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. 2013 Jun 3;15(4):461–465. doi: 10.1038/aja.2013.22

Analysis of circulating regulatory T cells (CD4+CD25+CD127−) after cryosurgery in prostate cancer

Tong-Guo Si 1,*, Jun-Ping Wang 2,*, Zhi Guo 1
PMCID: PMC3739231  PMID: 23728588

Abstract

This study was performed to assess the response of regulatory T cells (Tregs) following cryosurgery in prostate cancer (PCa) patients by measuring their frequency and immune function. Blood was collected prior to and at 4 and 8 weeks after treatment in 30 patients with high-risk PCa who underwent cryosurgery and from 15 healthy volunteers. Circulating CD4+CD25+CD127− Tregs were isolated. Their frequency was detected by flow cytometry, and immune suppressive function was evaluated by measuring the proliferation of CD4+CD25− T cells cocultured with Tregs. The results showed that the percentage of circulating CD4+CD25+CD127− Tregs was increased in PCa patients compared to healthy volunteers (7.6%±0.73% vs. 5.8%±0.54%, P<0.001). The frequency of circulating CD4+CD25+CD127− Tregs was reduced 4 weeks after cryosurgery compared to before surgery (6.3%±0.58% vs. 7.6%±0.73%, P<0.001), and the decrease persisted for 8 weeks. However, the suppressive function of Tregs was increased in eight of 12 patients, which might contribute to cancer recurrence. Then the response of circulating Tregs is complicated after cryosurgery for PCa, and further studies are warranted.

Keywords: cryosurgery, immune response, prostate cancer (PCa), regulatory T cell (Tregs)

Introduction

Cryosurgery is a minimally invasive technique that has proven to be effective in the treatment of patients with prostate cancer (PCa).1 This treatment destroys tumour tissue in situ by freezing, leaving tumour proteins and tumour-associated antigens intact, which can potentially modulate the host's immune response.2 In a mouse melanoma model, den Brok and collaborators3 demonstrated that tumour debris generated by cryosurgery could be captured by dendritic cells (DCs) and reach the draining lymph nodes, leading to a mild tumour-specific immune response. Further studies showed that the immune response was enhanced by the coadministration of anticytotoxic T-lymphocyte-associated antigen 4 blocking antibodies. Blocking anticytotoxic T-lymphocyte-associated antigen 4 abolishes the function of naturally occurring CD4+CD25+Foxp3+ T-regulatory cells.4,5 To the best of our knowledge, no direct assessment of the effect of cryosurgery on the Treg population and Treg function has been reported in clinical studies.

CD4+CD25+FoxP3+ regulatory T cells (Tregs) play an important role in immune homeostasis because of their ability to suppress the activation of T cells, and an increase in the number or functionality of Tregs could thus favour tumour development. Increased levels of CD4+CD25+FoxP3+ Tregs have been detected in peripheral blood mononuclear cells, the tumour microenvironment, and the draining lymph nodes in patients with PCa,6,7,8 other solid tumours9 and haematological malignancies.10 Clinical studies have demonstrated that Tregs can inhibit both antigen-specific and nonspecific T-cell responses11,12 and that an increase in FoxP3+ Tregs is associated with an increased risk of recurrence.13,14

Currently, the isolation and expansion of human Treg subsets into functionally active, disease-specific T cells is difficult due to the paucity of Tregs in the peripheral blood and the lack of specific identity markers for Tregs. In humans, CD4+CD25+ T cells are a mixed population, including suppressor CD4+CD25high T cells as well as CD4+CD25low T cells, which are non-suppressive, activated CD4+ T cells. Furthermore, the expression of Treg markers such as cytotoxic T–lymphocyte antigen 4 (CTLA-4) or glucocorticoid-induced tumour-necrosis factor receptor (GITR) can vary depending on cell activation, and these markers have not been useful for discriminating Tregs from effector T-cell populations. Similarly, Foxp3 expression, although more specific for Tregs, may also be upregulated on effector cells following activation.15 Furthermore, due to its intracellular localisation, Foxp3 cannot be used for the isolation of live Tregs. Recently, two groups have independently shown that CD127 expression, which is the α chain of the interleukin-7 receptor, discriminates CD127low Tregs from CD127high conventional T cells within the CD25+CD45RO+/RA− effector/memory and the CD45RA+RO− naive compartments in human peripheral blood and lymph nodes.16,17

In this study, we used these methods to evaluate changes in qualitative and quantitative parameters of Treg populations in patients with PCa after cryosurgery.

