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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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LNSCs acquire MHCII molecules from hematopoietic cells. (A) MHCII MFI on splenic macrophages and BMDCs from WT, pI−/−, and CIITA−/− mice. Error bars indicate mean + SEM. Data are representative of 4 independent experiments, with 2–3 mice per group. *, P < 0.05; ***, P < 0.001. (B) MHCII MFI on LEC, BEC, and FRC from indicated control mice and WT:MHCII−/− or MHCII−/−:pIV−/− BM chimeric mice which were generated as described in Materials and methods. Graphs are representative of 3 independent experiments with 5 mice per group. ***, P < 0.001. Error bars depict mean ± SEM. (C) Histograms show GFP expression gated on MHCII+ LEC, BEC, and FRC from indicated BM chimeric mice generated in MHCII−/− recipients. Graphs are representative of 2 independent experiments, with 3–4 mice per group. *, P < 0.05; **, P < 0.01. Error bars depict mean ± SEM. (D) Mice were either sublethally irradiated or untreated, and LNs harvested 3 d later. Data show MHCII MFI on LEC, BEC, and FRC and are representative of 2 independent experiments, with 7 mice per group. Each symbol represents an individual mouse. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars depict mean ± SEM. (E) LN cells were cultured and nonadherent hematopoietic cells were removed daily. LEC and FRC were analyzed at indicated times for MHCII expression. Data are representative of at least 3 independent experiments with minimum 3 mice per group.
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fig2: LNSCs acquire MHCII molecules from hematopoietic cells. (A) MHCII MFI on splenic macrophages and BMDCs from WT, pI−/−, and CIITA−/− mice. Error bars indicate mean + SEM. Data are representative of 4 independent experiments, with 2–3 mice per group. *, P < 0.05; ***, P < 0.001. (B) MHCII MFI on LEC, BEC, and FRC from indicated control mice and WT:MHCII−/− or MHCII−/−:pIV−/− BM chimeric mice which were generated as described in Materials and methods. Graphs are representative of 3 independent experiments with 5 mice per group. ***, P < 0.001. Error bars depict mean ± SEM. (C) Histograms show GFP expression gated on MHCII+ LEC, BEC, and FRC from indicated BM chimeric mice generated in MHCII−/− recipients. Graphs are representative of 2 independent experiments, with 3–4 mice per group. *, P < 0.05; **, P < 0.01. Error bars depict mean ± SEM. (D) Mice were either sublethally irradiated or untreated, and LNs harvested 3 d later. Data show MHCII MFI on LEC, BEC, and FRC and are representative of 2 independent experiments, with 7 mice per group. Each symbol represents an individual mouse. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars depict mean ± SEM. (E) LN cells were cultured and nonadherent hematopoietic cells were removed daily. LEC and FRC were analyzed at indicated times for MHCII expression. Data are representative of at least 3 independent experiments with minimum 3 mice per group.

Mentions: Even though pI mRNA was undetectable in WT LNSCs, pI−/− LNSCs exhibited a dramatic decrease in MHCII expression. We hypothesized that although LNSCs endogenously up-regulated MHCII molecules in an IFN-γ–inducible pIV-dependent manner (Fig. 1 C), they may also acquire MHCII from hematopoietic cells under steady state. Accordingly, MHCII expression by macrophages and DCs was not totally abrogated in pI−/− mice (Fig. 2 A) and may explain the remaining low MHCII expression by pI−/− LNSCs (Fig. 1 E). An alternative explanation is that pI−/− mice are pIV sufficient, and as mentioned previously, low levels of IFN-γ in vivo promote slight endogenous MHCII expression by LNSCs (Fig. 1 D). The hypothesis of MHCII acquisition by LNSCs from hematopoietic cells was tested by generating BM chimeric mice in which only the hematopoietic cell