GITR Agonism Triggers Antitumor Immune Responses through IL21-Expressing Follicular Helper T Cells

Glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) is a costimulatory molecule of the tumor necrosis factor receptor superfamily and is expressed in a range of immune cells, including T cells, natural killer (NK) cells, and B cells (1). Administration of GITR agonists inhibits tumor growth in tumor models and CD4⁺ T cells initiate the antitumor immunity induced by the GITR agonistic antibody, DTA-1 (2, 3). Treatment with GITR agonists reduced the number and suppressive effects of regulatory T cells (Treg; refs. 4, 5). B-cell deficiency disabled the capability of DTA-1 to generate cytolytic CD8⁺ T cells and reduced cytokine production in tumor-bearing mice (6). On the other hand, GITR costimulation increased the number of effector CD8⁺ T cells in the tumor microenvironment and upregulated their cellular metabolism (7–9). The in-human phase I trial of GITR agonistic antibodies (TRX518) explored the safety profile and immune effects in patients (10). Our group has demonstrated that IL9 plays a role in the antitumor activity of DTA-1 and that GITR costimulation promoted the differentiation of Th9 cells (11). We also found that DTA-1 treatment induced IL21 expression in CD4⁺ T cells. Questions arose concerning whether IL21 mediates the antitumor immunity that is induced by DTA-1.

IL21 is a member of the common-γ-chain cytokine family that includes IL2, -4, -7, -9, and -15 (12). IL21 is produced by NKT cells, Th17 cells, and follicular helper T cells (Tfh cells; ref. 12) and stimulates B cells, NK cells, and CD8⁺ T cells. IL21 is a pleiotropic cytokine known for its antitumor activities. Treatment with recombinant IL21 induces NK cell–mediated antitumor activity via NKG2D (13). In addition, IL21 induced expansion of cytotoxic CD8⁺ T cells without collateral expansion of Tregs (14). In breast cancer patients, CCR4^–CCR6^–CXCR3^– CD4⁺ T cells expanded in the peripheral blood promoted cytotoxicity caused by autologous CD8⁺ T cells via IL21 (15). Recombinant IL21 combined with rituximab showed a synergistic effect on tumor regression in cynomolgus monkeys and improved antibody-dependent cellular cytotoxicity (ADCC) in human NK cells (16). With PD-1 blockades, IL21 exhibited antitumor activity against H22 murine hepatocellular carcinoma by increasing cytotoxic T-lymphocyte (CTL)–induced cytotoxicity (17). We have also shown that IL21 exerts antitumor activity by restoring the function of Tim-3⁺PD-1⁺–exhausted NK cells in MHC class-I–deficient tumors (18).

Tfh cells have been defined by the expression of CXCR5, PD-1, and ICOS and production of IL21 as an effector cytokine (19). Although the master transcription factor for Tfh cells has not been defined yet, Bcl6 is considered to be a key transcription factor for Tfh cell development (20–22). Tfh cells play a role in humoral immune responses by inducing high-affinity antibody production, but the role of Tfh cells in tumor immunity is still controversial (23–25). Although tumor-infiltrating Tfh cells are positively correlated with clinical outcome in breast cancer patients (23), IL4 produced by Tfh cells compromises antitumor immunity by driving accumulation of CD11b⁺ immunosuppressive myeloid cells in the tumor microenvironment, and Tfh cells have protumorigenic activity in diffuse large B-cell lymphoma (DLBCL; refs. 24, 25).

In this study, we demonstrate that the IL21-producing Tfh cells induced by DTA-1 treatment have antitumor functions. Blocking IL21 signaling reversed DTA-1–mediated tumor growth inhibition and the antitumor CTL responses incited by DTA-1 treatment. DTA-1 induced IL21-producing CD4⁺ T cells in an antigen-specific manner. Most of the induced IL21⁺ CD4⁺ T cells were CXCR5⁺PD-1⁺ Tfh cells. GITR costimulation also induced IL21 production through the c-Maf pathway, in IL4-stimulated naïve CD4⁺ T cells. Using T cell–specific Bcl6 conditional-knockout mice and a chemical inhibitor of Bc16 (79-6), we investigated how DTA-1 inhibits tumor growth through the IL21-producing Tfh cells.

Six- to 8 week-old female C57BL/6 and BALB/c mice were purchased from the Charles River Laboratories. Bcl6^fl/fl, OT-I, and OT-II mice were purchased from the Jackson Laboratory. CD45.1 and IL4Ra^−/− mice were kindly provided by J.-O. Kim (International Vaccine Institute) and Y.-K. Kim, respectively. The CD4-cre mice were kindly provided by Y.-S. Chung. All mice were maintained under specific pathogen-free conditions in the animal facility of the Pharmaceutical Research Institute at Seoul National University. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Seoul National University.

