Glucocorticoid-induced TNFR-related Protein Research Articles

In vitro assays for effector T cell functions and activity of immunomodulatory antibodies.

The recent clinical success of cancer immunotherapy with checkpoint blockade has led to renewed interest into the development of immune modulatory agents with the capacity to activate anti-tumor T cell responses. Standardization of optimized in vitro assays for efficient assessment of immune function of such new drugs is thus needed to facilitate clinical development of the optimal drug candidates. Here, we describe an optimized version of T cell suppression assay designed to test the effect of immunomodulatory agents on T cell function and activation. We apply this assay to investigate the agonist activity of the T cell co-stimulatory molecule glucocorticoid-induced TNFR-related protein (GITR). We detail a protocol for concurrent assessment of multiple levels of T cell functional modulation upon GITR engagement, including T cell priming, activation and effector function, in a single assay. As human GITR agonist antibodies are currently under development, availability of standardized cell-based functional assays of GITR agonism is instrumental to translate anti-GITR therapy into the clinical setting.

Abstract 4155: Identification and characterisation of Affimer Proteins that are able to bind and agonise human co-stimulatory cell surface targets

Abstract Despite the impressive success of PD-1 pathway and CTLA-4 targeted immunotherapies, there remains a large proportion of cancer patients with many tumor types, that fail to benefit or acquire resistance during therapy. Co-stimulation of the tumor microenvironment may prove to enhance current checkpoint inhibitor therapies and kick start T-cell activity in tumors that have poor immune infiltrate. CD40 is a co-stimulatory protein and Glucocorticoid-induced TNFR-related protein (GITR) is a checkpoint inhibitory protein, both a member of the TNF-receptor superfamily. CD40 is expressed on a range of antigen presenting cells (APCs) binding to its ligand CD40L expressed on CD4+ T-cells, whilst GITR is expressed on T-cells. CD28 is also expressed on T-cells and provides co-stimulatory signals for T-cell activation and survival. CD40, GITR and CD28 are currently being investigated to provide complementary immune-modulation of the tumor micro environment alongside checkpoint inhibitor blockade therapy. Affimer biotherapeutics are a new protein scaffold with great potential for the generation of biotherapeutics. Based on the human protease inhibitor Stefin A, the scaffold is small (14kDa), lacks any post translational modifications such as disulfide bonds and expresses at high levels recombinantly. The Affimer biotherapeutic platform which is based on the human protein Stefin A, has been used to generate binders to members of the TNFR superfamily (CD40, and GITR) and CD28 using phage display. Selections were carried out with an Affimer phage library of size 6x 1010 made with two random loops each of size nine amino acids. With the Affimers raised against huCD40, we were able to encode those molecules as DNA with a membrane anchor sequence. These molecules were expressed transiently in HEK293 cells in vitro, shown to be correctly folded and anchored on the cell surface. The engineered cells were shown to be able to agonise huCD40 in the HEK Blue reporter gene assay. GITR binding Affimer proteins were able to agonise huGITR in a NF-κB Luciferase assay and huCD28 binding Affimer proteins were able to agonise CD28 in a IL-2 Luciferase gene reporter assay. We have demonstrated that Affimers can be used to agonise important co-stimulatory cell surface targets, either as recombinant protein or when displayed on a cell surface. Encoding the genes for these Affimers and delivering them directly to a tumour for expression (e.g. using an oncolytic virus) could offer a novel method of safely agonising these important co-stimulatory targets in cancer patients without generating the toxicity seen with using mAbs administered systemically. Citation Format: Amrik Basran. Identification and characterisation of Affimer Proteins that are able to bind and agonise human co-stimulatory cell surface targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4155.

Novel Therapeutic Approaches and Targets Currently Under Evaluation for Renal Cell Carcinoma: Waiting for the Revolution.

