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Lymph node stromal cells acquire peptide-MHCII complexes from dendritic cells and induce antigen-specific CD4⁺ T cell tolerance.

Dubrot J, Duraes FV, Potin L, Capotosti F, Brighouse D, Suter T, LeibundGut-Landmann S, Garbi N, Reith W, Swartz MA, Hugues S - J. Exp. Med. (2014)

Bottom Line: Although LNSCs express MHCII, it is unknown whether they can also impact CD4(+) T cell functions.We show that the promoter IV (pIV) of class II transactivator (CIITA), the master regulator of MHCII expression, controls endogenous MHCII expression by LNSCs.Our data reveals a novel, alternative mechanism where LN-resident stromal cells tolerize CD4(+) T cells through the presentation of self-antigens via transferred peptide-MHCII complexes of DC origin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Immunology, University of Geneva Medical School, 1211 Geneva, Switzerland.

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Mechanisms of MHCII transfer. (A) LECs/FRCs were co-cultured overnight with BMDCs previously treated (DC) or not with PFA (PFA DC). Histograms show MHCII levels on LEC and FRC. Data are representative of 3 independent experiments. (B) LECs/FRCs were co-cultured overnight with BMDCs in the same compartment or in two compartments separated by a transwell (TW) membrane. Histograms show MHCII levels on LEC and FRC. Graphs show MHCII MFI on LEC (left) and FRC (right). Data are representative of 3 independent experiments. Error bars depict mean ± SEM. Each symbol represents experimental replicates. (C) Expression of MHCII, CD80, CD86, and CD40 by DC-derived exosomes. Histograms are representative of 2 independent experiments. (D) 5 × 104 LECs/FRCs were co-cultured overnight with 5 × 104 (1:1) LPS-treated BMDCs or exosomes purified from 106 (1:20), 2 × 106 (1:40), or 3 × 106 (1:60) LPS-treated BMDCs. Data show MHCII MFI on LEC and FRC and are representative of 2 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant. Error bars depict mean ± SEM. Each symbol represents experimental replicates. (E) Levels of CD80, CD86, CD40, and CD11c expression by LEC and FRC cultured alone or co-cultured overnight with LPS-treated BMDCs. Red histograms represent expression of indicated molecules by LEC and FRC compared with isotype (gray histograms). Dotted histograms represent expression of the molecules by BMDCs. Data are representative of 3 independent experiments. (F) Expression of CD80, CD86, CD40, and CD11c by LEC, BEC, and FRC isolated from LN (red histograms) compared with isotype (gray histograms). Histograms are representative of 2 independent experiments with 2–4 mice per group.
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fig5: Mechanisms of MHCII transfer. (A) LECs/FRCs were co-cultured overnight with BMDCs previously treated (DC) or not with PFA (PFA DC). Histograms show MHCII levels on LEC and FRC. Data are representative of 3 independent experiments. (B) LECs/FRCs were co-cultured overnight with BMDCs in the same compartment or in two compartments separated by a transwell (TW) membrane. Histograms show MHCII levels on LEC and FRC. Graphs show MHCII MFI on LEC (left) and FRC (right). Data are representative of 3 independent experiments. Error bars depict mean ± SEM. Each symbol represents experimental replicates. (C) Expression of MHCII, CD80, CD86, and CD40 by DC-derived exosomes. Histograms are representative of 2 independent experiments. (D) 5 × 104 LECs/FRCs were co-cultured overnight with 5 × 104 (1:1) LPS-treated BMDCs or exosomes purified from 106 (1:20), 2 × 106 (1:40), or 3 × 106 (1:60) LPS-treated BMDCs. Data show MHCII MFI on LEC and FRC and are representative of 2 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant. Error bars depict mean ± SEM. Each symbol represents experimental replicates. (E) Levels of CD80, CD86, CD40, and CD11c expression by LEC and FRC cultured alone or co-cultured overnight with LPS-treated BMDCs. Red histograms represent expression of indicated molecules by LEC and FRC compared with isotype (gray histograms). Dotted histograms represent expression of the molecules by BMDCs. Data are representative of 3 independent experiments. (F) Expression of CD80, CD86, CD40, and CD11c by LEC, BEC, and FRC isolated from LN (red histograms) compared with isotype (gray histograms). Histograms are representative of 2 independent experiments with 2–4 mice per group.

Mentions: To investigate whether MHCII transfer from DCs to LNSCs relies on active processes, isolated LN LEC/FRC cultures were co-cultured with paraformaldehyde (PFA)-pretreated DCs. These paralyzed DCs entirely lost their ability to transfer MHCII to LECs and FRCs (Fig. 5 A), suggesting an active transfer of MHCII from donor to recipient cells. To investigate whether this process was cell–cell contact dependent, we co-cultured DCs and LEC/FRCs in different compartments separated by a culture insert membrane, and observed a dramatic reduction of MHCII expression on LECs and FRCs (Fig. 5 B). However, small amounts of MHCII were detectable on these cells (Fig. 5 B), suggesting that DC-derived vesicles might also be involved in transferring MHCII molecules in vitro. Indeed, DC-derived exosomes have been previously described to mediate intercellular transfer of surface proteins (Davis, 2007). To determine whether exosomes were involved in the process of MHCII transfer from DCs to LNSCs, we isolated and purified exosomes from cultured DCs as previously described (Théry et al., 2002). We found that DC-derived exosomes were positive for MHCII expression (Fig. 5 C). When DC-derived exosomes were added to LEC/FRC cultures, MHCII expression increased in a dose-dependent manner on both LECs and FRCs (Fig. 5 D), but to a lesser extent compared with DCs (Fig. 5 D).


