bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Virulence and Virulence Regulatory Factors in Escherichia coli using Dictyostelium discoideum as a Model Phagocyte
Authors: Snyder Michelle , Weichseldorfer Matthew , Lane Jorden , Kazmierski Rachel , Bains Harpal , White Theresa , Parker Dominique , Aklkharouf Nadim , Hemm Matthew
Published: January 2023

Escherichia coli is normally found as part of the human gut microbiota, however there exist a number of diarrheagenic strains. Identification and disruption of the virulence factors that promote infection could lead to a reduction in infection period of these E. coli strains and potentially of other species with similar means of virulence. To identify E. coli strains with altered virulence, we used Dictyostelium discoideum, a social amoeba that phagocytizes bacteria for nutritional purposes. Using random mutagenesis, we created mutant stocks of E. coli, each. These stocks were subjected to several rounds of phagocytosis by D. discoideum, enriching the culture and selecting for mutants resistant to intracellular killing. Resistant mutants were isolated and we found that some of the resistant strains showed increased survival when compared to the wild-type strain. Once isolated, the full genomes of our resistant mutant strains were sequenced using Illumina’s next generation MiSeq technology. While analyzing the sequencing data from these mutant strains, we found several potential genes of interest, including genes related to flagella, oxidoreductases, and acid resistance. We have begun to characterize genes of interest through various qualitative and quantitative assays using individual gene knockout strains. These studies should allow for identification of novel factors associated with regulation of virulence in E. coli. In addition, given that the development of mechanisms to resist D. discoideum predation may have contributed to the selection and maintenance of bacterial virulence factors against mammalian hosts, these studies may provide insight on the evolution of host-pathogen interactions.
Ccr7-dependent amelioration of renal tubular injury protects against infection-induced acute kidney injury
Authors: Break Timothy J. , Green Nathaniel G. , Kojima Hiroshi , Yuen Peter S. T. , Lee Chyi-chia “Richard” , Swamydas Muthulekha , Star Robert A. , Lionakis Michail S.
Published: January 2023

Systemic candidiasis (SC) is a leading cause of nosocomial bloodstream infections, with mortality of >40% despite therapy. In a mouse model of SC, the most highly infected organ is the kidney, and myeloid cells, including dendritic cells (DCs), are integral for host defense. We hypothesized that Ccr7, which mediates DC trafficking and survival, is critical for host defense during SC. Ccr7 and its ligands, Ccl19 and Ccl21, were highly up-regulated in the Candida-infected kidney. All Ccr7−/− mice rapidly succumbed during SC, whereas WT mice had 80% survival at day 14. Infected Ccr7−/− mice had significantly greater kidney fungal burden, with greater tissue damage, and more severe renal failure. Infection in Ccl19−/− mice and in plt/plt mice (that lack Ccl19 and the CCL21a isoform of Ccl21) did not phenocopy the susceptibility of Ccr7−/− mice, indicating that the CCL21b-Ccr7 axis is critical for protection. Surprisingly, bone marrow chimeras revealed that the protective effects of Ccr7 are solely mediated by non-hematopoietic cells. Studies using FACS, IHC, and mRNA from sorted renal cells showed that Ccr7 is expressed by renal tubular cells (RTCs), not other renal stromal cells. Our preliminary data show the Ccr7 is a survival factor for RTCs; indeed, Ccr7 deficiency resulted in significantly greater tubulitis and tubular necrosis during SC. The renal protective effects of Ccr7 were also evident during bacterial sepsis, indicating that Ccr7 is broadly protective against infection-induced acute kidney injury. In summary, our data are the first to implicate Ccr7 in innate control of any pathogen and to uncover the contribution of Ccr7 on non-hematopoietic cells in protection against acute kidney injury and systemic fungal infection.
The contribution of acidic mammalian chitinase to disease severity in experimental fungal asthma
Authors: Mackel Joseph J , Garth Jaleesa M , Reeder Kristen M , Blackburn Jonathan , Steele Chad
Published: January 2023

Chitin is a polysaccharide that provides structure and rigidity to the fungal cell wall. Exposure to A. fumigatus cell wall moieties such as chitin has been linked to fungal sensitization and allergic inflammation. Chitin is not present in mammals, however, mammals possess enzymes that function to degrade chitin known as chitinases. Mammals possess two functional chitinases, acidic mammalian chitinase (AMCase) and chitotriosidase. Although chitinase expression has been linked to Th2 respiratory disorders, their exact role in fungal sensitization and fungal asthma has not been determined. In an allergic asthma model associated with chronic A. fumigatus exposure, mice deficient in AMCase displayed attenuated levels of the proallergic/pro-type 2 factors CCL22, CCL17, and IL-33. Furthermore, a deficiency in AMCase led to improved total lung function. Surprisingly, examination of inflammatory responses revealed attenuated IL-17 and IL-22 responses, but not type 2 responses (IL-4, IL-5, IL-13), in the absence of AMCase. Collectively, these data suggest that expression of AMCase during allergic fungal asthma is associated with increased severity that is putatively associated with IL-17A and IL-22 responses.
We Are What We Eat ___ Bioinformatic Prediction of microRNAs in JPS Water Decoctum of Prescription in Traditional Chinese Medicine and Their Mammalian Target Genes
Authors: Shen Chao-Bin , Yu Lan , Cai Hong
Published: January 2023

Objective

To provide a good start for understanding the roles of microRNA (miRNAs) in Jade Screen Powder (JSP). JSP has been clinically used in China for over 700 years to treat RRTIs, asthma, and ephidrosis in childhood.

