bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell exhaustion is Reinforced by Progressive De novo DNA Methylation Programming
Authors: Youngblood Ben A , Ghoneim Hazem E. , Abdelsamed Hossam Aly , Carter Robert , Hale Jeffery Scott , Ahn Eunseon , Im Sejin , Ahmed Rafi
Published: January 2023

Antigen-specific CD8 T cells play a critical role in controlling chronic infections and cancer, but progressively lose their effector functions during prolonged antigen exposure. Repression of CD8 T cell effector functions, commonly referred to as T cell exhaustion, limits the ability of the immune system to purge the chronic pathogen from the host. It has recently become recognized that CD8 T cell exhaustion programs can be reinforced and heritably maintained. Therefore in order to develop and/or improve current therapeutic approaches that utilize host antigen-specific T cells to treat chronic infections or cancer a major challenge for the field is to identify mechanisms that stabilize T cell exhaustion. We have recently found that epigenetic modifications acquired in pathogen-specific CD8 T cells during prolonged antigen exposure reinforce T cell exhaustion. Using the LCMV model system of chronic viral infection we investigated the role of Dnmt3a mediated de novo DNA methylation in regulating CD8 T cell exhaustion. Strikingly, conditional deletion of Dnmt3a in activated CD8 T cells blocked the cells from becoming exhausted. Longitudinal analysis of whole-genome methylation programming of WT and Dnmt3a cKO CD8 T cells from chronically infected animals reveals progressive acquisition of Dnmt3a-dependent DNA methylation programs in genes, including interferon gamma, that are coupled to the progressive decline of effector functions. These results have significant implications for therapeutic strategies that utilize reactivation of host pathogen-specific CD8 T cells to control chronic viral infections or cancer and provide a nucleotide-resolution map of epigenetic programs progressively acquired during T cell exhaustion.
Intracellular delivery of anti-pPKCθ (T538) via protein transduction domain mimics for T cell immunomodulation
Authors: Ozay Emrah Ilker
Published: January 2023

Humanized antibodies are increasingly gaining attention for their therapeutic potential in immunotherapy but, to date, the collection of cellular targets has been limited to those that are secreted from cells or are expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as potential targets. We evaluated the potential for delivering a phospho-specific antibody into human peripheral blood mononuclear cells (hPBMCs) as a means to interrogate and modulate their response to activating stimuli. Protein kinase C-theta (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic (PTDM) capable of efficiently delivering the antibody against phosphorylated PKCθ (T538) into hPBMCs. We designed an in vitro delivery method for the PTDM:anti-pPKCθ complex then assessed T cell activation and the expression of various downstream indicators of T cell activation and differentiation. In proof-of-principle experiments using a humanized, lymphocyte transfer model of graft-vs-host disease, we evaluated the durability of PTDM:anti-pPKCθ modulation, when delivered into hPBMCs ex vivo. Altogether, these results demonstrate that PTDM:antibody complexes can be readily introduced with high efficacy into traditionally hard-to-transfect human PBMCs to modulate immune function, eliciting a biological response sufficient to alter disease progression. Thus, PTDM:antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.
Amplifying T cell responses to tumor antigens using a synthetic CD8-MyD88 co-receptor
Authors: Kaczanowska Sabina , Davila Eduardo
Published: January 2023

T cell–based immunotherapies are among the most promising approaches for patients with advanced melanoma and other cancers. The goal of this study is to boost T cell anti-tumor responses by harnessing the costimulatory effects of MyD88 signaling in T cells. We previously demonstrated that Toll-like receptor (TLR) engagement on T cells increases their proliferation, effector function, and survival in a MyD88-dependent manner. However, a challenge to stimulating TLRs on T cells in vivo is inefficiencies in delivering TLR ligands to the tumor site or adverse effects of TLR signaling in cancer cells. We hypothesized that coupling TLR and T cell receptor (TCR) signaling would provide antigen-dependent co-stimulation resulting in enhanced anti-tumor function. We developed a novel synthetic co-receptor consisting of a CD8a and MyD88 fusion protein to induce MyD88 signaling in a TCR–dependent but TLR ligand–independent mechanism. We have compelling data indicating that CD8-MyD88 stimulation improves T cell expansion and function in a tumor antigen–dependent manner. Furthermore, CD8-MyD88 T cells exhibit promising anti-tumor responses in mice with established melanoma tumors. Our strategy represents a universal approach since the CD8-MyD88 is not limited to a specific tumor antigen nor MHC haplotype. CD8-MyD88 also improves T cell responses to low concentrations of antigen, providing the opportunity to expand a large repertoire of tumor-reactive T cells. Furthermore, CD8-MyD88 activates MyD88 signaling in the engineered T cells exclusively, thus eliminating undesirable responses of TLR signaling in tumor cells. These studies represent a novel and versatile therapeutic approach for boosting T cell responses to a variety of cancers.
Selected TLR agonists act in synergy to reprogram DC-NK cross-talk and generate effector T cells in nicotinic environment
Authors: Nouri-Shirazi Mahyar A , Tamjidi Saba , Nourishirazi Erika
Published: January 2023

Background

The magnitude of immune responses to vaccination is a critical factor in determining protection from clinical disease. It is recognized that cigarette smoke dampens the immune system and increases the risk of vaccine-preventable diseases. We reported that the nicotine component of cigarette smoke disrupts the differentiation and functional properties of DCs that are pivotal in the initiation of immune response to vaccines.

Objective

In this study, we investigated whether TLR agonist(s) could potentially overcome the degrading effects of nicotine on human DC and DC-NK cross-talk essential for effector T cell generation.

