T cell functional phenotyping on a single cell scale using hydrogel microparticles.

Abstract Engineered cell therapies, especially chimeric antigen receptor (CAR) T cell therapies, have shown much promise in treating various cancers, most notably hematologic malignancies. However, the success of these therapies is highly variable between cancer types and across patient populations. A growing body of literature suggests that certain T cell functional properties are key drivers of response; thus, it is imperative to develop a more thorough understanding of the functional heterogeneity in T cell populations in order to inform the design of the next generation of cancer immunotherapies. Here, we used a new technology our team developed known as nanovials, which are microfluidically manufactured hydrogel microparticles, to enable the specific activation and capture of secreted products of T cells on a single-cell level. Leveraging this platform, we characterized the secretion of IFN-γ and TNF-α by individual CD8 +T cells harvested from OT-I mice. After initial expansion after isolation, T cells were loaded into functionalized nanovials and specifically activated with OVA peptide. The cells loaded in the nanovials were then separated based on cytokine secretion levels via fluorescence-activated cell sorting (FACS). Unlike traditional cytokine secretion assays which require fixation and permeabilization, cells remain viable throughout our process, allowing for sorted subpopulations to be dissociated from the microparticles, expanded, and further analyzed via cytotoxicity, co-culture, or phenotypic studies. Collectively, our efforts introduce a new paradigm for elucidating the functional properties of immune cells, providing critical insights that will guide the future design of engineered cell therapeutics. M.K. is a recipient of a National Science Foundation Graduate Research Fellowship Program award.