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Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that play a crucial role in the tumor surveillance. The activation of iNKT cells by their specific ligand α-galactosylceramide (α-GalCer) induces the activation of dendritic cells (DCs) via reciprocal interaction, which results in the generation of cellular immunity against cancer. Here we describe a method to detect DC-mediated cellular adjuvant properties of human iNKT cells in vitro.

In this chapter, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from non-T cells, followed by the differentiation of the T-cell lineage. Derivation of T cells from PSCs involves three steps: induction of PSCs to hematopoietic progenitor cells (HPCs), differentiation of HPCs into progenitor T cells, and maturation of progenitor T cells into mature T cells (CD8 single-positive (SP) or CD4 SP).

The use of induced pluripotent stem cells (iPSCs) as a cell source for producing cytotoxic T lymphocytes (CTLs) is expected to have advantages in the antigen specificity, rejuvenation profile, and reproducible number of CTLs. We have developed the way to differentiate CD8αβ T cells from TCR-transduced iPSCs (TCR-iPSCs). These T cells express monoclonal expression of the transduced TCR. Generating CD8αβ CTLs from TCR-iPSC could contribute to safe and effective allogeneic regenerative T cell immunotherapies.

In this chapter, we describe redifferentiation procedures from iPSCs to CD8αβ + cytotoxic T cells in 10 T1/2 and OP9/DL1 feeder condition. iPSC used here is derived from T-cell clone (T-iPSC), which has lost naïve phenotype and acquired exhaustion/senescence phenotype during cloning process (Note 1). On the other hand, redifferentiated T cells (T-iPSC-Ts) reacquire naïve phenotype (CD45RA + CD45RO - CCR7 + CD62L + ), which are reportedly critical for in vivo persistence of infused T cells and greatly affect therapeutic efficacy of adoptive immunotherapy. Indeed, T-iPSC-Ts exhibit much superior proliferative capacity while retaining equivalent effector function compared to parental T-cell clones. Here, we demonstrate the methodology to produce naïve-like T-iPSC-Ts, which could be potent cell source for adoptive immunotherapy.

In this chapter, we describe a protocol for hematopoietic differentiation of nonhuman primate (NHP)-induced pluripotent stem cells (iPSCs) derived from T cells and generation of T cells. Derivation of T cells from PSCs involves three steps: induction of PSCs to hematopoietic progenitor cells (HPCs), differentiation of HPCs into progenitor T cells, and maturation of progenitor T cells into mature T cells, in particular CD8 single-positive (SP) T cells.

T cells engineered to express chimeric antigen receptor (CAR) against the B cell antigen CD19 are achieving remarkable clinical effects on hematological malignancies. Allogeneic transplantation approach is promising for broaden application of CART therapy. iPSCs are one of the ideal cell sources for this approach. CAR-engineered iPSCs are demonstrated to give rise to CAR-engineered T cell and exert their effector function. In this section, we describe the method to generate CAR-engineered iPSCs and differentiate them into T cells.

In order to differentiate T cells in vitro, co-culture systems with Notch ligand-expressing feeder cells have been in use for a long time. Here we describe a feeder-free culture condition for differentiating T cells from hematopoietic cells that are cultured on Fc-DLL4-coated plate with T-lineage cytokines. This condition is capable of efficiently differentiating hematopoietic progenitor cells (HPCs) to immature T cells expressing both CD4 and CD8. To mature those cells into functional T cells, further stimulation and culture is necessary.