bluuebunny/crossref_metadata_2025 huggingface dataset:
WC1 is a hybrid gammadelta TCR coreceptor and pattern recognition receptor for pathogenic bacteria
Authors: Telfer Janice C , Le Page Lauren , Nandi Dipika , Buck Jessica , Boisvert Natalie , Gillespie Alexandra , Damani-Yokota Payal , Amir Maria , Yirsaw Alehegne , Baldwin Cynthia L
Published: January 2023

WC1 proteins are uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily. Present in variable, and sometimes high, numbers in most animals with adaptive immune systems, in cattle there are 13 distinct genes (WC1-1 to WC1-13). The structure of a multigenic WC1 gene array, with each WC1 protein containing multiple SRCR domains in its extracellular domain, has been conserved over millions of years of evolutionary time, suggesting that they act as a panel of pattern recognition receptors for conserved pathogen ligands. All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation-dependent manner and some are required for the γδ T cell response to Leptospira. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is comprised of amino acids in three discontinuous regions. In addition, recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. WC1 SRCR domains also bind to Borrelia burgdorferi and Mycobacterium spp. WC1 in other artiodactyls such as goat, sheep and swine are closely related, but show some divergence that may have been driven by natural selection by species-specific pathogens. Our data suggest that WC1 gene arrays play a multi-faceted role in the γδ T cell response to bacteria in multiple species, including acting as hybrid pattern recognition receptors and TCR coreceptors, and may function as anti-microbials.
Canine CD4+CD8+ double-positive T cells participate in immunity to Ehrlichia chaffeensis infection
Authors: McGill Jodi L , Boorgula Gunavanthi Devi Yadav , Wang Ying , Ganta Roman R
Published: January 2023

Ehrlichia chaffeensis is an obligate intracellular gram-negative bacteria and a frequent cause of severe and fatal tick-borne infection in people in North America. The reservoir host for E. chaffeensis is the white-tailed deer, while humans, dogs, coyotes and goats are regarded as common incidental hosts. In dogs, we have shown that E. chaffeensis establishes a chronic infection that persists for at least 42 days, while promoting development of Th1 and Th17 cellular responses and pathogen-specific humoral immunity. To date, little is known about the immune responses that correlate with control or successful clearance of the infection in dogs or humans. In dogs, a population of mature CD4+CD8+ double-positive (DP) T cells exist that have features of activated T cells and share some similarities to the CD4+CD8+ DP populations which have been described in pigs and humans. However, little is known about the function of these cells in dogs, particularly in the context of infectious diseases. Here, we demonstrate that canine CD4+CD8+ DP T cells expand significantly in the blood and spleen in response E. chaffeensis infection, and upregulate markers of T cell activation and antigen-experience. Recall responses after in vitro restimulation with E. chaffeensis host-cell free lysate showed that CD4+CD8+ DP T cells undergo multiple rounds of antigen-specific proliferation, produce IFNγ and IL-17, and upregulate expression of the cytotoxic molecules granzyme B and granulysin. Together, our results demonstrate that CD4+CD8+ DP T cells contribute to the immune response to E chaffeensis in the physiologic host, and suggest the importance of alternative lymphocyte populations in promoting protection and control of tick-borne diseases in the dog.
TAGLN2 inhibits Arp2/3 complex-nucleated actin branching and controls microvilli formation in T cells
Authors: Jun Chang-Duk , Kim Hye-Ran , Lee Kyung-Sik
Published: January 2023

Microvilli are abundant on the surfaces of circulating lymphocytes. However, factors that regulate microvilli formation on lymphocytes are not completely understood. Moreover, definitive roles of microvilli have not been fully addressed. Here we identified that TAGLN2—an actin-binding protein predominantly expressed in T cells— specifically localizes at the microvilli on T cells. Ectopic expression of TAGLN2 enhanced microvilli protrusion at the initial development of immunological synapse, and the opposite results were obtained by TAGLN2 depletion. Obviously, T-cell activation was influenced by the microvilli structures on T cells. Of interest was that TAGLN2 blocks Arp2/3 complex binding to actin filaments that leads to the inhibition of actin branch. Electron microscopy-based helical image analysis revealed that the main sites of TAGLN2 binding on actin share the Arp2/3 complex binding sites. These results demonstrate a novel function of TAGLN2 that contributes to enhance T-cell antigen recognition, and hence T-cell activation.
‘The role of phagocytic NADPH oxidase and innate immune cells in the immune activation in toluene diisocyanate (TDI)-induced lung inflammation’
Authors: CHENG CHIA YING , Shieh Chi-Chang K
Published: January 2023

Innate lymphoid cells (ILCs) have been known to play an important role in development of lymphoid tissues and in the initiation of inflammation at barrier surfaces in response to infection or tissue damage. Isocyanates are low-molecular-weight compounds noted for inducing an oxidant stress-dependent pulmonary inflammation, which is the leading cause of occupational asthma. Our previous studies indicated that phagocytic NADPH oxidase is an essential regulator in TDI-induced airway inflammation characterized by a server lung inflammation with both Th17 and Th2 immune components. In this study, we hypothesized that ILCs activation may play an important role in phagocytic NADPH oxidase-mediated downstream immune mechanism in TDI-induced airway inflammation. The wild type (WT) and phagocytic NADPH oxidase deficient (Ncf1−/−) mice were sensitized and challenged with 3% TDI to induce lung inflammation. Our results showed that type 2 inflammatory cytokines, including IL-33 and IL-5, increased in both WT and Ncf1−/−mice but more significantly in TDI-induced lung inflammation. Moreover, the pro-inflammatory cytokine IL-1b and IL-17A production was only induced by TDI-exposure in WT but not in Ncf1−/− mice. We also found that the population of lineage negative and CD45 positive cells was identified in inflammation lung tissue. Comparison of ILCs population between TDI-induced WT mice and Ncf1−/− mice showed that the number of ILC2s and ILC3s were significantly higher in WT mice. These results suggested that ROS may modulate the activation of ILC2s, ILC3s and may determine the Th2 and Th17 cytokines production in the TDI-induced lung inflammation.
IL-1R and IL-1RA differentially contribute to immunopathogenesis during fungal asthma
Authors: Godwin Matthew S , Blackburn Jonathan , Steele Chad
Published: January 2023

Severe asthma with fungal sensitization defines a subset of asthmatics with allergy to multiple fungal species, including Aspergillus fumigatus. We have previously shown genetic deficiency in or neutralization of IL-22 improves fungal asthma severity, which correlated with lower levels of IL-1α and IL-1β. Here, we show that bronchoalveolar lavage fluid (BALF) and induced sputum from human asthmatics sensitized to fungi demonstrate increased levels of IL-1α, IL-1β and IL-1 Receptor Antagonist (IL-1RA) compared to fungal negative asthmatics, suggesting that members of the IL-1 cytokine family may contribute to fungal asthma severity. To this end, we examined fungal asthma severity in mice deficient in IL-1R and IL-1RA with the hypothesis being that severity would be decreased and increased in these animals respectively. Surprisingly, the absence of IL-1R signaling had no impact on biomarkers of asthma severity (i.e. levels of proallergic CCL17, CCL22, and IL-33), yet Il1r−/−mice demonstrated better lung function compared to WT mice. Interestingly, the absence of IL-1R signaling resulted in a profound defect in the production of IL-22, suggesting a mechanism of improved lung function in Il1r−/− mice. In direct contrast, Il1rn−/− mice exhibited elevated proallergic biomarker levels, but no changes in the levels of IL-22. These preliminary results identify differential roles for IL-1R signaling (IL-22 mediated immunopathogenesis) and IL-1RA signaling (type 2 mediated immunopathogenesis) in fungal asthma severity.
Inhibition of tight junction protein ZO-1 expression by estrogen in human gut mucosa
Authors: Zhou Zejun , Gilkeson Gary , Jiang Wei
Published: January 2023

Background

Females have stronger cellular and humoral immunities compared to males. Sex hormones are playing roles in these differences; however, the role of sex hormones in human gut permeability is essentially unknown.

Methods

The expression of tight junction ZO-1, estrogen receptor (ER)-α and β in male and female primary gut tissues were analyzed using immunohistochemistry. The inhibition of estrogen on ZO-1 was evaluated in primary gut tissues by qPCR. ZO-1 expression and NF-κB activation in Caco-2 cells was evaluated by western blot, immunofluorescent staining and reporter activity assays. Data are presented as median (interquartile range [IQR]).

Results

The expression of ZO-1 and ER-β but not ER-α were present in both male and female gut tissues, the median immunofluorescence intensities (MFI) of ZO-1 expression were 2.48 (2.40–3.11) and 3.59 (3.11–4.23) for females and males respectively (P = 0.02, non-parametric Mann-whitney test). No sex difference in MFI expression of ER-β was found (P = 0.79). After 24 h incubating with physiologic dose of estrogen (200 pg/mL) in primary gut tissues in vitro, the median ZO-1 mRNA relative expression to GADPH by qPCR was 1.002 (1.000–1.003) and 0.80 (0.21–0.84) for treatment with medium alone and estrogen respectively. The median MFI expression of ZO-1 in Caco-2 cells assessed by microscopy was 0.97 (0.77–1.14) and 1.16 (0.76–1.37) for treatment with estrogen and medium alone respectively (P = 0.01). The median luciferase activity of NK-κB in Caco-2 cells assessed by NK-κB reporter activity assay was 0.58 (0.53–0.60) and 0.98 (0.87–1.05) for treatment with estrogen and medium alone respectively (P = 0.002).

Conclusion

Estrogen inhibits the ZO-1 expression and NF-κB activity in human gut mucosa.
A novel method of assessing ASC specks and caspase-1 activity by imaging flow cytometry
Authors: Nagar Abhinit , DeMarco Richard A , Harton Jonathan A
Published: January 2023

NLRP proteins interact with the adaptor ASC to recruit and activate caspase-1-dependent maturation of IL-1β and IL-18 in a multiprotein ‘inflammasome’ complex. Inflammasome activation occurs in response to structurally diverse agonists triggering NLRP activation leading to rapid localization of NLRP:ASC complexes in a typically singular perinuclear ‘speck’. At present, formation of inflammasome specks can be assayed by microscopy, which is time consuming, or a flow cytometric analysis technique evaluating altered width and height of ASC pulse (TOFIE), which provides no visualization of the speck. We have developed a technique utilizing the advantages of imaging flow cytometry that combines high-throughput sample collection and high magnification (60×) microscopic visualization. This technique not only detects the presence of ASC specks but also determines the distribution of active caspase-1 within the cell using the fluorescent caspase-1 substrate FLICA. This assay allows for high-throughput imaging of ASC-containing specks and characterization of these specks in terms of number and area. Additionally, this technique also determines the location of active caspase-1 and enumerates aggregates of active caspase-1. This method is more sensitive than the present flow cytometric method, TOFIE, which fails to differentiate between active inflammasome specks and false positive signals due to accumulation/aggregation of ASC. The capacity of this method to detect and quantify native as well as reconstituted inflammasome specks with associated caspase-1 activity renders it more powerful for quantifying the impact of mutations on the molecular interaction of NLRP proteins with ASC and accompanying caspase-1 activation.
Aryl hydrocarbon receptor: a novel target for the anti-inflammatory activity of statin therapy
Authors: Turco Antonella , Scalisi Giulia , Gargaro Marco , Pirro Matteo , Fallarino Francesca
Published: January 2023