Management of metastatic renal cell carcinoma has drastically changed in the last few years, witnessing the advent of more and more target therapies and, recently, of immune-checkpoint inhibitors. On the other hand, the adjuvant setting still lacks a clear beneficial treatment. Medical treatment still remains a compelling challenge. A large number of clinical trials is ongoing with the aim to identify new therapeutic approaches to expand the options in our repertoire. Several strategies are under investigation in renal cell carcinoma (RCC). These include new targeted agents and combinations of target therapy and immunotherapy. Programmed death receptor-1 (PD-1), programmed death receptor ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen 4 (CTLA4) are just part of the intricate network that regulates our immune response to cancer cells. Co-stimulators, such as glucocorticoid-induced TNFR-related protein (GITR) and tumor necrosis factor receptor superfamily, member 4 (OX40), and co-repressors, example.g. T cell immunoglobulin and mucin domain 3 (TIM-3) and lymphocyte-activation gene 3 (LAG-3), also take part. As knowledge of the functioning of the immune system grows, so do these pathways to target with new drugs. This review is an overview of the current state of the clinical research, providing a report of ongoing Phase I, II and III clinical trials for localized and metastatic RCC, including novel target therapies, novel immunotherapy agents and new combinations strategies.

Abstract PR01: Mechanistic rationale to combine GITR agonism with PD-1 blockade in cancer patients

Abstract Immune checkpoint blockade has evidenced the therapeutic activity of modulating T-cell co-inhibition/co-stimulation processes. However, many patients are refractory to these therapies, highlighting the need for developing additional forms of immunotherapy targeting alternative immune pathways. In this regard, the T-cell co-stimulatory receptor glucocorticoid-induced TNFR-related protein (GITR, TNFRSF18) is an attractive target for agonist antibodies (Abs). By promoting effector T-cell (Teff) function and hampering regulatory T-cell (Treg) suppression, GITR engagement may exert a dual positive effect on anti-tumor immune responses. We and others have reported potent antitumor effects of anti-GITR Abs in preclinical mouse models. Based on this rationale, we initiated the first-in-human phase-I trial of GITR stimulation with the GITR agonist monoclonal Ab (mAb) TRX518 (NCT01239134). TRX518 is a humanized aglycosylated IgG1κ mAb that binds and stimulates human GITR without engaging Fc effector functions. Here, we report the immune effects of a single ascending dose of TRX518 monotherapy in advanced cancer patients and provide mechanistic preclinical evidence to rationally combine GITR agonism with checkpoint blockade in future clinical trials. Analysis of peripheral blood mononuclear cells (PBMCs) from 37 advanced refractory solid cancer patients treated with >/= 0.005 mg/kg TRX518 (cohorts 3-9) revealed frequent reductions in circulating Tregs after treatment, with GITR+ Tregs and activated CD45RA-Foxp3hi effector Tregs (eTregs) being preferentially affected. In 8 patients for whom pre- and post-treatment PBMC samples and tumor biopsies were available, reductions in intratumor and circulating Tregs after TRX518 were positively correlated. However, coincident down-regulation of circulating and intratumor Tregs upon TRX518 was not sufficient to achieve a clinical benefit. To clarify the mechanisms underlying this outcome, we modeled tumor sensitivity and refractoriness to anti-GITR therapy by treating B16F10-melanoma-bearing mice with the mAb DTA-1 on day 4 (curative regimen, early tumors) or day 7 (refractory regimen, advanced/established tumors) after tumor implantation respectively. Time course analysis of T-cell infiltrates revealed that intratumor Tregs were significantly reduced and Teff:Treg ratios increased in both responding and refractory tumors. However, in responding tumors, Tregs completely failed to accumulate, suggesting that the presence of Tregs during tumor formation and progression could affect T-cell functionality. Gene expression analysis of intratumor CD8+ T-cells showed up-regulation of activation/memory T-cell markers and down-regulation of exhaustion markers in responding but not in refractory tumors. To overcome resistance to anti-GITR, we thus combined the anti-GITR refractory regimen (day 7 treatment) with PD-1 blockade starting on day 7 after tumor implantation. This combination controlled tumor growth similar to the curative anti-GITR monotherapy (day 4 treatment) and achieved 50% long-lasting complete response. This was associated with more activated and less exhausted profiles of intratumor CD8+ T-cells, which showed enhanced anti-tumor cytotoxicity compared to CD8+ T-cells from nonresponding tumors treated with each agent alone. These results indicate for the first time that Treg reduction may be a pharmacodynamic biomarker of anti-GITR therapy in patients. However, Treg elimination from advanced tumors is not sufficient to activate cytotoxic CD8+ T-cell responses unless the T-cell exhaustion process is concurrently blocked. This underscores the need to combine Treg-inhibiting/depleting immunotherapies with strategies able to counteract exhaustion, such as PD-1 blockade, to regress advanced tumors. Based on these observations, we have initiated a clinical trial exploring TRX518 in combination with PD-1 pathway blockade in patients with advanced solid tumor malignancies (NCT02628574). Citation Format: Roberta Zappasodi, Cynthia Sirard, Yanyun Li, Sadna Budhu, Moshen Abu-Akeel, Cailian Liu, Xia Yang, Hong Zhong, Walter Newman, Jinjin Qi, Phillip Wong, David Schaer, Henry Koon, Vamsidhar Velcheti, Michael Postow, Margaret K Callahan, Jedd D. Wolchok, Taha D. Merghoub. Mechanistic rationale to combine GITR agonism with PD-1 blockade in cancer patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR01.

Structure-Function Analysis of Immune Checkpoint Receptors to Guide Emerging Anticancer Immunotherapy.

The modulation of immune checkpoint receptors has been one of the most successful, exciting, and explored approaches for cancer immunotherapy. Currently, several immune checkpoint modulators, mainly monoclonal antibodies, are showing remarkable results. However, the failure to show a response in most patients and the induction of severe immune-related adverse effects are the major drawbacks. Novel approaches concerning the development of immune modulatory small molecules have emerged as an alternative. Nevertheless, the lack of structural information about immune checkpoint receptors has hindered the rational design of those small-molecule modulators by preventing the use of methodologies such as computer-aided drug design. Herein, we provide an overview and critical analysis of the structural and dynamic details of immune checkpoint receptors (cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and glucocorticoid-induced TNFR-related protein (GITR)) and their interaction with known modulators. This knowledge is essential to advance the understanding of their binding mode and guide the design of novel effective targeted anticancer medicines.

Influenza-Activated ILC1s Contribute to Antiviral Immunity Partially Influenced by Differential GITR Expression.

Innate lymphoid cells (ILCs) represent diversified subsets of effector cells as well as immune regulators of mucosal immunity and are classified into group 1 ILCs, group 2 ILCs, and group 3 ILCs. Group 1 ILCs encompass natural killer (NK) cells and non-NK ILCs (ILC1s) and mediate their functionality via the rapid production of IFN-γ and TNF-α. The current knowledge of ILC1s mainly associates them to inflammatory processes. Much less is known about their regulation during infection and their capacity to interact with cells of the adaptive immune system. The present study dissected the role of ILC1s during early influenza A virus infection, thereby revealing their impact on the antiviral response. Exploiting in vitro and in vivo H1N1 infection systems, a cross-talk of ILC1s with cells of the innate and the adaptive immunity was demonstrated, which contributes to anti-influenza immunity. A novel association of ILC1 functionality and the expression of the glucocorticoid-induced TNFR-related protein (GITR) was observed, which hints toward a so far undescribed role of GITR in regulating ILC1 responsiveness. Overexpression of GITR inhibits IFN-γ production by ILC1s, whereas partial reduction of GITR expression can reverse this effect, thereby regulating ILC1 functionality. These new insights into ILC1 biology define potential intervention targets to modulate the functional properties of ILC1s, thus contributing toward the development of new immune interventions against influenza.

