Authentic GITR Signaling Fails To Induce Tumor Regression unless Foxp3+ Regulatory T Cells Are Depleted

Young H. Kim, Su M. Shin, Beom K. Choi, Ho S. Oh, Chang H. Kim, Seung J. Lee, Kwang H. Kim, Don G. Lee, Sang H. Park and Byoung S. Kwon
J Immunol November 15, 2015, 195 (10) 4721-4729; DOI: https://doi.org/10.4049/jimmunol.1403076

Abstract

The glucocorticoid-induced TNFR family–related protein (GITR, TNFRSF18, CD357) is expressed on effector and regulatory T (Treg) cells. Previous studies demonstrated that GITR triggering by anti-GITR mAb enhanced T and B cell–mediated immune responses. GITR-deficient T cells, however, also proliferate more than normal T cells, and this effect is unexplained. Because the activities of mAbs are controlled by their Fc regions, the true effect of GITR signaling needs to be determined by examining its interaction with authentic ligand. Therefore, we generated a pentamerized form of the GITRL extracellular domain (pGITRL) for ligation to GITR and compared its effect on T cells with that of anti-GITR mAb. The pGITRL was more effective than anti-GITR mAb in enhancing the proliferation of effector and regulatory cells in vitro and in vivo. Nonetheless, the growth of MC38 adenocarcinoma cells in vivo was only suppressed for initial 15 d by pGITRL, whereas it was suppressed indefinitely by anti-GITR mAb. Detailed analysis revealed that pGITRL induced extensive proliferation of Foxp3+CD4+ Treg cells and led to the accumulation of activated Treg cells in tumor tissue and draining lymph nodes. Because GITR signaling could not neutralize the suppressive activity of activated Treg cells, pGITRL seems to lose its adjuvant effect when sufficient activated Treg cells have accumulated in the lymph nodes and tumor tissue. Indeed, the antitumor effects of pGITRL were markedly enhanced by depleting CD4+ cells. These results suggest that GITR signaling has stimulatory effects on effector T cells and inhibitory effects through Treg cells.

Introduction

The glucocorticoid-induced TNFR family–related protein (GITR, TNFRSF18, CD357) and its ligand GITRL are members of the TNF/TNFR superfamily, and have been implicated in both innate and adaptive immune responses (1, 2). GITR is constitutively expressed at a high level on CD4+CD25+ regulatory T (Treg) cells and at a moderate level on various types of immune cells, and it is upregulated rapidly after activation (1–3). GITRL is found on APCs, including dendritic cells (DCs), macrophages, and B cells, and its expression is increased upon stimulation (4, 5). Cross-linking of GITR using agonistic anti-GITR mAb (DTA-1) generally enhances immune responses in animal models involving viral infection, tumors, and autoimmune disease (6–12). GITR is an excellent adjuvant for humoral and cellular responses using agonistic mAb (13).

Because the functions of mAbs are controlled by their Fc regions, anti-GITR mAb DTA-1 (rat IgG2b) essentially requires activating FcγR, but not inhibitory FcγRIIB, to suppress tumor growth and to eliminate intratumoral Treg cells in vivo (14). However, another anti-GITR mAb, G3c (rat IgM), had a higher costimulatory potential than DTA-1 for both conventional CD25‑CD4+ (cCD4+) T and Treg cells, but failed to cure tumor-bearing mice and to remove Treg cells in vivo (15). GITR signaling by Fc-GITRL dimers has been also reported to enhance CD8+ T cell–mediated antitumor responses (16), but others have found that Fc-GITRL protein preferentially induces the proliferation of Treg cells rather than effector T cells (17). Each form of GITR agonist has its own curative effects in vivo, and the modulation or elimination of Treg cells seems to be crucial for GITR-mediated antitumor responses (18, 19). Consequently, the effects of GITR triggering in the absence of Fc-mediated signaling need to be determined. We therefore generated a pentamerized form of the GITRL extracellular domain (pGITRL) as a substitute for the natural ligand, and one that may trigger GITR signals more strongly than the natural ligand because of its greater avidity for GITR in the absence of FcR activation, and examined the immunological effects of GITR triggering by pGITRL in vitro and in vivo.

Materials and Methods

Mice and reagents

Six- to 8-wk-old C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained under specific pathogen-free conditions in the animal facility of the National Cancer Center (Goyang, Korea). Anti-mGITR mAb-producing hybridoma cells (DTA-1) were a gift of S. Sakaguchi (Kyoto University, Kyoto, Japan). Hybridomas producing anti-murine CD4 mAb (GK1.5), anti-murine CD8 mAb (2.43), and anti-NK1.1 mAb (PK136) were obtained from the American Type Culture Collection. Anti-mouse GITRL mAb-producing hybridomas (clones 40 and 54) were generated in our laboratory. The Abs were produced from hybridoma culture supernatants and purified with protein G-Sepharose 4 Fast Flow (GE Healthcare, Piscataway, NJ). Purified rat IgG was obtained from Sigma-Aldrich and served as control Ab. The following mAbs for flow cytometry were purchased from BD Pharmingen (San Diego, CA): anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD44, anti-CD62L, anti-B220, anti-CD11b, anti-CD11c, anti-NK1.1, anti-Gr-1, anti-IFN-γ, anti-GITR, and as isotype controls rat IgG2a and rat IgG2b. PE anti-mouse/rat Foxp3 (FJK-16s) staining sets and anti–GITRL-PE mAb were purchased from eBioscience (San Diego, CA).

