Polanco, L. | bioRxiv Science

bioRxiv2025

Generation of effector CD4+ T cells from Human iPSC

Off the shelf CD4+ T cell therapies, particularly those with immunoregulatory or cell repair functions, could be transformative in CAR therapies for cancers and treatment of chronic inflammatory diseases. However, progress is stunted in this area due to challenges generating human CD4+ T cells from induced pluripotent stem cells. Here we describe a key role for the withdrawal of Notch ligand during the final step of stimulation through the T cell receptor to prompt T cell maturation allowing access to the CD4 lineage in iPSC T cells (iCD4+ T cells). Functional analyses of iCD4+ T cells using a novel high-parameter CyTOF intracellular cytokine staining panel revealed both canonical Th1 cytokine signatures and cells producing varying combinations of other cytokines including IL-4, IL-8, and IL-13. Single cell RNA sequencing of iCD4+ T cells demonstrated a transcriptional signature similar to human blood CD4+ T cells. We believe this robust yet simple platform represents a key step towards the generation of off the shelf iCD4+ T cell therapies with utility for the treatment of a panoply of diseases including cancer and inflammatory autoimmune disorders. HIGHLIGHTST cell receptor stimulation of iPSC CD4+/CD8+ T cell progenitors on coating without Notch ligand allows access to the CD4+ T cell lineage iPSC derived CD4+ T cells express varied cytokines in response to stimulation

immunology↗

bioRxiv2025

Superior intracellular detection of cytokines, transcription factors, and phosphoproteins by CyTOF compared with fluorescent cytometry

Unraveling biological complexity, whether it be immune subset distribution in infectious disease(s), autoimmunity or tumor heterogeneity, requires technologies capable of single-cell proteomic analysis, such as flow cytometry. Surface immunophenotyping alone is often insufficient, as interrogating functional capacity is required to determine cellular mechanisms and effectively inform diagnostic biomarker discovery, therapeutics and vaccine development. However, large panels with intracellular markers are subject to numerous challenges, including spectral overlap and background cellular autofluorescence, reducing resolving power for rare subsets or populations defined by low-abundance expression. We posited that mass cytometry may overcome such limitations; to address this, three small (11-12-plex) clone-matched antibody panels were evaluated by spectral flow and mass cytometry. Panels were comprised of surface and intracellular targets (phospho-epitopes, transcription factors or cytokines) and designed to minimize fluorescence spectral overlap. CyTOF technology offered superior signal resolution across the range of intracellular targets. Improved signal-to-noise provided better resolution of phospho-events and transcription factor expression, in particular TOX and T-bet. Most strikingly, stimulation-specific IL-10+ and IL-13+ cells were only detected by CyTOF. Superior resolution of these cytokines enabled accurate population clustering, permitting more unique immune cell signatures to be found, including Tr1 and Tc2 populations, thus providing a more comprehensive picture of the immuno-diversity present. Our findings indicate that CyTOF technology could catalyze seminal discoveries in functional immune profiling, driving therapeutic design and diagnostics.

immunology↗