NOTCH activation at the hematovascular mesoderm stage facilitates efficient generation of T cells with high proliferation potential from human pluripotent stem cells

Abstract

Human pluripotent stem cells (hPSCs) offer the potential to serve as a versatile and scalable source of T cells for immunotherapies, which could be coupled with genetic engineering technologies to meet specific clinical needs. In order to improve T cell production from hPSCs it is essential to identify cell subsets that are highly enriched in T cell progenitors, and those stages of development at which NOTCH activation induces the most potent T cells. Here we evaluated the efficacy of T cell production from cell populations isolated at different stages of hematopoietic differentiation, including mesoderm, hemogenic endothelium (HE) and multipotent hematopoietic progenitors (MHPs). We demonstrate that KDR^hiCD31^- hematovascular mesodermal progenitors (HVMP) with definitive hematopoietic potential produce the highest numbers of T cells when cultured on OP9-DLL4 as compared to downstream progenitors, including HE and MHPs. In addition, we found that T cells generated from HVMPs have the capacity to expand for 6–7 weeks in vitro, in comparison to T cells generated from HEs and HPs, which could only be expanded for 4–5 weeks. Demonstrating the critical need of NOTCH activation at HVMP stage of hematopoietic development in order to establish a robust T cell production from hPSCs, may aid in establishing protocols for the efficient off-the shelf production and expansion of T cells for treating hematologic malignancies.

Introduction

Adoptive T cell therapies show promise in the treatment of several types of blood cancers. Recent clinical trials demonstrated remarkable clinical outcomes in relapse and refractory lymphoma patients treated with chimeric antigen receptor (CAR)-redirected T cells (1, 2). However, complicated logistics and impaired T cell function in patients with cancers or infection increases the costs and limits the utility of autologous T cell therapies (3). Human pluripotent stem cells (hPSCs) offer the potential to serve as a versatile and scalable source of the off-shelf T cells for immunotherapies, which could be coupled with genetic engineering technologies to meet specific clinical needs (3). In addition, generation of T-iPSCs from antigen-specific cytotoxic T cells and their re-differentiation into functional cytotoxic T lymphocytes (CTLs) provides opportunity to “rejuvenate” and enable scalable production of CTLs (4, 5). Although multiple reports demonstrated T cells generation from PSCs (6, 7) and feasibility of iPSC-based CAR-T cell therapies (8), increasing scalability of T cell production from iPSCs is critical for advancing these technologies to clinic.

Previously, we identified major stages of hematopoietic differentiation from hPSCs and showed the critical role of NOTCH signaling specification of definitive hematopoiesis and T cells from hPSCs (9–12). Following differentiation in coculture with OP9 or in defined conditions, hPSCs undergo stepwise progression towards APLNR⁺PDGFRα⁺ primitive posterior mesoderm with hemangioblast potential (day 3 of differentiation); KDR^hiCD31- hematovascular mesodermal progenitors (HVMP) with definitive hematopoietic potential (day 4 of differentiation); VE-cadherin (VEC)⁺CD43^-CD73^- hemogenic endothelium (HE), VEC⁺CD43^loCD235⁺CD73^- angiohematopoietic progenitors (AHP) and VEC⁺CD43^-CD73⁺ non-HE (days 4–5 of differentiation); and CD43⁺ hematopoietic progenitors (HPs; days 6–8 of differentiation) that include CD235⁺CD41⁺CD45^- erythromegakaryocytic progenitors (E-MkP) and CD235/41^-CD45^+/− multipotent hematopoietic progenitors (MHP) (Figure 1) with a lin^-CD34⁺CD90⁺CD38^-CD45RA^- hematopoietic stem progenitor cells (HSPC) phenotype (9, 11–13).

Fig.1.

Schematic diagram shows the progenitor subsets formed following hematopoietic differentiation of hPSCs and protocol used for their lymphoid differentiation. Hematopoietic differentiation of hPSC was induced in coculture with OP9 (Step I). Hemogenic progenitors were collected from OP9/hPSC coculture at different days of differentiation and cultured on OP9-DLL4 to induce T cell differentiation (Step II). HVM is hematovascular mesodermal progenitor; HE is hemogenic endothelium; AHP is angiohematopoietic progenitors; MHP is multipotent hematopoietic progenitors; E-MkP is erythromegakaryocytic progenitors.

