Dynamic cross-regulation of antigen-specific effector and regulatory T cell subpopulations and microglia in brain autoimmunity

Abstract

Background: Multiple Sclerosis (MS) is considered a T-cell-mediated autoimmune disease with a prototypical oscillatory behavior, as evidenced by the presence of clinical relapses. Understanding the dynamics of immune cells governing the course of MS, therefore, has many implications for immunotherapy. Here, we used flow cytometry to analyze the time-dependent behavior of antigen-specific effector (T(eff)) and regulatory (T(reg)) T cells and microglia in mice model of MS, Experimental Autoimmune Encephalomyelitis (EAE), and compared the observations with a mathematical cross-regulation model of T-cell dynamics in autoimmune disease.

Results: We found that T(eff) and T(reg) cells specific to myelin olygodendrocyte glycoprotein (MOG) developed coupled oscillatory dynamics with a 4- to 5-day period and decreasing amplitude that was always higher for the T(eff) populations, in agreement with the mathematical model. Microglia activation followed the oscillations of MOG-specific T(eff) cells in the secondary lymphoid organs, but they were activated before MOG-specific T-cell peaks in the CNS. Finally, we assessed the role of B-cell depletion induced by anti-CD20 therapy in the dynamics of T cells in an EAE model with more severe disease after therapy. We observed that B-cell depletion decreases T(eff) expansion, although its oscillatory behavior persists. However, the effect of B cell depletion was more significant in the T(reg) population within the CNS, which matched with activation of microglia and worsening of the disease. Mathematical modeling of T-cell cross-regulation after anti-CD20 therapy suggests that B-cell depletion may influence the dynamics of T cells by fine-tuning their activation.

Conclusions: The oscillatory dynamics of T-cells have an intrinsic origin in the physiological regulation of the adaptive immune response, which influences both disease phenotype and response to immunotherapy.

Figure 1

MOG-specific T cells and microglia subpopulations analyzed by flow cytometry in mice with EAE. Leukocytes were obtained from the spleen and the CNS from MOG_35-55 immunized C57BL/6 mice at different stages of EAE as indicated. A) T_eff and T_reg lymphocytes are shown. Numbers indicate the percentage of cells within the CD4+ cell gate. The histogram on the right shows the expression of CD69 on the surface of T_eff and T_reg cell subsets in the spleen. Numbers indicate the percentage of CD69+ cells. B) CD4+ cells stained with MOG_35-55/IA^b tetramer. MOG-specific T_eff and T_reg cells are shown. C) Dot-plots showing CD45⁺CD11b⁺MCH-II(IA^b)⁺ microglia population. D) Antigen-specific T_eff and T_reg oscillate over the course of EAE. Numbers indicate the percentage within the CD4+ cell gate. MOG-specific T cells from spleen (a, b) and CNS (c, d) in the onset, peak and recovery stages of EAE are shown. Animals were not treated (a, c) or treated with anti-CD20 antibody before immunization (b, d).

Figure 2

Dynamics of activated MOG-T_eff, MOG-Treg cells and microglia in secondary lymphoid organs and CNS during the course of EAE. A) Percentage of MOG-specific T_eff (red) or T_reg (blue) in the spleen after immunization. The upper graph shows the mean of the measurements from 3 animals each day for both populations (T_eff and T_reg) together. The bottom panels show the distribution of all measurements (raw data) from 3 animals per day for each subpopulation as dots, with the mean in black; B) percentage of MOG-specific T_eff (red) or T_reg (blue) in the CNS from day 9 after immunization. The upper graph shows the mean of the measurements from 3 animals each day, while the bottom panels show the distribution of all measurements for each subpopulation as dots, with the mean in black; C) Clinical score of animals suffering EAE after immunization (mean + SD); D) Percentage of activated microglia in the CNS from day 9 after immunization.

Figure 3

Analysis of the time dependence between microglia activation and MOG-specific T-cell infiltration of the CNS. Distribution of the average of incursion of MOG-specific T cells into the CNS (Teff and Treg) versus microglia phase cycle.

Figure 4

Simulations from the cross-regulation T-cell model. A) Graphical representation and the equations of the T-cell cross-regulation model as described in [10]. Parameters of the model are described in Additional file 1: Table S1. Simulation of the time course of the number of MOG-specific T_eff (red) or T_reg (blue) cells in the spleen (y-axis) after immunization (x-axis, in days) by the cross-regulation model in ‘healthy’ (B) and in ‘EAE’ configuration regimes (C). Simulations are shown by mean ± SD of 3 simulations for 30 days (discretized by days) to express average points similarly to experimental data in Figure 2A; D) Phase-space plot shows T_eff/T_reg trajectories in simulations (30 days) for both configurations (‘healthy’ in black and ‘EAE’ in red).

Figure 5

Dynamics of activated MOG-T_eff, MOG-T_reg cells and microglia in secondary lymphoid organs and CNS during the course of EAE after B cell depletion. A) Percentage of MOG-specific T_eff (red) or T_reg (blue) cells in the spleen after immunization. The upper graph shows the mean of measurements from 3 animals each day, and the bottom panels show the distribution of all measurements for each subpopulation as dots, with the mean in black; B) percentage of MOG-specific T_eff (red) or T_reg (blue) cells in the CNS from day 9 after immunization. The upper graph shows the mean of measurements from 3 animals each day, and the bottom panels show the distribution of all measurements for each subpopulation as dots, with the mean in black; C) clinical score of animals suffering EAE after B cell depletion (mean + SD); D) percentage of activated microglia in the CNS from day 9 after immunization.

Figure 6

Modeling the effect of anti-CD20 mediate B-cell depletion in the cross-regulation T-cell model. A) Simulation of the time course of the number of MOG -specific T_eff (red) and Tr_eg (blue) cells in the spleen (y-axis) after immunization (x-axis, in days) by the cross-regulation model using values for α_reg and K_eff from the ‘EAE’ regime (see Results section). B) Phase-space plots showing T_eff/T_reg cell counts trajectories in simulations of 100 (B) and 30 (D) days for ‘healthy’ (black), EAE (blue) and B-cell depletion (red) simulations. The 30-day simulation (D) was performed in order to compare with experimental data in C (in C, points ‘a’ and ‘b’ are marked as first peaks recorded in experiments for both groups compared). Simulations were discretized in days to show data points similar to experimental data in Figure 5A.