Unmethylated CpG Research Articles

ROLE OF TOLL-LIKE RECEPTOR POLYMORPHISMS IN THE DEVELOPMENT OF EXTREMELY SEVERE COVID-19 WITH FATAL OUTCOME

COVID-19 is a respiratory disease caused by the Sars-Cov-2 virus that can proceed to acute respiratory distress syndrome (ARDS) and trigger immunopathological mechanisms that lead to excessive inflammation. Toll-like receptors, as pattern recognition receptors, play an indispensable role in virus identification and activate the innate immune system, which can lead to the secretion of proinflammatory cytokines such as IL-1, IL-6, TNFα, and type I IFN. Antiviral toll-like receptors, including TLR3, TLR7, and TLR9, can activate the TRIF-dependent pathway, induce the production of proinflammatory cytokines, chemokines, and type I and type III interferons, participate in recognition of dsRNA and detection of unmethylated CpG DNA. In addition, an important role of TLR2 in COVID-19, which recognizes peptide glycans on the surface of gram-positive bacteria, and TLR4, which recognizes gram-negative bacteria lipopolysaccharide and plays a role in hyperinflammation in COVID-19, has been shown. It was confirmed that TLRs can be involved both in the initial failure of virus clearance and in the subsequent development of severe COVID-19 clinical manifestations mainly acute respiratory distress syndrome (ARDS) with fatal respiratory failure. The aim of this work was to study the polymorphisms of TLR3 (rs3775291), TLR9 (rs352140), TLR7 (rs3853839), TLR4 (rs4986790), TLR4 (rs4986791), TLR2 (rs574708) in patients with COVID-19 depending on disease outcome. The study included 187 patients, of whom 157 recovered and 30 deceased. Genetic analysis for polymorphisms of the TLR3 (rs3775291), TLR9 (rs352140), TLR7 (rs3853839), TLR4 (rs4986790), TLR4 (rs4986791), TLR2 (rs574708) genes was performed by real-time PCR. Statistical processing of the obtained results was performed using STATISTICA 12.0 (USA) software. The inter-group difference was considered statistically significant at p 0.05. The strength of associations was estimated and presented as odds ratio (OR) and 95% confidence interval. It was noted that the genotypes GC TLR7 (rs3853839), AG TLR2 (rs574708), GG TLR4 (rs4986790), TT TLR4 (rs4986791) can be predictors of a fatal COVID-19 outcome. On the contrary, the genotypes GG TLR7 (rs3853839), AA TLR2 (rs574708), AA TLR4 (rs4986790), CC TLR4 (rs4986791) can be protective and contribute to the recovery of patients.

Abstract A035: A real-time PCR method for the detection of cancer-specific methylation patterns in cfDNA

Abstract Non-invasive liquid biopsy tests are proving to be the next frontier in cancer diagnostics with methylation emerging as a reliable biomarker for low levels of circulating tumor DNA (ctDNA). There is a rising interest in sensitive, low-cost liquid biopsy assays to detect low copies of hypermethylated ctDNA in an excess background of non-cancer-associated hypomethylated cell- free DNA (cfDNA). Real-time quantitative methylation-specific PCR (qMSP) allows for the detection of low abundance methylated fragments. qMSP approaches have previously been developed but are predominantly designed to target a specific CpG site. To date we are not aware of any qMSP approaches that target pan-cancer-associated methylation patterns across multiple CpG sites. Harbinger Health has developed a qMSP method with locked nucleic acid (LNA) blockers that target methylation haplotypes, allowing for multiplex enrichment and quantification of cancer-specific methylation patterns. Using targeted bisulfite sequencing, we identified ten pan-cancer-informative CpG-dense regions which showed consistent contiguous elevated methylation states in cancer cfDNA and low levels of methylation in non-cancer cfDNA. We designed qMSP assays for these regions and LNA blockers which bind to unmethylated target CpG sites, outcompeting the hydrolysis probe through a high Tm differential. This prevented non-specific probe hybridization. The qMSP-LNA assays were evaluated in simplex and multiplex using cell line DNA in a titration of hypermethylated “ctDNA” spiked into hypomethylated “cfDNA” to simulate varying ctDNA levels, and then validated with cancer and non-cancer cfDNA samples at 5 ng input, equivalent to 1450 haploid copies. The percentage of methylated DNA was estimated by the ratio of methylated copies (qMSP-LNA) to total copies (internal reference), which were quantified by standard curves. The qMSP-LNA assays detected low abundance methylated DNA fragments down to 23 target copies. LNA blockers completely depleted background signal originating from unmethylated DNA with high specificity, showing no inhibition of methylated DNA detection. When tested in cfDNA samples which varied in conversion efficiency, discordant methylation states, and methylation abundance, the assays reproducibly amplified and detected highly methylated fragments which correspond to cancer signal. Harbinger Health has developed qMSP-LNA assays for contiguous CpG biomarkers which differentiate cancer and non-cancer samples from only ten pan-cancer-informative regions. The qMSP-LNA method detected low abundance fragments with specific methylation patterns with high specificity and sensitivity. The assay reproducibly detected methylated DNA in cfDNA samples that represent a clinically relevant range of methylation signal and will be further validated in a larger cohort of cancer and non-cancer cfDNA samples. Due to the high specificity and sensitivity, this cost-effective PCR method may provide utility and improve access for early cancer detection and monitoring. Citation Format: Emily Neaga, Caitlin Gilley, Sarah Falotico, Mayur Gurnani, Zhenyu Zhang, Jocelyn Charlton, Miguel Williams, Anthony Shuber. A real-time PCR method for the detection of cancer-specific methylation patterns in cfDNA [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A035.

