bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenosine DeAminase (ADA) as an adjuvant molecule for human HIV-1 vaccine
Authors: TARDIF Virginie , Gary Ebony , Muir Roshell , Chakhtoura Marita , Cubas Rafael , Metcalf Talibah , Kutzler Michele , Haddad Elias K
Published: January 2023

Follicular helper T cells (Tfh) play critical role in shaping, instructing, and initiating T-cell dependent antibody responses. Understanding the underlying mechanisms that enhance their function is therefore critical for vaccine development. Using a unique gene array analysis, we have identified adenosine deaminase (ADA), as a novel key molecule that drives Tfh helper program in proliferating Germinal Centers Tfh (GC Tfh) and circulatory Tfh (cTfh) cells following their interactions with B cells. In fact, our gene array analysis showed that CD26 and ADA were exclusively up-regulated within the less efficient cTfh1 (CD4+CD45RA−CXCR5+CXCR3+) and cTfh2-17 (CD4+CD45RA−CXCR5+CXCR3+CCR6+/−) subsets, respectively. ADA enzymatic activity is significantly higher, as well, in cTfh2-17 than in the less-efficient cTfh1 cells. Exogenous ADA enhances the ability of Tfh cells to provide B cell help while inhibition of ADA activity by specific inhibitors impeded Tfh function and blunted antibody response. Our results further demonstrated that enhancement of Tfh function by ADA pathway could be due to increase in IL-6 and decrease in IL-2 production in the co-culture, and maintenance of low extracellular expression of CD26. Moreover, blocking IL-2 in cTfh2-17 co-culture from virally suppressed HIV subjects (ST) showed a significant decrease of CD26 expression associated with a rescued helper capacity. Finally, in vivo use of recombinant ADA as an adjuvant in a DNA based HIV vaccine enhanced Tfh cells differentiation and enhanced anti-Env humoral response and isotype class-switch.

Thus, ADA activity fine-tunes Tfh helper program and deciphering how ADA regulate the function of Tfh subsets would benefit future vaccine adjuvant design.
Vaccine-induced temporal cross-talk between myeloid cells reduces the risk of SIVmac251 acquisition
Authors: Vaccari Monica , Gordon Shari N , Fourati Slim , Brown Dallas , Bissa Massimiliano , Doster Melvin N , Keele Brandon K , Schifanella Luca , Lyianage N , Foulds Kathryn , Roederer Mario , Venzon David , Felber Barbara K. , Rosati Margherita , Pavlakis George N , Barouch Dan H , Sekaly Rafick P , Franchini Genoveffa
Published: January 2023

Vaccination with recombinant ALVAC + gp120 alum boosts has decreased the risk of acquisition of HIV/SIV in humans and in macaques. We tested whether the substitution of the first ALVAC-SIV immunization with either a DNA- or an Ad26-based SIV vaccine would affect vaccine efficacy. We found that the DNA prime was associated with 52% vaccine efficacy whereas the Ad26 prime regimen was ineffective.

The two primes differently altered the monocytes levels of CD14+DR+ classical monocytes and myeloid-derived suppressor cells (MDSC) in blood. The DNA resulted in a more pronounced expansion of CD14+DR+ than the Ad26 prime. In this group the frequency of classical monocytes was associated with higher levels of SIV specific CD4+ T cell responses and delayed SIVmac251 acquisition, whereas MDSC correlated with lower levels of T cell responses and early acquisition. Accordingly, plasma levels of arginase, an enzyme produced by MDSC and linked to the suppression of immune responses, also correlated with early SIVmac251 acquisition. Systems biology studies revealed a correlation between the expression of distinct gene expression signatures associated to monocytes which correlated either with acquisition or protection.

Altogether these data indicate that the priming may shape the cross-talk between monocytes and MDSCs and highlights the importance of vaccine induced innate responses in protection from virus acquisition.
Efficacy and immunogenicity of a novel polyanhydride nanovaccine for use against RSV infection in the neonate
Authors: McGill Jodi L , Kelly Sean , Narasimhan Balaji , Henningson Jamie , Kumar Pankaj , Sacco Randy E
Published: January 2023

Respiratory syncytial virus (RSV) is a leading cause of severe acute lower respiratory tract infection in infants and children worldwide. Bovine RSV (BRSV) is closely related to human RSV and a significant cause of morbidity in young cattle. BRSV infection in calves displays many similarities to RSV infection in humans, including similar age dependency, gross and microscopic pathology, and innate and adaptive immune responses. The poor immunogenicity of RSV has been a complicating factor in the development of a safe and effective vaccine in both humans and calves. Polyanhydride nanovaccines (i.e. nanoparticle-based vaccines) have shown promise as adjuvants and vaccine delivery vehicles in rodent models due to their ability to promote enhanced immunogenicity through both the route of administration and the ability to provide sustained antigen exposure. In these studies, we determined the efficacy of a novel polyanhydride nanovaccine containing the stable, post-fusion F and G glycoproteins from BRSV, against RSV infection using a neonatal calf model. In vitro, the BRSV nanovaccine induced activation and cytokine secretion by monocyte-derived dendritic cells and alveolar macrophages. In vivo, calves receiving the BRSV F/G nanovaccine, followed by aerosol challenge with BRSV, demonstrated increased virus-specific IgA in the nasal and bronchoalveolar lavage fluid, an increase in RSV-specific cellular responses in the peripheral blood and airways, and a reduction in BRSV shedding compared to unvaccinated control calves. Our results suggest that the RSV-F/G nanovaccine is highly immunogenic and with optimization, has the potential to significantly reduce the disease burden associated with RSV infection in both humans and animals.
Understanding fetal immune responses to congenital porcine reproductive and respiratory syndrome virus infection
Authors: Venner Laura C , Hong Linjun , Ladinig Andrea , Harding John C S , Lunney Joan
Published: January 2023

Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most economically significant diseases in the global swine industry, causing late-term abortions, early farrowing, and respiratory disease. Few studies have previously explored the immune pathways that alter fetal PRRS resistance/ susceptibility. Using a model where 3rd trimester pregnant gilts were euthanized at 2, 5, 8, 12, or 14 days post infection (DPI) with PRRS virus (PRRSV), samples from the fetal thymus and placenta, and maternal endometrium were collected and viral loads measured (U Saskatchewan) to determine when the virus crosses the placental barrier and infects each fetus. RNA was extracted with a Qiagen RNA Isolation kit, and gene expression determined using a 220 gene NanoString array to evaluate differential expression (DE) of genes and biomarkers previously predicted to alter PRRS resistance and susceptibility. Selected biomarkers that were measured included interferon signaling pathways, TREM1, HMGB1, B and T cell receptors, cell division, apoptosis, tissue remodeling and epithelial integrity, based on pathways identified using Ingenuity Pathway Analysis. Fetuses from the control gilt will be compared to those from three PRRSV infected gilts from the same DPI based on serum and thymus viral levels, comparing their viral negative (uninfected) and viral positive (infected) fetuses from the same litter. Comparisons of DE genes will be made between neighboring fetuses and across DPI. Data should reveal immunological pathways that contribute to fetal susceptibility. Understanding these mechanisms will benefit future research dedicated to exploring targeted approaches to halt the congenital spread of this disease.
Equine B lymphocytes exhibit TLR4 expression similar to humans
Authors: Kasmark Leah , Wagner Alayna , Zhu Jing , Creger Lindsay , Leeth Caroline McPhee
Published: January 2023

Toll-like receptor 4 (TLR4) is a well-documented Toll-like receptor that recognizes bacterial lipopolysaccharide (LPS) as its ligand. Expression of TLR4 has been well described in murine and human B cells. Murine B lymphocytes have relatively constant TLR4 expression, where in humans, expression is present, but low. However, systemically mice show low sensitivity to LPS and humans show high sensitivity. Horses share this high sensitivity to LPS with humans. Here, we look at TLR4 presence and activation in equine B lymphocytes. Research in this area has been hampered due to the lack of quality B lymphocyte markers. Anti-human CD21 was previously found to mark peripheral equine B cells. We show CD45R (B220) also accurately labels equine B lymphocytes. We also demonstrate TLR4 is expressed on equine B lymphocytes using the TLR4 antibody, clone 76B357.1. By using the density gradient, Lympholyte, equine lymphocytes can be separated out of peripheral blood and stimulated with LPS. We demonstrate equine B lymphocytes fail to proliferate under LPS challenge as opposed to highly proliferative mouse B lymphocytes. This lack of response is similar to that seen in humans and may be a clue to LPS sensitivity shared by horses and humans.
The Slam/SAP signaling pathway regulates γδ T cell effector functions
Authors: Dienz Oliver , DeVault Victoria L. , Spear Camarie C. , Mei Linda , Boyson Jonathan E
Published: January 2023

The Slam/SAP (SLAM-associated protein) signaling pathway plays a central role in the host immune response to infections. Males that inherit an inactivated SAP gene develop X-linked lymphoproliferative syndrome (XLP), a serious immunodeficiency that often results in enhanced susceptibility to a variety of pathogens. SAP is a cytosolic adapter protein that transduces signals from Slam family receptors, a family of nine (SLAMf1 to SLAMf9) cell surface receptors that are expressed only on hematopoietic cells. Although the Slam/SAP axis is known to regulate the development and function of several lymphocyte subsets, their role in γδ T cell biology is less well understood. We analyzed simultaneously the expression of multiple Slam receptors on γδ T cells and observed significant heterogeneity among γδ T cell subsets. Interestingly, Slam expression patterns marked functional γδ T cell subsets in both naïve and influenza-infected mice. SLAMf6 expression on lung γδ T cells correlated with IFNγ-producing CD27+ cells while SLAMf1 expression characterized IL-17-producing CD27− RORγt+ γδ T cells. The heterogeneous Slam receptor expression patterns of γδ T cell subsets can already be seen during embryonic thymic development, suggesting a possible role in the developmental programming of γδ T cell effector function. Disruption of Slam signaling through deletion of SAP affected the development of RORγt+ CD44hi Vγ4 T cells in the embryonic thymus, and the frequency of RORγt+ Vγ4 T cells in the periphery was still diminished in the adult. These results suggest that the Slam/SAP signaling pathway is an important regulator of the functional programming γδ T cells undergo during thymic development which affects mucosal immune responses later in life.
Intracellular protein degradation of BRD4 by dBET1 reveals conserved in vivo cereblon function in human and mouse T-cells
Authors: Akuffo Afua Adutwumwa , Alontaga Aileen , Metclaf Rainer , Beatty Matthew S , McDaniel Jessica , Gunawan Steven , Ayaz Muhammad , Becker Andreas , Hesterberg Rebecca S , Goodheart William E , Yang Yan , Daniel Kenyon G , Schonbrunn Ernst , Guida Wayne , Lawrence Harshani , Lawrence Nicholas , Epling-Burnette Pearlie
Published: January 2023

