bluuebunny/crossref_metadata_2025 huggingface dataset:
The Enhanced Sensitivity Cytometric Bead Array, a method for detecting cytokines present in low concentrations. (117.11)
Authors: Luan Fengjun , Rabenstein Jacob , Chau Vicki , Chen Yu , Maher Patricia , Zhang Victor , Sepulveda Homero
Published: January 2023

Bead-based immunoassays enable users to measure multiple proteins in the same sample, significantly increasing the amount of data generated from a single sample. For samples that have significant levels of the cytokines of interest, bead-based immunoassays allow users to get a complete cytokine profile from a single sample in a single experiment. As powerful as bead-based assays are, there are still situations where the levels of cytokines fall below the assay range. Supernatants from un-stimulated cells, samples taken from early time-points in a culture stimulation, and numerous biological samples contain very low levels of some cytokines. Results obtained from these samples, where measurements are taken from below the standard curve range, are often ambiguous, variable, and can be misleading. Advances in technology have enabled the development of more sensitive bead-based immunoassays that can help to more reliably measure samples containing low levels of cytokines. Here, we evaluate the Enhanced Sensitivity (ES) Cytometric Bead Array (CBA) Assays for their use measuring low levels of cytokines in various sample types. Using the ES CBA assays, we were able to detect many cytokines earlier in the response than when using the regular CBA assays. The ES CBA Assays were able to detect cytokines when the regular CBA assays could not. For cytokines that were able to be measured using both the ES and the regular CBA Assays, the results obtained by both assays were comparable.
Alternatively spliced Interleukin-4 protein is naturally produced in asthmatics, and elicits immune inflammation through a STAT6- and STAT3-independent mechanism (117.14)
Authors: Luzina Irina , Lockatell Virginia , Kang Phillip , Bashkatova Yulia , Andreev Sergey , Atamas Sergei
Published: January 2023

A natural splice variant of IL-4 mRNA omits exon 2. It is unknown whether a corresponding protein, IL-4δ2, is also naturally produced. We have developed an mAb against IL-4δ2 with no cross-reactivity against IL-4. Freshly purified T cells from 8 patients with mild or moderate asthma and from 4 healthy controls were tested after up to 96 h activation with PMA/ionomycin. Levels of IL-4 and IL-4δ2 mRNAs were measured by RT-Q-PCR, and secretion of IL-4 and IL-4δ2 proteins was measured by ELISA. All cells expressed IL-4 and IL-4δ2 mRNAs; stimulation caused a 5-12 fold increase in IL-4 mRNA, and a 20-70 fold increase in IL-4δ2 mRNA at 12-24 h of activation. T cells from controls secreted IL-4 but not IL-4δ2 protein, whereas T cells from asthmatics produced both IL-4 and IL-4δ2 proteins in comparable amounts. Levels of IL-4 protein peaked at 24-48 h of activation, whereas IL-4δ2 protein peaked at 72 h. Adenoviral gene delivery of either mouse IL-4 or mouse IL-4δ2 to mouse lungs in vivo induced immune inflammation with accumulation of T and B cells in both cases. Delivery of IL-4 but not IL-4δ2 induced pulmonary eosinophilia, suggesting that IL-4 does and IL-4δ2 does not engage STAT6 signaling. Experiments in primary human T cell cultures revealed that, indeed, IL-4 but not IL-4δ2 induces phosphorylation of STAT6 or STAT3 in a time- and dose-dependent fashion. Thus, IL-4δ2 is naturally secreted as a protein; it is biologically active in vivo without engaging STAT6 or STAT3 signaling.
Antipsychotics affect DC differentiation and maturation with a skewed T-cell polarization (117.20)
Authors: Moins-Teisserenc Hélène , Aoki Masayuki , Hamdani Nora , Charron Dominique , Leboyer Marion , Tamouza Ryad
Published: January 2023

Schizophrenia is a complex disorder encompassing positive, negative symptoms and cognitive deficits. The immune system is implicated in the multifactorial pathogenesis of the disease through a cytokine network dysregulation leading to inflammation. Dendritic cells play a critical role in both innate and adaptive immune responses and generation of Th1 and Th2 cells. We hypothesized that some anti-psychotics could regulate DCs, resetting the equilibrium between the Th1 and Th2 pathways in addition to their anti-dopaminergic effect. The immunomodulatory activity of atypical antipsychotics on monocyte-derived dendritic cells (MoDCs) from donors and patients was examined by studying cell subsets, cytokine production and T-cell polarization. In vitro addition of antipsychotics delayed differentiation of CD14+ cells to CD1a+ DCs but increased expression of costimulatory molecules. MoDC+antipsychotics exhibited markedly different cytokine profiles after 2 days of maturation with a 12 fold reduction of IL-12p40 and high levels of IFN-a transcripts. Functional assays showed naïve T-cell polarization toward the Th2 phenotype. These data were supported by the ex vivo profile of Th2 lymphocyte polarization induced by MoDCs from schizophrenic patients after antipsychotic therapy. Our data provide new insights in the pathogenesis of schizophrenia and the beneficial role of antipsychotics, suggesting that the influence of these drugs on the cytokine network contribute to clinical efficacy.
IL30—a novel anti-inflammatory cytokine candidate for preventing and treating inflammatory cytokine-induced liver injury in mice. (117.21)
Authors: Dibra Denada , Cutrera Jeffry , Xia Xueqing , Li Shulin
Published: January 2023