Statin therapy is associated with an attenuation of the systemic inflammatory response. Aryl hydrocarbon receptor (AhR) is believed to play an active role in the control of inflammation. Specific statins promote the transcription of Cyp1a1, which induction involves AhR activation1. We investigated whether AhR might be involved in the anti-inflammatory effect of statins treatment.

We performed luciferase assay to test the ability of murine hepatocytes overexpressing AhR and the xenobiotic responsive elements (XRE) and the ability of murine embryonic fibroblast AhR KO, transfected with different mutants of AhR to activate receptor in response to statins. Lipopolysaccharide (LPS)-treated RAW 264.7 macrophage cells were tested for the expression of AhR and for pro-inflammatory/anti-inflammatory response to different statins. Activated AhR WT and KO macrophages were tested for the expression of M1 and M2 markers and for pro-inflammatory/anti-inflammatory cytokines production. Luciferase assays showed that atorvastatin and pravastatin activated AhR, but no AhR mutants. LPS treatment of RAW 264.7 cell line induced AhR. Exposure of RAW 264.7 to both pravastatin and atorvastatin concomitantly to LPS reduced IL-6 production and increased IL-10 secretion. Atorvastatin and pravastatin potentiated arginase expression and anti-inflammatory response in activated macrophages in AhR dependent manner.

These data show that atorvastatin and pravastatin activate AhR, attenuate the inflammatory response induced by LPS and promote M2 phenotype in AhR dependent manner. AhR might represent a mediator of the anti-inflammatory effects of statin treatment.
Attenuation of Staphylococcal enterotoxin-B (SEB)-induced lung injury (ALI) by resveratrol results from miR-193a down-regulation and alterations in metabolic profile
Authors: Alghetaa Hasan F , Mohammed Amira K , Nagarkatti Prakash , Nagarkatti Mitzi
Published: January 2023

Resveratrol, (RES), a phytoalexin, is well-known for its anti-inflammatory and anti-oxidant properties. SEB, a superantigen, is known to trigger ALI and cause mortality. In the current study, we tested the effect of RES in a dual-dose model of SEB exposure that triggers ALI and causes 100% mortality in C3H/HeJ-mice. The data revealed RES attenuated SEB-induced ALI and prevented mortality. Forty eight hours post-SEB exposure, lung-infiltrating mononuclear cells were tested for microRNA expression profile to determine the epigenetic regulation by resveratrol. SEB-activated splenocytes were pre-treated with 50 μM of RES or vehicle for metabolic profile analysis by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). We also noted significant decline in miR-193a in the lungs of RES-treated SEB group, which targeted and caused an increase in TGFβ2 and TGFβR3, potent inhibitors of T-cell proliferation, by using RT-PCR and validation by transfection studies. RES-induced downregulation of miR-193a also influenced the activity of mechanistic target of rapamycin (mTOR) as well as pyruvate kinase muscle isozyme2 (PKM2) genes, and caused RES-treated SEB-activated T cells to be quiescent metabolically in comparison to the energetic vehicle-treated SEB-activated T cells. Together, RES caused inhibition in the proliferation of SEB-activated T-cells by alterations in miR expression and metabolic profiles. (Supported by NIH grants P01AT003961, R01AT006888, R01ES019313, R01MH094755, P20GM103641 and R01AI129788).
The role of microglia and monocyte-derived macrophages in Toxoplasma gondii infection of the brain
Authors: Thompson Jeremy , Hayes Nikolas , Gevaria Anjana , Harris Tajie
Published: January 2023

In the brain, microglia are the resident immune cells. Despite their location in the brain parenchyma and expression of machinery capable of responding to pathogens, little is known about the role microglia play during infection. Toxoplasma gondii is an intracellular, protozoan parasite that establishes a chronic infection in the brain. T cell-derived IFN-g is central to control parasite replication and prevent host mortality by activating anti-parasitic mechanisms. In addition, during brain infection monocyte-derived macrophages (MDMs) enter the brain. Deciphering the contribution of these two cell populations during T. gondii infection has been confounded by their phenotypic and morphologic similarity. However, these cells likely contribute differently to parasite control. Using a reporter mouse to label microglia we have found that MDMs are the primary producers of nitric oxide, a key anti-parasitic molecule. Therefore, to explore the role of microglia and MDMs in the control of T. gondii in vivo we are taking advantage of constitutive or inducible cre recombinase activity driven from the CX3CR1 promoter which allow both microglia and MDMs or solely microglia, respectively, to be targeted genetically. By removing the gene STAT1 from these cells we have rendered them unable to upregulate key IFN-g induced genes necessary for controlling T. gondii. Mice in which MDMs and microglia are unresponsive to IFN-g are unable to control parasite replication and succumb to infection by 19dpi. Conversely, mice in which only microglia are deficient in IFN-g signaling do not succumb to infection and resemble infected control mice. These results suggest that microglia are not a key cell type responsible for controlling T. gondii in the CNS.
The immune environment dictates the dominance of licensed versus unlicensed natural killer cell subsets during viral infection
Authors: Aguilar Ethan George , Zamora Anthony Elston , Dunai Cordelia , Khuat Lam , Murphy William J
Published: January 2023