Protein degraders have been developed that can aide in understanding gene function. These chemicals contain complementary bifunctional components that 1) interact with specific proteins, and 2) engage an E3 ligase which leads to polyubiquitination and intracellular degradation by the proteasome. Thalidomide, infamously known for its teratogenic effects, is central to this new technology since it binds cereblon (CRBN) which promiscuously recruits the DDB1/Cul4A/Rbx1 complex to trigger target destruction. Thalidomide, and especially its immunomodulatory derivatives lenalidomide and pomalidomide, induce NK and T-cell activation by degrading IKZF1 which is a transcription factor that suppresses IL-2. Currently, it is debated whether mouse cereblon has conserved function due to a single non-conserved amino acid in mouse CRBN, Ile390 (equivalent to Val388 in human CRBN). First, our work confirms that lenalidomide induces IL-2 and the ubiquitin-dependent degradation of IKZF1 only in human and not mouse T-cells treated with lenalidomide. To further test for species-related effects, a series of theoretical and physical binding assays using mutant CRBN proteins show that the binding of IMiD compounds is not impaired by amino acid differences in mouse CRBN. Using dBET1, a novel thalidomide-JQ1 protein degrader, we show for the first time that mouse CRBN can trigger CRBN-dependent degradation of BRD4 and confirm that the CRBN/DDB1/Cul4A/Rbx1 complex is functional in mouse T-cells. Collectively, our results suggest that IKZF1 has divergent regulation in mouse cells rather than the IMiD complex and provide information relevant to the development of chemical conjugates that induce targeted intracellular protein degradation.
Sodium chloride inhibits IFN-γ, but not IL-4, production by invariant NKT cells
Authors: Jeong Dongjin , Chung Doo Hyun
Published: January 2023

Invariant NKT (iNKT) cells are a distinct subset of T cells that exert Janus-like functions in vivo by producing IFN-γ and IL-4. Sodium chloride modulates the functions of various immune cells, including conventional CD4+ T cells and macrophages. However, it is not known whether sodium chloride affects iNKT cell function, so we addressed this issue. Sodium chloride inhibited IFN-γ, but not IL-4, production by iNKT cells upon TCR stimulation in a dose-dependent manner. Consistently, sodium chloride reduced the expression level of t-bet, but not gata-3, in iNKT cells stimulated with TCR engagement. Sodium chloride increased phosphorylated p38 expression in iNKT cells and inhibitors of p38 and NFAT5, and SGK1 restored IFN-γ production by iNKT cells stimulated with sodium chloride and TCR engagement. Furthermore, adoptive transfer of iNKT cells pretreated with sodium chloride restored antibody-induced joint inflammation to a lesser extent than for untreated iNKT cells in Jα18 knockout mice. These findings suggest that sodium chloride inhibits IFN-γ production by iNKT cells upon TCR stimulation, which is dependent on p38, NFAT5, and SGK1. These findings highlight the functional role of sodium chloride in iNKT cell-mediated inflammatory diseases.
Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin
Authors: Liu Chaohong , Huang Lu , Zhang Yongjie , Zhao Xiaodong
Published: January 2023

As the central hub of the metabolism machinary, mTORC2 has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. It has been reported that Rictor is involved in B cell development, especially the peripheral development. But the role of Rictor on BCR signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell specfic Rictor knockout mice (cd19Cre) to investigate how Rictor regulates BCR signaling. We found that for the key positive and negative BCR signaling molecules, pBtk is reduced and pSHIP is enhanced in Rictor KO B cells. This indicates Rictor positively regulates the early events of B cell receptor (BCR) signaling. We found that the cellular F-actin is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing the BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration leads to decreases in the humoral immune response in Rictor KO mice. The inhibition of actin polymerization with Latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, where Rictor regulates BCR signaling via actin reorganization.
Itk independent development of natural Th17 cells and IL17A-driven hypersensitivity pneumonitis
Authors: Elmore Jessica , Huang Weishan , Carter Chavez , Koylass Nicholas , Bennett Amelia , August Avery
Published: January 2023

The tyrosine kinase, Itk, plays a critical role in T cell receptor signaling, and we have previously shown that Itk is required for the development of Th17 cells and their production of the pro-inflammatory cytokine IL17A. IL17A has been implicated in numerous pulmonary inflammatory diseases. We have previously shown that Itk−/− mice do not develop allergic airway disease coupled with protected reduced production of IL17A in the lungs in a model of allergic asthma. Farmer’s lung is a subset of hypersensitivity pneumonitis caused by repeated exposure to the bacteria Saccharopolyspora rectivirgula (SR). This IL17A-driven disease is characterized by an influx of neutrophils and T lymphocytes. Here we show that a population of natural Th17 cell develop in the absence of Itk. Furthermore, exposure to SR drives robust IL17A response produced by CD4+ T cells even in the absence of Itk, accompanied by the development of pulmonary inflammation. These studies suggest that TCR signaling through Itk differentially regulates the development of conventional and natural Th17 cells. Understanding how Itk modulates the Th17 cytokine responses will allow us to better understand the precise role of Itk in the production of IL17A in airway inflammation.
Targeting class switch recombination plays a potential role in retarding systemic lupus erythematosus (SLE)
Authors: Zhu Jing , Wagner Alayna , Kasmark Leah , Woodward Emily , Sproule Tom , Roopenian Derry C , Leeth Caroline McPhee
Published: January 2023

Systemic lupus erythematosus (SLE) is an autoimmune disease mediated mainly by autoantibodies reactive to nuclear acids and proteins. These autoantibodies are mostly of IgG isotype, indicating that class-switch recombination (CSR) may play an important role in the development of SLE. CSR is initiated by the enzyme, activation-induced cytidine deaminase (AID) which induces double strand DNA breaks then repaired by homologous recombination using the RAD51 complex. We hypothesize that therapeutic targeting of CSR will retard the development of SLE by inducing cell death in AID-expressed B cells and reducing pathogenic IgG antibodies. The BXSB/MpJ mouse is a well characterized model of human SLE, exhibiting a robust germinal center response and copious production of immunoglobulins. 4,4’-diisothiocyanatostilbene-2-2’-disulfonic acid (DIDS) is a molecule which impairs homologous recombination by inhibiting the binding of RAD51 complex to DNA. In this study, male BXSB/MpJ mice were injected with DIDS or control. Treated mice showed a reduction in the germinal center B cell population and a corresponding decrease in follicular T helper cells. T1 B cell and follicular B cell populations were significantly decreased in DIDS treated mice but no changes were found in marginal zone B cells. Sera were analyzed for the immunoglobulin levels and antinuclear antibodies. These findings demonstrate that targeting CSR by DIDS reduces potentially pathogenic B lymphocyte populations and may be a viable novel therapeutic option in the treatment of SLE.
Autoimmune responses are dependent on the MAVS innate pathway due to its natural co-stimulating effect on B cell activation and maturation
Authors: Sun Wenxiang , Qi Chen-Feng , Scott Bethany , Bolland Silvia
Published: January 2023

There is evidence that autoimmune disease might be induced by deficient regulation of innate immune pathways that normally induce type I interferon in response to pathogen-derived nucleic acids. To investigate a potential role for nucleic acid sensing pathways with cytosolic localization and more ubiquitous expression, we tested the requirement for MAVS (dsRNA-sensing) or STING (dsDNA-sensing) in the lupus autoimmune disease developed by FcγRIIB-KO (R2−/−) mice. These experiments revealed that STING was mildly protective, while MAVS was an essential factor for autoimmune disease in R2−/− mice. We found that MAVS deficiency, but not type I IFN receptor deficiency, completely eliminated autoantibodies and autoimmune pathology in R2−/− mice. MAVS deficiency also eliminated spontaneous activation of B and T cells, germinal center formation and follicular helper T cell development. Using mixed bone marrow reconstitution experiments, we determined that MAVS was required for autoreactivity in a B cell intrinsic manner, with a profound effect on B cell survival in the germinal center. MAVS deficiency did not alter BCR-proximal signaling, but resulted in diminished long term proliferative capacity of B cells, with lower cyclin D2 and Bcl-xL expression 24 hours after anti-IgM stimulation. MAVS deficiency also resulted in B cell developmental defects, fewer total B cells but a skewing toward marginal zone and against the follicular B cell program. Overall MAVS seems to provide its costimulatory effect independent of type I IFN, by enhancing the activation of NFκB in situations where T cell help or other modes of costimulation are not present and most importantly, by providing survival stimulus for autoreactive B cells.
Comprehensive proteomic profiling of Sjogren’s syndrome revealed dysregulation of interferon and other immunologic pathways
Authors: Wang Jingya , Wang Shu , Rahman Saifur , Kumar Varsha , Karnell Jodi L. , Voynova Elisaveta Ninova , Sanjuan Miguel A , Kolbeck Roland , Alevizos Ilias , Ettinger Rachel
Published: January 2023

Sjögren’s syndrome (SS) is the second most common rheumatic autoimmune disease with a broad spectrum of clinical and serological manifestations and no approved biologics. To better understand the pathogenesis of SS and explore potential soluble biomarkers and therapeutic targets, sera from 109 SS and 50 healthy controls (HC) were examined. Comprehensive profiling was performed using autoantigen and proteomic arrays (UTSW, 95 autoAb’s; RBM’s, 165 proteins; and SOMAscan, 1129 protein). Data showed that autoAb’s against U1sn-RNP-BB’, U1sn-RNP-C and Ribo phaspho protein P1 significantly increased in SS compared to HC (Fold change >1.5 and FDR <0.05) that positively correlate with FOCUS disease score (FDR<0.05). RBM and SOMAscan was highly consistent and detected 123 unique proteins up-or down-regulated in SS compared to HC. Strikingly, 71 were interferon-regulated proteins, with IP10 correlating best with FOCUS score (r=0.65). Pathway and network analysis also confirmed strong activation of type I interferon signaling. Type I interferon signature genes were also observed in salivary gland of the NOD.H-2h4 mice, a model that closely resembles human SS. Additional proteins dysregulated in SS patent sera include the TNF signaling, antimicrobial proteins A2M and B2M, and multiple SLAMF family members, which may shed light on disease mechanisms and novel therapeutic strategies.
Human MEMs are a distinct high IL-6 producing macrophage subset with anti-inflammatory and radioprotective properties in vivo
Authors: Capitini Christian M. , Bouchlaka Myriam N , Moffitt Andrea , Kim Jaehyup , Kink John A , Bloom Debra D , Love Cassandra , Dave Sandeep , Hematti Peiman
Published: January 2023