Abstract A014: A methylation state specific targeted background depletion technique for enrichment of ctDNA fraction

Abstract Liquid biopsy continues to be a focus of the cancer diagnostics industry, primarily due to the non-invasive nature of sample collection, however the low levels of circulating tumor DNA (ctDNA) remain a challenge. Methylation has emerged as a promising biomarker for detecting ctDNA, yet detecting the cancerous signal in an excess of non-cancerous, unmethylated cfDNA in the background has been proven difficult. Harbinger Health has developed a novel method that depletes unmethylated background at the CpG-level from specific genomic regions of interest, resulting in an enrichment of the ctDNA fraction. We developed a background cfDNA depletion method that utilizes the endonuclease activity and the specificity of CRISPR/Cas12a to remove unmethylated DNA fragments at ctDNA methylation biomarker sites. First, we identified cancer-informative regions that showed consistent contiguous elevated methylation states in cancer compared to normal samples. We then designed guide RNAs that specifically bind and cut unmethylated CpG sites within the target regions. Cas depletion of bisulfite-converted DNA enriches cancer informative signal for bioanalysis. To test validity of our Cas complex designs, we created model samples that mimic fully unmethylated and fully methylated species. We spiked these cfDNA models into a background of genomic DNA from 5% to 0.01%. In triplicate, we examined cutting efficiency, multiplexing and specificity of Cas to unmethylated species of model DNA. Results were assessed using qPCR by comparing differences in Ct with and without Cas treatment. Additionally, we applied this depletion step during our library preparation process to show utility in methylation analysis of low tumor content liquid biopsy samples. Using both the model sample dilution series and bisulfite converted cfDNA samples, which represent a range of cancerous and background methylation signal, we deplete unmethylated targets using Cas prior to indexing PCR. This rendered unmethylated targets non-amplifiable, removing them from analysis. Results were assessed using both qPCR and sequencing data, such as methylated read counts over the target region. We demonstrated our analytical approach efficiently depletes non-cancer cfDNA by on average 9-fold, which increases the fraction of ctDNA signal and can be multiplexed. This background signal depletion driven by CRISPR is sensitive and specific, maintaining methylated DNA signal detection down to 0.01% at inputs of 10ng DNA. Our Cas-mediated depletion of background signal method has proven useful for enriching rare methylated fragments over background by improving the signal to noise ratio. This technique is novel by harnessing the specificity of CRISPR to target DNA in both a sequence and methylation state specific manner. The depletion of precise methylation states at the CpG-level make it suitable for use in multiple bioassays. Citation Format: Caitlin Gilley, Miguel Williams, Emily Neaga, Sarah Falotico, Kade Pettie, Anthony Shuber. A methylation state specific targeted background depletion technique for enrichment of ctDNA fraction [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A014.

