Generation of antigen-specific mature T cells from RAG1-/-RAG2-/-B2M-/- stem cells by engineering their microenvironment

Abstract

Pluripotent stem cells (PSCs) are a promising source of allogeneic T cells for off-the-shelf immunotherapies. However, the process of differentiating genetically engineered PSCs to generate mature T cells requires that the same molecular elements that are crucial for the selection of these cells be removed to prevent alloreactivity. Here we show that antigen-restricted mature T cells can be generated in vitro from PSCs edited via CRISPR to lack endogenous T cell receptors (TCRs) and class I major histocompatibility complexes. Specifically, we used T cell precursors from RAG1^-/-RAG2^-/-B2M^-/- human PSCs expressing a single TCR, and a murine stromal cell line providing the cognate human major histocompatibility complex molecule and other critical signals for T cell maturation. Possibly owing to the absence of TCR mispairing, the generated T cells showed substantially better tumour control in mice than T cells with an intact endogenous TCR. Introducing the T cell selection components into the stromal microenvironment of the PSCs overcomes inherent biological challenges associated with the development of T cell immunotherapies from allogeneic PSCs.

Fig. 1. Inhibition of T cell development at the DP stage of T cell differentiation by DKO of RAG1 and RAG2.

a, Schematic showing the generation of clonal RAG1 and RAG2 DKO PSCs derived from the H1 or ESI017 parent embryonic stem cell lines and their differentiation within the ATO system. b, Representative flow cytometry analysis of ATOs generated from unedited (WT) and DKO PSCs from the HLA-A*02:01 positive (A*0201^pos) H1 and HLA-A*02:01 negative (A*0201^neg) ESI017 parent lines. Surface expression of TCRαβ and CD3 from T lineage-committed cells is shown at the indicated time points. c, Temporal dynamics of CD8α and CD4 expression in T lineage-committed cells from WT and DKO PSCs differentiated in the ATO system are shown at the indicated time points. In b and c, the gating strategy is shown above the plots, and the numbers in the plots indicate the percentage of cells within each gate. Gates for individual populations are drawn on flow cytometry plots. Fluorophores are as follows: DAPI (4′,6-diamidino-2-phenylindole); PE (phycoerythrin); APC (allophycocyanin); BV (Brilliant Violet). d, The percentage of T cell populations from the DAPI⁻mCD29⁻CD56⁻CD45⁺CD5⁺CD7⁺ gate in c at the indicated time points (data shown as mean ± s.e.m.; H1, n = 3 independent experiments; ESI017, n = 4 independent experiments). Populations are defined as DP (CD8α⁺CD4⁺) and mature SP8 (CD8α⁺CD4⁻TCRαβ⁺CD3⁺). Chr 11, chromosome 11. Source data

Fig. 2. Class I MHC-restricted rescue of T cell development by 1G4 TCR in RAG1 and RAG2 DKO PSCs.

a,b, Representative flow cytometry analysis of WT and RAG1 and RAG2 DKO PSCs from both parent lines, H1 (A*0201^pos) and ESI017 (A*0201^neg), stably transduced to express the HLA-A*02:01-restricted 1G4 TCR (WT + TCR and DKO + TCR). a, Surface expression of the transgenic V_β13.1 and CD3 (gated as shown) during ATO differentiation of PSC-derived cells at the indicated time points. b, Differentiation kinetics of V_β13.1⁺CD3⁺ T cells based on CD8α and CD4 expression at the indicated time points of ATO differentiation. The gating strategy is indicated above the plots, and the numbers in the plots indicate the percentage of cells within each gate. c, At 7 weeks of T cell differentiation, ATO-derived H1 WT + TCR and DKO + TCR SP8 T cells (gated as shown) were analysed for maturation markers of conventional T cells as shown; the numbers indicate the percentage of cells within each gate. d, The percentage of T cell populations from the DAPI⁻mCD29⁻CD56⁻CD45⁺V_β13.1⁺CD3⁺ gate in b at the indicated time points (data shown as mean ± s.e.m.; H1, n = 3 independent experiments; ESI017, n = 4 independent experiments). Populations are defined as double negative (DN; CD8α⁻CD4⁻), DP (CD8α⁺CD4⁺) and SP8 (CD8αβ⁺CD4⁻). e, Maximum SP8 T cell output per ATO reached over the 7 week course of T cell differentiation (SP8_max). The mean ± s.e.m. (*P < 0.05, **P < 0.01, two-tailed Mann–Whitney U-test) is shown for each group (H1 WT, n = 3 independent experiments; ESI017 WT, n = 4 independent experiments; H1 DKO + TCR and ESI017 DKO + TCR, n = 5 independent experiments). NS, not significant; FITC, fluorescein isothiocyanate; PerCP, peridinin-chlorophyll-protein. Source data

