Limits...
Ex vivo generation of myeloid-derived suppressor cells that model the tumor immunosuppressive environment in colorectal cancer.

Dufait I, Schwarze JK, Liechtenstein T, Leonard W, Jiang H, Escors D, De Ridder M, Breckpot K - Oncotarget (2015)

Bottom Line: This resulted in the generation of high numbers of CD11b(+) Ly6G(+) granulocytic and CD11b(+) Ly6C(+) monocytic MDSC, which closely resemble those found within the tumor but not the spleen of CT26 tumor-bearing mice.We confirmed that inhibition of arginase-1 or iNOS in vivo resulted in the stimulation of cytotoxic T-cell responses.These data confirm the role of MDSC as inhibitors of T-cell-mediated immune responses in CRC.

View Article: PubMed Central - PubMed

Affiliation: UZ Brussel, Department of Radiotherapy, Vrije Universiteit Brussel, Brussels, Belgium.

ABSTRACT
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate in tumor-bearing subjects and which strongly inhibit anti-cancer immune responses. To study the biology of MDSC in colorectal cancer (CRC), we cultured bone marrow cells in conditioned medium from CT26 cells, which are genetically modified to secrete high levels of granulocyte-macrophage colony-stimulating factor. This resulted in the generation of high numbers of CD11b(+) Ly6G(+) granulocytic and CD11b(+) Ly6C(+) monocytic MDSC, which closely resemble those found within the tumor but not the spleen of CT26 tumor-bearing mice. Such MDSC potently inhibited T-cell responses in vitro, a process that could be reversed upon blocking of arginase-1 or inducible nitric oxide synthase (iNOS). We confirmed that inhibition of arginase-1 or iNOS in vivo resulted in the stimulation of cytotoxic T-cell responses. A delay in tumor growth was observed upon functional repression of both enzymes. These data confirm the role of MDSC as inhibitors of T-cell-mediated immune responses in CRC. Moreover, MDSC differentiated in vitro from bone marrow cells using conditioned medium of GM-CSF-secreting CT26 cells, represent a valuable platform to study/identify drugs that counteract MDSC activities.

No MeSH data available.


Related in: MedlinePlus

Differentiated bone marrow cells possess strong suppressive capacities and can be subdivided into both MDSC subsets(A) Expression of CD11b by bone marrow cells after a 6-day incubation period in CM as measured by flow cytometry. (B) Summarizing graph of ratio of MDSC subsets (C) Flow cytometry contour plots of in vitro MDSC before and after MACS sort. Underneath the contour plots of the sorted MDSC, representative pictures showing the morphology of these subsets are depicted. Pictures were taken with a light microscope at 64 times magnification. (D) Representative experiment showing suppression of CD8+ T cells by sorted in vitro MDSC (1:4 ratio MDSC to T cell). (E) The graph on the left represents the proliferation inhibition of CD3/CD28-activated CD8+ T cells (ratio MDSC to T cell as indicated in the graph). The graph on the right shows changes in IFN-γ secretion measured during the same experiment. Control represents T cells incubated without MDSC. Mean of at least 3 experiments +/− SEM is shown in all graphs. Number of asterisks in the figures indicates the level of statistical significance as follows: ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4494944&req=5

Figure 2: Differentiated bone marrow cells possess strong suppressive capacities and can be subdivided into both MDSC subsets(A) Expression of CD11b by bone marrow cells after a 6-day incubation period in CM as measured by flow cytometry. (B) Summarizing graph of ratio of MDSC subsets (C) Flow cytometry contour plots of in vitro MDSC before and after MACS sort. Underneath the contour plots of the sorted MDSC, representative pictures showing the morphology of these subsets are depicted. Pictures were taken with a light microscope at 64 times magnification. (D) Representative experiment showing suppression of CD8+ T cells by sorted in vitro MDSC (1:4 ratio MDSC to T cell). (E) The graph on the left represents the proliferation inhibition of CD3/CD28-activated CD8+ T cells (ratio MDSC to T cell as indicated in the graph). The graph on the right shows changes in IFN-γ secretion measured during the same experiment. Control represents T cells incubated without MDSC. Mean of at least 3 experiments +/− SEM is shown in all graphs. Number of asterisks in the figures indicates the level of statistical significance as follows: ***, p < 0.001.

Mentions: CRC expands MDSC in vivo, which seem to contribute to tumor staging and poor prognosis [4, 29-31]. Usually, a large tumor burden is required to divert physiological myeloid differentiation towards MDSC expansion, possibly due to local and systemic GM-CSF accumulation. As we aimed to develop an in vitro culture system to differentiate bone marrow cells to MDSC resembling those found within CRC tumors, we first evaluated using ELISA whether the CRC cell line CT26 produced high levels of GM-CSF. CT26 tumor cells produced barely any GM-CSF (Fig. 1A). Therefore, we decided in analogy to our previous study on in vitro-generated melanoma MDSC [28], to transduce CT26 tumor cells with lentiviral vectors encoding GM-CSF. This resulted in secretion of high levels of GM-CSF (Fig. 1A). To examine whether the secreted GM-CSF was biologically active, GM-CSF-dependent FDCP-1 cells were labeled with CFSE and consequently cultured in the presence or absence of recombinant murine GM-CSF, as well as in conditioned medium (CM) of CT26-GM-CSF and CM of non-modified CT26 tumor cells. In this assay the proliferation of FDCP-1 cells incubated in recombinant GM-CSF was comparable to that of FDCP-1 cells incubated with CM of CT26-GM-CSF (Fig. 2B-2C). This CM was subsequently used to culture bone marrow cells, demonstrating that after 6 days of culture, cell yields were consistently higher in the high GM-CSF condition (Fig. 1D). Moreover, the majority of these cells expressed CD11b. This was not the case in cultures with CM of non-modified CT26 tumor cells (Fig. 1E). To identify the concentration of GM-CSF necessary to generate CD11b+ cells, we used CM of non-modified CT26 tumor cells supplemented with different concentrations of recombinant GM-CSF to culture bone marrow cells. High percentages of CD11b+ cells were generated in the presence of recombinant GM-CSF, without significant differences when using relative high (320 ng/ml) or low GM-CSF concentrations (20 ng/ml) (Fig. 1E). However, a significant difference was observed in the yield of CD11b+ cells between the conditions where recombinant GM-CSF or CM of transduced CT26 tumor cells was used (Fig. 1F). Since the yield and purity of CD11b+ cells was highest after differentiation in CM from CT26-GM-CSF cells (Fig. 2A), we continued with these culture conditions. MDSC are known to be a very heterogeneous population of cells but can be generally divided into a monocytic (Ly6C+) and a granulocytic (Ly6G+) subset. We examined the appearance of these subsets in the generated CD11b+ population (Fig. 2B). The ratio of the different subsets in the in vitro system coincides with the in vivo situation. Next, we examined their suppressive capacity as it is widely accepted that functionality and more specifically suppression of T-cell responses, is the single most important marker to identify MDSC. We showed that sorted CD11b+ Ly6C+ as well as CD11b+ Ly6G+ cells (Fig. 2C) had a high T-cell suppressive capacity (Fig. 2D-2E). Consequently, the CD11b+ cells obtained through the culture of bone marrow cells in CM of CT26-GM-CSF tumor cells could be considered as MDSC.


Ex vivo generation of myeloid-derived suppressor cells that model the tumor immunosuppressive environment in colorectal cancer.

Dufait I, Schwarze JK, Liechtenstein T, Leonard W, Jiang H, Escors D, De Ridder M, Breckpot K - Oncotarget (2015)

Differentiated bone marrow cells possess strong suppressive capacities and can be subdivided into both MDSC subsets(A) Expression of CD11b by bone marrow cells after a 6-day incubation period in CM as measured by flow cytometry. (B) Summarizing graph of ratio of MDSC subsets (C) Flow cytometry contour plots of in vitro MDSC before and after MACS sort. Underneath the contour plots of the sorted MDSC, representative pictures showing the morphology of these subsets are depicted. Pictures were taken with a light microscope at 64 times magnification. (D) Representative experiment showing suppression of CD8+ T cells by sorted in vitro MDSC (1:4 ratio MDSC to T cell). (E) The graph on the left represents the proliferation inhibition of CD3/CD28-activated CD8+ T cells (ratio MDSC to T cell as indicated in the graph). The graph on the right shows changes in IFN-γ secretion measured during the same experiment. Control represents T cells incubated without MDSC. Mean of at least 3 experiments +/− SEM is shown in all graphs. Number of asterisks in the figures indicates the level of statistical significance as follows: ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494944&req=5

