(C) Mice were challenged with CT26 cells i.v. contrast, NKT cell-deficiency in CD1d?/? mice conferred significant protection against lung tumors but had no effect on the growth of tumors in the skin, and tumor rejection induced against the CT26 in the lung did not confer protection for the same tumor cells in the skin. Thus, effector cells against the same tumor do not work in all tissues, and the induction site of the effector T cells is critical to control metastasis. Further, the regulation of tumor immunity may be different for the same tumor in different anatomical locations. a nylon membrane and cells were washed before leukocytes were fractionated Rabbit polyclonal to OAT using Percoll (Sigma-Aldrich, St. Louis, MO). Flow cytometry Single cell suspensions were stained with LIVE/DEAD? Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) followed by incubation with anti-CD16/CD32 and PBS57-loaded CD1d-tetramer (NIH tetramer core facility) before surface and intranuclear staining. Fluorescently labeled monoclonal antibodies against CD45, TCR- chain, CD8, CD4, CD25 and FoxP3 were obtained from BioLegend, San Diego, CA. True-Nuclear staining buffer (BioLegend) was utilized for intranuclear staining. Cells were analyzed for fluorescence by FACSymphony (BD Bioscience) and Flowjo (FlowJo, LLC, Ashland, Oregon). Statistics The Gonadorelin acetate data were analyzed using the nonparametric MannCWhitney test, Kruskal-Wallis with post hoc Dunn’s test or 2-way ANOVA with Tukey post hoc using GraphPad Prism (version 5 and 7; GraphPad software). Significance was decided at 0.05. All experiments were repeated at least twice to confirm reproducibility of results, and representative data from impartial experiments are shown. Results CD25+ cells suppress anti-tumor immunity in the skin, not lung We first assessed the role of CD25+ cells in regulation of anti-tumor immunity in the skin and lungs. Depletion of CD25+ cells resulted in s.c. tumor rejection in all mice (Fig. 1A). All mice developed palpable tumors that completely regressed beginning at day 10. In contrast, there was no effect on tumor growth in the lungs (Fig. 1B) although we confirmed a significant reduction of the number of CD4+CD25+Foxp3+T cells in the lungs of anti-CD25 treated mice (Fig. 1C). Flow cytometry analysis of leukocytes in tumors in the lung and skin and the normal lung showed that skin tumors contain a significantly higher frequency of CD4+Foxp3+ Treg cells than the lungs with tumors (Fig. 1D). This result is usually consistent with the observation that anti-CD25 treatment had no effect on tumor progression in the lung. It was also interesting to find that tumors in both tissues contained significant numbers of CD1d-restricted type I NKT cells. The majority of these NKT cells were a CD4?CD8? subset that has been suggested to be protective against lung metastasis of B16 melanoma.22 There was virtually no infiltation of CD8+ T cells in pre-necrotic tumors in the skin, and no increase in these cells in the tumor-bearing lung even though CT26 is known Gonadorelin acetate to express the immunogenic retrovirus-derived antigen, gp70. When investigating the dependence of subcutaneous CT26 tumor rejection on CD8+, CD4+ T or NK cells depletion and depletion experiments may be due to differences in the depletion method vs in spleen cell preparations (although in both cases full depletion was verified by flow cytometry) and to the fact that this cells in Fig. 3C were adoptively transferred into RAG1?/? mice that have no T or B cells of their own, a very different environment from the intact mouse. Nevertheless, the same overall finding that both CD4+ and CD8+ T cells are involved in the protection was true in both models. Open in a separate window Figure 3. Rejection of s.c. tumors following CD25-depletion induces a memory response that protects from subsequent tumor rechallenge. (A) Mice were injected with 0.5?mg anti-CD25 (PC61) i.v., and 5?days later, mice were challenged with 50,000 CT26 cells s.c.. At 3 months after tumors were rejected, the mice were re-challenged with CT26 cells either s.c. on the same flank as the original challenge or on the opposite flank or i.v.. Survival data plots are shown as representatives of two independent experiments.In contrast to the memory T cell induction induced by the clearance of s.c. protect against lung metastases of the same tumors. In contrast, NKT cell-deficiency in CD1d?