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MR1 uses an endocytic pathway to activate mucosal-associated invariant T cells.

Huang S, Gilfillan S, Kim S, Thompson B, Wang X, Sant AJ, Fremont DH, Lantz O, Hansen TH - J. Exp. Med. (2008)

Bottom Line: Like CD1d-restricted iNKT cells, mucosal-associated invariant T cells (MAITs) are "innate" T cells that express a canonical TCRalpha chain, have a memory phenotype, and rapidly secrete cytokines upon TCR ligation.Furthermore, inhibiting the acidification of the endocytic compartments reduced MR1 surface expression and ablated MAIT cell activation.The importance of the late endosome for MR1 antigen presentation was further corroborated by the localization of MR1 molecules in the multivesicular endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University, St. Louis, MO 63110, USA.

ABSTRACT
Like CD1d-restricted iNKT cells, mucosal-associated invariant T cells (MAITs) are "innate" T cells that express a canonical TCRalpha chain, have a memory phenotype, and rapidly secrete cytokines upon TCR ligation. Unlike iNKT cells, MAIT cells require the class Ib molecule MHC-related protein I (MR1), B cells, and gut flora for development and/or expansion, and they preferentially reside in the gut lamina propria. Evidence strongly suggests that MAIT cell activation is ligand-dependent, but the nature of MR1 ligand is unknown. In this study, we define a mechanism of endogenous antigen presentation by MR1 to MAIT cells. MAIT cell activation was dependent neither on a proteasome-processed ligand nor on the chaperoning by the MHC class I peptide loading complex. However, MAIT cell activation was enhanced by overexpression of MHC class II chaperones Ii and DM and was strikingly diminished by silencing endogenous Ii. Furthermore, inhibiting the acidification of the endocytic compartments reduced MR1 surface expression and ablated MAIT cell activation. The importance of the late endosome for MR1 antigen presentation was further corroborated by the localization of MR1 molecules in the multivesicular endosomes. These findings demonstrate that MR1 traffics through endocytic compartments, thereby allowing MAIT cells to sample both endocytosed and endogenous antigens.

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Late endosomal distribution of MR1 molecules in B cells visualized by confocal and cryoimmunoelectron microscopy. (A) CH27.mMR1.EGFP was stained with antibodies against Lamp1 and CRT. The quantitation of colocalization between MR1 and Lamp1 or CRT was plotted to the right. The r and P values were obtained with the calculation used in Fig. 5. Staining was repeated three times with similar results. (B) Percentage of MR1 colocalization in different compartments was plotted as the mean ± the SEM (C) CH27.mMR1.EGFP were stained with rabbit anti-EGFP antibody visualized with 18-nm colloidal gold using electron microscopy. N, nuclear region. MR1 labeled by gold particles is shown at the tubular structures (arrow), plasma membrane (PM), or Golgi complex (G). (D) Some MR1 particles reside at the single vesicle (arrow), indicating an endosomal compartment. (E) More particles (arrow) localize in the multivesicular body (MVB) representing the late endosome compartments. (F) Schematic model of MR1 endocytic trafficking. MR1 egresses from the ER with either a surrogate ligand or association of Ii. The Ii facilitates MR1 traffic to late endocytic compartments to obtain a hypothetical ligand and further express onto the cell surface for MAIT cell activation. Bars: (A) 5 μm; (C–E) 200 nm.
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fig8: Late endosomal distribution of MR1 molecules in B cells visualized by confocal and cryoimmunoelectron microscopy. (A) CH27.mMR1.EGFP was stained with antibodies against Lamp1 and CRT. The quantitation of colocalization between MR1 and Lamp1 or CRT was plotted to the right. The r and P values were obtained with the calculation used in Fig. 5. Staining was repeated three times with similar results. (B) Percentage of MR1 colocalization in different compartments was plotted as the mean ± the SEM (C) CH27.mMR1.EGFP were stained with rabbit anti-EGFP antibody visualized with 18-nm colloidal gold using electron microscopy. N, nuclear region. MR1 labeled by gold particles is shown at the tubular structures (arrow), plasma membrane (PM), or Golgi complex (G). (D) Some MR1 particles reside at the single vesicle (arrow), indicating an endosomal compartment. (E) More particles (arrow) localize in the multivesicular body (MVB) representing the late endosome compartments. (F) Schematic model of MR1 endocytic trafficking. MR1 egresses from the ER with either a surrogate ligand or association of Ii. The Ii facilitates MR1 traffic to late endocytic compartments to obtain a hypothetical ligand and further express onto the cell surface for MAIT cell activation. Bars: (A) 5 μm; (C–E) 200 nm.

Mentions: To visualize the MR1 trafficking pathway and further confirm the importance of the late endosomal compartment, the fusion protein mMR1.EGFP expressed in B cells was localized using confocal microscopy in the context of ER and late endocytic markers, CRT or Lamp1, respectively. Of note, the intact mMR1 molecule on CH27.mMR1 and fusion protein on CH27.mMR1.EGFP activated MAIT cells similarly (Fig. S5, A and B, available at http://www.jem.org/cgi/content/full/jem.20072579/DC1). Microscopy examination revealed that mMR1.EGFP mainly resided in the Lamp1+ late endosome/lysosome, and the CRT+ ER (Fig. 8, A and B). Both of these associations were found to be statistically significant when assessed using Pearson's coefficient calculation (Fig. 8 A). ATPase inhibitor CMA-treated CH27.mMR1.EGFP cells displayed a “donutlike” topology of swelled late endocytic compartments with intensive mMR1.EGFP staining, further confirming that a major subcellular location of MR1 is in the late endocytic compartments (Fig. S6). In addition, immunoelectron microscopy demonstrated that MR1 primarily localizes to ER tubular structures (Fig. 8 C) and endocytic compartments (Fig. 8 D), especially late endosomes as exemplified by their multivesicular bodies (Fig. 8 E). MR1 localization in multivesicular endosomes corroborates its dysfunction upon blocking of endosomal acidification. These data demonstrate that MR1 traffics through and likely binds ligand in a late endocytic compartment, as shown in the schematic model (Fig. 8 F), but do not exclude the possibility of a direct surface expression for the activation of other MAIT cell clones.


MR1 uses an endocytic pathway to activate mucosal-associated invariant T cells.

Huang S, Gilfillan S, Kim S, Thompson B, Wang X, Sant AJ, Fremont DH, Lantz O, Hansen TH - J. Exp. Med. (2008)

Late endosomal distribution of MR1 molecules in B cells visualized by confocal and cryoimmunoelectron microscopy. (A) CH27.mMR1.EGFP was stained with antibodies against Lamp1 and CRT. The quantitation of colocalization between MR1 and Lamp1 or CRT was plotted to the right. The r and P values were obtained with the calculation used in Fig. 5. Staining was repeated three times with similar results. (B) Percentage of MR1 colocalization in different compartments was plotted as the mean ± the SEM (C) CH27.mMR1.EGFP were stained with rabbit anti-EGFP antibody visualized with 18-nm colloidal gold using electron microscopy. N, nuclear region. MR1 labeled by gold particles is shown at the tubular structures (arrow), plasma membrane (PM), or Golgi complex (G). (D) Some MR1 particles reside at the single vesicle (arrow), indicating an endosomal compartment. (E) More particles (arrow) localize in the multivesicular body (MVB) representing the late endosome compartments. (F) Schematic model of MR1 endocytic trafficking. MR1 egresses from the ER with either a surrogate ligand or association of Ii. The Ii facilitates MR1 traffic to late endocytic compartments to obtain a hypothetical ligand and further express onto the cell surface for MAIT cell activation. Bars: (A) 5 μm; (C–E) 200 nm.
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Related In: Results  -  Collection

