immune response. In the Trichuris muris (Tm) infection model in mice, resistant strains expel worms via a Th2 cell-mediated response whereas susceptible strains produce high levels of Th1 cell-associated cytokines and become chronically infected. Myeloid-cell specific deletion of Ship1 (SHIPΔLysM) in a normally resistant mouse strain (C57Bl6) results in chronic infection because of inappropriate production of IL12 in the intestine and mesenteric lymph nodes (mLNs) in Tm-infected mice. Although Ship1-deficient macrophages overproduce IL12 in vitro and in vivo and thus likely contribute to the Tm-susceptibility of SHIPΔLysM mice; surprisingly, deletion of Ship1 in the neutrophil lineage alone (SHIPΔPMN) is sufficient to render a resistant strain susceptible. Chronic worm burden in SHIPΔPMN mice is associated with elevated IL12, IFNγ and decreased IL13 production in the intestine and mLNs. These data suggest that regulation of neutrophil function by Ship1 is required for parasite clearance in response to Tm infection. Although neutrophils are rapidly recruited to the intestine following Tm infection, neutrophils are not required for clearance of parasites. However, our results suggest that dysregulated neutrophil activity can contribute to chronic parasitic infection.
Role of heme oxygenase in the immune response to Mycobacterium tuberculosis in humans (P3095)
Authors: Collins Angela , Marciano Denise , Graviss Edward , Shiloh Michael
Published: January 2023

Mycobacterium tuberculosis, the causative agent of tuberculosis, is the cause of over two million deaths every year. We have previously shown that in mice, Mycobacterium tuberculosis infection induces the production of heme oxygenase, and the subsequent production of carbon monoxide gas is sensed by the bacteria to initiate a dormancy program. Mouse macrophages deficient in heme oxygenase are defective in controlling intracellular Mycobacterium tuberculosis infection and mice deficient in heme oxygenase succumb to disease more readily than wild type mice. While the mechanisms used by mouse macrophages to control intracellular Mycobacterium tuberculosis infection, including nitric oxide synthase, the respiratory burst, acidification and heme oxygenase are well studied; how human macrophages control Mycobacterium tuberculosis infection remains poorly understood. We now show that a polymorphism in the heme oxygenase promoter confers susceptibility to human tuberculosis, that heme oxygenase is induced in human tuberculosis lesions, and that Mycobacterium tuberculosis infection of human macrophages in vitro also induces heme oxygenase. We also show by confocal microscopy that heme oxygenase colocalizes with Mycobacterium tuberculosis in human macrophages, and hee oxygenase enzymatic activity in human macrophages is necessary for inflammatory cytokine production. Thus, we demonstrate an important role for heme oxygenase in controlling Mycobacterium tuberculosis infection.
Nonreplicating Toxoplasma gondii elicits potent antitumor responses (P3100)
Authors: Bzik David , Fox Barbara , Sanders Kiah
Published: January 2023

Toxoplasma gondii stimulates a dramatic host response marked by early IL-12 production and development of IFN-γ and CD8 T cell dependent immune control of natural infection. We demonstrate that intratumoral injection of an engineered nonreplicating nonreverting uracil auxotroph T. gondii strain (cps) elicited potent antitumor responses against established aggressive orthotopic ovarian cancer. IL-12 and IFN- γ were required for this antitumor response and the response was elicited by both type I and type II strains. The antitumor response was not diminished by pre-existing CD8 T cell dependent immunity to T. gondii, and was boosted by additional sequential treatments, suggesting that early IL-12 production was required. However, heat-killed parasites that do not actively invade host cells, parasite secreted molecules, parasite cytosol, and whole parasite extracts failed to stimulate any detectable antitumor response when exogenously introduced into the tumor environment. These results implicate active invasion and intracellular manipulation of host cells by the introduction of parasite-secreted molecules directly into the host cell cytosol as mediators of this antitumor response. To explore this mechanism we developed genetically defined cps strains that lack injected parasite molecules associated with manipulation of cells or host cell IL-12 production. The role of these novel parasite injected molecules in stimulating antitumor responses will be presented.
Neutrophils in pulmonary granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques upregulate granzyme B expression in response to mycobacterial products and pro-inflammatory signals (P3058)
Authors: Mattila Joshua , Sun Tao , Flynn JoAnne
Published: January 2023

