bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycolipid-peptide vaccination induces liver-resident memory CD8+ T cells that protect against malaria
Authors: Holz Lauren , Chua Yu Cheng , Anderson Regan J , Draper Sarah , Compton Benjamin , Li Jasmine , Kedzierski Lukasz , Wang Zhongfang , Osmond Taryn , Farrand Kathryn , Bertolino Patrick J , Bowen David , Cozijnsen Anton , Mollard Vanessa , McFadden Geoffrey , Caminschi Irina , Lahoud Mireille , Kedzierska Katherine , Turner Stephen John , Godfrey Dale , Hermans Ian , Painter Gavin , Heath William R
Published: January 2023

Liver resident-memory CD8+ T cells (TRM cells) provide important protection against liver-stage Plasmodium infections. Here, we show that a glycolipid-peptide conjugate vaccine designed to be cleaved in vivo to release the NKT cell agonist a-galactosylceramide (a-GalCer), together with a CD8+ T cell malarial epitope, efficiently induced intrahepatic malaria-specific T cells expressing canonical markers of liver TRM (CD69, CXCR6 and CD101). While sterile protection was achieved in a proportion of mice using a single vaccine dose, a combined prime-boost regimen induced higher numbers of liver TRM cells and more robust protection. Improved liver TRM cell generation and sterile immunity using a single dose of the vaccine were obtained in all mice by extending the C-and N-terminal residues of the minimal epitope, and by modifying the linker to enhance a-GalCer release. Our findings describe an ideal synthetic vaccine platform to generate large numbers of liver TRM cells that is potentially valuable for controlling a variety of hepatotropic infections.
Adiposity augments toxicity of mouse cytomegalovirus
Authors: Wang Ziming , Dunai Cordelia , Le Catherine , Murphy William J
Published: January 2023

Obesity, a meta-inflammatory state defined by BMI≥30kg/m2, is reaching pandemic proportions. Generally, the impact of obesity on immune responses is poorly understood. Obesity is associated with chronic low-grade systemic pro-inflammatory cytokines. Similar to aging, obesity can affect immune responses and cause susceptibility to infection and pathology. Cytomegalovirus (CMV) is a prevalent herpes virus that can remain asymptomatic for years but causes significant morbidity and mortality in immunosuppressed patients. Our goal was to evaluate the impact of obesity on immune responses to CMV infection with a focus on toxicity. We reported that systemic immunotherapy results in elevated levels of pro-inflammatory cytokines in obese mice, leading to TNF-mediated pathological responses and death. We hypothesized that obesity will augment toxicity in mouse CMV (MCMV) infection as a consequence of heightened pro-inflammatory cytokine production. C57BL/6 and Balb/c were fed either a 60% fat (DIO) or 10% fat (Control) diet starting at 6-weeks old until 6-months old. DIO mice had significantly more subcutaneous and visceral adipose tissue than control mice assessed by MRI. Although ob/ob mice were fed a regular diet, they were significantly fatter than WT mice. Mice (DIO vs control and ob/ob vs WT) infected with 1–5×104 p.f.u. MCMV were assessed for clinical symptoms, serum cytokines, and survival. We observed that 50% of both DIO and ob/ob mice succumbed starting 2 days post-infection, which was associated with increased pro-inflammatory cytokine production. The augmented toxicity induced by MCMV in obese models demonstrates that adiposity is a critical factor in immune-mediated pathological responses to infections.
A PML-Synonymous Mutation in MuPyV Results in Viral Escape from a Neutralizing Monoclonal Antibody
Authors: Lauver Matthew D , Ren Heather , Goetschius Daniel , Hafenstein Susan , Lukacher Aron
Published: January 2023

Progressive Multifocal Leukoencephalopathy (PML) is a fatal demyelinating brain disease caused by JC polyomavirus (JCPyV). Most individuals are asymptomatically infected with JCPyV, but those with immunological perturbations are at risk of developing PML. PML pathogenesis is closely linked with the emergence of JCPyV mutants bearing mutations in regions of the VP1 major capsid protein that bind host cell receptors; these mutations may mediate immune escape or result in altered cell tropism/neurovirulence. A frequent PML mutation in VP1, S269F, is located in the receptor-binding pocket of the virus capsid. To investigate the effects of the S269F mutation on viral infection and the immune response, we utilized mouse polyomavirus (MuPyV) as a natural pathogen-host model of JCPyV infection. We generated a MuPyV mutant containing a V296F substitution in VP1, which maps to the S269F mutation in JCPyV, to ask whether this mutation on immune evasion and host cell tropism. The MuPyV DNA genome incorporates cellular histones; thus we generated infectious fluorescent MuPyV by infecting a host cell line expressing an H2B histone-GFP protein. Using this fluorescently-labeled virus, we determined that a VP1 monoclonal antibody (mAb), which neutralizes wild type virus, binds the V296F mutant, but is unable to block viral attachment and infection. We are currently generating cryo-EM reconstructions to visualize the interaction of this mAb with the wild type and V296F mutant viruses. This data suggests that this PML-synonymous mutation in MuPyV may allow escape from antiviral humoral immunity.
Whole Genome Evolutionary Fingerprinting Suggests Novel Primate Gene Functions in Evolutionary Arms Races
Authors: Tsu Brian V , Murrell Benjamin , Daugherty Matthew , Wertheim Joel
Published: January 2023