The liver is subjected to the lifetime attacks from chronic viral infection, uptake of the therapeutic drugs, life behavior (alcoholic), and environmental contaminants, results in chronic inflammation and fibrosis. It is urgent to discover effective therapeutic agents for preventing and treating livery injury and the ideal drug will be naturally occurring biological inhibitors. Here, we show that IL30 is a potent anti-inflammatory cytokine that inhibits liver toxicity. IL27, which contains IL30 subunit, does not have such a property. Interestingly, IL30 is induced by pro-inflammatory cytokines such as IFNγ. Administration of IL30 via a gene therapy approach prevents and treats both IL12- and IFNγ-induced liver toxicity. These novel observations shed the light on a novel role of IL30 as a therapeutic cytokine that suppresses pro-inflammatory cytokine-associated liver toxicity.
Phenotypic and functional alterations in peripheral blood monocyte subpopulations following short-term physical training (117.32)
Authors: Rhind Shawn , Shiu Maria , Hamilton Christy , Currie Katherine , Thomas Scott , Goodman Jack , Locke Marius
Published: January 2023

Regular exercise can reduce low-grade systemic inflammation contributing to the prevention of chronic disease. Phenotypic and functional alterations in monocyte (MO) subpopulations are implicated in antiinflammatory mechanisms of physical training. Peripheral blood was sampled from 10 healthy males (24.8±4.3 y) before and after short-term endurance training (6-d cycling 2-h/d, 65% VO2peak). Samples were examined by multicolour immunofluorescence staining and flow cytometry. CD14 and CD16 surface markers characterized 3 distinct MO subsets: classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++); the latter two being potent producers of inflammatory proteins. Coexpression of toll-like receptors (TLR2/4), intracellular cytokines (TNF-α, IL-8, IL-1ra, IL-10), nuclear factor (NF)-κB, and oxidative burst (Rho123) were quantified as changes in fluorescence intensity. Total MO counts dropped after training, with significant reductions in intermediate and nonclassical subsets. TNF-α and IL-8 expression decreased in nonclassical and intermediate MO, respectively, with no change in classical. Conversely, IL-10 and IL-1ra increased in classical and intermediate, but were unchanged in nonclassical MO. TLR2/4 were upregulated on all MO, whereas, NF-κB translocation and oxidative burst were suppressed. Our data indicate short-term training modulates the distribution and functional activity of specific MO subsets, consistent with a net antiinflammatory response.
Differential mucosal expression of IL-34 gene in acute and chronic intestinal inflammation (117.8)
Authors: Das Lopa , Kossak Sarah , Lloyd Benjamin
Published: January 2023

An imbalance of mucosal pro- and anti-inflammatory cytokines is crucial in the pathogenesis of inflammatory bowel disease (IBD). IL-34, a newly discovered cytokine involved in macrophage differentiation, induces the production of macrophage chemoattractant protein (MCP)-1 and eotaxin-2 from primary macrophages. The present ongoing study compared mRNA and protein levels of IL-34, mRNA expression of its cognate receptor colony stimulating factor-1 receptor (CSF-1R) and MCP-1 in intestinal biopsies from controls, Crohn’s disease (CD) and ulcerative colitis (UC) patients. Evaluation of mRNA levels revealed that unlike CD, IL-34 mRNA was 5-fold elevated in UC samples, suggesting that IL-34 could be a potential marker for UC pathogenesis. IL-34 protein levels however, did not mirror mRNA levels suggesting post-transcriptional modification. Interestingly in UC, CSF-1R mRNA varied reciprocally to IL-34, suggesting that like IL-34, CSF-1R could be subjected to post transcriptional modification. Micro RNA database revealed that miR-155 was a predicted candidate to block CSF-1R translation. Low CSF-1R expression correlated with very high levels of miR-155 emphasizing the concept of microRNA mediated regulation. MiR-155 levels in CD were down regulated and varied reciprocally with CSF-1R. MCP-1 was elevated in both IBD patients. Our findings suggest that IL-34 mediated immune regulation is different in UC and CD, which might have important consequences for therapeutic intervention.
NK cell IFNγ response to HLA-G: a role for myeloid DCs? (147.10)
Authors: Le Page Michael , John Elisabeth , Christiansen Frank , Witt Campbell
Published: January 2023

Previous studies report that stimulation of freshly isolated Natural Killer (NK) cells with soluble (but not solid-phase) ligand to KIR2DL4 results in IFNγ production. We have found that stimulation of 13-day cultured, activated, KIR2DL4-expressing NK cells with solid-phase (but not soluble) ligand to KIR2DL4 also results in NK cell IFNγ production. Human Leukocyte Antigen-G (HLA-G) has been reported to be the ligand for KIR2DL4, but controversy persists due to the irreproducibility of interactions between HLA-G (in soluble or solid-phase form) and KIR2DL4. In this study, we took freshly isolated NK cells purified by two methods, stimulated with soluble recombinant HLA-G or control, and measured IFNγ response. We observed an IFNγ response only from RosetteSep purified NK cells, and not from Easysep purified cells. Antibody cocktail analysis predicted that dendritic cells would be removed by the Easysep method but not the RosetteSep method. We confirmed the presence of CD11c+ myeloid dendritic cells (mDCs) in the RosetteSep NK cell purification and showed that removal of mDCs by cell sorting negated the IFNγ response. We further found that addition of mDCs to 13-day cultured, activated, KIR2DL4-expressing NK cells enabled IFNγ production in response to soluble HLA-G. Lastly, we have observed HLA-G tetramers binding to mDCs but not NK cells. We therefore hypothesize that mDCs bind and subsequently present “soluble” HLA-G to NK cells in order to induce an IFNγ response.
Involvement of mFPR2 in dendritic cell maturation and trafficking (147.20)
Authors: Chen Keqiang , Liu Ying , Xiang Yi , Huang Jiaqiang , Le Yingying , Gong Wanghua , Yoshimura Teizo , Tessarollo Lino , Gao Ji-liang , Wang Ji Ming
Published: January 2023

