bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Nanogel-based Nasal Vaccine against Pneumonia
Authors: Nakahashi Rika , Yuki Yoshikazu , Fujihashi Kohtaro , Kiyono Hiroshi
Published: January 2023

Streptococcus pneumoniae (S. pneumoniae) cause pneumococcal disease in children and adults all over the world. Although there are vaccines comprising several pneumococcal polysaccharides in clinical use, they cannot cover the broad protections. Recently we established the cationic nanogel-based nasal vaccine against Pneumonia using a recombinant protein of pneumococcal surface protein A (PspA). We have designed three PspA chimeric antigens for trivalent vaccine and combined them with a cationic nanogel, a novel drug delivery system which adhere to the epithelial layer of the nasal cavity after nasal immunization and elicit the effective immunity by sustained antigen release. Indeed, the PspA nanogel vaccine induced not only the antigen-specific IgG in the body but also the antigen-specific secretory IgA (SIgA) at the mucosal surfaces such as nasal cavity where the entrance of pathogens. Both induced antigen-specific IgG and SIgA could bind to various serotypes of S. pneumoniae, indicating that these antibodies have neutralizing activity against broad types of S. pneumoniae. Moreover, the vaccinated mice were protected from infections by a several types of S. pneumoniae, and the nasal vaccine exhibited protective efficacy in some strains which cannot be covered by the current vaccines. Thus, our findings demonstrated the nasally administered PspA nanogel vaccine effectively elicit a protective immunity and provide a broad protection against S. pneumoniae infections.
The potential role of HLA-I associated adaptation in elevated risk of infection among HIV-1 STEP study vaccine recipients
Authors: Qin Kai , Carlson Jonathan , Boppana Sushma , Bansal Anju , Goepfert Paul
Published: January 2023

The biological mechanism(s) underlying the elevated risk of HIV-1 acquisition among vaccine recipients in Merck STEP study remain unclear. HIV-1 frequently adapts to CD8 T cell responses, leading to the accumulation of adapted epitopes (AE). Recently, we demonstrated that in chronic HIV infection, CD8 T responses skewed by HLA-I associated adaptation enhanced HIV trans-infection of CD4 T cells by dendritic cells (DCs). However, whether adaptation in HIV vaccines induces a pro-inflammatory status and aids in potentiating infection is unknown. Using in-vitro studies, we evaluated whether an enhanced CD4 T cell trans-infection mediated by AE-specific CD8 T cells could be a potential biological mechanism for the observed increased risk of HIV acquisition in the Step Study. We found that CD8 T cells responding to AE demonstrated a higher capacity in promoting DCs maturation than non-adapted epitope (NAE) (p=0.007). Additionally, DCs matured by AE-specific CD8 T cells, showed a higher trend in inducing HIV trans-infection of CD4 T cells in each of the four vaccinee samples tested. We also analyzed the potential in-vivo impact of adaptation on the rate of infection among vaccine recipients in the STEP study. Our data showed that vaccine recipients with high adaptation to the vaccine immunogen were at an increased risk of HIV acquisition compared to recipients with medium/low adaptation or placebos (HR=2.98, p=0.02, high (n=21), medium (n=639), low (n=192) and placebo (n=865)). Taken together, our results show that in STEP study, AE-specific CD8 T cells may have contributed to an enhanced risk of HIV-1 acquisition by promoting viral trans-infection of CD4 T cells by DC. These findings have important implications for future HIV vaccine design.
Generation of clinically-relevant vaccine vectors for use in schistosome-endemic areas
Authors: Norwood Stephanie K , Bement Zachary James , Shollenberger Lisa M
Published: January 2023

Schistosomiasis is a neglected tropical disease with 200–300 million people infected worldwide. Schistosome infections are immunomodulatory and induce a Th2 biased immune response. Chronic helminth infections, such as schistosomiasis, can cause Th1/Tc1 type vaccine failure. Schistosomiasis endemicity is geographically coincident for other diseases lacking effective vaccines (HIV, TB, and Malaria). New vaccine vectors are needed to counter the immune suppression displayed during this infection. Listeria monocytogenes has shown to function as an effective vaccine vector to induce cell-mediated immune responses. Wild-type Listeria expressing HIV-Gag was previously proven to be an effective bacterial vector, however there are safety concerns with using a pathogenic vaccine vector in humans. Therefore, it is important to develop a clinically relevant vaccine that instead uses an attenuated strain to overcome safety concerns. There are genetically attenuated strains of Listeria currently in use in human clinical trials as therapeutic cancer vaccine vectors. Here, we are cloning HIV-Gag into the attenuated strains to induce strong cell mediated immunity without risk of adverse effects from the pathogenic wild-type vector. We will confirm appropriate Gag protein expression from the novel vectors using standard methodology. Further, we will confirm the functionality of these vectors in naïve and schistosome-infected animals. Our aim is that these non-pathogenic vectors will be clinically relevant for use in schistosome-endemic areas.
Antibody therapeutics for emerging infectious diseases: Ebola virus and others
Authors: Dye John M , Chandran Kartik , Wec Anna , Walker Laura , Zeitlin Larry , Bornholdt Zachary , Herbert Andrew
Published: January 2023