GITR domain inside CAR co-stimulates activity of CAR-T cells against cancer.

T cells expressing Chimeric antigen receptors or CAR-T cells are used as a novel treatment against hematological and solid cancers. In this report, we designed CAR with glucocorticoid-induced TNFR-related protein (GITR) co-stimulatory domain to study its ability to co-activate CAR-T cells. EGFR-GITR-CD3 CAR-T cells were cytotoxic against EGFR-positive: pancreatic and ovarian cancer cells but not against EGFR-negative cancer cells. The cytotoxic activity of EGFR-GITR-CD3 CAR-T cells was comparable or better than EGFR-28-CD3 or EGFR-41BB-CD3 CAR-T cells. We designed also EGFR-CD3-GITR-CAR and EGFR-ΔGITR-CD3 with deleted 184-192 amino-acids of co-stimulatory GITR domain, and showed that EGFR-GITR-CD3 had significantly higher cytotoxic activity against EGFR-positive cells. The EGFR-GITR-CD3 cells secreted significantly higher levels of IFN-gamma than EGFR-CD3-GITR and EGFR-ΔGITR-CD3 cells. In addition, Mesothelin-GITR-CD3 CAR-T cells also killed mesothelin-positive ovarian cancer cell lines, and pancreatic cancer cells. Moreover, CD19-GITR-CD3 CAR-T cells had significant cytotoxic activity against CD19-positive cancer cells in vitro and in Raji xenograft tumors in vivo. Thus, our results clearly show that GITR co-stimulatory domain can be used as a novel co-stimulatory domain in CAR-T cells.

Induction of Regulatory T Cells and Its Regulation with Insulin-like Growth Factor/Insulin-like Growth Factor Binding Protein-4 by Human Mesenchymal Stem Cells.

Human mesenchymal stem cells (MSCs) are multipotent and exert anti-inflammatory effects, but the underlying mechanism remains to be elucidated. In the current study, we investigated the regulatory mechanism of regulatory T cell (Treg) induction through the growth factors released by human MSCs. Human naive CD4+ T cells were stimulated with anti-CD3/28 Abs and cocultured with human MSC culture supernatant for 48 h. The proliferation and cytokine production of CD4+ T cells and surface molecule expression on CD4+ T cells were evaluated. The proliferation of anti-CD3/28 Abs-stimulated CD4+ T cells was suppressed by the addition of human MSC culture supernatant; in addition, the production of IL-10 and IL-4 increased. The human MSC culture supernatant induced CD4+FOXP3+ Tregs that expressed CD25, CTLA-4, glucocorticoid-induced TNFR-related protein, insulin-like growth factor (IGF)-1R, and IGF-2R, showing antiproliferative activity against CD4+ T cells. In addition, the induction of Tregs by human MSC culture supernatant was enhanced by the addition of IGF and suppressed by the inhibition of IGF-1R. In contrast, a significant amount of IGF binding protein (IGFBP)-4, an inhibitor of IGF action, was detected in the human MSC culture supernatant. After neutralization of IGFBP-4 in the human MSC culture supernatant by anti-IGFBP-4 Ab, Treg numbers increased significantly. Thus, our results raise the possibility that human MSC actions also involve a negative-regulatory mechanism that suppresses Treg proliferation by releasing IGFBP-4. The results of this study suggest that regulation of IGF may be important for treatments using human MSCs.

Anti-GITR therapy promotes immunity against malignant glioma in a murine model.