The role of neutrophils in tuberculosis (TB) and whether neutrophils can express granzyme B (grzB), a pro-apoptotic enzyme associated with cytotoxic T cells, are controversial. We compared neutrophil and T cell grzB expression in peripheral blood and pulmonary granulomas from cynomolgus macaques to better understand the biology of neutrophils in TB. Flow cytometric analysis indicated that T cells were the dominant grzB-producing cell in unstimulated peripheral blood whereas very few neutrophils expressed grzB. Similarly, neutrophil-depleted PBMCs had significantly more grzB activity than neutrophils. Stimulation with M. tuberculosis culture filtrate, but not peptide antigens, caused neutrophil upregulation of grzB to levels equivalent to stimulation with LPS from E. coli. In contrast to peripheral blood, we found that more neutrophils than T cells expressed grzB in granulomas. Granuloma neutrophils contained significantly more grzB than T cells with respect to the cross-sectional area of the cell occupied by grzB and number of granules per cell. Perforin, a protein required for target cell pore formation and grzB-mediated cytotoxicity, was found on T cells but not neutrophils, suggesting that neutrophils in granulomas may not use grzB as a cytotoxic effector. These data indicate mycobacterial products in the pro-inflammatory environment of granulomas upregulate neutrophil grzB expression and suggest a previously unappreciated aspect of neutrophil biology in TB.
Efficient sensing of infected cells in absence of virus particles by plasmacytoid dendritic is blocked by the viral ribonuclease Erns (P6114)
Authors: Summerfield Artur , Python Sylvie , Ruggli Nicolas
Published: January 2023

Plasmacytoid dendritic cells (pDC) have been shown to efficiently sense HCV- or HIV-infected cells. Here we demonstrate for classical swine fever virus, a member of the Flaviviridae, that this process is much more efficient in terms of interferon-alpha induction when compared to direct stimulation by virus particles. By employment of virus replicon particles which can replicate but not form de novo virions, we exclude a transfer of virus from the donor cell to the pDC. pDC activation by infected cells to pDC was mediated by a contact-dependent RNA transfer to TLR7, requiring intact cytoskeleton, lipid rafts, but not involving membrane vesicles such as exosomes. We further demonstrate that a unique viral protein with ribonuclease activity, the Erns protein of pestiviruses, efficiently prevented this process. This required intact ribonuclease function in intracellular compartments. The present study underlines the importance of pDC activation by infected cells and identifies a novel pathway of virus escaping the interferon system. This pathway of activation could be of particular importance for viruses which are cell-associated and cause persistent infection. Considering that Erns is required for pestiviruses to establish persistent infection of foetuses after transplacental virus transmission resulting in the development of immunotolerant animals, this report also points on a possible role of pDC in preventing immunotolerance after viral infection of foetuses.
Nonclassical monocyte-derived macrophages are essential for preventing pyelonephritis upon urinary tract infection in mice (P4229)
Authors: Partida-Sanchez Santiago , Cortado Hanna , Horvath Dennis , Li Rollin , Flaño Emilio , Justice Sheryl
Published: January 2023

Phagocytes protect the urinary tract from bacterial infections (UTI). Neutrophils are required early upon infection for clearing off bacteria, however much less is known regarding the function of monocyte-derived macrophages during UTI. Two main subsets of blood monocytes can be distinguished by differential expression of the chemokine receptors CCR2 and CX3CR1. To determine the functional role of monocyte-derived phagocytes, we used CX3CR1 and CCR2 deficient mice and monitored the trafficking of monocyte-derived macrophages during the course of uropathogenic Escherichia coli (UPEC) induced UTI. We found that Ly6+hi/CCR2+hi/CX3CR1+low, classical inflammatory monocytes were recruited from the blood to the bladder and kidney at 8-24h after infection. A subset of Ly6+low/CCR2+low/CX3CR1+hi nonclassical blood monocytes was recruited to urinary organs early (4h) after infection. UPEC induced bacterial burden was significantly elevated in the bladder of CCR2 KO but not CX3CR1 KO mice at 16h post-infection, notoriously, CX3CR1 KO mice were unable to clear UPEC at 48h post-infection; instead, bacteria spread from the bladder to the kidney causing pyelonephritis in these mice but not in WT controls. Our studies indicate that classical monocyte derived macrophages are required to clear bacteria from the bladder during early UTI infection, in contrast, nonclassical macrophages are required for preventing later bacterial spreading, and likely the clearance of bacteria in the kidney.
FcγRI-mediated production of superoxide, induction of autophagy and intracellular reduction of Rhodococcus equi load (P4231)
Authors: Bhandari Mahesh , Shivalingappa Prashanth , Gilbertie Jessica , Wiechert Sarah , Jones Douglas , Sponseller Brett
Published: January 2023