In the present studies we focused on identifying the stage of hematopoietic development at which NOTCH activation allows for the highest efficacy of T cell production with robust expansion potential. We found that Day 3 APLNR⁺PDGFRa⁺ primitive posterior mesodermal cells did not produce T cells, while all downstream subsets except VEC⁺CD43^-CD73⁺ non-HE and CD235a⁺CD41a⁺CD45^- E-MkPs do produce T cells when cultured on OP9-DLL4. As determined by limiting dilution assay, the highest frequency of T cell precursors was detected from day 4 HVMPs. In addition, we found that T cells generated from HVMPs have the capacity to proliferate for 6–7 weeks, in comparison to T cells generated from HE and MHPs, which could only be expanded for 4–5 weeks. T cell differentiation from hPSCs proceeded through a CD5⁺CD7⁺ progenitor stage that eventually transitions into CD8⁺CD4⁺ double positive cells. To confirm T cell development, we analyzed the genomic DNA for the presence of T cell receptor (TCR) rearrangements. In vitro generated T-cells were functionally active and proliferated upon stimulation with PMA and IL-2. Upon activation, the cells express CD25⁺CD69⁺ markers, IFN-γ and cytolytic protein perforin.

These studies should improve our understanding of the early steps of lymphopoiesis from hPSCs and pave the way for developing of robust T cell differentiation protocols from hPSCs for disease modeling and adoptive T cell therapies.

Materials & methods

Cell culture and hPSCs maintenance

Irradiated mouse embryonic fibroblasts (MEFs), human embryonic stem cell (hESCs) line H1 (WA01) and, human fibroblast-derived DF-19–9-7T hiPSC line were obtained from WiCell (Madison, WI). hESCs and hiPSCs were maintained on MEFs as described previously (14). Mouse OP9 stromal cells were provided by Dr. Toru Nakano (Osaka University, Japan). The OP9 cell line expressing human DLL4 (OP9-DLL4) was established by using lentivirus expressing human DLL4 under the EF1α promoter. Wild and engineered OP9 cells were cultured in αMEM media containing 20% FBS and passaged every 3–4 days on 0.1% gelatin coated dishes. Overgrown OP9 cultures were prepared by prolonged culture of confluent OP9 monolayer for additional 4–8 days (15). Wild type OP9 and OP9-DLL4 cells used for hematopoietic and lymphoid differentiation were used for up to 50 passages. OP9 cells transduced with DLL1 (OP9-DLL1) were also generated and maintained as described above.

Hematopoietic differentiation in OP9 coculture and isolation of hemogenic cell subsets

hESC/iPSCs were differentiated in coculture with OP9 stromal cells and depleted of OP9 cells using anti-mouse CD29 antibodies (Serotec) as described (16, 17). Indicated cell subsets were isolated at days 3, 4, 5, and 8.5, of hematopoietic differentiation in OP9 coculture and used for T lymphoid differentiation. At day 3, ALPNR⁺ cells were isolated by MACS using ALPNR-APC antibody and anti-APC magnetic beads (Miltenyi Biotec). For isolation of HVMPs on day 4 of differentiation, cells were isolated by MACS using KDR-PE antibody and anti-PE magnetic beads (Miltenyi Biotec). Following enrichment KDR^hiCD31⁺ and KDR^hiCD31^- cells were isolated by FACS. For isolation of day 5 HE, AHP and non-HE subsets CD144⁺ cells were isolated by MACS using CD144-FITC antibody and anti-FITC magnetic beads. Positively selected cells were stained further with CD73-APC, CD43-PE, and CD235a-PE antibodies, and then sorted into HE, AHP and non-HE subsets using FACS Aria cell sorter. Day 8.5 CD43+ HPs were isolated with CD43-FITC antibody and anti-FITC magnetic bead by MACS. Positively selected cells were stained with CD235a/CD41a-PE antibody and CD45-APC antibody and sorted into E-MkP and MHP subsets using a FACSAria™ cell sorter (BD Biosciences).

