Normal Th1 development following long-term therapeutic blockade of CD154-CD40 in experimental autoimmune encephalomyelitis

Abstract

Experimental autoimmune encephalomyelitis (EAE) is a Th1-mediated demyelinating disease of the CNS with similarities to multiple sclerosis. We and others have shown that a short-term course of anti-CD154 mAb treatment to block CD154-CD40 interactions can be used to prevent or even treat ongoing PLP139-151-induced relapsing EAE. However, little is known of the long-term effects of CD154 blockade on the development of antigen-specific T cell function. Here, we show that short-term treatment with anti-CD154 at the time of PLP139-151/CFA immunization inhibits clinical disease for up to 100 days after immunization. At this point, comparable numbers of Th1 cells are observed in anti-CD154 and control Ig-treated mice, as assessed by antigen-specific ELISPOT assays. Thus, the long-term Th1/Th2 balance is largely unaffected. Inflammatory responses are diminished in anti-CD154-treated mice, as indicated by reduced in vivo delayed-type hypersensitivity and reduced levels of splenic IFN-gamma secretion in vitro. However, upon adoptive transfer of T cells isolated from the spleens of anti-CD154-treated mice, these cells contributed as effectively to clinical disease as those obtained from control-treated mice. Thus, anti-CD154 therapy leads to long-term therapeutic efficacy without exerting a long-term influence on Th1 development.

Figure 1

Short-term anti-CD154 mAb therapy causes long-term inhibition of EAE induction. (a) The indicated numbers of SJL mice were immunized with PLP139-151/CFA on day 0 and treated with 200 μg of either hamster IgG or anti-CD154 antibody (MR-1). Data represent combined results obtained from two separate experiments. *Disease incidence was significantly less (P < 0.0001) in anti-CD154–treated mice. (b and c) Mice immunized and treated with control Ig (b) or anti-CD154 (c) were sacrificed 105 days after immunization and sections were taken from lumbar and thoracic spinal cord sections, as described in Table 1. Sections demonstrate the continued normal appearance of spinal cord from anti-CD154–treated versus extensive immune cell infiltration, demyelination, and scarring observed in control Ig–treated mice.

Figure 2

Short-term anti-CD154 therapy inhibits in vivo PLP139-151–specific antibody and DTH responses. Mice were immunized with PLP139-151/CFA and treated with control Ig or anti-CD154 as described in Figure 1. (a) Sera obtained from mice 25 days after immunization were analyzed for anti–PLP139-151–specific IgG levels. Data were obtained from five serological samples combined from two separate experiments. (b) PLP139-151–specific DTH responses were measured at 100 days after immunization. Data presented are the combined results from two separate experiments, and the number of mice in each group is marked below each bar. *DTH response was significantly less (P < 0.05) in anti-CD154–treated mice.

Figure 3

Short-term anti-CD154 therapy has no long-term effect on specific proliferative responses. (a) Splenic and lymph node (LN) cells were obtained 100 days after immunization, and in vitro PLP139-151–specific proliferative responses were determined by ³H-TdR incorporation. Data are representative of two separate experiments. (b) Supernatants from proliferative cultures described above were analyzed for secretion of IL-2, IL-4, IL-5, and IFN-γ by capture ELISA. IFN-γ secretion from splenic T cells was significantly reduced in anti-CD154–treated mice. Data are representative of two separate experiments with three mice per experimental group. There was no detectable IL-4 or IL-5 secretion observed from any culture supernatants.

Figure 4

Frequencies of PLP139-151–specific Th1, Th2, and Th0 cells are comparable 100 days after immunization in control- and anti-CD154–treated mice. Splenic and lymph node CD4⁺ T cells were taken 100 days after immunization, and ELISPOT was used to determine the frequencies of Th1 (IFN-γ–secreting) cells, Th2 (IL-5–secreting) cells, and Th0 (IL-2–secreting) cells from lymph node and splenic compartments of control- and anti-CD154–treated mice. Data, shown as mean ± SEM, were obtained from three pooled samples and represent two separate experiments.

Figure 5

Short-term anti-CD154 therapy does not affect the early expression of homing receptors on OVA323-339–specific transgenic T cells in the draining lymph node. Five million DO11.10 transgenic OVA323-339–specific T cells were transferred to naive BALB/c recipients, and the recipients were immunized with 500 μg of OVA323-339/CFA and treated with either control Ig or anti-CD154 as described in Methods. Three days after immunization, lymph node cells were obtained and flow cytometric analysis of homing receptors evaluated ex vivo on KJ1-26 cells. Antigens critical for T cell entry into the CNS (CD44 and CD49d; a and b) and into the lymph node (CD62L; c), and IL-12–dependent homing receptors (E- and P-selectin ligands; d and e), were evaluated. Control antibody staining is indicated by the dashed line, staining in control Ig–treated mice by the black line, and staining in anti-CD154–treated mice by the gray line. Data are representative of three separate experiments.

Figure 6

Peripheral T cells from asymptomatic anti-CD154–treated mice demonstrate full encephalitogenic capacity. Splenic (Spl) T cells were isolated from anti-CD154– and control Ig–treated mice 30 days after PLP139-151/CFA immunization. These cells (5 × 10⁶ per recipient) were then transferred to naive recipient mice with or without cotransfer of 2 × 10⁶ encephalitogenic Th1 blasts. The Th1 blasts were obtained from the draining lymph nodes of mice 10 days after PLP139-151/CFA immunization and 4 days of in vitro reactivation in the presence of PLP139-151 peptide. Recipient mice were observed for clinical signs of disease for 31 days after transfer. Data are representative of two separate experiments with identical results.