WO2025213245A1 - Methods and compositions for the tunable differentiation and production of single positive cd4+ and cd8+ t cells and cells derived from same - Google Patents

Abstract

Described herein are serum-free and feeder-free methods and compositions for the tunable differentiation and production of CD4+ (single positive) T cells and CD8+ (single positive) T cells, by adjusting T cell receptor (TCR) stimulation and notch activation (or notch signaling activation, stimulation or inducement). In some other aspects the invention provides a method and in vitro niche and cell culture media that results in enhancing CD4+ (SP) T cell generation, including naive mature CD4+ single positive T cells from double positive (DP) CD4+8+cells or CD8+(SP) T cells by TCR stimulation while having low or no notch signaling activation, stimulation or inducement. In yet other aspects, the invention provides a method, niche and culture media for producing cells derived from CD4+ (SP) T cells, including Treg cells.

Description

METHODS AND COMPOSITIONS FOR THE TUNABLE DIFFERENTIATION AND

PRODUCTION OF SINGLE POSITIVE CD4+ AND CD8+ T CELLS AND CELLS DERIVED

FROM SAME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Pat App. Ser. No. 63/632,148, filed April 10, 2024 and U.S. Pat. App. Ser. No. 63/712,856, filed October 28, 2024, which are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of stem cell differentiation, cell culture and the production of Single Positive T cells. More particularly, in some aspects, the invention relates to methods and compositions for the tunable differentiation and production of single positive CD4+ T cells and CD8+ T cells and cells derived from same.

BACKGROUND OF THE INVENTION

[0003] Engineered T cells have an enormous potential to treat cancer, autoimmunity, and infectious disease (Rosado-Sanchez and Lcvings, 2020; Schott ct al., 2024; Aghajanian ct al., 2022). However, the high cost and logistical complexity of manufacturing and administering autologous (patient-derived) T cells significantly limit access to T cell therapies. There has therefore been strong interest in scalable, cost-effective production of allogeneic (off-the-shelf) T cells(Martin et al., 2024; Depil et al., 2020; Michaels et al., 2023).

[0004] The production of scalable, cost-effective production of allogeneic T cells has proven challenging. The molecular and cellular signals that guide T cell development from hematopoietic stem and progenitor cells (HSPCs) remain poorly understood. The thymic microenvironment (i.e. the “thymic niche”), integrates multiple thymic niche molecules to potentiate T-cell development in vivo. Feeder cells are often used in T cell culture media to recapitulate these signals or the thymic niche in vitro. However, use of feeder cells or serum environments pose a greater risk of viral or other pathogenic agent contamination, result in an undefined culture medium and is not optimal for in vivo therapeutic applications. Recapitulating these signals in vitro in a stromal cell-free system has been challenging and limits T-cell generation technologies.

[0005] A promising source of allogeneic T cells is human pluripotent stem cells (PSCs), which are capable of indefinite expansion and more amenable to genetic engineering than primary cells, and can be differentiated in vitro to cytotoxic (CD8⁺) T cells (Timmermans et al., 2009; Themeli et al., 2013; Nishimura et al., 2013; Viscardo et al., 2013). Recently, the present inventors and others identified clinically -relevant, fully -defined (feeder- and serum-free) conditions to differentiate PSCs into CD8⁺ T cells (Iriguchi et al., 2021 ; Trotman-Grant et al., 2021 ; Michaels et al., 2022; Jing et al., 2022). However, a long-standing challenge for PSC-to-T cell manufacturing, especially in feeder-free systems, has been the inability to make substantial numbers of mature helper (CD4⁺) T cells (Michaels et al., 2023) and thus also cells derived from CD4⁺ T cells. Another issue is how to control or tune the cell culture “niche” or microenvironment to generate more of the cell type that is desired, whether it be CD4+ or CD8+ single positive T cells.

[0006] CD4⁺ T cells are key orchestrators of the immune system and their absence from PSC-derived T cell populations may limit clinical efficacy. CD4⁺ T cells play an essential role in “helping” CD8⁺ T cells eliminate cancer in the context of both engineered T cell therapies (Sommermeyer et al., 2016; Melenhorst et al., 2022; Wang et al., 2018) and immunotherapies (He et al., 2023; Zuazo et al., 2019; Zuazo et al., 2020; Van Hoecke et al., 2018). Moreover, through their ability to differentiate into different helper T (Th) cell subsets, such as Thl, Th2, and Th 17 cells, and regulatory T (Treg) cells, CD4⁺ T cells can orchestrate and regulate diverse immune responses.

[0007] Natural differentiation of CD4⁺ vs CD8⁺ T cells is well-studied and primarily driven by the dynamics of T cell receptor (TCR) signaling²(Shinzawa et al., 2022; Singer et al., 2008).

[0008] In the thymus, T cell progenitors develop into double-positive (DP) CD4⁺CD8⁺ cells, then undergo V(D)J recombination and negative and positive selection, ultimately resulting in cells with functional TCRs that complex with CD4 or CD8 co-rcccptors to recognize peptide -loaded major histocompatibility (MHC) Class I (CD8⁺ T cells) or Class II (CD4⁺ T cells) proteins. Commitment to the CD4⁺ or CD8⁺ lineage is controlled by TCR signaling duration(Iwata et al., 1996; Yasutomo et al., 2000); in selecting thymocytes, TCR interactions with MHC Class I generate shorter signals due to transient downregulation of CD8, interrupting TCR-MHC Class I interactions (Shinzawa et al., 2022; Singer et al., 2008. Differences in TCR signaling dynamics lead to selective activation of RUNX3 (CD8) or ThPOK (ZBTB7B, CD4), mutually-repressive lineage -defining transcription factors(Steier et al., 2024; He et ah, 2005; Sun et al., 2005).

[0009] An outstanding question is why current PSC-to-T cell differentiation protocols lead to poor CD4⁺ T cell development. One explanation could be that differentiating cells have limited MHC Class II expression (Montel-Hagen et al., 2019). However, since most protocols replicate positive selection through MHC-independent TCR stimulation with anti-CD3 antibodies, this seems an unlikely explanation.

[0010] The ability to produce CD4+ T cells could have a number of important implications. First, it could enable so-called helper function for cytotoxic CD8+ T cells - making for more effective anticancer cell therapy products (Melenhorst et al., 2022; Sommermeyer et al., 2016). Further, CD4⁺ T cells are progenitors for a number of Th cell types, including Treg cells that could be used as cell therapies for autoimmune diseases (Rosado-Sanchez and Levings, 2020).

[0011] State-of-the art iPSC-CD4 T cell induction among published papers involves growing human PSC-derived HSPCs in an “artificial thymic organoid” (ATO) with MS5 feeder cells expressing the Notch ligand DLL 1 (Montel-Hagen et al., 2019). but the ATO system has relatively limited commercial potential due to the use of MS5 feeder cells, which introduces uncontrolled xenogenic material to the T cell production process. Feeder- and serum-free methods for CD4⁺ T cell induction would be more clinically translatable while also allowing for precise control over parameters such as the levels of cytokines and Notch ligands to which differentiating T cells are exposed.

[0012] Thus there is a need to better generate (in greater volumes) in vitro CD4⁺ T cells, and thus CD4+ T cell derived cells. There is also a need to better understand the factors that may skew development and generation of CD4+ versus CD8+ T cells and thus cells derived from same. More specifically, production of T cells from human induced pluripotent stem cells (hiPSCs) has great potential for producing safe, effective, and affordable new therapies. While prior work has been able to efficiently convert human iPSCs to CD8+ T cells, the ability to convert hiPSCs to CD4⁺ T cells has been challenging, resulting in highly inefficient generation of cells and/or limited maturation or functionality (Edgar et al., 2022; Iriguchi et al., 2021; Jing et al., 2022; Kawai et al., 2021; Michaels et al., 2022; Montel-Hagen et al., 2019; Nishimura et al., 2013; van der Stegen et al., 2022).

[0013] A recent pre-print on bioRxiv described die ability to produce CD4⁺ T cells using commercial serum- and feeder-free media. CD4⁺ T cell induction was done by stimulating the cells with a relatively low concentration of the T cell receptor (TCR) pathway -stimulating reagents phorbol 12-myristate 13- acetate (PMA — 0.2-100 ng/mL) and ionomycin (lono — 60 — 2000 ng/mL) (Fong et al., 2022, 2025). Though the authors showed some functional characteristics of the produced cells and the ability to polarize to Tregs, they did not clearly demonstrate the ability to produce CD4⁺ T cells with a mature, naive phenotype that can be polarized into multiple different Th subtypes.

SUMMARY OF THE INVENTION

[0014] The present disclosure describes the combined effects of Notch and TCR signaling levels on in vitro differentiation of PSCs to CD4⁺ vs CD8⁺ T cells. The present invention in certain aspects comprise the optimization of Notch and TCR stimulation to produce clonally diverse, pheno typically mature, and functional PSC-CD4⁺ T cells, with yields similar to those previously reported for PSC-CD8⁺ T cells. Importantly, these results were obtained using fully -defined media in the absence of feeder cells. The ability to reliably produce controlled proportions of CD4⁺ and CD8⁺ T cells paves the way for improved in vitro isogenic immune models and improved off-the-shelf T cell therapies.

[0015] As such in some embodiments of the invention, the invention pertains to a method of obtaining a population of cells enriched for CD4 single positive (CD4+) naive T cells or CD8 single positive (CD8+) naive T cells in vitro. In some embodiments, the cells or population of cells obtained by the method comprises culturing double positive T cells under SP induction conditions comprising no or low Notch signaling. In some other embodiments, the method comprises further culturing the cells in a niche or culture medium comprising TCR stimulating agents. [0016] In some other aspects, the method comprises: a. providing CD4⁺ CD8⁺ T cells or a cell population comprising CD4⁺CD8⁺ double positive T cells optionally wherein the immature T cells are engineered to express and to generate cells that express one or more CAR, TCR, or other therapeutic modality OR engineered to express one or more transcription factor/cofactor, signaling protein, receptor, or secreted factor; b. culturing the cells in a serum-free, feeder cell -free T cell maturation niche and/or in a serum-free, feeder cell-free T cell maturation medium comprising: i. to enrich for CD4+ T cells no Notch ligand or Notch stimulating reagent or conditions or lower Notch stimulation than for generating CD8⁺ cells and optionally a Notch signaling inhibitor or suppressor. ii. to enrich for CD8+ T cells a reagent or conditions that promote the Notch stimulation; wherein the adjusting the level of Notch signaling stimulation in the culture medium biases the enhancement of CD4+ T cells (low or no Notch signaling) or CD8+ T cells (conditions of higher or stronger Notch signaling) in the resulting cell population.

In some other embodiments the method comprises culturing the cells in a niche or culture medium comprising cytokines as noted herein. In some other aspects the invention comprises niches comprising components of the niche for creating the desired notch ligand and TCR stimulation environment. In other aspects the invention provides culture media for same. In yet other embodiments the invention provides a method of culturing cells derived form the cells or cell populations comprising said single - positive or enriched with the desired single positive T cell (CD4 + or CD8+). In yet some other embodiments the invention provides a population of the cells resulting form the methods of the invention and cells isolated from said population. In some other embodiments the invention provides naive, and some aspects mature SPs capable of further differentiation or development. In yet some other aspects the invention provides uses and method for using the resulting cells and cell populations in medical treatment or in the treatment of various diseases or conditions. In yet some other embodiments, the invention provides pharmaceutical or biological compositions comprising the cells and cell populations of the invention.

[0017] Other features, objects, and advantages of the invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the invention, is given by way of illustration only, not limitation. Various changes and modification within the scope of the invention will become apparent to those skilled in the art from the detailed description. DESCRIPTION OF DRAWINGS

[0018] These and other features of the disclosure will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:

[0019] FIGURE 1 illustrates the approach for feeder-free T cell differentiation from pluripotent stem cells: A) Schematic overview of the stages of T cell differentiation from pluripotent stem cells to mature T cells. The approach for the present invention comprises a 5-stage process where pluripotent stem cells are aggregated and directed to become hemogenic endothelial (HE) cells in published conditions (Sturgeon et al., 2014; Michaels et al., 2022) and then cultured on a cell culture substrate functionalized with recombinant DLL4 and VCAM1 in a media designed to support endothelial to hematopoietic transition (EHT) in published conditions (Sugimura et al., 2017; Michaels et al., 2022) to produce hematopoietic stem/progenitor cells (HSPCs). Next, the hematopoietic stem/progenitor cells are directed into becoming T cell progenitors, then DP T cells in two-part serum- and feeder-free engineered thymic niche (ETN) comprising immobilized DLL4 and VCAM1 proteins and two staged formulations of soluble cytokines. Finally, DP T cells are matured into SP T cells. B) Soluble cytokines and concentrations of DLL4/VCAM I needed for each step of differentiation protocol. C) Representative flow cytometry plots of key cell types in each of the differentiation stage.

[0020] FIGURE 2 illustrates that conversion of CD3" human thymocytes into CD4⁺ T cells requires high TCR and low Notch stimulation levels, while conversion into CD8⁺ T cells requires intermediate TCR and low Notch stimulation levels. A) Schematic overview of the experimental design used to assess the effects of varying Notch and TCR stimulation on CD3’ human thymocytes isolated from postnatal human thymus. B) Quantification of percent and logio yield of 4SP, DP, and 8SP subsets within CD3⁺TCRab⁺CD27⁺ population after 7 days of stimulation with varying TCR (via anti- CD2/3/28) and Notch (coated Fc-DLL4) signaling levels.

[0021] FIGURE 3 illustrates that conversion of cord blood-derived hematopoietic stem/progenitor cells into CD4⁺ T cells requires low TCR and low-to-medium Notch stimulation levels, while conversion into CD8⁺ T cells requires high TCR and medium-to-high Notch stimulation levels. A) Schematic overview of the experimental design used to assess the effects of varying Notch and TCR stimulation on human cord blood-derived hematopoietic stem/progenitor cells. B) Quantification of percent and log™ yield of 4SP, DP, and 8SP subsets within CD3⁺TCRab⁺CD27⁺ population after 7 days of stimulation with varying TCR (via anti-CD2/3/28) and Notch (coated Fc-DLL4) signaling levels.

[0022] FIGURE 4 illustrates that conversion of hiPSC-derived HSPC into CD4⁺ T cells requires low Notch stimulation levels and TCR stimulation by (i) intermediate levels of anti-CD2/3/28, (ii) intermediate levels of PHA (phytohemagglutinin), or (iii) low levels of PMA (phorbol myristate acetate); while conversion into CD8⁺ T cells requires high Notch stimulation levels and TCR stimulation by high levels of anti-CD2/3/28, (ii) high levels of PHA (phytohemagglutinin), or (iii) very low levels of PMA (phorbol myristate acetate). A) Schematic overview of the experimental design used to assess the effects of varying Notch and TCR stimulation on hiPSC-derived HSPC. B) Quantification of percent and lo io yield per HSPC of live CD3⁺TCRab⁺, as well as 4SP, DP, and 8SP subsets within CD3⁺TCRab⁺CD27⁺ population, after 14 days of stimulation with varying TCR (via anti-CD2/3/28, PHA, or PHA) and Notch signaling levels (n = 4 independent differentiations).

[0023] FIGURE 5 directly compares percents of DP, 4SP, and 8SP within the CD3⁺TCRab⁺CD27⁺ population as quantified in FIGURE 4, as well as CD45RA⁺CD62L⁺ cells within the CD3⁺TCRab⁺ population. A) Schematic of the experiment design (from FIGURE 4). B) Line plots comparing percents of each population as a function of TCR (x-axis) and Notch (different lines) stimulation inputs; error bars represent mean ± standard deviation.

[0024] FIGURE 6 directly compares yields of DP, 4SP, and 8SP within the CD3⁺TCRab⁺CD27⁺ population as quantified in FIGURE 4, as well as CD45RA⁺CD62L⁺ cells within the CD3⁺TCRab⁺ population. A) Schematic of the experiment design (from FIGURE 4). B) Line plots comparing logic yield per HSPC of each population as a function of TCR (x-axis) and Notch (different lines) stimulation inputs; error bars represent mean ± standard deviation.

[0025] FIGURE 7 illustrates representative populations of cells from FIGURE 4-6, highlighting that hiPSC-derived CD4⁺ T cells generated via stimulation with cither anti-CD2/3/28 or PHA express the naive markers CD27, CD45RA, and CD62L much more highly than cells generated via stimulation with PMA. A) Schematic overview of the experimental design (from FIGURE 4). B-D) Representative flow cytometry contour plots showing populations of naive-like CD27⁺CD45RA⁺CD62L⁺ CD4⁺ T cells generated in the absence of DLL4, and with TCR stimulation via B) 0.1% anti-CD2/3/28, C) 0.5 ug/mL PHA, or D) 0.25 ng/mL PMA.

[0026] FIGURE 8 summarizes data from FIGURE 4-6 to illustrate that the percentage and yields of hiPSC-derived CD4⁺ vs CD8⁺ T cells are tunable depending on levels of Notch and TCR stimulation inputs. A) Schematic overview of the experimental design (from FIGURE 4). B) Percentage of 4SP vs 8SP among CD3⁺TCRab⁺CD27⁺ cells. C) Log yield per HSPC of 4SP vs 8SP. Darker colors indicate increasing concentrations of coated DLL4, different symbols represent different TCR stimulating reagents (anti-CD2/3/28, PHA, PMA), error bars represent mean ± standard deviation.

[0027] FIGURE 9 illustrates that Notch and TCR stimulation finetunes induction of naive T cell markers. A) Schematic overview of the experimental design (from FIGURE 4). B) Quantification of percents and log™ yield per HSPC of subsets (within CD3⁺TCRab⁺ population), after 14 days of stimulation with varying TCR (via anti-CD2/3/28, PHA, or PMA) and Notch signaling levels. Higher levels of naive T cell markers (CD27, CD45RA, CD62L) are seen in samples stimulated with low levels of Notch and medium -to-high levels of anti-CD2/3/28 or PHA, or low-to-medium levels of PMA.

[0028] FIGURE 10 illustrates that weaker Notch ligands are more permissive to 4SP induction from hiPSCs, and that Notch inhibition can rescue 4SP induction when cells are exposed to Notch ligands. A) Schematic overview of the experimental design used to assess the effects of varying Notch ligands and Notch signaling inhibitor, DAPT, in generating 4SPs from hiPSC-derived HSPCs. B-C) hiPSC- DPs are stimulated for 14 days with 0.1% anti-CD2/3/28 in the presence of DLL4 and varying DAPT concentrations, and assessed for B) percentage (%), or C) yield per HSPC input (computed as log? foldchange relative to no DAPT), of 4SP vs 8SP (within CD3⁺TCRab⁺CD27⁺ population). D-E) hiPSC- DPs are stimulated for 14 days with 0.1% anti-CD2/3/28 in the presence of different coated Notch ligands (Fc-DLL4, Fc-DLLl, Fc-JAGl, Fc-JAG2, all with Fc-VCAMl, or VCAM1 alone, or uncoated wells) and with either DMSO or 5 pM DAPT, and assessed for D) percentage (%), or E) yield per HSPC input (corrected for baseline batch-to-batch variation via log2 fold-change to each batch mean), of 4SP vs 8SP (within CD3⁺TCRab⁺CD27⁺ population).

[0029] FIGURE 11 illustrates representative populations of cells from FIGURE 10, highlighting how the addition of DLL4 affects the relative frequency of CD4⁺ vs CD8⁺ T cells and expression of naive T cell markers CD27, CD45RA, and CD62L. A) Schematic overview of the experimental design (from FIGURE 9). B) Representative flow cytometry contour plots showing populations of naive-like CD27⁺CD45RA⁺CD62L⁺ CD4⁺ T and CD8⁺ T cells generated respectively without and with 10 pg/mL coated Fc-DLL4, both with TCR stimulation via 0.1% anti-CD2/3/28.

[0030] FIGURE 12 illustrates that CD4⁺ T cells can be generated from both hiPSC-derived DP and CD8⁺ T cells, though with much higher efficiency from DP cells. A) Schematic overview of the experimental design used to assess 4SP vs 8SP generation from different sorted starting populations generated from hiPSC-derived HSPCs. B) Representative flow cytometry scattcrplots showing the percent of 4SP vs 8SP cells among CD3⁺TCRαβ⁺ T cells after initial sorting for (i) all CD3⁺ cells, (ii) CD3⁺TCRαβ⁺ 8SP cells, or (iii) CD3⁺TCRαβ⁺ DP cells; all followed by a week of rest with no TCR stimulation, followed by one day of TCR stimulation by 3 μg/mL coated anti-CD3 [clone OKT3] and six days of expansion in PSC4 (an IL-7 + IL-15 based media). C) Quantitation of population distributions in B). D) Representative flow cytometry scatterplots showing the percent of 4SP vs 8SP cells among CD3⁺TCRαβ⁺ T cells after initial sorting for DP cells, followed by a week rest or TCR stimulation by 0.5% or 1.25% anti-CD2/3/28, followed by one day of TCR stimulation by 3 pg/mL coated anti-CD3 [clone OKT3] and six days of expansion in PSC4.

[0031] FIGURE 13 illustrates hiPSC-derived CD4⁺ T cells can be generated in different media formulations. A) Schematic overview of the experimental design used to assess 4SP vs 8SP generation from hiPSC-derived HSPCs in different maturation media variants. Zandstra developed PSC2 and Zandstra and StemCell technologies (SCT) developed StemSpan progenitor maturation media (SCT mat). B) Representative flow cytometry scatterplots showing the percent of 4SP vs 8SP cells among CD3⁺TCRαβ⁺ T cells after 7 days of stimulation with 0.1% anti-CD2/3/28 in the absence of Notch ligands.

[0032] FIGURE 14 illustrates new DP maturation medias based on combinations of Zandstra and commercial StemCell Technology (SCT) reagents, specifically showing that SCT-developed StemSpan progenitor maturation supplement (SCT Mat, shortened to SCT in the figure) combined with Zandstra- developed JAC Ultra base media (referred to as SCT JAC) generates the highest yields and purities of DP T cells among all combinations tested. Selective differentiation to CD4+ vs CD8+ T cells is confirmed when growing cells in SCT JAC. A) Schematic overview of the experimental design used to assess DP and 4SP vs 8SP generation from hiPSC-derived HSPCs in different maturation media variants. B) Schematic of different media combinations used for DP T cell maturation. C) Line plots showing yield per HSPC of DP and CD3⁺TCRαβ⁺ cells generated in different maturation medias at different maturation timepoints. D) Stacked bar charts showing the percent of cells differentiated into different populations at each timepoint (within the live population); note that all populations including 4SP and 8SP (8ab: CD8ot[i , 8aa: CD8aa⁺) are not necessarily CD3⁺TCRαβ⁺ at this point - overall frequencies of which are shown in the lower plots. E) Bar charts showing the percent of 4SP vs 8SP (within CD3⁺ TCRαβ⁺CD27⁺ population) after 14 days of stimulation. Cells were grown in the indicated media throughout both the DP maturation phase (MO - M21) and SP induction phase (SO - S14). For C-E), error bars represent mean ± standard deviation (n=4 independent differentiations).

[0033] FIGURE 15 illustrates that the identified CD4⁺ T cell induction conditions are applicable to different hPSC lines. A) Schematic overview of the experimental design used to assess 4SP vs 8SP generation from Hl hPSC-derived HSPCs. B) Flow cytometry contour plots confirming differentiation towards CD7⁺CD5⁺ Pro-T cells (left) and DP cells (right). C) Flow cytometry contour plots confirming differentiation towards CD3⁺TCRαβ⁺CD27⁺ 4SP cells after 7 days of stimulation by 0.1% anti- CD2/3/28 in the absence of Notch ligands.

[0034] FIGURE 16 illustrates hiPSC-derived CD4⁺ cells express naive T cell markers and the lineagespecific transcription factor ThPOK. A) Schematic for skewing hiPSC-DPs into DP-, 4SP-, or 8SP- skewing conditions. B) Representative flow cytometry contour plots of CD3 TCRa[i cells and their CD4 vs CD8a expression pattern after stimulating hiPSC-DPs for 7 days. C) Top: Representative histograms of expression of naive CD4⁺ T cell markers and lineage-specific transcription factors of hiPSC-DPs, -4SPs, and -8SPs after 7 days of stimulation; Bottom: log2-transform of each marker’s relative geometric mean fluorescence intensity (gMFI) of hiPSC-4SP/-8SP normalized to hiPSC-DPs. D) hiPSC-DPs were skewed into 4SP-skewing condition, and expression of CD45RA vs CD45RO are measured 1-2 weeks later. For C-D), error bars represent mean ± standard deviation; n=3-4 independent differentiations; statistical significance was determined using ordinary one-way ANOVA with p values shown.

[0035] FIGURE 17 illustrates hiPSC-derived CD4⁺ cells expand and express relevant markers upon stimulation. A) Sorted hiPSC-CD4⁺ T cells were expanded for 14 days in Immunocult-XF + 100 lU/mL IL-2, with stimulation on day 0 and 7 using 2.5% anti-CD2/3/28. B) The left plot shows the foldexpansion and the right plot shows the percent 4SP cells (CD4⁺CD8a⁺ within CD3⁺TCRαβ⁺) throughout expansion. Data is shown with mean ± standard deviation; n=4 independent differentiations. C) Expanded cells were rested overnight in 10 lU/mL IL-2, restimulated with anti-CD2/3/28 for 48 hours, and measured for T cell activation markers using flow cytometry. Data is shown with mean ± standard deviation; n=3-4 independent differentiations, or n=3-4 thymic- or blood-donors collected in 2-3 experiments. Statistical significance was determined using unpaired t-tests and ordinary one-way ANOVA with p values shown.

[0036] FIGURE 18 illustrates that hiPSC -derived CD4⁺ T cells express low levels of cytotoxicity- associated genes. A) Schematic overview for experimental design to produce, expand, and sequence hiPSC-derived T cells with scRNA-seq, scTCRseq, and CITEseq. B) UMAP visualization and RNA leiden-based clusters identified immature/mature CD4⁺ T cells, CD8⁺ T cells, DP cells (P: proliferating), ILC3-like cells, and y5 T cells. C) Dotplots showing the percent of cells in the CD4, CD8, and DP populations expressing key maturation, lineage-specific, and differentially -expressed genes, as well as the mean expression levels of the genes within each population group.

[0037] FIGURE 19 illustrates gene expression levels from FIGURE 18 overlayed on UMAP embeddings. A-B) Scatterplots showing expression levels per cell of markers differentially expressed in A) 4SP and B) 8SP populations.