Developing medical countermeasures against emerging infectious diseases remains a challenging task. Ebola virus, Crimean CongoHemorrhagic Fever Virus, and Hantaviruses are ever growing threats on the global landscape. A consortium of worldwide researchers has developed a platform for novel monoclonal antibody discovery. This platform is predicated on the understanding that harvesting antibody sequences from convalescent survivors of previous outbreaks may lead to the most effective treatments for future outbreaks. Utilizing flow cytometric sorting of antigen-specific memory B cells for the elucidation of heavy and light chain sequences, novel biologics were identified and generated for evaluation. Specifically, a cocktail of two monoclonal antibodies was discovered and developed for Sudan virus, Ebolavirus, and Bundibugyo virus. Previously, published antivirals have been virus-specific with no cross reactivity against additional species of virus beyond the original agent. This cocktail of monoclonals neutralized and provided efficacy against infection with each of these viruses in animal models (rodent and non-human primate studies). This cocktail is currently being moved into advanced development and phase I clinical trials. More generally, this successful discovery platform is currently being utilized for developing novel monoclonal treatments for alphaviruses, hantaviruses, arenaviruses and other filoviruses. New data on the development of biologics against these agents will be discussed as well.
Bacillus Calmette-Guérin (BCG) vaccine reprograms human neonatal metabolism in vivo and in vitro
Authors: Arce Joann Diray , Jensen Kristoffer Jarlov , Angelidou Asimenia , Conti Maria Giulia , Kelly Rachel S. , Pettengill Matthew , Liu Mark , van Haren Simon D. , Benn Christine Stabell , Ozonoff Al , Steen Hanno , Lasky-Su Jessica , Levy Ofer
Published: January 2023

Immunization has significantly reduced infectious morbidity and mortality, but vaccine optimization and development is limited by incomplete understanding of how early life vaccines engage the distinct infant immune system to induce protection. We used plasma metabolomics to gain immunometabolic insight into the effects of Bacille Calmette-Guérin (BCG) which prevents disseminated TB and induces heterologous immunity to unrelated pathogens. For in vivo studies, low-birth weight newborns in Guinea-Bissau received a neonatal dose of Bacille Calmette-Guérin (early BCG) at birth or a catch-up after blood collection at four weeks post-birth (delayed BCG). For in vitro studies, human neonatal cord blood samples were stimulated for 18 hours with either vehicle (saline) or BCG. In vivo and in vitro plasma samples were profiled for global untargeted metabolomics and complex lipid panel lipidomics.

BCG-induced perturbation of neonatal metabolic pathways was observed in vivo and confirmed in vitro. Lipid biomarkers of early BCG-induced response identified increased free fatty acids (FFA), and decreased lysoPC (LPC), lysoPE (LPE), monoacylglycerol (MAG), phosphatidylcholine (PC), phosphoethanolamine (PE) and triacylglycerol (TAG). BCG identified decrease on plasma levels of LPC, LPE, PC and MAG in vitro. Nucleotide purine and pyrimidine pathways are perturbed both in vivo and in vitro. Neonatal BCG vaccination alters the early life metabolism, in particular lipids implicated in immune response. This suggests that BCG-induced alterations in lipid pathways may contribute to its protective effect. Further studies may identify biomarkers of vaccine immunogenicity to inform development and optimization of early life vaccines.
Arenavirus vectors for massive tumor self-antigen-specific CD8 T cell attack
Authors: Pinschewer Daniel D. , Bonilla Weldy V. , Kallert Sandra , Kirchhammer Nicole , Marx Anna-Friederike , Krzyzaniak Magdalena , Schmidt Sarah , Raguz Josipa , Berka Ursula , Günther Stephan , Magnus Carsten , Orlinger Klaus , Zippelius Alfred
Published: January 2023

Therapeutic vaccination regimens inducing high frequencies of clinically effective tumor-targeting CD8 T cells represent an unmet need in cancer immunotherapy.

We exploit cutting-edge viral reverse genetics to harness the immunostimulatory properties of the arenaviruses Pichinde virus and lymphocytic choriomeningitis virus for therapeutic cancer vaccination. When administering to mice life-replicating vector formats of these two viruses, both delivering the same cancer testis self-antigen in a heterologous prime-boost regimen, tumor-specific CD8 T cell responses reached up to 50% of the circulating CD8 T cell pool. This massive CD8 T cell attack eliminated established solid tumors in a significant proportion of animals, accompanied by complete protection against tumor re-challenge. The magnitude of CD8 T cell responses was driven by alarmin signals and depended on the combined use of two genealogically distantly related arenaviruses. Combinations of closely related vectors or the repeated administration of the same vector were not inhibited by vector-neutralizing antibodies, but CD8 T cell immunodominance hierarchies favored vector backbone-targeted responses at the expense of self-reactive T cell specificities.

These findings establish a powerful arenavirus-based tumor immunotherapy regimen exploiting key innate signaling pathways and reshuffling immunodominance hierarchies to break tumor self-directed tolerance
Pharmacological inhibition of protein kinase C-eta inactivates the contact-dependent suppressive activity of regulatory T cells (Tregs): A novel approach for cancer immunotherapy?
Authors: Xie Jiji , Liu Hsin-Yu , Pedros Christophe , Kong Kok-Fai , Altman Amnon
Published: January 2023

Our earlier work has revealed a novel, Treg-intrinsic signaling pathway consisting of a CTLA4 and PKC-eta complex, which is required for Treg function to suppress immune responses, including tumor-specific immunity, in a contact-dependent manner, but is dispensable for suppressions mediated by suppressive cytokines (Nat. Immunol. 2014; J. Clin. Invest. Insight. 2017). Furthermore, we found PKC-eta to be dispensable for the activation and effector function of mouse and human CD8+ T cells, implying that PKC-eta is a promising cancer immunotherapy target, whose pharmacological inhibition would inhibit the suppressive activity of tumor-specific Tregs without impairing function of tumor-specific cytotoxic T cells. We therefore initiated, in collaboration with Takeda Oncology, the testing of two lead small molecule compounds previously reported to selectively inhibit the catalytic activity of Ca2+-independent PKC subfamily members, including PKC-eta. Using in vitro assays, we titrated these compounds and established a range of compound concentrations that significantly inhibited the in vitro contact-dependent suppressive activity of human Tregs, while only minimally inhibiting the activation and proliferation of conventional human T cells. Furthermore, they did not inhibit the production of IL-10 by stimulated human Tregs, consistent with our hypothesis that the CTLA4-PKC-eta signaling pathway is selectively required for contact-dependent suppression. Such compounds could potentially serve as a novel form of CTLA4-based checkpoint blockade for cancer immunotherapy and, at the same time, minimize immune-related adverse effects associated with clinically used anti-CTLA4 antibodies.
Exploiting tumor necrosis with novel immunotherapies
Authors: Franks S. Elizabeth , Solocinski Kristen , Schlom Jeffrey , Hodge James
Published: January 2023