Patients and methods

Patient characteristics

Thirty PCa patients, with a median age of 67 years, who underwent cryosurgery of the prostate for clinically localized, T1c–T2b PCa with high-risk features were identified. The inclusion criteria were as follows: patients with prehormone therapy high-risk features of PCa, which were defined as either a prostate-specific antigen (PSA) level ≥10 ng ml−1, a pathology report indicating a Gleason sum score ≥8, or both; no previous hormonal, immunosuppressive, or radiation therapy before this treatment; and no evidence of metastatic disease by bone scan, computer-assisted tomography, or magnetic resonance imaging. The patient characteristics are summarized in Table 1.

Table 1. Patient characteristics and treatment outcome.

Characteristic Value, mean (range)
Total no. 30
Median age (year) 67 (55–74)
TNM  
 T1c 11
 T2a 12
 T2b 7
Gleason score  
 6 5
 7 8
 8 13
 9 4
Mean PSA (ng ml−1)  
 Before treatment 14.5 (9.2–31.4)
 1 month after treatment 0.12 (0–0.3)
 3 months after treatment 0.17 (0–0.8)
 6 months after treatment 0.51 (0–6.7)a
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Abbreviations: PSA, prostate-specific antigen.

a

The values of PSA in patient 6 and patient 18 were 6.7 and 4.7 ng ml−1, respectively; cancer recurrence was proven by biopsy.

Cryosurgery treatment

All patients received a rectal enema the night before the procedure. Under spinal anaesthesia, the patients were placed in a lithotomy position. Cystoscopy was performed, and a suprapubic bladder catheter was placed under direct vision. All procedures were performed according to the modified Onik technique using an ENDOCare unit with argon and helium gas for freezing and thawing, respectively, for a total of two freeze–thaw cycles. Under transrectal ultrasound guidance, 5–7 cryoprobes were introduced into the prostate, and four thermoprobes were located bilaterally in the neurovascular bundles, one in Denonvilliers' fascia and the other at the sphincter. The freezing process was monitored in real time by transrectal ultrasonography and using thermoprobes to enable the direct visualisation of the ice ball and avoid creating lesions on adjacent tissues. During the procedure, the urethra was protected with a warming device at 37 °C degrees that was maintained in place until the patient left the operating room. All patients were discharged within 24 h, and the suprapubic catheter was removed after 1 week.

Blood sample preparation

Blood samples were obtained from 30 PCa patients prior to and 1 month after the treatment and from 15 age-matched healthy volunteers as controls (NCs). All subjects signed an informed consent approved by the Institutional Review Board of Tianjin Medical University. Peripheral blood mononuclear cells were isolated by centrifugation on Ficoll-Paque (Amersham Pharmacia Biotech). The cells were washed twice in phosphate-buffered saline without calcium and magnesium (pH 7.2) and resuspended in X-VIVO 15 medium (BioWhittaker and In Vitro) for further analysis.

Flow cytometry

Four-colour flow cytometry was performed on an FACSCalibur (BD Biosciences) with CellQuest Pro software. All monoclonal antibodies and isotype controls used were purchased from eBiosciences (San Diego, CA, USA): PE-Texas Red (ECD)-labelled anti-CD3, PC5-labelled anti-CD4, FITC-labelled anti-CD25 and PE labelled anti-CD127. After red blood cell lysis (Q-prep System; Beckman Coulter, Hialeah, FL, USA), naturally occurring Tregs were characterized by the expression of CD4 and CD25 and the lack of expression of CD127. The results are expressed as percentages of the CD4+ lymphocyte population and as the number of cells per microlitre of whole blood. In addition, CD56+/CD16+ cells were identified as natural killer (NK) cells.