Methods

The miRNA profile of JSP have used the next generation high-through put Illumina RNA-seq technology, the miRNAs and their target genes were predicted using bioinformatic approach . We applied a series of transcriptome analysis method, including miRNA differential expression analysis, de novo identification of miRNAs as well as co-expression target reconstruction, to identify prediction epigenetic molecular mechanisms of JSP in mammalian.

Results

We sequenced ~ 8,566,120 small RNA reads from the of JSP Water Decoctum and conserved miRNAs based on similarity to miRNA known in whole plant species and novel miRNAs based on potential hairpin-forming precusors with in miRBase 21. A general transcriptome database with 21,640 sequences was obtained. Based on the transcriptomic sequences, 53 miRNA, classified into 21 families. A total of 149 genes were predicted to be regulated by these high expression 8 miRNAs. Multiple targets were perturbed and the immunoregulatory was identified as the potential primary target pathway of JSP in mammalian.

Conclusion

Our results provide the first clue to the elucidation of miRNA profiles in prescription of JSP by thermodynamic treated. These miRNAs and the corresponding target genes identified in this study will provide a solid basis for understanding their biological functions in JPS Decoctum. Immunoregulation of herbal microRNAs in mammals are a new concept.
Commitment of mitosis in B cells: “before and after”
Authors: Singh Amit K , Spitzer Matthew , Nolan Garry P , Mattson Mark P , Sollott Steven , Sen Ranjan
Published: January 2023

Naïve B cells proliferate differentially in response to various mitogenic signals. However, regardless of the initiating stimulus, B cells that progressed through one mitotic division gain the ability to divide up to 3–4 times in the absence of renewed stimulation. In contrast, cells that had not progressed through mitosis did not divide except when re-stimulated. These observations go against accepted dogma about cell cycle regulation, which posits that cells need to be re-stimulated after each division to progress through G1. To identify the basis for signal-independent cell division we compared cells that had undergone one division and those that had not by several assays. Our findings indicate that divided cells are already pre-programed to cross restriction (R) point. The characteristics of cells that have divided once include bigger cell size in G1, low levels of p27, hyper-phosphorylated Rb, high c-Myc and phospho-CDK2. High throughput CyTOF analysis at single cell level with 25 parameters suggested that the G1 state of once-divided cells differs significantly from the G1 phase of cells progressing through the first cell division cycle. RNA-seq analysis identified Birc5 (Survivin) which is an E2F target gene as highly expressed in the cells that have underwent signal-independent proliferation. Survivin as exclusively present in G1 phase of divided B cells and blocking it with its inhibitor (LLP3) halted the G1-S progression and therefore blocked signal independent proliferation. This in vitro model may recapitulate aspects of signal independent cell division seen in the dark zone of the germinal center (GC) and resolve mechanisms involved in various GC specific B cell lymphomas.
Zymosan tolerizes primary pig monocytes to lipopolysaccharide stimulation
Authors: Byrne Kristen A , Loving Crystal L
Published: January 2023

Exposure of monocytes to β-1,3 glucans from yeast cell wall can enhance the immune response to heterologous innate agonists, a phenomenon termed innate training. This is in contrast to tolerance, which is well described in relation to lipopolysaccharide (LPS) responses. Yeast extracts are fed to food animals for production and immune benefits, but the mechanism of immune modulation provided by dietary yeast is not clearly defined. With the recent limitations to antibiotic use in food production animals, alternatives are sought to maintain animal health and production. To elucidate the immunological effect of yeast β-glucans on swine monocytes when re-exposed to a heterologous agonist, we primed primary swine monocytes with varying amounts and sources of β-glucan for 24h in vitro and restimulated the cells 5d later to LPS. Monocytes primed with media only or LPS, and exposed to LPS 5d later, served as controls to delineate trained versus tolerant responses. Swine monocytes primed with LPS produced IL-1β and TNF-α, and transitioned to a tolerant state, with decreased production of IL-1β and TNF-α upon re-stimulation with LPS. Interestingly, monocytes primed with the commercial β-glucan, zymosan (S. cerevisiae), at 10mg/mL or higher and restimulated with LPS exhibited a tolerized state. Specifically, zymosan primed cells did not produce IL-1β or TNF-α in response to LPS restimulation. Monocytes primed with laminarin (L. digitata) and restimulated with LPS did not exhibit a trained or tolerant state. These data indicate that dosage and source of β-glucan play an important role in the priming of monocytes to secondary LPS exposure, and warrants further study to elucidate differential responses as they relate to commercial dietary β-glucans.
Helminth-induced B lymphocytes mediate control of emphysema through production of RELMa
Authors: Chen Fei , Wu Wenhui , Millman Ariel , Palma Mark J , Gause William C
Published: January 2023

The intestinal nematode parasite, Nippostrongylus brasiliensis (Nb), can cause emphysema and the mechanisms remain poorly defined. Here we show that emphysema develops as early as 7 days after Nb inoculation and is associated with increased gene expression of Il17a, neutrophil elastase (Ela2), and macrophage matrix metalloproteinase 12 (Mmp12) in the lung. Transfer of Nb-primed neutrophils or macrophages to naïve recipients results in emphysema. Nb infection of Il17a−/−mice results in a less severe emphysematous pathology, decreased neutrophil influx to the lung, and lower gene expression of Ela2. Surprisingly, Nb infection in B cell deficient Jh−/− mice results in a more severe emphysema when compared to WT mice, and is associated with higher numbers of neutrophils and macrophages, as well as increased expression of Il17a, Ela2 and Mmp12 in the lung. Transfer of B lymphocytes from WT mice or Il10−/− mice, but not from Il4Rα−/− mice, or Relmα−/− mice to recipient Jh−/−mice can restore control of emphysema, decrease influx of neutrophils, macrophages, and downregulate IL-17A. B cells from lung tissue of Nb-primed WT mice, but not Il4Rα−/− mice express high levels of Relmα. Co-culture of Nb-primed WT B cells but not Relmα−/− B cells with Nb-primed neutrophils or Nb-primed macrophages decreased ELA-2 and MMP-12 activity, respectively. Taken together, these data suggest that IL-17A promotes emphysema through recruitment and activation of protease secreting neutrophils and macrophages in the lung. IL4R-dependent B regulatory cells control emphysema through two independent mechanisms, downregulation of IL-17A and suppression of myeloid cell protease production, both of which require secretion of RELMα.
Inositol kinase Itpkb is a novel therapeutic target for chronic graft versus host disease (cGVHD)
Authors: Du Jing , Paz Katelyn , Flynn Ryan , Taylor Patricia , Miller Andrew , Dahlberg Carol , Blazar Bruce
Published: January 2023

Chronic graft versus host disease (cGVHD) is a common and life-threatening complication of allogeneic hematopoietic stem cell transplantation. cGVHD is mediated by donor T cells recognizing and attacking host tissues followed by activation of effector cells such as B cells to product pathogenic autoantibodies and macrophages to mediate fibrotic responses. Safer and more effective therapies are needed for cGVHD treatment. Inositol 1,4,5-trisphosphate 3-kinase B (Itpkb) is a Ca2+ dependent kinase downstream of lymphocyte receptor signaling that regulates T and B cell homeostasis and function by controlling the intracellular Ca2+ level, thus regulating apoptotic responses. Genetic deletion of Itpkb results in apoptosis. GNF362, a LMW Itpkb inhibitor blocks T cell driven autoimmunity. In this study, we explore the role of Itpkb in cGVHD pathogenesis and hypothesize that cGVHD can be prevented by disrupting the Itpkb signaling pathway. We test the hypothesis in two preclinical cGVHD models with distinct clinical manifestations and pathophysiology, including a multi-organ system cGVHD mouse model that is characterized by bronchiolitis obliterans (BO) driven by spontaneous germinal center reactions and antibody deposition, and a scleroderma model that is characterized by macrophage infiltration and skin fibrosis. This study demonstrates that depleting Itpkb in donor T cells, but not in donor bone marrow blocked cGVHD lung disease and germinal center reaction in the BO model. In addition, pharmaceutical inhibition of Itpkb with GNF362 significantly reduced cGVHD manifestations in both models. These data identify Itpkb as an essential mediator of cGVHD pathogenesis and suggest Itpkb inhibition as a novel approach to treat cGVHD.
The p38 MAPK inhibitor SB203580 abrogates TNF-mediated expansion of regulatory T cells
Authors: Chen Xin , He Tianzhen
Published: January 2023

CD4+FoxP3+ regulatory T cells (Tregs) represent a major cellular mechanism of immune evasion of tumor. There is compelling evidence that TNF is able to activate and expand Tregs through TNFR2. However, it remains unclear which TNFR2 signaling pathway(s) is required for the activation of Tregs. In this study, we determined the identity of the TNFR2 signaling components by using specific small molecule inhibitors. To this end, MACS purified CD4+ T cells from WT C57BL/6 mice were labeled with CFSE and stimulated with TNF in the presence of IL-2. The proliferation of Tregs was analyzed by FACS, gating on FoxP3+cells. Treatment with the p38 MAPK inhibitor SB203580, rather than NF-κB inhibitors, abrogated TNF-mediated expansion of Tregs. In addition, administration of SB203580 blocked LPS-induced expansion of Tregs and up-regulation of TNFR2 expression on Tregs in vivo. Importantly, treatment with SB203580 potently inhibited lung metastasis of mouse 4T1 breast cancer, which presumably resulted from reduction of Treg activity in tumor bearing mice. Therefore, our data suggest that activation of p38 MAPK signaling pathway is required for TNF-mediated proliferative expansion of Tregs. Targeting p38 MAPK pathway may represent a novel strategy to enhance the efficacy of cancer immunotherapy through suppression of Treg activity.

[Supported by the University of Macau Research Grants (MYRG2016-00023-ICMS-QRCM) and a grant from the Science and Technology Development Fund (FDCT); Macao S.A.R. (014/2015/A1)]
Engraftment of Human Immune System and Human Tumor Xenograft in Murine Models: Applications in Oncology Drug Discovery
Authors: Tanaka Azusa , Connerney Mona
Published: January 2023

The development of animal models capable of mimicking human immune responses and function as a host of human tumor xenograft are crucial to study the safety and efficacy of immune checkpoint inhibitors, functionality of CAR-T therapies, and immuno-oncology treatment including gene and cell therapies. Mice reconstituted with human hematopoietic stem cells (HSC) are able to stably differentiate and repopulate human immune cells sufficient to model many aspects of tumor immunity. Taconic’s CIEA NOG mouse ® (NOG) portfolio is one of such powerful tools: capable of accepting human HSC which develops into multiple immune cells including T cells, as well as ideal hosts for human tumor xenografts due to its hyper immune deficiency caused by IL-2Rg mutation and NOD/Scid background.