Natural Killer (NK) cells are innate lymphocytes involved in anti-viral and anti-cancer responses. They can be functionally and phenotypically divided based on expression of inhibitory receptors capable of binding to self-MHC class I, a concept termed licensing/education. NK cells capable of binding to self-MHC (licensed) have been shown to be more functionally competent compared to those that cannot (unlicensed). It is still unclear which subset is more critical during an anti-viral response. Using two models of murine cytomegalovirus infection (MCMV), we show distinct subsets of NK cells that can both directly and indirectly contribute to the anti-viral response. We have previously shown that after hematopoietic stem cell transplantation (HSCT), the licensed effector NK population was predominantly responsible for resistance to MCMV, which was in part due to the absence of T cells present immediately after transplant. However, MCMV infection in non-HSCT mice yielded an opposite pattern with the unlicensed NK subset augmenting resistance to MCMV. We found preferential localization patterns of the different NK subsets which correlated with increased antigen specific T cell expansion and viral clearance. Interestingly, by depleting the different NK subsets in HSCT vs non-HSCT mice, we demonstrated differences in subset roles. The unlicensed NK cells, which promote antigen specific T cells, are more critical during normal conditions, while the licensed NK cells serve a better direct anti-viral role following HSCT where T cells take longer to repopulate. Thus, the contributions of these different NK subsets and their dependence on the immune environment should be taken into consideration when applying NK cells therapeutically.
Innate immune responses in fungal asthma enhance antiviral immunity during influenza virus infections
Authors: Samarasinghe Amali , LeMessurier Kim , Doorley Laura , Palipane Maneesha
Published: January 2023

Fungal allergic asthma is a subtype of asthma with high incidence. Although asthmatics are considered an ‘at risk’ group for severe influenza, these patients suffered less severe morbidity and mortality compared to non-asthmatics during the 2009 influenza pandemic. We developed a mouse model of fungal asthma and influenza comorbidities to recapitulate these epidemiologic findings and provide a means to explore mechanisms by which this protection may occur. We hypothesized that eosinophils guide antiviral responses against influenza virus infections in the allergic host. We characterized the immunophenotype of eosinophils in various niches including bone marrow, lymphoid organs, and lungs, in asthma and influenza comorbid conditions in comparison with individual disease conditions. Eosinophils from comorbid mice had high expression of MHCI, PIR-A/B, and CD62L in the lungs and spleens compared to influenza alone. The adoptive transfer of eosinophils led to a reduction in viral burden and increase in airway CD8+ T cells in virus-infected recipients. In addition, eosinophils were able to promote CD8+ T cell activation in vitro and ex vivo, and antigen-loaded eosinophils interacted with CD8+ T cells in vitro. Comorbid mice had more plasma cells in lymphoid organs although mature B cell numbers were similar to influenza alone. Antibody production in these cells was more prominent at early time points in comorbidity but was similar between the groups at late time points. Since eosinophils have been previously shown to regulate B cells and IgA production in the gut, we are now exploring the hypothesis that eosinophil-mediated B cell responses contribute to the increase in neutralizing antibodies noted in mice with asthma and influenza.
Testing the hypothesis that malaria-induced immune remodeling of monocytes plays a role in immunity to malaria
Authors: GUHA RAJAN , Crompton Peter D.
Published: January 2023

In malaria-naïve individuals, Plasmodium falciparum infection results in high levels of parasite-infected red blood cells (iRBCs) that trigger the systemic inflammation and fever characteristic of malaria. But, individuals in endemic areas who are repeatedly infected with P. falciparum are often asymptomatic and have low levels of iRBCs, even young children who have yet to acquire fully protective antibodies. The molecular mechanisms underlying these clinical observations remain unclear. We earlier showed that PBMCs collected from uninfected Malian children before the malaria season responded to iRBCs by producing pro-inflammatory mediators such as IL-1β, IL-6. However, following febrile malaria there was a marked shift in the response to iRBCs with the same children’s PBMCs producing lower levels of those cytokines. Moreover, genome-wide expression analysis showed that genes involved in phagocytosis and intracellular killing were upregulated in PBMCs after malaria as compared to before. Together, these data suggest that malaria-induced remodeling of innate immune cells might play a role in immunity to malaria. In ongoing work we are dissecting the underlying molecular pathways involved in P. falciparum-induced changes in monocyte/macrophages. We are studying monocytes ex vivo from malaria-exposed children, as well as monocytes stimulated with iRBCs in vitro. Initial data support the hypothesis that malaria-induced immune-remodeling of monocytes dampens pathogenic inflammation while enhancing anti-parasite effector mechanisms, consistent with our observation that children in malaria endemic areas are often afebrile and tend to control parasite replication in the face of repeated P. falciparum exposures.
Microbiota-independent mechanisms shape Th17 homeostatic responses at the oral barrier.
Authors: Dutzan Nicolas , Abusleme Loreto , Bridgeman Hayley , Greenwell-Wild Teresa , Zangerle-Murray Tamsin , Fife Mark E , Bouladoux Nicolas , Linley Holly , Brenchley Laurie , Wemyss Kelly , Trinchieri Giorgio , Diaz Patricia I , Belkaid Yasmine , Konkel Joanne E , Moutsopoulos Niki M
Published: January 2023