Immunosuppression assays

CD4+CD25− T cells (1×104 cells per well) were cultured alone or with Tregs in three different ratios with 1 µg ml−1 of anti-CD3 antibody (OKT3; eBiosciences) in the presence of irradiated (3500 rad) T-cell-depleted peripheral blood mononuclear cells (1×105 cells per well) in a 96-well flat-bottomed plate at 37 °C and 5% CO2. Cells were cultured in RPMI 1640 (Mediatech; Manassas, VA, USA) supplemented with 10% heat-inactivated human AB serum (Gemini BioProducts; West Sacramento, CA, USA), 100 units per ml of penicillin, 100 µg ml−1 of streptomycin (Mediatech) and 2 mmol l−1 of L-glutamine (Mediatech). Proliferation was measured by [3H] thymidine (PerkinElmer, Waltham, MA, USA) incorporation at 1 µCi (0.037 MBq) per well. Cells were pulsed on day 4 and quantified 18 h later using a liquid scintillation counter (PerkinElmer). All experiments were performed in triplicate. Proliferation of CD4+CD25− T cells without coculturing with Tregs was considered 100% proliferation.

Statistical analysis

The data are presented as the mean±s.d. because all variables were normally distributed. The statistical significance of differences between two groups was assessed by a two-tailed t-test. When comparisons were performed at multiple time points, a one-way analysis of variance followed by Bonferroni's post-test was used. P<0.05 was considered statistically significant.

Results

The PSA scores were all significantly decreased 4 weeks after treatment, and no cases of PSA failure were observed within 2 months. After 6 months, an elevated PSA score was found in two patients, and cancer recurrence was proven by biopsy (Table 1); hormone therapy was recommended. The observed complications were mostly minor, with 20% (4/20) reporting mild haematuria, and 15% (3/20) reporting perineal/scrotum haematoma with no need for further treatment. Rectal injury and urinary incontinence, which was defined as the need for one or more pads per day, were not observed.

We evaluated the levels of Tregs in the peripheral blood of PCa patients and healthy volunteers. CD4 cells were gated for CD25+ and CD127− cells. There was a significantly increased percentage of CD4+CD25+CD127− cells in PCa patients compared to healthy volunteers (7.6%±0.73% vs. 5.8%±0.54%, respectively, P<0.001) (Figure 1). Although the absolute count of CD4+ cells in the patients was decreased compared to healthy volunteers, the absolute count of CD4+CD25+CD127− cells in patients was also increased significantly compared to healthy volunteers. However, there was no significant difference between the two groups in either the percentage or absolute number of NK cells found (Table 2).

Figure 1.

Figure 1

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Tregs (CD4+CD25+CD127−) were isolated before cryosurgery (a, 7.2%) and 4 weeks after cryosurgery (b, 6.4%). Tregs, regulatory T cells.

Table 2. The lymphocyte sub-populations on admission.

  Patients Healthy volunteers t P
CD4+ lymphocytes (103 µl−1) 0.67±0.06 0.72±0.05 2.11 0.04
Tregs (cell µl−1) 51.5±6.81 41.7±5.43 4.85 <0.001
Tregs (% of CD4) 7.6±0.73 5.8±0.54 8.57 <0.001
NK cells (cell µl−1) 64.3±5.13 66.9±4.28 1.73 0.09
NK cells (% of lymphocytes) 6.3±0.42 6.6±0.52 1.83 0.07
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Abbreviations: NK, natural killer; Tregs, regulatory T cells.

The effects of cryosurgery on peripheral CD4+CD25+CD127− lymphocytes were evaluated, and the results showed that the percentage of CD4+CD25+CD127− cells in the patients was decreased significantly (6.3%±0.58% vs. 7.6%±0.73%, P<0.001) at 4 weeks after cryosurgery. Although the absolute count of CD4+ lymphocytes did not change markedly, the absolute count of CD4+CD25+CD127− cells did decrease significantly (42.3±5.01 µl−1 vs. 51.5±6.81 µl−1, P<0.001). Moreover, the decrease persisted up to 8 weeks after treatment (Table 3).

Table 3. The effect of cryoablation on the frequency of Tregs.

  Before treatment 4 weeks after treatment 8 weeks after treatment
CD4+ lymphocytes (103 µl−1) 0.67±0.07 0.70±0.06* 0.68±0.05*
Tregs (cell µl−1) 51.5±6.81 42.3±5.01* 44.1±5.06*
Tregs (% of CD4) 7.6±0.73 6.3±0.58* 6.4±0.46*
CD4+CD25− (% of CD4) 73.2±7.2 74.6±6.3 75.2±8.1
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Abbreviation: Tregs, regulatory T cells.

