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Published in final edited form as: Clin Genitourin Cancer. 2017 Oct 24;16(2):e469–e476. doi: 10.1016/j.clgc.2017.10.010

Immune Analysis of Radium-223 in Patients with Metastatic Prostate Cancer

Joseph W Kim 1,2, Min Sun Shin 1, Youna Kang 1, Insoo Kang 1,*, Daniel P Petrylak 1,2,*
PMCID: PMC5878980  NIHMSID: NIHMS915423  PMID: 29137877

Abstract

Background

Radium223 (Ra223) delivers high-energy radiation to osteoblastic metastasis of prostate cancer resulting in irreparable double-stranded DNA damage. The effects of Ra223 on CD8+ T cell subsets in prostate cancer patients is unknown.

Patients and methods

Fifteen men with metastatic prostate cancer with clinical indication for Ra223 without any autoimmune or immune deficiency conditions were enrolled. Patients received a course of Ra223 50kBq/kg. Concurrent use of prednisone ≤ 10mg a day, was allowed. Peripheral blood samples were collected before and 3 to 4 weeks after the first dose of Ra223 50kBq/kg. Peripheral blood mononuclear cells (PBMCs) were purified and analyzed for the phenotypic and functional characteristics of CD8+ T cells using flow cytometry.

Results

One Ra223 treatment did not result in significant change in the overall frequencies of CD8+ T cells and their subsets including naïve, central memory and effect memory (EM) cells. However, the mean frequency of PD-1 expressing EM CD8+ T cells decreased after one Ra223 treatment from 20.6% to 14.6% (p=0.020), while no significant change was observed in the frequencies of CD27, CD28 or CTLA4-expressing T cells. One Ra223 treatment was not associated with any significant change in the frequencies of CD8+ T cells producing IFN-γ, TNF-α and IL-13.

Conclusion

One Ra223 treatment is associated with a decreased mean frequency of PD-1 expressing EM CD8+ T cell without affecting other immune checkpoint molecules or cytokine production. Further investigations are warranted to elucidate the immunological and clinical significance of our observations and its long-term effects after multiple treatments.

Keywords: Survival, Radioisotope, Xofigo, Immune effect, prostate cancer, PD-1

INTRODUCTION

Radium223 (Ra223) is an alpha-radiation emitting radioisotope approved in treatment of metastatic castration resistant prostate cancer (mCRPC). A Phase III randomized, placebo-controlled study demonstrated that Ra223 improves survival in men with metastatic castration resistant prostate cancer (mCRPC) by 3.6 months (median, 14.9 months vs. 11.3 months, in Ra223 treated group vs placebo group, respectively) with a hazard ratio of death, 0.70 (95% CI, 0.58 to 0.83; P<0.001), and delays time to first skeletal event by 5.8 months with a HR=0.66, 95% CI 0.52–0.83; p=0.00037) with a favorable safety profile.1,2 Ra223, as a calcium mimetic, localizes to osteoblastic metastases of prostate cancer, and emits alpha particles leaving irreparable double-stranded DNA damage and eventual cell death.

In addition to its conventional cytotoxic effect via damaging DNA, radiation induces immunogenic cell death by affecting various compartments of the immune system.3,4 Preclinical studies have shown that radiation upregulates the expression of antigen-presenting MHC molecules and affects cytokine production and various subpopulations of immune cells as well as stromal cells.5–8 A beta particle-emitting radioisotope, Samarium-153-EDTMP, has been shown to induce immunogenic modulation by altering the phenotype of tumor cells such that they are more susceptible to T-cell mediated killing.7 A randomized phase II study of Sm-153 in combination with a PSA-targeted vaccine, versus Sm-153 alone, showed a trend toward improvement in 4-month progression free rate (23.8% versus 11.1%) (p=0.27), and an improvement in median PFS of 3.7 months vs 1.7 months (p=0.046).9 In addition, several other clinical trials incorporated the external beam radiation therapy, in combination with an immune-based therapy to capitalize on its immune modulating effects.10–13

The effect of Ra223 on human immune systems is unknown. We hypothesized that Ra223, would affect the CD8+ T cell and its subsets in patients with metastatic prostate cancer. We investigated the immunologic effect of Ra223 in patients with mCRPC by examining the phenotypic and functional characteristics of CD8+ T cells that include the expression of co-stimulatory, co-inhibitory molecules and cytokines.

