bluuebunny/crossref_metadata_2025 huggingface dataset:
Germinal Center Immune Dynamics in Secondary Lymphoid Organs after Influenza Vaccination in Children
Authors: Petrovas Constantinos , Amodio Donato , Macchiarulo Giulia , Rocca Salvatore , Cotugno Nicola , Rossi Paolo , Koup Richard , Palma Paolo
Published: January 2023

Introduction

The germinal center immune dynamics leading to protective antibody responses after vaccination in humans remains largely unknown. Investigation of such dynamics would provide valuable information regarding the cellular and molecular mechanisms governing the generation of these responses and lead to novel vaccine strategies

Materials and methods

Paraffin embedded tonsil tissue sections were analyzed by multiplexed confocal microscopy assays and Histo-cytometry. The phenotype of tonsil derived cells was analyzed with polychromatic flow cytometry, flu-specific B cell-responses were detected by ELISPOT to H1N1, H3N2, and B strain and plasmas analyzed with a Luminex multiplex assay.

Results

36 children (24 vaccinated with 2015/2016 seasonal influenza vaccine and 12 controls) scheduled for elective tonsillectomy were included. Flow cytometry and Histo-Cytometry analysis showed that vaccination induced the frequency of tonsil CD4 Tfh, especially the PD-1hiCD57lo ones. This profile was associated with a significant reduction of potential tissue suppressor CD4 T cells and positively correlated with all flu-antigen specific B cells. An induction of IL-4R on plasma cells was found after vaccination implying a further mobilization of these cells. Among the circulating cytokines/chemokines tested, CXCL-13 was selectively induced after vaccination, in line with the increased frequency of CD4 Tfh found.

Conclusions

Considered that children are continuously scheduled for tonsillectomy, tonsils could represent a valuable and approachable secondary lymphoid organ for the investigation of germinal center reactions after vaccination compared to lymph node not easily accessible in this population.
In vitro induction of an atypical memory B cell phenotype in human B cells
Authors: Sohn Hae Won , Ambegaonkar Abhijit A , Obeng-Adjei Nyamekye , Crompton Peter D. , Pierce Susan K
Published: January 2023

Several chronic human infectious diseases including malaria are associated with a large expansion of an unusual population of memory B cells(MBCs) which is referred to as atypical MBCs in malaria. Atypical MBCs in malaria are CD21−, CD27−, CD11c+, FcRL5+, high expression of the transcription factor, T-bet, and have accumulated somatic hyper mutations. These atypical MBCs showed poor B cell receptors signaling and B cell function, ex vivo. These studies suggest that atypical MBCs may contribute to the slow, inefficient acquisition of immunity to malaria in African children. However, the mechanisms by which atypical MBCs are induced during chronic infectious diseases are not known. Recently acute febrile malaria was correlated in African children with increases in peripheral blood TH1-type T follicular helper T cells that secrete inflammatory cytokines, including IFN-γ, and are impaired in their helper function. These results suggested that such T cells or their products may contribute to generation of atypical MBCs. We investigated the conditions under which human B cells can induce an atypical MBC phenotype in vitro. Studying tonsillar B cells we showed that triple combined signaling pathway, BCR, TLR and IFNγ is required for the induction of the malaria-associated atypical MBCs in vitro. In addition, human peripheral blood B cells were induced to express T-bet when incubated anti-Ig and the supernatants of peripheral blood mononuclear cells stimulated by parasite-infected red blood cells. Taken together these results suggest that atypical MBCs may be the product of B cell antigen engagement in the highly inflammatory environments that develop during chronic infections.
IL-17A-producing CD4+Foxp3+ T cells in chronic pulmonary aspergillosis
Authors: Jung Min Kyung , Kang Shin Myung , Kwak Jeong-Eun , Shin Eui-Cheol
Published: January 2023

Chronic pulmonary aspergillosis (CPA) is a severe pulmonary disease caused by aspergillus fumigatus infection. However, immune responses in CPA patients remained to be elucidated. In humans, the CD4+Foxp3+regulatory T cell (Treg) population are classified into the following three subsets based on their expression of CD45RA and Foxp3: CD45RA+Foxp3lo resting Treg cells (subset I), CD45RA−Foxp3high activated Treg cells (subset II), and CD45RA−Foxp3locytokine-secreting non-suppressive cells (subset III). In the present study, we investigated these three CD4+Foxp3+cell subsets and their clinical implication in CPA patients.

The frequency of total CD4+Foxp3+ cells did not differ between CPA patients and healthy controls. Whereas the frequency of subset I or II also did not differ between the two groups, the frequency of subset III was significantly increased in CPA patients compared with healthy controls. Interestingly, IL-17A production was observed in the subset III CD4+Foxp3+ T cells, and the frequency of subset III significantly correlated with IL-17A production from the CD4+ T cell population. Moreover, we found that better clinical prognosis, evaluated by less hospitalization incident, was associated with higher IL-17A production from the CD4+ T cell population and higher frequency of subset III CD4+Foxp3+ cells among CPA patients.