The CT26, TC-1, and B16F10 cells were purchased from the ATCC (CT26 was purchased in 2002, TC-1 was purchased in 2006, and B16F10 was purchased in 2016), and MC38 cells were kindly provided from Genentech in 2018 (26). These cells were negative for Mycoplasma contamination (e-Myco Mycoplasma PCR Detection Kit; iNtRON in 2016). Tumor cell lines were validated by their morphology, growth kinetics, and antigen expression before injection and tumor cells with three to four passages were used for inoculation. These cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin (DF10). The B16F10-OVA cells (kindly provided in 2006 by Dr. K. Rock, University of Massachusetts Medical School, Boston, MA) were cultured in DF10 medium supplemented with 200 μg/mL of geneticin (Gibco) and 60 μg/mL of hygromycin (Invitrogen).

The fluorochrome-conjugated antibodies to mouse CD3ϵ PerCP (145-2C11, 100326), CD4 APC/Cy7 (RM-4-5, 100526), CD8α pacific blue (53-6.7, 100725), CXCR5 biotin (L138D7, 145510), APC streptavidin (405207), IFNγ PerCP (XMG1.2, 505822), TNFα APC (MP6-XT22, 506308), CD107a FITC (1D4B, 121606), CD25 APC (PC61, 102012), CD44 PE/Cy7 (IM7, 103030), CD62L PerCP (MEL-14, 104430), CD45.1 PE (A20, 110708), and CD45.2 PE/Cy7 (104, 109830), and anti-human IgG PE (HP6017, 409304) were purchased from BioLegend. The antibodies to mouse anti–PD-1 FITC (J43, 11-9985-82) were purchased from eBioscience. The mIL21R-hIgG-Fc (596-MR-100) was purchased from R&D Systems. The chemical inhibitor of Bcl6 (79-6, 197345) was purchased from Calbiochem. The CellTraceTM CFSE cell proliferation kit (C34554) was purchased from Invitrogen.

Cells were stained with dye-conjugated antibodies in 1% FBS and 0.02% azide containing PBS buffer. For intracellular staining, cells were restimulated with brefeldin A (GolgiPlug; BD Biosciences) and monensin (GolgiStop; BD Biosciences) for 4 hours. Cytofix-Cytoperm kits (BD Biosciences) were used for fixation and permeabilization according to the manufacturer's instructions. An antibody against CD107a was added during restimulation. For staining transcription factors, cells were fixed and permeabilized with a Foxp3 staining kit (eBioscience).

A total of 2 × 10⁵ CT26 cells were subcutaneously (s.c.) injected into the left flank of the BALB/c mice, and 2 × 10⁵ TC-1, MC38, and B16F10 cells were s.c. injected into the left flank of the C57BL/6 mice. Five days later, 600 μg of DTA-1 or the rIgG2b control (LTF-2; Bio X Cell) was intraperitoneally (i.p.) administered. For blocking IL21 signaling, 300 μg of anti-mouse IL21 receptor (4A9; Bio X Cell) or the rIgG2a control (2A3; Bio X Cell) was administered every 2 or 3 days starting 8 days after tumor inoculation. For the Bcl6 inhibition, 1 mg of 79-6 (Calbiochem) was i.p. injected daily between 8 and 14 days after the tumor challenge. Bcl6^fl/flCd4^Cre mice were s.c. injected with 2 × 10⁵ TC-1 cells and 600 μg of DTA-1 was i.p. administered. The tumor sizes were measured with calipers every 2 or 3 days when the tumor cells were palpable.

Eight days after DTA-1 injection, the spleen and tumor-draining lymph nodes (TdLN) from CT26 tumor-bearing mice were harvested and cultured with the CT26 epitope AH-1 for 5 days. The CT26 cells were labeled with 1 μmol/L Calcein-AM (C3100MP; Life Technologies) and cocultured with AH-1–stimulated effector cells for 4 hours. Using a SpectraMAX M5 (Molecular Devices), the fluorescence of the supernatant from the wells with cocultured cells was analyzed.

CD4⁺CD25^–CD44^loCD62L^hi naïve T cells or CD4⁺CD25^– T cells isolated from the pooled spleen and lymph node cells of BALB/c mice were purified by flow cytometry and stimulated for 4 days with plate-bound anti-mouse CD3ϵ (2 μg/mL, 145-2C11; BioLegend) and anti-mouse CD28 (1 μg/mL, 37.51; BioLegend) or T cell–depleted splenocytes (1: 5), supplemented with IL2 (10 ng/mL). To induce IL21, IL4 (10 ng/mL; R&D Systems), IL6 (10 ng/mL; PeproTech), IL12 (10 ng/mL; PeproTech), and IL27 (10 ng/mL; PeproTech) were added to culture well of naïve CD4⁺ T cells. For some experiments, 2 μg of anti-mIL4 (11B11; ATCC), anti-mIL6 (MP520F3; R&D Systems), anti-mIL12/IL23p40 (C17.8; R&D Systems), and anti-mIL27 p28/IL30 (AF1834; R&D Systems) were added during culture. The IL21 concentration was determined using an IL21 ELISA kit (Invitrogen) according to the manufacturer's instructions.

