Up-regulation of proliferative and migratory genes in regulatory T cells from patients with metastatic castration-resistant prostate cancer

Abstract

A higher frequency of regulatory T cells (Tregs) has been observed in peripheral blood mononuclear cells (PBMC) of patients with different types of solid tumors and hematological malignancies as compared to healthy donors. In prostate cancer patients, Tregs in PBMC have been shown to have increased suppressive function. Tumor-induced biological changes in Tregs may enable tumor cells to escape immunosurveillance. We performed genome-wide expression analyses comparing the expression levels of more than 38,500 genes in Tregs with similar suppressive activity, isolated from the peripheral blood of healthy donors and patients with metastatic castration-resistant prostate cancer (mCRPC). The differentially expressed genes in mCRPC Tregs are involved in cell cycle processes, cellular growth and proliferation, immune responses, hematological system development and function and the interleukin-2 (IL-2) and transforming growth factor-β (TGF-β) pathways. Studies revealed that the levels of expression of genes responsible for T-cell proliferation (C-FOS, C-JUN and DUSP1) and cellular migration (RGS1) were greater in Tregs from mCRPC patients as compared to values observed in healthy donors. Increased RGS1 expression in Tregs from mCRPC patients suggests a decrease in these Tregs' migratory ability. In addition, the higher frequency of CD4(+) CD25(high) CD127(-) Tregs in the peripheral blood of mCRPC patients may be the result of an increase in Treg proliferation capacity. Results also suggest that the alterations observed in gene expression profiles of Tregs in mCRPC patients may be part of the mechanism of tumor escape from host immune surveillance.

Figure 1

Analysis of suppressive function of CD4⁺CD25^high Tregs. (a) Example of suppression of CD4⁺CD25⁻ T-cell proliferation by CD4⁺CD25^high Tregs in a healthy donor. Isolated effectors (CD4⁺CD25 ) and Tregs (CD4⁺CD25^high) were cultured separately, or cocultured at 3 different ratios (1:1, 1:0.5, and 1:0.2) upon stimulation with anti-CD3 and irradiated T cell-depleted PBMCs. Experiments were performed in triplicate. Columns: average count per minute (cpm); bars: SD. Percentages indicate level of inhibition of CD4⁺CD25⁻ T-cell proliferation. (b) Expression of FoxP3 in CD4⁺CD25⁺CD127^lo/− Tregs in a healthy donor. PBMCs from 3 healthy donors were analyzed by flow cytometry after cell surface staining with APC-Cy7-conjugated anti-CD4, PE-Cy7-conjugated anti-CD25, PerCP Cy5.5-conjugated anti-CD127, and intracellular staining with APC-conjugated FoxP3. The figure represents results from 1 healthy donor and similar results were also observed in other healthy donors and prostate cancer patients. Percentage indicates proportion of cells expressing FoxP3 in the CD4⁺CD25⁺CD127^lo/− Treg population.

Figure 2

PCA model showing the global gene expression difference between healthy donors (red balls; n = 3) and mCRPC patients (blue balls; n = 3).

Figure 3

Verification of gene expression by NanoString analysis and flow cytometry. Expression of (a) C-FOS, (b) C-JUN, (c) DUSP1, and (d) RGS1 in CD4⁺CD25^highCD127^lo/− Tregs from healthy donors (n = 10) and mCRPC patients (n = 9) was analyzed by NanoString analysis. C-JUN protein expression in CD4⁺CD25^highCD127^lo/− Tregs was further examined by flow cytometry analysis. (e) Percentage of c-Jun⁺ cells from CD4⁺CD25^highCD127^lo/− Tregs was compared between healthy donors (n = 8) and mCRPC patients (n = 8). (f) Percentage of c-Jun⁺ cells in CD4⁺CD25^highCD127^lo/− Foxp3⁺ Tregs. A statistically significant difference was seen between healthy donors and mCRPC patients in all analyses. *, p < 0.05, **, p < 0.01, and ***, p < 0.001.

Figure 3

Verification of gene expression by NanoString analysis and flow cytometry. Expression of (a) C-FOS, (b) C-JUN, (c) DUSP1, and (d) RGS1 in CD4⁺CD25^highCD127^lo/− Tregs from healthy donors (n = 10) and mCRPC patients (n = 9) was analyzed by NanoString analysis. C-JUN protein expression in CD4⁺CD25^highCD127^lo/− Tregs was further examined by flow cytometry analysis. (e) Percentage of c-Jun⁺ cells from CD4⁺CD25^highCD127^lo/− Tregs was compared between healthy donors (n = 8) and mCRPC patients (n = 8). (f) Percentage of c-Jun⁺ cells in CD4⁺CD25^highCD127^lo/− Foxp3⁺ Tregs. A statistically significant difference was seen between healthy donors and mCRPC patients in all analyses. *, p < 0.05, **, p < 0.01, and ***, p < 0.001.

Figure 3

Verification of gene expression by NanoString analysis and flow cytometry. Expression of (a) C-FOS, (b) C-JUN, (c) DUSP1, and (d) RGS1 in CD4⁺CD25^highCD127^lo/− Tregs from healthy donors (n = 10) and mCRPC patients (n = 9) was analyzed by NanoString analysis. C-JUN protein expression in CD4⁺CD25^highCD127^lo/− Tregs was further examined by flow cytometry analysis. (e) Percentage of c-Jun⁺ cells from CD4⁺CD25^highCD127^lo/− Tregs was compared between healthy donors (n = 8) and mCRPC patients (n = 8). (f) Percentage of c-Jun⁺ cells in CD4⁺CD25^highCD127^lo/− Foxp3⁺ Tregs. A statistically significant difference was seen between healthy donors and mCRPC patients in all analyses. *, p < 0.05, **, p < 0.01, and ***, p < 0.001.

Figure 4

Differential gene expression between mCRPC patient and healthy donor Tregs is not applicable to all CD4⁺ cells. Expression of (a) c-Fos, (b) c-Jun, (c) DUSP1 and (d) RGS1 was examined at the protein level in CD4⁺CD25⁻ cells isolated from mCRPC patients (n = 5) and healthy donors (n = 5). No significant differences were seen in the percentage of positive cells between the 2 groups (p > 0.05).

Figure 4

Differential gene expression between mCRPC patient and healthy donor Tregs is not applicable to all CD4⁺ cells. Expression of (a) c-Fos, (b) c-Jun, (c) DUSP1 and (d) RGS1 was examined at the protein level in CD4⁺CD25⁻ cells isolated from mCRPC patients (n = 5) and healthy donors (n = 5). No significant differences were seen in the percentage of positive cells between the 2 groups (p > 0.05).

Figure 5

Percentage of CD4⁺CD25^highCD127^lo/− Tregs in peripheral blood of healthy donors (n = 17) and mCRPC patients (n = 17). (a) Levels of CD4⁺CD25^highCD127^lo/− Tregs are presented as a percentage of total CD4⁺ T cells. A statistically significant difference was observed between the 2 groups (p < 0.05). (b) Percentage of FoxP3 expression in CD4⁺CD25^highCD127^lo/− Tregs from healthy donors (n = 8) and mCRPC patients (n = 8). No statistically significant difference was observed between the 2 groups (p > 0.05).