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fig8: Late endosomal distribution of MR1 molecules in B cells visualized by confocal and cryoimmunoelectron microscopy. (A) CH27.mMR1.EGFP was stained with antibodies against Lamp1 and CRT. The quantitation of colocalization between MR1 and Lamp1 or CRT was plotted to the right. The r and P values were obtained with the calculation used in Fig. 5. Staining was repeated three times with similar results. (B) Percentage of MR1 colocalization in different compartments was plotted as the mean ± the SEM (C) CH27.mMR1.EGFP were stained with rabbit anti-EGFP antibody visualized with 18-nm colloidal gold using electron microscopy. N, nuclear region. MR1 labeled by gold particles is shown at the tubular structures (arrow), plasma membrane (PM), or Golgi complex (G). (D) Some MR1 particles reside at the single vesicle (arrow), indicating an endosomal compartment. (E) More particles (arrow) localize in the multivesicular body (MVB) representing the late endosome compartments. (F) Schematic model of MR1 endocytic trafficking. MR1 egresses from the ER with either a surrogate ligand or association of Ii. The Ii facilitates MR1 traffic to late endocytic compartments to obtain a hypothetical ligand and further express onto the cell surface for MAIT cell activation. Bars: (A) 5 μm; (C–E) 200 nm.
Mentions: To visualize the MR1 trafficking pathway and further confirm the importance of the late endosomal compartment, the fusion protein mMR1.EGFP expressed in B cells was localized using confocal microscopy in the context of ER and late endocytic markers, CRT or Lamp1, respectively. Of note, the intact mMR1 molecule on CH27.mMR1 and fusion protein on CH27.mMR1.EGFP activated MAIT cells similarly (Fig. S5, A and B, available at http://www.jem.org/cgi/content/full/jem.20072579/DC1). Microscopy examination revealed that mMR1.EGFP mainly resided in the Lamp1+ late endosome/lysosome, and the CRT+ ER (Fig. 8, A and B). Both of these associations were found to be statistically significant when assessed using Pearson's coefficient calculation (Fig. 8 A). ATPase inhibitor CMA-treated CH27.mMR1.EGFP cells displayed a “donutlike” topology of swelled late endocytic compartments with intensive mMR1.EGFP staining, further confirming that a major subcellular location of MR1 is in the late endocytic compartments (Fig. S6). In addition, immunoelectron microscopy demonstrated that MR1 primarily localizes to ER tubular structures (Fig. 8 C) and endocytic compartments (Fig. 8 D), especially late endosomes as exemplified by their multivesicular bodies (Fig. 8 E). MR1 localization in multivesicular endosomes corroborates its dysfunction upon blocking of endosomal acidification. These data demonstrate that MR1 traffics through and likely binds ligand in a late endocytic compartment, as shown in the schematic model (Fig. 8 F), but do not exclude the possibility of a direct surface expression for the activation of other MAIT cell clones.

Bottom Line: Like CD1d-restricted iNKT cells, mucosal-associated invariant T cells (MAITs) are "innate" T cells that express a canonical TCRalpha chain, have a memory phenotype, and rapidly secrete cytokines upon TCR ligation.Furthermore, inhibiting the acidification of the endocytic compartments reduced MR1 surface expression and ablated MAIT cell activation.The importance of the late endosome for MR1 antigen presentation was further corroborated by the localization of MR1 molecules in the multivesicular endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University, St. Louis, MO 63110, USA.

ABSTRACT
Like CD1d-restricted iNKT cells, mucosal-associated invariant T cells (MAITs) are "innate" T cells that express a canonical TCRalpha chain, have a memory phenotype, and rapidly secrete cytokines upon TCR ligation. Unlike iNKT cells, MAIT cells require the class Ib molecule MHC-related protein I (MR1), B cells, and gut flora for development and/or expansion, and they preferentially reside in the gut lamina propria. Evidence strongly suggests that MAIT cell activation is ligand-dependent, but the nature of MR1 ligand is unknown. In this study, we define a mechanism of endogenous antigen presentation by MR1 to MAIT cells. MAIT cell activation was dependent neither on a proteasome-processed ligand nor on the chaperoning by the MHC class I peptide loading complex. However, MAIT cell activation was enhanced by overexpression of MHC class II chaperones Ii and DM and was strikingly diminished by silencing endogenous Ii. Furthermore, inhibiting the acidification of the endocytic compartments reduced MR1 surface expression and ablated MAIT cell activation. The importance of the late endosome for MR1 antigen presentation was further corroborated by the localization of MR1 molecules in the multivesicular endosomes. These findings demonstrate that MR1 traffics through endocytic compartments, thereby allowing MAIT cells to sample both endocytosed and endogenous antigens.

Show MeSH
Related in: MedlinePlus