Tumor necrosis prior to therapy historically correlates with aggressive tumor phenotypes. However, necrosis in response to chemotherapy leads to increased progression-free and overall survival in several cancer types. Chemotherapy with docetaxel results in inhibition of mitosis, leading to necrosis. To exploit the therapeutic potential of docetaxel, a bifunctional molecule consisting of a mAb specific for histone H1 and two molecules of IL-12 (NHS-IL-12), was engineered to deliver rIL-12 to the tumor microenvironment (TME) through specific targeting of necrotic regions in the tumor. IL-12 is a proinflammatory cytokine that increases lytic capacity of T and NK cells. Although promising preclinically, rIL-12 when delivered systemically leads to severe toxicity. We hypothesize inducing necrosis leads to immunogenic modulation that can be exploited with targeted therapies resulting in increased tumor cell killing.

Here, we show pathologist confirmed increase in tumor necrosis post-delivery of docetaxel, leading to improved retention of NHS-IL-12 in the TME. Significant anti-tumor activity is observed when combining docetaxel and NHS-IL-12, but there is elevated PD-L1 expression on tumor cells and serum TGFb levels, which correlate with poor prognosis. M7824 is a fusion protein containing a mAb against PD-L1 linked to the extracellular domain of TGFbR2, sequestering TGFb. Following the strategic addition of M7824, TGFb levels in mice receiving the triple combination had significantly reduced TGFb levels. Additionally, the greatest anti-tumor activity and increase in survival was observed in cohorts receiving docetaxel, NHS-IL-12 and M7824. Data presented herein have aided in the rationale design of a clinical trial at NCI.
Investigating the pathogenesis of chronic chikungunya virus infection
Authors: Locke Marissa Christine , Young Alissa , Cook Lindsey E. , Monte Kristen , Lenschow Deborah
Published: January 2023

Chikungunya virus (CHIKV) is an arthritogenic alphavirus that acutely causes fever as well as severe joint and muscle pain. Chronic musculoskeletal pain persists in a substantial fraction of patients for months to years after infection. While replicating virus is not detected in joint-associated tissues of patients nor in various animal models at convalescent time points, viral RNA is detected months after acute infection.

To identify the cells that might contribute to chronic pathogenesis, we developed a recombinant CHIKV that expresses Cre recombinase (CHIKV-3′-Cre). CHIKV-3′-Cre infection of tdTomato reporter mice resulted in a population of tdTomato+ cells that persisted for at least 112 days. Immunofluorescence and flow cytometric profiling 28 days post infection revealed that these tdTomato+ cells were predominantly myofibers and fibroblasts as well as a small population of macrophages. Further, tdTomato+ cells are enriched for CHIKV RNA. Interestingly, the population of surviving cells was dynamic over time with macrophages making up a high percentage of the cells early and then tapering off.

We applied this system to determine the differential roles of the type I interferon (IFN) subtypes during chronic CHIKV infection. Results using IFN blocking antibodies revealed that mice lacking the IFNαs, but not IFNβ, show an increase in chronic RNA levels and the number of fibroblasts that survive CHIKV infection in distal sites, indicating that type I IFNs have divergent roles during chronic CHIKV disease. Ongoing studies will explore the differential functions of IFNαs and IFNβ, will determine why the surviving cells and viral RNA are not cleared by the immune system, and will elucidate their contribution to chronic inflammation.
CD4+ T cell mediated HLA class II cross-restriction in HIV controllers
Authors: Gras Stephanie
Published: January 2023

Rare individuals, termed HIV controllers, spontaneously control HIV infection by mounting efficient T cell responses against the virus. Protective CD4+ T cell responses from HIV controllers involve high affinity public T cell receptors (TCRs) recognizing an immunodominant capsid epitope (Gag293) presented by a remarkably broad array of HLA class II molecules. Here we determine the structures of a prototypical public TCR bound to HLAD-R1, –DR11, and –DR15 molecules presenting the Gag293 epitope. TCR recognition was driven by contacts with the Gag293 epitope, a feature that underpinned the extensive HLA cross-restriction. These high affinity TCRs promoted mature immunological synapse formation and cytotoxic capacity in both CD4+ and CD8+ T cells. The public TCRs suppressed HIV replication in multiple genetic backgrounds ex vivo, emphasizing the functional advantage conferred by broad HLA class II cross-restriction.
Serine phosphorylation of the STAT1 transactivation domain promotes autoreactive B cell and systemic autoimmunity development
Authors: Fike Adam J , Chodisetti Sathi Babu , Domeier Phillip P , Singh Harinder , Schell Stephanie L , Mockus Taryn E , Choi Nicholas M , Corradetti Chelsea , Kawasawa Yuka Imamura , Hou Baidong , Atkins Hannah M , Caricchio Roberto , Decker Thomas , Lukacher Aron E , Olsen Nancy J , Rahman Ziaur S.M.
Published: January 2023