Macrophages are the host cells for Rhodococcus equi. Activation of FcγRI can induce phagocytosis, antigen presentation, cytokine production, antibody-dependent cell mediated cytotoxicity, reactive oxygen species (ROS) production, and induction of autophagy machinery in macrophages. Here we demonstrate enhanced macrophage bactericidal activity against intracellular R. equi concurrent with superoxide production and induction of autophagy. Following ligation of IFN-γ, TLR4 and FcγRI receptors, murine bone marrow-derived macrophages, equine pulmonary-alveolar macrophages and monocyte-derived macrophages were tested for superoxide production, induction of autophagy and bacterial load through Image Stream, flow cytometry, western blot and confocal microscopy. Results show that ligation of these receptors alone or in combination enhanced superoxide production and induction of LC3II. Importantly, however, FcγRI activation with a synthetic homodimeric peptide resulted in a sustained effect leading to a profound reduction in bacterial load as compared with other receptor agonists. These data are consistent with the notion that production of superoxide enhances autophagy resulting in reduction of bacterial load in infected macrophages.
Innate immune detection of flagellin positively and negatively regulates Salmonella infection. (P4001)
Authors: Lai Marvin , Quarles Ellen , Lopez-Yglesias Americo , Zhao Xiaodan , Hajjar Adeline , Smith Kelly
Published: January 2023

Salmonella enterica Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is critical for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by at least two independent pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems in innate immunity to wild type Salmonella infection is complex, and contribute to both protection and susceptibility. We hypothesized that this was due to efficient modulation of flagellin expression in vivo and evasion of innate immune detection. Salmonella deficient in the anti-sigma factor flgM overexpress flagellin, are attenuated in vivo, and this attenuation is dependent on flagellin expression. In this study, we used flgM- Salmonella to determine if flagellin recognition by the innate immune system is responsible for the attenuation of flgM- S. typhimurium, and to dissect the contribution of each flagellin recognition pathway to bacterial clearance, inflammation and infection. We demonstrate that caspase-1 controls systemic infection of flgM- S. Typhimurium, and also limits intestinal inflammation and injury. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum, and attenuates intestinal inflammation. Our results indicate that Salmonella evasion of caspase-1 dependent flagellin recognition is critical for establishing systemic infection and that evasion of TLR5 and caspase-1 dependent flagellin recognition help Salmonella induce intestinal inflammation and establish a niche in the inflamed gut.
Low-dose amphotericin B with murine dialyzable spleen extracts protects against systemic Candida infection in mice (P4053)
Authors: Robledo Avila Frank , Limón Flores Alberto , Pavón Romero Lenin , Hernández Pando Rogelio , Estrada Parra Sergio , Estrada García Iris , Pérez Tapia Sonia
Published: January 2023

Candida albicans is a fungus that causes opportunistic mucocutaneaous and systemic infections. Despite the development of several antifungal agents, mortality of systemic Candida infection remains high. Dialyzable spleen extracts (mDSE) are low molecular weight dialyzable products from immune cells which transmit the ability to express delayed-type hypersensitivity and cell mediated immunity from sensitized donors to non immune recipients. mDSE have been successfully used to treat patients with mucocutaneous candidiasis, but their effectiveness in systemic Candida infection has not been evaluated. In this study low-dose amphotericin B (AmB) was supplemented with mDSE to treat systemic candidasis in mice. We report a marked efficacy of this combined treatment on improvement of survival and reduction of the fungal burden in the kidney, correlating with high levels of serum hepcidin. Histopathological analyses of kidneys showed decrease damage, no inflammation and lower fungal burden than control kidneys. Combined treatment of low dose AmB and mDSE caused an immunomodulatory effect, increasing serum IL-6, as well as kidney IFN-γ and TGF-β1 levels, but downregulating TNF-α and IL-6. In conclusion, our data suggest that low dose AmB plus mDSE help to clear the infectious agent and reduce tissue damage due to excess inflammation, possible by the action of hepcidin.
Persistent inflammation and fibrosis of the lung are long-term complications of sepsis in baboons (P4015)
Authors: Lupu Florea , Silasi-Mansat Robert , Keshari Ravi , Peer Glenn , Popescu Narcis , Lupu Cristina , Taylor Fletcher , Lambris John , Kinasewitz Gary
Published: January 2023