Methods

nicDC, nicDC-NK, and nicDC-NK-T cultures exposed to individual and combined TLR agonists were evaluated for expression of corresponding costimulatory and activation molecules, cytokines, and intracellular cytokine IFN-g using ELISA and flow cytometry.

Results

Our data shows that among the panel of TLR agonists tested, TLR3 and TLR8/7 synergistically optimize nicDC maturation required for IFN-g production by co-cultured NK cells. Importantly, similar to DC-NK, nicDC-NK treated with TLR3 and TLR8/7 and co-cultured with naïve T cells promoted comparable number of effector T cells producing IFN-g.

Conclusion

Our data suggest that the addition of appropriate TLR agonist(s) to vaccine formulation could potentially improve the smokers’ immune response to vaccination. NIH-NIAID 1R03AI103750-01A1
In vivo characterization of the immune response towards the pathogenic Escherichia coli antigen SslE and modulation of the intestinal microbiota
Authors: Naili Ilham , Buonsanti Cecilia , Soriani Marco , Desvaux Mickael , Jubelin Gregory , Vieru Miguela , Baudner Barbara C. , D’Oro Ugo
Published: January 2023

Pathogenic E. coli, both intestinal (InPEC) and extraintestinal (ExPEC), account for a wide range of diseases, which can be fatal, across developing and developed countries. The vast array of virulence factors they can acquire may dramatically increase the severity of the infection. Emergence of resistant strains often renders antibiotics inefficient; in the case of Shiga toxin-producing E. coli, antibiotics can even be detrimental. Hence, a broad spectrum E. coli vaccine could be a promising alternative to prevent the spread of such diseases, while offering the potential for covering against several InPEC and ExPEC at once.

Using the «reverse vaccinology» approach on an ExPEC strain, nine antigens were identified as protective against a mouse sepsis model. With 82% protective efficacy, SslE (Secreted and Surface-associated Lipoprotein of E. coli) was the most potent candidate; additional models demonstrated that SslE was also cross-protective against other ExPEC strains. Functional assays have demonstrated in vitro and in vivo that SslE is a mucinase which plays an important role for colonization and virulence of E. coli.

To better understand the mechanism behind such protection, particularly at the mucosa, we are investigating the intestinal immune response obtained after immunizations with SslE using different routes of injection. In-depth analysis of each regimen will allow us to determine the most efficient method of immunization and adjuvant choice for SslE to be protective. Further, using this model of immunization, we will evaluate the potential perturbation caused by SslE on a human intestinal microbiota. These findings will be important to help us optimize the development of a potential broad spectrum vaccine.
Finding the evolutionary precursor of vertebrate hematopoietic lineage: Functional and molecular characterization of B. schlosseri immune system.
Authors: Rosental Benyamin , Kowarsky Mark A. , Corey Daniel M. , Ishizuka Katherine J. , Palmeri Karla J. , Chen Shih-Yu , Sinha Rahul , Voskoboynik Ayelet , Weissman Irving L
Published: January 2023

We are characterizing the immune system and cell populations of the tunicate model Botryllus schlosseri on functional and molecular levels. The chordate B. schlosseri belongs to a group that is considered the closest living invertebrate relative of vertebrates. Using FACS analysis we can isolate 12 populations of B. schlosseri cells by size, granularity and auto fluorescence. In order to increase the number of isolated cell populations, we used Cytof to scan large numbers of antibodies. Antibodies that differentially bind to B. schlosseri cells were validated by flow cytometry. Serum against the Botryllus Histocompatibility Factor, and lectins and fluorescent reagents activated by enzymatic activity were also used to differentiate live B. schlosseri cell types. Using these markers we isolated 34 cell populations and created RNA libraries for deep sequencing. Additionally, we developed immunological assays for cytotoxicity, phagocytosis and stem cell tracking. Using gene expression analysis we found hematopoietic stem cells and progenitors; in transplantation assays we showed their capability to induce chimerism of pigmentation and differentiation to other cell types, as well as localization to the stem cell niches. We characterized 3 different phagocytic cell types including a previously undescribed myeloid derived cell population. We describe the B. schlosseri cytotoxic cell population that originates as a large granular lymphocyte-like cell type that become morula cells upon allogeneic challenge activation. Altogether, it seems that the common ancestor of tunicates and vertebrates had a true hematopoietic myeloid lineage, while the cytotoxic cells are a convergent evolutionary system.
Polyfunctional cytokine production by central memory T cells from cattle in response to Mycobacterium bovis infection and BCG vaccination
Authors: Maggioli Mayara Fernanda , Palmer Mitchell V. , Thacker Tyler T , Vordermeier Martin , McGill Jodi L , Larsen Michelle H. , Jacobs William R. , Waters W. Ray
Published: January 2023