Previous positive selection analyses on primate host restriction factors have helped to elucidate differences in primate adaptation to viral infection. However, many of these studies have been focused on small gene families and genes known or suspected to play a role as primate host restriction factors. With the genome sequencing of numerous primate species, we are now capable of comparing the evolutionary profiles of primate genes across the whole genome. Using these evolutionary profiles as guides, we characterize evolutionary similarities and differences between known primate restriction factors and sets of other genes with diverse functions to provide insight on new candidate restriction factors and the biological pressures that affect them.
Interaction between HIV-1 integrase and viral RNA drives proper virion morphogenesis and is necessary for successful infection
Authors: Elliott Jennifer L , Eschbach Jenna E. , Lawson Dana Q. , Puray-Chavez Maritza , Townsend Dana , Li Wen , Koneru Pratibha , Engelman Alan N. , Kvaratskhelia Mamuka , Kutluay Sebla B.
Published: January 2023

Recent findings indicate a key role for the HIV-1 integrase (IN) enzyme in proper virion morphogenesis which involves its binding to viral RNA. Mutations within the IN C-terminal domain potently block IN-RNA interactions leading to the generation of non-infectious particles with “eccentric” morphology with the viral genomic RNA mislocalized outside the capsid core. Despite containing all the components necessary for replication, such particles cannot successfully infect target cells. Interestingly, mutations within IN at residues outside the RNA-binding site cause similar defects. To determine how multiple mutations within IN can lead to similar defects in virion morphology and viral replication, we characterized a panel of IN mutant viruses for defects in the viral lifecycle. Human monocyte-derived macrophages or cultured cells were infected with IN mutant viruses and viral RNA, DNA, and proteins were analyzed by either immunoblot, qPCR, or confocal microscopy. All of the mutant viruses screened were severely attenuated in infectivity, and encountered a block early after entering target cells. Viral RNA and IN were lost from target cells, and reverse-transcribed viral DNA did not accumulate. These findings support a model in which the viral capsid core is critical for protecting entering viral RNA and replicative enzymes from the host environment.
NLRX1 Facilitates HIV-1 Infection of CD4 T Cells by Promoting Oxidative Phosphorylation
Authors: Guo Haitao , Wang Qi , Wang Li , Rampanelli Elena , Chen Xian , Chanda Sumit , Su Lishan , Ting Jenny P
Published: January 2023

The nucleotide-binding, leucine-rich-repeat-containing protein, NLRX1 is recognized as a host factor which promotes HIV-1 infection of myeloid cells by suppressing HIV-1 reverse transcribed DNA induced and STING-mediated innate immune response. However, the function of NLRX1 in HIV-1 infection of CD4 T cells, the major cells responsible for HIV-1 replication in vivo, is still unrevealed. Here we found NLRX1 still acted as a host factor required for HIV-1 infection of the human T cell line, primary CD4 T cells, and CD4 T cell-reconstituted humanized mice through a mechanism involved in cell intrinsic pathways but not innate immune cytokines. In the perspective of virology, we found NLRX1 facilitated both of the integration of HIV-1 cDNA and HIV-1 LTR-mediated viral gene transcription. By using SILAC-based quantitative proteomics, we characterized that HIV-1 infection-induced expression of genes involved in the electron transport chain (ETC) of mitochondria through an NLRX1 dependent mechanism. Consistent with the proteomics data, HIV-1 infection led to increased oxidative phosphorylation in Jurkat T cell line but not in the counterpart containing shRNAs against NLRX1. In addition, inhibition of oxidative phosphorylation by mitochondrial complex I inhibitors suppressed HIV-1 infection in Jurkat cells, while forcing expression of genes involved in ETC rescued the deficiency of HIV-1 replication in NLRX1-deficient cells. Importantly, treating humanized mice with FDA-approved drug, metformin which targets mitochondrial complex I, significantly reduced HIV-1 viral load. This study demonstrated the potential of targeting NLRX1 and applying metformin in suppressing HIV-1 replication in patients.
Sphingosine Kinase 2 mediates LCMV-induced CD4+ T cell suppression and instigates viral persistence while preventing immunopathology
Authors: Studstill Caleb J , Pritzl Curtis J. , Seo Young-Jin , Kim Dae Y. , Xia Chuan Z. , Wolf Jennifer J , Vijayan Madhuvanthi , Hahm Bumsuk
Published: January 2023

Viruses often establish persistent infections by causing dysfunctional T cell responses. Little is known about the role of sphingosine kinase 2 (SphK2) in the immune response to viral infections; even though, its enzymatic product, sphingosine 1-phosphate, is well-known to regulate versatile cellular processes. In this work, we demonstrate that during lymphocytic choriomeningitis virus clone 13 (LCMV Cl 13) infection in mice, SphK2 functions to limit CD4+ T cell responses, which aids in the establishment of virus-induced immunosuppression and viral persistence. The infection of SphK2-deficient (SphK2−/−) mice with LCMV Cl 13 resulted in kidney disease and ultimately mortality, which was not observed with acute infection by the Armstrong strain of LCMV. Following infection, SphK2−/− mice were shown to have increased LCMV-specific CD4+ and CD8+ T cell responses. Depletion of CD4+ or CD8+ T cells prevented the infection-induced death of SphK2−/− mice, which indicates T cell-mediated immunopathology. With the use of LCMV epitope-specific TCR transgenic mouse lines for adoptive transfer studies, SphK2 was shown to have intrinsic negative function in CD4+ T cells, but not CD8+ T cells. Furthermore, SphK2−/− CD4+ T cells were able to promote endogenous, virus-specific CD8+ T cell responses. Importantly, oral treatment of LCMV Cl 13-infected mice with an SphK2-selective inhibitor increased the number of LCMV-reactive CD4+ and CD8+ T cells, and led to the accelerated termination of LCMV Cl 13 persistence, without causing mortality. Our results suggest that transient SphK2 inhibition is a promising novel immunotherapeutic strategy for the control of persistent viral infections.
A Brazilian Zika virus isolate suppresses type I interferon to establish a sustained infection in immunocompetent mice
Authors: Pardy Ryan D , Richer Martin J
Published: January 2023