Mouse formylpeptide receptor 2 (mFPR2) is a homologue of the human G-protein coupled chemoattractant receptor FPR2 (FPRL1), which interacts with many pathogen and host-derived agonists. Our previous study revealed reduced allergic airway inflammatory and immune responses in mFPR2 deficient (mFPR2-/-) mice in association with diminished dendritic cell (DC) recruitment into the airway and draining lymph nodes. These defects prompted us to investigate the maturation and function of DCs in mFPR2-/- mice. We found that DC maturation, trafficking and induction of T-cell proliferation are impaired in mFPR2-/- mice. In addition, DCs from mFPR2-/- mice exhibited diminished signaling of p38 MAPK and NF-κB triggered by maturation stimulants. Our observations indicate a non-redundant role for mFPR2 in DC maturation and function in immune responses.
Role of co-stimulatory molecules in macrophage regulation of T cell activation (147.3)
Authors: Riggs James , Bartlett Tom , Silberman Dan , Composto Gabriella , Werda Amy , Cua Jackelyn
Published: January 2023

Murine peritoneal cavity (PerC) T cells from C57BL/6J mice respond poorly in vitro to CD3 ligation due to the high concentration of macrophages (Mϕs) found in this cell source. Mϕs suppressed T cell activation primarily by iNOS catabolism of arginine. Rather than costimulating T cell activation, CD28 ligation increased T cell suppression (“cosuppressed”). IFNγ is necessary for suppression as evidenced by the absence of co-suppression with IFNγRKO PerC Mϕs. To further investigate the co-suppression biology of this system, PerC cells from CD28KO, CD80KO, CD86KO, & CD80/86KO (B7KO) mice were studied. PerC T cells from CD28KO & B7KO mice had the lowest number of IFNγ-secreting cells (IFNγSC) & were the least susceptible to Mϕ-mediated suppression. CD86KO PerC cells included more IFNγSC & their T cells were more suppressed than CD80KO T cells. CD28 ligation costimulated B7KO PerC T cells yet cosuppressed both CD80KO & CD86KO PerC T cells, generating more IFNγSC in the latter. These data, consistent with the higher affinity of CD80 for CD28, suggest that CD80 might represent a biomarker for tumor progression, particularly when considered in conjunction with the density of Mϕs/myeloid-derived suppressor cells.
Responsiveness to IL-2 characterizes a stable Foxp3+ subset in mice (148.16)
Authors: Bautista Jhoanne , Gilfillan Susan , Colonna Marco , Hsieh Chyi-Song
Published: January 2023

It has recently been suggested that lymphopenic conditions result in the loss of Foxp3 expression in CD4+ regulatory T (Treg) cells, thereby switching their function from suppressors to autoimmune effectors. To assess the factors that influence stable Foxp3 expression, we followed the fate of highly purified populations of Foxp3hi cells obtained by serial flow cytometric sorting from Foxp3gfp mice. Consistent with previous reports, we observed that a substantial frequency of Foxp3- cells could be generated from 10^4 Foxp3hi cells after transfer into lymphopenic hosts. This frequency was reduced to approximately 2% in the presence of 10^5 co-injected CD4+ filler cells. However, Foxp3hi cells deficient in IL-2 signaling, or cells expressing intermediate levels of Foxp3 (Foxp3int) were much less stable. In fact, Foxp3int cells that fail to upregulate Foxp3 upon in vitro IL-2 stimulation were the least stable. These data therefore suggest that IL-2 and Foxp3 are part of a positive feedback loop that maintains Treg cell lineage stability. Furthermore, it appears that the minimal level of instability in Foxp3hi Treg cells is unlikely to substantially contribute to spontaneous autoimmunity under clinically relevant lymphopenic states such as bone marrow transplantation.
Myelin-specific B cell receptor transgenic mice reveal critical role for B cell accessory function independent of antibodies in pathogenesis of CNS autoimmune disease (148.2)
Authors: Molnarfi Nicolas , Prod'homme Thomas , Weber Martin , Patarroyo Juan , Linington Christopher , Slavin Anthony , Bernard Claude , Wekerle Hartmut , Shlomchik Mark , Zamvil Scott
Published: January 2023

Human myelin oligodendrocyte glycoprotein (MOG) induces experimental autoimmune encephalomyelitis (EAE) in a B cell-dependent manner, and is associated with MOG-specific antibodies (Abs). Whether B cells serve as antigen presenting cells (APC) for activation of encephalitogenic MOG-specific T cells in EAE induced by MOG is unknown. To distinguish the role of APC function by MOG-specific B cells from MOG-specific Abs, we created MOG-specific B cell receptor (BCR) transgenic (Tg) mice containing B cells that express membrane MOG-specific Ig only (MOGmIg), but cannot secrete Abs. EAE was compared in MOGmIg mice, MOG-specific BCR knock-in (Th) mice that can secrete all isotypes, in B cell-deficient and wild-type (WT) mice. B cell-deficient mice were resistant, confirming the requirement for B cells in human MOG-induced EAE. MOG induced EAE in MOG BCR knock-in mice, WT mice and MOGmIg Tg mice, indicating that B cells provide an accessory (i.e. APC) function, independent of Ab secretion in MOG-induced EAE. To further evaluate B cell function, mixed bone marrow (bm) chimera mice were created that contained B cells that were selectively MHC II-deficient. Mixed bm chimera mice that contained MHC II+ B cells were susceptible to MOG-induced EAE, whereas mice containing MHC II- B cells were resistant, despite MHC II expression on other APC. Our results conclusively demonstrate that independent of their humoral role, B cells provide a critical cellular function in EAE pathogenesis.
Substance P, a key intermediate in the pathogenes Herpetic Stromal Keratitis (148.23)
Authors: Twardy Brandon , Channappanavar Rudragouda , Suvas Susmit
Published: January 2023