Sepsis-induced inflammation of the lung leads to acute respiratory distress syndrome (ARDS), which may trigger persistent fibrotic responses. We have developed a baboon model of E. coli sepsis-induced ARDS and characterized the long-term (6-24 months) effects of sepsis on the lung. By immunocytochemistry, we detected focal accumulation of neutrophils, M2 macrophages, IL17 and TGF-β positive cells, which indicates persistent inflammation in the lung, months after clinical recovery from the acute sepsis episode. Microscopical and biochemical quantification demonstrated an almost doubling of collagen content amount in the lungs of animals that survived sepsis as compared to age-matched nonexposed controls. Electron microscopy showed myofibroblast accumulation within the alveolar wall. Co-staining for fibroblast and cell proliferation markers demonstrated active cell proliferation in fibroblastic foci. Immunostaining with antibodies to procollagens showed active collagen synthesis within the fibroblastic foci and interalveolar septa. These fibroblasts were also stained with phospho Smad-2 and CTGF antibodies, suggesting that TGF-β signaling was one of the main mediators of fibrosis. In conclusion, we show that E. coli sepsis-induced ARDS triggers chronic inflammation, fibroblast proliferation and collagen deposition in the primate. These long-lasting fibrotic responses perpetuate the lung injury and may contribute to post ARDS morbidity and mortality observed in humans.
Induction of cytotoxic anti-tumor antibodies against N Glycolyl containing gangliosides through idiotypic vaccination: clinical data (P2022)
Authors: Hernandez Anita
Published: January 2023

N glycolylated (NeuGc) gangliosides are not normal components of the cytoplasmic membrane in humans, but their expression has been demonstrated in several human malignant tumors. We demonstrated in healthy donors the existence of naturally occurring antibodies that bind NeuGc containing gangliosides and are able to kill NeuGcGM3 expressing tumor cells. Both the levels and frequency of these anti NeuGcGM3 antibodies decreased with age. Interestingly, this reactivity was almost absent in the sera of non-small cell lung cancer patients. 1E10 is an anti-idiotype mAb specific for an antibody which reacts to NeuGc gangliosides. A phase 2 multicenter, controlled, randomized, double blind clinical trial was conducted to evaluate the effect of aluminum hydroxide precipitated 1E10 vaccine in the overall survival of patients with advanced NSCLC who have completed onco specific treatment. The clinical results of this study showed a significant increase in the overall survival, according to both the intent to treat and the per protocol analysis, as well as in the long term survival rates, for the 1E10 treated patients in comparison with the placebo group. The immunological studies performed showed that the patients that elicited anti NeuGcGM3 antibodies with cytotoxic capacity had a statistically significant increase in the survival times.
IL-2 produced by CD8+ immune T cells can augment their IFN-γ production independently from their proliferation in the secondary response to an intracellular pathogen (P3336)
Authors: Sa Qila , Woodward Jerold , Suzuki Yasuhiro
Published: January 2023