Polyfunctional T cells simultaneously produce IFN-γ, IL-2 and TNF-α and play relevant roles in several chronic infections, including TB. Mycobacterium bovis infection of cattle elicits ex vivo polyfunctional T cell responses. Vaccine-elicited IFN-γ Tcm (CD4+ CD45RO+ CCR7+) responses correlate to protection in cattle. The objective was to assess polyfunctional Tcm responses to vaccination with BCG or BCG mutants and subsequent M. bovis challenge in cattle. BCG induced polyfunctional Tcm to PPD and Ag85A/TB10.4. After challenge, TNF-α/IFN-γ Tcm responses to PPD, Ag85A/TB10.4 or ESAT-6:CFP10 were greater in non-vaccinates in comparison to vaccinates; while TNF-α/IFN-γ/IL-2 Tcm responses were lower. Both vaccines showed similar protection; however, vaccinated animals that were M. bovis culture negative had lower TNF-α/IFN-γ polyfunctional Tcm responses to ESAT-6:CFP10 3wpi than did culture positive vaccinates. In conclusion, BCG vaccination elicited Tcm polyfunctionality, mainly TNF-α/IFN- γ co-producers. Dampened TNF-α/IFN-γ and greater TNF-α/IFN-γ/IL-2 Tcm responses after challenge may indicate enhanced pathogen control and correlate with vaccine elicited protection.
NK cell activation and metabolic syndrome in a small nonhuman primate model of Hepacivirus infection
Authors: Manickam Cordelia , Reeves Roger Keith
Published: January 2023

Hepatitis C virus (HCV) infects greater than 170 million people worldwide. The disease remains a major source of global morbidity and mortality, but factors leading to resolution or chronicity remain unclear. In this study, we evaluated acute and longitudinal immune responses in an understudied model of HCV infection, GBV-B infection of common marmosets. Plasma viremia was detectable by day 3 post infection (pi) generally peaking by day 14 pi, at which point high virus titers were also found in the liver. Virus clearance was observed in all animals by day 112 pi. Elevated serum levels of ALT, AST and GGT, along with microscopic liver lesions of inflammation and fibrosis were indicative of hepatitis. Increases in serum creatine kinase, triglycerides and glucose, and decreased amylase levels suggested metabolic dysregulation. Interestingly, natural killer cells, which have been linked to bystander damage in the liver, demonstrated increased expression of perforin, Ki-67 and CXCR3 following infection, and were increased up to 5-fold in the liver by day 14 and remained elevated even after viral clearance. Generalized T cell activation was observed by day 28 pi, and GBV-B-specific responses were generally focused on NS3 peptides (IFN-γ ELISPOT) and were up to 10-fold higher in liver compared to peripheral organs. Our results suggest that early NK cell activation could play a major role in limiting hepaciviral infection in marmosets, or alternatively contribute to tissue inflammation and liver damage. Regardless, these data highlight the recapitulation of inflammation and metabolic dysfunction in a tangible small animal model for HCV and suggest innate cell targeting could be an attractive target for future therapeutics.
A role for Mitochondrial Antiviral Signaling molecule (MAVS) in the TCR pathway.
Authors: Thomas Sunil , Sekine Aimee , Gale Michael
Published: January 2023

Mitochondrial Antiviral Signaling molecule (MAVS) is an adaptor that functions downstream of the viral RNA sensors RIG-I and MDA5 to translate viral RNA sensing into effector responses such as the production of Type I IFN and inflammatory cytokines that result in the anti viral state that functions to limit viral spread. However a precise role for MAVS in CD8 T cells has not been delineated.

Using an in vivo adoptive transfer system with Lymphocytic choriomeningitis virus (LCMV) and transgenic LCMV- GP33 specific T cells (P14) and OVA-specific T cells (OT1) in vitro, we show that there is a cell-intrinsic role for MAVS in transducing signals proximal to the T cell receptor in both in vivo and in vitro settings. MAVS-dependent defects in CD8 T cell effector functions were identified in cells responding to in vivo LCMV infection in mice. At the peak of the adaptive response to LCMV, MAVS-deficient CD8 T cells were smaller in size, had deficient intracellular calcium, decreased mitochondrial membrane potential, decreased NFATc1 as well as defective production of the effector cytokines IFN γ, TNF α and IL-2. We found that MAVS is involved in the TCR signaling pathway by regulating the levels of phosphorylated zeta chain of protein kinase 70 (pZAP70), an enzyme that functions in the initial step of TCR-mediated signal transduction. The levels of pZAP70 were lower in the absence of MAVS in vivo in P14 CD8 T cells as well as in in vitro stimulated OT1 CD8 T cells. The degree of defect was dynamic and varied as a function of time after TCR ligation. The precise mechanisms by which MAVS regulates pZAP70 levels will be presented.
Failure to control measles virus spread in the neonatal CNS despite immune cell infiltration and cytokine production.
Authors: Ganesan Priya , Bohn Larissa , O’Donnell Lauren
Published: January 2023

Viral infections in the central nervous system (CNS) are associated with devastating neurological consequences in newborns. This may be due, in part, to deficiencies in the immature innate and adaptive immune response. To study viral CNS infections in neonates, we use a transgenic mouse model of neuronally-restricted measles virus (MV) infection (NSE-CD46 mice), where the human isoform of CD46, a MV receptor, is expressed under the control of the neuron-specific enolase promoter. Adult CD46+ mice survive infection and clear MV in an interferon gamma (IFNg)- and T cell-dependent manner. In contrast, neonatal CD46+ mice succumb to the infection despite T cell infiltration into the brain parenchyma. Neonatal mice lacking IFNg succumb more rapidly than wildtype pups despite higher T cell infiltration, equivalent natural killer cell infiltration, and microglial activation. Quantitative RT-PCR analysis demonstrated upregulation of pro-inflammatory cytokines and chemokines such as IFNg, IL-1β, and CXCL10, as well as the anti-inflammatory interleukin 1 receptor antagonist in infected adults and neonates. IL-12β, IL-23, CCL2, and TNF were significantly upregulated in infected neonates only. These results suggest the neonates are capable of cytokine production with a Th1-like profile within the CNS, but that the cytokine milieu is ineffective at controlling MV spread. Current experiments aim to inhibit inflammatory mediators such as CXCL10 and CCL2 that are expressed in an age-dependent manner during infection to determine if there is an improvement in neuropathology and survival of neonates.
Expression and regulatory effects of Schlafen genes in flavivirus infection
Authors: Kumar Mukesh , Hughes Laura , Nerurkar Vivek R
Published: January 2023

