bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of the endocrine system leads to local mucosal responses favoring allergic sensitization (MUC2P.919)
Authors: Berdnikovs Sergejs , Loffredo Lucas , Abdala-Valencia Hiam
Published: January 2023

Estrogen receptors play fundamental roles in health and disease by integrating systemic hormonal signals and local factors for maintenance of proper tissue homeostasis. Using a bioinformatics approach, we identified a genetic network reflecting aberrant regulation of mucosal homeostasis by estrogen receptors in allergic disease. To determine whether disruption of the estrogen system directly affects mucosal immune responses, we exposed mice to environmentally relevant doses of estrogen mimetic bisphenol A (BPA) in drinking water. Remarkably, systemic exposure to BPA facilitated allergic sensitization to inhaled ovalbumin and subsequent development of airway allergic inflammation without the use of adjuvants. Consistent with this effect, we identified a tissue stress response signature to endocrine disruption in the absence of tissue damage or noticeable inflammation, which persists for several weeks after cessation of BPA treatment. This includes increased tissue levels of chemokines CCL2, CXCL9, CXCL10 and CXCL11 at multiple barrier sites and elevated systemic levels of alarmins. Moreover, systemic exposure to BPA during postnatal development reduced differentiation potential of primary lung mesenchymal cells in adulthood, further supported by decreased expression of epithelial differentiation factors Fgfr2 and Sox2 in vivo. These findings demonstrate that environmental estrogens can induce tissue stress favoring allergic response in the absence of tissue damage.
High-throughput direct cell counting-based natural killer cell-mediated cytotoxicity assay using Celigo Imaging Cytometry (TUM2P.1027)
Authors: Chan Leo , Somanchi Srinivas , Rosbach Kelsey , Lee Dean
Published: January 2023

Cytotoxicity assays play a central role in studying the function of immune effector cells such as cytolytic T lymphocytes (CTL) and natural killer (NK) cells. Traditionally, cytotoxicity assays have been performed using 51Chromium (51Cr) and Calcein release assays. The assays involve labeling tumor cells (target) with radioisotope or fluorescent dyes, when the target cells are subjected to cytolysis by CTLs or NK cells, they releases the entrapped labels into the media upon lysis. These traditional methods may generate inconsistent results due to low sensitivity caused by poor loading efficiency and high spontaneous release of the reagents. In this work, we demonstrate a novel high-throughput direct cell counting method for cytotoxicity assay using the Celigo imaging cytometry method. Human NK cells from one healthy donor were used as effectors, and K562 and IMR32 were used as the target cells. Both target cells were first stained with Calcein AM, and seeded at in a 96-well microplate. NK cells were then added to each well at various Effector-to-Target (E:T) ratios. The 96 well plate was then scanned and analyzed using the Celigo to measure the % lysis of target cells at different time points. The results showed increasing % lysis as incubation time and E:T ratio increased. The propose Celigo imaging cytometry is an accurate and simple method for direct quantification of cytotoxicity, which can be an attractive method for both academic and clinical research.
Immunotherapeutic targeting of HSP90 client proteins overexpressed in chemo-/radio-resistant melanoma (TUM2P.1012)
Authors: Raveendran Subhara , Rao Aparna , Storkus Walter
Published: January 2023

Amongst the numerous genetic alterations that occur in cancer cells, is overexpression of DNA repair proteins (DNA-RP) in a disease-stage associated manner which has also been correlated to chemo/radiotherapy (CRT) resistance and poor overall prognosis. DNA-RP overexpression has been reported to involve post-translational protein stabilization mediated by the molecular chaperone Heat shock protein 90(HSP90), thereby preventing proteasome-dependent degradation of these HSP90 “client” proteins. Hence, HSP90 inhibitors (HSP90i) have been heralded as co-therapy agents for cancer patients that have developed resistance to first-line treatments such as temozolomide (TMZ). Under conditions in which its function is blocked by HSP90i, HSP90 client proteins rapidly become (poly)ubiquinated and undergo degradation resulting in improved sensitization of cancer cells to CRT. We observed that HSP90i promote the proteasome-dependent (i.e. blocked by inhibitors PS-341 and MG-132) degradation of a range of DNA-repair client proteins. We have also identified H-2Kb/Db-presented peptide epitopes derived from these DNA-RPs that may be recognized by Type-1 CD8+ T cells after specific vaccination in C57BL/6 mice. We are currently investigating the capacity of HSP90i to conditionally sensitize melanoma cells to the tumoricidal action of CRT as well as CD8+ T cells reactive against overexpressed DNA-RP that constitute HSP90 client proteins.
Humanized immune system mice: Improving the predictability of mouse models in immuno-oncology drug development (TUM3P.1044)
Authors: Hall Leon , Paz Keren , Kalbfliesh Megan , Pei Jiong , Baia Gilson
Published: January 2023

Mouse models are widely used in preclinical oncology research but species differences can limit efficacy predictions for clinical translation. Taconic Biosciences, Inc. is leveraging the super immunodeficient status of the CIEA NOD.Cg-Prkdcscid Il2rgtm1Sug/JicTac (NOG) mouse, which allows it so support the engraftment of human cells and tissues, to accelerate efficacy and safety testing of novel immunotherapies. We have optimized this model for efficient engraftment of the human immune system, and co-engraftment of patient derived xenografts (PDX). In a collaboration with Champions Oncology, the PDX engrafted humanized NOG mouse responded to immune checkpoint inhibition, by activating T cells which are able to infiltrate the tumor. An overview of Taconic's humanization program will be presented, including data showing the utility of humanized mice in PDX applications for immuno-oncology drug development, and recent advances in the development of next generation humanized mice
Characterization of the Anti-CD22 targeted therapy, moxetumomab pasudotox, for BCP-ALL (TUM3P.1053)
Authors: Kinjo Ichiko , Matlawska Ksenia , Winter Stuart , Burke Patricia , Wilson Bridget
Published: January 2023