Chronic infection with Toxoplasma gondii induces a potent resistance to re-infection, and IFN-γ production by CD8+ T cells is crucial for the resistance. Here we examined the role of IL-2 in IFN-γ production by CD8+ immune T cells in their secondary responses using T. gondii-specific CD8+ T cell hybridomas and splenic CD8+ immune T cells from chronically infected mice. The majority of CD8+ T cell hybridomas produced large amounts of IFN-γ only when a low amount (0.5 ng/ml) of exogenous IL-2 was provided in combination with T. gondii antigens. Inhibition of cell proliferation by mitomycin C (MMC) did not affect the enhancing effect of IL-2 on the IFN-γ production, and significant increases in transcription factor T-bet expression were associated with the IL-2-mediated IFN-γ amplification. Splenic CD8+ immune T cells produced similar low levels of IL-2 in the secondary response to T. gondii, and a blocking of IL-2 signaling by anti-IL-2Rα antibody or inhibitors of JAK1 and JAK3 significantly reduced IFN-γ production of the T cells. This IL-2-mediated upregulation of IFN-γ production was observed in MMC-treated CD8+ immune T cells, thus independent from their cell division. Therefore, endogenous IL-2 produced by CD8+ immune T cells can play an important autocrine enhancing role on their IFN-γ production in the secondary responses to T. gondii, suggesting an importance of induction of CD8+ immune T cells with an appropriate IL-2 production for vaccine development.
Genetically-determined cytokine programs of human NK cells uncover a novel mechanism of NK cells and Killer cell IgG-like Receptor genes in Crohn’s Disease susceptibility (P3262)
Authors: Lin Lin , Ma Chao , Wei Bo , Rajalingam Raja , Yusung Susy , Trachtenberg Elizabeth , Targan Stephan , McGovern Dermot , Braun Jonathan
Published: January 2023

Mucosal innate lymphocyte subsets have emerged as an important new factor in CD pathogenesis, but the role of NK cells is poorly understood. A recent genetic study identified the KIR gene KIR2DL2/3 as a risk factor for CD in the genetic context of its ligand HLA-C1. However, the biologic mechanism of this genetic contribution is unknown. NK cells are mainly known for their cytolytic function, but recent work indicates that strong KIR/HLA interactions that induce NK “licensing” augment NK cytotoxicity but also IFN-γ production. We therefore tested the hypothesis that human NK licensing by KIR2DL2/3 and HLA-C1 induces a broader cytokine program that modifies the threshold of pro-inflammatory T cell activation. Single cell multiplex functional proteomics revealed that licensed and unlicensed NK cells had strikingly distinct cytokine programs. Licensed NK cells were distinguished by high-output, proinflammatory, poly-cytokine expression. Select cytokine among this output fully accounted for their unique capacity, unlike unlicensed NK cells, to efficiently augment antigenic CD4+ T cell proliferation and induce CD4+ T cell IL-17A production. In conclusion, NK licensing mediated by KIR2DL2/3 and HLA-C1 elicits a novel cytokine program of NK cells that activates and induces pro-inflammatory CD4+ T cells, and therefore provides a predisposing biologic mechanism for this KIR-associated susceptibility to CD and other chronic inflammatory syndromes.
Innate lymphoid cells promote airway epithelial repair through the amphiregulin-EGFR pathway (P3250)
Authors: Monticelli Laurel , Sonnenberg Gregory , Abt Michael , Osborne Lisa , Tait Wojno Elia , Alenghat Theresa , Ziegler Carly , Wherry E. , Artis David
Published: January 2023

Innate lymphoid cells (ILCs) are a recently described group of immune cells that regulate immunity and inflammation at barrier surfaces. While recent studies have implicated Group 2 ILCs as initiators of pathologic Type 2 airway inflammation, the tissue-protective roles of ILCs in the respiratory tract remain poorly understood. Using genome-wide transcriptional profiling, we identify that naïve lung-resident group 2 ILCs express multiple genes associated with tissue repair pathways, including the epidermal growth factor family member amphiregulin. Amphiregulin was increased in a model of influenza virus-induced lung injury and ILC depletion during infection resulted in decreased amphiregulin expression, reduced lung function and impaired airway remodeling. IL-33 promoted expression of amphiregulin in lung ILCs and administration of amphiregulin effectively restored airway tissue remodeling in ILC-depleted mice, suggesting that ILC-intrinsic amphiregulin is a central mediator of epithelial repair. Supporting this, chemical or genetic inhibition of the EGFR-amphiregulin pathway resulted in severely impaired lung function and a failure to restore airway tissue homeostasis, leading to increased host mortality. Taken together, these data indicate a critical role for group 2 ILCs in mediating lung tissue homeostasis though an IL-33-amphiregulin-EGFR axis and suggest that ILCs serve as a key link between the signaling pathways of Type 2 inflammation and tissue repair.
Dermal γδ T cells and their role in contact hypersensitivity (P3252)
Authors: Jiang Xiaodong , Geddes Jenna , Gaide Olivier , Yoo Min Jae , Kupper Thomas
Published: January 2023

