Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovarian tumor microenvironment

Amorette Barber; Agnieszka Rynda; Charles L Sentman

doi:10.4049/jimmunol.0902000

. Author manuscript; available in PMC: 2010 Dec 1.

Published in final edited form as: J Immunol. 2009 Nov 13;183(11):6939–6947. doi: 10.4049/jimmunol.0902000

Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovarian tumor microenvironment

Amorette Barber ^*, "VSports手机版" Agnieszka Rynda ^*, Charles L Sentman ^*

PMCID: PMC2825039 NIHMSID: NIHMS176824 PMID: 19915047

"VSports注册入口" Abstract

Adoptive transfer of T cells expressing chimeric NKG2D receptors (chNKG2D), a fusion of NKG2D and CD3ζ, can lead to long-term, tumor-free surv ival in a murine model of ovarian cancer. To determine the mechanisms of chNKG2D T cell anti-tumor efficacy, we analyzed how chNKG2D T cells altered the tumor microenvironment, including the tumor-infiltrating leukocyte populations. ChNKG2D T cell treatment of mice bearing ID8 tumor cells increased the number and activation of NK cells, and activation of host CD8+ T cells within the tumor. Foxp3+ regulatory T cells at the tumor site decreased more than 300-fold after chNKG2D T cell treatment. Tumor-associated regulatory T cells expressed cell-surface NKG2D ligands and were killed by chNKG2D T cells in a perforin-dependent manner. ChNKG2D T cells also altered the function of myeloid cells at the tumor site, changing these cells from being immunosuppressive to enhancing T cell responses. Cells isolated from the tumor produced elevated amounts of IFNγ, nitric oxide, and other proinflammatory cytokines after chNKG2D T cell treatment. ChNKG2D T cells required perforin, IFNγ, and GM-CSF to induce a full response at the tumor site VSports最新版本. In addition, transfer of chNKG2D T cells into mice bearing tumors that were established for five weeks led to long-term survival of the mice. Thus, chNKG2D T cells altered the ovarian tumor microenvironment to eliminate immunosuppressive cells and induce infiltration and activation of anti-tumor immune cells and production of inflammatory cytokines. This induction of an immune response likely contributes chNKG2D T cells’ ability to eliminate established tumors.

Keywords: Tumor immunity, NK cells, Ovarian cancer, Gene therapy, Adoptive cell therapy

INTRODUCTION

Adoptive transfer of T cells has shown therapeutic potential in some types of cancer, such as melanoma and EBV-derived tumors (1–3). However, the response rate of T cell immunotherapy is relatively low in other types of cancer, including ovarian cancer (4). One contributing factor for the poor response to T cell immunotherapy may due to be the types of immune cells present at the tumor site VSports注册入口. Leukocytes that may have anti-tumor activities can be found at the tumor site, including CD8+ T cells and natural killer cells (5). However ovarian cancer also has many types of immunosuppressive cells in the tumor microenvironment, including myeloid derived suppressor cells, vascular leukocytes, and regulatory T cells (5–7). Elevated levels of immunosuppressive molecules such as PD-L1, IDO, PGE2, IL-10, and arginase may also inhibit the immune response against tumors (8–10). Therapies that alter leukocyte populations in the tumor microenvironment to prevent the function of immunosuppressive populations and induce the recruitment and activation of immune cells may lead to the development of long-lived anti-tumor immune responses and improve cancer therapy.

Therapeutic efforts to induce immune responses in ovarian cancer include administering inflammatory cytokines, such as IFNγ or GM-CSF alone or in combination with chemotherapy, and these treatment strategies have shown some therapeutic success (11–14) V体育官网入口. Additionally, treatments that inhibit immunosuppressive populations, such as inhibiting regulatory T cells using Ontak, which consists of IL-2 fused to diptheria toxin, or using CTLA-4 blocking antibodies, have increased anti-tumor immune responses in ovarian cancer patients (15, 16).

Transfer of tumor-reactive T cells has the potential to both kill tumor cells and activate the immune response through proinflammatory cytokine secretion VSports在线直播. It has been shown that adoptive transfer of T cells expressing chimeric NKG2D receptors (chNKG2D), which consist of the NKG2D receptor fused to the cytoplasmic region of the CD3ζ chain, into mice bearing ovarian tumors established for one week leads to long-term, tumor free survival (17). Complete anti-tumor efficacy required not only killing of the tumor cells by chNKG2D T cells through perforin, but also secretion of IFNγ and GM-CSF by the transferred T cells (17, 18). Additionally, CD8+ T cells isolated from human ovarian cancer samples transduced with chNKG2D receptors secreted proinflammatory cytokines, including IFNγ, GM-CSF, CCL3, and CCL5 when cultured with autologous tumor cells (19). This indicated that chNKG2D T cells may induce a proinflammatory immune response at the tumor site through the secretion of cytokines and release of tumor antigens. This current study determined how treatment with chNKG2D T cells altered the tumor-infiltrating leukocyte populations to promote anti-tumor immunity and which chNKG2D T cell effector mechanisms were required for these changes. The therapeutic efficacy of chNKG2D T cells against ovarian tumors established for five weeks in the mice was also determined.

MATERIALS AND METHODS

Mice (V体育安卓版)

Female C57BL/6 (B6) mice and B6-LY5. 2/Cr(CD45. 1 +)mice were purchased from the National Cancer Institute (Frederick, MD). C57BL/6 mice, interferon γ deficient mice B6. 129S7-Ifngtm1Ts/J (IFNγ−/−), interferon γ receptor 1 deficient mice B6. 129S7-IfngR1tm1Agt/J (IFNγR−/−), perforin deficient mice C57BL/6-Prf1tm1Sdz/J (Pfp−/−), Fas ligand deficient mice B6Smn. C3-Faslgld/J (FasL−/−), and C57BL/6-TgTcraTcrb1100Mjb/J (OT-I) micewere purchased from the Jackson Laboratory (Bar Harbor, ME). Mice deficient for both perforin and FasL (Pfp−/−FasL−/−) weregenerated as previously described (18). GM-CSF deficient mice (GM-CSF−/−) on a C57BL/6 background were kindly provided by Dr. Jeff Whitsett of the University of Cincinnati. Mice used were between 7 to 10 weeks of age at the start of each experiment V体育2025版. All animal work was performed in the Dartmouth Medical School Animal Facility in accordance with Institutional guidelines.