Regulatory T cells (Tregs) are potently immunosuppressive cells that accumulate within the glioma microenvironment. The reduction in their function and/or trafficking has been previously shown to enhance survival in preclinical models of glioma. Glucocorticoid-induced TNFR-related protein (GITR) is a tumor necrosis factor superfamily receptor enriched on Tregs that has shown promise as a target for immunotherapy. An agonistic antibody against GITR has been demonstrated to inhibit Tregs in a number of models and has only been recently addressed in glioma. In this study, we examined the modality of the antibody function at the tumor site as opposed to the periphery as the blood-brain barrier prevents efficient antibody delivery to brain tumors. Mice harboring established GL261 tumors were treated with anti-GITR monotherapy and were shown to have a significant increase in overall survival (p<0.01) when antibodies were injected directly into the glioma core, whereas peripheral antibody treatment only had a modest effect. Peripheral treatment resulted in a significant decrease in granzyme B (GrB) expression by Tregs, whereas intratumoral treatment resulted in both a decrease in GrB expression by Tregs and their selective depletion, which was largely mediated by FcγR-mediated destruction. We also discovered that anti-GITR treatment results in the enhanced survival and functionality of dendritic cells (DCs)-a previously unreported effect of this immunotherapy. In effect, this study demonstrates that the targeting of GITR is a feasible and noteworthy treatment option for glioma, but is largely dependent on the anatomical location in which the antibodies are delivered.

Abstract 3220: A novel agonist antibody (INCAGN01876) that targets the costimulatory receptor GITR

Abstract Activation of costimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily in T cells is considered a promising alternative approach to potentiate anti-tumor immunity that may complement strategies focused on the blockade of co-inhibitory pathways such PD-1/PDL1. Glucocorticoid-induced TNFR-related protein (GITR, CD357 or TNFRSF18) is an important T cell costimulatory receptor that can potentiate T cell receptor (TCR) signaling during CD4+ and CD8+ T cell priming, effector cell differentiation and memory T cell recall responses. In humans GITR expression is generally restricted to subsets of T cells responding to TCR stimulation, and is co-expressed with OX40. Like other TNFR family members, GITR co-stimulation can enhance T cell responsiveness to suboptimal TCR signaling by activating the NFκB pathway, leading to enhanced cytokine responses and survival. GITR signaling in T cells may also promote resistance to the immune suppressive effects of regulatory T cells, thereby enhancing T cell responsiveness to weakly immunogenic tumor-associated antigens. INCAGN01876 is a humanized IgG1 monoclonal antibody being developed for the treatment of advanced malignancies. INCAGN01876 potently binds to human and non-human primate GITR but does not cross-react with related TNFR family members. INCAGN01876 has been optimized to mediate receptor forward signaling under suboptimal TCR stimulatory conditions, leading to increased production of TNFα and IFNγ by both CD4+ and CD8+ T cells. INCAGN01876 achieves this functionality by virtue of its ability to facilitate GITR clustering in TCR-stimulated T lymphocytes. In mouse preclinical tumor models, GITR was found to be selectively overexpressed by intratumoral regulatory T cells, a finding that was also observed in primary human tumor samples from diverse tumor types. In mouse models, this feature enabled a surrogate anti-GITR antibody to co-engage activating Fcγ receptors expressed by tumor-associated effector cells, and mediate the selective depletion of intratumoral regulatory T cells. Consistent with this, INCAGN01876 was designed to co-engage activating Fcγ receptors and was shown to efficiently mediate immune effector cell mechanisms, including ADCC and ADCP. Taken together, the biophysical and functional attributes of INCAGN01876 make it ideally suited for clinical development, both as a single agent and in combination with other immunomodulatory agents. Citation Format: Ana Maria Gonzalez, Ekaterina Breous, Mariana L. Manrique, David Savitsky, Jeremy Waight, Randi Gombos, Yuqi Liu, Shiwen Lin, Olivier Leger, Volker Seibert, Takemasa Tsuji, Taha Merghoub, Sadna Budha, Roberta Zappasodi, Gerd Ritter, Jedd Wolchok, Peggy Scherle, Gregory Hollis, Reid Huber, Marc Van Dijk, Robert Stein, Nicholas Wilson. A novel agonist antibody (INCAGN01876) that targets the costimulatory receptor GITR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3220.