Zika virus (ZIKV) is a mosquito-borne pathogen that recently gained international attention following a major epidemic in South and Central America. The most striking aspect of this epidemic was the novel association of ZIKV infection with neurological symptoms, such as fetal microcephaly. In addition, ZIKV may persist in humans for 15- and 42-days post-symptom onset in serum and semen, respectively. Therefore, our objective was to investigate whether recent ZIKV isolates have an enhanced capacity to evade host immunity. To address this objective, we have established an immunocompetent mouse model of ZIKV infection, in which infection with pre-epidemic, early Asian lineage ZIKV isolate (ZIKV-CDN) induces robust type I interferon (IFN) production and prototypical Th1 CD4 and effector CD8 T cell responses. We are now comparing this baseline to the immune response induced by a ZIKV isolate from the Brazilian epidemic (ZIKV-BR). Our data demonstrate a dramatic suppression of type I IFN production during ZIKV-BR infection, which correlates with a severe reduction in the CD8 T cell response. Co-infection reduces the magnitude of the CD8 T cell response when compared to infection with ZIKV-CDN alone, suggesting ZIKV-BR actively interferes with the host immune response. As a result, ZIKV-BR establishes a sustained infection, with high viral loads detected as late as 7 days post-infection. Future studies will aim to determine why less type I interferons are produced, and the impact of ZIKV-BR infection on the CD4 T cell response. Together, our data suggest that contemporary strains of ZIKV have evolved novel ways of countering host immunity, which could explain, at least in part, the increased pathogenicity observed in recent outbreaks.
The Influence of The Lung Virome on Pulmonary B cells
Authors: Aihara Fumiaki , Breen Michael P. , Feng Feng , Fearns Rachel , Mizgerd Joseph P. , Kepler Thomas B.
Published: January 2023

The human microbiome is a complex and diverse environment that is implicated in human health and disease. Major locations that host a microbiome include the gut, respiratory system, and the urogenital tracts. While there have been steady advances of the effect of our commensal flora in regions such as the gut microbiome, the impact of the pulmonary microbiome, specifically the viral arm of the microbiome (virome), remains unclear and under-researched. Recent studies have shown that pulmonary viruses can influence disease susceptibility to the benefit, or detriment, to the host. Despite increasing evidence that the lung virome influences human health through the immune system, the direct effects on the host immune system remain unclear. We hypothesize that the chronic presence of the lung virome influences the pulmonary B cell repertoire. The constant exposure to pulmonary virus can generate a unique B cell repertoire independent of peripheral B cells that is tailored specifically towards pulmonary viruses. To address this hypothesis, we have utilized a single-cell B cell culture system to amplify human lung-derived memory B cells (MBCs) from limited human donor samples. By analyzing these MBCs at the genetic level by RNA sequencing and protein level by antigen affinity from secreted antibodies, we can begin to understand the impact of the lung virome on the pulmonary B cell repertoire.
Blimp-1 transcriptionally induced by growth factors, TNF-α and TLR ligands inhibits keratinocyte migration and inflammation but enhances cell migration in squamous cell carcinoma
Authors: Lin Wan-Wan , Huang Duen-Yi
Published: January 2023

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor, and plays a crucial role in the regulation of development and functions of various immune cells. Currently, there is limited understanding about the regulation of Blimp-1 expression and cellular functions in keratinocytes and cancer cells. The aim of the study was to investigate the regulation and functional link between EGFR and Blimp-1 in human epidermal keratinocytes and squamous cell carcinoma (SCC). We found that EGF, PMA, TNF-α, LPS (TLR4 activator) and polyIC (TLR3 activator) can upregulate the protein and mRNA levels of Blimp-1 in NHEK and/or HaCaT cells. This effect of EGF was dependent on PKC, p38, and ERK activation, and Syk is also involved in the action of PMA. Additionally, the stability of Blimp-1 protein was under the control of the proteasome and lysosome degradation systems. PMA, LPS and polyIC also can activate EGFR and Syk in HaCaT cells. In addition, Blimp-1 knockdown enhanced the EGFR-mediated IL8, CXCL5 and IL6 gene expression and keratinocyte migration, but reduced the EGFR-mediated suppression of differentiation marker K10. On the other hand, EGF, TNF-α, TGF-β and PMA could also upregulate Blimp-1 expression in SCC, and EGFR activation by TNF-α and PMA was observed. Unlike the action of Blimp-1 in keratinocytes, Blimp-1 silencing reduces SCC cell migration either in the absence or presence of TNF-α and PMA. Taken together, Blimp-1 can be upregulated by several growth factors, innate immune receptors and PKC signaling pathway. Blimp-1 is a negative regulator of EGF-induced inflammation and migration in keratinocytes, but mediates cell migration in SCC.
Follicular dendritic cell migration is promoted by the expression of matrix metalloproteinase 3 through TNFα-ERK1/2-AP-1 signaling
Authors: Yong-Woo Kim , Pak Hyo-Kyung , Park Chan-Sik
Published: January 2023

