Chimeric Receptor Research Articles

Rational design of an influenza-COVID-19 chimeric protective vaccine with HA-stalk and S-RBD

ABSTRACT Highly contagious respiratory illnesses like influenza and COVID-19 pose serious risks to public health. A two-in-one vaccine would be ideal to avoid multiple vaccinations for these diseases. Here, we generated a chimeric receptor binding domain of the spike protein (S-RBD) and hemagglutinin (HA)-stalk-based vaccine for both SARS-CoV-2 and influenza viruses. The S-RBD from SARS-CoV-2 Delta was fused to the headless HA from H1N1 (H1Delta), creating a chimera that forms trimers in solution. The cryo-electron microscopy structure of the chimeric protein complexed with the RBD-targeting CB6 and the HA-stalk-targeting CR9114 antibodies shows that the trimeric protein is stable and accessible for neutralizing antibody binding. Immunization with the vaccine elicited high and long-lasting neutralizing antibodies and effectively protected mice against the challenges of lethal H1N1 or heterosubtypic H5N8, as well as the SARS-CoV-2 Delta or Omicron BA.2 variants. Overall, this study offers a two-in-one universal vaccine design to combat infections caused by both SARS-CoV-2 variants of concern and influenza viruses.

Chimeric HLA antibody receptor T cells to target HLA-specific B cells in solid organ transplantation.

HLA-sensitized patients on the transplant waiting list harbor antibodies and memory B cells directed against allogeneic HLA molecules, which decreases the chance to receive a compatible donor organ. Current desensitization strategies non-specifically target circulating antibodies and B cells, warranting the development of therapies that specifically affect HLA-directed humoral immune responses. We developed Chimeric HLA Antibody Receptor (CHAR) constructs comprising the extracellular part of HLA-A2 or HLA-A3 coupled to CD28-CD3ζ domains. CHAR-transduced cells expressing reporter constructs encoding T-cell activation markers, and CHAR-transduced CD8+ T cells from healthy donors were stimulated with HLA-specific monoclonal antibody-coated microbeads, and HLA-specific B cell hybridomas. CHAR T cell activation was measured by upregulation of T cell activation markers and IFNγ secretion, whereas CHAR T cell killing of B cell hybridomas was assessed in chromium release assays and by IgG ELISpot. HLA-A2- and HLA-A3-CHAR expressing cells were specifically activated by HLA-A2- and HLA-A3-specific monoclonal antibodies, either soluble or coated on microbeads, as shown by CHAR-induced transcription factors. HLA-A2 and HLA-A3 CHAR T cells efficiently produced IFNγ with exquisite specificity and were capable of specifically lysing hybridoma cells expressing HLA-A2- or HLA-A3-specific B-cell receptors, respectively. Finally, we mutated the α3 domain of the CHAR molecules to minimize any alloreactive T-cell reactivity against CHAR T cells, while retaining CHAR activity. These data show proof of principle for CHAR T cells to serve as precision immunotherapy to specifically desensitize (highly) sensitized solid organ transplant candidates and to treat antibody-mediated rejection after solid organ transplantation.

An Extracellular miRNA‐Responsive Artificial Receptor via Dynamic DNA Nano‐assembly for Biomarker‐Driven Therapy

AbstractMicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers and therapeutic targets in various diseases. However, there is currently a lack of molecular strategies that can effectively use disease‐associated extracellular miRNAs as input signals to drive therapeutic functions. Herein, we present a modular and programmable miRNA‐responsive chimeric DNA receptor (miRNA‐CDR) capable of biomarker‐driven therapy. By grafting a miRNA‐responsive DNA nanodevice on a natural membrane receptor via aptamer anchoring, miRNA‐CDR can sense extracellular miRNA levels and autonomously induce dimerization‐mediated receptor activation via the complementary‐mediated strand displacement reaction‐induced dynamic DNA assembly. The sequence programmability of miRNA‐CDR allows it to sense and respond to a user‐defined miRNA with tunable sensitivity. Moreover, the miRNA‐CDR is versatile and customizable to reprogram desirable signaling output via adapting a designated receptor, such as MET and FGFR1. Using a mouse model of drug‐induced acute liver injury (DILI), we demonstrate the functionality of a designer miRNA‐CDR in rewiring the recognition of the DILI‐elevated miR‐122 to promote MET signaling of hepatocytes for biomarker‐driven in situ repair and liver function restoration. Our synthetic miRNA‐CDR platform provides a novel molecular device enabling biomarker‐driven therapeutic cellular response, potentially paving the way for improving the precision of cell therapy in regenerative medicine.

