bluuebunny/crossref_metadata_2025 huggingface dataset:
MINK1 kinase suppresses TH17 differentiation and autoimmune inflammation via inhibition of the TGF-β pathway
Authors: Lu Linrong
Published: January 2023

TH17 cells (interleukin-17-producing helper T cells) are present at the sites of tissue inflammation and associated with the induction of multiple autoimmune diseases. The differentiation of TH17 cells is instructed and critically regulated by various cytokines in the microenvironment, which drive intracellular signaling pathways and transcription profiles that determine the cell fate. Here we show that MINK1, a germinal center kinase (GCK) family kinase, plays as a negative regulator for TH17 cell differentiation. Mink1−/− mice has higher frequency of TH17 cells in vivo and developed more severe experimental autoimmune encephalomyelitis (EAE) than wild type (WT) mice. Further experiments revealed that MINK1 directly interacts with SMAD2 and phosphorylates SAMD2 at T324 residue in its α-helix 1 region, which inhibits the C-terminal phosphorylation (activation) of SMAD2. Moreover, we found that MINK1 kinase is activated by T cell receptor stimulation in a ROS (reactive oxygen species)-dependent manner, which can be compromised by ROS scavenger N-acetyl cysteine (NAC). Accordingly, NAC treatment markedly boost the in vitro induction of TH17 cells in a MINK1-dependent manner and potentiates TH17 cells generation and development of EAE in vivo. In sum, our data not only demonstrate a regulatory role of MINK1 kinase in TH17 cell differentiation, but also provide the molecular insight into a possible mechanism, by which increased dietary antioxidants supplement might represent a health risk factor for the development of autoimmune diseases through increased induction of TH17 cells.
NFAT transcription factors promote the generation of follicular helper T cells in response to acute viral infection
Authors: Martinez Gustavo J , Hu Joyce , Pereira Renata , Crampton Jordan , Togher Susan , Bild Nicholas , Crotty Shane , Rao Anjana
Published: January 2023

Follicular helper T (Tfh) cells provide B cell help in germinal center reactions that support class switching, somatic hypermutation, and the generation of high-affinity antibodies. Here we show that deficiency in NFAT1 and NFAT2 in CD4+ T cells leads to impaired germinal center reactions upon viral infection due to reduced Tfh cell differentiation and defective expression of proteins involved in T:B interactions and B cell help, including ICOS, PD-1, and SLAM family receptors. ChIP-seq data suggest that NFAT proteins likely directly participate in regulation of genes important for Tfh cell differentiation and function. NFAT proteins are important TCR and Ca2+-dependent regulators of T cell biology, and here we demonstrate a major positive role of NFAT family members in Tfh differentiation.
Differential regulation of TH1, TFH, and TCM gene programs by cytokine-mediated events
Authors: McDonald Paul W , Read Kaitlin A. , Baker Chandra E. , Anderson Ashlyn E. , Powell Michael D. , Ballesteros-Tato Andre , Oestreich Kenneth J
Published: January 2023

During the course of an immune response, multiple effector T helper cell types emerge to combat the initial infection, with subsequent emergence of long-term CD4+ T memory cells to ensure that a second exposure to the same pathogen will not cause disease. CD4+ T helper cell populations that are critical for this process include T helper 1 (TH1) and T follicular Helper (TFH) effector cells and long-lasting central memory T (TCM) cells. Previous work has demonstrated a level of flexibility between effector TH1 and TFH-like cell populations, with the respective gene programs regulated by alterations in interleukin 2 (IL-2) signaling. Here, we demonstrate that in response to decreased IL-2 signaling, effector TH1 cells are capable of initiating the expression of both TFH and TCM-like gene programs, including cell surface expression of IL-6Ra and IL-7R. Importantly, exposure of these cells to either IL-6 or IL-7 augments or inhibits the TFH gene program, respectively, suggesting a potential mechanism whereby cytokine availability may influence TFH or TCM cell function. Consistent with this model, we have identified a novel population of IL-6Ra+IL-7R+ cells that emerge at late time points post-infection in an in vivo mouse model of influenza infection. Further research into the temporal and spatial expression of IL-2, IL-6, and IL-7 in the modulation of these cells represents an exciting avenue of research with wide-ranging therapeutic implications.
Mis-acylation of tRNAs suppresses protective immune responses which control cutaneous inflammation by infection
Authors: HWANG HyunJu , Lee Byunghyuk , Lee Jeong Woong , Hong Changwan
Published: January 2023

The mouse sticky mutation which is a missense mutation in editing domain of the alanyl-tRNA synthetase gene impairs the proofreading activity during amino-acylation of tRNAs. An editing-defective tRNA synthetase induces the accumulation of mis-folded proteins in the neuron which is associated with neuro-degenerative disorder. Although the sticky mice were characterized by the rough, unkempt, sticky appearance, it is not clear whether the sticky mutation is associated with inflammatory disease. The protective role of T and B cells during cutaneous infection by a variety of pathogens has been known well. We therefore investigated how sticky mutation affects immune responses and linked to sticky phenotype. First of all, we examined immune cell numbers in sticky mice. LN cell number in sticky mice was significantly reduced compared to Sti/+ and WT mice, while LN profiles of sticky mice were not big different from control groups. Reduced cellularity by sticky mutant results from impaired proliferation, not by IL-7 signaling which is critical in T cell development and homeostasis. Interestingly, we found that mutation in tRNA processing reduced IFNγ and IL-17 production from stimulated T cells. And it also reduced Immunoglobulin production in B cells, particularly IgG1 and IgE which has an important role in resistance to bacterial and parasite infection. These findings indicate that disruption of translational fidelity induces deterioration of immune functions such as impaired cytokine and antibody production, resulting in reduction of the resistance to various infections. Furthermore, it provides a novel mechanism which the alteration of tRNA pool in inflammatory diseases affects the cellular and humoral immune responses.
IL-22 Increases Metastatic Properties in Androgen-Independent Prostate Cancer
Authors: Shafran Jordan , Andrieu Guillaume , Denis Gerald V
Published: January 2023