Macrophages (MØs) can polarize into subsets of “pro-inflammatory” (M1) MØs or “anti-inflammatory” (M2) MØs. Previously we described a unique population of MØs, termed human mesenchymal stem cell (MSC)-educated MØs (MEMs). In this study, we hypothesize that MEMs have potential to attenuate inflammation and promote tissue repair in vivo based on their immunophenotype and secretome. MEMs were generated by isolating monocytes from human peripheral blood and culturing them into MØs, followed by a co-culture with bone marrow-derived MSCs. RNA-Seq analysis showed genes associated with epithelial-mesenchymal transition, collagen formation, tissue development, and cell-cell adhesion were positively correlated with MEMs, while genes associated with oxidative phosphorylation were negatively correlated as compared to M1 MØs. We found increased expression of 8 genes implicated in wound healing, as well as genes representing remodeling enzymes, growth factors and the cytoskeleton. RT-PCR verified MEMs have increased CD206 (p<0.0001), IL-1β (p<0.001), TGFβ (p<0.001) and arginase-1 (p< 0.05), as well as increased IL-6 (p<0.0001) and CD73 (p<0.001) expression. MEMs produced over 4x greater amounts of IL-6 as compared to M2 MØs by ELISA (p<0.001), which could be enhanced with LPS stimulation (p<0.0001) or attenuated by inhibition of COX-2 (p<0.05), arginase-1 (p<0.01) or JAK1/JAK2 (p<0.05). In vivo, MEMs improved survival against lethal GVHD (p<0.01), in part by reducing T cell proliferation (p<0.001). MEMs also enhanced survival of mice from lethal radiation injury (p<0.01), in part by inducing host fibroblast proliferation (p<0.01). MEMs are a unique MØ subset with therapeutic potential for inflammatory disorders.
Molecular-based diagnosis of Multiple Sclerosis and its progressive stage
Authors: Kosa Peter , Barbour Christopher , Komori Mika , Tanigawa Makoto , Wu Tianxia , Johnson Kory , Masvekar Ruturaj , Fossati Valentina , Douvaras Panagiotis , Herbst Ronald , Wang Yue , Tan Keith , Greenwood Mark , Bielekova Bibiana
Published: January 2023

Biomarkers aid diagnosis, allow inexpensive screening of therapies and guide selection of rational patient-specific polypharmacy regimens in most internal medicine disciplines. In contrast, there are no clinically-validated measurements of the physiological status, or dysfunction(s) of cellular components of the central nervous system (CNS). Accordingly, patients with chronic neurological diseases are generally treated with a single disease-modifying therapy without understanding patient-specific drivers of disability.

Therefore, using multiple sclerosis (MS) as an example of a complex polygenic neurological disease, we sought to determine if cerebrospinal fluid (CSF) biomarkers are intra-individually stable, cell type-, disease- and/or process-specific and responsive to therapeutic intervention.

Using DNA-aptamer-based multiplex assay, in-vitro- and statistical models, we developed in a training cohort (n=225) and validated in an independent-cohort (n=85) two biomarker-based classifiers that differentiate: 1. MS from CNS diseases that mimic MS clinically, pathophysiologically and on imaging, and 2. relapsing-remitting from progressive MS with ~90% accuracy. Treatment-induced changes in biomarkers greatly exceeded intra-individual-and technical variabilities of the assay.

The observation that CNS biological processes reflected by combinatorial CSF biomarkers are robust, stable and disease- or even disease-stage specific opens opportunities for broad utilization of CSF biomarkers in drug development and precision medicine for CNS disorders.
Detection of fg/mL Levels of IL-2, IL-4, IL-6, IL-10, and IL-17A in Serum, Plasma, and Supernatant of Established Cell Models
Authors: Georlette Daphne , Wheeler Sarah E , Ranganathan Sripriya , Wang Su , Nikolenko Galina N , Shukla Animesh , Miller Thomas C , Aghvanyan Anahit , Stengelin Martin , Mathew Anu , Kuklis Catlin , Sharma Deepak , Bess Matthew , Davydov Ilia V , Wohlstadter Jacob N
Published: January 2023

Most immunoassays quantify cytokines with pg/mL sensitivity, which is insufficient to detect those present at low levels. MSD’s next-generation S-PLEXSM assay format was developed to quantify cytokines with fg/mL sensitivity. Here we describe measuring IL-2, IL-4, IL-6, IL-10, and IL-17A levels in normal sera and the supernatants of model cell lines using S-PLEX assays.

The assays demonstrated a dynamic range of approximately four orders of magnitude. Standard intra-plate coefficients of variation (CVs) ranged from 3%-15% and inter-plate CVs ranged from 8%–18%. The lower limit of detection (LLOD) was <1 fg/mL for all assays except IL-17A (11 fg/mL). IL-2, IL-6, IL-10, and IL-17A were detectable in 100% of normal sera samples (n=36–75) and IL-4 was detectable in 40% of normal sera samples (n=75). The average concentrations were <1 pg/mL for normal samples.

To confirm the sensitivity of these assays and their ability to detect native analytes, we characterized a panel of 23 cell lines that are models for cytokine secretion. As an example, the MOLT-4 cell line derived from acute lymphoblastic leukemia natively expressed detectable levels of IL-2 (1,031 fg/mL), IL-4 (221 fg/mL), IL-10 (60 fg/mL), and IL-17A (30 fg/mL). The expression profiles of these cell lines confirmed the sensitivity of the S-PLEX assays.

