Regulatory T cells and IL-10 independently counterregulate cytotoxic T lymphocyte responses induced by transcutaneous immunization

Abstract

Background: The imidazoquinoline derivate imiquimod induces inflammatory responses and protection against transplanted tumors when applied to the skin in combination with a cognate peptide epitope (transcutaneous immunization, TCI). Here we investigated the role of regulatory T cells (T(reg)) and the suppressive cytokine IL-10 in restricting TCI-induced cytotoxic T lymphocyte (CTL) responses.

Methodology/principal findings: TCI was performed with an ointment containing the TLR7 agonist imiquimod and a CTL epitope was applied to the depilated back skin of C57BL/6 mice. Using specific antibodies and FoxP3-diphteria toxin receptor transgenic (DEREG) mice, we interrogated inhibiting factors after TCI: by depleting FoxP3(+) regulatory T cells we found that specific CTL-responses were greatly enhanced. Beyond this, in IL-10 deficient (IL-10(-/-)) mice or after blocking of IL-10 signalling with an IL-10 receptor specific antibody, the TCI induced CTL response is greatly enhanced indicating an important role for this cytokine in TCI. However, by transfer of T(reg) in IL-10(-/-) mice and the use of B cell deficient JHT(-/-) mice, we can exclude T(reg) and B cells as source of IL-10 in the setting of TCI.

Conclusion/significance: We identify T(reg) and IL-10 as two important and independently acting suppressors of CTL-responses induced by transcutaneous immunization. Advanced vaccination strategies inhibiting T(reg) function and IL-10 release may lead the development of effective vaccination protocols aiming at the induction of T cell responses suitable for the prophylaxis or treatment of persistent infections or tumors.

Figure 1. FoxP3⁺ regulatory T cells inhibit TCI-induced immune response.

(A) Transcutaneous immunizations with imiquimod were performed at days 0 and 1 with Aldara (50 mg together with 100 µg SIINFEKL peptide). Mice were additionally treated with diphtheria toxin (days -1, 1 and 3), anti-CD25 (day -5) or anti-IL-10R (day -2) as indicated. DEREG mice were depleted of FoxP3⁺ regulatory T cells by intraperitoneal administration of diphtheria toxin (1 µg). Percentages of CD4⁺FoxP3⁺ T_reg were assessed at days 0 and 7 (B). Specific CD8⁺ T cells (C) and cytolytic activity (D) were quantified by flow cytometric analysis 7 days after immunization. The depicted results are cumulative from three independent experiments with three mice per group. (*) Significant difference (p<0.05) by Mann-Whitney test.

Figure 2. Reduction of regulatory T cell numbers results in enhanced immune responses.

C57BL/6 mice were transcutaneously immunized as described before. Were indicated mice received anti-CD25 (clone PC61, 500 µg i.p., day -5) for reduction of regulatory T cell numbers. Percentages of CD4⁺FoxP3⁺ T_reg were assessed at days 0 and 7 (A). Specific CD8⁺ T cells (B) were quantified by H2-K^b-Tetramer staining (percentage of CD8⁺H2-K^b-SIINFEKL⁺ cells). (C) Cytolytic activity was determined 8 hours after adoptive transfer of peptide-loaded splenocytes. Stated percentage indicates specific lysis of target cells. A cumulative analysis of two independent experiments with three mice per group is shown.

Figure 3. Prevention of IL-10 signalling enhances the induced immune response.

IL-10^-/- and wild type mice were immunized as described before. (A) Peptide specific T cells as well as (B) lysis of peptide loaded target cells were assessed. C57BL/6 mice were immunized with TCI. Where indicated mice received an intraperitoneal injection of a blocking anti-IL-10-receptor antibody (aIL-10R, 250 µg, d-2). Shown are the results for (C) the quantity of peptide specific T cells, and (D) in vivo lysis of transferred target cells. A cumulative analysis of three independent experiments with three mice per group is shown. (*) Significant difference (p<0.05) by Mann-Whitney test.

Figure 4. The suppressive capacity of regulatory T cells is not mediated via IL-10.

C57BL/6 and IL-10^-/--mice were immunized with TCI as described before. Where indicated mice received purified CD4⁺CD25⁺ or CD4⁺CD25^- T cells or splenocytes (containing CD4⁺CD25⁺ and CD4⁺CD25^-)(1×10⁶, d-2) from wild type donors by intravenous injection. After 7 days the amount of H2-K^b-SIINFEKL⁺CD8⁺ T cells from blood samples was assessed. A cumulative analysis of two independent experiments with three mice per group is shown. (*) Significant difference (p<0.05) by Mann-Whitney test.

Figure 5. B cells do not have an impact on TCI induced immune response.

B cell deficient JHT^-/- and C57BL/6 wild type mice were immunized with TCI. Specific CD8⁺ T cells (A) and cytolytic activity (B) were quantified by flow cytometric analysis 7 days after immunization. To assess the role of B cell-derived IL-10 C57BL/6 recipients received bone marrow transplantation of JHT^-/- (80 %) and IL-10^-/--mice (20 %). Control C57BL/6 recipients were reconstituted with Ly5.1 (80 %) and IL-10^-/- (20 %) bone marrow. After complete reconstitution mice were immunized as indicated and the amount of specific CD8⁺ T cells were determined (C). A cumulative analysis of two independent experiments with three mice per group is shown.