[0038] FIGURE 20 illustrates that hiPSC-derived CD4⁺ and CD8⁺ T cells express expected signature genes. A) CD4 and CD8 signature scores derived from human scRNAseq CD4- or CD8-annotated T cell clusters from Chopp et aZ. ⁽2020) and Park et al.(2020) B) CD4 and C) CD8 signature scores for subsets found in FIGURE 18. D) Scatterplots showing CD4 and CD8 signature scores per cell overlayed on UMAP embeddings. CD4 signature genes: ADD3, AP3M2, ARMH1, ATP5H, BACH2, Clorf228, CAMK4, CCR6, CD40LG, CLDN1, CORO1B, CTSL, CYLD, DENND2D, EMP3, FCGRT, FXYD5, GIMAP4, GPR183, GSTK1, HPGD, IFITM2, IL27RA, ITM2A, KLF2, KLRB1, LTB, MAL, MSL3, NDFIP1, NOSIP, PRMT2, RIPOR2, RNASET2, RORA, RPL17, RPL36A, SAMD3, SATB1, SCML1, SCML4, SELENOW, SLC2A3, STAT5A, SYTL2, TC2N, THOC7, TMSB10, TTC39C; CD8 signature genes: ACTN1, AOAH, C6orf48, CCL4, CCL5, CD248, CD55, CD7, CD8A, CD8B, CLEC2D, COTL1, CRTAM, CST7, CTSW, CYTIP, DUSP2, EGR1, FAM173A, FTL, GZMK, GZMM, HCST, HLA-DPB1, ICOS, ID3, LDLRAP1, LINC00861, LINC02446, LRRN3, LYAR, MT2A, MYC, NAB2, NELL2, NKG7, NR4A1, PASK, PAXX, PDE3B, PECAM1, PLAC8, PTGER4, PTPN6, RAP1A, RP11-291B21.2, RPL13, RPLP0, RPLP1, RPS10, RPS18, RPS19, RPS2, RPS5, S100B, SAT1, SH2D2A, SNX9, SPINK2, STAG3, STK17A, ZFP36, ZFP36L2.

[0039] FIGURE 21 illustrates that hiPSC-derived CD4⁺ T cells have rearranged TCRa- and TCRb- chains. A) TCR loci rearrangement is positionally -biased (adapted from Park et al; 2020); TCR 0 chain rearrangements initially prefer V segments at the ends of the V0 array in early developmental stages (Pro-T), and access interior V segments in later stages (DP, SP); a chain rearrangements initially prefer 3’ Va and 5’ Ja segments in earlier stages (Pro-T, DP), and access increasingly dispersed segments through serial recombination during SP induction and positive selection. B-C) B) TCR0 and C) TCRa chain segment usages across hiPSC-, thymic-, and blood-T cells. Segments are ordered based on genomic position, frequencies of each row sum to 1.

[0040] FIGURE 22 illustrates that hiPSC-derived CD4¹ and CD8¹ T cells can be derived from cells engineered with both class I- and class Il-targeted ectopic TCRs (eTCRs). A) Schematic overview of engineering hiPSCs with class I TCR (targeting EBV LMP2 peptide) or class II TCR (targeting glutamic acid decarboxylase 65 (GAD65)) via knockin to the TRAC locus, ablating expression of endogenous TCR sequences. Polyclonal cells were differentiated towards HE, HSPC, and Pro-T cells in standard conditions, into DP cells in SCTmat media, and finally assayed in 4SP or 8SP-skewing conditions. B- C) Representative flow cytometry scatterplots showing CD4 vs CD8b expression in CD3 cTCR CD27 cells from both lines 14 days post-SP induction in both B) 8SP-baised (TCR stimulation with 1.25% anti-CD2/3/28, Notch stimulation on wells coated 10 μg/mL DLL4 + 2.5 μg/mL VCAM1) and C) 4SP- biased conditions (TCR stimulation with 0.3% anti-CD2/3/28 on uncoated wells).

[0041] FIGURE 23 illustrates that the hiPSC-derived CD4⁺ T cells polarized into Thl , Th2, and Thl7- like states express relevant transcription factors and chemokine receptors. A) Schematic of polarization and the relevant Thl/2/Thl7 transcription factors, chemokine receptors, and cytokines. iPSC- and thymus-derived Th cells were polarized for two weeks, while peripheral blood-derived Th cells were polarized for one week. B-C) After polarization, cells were rested overnight in 10 lU/mL IL -2, and then measured for B) transcription factors and C) chemokine receptors. Data were acquired by flow cytometry and gated on live CD4 CD8a . Relative gMFI (geometric mean fluorescent intensity) values normalized to ThO and then logz-transformed. Errorbars represent mean ± standard deviation of log - transformed values (n = 4 independent differentiations).

[0042] FIGURE 24 illustrates that the hiPSC-derived CD4⁺ T cells polarized into Thl, Th2, and Thl 7- like states express relevant cytokines. A-B) Rested cells from Figure 23 were assessed for intracellular cytokine expression after 4 hours of stimulation with PMA, ionomycin, and Brefeldin A. Data were acquired by flow cytometry and gated on live CD4 CD8a . A) Representative flow cytometry contour plots and B) percent of polarized hiPSC-, thymus-, or primary peripheral blood-derived T cells (CD4 CD8ct subset) expressing IFNg, IL-4, IL-17A/F after stimulation with PMA+Iono for 4 hours. Error bars represent mean ± standard deviation; n=4 independent differentiations, or n=4 thymic- or blood-donors collected in 3-4 experiments; statistical significance was determined using ordinary oneway ANOVA with p values shown.

[0043] FIGURE 25 illustrates that the hiPSC-derived Th cells from Figures 23-24 maintain their 4SP phenotype following polarization. A) Representative flow cytometry contour plots showing CD4 vs CD8a expression on hiPSC-Th cells after two weeks of polarization. B) Quantification of DN/DP/4SP/8SP subsets in A). C) Representative flow cytometry contour plots showing CD4 vs CD8a expression in thymus-derived Th cells after two weeks of polarization. B) Quantification of DN/DP/CD4SP/CD8SP subsets in C). Errorbars represent mean ± standard deviation; n=4, collected in 3-4 individual experiments; statistical significance was determined using ordinary one-way ANOVA with p values shown.

[0044] FIGURE 26 illustrates that the hiPSC-derived Tregs express Treg-relevant markers. A) Timeline and molecules used to generate hiPSC -Tregs from hiPSC-DPs. B) Flow cytometry contour plots showing the phenotype of cells at the end of Treg induction stage, indicating high expression of key Treg markers CD25, FOXP3, and HELIOS. C) Flow cytometry contour plots showing the phenotype of sorted hiPSC-Tregs and hiPSC-Tconvs compared to ex vivo human postnatal thymic Tregs. D) Histograms showing expression levels of Treg -related markers CD39, ICOS, and intracellular CTLA-4 (iCTLA-4).

[0045] FIGURE 27 illustrates that low-level TCR stimulation increases the yield of hiPSC-derived DP TCRctfl cells. A) Schematic overview for experimental design to test effect of TCR stimulation reagents on DP T cell generation from hiPSC-derived HSPCs. B) Line plots showing the number (left) and percent (right) of CD3⁺TCRαβ⁺ cells per 96-well as a function of different TCR stimulating reagents applied between M14 and M21, with concentrations per dose level indicated in C). D) Line plots showing the number (left) and percent (right) of CD3 TCRαβ⁺ DP cells per 96-well as a function of different TCR stimulating reagents applied between M21 and M28, with concentrations per dose level indicated in E). Cells treated with a given dose level between M21-M28 received the same dose level between M14-M21, which was approximately V the concentration. The concentration was therefore “ramped up” between the first and second week.

[0046] FIGURE 28 builds upon FIGURE 27 to illustrate that low-level TCR stimulation increases the yield of hiPSC-derived DP TCRafJ cells. A) Schematic overview for experimental design to test effect of TCR stimulation reagents on DP T cell generation from hiPSC-derived HSPCs. Compared to FIGURE 27, this experiment added Thapsigargin, lonomycin, and soluble anti-CD3 (clone OKT3). B- D) Line plots showing B) the number of CD3⁺TCRαβ⁺ DP cells per 96-well and C) the expression level of TCR stimulation marker CD69 as a function of different TCR stimulating reagents applied between M14 and M21, with concentrations per dose level indicated in D). The doses that optimize DP T cell production yields are 4-16x lower than that needed to induce CD69, causing differentiation towards SP T cells.

[0047] FIGURE 29 illustrates that low doses of PMA increase production of cord blood-derived DP TCRafl cells. A) Schematic overview for experimental design to test effects of PMA and ionomycin on DP T cell generation from cord blood-derived HSPCs. B-C) Flow cytometry scatterplots showing the percent of B) CD3+TCRa0+ cells and C) DP cells in wells treated with different doses of PMA and ionomycin for one week between days 35 and 42. DP and TCR⁺ population frequencies are optimized with 0.2 ng/mL PMA + 1 ng/mL lono.

[0048] FIGURE 30 illustrates the effects of IL4, IL5, IL6, IL7, IL9, IL12, IL18, and anti-CD2/3/28 complexes on hiPSC-derived T cell maturation and CD4⁺ vs CD8⁺ T cell commitment. A) Schematic overview for experimental design to identify effects of and optimize levels of IL4, IL5, IL6, IL7, IL9, IL 12, IL 18, and anti-CD2/3/28 on DP T cell generation from cord blood-derived HSPCs. Cells were treated with the eight factors with three levels each in a fractional factorial response surface model (RSM) design-of-experiments approach. Resulting population measurements were fit with a 2^nd order polynomial with all interaction terms between each factor. B) Dotplot showing the coefficient and p- value estimated for each term in the polynomial model. * between factor names indicates an interaction term. Darker and larger circles indicate stronger and more significant effects predicted by the model, respectively. C) Model prediction for individual factor effects on the number of DP (± TCR), CD3⁺TCRαβ⁺ 8SP, CD3⁺TCRαβ⁺ 4SP, CD3⁺TCRαβ⁺CD27⁺, CD8aa⁺, and TCR 6 cells when sweeping across concentration levels and holding all other factors constant at level 0. D) Table of factor concentrations per level.

[0049] FIGURE 31 illustrates custom media formulations derived by using the model in Figure 30 to optimize factor levels for DP, 4SP, and 8SP cells. A) Optimization objectives for DP, 4SP, and 8SP cells that maximize target cell types (DP: DP cells (± TCR); 4SP: CD3⁺TCRαβ⁺ 4SP cells and CD3⁺TCRαβ⁺CD27⁺ cells; 8SP: CD3⁺TCRαβ⁺ 8SP cells and CD3⁺TCRαβ⁺CD27⁺ cells) while minimizing undesirable innate cell types (CD8aa⁺ and TCRy8⁺ cells). B) Distribution of factor levels within the top 16 (DP), 126 (4SP), or 24 (8SP) optimal media combinations identified via a basin hopping algorithm that uses a desirability function based on the optimization objectives in A). C-D) Similar approach to A-B) but using optimization objectives that do not penalize for the innate cell types. E) Novel media formulations based on distributions of optimal factor levels in B) and D). The factors are added to JAC Ultra in addition to standard concentrations of the other factors in PSC2: SCF, Flt3L, CXCL12, IL3, TNFa.

[0050] FIGURE 32 illustrates the effects of custom media fonnulations on maturation to DP cells and induction to 4SP vs 8SP cells. A) Schematic overview for experimental design to test effects of media optimized for DP (PSC2.1), 4SP (PSC2.4) and 8SP (PSC2.8) induction from hiPSC-derived HSPCs. Cells were grown for 3 weeks in PSC2, three weeks in PSC2.1 (indicated as PSC2.1 M0), one week in PSC2 followed by 100% media exchange (MX) to PSC2.1 for two weeks (indicated as PSC2.1 M7), or two weeks in PSC2 followed by 1005 MX to PSC2.1 , PSC2.4, or PSC2.8 for one week (indicated with M14). B) Percents and number of cells per 96 well generated in different media conditions for various cell sub-populations of interest. PSC2.1 addition at M0 or M7 generates a relatively high frequency of CD3⁺TCRαβ⁺CD27⁺ T cells

[0051] FIGURE 33 illustrates serial combinations of media formulations that enhance differentiation towards TCR+, DP, 4SP, and 8SP cells. A) Schematic overview for experimental design to test serial combinations of media for synergies in production of different hiPSC-derived T cell subpopulations. B) Dotplots showing the percent and number of cells per 96 well that differentiated into CD3⁺TCRαβ⁺ cells overall, DP (CD3⁺TCRαβ⁺) cells, and 4SP vs 8SP cells (both CD3⁺TCRap⁺CD27⁺) after being cultured in different combinations of media from S0-S7 (Y-axis) and from S7-S14 (X-axis). PSC2 X% refers to % anti-CD2/3/28 supplementation. SCT refers to StemSpan maturation media (SCT mat). The highest percent of CD3⁺TCRαβ⁺ cells is achieved when cells were rested (no TCR stimulation) after initial stimulation, connecting to FIGURES 27-28 and the stimulation-rest regime in FIGURES 4-11. The highest rate of 4SP induction generally occurs when using PSC2.1 or PSC2.4 media in week one or two, as well as when using low (0.1%) anti-CD2/3/28 input (the latter as seen in FIGURES 4-7).

[0052] FIGURE 34 illustrates the synergistic effects of PMA and IL-4 in generating highly pure DP cells from hiPSC-derived HSPCs. A) Schematic overview for experimental design to test combinations of PMA, SCF, IL-4, and base media (PSC2 vs C37 media) for generation of pure populations of DP T cells from hiPSC-derived HSPCs. Cells were cultured from MO to M7 in either (i) PSC2 media modified to remove SCF or (ii) C7 media, which supplements CXCL12 and IL-7 (at the same concentrations as in PSC2) into JAC Ultra base media. After, cells were transferred into downstream media combinations either in PSC2 -SCF or C37 media, which is like C7 media but further contains IL-3 at the concentration used in PSC2. In both such medias, cells were further supplemented with combinations of [0 or 0.04 ng/mL PMA] x [0, 1, or lOng/mL SCF] x [0 or lOng/mL IL-4]. B) Dotplots showing percent and logw yield per HSPC input of DP, CD3⁺TCRap⁺, CD8aa⁺, CD3 TCRv8 , and CD117 high cells (mast/progenitors). The highest rate of DP cells is achieved in conditions with PMA. The lowest rates of CD8aa⁺ and TCRy8⁺ cells are achieved in conditions with IL-4, with a slight trade-off in DP yield. Transient SCF removal in the media from M0-M7 prevents emergence of CD117 high cells, even at high dosages of SCF and IL-4, which together led to significant CD117 high induction in prior formulations (e.g. FIGURE 32). C) Dotplots showing percent and logic yield per HSPC input of the same populations in reference media variants: PSC2 and SCT mat supplements added to either SFEM II or JAC Ultra base. New media formulations outperform commercial media (SCT mat + SFEM II), but the highest performance comes from the combination of SCT mat supplement and JAC Ultra base media (see also FIGURE 14).

[0053] FIGURE 35 illustrates media formulations and timelines used for induction of HSPCs from hPSCs in the examples of the invention.

[0054] FIGURE 36 illustrates media formulations and timelines used for induction of T cells from hPSCs-derived HSPCs in the examples of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0055] It is herein disclosed serum-free and feeder cell-free in vitro methods and compositions for the tunable production of single positive T cells. The inventors have developed an optimized niche, cell culture media and methods, including staged media and culture protocols for double positive (CD4+CD8+) T cell maturation and single positive (SPCD4⁺ (4SP) and SPCD8⁺ (8SP) T cell induction, and expansion. Overall, from an isolated subset or mixture of pluripotent stem cell-derived cells (e.g. iPSC, or in some embodiments hiPSC-derived cells), the invention in some aspects provides the ability to tune for the generation of different ratios of CD4⁺ vs CD8⁺ single positive T cells based on the initial population composition, TCR stimulation reagent and level, Notch ligand and level, and media factor components and levels.

[0056] In some other aspects, the invention provides the ability obtain predicable ratios of CD4⁺ vs CD8⁺ T cells generated from double positive (CD4+, CD8+) T cells by controlling Notch and TCR pathway stimulation. In yet some other aspects of the invention, it is shown that optimal levels of multiple TCR pathway stimulators are compatible with PSC-CD4⁺ T cell production. In yet some further aspects, the invention provides the ability to enhance CD4+ T cell production or generation from a population of double positive T cells. In other aspects, the same niche and media can be used to generate CD4⁺ T cells from CD8⁺ T cells. Overall, these results suggest removal, suppression, inhibition or down regulation of Notch signaling, such as using no or lower affinity notch ligands and/or Notch inhibitors, such as DAPT, and optimization of TCR stimulation is a broadly-applicable strategy to enhance CD4⁺ T cell production or generation from PSCs and PSCs derived double positive cells. In some preferred embodiments, the double positive cells are TCR⁺/CD697CD27^_ double positive CD4⁺CD8⁺ T cells. In yet some further embodiments, the CD4 single positive (4SP) generated using the methods, niche, cell culture media of the invention are functional naive mature T cells that are capable of generating (e.g through polarization) to various subtype of 4SP derived cells, such as T helper (Thl, Th2 and Thl7) cells and that express diverse V(D)J recombination-generated T cell receptor (TCR) sequences.. In addition, the invention provides novel methods, novel niches and media of generating Treg cells from the 4SP cells using media comprising TGF-P but does not require all-trans retinoic acid (ATRA).

[0057] The approaches and conditions disclosed here have significant potential for advancing allogeneic T cell-based therapies

[0058] In some aspects the invention provides a means where the ratio of hiPSC-derived CD4⁺ vs CD8+ T cells, a key parameter associated with clinical efficacy of engineered CAR-T cell therapies (Melenhorst et al., 2022; Sommermeyer et al., 2016), can be tuned by setting specific levels of TCR (PMA -0.2-1 ng/mL, lono 0 - 200 ng/mL, PHA 0.5 - 2.5 ug/mL, and/or anti-CD2/3/28 0.1 - 0.5%) and Notch stimulation (0 - 10 ug/mL). Going beyond the results demonstrated by Sangamo (Fong et al., 2022, 2023), it is demonstrate that alternate TCR stimulation reagents (phytohemmag lutinin [PHA] and anti-CD3 complexes) surprisingly produce higher yields of CD4+ T cells than PMA/Iono. Going beyond the results demonstrated by Montel-Hagen et al. (2019), it is herein shown that weaker Notch ligands JAG1 and JAG2 are more permissive than both DLL1 and DLL4 to CD4+ T cell induction from hiPSCs, though the best induction occurs in conditions without Notch ligands or when Notch signaling is blocked by inhibitors. It is herein shown that Notch inhibition with DAPT (0.5 - 10 uM), and to a lesser degree with soluble DLL4 (>10 ug/mL) (when using a niche with immobilized Notch ligancs), can rescue CD4+ T cell induction for hiPSC-derived cells grown in the presence of Notch ligands. This result has consequences for generalizing the method of Notch removal to contexts such as ATOs where Notch ligands presented on feeder cells may be otherwise difficult or impractical to control, enabling tunable control of CD4+ vs CD8+ T cells in diverse differentiation systems.

[0059] It is also herein surprisingly shown that Notch and TCR stimulation dose -dependencies in the relative conversion to CD4+ and CD8+ T cells, depending on the reagent used and input cell type (thymocyte, cord blood HSPC-derived T cell, hiPSC-derived T cell). Although, longer/stronger and shorter/weaker TCR stimulation during in vivo positive selection yield CD4+ and CD8+ T cells, respectively (Taniuchi, 2018), prior to the present invention, it was unclear how artificial TCR stimulation modalities used for in vitro T cell maturation map onto this behavior. Unexpectedly, for some TCR pathway stimulating reagents, it was found herein that higher levels of input bias differentiation towards CD8⁺ T cells, rather than the expected CD4⁺ T cells. This helps to explain the lack of CD4¹ T cells in many developed protocols even where Notch ligands are not present in the DP- to-SP induction conditions (Iriguchi et al., 2021; Kawai et al., 2021; Maeda et al., 2016). Less well- understood overall was how Notch, a key inducer of early T cell development (Michaels et al., 2022), affects later decisions including the CD4⁺ vs CD8⁺ T cell induction. The present invention, , provides a clear demonstration that Notch signaling specifically suppresses CD4⁺ T cell induction in vitro from CB- or hiPSC-derived HSPCs and provides methods and niches and cell culture media to counteract that.

[0060] While confirming that CD4⁺/CD8⁺ double-positive (DP) T cells are the best at being converted to CD4⁺ T cells, it was also herein found that it is possible (but less efficient) to convert CD8⁺ T cells to CD4⁺ T cells in our optimized TCR and Notch stimulation conditions (see above). It is herein demonstrated the ability to produce CD4+ T cells from HSPCs generated from hiPSCs using both AggreWell-based differentiation conditions (Michaels et al., 2022) In combination with experiments identifying conditions to generate CD4+ T cells from human CB-derived HSPCs and human postnatal thymocytes, it is herein demonstrated that our methodology for identifying CD4+ T cell induction conditions is applicable to diverse input cell sources and types.

[0061] In addition, the present invention disclose media factors to improve production of DP, CD4⁺, and CD8⁺ T cells. First, the findings on CD4⁺T cell induction shows that a very low-dose of PMA (0.01 - 0.4 ng/mL) is effective for expanding and/or stabilizing hiPSC- and CB-derived DP T cells, while avoiding differentiation to CD8⁺ cells as seen with other TCR stimulating reagents (PHA and anti- CD2/3/28 complexes). Also shown are the discovery of niche factors that purify for and/or expand DP, CD4⁺, and CD8⁺ T cells in vitro, including IL4, IL5, IL6, IL 9, IL 12, and IL 18. While many of these have been identified as growth factors for thymocytes (Shichkin and Antica, 2022), they were not before known to play a role in the CD4+ vs CD8+ T cell switch. IL6 and other common y chain cytokines are known to be essential for CD8+ differentiation (Etzensperger et al., 2017) and IL12/18 have been demonstrated to improve hiPSC-derived CD8⁺ T cell expansion (Kawai et al., 2021); however, the effects of IL4, IL5, IL6, IL9, IL12, and IL18 have not been described for induction and maintenance of hiPSC-derived DP or CD4+ T cells, nor induction of hiPSC-derived CD8⁺ T cells. The present invention provides media comprising same in the maturation, induction and maintenance protocols herein. The screening and optimization of these factors, enabled the establishment of optimized niche and culture media for in vitro CD4+ T cell production.

Definitions

[0062] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0063] Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), Fields Virology, 6¹¹¹ Edition, published by Lippincott Williams & Wilkins, Philadelphia, PA, USA (2013), Knipe, D.M. and Howley, P.M. (ed.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1- 56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4* ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. [0064] As used herein the terms, “administration,” “administering” and variants thereof refers to introducing a composition or agent (e.g., nucleic acids, in particular ceDNA) into a subject and includes concurrent and sequential introduction of one or more compositions or agents. “Administration” can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. “Administration” also encompasses in vitro and ex vivo treatments. The introduction of a composition or agent into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intralymphatically, intratumorally, or topically. Administration includes self-administration and the administration by another. Administration can be carried out by any suitable route. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

[0065] As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce a toxic, an allergic, or similar untoward reaction when administered to a host.

[0066] The term “cell-derived cell” refers to a cell that differentiated from a different cell type. For instance, “CD4⁺-T cell-derived cells” refers to a cell that evolved from a “CD4⁺ T cell in the context of lineage of cell development, such as a T helper cells (e.g. Thl, Th2, Thl7) and Treg.

[0067] “Double positive cells”, “double positive T cells”, “DP”, “CD4⁺CD8⁺ cells or T cells” are T cells that express both CD4 and CD8 cell surface markers. There can be various maturation stages of DPs identified by their cell surface markers. In one embodiment the DP cells are TCR⁺CD69 CD27^_ double positive CD4⁺,CD8⁺ T cells.

[0068] As used herein, the phrase an “effective amount” or “therapeutically effective amount” of an active agent or therapeutic agent, such as a therapeutic nucleic acid, is an amount sufficient to produce the desired effect, e.g., inhibition of expression of a target sequence in comparison to the expression level detected in the absence of a therapeutic nucleic acid. Suitable assays for measuring expression of a target gene or target sequence include, e.g. , examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

[0069] As used herein, the term “exogenous” is meant to refer to a substance present in a cell other than its native source. The term “exogenous” when used herein can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, as used herein, the term “endogenous” refers to a substance that is native to the biological system or cell.

[0070] The term “feeder - free” refers to a cell culture niche/sy stem/culture media that is “free” (does not comprise) from cells (i.e. feeder cells, in certain aspects stromal cells ) that provide unknown collection of factors that allow cells to grow or be cultured or differentiate. In a feeder- free niche/system/culture media, the niche/sy stem/culture media comprises the critical factors or other activators required for the particular purpose. As the niche/system/media is well defined, it poses a lesser risk of transferring animal/human viruses or other contaminants to the cells/culture which is extremely important in in vivo applications. The term “serum-free”, analogously refers to a niche/system/media designed to grow a specific cell type or perform a specific application in the absence of serum. The term “stromal cell free”, means the absence of stromal cells.

[0071] The term “hemogenic endothelial cell” means an endothelial cell that has the potential to become a blood cell, is characterized by an endothelial-specific gene expression signature and endothelial-specific cell morphology, and is localized within the endothelial layer of a blood vessel. It is a specialized subset of the developing vascular endothelium that acquires hematopoietic potential and can give rise to multilineage hematopoietic stem and progenitor cells. In the present invention the presence of hemogenic endothelial cells was identified by the following cell surface marker profile: CD34+ and lack of CD43 (-). “Hemogenic endothelium cells” as used herein has the same meaning and are used interchangeably.

[0072] The term “hematopoietic stem/progenitor cells” or “HSPCs” or “blood progenitor cell” are repopulating progenitor cells, which give rise to lineage-specific cell types. As used herein it is a hematopoietic cell that has properties of either a hematopoietic stem cell or a hematopoietic progenitor cell. It can be identified by simultaneous expression of the cell surface markers CD34 and CD43. It is capable of differentiating into cells belonging to multiple hematopoietic lineages including, but not limited to, myeloid cells, erythroid cells, megakaryocytes, lymphoid cells, mast cells, basophils and eosinophils. It has the ability to differentiate into cells from at least two of these lineages. It may or may not be capable of self-renewal. It may be used interchangeably with the term hematopoietic stem/progenitor cell or hematopoietic stem and progenitor cells (HSPC).

[0073] The term “in vivo” refers to assays or processes that occur in or within an organism, such as a multicellular animal. In some of the aspects described herein, a method or use can be said to occur “in vivo” when a unicellular organism, such as a bacterium, is used. The term “e vivo” refers to methods and uses that are performed using a living cell with an intact membrane that is outside of the body of a multicellular animal or plant, e.g., explants, cultured cells, including primary cells and cell lines, transformed cell lines, and extracted tissue or cells, including blood cells, among others.

