Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Mar 1;190(5):2455-63.
doi: 10.4049/jimmunol.1201314. Epub 2013 Jan 25.

Mouse tumor vasculature expresses NKG2D ligands and can be targeted by chimeric NKG2D-modified T cells

Tong Zhang  1 , Charles L Sentman
Affiliations

Mouse tumor vasculature expresses NKG2D ligands and can be targeted by chimeric NKG2D-modified T cells

Tong Zhang et al. J Immunol. .

Abstract

Tumor angiogenesis plays an important role in the development of solid tumors, and targeting the tumor vasculature has emerged as a strategy to prevent growth and progression of solid tumors. In this study, we show that murine tumor vasculature expresses Rae1, a ligand for a stimulatory NK receptor NKG2D. By genetic modification of T cells with an NKG2D-based chimeric Ag receptor, referred to as chNKG2D in which the NKG2D receptor is fused to the signaling domain of CD3ζ-chain, T cells were capable of targeting tumor vasculature leading to reduced tumor angiogenesis and tumor growth. This occurred even in tumors where the tumor cells themselves did not express NKG2D ligands. H5V, an endothelial cell line, expresses Rae1 and was lysed by chNKG2D-bearing T cells in a perforin-dependent manner. In vitro capillary tube formation was inhibited by chNKG2D T cells through IFN-γ and cell-cell contact mechanisms. The in vivo antiangiogenesis effects mediated by chNKG2D-bearing T cells at the tumor site were dependent on IFN-γ and perforin. These results provide a novel mechanism for NKG2D-based targeting of solid tumors.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Intratumoral injection of chNKG2D T cells inhibits the growth of B16F10 tumors and tumor-associated angiogenesis
A schematic diagram of tumor inoculation and treatment is shown in (A). B16F10 tumor cells (5×105) were given subcutaneously to B6 mice. Either wtNKG2D- or chNKG2D-modified T cells (2×106) were intratumorally injected on days 7 and 9 post-tumor inoculations. (B) Tumor diameters were measured every 2 days starting 7 days post-tumor injection (n = 10 mice per group). The tumor areas are represented as Mean + SEM. ** p<0.01 (from day 9 to 15). (C) The tumor-inoculated sites in wtNKG2D T cell- or chNKG2D T cell–treated mice on day 15. (D) Tumor-inoculated sites were isolated from wtNKG2D T cell- or chNKG2D T cell–treated mice on day 15 and tumor-supplying vessels were counted. Data are represented as the mean + SEM of 10 mice in each group pooled from 2 independent experiments. ** p<0.01 (E) Matrigel (250 μl) was mixed with an equal volume of B16F10 cells (5×105) before subcutaneous injection into the flanks of B6 mice. Either wtNKG2D or chNKG2D T cells (2×106) were injected in matrigel plugs 5 and 7 days after Matrigel implantation. On day 9, Matrigel plugs were isolated and the hemoglobin content was determined, as a measure of the degree of vascularization of the Matrigel explants. Results are pooled data from two independent experiments. ** p<0.01
Figure 2
Figure 2. Tumor vasculature expresses Rae1 and can stimulate chNKG2D T cells to produce IFN-γ
Large (~12mm diameter) B16F10 tumors (A) or MC-38 tumors (B) were dissected and digested with collagenase and DNAse I. Viable cells were collected from interfaces by Histopaque-density gradient centrifugation. Cells were stained with anti-CD45-PE, anti-CD31-FITC, and anti-Rae1-APC mAbs (or isotype controls). Rae1 expression on tumor vessels (CD45−CD31+) is shown in histograms. The results shown are representative one of 4 independent experiments. Isotype control is shown in dashed lines, and anti-Rae1 staining is shown in solid lines. (C) Primary endothelial cells derived from B6 heart endothelial and aortas were isolated by digestion with collagenase and DNAse I, followed by density gradient. Cells were stained with anti-CD31-FITC mAbs and NKG2D-IgG-APC (or isotype controls). The results shown are representative one of 2 independent experiments. The isotype control is shown as a dashed line. NKG2D-IgG-APC staining is shown as a solid line. (D) B16F10 and MC-38 tumor-associated endothelial cells (CD31+) were isolated using MACS sorting. Endothelial cells (104) were co-cultured with either wtNKG2D (grey bar) or chNKG2D T cells (black bar) in 96-well plates for 24h. Endothelial cells alone (unfilled) were used as negative controls. The results shown are representative from three independent experiments and are shown as mean + SD. ** p<0.01
Figure 3
Figure 3. Oxidative stress induces the expression of Rae1 on mouse endothelial cells
(A) B6 mouse-derived endothelial cell line H5V cells were treated with 0.3 mM H2O2 or PBS for 48 h. Rae1 and CD31 cell surface expression were assessed by flow cytometry. Shaded histograms represent isotype controls, solid-line histograms represent PBS-treated cells, and dashed-line histograms represent H2O2-treated cells. Histograms are representative of at least 3 independent experiments. (B) H5V cells were incubated with 0.3 mM H2O2 in the presence of either 1mM caffeine (an inhibitor of ATM and ATR) or 10μM KU55933 (an ATM inhibitor) for 48 h. DMSO (0.02%) was used as a vehicle control. Surface expression of Rae1 and CD31 was analyzed by flow cytometry. The relative MFI values of Rae1 and CD31 expression were set as 100 in the vehicle control group. The results shown are pooled data from 3 independent experiments, and they are represented as mean + SD. * p<0.05, compared with DMSO controls or H2O2 plus ATM/ATR inhibitors. (C) ChNKG2D T cells use perforin to kill Rae1-positive H5V in vitro. Effector T cells derived from WT B6 or Pfp−/− mice that were modified with either wtNKG2D (open bars) or chNKG2D (filled bars) were cocultured with H5V cells at an E:T ratio of 5:1 in 5-hr LDH release assays. The data are presented as mean + SD of triplicates and are representative from 2 independent experiments. ** p<0.01
Figure 4
Figure 4. ChNKG2D T cells inhibit H5V tube formation through both cell contact and soluble factors
(A) H5V cells (4 × 104) were cultured on Matrigel-coated plates in the presence of 1:2 diluted condition media (CM) derived from immobilized anti-NKG2D mAb-stimulated T cells (either wtNKG2D or chNKG2D T cells). H5V cells alone were used as positive controls. The images shown are representative of 3 independent experiments. (B) H5V cells (4 × 104) were co-cultured with equal numbers of T cells (either wtNKG2D T cells or chNKG2D T cells) on Matrigel-coated plates for 6 hr. The images shown are representative of 2 independent experiments. (C & D) Scoring of H5V tube formation after addition of CM (C) or co-culture with T cells (D). The results shown are representative from two or three independent experiments, and they are shown as mean + SD. ** p<0.01, compared with the treatment with B6 wtNKG2D T cells (D) or its CM (C).
Figure 5
Figure 5. Perforin and IFN-γ are involved in chNKG2D T cell-mediated inhibition of H5V tube formation
(A) H5V cells (4 × 104) were co-cultured with either T cells (wtNKG2D, chNKG2D, or Pfp−/− chNKG2D T cells at T: H5V ratios 1:1) or diluted CM (1:4) derived from immobilized anti-NKG2D mAb-stimulated T cells on Matrigel-coated plates for 6 hr. Representative images of H5V tube formation are shown. (B) H5V cells were co-cultured with either wtNKG2D T cells (◇), chNKG2D T cells (▲), or Pfp−/− chNKG2D T cells (■) at T: H5V ratios from 0.1:1 to 1:1. H5V cells alone were used as controls. H5V tube formation was scored and set to 100 for H5V cells alone. The mean value of H5V tube formation in the absence of T cells is set to 100. Cumulative data from two independent experiments are shown as mean + SD. ** p<0.01, compared with wtNKG2D T cells; # p<0.01, compared with chNKG2D T cells. (C) H5V cells were cultured in the presence of diluted CM (1:4) on Matrigel-coated plates. The mean value of tube formation of H5V cells in the absence of T cells was set to 100. Data from two independent experiments are shown as mean + SD. ** p<0.01 (D) ChNKG2D T cells (105) or wtNKG2D T cells were cultured with H5V cells (4:1, filled bars) or with media alone (open bars), and IFN-g production was measured after 24h cultures by ELISA. Data are shown as IFN-γ(pg/ml) + SD, and they are representative of three experiments.
Figure 6
Figure 6. IFN-γ and perforin are involved in chNKG2D T cell-mediated anti-angiogenesis in vivo
(A) Tumor-bearing mice that were inoculated s.c. with 5×105 B16F10 cells on day 0 were treated with two doses of chNKG2D T cells (2×106) derived from either WT B6 (■), IFN-γ−/−(▲) and Pfp−/−(•) mice on days 7 and 9. WtNKG2D transduced T cells (◇) from B6 mice were used as negative controls. Tumor diameters were measured every 2 days starting 7 days post-tumor injection. The results shown are pooled data from 2 independent experiments. The tumor areas are represented as Mean + SEM. (B) Representative images of tumor-inoculated sites in T cell–treated mice on day 15. (C) Tumor-inoculated sites were isolated from T cell–treated mice at day 15 and tumor-supplying vessels were counted. Data are shown as the individual values of 8 mice in each group, pooled from 2 independent experiments.
Figure 7
Figure 7. Summary of mechanisms of chNKG2D T cell-mediated anti-tumor angiogenesis
Inside the tumor, chNKG2D T cells can directly target NKG2D ligand-positive tumor cells, tumor vasculature as well as angiogenesis-promoting MDSC and Treg, leading to less angiogenesis and reduced tumor growth. Both chNKG2D T cell-derived IFN-γ and perforin are involved in the inhibition. See text for details.
See this image and copyright information in PMC

