bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of CD8+ T cell antitumor function by an immune inhibitory molecule sushi domain containing 2
Authors: Zhao Bao , Gong Weipeng , Wen Haitao
Published: February 2023

A central mechanism by which the immunosuppressive tumor microenvironment (TME) facilitates tumor growth is the inhibition of CD8+ effector T cell function. Indeed, restoration of CD8+ T cell antitumor functions by immune checkpoint blockade (ICB) treatment represents a significant breakthrough in cancer therapy. However, a major challenge remaining in ICB immunotherapy is the existence of multiple immune checkpoint molecules. As a result, targeting of a single molecule cannot override compensatory signals from other immune inhibitory molecules. Therefore, there is an urgent need to identify new therapeutic target(s) to rejuvenate CD8+ T cell antitumor immunity for cancer therapy. Through unbiased gene profiling assay, our recent study has identified SUSD2 (sushi domain containing 2) as a highly expressed immune cell-associated cell surface molecule in an experimental cancer model. Using a newly developed Susd2 gene-deletion (Susd2−/−) mouse model, we found that genetic deletion of Susd2 led to an improved control of tumor growth in syngeneic tumor models including colorectal carcinoma MC38 and thymoma EG7. This improved tumor control phenotype of Susd2−/− mice required the presence of CD8+ T cells. Single-cell RNA sequencing assay revealed increased production of effector molecules such as granzyme B (Gzmb) and interferon (IFN)-γ in tumor-infiltrating CD8+ T cells from Susd2−/− mice compared to those from wild-type (WT) mice. Mechanistically, we found that SUSD2 negatively regulated IL-2 receptor (IL-2R)-mediated generation of Gzmb and IFN-g in CD8+ T cells. In sum, our results identify SUSD2 as a new immune inhibitory molecule which can be targeted to augment CD8+ T cell antitumor response.
High grade gliomas impact cell fate differentiation of hematopoietic stem and progenitor cells in the bone marrow
Authors: Dean Bayli DiVita , Wildes Tyler , Dean Joseph , Shin David , Francis Connor , Font Laura Falceto , Moore Ginger , Fernandez Brandon , Wummer Brandon , Kuizon Carmelle , Mitchell Duane , Flores Catherine
Published: February 2023

Glioblastoma (GBM) remains a deadly disease with an overall survival of 18 months. Despite advances in cancer immunotherapy, especially in the context of solid tumors derived outside of the central nervous system, GBM remains difficult to treat. This may be due, in part, to reduced T cell function and expansion of suppressive myeloid cells within the periphery. Here we sought to determine if GBM impacts the phenotype and gene expression of progenitor populations within the bone marrow where hematopoietic stem and progenitor cells (HSPCs) originate.

Using RNAseq, we found HSPCs derived from intracranial glioma-bearing mice possess altered gene expression relative to HSPCs derived from non-tumor-bearing mice. In addition, we found glioma-bearing mice possess an expansion of myeloid-derived suppressor cells (MDSCs) within their bone marrow and are significantly more suppressive on T cell proliferation and T cell-mediated tumor cell killing than MDSCs isolated from non-tumor-bearing mice. We also determined HSPCs derived from glioma-bearing mice are more likely to differentiate into MDSCs than HSPCs derived from non-tumor-bearing mice.

Interestingly, we found components of immunotherapy are capable of redirecting cell fate differentiation of glioma-bearing HSPCs. We determined HSPCs cultured in T cell supernatants are capable redirecting their differentiation from suppressive MDSCs towards stimulatory DCs. Finally, using two murine glioma models, we found adoptive cellular therapy using HSPCs derived from glioma-bearing mice is capable of providing a similar survival benefit as adoptive cellular therapy using HSPCs derived from non-tumor-bearing mice.
Probing the naturally acquired immune response to malaria for broadly reactive antibodies targeting P. falciparum antigens linked with severe disease
Authors: Reyes Raphael , Turner Louise , Ssewanyana Isaac , Rek John , Greenhouse Bryan , Bol Sebastiaan , Lavstsen Thomas , Bunnik Evelien
Published: February 2023

Severe malaria is caused by the adhesion of Plasmodium falciparum-infected erythrocytes to host receptors on vascular endothelium. Specifically, binding of the parasite variant surface antigen domain CIDRa1 to host endothelial protein C receptor (EPCR) is strongly associated with severe disease. In malaria-endemic regions, children quickly develop immunity against severe malaria, indicative of the development of an effective immune response against CIDRa1 domains. Indeed, serum IgG from naturally immune individuals exhibited reactivity against a diverse panel of CIDRa1 variants. In an effort to analyze CIDRa1 antibody responses at a monoclonal level, we isolated CIDRa1-specific memory B cells from malaria-experienced individuals living in Uganda and expressed the corresponding monoclonal antibodies (mAbs). Our approach has yielded anti-CIDRa1 mAbs ranging in breadth from variant-specific to reactive with the full spectrum of CIDRa1 domain variants, irrespective of their extensive sequence diversity. The broadest antibodies can be separated into two categories, those that bind outside the EPCR binding site and display exceptional breadth with modest inhibition of EPCR binding and those that target the EPCR binding site and show potent EPCR-binding inhibition. Two EPCR-binding site-targeting mAbs isolated from two different donors showed signs of convergent evolution. Our results demonstrate that natural P. falciparum infection can induce a broad and inhibitory antibody response against CIDRa1. Current experiments are aimed at understanding the structural basis of antibody-mediated inhibition of CIDRa1-EPCR binding to inform the design of a vaccine that protects against severe malaria.
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

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

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>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

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

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

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

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

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

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

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

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

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

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

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.
Fam49b-deficient lymphocytes modulate whole body energy metabolism.
Authors: Park Chansu , Guan Jian , Rhee Peter , Sadegh-Nasseri Scheherazade , Shastri Nilabh
Published: February 2023

TCR signal strength is determined by the balance of positively regulating, and negatively regulating proteins. Fam49b is identified as new negative regulator of TCR signaling that functions by binding to active rac-1 GTPase. In the absence of Fam49b, thymocytes receive higher TCR signaling and are defective in development of conventional T cell as well as agonist selected T cells such as CD8αα+ αβ T cells. Obesity is characterized by chronic inflammation and immune dysregulation, but the link between obesity and T cells has remains unclear. We show that Fam49b-deficient mice have a high level of energy expenditure and are resistant to high fat diet induced obesity. Furthermore, the higher energy expenditure characteristic was also observed in Fam49b-deficient chimera mice. The study reveals that Fam49b deficient lymphocytes play an important role in whole body energy metabolism. [This research was supported by NIH grants R01 AI130210 and R37 AI060040]
Myeloid Mayhem: How sleep alters the myeloid compartment and promotes plaque rupture in atherosclerosis
Authors: Moriarty Alina K , Keeter W. Coles , Waseem Tayab , Ma Shelby , Butcher Matthew J , Mussbacher Marion , Wellman Laurie , Sanford Larry , Galkina Elena
Published: February 2023

Adequate sleep is vital for overall health; however, today’s fast-paced society often neglects the importance of sleep. Poor sleep quality is tightly correlated with atherosclerosis and increased risk of mortality due to a cardiovascular event. Evidence demonstrates neutrophilia and monocytosis following chronic sleep fragmentation, but their effects on aortic inflammation and atherosclerotic plaque stability are not well understood. We found that chronic sleep fragmentation accelerated atherogenesis and decreased plaque stability in plaques of high fat diet (HFD) fed female apoliportein E-deficient (Apoe−/−) mice. Sleep fragmentation increased populations of pro-inflammatory monocytes and neutrophils in the bone marrow, blood, spleen, and small intestines of HFD fed female Apoe−/− mice. Additionally, sleep fragmentation increased gut permeability and circulating LPS in HFD fed female Apoe−/− mice. We also observed increased percentage of neutrophils in carotid arteries of sleep fragmented Apoe−/− mice. Importantly, these plaques exhibited increased necrotic areas, altered composition of extracellular matrix, and reduced production of stabilizing collagen. Taken together, our data indicates that sleep fragmentation accelerates plaque formation, and destabilizes atherosclerotic plaques by altering the local and systemic immune landscape. Sleep fragmentation contributes to systemic inflammation and likely activates the innate immune response at least partially, through the increase in circulating LPS mediated by inflammation in small intestines.
Autoimmunity against Cardiac Self-Antigen Drives Immune Checkpoint Inhibitor-Associated Myocarditis Development
Authors: Won Taejoon , Kalinoski Hannah M , Wood Megan K , Hughes David M , Talor Monica V , Reddy Jay , Cihakova Daniela
Published: February 2023

