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. 2011 Dec 1;187(11):6120-9.
doi: 10.4049/jimmunol.1101225. Epub 2011 Oct 28.

Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b+Gr1+ cells

Sergey Ryzhov  1 , Sergey V Novitskiy, Anna E Goldstein, Asel Biktasova, Michael R Blackburn, Italo Biaggioni, Mikhail M Dikov, Igor Feoktistov
Affiliations

Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b+Gr1+ cells

Sergey Ryzhov et al. J Immunol. .

Abstract

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A(2B) adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b(+)Gr1(high) cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b(+)Gr1(high) subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b(+)Gr-1(high) subset had the highest levels of CD73 (ecto-5'-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b(+)Gr-1(int) (ΔMFI of 57.9 ± 6.8) and CD11b(+)Gr-1(-/low) (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b(+)Gr-1(high) cells correlated with high levels of ecto-5'-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5'-nucleotidase substrate 5'-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.

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Figures

Figure 1
Figure 1. Ablation of A2B adenosine receptors reduces the percentage of CD11b+Gr-1high cells in the population of tumor-infiltrating host immune cells
(A) Representative cytofluorographic dot plots showing the percentage of immune host cells (CD45+) in total tumor cell population. Single cell suspensions were prepared from tumors extracted from A2BKO and WT mice on day 14 after inoculation with LLC cells. (B) Aggregate data from flow cytometry analysis of CD45+ cells obtained from 3 A2BKO and 3 WT animals. Values are expressed as mean±SEM; ns indicates non-significant difference (unpaired two-tail t-tests). (C) Representative example of flow cytometry analysis of tumor-infiltrating immune host cells (gated for CD45) from A2BKO and WT mice using anti-CD11b and anti-Gr-1 antibodies. (D) The percentage of CD11b+Gr-1−/low, CD11b+Gr-1int and CD11b+Gr-1high subsets in the populations of tumor-infiltrating immune host cells from 3 A2BKO and 3 WT mice measured by flow cytometry. Values are expressed as mean±SEM; the asterisks indicate significant difference (* P<0.05, unpaired two-tail t-tests) and ns indicates non-significant difference, compared to corresponding A2BKO values. (E) Representative example of flow cytometry analysis of tumor-infiltrating immune host cells from WT chimeric mice transplanted with A2BKO bone marrow cells (A2BKO→WT) and with WT bone marrow cells (WT→WT). (F) The percentage of CD11b+Gr-1−/low, CD11b+Gr-1int and CD11b+Gr-1high subsets in the populations of tumor-infiltrating immune host cells from 3 A2BKO→WT and 3 WT→WT bone marrow chimeric mice measured by flow cytometry. Values are expressed as mean±SEM; the asterisks indicate significant difference (* P<0.05, ** P<0.01, unpaired two-tail t-tests), compared to corresponding A2BKO→WT values.
Figure 2
Figure 2. Stimulation of A2B adenosine receptors promotes expansion of CD11b+Gr-1high cells in vitro
(A) Cytofluorographic dot plots of MDSCs generated from mouse bone marrow hematopoietic progenitors in the presence of the non-selective agonist NECA or selective concentrations of receptor-specific agonists. Representative results of three experiments are shown. NECA, but not the selective agonists to A1, A2A, and A3 adenosine receptors increased the proportion of CD11b+Gr-1high cell subpopulation. (B) Effect of addition of NECA (1 μM) at different time points during generation of MDSCs (starting in the absence of NECA) on the percentage of CD11b+Gr-1high cells assessed by flow cytometry on day 5. Values are expressed as mean±SEM, n=3. Asterisks indicate significant difference (** P<0.01, one-way ANOVA with Dunnett’s postest), compared to the value obtained with NECA added at the beginning of MDSC generation (time 0). (C) Selective antagonists at the A2B receptor (IPDX and CVT-6883) but not selective antagonists at A1, A2A, and A3 adenosine receptors (N0861, SCH58261, and MRS1191, respectively) inhibit NECA-induced expansion of CD11b+Gr-1high subset. MDSCs were generated from mouse bone marrow hematopoietic progenitors in the absence (Basal) or presence of 1 μM NECA and antagonists at their selective concentrations as indicated in Results. The proportion of CD11b+Gr-1high cells was measured by flow cytometry. Values are expressed as mean±SEM, n=3. Asterisks indicate significant difference (* P<0.05, ** P<0.01, one-way ANOVA with Dunnett’s postest), compared to NECA, and pounds indicate significant difference (# P<0.05, ## P<0.01, one-way ANOVA with Dunnett’s postest), compared to basal values. (D) NECA-induced expansion of CD11b+Gr-1high subset is not reproduced only in cells from A2BKO animals. MDSCs were generated from mouse bone marrow hematopoietic progenitors obtained from A1, A2A, A2B, A3 adenosine receptor KO or WT mice in the absence or presence of increasing concentrations of NECA. Values are expressed as mean±SEM, n=3.
Figure 3
Figure 3. Adenosine receptors promote preferential expansion of granulocytic MDSCs characterized by CD11b+Gr-1high/Ly-6Clow Ly-6Ghigh phenotype
