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.
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.
1. Cell Culture
2. In vitro Programming
3. In vivo Programming
4. Representative Results
CD3 and TCRβ are used as markers of T cells. To determine whether stimulation of iPS cells with the Notch ligand DL1 could contribute to T cell differentiation, we assessed expression of CD3 and TCRβ+ on iPS cell-derived cells, and further analyzed expression of CD4 and CD8, gating on CD3+ and TCRβ+ population. As shown here, on day 22, CD3+ TCRβ+ CD4– CD8+ single positive (SP) T cells were generated from iPS cells in vitro. In addition, the iPS cell-derived SP cells were able to produce IL-2 and IFN-γ when stimulated in vitro by plate-coated anti-CD3 and soluble anti-CD28 antibodies (Fig. 2), suggesting the iPS cell-derived T cells are functional.
After adoptive transfer into recipient mice, the majority of TCR gene-transduced iPS cells underwent differentiation into CD8+ CTLs, which responded in vitro to peptide stimulation by secreting IL-2 and IFN-γ (Fig. 3). Most importantly, adoptive transfer of TCR-transduced iPS cells triggered infiltration of OVA-reactive CTLs into tumor tissues and protected animals from tumor challenge (Fig. 5-6). Thus, TCR gene-transduced iPS cells can differentiate into functional antigen-specific CTLs in vivo.
Figure 1. Morphology of iPS cell differentiation. At various days, mouse iPS cells were co-cultured with OP9-DL1 cells in α-MEM medium supplemented with 20% FCS and 2.2 g/L sodium bicarbonate in the presence of 5 ng/ml mFlt3L and 1 ng/ml mIL-7.
Figure 2. T cell differentiation from iPS cells. Mouse iPS cells were co-cultured with OP9-DL1 cells as described in Figure 1. On day 22, iPS cell-derived cells were isolated and analyzed. A) CD4+ CD8– or CD4– CD8+ cells after gating on CD3+ and TCRβ+ populations. B) Cells were stimulated with plate-coated anti-CD3 and soluble anti-CD28 antibodies for 5 hours at 37 °C at 5% CO2. IL-2 and IFN-γ were analyzed by intracellular staining, after gating on live CD4– CD8+ T cells.
Figure 3. Antigen-specific CD8+ T-cell development from iPS cells in vivo. OT-I TCR gene-transduced iPS cells were injected i.v. into C57BL/6 mice. After six to ten weeks, OVA-specific CD8+ Vβ5+ T cell development was determined. A) CD8+ Vβ5+ T cells from pooled LNs and spleen were analyzed by flow cytometry, after gating on CD8+ populations. B) IL-2 and IFN-γ production (dark lines; shaded areas indicate isotype controls) were determined by intracellular cytokine staining, after gating on the CD8+ Vβ5+ populations. C) In vivo proliferation/cytotoxicity assay. CFSEhi (right peaks) and CFSElo (left peaks) target cells were pulsed with OVA257-264 peptide and the control, respectively, and were injected into mice ten weeks after iPS cell transfer or one day after OT-I CTL transfer.
Figure 4. Adoptive transfer of OT-I TCR gene-transduced iPS cells suppresses tumor growth. OT-I TCR gene-transduced iPS cells were adoptively transferred into C57BL/6 mice. One group of mice was injected with OVA-reactive CD8+ T cells from OT-I TCR transgenic mice, and one group of mice had no cell transfer. After either six weeks or on the following day after the cell transfer, mice were subjected to challenge with E. G7 tumor cells. On day 20, tumor cells in the peritoneal cavity were enumerated.
Figure 5. iPS cell-derived antigen-specific CTLs infiltrate into tumor tissues. On day 30 to 35 after tumor challenge, tumor tissues were examined for tumor-reactive T cell infiltration. A) H&E staining. Inflammatory cells infiltrated in tumor tissues (↓). B) Immunohistological staining. OVA-specific Vα2+ CTLs (red) infiltrated in OVA-expressing tumor tissues (green). C) Single-cell suspensions from tumor tissues were analyzed for expression of Vα2+ and Vβ5+ by flow cytometry, after gating on the CD8+ population.
Figure 6. Adoptive transfer of OT-I TCR gene-transduced iPS cells sustains mouse survival. OVA TCR gene-transduced iPS cells were adoptively transferred into C57BL/6 mice that were subjected to challenge with E. G7 tumor cells as described in Fig. 4. Mouse survival on day 50 was shown by Kaplan-Meier survival curves (n=6).
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 progenitor T cells, other survival factors may be cooperative to regulate these cell maturation. TCR gene transduction and in vitro stimulation with the Notch ligand largely direct T cell differentiation from iPS cells, however, the iPS cell-derived antigen-specific T cells still cannot survival longer, which prevents to obtain appropriate numbers of iPS cell-derived antigen-specific T cells for ACT-based immunotherapy.
