Cancer immunotherapies are highly potent and are gaining wide clinical usage. However, severe side effects require focusing effector immune cell activities on the tumor microenvironment (TME). We ...recently developed a chimeric antigen receptor tumor-induced vector (CARTIV), a synthetic promoter activated by TME factors. To improve CARTIV functions including background, activation levels, and synergism, we screened a library of promoters with variations in key positions. Here, we present a screening method involving turning ON/OFF stimulating TNFα and IFNγ cytokines, followed by sequential cell sorting. Sequencing of enriched promoters identified seventeen candidates, which were cloned and whose activities were then validated, leading to the identification of two CARTIVs with lower background and higher induction. We further combined a third hypoxia element with the two-factor CARTIV, demonstrating additional modular improvement. Our study presents a method of fine-tuning synthetic promoters for desired immunotherapy needs.
IL-2 is the master-regulator cytokine for T cell dependent responses and is crucial for proliferation and survival of T cells. However, IL-2-based treatments remained marginal, in part due to short ...half-life. Thus, we aimed to extend IL-2 half-life by flanking the IL-2 core with sequences derived from the extensively glycosylated hinge region of the NCR2 receptor. We termed this modified IL-2: "S2A". Importantly, S2A blood half-life was extended 14-fold compared to the clinical grade IL-2, Proleukin. Low doses inoculation of S2A significantly enhanced induction of Tregs (CD4
Regulatory T cells) in vivo, as compared to Proleukin, while both S2A and Proleukin induced low levels of CD8
T cells. In a B16 metastatic melanoma model, S2A treatment was unable to reduce the metastatic capacity of B16 melanoma, while enhancing induction and recruitment of Tregs, compared to Proleukin. Conversely, in two autoimmune models, rheumatoid arthritis and DSS-induced colitis, S2A treatment significantly reduced the progression of disease compared to Proleukin. Our results suggest new avenues for generating long-acting IL-2 for long-standing treatment and a new technique for manipulating short-life proteins for clinical and research uses.
Abstract
Lysine acetylation has been discovered in thousands of non-histone human proteins, including most metabolic enzymes. Deciphering the functions of acetylation is key to understanding how ...metabolic cues mediate metabolic enzyme regulation and cellular signaling. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is acetylated on multiple lysine residues. Using site-specifically acetylated G6PD, we show that acetylation can activate (AcK89) and inhibit (AcK403) G6PD. Acetylation-dependent inactivation is explained by structural studies showing distortion of the dimeric structure and active site of G6PD. We provide evidence for acetylation-dependent K95/97 ubiquitylation of G6PD and Y503 phosphorylation, as well as interaction with p53 and induction of early apoptotic events. Notably, we found that the acetylation of a single lysine residue coordinates diverse acetylation-dependent processes. Our data provide an example of the complex roles of acetylation as a posttranslational modification that orchestrates the regulation of enzymatic activity, posttranslational modifications, and apoptotic signaling.
Adult hematopoietic stem cells (HSCs) in the bone marrow (BM) are quiescent. Following perturbations, such as blood loss or infection, HSCs may undergo activation. Surprisingly, little is known about ...the earliest stages of HSCs activation. We utilize surface markers of HSCs activation, CD69 and CD317, revealing a response as early as 2 h after stimulation. The dynamic expression of HSCs activation markers varies between viral-like (poly-Inosinic-poly-Cytidylic) or bacterial-like (Lipopolysaccharide) immune stimuli. We further quantify dose response, revealing a low threshold, and similar sensitivity of HSCs and progenitors in the BM. Finally, we find a positive correlation between the expression of surface activation markers and early exit from quiescence. Our data show that the response of adult stem cells to immune stimulation is rapid and sensitive, rapidly leading HSCs out of quiescence.