Follicular dendritic cells (FDCs) are the major stromal components in the germinal center and are developed and maintained by lymphotoxin α1/β2 and tumor necrosis factor-α (TNFα). These cells have the pivotal role in high-affinity antibody production. However, many functions of FDCs are still unknown, such as its migration. Here, we demonstrate for the first time that TNFα increased matrix metalloproteinase 3 (MMP3) expression in human FDCs and induced migration of these cells. Normal human germinal centers expressed of MMP3, and MMP3 in the FDCs are increased by TNFα. The induction of MMP3 in FDCs is dependent on ERK1/2 activation. TNFα stimulation induced the promoter activity of the AP-1 binding sites in Mmp3 proximal promoter region. The induced expression of MMP3 increased FDC migration and the reduction of MMP3 expression decreased FDC migration. Overall, we identified a novel mechanism for the expression of MMP3 in FDCs to modulate the migration, a process of lymphoid follicle development. Lymphoid follicles are observed in the lesion of many autoimmune and autoantibody-associated diseases, thereby control of TNFα-mediated migration of FDCs may suppress those diseases.
TSPAN33 regulates endocytosis and migration of human B lymphocytes by regulating the plasma membrane tension
Authors: Navarro-Hernandez Itze Cecilia , Acevedo-Ochoa Ernesto , López-Ortega Orestes , Fragoso-Soriano Rogelio , Galván-Hernández Arturo , Antillón Armando , Ortega-Blake Iván , Hernández-Hernández José Manuel , Maravillas-Montero José Luis
Published: January 2023

B lymphocytes develop different immune-related functions apart from differentiating into antibody-secreting cells. These processes are triggered by external stimuli that activate them and induce changes in the physicochemical properties of their plasma membrane. These changes are regulated by the formation of discrete lipid-bilayer compartments, including the tetraspanin-enriched microdomains. Tetraspanins compose a superfamily of transmembrane proteins that establish lateral associations with other molecules, determining its activity. In this study, we identified TSPAN33 as an active player during B lymphocyte cytoskeleton and plasma membrane-related phenomena. By using an overexpression model of TSPAN33 in human Raji cells, we located this protein on different membrane organelles including Golgi and intracellular vesicles as observed by confocal microscopy. Additionally, these cells displayed diminished phagocytic and cell adhesion properties, due to aberrant surface expression of integrins. This phenotype also prove to enhance 2D and 3D migration in response to CCL21. Strikingly, when we evaluated the mechanical plasma membrane response of the cells using atomic force microscopy, during fibronectin-induced spreading, we found that TSPAN33 expression inhibited normal changes in membrane tension and roughness parameters. For that reason, we generate TSPAN33 knock-down cells that showed enhanced adhesive and phagocytic capacities when compared with control cells. Altogether, our data indicate that TSPAN33 represents an inhibitory element of the adhesion and migration of B cells, suggesting also a novel implication of this protein in the regulation of mechanical properties of the plasma membrane.
Selective Recruitment of Lethal Pro-inflammatory Macrophages in Sepsis by MIF but not D-DT (MIF-2)
Authors: Tilstam Pathricia Veronica , Schulte Wibke , Holowka Thomas , Kim Bong-Sung , Piecychna Marta , Pantouris Georgios , Lolis Elias , Leng Lin , Bernhagen Juergen , Bucala Richard
Published: January 2023

Sepsis is a leading cause of mortality with an estimated global incidence of 30 million cases annually. Current paradigms emphasize the role of an overwhelming or dysregulated inflammatory response in sepsis lethality, but to date interventions aimed at particular inflammatory pathways have been unsuccessful. Macrophage migration inhibitory factor (MIF) is of interest because MIF promoter polymorphisms predict mortality in different infections and anti-MIF antibody improves survival in experimental models when administered eight hours after infectious insult. The recent description of a second MIF superfamily member, D-dopachrome tautomerase (D-DT/MIF-2), prompts closer investigation of MIF-dependent responses. This study provides new insight into the pathophysiology of polymicrobial sepsis by revealing a direct role for MIF, but not its close structural homolog D-DT/MIF-2, in activating and recruiting a lethal macrophage subset. We subjected Mif −/− and Mif-2−/− mice to polymicrobial sepsis and observed a survival benefit with Mif but not Mif-2 deficiency. Survival was associated with lower levels of small peritoneal macrophages (SPMs) that, in contrast to large peritoneal macrophages (LPMs), are recruited to site of peritoneal inflammation during sepsis. LPMs produce higher quantities of MIF than SPMs, but SPMs express higher levels of inflammatory cytokines and MIF receptors CD74 and CXCR2. Adoptive transfer of SPMs into Mif −/− hosts abrogated the protective effect of Mif deficiency. Notably, MIF-2 lacks the pseudo-(E)LR motif present in MIF that mediates CXCR2 engagement and SPM migration, supporting MIF’s selective role in the recruitment and accumulation of lethal inflammatory SPMs.
A critical role for IL-37 in regulatory T cell maintenance and CD4+ T cell inhibition
Authors: Osborne Douglas Grant , Domenico Joanne , Dinarello Charles A , Fujita Mayumi
Published: January 2023

Regulatory T (Treg) cells express the transcription factor Foxp3 and play a vital role in suppressing immune responses and maintaining tolerance and homeostasis. Foxp3 expression is induced and maintained in Treg cells during thymic development and adaptive immune insults through three signaling pathways including TCR signaling and costimulation, IL-2 signaling, and TGF-β signaling, as well as Foxp3 expression itself. Recent studies, however, have suggested that these signaling pathways involved in the induction and maintenance of Foxp3 expression in the thymus and during activation are more dispensable in a steady state. To identify additional intracellular signaling molecules that induce/maintain Foxp3 expression and support Treg cell homeostasis and stability, we have screened proteins interacting with the three Foxp3 transcription factors NFAT, Stat5 and Smad3 for their expression in Treg cells and regulation of Foxp3 transcription. Using this protein screen we discovered IL-1 family member IL-37 plays a significant role in Foxp3 expression. By using human T cells from healthy donors and transgenic mice expressing IL-37, we uncovered a novel role for endogenous (expressed) and extracellular (secreted) IL-37 in Foxp3 expression and CD4+ T cells immunity. Treg cells that expressed IL-37 had elevated Foxp3 expression and were more suppressive in vitro and in vivo compared to Treg cells lacking IL-37. Extracellular IL-37, secreted by Treg cells, directly inhibited the canonical T cell signaling pathways through surface receptors. These results characterize an important mechanism for Treg directed immune suppression and Foxp3 expression.
Development of a high sensitivity 10-plex human cytokine assay using Simoa Planar Array technology
Authors: Lambert Jeremy , Zarozinski Christopher , Johnson Joseph , Yan Yu-xin , Rissin David , Stadler Patrizia , Ball Andrew
Published: January 2023

Approaches to quantify multiple cytokines have potential to improve understanding of mechanisms and progression of disease, including cancer pathogenesis. However, in healthy humans many cytokines are present at levels below the limits of quantification for established multiplex assays.

We developed the Simoa Planar Array to enable protein quantification in biological samples with greater sensitivity than traditional ELISAs. In this study we applied Simoa Planar technology to simultaneous multiplex detection of cytokines, using minimal sample volumes, to achieve sensitivity greater than established multiplex immunoassays.

Capture antibodies were printed in a circular pattern in microwells, with biotinylated detector antibodies and chemiluminescent detection used in a sandwich format. Antibody pairs were screened for ability to quantify analytes with minimal cross-reactivity. Pairs targeting IFNγ, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-22 and TNFα were chosen, then assay conditions optimized to minimize matrix interferences. Analytical validation was performed, and circulating cytokine levels measured in normal human serum and plasma.

12.5 μL of human serum/plasma provided sufficient sample volume per replicate. Sub-picomolar LOD was achieved for all analytes; LLOQs ranged from 0.1 – 1.4 pg/mL. In normal human serum and plasma, > 80% normal samples were quantifiable (above LLOQ) for most analytes. Analytical validation confirmed sensitivity, precision, specificity and dilution linearity.

The 10-plex Simoa Planar assay described here combines ultra-sensitivity with powerful multiplexing, enabling simultaneous detection of 10 cytokines in normal biological samples with superior sensitivity.
Bone Morphogenic Protein Receptor 1α and its ligands as a signaling circuit modulating immune response
Authors: Kraj Piotr , Kuczma Michal , Simms Colin , Pietrzak Maciej , Ignatowicz Leszek , Muranski Pawel , Browning Lauren
Published: January 2023

Despite increased understanding of how the transforming growth factor-β (TGF-β) regulates T cell functions, the immunomodulatory roles of other members of the TGF-β cytokine family, especially bone morphogenetic proteins, remain largely unknown. We have found that Bone Morphogenic Protein Receptor 1α (BMPR1α, Alk-3) expressed by activated effector and regulatory CD4+ T cells, modulates functions of both of these cell types by promoting generation of adaptive TR and inhibiting generation of Th17 cells from naive CD4+ T cells. Mice where BMPR1α is deleted in T cells (BMPR1αT− mice) had a decreased proportion of thymic derived TR cells. Activation of BMPR1α deficient (BMPR1α−) CD4+ T cells leads to generation of Th1/Th17-like effector cells expressing high levels of inflammatory cytokines, including IFN-γ, IL-17 and TNF family proteins. Effector CD4+ T cells activated by dendritic cells expressing BMP inhibitors expressed lower levels of PD1. Immunization of BMPR1αT− mice induced vigorous inflammatory response and mice were able to better control B16 melanoma tumors. Tumor infiltrate had very few TR cells and higher proportion of CD8+ T cells compared to tumors in wild type mice. These data demonstrate that TGF-β mediated immunosuppression in the course of tumor growth can be bypassed by inhibition of BMPR1α signaling.

Transcriptome analysis of wild type and BMPR1α− CD4+ Th cells revealed differential expression of transcription factors, cytokines and cytokine receptors and signaling molecules essential to establish regulatory networks supporting Th17 or TR cells. This suggests that BMPR1α is a potential target to augment effector Th cell responses.
Development of group-3 innate lymphoid cells via Tox2-dependent pathway in gut
Authors: Das Arundhoti , Wang Yueqiang , Lu Xiaoxiao , Harly Christelle , Kaye Jonathan , Bhandoola Avinash
Published: January 2023

Innate lymphoid cells (ILCs) are immune cells that lack specific antigen receptors but possess similar effector functions as T cells. Interestingly, ILCs and T cells express many of the same transcription factors. One such factor is TOX, a sequence non-specific nuclear transcriptional regulator that is crucial for development of both CD4 T cells and ILCs. TOX-deficient mice have significantly reduced number of early innate lymphoid progenitors (EILP) and ILC progenitors (ILCP). Consequently, the mature NK cells, ILC1, ILC2 and ILC3 subsets are greatly reduced in Tox−/− mice. Interestingly ILC3 in gut are not affected by the absence of TOX. We have discovered that TOX2, another member of the TOX family of HMG-box proteins, is highly expressed in ILC precursors. We generated Tox2−/− mice and found they have reduced ILC3 in gut; however, ILC3 cells in spleen or lymph node are intact. We also observed putative ILCP in fetal gut were significantly reduced in Tox2−/− mice. Hence our results indicate the existence of at least two pathways of ILC development: a central pathway in bone marrow and fetal liver that is TOX dependent; and a peripheral pathway of development of ILC3 in gut that requires TOX2.

Additionally, we observed that TOX2 is not required for development of T cells. However, Tox2−/− mice showed higher frequency of PD1 expressing CD8 effector memory cells. Tox2−/− mice also showed greater Th1 responses in vitro. Together, our results suggest that TOX is important centrally, for T cells and ILCs; whereas TOX2 is important for peripheral ILC3 generation or maintenance, and may further play an important role in effector T cells in tissues.
Distinct CD28 signaling requirements determine the alternative fates of thymic agonist selection
Authors: Watanabe Masashi , Alkhaleel Farrah , Hodes Richard J
Published: January 2023

During thymic T cell development, T cells strongly reactive to self-Ag are eliminated from the conventional T cell repertoire by clonal deletion. Alternatively, however, a number of T cell subsets are positively selected by strong recognition of self-Ag/MHC by TCR in thymus, termed agonist selection. Those T cell subsets include 1) Foxp3+ Treg cells, 2) invariant NKT (iNKT) cells, and 3) αβTCR+ PD-1+ CD4− CD8− precursors of CD8αα IELs (pIEL). In addition to a strong TCR signal, B7-CD28 co-stimulation has a role in these agonist selections. This role is context dependent. For instance, B7-CD28 has a positive role for thymic Treg and iNKT cell generation, while it has a negative role for thymic pIEL generation. These differences may indicate involvement of distinct CD28 signaling in these events. To test this hypothesis, we analyzed CD28 cytoplasmic tail mutant mice in which tyrosine 170 residue was substituted to phenylalanine (Y170F), proline residues in PYAP motif were substituted to alanine (AYAA), or both mutation were present (Y170F/AYAA). Generation of self-Ag-specific Treg, defined by pMHCII tetramer binding, was attenuated in AYAA mice but not in Y170F mice, consistent with the reported role of the PYAP motif for total Treg generation. Meanwhile, interestingly, the negative role of CD28 for self-Ag-specific pIEL generation was not affected by Y170F and/or AYAA substitution, indicating that this CD28 effect was mediated by motif(s) other than Y170 and PYAP. Analysis of thymic iNKT cell (NKT1, 2 and 17) development in CD28 mutant mice is currently in progress. These results suggest that the differing roles of B7-CD28 costimulation for agonist selection fates are mediated via qualitatively distinct CD28 signaling.
Regulation of thymic type I IFN expression and its role in T cell development
Authors: Salgado Barrero Oscar C , Lee Seungeun Thera , Breed Elise R , Hogquist Kristin Ann
Published: January 2023

Thymocytes undergo a complex maturation process after being positively selected. Recently, the involvement of type I IFN signaling during these late stages was suggested; however, the regulation of intra-thymic type I IFN production and its role in T cell development and central tolerance are relatively unknown. We found that type I IFN, especially IFN-beta, regulate the expression of the maturation marker Qa2, the transcription factor STAT1 and the cell surface protein Ly6A in mature thymocytes. Using a published luciferase reporter mouse strain, Ifnbluc, we found that IFN-beta is expressed in the thymus in the steady state, and its levels peak at around 2–3 weeks of age, when the expression quickly returns to basal levels. The cell source of IFN-beta was mainly medullary thymic epithelial cells (mTEC) expressing high levels of MHC-II and CD80. We showed that IFN-beta expression on mTEC was completely dependent on AIRE and partially dependent on RANKL and CD40 signaling pathways. Additionally, we evaluated the impact of thymic IFN-beta expression on T cell tolerance. Our data suggest that T cells from mice lacking IFN-beta or AIRE show a higher reactivity to IFN-stimulated antigen presenting cells compared to T cells from wild-type mice. We hypothesize that thymic type I IFN provide a mechanism to promote T cell tolerance to IFN-induced peptides.
In vitro modeling of human B cell development can identify distinct physiopathological patterns in primary antibody deficiency (PAD)
Authors: Troilo Arianna , Wehr Claudia , Janowska Iga , Rawluk Justyna , Thiel Jens , Venhoff Nils , Herget Georg , Ochs Björn Gunnar , Konstantinidis Lukas , Warnatz Klaus , Salzer Ulrich , Rizzi Marta
Published: January 2023

Bone marrow (BM) analysis show that 20% of PAD patients harbor a block at an early stage of B cell development. The aim of this work is to study the mechanisms of this developmental block. We set up a feeder free cultivation system that in healthy donors leads to the development of lymphocyte progenitors until the stage of Immature B cells.

BM derived CD34+ cells are expanded in cytokine cocktail for 2 weeks. From day 14 to day 49 cells are cultivated in cytokine-free medium and analyzed once a week by flow cytometry and RNA is collected for PCR assays.

BM derived CD34+ cells from 15 PAD patients and 2 Btk-deficient patients were tested in vitro for their capability to develop into IgM+ Immature B cells. CD34+ cells from patients showing a normal B cell development in vivo (6/17) developed in vitro until the stage of Immature B cells. Among PAD patients presenting a block at early B cell stage, 5/11, including the Btk-deficient patients, could not reach the Immature B cell stage, while 6/11 could develop in vitro until the stage of IgM+ B cells. Among this last group, reaching Immature B cell stage in vitro but not in vivo, 2/6 patients showed a rapid in vitro development of Immature B cells at day 14, but presented a progressive exhaustion of the common lymphoid progenitors (CLP) compartment. In patients with a block in B cell development (in vivo and in vitro), PCR analysis revealed adelayed expression of transcription factors E2A, CD79a and PAX5, reflecting an impaired lineage commitment.

