The endothelial basement membrane acts as a checkpoint for entry of pathogenic T cells into the brain

Abstract

The endothelial cell basement membrane (BM) is a barrier to migrating leukocytes and a rich source of signaling molecules that can influence extravasating cells. Using mice lacking the major endothelial BM components, laminin 411 or 511, in murine experimental autoimmune encephalomyelitis (EAE), we show here that loss of endothelial laminin 511 results in enhanced disease severity due to increased T cell infiltration and altered polarization and pathogenicity of infiltrating T cells. In vitro adhesion and migration assays reveal higher binding to laminin 511 than laminin 411 but faster migration across laminin 411. In vivo and in vitro analyses suggest that integrin α6β1- and αvβ1-mediated binding to laminin 511-high sites not only holds T cells at such sites but also limits their differentiation to pathogenic Th17 cells. This highlights the importance of the interface between the endothelial monolayer and the underlying BM for modulation of immune cell phenotype.

Figure 1.

Primary encephalitogenic CD4⁺ T cells show high affinity binding but low migration on laminin 511. (A) Binding of encephalitogenic CD4⁺ T cells to laminins 411 and 511, nonendothelial laminin 111, and the positive control VCAM-1. Data are means ± SD of six experiments, n = 3 mice/experiment and three to four replicates/group. Statistical analysis used a Mann–Whitney U test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Optical tweezer experiments showing displacement distance of CD4⁺ T cells plated on high and low concentrations of laminin 411 or 511 under 70 pN laser strength. Data are means of three experiments ± SD, n = 3 mice/experiment and 10 cells/group. Statistical analysis used a Mann–Whitney U test; *, P < 0.05; **, P < 0.01. (C and D) Representative migration trajectories of CD4⁺ T cells on laminin 411 or 511 in response to CCL21 (C) and corresponding mean velocities of all cells measured (D); two independent experiments were performed, n = 3 mice/experiment and20 cells/group. Statistical analysis used a Mann–Whitney U test; ***, P < 0.001. (E) Binding of encephalitogenic Th1 and Th17 cells to 10 µg/ml laminins 411, 511, and 111 and VCAM-1. Data are means of six experiments ± SD, n = 3 mice/experiment and three to four replicates/group. Statistical analysis used a Mann–Whitney U test; **, P < 0.01; ***, P < 0.001.

Figure 2.

Integrins α6β1 and αvβ1 mediate encephalitogenic CD4⁺ T cell adhesion and migration across endothelial laminins. (A and B) WT CD4⁺ T cell adhesion to laminins 411 (A) or 511 (B) in the presence or absence of function blocking antibodies to integrins α6 (GoH3), β1 (Ha/5), or β3 (2C9.G2) or high and low concentrations of linear RGD. Data are percentage of total cells added; means ± SD of six experiments are shown, n = 3 mice/experiment and five replicates/group. Statistical analysis used a Mann–Whitney U test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C and D) Binding of WT, Itga6^−/−, or Itgav^−/− CD4⁺ T cells to laminin 411 or 511 (C) and mini-laminin 511 or laminin 511 domain IVa (D). Data are percentage binding relative to binding in the absence of blocking agents or to WT cells; means ± SD of six experiments are shown, n = 3 mice/experiment and three to four replicates/group. Statistical analysis used a Mann–Whitney U test; **, P < 0.01; ***, P < 0.001. (E) Chemotaxis of WT, Itga6^−/−, or Itgav^−/− CD4⁺ T cells across mini-laminin 511, laminin 511 domain IVa, or laminin 411. Data are percentage of total cells added; means ± SD of six experiments are shown, n = 3 mice/experiment and three to four replicates/group. Statistical analysis used a Mann–Whitney U test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (F and G) Representative flow cytometry of TAMRA⁺ Itgav^−/− and CFSE⁺ WT CD4⁺ T cells in WT hosts 24 h after transfer (F) and corresponding quantification of Itgav^−/− relative to WT cells (G). Data are values from three independent experiments with n = 3–4 mice/group/experiment; mean values ± SD are shown. Statistical analysis used a Mann–Whitney U test; ***, P < 0.001. (H and I) Representative immunofluorescence staining of TAMRA⁺ Itgav^−/− and CFSE⁺ WT CD4⁺ T cells in WT lymph nodes and brains (scale bar = 50 µm; H) and quantification of the data from at least five sections/organ/host and four to five hosts (I). Data are means ± SD; statistical analysis was done by a Mann–Whitney U test; **, P < 0.01.

Figure S1.

Flow cytometry for integrins and MCAM expression levels on CD4⁺ T cells in naive and EAE conditions. (A and B) Representative flow cytometry of integrin profiles and MCAM on Itga6^−/− (A) and Itgav^−/− (B) CD4⁺ T cells compared with corresponding WT and isotype controls (Iso-ctrl). Data are representative of three independent experiments, n = 3–4 mice/group/experiment. (C) Representative flow cytometry of integrin α6, αv, and β1 levels on CD4 T cells in brain, spinal cord, spleen, and lymph node under naive and EAE conditions. Data are representative of three independent experiments; n = 3–4 mice/group/experiment. Very few cells are present in the brain and spinal cord under naive conditions, resulting in the low maximum counts.

Figure S2.

Characterization of mini laminin 511 and laminin 511 domain IVa for use in adhesion and transmigration assays. (A) Concentration-dependent, saturable binding of WT encephalitogenic CD4⁺ T cells to mini-laminin 511 and laminin 511 domain IVa. (B) Transmigration of WT encephalitogenic CD4⁺ T cells across mini-laminin 511, laminin 511 domain IVa, and laminin 411. Data in A and B are expressed as a percentage of total cells added and are means ± SD of five experiments, n = 3 replicates/group/experiment. Statistical analysis used a Mann–Whitney U test; **, P < 0.01; ***, P < 0.001.

Figure 3.

Integrin α6^highαv^low marks pathogenic Th17 cells, the differentiation of which is altered by endothelial laminins. (A and B) Representative flow cytometry of integrins α6 and αv on CD4⁺ T cells in the blood, lymph nodes, spleens, and brains of WT mice at peak EAE (A), and corresponding quantification of integrin α6–high and –low populations (B). Data are means ± SD of three experiments, n = 3 mice/group/experiment. Statistical analysis used a Mann–Whitney U test. (C) qPCR for the regulatory T cell marker, Foxp3, and Th17 pathogenicity markers in integrin α6^highαv^high and α6^highαv^low CD4⁺ T cells isolated from brains of WT mice at peak EAE. Data are means ± SD of four experiments, n = 3–6 mice/experiment and triplicates/group. Statistical analysis used a Mann–Whitney U test; **, P < 0.01; ***, P < 0.001. (D and E) Representative flow cytometry for CD4 and IFN-γ or IL-17 to identify Th1 and Th17 cells derived from in vitro differentiated MOG-specific CD4⁺ T cells cultured on laminin 411, laminin 511, or no coating (D), and corresponding quantification of the data expressed as percentage change from cells plated on uncoated plastic (E). Data are means ± SD of four experiments, n = 3–6 mice/experiment and triplicates/group. Statistical analysis used a Mann–Whitney U test; **, P < 0.01; ***, P < 0.001. (F) Corresponding qPCR for pathogenicity markers in Th17 cells performed in two experiments. Data are means ± SD, n = 4 mice/experiment with triplicates/group. Statistical analysis used a Mann–Whitney U test; *, P < 0.05; **, P < 0.01.

Figure 4.