Substance P (SP) is a pro-inflammatory neuropeptide that promotes chronic inflammation. In the current study, we ascertain the effects of blocking SP interaction with its receptor NK1R on the development of HSK lesion severity. Spantide I, a NK1R antagonist, is used for blocking SP-NK1R interaction in C57BL/6 mice after ocular HSV-1 infection. Infected mice were treated with Spantide I, either intra-peritoneally (IP) during pre-clinical (day 0-6) and clinical phases (day 7-15) or subconjuntivally (SC) during the clinical phase of the disease. Mice receiving IP or SC treatments during the clinical phase had a significant reduction in the development of corneal lesions. Corneas excised from these mice had a reduced number of neutrophils yet the number of CD4 T cells remained unchanged when compared to control mice. Additionally, IP administration of Spantide I reduced the frequency of IFNγ producing CD4 T cells in the spleen and cervical lymph nodes of infected mice, whereas SC treatments resulted in a lesser proportion of IFN-γ secreting CD4 T cells in the corneal stroma. Moreover, SC inoculation also resulted in a reduced proportion of IL-2 receptor (CD25) expressing CD4 T cells in the infected corneal tissue when compared to control mice. Taken together, our results show that blocking SP-NK1R interaction during the clinical phase of HSK inhibits the development of severe lesions by reducing CD4 T cell activation and number of neutrophils into the corneal stroma.
The effect of exercise on Th migration within an asthmatic lung. (149.11)
Authors: Dugger Kari , Estell Kim , Schwiebert Lisa
Published: January 2023

Exercise greatly influences the immune system; this includes an exercise-mediated redistribution of T cells in vivo. Increasing evidence demonstrates that Th cells play a central role in the pathogenesis of asthma. We reported previously that repeated bouts of aerobic exercise at a moderate intensity ameliorate asthma-related responses, e.g., airway inflammation and hyperresponsiveness. Studies using OVA-sensitized/challenged mice with luciferase expressing T cells provide a novel method to track T cell trafficking patterns in vivo in real-time. We found a significant reduction in proliferation and presence within the asthmatic lung of exercised mice in comparison with sedentary controls. Further, flow cytometry analysis demonstrated a difference in chemokine receptor profiles on the surface of T cells. The presence of CCR7, which mediates the exit out of lungs, was increased on exercised T cells. In addition, transmigration studies show exercised T cells have a decrease in response to chemokine gradients created by the asthmatic lung that arbitrate entry into the site of inflammation, CCL1 and CCL17. These data demonstrates that aerobic exercise training at a moderate intensity decreases Th cell migration into and increases Th cell exit out of the asthmatic lung; thereby, reducing the subsequent inflammation within the asthmatic lung. Future studies will investigate the underlying mechanism of this exercise-induced shift in homing receptor expression patterns on Th cells.
Regulation of Chemokine Receptor 7 expression by NF-κB in Squamous Cell Carcinoma of the Head and Neck (149.3)
Authors: Mburu Yvonne , Egloff Ann Marie , Wang Lin , Seethala Raja , Walker William , Van Waes Carter , Ferris Robert
Published: January 2023

Aberrant expression of CCR7 has been detected in a number of human malignancies and been linked to pro-survival, invasive and metastatic pathways. In squamous cell carcinoma of the head and neck (SCCHN), we previously described the selective upregulation of CCR7 in metastatic tumors. However, the mechanism(s) of CCR7 expression have not been fully determined. In immune cells, inflammatory cytokines are known to upregulate CCR7 through NF-κB dependent mechanisms. Interestingly, NF-κB has been associated with CCR7 expression and an aggressive SCCHN phenotype. Thus, we hypothesized that NF-κB may be a key mediator of invasive and metastatic disease by promoting CCR7 expression in SCCHN tumors. Immunohistochemical staining of SCCHN tumors showed a strong positive correlation between NF-κB and CCR7 expression. Subsequently, we identified several NF-κB binding sites in the CCR7 promoter and investigated their activity using various assays. Our studies show that each κB motif displayed differential protein binding and transcriptional activation. The induction of NF-κB activity significantly increased CCR7 expression, while blocking NF-κB resulted in a corresponding decrease in CCR7 expression. Our findings support a role for NF-κB in the regulation of CCR7 expression in metastatic SCCHN. The observed NF-κB induction of CCR7 and its subsequent downstream pathways provide clinically important therapeutic targets to control the progression and metastasis of SCCHN tumors.
Expression of chemokine receptor CX3CR1 characterizes effector and effector memory CD8+ T cells after viral infection (150.4)
Authors: Moseman E. , von Andrian Ulrich
Published: January 2023