[0074] The term “in vitro” refers to assays and methods that do not require the presence of a cell with an intact membrane, such as cellular extracts, and can refer to the introducing of a programmable synthetic biological circuit in a non-cellular system, such as a medium not comprising cells or cellular systems, such as cellular extracts.

[0075] The term “naive T cell” or “naive CD4+ T cell” or “naive CD8+ T cell” refers to a T cell that has differentiated and undergone positive and negative selection in the thymus but have not encountered its cognate antigen within the periphery and thus has not been activated to differentiate into memory or effector cells. Thus, they can respond to novel pathogens/antigens that they have not before encountered. In other words they are mature circulating T cells that have not yet encountered their antigens.

[0076] The term “niche' as used herein refers to the microenvironment of the cells cultured pursuant to this invention, including any culture media, substrate in that environment or components , such as integrins or notch ligands any immobilized components, such as integrins (e.g. VCAM-1) or notch ligands (e.g., JAG1, JAG2, DLL1 or DLL4) or cytokines.

[0077] The term “pluripotent stem cell” or “PSCs” as used herein are cells that can self-renew. Selfrenewal is the capacity of the stem cells to divide indefinitely, producing unaltered cell daughters maintaining the same properties of the progenitor cell. In particular conditions or under specific signals, a stem cell is able to exit from self renewal and engage a program leading to differentiate into specialized cell types deriving from the three germ layers (ectoderm, endoderm, and mesoderm). In general, there are two types of PSCs, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs are derived from the inner cell mass (ICM) of preimplantation embryos and can be indefinitely maintained and expanded in the pluripotent state in vitro. Pluripotent stem cells can also be obtained by inducing dedifferentiation of adult somatic cells through an in vitro technology, known as cell reprogramming. Similarly, to ESC, iPSC can be expanded indefinitely and they are capable or differentiating in all the derivatives of the three germ layers. iPSCs are derived from somatic skin or blood cells.( e.g., from adult umbilical cord blood and neonatal keratinocytes) that are “induced” or reprogrammed back into an embryonic -like pluripotent state. “Cord blood-derived PSCs (“CB PSCs”) and “Thymocyte-derived PSCs” are PSCs, or iPSCs derived from cord blood and thymocyte stem cells, respectively, cord blood cells and thymocyte, respectively.

[0078] As used herein a “precursor cell or cells” are an intermediate cell before they become differentiated after being a stem cell. Usually, a precursor cell is a stem cell with the capacity to differentiate into only one cell type. [0079] As used herein a “progenitor cell or cells” descend from stem cells that then further differentiate into specialized cell types (one or more types of cells). They are more specific than a stem cell and can be pushed to differentiate into its "target" cell. There are many types of progenitor cells throughout the human body. Each progenitor cell generally is only capable of differentiating into cells that belong to the same tissue or organ and typically do not have the ability for self-renewal. “Pro T cells” are T cell progenitors.

[0080] The main difference between progenitor and precursor cells is that progenitor cells are mainly multipotent cells that can differentiate into many types of cells, whereas precursor cells are unipotent cells that can only differentiate into a particular type of cells.

[0081] “Single Positive”, “SP”, single positive cells”, single positive T cells” are T cells that express only one of CD4 or CD8 cell surface markers. Thus the above terms are sometimes used in conjunction with the cell surface marker they express, such as CD4 single positive cell, or CD4⁺ SP or 4SP, and analogously when referring to single positive CD8 cells. They are generated from DP cells. The terms 4SP and 8SP refer to single positive T cells that express either CD4 (“4”) or CD8 (“8”) cell surface markers.

[0082] As used herein, a “stem cell(s)” is an undifferentiated cell that can divide to produce some offspring cells that continue as stem cells and some cells that are destined to differentiate (become specialized). They can differentiate into more specialized cells but also have the capacity for selfrenewal. Stem cells are an ongoing source of the differentiated cells that make up the tissues and organs of animals and plants. Stem cells include pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and multipotent stem cells, such as cord blood stem cells, and adult stem cells, which are found in various tissues.

[0083] The term “subject” as used herein refers to a human or animal, to whom treatment, including prophylactic treatment, with the closed-end DNA (“ceDNA”) vector according to the present invention, is provided. Usually the animal is a vertebrate such as, but not limited to a primate, rodent, domestic animal or game animal. Primates include but are not limited to, chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, but are not limited to, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate or a human. A subject can be male or female. Additionally, a subject can be an infant or a child. In some embodiments, the subject can be a neonate or an unborn subject, e.g., the subject is in utero. Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of diseases and disorders. In addition, the methods and compositions described herein can be used for domesticated animals and/or pets. A human subject can be of any age, gender, race or ethnic group, e.g., Caucasian (white), Asian, African, black, African American, African European, Hispanic, Mideastem, etc. In some embodiments, the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject is already undergoing treatment.

[0084] As used herein, the term “suppress”, “decrease”, “interfere”, “inhibit” and/or “reduce” (and like terms) generally refers to the act of reducing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.

[0085] As used herein, the terms “treat”, “treating”, and/or “treatment” include abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms of a condition, obtaining beneficial or desired clinical results. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder) s).

[0086] Beneficial or desired clinical results, such as pharmacologic and/or physiologic effects include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (z.e. , not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0087] As used herein, the terms “therapeutic amount”, "therapeutically effective amount", an "amount effective", or “pharmaceutically effective amount” of an active agent (e.g. a CeDNA lipid particle as described herein) are used interchangeably to refer to an amount that is sufficient to provide the intended benefit of treatment. However, dosage levels are based on a variety of factors, including the type of injury, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular active agent employed. Thus, the dosage regimen may vary widely, but can be determined routinely by a physician using standard methods. Additionally, the terms “therapeutic amount”, “therapeutically effective amounts” and “pharmaceutically effective amounts” include prophylactic or preventative amounts of the compositions of the described invention. In prophylactic or preventative applications of the described invention, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of, a disease, disorder or condition in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, disorder or condition, including biochemical, histologic and/or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes presenting during development of the disease, disorder or condition. It is generally preferred that a maximum dose be used, that is, the highest safe dose according to some medical judgment. The terms “dose” and “dosage” are used interchangeably herein.

[0088] As used herein the term “therapeutic effect” refers to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.

[0089] For any therapeutic agent described herein therapeutically effective amount may be initially determined from preliminary in vitro studies and/or animal models. A therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan. General principles for determining therapeutic effectiveness, which may be found in Chapter 1 of Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.

[0090] As used herein the term “tunable” in the context of cellular differentiation refers to the ability to control the cellular environment to favour one cellular differentiation outcome or path.

[0091] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective components) thereof, that are essential (subject to interpretation of equivalents and variations as would be apparent to person of skill in the art as may be permitted by rules of interpretation of applicable law), to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0092] As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0093] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0094] As used in diis specification and the appended claims, the singular fonns “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. Similarly, the word “or” is intended to include "and" unless the context clearly indicates otherwise. The phrase “and/or” should be understood to mean “either or both” of the elements so conjoined, i.e. , elements that are conjunctively present in some cases and disjunctively present in other cases. Thus, as a nonlimiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[0095] As used herein, the terms “such as”, “for example” and the like are intended to refer to exemplary embodiments and not to limit the scope of the present disclosure.

[0096] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, preferred materials and methods are described herein.

[0097] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%. In some other embodiments it could be up to +/- 10% a person of skill in the art would appreciate that the exact values would not impact the invention. The present invention is further explained in detail by the following examples, but the scope of the invention should not be limited thereto.

[0098] As used herein, the phrases “one or more” or “at least one” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “one or more” or “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “one or more of A and B” (or, equivalently, “one or more of A or B,” or, equivalently “one or more of A and/or B”) or “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0099] When a range of values is listed herein, it is intended to encompass each value and sub-range within the range. For example, “1-5 ng” is intended to encompass 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5 ng. Taken further, also includes, 1.1, 1.2, 1.3, etc., or 1.01, 1.02, 1.03, 1.04, 1.05 , etc... and all values and ranges within the range l-5ng.

[00100] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

Description

T Cell Development and Double Positive (CD4⁺ and CD8⁺) T Cells

[00101] In some aspects, the focus of the present invention is to control or “tune” the generation of single positive (SP) CD4⁺ (4SP) and CD8⁺ (8SP) from DP cells. In some aspects the starting DP cells are TCR⁺CD69'CD27' double positive CD4⁺, CD8⁺ T cells. There are very many ways in which to obtain, generate DP cells known in the art, including as described in WO/2022/241558 published November 24, 2022. The methods, niches and culture media which are the subject of the present invention have been shown to work irrespective of the source of the DPs (i.e., iPSCs, cord blood, Thymocytes) or the known ProT induction media (preferably serum-free and feeder-free), such as PSC1 (see for example Figure 36). The methods of the present invention include any method that incorporates the methods, niches and culture media and kits or components thereof into the T cell development process, such as methods for generating pro-T or DP cells from pluripotent stem cells or the subsequent induction and expansion steps post-SP production using the methods, niches, culture media, kits and components of the present invention.

[00102] In some aspects of the invention Pro-T cells are collected and re-seeded into feeder-/serum- free conditions that mature the cells towards CD4⁺CD8⁺ (double positive, DP) cells. The input population does not need to be pure CD7⁺CD5⁺ Pro-T cells, and can include earlier-stage progenitors and non-target cell types. Preferred, but not strict, qualifications of the input population include >70% CD7+, >10% CD5+, >20x expansion from iPSC-HSPCs.

[00103] Standard DP maturation media include (i) Zandstra-developed media (“PSC2”), (ii) StemSpan T cell Progenitor Maturation Media (SCT Mat), or (iii) a hybrid formulation of both (SCT JAC). In all cases, cells are seeded onto wells coated with 10-15 ug/mL DLL4 (Sino Bio) + 2.5 ug/mL VCAM1 (R&D Systems). Cells are fed with a 1 :1 media top up after 3-4 days, then are fed with 50% media exchanges every 3-4 days thereafter, for a total of 2-3 weeks. DP Maturation

[00104] In some aspects the DP cells are cultured in a maturation niche, including maturation media for 2 - 3 weeks. Again, many maturation niches or culture media may be used. In addition to the DP maturation media, this may also include functionalized substrates, wherein ligands such as Notch ligands and integrin ligands, such as VCAM-1 are immobilized. The maturation media may include base formulations further supplemented to enhance maturation. In some other embodiments factors may be removed to improve DP maturation. Examples of DP Maturation media include:

(i) PSC2 (Zandstra/UBC) - Michaels et al. (2022)

• Base media (“JAC Ultra”) : IMDM with GlutaMax (Thermo) + 4% B27 without vitamin A (Gibco) + 0.5% Pen/Strep (Invitrogen) + 24 uM 2-mercaptoethanol (Sigma) + 60 uM Ascorbic Acid (Sigma)

• Cytokines: SCF (9.76 ng/mL), Flt3L (8.49 ng/mL), CXCL12 (15.22 ng/mL), TNFa (0.04 ng/mL), IIL-3 (2.55 ng/mL), and IL-7 (71.93 ng/mL). All cytokines from R&D Systems.

(ii) SCT Mat (StemCell)

• Base media: SFEM II (StemCell)

• Cytokines: A 2017 poster indicated Flt3L and IL-7 without specifying concentrations; others may have been added or removed since

• Usage: Fong et al., (2025); Heinze et al., (2022); Trotman-Grant et al., (2021)

(iii) SCT JAC (developed by Zandstra/UBC)

• Base media: JAC Ultra (from Zandstra lab)

• Cytokines: The same as SCT Mat (1 Ox cytokine supplement)

[00105] The methods of the present invention are not limited to the above and other serum- /feeder-free could be used, such as:

(iv) Jing et al (2022)

• Base media: SFEM II (StemCell) + 10% BIT (StemCell)

• Cytokines: 20 ng/mL Flt3L (R&D Systems) and 15 ng/mL IL-7 (R&D Systems)

• Coating: 10 ug/mL DLL4 (Life Tech) + 2 ug/mL VCAM1 (R&D Systems)

(v) Iriguchi et al (2021)

• Base media - one of the following, supplemented with 55 uM 2-merceptoethanol (ThermoFisher), 50 ug/mL ascorbic acid (Sigma), and 2 mM Glutamax (ThermoFisher)

■ SFEM II (StemCell)

■ aMEM (ThermoFisher) + 20% BIT (StemCell)

■ IMDM (ThermoFisher) + 20% BIT (StemCell)

• Cytokines: 50 ng/mL SCF (R&D Systems), 50 ng/mL TPO (PeproTech), 50 ng/mL IL-7 (not indicated but likely PeproTech), 50 ng/mL Flt3L (PeproTech), and 30 nM CXCL12 (referred to as alt name SDF- l a within paper, PeproTech)

• Other supplements: 15 uM SB203580 (Tocris Bio) Coating: 10 ug/mL DLL4 (Sino Bio) + 10 ug/mL Retronectin (Takara)

[00106] The present inventors have developed a niche and culture media that further includes other cytokines and TCR stimulating reagents that can optimize DP maturation, such as one or more of the following:

- Cytokines: IL-4, IL-5, IL-6, IL-9, IL-12, IL-18

TCR stimulating reagents: PMA, lonomycin, PHA, anti-CD2/3/28

Induction of DP cells to SP cells

[00107] After the 2 - 3 weeks of maturation towards DP cells, cells are collected and res-seeded into feeder-serum-free conditions that induce differentiation towards single positive (CD4+CD8- (CD4 single positive, 4SP) or CD4-CD8+ (CD8 single positive, 8SP) cells.

[00108] Prior to the present invention the niche and culture media and methods uses resulted in skewing of production of much more 8SP than 4SP, or production of 4SP cells that predominantly lacked expression of naive T cell markers.. Prior to SP induction, the harvested DP cells can be optionally subject to a supplementation step comprising culturing the cells in one or more reagents to stimulate the TCR signaling pathway at a sufficiently low level to improve cell viability and development while not trigger positive selection and differentiation towards SP cells. This is usually at relatively low TCR stimulating reagent levels and for a limited period of time, (see for example Figure 36).

[00109] Although various base media can be used, the present inventors have optimized the SP induction niche and culture media to be able to control generation and ratios of 4SP and 8SPs. Wherein for enhancing 4SP in a cell population, it is desired, to lower or inhibit or suppress activation of the Notch signaling pathway using lower affinity ligands such as JAG 1 or JAG 2 (wherein JAG1 and JAG2 are better than DLL1 which in turn is better than the strong affinity Notch ligand DLL4; or preferably having no Notch signaling pathway activators or stimulants in the SP induction niche or culture media, and/or including a Notch signaling pathway inhibitor in the niche and/or culture media, such as a y secretase inhibitor, such as DAPT. One advantage of using a Notch inhibitor is to the extent that the a niche is used wherein Notch ligands with or without VCAM or immobilized on a surface of the environment (e.g. in the well or plate or on a microbead), one could potentially use that same well or plate during SP induction by adding the Notch inhibitor to the environment (e.g. DAPT or soluble DLL4, etc.. ). Conversely, to enhance 8SP generation in the cell population one can culture the cells under Notch stimulating conditions, (such as the use of Notch ligands and no inhibitors).

[00110] In addition, the present inventors have identified that the presence of TCR stimulating reagents are useful in optimizing and skewing the generation of 4SP or 8SPs during SP induction. In the present invention, the TCR stimulating reagents may be selected from one or more of: PHA, anti anti-CD2/3/28; and PMA/ionomycin. However, for tuning the SP induction niche to enhance the production of 4SPs, it has been surprisingly found that phytohemagglutinin (PHA) and anti-CD2/3/28 soluble complexes significantly enhance the generation and production of 4SPs over prior art methods. In some other embodiments of the invention, the niche or culture media is supplemented with cytokines, such as IL-4, IL-5, IL-6, IL-9, IL012, and IL-18. So in some embodiments, the present invention provides a SP induction niche or culture medium comprising: no or low affinity Notch Ligands, such as JAG1 or JAG2 and/or a Notch inhibitor, such as DAPT, a TCR simulating agent, such as PHA or anti CD2/3/28, but in some preferred embodiments PHA. In yet other embodiments, the niche and or culture media comprise one or more cytokines: IL-4, IL-5, IL-6, IL-9, IL-12 and IL-18.

[00111] Please refer to Figure 36 and the following tables for various ranges for optimizing DP maturation, or 4SP or 8SP induction. The components may be added separately or one or more can be added in pre-mixed form to the niche or in a culture media. These components can form part of a kit comprising the components together with instructions for use in the kit or provided separately. The components can form all or part of the DP maturation and/or SP induction kit. The kit can further optionally include the culture plates or wells and any pre-loaded (including with culture media) or immobilized components on the substrate or functionalized substrate.

[00112] for examples of media and niche components, as well as the below table.

[00113] In other embodiments of the invention, in addition or in replacement of DP cells, 8SP cells can also be used with the same niche and media and methods to generate 4SPs. In either case, culturing the DP and 8SP cells under the conditions that properly balance TCR stimulation and Notch signaling enhances the production of CD4⁺ T cells.

[00114] The following include 8SP and 4SP induction media described in the prior art, although some of the base media can be used in the present invention, the methods, niche and culture media described below, unlike the present invention, are not optimized for 4SP generation or production or to get a population of cells enhanced with 4SP or the ability to obtain a desired ratio or production of 4SP and 8SP from DP cells:

(i) 8SP induction - Vizcardo et al (2013) [Serum+/Feeder+]

• Base media: aMEM (Invitrogen) +20% FCS (Nichirei Bio)

• TCR stimulation: 50 ng/mL anti-CD3 (clone OKT3, eBioscience) + 2 ug/mL anti- CD28 (clone CD28.2, eBioscience)

• Other supplements: 5 ng/mL IL-7, 5 ng/mL Flt3L, 10 ng/mL SCF, 200 U/mL IL-2 (source of each unstated)

• Coating: Unclear but likely re-seeded onto OP9-DLL1 cells

(ii) 8SP induction - Maeda et al (2016) [Serum+/Feeder+]

• Base media: aMEM (Invitrogen) +20% FCS (Nichirei Bio)

• TCR stimulation: 15 ng/mL anti-CD3 (clone OKT3, eBioscience)

• Other supplements: 100 U/mL IL-2 and 5 ng/mL IL-7 (source of each unstated)

• Coating: Unclear but likely re-seeded onto OP9-DLL1 cells

(iii) 8SP induction - Michaels et al (2022) [Scrum-/Fccdcr-] • Base media: PSC2 (includes SCF, Flt3L, CXCL12, TNFa, IL-3, and IL-7)

• TCR stimulation: 1.25% anti-CD2/3/28 (Stem Cell)

• Other supplements: 20 ng/mL IL-21 (R&D Systems)

• Coating: 15 ug/mL DLL4 + 2.5 ug/mL VCAM1

(iv) 8SP induction - StemCell (per Technical Manual) [Serum-/Feeder-]

• Base media: SCT Mat (likely includes Flt3L and IL-7)

• TCR stimulation: 1.25% anti-CD2/3/28 (StemCell)

• Other supplements: 10 ng/mL IL- 15 (StemCell)

• Coating: StemSpan T Cell Differentiation Coating Material (StemCell)

(v) 8SP induction - Iriguchi et al (2021) [Serum +/Feeder-]

• Base media: aMEM (ThermoFisher) + 15% FBS (Coming), lx ITS-G (ThermoFisher), lx PSG (Sigma), 50 ug/mL ascorbic acid (Sigma)

• TCR stimulation: 0.5 ug/mL anti-CD3 (clone OKT3, eBioscience),

• Other supplements: 10 ng/mL IL-7 (not indicated but likely PeproTech), 10 ng/mL IL-

2 (PeproTech), and 10 nM dexamethasone (Fuji Pharma)

• Coating: None

(vi) 8SP induction - Jing et al (2022) [Serum-/Feeder-]

• Base media: complete maturation media (base + supplement from above), includes Flt3L and IL-7

• TCR stimulation: anti-CD3/28 (unstated concentration but likely 1.25-2.5%, StemCell)

• Other supplements: 5 ng/mL IL- 15 (StemCell)

• Coating: 10 ug/mL DLL4 (Life Tech) + 2 ug/mL VCAM1 (R&D Systems)

(vii) 8SP induction - Heinze et al (2022) [Serum+/Feeder+]

• Base media: “OP9 media”

• TCR stimulation: 2.5% anti-CD2/3/28, or 25 ng/mL PMA + 250 ng/mL lonomycin

• Other supplements: 5 ng/mL IL-7 (R&D Systems), 5 ng/mL Flt3L (Bio X Cell), 10 uM Dexamethasone (Sigma), 5 ng/mL IL-2 (source unstated)

• Coating: OP9-DLL4:MHCII feeder cells

(viii)8SP induction - Heinze et al (2022) [Serum-ZFeeder-]

• Base media: SCT Mat

• TCR stimulation: 1.25 anti-CD2/3/28

• Other supplements: 10 ng/mL IL-15

• Coating: StemSpan T Cell Differentiation Coating Material (StemCell)

(ix) 4SP induction - Fong et al (2022, 2025) [Serum-/Feeder-]

• Base media: SCT Mat

• TCR stimulation: 0.01 - 0.0405 uM (-6 - 25 ng/mL) PMA, 0.16 - 0.67 uM (-113 - 475 ng/mL) lonomycin

■ Both applied for one day, then removed the next day

• Other supplements: None

• Coating: StemSpan T Cell Differentiation Coating Material (StemCell)

[00115] Novel Compositions and Niche Components

The following table outlines various components that are used in the culture media and niche of the present invention, they may be substituted or combined with an equivalent or similar component of the same or similar function as noted on the far right column. The table notes the concentration ranges for use and preferred concentration or ranges for each of DP maturation (DP) and SP induction for both enhancing 4SP or 8SP generation during the SP induction process. One can time ratios by tuning amounts of the components used. Any qualification of the amounts by the term “about” also includes the exact concentrations or numbers noted as an embodiment. All compositions, niches and methods of the invention are serum-free and cell-free. % indicated are v/v.

(a) Cytokines to support DP maturation and SP induction

[00116] (b) TCR and Notch pathway stimulation/inhibition factors for DP maturation and SP induction

(c) Example compositions:

The following are example compositions as supported by the Examples and are included as some aspects of the invention.

DP maturation

Note that in some aspects of the inventon the preferred target population for DP induction (or maturation) are pre-selection TCR⁺CD69'CD27~ DP cells. SP induction SP induction

RORSP T Cell Population Generated Using the Invention

[00117] The SP induction methods of the present invention generate naive mature T cells, such as 8 SPs and 4SPs and are capable of further induction or polarization into other cell types. For instance, the present invention in some aspects produce 4SPs that can be expanded, maintained, cultured. In other aspects they can be polarized to generate Thelper cells, including Thl, Th2, and Thl7. In other aspects the 4SPs can be induced using cell expansion media. In other aspects they can be used to generate Treg cells.

[00118] In some other aspects, as used herein a mature T cell is a T cell that has developed its own T cell receptor (TCR⁺), or expresses an engineered TCR on the cell surface, or is a CD8⁺ single positive or CD4⁺ single positive T cell. The cells generated by the invention can be engineered cells to express a chimeric antigen receptor (CAR) or a CAR expressing T cell engineered to lack TCR. Such cells may be useful in various therapies, including cancer therapy.

[00119] In the present invention, the presence of mature T cells was identified by markers known in the art.

[00120] In some aspects, the invention provides T cells and T cell populations enhanced for a 4SP, 8SP or a ratio of 4SP and 8SP cells and/or cells derived from same. The use of the T cells produced by the method or the isolated cells or cell population(s) can be used for immunotherapy, such as CAR-T, engineered TCR T cells, Tregs, genetic modification therapy or other uses.

[00121] In some embodiments of the invention, the cell population (the SP or their precursors or progenitors (e.g. DP, pro-T, HSPC, PSC) is or are engineered to provide the cells with additional functionality such as hypoimmunity or to add cancer targeting moieties such as engineered TCR or CAR. In other embodiments the engineered TCR or CAR are added at specific stages of the process including during the EHT step, the CD34 HSPC step, the progenitor T cell step or even directly to mature T cells. Thus they can be used for such.

Generating Treg Cells from CD4+ T Cells (4SPs) [00122] The present inventors have shown that the 4SPs generated using the method of the present invention are capable of generating Treg cells. The inventors have developed a method and cell culture media to tune or skew the generation of Treg cells from the 4SPs.

[00123] The inventors used 4SP cells at a starting concentration of 2xl0⁶ cells/mL (200,000 cells per 96 well flat-bottom plate) and cultured the cells in a serum-free and feeder cell free niche with Immunocult-XF T Cell Expansion Media (see above) supplemented with 2000 lU/mL IL-2 (Proleukin), 20ng/mL TGF-01, and 5% CD2/3/28 T Cell (STEMCELL Technologies) (so final concentration is 1000 lU/ml IL-2, lOng/ml TGF-pi and 2.5% CD2/3/28 T Cell Activators). Cells are kept at a concentration of 0.5xl0⁶ cells/mL, splitting every 2 days in Immunocult-XF T Cell Expansion Media with 5 ng/mL TGF-pi and with 1000 lU/mL IL-2, at temperature (37°C) in static culture for seven (7) days. No all trans-retinoic acid was required in the culture medium.

The end concentration of Treg cells obtained was about: 0.5x10⁶ cells/mL The cells generated had Treg cell markers TCRap CD4 CD8a CD25'^,e?

[00124] As such, in some aspects, the present disclosure provides a population of cells enriched for CD4⁺ Treg cells obtained herein. In related aspects, the present disclosure provides a method of treating a patient in need of immunosuppression, comprising administering the population of cells enriched for CD4⁺ Treg cells obtained by the present methods; the use of the population of cells enriched for CD4⁺ Treg cells obtained by the present methods in the manufacture of a medicament in treating a patient in need of immunosuppression; and a population of cells enriched for CD4⁺ Treg cells obtained by the present methods for use in treating a patient in need of immunosuppression. In some embodiments, the patient has an autoimmune disease; or has received or will receive tissue transplantation. The Treg cells generated using the invention (unedited and edited PSC-derived Tregs (such as CAR-Tregs, transgenic TCR-Tregs) has various clinical and non-clinical uses including as a cell therapy for preventing and/or suppressing inflammation in multiple disease.

Methodology and Cell Density

[00125] In some aspects one cell density can be optimized to optimize the methods, niches and culture media of the present inventions.