*Significant differences were observed between before and 4 weeks after treatment or 8 weeks after treatment (P<0.001), but no difference was observed between 4 weeks after treatment and 8 weeks after treatment.

To determine whether cryosurgery could affect the levels of Treg suppressive function, in vitro functional studies were performed using CD4+CD25+CD127− cells collected from patients before and 4 weeks after cryosurgery (Figure 2). Because large volumes of peripheral blood were required to purify Tregs and evaluate Treg function, only 12 patients were included in this part of the study. As shown in Table 4, the suppressive activity of CD4+CD25+CD127− cells increased in seven patients, and cancer recurred in two of the patients 6 months after treatment (Table 4).

Figure 2.

Figure 2

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Suppression of CD4+CD25− T-cell proliferation by CD4+CD25+CD127− Tregs in a patient with PCa. Isolated effectors (CD4+CD25−) and Tregs (CD4+CD25+CD127−) were cultured alone or at three different ratios: column C 62.3% at 1∶1, column D 42.6% at 1∶0.5 and column E 15.2% at 1∶0.1. PCa, prostate cancer; Tregs, regulatory T cells.

Table 4. The effect of cryoablation on the suppressive function of Tregs.

Patients % Treg suppression Δ Suppression (post–pre)
  Pre–treatment Post-treatment  
#2 54.6 63.2 8.6
#3 38.4 28.9 −9.5
#5 46.5 31.4 −15.1
#6a 35.5 52.4 16.9
#9 50.6 40.2 −10.4
#11 47.6 43.4 −4.2
#12 45.2 67.3 22.1
#15 66.2 42.5 −23.7
#18a 35.4 51.3 15.9
#21 24.6 33.5 8.9
#24 43.2 52.6 9.4
#27 35.6 49.0 13.4
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Abbreviation: Tregs, regulatory T cells.

a

Cancer recurrence was proven by biopsy 6 months after treatment.

Discussion

PCa is the most common noncutaneous malignancy diagnosed in males, and it has become a large threat for ageing patients in Asia.18 Cryosurgery is an effective treatment strategy for PCa that destroys tumour and prostate gland tissue through freezing. The development of cryotherapy for localized PCa provides a potentially curative option for patients with primary or recurrent disease, with less morbidity than radical surgery.19 However, the American Urological Association has only recently accepted cryosurgery as a primary modality in the treatment of localized PCa, as published in the ‘Best practice statement on cryosurgery for the treatment of localized PCa'.20 Nonetheless, cryotherapy is currently indicated as a primary therapy for low-risk patients as an alternative to prostatectomy or radiotherapy in higher surgical risk patients and as a salvage procedure for patients who have not responded to radiation therapy. The Cryotherapy On-Line Data Registry (COLD Registry) used the Phoenix definition (nadir+2 ng ml−1 definition) to report 5-year biochemical disease-free survival rates in 1198 patients: 91% in low-risk patients, 78% in intermediate-risk patients and 62% in high-risk patients. These biochemical results compare quite favourably to the results of radiation therapy as a monotherapy.21 A recent randomized trial comparing cryotherapy with radiation therapy reported similar biochemical outcomes and a significantly lower rate of positive post-treatment biopsies in the cryotherapy-treated patients.22

Cryosurgery leaves tumour proteins and tumour-associated antigens intact. The presence of residual tumour antigens in an inflammatory microenvironment can stimulate anti-tumour immune responses.23 The immunological effects of cryosurgery were first documented by demonstrating the production of antibodies against rabbit male reproductive tissues after cryosurgery in the 1960s.24 Unfortunately, immunological assays at that time were limited. Therefore, the existence of a cryoimmunological response remained controversial, and the mechanisms by which this may occur were unknown. However, increased interest in the clinical potential of cryosurgery and a more detailed understanding of the mechanisms by which the immune system recognizes and targets tumour antigens have generated a renewed interest in the field of cryoimmunology. In experiments in several animal models of cancer including melanoma,3 colon cancer25 and breast cancer,26 the tumour debris generated by tumour cryosurgery was captured by DCs and transported to the draining lymph nodes, resulting in a weak but tumour-specific immune response. In a PCa study, Lubaroff et al.27 demonstrated that cryosurgery alone was not effective in producing an immune response that was protective against rechallenge in the Dunning R3327 adenocarcinoma model, but a recent clinical study showed that cryosurgery for PCa could induce limited tumour-specific cytotoxic T-cell stimulation.28 Several studies have demonstrated that the cryoimmunological response could be enhanced by other adjunct treatments such as the intratumour administration of DCs,25,29 Toll-like receptor stimulation30 and Treg depletion.3

