Human Head and Neck Squamous Cell Carcinoma-Associated Semaphorin 4D Induces Expansion of Myeloid-Derived Suppressor Cells

Abstract

One of the mechanisms by which malignancies can induce immune suppression is through the production of cytokines that affect the maturation and differentiation of inflammatory cells in the tumor microenvironment. Semaphorin 4D (Sema4D) is a proangiogenic cytokine produced by several malignancies, which has been described in the regulation of the immune system. In the present study, we examined the role of human head and neck squamous cell carcinoma (HNSCC)-secreted Sema4D on myeloid cell differentiation. CD33(+) cells cultured in HNSCC cell line-derived conditioned medium differentiated into myeloid derived suppressor cells (MDSC) (CD33(+)CD11b(+)HLA-DR(-/low)). The addition of anti-Sema4D Ab to HNSCC conditioned medium significantly reduced the expansion of the MDSC population. Similarly, knockdown of Sema4D in an HNSCC cell line resulted in a loss of MDSC function as shown by a decrease in the production of the immune-suppressive cytokines arginase-1, TGF-β, and IL-10 by MDSC, concomitant with recovery of T cell proliferation and IFN-γ production following stimulation of CD3/CD28. Importantly, CD33(+) myeloid and T cells cultured in conditioned medium of HNSCC cells in which Sema4D was knocked down promoted antitumor inflammatory profile, through recovery of the effector T cells (CD4(+)T-bet(+) and CD8(+)T-bet(+)), as well as a decrease in regulatory T cells (CD4(+)CD25(+)FOXP3(+)). We also showed that Sema4D was comparable to GM-CSF in its induction of MDSC. Collectively, this study describes a novel immunosuppressive role for Sema4D in HNSCC through induction of MDSC, and it highlights Sema4D as a therapeutic target for future studies to enhance the antitumorigenic inflammatory response in HNSCC and other epithelial malignancies.

Copyright © 2016 by The American Association of Immunologists, Inc.

Figures

FIGURE 1.

HNSCC conditioned media polarizes myeloid cells toward an MDSC phenotype. (A) CD33+ cells were cultured in NM or HNSCC conditioned media (HN6, HN13) for 72 h and then analyzed by flow cytometry. (B) Dose-dependent induction of MDSC following culture in HNSCC conditioned media. CD33+ cells were cultured in serial dilutions of HN6 conditioned medium (CM) and (C) HN13 CM. Cells were first gated for CD33+ then for CD11b+, followed by a third gate for CD11b+HLA-DR−/low. CD33 cells were isolated using MACS from PBMC separated by centrifugation gradient of peripheral blood. All samples were run in duplicates. CM, conditioned medium.

FIGURE 2.

Sema4D produced by HNSCC plays a role in MDSC induction. (A) Sema4D is secreted by HN6 and HN13 cell lines. Conditioned medium (CM) and total cell lysates from HN6 and HN13 HNSCC cell lines were analyzed by Western blot. Ponceau staining was used as a control for protein loading prior to immunoblotting. β-Actin served as control for cell lysates. (B) Anti-Sema4D Ab treatment downregulates MDSC induction by HNSCC cells. CD33+ cells were cultured in NM, NOK, HN6, and HN13 CM in the presence or absence of anti-Sema4D mAb (10 μg/ml) or isotype control mAb for 72 h. Cells were analyzed by flow cytometry by gating on CD33+CD11b+, then analyzed for CD11b+HLA-DR−/low. The experiment was independently repeated twice. (C) Graphical presentation of two combined experiments showing MDSC reduction (CD33+CD11b+HLA-DR−/low) upon anti-Sema4D Ab treatment of HN6 and HN13 CM. Data were normalized to MDSC in NM treated with IgG isotype. CM, conditioned medium.

FIGURE 3.

Inhibition of Sema4D produced by HN6 rescues MDSC-mediated T cell suppression. (A) Recovery of T cell proliferation following inhibition of Sema4D in HN6 cell line using lentivirus shRNA. T cells stained with CFSE and myeloid cells were added at the indicated ratios (myeloid/T cell [M:T]). Cells were cocultured in conditioned medium (CM) from HN6 Sema4D-shRNA or HN6 Ctl-shRNA and activated by anti-CD3/CD28 microbeads for 72 h. CFSE dilution in T cells was analyzed by FACS. (B) Graphical representation of flow cytometry data shown in (A). Data were normalized to activated T cells cultured alone in NM. The supernatants were collected to assess IFN-γ (C) and IL-4 (D) production using ELISA. Error bars indicate SD of triplicate assays.

