SupT1 Cells Research Articles

Bis-Chalcones: Recent Reports of Their Diverse Applications in Biological and Material Sciences.

Originating from the basic chalcone structure, bis-chalcones are characterized by their dual α,β-unsaturated carbonyl systems and carry a range of biological activities that include antimicrobial, antiviral, antiparasitic, antioxidant, antiproliferative, and chemical reactivities that warrant a review to cover recent progress. Thus, this review presents the significant potential demonstrated by bis-chalcones in various biological applications. For example, compounds 2.3.1 showed excellent antiparasitic activity against leishmania with good selectivity index, and compounds 2.2.1-2.2.3 showed submicromolar activity against SupT1 cells. Compound 2.6.22 stood out in its antiproliferative activity against a panel of 60 different cell lines. Compounds 2.6.4 and 2.6.9 have been shown as submicromolar noncompetitive xanthine oxidase inhibitors. We also present their recent applications in material science, for example, as photosensitizers and photoinitiators, to showcase their broader potential for innovation in both medicinal chemistry and industrial applications.

Interactome of the HIV-1 proteome and human host RNA

The human immunodeficiency virus (HIV-1) is highly dependent on a variety of host factors. Beside proteins, host RNA molecules are reported to aid HIV-1 replication and latency maintenance. Here, we implement multiple workflows of native RNA immunoprecipitation and sequencing (nRIPseq) to determine direct host RNA interaction partners of all 18 HIV-1 (poly)proteins. We identify 1,727 HIV-1 protein – human RNA interactions in the Jurkat cell line and 1,558 interactions in SupT1 cells for a subset of proteins, and discover distinct cellular pathways that seem to be used or controlled by HIV-1 on the RNA level: Tat binds mRNAs of proteins involved in the super elongation complex (AFF1-4, Cyclin-T1). Correlation of the interaction scores (based on binding abundancy) allows identifying the highest confidence interactions, for which we perform a small-scale knockdown screen that leads to the identification of three HIV-1 protein binding RNA interactors involved in HIV-1 replication (AFF2, H4C9 and RPLP0).

Engineering PD-1-targeted small protein variants for in vitro diagnostics and in vivo PET imaging

BackgroundProgrammed cell death 1 (PD-1) belongs to immune checkpoint proteins ensuring negative regulation of the immune response. In non-small cell lung cancer (NSCLC), the sensitivity to treatment with anti-PD-1 therapeutics, and its efficacy, mostly correlated with the increase of tumor infiltrating PD-1+ lymphocytes. Due to solid tumor heterogeneity of PD-1+ populations, novel low molecular weight anti-PD-1 high-affinity diagnostic probes can increase the reliability of expression profiling of PD-1+ tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies and in vivo mapping efficiency using immune-PET imaging.MethodsWe designed a 13 kDa β-sheet Myomedin scaffold combinatorial library by randomization of 12 mutable residues, and in combination with ribosome display, we identified anti-PD-1 Myomedin variants (MBA ligands) that specifically bound to human and murine PD-1-transfected HEK293T cells and human SUP-T1 cells spontaneously overexpressing cell surface PD-1.ResultsBinding affinity to cell-surface expressed human and murine PD-1 on transfected HEK293T cells was measured by fluorescence with LigandTracer and resulted in the selection of most promising variants MBA066 (hPD-1 KD = 6.9 nM; mPD-1 KD = 40.5 nM), MBA197 (hPD-1 KD = 29.7 nM; mPD-1 KD = 21.4 nM) and MBA414 (hPD-1 KD = 8.6 nM; mPD-1 KD = 2.4 nM). The potential of MBA proteins for imaging of PD-1+ populations in vivo was demonstrated using deferoxamine-conjugated MBA labeled with 68Galium isotope. Radiochemical purity of 68Ga-MBA proteins reached values 94.7–99.3% and in vitro stability in human serum after 120 min was in the range 94.6–98.2%. The distribution of 68Ga-MBA proteins in mice was monitored using whole-body positron emission tomography combined with computerized tomography (PET/CT) imaging up to 90 min post-injection and post mortem examined in 12 mouse organs. The specificity of MBA proteins was proven by co-staining frozen sections of human tonsils and NSCLC tissue biopsies with anti-PD-1 antibody, and demonstrated their potential for mapping PD-1+ populations in solid tumors.ConclusionsUsing directed evolution, we developed a unique set of small binding proteins that can improve PD-1 diagnostics in vitro as well as in vivo using PET/CT imaging.Graphical

Artificial Extracellular Vesicles Generated from T Cells Using Different Induction Techniques.