In summary, the frequency of IL-17A-producing CD4+Foxp3+ T cells was increased and correlated with the frequency of IL-17A+ CD4+ T cells, and the higher frequency of IL-17A-producing CD4+Foxp3+ T cells was related with better clinical prognosis. We suggest that IL-17A-producing CD4+Foxp3+ T cells (subset III) may have a role in Th17 regulation and antifungal immune responses in CPA patients.
Sequence and structural analyses reveal distinct and highly diverse human CD8+ TCR repertoires to immunodominant viral antigens
Authors: Chen Guobing , Yang Xinbo , Go Annette , Gao Mingming , Zhang Yongqing , Shi Alvin , Sun Xiaoping , Mariuzza Roy A. , Weng Nan-ping
Published: January 2023

A diverse TCR repertoire is considered as an essential property of T cell immunity to efficiently contain and eliminate viral invasion. However, limited information is available regarding the characteristics of TCR repertoire to a defined viral antigen peptide in human. Here, we report a comprehensive analysis of CD8 TCR repertoires, consensus binding motifs in CDR3, affinities of selected TCR/pMHCs, and crystal structures of two dominant viral antigens (CMV pp65-NLV and IAV M1-GIL) from 8 healthy adults. A few thousands of distinct TCR alpha and beta chains for NLV and GIL were identified containing highly selected public TCRs. The repertoire of NLV-specific TCR was substantially more diverse than that of GIL-specific TCR for both alpha and beta chains. Further analysis of CDR3 amino acid sequences revealed dozens of distinct consensus motifs in alpha and beta chains of both antigen specific TCRs. Single cell analysis showed preference of the CDR3 motifs paring between TCR alpha and beta. The affinity of TCR/pMHC ranged more widely for GIL (Kd, 1.8–191 μM) than for NLV (4.7–28 μM). Structural analysis exhibited similar patterns of three TCR-GIL-HLA-A2 interactions, while two distinct binding patterns among three TCR-NLV-HLA-A2 interactions. Our findings revealed highly diverse viral antigen-specific CD8 TCR repertoire, the consensus binding motifs in CDR3, binding affinity of TCR and pMHC, and different patterns of TCR-pMHC interactions. Such information will be essential to understand the make-up of T cell immunity and to guide the potential immune based clinical intervention such as cell therapy.
Suppressing type I interferon enhances both autoimmunity and anti-tumor immunotherapy
Authors: Riding Rebecca L. , Fukuda Keitaro , Richmond Jillian M , Harris John E
Published: January 2023

Vitiligo is an autoimmune disease of the skin in which melanocytes are targeted for destruction by autoreactive CD8 T cells specific for melanocyte/melanoma-shared antigens, such as premelanosome protein (PMEL). We developed a mouse model of vitiligo through adoptive cell transfer (ACT) using PMEL-specific TCR transgenic CD8+ T cells (PMELs) and found that vitiligo pathogenesis is primarily driven by IFNγ. There is much crosstalk between IFNγR signaling and type I interferon signaling as type I interferons, including interferon α and β proteins, can directly regulate shared IFNγ target genes. Therefore, we sought to understand the role of type I interferons in vitiligo. We discovered that type I interferon α-receptor deficient (IFNAR−/−) host mice develop accelerated and more severe vitiligo compared to WT mice. Subsequent studies showed that IFNAR expression was not required on PMELs or the majority of other immune cells to suppress vitiligo, but rather on radioresistant cells. To determine whether this observation could be useful in melanoma immunotherapy, we tested ACT of PMELs in B16F10 melanoma-inoculated IFNAR−/− and WT mice and found that IFNAR−/− mice were relatively protected from the tumor, with a lower tumor burden and an elevated number of tumor-infiltrating PMELs compared to WT mice. Collectively, these results strongly suggest that type I interferon signaling suppresses anti-melanocyte immune responses in both autoimmunity and tumor immunity. We are currently investigating the mechanism of type I interferon suppression, specifically what cell types are driving this response and what mediators are involved.
Squamous cell carcinomas escape immune surveillance via inducing co-expression of PD-1 and LAG-3 inhibitory receptors on tumor infiltrating T lymphocytes
Authors: Wang Jing H , Mishra Ameet K , Kadoishi Tanya , Wang Xiaoguang , Driver Emily , Chen Zhangguo , Wang Xiaojing
Published: January 2023

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer. About 90% of head and neck cancers are SCCs. It remains largely unknown how SCCs evade immune recognition. We recently established a mouse model by injecting tumor cells derived from primary SCCs harboring KrasG12D mutation and Smad4 deletion into wild-type (wt) or CD8−/− recipients. We found comparable tumor growth between wt and CD8−/− recipients, indicating a complete escape of CD8+ T cell-mediated anti-tumor responses by these SCCs. Mechanistically, CD8+ T cells were not defective in infiltrating tumors given their relatively increased percentage among tumor infiltrating lymphocytes (TILs). CD8+ TILs exhibited phenotypes of chronic activation and exhaustion, manifested with co-expression of programmed death 1 (PD-1) and lymphocyte activation gene-3 (LAG-3). Among CD4+ TILs, T regulatory cells (Tregs) were preferentially expanded. Contradictory to prior findings in melanoma, Treg expansion was independent of CD8+ T cells in our SCC model. Unexpectedly, CD8+ T cells were required for promoting NK cell infiltration within SCCs. Furthermore, we uncovered AKT-dependent lymphocyte-induced PD-L1 upregulation on SCCs, which was contributed greatly by combinatorial effects of CD8+ T and NK cells. Lastly, dual blockade of PD-1 and LAG-3 inhibited the tumor growth of SCCs. Thus, our findings identify novel immune evasion mechanisms of SCCs and suggest that immunosuppressive mechanisms operate in a cancer-type specific and context-dependent manner.
Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumor microenvironment
Authors: Kim Kyun Do , Bae Seyeon , Capece Tara , Nedelkovska Hristina , de Rubio Rafael G , Smrcka Alan V , Jung Woojin , Park Byeonghak , Kim Tae-il , Srikanth Sonal , Gwack Yousang , Kim Minsoo
Published: January 2023

