Volume 124, Issue 5, Pages 1420-1431 (May 2003) Delineation of a CD1d-restricted antigen presentation pathway associated with human and mouse intestinal epithelial cells  Yvonne van de Wal, Nadia Corazza, Matthieu Allez, Lloyd F Mayer, Hideki Iijima, Mark Ryan, Steven Cornwall, Dominique Kaiserlian, Robert Hershberg, Yasuhiko Koezuka, Sean P Colgan, Richard S Blumberg  Gastroenterology  Volume 124, Issue 5, Pages 1420-1431 (May 2003) DOI: 10.1016/S0016-5085(03)00219-1

Figure 1 CD1d and MHC class I expression by IEC MODE-K cells. (A) MODE-K cells were stained with either the rat-anti-mouse CD1d-specific mAb 3C11 or a mouse class I-specific mAb for comparison for 30 minutes at 4°C in PBS containing 2% FBS (staining buffer) either in the absence (−) or presence (+) of 1% saponin (for intracellular staining). Cells were washed twice in staining buffer, after which the secondary Ab was added for 30 minutes at 4°C. Cells were washed twice and subsequently fixed with 1% paraformaldehyde in PBS. Analysis was performed on a FACScan flow cytometer (Becton Dickinson). Closed histogram represents staining with the control rat and mouse antibodies in the case of the CD1d and MHC class I staining, respectively. Specific staining is indicated by the open histogram. Note the expression of CD1d by MODE-K cells. (B) MODE-K, spleen mononuclear cells, and PECs were stained with the mouse anti-mouse CD1d-specific mAb, 1B1 (open histogram), in comparison with an isotype-matched control mAb (closed histogram). In the case of the spleen mononuclear cells, staining is shown after gating on the B-cell marker, CD19. Note that the mean fluorescence intensity (MFI) of staining for CD1d on spleen B cells (MFI = 38.8 vs. 9.8 for control mAb) and PECs (MFI = 36.7 vs. 13.9 for control mAb) is 3-fold greater than MODE-K cells (12.9 vs. 9.5 for control mAb). Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)

Figure 2 Dose-dependent recognition of αGalCer, but not βGalCer, by the mouse invariant NK-T cell hybridoma DN32.D3 using mouse MODE-K cells as APC. A total of 105 MODE-K cells were pulsed overnight with either α or βGalCer in a 96-well, flat-bottom plate at the indicated concentrations, and washed twice with PBS preceding the addition of 50,000 T cells. IL-2 concentration in the culture supernatants was determined after a 24-hour incubation by ELISA. Note the dose-dependent increase in IL-2 production with αGalCer, but not βGalCer. Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)

Figure 3 The mouse NK-T cell hybridoma DN32.D3 can recognize human CD1d molecules in the presence of αGalCer and β2m. A total of 105 FO-1 cells were pulsed overnight with 0.1 μg/mL of either αGalCer or βGalCer in a 96-well, flat-bottom plate, and washed twice with PBS preceding the addition of 50,000 T cells. IL-2 concentration in the culture supernatants was determined after a 24-hour incubation by ELISA. No recognition of αGalCer by the invariant NK-T cell hybridoma was observed using the wild-type melanoma cell line FO-1 (which lacks both endogenous CD1d and β2m). The T-cell hybridoma was found not to significantly respond to the FO-1 CD1d single transfectant, FO-1d. However, double transfection of FO-1 with both CD1d and β2m (FO-1d/β2m), resulted in efficient T-cell recognition in the presence of αGalCer. Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)

