bluuebunny/crossref_metadata_2025_split huggingface dataset:
Allelic polymorphism gates vaccine amplification of a human broadly neutralizing response against influenza virus
Authors: Sangesland Maya , de la Pena Alba Torrents , Ronsard Larance , Ghebremichael Musie , Ward Andrew B , Lingwood Daniel 
Published: February 2023

Abstract
A major goal of universal influenza vaccine design is to elicit high titer antibody responses against functionally conserved but immunologically subdominant sites of vulnerability. Using a transgenic mouse model where antibodies develop with human-like CDRH3 diversity but are otherwise restricted to single VH-genes of interest, we have demonstrated that human IGHV1-69 endows for pathway-amplifiable broadly neutralizing antibody (bnAb) responses targeting the hemagglutinin (HA) stalk of group 1 influenza A viruses following vaccination with an engineered HA nanoparticle. However, IGHV1-69 is polymorphic with alleles containing either a phenylalanine (F54) or leucine (L54) within the contacting CDRH2 loop at germline, the latter which is correlated with dampened IGHV1-69 usage in human stalk bnAbs. To experimentally test the consequence of this, we applied our transgenic model to assess the capacity for both alleles to vaccine expand bnAbs through pathway-amplification. We found that while both VH forms endow germline targeting solutions to the same bnAb site on HA, only the F54 allele is productive following sequential immunization with a stalk-only nanoparticle immunogen. By contrast, L54 BCRs, while initially expanded, were autoreactive and rapidly tolerized upon boosting. While globally most humans utilize at least one copy of the F54 allele and would be predicted to support bnAb expansion, certain ethnicities show heightened usage of the L54 allele and may be unable to support such pathway-amplification. Thus, we find that allele usage gates the ability to pathway expand productive bnAb responses through immune tolerance.
Sensing of antigen affinity and kinetic rates by B cells expressing receptors of HIV-1 broadly neutralizing antibodies.
Authors: Hossain Md Alamgir , Anasti Kara , Ortiz Yaneth , Cronin Kenneth , Watts Brian , Khanore Advaiti , Verkoczy Laurent , Reth Michael , Alam S. Munir 
Published: February 2023

Abstract
B cell activation is dependent on the productive engagement of an antigen (Ag) with the B cell receptor (BCR). How complex protein Ags that bind with different affinities and kinetic rates affect the strength of B cell activation is not fully understood. We used a panel of HIV-1 Env proteins of varying affinities and each expressed in either monomeric or multimeric forms to investigate the role of binding rates on B cell activation and Ag-induced BCR down-modulation. Ramos cells expressing the CD4 binding-site CH31 IgM BCRs were functional for Ag-specific activation and gave Ca-flux responses that was not dependent on the overall affinity or dissociation rates (off-rates) but on the association rates (on-rates) of Ag binding. Monomeric Ags did not induce flux responses and required multimerization, and the strength of activation was dependent on the on-rate of the tetrameric Ags. Comparison of half-life of the tetrameric Ags indicated a requirement of a half-life threshold for both activation and BCR down-modulation. In contrast, trimeric Ags that bound with faster on-rates (ka&gt;104 M−1s−1) did not require higher-order multimerization (6-mer or 20-mer) for Ca-flux responses. These results provide support to a kinetic model in which B cell activation is dependent on the rates of receptor occupancy, and an above threshold half-life of the Ag-BCR complex.
Pathogenic ILC1 and NKp46+ ILC3 Are Defined by Core and Tissue-Specific Transcriptional Signatures in Autoimmune Neuroinflammation
Authors: Spinner Camille A , Guan Yukun , Fan Yu , Magen Assaf , Ciucci Thomas , Bosselut Rémy , Meerzaman Daoud , Lazarevic Vanja 
Published: February 2023

