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Acute Myeloid Leukemia (AML) is a severe disease with a very high relapse rate. AML relapse may be attributable to leukemic stem cells (LSC). Notably, the “cancer stem cell” theory, which relates to LSCs, is controversial and criticized due to the technical peculiarities of the xenotransplant of human cells into mice. In this study, we searched for possible LSCs in an immunocompetent synergetic mice model. First, we found phenotypic heterogeneity in the ML23 leukemia line. We prospectively isolated a sub-population using the surface markers cKit+CD9−CD48+Mac1−/low, which have the potency to relapse the disease. Importantly, this sub-population can pass in syngeneic hosts and retrieve the heterogeneity of the parental ML23 leukemia line. The LSC sub-population resides in various organs. We present a unique gene expression signature of the LSC in the ML23 model compared to the other sub-populations. Interestingly, the ML23 LSC sub-population expresses therapeutic targeted genes such as CD47 and CD93. Taken together, we present the identification and molecular characterization of LSCs in a syngeneic murine model.

Vaccination and natural infection both elicit potent humoral responses that provide protection from subsequent infections. The immune-history of an individual following such exposures is in part encoded by antibodies. While there are multiple immunoassays for measuring antibody responses, the majority of these methods measure responses to a single antigen. A commonly used method for measuring antibody responses is the enzyme-linked immunosorbent assay (ELISA) - a semi-quantitative assay that is simple to perform in research and clinical settings. Here, we present FLU-LISA (fluorescence-linked immunosorbent assay) - a novel antigen microarray based assay for rapid high-throughput antibody profiling. The assay can be used for profiling IgG, IgA and IgM responses to multiple antigens simultaneously, requiring minimal amounts of sample and antigens. Using several influenza and SARS-CoV-2 antigen microarrays, we demonstrated the specificity and sensitivity of our novel assay and compared it to the traditional ELISA, using samples from mice, chickens and humans. We also showed that our assay can be readily used with dried blood spots, which can be collected from humans and wild birds. The FLU-LISA can be readily used to profile hundreds of samples against dozens of antigens in a single day, and therefore offers an attractive alternative to the traditional ELISA.

Vaccination and natural infection both elicit potent humoral responses that provide protection from subsequent infections. The immune-history of an individual following such exposures is in part encoded by antibodies. While there are multiple immunoassays for measuring antibody responses, the majority of these methods measure responses to a single antigen. A commonly used method for measuring antibody responses is the enzyme-linked immunosorbent assay (ELISA) assay - a semi-quantitative assay that is simple to perform in research and clinical settings. Here we present the ELISA-on-Chip assay - a novel antigen microarray based assay for rapid high-throughput antibody profiling. The assay can be used for profiling IgG, IgA and IgM responses to multiple antigens simultaneously, requiring minimal amounts of sample and antigens. Using three different types of influenza antigen microarrays, we demonstrated the specificity and sensitivity of our novel assay and compared it to the traditional ELISA assay, using samples from mice, chickens and humans. We also showed that our assay can be readily used with dried blood spots, which can be collected from wild birds, as well as from newborns and children. The ELISA-on-Chip assay can be readily used to profile hundreds of samples against dozens of antigens in a single day, and therefore offers an attractive alternative to the traditional ELISA assay.

Translation of the unprecedented efficacy of chimeric antigen receptor (CAR) T-cell technology into solid tumors requires a solution to the problem of on-target damage of vital organs. We have developed an inhibitory CAR platform (iCAR) that imposes self-regulation in CAR T-cells to restrict inappropriate activation against normal cells without compromising tumor efficacy. The system consists of an iCAR that is co-expressed with a conventional activating CAR (aCAR) designed to activate T-cells. The iCAR and aCAR scFvs bind distinct cell-surface antigens that are ubiquitously expressed across all solid tumor histologies and normal tissues. The iCAR is allele-specific whereas the aCAR is pan-allelic. The iCAR technology was validated in functional assays and NSG models with cancer cell lines that mimic loss-of-heterozygosity (LOH) which is common in most human cancers due to increased chromosomal instability. In this context, loss of iCAR target expression due to LOH generates irreversible target specificity. Tumor-specific overexpression of aCAR and iCAR targets, a common barrier to the clinical translation of potent agents targeting the cell-surface proteins is not required. We have shown using in vitro and in vivo models that CAR T-cell activation and target killing are fully inhibited by dual iCAR + aCAR antigen engagement. This outcome validates the concept of iCAR-mediated protection of normal cells in vital organs. In contrast, tumors that mimic iCAR target LOH through CRISPR editing were fully eradicated with aCAR engagement alone. T-cell activation, proliferation, and cytotoxic activity after exposure to target cells lacking iCAR target antigen was found to be quantitatively indistinguishable from the results obtained with single input aCAR CAR T-cells. These results suggest that iCAR technology can enable treatment of cancer patients with solid tumors using CAR T-cells that maximize potency and efficacy by mitigating on-target CRS, neurotoxicity, and inevitable organ damage. The restriction of CAR T activation to the local tumor environment by iCAR-mediated self-regulation directly addresses a fundamental technology gap that cannot be solved by CAR T dosing or aCAR modification alone. Citation Format: Michael R. Weist, Adi Sharbi-Yunger, Jason Yi, Caitlin Schnair, David Bassan, Tanya Kim, Sarit Tabak, Yael Lopesco, Leehee Weinberger, Nir Bujanover, Neta Chaim, Kristina Vucci, Orit Foord, Frank J. Calzone, Rick Kendall, Gregor B. Adams. Rewiring CAR T-cells for absolute solid tumor specificity and safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2826.

Chimeric antigen receptor (CAR)-T cell therapy is a groundbreaking cancer treatment that has produced remarkable clinical efficacy for hematopoietic malignancies, yet “on-target off-tumor” toxicity due to lack of target specificity limits the therapeutic potential of CAR-T cells in solid tumors. We are developing dual CAR-T cells composed of a canonical activator CAR (aCAR), intended to elicit efficacy in solid tumors, and an inhibitory CAR (iCAR) designed to efficiently inhibit the aCAR activity in normal tissue and vital organs. The iCAR and aCAR scFvs of this system bind distinct cell-surface antigens that are widely co-expressed on the normal epithelial from which solid tumors originate. Upon antigen binding, signal propagation through iCAR cytoplasmic domain (iDomain) counteracts aCAR induction of CAR T-cell activation and killing; thus protecting normal tissue. Dual CAR-T cells are activated and kill when exposed to tumor cells that have lost iCAR target expression due to chromosomal loss-of-heterozygosity (LOH) which is common in all cancers. iCAR-targeted LOH generates absolute and irreversible tumor specificity therefore tumor-specific overexpression of the iCAR or aCAR targets is not required. We have carried out a comprehensive screen using human T-cells to identify the most effective inhibitory iDomains derived from cell-surface receptors that naturally inhibit or moderate immune cell activation. The cell-based assay suite developed for this screen consisted of cell lines that co-express iCAR and aCAR target at clinically relevant levels and isogenic partners that mimic LOH through CRISPR editing of the iCAR target. The expression of dual CARs in T cells as biscistronic constructs introduced with lentiviral vectors was successful in a limited number of cases, and it was necessary to develop alternative methods of iCAR aCAR co-expression to complete the screen. Combining aCAR transduction with iCAR mRNA Electroporation (T-REP) proved to be a useful method to disentangle iDomain potency and expression level. The screen identified new iDomains that may expand the potential of iCAR technology in developing strategies to treat solid tumors without compromising efficacy for safety. Citation Format: David Bassan, Jason Yi, Neta Chaim, Nir Bujanover, Sarit Tabak, Tanya Kim, Yael Lopesco, Leehee Weinberger, Kristina Vucci, Michael Weist, Caitlin Schnair, Gregor B. Adams, Orit Foord, Frank J. Calzone, Rick Kendall, Adi Sharbi-Yunger. Incorporation of inhibitory signaling domains into chimeric antigen receptors (iCAR) designed for self-regulation of canonical CAR-T to treat solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2848.

Haematopoietic stem cells (HSCs) have the potential for lifetime production of blood and immune cells. The introduction of transgenes into HSCs is important for basic research, as well as for multiple clinical applications, because HSC transplantation is an already established procedure. Recently, a major advancement has been reported in the use of cyclosporine H (CsH), which can significantly enhance the lentivirus (LV) transduction of human haematopoietic stem and progenitor cells (HSPCs). In this study, we employed CsH for LV transduction of murine HSCs and defined haematopoietic progenitors, confirming previous findings in more specific subsets of primitive haematopoietic cells. Our data confirm increased efficiencies, in agreement with the published data. We further experimented with the transduction with the simultaneous use of several vectors. The use of CsH yielded an even more robust increase in rates of multi-vector infection than the increase for a single-vector. CsH was reported to reduce the innate resistance mechanism against LV infection. We indeed found that additional pretreatment could increase the efficiency of transduction, in agreement with the originally reported results. Our data also suggest that CsH does not reduce the efficiency of transplantation into immune-competent hosts or the differentiation of HSCs while enhancing stable long-term expression in vivo. This new additive will surely help many studies in animal models and might be very useful for the development of novel HSC gene therapy approaches.