MDSCs were generated from mouse bone marrow hematopoietic progenitors in the absence (Control) or presence of 1 μM NECA. (A) Representative example of flow cytometry analysis using anti-CD11b and anti-Gr-1 antibodies. (B) The percentage of CD11b+Gr-1−/low, CD11b+Gr-1int and CD11b+Gr-1high subsets generated in the absence or presence of NECA measured by flow cytometry. Values are expressed as mean±SEM, n=8. Asterisks indicate significant difference (** P<0.01, unpaired two-tail t-tests) and ns indicates non-significant difference, compared to control. (C) Staining with Diff-Quik to evaluate subset morphology. CD11b+Gr-1−/low, CD11b+Gr-1int and CD11b+Gr-1high subsets were generated in the absence or presence of 1 μM NECA and sorted by flow cytometry. (D) Representative example of flow cytometry analysis using anti-Ly-6C and anti-Ly-6G antibodies. (E) The percentage of Ly-6ChighLy-6Glow and Ly-6ClowLy-6Ghigh subsets generated in the absence or presence of NECA measured by flow cytometry. Values are expressed as mean±SEM, n=3. Asterisks indicate significant difference (** P<0.01, unpaired two-tail t-tests) and ns indicates non-significant difference, compared to control.
Figure 4
Figure 4. Production of reactive oxygen species in MDSC subsets and the expression of enzymes relevant to their suppressive activity
(A) MDSCs were generated from mouse bone marrow hematopoietic progenitors in the absence (Control) or presence of 1 μM NECA. Real-time RT-PCR analysis of Arg 1 mRNA was performed in CD11b+Gr-1−/low, CD11b+Gr-1int and CD11b+Gr-1high subsets isolated by flow cytometry. Values are expressed as mean±SEM, n=3. The asterisk indicates significant difference (* P<0.05, unpaired two-tail t-tests) and ns indicates non-significant difference, compared to control. (B) MDSCs were generated from mouse bone marrow hematopoietic progenitors in the absence (Control) or presence of 1 μM NECA. Real-time RT-PCR analysis of iNOS mRNA was performed in CD11b+Gr-1−/low, CD11b+Gr-1int and CD11b+Gr-1high subsets isolated by flow cytometry. Values are expressed as mean±SEM, n=4; ns indicates non-significant difference, compared to control. (C) ROS production in CD11b+Gr-1−/low, CD11b+Gr-1int and CD11b+Gr-1high subsets at rest or after stimulation with 100 nM PMA was evaluated as described in Methods. Values represent a difference between mean fluorescence intensity (ΔMFI) of cells stained with the oxidation-sensitive dye CM-H2DCFDA and unstained control. Values are expressed as average of two determinations. (D) Effect of increasing concentrations of PMA on ROS production in CD11b+Gr-1high subsets of MDSCs generated in the absence (Control) or presence of 1 μM NECA. Values are expressed as mean±SEM, n=3.
Figure 5
Figure 5. Granulocytic MDSCs express high levels of functional ecto-5′-nucleotidase
(A) Representative flow cytometry histograms of CD73 expression on the surface of LLC tumor cells and CD11b+Gr-1−/low, CD11b+Gr-1int and CD11b+Gr-1high subsets of MDSCs generated from mouse bone marrow hematopoietic progenitors. (B) Graphic representation of data from flow cytometry analysis of CD73 expression on the surface of LLC tumor cells and CD11b+Gr-1−/low, CD11b+Gr-1int and CD11b+Gr-1high subsets of MDSCs generated from mouse bone marrow hematopoietic progenitors. Values are expressed as mean±SEM, n=4. Asterisks indicate significant difference (* P<0.05, ** P<0.01, *** P<0.001 one-way ANOVA with Bonferroni’s postest) between subsets. (C) Enzymatic activity of ecto-5′-nucleotidase expressed on CD11b+Gr-1−/low, CD11b+Gr-1int and CD11b+Gr-1high subsets of MDSCs generated from mouse bone marrow hematopoietic progenitors in the absence (Control) or presence of 1 μM NECA (NECA-treated). Values are expressed as mean±SEM, n=3. (D) Effect of ecto-5′-nucleotidase inhibition with APCP on AMP-induced expansion of granulocytic MDSCs. The percentage of CD11b+Gr-1high cells generated in the absence (Basal, APCP) or presence of 100 μM AMP (AMP, AMP+APCP) and in the absence (AMP, Basal) or presence of 100 μM APCP (AMP+APCP, APCP) was measured by flow cytometry. Values are expressed as mean±SEM, n=3. Asterisks indicate significant differences (* P<0.05, ** P<0.01, one-way ANOVA with Bonferroni’s postest) and ns indicates non-significant difference between values.
Figure 6
Figure 6. Ecto-5′-nucleotidase activity facilitates the suppression of T cell proliferation by granulocytic MDSCs
(A) MDSCs were generated from mouse bone marrow hematopoietic progenitors and CD11b+Gr-1high cells were enriched (>70%) by positive immunomagnetic selection with anti-Ly-6G antibody. T cells were stimulated with anti-CD3–anti-CD28–coupled microbeads and cultured without (0) or co-cultured together with CD11b+Gr-1high cells added at numbers corresponding to 3, 6 or 12% of T cell numbers in the presence of increasing concentrations of 5′-AMP. Values are expressed as mean±SEM, n=3. (B) MDSCs were generated from mouse bone marrow hematopoietic progenitors in the absence (Control) or presence of 1 μM NECA (NECA-treated) and CD11b+Gr-1high cells were purified (>95%) by flow cytometry. T cells were stimulated with anti-CD3–anti-CD28–coupled microbeads and cultured without (0) or co-cultured together with CD11b+Gr-1high cells added at numbers corresponding to 6 or 12% of T cell numbers in the absence (Control, NECA-treated) or presence of 300 μM 5′-AMP (Control+AMP, NECA-treated+AMP). Data are presented as mean±SEM (n=3) of maximal thymidine incorporation.
Figure 7
Figure 7. Model of adenosineric regulation of MDSC expansion and function
Generation of adenosine by ecto-5′-nucleotidase (CD73) expressed at high levels on granulocytic MDSCs (CD11b+Gr-1high) may promote their expansion by stimulation of A2B receptors on myeloid progenitors (MPs) and facilitate their suppressive activity by acting on A2A receptors of T cells, thus limiting immune response.
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Comment in

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