Tumor tissues from tumor-bearing mice were cut into small pieces and dissociated with gentle MACS Dissociator (Miltenyi Biotec). The dissociated tumor tissues were digested in 2% FBS RPMI medium containing 1 mg/mL collagenase D (Roche), 100 μg/mL hyaluronidase (Sigma-Aldrich), and 100 μg/mL DNase I (Sigma-Aldrich) at 37°C for 30 minutes. From the digested tumor tissues, the lymphocytes were isolated by lymphocyte separation medium (MP Biomedicals) and used for experiments.

The scrambled vector used as a control and the c-Maf shRNA vector were kindly provided by S.-H. Im [Division of Integrative Biosciences and Biotechnology, Department of Life Sciences, Pohang University of Science and Technology (POSTECH)]. Retroviral supernatants were generated by Platinum-E (Plat-E) retroviral packaging cell lines with FuGene HD transfection reagent (Promega) according to the manufacturer's instructions. FACS-sorted naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) with 10 ng/mL of IL2 for 24 hours and the cells were transduced with retroviral supernatants containing 4 μg/mL of polybrene by means of spin infection (800 × g for 90 minutes at 37°C). After infection, the CD4⁺ T cells were cultured in the presence of plate-bound anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) with IL4 (10 ng/mL) and DTA-1 (2 μg/mL). Eighteen or 48 hours later, the GFP⁺ cells were sorted and resuspended with TRIzol reagent (Invitrogen) for qPCR analysis.

Human peripheral blood cells were obtained from healthy volunteers in compliance with Institutional Review Board protocols. Donors were previously informed and provided written consent to experimental procedures using their samples in accordance with the Declaration of Helsinki. Mononuclear cells were isolated by Ficoll-Histopaque (Sigma-Aldrich) density gradient centrifugation. The collection of human samples and all human experiments were approved by the ethical committee of Seoul National University (IRB No. 1712/001-003).

A 1:1 mixture of CD4⁺ T cells (2 × 10⁶ each) from the CD45.1⁺CD45.2⁺ OT-II mice and CD45.1⁺ B6/SJL mice was intravenously injected into the B16F10-OVA tumor–bearing C57BL/6 mice 9 days after tumor challenge. The next day, 600 μg of DTA-1 or rIgG2b control antibody was administered. TdLNs were analyzed 7 days after antibody treatment.

Total RNA was extracted with TRIzol reagent (Invitrogen) and reverse transcribed using AmfiRivert cDNA Synthesis Platinum Master Mix (GenDEPOT). The total RNA was measured using Multiskan Go microplate spectrophotometer (Thermo Scientific) and we used 1 μg of total RNA for cDNA synthesis. The synthesized cDNA was quantified with a TB Green Premix Ex Taq (TaKaRa) and the AriaMx 96 Real-Time PCR System (Agilent). The relative expression data of the target genes were normalized to the amount of mHprt expression (2^{−(CT_{Target gene}–CT_mHPRT)}). Primers used in analyses were as follows: Mouse Il21 forward; 5′-GCT CCA CAA GAT GTA AAG GGG C-3′, mouse Il21 reverse; 5′-CCA CGA GGT CAA TGA TGA ATG TC-3′, mouse Il4 forward; 5′-ATC CAC GGATGC GAC AAA AA-3′, mouse Il4 reverse; 5′-GTG GTG TTC TTC GTT GCT GTG A-3′, mouse Il6 forward; 5′-ATC CAG TTG CCT TCT TGG GAC-3′, mouse Il6 reverse; 5′-TAA GCC TCC GAC TTG TGA AGT-3′, mouse Il9 forward; 5′-AAC GTG ACC AGC TGC TTG TGT-3′, mouse Il9 reverse; 5′-CTT GAT TTC TGT GTG GCA TTG G-3′, mouse Il12a forward; 5′-ACG AGA GTT GCC TGG CTA CTA G-3′, mouse Il12a reverse; 5′-CCT CAT AGA TGC TAC CAA GGC AC-3′, mouse Il12b forward; 5′-TTG AAC TGG CGT TGG AAG CAC G-3′, mouse Il12b reverse; 5′-CCA CCT GTG AGT TCT TCA AAG GC-3′, mouse Il27 forward; 5′-TCT CGA TTG CCA GGA GTG AAC C-3′, mouse Il27 reverse; 5′-AGT GTG GTA GCG AGG AAG CAG A-3′, mouse Nfatc1 forward; 5′-CAA CGC CCT GAC CAC CGA TAG-3′, mouse Nfatc1 reverse; 5′-GGC TGC CTT CCG TCT CAT AGT-3′, mouse Nfatc2 forward; 5′-ATC TAC CCA GAT CAG TAT GG-3′, mouse Nfatc2 reverse; 5′-TCA GGA GTA TAC CAT TTC TC-3′, mouse Nfatc3 forward; 5′-CTT TCA GTT CCT TCA CCC TTT ACC T-3′, mouse Nfatc3 reverse; 5′-TGC CAA TAT CAG TTT CTC CTT TTC-3′, mouse Jun forward; 5′-ACG ACC TTC TAC GAC GAT GC-3′, mouse Jun reverse; 5′- CCA GGT TCA AGG TCA TGC TC-3′, mouse Junb forward; 5′-AGG CAG CTA CTT TTC GGG TC-3′, mouse Junb reverse; 5′-TTG CTG TTG GGG ACG ATC AA-3′, mouse Fos forward; 5′-TAC TAC CAT TCC CCA GCC GA-3′, mouse Fos reverse; 5′-GCT GTC ACC GTG GGG ATA AA-3′, mouse Maf forward; 5′-CGC CTA CAA GGA GAA ATA CGA GAA-3′, mouse Maf reverse; 5′-GAC CCC CAC GGA GCA TTT-3′, mouse Hprt forward; 5′-AAG ACT TGC TCG AGA TGT CAT GAA-3′, mouse Hprt reverse; 5′-ATC CAG CAG GTC AGC AAA GAA-3′.