An Extracellular miRNA-Responsive Artificial Receptor via Dynamic DNA Nano-assembly for Biomarker-Driven Therapy.

MicroRNAs (miRNAs) have emerged as promising diagnostic biomarkers and therapeutic targets in various diseases. However, there is currently a lack of molecular strategies that can effectively use disease-associated extracellular miRNAs as input signals to drive therapeutic functions. Herein, we present a modular and programmable miRNA-responsive chimeric DNA receptor (miRNA-CDR) capable of biomarker-driven therapy. By grafting a miRNA-responsive DNA nanodevice on a natural membrane receptor via aptamer anchoring, miRNA-CDR can sense extracellular miRNA levels and autonomously induce dimerization-mediated receptor activation via the complementary-mediated strand displacement reaction-induced dynamic DNA assembly. The sequence programmability of miRNA-CDR allows it to sense and respond to a user-defined miRNA with tunable sensitivity. Moreover, the miRNA-CDR is versatile and customizable to reprogram desirable signaling output via adapting a designated receptor, such as MET and FGFR1. Using a mouse model of drug-induced acute liver injury (DILI), we demonstrate the functionality of a designer miRNA-CDR in rewiring the recognition of the DILI-elevated miR-122 to promote MET signaling of hepatocytes for biomarker-driven in situ repair and liver function restoration. Our synthetic miRNA-CDR platform provides a novel molecular device enabling biomarker-driven therapeutic cellular response, potentially paving the way for improving the precision of cell therapy in regenerative medicine.

Determination of CAR T cell metabolism in an optimized protocol.

Adoptive transfer of T cells modified to express chimeric antigenic receptors (CAR) has emerged as a solution to cure refractory malignancies. However, although CAR T cell treatment of haematological cancers has now shown impressive improvement in outcome, solid tumours have been more challenging to control. The latter type is protected by a strong tumour microenvironment (TME) which might impact cellular therapeutic treatments. Indeed, the milieu around the tumour can become particularly inhibitory to T cells by directly affecting their metabolism. Consequently, the therapeutic cells become physically impeded before being able to attack the tumour. It is therefore extremely important to understand the mechanism behind this metabolic break in order to develop TME-resistant CAR T cells. Historically, the measurement of cellular metabolism has been performed at a low throughput which only permitted a limited number of measurements. However, this has been changed by the introduction of real-time technologies which have lately become more popular to study CAR T cell quality. Unfortunately, the published protocols lack uniformity and their interpretation become confusing. We herein tested the essential parameters to perform a metabolic study on CAR T cells and propose a check list of factors that should be set in order to draw sound conclusion.

DNAM-1 chimeric receptor-engineered NK cells: a new frontier for CAR-NK cell-based immunotherapy.

DNAM-1 is a major NK cell activating receptor and, together with NKG2D and NCRs, by binding specific ligands, strongly contributes to mediating the killing of tumor or virus-infected cells. DNAM-1 specifically recognizes PVR and Nectin-2 ligands that are expressed on some virus-infected cells and on a broad spectrum of tumor cells of both hematological and solid malignancies. So far, while NK cells engineered for different antigen chimeric receptors (CARs) or chimeric NKG2D receptor have been extensively tested in preclinical and clinical studies, the use of DNAM-1 chimeric receptor-engineered NK cells has been proposed only in our recent proof-of-concept study and deserves further development. The aim of this perspective study is to describe the rationale for using this novel tool as a new anti-cancer immunotherapy.

T cells with split CARs specific for NKG2D ligands and PD-L1 exhibit improved selectivity towards monocyte-derived cells while effective in eliminating acute myeloid leukaemia in vivo.

