Reconstitution of a human adaptive immune system in B2m-NOG mice engrafted with PBMC from MS patient and healthy donors.

(A) Progressive engraftment of human (h) CD45+ leukocytes from donors in groups of B2m-NOG mice, non-immunized (left) and immunized using an EAE protocol (right), measured in peripheral blood samples taken at different time points, and in spleen recovered at sacrifice 42 days post-transplantation (dpt 42). (B) Proportions of hCD4+ and hCD8+ T cells in blood hCD45+CD3+ T cells at different time points. (C) Proportions of human immune cell subpopulations in spleens of immunized and non-immunized mice at dpt 42 (see also Supplementary Table 3). (D) Antigen-specific T cell proliferation responses to the immunizing antigens (mMOG35-55, hMOG35-55, MOG1-20, MBP83-99), anti-hCD3 (positive control) and medium (unstimulated; US), in splenocytes recovered from non-immunized and immunized DR13 MS PBMC humanized mice at dpt 42. Results are expressed as a cell division index. (E) Proportions of IL-17A-producing CD4+ (left graph), and interferon-γ-producing (right graph) CD4+ and CD8+ T cells in splenocytes recovered from mice in the different groups at dpt 42. (F) Draining lymphoid structures in inguinal fat of mice engrafted with DR13 MS and DR15 MS PBMC and immunized with myelin peptides, recovered at dpt 42. Immunohistochemistry revealed the presence of hCD45- and mCD45-positive leukocytes, hCD4- and hCD8-positive T cells, and hCD20-positive B cells (latter only DR13 MS mice). All results are depicted as mean ± SEM. Statistical significance is shown after pairwise comparisons between groups using Student’s t test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Human T cells accumulate at brain borders in non-immunized DR15 MS and DR15 HI mice, and form parenchymal lesions in brain and spinal cord of DR15 MS mice

Immunohistochemical analysis of the brain from non-immunized PBMC B2m-NOG mice showing infiltration by human (h) and mouse (m) CD45-positive leukocytes, and hCD8-positive T cells, in brain border regions (denoted in diagram A) and in brainstem parenchyma (denoted in diagram B). (B) Parenchymal lesion containing hCD8-positive T cells in brainstem of a DR15 MS mouse. (C, D) Accumulation of hCD45- and mCD45-positive leukocytes and hCD8-positive T cells, together with local activation of Iba1-positive microglia and GFAP-positive astrocytes, at border regions in the brains of DR15 HI and DR15 MS mice, specifically in the connective tissue of the interventricular foramen joining the lateral and third ventricles (C, arrowheads), and at meninges close to the optic tract (D, arrowheads). Scattered hCD45-positive leukocytes and hCD8 T cells in brain parenchyma of DR15 MS mice (C, arrows). (E) Counting of barrier-associated and parenchymal hCD8-positive T cells in whole coronal sections of brain (shown in A) from DR13 MS, DR15 MS and DR15 HI mice. Scale bar 100 μm; x20 objective (C, D). All results are depicted as mean ± SEM. Statistical analysis was performed by pairwise comparisons between different groups of mice using Student’s t test.

Human T cells infiltrate spinal cord white matter in non-immunized DR15 MS and DR15 HI mice, and form grey matter lesions in DR15 MS mice

Immunohistochemical analysis of spinal cord from non-immunized PBMC B2m-NOG mice showing infiltration by human (h) CD3-positive T cells in the grey and white matter regions. (A) Infiltrating hCD3-positive T cells were scattered individually throughout spinal cord grey and white matter and formed small lesions in the white matter (WM) of both DR15 MS and DR15 HI mice (lower panels, arrowheads), and grey matter lesions only in DR15 MS mice (upper panels, arrowheads). The dotted lines mark the boundary between grey matter and white matter. (B) Counting of hCD3-positive T cell lesions (≥ 3 adjacent cells) in grey and white matter in whole spinal cord sections from DR15 HI and DR15 MS mice. (C) Semi-quantitative estimation of hCD3-positive T cells in whole spinal cord sections from DR15 HI and DR15 MS mice. (D) Double immunofluorescence staining for hCD45-positive leukocytes (green, arrowheads) and MBP-positive myelin (red), with DAPI counterstained nuclei (blue), in spinal cord white matter (WM) of DR15 MS mice. Grey matter (GM). Scale bars 100 μm; x20 objective (A, upper panels); x40 objective (A, lower panels). All results are depicted as mean ± SEM. Statistical analysis was performed by pairwise comparisons between different groups of mice using Student’s t test.