A number of changes in surface marker expression characterize the differentiation of naïve to effector and memory CD8+ T cells. However, no single surface marker has been reported that durably characterizes effector memory (CD62L- and CCR7-) CD8+ T cells and allows distinction from other T cell subsets, in particular central memory CD8+ T cells (CD62L+ CCR7+). We report here the robust surface expression of chemokine receptor CX3CR1 by effector and memory CD8+ T cells following viral challenge. CX3CR1 expression by CD8+ T cells occurred relatively late after activation, appearing on virus specific T cells 4-5 days after LCMV infection. Expression of CD127 increased on CX3CR1hi cells over time and was markedly enriched during the memory phase. Memory CX3CR1hi CD8+ cells did not express central memory associated LN homing molecules CCR7 or CD62L, and failed to enter LNs, while preferentially localizing to peripheral tissues. Memory CX3CR1hi possessed potent cytolytic function while maintaining a modest proliferative capacity following viral rechallenge. These findings characterize CX3CR1 as a robust and distinctly expressed marker to identify long-lived effector memory CD8+ T cell, which to date could only be approximated as cells that express memory markers, but lack lymph node homing receptors. Expression of CX3CR1 by effector memory CD8+ T cells may provide a potent tool for differentially visualizing their in vivo behavior alongside central memory T cells after rechallenge.
Anandamide inhibits FcϵRI-dependent degranulation through a mechanism involving receptor-operated, but not store-operated calcium entry in mast cells (151.7)
Authors: Cervantes Villagrana Rodolfo , Cruz Silvia , González Espinosa Claudia
Published: January 2023

IgE/Antigen-mediated crosslinking of the high affinity IgE receptor (FcϵRI) in mast cells leads to the release of preformed, granule-stored mediators in a calcium-dependent process known as anaphylactic degranulation. Inhibition of degranulation has been proposed for the control of allergic reactions. Cannabinoid (CB) receptors have been recently found to modulate distinct immune responses. The aim of this work was to analyze the effects of the CB agonist anandamide (AEA) on the anaphylactic degranulation triggered by FcϵRI activation in murine bone marrow-derived mast cells. AEA inhibited FcϵRI-induced degranulation in a concentration-dependent manner. The inhibitory effect of AEA was not observable when degranulation was triggered by A23187+PMA or by thapsigargin (Thap). AEA inhibited maximal intracellular calcium increase in response to IgE/Ag, but intracellular calcium riseafter Thap addition was not changed. Results indicate that CB receptor activation inhibits IgE/Ag-dependent degranulation in mast cells modifying the coupling of FcϵRI with a calcium entry distinct from that dependent on intracellular store depletion
Dimeric galectin-1 alters the plasticity and commitment of Th1 and Th17 cells by promoting IL-10 expression.(152.20)
Authors: Cedeno-Laurent Filiberto , Opperman Matthew , Barthel Steven , Dimitroff Charles
Published: January 2023

Plasticity and commitment of T helper (Th) cell lineages are concepts still under debate. Galectin-1 (Gal-1), a β-galactoside-binding protein, was originally thought to mediate immunoregulation exclusively through its pro-apoptotic effects on activated leukocytes; however, new data suggests that Gal-1 also promotes Th2 skewing, and synthesis of tolerogenic dendritic cells driving differentiation of Type 1 regulatory (Tr1) cells. In this report, using our dimeric Gal-1 hFc, we demonstrate that Th1 and Th17 cells express similar Gal-1-binding glycoprotein repertoires and specifically, CD45, as a predominant binding partner. Furthermore, we show that, at high micromolar concentrations, Gal-1hFc drives caspase-independent apoptosis on Th1 and Th17 cells characterized by nuclear translocation of mitochondrial endonuclease G, but not cytochrome c release. Additionally, at a 10-fold lower concentration, Gal-1hFc incubations during Th1 and Th17 cell priming or after full differentiation caused both pro-inflammatory subsets to synthesize IL-10. In vivo confirmation using murine B16 melanoma cells overexpressing (B16 Gal-1hi) or deficient in Gal-1 (B16 Gal-1KD) showed that mice inoculated with B16 Gal-1hi cells contained higher levels of IL-10+ Th1 and Th17 cells than mice inoculated with B16Gal-1KD cells. This novel property reinforces Gal-1’s role in immune evasion of cancer and highlights its importance as a molecular ‘thermostat’ to help control T cell-mediated inflammation.
E47 regulates hematopoietic stem cell proliferation and energetics but not myeloid lineage restriction (153.1)
Authors: Yang Qi , Esplin Brandt , Borghesi Lisa
Published: January 2023

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins, the widely expressed basic helix-loop-helix (bHLH) transcription factors, contribute to HSC and MPP activity but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47 deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However, long-term myeloid and lymphoid differentiation is compromised due to a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyper-proliferation. The activity of E47 is shown to be cell-intrinsic. Moreover, E47 deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism, and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is partially rescued in vivo by anti-oxidant. Together, these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism, and demonstrate that E47 is not required for short-term myeloid differentiation.
Negative regulation of eosinophil production by TLR4 ligand (153.26)
Authors: Fulkerson Patricia , Schollaert Kaila , Rothenberg Marc
Published: January 2023

It has long been appreciated that a drop in eosinophils is associated with acute bacterial infection while, in contrast, blood levels of monocytes and neutrophils rise. The initial eosinopenic response is believed to be secondary to margination of circulating eosinophils to blood vessel walls, but the mechanism for prolonged eosinophil depletion with bacterial infection remains undefined. In a culture system developed to differentiate mature eosinophils from low density bone marrow progenitors, IL-5 stimulation of progenitors resulted in induced expression of six TLRs with highest expression of TLR2 and TLR4 noted throughout eosinophil development. Surface expression of TLR4 was confirmed on CD34+ progenitors by FACS. Stimulation of progenitors with LPS markedly inhibited IL-5-mediated eosinophil production. Further, LPS administration in vivo specifically reduced numbers of eosinophil progenitors in the bone marrow. Treatment of developing eosinophils with LPS induced the generation of pro-inflammatory mediators, including IL-6, IL-10, IFN-γ and TNF-α, but mechanistic studies demonstrated that suppression of eosinophil production by LPS was independent of these mediators. Taken together, these findings suggest a direct effect of LPS on eosinophil progenitors as an explanation for eosinopenia following bacterial infections and a potentially novel therapeutic strategy for depleting eosinophil progenitors and inhibiting peripheral eosinophilia in eosinophil-associated diseases.
ADAM10 overexpression dysregulates S2/S3 cleavage of Notch, shifting hematopoietic lineage commitment in favor of myelopoiesis (153.3)
Authors: Saleem Sheinei , Gibb David , Conrad Daniel
Published: January 2023

