bluuebunny/crossref_metadata_2025 huggingface dataset:
Disease manifestations with immune alteration by bacterial neuraminidases in influenza virus infection with Streptococcus pneumoniae superinfection
Authors: Dutta Avijit , Huang Ching-Tai , Hseih Yu-Chia , Chen Tse-Ching , Lin Chun-Yen , Lin Yung-Chang , Chang Chia-Shiang , He Yueh-Chia , Huang Yu-Lin , Chen Ting-An
Published: January 2023

Streptococcus pneumoniae often causes superinfection in influenza and exacerbates the disease with more severity and even mortality. In our influenza hemagglutinin (HA) antigen-specific transgenic mouse model, mice suffered from severe disease and died with increased virus titer and pathology in the lungs upon serotype-3 Streptococcus pneumoniae superinfection on day 4- post PR8 strain H1N1 influenza virus infection. Same dose viral or bacterial infection caused mild disease in control mice. Streptococcus pneumoniae supreinfection attenuated anti-influenza T cell immunity in the lungs. There was less clonotypic expansion, T-bet induction and effector cytokines IFN-γ and TNF-α production in HA-specific CD4+ T cells on day 2- and 3- post bacterial superinfection, with impaired viral clearance. Streptococcus pneumoniae strains with different combinations of bacterial neuraminidase (NA) demonstrated varied degrees of T cell immunity attenuation. Streptococcus pneumoniae with NA-B only was the most suppressive with up-regulated active TGF-β and down-regulated IFN-γ expression in HA-specific CD4+ T cells, followed by those with both NA-A and NA-B. Streptococcus pneumoniae with both NA-A and NA-B and inserted NA-C was least suppressive. With in vitro experiments, recombinant bacterial NA-A or NA-B augmented TGF-β and attenuated IFN-γ in HA-specific CD4+ T cells in overnight culture with PR8 virus. Recombinant bacterial NA-C mitigated TGF-β and increased IFN-γ in HA-specific CD4+ T cells. Our results demonestrates distinct immune alteration by different bacterial neuraminidases, which may play a significnt role of disease manifestation in influenza with bacterial superinfection.
IFITM3 is cardioprotective during influenza virus infection
Authors: Kenney Adam , Imas Alexander , Rajaram Murugesan , Yount Jacob
Published: January 2023

Influenza virus annually infects more than one million individuals worldwide and causes severe pandemic outbreaks. Myocarditis and cardiac dysfunction can be observed in particularly severe cases of influenza. Humans with polymorphisms in the gene encoding interferon-induced transmembrane protein 3 (IFITM3), an antiviral restriction factor, experience severe influenza virus infections. Thus, we hypothesized that IFITM3 deficiency leads to cardiac dysfunction during influenza virus infection. We found that Ifitm3−/− mice generated in our laboratory exhibited increased morbidity, mortality, and viral lung titers compared to WT mice when infected with influenza A virus. Ifitm3−/− and WT mice showed identical cardiac function and electrical activity prior to infection as confirmed by echocardiogram and electrocardiogram. While major alterations were not observed early in infection for WT mice, Ifitm3−/− mice exhibited aberrant electrical activity, including decreased heart rate, decreased QRS interval, and irregular R/R interval. These changes were further exacerbated as infection progressed, likely contributing to the complete lethality observed in knockout mice. In contrast, WT mice exhibited only mild cardiac dysfunction late in infection that resolved following recovery. Minimal viral replication was observed in the hearts of WT and Ifitm3−/− mice. Heart sectioning and staining suggests a role for lymphocyte infiltration and tissue damage underlying the cardiac dysfunction observed in Ifitm3−/− mice. These data identify a novel cardioprotective role for IFITM3 during influenza virus infection and establish Ifitm3−/− mice as a tool for studying molecular events leading to virus-induced cardiac dysfunction.
Characterization of Peptides Associated with Molecules of the Major Histocompatibility Complex using Mass Spectrometry
Authors: Ford Michael J. , Jones Richard , Amunugama Ravi , Allen David , Pisano Michael , Mobley James L. , Domanski Paul , Bochar Daniel
Published: January 2023

The major histocompatibility complex (MHC) is a region of highly polymorphic genes encoding for glycoproteins (MHC molecules) that form part of the cell-mediated branch of the acquired immune system. In the cytosol, cellular self and foreign (non-self) proteins are constantly being degraded; it is the peptides generated that are presented, non-covalently bound to MHC molecules, on the surface of cells for inspection by cytotoxic T-lymphocytes (CTLs). Non-recognition of the presented peptide ultimately leads to cell destruction.

Characterizing the factors associated with non-recognition is an attractive proposition for anyone interested in generating tools for targeted cell destruction. In the field of oncology the obvious application then is the targeted destruction of cancerous cells. To enable the molecular level characterization of peptides associated with molecules of the major histocompatibility complex requires a targeted protein complex enrichment, an unbiased peptide elution and finally a peptide analysis method. Most frequently an immunoprecipitation is used to isolate the target complex. The peptide elution is performed under conditions minimizing protein contamination and finally peptide analysis is accomplished by mass spectrometry.

