The Crooks Lab has published its most recent research in Nature Methods (Online publication 03 April 2017):
Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids
Christopher S Seet, Chongbin He, Michael T Bethune, Suwen Li, Brent Chick, Eric H Gschweng, Yuhua Zhu, Kenneth Kim, Donald B Kohn, David Baltimore, Gay M Crooks & Amelie Montel-Hagen
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".
Chris was also awarded the American Association of Immunologists Young Investigator Award at Immunology2015 for best poster presentation.
- 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.
KTCC 2017 13-17 MARCH KyotO
Dr. Stephanie C. De Barros from the Crooks Lab presented her work at KTCC this year. Here is her abstract selected for oral presentation:
VEGF regulates neonatal thymic epithelium indirectly through NRP1 signaling on thymic mesenchyme.
AAAAI 2017 3-6 MARCH ATLANTA
Dr. Vanessa Bundy from the Crooks Lab presented her work at the ASH meeting. Here is her abstract selected for poster presentation:
The Opposing Roles of Let-7c and Mir-125-b2 in Human Hematopoietic Stem Cell Maintenance and Proliferation
RATIONALE: Hematopoietic stem cells (HSCs) possess extensive selfrenewal, proliferation and differentiation capacities that enable them to
sustain long-term repopulation of all hematopoietic cell types. This process requires an intricate balance between self-renewal and differentiation.
Micro RNAs (miRNAs) are known to regulate transcription, but precise molecular networks affecting HSC maintenance remain poorly understood.
METHODS: Our laboratory used microarray to identify a genomic cluster of miRNAs (miR-99a/let-7c/miR-125b2) that is highly expressed in hematopoietic stem cells and almost absent in progenitors and mature cells. To elucidate the impact of miRNA expression in human HSCs, we generated lentiviral overexpression vectors for let-7c, miR-125b2, miR99a and the entire cluster. ‘‘Sponge’’ inhibition vectors were generated for let- 7c and miR-125b2. Vector mediated gene transfer studies included in vitro assays of differentiation, long-term culture initiation cells (LTC-ICs), proliferation and survival assays and in vivo mouse transplantation assays.
RESULTS: Let-7c overexpression decreased CD34+ cells, reduced LTCICs and increased myeloid cell output. MiR-125b2 overexpression
expanded CD34+ cells and LTC-ICs. Cluster overexpression revealed an intermediate phenotype between Let-7c and mir-125b using in vitro and in vivo (primary and secondary transplant) assays. Let-7c inhibition showed markedly increased cell proliferation and CD34+ cell output, with decreased myeloid differentiation.
CONCLUSIONS: Our data suggest that let-7c may function within the cluster to modulate the profound proliferative effects of miR-125b2
activity in HSCs. We hypothesize that sponge inhibition of let-7c enhances HSC proliferation due to unopposed miR-125b2. Future studies will use RNA-Seq to explore the transcriptional networks through which let-7c and miR-125b2 modify the behavior of HSCs.
ASH 2016 3-6 DECEMBER SAN DIEGO
Dr. Chris Seet from the Crooks Lab presented his work at the ASH meeting. Here is her abstract selected for poster presentation:
Artificial Thymic Organoids Permit Allelic Exclusion and Efficient Generation of Naïve TCR-Engineered T-Cells from Human Hematopoietic Stem Cells In Vitro
Engineered adoptive immunotherapies have shown unprecedented activity in the treatment of cancer and chronic viral infections. Current approaches rely on individualized ex vivo genetic modification of autologous T cells due to the risk of graft-versus-host disease from allogeneic T cells. These processes furthermore require activation and prolonged expansion of T cells, which may reduce in vivo efficacy and persistence. Direct in vitro differentiation of engineered T cells from hematopoietic stem and progenitor cells (HSPCs) may overcome these problems by permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. Existing methods of in vitro human T cell differentiation are subject to wide experimental variability and do not adequately support the positive selection of immature T cell precursors to mature T cells, and thus have not been suitable for clinical-scale production of engineered T cells.
We report here the preclinical development of an artificial thymic organoid (ATO) system using off-the-shelf, serum-free components and a standardized stromal cell line that supports highly efficient in vitro differentiation and positive selection of native and TCR-engineered human T cells from cord blood (CB), bone marrow, and mobilized peripheral blood CD34+ HSPCs, and purified CD34+CD38- hematopoietic stem cells. ATOs closely recapitulated thymic T cell commitment and differentiation, resulting in greater than 80% CD7+CD5+ T-lineage cells and 50% CD4+CD8+ double positive (DP) T cell precursors by 4 weeks. By 6 weeks, 30-40% of ATO cells were CD3+TCRαβ+ T cells, of which 20-30% were mature CD8 single positive (SP) T cells. CD4SP cells were generated at a lower frequency and later in culture (2-14% of CD3+TCRαβ+ cells). ATO-derived T cells exhibited a naïve CD45RA+CD27+CCR7+CD62L+ phenotype, a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation.
Transduction of CB CD34+ HSPCs with an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell output per ATO (>400-fold, relative to input HSPCs) and enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. Positive selection of TCR-engineered naïve T cells could be further enhanced by expression of cognate HLA-A*02:01 in ATO stromal cells. ATO-derived TCR-engineered T cells exhibited a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. ATO-derived engineered T cells underwent antigen-specific cytotoxic priming, polyfunctional cytokine release, and proliferation in response to artificial APCs; and exhibited antigen-specific killing of NY-ESO-1+ tumor cells in vitro and in vivo.
ATOs thus present a highly efficient off-the-shelf platform for the generation of clinically relevant numbers of naïve and potentially non-alloreactive engineered T cells for adoptive immunotherapy. Clinical translation of the ATO system will be aided by its simplicity, scalability, use of serum-free components, and compatibility with irradiated stromal cells. In addition, genetic manipulation of stem or stromal cell components can be easily incorporated into the system to further enhance downstream T cell engraftment or function.