A T helper cell 2 (Th2) immune response against non-self antigens modifies the cytokine profile of autoimmune T cells and protects against experimental allergic encephalomyelitis

Abstract

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS), and the most commonly used experimental model for multiple sclerosis. It is mediated by autoreactive T cell clones exhibiting a T helper cell (Th) 1 cytokine profile. Nonencephalitogenic T lymphocytes specific for self or exogenous antigens have been found to suppress encephalitogenic T cell responses and to protect against autoimmune disease. The mechanisms by which exogenous antigens modulate autoimmunity are not fully understood. In this study, we tested the hypothesis that a Th2-type immune response against an exogenous, nonself antigen, keyhole limpet hemocyanin (KLH), by releasing IL-4 in the microenvironment, could shift the cytokine profile of encephalitogenic T cells from an inflammatory Th1 to a protective Th2 type. SJL/J mice were preimmunized with the KLH in incomplete Freund's adjuvant to induce a population of Th2 memory cells that would be expected to release Th2 cytokines when activated by the specific antigen at the time of EAE induction. Four weeks later, mice received an encephalitogenic challenge containing guinea pig myelin in complete Freund's adjuvant with or without KLH. All KLH primed animals not receiving the exogenous antigen at the time of EAE induction developed a severe clinical disease indistinguishable from control mice not KLH primed. In contrast, animals preimmunized and challenged with the encephalitogenic inoculum containing KLH showed either no, or markedly reduced, clinical signs. Enzyme-linked immunospot analysis demonstrated that KLH-specific T cells in the primed mice were producing IL-4 characteristic of Th2 cells. In the KLH-primed and restimulated mice, the cytokine profile of the autoreactive, myelin basic protein-specific T cells was shifted from an inflammatory Th1 towards a protective Th2 type. We infer that the presence of IL-4 secreted by KLH-specific memory Th2 cells in the lymphoid system microenvironment in which the autoreactive T cells were engaged by the encephalitogenic stimulus were able to bias their cytokine profile towards a protective Th2 phenotype. This interpretation is supported by the observation that the protective effect of preimmunization with KLH was overcome by rm-IL-12, which inhibited the production of IL-4 by the Th1 cells and biased the autoimmune response to a predominantly Th1 type. Since IL-4 mRNA could not be detected by reverse transcriptase PCR in the CNS, the protective effect was inferred to be mediated by Th2 cells in the lymphoid system, and not the target organ. We conclude that exogenous, nonself antigens that can induce Th2 responses, can modify the cytokine environment sufficiently to alter the cytokine phenotype of inflammatory, autoreactive T cell clones, and ultimately, to provide significant protection against EAE and possibly other T cell-mediated autoimmune diseases.

Figure 1

Effect of the KLH preimmunization and challenge at the time of EAE induction on the incidence and clinical score of the disease. Two groups of SJL/J mice were injected intraperitoneally with an emulsion of KLH in IFA. One control group was injected with PBS. After 4 wk, EAE was induced by immunizing the animals with guinea pig myelin in CFA. One of the two groups of mice pretreated with KLH received the foreign antigen with the encephalitogenic inoculum. This group of mice was almost completely protected (filled circles) against EAE. The control group that was not preimmunized with KLH (open circles) or the group that was primed, but not challenged a second time with KLH (open squares) both showed high incidences and clinical scores for the disease.

Figure 2

rmIL-12 at the time of immunization overcomes protection engendered by pretreatment and reticulation with KLH. One group of mice that was pretreated with KLH in IFA received the antigen again with the encephalitogenic inoculum together with rmIL-12 (0.3 mg/ mouse), given on days 0, 1, and 2 after immunization (filled squares). The mice receiving the minimal treatment with rmIL-12 were not protected against EAE and showed a significantly higher mean clinical score compared to the KLH-protected mice (filled circles).

Figure 3

Determination of cytokine profiles of KLH (a) and MBP (b) specific short-term T cell lines. Two mice of each group were killed at day 10 p.i. and lymph node cells stimulated in vitro with KLH or MBP for 6 d. The number of IL-4 (crosshatched bars) and IFN-γ (solid bars) was determined by ELISPOT analysis. The KLH-specific lymph node T cells of mice immunized with the antigen in IFA showed a Th2 cytokine profile. When the mice were treated with rmIL-12, the number of IL-4–producing cells was decreased. In control mice receiving only PBS, the primary antigenic stimulation in vitro with KLH induced a Th1 phenotype. The MBP-specific T cells showed a Th1 inflammatory profile in nonprotected controls (not pretreated with, or not restimulated with KLH) while in KLH-protected mice, the cytokine profile was shifted to a Th2 type. Treatment with rmIL-12 for only 3 d was sufficient to modify the phenotype of the MBP-specific T cells in the KLH/IFA-treated mice from Th2 from Th1.

Figure 3

Determination of cytokine profiles of KLH (a) and MBP (b) specific short-term T cell lines. Two mice of each group were killed at day 10 p.i. and lymph node cells stimulated in vitro with KLH or MBP for 6 d. The number of IL-4 (crosshatched bars) and IFN-γ (solid bars) was determined by ELISPOT analysis. The KLH-specific lymph node T cells of mice immunized with the antigen in IFA showed a Th2 cytokine profile. When the mice were treated with rmIL-12, the number of IL-4–producing cells was decreased. In control mice receiving only PBS, the primary antigenic stimulation in vitro with KLH induced a Th1 phenotype. The MBP-specific T cells showed a Th1 inflammatory profile in nonprotected controls (not pretreated with, or not restimulated with KLH) while in KLH-protected mice, the cytokine profile was shifted to a Th2 type. Treatment with rmIL-12 for only 3 d was sufficient to modify the phenotype of the MBP-specific T cells in the KLH/IFA-treated mice from Th2 from Th1.

Figure 4

IFN-γ and IL-4 mRNA in the CNS of KLHprotected and control mice sensitized to myelin. The RT-PCR was performed on RNA samples extracted from brain and spinal cord of the mice at day 16 after immunization using specific primers for IFN-γ and IL-4. The mRNAs for IFN-γ and IL-4 were measured by quantitative PCR using a competitor DNA fragment for each, and the quantities expressed as attomoles of cDNA per microgram of total RNA.