Moxetumomab pasudotox (moxe, HA22, CAT-8015) is a recombinant, affinity optimized anti-CD22 immunotoxin. The demonstrated antitumor activity of moxe in patients with relapsed/refractory hairy cell leukemia supports its development in hematologic malignancies expressing CD22. Anti-leukemic activity has also been demonstrated in children with chemotherapy-refractory precursor B cell acute lymphoblastic leukemia (BCP-ALL) at various dose levels. Here, we explore potential mechanisms contributing to BCP-ALL sensitivity to moxe in vitro and in vivo. Binding, internalization and apoptosis studies were conducted using fluorescently-tagged moxe and two BCP-ALL cell lines (697, Nalm6), as well as blast cells from patients. Moxe cytotoxicity was only weakly correlated to CD22 expression levels, implicating additional processes after CD22-binding in the trafficking and processing of moxe also contribute to cytotoxicity. Studies in NSG mice engrafted with human BCP-ALL cell lines or patient blasts show that fluorescently-tagged moxe is taken up differentially by leukemia cells disseminated to different organs in the mouse (bone marrow, spleen, liver, brain). Studies were performed to evaluate the site-specific leukemia burden of cell lines and patient blasts in xenografted mice, and their correlation with in vitro uptake and cytoxicity assays. Our results suggest additional studies may be valuable to evaluate additional dosing strategies to maximize efficacy in these models.
Differential antibody induction to surface textured silica nanoparticle adjuvants (VAC3P.1055)
Authors: Tsirikis Peter , Wilson Kirsty , Kong Ying , Xiang Sue , Selomulya Cordelia , Plebanski Magdalena
Published: January 2023

Nanoparticles have been shown to be a potent adjuvant in vaccine design. Previous studies demonstrated that carboxylated 40-50 nm polystyrene nanoparticles (PSNPs) with covalently bound antigen offer a new class of vaccine with only two elements (antigen and particle) but with no added inflammatory stimuli. Herein, we report on a deconstructed two-part vaccine whereby the adjuvant and protein carrier are co-administered to elicit potent antibody responses in vivo. Recent works indicate that particle shape can also influence the immune response. As such, we investigate the influence of surface morphology using smooth and rough surfaced silica nanoparticle adjuvants, showing smooth nanoparticles as the more potent antibody inducers. MTT assay confirms that both silica nanoparticle adjuvant and polystyrene protein carrier display excellent biocompatibility with over 95% viability with COS-7 cells. Preliminary in vitro dendritic cell (DC) culture experiments indicate that silica nanoparticle adjuvants, similarly to PSNPs, do not induce the up-regulation of maturation markers, CD80, CD86 and MHC II on CD11c+ gated DCs. This suggests the particle carrier itself is non-inflammatory, a required feature for our vaccine approach. Ongoing studies are focusing on the cytokines induced in vitro and in vivo by these novel constructs. The outcomes from this study provide key design criteria in the development of novel nanoparticle immunotherapeutics for the treatment of disease.
Cell-mediated immune responses to ZOSTAVAX© are characterized by a boost in polyfunctional central memory T cells (VAC4P.1115)
Authors: Sei Janet , Cox Kara , Dubey Sheri , Antonello Joseph , Vora Kalpit
Published: January 2023

Herpes Zoster (HZ) is a debilitating disease caused by reactivation of latent varicella-zoster virus (VZV). ZOSTAVAX is approved for the prevention of HZ and its complications in individuals 50 years and older in many countries around the world. The mechanistic immune correlates of protection and VZV antigens responsible for induction of protective CMI are not defined. To screen for dominant VZV antigens, whole blood from subjects pre-and-post ZOSTAVAX vaccination were assessed for production of IFN-ϒ, IL-2 and, TNF-α. VZV antigens eliciting high cytokine levels were used to stimulate PBMC and intracellular cytokine staining performed. We show that the VZV antigens that led to a boost in the frequency of polyfunctional CD4+ T cells (TCD4+) were IE63>IE62>gB>ORF9>gE. Polyfunctional TCD4+ produced higher levels of cytokines than monofunctional cells, and a boost in expression of cytokines was observed, post-ZOSTAVAX. Phenotypic characterization of polyfunctional TCD4+ demonstrated that they are TCM cells. Notably, we detected ORF9-specific CD8+ T cells in 52% of subjects tested, however whether these responses are cross-functional or VZV-specific needs to be determined. Overall, these studies outline the CMI that may correlate with ZOSTAVAX protection.
Inhibition of GSK3beta leads to increased survival, proliferation and memory phenotype generation of GBM-specific CAR T cells (VAC5P.1125)
Authors: Sengupta Sadhak , Guha Prajna , Katz Steven , Sampath Prakash
Published: January 2023

Application of chimeric antigen receptors (CAR), where an antibody or ligand-binding domain is fused with the zeta signaling chain of the T cell receptor, is a novel and alternative strategy to bypass the failure of cytotoxic immune response induction by most tumor cells. Although CARs have shown impressive preclinical efficacy, translational success in solid tumors like glioblastoma (GBM) is often restricted by the limited viability of the T cells either due to the absence of IL2 in the central nervous system and/or because of mandated chemotherapy. Although multiple doses of T cell transfer and systemic administration of IL2 are effective for short time periods, unregulated overdose of T cells and IL2 result in immune toxicity. Inhibition of glycogen synthase kinase 3 beta (GSK3β) in GBM-specific IL13CAR T cells increased IL2-independent survival and proliferation of the CAR-positive T cells upon activation with IL13Rα2-expressing GBM tumor cells or soluble target antigen, when compared to bystander CAR-negative T cells or untreated IL13CAR T cells. GSK3β-inhibited IL13CAR T cells also showed increased IL7R and CD45RO expression compared to the bystanders as well as untreated IL13CAR T cells indicating a possible development of memory population. Exposure to target antigen increased cytotoxic efficacy and tumor-killing ability specifically in GSK3β-inhibited IL13CAR T cells. These observations suggest an innovative advancement in anti-GBM CAR T cell-mediated immunotherapy.
Critical role of ITK in mouse γ-herpes virus 68 infection as a model for human immunodeficiency (VIR1P.1140)
Authors: Cho Hyoung-Soo , Shin HyunMu , Berg Leslie
Published: January 2023

IL-2-inducible T cell kinase (ITK) is a Tec family tyrosine kinase predominantly expressed in T cells. In the absence of ITK, the magnitude of T cell receptor (TCR) signaling is greatly reduced, but not abolished, leading to an overall decrease in TCR signaling strength. Recently, various mutations in ITK were identified in patients suffering from a fatal lymphoproliferation due to a failure to control Epstein-Barr virus (EBV) infection. Interestingly, Itk-/- mice have been shown to mount protective responses to several acute virus infections. Thus, we hypothesized that ITK plays an essential role in long-term anti-viral immunity against latent γ-herpes virus infection. To study this, we infected WT and Itk-/- mice with mouse γ-herpes virus 68 (MHV68). Despite a reduced response at D7 post-infection, Itk-/- mice ultimately controlled MHV68 replication in the spleen by D14. After the initial response, both WT and Itk-/- mice showed no difference in their viral titers in the spleen at later time points. However, Itk-/- mice developed severe gastrointestinal pathology following LPS-mediated reactivation of latent MHV68, accompanied by a high viral titer in the intestine. Furthermore, the number of lymphocytes in the intestinal epithelium and lamina propria in naïve Itk-/- mice was substantially less than WT mice. Overall, these data suggest that ITK is essential for anti-viral immunity against γ-herpes virus infection in the gut following reactivation from latency.
Recruitment of GL7+ B cells to the inflamed CNS prior to peripheral germinal center formation (VIR1P.1151)
Authors: DiSano Krista , Phares Timothy , Bergmann Cornelia
Published: January 2023

Central nervous system (CNS) inflammation induced by viral infection and autoimmune disease results in the accumulation of various B cell subsets, including antibody secreting cells (ASC). However, temporal dynamics and precursor relationships of B cell subsets between the periphery and the inflamed CNS remain unclear. Encephalomyelitis induced by gliatropic coronavirus JHMV resolves into a persistent CNS infection that is controlled by local humoral immunity. Prior to the accumulation of essential CD138+ ASC, CD138- B cells traffic to the CNS by day 5 post infection (p.i.). Analysis of these early accumulating B cells by flow cytometry shows they comprise several phenotypes, which typically reside within the cervical lymph node (CLN), including naïve, activated, germinal center (GC)-like (GL7+), and unswitched memory B cells. B cell activation within the CLN occurs by day 7 p.i., but GC formation is not robust until day 14 p.i.. Unexpectedly, GL7+ B cells are detected within the CNS prior to GC formation. Moreover, in contrast to naïve/early activated B cells, which decline in number, GL7+ B cells increase differentially in brain and spinal cord throughout persistence. Maintenance of more differentiated B cell subsets within the CNS suggests either selective ongoing recruitment from the periphery or preferential local CNS maintenance. The lack of evidence for ectopic follicles suggests activated B cells, including GC-like B cells, can be recruited from the CLN.
The role of KIR and HLA polymorphism in HIV acquisition: association and in vitro functional studies (VIR1P.1159)
Authors: erlich henry , Wolinsky Steven , Penugonda Sudhir , Hayes Elizabeth , Nichols Robert , Saeteurn Koy , Duggal Priya , Maljkovic Berry Irina , Reddy Susheel , Swantek Peter , Ladner Martha , Lin Lin , Braun Jonathan , Trachtenberg Elizabeth
Published: January 2023

Polymorphism in the genes that encode the HLA antigens and the KIR receptors on NK cells influence both adaptive and innate immunity. We analyzed the role of HLA and KIR polymorphisms in HIV acquisition by comparing 405 HIV+ and 523 HIV- men in the Chicago MACS cohort. We found no significant association with any HLA allele, in contrast to the well established strongly protective effect of HLA-B*57:01 for HIV disease progression. The inhibitory KIR ligands are HLA class I epitopes and fall into the groups: HLA-C1,C2, HLA-Bw4, and HLA-A3/A11. Association analyses of individual KIR genes in the presence/absence of their cognate HLA ligand revealed that one KIR/HLA ligand genotype combination was protective for HIV acquisition. The HLA ligand genotype C1/C1 was protective compared to C1/C2 and C2/C2 but only with genotype KIR2DL3+/KIR2DL2- (OR = 1.76, p = 0.007). This KIR genotype marks homozygosity for the centromeric KIR A haplotype, which is more inhibitory, has fewer genes, and less diverse than KIR B haplotypes. Since C2 is the ligand for the KIR2DL1 receptor, and C1 is the ligand for the KIR2DL3 receptor, the protective association observed with C1/C1 homozygotes suggests that a lack of signaling via the strongly inhibitory KIR2DL1 receptor in addition to signaling via the KIR2DL3 receptor is protective. In vitro assays of NK cells from KIR2DL3 homozygous individuals demonstrate that C1/C1 vs C1/C2 and C2/C2 (C2+) genotypes are more active in terms of cytokine release.
Ly6C+ monocyte efferocytosis and cross-presentation of cell-associated antigen
Authors: Larson Sandy R , Thomas Stacey , Atif Shaikh M , Gibbings Sophie , Prabagar Miglena , Henson Peter M , Jakubzick Claudia
Published: January 2023