FIGURE 4.

Sema4D produced by HN6 induces an immune-suppressive T cell phenotype. (A) T cells were cocultured with myeloid cells at the indicated ratios, followed by CD3/CD28 activation for 72 h. Then cells were analyzed by flow cytometry. (A) The cells were gated on the T cell subpopulation, followed by a second gate on CD3+ cells. CD3+ cells were then gated on (B) CD4+T-bet+ and (C) CD8+T-bet+. The CD4+ population was gated on CD25+FOXP3+. Representative flow cytometry plots displaying percentage of recovery of (B) CD3+CD4+T-bet+ effector cells, (C) CD3+CD8+T-bet+ effector cells, and (D) decrease in CD4+CD25+FOXP3+ Tregs in HN6 conditioned medium (CM) of Sema4D-shRNA versus Ctl-shRNA.

FIGURE 5.

Sema4D enhances GM-CSF plus IL-6–mediated induction of MDSC. (A) The percentage of CD33+HLA-DR−/low MDSC induced by culture in HN6 conditioned medium (CM) is reduced following Sema4D or GM-CSF immunoprecipitation (IP) HN6 CM. (B) Graph shows comparable decrease in the CD33+HLA-DR−/low population following immune depletion of Sema4D or GM-CSF, compared with the control HN6 CM. (C) HN6 CM was depleted of Sema4D or GM-CSF by IP and then used to culture myeloid cells and T cells, followed by anti-CD3/CD28 stimulation. After 72 h, the cells were analyzed by flow cytometry analysis, and the supernatants were collected for ELISA. CFSE dilution in T cells was analyzed by flow cytometry. (D) Graph shows comparable recovery of T cell proliferation upon immune depletion of Sema4D or GM-CSF in HN6 CM. (E) IFN-γ production following immune depletion of Sema4D is comparable to GM-CSF–depleted CM. (F) Sema4D is synergistic to GM-CSF plus IL-6 in MDSC induction. CD33+ cells separated from PBMC of normal donors were cultured in NM with the recombinant protein GM-CSF alone, GM-CSF plus IL-6, or the cytokine induction mixture of GM-CSF plus IL-6 plus Sema4D. (G) CD33+HLA-DR−/low induction in response to treatment with Sema4D, IL-6, and GM-CSF alone or in combination. For (F) and (G), each of the recombinant proteins was used at a concentration of 10 ng/ml. CM, conditioned medium; IP, immunoprecipitation.

FIGURE 6.

HN6-secreted Sema4D induces production of immune-suppressive mediators by MDSC. (A) Sema4D promotes proliferation of total myeloid cells. Bulk CD33+ cells cultured in HN6 control or Sema4D-shRNA conditioned medium (CM) for 24 h were analyzed using the WST-1 proliferation assay. (B) Sema4D promotes proliferation of CD33+HLA-DR−/low cells. CD33+HLA-DR−/low cells were sorted using magnetic beads and analyzed by WST-1 proliferation assay. All readings were normalized to cells growing in NM. (C) Reconstitution of HN6 Sema4D-shRNA CM with human recombinant Sema4D rescues CD33+HLA-DR−/low MDSC proliferation. CD33+HLA-DR−/low cells labeled with CFSE were analyzed by FACS. (D) Sema4D inhibition in HN6 decreases arginase-1 expression in myeloid cells. CD33+ cells were cultured in nontransfected (NT), Ctl-shRNA, or Sema4D-shRNA HN6 CM for 24 h and then analyzed by Western blot for arginase-1 expression. GAPDH served as a loading control. Densitometric analyses included for Western blot data were determined using ImageJ. (E) Sema4D inhibition in HN6 decreases NO production by myeloid cells. HN6 Sema4D-shRNA CM inhibits nitrite production by myeloid cells. Total CD33+ cells were cultured in CM from HN6 Ctl-shRNA or HN6 Sema4D-shRNA for 72 h, and then the media were collected to detect the nitrite concentration using Griess reagent. (F) CD33+ cells were cultured in HN6 Sema4D-shRNA or Ctl-shRNA CM, and cytokine production was assessed by ELISA. Knockdown of Sema4D results in a decrease in IL-10 production by CD33+ cells. (G) A reduction in Sema4D results in a concomitant decrease in TGF-β production by CD33+ cells. Data are shown with background levels of cytokine subtracted to demonstrate myeloid cell–specific cytokine production. CM, conditioned medium; NT, nontransfected.