Injection of ID8-GFP cells and treatment of mice with genetically modified T cells

Mouse spleen cells were stimulated with ConA for 18 hours (1μg/ml) and transduced as previously described (19, 20). Two days after transduction, T cells were selected in media containing G418 (0. 5 mg/mL) and 25 U/mL recombinant human IL-2 for three days. Viable cells were isolated using Histopaque-1083 (Sigma, St Louis, MO) and expanded for two days without G418 (19, 20). At the time of transfer, wtNKG2D and chNKG2D transduced splenocytes were 99% CD3+NK1 VSports. 1−, were a mixture of CD8+ (80–90%) and CD4+ (10–20%) cells, and had increased expression of NKG2D (21). ID8-GFP cells (2 × 106) were injected intraperitoneally (i. p. ) into B6 or IFNγR1 deficient mice. WtNKG2D or chNKG2D T cells (5 × 106) were transferred i. p. at one or five weeks after tumor injection. Mice were sacrificed and peritoneal washes were performed using 10ml PBS. Red blood cells in the peritoneal washes were lysed with ACK lysis buffer and the number of cells was counted. For survival experiments, ID8-GFP cells (2 × 106) were injected i. p. into B6 mice and wtNKG2D or chNKG2D T cells (5 × 106) were transferred i. p. five, six, and seven weeks or five, seven, and nine weeks after tumor injection. Mice were weighed at least once a week and were sacrificed if they had gained more than 80% of their original body weight.

Detection of tumor infiltrating populations by flow cytometry

Cells isolated by peritoneal wash were incubated with anti-CD16/CD32 and mouse γ globulin (Jackson ImmunoResearch Laboratories, Westgrove, PA), and stained with FITC-conjugated anti-CD3 (clone 145-2C11), anti-CD11c (clone N418), or anti-CD4 (clone GK1. 5), PE-conjugated anti-CD3, anti-MHC class II (clone M5/114. 15. 2), or anti-CD19 (clone 1D3), APC-conjugated anti-CD45. 1 (clone A20), anti-F4/80 (clone BM8), or anti-NK1. 1 (clone PK136), and biotin-conjugated anti-CD69 (clone H1. 2F3), or anti-GR-1 (clone RB6-8C5, BD Pharmingen) with a secondary incubation with PE-Cy5. 5 conjugated streptavidin. To detect cell surface NKG2D ligand expression, cells were stained with mouse NKG2D-human IgG1 fusion protein (R & D Systems) with a APC- labeled goat anti-human IgG secondary (Jackson ImmunoResearch Laboratories). For detection of Foxp3, cells were stained with antibodies for cell surface molecules, and then cells were fixed with 1% paraformaldehyde, permeabilized with 0. 1% saponin, and stained with PE-conjugated anti-Foxp3 (clone MF-14, BioLegend) or isotype control antibodies. All antibodies were purchased from eBiosciences (San Diego, CA) unless otherwise noted VSports app下载. Cell fluorescence was monitored using a FACSCalibur cytometer (Becton Dickinson, San Jose, CA).

To test for in vitro killing of CD4⁺Foxp3⁺ cells and CD19⁺ cells, peritoneal wash cells were isolated from mice bearing ID8-GFP cells for 8–9 weeks. Peritoneal wash cells (2 × 10⁶) were cultured with 1 × 10⁶ wtNKG2D or chNKG2D T cells generated from a B6 mouse, or chNKG2D T cells generated from mice deficient in perforin, FasL, or both perforin and FasL. After 24 hours, the percent CD4⁺Foxp3⁺ cells and CD19⁺ cells was evaluated by flow cytometry.

T cell proliferation assay

Three days after T cell transfer, tumor-bearing mice were sacrificed and a peritoneal wash was performed. Peritoneal wash cells from naïve mice were used as a control. F4/80⁺ cells were isolated from peritoneal washes using biotin-conjugated anti-F4/80 antibodies and magnetic bead selection (Miltenyi Biotec) according to the manufacturer’s instructions. CD8⁺ OT-I T cells were magnetically purified from spleen and lymph node cells using FITC-conjugated anti-CD8β antibodies. OT-I T cells (10⁵) were CFSE-labeled and cultured with F4/80⁺ cells (2 × 10⁵) and OVA_257–264 peptide (10⁻¹⁰ M). Proliferation of OT-I T cells was determined by flow cytometry after four days of culture.

Cytokine secretion and intracellular cytokine staining

Peritoneal wash cells (10⁶) from tumor-bearing mice treated with wtNKG2D or chNKG2D T cells were cultured in 48-well plates in complete media. Twenty-four hour cell free conditioned media were assayed for IFNγ by ELISA using mouse Duoset ELISA kits (R&D systems) and for nitric oxide using Griess’s Reagent for nitrite (Sigma) according to manufacturers’ protocols. Seventy-two hour conditioned media were assayed for additional cytokines using multiplex analysis (Bio-Rad) by the Immune Monitoring Laboratory of the Norris Cotton Cancer Center (Lebanon, NH). For intracellular staining, peritoneal wash cells (10⁶) or spleen cells (2.5 × 10⁶) were cultured in complete media for 24 hours. Brefeldin A (10μg/ml, Sigma) was added during the last five hours of culture. Cells were incubated with FcR block and stained with FITC-conjugated anti-CD8β (clone CT-CD8β), APC-conjugated anti-NK1.1 (clone PK136), or APC-conjugated anti-CD45.1 (clone A20), and biotin-conjugated anti-CD3 (clone eBio500A2), with a PE-Cy5.5 conjugated-streptavidin secondary. Cells were fixed with 1% paraformaldehyde, permeabilized with 0.1% saponin, and stained with PE-conjugated anti-IFNγ (clone XMG12), or PE-conjugated anti-rat IgG1 isotype control.