Adoptive cell transfer utilizing tumor-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against hematological malignancies, but significant clinical success has not yet been achieved in solid tumors due in part to the strong immunosuppressive tumor microenvironment. Systemic or intratumoral delivery of an immune boosting molecule to overcome local suppression has been proposed, but the full potential is limited by non-specific stimulation of tumor growth, metastasis, and angiogenesis. Here, we show that suppression of CTL killing by CD4+CD25+Foxp3+ regulatory T cell (Treg) is mainly mediated by TGFβ-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Both in vitro and in vivo assays revealed that highly selective optical control of Ca2+ signaling in adoptively transferred CTLs was sufficient to overcome immunosuppression at the tumor site by enhancing T cell activation, IFN-γ production and antitumor cytotoxicity, leading to a significant reduction in tumor growth in mice. Together, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses only at the targeted tumor sites.
ID2-dependent anti-tumor immunity promotes anti-cancer therapies effects
Authors: Guo Xiaohuan
Published: January 2023

More and more studies show that the efficacy of many kinds of anti-cancer therapies, including chemotherapy, radiotherapy, targeting therapy and even immunotherapy, require host activated anti-tumor immune responses. However, the regulation mechanisms of anti-tumor immunity are not fully understood yet. Here, we show that a transcription regulator, Inhibitor of DNA binding 2 (ID2), could positively regulate the anti-tumor immunity. E and ID proteins are both HLH proteins and play important roles in the development of various immune cells. E proteins can form homodimers or heterodimers with HLH proteins and function as transcription activators or repressors, while ID proteins can prevent E proteins from binding to DNA. With a T cell specific ID2 knockout mice (Cd4-cre; Id2 fl/fl), we found that the anti-cancer efficacy of oxaliplatin and anti-PD-L1 treatment for subcutaneous transplant tumor model required ID2 expression in T cells. Further experiments showed that these anti-cancer therapies could induce IFNg production in wild type mice, but failed in ID2 deficient mice. Moreover, the tumor infiltrating cytotoxic CD8+ T cells, but not CD4+ T cells, were dramatically reduced in ID2 deficient mice compared their littermate controls. Our hypothesis is that post the anti-cancer therapy, ID2 could be upregulated and promote CD8+ T cell activation and migration into the tumor sites, then mediate the anti-tumor effect. Since E proteins are main targets of ID2, small molecules targeting E protein may be new choices for boosting anti-tumor immunity to enhance the efficacy of anti-cancer therapies.

Supported by grants from Tsinghua University and The Recruitment Program for Young Professionals (to X.G.).
Prevention and reversal of adverse humoral immune response by BAR-transduced regulatory T cells
Authors: Zhang Ai-Hong , Yoon Jeongheon , Kim Yong Chan , Wang Hong , Scott David W.
Published: January 2023

Antigen-specific elimination of unwanted immune response, such as anti-FVIII neutralizing antibody formation in hemophilia A patients, is highly desirable but remains a challenge. Analogous to the chimeric antigen receptor (CAR) strategy successfully used in cancer immunotherapy, we have created a chimeric receptor comprising a protein antigen or its domain, linked with the transmembrane and signal transduction domains, CD28-CD3ζ. We termed it “BAR” for B-cell-targeting antibody receptor. CD4+CD25hiCD127low human Tregs were retrovirally transduced to express a BAR containing the immunodominant FVIII C2 (C2 BAR) or A2 domain (A2 BAR). Control human Tregs were transduced to express a BAR containing chicken ovalbumin (OVA BAR). FACS sorted, BAR transduced human CD4+ Tregs have been successfully expanded in vitro, and shown to maintain phenotypic Treg markers in terms of co-expression of Foxp3 and Helios. Pretreatment of naïve hemophilia A mice with a mixture of C2 BAR plus A2 BAR human Tregs completely prevented anti-FVIII antibody development in response to FVIII/IFA immunization for as long as 8 weeks. More importantly, BAR human Tregs also effectively suppressed anti-FVIII antibody formation in primed animals with detectible pre-existing anti-FVIII antibodies. Further mechanistic studies suggest that the FVIII-specific B cell response were directly suppressed while the T cell response remained intact. Taking together, we report here a successful proof of principle strategy utilizing FVIII-specific BAR Tregs to directly target FVIII-specific B cells, an approach which could be adapted to address other adverse immune responses as well.
Macrophages play a pathogenic role in IL-23 mediated psoriasiform skin inflammation
Authors: Wang Yibing , Edelmayer Rebecca , Wetter Joe , Salte Katherine , Gauvin Donna , Leys Laura , Lippert Stephanie , Su Zhi , Miller Loan , Huang Susan , Honore Prisca , Kort Mike , McGaraughty Steve , Scott Victoria , Gauld Stephen B
Published: January 2023

Psoriasis (Ps) is an immune-mediated inflammatory skin disease that affects millions worldwide. Studying immune cells involved in Ps pathogenesis is essential to identify effective and safe therapeutics for the disease. Recent studies have demonstrated the involvement of macrophages in human Ps skin. Using an IL-23 murine model of psoriasiform dermatitis, which closely models human Ps, we have characterized the cellular lineage during disease progression and found continuous infiltration of macrophage as dominant immune population. Depletion of macrophages in the IL-23 murine model significantly reduced the inflammation as well as inflammatory mediators, such as TNFa, in the skin. This strongly suggests a pathogenic role of macrophages in the psoriasiform skin inflammation. Further characterization of this model revealed a number of phenotypically distinct macrophage populations suggesting they may play different roles in disease progression. Collectively, our data suggests that targeting macrophages may have therapeutic benefit for Ps patients.
Novel immunomodulatory proteins generated via directed evolution of variant IgSF domains
Authors: Evans Lawrence S , Levin Steven D , Rickel Erika , Wolfson Martin F , Dillon Stacey R , Kornacker Michael G , Swanson Ryan , Peng Stanford L
Published: January 2023