Although STAT1 tyrosine-701 phosphorylation (STAT1-pY701) is indispensable for STAT1 function, the requirement for STAT1 serine-727 phosphorylation (STAT1-pS727) during autoimmune and anti-pathogen responses remains unclear. Here we report that STAT1-pS727 promotes autoimmune antibody-forming cell (AFC) and germinal center (GC) responses, driving systemic lupus erythematosus (SLE) development. STAT1-pS727, however, is not required for GC and antibody responses to foreign-antigens including pathogens or gut microbiota. STAT1-pS727 plays an important B cell-intrinsic role in driving autoimmunity. Transcriptomic analysis of B cells from TLR7-accelerated SLE-prone mice reveals STAT1-pS727-mediated gene regulation of cellular pathways known to be involved in autoimmune GC and AFC responses. Mechanistically, TLR7 activation in B cells induces STAT1-pS727 and during autoimmune responses TLR7 signaling converges with IFNγ-STAT1 signaling in B cells by recruiting STAT1 into the MyD88 signaling complex. Together, we identify previously unappreciated differential regulation of autoimmune and anti-pathogen responses by STAT1-pS727, and implicate STAT1-pS727 as a therapeutic target for SLE.
Food allergens could promote the Th2 mediated autoantibody development in an autoimmune skin disease bullous pemphigoid
Authors: Qian Ye , Lin Lan , Moran Timothy Paul , Yang Jinsheng , Liu Zhi , Culton Donna Aline
Published: January 2023

Bullous pemphigoid (BP) is the most common autoimmune skin blistering disease and is characterized by the presence of autoantibodies (autoAbs) against skin hemidesmosomal adhesion molecules. The predominant antigenic target is the non-collagenous domain 16 (NC16A) of BP180. AutoAbs against NC16A lead to loss of adhesion and subsequent subepidermal blistering. It is unclear what triggers the development of autoAbs. Food allergens are known to induce T helper 2 (Th2) antibody responses and our previous studies have shown the possible role of environmental antigens/allergens in promoting autoAb development. The goal of this study is to investigate whether Th2 mediated immune responses against food allergens drive the autoAb development in BP. BP serum antibodies were tested against a panel of common environmental and food allergens. Anti-NC16A IgE and IgG4 monoclonal antibodies (mAbs) and their naïve counterparts were generated from a BP patient to determine their specificity or cross-reactivity. We found that antibodies from BP patients recognized food allergens, such as walnut, but not other types of allergens, such as insects, epithelia, fungi, or pollens. The levels of serum IgE and IgG4, but not IgG1, against selected food allergens were significantly higher than controls. The anti-NC16A IgE and IgG4 mAbs cross-reacted to food allergens, and their naïve mAb counterparts had reduced reactivity to NC16A, but still reacted strongly to food allergens. In addition, mice immunized with walnut allergen produced anti-NC16A antibodies. Our results indicate that Th2-associated autoAbs from BP subjects might be linked to antibody responses toward food allergens and may provide a target for the management and treatment of BP.
Autoreactive B cells have a specific metabolic response during humoral responses
Authors: Choi Seung-Chul , Li Wei , Zhang Xiaojuan , Teng Xiangyu , Morel Laurence
Published: January 2023

Germinal centers (GC) provide a unique microenvironment for B-cell affinity maturation and class-switching recombination to occur. We compared for the first time the metabolism of B cells between lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice and B6 controls at steady state, as well as during T cell-dependent (TD) and T cell-independent (TI) immunizations. B cells from TC mice showed an elevated glycolysis and mitochondrial oxidative metabolism, which was normalized by inhibiting glycolysis with in vivo 2-deoxy-D-glucose (2DG) treatment. 2DG greatly reduced the production of TI-antigen-specific antibodies, but showed minimal effect with TD-antigens. In contrast, glutaminolysis inhibition with 6-Diazo-5-oxo-L-norleucine (DON) had a greater effect on TD than TI Ag-specific antibodies in both strains. Interestingly, 2DG, but not DON reduced TI-Ag-specific IgM in TC mice, whereas both 2DG and DON prevented Ag-specific IgM production in B6 mice. Thus, autoreactive and control B cells have different intrinsic metabolic requirements. Autoreactive B cells are more glycolytic, which mirrors our previous results showing that autoreactive TFH cells have opposite glucose and glutamine requirements. The requirement for glutamine in TD-responses could be due to a direct effect on GC B cells, or an indirect effect through TFH cells. Overall, these results predict that targeting glucose metabolism provides an effective therapeutic approach for systemic autoimmunity by eliminating both autoreactive TFH and B cells, although TI-responses may be reduced in lupus-prone mice.
Deletion of von-Hippel Lindau in Dmp1-expressing cells impairs B cell development
Authors: Chicana Betsabel , Burns Christian , Spencer Joel , Manilay Jennifer
Published: January 2023

Our objective is to characterize how altered bone homeostasis influences B lymphocyte development. The von-Hippel Lindau protein (VHL) regulates hypoxia-inducible factor degradation and is involved in responses to low oxygen environments. Osteoblasts (OBs) support B cell development and mature into osteocytes (OCYs). We utilized Dmp1-Cre;Vhl conditional knockout mice (cKO), in which Vhl is deleted in OBs and OCYs. The cKO display dysregulated bone growth, high bone density, smaller bone marrow (BM) cavity volume, and an overall decrease in BM cellularity compared to wild-type (WT) mice. B cell progenitors is significantly increased in frequency and numbers whereas Hardy Fractions B, C, D, E and F were significantly decreased in the BM of cKO mice. Analysis of BM B cells 16 weeks after whole BM transplantation, showed a significant reduction in Fractions B-C and later Fractions in the WT→cKO mice, while B cell development was normal in the cKO→WT chimeras, indicating a cell-extrinsic effect of Vhl on B cells within the cKO BM microenvironment. We hypothesize that B cell development is not supported due to reduction of key niche cells and decreased production of IL7, SCF, CXCL12, and Flt3. To define the defective niche cells, we will use in vitro B cell development assays in which WT B cells will be co-cultured with sorted cKO OBs, MSCs, and OCYs. B cell apoptosis, proliferation and turnover rates will be measured. We have begun preliminary studies using intravital and ex vivo imaging of the cKO BM to identify changes in oxygen tension, blood vessel type and structure, the presence and location of stromal cells. These studies have the potential to reveal novel molecular mechanisms by which Vhl in Dmp1 expressing cells may serve as a B cell niche.
Immunological characteristics and clinical significance of urinary CD11c+ macrophages in lupus nephritis
Authors: Kim Jihye , Hong Seokchan , Lee Chang-Keun
Published: January 2023

