bluuebunny/crossref_metadata_2025 huggingface dataset:
Discovery of a novel human anti-IL-2 antibody that potentiates Regulatory T cells by a structure-based mechanism
Authors: Trotta Eleonora , Bessette Paul H. , Silveria Stephanie L. , Ely Lauren K. , Jude Kevin , Holst Charles R. , Coyle Anthony , Garcia Christopher K. , Crellin Natasha K. , Rondon Isaac J. , Bluestone Jeffrey A.
Published: January 2023

Interleukin-2 (IL-2) has been shown to suppress immune pathologies by preferentially expanding regulatory T cells (Treg). However, this therapy has been limited by off-target complications due to pathogenic cell expansion. Recent efforts have been focused on developing a more selective IL-2. It is well documented that certain anti-mouse IL-2 antibodies induce conformational changes that result in selective targeting of Treg cells. We report the generation of the first fully human anti-IL-2 antibody, F5111.2 that stabilizes IL-2 in a conformation that results in the preferential STAT5 phosphorylation of Tregs in vitro and selective expansion of Tregs in vivo. When complexed with human IL-2, F5111.2 induced remission of type 1 diabetes in the NOD mouse model, reduced disease severity in a model of experimental autoimmune encephalitis, and protected mice against xenogeneic Graft-versus-Host-Disease (GvHD). These results suggest that IL-2-F5111.2 may provide a new immunotherapy to treat autoimmune diseases and GvHD.
APD588, a novel, selective S1P- receptor modulator, inhibits inflammation and airway hyperresponsiveness in the ovalbumin-induced model of allergic asthma
Authors: Crosby Catherine Marie , Komori H. Kiyomi , Christopher Ronald , Adams John W
Published: January 2023

Background

FTY720 is a non-selective sphingosine-1-phosphate (S1P) receptor modulator that has shown efficacy in mouse asthma models. APD588 is an investigational stage S1P receptor modulator with increased selectivity and potential for an improved safety profile. We hypothesized that the optimized S1P receptor profile of APD588 would confer beneficial effects in ovalbumin (OVA)-induced asthma.

Aim

To evaluate efficacy of prophylactic APD588 in the OVA-induced allergic asthma model

Methods

Receptor potency and selectivity profile of APD588 was determined (b-arrestin, GTPgS) in cell lines expressing recombinant human S1P1–5. Efficacy in allergic asthma was assessed in BALB/c mice which were sensitized with intraperitoneal OVA on days 0, 7, and 17, then exposed to intratracheal (IT) vehicle or OVA on days 21–23. From day 20, vehicle, APD588 or FTY720 were dosed IT, QD. On day 24, methacholine-induced airway hyperresponsiveness (AHR), immune cell infiltration and BAL cytokines were measured.

Results

APD588 was a potent modulator of S1P1, with less potency at S1P4, 5, and no detectable activity at S1P2,3. IT administration of APD588 produced a dose-dependent inhibition of AHR, significantly inhibited airway eosinophil and lymphocyte infiltration, and BAL cytokines IL-4 and IL-5. FTY720 also reduced AHR and neutrophil and lymphocyte BAL cell counts, but had no effect on eosinophil counts or cytokines.

Conclusions

APD588 was an effective IT therapy for OVA-induced asthma. Lung function improvements correlated with reduced Th2 cytokines and lymphocyte and eosinophil infiltration in the BAL. Based on these results, APD588 may have therapeutic potential in asthma, and broadly indicate potential in other atopic Th2 diseases.
Sox9 deletion in the intestinal epithelium increases house dust mite induced allergic asthma symptoms in mice
Authors: Joldrichsen Marisa R , Kim Eunsoo , Attia Zayed , Davis Ian C , Comet-Boyaka Estelle , Boyaka Prosper N
Published: January 2023

Paneth cells regulate many key aspects of gastrointestinal health through the antimicrobial products and cytokines they produce. Loss or defective Paneth cell functions leads to dysbiosis and is one of the causes of Inflammatory Bowel Disease (IBD). Mice with Sox9 gene deletion within the intestinal epithelium (Sox9 ΔIEC mice) lack Paneth cells making these mice a great model for the study or IBD and other consequences of dysbiosis. Our separate studies have shown that dysbiosis of the gut microbiota could enhance allergic sensitization in the gut and subsequent allergic responses to nasal allergen challenge. We investigated whether dysbiosis caused by a Sox9 deletion in the intestinal epithelium of mice could affect allergic sensitization in the airway and development of asthma responses. For this purpose, control wild-type and Sox9 ΔIEC mice were sensitized tohouse dust mite (HDM) by daily intranasal administration over two weeks period. Upon nasal HDM challenge, Sox9 ΔIEC mice showed more severe signs of allergic asthma than wild-type C57 BL/6 mice as indicated by their higher hypothermia. We also established that the increased allergic symptoms in Sox9 ΔIEC mice correlated with higherIgE levels in serum and bronchoalveolar lavage fluid, and higher frequency of HDM-specific Th2 cells but no difference in Th1 or Th17 responses. These results further illustrate that the gut immune homeostasis plays an important role in allergic responses at distant mucosal sites.
Release of transglutaminase 2 from respiratory syncytial virus-infected airway epithelial cells and its role in disease pathogenesis
Authors: Hosakote Yashoda Madaiah , Rayavara Kempaiah , Kurosky Alexander
Published: January 2023

Respiratory syncytial virus (RSV) is the common cause of severe lower respiratory tract infections in young children, the elderly and the immunocompromised, for which no effective treatment or vaccine is currently available. RSV infections have also been linked to both the development and severity of asthma. Oxidative stress plays a major role in the pathogenesis of many lung inflammatory diseases. Our studies have shown that oxidative stress responses in the airways play a major role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium levels, which in turn activate calcium dependent enzymes, including transglutaminase 2 (TG2). TG2 is a transamidating acyltransferase that catalyzes Ca2+-dependent protein modifications and has been implicated in the pathogenesis of many diseases. This study’s objective was to investigate the release of TG2 in airway epithelial cells in response to RSV infection and its potential mechanism. Studies were conducted in airway epithelial cells, a human lung carcinoma cell line and normal small alveolar epithelial cells. The changes in the TG2 levels were measured by Western blot and RT-PCR after cells were pretreated with antioxidants and infected with RSV. TG2 levels were significantly increased in the secretome with a concomitant decrease in the cell lysates, cytoplasmic and nuclear extracts in response to RSV infection. Antioxidants treatment significantly inhibited RSV-induced TG2 release. TG2 release was induced only by RSV but not RSV-induced cytokines or chemokines. These results suggest that modulation of oxidative stress represents a potential novel pharmacological approach to ameliorate virus-induced acute airway disease through TG2 release blockade.
T follicular helper cell recruitment to the ischemic brain
Authors: Kissel Lee T , Rayasam Aditya , Kijak Julie , Choi Yun Hwa , Hsu Martin , Kovacs Krizstian , Sandor Matyas , Fabry Zsuzsanna
Published: January 2023

Stroke is one of the leading causes of death and disability worldwide. Both clinical and preclinical studies suggest the importance of inflammation in acute and long-term neuronal tissue damage following ischemic stroke. However, the mechanisms of how components of the adaptive immune system contribute to neuroinflammation after stroke are still not fully understood. Previous work in our lab demonstrated that CD4+ T cells accumulate in the brain after cerebral ischemia and Interleukin-21 (IL-21) plays a significant role in T cell-mediated reperfusion damage following ischemic stroke. Given that the major producer of IL-21 is follicular helper T (TFH) cells, we sought to delineate the contribution of these IL-21 producing cells to ischemic damage. Here, we identify that pro-inflammatory CD4+CXCR5+IL21+ TFH cells are recruited to the ischemic brain during the reperfusion phase of transient middle cerebral artery occlusion (tMCAO) in mice. We also show that in human stroke tissue, CD4+CXCR5+ cells adhere to CXCL13-expressing inflamed vessel within the stroke lesions. Additionally, we found that inflamed endothelial cells in mice upregulate CXCL13 following tMCAO, indicating a possible mechanism contributing to TFH cell recruitment to the ischemic brain. Most importantly, administration of blocking antibodies against CXCL13 or BCL6, a TFH cell transcription factor, after tMCAO resulted in a significant decrease in infarct volume and TFH cell frequency in the brain. Overall, these data identify a novel mechanism for how pro-inflammatory T cells are recruited to the ischemic brain to propagate stroke damage and identify potential novel targets for stroke therapy.
TET enzymes augment AID expression via 5hmC modifications at the Aicda superenhancer
Authors: Lio Chan-Wang Jerry , Shukla Vipul , Samaniego-Castruita Daniela , Avalos Edahi Gonzalez , Chakraborty Abhijit , Yue Xiaojing , Schatz David G , Rao Anjana
Published: January 2023

TET enzymes are dioxygenases that promote DNA demethylation by oxidizing the methyl group of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Here we report a close correspondence between 5hmC-marked regions, chromatin accessibility and enhancer activity in B cells, and a strong enrichment for consensus binding motifs for basic region-leucine zipper (bZIP) transcription factors at TET-responsive genomic regions. Functionally, Tet2 and Tet3 regulate class switch recombination (CSR) in murine B cells by enhancing expression of Aicda, encoding the cytidine deaminase AID essential for CSR. TET enzymes deposit 5hmC, demethylate and maintain chromatin accessibility at two TET-responsive elements, TetE1 and TetE2, located within a superenhancer in the Aicda locus. Our data identify BATF as a key bZIP transcription factor involved in TET-dependent Aicda expression. 5hmC is not deposited at TetE1 in activated Batf-deficient B cells, indicating that BATF facilitates TET recruitment to this Aicda enhancer. Our study emphasizes the importance of TET enzymes for bolstering AID expression, and highlight 5hmC as an epigenetic mark that captures enhancer dynamics during cell activation.
Direct activation of Toll-like receptors induce differential B cell responses
Authors: Nelson Alexander J , Wu Yee Ling
Published: January 2023

B cells coordinate host defense against microbes by producing antibodies, secreting cytokines, and presenting antigens to T cells. Recognition of pathogen-associated molecular patterns (PAMPs) by Toll-like receptors (TLRs) plays a crucial role on multiple cell types to initiate protective immune responses. This study is aimed at delineating the direct effects of each TLR signaling on B cells.

Multiple toll-like receptors (TLR1, 2, 3, 4, 6, 7 and 9) are expressed on B cells. We isolated mature, naïve B cells from mouse spleens and stimulated them in vitro with individual TLR agonists alone or in combination with T cell help (via ligation of CD40) and TH2 cytokine interleukin IL-4. B cell responses including proliferation, antibody class switch recombination (CSR) and cytokine production were assayed. We confirmed that stimulation of TLR4 on B cells by lipopolysaccharide (LPS) potently induced robust proliferation and CSR to IgG3. Stimulation of TLR1/2 and 2/6 by agonists Pam3CSK4 and FSL-1 respectively, induced proliferation and CSR to IgG3 albeit to a lesser degree compared to LPS. Stimulation of TLR9 by CpG oligodeoxynucleotides (ODNs) induced robust proliferation, but did not promote CSR. In the presence of anti-CD40 and IL-4, stimulation of TLR1/2, TLR2/6, or TLR9 enhanced proliferation and CSR to IgG1. In contrast, TLR3 agonist (poly I:C) and TLR7 agonist (single-stranded RNA) had no detectable effects on proliferation or CSR. Investigations of TLR signaling on cytokine production by B cells are ongoing. Altogether, these results demonstrate that stimulation of TLRs differentially induce B cell proliferation and modulate CSR.
Physiological V(D)J Recombination is Mediated by RAG Scanning of Loop-extruded Chromatin
Authors: Zhang Yu , Zhang Xuefei , Liang Zhuoyi , Ba Zhaoqing , Dring Eddie , Zurita Jeffrey , Aiden Aviva Presser , Aiden Erez Lieberman , Alt Frederick W.
Published: January 2023

RAG endonuclease initiates V(D)J recombination by cleaving paired V, D, and J gene segments flanked by complementary recombination signal sequences (RSSs). We address physiological relevance of RAG scanning and the underlying mechanisms through studies of the ~300 kb 3′-proximal IgH locus CBE-anchored loop domain. This domain contains nine Ds and four JHs within a 57-kb region between the upstream IGCR1 loop anchor and the V(D)J recombination center (RC). IgH D to JH-rearrangements occur in deletional orientation, mediated by convergent downstream D-RSSs and JH-RSSs, despite Ds having upstream RSSs in the same orientation as JH-RSSs that could promote inversion. Extra-chromosomal V(D)J recombination substrate studies attributed this deletional orientation-specific joining bias to D-RSS sequence differences. However, we find that deletional joining of 8 of 9 chromosomal IgH Ds, results from RAG linear scanning of chromatin upstream of the RAG-bound RC, a process that detects convergent D-RSSs but to which D-RSSs in the same orientation are invisible. The exceptional D employs an RSS-based mechanism to counteract proximal location to JHs in the RC. By deleting JHs, we reveal that RAG scans directionally downstream from 3′D-RSSs until reaching the IgH 3′CBE loop anchor. Targeting catalytic-dead Cas9 (dCas9) binding in this scanning path impedes RAG scanning in association with cohesin accumulation and loop formation between the block site and RC. Strong transcription through a repetitive downstream switch region blocks RAG scanning similarly to the dCas9 block. We demonstrate a critical role for RAG scanning in physiological V(D)J recombination and implicate cohesin-mediated loop extrusion as a driving force.
The role of ERK2 in germinal center B cell selection
Authors: Negron Austin Joseph , Jeffreys Sean , Perez Rebecca , Abdul-Baki Nawal , Forsthuber Thomas
Published: January 2023

The regulation of B cell activation and survival is a crucial control point for the maintenance of B cell tolerance in the germinal center (GC) and involves the coordination of several intracellular signaling pathways. Dysregulation of these pathways can lead to the production and survival of autoreactive memory B cells and plasma cells (PCs), resulting in autoimmune disease. The mitogen-activated protein kinase (MAPK) pathway is involved in the transduction of stimulating and apoptotic signals such as those received through the B cell receptor (BCR) and the Fas receptor (CD95), respectively. Extracellular signal regulated kinases 1 and 2 (ERK1/2), important effectors of this pathway, play a major role in B cell activation and survival. Though ERK2 specifically regulates the pro-apoptotic protein, Bmf, its role in regulating GC B cell selection is unknown. Our lab has developed a novel mouse model in which the deletion of Erk2 is accompanied by the expression of the fluorescent reporter protein, eYFP, allowing us to identify and isolate viable Erk2Δ B cells using flow cytometry. Preliminary data using this model has shown that peripheral Erk2Δ B cells express several surface markers associated with follicular T helper cell interactions and immunoglobulin class switching, such as DEC205 and CD80, respectively. Thus we hypothesize that ERK2 is a critical regulator of GC B cell selection. We are currently investigating the ability of Erk2Δ B cells to respond to various activating stimuli in vitro as well as to differentiate into antigen-specific PCs. Our results will contribute to a better understanding of the molecular programs involved in B cell selection, potentially offering new approaches to treating B cell-driven autoimmune diseases.
T-cell signaling in Th17-mediated inflammation
Authors: Tan Tse-Hua , Chuang Huai-Chia
Published: January 2023

MAP kinase kinase kinase kinases (MAP4Ks) are a subfamily of mammalian Ste20-like serine/threonine kinases that activate the JNK kinase cascade. We have cloned and characterized three MAP4Ks, namely HPK1 (MAP4K1), GLK (MAP4K3) and HGK (MAP4K4). Epigenetic downregulation of HGK (MAP4K4) in T cell results in differentiation of IL-6+ Th17 cells, which play important roles in the pathogenesis of Asia-prevalent non-obese T2D.

HPK1 (MAP4K1) is a negative regulator of T-cell signaling through phosphorylation and sequential ubiquitination of the T-cell adaptor SLP-76. GLK (MAP4K3) also interacts with SLP-76 but does not negatively regulate SLP-76. GLK activates PKC-θ during TCR signaling; GLK-deficient mice showed impaired Th17 differentiation. T-cell specific GLK transgenic mice spontaneously developed autoimmune diseases with an induction of systemic inflammation. We found that GLK signaling specifically induced IL-17A transcription in the T cells of GLK transgenic mice. We will present the data on a novel signal transduction mechanism of IL-17A transcriptional activation of AhR and RORγt by GLK in inflammatory T cells and activated T cells. In addition, we found that GLK negatively regulated differentiation and activity of Treg cells through IKKβ. Collectively, MAP4K3/GLK plays a critical role in IL-17A-induced inflammation.
Choline uptake and metabolism modulate macrophage immunity
Authors: Sanchez-Lopez Elsa , Zhong Zhenyu , Stubelius Alexandra , Sweeney Shannon Renee , Booshehri Laela , Antonucci Laura , Liu-Bryan Ru , Lodi Alessia , Terkeltaub Robert , Lacal Juan Carlos , Murphy Anne N. , Hoffman Hal M , Tiziani Stefano , Guma Monica , Karin Michael
Published: January 2023

Choline is a vitamin-like nutrient that is taken up via specific transporters and metabolized by choline kinase (ChoKα) which converts it to phosphocholine needed for de novo synthesis of phosphatidylcholine (PC), the main phospholipid of cellular membranes. We found that Toll-like receptor (TLR) activation enhances choline uptake by macrophages through induction of the choline transporter CTL1. Choline is rapidly phosphorylated and mobilized to the Kennedy pathway leading to increased cellular phosphatidylcholine production. Inhibition of choline uptake or phosphorylation attenuated NLRP3 inflammasome activation and IL-1β and IL-18 production in stimulated macrophages. Mechanistically, reduced choline uptake altered mitochondrial lipid profile, attenuated mitochondrial ATP synthesis and activated the energy sensor AMP-activated protein kinase (AMPK). By potentiating mitochondrial recruitment of DRP1, AMPK stimulates mitophagy, which contributes to termination of NLRP3 inflammasome activation. Correspondingly, pharmacological ChoKα inhibition ameliorated acute and chronic macrophage-dependent inflammation in vivo.
Dual function of galectin-1 in microglial activation of the brain
Authors: jeon saebom , yoon hee jung , park eun jung
Published: January 2023

Galectins-1, one of the β-galactosides-binding soluble lectin family, exert various roles in immune responses, and have been reported to be closely linked with several inflammation-associated diseases including cancer. However, their roles and underlying mechanisms remain largely unknown in the brain inflammation. In this study, we provide that galectin-1 could act as a regulatory molecule in the brain pathological conditions. Galectin-1 expression is markedly induced in IFN-g-treated microglial cells. In intracranial brain tumor model, we show that expression level of galectin-1 is significantly higher in B35 brain tumor cells and tumor neighboring microglial cells. Treatment of exogenous galectin-1 changes morphology of microglia, and induces expression of ICAM-1, adhesion molecule. In addition, galectin-1 triggers production of both pro-inflammatory mediators and anti-inflammatory mediators. Galectin-1-treated microglial cells activate lymphocytes. These findings indicate that galectin-1 involved in regulation of M1 and M2 phenotype of microglial cells. Therefore, we strongly suggest that galectin-1 may be dual roles in microglial activation of the brain pathophysiological environment.
PD-1 is not expressed on highly activated Natural Killer cells in human and murine models
Authors: Judge Sean J , Dunai Cordelia , Sturgill Ian , Stoffel Kevin , Darrow Morgan , Canter Robert , Murphy William J
Published: January 2023

PD-1 expression is a hallmark of acute T cell priming and response to chronic antigen exposure with a key role in T cell exhaustion. Though identified on NK cells in select populations, the role of PD-1 on NK cells is less clear. Our objective was to determine the expression of PD-1 on resting and activated NK cells in human and murine models. Human NK cells were isolated and expanded ex vivo using a feeder line or cytokine culture. Murine NK cells were evaluated in vivo in tumor-bearing mice and post-MCMV infection in both immunocompetent and immunodeficient mice. We assessed NK cell surface phenotype, activation markers, and intracellular cytokine by flow cytometry and qRT-PCR. Throughout ex vivo human NK activation and proliferation, NK PD-1+ frequency was consistently less than 1% by flow cytometry and negligible by PCR, with upregulation of TIGIT by PCR and flow cytometry (P<0.05). In cytokine stimulated human PBMCs, NK PD-1 expression was 0–2%. In vivo studies with 4T1-bearing mice showed negligible PD-1 from intra-tumoral and splenic NK cells. NK cells isolated from livers in C57BL/6 mice after acute MCMV infection showed high activation by CD69 (>90% positive) but <2% PD-1 expressing NK cells, while T cells exhibited high activation and a 5-fold increase in PD-1 expression. In Rag2−/− mice exposed to MCMV, NK cells isolated from the liver on post-infection day 3 showed minimal PD-1 expression, but positive PD-L1 and TIGIT expression. Unlike T cells, our results show that highly activated human and murine NK cells do not significantly upregulate PD-1. Our data suggest that PD-1 is not a consistent marker for NK exhaustion, though TIGIT appears to demonstrate consistent upregulation and may represent a more useful marker for NK exhaustion.
Characterization of mouse peritoneal macrophages using novel flow cytometry antibodies
Authors: Stepniak Dariusz , Zahner Sonja , Gerhart Ryan , Navert Robert , Schrantz Nicolas
Published: January 2023

Peritoneal resident macrophages are one of the most extensively studied macrophage populations in mouse. These cells can be divided into at least two discrete populations: small peritoneal macrophages (SPM) and large peritoneal macrophages (LPM). Under steady state conditions the monocyte-derived SPM constitute a minor population. They have a F4/80 (low) MHC-II (high) phenotype and constitutively produce cytokine RELM-alpha, known to be involved in anti-helminth response. LPM originate from embryonic progenitors and are normally more abundant. They express high levels of F4/80 and low levels of MHCII, and constitutively produce the B-cell attracting chemokine CXCL13. Here, we examined the homogeneity of each of the populations in C57Bl/6 and Balb/c mice. Our flow cytometric analysis with an array of novel antibodies, including anti-VSIG4 (CRIg), arginase 1, CD206, and CD163, revealed diversity within the LPM population. This phenotypic diversity most likely reflects differences in function and activation status, and will need further investigation.
mGBP7 interacting proteins in Toxoplasma gondii infection and biochemical characteristics of mGBP7
Authors: Legewie Larissa , Loschwitz Jennifer , Smits Sander , Prescher Martin , Wang Xue , Steffens Nora , Degrandi Daniel , Strodel Birgit , Schmitt Lutz , Pfeffer Klaus
Published: January 2023

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite and the causative agent of toxoplasmosis. During host cell invasion, T. gondii forms an unique membranous compartment called the parasitophorous vacuole (PV). Members of the murine guanylate binding protein (mGBP) family localize around the PV leading to its disruption, but the underlying molecular mechanisms are poorly understood. The aim of this project is to provide a better understanding of the biochemistry of mGBPs as well as to elucidate the molecular mechanisms by which this IFNγ-inducible protein family is able to attack the parasite directly.

The GTPase activity of mGBP7 and its oligomerization status were analyzed using a colorimetric assay and multi-angle light scattering (MALS). The structure model of mGBP7 was obtained using I-TASSER homology modeling. For the identification of potential mGBP7 interaction partners co-immunoprecipitation (IP) and mass spectrometry (MS) experiments were performed.

The obtained biochemical data for mGBP7 display a GTPase activity in the μM range and a positive cooperativity of GTP hydrolysis. Furthermore, the MALS results indicate a transient oligomer formation independent of the presence of nucleotides. Homology modeling reveals an extended C-terminal domain for mGBP7 probably responsible for its oligomerization pattern. The initial mGBP7 IP-MS results offer hints about potential mGBP7 interaction partners and indicate that mGBP7 is ubiquitinated and ISGylated even in the absence of T. gondii infection.

The biochemical characterization of mGBP7 as well as the identification of potential mGBP7 interacting proteins will provide new insights into the dynamic interactions at the interface of parasite and host.
TCF1 and LEF1 control Treg competitive survival to maintain immune tolerance
Authors: Fu Wenxian , Yang Bi-Huei
Published: January 2023

CD4+Foxp3+T regulatory (Treg) cells are key players in preventing lethal autoimmunity and deleterious tissue inflammation. To fulfill this role, Treg cells undertake activation and differentiation processes and acquire diverse functional properties. However, how Treg’s differentiation and functional specifications are regulated remains incompletely understood. Here we show that gradient expression of two TCF/LEF family transcription factors, TCF1 and LEF1, distinguishes Treg cells into three distinct populations. Treg-specific ablation of TCF1 and LEF1 renders the mice susceptible to tissue immunopathology and aberrant activation of cellular and humoral responses. TCF1 and LEF1 are essential for promoting Treg’s competitive survival. In addition, the development of T follicular regulatory (Tfr) cells is completely abolished in TCF1/LEF1-conditional knockout mice, leading to unrestrained T follicular helper (Tfh) and germinal center B cell responses. Thus, TCF1 and LEF1 act redundantly to control the maintenance and functional specification of Treg subsets to prevent autoimmune and inflammatory diseases.
Profile of Double Positive T Cells (CD45+CD3+CD4+CD8+) in HIV-Infected Population in Santos City - Brazil
Authors: da Silva Silvano Aparecido , de Almeida Caroline Rossetti , dos Santos Barros Cláudia Renata
Published: January 2023

Background

Double Positive T Cells (CD45+CD3+CD4+CD8+) (DP) are characterized by the presence of CD4 and CD8 receptors during the development of T lymphocytes in thymus. The presence of these immature cells in peripheral blood have been shown to be present in several diseases, including in advanced stages in HIV infections. The objective of this research was to characterize the populations of Double Positive T cells in patients infected with HIV in the city of Santos, Brazil.

Methodology

Analysis of 12350 peripheral blood samples from HIV patients in the city of Santos between the years 2014 and 2017. The identification of the DP cells was performed by flow cytometry with CD3, CD4, CD8 and CD45 receptor markers.

Results

The analysis determined the prevalence of 4.7% (585 samples) of DP T cells in the population of Santos. The mean T-lymphocyte population is 1 cell. The absolute means of this population of helper and suppressor lymphocytes are 858.43 cells/μl - 31.33% and 1243.03 cells/μl - 45.39%. Samples with values greater than 5% of DP cells represented 0.11% (14 samples), which presented mean values higher than 1000 cells/μl of suppressor lymphocytes. Only half the samples had viral copies, the others were undetectable. There was a predominance of Double Negative T cells (CD45+CD3+CD4−CD8−) 95.4% (11,784 samples).

Conclusions

The presence of DP T cells was not superior to Double Negative T cells. No changes were found in the absolute counts of helper lymphocytes and only half had viral copies, with lymphocyte counts without compromising. The immune status of people living with HIV is important for understanding the course of the disease and for future therapeutic interventions.
Regulation of cancer playmaker survival: Increased neutrophil life span by the action of cancer derived a2 V-ATPase
Authors: Ibrahim Safaa A. , Kulshrestha Arpita , Katara Gajendra K , Sahoo Manoranjan , Beaman Kenneth D.
Published: January 2023

Pro-tumorigenic tumor associated neutrophils (TAN) have a long life span and are playmakers “supporter” in cancer progression. Identifying cancer associated factors that regulate neutrophil survival will provide new targets for cancer therapy. A peptide (a2NTD) derived from a2 vacuolar ATPase (a2V) in cancer cells promotes the neutrophil pro-tumorigenic properties that enhance angiogenesis and cancer invasion. Here, Immunohistochemical analysis of different human cancer tissues demonstrated a positive correlation between a2NTD expression and neutrophil count with lung cancer showing the highest correlation. Tissues that are heavily infiltrated by neutrophils are either poorly or moderately differentiated that indicates highly aggressive tumors. Furthermore, treatment of freshly isolated neutrophils from blood of healthy volunteers with recombinant a2NTD led to 2 fold increase in neutrophil survival. This enhancement of survival was dependent on NF-kB activation and partially dependent on TLR-2 activation. Further analysis revealed that a2NTD treatment increased the expression of anti-apoptotic proteins as Bcl-xL, Bcl-2-A1 and decreased the expression of pro-apoptotic proteins as Bax, Apaf-1 and caspase-3,-6, -7. Furthermore, a2NTD treatment stabilized the mitochondrial membrane potential and preserved the Cyt-C in the mitochondria that led to decreased caspase-9 activation. Autocrine secretion of pro-survival cytokines as TNF-a and IL-8 from a2NTD treated cells participated in this increased survival. These data provide evidence that cancer associated a2V is regulating the apoptosis of pro-tumorigenic neutrophils by influencing the intrinsic apoptosis pathway.
Reprograming human Treg metabolism via innate TLR signaling for tumor immunotherapy
Authors: Liu Xia , Li Lingyun , Sanders Katherine L. , Edwards James L. , Hsueh Eddy C. , Ford David A. , Hoft Daniel F. , Peng Guangyong
Published: January 2023

Regulatory T (Treg) cells induce an immunosuppressive microenvironment that is a major obstacle to successful tumor immunotherapy. Dissecting the regulations between energy metabolism and functionality in human Treg cells will provide insights for developing novel immunotherapies against cancer. We discovered that human naturally occurring Treg (nTreg) and tumor-associated Treg cells exhibit distinct metabolic profiles with predominant dependence on glucose metabolism compared with effector T cells. Activated nTreg and tumor-associated Treg cells depend on accelerated glucose consumption for their cellular senescence induction and suppressive activity in responder T cells during their cross-talk. We further identified that TLR8 signaling selectively inhibits glucose uptake and suppresses metabolic processes of glycolysis in human Treg cells, resulting in reversal of Treg suppressive function and cellular senescence induction. In addition, TLR8 signaling activation downregulates mTORC1-HIF1α signaling in Treg cells that control molecular processes of Treg glucose metabolism and suppressive functions. Our in vivo studies further demonstrated that TLR8 signaling-mediated reprogramming of glucose metabolism and function in human Treg cells can enhance anti-tumor immunity and immunotherapy efficacy in a melanoma adoptive transfer T cell therapy model. These studies identify novel mechanistic links between innate signaling and metabolic regulation of human Treg suppression, and provide a new strategy and concept for tumor immunotherapy.
Robust immune response and protection generated by a polyanhydride-based nanoparticle vaccine utilizing the RSV prefusion F protein and/or M protein
Authors: Stephens Laura , Ross Kathleen A. , Hartwig Stacey M. , McLellan Jason S. , Narasimhan Balaji , Legge Kevin L. , Varga Steven M.
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory infections in young children, resulting in 34 million new infections each year. Natural RSV infection is insufficient to establish long-lived and complete protective immunity from reinfection, and there is no licensed vaccine for RSV. We developed a vaccine utilizing a prefusion-stabilized variant of the RSV fusion (F) protein (DS-Cav1) and/or the RSV matrix (M) protein. The vaccine is encapsulated within 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxoctane (CPTEG):1,6-bis(p-carboxyphenoxy) hexane (CPH) copolymer nanoparticles with CpG 1668 ODN. The prefusion conformation of RSV F allows for better exposure of the major antigenic site, termed site Ø, a primary target of neutralizing antibody responses found in human serum. The RSV M protein contains epitopes that are known targets of both CD4 and CD8 T cells. To evaluate this vaccine candidate, BALB/c mice were vaccinated intranasally and subsequently boosted intranasally 4 weeks later. Vaccination with the prefusion RSV F nanovaccine significantly ameliorated weight loss and pulmonary dysfunction compared to unvaccinated mice in response to an RSV challenge. This protection remained until at least 100 days post prime vaccination. Additionally, peak viral gene copy numbers in the lung were reduced in vaccinated mice. The prime/boost prefusion RSV F vaccination induced activated lung-localized T cells, as well as tissue-resident memory CD4 and CD8 T cells in the lungs. Vaccination also induced lung tissue-resident B cells. Given the strong immune response generated as well as the protection provided from challenge, this prefusion RSV F nanoparticle formulation may be a promising vaccine candidate.
The role of mTOR, TRAF1, and local antigen in 4-1BB-dependent establishment of Trm in the lung
Authors: Batista Nathalia Vieira , Zhou Angela C , Watts Tania H
Published: January 2023

The tumour necrosis factor receptor (TNFR) superfamily receptor 4-1BB is important in the establishment of a tissue-resident memory (Trm) population in the lung tissue following influenza infection. Moreover, supraphysiological boosting of 4-1BB during the boost phase of a prime-boost immunization regimen can greatly enhance the establishment of a long-lived Trm population that protects against lethal heterotypic challenge (Zhou et al. Mucosal Immunology 2017). However, little is known about how 4-1BB contributes to the establishment of the lung Trm population. Here, we investigated the mechanism by which 4-1BB induces lung Trm cells. Using competitive mixed bone marrow chimeras, we found that the effect of 4-1BB on lung resident influenza-specific T cells does not substantially change between day 9 and 45 post-infection, suggesting that the main effect of 4-1BB is to allow the persistence of CD8 T effector cells as they transition to Trm. The signaling adaptor TRAF1, downstream of 4-1BB was also shown to be important in enhancing the numbers of Teffectors and Trm in the lung. Using supraphysiological stimulation of 4-1BB in the boost phase of a prime boost immunization, we show 4-1BB-mediated Trm generation is dependent on local delivery of both antigen and costimulation and that this process is inhibited by rapamycin, suggesting a role for mTOR in this process. Also, using this prime-boost model we demonstrate that TRAF1 contributes to the effect of 4-1BB on Trm generation. Taken together, these data point to an important role for 4-1BB, TRAF1 and mTORC1 in allowing the lung effector T cells in the lung parenchyma to survive through the transition to tissue resident memory T cells.

This work was funded by CIHR grant FDN-143250 to THW
Gut IgA abundance in adult life can protect from DSS colitis even if maternal IgA received in early life is inadequate
Authors: Gupta Suman , Basu Srijani , Bal Vineeta , Rath Satyajit , George Anna
Published: January 2023

Susceptibility to DSS induced colitis has been shown to depend on various host and microbial factors, the caveat being that most of these studies have used gene-deficient or gnotobiotic mouse models. In order to study specific factors contributing to colitis susceptibility under more physiological conditions we used inbred mouse strains that differ in their IgA responses but are otherwise healthy. We found that the IgA high strain CBA/CaJ (CBA) is resistant to DSS-colitis unlike the IgA low strain C57BL/6 (B6) strain and resistance correlated with extensive IgA-coating of fecal bacteria and lower fecal bacterial loads. Additionally, we found a more robust intestinal barrier in CBA mouse, along with lower intestinal inflammation and bacterial translocation during colitis induction. Through homologous fecal microbial transplants we found that the IgA-uncoated fraction of B6 flora was relatively more colitogenic. Further, experiments with F1 and F1xParent ‘backcrossed’ progeny generated separately with CBA and B6 dams indicated that pups born to IgA high proximal dams were resistant to colitis. These results indicate a role of passive maternal IgA and active IgA in colitis resistance. In order to understand whether early exposure to passive IgA alone can confer resistance to colitis, we foster-nursed B6 pups on CBA dams and vice-versa but it did not have an effect on DSS susceptibility. Interestingly, oral administration of immunoglobulins to B6 adults did decrease DSS associated disease. Collectively, our data indicate that higher gut IgA in adult life plays a more important role as compared to maternal IgA received during early life in conferring resistance to DSS colitis.
JAML-CAR – A Mechanism of T Cell Antitumor Immunity
Authors: McGraw Joseph Michael , Witherden Deborah , Havran Wendy L
Published: January 2023

The junctional adhesion molecule-like protein (JAML) is expressed by a variety of immune cell subsets and binds the Coxsackie and adenovirus receptor (CAR), an adhesion molecule expressed in epithelial tissues. Previous work from our lab demonstrated that JAML-CAR interactions are a unique costimulatory mechanism that controls activation of tissue-resident γδ T cells. High expression of JAML within tumors is associated with improved patient survival in a variety of epithelial cancers, yet the role of JAML-CAR interactions in antitumor immunity has not been characterized. Based on this evidence and the fact that CAR is expressed in epithelial tissues where many tumors arise, we hypothesized that JAML-CAR interactions are critical for antitumor immunity. Initial results utilizing the B16F10 melanoma model show that JAML−/− mice are more susceptible to tumor growth and have fewer γδ and CD8 T cells within tumors compared to WT mice. In WT mice, JAML is upregulated on γδ and CD8 T cells following activation, and a large fraction of γδ and CD8 tumor-infiltrating lymphocytes express JAML. Expression of CAR in mouse B16F10 melanoma is detectable within tumor margins but not within the tumor interior, and in human melanoma, CAR expression decreases with increased malignancy. This data suggests that tumors can downregulate CAR to escape immune responses. In support of this hypothesis, overexpression of CAR on B16F10 melanoma results in slower tumor formation and growth in WT mice but not RAG−/− or JAML−/− mice, which indicates that expression of CAR sensitives tumors to immune detection. These results show that JAML-CAR interactions are critical for T cell antitumor immunity and may be a novel therapeutic target to treat epithelial cancers.
Deep characterization of tumor microenvironments using single cell multi-omics analysis
Authors: Montesclaros Luz , Boutet Stephane C , Taylor Sarah E B , Stubbington Michael J T , Giangarra Valeria , Lau Julia K , Sapida Jerald , Ziraldo Solongo , Pfeiffer Katherine A. , Zheng Grace , Barrio Alvaro Martinez , Lee Josephine Y , Marrs Samuel , Wu Kevin , Mikkelsen Tarjei S
Published: January 2023

Understanding the complexity of cell interactions in the tumor microenvironment (TME) requires the ability to distinguish each cell type, and is a prerequisite of personalized cancer treatments. Here, we use a fully integrated system for single cell RNA sequencing, to simultaneously profile the transcriptome, cell surface proteins and immune repertoire of cells from primary colorectal cancer (CRC), non-small cell lung cancer (NSCLC), and mucosa-associated lymphoid tissue (MALT) lymphoma. Each tumor varied in type and proportion of its cellular components, and in particular in the proportion of immune cells. The CRC tumor consisted of T (3% CD4+, 3% CD8+), B lymphocytes (5% CD79A+) and plasma B cells (11% IGH high). Repertoire sequencing identified a clonal expansion (>4% of B cell clonotypes) suggesting a strong B cell response in this tumor. The NSCLC tumor displayed a marked immune cell infiltration containing predominantly B cells (30% CD79A+ and 8% IGH high plasma B) with a very limited clonal expansion. The MALT lymphoma consisted of only T and B lymphocytes (31% CD4+, 8% CD8+, 57% CD79A+). In addition to the activated CD4+ and CD8+ T cells, Tfh and Treg cells were clearly identified as well as two distinct large B cell populations showing plasmacytic differentiation. Analysis of the B cell repertoire revealed a large expanded clone bearing an IGHV segment associated with parotid MALT lymphoma. These findings emphasize the importance of combining repertoire and gene expression sequencing data to determine the nature and clonality of an immune response. This method enables full characterization of tumor heterogeneity and the adaptive immune response to the TME and will be key in the development of successful immunotherapies.
Irreversible electroporation stimulates a pro-inflammatory tumor microenvironment in pancreatic cancer
Authors: Brock Rebecca Michaela , White Natalie B , Ringel-Scaia Veronica Marie , Coutermarsh-Ott Sheryl L , Eden Kristin , Coutri Jenna , Manuchehrabadi Navid , Davalos Rafael V , Allen Irving C
Published: January 2023