Construction, expression, and purification of pGITRL fusion proteins

pD18 vector contains a secretory signal sequence for CD5L (MPMGSLQPLATLYLLGMLVASV) and a FLAG tag (DYKDDDDK), and was used to express the coiled-coil domain of human cartilage oligomeric matrix protein29–73 (COMP; DLAPQMLRELQETNAALQDVRELLRQQVKE ITFLKNTVMECDACG) fused to the extracellular domain of mouse GITRL104–309. Recombinant pGITRL is composed of signal sequence, FLAG, COMP, and mouse GITRL extracellular domain. The DNA construct was introduced into Chinese hamster ovary (CHO) cells by electroporation (500 V, 825 μF), and the cells were then exposed to increasing amounts of methotrexate. Recombinant pGITRL was affinity purified from the supernatant of the CHO cells with protein G–agarose resin cross-linked with anti-mouse GITRL mAb. The biological activity of the purified pGITRL was assessed by staining naive and activated T cells with various concentrations of the protein.

Binding of pGITRL

To measure the binding of pGITRL to GITR, lymphocytes were isolated from lymph nodes and spleens of C57BL/6 mice, and single-cell suspensions were stimulated with 0.5 μg/ml anti-CD3 mAb for 24 h. The activated or naive lymphocytes were first incubated with 0, 0.1, 0.5, 1.0, and 5.0 μg/ml pGITRL or equimolar amount of heat-inactivated pGITRL and then stained with anti–GITRL-PE as well as anti–CD4-FITC and anti–CD8-PE-Cy5.

Cell isolation

T cell subpopulations were purified using the MACS magnetic separation system, according to the manufacturer’s instructions (Miltenyi Biotec). In brief, single-cell suspensions were prepared from the spleens and lymph nodes of C57BL/6 mice and preincubated with Fc blocker 2.4G2 for 10 min at 4°C. The cCD4+ T, CD8+ T, or Treg cells were isolated using CD4 or CD8 microbeads or a CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec), according to the manufacturer’s instructions. cCD4+ T cells were isolated from the CD25-depleted cell fraction using CD4 microbeads. Purity of the cCD4+, CD8+, and Treg cells was routinely >92% by flow cytometry.

T cell proliferation assay

The purified T cells were incubated with 0.5 μg/ml anti-CD3 mAb at 2 × 105 cells/well in 96-well, round-bottom microplates in the presence of varying concentrations of soluble pGITRL or anti-GITR mAb. The cells were cultured for 72 h and labeled with 1 μCi [3H]thymidine for the last 8 h. Cellular DNA was harvested and counted by liquid scintillation spectroscopy. Results are expressed as mean ± SD cpm of triplicate wells. For assay of Treg cell suppression, Treg cells (0.5 or 1.0 × 105 cells/well) and cCD4+ T cells (1.0 × 105 cells/well) were cocultured with irradiated splenocytes (20 Gy; 5% with respect to total cells/well) for 3 d in the presence of 0.5 μg/ml anti-CD3 mAb alone or in combination with pGITRL or anti-GITR mAb.

Measurement of cytokines

Cytokines produced from T cells after 3 d of in vitro stimulation were quantified in culture supernatants using a cytometric bead array kit (BD Biosciences, San Jose, CA) on a FACSCalibur cytometer equipped with CellQuestPro and cytometric bead array software, according to the manufacturer’s instructions.

Tumor challenge and treatment

Mice were injected s.c. with 5 × 105 MC38 colon adenocarcinoma cells on the dorsal surface. The tumors were grown for 5 d until they reached 2∼3 mm3 in diameter. Five days after tumor challenge, the mice were treated i.p. with 200 μg pGITRL or anti-GITR mAb five times at 2-d intervals. Heat-inactivated pGITRL were used as isotype control. The mice were monitored every day for tumor growth and survival. In separate experiments, some mice treated with pGITRL or anti-GITR mAb received simultaneously 400 μg depleting anti-CD4, anti-CD8, or anti-NK1.1 mAb on postinjection (PI) days 5, 9, and 13.

Flow cytometry and calculation of absolute cell numbers

Single-cell suspensions were prepared from draining lymph nodes (DLNs), spleens, and tumor tissues. They were incubated with the Fc blocker 2.4G2 for 10 min at 4°C and stained with specific Abs for surface markers. To stain intracellular IFN-γ cytokine, DLN cells were stimulated for 5 h with 50 ng/ml PMA and 500 ng/ml ionomycin along with brefeldin A. The cells were then incubated with 2.4G2 for 10 min at 4°C, stained with FITC-conjugated anti-CD4 or anti-CD8, fixed, permeabilized with a Cytofix/Cytoperm kit (BD Pharmingen), and incubated with PE-conjugated anti–IFN-γ. All samples were analyzed on a FACSCalibur (BD Biosciences). To detect Foxp3, DLN cells were first surface stained with PE-Cy5 anti-CD4 and FITC anti-CD25 mAb, and then intracellularly stained with PE anti-Foxp3 mAb, according to the manufacturer’s instructions (eBioscience). Absolute numbers of each population were calculated by multiplying the percentages measured by flow cytometry by the total number of viable cells (absolute number = % × total cells recovered).

Analysis of tumor-infiltrating lymphocytes

Tumor tissues of tumor-bearing mice were dissected, cut into pieces, and digested with collagenase/DNase I for 30 min at 37°C. Single-cell suspensions were analyzed by flow cytometry.

Statistical analysis

Data are expressed as mean ± SD. Student t test and one-way ANOVA were performed using the Prism software program (GraphPad Software).

Results

Binding of recombinant pentamerized GITRL to GITR on T cells

Recombinant pGITRL was designed as shown in Fig. 1A. The extracellular domain of mouse GITRL was fused to FLAG and COMP to form a five-stranded structure. Recombinant pGITRL proteins produced from CHO cells were purified with an anti-GITRL mAb cross-linked affinity column. The purified pGITRL was ∼25 kDa in size in monomeric form and ∼125 kDa in pentamerized form, on SDS-PAGE and Western blot analysis (Fig. 1B).

FIGURE 1.
FIGURE 1.

Construction, expression, and characterization of recombinant pGITRL. (A) Schematic diagram of the pGITRL (penta-mGITRL) fusion protein construct. (B) SDS-PAGE (left) and Western blot analysis (right) of purified pGITRL protein using anti-FLAG mAb in the presence or absence of 2-ME. (C and D) To measure the binding of pGITRL to GITR on CD4+ T and CD8+ T cells, lymphocytes of C57BL/6 mice were stimulated with anti-CD3 mAb for 24 h. Naive (C) or anti-CD3–activated lymphocytes (D) were first incubated with the indicated doses of pGITRL, or equal amounts of heat-inactivated pGITRL, and then stained with anti–GITRL-PE, anti–CD4-FITC, and anti–CD8-PE-Cy5. All samples were immediately analyzed by FACScan (BD Biosciences). GITR expression on T cells was confirmed using anti–GITR-PE as a positive control (C and D, left panels). Results are representative of two independent experiments.

To assess the ability of pGITRL to bind to GITR, naive and activated CD4+ and CD8+ T cells were first stained with pGITRL, and binding was detected with fluorescence-conjugated anti-GITRL mAb. As previously reported (2), staining of T cells with anti-GITR mAb followed by flow cytometric analysis showed that naive CD4+ T and CD8+ T cells expressed GITR at moderate levels, and expression was increased by 24-h stimulation with anti-CD3 mAb (Fig. 1C, 1D; left panels). GITR expression of T cells was detected by pGITRL in a dose-dependent manner (Fig. 1C, 1D). These results indicated that recombinant pGITRL is able to bind to native GITR protein on T cells.

Recombinant pGITRL is superior to agonistic anti-GITR mAb in inducing proliferation of effector and Treg cells in vitro

To compare the effects of pGITRL and agonistic anti-GITR mAb on T cell proliferation, various types of T cells, including cCD4+ T, CD8+ T, and Treg cells, were isolated from the lymph nodes and spleens of B6 mice and stimulated with anti-CD3 mAb in the presence of agonistic anti-GITR mAb (DTA-1) or pGITRL for 3 d. Proliferation of cCD4+ T, CD8+ T, and Treg cells was increased 1- to 3-fold by agonistic anti-GITR mAb, and 3- to 11-fold by pGITRL (Fig. 2A). pGITRL was particularly effective in enhancing the proliferation of Treg and cCD4+ T cells compared with CD8+ T cells (Fig. 2A).

FIGURE 2.
FIGURE 2.