Immunization with myelin peptides increases T cell infiltration of brain parenchyma and promotes development of brain lesions in both DR15 MS and DR15 HI mice

Immunohistochemical analysis of the brain from PBMC B2m-NOG mice immunized for EAE showing infiltration by human (h) and mouse (m) CD45-positive leukocytes, hCD8- and hCD4-positive T cells, and local activation of Iba1-positive microglia and GFAP-positive astrocytes, in brain regions denoted in the diagram (A). (B, C) Individual hCD45-positive leukocytes and hCD8-positive T cells scattered in the corpus callosum white matter (B) and small lesions in the optic tract (C, arrowhead) in the presence of locally activated Iba1-positive microglia and GFAP-positive astrocytes in immunized DR15 HI mice. (D) Prominent lesions in the corpus callosum white matter of two of the three DR15 MS mice (i, ii, arrowheads), containing hCD45- and mCD45-positive leukocytes, hCD4- and hCD8-positive T cells and locally activated Iba1-positive microglia and GFAP-positive astrocytes. (E) Counting of barrier-associated and parenchymal hCD8-positive T cells in whole coronal sections of brain from DR13 MS, DR15 MS and DR15 HI mice. (F) Double immunofluorescence staining for hCD45-positive leukocytes (green, arrowhead) and Iba1-positive microglia/border macrophages (red, yellow arrowhead; non-specific red staining of erythrocytes in vessel lumen), with DAPI counterstained nuclei (blue), showing a small perivascular lesion in the thalamus grey matter (GM) of immunized DR15 MS mice. (G) Double immunofluorescence staining for hCD45-positive leukocytes (green, arrowheads) and MBP-positive myelin (red), with DAPI counterstained nuclei (blue), in corpus callosum in immunized DR15 MS mice, showing inflammatory lesion without demyelination. Scale bars 100 μm; x20 objective (B, C, D, G); x40 objective (F). All results are depicted as mean ± SEM. Statistical analysis was performed by pairwise comparisons between different groups of mice using Student’s t test.

Immunization with myelin peptides induces T cell lesions in spinal cord white matter of both DR15 MS and DR15 HI mice

Immunohistochemical analysis of spinal cord from PBMC B2m-NOG mice immunized for EAE showing infiltration by human (h) CD3-positive T cells in the grey and white matter regions. (A) Infiltrating hCD3-positive T cells scattered throughout spinal cord grey and white matter and forming small lesions in the white matter (WM) of both DR15 MS and DR15 HI mice (lower panels, arrowheads) The dotted lines mark the boundary between grey matter and white matter. (B) Counting of hCD3-positive T cell lesions (≥ 3 adjacent cells) in grey and white matter in whole spinal cord sections from DR15 HI and DR15 MS mice. (C) Double immunofluorescence staining for hCD45-positive leukocytes (green, arrowheads) and MBP-positive myelin (red), with DAPI counterstained nuclei (blue), in white matter (WM) of immunized DR15 MS spinal cord. Grey matter (GM). (D) Semi-quantitative estimation of hCD3-positive T cells in whole spinal cord sections from DR15 HI and DR15 MS mice. All results are depicted as mean ± SEM. Scale bars 100 μm; x40 objective (A). Statistical analysis was performed by pairwise comparisons between different groups of mice using Student’s t test.