Abstract
During multiple sclerosis, myelin-specific CD4+T cells invade the central nervous system (CNS) where they initiate a chronic inflammatory disease, leading to demyelination and neurodegeneration. Therefore, it is crucial to identify immunological factors allowing disease-inducing T cells to enter the CNS. Our previous study revealed a novel function of T-bet-expressing NKp46+ innate lymphocytes (ILC) as CNS gatekeepers, which control functional stability and access of autoreactive Th17 cells to the CNS. However, the identity of NKp46+ ILC subset(s) that regulate neuroinflammation and their mechanism of action are still poorly characterized. T-bet-expressing NKp46+ ILC encompass three subsets – conventional natural killer (NK) cells, ILC1 and NKp46+ ILC3. Here, we demonstrate that the pathogenic role of T-bet-expressing NKp46+ ILC maps to ILC1 and NKp46+ ILC3 but not NK cells. Single cell RNA-Seq analysis revealed that meninges-infiltrating NK cells exhibit undifferentiated phenotype devoid of expression of effector molecules and reminiscent of blood NK cells, explaining the functional redundancy of NK cells in the disease process. By contrast, NKp46+ ILC1/3, which directly interact with myelin-specific CD4+ T cells, were defined by the core set of 13 genes, which was not shared by splenic or meningeal NK cells. A subset of the ILC-specific genes was highly expressed by meningeal, but not splenic, ILCs, suggesting their importance in disease pathology. Thus, our transcriptional analysis identified modules of gene expression that coordinate functional polarization of ILCs in meningeal microenvironment and provides a blueprint for the discovery of ILC-specific targeted therapy for the treatment of CNS autoimmune disorder.
EGR2 plays a differential immune regulatory role in normal C57BL/6 mice and autoimmune-prone B6/lpr mice
Authors: Dai Rujuan , Heid Bettina , Wang Zhuang , Reilly Christopher M , Ahmed S. Ansar 
Published: February 2023

Abstract
Early growth response protein 2 (EGR2) is a zinc-finger transcription factor that belongs to the early growth response (Egr) gene family. Recent studies have shown that EGR2 is required to induce T cell anergy, and it plays an important role in T cell immunity and autoimmunity. Conditional depletion of EGR2 in T lymphocytes in C57BL/6 (B6) mice (EGR2−/−B6) was able to break immune tolerance and induce T cell-driven, a lupus-like autoimmune disease. In EGR2−/−B6 mice, there was also enhanced GC response and humoral responses. However, increased EGR2 expression has been observed in lupus and autoimmune disorder systemic sclerosis. These observations seem to contradict the autoimmune suppressive role of EGR2 in B6 mice. To investigate further the immune regulatory role of EGR2 in an autoimmune context, we derived EGR2−/−B6/lpr mice with EGR2 deficiency in both T and B cells. We found that EGR2 depletion promoted splenomegaly and increased IFNγ, IL-10, IL-2 in both B6 and B6/lpr mice. Significantly, we found that in B6/lpr mice, EGR2 depletion reduced serum levels of anti-dsDNA autoantibodies, total IgG, IgG1, IgG2a, and IgM. Furthermore, in B6/lpr mice, depletion of EGR2 promoted germinal center B (GCB) cell development, but suppressed plasma cell (PC) differentiation. However, in wild-type B6 mice, EGR2 depletion significantly increased anti-dsDNA autoantibodies, accompanied by a significant increase of GCB cell development and a trend of increase in PC differentiation. Together, we demonstrated that EGR2 has a differential role in the normal and autoimmune state, particularly in regulating GCB transition into plasma cell during late B cell development.
SARS-CoV2 spike protein induces inflammatory molecules through TLR2 in macrophages and monocytes
Authors: Zaki Hasan , Khan Shahanshah 
Published: February 2023

Abstract
COVID-19 pandemic caused by SARS-CoV2 infection has emerged as a major public health problem. Excessive inflammation or cytokine storm is considered a major cause of respiratory failure, organ damage, and death of COVID-19 patients. However, the precise mechanism of inflammatory response induced by SARS-CoV2 is poorly understood. We investigated whether the structural proteins of SARS-CoV2 including spike (S), membrane (M), envelope (E), and neucleocapsid (N) are involved in inducing inflammation. Human monocytic cells THP-1 derived macrophages or human peripheral blood mononuclear cells were stimulated with the recombinant structural proteins and measured for the induction of inflammatory cytokines and chemokines. We observed that S protein potently induces IL-6, IL-1b, TNFa, CXCL1, and CCL2 in macrophages and monocytes, while other structural proteins were non-stimulatory. Notably, human lung epithelial cells A549 and human embryonic kidney epithelial cell HEK293T, which express S protein receptor ACE2, did not express cytokines and chemokines in response to S and other structural proteins. We dissected signaling pathways involved in inducing inflammatory molecules in response to S protein. S protein activates the NF-kB pathway in a MyD88 dependent manner. Further, S protein-mediated activation of NF-kB and induction of inflammatory responses were completely abrogated in TLR2-deficient macrophages, suggesting that TLR2 is an innate immune receptor for SARS-CoV2 S protein. Together these data reveal a mechanism of cytokine storm during SARS-CoV2 infection, and points to TLR2 as a potential therapeutic target for COVID-19.
Tolerance Induction by Exopolysaccharide from the Probiotic Bacillus subtilis
Authors: Zamora-Pineda Jesus Bernabe , Knight Katherine 
Published: February 2023

