Fig 1.
EAE induction causes expansion of the Th40 cell subset and infiltration of Th40 cells into brain and spinal cord tissues.
Female C57BL/6 mice were EAE induced. (A) Representative plots of CD4 and CD40 stained cells (blue) in peripheral draining lymph nodes (dLN) and spleens from age- and sex-matched, wild type, control (without injection of any EAE induction component) and EAE induced mice (disease score of at least 2). Gate was set from isotype (red) and FMO (separate plots) controls. CD3 expression in CD4+CD40+ quadrant was confirmed (histograms). (B) Graph depicting percent Th40 cells from cumulative data in B (Control, n = 11; EAE, n = 8). Data are represented as mean ± SEM. Significance was calculated by two-tailed t-test. (C) Graph depicting absolute number of T cells recovered from brains and spinal cords of EAE (n = 9; disease score of at least 2) and control (n = 5) mice. Cells were MHCII and CD11b depleted to remove B cells, macrophages and contaminating microglial cells. Data is represented as mean +/- SEM. Significance was calculated using one-tailed t-test. (D) CD40 and CD3 western blots on T cells recovered from brains and spinal cords of control and EAE induced mice as in D. Disease scores are noted below each lane. Control samples from both brain and spinal cord were pooled from 3 mice to achieve enough cells for western blot. EAE samples from spinal cord were pooled from 3 mice 10 (10 d), 14 (14 d), 21 (21 d), and 28 (28 d) days post induction. As an internal control, membranes were stripped and stained with Coomassie blue and a representative band is shown below each blot (Standard). Graphs depict densitometry for CD40 and CD3. It should be noted that the exposure times in the western blots are different between brain and spinal cord samples in order to better visualize spinal cord samples. However, densitometry was done on the same exposures for brain and spinal cord. Data in figure represent at least 3 experiments.
Fig 2.
Draining LN lymphocytes from EAE mice secrete high levels of IFNγ.
dLN were isolated from control (n = 2) and EAE induced mice (10 days post-EAE-induction; n = 3; EAE scores 2, 2, and 0) and lymphocytes purified. Lymphocytes were cultured in the absence/presence of CD3, CD28, and CD40 stimulation for 5 days. Cytokines in the supernatant were assayed. There were significant differences in cytokine production between lymphocytes from EAE and control mice (Two-way ANOVA) as well as between stimulated and isotype treated EAE lymphocytes (two-tailed t-test). Data represent 2 experiments.
Fig 3.
CFA induces expansion of Th40 cells.
Mice were challenged with the EAE induction regimen with (CFA+MOG) or without (CFA-contol) MOG35-55. Alternatively, mice were challenged with LPS or poly I:C. After 3, 7, and 12 days (n = 3 per time point in each group; disease scores for all mice was 0, except in the 3 CFA+MOG mice at 12 days where the scores were 2, 3, and 4 respectively) lymphocytes were purified from (A) spleens and (B) dLN and stained for CD3, CD4, and CD40 for flow cytometry. Cells were first gated on CD3 then CD4 and CD40 levels were assessed in that gate. All gates were set from isotype/FMO controls. T cells were recovered from (C) brains and (D) spinal cords and enumerated. T cells from (E) brains and (F) spinal cords were stained and gated as above and absolute numbers of Th40 cells calculated. Percent Th40 cells of total CD4+ T cells was assessed in (G) brains and (H) spinal cords. Statistical differences were calculated by One-Way ANOVA with Bonferroni post-test. Data in figure represent at least 3 experiments.
Fig 4.
CFA alters CD69 and CD62L expression on Th40 cells in EAE.
Mice were challenged with the EAE induction regimen with (CFA+MOG) or without (CFA-control) MOG35-55. Alternatively, mice were challenged with LPS or poly I:C for 12 days or were left completely untreated (Un-induced). After 3, 7, and 12 days (n = 3 per time point in each group; disease scores for all mice was 0, except in the 3 CFA+MOG mice at 12 days where the scores were 2, 3, and 4 respectively) lymphocytes were purified from spleens and dLN and stained for CD4, CD40, CD69, and CD62L. All gates were set from isotype and FMO controls. CD69 and CD62L expression in Th40 cells was assessed in spleen (A and C respectively) and dLN (B and D respectively). Statistical differences were calculated by One-Way ANOVA with Bonferroni post-test. Data in figure are representative of 3 experiments.
Fig 5.