The immunoglobulin superfamily (IgSF) is a large, diverse family of proteins expressed on immune cells that have been extensively targeted for treatment of cancers and autoimmune diseases. Most of the therapeutic strategies targeting this family have focused on high affinity antibodies that bind a single receptor. Moreover, wild-type IgSF receptors typically exhibit low affinities for their counter-structures, limiting their utility in therapeutic modulation of immune responses. We have developed a novel variant Ig domain (vIgD™) platform using directed evolution and yeast display to affinity mature human IgSF extracellular domains. In this platform, libraries of mutagenized IgSF domains were selected for enhanced or altered affinity to specific recombinant proteins. Fc fusion proteins incorporating these evolved immunomodulatory IgSF domains were then tested in vitro for their ability to either agonize or antagonize T cell responses. Multiple novel vIgD™ fusion proteins have been generated that significantly attenuate T cell activation in vitro as assessed by proliferation and cytokine production. Lead molecules also exhibited in vivo efficacy in the human PBMC-NSG™ GVHD mouse model. Efficacy in vitro and in vivo was superior to wild-type IgSF domains due to the induced alterations in affinity for cognate ligand and through specifically directed changes in their ability to bind additional counter-structures. Our results demonstrate that vIgDs™ can be developed to acquire unique biochemical properties that significantly enhance therapeutic utility as immunomodulatory agents. This vIgD™ therapeutic platform has broad potential to enhance the activity of biologics in treatment of both autoimmune disease and cancer.
Therapeutic effect of Lactobacillus reuteri DSM 17938 on Treg-deficiency-induced autoimmunity (IPEX syndrome) via the inosine-adenosine 2A receptors
Authors: Liu Yuying , He Baokun , Hoang Thomas K. , Wang Ting , Taylor Christopher M. , Tian Xiangjun , Luo Meng , Tran Dat Q , Zhou Jain , Tatevian Nina , Luo Fayong , Molina Jose G. , Blackburn Michael R. , Gomez Thomas H. , Roos Stefan , Rhoads J Marc
Published: January 2023

Regulatory T-cell (Treg) deficiency causes lethal, CD4+T cell-driven autoimmune diseases. Stem cell transplantation is used to treat these diseases, but this procedure is limited by the availability of a suitable donor. The intestinal microbiota drives host immune homeostasis by regulating the development of Treg, Th1 and Th2 cells. It is currently unclear if Treg-deficiency autoimmune disorders can be treated by targeting the enteric microbiota. Our aims are to determine the autoimmunity, gut microbiota, and plasma metabolomics affected by probiotic Lactobacillus reuteri DSM 17938 (LR), and to further identify the mechanism of modulated metabolite(s) in suppressing autoimmunity in Treg-deficient scurfy (SF) mice. We demonstrated that Foxp3+Treg deficiency results in gut microbial dysbiosis and autoimmunity over the lifespan of SF mouse. Remodeling microbiota with LR prolonged survival and reduced multi-organ inflammation in SF mice. LR changed the metabolomics profile disrupted by Treg-deficiency with a major effect of restoring levels of the purine metabolite inosine. Feeding inosine itself prolonged life and inhibited multi-organ inflammation by reducing Th1/Th2 cells and their associated cytokines. Mechanistically, the inhibition of inosine on the differentiation of Th1 and Th2 cells in vitro depended on adenosine A2A receptors. Both A2A receptor specific antagonist or genetically knockout A2A to SF mice reversed the anti-inflammatory effects of both inosine and LR in vivo. In conclusions, A2A receptors mediate a substantial protective effect of inosine and LR. The LR-modulated-microbiota-inosine-A2A axis might represent a potential avenue for combatting autoimmune diseases mediated by Treg dysfunction.
MicroRNA-21a-5p/PDCD4 axis Regulates Mesenchymal-Stem-Cell-Induced Neuroprotection in Acute Glaucoma
Authors: Wang Julie , Su Wenru , Zheng Song Guo
Published: January 2023

The clinical application of mesenchymal stem cells (MSCs) yields promising therapeutic benefits for a variety of neurological diseases, but underlying mechanisms are poorly understood. Here we show that intravitreal infusion of MSCs promotes retinal ganglion cell (RGC) survival in a mouse model of acute glaucoma, with significantly decreased microglial activation, decreased production of TNF-α, IL-1β, and reactive oxygen species, and reduced caspase-8 and caspase-3 activation. In vitro, MSCs inhibit both caspase-8-mediated RGC apoptosis and microglial activation in part through stanniocalcin 1 (STC1). Furthermore, we found that microRNA-21a-5p (miR-21) and its target, PDCD4, are essential for STC1 production and neuroprotective properties of MSCs in vitro and in vivo. Importantly, our results also demonstrate that miR-21 overexpression or PDCD4 knockdown enhances MSC-induced neuroprotective effects in an acute glaucoma model. These data therefore uncover a previously unrecognized neuroprotective mechanism, by which the miR-21/PDCD4 axis induces MSCs to secrete STC1 and other factors exerting neuroprotective effects. Furthermore, the miR-21/PDCD4 axis is a promising new target for neuroprotection in IOP-induced retinal damage.
Synergistic enhancement of ISG54/ISRE promoter activity with combinations of Pattern Recognition Receptor agonists and Interferon-gamma
Authors: Guinn Zachary P , Brown Deborah M , Petro Thomas M
Published: January 2023