Effects of pGITRL and anti-GITR mAb on the proliferation of T cell subpopulations in vitro and their cytokine production. (A) CD25−CD4+ T (cCD4+ T), CD8+ T, and CD25+CD4+ Treg cells were purified using the MACS magnetic system. Each cell population was cultured with 0.5 μg/ml anti-CD3 mAb and the indicated doses (μg/ml) of anti-mGITR mAb (DTA-1) or pGITRL for 72 h. Heat-inactivated pGITRL was used as an isotype control (n = 3). The cells were labeled with [3H]thymidine during the last 8 h of culture, harvested onto glass fiber filter mats, and analyzed for [3H]thymidine incorporation by liquid scintillation counting. (B) Cytokine production by T cells was measured in culture supernatant with a cytometric bead array kit (BD Biosciences) after 3 d of culture. (C) Treg cells (0.5 or 1.0 × 105 cells/well) and cCD4+ T cells (1.0 × 105 cells/well) were cocultured with 5% irradiated splenocytes for 3 d in the presence of 0.5 μg/ml anti-CD3 mAb alone or in combination with pGITRL or anti-GITR mAb. (D–F) CFSE-labeled cCD4+ T or Treg cells were mixed with nonlabeled Treg or cCD4+ T cells in a 2:1 ratio along with 5% irradiated splenocytes and cultured in the presence of 0.5 μg/ml anti-CD3 mAb alone or in combination with pGITRL or anti-GITR mAb, as described above. On day 3, live cells were counted and CFSE dilutions were assessed by FACSCalibur (BD Biosciences). (D) CFSE dilution of cCD4+ T or Treg cells. (E and F) Fold increases of dividing cCD4+ T or Treg cells relative to anti-CD3 mAb plus isotype calculated using percentages of dividing cell and their absolute numbers. (E and F) Average fold increases relative to anti-CD3 mAb plus isotype (CD3+ iso) are indicated at the top of each column (A–C; E and F). Plotted data are means ± SDs (*p < 0.05, **p < 0.01), and results are representative of three independent experiments.

We compared IFN-γ levels in culture supernatants of anti-CD3–stimulated cCD4+ T and CD8+ T cells incubated in the presence of anti-GITR mAb or pGITRL for 3 d. IFN-γ levels were higher in the supernatants of cells exposed to pGITRL than in those exposed to anti-GITR mAb. pGITRL increased IFN-γ production 4- to 5-fold in cCD4+ T cells, and 8- to 14-fold in CD8+ T cells (Fig. 2B). These results indicated that activation of GITR signaling by pGITRL promotes the proliferation of CD4+ T, CD8+ T cells, and Treg cells, as well as IFN-γ production by CD8+ T cells.

Because GITR signaling had been reported to neutralize the suppressive function of naive Treg cells (1, 16), we examined whether pGITRL had the same effect. Freshly isolated Treg cells suppressed the proliferation of anti-CD3 mAb-treated cCD4+ T cells (Fig. 2C). The suppression of proliferation of the cCD4+ T cells by Treg cells was reversed by treatment with agonistic anti-GITR mAb and more effectively by pGITRL (Fig. 2C). To separately assess the proliferation of the cCD4+ T and Treg cells during their coculture, CFSE-labeled cCD4+ T or Treg cells were cocultured with nonlabeled cCD4+ T or Treg cells and activated with anti-CD3 mAb as well as with anti-GITR mAb or pGITRL for 3 d, as described above. Although the division of cCD4+ T cells was increased by anti-GITR mAb or pGITRL, as expected, the division of Treg cells was instead decreased by anti-GITR mAb or pGITRL (Fig. 2D). Statistical analysis confirmed that both anti-GITR mAb and pGITRL significantly increased the frequency of dividing cCD4+ T cells but reduced that of dividing Treg cells (Fig. 2E). Moreover, when the absolute numbers of dividing cCD4+ T and Treg cells were calculated, anti-GITR mAb and pGITRL significantly increased the number of dividing CD4+ T, but not that of Treg cells (Fig. 2F). Given that GITR triggering enhances IFN-γ production from the activated T cells (Fig. 2B) and IFN-γ signaling directly causes cell cycle arrest of Treg cells (20), GITR signaling appears to preferentially enhance the proliferation of conventional T cells rather than Treg cells in cocultures.

We also tested whether pGITRL would affect Foxp3 expression in Treg cells, but Foxp3 transcript and protein levels did not change (Supplemental Fig. 1). These results indicate that pGITRL is superior to the DTA-1 anti-GITR mAb in enhancing the proliferation of Treg and T cells, promoting IFN-γ production from CD8+ T cells, and inhibiting the suppressive activity of naive Treg cells.

Recombinant pGITRL and agonistic anti-GITR mAb induce transient and persistent suppression of in vivo tumor growth, respectively

To determine the t1/2 of pGITRL in vivo, 200 μg purified pGITRL was injected i.v. into C57BL/6 mice, and blood was sampled regularly. Serum concentrations of pGITRL were determined by ELISA with anti-GITRL mAb, and this demonstrated that the in vivo t1/2 of pGITRL was ∼48 h (Supplemental Fig. 2).

To assess the antitumor effects of pGITRL in vivo, C57BL/6 mice were first injected s.c. with MC38 adenocarcinoma cells and then treated i.p. with 200 μg purified pGITRL or anti-GITR mAb five times at 2-d intervals from the fifth day after tumor injection. Unexpectedly, although pGITRL was more effective in enhancing the proliferation of T cells and their production of IFN-γ than anti-GITR mAb in vitro (Fig. 2), pGITRL only suppressed the growth of MC38 tumor cells for the initial 15 d, whereas anti-GITR mAb caused sustained inhibition of the growth of the MC38 tumor cells (Fig. 3A).

