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. 2013;8(1):e55818.
doi: 10.1371/journal.pone.0055818. Epub 2013 Jan 31.

Increased level of myeloid-derived suppressor cells, programmed death receptor ligand 1/programmed death receptor 1, and soluble CD25 in Sokal high risk chronic myeloid leukemia

Lisa Christiansson  1 , Stina Söderlund, Emma Svensson, Satu Mustjoki, Mats Bengtsson, Bengt Simonsson, Ulla Olsson-Strömberg, Angelica S I Loskog
Affiliations

Increased level of myeloid-derived suppressor cells, programmed death receptor ligand 1/programmed death receptor 1, and soluble CD25 in Sokal high risk chronic myeloid leukemia

Lisa Christiansson et al. PLoS One. 2013.

Abstract

Immunotherapy (eg interferon α) in combination with tyrosine kinase inhibitors is currently in clinical trials for treatment of chronic myeloid leukemia (CML). Cancer patients commonly have problems with so called immune escape mechanisms that may hamper immunotherapy. Hence, to study the function of the immune system in CML is of interest. In the present paper we have identified immune escape mechanisms in CML with focus on those that directly hamper T cells since these cells are important to control tumor progression. CML patient samples were investigated for the presence of myeloid-derived suppressor cells (MDSCs), expression of programmed death receptor ligand 1/programmed death receptor 1 (PD-L1/PD-1), arginase 1 and soluble CD25. MDSC levels were increased in samples from Sokal high risk patients (p<0.05) and the cells were present on both CD34 negative and CD34 positive cell populations. Furthermore, expression of the MDSC-associated molecule arginase 1, known to inhibit T cells, was increased in the patients (p = 0.0079). Myeloid cells upregulated PD-L1 (p<0.05) and the receptor PD-1 was present on T cells. However, PD-L1 blockade did not increase T cell proliferation but upregulated IL-2 secretion. Finally, soluble CD25 was increased in high risk patients (p<0.0001). In conclusion T cells in CML patients may be under the control of different immune escape mechanisms that could hamper the use of immunotherapy in these patients. These escape mechanisms should be monitored in trials to understand their importance and how to overcome the immune suppression.

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Conflict of interest statement

Competing Interests: Satu Mustjoki receives honoraria from Novartis and Bristol-Myers Squibb, Angelica Loskog is the CEO of Lokon Pharma AB, a scientific advisor to NEXTTOBE AB and has a royalty agreement with Alligator Biosciences AB. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Myeloid derived suppressor cell (MDSC) and arginase 1 (Arg1) levels.
(A) Levels of MDSCs (CD11b+CD14−CD33+) in different CML patient risk groups (high risk (HR) n = 10, low risk (LR) n = 7) and healthy control subjects (HC, n = 21) as shown by flow cytometry. (B) Levels of MDSCs expressing CD34 in CML patient risk groups (HR n = 10, LR n = 7) and HCs (n = 21). (C) Arg1 mRNA expression in HCs (n = 9) and CML patients (HR n = 4, LR n = 2) assessed by real time PCR. Statistically significant differences between groups are indicated by P-values in the figures. (D) Arg1 concentration in CML patient plasma (HR n = 7, LR n = 3) measured by ELISA. (E) Arg 1 mRNA expression in CML cell lines. The plot shows mean values with standard error of the mean.
Figure 2
Figure 2. Programmed death receptor ligand 1 (PD-L1) expression levels on patient cells.
(A) PD-L1 expression on myeloid (CD11b+) cells from patients (HR n = 11, LR n = 6) and HCs (n = 21) as assessed by flow cytometry. (B) Median fluorescence intensity (MFI) of PD-L1 on CD11b cells. (C) MFI of PD-L1 expression on CD11b cells from a representative healthy control (filled histogram) and CML patient (thick line), the thin line represents the isotype control. (C) Percent PD-L1 expressing CD34 cells of all live cells in HCs (n = 21), LR (n = 6) and HR (n = 11) patients. Statistically significant differences between groups are reported as P-values in the figures.
Figure 3
Figure 3. Programmed death receptor 1 (PD-1) expression on T cells.
(A) The percentage of PD-1 positive cytotoxic T cells (CD3+CD8+) in HCs (n = 21), LR (n = 7) and HR (n = 11) as determined by flow cytometry. (B) The PD-1 MFI is shown for cytotoxic T cells. (C) The percentage of PD-1 positive T helper cells (CD3+CD8−) in HCs (n = 21), LR (n = 7) and HR (n = 11) patients was determined by flow cytometry. (D) The PD-1 MFI for helper T cells. Statistically significant differences between groups are indicated as P-value in the figure.
Figure 4
Figure 4. Blocking of programmed death receptor ligand 1 (PD-L1) in co-cultures.
(A) PD-L1 expression on the CML cell lines K562, BV-173 and CML-T1 was evaluated by flow cytometry. The thick line represents staining with specific antibody and the filled histograms represent the staining with isotype control antibody. (B) Proliferative response of healthy donor PBMCs to CML cell lines with or without addition of PD-L1 blocking antibody. The bars show the proliferative response in different wells after subtraction of proliferation seen when cells lines were cultured alone. The experiment was repeated twice with four donors. Proliferation was measured by thymidine H3 incorporation on triplicate samples. (C) T cell-depleted leukocytes from CML patients (HR n = 7, LR n = 4)) and HCs (n = 15) were co-cultured with PBMCs from a control subject (responder) with addition of an irrelevant antibody (iso) or an antibody blocking PD-L1 (αPD-L1). The percentage of proliferating T cells in the co-culture is shown. (D) The concentration of IL-2 in co-culture supernatants from HCs (n = 15), LR (n = 4) and HR (n = 7) was assessed by ELISA. The increase/decrease in IL-2 concentration for the individual control subjects and CML patients are shown in the figure.
Figure 5
Figure 5. Soluble CD25 concentration.
The concentration of soluble CD25 in blood plasma from CML patients (LR n = 6, HR n = 9) and HCs (n = 18) was investigated with ELISA. The figure shows soluble CD25 concentration expressed as pg/ml in blood plasma. Statistically significant difference between groups is reported as P-value in the figure.
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The study was supported by the Nordic CML study group (http://www.nordiccml.org/), EuropeanLeukemiaNet (http://www.leukemia-net.org/content/home/), Uppsala University Hospital and the Medical Faculty at Uppsala University (http://www.akademiska.se/, http://www.medfarm.uu.se/medicinska_fakulteten/index.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.