Mitomycin C-treated dendritic cells inactivate autoreactive T cells: toward the development of a tolerogenic vaccine in autoimmune diseases

Abstract

Treatment of autoimmune diseases remains a challenge for immunological research. An ideal therapy should inhibit the immune reaction against the diseased organ and leave the rest of the immune response intact. Our previous studies showed that donor-derived dendritic cells (DCs) treated in vitro with mitomycin C (MMC) suppress rat heart allograft rejection if injected into recipients before transplantation. Here we analyze their efficacy in controlling autoimmunity. MMC-DCs loaded with myelin-basic-protein (MBP) inhibited specific T cells derived from multiple sclerosis patients in vitro. If coincubated with MMC-DCs, T cells were arrested in the G(0)/G(1) cell cycle phase. Microarray gene scan showed that MMC influences the expression of 116 genes in DCs, one main cluster comprising apoptotic and the second cluster immunosuppressive genes. Apparently, the combination of apoptosis with expression of tolerogenic molecules renders MMC-DCs suppressive. MBP-loaded MMC-DCs also inhibited mouse T cells in vitro and, in contrast to MBP-loaded naïve DCs, did not induce experimental autoimmune encephalitis. Most importantly, mice vaccinated with inhibitory DCs became resistant to the disease. Whereas this is not the first report on generation of suppressive DCs, it delineates a method using a clinically approved drug at nontoxic concentrations, which yields irreversibly changed DCs, effective across species in vitro and in vivo.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Effect of MMC-treated DCs loaded with MBP on resting or activated T cells from MS patients. (A and B) Primary stimulation. DCs loaded with MBP and matured were treated with MMC in various concentrations and coincubated with resting (A) (n=8) or activated peripheral lymphocytes (clone ES-BP8T) (B) (n=7) at a ratio of ≤1:10. Mature DCs were CD14−, HLA-DR2+, CD802+, CD862+, CD83+ and constituted >80% of dendritic cells. Negative controls were MBP-loaded DCs or lymphocytes only, whereas positive controls were MBP-loaded MMC-untreated DCs coincubated with lymphocytes. Additional negative controls of experiment B (not shown) consisted of DCs only or DCs loaded with an irrelevant peptide, both coincubated with lymphocytes (none of them inducing T cell stimulation). (C and D) Restimulation. CD4+ cells were isolated from previous culture settings and restimulated with freshly prepared MBP-loaded DCs of the same donor. Legend to abscissa shows how CD4 cells were pretreated. Ordinate shows T cell proliferation. The first column represents DCs only (negative control). Cell proliferation was assessed by [3H]thymidine incorporation. Data represent mean ± SD and are expressed as percentage of positive control values (MBP-loaded DCs plus lymphocytes = 100%) (for all MMC-treated cells vs. untreated controls P < 0.05).

Fig. 2.

Flowcytometric analysis of apoptosis following treatment of DCs with MMC. DCs were treated with 50 μg/ml MMC and labeled with annexin-V-FITC and 7-AAD after 2, 6, and 24 h of incubation. Controls consisted of untreated DCs (− MMC). The lower and upper right quadrants show apoptosis. Percentages of apoptotic cells are displayed.

Fig. 3.

Effect of MMC-treated, MBP-presenting dendritic cells in vivo. (A) Tg4 mice were injected intravenously with either MBP-DCs (♦) or MMC-treated MBP-DCs (■). (B) Mice immunized in experiment A with MMC-treated MBP-DCs (■) were challenged on day 28 with MBP-loaded DCs. Mice immunized in experiment A with MBP-DCs (♦) served as controls. Evaluation of EAE was performed according to the Coligan score. Data are shown as mean values ± SEM (n = 8 per group).

Fig. 4.

Prophylactic vaccination against EAE with MBP-loaded MMC-treated dendritic cells. (A) Tg4 mice were repetitively immunized with MBP-MMC-DCs. On day 0 these (■), as well as nonvaccinated mice (♦), were challenged with MBP-pulsed DCs. (B) The EAE severity (Coligan score) is shown, starting from day 10 after MBP-DC challenge. Data are displayed as mean ± SEM (n = 8 nonvaccinated, n = 10 vaccinated group).