The expression of NKG2D ligands and PD-L1 has been detected on acute myeloid leukaemia (AML) cells, as well as normal cells of the myeloid lineage. To target leukemic cells while minimizing collateral damage to normal cells, we constructed a split dual CAR system based on the AND-gate logic. The NKG2D extracellular domain linked with DAP12 without a co-stimulatory signal was used for the basal activation of T cells, and used together with the PD-L1-specific chimeric costimulatory receptor containing the 4-1BB activating domain for co-stimulatory signal 2 input. This dual CAR displayed cell-type specificity and activity similar as a 2nd generation NKG2D ligand-specific CAR. When compared to CD64 and PD-L1-specific 2nd generation CARs, we observed that the split dual CAR offered an improved myeloid cell type selectivity. For example, PD-L1-specific CAR-T cells lysed all tested myeloid cell types that expressed PD-L1, including M0 macrophages (Mø0), LPS-polarized Mø1, IFN-γ polarized Mø1, IL-4 polarized Mø2, monocytes, immature dendritic cells (imDCs), mature DCs, as well as KG-1 AML cells, while the dual CAR-T cells displaying killing activity only towards LPS polarized Mø1, mature DCs and KG-1 cells that expressed both NKG2D ligands and PD-L1. In a mouse liquid tumor model, the dual CAR-T cells were effective in eradicating established KG-1 AML xenografts. The improved cell type specificity offered by our split dual CAR-T cell system targeting paired antigens would favour the reduction of the on-target off-tumor toxicity towards normal myeloid cells during the treatment of myeloid leukaemia.

Intratumoral pan-ErbB targeted CAR-T for head and neck squamous cell carcinoma: interim analysis of the T4 immunotherapy study

BackgroundLocally advanced/recurrent head and neck squamous cell carcinoma (HNSCC) is associated with significant morbidity and mortality. To target upregulated ErbB dimer expression in this cancer, we developed an autologous CD28-based...

"Armed" CEA CAR-T with a SIRPγ-CD28 chimeric co-receptor to exhibit the enhanced antitumor activity in preclinical study of colorectal cancer.

e14530 Background: Nowadays, CAR-T cell therapy still faces limited therapeutic efficacy for solid tumors. SIRPγ is known as a ligand for a well-known immune checkpoint molecule CD47. It is distinguished from other members of its family in that it has a very short intracytoplasmic tail and is incapable of transducing signals on its own. Of note, the role of SIRPγ still largely unknown to date. Preliminary data indicated SIRPγ plays a key role in T-cell transendothelial migration and promotes antigen-specific T-cell proliferation. We thus designed a novel SIRPγ-CD28 chimeric receptor comprising the extracellular part of SIRPγ, the transmembrane and intracellular domains of CD28 and used as the co-receptor for a CEA-targeting CAR. Here, we report on the activity of this "armed" CAR-T with the SIRPγ-CD28 co-receptor in colorectal tumor (CRC) and its anti-tumor efficacy in mice xenograft model. Methods: In vitro luciferase-based cytotoxicity assay were used by coculturing CAR-T and DLD1-CEA CRC cells to assess the activities of CAR-T w/wo the co-receptor. The incubation supernatants were collected for detecting the release of cytokines. In vivo evaluation of the anti-tumor efficacy of CAR-T with the SIRPγ-CD28 co-receptor versus control T cell, and control CAR-T was performed in NOG mice CRC xenograft model. DLD1-CEA cells expressing luciferase reporter were s.c. implanted and T cells were i.v. injected. Anti-tumor efficacy was assessed by in vivo system. Results: CAR-T with the SIRPγ-CD28 co-receptor exhibited specific cytotoxicity only to CEA+ CRC cells, and the efficacies of CAR-T w/wo the co-receptor were comparable (88.6% vs 87.8%, p > 0.05). However, cytokines were significantly higher in the CAR-T group with the co-receptor. The levels of IFNγ, IL2 and TNFα in groups of CAR-T w/wo the co-receptor were 72,793.33 vs 19,013.33 pg/ml (p < 0.05), 1834.67 vs 25.15 pg/ml (p < 0.05) and 79.65 vs 0 pg/ml (p < 0.05), respectively. In mice CRC xenografts model, the tumors were totally eliminated in the CAR-T group with the co-receptor at 21 days and lasted until the end of experiment, while the tumor growth was suppressed in the control CAR-T group at first and out-of-control in the end (Table). Conclusions: These preclinical studies demonstrated the potential of SIRPγ-CD28 co-receptor as a novel T cellular activation signals for CAR-T. The "armed" CAR-T exhibits a fabulous anti-tumor efficacy in CRC and has great potential for applications in other solid tumors. [Table: see text]

CAR-T Cells Immunotherapies for the Treatment of Acute Myeloid Leukemia-Recent Advances.