Induction of interferon stimulated genes (ISG)-54, which depends on activation of Interferon Regulatory Factor 3 (IRF-3), is critical for innate immune responses of virus-infected cells by reducing cell viability through the intrinsic apoptosis pathway. Using wild type (WT) and IRF3 deficient (IRF3KO) RAW 264.7 cell lines, genetically engineered to express secreted Lucia luciferase under control of an ISG54/ISRE promoter (RAW-Lucia), we saw that ISG54 promoter activity is IRF3 dependent in response to IFN-γ. To investigate IRF3 in responses to IFN-γ, WT and IRF3KO RAW-Lucia cells were stimulated with IFN-γ in the presence or absence of the IRF-3 agonist, Polyinosinic-Polycytidylic acid (PolyI:C). IFN-γ and PolyI:C synergistically enhanced ISG54/ISRE promoter activity in both WT and IRF3KO RAW-Lucia cells, however, this activity was 91% lower in IRF3KO compared to WT cells. Moreover, ISG54 promoter activity was negatively correlated with viability of WT RAW-Lucia. RAW-Lucia cells also responded in a similar fashion when stimulated with IFN-γ plus transfected PolyI:C or dA:dT, which are RIG-I/MDA-5 and STING agonists, respectively. Co-stimulation with IFN-γ and TLR7 agonist, Loxoribine-which activates IRF7 but not IRF3-also synergistically enhanced ISG54/ISRE promoter activity in WT and IRF3KO RAW-Lucia cells, but IRF3 deficiency reduced the synergistic enhancement of ISG54/ISRE promoter activity by 92%. In contrast, the TLR9 agonists, ODN1585, which also activates IRF7 but not IRF3, failed to stimulate ISG54/ISRE promoter activity in RAW-Lucia cells and only enhanced the response to IFN-γ by 30%. These studies further reveal a previously unnoticed IFN-γ/IRF3/ISG54 nexus that can be exploited for antiviral therapy.
Trehalose-6, 6′-dibehenate inhibits lipopolysaccharide-induced NF-κB activation via PLC-γ1-PKC-ERK signaling and promotes M2 microglial polarization
Authors: Lin Wan-Wan
Published: January 2023

Activation of microglia triggers a robust amount of pro-inflammatory cytokines, which are involved in the pathogenesis of central nervous system tuberculosis (CNS-TB). The mycobacterial cord factor (Trehalose-6, 6-dimycolate [TDM]), and its synthetic analogue Trehalose-6, 6-dibehenate (TDB) bind to C-type lectin receptors Mincle to activate macrophages and dendritic cells via the Syk/Card9 pathway. However, there are no studies have been conducted to determine the effect of TDB on inflammatory responses in microglia and its underlying signalling pathways. Here using BV2 microglial cells we found that TDB can inhibit NO production, iNOS, pro-IL-1β, and the pro-inflammatory gene expression of TNF-a, pro-IL-1β, IL-6, Mincle, IFNb and MIP2 in LPS-stimulated BV-2 microglial cells. TDB-induced ERK-1/2 activation in LPS-activated microglial cells, whereas TDB had no effect on p38 and JNK phosphorylation induced by LPS. Interestingly, TDB suppressed LPS-induced NF-κB nuclear translocation but did not affect the IKK activity, and these actions were reversed by ERK inhibitor. In addition, TDB caused PLC-γ1 and PKC activation in BV-2 cells, and the TDB-mediated ERK upregulation could be attenuated by PLC-γ1 and PKC inhibitors. TDB also induced AMPK activation via calcium/CaMKK dependent pathway. Moreover, TDB induced M2 gene expression in LPS-stimulated BV-2 cells, and this action was prevented by the AMPK inhibitor, indicating that AMPK activation participates in TDB-mediated M2 polarisation effects. Therefore, TDB may have therapeutic potential for mitigating microglia-mediated neuroinflammation.
Critical Role for NLRC3 in Cell Proliferation and Cancer
Authors: Karki Rajendra , Kanneganti Thirumala-Devi
Published: January 2023

The nucleotide-binding oligomerization domain-like receptors (NOD-like receptors, NLRs) are intracellular innate immune sensors of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), and are critical in regulating diverse range of biological functions. Genetic mutations in these cytosolic receptors are associated with chronic inflammatory or autoimmune disorders. NLRs are known to modulate inflammatory responses, cell death, proliferation, and/or the gut microbiota to attenuate tumorigenesis. We identified NLRC3, a poorly defined member of the NLR family, as a negative regulator of PI3K-mTOR signaling, and characterized the potential tumor suppressor function of NLRC3 in colorectal cancer. We showed that NLRC3 is critical in restricting tumorigenesis in both colitis-associated colorectal cancer (CAC) model and spontaneous mice model ApcMin/+. Mice lacking NLRC3 were hyper-susceptible to colitis and colitis-associated cancer (CAC). Likewise, ApcMin/+ mice lacking NLRC3 developed more tumors in the colon than that of ApcMin/+ mice. We further showed that NLRC3 pre-dominantly functions in intestinal epithelial cells. Mechanistically, enterocytes lacking NLRC3 were unable to control cellular proliferation owing to a failure to suppress activation of the PI3K-mTOR signaling pathways.
Polyamine blockade alters the neuroblastoma microenvironment to favor restriction of tumor growth
Authors: Bassiri Hamid , Vu Annette , Hogarty Michael D , Benavides Adriana D
Published: January 2023

Despite improved treatments, neuroblastoma (NB) still accounts for ~15% of childhood cancer deaths. Amplification of MYCN results in NB with high-risk features and poor 5yr survival. Interestingly, MYCN-amplified NBs have elevated polyamine (PA) levels, as the gate-keeper enzyme in PA synthesis, ornithine decarboxylase (ODC1), is a direct Myc target. Inhibition of ODC1 activity by the FDA-approved medication difluoromethylornithine (DFMO) results in modest growth reduction of NB cell lines in vitro, whereas DFMO administration to mice that are predisposed to developing MYCN-driven NBs (TH-MYCN+/+ mice) leads to survival extensions that were more profound than that predicted by the tumor-intrinsic activity of the drug alone. These observations implied that DFMO might also exert tumor-extrinsic effects on the tumor microenvironment (TME). Indeed, DFMO treatment of TH-MYCN+/+ mice alters the NB TME in several ways, the most profound of which is an increase in NK cell frequency. We also find an increase in the percentage of NB cells expressing NKG2D ligands, and alterations in the expression of NK cell stimulatory and inhibitory receptors to favor activation. Collectively, these findings suggest that PA blockade induces distinct TME changes that allow for more efficient immune control of NB growth. We are now testing whether the activity of DFMO is dependent on NK cells, and are delineating how DFMO alters the transcriptional profiles of NBs and immune cells in the TME. We hope that these studies will complement the data from phase I/II clinical studies using DFMO in various therapeutic strategies for NB, and will allow for an increased understanding of how to more effectively combine PA blockade with other immunotherapies for this disease.
Inflammasomes/IL-1 pathways in myeloid cells modulate PD-1/PD-L1 checkpoint molecules
Authors: Guo Beichu , Zhang Jinyu , Fu Shunjun
Published: January 2023