Expression of 4-1BB and its ligand on blasts correlates with prognosis of patients with AML

Costimulatory ligands (COLs) and their receptors (COR) regulate immune reactions and cellular survival and might be relevant in acute myeloid leukemia (AML). This study evaluated the clinical relevance of 4-1BBL,...

Role of Thalidomide on the Expression of OX40, 4-1BB, and GITR in T Cell Subsets

BackgroundThalidomide (TM) is known to have anti-cancer and anti-inflammatory properties; however, its mechanism on T cells is still unclear. We previously showed the immune modulatory effect of TM on T cells and its therapeutic effect on lupus nephritis models. Here we examined the changes in the expression of tumor necrosis factor receptor superfamilies (TNFRSFs), including OX40, 4-1BB, and glucocorticoid-induced TNFR-related protein (GITR) in T cell subsets by TM treatments. MethodsSplenic naïve T cells (Tnaives) from C57BL/6 mice were sort-purified and cultured for CD4+ T cell proliferation and regulatory T cells (Tregs) conversion with TM treatments. All samples were analyzed by flow cytometry after stained with anti-mouse CD4, Foxp3, OX40, 4-1BB, or GITR antibodies. ResultsExpressions of OX40, 4-1BB, and GITR on CD4+ T cells showed a decreasing tendency by TM treatments. Especially, downregulation of these molecules on CD4+CFSElow T cells was significant in TM treatment groups. On the condition of Treg conversion, OX40 was downregulated significantly. In contrast, the expression of GITR was increased, and that of 4-1BB had shown no particular change under the condition of Treg. ConclusionConsidering these results, TM may have an immune modulatory role through the T cell subset-specific change of OX40, 4-1BB, and GITR expression. Further study is required to elucidate the effect of thalidomide on T cells.

Serum Levels of TNF Receptor Ligands Are Dysregulated in Sepsis and Predict Mortality in Critically Ill Patients.

IntroductionTNF superfamily members, including TNF-related weak inducer of apoptosis (TWEAK) and Glucocorticoid-Induced TNFR-Related Protein Ligand (GITRL) have been described as serum based biomarkers for inflammatory and immune mediated diseases. However, up to now the role of TWEAK and GITRL has not been analyzed in critical illness and sepsis.MethodsGITRL and TWEAK serum concentrations were measured in 121 critically ill patients (84 fulfilled with septic disease), in comparison to 50 healthy controls. Results were correlated with clinical data.ResultsSerum levels of TWEAK and GITRL were strongly decreased in critically ill patients compared with healthy controls. Concentrations of TWEAK (but not GITRL) were further decreased in patients with sepsis and correlated with routinely used markers of inflammation and bacterial infection such as C-reactive protein, procalcitonin and Interleukin-6. Notably, we failed to detect a correlation to other TNFR ligands such as TNF or APRIL. Finally, TWEAK levels of the upper quartile of the cohort were prognostic for mortality during ICU treatment.ConclusionTWEAK and GITRL levels were lower in intensive care unit medical patients. Levels of TWEAK were further decreased in septic patients, and alterations in TWEAK concentrations were linked to an unfavorable outcome. Together with recently published results on other TNFR ligands, these data indicate specific functions of the different TNFR ligands in septic diseases.

GITR subverts Foxp3(+) Tregs to boost Th9 immunity through regulation of histone acetylation.

Glucocorticoid-induced TNFR-related protein (GITR) is a costimulatory molecule with diverse effects on effector T cells and regulatory T cells (Tregs), but the underlying mechanism remains poorly defined. Here we demonstrate that GITR ligation subverts the induction of Foxp3+ Tregs and directs the activated CD4+ T cells to Th9 cells. Such GITR-mediated iTreg to Th9 induction enhances anti-tumour immunity in vivo. Mechanistically, GITR upregulates the NF-κB family member p50, which recruits histone deacetylases to the Foxp3 locus to produce a ‘closed' chromatin structure. Furthermore, GITR ligation also activates STAT6, and STAT6 renders Il9 locus accessible via recruitment of histone acetyltransferase p300, and together with inhibition of Foxp3, GITR induces strong Th9 responses. Thus, Th9 cells and iTregs are developmentally linked and GITR can subvert tolerogenic conditions to boost Th9 immunity.

