Fig. 7
Exosomal galectin-1 and RNA factors contribute to the induction of CD8+ Ts cells. a. Conditioned media from Tu167 and Caco-2 cells was harvested after 48 h, centrifuged and filtered to remove cellular debris, and then ultracentrifuged for 18 h to pellet the exosome fraction. Protein was isolated by extraction in RIPA buffer and used in western blotting for the indicated proteins. For comparison, whole cell lysates were prepared from the same cell lines. b. Galectin-1 was then knocked-out in Tu167 cells by CRISPR/Cas9 targeting constructs, which was then verified by Western blot of whole-cell lysates. c. Conditioned growth media from Tu167 wildtype and Gal-1ko cells, as well as Caco-2 control cells was then collected as previously described, and exosomes were enriched via ultracentrifugation and incubated with normal donor T cells for 6 h. Cells were then washed and cultured for an additional 6 days before assessing for CD27 and CD28 expression on CD8+ T cells by flow cytometry. Indicated plots are gated on live CD3+ CD8+ cells. d. Treated CD8+ T cells were then suppression assays with CFSE-labeled, normal donor T cells as responders similar to previous experiments. e. RNA was isolated from purified exosomes by Trizol extraction and introduced into freshly purified T cells by electroporation using a Nucleofector™ device following manufacturer’s instructions (Amaxa). Successful Nucleofection was verified by the addition of a GFP-expressing plasmid followed by analysis by flow cytometry. Nucleofected T cells were then incubated for 6 days and then used in an in vitro suppression assay as previously described. f. 3H-thymidine incorporation suppression assay was done for 72 h with 3H-thymidine being added 22 h before the end of the experiment. All data represents the mean +/− SD of 3. To compare means, a one-way analysis of variance was used with a Bonferroni multiple comparison test. *p < 0.05, ***p < 0.001 for the indicated groups vs. incubation of T cells in cRPMI followed by incubation with responder T cells at 1:2 ratio