New evidence is emerging that suggests that a subpopulation of individuals characterized as metabolically abnormal obese (“MAO”; i.e.: co-morbid Type 2 diabetes (T2D), hypertension and systemic inflammation) have a much greater risk for cancer mortality compared to obese but metabolically healthy individuals (“MHO”; i.e.: non-diabetic, lack hypertension, non-inflamed). Because MAO individuals have systemic and local adipose inflammation associated with obesity co-morbidities, it is now hypothesized that alterations to signal transduction pathways in the tumor microenvironment (which come as a result of increased T2D-associated Th17 cytokines (IL-17A, IL-17F, IL-21, IL-22, etc.)), lead to a more advanced, pro-metastatic cancer phenotype. Furthermore, additional observations have also shown that Bromodomain and Extraterminal domain (BET) proteins (BRD2, BRD3 and BRD4) act as transcriptional effectors for these Th17 cytokines. Based on these data, we sought to determine whether (i); T2D-associated Th17 cytokines increase metastatic properties in androgen-independent prostate cancer cells, (ii) signaling pathways utilize Th17 cytokines to exacerbate metastatic properties and (iii) BET proteins act as effectors for Th17 cytokine signaling. Here we report that IL-22 increases migration and invasion in androgen-independent prostate cancer. This phenotype is achieved, in part, through the activation of STAT3 in a BRD4-dependent manner. Moreover, BRD2 opposes BRD4 in this process. Overall, these results identify a novel mechanism that is critical for prostate cancer metastasis.
Unique roles of TECs, DCs and B cells in Treg repertoire formation
Authors: Watanabe Masashi , Breen Michael , Alkhaleel Farrah , Hodes Richard J
Published: January 2023

Foxp3+ CD4+ regulatory T cells (Treg) are an essential component in maintenance of immunological tolerance against self. Treg are generated in the thymus by relatively strong TCR recognition of self-peptide-MHCII complex (pMHCII) on antigen presenting cells (APCs). In addition to pMHCII dependent TCR signaling, B7 expression on APC is critical for Treg generation through CD28 co-stimulation. APCs that might be capable of providing such TCR/CD28 signaling to developing thymocytes include thymic epithelial cells (TECs), dendritic cells (DCs) and B cells. To date, however, how each APC contributes to the generation of the Treg repertoire specific for self-antigen under physiological condition is not fully understood. To address this question, we generated B7 conditional knockout mice enabling selective deletion of B7 from each APC type and assessed the effect of this cell type-specific perturbation of APC function on Treg generation. We first observed that B7 deletion from DCs, but not TECs or B cells, resulted in a substantial decrease in total Treg numbers. Interestingly combined B7 deletion from two (TECs + DCs) or three (TECs + DCs + B cells) APCs showed additive effects in decreasing the total Treg population size. In contrast, we found that the pMOG-specific Treg population depended on B7 on DCs, with no individual or additive effect of B7 deletion from TECs or B cells. These results suggested that TECs, DCs and B cells have unique and non-redundant roles in Treg repertoire formation. Further studies are currently underway to determine the role of TECs, DCs and B cells for the generation of Treg repertoires with distinct self-antigen specificities by utilizing panel of pMHCII-tetramers.
Early differences in TCR signal strength modulate effector and memory differentiation of CD4+ T cells responding to viral infection.
Authors: Snook Jeremy , Williams Matthew A
Published: January 2023

Heterogeneity in TCR-dependent activation signals influences both effector differentiation and memory formation of CD4+ T cells. Our previous studies found that the formation of CD4+ memory T cells specific for lymphocytic choriomeningitis virus (LCMV) was associated with the selection of TCRs with sustained antigen binding properties. The goal of the current study was to define early TCR-dependent activation events that promote memory differentiation. We found that early in vitro and in vivo activation of distinct T cell clones specific for the same LCMV antigen demonstrated highly variable NFAT transcriptional activity and expression of early activation markers such as CD25. Clones with enhanced memory potential had reduced NFAT activity in vitro and rapid down-regulation of CD25 in vivo. We further observed decreased CD3ζ and Zap-70 phosphorylation, as well as a decrease in NFAT-dependent gene transcription, in virus-specific CD4+ T cells that displayed decreased CD25 surface expression early after LCMV infection. Adoptive transfer of CD25hi or CD25lo effector T cells (d3 or d5 post-infection) into infection-matched hosts revealed that the vast majority of both Th1-like and Tfh-like memory cells derived from CD25lo effector cells. RNASeq analysis further confirmed enrichment of genes associated with enhanced memory development within CD25lo effector cells, including Tcf-1 and OCA-B, but decreased expression of genes associated with enhanced effector activity. We conclude that differences in TCR signal strength account for clonal differences in CD4+ memory T cell differentiation, with weaker early TCR signals permitting long-term memory formation without terminal effector differentiation.
Human memory CD8 T cell effector potential is epigenetically preserved during in vivo homeostasis
Authors: Abdelsamed Hossam Aly
Published: January 2023

Maintenance of memory CD8 T cell quantity and quality through antigen-independent homeostatic proliferation is vital for sustaining long-lived T cell-mediated immunity, yet the underlying mechanisms that preserve memory T cell functions during homeostasis remain largely unexplored. Here we show that preservation of effector-potential among human memory CD8 T cells during in vitro and in vivo homeostasis is coupled to maintenance of memory-associated DNA methylation programs. Whole-genome bisulfite sequencing of primary human naïve, short-lived effector memory (Tem), and longer-lived central memory (Tcm) and stem cell memory (Tscm) CD8 T cells identified demethylated promoters of effector molecules that are poised for rapid expression among all memory cell subsets. Effector-loci demethylation was heritably preserved during IL-7 and IL-15 mediated in vitro cell proliferation. In contrast to the effector-potential, antigen-independent proliferation induced a phenotypic conversion of Tcm and Tscm memory cells into Tem cells that was coupled to increased methylation of the CCR7 locus. Furthermore, in vivo proliferation of haploidentical donor memory CD8 T cells in lymhodepleted recipients resulted in a similar preservation of effector-associated methylation programs while enriching for Tem-associated programs. These data demonstrate that long-lived human memory CD8 T cells retain the ability to undergo antigen-independent epigenetic reprogramming during their developmental conversion into other memory subsets while at the same time preserving the poised effector state utilized by all memory T cells.
Defining the role of CD69 in the formation of resident memory CD8+ T cells
Authors: Walsh Daniel , da Silva Henrique Borges , Hamilton Sara , Masopust David , Jameson Stephen
Published: January 2023

Resident memory CD8+ T cells (TRM) occupy parenchymal tissues and have a well-defined role in protecting from reinfection. CD69 is often considered a definitive marker of TRM, and CD69 can regulate T cell trafficking through its interaction with the G-protein-coupled receptor S1PR1. However, the functional requirement for CD69 in controlling the generation and maintenance of CD8+ TRM is unclear. We tested this using co-transfer of CD69 sufficient and deficient P14 CD8+ T cells specific for gp33/Db in the context of acute LCMV and Flu-GP33 infections. Surprisingly, we found that CD69 was not necessary for TRM establishment in most tissues. However, we found that depending on the infectious agent, CD69 could promote TRM localization in certain sites. We propose that the requirement for CD69 is context dependent, and that a combination of factors including the tissue microenvironment and infectious agent dictate its influence on development of resident memory.
ATP receptor P2rX7 direct the generation of long-lived memory CD8+ T cells
Authors: da Silva Henrique Borges , Wang Haiguang , Fonseca Raissa , Block Katharine E. , Masopust Davd , Hogquist Kristin Ann , Jameson Stephen C.
Published: January 2023

P2rX7 is an ATP-sensitive purinergic receptor often known for its role in neuropathic pain and innate immunity. P2rX7 is also known to mediate CD4+ T cell death upon stimulation. To evaluate whether CD8+ T cell immune responses are also regulated by P2rX7, we infected mice with LCMV and tracked gp33-specific P14 CD8+ T cells. Unexpectedly, we found that P2rX7 expression on memory precursors (MPEC) positively correlated with generation of central memory CD8+ T cells (TCM). P2rX7KO CD8+ T cells also had reduced memory generation, especially TCM and resident memory (TRM). This defect was cell-intrinsic, as revealed by bone marrow chimeras and co-adoptive transfer of P2rX7KO and WT P14 T cells. Defective memory generation by P2rX7KO CD8+ T cells was preceded by impaired MPEC generation as well as diminished Tcf1 expression. Strikingly, P2rX7KO CD8+ T cells had an early and sustained impairment of proper mitochondrial function, as observed by flow cytometry (lower membrane potential and mitochondrial mass) or seahorse assays (lower SRC, higher proton leak). These defects were observed specifically on TCM, but not on effector memory (TEM) or long-lived effector cells (LLEC). P2rX7KO CD8+ T cells also showed reduced recall proliferation following secondary antigen stimulation. Importantly, treatment with P2rX7 pharmacological inhibitors during the immune response effector phase lead to similar defects in TCM and TRM. These findings show that P2rX7 is critical for development of a long-lived memory CD8+ T cell response. Also, they have important implications for the immunological effects of P2rX7 inhibitors used for pain control, and the impact of loss-of-function P2rX7 polymorphisms on immunity and the response to vaccines.
ADAMs10/17 deficient mast cells exhibit decreased IgE-mediated activation in vitro.
Authors: Luker Andrea , Conrad Daniel
Published: January 2023

Allergies and asthma are common ailments that are on the rise around the world. Mast cells play a direct role in the signs and symptoms characteristic in allergic patients. The family of A Disintegrin And Metalloproteinases (ADAMs) are involved in regulating many cellular processes by cleaving surface receptors, ligands, and signaling molecules. We sought to determine the role of ADAMs10/17 in bone marrow-derived mast cell (BMMC) activity. In dual inhibitor-based studies, percent degranulation and cytokines released by IgE-mediated activation were significantly reduced. Interestingly, ionomycin-activation was unchanged with inhibitor treatment, suggesting that ADAMs10/17 may be involved in IgE-mediated mast cell activation upstream of calcium influx. Preliminary results with ADAM17MC−/−BMMCs have confirmed the inhibitor data and provided interesting results regarding increased mast cell survival and proliferation. These data suggest that targeting ADAMs10/17 could attenuate mast cell-related TH2 responses.
Mechanistic insight into the suppressive effects of stem cell factor during IgE-mediated activation in bone marrow-derived mast cells.
Authors: Lyons David O , Pullen Nicholas A
Published: January 2023

