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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.

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

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CIITA pIV controls IFN-γ–mediated up-regulation, but not steady-state expression of MHCII by LNSCs. (A) LNSCs were defined by FACS as follows: LEC, CD45− CD31+ gp38+; BEC, CD45− CD31+ gp38−; FRC, CD45− CD31− gp38+; DN, CD45− CD31− gp38−. Histograms show MHCII expression levels by each LNSC population (gray histograms, isotype control). Data are representative of 4 independent experiments with at least 3 mice each. (B) Ciita promoters pI, pIII, and pIV mRNA levels were quantified by qPCR from FACS-sorted LEC, BEC, or FRC. Indicated control cells (gray bars) were used as reference. Data are representative of 3 independent experiments, with a pool of 10 mice each. (C) Mice were injected subcutaneously (green) or not (blue) with IFN-γ and draining LNs were collected 24 h later. Histograms show MHCII expression levels on LEC, BEC, and FRC. Graphs depict MHCII MFI where each symbol represents individual mouse. Data are representative of 3 independent experiments, with 4 mice per group. ***, P < 0.001, n.s. = not significant. Error bars depict mean ± SEM. (D and E) Histograms show MHCII expression levels on LEC, BEC, and FRC from indicated mice. Histograms are representative of at least 2 independent experiments with 2–3 mice per group. (E) Graphs depict MHCII MFI where each symbol represents individual mouse. Data are pooled from 3 independent experiments with 2–3 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant. Error bars depict mean ± SEM.
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fig1: CIITA pIV controls IFN-γ–mediated up-regulation, but not steady-state expression of MHCII by LNSCs. (A) LNSCs were defined by FACS as follows: LEC, CD45− CD31+ gp38+; BEC, CD45− CD31+ gp38−; FRC, CD45− CD31− gp38+; DN, CD45− CD31− gp38−. Histograms show MHCII expression levels by each LNSC population (gray histograms, isotype control). Data are representative of 4 independent experiments with at least 3 mice each. (B) Ciita promoters pI, pIII, and pIV mRNA levels were quantified by qPCR from FACS-sorted LEC, BEC, or FRC. Indicated control cells (gray bars) were used as reference. Data are representative of 3 independent experiments, with a pool of 10 mice each. (C) Mice were injected subcutaneously (green) or not (blue) with IFN-γ and draining LNs were collected 24 h later. Histograms show MHCII expression levels on LEC, BEC, and FRC. Graphs depict MHCII MFI where each symbol represents individual mouse. Data are representative of 3 independent experiments, with 4 mice per group. ***, P < 0.001, n.s. = not significant. Error bars depict mean ± SEM. (D and E) Histograms show MHCII expression levels on LEC, BEC, and FRC from indicated mice. Histograms are representative of at least 2 independent experiments with 2–3 mice per group. (E) Graphs depict MHCII MFI where each symbol represents individual mouse. Data are pooled from 3 independent experiments with 2–3 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant. Error bars depict mean ± SEM.

Mentions: We first characterized steady-state MHCII expression by primary murine LNSCs. As previously described (Malhotra et al., 2012), LECs, BECs, and FRCs, but not DN cells (Fig. S1), expressed low basal levels of MHCII molecules (Fig. 1 A). MHCII expression is almost exclusively controlled by a single master regulatory factor, CIITA (Reith et al., 2005). Expression of CIITA is regulated mainly at the transcriptional level by a large regulatory region that contains three distinct promoters in mice, pI, pIII, and pIV (Fig. S2; Reith et al., 2005). We quantified pI, pIII, and pIV mRNA in FACS-sorted FRCs, BECs, and LECs from CD45neg-enriched LN fractions. Although pI and pIII mRNAs were undetectable, all three LNSC subpopulations expressed pIV mRNA (Fig. 1 B). Because pIV is induced by IFN-γ, we injected WT and pIV knockout (pIV−/−) mice with IFN-γ and observed that LECs, BECs, and FRCs isolated from WT mice strongly up-regulated MHCII compared with untreated WT mice (Fig. 1 C). In contrast, LECs, BECs and FRCs isolated from pIV−/− mice did not increase MHCII after IFN-γ treatment (Fig. 1 C), demonstrating that IFN-γ–mediated MHCII up-regulation by LNSC is pIV dependent. Surprisingly, although only pIV mRNA was detected in LNSC (Fig. 1 B), basal MHCII expression was slightly reduced but not abrogated in cells isolated from untreated pIV−/− mice. Similarly, this reduced but clear expression of MHCII was detectable in IFN-γR−/− LNSCs (Fig. 1 D), indicating that although steady-state IFN-γ is partially responsible for pIV-dependent basal MHCII expression, other mechanisms driving MHCII expression must also exist.