S-PLEX cytokine assays provide a 100 to 1,000-fold lower LOD than standard ELISAs, allowing the determination of baseline cytokine levels in many sample types.
Molecular chaperone is required for gut tolerogenic dendritic cell development and function
Authors: Iwanowycz Stephen , Yang Yi , Li Zihai , Liu Bei
Published: January 2023

The intestinal mucosa is continuously inundated with foreign antigens. Therefore, immune cells need to be tightly regulated to maintain tolerance against harmless dietary antigens and microbiota while responding to pathogens. Intestinal dendritic cells (DCs) play an integral role in regulating immunity and tolerance because of their unique position at the interface of innate and adaptive immunity. Multiple populations of intestinal DCs have been reported based on the expression of CD103, CD11b, and CX3CR1. However, the mechanism by which different DC subsets regulate intestinal homeostasis is still unclear. Heat shock protein gp96 is an essential chaperone for most TLRs, integrins, and other proteins important for DC functions. However, the role of DC-intrinsic gp96 in regulating gut tolerance has not been studied. By a genetic strategy, we discovered that selective deletion of gp96 from CD11c+ cells in mice results in alteration of DC and T cell subsets in the gut as well as the loss of antigen-specific regulatory T cell (Treg) induction. Strikingly DC-specific gp96 knockout mice develop spontaneous colitis by 24 weeks of age. Furthermore, differentiation of murine bone marrow-derived DCs revealed that gp96 depletion selectively decreased cell maturation in response to FLT3L compared to CSF2. Also, gp96 depletion reduced DC migration and cross-presentation. Taken together, our results demonstrated that gp96 depletion from CD11c+ cells selectively inhibits tolerogenic DCs in the gut leading to decreased Treg cells, which indicates that DC-intrinsic gp96 plays essential roles in maintaining gut tolerance.
The intestinal lamina propria is a unique niche for regulatory T cells
Authors: Morales Elisa Cruz , Laufer Terri M.
Published: January 2023

Foxp3+ regulatory T cells (Tregs) are critical to mantain intestinal homeostasis. Like all CD4+ T cells, Tregs require TCR-MHCII interactions for their development in thymus and differentiation in periphery. Past studies suggest that the TCR repertoire of intestinal lamina propria (siLP) Tregs is skewed towards food and commensal bacteria antigens. However, by using a transgenic mouse model that lacks peripheral MHCII expression and peripheral Tregs induction (K14 mice), we have demonstrated that thymically generated Tregs could enter to the siLP of weanlings and proliferate independently of MHCII to fill and maintain the compartment.

Newly-generated Tregs egress from thymus as central Tregs and acquire an effector phenotype after TCR signals in periphery. However, despite the lack of MHCII in the siLP, K14 Tregs are activated and phenotypically similar to effector Tregs in WT siLP. Interestingly, transcriptional profiling reveals that many components of a TCR-dependent expression module are present in both K14 and WT siLP Tregs. Furthermore, differently to splenic Tregs, K14 siLP Tregs highly express Nur77 and IRF4, often regulated through TCR signaling.

Tregs from K14 siLP localize mainly at isolated lymphoid follicles, where they are in proximity to B cells and dendritic cells; suggesting that siLP Tregs may still receive MHCII-independent signals from APCs, perhaps via costimulation molecules and/or paracrine cytokine stimulation. Indeed a short-term costimulation blockade with CTLA4-Ig reduces Nur77 expression on K14 siLP Tregs.

Taken together, these results suggest that the intestinal microenvironment uniquely maintains the differentiation and homeostasis of effector Tregs independent of MHCII-TCR interactions.
B1 cell IgE impedes B2 cell IgE-mediated parasite expulsion
Authors: Martin Rebecca , Damle Sheela R. , Valentine Yolander A. , Luker Andrea J. , Lownik Joseph C. , Urban Joseph F. , Conrad Daniel H.
Published: January 2023

Helminth infection is known for generating large amounts of poly-specific IgE. Previously we reported that innate-like B1 cells are responsible for poly-specific IgE production during infection with the nematode parasites Nippostrongylus brasiliensis and Heligmosomoides polygyrus bakeri. We showed that B1 cells make large amounts of IgE. In vitro analysis of factors critical for B1 cell immunoglobulin class switch recombination to IgE demonstrated a requirement for anti-CD40 and IL-4 that further enhanced IgE production when IL-5 was added to culture or when the B1 source was helminth infected mice. We additionally reported that there was also a novel IL-25-induced upregulation of IgE in B1 cells isolated from helminth infected mice. Here, we report that in T cell reconstituted RAG1−/− mice further reconstituted with or without B1 cells and/or B2 cells and followed by infection with N. brasiliensis only B2-reconstituted mice had significantly reduced fecal egg production that was IgE dependent. Parasite clearance was impeded by reconstitution with B1 cells and was also IgE dependent. Helminth-induced B1 cell IgE was not specific for N. brasiliensis because it blocked mast cell degranulation to N. brasiliensis excretory secretory antigens in infected mice. The data supports a hypothesis that B1 cell IgE secretion has evolved as a protective response used by the helminth. In searching for a human B cell equivalent, we additionally identified a novel population of human tonsilar B cells that respond to IL-25 stimulation with increased proliferation and IgE production. We hypothesize that these cells may be important in generation of human poly-specific IgE.
Distinct mechanisms govern resident memory T cell differentiation and survival in different tissues
Authors: Wynne-Jones Erica , Freestone David , Christo Susan N , Yang Kun , Reddiex Scott JJ , Pellicci Daniel G , Carbone Francis R , Kallies Axel , Mackay Laura K
Published: January 2023