Immune checkpoint inhibitor (ICI) blocking PD-1 has been approved by the Food and Drug Administration for treatment of several types of cancers. However, fulminant myocarditis has occurred in the ICI-treated cancer patients, with low incidence but with 50 percent mortality. In this study, we investigated if PD-1 blockade can induce myocarditis in the mouse mimicking ICI-associated myocarditis in the human. We found that anti-PD-1 mAb treatment caused myocarditis development in A/J mice exhibiting enlarged heart and increased cardiac infiltration. In the cardiac infiltration, CD8+ T cells, CD4+ T cells, monocytes and macrophages were increased in the PD-1 blockade group compared to isotype control. Cardiac damage was confirmed by elevated serum level of cardiac troponin I and anti-myosin IgG. In addition, PD-1 blockade caused arrhythmia, right bundle branch block, while controls showed a normal ECG trace. The A/J mice with PD-1 blockade also developed systemic autoimmune responses such as colitis, sialadenitis, pneumonitis and myositis like immune-related adverse events in the human, supporting the immunotoxicity of PD-1 blockade. Next, using myosin-MHC tetramer staining, we found the increase of autoreactive T cells specific for cardiac myosin in the mouse heart with ICI-associated myocarditis compared to control. In the naïve heart, we were able to detect myosin-specific T cells, which number was very few but expressed a high level of PD-1. Collectively, we have developed the mouse model for human ICI-associated myocarditis and showed that the pathogenesis of this disease is an autoimmune reaction against heart-specific antigens.
Enhanced suppressive phenotype of Tim-3+ Treg during chronic infection
Authors: Nieves-Rosado Hector M , Banerjee Hridesh , Kane Lawrence P
Published: February 2023

T cell (or transmembrane) immunoglobulin and mucin-domain containing protein-3 (Tim-3) is a cell-surface protein expressed during T cell exhaustion, a process that leads to a progressive loss of effector function due to chronic T cell receptor (TCR) stimulation. In humans, Tim-3 expression has also been found to be increased on Treg isolated from tumors and during chronic viral infection. However, the role of Tim-3 on Treg during chronic viral infection remains largely unknown. Our main hypothesis is that Tim-3 expression on Treg will increase their proliferation and suppressive capacity during LCMV infection. To test this, we infected C57/BL6 mice for 30 days with LCMV clone-13, a well-established model for chronic infection. We found that at 16 days post-infection there is an increase in the frequency and number of Tim-3+Foxp3+ Treg in the spleen. By flow cytometry we found that Tim3+ Treg have increased expression of CD44, Ki67, Helios, ICOS, KLRG-1, CD39 and CD25, compared with Tim-3− Treg. Therefore, our data suggest that Tim-3+ Treg have a phenotype associated with higher activation, proliferation and suppression during chronic infection. Understanding the contributions of Tim-3 to Treg activation and function may provide new insights into the role of Tim-3 during disease progression and response to therapy.
MHCII-mediated antigen presentation by thymic stromal cells is required to enforce central T-cell tolerance
Authors: Proekt Irina , Miller Corey N , Chan Jackie Y , Hsieh Chyi-Song , Anderson Mark S
Published: February 2023

Central T-cell tolerance relies on elimination of self-reactive CD4+ T-cell clones in the thymus by either deletion or conversion to regulatory T cells (tTregs). Developing thymocytes are selected by exposure to tissue-specific antigens (TSAs) expressed by thymic medullary epithelial cells (mTECs) under the control of Autoimmune Regulator (Aire). TSA-derived peptides-MHCII complexes can be presented either directly by mTECs or indirectly by thymic DCs. Dissecting the contributions of these antigen presenting cell (APC) subsets to thymic tolerance has been hindered by the lack of appropriate genetic tools to selectively target presentation by mTECs. To address this, we created a mouse model in which MHCII can be inducibly ablated in Aire-expressing mTECs (iAire-CreERT2;MHCIIfl/fl). We show that treatment of these mice with tamoxifen leads to a complete loss of MHCII expression by Aire+ and post-Aire mTECs without affecting other thymic APCs or perturbing thymic homeostasis. In a TCR-restricted mouse model of negative selection, ablation of MHCII on mTECs led to a failure to delete self-reactive T cells, allowing escape and activation in the periphery. Surprisingly, we also found a defect in conversion of self-reactive CD4+ T cells to FoxP3+ tTregs. Similarly, in the diverse repertoire, loss of direct antigen presentation by mTECs resulted in tTreg reduction. In contrast, using CD11c-Cre;MHCIIfl/fl mice, in which MHCII is absent on thymic DCs but not mTECs, we found no disruption to negative selection or Treg generation. In sum, our data support a distinct and non-redundant role for direct antigen presentation by mTECs in shaping thymic T cell selection.
Integrative profiling of chromatin accessibility and gene expression elucidate specific transcriptional network in porcine neutrophils
Authors: Herrera-Uribe Juber , Daharsh Lance , Byrne Kristen A , Liu Haibo , Loving Crystal , Tuggle Christopher K
Published: February 2023

Pigs are an important component of global food security, and serve as a human biomedical model. Infectious diseases impact pig and human health and global food productivity. Components of the pig innate immune system, such as neutrophils, have not been fully described, and epigenetic mechanisms controlling gene expression remain unclear in pigs. We performed RNA- and assay for transposase-accessible chromatin (ATAC)-sequencing on two porcine neutrophil samples to characterize epigenetic regulation of gene expression in these differentiated cells. A deep neutrophil transcriptome detected expression of 10,974 genes. In comparison to previously reported RNA-seq data on eight sorted immune cell types from the same animals, we predicted 833 enriched (significantly higher and 2-fold higher than average across all other cell types) genes in neutrophils. The 833 gene list was significantly enriched for biological processes related to typical neutrophil functions using Gene Ontology and in comparison to human neutrophil RNA-seq data. ATAC-seq detected open chromatin (OP) regions from ~227 million total reads that identified on average 151,789 peaks located in intergenic (67%), intron (24%), promoter-TSS (6%), exon (3%) or TTS (1%) regions. OP regions whose signal is correlated with, and nearby to, genes with enriched neutrophil expression are being analyzed for sites predicted to be bound by transcription factors that could explain neutrophil-enriched expression patterns. The data reported here is the first integrative study of the epigenetics of porcine neutrophil gene expression and provide a valuable resource to elucidate neutrophil transcriptional regulatory networks.
Rapid cell cycling of both circulating and resident memory CD8+ T cells driven by common gamma chain-dependent cytokines
Authors: Jarjour Nicholas N , Wanhainen Kelsey M , Silva Henrique Borges da , Peng Changwei , Martinez Ryan J , Huggins Matthew A , Urban Joseph F , Hamilton Sara E , Jameson Stephen C
Published: February 2023

Diverse populations of resident leukocytes live and self-renew within the tissues of the body, but the mechanisms controlling their proliferation are poorly understood. Among these populations are many memory CD8+ T cells, which are found throughout the body and can locally control reinfection. A poor ability of vaccines to elicit stable and appropriate tissue-resident CD8+ T cell populations has likely contributed to long-standing challenges in vaccinating against several important pathogens. The factors required to support TRM proliferation in homeostasis are not well understood. We propose that proliferating TRMs flexibly use the available members of the common gamma chain-(γC)-dependent cytokine family to persist as bystanders during other immune responses. We have found that many γC-dependent cytokines can mediate cell-intrinsic signaling to drive proliferation of both circulating and resident memory CD8+ T cells of known antigen specificities without cognate antigen. Via transcriptional analysis of resting and in vivo cytokine-stimulated TRMs, we are elucidating downstream signaling and the gene expression signature of cytokine-driven TRM proliferation. We have also found that memory CD8+ T cells proliferate similarly in response to exogenous cytokines or unrelated pathogens, consistent with pathogen-elicited γC-dependent cytokine signals driving memory CD8+ T cell proliferation. These data support the concept that TRMs use γC-dependent cytokines produced during infections to support enhanced proliferation, likely to promote their maintenance. Our findings may lead to novel methods to therapeutically enhance TRM immunity.
Dietary fish oil directly impact viral immunity by modifying CD8 T cell membrane flexibility and bond lifetime under force
Authors: Kolawole Elizabeth Motunrayo , Evavold Brian D
Published: February 2023

Fish oil supplements enriched in omega-3s are increasingly consumed for their health benefits and exhibit immunosuppressive effects on both innate and adaptive immunity. While omega-3s have been shown to reduced inflammatory metabolites, we focused on the direct effects of a high omega-3 diet on T cell activation during viral infection. Using lymphocytic choriomeningitis (LCMV) infection we analyzed CD8 T cells response to antigen. The omega-3 based diet manipulated structural lipid content in plasma membranes leading to stiffer T cells which subsequently resulted in reduced frequency and activation of the prevalence immunodominant epitopes. We further found that a high omega-3 diet decreased 2D affinity and number of tetramer positive polyclonal as well as CD8 T cell receptor transgenic cells at peak anti-viral immunity. In addition to affinity, the bond lifetime under force of antigen recognition was also decreased. This resulted in decreased numbers of SLEC and effector cytokines without impeding viral clearance. Our work indicates that a major effect of dietary omega-3 fatty acids on T cells is the modification of the cell membrane to dampen the initial recognition of antigen leading to decreased but effective immunity.
Lung microbiome influences Coccidioides colonization and invasiveness
Authors: Tejeda-Garibay Susana , Diep Anh L. , Delgadillo Nadia Miranda , Hoyer Katrina K.
Published: February 2023