compartment or, conversely, the radioresistant stromal cells originate from WT. As mentioned above (Fig. 1), control pIV−/− LNSCs exhibited a reduced but significant MHCII expression compared with WT LNSCs (Fig. 2 B). Because there is no endogenous MHCII expression in pIV−/− LNSCs, we considered that this expression reflects only acquired MHCII molecules. Consistently, MHCII expression by LNSCs from WT BM: MHCII−/− chimeric mice was restored to similar levels compared with pIV−/− LNSCs, demonstrating that steady-state MHCII at LNSC surfaces were acquired from hematopoietic cells (Fig. 2 B). An almost total lack of MHCII expression by LNSCs from MHCII−/− BM: pIV−/− chimeric mice was observed (Fig. 2 B), suggesting that LNSCs do acquire MHCII from hematopoietic cells. In addition, we reconstituted irradiated MHCII−/− recipient mice with a mixture (1:1) of WT and I-Ab GFP+ BM cells. Levels of GFP expression by MHCII+ LECs, BECs, and FRCs were intermediate in mixed BM chimeras compared with control MHCII−/− recipient mice reconstituted with exclusively WT or I-Ab GFP+ BM cells (Fig. 2 C). Thus, LNSCs from mixed BM chimeric mice express both nonfluorescent (from WT BM) and GFP+ (from I-Ab GFP+ BM) MHCII molecules, reinforcing the idea that nonhematopoietic radioresistant LECs, BECs, and FRCs acquire MHCII from hematopoietic cells. To further test our hypothesis, we sublethally irradiated WT mice to eliminate hematopoietic cells. pIV−/− mice were included to avoid any possible endogenous MHCII up-regulation after irradiation-induced inflammation. 3 d after irradiation, the number of hematopoietic cells in LN was dramatically reduced (not depicted) and correlated with a significant decrease in MHCII expression by both WT and pIV−/− LNSCs (Fig. 2 D). These results indicated that LNSCs transiently acquire MHCII molecules from hematopoietic cells in vivo. To analyze this phenomenon in vitro, we generated LEC/FRC cultures (Lukacs-Kornek et al., 2011). In brief, LN cells were cultured for 5–7 d and washed daily to remove nonadherent hematopoietic cells. Consistent with our hypothesis that LNSCs acquire most MHCII from hematopoietic cells in steady state, removal of hematopoietic cells correlated with the disappearance of MHCII expression levels on LECs and FRCs over time (Fig. 2 E). This also reinforced our in vivo results showing that transferred MHCII are not stable at the surface of LNSC (Fig. 2 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)

LNSCs acquire MHCII molecules from hematopoietic cells. (A) MHCII MFI on splenic macrophages and BMDCs from WT, pI−/−, and CIITA−/− mice. Error bars indicate mean + SEM. Data are representative of 4 independent experiments, with 2–3 mice per group. *, P < 0.05; ***, P < 0.001. (B) MHCII MFI on LEC, BEC, and FRC from indicated control mice and WT:MHCII−/− or MHCII−/−:pIV−/− BM chimeric mice which were generated as described in Materials and methods. Graphs are representative of 3 independent experiments with 5 mice per group. ***, P < 0.001. Error bars depict mean ± SEM. (C) Histograms show GFP expression gated on MHCII+ LEC, BEC, and FRC from indicated BM chimeric mice generated in MHCII−/− recipients. Graphs are representative of 2 independent experiments, with 3–4 mice per group. *, P < 0.05; **, P < 0.01. Error bars depict mean ± SEM. (D) Mice were either sublethally irradiated or untreated, and LNs harvested 3 d later. Data show MHCII MFI on LEC, BEC, and FRC and are representative of 2 independent experiments, with 7 mice per group. Each symbol represents an individual mouse. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars depict mean ± SEM. (E) LN cells were cultured and nonadherent hematopoietic cells were removed daily. LEC and FRC were analyzed at indicated times for MHCII expression. Data are representative of at least 3 independent experiments with minimum 3 mice per group.