Tregs (5%–10% of peripheral CD4+ T cells) are primarily generated in the thymus and represent an essential mechanism of peripheral tolerance to self-antigens.31 These cells may suppress or kill CD8+ or CD4+ T cells, resulting in suppressed antitumour immunity. Increased Treg numbers have been observed in patients with malignant tumours, including PCa, gastric cancer, lung cancer, pancreatic cancer and breast cancer.32 Additionally, some evidence suggests that increased Treg numbers are correlated with a poor prognosis for patients with malignant tumours.33,34 Furthermore, the elimination of these cells or the inhibition of their function may increase the antitumour immune response.35,36

Previous studies have suggested that cryosurgery can reduce the release of immunosuppressive factors and that tumour antigens are released into the circulation by necrotic tumour cells, leading to increased antitumour immunity.37 The present study indicated that the frequency and absolute number of peripheral Tregs were increased in patients with PCa, which is consistent with a previous study.6 More importantly, this study provided clinical evidence that cryosurgery for PCa can affect the peripheral Treg pool. Indeed, 4 weeks after treatment, the number of these cells was reduced significantly, and this effect lasted for at least 8 weeks after treatment. Surprisingly, the results showed that the suppressive effects of Tregs were increased in most patients (8/12) after cryosurgery. Moreover, cancer recurrence was observed in two of these patients. These results appear to be controversial because no direct evidence of an influence of cryosurgery on the frequency or function of Tregs in PCa patients was found.

Several studies have found that the carcinoma microenvironment can induce, recruit, and/or activate and expand Tregs.38 It is possible that the tumour cells or other cells inside the tumour secrete chemokines that attract Tregs to migrate into the tumour. Tregs have been shown to express a variety of chemokine receptors, including CCR4, CCR7, CCR8, CXCR4 and CXCR5, depending on their activation status and tissue location.38 A previous study on PCa demonstrated that some PCa cell lines, malignant ascites fluid and prostate tumour biopsies in culture contain or secrete CCL22 and chemoattract Tregs in an in vitro migration assay.6 Cryosurgery of tumour tissue leads to cellular coagulative necrosis and causes cellular breakdown and the release of intracellular contents and proinflammatory cytokines that initiate the innate immune response and attract granulocytes and/or macrophages.40 We therefore hypothesized that some cytokines released after cryosurgery attract Tregs into the tumour microenvironment and trigger the suppressive function simultaneously. The frequency of Tregs in the circulation would therefore be reduced, but increased suppressive function would be detected. To prove this hypothesis, further studies of the molecular mechanisms involved in the origin, function and interactions of Tregs after cryosurgery are urgently required. As animal studies have shown that cryosurgery combined with Treg depletion can enhance the antitumour immunological response to reduce cancer recurrence,3 our findings suggest that monitoring Treg frequency and function in addition to administering anti-Treg treatment, such as cyclophosphamide 41 or ipilimumab,42 might be crucial to allow the development of effective antitumour immunity after cryosurgery. Further studies are warranted.

In this study, we attempted to measure the function of Tregs in the peripheral blood of patients by evaluating the ability of peripheral blood cells to facilitate CD4+CD25− T-cell proliferation. There was the potential concern of artefacts induced by peripheral blood handling, and the results do not represent the same sub-population present at the site of tumour ablation.

Author contributions

TGS and JPW performed the blood collection and flow cytometry analysis and drafted the manuscript. ZG designed the study and collected the clinical features. All authors read and approved the final manuscript.

Acknowledgments

This work was supported in part by the National Natural Science Foundation of China (No. 81001002 and No. 30973438) and Tianjin Health Bureau Foundation of China (No. 2010KZ7).

The authors declare that they have no competing financial interests.

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