Kidney-infiltrating immune cells may be involved in the pathogenesis of lupus nephritis (LN). We aimed to analyse the immunological characteristics and clinicopathologic relevance of urinary CD11c+ macrophages in patients with LN. Compared with patients with no LN or non-proliferative LN, those with proliferative LN had significantly higher urinary levels of CD11c+ macrophages, which were strongly correlated with their anti-dsDNA antibody titer. Urinary CD11c+ macrophages expressed pro-inflammatory cytokines, such as interleukin (IL)-6 and IL-1β, and resembled infiltrated monocytes rather than tissue-resident macrophages in terms of surface marker expression. They showed high expression of the chemokine receptor CXCR3, whose cognate chemokine IP-10 was expressed by urinary tubular epithelial cells (TECs). When treated with sera from systemic lupus erythematosus patients, peripheral monocytes acquired the morphological and functional properties of urinary CD11c+ macrophages, which was blocked by DNase treatment. Finally, SLE sera treated monocytes induced fibronectin expression, apoptosis, and cell detachment in HK-2 cells via IL-6 production. Importantly, the number of urinary CD11c+ macrophages was significantly associated with chronicity indices, including tubular atrophy and interstitial fibrosis, and renal response in LN patients. CD11c+ macrophages may be involved in the pathogenesis of tubulointerstitial damage in LN.
High dimensional single cell characterization of autoreactive CD8 T-cells reveals heterogeneous phenotypes that play a role in disease progression in Type 1 Diabetes
Authors: Nguyen Hai T , Dufort Mathew T. , Speake Cate , Greenbaum Carla , Linsley Peter S , James Eddie A
Published: January 2023

Type 1 Diabetes (T1D) is caused by beta cell destruction, eventually resulting in loss of glycemic control. Following onset, subjects with T1D exhibit diverse amounts of residual c-peptide, indicating varying levels of beta cell function. Persistence of c-peptide is correlated with reduced risk of complications and its preservation has been used as an endpoint in clinical trials. However, the immunologic factors that differentiate subjects who retain measurable c-peptide from those who do not are not well characterized. We investigated the number and phenotype of beta cell specific CD8+ T-cells in peripheral blood samples from a cohort of T1D subjects, stratified into slow progressors (c-peptide > 0.1ng/ml) and rapid progressors (undetectable c-peptide), including longitudinal samples. Using tetramers corresponding to HLA-A2 epitopes, we enumerated epitope specific T-cells and sorted single cells for RNA-Seq. Differential gene expression between slow and rapid progressors indicated differences in effector, exhaustion, and regulatory pathways which were consistent with a recently published high dimensional CyTOF data set. Assembly of autoantigen specific TCRs revealed differences in clonal expansion for specific cell clusters. Pseudotime trajectory analysis of the scRNAseq data revealed that CD8+ T cells expressing the same TCR resided in different phenotypic end states. Furthermore, flow cytometric analyses demonstrated differential expression of lineage-specific cell surface markers, which correlated well with the transcriptional profiles within each cluster. Cumulatively, we discovered specific aspects of T cell function and repertoire that differ between slow- and rapid-progressing T1D subjects.
Role of ICOS expressing CXCR5+PD-1+ T helper cells in pemphigus vulgaris
Authors: Kim A Reum , Han Dawoon , Choi Ji Young , Seok Joon , Kim Song-Ee , Seo Seong-Hoon , Park Su-Hyung , Kim Soo-Chan , Shin Eui-Cheol , Kim Jong Hoon
Published: January 2023

Pemphigus vulgaris (PV) is an autoimmune bullous disease mediated by autoantibodies against desmoglein (DSG) 3 which is the adhesive molecule in the desmosomes. DSG3-specific CD4+ helper T cells have been demonstrated to be involved in autoantibody production and pathogenesis in mouse models and human patients. However, it remains unknown if T follicular helper (Tfh) cells expressing ICOS play a role in the pathogenesis of PV although ICOS+ Tfh cells have a critical role in various autoimmune diseases. The aim of this study was to analyze the immunological characteristics and pathogenic roles of ICOS+ Tfh cells using PV mouse model and peripheral blood mononuclear cells (PBMCs) from pemphigus patients. In addition, we tried to investigate the therapeutic effect of ICOS-ICOSL blockade in PV mice. We immunized the DSG3−/− mice by grafting skin of wild-type mice, then we adoptively transferred CD4+ T and B cells from the DSG3-primed DSG3−/− mice to RAG1−/− mice. We found that ICOS+ Tfh cells were associated with B cell development in the mouse model. By using an MHC class II tetramer, we showed that DSG3-specific ICOS+ Tfh cells were increased and significantly related to the level of anti-DSG3 antibody. In PBMCs from the patients with pemphigus vulgaris, the frequency of ICOS+CXCR5+PD-1+ memory CD4+ T cells was positively correlated with DSG3 autoantibody levels. Furthermore, the administration of anti-ICOS blocking antibody to PV mice in vivo decreased the production of anti-DSG3 antibody and prevented disease activation. Therefore, ICOS+CXCR5+PD-1+ T helper cells are associated with the disease progression and can be a therapeutic target for PV.
Glucocorticoid receptor modulates myeloid-derived suppressor cell function via mitochondria respiration and metabolism in immune thrombocytopenia
Authors: Wang Shuwen , Xie Jie , Liu Yan , Peng Jun
Published: January 2023

Methods

We collected 27 immune thrombocytopenia (ITP) patients who had received high-dose dexamethasone treatment. The myeloid-derived suppressor cells (MDSCs) were induced in vitro and CHIP-qPCR assays were performed. Seahorse XF analyzer was used to analyze oxygen consumption rate and parameters of mitochondrial function of MDSCs. MDSCs were treated with CPT-1 inhibitor, etomoxir, to explore the effect of inhibiting fatty acid oxidation. Glucocorticoid receptor (GR) expression was blocked in murine MDSCs which were transplanted into ITP mice.