The continuing spread of flaviviruses, combined with the lack of specific therapeutics and vaccines, imparts a pressing need to identify the viral and host factors that control replication and outcome of infection. The Schlafen (SLFN) family includes several mouse and human genes that have been investigated for their role in cell proliferation, regulation of immune response, and control of virus replication. Role of SLFN family genes in flavivirus infections is yet to be determined. Herein, we demonstrate that infection with lethal West Nile virus (WNV) NY99 strain induces the expression of SLFN family genes; SLFN2, SLFN3, SLFN4, SLFN5, SLFN8, and SLFN9, in mice brain, and mouse primary cells including mouse embryonic fibroblasts (MEFs), macrophages, astrocytes, neurons and microglia. Among SLFN family genes, highest induction was observed in the expression of SLFN4. Interestingly, infection with non-lethal WNV Eg101 strain does not induce the expression of SLFN family genes in mice brain. Moreover, WNV NY99 infection of primary human cells including neurons, astrocytes, brain endothelial cells induced dramatic up regulation of SLFN12, SLFN12L (human orthologs of SLFN4), and SLFN14. Similarly, infection with dengue virus-1 (DENV-1), DENV-2, DENV-3, and DENV-4 induced the expression of SLFN12, SLFN12L and SLFN14 in Vero cells and human monocytes. Furthermore, using siRNA, we demonstrate that knockdown of SLFN4 results in significant decrease of WNV NY99 titers in MEFs. Additionally, mRNA and protein expression of WNV-induced interferons, chemokines, and cytokines were significantly decreased in SLFN4-knockdown MEFs. Collectively our data for the first time indicate the novel role of SLFN genes in flavivirus pathogenesis.
Interactions between the Ranavirus Frog virus 3 and mammalian macrophage cell lines
Authors: Torres-Luquis Odalys Joan , Edholm Eva-Stina , De Jesús-Andino Francisco , Elliott Michael Rusty , Robert Jacques
Published: January 2023

Infections caused by Ranaviruses (RVs, Irridoviridae) are increasing at alarming rates in prevalence and the wide range of host species infected, (from amphibians to fishes and reptiles), which suggests that RVs possess potent immune evasion strategies. Growing evidence indicated that RVs host infection strategies and disseminations are critically linked to the host immune response, specifically macrophage-lineage subsets. Indeed, we have shown that in the amphibian Xenopus laevis the RV Frog virus 3 (FV3) can persist in a quiescent state in macrophages of otherwise asymptomatic hosts and can be reactivated following inflammation-induced stimulation. To further explore the cellular and molecular mechanisms involved in these processes we have developed an in vitro culture system using FV3 in combination with five murine macrophage cell lines that were adapted to 30°C culture condition necessary for FV3 replication. FV3 infectivity was determined using a knock-in recombinant FV3 expressing a GFP reporter under the control of the 18K immediate-early FV3 promoter. FV3 infected all murine cell lines. However, viral replication varied depending on the cell type: it was similar to that detected in control BHK cells with RAW (peritoneal) cells, comparably modest with J774 (peritoneal) and BV-2 (microglial) cells, whereas no FV3 replication was detected with TM4 (Sertoli) and P338D1 (monocytic) cells. This lack of FV3 replication in TM4 and P338D1 despite successful viral entry and expression of FV3 immediate-early genes in TM4 is similar to what is observed with X. laevis macrophages and suggests that FV3 triggers similar cellular and molecular mechanisms in mammalian as in X. laevis macrophages resulting in viral quiescence.
Resveratrol (RES) and its antagonist, sirtinol suppress human cytomegalovirus (hCMV) replication and hCMV induced transcription of proinflammatory cytokines and chemokines in THP-1 derived macrophages
Authors: Li Huifen , Ning Xiaoxuan , Wang Jintang , Ouyang Min , Mao Genxiang , Leng Sean X
Published: January 2023

Background

Mounting evidence suggests significant contribution of chronic hCMV infection to T-cell immunosenenscence and chronic inflammation in old adults. THP-1, a human monocytic cell line and its derived macrophages serve as a useful cell culture model to study hCMV infection and its biology. Novel anti-CMV agents are needed as current anti-CMV treatment is limited in efficacy with significant side effects. We and others observed that Resveratrol (RES) and sirtinol, suppressed CMV replication in human fibroblasts. Here we report their inhibition on hCMV replication and hCMV induced expression of IL-6, IP-10, MCP-1 and TNF-a in THP-1 derived macrophages.

Methods

THP-1 cells were differentiated into macrophages after incubation with PMA (50 ng/ml) in RPMI 1640 with 10%FBS for 24 hrs. RES or sirtinol was added 2 h followed by hCMV (Towne strain) inoculation at multiplicity of infection of MOI 2 for 24 hrs. Cells were then cultured in hCMV-free medium for indicated times. qPCR and RT-PCR were performed to evaluate hCMV replication and transcription of IL-6, IP-10, MCP-1 and TNF-a, respectively.

Results

Both RES and sirtinol suppressed hCMV replication. They also suppressed hCMV induced transcription of proinflammatory mediators except for TNF-a.