Synergistic antitumor responses by combined GITR activation and sunitinib in metastatic renal cell carcinoma.

Sunitinib, a multitargeted tyrosine kinase inhibitor, is the frontline therapy for renal and gastrointestinal cancers. In view of its well-documented proapoptotic and immunoadjuvant properties, we speculate that combination of Sunitinib and immunotherapy would provide a synergistic antitumor effect. Here, we report that a remarkably synergistic antitumor responses elicited by the combined treatment of Sunitinib and an agonistic antibody against glucocorticoid-induced TNFR related protein (GITR) in a model of metastatic renal cell carcinoma. Sunitinib significantly increased the infiltration, activation, and proliferation and/or cytotoxicity of CD8(+) T cells and NK cells in liver metastatic foci when combined with the anti (α)-GITR agonist, which was associated with treatment-induced prominent upregulation of Th1-biased immune genes in the livers from mice receiving combined therapy versus single treatment. Sunitinib/α-GITR treatment also markedly promoted the maturation, activation and cytokine production of liver-resident macrophages and DCs compared with that achieved by α-GITR or Sunitinib treatment alone in mice. Cell depletion experiments demonstrated that CD8(+) T cells, NK cells and macrophage infiltrating liver metastatic foci all contribute to the antitumor effect induced by combined treatment. Furthermore, mechanistic investigation revealed that Sunitinib treatment reprograms tumor-associated macrophages toward classically activated or "M1" polarization upon GITR stimulation and consequently mounts an antitumor CD8(+) T and NK cell response via inhibiting STAT3 activity. Thus, our findings provide a proof of concept that Sunitinib can synergize with α-GITR treatment to remodel the tumor immune microenvironment to trigger regressions of an established metastatic cancer.

Interleukin-17A-Induced Human Mesenchymal Stem Cells Are Superior Modulators of Immunological Function.

Interferon-γ (IFN-γ)-preactivated mesenchymal stem cells (MSC-γ) are highly immunosuppressive but immunogenic in vivo due to their inherent expression of major histocompatibility (MHC) molecules. Here, we present an improved approach where we modified human bone marrow-derived MSC with interleukin-17A (MSC-17) to enhance T cell immunosuppression but not their immunogenicity. MSC-17, unlike MSC-γ, showed no induction or upregulation of MHC class I, MHC class II, and T cell costimulatory molecule CD40, but maintained normal MSC morphology and phenotypic marker expression. When cocultured with phytohemagglutinin (PHA)-activated human T cells, MSCs-17 were potent suppressors of T cell proliferation. Furthermore, MSC-17 inhibited surface CD25 expression and suppressed the elaboration of Th1 cytokines, IFN-γ, tumor necrosis factor-α (TNF-α), and IL-2 when compared with untreated MSCs (UT-MSCs). T cell suppression by MSC-17 correlated with increased IL-6 but not with indoleamine 2,3-dioxygenase 1, cyclooxygenase 1, and transforming growth factor β-1. MSC-17 but not MSC-γ consistently induced CD4(+) CD25(high) CD127(low) FoxP3(+) regulatory T cells (iTregs) from PHA-activated CD4(+) CD25(-) T cells. MSC-induced iTregs expressed CD39, CD73, CD69, OX40, cytotoxic T-lymphocyte associated antigen-4 (CTLA-4), and glucocorticoid-induced TNFR-related protein (GITR). These suppressive MSCs-17 can engender Tregs to potently suppress T cell activation with minimal immunogenicity and thus represent a superior T cell immunomodulator for clinical application.