Cell therapy is at the forefront of biomedicine in oncology and regenerative medicine. However, there are still significant challenges to their wider clinical application such as limited efficacy, side effects, and logistical difficulties. One of the potential approaches that could overcome these problems is based on extracellular vesicles (EVs) as a cell-free therapy modality. One of the major obstacles in the translation of EVs into practice is their low yield of production, which is insufficient to achieve therapeutic amounts. Here, we evaluated two primary approaches of artificial vesicle induction in primary T cells and the SupT1 cell line-cytochalasin B as a chemical inducer and ultrasonication as a physical inducer. We found that both methods are capable of producing artificial vesicles, but cytochalasin B induction leads to vesicle yield compared to natural secretion, while ultrasonication leads to a three-fold increase in particle yield. Cytochalasin B induces the formation of vesicles full of cytoplasmic compartments without nuclear fraction, while ultrasonication induces the formation of particles rich in membranes and membrane-related components such as CD3 or HLAII proteins. The most effective approach for T-cell induction in terms of the number of vesicles seems to be the combination of anti-CD3/CD28 antibody activation with ultrasonication, which leads to a seven-fold yield increase in particles with a high content of functionally important proteins (CD3, granzyme B, and HLA II).

Abstract 100: Blockade of CCL5 and CXCL10 signaling as a novel immunotherapy for epithelial ovarian cancer

Abstract Background: Cancer immune evasion remains a major barrier in developing enduring therapies for epithelial ovarian cancer (EOC). Expression of programmed death-ligand 1 (PD-L1) is known for its immunosuppressive role in cancer, thus interferences to PD-L1 have shown marked efficacy against many advanced cancers. However, the mechanisms by which PD-L1-negative cancers evade immune response remain unelucidated. We have found chemokine (C-C motif) ligand 5 (CCL5) and (C-X-C motif) ligand 10 (CXCL10) to up-regulate in PD-L1-negative EOC. Therefore, the present study aims to investigate blockade of CCL5/CXCL10 signaling to overcome immune evasion of EOC. Methods: Isogenic BRCA2-mutated PEO1 and BRCA2-wild type PEO4 EOC cells were treated with IFNγ, olaparib, or both in combination for western blot and flow cytometric analyses of PD-L1 expression. The conditioned medium of PEO1 and PEO4 cells was analyzed for immune array analysis to determine and compare the levels of 36 cytokines. PEO1 and PEO4 cells were co-cultured with CD3/CD28 antibodies-activated human peripheral blood mononuclear cells (PBMCs). Then, PBMCs were stained with anti-CD8 or CD4 and CD25 antibodies for flow cytometric analyses of cytotoxic or helper T cells, respectively. PEO1 and PEO4 cells were co-cultured with activated PBMCs in the presence and absence of TAK-779 or AMG-487. Thereafter, PBMCs were stained with anti-CD4, CD25, and FOXP3 antibodies for flow cytometric analyses of regulatory T cells (Tregs). PEO1 and PEO4 cells were transfected with CCL5- or CXCL10-siRNA and subsequently co-cultured with activated SUPT1 cells or PBMCs. The conditioned medium was collected and analyzed for the levels of IFNγ by ELISA. Results: PEO1 cells exhibited an up-regulation of basal and IFNγ-induced PD-L1 compared with PEO4 cells. PEO4 cells displayed a pronounced increase in the CCL5 and IFNγ-induced CXCL10 compared with PEO1 cells. Co-culture of PBMCs with PEO1 and PEO4 cells led to a significant decrease in activated CD8+ effector T and CD4+ helper T cells. However, co-culture of PBMCs with PEO4 cells caused a significant increase in activated CD4+ helper T cells. Co-culture of PBMCs with PEO4 cells caused a significant increase in FOXP3+CD4+CD25+ Tregs. This increase in Tregs was significantly reduced by the treatment with the CCL5/CCR5 antagonist TAK-779 or the CXCL10/CXCR3 antagonist AMG-487. Silencing of CCL5 or CXCL10 in PEO4 cells by siRNA significantly increased IFNγ production by SUPT1 cells and PBMCs. Conclusions: PEO4 cells secrete CCL5 and IFNγ-induced CXCL10 to activate Tregs, enabling tumor cells to evade host immune response by dysregulating their microenvironment. PEO4 cells-induced Tregs are reduced by CCL5/CXCL10 signaling blockade with TAG-779 or AMG-487. Furthermore, silencing of CCL5 or CXCL10 in PEO4 cells with siRNA abolishes Treg-mediated immunosuppression and restores the antitumor activity of effector T cells. Citation Format: Annabelle C. Lin, Jake Moscarelli, Z. Ping Lin, Elena S. Ratner. Blockade of CCL5 and CXCL10 signaling as a novel immunotherapy for epithelial ovarian cancer [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 100.