T cell lymphoid differentiation

OP9-DLL4 cells were maintained in αMEM media containing 20% FBS on 0.1% gelatin coated 10cm cell culture dish. For lymphoid differentiation, OP9-DLL4 monolayer cultures were prepared in 6 well plates. Cells were isolated at different stages of differentiation and seeded on confluent OP9-DLL4 cells in αMEM, 20% FBS, IL-7 (5ng/ml), Flt3L (5ng/ml) and SCF (10ng/ml) at 37°C and 5% CO2 for 3–4 weeks with weekly passage. Every 6–7 days, cells were collected by vigorous pipetting, filtered through a 40μm cell strainer and transferred onto fresh OP9-DLL4 monolayer. For antibody staining, cells were prepared in PBS containing 2% FBS, 1 mM EDTA, and 0.1% sodium azide and stained with CD45-APC, CD4-APC, CD5-PE, CD7-FITC, and CD8-PE T cell specific antibodies. 7AAD (5μg/ml) was added 10 minutes before flow cytometry to exclude dead cells. Expression of T lymphoid markers was evaluated following gating CD45⁺ cells. Cell analysis was performed with the FACSCalibur flow cytometer (BD Biosciences) and MACSquant (MiltenylBiotech), and acquired data was analyzed by Flowjo software.

Limiting dilution assay

For Limiting Dilution Assays (LDA), floating cells were collected from day 14 cultures of various hemogenic subsets on OP9-DLL4 in T cell conditions. Row A of a 96-well plate received 500 cells/well, and each subsequent row afterwards had half the previous row (Row B contained 250, Row C contained 125... Row H contained 3–4 cells). The wells were scored 2 weeks later by eye and flow-cytometry for CD5⁺CD7⁺ containing cells. Extreme limiting dilution analysis was conducted using the previously established algorithm (18).

Functional assay

hPSC-derived T cells were stimulated with Cell Activation Cocktail according to manufacturer’s instructions (Biolegend). Briefly, T cells were resuspended in cell culture medium (1×10⁶ cells/ml) along with Cell Activation Cocktail ( 2 μl/ml; Biolegend), IL2 (10ng/ml; Peprotech) and Brefeldin A (3 μg/ml; eBioscience) for 24 hours, then surface stained with CD25 APC and CD69 FITC antibodies. Evaluation of perforin and IFNγ expression was performed using intracellular staining.

Antibodies

Antibodies used include the following: CD3(SK7), CD4(RPA-T4), CD5(UCHT2), CD7(M-T701), CD8 (HIT8a), CD31(WM59), CD41a(HIP8), CD43(1G10), CD45(HI30, 2D1), CD73(AD2), CD144(557H1,16B1), CD235a(GA-R2), KDR(89106),TCR αβ(T10B9.1A-BD), and Perforin(δG9) from BD Biosciences; CD144(16B1) and IFNγ(4S.B3) from eBioscience; APJ (72133)and DLL4 (447506) from R&D System and TCRγδ(5A6.E9) from Invitrogen.

TCR rearrangement

For TCR rearrangement assay, DNA was isolated by Flexigene DNA kit (QIAGEN, Germany). TCRβ and TCRγ clonality detection was performed by PCR amplification kit (Invivoscribe, San Diego) with AmpliTaq Gold DNA polymerase (Applied Biosystems, CA).