Here we present a case study of our recent work optimizing and performing a workflow for the analysis of peptides associated with Class I and Class II MHC molecules.
Extraction and intracellular trafficking of membrane associated antigen distinguishes human germinal center B cells from naïve B cells and memory B cells
Authors: Kwak Kihyuck , Saniee Avva , Quizon Nicolas , Sohn Haewon , Pierce Susan
Published: January 2023

Protective antibody responses to vaccination or infection in humans depend on the competitive selection of high-affinity germinal-center (GC) B cells into the long-lived memory B cell compartment. Selection is determined in large part by the ability of GC B cells to gather, process and present antigen to T follicular helper T cells (TFH cells). We have shown that human GC B cells are better able to distinguish their affinity for antigen as compared to naïve B cells in the gathering and internalization of antigen. Here we provide evidence that human naïve B cells and GC B cells rely on different endocytic processes for antigen internalization and trafficking. We found that the Microtubule Organizing Centers in activated GC B cells were not polarized toward the immune synapse with the antigen-containing membrane as in naïve B cells but rather dispersed large distances from the immune synapse in GC B cells. GC B cells trafficked antigen-bound BCRs from the synapse outside the sides of the cells to distal sites for internalization whereas naïve B cells internalized antigen from the immune synapse. Moreover, as compared to naïve B cells, LAMP-1+ vesicles in GC B cells were less polarized and less internalized antigen was colocalized with LAMP-1+ vesicles. We also found that the expression of SNX9 and SNX18, key components of the endocytosis machinery and modulators of clathrine mediated endocytosis, was significantly lower in GC B cells as compared to naïve B cells and SNX9 and SNX18 were poorly polarized toward the immune synapse in activated GC B cells. We propose that these differences in endocytosis processes for antigen internalization contribute the higher affinity thresholds of GC B cells.
Subversion of antigen presentation by Adenoviruses
Authors: Bouvier Marlene , Li Lenong , Deng Hui
Published: January 2023

The cell-surface presentation of viral antigens by MHC class I molecules to CD8+ T-cells is critical for eliminating virus-infected cells. In turn, viruses have evolved numerous strategies to interfere with the generation and presentation of class I antigens. Human adenoviruses (Ads) comprise more than 70 different types classified into seven species (A to G); Ad infections cause diseases of the respiratory track (species B, C, and E), eyes (species D), and gastrointestinal track (species F). Ads encode the E3-19K protein that targets MHC class I molecules for retention in the endoplasmic reticulum of infected cells, thereby suppressing cell-surface presentation of Ad-derived antigens. In recent years, we determined the x-ray structures of Ad2 (species C) and Ad4 (species E) E3-19K bound to HLA-A2, which together with biochemical and functional data allowed to explain the mechanism of immunomodulation. Interestingly, in comparison to other Ads, species F Ads lack a gene for the common E3-19K protein and instead have two unique and uncharacterized genes, E3-19.4K and -31.6K. We are using a biochemical and functional approach to investigate how species F Ads modulate various cell surface immune proteins, including MHC I molecules, and what role of E3-19.4K and -31.6K proteins play in these effects.
Synthetic Peptide Nanofiber Adjuvants Engage Mechanisms of Autophagy in Antigen Presenting Cells
Authors: Rudra Jai S. , Khan Arshad , Clover Tara , Endsley Janice , Zloza Andrew , Wang Jin , Jagannath Chinnaswamy
Published: January 2023

A crucial challenge in subunit vaccine development is to minimize adjuvant-associated toxicity and balance immunogenicity with safety. Due to the chemical heterogeneity and toxicity associated with most plant- or pathogen-derived adjuvants, those clinically approved in the U.S. are limited to alum and AS04 (alum combined with MPLA). Therefore, there is a compelling clinical need for new adjuvant strategies that potentiate strong immune responses and are chemically defined and minimally toxic. We have previously demonstrated that supramolecular peptide nanofibers are potent adjuvants that elicit protective antibody and cellular immune responses in animal models of infectious diseases, cancer, and addiction. Critically, the adjuvant potential was non-inflammatory and the exact mechanism of action was unknown. Here, we report that self-assembling peptide nanofibers bearing CD4+ or CD8+T cell epitopes are processed through mechanisms of autophagy in antigen presenting cells (APCs). Using in vitro antigen presentation assays, we confirmed loss and gain of adjuvant function using pharmacological modulators of autophagy and APCs deficient in multiple autophagy proteins. Incorporation of microtubule associated protein 1A/1B-light chain 3 (LC3-II) into the autophagosomal membrane, a key biological marker for autophagy, was confirmed using microscopy. Our findings indicate that autophagy plays an essential role in the mechanism of adjuvant action of supramolecular peptide nanofibers.
Comprehending the phenotypic and functional diversity of γδ T cell subtypes in autoimmune skin disease Pemphigus Vulgaris
Authors: Das Dayasagar , Santosh KV , Sharma Alpana , Arava Sudheer
Published: January 2023

Pemphigus vulgaris (PV) is an atypical form of mucocutaneous autoimmune disease caused due to the formation of auto-antibodies against desmoglein3 (Dsg3) and anomalous T cell function. Gamma Delta T cells (γδ T cells) are evolutionary conserved distinctive cells that maintain the immune surveillance at the epithelial surface. The role of γδ T cells and their plasticity is not yet illustrated in the immunopathogenesis of PV. γδ T cells isolated from the PV patients showed diverse phenotypes (γδ1, γδ2, γδ17, γδreg) and functionality, such as migration, and multiple cytokine productions. Significantly higher frequency of γδ T cells producing IFN-γ & IL-17 (8.5% vs. 5.2%) was found in the circulation PV patients compared to control. Circulation of PV patients indicated higher number of CCR6+ γδ T cells suggesting their migratory potential. Cytotoxic activity of γδ T cells from PV patients (30±8%) was observed to be higher as compared to control (16±5%). Primary culture of γδ T cells from PV patients showed increased mRNA expression of inflammatory cytokines (IL17, ROR γt, IL-23) and co-stimulatory markers (CD27 and CD70). The regulatory markers associated with regulatory γδ T cells (TGFβ, FoxP3) were downregulated. At the tissue level, γδ 17 T cells in patient skin showed increased IL-17, CCR6 expression while CCR4 receptor associated with γδ reg T cells has shown lower expression indicating their imbalance and possible involvement in the immunopathogenesis of PV. These maiden findings suggest the versatile nature of γδ T cells that will help to find a better approach for novel therapeutics for PV.
Thpok suppresses pathogenic cytokine production by Th17 cells in autoimmune disease
Authors: Marks Kathryne E , Flaherty Stephanie , Reynolds Joseph M
Published: January 2023

