Reprogramming of Melanoma Tumor-Infiltrating Lymphocytes to Induced Pluripotent Stem Cells

Abstract

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients hold great promise for autologous cell therapies. One of the possible applications of iPSCs is to use them as a cell source for producing autologous lymphocytes for cell-based therapy against cancer. Tumor-infiltrating lymphocytes (TILs) that express programmed cell death protein-1 (PD-1) are tumor-reactive T cells, and adoptive cell therapy with autologous TILs has been found to achieve durable complete response in selected patients with metastatic melanoma. Here, we describe the derivation of human iPSCs from melanoma TILs expressing high level of PD-1 by Sendai virus-mediated transduction of the four transcription factors, OCT3/4, SOX2, KLF4, and c-MYC. TIL-derived iPSCs display embryonic stem cell-like morphology, have normal karyotype, express stem cell-specific surface antigens and pluripotency-associated transcription factors, and have the capacity to differentiate in vitro and in vivo. A wide variety of T cell receptor gene rearrangement patterns in TIL-derived iPSCs confirmed the heterogeneity of T cells infiltrating melanomas. The ability to reprogram TILs containing patient-specific tumor-reactive repertoire might allow the generation of patient- and tumor-specific polyclonal T cells for cancer immunotherapy.

Figure 1

Freshly isolated CD8⁺ tumor-infiltrating lymphocytes (TILs) showed distinct phenotypical difference compared with CD8⁺ peripheral blood T cells (PBTCs). Phenotypic characterization of freshly isolated CD8⁺ T cells in TILs and PBTCs from patient (A) and patient (B). Expression of CCR7 and CD45RO (a) and PD-1, LAG-3, and TIM-3 (b) in CD8-gated live cells in TILs and PBTCs are shown. Number indicates the percentage of cells shown in each quadrant (a) or indicated gated regions (b).

Figure 2

Generation of iPSCs from melanoma tumor-infiltrating lymphocytes. (a) Time schedule outlining expansion, activation, and reprogramming of TILs to generate iPSCs. (b) Morphologies of TILs when they started to expand 2-3 weeks after initiation of culture. (c) Efficient GFP introduction by Sendai virus (SeV) in TILs transfected at a MOI of 20. (d) Typical ESC-like iPSC colonies on day 21 after SeV infection. (e) Examples of 6-well plate containing SeV-reprogrammed iPSC clones stained for alkaline phosphatase (ALP), showing numerous ALP-positive colonies. (f) Cytogenetic analysis was performed on twenty G-banded metaphase cells from one of TIL-derived iPSCs (TIL-iPSCs). All twenty cells demonstrated a normal karyotype. (g) Immunofluorescence staining for pluripotency and surface markers (SSEA3, SSEA4, TRA-1-81, TRA-1-60, and OCT3/4) in iPSCs derived from melanoma TILs. Scale bars represent 100 μm. (h) RT-PCR analysis for the human ES cell marker genes NANOG, OCT3/4, SOX2, KLF4, and c-MYC in TIL-iPSCs and ESCs (H1). (i) Scatter plots comparing the global gene expression profiles of TIL-iPSCs and ESCs and TIL-iPSCs and TILs. (j) Immunofluorescence staining for SOX17 (endodermal marker), βIII tubulin (ectodermal marker), and αSMA (mesodermal marker) in TIL-iPSC-derived differentiated cell. Nuclei were counterstained with DAPI. Scale bars represent 100 μm. (k) Hematoxylin and eosin-stained representative teratoma sections of TIL-iPSC clones from patients (A) and (B) (6 weeks after injection into NOD/SCID mice).

Figure 3

A wide variety of TCR-β gene arrangement patterns in TIL-iPSCs. Characterization of the TCR-β gene arrangement in TIL-iPSCs from patients (A) and (B) by capillary electrophoresis. The green line is derived from the band for the Jβ1 gene, and blue line is derived from the band for the Jβ2 gene.