Treatment of bone marrow derived mast cells (BMMCs) with stem cell factor (SCF) is common practice when differentiating mast cells for experimentation. The enhanced growing potential from including SCF during mast cell culture and differentiation allows for easier maintenance and quicker experiments. Recent publications have begun to identify potentially negative effects of treatment with SCF regarding mast cell activation. The mechanism of SCF inhibition on mast cell activation is mostly unknown, but BMMCs derived with the addition of SCF function differently than untreated cells when mast cells are primed and subsequently activated. This study provides insight into the specific targets of SCF treatment and the impact SCF has on the ability of mast cells to activate. Running ELISAs in conjunction with real-time qPCR, we identified differentially expressed proteins involved in FCɛRI signaling and mast cell activation in BMMCs exposed to SCF. We examine the effects of SCF on Fyn, Lyn, Syk and Stat5 protein expression compared to mast cells raised without SCF. These data are also placed in the context of other immunosuppressive mechanism, (e.g. TGFβ). The presented data allow researchers to better understand the implications that may result from SCF treatment of BMMCs, especially with respect to examining IgE-mediated functions.
Suppression of mast cell activation by exopolysaccharide produced by Bacillus subtilis.
Authors: Swartzendruber Julie A , Tolia Sanika , Knight Katherine L
Published: January 2023

The incidence of allergies is on the rise, and to prevent or treat allergic diseases, we need a means of inhibiting immune pathways leading to allergic responses. Reduced exposure to microbes, increased use of antibiotics and changes in diet all lead to an increase in allergies. The consequences of these changes in lifestyle include an alteration of intestinal bacteria that are important for development of the immune system. Our work seeks to utilize the beneficial effects of gut microbiota to manipulate allergic responses. A key cell responsible for the symptoms of allergies is the mast cell. We are using the probiotic bacterium Bacillus subtilis which produces an exopolysaccharide (EPS) with anti-inflammatory activity. We found that EPS treatment alone was unable to alter mast cell degranulation following IgE-mediated activation, however treatment with peritoneal cells from EPS-injected mice prevented mast cell degranulation. We hypothesize that EPS induces a suppressive cell in the peritoneal cavity that leads to the suppression of mast cell activation and could be used to suppress the immune response against common food or environmental factors.
The E3 ligase Itch suppresses the development of pathogenic Basophils
Authors: Rathinam Chozha Vendan , Thummar Keyur
Published: January 2023

Basophils are one of the major players in the pathophysiology of allergy related immune disorders. In spite of their key roles in autoimmune and inflammatory disorders in humans, molecular mechanisms that control basophils remain largely unknown. Therefore we have been involved in elucidating the biochemical and molecular mechanisms through which development and functions of basophils are controlled in the immune system. In this study, we utilized the Itch deficient mouse as a model system for understanding the biology of Basophils. Itch is an E3 ubiquitin ligase that belongs to the HECT family. Mice deficient for itch develop severe autoimmune defects (‘itchy’ phenotype) and patients with ITCH mutations show characteristic clinical features, including multisystem autoimmune disease. Our preliminary analysis indicated that itch deficiency leads to Basophilia, hyperactivation of basophils, elevated IL4 expression and Th2 priming by basophils, in a cell intrinsic manner. Strikingly, depleting basophils in itch deficient mice resulted in rescue of the ‘itchy’ phenotype. Our mechanistic studies demonstrated that notch signals are elevated in itch deficient basophils, due to reduced ubiquitylation of notch. Interestingly, ablation of notch signals in itch deficient mice resulted in normal frequencies of basophils and rescue of the ‘itchy’ phenotype. At this annual meeting, we would be discussing the precise involvement of the signaling pathways and molecular mechanisms in the restriction of pathogenic basophils.
Fluvastatin enhances IL-33-mediated inflammatory functions in mast cells
Authors: Taruselli Marcela T , Kolawole Elizabeth Motunrayo , Ryan John J
Published: January 2023

Statins are commonly used HMG-CoA reductase (HMGCR) inhibitors prescribed for treating cardiovascular diseases by lowering cholesterol levels. Independent of lowering cholesterol, statins have been shown to inhibit proinflammatory functions in macrophages and T cells. Our lab has previously found that fluvastatin suppresses IgE activation of mouse and human mast cells in vitro, while also protecting C57BL/6 mice from passive systemic anaphylaxis. However, a recent study found that statins can induce proinflammatory cytokines in LPS stimulated macrophages. Due to the conflicting nature of statins, we extended our studies to IL-33, a strong mast cell activator with signaling similar to LPS. In contrast to our findings with IgE signaling, we found that fluvastatin augmented TNF and IL-6 production induced by IL-33 in primary mouse and human mast cells. These effects were dependent on the mitogen SCF, since in its absence the enhancing effects were absent. Mevalonic acid, the product of the HMGCR reaction, reversed fluvastatin effects. Additionally, fluvastatin effects required loss of isoprenoids downstream of HMGCR, since geranylgeranyl pyrophosphate and farnesyl pyrophosphate reduced fluvastatin enhancement of TNF and IL-6 secretion. Fluvastatin enhanced NFκB transcriptional activity in IL-33 stimulated BMMC, and by using an NFκB inhibitor, we found that fluvastatin enhancement of IL-6 and TNF induced by IL-33 is NFκB dependent. This study demonstrates that HMGCR blockade can have either anti or proinflammatory effects on mast cell function, dependent upon the stimulus. These data further reveal the divergent roles for isoprenoid lipids in mast cell function, which may offer new clinical targets for inflammation.
IL-10 enhances IgE-independent IL-33-mediated mast cell cytokine production
Authors: Mathias Clinton B , Rovatti Jeffrey , Polukort Stephanie
Published: January 2023

IgE antibodies play a critical role in antigen-mediated mast cell activation. However, mast cells can be activated via IgE-independent mechanisms including signaling via the IL-33 receptor ST2. We have previously shown that the Th2-derived cytokine IL-10 can enhance IgE-dependent mucosal mast cell activation and promote mast cell function and survival. However, whether the effects of IL-10 on mast cells are global in nature and not limited to IgE-mediated signaling is not clear. To determine whether IL-10 can prime the activation of mast cells mediated by IgE-independent signals such as IL-33, we assessed the effects of rIL-10 exposure on IL-33-stimulated mast cells and their subsequent activation via IgE. Bone marrow-derived mast cells (BMMCs) from wild-type (WT) and IL-10−/− mice were cultured with rIL-33 with or without rIL-10 and activated with DNP-IgE and antigen. Exposure to IL-10 enhanced cytokine production in both WT and IL-10−/− BMMCs, and this was significantly increased after activation via IgE. IL-10−/−BMMCs exhibited decreased cytokine production, but this was restored upon culture with rIL-10. Interestingly, IL-33 stimulation (and subsequent activation with IgE) induced comparably enhanced levels of cytokine production, specifically IL-13 and IL-6, in both WT and IL-10−/−cells, suggesting that ST2-mediated signals can overcome the lack of IL-10. However, the addition of exogenous IL-10 to IL-33-stimulated cultures further enhanced the levels of these cytokines not only in IgE-activated cells, but also in cells stimulated with IL-33 alone. Our observations therefore suggest that IL-10 can play critical roles in enhancing mast cell function both via IgE-dependent and independent mechanisms.
Unlocking mast cell diversity in human nasal polyps
Authors: Dwyer Daniel F , Ordovas-Montanes Jose M , Buchheit Kathleen M. , Lai Juying , Katz Howard R , Bhattacharyya Neil , Shalek Alex K , Laidlaw Tanya M , Boyce Joshua A , Barrett Nora A
Published: January 2023

Mast Cells (MCs) infiltrate the nasal polyps of patients with chronic rhinosinusitis (CRS) and the bronchial mucosa of patients with mild, moderate, and severe asthma. MCs are a significant source of IL-33-elicited Th2 cytokines in the sputum of asthmatics and upon activation generate or release high levels of mediators including tryptase, histamine, and prostaglandin D2. Although airway MC heterogeneity can readily identified through protease immunostaining and tissue sub-localization, the functional distinctions and developmental relationships between MC subsets remain uncertain, in part because of the lack of flow cytometric markers to distinguish them and technical difficulties in isolating MCs. Using a novel flow cytometric approach we have identified several distinct MC subsets in the nasal polyps of patients with CRS that vary in expression of FcER1, c-kit, B7, CD203 and chymase. Nasal polyp MCs were further characterized through unbiased single cell RNA sequencing, revealing heterogeneity within the MC population and providing novel insight into transcriptional differences between these subsets.
Natural IgM initiates the immunological cascade against minor antigen-mismatched cells, illustrating the dependency and unique role of APC subtypes
Authors: Atif Shaikh M , Gibbings Sophie , Torres Raul M , Kedl Ross , Jakubzick Claudia V
Published: January 2023

In organ transplantation a major obstacle is the immunological response against mismatches of major and minor histocompatibility antigen (Ags). Even when MHC Ags are matched between donor and recipient, minor Ags can elicit graft rejection. To date, it is unclear how endogenous antigen-presenting cells (APCs) recognize, coordinate and induce immunological responses against minor Ag-mismatched cells, particularly in the absence of pathogen-associated molecular patterns (PAMPs). We hypothesized that each APC subtype is dependent on each other to complete an immunological response against mismatches of minor Ags. Using a well-established minor Agmismatch model, we demonstrated that the elimination of any one APC subtype (Batf3+ DCs, Irf4+ DCs, Ly6C+ monocytes or B cells) resulted in a diminished or abolished immune response against minor Ag-mismatches. Specifically, we demonstrated that this immunological response began with the recognition of minor Ag-mismatched cells by natural IgM, which resulted in an immune complex formation. This cellular immune complex was then acquired by MHC II presenting APCs, of which one was MHC II expressing Ly6C+ monocytes. Subsequently, Ly6C+ monocyte induced Ag-specific CD4+ T cells licensed Batf3+ DCs via CD40 to present mismatches of minor antigen to Ag-specific CD8+T cells, which were required for the cytotoxic elimination of minor-Ag mismatched cells. Overall, our in vivo findings suggest a sequential, coordinated immunological event is required for the rejection of minor Ag-mismatched cells.
Rab11a Regulates MHC Class II Recycling and Promotes Antigenic Peptide Exchange in Recycling Endosomes of Mature Dendritic Cells
Authors: Cho Kyung Jin , Ishido Satoshi , Eisenlohr Lawrence , Roche Paul
Published: January 2023