Cytoplasmic fractions of cells were prepared as follows: in vitro cultured CD4⁺ T cells were washed once with ice-cold PBS and collected by centrifugation at 5000 r.p.m. for 10 minutes. The cells were resuspended in RIPA buffer (R0278, Sigma-Aldrich) with 1 mmol/L DTT and 0.25 mmol/L PMSF and a proteinase inhibitor cocktail. The mixtures were vortexed at high speed for 30 minutes. The extracts were collected by centrifugation at 13,000 r.p.m. for 10 minutes. The supernatants were collected as cytoplasmic extracts. These cell extracts were loaded onto SDS-PAGE gels and then transferred to PVDF membranes using iBlot 2 PVDF Mini Stacks (Invitrogen). The membranes were stained with specific antibodies, and chemiluminescence was visualized using an LAS-3000 LuminoImage analyzer (Fuji film).

Statistical analyses were performed using Prism 5.0 software (GraphPad). Unpaired two-tailed Student t tests and two-way ANOVA were used for statistical analyses. The results with a P value of < 0.05 were considered statistically significant. Data are presented as the means ± SEM.

All animal studies adhered to the approved IACUC protocols (SNU-181214-1) at Seoul National University. Human experiments were approved by the ethical committee of Seoul National University (IRB No. 1712/001-003).

To examine the role of IL21 in DTA-1–induced antitumor immune responses, we used CT26 and TC-1 tumor models. Mice were subcutaneously injected with CT26 or TC-1 cells and were given DTA-1 or rIgG2b control antibodies 5 days after tumor inoculation. Consistent with earlier studies (2, 3, 6, 11), the DTA-1 treatment suppressed tumor growth; however, administration of the anti-IL21 receptor (anti-IL21R) significantly abrogated this antitumor effect in CT26 and TC-1 models (Fig. 1A; Supplementary Fig. S1). Next, we investigated the cellular mechanism by which DTA-1 administration triggers antitumor immunity via IL21. Given that NK and CD8⁺ T cells play a role in eliminating tumors and are also targets of IL21 (27–32), we analyzed the production of effector molecules by CD8⁺ T cells and NK cells in CT26 tumor–bearing mice. Eight days after the DTA-1 treatment, the production of IFNγ and granzyme B by NK cells was unchanged (Fig. 1B and C). On the other hand, the expression of effector molecules and degranulation marker was upregulated by DTA-1 in the CD8⁺ T cells but was subsequently downregulated by anti-IL21R treatment (Fig. 1D). We observed target cell killing activity of CD8⁺ T cells from the DTA-1–treated mice which was significantly decreased by anti-IL21R treatment (Fig. 1E). In addition to its impact on effector cells, IL21 is a negative regulator of suppressive Tregs (33, 34). We also previously reported that DTA-1 hampers the differentiation of naïve CD4⁺ T cells into Tregs, which resulted in reduced Treg populations in tumor tissue (11). To examine whether DTA-1 induced Treg decrement is IL21 dependent, we analyzed the Foxp3⁺ tumor-infiltrating lymphocyte (TIL) Treg population in tumor-bearing mice treated with DTA-1 and anti-IL21R. We found that blocking IL21 signaling had no effect on the TIL Treg population (Supplementary Fig. S2). Collectively, these results demonstrate that DTA-1–induced IL21 facilitates antitumor immune responses by activating CD8⁺ T cells.