Abstract 3111: Preclinical development of a peptide-based immune-surveillance PET tracer for immuno-oncology stratification

Abstract Introduction: Cancer immunotherapies, such as immune checkpoint inhibitors (ICIs), have become an integral part of cancer treatment. However, significant differences in their effectiveness persist among individual patients. This variability partly stems from poorly immunogenic tumors, referred to as "cold" tumors, characterized by low levels of tumor-infiltrating leukocytes (TILs). Studies indicate that lower levels of TILs, specifically CD4+ and CD8+ T-cells, lead to reduced efficacy of certain immunotherapies. Therefore, the imaging of these cell types could be invaluable in screening cancer patients to predict the effectiveness of a prescribed immunotherapeutic agent before and during treatment. CD4+ T-cells, identified by their high expression of CD4 receptors, could serve as a potential molecular imaging target for an immuno-oncology relevant PET tracer. For this purpose a peptide-based PET tracer offer several beneficial characteristics such as relatively low circulation time, cell penetration, and low development costs. Methods: All peptides were identified through peptide phage display (PPD) and commercially synthesized (>95% purity). These peptides underwent screening for their binding affinity to recombinant human CD4 and a negative control protein using surface plasmon resonance (SPR) on a Biacore X100. Peptides exhibiting specific binding to CD4, as determined by SPR, were further selected for FITC-labeling (>95% purity). FITC-labeled peptides were applied in fluorescence-based cell assays conducted on the SUP-T1 (CRL-1942) cell line and an established SUP-T1 CD4 knockout (KO) cell line. Flow cytometry assays were performed using a Fortessa X-20, while live cell imaging was conducted on either a LSM980 or LSM900 confocal microscopy. Simultaneously, an in silico docking method was employed, utilizing ColabFold—an online complex predictor powered by AlphaFold2—for CD4-peptide docking analysis. Results: Several peptides successfully identified via PPD exhibited binding to recombinant human CD4 as shown by SPR screening. The FITC-labeling and assessment against SUP-T1 and corresponding CD4 KO cell line unveiled specific CD4 binding within a subset of peptides, as observed in both flow cytometry and confocal microscopy. In vitro data was compared to CD4-peptide docking results to create a list of peptides for future in vivo studies. Conclusions: Multiple peptides successfully identified via PPD showed robust in vitro CD4 binding through SPR screening. Select peptides also demonstrated specific CD4 binding in cell assays, corroborated by docking results. Future prospective for the high performing peptides include in vivo PET/CT Ga-68 or Cu-64 imaging in tumors with high and low CD4+ T-cell infiltration. Citation Format: Anders W. Sinkjaer, Danna K. Sell, Ida M. Dawoodi, Babak Bakhshinejad, Camilla Stavnsbjerg, Andreas Kjaer. Preclinical development of a peptide-based immune-surveillance PET tracer for immuno-oncology stratification [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 3111.

The interaction of human immunodeficiency virus-1 and human endogenous retroviruses in patients (primary cell cultures) and cell line models.

Human endogenous retroviruses (HERVs) integrated into the human genome millions of years ago, and their expression has been described both in healthy tissues and in pathology. In the present study, we study the differential expression of multiple HERV families in the presence of human immunodeficiency virus (HIV) infection in the peripheral blood mononuclear cells (PBMCs) of HIV-positive individuals, in HIV-infected cell lines, and in vitro HIV-infected macrophages/monocytes of healthy donors, utilizing publicly available data sets for secondary analysis using a pipeline appropriate for HERV transcription detection and comparison. We recognized significantly decreased expression of HERV-H in treatment-naïve HIV patients with viremia and increased expression of multiple HERV families in the PBMCs of HIV-infected individuals under combination antiretroviral therapy compared to healthy donors, in in vitro HIV-infected Stanford University pediatric T (SUP-T1) cells at 24 hours after HIV infection compared to non-infected SUP-T1 cells, and in in vitro HIV-infected MT4 T cells at 3 days after HIV infection compared to non-infected MT4 T cells. We recognized significantly increased expression of HERV-W, after HIV infection in monocyte-derived macrophage cell culture; no difference was found in HERV expression after HIV infection of monocyte cultures. We found a significantly decreased expression of HERV-K (HML-6) in donor-derived macrophage cultures at 36 hours after HIV infection, which does not persist at 6 days after infection. HIV infection modifies the expression of multiple HERV families in the PBMCs of HIV patients and in T cell-derived cultures, while the effect of HIV infection on HERV expression appears to be more restricted in cells of monocytic origin.IMPORTANCEIn this work, we demonstrated that human immunodeficiency virus (HIV) infection leads to the modification of the human endogenous retrovirus (HERV) expression. Differential expression of multiple HERVs was found in peripheral blood mononuclear cells derived from HIV-infected patients compared to healthy donors and HIV-infected T cell cultures compared to non-infected. The effect of HIV presence on HERV expression appears to be more restricted in cells of monocytic origin, as only deregulation of HERV-W and HERV-K (HML-6) was found in these cell cultures after their infection with HIV. Multiple factors contribute to this aberrant HERV expression, and its levels appear to be modified in a time-dependent manner. Further studies and the development of optimized in vitro protocols are warranted to elucidate the interactions between HIV and HERVs in detail.

HIV-1 active and latent infections induce disparate chromatin reorganization and transcriptional regulation of mRNAs and lncRNAs in SupT1 cells.