Fig. 3. Engineering the ATO stroma to express HLA-A*02:01 rescues differentiation of 1G4 TCR-transduced RAG1, RAG2 and B2M TKO PSCs.

a, Schematic showing ESI017 TKO + TCR PSC generation. ESI017 DKO + TCR PSC clones were gene edited to KO B2M to generate the polyclonal ESI017 TKO + TCR line. Following haematopoietic induction, EMOs were collected and then aggregated with either hDLL4 or hDLL4-A02BI stroma for T cell differentiation in ATOs. b, Differentiation kinetics of WT + TCR and TKO + TCR T cells with hDLL4 or hDLL4-A02BI at the indicated time points of ATO differentiation. The gating strategy is indicated above the plots, and the numbers within the plots indicate the percentage of cells within each gate. c, The percentage of T cell populations from the DAPI⁻mCD29⁻CD56⁻CD45⁺V_β13.1⁺CD3⁺ gate in b at the indicated time points (data are shown as mean ± s.e.m.; WT + TCR with hDLL4 stroma, n = 4 independent experiments; WT + TCR with hDLL4-A02BI stroma, n = 3 independent experiments; TKO + TCR with hDLL4 stroma, n = 4 independent experiments; TKO + TCR with hDLL4-A02BI stroma, n = 5 independent experiments). The populations are defined as DN (CD8α⁻CD4⁻), DP (CD8α⁺CD4⁺) and SP8 (CD8αβ⁺CD4⁻). Source data

Fig. 4. Output and phenotypic characterization of SP8 T cells from RAG1, RAG2 and B2M TKO PSCs.

a, Output of SP8 T cells per WT + TCR or TKO + TCR PSC ATO aggregated with hDLL4 or hDLL4-A02BI stroma over the 7 week course of T cell differentiation (mean ± s.e.m.; P values are shown compared with TKO + TCR with hDLL4-A02BI stroma; *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired t-test; test is not significant if no P value is indicated; WT + TCR with hDLL4 stroma, n = 4 independent experiments; WT + TCR with hDLL4-A02BI stroma, n = 3 independent experiments; TKO + TCR with hDLL4 stroma, n = 4 independent experiments; TKO + TCR with hDLL4-A02BI stroma, n = 5 independent experiments). b, SP8_max T cell output per ATO reached over the 7 week course of T cell differentiation. The mean ± s.e.m. (*P < 0.05, two-tailed Mann–Whitney U-test) is shown for each group (WT + TCR with hDLL4 stroma, n = 4 independent experiments; WT + TCR with hDLL4-A02BI stroma, n = 3 independent experiments; TKO + TCR with hDLL4 stroma, n = 4 independent experiments; TKO + TCR with hDLL4-A02BI stroma, n = 5 independent experiments). c, At 7 weeks of T cell differentiation, TKO + TCR PSC-derived SP8 T cells were analysed for maturation markers of conventional T cells. The gating strategy is indicated above the plots, and the numbers within the plots indicate the percentage of cells within each gate. Source data

Fig. 5. scRNA-seq profiling of PSC-derived, peripheral and thymus SP8s.

a, UMAP visualization of ATO-derived SP8 T cells from WT PSCs with hDLL4 stroma (n = 1 independent experiment, 4,710 cells total), WT + TCR PSCs with hDLL4 (n = 2 independent experiments, 8,618 cells total) and hDLL4-A02BI (n = 3 independent experiments, 12,797 cells total) stroma, and TKO + TCR PSCs with hDLL4-A02BI stroma (n = 3 independent experiments, 7,930 cells total) compared with thymic SP8 (n = 2 independent experiments, 4,793 cells total), PB SP8 (n = 1 independent experiments, 2,083 cells total), PB NK (n = 2 independent experiments, 684 cells total) and PB monocytes (n = 2 independent experiments, 2,630 cells total). b, Average expression of specific genes from each scRNA-seq sample group. c, Dendrogram of hierarchical clustering analysis and heatmap showing Pearson’s correlation of global gene expression for all pairwise combinations between each sample, listed individually to the right of the heatmap. Source data

Fig. 6. Mispairing between endogenous TCR chains and transgenic TCR chains is absent in SP8s derived from TKO + TCR PSCs.

