Figure 3.

Human Treg Cell Proliferation In Vitro Requires Both Glycolysis and FAO

(A) In vitro proliferation of Treg cells upon 72 hr anti-CD3 and anti-CD28 stimulation in the presence or absence of leptin-neutralizing antibody. The data are shown as mean ± SEM (n = 6).

(B) Immunoblot for P-ERK1/2 on human Treg cells upon 12 hr anti-CD3 and anti-CD28 stimulation in the presence or absence of leptin-neutralizing antibody. Total ERK1/2 served as a loading control. One representative out of three independent experiments is shown.

(C) In vitro proliferation of Treg cells upon 72 hr anti-CD3 and anti-CD28 stimulation in the presence or absence of leptin-neutralizing antibody, 2-DG, or Etomoxir, alone or in combination. The data are shown as mean ± SEM (n = 3).

(D) Fold inhibition of Treg cell proliferation upon leptin neutralization in the presence of 2-DG or Etomoxir. The data are shown as mean ± SEM (n = 3).

(E) Kinetic profile of ECAR in Treg cells stimulated or not with anti-CD3 and anti-CD28 for 12 hr, in the presence or absence of leptin-neutralizing antibody (one representative out of three independent experiments). The data are shown as mean ± SEM of duplicates. ECAR was measured in real time, under basal conditions and in response to glucose, oligomycin and 2-DG. Indices of glycolytic pathway activation, calculated from Treg cells ECAR profile: basal ECAR (F), maximal ECAR (G), and glycolytic capacity (H) in Treg cells stimulated or not with anti-CD3 and anti-CD28 for 12 hr, in the presence or absence of leptin-neutralizing antibody (one representative out of three independent experiments). Data are expressed as mean ± SEM of three measurements, each of them in duplicates.

(I) ECAR profile of unstimulated Treg cells before and after leptin-neutralizing antibody injection (one representative out of three independent experiments). The data are shown as mean ± SEM of triplicates.

(J) Kinetic profile of OCR in Treg cells stimulated or not with anti-CD3 and anti-CD28 for 12 hr, in the presence or absence of leptin-neutralizing antibody (one representative out of three independent experiments). The data are shown as mean ± SEM of duplicates. OCR was measured in real time, under basal conditions and in response to indicated mitochondrial inhibitors: oligomycin, FCCP, Antimycin A and Rotenone. Indices of mitochondrial respiratory function, calculated from Treg cells OCR profile: basal OCR (K), ATP-linked OCR (L), and maximal OCR (M) in Treg cells stimulated or not with anti-CD3 and anti-CD28 for 12 hr, in the presence or absence of leptin-neutralizing antibody (one representative out of three independent experiments). Data are expressed as mean ± SEM of three measurements, each of them in duplicates.

(N and O) Percentage of FAO in Treg cells stimulated or not with anti-CD3 and anti-CD28 for 12 hr in the presence or absence of leptin-neutralizing antibody. FAO was evaluated in basal conditions (N) and during maximal respiration (O) (one representative out of three independent experiments). Data are expressed as mean ± SEM of three measurements, each of them in duplicates.

(P) Immunoblot for aldolase, enolase, hexokinase, and PKM1/2, FAS, ApoA4, HADHA, ACAD9, CPT1A, on Treg cells upon 12 hr anti-CD3 and anti-CD28 stimulation in the presence or absence of leptin-neutralizing antibody. Actin and total ERK 1/2 served as a loading control. One representative out of two independent experiments is shown. The graphs show the relative densitometric quantitation of aldolase, enolase, hexokinase, PKM1/2, FAS, ApoA4, HADHA, ACAD9, and CPT1A normalized on actin in unstimulated (white columns), anti-CD3, and anti-CD28-stimulated (gray columns) and leptin-neutralized Treg cells (black columns) and shown as fold over unstimulated Treg cells (n = 6; data are shown as mean ± SEM of at least two independent experiments, in triplicates). All statistical analysis by paired two-tailed Student’s t test. (^∗p < 0.05, ^∗∗p < 0.001, ^∗∗∗p < 0.0001).