RNA Extraction and Quantitative RT-PCR

Day 4 HVMP, day 4 HE and day 5 HE were isolated from H1 hESC/OP9 cocultures, and subsequently cocultured on OP9 or OP9-DLL4 for an additional 2 days. After 2 days of secondary coculture, OP9 cell were depleted by MACS using anti-mouse CD29 antibody (19). RNA was extracted with PureLinkTM RNA Micro Scale kit (ThermoFisher) and reverse transcribed using Advantage RT-for PCR kit (Takara). Quantitative PCR analysis was performed on all cDNA samples using Power SYBR Green PCR master mix (Life Technologies) and the following primers: NOTCH1, 5’- CAATGTGGATGCCGCAGTTGTG-3’(forward) and 5’-CAGCACCTTGGCGGTCTCG- TA-3’(reverse), HEY2, 5’-TTCAAGGCAGCTCGGTAACTGAC-3’ (forward) and 5’- CATACTGATGCACTGCTGGATGG-3’(reverse) and HES1, 5’-TACCCCAGCCAGTG- TCAAC-3’ (forward) and 5’- TCAGCTGGCTCAGACTTTCA-3’ (reverse). PCR was performed using the Mastercycler realplex thermal cycler (Eppendorf). Expression levels were calculated by minimal cycle threshold values (Ct) normalized to GAPDH.

Statistical Tests

The significance of differences between the mean values was determined by one -way ANOVA followed by Tukey post hoc test as appropriate using GraphPad Prism software (GraphPad, San Diego, CA).

Results

Analysis of T cell potential of hemogenic subsets generated at different stages of development.

To identify stage of development and cell population with the most robust potential, we isolated hemogenic populations obtained from H1 hESCs in coculture with OP9 on days 3, 4, 5 and 8.5 of differentiation and cultured them in T cell conditions on OP9-DLL4 (Figure 1). After 3–4 weeks of culture, presence of T lymphoid cells was detected by flow cytometry. As we previously demonstrated, the most primitive APLNR⁺PDGFRa⁺ mesodermal precursor with hemogenic potential arise in OP9/hPSC coculture on day 3 of differentiation (11, 17). These cells have features of posterior primitive streak and hemangioblast potential which reflects primitive hematopoiesis. We found that day 3 mesodermal cells failed to produce T cells consistent with their primitive hematopoietic characteristics (Figure 2A). In contrast, KDR^hiCD31^- HVMPs isolated on day 4 of differentiation, efficiently generated T cells in OP9-DLL4 cultures (Figure 2B and2C ), thereby confirming that day 4 HVMP possess a definitive hematopoietic potential. Similarly T cells were generated from day 4 and day 5 VEC⁺CD235a/43^-CD73^- HE and VEC⁺CD43/CD235a⁺CD73^- AHP subsets (Figure 2D and2E), although AHP produced significantly less CD4⁺CD8⁺ cells compared to HE. Consistent with their non-hemogenic nature, VEC⁺CD43/235a^-CD73⁺ cells failed to produce T cells (Figure 2D). Assessment of T cell potential of CD43⁺ cells generated on day 8.5 differentiation, revealed that CD235a/CD41a^- CD45^+/− MHP could efficiently generate T lymphoid cells. As expected CD235a/CD41a⁺ CD45^- EMkPs were essentially lacking T cell potential (Figure 2F). Similar results were obtained with hemogenic subsets obtained from fibroblast derived DF-19–9-7T iPSCs (Supplemental Figure S1A-C).

Fig.2.

T cell potential of hemogenic subsets isolated at different stages of H1 hPSC differentiation in OP9 coculture. Indicated cell subsets were obtained at different days of hematopoietic differentiation in hPSC/OP9 coculture and subsequently cultured in T cell conditions on OP9-DLL4. (A) APLR⁺ mesoderm isolated on day 3 of differentiation. (B) and (C) KDR^hiCD31^- and KDR^hiCD31⁺ HE isolated on day 4 of differentiation. (D) and (E) VEC⁺ subsets isolated on day 5 of differentiation. (F) and (G) CD43⁺ progenitor subsets isolated on day 8.5. Gates used for sorting hemogenic populations are numbered from I through VII. Bars in (C), (E) and (G) show percentage of CD5⁺CD7⁺ and CD4⁺CD8⁺ cells generated in cultures (mean + SD) for three independent experiments).

