Figure 2. Autophagy is induced in lymphocytes following homeostatic expansion.

(A,B) WT (CD45.1) liver lymphocytes were adoptively transferred into Rag2^−/− × Il2rg^−/− (homeostatic proliferation) (A) Representative histograms and (B) quantification of Cyto-ID mean fluorescence intensity (MFI) in adoptively transferred NK, ILC1, NKT, and CD8⁺ T cells harvested from livers of recipient mice on day 5 and 7 after transfer. (C) WT (CD45.1) liver lymphocytes were adoptively transferred into WT (CD45.1 × CD45.2) recipients (basal homeostasis) and quantified for Cyto-ID staining on day 5 and 7 after transfer from recipient livers. (D,E) LC3-GFP (CD45.2) transgenic liver lymphocytes were adoptively transferred into Rag2^−/− × Il2rg^−/− recipients, and livers were harvested on either day 5 or 7 post-transfer. (D) Representative histograms and (E) quantification of LC3-II MFI for indicated adoptively transferred cell subsets. (F) LC3-GFP transgenic liver lymphocytes were adoptively transferred into WT (CD45.1 × CD45.2) recipients and quantified for LC3-II staining for each indicated cell subset on day 5 and 7 after transfer from recipient livers (G–I) LC3-GFP transgenic splenocytes were adoptively transferred into Rag2^−/− × Il2rg^−/− recipients. (G) mNK and CD8⁺ T cells were sorted at the indicated time points from recipient mice, and analyzed for Map1lc3b (LC3) mRNA levels by qRT-PCR (n=3 for each time point) (H) Confocal microscopy (original magnification, 63×) of sort-purified mNK or CD8⁺ T cells and (I) quantification of LC3-II⁺ puncta on either day 0 or 6 post-transfer (presented as the frequency of cells with more than one LC3-II⁺ punctum). Data are representative of two independent experiments, with n=3–4 per time point. Samples were compared using an unpaired, two-tailed Student’s t test, and data presented as the mean ± s.e.m. (*p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant).