bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor cell-intrinsic programmed death protein 1 expression and induction in human cancer cell lines
Authors: Osta Erica , Gupta Harshita B. , Zhang Deyi , Kornepati Anand , Clark Curtis A. , Curiel Tyler J.
Published: January 2023

The programmed death protein 1 (PD-1) and ligand (PD-L1) axis inhibits T cell activation and reduces anti-tumor immunity. Most studies of the PD-1/PD-L1 axis test extrinsic, immune-dependent effects. However, tumor cell-intrinsic PD-L1 regulates immune-independent tumor mTOR signals, autophagy, and tumor initiating cell generation. Melanoma cell-intrinsic PD-1 regulates melanoma growth and mTOR. We tested PD-1 expression and induction in multiple human tumor cell lines. We detected PD-1 in human cell lines in vitro from ES2 ovarian cancer, VMM122 melanoma, and RT4 bladder cancer by flow cytometry, Western blot and RT-PCR. All lines were negative for the other PD-L1 receptor, CD80, by flow cytometry. Incubating lines with IFNα or IFNγ for 48 hours further augmented PD-1 expression as detected by flow, Western blot and RT-PCR. αPD-1 antibody (50 μg/ml) reduced proliferation of RT4 bladder cancer cells in vitro, demonstrating a direct PD-1 signal effect in these cells, and αPD-L1 similarly reduced in vitro proliferation. Interestingly, anti-PD-1 effects on reducing proliferation were similar: 41.5% reduction; p<0.0001. We used CRISPR/Cas9 to generate PD-L1KO RT4 cells. αPD-L1 did not reduce in vitro proliferation as expected, but unexpectedly, αPD-1 also had no anti-proliferative effect on PD-L1KO RT4 cells, suggesting a PD-L1/PD-1 interaction. In murine B16 melanoma, we generated PD-L1KO and PD-1KO cells using CRISPR/Cas9. Cell-intrinsic PD-L1 and PD-1 downregulate chemokine production. Among chemokines analyzed, PD-L1 suppressed CCL2 and CCL5, PD-1 suppressed CCL4 and CCL7, and both suppressed CCL3 and CXCL2. Understanding intrinsic PD-1 and PD-L1 effects on tumor cells can be exploited to improve cancer immunotherapy
Focused ultrasound ablation as an immunomodulatory strategy for metastatic breast cancer therapy
Authors: Sheybani Natasha Diba , Witter Alexandra R. , Stevens Aaron D. , Bullock Timothy N. , Price Richard J.
Published: January 2023

Metastatic breast cancer (BrCa) is incurable, with a 5 year survival of only 22%. While immunotherapies are capable of generating durable responses in many forms of cancer, immune rejection of BrCa is very rare. Indeed, immunotherapies (e.g. checkpoint blockade) for BrCa are limited by poor functional CD8+ T cell infiltration and immunosuppression in the tumor microenvironment. Thus, adjunct strategies that render BrCa tumors responsive to immunotherapies are desperately needed. Focused ultrasound (FUS) mediates noninvasive acoustic energy deposition into tumors, conferring localized thermal and mechanical damage to targeted tissue. Here, we tested the hypothesis that FUS thermal ablation (TA) can serve as an auto-vaccine for treatment of BrCa with immunotherapy. Murine mammary carcinoma (4T1-HA) tumors were partially ablated with a 3 MHz ultrasound-guided FUS system. In tumor-draining lymph nodes, 1 week after FUS, we observed a ~3-fold increase in mature dendritic cells (CD11c-hi/CD86+), as well as an increasing trend in M1 macrophage representation by flow cytometry. While FUS did not confer changes in intratumoral CD8+ and CD4+ T cells, likely due to an overwhelming immunosuppressive burden imposed by myeloid derived suppressor cells (MDSCs), infiltrating T cells did express PD1. Therefore, we developed a combination treatment approach of FUS + gemcitabine and/or anti-PD1. This combination treatment elicited an ~50% reduction in 4T1-HA tumor growth at 5 days post-FUS ablation, suggesting that partial TA of BrCa tumors with FUS may represent a promising immunoadjuvant approach. Taken together, these results suggest immunoadjuvants abrogating immunosuppression may enable FUS to stimulate anti-tumor immunity in BrCa.
A delicate interplay between adaptive and innate immunity caused by immunotherapy triggers tumor immunity and aseptic inflammation
Authors: Hirschhorn Daniel , Ricca Jacob , Gasmi Billel , DeHanau Olivier , Mangarin Levi Mark , Budhu Sadna , Li Yanyn , Cortez Czarina , Liu Cailian , Zappasodi Roberta , Houghton Sean , Betof Allison , Lacouture Mario , Hollman Travis , Albrengues Jean , Egeblad Mikala , Wolchok Jedd D. , Merghoub Taha
Published: January 2023

Successful immune-based therapies that treat tumors are often accompanied by immune-related adverse events (irAE). Immunotherapy-treated patients who experience favorable anti-tumor responses typically manifest irAEs. The primary immunotherapies currently in clinic include agents that activate T cell responses such as checkpoint blockade of inhibitory pathways and infusion of ex-vivo tumor-derived or T cell receptor (TCR)-transgenic or chimeric antigen receptor (CAR)-modified T cells. While the beneficial and toxic effects of T cell-based immunotherapies in the clinic are being extensively explored, the precise mechanisms of tumor elimination and irAE remain the subject of intense investigation. In the present study, we treated established tumors with melanoma-specific adoptive CD4+ T cell transfer and costimulation via anti-OX40 or anti-CTLA-4. We found that, in spite of co-opting the adaptive immune response, acute local inflammation plays a fundamental role in tumor elimination and related toxicities in a model irAE. While stimulated T cells are necessary for initiating a therapeutic response, activation of endogenous neutrophils constitute an important and necessary effector mechanism of tumor destruction and irAE. Extensive neutrophils extracellular traps (NETs) were associated with irAE. Melanoma patients treated with checkpoint blockade, suffering from skin rashes equivalent to irEA found in mice, showed increased survival and NETois in biopsies from rashes and tumors. Our results bring forward a novel paradigm where T cells prime an anti-tumor immune response followed by an inflammatory effector mechanism, provided by the innate immune system with curative as well as morbid effects.
Differential abundance of mDC subsets predict response to Hepatitis B vaccination
Authors: Scheuermann Richard H. , Novotny Mark , Aevermann Brian , Ben-Othman Rym , Liu Aaron , Sadarangani Manish , Kollmann Tobias
Published: January 2023

Vaccination as a strategy for the prevention of infectious disease has been one of the greatest success stories in modern medicine, resulting in the complete eradication of smallpox, the near eradication of polio, and the drastic lowering in the incidences of measles, mumps, influenza and other common diseases. In contrast to these remarkable successes, recent failures in the development of effective vaccines against other major public health threats, including HIV, TB and malaria have highlighted the limitations to the current approach of empirical vaccine design and the need to better understand the basic principles of how to recognize and elicit immune responses that generate effective and durable protective immunity. Single cell transcriptional profiling by RNA sequencing (scRNAseq) is a powerful tool for exploring the phenotypic diversity of cell populations present in peripheral blood and other tissue specimens in an unbiased fashion. We applied scRNAseq to FACS sorted blood samples collected from participants before and after challenge with the licensed Hepatitis B vaccine, and identified several innate cell subtypes using unbiased clustering analysis. Marker genes specific for each cell subtype were identified using random forest machine learning for use in qPCR. Our results suggest that the relative levels of myeloid dendritic cells (mDCs) specifically expressing the N-myc target gene, NDRG2, prior to vaccination predict serum antibody responses to the HepB vaccine.
Intranasal immunization with an acellular pertussis vaccine containing a Th1/17 skewing adjuvant, BcfA, improves B. pertussis clearance from the mouse respiratory tract.
Authors: Yount Kacy S. , Jennings-Gee Jamie , Quataert Sally , Deora Rajendar , Dubey Purnima
Published: January 2023

Bordetella pertussis is the causative agent of whooping cough, a resurging vaccine-preventable disease. This highly contagious disease is most severe in infants and young children. Current alum-adjuvanted acellular pertussis vaccines (aPV) prevent severe disease but do not prevent nasal carriage and subsequent person-to-person pathogen transmission. The Th1/2 skewed immune response induced by aPV is one potential explanation for this failure. We are testing the hypothesis that modification of current aPV by the addition of an adjuvant, BcfA, that skews immune responses towards the more protective Th1/17 phenotype will improve protection. We also hypothesize that changing the delivery route from intramuscular (i.m) to intranasal (i.n) administration will generate mucosal immunity and further improve protection. In the established mouse model of B. pertussis infection, we found that i.m. immunization of mice with an experimental aPV containing BcfA and alum as the adjuvants significantly reduced bacterial numbers in the lungs compared to an alum-adjuvanted vaccine alone. Further, i.n administration of the BcfA+alum containing vaccine provided better protection against colonization of the trachea and lungs than i.m. immunization. Alum-induced Th2 cytokine responses were reduced by addition of BcfA, and were further reduced by i.n. vaccine delivery. Thus, a revised aPV that includes BcfA, administered i.n, may improve protection against B. pertussis infection compared with current formulations and delivery method.
Efficient Isolation of Monoclonal Antibodies from Antigen-specific Human Plasmablasts and Memory B Cells from a Pneumococcal Conjugate Vaccinee
Authors: Cox Kara S. , Chen Zhifeng , Tang Aimin , Fink Malorie , Guan Liming , Roman Jae , Verch Thorsten , Retzlaff Mary , Vora Kalpit A.
Published: January 2023

Community-acquired pneumonia is a leading infectious cause of hospitalization, the annual incidence of Pneumococcal pneumonia is 24.8 cases per 10,000 adults in USA. Pneumococcal conjugate vaccine (PCV13) is currently used in clinic to prevent pneumococcal disease. We isolated plasmablast cells from a Pneumococcal conjugate vaccinee 7 days post vaccination to understand humoral responses. The plasmablast cells were single-sorted into 96 well PCR plates and applied to direct molecular cloning for PCR amplification of antibody sequences. Paired antibody heavy and light chain variable region sequences were achieved from 35 out of 48 wells with molecular cloning success rate of 72.9%. The antibody sequences were recombinantly expressed as human IgG1, and 17 out of 35 hits were positive for vaccine antigen by ELSIA s giving a positive rate of 48.9%. We also isolated memory B cells from the same donor 28 days post vaccination. The memory B cells were single-sorted into 96 well cell culture plates pre-seeded with CD40L-expressing feeder cells in complete RPMI 1640 medium complemented with IL21 and cultured for two weeks. The culture supernatants were tested in ELISA and 7 wells were identified as antigen-specific hits. Paired heavy and light chain antibody sequences were amplified from all of the 7 hits, all of them were successfully cloned and recombinantly produced as human IgG1 and tested in ELISA. 6 out of the 7 hits (85.7%) turned out to be vaccine antigen specific. We will further compare and discuss the clonal specificities isolated from plasmablast and memory B cells to understand the clone’s participation in immune responses to vaccination.
Generating Neutralizing Antibody responses using Recombinant Infectious Rhinovirus
Authors: Lee Sujin , Tang Roderick , Currier Michael , Moore Martin
Published: January 2023

Human Rhinovirus (RV), a leading causative agent for common colds and the subsequent development of pneumonia, can exacerbate both asthma and chronic obstructive pulmonary disease (COPD). There is no vaccine against RV yet. As previous studies demonstrated that RV-neutralizing antibodies (nAb) correlates with protection, eliciting robust nAb responses might be a key factor for ideal RV vaccine development. Five neutralizing immunogenic (NIm) sites were identified. NIm-IA, IB, and IV are located on VP1, while NIm-II and III are located on VP2 and VP3, respectively. Since the capsid proteins (VP1, 2, 3 and 4) seem to be immunogenic, we hypothesize that a chimeric virus expressing the capsid proteins is sufficient to elicit nAb responses. The HRV-A76 insert expressing the four capsid proteins and 2A protein was cloned into a bacterial artificial chromosome (BAC) harboring cDNA expressing the full-length genome of HRV-A33. Utilizing reverse genetics, a capsid-chimeric recombinant RV strain, BAC HRV33cap76, was generated using the four capsid proteins and 2A protein from HRV-A76, and the remaining part of the genome from HRV-A33. Following RNA transfection and recovery, we found that the titer of BAC HRV33cap76 was similar to biological HRV33 or HRV76. We next immunized BALB/c mice intramuscularly with inactivated BAC HRV33cap76 mixed with alum adjuvant to determine its immunogenicity. The BAC HRV33cap76 immunization resulted in nAb against only HRV-A76. Our findings demonstrated that the capsid proteins are responsible for the generation of nAb against RV. As a capsid-chimeric recombinant RV was capable of eliciting robust nAb, it could be a novel vaccine platform to generate highly effective and safe vaccine for preventing RV infection.
Vaccination against Follicle Stimulating Hormone Receptor delays ovarian cancer progression by breaking immune tolerance
Authors: Puchalt Alfredo Perales , Wojtak Kryzstof , Duperret Elizabeth K. , Yan Jian , Weiner David B.
Published: January 2023

Ovarian cancer is a frequent disease with poor prognosis. Improvements in surgery and chemotherapy have not been able to improve the 5-year rate survival. The follicle stimulating hormone receptor (FSHR) is expressed in approximately 50% of ovarian cancers but not in healthy tissues except for the gonads. We generated a vaccine against FSHR consisting of a consensus sequence of FSHR from other species that has a 95% homology with murine FSHR. The FSHR vaccine was able to break immune tolerance generating a strong FSHR-specific CD8 and CD4 response. This response was long lasting and able to delay progression of FSHR+ tumors in an immunocompetent mouse. Vaccination generated an increased T-cell infiltration in the tumor microenvironment and was able to synergize with previously inactive anti-PD1 antibodies. We found that the newly generated FSHR-specific CD8 T cells were sufficient and necessary for the effect of our vaccine. In conclusion, vaccination against FSHR is a promising new immunotherapy against ovarian cancer and can increase the susceptibility of ovarian cancer to checkpoint blockade.
Tumor membrane vesicle (TMV)-based cancer vaccine induces dendritic cell maturation and downstream T cell activation
Authors: Munoz Luis E. , Shafizadeh Yalda , Margaroli Camilla , Bommireddy Ramireddy , Selvaraj Periasamy
Published: January 2023

Breast cancer remains the most commonly diagnosed cancers among American women, with over 250,000 new cases expected per year. Breast cancer can be subdivided into different categories depending on the expression of receptors, most commonly into ER+, PR+, HER2+, and TNBC. Clinically, small molecule inhibitors of the hormone receptors and antibodies for HER2 have been developed to target these receptors and inhibit tumor signaling or eliminate these cells via phagocytosis and ADCC. Despite this, tumor cells that develop resistance to therapy remain a significant unmet need as they tend to be more aggressive and metastasize. We developed a novel immunotherapy approach that addresses the high degree of inter-patient and intra-tumor heterogeneity by making tumor membrane vesicle (TMV) vaccines out of individual tumor samples and modifying them with GPI-linked molecules like IL-12 and B7-1 to induce an antitumor immune response. In the present study, we investigated whether GPI-GM-CSF and GPI-IL-12 on TMVs from HER2+ murine breast cancer can target and activate antigen presenting cells. Here, we show that TMVs containing with GPI-GM-CSF and GPI-IL-12 can be phagocytosed by both macrophages and dendritic cells, induce upregulation of MHC-I, MHC-II and CD86, and inflammatory cytokine production in vitro. Further, when the DCs activated with our TMV vaccine were co-cultured with syngeneic T cells, they induced the upregulation of T cell activation markers. In summary, our results indicate that TMVs containing GPI-IL-12 and GPI-GM-CSF can enhance uptake and maturation of APCs and induce a strong T cell response downstream making it a potential therapeutic approach for HER2+ breast cancer.
Vaccine targeting antigens associated with cancer stem cells/epithelial to mesenchymal transition inhibits triple negative breast cancer growth
Authors: Cecil Denise L. , Curtis Benjamin , Gad Ekram , Corulli Lauren , Disis Mary L.
Published: January 2023