In partnership with the Central Institute for Experimental Animals (CIEA), Taconic has developed a variety of next generation immune-deficient NOG mice that are suitable and highly effective at modeling the diverse mechanistic functions of human immunity as well as being the host for human tumor xenograft including patient derived xenograft (PDX). This presentation will focus on the current state of human immune system engraftment models (huNOG and huNOG-EXL) and utilization of wide range of NOG mice portfolio as the next generation models that diversify the mechanistic functionality of immune engraftment and recent advances in modeling human immunity in immuno-oncology applications.
MyD88-dependent activation of protein kinase D1 is essential for Group B streptococci-induced proinflammatory responses
Authors: Yi Ae-Kyung , Yoon Tae Won , Upadhyay Kirtikumar , Park Jeoung-Eun , Kim Young-In , Metcalfe Victoria , Halder Priyanka , Talati Ajay J.
Published: January 2023

Group B streptococcus (GBS) causes life-threatening diseases, like pneumonia, septicemia and meningitis, in neonates. It has been reported that the pathogen-associated molecular patterns in GBS are recognized by pattern-recognition receptors, including Toll-like receptors (TLR), in the host innate immune cells. This recognition results in the cascades of inflammatory reactions. However, the molecular basis of GBS-induced proinflammatory responses are not yet completely elucidated. Protein kinase D1 (PKD1) has been identified as one of key signaling intermediaries that plays an indispensable role in proinflammatory responses mediated by TLRs. However, it is currently unknown whether PKD1 is activated, and contributes to proinflammatory responses induced by GBS. In the present study, the role of PKD1 in the proinflammatory responses to GBS is investigated. We found that both live and antibiotic-killed GBS activate PKD1 in human and murine cells. This PKD1 activation is dependent on the TLR signaling adaptor MyD88 and its downstream kinase IRAK1, but not TRAF6. Inhibition of PKD using a pharmacological inhibitor revealed that PKD1 is indispensable for GBS-mediated expression of various cytokines/chemokines in vitro and in vivo. Furthermore, systemic administration of PKD inhibitor protects D-galactosamine-sensitized mice from shock-mediated death caused by antibiotic-killed GBS. Our findings imply that PKD1 is one of the critical factors that play a regulatory role in GBS-induced proinflammatory reactions, and inhibition of PKD1 activation together with antibiotic treatment in GBS-infected neonates might be an effective way to control GBS diseases.
Molecular Basis Of Tick Saliva Proteins Effects On Macrophage Function
Authors: Bakshi Mariam , Mwangi Waithaka , Mulenga Albert
Published: January 2023

Ticks are medically important ectoparasites and transmit many tick borne diseases. Tick feeding and transmission of TBD agents start with the tick penetrating host skin and is aided by immune-modulatory tick salivary components. Macrophages are critical early responders in host defense after neutrophils and have profound effect on host defense mechanisms against ticks. Combatting the inflammatory mechanisms directed by macrophages is one task that ticks face during feeding. To address the possibility that ticks affect macrophage function, we previously screened effects of 15 recombinant TSPs. This analysis found pro-inflammatory and anti-inflammation TSPs. Of significant interest anti-inflammation rTSPs reversed pro-inflammation rTSP activation of macrophages. To investigate the molecular basis of this response, we carried out a systematic examination of the transcriptional gene expression in these macrophages in response to the rTSPs. We found a remarkable program of gene expression induced/suppressed specifically by pro- and anti-inflammatory rTSPs. An intricately choreographed expression pattern devoted to immunity, locomotion, response due to stimulus and adhesion was a prominent feature of this response. Our finding indicate that meshwork of host molecules respond to pro- and anti-inflammation rTSPs. Our analysis reveal a complex and intimate relationship between host and ticks.
Functionality of tissue-resident CD8+ T-cells in the human gastrointestinal tract during chronic HIV-1 infection.
Authors: Kiniry Brenna , Ganesh Anupama , Hunt Peter W , Somsouk Ma , Deeks Steven G , Shacklett Barbara L
Published: January 2023

The gastrointestinal tract (GIT) is an important site of HIV transmission and pathogenesis. Preventing or curing HIV infection will likely entail maintenance of protective anti-HIV immune responses in the GIT. Tissue resident memory T-cells (TRM) are long-lived, non-recirculating memory T-cells localized in tissues including the GIT. Positioned near sites of infection, TRM have protective efficacy against several pathogens. The precise role CD8+ TRM in HIV infection is not known. Blood and GIT CD8+ TRM cells from HIV+ and HIV- adults were identified by flow cytrometry using CD45RO, CD69, CD103, S1PR1, T-bet, and Eomesodermin (Eomes). Functionality was assessed in a 6-hour ex vivo stimulation assay. Cells were stimulated with DMSO, Gag, or Staphylococcal Enterotoxin B, and stained for CD107a, IFNγ, Mip-1β, TNFα, and IL-2. The frequency of CD103+CD69+S1PR1− CD8+ T-cells was significantly greater (P=0.005) in gut compared to blood (medians 54.8% and 0.10% respectively), indicative of tissue residency. CD45RO+ cells in this subset were classified as TRM. Consistent with murine studies, CD8+TRM were mainly T-betLowEomesNeg; however a population of EomesHigh CD8+ TRM emerged in HIV+ individuals not on antiretroviral therapy. Although polyfunctional Gag-specific CD8+ TRM responses (CD107a+, IFNγ+, Mip-1β+) appeared strongest in participants who control HIV without therapy (“controllers”), they constituted a smaller proportion of the total rectal Gag-specific CD8+ T-cell response compared to viremic participants not on ART (approximately 15% and 29% respectively). In summary, a large subset of CD8+ T-cells in rectal mucosa are TRM and likely play an important role in HIV-1 containment.
Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4
Authors: Gomez-Rodriguez Julio Washington , Meylan Francoise , Handon Robin , Hayes Erika T , Anderson Stacie , Kirby Martha R , Siegel Richard , Schwartzberg Pamela L.
Published: January 2023