In order to increase the effectiveness of cancer therapies and extend the long-term survival of patients, more and more often, in addition to standard treatment, oncological patients receive also targeted therapy, i.e., CAR-T cells. These cells express a chimeric receptor (CAR) that specifically binds an antigen present on tumor cells, resulting in tumor cell lysis. The use of CAR-T cells in the therapy of relapsed and refractory B-type acute lymphoblastic leukemia (ALL) resulted in complete remission in many patients, which prompted researchers to conduct tests on the use of CAR-T cells in the treatment of other hematological malignancies, including acute myeloid leukemia (AML). AML is associated with a poorer prognosis compared to ALL due to a higher risk of relapse caused by the development of resistance to standard treatment. The 5-year relative survival rate in AML patients was estimated at 31.7%. The objective of the following review is to present the mechanism of action of CAR-T cells, and discuss the latest findings on the results of anti-CD33, -CD123, -FLT3 and -CLL-1 CAR-T cell therapy, the emerging challenges as well as the prospects for the future.

Chimeric TIM-4 receptor-modified T cells targeting phosphatidylserine mediates both cytotoxic anti-tumor responses and phagocytic uptake of tumor-associated antigen for T cell cross-presentation

To leverage complementary mechanisms for cancer cell removal, we developed a novel cell engineering and therapeutic strategy co-opting phagocytic clearance and antigen presentation activity into Tcells. We engineered a chimeric engulfment receptor (CER)-1236, which combines the extracellular domain of TIM-4, a phagocytic receptor recognizing the "eat me" signal phosphatidylserine, with intracellular signaling domains (TLR2/TIR, CD28, and CD3ζ) to enhance both TIM-4-mediated phagocytosis and Tcell cytotoxic function. CER-1236 Tcells demonstrate target-dependent phagocytic function and induce transcriptional signatures of key regulators responsible for phagocytic recognition and uptake, along with cytotoxic mediators. Pre-clinical models of mantle cell lymphoma (MCL) and EGFR mutation-positive non-small cell lung cancer (NSCLC) demonstrate collaborative innate-adaptive anti-tumor immune responses both invitro and invivo. Treatment with BTK (MCL) and EGFR (NSCLC) inhibitors increased target ligand, conditionally driving CER-1236 function to augment anti-tumor responses. We also show that activated CER-1236 Tcells exhibit superior cross-presentation ability compared with conventional Tcells, triggering E7-specific TCR T responses in an HLA class I- and TLR-2-dependent manner, thereby overcoming the limited antigen presentation capacity of conventional Tcells. Therefore, CER-1236 Tcells have the potential to achieve tumor control by eliciting both direct cytotoxic effects and indirect-mediated cross-priming.

Chimeric cytokine receptor provides IL-2 signaling for engineered regulatory T cell therapy

Abstract Regulatory T cells (Tregs) are potent immune suppressors and critical to the maintenance of immunological tolerance. Interleukin 2 (IL-2) is essential for Treg survival, expansion, phenotypic stability, and suppressive function. However, Tregs do not produce IL-2 and are dependent on exogenous sources of IL-2. This poses a significant challenge for Treg cell therapies in tissues with no or limited IL-2 availability in the microenvironment. Therefore, we have developed engineered Treg cells containing a chimeric cytokine receptor that provides an IL-2 signal. The chimeric cytokine receptor, termed IL-9 tethered switch receptor (IL-9TSR), consists of interleukin 9 (IL-9) tethered to the extracellular domain of IL9RA paired with the intracellular domain of IL2RB. We have shown that Tregs expressing IL-9TSR can survive and expand in the absence of exogenous IL-2 in vitro and in vivo but require TCR stimulation for prolonged persistency. IL-9TSR Tregs have a stable FOXP3 +HELIOS +phenotype and superior suppressive function when compared to control Tregs. In conclusion, we have generated Treg cells engineered to survive and function in a low IL-2 environment. Treg cell therapies equipped with IL-2 signaling enable increased persistency and allow for potential use in more clinical indications than previously possible.