Recently it was shown that circulating Ly6C+ monocytes traffic from tissue to the draining lymph nodes (LNs) with minimal alteration in their overall phenotype. Furthermore, in the steady state, Ly6C+ monocytes are as abundant as classical dendritic cells (DCs) within the draining LNs, and even more abundant during inflammation. However, little is known about the functional roles of constitutively trafficking Ly6C+ monocytes. In this study we investigated whether Ly6C+ monocytes can efferocytose (acquire dying cells) and cross-present cell-associated antigen, a functional property specially attributed to Batf3+ DCs. We demonstrated that Ly6C+ monocytes intrinsically efferocytose and cross-present cell-associated antigen to CD8+ T cells. In addition, efferocytosis was enhanced upon direct activation of the Ly6C+ monocytes through its corresponding TLRs, TLR4 and TLR7. However, only ligation of TLR7, and not TLR4, enhanced cross-presentation by Ly6C+ monocytes. Overall, this study outlines two functional roles, among others, that Ly6C+ monocytes have during an adaptive immune response.
Loss of phagocytic and antigen cross-presenting capacity in aging DCs is associated with mitochondrial dysfunction
Authors: Janssen Edith , Thacker Robert , Shehata Hesham M , Lages Celine Silva , Hennies Cassandra , Lehn Maria , Chougnet Claire
Published: January 2023

Impaired functionality of dendritic cells (DCs) significantly contributes to decreased adaptive immune responses in aged hosts. The expression of MHC-peptide on the DC surface is the critical first step in T cell priming, but few studies have addressed the effect of aging on antigen (Ag) acquisition, processing, and presentation by DCs. Here we show that aged murine DCs were less efficient in the cross-presentation of cell-associated Ag and subsequently in the cross-priming of CD8+ T cells than their young counterparts.

The decreased cross-presentation was associated with a reduction in the frequency of CD8αDCs and merocytic (CD8α-CD11b-)DCs that could endocytose cell-associated Ag, as well as the number and the size of the endocytosed particles in the DC that did internalize cell-associated materials.

Mechanistically, phagocytic capacity has been associated with mitochondrial activity and membrane potential (Δψm). Aged DCs exhibited profound signs of mitochondrial dysfunction, illustrated by lower Δψm, reduced ATP turnover and coupling efficiency, decreased baseline oxidative phosphorylation (OXPHOS), and greater proton leak and ROS production. Mimicking the aged metabolic phenotype in young DCs by pharmacologic manipulation indicated that the reductions in Δψm and ATP impeded the phagocytic capacity while ROS interfered with a later step in the cross-presentation process. Conversely, in vitro scavenging of ROS partially restored cross-presentation by aged DCs.

Together, these data suggest that improvement of aged DC functionality might be feasible in the elderly, by targeting metabolic dysfunction, or its downstream sequelae, thereby opening new avenues for enhancing vaccine efficiency in this population.
Potential interaction of WT and Mutant TNF-Receptor 1 and Pyruvate Kinase M2 in TNF-Receptor-Associated Periodic Fever Syndrome (TRAPS)
Authors: Ploch Michelle , Pelletier Martin , Billingham Leah , Siegel Richard M
Published: January 2023

Tumor Necrosis Factor (TNF) is a trimeric cytokine induces innate inflammatory responses or cell death through TNFR1. Extracellular mutations in TNFR1 are associated with Receptor-Associated Periodic Syndrome (TRAPS), an autosomal dominant autoinflammatory disorder characterized by recurring fevers, dermal and synovial inflammation and elevated risk of amyloidosis. Previous work by our group has demonstrated that a TRAPS-like mutation in TNFR1 leads to accumulation of the receptor in the ER, leading to abnormal MAPK activation and correlated with reduced glucose metabolism. To better understand how the mutant TNFR1 signals to induce these abnormal cellular phenotypes, we immunoprecipitated the wild-type and TRAPS-associated mutant receptors and performed mass spectroscopy to identify associated proteins. Both WT and Mutant were able to bind the known adapter protein TRADD, but not the death domain (DD) deleted mutant. In addition, we also identified a novel association of WT and mutant TNFR1 with pyruvate kinase M2 (PKM2). Pyruvate kinase is the rate limiting enzyme in glycolysis and PKM2, and has been shown to be more activated in cancer cells, leading to abnormal metabolism known as the Warburg effect where cells undergo aerobic glycolysis. In addition, other studies suggest a link between activated PKM2 and induction of inflammatory responses, increasing TNF and IL1-b production. To confirm this interaction, we utilized colocalization microscopy techniques and IP/Western blot analysis which inferred a link between PKM2 and mutant TNFR1 more than the wild-type. Association between PKM2 TNFR1 may be a novel link between TNFR1 signaling and metabolism and explain some of the metabolic abnormalities in TRAPS cells.
Superoxide deficiency protects against Coxsackievirus-induced Type 1 diabetes through dampened macrophage anti-viral responses
Authors: Burg Ashley R , Das Shaonli , Padgett Lindsey E , Tse Hubert Ming
Published: January 2023