Tissue-resident memory T (Trm) cells are a recently defined population of memory T cells that persist at the site of previous infection and contribute to protective local immunity. Trm cells have been identified in both epithelial and solid organs; however, despite their ubiquity, they share molecular commonalities that are distinct from circulating memory T cells. Therefore, we sought to explore the transcriptional regulation of Trm cells in a number of different organs. Our earlier data demonstrated that Trm cells share a core transcriptional signature regulated by the two related transcription factors Hobit and Blimp1. Here we show that Trm cells also have distinct molecular requirements according to anatomic location. While Hobit was uniquely required for the development of Trm cells in all organs tested, Blimp1 was required in some organs, but dispensable in others. Similar to Blimp1, the T-box transcription factor T-bet was also required for Trm cell development in only a subset of organs. This differential transcription factor dependency was linked to distinct cytokine requirements for Trm cell development in different organs. Trm cell defects were enhanced in the absence of multiple transcription factors, suggesting functional redundancy and implicating a role for distinct transcriptional networks as drivers of Trm cell development in different organs. Together, these data demonstrate the adaptation of Trm cells to specific tissue microenvironments and highlight the importance of studying these cells in a variety of organs.
Inflammasome Independent Leukotriene B4 Production Drives Crystalline Silica Induced Sterile Inflammation in Lungs
Authors: Hegde Bindu , Satpathy Shuchismita , Bodduluri Sobha Rani , Jala Venkatakrishna R , Bodduluri Haribabu
Published: January 2023

Occupational exposure to Crystalline Silica (CS) is a major public health concern with millions of workers continuously exposed to CS. Deposition of fine silica particles in the lungs can lead to severe lung inflammation and cancer. Lipid chemokine Leukotriene B4 (LTB4) and IL-1b that mediate leukocyte recruitment to the site of injury, are the key mediators of CS-induced sterile inflammation. Our previous studies have shown that CS exposure increases lung tumor burden in K-rasLA1 mice and that is abrogated in the LTB4 receptor, BLT1 deficient mice. Here, we show that phagocytosis of CS sequentially activates a series of pathways leading to LTB4, IL-1b and CXC chemokine production. CS uptake induces rapid formation of lipid bodies termed “lipidosome” independent of inflammasome activation. Studies with inhibitors suggested that CS-induced lipidosome formation and LTB4 production is linked to the phagocytosis pathway, an observation confirmed by confocal microscopy. We further demonstrated the SiRNA knockdown of cPla2, JNK or Rab40c prevents the formation of lipidosome and production of LTB4. Lastly, we demonstrate using an air pouch model in mice that JNK inhibitor, BI-78D3 is effective in reducing CS induced sterile-inflammation in-vivo. In summary, our studies highlight a novel inflammasome independent and JNK activation dependent lipidosome pathway leading to the production LTB4 as a major regulator of CS-induced sterile inflammation.
IL-1β and caspase-1 drive autoinflammatory disease independently of IL-1α or caspase-8 in a mouse model of Familial Mediterranean Fever
Authors: Sharma Deepika , Sharma Bhesh Raj , Kanneganti Thirumala-Devi
Published: January 2023

Mutations in the gene encoding pyrin (MEFV) are associated with autoinflammatory disorder Familial Mediterranean Fever (FMF). A “FMF-knock-in” (FMF-KI) mouse strain that expresses chimeric pyrin protein with a V726A mutation (MefvV726A/V726A) was generated to model human FMF. This mouse strain exhibits an autoinflammatory disorder that is prevented by genetic deletion of IL-1 receptor (IL-1R) or apoptosis-associated speck-like protein containing a CARD (ASC). ASC-mediated cell death leads to the release of IL-1α and IL-1β, both of which signal through IL-1R. Further, caspase-1 and caspase-8 can interact with ASC to mediate secretion of IL-1 cytokines. The specific IL-1 cytokine instigating development of FMF and the enzymatic caspase involved in its secretion are currently unknown. In this study, we demonstrate that the autoinflammation observed in MefvV726A/V726A mice is specifically mediated by IL-1β and not IL-1α. Furthermore, the disorder is dependent on the caspase-1-ASC axis, while caspase-8 is dispensable. Concurrently, aberrant IL-1β release by MefvV726A/V726A monocytes in response to stimulation with lipopolysaccharide is also dependent on the caspase-1-ASC axis. In conclusion, our studies have uncovered a specific role for caspase-1-mediated IL-1β release in the manifestation of FMF.
IL-4 Signal Regulates IL-9-producing Mucosal Mast Cell Differentiation And Function During The Development of Experimental Food Allergy
Authors: Tomar Sunil , Shik Dana , Ganesan Varsha , Smith Andrew , Wang Yui-Hsi
Published: January 2023

The prevalence of food allergy is increasing significantly in developed nations. Accumulating evidences suggest a pivotal role of intestinal mast cells (MCs), TH2 cytokines, and IgE in the pathophysiology of food allergy. We have recently identified the novel IL-9 producing mucosal mast cells (MMC9s), which can produce high levels of IL-9, IL-13, and mast cells mediators. Mice deficient of IL-4 signals fail to develop MMC9s and become resistant to IgE-mediated food allergy. However, it remains unclear whether IL-4 can directly regulate the development and function of MMC9s. In a skin sensitization induced food allergy model, Il4RaF709 mice which have gain-of-function mutations in IL-4Ra were found to produce much higher levels of ovalbumin (OVA) specific IgE and mast cell protease-1 in blood and became more susceptible to experimental food allergy. While the occurrence of MMC9s and type-2 innate lymphoid cells (ILC2s) were comparable between wild type (WT) and Il4RaF709 mice, repeated OVA ingestions induced significant increase of CD4+TH2 cell occurrence and MC numbers in the small intestine of Il4RaF709 mice than those in WT mice. Furthermore, IL-4 treatments induced bone marrow derived mast cells (BMMCs) from Il4RaF709 mice to produce much more IL-13 and TNF-α than those by WT BMMCs. These results suggest that IL-4 signal provided by CD4+TH2 cells may directly potentiate TH2 effector function of MMC9s and promote the differentiation of MMC9s into intestinal mature MCs, resulting in the increased intestinal hypersensitivity to ingested food allergens. Detailed mechanistic studies of IL-4 signaling in MMC9 development and function may provide therapeutic insights into the preventions and treatments of food allergy.
Carbonic anhydrase enzymes regulate mast cell-mediated inflammation
Authors: Henry Everett Kincie , Sy Chandler B , Inclan-Rico Juan M , Siracusa Mark C
Published: January 2023