Although the mechanism by which A Disintegrin and Metalloproteinase 10 (ADAM10) regulates hematopoiesis remains unclear, recent reports indicate its essential role in performing S2 cleavage of Notch receptors. Thus to determine the role of ADAM10 activity in hematopoiesis, we generated ADAM10 overexpressing mice (A10Tg). Transgene expression alters hematopoiesis post-hematopoietic Lineage-Sca-1+c-kit+ (LSK) subset differentiation but prior to lineage commitment of progenitor populations, which are dramatically affected in A10Tg mice. This results in delayed T cell development, abrogated B cell development, and expansion of myeloid derived suppressor cells (MDSCs). Albeit being in a tumor-free host, A10Tg MDSCs are functionally and phenotypically analogous to tumor-derived MDSCs. Given ADAM10’s role in Notch signaling, we hypothesized that the observed hematopoietic alterations may be a consequence of perturbed Notch signaling. In fact, blockade of ADAM10 (S2) rescues B cell development and reduces myeloid cells in A10Tg LSKs. Additionally, inhibition of γ-secretase (S3) in wild type LSKs results in enhanced myelopoiesis, mimicking the phenotype of A10Tg mice. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical to hematopoietic cell-fate determination. Additionally, A10Tg mice provide a novel model for the examination of MDSC-mediated immune suppression in a tumor free environment.
Role of IL-17 and Th17 cells in HSV-1 induced corneal immunopathology (154.10)
Authors: Suryawanshi Amol , Veiga-Parga Tamara , Rajasagi Naveen , Rouse Barry
Published: January 2023

Herpes Simplex Virus-1 (HSV) infection of the cornea leads to a blinding immuno-inflammatory lesion of the eye termed stromal keratitis (SK). Recently, IL-17 producing CD4 T cells (Th17) were shown to play an important role in many autoimmune as well as chronic inflammatory conditions, but the role of IL-17 and/or of Th17 cells in HSV mediated immunopathology is unclear. Here we show that, after HSV infection of the cornea, IL-17 is upregulated in a biphasic manner with an initial peak production around day 2 pi and a second peak at day 21 pi, a time point when clinical lesions are evident. Further studies demonstrated that innate cells particularly, γδ T cells, were the major producers of IL-17 early after HSV infection. However, during the clinical phase of SK, the predominant source of IL-17 was Th17 cells which infiltrated the cornea only after the entry of Th1 cells. The delayed influx of Th17 cells in the cornea was partly attributed to the local chemokine and cytokine milieu as well as the inhibitory effects of the cytokines produced by Th1 cells. Finally, HSV infection of the IL-17 receptor knockout mice, as well as IL-17 neutralization in WT mice showed diminished SK severity. This was attributed to the reduced level of pro-inflammatory cytokines and MMPs as well as reduced infiltration of neutrophils. In conclusion, our results show that IL-17 and Th-17 cells contribute to the pathogenesis of SK, the most common cause of infectious blindness in the western world.
Viral variant evades CD8+ T cell immunity following suboptimal antigen recognition and activation of responding effectors (154.26)
Authors: Shorter Shayla , Schnell Frederick , Edwards Lindsay , Evavold Brian
Published: January 2023

Evasion of the CD8+ T cell response by mutating a dominant epitope is a common evasion strategy employed by many viruses. Citing the lack of a measurable antigen-specific CTL response, it is thought that this strategy allows for the virus to persist undetected by anti-viral effectors. Using an LCMV viral escape mutant, 35A, which contains a single amino acid mutation in the H-2Db restricted, gp33-43 CD8+ T cell epitope, we show that antigen specific P14 CD8+ T cells recognize the mutant antigen and mount an early response. Despite showing greatly reduced affinity for the presenting MHC class I molecule by RMAS assay, both in-vivo and in vitro data show that P14s undergo early proliferation in response to the mutated epitope but are sub-optimally stimulated as evidenced by lower levels of cell markers CD25 and CD28, increased apoptosis, and negligible IL-2 production. P14s responding to 35A also maintain a sustained level of SHP-1 phosphatase activity pointing to the initiation of a negative regulatory pathway that aborts full activation. In contrast to the viral escape mutation being ignored by antigen specific T cells, these observations show an active mechanism by which epitope mutation prevents the accumulation of a robust antigen specific CD8+ T cell response.
Early CD4 T cell help fails to rescue CD8 T cell-mediated control of neurotropic coronavirus infection (154.8)
Authors: Phares Timothy , Stohlman Stephen , Bergmann Cornelia
Published: January 2023

