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Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro.

ABSTRACT: Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs. The approach takes advantage of natural Tcrb gene rearrangement to generate diversity in the length and composition of CDR3?. In vitro differentiation of progenitors transduced with a known Tcra gene in the presence of antigen drives differentiation of cells with a distinct agonist-selected phenotype. We purified these cells to generate TCR? chain libraries pre-enriched for target antigen specificity. Several TCR? chains paired with a transgenic TCR? chain to produce a TCR with higher affinity than the parental TCR for target antigen, without evidence of cross-reactivity.

SUBMITTER: Schmitt TM

PROVIDER: S-EPMC5722674 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro.

Schmitt Thomas M TM Aggen David H DH Ishida-Tsubota Kumiko K Ochsenreither Sebastian S Kranz David M DM Greenberg Philip D PD

Nature biotechnology 20171106 12

Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells ...[more]

PMID: 29106410

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Project description:Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4? and ?-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

Project description:Unlabelled: Huntington's disease (HD) results from a CAG repeat expansion in the gene encoding the huntingtin protein. This inherited disorder is characterized by progressive neurodegeneration. In particular, HD progression involves the loss of striatal projection neurons. The limited availability of reliable sources of human striatal projection neurons currently hampers our understanding of HD mechanisms and hinders the development of novel HD treatments. In this paper, we described two- and three-step methods for differentiating human neural progenitor cells toward striatal projection neurons. In the two-step differentiation protocol, 90%, 54%, and 6% of MAP2-positive cells were immunopositive for GABA, calbindin (CALB1), and DARPP-32/PPP1R1B, respectively. In the three-step differentiation protocol, 96%, 84%, and 21% of MAP2-positive cells were immunopositive for GABA, calbindin, and DARPP-32/PPP1R1B, respectively. In line with a striatal projection neuron phenotype, cells differentiated with our protocols displayed significantly increased expression of MAP2, CALB1, DARPP-32/PPP1R1B, ARPP21, and CTIP2. Application of glutamate receptor agonists induced calcium influx; accordingly, the cells also expressed various ionotropic glutamate receptor subunits. Differentiated cells also released GABA on stimulation. We suggest that our three-step differentiation protocol presents a reliable and simplified method for the generation of striatal projection neurons, yielding a critical resource for neuronal physiology and neurodegenerative disorder studies.SignificanceThe earliest changes in the neurodegenerative disorder Huntington's disease affect a specific type of brain neurons, the so-called medium spiny neurons of the striatum. In this study, two protocols were developed for the differentiation of neural progenitor cells into striatal medium spiny neurons, and the differentiated neurons were extensively characterized. The data indicate that the three-step differentiation protocol presents a reliable and simplified method for the generation of striatal medium spiny neurons. The generated striatal medium spiny neurons could represent a critical resource for the study of neurodegenerative disorders, a model system for drug discovery, and a step toward cell-based regeneration therapies.

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Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro.

Publications

Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro.

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