Generation of novel anti-tumor chimeric antigen receptors incorporating downstream T-cell signaling proteins

Abstract Chimeric antigen receptors (CAR) are molecules with an antibody-derived extracellular domain combined intracellularly with T cell receptor (TCR) signaling proteins. Although CAR-T cells have been a breakthrough clinically, several challenges persist: adverse effects from cytokine secretion, CAR-T cell exhaustion, and limited sensitivity in tumor microenvironments with low density of target antigen (i.e. tumor relapse and solid tumors). While many efforts have focused on identifying new cell surface targets, we have modified CAR intracellular domains. This idea arose from our super-resolution microscopy study that revealed that adapter molecules form clusters distinct from the TCR complex and that full activation necessitates passing a signaling threshold. From these findings, novel Chimeric Adapter Proteins (CAPs) were designed to trigger signaling downstream of the TCRζ chain. CAPs replace the TCRζ with ZAP70 intracellularly and are fused to an extracellular targeting domain. Thus, the potency problem associated with CARs would be ameliorated as CAPs would bypass kinetic proofreading steps defining the signaling threshold and the inhibitory regulation of upstream molecules. Indeed, CAPs exhibited high anti-tumor efficacy, and significantly enhanced long-term in vivo persistence of tumor clearance in leukemia-bearing NSG mice as compared with conventional CD19–28ζ CAR-T. Mechanistically, CAPs were activated in an Lck-independent manner and displayed slower phosphorylation kinetics and a longer duration of signaling compared with 28ζ-CAR. The unique signaling properties of CAPs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an anti-tumor chimeric receptor.

Abstract PR03: ADCLEC.syn1 CAR T cells detect combinatorial target signatures to safely prevent antigen-low AML escape

Abstract CAR therapy is efficacious in relapsed/refractory CD19-positive malignancies including B-ALL. In AML, conventional 2nd-generation CAR therapies are being evaluated clinically. Some of the potential obstacles are antigen-low leukemia escape and on-target toxicities due to widespread CAR target expression in normal hematopoietic cells. We hypothesized that novel targets and fine-tuned combinatorial CAR approaches may enable distinctive target elimination based on differential antigen co-expression patterns in AML vs normal cells, thereby maintaining anti-leukemic efficacy while limiting toxicity on normal cells. Here, we present our target phenotypic profiling of primary AML (n>30 r/r AML patients) and normal hematopoietic cells, and link it with CAR efficacy and changes in normal hematopoiesis. Using spectral flow cytometry on bone marrow and peripheral blood from AML patients and healthy controls, we quantified differential target co-expression of several AML targets, including CD33, CD123, CLEC12A and the novel target ADGRE2. We estimated the numbers of surface molecules per cell and created two-dimensional maps of combinatorial target densities in AML vs normal. In this analysis, the combination of ADGRE2+CLEC12A offered the best therapeutic window. We developed a combinatorial CAR+CCR platform, ADCLEC.syn1, consisting of an ADGRE2-targeting 28z1XX-CAR and a CLEC12A-targeting chimeric costimulatory receptor (CCR) providing additional 4-1BB costimulation. This CAR+CCR configuration triggers killing of cells with high CAR target density alone, whereas cells with low CAR target density are only killed if the CCR target is co-expressed (IF-BETTER gate). Both in vitro and in vivo, we used MOLM13 AML cell line variants matching a variety of different ADGRE2 and CLEC12A target density combinations found in AML or normal hematopoietic cells. The observed target density thresholds for ADCLEC.syn1-mediated cell lysis demonstrated potential for sparing vital normal hematopoietic cells. In addition, we evaluated ADCLEC.syn1 in several molecularly distinct AML PDX models with clinically representative target phenotypes, achieving long-term AML remissions in all studied PDX models. In a humanized mouse model co-engrafted with MOLM13 AML cells, ADCLEC.syn1 T cells rapidly induced complete and durable AML remissions in all mice, with minimal toxicity of normal hematopoietic cells compared to control CARs. Overall, we profiled antigen densities of several CAR target combinations in AML and normal hematopoietic cells, which informed the development of the ADCLEC.syn1 platform. Pre-clinical in vivo models based on AML with clinically representative target densities demonstrated high anti-leukemic efficacy of ADCLEC.syn1 and established killing thresholds with potential for sparing normal hematopoietic cells. A first-in-human phase 1 clinical trial evaluating ADCLEC.syn1 T cells is anticipated to be initiated at MSKCC in 2023. Citation Format: Sascha Haubner, Jorge Mansilla-Soto, Sarah Nataraj, Friederike Kogel, Qing Chang, Elisa De Stanchina, Kathryn Fraser, Jae H. Park, Xiuyan Wang, Isabelle Rivière, Michel Sadelain. ADCLEC.syn1 CAR T cells detect combinatorial target signatures to safely prevent antigen-low AML escape [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr PR03.

