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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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LECs do not present endogenous β-gal on MHC-II.(a) Representative and (b) cumulative data of CTV-labelled Thy1.1+ Bg2 cells adoptively transferred into the indicated recipients. CTV-labelled Thy1.1neg cells were co-transferred as an injection control. Skin-draining LNs were analysed 3 (a) or 7 (a,b) days later, and plots are gated on CD4+ T cells. Data representative of 1–3 experiments with 1–4 mice each. Indicated groups were compared using a one-way analysis of variance (ANOVA) with Bonferroni post-test. (c) CTV-labelled Bg2 cells were adoptively transferred into B6 and MHC-II−/−→Prox1xβ-gal mice treated with PBS, αCD28 or IFN-γ, and proliferation was analysed 3 days later. Plots are gated on Thy1.1+CD4+ cells. Data representative of 2 experiments with 2 mice each. (d) CTV-labelled Thy1.1+ Bg2 cells were transferred into the indicated recipients and activation markers were analysed 16 h later. Plots are gated on Thy1.1+CD4+ cells. Data from one experiment. (e) LNs from MHC-II−/− and PBS- or IFN-γ-treated B6 mice were enzymatically digested 24 h after treatment, and MHC-II on LECs was analysed by flow cytometry. Data representative of 3 experiments with 1–2 mice each. (f) LNSCs, DCs and macrophages from B6 mice were sorted by flow cytometry, pulsed with 50 μM Bg2 peptide for 3 h, washed and co-cultured with CPD eF670-labelled Thy1.1+ Bg2 T cells for 4 days. (g) LNSCs, DCs and macrophages from Prox1xβ-gal mice were sorted by flow cytometry and co-cultured with CPD eF670-labelled Thy1.1+ Bg2 T cells for 4 days. (f,g) Plots are gated on DAPInegCD4+ Thy1.1+ cells. Data representative of 2 experiments with pooled LNs from 5 to 9 mice. All data shown as mean±s.e.m.
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f4: LECs do not present endogenous β-gal on MHC-II.(a) Representative and (b) cumulative data of CTV-labelled Thy1.1+ Bg2 cells adoptively transferred into the indicated recipients. CTV-labelled Thy1.1neg cells were co-transferred as an injection control. Skin-draining LNs were analysed 3 (a) or 7 (a,b) days later, and plots are gated on CD4+ T cells. Data representative of 1–3 experiments with 1–4 mice each. Indicated groups were compared using a one-way analysis of variance (ANOVA) with Bonferroni post-test. (c) CTV-labelled Bg2 cells were adoptively transferred into B6 and MHC-II−/−→Prox1xβ-gal mice treated with PBS, αCD28 or IFN-γ, and proliferation was analysed 3 days later. Plots are gated on Thy1.1+CD4+ cells. Data representative of 2 experiments with 2 mice each. (d) CTV-labelled Thy1.1+ Bg2 cells were transferred into the indicated recipients and activation markers were analysed 16 h later. Plots are gated on Thy1.1+CD4+ cells. Data from one experiment. (e) LNs from MHC-II−/− and PBS- or IFN-γ-treated B6 mice were enzymatically digested 24 h after treatment, and MHC-II on LECs was analysed by flow cytometry. Data representative of 3 experiments with 1–2 mice each. (f) LNSCs, DCs and macrophages from B6 mice were sorted by flow cytometry, pulsed with 50 μM Bg2 peptide for 3 h, washed and co-cultured with CPD eF670-labelled Thy1.1+ Bg2 T cells for 4 days. (g) LNSCs, DCs and macrophages from Prox1xβ-gal mice were sorted by flow cytometry and co-cultured with CPD eF670-labelled Thy1.1+ Bg2 T cells for 4 days. (f,g) Plots are gated on DAPInegCD4+ Thy1.1+ cells. Data representative of 2 experiments with pooled LNs from 5 to 9 mice. All data shown as mean±s.e.m.

Mentions: To test whether LECs from Prox-1xβ-gal and Lyve-1xβ-gal mice present β-gal epitopes on MHC-II molecules, we used Bg2 CD4 T-cells25, which recognize β-gal721-739 presented by I-Ab. Bg2 cells transferred into Lyve-1xβ-gal or Prox-1xβ-gal mice proliferated by day 3 and continued to proliferate and accumulate by day 7 (Fig. 4a,b). However, they did not proliferate in MHC-II−/−→Lyve-1xβ-gal or MHC-II−/−→Prox1xβ-gal chimeras, in which radioresistant LECs synthesize MHC-II molecules but bone marrow-derived cells do not. This indicates that Bg2 proliferation in non-chimeric mice is induced by bone marrow-derived cells, but not LECs. We considered that Bg2 T cells might recognize antigen but not proliferate because of the lack of costimulatory molecules on LECs12. However, Bg2 cells adoptively transferred into MHC-II−/−→Prox1xβ-gal chimeras treated with αCD28 agonistic antibodies also did not proliferate (Fig. 4c). We also considered that LECs might present β-gal, but rapidly induce anergy or suppress T-cell proliferation through nitric oxide2627. Therefore, we examined Bg2 cells for upregulation of CD69, CD25 and CD44, and downregulation of CD62L, one day after adoptive transfer. After transfer into non-chimeric Prox1xβ-gal mice, CD69, CD25 and CD44 were all upregulated and CD62L was downregulated (Fig. 4d). However, these markers remained unchanged and identical on Bg2 cells transferred into MHC-II−/−→Prox1xβ-gal chimeras and antigen-free B6 mice (Fig. 4d), indicating the Bg2 cells were not activated. We conclude that LECs do not present MHC-II-restricted β-gal epitopes to Bg2 cells in vivo, even though they express the source protein and the restriction element.