Lymph node stromal cells acquire peptide-MHCII complexes from dendritic cells and induce antigen-specific CD4⁺ T cell tolerance.

Dubrot J, Duraes FV, Potin L, Capotosti F, Brighouse D, Suter T, LeibundGut-Landmann S, Garbi N, Reith W, Swartz MA, Hugues S - J. Exp. Med. (2014)

Mechanisms of MHCII transfer. (A) LECs/FRCs were co-cultured overnight with BMDCs previously treated (DC) or not with PFA (PFA DC). Histograms show MHCII levels on LEC and FRC. Data are representative of 3 independent experiments. (B) LECs/FRCs were co-cultured overnight with BMDCs in the same compartment or in two compartments separated by a transwell (TW) membrane. Histograms show MHCII levels on LEC and FRC. Graphs show MHCII MFI on LEC (left) and FRC (right). Data are representative of 3 independent experiments. Error bars depict mean ± SEM. Each symbol represents experimental replicates. (C) Expression of MHCII, CD80, CD86, and CD40 by DC-derived exosomes. Histograms are representative of 2 independent experiments. (D) 5 × 104 LECs/FRCs were co-cultured overnight with 5 × 104 (1:1) LPS-treated BMDCs or exosomes purified from 106 (1:20), 2 × 106 (1:40), or 3 × 106 (1:60) LPS-treated BMDCs. Data show MHCII MFI on LEC and FRC and are representative of 2 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant. Error bars depict mean ± SEM. Each symbol represents experimental replicates. (E) Levels of CD80, CD86, CD40, and CD11c expression by LEC and FRC cultured alone or co-cultured overnight with LPS-treated BMDCs. Red histograms represent expression of indicated molecules by LEC and FRC compared with isotype (gray histograms). Dotted histograms represent expression of the molecules by BMDCs. Data are representative of 3 independent experiments. (F) Expression of CD80, CD86, CD40, and CD11c by LEC, BEC, and FRC isolated from LN (red histograms) compared with isotype (gray histograms). Histograms are representative of 2 independent experiments with 2–4 mice per group.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig5: Mechanisms of MHCII transfer. (A) LECs/FRCs were co-cultured overnight with BMDCs previously treated (DC) or not with PFA (PFA DC). Histograms show MHCII levels on LEC and FRC. Data are representative of 3 independent experiments. (B) LECs/FRCs were co-cultured overnight with BMDCs in the same compartment or in two compartments separated by a transwell (TW) membrane. Histograms show MHCII levels on LEC and FRC. Graphs show MHCII MFI on LEC (left) and FRC (right). Data are representative of 3 independent experiments. Error bars depict mean ± SEM. Each symbol represents experimental replicates. (C) Expression of MHCII, CD80, CD86, and CD40 by DC-derived exosomes. Histograms are representative of 2 independent experiments. (D) 5 × 104 LECs/FRCs were co-cultured overnight with 5 × 104 (1:1) LPS-treated BMDCs or exosomes purified from 106 (1:20), 2 × 106 (1:40), or 3 × 106 (1:60) LPS-treated BMDCs. Data show MHCII MFI on LEC and FRC and are representative of 2 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant. Error bars depict mean ± SEM. Each symbol represents experimental replicates. (E) Levels of CD80, CD86, CD40, and CD11c expression by LEC and FRC cultured alone or co-cultured overnight with LPS-treated BMDCs. Red histograms represent expression of indicated molecules by LEC and FRC compared with isotype (gray histograms). Dotted histograms represent expression of the molecules by BMDCs. Data are representative of 3 independent experiments. (F) Expression of CD80, CD86, CD40, and CD11c by LEC, BEC, and FRC isolated from LN (red histograms) compared with isotype (gray histograms). Histograms are representative of 2 independent experiments with 2–4 mice per group.
Mentions: To investigate whether MHCII transfer from DCs to LNSCs relies on active processes, isolated LN LEC/FRC cultures were co-cultured with paraformaldehyde (PFA)-pretreated DCs. These paralyzed DCs entirely lost their ability to transfer MHCII to LECs and FRCs (Fig. 5 A), suggesting an active transfer of MHCII from donor to recipient cells. To investigate whether this process was cell–cell contact dependent, we co-cultured DCs and LEC/FRCs in different compartments separated by a culture insert membrane, and observed a dramatic reduction of MHCII expression on LECs and FRCs (Fig. 5 B). However, small amounts of MHCII were detectable on these cells (Fig. 5 B), suggesting that DC-derived vesicles might also be involved in transferring MHCII molecules in vitro. Indeed, DC-derived exosomes have been previously described to mediate intercellular transfer of surface proteins (Davis, 2007). To determine whether exosomes were involved in the process of MHCII transfer from DCs to LNSCs, we isolated and purified exosomes from cultured DCs as previously described (Théry et al., 2002). We found that DC-derived exosomes were positive for MHCII expression (Fig. 5 C). When DC-derived exosomes were added to LEC/FRC cultures, MHCII expression increased in a dose-dependent manner on both LECs and FRCs (Fig. 5 D), but to a lesser extent compared with DCs (Fig. 5 D).

Bottom Line: Although LNSCs express MHCII, it is unknown whether they can also impact CD4(+) T cell functions.We show that the promoter IV (pIV) of class II transactivator (CIITA), the master regulator of MHCII expression, controls endogenous MHCII expression by LNSCs.Our data reveals a novel, alternative mechanism where LN-resident stromal cells tolerize CD4(+) T cells through the presentation of self-antigens via transferred peptide-MHCII complexes of DC origin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Immunology, University of Geneva Medical School, 1211 Geneva, Switzerland.

Show MeSH
Related in: MedlinePlus