Notch-mediated conversion of activated T cells into stem cell memory-like T cells for adoptive immunotherapy

Adoptive T-cell immunotherapy is a promising approach to cancer therapy. Stem cell memory T (TSCM) cells have been proposed as a class of long-lived and highly proliferative memory T cells. CD8+ TSCM cells can be generated in vitro from naive CD8+ T cells via Wnt signalling; however, methods do not yet exist for inducing TSCM cells from activated or memory T cells. Here, we show a strategy for generating TSCM-like cells in vitro (iTSCM cells) from activated CD4+ and CD8+ T cells in mice and humans by coculturing with stromal cells that express a Notch ligand. iTSCM cells lose PD-1 and CTLA-4 expression, and produce a large number of tumour-specific effector cells after restimulation. This method could therefore be used to generate antigen-specific effector T cells for adoptive immunotherapy.


Supplementary Figure 3 Single cell-based analysis of iT SCM cell generation.
OT-II naïve CD4 + T cells isolated from OT-II Ifng Venus mice were primed with OVA-DCs under Th1 conditions (upper). Four days later, CD44 hi IFN-γ hi that fully differentiated into Th1 cells were single cell-sorted into 96-well plates, and then cocultured with OP9 or OP9-DL1 cells for 12 days. Dot plots show CD4 + T cells generated from single Th1 cell (lower). Data are representative of two independent experiments.

Supplementary Figure 4 Characterization of CD44 lo CD62L + T cells cocultured with OP9 control feeder cells
Flow cytometry analysis for the expression of surface markers and intracellular Bcl-2 in CD44 hi and CD44 lo CD62L + T cells cocultured with OP9 control feeder cells. Data are representative of two independent experiments.

Supplementary Figure 5
Comprehensive gene expression analysis of naïve CD4 + T, CD4 + iT CM , and CD4 + iT SCM cells. (a, b) Microarray analysis of wild-type naïve CD4 + T, CD4 + iT CM , and CD4 + iT SCM cells. Data are presented as a clustering analysis of the gene expression (a). Heatmap images of expression of effector molecules, cytokines, and transcription factors (b). Data are acquired from single experiment.

Supplementary Figure 7
Proliferative capacity of CD4 + and CD8 + iT SCM cells (a) CFSE dilution assays. OT-I naïve T, iT CM , and iT SCM cells were analyzed by flow cytometry 36 h after OVA-DCs restimulation (n = 3 per group). (b) Number of restimulated CD44 hi and CD44 lo CD62L + cells cocultured with OP9 cells. CD4 + T cells cocultured with OP9 cells were sorted using a CD44 hi CD62L + gate and CD44 lo CD62L + gate (left), stimulated with OVA-DCs for four days (n = 3 per group). Graph shows cell number of T cells. (c) Secondary culture with OP9-DL1. CD62L + cells, iT CM , and iT SCM cells induced from in vitro activated CD4 + T cells were restimulated with OVA-DCs, and then cultured with OP9-DL1 cells. The number of T cells were scored (n = 3 per group). (d, e) Cell division of CD4 + T cells after stimulation with OVA-DCs in vitro assessed by CFSE-dilution assay. Naive CD4 + T cells, T EM , and T CM cells were isolated from immunized CD45.1 + OT-II mice, and iT CM , and iT SCM cells were generated from in vitro activated CD45.1 + OT-II CD4 + T cells. These cells were labeled by CFSE, then 2 × 10 5 cells were transferred into sublethally irradiated CD45.2 + wild-type mice, and then OVA/IFA was subcutaneously injected into the mice. Six days later, cell numbers in the spleens and lymph nodes were counted (n = 6 for naïve, T EM , T CM , activated, iT CM , and iT SCM ; n = 8 for CD62L + ). Bar graphs show recovered cell number (d). Contour plots show CFSE dilution of transferred cells gated as CD45.1 + (e). **P < 0.01 (One-way ANOVA). Data are representative of two independent experiments.Error bars show SEM.