Statistical analysis

Differences between groups were analyzed using the Student’s ttest or ANOVA using Prism software (GraphPad Software, San Diego, CA). For survival studies, Kaplan-Meier curves were plotted and analyzed using the log rank test and Prism software. Valuesof p<0.05 were considered significant.

RESULTS (VSports注册入口)

"VSports注册入口" Treatment with chNKG2D T cells induced activation of the tumor-infiltrating leukocyte populations

To determine how treatment with chNKG2D T cells alters the tumor microenvironment, ID8 tumor cells were injected into B6 mice and mice were treated with chNKG2D or wtNKG2D T cells that were congenically marked with Ly5.1⁺ after one week. Multiple host leukocyte populations were altered at the tumor site after chNKG2D T cell injection. The number of NK cells increased after chNKG2D T cell injection, with the peak response three days after T cell transfer (Figure 1A). An increased percentage of NK cells expressed CD69, indicating the infiltrating NK cells were more activated in mice treated with chNKG2D T cells. The number of host Ly5.1⁻ CD8⁺ T cells in the peritoneal wash did not change after chNKG2D T cell treatment, however the host CD8⁺ T cells were more activated, as shown by an increased percentage of CD8⁺CD69⁺ cells (Figure 1C). ChNKG2D T cell treatment also increased the number of GR1⁺F4/80⁻ cells, likely neutrophils, in the peritoneal wash (Figure 1D). There was a significant difference in CD19⁺ B cells (Figure 1E). ChNKG2D T cells resulted in rapid decrease in the number of ID8 tumor cells just one day after T cell injection, and it was previously shown that chNKG2D T cells require perforin to directly kill ID8 tumor cells (Figure 1F) (17). These data indicate that treatment with chNKG2D T cells induced a proinflammatory immune response at the tumor site, resulting in an infiltration and activation of immune cells that can decrease tumor burden, including NK cells and CD8⁺ T cells.

ID8-GFP cells were injected and mice were treated with Ly5.1⁺ wtNKG2D (triangles) or chNKG2D (circles) T cells i.p. seven days later. (A) The number of NK1.1⁺CD3⁻ NK cells (B) CD69⁺NK1.1⁺CD3⁻ activated NK cells, (C) Ly5.1⁻ CD8⁺ CD69⁺ T cells, (D) F480⁻ GR1⁺ neutrophils, (E) CD19⁺ CD3⁻ B cells, or (F) GFP⁺ ID8 tumor cells was determined prior to T cell injection (day 0), and one, three, and seven days after T cell injection. The average of each group (n=4) is shown. Treatment with chNKG2D T cells significantly changed the number of the different cell populations compared to control treated mice (*-p<0.05). Data are representative of at least 5 independent experiments.

ChNKG2D T cells eliminate suppressive cells

CD4⁺ regulatory T cells can be found in ovarian tumors and have been previously shown to be present in mice bearing established ID8 tumors (8 weeks) (5, 7, 22). The effect of chNKG2D T cell treatment on the number of host Foxp3⁺CD4⁺ T cells in the tumor was determined. Mice with established tumors (7 to 9 weeks) were treated with wtNKG2D or chNKG2D T cells and the number of Foxp3⁺ T cells at the tumor site was determined three days after T cell transfer. Treatment with chNKG2D T cells decreased the number of Foxp3⁺CD4⁺ T cells at the tumor site by more than 300-fold compared to mice treated with wtNKG2D T cells or that received no treatment (Figure 2A). CD8⁺Foxp3⁺ T cells were not detected in the peritoneal washes of the tumor-bearing mice (data not shown). Analyses with a soluble NKG2D receptor showed that Foxp3⁺CD4⁺ T cells from the tumor site expressed low levels of NKG2D ligands in all mice tested, indicating that these cells may be direct targets of chNKG2D T cells (Figure 2B). While Foxp3⁺CD4⁺ T cells at the tumor site were eliminated, the number of Foxp3⁺CD4⁺ T cells in the spleen was not altered after i.p. injection of chNKG2D T cells, suggesting that the depletion of regulatory cells was a local effect (Figure 2C). Foxp3⁺CD4⁺ T cells in the spleen of tumor-bearing mice or naïve mice did not express NKG2D ligands on the cell surface in all mice tested (Figure 2D and data not shown), indicating that regulatory T cells increase expression of NKG2D ligands within the tumor environment.

ID8-GFP cells were injected i.p. into mice. After eight weeks mice received no treatment (-, grey bars), or were treated with Ly5.1⁺ wtNKG2D (white bars) or chNKG2D (black bars) T cells i.p. Three days after wtNKG2D or chNKG2D T cell injection, the number of Ly5.1⁻ Foxp3⁺CD4⁺ T cells was determined in (A) the peritoneal wash or (C) the spleen. The average of each group + SD (n=4) is shown. Treatment with chNKG2D T cells significantly decreased the number of the Foxp3⁺CD4⁺ T cells in the peritoneal cavity compared to control treated mice (***- p<0.001). (B and D) The expression of NKG2D ligands on Foxp3⁺CD4⁺ T cells was determined by staining (B) peritoneal wash cells or (D) spleen cells with sNKG2D-hIgG (filled) or human IgG isotype control (grey) and with anti-human IgG secondary antibodies. The FACS histograms show NKG2D ligand expression on Ly5.1⁻ CD3⁺CD4⁺Foxp3⁺ cells and are representative of three separate experiments, with n=4 for each experiment. (E and F) ID8-GFP cells were injected i.p. into mice. After eight weeks, peritoneal wash cells were cultured with media, wtNKG2D T cells, chNKG2D T cells, or chNKG2D T cells deficient in perforin (Pfp), FasL, or perforin and FasL (P/F). After 24 hours, the percent of (E) Foxp3⁺CD4⁺ T cells or (F) CD19⁺CD3⁻ cells was determined. Data are representative of two independent experiments. Culture with chNKG2D T cells significantly decreased the percent of Foxp3⁺CD4⁺ T cells (*- p<0.001).