During murine neurotropic coronavirus infection CD8 T cells are critical anti-viral effectors, while CD4 T cells play a prominent role in the disease and demyelination associated with the infection. The failure of CD8 T cells to control infection in the absence of CD4 T cells implied that CD8 T cells require CD4 T help in this encephalomyelitis model. To explore whether CD4 T cell help was imprinted early during the priming and CD8 T cell expansion phase, CD4 T cells were not depleted until days 4 and 6 post-infection. Disease onset and progression was comparable between depleted and control groups, with CD4 depleted mice displaying slightly less severe clinical symptoms. Peripheral expansion of virus-specific CD8 T cells was also comparable to controls and recruitment of virus-specific CD8 T cells into the CNS was only slightly diminished. Nevertheless, control of infectious virus was impaired following CD4 T cell depletion and mice succumbed to infection. Failure to control virus correlated with reduced IFN-gamma in the CNS, as well as lower granzyme B and perforin mRNA levels in CD8 T cells. Impaired CD8 T cell activity coincided with the absence of CD4 dependent IL-21 expression in the CNS. These results indicate that the presence of CD4 T cells during priming does not rescue impaired CD8 T cell function within the CNS. The data rather suggest that CD4 T cell play a critical role in promoting CD8 T cell function and accumulation at the site of infection.
Efficient mucosal vaccination mediated by the neonatal Fc receptor (155.9)
Authors: Zhu Xiaoping , Ye Lilin , Zeng Rongyu , Bai Yu , Roopenian Derry
Published: January 2023

Almost all infectious diseases are initiated at mucosal surfaces, yet intramuscular or subcutaneous vaccination usually provides only minimal protection at sites of infection owing to suboptimal activation of the mucosal immune system. The neonatal Fc receptor (FcRn) mediates the transport of IgG across polarized epithelial cells lining mucosal surfaces. We mimicked this process by fusing a model antigen, herpes simplex virus type-2 (HSV-2) glycoprotein gD, to an IgG Fc fragment. Intranasal immunization, together with the adjuvant CpG, completely protected wild-type, but not FcRn knockout, mice after intravaginal challenge with virulent HSV-2 186. This immunization strategy induced efficient mucosal and systemic antibody, B- and T-cell immune responses, with stable protection for at least 6 months after vaccination in most of the immunized animals. The FcRn-IgG transcellular transport pathway may provide a general delivery route for subunit vaccines against many mucosal pathogens.
Adoptive transfer of multiple CTL clones limits antigen gene loss and tumor escape (156.22)
Authors: Kaluza Karen , Thompson Jill , Vile Richard
Published: January 2023

We study mechanisms of tumor escape from adoptive therapy in the mouse melanoma model B16ova. In this model, transfer of OT-I T cells can significantly prolong the survival of mice. However, intense OT-I pressure promotes tumor cell genomic instability, and most mice eventually develop recurrent ova gene-loss tumors. We hypothesized that simultaneous transfer of two CTL clones would limit tumor recurrence by preventing the selection of single gene-loss cells. To accomplish this, we co-transferred OT-I T cells and Pmel T cells. Co-transfer three days following tumor challenge prevented tumor growth, while neither clone alone could. And in mice with established tumors, co-transfer provided significantly better therapy than either clone alone. This improvement was due to both fewer tumors recurring and the delayed appearance of those that did recur. Additionally, we found that recurrent tumors from mice treated with co-transfer continued to express both target antigens, and the mice maintained both T cell populations. We therefore hypothesized that it should be possible to exploit those cells to further prolong the survival of mice. We used cyclophosphamide to stimulate reactivation of the immune system in mice with recurrent tumors and found that this supplemental therapy significantly prolonged the lives of only those mice which had previously received co-transfer. Overall, our results reveal several benefits of targeting multiple tumor antigens.
Selective expansion of NK cells from PBMCs for cancer immunotherapy (156.28)
Authors: Lim Seon Ah , Kim Tae-Jin , Kim Kwanghee , Lee Kyung-Mi
Published: January 2023

NK cells have the ability to kill transformed or tumor cells without any prior stimulation. Despite this ability, the function of NK cells in tumor patients is greatly suppressed due to multiple factors. Therefore, efforts have been focused on isolating and expanding NK cells from tumor patients and re-infusing them after in vitro activation. These processes require multiple procedures to isolate and purify NK cells from the patients' PBMCs and high cost cytokines. To devise a simple and low-cost method to expand and activate NK cells with high purity, we have screened numerous feeder cell lines. Among these cell lines, we discovered KL-1 cell line, selectively enriched NK cells from whole PBMCs. After 15 days of culture, the number of CD56+ CD3- NK cells increased approximately 130-fold with the NK cell purity of over 90%. Expanded NK cells mounted superior cytotoxicity against NK-sensitive K562 cells as well as KL-1 cells. These results indicated that KL-1 feeder cell line could be exploited to generate NK cells for adoptive transfer with this cost-effective yet highly functional NK expansion method.
SA-4-1BBL as an adjuvant component of tumor vaccines achieves better therapeutic efficacy than an agonistic 4-1BB antibody by limiting the frequency of regulatory T cells (156.8)
Authors: Sharma Rajesh , Srivastava Abhishek , Yolcu Esma , Shirwan Haval
Published: January 2023

Costimulation through 4-1BB receptor is important for the generation of CD8+ T cell responses critical for the control of tumors. As such, this pathway has been targeted for tumor immunotherapy using agonistic 4-1BB Abs. However, the use of Abs is associated with severe toxicity. We recently developed a nontoxic form of soluble 4-1BBL, SA-4-1BBL, and demonstrated that it delivers quantitatively and qualitatively improved signals to T cells than an agonistic 4-1BB Ab (3H3). We herein demonstrate that SA-4-1BBL as the adjuvant component of HPV E7 and survivin TAA-based vaccines shows better therapeutic efficacy than the 3H3 Ab in E7 expressing TC-1 cervical and survivin over expressing 3LL lung cancer mouse models. The better therapeutic efficacy of SA-4-1BBL was associated with its ability to restrict the frequency of CD4+CD25+Foxp3 Treg cells. In marked contrast, mice vaccinated with 3H3 Ab had dramatically increased numbers of Treg cells. Importantly, in vivo depletion of Treg cells using an Ab to CD25 significantly enhanced the efficacy of 3H3, but not SA-4-1BBL, based vaccine. Taken together, these data demonstrate that SA-4-1BBL serves as a better alternative to agonistic Abs to 4-1BB receptor for the development of cancer vaccines due to its safety and ability to diminish the incidence of regulatory T cells.
Essential role of NLRP3 and AIM2 inflammasomes in IL-1β production induced by the extracellular pathogen, enterohemorrhagic Escherichia coli (157.1)
Authors: Kailasan Vanaja Sivapriya , Rathinam Vijay , Kalantari Parisa , Fitzgerald Katherine , Leong John
Published: January 2023

