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AbstractChromobox 2 (CBX2),an epigenetic reader and component of polycomb repressor complex 1,is highly expressed in >75% of high-grade serous carcinoma. Increased CBX2 expression is associated with poorer survival,whereas CBX2 knockdown leads to improved chemotherapy sensitivity. In a high-grade serous carcinoma immune-competent murine model,knockdown of CBX2 decreased tumor progression. We sought to explore the impact of modulation of CBX2 on the tumor immune microenvironment (TIME),understanding that the TIME plays a critical role in disease progression and development of therapy resistance. Exploration of existing datasets demonstrated that elevated CBX2 expression significantly correlated with specific immune cell types in the TIME. RNA sequencing and pathway analysis of differentially expressed genes demonstrated immune signature enrichment. Confocal microscopy and co-culture experiments found that modulation of CBX2 leads to increased recruitment and infiltration of macrophages. Flow cytometry of macrophages cultured with CBX2-overexpressing cells showed increased M2-like macrophages and decreased phagocytosis activity. Cbx2 knockdown in the Trp53-null,Brca2-null ID8 syngeneic murine model (ID8 Trp53−/−Brca2−/−) led to decreased tumor progression compared with the control. NanoString immuno-oncology panel analysis suggested that knockdown in Cbx2 shifts immune cell composition,with an increase in macrophages. Multispectral immunohistochemistry (mIHC) further confirmed an increase in macrophage infiltration. Increased CBX2 expression leads to recruitment and polarization of protumor macrophages,and targeting CBX2 may serve to modulate the TIME to enhance the efficacy of immune therapies.Significance:CBX2 expression correlates with the TIME. CBX2 modulation shifts the macrophage population,potentially leading to an immunosuppressive microenvironment,highlighting CBX2 as a target to improve efficacy of immunotherapy. View Publication

IntroductionHeme oxygenase-1 (HO-1) is a stress-inducible heat shock protein (HSP32) that exerts cytoprotective effects against oxidative stress and inflammation,and is involved in the maintenance of cellular homeostasis. This study aimed to evaluate the expression of HO-1 in natural killer (NK) cells from individuals of different age groups after stimulation with various factors,and to analyze the relationships between the concentration of this cytoprotective protein and parameters corresponding to oxidative stress and inflammation,that is,NOD-like receptor protein 3 (NLRP3),glutathione (GSH),GSH disulfide (GSSG),and interleukin 6 (IL-6).MethodsThe study population comprised three age groups: young adults (age range,19–23 years),older adults aged under 85 years (age range,73–84 years),and older adults aged over 85 years (age range,85–92 years). NLRP3,GSH,and GSSG concentrations were measured in serum,whereas the HO-1 concentration and IL-6 expression were studied in NK cells cultivated for 48 h and stimulated with IL-2,lipopolysaccharide (LPS),or phorbol 12-myristate 13-acetate (PMA) with ionomycin.ResultsThe analysis of serum NLRP3,GSH,and GSSG concentrations revealed no statistically significant differences among the studied age groups. However,some typical trends of aging were observed,such as a decrease in GSH concentration and an increase in both GSSG level,and GSSG/GSH ratio. The highest basal expression of IL-6 and lowest basal content of HO-1 were found in NK cells of adults over 85 years of age. The NK cells in this age group also showed the highest sensitivity to stimulation with the applied factors. Moreover,statistically significant negative correlations were observed between HO-1 and IL-6 expression levels in the studied NK cells.ConclusionsThese results showed that NK cells can express HO-1 at a basal level,which was significantly increased in activated cells,even in the oldest group of adults. The reciprocal relationship between HO-1 and IL-6 expression suggests a negative feedback loop between these parameters. View Publication

