Extrathymic generation of tumor-specific T cells from genetically engineered human hematopoietic stem cells via Notch signaling

Abstract

Adoptive cell transfer (ACT) of tumor-reactive lymphocytes has been shown to be an effective treatment for cancer patients. Studies in murine models of ACT indicated that antitumor efficacy of adoptively transferred T cells is dependent on the differentiation status of the cells, with lymphocyte differentiation inversely correlated with in vivo antitumor effectiveness. T-cell in vitro development technologies provide a new opportunity to generate naive T cells for the purpose of ACT. In this study, we genetically modified human umbilical cord blood-derived hematopoietic stem cells (HSCs) to express tumor antigen-specific T-cell receptor (TCR) genes and generated T lymphocytes by coculture with a murine cell line expressing Notch-1 ligand, Delta-like-1 (OP9-DL1). Input HSCs were differentiated into T cells as evidenced by the expression of T-cell markers, such as CD7, CD1a, CD4, CD8, and CD3, and by detection of TCR excision circles. We found that such in vitro differentiated T cells expressed the TCR and showed HLA-A2-restricted, specific recognition and killing of tumor antigen peptide-pulsed antigen-presenting cells but manifested additional natural killer cell-like killing of tumor cell lines. The genetic manipulation of HSCs has broad implications for ACT of cancer.

Figure 1

T cells committed from genetically engendered HSCs. A, flow cytometry analysis for T-cell markers for MSGV-p53-AIB–transduced HSCs (p53 TCR) or nontransduced HSCs (NV) cocultured with OP9-DL1 cell line for 29 d. Representative of four experiments. B, flow cytometry analysis for T-cell markers for MSGV-p53-AIB–transduced HSCs cocultured with OP9-DL1 or OP9 for 25 d. Data were gated as in (A). Representative of two experiments. C, HSCs were cocultured with OP9-DL1 for 37 d and stained for human TCR α/β (TCR α/β) or human TCR γ/δ (TCR γ/δ). D, flow cytometry analysis for T-cell markers for MSGV-p53-AIB–transduced HSCs (TCR) or nontransduced HSCs (NV) before and after OKT3/IL-2 treatment. HSCs were cocultured with OP9-DL1 cell line for 44 d (top), and then cells were cultured in medium supplemented with OKT3 + IL-2 (middle) or IL-2 alone (bottom) for additional 5 d. Representative of three experiments.

Figure 2

Expansion of transgene expression of in vitro developed T cells. A, cell number was counted at different time intervals after MSGV-p53-AIB–transduced HSCs (p53 TCR) were cocultured with OP9-DL1 as indicated. The fold expansion was based on the starting HSC numbers. Nontransduced HSCs (NV) cocultured with OP9-DL1 was used as control. B, expression of CD34 and GFP on transduced HSCs (top) and following coculture for 24 d with OP9-DL1 (bottom). C, PCR detection of TREC in T cells generated from HSCs cocultured with OP9-DL1 for 15 and 35 d. As a control, DNA from purified CD3⁺ CB was used as a positive control (P). DNA from adult PBLs was used as a negative control (N). DNA isolated from CD34⁺ HSCs before coculture with OP9-DL1 was shown as input cells (I). D, flow cytometry analysis of MSGIN or MSGV-p53-AIB (p53 TCR)–transduced HSCs cultured with OP9-DL1 for 24 d. Nontransduced HSCs (NV) cocultured with OP9-DL1 cell line for 24 d were used as a control. Data were gated as in (A).

Figure 3

Antigen-specific functional responses of the T cells in vitro developed from tumor antigen TCR–transduced HSCs. A, flow cytometry analysis of CD3 and transduced TCR for MSGV-p53-AIB (p53 TCR, detected by anti-murine TCR), MSGE1APB (ESO TCR, detected by anti-human TCR), or nontransduced (NV) HSCs cocultured with OP9-DL1 for 30 d. Representative of two experiments. B, T cells generated by coculturing MSGV-p53-AIB (p53 TCR)–, MSGE1APB (ESO TCR)–, or MSGIN (MSGIN)–transduced HSCs with OP9-DL1 for 24 d [nontransduced HSCs (NV) were used as control] were cocultured with serial diluted p53 peptide–pulsed T2 cells (p53) or NY-ESO-1 peptide–pulsed T2 cells (NY-ESO-1). Twenty hours later, the supernatant collected from the coculture was subjected to ELISA for the detection of GM-CSF secretion. Representative of two experiments. C, T cells generated by coculturing MSGV-p53-AIB (p53 TCR)–transduced HSCs with OP9-DL1 for 42 d [nontransduced HSCs (NV) were used as control] were cocultured with p53 peptide–pulsed T2 cell line (T2-p53); NY-ESO-1 p156-165V peptide–pulsed T2 cells (T2-ESO) were used as a control. Production of IL-2 and GM-CSF was determined by ELISA. Representative of three experiments. D, T cells generated by coculturing MSGV-p53-AIB–transduced HSCs (p53 TCR) or nontransduced HSCs (NV) with OP9-DL1 for 44 d were cocultured with ⁵¹Cr-labeled, p53 peptide–pulsed T2 (T2-p53) or NY-ESO-1 peptide–pulsed T2 (T2-ESO) for 4 h, and ⁵¹Cr release was measured. Representative of two experiments.

Figure 4

Killing of tumor cell lines by in vitro generated T cells. A, TCR-transduced (p53) or nontransduced (NV) T cells generated by coculture of HSCs with OP9-DL1 for 40 d were cocultured with ⁵¹Cr-labeled tumor cell line p53⁺ MDA231 or p53⁻Saos-2. ⁵¹Cr release was measured after 4 h of coculture. B, in vitro developed T cells from a 40-d coculture with OP9-DL1 were cultured with OKT3/IL-2 (14 d) to stimulate differentiation to CD8 single-positive cells. Fourteen d later, these cells were cocultured with ⁵¹Cr-labeled cells as indicated, and the percent lysis was determined at 4 h.