The inflammatory microenvironment is not only critical for tumor growth and progression, but also associated with immunosuppression. Expression of immune checkpoint molecules, such as PD-L1, in tumor cells and innate immune cells has been linked to diminished anti-tumor response. However, the connection between inflammation and expression of checkpoint molecules in myeloid cells and tumor cells is poorly characterized. Our recent studies have demonstrated that the inflammasome and IL-1β pathway plays a critical role in promoting tumor growth and metastasis. Our data show that inflammasome activation promoted the infiltration of myeloid cells, such as myeloid-derived suppressor cells and tumor-associated macrophages, into tumor microenvironments. Interestingly, we found that inflammasome/IL-1 pathways induced expression of immune checkpoint molecule PD-L1 on infiltrated myeloid cells and tumor cells. Furthermore, blocking IL-1R with IL-1R antagonist (IL-Ra) or anti-IL-1R antibody inhibits tumor growth and metastasis accompanied by decreased accumulation of myeloid cells and expression of the PD-L1 molecule. Together, our results demonstrate that inflammasomes/IL-1 pathways promote infiltration of myeloid cells and expression of PD-L1, contributing to tumor immune evasion and tumor progression.
Defective STING signaling in ovarian cancer cells favor oncolytic virus action
Authors: de Queiroz Nina Marí Gual Pimenta , Xia Tianli , Barber Glen N.
Published: January 2023

Ovarian cancer is the sixth most prevalent cancer in women and the most lethal of the gynecologic malignancies. Our group has recently shown that STING signaling is often suppressed in a wide variety of cancers, including colorectal carcinoma and melanoma. In this work, we examined the expression and activation of STING pathway in 11 different human ovarian cancer cell lines (CA), generated from the diagnosis of ovarian cancer in varied stages and cultured in vitro. We evaluated STING expression by Western blot in these cell lines and showed that STING is present in 8 of the 11 cell lines. Signaling via STING requires the activation of cGAS synthase enzyme, which was absent in 7 cell lines. In the absence of both STING and cGAS all STING pathway was affected with no IRF-3 and NFκB-p65 translocation and activation. In order to correlate expression levels with STING function in the production of IFN-β, we transfected cells with dsDNA90 to activate STING or with Poly(I:C) for activation via RIG-I. ELISA and Real Time-PCR analyses showed that all 11 CA responded poorly to dsDNA90 stimulation. In contrast, majority of CA were able to produce IFN-β and CXCL10 in response to Poly(I:C), indicating that RIG-I pathway was preserved. Defective production of these cell death control cytokines after DNA stimulus may favor CA proliferation, but also facilitates viral replication and make this CA highly susceptible to HSV1γ34.5 (Herpes simplex virus). Analyzes in vivo by injection of human ovarian cancer cells subcutaneously in Nu/Nu mice followed by HSV1γ34.5 treatments correlates with in vitro findings. The results suggest that STING pathway can be suppressed during ovarian cancer development and these defects let CA more susceptible to oncolytic viral therapy.
Characterize the role of cancer associated fibroblast (CAFs) derived IL-32 in esophageal squamous cell carcinoma (ESCC)
Authors: CHEN Miao , GUAN Xin-yuan
Published: January 2023

Cancer-associated fibroblasts (CAFs) are localized adjacently to cancer cells at all stages of esophageal squamous cell carcinoma (ESCC) progression, and their functional contributions to this process are emerging. Our previous studies have identified FGFR2 as CAFs specific marker in ESCC. In this study, using microarray analysis, we identified IL-32 was the most differentially expressed cytokine in CAFs in patients with ESCC. Flow cytometric analysis and western blot have revealed markedly increased IL-32 in FGFR2+ CAFs from ESCC tumor specimens, compared with non-tumor epithelial tissues. IL-32 treatment could promote prosurvival effect in tumor cell from upon cisplatin or 5-Fu mediated apoptosis in culture and human ESCC tumor-bearing nude mice. In contrast, knockdown of IL-32 expression in CAFs by shRNA reduce tumor cell viability in reconstituted mouse model upon cisplatin or 5-Fu treatment. Mechanistic study shows that IL-32 treatment significantly downregulated caspase 3/9 and upregulated Bcl-2 expression by ESCC tumor upon Cisplatin or 5-Fu stimulation. In contrast, blockage of type II IFN signaling in CAFs resulted in significantly reduced IL-32 secretion and increased apoptotic cell death of ESCC. Together, these findings have suggested a critical role of CAFs derived IL-32 in prosurvival effect ESCC and thereby exacerbating cancer development, which suggested that IL-32 blockade may serve as potential therapeutic target for ESCC.
Rise in Interleukin-17-producing invariant Natural Killer T Cells during Bordetella Pertussis Infection in Mice
Authors: Santos Daniel Rebelo , Nguyen Alana , Bui Khoa , Abramson Tzvia
Published: January 2023

