Volume 142, Issue 7, Pages 1493-1503.e6 (June 2012) Autophagy Attenuates the Adaptive Immune Response by Destabilizing the Immunologic Synapse  Manon E. Wildenberg, Anne Christine W. Vos, Simone C.S. Wolfkamp, Marjolijn Duijvestein, Auke P. Verhaar, Anje A. Te Velde, Gijs R. van den Brink, Daniel W. Hommes  Gastroenterology  Volume 142, Issue 7, Pages 1493-1503.e6 (June 2012) DOI: 10.1053/j.gastro.2012.02.034 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Immunologic synapse formation induces autophagy in both human and murine DCs. (A) GFP-LC3–transfected human DCs were incubated in the absence (black bars) or presence (white bars) of allogeneic lymphocytes, samples then were blotted for GFP and the LC3II/LC3I ratio was calculated by image analysis. Western blot representative of 3 independent experiments. Data normalized against control samples, bars represent mean and SEM of 3 individual experiments. (B) GFP-LC3–transfected human DCs co-incubated with allogeneic T cells and analyzed by fluorescent microscopy, picture shows puncta formation in a conjugated DC (arrow) and dispersed LC3 in a single DC (arrowhead). (C) Interactions were induced between GFP-LC3–transfected human DCs and autologous or allogeneic T cells (left), or between murine BMDCs and OT-II cells in the presence of control or cognate antigen (right) for 60 minutes. Number of LC3 puncta/DCs then was scored in unconjugated DCs (black bars) and DCs conjugated to T cells (white bars) in the same image fields. Data are representative of 3 individual experiments, n > 40 conjugates per experiment. Bars represent mean ± standard error of the mean. *P < .05, **P < .01. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Autophagosomes contain synaptic components. (A) Conjugates of human DCs and allogeneic T cells were induced and stained for expression of HLA-DR or ICAM-1 (red) and LC3 (green). Subcellular localization of positive staining was determined by confocal imaging. Two individual DC–T-cell conjugates are shown. (B) Autophagosome diameter was determined by image analysis in more than 50 cells. (C) Position of LC3+/MHC+ and LC3+/MHC- spots were calculated as relative to the maximum width of the cell (n = 13 conjugates). Data are representative of 3 independent experiments in 4 donors. **P < .01, ***P < .001. DIC, differential interference contrast microscopy; DAPI, 4',6-diamidino-2-phenylindole. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Decreased autophagy leads to increased cytoskeletal rearrangements in DCs and prolonged DC–T-cell interactions. (A) DCs generated from siRNA-treated monocytes show an elongated phenotype. Cell length was measured by image analysis. Mean and standard error of the mean (SEM) of more than 50 cells per condition is shown. Image shows representative picture of 3 experiments. (B and C) DC–T-cell interactions were generated by centrifugation and maintained for 30 minutes. The actin cytoskeleton was visualized by phalloidin staining and scored for actin polarization on a 3-point scale (0–2, n > 50). Bars represent mean and SEM, representative figure of at least 4 independent experiments in individual donors. (C) DCs were pretreated with 3-MA for 2 hours and washed carefully before clustering with T cells. (D) Time-lapse analysis of DC–T-cell interactions. Allogeneic T cells were added to adherent siRNA-treated human DCs (ratio 5:1, left panel) and OT-II cells were added to adherent siRNA-treated BMDCs in the presence of cognate antigen (OVA323-339 peptide; 1 μg/mL; ratio, 1:5, right panel). All samples were analyzed for 30 minutes by time-lapse microscopy and the total duration of individual interactions was calculated in duplicate samples (>45 interactions/experiment). Interactions were categorized as short (<3 min, black), intermediate (3–15 min, white), or long (>15 min, grey). Results are representative of 5 independent experiments *P < .05, ***P < .001. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Decreased autophagy results in increased organization of the immunologic synapse. (A and B) Interactions between siRNA-treated DCs and allogeneic T cells were induced and conjugates were stained for actin (green) and HLA-DR or ICAM-1 (red). Conjugates then were analyzed by confocal microscopy. Orthogonal sections are shown to the right (YZ) and bottom (XZ) of the main image (XY). On the right, an overlay of the en face view of the 2 stainings is depicted. (C) For quantification purposes, en face images were obtained and the relative fraction of staining present in the periphery and the center were calculated. Each dot represents an individual cell analyzed. *P < .05. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Decreased DC autophagy results in increased T-cell proliferation without phenotypic maturation. (A) Inhibition of ATG16L1 or IRGM in DCs results in increased allogeneic T-cell stimulation in an mixed lymphocyte reaction (MLR) culture. Cells were co-cultured for 72–96 hours and data were normalized against proliferation in the control sample of the same donor (n = 10 and n = 9, respectively). (B) Decreased autophagy in BMDCs pulsed with OVA results in increased OT-II proliferation. Cells were co-cultured for 96–120 hours and data were normalized against control sample. Mean and standard error of the mean (SEM) of 3 independent experiments are shown. (C) Inhibition of ATG16L1 expression does not alter DC maturation. DCs were treated with ATG16L1 (green line) or control (red line) siRNA for 48 hours and analyzed by flow cytometry. Blue lines indicate control staining. Data are representative of 4 independent experiments. (D) Isolated CD45RO+ T cells were co-cultured with autologous siRNA-treated DCs for 4 days, restimulated by phorbol 12-myristate 13-acetate (PMA)/ionomycin, and analyzed by flow cytometry Plots show representative data, bars represent mean (normalized against control sample in each donor) and SEM of duplicate experiments in 5 different donors. (E) Isolated CD45RO+ T cells were co-cultured with autologous DCs for 4 days in the presence of blebbistatin (5 μmol/L) or vehicle control (dimethyl sulfoxide), restimulated by phorbol 12-myristate 13-acetate (PMA)/ionomycin, and analyzed by flow cytometry. Data are normalized against vehicle control for each donor and represent the mean and SEM (n = 5). *P < .05, **P < .01. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 DCs generated from patients carrying an ATG16L1 risk allele display similarly altered morphology and hyperpolarization. (A) Monocytes isolated from peripheral blood were cultured in the presence of granulocyte-macrophage colony–stimulating factor and IL-4 for 6 days. Two images were taken from each well at random locations and processed by image analysis (n = 9), representative images from 2 donors are shown (right panel). (B) Monocyte–T-cell interactions were induced by centrifugation, actin was stained using phalloidin, and actin polarization was scored on a 3-point scale (0–2, n = 9). (C) DCs were co-cultured with allogeneic T cells for 96 hours. Supernatant then was collected for cytokine measurements by enzyme-linked immunosorbent assay. Cell length as measured in panel A was correlated to IL-17 concentration in supernatants using Spearman rho (n = 9). *P < .05. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 Immunologic synapse formation induces autophagy through LKB1. (A and B) Interactions between DCs and either autologous (auto) or allogeneic (allo) lymphocytes were induced and maintained for 90 minutes. DCs were isolated and lysates were analyzed for expression of phosphorylated proteins by Western blotting. Bars represent mean and standard error of the mean (SEM) of 4 individual experiments. (C) Conjugates were stained for LKB1 (green) and actin (red). Two individual conjugates are shown, blue represents nuclear 4′,6-diamidino-2-phenylindole (DAPI) staining. Images are representative of 3 independent experiments. (D–F) DCs were treated with either LKB1-specific or control siRNA and co-incubated with lymphocytes. Cell length was measured (n > 100) and polarization of the actin skeleton was scored on a 3-point scale (0–2, n > 100 conjugates). Bars represent mean and SEM. Figure is representative of 3 independent experiments. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Clusters of GFP–LC3–transfected DCs and allogeneic T cells were induced for 60 minutes and stained for expression of HLA-DR (red) and LAMP2 (blue). Subcellular localization of positive staining was determined by confocal imaging. Arrows indicate location of the T cell in cluster. Arrowheads indicate the line of analysis shown in line graphs. Graphs show the relative signal intensity along the same line for all 3 stainings. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 (A) DCs were treated with specific or control siRNA for 48 hours and analyzed by quantitative polymerase chain reaction (PCR). All data was normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars indicate mean expression and standard error of the mean (SEM), representative of more than 10 experiments. (B) siRNA-treated DCs were stimulated with rapamycin for 12 hours (20 μg/mL) and immunoblotted for expression of p62. Data are representative of 3 donors. (C) siRNA-treated DCs were cultured for 3 days in RPMI culture medium containing 10% fetal calf serum (FCS) and tested for viability by MTS assay. Bars depict mean and SEM of 3 individual donors. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 Clusters were formed between (A) OT-II T cells and bone marrow–derived DCs transfected with ATG16L1 siRNA, or (B) peripheral lymphocytes and monocyte-derived DCs transfected using NOD2 siRNA. Actin skeleton was visualized by phalloidin staining and scored for actin polarization on a 3-point scale (0–2). Data are representative of 3 individual experiments. Bars represent mean and standard error of the mean of more than 100 cells/experiment (n > 100). **P < .01. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 4 (A) Cytokine secretion of DCs treated with ATG16L1 siRNA co-cultured with allogeneic lymphocytes (ratio, 1:5) was determined in supernatants by cytometric bead array. Data were normalized against control of the individual donors. Mean and standard error of the mean (SEM) of 3 individual experiments are shown. (B) Isolated CD45RO+ T cells were co-cultured with autologous DCs treated with ATG16L1 siRNA for 4 days, restimulated by PMA/ionomycin, and analyzed by quantitative polymerase chain reaction (PCR) (left and right panels) or enzyme-linked immunosorbent assay (ELISA) (middle panel). Bars represent mean relative messenger RNA expression and SEM (left and right panel, n = 3 and n = 4, respectively) normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and CD11c expression in each individual donor. Middle panel represents the mean protein level and SEM of 4 individual donors. Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 5 Examples of actin polarization and scoring. DC–T-cell clusters were plated on coated slides and stained for F-actin using phalloidin. For quantification, clusters of a DC and a single lymphocyte were scored on a 3-point scale as shown (0 = no actin polarization visible, 1 = partial polarization, 2 = strong polarization toward site of interactions). Gastroenterology 2012 142, 1493-1503.e6DOI: (10.1053/j.gastro.2012.02.034) Copyright © 2012 AGA Institute Terms and Conditions