Results

The mRNA expression of GR and mtDNA-encoded genes (ATP8, COX1, CYTB, ND1, ND5), as well as the binding of GR to mtDNA in MDSCs were decreased in steroid-resistant ITP group compared to steroid-responsive ITP group and healthy controls. MDSCs in steroid-resistant patients had decreased levels of basal respiration, maximal respiration and ATP production, suggesting that the mitochondria of MDSCs in steroid-resistant group had lower activities of electron transport chains and oxidative phosphorylation. The function of MDSCs to inhibit T-cell proliferation was decreased in steroid-resistant group. Etomoxir reduced the ability of MDSCs to block T-cell proliferation in steroid-responsive group, but did not alter the suppressive function of MDSCs on T cells in steroid-resistant group. ITP mice transplanted with GR siRNA-treated MDSCs showed lower platelet counts than those with control siRNA-treated MDSCs, suggesting that the function of MDSCs to ameliorate ITP relied on GR.

Conclusions

This study demonstrated that GR-associated MDSCs impairments are involved in the pathogenesis of ITP. It might be related to the modulation of mitochondrial respiration and metabolism by GR.
Anti-idiotypic network involved in restoring ADAMTS13 activity in immune-mediated thrombotic thrombocytopenic purpura (iTTP)
Authors: Heeb Silvan Rolf , Schaller Monica , Hovinga Johanna Anna Kremer
Published: January 2023

The culprit of iTTP, a fatal autoimmune disease is a severe deficiency of the von Willebrand factor-cleaving protease ADAMTS13, caused by autoantibodies inhibiting its enzymatic activity. The pathophysiology upholding remission in certain patients but leading to relapse in others is unknown. We hypothesize that one mechanism keeping pathogenic autoantibodies in check is a network of anti-idiotypic antibodies, which we aimed to identify and characterize.

From splenic mononuclear cells of two frequently relapsing iTTP patients (A and C), the anti-idiotypic IgG1 Fab κ/λ repertoire was amplified by phage display technology on previously generated selecting monoclonal anti-ADAMTS13 Fabs (Schaller et al., Blood 2014). Purified Fabs were pooled (pools A (n=4) and C (n=4)) and each pool was tested for its ability to neutralize anti-ADAMTS13 antibodies thereby restoring ADAMTS13 activity in plasma of 22 iTTP patients using the FRETS-vWF73 assay.

Single anti-idiotypic clones revealed a highly diverse IGHV gene usage within and between the two iTTP patients, but four IGHV genes were shared among them. Partial neutralization of functional ADAMTS13 inhibitors of >2BU/ml in 3/11 (27%) and 5/11 (45%) patients and full neutralization in 2/11 (18%) and 3/11 (27%) patients with lower inhibitor titer (1–2BU/ml) were shown for pools A and C, respectively.

To summarize, we have identified functional anti-ADAMTS13 anti-idiotypes in the splenic IgG repertoire of two patients underlining the role of an anti-idiotypic response involved in remission of iTTP. The contribution of single anti-idiotypic Fabs to the neutralization potential and plasma mixing studies investigating acute and remission phase antibody repertoires are underway.
Increased expression of EZH2 is mediated by higher glycolysis and mTORC1 activation in lupus CD4+ T cells
Authors: Sawalha Amr , Zheng Xiaoqing , Tsou Pei-Suen
Published: January 2023

Objective

EZH2 is overexpressed in lupus CD4+ T cells. Increased disease activity in lupus patients is associated with a pro-inflammatory epigenetic shift in naïve CD4+ T cells, likely mediated by EZH2. Here we aim to understand the upstream mechanisms underlying EZH2 overexpression in lupus CD4+ T cells.

Methods

Naïve CD4+ T cells were isolated from lupus patients and then stimulated with anti-CD3/anti-CD28. qPCR and Western blotting were used to measure mRNA and protein expression levels, respectively. 2-Deoxy-D-glucose (2-DG) was used to inhibit glycolysis. mTORC1 signaling was inhibited using rapamycin. Oxidative stress was induced by H2O2.

Results

Because glycolysis is increased in lupus CD4+ T cells and glycolysis regulates miR-101 and miR-26a, which target EZH2, we examined the effect of inhibiting glycolysis on EZH2 expression. 2-DG significantly inhibited EZH2 expression in lupus CD4+ T cells. In addition, 2-DG restored the expression of miR-101 and miR-26a, suggesting that suppression of EZH2 by 2-DG might be mediated by miR-101 and miR-26a. Because mTORC1 is activated in lupus CD4+ T cells in part due to increased oxidative stress, and mTORC1 activation increases glycolysis, we hypothesized that mTORC1 mediates increased EZH2 expression. Indeed, inhibiting mTORC1 increased miR-101 and miR-26a and suppressed EZH2 expression in lupus CD4+ T cells. Further, H2O2 treatment increased EZH2 expression, however, this effect appears to be independent of miR-101 and miR-26a.

Conclusion

Increased EZH2 is mediated by activation of mTORC1 and increased glycolysis in lupus CD4+ T cells. Therapeutic effects from mTORC1 inhibitors and glycolysis inhibitors in lupus might be in part mediated by suppression of EZH2.
Aryl hydrocarbon receptor inhibition and non-canonical signaling activation impact Treg function in autoimmune hepatitis
Authors: Vuerich Marta , Harshe Rasika , Abrahão-Frank Luiza , Mukherjee Samiran , Gromova Barbora , Bonder Alan , Patwardhan Vilas , Robson Simon C. , Longhi Maria Serena
Published: January 2023

Treg are dysfunctional in autoimmune hepatitis (AIH), a severe hepatopathy characterized by hypergammaglobulinemia, presence of serum autoantibodies and interface hepatitis on histology. AIH-derived Treg display impaired expression of CD39, an ectonucleotidase that scavenges extracellular ATP to generate immunosuppressive adenosine. CD39 can be regulated upon activation of aryl-hydrocarbon-receptor (AHR), a mediator of toxin responses that also modulates T-cell immunity. Upon binding to endogenous or exogenous ligands, AHR dimerizes with aryl-hydrocarbon-receptor-nuclear-translocator (ARNT) or other non-canonical factors like estrogen-receptor-alpha (Era) or Kruppel-like-factor-6 (KLF6) to modulate gene expression. AHR is in turn regulated by the aryl-hydrocarbon-receptor-repressor (AHRR) that inhibits the transcriptional activity of the AHR/ARNT complex. We note that, compared to healthy subjects (HS), Treg obtained from AIH patients display increased AHRR levels and heightened expression of non-canonical binding factors Era and KLF6, but comparable AHR and ARNT levels. Differently from HS, AIH Treg do not upregulate CD39 expression and activity upon exposure to the AHR ligands unconjugated bilirubin (UCB), L-Kynurenine and quercetin. Silencing of AHRR and Era via siRNA boosts CD39 in AIH Treg in the presence of UCB and quercetin. We further note that, in AIH Treg, AHR binds Era with higher affinity than ARNT. In conclusion, multiple AHR signaling alterations might impact CD39 and Treg function in AIH. Targeted blockade of AHRR and Era represents a potential therapeutic approach to restore immune homeostasis in AIH and other chronic inflammatory conditions by boosting CD39.
Genetic and infectious regulators of baseline and acute immunity across ancestrally distinct human populations
Authors: Souquette Aisha , Allen Kaity , Oshansky Christine , Tang Li , Wong Sook-san , Jeevan Trushar , Ogunjimi Benson , Schultz-Cherry Stacey , Webby Richard , Gordon Aubree , Rothman Richard , Pekosz Andrew , Thomas Paul Glyndwr
Published: January 2023

Studies have identified genetic, viral, and immunological associations with the quality of anti-influenza immunity and/or influenza disease severity, however, there has not been an integrative analysis of these factors, both at baseline and during acute infection. Using samples from healthy or flu infected human cohorts across 5 countries, we found that herpesviruses (HVs) have unique and interactive effects on cytokine levels specific to anatomic location during flu infection. Similar cytokine associations were also observed in healthy controls. Additionally, HV infection is also associated with decreased flu severity and virus shedding, and increased antibody titers. Associations between cytokine levels and flu severity were consistent in cohorts of similar ancestral and environmental backgrounds, however unique correlates were observed in populations from distinct backgrounds. Further, we identified ~100 variants in immune related genes either enriched in or absent from a given ancestral population, a portion of which are eQTLs at baseline, supporting the potential of host genetics to impact immune variation. Ongoing work focuses on assessing which SNPs are eQTLs in response to bacterial and viral TLR agonists. Ancestry informative marker PCA values and eQTLs will be included in the statistical models to account for the collective effects of infectious, biological, and genetic factors. These results will provide insight into which factors predominantly affect a given immune measure and how this contributes to immune competence and variation across distinct populations. Understanding these interactions will have implications for other infectious diseases, autoimmunity, clinical study design, and immunotherapy.
Synergic effect of combined intervention with clindamycin and PPARγ agonist in an HLA-II mouse model of Group A Streptococcal Necrotizing Fasciitis
Authors: Nookala Suba , Mukundan Santhosh , Combs Colin , Satpati Abhijit , Kotb Malak
Published: January 2023

Group A Streptococcus (GAS, Streptococcus pyogenes) is an important human pathogen that can cause a broad spectrum of diseases ranging from self-limiting pharyngitis and impetigo to deeper, life-threatening invasive infections such as streptococcal toxic shock syndrome and necrotizing fasciitis (NF). An overarching pro-inflammatory cytokine storm and a rapidly progressive tissue destruction lead to significant morbidity and lethal outcomes of GAS NF. Transcriptome profiling of uninfected and GAS infected skin from HLA-II transgenic mouse models of GAS NF revealed strong activation of inflammatory signaling and inhibition of peroxisome proliferator-activated receptor (PPAR) signaling pathways. Specifically, PPARgamma (PPARγ), a lipid ligand-activated transcription factor driving adipogenic and anti-inflammatory effects, was a significantly inhibited upstream regulator and this inhibition was reflected in downregulation of PPARγ-dependent genes. So far, there are no reports on the role of PPARγ in GAS infections. Here, we explored the potential use of the PPARγ selective synthetic agonist, pioglitazone (PZ), in combination with clindamycin (CLN), as a combined therapeutic approach in vivo in an HLA-II mouse model of GAS NF. Although the mechanism of synergy is not understood, our data demonstrate that combined CLN PZ intervention offers significant advantages over treatment with CLN alone, and uncover a new role for PPARγ in attenuating skin lesions, GAS burden, IL-6 and IFN-γ levels, and weight loss during GAS NF. To our knowledge, this is the first report to demonstrate the benefits of a combined host- and pathogen-directed treatment approach to ameliorate severity and inflammation during GAS NF.
HIV-1 infection is associated with increased prevalence of immature neutrophils and alterations in total neutrophil phenotype and metabolic profile
Authors: Connelly Ashley N , Pal Harish , Davis Marcus , Ong Krystle , Kuznetsova Valeriya , Fay Christian , Overton E. Turner , Hel Zdenek
Published: January 2023