To determine if chNKG2D T cells were directly killing Foxp3⁺CD4⁺ T cells, peritoneal wash cells from mice with established tumors (8–9 weeks) were cultured with wtNKG2D or chNKG2D T cells for 24 hours (Figure 2E). Culture with chNKG2D T cells resulted in much fewer live Foxp3⁺CD4⁺ T cells compare to those cultured with wtNKG2D T cells or medium alone. ChNKG2D T cells required expression of perforin but not FasL to remove the Foxp3⁺CD4⁺ T cells because there was no loss of Foxp3⁺CD4⁺ T cells when they were cultured with chNKG2D T cells deficient in perforin, or both perforin and FasL. These data are consistent with the idea that chNKG2D T cells directly kill tumor-associated Foxp3⁺CD4⁺ T cells using perforin. However, this reduction in vitro was not as large as observed in vivo suggesting that other mechanisms may also be involved to eliminate Foxp3⁺CD4⁺ T cells in vivo.

In addition to regulatory T cells, other populations present in the pertioneal wash of mice with large ascites at 8–9 weeks included ID8 tumor cells (10–15%) and leukocytes including CD11c⁺ cells, F4/80⁺GR1⁺ cells, F4/80⁻GR1⁺ cells, and CD19⁺ B cells. The number of CD19⁺ cells also decreased in vivo when mice were treated with chNKG2D T cells. However in vitro, the percent of CD19⁺ cells did not decrease when cultured with chNKG2D T cells (Figure 2F). Thus, the change in B cell numbers in vivo is likely not due to direct killing by chNKG2D T cells.

Treatment with chNKG2D T cells also induced changes in the antigen presenting cells at the tumor site. There was an increase in CD11c⁺ MHC class II^hi dendritic cells (DCs) (Figure 3A). ChNKG2D T cells also altered the phenotype of the tumor infiltrating macrophages. Macrophages in wtNKG2D T cell treated mice expressed high levels of F4/80 and had low MHC class II expression. After chNKG2D T cell treatment, the macrophages decreased F4/80 expression and increased GR1 and MHC class II expression (Figure 3B). Macrophages from both wtNKG2D and chNKG2D T cell treated mice expressed CD11b. While some previous studies have shown that tumor associated, GR1-expressing macrophages can have an immunosuppressive phenotype, other studies have shown that this is an activated cell phenotype (10, 23). To investigate whether chNKG2D T cells altered the function of macrophages to become immunostimulatory or immunosuppressive, F4/80⁺ cells from mice treated with wtNKG2D or chNKG2D T cells were isolated from peritoneal washes and cultured with CFSE-labeled OT-I T cells and Ova peptide. OT-I T cells cultured with F4/80⁺ cells isolated from chNKG2D T cell treated mice proliferated more than when cultured with F4/80⁺ cells from wtNKG2D T cell treated mice or from naïve mice (Figure 3C-D). The OT-I T cells cultured with F4/80⁺ cells from wtNKG2D T cell treated mice had little proliferation, which is consistent with an immunosuppressive function of these tumor-associated myeloid cells. Thus chNKG2D T cell treatment of tumor-bearing mice induced an activation of the tumor-infiltrating antigen presenting cells, so that they stimulated rather than inhibited antigen-specific T cells.

ID8-GFP cells were injected i.p. into mice. After seven days mice were treated with wtNKG2D (triangles) or chNKG2D (circles) T cells i.p. (A) The number of CD11c⁺MHC class II^hi cells or (B) the number of F4/80⁺GR1⁺ cells was determined prior to T cell injection (day 0), and one, three, and seven days after T cell injection. (C-D) F4/80⁺ cells were isolated from the peritoneal washes three days after injection of wtNKG2D (white bars) or chNKG2D (black bars) T cells or from naïve mice (grey bars) and were cultured with CFSE-labeled OT-I T cells and Ova peptide. OT-I T cell proliferation was measured after four days of culture. (C) The average of each group + SD (n=4) and (D representative histograms of OT-I T cell proliferation are shown. Treatment with chNKG2D T cells significantly changed the number of the different cell populations compared to control treated mice (*-p<0.05). Data are representative of at least 2 separate experiments.

ChNKG2D T cell treatment leads to production of IFNγ and nitric oxide by tumor infiltrating host cells

IFNγ has multiple anti-tumor properties and previous studies have shown that chNKG2D T cells secrete IFNγ when cultured with ID8 tumor cells in vitro and that IFNγ is an important effector molecule for anti-tumor efficacy in vivo (17, 19). To determine whether chNKG2D T cell treatment induced IFNγ production at the tumor site, cytokine production was measured in peritoneal cells isolated from tumor-bearing mice treated with wtNKG2D or chNKG2D T cells. Peritoneal wash cells from chNKG2D T cell treated mice secreted more IFNγ compared to cells isolated from wtNKG2D T cell treated mice (Figure 4A). Additionally, the peritoneal wash cells from chNKG2D T cell treated mice secreted more nitric oxide (Figure 4A), possibly due to the increased IFNγ production as this cytokine can induce macrophage activation. The significantly increased cytokine response was observed as early as one day after chNKG2D T cell injection with a peak occurring seven days after T cellinjection. Intracellular staining for IFNγ was performed to determine which cells at the tumor site were producing IFNγ. Ly5.1⁺ chNKG2D T cells secreted significant levels of IFNγ one and three days after injection, however after seven days, the transferred T cells were no longer found in the peritoneal cavity (Figure 4B). In addition to the transferred T cells, host NK cells, CD4⁺, and CD8⁺ T cells produced significantly more IFNγ in chNKG2D T cell treated mice compared to mice treated with wtNKG2D T cells. Host cell production of IFNγ began one day after chNKG2D T cell injection and this host immune response continued to increasefor seven days. This indicated that chNKG2D T cells induced a host immune response at the tumor site.

(A) Peritoneal wash cells from wtNKG2D (white bars) or chNKG2D T cell (black bars) treated tumor-bearing mice from each timepoint were cultured in media for 24 hours. Cell-free supernatants were assayed for (A) IFNγ or nitric oxide. (B) Intracellular staining was performed on peritoneal wash cells cultured in media for 24 hours. Cells were evaluated for IFNγ production and were gated on either Ly5.1⁺CD3⁺, CD8⁺CD3⁺, CD4⁺CD3⁺, or NK1.1⁺CD3⁻ as indicated. The average of each group + SD (n=4) is shown. Treatment with chNKG2D T cells significantly increased IFNγ and nitric oxide secretion compared to control treated mice (*- p<0.05, **-p<0.01). Data are representative of at least 5 separate experiments.