[00126] In some aspects, cells are cultured on flat-bottom tissue-culture treated plates. Coatings are applied by diluting proteins (e.g. DLL4, VCAM1, anti-CD3) into PBS, adding to the plate/wells, and incubating for 2 hours at 37C or overnight at 4C. Prior to seeding cells, the coating is aspirated and plate/wells are washed with PBS. 15 cm plate 152 cm²

[00127] For each stage, PSC -derived cell inputs are counted and seeded at tire following densities, yielding the indicated number of output cells. Cells are grown in a static, normoxic (20% O2) incubator at 37C and with 5% CO2

Uses

[00128] As noted in this application, there arc many uses of the methods, niches, and culture media of the present invention, including in the generation of cells for medical therapies or other purposes. Non-CAR engineered T cells/Tregs can for instance be useful in immune system modeling, TCR repertoire diversity studies, TCR screening, and GVHD modeling. In some other aspects, CAR-T and TCR-T cells can be useful in cancer therapy, anti-viral therapy, autoimmune therapy (especially viral- driven autoimmune disorders), allergen desensitization, and immune system modeling. The methods and niches and culture media and kits of the invention can be useful in the generation of CAR-Treg and TCR-Treg cells, autoimmune therapy, graft-vs-host-disease, transplant, allergen desensitization, immune system modeling.

[00129] Allogeneic T cell therapies are a highly desirable option to circumvent the cost and complexity of using autologous T cells to treat diseases. Allogeneic CD8⁺ T cells can be made from pluripotent stem cells (PSCs), but deriving CD4⁺ T cells from PSCs remained a significant challenge. Using feeder- and serum-free conditions, we found that CD4⁺ versus CD8⁺ T cell commitment from PSCs can be controlled by fine-tuning the dynamics of Notch and T cell receptor signaling delivered to CD4⁺CD8⁺ double positive T cells. Notch signaling negatively impacts CD4⁺ T cell commitment, and its timed removal allows generation of clonally-diverse and expandable CD4 T cells from PSCs. The resulting CD4⁺ T cells respond to cytokine -mediated polarization by differentiating into Thl, Th2, or Th 17 cells, recapitulating canonical helper cell function. These findings represent a significant step towards using PSC-derived CD4⁺ T cells as a low cost, off-the-shelf, cell therapy.

Kits for the Tunable Differentiation and Production of CD4+ Cells and CD8+- AND Cells Derived From Same

[00130] The present disclosure contemplates kits for carrying out the methods provided herein. Such kits typically comprise two or more components required for generation of CD4+ and/or CD8+ cells and/or cells derived from the foregoing. Components of the kit include, but are not limited to, one or more of compounds, reagents, containers, equipment and instructions for using the kit. Any precursor cell populations (such as DP cells). Accordingly, the methods described herein may be performed by utilizing pre-packaged kits provided herein.

[00131] In an embodiment, a kit for use to generate 4SPs, 8SPs or a desired ratio of same or cells derived from same, such as Thl, Th2, Th 17 or Treg cells from DP, or some other aspects 8SP cells, in vitro in a serum-free, feeder-free manner can be provided. The kit comprises one or more of TCR signaling reagents, Notch ligands, Notch ligand inhibitors, various cytokines. In some embodiments they can provide other components or base media or pre-adsorbed or immobilized components on functionalized substrates, such as Notch ligand and VCAM-1. The instructions may comprise one or more protocols for: preparing the desired niche, culture media and, optionally, components to a culture system; culture conditions, such as time, temperature, and/or gas incubation concentrations; harvesting protocols; and protocols for identifying the generated T cells and, optionally, more mature T cells or cells derived from same. [00132] The kit may further include materials useful for conducting the present method such as, for example, culture plates, welled plates, petri dishes and the like.

EXAMPLES

[00133] The following examples are provided by way of illustration not limitation.

Standard Methodology

Production of HE cells from hiPSCs

[00134] In this process, starting PSCs are either the iPSl 1 cells (ALSTEM, episomal human foreskin fibroblast-derived) or Hl cells (WiCell). PSCs were maintained in mTeSR™l or mTeSR™ Plus media (STEMCELL Technologies) supplemented with 0.5% penicillin/streptomycin (GIBCO) and in normoxic conditions (20% O2, 37°C, 5% CO2). Cells were grown on tissue culture-treated plastic plates coated for 1 hour at 37°C with Geltrex (GIBCO) or Cultrex (Bio-Techne). For passaging and seeding HE differentiations, PSCs were dissociated to small clusters by incubating in TrypLE Express (GIBCO) for 2-4 minutes at 37°C, followed by quenching with mTeSR™l or mTeSR™Plus and light pipetting. On thaw or passage, cells were treated with 5 pM ROCK inhibitor (ROCKi) Y-27632 (STEMCELL Technologies).

[00135] Differentiations of PSCs to CD34⁺ HE cells followed the inventors published AggreWell- based protocol (Michaels et al., 2022) with minor modifications. AggreWell 400 6-well plates (STEMCELL Technologies) were prepared by coating with 2 mL Anti -Adherence Rinsing Solution (STEMCELL Technologies), spinning down for 5-10 min at 1300 g (to remove bubbles), incubating for 2 hours at room temperature, then washing with 2 mL PBS. PSCs were grown to 50-75% confluency , then dissociated to single cells by incubating in TrypLE Express for 3-5 minutes at 37°C, followed by quenching with mTeSRl or mTeSR Plus and moderate pipetting. Collected cells were spun down at 200 g for 5 min, then resuspended to 2.21 million cells/mL in “TO” media. TO media comprises 0.0039% 1 -thioglycerol (MTG) (Sigma- Aldrich), 50 μg/mL ascorbic acid (AA) (Sigma- Aldrich), 150 pg/mL Transferrin (Sigma-Aldrich), 10 ng/mL BMP4 (R&D Systems), and 5 pM ROCKi Y-27632 supplemented into StemPro “Complete” base media. StemPro Complete comprises StemPro 34 (Thermo Fisher Scientific) supplemented with 1% Glutamax (Thermo Fisher) and 0.5% Pen/Strep. Aiming for 75 cells/microwell, we added 2 mL TO-cell suspension per 6-well of the AggreWell plate. To achieve uniform aggregation, we allowed cells to evenly settle at RT for 5 minutes, then spun down for 5 minutes at 200 g. Throughout the HE induction, cells were grown in hypoxic conditions (5% O2, 37°C, 5% CO2).

[00136] Differentiating cells were subsequently fed at 24, 42, 72, 96, and 144 hours with “Tl”, “T1.75”, “T3”, “T4”, and “T6” media, respectively. The Tl, T3, and T6 feeds were each 2 mL media top-ups, while T1.75 and T4 feeds involved carefully aspirating the existing media and adding 2 mL fresh media. Media aspirations and additions were done slowly and at the same edge of each well to minimize disturbances to the aggregates. Each media formulation uses StemPro compete as base and contains the same concentrations of MTG, AA, and Transferrin as in TO media. T1 media further contains 10 ng/mL BMP4 and 10* ng/mL bFGF (Thermo Fisher) [*the latter achieving a final concentration of 5 ng/mL after topping-up the existing TO media in each well]. T1.75 and T3 media (equivalent) further contain 10 ng/mL BMP4, 5 ng/mL bFGF, 6 pM SB-431542 (Sigma-Aldrich), and 4 pM CHIR-99021 (Tocris Bioscience). T4 media further contains 5 ng/mL bFGF, 15 ng/mL VEGF (R&D Systems), 10 ng/mL IL6 (R&D Systems), and 5 ng/mL IL11 (R&D Systems). T6 media contains the same factors as T4 media along with 50* ng/mL SCF (R&D Systems), 4* U/mL EPO (Thermo Fisher), and 50* ng/mL IGF-1 (R&D Systems) | *each achieving half the stated concentrations after topping-up the existing T4 media in each well].

[00137] CD34⁺ HE cells were collected 192 hours (day 8, “T8”) after initiation of differentiation. Aggregates were collected, spun down at 200 g for 5 min, then dissociated to single cells by incubating in 0.5 mL (per pooled 6-well) TrypLE supplemented with 100 U/mL DNase I (Sigma-Aldrich) for 15 minutes at 37°C, vigorously pipetting every 5 minutes. The TrypLE was quenched with a 50:50 mix of HBSS (GIBCO) and FBS (GIBCO). Cells were spun down for 5 min at 200 g, washed in 3 mL HBSS +2% FBS, then resuspended in 1 mL HBSS +2% FBS for pre -enrichment counting; a subset of cells was set aside for flow cytometry. CD34⁺ cells were isolated using the human CD34-MicroBead kit (Miltenyi Biotec), as per the manufacturer’s instructions. Post-enrichment cells were counted and frozen down in CryoStor CS10 (STEMCELL Technologies) at 1 million cells/mL; a subset of cells was set aside for flow cytometry. The set-aside pre- and post-enrichment cells were stained with antibodies against key markers: CD34, CD43, CD73, and CD184 to validate HE induction.

Production ofHSPCs from hiP SC-derived HE cells

[00138] HSPC differentiation followed the inventors’ published protocol²(Michaels et al., 2022) . For PSC-HE to HSPC induction, wells were prepared by coating TC-treated plates with 10-15 (typically 15) μg/mL hDLL4-Fc (Sino Biological) + 2.5 μg/mL mVCAMl (R&D Systems) for 2 hours at RT or 37°C, or overnight at 4°C. PSC-HE cells were thawed and seeded at 30-100 (typically 100) x 10³ cells/mL (3-10 x 10³ cells/100 pL per 96 well) in EHT media. EHT media contains the same factors as T4 media (see above) with VEGF reduced to 5 ng/mL and additionally supplemented with 10 ng/mL BMP4, 50 ng/mL SCF, 30 ng/mL TPO (R&D Systems), 25 ng/mL IGF-1, 10 ng/mL IL3 (R&D Systems), 10 ng/mL Flt3L (R&D Systems), and 10 pM ROCKi Y-27632. Cells were incubated in nonnoxic conditions (20% O2, 37°C, 5% CO2). Cells were collected via light pipetting and passaged to Pro-T induction conditions at EHT day 4-7 (annotated E4-7; typically E5).

Production ofPro-T cells from hiPSC-derived HSPCs

[00139] For PSC-HSPC to Pro-T induction, wells were prepared by coating with 10-15 (typically 15) μg/mL hDLL4-Fc + 2.5 μg/mL mVCAMl for 2 hours at RT or 37°C, or overnight at 4°C. Cells output from the HSPC induction stage were seeded at 30-100 (typically 100) x 10³ cells/mL (3-10 x 10³ cells/100 |iL per 96 well) in “PSC1” media. PSC1 comprises 12.37 ng/mL SCF, 8.61 ng/mL Flt3L, 97.4 ng/mL CXCL12 (R&D Systems), 0.07 ng/mL TNFa (R&D Systems), 0.97 ng/mL IL3, and 65.25 ng/mL IL7 (R&D Systems) supplemented into “JAC Ultra” base media. JAC Ultra comprises IMDM with GlutaMAX (GIBCO) supplemented with 4% B27 without Vitamin A (Thermo Fisher), 0.5% Pen/Strep, 24 pM BME (Sigma- Aldrich) and 60 pM AA. Cells were incubated in normoxic conditions (20% O2, 37°C, 5% CO2). Cells were fed by top-up with an equal volume of fresh media at Pro-T induction day 3 or 4 (annotated P3 or P4). Cells were sampled for flow cytometry and passaged to DP induction conditions between day P7-9 (typically P7).

Production of DP cells from hiP SC-derived Pro-T cells

[00140] For hiPSC-Pro-T to DP maturation, wells were prepared by coating with 10-15 (typically 10) μg/mL hDLL4-Fc + 2.5 μg/mL mVCAMl for 2 hours at RT or 37°C, or overnight at 4°C. Cells output from the Pro-T induction stage were seeded at 0.5-4 (typically 2) x 10⁶ cells/mL (50-400 x 10³ cells/100 pL per 96 well) in “PSC2” media. PSC2 comprises 9.76 ng/mL SCF, 4.96 ng/mL Flt3L, 15.22 ng/mL CXCL12/SDF-1, 0.04 ng/mL TNFa, 2.55 ng/mL IL3, and 71.93 ng/mL IL7 supplemented into “JAC Ultra” base media. Where indicated, cells were instead seeded into media comprising StemSpan T cell Progenitor Maturation Supplement (STEM CELL Technologies) diluted 1:10 into either JAC Ultra or SFEM II (STEMCELL Technologies). Where indicated, cells were instead seeded into other custom media variations. Cells were fed by top-up with an equal volume of fresh media at DP maturation day 3 or 4 (annotated M3 or M4). Thereafter, cells were fed by 50% media exchange with an equal volume of fresh media every 3-4 days. Cells were typically sampled for flow cytometry every ~7 days (e.g. days M7, M14, and M21). Cells were induced to SP cells starting between day M14-28 (typically M14 or M21), aiming for the population of CD3⁺TCRαβ⁺ cells to be >10% among live cells.

Flow cytometry

[00141] HBSS or PBS supplemented with 2% FBS was used as Flow buffer. Staining was done entirely in 96 well V-bottom plates. Cells were collected and washed once with PBS. Cells were stained with Fc-blocking antibodies (BD Biosciences or Thermo Fisher) to reduce non-specific binding, Fixable Viability Dye (BioLegend or Thermo Fisher) to exclude dead cells. Cells were stained for surface proteins in PBS or Flow buffer for 30 min in the dark at 4°C or room temperature. Brilliant Plus Buffer was included in staining mixes when using at least three BV or BUV antibodies. Cells were washed with PBS or Flow buffer, resuspended in Flow buffer, and acquired on FACSymphony A5 (BD Biosciences) or CytoFLEX LX N3-V5-B3-Y5-R3-I0 (Beckman Coulter).

[00142] For detection of intracellular proteins, cells were washed with PBS of Flow buffer, fixed and permeabilized with eBioscience Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher) for 45 min in the dark at room temperature or 4°C overnight, then stained for intracellular proteins for 45 min in the dark at 4°C or room temperature. Brilliant Plus Buffer was included in intracellular staining mixes when using at least three BV or BUV antibodies. Cells were washed and resuspended in Perm Buffer from the eBiosciences kit and acquired on the FACSymphony or CytoFLEX.

[00143] Data were analyzed on Flow Jo software (vlO.X; BD Biosciences) or CytExpert (v2.6; Beckman Coulter), then exported for plotting and statistical analysis with GraphPad Prism (vlO, GraphPad Software) or Python (v3.11.4).

EXAMPLE 1: Conversion of human postnatal CD3-Thymocytes to 4SPs

[00144] This example describes processes in which human CD3-Thymocytes to were differentiated into CD4⁺ T cells in tissue culture. This process is illustrated in FIGURE 2.

[00145] Human postnatal thymus tissue was collected in UW Solution (Bridge to Life) with consent from infants undergoing cardiac surgeries in BC Children’s Hospital. A piece of the thymus tissue was mechanically dissociated in Immunocult-XF T cell Expansion Medium using GentleMACS Dissociator (Miltenyi Biotec). The dissociated bulk thymocytes were filtered and subsequently frozen for further use.

[00146] On day of experiment, frozen bulk thymocytes were thawed, resuspended in EasySep Buffer (IX PBS solution supplemented with 2% Fetal Bovine Serum and 1 mM EDTA), and magnetically- depleted of CD3⁺ thymocytes (EasySep™ Human CD3 Positive Selection Kit II, STEMCELL Technologies). 100,000 CD3- thymocytes were seeded in 96 well flat-bottom plates that were left uncoated, or coated with VCAM1 (2.5 pg/mL) and different concentrations of DLL4 (0, 1.5, 15 pg/mL). The cells were stimulated with anti-CD2/3/28 (STEMCELL Technologies) at a range of concentrations (0%, 0.0625%, 0.125%, 0.25%, 0.5%, 1.0%, all v:v) in PSC2 media. After 7 days, the cells were harvested and measured for population numbers and frequencies with flow cytometry.

[00147] The data shows that conversion of CD3 human thymocytes into lineage-committed CD3⁺TCRαβ⁺CD27⁺ CD4⁺ T cells requires high TCR and low Notch stimulation levels, while conversion into CD8⁺ T cells requires intermediate TCR and low Notch stimulation levels. The data further shows that high Notch stimulation suppresses conversion of DP T cells into both 4SP and 8SP T cells.

EXAMPLE 2: Production of 4SPs from human cord blood-derived HSPCs

[00148] This example describes a process in which human cord blood (CB)-derived HSPCs were differentiated into CD4⁺ T cells in tissue culture. This process is illustrated in FIGURE 3.

[00149] CB HSPCs were differentiated into DP T cells using media formulations described in tire inventors’ published protocol (Edgar et al., 2022). Briefly, CB HSPCs were first induced towards Pro- T cells by seeding at 30,000 cclls/mL into 96 well plates coated with 15 μg/mL DLL4 + 2.5 pg/mL VCAM1. The media for Pro-T induction was “CB1” media, comprising JAC Classic (IMDM + 10% BIT 9500 [STEMCELL Technologies] supplemented with 0.05% LDL [STEMCELL Technologies], 1% penicillin/streptomycin, 24 pM 2-mercaptoethanol, and 60 M ascorbic acid), further supplemented with 23.9 ng/mL SCF, 8.7 ng/mL Flt3L, 5.3 ng/mL IL-3, 10 ng/mL IL-7, 4.9 ng/mL TNFa, and 9.7 ng/mL CXCL12. Cells were fed by top-up addition of an equivalent amount of the same media after 3 days. After one week of Pro-T induction (D7), cells were collected and induced towards DP cells in a two-stage, three-part process. In maturation stage 1, cells were seeded at 2 x 10⁶ cells/mL into 96 well plates coated with 10 μg/mL DLL4 + 2.5 μg/mL VCAM1. Cells in this stage were cultured in “CB2” media, comprising JAC Classic supplemented with 120.5 ng/mL SCF, 8.0 ng/mL Flt3L, 1.2 ng/mL IL- 3, 44.5 ng/mL IL-7, 0.4 ng/mL TNFa, and 14.8 ng/mL CXCL12. Cells were fed by top-up addition of an equivalent amount of the same media after 3 days. After 7 days (DI 4), the cells were collected and re-seeded at 3 x 10⁶ cells/mL into freshly -coated plates with the same CB2 media. Cells were fed again 3 days later. After 7 days (D21), cells were collected and re-seeded at a 1 : 1 split ratio into wells freshly coated with 3 μg/mL DLL4 + 2.5 μg/mL VCAM1. Cells in this stage were cultured in “CB3” media, comprising JAC Classic supplemented with 77.1 ng/mL SCF, 9.8 ng/mL Flt3L, 1.0 ng/mL IL-3, 33.5 ng/mL IL-7, 0.1 ng/mL TNFa, and 15.7 ng/mL CXCL12. Cells were fed by top-up addition of an equivalent amount of the same media after 3 days.

[00150] At day 28 (D28), the produced CB HSPC-derived DP cells induced towards SP T cells. Cells were collected and re-seeded at a 1 : 1 split ratio into wells coated with 2.5 μg/mL VCAM 1 and different concentrations of DLL4 (0, 1, 3, 10 pg/mL). Cells in this stage were cultured in CB3 media supplemented with anti-CD2/3/28 at a range of concentrations (0%, 0.0625%, 0.125%, 0.25%, 0.5%, 1.0%, all v:v). Cells were fed by top-up addition of an equivalent amount of the same media, without anti-CD2/3/28, after 3 days. At D35, cells were harvested and measured for population numbers and frequencies with flow cytometry.

[00151] The data shows that conversion of cord blood-derived hematopoietic stem/progenitor cells into CD4⁺ T cells requires low TCR and low-to-medium Notch stimulation levels, while conversion into CD8⁺ T cells requires high TCR and medium-to-high Notch stimulation levels. As seen in EXAMPLE 1 and FIGURE 2, higher levels of DLL4 is seen to suppresses conversion of DP T cells into both 4SP and 8SP T cells.

EXAMPLE 3: Tunable production of 4SPs vs 8SPs from hPSC-derived HSPCs

[00152] This example describes processes in which hiPSC -derived HSPCs were differentiated into CD4⁺ T cells in tissue culture. This process is illustrated in FIGURES 4-11.

[00153] hiPSCs were differentiated towards DP cells as described in STANDARD METHODOLGY herein, using PSC2 media and seeding 2 x 10⁶ cells/mL into 96 well plates coated with 10 pg/mL DLL4 + 2.5 μg/mL VCAM1.

[00154] For hiPSC-DP to SP induction, wells were prepared by coating with 0-10 pg/mL hDLL4-Fc, hDLLl-Fc (R&D Systems), hJAGl-Fc (R&D Systems), or hJAG2-Fc (R&D Systems) + 2.5 ug/mL mVCAM-1 for 2 hours at RT or 37°C, or overnight at 4°C. Where indicated, the wells were left uncoated. In FIGURES 10-11, cells were treated with 0-10 iM DAPT (4',6-Diamidine-2'-phenylindole dihydrochloride, Sigma-Aldrich), a y secretase/Notch pathway inhibitor. Cells output from the DP induction stage were seeded 1:1 in PSC2 supplemented with TCR pathway stimulants: 0-2.5% Immunocult anti-CD2/3/28 complexes (STEMCELL Technologies), 0-2.5 μg/mL phytohemagglutinin (PHA)-M (ChemScene), or 0-0.25 ng/mL PMA (phorbol 12-myristate 13-acetate, Sigma-Aldrich) + 100 ng/mL lonomycin (Sigma-Aldrich). Cells were fed by top-up with an equal volume of fresh media (without stimulating reagents, and with DAPT or DMSO as indicated) at SP induction day 3 or 4 (annotated S3 or S4). For 14-day stimulations, cells were either passaged 1:1 onto wells freshly coated with 2.5 μg/mL mVCAMl only (FIGURES 4-9), or fed by 50% media exchange (FIGURES 10-11), both with fresh media without TCR pathway stimulants. In both cases, cells were fed again with fresh media (without stimulation) at day S 10 or S 11. Cells were typically sampled for flow cytometry on days S7 and S14.

Tunability of CD4⁺ vs CD8⁺ T cell generation

[00155] The data collectively shows that Notch signaling suppresses CD4⁺ T cell differentiation from hiPSC-derived DP cells, and that removal or inhibition of Notch, along with optimization of TCR pathway stimulation reagents, enables biasing of in vitro differentiations towards CD4⁺ T cells. Tuning the relative levels of TCR and Notch pathway inputs is shown to tune the ratio of CD4⁺ vs CD8⁺ T cells that are produced (FIGURES 8 & 10). Both cell types are shown to acquire naive T cell markers including CD27, CD45RA, and CD62L (FIGURES 7 & 11), with higher rates of mature marker expression in cells cultured with low/no Notch stimulation (FIGURE 9).

Anti-CD2/3/28 and PHA induce higher yields and more mature CD4+ T cells than PMA

[00156] Comparisons of TCR pathway stimulation to induce positive selection and SP T cell differentiation by anti-CD2/3/28, PHA, and PMA show that while PMA can induce the highest frequency of CD4+ T cells, yields and expression of naive T cell markers including CD27, CD45RA, and CD62L are all lower than when cells are stimulated with anti-CD2/3/28 or PHA (FIGURES 4-9).

Different Notch ligands have different SP induction biases

[00157] In FIGURE 10, Notch stimulation by DLL4 and DLL1 are shown to induce the strongest bias towards differentiation of CD8+ T cells, while JAG1 and JAG2 are shown to have more CD4⁺ T cell bias. Inhibiting Notch pathway stimulation by all such ligands with DAPT restores CD4⁺ T cell bias. Notch ligands are shown to predominantly reduce yields of CD4⁺ T cells, and to a lesser extent increase yields of CD8⁺ T cells (see also FIGURE 6). EXAMPLE 4: Generation of 4SPs from sorted DPs and 8SPs

[00158] While DPs are the common precursor of 4SP and 8SP cells, 4SP and 8SP cells are known to traverse several stages prior to lineage commitment, thus causing some 8SPs to retain 4SP potential. To assess this, cells were sorted into different populations and induced towards SP T cells (FIGURE 12). First, cultured in PSC2 from maturation days M0-M14 were first transferred 1:1 onto plates freshly coated with 10 μg/mL DLL4 + 2.5 μg/mL VCAM1 and treated with low doses of anti-CD2/3/28 (0.1% for one week, then 0.3%) to promote survival while minimizing differentiation towards SPs (see FIGURES 27-28 & EXAMPLE 10). At day M28, cells were harvested and sorted via magnetic bead enrichment for CD3 (CD3 EasySep kit, STEMCELL Technologies), or FACS for CD3 TCRap 8SP or DP phenotypes, the former of which appeared at frequencies between 10-50%. Cells were then induced towards SP T cells by treatment with 0%, 0.5%, or 1.25% anti-CD2/3/28 for one week. Cells were fed with fresh media (without stimulation) at day S3.

[00159] After 7 days of SP induction (day S7), cells were harvested and transferred into expansion conditions (annotated day X1.0). Expansion conditions comprised one day of stimulation on plates coated with 3 μg/mL anti-CD3 (clone OKT3, Ultra-LEAF, BioLegend) + 150 pg/mL RetroNectin, in “PSC4 Stim” media (Kawai et al., 2021; Michaels et al., 2022), which is JAC Ultra media supplemented with 5 ng/mL IL-7, 5 ng/mL IL-15, 50 ng/mL IL-12, 50 ng/mL IL- 18, 20 ng/mL IL-21, 10 pM Z-VAD-FMK (R&D Systems), and 3 μg/mL anti-CD28 (clone CD28.2, Ultra-LEAF, BioLcgcnd). After one day of stimulation (day Xl.l), cells were transferred to plates coated with 150 μg/mL RetroNectin only in “PSC4” media, which is JAC Ultra supplemented with 5 ng/mL IL-7 and 5 ng/mL IL-15. Cells were fed by top-up with an equivalent volume of PSC4 media on day X1.4.

[00160] The data shows that sorted CD3⁺TCRa[3⁺ 8SP and DP cells are both capable of differentiating into both CD4⁺ and CD8⁺ T cells, with a higher frequency of 4SP generation from sorted DP cells, as expected. Among DP-sorted cells pre-treated with TCR stimulating reagents, the highest frequency and yield of 4SP cells post-expansion was observed when inducing SP differentiation with 1.25% anti-CD2/3/28. While initially surprising that the higher level of anti-CD2/3/28 input did not bias the differentiation towards 8SP cells (as in FIGURES 4-8), the discrepancy is likely explained by the low-level pre-stimulation regime, which will initialize the TCR signaling network differently than unstimulated cells, and post-SP induction expansion process, which may differentially amplify cells based on prior stimulation experience.