At barrier sites, resident immune cell populations help to maintain tissue homeostasis and function. These cells receive and integrate key signals from the local environment including stromal/epithelial cells and the commensal microbiome. Studies of the skin and gastrointestinal tract have revealed the importance of these signals for the development of host immune response. However, which commensal or tissue-specific cues are important for the immune system at the oral barrier remains minimally explored. Th17 cells have been described as key mediators of immunity at the oral barrier but also essential for periodontitis, a highly prevalent inflammatory pathology that affects the gingiva. In this study we focused in the identification of the mechanisms controlling the induction and regulation of Th17 cells in the gingiva. Our data show that IL-17-producing CD4+ T cells increase with age and their accumulation at the oral barrier occurs independently of commensal colonization. Moreover, we demonstrate that IL-6 elicited by physiological mechanical damage during mastication shapes the function of T cells at the oral mucosa, promoting Th17 differentiation. Finally, we observe that long-term mechanical damage through mastication induces IL-17 mediated bone loss at the gingival barrier. Our data highlight the notion that a variety of signals may be essential to shape the immune responses at different barrier sites, and particularly at the oral cavity, unique mechanisms modulates homeostatic and also pathogenic Th17 responses.
A donor-specific primary monocyte-derived dendritic cell maturation model for high throughput screening of immunostimulatory compounds
Authors: Kelly Erin , Lawrenz Anthony , Wong Alan , Harberts Erin M , Ernst Robert K , Shirazian Afshin , Chen Qin
Published: January 2023

Dendritic cell (DC)-based immunotherapy is a promising treatment alternative for drug-resistant malignancies. Identifying candidate immunostimulatory compounds that can reliably facilitate DC maturation in the context of antigen-specific therapies is a significant hurdle in the development of such immunotherapies. In this study, we present a high throughput in vitro primary cell assay to evaluate dose-dependent DC maturation in a panel of healthy donor primary cells. CD14+ monocytes from four healthy donors were isolated from leukapheresis (Leuko Pak)-derived peripheral blood mononuclear cells (PBMC), differentiated by culture with GM-CSF and IL-4 cytokines, and then stimulated with lipopolysaccharide (LPS), CpG oligonucleotides, or a panel of novel Toll-like receptor 4 (TLR4) ligands. To quantify the resultant maturation, flow cytometry was used to measure the expression of cell surface biomarkers MHC-II, CD40, CD80, CD83, and CD86. While monocytes from all donors responded to the ligands tested, the intensity of activation marker expression varied between donors. LPS-induction of CD83 expression ranged from 27 to 82% of DCs amongst the 4 donors tested, highlighting the importance of multi-donor screening in interpreting quantitative results. In conclusion, a high throughput in vitro primary monocyte-derived DC differentiation assay reveals donor to donor variations that may go unobserved in screening experiments completed in cell lines or single donor primary cells sources. This assay can be used to assist researchers in lead generation, lead optimization, and mechanism of action studies of immunostimulatory compounds.
The role of Krueppel-like factor 2 in B cell activation and plasma cell homeostasis
Authors: Schuh Wolfgang , Schubert Tina , Knipfer Lisa , Wallrapp Antonia , Jäck Hans-Martin
Published: January 2023

Upon contact to antigen, B cells differentiate into memory B cells or antibody secreting short-lived or long-lived plasma cells (PC). The survival of long-lived PCs depends on interaction with survival niches in the spleen or in the bone marrow. However, the molecular mechanisms controlling PC generation, migration, homing and survival are poorly understood. We recently reported that Krueppel-like factor 2 (KLF2) is an important regulator of PC homeostasis. KLF2-deficient B cells express lower levels of homing factors, such as beta7-Integrin and L-Selectin. Numbers of antigen-specific PCs in the bone marrow of immunized KLF2-deficient mice are drastically reduced, indicating that KLF2 plays a crucial role in controlling PC homing and/or survival. Based on these findings we wanted to further characterize the functional impact of KLF2 on B cell activation and PC homeostasis. We observed that KLF2-deficient follicular B cells show a lower activation threshold and differentiate faster into plasma blasts in response to lower LPS concentrations in vitro compared to wildtype B cells. Furthermore, KLF2-deficient B cells proliferate faster as measured by CFSE. Target genes and signalling pathways modulated by KLF2 in B cells and PCs are currently analysed by gene expression profiling. We conclude that KLF2 acts as a quiescence factor in mature B cells by setting their activation threshold. In addition, KLF2 is crucial for PC homeostasis. KLF2-mediated signalling pathways and KLF2-regulated target genes which set the threshold for B cell activation, regulate plasma blast homing and mediate plasma cell survival will be identified by transcriptome analyses. This study was supported by DFG grants FOR832 and TRR130 to H.-M.J. and ELAN grant to W.S.
Role of Trim13 in toll-like receptor 2-mediated NF-κB activation
Authors: Baek Suk-Hwan , Huang Bin , Pei Han Zhong
Published: January 2023