Combining BM phenotyping and in vitro modeling we identified four patients’ groups characterized by (1) an intrinsic B cell defect, (2) a defect in the BM microenvironment impairing early stages of B cell development, (3) a defect in repopulation of CLP and (4) normal B cell development.
TRAF3IP3 at the trans-Golgi network regulates NKT2 maturation via MEK/ERK signaling pathway
Authors: Ge Qing , Zhang Xinwei
Published: January 2023

Thymic natural killer T (NKT)2 cells are a subset of invariant NKT cells with PLZFhiGATA3hiIL-4+. The differentiation of NKT2 cells is not fully understood. We report in the present study an important role of TRAF3-interacting protein 3 (TRAF3IP3) in the functional maturation and expansion of committed NKT2s in thymic medulla. Mice with T cell-specific deletion of TRAF3IP3 had decreased thymic NKT2 cells, decreased IL-4 producing peripheral iNKTs, and defects in response to α-galactosylceramide. Positive selection and high PLZF expression in CD24+CD44− and CCR7+CD44− immature iNKTs were not affected. Only CD44hiNK1.1− iNKTs in Traf3ip3−/− mice showed reduced expression of Egr2, PLZF, IL-17RB, decreased proliferation, and decreased IL-4 production upon stimulation. Such Egr2 and IL-4 expression were augmented by MEK1/ERK activation in iNKTs and TRAF3IP3 at the trans-Golgi network recruited MEK1 and facilitated ERK phosphorylation and nuclear translocation. These data demonstrate an important functional maturation process in NKT2 differentiation that is regulated by MEK/ERK signaling at the trans-Golgi network.
Selective Serotonin Reuptake Inhibitors Suppress Mast Cell Function
Authors: Haque Tamara , Ryan John
Published: January 2023

Mast cells are tissue resident innate immune cells activated by IgE and many other ligands, including purines. Upon activation, they release histamine, cytokines, chemokines, proteases, and lipid mediators evoking allergic symptoms. New ways of targeting mast cells could greatly benefit allergic disease therapy. One plausible target is activation by purinergic receptors binding ligands such as ATP. However, there are currently no clinically available therapies targeting purinergic signaling. Because anti-depressants in the SSRI family can have off-target effects on purinergic receptors, we tested the hypothesis that SSRIs can attenuate mast cell function. We found that the SSRI fluoxetine blocks IgE- and ATP-induced cytokine production, degranulation, and calcium influx in murine bone marrow derived mast cells (BMMC). Furthermore, fluoxetine suppresses IgE-mediated anaphylaxis in vivo, and reduces ATP cellular uptake. Because adding serotonin to BMMC or deleting the serotonin transporter has no effect on fluoxetine-mediated suppression, an off-target effect seemed likely. We show that inhibitors of the ATP receptor P2X3 attenuate IgE-mediated activation similarly to fluoxetine, and combining fluoxetine with these inhibitors does not result in additive effects. Using siRNAs to knock down P2X3, we find that P2X3 is needed for suppression by fluoxetine. These data support the possibility of targeting purinergic signaling to treat allergic diseases, and suggest that this may be possible by repurposing FDA-approved SSRIs.
The HMGCR Inhibitor Fluvastatin Induces Apoptosis and Autophagy in Primary and Neoplastic Mast Cells
Authors: Barnstein Brian O , Paez Patrick A , Dailey Jordan M. , Finnegan Ryan M. , Ryan John J
Published: January 2023

Mast cell hyperplasia is noted in many inflammatory and allergic diseases. Mast cells can also generate a subtype of acute myelogenous leukemia. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase (HMGCR) inhibitors to lower serum cholesterol, have been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and mastocytoma cells. Apoptosis was suppressed in p53 KO and Bcl-2 Tg BMMC, consistent with detectable mitochondrial damage and Caspase-9 activation. Because apoptosis induction could be mimicked by suppressing geranylgeranyl transferase (GGT), and fluvastatin effects were reversed by adding the product of the GGT reaction, loss of GGT-mediated protein lipidation is the likely mechanism of cell death. In addition to apoptosis, fluvastatin also induced autophagy. Chemical blockade of autophagy enhanced fluvastatin-mediated apoptosis in primary mast cells, but reduced it in mastocytomas. These data suggest that the FDA-approved statin drugs may be useful in suppressing mast cell hyperplasia and leukemia.
Comparing mast cell immunometabolism shifts induced by IgE mediated and non-IgE mediated degranulation
Authors: Mendoza Ryan P , Anderson Colin C , Roede James R , Brown Jared M
Published: January 2023

Mast cells are immune effector cells with the ability to immediately release an array of pre-formed mediators in response to various stimuli, a process termed degranulation. Degranulation is generally divided into two categories: IgE mediated and non-IgE mediated. Non-IgE mast cell degranulation occurs in response to various environmental agents without prior IgE sensitization often making it difficult to identify the causative agent in patients. To better understand non-IgE mast cell degranulation, we characterized and compared metabolic shifts in response to both mechanisms of degranulation (IgE vs non-IgE) in bone marrow-derived mast cells (BMMCs) grown from C57BL/6 mice. Compound 48/80 and silver nanoparticles were used to trigger non-IgE degranulation. To explore metabolic changes in BMMCs, we used Seahorse XF technology to measure: 1) respiratory mitochondrial metabolism, 2) anaerobic glycolytic metabolism, and 3) performed a phenotype stress test to define metabolic pathways. The mito stress test revealed a decrease in respiration for both mechanisms of degranulation but was consistently lower for non-IgE triggers. Secondly, we observed an increase in glycolysis from both mechanisms which was more prominent in non-IgE degranulation. However, there was a complete depletion of glycolytic reserve with non-IgE degranulation that did not occur with IgE mediated degranulation. The cell phenotype test revealed that BMMCs shift towards glycolysis when activated via a non-IgE pathway more prominently than via an IgE-pathway. In conclusion, mast cell metabolism varies significantly between IgE and non-IgE degranulation with an apparent shift towards glycolytic dependence for non-IgE activated mast cells.
Perinatal androgens drive sex differences in mast cell phenotype and severity of mast cell disease
Authors: Mackey Emily , Moeser Adam J.
Published: January 2023