Abstract
Intestinal microbiota can lessen the severity of inflammatory diseases, although the mechanisms by which this occur are poorly understood. We showed that exopolysaccharide (EPS) from the probiotic Bacillus subtilis induces anti-inflammatory macrophages and protects mice from enteric infection and sepsis. Here, we examined the molecular mechanisms by which EPS induces an anti-inflammatory response. In co-cultures of OVA-loaded bone marrow-derived dendritic cells (BMDC) and OT-II T cells, EPS inhibits T cell activation in a TLR4-dependent manner. We hypothesize that EPS engages TLR4 and activates the noncanonical NF-kB signaling pathway, which up-regulates the expression of indoleamine 2,3-dioxygenase (IDO). IDO degrades the essential amino acid tryptophan into kynurenine. The subsequent tryptophan depletion and kynurenine formation leads to suppression of T-cell activation and induction of tolerance. Western blot analysis of BMDC treated with EPS showed nuclear translocation of the transcriptional factor RelB, which is central to the noncanonical pathway, and upregulation of IDO. Inhibition of IDO using the inhibitor, 1-methyl tryptophan and deletion of IDO in BMDCs reverses the effect of EPS. We conclude that EPS inhibition of T cell activation and proliferation is mediated by IDO.
Investigating the impact of TLR ligand induced Id2 expression in dendritic cell development and function
Authors: Babcock Rachel Lauren , Patel Bhakti , Zhou Yifan , Chrisikos Taylor T , Kahn Laura M. , Medik Yusra B. , Watowich Stephanie S 
Published: February 2023

Abstract
The inhibitor of DNA binding (Id) proteins and E proteins are basic helix-loop-helix proteins that act as mutual antagonists to direct immune cell development and function. In developing dendritic cells (DCs), Id2 specifies type 1 conventional DCs (cDC1s) and represses E2-2, an E protein and master regulator of plasmacytoid DCs (pDCs). DCs detect viruses, bacteria, and tumor cells via pattern recognition receptors, such as Toll-like receptors (TLRs), to elicit immune responses. Previously, we found Id2 mRNA and protein was induced in TLR agonist-stimulated murine pDCs. These data suggest TLR signaling alters the balance of Id and E proteins in DCs, however it is unknown whether TLR signaling affects Id2 mRNA in common DC progenitors (CDPs) and what effect this has on DC lineage development. We hypothesize TLR stimulation induces Id2 in DC progenitors thereby affecting generation of DC subsets. We found TLR agonist-stimulated CDPs expressed higher amounts of Id2 mRNA compared to non-treated controls. TLR ligand-treated mice exhibited a rapid decrease in CDPs that recovered within 24-hours, consistent with prior work, as well as changes in peripheral DC subset frequencies. Interestingly, Id2-deficient and -sufficient pDCs and cDC1s exhibited similar phenotypic and functional changes to TLR agonist stimulation, including co-stimulatory molecule expression and soluble factor production. These data suggest induction of Id2 occurs in CDPs in response to TLR stimulation and affects DC development, while Id2 appears to be dispensable for pDC and cDC1 maturation. Future work will test the requirement of Id2 in TLR agonist-induced DC lineage differentiation, and analyze the effect of cytokines on Id2 expression in DC progenitors.
Regulation of Th17 CD4 T Cell Responses During Pulmonary Group A Streptococcus (GAS) Infections by Dendritic cells
Authors: Lopez Christopher , Zacharias Zeb , Waldstein Kody , Hornick Emma , Legge Kevin L 
Published: February 2023

Abstract
The respiratory mucosa serves as the first line of cellular immune defense against respiratory pathogen exposure. Prior to the use of antibiotics, GAS accounted for 2–5% of pulmonary bacterial infections and has been associated with secondary bacterial pneumonia following influenza virus infections particularly during pandemics. While studies examining the immune response within the lower airways of the lung during GAS pneumonia have been limited, current findings suggest that nasal infections that elicit both Th17 and antibody (Ab) responses reduce disease and colonization. Given the role that local dendritic cell (DC): T cell interactions within the lungs have been demonstrated to have in other infections, our hypothesis is that pulmonary DC contribute to protective Th17 CD4 T cell responses during GAS infections. Our findings suggest that GAS infection induces the recruitment of a novel dendritic cell subset into the lungs from the blood whose accumulation correlates with local Th17 responses. Depletion of the DC subset results in reduced pulmonary Th17 responses and increased mortality. Therefore, our result suggest that this DC subset is critical for local Th17 responses and control of pulmonary GAS infections. An improved understanding of the contribution of these DC to the regulation of Th17 and other CD4 T cell responses during GAS infection may allow targeted therapy to boost and shape the local CD4 T cell response and anti-GAS immunity.
Interleukin-10 suppression enhances CD8+ T cell responses to checkpoint blockade immunotherapy in Chronic Lymphocytic Leukemia
Authors: Rivas Jacqueline R , Alhakeem Sara S , Liu Yang , Eckenrode Joseph M , Collard James P , Zhang Yinan , Shaaban Khaled A , Muthusamy Natarajan , Hildebrandt Gerhard C , Fleischman Roger A , Chen Li , Thorson Jon S , Leggas Markos , Bondada Subbarao 
Published: February 2023

Abstract
T-cell-based immunotherapy revolutionized the treatment of immune suppressive cancers. Blocking T cell inhibitory receptors such as programmed death 1 (PD1) or their ligands (like PD-L1) enhances antitumor T cell responses. However, many cancers employ multiple methods of immune suppression, including the production of potent regulatory cytokines like Interleukin-10 (IL-10). B cell Chronic Lymphocytic Leukemia (CLL) is one such B cell malignancy that elicits widespread immune dysfunction including numerous functional deficits in T cells. Human and mouse CLL cells (Eμ-Tcl1) produce IL-10 and PD-L1, which dampen antitumor immunity. Since immune checkpoint blockade experienced limited success in CLL, we investigated IL-10 suppression as a novel combination therapy. Exogenous and CLL-derived IL-10 decreased human and mouse T cell functionality in vitro. Further, CLL-derived IL-10 can be blocked with antibodies or can be reduced with small molecule inhibitors to improve CD8+ T cell functionality in vivo. CLL IL-10 production depends on the transcription factor Sp1. Hence, we utilized a novel analogue of the Sp1 inhibitor mithramycin to reduce CLL IL-10 secretion. IL-10 inhibition improved responses to anti-PD-L1 therapy, with increased survival and decreased CLL burden compared to anti-PD-L1 alone. This method improved CD8+ T cell proliferation, effector-memory cell frequency, and interferon-γ production. CD8+ T cells were also more abundant, with fewer exhausted T cells even at very advanced stages of disease. Current cancer therapies do not target IL-10 and our studies provide evidence for targeting IL-10 to increase the efficacy of T cell immunotherapies in human CLL, and possibly other cancers with T cell suppression.
Fiber-associated Lachnospiraceae reduce colon tumorigenesis by modulation of the tumor-immune microenvironment
Authors: Almeida Ana S , O’Toole Paul W 
Published: February 2023

