Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids

Abstract

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3⁺TCR-αβ⁺ single-positive CD8⁺ or CD4⁺ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

Figure 1. Efficiency and reproducibility of human T cell development in the ATO system

(a) Schematic of the ATO model. Inset: appearance of a typical ATO attached to cell culture insert at 6 weeks (shown after removal from culture well). (b) Kinetics of T cell differentiation from CB CD34+CD3- HSPCs at the indicated weeks, gated on CD14-CD56- cells to exclude monocytes and NK cells respectively. (c) Maintenance of early CD34+ thymic T cell progenitor phenotypes in ATOs based on two classification schemes, both gated on CD34+ cells as shown in (b). (d) Frequencies of cell types in ATOs at 6 weeks. Top panel: frequencies of monocytes (CD14+), NK cells (CD56+), B cells (CD19+), HSPCs (CD34+), or T lineage cell (CD7+CD5+) (gated on total live cells). Middle panel: T cell precursor and TCR+ T cell frequencies (gated on CD14-CD56- cells). Bottom panel: frequency of DP and mature CD8 and CD4 single positive (SP) T cells (gated on CD3+TCRαβ+ cells). (e) CD3 expression by immunofluorescence analysis in week 4 organoids generated with CB HSPCs and MS-5 cells (left) or MS5-hDLL1 cells (i.e. ATO) (right). (f) Numbers of total live cells and CD3+TCRαβ+CD8SP T cells generated per ATO at week 6 from 7.5–22.5 ×10³ CB HSPCs per ATO. Data are shown for 11 independent experiments (error bars indicate standard deviation).

Figure 2. Enhanced positive selection in the ATO system compared to OP9-DL1 monolayers

CD34+CD3- HSPCs from the same cord blood donor were used to initiate ATOs or standard OP9-DL1 monolayer cultures (containing 20% FBS) in parallel, and analyzed by flow cytometry and cell counting. Shown are data from 6 week cultures. (a,b) Representative flow cytometry profiles of cells gated on (a) total CD14-CD56- cells and (b) CD3+TCRαβ+ cells. (c,d) Summary of data from three different cord blood donors used to initiate parallel ATO and OP9-DL1 cultures. (c) Frequencies of monocytes (CD14+), NK cells (CD56+), B cells (CD19+), HSPCs (CD34+), or T lineage cells (CD7+CD5+) (gated on total CD45+ cells); CD4ISP and DP T cell precursors, and CD3+TCRαβ+ and CD3+TCRαβ+CD8SP mature T cells (gated on CD14-CD56- cells) in OP9-DL1 monolayer co-cultures versus ATOs at 6 weeks. Error bars represent the SD of three independent experiments. (d) Absolute numbers of T cell subsets at week 6 in OP9-DL1 co-cultures versus ATOs using the frequency data shown in (c). OP9-DL1 cultures were each initiated with 1.5×10⁴ CD34+CD3- CB HSPCs cells, and ATOs were each initiated with 7.5×10³ HSPCs from the same cord blood unit with technical duplicate ATOs harvested and pooled at 6 weeks for comparison of cell counts. Bars represent the mean and SD of three independent experiments.

Figure 3. Recapitulation of thymopoiesis and naïve T cell development in ATOs

Comparison of T cell differentiation in CB ATOs at 12 weeks and human postnatal thymocytes, gated on (a) total CD14-CD56- and (b) CD3+TCRαβ+ cells. (c) Generation of immature (CD45RA-CD45RO+) and mature (CD45RA+CD45RO-) naïve T cells in ATOs or thymus (gated on CD3+TCRαβ+ cells, with CD8SP or CD4SP subgates indicated). Data are representative of three independent experiments.

Figure 4. Generation of T cells from multiple HSPC sources and subsets

Efficient T cell development in week 6 ATOs initiated with CD34+CD3- HSPCs from human cord blood (CB), adult bone marrow (BM), G-CSF mobilized peripheral blood (MPB), or non-mobilized peripheral blood (PB). Gated on (a) total CD14-CD56- cells, and (b) CD3+TCRαβ+ T cells. (c) T cell differentiation kinetics over 12 weeks in ATOs generated from 7500 CD34+CD3- cells isolated from CB, neonatal thymi, BM, or MPB. Mean and SD of T cell precursor and mature T cells frequencies are shown from three technical replicates per tissue. Data are representative of two different experiments. (d) Numbers of total cells and CD3+TCRαβ+CD8SP T cells from ATO experiments shown in (c). (e) T cell differentiation from hematopoietic stem cell (HSC)-enriched (Lin-CD34+CD38-) fractions from CB, BM, and MPB in week 6 ATOs, gated on CD14-CD56- cells and (f) CD3+TCRαβ+ T cells. Data are representative of independent experiments (CB n=3, BM n=2, and MPB n=1). (g) T cell differentiation potential of adult BM total HSPCs (CD34+lin-) and purified progenitor (LMPP and CLP) subsets in ATOs at week 6; frequencies of CD34+ HSPCs, total T lineage cells (CD5+CD7+) and different T cell subsets are shown. (h) Numbers of total cells and CD3+TCRαβ+CD8SP T cells from ATOs shown in (g). Mean and SD of technical triplicates are shown for (g) and (h), and data are representative of three independent experiments.

