BackgroundPlasmacytoid Dendritic Cells (pDCs) - sometimes referred to as type I interferon producing innate lymphocyte cells - are known as a master regulator of the immune system. The pDCs are ...responsible for the body’s Interferon (IFN) α production, and contain multiple direct effector functions that can mount strong anti-tumor responses. The low number of pDCs in tissues and their frailness have, however, impeded their use in the clinic as adoptive cell therapy. Here we present a first-in-class pDC cell therapy engineered for antigen-dependent activation that results in release of IFNs, direct killing of tumor cells, and recruitment of other innate and adaptive immune cells.MethodsHematopoietic stem and progenitor cells (HSPCs) were used as source material in a GMP-compliant manufacturing process of human pDCs. During early differentiation, HSPCs were genetically engineered with a Synthetic Notch (SynNotch) receptor by lentiviral transduction. In this system, tumor specificity was provided by a single-chain fragment variant (scFv) targeting tumor antigen (here used CD19) and antigen-dependent activation of using a genetic response element, based on gain-of-function STING gene (STINGGoF). In vitro co-culture assays including different CD19+cancer cell lines (REH, NALM6, RAJI) with genetic modified pDCs were used to assess functionality. An in vivo xenograft model of NOG (NOD.Cg-PrkdcscidIl2rgtm1Sug/JicTac) mice subcutaneously engrafted with NALM6 cells to mimic a solid tumor, was used to study the potency of the cell product.ResultsFrom bulk RNA-seq we demonstrate that in vitro co-cultures of pDC-STINGGoF with cancer cell lines elicit a strong and broad gene signature including Type I IFNs and a spectrum of inflammatory cytokines. In cytotoxicity assays, pDC-STINGGoF elicit antigen-dependent killing of all tested cancer cell lines to levels beyond that of NK cells (up to 80% in a 1:2 (T:E) ratio). In NOG mice xenografted with NALM6 tumors and receiving a single intravenous infusion of pDC-STINGGoF, we observed pDC tumor infiltration 48hrs post-injection which was supported by strong tumor regression. Mice treated with non-targeted pDCs exhibit tumor growth similar to control mice.ConclusionsOur genetic engineered pDC is a novel cell therapy that may change the paradigm of solid tumor treatment by the inhibitory effects on tumor cells of Type I interferons, combined with the release of pro-inflammatory cytokines, resulting in recruitment and engagement of other immune cells. This activity is further complemented by pDCs direct tumor cell killing effects. We are currently moving toward phase I to evaluate the safety and efficacy of autologous pDCs.Ethics ApprovalHuman cord blood was collected from Aarhus University Hospital by consent from donors that the material could be used for research under complete anonymisation. The animal experiments have been approved by Danish ethics committee in Region Midt with the ID#2020-15-0201-00394
Hematopoiesis relies on a well-regulated balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) to maintain the lifelong regeneration of blood cells. This homeostasis is ...highly dependent on regulated gene expression both at transcriptional and post-transcriptional levels. Epigenetic regulation has been shown to significantly affect regulation and maintenance of HSC potential. Through an shRNA screen, we were able to identify KDM5C, a H3K4me2/3-demethylase, as a tumour suppressor in acute myeloid leukemia. However, the role of KDM5C in normal hematopoiesis has yet to be investigated.
To explore this question, we generated a conditional Kdm5c knock-out (KO) mouse. Preliminary data shows that loss of Kdm5c perturbs normal hematopoiesis via reduced HSC-fitness. This project aims to gain insight into the mechanisms underlying this phenotype. However, loss of HSCs resulting from Kdm5c-KO, limits the amount material available for downstream analysis, such as ChIPseq, ATACseq etc. To counteract this limitation, we have successfully expanded Kdm5c-WT and -KO HSCs ex vivo. Although both Kdm5c-KO and WT cells were able to expand, expanded KO HSCs presented significantly higher numbers of apoptotic cells, thus reflecting the above-mentioned reduced in vivo fitness. Additionally, upon transplantation, cultured Kdm5c KO cells display significantly reduced engraftment capacity.
Briefly, in our study, we use a promising tool to elucidate the role of KDM5C in HSC regulation in normal hematopoiesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting ...AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Studies reporting beneficial effects of B lymphocytes in autoimmune diseases have been accumulating and a regulatory role for certain B cell subsets is hence getting more and more recognition. ...Recently, B cells were shown to exhibit a regulatory effect in a T cell transfer model of colitis. Here, B cells exposed to enteroantigen (eAg) ex vivo abrogated the colitogenic effect if co‐transplanted with Treg‐depleted (CD4+CD25−) T cells into severe combined immune deficiency (SCID) mice. These data may imply a role for B cells that bind eAg (eAg+ B cells) in the immunopathology of colitis. Here, we report the detection of a subset of eAg+ B cells, including both B2 and B1 lineages, and show that these cells are present in all peripheral lymphoid organs of the mouse including the peritoneal cavity. eAg+ B cells are far more efficient as eAg‐presenting cells than unfractionated splenocytes or eAg− B cells in causing proliferation of eAg‐specific T cells. In comparison with eAg− B cells, eAg+ B cells secrete a significant amount of IL‐10 in vitro, suggesting an anti‐inflammatory potential. Compared with wild‐type B cells, B cell receptor (BCR) transgenic, hen egg lysozyme‐specific B cells show inferior eAg binding and T cell stimulatory activity suggesting involvement of the BCR in eAg binding and processing. After activation of CD19+ B cells by eAg and hybridization with hypoxanthine‐aminopterin‐thymidine (HAT) sensitive ×63 lymphoma cells followed by cloning at limiting dilution conditions, around 10% of the hybridoma cells secrete eAg‐specific antibodies.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
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