Abstract
Several studies have highlighted the role of the gut microbiota in colorectal cancer (CRC) progression. Patients with CRC harbor gut microbiomes that differ in structure and function from those of healthy individuals, suggesting this altered microbiome could contribute to carcinogenesis. Despite the increasing evidence implicating the gut microbiome in CRC, the collective role of different microbial consortia in CRC carcinogenesis is unclear. Using a combination of 16S rRNA amplicon sequencing, transcriptomics, and metagenomics, we assessed the capacity of the gut microbiota to shape the tumor microbiome, modulate the immune system and, ultimately, affect tumor growth. Here, we found that tumor biopsy tissue from patients with a “high-risk” Pathogen-type microbiome had a different immune transcriptome from those with a “low-risk” Lachnospiraceae-type microbiome. Transplantation from patients of the two fecal microbiome types into mice with an orthotopic tumor differentially affected tumor growth and the systemic anti-tumor immune response. The differences in tumor volume and immunophenotype between mice receiving the high-risk and the low-risk microbiome correlated with differences in the engrafted human microbial species and predicted microbiome-encoded metabolites in the two groups. These data suggest that the configuration of the gut microbiome may influence colon cancer progression and disease outcome by modulating the anti-tumor immune response.
CRISPR-Cas9–mediated TIM3 knockout in human Natural Killer Cells Enhances Growth Inhibitory Effects on Human Glioma Cells
Authors: Morimoto Takayuki , Nakazawa Tsutomu , Matsuda Ryosuke , Nishimura Fumihiko , Nakamura Mitsutoshi , Yamada Shuichi , Nakagawa Ichiro , Park Young-Soo , Tsujimura Takahiro , Nakase Hiroyuki 
Published: February 2023

Abstract
Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based-immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA-Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell–mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM.
Defining the mechanisms of GSK3 and Med16 in the regulation of MHCII
Authors: Ankley Laurisa M , Kiritsy Michael , Trombley Justin , Huizinga Gabrielle , Olive Andrew J 
Published: February 2023

Abstract
The IFNg-dependent induction of MHCII expression is critical for CD4+ T cell function. Dysregulation of MHCII is associated with autoimmunity, graft versus host disease, and increased susceptibility to cancers and chronic infections. Despite its central role in host immunity, the complex and dynamic regulation of IFNg-induced MHCII is poorly understood. We hypothesize that modulating MHCII regulatory pathways in macrophages will directly alter T cell effector function. Our goal is to dissect MHCII regulation to identify new therapeutic targets that will improve MHCII disease states, antigen presentation, and host immune responses against persistent infections. Using a CRISPR screen we identified new MHCII regulators including the multifunctional kinase GSK3 and the mediator complex subunit Med16. Mechanistic studies found that both GSK3 and Med16 are essential for IFNg-mediated Ciita induction and for macrophages to serve as antigen presenting cells for CD4+ T cells. Using the GSK3 inhibitor CHIR99021 in Med16 KO macrophages we observed further inhibition of MHCII expression suggesting GSK3 and Med16 function in parallel networks. To test this directly, RNAseq was used to globally profile the transcriptomes in Med16 and GSK3 KO cells in the presence and absence of IFNg. We found that Med16 and GSK3 control distinct transcriptional networks without disrupting canonical IFNg transcription factors like Stat1, Stat3 and IRF1. The RNAseq also suggested IFNg dependent control of cytokines are disrupted in the absence of GSK3 and Med16 which we confirmed using Mycobacterium tuberculosis infection. Thus, GSK3 and Med16 are parallel regulators that balance IFNg responses in macrophages and modulate adaptive immune responses.
Dendritic cell PIK3C3/VPS34 controls the pathogenicity of CNS autoimmunity independently of LC3-associated phagocytosis
Authors: Yang Guan , Postoak J. Luke , Song Wenqiang , Martinez Jennifer , Zhang Jianhua , Wu Lan , Van Kaer Luc 
Published: February 2023

Abstract
The PIK3C3/VPS34 subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is a key player in macroautophagy/autophagy and MAP1LC3/LC3-associated phagocytosis (LAP), both of which play critical roles in mediating dendritic cell (DC) function. In this study, we assessed the contribution of PIK3C3 to DC function in the context of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We found that Pik3c3-deficient DCs exhibited attenuated ability in reactivating encephalitogenic T cells in the central nervous system, leading to reduced incidence and severity of EAE in DC-specific Pik3c3-deficient mice. Additionally, animals with DC-specific deficiency of Rb1cc1 (essential for autophagosome nucleation but not LAP) but not Rubcn (required for LAP but not autophagy) were protected against EAE, suggesting that the EAE phenotype of DC-specific Pik3c3-deficient mice is associated with the lack of canonical autophagy rather than LAP. Collectively, our studies have revealed a critical role of PIK3C3 in DC function and in the pathogenicity of these cells during EAE. Our findings also have important implications for the development of immunotherapies to treat autoimmune diseases such as MS by targeting PIK3C3-containing complexes.