Research ArticleAutoimmunity

Brain-resident memory T cells generated early in life predispose to autoimmune disease in mice

See allHide authors and affiliations

Science Translational Medicine  26 Jun 2019:
Vol. 11, Issue 498, eaav5519
DOI: 10.1126/scitranslmed.aav5519
  • Fig. 1 Transient cerebral infection in EL predisposes to the development of brain inflammation by autoantigen-primed T cells.

    (A) Experimental setup: At 1 week (EL) or 3 to 4 weeks of age (YA), WT mice were injected intracerebrally (i.c.) with rLCMV or vehicle, respectively. At least 5 weeks later, in vitro–primed 2D2 T cells were transferred intraperitoneally (i.p.) to induce EAE. (B) Incidence, (C) scores, and (D) maximal scores of atypical symptoms for mice infected in EL in comparison to age-matched mock-infected controls. Maximal scores >1 (dashed line) were considered as atypical EAE. (E to G) As in (B) to (D), for mice infected as YA. ns, not significant. (H) 2D2 T cell infiltration into the cerebrum of mice infected in EL or as YA. (I) Left: Representative immunofluorescence images of cerebral (periventricular) CD4+ T cells and Mac3+ activated macrophages in mice infected in EL in comparison to age-matched mock-infected controls. Right: Quantification of Mac3+ inflammatory lesions. (J) As in (I), for mice infected as YA. Symbols represent individual mice, except for (C) and (F), where data represent means ± SEM (n = 12 to 23 mice per group). Data are pooled (B to G, I, and J) or representative (H) of two independent experiments. *P < 0.05, **P < 0.01, log-rank (Mantel-Cox) test (B and E), Mann-Whitney U test (C, D, F, and G), and Student’s t test (H to J).

  • Fig. 2 Brain autoimmune lesions in mice infected in EL develop preferentially close to sites of resolved virus infection.

    (A) Experimental setup: St-RFP mice were infected intracerebrally with rLCMV-Cre in EL, and in vitro–primed 2D2 T cells were transferred >5 weeks after infection. (B) Representative images of lesions containing T cells (CD3) and activated macrophages (Mac3) with RFP+ reporter cells 2 weeks after transfer of 2D2 T cells. Numbers indicate the percentage of lesions found colocalized with or in indicated distance to RFP+ cells. (C) Spatial correlation analysis of lesions and RFP+ cells. Number of lesions in correlation to distances to the closest RFP+ cells are presented in comparison to a computer-simulated random distribution. Data show a spatial analysis of a total of n = 351 lesions (n = 10 mice) compared to n = 354 simulated random lesions. Distances are represented as boxplots (with boxes indicating the 25 to 75% interquartile range) and surrounded by violin plots. (D) Experimental setup for (E) to (H): Cre recombinase–inducible DTX receptor (iDTR) mice were infected intracerebrally with rLCMV-Cre. Virus-experienced cells were ablated by diphtheria toxin (DTX) administration 3 weeks before transfer of 2D2 T cells. (E) Incidence, (F) scores, and (G) maximal scores of atypical symptoms of ablated iDTR mice in comparison to DTX-treated infected WT littermates and mock-infected controls. Maximal scores >1 (dashed line) were considered as atypical EAE. Data in (F) represent means ± SEM (n = 15 to 17 mice per group). (H) Histological analysis of cerebral inflammation (Mac3+ lesions) 2 weeks after 2D2 T cell transfer. Symbols represent individual mice, and means ± SEM are shown. All data are pooled from two independent experiments. *P < 0.05, **P < 0.01, Mann-Whitney U test (C), log-rank (Mantel-Cox) tests against mock-infected controls (E), Kruskal-Wallis H test (F and G), and one-way analysis of variance (ANOVA) (H).

  • Fig. 3 Clustering of CCL5+ TRMat sites of previous infection leads to high local CCL5 expression that confers enhanced vulnerability to autoimmune attack.

