Analysis of circulating regulatory T cells in patients with metastatic prostate cancer pre- versus post-vaccination

Abstract

We have previously shown that the suppressive function of regulatory T cells (Tregs) from peripheral blood mononuclear cells (PBMCs) is enhanced in patients with prostate cancer when compared with healthy individuals. Two phase II studies using the PSA-TRICOM vaccine in patients with metastatic castration-resistant prostate cancer (mCRPC) showed evidence of patient benefit in terms of enhanced survival. The Halabi nomogram has been used to predict survival (HPS) of patients with mCRPC treated with conventional chemotherapy or second-line hormonal therapy. Tregs from PBMCs of patients (n = 23) with mCRPC were obtained pre- and post-three monthly vaccinations, and analyzed for number, phenotype, and suppressive function. Changes post- versus pre-vaccination in these parameters were compared with 3-year survival and HPS. No differences in Treg numbers were observed post- versus pre-vaccination. Trends (P = 0.029) were observed between overall survival (OS) and a decrease in Treg suppressive function post- versus pre-vaccination. Trends were also observed in analyzing effector:Treg (CD4(+)CD25(+)CD127(-)FoxP3(+)CTLA4(+)) ratio post- versus pre-vaccination with OS versus HPS. These data provide preliminary evidence for a possible association between improved OS and a decrease in Treg function when PBMCs are analyzed after three monthly vaccinations. Patients with an OS > HPS were more likely to have decreased Treg function following vaccine. Larger studies to confirm and extend these findings are warranted.

Fig. 1

Expression of FoxP3 in CD4⁺CD25⁺CD127⁻ Tregs in patients receiving the PSA-TRICOM vaccine. a Expression of FoxP3 in CD4⁺CD25⁺CD127⁻ Tregs. PBMCs from a patient were analyzed by flow cytometry after cell surface staining with FITC-conjugated anti-CD4, phycoerythrin-conjugated anti-CD127, PECy7-conjugated anti-CD25, and intracellular staining with APC-conjugated anti-FoxP3. b Percentage of CD4⁺CD25⁺CD127⁻Foxp3⁺ Tregs in CD4 population pre- and post-vaccination in the peripheral blood of all patients enrolled on study. c Percentage of CD4⁺CD25⁺CD127⁻Foxp3⁺ Tregs in CD4 population pre- and post-vaccination in the peripheral blood of patients with OS > HPS. d Percentage of CD4⁺CD25⁺CD127⁻Foxp3⁺ Tregs in CD4 population pre- and post-vaccination in the peripheral blood of patients with OS < HPS. There were no significant differences in levels of Tregs pre- and post-vaccination among patients on study, patients with OS > HPS, and patients with OS < HPS (P = 0.1, P = 0.107, and P = 0.745, respectively)

Fig. 2

Suppression of CD4⁺CD25⁻ T-cell proliferation by CD4⁺CD25^high Tregs in a patient with PCa. Isolated effectors (CD4⁺CD25⁻) and Tregs (CD4⁺CD25^high) were cultured alone or at three different ratios (1:1, 1:0.5, and 1:0.1) upon stimulation with anti-CD3 and irradiated T cell-depleted PBMCs. Experiments were performed in triplicate. Columns mean (cpm), bars SD. Percentages indicate level of inhibition

Fig. 3

Treg suppressive function was not influenced by effector T cells. CD4⁺CD25⁻ T cells from a PCa patient pre-vaccination (a) and post-vaccination (b) were cultured alone or at a 1:1 ratio with Tregs isolated from the same PCa patient post-vaccination, upon stimulation with anti-CD3 and irradiated T cell-depleted PBMCs. CD4⁺CD25⁻ T cells from a PCa patient pre-vaccination (c) and post-vaccination (d) were cultured alone or at a 1:1 ratio with Tregs isolated from the same PCa patient pre-vaccination, upon stimulation with anti-CD3 and irradiated T cell-depleted PBMCs. Experiments were performed in triplicate. Columns means (cpm), bars SD. Percentages indicate level of inhibition

Fig. 4

Expression of CTLA-4 on Tregs. a Levels of CTLA-4-expressing CD4⁺CD25⁺CD127⁻FoxP3⁺ Tregs in PBMCs of all PCa patients on study. No statistically significant differences were detected pre- and post-vaccination (P = 0.380). b Levels of CTLA-4-expressing CD4⁺CD25⁺CD127⁻FoxP3⁺ Tregs in PBMCs of PCa patients with OS > HPS. No statistically significant differences were detected pre- and post-vaccination (P = 0.184). c Levels of CTLA-4-expressing CD4⁺CD25⁺CD127⁻FoxP3⁺ Tregs in PBMCs of PCa patients with OS < HPS. Statistically significant differences were detected pre- and post-vaccination (P = 0.019)

Fig. 5

Effector:CTLA4⁺ Treg ratios post- versus pre-vaccination. a There were no significant differences in effector:CTLA-4⁺ Treg ratio pre- and post-vaccination among PCa patients enrolled on study (P = 0.894). b There was a statistically significant increase in effector:CTLA-4⁺ Treg ratio post-vaccination in PCa patients with OS > HPS (P = 0.029). c There was a statistically significant decrease in effector:CTLA-4⁺ Treg ratio post-vaccination in PCa patients with OS < HPS (P = 0.027)