Figure 4 CD1d-transfected human IEC T84 (T84d) and freshly isolated human IECs, but not untransfected T84, can present αGalCer to the mouse invariant NK-T cell hybridoma DN32.D3. (A) A total of 105 cells were pulsed overnight with 0.1 μg/mL of either αGalCer or βGalCer in a 96-well flat-bottom plate, washed twice with PBS, and incubated with either media or 51.1.3 anti-human CD1d mAb for 2 hours at 37°C preceding the addition of 50,000 T cells. IL-2 concentration in the culture supernatants was determined after a 24-hour incubation by ELISA. Note recognition of αGalCer by the DN32.D3 clone when presented by the T84d cell line, but not the T84 cell line, and its blockade by the 51.1.3 mAb (∗P = 0.005 for αGalCer vs. αGalCer plus 51.1.3 mAb). (B) A total of 105 freshly isolated human IECs were pulsed for 12 hours with αGalCer at the indicated concentrations (100 and 400 ng/mL), and washed with PBS preceding the addition of 105 NK-T cells (DN32.D3) as indicated and mIL-2 concentrations determined in the supernatants after 24 hours. Note the dose-dependent presentation of αGalCer to the DN32.D3 cell line by fresh IECs in comparison with the T84d cell line. (C) Experimental protocol was established as in panel A with freshly isolated human IECs in the presence or absence of either the 51.1.3 (anti-CD1d), W6/32 (anti-MHC class I), or IgG2b (control) mAbs. Note the inhibition by the 51.1.3 but not the W6/32 mAbs (∗P ≤ 0.001). Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)

Figure 5 Basolateral greater than apical presentation of αGalCer to invariant NK-T cells. Stimulation of the NK-T cell hybridoma DN32.D3 by T84 transfectants T84d and T84d/CIITA on transwells pulsed with 0.1 μg/mL of either αGalCer or βGalCer on the apical or basolateral surface as indicated. Incubation with antigens was performed overnight. Subsequently, the transwells were washed twice with PBS, and incubated with either media or 51.1.3 anti-human CD1d mAb for 2 hours at 37°C preceding the addition of 50,000 T cells. After a 24-hour coculture, culture supernatants of the upper chamber were harvested and IL-2 content was determined by ELISA. Note the significant production of IL-2 when the T cells were exposed to αGalCer on the basolateral and less so on the apical cell surface. , T cells; , αGalCer antigen in the schematic diagrams describing the experimental setups (∗P = 0.006 for T84d ± 51.3.3 mAb; ∗P = 0.02 for T84d/CIITA ± 51.1.3 mAb). Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)

Figure 5 Basolateral greater than apical presentation of αGalCer to invariant NK-T cells. Stimulation of the NK-T cell hybridoma DN32.D3 by T84 transfectants T84d and T84d/CIITA on transwells pulsed with 0.1 μg/mL of either αGalCer or βGalCer on the apical or basolateral surface as indicated. Incubation with antigens was performed overnight. Subsequently, the transwells were washed twice with PBS, and incubated with either media or 51.1.3 anti-human CD1d mAb for 2 hours at 37°C preceding the addition of 50,000 T cells. After a 24-hour coculture, culture supernatants of the upper chamber were harvested and IL-2 content was determined by ELISA. Note the significant production of IL-2 when the T cells were exposed to αGalCer on the basolateral and less so on the apical cell surface. , T cells; , αGalCer antigen in the schematic diagrams describing the experimental setups (∗P = 0.006 for T84d ± 51.3.3 mAb; ∗P = 0.02 for T84d/CIITA ± 51.1.3 mAb). Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)

Figure 5 Basolateral greater than apical presentation of αGalCer to invariant NK-T cells. Stimulation of the NK-T cell hybridoma DN32.D3 by T84 transfectants T84d and T84d/CIITA on transwells pulsed with 0.1 μg/mL of either αGalCer or βGalCer on the apical or basolateral surface as indicated. Incubation with antigens was performed overnight. Subsequently, the transwells were washed twice with PBS, and incubated with either media or 51.1.3 anti-human CD1d mAb for 2 hours at 37°C preceding the addition of 50,000 T cells. After a 24-hour coculture, culture supernatants of the upper chamber were harvested and IL-2 content was determined by ELISA. Note the significant production of IL-2 when the T cells were exposed to αGalCer on the basolateral and less so on the apical cell surface. , T cells; , αGalCer antigen in the schematic diagrams describing the experimental setups (∗P = 0.006 for T84d ± 51.3.3 mAb; ∗P = 0.02 for T84d/CIITA ± 51.1.3 mAb). Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)