FIGURE 3.
FIGURE 3.

Antitumor effects of pGITRL and anti-GITR mAb in vivo. C57BL/6 mice were challenged s.c. with 5 × 105 MC38 colon adenocarcinoma cells. Five days after tumor injection, the mice were treated i.p. with 200 μg pGITRL or anti-GITR mAb five times every 2 d (n = 5). Heat-inactivated pGITRL was used as an isotype control. (A) The mice were monitored every day for tumor growth and survival. (B) Absolute number of total, CD62LlowCD4+ T and CD8+ T, and IFN-γ+ T cells in TDLNs on PI day 16 (n = 3). Single-cell suspensions from inguinal LNs were counted and stained with fluorescence-conjugated Ab against cell surface Ag. The surface-stained cells were also intracellularly stained with anti–IFN-γ-PE. (C and D) Percentages, absolute numbers, and phenotypes of Treg cells in TDLNs and spleens on PI day 16 (n = 3). Single-cell suspensions of TDLNs and spleens were counted and stained with specific Abs for surface markers and intracellularly with anti–Foxp3-PE. (C) The gated Foxp3+CD4+ T cells are plotted as CD4 versus CD62L or CD4 versus CD69 (D). Plotted data are means ± SD, and results are representative of three independent experiments.

Cellular analysis of pGITRL- or anti-GITR mAb-injected mice revealed that pGITRL was more effective in increasing the total number of lymphocytes in tumor DLNs (TDLNs) than anti-GITR mAb (Fig. 3B). Similar to total lymphocyte numbers, CD62Llow-activated CD4+ T cells were increased 2.3-fold by pGITRL and 1.4-fold by anti-GITR mAb, and CD62Llow activated CD8+ T cells were increased 2.0-fold by pGITRL and 1.7-fold by anti-GITR mAb (Fig. 3B). IFN-γ–producing CD4+ and CD8+ T cells were increased 2.7-fold and 2.4-fold by pGITRL, respectively, and ∼1.7-fold by anti-GITR mAb (Fig. 3B). It is noteworthy that the frequency of Foxp3+CD4+ Treg cells in TDLNs and spleens was increased somewhat only by pGITRL, not by anti-GITR mAb (Fig. 3C). As a consequence, the absolute number of Foxp3+CD4+ Treg cells was increased >2-fold by pGITRL (Fig. 3C). In addition, when we analyzed the activation status of Foxp3+CD4+ Treg cells by measuring the expression of CD62L and CD69, half of the Treg cells were in an activated state, whether they were exposed to pGITRL or anti-GITR mAb (Fig. 3D).

Taken together, these results indicate that pGITRL strongly enhances the activation of effector T cells and increases the numbers of activated Treg cells in secondary lymphoid organs.

Depletion of CD4+ T cells enhances the antitumor effects of pGITRL

Because Treg cells have a greater suppressive effect following activation (21), we suspected that the increase of Treg cells might interfere with the antitumor effects of pGITRL in vivo. Therefore, we next examined whether the antitumor effects of pGITRL were enhanced by depleting CD4+ T cells, both cCD4+ T cells and Treg cells. Again pGITRL transiently suppressed tumor growth, and the depletion of CD4+ T cells, including Treg cells, also somewhat suppressed tumor growth (Fig. 4A) (17, 18). However, treatment with pGITRL in the absence of cCD4+ T and Treg cells suppressed tumor growth almost completely and even generated tumor-free mice (Fig. 4A, upper panel). In contrast, the elimination of CD8+ T cells or NK cells did not boost the antitumor effect of pGITRL at all (Fig. 4A, upper panel). Several groups have reported that DTA-1 agonistic anti-GITR mAb strongly suppresses tumor growth in vivo and that this effect is mediated by CD4+ T, CD8+ T, and NK cells (7, 22) and is due to a decrease of Treg cells in tumor tissue (6, 14, 23). Indeed, anti-GITR mAb strongly suppressed the growth of MC38 tumors, and this effect was partially reversed by treating the mice with depleting anti-CD4 or anti-NK1.1 mAbs, and completely reversed by depleting them with anti-CD8 mAb (Fig. 4A, lower panel). We note that depletion of CD4+ cells had the opposite therapeutic effect as those of pGITRL and anti-GITR mAbs, indicating that the therapeutic effects of anti-GITR mAb and pGITRL are primarily determined by whether Treg cells are depleted or activated.

FIGURE 4.
FIGURE 4.

CD4+ T cell depletion enhances pGITRL-mediated antitumor effects. C57BL/6 mice were challenged s.c. with 5 × 105 MC38 colon adenocarcinoma cells (n = 5). Five days after the tumor injection, the mice were treated i.p. with 200 μg pGITRL or heat-inactivated (HI) pGITRL as a control, or alternatively 200 μg DTA-1 anti-GITR mAb. Some mice received 400 μg depleting anti-CD4, anti-CD8, or anti-NK1.1 mAb on PI days 5, 9, and 13. (A) The mice were monitored every 2–3 d for tumor growth and survival. (B and C) On PI day 15, single-cell suspensions of TDLNs and spleens from pGITRL-treated mice were counted, surface stained with FITC–anti-CD25 and PE–Cy5-anti-CD4, and then intracellularly stained with PE–anti-Foxp3 (n = 3). Samples were subsequently analyzed by FACSCalibur (BD Biosciences). Representative results of flow cytometric analysis (B). Percentages (C) and absolute numbers (D) of Foxp3+ Treg cells in TDLNs and spleens. The plotted data are means ± SD, and results are representative of three independent experiments.

To confirm the absence of Treg cells following treatment with depleting anti-CD4 mAb, lymphocytes from TDLNs and spleens were prepared from each group of mice 15 d after tumor injection, and the expression of CD4 and Foxp3 was assessed by flow cytometry. pGITRL increased moderately the frequency of Treg cells in TDLNs and spleens, and the depleting anti-CD4 mAb completely removed both cCD4+ T cells and Tregs (Fig. 4B, 4C). The absolute number of Treg cells was increased 2-fold by treatment with pGITRL (Fig. 4D).

These results suggest that the antitumor effects of pGITRL may be compromised by the increase in activated Treg cells even though conventional CD4+ T and CD8+ T cells increase in parallel.

pGITRL has a strong adjuvant effect in inducing antitumor immunity in the absence of Treg cells

Because pGITRL had a strong antitumor effect in the absence of cCD4+ T and Treg cells (Fig. 4A), we next examined whether it enhanced the activation of CD8+ T cells that are crucial for the rejection of tumor cells. When MC38 tumor-challenged B6 mice were treated with pGITRL and/or anti-CD4 mAb, pGITRL alone increased total lymphocyte numbers in TDLNs 1.5-fold, anti-CD4 mAb alone 2.6-fold, whereas pGITRL plus anti-CD4 mAb increased them 4.6-fold (Fig. 5A). Moreover, the combined treatment significantly increased many types of immune cells in TDLNs, including CD3+ T, CD8+ T, B220+ B, CD11c+ DCs, CD11b+ monocytes, and NK1.1+ NK cells (Fig. 5B).

FIGURE 5.
FIGURE 5.

Increased effector/memory CD8+ T cells induced by pGITRL in the absence of Treg cells. C57BL/6 mice were challenged s.c. with 5 × 105 MC38 colon adenocarcinoma cells and treated i.p. with pGITRL and/or depleting anti-CD4 mAb, as described above (n = 3). On PI day 15, lymphocytes of inguinal LNs were counted and stained with fluorescence-conjugated Ab against cell surface Ag, as indicated. (A) Absolute numbers of total lymphocytes in inguinal LN. Numbers at the head of each column indicate average fold increase relative to isotype control. (B) Absolute numbers of each subpopulation in inguinal LN. (C) Representative results of flow cytometric analysis of the expression of CD44 and CD62L on CD4+ T and CD8+ T cells. (D) Absolute numbers of effector (CD62LlowCD44high), memory (CD62LhighCD44high), and naive (CD62LhighCD44low) CD4+ T and CD8+ T cells in inguinal LNs. The plotted data are means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001), and results are representative of three independent experiments with similar results.

To further characterize the activation status of the CD8+ T cells, we analyzed the expression of CD44 and CD62L on CD4+ T and CD8+ T cells by flow cytometry in the TDLN cells of each group of mice on PI day 15. Treatment with pGITRL alone marginally increased CD62LlowCD44high effector and CD62LhighCD44high memory T cells, among both CD4+ T and CD8+ T cells (Fig. 5C). Depletion of CD4+ T cells, including Tregs, increased effector and memory CD8+ T cells rather more effectively; however, this increase was massively amplified by combining pGITRL with depletion of CD4+ T cells (Fig. 5C). When the absolute numbers of naive (CD62LlowCD44low), effector (CD62LlowCD44high), and memory (CD62LhighCD44high) CD4+ T and CD8+ T cells in each group of mice were calculated, the results clearly demonstrated that pGITRL induced extensive increased effector and memory CD8+ T cells in the absence of Treg cells (Fig. 5D).

These results support the view that pGITRL amplifies the activation of innate and adaptive immune cells in the absence of Treg cells.