Supplementary Figure 8
In vivo long-term survival of CD4 + iT SCM cells. The long-lived capacity of iT SCM cells. Freshly isolated Rag2 -/-OT-II naïve T cells, iT CM or iT SCM cells (5 × 10 4 ) were transferred into sublethally irradiated CD45.1 + congenic mice. At 30, 75, and 150 days after transfer, cells in the secondary lymphoid organs were subjected to flow cytometry analysis. CD44 and CD62L expression on transferred T cells that gated on CD4 + T cells in the spleen on day 150. Numbers in the plots show percentages of the transferred CD4 + T cells (upper), and iT CM and iT SCM cell populations (lower). Graphs show percentages of the transferred CD4 + T cells in the spleen, the peripheral LN (pLN), and the mesenteric LN (mLN) at 30, 75, and 150 days after T cell transfer (n = 3 for naïve, iT CM and iT SCM at day30; n = 5 for naïve at day 75 and naïve, iT CM and iT SCM at day150; n =6 for iT CM and iT SCM at day 150 ). ND, not detectable. **P < 0.01 (one-way ANOVA). Data are representative of at least two independent experiments. Error bars show SEM.

Supplementary Figure 9
IL-2 production and its signal transduction of naïve CD4 + T, CD4 + iT CM , and iT SCM cells.
(a) IL-2 concentrations in the culture supernatants of OT-II naïve CD4 + T, CD4 + iT CM , and iT SCM cells at 12, 24, 36, and 48 h after TCR stimulation with OVA-DCs. (b) Flow cytometry analysis of Stat5 and Akt phosphorylation upon IL-2 stimulation. Naïve CD4 + T, CD4 + iT CM , and iT SCM cells were stimulated with IL-2 (20 ng ml -1 ) for 30 min, then stained with these specific Abs. Solid and dashed lines in the histograms are from IL-2-stimulated and unstimulated cells, respectively. n = 3 per group of all experiment. *P < 0.05, **P < 0.01 (one-way ANOVA). Data are representative of at least two independent experiments. Error bars show SEM.

Supplementary Figure 10 Lower expression of p53 allows CD4 + iT SCM cells to evade cell cycle arrest and apoptosis.
(a-c, e, f) Naïve CD4 + T, CD4 + iT CM , and iT SCM cells that were all derived from OT-II mice were subjected to the following experiments after stimulation with anti-CD3 and anti-CD28 mAbs (a, b, f) or with OVA-pulsed DC (c, e). (a) Cell cycle analysis by determining DNA content in the cells. Before or one, two, and three days after the TCR stimulation, propidium iodide (PI) was added, and then cells were subjected to flow cytometry analysis. Representative histogram (left). The bar graphs show the percentages of G2/M and S phases (n = 3) (right). (b) Apoptotic cell analysis. Two days after the TCR stimulation, cells were stained with AnnexinV. PI was added, and then cells were subjected to flow cytometry analysis. Representative contour plots (left). The bar graphs show the percentages of apoptotic cells (n = 3) (right). (c, d) Flow cytometry analysis for intracellular p53 expression. (c) At the indicated time points, cells were stained with anti-p53 Ab and then subjected to flow cytometry analysis. Representative histogram (top). The bar graphs show p53 expression levels that are indicated by the differences in mean fluorescence intensity (MFI) between p53-specific staining and isotype control staining (bottom) (n = 3). (d) CD45.1 + cells were transferred into CD45.2 + wild-type mice. Three days after immunization with OVA/IFA, CD4 + T cells in the spleens were stained with anti-p53 Ab and then subjected to flow cytometry analysis (n = 5 for naïve T and iT CM ; n = 4 for iT SCM ). (e) Quantitative RT-PCR analysis of expression of p53 target genes. Thirty-six hours after the TCR stimulation, total RNA was extracted from the cells (n = 3). (f) Effects of Nutlin3a on cell cycle and apoptosis of CD4 + iT SCM cells. Cells were cocultured with OVA-DCs in the presence or absence of 5µM Nutlin3a. Two days later, cells were subjected to the cell cycle and apoptosis analysis (n = 3). *P < 0.05, **P < 0.01 (two-way ANOVA [a, c] or one- way ANOVA [b, d, e, f]). Green, blue, and red markers indicate naïve CD4 + T, CD4 + iT CM , and iT SCM cells, respectively. Data are representative of at least two independent experiments. Error bars show SEM.

