Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Summary

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

Abstract

Adoptive cell transfer (ACT) of antigen-specific CD8⁺ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies ¹. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines ^2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.

TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases ^8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic ^11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture ^14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.

Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Protocol

1. Cell Culture

1. Preparation of irradiated SNL76/7 (irSNL76/7) feeder cells for culture.
   SNL76/7 cells are generally maintained in 10% fetal bovine serum (FBS) Dulbecco's Modified Eagle Medium (DMEM) media.
   1. A culture dish or flask will be coated with 0.1% gelatin solution in 37 °C; incubator for 30 minutes before recovering SNL76/7 cells from liquid nitrogen.
   2. When SNL76/7 cells reach confluency, cells will be trypsinized off, centrifuged at 400 g for 5 min and resuspended in fresh media.
   3. Resuspended SNL76/7 cells will be irradiated in a ⁶⁰Co irradiator with a dose of 5000 Rads.
      Alternative ap....

Discussion

For ACT-based therapies, the in vitro generation of large numbers of highly reactive Ag-specific T cells for in vivo re-infusion is an optimal approach. Although our in vitro method gives rise of functional T cells from iPS cells, large numbers of iPS cell-derived cells die in four weeks, especially in the fourth week. We conclude that the survival signals from Notch signaling mediated by the DL1 as well as IL-7 and FLt3L are not sufficient to maintain the survival of iPS cell-derived prog.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL/6J mice	Jackson Laboratory	000664
B6.129S7-Rag1tm1Mom/J	Jackson Laboratory	002216
Anti-CD3 (2C11) antibody	BD Biosciences	553058
Anti-CD28 (37.51) antibody	BD Biosciences	553295
Anti-CD3 (17A2) antibody	BioLegend	100202
Anti-CD4 (GK1.5) antibody	BioLegend	100417
Anti-CD8 (53-6.7) antibody	BioLegend	100714
Anti-CD25 (3C7) antibody	BioLegend	101912
Anti-CD44 (1M7) antibody	BioLegend	103012
Anti-CD117 (2B8) antibody	BioLegend	105812
Anti-TCR-β (H57597) antibody	BioLegend	109220
Anti-IL-2 (JES6-5H4) antibody	BioLegend	503810
Anti-IFN-γ (XMG1.2) antibody	BioLegend	505822
DMEM	Invitrogen	ABCD1234
α-MEM	Invitrogen	A10490-01
FBS	Hyclone	SH3007.01
Brefeldin A	Sigma-Aldrich	B7651
Polybrene	Sigma-Aldrich	107689
GeneJammer	Integrated Sciences	204130
RNA kit	Qiagen	74104
DNA kit	Qiagen	69504
CD8 Isolation Kit	Miltenyi Biotec	130-095-236
ACK lysis buffer	Lonza Inc.	10-548E
mFlt-3L	PeproTech Inc	250-31L
mIL-7	PeproTech Inc	217-17
Gelatin	Sigma-Aldrich	G9391
FITC-anti-OVA antibody	Rockland Immunochemicals	200-4233
Permeabilization buffer	Biolegend	421002
BSA	Sigma-Aldrich	A7906
Formaldehyde	Sigma-Aldrich	F8775
0.4 μm filter	EMD Millipore
Moflo Cell Sorter	Dako
Calibur Flow Cytometer	BD Biosciences
LSR II Flow Cytometer	BD Biosciences
Mouse restrainer	Braintree Scientific, Inc.