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fig2: LNSCs acquire MHCII molecules from hematopoietic cells. (A) MHCII MFI on splenic macrophages and BMDCs from WT, pI−/−, and CIITA−/− mice. Error bars indicate mean + SEM. Data are representative of 4 independent experiments, with 2–3 mice per group. *, P < 0.05; ***, P < 0.001. (B) MHCII MFI on LEC, BEC, and FRC from indicated control mice and WT:MHCII−/− or MHCII−/−:pIV−/− BM chimeric mice which were generated as described in Materials and methods. Graphs are representative of 3 independent experiments with 5 mice per group. ***, P < 0.001. Error bars depict mean ± SEM. (C) Histograms show GFP expression gated on MHCII+ LEC, BEC, and FRC from indicated BM chimeric mice generated in MHCII−/− recipients. Graphs are representative of 2 independent experiments, with 3–4 mice per group. *, P < 0.05; **, P < 0.01. Error bars depict mean ± SEM. (D) Mice were either sublethally irradiated or untreated, and LNs harvested 3 d later. Data show MHCII MFI on LEC, BEC, and FRC and are representative of 2 independent experiments, with 7 mice per group. Each symbol represents an individual mouse. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Error bars depict mean ± SEM. (E) LN cells were cultured and nonadherent hematopoietic cells were removed daily. LEC and FRC were analyzed at indicated times for MHCII expression. Data are representative of at least 3 independent experiments with minimum 3 mice per group.
Mentions: Even though pI mRNA was undetectable in WT LNSCs, pI−/− LNSCs exhibited a dramatic decrease in MHCII expression. We hypothesized that although LNSCs endogenously up-regulated MHCII molecules in an IFN-γ–inducible pIV-dependent manner (Fig. 1 C), they may also acquire MHCII from hematopoietic cells under steady state. Accordingly, MHCII expression by macrophages and DCs was not totally abrogated in pI−/− mice (Fig. 2 A) and may explain the remaining low MHCII expression by pI−/− LNSCs (Fig. 1 E). An alternative explanation is that pI−/− mice are pIV sufficient, and as mentioned previously, low levels of IFN-γ in vivo promote slight endogenous MHCII expression by LNSCs (Fig. 1 D). The hypothesis of MHCII acquisition by LNSCs from hematopoietic cells was tested by generating BM chimeric mice in which only the hematopoietic cell compartment or, conversely, the radioresistant stromal cells originate from WT. As mentioned above (Fig. 1), control pIV−/− LNSCs exhibited a reduced but significant MHCII expression compared with WT LNSCs (Fig. 2 B). Because there is no endogenous MHCII expression in pIV−/− LNSCs, we considered that this expression reflects only acquired MHCII molecules. Consistently, MHCII expression by LNSCs from WT BM: MHCII−/− chimeric mice was restored to similar levels compared with pIV−/− LNSCs, demonstrating that steady-state MHCII at LNSC surfaces were acquired from hematopoietic cells (Fig. 2 B). An almost total lack of MHCII expression by LNSCs from MHCII−/− BM: pIV−/− chimeric mice was observed (Fig. 2 B), suggesting that LNSCs do acquire MHCII from hematopoietic cells. In addition, we reconstituted irradiated MHCII−/− recipient mice with a mixture (1:1) of WT and I-Ab GFP+ BM cells. Levels of GFP expression by MHCII+ LECs, BECs, and FRCs were intermediate in mixed BM chimeras compared with control MHCII−/− recipient mice reconstituted with exclusively WT or I-Ab GFP+ BM cells (Fig. 2 C). Thus, LNSCs from mixed BM chimeric mice express both nonfluorescent (from WT BM) and GFP+ (from I-Ab GFP+ BM) MHCII molecules, reinforcing the idea that nonhematopoietic radioresistant LECs, BECs, and FRCs acquire MHCII from hematopoietic cells. To further test our hypothesis, we sublethally irradiated WT mice to eliminate hematopoietic cells. pIV−/− mice were included to avoid any possible endogenous MHCII up-regulation after irradiation-induced inflammation. 3 d after irradiation, the number of hematopoietic cells in LN was dramatically reduced (not depicted) and correlated with a significant decrease in MHCII expression by both WT and pIV−/− LNSCs (Fig. 2 D). These results indicated that LNSCs transiently acquire MHCII molecules from hematopoietic cells in vivo. To analyze this phenomenon in vitro, we generated LEC/FRC cultures (Lukacs-Kornek et al., 2011). In brief, LN cells were cultured for 5–7 d and washed daily to remove nonadherent hematopoietic cells. Consistent with our hypothesis that LNSCs acquire most MHCII from hematopoietic cells in steady state, removal of hematopoietic cells correlated with the disappearance of MHCII expression levels on LECs and FRCs over time (Fig. 2 E). This also reinforced our in vivo results showing that transferred MHCII are not stable at the surface of LNSC (Fig. 2 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