The Crooks Lab has published its most recent research in Cell Stem Cell: 2019 Jan 4. pii: S1934-5909(18)30601-5. doi: 10.1016/j.stem.2018.12.011.
organoid-induced differentiation of conventional t cells from human pluripotent stem cells
Montel-Hagen A, Seet CS, Li S, Chick B, Zhu Y, Chang P, Tsai S, Sun V, Lopez S, Chen HC, He C, Chin CJ, Casero D, Crooks GM.
Christopher Seet was awarded a Tower Cancer Research Foundation Career Development Award for a project to identify novel mechanisms of immune evasion in acute myeloid leukemia. This provides $100,000 in research funding to "physician scientists who are pursuing a career in academic medicine focusing on a bench-to-bedside approach".
The Crooks Lab was awarded a new grant from CIRM (DISC2-10134): The grant provides funding for the development of a Platform Technology for Pluripotent Stem Cell-Derived T cell Immunotherapy.
Kite Pharma, Inc. (Santa Monica) has entered into an exclusive, worldwide license agreement with The Regents of the University of California, on behalf of the University of California, Los Angeles (UCLA), for technology developed in the Crooks Lab to advance the development of off-the-shelf allogeneic T-cell therapies from renewable pluripotent stem cells . Kite has entered into a Sponsored Research Agreement with UCLA to support ongoing preclinical research in Dr. Crooks' laboratory.
The Crooks lab, in collaboration with Hanna Mikkola and Yi Xing at UCLA was awarded a new grant from the CIRM Stanford/Salk Center of Excellence in Stem Cell Genomics (CESG). The grant provides $750,000 to the team who will isolate a range of cell types, from the most primitive or early stage stem cells to the many diverse blood cells produced by the stem cells, and then perform genomic analysis to understand how the gene networks function together to control normal blood formation during life. These studies will also provide the knowledge to generate blood from pluripotent stem cells and to understand how leukemia develops when genes become mutated.
ASH 2017 9-12 DECEMBER Atlanta
Dr. Chris Seet from the Crooks Lab presented his work at the ASH meeting. Here is his abstract selected for oral presentation:
Notch Signaling Promotes the Differentiation of Human CLEC9A+ Dendritic Cells from Hematopoietic Stem and Progenitor Cells
Human CLEC9A+ dendritic cells (also known as CD141+ DCs, XCR1+ DCs, and cDC1) are an endogenous DC subset with constitutive ability to traffic to secondary lymphoid tissues, efficiently crosspresent cellular antigens, and prime CD4+ and CD8+ antiviral and antitumor T cell responses. As such, CLEC9A+ DCs have been proposed as ideal candidates for adoptive cell immunotherapy, however their rarity in vivo and a lack of knowledge regarding their development have thus far precluded their therapeutic use. We used in vitro differentiation approaches to identify novel microenvironmental cues that regulate the development of CLEC9A+ DCs from human CD34+ hematopoietic stem and progenitor cells (HSPCs). Our findings reveal a previously unknown role for Notch signaling as a key regulatory pathway in CLEC9A+ DC differentiation from HSPCs. Exposure of HSPCs to the Notch ligand DLL1 expressed by MS5 stromal cells, together with the DCpromoting cytokines FLT3L and GMCSF, markedly enhanced the generation of CLEC9A+ DCs relative to control cultures lacking DLL1 expression. Resulting CLEC9A+ DC frequency was on average 66% of total cells in DLL1 cultures, compared with 6% in control cultures at 20 days (p < 0.0001). Promotion of CLEC9A+ DC development was accompanied by statistically significant decreases in monocyte, CD1c+ DC, and plasmacytoid DC frequencies and cell numbers, with granulocytic differentiation relatively unaffected. Similar changes were seen when DLL1 was substituted for the Notch ligands DLL4 or JAG1, and abrogated by gamma secretase inhibition. Furthermore, promotion of CLEC9A+ DC differentiation was mimicked by transduction of HSPCs with intracellular Notch1, consistent with a cellintrinsic role for Notch in CLEC9A+ DC development. The effect of Notch signaling on CLEC9A+ DC development was conserved between HSPCs isolated from human cord blood (CB), bone marrow (BM), or GCSF mobilized peripheral blood (MPB). Furthermore, highly purified BM monocyte/dendritic cell progenitors (MDP) or common dendritic cell progenitors (CDP) cultured in the presence of DLL1 preferentially generated CLEC9A+ DCs with loss of alternative lineage outputs, supporting a mechanism by which Notch signaling skews lineage commitment to the CLEC9A+ DC lineage at the level of multipotent DC progenitors.
