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Abstract Cytokines, crucial in immune modulation, impact disease progression when their secretion is dysregulated. Existing methods for profiling cytokine secretion suffer from time‐consuming and labor‐intensive processes and often fail to capture the dynamic nature of immune responses. Here, iSECRETE, an integrated platform that enables synchronous cell activation, wash‐free, and target‐responsive protein detection for single‐cell IFN‐γ cytokine secretion analysis within 30 min at room temperature is presented. By incorporating a DNA proximity assay (DPA) into a multifunctional microfluidic system, one‐pot homogenous cytokine signal amplification, with a limit of detection of ≈50 secreted molecules per cell is achieved. iSECRETE can robustly handle various sample types that are shown. Two distinct immune activation assay modalities are demonstrated on iSECRETE. Finally, the detection of single‐cell IFN‐γ secretion as an activation hallmark of chimeric antigen receptor T cells within 6 h of exposure to cancer targets is shown. iSECRETE represents the fastest single‐cell sample‐to‐result cytokine secretion assay to date, providing a powerful tool for advancing the understanding of biological phenotypes, functions, and pathways under in vivo‐like conditions.

Abstract In keeping with the rule of “form follows function”, morphological aspects of a cell can reflect its role. Here, it is shown that the cellular granularity of a lymphocyte, represented by its intrinsic side scatter (SSC), is a potent indicator of its cell state and function. The granularity of a lymphocyte increases from naïve to terminal effector state. High‐throughput cell‐sorting yields a SSChigh population that can mediate immediate effector functions, and a highly prolific SSClow population that can give rise to the replenishment of the memory pool. CAR‐T cells derived from the younger SSClow population possess desirable attributes for immunotherapy, manifested by increased naïve‐like cells and stem cell memory (TSCM)‐like cells together with a balanced CD4/CD8 ratio, as well as enhanced target‐killing in vitro and in vivo. Altogether, lymphocyte segregation based on biophysical properties is an effective approach for label‐free selection of cells that share collective functions and can have important applications for cell‐based immunotherapies.