    (A) Schematic representation of tissue sampling around RFP+ reporter cells for transcriptome analysis. (B) Gene expression analysis of 256 inflammatory genes represented as heatmap. (C) Volcano plot of gene expression data presented in (B). Mice that had been infected in EL are compared to the pool of both control groups (mock-infected and infected as YA). (D) Representative image illustrating CCL5 expression by CD3+ T cells. (E to H) Ex vivo flow cytometric analysis of CCL5 expression. (E) Representative histograms of intracellular staining for CCL5 in brain CD8+CD69+CD44+ TRM and CD4+CD69+CD44+ TRM >5 weeks after EL intracerebral rLCMV infection in comparison to CD11b+ microglia/macrophages in adult mice infected in EL. (F) Numbers of CCL5+ brain TRM persisting at 5 weeks after intracerebral rLCMV infection. (G and H) Comparison of CCL5 expression in CD8+ bTRM from adult mice infected in EL or as YA. Fmo, fluorescence minus one. (I) Left: Representative images of CCL5+CD8+ T cells in areas with (RFP+) and without (RFP) reporter cells. Right: Quantification of CD8+ T cell density in RFP+ areas (<200 μm to RFP+ cells) in correlation to RFP areas. (J) Left: Representative images of CD8+CCL5+ T cell clusters in adult mice infected in EL (top) and as YA (bottom). Right: Quantification of CD8+ T cell clusters (>4 CD8+ T cells in a field of view of 0.04 mm2) on brain tissue sections in three different anatomical areas. DAPI, 4′,6-diamidino-2-phenylindole. (K) Experimental setup for (L) to (O): Mice were infected intracerebrally with rLCMV in EL or as YA, and in vitro–primed 2D2 T cells were transferred >5 weeks later. Mice were treated intraperitoneally with 10 μg of 5P12-RANTES or phosphate-buffered saline (PBS) daily after transfer of 2D2 T cells. (L) Incidence, (M) scores, and (N) maximal scores of atypical EAE in 5P12-RANTES–treated mice in comparison to PBS-treated littermates or mock-infected controls. (O) Histological analysis of cerebral inflammation (Mac3+ lesions) 2 weeks after 2D2 T cell transfer of mice in (L) to (N). (F, H, I, J, N, and O) Symbols represent individual mice. (M) Means ± SEM is shown (n = 4 to 6 mice per group). Gene expression profiling (B and C) was performed once. Other data are either pooled (F, H, and J) or representative (D, E, I, L, and O) of two independent experiments. *P < 0.05, **P < 0.01, Student’s t test (F and H), paired Student’s t test (I), Mann-Whitney U test (J), log-rank (Mantel-Cox) test (L), Kruskal-Wallis H test (M and N), and one-way ANOVA (O).

  • Fig. 4 bTRM cluster with MHCII+APCs in postinfectious mice.

    WT mice were infected with rLCMV intracerebrally in EL or as YA and colocalization of CD8+ T cells, and MHCII+ APCs was analyzed >5 weeks later. Mock-infected control mice were injected with vehicle only. (A) Left: Representative images of cerebral MHCII expression in the choroid plexus and periventricular brain parenchyma. Right: Quantification of parenchymal MHCII expression, persisting >5 weeks after infection (n = 4 to 6). (B) Representative image and quantification of iba1+ MHCII+ cells in proximity to virus-purged RFP+ cells (n = 4). (C) Left: Representative images of MHCII+ cells in areas with (RFP+) and without (RFP) reporter cells. Right: Quantification of MHCII expression in RFP+ areas (<200 μm to RFP+ cells) in correlation to RFP areas (n = 6). (D) Left: Representative images of CD8+ T cell clustering with MHCII+ APCs in adult mice infected in EL and as YA. Right: Quantification of MHCII+ CD8+ T cell clusters (>4 CD8+ T cells in an MHCII+ field of view of 0.04 mm2) on brain tissue sections on three different anatomical areas (n = 11 to 15). Symbols represent individual mice, and means ± SEM is indicated in (A), (B), and (D). Data are either pooled (D) or representative (A to C) of two independent experiments. **P < 0.01, one-way ANOVA (A), followed by Tukey’s multiple comparisons test, paired Student's t test (C), and Mann-Whitney U test (D).