Notch induced CLEC9A+ DCs derived from HSPCs were phenotypically and functionally similar to primary CLEC9A+ DCs isolated from the blood. Whole transcriptome profiling by RNAseq of Notchinduced CLEC9A+ DCs confirmed lineagespecific upregulation of key CLEC9A+ DC genes including XCR1, CADM1, BATF3, and IRF8. Furthermore, global gene expression profiles were similar between Notchinduced CLEC9A+ DCs generated from HSPCs isolated from CB or MPB. Notchinduced CLEC9A+ DCs expressed high levels of CD62L and CCR7, and underwent efficient chemotaxis in response to CCL21 in transwell migration assays, suggesting lymph nodehoming capacity. Compared to primary blood CLEC9A+ DCs, Notchinduced CLEC9A+ DCs exhibited higher basal surface expression of T cell costimulatory molecules including CD80, CD83, and CD86, reminiscent of dermal CLEC9A+ DCs, and these were further upregulated in response to the TLR agonists poly(I:C) or R848. Notchinduced CLEC9A+ DCs exhibited potent CD4+ and CD8+ immunostimulatory activity in mixed lymphocyte reactions, and induced antigenspecific CD8+ T cell responses to an HLAA*0201restricted NYESO1 epitope through either
crosspresentation of cellular antigen acquired from necrotic tumor cells, or endogenous presentation of antigen transduced at the CD34+ HSPC stage.
In conclusion, we have identified a novel role for Notch signaling in the differentiation of CLEC9A+ DCs from HSPCs. We propose Notch signaling enforces CLEC9A+ DC lineage commitment in multipotent DC progenitors, and RNAseq studies addressing the molecular basis of Notch induced CLEC9A+ DC commitment are in progress. Importantly, the identification of Notch as a critical regulatory pathway in human CLEC9A+ DC development allows robust in vitro differentiation of large numbers of highly functional CLEC9A+ DCs. Taken together our findings provide insight into the development of this important DC lineage and permit the preclinical development of next generation cellular vaccine strategies using in vitro derived CLEC9A+ DCs.
ASH 2017 9-12 DECEMBER Atlanta
Dr. Amelie Montel-Hagen from the Crooks Lab presented her work at the ASH meeting. Here is her abstract selected for oral presentation:
In vitro Generation of human pluripotent stem cell-derived T cells for immunotherapy
Adoptive cell therapy using T cells engineered to express antigen-specific T cell receptors (TCR-T) or chimeric antigen receptors (CAR-T) offer targeted and potentially curative treatments for malignancy. Current approaches rely on the genetic modification and expansion of mature circulating T-cells. Such processes are limited to autologous T cells due to the risk of graft-versus-host (GvHD) disease from allogeneic T cells through endogenous TCR expression as well as rejection through MHC incompatibility. Furthermore, prolonged ex-vivo expansion of T cells may reduce in vivo efficacy and harvesting sufficient T cells from lymphopenic patients is challenging. Direct in vitro differentiation of engineered T cells from human pluripotent stem cells (HSPCs) may overcome these problems by providing an unlimited source of cells that can be genetically edited, permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. We have developed an in vitro Artificial Thymic Organoid (ATO) system that induces highly efficient and reproducible production of mature naïve T cells from human hematopoietic stem cells and progenitor cells (HSPC). Here, we
report the preclinical development of a modified ATO system that supports highly efficient in vitro differentiation and positive selection of naive human T cells from at least 5 different lines of human pluripotent stem cells (PSC), including Embryonic stem cells (ESC) and induced Pluripotent Stem Cells (iPSC). T cell differentiation from PSC was very similar phenotypically to that from HSPC. As in normal human thymopoiesis, the first evidence for T cell commitment was expression of CD7 and CD5, followed by the CD3-CD8lo “ISP8” stage, then CD4+CD8+ “DP” stage and finally production of CD3+CD8+CD4- “CD8SP” and Cd3+CD4+CD8- “CD4SP”. As is typical with both monolayer cultures and ATOs (and opposite to normal thymus), CD8SP predominated over CD4SP. Surprisingly, differentiation occurred more rapidly from PSC than with HSPC. As with HSPC-ATOs, CD8SP from PSC ATOs showed a mature naïve conventional T cell phenotype i.e. CD3+TCRab+CD4- CD45RA+CD62L+CD27+ and exhibited a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation. The differentiation in ATOs of an ESC line that expresses an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell yield with an enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. TCR-engineered T cells produced from PSC in ATOs displayed a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. The TCR engineered T cells underwent polyfunctional cytokine release, and proliferation in response to artificial APCs. Moreover, the differentiation in ATOs of an ESC line that expresses a CD19-specific 2nd generation (CD28/CD3zeta) CAR construct resulted in the production of CD5+CD7+ CD45RA+ CAR T cells. As reported previously, the ESC-
derived CAR T cells did not express CD4, CD8 or CD3; however, they responded to PMA/ionomycin and underwent specific cytokine release and degranulation in response to target cells expressing CD19.
PSC-derived ATOs thus present a highly efficient platform for the generation of clinically relevant mature naïve and potentially non-alloreactive TCR and CAR engineered T cells for adoptive immunotherapy.