a, After 6 weeks of T cell differentiation, mature, conventional SP8 T cells were isolated for TCR repertoire analysis at a single-cell resolution from the following ATO conditions: WT PSCs with hDLL4 stroma (n = 1 independent experiment), WT + TCR PSCs with hDLL4 stroma (n = 2 independent experiments), WT + TCR PSCs with hDLL4-A02BI stroma (n = 3 independent experiments) and TKO + TCR PSCs with hDLL4-A02BI stroma (n = 3 independent experiments). Pie charts showing the frequency of barcoded cells that expressed full contigs of only endogenous TCR chains (grey), only 1G4 TCR chains (black), 1G4 TCR and endogenous TRAV chains (pink), 1G4 TCR and endogenous TRBV chains (green), and 1G4 TCR and both endogenous TRAV and TRBV chains (purple). b,c, TCR V_α (b) and TCR V_β (c) diversity from mature, conventional SP8 T cells at single-cell resolution. WT PSCs with hDLL4 stroma (grey; n = 1 independent experiment, 4,710 cells in total), WT + TCR PSCs with hDLL4 stroma (orange; n = 2 independent experiments, 9,619 cells in total), WT + TCR PSCs with hDLL4-A02BI stroma (green; n = 3 independent experiments, 12,797 cells in total) and TKO + TCR PSCs with hDLL4-A02BI stroma (red; n = 3 independent experiments, 7,930 cells in total). Source data

Fig. 7. Functional characterization of PSC-derived, antigen-specific T cells in vitro.

a–f, WT + TCR and TKO + TCR (1G4 TCR) SP8 T cells were isolated from week 6 ATOs, both generated with hDLL4-A02BI stroma and expanded with K562 aAPCs expressing the cognate antigen (NYESO), IL-2 and IL-7 before in vitro functional assays. The numbers indicate the percentage of cells within each gate. a, Expanded T cells were rested and then stimulated for 6 hours with K562 aAPCs presenting non-specific (MART1) or cognate (NYESO) antigen as an SCT to assay cytokine production and CD107α upregulation (gated on Zombie NIR⁻mStrawberry⁻TCRαβ⁺CD3⁺CD8α⁺ cells). Data are representative of three independent experiments. b, Upregulation of activation markers CD25 and 4-1BB on WT + TCR and TKO + TCR SP8 T cells in response to stimulation with MART1 or NYESO aAPCs for 24 hours (gated on DAPI⁻V_β13.1⁺CD3⁺CD8α⁺ cells). c, Proliferation of SP8 T cells, measured by carboxyfluorescein succinimidyl ester (CFSE), in response to stimulation with MART1 or NYESO aAPCs for 5 days (gated on DAPI⁻mStrawberry⁻CD8α⁺ cells). d, Staining with 1G4 tetramer and V_β13.1 from 1G4 TCR on SP8 T cells, after 6 days of expansion with NYESO aAPCs (gated on DAPI⁻CD8α⁺ cells). e, Cell-surface expression of transgenic 1G4 TCR (measured by V_β13.1) and CD3 on SP8 T cells in response to co-culture with MART1 or NYESO aAPCs for 24 hours (gated on DAPI⁻CD8α⁺ cells). f, In vitro cytotoxicity of WT + TCR and TKO + TCR SP8 T cells against MART1 or NYESO aAPCs based on Apotracker Green assay at 6 hours of co-culture (data are representative of n = 3 independent experiments). g, Fold expansion (from initial input) of WT + TCR and TKO + TCR SP8 T cells immediately after isolation from ATOs in response to NYESO aAPCs, IL-2 and IL-7 (data are shown as mean ± s.e.m.; n = 2 independent experiments). Source data

Fig. 8. In vivo function of 1G4-expressing, class I MHC-null, RAG1- and RAG2-null SP8 T cells.

a, Experimental design for in vivo tumour challenge in NSG mice I.V. engrafted with NALM6 tumour cells (5 × 10⁵ per mouse) expressing the cognate NYESO SCT and firefly luciferase. Five days after tumour engraftment, mice were injected I.V. with PBS, WT + TCR SP8 T cells (1 × 10⁷ per mouse) or TKO + TCR SP8 T cells (1 × 10⁷ per mouse). Tumour bioluminescence was measured every 3–4 days; IL-2 (10,000 IU per dose) was administered on the indicated days. b, Stratification of mice with equivalent tumour bioluminescence signal at day 5 into treatment groups. The mean ± s.e.m. for each group is shown; two-tailed Mann–Whitney U-test for each group showed NS difference (n = 9 individual mice, all groups). c, Representative imaging of mice from each treatment group at the indicated time points. d, Summary of bioluminescence tumour signal for all animals in each treatment group at the indicated time points. The mean ± s.e.m. (*P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Mann–Whitney U-test) for each group is shown (n = 9 individual mice, all groups). Source data