As we previously described (11), treatment with DTA-1 increased IL21 transcription in CD4⁺ T cells from TdLN (Fig. 2A). Next, we examined the kinetics of IL21 expression in CD4⁺ T cells following DTA-1 treatment. We defined IL21-expressing CD4⁺ T cells as CD3ϵ⁺CD4⁺IL21⁺ (Supplementary Fig. S3). We found that numbers of IL21⁺ CD4⁺ T cells increased and peaked at day 7 and then decreased thereafter (Supplementary Fig. S4). To determine the structural changes induced by DTA-1 in TdLN at day 7, we performed IHC staining. The immunohistological analysis showed that treatment with DTA-1 enhanced germinal center responses in vivo (Supplementary Fig. S5). Given that Tfh cells, which mediate germinal center responses, are a known source of IL21, we investigated whether DTA-1–induced IL21 production is found in Tfh cells. As we expected, DTA-1 treatment increased the frequency of IL21-producing cells and most of these cells coexpressed CXCR5 and PD-1, which are typical surface markers for Tfh cells (Fig. 2B). Coculture experiments revealed that DTA-1–induced CXCR5⁺PD-1⁺CD4⁺ T cells were genuine Tfh cells as they increased IgG production from B cells in vitro (Supplementary Fig. S6). In addition to frequency, the absolute number of IL21-expressing Tfh cells was significantly increased in DTA-1–treated mice compared with rIgG2b-treated mice (Fig. 2C). The frequency of IL21-expressing cells in Tfh cells was also increased by DTA-1 treatment (Supplementary Fig. S7). We also observed that DTA-1 treatment increased IL21-expressing Tfh cells in TdLN in other tumor models such as B16F10, MC38, and TC-1 cells (Supplementary Fig. S8). Treatment with DTA-1 controlled tumor growth in these models and increased IL21-expressing Tfh cells in the tumor tissue of the B16F10 and MC38 models (Supplementary Fig. S9). The number of CXCR5⁺PD-1⁺ cells also peaked at 7 days after DTA-1 treatment as the IL21⁺ CD4⁺ T cells did (Fig. 2D). We further analyzed tumor-infiltrating lymphocytes and found that IL21-producing Tfh cells were also increased by DTA-1 treatment in tumor tissue (Fig. 2E and F). Inhibiting the egression of T cells from lymphoid organs with FTY720 decreased the increase in IL21-producing CD4⁺ T cells induced by DTA-1 in tumor tissues (Supplementary Fig. S10). We therefore concluded that DTA-1–induced IL21 is produced mainly by Tfh cells.

Next, we asked whether the induction of IL21-expressing Tfh cells by DTA-1 treatment is antigen specific. We first compared the frequencies of IL21-expressing Tfh cells in the distal lymph nodes (distal LN), which is the opposite side from tumor injected flank, and TdLNs. We found that IL21-producing Tfh cells were increased only in TdLNs after DTA-1 treatment (Fig. 3A–C). To directly determine the antigen specificity, we s.c. injected mice with B16F10-OVA tumor cells and then adoptively transferred a 1:1 mixture of CD45.1⁺CD45.2⁺ OVA-specific CD4⁺ T cells and CD45.1⁺CD45.2⁻ polyclonal CD4⁺ T cells 9 days after tumor inoculation. Compared with the polyclonal CD4⁺ T cells, the IL21-secreting CXCR5⁺PD-1⁺ Tfh cells were significantly increased in OVA-specific CD4⁺ T cells upon DTA-1 treatment (Fig. 3D–G). Taken together, these data suggest that the induction of IL21⁺ Tfh cell populations by GITR stimulation is an antigen-specific process.

Next, we investigated the mechanism by which DTA-1 induces IL21 production in CD4⁺ T cells. First, we examined whether DTA-1 stimulation directly induces IL21 production from CD4⁺ T cells in vitro. We found that DTA-1 induced IL21 production from total CD4⁺ T cells (Supplementary Fig. S11). To determine whether IL21 is produced from effector or naïve CD4⁺ T cells, we observed IL21 production with different initial ratios of naïve and effector CD4⁺ T cells. Neither naïve nor effector CD4⁺ T cells alone produced IL21 in response to DTA-1 stimulation, whereas the combination of naïve and effector CD4⁺ T cells synergistically produced a substantial amount of IL21 (Supplementary Fig. S12). To clarify the source of IL21, we cocultured Thy1.1⁺CD44⁺CD62L⁻ cells and Thy1.2⁺CD44⁻CD62L⁺ cells stimulated with anti-CD3 and anti-CD28 in the presence of DTA-1. We found that IL21 was produced by only restimulated Thy1.2⁺ CD4⁺ T cells that were derived from CD44⁻CD62L⁺ naïve CD4⁺ T cells (Supplementary Fig. S13). We conclude that DTA-1 stimulation differentiated naïve CD4⁺ T cells into IL21-producing CD4⁺ T cells.