CD4+ T cells, including Th17 cells, are instrumental in the development and progression of autoimmune diseases such as multiple sclerosis. While pathogenic Th17 cells drive inflammation in autoimmunity, the molecular programming underlying their pathogenicity remains insufficiently understood. Through RNA-sequencing, we have identified the transcription factor Thpok (Zbtb7b), also known as the CD4 lineage determination factor, as an apparent negative regulator of Th17 pathogenicity. Thpok expression is maintained in peripheral CD4+ T cells and assists in formation of memory CD4+ T cells. Furthermore, Thpok expression in CD4+ T cells represses cytotoxic genes and contributes to the Th17 response during intestinal inflammation. Herein we show Thpok is upregulated in pathogenic Th17 cells both in vitro and in vivo. In addition, we show increased expression of Thpok results in reduced production of IL-17, while conversely, loss of Thpok results in higher inflammatory potential of Th17 cells. Moreover, we have demonstrated that Thpok expression in Th17 cells in vivo functions to inhibit the development of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Altogether this study establishes Thpok as a suppressor of the autoimmune inflammatory function of Th17 cells.
CNS-specific autoregulatory CD8 T cells rely on IFNγ signaling for optimal suppression of pathogenic CD4 T cell responses during inhibition of demyelinating disease
Authors: Boyden Alexander , Brate Ashley A. , Laageide Leah G. , Karandikar Nitin J.
Published: January 2023

Investigating the complex cellular interplay controlling immunopathogenic and immunoregulatory responses is critical for understanding multiple sclerosis (MS) pathogenesis and for developing successful immunotherapies for patients. We have shown previously that CNS-specific CD8 T cells (CNS-CD8) have an unexpected regulatory role in the MS mouse model experimental autoimmune encephalomyelitis (EAE). CNS-CD8 fail to transfer or exacerbate disease but rather protect mice from EAE, and use IFNγ and perforin production to exert suppression. However, the cellular target and downstream effects of IFNγ are less well understood. Here, we evaluated IFNγ dynamics, its receptor and perforin during inhibition of CNS-specific DTH and EAE and show that both IFNγ and perforin production by CNS-CD8 were required at different times to suppress CD4 responses and inhibit EAE disease. Interestingly, while WT CNS-CD8 suppressed CD4 responses in IFNγR-deficient mice, IFNγR-deficient CD8 T cells showed suboptimal inhibition of CD4 responses in WT mice. Likewise, IFNγR-deficient CD8 T cells also exhibited delayed and suboptimal EAE suppression. These data suggest that autoregulatory CD8 T cells’ ability to suppress pathogenic responses during demyelinating disease is influenced by different mechanisms during different phases of disease. Moreover, the results suggest that IFNγ may have an effect on the CD8 T cells themselves, revealing a potential previously unappreciated autocrine/paracrine IFNγ-mediated pathway. Results from these studies have mechanistic implications for the interaction between IFNγ-mediated and perforin-mediated suppression pathways, which may help educate future adoptive immunotherapeutic approaches for MS.
Keratinocyte-derived TGF-β is dispensable in skin autoimmunity in mice
Authors: YANG YI , Zenke Yukari , Hirai Toshiro , Kaplan Daniel H.
Published: January 2023

Transforming growth factor beta 1 (TGF-β) required to suppress spontaneous autoimmunity as global deletion of TGF-β resulted in severe multi-organ autoimmunity in mice. Some TGF-β dificient mice showed skin phenotype. However, the effect of TGF-β on the autoimmunity in skin is not clear. In the epidermis of the skin, keratinocytes(KCs) are the major source of TGF-β. To test whether KCs are an obligate source of TGF-β, we generated K14-Cre-ERT2 TGF-βfl/fl mice. Topical application of tamoxifen could result in efficient deletion of TGF-β. We noted increase epidermal thickness but failed to observe evidence of cutaneous inflammation as measured by histology and flow cytometry. Notably, numbers of regulatory T cells were unaffected by the absence of KC-derived TGF-β. Since αvβ6- and αvβ8-integrins (Itgb6 and Itgb8) are principal source of activation of TGF-β in skin and then we generated Itgb6−/− Itgb8ΔKC. Similarly, we did not observe the development of spontaneous inflammation in Itgb6−/− Itgb8ΔKC mice which lack KC-derived TGFβ-activating integrins in the epidermis. From these data we concluded that KC-derived TGF-β and epidermal activation of TGF-β are not required to suppress spontaneous inflammation.
Regulation of FOXP3 Expression by c-Rel O-GlcNAcylation
Authors: de Jesus Tristan J , Tomalka Jeffery , Shukla Sudhanshu , Staback Franklin , Agarwal Ruchira , Centore Joshua , Ramakrishnan Parameswaran
Published: January 2023

