Impact of curative radiotherapy on the immune status of patients with localized prostate cancer

Abstract

Combination of radiotherapy with immunotherapy has become an attractive concept for the treatment of cancer. The objective of this study was to assess the effect of curative, normofractionated radiotherapy on peripheral immune lymphocytes in prostate cancer patients, in order to propose a rationale for scheduling of normofractionated radiotherapy with T-cell based immunotherapy. In a prospective study (clinicaltrials.gov: NCT01376674), eighteen patients with localized prostate cancer were treated with radiotherapy with or without hormonal therapy. Irradiation volumes encompassed prostate and, in select cases, elective pelvic nodal regions. Blood samples were collected from all patients before, during, and after radiotherapy, as well as from 6 healthy individuals as control. Normofractionated radiotherapy of prostate cancer over eight weeks had a significant influence on the systemic immune status of patients compared to healthy controls. Absolute leukocyte and lymphocyte counts decreased during treatment as did peripheral blood immune subsets (T cells, CD8+ and naïve CD4+ T cells, B cells). Regulatory T cells and NK cells increased. Proliferation of all immune cells except regulatory T cells increased during RT. Most of these changes were transient. Importantly, the functionality of T lymphocytes and the frequency of antigen-specific CD8+ T cells were not affected during therapy. Our data indicate that combination of normofractionated radiotherapy with immunotherapy might be feasible for patients with prostate cancer. Conceptually, beginning with immunotherapy early during the course of radiotherapy could be beneficial, as the percentage of T cells is highest, the percentage of regulatory T cells is lowest, and as the effects of radiotherapy did not completely subside 3 months after end of radiotherapy.

Keywords: Prostate cancer; T cells; immunotherapy; peripheral lymphocytes; radiotherapy.

Figures

Figure 1.

Flow chart of the study and white blood cells counts. (A) HLA-A*02+ patients undergoing primary RT for prostate cancer were included (n = 18). RT regimens are shown (SV = seminal vesicles). Blood samples were obtained before start of treatment (timepoint A), twice during therapy at 1 month intervals (timepoints B, C) and three months after the end of treatment at a follow-up visit (timepoint D). As controls, three consecutive blood samples (A, B, C) were obtained at one month intervals from HLA-A*02+ healthy donors (n = 6). (B) Absolute numbers of leucocytes (left) and lymphocytes (right) are shown before (A), during (B, C) and post-therapy (D) for four patients (four timepoints each, except for one patient for whom only 3 samples were available).

Figure 2.

Impact of RT on T lymphocyte subsets. (A) Percentage of total T cells among viable lymphocytes defined as CD3+ positive cells, (B, C) CD8+ and CD4+ T cells and (D) CD4+CD25+Foxp3+ (Treg) cells within CD4+ cells are shown for the 4 timepoints A-D; n = 18 RT patients are included. Healthy donors are shown in comparison (n = 6, mean of three timepoints per donor). Bars indicate means. Significant differences: * p < 0.05; ** p < 0.01.

Figure 3.

Impact of RT on B cells and NK cells. Percentage of (A) B lymphocytes, and (B) NK cells within viable lymphocytes for the 4 timepoints A-D; n = 18 RT patients are included. Healthy donors are shown in comparison (n = 6, mean of three timepoints per donor). Bars indicate means. Significant differences: ** p < 0.01; *** p < 0.001.

Figure 4.

Proliferation of lymphocyte subsets. (A) Proliferation was assessed by intracellular Ki67 staining in T cells, B cells and NK cells. Bars indicate means. (B) Mean ± SEM of Ki67 expressing cells in total CD4+, total CD8+ and Treg subsets for n = 18 RT patients and n = 6 HD. Significant differences are shown as * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5.

Differentiation status and function of T cells. (A) Naïve cells (CD45RA+CD28+) and effectors (CD45RA+CD28−) are shown. n = 16 RT patients. Bars indicate means and significant differences are shown as * p < 0.05; *** p < 0.001. (B) Viral-specific CD8+ T cells stained with four different HLA-A*02 multimers over time. Intermediate to high frequencies (> 0.1% of the CD8+ subset) are shown on the left panel and low frequencies (< 0.1% of the CD8+ subset) on the right panel. Altogether 20 specificities were detected in n = 9 RT patients. (C) Example of EBV-BMLF1 multimer stainings in one patient. Cells are gated on living CD4− lymphocytes; timepoints and % CD8+multimer+ cells are indicated. (D) Intracellular cytokine production of CD4+ (left, TNF and IL-2) and CD8+ (right, TNF and IFNγ) T cells after activation with SEB. Timepoints A and C are shown for n = 7 RT patients, mean and SD are indicated. The lower panels show the pairwise percentage of TNF+ and IL-2+ and TNF+ and IFNγ+ T cells in the subset of CD4+ and CD8+ T cells, respectively.

Figure 6.

The ratio of CD8+ T cells to Tregs within CD4+ cells at the four timepoints for patients receiving standard (n = 8) vs extended (n = 5) RT. Mean and SEM are shown. Significant differences were detectable in the group treated with extended RT volumes at timepoint C compared to initial CD8+/Treg ratio, as well as between the treatment groups at timepoints C and D. * p < 0.05.