Bordetella pertussis is a Gram-negative bacterium known for causing the respiratory disease whooping cough. Whooping cough has recently seen resurgence, especially among children, with the disease’s resolution being delayed. Here, we observed the contribution of invariant Natural Killer T (iNKT) cells in the response to B. pertussis infections. Invariant NKT cells are so called for their invariant chain (Vα14Jα18 in mice) within the T cell receptor, which allows these cells to respond to lipid antigens presented by CD1d molecules. iNKT cells are known to express a variety of transcription factors and release different cytokines, as well as interact with numerous cells of the immune system. Here we hypothesize that IL-17 producing iNKT (iNKT17) cells rise in number during B. pertussis infections to aid in the resolution via an IL-17 driven inflammation, as well as to activate antigen presenting cells. The population of iNKT17 cells was assessed via comparison of uninfected to B. pertussis-infected C57BL/6 mice, using a multicolor flow cytometry analysis. The mice infected with pertussis displayed a significant rise in iNKT cells when compared to uninfected mice, while being positive for RORγt and IL-17 secretion. Immunohistochemical analysis displays the interaction of iNKT cells with resident dendritic cells during infection. These findings indicate a role for iNKT17 cells in the pathogenesis and potential resolution of B. pertussis infection, with the potential for future analysis into iNKT cell knockout studies to determine further understanding of the cell’s function during the infection.
Early-life respiratory bacterial infection-induced chronic lung disease is driven by a novel TLR2/IL-13/miR-21/PI3K-dependent, but MyD88-independent signalling pathway
Authors: Starkey Malcolm Ronald , Nguyen Duc H , Kim Richard Y , Nair Prema M , Haw TattJhong , Horvat Jay C , Godfrey Dale I , McKenzie Andrew N , Hansbro Philip M
Published: January 2023

There is a critical window in early-life where the lung is still maturing and susceptible to infection. Indeed, severe respiratory infections in early-life are a risk factor for the development of chronic lung diseases. The aim of this study was to identify the mechanisms involved.

Neonatal wild-type (WT), TLR2 deficient (−/−), IL-13−/−, MyD88−/− and STAT6−/− mice were infected with the natural mouse bacterial pathogen Chlamydia muridarum, as a model of severe respiratory tract infection in early-life. In some experiments WT mice were treated with miR-21 antagomirs, PI3K inhibitors, or relevant controls during early-life infection. The impact of targeting these specific immune molecules during early-life on infection-induced impairment of lung function and structure were assessed.

Neonatal Chlamydia respiratory infection increased TLR2, IL-13-receptor, miR-21 and PI3K expression and/or activity in the lung. TLR2 signalling induced IL-13-receptor expression, IL-13 signalling induced miR-21 expression and miR-21 increased PI3K activity. TLR2 signalling also increased IL-13+ ILC2s in the lung. TLR2−/− and IL-13−/− mice were protected against infection-induced airway hyperresponsiveness (AHR), but not emphysema-like alveolar enlargement. This TLR2/IL-13 mediated phenotype was independent of MyD88, but dependent on STAT6. Specific targeting of miR-21 prevented AHR but not emphysema. Pan-PI3K inhibition did not affect AHR, but protected against emphysema. Interestingly, early-life infection-induced AHR was steroid insensitive.

This study identifies a novel signalling network that may be targeted for the prevention of the long-term deleterious effects of early-life infection on lung function and structure.
Differential regulation of development of experimental autoimmune encephalomyelitis by Akt-1 and Akt-2
Authors: Zeng Qiuming , Ouyang Song , Guo Hui , Tang Rong , Liu Jishi , Zhang Jian
Published: January 2023

Akt-1 and -2 are major members of serine/threonine Akt family kinases that play a key role in multiple cellular processes. Previously, we have shown that Akt-2 but not Akt-1 is involved in the inhibition of inducible regulatory T cell development. However, the role of both isoforms of Akt in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is completely unknown. In this study, we showed that while Akt1−/− mice develop ameliorated EAE, Akt2−/− mice develop severe disease. At the cellular levels, Akt-1 appears to inhibit proliferation of thymic-derived CD4+CD25+Foxp3+ regulatory T cells (tTregs), but facilitates antigen-specific TH1/TH17 responses during EAE induction. In a sharp contrast to Akt-1, Akt-2 potentiates tTreg proliferation, and suppresses antigen-specific TH1/TH17 responses. Our data demonstrate Akt-1 and Akt-2 differentially regulates the susceptibility of mice to EAE possibly by controlling tTreg proliferation in the periphery, thus controlling TH1/TH17 responses during EAE development.
Plasma cells and not memory cells, constitute the majority of human IgE Cells
Authors: Saunders Sean P , Narayanan Sriram , Narang Viping N. , Wong Glenn , Aw Derrick C.W. , Chandran Nisha , Navin Ishwar , Reibman Joan , Poidinger Michael , Zolezzi Francesca , Curotto De Lafaille Maria A.
Published: January 2023

Immunoglobulin E (IgE) is central in allergy pathology as it binds high-affinity receptors on mast cells, inducing degranulation upon allergen crosslinking. However, IgE has the lowest serum concentration and shortest half-life of all Ig’s, and IgE+ B cells are extremely rare. This work aims to uncover characteristics of IgE+B cells that could be used in the design of new therapeutic strategies for allergic diseases.

IgE+ cells were identified in Atopic Dermatitis (AD) patients, and were also generated in vitro by stimulating with aCD40 and IL-4. Naïve, IgG+, IgA+ and IgE+ cells from AD donors were phenotyped and sorted by flow cytometry, and their transcriptomes were determined by RNAseq. Inter-sample similarity was determined using multidimensional scaling (MDS). Gene ontology (GO) of differentially expressed genes (DEG) identified molecular pathways enriched in IgE+ cells.

