Figure 3. AQP9 Deficiency Impairs TAG Synthesis and Storage in CD8+ T Cells.
(A) Aqp9−/− or Aqp9+/+ P14 CD8+ T cells were cultured in vitro with GP33–41 peptide for 3 days and then in IL-7 for two days. The amount of free glycerol was measured in total cell lysates by a coupled enzymatic reaction system.
(B) Aqp9−/− or Aqp9+/+ P14 memory CD8 T cells from 40 dpi were stained with the neutral lipid indicator Bodipy493/503 and analyzed by flow cytometry.
(C) P14 CD8+ T cells described in (A) were pulsed with 0.1 μcurie per milliliter (Ci/ml) [1,2,3-14C]-Glycerol for 4 hr, then lipids were extracted and resolved by TLC. The bar graphs on the right show densitometry quantification of TAG and DAG autoradiography bands after a 2-week exposure.
(D) P14 CD8+ T cells described in (A) were cultured in the presence or absence of glycerol (Gly) or OA for 2 days before lipid extraction and TLC assay. Standards for TAG and CHO were loaded on the left- and right-most lanes. The bar graph on the right shows densitometry quantification of TAG band.
(E) Lipids were extracted from Aqp9+/+ and Aqp9−/− P14 CD8+ T cells described in (A) for LC-MS analysis of TAG isobaric species.
Data are representative of two (B and E) and three (A, C, and D) independent experiments (n = 3–7 mice/group): *p < 0.05, **p < 0.01, and not significant (n.s.) (see also Figure S3).