HIV-1 infection of T-lymphocytes depends on co-opting cellular transcriptional and translational machineries for viral replication. This requires significant changes in the cellular microenvironment. We have characterized and compared the changes in cellular chromatin structures as well as gene expression landscapes in T cells that are either actively or latently infected with HIV-1. Our results reveal that chromatin accessibility and expression of both protein-coding mRNAs and non-coding lncRNAs are uniquely regulated in HIV-1-infected T cells, depending on whether the virus is actively transcribing or remains in a transcriptionally silent, latent state. HIV-1 latent infection elicits more robust changes in the cellular chromatin organization than active viral infection. Our analysis also identifies the effects of such epigenomic changes on the cellular gene expression and subsequent biological pathways. This study comprehensively characterizes the cellular epigenomic and transcriptomic states that support active and latent HIV-1 infection in an in vitro model of SupT1 cells.

In Vitro and In Vivo Specificity and Biodistribution of a Novel CD8-Targeted Fusosome

Autologouschimeric antigen receptor (CAR) T cells represent a groundbreaking therapy for treating B-cell malignancies. However, the complexity, cost, time and toxicity associated with manufacturing and administering current CAR T cell therapies hinder their broader applications. To overcome CAR T limitations, Sana has developed a novel fusosome platform that can deliver a T cell targeted CAR gene therapy via systemic administration, without ablative pre-conditioning. The fusosome is a lentiviral vector pseudotyped with a targeted fusogen that specifically directs gene transfer into CD8+ cells. Our CD8-targeted CD19-directed CAR fusosome, SG299, transduces CD8+ cells in lymphoid tissues, showing preclinical safety and high selectivity, laying the groundwork for clinical evaluation of an in vivo CAR gene therapy. In vitro tissue cross reactivity (TCR) study was assessed to evaluate binding of the CD8 fusogen binder, a CD8-binding scFv, on tissues from Nemestrina macaques and compared these with tissues from human donors. SG299 specificity was also evaluated in vitro using panel of cell lines representing a breadth of receptor types. Cells were exposed to 8 IU/cell SG299 and vector integration was evaluated using ddPCR. Reverse transcriptase inhibitor nevirapine was used as control for the in vitro studies. To evaluate in vivo specificity and biodistribution, healthy nemestrina macaques were treated with a single dose of SG299 at 1 or 4 x 10 9 IU/kg (n=2 or 4) or saline (n=1 or 2) via intravenous infusion and evaluated for up to 35 days for vector biodistribution in CD8 enriched PBMCs and at terminal timepoint in panel of 17 tissues using ddPCR. In addition, we also demonstrated SG299 specificity with help of flow cytometry, in NALM6 tumor model in NSG mice. Briefly, NSG mice engrafted with human PBMCs were challenged with NALM6 tumors and treated with either SG299 or saline. The TCR study demonstrated that the CD8 fusogen binder is specific for the target tissues containing T cells and similar binding across multiple human and Nemestrina tissues. SG299 fusosome demonstrated CD8-specificity evaluated by transduction of cell types with CD8 receptor (SupT1 and PanT cells). There was no detectable off-target transduction in sets of primary as well as immortalized cell lines evaluated including pulmonary cells, renal epithelial cells, lymphoblast, endothelial cells and CD8KO cells. VCN analysis of nemestrina macaques PBMC and tissue samples following treatment with SG299 demonstrated dose dependent integrations per diploid genome through Day 35. All animals dosed 4 x 10 9 IU/kg showed quantifiable VCN signal through Day 34 in PBMC population enriched for CD8+ cells (0.0032 - 0.6544 vc/dg, vector copy per diploid genome). One out of 4 animals dosed at 1 x 10 9 IU/kg showed quantifiable VCN signal at Day 34 (0.0276 vc/dg). Tissues collected at necropsy on Day 35 demonstrated quantifiable VCN signal in spleen, lymph node, CD8+ splenocyte and lymphocyte, draining lymph node, heart ventricle, and injection site, for animals dosed at 4 x 10 9 IU/kg. One of 4 animals dosed 1 x 10 9 IU/kg showed quantifiable VCN in spleen, CD8+ splenocyte, and injection site. Flow cytometric analysis of peripheral blood of mice treated with SG299 showed a dose dependent increase in CAR T cells, and the CAR T cells were CD8+ T cells specific with no CAR T cell detection amongst the CD4+ T cells. Collectively, these studies demonstrate high on target specificity of SG299 fusosome to transduce CD8+ T cells in vitro and in vivo. Therefore, SG299 represents a novel therapeutic opportunity to generate CAR T cells in vivo and potentially overcome challenges associated with ex vivo CAR T therapies.