The NF-kappaB protein c-Rel plays a critical role in controlling autoimmunity through regulating T cell function and T regulatory cell development. Type 1 diabetes is a T cell-mediated autoimmune disease associated with hyperglycemia and chronic inflammatory complications. Previously, we discovered that hyperglycemia induces hyper O-GlcNAcylation of c-Rel at serine residue 350 and enhances the transcription of c-Rel-dependent pro-autoimmune cytokines interleukin-2 (IL-2), interferon gamma (IFNG) and granulocyte macrophage colony-stimulating factor (GM-CSF). In contrast, our recent results show that c-Rel O-GlcNAcylation decreases forkhead boxP3 (FOXP3) expression in T cells. Chemically enhancing cellular O-GlcNAcylation decreases the DNA binding ability of c-Rel at the FOXP3 promoter as demonstrated both by oligonucleotide pulldown and chromatin immunoprecipitation assays. Moreover, mutation of serine 350 in c-Rel to alanine augments T cell receptor-induced FOXP3 expression. O-GlcNAcylation-dependent suppression of FOXP3 was also found in vivo in streptozotocin-induced diabetic mouse model. This study reveals a novel molecular mechanism that regulates hyperglycemia-dependent c-Rel function in autoimmune diabetes, with potential to develop novel therapeutics targeting c-Rel O-GlcNAcylation.
Activation of naive CD4+CD25+FOXP3+Treg by specific autoantigen and Th2 cytokines IL-4 and IL-5 induces autoantigen specific Treg that inhibit EAE
Authors: Hall Bruce Milne , Tran Giang , Verma Nirupama Darshan , Robinson Catherine Margaret , Wilcox Paul , Hodgkinson Suzanne Jean
Published: January 2023

CD4+CD25+FOXP3+Treg (tTreg) protect against induction of autoimmunity, but fresh naïve or ex vivo expanded tTreg have limited ability to control established autoimmunity. We have examined pathways of naïve tTreg activation by antigen and Th2 cytokines IL-4 and IL-5. First, IL-4 and antigen induce Ts2 cells that express IL-5Ralpha. In a second step, IL-5, not IL-4, and antigen induce Ts2 cells to Th2-like Treg.

EAE was induced in Lewis rats by immunization with myelin basic protein (MBP) in Freund’s complete adjuvant. Naïve CD4+CD25+Treg cultured with MBP and IL-4 for 4d produced Ts2 cells. To produce Th2-like Treg, Ts2 cells were cultured with IL-5 and MBP for 4d. Cells were assessed by FACS, RT-PCR and ability to suppress EAE.

5×106 Ts2 or 1×106 Th2-like Treg were given ivi 9d post-immunization. Ts2 cells activated by MBP, but not those activated by renal tubular antigen, suppressed EAE between 11 and 16d post-immunization. Clinical score: 0.1±0.5(n=6) in treated vs 1.5±2.7(n=13) in controls at 12d (p<0.01); 1.3±0.7 vs 2.5±0.8 at 13d (p<0.001). Onset of EAE was delayed, peak disease score reduced, as was weight loss. Naïve tTreg had no effect on EAE.

Th2-like Treg had no effect on the onset until 14d when there was rapid recovery with no weakness from 15–21d. Controls did not recover until 22d.

The Ts2 and Th2-like Treg were 99% CD4+, 98% CD25+ and 70–80% FOXP3+. Naïve tTreg expressed no il5ra whereas Ts2 and Th2-like Treg expressed Il5ra. Th2-like Treg had higher Foxp3, irf4 and gata3.

Ex vivo culture of naïve tTreg with IL-4 and autoantigen induces antigen-specific Treg that can control established autoimmune responses. These Ts2 cells further activated by culture with IL-5 and MBP also control autoimmunity. Both cells may have therapeutic potential.
sCD83 alleviates experimental autoimmune uveitis through disrupting the regulation of Rab1a/LRRK2 on F-actin rearrangements
Authors: Lin Wei
Published: January 2023

A soluble form of CD83, generated from membrane CD83 by splice variants or by shedding, inhibites T-cell proliferation to regulatory the immune response in many autoimmune disease and organ-rejection treatment. However, the role of sCD83 in the autoimmune uveitis is still not clear. In our study, we show that sCD83 alleviates experimental autoimmune uveitis (EAU) through a novel mechanism. During onset and recovery of EAU, the level of sCD83 rises in the serum and aqueous humor. Systemic or topical application of sCD83 application alleviates the symptom of EAU and decreases the infiltrated inflammatory cells and cytokines. Mechanistically, sCD83 induces tolerogenic DCs by decreasing the synaptic expression of co-stimulatory molecules and hampering the cell contact between DCs and T cells. sCD83 disruptes the cytoskeletal rearrangements at the DC-T cell contact zone, leading to alter localization of co-stimulatory molecules and suppress T-cell activation. Furthermore, sCD83 is proved to enter into DCs through caveolin-dependent endocytic path-way. And then, sCD83 can bind with Rab1a to inhibit Rab1a-GTP activation, which further influence the regulation of LRRK2 on F-actin rearrangements. This pathway is different from the sCD83/TLR4/MD2 path-way. Thus, the ability of sCD83 to modulate DC-mediated inflammation in the eye could be harnessed to develop new immunosuppressive therapeutics for autoimmune uveitis.
The cellular basis of the PD-1/PD-L1 regulatory pathway in diabetic autoimmunity in the NOD mouse
Authors: Hu Hao , Wan Xiaoxiao , Zakharov Pavel , Unanue Emil R
Published: January 2023