Dendritic cells (DCs) specialize in antigen capture and maintain a highly dynamic pool of peptide-MHC class II complexes (MHC-II) that continuously internalized and degraded in immature DCs. By contrast, in mature DCs pMHC-II internalizes but is efficiently recycled back to the plasma membrane for recognition by CD4 T cells. Regulation of pMHC-II biosynthesis and degradation is essential for optimal DC function, however the molecular basis of pMHC-II recycling and the cellular machinery that orchestrates pMHC-II trafficking are incompletely understood. In the present study we found that internalized pMHC-II on mature DCs, but not immature DCs, colocalizes with the regulator of endosome recycling Rab11a. Using a lentivirus encoded shRNA, we show that Rab11a knock-down inhibits recycling of pMHC-II in mature DCs. Moreover, Rab11a knock-down results in the intracellular accumulation of internalized surface pMHC-II. We also show that Rab11a knock-down in mature DCs specifically inhibits presentation of a “recycling endosome” epitope of internalized influenza hemagglutinin only in mature DCs. Our study thus identifies Rab11a as an important regulator of pMHC-II recycling that can enhance presentation of certain antigenic peptides in mature DCs.
Defining the Impact of Antigen Presentation by Dendritic Cells and Macrophages in CNS Cancer Using a Novel H-2Kb MHC Class I Conditional Knockout Mouse
Authors: Malo Courtney Sarah , Goddery Emma N , Huggins Matthew A , Jin Fang , Hansen Michael J , Pavelko Kevin D , Johnson Aaron J
Published: January 2023

Picornavirus vaccination holds great promise in generating tumor-specific CD8 T cell responses. However, the central question remains regarding which cell types are required to present antigen to CD8 T cells to initiate tumor immunity. Previously, studies addressing antigen presentation in the central nervous system (CNS) have relied on ablation of entire cellular compartments. In order to address this critical question without elimination of critical cell types, we generated a novel H-2Kb LoxP transgenic animal. This allows us to conditionally delete the H-2Kb major histocompatibility complex (MHC) class I molecule using cre recombinase driven by cell-specific promoters. We hypothesized that dendritic cells (DCs), but not macrophages (MΦs), are required to prime tumor-specific CD8 T cells, and that DC-specific deletion of H-2Kb would abrogate efficacy of glioma-specific picornavirus vaccination. We assessed tumor burden and immune infiltration in CMV-cre Kb cKO animals (ubiquitous Kb deletion), CD11c-cre Kb cKO animals (DC-specific deletion), LysM--cre Kb cKO animals (MΦ-specific deletion) and cre− littermates. We demonstrated that DC-specific deletion of H-2Kb increased the proportion of glioma-bearing animals. Additionally, we determined that DC-specific deletion, but not MΦ-specific deletion, of H-2Kb reduced the proportion of tumor-specific CD8 T cells and increased tumor burden, but not as severely as ubiquitous H-2Kb deletion. Thus, DCs, but not MΦs, are critical for generation of a complete anti-glioma T cell response. To our knowledge, this is the first report of a model of conditional MHC class I deletion, and provides unique insight into the impact of antigen presentation in the CNS.
HLA–DM senses peptide-MHC class II interactions throughout the peptide binding groove.
Authors: Reyes-Vargas Eduardo , Barker Adam P , Zhou Zemin , He Xiao , Jensen Peter E
Published: January 2023

HLA-DM is an enzyme-like molecule that edits the MHC class II peptide repertoire by catalyzing multiple rounds of peptide exchange on MHCII molecules. It differentially edits peptide-MHCII complexes and thereby skews the foreign and self-peptide repertoire available for activation or tolerance induction of CD4 T-cells. Though it has important implications for the adaptive immune response, the determinants of susceptibility to DM mediated editing remain undefined. It has been suggested that unstable interactions at the N-terminus of a bound peptide is a key determinant for DM editing and that partial dissociation of the peptides N-terminus is necessary for this sensitivity. Herein, we assessed individual components of the catalytic mechanism in real-time kinetic measurements of DM activity. Using a series of amino acid substituted peptides, we analyzed the effects of disfavored interactions on the catalytic turnover and affinity of DM editing and find that disruptions throughout the peptide binding groove contribute to both the affinity of DM for a given peptide complex and to the rate of peptide dissociation from the catalytic complex. Moreover, disruptions in the conserved hydrogen bond network or disfavored amino acid residues near a peptides C-terminus result in increased DM affinity and catalytic turnover. Furthermore, complexes with N-terminal truncated peptides show increased catalytic turnover when compared to parent complexes, indicating that the functionally active DM-peptide-MHCII catalytic complex can operate on a peptide-MHCII complex with full occupancy. Together, this implies that interactions at the DM-MHCII interface are intimately linked to unstable interactions throughout the peptide-binding groove.
ERAP1 deficiency mitigates HLA-B27 misfolding and ER stress, and protects HLA-B27 transgenic rats from arthritis and orchitis
Authors: Tran Tri M , Gill Tejpal , Bennett Joshua , Holt Vance , Hong Sohee , Taurog Joel D , Colbert Robert A
Published: January 2023

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is associated with ankylosing spondylitis (AS) in HLA-B27-positive individuals. ERAP1 optimizes peptide length for binding to MHC class I molecules. However, the role of HLA-B27-bound peptides in AS pathogenesis remains unclear. We investigated epistasis between ERAP1 and HLA-B27 in a rat model of spondyloarthritis (SpA). ERAP1-deficient rats generated by TALEN-mediated genome editing were crossed with disease prone HLA-B27 transgenic (B27 Tg) rats (33-3 transgene). ERAP1 deficiency reduced the prevalence of arthritis (4/34 in ERAP1−/− vs. 12/37 in ERAP1+/+; p<0.05) and testicular inflammation (6/34 vs. 15/37, p<0.05) in male B27 Tg rats. Wild type and HLA-B7 transgenic (B7 Tg) rats with and without ERAP1 remained healthy. Bone marrow-derived macrophages (BMDMs) from B27 Tg ERAP1−/− rats exhibited a 12-fold (p<0.05) increase in MARB4-reactive cell surface B27 complexes containing long peptides, a 30% reduction of total cellular unfolded B27 heavy chains (HC) (p<0.01), and a 32% increase in the ratio of folded/unfolded B27 HC (p=0.01). ERAP1 deficiency also reduced by 50% the formation of disulfide-linked dimers and B27-BiP complexes (p<0.01). ERAP1-deficiency attenuated BiP and CHOP induction and XBP1 splicing (50%, 27%, and 25%, respectively; p<0.01) caused by IFNg-induced B27 upregulation. These results indicate that ERAP1-deficiency is protective for arthritis development in 33-3 B27 Tg rats, and raise the possibility that alleviation of ER stress may contribute to protection, although further investigation is needed to define the mechanism.
Lipophilic air pollutants inhibit endocytic lipid antigen presentation
Authors: Huang Shouxiong , Sharma Manju , Zhang Xiang , Zhang Shuangmin , Niu Liang , Ho Shuk-mei , Chen Aimin
Published: January 2023