Changes in the tumor infiltrating populations require chNKG2D T cell derived cytokines and perforin

Expression of perforin, GM-CSF, and IFNγ by chNKG2D T cells is required for complete reduction in tumor burden (17, 18). As these effector molecules likely contribute to the activation of the host immune response, the requirement of these molecules for chNKG2D T cell-induced changes at the tumor site was determined. Tumor-bearing mice were treated with wtNKG2D or chNKG2D T cells derived from B6 mice or mice deficient in GM-CSF, IFNγ, or perforin. Three days after T cell injection, the tumor infiltrating populations were measured (Table I). ChNKG2D T cell-derived GM-CSF was involved in inducing a significant increase in NK cells and NK cell activation, CD8⁺ T cell activation, DCs, and neutrophils, and fewer B cells, because mice treated with chNKG2D T cells deficient in GM-CSF did not have these changes in these leukocyte populations.

Table I.

Leukocyte and tumor cell numbers at the tumor site three days after T cell

	WT	CH	GMCSF^−/− CH	IFNγ^−/− CH	IFNγ R^−/−	Pfp^−/− CH
NK1.1⁺CD⁻	12^a	80^*	46^*^δ	13^δ	12^δ	62^*
NK1.1⁺CD69⁺	5	55^*	25^*^δ	7^δ	7^δ	33^*
CD8⁺CD69⁺	4	11^*	6^δ	3^δ	5^δ	10^*
CD11c⁺MHCII⁺	13	208^*	94^*^δ	21^δ	59^δ	179^*
F480⁺GR1⁺	49	123^*	91^*	44^δ	26^δ	106^*
F480⁻GR1⁺	65	381^*	126^*^δ	73^δ	73^δ	235^*
CD4⁺CD3⁺	120	53^*	72^*	76^*	13^*	78^*
CD19⁺	291	56^*	242^δ	193^*^δ	58^*	171^*^δ
ID8-GFP	10	2^*	3^*	6^δ	6^δ	8^δ

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WT- wfNKG2D T cell treated mice, CH- chNKG2D T cell treated mice

Cell numbers in the peritoneal wash × 10⁴, averages of four mice per group. Data are representative of two to five experiments.

Significantly different than WtNKG2D T cell treated mice, p<0.05.

^δ

Significantly different than ChNKG2D T cell treated mice, p<0.05.

ChNKG2D T cell-derived IFNγ was required for a significant increase in NK cells and in NK cell activation, CD8⁺ T cell activation, neutrophils, and activation of DCs and macrophages. ChNKG2D T cell-derived IFNγ was also required for the alteration in B cells because mice treated with chNKG2D T cells deficient in IFNγ did not have this decrease. To determine whether the chNKG2D T cell-derived IFNγ had a direct effect on host cells, ID8-GFP cells were injected into mice deficient in IFNγR1 and the mice were treated with wtNKG2D or chNKG2D T cells. Similar to mice treated with IFNγ deficient chNKG2D T cells, mice deficient in IFNγR1 treated with chNKG2D T cells did not have an increase in NK cells and NK cell activation, CD8⁺ T cell activation, neutrophils, or maturation and activation of DCs and macrophages. Thus, IFNγ derived from chNKG2D T cells acts on cells of the host to induce changes in the tumor microenvironment. ChNKG2D T cell-derived perforin was required for the decrease in CD19⁺ cells, while all other changes in leukocytes were observed when mice were treated with chNKG2D T cells deficient in perforin. However CD19⁺ cells did not express NKG2D ligands, and were shown to not be direct targets of chNKG2D T cells (Figure 2F). ChNKG2D T cell-derived perforin was also required for the decrease in ID8 tumor cells.

The requirement of chNKG2D T cell-derived molecules for the induction of cytokine secretion at the tumor site was also determined. Peritoneal cells had increased secretion of IFNγ after injection of chNKG2D T cells, and this increase was significantly diminished when chNKG2D T cells lacked GM-CSF, IFNγ, or perforin (Figure 5A). While all three effector molecules from chNKG2D T cells were required for the induction of a host IFNγ response, only chNKG2D T cell-derived IFNγ was required for production of nitric oxide at three and seven days after T cell injection. Mice deficient in IFNγR1 also did not have an increase in IFNγ or nitric oxide secretion after treatment with chNKG2D T cells, demonstrating that host cells need to be responsive to IFNγ in order to increase production of these cytokines at the tumor site (Figure 5B). Intracellular staining was performed to determine which of the IFNγ-producing cells the chNKG2D T cell-derived molecules affected. There were lower percentages of NK cells and CD4⁺ T cells secreting IFNγ when chNKG2D T cells were deficient in GM-CSF, IFNγ, or perforin, indicating that all three molecules were involved in the induction of NK cell and CD4⁺ T cell production of IFNγ (Figure 5C). While there was a reduced percentage of CD8⁺ T cells producing IFNγ when chNKG2D T cells were deficient in GM-CSF, IFNγ, or perforin, this reduction was not significant.

(A) Peritoneal wash cells from B6- derived wtNKG2D (white bars) or chNKG2D T cells (black bars), or chNKG2D T cells deficient in GM-CSF (hashed), IFNγ (striped), or perforin (grey) treated tumor bearing mice from each timepoint were cultured in media for 24 hours. Cell-free supernatants were assayed for (A) IFNγ or nitric oxide. (B) Three days after T cell injection, peritoneal wash cells from B6 mice treated with wtNKG2D (white bars) or chNKG2D T cells (black bars), or IFNγR^−/− mice treated with wtNKG2D (hashed bars) or chNKG2D T cells (grey) were cultured in media for 24 hours. Cell-free supernatants were assayed for IFNγ or nitric oxide. (C) Intracellular staining was performed on peritoneal wash cells cultured in media for 24 hours. Cells were evaluated for IFNγ production and were gated on either CD8⁺CD3⁺, CD4⁺CD3⁺, or NK1.1⁺CD3⁻ as indicated. The average of each group + SD (n=4) is shown. Treatment with chNKG2D T cells significantly increased IFNγ and nitric oxide secretion compared to control treated mice (*p<0.05) and mice treated with chNKG2D T cells deficient in effector molecules produced significantly less IFNγ and nitric oxide compared to chNKG2D T cell treated mice (§- p<0.05). Data are representative of at least 2 separate experiments.

