Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 70 (6), 375-82

Secretion of Vascular Endothelial Growth Factor by Oral Squamous Cell Carcinoma Cells Skews Endothelial Cells to Suppress T-cell Functions

Affiliations

Secretion of Vascular Endothelial Growth Factor by Oral Squamous Cell Carcinoma Cells Skews Endothelial Cells to Suppress T-cell Functions

Jennifer K Mulligan et al. Hum Immunol.

Abstract

Patients with oral squamous cell carcinoma (OSCC) have severe defects in antitumor immune function. Endothelial cells are potential regulators of immune cell function and have therefore been examined to determine their role in tumor-induced immune suppression. The present studies demonstrated that supernatants from endothelial cells exposed to OSCC-conditioned media (endo(OSCC-sup)) exhibited elevated levels of the immune suppressive products prostaglandin E(2) (PGE(2)) and vascular endothelial growth factor (VEGF) compared with supernatants from endothelial cells treated with medium alone (endo(medium)) or with keratinocyte-conditioned medium (endo(ker-sup)). Antibody neutralization of OSCC-derived VEGF prevented tumor-conditioned media from inducing endothelial cells to increase production of PGE(2)and VEGF. Furthermore, treatment of T-cells with supernatants from endo(OSCC-sup) resulted in diminished T-cell proliferation and decreased interferon-gamma (IFN-gamma) production compared with T-cells treated with medium or supernatants from endo(medium) or endo(ker-sup) controls. T-cell levels of granzyme B and perforin were reduced after treatment with supernatant from endo(OSCC-sup) compared with control treatments. The addition of VEGF neutralizing antibody to the OSCC-conditioned medium prevented endothelial cells from being skewed to downregulate T-cell proliferation and production of IFN-gamma, perforin, and granzyme B. Taken together, these studies provide support for the use of VEGF-targeting therapies as an immunotherapeutic agent to block induction of immune suppressive endothelial cells in patients with OSCC.