NF-κB regulates the expression of a various genes involved in diverse cellular processes including inflammation and immunity. Ubiquitination is a versatile post-translational modification involved in NF-κB activation of toll-like receptor (TLR) signaling. Here, we demonstrated that Trim13, an E3 ubiquitin ligase, is up-regulated in macrophages upon stimulation with TLR2 ligand. Knock-down of Trim13 attenuated TLR2-mediated production of cytokines/chemokines and formation of foam cells, as well as activation of NF-κB. Trim13 interacts with TRAF6 and potentiates NF-κB activity via ubiquitination of TRAF6. Overexpression of inactive mutant (C10/13A) or RING deletion mutant of Trim13 did not potentiate ubiquitination of TRAF6 or activation of NF-κB. These results suggest that the effects of Trim13 are dependent on its E3 ligase activity. Trim13 used K29-linked polyubiquitin chains for TRAF6 ubiquitination to promote NF-κB activity and thus potentiated activation of TLR2-mediated immune responses. Our data identify Trim13 as a positive regulator of NF-κB activation and suggest that K29-linked polyubiquitination is a specific ubiquitin-linked pattern involved in the control of TLR2 signaling.
APOBEC gene family expression and hallmarks in chronic lymphocytic leukemia
Authors: Chu Charles C , Vergani Stefano , Yan Xiao-Jie , Dhayalan Arvind , Patten Piers E.M. , MacCarthy Thomas , Yuan Chaohui , Barrientos Jacqueline C. , Kolitz Jonathan E. , Allen Steven L. , Rai Kanti R. , Chiorazzi Nicholas
Published: January 2023

The hallmark activity of APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) family of cytidine deaminases, including activation-induced deaminase (AID) and APOBEC3 genes, has been detected in somatic mutation signatures by ultra-deep sequencing of the genomes of many cancers, including chronic lymphocytic leukemia (CLL). The acquisition of these mutations is hypothesized to lead to the progression towards aggressive disease in cancer. To examine this in CLL, we tested if increased APOBEC family member gene expression in CLL cells, as measured by microarray and quantitative real time PCR, correlated with worse patient outcome. Higher levels of AID, APOBEC3B, APOBEC3F and APOBEC3H in CLL cells correlated with worse patient outcome, whereas APOBEC3G did not. Interestingly, higher levels of a truncated splice variant of APOBEC3F tended to correlate with better patient outcome. The expression of truncated APOBEC3F may possibly interfere with APOBEC family member mutation activity. To test mutation activity, CLL cells were activated by transfer into NOD-scid IL2Rγnull mice, a xenograft model of human CLL, and hallmark mutation signatures in the expressed immunoglobulin variable region (IGHV) of CLL cells were analyzed by targeted ultra-deep sequencing. Induced IGHV mutation hallmarks consistent with AID were found. These data support the hypothesis that expression and mutation activity of APOBEC family members, such as AID, in CLL cells could lead to adverse patient consequences.
Antibody repertoire analysis in sera of breast cancer patients using a random peptide microarray differentiates cases from controls with high specificity and sensitivity
Authors: Chapoval Andrei I , Podlesknyh Stepan , Kolosova Evgenia , Anisimov Daniil , Ryzanov Mikhail , Petrova Valentina , Shcherbakov Dmitry , Johnston Stephen , Lazarev Aleksander
Published: January 2023

Breast cancer (BC) is the most common malignancy and the second most common cause of cancer-related mortality in women. Early detection of BC using different biomarkers is a key factor for successful BC therapy. Many studies have shown that serum antibodies (Ab) against tumor-associated antigens (TAAs) could be used as potential biomarkers in the detection of BC. However in some cases the most of informative TAAs for circulating Ab detection, at early stages of BC, might be unknown. Even if informative Ab targets are currently unknown it is possible to analyze Ab repertoire (immunosignature) using random peptide arrays. In the present study, we probe a custom 330,000 random 12-mer peptide microarray with sera from 42 BC cases and 41 controls to develop a diagnostic signature. We determined that differentiation of BC cases from controls with high sensitivity and specificity requires a panel of at least 100–150 peptides. Basic Local Alignment Search Tool (BLAST) searches suggest that the identified peptides have no similarity with known TAAs. However, despite the random nature of the amino acid sequence that were used in microchip design, common amino acid motif is present in the groups of peptides that react with sera Ab from patients with BC but not healthy control. Thus the evaluation of Ab repertoire (immunosignature) using microarray containing peptides with random amino acid sequences has a potential value in the early diagnosis of cancer.
Dietary Early Glycation Products Promote Prostate Tumorigenesis through Modulating Macrophages
Authors: Chen Yingjia , Guo Tai L
Published: January 2023

Glycation products are ubiquitous in food. Moreover, the well-controlled glycation, that results in the production of early glycation products (EGPs), has been proposed as a strategy to improve the physicochemical properties of the food proteins. However, the health effects of EGPs are rarely known. Since there is evidence showing that Western diet (glycation prone) is associated with higher mortality of prostate patients than Prudent diet, we investigated the role of EGPs in prostate tumorigenesis.

EGPs were produced from a whey protein isolate-glucose model system, with non-reacted samples (NR) and advanced glycation end-products (AGEs) as controls. They were used to gavage male C57BL6 mice, and the mice were then subcutaneously injected with TRAMP-C2 cells (a mouse prostate cancer (PCa) cell line) in the flank area. The tumor length, width, and the proliferation index were measured, and the splenic leukocytes were studied. In addition, the mouse TRAMP-C2 cells and two human PCa cell lines LNCaP and PC-3 were cultured with NR, EGPs and AGEs, and LNCaP and PC-3 were further cultured with the conditioned medium generated from human macrophages. The PCa cell proliferation, and the cytokine/chemokine production and iNOS expression by macrophages were determined. The results showed that EGPs promoted prostate tumor growth most in vivo, and PCa cell proliferation only when the presence of macrophages in vitro. In addition, EGPs activated the production of most cytokines/chemokines examined, with IL-10 and MIP-1β unregulated most, and decreased the iNOS expression in macrophage.

In conclusion, EGPs promoted PCa possibly by modulating macrophages to become prostate tumor-associated M2, which produced high levels of IL-10 and MIP-1β.
Enhanced inhibition of chemotaxis by adenosine in peripheral blood CD8+ T cells of head and neck cancer patients
Authors: Chimote Ameet A , Arnold Michael J , Balajthy Andras , Newton Hannah , Wise-Draper Trisha , Conforti Laura
Published: January 2023

Adenosine (Ado) accumulates in high concentrations in solid tumors where it contributes to the failure of the immune cells to eliminate cancer cells. We have previously shown in healthy T cells that Ado acts via A2A receptors and inhibits T cell motility by suppressing KCa3.1 channel function. Herein, we conducted experiments to elucidate the effect of Ado on the chemotactic abilities of peripheral blood T cells (PBTs) from head and neck squamous cell carcinoma (HNSCC) patients. CD8+ PBTs were isolated from HNSCC patients prior to any form of treatment and healthy donors (HD), and activated with CD3/CD28 antibodies. CD69 expression indicated that comparable activation was achieved in HNSCC and HD T cells. Experiments to measure chemotaxis towards a CXCL12 gradient were performed on T cells suspended in a collagen matrix, which resembles the fibrous network within solid tumors. Chemotaxis was evaluated by determining the center of mass along the Y-axis (Y-COM), which reflects the location of the cells at the end of the experiment and is indicative of their ability to move towards the chemokine gradient. PBTs of HNSCC patients and HDs underwent comparable chemotaxis in the CXCL12 environment. Next, we compared their chemotaxis when Ado was added to the chemokine gradient. Ado was responsible for a significantly greater reduction in the Y-COM in HNSCC (~82%, n=16) compared to HD (~26%, n=7; p=0.012). This effect was mimicked by the A2A receptor agonist CGS21680 and was reversed by the A2A receptor antagonist SCH5826. Our data suggest that Ado acts via A2A receptors and suppresses chemotaxis in HNSCC patients to a greater degree than their healthy equivalents, which may limit their ability to effectively penetrate the tumor.
Bacterial modulation of noncanonical inflammasome.
Authors: Vanaja Sivapriya Kailasan , Nair Meera Surendran , George Mariam , Rathinam Vijay
Published: January 2023

Sensing of microbial products by pattern recognition receptors to mount effective antimicrobial responses is an evolutionarily conserved immune strategy. Cytosolic LPS sensing by the noncanonical inflammasome via caspase-11 and the ensuing IL-1 and cell death responses are emerging as a key host defense strategy that play a crucial role in the clearance of infectious agents. A strong selection pressure from the host such as this drives pathogens to develop strategies to actively antagonize or evade inflammasome responses. However, it is not known if bacteria antagonize noncanonical inflammasome pathway. This study addresses this knowledge gap and examines the modulation of noncanonical inflammasome by bacteria utilizing Enterohemorrhagic E. coli (EHEC) as a model organism. Supporting the idea of inflammasome suppression by EHEC, our results indicate that compared to E. coli K12, an avirulent laboratory strain of E. coli, EHEC induces substantially weaker cell death and IL-1 secretion. Importantly, by screening a library of EHEC mutants lacking known and putative virulence factors, we found that a mutant that lacks an EHEC-specific genomic island triggered markedly higher cell death and IL-1 responses compared to wild-type EHEC revealing that this genomic island harbors a suppressor of noncanonical inflammasome. Altogether, this study attributes an inflammasome modulatory function to an EHEC genomic island, whose function was previously unknown. Identifying novel mechanisms by which pathogenic bacteria silence noncanonical inflammasome is crucial as it may aid in designing novel therapeutic approaches against Gram-negative infections.
Analysis of immunogenicity of different sizes of HER2/neu antigen
Authors: Jeon Insu , Seo Hyungseok , Park Myung Hwan , Shin Kwang-Soo , Lee Jeong-mi , Choi Jeongwon , Song Boyeong , Koh Choong-Hyun , Bae Eun-Ah , Kang Chang-Yuil
Published: January 2023

One of the most important factors of cancer immunotherapeutic vaccine is to induce tumor antigen specific T cell response. Prior to considering how to induce T cell response efficiently, researchers should select tumor antigen with careful consideration because some tumor antigen show oncogenic property that may lead to safety problems. To reduce these concerns, tumor antigens are used in artificially mutated or truncated form so that oncogenic function would be eliminated. On the other hand, as the size of antigen determines T cell receptor epitope repertoire, it is important to maintain the original size as much as possible. In this study, we tested immunogenicity of four different sizes of HER2/neu antigens in B cell and monocyte based vaccine models. In murine models, we observed antigen specific T cell response in the antigen size dependent manner and antigen specific antibody response in the antigen expression dependent manner. In addition, all of four different sizes of antigen showed suppression of established tumor growth but the efficacy was depending on the size. Moreover, in human PBMC studies, the bigger antigen showed better CD4 and CD8 T cell responses than the others. These data demonstrate that if the oncogenic potential of antigen could be eliminated, the biggest form of antigen would show better therapeutic effects.
Measuring fg/mL Concentrations of Cytokines in Cerebrospinal Fluid (CSF)
Authors: Georlette Daphne , Suschak Jessica , Aghvanyan Anahit , Navaratnam Manjula , Lockney Dustin , Biesso Arianna , Gardner Louis , Bartlett Dan , Porter Erica , Tamot Banita , Shukla Animesh , Miller Thomas , Mathew Anu , Wheeler Sarah E , Stengelin Martin , Wohlstadter Jacob N
Published: January 2023

Measuring low-abundance cytokines in CSF may be useful when studying brain and nervous system disorders. We recently developed novel immunoassays with fg/mL and sub-fg/mL detection limits that enable quantitation of low-abundance biomarkers in CSF. S-PLEXSM assays, utilizing MSD’s MULTI-ARRAY® electrochemiluminescence technology, were developed and analytically characterized and the concentrations of cytokines in CSF were measured using these assays.