Viral infections are highly-suspected environmental triggers of autoimmune Type 1 diabetes (T1D), but the mechanism remains unclear. Many innate immune pro-inflammatory anti-viral factors, including IFN-α/β, TNF-α, CXCL10 and CCL5, are pathogenic in T1D. We recently demonstrated a crucial role for reactive oxygen species (ROS) production in modulating these factors, as Non-Obese Diabetic (NOD) mice deficient in NADPH oxidase (NOX)-derived superoxide (NOD.Ncf1m1J) are highly protected against T1D, partly due to impaired inflammatory macrophage responses. Therefore, we hypothesize that NOD.Ncf1m1J mice will be protected from virus-induced diabetes due to dampened pro-inflammatory anti-viral macrophage responses. Upon infection with Coxsackie B3 (CB3), a suspected diabetogenic virus, NOX-derived superoxide is rapidly generated by NOD macrophages, but is significantly decreased in NOD.Ncf1m1J macrophages. Loss of redox-dependent pro-inflammatory cytokine and chemokine synthesis significantly protected NOD.Ncf1m1J mice from virus accelerated T1D, as infected NOD.Ncf1m1J pancreata revealed significant decreases in Tnf, Cxcl10 and Ccl5 mRNA accumulation. This corresponded with 2.5-fold decreases in TNF-α- and IL-1β-producing pancreas-infiltrating macrophages, despite having no significant defect in CB3 viral clearance. Mechanistically, we determined that MDA5, a viral RNA sensor, was regulated by ROS. CB3-infected NOD.Ncf1m1J macrophages displayed a decrease in MDA5 expression, but was rescued by addition of exogenous ROS. Therefore, our results provide evidence that ROS can synergistically influence anti-viral responses to promote autoimmune diabetes in response to diabetogenic viral infections.
NKG2D-H60a interaction via homotypic T cell contact delays NOD diabetes development.
Authors: Trembath Andrew P , Sharma Neekun , Matocha Kimberly Nicole , Markiewicz Mary A
Published: January 2023

Type I diabetes is an autoimmune disorder mediated by T cells that destroy the insulin-secreting β-islet cells in the pancreas. Both human and murine studies implicate the immune receptor natural killer group 2 member D (NKG2D) in enhancing type 1 diabetes development. However, a mechanism for NKG2D influence in type 1 diabetes development is not clear. Utilizing NKG2D-deficient non-obese diabetic (NOD) mice, we demonstrate that NKG2D expression affects diabetes development via multiple mechanisms in multiple anatomical locations, with both pro- and anti-diabetic effects. First, we found NKG2D influences NOD diabetes development via alteration in the microbiota composition. When we eliminated this microbiome effect, NKG2D-deficient mice had enhanced diabetes development, demonstrating a protective role for NKG2D in this disease. Our in vitro studies suggest this protection is mediated via a previously undescribed interaction between NKG2D and the NKG2D ligand H60a, both expressed by activated NOD CD8+ T cells, during diabetogenic cytotoxic T cell (CTL) generation. We show that during CD8+ T cell differentiation into CTLs, NKG2D-H60a interaction generally decreases the subsequent CTL effector cytokine response. Taken together, these findings demonstrate there is a protective role for NKG2D during diabetes development that is likely mediated by NKG2D-ligand interaction during diabetogenic CTL differentiation.
Loss of intrasplenic 33D1+DC migration impairs alloimmune responses against RBC transfusion
Authors: Liu Dong , Calabro Samuele , Gallman Antonia , Nascimento Manuela , Chen Pei , Krishnaswamy Kumar , Uthaman Gowthaman , Xu Lan , Xu Lan , Williams Adam , Eisenbarth Stephanie C
Published: January 2023

Although the migration of tissue DCs from periphery to draining LNs is the crucial step for the induction of T cell responses, it is not clear whether the same holds true within the spleen. Here we use red blood cell (RBC) transfusion model to specifically study intrasplenic DC migration in the induction of alloantibodies to blood group antigens present on the donor RBCs. We observed that mice deficient in DOCK8 (dedicator of cytokinesis 8), a guanine nucleotide exchange factor that regulates the activity of Rho GTPase family members, fail to produce alloantibodies following transfusion. Two major splenic conventional DC subsets, the Batf3-dependent CD8+ XCR1+ subset and the IRF-4-dependent CD11b+33D1+ subset, prime CD4+ and CD8+ T cells, respectively. Our data demonstrate that only bridging channel 33D1+ DCs migrate to the T cell zone to initiate CD4+ T cell responses during RBC transfusion. Using in vivo antibody labeling method, we are able to differentiate marginal zone/red pulp dendritic cells from T cell zone/white pulp dendritic cells. In vivo labeling reveals that the failure of generating alloantibodies to RBCs in DOCK8 deficient mice is due to impaired 33D1+ DC migration. Interestingly, although the XCR1+ DCs in DOCK8 deficient mice migrated normally, they played no role in alloimmune responses. Taken together, our data demonstrate that subset specific migration of DCs within spleen is critical for initiation of CD4 T cell response and generation of alloantibodies. As equivalent populations of DC subsets exist in humans, our work suggests that blocking certain DC subset migration therapeutically during RBC transfusion could potentially prevent the harmful sensitization that occurs in patients who require lifelong RBC support.
Proprotein convertases generate a highly functional heterodimeric form of thymic stromal lymphopoietin in chronic rhinosinusitis
Authors: Kato Atsushi , Poposki Julie A , Klingler Aiko I , Stevens Whitney W , Peters Anju T , Hulse Kathryn E , Grammer Leslie C , Schleimer Robert P , Welch Kevin C , Smith Stephanie S , Sidle Douglas M , Conley David B , Tan Bruce K , Kern Robert C
Published: January 2023