PATIENTS AND METHODS

Patient population

A total of 15 men with castration resistant prostate cancer with osseous metastases with clinical indication for Ra223 were enrolled. Patients with active autoimmune disease requiring therapy, or with known immunodeficiency syndrome were excluded. Patients who had been on low dose corticosteroids (≤ 10mg prednisone daily) were allowed. Patients received a course of Ra223 50kBq/kg intravenously every 4 weeks as a standard practice. Table 1 shows clinical characteristics of the study subjects. Research blood was collected at two time points: (a) before and (b) 3 to 4 weeks after the first dose of Ra223 50kBq/kg.

Table 1.

Baseline Patient Characteristics

Patient Characteristics Median (Range)
N = 12
Age 72 (55–85)
PSA at entry 78 (10.6 – 808)
Number of prior therapies for mCRPC 3 (1–4)
Prior Chemotherapy YES 8
NO 4
Concurrent use of prednisone YES 4
NO 8
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Purification and stimulation of peripheral blood mononuclear cells (PBMCs)

PBMCs were isolated from heparinized peripheral venous blood using Ficoll-Hypaque gradient (GE Healthcare, Piscataway, NJ). PBMCs were washed with phosphate buffered saline (PBS) and resuspended in RPMI 1640 media supplemented with 10% fetal calf serum and 1% glutamine/penicillin/streptomycin. Cells were stimulated for 4 hours with PBS (control) or PMA (50 ng/ml; Sigma, St. Louis, MO) and ionomycin (1 μg/ml; Sigma) in the presence of Golgiplug (BD Biosciences, San Jose, CA) in a tissue culture incubator at 37°C as previously done.14–16

Flow cytometry

Fresh PBMCs were stained with anti-CD3-APC-Cy7, CD8-APC, CD45RA-PE-Cy5, CCR7-PE-Cy7, PD-1-Pacific Blue, IL-7Rα-FITC, CD27-PE, CD28-PE, CTLA4-PE, CX3CR1-PE antibodies or isotype control (all from BioLegend, San Diego, CA). PBMCs that had been stimulated with PMA/ionomycin were stained with anti-CD3-APC-Cy7, CD8-APC, CD45RAPE-Cy5 and CCR7-PE-Cy7 antibodies followed by fixation, permeabilization (Cytofix/Cytoperm Kit, BD Biosciences) and staining with anti-IFNγ-PE, TNF-α-FITC or IL-13-PE antibodies (BioLegend).14 Stained cells were analyzed on an LSRII® flow cytometer (BD Biosciences). Collected data were analyzed using FlowJo software (Tree Star, Ashland, OR).

Statistical analysis

Statistical analysis was done using the GraphPad Prism 6 (GraphPad Software, La Jolla, CA). P values less than 0.05 were considered statistically significant.

RESULTS

The frequency of PD-1 expressing effector memory (EM) CD8+ T cells decreased in patients with mCRPC after a treatment of Ra223

To determine the possible effect of Ra223 on the expression of co-stimulatory and -inhibitory molecules by CD8+ T cells in patients with mCRPC, we analyzed the expression CD27, CD28, PD-1 and CTLA-4 on different subsets of CD8+ T cells in these patients before and after Ra223 administration. Based on the expression of the T cell receptor co-receptor CD45RA and lymphoid tissue homing chemokine receptor CCR7, we identified naïve (CD45RA+CCR7+), central (CD45RA−CCR7+) and effector memory (CD45RA+/− CCR7−) CD8+ T cells in peripheral blood (Fig 1A). The overall frequency of lymphocytes, total CD8+ T cells, naïve, CM and EM CD8+ T cells did not change after Ra223 treatment (Table 2). Most naïve and CM CD8+ T cells expressed CD27 and CD28 but not PD-1 (data not shown). In EM CD8+ T cells, some cells expressed CD27, CD28 and/or PD-1 (Fig 1B). The frequency of PD-1+ EM CD8+ T cells decreased after Ra223 treatment (mean frequency (%) ± SEM, 20.6 ± 2.13 vs. 14.6 ± 2.37, P = 0.020) while the frequency of CD27+ and CD28+ EM CD8+ T cells remained unchanged (Fig 1C). CTLA-4 expressing naïve, CM and EM CD8+ T cells were hardly detected in most samples (data not shown). In naïve and CM CD8+ T cells, most cells continued to express CD27 and CD28 at similar levels after Ra223 (mean frequency (%) ± standard error of the mean (SEM), naïve CD27, 96 ± 1.53 vs. 94.8 ± 1.76; naïve CD28, 95.0 ± 1.74 vs. 93.0 ± 2.11; CM CD27, 89.0 ± 1.64 vs. 86.3 ± 2.51; CM CD28, 98.0 ± 0.94 vs. 95.1 ± 1.95, P > 0.05 for all).