It has long been thought that the lung is a sterile environment; however, recent studies revealed that healthy lungs are colonized by bacteria, fungi, and viruses. Coccidioidomycosis, also known as Valley fever, is a lung infection caused by the fungal species Coccidioides. Because of poor testing strategies 60–80% of patients are treated with antibiotics prior to accurate diagnosis of coccidioidomycosis. This potentially depletes bacteria with symbiotic relationships within the lung microenvironment and promotes susceptibility to invasion by Coccidioides. Coccidioides immitis is a dimorphic fungal pathogens whose fungal life cycle alternates between the saprophytic phase (soil mycelia) and parasitic phase (tissue infecting spherules). Natural microbial antagonists that inhibit soil Coccidioides immitis have been identified; Bacillus subtilis displays antifungal activity against Coccidioides growth through a clear zone of inhibition between fungi and bacteria. Whether these findings hold true in the host phase of the Coccidioides life cycle is unknown but have major implications for patient treatment. Our preliminary data suggest that host microbiome inhibits Coccidioides growth, and antibiotic treatment shifts or depletes the lung microbiome allowing a niche for Coccidioides growth. We are also exploring how this interface between the lung, Coccidioides, and the microbiome influences host innate and adaptive immune responses. Furthermore, these findings could shape the way physicians assess prescription of antibiotics prior to fungal diagnosis.
Single-Nucleotide Polymorphisms in Glucose-6-Phosphate Dehydrogenase and their Relevance for the Deployment of Primaquine as a Radical Cure for Malaria
Authors: Nain Minu , Gill Jasmita , Mohan Mradul , Sharma Amit
Published: February 2023

Malaria remains an important public health problem despite efforts to control it. Besides active transmission, relapsing malaria caused by dormant liver stages of Plasmodium vivax and Plasmodium ovale hypnozoites is a major hurdle in malaria control and elimination programs. Primaquine (PQ) is the most widely used drug for radical cure of malaria. Due to its anti-hypnozoite and gametocidal activity, PQ plays a key role in malaria relapse and transmission. The human enzyme glucose-6-phosphate dehydrogenase (G6PD) is crucial in determining the safety of PQ because G6PD-deficient individuals are prone to hemolysis if treated with PQ. Therefore, there is a need to study the prevalence of G6PD-deficient genetic variants in endemic populations to assess the risk of PQ treatment and the necessity to develop alternative treatments. In this work, we discuss the common G6PD variants, their varying enzymatic activity, and their distribution on the three-dimensional structure of G6PD. Our work highlights the important G6PD variants and the need for large-scale G6PD gene polymorphism studies to predict populations at risk of PQ-induced toxicity.
Missio Dei’s Pleromatic Disposition: The Infinite Missionary God
Authors: Thinane Dr. Jonas Sello
Published: February 2023

What characterized the missiological shift from pre- to post-1952 Willingen and its resulting perspectives is the recognition of the pleromatic (Greek for “Fullness”) nature of the mission, denoting the totality of the Trinitarian God constantly or coherently at work saving the entire universe. Although the missiological literature commendably continues to emphasize total salvation and the attainment of the all-encompassing kingdom of God as the fundamental objectives of the missio Dei, its pleromatic character, that of the missionary triune God, so to speak, remains less obvious—and therefore in need of explanation. Accordingly, this article relies upon literature to highlight the importance of the pleroma as a theological concept, with the intention of contextualizing its use along with efforts to deepen the understanding of the missio Dei. Although this terminology is more associated with Gnosticism, it is intentionally used here in a different sense from that of the Gnostics, but merely streamlined to emphasize the infinite nature of the missio Dei. Consequently, it is here concluded that the scope of the missio Dei, when understood through its pleromatic character, transcends conceivable human and ecclesiastical limits, religious parameters, and even the limits of the universe or the cosmos itself. Therefore, the scope of this paper justifies further research aimed at uncovering the pleromatic character of the missio Dei, which is constantly at work and inhabits the universe in its entirety.