/? mice conferred significant protection against lung tumors but had no effect on the growth of tumors in the skin, and tumor rejection induced against the CT26 in the lung did not confer protection for the same tumor cells in the skin. Thus, effector cells against the same tumor do not work in all tissues, and the induction site of the effector T cells is critical to control metastasis. Further, the regulation of tumor immunity may be different for the same tumor in different anatomical locations. a nylon membrane and cells were washed before leukocytes were fractionated using Percoll (Sigma-Aldrich, St. Louis, MO). Flow cytometry Single cell suspensions were stained with LIVE/DEAD? Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) followed by incubation with anti-CD16/CD32 and PBS57-loaded CD1d-tetramer (NIH tetramer core facility) before surface and intranuclear staining. Fluorescently labeled monoclonal antibodies against CD45, TCR- chain, CD8, CD4, CD25 and FoxP3 were obtained from BioLegend, San Diego, CA. True-Nuclear staining buffer (BioLegend) was utilized for intranuclear staining. Cells were analyzed for fluorescence by FACSymphony (BD Bioscience) and Flowjo (FlowJo, LLC, Ashland, Oregon). Statistics The data were analyzed using the nonparametric MannCWhitney test, Kruskal-Wallis with post hoc Dunn’s test or 2-way ANOVA with Tukey post hoc using GraphPad Prism (version 5 and 7; GraphPad software). Significance was determined at 0.05. All experiments were repeated at least twice to confirm reproducibility of results, and representative data from independent experiments are shown. Results CD25+ cells suppress anti-tumor immunity in the skin, Gonadorelin acetate not lung We first assessed the role of CD25+ cells in regulation of anti-tumor immunity in the skin and lungs. Depletion of CD25+ cells resulted in s.c. tumor rejection in all mice (Fig. 1A). All mice developed palpable tumors that completely regressed beginning at day 10. In contrast, there was no effect on tumor growth in the lungs (Fig. 1B) although we confirmed a significant reduction of the number of CD4+CD25+Foxp3+T cells in the lungs of anti-CD25 treated mice (Fig. 1C). Flow cytometry analysis of leukocytes in tumors in the lung and skin and the normal lung showed that skin tumors contain a significantly higher frequency of CD4+Foxp3+ Treg cells than the lungs with tumors (Fig. 1D). This result is consistent with the observation that anti-CD25 treatment had no effect on tumor progression in the lung. It was also interesting to find that tumors in both tissues contained significant numbers of CD1d-restricted type I NKT cells. The majority of these NKT cells were a CD4?CD8? subset that has been suggested to be protective against lung metastasis of B16 melanoma.22 There was virtually no infiltation of CD8+ T cells in pre-necrotic tumors in the skin, and no increase in these cells in the tumor-bearing lung even though CT26 is known to express the immunogenic retrovirus-derived antigen, gp70. When investigating the dependence of subcutaneous CT26 tumor rejection on CD8+, CD4+ T or NK cells depletion and depletion experiments may be due to differences in the depletion method vs in spleen cell preparations (although in both cases full depletion was verified by flow cytometry) and to the fact that the cells in Fig. 3C were adoptively transferred into RAG1?/? mice that have no T or B cells of their own, a very different environment from the intact mouse. Nevertheless, the same overall finding that both CD4+ and CD8+ T cells are involved in the protection was true in both models. Open in a separate Gonadorelin acetate window Figure 3. Rejection of s.c. tumors following CD25-depletion induces a memory response that protects from subsequent tumor rechallenge. (A) Mice were injected with 0.5?mg anti-CD25 (PC61) i.v., and 5?days later, mice were challenged with 50,000 CT26 cells s.c.. At 3 months after tumors were rejected, the mice were re-challenged with CT26 cells either s.c. on the same flank as the original challenge or on the opposite flank or i.v.. Survival data plots are shown as representatives of two independent experiments (n = 5). Statistical significance was determined by Log-rank test *p 0.05, **p 0.01. (B) Depletion antibodies were given the day before, the day of, and 5?days after the second tumor challenge. S.c. tumor area was measured, and tumor-free survival data plots are shown as representatives of two independent experiments (n = 5) (left panel), and survival data plots are shown (right panel). (C) 1? 107 T cells from.
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