IgE+ cells were CD38hiCD27hi, indicating a high content of plasma cells in IgE+ B cells. IgE+ cells expressed high levels of proliferation marker Ki67. MDS comparison of datasets of naïve, IgG+, IgA+ and IgE+ cells showed that IgE cells were distant from both IgG and IgA cells. GO analysis of IgE DEG demonstrated that IgE cells expressed genes associated with N-glycosylation, the IRE1-XBP1 endoplasmic reticulum unfolded protein response, and mitochondrial oxidative phosphorylation, synonymous with plasma cells.

Our data indicates that IgE+ cells in AD patients are predominantly plasmablasts and plasma cells, and not memory B cells. Ongoing studies aim to characterize human IgE cell diversity using single cell analysis, and to identify non-IgE precursor memory cells that give rise to IgE+ cells upon allergen activation.
Impact of Nur77 on the initiation and development of E. coli induced acute lung injury
Authors: Hou Jinchao
Published: January 2023

Objectives

Orphan nuclear receptor Nur77 (Nur77) has been emerged as a regulator of gene expression in aseptic inflammation, but its role in regulating macrophage response to bacterial infection remains unclear. So, this study was designed to investigate the impact of Nur77 in E. coli induced acute lung injury.

Methods

Mice with different genotypes (WT, Nur77−/−) were challenged with E. coli intratracheal inoculation. Mortality was assessed hourly. Bronchoalveolar lavage fluid (BALF) and blood were obtained and bacterial burdens were assayed using agar plating method. Lung morphology changes and pulmonary vascular permeability were assessed by HE staining or BALF protein accumulation, separately. The TNF-α level in the BALF were determined by ELISA. Ex vivo, the Nur77 mRNA level expressed on the alveolar macrophages (AMs) under rest or E. coli stimulation were detected by qPCR. The phagocytosis ability of AMs from WT and Nur77−/− mice were further assessed.

Results

The survival rate of the Nur77−/− mice was 93.3%, while only 31.3% of WT mice survived more than 48 hours. Bacterial counts in the BALF collected from the WT mice were higher than those from the Nur77−/− mice by 10 folds. Along with reduced pulmonary bacterial load, diminished lung injury and permeability were observed in the Nur77−/− group. In addition, we found largely decreased TNF-α level in Nur77−/− mice compared with WT mice in BALF. The mRNA level of Nur77 was largely up-regulated in AMs under E. coli stimulation ex vivo. Functionally, Nur77−/− AMs exhibited increased rates of bacterial engulfment.

Conclusions

The absence of Nur77 enhances lung immune defense to eliminate invading E. coli and prevents the pathogen from spreading, ultimately improving the outcome.
Human neutrophils alter IL-20 receptor expression and IL-20 modulates human neutrophil function during in vitro infection with Staphylococcus aureus
Authors: Datta Sandip K , Gough Portia
Published: January 2023

Neutrophils are critical to a protective immune response to infection with Staphylococcus aureus, a leading cause of skin and soft tissue infections. The responses of neutrophils are shaped by environmental cues in the form of the type of pathogen encountered and host signals in the form of cytokines and chemokines. Yet, there is an incomplete understanding of signaling at the site of infection and how signals work in concert to influence neutrophil function and the biological tug-of-war that occurs between host and pathogen. Previous work showed that IL-20 is produced by human keratinocytes infected with S. aureus in vitro, and during a subcutaneous model of infection in mice in vivo. Signaling through an IL-20 receptor reduced neutrophil recruitment via reduced levels of IL-1β and IL-17A, thereby contributing to the pathogenesis of infection. Since IL-20 is produced by infected epithelial cells, we investigated if this cytokine could affect neutrophils directly. We found that neutrophils alter IL-20 receptor expression in response to in vitro infection with S. aureus and exposure to pro-inflammatory host factors, TNFα and IL-8. In these conditions, IL-20 altered actin dynamics in neutrophils and affected functions that rely on this activity: migration, phagocytic uptake and tertiary granule exocytosis.
Control of regulatory T (Treg) cell function by protein kinase C-eta (PKCη): A novel target for cancer immunotherapy
Authors: Pedros Christophe , Kong Kok-Fai , Altman Amnon
Published: January 2023

Modulating regulatory T cell (Treg) function to promote anti-tumor immunity is a desirable strategy for cancer immunotherapy. Blockade of checkpoint molecules CTLA-4 and PD-1 has shown clinical efficacy in metastatic melanoma and other malignancies. We recently reported that Treg stimulation induced the recruitment of protein kinase C-eta (PKCη), together with the GIT-PAK-PIX signaling complex that mediates focal adhesion disassembly, to CTLA-4, an inhibitory receptor highly expressed on Foxp3+ Tregs and important for Treg suppression. As a result, PKCη was recruited to the immunological synapse of stimulated Treg. These events promoted Treg-mediated contact-dependent suppression, including suppression of tumor immunity, via a mechanism likely involving depletion of costimulatory CD80/86 ligands from APCs (Nat Immunol. 2014;15:465–72).

PKCη-deficient (PKCη−/−) Tregs were defective in the suppression of anti-tumor immunity against B16 melanoma and the prostate adenocarcinoma TRAMP-C1 but retained the ability to inhibit experimental colitis, a disease that often occurs in patients treated with anti-CTLA-4 antibodies. PKCη loss in mouse Treg cells increased both the number of intratumoral CD4+ and CD8+ effector T cells and their ability to produce cytokines upon restimulation. Moreover, shRNA-mediated knockdown of PKCη reduced the suppressive activity of human Tregs. Impaired activation of the GIT-PAK-PIX complex and the resulting defect in CD80/86 depletion are likely responsible for the defective suppressive activity of PKCη−/− Tregs. Altogether, our findings establish the CTLA-4-PKCη signaling pathway as critical for suppressing tumor immunity, and implicate it as an attractive cancer immunotherapy target.