The HIV-2 OGH double reporter virus shows that HIV-2 is less cytotoxic and less sensitive to reactivation from latency than HIV-1 in cell culture

A better understanding of HIV-1 latency is a research priority in HIV cure research. Conversely, little is known about the latency characteristics of HIV-2, the closely related human lentivirus. Though both viruses cause AIDS, HIV-2 infection progresses more slowly with significantly lower viral loads, even when corrected for CD4+ T cell counts. Hence a direct comparison of latency characteristics between HIV-1 and HIV-2 could provide important clues towards a functional cure.Transduction of SupT1 cells with single-round HIV-1 and HIV-2 viruses with an enhanced green fluorescent protein (eGFP) reporter showed higher levels of eGFP expression for HIV-2 than HIV-1, while HIV-1 expression appeared more cytotoxic. To compare HIV-1 and HIV-2 gene expression, latency and reactivation in more detail, we have generated HIV-2 OGH, a replication deficient, near full- length, double reporter virus that discriminates latently and productively infected cells in cell culture. This construct is based on HIV-1 OGH, and to our knowledge, first of its kind for HIV-2. Using this construct we have observed a higher eGFP expression for HIV-2, but higher losses of HIV-1 transduced cells in SupT1 and Jurkat cells and a reduced sensitivity of HIV-2 for reactivation with TNF-α. In addition, we have analysed HIV-2 integration sites and their epigenetic environment. HIV-1 and HIV-2 share a preference for actively transcribed genes in gene-dense regions and favor active chromatin marks while disfavoring methylation markers associated with heterochromatin. In conclusion the HIV-2 OGH construct provides an interesting tool for studying HIV-2 expression, latency and reactivation. As simian immunodeficiency virus (SIV) and HIV-2 have been proposed to model a functional HIV cure, a better understanding of the mechanisms governing HIV-2 and SIV latency will be important to move forward. Further research is needed to investigate if HIV-2 uses similar mechanisms as HIV-1 to achieve its integration site selectivity.

Abstract LB311: Increased potency of CD8-targeted fusosomes enhances CAR gene delivery to resting primary T cells

Abstract Introduction: Fusosomes are viral vectors pseudotyped with modified paramyxovirus envelopes targeting specific cell types. A CD8-targeted fusosome delivering CD19CAR transgene has the potential to provide an off-the-shelf therapeutic approach to generate CD19-directed chimeric antigen receptor (CAR) T cells in patients after direct intravenous delivery or extracorporeal delivery (ECD), i.e., a short-term exposure of apheresis product to fusosome before re-infusion into the patient. Here, we demonstrate that an improved manufacturing process resulted in higher fusosome titer and quality, increasing the potency of CAR T generation from resting CD8 T cells. This potency is also observed when fusosomes are evaluated in a clinically relevant mock ECD approach. Methods: Fusosome titer was quantified by ddPCR directly for physical titer (vector genomes, GQA) or after infections of SupT1 cells for functional titer (IU/mL). Transduction of CD8 T cells was measured by flow cytometry and vector copy number analysis after short-term (1-4 hour) incubation of PBMCs with fusosome followed by wash and culture in resting or CD3/CD28-activated conditions. To assess CD19CAR T cell generation at a mock clinical setting and scale, a healthy donor was apheresed to collect PBMCs, fusosome was administered into the apheresis bag and PBMCs were sampled from the bag over a 2.5-hour time course for further analysis. In vivo anti-tumor efficacy of the fusosome was measured in immune-deficient NSG mice engrafted intravenously with Nalm6, then PBMCs followed by fusosome one day later. Tumor growth was monitored by bioluminescence imaging. Results: Fusosomes produced by the improved process had both a higher functional concentration (>14-fold higher IU/mL) and better infectivity (>10-fold higher IU/GQA) in comparison to fusosomes produced by the original process. Exposure of PBMCs to fusosomes produced by the improved process led to increased fusosome binding to CD8 T cells at high IU/PBMC doses and higher transduction efficiency in CD8 T cells at the same calculated dose per cell compared to fusosomes produced by the original process (>3-fold higher mean CAR+ percentage after 2-hour exposure to 2 IU/PBMC, n=8). Furthermore, fusosomes produced by the improved process generated higher frequencies of CAR+ CD8 T cells and resulted in increased ability to control tumor growth at lower doses in vivo in a Nalm6 model. Importantly, the ECD approach translated well to a mock clinical setting, resulting in successful generation of CAR T cells (12.6% of CD8 T cells after a 2.5-hour incubation). Conclusion: CD8-targeted fusosome encoding a CD19CAR transgene represents a potential novel therapeutic approach for cancer. This study demonstrates that improvements in the manufacturing process can significantly increase the potency of the fusosome in T cells, and we intend to move forward to clinical trials using this improved process. Citation Format: Jesse Green, Brian Granger, Ouwen Liang, Jesus Moreno, Andrew Tucker, Aesha Vakil, Reuben Burch, Nicholas Mozdzierz, Matthew Scott-Skandera, Stephanie Riofrio, Mike DeCosta, Amey Gaikwad, Sanket Phadke, Kaely Gallagher, Adam Charlton, Sarah Miller, Hosein Kouros-Mehr, Misha Shamashkin, Neal Van Hoeven, Kutlu Elpek, Terry Fry. Increased potency of CD8-targeted fusosomes enhances CAR gene delivery to resting primary T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB311.

Gluten Exorphins Promote Cell Proliferation through the Activation of Mitogenic and Pro-Survival Pathways.

Celiac disease (CD) is a chronic and systemic autoimmune disorder that affects preferentially the small intestine of individuals with a genetic predisposition. CD is promoted by the ingestion of gluten, a storage protein contained in the endosperm of the seeds of wheat, barley, rye, and related cereals. Once in the gastrointestinal (GI) tract, gluten is enzymatically digested with the consequent release of immunomodulatory and cytotoxic peptides, i.e., 33mer and p31-43. In the late 1970s a new group of biologically active peptides, called gluten exorphins (GEs), was discovered and characterized. In particular, these short peptides showed a morphine-like activity and high affinity for the δ-opioid receptor (DOR). The relevance of GEs in the pathogenesis of CD is still unknown. Recently, it has been proposed that GEs could contribute to asymptomatic CD, which is characterized by the absence of symptoms that are typical of this disorder. In the present work, GEs cellular and molecular effects were in vitro investigated in SUP-T1 and Caco-2 cells, also comparing viability effects with human normal primary lymphocytes. As a result, GEs treatments increased tumor cell proliferation by cell cycle and Cyclins activation as well as by induction of mitogenic and pro-survival pathways. Finally, a computational model of GEs interaction with DOR is provided. Altogether, the results might suggest a possible role of GEs in CD pathogenesis and on its associated cancer comorbidities.

Cyclic peptides target the aromatic cage of a PHD-finger reader domain to modulate epigenetic protein function.

Plant homeodomain fingers (PHD-fingers) are a family of reader domains that can recruit epigenetic proteins to specific histone modification sites. Many PHD-fingers recognise methylated lysines on histone tails and play crucial roles in transcriptional regulation, with their dysregulation linked to various human diseases. Despite their biological importance, chemical inhibitors for targeting PHD-fingers are very limited. Here we report a potent and selective de novo cyclic peptide inhibitor (OC9) targeting the Nε-trimethyllysine-binding PHD-fingers of the KDM7 histone demethylases, developed using mRNA display. OC9 disrupts PHD-finger interaction with histone H3K4me3 by engaging the Nε-methyllysine-binding aromatic cage through a valine, revealing a new non-lysine recognition motif for the PHD-fingers that does not require cation-π interaction. PHD-finger inhibition by OC9 impacted JmjC-domain mediated demethylase activity at H3K9me2, leading to inhibition of KDM7B (PHF8) but stimulation of KDM7A (KIAA1718), representing a new approach for selective allosteric modulation of demethylase activity. Chemoproteomic analysis showed selective engagement of OC9 with KDM7s in T cell lymphoblastic lymphoma SUP T1 cells. Our results highlight the utility of mRNA-display derived cyclic peptides for targeting challenging epigenetic reader proteins to probe their biology, and the broader potential of this approach for targeting protein-protein interactions.

Validation of Promoters and Codon Optimization on CRISPR/Cas9-Engineered Jurkat Cells Stably Expressing αRep4E3 for Interfering with HIV-1 Replication

Persistent and efficient therapeutic protein expression in the specific target cell is a significant concern in gene therapy. The controllable integration site, suitable promoter, and proper codon usage influence the effectiveness of the therapeutic outcome. Previously, we developed a non-immunoglobulin scaffold, alpha repeat protein (αRep4E3), as an HIV-1 RNA packaging interference system in SupT1 cells using the lentiviral gene transfer. Although the success of anti-HIV-1 activity was evidenced, the integration site is uncontrollable and may not be practical for clinical translation. In this study, we use the CRISPR/Cas9 gene editing technology to precisely knock-in αRep4E3 genes into the adeno-associated virus integration site 1 (AAVS1) safe harbor locus of the target cells. We compare the αRep4E3 expression under the regulation of three different promoters, including cytomegalovirus (CMV), human elongation factor-1 alpha (EF1α), and ubiquitin C (UbC) promoters with and without codon optimization in HEK293T cells. The results demonstrated that the EF1α promoter with codon-optimized αRep4E3mCherry showed higher protein expression than other promoters with non-optimized codons. We then performed a proof-of-concept study by knocking in the αRep4E3mCherry gene at the AAVS1 locus of the Jurkat cells. The results showed that the αRep4E3mCherry-expressing Jurkat cells exhibited anti-HIV-1 activities against HIV-1NL4-3 strain as evidenced by decreased capsid (p24) protein levels and viral genome copies as compared to the untransfected Jurkat control cells. Altogether, our study demonstrates that the αRep4E3 could interfere with the viral RNA packaging and suggests that the αRep4E3 scaffold protein could be a promising anti-viral molecule that offers a functional cure for people living with HIV-1.

Rab11-FIP1C Is Dispensable for HIV-1 Replication in Primary CD4+ T Cells, but Its Role Is Cell Type Dependent in Immortalized Human T-Cell Lines.