Gastroenterology 2003 124, 1420-1431DOI: (10 Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)

Gastroenterology 2003 124, 1420-1431DOI: (10 Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)

Figure 6 IECs can present but not process glycolipid antigens. (A) C1R cells transfected with CD1d that were either fixed (hatched bar) or unfixed (closed bar) with paraformaldehyde as described in the Materials and Methods section, and incubated with 100 ng/mL αGalCer, which does not require antigen processing, or 2 glycolipid antigens that do require processing for presentation to the DN32.D3 cell line (GalNAC [α1-3]Glc [α1-2] Galcer and Gal[α1-2]GalCer). The nonfunctional glycolipid antigen, βGalCer, served as a negative control. Note the ability of the C1R-CD1d cell line to present αGalCer but not the other diglyceride glycolipid antigens after fixation. (B) Stimulation of the invariant NK-T cell hybridoma DN32.D3 by the IEC MODE-K cell line loaded with either αGalCer (0.1 μg/mL), βGalCer (0.1 μg/mL), Gal(α1-6)GalCer (1 μg/mL), or Gal(α1-2)GalCer (1 μg/mL) as previously described.42 The latter glycolipid Ag is known to require internalization and processing by the lysosomal enzyme, α-galactosidase, to stimulate invariant NK-T cells.42 For loading, 105 IECs were pulsed overnight with the indicated glycolipid antigens in a 96-well, flat-bottom plate at the indicated concentrations, washed twice with PBS, and subsequently incubated with either media or blocking mAb solutions (20 μg/mL of the mouse CD1d-specific 19G11 mAb) for 2 hours at 37°C preceding the addition of 50,000 T cells. IL-2 concentration in the culture supernatants was determined after a 24-hour incubation by ELISA. Note the recognition of αGalCer and Gal(α1-6)GalCer but not of either βGalCer or Gal(α1-2)GalCer and its blockade by the CD1d-specific mAb (∗P = 0.001 for αGalCer ± 19G11; ∗P = 0.0006 for Gal(α1-6)GalCer ± 19G11). (C) Similar type of analysis as in panel A for the T84d/HLA-DR4 transfectant (∗P = 0.033 for αGalCer ± 51.3.1 mAb; ∗P = 0.05 for Gal(α1-6)GalCer ± 51.1.3 mAb). (D) T84d and T84/CIITA transfectants analyzed similarly to panel A. Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)

Figure 7 Efficient glycolipid-specific, CD1d-restricted recognition by fresh mouse liver T cells presented by MODE-K cells. Liver T cells were isolated from C57/BL6 mice that had been injected twice with αGalCer (2 μg per mouse intraperitoneally) to increase the proportion of invariant NK-T cells. Subsequently, these cells were tested against a panel of glycolipid Ags. A total of 105 MODE-K cells were loaded overnight with either αGalCer (0.1 μg/mL), βGalCer (0.1 μg/mL), Gal(α1-6)GalCer (1 μg/mL), or Gal(α1-2)GalCer (1 μg/mL) in a 96-well, flat-bottom plate, washed twice with PBS, and subsequently incubated with either media or 20 μg/mL of the mouse CD1d-specific mAb 19G11 for 2 hours at 37°C, preceding the addition of 250,000 fresh mouse liver T cells. Mouse IL-4 (mIL-4) concentration in the culture supernatants was determined after a 24-hour incubation by ELISA. Note recognition of αGalCer and Gal(α1-6)GalCer but not βGalCer or Gal(α1-2)GalCer by the fresh T cells when presented by MODE-K cells, and blockade of the recognition by the anti-mouse-CD1d-specific 19G11 mAb (∗P = 0.0017 for αGalCer ± 19G11 mAb; ∗P = 0.034 for Gal(α1-6)GalCer ± 19G11 mAb). Gastroenterology 2003 124, 1420-1431DOI: (10.1016/S0016-5085(03)00219-1)