Figure 1. Ra223 treatment decreases circulating PD-1-expressing EM CD8+ T cells in patients with mCRPC.

Figure 1

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PBMCs were purified from the peripheral blood of patients with mCPRC and stained with antibodies to CD3, CD8, CD45RA, CCR7 and PD-1, CD27 or CD28. (A) Gating strategies for flow cytometry analysis of CD8+ T cells of PMBCs from patients with mCRPC. Lymphocytes were gated based on forward and side scatters. The CD3+ CD8+ T cells were selected for further analysis. Naïve (N), central memory (CM) and effector memory (EM) CD8+ T cells were gated based on the expression of CD45RA and CCR7. (B) Representative histograms showing the expression of PD-1, CD27 and CD28 on EM CD8+ T cells. Dotted lines represent isotype control staining and bold lines represent specific antibody staining. (C) Frequency of each PD-1, CD27 or CD28 expressing cells in EM CD8+ T cells of patients with mCRPC before and after Ra223 treatment measured by flow cytometry. Bars and error bars indicate mean ± SEM. P-values were obtained by the paired Student t-test.

Table 2.

Frequencies of CD8+ T cell subsets before and 1 month after Ra223 treatment

Cells Before After P value
Lymphocytes in PBMCs *27.3 ± 3.4 31.9 ± 4.5 0.149
CD8+ T cells in lymphocytes 28.4 ± 4.0 24.8 ± 2.5 0.357
Naïve CD8+ T cells 13.9 ± 3.9 17.8 ± 5.4 0.201
Central memory CD8+ T cells 6.6 ± 2.4 6.2 ± 1.5 0.769
Effector memory CD8+ T cells 74.9 ± 4.0 70.0 ± 5.3 0.238
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*

values are mean frequency (%) ± standard error of the mean (n = 12)

A decrease in the frequency of PD-1 expression was also observed among IL-7Rαhigh EM CD8+ T cells

The cytokine IL-7 and its receptor IL-7 receptor alpha (IL-7Rα) play a major role in the generation and maintenance of memory T cells by promoting cell survival.17 We previously showed the presence of two distinct cells expressing high and low levels of IL-7Rα in human EM CD8+ T cells.18 A recent study reported that tumor-specific effector CD8+ T cells with the capacity to establish immunological memory in humans after adoptive transfer are marked by IL-7Rα.19 Thus, we analyzed the frequency of IL-7Rαhigh and IL-7Rαl°w cells in EM CD8+ T cells as well as their expression of PD-1. Both IL-7Rαhigh and IL-7Rαl°w cells in EM CD8+ T cells were detected in PBMCs of the patients, and their frequencies did not change after the treatment (Fig 2A–B). PD-1-expresing cells were found in both IL-7Rαhigh and IL-7Rαl°w EM CD8+ T cells (Fig 2C). Interestingly, a decrease in the frequency of PD-1+ cells was found in IL-7Rαhigh EM CD8+ T cells (p<0.001) (Fig 2D). A similar observation was noticed in IL-7Rαl°w EM CD8+ T cells although the difference did not reach the level of statistical significance (p=0.056) (Fig 2D).

Figure 2. Ra223 treatment has no effect on the frequency of IL-7 receptor alpha (IL-7Rαhigh and low expressing EM CD8+ T cells in patients with mCPRC.

Figure 2

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(A) Representative histogram showing IL-7Rαhigh and low cells in EM CD8+ T cells of a patient with mCPRC. (B) Frequency of IL-7Rαhigh and IL-7Rαl°w cells in EM CD8+ T cells of patients with mCPRC before and after Ra223 treatment as measured by flow cytometry. (C) Naïve (N), central memory (CM) and effector memory (EM) CD8+ T cells were gated based on the expression of CD45RA and CCR7. Representative histograms showing PD-1 expressing cells in IL-7Rαhigh and low EM CD8+ T cells of a patient with mCPRC. (D) Frequency of PD-expressing cells in IL-7Rαhigh and low EM CD8+ T cells of patients with mCPRC before and after Ra223 treatment as measured by flow cytometry. Bars and error bars indicate mean ± SEM. P-value was obtained by the paired Student t-test. Dotted lines represent isotype control staining and bold lines represent specific antibody staining (A and C).

Ra223 treatment does not alter the frequency of CD8+ T cells expressing IFN-γ, TNF-α and IL-13

T helper (Th) 1 cytokines, such as IFN-γ, and TNF-α can promote anti-tumor immunity by activating T cells, natural killer (NK) cells and monocytes while Th2 cytokines such as IL-13 can be associated with cancer progression.20–22 The expression of cytokines by CD8+ T cells were also examined in patients with mCRPC before and after Ra223 treatment using flow cytometry (Fig 3A). The frequency of CD8+ T cells expressing IFN-γ, TNF-α and IL-13 were similar in blood samples collected before and after Ra223 (Fig 3B).