Triple negative breast cancer (TNBC) is characterized by higher relapse and poor survival rates compared to other breast cancer types and is marked by a cancer stem cell (CSC)/epithelial to mesenchymal transition (EMT) signature. We questioned whether a vaccine targeting CSC/EMT associated proteins could prevent development of TNBC. We identified five overexpressed proteins found in TNBC and associated with different CSC/EMT pathways. We used a scoring system derived from three common algorithms for predicting class II MHC binding to select Th epitopes for CD105, CDH3, MDM2, SOX-2 and YB-1. Those epitopes that induced a selective Th1 (IFN-gamma) immune response in human population based screening were included in a multi-antigen vaccine. Epitopes were highly homologous between mouse and man. Immunization with the multi-antigen vaccine induced Th1 specific for all five antigens compared to adjuvant control (p<0.05 for all). Immunity persisted 3 months after the final vaccine (p<0.05 for all). Vaccination generated an antigen-specific cytotoxic T-cell (CTL) immune response to three antigens (p<0.05). The vaccine increased effector memory T-cells by 2-fold compared to control (p=0.03). Tumor growth in vaccinated FVB-Tg(C3-1-TAg) mice, a basal breast cancer model, was inhibited by over 80% compared to control mice (p<0.0001). We observed significantly more CTL (p=0.04) and Th1 (p<0.001) infiltrating the tumor with a concomitant reduction in cells expressing the stem cell marker Sca-1 (p=0.003) after immunization with vaccine compared to control. This multi-antigen vaccine targeting CSC/EMT antigens is currently being investigated in a Phase I clinical trial (NCT02157051).
Development of vaccines for treatment of opioid abuse using carrier proteins suitable for pharmaceutical manufacturing
Authors: Huseby Kelcher April M. , Gradinati Valeria , Limkar Ajinkya , Baruffaldi Federico , Lees Andrew , Pravetoni Marco
Published: January 2023

In 2016, over 50,000 people died in the US from heroin and prescription opioid related overdoses. Current standard of care is not sufficient. Therapeutic vaccines against opioids are safe, long-lasting, and cost-effective alternatives to opioid-based pharmacotherapy. Opioid vaccines elicit opioid-specific antibodies that prevent opioid distribution to the brain and opioid-induced behavior and toxicity. To ensure translation, we have developed candidate vaccines that include components suitable for good manufacturing practices and have the ability to be characterized according to FDA guidelines. In this study, we synthesized three oxycodone-based (OXY) haptens to assess linker and bioconjugation chemistry in vaccine efficacy against oxycodone in mice. Then, we tested efficacy of lead OXY haptens conjugated to novel carrier proteins consisting of E. coli-expressed diphtheria cross-reactive material (EcoCRM™) and nontoxic tetanus toxin fragment (rTTHc). Haptens using tetraglycine linkers coupled with carbodiimide chemistry elicited the greatest oxycodone specific serum IgG antibody titers and significantly reduced oxycodone distribution to the brain. Oxycodone vaccines containing EcoCRM™ and rTTHc, adsorbed on alum adjuvant, were effective in inducing expansion of oxycodone-specific plasma and germinal center B cells, blocking oxycodone distribution to the brain, and were easily characterized by mass spectrometry. The OXY-EcoCRM™ and OXY-rTTHc were equivalent, or superior, to vaccines containing keyhole limpet hemocyanin protein, tetanus toxoid and CRM197, making them candidate immunogens for further development.
Combination of a DepoVax™ peptide vaccine with a lentivector vaccine induces strong antigen-specific immune responses and provides effective tumor control in murine models
Authors: Hrytsenko Olga , Weir Genevieve M. , Stanford Marianne M.
Published: January 2023

Many strategies are being investigated to induce and sustain anti-tumor immune responses through vaccination. Viral vectors have been used to induce robust cellular immune responses, however due to immune responses against the vector they loose potency with boosting vaccinations. In this study, we investigated a heterologous prime boost strategy that combines lentivirus and peptide vaccination to induce robust anti-tumour immune responses in a preclinical model.

DepoVax™ is a water-free vaccine formulation that facilities strong and sustained immune responses to peptide antigens. We showed that while homologous immunizations with DepoVax peptide vaccine containing HPV16 E749–57 T cell peptide antigen (DPX-FP) elicit potent antigen-specific responses, heterologous prime-boost immunizations with DPX-FP and lentivector vaccine expressing HPV16 E7 protein could further enhance systemic immune responses detected by IFN-γ ELISPOT. The synergy between two vaccines is influenced by the order of vaccine administration, dose, and delivery route. Using an HPV-expressing murine tumor model (C3), we found that heterologous priming with the DPX-FP vaccine and boosting with E7 lentivector vaccine enhanced tumor control and increased survival rates as compared to the treatments with either vaccine alone. RT-qPCR profiling demonstrated that the vaccine-draining lymph node cells of mice primed with DPX-FP and boosted with E7 lentivector vaccine had an altered immune gene signature with an increased expression of cytotoxic T lymphocyte markers (Gzmb and Tbx21). Taken together our results support a rationale for combining DepoVax based therapy with lentivector vaccines in future clinical trials.
Development of an Effective Cancer Vaccine Platform Using Attenuated Salmonella typhi
Authors: Xu Xin , Metelitsa Leonid S.
Published: January 2023

Poor antigen delivery is one of the major limitations of modern cancer vaccine vectors. To overcome this challenge, we exploited Salmonella Pathogenicity Island 2 and type III secretion system to deliver tumor-associated antigen (TAAs) into the cytosol of antigen-presenting cells in situ. In a recent report, we demonstrated that an attenuated strain of S. typhimurium, which was engineered to express SPI2-regulated oncoprotein survivin (SVN), induced potent CD8 T-cell-mediated antitumor response that was curative in a murine model of highly aggressive lymphoma. In further development of this technology for clinical use, the vaccine has been transferred from an experimental strain to a clinically validated strain of S. typhi, CVD908. To adapt CVD908 to stably express recombinant antigens without antibiotic-dependent selection, we used a recently reported plasmid stabilization system that encodes the single-stranded binding protein (SSB), an essential protein in DNA metabolism, which was deleted from the bacterial chromosome. The SPI2-regulated expression cassette was then cloned into the SSB plasmid, so that the resultant construct maintained bacterial vector stability while expressing and translocating TAAs. We found that CVD908Δssb vector effectively infects human dendritic cells in vitro and translocates recombinant SVN and MYCN oncoproteins into their cytosol. Furthermore, CVD908Δssb remains stable in mice and induces generation of antigen-specific CD8 T cells. Therapeutic vaccination of tumor-bearing mice with SVN or MYCN vaccine produced potent antitumor activity in murine models of lymphoma or neuroblastoma. The results justify clinical testing of CVD908Δssb-based SVN and MYCN vaccines in cancer patients.
Molluscum contagiosum virus subverts MHC-I antigen presentation by targeting tapasin for degradation
Authors: Harvey Ian B. , Wang Xiaoli , Fremont Daved H.
Published: January 2023

Molluscum contagiosum virus (MCV) is an extremely contagious poxvirus and one of the most common human skin pathogens. It is capable of producing persistent skin lesions that lack an inflammatory response, suggesting potent immune evasion strategies. However, the lack of an animal model or cell line to propagate the virus hampers investigations of the underlying mechanisms of pathogenesis. Here we demonstrate that MCV-encoded MC80 subverts MHC-I antigen presentation to evade CTL recognition. The MC80 ORF is conserved in the genome of all known MCV strains and encodes a type I TM protein with sequence similarity to classical MHC-I proteins. We find that expression of MC80 in human and mouse cells results in retention of host MHC-I in the ER and consequent surface MHC-I downregulation. Mechanistically, we find that MC80 can directly interact with tapasin via its ectodomain and target Tpn for ER-associated degradation in a TM and cytoplasmic tail dependent manner. The degradation of Tpn leads to a loss of TAP and impedes assembly of MHC-I with peptide. Our findings reveal a novel strategy for viral sabotage of the peptide loading complex and provide evidence that MCV actively subverts adaptive immune surveillance.
CRABP1 and 2 differentially regulate HCV infection via modulation of lipid droplet abundance.
Authors: Bang Bo R. , Li Meng , Tsai Kuen-Nan , Aoyagi Haruyo , Machida Keigo , Aizaki Hideki , Ou Jing-Hsiung James , Saito Takeshi
Published: January 2023

Vitamin A is an essential nutritional constituent that governs a broad spectrum of cell biology through the activation of its nuclear receptor complexes. The active metabolite of vitamin A, all-trans retinoic acid (ATRA), binds to the chaperone molecule, cytoplasmic retinoic acid binding protein (CRABP) 1 or 2 to regulate its signaling pathways. Given the extremely high homology, CRABP1 and 2 have been predicted to play a redundant role. However, a few studies by others suggested that CRABP1 and 2 might differentially regulate the cellular response to ATRA.

Although the antiviral effect of ATRA is well recognized, the roles of CRABPs in the regulation of viral pathogens have never been studied. In this study, we employed hepatitis C virus (HCV) as a study model to assess the importance of CRABPs. Of great interest, CRABP1 expression enhanced HCV infection while CRABP2 expression resulted in the suppression of HCV lifecycle. Our follow up studies collectively indicated that CRABPs regulate the efficiency of HCV infection through the modulation of lipid droplet (LD) abundance wherein HCV establishes the replicase complex.

The transcriptome analysis revealed that CRABP1 expression downregulated the genes regulated by ATRA and is associated with the expression of genes involved in the steatosis of the liver. In contrast, CRABP2 expression augmented ATRA response, resulting in the inhibition of LD accumulation. Moreover, the chemical inhibition of the CRABP1 and 2 pathways resulted in the abrogation of their pro- and antiviral effect, respectively.

In conclusion, our study revealed a novel link between the cellular response to the micronutrients and its influence on the viral infectious disease.
HIV specific B-cell memory persists in seronegative early treated children and is dominated by IgM-memory responses
Authors: Cotugno Nicola , Morrocchi Elena , Pepponi Ilaria , Rocca Salvatore , Bernardi Stefania , Rinaldi Stefano , Di Cesare Silvia , Cameron Mark J. , Pallikkuth Suresh , Rossi Paolo , Ananworanich Jintanat , Pahwa Savita , Palma Paolo
Published: January 2023

Early initiation of ART in HIV vertically infected infants influences specific immunity by limiting Ag exposure and reducing the viral reservoir size. Currently these patients represent ideal candidates for testing immune therapeutic strategies towards HIV-remission. However a proportion of these early ART treated children (ET) show an undetectable HIV Ab response. It is still unknown whether such profile is associated with a lower ability of these children to mount HIV specific responses once the Ag is re-encountered.