Ligation of GITR by pGITRL enhances tumor infiltration by CD8+ T cells in the absence of Treg cells

Because pGITRL enhances the activation of CD8+ T cells in the absence of Treg cells (Fig. 5), we next asked whether pGITRL increased the effector activity of CD8+ T cells and their infiltration into tumor tissue. B6 mice challenged with MC38 tumors were treated with pGITRL and/or anti-CD4 mAb, as described above, and the lymphocytes in TDLNs and tumor tissue were analyzed on PI day 15. Consistent with the results in Fig. 5, IFN-γ–producing CD4+ T and CD8+ T cells were marginally increased by pGITRL alone, and IFN-γ–producing CD8+ T cells were moderately increased by depletion of conventional CD4+ T and Treg cells; however, the combined treatment with pGITRL plus anti-CD4 mAb caused a major increase in IFN-γ–producing CD8+ T cells (Fig. 6A). The combined treatment also increased the absolute numbers of IFN-γ–producing CD8+ T cells by >30-fold compared with rat IgG or pGITRL on its own (Fig. 6B).

FIGURE 6.
FIGURE 6.

Increased CD8+ T cell infiltration induced by pGITRL in the absence of Treg cells. MC38 tumor-challenged mice (n = 3) were treated i.p. with pGITRL and/or depleting anti-CD4 mAb, as described above. (A and B) On PI day 15, lymphocytes of inguinal LNs were counted and intracellularly stained with anti–IFN-γ-PE following staining with anti–CD4-FITC or anti–CD8-FITC. All samples were subsequently analyzed by FACScan (BD Biosciences) (A), and absolute numbers of IFN-γ–producing CD4+ T and CD8+ T cells were calculated (B). (C and D) Single-cell suspensions were prepared from tumor tissue on PI day 15. (C) Cells were stained with fluorescence-conjugated anti-CD3, anti-NK1.1, anti-CD4, and anti-CD8 mAb. (D) Cells were intracellularly stained with anti–Foxp3-PE following staining with fluorescence-conjugated anti-CD4 and anti-CD25 mAb for Treg cells, or surface stained with anti–Gr-1 and anti-CD11b mAb for myeloid-derived suppressor cells. (E) Suppression assay of Treg cells (n = 3). CD25−CD4+ T and CD25+CD4+ Treg cells were purified using the MACS magnetic system. CD25−CD4+ T cells (2 × 105) were mixed with naive or activated CD25+CD4+ Treg cells (1 × 105) and stimulated with 0.5 μg/ml anti-CD3 mAb in the presence of 5.0 μg/ml pGITRL or anti-mGITR mAb for 72 h. Heat-inactivated pGITRL was used as an isotype control. Proliferation of T cells was assessed by [3H]thymidine incorporation. The plotted data are means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001), and results are representative of three independent experiments.

To analyze tumor-infiltrating lymphocytes, single-cell suspensions were prepared from tumor tissues 15 d after tumor injection. Flow cytometric analysis showed that pGITRL alone did not significantly affect the infiltration of CD8+ T, NK, Treg, and myeloid-derived suppressor cells (Fig. 6C, 6D); treatment with anti-CD4 mAb did increase CD8+ T and NK cells somewhat and deplete Treg cells (24, 25); again, the combined treatment with pGITRL plus anti-CD4 mAb markedly enhanced these effects (Fig. 6C, 6D).

Because GITR signaling inhibited the suppressive activity of naive but not activated Treg cells (1, 21), we examined the effect of pGITRL on the suppressive activity of Treg cells in vitro using purified cCD4+ T and Treg cells. When the cCD4+ T cells were treated with pGITRL or anti-GITR mAb following anti-CD3 mAb stimulation, the proliferation of CD4+ T cells was more efficiently stimulated by pGITRL than anti-GITR mAb (Fig. 6E). When the cCD4+ T cells were cocultured with freshly isolated naive Treg cells or activated Treg cells, both pGITRL and anti-GITR mAb rescued the proliferation of CD4+ T cells from suppression by naive Treg cells; however, they failed to inhibit the suppressive function of activated Treg cells (Fig. 6E).

Taken together, these results indicate that activated Treg cells have stronger suppressive activity than naive Treg cells and are insensitive to GITR-mediated inhibition of their suppressive activity. It seems therefore that pGITRL gradually loses its antitumor adjuvant activity as GITR-insensitive activated Treg cells accumulate in TDLNs.

Discussion

The engagement of TNFR superfamily costimulatory receptors with agonistic mAb or their endogenous ligand helps to protect against autoimmune diseases and cancer (26). Such therapeutic effects have been mainly studied using agonistic mAb, because the authentic soluble ligands have not as yet been found to generate signals through the corresponding receptors (26). However, treatment with agonistic mAb in mice and humans is often associated with severe toxicity resulting from nonspecific inflammation. Thus, it is desirable to generate agonists that provide TNFR costimulatory signals without global deleterious effects on the host immune system.