Assays for the following cytokines were used: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, IL-21, IFN-γ, GM-CSF, TSLP, and TNF-α. The lower limits of detection achieved for S-PLEX assays for these analytes ranged from ~0.2 fg/mL to ~50 fg/mL. Spike recovery and dilution linearity were between 80% and 120%. Depletion experiments demonstrated specificity for each analyte. Cytokine levels in the majority of apparently healthy CSF samples were measurable for 8 out of the 12 analytes tested.

We have developed a next-generation assay format that is 100 to 1,000 times more sensitive than the current limits of standard ELISA technology. This increased sensitivity enables accurate quantitation of cytokine levels in the CSF of apparently healthy donors. These assays will allow for a better understanding of the role these cytokines play in brain and nervous system disorders.
Missing KIR ligand in the host-versus-graft (HvG) direction is associated with second cell infusion in children with NK+ severe combined immunodeficiency (SCID) receiving hematopoietic stem cell transplantation.
Authors: Wahlstrom Justin , Bender Henry , Yin Ziyan , Logan Brent , Rajalingam Raja , Dvorak Christopher C. , Haddad Elie , Cowan Morton J
Published: January 2023

Functional NK cells are thought to mediate graft rejection in allogeneic hematopoietic stem cell transplantation (HSCT) for SCID. We hypothesized that missing KIR ligand (MKL) in the HvG direction mediates NK alloreactivity and is associated with increased rates of 2nd cell infusion requirement in NK+ SCID patients treated with HSCT.

Data collected by the Pediatric Immune Deficiency Treatment Consortium (PIDTC) from 1982 to 2012 were reviewed. A total of 174 NK+ SCID transplant recipients were identified. Of these, HLA-B typing was available for 172 recipient/donor pairs, and HLA-C typing was available for 131 pairs. MKL was identified if Bw4, C1, or C2 inhibitory ligands were present in the recipient but not the donor.

MKL was identified for Bw4 in 18 of 172 donors; for C1 in 11 of 131 donors; and for C2 in 9 of 131 donors. Combined Bw4, C1, and C2 MKL analysis was feasible for 129 transplants; 38 of these donors were missing one or two ligands in the HvG direction. Missing Bw4 ligand alone was not associated with decreased EFS or increased need for 2nd cell infusion. The incidence of 2nd infusion at 2 years was significantly lower for pairs without missing HLA-B or HLA-C ligands (19.3%, 95% CI 11.8% – 28.2%) compared to those with MKL (28.9%, 95% CI 15.5% – 43.9%; Gray’s p=0.04). EFS was 63.2% in patients with MKL (95% CI 45.9% – 76.3%) and 75.2% in those without MKL (95% CI 64.7% – 82.9%; Gray’s p=0.05).

Missing KIR ligand in the HvG direction is associated with 2nd cell infusion in HSCT for NK+ SCID and may represent a mechanism for NK alloreactivity in this setting. Multivariate analysis and biologic studies of NK reactivity in SCID patients are needed to better understand the role of recipient NK cells in HSCT.
Deletion of AMP-activated protein kinase (AMPK) in donor T cells mitigates graft-versus-host disease without impacting graft-versus-leukemia effects
Authors: Byersdorfer Craig A , Beezhold Kevin J , Moore Nathan , Chiaranunt Pailin , Brown Rebecca Audrey
Published: January 2023

Allogeneic blood and marrow transplantation cures a variety of hematologic disorders, but its broader use remains limited by acute graft-versus-host disease (GVHD), where donor T cells attack and destroy host tissues. To explore this process mechanistically, we elucidated the role of AMP-activated protein kinase (AMPK), an intracellular energy sensor, in GHVD-causing T cells. Alloreactive T cells increased phosphorylation of AMPK as early as day 3 post-transplant (8-fold higher than naïve cells, p=0.0003), while T cells lacking AMPK caused less GVHD in two separate models. Importantly, AMPK−/− cells demonstrated equivalent cytotoxicity, with equal clearance of p815 leukemia cells, in graft-versus-leukemia experiments.

We next sought to explain our lower rates of GVHD. Surprisingly, fat oxidation, T cell autophagy, and mTOR signaling were identical between wildtype (wt) and AMPK−/− cells. However, the number of donor T cells decreased following transplantation of AMPK−/− cells (3.15 ±0.49×106vs. 1.87 ±0.53×106, p=0.0006, wt vs. AMPK−/− respectively), with fewer CD3+ cells infiltrating into GVHD target organs (e.g. liver, 206.1 ± 26.72 vs. 83.58 ± 11.10 CD3+ cells/hpf, p=0.0001). In contrast, both the percentage and total number Treg increased in recipients of AMPK−/− cells (0.85 ±0.32×104 vs. 1.69 ± 0.34×104, wt vs. AMPK−/−, p=0.004).

Thus, deletion of AMPK decreases GVHD severity but spares anti-leukemia responses. This occurs through impaired effector T cell survival, preservation of Tregs, and reduced infiltration of CD3+ cells into target organs. From these data, we conclude that targeting AMPK in donor T cells represents an attractive strategy to mitigate GVHD while still preserving graft-versus-leukemia effects.