Enterohemorrhagic Escherichia coli (EHEC) induces elevated levels of IL-1β in infected patients. However, the molecular mechanisms involved in the activation of IL-1β by EHEC are unknown. Inflammasomes are multiprotein complexes in the cytosol that process proIL-1β to bioactive IL-1β. To identify the inflammasomes involved in IL-1β induction by EHEC, we infected macrophages derived from wild type and mice deficient in inflammasome components with EHEC and analyzed the processing and release of mature IL-1β. EHEC-induced IL-1β production was dependent on caspase-1 and the adaptor ASC. Notably, both NLRP3, a cytosolic sensor for microbial and endogenous products, and AIM2, a cytosolic sensor for DNA, were absolutely essential for EHEC-induced IL-1β production. Though EHEC is a non-cytosolic pathogen, bacterial products such as DNA were observed in the cytosol of infected cells, providing a potential means by which cytosolic inflammasomes might be activated. This also indicated that phagosomal degradation of bacteria is a prerequisite for inflammasome activation by EHEC. Supporting this hypothesis, blocking lysosomal acidification and type I interferon signaling that are essential for bacterial lysis in phagosome, led to a marked decrease in EHEC-induced IL-1β secretion. Thus, our results demonstrate that EHEC potently activates multiple inflammasomes as a result of release of bacterial products from phagosomes into the cytosol.
IL-22 enhances Calgranulin A expression by human macrophages to inhibit virulent Mycobacterium tuberculosis growth (158.21)
Authors: Dhiman Rohan , Paidipally Padmaja , Barnes Peter , Tvinnereim Amy , Vankayalapati Ramakrishna
Published: January 2023

Previously, we found that IL-22 produced by NK cells inhibits intracellular mycobacterial growth in human macrophages (Mϕ) by enhancing phagolysosomal fusion. In the current study, we determined the mechanism/s by which IL-22 inhibits virulent M. tuberculosis H37Rv (M. tb) growth in Mϕ. First we asked whether IL-22 restricts M. tb growth in phagosomes that leads to enhanced phagolysosomal fusion. W7, a phagolysosomal fusion inhibitor abrogated IL-22 dependent M. tb growth in Mϕ (11.38 ± 3.3 x 106 vs 2.95 ± 1.7 x 106 CFU per well, p=0.05) suggesting IL-22 inhibits M. tb growth after phagolysosomal fusion. In microarray analysis we found that mRNA expression for 41 genes was >1-fold higher in rIL-22 cultured infected Mϕ compared to infected Mϕ. Of these genes, we selected Indolamine 2,3-dioxegenase (IDO1) and Calgranulin A for further study. rIL-22 enhanced Calgranulin A expression in M. tb infected Mϕ by three fold (3.3 ± 0.83 vs 1.0 arbitrary units, p=0.04) compared to infected Mϕ as measured by real time PCR and confirmed by confocal microscopy. In contrast, IDO1 expression of infected Mϕ was not effected by rIL-22. Calgranulin A siRNA abrogated rIL-22 dependent growth inhibition of M. tb in macrophages (18.4 ± 1.8 x 106 vs 7.97 ± 0.8 x 106 CFU per well, p=0.0005). Studies are underway to determine the effect of Calgranulin A siRNA on phagolysosomal fusion using early endosomal, late endosomal and lysosomal markers.
Autophagy protects against infection and inflammation in a murine model of tuberculosis (158.9)
Authors: Castillo Eliseo , Dekonenko Alex , Delgado-Vargas Monica , Arko-Mensah John , Lyons Rick , Virgin Herbert , Deretic Vojo
Published: January 2023

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, infects approximately one-third of the world’s population. Recently, autophagy, an intracellular process that sequesters portions of the cytoplasm, including damaged or surplus organelles into autophagosomes and degrades them upon fusion with lysosomes, was found to be crucial in macrophages for controlling MTB ex vivo. Additionally, the autophagy pathway and proteins have been shown to be critical in immunity and inflammation. Despite the known roles of autophagy in killing intracellular bacteria and regulating the immune system it is unknown how autophagy controls MTB in vivo. Using Atg5f/f LyzM-Cre mice, we investigated the in vivo role of autophagy in macrophages in controlling MTB infection and shaping the immune response. Compared to wild-type mice, Atg5f/f LyzM-Cre mice displayed greater weight loss, lung pathology and death in a dose-dependent manner. An increase in neutrophils and macrophages was found in the lung of infected Atg5f/f LyzM-Cre mice whereas the wild type mice had an increase in CD4 and CD8 T cell infiltrates. In this context, luminex assay also revealed an increase in IL-1α, GM-CSF, IL-17 and CXCL1 in the lung lavage of the infected Atg5f/f LyzM-Cre mice when compared to the wild type mice. Taken together, these results show the importance of the autophagy pathway in macrophages in vivo in controlling MTB infection, and indicate a broader role in shaping the immune response.