Air pollutants as non-heritable factors are recognized as triggers for various human inflammatory diseases, in which T cells are central in producing cytokines and determining disease outcomes. We postulated that lipid antigen presentation mediated by cluster of differentiation 1 (CD1) proteins for T cell activation is susceptible to lipophilic air pollutants. To test this notion, we determined whether common lipophilic pollutants benzo[a]pyrene and diesel exhaust particles impact on the activation of lipid-specific T cells. Our results demonstrated that the expression of CD1a and CD1d proteins, and the activation of CD1a- and CD1d-restricted T cells were sensitively inhibited by benzo[a]pyrene even at low concentrations detectable in exposed human populations. Similarly, diesel exhaust particles showed marginal inhibitory effect. Using transcriptomic profiling, we discovered that gene expression for regulating endocytic and lipid metabolic pathways was perturbed by benzo[a]pyrene. Imaging flow cytometry also showed that CD1a and CD1d proteins were respectively retained in early and late endosomal compartments, supporting an impaired endocytic lipid antigen presentation for T cell activation upon benzo[a]pyrene exposure. This work conceptually demonstrates that lipid antigen presentation for T cell activation is inhibited by lipophilic pollutants through profound interference with gene expression and endocytic function, likely further disrupting regulatory cytokine secretion and ultimately exacerbating inflammatory diseases.
Rationally designed TLR4 ligands for vaccine adjuvant discovery.
Authors: Harberts Erin M , Gregg Kelsey A , Gardner Francesca M , Pelletier Mark R , Yu Li , McCarthy Michael P , Marshall Jason D , Ernst Robert K
Published: January 2023

Adjuvant properties of bacterial cell wall components like MPLA are well described, and have gained FDA approval for use in vaccines such as Cervarix. MPLA is the product of chemically modified lipooligosaccharide (LOS), altered to diminish toxic proinflammatory effects while retaining adequate immunogenicity. Despite the virtually unlimited number of potential source bacterial strains, useable compounds within this promising class of adjuvants remain unfortunately low. We have developed bacterial enzymatic combinatorial chemistry (BECC) as a method to generate functionally diverse and rationally designed lipid A. BECC removes endogenous, or introduces exogenous, lipid A-modifying enzymes to bacteria, effectively reprogramming the lipid A biosynthetic pathway. In this study, BECC is applied within an avirulent strain of Yersinia pestis (Yp), to develop structurally distinct LOS molecules that elicit differential TLR4 activation. Using reporter cell lines that measure innate immune activated NF-κB transcription, BECC-derived molecules were screened for the ability to induce a lower pro-inflammatory response than E. coli LOS. Structures exhibit varied, dose-dependent TLR4-driven NF-κB activation with both human- and mouse-TLR4 complexes. Additional cytokine secretion screening identified molecules that induce comparable levels of TNF-α and IL-8, as compared to PHAD. Lead candidates demonstrated potent immunostimulation in mouse splenocytes, human primary PBMCs, and human monocyte-derived DC. This newly described BECC system allows for directed programming of lipid A synthesis and has the potential to generate a diverse array of TLR4 agonist candidates comparable if not preferable to PHAD and MPLA.
Identification of microRNA-21 as a biomarker in live attenuated Leishmania vaccine induced protective immunity
Authors: Bhattacharya Parna , Ismail Nevien , Kaul Amit , Gannavaram Sreenivas , Nakhasi Hira L
Published: January 2023

No vaccine exists against visceral leishmaniasis. Towards developing an effective vaccine, we have reported extensively on the immunogenicity of live attenuated LdCentrin−/− mutants in animal models. However, for the use of LdCen1−/− in humans there is a need to develop biomarkers that can predict the immunogenicity of LdCen−/− at an earlier stage of vaccination. Unbiased gene expression profiling of US blood donor’s PBMCs infected with Leishmania parasites (LdWT or LdCen−/−) ex-vivo resulted in the identification of several miRs that regulate important cytokine genes. Importantly, we found a strong induction of miR-21, known to control the expression of IL-12 mRNA, in LdWT infection compared to LdCen−/− infection. IL-12 produced by DCs is critical for priming a host protective Th1 cell response during Leishmania infection. Validation of human PBMC data in a mouse model revealed an enhanced expression of miR-21 in virulent LdWT infected dendritic cells (DCs) compared to LdCen−/−. This correlated with low levels of IL-12 mRNA in WT compared to LdCen−/− infected DCs. Silencing of miR-21 resulted in an augmented induction of IL12 in LdWT infected DCs both in vitro and in vivo, illustrating the role of miR-21 in LdWT infection mediated suppression of IL12. In contrast, LdCen−/− infected DCs showed a strong induction of IL12, and miR-21 silencing resulted in a further increase in IL12 levels. Additionally, blocking of miR-21 enabled strong proliferation of CD4+ T cells in mice infected with LdWT parasites. Thus lower level of miR-21 in a vaccinee may indicate the safety and immunogenicity consistent with a protective response of the vaccine. Our data suggests that miR-21 could be used as a biomarker of protection in anti-leishmanial vaccines.
Utilizing previously identified in vitro correlates of protection to predict the efficacy of a novel vaccine candidate against the intracellular bacterium Francisella tularensis
Authors: De Pascalis Roberto , Brook Helen M , Frey Blake , Conlan Wayne , Sjostedt Anders , Elkins Karen L
Published: January 2023