  • Fig. 5 bTRM cluster in HLA-DR high-expressing preactive MS lesions.

    (A) Cerebral tissue sections of patients with MS and controls (NND) were stained for myelin basic protein (MBP) and HLA-DR to identify HLA-DR high-expressing (HLA-DRhigh) and low-expressing (HLA-DRlow) NAWM. (B) Prevalence of HLA-DRhigh NAWM in investigated cases. (C) Quantification of CD8+ T cells in HLA-DRhigh areas in comparison to HLA-DR low-expressing (HLA-DRlow) areas in NAWM of patients with MS and NND control samples. Symbols represent individually analyzed tissue areas of n = 9 MS tissue samples and n = 7 NND controls. (D) Representative images illustrating the identification of CCL5+ TRM (CD8+CD69+BCL-2+) in HLA-DRhigh areas using an elution and restain approach (for details, see Material and Methods). Left: MS NAWM; right: NND NAWM. (E) Quantification of TRM (CD8+CD69+BCL-2+) among CD8+ cells. (F) Quantification of CD8+ (non-TRM, CD8+CD69/BCL-2), CCL5, and CCL5+ TRM in HLA-DRhigh and HLA-DRlow areas in NAWM of patients with chronic MS. (G) Quantification of CD8+ (non-TRM), CCL5, and CCL5+ TRM (CD8+CD69+BCL-2+) in HLA-DRhigh and HLA-DRlow areas in NAWM of control samples (NND). **P < 0.01, *P < 0.05, one-way ANOVA, followed by Tukey’s multiple comparisons test (C), and Student’s t test (E). (F and G) HLA-DRhigh and HLA-DRlow areas of the same tissue sample have been compared in a matched analysis (two-way repeated-measures ANOVA, followed by Sidak’s multiple comparisons test). See also table S1.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/498/eaav5519/DC1

    Fig. S1. Transient intracerebral infection with rLCMV in EL and as YA.

    Fig. S2. Transient intracerebral infection with rLCMV does not alter CD4+ T cell–driven spinal cord inflammation and classical EAE symptoms.

    Fig. S3. Virus-purged cells in adult mice that had been infected in EL mark sites of previous infection.

    Fig. S4. DTX-mediated ablation of virus-purged cells.

    Fig. S5. T cells are the major producers of CCL5.

    Fig. S6. Characterization of TM subsets in mice infected in EL and as YA.

    Fig. S7. 5P12-RANTES is a potent and selective inhibitor of CCR5.

    Fig. S8. CCR5 antagonization does not alter classical EAE symptoms and spinal cord inflammation.

    Fig. S9. Ablation of purged cells does not reduce MHCII expression in the brain.

    Table S1. Human brain tissue samples for CD8 quantification and multiplex staining.

    Table S2. Raw data.

  • The PDF file includes:

    • Fig. S1. Transient intracerebral infection with rLCMV in EL and as YA.
    • Fig. S2. Transient intracerebral infection with rLCMV does not alter CD4+ T cell–driven spinal cord inflammation and classical EAE symptoms.
    • Fig. S3. Virus-purged cells in adult mice that had been infected in EL mark sites of previous infection.
    • Fig. S4. DTX-mediated ablation of virus-purged cells.
    • Fig. S5. T cells are the major producers of CCL5.
    • Fig. S6. Characterization of TM subsets in mice infected in EL and as YA.
    • Fig. S7. 5P12-RANTES is a potent and selective inhibitor of CCR5.
    • Fig. S8. CCR5 antagonization does not alter classical EAE symptoms and spinal cord inflammation.
    • Fig. S9. Ablation of purged cells does not reduce MHCII expression in the brain.
    • Table S1. Human brain tissue samples for CD8 quantification and multiplex staining.
    • Legend for table S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Raw data.

Stay Connected to Science Translational Medicine

Navigate This Article