Volume 118, Issue 1, Pages 128-137 (January 2000) Modulation of antigen trafficking to MHC class II–positive late endosomes of enterocytes  Klaus–Peter Zimmer, Jürgen Büning, Peter Weber, Dominique Kaiserlian, Stephan Strobel  Gastroenterology  Volume 118, Issue 1, Pages 128-137 (January 2000) DOI: 10.1016/S0016-5085(00)70421-5 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 BALB/c enterocytes rapidly endocytose and transport OVA to the paracellular space. Thin frozen sections of small bowel biopsy specimens were taken from BALB/c mice after OVA administration. They are labeled by a polyclonal antibody against OVA and 12-nm large immunogold particles. Bars = 0.1 μm. (A) OVA is present on the enterocyte microvilli (Mi), within an endocytic invagination (arrow) of the apical membrane, and an early endosome (E) 5 minutes after an OVA feed. (B) OVA is detected on the microvilli and in the paracellular space 5 minutes after its administration. The junctional complex (JC) is intact. BL, basolateral membrane. (C) OVA is found in the trans-Golgi network (TGN) of enterocytes at 10 minutes after a feed. N, nucleus; G, Golgi stacks. (D) At the 10-minute time point, OVA is targeted to late endosomes (LE) containing electron-dense material. An early endosome (EE) with a low amount of OVA seems to fuse with this compartment, which is enriched with OVA. (E) Sections of biopsy specimens obtained at the 10-minute time point reveal no staining after omitting the primary anti-OVA antibody and performing the same labeling procedures as in A-D. D, desmosome; M, mitochondrion; PS, paracellular space. (F) There is negligible background labeling in biopsy specimens of unfed mice. L, lysosome. Gastroenterology 2000 118, 128-137DOI: (10.1016/S0016-5085(00)70421-5) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Rapid endocytosis and vesicular transport of OVA to the paracellular space within SCID enterocytes without detection inside the Golgi region. Thin frozen sections of small bowel biopsy specimens taken from SCID mice after an OVA feed are labeled by the polyclonal antibody against OVA and 12-nm large immunogold particles. Bars = 0.1 μm. (A) Uptake of OVA by endocytic invagination (arrowhead) of the apical membrane at 5 minutes after administration. OVA is also present on microvilli (Mi). (B) OVA is located within vesicles in the apical region of SCID enterocytes 5 minutes after a feed. Microvilli are also labeled by the OVA antibody. Because OVA, as a ligand, is present in vesicles close to the cell surface already 5 minutes after its uptake, an early endosomal (EE) nature of these structures is suggested. Lu, lumen. (C) OVA is already detected in the paracellular space (PS) 5 minutes after oral administration. The junctional complex is preserved. TJ, tight junction; M, mitochondrion; D, desmosome. (D) There is no OVA labeling within the Golgi (G) region at any time point including 10 minutes after the OVA feed within SCID enterocytes. N, nucleus. Gastroenterology 2000 118, 128-137DOI: (10.1016/S0016-5085(00)70421-5) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 OVA is targeted to LAMP-1–positive vacuoles within enterocytes of BALB/c but not SCID mice. Time-matched labeling patterns of LAMP-1 and OVA within enterocytes of (A, C, E, G) BALB/c and (B, D, F, H) SCID enterocytes. (C-H) Thin frozen sections with simultaneous labeling by the polyclonal anti-OVA and the monoclonal anti–LAMP-1 antibody. The binding sites of the OVA antibody are indicated by 6-nm small immunogold particles and those of the LAMP-1 antibody by 12-nm large immunogold particles. Bars = 0.1 μm. (A and B) LAMP-1 is localized on a semithin frozen section in the fluorescence microscope, indicating an increased number of LAMP-1–positive vacuoles within (B) SCID in contrast to (A) BALB/c enterocytes. (C and D) Five minutes after OVA administration, OVA is detectable on microvilli (Mi) and early endosomes (EE). There are no colocalization sites with LAMP-1–positive late endosomes (LE) either in (C) BALB/c or (D) SCID enterocytes. TJ, tight junction; D, desmosome; M, mitochondria. (E and F) In BALB/c enterocytes, vacuoles were found close to the paracellular space (PS) 10 minutes after an OVA feed containing OVA and LAMP-1 (E). In enterocytes of SCID mice, the LAMP-1–positive late endosomes of this ultrastructural site do not contain any OVA (F ), whereas OVA can be detected in the paracellular space (arrows). (G and H) Ten minutes after its administration, OVA is transported to LAMP-1–positive late endosomes of BALB/c enterocytes (G). It is also present in an electron-dense vacuole containing a low number of LAMP-1 binding sites that corresponds to a lysosome (L). In addition, OVA is found in a LAMP-1–negative vacuole that represents an early endosome (on the top of the micrograph). In contrast to BALB/c mice, the LAMP-1–positive late endosomes within enterocytes of SCID mice do not contain any OVA at any time after OVA administration (H). Gastroenterology 2000 118, 