Repetitive injections of dendritic cells matured with tumor necrosis factor alpha induce antigen-specific protection of mice from autoimmunity

Abstract

Mature dendritic cells (DCs) are believed to induce T cell immunity, whereas immature DCs induce T cell tolerance. Here we describe that injections of DCs matured with tumor necrosis factor (TNF)-alpha (TNF/DCs) induce antigen-specific protection from experimental autoimmune encephalomyelitis (EAE) in mice. Maturation by TNF-alpha induced high levels of major histocompatibility complex class II and costimulatory molecules on DCs, but they remained weak producers of proinflammatory cytokines. One injection of such TNF/DCs pulsed with auto-antigenic peptide ameliorated the disease score of EAE. This could not be observed with immature DCs or DCs matured with lipopolysaccharide (LPS) plus anti-CD40. Three consecutive injections of peptide-pulsed TNF/DCs derived from wild-type led to the induction of peptide-specific predominantly interleukin (IL)-10-producing CD4(+) T cells and complete protection from EAE. Blocking of IL-10 in vivo could only partially restore the susceptibility to EAE, suggesting an important but not exclusive role of IL-10 for EAE prevention. Notably, the protection was peptide specific, as TNF/DCs pulsed with unrelated peptide could not prevent EAE. In conclusion, this study describes that stimulation by TNF-alpha results in incompletely matured DCs (semi-mature DCs) which induce peptide-specific IL-10-producing T cells in vivo and prevent EAE.

Figure 1.

Surface phenotype and cytokine production of DCs matured with TNF-α. (a) DCs were generated from C57BL/6 mice until day 8 in the presence of IL-10 to inhibit their maturation, or in the absence of IL-10 and additional stimulation with TNF-α or anti-CD40 plus LPS overnight. FACS^® analysis shows the expression of the indicated markers (straight line) or isotype controls (dotted line) of CD11c⁺ cells within a life gate. Numbers within histograms represent the mean fluorescence of the marker with subtracted isotype values. (b) DCs were cultured in a 24-well plate until day 8. Then, cells were stimulated without transfer for 24 h with TNF-α, anti-CD40, LPS, or their combinations, before supernatants were tested for their cytokine content by ELISA. (c) DCs were stimulated at day 8 as indicated and harvested after 24 h. The levels of mRNA expression were detected by RNase protection assay. The data for (a) is representative of more than six, and the data for b and c are each representative of three independent experiments with similar results.

Figure 2.

Repetitive injections of TNF/DCs protect mice from EAE DCs were simultaneously pulsed with MOG peptide and treated with TNF-α for 4 h (MOG/TNF/DC), washed in PBS, and 2.5 × 10⁶ cells were injected once (1×) or three times (3×) intravenously into 3–4 C57BL/6 mice per group. Control mice were left without DC injections (untreated). 3 d after the last/only DC injection EAE was induced and the mice observed for paralysis. (a) Injection of immature DCs, generated in the presence of IL-10, pulsed with MOG peptide, and injected 3 d before EAE induction, were not protective. (b) DCs matured with LPS plus anti-CD40 and pulsed with MOG peptide and injected before EAE induction were not protective. (c) Single injections of MOG pulsed and TNF-α matured DCs, given 7 d before EAE induction, could ameliorate the disease. (d) Three injections of MOG-pulsed TNF/DC but not unpulsed TNF/DC given at days −7, −5, and −3 before EAE induction completely protected mice from EAE. (e) OVA/TNF/DCs could not protect from EAE. As a peptide specificity control, three injections of OVA-pulsed TNF/DC were given at days −7, −5, and −3 before EAE induction or mice were left untreated. OVA peptide was additionally emulsified together with the MOG peptide in CFA at day 0 in this experiment. The data for a–d are each representative of three independent experiments with similar results.

Figure 2.

Repetitive injections of TNF/DCs protect mice from EAE DCs were simultaneously pulsed with MOG peptide and treated with TNF-α for 4 h (MOG/TNF/DC), washed in PBS, and 2.5 × 10⁶ cells were injected once (1×) or three times (3×) intravenously into 3–4 C57BL/6 mice per group. Control mice were left without DC injections (untreated). 3 d after the last/only DC injection EAE was induced and the mice observed for paralysis. (a) Injection of immature DCs, generated in the presence of IL-10, pulsed with MOG peptide, and injected 3 d before EAE induction, were not protective. (b) DCs matured with LPS plus anti-CD40 and pulsed with MOG peptide and injected before EAE induction were not protective. (c) Single injections of MOG pulsed and TNF-α matured DCs, given 7 d before EAE induction, could ameliorate the disease. (d) Three injections of MOG-pulsed TNF/DC but not unpulsed TNF/DC given at days −7, −5, and −3 before EAE induction completely protected mice from EAE. (e) OVA/TNF/DCs could not protect from EAE. As a peptide specificity control, three injections of OVA-pulsed TNF/DC were given at days −7, −5, and −3 before EAE induction or mice were left untreated. OVA peptide was additionally emulsified together with the MOG peptide in CFA at day 0 in this experiment. The data for a–d are each representative of three independent experiments with similar results.

Figure 3.

Repetitive injections of TNF/DCs induce peptide-specific CD4⁺ T cells producing IL-10 which is partially involved in EAE protection. (a) TNF/DCs induce IL-10–producing cells from restimulated spleen cells. C57BL/6 mice received three intravenous injections of MOG-pulsed TNF/DCs or LPS/CD40/DCs at days −7, −5, and −3. Spleen cells from these mice were restimulated at day 0 with 10 μM MOG peptide or 10 μM unrelated OVA peptide. Cell supernatants were taken after 24, 48, 72, and 96 h and tested for their cytokine content by ELISA. (b) The IL-10 production from spleen cells induced by DC injections is derived from CD4⁺CD3⁺ T cells, but not CD11c⁺ DCs. C57BL/6 mice received three injections of MOG pulsed TNF/DC at days −7, −5, and −3. Spleen cells from these mice were sorted for CD4⁺ and CD11c⁺ cells at day 0 and then stimulated for 24 h with PMA plus Ionomycin before supernatants were tested by ELISA for their IL-10 content. From the CD4⁺ enriched cells 97% coexpressed CD3, and the contamination CD11c⁺ cells within the CD4⁺ cells was 0.8–1.4%. (c) The protection from EAE partially depends on IL-10. C57BL/6 mice (3–4 per group) were injected three times with MOG/TNF/DCs at days −7, −5, and −3 (3×) and EAE was induced at day 0. Mice were injected intraperitoneally with anti–IL-10R mAb or isotype mAb at the same days as TNF/DCs and additionally at day −1 and day 1. Control mice remained without pretreatment by DCs and were not injected with mAb. The data for (a) is representative of three, and the data for b and c are each representative of two independent experiments with similar results.