Deletion of mitochondrial DNA methyltransferase 1 contributes the development of murine heart failure under pressure overloaded

Abstract Background Mitochondrial DNA (mitDNA) has similarities to bacterial DNA, which contains inflammatogenic unmethylated CpG motifs. We found that mitDNA that escapes from autophagy causes inflammatory response and heart failure under pressure overload. DNA methyltransferase 1 (DNMT1) maintains methylation patterns in somatic cells. Deletion of DNMT1 results in embryonic lethality in mice and mitotic catastrophe in cultured cells. Additional possible translational start sites (ATG1, ATG2) are located upstream of the commonly accepted nuclear DNMT1 translational start site (ATG3) and there is a conserved mitochondrial translocation signal sequence between ATG1 and ATG3. The role of mitochondrial DNMT1 in the development of heart failure remains to be elucidated. Purpose The purpose of this study is to examine the in vivo role of mitochondrial DNMT1 in the heart under pressure overload. Methods We generated HA-tagged DNMT1 constructs, 1) starting from ATG1 (termed as whole) 2) from ATG3 (nuclear) and 3) from ATG3 and mutated in the nuclear translocation sequence (mitochondrial) and transfected HEK293 cells with the constructs. Localization of HA-tagged proteins was assessed by immunocytochemistry. We also generated mitochondrial DNMT1-deficient mice by inserting mutations into the ATG1 and ATG2. The mice were subjected to pressure overload by means of transverse aortic constriction (TAC) to induce heart failure. Cardiac remodelling was assessed by echocardiography as well as histological analyses 4 weeks after operation. Results Whole, nuclear and mitochondrial DMNT1 translocated to both mitochondria and nuclei, only nucleus and only mitochondria, respectively. Mitochondrial DNMT1-deficient mice were born at Mendelian frequency and grew to adulthood. Those showed no cardiac phenotypes under baseline conditions. However, the mice exhibited severe systolic dysfunction, indicated by left ventricular fractional shortening, 2 weeks after surgery compared with control littermates (control littermates 47.9% versus Mitochondrial DNMT1-deficient mice 32.3%, p<0.05). Intermuscular cell infiltration was observed in TAC-operated mitochondrial DNMT1-deficient hearts. Conclusions Mitochondrial DNA methyltransferase 1 plays a protective role in failing hearts and mitDNA methylation might be a therapeutic target to treat patients with heart failure.

MethylSeqLogo: DNA methylation smart sequence logos

BackgroundSome transcription factors, MYC for example, bind sites of potentially methylated DNA. This may increase binding specificity as such sites are (1) highly under-represented in the genome, and (2) offer additional, tissue specific information in the form of hypo- or hyper-methylation. Fortunately, bisulfite sequencing data can be used to investigate this phenomenon.MethodWe developed MethylSeqLogo, an extension of sequence logos which includes new elements to indicate DNA methylation and under-represented dimers in each position of a set binding sites. Our method displays information from both DNA strands, and takes into account the sequence context (CpG or other) and genome region (promoter versus whole genome) appropriate to properly assess the expected background dimer frequency and level of methylation. MethylSeqLogo preserves sequence logo semantics—the relative height of nucleotides within a column represents their proportion in the binding sites, while the absolute height of each column represents information (relative entropy) and the height of all columns added together represents total informationResultsWe present figures illustrating the utility of using MethylSeqLogo to summarize data from several CpG binding transcription factors. The logos show that unmethylated CpG binding sites are a feature of transcription factors such as MYC and ZBTB33, while some other CpG binding transcription factors, such as CEBPB, appear methylation neutral.ConclusionsOur software enables users to explore bisulfite and ChIP sequencing data sets—and in the process obtain publication quality figures.

Structural basis of DNA recognition by BEN domain proteins reveals a role for oligomerization in unmethylated DNA selection by BANP.

The BEN domain is a newly discovered type of DNA-binding domain that exists in a variety of species. There are nine BEN domain-containing proteins in humans, and most have been shown to have chromatin-related functions. NACC1 preferentially binds to CATG motif-containing sequences and functions primarily as a transcriptional coregulator. BANP and BEND3 preferentially bind DNA bearing unmethylated CpG motifs, and they function as CpG island-binding proteins. To date, the DNA recognition mechanism of quite a few of these proteins remains to be determined. In this study, we solved the crystal structures of the BEN domains of NACC1 and BANP in complex with their cognate DNA substrates. We revealed the details of DNA binding by these BEN domain proteins and unexpectedly revealed that oligomerization is required for BANP to select unmethylated CGCG motif-containing DNA substrates. Our study clarifies the controversies surrounding DNA recognition by BANP and demonstrates a new mechanism by which BANP selects unmethylated CpG motifs and functions as a CpG island-binding protein. This understanding will facilitate further exploration of the physiological functions of the BEN domain proteins in the future.

