Prophylactic DNA vaccine targeting Foxp3+ regulatory T cells depletes myeloid-derived suppressor cells and improves anti-melanoma immune responses in a murine model

Abstract

Regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC) are the two important and interactive immunosuppressive components of the tumor microenvironment that hamper anti-tumor immune responses. Therefore, targeting these two populations together might be beneficial for overcoming immune suppression in the tumor microenvironment. We have recently shown that prophylactic Foxp3 DNA/recombinant protein vaccine (Foxp3 vaccine) promotes immunity against Treg in tumor-free conditions. In the present study, we investigated the immune modulatory effects of a prophylactic regimen of the redesigned Foxp3 vaccine in the B16F10 melanoma model. Our results indicate that Foxp3 vaccination continuously reduces Treg population in both the tumor site and the spleen. Surprisingly, Treg reduction was associated with a significant decrease in the frequency of MDSC, both in the spleen and in the tumor environment. Furthermore, Foxp3 vaccination resulted in a significant reduction of arginase-1(Arg-1)-induced nitric oxide synthase (iNOS), reactive oxygen species (ROS) and suppressed MDSC activity. Moreover, this concurrent depletion restored production of inflammatory cytokine IFN-γ and enhanced tumor-specific CTL response, which subsequently resulted in the reduction of tumor growth and the improved survival rate of vaccinated mice. In conclusion, our results revealed that Foxp3 vaccine promotes an immune response against tumor by targeting both Treg and MDSC, which could be exploited as a potential immunotherapy approach.

Keywords: Foxp3; Melanoma; Myeloid-derived suppressor cells; Regulatory T cells.

Fig. 1

Construction and generation of Foxp3 plasmids and recombinant protein. a, b Schematic representation of the construction of Fox-GFP and Fox-pET plasmids is shown, respectively. c SDS-PAGE analysis of an Foxp3 recombinant protein expressed in BL21 E. coli. M, protein marker; lanes 1 and 2, Fox-pET-transfected BL21 E. coli cells 3 h after IPTG induction; lane 3, Fox-pET-transfected BL21 E. coli cells before induction; lane 4, non-transfected BL21 E. coli cells. d SDS-PAGE analysis of various steps of Foxp3 recombinant protein purification using Ni–NTA resin. Lanes 1–4, final fraction elution of the column (pH 4.5) consists of Foxp3 recombinant protein; lane 5, flow through of the column at pH 6.3; lane 6, flow through of the column at pH 8; M, protein marker; lane 7, Fox-Pet-transfected BL21 E. coli lysate before Ni–NTA resin mixing; lane 8, transfected BL21 E. coli after IPTG induction; and lane 9, transfected BL21 E. coli before IPTG induction. e Western blot analysis of the pooled fraction 2 and 3 elution (pH 4.5) from the Ni–NTA column (lane 1) and protein marker (M) represents the band at the molecular weight of approximately 40 kDa. IPTG isopropyl β d-thiogalactoside

Fig. 2

Foxp3 vaccine reduces CD4⁺Foxp3⁺ Treg. a Schematic representation of vaccination and tumor challenge timeline. C57BL/6 mice (n = 11 per group) were immunized i.m. with pEGFP-N1 or Fox-GFP plasmids on day − 38. Mice were then injected i.d. with molecular grade bovine serum albumin or Foxp3 recombinant protein emulsified in IFA on days − 24 and − 10, as a prime-boost regimen. A week after the boost injection, mice (n = 3 per group) was sacrificed and examined for the frequency of CD4⁺Foxp3⁺ T cells in splenocytes before tumor challenge. On day 0, B16F10 tumor cell line was injected s.c., and on day 13, the final analyses were assessed on spleens and tumors of mice (n = 3 mice per group). b Representative dot plots showing the frequency of CD4⁺Foxp3⁺ T cells in splenocytes and cells isolated from tumor tissues. c Percentages of CD4⁺Foxp3⁺ T cells in splenocytes before tumor challenge. d Percentages of CD4⁺Foxp3⁺ T cells in spleen and tumor tissue obtained from Foxp3 vaccinated versus control mice 13 day post-tumor challenge. e Foxp3 gene expression in splenocytes and tumor tissues of Foxp3 vaccinated versus control mice 13 day post-tumor challenge (β-actin was used as housekeeping gene). Data represent mean ± one standard error of two independent experiments

Fig. 3

Foxp3 vaccine reduces MDSC. The splenocytes and tumor cells from Foxp3-vaccinated or control mice 13 day post-tumor challenge were analyzed for Gr-1 and CD11b using flow cytometry (n = 6 mice per group). a Representative dot plots indicating Gr1⁺CD11b⁺ cells in Foxp3 vaccinated versus control mice. b Percentages of Gr1⁺CD11b⁺ cells in splenocytes versus tumor cells. Data represent mean ± one standard error of two independent experiments

Fig. 4

Foxp3 vaccine strongly modulates the suppressive mechanisms of MDSC. Gr1⁺ cells isolated from splenocytes of Foxp3-vaccinated or control mice 13 day post-tumor challenge and CFSE-labeled naïve T cells isolated from splenocytes of healthy C57BL/6 mice were co-cultured, as described in the “Materials and methods” (n = 6 mice per group). a Representative dot plots showing percent proliferation of CFSE-labeled T cells in the presence of MDSC isolated from Foxp3-vaccinated versus control mice. b Percentages of splenocyte MDSC mediated suppression from Foxp3-vaccinated versus control mice. c Nitrite levels recovered from the supernatant of MDSC-T-cell co-culture. d MFI for ROS among Gr1⁺CD11b⁺ cells in splenocytes and tumor-infiltrated cells obtained from Foxp3-vaccinated and control mice. e, f Expression of iNOS and Arg-1 genes in the splenocytes and tumor tissue of Foxp3-vaccinated versus control mice. β-actin was used as housekeeping gene. Data represent mean ± one standard error of two independent experiments. MFI mean fluorescence intensity

Fig. 5

Foxp3 vaccine promotes CTL responses. Splenocytes from Foxp3-vaccinated and control mice were analyzed for CD107a and IFN-γ expression in CD8⁺ T cells, as described in the “Materials and methods”. a Representative dot plots showing percent CD8⁺CD107a⁺ T cells from the Foxp3-vaccinated and control mice. b Percentages of CD8⁺CD107a⁺ T cells in Foxp3-vaccinated versus control mice. c Representative dot plots showing percentages of CD8⁺ IFN-γ⁺ T cells from the Foxp3-vaccinated and control mice. d Percentages of CD8⁺ IFN-γ⁺ T cells in Foxp3-vaccinated versus control mice (n = 6 mice per group). Data represent mean ± one standard error of two independent experiments

Fig. 6

Foxp3 vaccine reduces the rate of tumor growth and enhances mice survival. a Rate of tumor growth reported in mm² is shown. b Kaplan–Meier curve represents the percentage of mice survival in each group (n = 5 mice per group). Data represent mean ± one standard error of two independent experiments