Highly prevalent and debilitating mast cell (MC)-associated immune disorders, such as allergy and irritable bowel syndrome exhibit a female sex bias in childhood and adulthood. The mechanisms underlying sex differences in MC disorders are unknown. Previously, we found adult female mice exhibited greater serum histamine and more severe MC-associated pathophysiology that coincided with an increased capacity to synthesize and store immune mediators, compared with males. Further, recent data showed sex differences in the MC exist prior to puberty, suggesting a mechanism independent of adult sex hormones. Here, we investigated the role of perinatal androgens, a key early life component of sexual differentiation, in sex differences in MC phenotype and MC disease. Testosterone proprionate (100 μg sc qd) or vehicle was given to pregnant female mice E16-P0 and directly to pups PN1-7. IgE-mediated anaphylaxis was performed in gonadectomized control male (CM), control female (CF) and perinatally androgenized female (AF) offspring at 8 wk of age. Serum histamine levels (CM 768 ± 106, CF 1406 ± 200, AF 676 ± 110 ng/mL) and hypothermia responses (Δ temp: CM −4.87 ± 0.45, CF − 7.28 ± 0.48, AF − 4.8 ± 0.39 °C) were ~2x lower in AF than CF mice, while AF and CM mice had similar responses. Further, bone marrow MC from AF mice had lower histamine content (AF 1305 ± 82: CF 2082 ± 94 ng/106cells) and 47% lower histamine release to IgE-FcɛRI stimulus compared to CF mice. Together, we demonstrate that sex differences in MC phenotype and disease susceptibility are established early in life by perinatal androgens. These findings provide insight into the mechanisms of sex differences in MC-associated immune disorders that could lead to new therapeutic targets for MC diseases.
Allergen-induced disulfide-linked dimers/oligomers of histamine-releasing factor enhance mast cell activation
Authors: Kawakami Yu , Kasakura Kazumi , Ando Tomoaki , Kawakami Yuko , Kawakami Toshiaki
Published: January 2023

Objective

We are investigating the role of disulfide-linked HRF dimers/oligomers in facilitating allergen-induced anaphylaxis.

Results

Our lab has previously shown that IgE can act as a receptor for histamine-releasing factor (HRF). Interestingly, HRF dimers and oligomers are able to activate mast cells in an HRF-reactive IgE-dependent manner, whereas HRF monomers cannot. HRF homo-dimerization occurs via disulfide bonding through its C-terminal residue, cysteine 172 (C172). We have found that secretion of HRF dimers/oligomers in the small intestine is more abundant in food allergic than non-allergic mice. To investigate the role of C172-mediated HRF di-/oligomerization in allergic reactions, we generated a transgenic HRF-C172A mouse in which HRF’s C172 was substituted with alanine. Using ovalbumin-induced food allergy and passive systemic anaphylaxis experiments, we showed that the HRF-C172A mice exhibit much weaker anaphylactic responses compared to their wild-type counterparts. In vitro experiments showed that antigen/IgE-mediated mast cell activation is maintained in the C172A mice, indicating that mast cell functionality is intact in the mutant mice. Since both of the in vivo experiments represent IgE/mast cell-dependent models of anaphylaxis, the differences in phenotype demonstrate the crucial role of disulfide linkage of HRF dimer/oligomers in IgE-mediated anaphylaxis. We also show that HRF dimerization is mediated by disulfide-bond-forming Ero family members and related enzymes.

Conclusion

Disulfide-linked HRF dimer/oligomers are a key factor in enhancing anaphylactic symptoms in the murine allergy models.
IL-33 with anti-FceRI Ab induces functional CD25 on human skin mast cells
Authors: Fukuoka Yoshihiro , Ward Brant R , Schwartz Lawrence B
Published: January 2023

Purpose

CD25 is expressed on the surface of clonal mast cells (MCs) in disorders such as mastocytosis, but not on normal MCs. IL-33, a new member of the IL-1 family of cytokines, is expressed primarily by epithelial and endothelial cells. IL-33 is released during cell necrosis, acting as “an alarmin” that can enhance T cell responses and activate MCs. We tests the hypothesis that treating MCs with IL-33 and anti-FceRI Ab induces cell-surface CD25.

Methods

MCs were dispersed from fresh surgical human skin and placed into culture with SCF (100 ng/ml) for 6–10 weeks. FACS analysis, western blotting, qRT-PCR and degranulation assays were performed, and cytokine, chemokine and sCD25 secretion was measured by ELISA.

Results

We found CD25 mRNA is expressed in primary MCs, and CD25 protein is detected within but not on the surface of resting MCs. When MCs were primed with IL-33 (10 ng/ml) and anti-FceRI Ab (10 ng/ml) for 2 days, surface CD25 was induced, as confirmed by flow cytometry and western blotting. Induction of CD25 was effectively inhibited by either anti-ST2 Ab or cyclosporine A. As C5a (10 nM) did not induce surface CD25 in combination with IL-33, degranulation of MCs is not critical for CD25 expression. MCs express CD132 mRNA and protein, and IL-33 plus anti-FceRI Ab induces CD122 mRNA. Consequently, the effect of IL-2 was examined. IL-2 (25–50 ng/ml) dose-dependently induced IL-6, IL-8, CCL4 and GM-CSF release by MCs only after priming with IL-33 and anti-FceRI Ab. Additionally, IL-33 with anti-FceRI Ab induces shedding of CD25, which can be suppressed by ADAM 17 inhibition to a greater extent than ADAM 10 inhibition.

Conclusion

Thus, polyclonal MCs from human skin, when primed, can express surface CD25 and a functional IL-2R complex.