We report our studies on the effects of checkpoint blockade on the development of diabetic autoimmunity in the NOD mouse. One of the complications of checkpoint blockade therapy in cancer patients is the development of acute type 1 diabetes (T1D). We confirm that the PD-1/PD-L1 regulatory pathway is highly involved in T1D in the NOD mouse. In NOD autoimmunity there is an entry of CD4+ T cells specific to insulin peptides shortly after weaning followed by CD103+ dendritic cells (DC), CD4+ T cells and CD8+ T cells recognizing broader islet antigens into the pancreatic islets. The islet resident macrophages and infiltrated DCs, as well as the endothelium and the beta cells, express PD-L1. Diabetes doesn’t develop in NOD mice until about 20 weeks of age or later. Administration of anti-PD-1 antibody rapidly induces diabetes in the young mice showing a control phase established very early in the autoimmune process. Anti-PD-1 treatment induces a high level of islet leukocytes infiltration including CD4+ T cells and CD8+ T cells. The regulatory effect of PD-1 is involved in both CD4+ T cells which recognize self-peptides via H2g7MHC haplotype, as well as in CD8+ T cells. The islet resident macrophage is an essential component for the PD-1/PD-L1 regulatory pathway to take effect. In their absence, no diabetes develops even after anti-PD-1 treatment. The same observation is found in the NOD.Batf-3−/− mice. We suggest that the control mechanism takes place once T cells enter islets. To establish PD-1 regulation, IFNγ signaling is required probably via up-regulating PD-L1 expression on various types of cells in islets. Together, our study has explored the cellular basis of PD-1 in regulating diabetic autoimmunity in the NOD mouse.
Essential role of IL-17A in Tregs induction in autoimmune uveitis
Authors: Chong Wai Po , Zhong Yajie , Mattapallil Mary , Chen Jun , Caspi Rachel R
Published: January 2023

The signature cytokine of Th17 cells, IL-17A, is a pro-inflammatory cytokine associated with various autoimmune diseases in patients and in animal models. However, clinical trials targeting IL-17A in autoimmune disease have been disappointing. We found that Foxp3+ T regulatory cells (Tregs), express both IL17A receptor subunits, i.e. IL17RA and IL17RC., We therefore hypothesized that IL-17A may regulate the induction and/or function of Tregs. Recombinant IL-17A significantly increased the in-vitro polarization of T cells into Tregs. We next investigated the number of Tregs in WT and IL-17A−/− mice that were immunized for experimental autoimmune uveitis. Although there was no significant difference in Treg frequency between WT and IL-17A−/− mice before disease onset, IL-17A− /− mice failed to induce Tregs as efficiently as WT mice when disease progressed. Interestingly, IL-17A−/− mice developed similar disease severity when compared to WT mice, which may in part be due to an impairment in Treg induction. Our data suggest that IL-17A is involved in Tregs induction and/or maintenance, and that this has a role in controlling the (auto)immune response.
Type-1 interferons inhibition of Interleukin-10 signaling in T cells in Type 1 Diabetes development
Authors: Lozano Marcos Iglesias , Chicco Maria , Bibicheff Darrel , Brandacher Gerald , Raimondi Giorgio
Published: January 2023

Type 1 Diabetes (T1D) is caused by T cell-mediated destruction of pancreatic beta cells. The “fertile field” hypothesis of T1D proposes that, in a genetically predisposed individual, an inflamed environment prompts the organ towards autoimmunity. In this context, the protective role of the cytokine IL-10 remains unclear and somewhat controversial. Here we tested whether chronic and localized accumulation of type-I interferons (TI-IFNs) creates a “fertile field” that releases diabetogenic T cells from IL-10 mediated regulation and prompts T1D development. In vitro incubation of mouse T cells with IFN-b induces a dramatic defect in the production of phospho-STAT3 in response to IL-10 (but not in response to IL-6). This alteration was NOT caused by reduction of IL-10 receptor expression, nor induction of SOCS-1 or 3. Instead, microarray analysis showed that IFN-b increases STAT1 levels causing a reversal of the STAT1/STAT3 protein ratio in T cells, suggesting a novel role for this transcription factor. In agreement, STAT1-KO T cells exposed to IFN-b did not show any defect in IL-10 signaling. In diabetes-prone NOD mice we discovered a persistent reduction of IL-10 signaling in T cells of pancreatic and mesenteric lymph nodes (but not in T cells of other lymphoid tissues), correlating with the reported aberrant and localized accumulation of TI-IFNs in these mice. Early treatment of NOD mice with an IFNRA blocking antibody (that delays the onset and reduces the incidence of T1D) showed a clear restoration of IL-10 signaling.

Overall, these data reveal a previously unknown molecular interplay between IL-10 and TI-IFNs that, if thoroughly elucidated, could reveal a novel target of intervention to enhance the efficacy of T1D immunotherapies.
Protein kinase C activates multiple pathways to induce dendritic cell differentiation
Authors: Chavel Colin A , Lee Kelvin P
Published: January 2023

Dendritic cell (DC) differentiation is a complex process that requires multiple signaling pathways to induce this response. Tumor derived factors can block this process, and one way in which this occurs is through repression of the protein kinase C (PKC) isoform PKCβ at the gene level. However, the role PKCβ plays in DC differentiation has yet to be fully characterized. To address this question, we used the K562 cell line, which our lab and others has shown to be capable of differentiating into DCs with PMA. Inhibition of either the ERK or NFκB pathways in the presence of PMA led to DCs that were unable to activate T-cells. Upon further examination of either pathway, we uncovered novel interactions for PKC signaling and ERK or NFκB during the early stages of DC differentiation. Multiple PKCs induced C-Raf activation, in part through RKIP repression. PKC signaling also induced changes in multiple ERK targets, including degradation of Foxo3a, an interaction not shown previously in DCs. PKCβ was the sole PKC isoform required for canonical NFκB signaling through a mechanism involving the CARD11 complex and PKCβ was required for CARD11 complex formation. However, the IKK kinase TAK1 was not required for downstream NFκB signaling, as opposed to its established role in B-cell and T-cell receptor induced NFκB signaling, indicating an alternative mechanism by which PKCβ induces NFκB signaling. PKCβ was also required for non-canonical NFκB signaling outside of the established TRAF and cIAP cascade. We also found crosstalk between the ERK and NFκB pathways at both protein-protein interaction and protein expression levels. Taken together, these results indicate PKC as an important signaling node for DC differentiation.
IL-9-secreting tissue-resident memory CD4+ T cells contribute to allergic airway recall responses
Authors: Ulrich Benjamin Joseph , Fu Yongyao , Koh Byunghee , Kharwadkar Rakshin , Kaplan Mark H
Published: January 2023

Asthma is a chronic intermittent inflammatory lung disease for which there is no cure. Allergen-specific CD4 T helper (Th) cells that secrete cytokines including IL-4, IL-13, and IL-9 mediate asthma pathogenesis. In a six week chronic model of allergic airway disease, we were able to detect a robust IL-9+CD4+ T cell population in the lungs. Interestingly, the in vivo derived IL-9+CD4+ T cells had a multi-cytokine profile including the production of IL-5, IL-13, and IL-10. IL-9-reporter-positive cells demonstrated high gene expression levels of innate cytokine receptors Il1rl1, Il17rb, and Crlf2 and the transcription factor Gata3. IL-9+CD4+ T cells were found to be tissue-resident through intravenous labeling and had CD4+ tissue-resident memory (Trm) cell markers CD69 and CD11a. To determine the Trm phenotype of lung resident IL-9+ T cells, we developed a memory model using a five week period of rest after six weeks of chronic challenge, before a recall allergen challenge. In the memory model, IL-9+ T cells maintained tissue residency and demonstrated increased cell frequency and IL-9 production, characteristic of a secondary response. Antibody blockade of IL-9 immediately prior to the recall challenge significantly reduced overall allergic lung inflammation, suggesting that IL-9 plays an obligate role in the allergic memory response following pulmonary allergen challenge. These observations further suggest that IL-9 from Trm cell populations plays a novel role in allergen recall responses and is a potential therapeutic target for patients suffering from chronic intermittent asthma.
The complex allergen house dust mite (HDM) acts directly on macrophages to stimulate noncanonical autophagy
Authors: Gao Hongjuan , Qi Xiulan , Jackson William , Keegan Achsah
Published: January 2023

Asthma is a chronic inflammatory disease that results in narrowing of the airways. Inhalation of house dust mites (HDM) is a main cause of allergic asthma. Macrophages (Mf) are abundant in the lung and are one of the first cells exposed to HDM, however little is known about the effects of HDM on Mf. Autophagy has been linked to Mf phenotypes and asthma but the mechanism by which autophagy contributes to these processes is unclear. Autophagy is a homeostatic process involving degradation of intracellular components, including proteins, organelles, and foreign bodies. To determine whether HDM stimulates autophagy, we treated bone marrow-derived Mf (BMM) with HDM and evaluated autophagosome foci formation. HDM induced foci formation that was similar to treatment with the autophagy inducer Torin 1. Further, electron microscopy revealed an increase in double membraned vesicles in response to HDM and Torin 1, which could be due to an increase in autophagosome formation or inhibition of their fusion with lysosomes. To differentiate these hypotheses, we investigated markers of autophagy. During canonical autophagy, LC3-I is converted to membrane-associated LC3-II by lipidation, while the cargo adapter protein SQSTM1/p62 is degraded. Surprisingly, treatment with HDM caused a general increase in LC3-I and LC3-II without a substantial change in the LC3-II to LC3-I ratio. As expected, Torin 1 induced autophagic degradation demonstrated by reduction of SQSTM1/p62. However, SQSTM1/p62 increased dramatically in response to HDM, indicating a block in autophagic degradation. We observed a similar trend in THP-1 and human CD14+ Mf. These results suggest that HDM engages autophagy machinery but does not stimulate degradative autophagy.
Impaired tumor-necrosis-factor-α-driven dendritic cell activation limits lipopolysaccharide-induced protection from allergic inflammation in infants
Authors: Kaur Kamaljeet , Bachus Holly , Papillion Amber M. , Marquez-Lago Tatiana T. , Yu Zhihong , Ballesteros-Tato Andre , Matalon Sadis , Leon-Ruiz Beatriz
Published: January 2023

Infants have a higher risk of developing allergic asthma than adults. However, the underlying mechanism remains unknown. We show here that sensitization of mice with house-dust mites (HDMs) in the presence of low-dose lipopolysaccharide (LPS) prevented T helper 2 (Th2) cell allergic responses in adult, but not infant, mice. Mechanistically, adult CD11b + migratory dendritic cells (mDCs) upregulated the transcription factor T-bet in response to tumor necrosis factor-α (TNF-α), which was rapidly induced after HDM + LPS sensitization. Consequently, adult CD11b+ mDCs produced interleukin-12 (IL-12), which prevented Th2 cell development by promoting T-bet upregulation in responding T cells. Conversely, infants failed to induce TNF-α after HDM + LPS sensitization. Therefore, CD11b+ mDCs failed to upregulate T-bet and did not secrete IL-12 and Th2 cell responses normally developed in infant mice. Thus, the availability of TNF-α dictates the ability of CD11b+ mDCs to suppress allergic Th2-cell responses upon dose-dependent endotoxin sensitization and is a key mediator governing susceptibility to allergic airway inflammation in infant mice.