The secretion of additional cytokines by peritoneal cells was altered after treatment with chNKG2D T cells. Many proinflammatory cytokines were increased after chNKG2D T cell treatment, including IL-1, IL-2, IL-12, CCL2, CCL3, and CCL5 (Table II). Anti-inflammatory cytokines IL-9 and IL-10 were decreased in chNKG2D T cell treated mice. The induction of these cytokines also required chNKG2D T cell-derived molecules. ChNKG2D T cell-derived IFNγ was essential for the production of IL-1, IL-2, IL-12, G-CSF, GM-CSF, CCL3, and CCL5, and also for decreasing the amount of IL-6. GM-CSF from chNKG2D T cells had a similar role in cytokine induction, and additionally increased CCL2 and decreased TNFα production. ChNKG2D T cell-derived perforin was necessary for the increase in IL-1, G-CSF, CCL2, and CCL3. Cytokines that did not change after chNKG2D T cell treatment included IL-3, IL-5, IL-13, IL-17, KC, and CCL4, and cytokines not found at the tumor site included IL-4 and Eotaxin (data not shown). Together, these data demonstrate that treatment of tumor-bearing mice with chNKG2D T cells induced a proinflammatory host immune response at the tumor site, decreased immunosuppressive regulatory cells, increased cell populations with anti-tumor capabilities, and the local production of proinflammatory cytokines.

Table II.

Cytokine secretion by peritoneal cells three days after T cell injection

	WT	CH	GM-CSF⁻/⁻ CH	IFNγ⁻/⁻CH	Pfp⁻/⁻ CH
IL-1α	7^a	156^*	13^δ	4^δ	9^δ
IL-1β	18	41^*	15^δ	14^δ	9^δ
IL-2	323	754^*	5<5l^*^δ	427^δ	865^*
IL-6	2216^b	87^*	902^*	523^*^δ	52^*
IL-9	38	13^*	20	20	10^*
IL-10	91	27^*	43^*	40^*	37^*
IL-12 p40	14	95^*	34^δ	31^δ	105^*
IL-12p70	180	55^*	66^*	108^*^δ	32^*
G-CSF	24	148^*	14^δ	18^δ	12^δ
GM-CSF	37	150^*	45^δ	50^δ	141^*
CCL2	3400	9931^*	937^*^δ	10123^*	2221^δ
CCL3	17	33^*	12^δ	18^δ	4^δ
CCL5	128	348^*	171^*^δ	287^*	254^*
TNFα	53	4^*	23^*^δ	4^*	4^*

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Values shown are pg/mL averages of three mice per group.

Values were above detection limit of assay

Significantly different than WtNKG2D T cell treated mice, p<0.05.

^δ

Significantly different than ChNKG2D T cell treated mice, p<0.05.

Treatment of advanced tumors with chNKG2D T cells induced activation of the tumor-infiltrating leukocyte populations and increased survival of tumor-bearing mice

Previous studies showed that treatment of ID8-tumor bearing mice with chNKG2D T cells one, two, and three weeks after tumor cell injection lead to long-term, tumor free survival in 100% of the mice (17). However the efficacy of chNKG2D T cells treating mice with established solid tumors has not been tested. First it was determined if chNKG2D T cells induced changes in the tumor microenvironment in mice bearing five week tumors, which is a time when many solid tumors have been established on the peritoneal wall. Compared to treating one week after tumor cell injection, chNKG2D T cell treatment induced similar changes in tumor-infiltrating populations in mice bearing tumors for five weeks, including an increase in the number of activated NK cells, CD8⁺ T cells, and macrophages, an increase in MHC class II ⁺ dendritic cells, an increase in neutrophils, a decrease in B cells, and a decrease in ID8-GFP tumor cells (Figure 6). Similar changes were also seen after treatment with chNKG2D T cells in mice with tumors established for 7–9 weeks (data not shown). There was also an increase in IFNγ and nitric oxide secretion from peritoneal wash cells in chNKG2D T cell treated mice. This indicated that chNKG2D T cells led to the activation of the tumor-associated leukocytes even in mice bearing tumors established for five weeks.

ID8-GFP cells were injected, and mice were treated with wtNKG2D (triangles) or chNKG2D (circles) T cells i.p. five weeks later. (A) The number of NK1.1⁺CD3⁻ NK cells, CD69⁺NK1.1⁺CD3⁻ activated NK cells, Ly5.1⁻CD8⁺ CD69⁺ T cells, CD19⁺ CD3⁻ B cells, CD11c⁺MHC class II^hi cells, F4/80⁺GR1⁺ cells, F480⁻ GR1⁺ neutrophils,or GFP⁺ ID8 tumor cells was determined prior to T cell injection (day 0), and one and three days after T cell injection. (B) Peritoneal wash cells from wtNKG2D (white bars) or chNKG2D T cell (black bars) treated tumor-bearing mice from each timepoint were cultured in media for 24 hours. Cell-free supernatants were assayed for IFNγ or nitric oxide. The average of each group (n=4) is shown. Treatment with chNKG2D T cells significantly changed the number of the different cell populations compared to control treated mice (*-p<0.05). Data are representative of 2 independent experiments.

To determine if treatment with chNKG2D T cells could increase survival in mice with established solid tumors, wtNKG2D or chNKG2D T cells were transferred to tumor bearing mice five, six, and seven weeks after tumor cell injection (Figure 7A). While mice treated with wtNKG2D T cells had a median survival of 88 days, treatment with chNKG2D T cells significantly increased the survival of tumor-bearing mice. All chNKG2D T cell-treated mice survived longer than the mice treated with wtNKG2D T cells, and seven out of twelve chNKG2D T cell-treated mice survived long-term and were tumor-free 225 days after tumor cell injection. The wtNKG2D T cell treated mice had large tumor burdens at the time of sacrifice, with all mice having over 100 solid tumors on the peritoneal cavity. However, the five chNKG2D T cell treated mice that were sacrificed due to tumor growth had fewer solid tumors on the peritoneal cavity, ranging from 8–40 large solid tumors. This indicated that while some chNKG2D T cell treated mice could not control tumor growth, their tumor burden was still decreased compared to wtNKG2D T cell-treated mice.

