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. 2016 Feb 16;44(2):406–421. doi: 10.1016/j.immuni.2016.01.028

Figure 6.

Figure 6

Human Tconv Cell Proliferation In Vitro Requires Glycolysis but Not FAO

(A) In vitro proliferation of Tconv 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 Tconv 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 Tconv 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 in Tconv cells proliferation upon TCR-mediated stimulation in the presence of 2-DG and etomoxir. The data are shown as mean ± SEM (n = 3).

(E) Kinetic profile of ECAR in Tconv 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 triplicates. 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 Tconv ECAR profile: basal ECAR (F), maximal ECAR (G), and glycolytic capacity (H) in Tconv 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 triplicates.

(I) Kinetic profile of OCR in Tconv 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 triplicates. 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 Tconv OCR profile: basal OCR (J), ATP-linked OCR (K), maximal OCR (L) in Tconv 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 triplicates.

(M and N) Percentage of FAO in Tconv 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 (M) and during maximal respiration (N) (one representative out of three independent experiments). Data are expressed as mean ± SEM of three measurements, each of them in duplicates.

(O) Immunoblot for Aldolase, Enolase, Hexokinase, and PKM1/2, Fatty acid synthase (FAS), ApoA4, HADHA, ACAD9, CPT1A on Tconv 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 three 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 Tconv cells (black columms) and shown as fold over unstimulated Tconv cells. (n = 6; data are shown as mean ± SEM of 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).