In the present study, we investigated whether HIV specific B-cells persist in seronegative (SN) patients, and the associated gene signatures after re-encountering the virus in vitro. We enrolled 20 ET (6 SN and 14 seropositive, SP), who initiated ART at a mean age of 4.4±3.6 mo. and under durable viral control (> 2 years). We found that gp140+ B-cells, analyzed by FACS, persist in SN patients with a similar frequency of SP. Further analysis revealed a higher percentage of HIV-specific IgM cells in SN CD27+ IgD+ memory subset compared to SP (P=0.002). In addition we investigated by multiplexed RT-PCR, gene expression dynamics after in vitro stimulation with HIV peptides mix. Our results on sorted HIV specific IgM+ cells showed up-regulation of Blimp1 expression only in SP (fold change=1.9; p=0.03), suggesting an impairment of plasma cell differentiation in IgM+gp140+ B-cells in SN patients. Our results firstly reveal that HIV specific B-cell response persists in SN ET, and predominantly resides in the IgM memory compartment. We hypothesize that such responses could be targeted by novel strategies aiming at HIV remission in HIV early treated children.
Establishing the HIV reservoir: HIV-susceptible cells and the signals that recruit them
Authors: Packard Thomas Alexander , Luo Xiaoyu , Grimmett Zachary W. , Herzig Eytan , Roan Nadia R. , Greene Warner C.
Published: January 2023

HIV infection rapidly causes localized inflammation, which contributes to viral pathogenesis by recruiting cells to become infected. At the same time, the stable HIV reservoir forms swiftly and persists over the life of the individual, likely due to infection of long-lived central memory CD4 T cells. The elimination/control of the reservoir is critical to an HIV cure. We sought to understand how host inflammatory responses to HIV drive the formation of the viral reservoir.

Our early studies found that CD4 T cells rapidly produce the chemoattractant CCL2 following HIV infection, and this host response is dependent on HIV reverse transcription but not integration of viral DNA. We proposed that CCL2 produced early in response to HIV lures cells bearing the receptor—CCR2—to the zone of infection. Using mass cytometry, we identified a novel population of human lymphoid CCR2+ CD4 T cells that co-express CCR5 and have a central memory phenotype, along with markers associated with the HIV reservoir. We found that CCR2/5+ cells migrate in response to CCL2, fuse to R5 and X4-tropic HIV and support productive and latent infection. To measure the HIV reservoir in vivo, we sorted CCR2/5+ cells from PBMCs of HIV-infected ART-suppressed individuals and measured integrated HIV proviral DNA. Our preliminary findings show that CCR2/5+ cells have significantly higher levels of integrated HIV than CCR-negative memory (12-fold, p = 0.046) or naïve cells (28-fold, p = 0.012).

We propose that HIV hijacks the host response to the virus to recruit CCR2+ cells, which may become latently infected to form the long-lived reservoir, providing a link between inflammation and reservoir seeding. We are now testing if blockade of CCL2 disrupts formation of the HIV reservoir.
T cells Protect the Brain from a Nasal Virus Infection by Engaging Local Myeloid Cells that Cross-Present Antigen
Authors: Moseman E. Ashley , Ciesinski Alexa F. , McGavern Dorian
Published: January 2023

Pathogens that gain entry into the CNS elicit a uniquely tailored response from the immune compartment by modulating cytolytic and inflammatory functions to confer viral clearance with minimal brain damage. Neurotropic viruses such as vesicular stomatitis virus (VSV) infect neurons in the olfactory epithelial barrier and pass via sensory axons directly into the brain. Within the olfactory bulb, VSV-infected neurons contact a variety of CNS neurons; however, the infection rarely invades the brain. While type I interferon responses are crucial for initial viral control, little is known about how the local adaptive immune response prevents fatal viral neuroinvasion. In this study, we sought insights into how cytotoxic lymphocytes (CTL) prevent the distal spread of infection from the olfactory bulb. Without T cells, virus containment is compromised and VSV traverses caudally to hindbrain regions. Utilizing intravital two-photon microscopy and other techniques, we characterized the mechanism by which T cell interactions within the olfactory bulb facilitate barrier protection. Interestingly, chimeric mice deficient in MHC class I on brain-resident cells, as well as microglia-depleted mice, showed reduced antiviral T cell calcium signaling upon engagement of target cells, suggesting that microglia can cross-present viral antigen and indirectly facilitate viral clearance from adjacent virally infected neurons. This study has revealed a novel mechanism of T cell-mediated viral control in neurons that involves engagement of resident myeloid cells that acquire antigen but are not infected by the virus. This in turn provides barrier protection against a virus that attempts to enter the CNS via the nasal route.
Eastern equine encephalitis virus evades induction of the host immune response through miR-142-3p restriction of myeloid cell replication
Authors: Trobaugh Derek , Sun Chengqun , Klimstra William
Published: January 2023

Eastern equine encephalitis virus (EEEV) is an alphavirus that causes the most virulent mosquito-borne disease in North America. We have previously determined that a myeloid cell-specific miRNA, miR-142-3p, binds to the 3′ non-translated region of EEEV suppressing virus replication and the induction of systemic IFN-α/β, promoting severe disease. Deletion of the miR-142-3p binding sites (virus 11337) rescues virus replication in myeloid cells, induces systemic IFN-α/β production, and leads to virus attenuation in vivo. We hypothesized that miR-142-3p restriction of EEEV replication in myeloid cells suppresses the adaptive immune response. Infection of C57BL6 mice with the myeloid tropic 11337 virus lead to increased cellularity in the popliteal lymph node (PLN) within 12 hours, predominantly comprised of CD11b+ F4/80+ Ly-6C+ inflammatory macrophages. Recruitment of inflammatory macrophages was significantly delayed in the non-myeloid tropic WT infected mice. NK cells and neutrophils were also recruited to the PLN after 11337 infection but not WT infection. In addition, 11337 infection of myeloid cells lead to increased cytokine and chemokine mRNA levels in the PLN compared to WT infection. In the spleen, 11337 infection lead to an increased frequency and number of epitope-specific CD8+ T cells compared to WT infection; however, the frequency was similar in the PLN. On day 6, epitope-specific CD8+ T cells expressed both KLRG1+ and CD127+during a WT infection while 11337 infection lead to the generation of short-lived effector cells (KLRG1+ CD127−). These results demonstrate that miR-142-3p restriction of EEEV replication in myeloid cells limits the induction of the host immune response and the generation of effector CD8+ T cells.
Tracing self-reactive B cells in normal mice
Authors: Kuraoka Masayuki , Nojima Takuya , Reynolds Alexander E. , Kitamura Daisuke , Kelsoe Garnett H.
Published: January 2023

Self-tolerance removes or inactivates autoreactive B cells in primary and secondary lymphoid organs. Studies have used transgenic mouse models or characterizing recombinant antibodies from single B cells to study B-cell tolerance. These approaches are limited, however, by the restricted diversity of transgenic B-cell repertoires or the effort necessary to generate significant numbers of recombinant antibodies. To overcome these difficulties, we used a single-cell culture approach, which supports high-throughput analysis of the B-cell repertoire in pre-tolerance, post-tolerance, and tolerizing anergic B-cell compartments. This approach revealed the affinity “setpoints” of the tolerance checkpoints, the stochastic nature of B-cell tolerance, and subsets of autoreactive B cells that are persistent in periphery. We propose that these anergic compartments may be the origin of systemic autoimmunity and a potential target for HIV-1 vaccines that elicit neutralizing antibodies cross-reactive with self-antigens.
Programmed death-1 restrains the germinal center reaction in type 1 diabetes
Authors: Martinov Tijana , Spanier Justin , Swanson Linnea , Fife Brian T.
Published: January 2023

Programmed death-1 (PD-1) is a T cell inhibitory receptor important for tolerance maintenance. PD-1 deficiency or blockade accelerates autoimmune diabetes in non-obese diabetic (NOD) mice, but the mechanism remains unclear. Autoantibody production is a hallmark of autoimmunity, and has also been reported in patients treated with PD-1 blockade, suggesting that PD-1 might regulate this process. Autoantibody production results from B cell:CD4 T cell interactions in the germinal center of the lymph node. The dynamics and regulation of the germinal center response in spontaneous autoimmunity and in the face of PD-1 deprivation are not well understood, primarily due to an inability to track self-specific lymphocytes. To bridge this knowledge gap, we used tetramers to phenotype islet-specific CD4 T cells and B cells in mice. PD-1-deficient mice, and NOD mice treated with anti-PD-1, had increased insulin autoantibodies, as well as increased insulin-specific germinal center T follicular helper CD4 T cells and germinal center B cells compared to controls. This work provides a mechanistic explanation for autoantibody onset following PD-1 blockade in the clinic, and has important implications for cancer immunotherapy and autoimmunity.
UVB induces early increases in IgG+ B cells in lupus-prone but not wild-type mice
Authors: Wolf Sonya , Theros Jonathan , Kahlenberg Michelle
Published: January 2023

At least 45% of patients with systemic lupus erythematousus (SLE) will experience photosensitivity, which is characterized by the development of cutaneous lesions following minimal sun exposure. Clinical and murine evidence support a correlation between ultraviolet (UV) light exposure and induction of systemic lupus flares, including autoantibody production, organ damage mediated by autoantibodies, and immune complex deposition. Therapies for preventing photosensitivity have remained elusive due to an incomplete understanding of the mechanisms involved. This study explores the question of how the B cell population changes after UVB exposure in a lupus prone mouse model (NZM2328) compared to wild-type (Balb/c) mice. In order to examine UVB responses, 10-week old female NZM2328 and Balb/c mice were treated on their dorsal skin with 100mJ/cm2 of UVB for 0,1, 3, or 5 days. No differences in the skin injury process (edema and scaling) were noted between the NZM2328 and Balb/c mice. Notably, however, only the NZM2328 mice showed an increase in the CD19+ IgG+ B cell population in the skin, draining lymph node and spleen within 3days. NZM2328, but not Balb/c mice also exhibited in increase in circulating anti-dsDNA IgG antibodies 2 weeks after 5 days of 100mJ UV treatment. These data suggest that NZM2328 mice have early activation of autoreactive memory B cell populations locally and systemically following UVB exposure compared to wild-type mice. This activation results in increased autoantibodies following UVB treatment. Thus, we propose memory B cell populations as important to photosensitive responses in lupus skin and suggest that targeting memory B cell activation could offer potential as a preventative treatment for SLE flares.
Regulation of Myeloid Derived Suppressor Cell (MDSC) dynamics by T regulatory cells
Authors: Tanwar Shalini , Shevach Ethan M.
Published: January 2023

Multiple sclerosis is an inflammatory demyelinating autoimmune disorder affecting the central nervous system whose severity is reduced using immune suppressive drugs. Therapeutic intervention with interferon-b reduces disease exacerbations and delays relapses. The receptor for type 1 interferon, IFNAR, is present on virtually all cell types making it difficult to dissect the mechanisms involved. We generated mice with a conditional deletion (cKO) of IFNAR in Treg cells (IFNARfl/flFoxp3cre). RNA seq analysis revealed little differences between steady state Treg cells from WT and cKO mice. Yet cKO mice developed severe EAE with an earlier onset than control mice. Although Treg cells from cKO mice appeared to be more activated, the activation status and effector cytokine production of CD4+ and CD8+ T cells in the draining lymph nodes (dLN) was similar in WT and cKO mice during the priming phase, but higher in cKO CD4 T cells in the effector phase. Interestingly, we noted a substantial reduction of myeloid derived suppressor cells (MDSCs) in the dLN of cKO mice, while generation of MDSCs in bone marrow and recruitment to spleen of WT and cKO mice was comparable. CD4+ T cells from cKO mice were found to be defective in chemokine secretion suggesting that IFNAR signaling on Treg cells modulates the capacity of CD4+ T cells to secrete MDSC recruiting chemokines during the priming phase. While modulation of Treg cell number and function by MDSCs has been documented, this study is one of the first to demonstrate that Treg cells may modulate MDSC homing and raises the possibility of a novel role for Treg cells in regulating the kinetics of MDSC recruitment during inflammatory conditions.
Fingolimod impacts innate lymphoid cell biology
Authors: Eken Ahmet , Yetkin Mehmet Fatih , Okus Fatma Zehra , Erdem Serife , Cakir Mustafa , Vural Alperen , Donmez-Altuntas Hamiyet , Mirza Meral , Canatan Halit
Published: January 2023