The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that small interfering RNA (siRNA)-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR-Cas9 technology was used to knock out the expression of this protein in several human T-cell lines-Jurkat E6.1, SupT1, and H9-and in primary human CD4+ T cells. FIP1C knockout caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T cells. In H9 cells, FIP1C knockout caused a cell density-dependent defect in virus replication. In primary CD4+ T cells, FIP1C knockout had no effect on HIV-1 replication. Furthermore, human T-cell leukemia virus type 1 (HTLV-1)-transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail (Y795W) implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. IMPORTANCE The incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp41, into virus particles is critical for virus infectivity. gp41 contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types-human T-cell lines and primary CD4+ T cells-we used CRISPR-Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4+ T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4+ T cells.

5-Aza-dC promotes T-cell acute lymphoblastic leukemia cell invasion via downregulation of DNMT1 and upregulation of MMP-2 and MMP-9

5-Aza-2'-deoxycytidine (5-Aza-dC) is a demethylation agent known to deplete DNA methyltransferases (DNMTs) in leukemia cancer cells, and can restore the expression of their target genes in Jurkat cells. The goal of this study was to discern the potential effect of 5-Aza-dC on the invasion of T-ALL cells in acute lymphoblastic leukemia (ALL). The role of matrix metallopeptidase (MMP)-2, MMP-9, and DNMT1 in cell invasion was determined using loss- and gain-of-function investigations in Jurkat- and Sup-T1-R cells. A nude mouse model of ALL was established for further exploration of their roles in vivo. MMP-2 and MMP-9 exhibited high expression and low DNA methylation levels in 5-Aza-dC-resistant T-ALL cells. DNMT1 was poorly expressed in 5-Aza-dC-resistant T-ALL cells and exhibited decreased enrichment in the promoter region of MMP-2 and MMP-9. Silencing of MMP-2 and MMP-9 or DNMT1 overexpression reduced T-ALL cell invasion. After treatment of Sup-T1 cells with 5-Aza-dC, MMP-2 and MMP-9 presented with reduced DNA methylation levels but increased expression, and DNMT1 expression was identified to be suppressed. Further, in vivo assays revealed that DNMT1 alleviated T-ALL by reducing the expression of MMP-2 and MMP-9 in vivo. All in all, 5-Aza-dC activates MMP-2 and MMP-9 expression by reducing DNMT1-dependent DNA methylation levels and, hence, promotes the invasion of T-ALL cells.

Developing RNA‐Seq and ChIP‐Seq Pipelines on an Oxford Nanopore Technologies Platform to Study Notch1 Transcriptional Dynamics

Notch signaling plays a crucial role in cell development and proliferation. The Notch receptor is an inducible transcription factor found on the cell surface which is cleaved resulting in the release of the intracellular domain (NICD). The NICD enters the nucleus and complexes with CBF1 and Mastermind (MAM) to activate target genes. CBF1 recognizes the binding motif RTGRGAR (R=G or A), and this is referred to an NRE (Notch Response Element). Recent studies have shown that a paired CBF1 binding site is also important for target gene activation. While individual genes regulated by SPS are known, the extent to which an SPS vs individual NREs are responsible for Notch‐mediated transcription on a genomic level remains an active area of research. It is hypothesized that the majority of Notch‐mediated transcription occurs as a result of a dimeric Notch complex in the promoters or enhancers of transcribed genes. Recently, high‐throughput techniques have allowed the study of Notch complex assembly and their effects on transcription. Here, we use an Oxford Nanopore Platform was used.To identify genes regulated by Notch transcriptional complexes, RNA‐Seq was performed. Total RNA was isolated from SUPT1 cells treated with 5 µM of the Notch inhibitor DAPT for 24 hours, or DMSO as a control. Poly‐A RNA was purified using an oligo‐dT column and double‐stranded cDNA generated. PCR adapters were nonspecifically ligated to cDNA and amplified to increase library yield. Sequencing was performed using an Oxford Nanopore Minion, and results were mapped to the human reference genome to identify genes significantly downregulated when Notch was inhibited. To identify NREs and SPS in the regulatory elements of these genes, ChIP‐Seq was performed for Notch. ChIP was performed in DAPT and DMSO treated cells, and ChIP DNA used to prepare a sequencing library as above. Sequenced fragments were aligned to a known regulatory elements dataset, and genes with significant Notch binding identified.RNA‐Seq data shows significant downregulation of known Notch targets genes, such as Hes1, Hey1, and Hes4. Sequenced mRNA fragments, however, averaged 1kb suggesting size selectivity during the purification or amplification steps of library preparation. Additionally, underrepresentation of other Notch known targets such as Hes5 may suggest that computational stringency may have to be adjusted. We are currently pursuing ChIP‐Seq experiments that will be presented at the conference to identify the locations of SPS in the genome. Identified SPS in regulatory elements will then be correlated with the genes they activate, and the downstream effects on transcription analyzed. It is expected that a large number of genes that are inactivated under Notch inhibition conditions will exhibit a SPS in a corresponding regulatory element.

