Adoptive immunotherapy with functional T cells is potentially an effective therapeutic strategy for combating many types of cancer and viral infection. However, exhaustion of antigen-specific T cells represents a major challenge to this type of approach. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8(+) T cells from an HIV-1-infected patient to pluripotency. The T cell-derived induced pluripotent stem cells were then redifferentiated into CD8(+) T cells that had a high proliferative capacity and elongated telomeres. These "rejuvenated" cells possessed antigen-specific killing activity and exhibited T cell receptor gene-rearrangement patterns identical to those of the original T cell clone from the patient. We also found that this method can be effective for generating specific T cells for other pathology-associated antigens. Thus, this type of approach may have broad applications in the field of adoptive immunotherapy.
SUMMARY
Understanding the relative contributions of genetic and epigenetic
abnormalities to acute myeloid leukemia (AML) should assist integrated design of
targeted therapies. In this study, we generated induced pluripotent stem cells
(iPSCs) from AML patient samples harboring MLL rearrangements and found that
they retained leukemic mutations but reset leukemic DNA methylation/gene
expression patterns. AML-iPSCs lacked leukemic potential, but when
differentiated into hematopoietic cells, they reacquired the ability to give
rise to leukemia in vivo and reestablished leukemic DNA methylation/gene
expression patterns, including an aberrant MLL signature. Epigenetic
reprogramming was therefore not sufficient to eliminate leukemic behavior. This
approach also allowed us to study the properties of distinct AML subclones,
including differential drug susceptibilities of KRAS mutant and wild-type cells,
and predict relapse based on increased cytarabine resistance of a KRAS wild-type
subclone. Overall, our findings illustrate the value of AML-iPSCs for
investigating the mechanistic basis and clonal properties of human AML.
Jawless vertebrates have acquired immunity but do not have immunoglobulin-type antigen receptors. Variable lymphocyte receptors (VLRs) have been identified in lamprey that consist of multiple leucine-rich repeat (LRR) modules. An active VLR gene is generated by the assembly of a series of variable gene segments, including many that encode LRRs. Stepwise assembly of the gene segments seems to occur by replacement of the intervening DNA between the 5' and 3' constant-region genes. Here we report that lamprey (Lethenteron japonicum) assemble their VLR genes by a process involving 'copy choice'. Regions of short homology seemed to prime copying of donor LRR-encoding sequences into the recipient gene. Those LRR-encoding germline sequences were abundant and shared extensive sequence homologies. Such genomic organization permits initiation of copying anywhere in an LRR-encoding module for the generation of various hybrid LRRs. Thus, a vast repertoire of recombinant VLR genes could be generated not only by copying of various LRR segments in diverse combinations but also by the use of multiple sites in an LRR gene segment for priming.
Highlights d AAV6 is an effective donor delivery vector for genome editing in hPSCs d Electroporation of Cas9 RNP prior to AAV6 transduction yields editing up to 90% d The Cas9 RNP/AAV6 method allows for specific modifications ranging from 1 to >3,000 bp d This method yields highly edited cells without selection markers or antibiotics
Graphical Abstract Highlights d EpiSCs injected into blastocysts rapidly disappear due to apoptosis d Induced BCL2 expression enables injected EpiSCs to survive and form chimeras d BCL2-expressing Sox17 + endoderm progenitors can also form region-specific chimeras d BCL2-expressing rat EpiSCs form interspecies chimeras that survive to adulthood In Brief Masaki et al. show that expression of the anti-apoptotic gene BCL2 allows developmentally incompatible cells such as epiblast stem cells and endoderm progenitors to engraft into mouse blastocysts. This approach can overcome stage-related barriers to cellular integration, allowing effective formation of chimeras within and between species.
SummaryThe discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.
Despite their rapidly-expanding therapeutic potential, human pluripotent stem cell (hPSC)-derived cell therapies continue to have serious safety risks. Transplantation of hPSC-derived cell populations into preclinical models has generated teratomas (tumors arising from undifferentiated hPSCs), unwanted tissues, and other types of adverse events. Mitigating these risks is important to increase the safety of such therapies. Here we use genome editing to engineer a general platform to improve the safety of future hPSC-derived cell transplantation therapies. Specifically, we develop hPSC lines bearing two drug-inducible safeguards, which have distinct functionalities and address separate safety concerns. In vitro administration of one small molecule depletes undifferentiated hPSCs >106-fold, thus preventing teratoma formation in vivo. Administration of a second small molecule kills all hPSC-derived cell-types, thus providing an option to eliminate the entire hPSC-derived cell product in vivo if adverse events arise. These orthogonal safety switches address major safety concerns with pluripotent cell-derived therapies.
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