Figure 3. Ra223 treatment does not alter the frequency of CD8+ T cells producing IFN-γ, TNF-α and IL-13 in patients with mCPRC.

Figure 3

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PBMCs were purified from the peripheral blood of patients with mCPRC and stimulated for 4 hours with or without PMA/ionomycin in the presence of Brefeldin A. Stimulated cells were stained with antibodies to CD3, CD8, CD45RA and CCR7, fixed and permeabilized followed by IFN-γ, TNF-α or IL-13 staining. Flow cytometric analysis was done to determine the frequency of cells producing IFN-γ, TNF-α or IL-13 in CD8+ T cells. (A) Representative contour plots showing IFN-γ, TNF-α or IL-13 producing CD8+ T cells stimulated for 4 hours with or without PMA/ionomycin. Numbers in the plots indicate the frequency of cytokine producing cells. (B) Frequency of IFN-γ, TNF-α or IL-13 producing cells in CD8+ T cells of patients with mCPRC before and after Ra223 treatment as measured by flow cytometry. Bars and error bars indicate mean ± SEM. P-value was obtained by the paired Student t-test.

DISCUSSION

To the best of our knowledge, this is the first study to investigate the effect of Ra223 treatment on CD8+ T cells in patients with mCPRC. While there was no significant change observed in overall frequencies of CD8+ T cell or their major subsets, it appears that Ra223 has an effect on expression on PD-1 on CD8+ effector memory (EM) T cells. Ra223 was associated with a statistically significant decrease in frequency of PD-1+ EM CD8+ T cells (20.6% pre-treatment vs 14.6% post-treatment; P=0.020). The decrease in PD-1 expression was also observed in IL-7Rαhigh EM CD8+ T cells (P<0.001). The expression of other immune checkpoint molecules including CD27, CD28, CTLA4 remained unchanged. Also, the expression of cytokines including IFN-γ, TNF-α and IL-13 remained unchanged. It is unclear if this selective decrease in the frequency of PD-1 expressing CD8+ EM cells is because of apoptosis of PD-1 expressing T cells, down-regulation of PD-1 receptor on these T cells, or other mechanisms. Our study does not provide explanation for any of these hypothetical mechanisms. Nevertheless, it generates hypotheses that are subject to further investigation.

Programmed death 1 (PD-1) is a transmembrane glycoprotein, a member of CD28/CTLA-4 immunoglobulin family and an inhibitory co-receptor expressed on T cells.23 PD-1 receptor is absent in resting T cells, and is present in activated T cells and up-regulated in exhausted T cells in the setting of chronic antigen stimulation.24,25 Upon its engagement with its ligands, PD-L1 or PD-L2, PD-1 negatively regulates T cell receptor signaling and is associated with decreased T cell proliferation and cytokine production leading to induction of T cell apoptosis, and facilitation of T cell anergy and exhaustion.23,26 PD-1 expressing tumor infiltrating CD8+ T cells are often found functionally impaired.27,28 In this context, our observation of decreased frequency of PD-1 expressing T cells generates a couple of hypotheses.

One speculated hypothesis is that Ra223, by undefined mechanisms, upregulates the PD-L1 or PD-L2 expression on antigen-presenting cells or metastatic prostate cancer cells allowing their engagement with PD-1 expressing T cells, leading to apoptosis of PD-1 expressing CD8+ EM T cells and thus leading to observed, decreased frequency of PD-1 expressing T cells. Evidence suggests that irradiated tumors are associated with increased expression of PD-L1.29–32 A study is warranted to investigate if the Ra223 is, in fact, associated with upregulation of PD-L1 expression in the tumor microenvironment of osteoblastic metastases.

Another speculation is that Ra223, by undefined mechanisms, causes relative expansion of resting T cells that are PD-1 negative, following the exhaustion and apoptosis of PD-1 expressing CD8+ EM T cells without further expansion of activated T cells. This speculation is supported by our observation that there was no significant increase in production of proinflammatory cytokines, such as IFN-γ, and TNF-α. However, this hypothesis is not so well-grounded because contrary evidence exists to suggest that radiation often increases antigen exposure and presentation to T cells by inducing the release of tumor antigens and upregulation of major histocompatibility complex.6,8,33 Nonetheless, this remains a hypothesis subject for further investigation.