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)

CIITA pIV controls IFN-γ–mediated up-regulation, but not steady-state expression of MHCII by LNSCs. (A) LNSCs were defined by FACS as follows: LEC, CD45− CD31+ gp38+; BEC, CD45− CD31+ gp38−; FRC, CD45− CD31− gp38+; DN, CD45− CD31− gp38−. Histograms show MHCII expression levels by each LNSC population (gray histograms, isotype control). Data are representative of 4 independent experiments with at least 3 mice each. (B) Ciita promoters pI, pIII, and pIV mRNA levels were quantified by qPCR from FACS-sorted LEC, BEC, or FRC. Indicated control cells (gray bars) were used as reference. Data are representative of 3 independent experiments, with a pool of 10 mice each. (C) Mice were injected subcutaneously (green) or not (blue) with IFN-γ and draining LNs were collected 24 h later. Histograms show MHCII expression levels on LEC, BEC, and FRC. Graphs depict MHCII MFI where each symbol represents individual mouse. Data are representative of 3 independent experiments, with 4 mice per group. ***, P < 0.001, n.s. = not significant. Error bars depict mean ± SEM. (D and E) Histograms show MHCII expression levels on LEC, BEC, and FRC from indicated mice. Histograms are representative of at least 2 independent experiments with 2–3 mice per group. (E) Graphs depict MHCII MFI where each symbol represents individual mouse. Data are pooled from 3 independent experiments with 2–3 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant. Error bars depict mean ± SEM.
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fig1: CIITA pIV controls IFN-γ–mediated up-regulation, but not steady-state expression of MHCII by LNSCs. (A) LNSCs were defined by FACS as follows: LEC, CD45− CD31+ gp38+; BEC, CD45− CD31+ gp38−; FRC, CD45− CD31− gp38+; DN, CD45− CD31− gp38−. Histograms show MHCII expression levels by each LNSC population (gray histograms, isotype control). Data are representative of 4 independent experiments with at least 3 mice each. (B) Ciita promoters pI, pIII, and pIV mRNA levels were quantified by qPCR from FACS-sorted LEC, BEC, or FRC. Indicated control cells (gray bars) were used as reference. Data are representative of 3 independent experiments, with a pool of 10 mice each. (C) Mice were injected subcutaneously (green) or not (blue) with IFN-γ and draining LNs were collected 24 h later. Histograms show MHCII expression levels on LEC, BEC, and FRC. Graphs depict MHCII MFI where each symbol represents individual mouse. Data are representative of 3 independent experiments, with 4 mice per group. ***, P < 0.001, n.s. = not significant. Error bars depict mean ± SEM. (D and E) Histograms show MHCII expression levels on LEC, BEC, and FRC from indicated mice. Histograms are representative of at least 2 independent experiments with 2–3 mice per group. (E) Graphs depict MHCII MFI where each symbol represents individual mouse. Data are pooled from 3 independent experiments with 2–3 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s. = not significant. Error bars depict mean ± SEM.
Mentions: We first characterized steady-state MHCII expression by primary murine LNSCs. As previously described (Malhotra et al., 2012), LECs, BECs, and FRCs, but not DN cells (Fig. S1), expressed low basal levels of MHCII molecules (Fig. 1 A). MHCII expression is almost exclusively controlled by a single master regulatory factor, CIITA (Reith et al., 2005). Expression of CIITA is regulated mainly at the transcriptional level by a large regulatory region that contains three distinct promoters in mice, pI, pIII, and pIV (Fig. S2; Reith et al., 2005). We quantified pI, pIII, and pIV mRNA in FACS-sorted FRCs, BECs, and LECs from CD45neg-enriched LN fractions. Although pI and pIII mRNAs were undetectable, all three LNSC subpopulations expressed pIV mRNA (Fig. 1 B). Because pIV is induced by IFN-γ, we injected WT and pIV knockout (pIV−/−) mice with IFN-γ and observed that LECs, BECs, and FRCs isolated from WT mice strongly up-regulated MHCII compared with untreated WT mice (Fig. 1 C). In contrast, LECs, BECs and FRCs isolated from pIV−/− mice did not increase MHCII after IFN-γ treatment (Fig. 1 C), demonstrating that IFN-γ–mediated MHCII up-regulation by LNSC is pIV dependent. Surprisingly, although only pIV mRNA was detected in LNSC (Fig. 1 B), basal MHCII expression was slightly reduced but not abrogated in cells isolated from untreated pIV−/− mice. Similarly, this reduced but clear expression of MHCII was detectable in IFN-γR−/− LNSCs (Fig. 1 D), indicating that although steady-state IFN-γ is partially responsible for pIV-dependent basal MHCII expression, other mechanisms driving MHCII expression must also exist.

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