High amphipathicity of α-helical peptides enhances unmethylated CpG DNA-induced activation of mouse macrophage-like RAW264.7 cells

The α-helical antimicrobial peptide Kn2-7 enhances the activation of mouse macrophage-like RAW264.7 induced by DNA containing unmethylated cytosine-guanine motifs (CpG DNA). This enhancement is related to increased cellular uptake of DNA by Kn2-7, but the relevant properties of Kn2-7 are unknown. Physicochemical property analysis revealed that Kn2-7 has high amphipathicity. In contrast, the α-helical antimicrobial peptide L5, which increases the cellular uptake of CpG DNA but does not enhance CpG DNA-induced activation, has low amphipathicity. Kn2-7 derivatives with decreased amphipathicity but the same amino acid composition as Kn2-7 did not enhance CpG DNA-induced activation. On the other hand, L5 derivatives with high amphipathicity but the same amino acid composition as L5 enhanced CpG DNA-induced activation. Cellular uptake of DNA was not increased by the L5 derivatives, indicating that high amphipathicity does not affect DNA uptake. Furthermore, α-helical peptides with reversed sequences relative to the Kn2-7 and L5 derivatives with high amphipathicity were synthesized. The reversed-sequence peptides, which had the same amphipathicity but different amino acid sequences from their counterparts, enhanced CpG DNA-induced activation. Taken together, these observations indicate that the high amphipathicity of α-helical peptides enhances the CpG DNA-induced activation of RAW264.7.

Recognition of Chlamydia trachomatis by Toll-like receptor 9 is altered during persistence.

Toll-like receptor 9 (TLR9) is an innate immune receptor that localizes to endosomes in antigen presenting cells and recognizes single stranded unmethylated CpG sites on bacterial genomic DNA (gDNA). Previous bioinformatic studies have demonstrated that the genome of the human pathogen Chlamydia trachomatis contains TLR9 stimulatory motifs, and correlative studies have implied a link between human TLR9 (hTLR9) genotype variants and susceptibility to infection. Here, we present our evaluation of the stimulatory potential of C. trachomatis gDNA and its recognition by hTLR9- and murine TLR9 (mTLR9)-expressing cells. Utilizing reporter cell lines, we demonstrate that purified gDNA from C. trachomatis can stimulate hTLR9 signaling, albeit at lower levels than gDNA prepared from other Gram-negative bacteria. Interestingly, we found that while C. trachomatis is capable of signaling through hTLR9 and mTLR9 during live infections in HEK293 reporter cell lines, signaling only occurs at later developmental time points. Chlamydia-specific induction of hTLR9 is blocked when protein synthesis is inhibited prior to the RB-to-EB conversion, exacerbated by the inhibition of lipooligosaccharide biosynthesis, and is significantly altered during the induction of aberrance/persistence. Our observations support the hypothesis that chlamydial gDNA is released during the conversion between the pathogen's replicative and infectious forms and during treatment with antibiotics targeting peptidoglycan assembly. Given that C. trachomatis inclusions do not co-localize with TLR9-containing vacuoles in the pro-monocytic cell line U937, our findings also hint that chlamydial gDNA is capable of egress from the inclusion, and traffics to TLR9-containing vacuoles via an as yet unknown pathway.

Antimetastatic effect of intratumoral Treg antagonists in mice with renal cancer.

The interplay of regulatory T cells (Tregs) within the tumour microenvironment presents a significant challenge in anticancer immunotherapy. This study investigates the potential of Treg blockade to enhance the efficiency of effector T cells. Two distinct treatment cocktails were examined: 3p-hpRNA (5' triphosphate hairpin RNA) combined with unmethylated CpG oligonucleotide (CpG); CpG in combination with OX40 receptor-specific monoclonal antibody (anti-OX40). Treatment efficacy was assessed using a murine model of kidney adenocarcinoma.Renca cells (renal cortical cells with adenocarcinoma) were subcutaneously engrafted in 30 BALB/c mice, then animals were allocated into three treatment groups: Group 1: CpG+anti-OX40, Group 2: CpG+3p-hpRNA, Group 3: untreated control. Treatment efficacy was evaluated based on tumour growth, the occurrence of metastases and overall survival.On day 28 post-implantation, experiments had to be terminated due to tumour progression. Although comparisons of survival times and primary tumour sizes thus became inconsequential, histological examinations provided valuable insights. We observed distinct variations in primary tumour characteristics among the different groups: Groups 1 and 2 displayed demarcations, while Group 3 exhibited diffuse tumours with necrosis. Lung metastases were evident in 70% of untreated mice, whereas none were observed in either of the treated groups.Our findings instil confidence in the potential efficacy of the treatments, thereby laying a solid foundation for future investigations.