T cells from CFA-control challenged mice have a similar cytokine production potential compared to those from CFA+MOG35-55 challenged mice.
dLN lymphocytes were purified from CFA-control and CFA+MOG challenged mice, 12 days after challenge, then the cells were cultured in the absence/presence of CD3, CD28, and/or CD40 stimulation or cultured with isotype antibodies for 3 days. Brefeldin A was added the last 2 hours, then the cells were stained for CD3, CD4, CD40, as well as for different cytokines (intracellularly). Cells were gated on CD4+CD40+ (Th40) and CD4+CD40- (Conv. CD4; CD3 expression was confirmed in both populations) then cytokine levels were assessed. Gates were set from isotype controls. (A) Graphs depicting cytokine production by Th40 cells from CFA-control and Th40 cells from CFA+MOG challenged mice. (B) Graphs comparing cytokine production by Th40 cells to cytokine production by conventional CD4 T cells within either CFA-control (left) or CFA+MOG (right) generated cells. Statistical differences in A were calculated by One-Way ANOVA with Bonferroni post-test. Statistical differences in B were calculated by Two-Way ANOVA. Data in figure are representative of 3 experiments.
Fig 6.
MOG35-55 shapes the TCR expression profile.
Mice were challenged with the full EAE regimen with (CFA+MOG) or without (CFA-control) MOG35-55 or were not challenged at all (Un-induced). After 12 days, lymphocytes were purified from dLN and stained for CD4, CD8, CD40, and TCR Vα, or TCR Vβ individually as indicated. Representative histograms/dot plots are shown (Red, isotype control; Blue, stained sample (CD3, CD4, CD40, or Vα/β)). Mean percent expression ±SEM in Th40 cells (A and B) and in CD8 T cells (C and D) is shown. All gates were set from isotype controls. Statistical difference was calculated by Two-Way ANOVA (A and B) or two-tailed t-test (D). Data in figure are representative of 3 experiments.
Fig 7.
Cells, 6 x 107, were transferred to C57BL/6.scid mice. (A) Graph depicting cumulative disease scores from Th40 cell recipients (n = 3 in each group) that received either CFA-control, full EAE regimen (CFA/MOG/PT), IFA/MOG/PT, PT alone, or nothing (Th40 cells alone). Statistical differences were calculated by One-Way ANOVA with Bonferroni’s multiple comparison test. (B) Graph depicting cumulative disease scores for further purified Th40 cell (further depleted on CD11b, CD11c, and CD19) or CD4+CD40- T cell (depleted of Treg) recipients with and without CFA injection. Statistical differences were calculated by One-Way ANOVA with Bonferroni’s multiple comparison test. (C) Flow cytometry histograms of donor cells, prior to transfer, stained for CD4 and CD40 extracellularly (left) or extracellularly and intracellularly (right). Thick black line, Th40 cells; Dotted line, CD4+(CD40(surface)-) cells; Tinted, isotype control. Below histograms, western blot for CD3, CD4, TCRβ (H57-597), and CD40 on donor cells prior to transfer. As a loading standard, membranes were stripped and stained for coomassie blue and a representative band is shown. Graph below western blot is densitometry of the CD40 band divided by the loading standard. (D) Cells recovered from spleen and dLN from Th40 or CD4+(CD40(surface)-) T cell recipient mice were stained for CD3, CD4, and CD40. The CD4/CD40 gate was set from isotype controls. CD3 expression was confirmed. Graph depicts mean ± SEM percent CD4+CD40(surface)+ cells from cumulative data in each group. Statistical differences were calculated by t-test. Data in figure are representative of at least 2 experiments.
Fig 8.
Th40 cells infiltrate brain and spinal cord in EAE disease transfer mice with resulting demyelination.
Splenic Th40 cells from EAE induced C57BL/6 mice were transferred to C57BL/6.scid mice. Brain and spinal cord sections from Th40 cell recipient and un-induced control mice are shown. (A) H&E stained Th40 EAE (at disease score 4) and control brain (B) CD3 immunohistochemistry on Th40 EAE (at disease score 4) and control brain. In both A and B numbered squares in the top left section are enlarged (40x; right panels). Arrow heads indicate CD3 positive areas. (C) H&E stained Th40 EAE (at disease score 4) and control spinal cord. (D) CD3 immunohistochemistry on Th40 EAE (at disease score 4) and control spinal cord (white matter, WM, and grey matter, GM, are indicated). (E) Representative Luxol Fast Blue stained coronal brain and longitudinal spinal cord (mid-thoracic to tail) sections from Th40 EAE (bottom panels; n = 3) compared to control mice (top panels; n = 2). Scale bars are 2 mm. Data in figure are representative of at least 3 experiments.