Increased EAE severity in Tek-cre::Lama5^−/− mice. (A) EAE scores versus days after low-dose MOG_35–55 immunization of Tek-cre::Lama5^−/− and WT littermates. Data are mean scores ± SD from three experiments, n = 14 mice/group. Statistical analysis used a Mann–Whitney U test; P value is shown. (B–E) Corresponding Kaplan–Meier curves showing EAE incidence (B) and survival (C) and bar graphs showing mean day of disease onset (D) and mean clinical scores (E) in mice immunized with high- or low-dose MOG_35–55. Statistical tests were log-rank tests; P values in B and C are shown; *, P < 0.05; **, P < 0.01; ns, not significant. (F) Flow cytometry quantification of CD4⁺ and CD8⁺ T cells, B220⁺ B cells, CD11b⁺ macrophages, and Th1 and Th17 CD4⁺ T cells in brains, lymph nodes, and blood of Tek-cre::Lama5^−/− and WT littermates at peak disease (day 17). Data are means ± SD of three experiments, n = 15 mice/group. Statistical analysis used a Mann–Whitney U test; **, P < 0.01. (G) Corresponding CD4, CD8, and CD11b immunofluorescence stainings; scale bars are 100 µm.

Figure S3.

Quantification of immune cells in spinal cords of WT and Tek-cre::Lama5^−/− mice at peak EAE and in vivo proliferation and migration of adoptively transferred encephalitogenic CD4⁺ T cells inWT and Tek-cre::Lama5^−/− mice with corresponding clinical scores over a 40 d period and quantification of transferred cells in the CNS and periphery. (A) Quantification of flow cytometry analyses of CD8⁺ and CD4⁺ T cells, B220⁺ B cells, CD11b⁺ macrophages, and Th1 and Th17 CD4⁺ T cells in spinal cords of WT and Tek-cre::Lama5^−/− mice at peak EAE. Data are means ± SD of three experiments, n = 5 mice/group/experiment. Statistical analysis used a Mann–Whitney U test; *, P < 0.05. (B) Representative flow cytometry of three experiments of BrdU⁺ CD4⁺ T cells in the brain, spleen, lymph node, and blood of WT and Tek-cre::Lama5^−/− mice at peak EAE. (C) WT CD45.1⁺ CD4⁺ T cells were adoptively transferred to CD45.2⁺ Tek-cre::Lama5^−/− and WT recipients and analyzed at day 3 by flow cytometry for infiltrating CD45.1⁺ CD4 T cells; representative flow cytometry of three experiments is shown. (D) The same adoptive transfer experiments were performed over a 40-d period, and mean clinical scores were measured. Data shown are means ± SD of two experiments, n = 7 mice/group. Statistical analysis used a Mann–Whitney U test; P value is shown. (E and F) Corresponding representative flow cytometry of adoptively transferred WT CD45.1⁺ CD4⁺ T cells in the brain, lymph node, spleen, and blood at peak EAE (E) and quantification of the data (F). Data are means ± SD of three experiments, n = 9 mice/group. Statistical analysis used a Mann–Whitney U test; *, P < 0.05.

Figure 5.

Integrin α6^highαv^low CD4⁺ T cells predominate in brains of Lama4^−/− mice at peak EAE. (A and B) Representative flow cytometry of integrins α6 and αv on CD4⁺ T cells in the periphery (lymph node) and brain (without meninges) of Lama4^−/−, Tek-cre::Lama5^−/−, and WT mice at peak EAE (A) and corresponding quantification of proportions of integrin α6^highαv^high and α6^highαv^low T cells (B). (C) Total CD4⁺ T cell infiltrates in Lama4^−/− and Tek-cre::Lama5^−/− and WT mice at peak EAE showing ratios of integrin α6^highαv^high to α6^highαv^low T cells. Data are means ± SD of four experiments, n = 6 mice/group. Statistical analysis used a Mann–Whitney U test; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (D) Scheme showing that laminin 511^high sites in the endothelial BM bind extravasating integrin α6^high/αv^high T cells, restricting their differentiation to the highly motile, pathogenic integrin α6^high/αv^low Th17 population and, thereby, acting as a checkpoint in neuroinflammation.