References

    1. Chouaib S, Kieda C, Benlalam H, Noman MZ, Mami-Chouaib F, Ruegg C. Endothelial cells as key determinants of the tumor microenvironment: interaction with tumor cells, extracellular matrix and immune killer cells. Crit Rev Immunol. 30:529–545. - PubMed
    1. Kerbel RS. Tumor angiogenesis. N Engl J Med. 2008;358:2039–2049. - PMC - PubMed
    1. Rakhra K, Bachireddy P, Zabuawala T, Zeiser R, Xu L, Kopelman A, Fan AC, Yang Q, Braunstein L, Crosby E, Ryeom S, Felsher DW. CD4(+) T cells contribute to the remodeling of the microenvironment required for sustained tumor regression upon oncogene inactivation. Cancer Cell. 18:485–498. - PMC - PubMed
    1. Schoenfeld J, Jinushi M, Nakazaki Y, Wiener D, Park J, Soiffer R, Neuberg D, Mihm M, Hodi FS, Dranoff G. Active immunotherapy induces antibody responses that target tumor angiogenesis. Cancer Res. 70:10150–10160. - PMC - PubMed
    1. Los M, Roodhart JM, Voest EE. Target practice: lessons from phase III trials with bevacizumab and vatalanib in the treatment of advanced colorectal cancer. Oncologist. 2007;12:443–450. - PubMed

Publication types

MeSH terms