We generated pentamerized GITRL protein to obtain authentic ligation of GITR and found that the in vitro proliferation of both conventional T cells and Treg cells was more robust in response to pGITRL than to agonistic anti-GITR mAb (Fig. 2). Nonetheless, the growth of MC38 tumor cells was only transiently suppressed by pGITRL but continuously inhibited by anti-GITR mAb (Fig. 3A). It was notable that pGITRL was more effective than anti-GITR mAb in increasing the numbers of activated T cells and total lymphocytes in TDLNs (Fig. 3B). Because pGITRL failed to continuously suppress tumor growth in vivo, we suspected that the activated Treg cells arising in response to pGITRL might be responsible for the transient nature of the suppression. Indeed, we found that depletion of Treg cells along with cCD4+ T cells markedly enhanced pGITRL-mediated antitumor immunity and completely suppressed tumor growth (Fig. 4). Consistent with these data, we observed an increase of both cCD4+ T and Treg cells in the GITRL transgenic mice, accompanied by multiorgan lymphocytic infiltration without any sign of autoimmune disease (27, 28), which suggests that the GITR/GITRL interaction regulates the balance between regulatory and effector CD4+ T cells. Our data also indicate that authentic GITR signaling has a dual effect on immune responses—a stimulatory effect on conventional T cells and an inhibitory effect via Treg cells.

The fact that anti-GITR mAb causes persistent tumor suppression in vivo could be due to Fc-dependent depletion of Treg cells (14, 23). Indeed, Bulliard et al. (14) have demonstrated that DTA-1 anti-GITR mAb completely loses its therapeutic effect when the Fc region is replaced by murine IgG2a with the N297A mutation (N297A), which eliminates binding to all murine FcγRs. The chimeric DTA-1 (N297A) not only fails to suppress tumor growth, but also does not eliminate Treg cells from tumor tissue. In contrast to the increase of Treg cells brought about by pGITRL, in vivo administration of anti-GITR results in depletion of Treg cells in the tumors but not in the TDLNs, where they are responsible for potent antitumor immunity (14, 23). Like pGITRL, GITRL fused to human IgG Fc and G3c anti-GITR mAb (IgM) did not deplete Treg cells in vivo due to their Fc types and therefore increased cCD4+ T and Treg cells in parallel (15–17). Taken together, our data and previous reports provide a solid platform for designing GITR-targeting mAb, which should enhance conventional T cell activities, but suppress or deplete Treg cells.

It is well known that GITR signaling enhances the proliferation of Treg cells and neutralizes the suppressive activity of naive, but not activated Treg cells (1, 21, 29). Recombinant pGITRL effectively enhanced the proliferation of naive Treg cells (Fig. 2A), but was not able to inhibit the suppressive function of activated Treg cells (Fig. 6E). Treg cells acquire stronger suppressive activity toward conventional T cells following activation and proliferation (21). Therefore, it is reasonable to suppose that the pGITRL-induced Treg cells arising in vivo have stronger suppressive activity than naive Treg cells and may be insensitive to GITR-mediated inhibition of their suppressive activity. As a consequence, pGITRL would lose its antitumor effect in vivo as the activated Treg cells accumulated.

Treg cells have a pivotal role in raising the threshold for triggering of T cell responses to prevent the activation of autoreactive T cells (30), and their suppressive effect generally requires cell–cell contact (31, 32). When DCs interact with conventional T cells to provide TCR triggering, Treg cells join in the formation of cell aggregates and may therefore receive various types of costimulatory signals from the DCs (33). Because DCs express the ligand for GITR (4), the conventional T and Treg cells in such aggregates would receive GITR signaling along with TCR triggering. Eventually, GITRL on mature DCs would block the suppressive function of the Treg cells and enhance activation of the conventional T cells, so initiating the development of adaptive immunity. Thereafter, other costimulatory factors such as 4-1BB appear to continuously block the suppressive function of the activated Treg cells until the pathogen is cleared (29).

T cells from GITR-deficient mice multiply more rapidly than wild-type T cells and are hypersensitive to activation-induced cell death (34). Ronchetti et al. (34) suggest that GITR is another tumor necrosis factor superfamily member delivering a negative signal to activated cells via an unknown mechanism. Our work provides evidence that authentic GITR signaling exerts a negative regulatory effect by increasing the number of activated Treg cells. Therefore, the role we have proposed for authentic GITR ligation suggests that GITR-mediated enhancement of antitumor responses could be achieved therapeutically by inhibiting or eliminating the activation of Treg cells.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Cancer Center, Korea, Grant NCC-1310430; National Research Foundation of Korea Grants NRF-2005-0093837 and NRF-2013R1A1A2008703; Ministry of Trade, Industry, and Energy Grant N0000901; and Ministry of Science, ICT and Future Planning, Ministry of Trade, Industry and Energy, Ministry of Health and Welfare Grant KDDF201408-1.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    cCD4
    conventional CD4
    CHO
    Chinese hamster ovary
    COMP
    human cartilage oligomeric matrix protein29–73
    DC
    dendritic cell
    DLN
    draining lymph node
    GITR
    glucocorticoid-induced TNFR family–related protein
    pGITRL
    pentamerized form of the murine GITRL extracellular domain
    PI
    postinjection
    TDLN
    tumor DLN
    Treg
    regulatory T.

  • Received December 10, 2014.
  • Accepted September 8, 2015.

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