The development of T lymphocytes in the thymus is a well-ordered process. The immature thymocytes lacking expression of CD4 and CD8 are referred to as double negative (DN) cells. DN precursors are divided into developmental subsets based on expression of CD44 and CD25: DN1 (CD44+CD25–), DN2 (CD44+CD25+), DN3 (CD44–CD25+) and DN4 (CD44–CD25–). Only DN3 cells that have generated a functional TCRβ chain, which pairs with the invariant pre-Tα and the CD3 chains to form a pre-TCR and are selected for further differentiation. This event, termed β-selection, represents the first checkpoint during T cell development. Pre-TCR formation signals proliferation, termination of TCRβ locus rearrangement, and differentiation of DN thymocytes to the CD4+CD8+ double positive (DP) stage17. Our in vitro stimulation with the Notch ligand DL1 drives iPS cells to pass through the β-selection checkpoint in 2 weeks, and become pre-T cells (CD3+TCRβ+; CD25–CD44–; CD4–CD8–). Additional 2-week stimulation allows pre-T cells to transit into mature CD8+ T cells (CD3+TCRβ+; CD4–CD8+; CD62L+CCR7+ CD27+CD127+). The mature SP T cells will die in the absence of a further stimulation by the TCR and CD3 complex.
In vivo programming of antigen-specific CTLs from iPS cells can overcome the deficiency to obtain sufficient numbers of T cells for ACT-based therapies. Despite the observed control of tumor growth, we identified some limitations of ACT with TCR gene-transduced iPS cells. First, at least six-weeks in vivo development is essential for T-cell differentiation derived from the transferred iPS cells. Second, we noticed fur loss, osteoporosis and other minor autoimmune manifestations in mice that received TCR-transduced iPS cells as observed in some clinical trials administrating T cell-based cancer immunotherapy. These effects may be caused by the generation of other immune cell types from the transferred iPS cells. However, how these cells are generated in vivo remains unknown. Third, adoptive transfer of TCR gene-transduced iPS cells has the risk of generating teratoma because of its stem phenotype. But so far in our study, we only identified extrathymic mass in one Rag1-/- mouse and have not observed abnormality in conventional C57BL/6 mice. Therefore, we suggest, to gain the maximal efficiency, it is better to get genetic background match for in vivo iPS differentiation.
In contrast to derivatives of ESCs, abnormal gene expression in some cells differentiated from iPS cells have the potential to induce T-cell-dependent immune response in syngeneic recipients 18. Therefore, the immunogenicity of cells derived from patient-specific iPS cells should be evaluated before any clinical application of these autologous cells is contemplated. By analyzing the gene-expression profiles of iPS-cell-derived cells, it has been shown that a group of 9 genes (Hormad1, Zg16, Cyp3a11, Lce1f, Spt1, Lce3a, Chi3L4, Olr1, Retn) were expressed at abnormally high levels. In addition, inducing expression of three of these genes (Hormad1, Zg16 and Cyp3a11) in ES cells significantly increased immunogenicity on transplantation into genetically matched recipients 18. Thus, the group of 9 proteins has the potential to cause immune rejection of the iPS cell-derived cells after adoptive transfer, and may represent immunogenic markers. Nevertheless, the potential immunogenicity of iPS cell-derived T lymphocytes has not been determined.
The authors have nothing to disclose.
We thank Dr. Shinya Yamanaka (Kyoto University) for providing iPS-MEF-Ng-20D-17 cell line, Dr. Dario Vignali (St. Jude Children’s Research Hospital) for supporting the OT1-2A•pMig II construct, Dr. Juan Carlos Zuniga-Pflucker (Department of Immunology, University of Toronto) for supporting the OP9-DL1 cell line, and Dr. Kent E Vrana (Department of Pharmacology, Penn State University College of Medicine) for helping the design of this study. This project is funded, under grants with the Grant Number K18CA151798 from the National Cancer Institute, the Barsumian Trust and the Melanoma Research Foundation (J. Song).
Name of the reagent | Company | Catalogue number |
C57BL/6J mice | Jackson Laboratory | 000664 |
B6.129S7-Rag1tm1Mom/J | Jackson Laboratory | 002216 |
Anti-CD3 (2C11) antibody | BD PharMingen | 553058 |
Anti-CD28 (37.51) antibody | BD PharMingen | 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 | B7651 |
Polybrene | Sigma | 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 | 10-548E |
mFlt-3L | PeproTech | 250-31L |
mIL-7 | PeproTech | 217-17 |
Gelatin | Sigma | G9391 |
FITC-anti-OVA antibody | Rockland Immunochemicals | 200-4233 |
Permeabilization buffer | Biolegend | 421002 |
BSA | Sigma | A7906 |
Formaldehyde | Sigma | F8775 |
0.4 μm filter | MIllipore | |
Moflo Cell Sorter | Dake Cytomation | |
Calibur Flow Cytometer | BD | |
LSR II Flow Cytometer | BD | |
Mouse restrainer | Braintree Scientific |