EXAMPLE 5: Generation of hiPSC-derived 4SPs in different maturation media

[00161] To ensure results for induction of 4SP vs 8SP cells from hiPSC-derived cells were not specific to the standard PSC2 media conditions, cells were cultured in PSC2, StemSpan T cell progenitor maturation media (SCT mat, STEMCELL Technologies), and combinations of components from both PSC2 and SCT mat for 14-21 days of DP maturation, followed by 7-14 days of SP induction. Cells were seeded into DP maturation conditions at 2-3 x 10^s cells/mL on plates coated with 10 μg/mL DLL4 + 2.5 μg/mL VCAM1, as normal. Media variants included PSC2 supplement in JAC Ultra (normal PSC2, referred to here as PSC2 JAC), PSC2 supplement in SFEM II base (PSC2 SFEM), SCT mat supplement in JAC Ultra (SCT JAC), and SCT mat supplement in SFEM II (normal SCT mat, referred to here as SCT SFEM). Results in FIGURES 13-14 show that CD8⁺ and CD4⁺ T cell induction is similar across all media variants that produced CD3⁺TCR⁺ DP cells (i.e. all but PSC2 SFEM).

EXAMPLE 6: 4SPs can be generated from alternate PSC lines

[00162] To ensure results for induction of 4SP vs 8SP cells from hiPSC -derived cells were not specific to the standard hiPSC line of choice (Alstem iPSl l cells), the human Hl ESC line was differentiated to Pro-T cells in standard conditions. For DP maturation, Hl -derived Pro-T cells were seeded into SCT mat media at 3 x 10⁶ cells/mL and cultured for 2 weeks as normal. At day M14, cells were collected and re-seeded at 2 x 10⁶ cells/mL into uncoated wells and stimulated with 0.1% anti-CD2/3/28 to induce SP differentiation. The outcome was successful generation of CD3+TCRaP+CD27+ 4SP cells at >65% frequency.

EXAMPLE 7: Characterization of hiPSC-derived CD4⁺ T cells iPSC-CD4⁺ T cells express naive T cell markers

[00163] 300,000 hiPSC-DP cells were transferred into different downstream conditions in 100 I IL StemSpan Maturation media for: DP-skewing: 15 mg/mL DLL4 and no stimulation; CD4- skewing: 0 mg/mL DLL4 and stimulation with 0.1% anti-CD2/3/28 or 0.5 pg/mL PMA-L (Sigma-Aldrich); or CD8-skewing: 15 mg/mL DLL4 and stimulation with 0.1% anti-CD2/3/28 or 0.5 μg/mL PHA-L. Expression of naive CD4⁺ T cell markers and lineage-specific transcription factors were measured after 7 days by flow cytometry (FIGURE 16).

[00164] FIGURE 16C shows that compared to DP cells, newly commited 4SPs and 8SPs upregulate CD27 and CCR7, but downregulate CD62L. 4SPs upregulate ThPOK more strongly than 8SPs, with the opposite patern for RUNX3 upregulation.

[00165] At the end of 4SP-skewing conditions (day S7), cells were rested an additional week. On days S7 and S10, cells underwent 50% media exchange with Immunocult-XF T Cell Expansion Media (STEMCELL Technologies) supplemented with 200 lU/mL IL-2 (Proleukin).

[00166] At the end of 4SP-skewing conditions (day S7/1 week post-stim) and resting (day S14, 2 weeks post-stim), cells were harvested for flow cytometry analysis for CD45RA and CD45RO expression. 4SPs significantly upregulated CD45RA and downregulated CD45RO after resting. IPSC-CD4+ T cells expand and express relevant markers upon stimulation

[00167] For this expansion experiment, during hiPSC-DP to 4SP induction, day M14 cells were collected, centrifuged, resuspended in StemSpan media supplemented with 0.5 pg/mL PHA-L for stimulation, and plated into uncoated 6 well plates (9 x 10³ cells/5mL per 6 well). Cells were fed by 50% media exchange with an equal volume of fresh StemSpan media (without stimulation) 3-4 days post-stimulation (S3-4). On days S7 and S10, cells were fed by 50% media exchange with Immunocult-XF T Cell Expansion Media (STEMCELL Technologies) supplemented with 200 lU/mL IL-2 (Proleukin). On day S14, cells were collected, stained for antibodies for 15 min at room temperature, and sorted for hiPSC-CD4⁺ T cells (TCRap⁺CD4⁺CD8a ) using MoFlo Astrios (Beckman Coulter) or FACSAria Fusion (BD Biosciences). Flow-sorted cells were subsequently used for experiments.

[00168] As a control, primary CD4⁺ T cells were isolated from human postnatal thymuses or adult leukapheresis products. Human postnatal thymus tissue was collected in UW Solution (Bridge to Life) with consent from infants undergoing cardiac surgeries in BC Children’s Hospital. A piece of the thymus tissue was mechanically dissociated in Immunocult-XF T cell Expansion Medium using GentleMACS Dissociator (Miltenyi Biotec). The dissociated bulk thymocytes were filtered and subsequently frozen for further use. On day of experiment, frozen bulk thymocytes were thawed, treated with DNAsel (1 mg/mL final concentration; STEMCELL Technologies) for 10 min at room temperature, stained with antibodies at 15 min at room temperature, and flow-sorted for thymic -CD4⁺ T cells (TCRa|3 CD25 CD4 CD8a ) using FACSAria Fusion (BD Biosciences). Flow-sorted cells were subsequently used for experiments.

[00169] Leukapheresis products were obtained from consented healthy adult donors and enriched for CD4⁺ cells prior to flow sorting. Blood samples were incubated with RosetteSep Human CD4⁺ T Cell Enrichment (STEMCELL Technologies) at room temperature for 20 minutes, diluted in 1:1 ratio with IX PBS (GIBCO), layered atop Lymphoprep (15 mL/tube, STEMCELL Technologies), and fractionated by centrifugation (582 x g, 25 min, no brake, room temperature). The buffy coat layer was collected using transfer pipettes, and red blood cells within the layer were lysed using Ammonium Chloride Solution (5 mL/donor, STEMCELL Technologies) for 5 min at room temperature. Platelets were then removed by centrifugation (129 x g, 10 min, room temperature). The purified CD4⁺ cells were minimally depleted of CD45RO cells using half of the recommended concentration of Easy Sep Human Naive CD4⁺ T Cell Isolation Kit and Magnet (STEMCELL Technologies). Negative fraction from the magnetic isolation were stained with antibodies for 15 min at room temperature, and flow- sorted for naive blood CD4⁺ T cells (CD4⁺CD25 CD127⁺CD45RA^luCD45RO CD62L¹¹¹) using FACSAria Fusion (BD Biosciences). Flow-sorted cells were frozen and thawed on the day of experiment.

[00170] For expansion, cells were incubated in normoxic conditions (20% O2, 37°C, 5% CO2), maintained at a concentration of 0.5 x 10⁶ cells/mL and cell density of 0.3 x 10⁶ cells/m², and expanded in Immunocult-XF T Cell Expansion Media (STEMCELL Technologies) supplemented with IX Pen/Strep (G1BCO), 100 IlJ/mL IL-2 (Proleukin).

[00171] On day 0, hiPSC-, thymic-, or blood-CD4⁺ T cells were stimulated with 2.5% anti-CD2/3/28 (STEMCELL Technologies). Cells were split every 2-3 days and replated at the above cell density/concentration, with cytokines replenished. hiPSC- and thymic-CD4⁺ T cells were restimulated on day 7 with 2.5% anti-CD2/3/28, and undergo an additional 7 days of expansion/polarization. On day 14 (iPSC- and thymic-CD4⁺ T cells) or day 7 (blood-CD4⁺ T cells), cells were collected, washed, and resuspended in fresh Immunocult-XF T Cell Expansion Media supplemented with only 10 lU/mL IL-2 (Proleukin), and rested for 1 -24 hours prior to running downstream assays.

[00172] The data shows that over 14 days, with restimulation at day 7, the cells proliferated ~15-fold while retaining the 4SP phenotype in >80% of cells (FIGURE 17B).

[00173] To assess for T cell activation markers: post-rested cells were washed twice with PBS, and plated at 100 x 10³ cells/200 pL in a 96 well flat-bottom, in Immunocult-XF T Cell Expansion Media supplemented with 10 lU/mL IL-2 and 2.5% anti-CD2/3/28 complexes (STEMCELL Technologies). Cells were incubated in normoxic conditions (5% O2, 37°C, 5% CO2) for 48 hours. Post-stimulation, cells were harvested, stained, and phenotyped for activation markers on FACSymphony A5 (BD Biosciences).

[00174] The data shows that iPSC-CD4⁺ T cells significantly upregulated all T cell activation markers tested: CD71, 4-1BB, 0X40, and CD40L (FIGURE 17C-D). scRNAseq confirms hiPSC-CD4+ T cells are mature and express low levels of cytotoxicity genes

[00175] To generate cells for sequencing, cells hiPSC-derived cells at day M14 of DP maturation were induced to SP cells over two weeks in conditions with approximately equal bias (0.1% [week 1], then 0.3% [week 2] anti-CD2/3/28 on 10 μg/mL DLL4 + 2.5 μg/mL VCAM1 [both weeks]). CD3+ cells were then magnetically enriched, rested for one week, and expanded by stimulation for one day on CD3 [clone OKT3] + RetroNectin in PSC4 stim and rest for 10 days in PSC4 on wells coated with RetroNectin only (see EXAMPLE 4 for details). After 11 days of expansion, the cells were collected and measured with CITE-seq, scRNAseq, and scTCRseq, with processed and analyzed data shown in FIGURES 18-21.

[00176] To prepare cells for sequencing, BioLegend TotalSeq-C CITE-seq antibody preparation and 10X Genomics CITE-seq cell staining protocols were followed. Briefly, one vial of TotalSeq-C Human Universal Antibody Cocktail vl.O (BioLegend) was equilibrated to room temperature for 5 min and then spun at 10,000g for 30 seconds. The lyophilized panel was resuspended in 27.5uL of HBSS + 4% FBS, vortexed for 10 seconds, and then incubated at room temperature for 5 min. The vial was vortexed again for 10 seconds and then spun at 10,000g for 30 seconds. The entire volume was transferred to a low protein binding PCR tube and then centrifuged at 14,000g for lOmin at 4°C.

[00177] Prior to CITE-seq antibody staining, 250,000 total cells were partitioned into a 12 x 75mm tube. Cells were spun at 500xg for 5 min at 4°C and resuspended directly in 12.5uL of HBSS + 4% FBS. 12.5uL of the antibody staining cocktail was added and the cells were incubated for 30min at 4°C. Cells were washed three times with 3.5mL HBSS + 4% FBS and resuspended in a final volume of 55uL. Cells were counted, stored on ice, and given immediately to the sequencing facility for 10X Genomics 5’ library preparation and sequencing.

[00178] The generation of single cell indexed libraries was performed by the Biomedical Research Center Next Generation Sequencing Core using the 10X Genomics Chromium Controller platform and the Chromium Single Cell 5’ Library and Gel Bead Kit vl .1 and Chromium Single Cell Library and Gel Bead Kit v2 reagents. The sequencing protocol provided by the supplier was followed without modification for CITE-seq and run with the NextSeq 2000. After run completion, the Binary base call (bcl) files were converted to fastq format using the Illumina bc!2fastq2 software, and data were received for furdrcr analysis.

[00179] Raw fastq files from all samples were aligned and quantified using CellRanger (CellRanger 6.0.1) and aligned with the human genome reference hg38. Following the suggested pipeline for quality control in Scanpy (v 1.10.2), CITE-seq data were filtered for dead cells, doublets, and red blood cells by excluding cells with greater than 5% mitochondrial genes and less than 1000, but no more than 30000 genes. Genes found in less than 3 cells were removed. Samples underwent normalization, scaling, dimensional reduction, and further downstream analysis using the standard Scanpy workflow. Leiden clustering was performed using 50 principal components and a resolution of 0.5. Cells were clustered and identified based on known marker genes.

[00180] Analysis of gene expression in the clusters confirmed the presence of both CD4⁺ and CD8⁺ T cells, along with quiescent and proliferating DPs and several populations of innate-like T cells, including ILC3-like cells and yb T cells (FIGURE 18B). Focusing on a T cell populations, cells in the CD4_mature cluster expressed maturation markers seen by flow cytometry (see EXAMPLE 7 & FIGURE 16) such as CD27, CCR7, and CD40LG, as well as others including CCR4, IL7RA, S1PR1, and KLF2. Cells in the CD4_mature cluster also expressed CD4 lineage induction-associated genes including LEF1 and TCF7 (TCF-1) and lacked expression of CD8⁺ T cell-specific genes such as RUNX3, CXCR6, GZMB, GZMK, and PRF1 (FIGURES 18C & 19).

Confirmation that CD4 and CD8 clusters express expected signature genes

[00181] Using two distinct human T cell development single-cell RNA sequencing datasets (Chopp et al: 2020 and Park et al., 2020), gene signatures of thymic CD4⁺ and CD8⁺ T cells were generated to measure the likeness of iPSC -derived populations. The top 20 marker genes for each relevant CD4⁺ or CD8⁺ T cell cluster were aggregated, then filtered to remove duplicate genes. Chopp et al. CD4 clusters: “hs-ImCD4”, “hs-MatCD4”; Park et al. CD4 clusters: “CD4+T”, “CD4+Tmem”; Chopp et al. CD8 clusters: “hs-ImCD8”, “hs-MatCD8”, “hs-CD8”; Park et al. CD8 clusters: “CD8+T”, “CD8+Tmem”. By taking the union of genes across these CD4- or CD8-related clusters, signatures were ensured to be robust to manual annotation and batch effects. Finally, genes present in both the CD4 and CD8 signatures were excluded. The final CD4 and CD8 signatures contain 49 and 63 genes, respectively (FIGURE 20A). Signature scores were generated with Scanpy’s gene set scoring function (scanpy.tl.score genes), which normalizes the average z-score of signature gene expression against a set of 50 genes in a standard reference set.

[00182] The data confirmed that cells in the CD4_mature and immature clusters scored highest on the CD4 signature while cells in the CD8 cluster scored highest on the CD8 signature (FIGURE 20B-D).

Confirmation ofTCR rearrangement at both alpha and beta chains

[00183] From the combined scRNAseq + CITEseq + TCRseq dataset, Signac (v0.17.0) was used to process scTCR-seq data. iPSC-T cell scTCRseq data were compiled with fetal and postnatal thymocyte data (Park et al., 2023) and PBMC data (Trofimov et al., 2022). TCR V(D)J rearrangement patterns show stage-specific biases in primary thymocytes and are hypothesized to be driven by genomic folding and proximity (FIGURE 21A).

[00184] The data showed that the TCR repertoire of iPSC-CD4⁺ and CD8⁺ T cells both used diverse V and J segments across the entire TCRa and p loci (FIGURE 21B-C). Notably, all iPSC-T cells showed a unique pattern of VP chain usages for several segments near the center of the VP array, in the range TRBV07-08 to TRBV25-01, but the overall density of VP chains was otherwise similarly dispersed throughout the array (FIGURE 21B). All iPSC-T cells except the CD4_mature cluster infrequently used middle jp segments compared to primary cell types. [00185] TCRa chain selection was similar between iPSC-T cells and all primary cell types except fetal DP thymocytes (FIGURE 21C). iPSC-T cell Va patterns were highly enriched for fragments near the center of the Va array and were most similar to patterns in postnatal thymocyte- and PBMC-CD4 T cells. iPSC-T cell Ja patterns were enriched for fragments near the 5’ end of the Ja array and were most similar to patterns in postnatal thymocyte-DPs and - CD4⁺ T cells.

EXAMPLE 8: Tunability of T cell differentiation: hiPSCs engineered with both Class I and Class n-targeted TCRs can be converted to either 4SP or 8SP on-demand

[00186] iPSl 1 cells were engineered via plasmid co-transfection of Cas9, sgRNA, and donor vectors to knock in ectopic TCRs (eTCRs) into the TRAC locus. Two eTCRs were chosen for comparison: a class I TCR targeting EBV LMP2 peptide, and a class II TCR targeting glutamic acid decarboxylase 65 (GAD65). Each eTCR was 2A-linked to the fluorescent protein mNeonGreen to report on expression throughout differentiation. Cells were selected as a polyclonal population for 1-2 weeks by a co-integrated pac (PuroR) resistance gene downstream of the eTCR insert in the TRAC locus. Polyclonal cells were subsequently differentiated towards HE, HSPC, and Pro-T cells in standard conditions, into DP cells in standard conditions with SCT JAC media, and finally assayed in 4SP or 8SP-skewing conditions. For 4SP-skewing, cells were stimulated in SCT JAC media supplemented with 0.3% anti-CD2/3/28 and seeded onto uncoated plates. For 8SP-skewing, cells were stimulated in SCT JAC media supplemented with 1.25% anti-CD2/3/28 and seeded onto plates coated with 10 pig/mL DLL4 + 2.5 μg/mL VCAM1. In both conditions, cells were cultured in the same wells for 14 days, feeding every 3-4 days with top-up (day S3) or 50% MX (days S7 and S10) with SCT JAC media without additional stimulation supplement.

[00187] The data shows that cells expressing both the class I and class II eTCRs can be differentiated into both CD4⁺ and CD8⁺ T cells (FIGURE 22), indicating that TCR stimulation by anti- CD2/3/28 induces SP differentiation mostly independently from the cell type origin and HLA specificity of the TCR. Notable other differences include a much higher rate of CD27⁺ cells among CD3 TCRαβ⁺ cells in the 4SP-skewing conditions, which likely results from prolonged exposure to DLL4 during this version of the 8SP-skewing conditions. DLL4 was seen to reduce expression of naive markers in FIGURE 9.

EXAMPLE 9: Differentiation of hiPSC-CD4⁺ T cells into T helper (Th) or regulatory T (Treg) cell lineages.

[00188] This example describes processes in which hiPSC-CD4⁺ T cells were differentiated into Th or Treg cell lineages in tissue culture. These processes are illustrated in FIGURES 23 & 26.

Differentiation into Thl, Th2, and Thl 7 cells [00189] Process for generating/isolating hiPSC-, thymic-, or blood-CD4⁺ T cells are described above in EXAMPLE 7.

[00190] For all polarization experiments, cells were incubated in normoxic conditions (20% O2, 37°C, 5% CO2), maintained at a concentration of 0.5 x 10⁶ cells/mL and cell density of 0.3 x 10⁶ cells/m², and expanded in Immunocult-XF T Cell Expansion Media (STEMCELL Technologies) supplemented with IX Pen/Strep (GIBCO), 100 lU/mL IL-2 (Proleukin). For polarization experiments, the cells were further supplemented with the following Thl/Th2/Thl7 polarization cocktails for the entirety of culture period. Thl cytokine cocktail: 10 ng/mL IL- 12, 1 μg/mL anti-IL-4; Th2 cytokine cocktail: 10 ng/mL IL-4, 1 pg/mL, 1 pg/mL anti-IFNy; and Th 17 cytokine cocktail: 10 ng/mL IL-ip, 10 ng/mL IL-6, 20 ng/mL IL-23, 10 ng/mL TGF-pi, 5 μg/mL anti-IFNy, and 5 μg/mL anti-IL-4

[00191] On day 0, hiPSC-, thymic-, or blood-CD4⁺ T cells were stimulated with 2.5% anti-CD2/3/28 (STEMCELL Technologies). Cells were split every 2-3 days and replated at the above cell density /concentration, with cytokines replenished.

[00192] On day 7, hiPSC- and thymic-CD4⁺ T cells were restimulated with 2.5% anti-CD2/3/28, and undergo an additional 7 days of expansion/polarization.

[00193] On day 14 (iPSC- and thymic-CD4⁺ T cells) or day 7 (blood-CD4⁺ T cells), cells were collected, washed, and resuspended in fresh Immunocult-XF T Cell Expansion Media supplemented with only 10 lU/mL IL-2 (Proleukin), and rested for 16-24 hours prior to running downstream assays. Post-resting, cells were sampled and phenotyped on FACSymphony A5 (BD Biosciences).

[00194] Polarized cells were phenotyped for Th-related transcription factors and chemokine receptors by flow cytometry and expression levels were normalized to unpolarized ThO (FIGURE 23B- C). Under Thl conditions, hiPSC-Thl significantly upregulated TBET expression, and downregulated GATA-3, similar to thymic- and blood-Thl cells. CXCR3 was upregulated compared to Th2 and Thl7 but not ThO conditions. Under Th2 conditions, hiPSC-Th2 cells upregulated GATA-3 similarly to blood-Th2 cells, but downregulated CCR4 unlike thymic - and blood-Th2 cells. Furthermore, while hiPSC-Th2s downregulated CXCR3, they did not downrcgulatc TBET. Under Thl7 conditions, hiPSC-Thl7 significantly upregulated CCR6, and displayed a trend towards increased RORyt that was not statistically significant. However, like thymic- and blood-Thl 7, hiPSC-Thl7 cells significantly upregulated CCR4 and downregulated TBET and CXCR3.

[00195] To assess for intracellular cytokine secretion, polarized post-rested cells were washed twice with PBS, and plated at 50 x 10³ cells/200 pL in a 96 well round-bottom, in Immunocult-XF T Cell Expansion Media. Unstimulated cells were supplemented with 10 μg/mL Brefeldin A. Stimulated cells were supplemented with 10 μg/mL Brefeldin A, 10 ng/mL PMA, and 500 ng/mL ionomycin. Cells were incubated in normoxic conditions (5% O2, 37°C, 5% CO2) for 4 hours. Cells were then harvested, stained, and phenotyped on FACSymphony A5 (BD Biosciences). Flow cytometry analysis confirmed the ability of hiPSC-Th cells to secrete relevant cytokines in response to stimulation (FIGURE 24A-B). hiPSC-Thl cells significantly upregulated IFNy expression compared to Th2 and Thl7 conditions. Neither hiPSC- nor thymic-Thl cells downregulated IL-4 expression, unlike blood-Thl cells. Under Th2 conditions, IL-4 expression was unchanged for all hiPSC-, thymic-, and blood-Th2. Under Thl7 conditions, hiPSC-Thl7 significantly upregulated IL-17A/F expression relative to ThO, and downregulated IFNy and IL-4 expression relative to their Thl and Th2 counterparts, with a similar overall pattern of cytokine production compared to thymic- and blood-Thl7 cells.

[00196] hiPSC-ThO, -Thl, -Th2, cells maintained 4SP phenotype at the end of polarization, while hiPSC-Th 17 started to destabilize into DN and 8SP populations (FIGURE 25).

Differentiation into Treg cells

[00197] For Treg generation, during hiPSC -DP to 4SP induction, day M14 cells were collected, and live cells are enriched by density centrifugation (Lymphoprep, STEMCELL Technologies). The resulting live DP cells are collected and resuspended in resuspended in StemSpan media supplemented with 0.5 μg/mL PHA-L or 0.1% anti-CD2/3/28 for stimulation, and plated at 200,000 cells per 96 well flat-bottom plate (2xl0⁶ cclls/mL). Cells arc fed by 50% media exchange with StemSpan media every 3-4 days for 7 days (FIGURE 26A).

[00198] After 7 days of 4SP induction, for Treg-skewing, cells are fed by 50% media exchange with Immunocult-XF T Cell Expansion Media (STEMCELL Technologies) supplemented with 2000 lU/mL IL-2 (Proleukin), 20 ng/mL TGF-bl, and 5% CD2/3/28 T Cell Activators (STEMCELL Technologies) (so final concentration is 1000 lU/ml IL-2, lOng/ml TGF-bl and 2.5% CD2/3/28 T Cell Activators). Cells are kept at a concentration of 0.5xl0⁶ cells/mL, splitting every 2 days in Immunocult-XF T Cell Expansion Media with 5 ng/mL TGF-bl and with 1000 lU/mL IL-2.

[00199] For control inflammatory Tconv-skewing condition, cells were fed by 50% media exchange with Immunocult-XF T Cell Expansion Media (STEMCELL Technologies) supplemented with 200 lU/mL IL-2 (Proleukin) and 5% CD2/3/28 T Cell Activators). Cells were kept at a concentration of 0.5x10⁶ cells/mL in Immunocult-XF T Cell Expansion Media with 100 lU/mL IL-2.

[00200] At the end of Treg induction, flow cytometry analysis showed that up to 68% of cells in the CD3⁺TCRa|3⁺CD4⁺CD8a^_ population are CD25⁺FOXP3⁺, and sub-gating on CD25⁺FOXP3⁺, showed up to 97% of cells co-expressing FOXP3 and HELIOS (FIGURE 26B). § [00201] At the end of Treg induction. hiPSC-Tregs can be further enriched by flow-sorting via expression of markers TCRαβ⁺CD4⁺CD8α‘CD25^hi or magnetic isolation via CD25 (FIGURE 26A). hiPSC-Tconvs are isolated via flow-sorting via expression of markers TCRαβ⁺CD4⁺CD8a‘CD25^ne®. Flow cytometric analysis on flow-sorted hiPSC-Tregs showed enrichment for FOXP3⁺HELIOS⁺ population, while flow-sorted hiPSC-Tconvs are FOXP3^negHELIOS⁺. Expression of FOXP3 and HELIOS in hiPSC-Tregs are comparable to human thymic Tregs (FIGURE 26C). Flow-sorted hiPSC-Tregs highly upregulated Treg- related markers such as CD39, ICOS, and intracellular CTLA-4 (iCTLA-4). (FIGURE 26D).

EXAMPLE 10: TCR stimulation ramp-up increases yield of DP TCR+ cells

TCR stimulation ramp-up increases yield of DP TCR+ cells from hiPSCs

[00202] hiPSC-derived Pro-T cells were seeded into standard DP maturation conditions and differentiated for 2 weeks in PSC2. At M14, the cells were transferred into either PSC2 or SCT mat media with varying dosages of TCR stimulating reagents PHA-M, PM A, anti-CD2/3/28, Thapsigargin (Sigma- Aldrich), lonomycin, and soluble anti-CD3 (clone OKT3, BioLegend Ultra-LEAF) (FIGURES 27 & 28). Cells were harvested and measured by flow cytometry at M28 (both) and M35 (FIGURE 27 only), with TCR stimulation reagent levels increased starting at M28 for the latter. Unlike in most other experiments, here the stimulating reagents were also added during mid-week feeds.

[00203] The data show that low doses of PHA-M, PMA, and anti-CD3 can improve production of CD3⁺TCRαβ⁺ DP cells. The doses that optimize DP T cell production yields are 4-16x lower than that needed to induce the TCR pathway stimulation marker CD69 and concomitantly induce differentiation towards SP T cells (FIGURE 28B-C).

Low-doses of PMA increases percent and yield of DP TCR⁺ cells from CB-dertved cells

[00204] CB-derived HSPCs were seeded into standard T cell induction conditions (see EXAMPLE 2) and differentiated for 5 weeks in CB1 (1 week), CB2 (2 weeks), and CB3 (2 weeks). Cells were subsequently collected and re-seeded 1 : 1 into fresh CB3 media on plates freshly coated with 3 μg/mL DLL4 + 2.5 μg/mL VCAM1. Cells were treated with combinations of [0, 0.1, 0.2, or 0.4 ng/mL PMA] x [0 or 1 ng/mL lonomycin]. At day D38, cells were fed by top-up with fresh CB3 media containing the same concentration of PMA/Iono. Ad day D42, cells were harvested and measured by flow cytometry (FIGURE 29). The data shows a similar effect as in FIGURES 27-28, with low-dose PMA-treated cells having higher rates of CD3 TCRa[l and DP cells. DP and TCR population frequencies are optimized with 0.2 ng/mL PMA + 1 ng/mL lonomycin. EXAMPLE 11 — Design of experiments-based optimization of media for generation of DP, 4SP and 8SP cells

Identification of cytokines and optimization of concentrations to improve DP, 4SP and 8SP induction from hiPSCs

[00205] hiPSC-derived Pro-T cells were seeded into standard DP maturation conditions at 2 x 10⁶ cells/mL on wells coated with 10 μg/mL DLL4 + 2.5 μg/mL VCAM1 and in PSC2 media. At day M14, cells were collected and re-seeded into a series of conditions for a response surface methodology (RSM) design-of-experiments study. The RSM was a 3-level, 8-factor fractional factorial design with a total of 51 conditions. The 8 factors were IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 12, IL- 18, and anti-CD2/3/28. Concentrations for each level are shown in FIGURE 30D. At day M 17, cells were fed by top-up addition of an equivalent volume of the same RSM conditions (including anti-CD2/3/28). On day M21 , cells were harvested for flow cytometry analysis.

[00206] The square root of the number of cells per population of interest and levels of each factor in each condition were fit with a 2^nd order polynomial model using the PolynomialFeatures class in the scikitleam Python package. The resulting coefficient values and p-values were computed for each model term (FIGURE 30B). Notable highly significant coefficients included across- the-board positive 1^st order terms for 11-7, and negative 1^st order terms for IL-4 or anti-CD2/3/28 levels and CD3⁺TCRy8⁺ cells, which is reversed when examining the 2^nd order IL4*anti- CD2/3/28 interaction. IL-12 and IL-18 likewise had negative 1^st order temis for many populations but stronger for CD8aa⁺ cells in particular. DP cells showed a high positive term for the IL-9*IL-9 2^nd order interaction, implying a non-linear dose-response, as well as positive terms for many 2^nd order IL-5 and IL-6 interactions with each other and other factors.

[00207] To optimize new media variants using the model, desirability functions were defined for DP, 4SP, and 8SP cells. In one case, the desirability score was penalized for the number of TCRyO or CD8aa⁺ cells, while in another case, the desirability score only accounted for on-target populations (FIGURE 31A, C). The on-target populations included DP: | CD3 and CD3⁺TCRap⁺ DP cells]; 4SP: [CD3⁺TCRa(3⁺ 4SP cells and CD3 TCRa|TCD27 cells]; and 8SP: [CD3⁺TCRαβ⁺ 8SP cells and CD3 TCRa[TCD27 cells]. A basin-hopping algorithm (Nelder-Mead) with 500 random starts and up to 10 basin hops was run to identify combinations and levels of factors that maximize the desirability function. Top media variants were sorted by desirability, and optimal factor levels were plotted and compared between the optimization strategies to choose combinations of factors and levels for new media variants that optimize for DP cells (“PSC2.1 ”), 4SPs (“PSC2.4”), or 8SPs (“PSC2.8”) (FIGURE 31 B, D-E). Validation of media variants and optimization of media sequencing

[00208] To validate the predicted optimal medias, cells were grown for 3 weeks in PSC2, three weeks in PSC2.1 (indicated as PSC2.1 MO), one week in PSC2 followed by 100% media exchange (MX) to PSC2.1 for two weeks (indicated as PSC2.1 M7), or two weeks in PSC2 followed by 100% MX to PSC2.1, PSC2.4, or PSC2.8 for one week (indicated with M14) (FIGURE 32).

[00209] The data show that seeding cells into PSC2.1 at day M0 or M7 reduces DP T cell production relative to PSC2, notably generating instead a high frequency and number of CD117 high (mast) cells. Seeding into PSC2.1 at day M7 also generated a surprisingly high percent of 4SP cells, though with low cellularity. Waiting to seed cells into PSC2.1, PSC2.4, or PSC2.8 until day M14 led to the expected selective induction of target DP, 4SP, and 8SP populations, respectively. Notably, all three new media variants reduce frequencies and numbers of CD8aa⁺ and CD3⁺TCRy5⁺ cells compared to PSC2, while increasing the frequency and number of CD117 high cells (FIGURE 32B).

[00210] Based on the results above, optimizing the sequence of media was predicted to improve SP induction rates and yields. hiPSC-dcrivcd Pro-T cells were seeded into standard DP maturation conditions and cultured for two weeks in PSC2. On day Ml 4, cells were collected and transferred into PSC2, PSC2.1, PSC2.4, or PSC2.8 media. Cells were fed by top-up with an equal volume of the same media variant at M17. On day M21, cells were collected from each media condition and transferred into different downstream media conditions to create all combinations of [M14: PSC2, PSC2.1, PSC2.4, PSC2.8] x [PSC2, PSC2 +0.1% anti-CD2/3/28, PSC2 +0.5% anti-CD2/3/28, PSC2.1, PSC2.4, PSC2.8, and SCT mat]. In each condition, wells were coated with 1 μg/mL DLL4 + 2.5 μg/mL VCAM1. Cells were fed by top-up with the equivalent downstream media on day M24. Cells were harvested for measurement with flow cytometry on day M28 (FIGURE 33).

[00211] The highest percent of CD3⁺TCRa0⁺ cells was achieved when cells were rested (no TCR stimulation) after initial stimulation (FIGURE 33B), connecting to FIGURES 27-28 and the stimulation -rest regime in FIGURES 4-11. The highest rate of 4SP induction generally occurred when using PSC2.1 or PSC2.4 media in week one or two, as well as when using low (0.1%) anti-CD2/3/28 input (FIGURE 33B - the latter as seen in FIGURES 4-7).

Addition of PMA + IL4 generates highly pure DP populations

[00212] hiPSC-derived Pro-T cells were seeded into DP maturation conditions in either (i) PSC2 media modified to remove SCF or (ii) C7 media, which supplements CXCL12 and IL-7 (at the same concentrations as in PSC2) into JAC Ultra base media. After one week (day M7), cells were transferred into downstream media combinations either in PSC2 -SCF or C37 media, which is like C7 media but further contains IL-3 at the concentration used in PSC2. In both such medias, cells were further supplemented with all combinations of [0 or 0.04 ng/mL PMA] x [0, 1, or 10 ng/mL SCF] x [0 or 10 ng/mL IL-4], Cells were harvested for flow cytometry at day M21.

[00213] The data show that the highest rate of DP cells is achieved in conditions with PMA. The lowest rates of CD8aa⁺ and TCRvd cells are achieved in conditions with IL-4, with a slight trade-off in DP yield. Transient SCF removal in the media from M0-M7 prevents emergence of CD117 high cells, even at high dosages of SCF and IL-4, which together led to significant production of CD117 high cells in prior formulations (e.g. FIGURE 32). A new media formulation comprising PSC2 without SCF + 0.04 ng/mL PMA + 10 ng/mL IL-4 was named “PSC2-xSM4”. xSM4 media outperforms commercial media (SCT mat + SFEM II) in terms of DP percent and yield, but performs worse on both metrics compared to the combination of SCT mat supplement and JAC Ultra base media (SCT JAC, see also FIGURE 14).

EXAMPLE 12 - Key media formulations

Media formula: HSPC induction from hPSCs

[00214] FIGURE 35 defines the sequence and composition of medias for induction of CD34+ HE cells and HSPCs from hPSCs.

Media Formulae: T cell induction from hP SC-derived HPSCs

[00215] FIGURE 36 defines all major media variants used and the typical sequences of usage in Pro- T induction, DP cell maturation, and SP cell induction and expansion.

[00216] Although, die disclosure has been described with reference to certain specific embodiments, various modifications thereof will be apparent to those skilled in the art. Any examples provided herein are included solely for the purpose of illustrating the disclosure and are not intended to limit the disclosure in any way. Any drawings provided herein are solely for the purpose of illustrating various aspects of the disclosure and are not intended to be drawn to scale or to limit the disclosure in any way. The scope of the claims appended hereto should not be limited by the preferred embodiments set forth in the above description, but should be given the broadest interpretation consistent with the present specification as a whole. The disclosures of all prior art recited herein are incorporated herein by reference in their entirety. REFERENCES

All references listed and disclosed in the specification and Examples, including patents, patent applications, international patent applications and publications are incorporated herein in their entirety by reference.

1. Rosado-Sanchez, I., and Levings, M.K. (2020). Building a CAR-Treg: Going from the basic to the luxury model. Cell. Immunol. 358, 104220. https://doi.Org/10.1016/j.cellimm.2020.104220.

2. Schett, G., Muller, F., Taubmann, J., Mackensen, A., Wang, W., Furie, R.A., Gold, R., Haghikia, A., Merkel, P.A., Caricchio, R., et al. (2024). Advancements and challenges in CAR T cell therapy in autoimmune diseases. Nat. Rev. Rheumatol. 20, 531-544. https://doi.org/10.1038/s41584-024-01139-z.

3. Aghajanian, H., Rurik, J.G., and Epstein, J.A. (2022). CAR-based therapies: opportunities for immuno-medicine beyond cancer. Nat. Metab. 4, 163-169. https://doi.org/10.1038/s42255- 022-00537-5.

4. Martin, K.E., Hammer, Q., Perica, K., Sadelain, M., and Malmberg, K.J. (2024). Engineering immune -evasive allogeneic cellular immunotherapies. Nat. Rev. Immunol. 24, 680-693. https://doi.org/10.1038/s41577-024-01022-8.

5. Depil, S., Duchateau, P., Grupp, S.A., Mufti, G., and Poirot, L. (2020). ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat. Rev. Drug Discov. 19, 185-199. https://doi.org/10.1038/s41573-019-0051-2.

6. Michaels, Y.S., Durland, L.J., and Zandstra, P.W. (2023). Engineering T Cell Development for the Next Generation of Stem Cell-Derived Immunotherapies. GEN Bioteclmol. 2, 106-119. https://doi.org/10.1089/genbio.2023.0008.

7. Timmermans, F., Velghe, I., Vanwalleghem, L., De Smedt, M., Van Coppemolle, S., Taghon, T., Moore, H.D., Lcclcrcq, G., Langcrak, A.W., Kcrrc, T., ct al. (2009). Generation of T Cells from Human Embryonic Stem Cell-Derived Hematopoietic Zones. J. Immunol. 182, 6879- 6888. https://doi.org/! 0.4049/jimmunol.0803670.

8. Themeli, M., Kloss, C.C., Ciriello, G., Fedorov, V.D., Perna, F., Gonen, M., and Sadelain, M. (2013). Generation of tumor -targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat. Biotechnol. 31, 928-933. https://doi.org/10.1038/nbt.2678.

9. Nishimura, T., Kaneko, S., Kawana-Tachikawa, A., Tajima, Y., Goto, H., Zhu, D., Nakayama- Hosoya, K., Iriguchi, S., Uemura, Y., Shimizu, T., et al. (2013). Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation. Cell Stem Cell 12, 114-126. https://doi.Org/10.1016/j.stem.2012.ll.002.

10. Vizcardo, R., Masuda, K., Yamada, D., Ikawa, T., Shimizu, K., Fujii, S.I., Koseki, H., and Kawamoto, H. (2013). Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD8+ T cells. Cell Stem Cell 12, 31-36. https : //do i .or g/ 10.1016/j . stem .2012.12.006. Iriguchi, S., Yasui, Y., Kawai, Y., Arima, S., Kunitomo, M., Sato, T., Ueda, T., Minagawa, A., Mishima, Y., Yanagawa, N., et al. (2021). A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nat. Commun. 12, 430. https://doi.org/10.1038/s41467-020-20658-3. Trotman-Grant, A.C., Mohtashami, M., De Sousa Casal, J., Martinez, E.C., Lee, D., Teichman, S., Brauer, P.M., Han, J., Anderson, M.K., and Zuniga-Pflucker, J.C. (2021). DL4-pbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system. Nat.

Commun. 12, 5023. https://doi.org/10.1038/s41467-021-25245-8. Michaels, Y.S., Edgar, J.M., Major, M.C., Castle, E.L., Zimmerman, C., Yin, T., Hagner, A., Lau, C., Ibanez-Rios, M.I., Knapp, D.J.H.F., et al. (2022). DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells. Sci. Adv. 8, eabn5522. Jing, R., Scarfo, I., Najia, M.A., Lummertz da Rocha, E., Han, A., Sanborn, M„ Bingham, T., Kubaczka, C., Jha, D.K., Falchetti, M., et al. (2022). EZH1 repression generates mature iPSC- derived CAR T cells with enhanced antitumor activity. Cell Stem Cell 29, 1181-1196. https://d0i.0rg/l 0.1016/j. stem .2022.06.014. Sommenneyer, D., Hudecek, M., Kosasih, P.L., Gogishvili, T., Maloney, D.G., Turtle, C.J., and Riddell, S.R. (2016). Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia 30, 492-500. https://doi.org/10.1038/leu.2015.247. Melenhorst, J.J., Chen, G.M., Wang, M., Porter, D.L., Chen, C., Collins, M.K.A., Gao, P., Bandy opadhy ay, S., Sun, H., Zhao, Z., et al. (2022). Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature 602, 503—509. https://doi.org/10.1038/s41586-021- 04390-6. Wang, D., Aguilar, B., Starr, R., Alizadeh, D., Brito, A., Sarkissian, A., Ostberg, J.R., Forman, S.J., and Brown, C.E. (2018). Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity. JCI insight 3. e99048. https://doi.org/10.1172/jci.insight.99048. He, Y., Hong, C., Huang, S., Kaskow, J.A., Covarrubias, G., Pires, I.S., Sacane, J.C., Hammond, P.T., and Belcher, A.M. (2023). STING Protein-Based In Situ Vaccine Synergizes CD4+ T, CD8+ T, and NK Cells for Tumor Eradication. Adv. Healthc. Mater. 12, 2300688. https://doi.org/10.1002/adhm.202300688. Zuazo, M., Arasanz, H., Bocanegra, A., Fernandez, G., Chocarro, L., Vera, R., Kochan, G., and Escors, D. (2020). Systemic CD4 Immunity as a Key Contributor to PD-L1/PD-1 Blockade Immunotherapy Efficacy. Front. Immunol. 11, 586907. https://doi.org/10.3389/fimmu.2020.586907. Zuazo, M., Arasanz, H., Femandez-Hinojal, G., Garcia-Granda, M.J., Gato, M., Bocanegra, A., Martinez, M., Hernandez, B., Teijeira, L., Morilla, L, et al. (2019). Functional systemic CD4 immunity is required for clinical responses to PD-L1/PD-1 blockade therapy. EMBO Mol. Med. 11, el0293. https://doi.org/10.15252/emmm.201910293. Van Hoecke, L., Van Lint, S., Roose, K., Van Parys, A., Vandenabeele, P., Grooten, J., Tavernier, J., De Koker, S., and Saelens, X. (2018). Treatment with mRNA coding for the necroptosis mediator MLKL induces antitumor immunity directed against neo-epitopes. Nat. Commun. 9, 3417. https://doi.org/10.1038/s41467-018-05979-8. Shinzawa, M., Moseman, E.A., Gossa, S., Mano, Y., Bhattacharya, A., Guinter, T., Alag, A., Chen, X., Cam, M., McGavern, D.B., et al. (2022). Reversal of the T cell immune system reveals the molecular basis for T cell lineage fate determination in the thymus. Nat. Immunol. 23, 731-742. https://doi.org/10.1038/s41590-022-01187-1. Singer, A., Adoro, S., and Park, J.H. (2008). Lineage fate and intense debate: Myths, models and mechanisms of CD4- versus CD8-lineage choice. Nat. Rev. Immunol. 8, 788-801. https://d0i.0rg/l 0.1038/nri2416. Iwata, M., Kuwata, T., Mukai, M., Tozawa, Y., and Yokoyama, M. (1996). Differential induction of helper and killer T cells from isolated CD4+CD8+ thymocytes in suspension culture. Eur. J. Immunol. 26, 2081—2086. https://doi.org/10.1002/eji.1830260918. Yasutomo, K., Doyle, C., Miele, L., and Germain, R.N. (2000). The duration of antigen receptor signalling determines CD4+ versus CD8+ T-cell lineage fate. Nature 404, 506-510. https://doi.org/10.1038/35006664. Steier, Z., Kim, E.J.Y., Aylard, D.A., and Robey, E.A. (2024). The CD4 Versus CD8 T Cell Fate Decision: A Multiomics-Informed Perspective. Annu. Rev. Immunol. 42, 235—258. https://d0i.0rg/l 0.1146/annurev-immunol-083122-040929. He, X., Dave, V., Zhang, Y., Hua, X., Nicolas, E., Xu, W., Roe, B., and Kappes, D. (2005). The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433, 826—833. Sun, G., Liu, X., Mercado, P., Jenkinson, S.R., Kypriotou, M., Feigenbaum, L., Galera, P., and Bosselut, R. (2005). The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection. Nat. Immunol. 6, 373—381. https://doi.org/10.1038/nill83. Montel-Hagen, A., Seet, C.S., Li, S., Chick, B., Zhu, Y., Chang, P., Tsai, S., Sun, V., Lopez, S., Chen, H.C., et al. (2019). Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells. Cell Stem Cell 24, 376-389. https://doi.0rg/lO.lOl6/j.stem.2Ol8.i2.Oll. Laky, K., Fleischacker, C., and Fowlkes, B.J. (2006). TCR and Notch signaling in CD4 and CD8 T cell development. Immunol. Rev. 209, 274-283. https://doi.0rg/lO.ll ll/j.0105- 2896.2006.00358.x. Laky, K., and Fowlkes, B.J. (2008). Notch signaling in CD4 and CD8 T cell development. Curr. Opin. Immunol. 20, 197—202. https://doi.Org/10.1016/j.coi.2008.03.004. Van De Walle, I., De Smet, G., Gartner, M., De Smedt, M., Waegemans, E., Vandekerckhove, B., Leclercq, G., Plum, J., Aster, J.C., Bernstein, I.D., et al. (2011). Jagged2 acts as a Deltalike Notch ligand during early hematopoietic cell fate decisions. Blood 117, 4449—4459. https://doi.org/! 0.1182/blood-2010-06-290049. Michaels, Y.S., Major, M.C., Bonham-Carter, B., Zhang, J., Heydari, T., Edgar, J.M., Greenstreet, L., Vilarrasa-Blasi, R., Kim, S., Castle, E.L., et al. (2023). Time- and lineage- resolved transcriptional profiling uncovers gene expression programs and clonal relationships that underlie human T lineage specification. bioRxiv, https://d0i.0rg/l 0.1101/2023.10.06.561277. Vanhecke, D„ Verhasselt, B., De Smedt, M., Leclercq, G., Plum, J., and Vandekerckhove, B. (1997). Human thymocytes become lineage committed at an early postselection CD69+ stage, before the onset of functional maturation. J. Immunol. 159, 5973-5983. Stankiewicz, L.N., Salim, K., Flaschner, E.A., Wang, Y.X., Edgar, J.M., Durland, L.J., Lin, B.Z.B., Bingham, G.C., Major, M.C., Jones, R.D., et al. (2024). Sex biased human thymic architecture guides T cell development through spatially defined niches. Dev. Cell, https://doi.Org/10.1016/j.devcel.2024.09.011. https://doi.Org/10.1016/j.devcel.2024.09.011. van der Stegen, S.J.C., Lindenbergh, P.L., Petrovic, R.M., Xie, H., Diop, M.P., Alexeeva, V., Shi, Y., Mansilla-soto, J., Hamieh, M., Eyquem, J., et al. (2022). Generation of T-cell- receptor-negative CD8aP-positive CAR T cells from T-cell-derived induced pluripotent stem cells. Nat. Biomed. Eng. 6, 1284-1297. https://doi.org/10.1038/s41551-022-00915-0. Mohtashami, M., Shah, D.K., Nakase, H., Kianizad, K., Petrie, H.T., and Zuniga-Pfliicker, J.C. (2010). Direct Comparison of Dill - and D114-Mediated Notch Activation Levels Shows Differential Lymphomycloid Lineage Commitment Outcomes. J. Immunol. 185, 867—876. https ://doi .or g/ 10.4049/j immunol.1000782. Cohen, A.D., Garfall, A.L., Stadtmauer, E.A., Melenhorst, J.J., Lacey, S.F., Lancaster, E., Vogl, D.T., Weiss, B.M., Dengel, K., Nelson, A., et al. (2019). B cell maturation antigenspecific CAR T cells are clinically active in multiple myeloma. J. Clin. Invest. 129, 2210- 2221. Baldwin, L, and Robey, E.A. (2024). Adjusting to self in the thymus: CD4 versus CD8 lineage commitment and regulatory T cell development. J. Exp. Med. 221, e20230896. https://doi.org/! 0.1084/jem.20230896. Trendel, N., Kruger, P., Gaglione, S., Nguyen, J., Pettmann, J., Sontag, E.D., and Dushek, O. (2021). Perfect adaptation of CD8+T cell responses to constant antigen input over a wide range of affinities is overcome by costimulation. Sci. Signal. 14, caay9363. https://doi.org/! 0.1126/scisignal.aay 9363. Naramura, M., Jang, I.K., Kole, H., Huang, F., Haines, D„ and Gu, H. (2002). C-Cbl and Cbl- b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nat. Immunol. 3, 1192—1199. https://doi.org/10.1038/ni855. Gimferrer, I., Hu, T., Simmons, A., Wang, C., Souabni, A., Busslinger, M., Bender, T.P., Hernandez-Hoyos, G., and Alberola-Ila, J. (2011). Regulation of GATA-3 Expression during CD4 Lineage Differentiation. J. Immunol. 186, 3892-3898. https://doi.org/! 0.4049/jimmunol.1003505. Wang, L., Wildt, K.F., Zhu, J., Zhang, X., Feigenbaum, L., Tessarollo, L., Paul, W.E., Fowlkes, B.J., and Bosselut, R. (2008). Distinct functions for the transcription factors GATA-3 and ThPOK during intrathymic differentiation of CD4+ T cells. Nat. Immunol. 9, 1122-1130. https://doi.org/10.1038/ni.1647. Xiong, Y., Castro, E., Yagi, R., Zhu, J., Lesoume, R., Love, P.E., Feigenbaum, L„ and Bosselut, R. (2013). Thpok-independent repression of Runx3 by Gata3 during CD4+ T-cell differentiation in the thymus. Eur. J. Immunol. 43, 918-928. https://d0i.0rg/l 0.1002/eji.201242944. Taniuchi, I. (2018). CD4 Helper and CD8 Cytotoxic T Cell Differentiation. Annu. Rev. Immunol. 36, 579—601. https://doi.org/10.1146/armurev-immunol-042617-053411. Chopp, L.B., Gopalan, V., Ciucci, T., Ruchinskas, A., Rae, Z., Lagarde, M., Gao, Y., Li, C., Bosticardo, M., Paia, F., et al. (2020). An Integrated Epigenomic and Transcriptomic Map of Mouse and Human αβ T Cell Development. Immunity 53, 1182—1201. https://d0i.0rg/l 0.1016/j .immuni.2020.10.024. Rui, J., Liu, H., Zhu, X., Cui, Y., and Liu, X. (2012). Epigenetic Silencing of Cd8 Genes by ThPOK-Mediated Deacetylation during CD4 T Cell Differentiation. J. Immunol. 189, 13 SO- 1390. https ://doi .org/ 10.4049/j immunol .1201077. Sato, T., Ohno, S.I., Hayashi, T., Sato, C., Kohu, K., Satakc, M., and Habu, S. (2005). Dual functions of runx proteins for reactivating CD8 and silencing CD4 at the commitment process into CD8 thymocytes. Immunity 22, 317—328. https://doi.Org/10.1016/j.immuni.2005.01.012. Park, J.E., Botting, R.A., Conde, C.D., Popescu, D.M., Lavaert, M., Kunz, D.L, Goh, L, Stephenson, E., Ragazzini, R., Tuck, E., et al. (2020). A cell atlas of human thymic development defines T cell repertoire formation. Science (80-. ). 367, caay3224. https://doi.org/! 0.1126/science.aay3224. Park, J.J., Lee, K.A.V., Lam, S.Z., Moon, K.S., Fang, Z., and Chen, S. (2023). Machine learning identifies T cell receptor repertoire signatures associated with COVID-19 severity. Commun. Biol. 6, 76. https://doi.org/10.1038/s42003-023-04447-4. Trofimov, A., Brouillard, P., Larouche, J.D., Seguin, J., Laverdure, J.P., Brasey, A., Ehx, G., Roy, D.C., Busque, L., Lachance, S., et al. (2022). Two types of human TCR differentially regulate reactivity to self and non-self antigens. iScience 25, 104968. https://doi.Org/10.1016/j.isci.2022.104968. Carico, Z.M., Roy Choudhury, K., Zhang, B., Zhuang, Y., and Krangel, M.S. (2017). Tcrd Rearrangement Redirects a Processive Tcra Recombination Program to Expand the Tcra Repertoire. Cell Rep. 19, 2157-2173. https://doi.Org/10.1016/j.celrep.2017.05.045. Dave, V.P. (2020). Control of CD4 Helper and CD8 Cytotoxic T cell Differentiation. J. Cell. Immunol. 2, 220-226. https://doi.org/10.1146/annurev-immunol-042617-053411. Maeda, T., Nagano, S., Ichise, H., Kataoka, K., Yamada, D., Ogawa, S., Koseki, H., Kitawaki, T., Kadowaki, N., Takaori-Kondo, A., et al. (2016). Regeneration of CD8aP T cells from T- cell-derived iPSC imparts potent tumor antigen-specific cytotoxicity. Cancer Res. 76, 6839- 6850. https://doi.org/10.1158/0008-5472.CAN-16-1149. Kawai, Y., Kawana-Tachikawa, A., Kitayama, S., Ueda, T., Miki, S„ Watanabe, A., and Kaneko, S. (2021). Generation of highly proliferative, rejuvenated cytotoxic T cell clones through pluripotency reprogramming for adoptive immunotherapy. Mol. Ther. 29, 3027—3041. https://d0i.0rg/l 0.1016/j.ym the.2021.05.016. Izon, D.J., Punt, J.A., Xu, L., Karnell, F.G., Allman, D., Myung, P.S., Boerth, N.J., Pui, J.C., Koretzky, G.A., and Pear, W.S. (2001). Notchl regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength. Immunity 14, 253-264. https://d0i.0rg/l 0.1016/S 1074-7613(01)00107-8. Steinbuck, M.P., Arakcheeva, K., and Winandy, S. (2018). Novel TCR-Mediated Mechanisms of Notch Activation and Signaling. J. Immunol. 200, 997—1007. https://d0i.0rg/l 0.4049/jimmunol.1700070. Laky, K., Evans, S., Perez-Diez, A., and Fowlkes, B.J. (2015). Notch Signaling Regulates Antigen Sensitivity of Naive CD4+ T Cells by Tuning Co-stimulation. Immunity 42, 80-94. https ://doi.org/10.1016/j.immuni.2014.12.027. Stcicr, Z., Aylard, D.A., McIntyre, L.L., Baldwin, I., Jeong Yoon Kim, E., Lutes, L.K., Ergen, C., Huang, T.-S., Robey, E.A., Yosef, N., et al. (2023). Single-cell multiomic analysis of thymocyte development reveals drivers of CD4/CD8 lineage commitment. Nat. Immunol. 24, 1579-1590. https://doi.org/10.1038/s41590-023-01584-0. Wan, Y.Y. (2014). GAT A3: A master of many trades in immune regulation. Trends Immunol. 35, 233-242. https://doi.Org/10.1016/j.it.2014.04.002. Aliahmad, P., and Kaye, J. (2008). Development of all CD4 T lineages requires nuclear factor TOX. J. Exp. Med. 205, 245-256. https://doi.org/10.1084/jem.20071944. Sakaguchi, S., Hainberger, D., Tizian, C., Tanaka, H., Okuda, T., Taniuchi, L, and Ellmeier, W. (2015). MAZR and Runx Factors Synergistically Repress ThPOK during CD8 + T Cell Lineage Development. J. Immunol. 195, 2879-2887. https://doi.org/ 10.4049/j immunol.1500387. Kastner, P., Chan, S., Vogel, W.K., Zhang, L.J., Topark-Ngann, A., Golonzhka, O., Jost, B., Le Gras, S., Gross, M.K., and Leid, M. (2010). Bell lb represses a mature T-cell gene expression program in immature CD4+CD8+ thymocytes. Eur. J. Immunol. 40, 2143—2154. https://doi.org/10.1002/eji.200940258. Steinke, F.C., Yu, S., Zhou, X., He, B., Yang, W., Zhou, B., Kawamoto, H., Zhu, J., Tan, K., and Xue, H.H. (2014). TCF-1 and LEF-1 act upstream of Th-POK to promote the CD4 + T cell fate and interact with Runx3 to silence Cd4 in CD8 + T cells. Nat. Immunol. 75, 646-656. https://doi.org/10.1038/ni.2897. Basson, M.A., Bommhardt, U., Cole, M.S., Tso, J.Y., and Zamoyska, R. (1998). CD3 ligation on immature thymocytes generates antagonist-like signals appropriate for CD8 lineage commitment, independently of T cell receptor specificity. J. Exp. Med. 187, 1249—1260. https://doi.Org/10.1084/jem.187.8.1249. Ohoka, Y., Kuwata, T., Tozawa, Y., Zhao, Y„ Mukai, M., Motegi, Y., Suzuki, R„ Yokoyama, M„ and Iwata, M. (1996). In vitro differentiation and commitment of CD4+CD8+ thymocytes to the CD4 lineage without TCR engagement. Int. Immunol. 8, 297-306. https://doi.Org/10.1093/intimm/8.3.297. Stankiewicz, L.N., Rossi, F.M. V., and Zandstra, P.W. (2024). Rebuilding and rebooting immunity with stem cells. Cell Stem Cell 31, 597—616. https://doi.org/ 10.1016/j . stem .2024.03.012. Michaels, Y.S., Buchanan, C.F., Gjorevski, N., and Moisan, A. (2023). Bioengineering translational models of lymphoid tissues. Nat. Rev. Bioeng. 7, 731-748. https://doi.org/10.1038/s44222-023-00101-0. Xue, D., Lu, S., Zhang, H., Zhang, L., Dai, Z., Kaufman, D.S., and Zhang, J. (2023). Induced pluripotent stem cell-derived engineered T cells, natural killer cells, macrophages, and dendritic cells in immunotherapy. Trends Biotechnol. 41, 907—922. https://doi.Org/10.1016/j.tibtcch.2023.02.003. Hosokawa, H., and Rothenberg, E. V. (2021). How transcription factors drive choice of the T cell fate. Nat. Rev. Immunol. 27, 162-176. https://doi.org/10.1038/s41577-020-00426-6. Etzensperger, R., Kadakia, T., Tai, X., Alag, A., Guinter, T.I., Egawa, T., Erman, B., and Singer, A. (2017). Identification of lineage-specifying cytokines that signal all CD8+- cytotoxic -lineage-fate “decisions” in the thymus. Nat. Immunol. 18, 1218-1227. https://doi.org/10.1038/ni.3847. Naito, T., and Taniuchi, I. (2010). The network of transcription factors that underlie the CD4 versus CD8 lineage decision. Int. Immunol. 22, 791—796. https://doi.org/! 0.1093/intimm/dxq436. Vacchio, M.S., and Bosselut, R. (2016). What Happens in the Thymus Does Not Stay in the Thymus: How T Cells Recycle the CD4+ - CD8+ Lineage Commitment Transcriptional Circuitry To Control Their Function. J. Immunol. 196, 4848—4856. https://doi.org/! 0.4049/jimmunol.1600415. Wang, L., and Bosselut, R. (2009). CD4-CD8 Lineage Differentiation: Thpok-ing into the Nucleus. J. Immunol. 183, 2903-2910. https://doi.org/10.4049/jimmunol.0901041. Tiffeau-Mayer, A. (2024). Unbiased estimation of sampling variance for Simpson’s diversity index. Phys. Rev. E 109, 064411. https://doi.org/10.1103/PhysRevE.109.064411. Shukla, S., Langley, M.A., Singh, J., Edgar, J.M., Mohtashami, M., Zuniga-Pflticker, J.C., and Zandstra, P.W. (2017). Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1. Nat. Methods 14, 531—538. https://doi.org/10.1038/nmeth.4258. Edgar, J.M., Michaels, Y.S., and Zandstra, P.W. (2022). Multi-objective optimization reveals time- and dose-dependent inflammatory cytokine-mediated regulation of human stem cell derived T-cell development, npj Regen. Med. 7, 11. https://doi.org/10.1038/s41536-022- 00210-1. Thomson, J.A. (1998). Embryonic stem cell lines derived from human blastocysts. Science (80-. ). 282, 1145-1147. https://doi.org/10.1126/science.282.5391.1145. Ditadi, A., Sturgeon, C.M., Tober, J., Awong, G., Kennedy, M., Yzaguirre, A.D., Azzola, L., Ng, E.S., Stanley, E.G., French, D.L., et al. (2015). Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages. Nat. Cell Biol. 17, 580-591. https://d0i.0rg/l 0.1038/ncb3161. Simpson, E.H. (1949). Measurement of Diversity. Nature 163, 688. Conway, A., Fong, H., and Kwong, G. (2021). Generation of engineered regulatory T cells. Ditadi, A., Sturgeon, C.M., Tober, J., Awong, G., Kennedy, M., Yzaguirre, A.D., Azzola, L., Ng, E.S., Stanley, E.G., French, D.L., et al. (2015). Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages. Nat. Cell Biol. 17, 580-591. Edgar, J.M., Michaels, Y.S., and Zandstra, P.W. (2022). Multi-objective optimization reveals time- and dose -dependent inflammatory cytokine-mediated regulation of human stem cell derived T-cell development. Npj Regen. Med. 7, 11. Etzensperger, R., Kadakia, T., Tai, X., Alag, A., Guinter, T.I., Egawa, T., Erman, B., and Singer, A. (2017). Identification of lineage-specifying cytokines that signal all CD8 + - cytotoxic-lineage-fate “decisions” in the thymus. Nat. Immunol. 18, 1218-1227. Fong, H., Conway, A., and Mendel, M.C. (2022). WO2022094154Al_Sangamo.pdf. WO 2022/094154 AL Fong, H., Mendel, M., Jascur, J., Najmi, L., Kim, K., Lew, G., Garimalla, S., Schock, S., Hu, J., Villegas, A., et al. (2023). A Serum- and Feeder-Free System to Generate CD4 and Regulatory T Cells from Human iPSCs. BioRxiv https://doi.org/10.1101/2023.07.01.547333. Iriguchi, S., Yasui, Y., Kawai, Y., Arima, S., Kunitomo, M., Sato, T., Ueda, T., Minagawa, A., Mishima, Y., Yanagawa, N., et al. (2021). A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nat. Commun. 12, 430. Izon, D.J., Punt, J.A., Xu, L., Karnell, F.G., Allman, D., Myung, P.S., Boerth, N.J., Pui, J.C., Koretzky, G.A., and Pear, W.S. (2001). Notchl regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength. Immunity 14, 253-264. Jing, R., Scarfo, I., Najia, M.A., Lummertz da Rocha, E„ Han, A., Sanborn, M., Bingham, T„ Kubaczka, C., Jha, D.K., Falchetti, M„ et al. (2022). EZH1 repression generates mature iPSC- derived CAR T cells with enhanced antitumor activity. Cell Stem Cell 29, 1181-1196.e6. Kawai, Y., Kawana-Tachikawa, A., Kitayama, S., Ueda, T., Miki, S., Watanabe, A., and Kaneko, S. (2021). Generation of highly proliferative, rejuvenated cytotoxic T cell clones through pluripotency reprogramming for adoptive immunotherapy. Mol. Ther. 29, 3027—3041. Laky, K., and Fowlkes, B.J. (2008). Notch signaling in CD4 and CD8 T cell development. Curr. Opin. Immunol. 20, 197—202. Maeda, T., Nagano, S., Ichise, H., Kataoka, K., Yamada, D., Ogawa, S., Koseki, H., Kitawaki, T., Kadowaki, N., Takaori -Kondo, A., et al. (2016). Regeneration of CD8aP T cells from T- cell-derived iPSC imparts potent tumor antigen-specific cytotoxicity. Cancer Res. 76, 6839- 6850. Melenhorst, J.J., Chen, G.M., Wang, M., Porter, D.L., Chen, C., Collins, M.K.A., Gao, P., Bandy opadhy ay, S., Sun, H., Zhao, Z., et al. (2022). Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature 602, 503—509 Michaels, Y.S., Edgar, J.M., Major, M.C., Castle, E.L., Zimmerman, C., Yin, T., Hagner, A., Lau, C., Ibanez-Rios, M.I., Knapp, D.J.H.F., et al. (2022). DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells. Sci. Adv. 8, eabn5522. Montel-Hagen, A., Sect, C.S., Li, S., Chick, B., Zhu, Y., Chang, P., Tsai, S., Sun, V., Lopez, S., Chen, H.C., et al. (2019). Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells. Cell Stem Cell 24, 376—389.

96. Nishimura, T., Kaneko, S., Kawana-Tachikawa, A., Tajima, Y., Goto, H., Zhu, D., Nakayama- Hosoya, K., Iriguchi, S., Uemura, Y., Shimizu, T., et al. (2013). Generation of rejuvenated antigen-specific T cells by reprogramming to pluripotency and redifferentiation. Cell Stem Cell 12, 114-126.

97. Rosado-Sanchez, I., and Levings, M.K. (2020). Building a CAR-Treg: Going from the basic to the luxury model. Cell. Immunol. 358.

98. Shichkin, V.P., and Antica, M. (2022). Key Factors for Thymic Function and Development. Front. Immunol. 13, 1-18.

99. Sommermeyer, D„ Hudecek, M., Kosasih, P.L., Gogishvili, T., Maloney, D.G., Turtle, C.J., and Riddell, S.R. (2016). Chimeric antigen receptor -modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia 30, 492-500.

100. van der Stegen, S.J.C., Lindenbergh, P.L., Petrovic, R.M., Xie, H., Diop, M.P., Alexeeva, V., Shi, Y., Mansilla-soto, J., Hamieh, M., Ey quern, J., et al. (2022). Generation of T-cell-receptor- negative CD8aP-positive CAR T cells from T-cell-derived induced pluripotent stem cells. Nat. Biomed. Eng. 6, 1284—1297.

101. Sturgeon, C.M., Ditadi, A., Awong, G., Kennedy, M., and Keller, G. (2014). Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat. Biotcchnol. 32, 554-561.

102. Taniuchi, I. (2018). CD4 Helper and CD8 Cytotoxic T Cell Differentiation. Annu. Rev. Immunol. 36, 579—601.

103. Wolfer, A., Bakker, T., Wilson, A., Nicolas, M., loannidis, V., Littman, D.R., Wilson, C.B., Held, W., MacDonald, H.R., and Radtke, F. (2001). Inactivation of Notchl in immature thymocytes does not perturb CD4 or CD8 T cell development. Nat. Immunol. 2, 235-241.

Aghajanian, H., Rurik, J.G., and Epstein, J.A. (2022). CAR-based therapies: opportunities for immuno-medicine beyond cancer. Nat. Metab. 4, 163-169. https://doi.org/10.1038/s42255-022- 00537-5.

Aliahmad, P., and Kaye, J. (2008). Development of all CD4 T lineages requires nuclear factor TOX. J. Exp. Med. 205, 245-256. https://doi.org/10.1084/jem.20071944.

Baldwin, I., and Robey, E.A. (2024). Adjusting to self in the thymus: CD4 versus CD8 lineage commitment and regulatory T cell development. J. Exp. Med. 221, e20230896. https://doi.org/10.1084/jem.20230896.

Basson, M.A., Bommhardt, U., Cole, M.S., Tso, J.Y., and Zamoyska, R. (1998). CD3 ligation on immature thymocytes generates antagonist-like signals appropriate for CD8 lineage commitment, independently of T cell receptor specificity. J. Exp. Med. 187, 1249—1260. https://doi.Org/10.1084/jem.187.8.1249.

Carico, Z.M., Roy Choudhury, K., Zhang, B., Zhuang, Y., and Krangel, M.S. (2017). Tcrd Rearrangement Redirects a Processive Tcra Recombination Program to Expand the Tcra Repertoire. Cell Rep. 19, 2157-2173. https://doi.Org/10.1016/j.celrep.2017.05.045.

Chopp, L.B., Gopalan, V., Ciucci, T., Ruchinskas, A., Rae, Z., Lagarde, M., Gao, Y„ Li, C., Bosticardo, M., Paia, F., et al. (2020). An Integrated Epigenomic and Transcriptomic Map of Mouse and Human a|3 T Cell Development. Immunity 53, 1182-1201. https ://doi.org/ 10.1016/j .immuni .2020.10.024.

Cohen, A.D., Garfall, A.L., Stadtmauer, E.A., Melenhorst, J.J., Lacey, S.F., Lancaster, E., Vogl, D.T., Weiss, B.M., Dengel, K., Nelson, A., et al. (2019). B cell maturation antigen-specific CAR T cells are clinically active in multiple myeloma. J. Clin. Invest. 129, 2210—2221.

Conway, A., Fong, H., and Kwong, G. (2021). Generation of engineered regulatory T cells.

Dave, V.P. (2020). Control of CD4 Helper and CD8 Cytotoxic T cell Differentiation. J. Cell. Immunol. 2, 220-226. https://doi.org/10.1146/armurev-immunol-042617-053411.

Depil, S., Duchateau, P., Grupp, S.A., Mufti, G., and Poirot, L. (2020). ‘Off-the-shelf’ allogeneic CART cells: development and challenges. Nat. Rev. Drug Discov. 19, 185-199. https://doi.org/! 0.1038/s41573 -019-0051 -2.

Ditadi, A., Sturgeon, C.M., Tober, J., Awong, G., Kennedy, M., Yzaguirrc, A.D., Azzola, L., Ng, E.S., Stanley, E.G., French, D.L., et al. (2015). Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages. Nat. Cell Biol. 17, 580-591. https ://doi.org/ 10.1038/ncb3161.

Edgar, J.M., Michaels, Y.S., and Zandstra, P.W. (2022). Multi-objective optimization reveals time- and dose-dependent inflammatory cytokine-mediated regulation of human stem cell derived T-cell development, npj Regen. Med. 7, 11. https://doi.org/10.1038/s41536-022-00210-l.

Etzensperger, R., Kadakia, T., Tai, X., Alag, A., Guinter, T.I., Egawa, T., Erman, B., and Singer, A. (2017). Identification of lineage-specifying cytokines that signal all CD8+-cytotoxic-lineage-fate “decisions” in the thymus. Nat. Immunol. 18, 1218-1227. https://doi.org/10.1038/ni.3847.

Fong, H., Conway, A., and Mendel, M.C. (2022). WO2022094154Al_Sangamo.pdf. WO 2022/094154 AL

Fong, H., Mendel, M., Jascur, J., Najmi, L., Kim, K., Lew, G., Garimalla, S., Schock, S., Hu, I, Villegas, A., et al. (2025). A Serum- and Feeder-Free System to Generate CD4 and Regulatory T Cells from Human iPSCs. Stem Cells. sxafOOl.

Gimferrer, I., Hu, T., Simmons, A., Wang, C., Souabni, A., Busslinger, M., Bender, T.P., Hernandez- Hoyos, G., and Alberola-Ila, J. (2011). Regulation of GATA-3 Expression during CD4 Lineage Differentiation. J. Immunol. 186, 3892-3898. https://doi.org/10.4049/jimmunol.1003505.

He, X., Dave, V., Zhang, Y., Hua, X., Nicolas, E., Xu, W., Roe, B., and Kappes, D. (2005). The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433, 826-833.

He, Y., Hong, C., Huang, S., Kaskow, J.A., Covarrubias, G., Pires, I.S., Sacane, J.C., Hammond, P.T., and Belcher, A.M. (2023). STING Protein-Based In Situ Vaccine Synergizes CD4+ T, CD8+ T, and NK Cells for Tumor Eradication. Adv. Healthc. Mater. 12, 2300688. https://d0i.0rg/l 0.1002/adhm.202300688.

Hosokawa, H„ and Rothenberg, E. V. (2021). How transcription factors drive choice of the T cell fate. Nat. Rev. Immunol. 21, 162-176. https://doi.org/10.1038/s41577-020-00426-6.

Iriguchi, S., Yasui, Y., Kawai, Y., Arima, S., Kunitomo, M., Sato, T., Ueda, T., Minagawa, A., Mishima, Y., Yanagawa, N., et al. (2021). A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy. Nat. Commun. 12, 430. https://doi.org/10.1038/s41467-020-20658-3.

Iwata, M., Kuwata, T., Mukai, M., Tozawa, Y., and Yokoyama, M. (1996). Differential induction of helper and killer T cells from isolated CD4+CD8+ thymocytes in suspension culture. Eur. J. Immunol. 26, 2081-2086. https://doi.org/10.1002/eji.1830260918.

Izon, D.J., Punt, J.A., Xu, L., Karnell, F.G., Allman, D., Myung, P.S., Boerth, N.J., Pui, J.C., Koretzky, G.A., and Pear, W.S. (2001). Notchl regulates maturation of CD4+ and CD8+ thymocytes by modulating TCR signal strength. Immunity 14, 253-264. https://doi.org/10.1016/S1074- 7613(01)00107-8.

Jing, R., Scarfo, I., Najia, M.A., Lummertz da Rocha, E., Han, A., Sanborn, M., Bingham, T., Kubaczka, C., Jlra, D.K., Falchetti, M., et al. (2022). EZH1 repression generates mature iPSC-derived CART cells with enhanced antitumor activity. Cell Stem Cell 29, 1181—1196. https://doi.Org/10.1016/j.stem.2022.06.014.

Kastner, P., Chan, S., Vogel, W.K., Zhang, L.J., Topark-Ngarm, A., Golonzhka, O., Jost, B., Le Gras, S., Gross, M.K., and Leid, M. (2010). Bell lb represses a mature T-cell gene expression program in immature CD4+CD8+ thymocytes. Eur. J. Immunol. 40, 2143-2154. https://doi.org/! 0.1002/eji.200940258.

Kawai, Y., Kawana-Tachikawa, A., Kitayama, S., Ueda, T., Miki, S., Watanabe, A., and Kaneko, S. (2021). Generation of highly proliferative, rejuvenated cytotoxic T cell clones through pluripotency reprogramming for adoptive immunotherapy. Mol. Ther. 29, 3027-3041. https://doi.org/ 10.1016/j .ymthe.2021.05.016. Laky, K., and Fowlkes, B.J. (2008). Notch signaling in CD4 and CD8 T cell development. Curr. Opin. Immunol. 20, 197—202. https://doi.Org/10.1016/j.coi.2008.03.004.

Laky, K., Evans, S., Perez-Diez, A., and Fowlkes, B.J. (2015). Notch Signaling Regulates Antigen Sensitivity of Naive CD4+ T Cells by Tuning Co-stimulation. Immunity 42, 80-94. https://doi.Org/10.1016/j.immuni.2014.12.027.

Laky, K., Fleischacker, C., and Fowlkes, B.J. (2006). TCR and Notch signaling in CD4 and CD8 T cell development. Immunol. Rev. 209, 274—283. https://doi.Org/10.ll ll/j.0105-2896.2006.00358.x. Maeda, T., Nagano, S., Ichise, H., Kataoka, K., Yamada, D., Ogawa, S., Koseki, H., Kitawaki, T., Kadowaki, N., Takaori-Kondo, A., et al. (2016). Regeneration of CD8a0 T cells from T-cell-derived iPSC imparts potent tumor antigen-specific cytotoxicity. Cancer Res. 76, 6839-6850. https://doi.org/10.1158/0008-5472.CAN-16-1149.

Martin, K.E., Hammer, Q., Perica, K„ Sadelain, M., and Malm berg, K.J. (2024). Engineering immune-evasive allogeneic cellular immunotherapies. Nat. Rev. Immunol. 24, 680-693. https://doi.org/10.1038/s41577-024-01022-8.Melenhorst, J.J., Chen, G.M., Wang, M„ Porter, D.L., Chen, C., Collins, M.K.A., Gao, P., Bandyopadhyay, S., Sun, H., Zhao, Z., et al. (2022). Decade-long leukaemia remissions with persistence of CD4+ CAR T cells. Nature 602, 503—509. https://doi.org/10.1038/s41586-021-04390-6.

Michaels, Y.S., Buchanan, C.F., Gjorevski, N., and Moisan, A. (2023). Bioengineering translational models of lymphoid tissues. Nat. Rev. Bioeng. 1, 731-748. https://doi.org/10.1038/s44222-023- 00101-0.

Michaels, Y.S., Durland, L.J., and Zandstra, P.W. (2023). Engineering T Cell Development for the Next Generation of Stem Cell-Derived Immunotherapies. GEN Biotechnol. 2, 106-119. https ://doi.org/ 10.1089/genbio .2023.0008.

Michaels, Y.S., Edgar, J.M., Major, M.C., Castle, E.L., Zimmerman, C., Yin, T., Hagner, A., Lau, C., Ibanez-Rios, M.I., Knapp, D.J.H.F., et al. (2022). DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells. Sci. Adv. 8, eabn5522. Mohtashami, M., Shah, D.K., Nakase, H., Kianizad, K., Petrie, H.T., and Zuniga-Pfliicker, J.C. (2010). Direct Comparison of Dill- and D114-Mediated Notch Activation Levels Shows Differential Lymphomyeloid Lineage Commitment Outcomes. J. hnmunol. 185, 867—876. https ://doi.org/ 10.4049/j immunol.1000782.

Montel-Hagen, A., Seet, C.S., Li, S., Chick, B., Zhu, Y., Chang, P., Tsai, S., Sun, V., Lopez, S., Chen, H.C., et al. (2019). Organoid-Induced Differentiation of Conventional T Cells from Human Pluripotent Stem Cells. Cell Stem Cell 24, 376-389. https://doi.Org/10.1016/j.stem.2018.12.011.

Naito, T., and Taniuchi, I. (2010). The network of transcription factors that underlie the CD4 versus CD8 lineage decision. Int. Immunol. 22, 791-796. https://doi.org/10.1093/intimm/dxq436.

Naramura, M., Jang, I.K., Kole, H., Huang, F., Haines, D., and Gu, H. (2002). C-Cbl and Cbl-b regulate T cell responsiveness by promoting ligand-induced TCR down-modulation. Nat. Immunol. 3, 1192-1199. https://doi.org/10.1038/ni855.

Nishimura, T., Kaneko, S., Kawana-Tachikawa, A., Tajima, Y., Goto, H., Zhu, D., Nakayama- Hosoya, K., Iriguchi, S., Uemura, Y., Shimizu, T., et al. (2013). Generation of rejuvenated antigenspecific T cells by reprogramming to pluripotency and redifferentiation. Cell Stem Cell 12, 114-126. https://doi.Org/10.1016/j.stem.2012.ll.002.

Ohoka, Y., Kuwata, T., Tozawa, Y., Zhao, Y., Mukai, M., Motegi, Y., Suzuki, R., Yokoyama, M., and Iwata, M. (1996). In vitro differentiation and commitment of CD4+CD8+ thymocytes to the CD4 lineage without TCR engagement. Int. Immunol. 8, 297-306. https://doi.Org/10.1093/intimm/8.3.297. Park, J.E., Botting, R.A., Conde, C.D., Popescu, D.M., Lavaert, M., Kunz, D.J., Goh, I., Stephenson, E., Ragazzini, R., Tuck, E., et al. (2020). A cell atlas of human thymic development defines T cell repertoire formation. Science (80-. ). 367, caay3224. https://doi.org/10.1126/science.aay3224.

Park, J.J., Lee, K.A.V., Lam, S.Z., Moon, K.S., Fang, Z., and Chen, S. (2023). Machine learning identifies T cell receptor repertoire signatures associated with COVID-19 severity. Commun. Biol. 6, 76. https://doi.org/10.1038/s42003-023-04447-4.

Rosado-Sanchez, L, and Levings, M.K. (2020). Building a CAR-Treg: Going from the basic to the luxury model. Cell. Immunol. 358, 104220. https://doi.Org/10.1016/j.cellimm.2020.104220.

Rui, J., Liu, H., Zhu, X., Cui, Y., and Liu, X. (2012). Epigenetic Silencing of Cd8 Genes by ThPOK- Mediated Deacetylation during CD4 T Cell Differentiation. J. Immunol. 189, 1380—1390. https ://doi.org/ 10.4049/j immunol.1201077.

Sakaguchi, S., Hainberger, D., Tizian, C., Tanaka, H., Okuda, T., Taniuchi, L, and Ellmeier, W. (2015). MAZR and Runx Factors Synergistically Repress ThPOK during CD8 + T Cell Lineage Development. J. Immunol. 195, 2879—2887. https://doi.org/10.4049/jimmunol.1500387.

Sato, T., Ohno, S.I., Hayashi, T., Sato, C., Kohu, K., Satake, M., and Habu, S. (2005). Dual functions of runx proteins for reactivating CD8 and silencing CD4 at tire commitment process into CD8 thymocytes. Immunity 22, 317—328. https://doi.Org/10.1016/j.immuni.2005.01.012.

Schett, G., Muller, F., Taubmann, J., Mackensen, A., Wang, W., Furie, R.A., Gold, R., Haghikia, A., Merkel, P.A., Caricchio, R., et al. (2024). Advancements and challenges in CAR T cell therapy in autoimmune diseases. Nat. Rev. Rheumatol. 20, 531-544. https://doi.org/10.1038/s41584-024-01139- z.

Shichkin, V.P., and Antica, M. (2022). Key Factors for Thymic Function and Development. Front. Immunol. 13, 1—18.

Shinzawa, M., Moseman, E.A., Gossa, S., Mano, Y., Bhattacharya, A., Guinter, T., Alag, A., Chen, X., Cam, M., McGavem, D.B., et al. (2022). Reversal of the T cell immune system reveals the molecular basis for T cell lineage fate determination in the thymus. Nat. Immunol. 23, 731-742. https ://doi.org/ 10.1038/s41590-022-01187-1.

Shukla, S., Langley, M.A., Singh, J., Edgar, J.M., Mohtashami, M., Zuniga-Pflucker, J.C., and Zandstra, P.W. (2017). Progenitor T-cell differentiation from hematopoietic stem cells using Delta- like-4 and VCAM-1. Nat. Methods 14, 531-538. https://doi.org/10.1038/nmeth.4258.

Singer, A., Adoro, S., and Park, J.H. (2008). Lineage fate and intense debate: Myths, models and mechanisms of CD4- versus CD8-lineage choice. Nat. Rev. Immunol. 8, 788-801. https ://doi.org/ 10.1038/nri2416.

Sommermeyer, D., Hudecek, M., Kosasih, P.L., Gogishvili, T., Maloney, D.G., Turtle, C.J., and Riddell, S.R. (2016). Chimeric antigen receptor-modified T cells derived from defined CD8+ and CD4+ subsets confer superior antitumor reactivity in vivo. Leukemia 30, 492-500. https://doi.org/10.1038/leu.2015.247.

Stankiewicz, L.N., Rossi, F.M. V., and Zandstra, P.W. (2024). Rebuilding and rebooting immunity with stem cells. Cell Stem Cell 31, 597-616. https://doi.Org/10.1016/j.stem.2024.03.012.

Stankiewicz, L.N., Salim, K., Flaschner, E.A., Wang, Y.X., Edgar, J.M., Durland, L.J., Lin, B.Z.B., Bingham, G.C., Major, M.C., Jones, R.D., et al. (2024). Sex biased human thymic architecture guides T cell development through spatially defined niches. Dev. Cell, https://doi.Org/10.1016/j.devcel.2024.09.011. https://doi.Org/10.1016/j.devcel.2024.09.011.Steier, Z., Aylard, D.A., McIntyre, L.L., Baldwin, L, Jeong Yoon Kim, E., Lutes, L.K., Ergen, C., Huang, T.-S., Robey, E.A., Yosef, N., et al. (2023). Single-cell multiomic analysis of thymocyte development reveals drivers of CD4/CD8 lineage commitment. Nat. Immunol. 24, 1579—1590. https://doi.org/10.1038/s41590-023-01584-0.

Steier, Z., Kim, E.J.Y., Aylard, D.A., and Robey, E.A. (2024). The CD4 Versus CD8 T Cell Fate Decision: A Multiomics-Informed Perspective. Annu. Rev. Immunol. 42, 235—258. https://doi.org/10.1146/annurev-immunol-083122-040929.Steinbuck, M.P., Arakcheeva, K., and Winandy, S. (2018). Novel TCR-Mediated Mechanisms of Notch Activation and Signaling. J. Immunol. 200, 997-1007. https://doi.org/10.4049/jimmunol.1700070.

Steinke, F.C., Yu, S., Zhou, X., He, B., Yang, W., Zhou, B., Kawamoto, H., Zhu, J., Tan, K., and Xue, H.H. (2014). TCF-1 and LEF-1 act upstream of Th-POK to promote the CD4 + T cell fate and interact with Runx3 to silence Cd4 in CD8 + T cells. Nat. Immunol. 15, 646—656. https://doi.org/10.1038/ni.2897.

Sturgeon, C.M., Ditadi, A., Awong, G., Kennedy, M., and Keller, G. (2014). Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells. Nat. Biotechnol. 32, 554-56 l.Sugimura, Ryohichi, Deepak Kumar Jha, Areum Han, Clara Soria-Valles, Edroaldo Lummertz Da Rocha, Yi Fen Lu, et al. (2017). Haematopoietic Stem and Progenitor Cells from Human Pluripotent Stem Cells, Nature, 545, 432-38

Sun, G., Liu, X., Mercado, P., Jenkinson, S.R., Kypriotou, M., Feigenbaum, L., Galera, P., and Bosselut, R. (2005). The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection. Nat. Immunol. 6, 373-381. https://doi.org/10.1038/nil 183. Taniuchi, I. (2018). CD4 Helper and CD8 Cytotoxic T Cell Differentiation. Annu. Rev. Immunol. 36, 579—601. https://doi.org/ 10.1146/annurev -immuno 1-042617-053411. Themeli, M., Kloss, C.C., Ciriello, G., Fedorov, V.D., Perna, F., Gonen, M., and Sadelain, M. (2013). Generation of tumor-targeted human T lymphocytes from induced pluripotent stem cells for cancer therapy. Nat. Biotechnol. 31, 928-933. https://doi.org/10.1038/nbt.2678.

Thomson, J. A. (1998). Embryonic stem cell lines derived from human blastocysts. Science (80-. ). 282, 1145-1147. https://doi.org/10.1126/science.282.5391.1145.Timmermans, F., Velghe, I., Vanwalleghem, L., De Smedt, M., Van Coppernolle, S., Taghon, T., Moore, H.D., Leclercq, G., Langerak, A.W., Kerre, T., et al. (2009). Generation of T Cells from Human Embryonic Stem Cell- Derived Hematopoietic Zones. J. Immunol. 182, 6879-6888. https ://doi.org/ 10.4049/j immunol.0803670.

Trendel, N., Kruger, P., Gaglione, S., Nguyen, J., Pettmann, J., Sontag, E.D., and Dushek, O. (2021). Perfect adaptation of CD8+T cell responses to constant antigen input over a wide range of affinities is overcome by costimulation. Sci. Signal. 14, eaay9363. https://doi.org/10.1126/scisignal.aay9363.

Trofimov, A., Brouillard, P., Larouche, J.D., Seguin, J., Laverdure, J.P., Brasey, A., Ehx, G., Roy, D.C., Busque, L„ Lachance, S., et al. (2022). Two types of human TCR differentially regulate reactivity to self and non-self antigens. iScience 25, 104968. https://doi.Org/10.1016/j.isci.2022.104968.

Trotman-Grant, A.C., Mohtashami, M., De Sousa Casal, J., Martinez, E.C., Lee, D., Teichman, S., Brauer, P.M., Han, J., Anderson, M.K., and Zuniga-Pfliicker, J.C. (2021). DL4-pbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system. Nat. Commun. 12, 5023. https://doi.org/10.1038/s41467-021-25245-8.

Vacchio, M.S., and Bosselut, R. (2016). What Happens in the Thymus Does Not Stay in the Thymus: How T Cells Recycle the CD4+ — CD8+ Lineage Commitment Transcriptional Circuitry To Control Their Function. J. Immunol. 196, 4848-4856. https://doi.org/10.4049/jimmunol.1600415.

Van De Walle, I., De Smet, G., Gartner, M., De Smedt, M., Waegemans, E., Vandekerckhove, B., Leclercq, G., Plum, J., Aster, J.C., Bernstein, I.D., et al. (2011). Jaggcd2 acts as a Delta-like Notch ligand during early hematopoietic cell fate decisions. Blood 117, 4449—4459. https://doi.org/10.1182/blood-2010-06-290049.van der Stegen, S.J.C., Lindenbergh, P.L., Petrovic, R.M., Xie, H., Diop, M.P., Alexeeva, V., Shi, Y., Mansilla-soto, J., Hamieh, M., Eyquem, J., et al. (2022). Generation of T-cell-receptor-negative CD8aP-positive CAR T cells from T-cell-derived induced pluripotent stem cells. Nat. Biomed. Eng. 6, 1284-1297. https://doi.org/10.1038/s41551-022- 00915-0.

Van Hoecke, L., Van Lint, S., Roose, K., Van Parys, A., Vandenabeele, P., Grooten, J., Tavernier, J., De Koker, S., and Saelens, X. (2018). Treatment with mRNA coding for the necroptosis mediator MLKL induces antitumor immunity directed against neo-epitopes. Nat. Commun. 9, 3417. https://doi.org/10.1038/s41467-018-05979-8.

Vanhecke, D., Verhasselt, B., De Smedt, M., Leclercq, G., Plum, J., and Vandekerckhove, B. (1997). Human thymocytes become lineage committed at an early postselection CD69+ stage, before the onset of functional maturation. J. Immunol. 159, 5973—5983.

Vizcardo, R., Masuda, K., Yamada, D., Ikawa, T., Shimizu, K., Fujii, S.I., Koseki, H., and Kawamoto, H. (2013). Regeneration of human tumor antigen-specific T cells from iPSCs derived from mature CD8+ T cells. Cell Stem Cell 12, 31-36. https://doi.Org/10.1016/j.stem.2012.12.006. Wan, Y.Y. (2014). GAT A3: A master of many trades in immune regulation. Trends Immunol. 35, 233-242. https://doi.Org/10.1016/j.it.2014.04.002.

Wang, D., Aguilar, B., Starr, R., Alizadeh, D., Brito, A., Sarkissian, A., Ostberg, J.R., Forman, S.J., and Brown, C.E. (2018). Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity. JC1 insight 3, e99048. https://doi.org/10.1172/jci.insight.99048.

Wang, L., and Bosselut, R. (2009). CD4-CD8 Lineage Differentiation: Thpok-ing into the Nucleus. J. Immunol. 183, 2903-2910. https://doi.org/10.4049/jimmunol.0901041.

Wang, L., Wildt, K.F., Zhu, I, Zhang, X., Feigenbaum, L., Tessarollo, L„ Paul, W.E., Fowlkes, B.J., and Bosselut, R. (2008). Distinct functions for the transcription factors GATA-3 and ThPOK during intrathymic differentiation of CD4+ T cells. Nat. Immunol. 9, 1122-1130. https://d0i.0rg/l 0.1038/ni.l647.

Wolfer, A., Bakker, T., Wilson, A., Nicolas, M., loannidis, V., Littman, D.R., Wilson, C.B., Held, W., MacDonald, H.R., and Radtke, F. (2001). Inactivation of Notchl in immature thymocytes does not perturb CD4 or CD8 T cell development. Nat. Immunol. 2, 235-241.

Xiong, Y., Castro, E., Yagi, R., Zhu, J., Lesoume, R., Love, P.E., Feigenbaum, L., and Bosselut, R. (2013). Thpok-independent repression of Runx3 by Gata3 during CD4+ T-cell differentiation in the thymus. Eur. J. Immunol. 43. 918-928. https://doi.org/10.1002/eji.201242944.

Xue, D., Lu, S., Zhang, H., Zhang, L., Dai, Z., Kaufman, D.S., and Zhang, J. (2023). Induced pluripotent stem cell-derived engineered T cells, natural killer cells, macrophages, and dendritic cells in immunotherapy. Trends Biotechnol. 41, 907-922. https://doi.Org/10.1016/j.tibtech.2023.02.003. Yasutomo, K., Doyle, C., Miele, L., and Germain, R.N. (2000). The duration of antigen receptor signalling determines CD4+ versus CD8+ T-cell lineage fate. Nature 404, 506-510. https://doi.org/10.1038/35006664.

Zuazo, M., Arasanz, H., Bocanegra, A., Fernandez, G., Chocarro, L., Vera, R., Kochan, G., and Escors, D. (2020). Systemic CD4 Immunity as a Key Contributor to PD-L1/PD-1 Blockade Immunotherapy Efficacy. Front. Immunol. 11, 586907. https://doi.org/10.3389/fimmu.2020.586907. Zuazo, M., Arasanz, H., Femandez-Hinojal, G., Garcia-Granda, M.J., Gato, M., Bocanegra, A., Martinez, M., Hernandez, B., Teijeira, L., Morilla, L, et al. (2019). Functional systemic CD4 immunity is required for clinical responses to PD-L1/PD-1 blockade therapy. EMBO Mol. Med. 11, el0293. https://doi.org/10.15252/emmm.201910293.

Claims

1. A method of obtaining a population of cells enriched for CD4 single positive (CD4+) naive T cells or CD 8 single positive (CD8+) naive T cells in vitro, comprising: c. providing CD4+ CD8+ T cells or a cell population comprising CD4⁺CD8⁺ double positive T cells optionally wherein the immature T cells are engineered to express and to generate cells that express one or more CAR, TCR, or other therapeutic modality OR engineered to express one or more transcription factor/cofactor, signaling protein, receptor, or secreted factor; d. culturing the cells in a serum-free, feeder cell -free T cell maturation niche and/or in a serum-free, feeder cell-free T cell maturation medium comprising: i. to enrich for CD4+ T cells no Notch ligand or Notch stimulating reagent or conditions or lower Notch stimulation than for generating CD8+ cells and optionally a Notch signaling inhibitor or suppressor. ii. to enrich for CD8+ T cells a reagent or conditions that promote the Notch stimulation; wherein the adjusting the level of Notch signaling stimulation in the culture medium biases the enhancement of CD4+ T cells (low or no Notch signaling) or CD8+ T cells (conditions of higher or stronger Notch signaling) in the resulting cell population.

2. The method of claims 1, wherein the T cell maturation niche or cell culture medium comprises: a. For enhancing the generation of CD4+ T cells, 0 - 10 ug/mL of coated/immobilized Notch ligand JAG1 or JAG2, optionally co-coated with an integrin ligand, such as VCAM1 (about 2.5 ug/mL) or about 0.01 ug/mL of a stronger Notch ligand such as coated/immobilized DLL4 or DLL1, optionally co-coated with an integrin ligand such as VCAM1 (about 2.5 ug/mL)l and optionally a Notch ligand inhibitor or suppressor, such as a y-secretase inhibitor , such as DAPT (about 0.3 - about 0 uM) thereby obtaining a population of cells enriched with CD4+ T cells; and b. For enhancing the generation of CD8+ T cells, greater than or equal to 10 ug/mL of coated/immobilized Notch ligand JAG1 or JAG2, optionally co-coated with an integrin ligand, such as VCAM1 (about 2.5 ug/mL) or about 1 - about 20 ug/mL of a stronger Notch ligand such as coated/immobilized DLL4 or DLL1, optionally co-coated with an integrin ligand such as VCAM1 (about 2.5 ug/mL), thereby obtaining a population of cells enriched with CD8+ T cells.

3. The method of claim 1 or 2 wherein the T cell maturation niche or cell culture medium, comprise components that stimulate TCR signaling.

4. The method of claim 3, wherein the components that stimulate TCR signaling are selected from: phorbol 12-myristate 13-acetate (PMA) and ionomycin; coated/immobilized anti-CD3; PHA; or anti-CD2/3/28; or any method of stimulating TCR signaling with anti-CD3 and different types of co-stimulation mediated through antibodies or recombination proteins.

5. The method of claim 4, wherein the niche or cell culture medium comprises the following amounts of components that stimulate TCR signaling:

. The method according to any one of claims 1 - 5, wherein the ratio of CD4 vs CD8 T cell development is tuned by modulating the level of Notch signaling via the concentration of Notch ligands in the niche, and/or via Notch inhibitors (e.g. gamma secretase inhibitor [GSI] DAPT) a. Low to high Notch ligand concentration: CD4 to CD8 T cell bias b. Low to high Notch ligand potency: CD4 to CD8 T cell bias c. Low to high Notch inhibition: CD8 to CD4 T cell bias

7. The method according to claim 1 - 6, wherein the ratio of CD4 vs CD8 T cell development is tuned by modulating the level and duration of TCR stimulation specific to each TCR stimulation modality c. Low to high PM A: CD8 to CD4 T cell bias d. Low to high PHA: CD4 T cell bias at intermediate level, otherwise CD8 bias e. Low to high anti-CD2/3/28 (soluble complexes and similar): CD4 to CD8 T cell bias f. Low to high anti-CD3 (plate immobilized and similar): CD4 to CD8 T cell bias

8. The method of any one of claims 1 — 8, wherein the niche or culture medium further comprises cytokines that support DP maturation and SP induction, selected from one or more of the following:

9. The method of any one of claims 1-8, wherein the cells are cultured in the niche or medium for about 5 - 7 days.

10. The method of any one of claims 1-9, further comprising culturing the CD4 single positive T cells in a second medium comprising TGF-0 but no all trans-retinoic acid (ATRA), thereby obtaining a population of cells enriched for CD4⁺ regulatory T (Treg) cells.

11. The method of claim 10, wherein the second medium further comprises IL-2, an anti-CD2 antibody , an anti-CD3 antibody, and an anti-CD28 antibody or stimulated with anti-CD3 and different types of co-stimulation mediated through antibodies or recombination proteins

12. The method of any one of claim 10 or 1, wherein the CD4 single positive T cells arc cultured in the second medium for about 5-10 days or about 7 days.

13. The method of any one of claims 1-12, further comprising isolating the CD4 single positive Tconv or Treg cells from the tissue culture by fluorescence-activated cell sorting (FACS) or magnetic- activated cell sorting (MACS).

14. The method of any one of claims 1-12, further comprising isolating the CD4 single positive Tconv or CD4⁺CD25^+/hlgh Treg cells from the tissue culture by fluorescence-activated cell sorting (FACS).

15. The method of any one of claims 1-14, further comprising isolating the CD4⁺CD25^+/lugh Treg and CD4⁺CD25^low Tconv cells from the tissue culture by FACS.

16. The method of any one of claims 1-14, wherein the starting population of CD4⁺CD8⁺ T cells are derived from human induced pluripotent stem cells (hiPSCs).

17. A population of cells enriched for CD4 single positive cells obtained by the method of any one of claims 1-15.

18. A population of cells enriched for CD4⁺ single positive T cells obtained by the method of any one of claims 1-15.

19. A method of treating cancer, an infectious disease, an allergy, asthma, or an autoimmune or inflammatory disease in a patient in need thereof, comprising administering the population of cells of claim 16 to the patient.

20. Use of the population of cells of claim 18 for the manufacture of a medicament for treating cancer, an infectious disease, an allergy, asthma, or an autoimmune or inflammatory disease in a patient in need thereof.

21. A population of cells of claim 16 for use in treating cancer, an infectious disease, an allergy, asthma, or an autoimmune or inflammatory disease in a patient in need thereof.

22. A population of cells enriched for CD4⁺ Treg cells obtained by the method of any one of claims 11 - 15 or 18 .

23. A method of treating a patient in need of immunosuppression, comprising administering the population of cells of claim 16 or 17 to the patient.

24. Use of the population of cells of claim 2 Ifor the manufacture of a medicament for treating a patient in need of immunosuppression.

25. A population of cells of claim 14-16 for use in treating a patient in need of immunosuppression.

26. The method, use, or population of cells for use of any one of claims 36-38, wherein the patient has an autoimmune disease, or has received or will receive tissue transplantation.

27. A pharmaceutical composition comprising the population of cells of claim 30, 31, or 35 and a pharmaceutically acceptable carrier.

28. A method that enhances the production of single positive CD4+ cells comprising culturing a population of double positive CD4+, CD8+ T cells or single positive CD8+ cells in a serum-free, stomal and feeder-free T-cell maturation niche that does not activate Notch, or has low Notch activation as compared to conditions that promote generation of CD8+ single positive T cells.

29. The method of claim 28, wherein the niche does not comprise Notch activation reagents.

30. The method of claim 28, wherein the niche comprises low affinity Notch activation reagents.

31. The method of claim 30, wherein the Notch activation reagents are JAG1, JAG2, or DLL1 Notch ligands.

32. The method of claim 31, wherein the Notch activation reagents are JAG 1 or JAG2.

33. The method of claim 30, wherein if the niche comprises a Notch ligand or a stonger affinity Notch ligand such as DLL4, it further comprises a Notch suppressor or inhibitor.

34. The method of claim 33, wherein the Notch inhibitor is a y-secretase inhibitor that indirectly blocks the Notch signaling.

35. The method of claim 34, wherein the Notch inhibitor is (N-[N-(3, 5-difluorophenacetyl)-l-alanyl]- s-phenylglycinet-butyl ester) (“DAPT”).

36. The method of any one of claims 1 - 35, wherein the T-cell maturation niche further comprises a TCR stimulating reagent.

37. The method of claim 36, wherein the TCR stimulating reagent is selected from the group consisting of phytohaemagglutinin P (PHA), anti-CD2/3/28, and phorbol myristate acetate (PMA)/ionomycin.

38. The serum-free, feeder-free method of claim 37, wherein the TCR stimulation reagent is PHA or CD2/3/28.

39. The method of any one of claims4 1 -38 wherein the cells are double positive CD4+, CD8+ T cells.

40. The method of any one of claims 28 - 39, wherein die double positive T cells arc TCR+/CD69- /CD27- double positive CD4+, CD8+ T cells.

41. The method of any one of claims 28 - 40, wherein the double positive T cells are derived from iPSCs, CD3- thymocytes, or cord blood-derived HSPCs.

42. The method of claim 41, wherein the double positive T cells are derived from human cells, or hiPSCs, hiPSCs-derived HSPCs or human embryonic stem cells.

43. The method of any one of claims 28 - 42 wherein the niche further comprises cytokines to support cell double positive cell maturation and single positive T cell induction.

44. The method of claim 45, wherein the cytokines are selected from one or more of the following: IL- 4, IL-5, IL-6, IL-7, IL-9, IL-12, and IL-18.

45. The method of any one of claims 28 -44, wherein the single positive CD4+ T cell is a naive single positive CD4+ T cell.

46. The method of any one of claims 28 - 45, wherein the single positive CD4+ T cell is a mature single positive CD4+ T cell.

47. The method of any one of claims281 - 46, wherein the culturing step increase the yield of single positive CD4+ T cells as compared to culturing the cells in a niche that more strongly promotes Notch signaling.

48. The method of claim 47, wherein the conditions that more strongly promote Notch signaling comprises a niche with a strong Notch ligand and no Notch inhibitor.

49. The method of claim 48, wherein the strong affinity Notch ligand is DLL4.

50. A method of generating Treg cells from the CD4+ T cells generated from any one of claims 28 — 49, comprising culturing the cells in a niche comprising T cell expansion media supplemented with IL-2 and TFG-bl and CD2/3/28 T cell activators.

51. The method of claim 50, wherein the T cell expansion medium is T Cell Expansion Media (STEMCELL 1 and CD2/3/28 T cell activators.

52. The method of claim 50, wherein the T cell expansion medium is T Cell Expansion Media (STEMCELL Technologies)

53. The method of claim 50 or 51 wherein IL-2 is supplemented with about 2000 lU/mL IL-2 (Proleukin), about 20 ng/mL TGF-pi, and about 5% CD2/3/28 T Cell Activators (STEMCELL Technologies) to a final concentration of about 1000 lU/ml IL-2, about lOng/ml TGF-pi and about 2.5% CD2/3/28 T Cell Activators).

54. The method of any one of claims 50 - 52 wherein the cells are cultured for 7 days at 37 C.

55. The method of any one of claims 50 — 53, wherein the generated Treg cells are TCRαβ⁺CD4⁺CD8a^_ CD25+^lugh .

56. The method of any one of claims 1 - 54, wherein the cells have been engineered to express one or more CAR, TCR, or other therapeutic modality.

57. The method according to any one of claims 1 55 wherein the cells have been engineered to express one or more transcription factor/cofactor, signaling protein, receptor, or secreted factor.

58. The method of any one of claims 28 - 56, wherein enhancing the Notch signaling pathway activation comprises culturing the cells on a surface functionalised with ligands that enhance activation of the Notch signaling pathway.

59. The method of claims57 wherein the ligands are adsorbed or immobilized on the surface.

60. The method of any one of claims 57 or 58 wherein the surface functionalised with ligands is selected from: a two- dimensional tissue culture surface; a tissue culture plate; the surface of beads; the surface of hydrogels; manufactured or human made surface; and other suitable surfaces.

61. The method of any one of claims 57 -59 wherein the surface is functionalised with ligands comprising a Notch ligand and an integrin ligand.

62. A differentiation niche for enhancing the product of 4SP cells , wherein the DP are generated from T cell progenitors including CD4-/CD8- (Double negative or DN), CD4+/CD8-/CD3- (4ISP), or CD4+/CD8+/CD3- (DPearly)

63. The differentiation niche according to claim 61, wherein the immature T cells include CD4+/CD8+/CD3+ (DPmature), CD4-/CD8+/CD3+ (8SP), or CD4+/CD8-/CD3+/CD27- (4SPearly)

64. The differentiation niche according to claim 61, wherein a gradual increase in TCR stimulation level over time increases the yield of immature T cells

65. The differentiation niche according to claim 61, wherein the differentiation niche differentiates immature T cells to CD4+/CD8-/CD3+/CD27+ (4SPmature) T cells

66. The differentiation niche according to claim 64, wherein the immature T cells are isolated or derived from pluripotent stem cells (hPSCs) or a primary sample (e.g. thymus, umbilical cord blood [UCB])

67. The differentiation niche according to claim 64, wherein the immature T cells have been engineered to express one or more CAR, TCR, or other therapeutic modality

68. The differentiation niche according to claim 64, wherein the immature T cells have been engineered to express one or more transcription factor/cofactor, signaling protein, receptor, or secreted factor

69. The differentiation niche according to claim 64, wherein the ratio of CD4 vs CD8 T cell development is tuned by modulating the level of Notch signaling via the concentration of Notch ligands in the niche, and/or via Notch inhibitors (e.g. gamma secretase inhibitor [GSI] DAPT) a. Low to high Notch ligand concentration: CD4 to CD8 T cell bias b. Low to high Notch ligand potency: CD4 to CD8 T cell bias c. Low to high Notch inhibition: CD8 to CD4 T cell bias

70. The differentiation niche according to claim 64, wherein the ratio of CD4 vs CD8 T cell development is tuned by modulating the level and duration of TCR stimulation specific to each TCR stimulation modality. a. Low to high PMA: CD8 to CD4 T cell bias b. Low to high PHA: CD4 T cell bias at intermediate level, otherwise CD8 bias c. Low to high anti-CD2/3/28 (soluble complexes and similar): CD4 to CD8 T cell bias d. Low to high anti-CD3 (plate immobilized and similar): CD4 to CD8 T cell bias.