Group 3 Innate lymphoid cells (ILC3) resemble to Th17 cells in their production of effector cytokines and tissue localization. Th17 cells have been shown to be pathogenic in mouse MS models. Previous studies from others and our lab have shown that Sphingosine 1 phosphate 1 (S1P1) receptor impacts Th17 cell biology. S1P1 receptor agonist Fingolimod is used in the treatment of Multiple sclerosis (MS). To this day, however, the effects of fingolimod on human ILC populations have not been studied. In this study, using PBMCs from Fingolimod-receiving or control relapsing-remitting MS patients, combined with ex vivo culture system we characterized the impact of S1P1 agonists on ILC populations via Flow cytometry, real-time qPCR and ELISA. We report that human ILCs express S1P1 receptor, respond to it, as evident in reduced peripheral ILC cellularity in MS patients receiving Fingolimod. Also in culture, exposure to S1P1 agonists reduced gene expression and secretion of GMCSF and IFN-g by human ILC3 and ILC1, respectively. To our knowledge, this is the first report to study the impact of fingolimod on ILC populations in MS patients.
λ-Carrageenan initiates inflammation via activation of heterodimers TLR2/6 and TLR4/6
Authors: Lewandowski Annie , Deaver Christine M. , Myers Michael J.
Published: January 2023

λ-Carrageenan (λCGN) is frequently used to model local inflammation, however, the mechanism by which λCGN functions has not been fully examined. Swine whole blood was stimulated with λCGN and biomarkers were evaluated. Monocyte Chemotactic Protein-1(MCP-1) and Interleukin-8 protein levels were increased, while Serum Amyloid A (SAA) and Tumor Necrosis Factor-α (TNFα) protein levels were unchanged. Gene expression of MCP-1, Phosphatidylinositol 3-Kinase, and Alveolar Macrophage Chemotactic–II were increased; gene expression of SAA, Interleukin-6, and TNFα were unchanged; and gene expression of Thrombospondin-2 Precursor were decreased. Assessment of which TLRs (Toll-Like Receptors) were involved in the λCGN activation pathway, HEK-293 cells transfected with one plasmid containing either TLR2 or TLR4, along with a second plasmid to release SEAP (secreted embryonic alkaline phosphatase) upon NFκB activation were stimulated with λCGN. TLR2 and TLR4 both showed increased SEAP release after λCGN stimulation. The lack of TNFα production after λCGN stimulation suggested that it was not working through a classic TLR4 homodimer. To determine if a common mechanism could explain these results, RNA silencing studies were initiated. TLR6 was silenced in SEAP HEK-293 cells containing a plasmid for either TLR2 or TLR4. In both cell lines where TLR6 was silenced, λCGN was unable to induce a SEAP response, while these cells maintained responsiveness to a TLR1/2 ligand and a TLR 4 ligand, respectively. These results suggest that λCGN initiates inflammation via TLR2/6 and TLR4/6 heterodimers.
Nitric oxide synthase 2 augments the survival of mice upon Salmonella Typhimurium infection induced sepsis: impact on innate immune responses & organ damage
Authors: Yadav Shikha , Nandi Dipankar
Published: January 2023

Sepsis is defined as infection induced deregulation of host immune responses that may lead to multi organ dysfunction. Enhanced inflammation and persistent organ damage often leads to impaired recovery and death of sepsis patients. Thus molecules that attenuate inflammatory responses are potential therapeutic targets for sepsis. We have established intra-peritoneal S. Typhimurium infection induced peritonitis model of sepsis in mice characterized by: enhanced amounts of nitric oxide (NO), ROS, cytokines, neutrophil recruitment at the site of infection, organ damage and death of mice. Nitric oxide (NO), a highly reactive molecule has pleiotropic effects in host ranging from cell signaling and gene modulation to antimicrobial effects. Enhanced activity of Nos2 and NO is seen in sepsis patients. However, the roles of NO in pathogenesis of sepsis are controversial. Using Nos2−/− mice we attempted to elucidate the roles of Nos2 derived NO in sepsis. Upon infection Nos2−/− mice showed attenuated induction of ROS, pro-inflammatory cytokines and neutrophil recruitment. Importantly, dampened innate immune responses in Nos2−/− mice correlated with increased microbial burden, liver damage, cardiac dysfunction and reduced survival upon sepsis. Clearly Nos2 derived NO promotes protective innate immune responses during sepsis. The precise role of Nos2 derived NO was further evaluated by exogenous supplementation of Nos2−/− mice with a NO donor. Administration of exogenous NO in Nos2−/− mice beneficially restored host innate immune responses and increased survival. Our findings are crucial in the context of therapeutic interventions for sepsis and the implications for NO as a target molecule are discussed.