Conclusions and discussion

These findings indicate potent inhibition of hCMV infection and hCMV induced transcription of proinflammatory mediators by RES and sirtinol in THP-1 derived macrophages. While being non-permissive, monocytes are important reservoir for chronic or latent CMV infection. Further investigation into the role and mechanisms of RES, sirtinol and other agents in control of chronic CMV infection and its adverse impact is indicated.
Markers of Chronic Immune Activation in HIV Patients Receiving Antiretroviral Therapy
Authors: Albino Elinette , Godoy Lenin , Hill Martin
Published: January 2023

Antiretroviral therapy (ART) prolongs the lifespan of HIV-infected individuals but does not reduce chronic immune activation and inflammation. While ART typically decreases both immune activation and inflammation markers, recent studies showed these markers remain elevated in many HIV-infected patients. Over time, this heightened inflammation state may increase the rate of cardiovascular disease in HIV-infected individuals. In this study we characterize the inflammatory monocyte subsets in HIV positive subjects taking HAART and correlate this information with innate and inflammatory cytokines levels in plasma. Blood samples were taken from 50 subjects including: HIV positive subjects under ART, untreated HIV positive subjects healthy subjects taking HAART and healthy controls. Nine cytokines were measured in plasma using a Magnetic Luminex technology. In addition, fresh monocytes were separated from peripheral blood mononuclear cells (PBMCs) by CD14/CD16 monoclonal antibodies. Levels inflammatory cytokines were associated with health status parameters and self reported lifestyles. HIV-1 infected subjects had increased levels of both CD14/CD16 cells and inflammatory cytokines compare to the rest of the subjects enrolled in the study. A plasma inflammatory biomarker signature consisting of CD14, IL-6, IL-15, CD163 and IL-12 p70 corresponded with immune activation after prolonged exposition to ART. These findings suggest that increased inflammatory cytokines levels could be a consequence of antiretroviral use, yet the specific role of these inflammatory markers in disease state remains to be determined.
RIPK3 restricts West Nile virus neuropathogenesis independently of programmed necrosis.
Authors: Daniels Brian , Loo Yueh-Ming , Gale Michael , Oberst Andrew
Published: January 2023

Programmed necrosis coordinated by receptor-interacting kinase-3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) is known to contribute to host immune responses to viral infections. However, the role of these proteins in host immunity during flavivirus infection remains poorly understood. Using a mouse model of West Nile virus encephalitis, we show that RIPK3 is required for the restriction of West Nile virus pathogenesis, independently of its role in programmed necrosis. While Ripk3−/− mice exhibited accelerated and enhanced mortality compared to WT controls, survival and clinical scores in mice lacking the executioner protein MLKL were unaffected. Moreover, cell death following WNV infection in primary cultures of myeloid and neuronal cells was unchanged in both Ripk3−/− and Mlkl−/− cultures. The enhanced susceptibility of Ripk3−/− mice arose, instead, from a failure to control infection within the central nervous system (CNS). While peripheral adaptive immune responses to WNV remained intact, Ripk3−/− mice exhibited decreased recruitment of inflammatory myeloid cells and lymphocytes to the CNS, despite higher CNS viral loads. RIP3 suppressed CNS viral burden via both CNS-intrinsic and –extrinsic mechanisms, as viral titers in the brain and spinal cord were higher in Ripk3−/− mice compared to controls following both subcutaneous and intracranial inoculation with WNV. Together, these data identify new roles for RIPK3 in the restriction of viral pathogenesis, and implicate RIPK3 as a key regulator of antiviral immunity within the CNS.
ZMPSTE24 is a novel intrinsic immune protein that restricts a broad-spectrum of viruses
Authors: Li Shitao , Wang Lingyan , Fu Bishi , Dorf Martin E
Published: January 2023

Virus entry begins with attachment to cell-surface receptors and ends with delivery of the viral genome into the cytoplasm. Many enveloped viruses are ferried deep into the cytoplasm through endocytic vesicles and utilize the low pH endosomal environment to activate the fusion process between viral and cellular membranes. Higher vertebrates evolved interferon-induced transmembrane proteins (IFITM) to impede endocytic penetration by a broad spectrum of RNA viruses. However, the molecular mechanism by which IFITM proteins hinder viral entry is not well elucidated. To address the antiviral mechanism of IFITM, we hypothesize that type I interferon (IFN) induces recruitment of co-factors to the IFITM3 protein complex. Proteomic analysis of IFITM3-FLAG protein complex in HEK293 cells revealed that IFN induced IFITM3 to recruit ZMPSTE24, the 7-pass transmembrane zinc metalloprotease whose function in cytoplasmic organelles is not defined. Contrary to IFITM, ZMPSTE24 mRNA and protein expression are not regulated by IFN. But consistent with IFITM, ZMPSTE24 restricts both RNA and DNA viruses using endocytic entry, including influenza A, vesicular stomatitis, sindbis, Ebola, cowpox and vaccinia viruses. Antiviral activity requires ZMPSTE24 endosomal localization and is independent of its protease activity. Furthermore, ZMPSTE24 is recruited to endosomes by IFITM and block viral endocytic entry. These findings identify a new role for ZMPSTE24 as a broad-spectrum intrinsic antiviral protein and the downstream antiviral effector of IFITM.
MHC class II antigen-processing chaperone H2-O shapes CD4 T cell receptor repertoire
Authors: Song Nianbin , Poluektov Yuri , Welsh Robin , Sadegh-Nasseri Scheherazade
Published: January 2023

