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2071 iPSC-Derived CD4 T Cell Generation and Investigation of CD4/CD8 T Cell Lineage Choice

Program: Oral and Poster Abstracts
Session: 703. Cellular Immunotherapies: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Research, Translational Research
Saturday, December 9, 2023, 5:30 PM-7:30 PM

Yoshiki Furukawa, MD1*, Midori Ishii2*, Ayaka Goto2*, Shintaro Kinoshita2*, Jun Ando2,3*, Hiromitsu Nakauchi4,5* and Miki Ando, MD, phD2

1Department of Hematology, Juntendo University School of Medicine, Hongo, Bunkyo-Ku, Japan
2Department of Hematology, Juntendo University School of Medicine, Tokyo, Japan
3Division of Cell Therapy & Blood Transfusion Medicine, Juntendo University School of Medicine, Tokyo, Japan
4Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford
5Stem Cell Therapy Laboratory, Advanced Research Institute, Tokyo Medical and Dental University, Tokyo, Japan

iPSC-derived functionally rejuvenated antigen-specific cytotoxic T lymphocytes generated from exhausted CTLs show promising antitumor effect towards refractory tumors. Currently, we are preparing for clinical trials using iPSC-derived CTLs for EBV-associated lymphomas. These iPSC-derived T cells (iPSC-Ts) all generated into CD8 T cells, and stable generation of CD4 single positive (SP) T cells from iPSCs has never been accomplished without enforced CD4 gene transduction even though many research groups have challenged to do so. Even when an iPSC is established from a CD4 T cell clone, differentiating this iPSC does not generate into CD4 T cells, but in fact it generates into CD8 T cells. Therefore, we aimed to investigate why iPSC-Ts only differentiate into CD8 T cells, and not into CD4 T cells. We focused on adult T cell leukemia (ATL), because HTLV-1 infected CD4 T cells clonally expand. We reprogrammed CD4 T cells obtained from acute-type ATL patients into iPSCs (ATL-iPSCs), then differentiated them into T cells using our established method.

Interestingly, ATL-iPSCs from different donors all successfully differentiated into CD4 SP T cells (ATL-iPSC-Ts). Upon characterization of ATL-iPSC-Ts we confirmed that they showed FOXP3 positive naive regulatory T cell (Treg) phenotype. To investigate the function of these cells, we performed 51Cr-release assay. The cytotoxicity of HTLV-1 Tax-specific CTLs (Tax-CTLs) with or without ATL-iPSC-Ts against primary ATL cells was measured. The percentages of ATL cells lysed by Tax-CTLs was 34 ± 3.2%, whereas by the addition of ATL-iPSC-Ts, the cytotoxicity decreased to 23.4 ± 1.0% revealing the functionally suppressive effect of ATL-iPSC-Ts.

In order to further understand the mechanism of CD4/CD8 T cell lineage choice, and discover the key regulators needed for generation of CD4 SP T cells from iPSCs, we performed single cell RNA-sequencing (scRNA-seq) on ATL-iPSC-Ts. As a control we used healthy donor (HD) CD4 SP T cell clone-derived iPSCs (HD-iPSCs), which differentiated into CD8 T cells (HD-iPSC-Ts [CD8+]). We identified several genes expressed at a significantly higher level on HD-iPSC-Ts (CD8+) than on ATL-iPSC-Ts (CD4+). We presumed these genes to be essential for CD8 T cell differentiation. Therefore, we used CRISPR/Cas9 technology to knock out the respective gene in HD-iPSCs and confirmed if these iPSCs could differentiate into CD4 SP T cells, instead of CD8 T cells. This resulted in successful generation of CD4 SP T cells. We confirmed that these CD4 SP T cells were FOXP3 negative and were not Tregs. On the other hand, overexpression of this gene on ATL-iPSCs differentiated into CD8 SP T cells, instead of CD4 T cells.

In conclusion, our work represents the first successful generation of natural CD4 SP T cells from iPSCs. The gene we identified appears to be a pivotal regulator of CD4/CD8 T cell lineage choice in our iPSC-T differentiation system. While more investigation is required, our method shows promise towards facilitating stable generation of CD4 T cells for T cell therapy.

Disclosures: Ando: AbbVie Inc.: Honoraria, Research Funding. Nakauchi: Century Therapeutics: Consultancy. Ando: Chugai Pharmaceutical: Honoraria, Research Funding; Daiichi Sankyo: Research Funding; Century Therapeutics: Research Funding; Novartis Pharma: Honoraria; Sumitomo Pharma: Research Funding; Kyowa Kirin: Research Funding; AstraZeneca: Honoraria; AbbVie: Honoraria, Research Funding; Astellas Pharma: Honoraria.

*signifies non-member of ASH