Thymic stromal lymphopoietin (TSLP) is known to be elevated and truncated in nasal polyps (NPs) of chronic rhinosinusitis and might play a significant role in type 2 inflammation in this disease. However, neither the structure nor the role of the truncated products of TSLP has been studied. We investigated the mechanisms of truncation of TSLP in NPs and the function of the truncated products. We incubated recombinant full-length 15.1 KDa human TSLP (methionine + residues 29–159, referred to as M29–159) with NP extracts. SDS-PAGE revealed two major truncated products of TSLP in NP extracts. Edman sequencing and MALDI-TOF MS results indicated that the longer truncation product was TSLP (M29–124) and the smaller product was TSLP (131–159). We found that PCSK (proprotein convertase subtilisin/kexin) family proteases were rate-limiting enzymes in the truncation and generation of TSLP (M29–130) and TSLP (131–159). Carboxypeptidases further digested 6 amino acids from the C-terminus of TSLP (M29–130) to generate TSLP (M29–124) in NPs. Interestingly, recombinant TSLP (M29–124) and TSLP (131–159) did not manifest TSLP activity, but PCSK-digested TSLP had significantly higher activity than full-length TSLP on myeloid dendritic cells and group 2 innate lymphoid cells. Finally, we identified a 14.2 KDa TSLP from incubation of TSLP with NP extracts under non-reducing conditions that was significantly reduced in the presence of DTT. This indicates that the two cleaved products are dimerized with disulfide bonds and the dimerized form, TSLP (29–124 + 131–159), is an active metabolite and may contribute to the amplification of type 2 inflammation in NPs.
MiR-205 Supports Thymopoiesis Following Stress by Positively Regulating Foxn1 Expression
Authors: van Oers Nicolai S , Hoover Ashley R , MacLeod Jessica , Dozmorov Igor , de la Morena M. Teresa
Published: January 2023

T cells are a critical component of the adaptive immune system, developing within the thymus. Immature thymocytes interact with an interconnected meshwork of thymic epithelial cells (TECs) to establish the T cell repertoire. The developmental process is extremely stress sensitive, as the thymus can undergo a rapid involution in response to diverse inflammatory conditions, and a more prolonged hypoplasia during aging. The type of stress predicates whether TECs, thymocytes, and/or myeloid cell populations are affected. Several microRNAs (miRs) have been identified in the thymus based on their ability to mitigate the stress damage. We identified miR-205 as a stress responsive miR specifically expressed in TECs. Mice generated with a conditional ablation of miR-205 in TECs exhibit an age and sex-dependent thymic hypoplasia beginning at 8 weeks. Under stress conditions involving a type I interferon response (dsRNA mimic; polyI:C), the TEC-miR-205 deficient mice had a severe thymic atrophy compared to littermate controls. The TEC-miR-205 deficient mice displayed a delayed recovery of single positive CD4 and CD8 thymocytes, likely resulting from a block in the cortical TEC expansion. qPCR and gene expression comparisons revealed that the miR-205 deficient TECs had significant changes in chemokine/chemokine receptor and antigen processing pathways. MiR-205 positively regulated Foxn1 transcription factor expression, the master regulator of TEC development and function. Interestingly, miR-205 is encoded within a long non-coding RNA (lncRNA), 4631405K08Rik. Current experiments will reveal the mechanisms by which the miR and lncRNA are transcriptionally regulated, and how these affect Foxn1 expression to support thymopoiesis.
Alteration of TCR Dβ sequence content alters Treg development.
Authors: Levinson Michael , Silva-Sanchez Aaron , Sleckman Barry P , Zhuang Yingxin , Schroeder Harry W
Published: January 2023

Diversity of the T cell receptor is mainly developed through V(D)J rearrangement and N addition. The product of this rearrangement is the CDRβ3, a region of high variability that recognizes peptide and includes all of the D gene. Interestingly, the Dβ sequence is highly conserved in animals that use an adaptive immune response. This suggests that there are naturally selected germline encoded constraints on the TCR; we hypothesize that these constraints are there to streamline the process of T cell generation by biasing the repertoire into one that can recognize the most amount of antigens while limiting deleterious T cells from reaching the periphery. Because of this conservation, we hypothesize that altering the D region will have an effect on the development of thymocytes and function of mature T cells.

To test this, we created Dβ altered mice: a replacement of the Dβ locus with a charged Dβ (DβDKRQ) and a replacement of the Dβ locus with a hydrophobic DH (DβYTL). Compared to WT, the mutant mice have an altered TCR repertoire in both CDRβ3 amino acid composition and length; these differences can be attributed to the changes in the germline sequence. Changing the Dβ also changes T cell numbers in developing and mature T cells, with an altered Dβ being selected against. We also found functional differences; there is decreased epitope recognition in the DβYTL mice after immunization with ovalbumin. These data supports our hypothesis that alteration from the preferred sequence will have effects on T cell development and function.
UTX controls lineage-specific epigenetic program and development of iNKT cells
Authors: Beyaz Semir
Published: January 2023

iNKT cells are innate-like lymphocytes that protect against infection, autoimmune disease, and cancer. However, the role of epigenetic regulation in iNKT cell biology remains elusive. Here, we show that the H3K27me3 histone demethylase UTX is an essential cell-intrinsic factor for iNKT cell development in the thymus. UTX-deficient iNKT cells exhibit impaired expression of iNKT signature genes, including NK cell receptors and the transcription factor Tbx21. Epigenetic profiling uncovers a decrease in activation-associated H3K4me3 and an increase in repressive H3K27me3 marks within the promoters of downregulated signature genes. UTX binds to these promoters and its enzymatic demethylase activity is important for establishing the iNKT gene expression program. Moreover, we identify the AP-1 transcription factor JunB as novel master regulator of the iNKT cell gene signature, highlighted by its molecular interaction with UTX. Finally, iNKT cells harbor super-enhancers for their key factors T-bet, PLZF, and JunB, underlining their role in lineage specification.
Conditional disruption of Raptor reveals an essential role for mTORC1 in B cell development, survival, and metabolism.
Authors: Iwata Terri , Ramirez Julita , Margineantu Daciana , Hockenbery David , Tsang Mark , Iritani Brian M
Published: January 2023