HLA-DO (DO) is a MHC class II-encoded, nonpolymorphic heterodimeric protein in humans (known as H2-O in mice). It is expressed only in thymic medulla, B lymphocytes and some subsets of dendritic cells. Although DO is found to form a stable complex with HLA—DM (DM), its physical function remains unknown. Some studies have shown that it inhibits the activity of DM to edit peptide presentation on MHC II molecules while others reported that the DO can up- or downmodulate presentation of certain peptides in association with DM on HLA-DR4 molecules. To solve this controversy, our lab recently proposed a model suggesting that the DO facilitates the binding of DM-resistant peptides and inhibits the binding of DM-sensitive peptides to the HLA-DR1 molecules in vitro. Based on this model, we expected to see lower density of pMHC expression on mTEC cells in thymus of H2-O-KO mice, which will lead to faulty negative selection and different CD4 TCR repertoires as compared to H2-O-WT mice. By developing a “Prime-Restim” strategy based on the principle of mixed lymphocyte reaction, we observed the proliferation of primed H2-O-WT CD4 T cells in responding to repeated in vitro stimulations by antigen presenting cells from H2-O-KO mice and vice versa. The results demonstrate that in the absence of H2-O, the peptide repertoire of MHC II in the H2-O knockout mice is different from the peptide repertoire of MHC II in the H2-O-WT mice. It establishes a role for DO in regulation of epitope selection for presentation by MHC class II molecules in vivo. These findings support the idea that the expression of DO in thymic medulla might lead to better thymic deletion of self-reactive CD4 T cells.
MARCH1 ubiquitin ligase rescues thymic dendritic cells from MHCII proteotoxicity and supports regulatory T cell development and repertoire expansion
Authors: Oh Jaehak , Perry Justin , Pua Heather , Hsieh Chyi-song , Shin Jeoung-Sook
Published: January 2023

Membrane-Associated RING-CH1 (MARCH1) is an ubiquitin ligase preferentially expressed in antigen-presenting cells and mediating ubiquitination and degradation of MHCII and CD86. MARCH1 deficiency results in a significant reduction in the number of thymic regulatory T (Treg) cells in mice, implicating an important role of this molecule in Treg cell development. However, neither the mechanism by which MARCH1 supports Treg cell development nor its impact on Treg cell function has been clearly defined. Here we report that the role of MARCH1 in Treg cell development critically depends on its expression in dendritic cells and its activity to ubiquitinate MHCII. Importantly, not the MHCII ubiquitination per se but its consequences to MHCII proteostasis is essential for dendritic cells to organize surface proteins appropriately, make a stable conjugate with thymocytes, and provide them with sufficient signal for Treg cell differentiation. When MARCH1-mediated MHCII proteostasis in dendritic cells fails, Treg cells develop to a markedly reduced number and a substantially restricted TCR repertoire in the thymus, resulting in the function of these cells to suppress graft-versus-host-disease severely impaired. Thus, MARCH1-mediated MHCII proteostasis plays an essential role for thymic dendritic cells to select regulatory T cells in abundance and diversity required for proper immune suppression.
The future of the immunopeptidome in health and disease: a comprehensive analysis of naturally processed ligand data
Authors: Vaughan Kerrie , Caron Etienne , Peters Bjoern , Sette Alessandro
Published: January 2023

The promise of big data (‘OMICS’) in fundamental and translational science is clear. In this respect, high-throughput analysis of HLA class I and II associated immunopeptidomes by mass spectrometry is an important tool to establish T cell response repertoires in the context of disease. The last decade has seen tremendous growth in the breadth and number of these studies. The time is therefore ideal for initial assessment of these data as a whole. For this, we made use of all naturally eluted peptide data captured to date within the IEDB, representing more than 100,000 class I and II peptides. The goal of this analysis was to compare and contrast the overall nature of naturally eluted peptides (NP) from several standpoints, including molecular and biological function, sub-cellular location and protein class. Further, we investigated the extent to which the NP data overlap with existing T cell epitope data, MHC binding data, as well as output from class I and II prediction algorithms. The comprehensive collection of NP data will allow us to examine and bench mark the correlation with independently determined immunological response and MHC binding, and might also be useful to develop novel methods for the identification of optimal target antigens and for epitope prediction. These data support the potential value of immunopeptidomics in understanding T cell immunity.
Systemic recognition of insulin by autoimmune T and B cells leads to production of antibodies targeting the islet of Langerhans
Authors: Wan Xiaoxiao , Thomas James W , Unanue Emil R
Published: January 2023

The presence of class-switched insulin autoantibodies (IAAs) is a consistent feature of patients with type 1 diabetes and non-obese diabetic mice. The mechanisms underlying their production and pathogenic role are poorly understood. IAA production is unexpected because insulin-reactive B cells are maintained in a tolerant state. Furthermore, the source of T cell help is limited because T cells recognizing insulin are deleted in the thymus, and those that escape thymic selection recognize insulin peptides but not native insulin. However, increased titers of IAAs to conformational insulin were found in a T cell receptor transgenic strain, 8F10, whose T cell reactivity is restricted to insulin peptides but not insulin protein. Furthermore, when an anti-insulin B cell receptor heavy chain transgene was introduced into the 8F10 mice, a marked increase in IAAs specific for native insulin was observed. Dynamic T-B cell interactions and spontaneous germinal center formation were observed in multiple lymphoid organs indicating widespread insulin presentation. But more pointedly we found that the germinal center B cells were activated and in contrast to naïve B cells, presented a broader repertoire of insulin epitopes. Finally, for the first time we show that IAAs localize in the islets of Langerhans, form deposits on β-cells, and enhance considerably the presentation of diabetogenic antigens. Thus, systemic recognition of a native circulating self-protein is sufficient for generation of autoantibodies that precipitate pathogenic outcomes in the target organ.
Role of the transcription factor Ikaros in development of autoimmune disease
Authors: Schjerven Hilde , Frietze Seth , Hai Si-Han , Hermiston Michelle , Kogan Scott , Muschen Markus
Published: January 2023

The etiology of autoimmune disease is not fully understood, but disease development is believed to be due to a combination of genetic predispositions and environmental factors. Ikaros is a transcription factor that is an important regulator of hematopoiesis and critical for development of B cells. Recent genome wide association studies have found that several autoimmune diseases are associated with SNPs annotated to the Ikaros gene (IKZF1), as well as the closely related Ikaros family member Aiolos (IKZF3). Mouse models of autoimmunity are important research tools, and we recently reported the generation of two new Ikaros-mutant mice, with targeted deletions of the exons encoding the DNA-binding zinc finger 1 (ZnF1) or ZnF4 (Schjerven et al., 2013). These deletions did not abolish B-cell development, but rather illuminated differential roles of Ikaros at different stages of early B-cell development. Interestingly, we found that one of these mouse strains displayed very high levels of serum Anti-Nuclear Antibodies (ANA), indicative of autoimmune disease. Furthermore, we found by ex vivo studies that while wt spleen B cells require a “second signal” such as CD40L in addition to BCR stimulation (by anti-IgM), Ikaros-mutant B cells became activated and proliferated upon BCR stimulation alone without any externally added “second signal”. We hypothesize that Ikaros is important to limit autoimmunity and prevent auto reactive B cells by setting a requirement for the “second signal” in adaptive immune-recognition. Results will be presented from ongoing investigation using in vivo analysis of the autoimmune-related phenotype, and ex vivo analysis of Ikaros-dependent molecular mechanisms underlying the response to stimuli in B cells.
Casapse-1 is not required for the induction of Silica-induced autoimmunity
Authors: Pawar Rahul D. , Mayeux Jessica , Cooper Timothy , Awad Alaa S. , Kono Dwight H , Pollard Kenneth M.
Published: January 2023

Occupational exposure to respirable crystalline silica induces silicosis in humans & has been associated with lupus (SLE), but the specific mechanisms & susceptibility factors are largely unknown. Recent studies have highlighted a prominent role for interleukin-1β in silica-induced lung inflammation. Caspase-1 is predominantly involved in the cleavage of pro-IL-1β to active IL-1β. Therefore, we investigated the role of IL-1β in the severity of lung damage & autoimmune response to silica by exposing B10.S and Caspase-1 deficient B10.S (B10.S-Casp1−/−) mice to crystalline silica (1 mg in 25 μl) in PBS or PBS only, by intranasal instillation twice a week for 4 weeks. When analyzed for autoimmunity at 28 weeks post-exposure, both B10.S and B10.S-Casp1−/− mice developed similar elevated levels of anti-chromatin & anti-nuclear antibodies in serum & broncho-alveolar lavage (BAL) compared to PBS controls. Correspondingly, both silica-treated B10.S and B10.S-Casp1−/−mice had a significant peribronchial & perivascular lymphocytic infiltrates (PLI), alveolar proteinosis (AP), and alveolitis, but the PLI and AP were significantly higher in B10.S mice than B10.S-Casp1−/−mice. Similarly, robust increases in the levels of total protein & cell numbers in BAL fluid, but total protein was significantly higher in B10.S mice than B10.S-Casp1−/−mice. Higher numbers of inflammatory M1 macrophages (CD11b+Ly6chi) & neutrophils (Ly6Ghi) were also observed in both groups of silica-treated mice. These findings indicate that caspase-1 and by inference IL-1β is not required for silica-induced autoimmunity in mice, but contributes to the lung inflammation, & suggests that NLRP3 inflammasome, cathepsin C, elastase, granzyme B may be more important.
Influenza vaccine induces anti-nuclear and anti-double strand DNA IgG antibodies and their relates to levels of microbial translocation in antiretroviral-treated aviremic HIV-infected patients but not in healthy controls
Authors: Luo Zhenwu , Martin Lisa , Kilby Michael , Liu Hui , Jin Ping , Stroncek David , Jiang Wei
Published: January 2023

Background

The mechanism of autoreactive antibody induction in HIV disease is unknown.

Methods

In this study, blood samples from healthy controls (n = 16) and antiretroviral therapy (ART)-treated HIV infected patients (n = 26) receiving 2013–2014 seasonal influenza vaccine were analyzed pre-vaccination (D0) and post-vaccination (D14). B cell function, LPS, 16S rDNA, anti-nuclear IgG (ANA, OD) and anti-dsDNA (IU/ml) IgG in plasma were evaluated. Data are presented as median (interquartile range [IQR]).

Results

At baseline, there were increased frequencies of B cell apoptosis and cycling expression, and increased plasma LPS [pg/mL, 18.0 (13.3–22.0) vs 13.4 (9.82–15.2), P = 0.006] in patients compare with controls. Bacterial 16S rDNA tended to be higher in patients compared with controls [copies/μL, 38.2 (30.3–44.9) vs 27.8 (2.3–53.6), P = 0.18]. B cells of patients exhibited increased TLR2 and 4 expression compared with controls, and TLR signaling pathway activation by microarray on D0. Plasma ANA levels on D0 and D14 were 0.31 (0.25–0.41) and 0.58 (0.31–1.29) versus 0.32 (0.23–0.40) and 0.31 (0.24–0.38), anti-dsDNA IgG levels on D0 and D14 were 15.2 (8.06–23.7) and 20.2 (13.0–50.7) versus 15.3 (12.3–37.1) and 15.5 (13.0–36.7) in patients and controls respectively. Only patients showed significant increase of both auto-IgG post-vaccination (P < 0.001). Direct correlations between plasma LPS and ANA (r = 0.48, P = 0.01), and plasma 16S rDNA and anti-dsDNA IgGs (r = 0.44, P = 0.04) were found only in patients.

