RESUMO
The transcription factor B cell CLL/lymphoma 11B (BCL11B) is indispensable for T lineage development of lymphoid progenitors. Here, we show that chimeric antigen receptor (CAR) expression during early phases of ex vivo generation of lymphoid progenitors suppressed BCL11B, leading to suppression of T cell-associated gene expression and acquisition of NK cell-like properties. Upon adoptive transfer into hematopoietic stem cell transplant recipients, CAR-expressing lymphoid progenitors differentiated into CAR-induced killer (CARiK) cells that mediated potent antigen-directed antileukemic activity even across MHC barriers. CD28 and active immunoreceptor tyrosine-based activation motifs were critical for a functional CARiK phenotype. These results give important insights into differentiation of murine and human lymphoid progenitors driven by synthetic CAR transgene expression and encourage further evaluation of ex vivo-generated CARiK cells for targeted immunotherapy.
Assuntos
Antígenos CD28/metabolismo , Células Matadoras Naturais/citologia , Linfócitos/citologia , Receptores de Antígenos Quiméricos/metabolismo , Proteínas Repressoras/metabolismo , Linfócitos T/citologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Antígenos CD19/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Separação Celular , Citotoxicidade Imunológica , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoterapia , Imunoterapia Adotiva , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Engenharia de Proteínas , Células-Tronco/citologia , TransgenesRESUMO
Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer.