Development of a Cell Line Containing the Chimeric ETV6-NTRK3 Gene. The Search for Mutations of the Tyrosine Kinase Chimeric Domain That Cause Resistance to Larotrectinib.

The development, registration, and further use of entrectinib and larotrectinib for the treatment of tumors resulting from oncogenic stimulation of chimeric neurotrophin receptors (TRK) attracted much interest to the mechanisms of tumor cells resistance to TRK inhibitors during treatment. In the presented study, a cell line carrying the chimeric gene ETV6-NTRK3 (HFF-EN) was created on the basis of human fibroblasts. The transcription level of the chimeric ETV6-NTRK3 gene in HFF-EN was comparable to the transcription level of the household ACTB gene, the expression of the ETV6-NTRKA protein was confirmed by immunoblotting. A comparison of the dose-effect curves of fibroblasts and HFF-EN cells showed a ~38-fold increase in the sensitivity of HFF-EN to larotrectinib. To obtain a cell model of the resistance to larotrectinib in NTRK-dependent cancer, we used cell passages with a gradually increasing concentration of larotrectinib and obtained six resistant clones. p.G623E c.1868G>A mutation was found in five clones, and p.R582W c.1744C>T mutation, previously not described as a resistance mutation, was found in one clone showing significantly less resistance. These results can be further used for more complete understanding of the mechanisms of the resistance to TRK inhibitors and for the development of new drugs.

Universal chimeric Fcγ receptor T cells with appropriate affinity for IgG1 antibody exhibit optimal antitumor efficacy.

Developing universal CARs with improved flexible targeting and controllable activities is urgently needed. While several studies have suggested the potential of CD16a in tandem with monoclonal antibodies to construct universal CAR-T cells, the weak affinity between them is one of the limiting factors for efficacy. Herein, we systematically investigated the impact of Fcγ receptor (FcγR) affinity on CAR-T cells properties by constructing universal CARs using Fcγ receptors with different affinities for IgG1 antibodies, namely CD16a, CD32a, and CD64. We demonstrated that the activities of these universal CAR-T cells on tumor cells could be redirected and regulated by IgG1 antibodies. In xenografted mice, 64CAR chimeric Jurkat cells with the highest affinity showed significant antitumor effects in combination with herceptin in the HER2 low expression U251 MG model. However, in the CD20 high expression Raji model, 64CAR caused excessive activation of CAR-T cells, which resulted in cytokine release syndrome (CRS) and the decline of antitumor activity, and 32CAR with a moderate affinity brought the best efficacy. Our work extended the knowledge about FcγR-based universal CAR-T cells and suggested that only the FcγRCAR with an appropriate affinity can offer the optimal antitumor advantages of CAR-T cells.