128-137DOI: (10.1016/S0016-5085(00)70421-5) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 OVA is targeted to LAMP-1–positive vacuoles within enterocytes of BALB/c but not SCID mice. Time-matched labeling patterns of LAMP-1 and OVA within enterocytes of (A, C, E, G) BALB/c and (B, D, F, H) SCID enterocytes. (C-H) Thin frozen sections with simultaneous labeling by the polyclonal anti-OVA and the monoclonal anti–LAMP-1 antibody. The binding sites of the OVA antibody are indicated by 6-nm small immunogold particles and those of the LAMP-1 antibody by 12-nm large immunogold particles. Bars = 0.1 μm. (A and B) LAMP-1 is localized on a semithin frozen section in the fluorescence microscope, indicating an increased number of LAMP-1–positive vacuoles within (B) SCID in contrast to (A) BALB/c enterocytes. (C and D) Five minutes after OVA administration, OVA is detectable on microvilli (Mi) and early endosomes (EE). There are no colocalization sites with LAMP-1–positive late endosomes (LE) either in (C) BALB/c or (D) SCID enterocytes. TJ, tight junction; D, desmosome; M, mitochondria. (E and F) In BALB/c enterocytes, vacuoles were found close to the paracellular space (PS) 10 minutes after an OVA feed containing OVA and LAMP-1 (E). In enterocytes of SCID mice, the LAMP-1–positive late endosomes of this ultrastructural site do not contain any OVA (F ), whereas OVA can be detected in the paracellular space (arrows). (G and H) Ten minutes after its administration, OVA is transported to LAMP-1–positive late endosomes of BALB/c enterocytes (G). It is also present in an electron-dense vacuole containing a low number of LAMP-1 binding sites that corresponds to a lysosome (L). In addition, OVA is found in a LAMP-1–negative vacuole that represents an early endosome (on the top of the micrograph). In contrast to BALB/c mice, the LAMP-1–positive late endosomes within enterocytes of SCID mice do not contain any OVA at any time after OVA administration (H). Gastroenterology 2000 118, 128-137DOI: (10.1016/S0016-5085(00)70421-5) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 Colocalization of OVA with MHC class II antigens in vacuoles of BALB/c but not SCID mice. Small bowel biopsy specimens are prepared for (A and B) semithin and (C-E ) thin frozen sections labeled by the polyclonal antibody against OVA and the monoclonal antibody against MHC class II antigens (CD311). Their binding sites were detected by a FITC-conjugated goat anti-rat antibody (A and B) and 6-nm (OVA) and 12-nm (MHC class II antigens) immunogold particles (C-E). Bars = 0.1 μm. (A) The enterocytes of the BALB/c mice are labeled with the MHC class II antibody and visualized by immunofluorescence microscopy. There is a strong reactivity of the basolateral membrane and within intracellular vacuoles. (B) Enterocytes of SCID mice are not labeled by the MHC class II antibody. The antibody only reacts with cells of the lamina propria. (C) OVA colocalizes with MHC class II antigens in late endosomes (LE) of BALB/c enterocytes 10 minutes after an OVA feed. OVA is also visible within vesicles (V) without any MHC class II antigens. A multivesicular body (MVB) contains only MHC class II antigens. This micrograph also shows that there is no cross-reactivity between the first and second labeling steps. (D) Fusion between an early endosome (EE) containing OVA and a late endosome labeled by the MHC class II antibody. The OVA-positive content of the early endosome is penetrating into the MHC class II–positive vacuole. Arrowheads indicate where the membranes of both vacuoles merge. (E) Endosomes (E) containing MHC class II antigens and OVA are occasionally found to fuse with the lateral membrane of enterocytes (arrowhead). Arrows indicate binding sites of the OVA antibody. D, desmosome; BL, basolateral membrane. (F) Enterocytes of BALB/c mice show OVA and MHC class II binding sites on the basolateral membrane in close spatial relationship (arrows). This is especially obvious where the basal membrane (B) of enterocytes (E) is in close contact to lymphocytes (L). PM, plasma membrane; N, nucleus; V, vesicle. Gastroenterology 2000 118, 128-137DOI: (10.1016/S0016-5085(00)70421-5) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 A model for the intracellular pathway of OVA within enterocytes of BALB/c and SCID mice. Five minutes after oral administration, OVA is taken up at the apical membrane of enterocytes and transported to the paracellular space by a transcellular route passing early endosomes. This transport process is observed in BALB/c and SCID mice. In contrast to SCID mice, OVA reaches the trans-Golgi network and (LAMP-1–positive) late endosomes of BALB/c enterocytes 10 minutes after the OVA feed. At this time point, OVA colocalizes with MHC class II antigens within late endosomes and on the basolateral membrane only in enterocytes of BALB/c mice. Gastroenterology 2000 118, 128-137DOI: (10.1016/S0016-5085(00)70421-5) Copyright © 2000 American Gastroenterological Association Terms and Conditions