Recent studies have shown that γδ T cells not only exist in murine epidermis (Dendritic Epidermal T cells, DETC) but also populate in the dermis. DETC have been well-studied for decades. However, little is known about the homeostasis of dermal γδ T cells and their roles in skin immune responses such as in contact hypersensitivity (CHS). In this study, phenotype analysis showed that dermal γδ T cells have heterogeneous γδ TCR repertoires (30% TCR Vγ2+), but have a memory phenotype (CD62L-CD44hi) and express high levels of CD69, CD103, and E- and P-selectin ligands. Upon activation, dermal γδ T cells can produce large amounts of IL-17 and IL-22. Parabiosis experiment further showed that dermal γδ T cells are constantly circulating, whereas DETC are non-recirculating. To explore their roles in CHS, we first sensitized and challenged TCRδ-/- mice with 2, 4-dinitrofluorobenzene (DNFB). A remarkable reduction of CHS responses in these pan-γδ T cell-deficient mice was observed, suggesting γδ T cells play a pivotal role in CHS responses. We next generated dermal γδ T cell-deficient chimeric mice and found CHS responses in these mice were significantly lower than that of wild type mice, demonstrating dermal γδ T cells do participate in CHS responses. Finally, depletion of IL-17-producing Vγ2+ dermal γδ T cells also resulted in a significant reduction of CHS responses. Therefore, our data suggest that γδ T cells in dermis play an important role in establishing CHS responses.
Viral antigen induces differentiation of Foxp3+ natural regulatory T cells in influenza virus-infected mice (P1043)
Authors: Bedoya Felipe , Cheng Guang-Shing , Leibow Abigail , Zakhary Nardine , Weissler Katherine , Garcia Victoria , Kropf Elizabeth , Aitken Malinda , Garlick David , Wherry E. , Erikson Jan , Caton Andrew
Published: January 2023

We have examined the formation, participation and functional specialization of virus-reactive Foxp3+ regulatory T cells (Tregs) in a mouse model of influenza virus infection. “Natural” Tregs generated intra-thymically based on interactions with a self-peptide proliferated in response to a homologous viral antigen in the lungs, and to a lesser extent in the lung-draining mediastinal LN (medLN), of virus-infected mice. By contrast, conventional CD4+ T cells with identical TCR specificity underwent little or no conversion to become “adaptive” Tregs. The virus-reactive Tregs in the medLN and the lungs of infected mice upregulated a variety of molecules associated with Treg activation, and also acquired expression of molecules (T-bet, Blimp-1 and IL-10) that confer functional specialization to Tregs. Notably, however, the phenotypes of the T-bet+ Tregs obtained from these sites were distinct, since Tregs isolated from the lungs expressed significantly higher levels of T-bet, Blimp-1 and IL-10 than did Tregs from the medLN. Adoptive transfer of antigen-reactive Tregs led to decreased proliferation of anti-viral CD4+ and CD8+ effector T cells in the lungs of infected hosts, while depletion of Tregs had a reciprocal effect. These studies demonstrate that thymically-generated Tregs can become activated by a pathogen-derived peptide and acquire discrete T-bet+ Treg phenotypes while participating in and modulating an antiviral immune response.
Efficient in vivo delivery of immunomodulating biomolecules and enhanced antitumor immunity using multifunctional hybrid nanoconjugates (P4275)
Authors: Lim Yong Taik , Noh Young-Woock , Heo Min Beom
Published: January 2023