There are several limitations in interpreting our data. The first is lack of data at multiple data points post Ra223 treatment. The study was originally planned to obtain the blood samples after 3 and after 6 treatments. However, most of the patients had early termination of Ra223 treatment due to the progressive disease. Therefore, the blood samples at later time points could not be collected as planned. Our patients were generally well-advanced in their disease status with median prior therapy of 3, 62% of patients with prior chemotherapy, 100% of patients with ECOG performance status of 2. Our observation may be related to the poor prognostic factors of our study patients. Secondly, the lack of control data from normal peripheral blood limit the interpretation of our data. Our data are the change of these immune subsets from before to after the treatment. The normal variance of these subsets in normal peripheral blood is unknown. Therefore, the immunological significance of these changes remains to be a subject of further investigations. Thirdly, our data analysis was exploratory from a small sample size. Albeit, it was interesting to observe that there was a mean decrease in a subset of PD-1 expressing T cells following a Ra223 therapy.

A phase I/II study of an anti-PD-L1 antibody, atezolizumab, in combination with Ra223 is ongoing, to study the safety and optimal sequencing of these therapies (NCT02814669). In this study, patients will be randomized to start Ra223 either before, after or concurrent with the first dose of atezolizumab. A phase II study of Ra223 with or without anti-PD-1 antibody, pembrolizumab, is also enrolling patients (NCT03093428). Both trials require a pair of tumor biopsies to interrogate the immune tumor microenvironment on Ra223 treated tumors. Among others, these biopsies will examine whether or not Ra223 upregulates PD-L1 expression. Further, a randomized phase II study of sipuleucel-T with or without Ra223 in men with mCRPC (NCT02463799) is ongoing. This study will examine if Ra223 makes a difference in mounting peripheral immune responses to sipuleucel-T.

Additionally, there is an ongoing clinical trial of glycosylated recombinant human interleukin-7 subcutaneous injection after a course of sipuleucel-T therapy for metastatic prostate cancer as an immune booster (NCT01881867). The cytokine IL-7 and its receptor IL-7 receptor alpha (IL-7Rα) play a critical role in the generation and maintenance of memory T cells.17 Our data indicate that both IL-7Rαhigh and IL-7Rαl°w cells in EM CD8+ T cells were detected in PBMCs of the patients and their frequencies were not affected after the Radium223 treatment. The aforementioned study will test if the memory response to sipuleucel-T would be maintained with IL-7 subcutaneous injection.

CONCLUSION

A Ra223 treatment was associated with a decrease in frequency of programmed death-1 (PD-1) expressing CD8+ effector memory T cells.

Supplementary Material

Supplemental Figure 1. Ra223 treatment decreases circulating PD-1-expressing EM CD8+ T cells in patients with mCRPC. (A) Changes of PD1 expressing EM CD8+ T cells before and after one treatment of Ra223. (B) Changes of PD1 expressing IL-7Rαhigh CD8+ T cells before and after one treatment of Ra223.

Clinical Practice Points.

  1. Radium223 (Ra223) is an alpha radiation-emitting radioisotope that results in irreparable DNA damage in osteoblastic metastases of prostate cancer and cell death.

  2. A Ra223 treatment has an impact on CD8+ T cells subset, selectively in PD-1 expressing effector memory cells.

  3. Further studies are warranted to investigate the clinical and biological implication of these findings.

  4. Several studies are ongoing to examine the clinical and immunological response of Ra223 in combination with an immunotherapy, namely atezolizumab, pembrolizumab and sipuleucel-T.

MicroAbstract.

Radium-223 delivers high-energy radiation to osseous metastases. Its effect on the immune system is known. We observed a decrease in the mean frequency of PD-1 expressing cytotoxic T cells after one treatment of Ra223 in prostate cancer patients. Further investigation is warranted to define the clinical significance of this finding and to elucidate immunological aspect of how radium-223 mediates its anti-tumor activity.

Acknowledgments

GRANT SUPPORTS:

This was funded by 2013 Conquer Cancer Foundation/American Society of Clinical Oncology Young Investigator Award (J Kim) and in part supported in part by National Institutes of Health Grant 2R56 AG028069-06A1 (I Kang).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

JWK, MSS, YK and IK reports no relevant conflict of interest. DPP reports consultant fees from Bayer.

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Associated Data

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Supplementary Materials

Supplemental Figure 1. Ra223 treatment decreases circulating PD-1-expressing EM CD8+ T cells in patients with mCRPC. (A) Changes of PD1 expressing EM CD8+ T cells before and after one treatment of Ra223. (B) Changes of PD1 expressing IL-7Rαhigh CD8+ T cells before and after one treatment of Ra223.

NIHMS915423-supplement-supplement_1.pdf (33.3KB, pdf)

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