A major function of the immune system is to mount specific responses to pathogens, while minimizing self-reactivity. To help orchestrate this, CD4+ T cells differentiate into distinct effector cell populations. Among these, T helper 9 (Th9) cells produce IL-9, a cytokine important for the eradication of type-II-inducing pathogens; however IL-9 has been also implicated in allergic asthma and autoimmunity. We show that Itk, a mediator of T-cell receptor signalling required for Th2 immune responses and the development of asthma, is a positive regulator of Th9 differentiation in vivo and in vitro. In a papain-induced model of allergic lung disease, Itk-deficient mice have reduced pulmonary inflammation and decreased IL-9 production by T cells and innate lymphoid type 2 cells (ILC2), despite normal early induction of ILC2s. In vitro, naïve Itk−/− CD4+ T cells essentially fail to produce IL-9 and have reduced levels of IRF4, a critical transcription factor for effector T-cell function. Both IL-9 and IRF4 expression are rescued by either IL-2 or expression of constitutively-active STAT5, but not NFATc1. We further find that STAT5 binds the Irf4 promoter, indicating one mechanism by which IL-2 can rescue weakly-activated T cells. Interestingly, while naïve WT CD4 T cells cultured in presence of the Itk inhibitor do not produce IL-9, addition of IL-2 leads to paradoxically high levels of IL-9, suggesting complex effects of Itk inhibitors that may prevent their use as therapeutics. Thus, inhibiting Itk as a therapeutic strategy for treating or preventing symptoms of asthma and other diseases in which IL-9 participates still remains an important and complex question.
The role of CD40 in calcitonin gene related protein (CGRP) mediated antiviral effect in glial cells
Authors: Grlickova-Duzevik Eliza , Cao Ling
Published: January 2023

LP-BM5 is a murine retrovirus that induces peripheral neuropathy and AIDS like immune deficiency in B6 mice. Upon infection, primary sensory neurons in the spinal cord express elevated levels of calcitonin gene related protein (CGRP). Previously, we showed that CGRP induced reduction of LP-BM5 viral loads in primary mixed glial cells. The purpose of this current study was to investigate possible CGRP downstream targets that may contribute to its antiretroviral response. We hypothesized that CGRP decreases the viral load in glial cells by modifying the CD40 expression; increasing in CD40 signaling may promote production of cytokines and chemokines with antiretroviral properties. Using CD40 knockout mice we showed that CD40 is required for CGRP mediated antiretroviral response in mixed glia. In microglial cell line, CD40 expression was elevated after treatment with CGRP. Similar effect was seen in microglia from spinal cord derived mixed glial cultures. While LP-BM5 increased microglial CD40 expression gradually over time up to 7 days post-infection in primary mixed glia, CGRP caused an early elevation (24 hr post-infection) of microglial CD40 expression in microglia and stayed relatively steady levels 7 days after infection. CGRP also promoted chemokine CXCL1(KC) production in mixed glia. Our data suggest that activation of CD40 mediated signaling may result in increased production of chemokines that may interfere with retroviral replication. Future studies will be directed to specifically delineate CD40 downstream target involved in antiretroviral response in glial cells.
Toll-like receptor 9 is required for the maintenance of CD25+FoxP3+CD4+ Treg cells during Listeria monocytogenes infection
Authors: Dolina Joseph S , Schoenberger Stephen P
Published: January 2023

It has long been appreciated, but not understood, that the CD8+ cytotoxic T lymphocyte (CTL) dependence on CD4+T cell help (Th) is conditional; needed for some immunogens but not others. One explanation for this phenomenon envisions Th requirement as an intrinsic property of the pathogen itself rather than its introduction to the immune system. Here we show that dependence of the optimal CD8+ T cell response to Listeria monocytogenes (Lm) on CD4+ T cells is a function of the immunogen dose used for priming, with low dose Lm (LD; 50 or 103 CFU WT or ΔActA, respectively) inducing a primary antigen-specific CTL response profoundly dependent on CD4+ Th cells while that induced by high dose Lm (HD; 4×103 or 106 CFU WT or ΔActA, respectively) is significantly inhibited by CD4+CD25+FoxP3+regulatory T cells (Treg). The Th-independence of HD immunization is not overcome by additional antigen but instead involves the inflammatory response to more bacteria. Evaluation of various TLR pathways as the relevant sensing mechanism showed that TLR2 is required for CTL responses to LD immunization, and that HD immunization in the absence of TLR9 results in a simultaneous loss of CD25+FoxP3+CD4+ Treg cells and increase in conventional CD4+ Th cells and CTLs. Our data thus reveal that the CTL response to the same pathogen is determined by distinct roles for CD4+ T cells as helpers versus regulators based on immunogen dose and demonstrate a previously undescribed role for TLR9 in the regulation of CD4+ Th and Treg cells.
Human T follicular regulatory cells (Tfr) able to inhibit antibody production are enriched at the lymph node T-B border and rare in germinal centers
Authors: Sayin Ismail , Radtke Andrea J , Vella Laura A , Buggert Marcus , Jin Wenjie , Wherry E. John , Germain Ronald N , Betts Michael R , Herati Ramin S , Canaday David H
Published: January 2023

