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Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction.

Rouhani SJ, Eccles JD, Riccardi P, Peske JD, Tewalt EF, Cohen JN, Liblau R, Mäkinen T, Engelhard VH - Nat Commun (2015)

Bottom Line: In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells.Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy.Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.

View Article: PubMed Central - PubMed

Affiliation: Carter Immunology Center, Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.

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Prox1-creERT2 is active in LECs but not other LNSCs or haematopoietically derived cells.Prox1-CreERT2x EYFPstop-flox (Prox1xEYFP) mice were maintained on tamoxifen chow for 2 weeks. Pooled inguinal, axillary and brachial LNs from B6 or Prox1xEYFP mice were enzymatically digested and EYFP expression in LECs (DAPInegCD45negCD31+gp38+), BECs (DAPInegCD45negCD31+gp38neg), FRCs (DAPInegCD45negCD31neggp38+), DCs (DAPInegCD11chigh), macrophages (DAPInegCD11cneg/lowCD11b+), B cells (DAPInegCD19+), CD4 T cells (DAPInegCD4+CD19neg) and CD8 T cells (DAPInegCD8+ CD19neg) was analysed by flow cytometry. Numbers refer to the percent of EYFP+ cells in the Prox1xEYFP mice. Data representative of two independent experiments with 1–2 mice each.
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f2: Prox1-creERT2 is active in LECs but not other LNSCs or haematopoietically derived cells.Prox1-CreERT2x EYFPstop-flox (Prox1xEYFP) mice were maintained on tamoxifen chow for 2 weeks. Pooled inguinal, axillary and brachial LNs from B6 or Prox1xEYFP mice were enzymatically digested and EYFP expression in LECs (DAPInegCD45negCD31+gp38+), BECs (DAPInegCD45negCD31+gp38neg), FRCs (DAPInegCD45negCD31neggp38+), DCs (DAPInegCD11chigh), macrophages (DAPInegCD11cneg/lowCD11b+), B cells (DAPInegCD19+), CD4 T cells (DAPInegCD4+CD19neg) and CD8 T cells (DAPInegCD8+ CD19neg) was analysed by flow cytometry. Numbers refer to the percent of EYFP+ cells in the Prox1xEYFP mice. Data representative of two independent experiments with 1–2 mice each.

Mentions: Because there are no CD4 TCR transgenic models directed against endogenous PTAs expressed by LECs, we transgenically expressed β-gal as a model PTA (Supplementary Table 1), as there are β-gal-specific CD4 and CD8 TCR transgenic mice available (Supplementary Table 2). Rosa26stop-LacZ (ref 20) mice express β-gal after cre-mediated excision of a floxed stop cassette. These mice were crossed with mice expressing either Lyve-1-cre21 or Prox1-creERT2 (ref. 22; Supplementary Fig. 1). Lyve-1 is used as a specific marker for LECs23. Lyve-1-cre is constitutively active in all LECs, and in limited subsets of BECs, lymphoid and myeloid cells21. Prox1 is the master transcriptional regulator inducing LEC differentiation23. Prox1-creERT2 is induced by tamoxifen, and mediates high-efficiency recombination in LECs but not in BECs, FRCs, DCs, macrophages, B or T cells (Fig. 2). Since Prox1-creERT2 can also lead to recombination in Prox1+ cells in the liver and heart22, we used skin-draining LNs in our analysis to eliminate potential effects of antigen draining from these sites. Because these two models, referred to hereafter as Lyve-1xβ-gal and Prox1xβ-gal, differ in their patterns of ectopic expression, they were used as complementary models to investigate LEC-induced tolerance.


Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction.