Summary Immune cells are generated from hematopoietic stem cells (HSCs) in the bone marrow (BM). Immune stimulation can rapidly activate HSCs out of their quiescent state to accelerate the generation of immune cells. HSCs’ activation follows various viral or bacterial stimuli, and we sought to investigate the hypersensitivity immune response. Surprisingly, the Ova-induced hypersensitivity peritonitis model finds no significant changes in BM HSCs. HSC markers cKIT, SCA1, CD48, CD150, and the Fgd5-mCherry reporter showed no significant difference from control. Functionally, hypersensitivity did not alter HSCs' potency, as assayed by transplantation. We further characterized the possible impact of hypersensitivity using RNA-sequencing of HSCs, finding minor changes at the transcriptome level. Moreover, hypersensitivity induced no significant change in the proliferative state of HSCs. Therefore, this study suggests that, in contrast to other immune stimuli, hypersensitivity has no impact on HSCs., Graphical abstract, Highlights 1. Hematopoietic stem cells (HSCs) can be activated by viral or bacterial immune stimuli 2. Hypersensitivity stimulation does not change the phenotype of HSCs 3. HSCs’ potency and quiescence are not affected by a hypersensitivity response, In this article, Bujanover and colleagues show that hematopoietic stem cells (HSCs) are not significantly affected by hypersensitivity immune response. This is surprising, as many other viral or bacterial immune stimuli elicit robust activation of HSCs. The lack of activation may suggest a decision mechanism by which HSCs retain quiescence and potency even through an acute hypersensitivity response.

Extensive neuronal cell death is among the pathological hallmarks of Alzheimer's disease. While neuron death is coincident with formation of plaques comprising the beta-amyloid (Aβ) peptide, a direct causative link between Aβ (or other Alzheimer's-associated proteins) and cell toxicity is yet to be found. Here we show that BIM-BH3, the primary proapoptotic domain of BIM, a key protein in varied apoptotic cascades of which elevated levels have been found in brain cells of patients afflicted with Alzheimer's disease, interacts with the 42-residue amyloid isoform Aβ42. Remarkably, BIM-BH3 modulated the structure, fibrillation pathway, aggregate morphology, and membrane interactions of Aβ42. In particular, BIM-BH3 inhibited Aβ42 fibril-formation, while it simultaneously enhanced protofibril assembly. Furthermore, we discovered that BIM-BH3/Aβ42 interactions induced cell death in a human neuroblastoma cell model. Overall, our data provide a crucial mechanistic link accounting for neuronal cell death in Alzheimer's disease patients and the participation of both BIM and Aβ42 in the neurotoxicity process.

Summary Hematopoietic stem cells (HSCs) are rare cells that generate all the various types of blood and immune cells. High-quality transcriptome data have enabled the identification of significant genes for HSCs. However, most genes are expressed in various forms by alternative splicing (AS), extending transcriptome complexity. Here, we delineate AS to determine which isoforms are expressed in mouse HSCs. Our analysis of microarray and RNA-sequencing data includes differential expression of splicing factors that may regulate AS, and a complete map of splicing isoforms. Multiple types of isoforms for known HSC genes and unannotated splicing that may alter gene function are presented. Transcriptome-wide identification of genes and their respective isoforms in mouse HSCs will open another dimension for adult stem cells., Graphical Abstract, Highlights • This study presents the splicing landscape in mouse hematopoietic stem cells • Many HSC genes express multiple isoforms, some with alternative isoforms • High-resolution analysis allows the discovery of unannotated splicing • HSC-expressed isoforms can have similar or divergent functions, Gazit and colleagues mapped all splicing within mouse hematopoietic stem cells by analysis of microarrays and meta-analysis of RNA-sequencing datasets. They validated co-expression of minor isoforms, variant isoform predominance, multiple isoforms from one gene, and discovery of unannotated splicing. Functionally, variant isoforms may alter the activity of HSC genes, or not. Whole-transcriptome splicing data at nucleotide resolution is providing a useful tool for the community.

Haematopoietic stem cells (HSCs) have the potential for lifetime production of blood and immune cells. The introduction of transgenes into HSCs is important for basic research, as well as for multiple clinical applications, because HSC transplantation is an already established procedure. Recently, a major advancement has been reported in the use of cyclosporine H (CsH), which can significantly enhance the lentivirus (LV) transduction of human haematopoietic stem and progenitor cells (HSPCs). In this study, we employed CsH for LV transduction of murine HSCs and defined haematopoietic progenitors, confirming previous findings in more specific subsets of primitive haematopoietic cells. Our data confirm increased efficiencies, in agreement with the published data. We further experimented with the transduction with the simultaneous use of several vectors. The use of CsH yielded an even more robust increase in rates of multi-vector infection than the increase for a single-vector. CsH was reported to reduce the innate resistance mechanism against LV infection. We indeed found that additional pretreatment could increase the efficiency of transduction, in agreement with the originally reported results. Our data also suggest that CsH does not reduce the efficiency of transplantation into immune-competent hosts or the differentiation of HSCs while enhancing stable long-term expression in vivo. This new additive will surely help many studies in animal models and might be very useful for the development of novel HSC gene therapy approaches.