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)

LECs do not present endogenous β-gal on MHC-II.(a) Representative and (b) cumulative data of CTV-labelled Thy1.1+ Bg2 cells adoptively transferred into the indicated recipients. CTV-labelled Thy1.1neg cells were co-transferred as an injection control. Skin-draining LNs were analysed 3 (a) or 7 (a,b) days later, and plots are gated on CD4+ T cells. Data representative of 1–3 experiments with 1–4 mice each. Indicated groups were compared using a one-way analysis of variance (ANOVA) with Bonferroni post-test. (c) CTV-labelled Bg2 cells were adoptively transferred into B6 and MHC-II−/−→Prox1xβ-gal mice treated with PBS, αCD28 or IFN-γ, and proliferation was analysed 3 days later. Plots are gated on Thy1.1+CD4+ cells. Data representative of 2 experiments with 2 mice each. (d) CTV-labelled Thy1.1+ Bg2 cells were transferred into the indicated recipients and activation markers were analysed 16 h later. Plots are gated on Thy1.1+CD4+ cells. Data from one experiment. (e) LNs from MHC-II−/− and PBS- or IFN-γ-treated B6 mice were enzymatically digested 24 h after treatment, and MHC-II on LECs was analysed by flow cytometry. Data representative of 3 experiments with 1–2 mice each. (f) LNSCs, DCs and macrophages from B6 mice were sorted by flow cytometry, pulsed with 50 μM Bg2 peptide for 3 h, washed and co-cultured with CPD eF670-labelled Thy1.1+ Bg2 T cells for 4 days. (g) LNSCs, DCs and macrophages from Prox1xβ-gal mice were sorted by flow cytometry and co-cultured with CPD eF670-labelled Thy1.1+ Bg2 T cells for 4 days. (f,g) Plots are gated on DAPInegCD4+ Thy1.1+ cells. Data representative of 2 experiments with pooled LNs from 5 to 9 mice. All data shown as mean±s.e.m.
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f4: LECs do not present endogenous β-gal on MHC-II.(a) Representative and (b) cumulative data of CTV-labelled Thy1.1+ Bg2 cells adoptively transferred into the indicated recipients. CTV-labelled Thy1.1neg cells were co-transferred as an injection control. Skin-draining LNs were analysed 3 (a) or 7 (a,b) days later, and plots are gated on CD4+ T cells. Data representative of 1–3 experiments with 1–4 mice each. Indicated groups were compared using a one-way analysis of variance (ANOVA) with Bonferroni post-test. (c) CTV-labelled Bg2 cells were adoptively transferred into B6 and MHC-II−/−→Prox1xβ-gal mice treated with PBS, αCD28 or IFN-γ, and proliferation was analysed 3 days later. Plots are gated on Thy1.1+CD4+ cells. Data representative of 2 experiments with 2 mice each. (d) CTV-labelled Thy1.1+ Bg2 cells were transferred into the indicated recipients and activation markers were analysed 16 h later. Plots are gated on Thy1.1+CD4+ cells. Data from one experiment. (e) LNs from MHC-II−/− and PBS- or IFN-γ-treated B6 mice were enzymatically digested 24 h after treatment, and MHC-II on LECs was analysed by flow cytometry. Data representative of 3 experiments with 1–2 mice each. (f) LNSCs, DCs and macrophages from B6 mice were sorted by flow cytometry, pulsed with 50 μM Bg2 peptide for 3 h, washed and co-cultured with CPD eF670-labelled Thy1.1+ Bg2 T cells for 4 days. (g) LNSCs, DCs and macrophages from Prox1xβ-gal mice were sorted by flow cytometry and co-cultured with CPD eF670-labelled Thy1.1+ Bg2 T cells for 4 days. (f,g) Plots are gated on DAPInegCD4+ Thy1.1+ cells. Data representative of 2 experiments with pooled LNs from 5 to 9 mice. All data shown as mean±s.e.m.
Mentions: To test whether LECs from Prox-1xβ-gal and Lyve-1xβ-gal mice present β-gal epitopes on MHC-II molecules, we used Bg2 CD4 T-cells25, which recognize β-gal721-739 presented by I-Ab. Bg2 cells transferred into Lyve-1xβ-gal or Prox-1xβ-gal mice proliferated by day 3 and continued to proliferate and accumulate by day 7 (Fig. 4a,b). However, they did not proliferate in MHC-II−/−→Lyve-1xβ-gal or MHC-II−/−→Prox1xβ-gal chimeras, in which radioresistant LECs synthesize MHC-II molecules but bone marrow-derived cells do not. This indicates that Bg2 proliferation in non-chimeric mice is induced by bone marrow-derived cells, but not LECs. We considered that Bg2 T cells might recognize antigen but not proliferate because of the lack of costimulatory molecules on LECs12. However, Bg2 cells adoptively transferred into MHC-II−/−→Prox1xβ-gal chimeras treated with αCD28 agonistic antibodies also did not proliferate (Fig. 4c). We also considered that LECs might present β-gal, but rapidly induce anergy or suppress T-cell proliferation through nitric oxide2627. Therefore, we examined Bg2 cells for upregulation of CD69, CD25 and CD44, and downregulation of CD62L, one day after adoptive transfer. After transfer into non-chimeric Prox1xβ-gal mice, CD69, CD25 and CD44 were all upregulated and CD62L was downregulated (Fig. 4d). However, these markers remained unchanged and identical on Bg2 cells transferred into MHC-II−/−→Prox1xβ-gal chimeras and antigen-free B6 mice (Fig. 4d), indicating the Bg2 cells were not activated. We conclude that LECs do not present MHC-II-restricted β-gal epitopes to Bg2 cells in vivo, even though they express the source protein and the restriction element.

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