Rouhani SJ, Eccles JD, Riccardi P, Peske JD, Tewalt EF, Cohen JN, Liblau R, Mäkinen T, Engelhard VH - Nat Commun (2015)

Prox1-creERT2 is active in LECs but not other LNSCs or haematopoietically derived cells.Prox1-CreERT2x EYFPstop-flox (Prox1xEYFP) mice were maintained on tamoxifen chow for 2 weeks. Pooled inguinal, axillary and brachial LNs from B6 or Prox1xEYFP mice were enzymatically digested and EYFP expression in LECs (DAPInegCD45negCD31+gp38+), BECs (DAPInegCD45negCD31+gp38neg), FRCs (DAPInegCD45negCD31neggp38+), DCs (DAPInegCD11chigh), macrophages (DAPInegCD11cneg/lowCD11b+), B cells (DAPInegCD19+), CD4 T cells (DAPInegCD4+CD19neg) and CD8 T cells (DAPInegCD8+ CD19neg) was analysed by flow cytometry. Numbers refer to the percent of EYFP+ cells in the Prox1xEYFP mice. Data representative of two independent experiments with 1–2 mice each.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403767&req=5

f2: Prox1-creERT2 is active in LECs but not other LNSCs or haematopoietically derived cells.Prox1-CreERT2x EYFPstop-flox (Prox1xEYFP) mice were maintained on tamoxifen chow for 2 weeks. Pooled inguinal, axillary and brachial LNs from B6 or Prox1xEYFP mice were enzymatically digested and EYFP expression in LECs (DAPInegCD45negCD31+gp38+), BECs (DAPInegCD45negCD31+gp38neg), FRCs (DAPInegCD45negCD31neggp38+), DCs (DAPInegCD11chigh), macrophages (DAPInegCD11cneg/lowCD11b+), B cells (DAPInegCD19+), CD4 T cells (DAPInegCD4+CD19neg) and CD8 T cells (DAPInegCD8+ CD19neg) was analysed by flow cytometry. Numbers refer to the percent of EYFP+ cells in the Prox1xEYFP mice. Data representative of two independent experiments with 1–2 mice each.
Mentions: Because there are no CD4 TCR transgenic models directed against endogenous PTAs expressed by LECs, we transgenically expressed β-gal as a model PTA (Supplementary Table 1), as there are β-gal-specific CD4 and CD8 TCR transgenic mice available (Supplementary Table 2). Rosa26stop-LacZ (ref 20) mice express β-gal after cre-mediated excision of a floxed stop cassette. These mice were crossed with mice expressing either Lyve-1-cre21 or Prox1-creERT2 (ref. 22; Supplementary Fig. 1). Lyve-1 is used as a specific marker for LECs23. Lyve-1-cre is constitutively active in all LECs, and in limited subsets of BECs, lymphoid and myeloid cells21. Prox1 is the master transcriptional regulator inducing LEC differentiation23. Prox1-creERT2 is induced by tamoxifen, and mediates high-efficiency recombination in LECs but not in BECs, FRCs, DCs, macrophages, B or T cells (Fig. 2). Since Prox1-creERT2 can also lead to recombination in Prox1+ cells in the liver and heart22, we used skin-draining LNs in our analysis to eliminate potential effects of antigen draining from these sites. Because these two models, referred to hereafter as Lyve-1xβ-gal and Prox1xβ-gal, differ in their patterns of ectopic expression, they were used as complementary models to investigate LEC-induced tolerance.

Bottom Line: In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells.Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy.Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.

View Article: PubMed Central - PubMed

Affiliation: Carter Immunology Center, Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Lymphatic endothelial cells (LECs) directly express peripheral tissue antigens and induce CD8 T-cell deletional tolerance. LECs express MHC-II molecules, suggesting they might also tolerize CD4 T cells. We demonstrate that when β-galactosidase (β-gal) is expressed in LECs, β-gal-specific CD8 T cells undergo deletion via the PD-1/PD-L1 and LAG-3/MHC-II pathways. In contrast, LECs do not present endogenous β-gal in the context of MHC-II molecules to β-gal-specific CD4 T cells. Lack of presentation is independent of antigen localization, as membrane-bound haemagglutinin and I-Eα are also not presented by MHC-II molecules. LECs express invariant chain and cathepsin L, but not H2-M, suggesting that they cannot load endogenous antigenic peptides onto MHC-II molecules. Importantly, LECs transfer β-gal to dendritic cells, which subsequently present it to induce CD4 T-cell anergy. Therefore, LECs serve as an antigen reservoir for CD4 T-cell tolerance, and MHC-II molecules on LECs are used to induce CD8 T-cell tolerance via LAG-3.

Show MeSH
Related in: MedlinePlus