TLR21 is involved in the NF-κB and IFN-β pathways in largemouth bass (Micropterus salmoides) and interacts with TRIF but not with the Myd88 adaptor

Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-β (IFN-β) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-β (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.

Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson–Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides—crucial elements for ASO therapeutics under development—have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5′-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Improvement of the Nuclease Resistance and Immunostimulatory Activity of CpG Oligodeoxynucleotides by Conjugation to Sugar-Immobilized Gold Nanoparticles.

Adjuvants are essential substances for vaccines and immunotherapies that enhance antigen-specific immune responses. Single-stranded oligodeoxynucleotides containing an unmethylated CpG motif (CpG ODNs) are agonistic ligands for toll-like receptor 9 that initiate an innate immune response. They represent promising adjuvants for antiviral and antitumor immunotherapies; however, CpG ODNs have some limitations, such as poor nuclease resistance and low cell membrane permeability. Therefore, an effective formulation is needed to improve the nuclease resistance and immunostimulatory effects of CpG ODNs. Previously, we demonstrated the selective delivery of a small molecule toll-like receptor 7 ligand to immune cells through sugar-binding receptors using sugar-immobilized gold nanoparticles (SGNPs), which significantly enhanced the potency of the ligand. In this study, we examined SGNPs as carriers for partially phosphorothioated A-type CpG ODN (D35) and an entirely phosphorothioated B-type CpG ODN (K3) and evaluated the functionality of the sugar moiety on SGNPs immobilized with CpG ODN. SGNPs immobilized with D35 (D35-SGNPs) exhibited improved nuclease resistance and the in vitro and in vivo potency was significantly higher compared with that of unconjugated D35. Furthermore, the sugar structure on the GNPs was a significant factor in enhancing the cell internalization ability, and enhanced intracellular delivery of D35 resulted in improving the potencies of the A-type CpG ODN, D35. SGNPs immobilized with K3 (K3-SGNPs) exhibited significantly higher induction activities for both humoral and cellular immunity compared with unconjugated K3 and D35-SGNPs. On the other hand, sugar structure on K3-SGNPs did not affect the immunostimulatory effects. These results indicate that the sugar moiety on K3-SGNPs primarily functions as a hydrophilic dispersant for GNPs and the formulation of K3 to SGNPs contributes to improving the immunostimulatory activity of K3. Because our CpG ODN-SGNPs have superior induction activities for antigen-specific T-cell mediated immune responses, they may be effective adjuvants for vaccines and immunotherapies.

CXXC5 drove inflammation and ovarian cancer proliferation via transcriptional activation of ZNF143 and EGR1

CXXC5, a zinc-finger protein, is known for its role in epigenetic regulation via binding to unmethylated CpG islands in gene promoters. As a transcription factor and epigenetic regulator, CXXC5 modulates various signaling processes and acts as a key coordinator. Altered expression or activity of CXXC5 has been linked to various pathological conditions, including tumorigenesis. Despite its known role in cancer, CXXC5's function and mechanism in ovarian cancer are unclear. We analyzed multiple public databases and found that CXXC5 is highly expressed in ovarian cancer, with high expression correlating with poor patient prognosis. We show that CXXC5 expression is regulated by oxygen concentration and is a direct target of HIF1A. CXXC5 is critical for maintaining the proliferative potential of ovarian cancer cells, with knockdown decreasing and overexpression increasing cell proliferation. Loss of CXXC5 led to inactivation of multiple inflammatory signaling pathways, while overexpression activated these pathways. Through in vitro and in vivo experiments, we confirmed ZNF143 and EGR1 as downstream transcription factors of CXXC5, mediating its proliferative potential in ovarian cancer. Our findings suggest that the CXXC5-ZNF143/EGR1 axis forms a network driving ovarian cell proliferation and tumorigenesis, and highlight CXXC5 as a potential therapeutic target for ovarian cancer treatment.

An innate immune response to adeno-associated virus genomes decreases cortical dendritic complexity and disrupts synaptic transmission

Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery to the nervous system, are widely used in neuroscience research, and are the basis of FDA-approved neuron-targeting gene therapies. Here we find that an innate immune response to the AAV genome reduces dendritic length and complexity and disrupts synaptic transmission in mouse somatosensory cortex. Dendritic loss is apparent three weeks after injection of experimentally relevant viral titers, is not restricted to a particular capsid serotype, transgene, promoter, or production facility, and cannot be explained by responses to surgery or transgene expression. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents and an increase in the proportion of GluA2-lacking, calcium permeable AMPA receptors. The AAV genome is rich in unmethylated CpG DNA, which is recognized by the innate immunoreceptor Toll-like receptor 9 (TLR9), and acutely blocking TLR9 preserves dendritic complexity and AMPA receptor subunit composition in AAV-injected mice. These results reveal unexpected impacts of an immune response to the AAV genome on neuronal structure and function, and identify approaches to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system.