Figure 2: Differentiated bone marrow cells possess strong suppressive capacities and can be subdivided into both MDSC subsets(A) Expression of CD11b by bone marrow cells after a 6-day incubation period in CM as measured by flow cytometry. (B) Summarizing graph of ratio of MDSC subsets (C) Flow cytometry contour plots of in vitro MDSC before and after MACS sort. Underneath the contour plots of the sorted MDSC, representative pictures showing the morphology of these subsets are depicted. Pictures were taken with a light microscope at 64 times magnification. (D) Representative experiment showing suppression of CD8+ T cells by sorted in vitro MDSC (1:4 ratio MDSC to T cell). (E) The graph on the left represents the proliferation inhibition of CD3/CD28-activated CD8+ T cells (ratio MDSC to T cell as indicated in the graph). The graph on the right shows changes in IFN-γ secretion measured during the same experiment. Control represents T cells incubated without MDSC. Mean of at least 3 experiments +/− SEM is shown in all graphs. Number of asterisks in the figures indicates the level of statistical significance as follows: ***, p < 0.001.
Mentions: CRC expands MDSC in vivo, which seem to contribute to tumor staging and poor prognosis [4, 29-31]. Usually, a large tumor burden is required to divert physiological myeloid differentiation towards MDSC expansion, possibly due to local and systemic GM-CSF accumulation. As we aimed to develop an in vitro culture system to differentiate bone marrow cells to MDSC resembling those found within CRC tumors, we first evaluated using ELISA whether the CRC cell line CT26 produced high levels of GM-CSF. CT26 tumor cells produced barely any GM-CSF (Fig. 1A). Therefore, we decided in analogy to our previous study on in vitro-generated melanoma MDSC [28], to transduce CT26 tumor cells with lentiviral vectors encoding GM-CSF. This resulted in secretion of high levels of GM-CSF (Fig. 1A). To examine whether the secreted GM-CSF was biologically active, GM-CSF-dependent FDCP-1 cells were labeled with CFSE and consequently cultured in the presence or absence of recombinant murine GM-CSF, as well as in conditioned medium (CM) of CT26-GM-CSF and CM of non-modified CT26 tumor cells. In this assay the proliferation of FDCP-1 cells incubated in recombinant GM-CSF was comparable to that of FDCP-1 cells incubated with CM of CT26-GM-CSF (Fig. 2B-2C). This CM was subsequently used to culture bone marrow cells, demonstrating that after 6 days of culture, cell yields were consistently higher in the high GM-CSF condition (Fig. 1D). Moreover, the majority of these cells expressed CD11b. This was not the case in cultures with CM of non-modified CT26 tumor cells (Fig. 1E). To identify the concentration of GM-CSF necessary to generate CD11b+ cells, we used CM of non-modified CT26 tumor cells supplemented with different concentrations of recombinant GM-CSF to culture bone marrow cells. High percentages of CD11b+ cells were generated in the presence of recombinant GM-CSF, without significant differences when using relative high (320 ng/ml) or low GM-CSF concentrations (20 ng/ml) (Fig. 1E). However, a significant difference was observed in the yield of CD11b+ cells between the conditions where recombinant GM-CSF or CM of transduced CT26 tumor cells was used (Fig. 1F). Since the yield and purity of CD11b+ cells was highest after differentiation in CM from CT26-GM-CSF cells (Fig. 2A), we continued with these culture conditions. MDSC are known to be a very heterogeneous population of cells but can be generally divided into a monocytic (Ly6C+) and a granulocytic (Ly6G+) subset. We examined the appearance of these subsets in the generated CD11b+ population (Fig. 2B). The ratio of the different subsets in the in vitro system coincides with the in vivo situation. Next, we examined their suppressive capacity as it is widely accepted that functionality and more specifically suppression of T-cell responses, is the single most important marker to identify MDSC. We showed that sorted CD11b+ Ly6C+ as well as CD11b+ Ly6G+ cells (Fig. 2C) had a high T-cell suppressive capacity (Fig. 2D-2E). Consequently, the CD11b+ cells obtained through the culture of bone marrow cells in CM of CT26-GM-CSF tumor cells could be considered as MDSC.

Bottom Line: This resulted in the generation of high numbers of CD11b(+) Ly6G(+) granulocytic and CD11b(+) Ly6C(+) monocytic MDSC, which closely resemble those found within the tumor but not the spleen of CT26 tumor-bearing mice.We confirmed that inhibition of arginase-1 or iNOS in vivo resulted in the stimulation of cytotoxic T-cell responses.These data confirm the role of MDSC as inhibitors of T-cell-mediated immune responses in CRC.

View Article: PubMed Central - PubMed

Affiliation: UZ Brussel, Department of Radiotherapy, Vrije Universiteit Brussel, Brussels, Belgium.

ABSTRACT
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells that accumulate in tumor-bearing subjects and which strongly inhibit anti-cancer immune responses. To study the biology of MDSC in colorectal cancer (CRC), we cultured bone marrow cells in conditioned medium from CT26 cells, which are genetically modified to secrete high levels of granulocyte-macrophage colony-stimulating factor. This resulted in the generation of high numbers of CD11b(+) Ly6G(+) granulocytic and CD11b(+) Ly6C(+) monocytic MDSC, which closely resemble those found within the tumor but not the spleen of CT26 tumor-bearing mice. Such MDSC potently inhibited T-cell responses in vitro, a process that could be reversed upon blocking of arginase-1 or inducible nitric oxide synthase (iNOS). We confirmed that inhibition of arginase-1 or iNOS in vivo resulted in the stimulation of cytotoxic T-cell responses. A delay in tumor growth was observed upon functional repression of both enzymes. These data confirm the role of MDSC as inhibitors of T-cell-mediated immune responses in CRC. Moreover, MDSC differentiated in vitro from bone marrow cells using conditioned medium of GM-CSF-secreting CT26 cells, represent a valuable platform to study/identify drugs that counteract MDSC activities.

No MeSH data available.


Related in: MedlinePlus