Pancreatic cancer is a major cancer with a survival rate of only 6% due to late diagnosis and the aggressiveness of the malignancy. There is also a severe lack of viable treatment options due to the localization of the primary tumor near major blood vessels and bile ducts as well as the highly immunosuppressive nature and metastatic potential of pancreatic cancer. New ablative techniques for pancreatic tumors are being developed; however most are thermal and can cause damage to the delicate surrounding structures. Non-thermal irreversible electroporation (IRE) uses short, high frequency electrical pulses to permeabilize cancer cell membranes and illicit cell death. IRE has shown promising results in clinical trials of late-stage, locally advanced cases. Here we utilize several models of pancreatic cancer in vitro, ex vivo, and in vivo to determine the effect of IRE on cell death pathways, cancer pathways, and immune signaling. IRE elicits pro-inflammatory cell death via necrosis and pyroptosis in immunocompetent murine and human patient-derived xenograft models. This pro-inflammatory environment can stimulate immune cell infiltration to the tumor site, halting tumor progression. Moreover, a non-thermal ablative approach also preserves cancer cell antigens that can lead to better immunosurveillance against reoccurrence and metastatic lesions. With the advent of these new techniques, the impact of such technologies to the tumor microenvironment and immune system need to be assessed to determine their clinical application. IRE is proving to be a safe and effective treatment for pancreatic cancer as it not only ablates the primary tumor site but also stimulates the immune system to recognize and combat the tumor throughout the body.
Inhibition of PI3K promotes anti-tumor immune responses to Mantle Cell Lymphoma
Authors: Shissler Susannah Claire , Webb Tonya J
Published: January 2023

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin’s lymphoma that is characterized by a chromosomal translocation resulting in overexpression of Cyclin D1, dysregulation of the cell cycle, and constitutive activation of phosphatidylinositol 3 kinase (PI3K). MCL patients also have decreased natural killer T (NKT) cells – an innate-like lymphocyte population involved in initiating the anti-tumor immune response. We hypothesize that constitutive activation of PI3K in MCL alters CD1d-mediated antigen processing and presentation, ultimately resulting in decreased anti-tumor NKT cell responses. To investigate the role of PI3K on MCL survival and immunogenicity in vitro and in vivo, we utilized a panel of PI3K inhibitors (PI3Ki). Pretreatment of human MCL cell lines with micromolar concentrations of PI3Ki resulted in increased apoptosis and enhanced immunogenicity. While dual PI3K/mTOR inhibitor Dactolisib (BEZ-235), completely abrogates NKT/T cell responses of in vitro, stimulated peripheral blood mononuclear cells, PI3K-specific inhibition allows immune cells to function. In a murine model of MCL, PI3Ki-treated animals had decreased tumor burden and increased NKT/T cell responses to ex vivo stimulation with the most significant effect seen in the Buparlisib (BKM-120)-treated group. To elucidate the mechanism, MCL cells were treated in vitro with BKM-120 and flow cytometric analysis indicated increased CD1d expression, but decreased MHC class I expression. These results suggest that selectively targeting PI3K can reduce toxicity, as well as have a significant impact on tumor burden and immune function.
Type I interferon suppresses tumor growth through activating the STAT3-granzyme B pathway in tumor-infiltrating cytotoxic T lymphocytes
Authors: Lu Chunwan , Klement John D. , Ibrahim Mohammed L. , Redd Priscilla S. , Zhou Gang , Liu Kebin
Published: January 2023

Background

Type I interferons (IFN-I) have recently emerged as key regulators of tumor response to chemotherapy and immunotherapy. However, IFN-I function in immune cells in the tumor microenvironment is largely unknown.

Methods

IFNAR1 knock out and mixed chimera mice, and mice with IFNAR1 deficiency only in T cells were used to determine IFN-I function in immune cell tumor infiltration and tumor development. IFN-I-regulated genes in CTLs were identified and functionally analyzed by genome-wide gene expression profiling and molecular approaches.

Results

IFNα13 and IFNβ are selectively expressed in colon carcinoma tissues in vivo. Although IFNAR1 KO mice exhibited increased susceptibility to carcinogen and inflammation-induced sarcoma and colon tumorigenesis, IFNAR1-sufficient tumors grow significantly faster in IFNAR1 KO mice and in mice with IFNAR1 deficiency only in T cells, suggesting that IFN-I functions in T cells to enhance host cancer immunosurveillance. Surprisingly, unlike in an anti-viral immune response, IFNAR1-deficient T cells exhibit no deficiency in generation of antigen-specific CTLs and IFNAR1-deficient CTLs infiltration level is similar as compared to that of WT CTLs in colon tumor-bearing mice. Gene expression profiling identified Gzmb as an IFN-I target gene in tumor-infiltrating CTLs and in antigen-specific CTLs in vivo. Mechanistically, we determined that IFN-I activates STAT3 that binds to the Gzmb promoter to activate Gzmb transcription in CTLs. IFN-I also regulates GZMB expression in human T cells, but IFNAR1 is not only significantly down-regulated in human colon carcinomas but also down-regulated in CTLs of human colon cancer patients as compared to normal colon and CTLs from healthy donors.