Mechanistic target of Rapamycin (mTOR) senses diverse environmental conditions including nutrient, growth factor and stress conditions, to regulate cellular growth, division, and differentiation. Studies examining the role of mTOR signaling in immune function have revealed critical roles for mTOR in regulating T-cell differentiation and function. However, there have been few studies investigating the role of mTOR in early B cell development and function. We found that mTORC1 activity is highly activated during the pro- and pre-B stages of mouse B cell development. Disruption of mTORC1 signaling in developing mouse B-cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B-cells and loss of antigen-specific antibody production. Pre-B cell survival and proliferation were also significantly reduced in B-cells from mTORC1-signaling deficient mice. mTORC1-signaling deficiency resulted in a significant decrease in both oxidative phosphorylation and glycolysis indicating that loss of mTORC1 signaling in B cells results in altered cellular metabolism. Forced expression of a transgenic B-cell receptor on mTORC1-signaling deficient B-cells failed to rescue the developmental block. Collectively, our data reveal a previously uncharacterized role of mTORC1 signaling in early B-cell development and metabolism.
SRT1720 inhibition of M1 macrophages and chronic inflammation in Chagas disease
Authors: Wan Xianxiu , Koo Sue-Jie , Wen Jianjun
Published: January 2023

Trypanosoma cruzi (T. cruzi) causes Chagas disease that is one of the most frequent causes of heart failure and sudden death in Latin America. We observed the therapeutic effect of SRT1720 and explored its immunological mechanisms especially its effect on macrophage phenotype and function in chronic Chagasic mouse. SRT1720, an SIRT1 agonist, was injected intraperitoneally in T. cruzi infected mice at 1 mg/mouse/dose during day 45–66 post-infection (pi, three times a week). Mice were harvested during chronic disease phase (~150 days pi). Left ventricular dysfunction was prevented in SIRT1720-treated chagasic mice. SRT1720 ameliorated the clinical severity of chronic Chagas diseases, decreased proinflammatory cytokine (IFN-γ, IL-1 β, TNF-α) gene expression and inflammatory infiltrate in chronic chagasic hearts. The expression of iNOS, a specific marker for M1, was decreased in SIRT1720-treated chagasic heart. Splenic evaluation by flow cytometry showed both M1 (CD80+ and CD64+) and M2 (CD200R+ and CD206+) macrophages were activated in chronically-infected mice, and SRT1720 inhibited T. cruzi -induced increase in M1 macrophages. These results demonstrated that SRT1720 has therapeutic potential in Chagas disease possibly through preventing the polarization of M1 macrophages and inhibiting inflammatory responses. In vitro experiments will be done to confirm these results. Spleen macrophages will be isolated from these mice and will be cultured for 24h. The release of NO and accumulation of iNOS and secretion of IFN-γ, IL-1 β, TNF-α in cultured macrophages will be examined.
IL-4 Rescues Surface IgM Expression in Chronic Lymphocytic Leukemia
Authors: Guo Benchang , zhang Lu , Chiorazzi Nicholas , Rothstein Thomas L
Published: January 2023

Chronic lymphocytic leukemia (CLL) cells express poor levels of surface Ig, and many are minimally activated or anergic in response to BCR crosslinking in vitro. Paradoxically, CLL cells in patients are highly activated through BCR signaling and expand in proliferation centers, suggesting that the function of sIg signaling is rescued. Here, we find that, compared with normal naïve B cells, CLL cells express an extremely low level of CD79b protein but relatively normal levels of CD79a and IgM protein. Association of both CD79a and CD79b to IgM is markedly reduced. We further find that IL-4 markedly rescues CD79b and sIgM protein levels in CLL cells. These changes significantly enhance signaling in response to BCR crosslinking. Furthermore, we find that these changes are more pronounced in IGHV-unmutated CLL than IGHV-mutated CLL cells. The results described herein reveal that reduced sIgM is due to low expression of CD79b protein in CLL cells. Crosstalk between the IL-4 receptor and the BCR rescues sIgM-BCR expression and signaling. The differential responses of IGHV-unmutated and -mutated CLL cells to IL-4 may reflect, or be involved in, the distinct clinical courses that characterize these populations.
Foxp3-driven miR-23~27~24 clusters control regulatory T cell-mediated immune regulation
Authors: Wu Cheng-Jang , Cho Sunglim , Cruz Leilani Ong , Lin Ling-Li , Lu Li-Fan
Published: January 2023