Frequency and expansion potential of T lymphoid cells generated from different hemogenic subsets

To determine which hPSC-derived hemogenic population possess the most robust T cell potential, we assessed the frequency of lymphoid progenitors in hematopoietic cells obtained from culture of various hemogenic subsets in T cells conditions on OP9-DLL4 after first 14 days of culture using limiting dilution assay (LDA). We found the highest frequency of T cell precursors from day 4 HVMPs (1 in 14 HVMP). The frequencies of T cell progenitors were slightly lower in day 4 and 5 HE and day 8 MHPs (Figure 3A). The most dramatic differences in T cell progenitors was observed in AHP cultures which revealed a very low 1/51 T cell progenitor frequency, thereby suggesting their limited T cell potential. We also evaluated the expansion potential of T cells generated from different subsets. We found that T cells generated from HVMPs have the capacity to proliferate for 6–7 weeks, in comparison to HEs and MHPs subsets, which could only be expanded for 4–5 weeks (Figure 3B). Similar expansion potential was observed in T cell cultures generated from fibroblast DF-19–9-7T iPSC-derived HVMPs (Supplemental Figure S1D). Based on these studies we concluded that the most efficient production of T cells with robust expansion potential could be achieved by culture of day 4 HVMPs in NOTCH-activating T cell conditions on OP9-DLL4.

Fig.3.

Frequency and expansion potential of T cell progenitors generated from different hemogenic subsets originating from H1 hESCs. (A) Limiting dilution assay to determine the frequency of the T lymphoid progenitors from different subsets. *p<0.005. (B) Expansion potential of T lymphoid progenitors generated from various hemogenic subsets.

To investigate differences in NOTCH signaling during initiation of T cell differentiation from HVMPs and HE cells, we compared changes in the expression of NOTCH associated molecules NOTCH1, HEY2 and HES1 after 2 days of secondary coculture of these cell subsets on OP9-DLL4 versus wild-type OP9 (Supplemental Figure S1E). We found that coculture on OP9-DLL4 versus wild-type OP9 induces more greater upregulation of NOTCH1 and HEY2 expression in HVMPs as compared to day 4 and 5 HE. Fold changes in HES1 expression were higher in day 5 HE, but no difference was observed between HVMPs and day 4 or 5 HE. These findings suggest that the favorable NOTCH signaling activation may occur when T lymphoid cultures were initiated from HVMPs.

Characterization of T cells derived from HVMPs.

As determined by flow cytometry, CD4⁺CD8⁺ T cells generated from HVMPs included populations expressing TCRα/β and TCRγ/δ (Figure 4A and4B). Analysis of TCR gene arrangement by PCR, revealed DNA rearrangement at the variable (V), joining (J), and diversity (D) regions in TCRβ and TCRγ loci (Figure 4C). Finally, to examine whether T cells derived from HVMP in vitro are functional, we stimulated these T cells using PMA-ionomycin cocktail and IL2 that are known to activate T cells and quantitated surface marker expression and cytokine production. After stimulation, CD25 and CD69 double positive T cells derived from HVMP increased from nothing to 78% (Figure 5A). These T cells also up-regulated the expression of IFN-γ after stimulation (Figure 5B). Perforin, which is secreted by both cytotoxic T cells and NK cells, was also observed after stimulation. These data suggest that T cells generated by our system are functional.

Fig.4.

Assessment of TCR in T cells generated from KDR^hiCD31^- HVMPs. (A) and (B) Flow cytometric analysis of CD3, TCRαβ and TCRγδ expression. (B) Bars shows percentage of CD3⁺ TCRαβ ⁺ and CD3⁺ TCRγδ ⁺ cells (mean + SD for three independent experiments). (C) Analysis of TCR rearrangement by genomic PCR. The PCR products were resolved on 2% agarose gel and visualized using ethidium bromide. The valid size range for rearranged TCR fragments is listed under the corresponding gel panel and by vertical lines on the gel. PB is peripheral blood (positive control) and H1 ESC is negative control. FiPSC is DF-19–9-7T iPSC line derived from fibroblasts.

Fig.5.

Functional analysis of T lymphoid cell generated from KDR^hiCD31^- HVMPs. Expression of CD25 and CD69 (A), IFNγ (B), and perforin (C) following T cell stimulation with PMA and ionomycin cocktail in the presence of Brefeldin A. Bars shows mean + SD for three independent experiments.

