The viral antigen (Ag)-specific CD8+ cytotoxic T lymphocytes (CTLs) derived from pluripotent stem cells (PSCs), i.e., PSC-CTLs, have the ability to suppress the human immunodeficiency virus (HIV) infection. After adoptive transfer, PSC-CTLs can infiltrate into the local tissues to suppress HIV replication. Nevertheless, the mechanisms by which the viral Ag-specific PSC-CTLs elicit the antiviral response remain to be fully elucidated. In this study, we generated the functional HIV-1 Gag epitope SL9-specific CTLs from the induced PSC (iPSCs), i.e., iPSC-CTLs, and investigated the suppression of SL9-specific iPSC-CTLs on viral replication and the protection of CD4+ T cells. A chimeric HIV-1, i.e., EcoHIV, was used to produce HIV replication in mice. We show that adoptive transfer of SL9-specific iPSC-CTLs greatly suppressed EcoHIV replication in the peritoneal macrophages and spleen in the animal model. Furthermore, we demonstrate that the adoptive transfer significantly reduced expression of PD-1 on CD4+ T cells in the spleen and generated persistent anti-HIV memory T cells. These results indicate that stem cell-derived viral Ag-specific CTLs can robustly accumulate in the local tissues to suppress HIV replication and prevent CD4+ T cell exhaustion through reduction of PD-1 expression.

Background/Objectives: Pediatric cancers are disorders of dysregulated development driven largely by genomic and epigenetic alterations. Precisely modeling these developmental differences is essential for understanding the unique biology of childhood cancers. Patient-derived organoids (PDOs) offer a powerful in vitro platform that recapitulates tumor heterogeneity, plasticity, microenvironment (including immune cells) and disease-relevant features. Methods: Here, we describe a step-by-step protocol for the establishment of PDOs from cells derived from pediatric brain tumors and extracranial solid tumor biopsies and bone marrow aspirates, including tumor processing, organoid culture/subculture, and cryopreservation. Results: Furthermore, we present the use of PDOs for further experimental analysis such as fluorescence imaging, Western blotting, flow cytometry, and immunohistochemistry (IHC) to investigate the underlying pathophysiology of tumorigenesis. Conclusions: Expanding the application of organoids to childhood malignancies holds exceptional promise for elucidating pediatric tumor biology and advancing therapeutic strategies, representing the long-overdue convergence of technology and clinical need.