Label-free lymphocytes reconstitution using side scatter for optimal T cell manufacturing

Abstract

SUMMARYLymphocyte biology research commonly involves purification of lymphocyte subpopulations by fluorescence-activated cell sorting (FACS) or immunomagnetic separation (IMS), both of which typically rely on antibody labeling of validated cell markers. Methods enabling label-free segregation of lymphocyte subpopulations would be invaluable with regard to less-perturbation, simplicity and cost-effectiveness. Here, we introduce TRuST, a label-free approach for T cell reconstitution using side-scatter (SSC). TRuST-sorted SSClow cells enrich for CD4+ T and naïve T cells, while SSChigh cells enrich for CD8+ T, NK and differentiated T cells. Enrichment purity can be improved by computational gate design. SSClow cells have superior expansion capacity and generate more central memory precursors with naïve-resembling cytokine responses. Moreover, we find that both T cell differentiation status and CD4/CD8 T ratio in the starting cellular material are critical attributes predicting T cell product quality and quantity. TRuST presents an effective and reliable technique for label-free lymphocytes selection and reconstitution.

Time-resolved assessment of single-cell protein secretion by sequencing

“…These IL-2 + TNF-α + or IL-2 + TNF-α + IFN-γ + had a dramatic upregulation of genes involved in IL2-STAT and TNF-NFKB signaling pathways, and genes favoring cell survival and proliferation/differentiation (e.g., CSF2, IER3, CCND3, EZH2, and Mycregulated genes). These data have extended our previous finding that the generation of TCMRA during the production of therapeutic T cells was wellcorrelated with the proportion of TN in cellular starting material 42 , and cell products containing more TCMRA were manifested by younger phenotypes and superior functions that are recognized to be good for therapeutic outcomes.…”

“…Thus, we further dissected the secretion potency of heterogeneous cell 12 subpopulations, especially T lymphocytes. We found that pre-existing central memory T cells with CD45RA expression (TCMRA) (Figure S4E) 42,43 regardless of CD4 + T or CD8 + T cells had the rapidest cytokine secretion, primarily TNF-α + IL-2 + and TNF-α + within one hour post activation, while those moredifferentiated counterparts like central memory without CD45RA expression (TCMO) and effector memory (TEM and TEM_late) tended to secret mono-cytokine such as TNF-α + or IL-2 during initial stimulation (Figure 3A). CD8 + TCMRA continued to release multiple cytokines, specifically IFN-γ-involved triple or double positive cytokines, whereas amongst CD4 + T cells, CD4 + TCMRA and CD4 + TCMO_late were the multi-cytokine releasing cells (Figure 3B, C).…”