Immunoreactivity of humanized single-chain variable fragment against its functional epitope on domain 1 of CD147

Domain 1 of CD147 participates in matrix metalloproteinase (MMP) production and is a candidate for targeted therapy to prevent cancer invasion and metastasis. A functional mouse anti-CD147 monoclonal antibody, M6-1B9, was found to recognize domain 1 of CD147, and its respective mouse single-chain variable fragment (ScFvM61B9) was subsequently generated. The EDLGS epitope candidate for M6-1B9 was identified using the phage display peptide technique in this study. For future clinical applications, humanized ScFv specific to domain 1 of CD147 (HuScFvM61B9) was partially adopted from the hypervariable sequences of parental mouse ScFvM61B9 and grafted onto suitable human immunoglobulin frameworks. Molecular modelling and simulation were performed in silico to generate the conformational structure of HuScFvM61B9. These results elucidated the amino acid residues that contributed to the interactions between CDRs and the epitope motif. The expressed HuScFvM61B9 specifically interacted with CD147 at the same epitope as the original mAb, M6-1B9, and retained immunoreactivity against CD147 in SupT1 cells. The reactivity of HuScFvM61B9 was confirmed using CD147 knockout Jurkat cells. In addition, the inhibitory effect of HuScFvM61B9 on OKT3-induced T-cell proliferation as M6-1B9 mAb was preserved. As domain 1 is responsible for cancer invasion and metastasis, HuScFvM61B9 would be a candidate for cancer targeted therapy in the future.

Assessment of T cell chemotaxis to S1P

Introduction: Migration of leukocytes in response to chemical gradients, chemotaxis, is dependent on many factors, including cell type, surface markers, the chemoattractant, etc. Sphinogsine-1-phosphate (S1P) is a chemoattractant playing a large role in migrating activated T cells out of lymph nodes by binding to their S1P receptor, S1P1. The importance of the egress in T cells from lymph nodes is highlighted by pharmacological disruption of this migration can lead to immune dampening and thus control of multiple sclerosis, an autoimmune disease. In the case of human immunodeficiency virus (HIV), it has been shown that HIV downregulates S1P1 surface expression, effectively inhibiting chemotaxis. Our experiments attempt to study a particular HIV-encoded protein, Nef in S1P-elicited T cell migration, and to optimize the conditions for assessing T cell migration in response to S1P.
 Methods: In our Transwell migration assays, migration of serum-starved SupT1 cells was induced using various concentrations of S1P bound to delipidated bovine serum albumin (BSA). Before migration, cells were labeled using Calcein AM. Cells were allowed to migrate for 2-4 hours at 37°C in serum-free media. After migration, fluorescence intensity was measured using a CLARIOstar microplate reader.
 Results: S1P showed a direct dose-dependent response to SupT1 cell migration from 0 to 100 nM S1P. Optimization of the migration showed that both number of trans-migrated cells and those still present within the transwell filter were significant indicators of SupT1 migration.
 Conclusion: S1P’s chemoattractant ability is prevalent in the migration of SupT1 cells in concentrations lower than 125nM. Because we have inducible systems for HIV-Nef expression established in this cell line, these data are useful for testing the role of Nef in HIV-mediated T cell retention.

The Antileukemic Effect of Xestoquinone, A Marine-Derived Polycyclic Quinone-Type Metabolite, Is Mediated through ROS-Induced Inhibition of HSP-90.

Xestoquinone is a polycyclic quinone-type metabolite with a reported antitumor effect. We tested the cytotoxic activity of xestoquinone on a series of hematological cancer cell lines. The antileukemic effect of xestoquinone was evaluated in vitro and in vivo. This marine metabolite suppressed the proliferation of Molt-4, K562, and Sup-T1 cells with IC50 values of 2.95 ± 0.21, 6.22 ± 0.21, and 8.58 ± 0.60 µM, respectively, as demonstrated by MTT assay. In the cell-free system, it inhibited the activity of topoisomerase I (Topo I) and II (Topo II) by 50% after treatment with 0.235 and 0.094 μM, respectively. The flow cytometric analysis indicated that the cytotoxic effect of xestoquinone was mediated through the induction of multiple apoptotic pathways in Molt-4 cells. The pretreatment of Molt-4 cells with N-acetyl cysteine (NAC) diminished the disruption of the mitochondrial membrane potential (MMP) and apoptosis, as well as retaining the expression of both Topo I and II. In the nude mice xenograft model, the administration of xestoquinone (1 μg/g) significantly attenuated tumor growth by 31.2% compared with the solvent control. Molecular docking, Western blotting, and thermal shift assay verified the catalytic inhibitory activity of xestoquinone by high binding affinity to HSP-90 and Topo I/II. Our findings indicated that xestoquinone targeted leukemia cancer cells through multiple pathways, suggesting its potential application as an antileukemic drug lead.