[The above abstract is a summary from our recently published paper under the same title – Immunity; published: Jan 8, 2019 DOI: https://doi.org/10.1016/j.immuni.2018.11.012]
Tissue driven influences on human NK cell development, function and residence
Authors: Dogra Pranay , Rancan Chiara , Ma Wenji , Toth Marta , Senda Takashi , Carpenter Dustin J. , Thapa Puspa , Szabo Peter Anthony , Shen Yufeng , Fong Lawrence , Lanier Lewis L , Farber Donna L
Published: January 2023

Natural killer (NK) cells are innate immune cells with the inherent ability to kill tumor and virus infected cells without any prior priming. Recent studies have demonstrated the ability of NK cells to develop adaptive memory and tissue residence. However, our understanding of human NK cells in tissues and their site-specific properties is limited. Here we use our unique tissue resource established in collaboration with LiveOnNY to elucidate the diversity of human NK cell phenotypic subsets, functional profile and transcriptional signatures in non-mucosal and mucosal tissue sites. We identified tissue-specific patterns of NK cell maturation and show that NK cell subset (immature and mature) and memory NK cell distribution is a function of tissue site which is not affected by age. Our results also show that while NK cells from different tissue sites express similar levels of granzyme B, NK cell antibody-dependent cellular cytotoxicity response and cytokine driven activation may be influenced by tissue site. Whole transcriptome profiling of NK cell subsets revealed that mature NK cells from all the sites (blood, bone marrow, spleen, lung and LN) have very similar transcriptional profiles. Interestingly, immature NK cells from all tissue sites have very different transcriptional profiles compared to their blood counterpart and are enriched for tissue resident memory T cell transcriptional signature. Furthermore, we show that immature NK cells show site-specific expression patterns of tissue residence surface markers. Overall, our data demonstrates the effects of tissue microenvironment on the developmental and function potential of tissue NK cells and provides insight into the development of future NK cell mediated immunotherapies.
DNA origami probes for single-cell paired BCR heavy and light chain immune repertoire analysis
Authors: Chaudhary Neelam , Schoettle Louis , Dubois Courtney , Jones Kylee , Ulrich Peaches Rebecca , Yan Hao , Blattman Joseph N
Published: January 2023

Antibodies are a diverse set of glycoproteins produced by B-cells in response to antigen exposure, infection, or certain malignancies, making it crucial to develop a high throughput and robust technique to obtain paired heavy and light chain information from individual B-cells. We have developed DNA origami-based capture probes to isolate paired heavy and light sequence information from single B-cells. The capture probes are designed to hybridize with the constant regions of heavy and light chains. Each capture probe also incorporates sequences for hybridization with DNA origami nanostructures. The nanostructures can be electroporated into B-cells and re-isolated by cell lysis and streptavidin affinity purification. Heavy and light chains are then reverse transcribed, appending the isolated sequences to the capture probes. Following PCR-amplification, each amplicon will contain BCR heavy or light chain sequence, as well as a unique barcode. Matching barcodes from complementary heavy and light chain amplicons can then be paired via next-generation sequencing. We have used this technique to analyze the antibody repertoire from splenic tissue of unimmunized C57BL/6J mice. We designed a single IgM-Igk specific capture probe and synthesized DNA origami nanostructures. The nanostructures were transfected into naïve B-cells, purified, reverse transcribed, and PCR-amplified. The isolated IgM and Igk amplicons were confirmed by sequencing. Through this approach, we present a novel DNA origami-based technique for single cell paired BCR repertoire analysis. This is a highly versatile technique that can be used to isolate any gene pair functioning in cellular dynamics such as signaling pathways or co-occurring mutations in cancer.
Development of Sialoglycan Recognizing Probes (SGRPs) With Defined Specificities: Towards Exploring the Dynamic Mammalian Sialoglycome
Authors: Srivastava Saurabh , Verhagen Andrea , Wasik Brian , Yu Hai , Sasmal Aniruddha , Bensing Barbara , Khan Naazneen , Khedri Zahra , Diaz Sandra , Sullam Paul , Varki Nissi , Chen Xi , Parrish Colin , Varki Ajit
Published: January 2023

Dense and complex glycans on vertebrate cell surfaces and secreted molecules often terminate with sialic acids (Sias), a diverse family of sugars involved in numerous biological processes, including cell-cell interactions, immune regulation, cellular homeostasis, host-pathogen interactions and microbial pathogeneses. Due to immense structural variations, a major fraction of sialoglycans remain poorly characterized by current inadequate conventional techniques. While traditional glycomic methods are useful, they can destroy or overlook crucial aspects of Sia diversity, or fail to elucidate native structures as they exist in biological systems. To date, in situdetection and analysis of sialoglycans have been largely based on plant lectins, sialidases or antibodies whose preferences are limited and/or uncertain.

We defined 9 classes of Sialoglycan Recognizing Probes (SGRPs: SGRP1- SGRP9) aiming to provide a simple and systematic approach to track mammalian sialome changes, using techniques common in most laboratories. Our approach utilizes molecules naturally evolved with maximized specificity towards sialoglycans such as bacterial adhesins, toxin subunits and viral hemagglutinin-esterases. We selected the experimentally proven best candidates for SGRPs and engineered each with a corresponding non-binding mutant as a negative control. Following sialoglycan microarray studies to confirm the binding specificity of our SGRPs, the optimized panel of SGRPs was subsequently applied in common detection methods such as ELISA, Western Blot and FACS analysis. While further work is needed to define a comprehensive suite of SGRPs, our efforts provide a reliable toolkit with specificity to track the mammalian sialoglycome.
Database of Immune Cell EQTLs, Expression, Epigenomics
Authors: Ha Brendan , Greenbaum Jason A , Shmiedel Benjamin J , Singh Divya , Madrigal Ariel , Valdovino-Gonzalez Alan G , White Brandie M , Zapardiel-Gonzalo Jose , Altay Gokmen , McVicker Graham , Seumois Gregory , Rao Anjana , Kronenberg Mitchell , Vijayanand Pandurangan , Peters Bjoern
Published: January 2023

Database of Immune Cell EQTLs, Expression, Epigenomics (DICE, dice-database.org) is a freely accessible web resource for diving deep into RNA-seq data across 13 human immune cell type (subsets of T cells, B cells, monocytes and NK cells), in a cohort of 91 healthy, genotyped donors1. The site is composed of four main modules that allow users to view the integrated data by gene, limit the scope to individual cell types, compare expression across cell types, and explore the effect of SNPs on gene expression. The site is optimized to be accessible on both desktop and mobile devices. DICE is an evolving resource with data for additional cell types being deposited on a continuous basis, with 3 additional B cell subsets being next in line. In addition to the core database website, an associated tools website (tools.dice-database.org) is actively being developed and currently hosts tools that operate on lists of genes to perform interactive GO enrichment analysis (GOnet), identify cell types most likely responsible for expression of a set of genes (CellTypeScorer), and examine GeneNetworks deduced from gene co-expression analysis.
Gene conversion identification and analysis in immunoglobulins
Authors: Bonissone Stefano R
Published: January 2023

Few, if any, somatic processes exhibit such sequence level diversity as the immunoglobulin rearrangement and somatic hypermutation (SHM) mechanisms. It is due to these processes that novel antibody receptors can be created against most pathogens.

Somatic hypermutation is attributable to the majority of non-junction diversity in studied repertoires, however, this has been largely due to the organisms most heavily studied in repertoire sequencing: human and mouse. Gene conversion is another mechanism that drives diversity in the immunoglobulin repertoire of other organisms, particularly in chicken and rabbit.

Confidently identifying gene conversion events has not been a focus for most repertoire sequencing studies, largely due to the organisms typically studied. However, their identification also poses an algorithmic and statistical problem: namely how to identify a tract of donor sequence from similar V genes, and subsequently assessing the significance of such an identification. The scant examples of analysis of immunoglobulin gene conversion in the literature have used two approaches: visual inspection and the use of GENECONV.

We propose two alternate approaches to identify the events: using a specialized pairwise alignment between the read, host, and donor references; and using a partial order alignment graph. These new approaches are validated on simulated antibody data, showing improved recall, and reduced false positive rates. The new approaches are more sensitive than GENECONV on smaller donor tract lengths (< 100nt), and are not sensitive to higher mutation load. We then apply the new approach to identifying gene conversion events in high throughput repertoire sequencing of rabbit, and public human datasets.
Antibody epitope mapping at single residue resolution for unpurified antigens
Authors: Pan Jingxi , Zhang Sara
Published: January 2023

The vast majority of antigen-antibody interactions rely upon binding to conformational epitopes, which are composed of amino acids that are discontinuous in the protein sequence but are brought together upon three-dimensional protein folding. This character has made conformational epitopes particularly difficult to map because they are only formed in the native structure of the antigen protein. X-ray crystallography is a gold-standard method for mapping conformational epitopes at high-resolution, but it only works for highly purified antigens and is time consuming, and is not applicable when crystals of antigen-antibody complex are unavailable. Here we present a novel middle-down HDX-MS technology with subzero temperature separation that effectively tackles these limitations. It successfully mapped the conformational epitopes in a VPF growth factor targeted by a therapeutic antibody, in a complex protein mixture where the antigen content is as low as 10%. It provided single amino acid resolution by online gas-phase fragmentation on the LC-MS time scale. It also uses directly the whole antibody, pre-digestion of the antibody to Fab fragments is no longer needed.

In conclusion, the subzero temperature separation based HDX-MS technology represents an alternative high-resolution epitope mapping approach to X-ray crystallography, and is also a fast and cost-effective solution for new antibody and vaccine development. Its potential in paratope mapping, as well as epitope mapping for other difficult targets and bispecific antibodies will be discussed.
C15 is actionable drug target for anti-arthritis via activation Nrf2 signaling
Authors: Li Ting , Liao Kangsheng , Zhuang Yuxin , Wu Jianlin , Liu Juan
Published: January 2023

SNM is a purified compound isolated from Chinese Medicinal plant. In the current study, we found that SNM not only ameliorated the progression of collagen induced arthritis (CIA), but protected joints from destruction in mice, indicating that SNM probably restricted the local inflammation of arthritic joints. As synovium in the joints is the major target tissue of rheumatoid arthritis, the synovium fibroblasts derived from RA patients (RASFs) were employed to validate the anti-arthritic effect and explore underlying mechanism of SNM. The results demonstrated that SNM significantly inhibited IL-6 and IL-33 secretion, COX-2 expression and ROS production in RASFs. Mass spectrometry results demonstrated that cysteine 26 (C26) and lysine 873 (K873) of C15 are a direct target of SNM. Underlying mechanistic study showed that knockdown C15 resulted in the accumulation and phosphorylation of p62 and aggregation of Nrf2 and HO-1 in cytoplasma of RASFs. Consistently, SNM activated p62/Nrf2 signaling in RASFs via covalently binding at C26 and K873 of C15. Furthermore, the collagen antibody-induced arthritis (CAIA) model was established in Nrf2−/− mice to verify the role of Nrf2 in the anti-arthritic effect of SNM, and the results explored that Nrf2−/− mice are resistant to the anti-arthritic effect of SNM. Our findings explored that C15 is an actionable drug target for anti-arthritis via activation Nrf2 signaling.