Modulation of calcium signaling “on demand” to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. Over the past two decades, major advances have been made in our understanding of the disease with the identification of germline or somatic mutations in ion channels and pumps. These mutations enhance calcium signaling, the main trigger of aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, thus mimicking some of the known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the a7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3a named a7-5HT3. Its activation by a selective agonist named PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular sodium and calcium concentrations, cell proliferation, steroid production electrophysiological properties, and gene expression. Treatment of a7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10–9 to 10–5 M) induced a significant increase in intracellular calcium concentrations. Interestingly, whereas cells were hyperpolarized in absence of stimulation (around –60 mV), PSEM-817 induced a strong depolarization, with cells rising to a membrane potential of around –10 mV. The stimulation of calcium signaling by PSEM-817 leads to an increase of CYP11B2 (encoding for aldosterone synthase) mRNA expression and aldosterone biosynthesis but did not affect cell proliferation measured at different time points (8 h, 24 h, 48 h, 72 h). Interestingly, calcium kinetic analyses revealed significant differences in response to the modulation of potassium or sodium entry into cells. Gene expression analyses of gene expression by RNAseq highlighted the activation of specific signaling pathways in response to treatment with Angiotensin II, potassium, and PSEM-817. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Identification of T cell Autoantigens in a mouse model of Lupus Nephritis

Abstract Lupus nephritis (LN) is the most common severe organ manifestation of Systemic lupus erythematosus (SLE). Autoreactive CD4+ T cells play a key role in SLE, but the mechanism of loss of self-tolerance and antigen specificity of CD4+ T cells is unclear. CD4+ αβ T cells use their T cell receptor (TCR) to recognize 12–20 residue-long epitopes presented on class II MHC molecules. Identifying CD4+ T cell epitopes has been challenging due to lack of efficient and easy-to-implement methods for epitope discovery. We developed a robust T cell antigen discovery technology that uses chimeric receptors called SABR-II (Signaling and Antigen-presenting Bifunctional Receptors) to express large peptide antigen libraries in the context of MHC molecules. Using SABR-IIs, we sought to identify the autoantigens recognized by kidney-infiltrating CD4+ T cells in a mouse mode of LN – MRL/lpr mice. We used a high-throughput single cell TCR cloning to clone and express 93 TCRs from CD4+ T cells sorted from kidneys of 26-week-old MRL/lpr mice. For antigen discovery, we constructed SABR-IIs with I-Ek and I-Ak (class II MHC allele of MRL/lpr mice) and confirmed their functionality. We built libraries of ~25,000 epitopes derived from nuclear proteins and validated them using a known antigen-specific TCR. We have identified novel putative autoantigens, which will be validated in vitro. This study will lead to identification of novel self-antigens, providing the groundwork for understanding the biology of LN and for development of preventative and therapeutic approaches in SLE.

Chimeric receptors broaden the therapeutic landscape for autoimmune disease.

Chimeric receptors broaden the therapeutic landscape for autoimmune disease.

Neurofibromatosis Symptom-Lacking B-Cell Lineage Acute Lymphoblastic Leukemia with Only an NF1 Gene Pathogenic Variant

Next-generation sequencing technology has improved molecular genetic analysis, and many molecular genetic studies have been utilized for diagnostic classification, risk stratification, and prognosis prediction of acute lymphoblastic leukemia (ALL). Inactivation of neurofibromin or Nf1, a protein derived from the NF1 gene, causes Ras pathway regulation failure, which is related to leukemogenesis. Pathogenic variants of the NF1 gene in B-cell lineage ALL are uncommon, and in this study, we reported a pathogenic variant that is not registered in any public database. The patient diagnosed with B-cell lineage ALL had no clinical symptoms of neurofibromatosis. Studies on the biology, diagnosis, and treatment of this uncommon disease, as well as other related hematologic neoplasms, such as acute myeloid leukemia and juvenile myelomonocytic leukemia, were reviewed. Biological studies included epidemiological differences among age intervals and pathways for leukemia, such as the Ras pathway. Diagnostic studies included cytogenetic, FISH, and molecular tests for leukemia-related genes and ALL classification, such as Ph-like ALL or BCR-ABL1-like ALL. Treatment studies included pathway inhibitors and chimeric antigen cell receptor T-cells. Resistance mechanisms related to leukemia drugs were also investigated. We believe that these literature reviews will enhance medical care for the uncommon diagnosis of B-cell lineage ALL.