Supplementary Figure 11
Lower expression of p53 allows CD8 + iT SCM cells to evade cell cycle arrest and apoptosis. Naïve CD8 + T, CD8 + iT CM , and iT SCM cells that were all derived from OT-I mice were subjected to the following experiments after stimulation with anti-CD3 and anti-CD28 mAbs (a, b) or with OVA-pulsed DC (c, d). (a) Cell cycle analysis by determining DNA content in the cells. Before or one and two days after the TCR stimulation, propidium iodide (PI) was added, and then cells were subjected to flow cytometry analysis. Representative histogram (left). The bar graphs show the percentages of G2/M and S phases (n = 3) (right). (b) Apoptotic cell analysis. Two days after the TCR stimulation, cells were stained with AnnexinV. PI was added, and then cells were subjected to flow cytometry analysis. Representative contour plots (left). The bar graphs show the percentages of apoptotic cells (n = 3) (right). (c) Flow cytometry analysis for intracellular p53 expression. At the indicated time points, cells were stained with an anti-p53 Ab and then subjected to flow cytometry analysis. The bar graphs show p53 expression levels that are indicated by the differences in mean fluorescence intensity (MFI) between p53-specific staining and isotype control staining (n = 3). (d) Quantitative RT-PCR analysis of expression of p53 target genes. Thirty-six hours after the TCR stimulation, total RNA was extracted from the cells (n = 3). **P < 0.01 (two-way ANOVA [a, c] or one- way ANOVA [b, d]). Green, blue, and red markers indicate naïve CD8 + T, CD8 + iT CM , and iT SCM cells, respectively. Data are representative of at least two independent experiments. Error bars show SEM.

Supplementary Figure 12
Antitumour effects of antigen-specific murine CD4 + iT SCM cells. (a) Numbers and CFSE dilution of CD8 + T cells in the spleens and lymph nodes. CFSE-labeled CD45.1 + OT-II CD8 + iT CM and iT SCM cells (1.5 × 10 5 ) that were primed with OVA-DCs were transferred into E.G7-OVA-bearing CD45.2 + wild-type mice. Three days later, the spleens and LNs were taken from the mice. CD45.1 + OT-I CD8 + T cells of the spleens and LNs were counted by flow cytometry analysis (n = 5 for no transfer; n = 8 for iT CM ; n = 7 for iT SCM ). Graphs indicate the percentage of CFSE diluted cells (top right) and the recovered cell number (bottom). (b) Numbers of CD4 + T cells infiltrating into the sentinel lymph nodes (LNs). CD45.1 + OT-II CD4 + iT CM and iT SCM cells (1.5 × 10 5 ) that were primed with OVA-DCs were transferred into E.G7-OVA-bearing CD45.2 + wild-type mice. Thirty-six hours later, the sentinel LNs were taken from the mice. CD45.1 + OT-II CD4 + T cells of the sentinel LNs were counted by flow cytometry analysis (n = 4 for no transfer; n = 5 for iT CM ; n = 5 for iT SCM ). (c) Tumour volumes (left) and survival rates (right) of E.G7-OVA-bearing mice into which the primed CD45.1 + OT-II CD4 + in vivo T EM , T CM , CD62L + , iT CM and iT SCM cells (3 x 10 5 ) were transferred (n = 10 for no transfer; n = 9 for T EM and T CM ; n = 5 for CD62L + ; n = 11 for iT CM ; n = 14 for iT SCM ). (ND, not detectable) **P < 0.01 (One-way

Supplementary Figure 15
Functions of human antigen-specific iT SCM cells (a) Generation of Mart-1-specific iT SCM cells from human peripheral blood T cells. CFSE-labeled CD8 + T cells were cocultured with Mart-1 peptide-pulsed autologous monocyte-derived DCs for seven days. Mart-1-specific activated T cells (Mart-1-Tet + CD45RA -CD45RO + CCR7 -CD95 + CFSE lo ) (Day 0) were sorted, and then co-cultured with OP9-hDL1 cells for 11 days. Cells on Days 0 and 11 were subjected to flow cytometry analysis. Prostate-specific antigen (PSA)-tetramer was used for a negative control. Each blue and red square indicates iT CM and iT SCM cells, respectively (left). (b) Expression of effector molecules in EBV-specific iT CM and iT SCM cells. Cells were restimulated by autologous LCL in the presence of breferdin A for 6 h and then permeabilized. Intracellular molecules were stained with the specific-Abs. (c) Cytotoxic responses of EBV-or Flu-specific CD8 + iT CM and iT SCM cells against autologous LCLs. LCLs were cocultured with CD8 + iT CM and iT SCM cells. Three hours later, cells were labeled with calcein AM and EthD-1, and were then subjected to flow cytometry analysis. LCLs were detected as CD8αcells by flow cytometry (n = 3 per group). **P < 0.01 (One-way ANOVA). Data are representative of two independent experiments. Error bars show SEM.