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 for EAE using a myelin peptide cocktail in repeat immunizations with 200 μg/each myelin peptide (EAE experiment 1), or single immunization with 100 μg/each myelin peptide (EAE experiment 2) (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) Proportions of interferon-γ-producing and IL-17A-producing CD4+ and CD8+ (or CD4−) T cells in splenocytes recovered from mice in the different groups at dpt 42. (E) 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. (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 spontaneous parenchymal lesions in brain 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 and parenchymal regions (denoted in diagrams of brain and brainstem A). (B, C) 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 at meninges close to the optic tract (B, arrowheads) and in the connective tissue of the interventricular foramen joining the lateral and third ventricles (C, arrowheads). Scattered hCD45-positive leukocytes and hCD8 T cells in brain parenchyma of DR15 MS mice (C, arrows). (D) Counting of barrier-associated and parenchymal hCD8- and hCD4-positive T cells at borders and in parenchyma of whole coronal sections of brain/ mm2 of humanized mice. (E) Ratios of hCD4/hCD8 T cells at borders and parenchyma of selected humanized mice. (F) Small hCD45-positive immune cell lesion in brainstem of DR15 MS1 mice (see also diagram A). (G) hCD45-positive immune cell lesions in grey matter of hippocampus (i, ii) and sub-hippocampus/thalamus (iii), and white matter of optic chiasm (H) of DR15 MS3 mice. (I, J) hCD45-positive immune cell lesions in grey matter of thalamus and hippocampus, respectively, of DR15 MS5 mice. Scale bars 100 μm; x20 objective (B, C, F), 200μm; 10x objective (G), 100μm; 40x objective (H, I, J). 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 parenchymal 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 GM and WM regions in whole spinal cord sections compared at equivalent cord regions from DR15 HI and DR15 MS mice. (D) Ratios of hCD4/hCD8 T cells at borders and parenchyma of DR15 HI and DR15 MS mice. (E) Double immunofluorescence staining for hCD45-positive leukocytes (green, arrowheads) and MBP-positive myelin (red), with DAPI counterstained nuclei (blue), in spinal cord WM of DR15 MS mice. 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 hCD4 T cell infiltration of brain parenchyma resulting in mixed hCD4/hCD8 T cell lesions in brain of 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) Individual hCD45-positive leukocytes and hCD8-positive T cells form small lesions in the optic tract (arrowhead) in immunized DR15 HI mice. (C) Counting of border-associated and parenchymal hCD45-positive T cells in whole coronal sections of brain from non-immunized and immunized DR15 HI and DR15 MS mice. (D) Counting of barrier-associated and parenchymal hCD4- and hCD8-positive T cells in whole coronal sections of brain from non-immunized and immunized DR13 MS, DR15 HI and DR15 MS mice. (E) Ratios of hCD4/hCD8 T cells at borders and parenchyma of non-immunized and immunized DR15 HI and DR15 MS mice. (F) Prominent lesions in the corpus callosum white matter of two of three DR15 MS1 mice (arrowheads), containing hCD45- and mCD45-positive leukocytes, hCD4- and hCD8-positive T cells and locally activated Iba1-positive microglia. Inset shows a serial section stained by Luxol fast blue showing absence of demyelination. (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 MS1 mice, showing inflammatory lesion without demyelination. (H) Small white matter lesion in DR15 MS2 mouse. (I) Prominent white and (J) grey matter lesions in DR15 MS3 mice containing both hCD4 and hCD8 T cells in DR15 MS3 mice. (K) Small human hCD45-positive immune cell lesion in sub-thalamic area of DR15 MS5 mice. (L) Correlation analysis between numbers of hCD4 or hCD8 with number of hCD45 lesions in brain parenchyma of combined immunized DR15 HI and DR15 MS1-5 mice.

Scale bars 100 μm; x20 objective (B,F); x40 objective (G,K). 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 hCD4 T cell infiltration of spinal cord white matter in 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 MS1 and DR15 HI mice (lower panels, arrowheads) The dotted lines mark the boundary between grey matter and white matter. (B) Semi-quantitative estimation of hCD3-positive T cells in whole spinal cord sections from DR15 HI and DR15 MS mice. (C) Counting of hCD3-positive T cell lesions (≥ 3 adjacent cells) in grey (GM) and white matter (WM) in whole spinal cord sections from DR15 HI and DR15 MS1 mice. (D) Ratios of hCD4/hCD8 T cells in spinal cord parenchyma of non-immunized and immunized DR15 HI and DR15 MS mice. (E) 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. 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.