Background & AimsType 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However,little is known about molecular effects of IL-13 in SPEM cells. We now sought to establish a reliable organoid model,Meta1 gastroids,to model SPEM cells in vitro. We evaluated cellular and molecular effects of ILC2s and IL-13 on maturation and proliferation of SPEM cells.MethodsWe performed single-cell RNA sequencing to characterize Meta1 gastroids,which were derived from stomachs of Mist1-Kras transgenic mice that displayed pyloric metaplasia. Cell sorting was used to isolate activated ILC2s from stomachs of IL-13-tdTomato reporter mice treated with L635. Three-dimensional co-culture was used to determine the effects of ILC2s on Meta1 gastroids. Mouse normal or metaplastic (Meta1) and human metaplastic gastroids were cultured with IL-13 to evaluate cell responses. Air-Liquid Interface culture was performed to test long-term culture effects of IL-13. In silico analysis determined possible STAT6-binding sites in gene promoter regions. STAT6 inhibition was performed to corroborate STAT6 role in SPEM cells maturation.ResultsMeta1 gastroids showed the characteristics of SPEM cell lineages in vitro even after several passages. We demonstrated that co-culture with ILC2s or IL-13 treatment can induce phosphorylation of STAT6 in Meta1 and normal gastroids and promote the maturation and proliferation of SPEM cell lineages. IL-13 up-regulated expression of mucin-related proteins in human metaplastic gastroids. Inhibition of STAT6 blocked SPEM-related gene expression in Meta1 gastroids and maturation of SPEM in both normal and Meta1 gastroids.ConclusionsIL-13 promotes the maturation and proliferation of SPEM cells consistent with gastric mucosal regeneration. Graphical Abstract View Publication

IntroductionHumanization is typically adopted to reduce the immunogenicity of murine antibodies generated by hybridoma technology when used in humans.MethodsTwo different strategies of antibody humanization are popularly employed,including “complementarity determining region (CDR) grafting” and “framework (FR) shuffling” to humanize a murine antibody against human programmed death-1 (PD-1),XM PD1. In CDR-grafting humanization,the CDRs of XM PD-1,were grafted into the human FR regions with high homology to the murine FR counterparts,and back mutations of key residues were performed to retain the antigen-binding affinities. While in FR-shuffling humanization,a combinatorial library of the six murine CDRs in-frame of XM PD-1 was constructed to a pool of human germline FRs for high-throughput screening for the most favorable variants. We evaluated many aspects which were important during antibody development of the molecules obtained by the two methods,including antibody purity,thermal stability,binding efficacy,predicted humanness,and immunogenicity,along with T cell epitope prediction for the humanized antibodies.ResultsWhile the ideal molecule was not achieved through CDR grafting in this particular instance,FR-shuffling proved successful in identifying a suitable candidate. The study highlights FR-shuffling as an effective complementary approach that potentially increases the success rate of antibody humanization. It is particularly noted for its accessibility to those with a biological rather than a computational background. DiscussionThe insights from this comparison are intended to assist other researchers in selecting appropriate humanization strategies for drug development,contributing to broader application and understanding in the field. View Publication

Activation-induced cytidine deaminase (AID) is a B cell-specific mutator required for antibody diversification. However,it is also implicated in the etiology of several B cell malignancies. Evaluating the AID-induced mutation load in patients at-risk for certain blood cancers is critical in assessing disease severity and treatment options. We have developed a digital PCR (dPCR) assay that allows us to quantify mutations resulting from AID modification or DNA double-strand break (DSB) formation and repair at sites known to be prone to DSBs. Implementation of this assay shows that increased AID levels in immature B cells increase genome instability at loci linked to chromosomal translocation formation. This includes the CRLF2 locus that is often involved in translocations associated with a subtype of acute lymphoblastic leukemia (ALL) that disproportionately affects Hispanics,particularly those with Latin American ancestry. Using dPCR,we characterize the CRLF2 locus in B cell-derived genomic DNA from both Hispanic ALL patients and healthy Hispanic donors and found increased mutations in both,suggesting that vulnerability to DNA damage at CRLF2 may be driving this health disparity. Our ability to detect and quantify these mutations will potentiate future risk identification,early detection of cancers,and reduction of associated cancer health disparities. Rates of Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) continue to rise in Hispanics and Latinos. Authors developed a digital PCR assay to quantify activation-induced cytidine deaminase activity at risk loci involved in cancer etiology that may contribute to this health disparity. View Publication

MIF is a ubiquitous protein involved in proinflammatory processes,which undergoes an oxidation-driven conformational change to oxidized (ox)MIF. We demonstrate that hypochlorous acid,produced by neutrophil-released myeloperoxidase (MPO) under inflammatory conditions,effectively oxidizes MIF into the oxMIF isoform,which is specifically recognized by the anti-oxMIF therapeutic antibody,ON104. NMR investigation of MIF oxidized by the MPO system revealed increased flexibility throughout the MIF structure,including at several catalytic and allosteric sites. Mass spectrometry of MPO-oxMIF revealed methionines as the primary site of oxidation,whereas Pro2 and Tyr99/100 remained almost unmodified. ELISA,SPR and cell-based assays demonstrated that structural changes caused by MPO-driven oxidation promoted binding of oxMIF to its receptor,CD74,which does not occur with native MIF. These data reveal the environment and modifications that facilitate interactions between MIF and its pro-inflammatory receptor,and a route for therapeutic intervention targeting the oxMIF isoform. View Publication

Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways. However,targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here,we have developed an mRNA therapeutic for the lower lung using one-component Ionizable Amphiphilic Janus Dendrimers (IAJDs) as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA,transforming growth factor-beta (TGF-β),to produce transient protein expression in the lower regions of the lung. This study highlights IAJD’s potential for precise,effective,and safe delivery of TGF-β mRNA to the lung. This delivery system offers a promising approach for targeting therapeutics to the specific tissues,a strategy necessary to fill the current clinical gap in treating parenchymal lung injury and disease. View Publication

Trained immunity may play a role in vaccine-induced protection against infections. We showed that the highly efficacious recombinant VZV-gE zoster vaccine (RZV) generated trained immunity in monocytes,natural killer (NK) cells,and dendritic cells (DCs) and that the less efficacious live zoster vaccine did not. RZV stimulated ex vivo gE-specific monocyte,DC and NK cell responses that did not correlate with CD4 + T-cell responses. These responses were also elicited in purified monocyte and NK cell cocultures stimulated with VZV-gE and persisted above prevaccination levels for ≥ 4 years post-RZV administration. RZV administration also increased ex vivo heterologous monocyte and NK cell responses to herpes simplex and cytomegalovirus antigens. ATAC-seq analysis and ex vivo TGFβ1 supplementation and inhibition experiments demonstrated that decreased tgfβ1 transcription resulting from RZV-induced chromatin modifications may explain the development of monocyte trained immunity. The role of RZV-trained immunity in protection against herpes zoster and other infections should be further studied. View Publication

Background & AimsThe immune tolerance induced by hepatitis B virus (HBV) is a major challenge for achieving effective viral clearance,and the mechanisms involved are not well-understood. One potential factor involved in modulating immune responses is mesencephalic astrocyte-derived neurotrophic factor (MANF),which has been reported to be increased in patients with chronic hepatitis B. In this study,our objective is to examine the role of MANF in regulating immune responses to HBV.MethodsWe utilized a commonly used HBV-harboring mouse model,where mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. We assessed the HBV load by measuring the levels of various markers including hepatitis B surface antigen,hepatitis B envelope antigen,hepatitis B core antigen,HBV DNA,and HBV RNA.ResultsOur study revealed that following HBV infection,both myeloid cells and hepatocytes exhibited increased expression of MANF. Moreover,we observed that mice with myeloid-specific MANF knockout (ManfMye-/-) displayed reduced HBV load and improved HBV-specific T cell responses. The decreased HBV-induced tolerance in ManfMye-/- mice was associated with reduced accumulation of myeloid-derived suppressor cells (MDSCs) in the liver. Restoring MDSC levels in ManfMye-/- mice through MDSC adoptive transfer reinstated HBV-induced tolerance. Mechanistically,we found that MANF promoted MDSC expansion by activating the IL-6/STAT3 pathway. Importantly,our study demonstrated the effectiveness of a combination therapy involving an hepatitis B surface antigen vaccine and nanoparticle-encapsulated MANF siRNA in effectively clearing HBV in HBV-carrier mice.ConclusionThe current study reveals that MANF plays a previously unrecognized regulatory role in liver tolerance by expanding MDSCs in the liver through IL-6/STAT3 signaling,leading to MDSC-mediated CD8+ T cell exhaustion. Graphical abstract View Publication

Mitochondria react to infection with sub-lethal signals in the apoptosis pathway. Mitochondrial signals can be inflammatory but mechanisms are only partially understood. We show that activation of the caspase-activated DNase (CAD) mediates mitochondrial pro-inflammatory functions and substantially contributes to host defense against viral infection. In cells lacking CAD,the pro-inflammatory activity of sub-lethal signals was reduced. Experimental activation of CAD caused transient DNA-damage and a pronounced DNA damage response,involving major kinase signaling pathways,NF-κB and cGAS/STING,driving the production of interferon,cytokines/chemokines and attracting neutrophils. The transcriptional response to CAD-activation was reminiscent of the reaction to microbial infection. CAD-deficient cells had a diminished response to viral infection. Influenza virus infected CAD-deficient mice displayed reduced inflammation in lung tissue,higher viral titers and increased weight loss. Thus,CAD links the mitochondrial apoptosis system and cell death caspases to host defense. CAD-driven DNA damage is a physiological element of the inflammatory response to infection. View Publication