Francisella tularensis (Ft) is an intracellular bacterium that causes tularemia, a disease with a low incidence in US. The only available vaccine, the Live Vaccine Strain (LVS), is investigational and is derived from Type B Ft, not the more virulent Type A Ft. Previous work produced potential correlates to predict successful vaccination. These were determined by in vitro stimulation of murine Ft LVS-immune cells and analyses of their gene expression. We used this approach to investigate correlates of protection for the novel ΔclpB vaccine, derived from Type A Ft SchuS4. Mice were vaccinated with ΔclpB as well as LVS-derived vaccines and subsequently challenged with a lethal dose of LVS, after which all mice vaccinated with ΔclpB and LVS survived. The in vivo survival data was compared with in vitro data obtained from PBLs and splenocytes from vaccinated mice. In general, the in vitro functions of leukocytes from ΔclpB-vaccinated mice were comparable or exceeded those of leukocytes from LVS-vaccinated mice, including control of LVS intramacrophage replication, IFN-gamma secretion, and NO production. Correlates up-regulated in cells from mice vaccinated with ΔclpB included IFN-gamma, IL-21, Nos2, LTA, T-bet, IL-12rbeta2, CCL5 and GzmB; in some cases up-regulation was higher than that from LVS-derived PBLs. Other genes were up-regulated in ΔclpB-derived but not LVS-derived leukocytes, suggesting that improved protection stimulated by the ΔclpB vaccine may be related to the change in strain and/or to stronger immune responses. Hence, this panel of correlates could contribute to the screening of new vaccine candidates, bridging from animal models to humans, and augmenting clinical trials.
Increase in Incidence of Clostridium Difficile infection in aged models and effective DNA vaccine strategies for the prevention of disease
Authors: Kutzler Michele A.
Published: January 2023

Clostridium difficile–associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Key toxin-neutralizing epitopes have been discovered within the carboxy-terminal receptor-binding domain (RBD) of the toxins, which has generated interest in developing the RBD as a vaccine target. The elderly mount an impaired humoral response to C. difficile toxins leading to more severe and recurrent disease; however, it has yet to be determined what underlying mechanisms lead to lower anti-toxin titers, thus higher incidence of severe and recurrent disease. While numerous platforms have been studied, very few data describe the potential of DNA vaccination against CDAD. We immunized C57BL/6 mice with plasmids encoding the RBD from TcdA and TcdB intramuscularly followed by in vivo electroporation. Vaccination induced significant levels of anti-RBD antibodies within the serum and feces that could neutralize toxins in an in vitro cytotoxicity assay. Aged animals had lower endpoint and anti-toxin neutralization titers, but aged mice still elicited functional antibody post vaccination. Both young and aged immunized mice were protected following orogastric challenge with strains of C. difficile spores that were homologous (VPI 10463; n=10/10) and heterologous (hypervirulent UK1; n=4/8) strains to our vaccine antigens. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical young adult and aged models of acute toxin-associated disease.
PspA-based sublingual vaccine provides IgA dependent protective immunity to nasopharyngeal carriage by Streptococcus pneumoniae
Authors: Nagano Hiromi , SUGITA Gen , SUGITA rinya , Ikeda Yorihiko , Fujihashi Keiko , Tsuruhara Akitoshi , Kurono Yuichi , Fujihashi Kohtaro
Published: January 2023

Pneumococcal infection caused by Streptococcus pneumoniae is a major upper respiratory tract disease which causes severe illness and mortality. It is important to develop safe and effective vaccines to prevent pneumococcal infections. Sublingual (SL) antigen (Ag) delivery is a non-invasive route which avoids possible entry into the olfactory region and the central nervous system. We examined the potential of Poly (I:C) as a SL adjuvant for the induction of specific immune responses against S. pneumoniae infection. C57BL/6 and IgA−/− mice were SL immunized with 1 μg of pneumococcal surface protein A (PspA) and 10 μg of Poly (I:C) four times at weekly intervals. One week after the final immunization, nasal washes (NWs), saliva and plasma samples were collected and subjected to a PspA-specific ELISA. Further, mice were challenged with S. pneumoniae strain EF3030, and the numbers of CFUs in NWs and nasal passages (NPs) were determined. Significantly increased levels of PspA-specific secretory IgA (SIgA) antibody (Ab) responses were seen in NWs of mice given SL PspA plus Poly (I:C) when compared with controls. Further, higher levels of PspA-specific IgG and IgA Abs were noted in plasma of vaccinated mice. Importantly, mice given SL PspA plus Poly (I:C) showed significantly lower numbers of bacteria in the NPs and NWs when compared with controls. Although IgA−/− mice given SL PspA-vaccine had significantly high levels of PspA-specific IgG Abs, high numbers of CFUs were detected in NWs and NP. These results show that SL immunization is effective mucosal immunization strategy for induction of protective pneumococcal-specific Ab responses and specific SIgA Abs play a necessary role in the blockade of S. pneumoniae colonization of the nasal cavity.