Fig. 1: T cell uptake of macropinocytosis probes.
From: Macropinocytosis drives T cell growth by sustaining the activation of mTORC1
a–f Murine splenocytes (a–f) were unstimulated or stimulated with CD3/28 mAb for 24 h (a–c) or 20 h (d–f). 70 kDa Fdex (a–c) or BSA-Alexa 488 (d–f) probes were added to cells for the last 4 h or 8 h of culture, respectively, at the indicated temperatures. a, b, d, e Representative flow cytometry histogram plots of CD4+ T cell probe uptake and contour plots of probe uptake vs. FSC-A. c, f Mean ± 1 SEM of the ratio of Fdex (c) or BSA (f) probe uptake in stimulated vs. unstimulated CD4+ and CD8+ T cells at 37 °C (a–c, n = 10 independent experiments; d–f, n = 15 and 12 independent experiments for CD4+ and CD8+ T cells, respectively). ****P < 0.0001 by Student’s 1-sample 2-sided t-test. g Images show temperature-dependent uptake of BSA-Alexa 488 by CD3/28 mAb-stimulated purified murine pan T cells (probe incubation from 12 to 20 h) into structures that resemble macropinosomes. Representative images of six repeat experiments are shown. h Quantitation of the number of macropinosomes per CD4 T cell (n = 169 and 196 cells at 37 °C and 4 °C, respectively). i Method used to assess uptake of BSA-Alexa 647 by OTII TCR Tg T cells in vivo following immunization with whole Ova protein. j, k Representative flow cytometry contour plot of probe uptake vs. FSC-A (j) and mean ± 1 SEM of median fluorescence intensity (MFI) of Alexa 647 fluorescence of OTII TCR Tg T cells from unimmunized and immunized mice (n = 4 mice for each condition) (k). ***P < 0.001 by Student’s 2-sample 2-sided t-test. l–n Human PBMC were unstimulated or stimulated with CD3/28 mAb for 20 h. BSA-Alexa 488 was added to cells for the last 8 h of culture at the indicated temperatures. l, m Representative flow cytometry histogram plots of CD4+ T cell probe uptake and contour plots of probe uptake vs. FSC-A. n Mean ± 1 SEM of the ratio of BSA uptake in stimulated vs. unstimulated CD4+ and CD8+ T cells at 37 °C (n = 3 independent experiments). *P < 0.05, **P < 0.01, by Student’s 1-sample 2-sided t-test. Source data are provided as Source Data file.
