RESUMEN
The quality of chimeric antigen receptor (CAR)-T cell products, namely, memory and exhaustion markers, affects the long-term functionality of CAR-T cells. We previously reported that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit a memory-rich phenotype that is characterized by the high proportion of CD45RA+/C-C chemokine receptor type 7 (CCR7)+ T-cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA- peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA- CAR, respectively), and compared their phenotypes and antitumor activity. RA+ CAR-T cells showed better transient gene transfer efficiency 24 h after transduction and superior expansion capacity after 14 days of culture than those shown by RA- CAR-T cells. RA+ CAR-T cells exhibited dominant CD8 expression, decreased expression of the exhaustion marker programmed cell death protein-1 (PD-1) and T-cell senescence marker CD57, and enriched naïve/stem cell memory fraction, which are associated with the longevity of CAR-T cells. Transcriptome analysis showed that canonical exhaustion markers were downregulated in RA+ CAR-T, even after antigen stimulation. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, the expression of CAR molecules on cell surface after antigen stimulation in RA+ CAR-T cells was controlled at a relatively lower level than that in RA- CAR-T cells. In the in vivo stress test, RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells compared with RA- CAR-T cells. CAR-T cells were not detected in the control or RA- CAR-T cells but RA+ CAR-T cells were expanded even after 50 days of treatment, as confirmed by sequential bone marrow aspiration. Our results suggest that PB-mediated RA+ CAR-T cells exhibit a memory-rich phenotype and superior antitumor function, thus CD45RA+ PBMCs might be considered an efficient starting material for PB-CAR-T cell manufacturing. This novel approach will be beneficial for effective treatment of B cell malignancies.
Asunto(s)
Antígenos CD19/genética , Elementos Transponibles de ADN/genética , Antígenos Comunes de Leucocito/genética , Leucocitos Mononucleares/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Animales , Antígenos CD19/inmunología , Línea Celular Tumoral , Femenino , Humanos , Inmunoterapia Adoptiva/métodos , Antígenos Comunes de Leucocito/inmunología , Ratones , Ratones Endogámicos NOD , Fenotipo , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunologíaRESUMEN
Cytosolic chaperonin containing t-complex polypeptide 1 (CCT)/TRiC is a group II chaperonin that assists in the folding of newly synthesized proteins. It is a eukaryotic homologue of the bacterial group I chaperonin GroEL. In contrast to the well studied functions of GroEL, the substrate recognition mechanism of CCT/TRiC is poorly understood. Here, we established a system for analyzing CCT/TRiC functions by using a reconstituted protein synthesis by using recombinant elements system and show that CCT/TRiC strongly recognizes WD40 proteins particularly at hydrophobic beta-strands. Using the G protein beta subunit (Gbeta), a WD40 protein that is very rich in beta-sheets, as a model substrate, we found that CCT/TRiC prevents aggregation and assists in folding of Gbeta, whereas GroEL does not. Gbeta has a seven-bladed beta-propeller structure; each blade is formed from a WD40 repeat sequence encoding four beta-strands. Detailed mutational analysis of Gbeta indicated that CCT/TRiC, but not GroEL, preferentially recognizes hydrophobic residues aligned on surfaces of beta-strands in the second WD40 repeat of Gbeta. These findings indicate that one of the CCT/TRiC-specific targets is hydrophobic beta-strands, which are highly prone to aggregation.
Asunto(s)
Chaperoninas/metabolismo , Proteínas de Unión al GTP/metabolismo , Pliegue de Proteína , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Chaperonina 60/metabolismo , Chaperonina con TCP-1 , Chaperoninas/química , Chaperoninas/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/metabolismo , Alineación de SecuenciaRESUMEN
Transfer RNA (guanosine-2')-methyltransferase (Gm-methylase) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to 2'-OH of G18 in the D-loop of tRNA. Based on their mode of tRNA recognition, Gm-methylases can be divided into the following two types: type I having broad specificity toward the substrate tRNA, and type II that methylates only limited tRNA species. Protein synthesized by in vitro cell-free translation revealed that Gm-methylase encoded in the Aquifex aeolicus genome is a novel type II enzyme. Experiments with chimeric tRNAs and mini- and micro-helix RNAs showed that the recognition region of this enzyme is included within the D-arm structure of tRNALeu and that a bulge is essentially required. Variants of tRNALeu, tRNASer, and tRNAPhe revealed that a combination of certain base pairs in the D-stem is strongly recognized by the enzyme, that 4 bp in the D-stem enhance methyl acceptance activity, and that the Py16Py17G18G19 sequence is important for efficient methyl transfer. The methyl acceptance activities of all the A. aeolicus tRNA genes, which can be classified into 14 categories on the basis of their D-arm structure, were tested. The results clearly showed that the substrate recognition mechanism elucidated by the variant experiments was applicable to their native substrates.