Conclusion

B cell function in HIV patients is not fully recovered by ART treatment, as reflected by producing autoreactive Abs in response to influenza vaccinations and its correlates with levels of microbial translocation.
GM-CSF is not required for EAE induction but contributes to both brain and spinal cord inflammation
Authors: Pierson Emily , Goverman Joan
Published: January 2023

Myelin-specific CD4+ T cells initiate inflammation in the central nervous system during induction of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. CD4+ T cell pathogenesis has been attributed to the secretion of various cytokines, including IL-17, GM-CSF, and IFN-γ. The relative importance of these CD4+ cytokines in EAE has been the subject of numerous studies, and in C57BL/6 and B10.PL mice, GM-CSF production by T cells is required to induce EAE, while IL-17 and IFN-γ are not required. However, our studies in C3Heb/Fej mice do not support an essential role for GM-CSF in the induction of EAE. Our model is unique in that C3Heb/Fej mice develop brain inflammation in addition to the spinal cord inflammation seen in most other EAE models. We have previously shown that IL-17 is important for (and IFN-γ inhibits) neutrophil recruitment and parenchymal inflammation in the brain of these mice, while IFN-γ promotes spinal cord inflammation. We found that GM-CSF−/− T cells were capable of inducing brain and spinal cord inflammation in wildtype recipient mice. In IL-17RA−/− recipients, wildtype T cells induce intermediate levels of brain inflammation and normal spinal cord inflammation. However, when GM-CSF−/− T cells were transferred into IL-17RA−/− recipient mice, there was a complete loss of clinical signs of brain inflammation, and spinal cord inflammation was significantly reduced, indicating that GM-CSF contributes to both brain and spinal cord inflammation. These data reveal that GM-CSF, IL-17, and IFN-γ all influence inflammation in the brain and spinal cord in C3Heb/Fej mice; while none are required, coordinately these cytokines shape CNS inflammation.
PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) regulates IL-6-STAT3 signaling to reduce neutrophil infiltration into the synovium of arthritic joints
Authors: Kim Young-Jun , Shin Jae Min , Yoon Sun Young , Kim Joo Heon , Kim Myung-Hwan , Sohn Ki-Young , Kim Heung-Jae , Han Yong-Hae , Chong Saeho , Kim Jae Wha
Published: January 2023

Inappropriate regulation of leukocyte trafficking can lead to impaired neutrophil clearance and increased tissue damage from the accumulation of neutrophil-secreted proteases and reactive oxygen species at the site of inflammation. In this study, we have discovered that 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) regulates the activity of signal transducer and activator of transcription 3 (STAT3), which is a master regulator of IL-6 expression. PLAG has been isolated from the antlers of Sika deer (Cervus nippon Temminck), which were known to have immunosuppressive and anti-arthritic activities. PLAG induced a significant decrease of IL-6 production in a macrophage cell line, RAW264.7, and rheumatoid arthritis-fibroblast-like synoviocyte (RA-FLS) via the regulation of STAT3 signaling. IL-6, a multifunctional pro-inflammatory cytokine, plays a critical role in the pathogenesis of joint and systemic inflammation in RA. In collagen-induced arthritis (CIA) mouse model, arthritic symptoms were recapitulated with an increase of IL-6 in the synovium, which was recuperated by the treatment of PLAG to the level comparable with commercial therapeutics (such as Remicade or Methotrexate). When the joint tissues were stained with neutrophil-specific antibodies, PLAG significantly reduced the infiltration of neutrophils into the joint synovium of CIA mouse. Therefore, PLAG inhibits the infiltration of destructive neutrophils into inflammatory sites, and can be utilized as a therapeutic agent for the treatment of sustained inflammation and joint destruction.
Dissecting the mechanisms underling acute lymphoblastic leukemia brain metastasis
Authors: Kinjo Ichiko , Bragin Denis E , Wilson Bridget S
Published: January 2023

Despite improved treatments, patient with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) experience relapse with devastating tumor cell dissemination into the central nervous system (CNS). As an “immune privilege site”, CNS is protected from infiltration of immune cells by specialized structure of blood-brain barrier with astrocyte endfeet that surround the blood vessels. To assess whether leukemia cells hijack mechanisms employed by inflamed immune cells to invade into the brain parenchyma, we set up leukemia mouse xenograft models with well-characterized human BCP-ALL cell lines and primary cells derived from patients. We found massive infiltration/colonization of GFP+ leukemia cells inside brain vessels and parenchyma addition to meningeal space. Immunohistochemistry staining showed that activation of astrocytes following the induction of VEGF-A in brains of leukemia burden mice. We address phenotype changes in leukemia cells, that are associated with brain metastasis by comparing the expression of chemokine receptors and cytokines in leukemia cells recovered from bone marrow and other extramedullary sites (spleen, liver) for comparison to the cells recovered from brain tissue. By investigation of extravasation steps in intact brain, we also try to figure how leukemia cells bleach the BBB and find new niche to colonize at brain parenchyma. Ongoing studies will provide new insights into the mechanisms that regulate leukemia CNS infiltration.