T follicular helper (Tfh) cells are a subset of CD4+ T cells that are critical for the formation of germinal centers (GCs) and the development of high affinity, class-switched antibodies. Tfh cells are differentiated in a Bcl6-dependent manner and are characterized by expression of the inhibitory receptor PD-1 along with CXCR5, a chemokine receptor that directs these cells to the follicle. More recently, T follicular regulatory cells (Tfr) have been shown to express regulatory T cell (Treg) markers and suppress humoral immunity. To date, much of our understanding of the function and distribution of Tfh and Tfr cells has been obtained from animal studies. Using highly multiplexed, quantitative imaging and functional assays, we examined the spatial distribution and suppressive function of human Tfr cells obtained from mesenteric lymph nodes. Here, we report that the majority of Tfr cells reside at the border between the T cell zone and B cell follicle and are rare within the GC, being far outnumbered by Tfh cells. In contrast to Tfh cells, Tfr cells expressed low to undetectable levels of PD-1 in situ yet potently suppressed antibody production in vitro. These findings reveal that Tfh and Tfr cells are spatially segregated within human lymph nodes and caution against using PD-1 expression levels as a phenotypic marker for human Tfr cells.

This research was supported in part by NIH R01, VA, and Intramural Research Program of the NIH, NIAID.
Outcomes of congenital Zika disease depend on timing of infection and maternal-fetal immunophysiological conditions
Authors: Chen Jinling , Liang Yuejin , Yi Panpan , Azar Sasha , Rossi Shannan , Sun Jiaren
Published: January 2023

Here, we examined outcomes of congenital Zika disease in mice at different times of infection. Female mice lacking interferon alpha receptor (IFNAR−/−) were crossed to their male counterparts (KO×KO), and were subsequently infected with ZIKV at embryonic day (E) 5.5, E10.5 and E15.5, respectively. Dams infected with ZIKV at E5.5 had nearly 100% fetal demise. Dams exposed to ZIKV at E10.5 exhibited fetal demise and IUGR, whereas infection at E15.5 did not result in these clinical outcomes. To gauge the effect of IFN signaling at the maternal-fetal interface on the pregnant outcomes, we established a mouse model in which IFNAR−/−female mice were susceptible to ZIKV infection and their offspring were capable of type I IFN signaling due to a heterozygous breeding scheme (KO×WT). No viral burden was detected in multiple organs; also, no fetal demise was observed in the KO×WT group when they were infected at mid- and late pregnancy. The immune resistance in the heterozygous group was accompanied by high expression of IFN-stimulated genes (ISGs). Type III IFN also induced ISGs similar to type I IFNs through a distinct receptor. To gauge the effect of type III IFN on the host resistance to ZIKV and pregnant outcomes, we i.p. treated the pregnant mice with IFN-λ prior to and after ZIKV infection at E5.5, E10.5, and E15.5. We found that IFN-λ treatment reduced viral burden in maternal and fetal organs, upregulated ISG expression, and relieved fetal growth restriction at mid pregnancy. Thus, our data demonstrated that IFN signaling at the maternal-fetal interface is critical for the outcomes of pregnancy, especially at the mid pregnancy stage, during maternal ZIKV infection. IFN-λ inhibits ZIKV infection and confers protection to infected fetuses.
Topical exposure to the quaternary ammonium compound didecyldimethylammonium chloride augments dermal type 2 innate lymphoid cell populations
Authors: Shane Hillary L , Marshall Nikki B , Lukomska Ewa , Long Carrie M , Anderson Stacey E
Published: January 2023

Didecyldimethylammonium chloride (DDAC) is a dialkyl-quaternary ammonium compound used in commercial and industrial products for its antimicrobial properties. Clinical reports suggest that topical exposure to DDAC can resultin immediate and delayed type hypersensitivity, and recently our laboratory has confirmed that DDAC is a strong non-IgE mediated sensitizer in mice at relevant human exposure concentrations. Here, we further investigate the mechanism of DDAC induced hypersensitivity in mice, focusing on dermal immune responses. Gene expression analysis shows significant reduction in epithelial Cdh1, and increases in Tslp and Ccl17 following DDAC application to the skin, indicating that dermal type 2 innate lymphoid cells (ILC2s) may be involved in DDAC-induced hypersensitivity. Phenotypic analysis of dermal ILC2s shows that these cells are rapidly activated with increased expression of CD25, ICOS, and KLRG1 occurring by 24 hours post DDAC exposure. Increases in ILC2 number were observed following 2 weeks of DDAC exposure and found to persist for up to 2 weeks in the absence of DDAC and Tslp. Additionally, the phenotype of these cells suggests a potential for longevity (reduced KLRG1 and increased CD127) and enhanced activity (increased ICOS), which may influence future sensitization events. ILC2s have been previously implicated to play a role in IgE-independent allergic responses, but to our knowledge have not been investigated in chemical allergy. These results indicate that skin resident ILC2s may play a role in the development of the hypersensitivity response to DDAC and raise concerns about the use of this chemical and other quaternary ammonium compounds that may elicit similar effects.
The formation of memory-like innate lymphoid cells 2 in allergic asthma
Authors: Jing Xiuli , Wang Meixiang , Zhuang Qiannan , Zhao Bin , Liu Jianguo , Yi Shuying , Song Wengang , Tang Hua
Published: January 2023