ID8-GFP cells were injected on day 0, and mice were treated three times with wtNKG2D (filled symbols) or chNKG2D (open symbols) T cells i.p. either (A) after five, six, and seven weeks or (B) after five, seven, and nine weeks. The survival of the mice was measured (n=11–12 per group). Treatment with chNKG2D T cells significantly increased the survival of tumor-bearing mice compared to control treated mice (***- p<0.001).

Transferring chNKG2D T cells at weekly intervals may not be an ideal therapeutic approach as the peak of the IFNγ immune response occured one week after chNKG2D T cell injection. Injecting the second dose of chNKG2D T cells during the peak of the immune response from the first chNKG2D T cell injection may not result in a stronger boost in the ongoing immune response. Therefore the timing of the chNKG2D T cell injections was altered to be administered five, seven, and nine weeks after tumor cell injection (Figure 7B). Using this treatment regimen, transfer of chNKG2D T cells led to long-term, tumor-free survival in 100% of the mice bearing tumors for five weeks. This demonstrates that successful treatment of established tumors did not require the administration of more chNKG2D T cells but an optimal timing of the T cell infusion based on what occurred in the microenvironment was necessary.

DISCUSSION

Therapies that alter leukocyte populations in the tumor microenvironment to decrease the effect of the immunosuppressive populations and induce the recruitment and activation of anti-tumor immune cells may lead to the development of long-lived anti-tumor immune responses and improve cancer therapy. These data show that treatment with chNKG2D T cells induced a proinflammatory immune response at the tumor site in a mouse model of ovarian cancer. ChNKG2D T cell treatment led to an increase in activated NK cells and activated CD8⁺ T cells, and a decrease in Foxp3⁺ regulatory T cells. Tumor-infiltrating macrophages and DCs also became activated after chNKG2D T cell treatment increasing their activation of antigen-specific T cells. Cells isolated from the peritoneal cavity produced increased amounts of IFNγ, NO, and other proinflammatory cytokines. These changes in tumor-infiltrating populations and cytokine secretion required chNKG2D T cell derived IFNγ, GM-CSF, and perforin, indicating that both cytotoxicity and cytokine secretion played a role in changing the tumor microenvironment. By understanding the immune response induced by chNKG2D T cells, a treatment regimen with chNKG2D T cells was successfully designed to lead to tumor-free survival in mice bearing advanced ovarian tumors.

Many immunosuppressive cells and molecules are found in advanced ovarian cancer, including myeloid derived suppressor cells and regulatory T cells. These cells can inhibit the immune response to ovarian cancer cells through multiple mechanisms. Tumor associated myeloid cells may express molecules that can inhibit T cell responses, including B7-H1 and B7-H4 (8, 24, 25). Tumor-infiltrating T cells can express PD-1, and interaction with PD-L1 and PD-L2 expressed by cells at the tumor site inhibits T cell proliferation, cytokine secretion, and cytotoxicity (8, 26, 27). Additionally, tumor associated macrophages may express IDO, PGE2, and arginase, which may inhibit T cell responses through downregulating CD3 expression, inhibiting T cell proliferation, or inducing apoptosis (9, 10, 28). While many studies suggest that CD11b⁺GR-1⁺ myeloid cells are suppressive in the tumor microenvironment, other studies in infection models find that GR-1 expressing macrophages are inflammatory monocytes (9, 10, 23). These F4/80⁺GR-1⁺ inflammatory monocytes secrete cytokines including NO, IL-12, and TNFα, and are involved in clearance of infections such as Listeria monocytogenes and Toxoplasma gondii (23, 29, 30). Treatment of tumor-bearing mice with chNKG2D T cells changes the phenotype of the tumor-associated macrophages, such that the cells display characteristics of inflammatory macrophages, expressing GR-1 and secreting NO. These cells also stimulated antigen-specific T cell proliferation. Thus chNKG2D T cells reversed the immunosuppressive phenotype of tumor-associated macrophages to become proinflammatory, tipping the balance in favor of developing a host immune response against the tumor. Activation of macrophages required chNKG2D T cell-derived IFNγ and host cell responsiveness to IFNγ, demonstrating that expression of IFNγ is essential for alteration of macrophages at the tumor site.

Foxp3⁺ regulatory T cells (T_reg) are found in human and murine ovarian tumors, and the presence of T_reg cells is inversely correlated with survival (31, 32). T_reg cells isolated from ovarian cancer ascites samples can inhibit the proliferation, cytokine secretion, and cytotoxicity of tumor-infiltrating T cells (5, 31). ChNKG2D T cell treatment of established tumors almost completely eliminated Foxp3⁺ T_reg cells at the tumor site. It has been shown that human adaptive regulatory T cells can express NKG2D ligands during infection with Mycobacterium tuberculosis, but that natural T_reg cells did not express NKG2D ligands (33). Similarly, this study showed that murine Foxp3⁺CD4⁺ regulatory T cells isolated from the tumor environment expressed NKG2D ligands, while T_reg cells from the spleen of tumor-bearing mice or from naïve mice did not express NKG2D ligands on their cell surface. Thus tumor-associated T_reg cells are potential direct targets for chNKG2D T cells, and chNKG2D T cells were shown to kill T_reg cells in vitro through a mechanism that required expression of perforin but not FasL. The elimination of this suppressive population from the tumor site would allow tumor-infiltrating immune cells to be more effective.

