Mechanisms of immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple sclerosis

Abstract

Relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL mouse is a Th1-mediated autoimmune demyelinating disease model for human multiple sclerosis and is characterized by infiltration of the central nervous system (CNS) by Th1 cells and macrophages. Disease relapses are mediated by T cells specific for endogenous myelin epitopes released during acute disease, reflecting a critical role for epitope spreading in the perpetuation of chronic central CNS pathology. We asked whether blockade of the CD40-CD154 (CD40L) costimulatory pathway could suppress relapses in mice with established R-EAE. Anti-CD154 antibody treatment at either the peak of acute disease or during remission effectively blocked clinical disease progression and CNS inflammation. This treatment blocked Th1 differentiation and effector function rather than expansion of myelin-specific T cells. Although T-cell proliferation and production of interleukin (IL)-2, IL-4, IL-5, and IL-10 were normal, antibody treatment severely inhibited interferon-gamma production, myelin peptide-specific delayed-type hypersensitivity responses, and induction of encephalitogenic effector cells. Anti-CD154 antibody treatment also impaired the expression of clinical disease in adoptive recipients of encephalitogenic T cells, suggesting that CD40-CD154 interactions may be involved in directing the CNS migration of these cells and/or in their effector ability to activate CNS macrophages/microglia. Thus, blockade of CD154-CD40 interactions is a promising immunotherapeutic strategy for treatment of ongoing T cell-mediated autoimmune diseases.

Figures

Figure 1

Anti-CD154 inhibits clinical progression of established R-EAE. Mice were primed with PLP139-151/CFA as described in Methods. Mice were treated with either anti-CD154 or control hamster IgG at time of priming (a), at the peak of acute disease (b) (square symbols), or during the remission following recovery from acute disease (b) (triangular symbols). The data are expressed as the mean clinical score versus days after immunization for mice treated at the time of immunization (a), or as relapse rate for those mice treated during ongoing disease (b). Data shown are representative of two similar experiments. Ab, antibody; R-EAE, relapsing experimental autoimmune encephalomyelitis.

Figure 2

Effect of anti-CD154 treatment on CNS histology. Lumbar spinal cord sections from representative animals of the treatment groups described in Fig. 1 were examined for CNS histopathology. (a) Section of spinal cord from a mouse treated with control Ig at the time of priming and sacrificed on day 25. (b) Section of spinal cord from a mouse treated with anti-CD154 at the time of priming and sacrificed on day 25. (c) Section of spinal cord from a mouse treated with control Ig at the peak of acute disease and sacrificed on day 40. (d) Section of spinal cord from a mouse treated with anti-CD154 at the peak of acute disease and sacrificed on day 40. Details of the degree of inflammation and demyelination are given in Table 1. Sections shown in a–d are 1-μm-thick Epon-embedded sections stained with toluidine blue. ×220. CNS, central nervous system.

Figure 3

Anti-CD154 treatment inhibits DTH responses to both the initiating and relapse-associated myelin epitopes. DTH responses to both the initiating PLP139-151 peptide and to the relapse-associated PLP178-191 peptide were evaluated in mice treated with either control Ig or anti-CD154 at the time of priming (a) or at the peak of acute disease (b). Data represent mean ± SD of the change in ear thickness 24 h after ear challenge with 10 μg of peptide. The numbers of mice in each group are shown below each bar. Data are representative of two experiments with similar results. DTH responses significantly less than those of control Ig-treated mice: *P < 0.05; **P < 0.01). Results shown above each bar show statistical comparisons to background ear-swelling levels, i.e., levels in naive mice challenged with each peptide. DTH, delayed-type hypersensitivity.

Figure 4

Myelin epitope-specific in vitro proliferative responses of spleen and lymph node cells from mice treated with anti-CD154 during disease induction or at the peak of acute disease. Splenic and lymph node lymphocytes from three mice each treated with control Ig or anti-CD154 either at the time of priming were harvested at day 10 after immunization (a and b) or at the peak of acute disease were harvested at day 25 after immunization (c and d). A total of 5 × 105 viable cells per well were cultured with varying concentrations of PLP139-151 or PLP178-191 for 4 days. Cultures were pulsed with 3H-TdR 18–24 h before harvest. Data are presented as ΔCPM (3H-TdR incorporation in cultures containing peptide antigen – 3H-TdR incorporation in cultures containing medium). Data shown are representative of two experiments. Mean ± SD of viable lymphocytes recovered from each treatment group (minimum of three mice each from three separate experiments) is shown in the legend (*P < 0.05). Stimulation indices (S.I.) are shown for each treatment group at the concentration of peptide that stimulated optimal proliferation. An S.I. ≥3 is considered significant.

Figure 5

Anti-CD154 treatment blocks the differentiation of PLP139-151-specific, IFN-γ–producing Th1 cells. Splenic and lymph node lymphocytes from three mice each treated at time of priming with either control Ig or anti-CD154 were harvested at day 10 after immunization. A total of 2 × 105 viable cells/well were cultured with an optimal dose of PLP139-151 (25 μM). Culture supernatants were harvested each day and analyzed for IL-2, IFN-γ, and IL-10 by ELISA, as described in Methods. Data shown are representative of two experiments. In all cases, the SD was less than 10% of the values. IFN, interferon; IL, interleukin.

Figure 6

Anti-CD154 treatment of donor mice diminishes the capacity of PLP139-151–primed T cells to transfer R-EAE. Female SJL/J mice were primed with PLP139-151/CFA. These donor mice treated with either 250 μg of control Ig (open squares) or anti-CD154 (closed squares) on days 0, 2, 4, and 6. Draining lymph nodes were harvested on day 8 and cultured with 70 μg/ml PLP139-151 in vitro. After 4 days in culture, naive female SJL/J recipient mice were injected intravenously with 107 (a), 5 × 106 (b), or 2.5 × 106 (c) viable cells. In b, some mice received cells from anti-CD154–treated donors that were also treated with 25 μg/ml of anti-CD154 in vitro (open triangles). In addition, some mice received cells from anti-CD154–treated donors that were exposed to anti-CD154 during the in vitro activation culture and transferred into anti-CD154 treated recipients (closed triangles). Recipients treated with anti-CD154 were injected with 250 μg intraperitoneally three times a week. Mice were examined for disease severity and scored as described in Methods.

Figure 7

Anti-CD154 treatment of recipient mice diminishes the capacity of PLP139-151–primed T cells to transfer R-EAE. Untreated female SJL/J mice were primed with PLP139-151/CFA, and draining lymph nodes were harvested on day 8 and cultured with 70 μg/ml PLP in vitro. After 4 days in culture, naive female SJL/J recipient mice were injected intravenously with 5 × 106 (a), 2.5 × 106 (b), or 1.25 × 106 (c) viable cells. Recipient mice were then treated with 250 μg of control Ig (open boxes) or anti-CD154 (closed boxes) administered intraperitoneally three times a week. Mice were examined for disease severity and scored as described in Methods.