Immunotherapy is regarded as a method of attractive treatment for cancer. Especially, dendritic cells (DCs) represent important targets for cell-mediated immunotherapy in cancer because they can capture tumor antigens that are released from tumor cells and migrate to the tumor-draining lymph nodes, where they present the antigens to T cells and secrete the pro-inflammatory cytokines that enhance T cell activation. However, immunosuppressive factors, such as signal transducer and activator of transcription-3 (STAT3), represent a major limitation for DCs-based cancer therapies. In this study, we have designed and synthesized an immunomodulatory hybrid nanoconjugates (HNC) system based on polymer nanocomposites containing quantum dots (QDs; as imaging tracers) that are decorated with CpG ODNs (as a TLR9 ligand) and STAT3 siRNAs (as an immunosuppressive gene silencer) for the efficient immunotherapeutic cancer therapy. These HNC efficiently targeted immune cells, induced TLR activation, and silenced immunosuppressive genes. Simultaneous in vivo delivery of STAT3 siRNA and CpG ODN to DCs in the tumor microenvironment induced both the inhibition of STAT3 and activation of DCs by CpG ODNs, and synergistically elicited anti-tumor effects. By using NIR-emitting QDs, the migration of in vivo DCs to lymph nodes was also tracked by real-time NIR fluorescence imaging. In the future, these studies are expected to facilitate the development of immune cell-based cancer therapy.
Natural killer T cell adjuvanted inactivated swine influenza virus vaccine enhanced the viral load and suppressed the host immune response to pandemic 2009 H1N1 virus in pigs (P6093)
Authors: Gourapura Renukaradhya , Manickam Cordelia , Dwivedi Varun , Ouyang Kang , Binjawadagi Basavaraj , Henn Kara , Crittenden Patrick , Hiremath Jagadish , Khatri Mahesh
Published: January 2023

Swine influenza virus (SIV) causes an acute respiratory disease in pigs, and pigs are infectable by both avian and mammalian influenza viruses. Activation of NKT cell induces heterologous protection against influenza viruses in rodent studies. We discovered CD1d-restricted NKT cell in pigs. Aim of this study was to determine the efficacy of UV-inactivated bivalent SIV vaccine, comprising of triple reassortant zoonotic H1N1 (Sw/OH/24366/07) and H3N2 (Sw/OH/04) viruses; coadministered intranasally with NKT cell adjuvants, phosphatidylinositolmannosides-2 (PIM2) and α-Galctosylceramide (α-GalCer). In vaccinated homologous H1N1 virus challenged pigs, reduced viral load in the lungs associated with increased IFN-γ+ lymphocytes in the lungs and tracheobronchial lymph nodes by ELISPOT, and an increase in IFN-γ+CD8+ and IFN-γ+ γδ T cells was observed. Further, increased virus specific IgA response in the BAL fluid and enhanced lung NK cell-cytotoxicity was detected. However, in vaccinated pandemic 2009 H1N1 virus challenged pigs, enhanced nasal viral shedding and lung viral load was detected. Immunologically, reduced frequency of total lymphocytes and CD8+ T cells, and reduced frequency of total IFN-γ+ T cells was detected in the lungs. In conclusion, NKT cell adjuvanted inactivated SIV vaccine in pigs enhanced the pandemic 2009 H1N1 replication and dampened the anti-viral immune response.
Inflammatory and regulatory T cells contribute to a unique immune microenvironment in tumor tissue of colorectal cancer patients (P2145)
Authors: Kemp Roslyn , Taylor Edward , Girardin Adam , Munro Fran , Black Mik , McCall John
Published: January 2023

Colorectal cancer (CRC) is a leading causes of cancer mortality. T cells play a crucial role in the elimination of cancer. Despite their importance little in known about the composition of the T cell subsets in CRC and how the cancer cells can affect these subsets. Using a novel analytical flow cytometric approach, we have analysed T cell subsets in CRC tumor tissue and matched non-transformed bowel tissue. We discovered that the tumor has a lower frequency of effector T cells, but a higher frequency of both regulatory and inflammatory T cells, compared with associated non-transformed bowel tissue. To study the functional interactions of these cell subsets, methods have been established to isolate live, unmanipulated T cell subsets from tumor tissue and non-transformed bowel tissue. Effector T cells (IFNγ +), regulatory T cells (CD25hi, FoxP3+) and inflammatory T cells (IL-17+) have been isolated from CRC tumor tissue or non-transformed bowel tissue. Ex vivo studies have shown that effector T cells from the tumor tissue proliferate less, and are unable to produce IL-2 upon stimulation, than effector T cells from non-transformed bowel tissue. In addition, regulatory T cells isolated from non-transformed bowel tissue actively inhibit proliferation of T cells from the same tissue. These data describe an immune microenvironment in colorectal cancer unique, in both phenotype and function, to the tumor tissue and distinct from the surrounding non-transformed bowel tissue.
Usurpation of a lymphocyte costimulatory molecule as a novel melanoma cell survival pathway (P2187)
Authors: Haymaker Cara , Chen Jie Qing , Bernatchez Chantale , Kale Charuta , Torres-Cabala Carlos , Radvanyi Laszlo
Published: January 2023