CD4+ Foxp3+ regulatory T (Treg) cells maintain immune homeostasis by regulating pathologic immune responses against self-, innocuous as well as dangerous foreign antigens. Previously, microRNA (miRNA)-mediated gene regulation has been shown to play a pivotal role in the maintenance and function of Treg cells. Here, we report that miR-23~27~24 clusters, which are abundantly expressed in Treg cells, are critical in controlling Treg cell biology. Foxp3 directly promotes elevated expressions of miR-23~27~24 clusters required for optimal Treg cell function through binding to the “enhancer element” at the miR-23~27~24 loci. Loss of miR-23~27~24 clusters in Treg cells lead to the development of spontaneous lympho-hyperactivation phenotypes over time. Moreover, upon allergen challenge, mice with Treg cell-specific ablation of miR-23~27~24 clusters exhibited elevated eosinophilic airway inflammation. Together, our results identify a Foxp3-driven miRNA family that plays important roles in controlling Treg cell-mediated immune regulation at both physiological and pathological settings.
Image based machine learning for identification of M1 and M2 macrophages
Authors: Rostam Hassan Muhammad , Reynolds Paul M. , Singh Sonali , Alexander Morgan R , Gadegaard Nikolaj , Ghaemmaghami Amir
Published: January 2023

Macrophages play a crucial role in orchestrating immune responses against foreign materials, pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at the two ends of the spectrum. Characterisation of M1 and M2 subsets is usually carried out by quantification of multiple cell surface markers, transcription factors and cytokine profiles. These approaches are time-consuming, require large numbers of cells and are resource intensive. In this study, we have used machine learning algorithms to develop a simple and fast imaging-based approach that enables automated identification of different macrophage functional phenotypes using their cell size and morphology. Macrophages were polarised to M1 and M2 phenotypes by GM-CSF+IFN-gamma and M-CSF+IL-4, respectively. Control cells were un-polarised (naïve) macrophages and monocytes. The efficiency of macrophage polarisation was confirmed by quantifying M1 and M2 specific surface markers, cytokine profile and transcription factors. Fluorescent microscopy was used to assess cell morphology of all cell types which stained only for nucleus and actin distribution using DAPI and phalloidin, respectively. Using only the analysis of the morphological features we were able to identify M1 and M2 phenotypes effectively and could distinguish them from naïve macrophages and monocytes. We were able to classify all 5 cell types with an average accuracy of 70%, whilst concentrating on classification of M1/M2 cells with a binary classifier was, on average, correct 88% of the time. Thus we suggest high content and automated image analysis can be used for fast phenotyping of functionally diverse cell populations with reasonable accuracy and without the need for using multiple markers.
Controlling macrophage phagocytosis towards healthy self cells: Two layers of activating and inhibitory mechanisms
Authors: Bian Zhen , Shi Lei , Lv Zhiyuan , Tremblay Alexandra V , Niu Shuo , Zen Ke , Zhang Yongliang , Liu Yuan
Published: January 2023

Rapid clearance of adoptively transferred CD47-null cells in mice suggests a critical self-recognition mechanism, in which CD47 is the ubiquitous marker of ‘self’, and its interaction with macrophage SIRPα triggers inhibitory signaling through SIRPα cytoplasmic ITIMs and tyrosine phosphatase SHP-1/2. However, instead of displaying self-destruction phenotype, the mice deficient of CD47 manifest no enhanced macrophage phagocytosis towards self, a phenomenon suggesting that additional mechanisms besides CD47-SIRPα command macrophage behavior. Studying our recently established SIRPα-knockout (SIRPα−/−) mice, as well as CD47−/− mice, we reveal that macrophage phagocytosis toward self is not only inhibited by CD47-SIRPα signaling but also requires an activation mechanism induced by unique inflammatory stimuli. SIRPα−/− mice and CD47−/− mice, though being healthy under normal condition, develop severe anemia and splenomegaly owing to splenic macrophages aggressively phagocytizing self RBC under chronic colitis, peritonitis, inflammatory cytokine treatments, or CFA and LPS-induced inflammation. Ex vivo phagocytosis assays demonstrate that macrophages from SIRPα−/− or CD47−/− mice, and even peritoneal macrophages from WT mice, are generally incompetent to attack self cells albeit their aggressiveness towards bacteria, fungi, apoptotic cells and immune complexes. However, treating all macrophages with IL-17, LPS, IL-6, IL-1β and TNFα, but not IFNg, dramatically initiates potent phagocytosis towards self cells providing an absence of the SIRPα-CD47 restraint. In addition, we identify that WT spleen red pulp is one specific environment that stimulates macrophage phagocytosis and that is lacking in deficient mice.
Regulation of Natural Killer cell cytotoxicity by shedding of the Fc receptor CD16
Authors: Srpan Katja , Cartwright Adam N. R. , Davis Daniel M
Published: January 2023

Natural Killer (NK) cells are cytotoxic lymphocytes that can recognize and kill virally infected or tumour transformed cells by the secretion of cytolytic granules containing perforin. An individual NK cell can kill several target cells sequentially. Each target cell can trigger NK cell activation via different activating ligands and here we report that the order in which ligands are encountered affects the NK cell response. When NK cells are repeatedly activated via their Fc receptor CD16, with the therapeutic antibody rituximab, perforin secretion decreases with each stimulation. However, perforin secretion is restored to its initial level upon subsequent activation by MICA, which ligates NKG2D. Repeated stimulation of NK cells via MICA also decreases the degranulation capacity of NK cells but, strikingly, this effect cannot be rescued by a subsequent stimulation with rituximab. We found that the mechanism underlying these differential outcomes involves shedding of CD16, which occurs upon NK cell activation through both CD16 and NKG2D. Shedding of CD16 renders the cells insensitive to further activation via that receptor but they remain competent for further activation by NKG2D. We also found that NK cells are more motile on rituximab-coated surfaces than on MICA-coated surfaces and their migration speed decreases upon inhibition of CD16 shedding. Thus, although CD16 shedding hinders NK cell responsiveness it can serve to augment NK cell motility to move between target cells. Together these data indicate that manipulation of CD16 shedding might be an important target for NK cell-based therapies including treatments with therapeutic antibodies.