Numerous studies have been suggested that ILC2s play a critical role in the initiation of allergic asthmatic airway inflammation, but their property and role in chronic allergic lung inflammation has not been well established. Our study found that purified allergen-experienced ILC2s produce more cytokines than naive ILC2s when stimulated by suboptimal amounts of IL-33 and IL-2 in vitro. Furthermore, upon allergen intranasal re-challenge, allergen-experienced ILC2s proliferate more vigorously and produce much greater amounts of type 2 cytokines than naive ILC2s. These results suggested that allergen-experienced ILC2s showed some memory-like properties in allergic asthma. To further characterize these memory-like ILC2s, we analyzed the expression of some activation surface markers, such as KLRG1, CD62L, CD69, ST2, ICOS, NKG2D, CD44, CD25 and CD122 on ILC2s in the lungs of naive and IL-33-treated mice at different time point. The results showed that during the initial expansion phase, IL-33 treatment resulted in a rapid increase in the number of CD25− ILC2s cells, which peaked at d6. And then the CD25− ILC2 population in the lung started to contract, the number of CD25− ILC2s cells was rapidly declined at d14, a time point that the numbers of CD25+ ILC2s reached their peak. Both of CD25− and CD25+ ILC2s rapidly declined at d30, however, the numbers of CD25+ ILC2s was much higher than CD25− ILC2. Thus, these results suggested that allergen-experienced ILC2s could differentiate into two different memory potential ILC2 subsets, namely CD25− short-term surviving effector ILC2s cells an CD25+ long-term surviving memory ILC2s cells. This work was supported by grants (2015CB943203, 31300730, 81272315, 2016GCC09)
Aortic B cells: Identifying antibodies specific for atherosclerosis in mice
Authors: Hutchinson Mark A , Maul Robert W , Gearhart Patricia J
Published: January 2023

Atherosclerosis is characterized by the development of arterial plaques containing lipid deposits and immune cells, such as B cells. Once recruited to the site of inflammation, B cells become activated and form germinal centers within the adventitia of the artery adjacent to the plaque. To determine if these B cells produce antibodies specific for atherosclerosis, abdominal aortas were collected, and germinal center B cells were isolated by flow cytometry. We characterized their heavy and light chain repertoires by next generation sequencing to compare non-diseased (C57BL/6) and diseased (ApoE−/−) tissues. Furthermore, the aortic repertoires were compared to the germinal center repertoires from spleens taken from the same mice to address whether local (aortic) and systemic (spleen) B cell responses were similar. Preliminary results suggest that all the splenic repertoires were similar, independent of diet or disease. However, aortic repertoires from ApoE−/− mice indicated that some VH and Vk genes were overexpressed, suggesting selection for particular antigens. We therefore performed single cell analysis and identified over 100 VH and Vk gene pairs, with different frequencies of expression and somatic hypermutation. Antibody gene pairs will then be cloned and expressed in culture using HEK293A cells. The purified recombinant antibodies will be used to identify antigens specific to atherosclerosis by protein array. These results may be useful in creating potential vaccines for its prevention, as well as treatment via antibody immunotherapy.
Inhibiting a mutagen: mitigating Activation-Induced Deaminase activity
Authors: Alvarez-Gonzalez Juan Antonio , Crawford Daniel J , Maul Robert W , Kohli Rahul M , Gearhart Patricia J
Published: January 2023

Activation-Induced Deaminase (AID) is a cytosine deaminase which converts cytosine into uracil within stretches of ssDNA. The presence of uracil in DNA initiates a cascade of mutagenic DNA repair to introduce point mutations and DNA double strand breaks. Specific targeting of AID to the immunoglobulin heavy chain loci (Igh) promotes mutagenesis in the antibody variable region for somatic hypermutation (SHM) or switch regions leading to class switch recombination (CSR). However, AID also plays a role in accumulating unnecessary mutations across the genome. Mis-targeting of AID results in the initiation of tumor development and leads to greater drug resistance among cancer cells when continually expressed. To identify an AID inhibitor, we screened ~400,000 compounds in conjunction with the NCATS core facility at NIH. Using FRET based analysis of cytosine deamination, we have identified approximately 150 potential inhibitors of AID catalytic activity. To confirm biological function, we have examined the compounds’ effects on CSR in an in vitro murine B-cell activation assay. Of 30 confirmed inhibitor candidates, we have selected the top 7 to proceed with further characterization to determine if they prevent uracil accumulation in primary mouse cells after ex vivo stimulation. Using these approaches, we hope to identify a small molecule with great efficacy and low toxicity for use as a molecular probe to further characterize AID’s intricacies, or even as a therapeutic.
Harnessing tissue resident memory T cells to combat solid tumors
Authors: Rosato Pamela , Manlove Luke , Nelson Christine , Pennell Christopher , Vezys Vaiva , Masopust David
Published: January 2023

The immunosuppressive tumor microenvironment is barrier to successful cancer therapies. Newly discovered tissue resident memory CD8+ T cells (TRM) function to create a potent immunostimulatory environment to protect against local reinfection. As TRM are present in abundance in nearly every tissue and can be triggered by cognate peptide alone, without adjuvant, we tested whether we could hijack infection-specific TRM in tumors to reverse the immunosuppressive tumor microenvironment and enhance existing immunotherapies. Mice with established vesicular stomatitis virus (VSV)-specific skin TRM were challenged with the transplantable B16 melanoma cell line. We find VSV-specific CD8+ T cells within established tumors and by 12 hours of cognate peptide delivery, these cells upregulate IFNγ, CD25, and granzyme B. This led to 1) a 10-fold increase in intratumoral NK cells, 2) bystander memory CD8+ T cell recruitment to the tumor, 3) tumor NK cell and CD8+ T cell activation through granzyme B upregulation and 4) DC activation in tumor draining lymph nodes through CCR7 and CD86 upregulation. Ongoing studies using transplantable and autochthonous melanoma models are testing whether TRM immunotherapy synergizes with CAR T cell and checkpoint blockade immunotherapies for eradication of recalcitrant tumors. These results demonstrate proof of principal efficacy for exploiting infection-specific TRM as a tumor immunotherapy.
Eomesodermin together with KIR/NKG2A membrane receptors and CD49d as an useful strategy to identify NK-like CD8(+) T cells with innate features in humans: focusing on cancer diseases
Authors: Herbelin Andre , Cayssials Emilie , Jacomet Florence , Nunez Nicolas Gonzalo , Basbous Sara , Lefevre Lucie , Abdallah Myriam , Piccirilli Nathalie , Lavoue Vincent , Catros Véronique , Piaggio Eliane , Gombert Jean-Marc , Barbarin Alice
Published: January 2023

Among the burgeoning family of unconventional T cells, innate-like CD8(+) T cells bear a memory phenotype without having encountered a foreign antigen and can respond to innate-like IL-12+IL-18 stimulation. Although the concept of innate memory (IM) CD8(+) T cells is now well-established in mice, whether an equivalent memory NK-like T-cell population exists in humans remains under debate. We recently reported that CD8(+) T cells responding to innate-like IL-12+IL-18 stimulation and co-expressing the transcription factor Eomesodermin (Eomes) and KIR/NKG2A membrane receptors with a memory/EMRA phenotype may represent a new, functionally distinct innate T cell subset in humans.

Here, we proposed Eomes together with KIR/NKG2A and CD49d as a signature to standardize the identification of this innate CD8(+) T-cell subset in humans. Focusing on cancer diseases, we have provided new insights into the potential role of these IM CD8(+) T cells in a physiopathological context in humans. Based on empirical data obtained in cases of chronic myeloid leukemia, a myeloproliferative syndrome controlled by the immune system, and in solid tumors, we have observed both the possible contribution of IM CD8(+) T cells to cancer disease control and their susceptibility to tumor immune subversion. Finally, we have investigated the involvement of IL-4 and IL-15 in the generation of the IM CD8(+) T cell pool and its dependency on promyelocytic leukemia zinc finger (PLZF)-expressing invariant Natural Killer T (iNKT) cells.

All in all, this study significantly contributes to understanding of the physiopathological role of NK-like CD8(+) T cells in humans and raises the question of the possible involvement of an iNKT/IM CD8(+) T cell axis in cancer.
Calnexin induces impairment of proliferation and effector function of CD4+ and CD8+ T cells and promotes tumor growth
Authors: Ma Da , Chen Yi Chen , Wang Zhi
Published: January 2023

Elucidation of molecular mechanism underlying the inhibited infiltration effector function of T cells induced is essential for improvement of anti-tumor immunotherapy. Here, we found that Calnexin, a ER chaperon protein, is significantly up-regulated in OSCC tumor cells and multiple other tumors. Up-regulation of Clanexin in tumor cell membrane links with limited infiltration of T cells in OSCC tumor core and poorer survival of OSCC patients. Importantly, we found that Calnexin mediates inhibition of proliferation of CD4+ and CD8+T cells and effector functions of expression of IFN-γ, TNF-α, IL-2 by T cells. While knock-down of Calnexin enhanced the infiltration and effector functions of T cells in tumor cores and conferred better control of tumor growth, treatment of recombinant Calnexin induced impairment of infiltration and effector functions of T cells in tumor cores and promotes tumor growth in mice. Thus, this work uncovers a previously unknown mechanism in which infiltration of T cells and effector functions of T cells are inhibited by Calnexin expressed in tumor cells, and implicates that Calnexin may serve as an important target for improvement anti-tumor immunotherapy.
NKG2D signaling certifies CD8 T cells to become memory cells
Authors: Perez Cynthia , Prajapati Kushal , Burke Brianna , Plaza-Rojas Lourdes , Guevara-Patiño Jose Alejandro
Published: January 2023

The ability to form memory is a major characteristic of the adaptive immune system. NKG2D, as a costimulatory receptor for CD8+ T cells, was shown to rescue memory formation in absence of CD4+ T cell help. However, the exact function of NKG2D on CD8+ T cells and the importance of NKG2D signaling for memory formation are not known. To recapitulate a full course of CD8+ T cell activation, from priming to memory recall, we immunized C57BL/6 mice with peptide-pulsed dendritic cells and followed the CD8+ T cell responses at priming, effector and memory phases. Using NKG2D deficient (NKG2D-KO) CD8+ T cells, we found that NKG2D signaling was not necessary to achieve CD8+ T cell priming. However, NKG2D-KO CD8+ T cells showed lower effector functions (reduced in vivo kill and reduced cytokine secretion) compared to wildtype CD8+ T cells. Blocking NKG2D signaling at the effector phase with a single injection of a non-depleting anti-NKG2D antibody recapitulated the results observed with NKG2D-KO CD8+ T cells. Importantly, the CD8+ T cells that lacked NKG2D signaling at effector phase failed to differentiate into protective memory cells. These results indicate that NKG2D signaling certifies CD8+ T cells to become memory cells and that this certification occurs during the effector phase. This study was supported by the PO1 CA154778, the RO1 CA188395 and the Vogol Foundation.