BTLA (B- and T- lymphocyte attenuator), binds to the TNF-R family member HVEM (herpes virus entry mediator). HVEM through its interaction with BTLA in trans or in cis has been shown to act as a pro-survival signal through activation of NF-κB. In contrast, signaling through BTLA following HVEM engagement has been found to induce a negative signaling pathway by activating SHP1/2 phosphatases. During an initial immunohistochemistry screen for the expression of BTLA in melanoma tumor sections, we unexpectedly found that a many tumor cells also exhibited a uniform and strong positive intracellular expression of BTLA. BTLA was expressed in both primary and metastatic melanoma cells with low to negative expression in benign nevi. PCR analysis also revealed that although most tumor lines expressed isoform 1 of BTLA, some lines expressed both isoform 1 and 2 suggesting BTLA by be secreted. Moreover, we also found that melanoma cells express both surface and intracellular HVEM. Intriguingly, significant pools of BTLA were found to be co-localized with intracellular HVEM in the melanoma cells. This co-localization suggests that tumor cells may use HVEM activation as a mechanism of cell survival. Blockade of BTLA or inhibition of BTLA or HVEM expression on melanoma cells may therefore be a novel approach to inhibit tumor cell survival as well as prevent negative signaling through BTLA in immune cells.
PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1/PD-L1 in airway epithelium (P3199)
Authors: Matsumoto Koichiro , Kan-o Keiko , Asai Yukari , Fukuyama Satoru , Hamano Saaka , Seki Nanae , Yanagi Shigehisa , Suzuki Akira , Nakanishi Yoichi , Inoue Hiromasa
Published: January 2023

B7-H1/PD-L1 is implicated in the escape of viruses from the host defense systems, leading to persistence of infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. We previously reported that stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC), upregulated the expression of B7-H1 via activation of NF-κB. The mechanism of upregulation was further investigated with regard to involvement of phosphatidylinositol 3-kinases. Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by a chemical inhibitor or a small interfering RNA for PI3Kδ. The expression of p110δ was detected by Western blot and suppressed by PI3Kδ siRNA. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. The transcriptional activity of NF-κB is regulated by histone deacetylase. The activity of histone deacetylase was suppressed by poly IC, which was protected by a PI3Kδ inhibitor. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 in airway mucosa and a causal link between oxidative stress and epigenetic regulation in virus-associated B7-H1 upregulation.
Identification of risk genes linking inflammation and fibrosis in passively acquired autoimmune disease. (P4170)
Authors: Reed Joanne , Clancy Robert , Zavadil Jiri , Buyon Jill
Published: January 2023

Fetuses exposed to maternal Ro60 autoantibodies can develop life threatening cardiac conduction disease and cardiomyopathy, manifestations of neonatal lupus (NL). Recent data support an injury model where immune complexes (IC) comprised of Ro60 autoantigen, hY3 single stranded RNA, and anti-Ro60 engage Toll-like receptor (TLR) 7 and promote secretion of inflammatory and fibrotic factors by macrophages. This study sought to identify candidate risk genes based on:1) expression arrays from IC-stimulated human macrophages with and without TLR7 inhibitor; and 2) single nucleotide polymorphism (SNP) genotyping of 116 children with cardiac NL. In IC stimulated macrophages, 891 upregulated genes were reduced by at least 50% with the TLR7 inhibitor. Among these, there was an enrichment of genes related to inflammation and fibrosis. Ingenuity Pathway Analysis was used to generate a list of 327 genes with fibrotic functions that showed a significant enrichment of associations with cardiac NL (P=2.27x10-9). Joint analysis of these fibrotic genes identified 20 candidates which were also overexpressed by macrophages in TLR7-dependent arrays. Among the dataset was Relaxin receptor 1 (RXFP1), a G-protein coupled receptor that binds relaxin and plays a role in collagen and connective tissue remodeling. Key inflammatory mediators included IL6 and TNF. These data support the applicability of a novel model which can help uncover biological pathways underlying autoantibody-mediated tissue injury.