Type 2 cytokine responses are necessary for the development of protective immunity to helminth parasites but also cause the inflammation associated with allergies and asthma. Recent studies have found that peripheral hematopoietic progenitor cells contribute to type 2 cytokine-mediated inflammation through their enhanced ability to develop into mast cells. Here we show that carbonic anhydrase (Car) enzymes are upregulated in type 2-associated progenitor cells and demonstrate that Car enzyme inhibition is sufficient to prevent murine mast cell responses and inflammation following Trichinella spiralis infection or the induction of food allergy-like disease. Further, we employed CRISPR/Cas9 technology and illustrate that genetically editing Car1 is sufficient to selectively reduce mast cell development. Finally, we demonstrate that Car enzymes can be targeted to prevent human mast cell development. Collectively these studies identify a previously unrecognized role for Car enzymes in regulating mast cell lineage commitment and suggest that Car enzyme inhibitors may possess therapeutic potential that can be employed to treat mast cell-mediated inflammation.
IL-33R signaling in CD4+ T cells regulates intratumoral myeloid cell recruitment.
Authors: Perez-Chanona Ernesto , Smith Carolyne K , Trinchieri Giorgio
Published: January 2023

The biological functions governed by IL-33 include the activation of TH1, TH2, CD8+ T cell and NK cell responses, however the contribution of IL-33 to anti-tumor responses remains controversial. One study showed that IL-33 drives the CD8+ T cell-mediated attenuation of lung metastasis of B16 melanomas. Another study, however, demonstrated that the depletion of the IL-33 receptor (IL-33R) enhanced anti-tumor responses of NK cells against 4T1 mammary carcinomas. By eliminating the IL-33R in specific immune compartments, including T cells and myeloid cells, we have begun to unravel the complexity of the intratumoral biology of IL-33. For example, we have observed that deletion of Il33r in T cells using Il33rfl/fl mice crossed with CD4-Cre expressing mice significantly impaired tumor growth of subcutaneous MC38 colorectal carcinomas compared with the floxed controls. After three weeks, CD4+ Gata3+ cells and neutrophils were notably more abundant in the tumors of Il33rΔTcells mice, while the CD8+ cell populations remained unaffected. Myeloid cell populations, such as CD11b+ Ly6Chi SSClo MHCII+ monocyte/macrophages, and CD11b+ Ly6C+Ly6G+ granulocytes infiltrated in larger numbers into the tumors growing in Il33rΔTcells mice than those in control mice. The contribution of IL-33 towards the anti-tumor activity or tumor-promoting capacity of these cells is under investigation. In addition, our ongoing studies suggest the possibility that not only IL-33R signaling mediates the extent of tumor clearance, but it also contributes to cancer-induced morbidities such as cachexia and the therapeutic efficacy of chemotherapies. Thus, our results suggest that IL-33R signaling may be a target for cancer immunotherapy.
Macrophage depletion decreases tumorigenesis and alters gut microbiota in the AOM/DSS mouse model of colon cancer
Authors: Bader Jackie E , Velazquez Kandy T , Enos Reilly T , Carson Meredith S , Cranford Taryn L , Nagarkatti Prakash , Nagarkatti Mitzi , Davis J Mark , Murphy E Angela
Published: January 2023

Macrophages are recognized as major players in the connection between inflammation and cancer; they represent up to 50% of the tumor mass and produce a wide array of inflammatory mediators with pro-tumoral functions. Emerging evidence links gut microbiota to the development of colorectal cancer and host-derived immune and inflammatory responses are important driving forces that can shape microbial community composition. We used a macrophage depletion technique to examine the role of macrophages on colon tumorigenesis and gut microbiota. To induce colorectal cancer, male C57BL/6 mice (n=32) received a single injection of AOM followed by three cycles of DSS supplemented water at concentrations of 2%, 1%, 1% at weeks 1, 4, and 7, respectively. Prior to the final DSS cycle (week 7) and twice weekly until sacrifice, mice (n=16/group) received 200ul i.p. of clodronate (CL2MDP) or PBS encapsulated liposomes to deplete macrophages. Mice were sacrificed at 12 weeks following the initial AOM injection and colon tissue was analyzed for tumor burden, macrophage markers, and inflammatory mediators. Stool samples were collected and DNA was isolated and subsequently sequenced for 16S rRNA. Macrophage depletion decreased tumor number by ~35% and specifically large (≥1mm) tumors by ~35% (p<0.05). This was consistent with a decrease in gene expression of EMR1 (overall macrophage marker) as well as expression of markers associated with M2 macrophages (IL-13, IL-10, TGFβ & CCL17) (p<0.05) but not M1 markers (NOS2, IL-12a & IL-23). Macrophage depletion also increased relative abundance of the Firmicutes phylum (p<0.05) in stool. We demonstrate that macrophages are important mediators of tumor growth and changes in gut microbiota in colorectal cancer.