Animal models are necessary to study cancer and develop treatments. After decades of intensive research, effective treatments are available for only a few types of leukemia, while others are ...currently incurable. Our goal was to generate novel leukemia models in immunocompetent mice. We had achieved abilities for overexpression of multiple driving oncogenes simultaneously in normal primary cells, which can be transplanted and followed in vivo. Our experiments demonstrated the induction of primary malignant growth. Leukemia lines that model various types of leukemia, such as acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL), were passaged robustly in congenic wild-type immunocompetent mice. These novel leukemia lines, which may complement previous models, offer the flexibility to generate tailored models of defined oncogenes of interest. The characterization of our leukemia models in immunocompetent animals can uncover the mechanisms of malignancy progression and offer a unique opportunity to stringently test anti-cancer chemotherapies.
Most human genes code for more than one transcript. Different ratios of transcripts of the same gene can be found in different cell types or states, indicating differential use of transcription start ...sites or differential splicing. Such differential transcript use (DTUs) events provide an additional layer of regulation and protein diversity. With the exceptions of PTPRC and CIITA, there are very few reported cases of DTU events in the immune system. To rigorously map DTUs between different human immune cell types, we leveraged four publicly available RNA sequencing datasets. We identified 282 DTU events between five human healthy immune cell types that appear in at least two datasets. The patterns of the DTU events were mostly cell-type-specific or lineage-specific, in the context of the five cell types tested. DTUs correlated with the expression pattern of potential regulators, namely, splicing factors and transcription factors. Of the several immune related conditions studied, only sepsis affected the splicing of more than a few genes and only in innate immune cells. Taken together, we map the DTUs landscape in human peripheral blood immune cell types, and present hundreds of genes whose transcript use changes between cell types or upon activation.
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.
The Shraga Segal department of Microbiology Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev.
Hematopoietic Stem Cells (HSCs) are the continuous source for all ...types of blood and immune cells. HSCs are mostly quiescent on healthy adult life, but they activate following immunological stimuli. We can prospectively isolate naïve-HSCs; however, there is relatively little understanding of immune-activated HSCs. Part of the difficulties come from usage of multiple surface-markers, some of which are changing drastically after stimuli. We found the Fgd5-mCherry reporter mouse to allow better identification of HSCs. Importantly, we revealed differently expressed surface markers, including CD317 (also known as Bst2) and CD69, being the first HSC-activation markers.
Interestingly, various types of immune stimuli cause different activation of HSCs, or even the lack of activation. Furthermore, chronic long-term immune stimulation exerts deleterious effects on HSCs, and may predispose toward malignancy, which is exposed using p53-mutated mice model. In the Myeloid Meeting 2022 we intend to present unpublished data quantifying dosage and timing of HSCs activation following various stimuli. Chronic bacterial infection can cause unexpected prolonged activation of HSCs with, or w/o, severe loss of transplantation potency. Finally, we do focus on the acute-phase of HSC activation, finding surprisingly fast and diverse responses to viral-type or bacterial-stimuli.
Taken together, our studies are providing insights into the acute- and chronic-activation of HSCs, with implications to pathogen infection and pre-disposition to malignancies. Understanding the regulation of HSCs will open opportunities to boost essential immune responses, or to prophylactic reduce risk of exhaustion.
Harnessing immune effector cells to benefit cancer patients is becoming more and more prevalent in recent years. However, the increasing number of different therapeutic approaches, such as chimeric ...antigen receptors and armored chimeric antigen receptors, requires constant adjustments of the transgene expression levels. We have previously demonstrated it is possible to achieve spatial and temporal control of transgene expression as well as tailoring the inducing agents using the Chimeric Antigen Receptor Tumor Induced Vector (CARTIV) platform. Here we describe the next level of customization in our promoter platform. We have tested the functionality of three different minimal promoters, representing three different promoters’ strengths, leading to varying levels of CAR expression and primary T cell function. This strategy shows yet another level of CARTIV gene regulation that can be easily integrated into existing CAR T systems.
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