Myeloid derived suppressor cells (MDSCs) are increased and exert immunosuppressive activity together with polymorphonuclear leukocytes (PMNs) in chronic myeloid leukemia patients

Cesarina Giallongo, Nunziatina Parrinello, Daniele Tibullo, Piera La Cava, Alessandra Romano, Annalisa Chiarenza, Ignazio Barbagallo, Giuseppe A Palumbo, Fabio Stagno, Paolo Vigneri, Francesco Di Raimondo, Cesarina Giallongo, Nunziatina Parrinello, Daniele Tibullo, Piera La Cava, Alessandra Romano, Annalisa Chiarenza, Ignazio Barbagallo, Giuseppe A Palumbo, Fabio Stagno, Paolo Vigneri, Francesco Di Raimondo

Abstract

Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs), able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML) patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs) we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Flow cytometry analysis of circulating…
Figure 1. Flow cytometry analysis of circulating Gr-MDSCs and Mo-MDSCs cells in PB from HD and CML patients at diagnosis (D) and during IM therapy.
A: Representative data from one HD and one CML at diagnosis. Flow cytometry analysis was performed with gates set on either CD11b+CD33+CD14-HLADR- or CD14+HLADR-cells populations. B: Figures show the quantitative data for Gr-MDSCs and Mo-MDSCs. Results are expressed either as percentage or absolute number. Statistically significant differences between groups are indicated by P-value in the figures.
Figure 2. Frequency of circulating T-reg cells…
Figure 2. Frequency of circulating T-reg cells in PB from HD and CML patients at diagnosis and during IM therapy.
A: Figures show the quantitative data expressed either as percentage or absolute number. Cytometric analysis was performed with gates set on CD4+ cells and the results presented as the percentage of CD25+Foxp3+cells in CD4+ cells. Statistically significant differences between groups are indicated by P-value in the figures. B: Correlation of the percentages of Gr-MDSCs cells and T-reg in PB from CML patients at diagnosis.
Figure 3. Increased Arg1 expression in CML…
Figure 3. Increased Arg1 expression in CML patients at diagnosis.
A: Arg1 mRNA expression in HD and CML patients at diagnosis (D) and during IM therapy assessed by real time PCR. Healthy donors (HD) vs D and D vs therapy: p<0.0001. B: Levels of Arg1 mRNA in Gr-MDSCs from HD and CML patients at diagnosis. The expression resulted higher in CML Gr-MDSCs in respect to HD (calculated value of 2∧-ΔΔCt in HD was 1; p<0.001). C: Arg1 concentration measured by ELISA. Circulating protein was increased in CML patients at diagnosis compared to HD (p<0.0001) and decreased during IM therapy (D vs 3–6 and 12 months: p<0.001 and p<0.05 respectively). D: Correlation of the percentage of Gr-MDSCs cells in PB and Arg1 levels in serum of CML patients at diagnosis.
Figure 4. Gr-MDSCs and PMNs exert immunosuppressive…
Figure 4. Gr-MDSCs and PMNs exert immunosuppressive activity in CML patients.
Arg1 expression (A) and activity (B) were significantly increased in CML PMNs more than autologous Gr-MDSCs isolated from patients at diagnosis. For analysis of western blot the optical density of the bands was measured using Scion Image software. Results represent the means of three independent experiments; error bars denote SEM. (C) The percentage of T cell proliferation was significantly reduced when T cells were cultured with CML Gr-MDSCs (p<0.05) and CML PMNs (p<0.001), while Gr-MDSCs and PMNs from HD did not exert any suppressive activity. Mean frequency of CD3+ CFSE dim cells ± SEM from four independent experiments in duplicate is shown.
Figure 5. CML serum has immunosuppressive activity…
Figure 5. CML serum has immunosuppressive activity linked to Arg1.
8 CML sera were analyzed on 4 different HD T cells with a reduction of T cell proliferation (pdim cells ± SEM is shown.

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