A large proportion of the cells at the tumor site were CD19⁺ B cells and this population was also altered after chNKG2D T cell treatment. These cells can express PD-L1 and PD-L2 and can secrete anti-inflammatory cytokines including IL-10 that may inhibit immune responses to tumors (34, 35). ChNKG2D T cells required the expression of not only GM-CSF and IFNγ, but also perforin for the decrease in B cells. IFNγ and TLR stimulation can induce the egress of B cells from the peritoneal cavity, thus IFNγ secretion from chNKG2D T cells and host cells may directly cause the trafficking of the B cells out of the peritoneal cavity (36, 37). Another possibility is that chNKG2D T cells lysed the B cells; however, we did not detect NKG2D ligand expression on these B cells nor did we observe killing of B cells in vitro, so direct killing was unlikely in vivo. Another hypothesis is that upon chNKG2D T cell lysis of the ID8 tumor cells, endogenous molecules that can stimulate TLRs were released from the dying tumor cells, such as heat shock proteins (38). These molecules may stimulate TLRs on the B cells, causing their activation and subsequent trafficking from the peritoneal cavity.

In addition to decreasing immunosuppressive populations at the tumor site, chNKG2D T cell treatment also increased cells that can potentially attack tumor cells. NK cells and CD8⁺ T cells both can lyse tumor cells, thus decreasing tumor burden and also releasing tumor antigens to promote antigen presentation. There was also an increase in the secretion of proinflammatory cytokines, including IFNγ, by host cells at the tumor site. IFNγ has many anti-tumor properties, including increasing antigen presentation, maturing antigen presenting cells, decreasing angiogenesis, having cytostatic effects directly on tumor cells, and IFNγ expression in human ovarian cancer is associated with a favorable prognosis (39–43). Components of the endogenous immune system may be involved in immune surveillance against the tumor even without chNKG2D T cell treatment. Previous work has shown that the host immune system responds to this tumor, but this response is not sufficient for tumor elimination (44). Specifically, wtNKG2D T cell-treated mice that were deficient in IFNγ or NK cell depleted had greater tumor growth compared with B6 mice. This indicates that these host mechanisms play a role in controlling tumor growth; however, these host mechanisms are not able to eliminate the tumor, which may be due to immune suppression at the tumor site. ChNKG2D T cells likely act in combination with host immune cells to overcome local immune suppression and result in tumor elimination. Additional work has shown that host-derived perforin, IFNγ, NK cells, and lymphocytes are all required for complete tumor reduction by chNKG2D T cells (44). This further indicates the importance of the activation of the host immune cells for chNKG2D T cells anti-tumor efficacy. Data show that host cells need to express IFNγR1 for the inflammatory response at the tumor site, indicating that IFNγ produced at the tumor site by chNKG2D T cells and host cells is acting directly on host cells. One possible action may be to activate tumor-associated macrophages thus changing the phenotype of the macrophages and inducing cytokine secretion. The activated macrophages may be secreting nitric oxide, which can have anti-tumor effects including direct killing of ID8 tumor cells, or may be secreting other cytokines that activate host NK cells and T cells (45–47).

Through developing a better understanding of the immune response generated by the chNKG2D T cells at the tumor site, this study helped to develop a treatment regimen that was successful at treating tumors established for five weeks, a time when many solid tumors are well-established on the peritoneal wall. Instead of administering an increased number of chNKG2D T cells to achieve long-term tumor free survival in all mice, we altered the scheduling of the chNKG2D T cell doses such that the second and third doses did not coincide with the peak of the ongoing host immune response after the initial chNKG2D T cell injection. The treatment regimen of administering chNKG2D T cells every other week led to long-term tumor-free survival in 100% of mice. This illustrates that one may need to analyze the kinetics and type of immune response induced by the transferred T cells and determine the best therapeutic regimen to obtain optimal efficacy.

In this study, chNKG2D T cells were able to reduce immunosuppressive cells and induce activation of host anti-tumor immune cells both in early and established tumors. This indicates that despite the increased prevalence of immunosuppressive cells in established tumors, chNKG2D T cells may potentially be able to induce immune responses in patients with early or late stage tumors. ChNKG2D T cell derived IFNγ and GM-CSF were required for many of the changes in the leukocyte populations and for the secretion of proinflammatory cytokines. Previous work has shown that chNKG2D T cell-derived IFNγ and GM-CSF were also required for complete anti-tumor efficacy, indicating that changing the tumor microenvironment and inducing a proinflammatory response at the tumor site is essential for cancer therapy (17, 18). ChNKG2D T cells may lyse tumor cells, thus decreasing tumor burden and increasing antigen presentation. Additionally, chNKG2D T cells reduce the immunosuppressive populations while concurrently secreting proinflammatory cytokines, which can recruit and activate host NK cells, T cells, and antigen presenting cells. While many current studies need to combine multiple different therapies to decrease immunosuppression and increase the activation of the immune response, this study shows that transfer of chNKG2D T cells is a novel approach to tip the balance of the immune response in favor of decreasing immune suppression and developing an inflammatory anti-tumor immune response in ovarian cancer, resulting in long-term, tumor-free survival in mice bearing established ovarian tumors.

Acknowledgments

The authors wish to thank Gary Ward and Alice Givan at the Englert Cell Analysis Laboratory for assistance with flow cytometry (Norris Cotton Cancer Center, Lebanon, NH), the Immune Monitoring Laboratory for assistance in luminex analysis (Norris Cotton Cancer Center), and the Animal Resource Center at Dartmouth Medical School for help with the animal studies.

Grant Support: This study was supported in part by grants from the Department of Microbiology and Immunology and the National Institutes of Health (T32 AI 07363, CA 130911). AB was supported by a John H. Copenhaver, Jr., and William H. Thomas, M.D., Fellowship from Dartmouth College. The contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH.

Footnotes

Publisher's Disclaimer: “This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third party. The final, citable version of record can be found at www.jimmunol.org.”

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[R44] 44.Barber A, Sentman CL. Chimeric NKG2D T cells require both T cell- and host-derived cytokine secretion and perforin expression to increase tumor antigen presentation and systemic immunity. J Immunol. 2009;183:2365–2372. doi: 10.4049/jimmunol.0900721. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Chimeric NKG2D expressing T cells eliminate immunosuppression and activate immunity within the ovarian tumor microenvironment

Amorette Barber (V体育官网)

Agnieszka Rynda (V体育平台登录)

Charles L Sentman

"VSports注册入口" Abstract

INTRODUCTION