Figures

Figure 1
Figure 1
Effects of tumor-exposed endothelial cell supernatant on T-cell proliferation. Healthy donor T-cell proliferation was assessed by MTS analysis in response to anti-CD3 stimulation and exposure to various endothelial cells supernatants. (*) indicates that p ≤ 0.002 compared to EndoMedia or EndoKer-sup treatments. There is no significant difference between media and EndoOSCC-sup treatments. Data presented is average optical density (OD) ± SD with n = 6 and is representative of 3 experiments with similar results.
Figure 2
Figure 2
T-cell IFN-γ production in response to anti-CD3 stimulation and endothelial cell supernatant treatment. (A-D) Representative histograms of immunostaining for total T-cell IFN-γ expression. Dark gray peak represents cells staining positive for IFN-γ, with the percent staining positive indicated by the number above the peak. (E) Mean percent of total T-cell, staining positive for IFN-γ+ ± SEM with n ≥ 4 per treatment group. (*) indicates that p < 0.0001 compared to EndoMedia or EndoKer-sup treatments. There is no significant difference between media alone and EndoOSCC-sup treatments. (F) Mean fluorescence intensity of CD8+ T-cells and CD4+ T-cells staining for IFN-γ ± SEM with n ≥ 3 per treatment group. (**) indicates that p ≤ 0.002 compared to EndoMedia or EndoKer-sup treatments. There is no significant difference between media alone and EndoOSCC-sup treatments. (NS) indicates no significant difference in CD4+ T-cell IFN-γ production among any of the treatment groups.
Figure 3
Figure 3
Neutralization of OSCC-secreted VEGF blocks the capacity of OSCC-conditioned media to stimulate endothelial cell PGE2 and VEGF secretion, but not IL-6 secretion. VEGF neutralizing antibody was added to OSCC-conditioned media prior to being used to treat endothelial cells. 24-hours after the addition of OSCC-conditioned media, endothelial cells were washed and new media was added. After this time, endothelial cell supernatants were collected and assayed by ELISA for presence of immune modulatory products. (*) indicates that p ≤ 0.01 compared to EndoMedia or EndoKer-sup treatments. (**) indicates that p ≤ 0.0001 compared to EndoOSCC-sup plus isotype. (#) indicates that p ≤ 0.005 compared to EndoMedia or EndoKer-sup treatments. (NS) Indicates that there is no significant difference between addition of isotype (EndoOSCC-sup + Isotype) versus anti-VEGF antibodies (EndoOSCC-sup + αVEGF) to the OSCC-conditioned media prior to exposure to endothelial cells. Data presented is mean + SEM with n ≥ 4 per treatment group.
Figure 4
Figure 4
Neutralization of OSCC-secreted VEGF blocks endothelial cells from being skewed to suppress CD8+ T-cell IFN-γ production. Mean fluorescent intensity of immunostaining of T-cells that are double positive for IFN-γ+ and CD8+ ± SEM (n ≥ 3 per treatment group). (*) indicates that p ≤ 0.0003 compared to EndoMedia or EndoKer-sup treatments. (**) indicates that p = 0.0008 compared to results of when isotype control antibodies were added to OSCC-conditioned media. There is no significant difference in T-cell IFN-γ production between EndoMedia or EndoKer-sup control treatments and results of when VEGF antibody was added to OSCC supernatants (EndoOSCC-sup + αVEGF).
Figure 5
Figure 5
Endothelial cell supernatant's modulation of T-cell activation to produce IFN-γ in response to anti-CD3 stimulation. T-cell secretion of IFN-γ was measured by ELISA. Mean levels ± SEM with n ≥ 4 of IFN-γ production following exposure to various endothelial cell conditioned medium treatments. (*) indicates that p < 0.0001 compared to EndoMedia or EndoKer-sup treatments. (**) indicates that p < 0.0001 compared to results of when isotype control antibodies were added to OSCC-conditioned media (EndoOSCC-sup + Isotype treatment).
Figure 6
Figure 6
Neutralization of OSCC-secreted VEGF blocks induction of endothelial cells that suppress T-cell proliferation in response to anti-CD3 stimulation. The effects of various endothelial cell supernatants on proliferation of healthy donor T-cells in response to anti-CD3 stimulation were assessed by MTS analysis. Data presented are average optical densities (OD) ± SD with n = 7 and is representative of four separate experiments. (*) indicates that p ≤ 0.003 compared to EndoMedia or EndoKer-sup treatments. (**) indicates that p = 0.002 compared to results of when isotype antibodies were added to OSCC-conditioned media (EndoOSCC-sup + Isotype).
Figure 7
Figure 7
Neutralization of OSCC-secreted VEGF prevents induction of endothelial cells that reduce T-cell levels of the cytotoxic mediators granzyme B and perforin. T-cell production of cytotoxic mediators was examined by flow cytometric analysis of intracellular immunostaining. Mean levels ± SEM of perforin and granzyme B following CD8+ T-cell exposure to endothelial cell conditioned media are in (A) and (B) respectively. (*) indicates that p = 0.01 compared to EndoMedia or EndoKer-sup treatments. (**) indicates that p = 0.04 and (#) indicates that p = 0.01 compared to results of when isotype control antibodies were added to OSCC-conditioned media (EndoOSCC-sup + Isotype). Results are means of analyses from three separate experiments to measure granzyme B and four different experiments to measure perforin.
Figure 8
Figure 8
Summary of results. OSCC-secretion of VEGF induces endothelial cells to increase production of VEGF and PGE2. Furthermore, OSCC-derived VEGF skews endothelial cells to produce factors that diminish endothelial cells' ability to heighten T-cell proliferative responses to anti-CD3 and to downregulate T-cell IFN-γ, granzyme B and perforin production. It is hypothesized that elevated secretion of PGE2 and VEGF by suppressor endothelial cells contributes to their disruption of T-cell functions. Additionally, other OSCC-secreted factors may contribute to induction of immune suppressive endothelial cells by inducing endothelial cells to increase production of IL-6, though this does not appear to be involved in altering T-cell functions.

Similar articles

See all similar articles

Cited by 21 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback