Distinct functions of autoreactive memory and effector CD4+ T cells in experimental autoimmune encephalomyelitis

Abstract

The persistence of human autoimmune diseases is thought to be mediated predominantly by memory T cells. We investigated the phenotype and migration of memory versus effector T cells in vivo in experimental autoimmune encephalomyelitis (EAE). We found that memory CD4(+) T cells up-regulated the activation marker CD44 as well as CXCR3 and ICOS, proliferated more and produced more interferon-gamma and less interleukin-17 compared to effector T cells. Moreover, adoptive transfer of memory T cells into T cell receptor (TCR)alphabeta(-/-) recipients induced more severe disease than did effector CD4(+) T cells with marked central nervous system inflammation and axonal damage. The uniqueness of disease mediated by memory T cells was confirmed by the differential susceptibility to immunomodulatory therapies in vivo. CD28-B7 T cell costimulatory signal blockade by CTLA4Ig suppressed effector cell-mediated EAE but had minimal effects on disease induced by memory cells. In contrast, ICOS-B7h blockade exacerbated effector T cell-induced EAE but protected from disease induced by memory T cells. However, blockade of the OX40 (CD134) costimulatory pathway ameliorated disease mediated by both memory and effector T cells. Our data extend the understanding of the pathogenicity of autoreactive memory T cells and have important implications for the development of novel therapies for human autoimmune diseases.

Figure 1

Adoptive transfer studies into TCRαβ^−/− mice. A: A representative experiment showing transfer of disease in TCRαβ^−/− mice. Spleens and lymph nodes from wild-type mice were harvested at 12 days (Effector, ▵) or more than 100 days (Memory, ▪) postimmunization; CD4⁺ MACS were negative-selected and cell-sorted for expression of CD44. Naïve CD4⁺ T cells were isolated from nonimmunized mice and used as control. The cells were resuspended in PBS at a concentration of 1 × 10⁶/100 μl and injected i.v. into TCRαβ^−/− mice. Recipients were immunized with MOG35-55 peptide on the day of transfer and graded for disease daily. The mean daily disease grade ± SEM for each group (n = 8 to 16 mice per group) is shown. B: Frequency of MOG-specific CD4⁺ T cells. Splenocytes isolated from short- or long-term MOG-immunized WT mice (n = 3 to 5) were activated in vitro, and the cell suspensions were enriched for T cells by Ficoll-Hypaque density gradient centrifugation and incubated with IA^b multimers (30 μg/ml) in Dulbecco’s modified Eagle’s medium supplemented with IL-2 (5 μmol/L) at 37°C for 3 hours. The cells were acquired using a FACSort, and the number of tetramer-PE (MOG35-55)-positive cells was then determined in live (7-AAD-negative) CD4-positive populations. Splenocytes from B6 mice immunized for 10 (effector) or 100 (memory) days with ovalbumin (Ova) were stained with MOG35-55 tetramer and used as control, confirming the specificity of the tetramer (representative of three independent experiments). C: Splenocytes from mice that received effector or memory T cells were cultured with MOG35-55 peptide at 1, 10, and 100 μg/ml and cell proliferation was measured by [³H]thymidine incorporation. The proliferation rate was significantly higher in cultures from the memory (•) T cell group compared to the effector (▴) group. D: Intracellular FACS staining of splenocytes from MOG35-55-immunized B6 mice. Spleen cells were isolated and activated in vitro with anti-TCR and anti-CD28 antibody for 6 hours and stained with anti-CD4-peridinin-chlorophyll-protein complex, anti-CD44-FITC, and a variety of cytokine-PE antibodies. **P < 0.001; ***P < 0.0001.

Figure 2

Competitive assay of MOG-specific memory, effector, and naïve activated CD4⁺ T cells. A: A total of 1 × 10⁶ MOG-TCR transgenic effector CD4⁺ (CD4⁺CD44^hi, from day 10 postimmunization, ▵) or 1 × 10⁶ memory CD4⁺ (CD4⁺CD44^hi, from day 100 postimmunization, ▪) were transferred into TCRαβ^−/− mice (n = 6 to 8) that were immunized with MOG35-55. Disease induced by MOG-TCR transgenic memory CD4⁺ cells was more severe than that induced by effector CD4⁺ cells (mean maximal score 3.8 ± 0.4 versus 2.8 ± 0.1, P = 0.004), although the time to onset was similar in the two groups. B: Phenotypic characterization of MOG TCR-specific memory, effector, and naïve CD4⁺ T cells. Cells were stained for different markers and analyzed by flow cytometry. Results are expressed as MFI. C: Time course- and tissue-specific analysis of cytokine profile of autoreactive memory (Thy1.1) and effector (Thy1.2) CD4⁺ T cells that were transferred into Thy1.1/Thy1.2 hybrid recipients. D: Another set of experiments was performed to compare memory (Thy1.1) to naïve-activated (Thy1.2) T cells in other hybrid recipients. Cells were collected from the peripheral organs (spleens and lymph nodes) and from the CNS compartment (meninges and spinal cords) and analyzed by flow cytometry before EAE onset (Presymptomatic) and at the peak of the disease (Onset) in the periphery and the CNS. Data represent the percentage of cytokine-positive cells and the mean fluorescence intensity of the surface molecule expression. Bars represent SEM values. *P < 0.05; **P < 0.001.

Figure 3

Inflammatory cell infiltrates in the CNS of TCRαβ^−/− mice recipients of memory versus effector CD4⁺ T cells. Spinal cord tissues from the recipients of memory CD4⁺ T cells (A–C, G–I, and M–O) or effector CD4⁺ T cells (D–F, J–L, and P–R) were harvested 32 days postimmunization, processed for frozen sectioning, and immunostained with rat anti-CD4 (A, D, G, J, M, and P; red), anti-lectin B4 (B and E; green), anti-GFAP (H and K; green), and mouse anti-SMI32 (N and Q; green). Sections were co-stained with the nuclear dye TOPO3 (blue). Merged pictures (C, F, I, L, O, and R) are shown of images captured using confocal imaging at ×63 magnification.

Figure 4

Differential role of costimulatory molecules in disease induced by CD4⁺ memory and effector T cells. A: Effects of CTLA4Ig on the clinical disease induced by the transfer of effector or memory cells into TCRαβ^−/−. B: ELISPOT data of the frequency of IFN-γ-, IL-10-, and IL-17-producing cells. Spleen cells were exposed to MOG peptide (50 μg/ml) for 48 hours and positive cells were quantified. C: Effects of anti-ICOS-L on the clinical disease induced by effector or memory cells. D: ELISPOT analysis of the frequency of cells producing IFN-γ, IL-10, and IL-17 was performed using spleen cells isolated from mice recipients of memory or effector T cell. E: Blockade of OX40 costimulatory pathway using anti-OX40L monoclonal antibody in EAE induced by the memory or effector T cells. F: ELISPOT data of the cytokine profile of memory and effector CD4⁺ T cells exposed to anti-OX40L or control IgG during EAE. Representative of two separate experiments (n = 8 mice per group). *P < 0.05; **P < 0.01.