Figure 8. Cell-intrinsic autophagy is required for metabolic adaptation and survival of intestinal Foxp3⁺ T_reg cells.

(A) Foxp3⁺ T_reg cell frequencies among CD4⁺ TCRβ⁺ T cells in Atg16l1^ΔFoxp3 and Atg16l1^fl/fl littermates and (B) representative FACS plots of Foxp3 expression in cLP CD4⁺ T cells from young Atg16l1^ΔFoxp3 and Atg16l1^fl/fl littermates (gated on CD4⁺ TCRβ⁺ T cells). (C) qPCR analysis of glycolytic gene levels in sorted Foxp3⁺ T_reg cells from spleen and cLP of young Atg16l1^ΔFoxp3 and Foxp3^Cre mice (sorted for CD4⁺ TCRβ⁺ YFP⁺). (D) qPCR analysis of FAS and FAO gene levels in Foxp3⁺ T_reg cells from the spleen and cLP of young Atg16l1^ΔFoxp3 and Foxp3^Cre mice (sorted for CD4⁺ TCRβ⁺ YFP⁺). FAS: fatty acid synthesis, FAO: fatty acid oxidation, Glut1: glucose transporter 1, Slc16ac: solute carrier family 16 member 3 (lactic acid and pyruvate transporter), Tpi1: triosephosphate isomerase 1, Aldo–α: aldolase α, Ldh-α: lactate dehydrogenase α, Gpi1: Glucose phosphate isomerase 1, Pgk1: Phosphoglycerate kinase 1, Acc1: acetyl-CoA carboxylase 1, Acc2: acetyl-CoA carboxylase 2, Srebf1: sterol regulatory element binding transcription factor 1, Srebf2: sterol regulatory element binding transcription factor 2, Fdft1: farnesyl-diphosphate farnesyltransferase 1, Fabp: Fatty acid-binding protein. Data are representative from two (C,D) or three independent experiments (A,B). Each dot represents individual mouse (A) or data are shown as mean ± s.e.m (C,D). Gene expression levels are shown as mean ± s.e.m of three technical replicates (C,D). Numbers indicate percentage of cells in gates (B). cLP – colonic lamina propria. Young mice: 8–12 weeks old.

DOI: http://dx.doi.org/10.7554/eLife.12444.019

Figure 8—figure supplement 1. Atg16l1-deficient colonic T_reg cells exhibit increased cytokine secretion.

(A) Frequencies of IFN-γ⁺, IL-17A⁺, IL-4⁺, IL-13⁺ Foxp3⁺ T_reg cells in the cLP of aged Atg16l1^ΔFoxp3 and Atg16l1^fl/fl littermates (gated on Foxp3⁺ CD4⁺ TCRβ⁺ T cells). (B) qPCR analysis of Bcl2, Bim and Bax levels in Foxp3⁺ T_reg cells from young cLP of Atg16l1^ΔFoxp3 and Foxp3^Cre mice (sorted for CD4⁺ TCRβ⁺ YFP⁺). Data are combined from three independent experiments with two to five mice per group (A) or are representative from two independent experiments (B). Each dot represents an individual mouse and horizontal bars denote means (A). Gene expression levels are shown as mean ± s.e.m of three technical replicates (B). cLP – colonic lamina propria. Young mice: 8–12 weeks old.

DOI: http://dx.doi.org/10.7554/eLife.12444.020

Figure 8—figure supplement 2. Increased lipid uptake by intestinal T_reg cells.

(A,B) Atg16l1^fl/fl and Atg16l1^ΔCD4 littermates were injected i.p. with 50 μg of fluorescent 16-carbon fatty acid analog BODIPY C-16 and culled 1 hr later and tissue was collected for analysis by flow cytometry. (A) Representative FACS plots and (B) quantification of C16-Bodipy uptake by Foxp3⁺ T_reg cells in the spleen, mLN and cLP (gated on Foxp3⁺ CD4⁺ TCRβ⁺ T cells). (C) Representative FACS plots and (D) quantification of CD36 expression by Foxp3⁺ T_reg cells in the spleen, mLN and cLP of Atg16l1^fl/fl and Atg16l1^ΔCD4 littermates (gated on Foxp3⁺ CD4⁺ TCRβ⁺ T cells). Data are combined from (B,D) or are representative of (A,C) two independent experiments with 3–5 mice per group. Each dot represents an individual mouse and horizontal bars denote means. Statistical significance was determined using the Mann–Whitney test, *p<0.05. mLN - mesenteric lymph nodes, cLP – colonic lamina propria. Young mice: 8–12 weeks old.

DOI: http://dx.doi.org/10.7554/eLife.12444.021

Figure 8—figure supplement 3. T_H2 cells exhibit an enhanced glycolytic metabolic profile that is independent of autophagy.

(A) Representative FACS plot and quantification of the cell size (FSC-H) of naïve (CD44^- CD62L⁺) CD4⁺ T cells from the spleen of Atg16l1^ΔCD4 or Atg16l1^fl/fl littermates. (B) Basal level of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in naïve (CD44^- CD62L⁺) unstimulated CD4⁺ T cells isolated from the spleen of Atg16l1^ΔCD4 or Atg16l1^fl/fl littermates measured using the Seahorse metabolic flux analyzer. (C,D) Expression of c-Myc (C) and cell size (D) was analyzed by FACS in Atg16l1^ΔCD4 or Atg16l1^fl/fl CD4⁺ T cells that were cultured in T_H2 or T_reg-polarizing conditions for 3 days and rested for one day in the presence of polarizing cytokines. (E) Basal level of OCR and ECAR were measured by Seahorse metabolic flux analyzer in Atg16l1^ΔCD4 or Atg16l1^fl/fl CD4⁺ T cells cultured in T_H2 or T_reg- polarizing conditions for 3 days and rested for 2 days in the presence of polarizing cytokines. (F) qPCR analysis of glycolytic gene levels in Atg16l1^ΔCD4 or Atg16l1^fl/fl CD4⁺ T cells cultured in T_H2 or T_reg polarizing conditions for 3 days and rested for 1 day in the presence of polarizing cytokines. Data are combined from two independent experiments (A), or are representative of two independent experiments (B-D), or are from one experiment (E,F). Each dot represents an individual mouse (A) or individual cell culture (D). ECAR and OCAR data represent mean ± s.e.m values of T cell populations that were assayed in triplicates or quadruplicates (B,E). Gene expression data of triplicate cultures represent normalized expression values for each gene that were scaled to a mean of 0 and a standard deviation of 1 (F). Statistical significance was determined using the Mann–Whitney test (A) or unpaired Student’s t –test (B,D,E), **p<0.01; **p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.12444.022