The differentiation of naïve CD4⁺ T cells into each Th subset is dependent on cytokine stimulation. Thus, we examined cytokines related to IL21 production in the TdLNs upon DTA-1 treatment. We found that IL4, -6, -9, and -27, but not IL12, were increased by DTA-1 treatment (Fig. 4A). Next, we analyzed IL21 production from CD4⁺ T cells upon DTA-1 stimulation in vitro. Neutralizing antibodies to each cytokine showed that the IL21 production increased by DTA-1 stimulation was reversed only by adding antibodies against IL4 (Fig. 4B). In the presence of IL4, DTA-1 stimulation increased IL21 production from naïve CD4⁺ T cells (Fig. 4C). In vitro DTA-1 stimulation increased the frequency of CD44⁺CD62L⁻ CD4⁺ T cells but did not change expression of CXCR5 and PD-1 in the presence of IL4 (Supplementary Fig. S14). GITR ligation also promoted IL21 production in human naïve CD4⁺ T cells in the presence of IL4 (Fig. 4D). However, we did not find any induction of IL21 in human CD4⁺ T cells in the absence of IL4 (Supplementary Fig. S15). As we demonstrated that both naïve and effector CD4⁺ T cells are required for DTA-1–induced IL21 production in vitro (Supplementary Fig. S12), we hypothesized that DTA-1–induced IL21 is produced by naïve CD4⁺ T cells stimulated with DTA-1 and IL4 that is produced by effector CD4⁺ T cells. Thus, we examined IL21 production from CD4⁺ T cells composed of IL4 receptor–deficient naïve or effector CD4⁺ T cells. DTA-1 stimulation on CD4⁺ T cells produced IL21 with IL4 receptor deficiency in effector CD4⁺ T cells. However, IL4 receptor deficiency in naïve CD4⁺ T cells resulted in significantly reduced IL21 production by DTA-1 stimulation (Supplementary Fig. S16). Using IL4 receptor–deficient mice, we found that the increased frequencies of IL21-producing CD4⁺ T cells as well as Tfh cells upon DTA-1 treatment were significantly reduced in IL4 receptor–deficient mice compared with littermate controls in vivo (Fig. 4E–I). Altogether, these results suggest that IL4 enhances IL21 upregulation by CD4⁺ T cells when GITR costimulation is supplemented.

Given that DTA-1–induced IL9 mediates antitumor immune responses (11) and that DTA-1–induced IL9 expression precedes IL21 expression, we tested whether the IL21 induction by DTA-1 is dependent on IL9. We found that neutralization of IL9 did not alter the frequencies of IL21-expressing CD4⁺ T cells or Tfh cells in DTA-1–treated mice (Supplementary Fig. S17A). Also, IL9 did not induce IL21 production in naïve CD4⁺ T cells in vitro (Supplementary Fig. S17B). Taken together, DTA-1–induced IL21 production is independent of IL9.

We further dissected the molecular mechanism by which IL4 and DTA-1 stimulation triggers the differentiation of naïve CD4⁺ T cells into IL21-expressing CD4⁺ T cells. To this end, we examined the mRNA expression of the transcription factors involved in transcription of IL21, including AP-1 transcription factors (35–38). We found that Maf mRNA was increased by stimulation with IL4 and DTA-1 to a peak at 18 hours of stimulation (Fig. 5A and B). The c-Maf protein was also increased after 2 days of stimulation with IL4 and DTA-1 (Fig. 5C). When c-Maf was downregulated using shRNA targeting c-Maf (Fig. 5D), the IL21 expression in CD4⁺ T cells was reduced compared with that of control vector–transduced T cells that were stimulated with IL4 and DTA-1 (Fig. 5E). Like in vitro–stimulated CD4⁺ T cells, DTA-1–induced Tfh cells expressed significantly more c-Maf than did Tfh cells in control rIgG2b-treated mice in B16F10, MC38, and TC-1 models (Supplementary Fig. S18). Collectively, these results suggest that IL4- and DTA-1–induced IL21 production in CD4⁺ T cells depends on the transcription factor c-Maf.

As we demonstrated the requirement of IL21 in the antitumor immunity induced by DTA-1, we next asked whether Tfh cells are responsible for antitumor immunity of DTA-1. To address this question, we harvested Tfh cells and non-Tfh cells from B16F10-OVA tumor–bearing mice treated with DTA-1. We cocultured these cells with OVA-specific OT-I CD8⁺ T cells for 2 days stimulated with T cell–depleted APC in the presence of OVA peptide (OVA_257-264 and OVA_329-337). As a result, Tfh cells promoted cell proliferation and IFNγ production in responder OT-I T cells as much as IL21 did (Fig. 6A). To inhibit Tfh cell responses, we used the small-molecule inhibitor 79-6, which inhibits transcription factor Bcl6 (39, 40). Treatment with 79-6 decreased IL21 production in IL4- and DTA-1–stimulated naïve CD4⁺ T cells without inducing cellular cytotoxicity (Fig. 6B; Supplementary Fig. S19). We next determined the role of Tfh cells in tumor growth inhibition by DTA-1. In the CT26 tumor model, administration of 79-6 significantly reduced the frequencies of Tfh cells in DTA-1–treated mice (Fig. 6C). IL21-producing CD4⁺ T cells and IL21-producing Tfh cells were also diminished in these mice (Fig. 6D and E). Consequently, tumor growth inhibition by DTA-1 was reversed by 79-6 treatment (Fig. 6F). To confirm the specific requirement of Tfh cells in DTA-1–induced antitumor immunity, we used Bcl6^fl/flCd4^Cre mice in an MC38 and a TC-1 tumor model. Although Bcl6^fl/flCd4^Cre mice also have a Bcl6 defect in CD8⁺ T cells, the CD8⁺ T cells in these mice produced sufficient IFNγ in response to IL21 (Supplementary Fig. S20). As expected, Bcl6^fl/flCd4^Cre mice showed reduced frequencies of IL21⁺ Tfh cells in the TdLN (Supplementary Fig. S21). Bcl6 deficiency in T cells resulted in impaired DTA-1–induced inhibition of tumor growth compared with the control mice (Fig. 6G; Supplementary Fig. S22). Collectively, these results suggest that Tfh cells are required for the antitumor activity induced by DTA-1.

GITR agonism is a promising strategy for cancer immunotherapy. Various studies have analyzed its cellular mechanism in the context of Tregs and CD8⁺ T cells (4, 7, 9, 10). Here, we focused on cytokines produced by CD4⁺ T cells. We showed previously that the GITR agonistic antibody exerts antitumor activity in an IL9–dependent fashion (11). However, neutralization of IL9 did not completely reverse the increased CTL activity induced by DTA-1, suggesting that DTA-1 has multiple arms of antitumor activity. In this study, we found that IL21 was essential for antitumor CTL responses and subsequent tumor growth inhibition by DTA-1 treatment and that production of IL21 was achieved mainly by Tfh cells in an antigen-specific manner. We demonstrated that GITR costimulation promoted IL21 expression via c-Maf in naïve CD4⁺ T cells stimulated with IL4. Thus, we conclude that DTA-1–induced antitumor immunity depends on the induction of IL21-producing CD4⁺ T cells.

Previous studies have demonstrated the mechanism of GITR agonism and found that CD4⁺ T cells and mature B cells are responsible for antitumor immunity induced by DTA-1 (2, 6). Depletion of CD4⁺ T cells inhibited the activation of CD8⁺ T, NK, and B cells after DTA-1 treatment and resulted in uncontrolled tumor growth (2). Another study demonstrated that DTA-1 treatment increased B-cell responses in tumor-bearing mice and failed to induce antitumor immunity in mature B cell–deficient JHD mice with low CTL activity (6). On the basis of the current study, we suggest that Tfh cells account for the role of CD4⁺ T cells and B cells in DTA-1–induced antitumor immunity. In this study, we focused on the effects of DTA-1–induced IL21-producing Tfh cells on CD8⁺ T cells. The effects of such Tfh cells on mature B cells remain a question for further study.

Several studies have reported the positive role of GITR on Tfh cell generation. GITR signaling plays a role in the pathogenesis of chronic lymphocytic choriomeningitis virus infection and collagen-induced arthritis by upregulating Tfh cell responses (41, 42). However, the role of GITR on Tfh cells in tumor microenvironments has not been defined yet. Consistent with other models, GITR costimulation increased antigen-specific Tfh cells in TdLNs and tumor tissues. Although clinical studies have shown that the frequency of circulating Tfh cells was increased in cancer patients, the role of Tfh cells in tumor immunity has been controversial (25, 43, 44). In this context, we suggest that Tfh cells as antitumorigenic participants since they activated antigen-specific CD8⁺ T cells. Furthermore, Bcl6-deficient model confirmed the requirement of Tfh cells in DTA-1–induced tumor rejection.

We observed that CD44⁺CD62L^– effector CD4⁺ T cells and CD44^–CD62L⁺ naïve CD4⁺ T cells each produced less IL21 than the coexisting naïve and effector CD4⁺ T cells did upon GITR costimulation, which was mediated by IL4 signal transduction in naïve CD4⁺ T cells. We assume from this result that, because most of effector CD4⁺ T cells are differentiated cells, effector CD4⁺ T cells are refractory to redifferentiate into IL21-producing T cells. Also, IL4 produced by GITR-costimulated naïve CD4⁺ T cells was not sufficient for IL21 induction. Both effector and naïve CD4⁺ T cells were responsible for optimal IL21 production by GITR costimulation: effector CD4⁺ T cells as IL4 providers and naïve CD4⁺ T cells as IL21 producers. A clinical trial reported that human GITR agonistic antibody was not sufficient to mediate clinical responses (10). On the basis of our results, the limited efficacy of GITR-targeting monotherapy might be due to an insufficient portion of naïve CD4⁺ T cells producing IL21 in the patients with the late-stage cancer.

IL4 is a typical type 2 cytokine and plays a role in humoral immune responses. Studies have shown that Tfh cells are also IL4 producers, although Th2 cells are considered the main producer of IL4 (45–47). Indeed, in a helminth infection model, IL4 secretion was restricted to Tfh cells in an ICOS-dependent manner (48). On the other hand, a study has also proposed IL4-committed Tfh cells as precursors of short-term effector Th2 cells upon house dust mite challenge (45). Our findings demonstrated that IL4 was required for the optimal induction of IL21-producing Tfh cells upon DTA-1 treatment and human naïve CD4⁺ T cells also produced IL21 in the presence of IL4 upon hGITR costimulation. In addition, we observed that the frequency of CXCR5⁺PD-1⁺ Tfh populations was not increased in CT26 tumor–bearing IL4 receptor knockout mice treated with DTA-1. In vitro–differentiated CD4⁺ T cells stimulated with IL4 and DTA-1 produced IL21 without inducing CXCR5 and PD-1 expression. The differentiation of Tfh cells is a complex process that requires contact with B cells, dendritic cells, appropriate TCR signals, costimulatory molecules, and cytokines (19, 49, 50). Given that IL4 and GITR signals induce IL21-expressing Tfh cells in vivo and IL21-expressing CD4⁺ T cells in vitro, we assume that IL4 and GITR are involved in the complex process of Tfh cell generation. We show that IL4 induces IL21-producing Tfh cells. Further studies are needed to define a requirement of IL4 and GITR costimulation for Tfh cell development and whether agonistic anti-hGITR treatment also induces IL21⁺ Tfh cells and has antitumor activity through this population in human cancer patients.

The roles of c-Maf in Tfh cell development and IL21 expression have been previously described. C-Maf binds to the promoter and CNS-2 regions of IL21 gene loci and activates IL21 expression in CD4⁺ T cells (38). Upon IL21 induction in vitro, c-Maf was transiently upregulated among IL21-regulating transcription factors. ShRNA targeting Maf gene indicated that c-Maf mediated IL21 production in naïve CD4⁺ T cells stimulated with IL4 and DTA-1. Whether GITR costimulation directly regulates c-Maf expression remains to be elucidated.

Our study revealed a mechanism by which GITR costimulation inhibits tumor growth through the induction of IL21-producing Tfh cells. IL4 ensures that Tfh cells produce IL21 upon GITR costimulation via c-Maf. Therefore, our findings provide a basis for the use of GITR agonists to treat cancer and suggest IL21 as a surrogate marker for the effectiveness of GITR agonists.

No potential conflicts of interest were disclosed.

Conception and design: C.-H. Koh, I.-K. Kim, C.-Y. Kang

Development of methodology: I.-K. Kim

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C.-H. Koh, I.-K. Kim, K.-S. Shin, I. Jeon, J.-M. Lee, E.-A. Bae, T.-S. Kang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C.-H. Koh, K.-S. Shin, B. Song, B.-S. Kim, C.-Y. Kang

Writing, review, and/or revision of the manuscript: C.-H. Koh, B. Song, B.-S. Kim, Y. Chung, C.-Y. Kang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C.-H. Koh, E.-A. Bae, H. Seo

Study supervision: C.-Y. Kang

Human GITR agonistic antibodies and c-Maf shRNA were kindly provided by Byoung S. Kwon and Sin-Hyeog Im. This work was supported by grants from the Basic Science Research Program (NRF-2015R1A2A1A10055844) and the Bio and Medical Technology Development Program (NRF-2016M3A9B5941426) through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning. The authors thank the staff of the National Center for Inter-University Research Facilities (NCIRF) at Seoul National University for assistance with the cell sorting by flow cytometry (FACSARIA II, BD Biosciences).

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