Discussion

NOTCH signaling is essential factor in determining T lymphoid fate of hematopoietic progenitors (20, 21). During development, NOTCH signaling is critical for arterial specification and HSC development (22–25). In present studies, we assessed the stage of development at which NOTCH activation could induce the most efficient T cell generation from hPSCs. We found that NOTCH activation at KDR^hiCD31^- mesodermal stage of development allows for the most efficient T cell production with robust expansion potential. Cultures of HE or already established MHPs on OP9-DLL4, produced fewer T cell progenitors and had more limited expansion potential. As we recently reported, NOTCH signaling is critical for specification of arterial HE, which is highly enriched in definitive lymphomyeloid progenitors (10). Thus, it is highly likely that robust T cell production from HVMPs can be explained by synergistic role of NOTCH signaling in induction of arterial HE and T cell generation. Activation of NOTCH signaling at HVMP stage promotes arterial HE formation with lymphoid potential, which subsequently produces T cells following continuous exposure to NOTCH signaling. When NOTCH signaling is activated at MHP stage, its enhancing effect on arterial HE generation is skipped, and therefore T cell production is reduced.

Several studies describe generation of T cells from hESCs (26, 27) and iPSCs obtained through reprogramming of peripheral blood T lymphocytes (6–8). While some researchers generated iPSCs with MART-1 specific TCR (6), others transduced iPSCs with second generation CD19 CAR (8). Other studies showed the generation of T cells from reprogrammed antigen specific CD8⁺ T cells from a HIV-1 infected patients (7), mucosal-associated invariant T cells from reprogrammed Vα7.2+ cord blood T cells (28) and NKT cells from reprogrammed peripheral blood CD4⁺ (29) and Vα24 NKT cells (30). The hESCs and iPSCs were differentiated to blood either using coculture with stromal cells (6, 7, 27–30) or embryoid body differentiation system in serum and feeder free conditions (8, 26). Blood cell generated in these conditions were subsequently differentiated into T cells using OP9-DLL1 or OP9-DLL4 cultures. In our study we used OP9 feeder cells to obtain hematopoietic progenitors and then used OP9-DLL4 cultures for lymphoid differentiation. We found that OP9-DLL4 cultures were more efficient than OP9-DLL1 in inducing lymphoid differentiation (Supplemental Figure S2A). In addition, sorting OP9-DLL4 by high and low DLL4 expression demonstrated that OP9 cells expressing high levels of DLL4 are essential for achieving efficient T cell production (Supplemental Figure S2B). While most prior studies have used iPSC-derived HPs for lymphoid differentiation, we have shown that more efficient T cell production could be achieved when coculture with OP9-DLL4 initiated using definitive hemogenic progenitors isolated at earlier stages of hematopoietic development, such as HVMP stage.

The promising clinical results with adoptive T cell therapies call for search of novel universal T cell sources to simplify logistics and reduce the costs of these therapies. Because hPSCs can be expanded indefinitely and engineered to express CARs, they potentially can serve as an endless supply for off-the-shelf CAR T cells. Alternatively, iPSC can be generated from T cells with particular TCR, and used to generate T cells expressing TCR of interest (6). However, several challenges for applying hPSC-based strategies for T cell therapies remain. It is important to develop chemically-defined conditions that allows for robust T cell production with optimal expansion potential. In addition, strategies to overcome potential GVHD and rejection of infused cells has to be developed. One of the most attractive strategy to prevent GVHD could be based on infusion of T cell progenitors rather than mature T cells. T cell progenitors undergo positive and negative selection in host thymus and become restricted to host MHC (31). Alternatively, TCR locus can be deleted in hPSCs to allow for production of TCR-less T cells. Immune rejection of infused T cells can be mitigated by establishing iPSC banks with most common HLA haplotypes or from HLA-homozygous donors. It is estimated that only 55 cell lines homozygous for highly conserved HLA haplotypes would provide a beneficial HLA match for 80% of the population in Japan (32) and only 150 homozygous lines would provide a match for more than 93% of the UK population (33). In conclusion, demonstration of the importance of NOTCH activation at hematovascular mesodermal stage of hPSC differentiation to amplify T cell generation in current studies offers an optimized strategy for achieving a robust T cell production from hPSCs and advancing their use for immunotherapy.