Despite successful viral control, HIV-1-infected individuals have a higher risk of developing life-threatening comorbidities including liver and cardiovascular diseases which are associated with inflammation and immune dysregulation. However, the specific mediators driving these pathologies are not yet understood. Neutrophils are increasingly recognized as a heterogeneous population with critical roles in immune regulation and disease pathogenesis. Methods of neutrophil characterization developed in our laboratory have facilitated the identification of novel neutrophil subpopulations including two subsets of low-density neutrophils (LDNs). LDNs are known to be expanded in inflammatory conditions including cancers and chronic infections. The LDN subpopulations identified in our laboratory represent different maturation states as indicated by their distinct nuclear morphologies and phenotypic differences [mature (mLDNs) CD16+CD64Low and immature (imLNDs) CD16−CD64+]. Our findings indicate that imLDNs have a reduced capacity for reactive oxygen species production and phagocytosis. We present data demonstrating that HIV-1/AIDS is associated with a higher frequency of imLDNs and significant alterations in total neutrophil phenotype (decreased CD16 and increased CXCR4 and CD10), function, and metabolism including increased expression of genes related to oxidative phosphorylation and mTOR signaling. Observed changes in neutrophil populations correlate with key clinical parameters including CD4+ T cell count, level of liver fibrosis, and arterial stiffness suggesting an important role for these cells and specific subsets in the pathogenesis of HIV-1/AIDS and potentially other inflammatory diseases.
Complement-driven inflammasome activation on circulating blood monocytes in immune reconstitution inflammatory syndrome in TB-HIV coinfected patients
Authors: Lage Silvia Lucena , Wong Chun-Shu , HSU Denise C , Sturdevant Daniel , Manion Maura , Naqvi Nuha S , Lisco Andrea , Laidlaw Elizabeth , Mejia Yolanda , Amaral Eduardo Pinheiro , Sereti Irini
Published: January 2023

Inflammasome and complement pathways have been independently implicated in the pathogenesis of Tuberculosis (TB) immune reconstitution inflammatory syndrome (IRIS), a clinical complication affecting individuals initiating antiretroviral therapy (ART). Here we sought to investigate whether dysregulated complement contributes to inflammasome activation during TB-IRIS. Caspase-1+ specks are cytosolic inflammasome aggregates responsible for IL-1β and IL-18 release. We detected higher numbers of monocytes with spontaneous caspase-1+ specks in IRIS patients (n=9, baseline median CD4 count= 46 cells/mL and VL= 606,754 copies/mL) in comparison with TB infected, non-IRIS controls (n=7, baseline median CD4 count= 47 cells/mL and VL= 85,831 copies/mL) at IRIS time-point (p<0.05). Patients experiencing IRIS also showed elevated C1q and C3 deposition on monocytes, followed by increased levels of the membrane attack complex (MAC) molecule, C9 (p<0.01). Levels of membrane-bound C9 (r= 0.69, p<0.05), and the cell-associated MAC inhibitor, CD59 (r = 0.6 p <0.05), correlated with IL-18 plasma levels and were associated with caspase-1 activation on patients-derived monocytes (r= 0.85, p<0.001 and r= 0.97, p<0.001, respectively to C9 and CD59), supporting a role for sublytic MAC-driven membrane pore formation in inflammasome activation on patient cells. Because the expression level of CD59 strongly correlated with caspase-1 activation within the CD14high monocyte subsets, our data suggest that those cells were able to prevent full MAC deposition, therefore undergoing sublytic MAC-driven inflammasome activation. This data may help identify new targets for prevention or treatment of TB-IRIS inflammatory syndrome.
The chemokine CCL17 is associated with protection against HIV-1 acquisition
Authors: Powell Timothy R , Duarte Rodrigo , Hotopf Matthew , Hatch Stephani , de Mulder Miguel , Marín-Hernández Daniela Marin , Breen Gerome , Lewis Cathryn , Nixon Douglas F.
Published: January 2023

Millions are exposed to the human immunodeficiency virus type 1 (HIV-1) every year, but not all exposed individuals become infected. This suggests a key role for host genetics in resilience or susceptibility to HIV-1 infection, but a comprehensive study of the genetic mechanisms contributing to HIV-1 acquisition risk is lacking. Here, we performed LD score regression analysis using summary statistics from the largest genome-wide association study of HIV-1 acquisition, consisting of 6,334 infected patients and 7,247 population controls. We found that HIV-1 acquisition is polygenic and highly heritable (28%). Genetic correlations performed alongside UK Biobank data revealed associations between HIV-1 acquisition and smoking, prospective memory and socioeconomic traits. Gene-level enrichment analysis identified EF-hand calcium binding domain 14 (EFCAB14) as a novel risk gene for HIV-1 acquisition. We also observed that risk variants for HIV-1 acquisition were significantly enriched for genes expressed in T-cells, but also those expressed in striatal and hippocampal neurons, which could explain the behavioral parameters genetically correlated with acquisition. Finally, we modeled how inter-individual variation in risk for HIV-1 acquisition influences the pre-exposed immune profile, by testing how individualized polygenic risk scores influence blood levels of 34 inflammatory markers in 406 HIV-1-negative individuals. We showed that higher genetic risk for HIV-1 acquisition was associated with lower levels of CCL17. Our findings corroborate a complex model for HIV-1 acquisition, whereby susceptibility is moderated by specific behavioral, cellular and immunological parameters.