Figure 5. TCR diversity and function of ATO-derived T cells

(a) Generation of TCR diversity in CD3+TCRαβ+CD8SP T cells from week 7 ATOs (n=5) or human thymi (n=4), as shown by flow cytometric analysis of the frequency of TCR Vβ family expression. (b) TCR clonotype diversity in CD3+TCRαβ+CD8SP T cells from ATOs, thymus, and peripheral blood (PB) naïve T cells by deep sequencing of TCR Vα and (c) TCR Vβ CDR3 regions. Frequency of individual clonotypes is shown. Data are representative of three independent experiments. (d) Polyfunctional cytokine production by ATO-derived CD3+TCRαβ+CD8SP T cells treated with PMA/ionomycin for 6h. Data are representative of three individual experiments. (e) Proliferation (CFSE dilution) and activation (upregulation of CD25 and 4-1BB) of ATO-derived CD3+TCRαβ+CD8SP cells after 5 days in response to anti-CD3/CD28 and IL-2. Data are representative of two individual experiments. (f) Post-ATO expansion of ATO-derived CD3+TCRαβ+CD8SP T cells relative to starting cell number in response to anti-CD3/CD28 and IL-2 after 7 and 14 days. Mean and SD of technical triplicates are shown, and data are representative of three independent experiments.

Figure 6. Differentiation and allelic exclusion of TCR-engineered T cells in ATOs

(a) Efficient generation of HLA-A*0201/NY-ESO-1_157-165 specific TCR-engineered T cells in week 7 ATOs initiated with TCR-transduced (top row) versus mock-transduced (bottom row) CB CD34+CD3- HSPCs. Cells are gated on CD14-CD56- cells with sequential sub-gates indicated above each plot. (b) Co-expression of CD8α and CD8β and lack of CD56 and CD16 expression on CD3+TCRαβ+tetramer+ T cells from TCR-transduced CB ATOs, indicating conventional T cell development. Data are representative of 3 independent experiments. (c) Enhanced total cell output and cell yield relative to starting number of HSPCs in TCR-transduced versus mock-transduced ATO at 6 or 7 weeks, generated with 7.5–18×10³ starting CB HSPCs. Mean and SD of independent experiments are shown (mock n=3, TCR n=8, **p=0.002). (d) Cytotoxic priming of ATO-derived TCR-engineered T cells by artificial antigen presenting cells (aAPCs). Cytokine production and CD107a membrane mobilization of tetramer+CD3+CD8SP T cells in response to K562 cells or K562 aAPCs that express CD80 and HLA-A*02:01 single chain trimers presenting an irrelevant (MART1_26-35) or cognate (NY-ESO1_156-165) peptide. Data are representative of three independent experiments. (e) Proliferation (CFSE dilution) and activation (CD25 upregulation) of ATO-derived CD3+tetramer+CD8SP T cells in response to irrelevant (MART1) or cognate (NY-ESO-1) aAPCs for 72h. Data are representative of two independent experiments. (f) Post-ATO expansion of CD3+TCRαβ+CD8SP T cells isolated from TCR-transduced ATOs relative to starting cell number, in response to anti-CD3/CD28 and either IL-2 or IL-7/IL-15 after 7 and 14 days. Mean and SD of technical triplicates are shown, and data are representative of three independent experiments. (g) Allelic exclusion of endogenous TCR Vβ in CD3+TCRαβ+tetramer+CD8SP cells isolated from TCR-transduced (n=3) compared with non-transduced (n=5) ATOs as shown by flow cytometric analysis of Vβ family frequency. Error bars represent SD. (h) In vitro cytotoxicity of ATO-derived TCR-engineered T cells. CD8SP T cells from HLA-A*02:01/NY-ESO-1_157-165-specific TCR-transduced ATOs were activated with anti-CD3/28 +IL-2 for 36h and co-incubated with K562 cells, K562 cells transduced with HLA-A*02:01 single chain trimers presenting an irrelevant (MART1_26-35) or cognate (NY-ESO1_156-165) peptide (K562-MART-1 and K562-ESO, respectively), or the HLA-A*02:01 U266 multiple myeloma cell line which expresses NY-ESO-1 endogenously. Apoptosis was determined by flow cytometry for annexin V+ cells at 9h. Effector:Target (E:T) ratios were calculated based on percent tetramer+CD3+ T cells at the start of co-cultures. Data are representative of two independent experiments. (i) In vivo tumor control by ATO-derived TCR-engineered T cells. CD8SP T cells from TCR-transduced ATOs were activated and expanded for 14 days. 5.7×10⁶ total T cells (4.5×10⁶ antigen-specific T cells by tetramer staining) or PBS were injected intravenously into NSG mice subcutaneously implanted 3 days earlier with 2.5×10⁵ luciferase-transduced K562-ESO tumor cells. Bioluminescence was recorded at the indicated timepoints. Mean and SD for each group is shown (PBS n=2, TCR-transduced ATO T cells n=3) (**p=0.00033, ****p=0.000066).