Fig 2: Pro-migratory Stimuli Induce Metabolic Reprogramming of Treg Cells(A and B) Expression of the indicated enzymes in Treg cells was measured 4 hr after antibody stimulation by western blotting. In (B) the mean relative expression measured by densitometric analysis in 3 independent experiments ± SD is shown.(C) Expression of the indicated enzymes by CD28- or LFA-1-stimulated Treg cells measured by western blotting at the indicated time points.(D–G) Relative mRNA expression of GCK (D) and GCKR (E) and cellular protein expression (F and G) by antibody-stimulated Treg cells was measured by RT-PCR and confocal microscopy, respectively. In (G) the mean MFI ± SEM measured using ImageJ software is shown. N = 3. Scale bar 20 µm.(H) AZD1656 (GCK activator, 1 µM) and vehicle-treated Treg cells (2 hr in insulin-free medium) were labeled with different intravital fluorescent dyes, and co-injected into syngeneic recipients that had received IFN-? i.p. 48 hr earlier. Cells were recovered from the peritoneum or spleen after 24 hr and analyzed by flow cytometry. Representative dot plots from 2 independent experiments are shown. The bar graphs indicate mean absolute number of labeled cells retrieved ± SD (n = 4, N = 2).(I and J) expression of PCNA by Treg cells stimulated with allogeneic DCs following treatment with either AZD1656 (I) or Clotrimazole (CLT, 1 µM, 2 hours, J) or vehicle alone was measured by flow cytometry. NS, non-stimulated control cells. Representative histograms from 3 independent experiments are shown. (n = 3, N = 3).(K) ECAR of Treg cells activated with recombinant ICAM-1 or Fc control. CLT or vehicle as well as other glycolysis-affecting drugs were added as indicated.(L) CLT- or vehicle-treated Treg cells underwent CD28 or isotype-matched antibody stimulation, labeled with different intravital fluorescent dyes, and injected i.v. in syngeneic recipients that had received IFN-? i.p. 48 hr earlier. Cells were recovered from the peritoneum (P) or spleen (S) after 24 hr and analyzed by flow cytometry. Representative dot plots from 2 independent experiments are shown. The column graphs indicate mean absolute number of labeled cells retrieved ± SD (n = 3, N = 2).*p < 0.05 ***p < 0.005; ****p < 0.001. Please see also Figure S4.

Fig 3: mTORC2 Controls Metabolic Reprogramming Induced by Pro-migratory Stimuli(A) Phosphorylation of AKT at Thr308 and Ser473 in Treg cells activated with CD28- or IsC-antibody ligation was measured by immunoblotting.(B) Treg cells were virally transfected with Rictor-specific or GCK-specific or non-sense (PLKO.1) sh-RNAs, as described in STAR Methods. Expression of GCK was measured by immunoblotting 24 hr later.(C–E) Expression of GCK and GCKR by control or Rictor-deficient Treg cells following CD28 or LFA-1 activation for 45 min. Bar graphs (D and E) show the mean protein expression (Total cell fluorescence) measured in 3 independent experiments by ImageJ software ± SD. Scale bar, 40 µm.(F–I) ECAR of CD28- or LFA-1-stimulated Rictor- and GCK-deficient and control T cells was measured with an extracellular flux analyzer. A glycolysis stress assay was performed by adding the indicated compounds at the time points indicated by the green lines. The basal and maximal glycolysis and the glycolytic reserve are shown in (G), (H), and (I), respectively (±SEM). N = 2.*p < 0.05 ***p < 0.005; ****p < 0.001. Please see also Figure S5.

Fig 4: Rictor-Deficient Treg Cells Display Impaired Motility(A–E) CD28-stimulated or IsC-treated PLKO.1, Rictor- or GCK-deficient Treg cells were labeled with PKH26 and co-injected i.v. with identical numbers of IsC-treated CFSE-labeled cells in syngeneic recipients treated with IFN-? 48 hr earlier. The presence of differently labeled cells in the indicated organs was assessed by flow cytometry 24 hr later. Representative dot plots from 3 independent experiments are shown on top in (A), (B), and (C). The bar graphs in (A), (B), and (D) indicate mean absolute number of labeled Treg cells recovered in the indicated tissues from 4 different recipients ± SD (N = 3). The bar graph in (E) shows the ratio of Treg cells recovered in the lung and the spleen (n = 4) ± SD.(F and K) Control and ShGCK Treg cell migration through 5 or 3 µm pore bare-filter transwells in response to CCL22 (F) or CXCL10 (K). Results are expressed as percentage of migrated cells at the indicated time points ± SD (n = 3).(H–J) BALB/c-derived skin was grafted onto C57BL/6 recipients who had received mock-transduced, Rictor- or GCK-depleted Treg cells or no cells 24 hr earlier. Graft rejection was monitored daily (H). Some grafts were removed 5 days post-grafting and the presence of fluorescently labeled Treg cells in the indicated tissue was assessed by widefield fluorescence microscopy. Representative images of grafts and spleens from 2 independent experiments are shown in (I). The bar graphs (J) indicate the mean number of labeled cells detected in at least 10 10× tissue images from each animal ± SD (n = 8).*p < 0.05 **p < 0.01; ***p < 0.005. Please see also Figure S6.

Fig 5: CD28-Induced Migration and Metabolic Reprogramming Require PI3K but Not mTORC-1 ActivationWT and Cd28Y170F mice received an i.p. injection of Zymosan. Samples were collected either before or 72 hr after the injection. The presence of Treg cells in the indicated tissues was measured by flow cytometry.(A) Representative dot plots from 2 independent experiments.(B) The mean percentage of cells measured in two experiments of identical design ± SD.(C and D) Ratio of cells retrieved in the indicated tissues over time ± SD (n = 3).(E) ECAR (±SD) of antibody-stimulated Cd28Y170F and WT Treg cells was compared using fluxometry. n = 4, N = 2.(F) Migration of antibody-stimulated Cd28Y170F and WT Treg cells through IFN-?-treated EC monolayers. Results are expressed as mean percentage of migrated cells after 24 hr ± SD. N = 4.(G–J) Equal numbers of antibody-stimulated PKH26-labeled WT or Cd28Y170F Treg cells were injected i.v. into syngeneic mice treated with IFN-? i.p. 48 hr earlier. Cells were harvested from the indicated tissues 24 hr later, counter-stained for Foxp3, and analyzed by flow cytometry. Representative dot plots of 2 independent experiments are shown in (G) and (I). The bar graphs in (H) and (J) indicate mean absolute numbers of labeled cells (n = 4, N = 2) ± SD.*p < 0.05; **p < 0.01; ***p < 0.005. Please see also Figure S3.