Type I interferon promotes the fate of Toll-like receptor 9-stimulated follicular B cells to plasma cell differentiation.

The activation and differentiation of B cells into plasma cells (PCs) play critical roles in the immune response to infections and autoimmune diseases. Toll-like receptor 9 (TLR9) responds to bacterial and viral DNA containing unmethylated CpG motifs and triggers immune responses in B cells; however, abnormal recognition of self-DNA by TLR9 can cause autoimmune diseases. When stimulated with TLR9 agonists, follicular (FO) B cells, a subset of B cells residing in the FO regions of secondary lymphoid organs, exhibit a propensity for activation but fail to give rise to PCs. The factors that enable the transition of TLR9-activated FO B cells from activation to differentiation into PCs remain unclear. In this study, we show that type I interferon-alpha (IFNα) signaling causes FO B cells activated by CpG stimulation to differentiate into PCs. Although CpG stimulation alone only temporarily increased interferon regulatory factor 4 (IRF4) expression in FO B cells, co-stimulation with both CpG and IFNα enhanced and maintained high IRF4 expression levels, ultimately enabling the cells to differentiate into PCs. Overexpression of IRF4 in FO B cells results in CpG-induced PC transition without IFN signaling. Furthermore, co-stimulation of TLR9 and IFNα receptors significantly enhanced mammalian target of rapamycin (mTOR) signaling, which regulates IRF4 expression and PC generation. These findings suggest that IFNα may play a key role in promoting the fate of PC differentiation in FO B cells activated by TLR9 stimulation.

Abstract 6746: A B-cell targeted TLR9 agonist antibody conjugate potentiates cancer vaccine efficacy and rejuvenates vaccine responses in the elderly

Abstract TLR9 agonists (unmethylated CpG oligodeoxynucleotides) are clinically validated as vaccine adjuvants and cancer therapeutics1,2. We developed a Toll-like Receptor Agonist Antibody Conjugate (TRAAC) comprised of a potent TLR9 agonist (T-CpG) conjugated to an antibody against CD22, a receptor restricted to B cells (TAC-001). Systemically administered TAC-001 delivers T-CpG to B cells, resulting in TLR9 activation, and innate and adaptive anti-tumor immunity3. TAC-001 is being evaluated as an immunotherapy in a Phase 1 clinical trial in patients with advanced solid tumors (NCT05399654)4. In this study, we investigated whether TAC-001 mouse surrogate, mCD22 TRAAC, can potentiate cancer vaccine efficacy using preclinical vaccination models. Human Nectin-4 ectodomain protein (N4, 94% homology to its mouse ortholog) was used as a neoantigen-like vaccine, alone and in combination with mCD22 TRAAC, MPLA (a TLR4 agonist), or unconjugated T-CpG. Mice were immunized, then challenged 2 months later with syngeneic CT26 tumors expressing N4. Mice in treatment combination cohorts that resisted tumor development were split into two groups and rechallenged 3.5 months later with a more aggressive tumor, syngeneic 4T1 or 4T1 expressing N4. The combination of vaccine with mCD22 TRAAC was the only treatment able to inhibit growth of the 4T1-N4 tumor. No efficacy was observed in mice inoculated with parental 4T1, thereby demonstrating neoantigen vaccine specific anti-tumor immunity. An MHCI restricted peptide of HER2/neu, GP2, fused to a B cell epitope peptide, P4, and conjugated to KLH, was used as another vaccine model5. GP2-P4-KLH was administered to mice alone or in combination with mCD22 TRAAC or unconjugated T-CpG. Ex vivo GP2/GP2-P4 stimulation of lymphoid organ immune cells from mice treated with vaccine plus mCD22 TRAAC resulted in elevated Granzyme B, IFNγ and IL17A levels, indicating that mCD22 TRAAC enhances T cell activation and cytotoxic activity. Lastly, to investigate the effect of mCD22 TRAAC in elderly mice, SARS-CoV-2 spike protein was used as a vaccine. Spike was administered alone or in combination with mCD22 TRAAC, MPLA or T-CpG. Combining mCD22 TRAAC with vaccine resulted in high levels of spike neutralizing IgG in both youth and elderly cohorts, showing that mCD22 TRAAC rejuvenates vaccine responses in elderly. In all studies, mCD22 TRAAC led to the most potent vaccine recall response, with high antigen specific IgG titers skewing towards a Th1 phenotype, as shown by elevated levels of effector function antibodies of IgG2a subclass and increased IFNγ production. These findings provide a strong rationale for combining TAC-001 with cancer vaccines. References1Hyer et al., Vaccine 2018; 36(19) 2Long et al., Annals of Oncology 2018; 29(8) 3Kuo et al., Cancer Research 2021; 81(13)4Perez et al., JITC 2023; 11(1) 5Nordin et al., Cancers 2021; 13(19) Citation Format: Maja Z. Bonacorsi, Amy Chen, Ons Harrabi, Min Li, Emma R. Sangalang, Danielle Fontaine, Janet Sim, Pavel Strop, Hong I. Wan, Maria Jose Costa. A B-cell targeted TLR9 agonist antibody conjugate potentiates cancer vaccine efficacy and rejuvenates vaccine responses in the elderly [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6746.

SPINK5 inhibits esophageal squamous cell carcinoma metastasis via immune activity.

Esophageal squamous cell carcinoma (ESCC) is a predominant subtype of esophageal cancer with relatively high mortality worldwide. Serine peptidase inhibitor Kazal-type 5 (SPINK5) is reported to be downregulated in ESCC. However, its explicit role in ESCC remains further investigation. The tumor tissues and adjacent non-cancerous tissues were obtained from 196 patients with ESCC for the determination of SPINK5 mRNA levels. Additionally, the relationship between SPINK5 mRNA levels and clinicopathological features of ESCC patients was explored. The effects of SPINK5 on the invasion and migration of ESCC cells were assessed using Transwell assays. Furthermore, SPINK5 mRNA and LEKTI protein were measured in ESCC cell lines after treatment with poly (I:C), lipopolysaccharide (LPS) or unmethylated CpG DNA. Moreover, the correlation between expression of SPINK5 and nuclear factor-kappa B (NF-κB) signaling pathway-related genes was analyzed in the TCGA-ESCC cohort, and the effects of SPINK5 on NF-κB transcription was analyzed using a luciferase reporter gene assay. Finally, the correlations between SPINK5 and infiltration of immune cells, immune scores, stromal scores and ESTIMATE (i.e., Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data) scores were explored. SPINK5 mRNA levels were downregulated in tumor tissues, which was significantly correlated with higher lymph node metastases. Overexpressed SPINK5 inhibited cell invasion and migration in ESCC cell lines. Mechanistically, LPS-induced activation of Toll-like receptor 4 (TLR4) decreased SPINK5 mRNA and LEKTI in KYSE150 and KYSE70 cells. Spearman correlation analysis revealed that SPINK5 mRNA was significantly negatively correlated with a total of seven NF-κB signaling pathway-related genes in TCGA-ESCC patients. Moreover, downregulation of SPINK5 increased and upregulation of SPINK5 decreased the activity of the NF-κB promoter in HEK293T cells. Finally, immune cells infiltration analysis revealed that SPINK5 was significantly correlated with the infiltration of various immune cells, stromal scores, immune scores and ESTIMATE scores. SPINK5 plays critical roles in the TLR4/NF-κB pathway and immune cells infiltration, which might contribute to the ESCC metastasis. The findings of the present study may provide a promising biomarker for the diagnosis and treatment of esophageal squamous cell carcinoma.

Light-Activatable MBD-Readers of 5-Methylcytosine Reveal Domain-Dependent Chromatin Association Kinetics In Vivo.

5-Methylcytosine (5mC) is the central epigenetic mark of mammalian DNA, and plays fundamental roles in chromatin regulation. 5mC is dynamically read and translated into regulatory outputs by methyl-CpG-binding domain (MBD) proteins. These multidomain readers recognize 5mC via an MBD domain, and undergo additional domain-dependent interactions with multiple additional chromatin components. However, studying this dynamic process is limited by a lack of methods to conditionally control the 5mC affinity of MBD readers in cells. Light-control of MBD association to chromatin by genetically encoding a photocaged serine at the MBD-DNA interface is reported. The authors study the association of MBD1 to mouse pericentromeres, dependent on its CxxC3 and transcriptional repressor domains (TRD) which interact with unmethylated CpG and heterochromatin-associated complexes, respectively. Both domains significantly modulate association kinetics, arguing for a model in which the CxxC3 delays methylation responses of MBD1 by holding it at unmethylated loci, whereas the TRD promotes responses by aiding heterochromatin association is studied. Theirapproach offers otherwise inaccessible kinetic insights into the domain-specific regulation of a central MBD reader, and sets the basis for further unravelling how the integration of MBDs into complex heterochromatin interaction networks control the kinetics of 5mC reading and translation into altered chromatin states.

10125-GGE-10 EVALUATION OF MGMT PROMOTER METHYLATION STATUS OF GBM CELL LINE CLONAL POPULATION.

Abstract In this study, the epigenetic heterogeneity of GBM was investigated by evaluating the methylation status of the O6-methylguanine methyltransferase (MGMT) promoter in individual clones of a single cell derived from GBM cell lines. Cell lines, U251 and U373, were used for the experiments from the Brain Tumour Research Centre of the Montreal Neurological Institute. To evaluate the methylation status of the MGMT promoter, pyrosequencing and methylation-specific PCR (MSP) were used. A total of 12 U251 and 12 U373 clones were isolated. The methylation status of 83 of 97 CpG sites in the MGMT promoter were evaluated by pyrosequencing, and 11 methylated CpG sites and 13 unmethylated CpG sites were evaluated by MSP. The results demonstrate tumor heterogeneity among individual clones derived from a single GBM cell.

Abstract A032: Antitumor activities of different RIG-I agonists in a murine model of kidney adenocarcinoma

Abstract Purpose: Activation of retinoic acid-inducible gene I (RIG-I) signaling induces tumor cell apoptosis and enhances NK, dendritic, and effector T cell activity. We applied two distinct RIG-I agonists: purified influenza virus RNA (IV RNA) in combination with an agonistic OX40 receptor-specific monoclonal antibody (anti-OX40) and a 3p-hpRNA (5' triphosphate hairpin RNA) combined with unmethylated CpG oligonucleotide (CpG). We also investigated the CpG and anti-OX40 combination. The study aimed to establish the efficacy of these compounds in a murine model of kidney adenocarcinoma (Renca). Methods: Renca cells were subcutaneously engrafted in 40 BALB/c mice. The animals were allocated into three treatment groups and a control group. Intratumoral administration of specific adjuvant combinations was carried out thrice as follows: Group 1: CpG+anti-OX40, Group 2: CpG+3p-hpRNA, Group 3: IV RNA+anti-OX40, and Group 4: PBS. Daily monitoring encompassed assessment of primary tumor sizes and physiological parameters. Deceased mice underwent dissection, and histological evaluations were performed. Treatment efficacy was ascertained based on tumor growth, distant metastases' occurrence, and cytokine levels' alterations. Statistical analysis was conducted, with p-values<0.05 considered statistically significant. Results: On day 38 post-implantation, the survival rates for the various groups were as follows: Group 4: 20%, Group 1: 70% (sig), Group 2: 50% (ns), and Group 3: 60% (sig). Measurements of primary tumor sizes were conducted following the final treatment on day 17 and subsequently every seven days until day 38. On day 17, no significant disparities in primary tumor sizes were observed between Groups 1, 2, 3, and 4. However, on day 24, Groups 1, 2, and 3; on day 31, Groups 1 and 2 exhibited significantly smaller primary tumors than Group 4. By day 38, no significant differences in tumor sizes were evident among the groups. Notably, discernible variations in primary tumor characteristics were observed: Groups 1, 2, and 3 demonstrated incipient demarcation, whereas Group 4 exhibited diffuse primary tumors accompanied by necrosis. Concerning metastatic development, the results are currently under evaluation and will be available for presentation at the conference, alongside the outcomes of cytokine ELISA tests. Discussion: After concluding the experiment, we deem the treatments to hold considerable promise. Notably, while the treatment effects were sustained, tangible outcomes were achieved: prolonged survival of the mice, reduction in primary tumor sizes, and mitigated metastatic formation. However, following the final treatment, there was a notable decline in the animals' overall quality of life. Consequently, we decided to terminate the experiment. In light of our findings, we ascertain that the treatments serve as a solid foundation for further exploration. By modifying the composition of the treatment cocktails and adjusting the treatment frequency, there exists the potential for further enhancement of the achieved results. Citation Format: Dominik Gulyás, Márta Lőrincz, István Jankovics, Béla Dénes, Dóra Földi, Rhiannon Rodgers, Katie Commins, Gábor Andócs, Gábor Kovács. Antitumor activities of different RIG-I agonists in a murine model of kidney adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A032.