Corticosterone Signaling in Central Memory CD8+ T Cells Triggers Apoptosis that is Inhibited by Interleukin-15

Immunological memory plays a vital role in monitoring the body against specific pathogens. CD8+ memory lymphocytes allow a rapid response and elimination of any threat previously encountered in addition to keeping latent some chronic viruses in the host. However, certain situations such as chronic stress decrease immune function and could thereby promote viral reactivation. We demonstrate here that the stress hormone corticosterone, a cortisol analog in mice, induces apoptosis in CD8+ memory T cells. Interleukin-15 (IL-15), a cytokine essential for the homeostasis of memory T cells rescues corticosterone-induced apoptosis in a dose-dependent manner. IL-15 and glucocorticoid signaling pathways were investigated to explain the inhibitory effect of this cytokine. NF-kB, IkB? and Bim protein expression did not vary significantly when memory lymphocytes were cultured with corticosterone. However, expression of the glucocorticoid-induced TNFR-related protein (GITR), a member of the TNF receptor family, did significantly increase in the presence of IL-15 in a dose-dependent manner. Since GITR is required for the survival of memory CD8 T cells in vivo, this suggests that it may be involved in concert with IL-15 to counter glucocorticoid-induced apoptosis of CD8 memory T cells.

Regulatory T cells in B-cell-deficient and wild-type mice differ functionally and in expression of cell surface markers.

NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) with chronic inflammation of thyroids by T and B cells. B-cell deficient (B(-/-) ) mice are resistant to SAT but develop SAT if regulatory T (Treg) cells are transiently depleted. We established a transfer model using splenocytes from CD28(-/-) B(-/-) mice (effector cells and antigen-presenting cells) cultured with or without sorted Treg cells from Foxp3-GFP wild-type (WT) or B(-/-) mice. After transfer to mice lacking T cells, mice given Treg cells from B(-/-) mice had significantly lower SAT severity scores than mice given Treg cells from WT mice, indicating that Treg cells in B(-/-) mice are more effective suppressors of SAT than Treg cells in WT mice. Treg cells from B(-/-) mice differ from WT Treg cells in expression of CD27, tumour necrosis factor receptor (TNFR) II p75, and glucocorticoid-induced TNFR-related protein (GITR). After transient depletion using anti-CD25 or diphtheria toxin, the repopulating Treg cells in B(-/-) mice lack suppressor function, and expression of CD27, GITR and p75 is like that of WT Treg cells. If B(-/-) Treg cells develop with B cells in bone marrow chimeras, their phenotype is like that of WT Treg cells. Addition of B cells to cultures of B(-/-) Treg and T effector cells abrogates their suppressive function and their phenotype is like that of WT Treg cells. These results establish for the first time that Treg cells in WT and B(-/-) mice differ both functionally and in expression of particular cell surface markers. Both properties are altered after transient depletion and repopulation of B(-/-) Treg cells, and by the presence of B cells during Treg cell development or during interaction with effector T cells.

Enhanced CD8 T cell responses through GITR-mediated costimulation resolve chronic viral infection.

Chronic infections are characterized by the inability to eliminate the persisting pathogen and often associated with functional impairment of virus-specific T-cell responses. Costimulation through Glucocorticoid-induced TNFR-related protein (GITR) can increase survival and function of effector T cells. Here, we report that constitutive expression of GITR-ligand (GITRL) confers protection against chronic lymphocytic choriomeningitis virus (LCMV) infection, accelerating recovery without increasing pathology. Rapid viral clearance in GITRL transgenic mice coincided with increased numbers of poly-functional, virus-specific effector CD8+ T cells that expressed more T-bet and reduced levels of the rheostat marker PD-1. GITR triggering also boosted the helper function of virus-specific CD4 T cells already early in the infection, as was evidenced by increased IL-2 and IFNγ production, and more expression of CD40L and T-bet. Importantly, CD4-depletion experiments revealed that the expanded pool of virus-specific effector CD8 T cells and the ensuing viral clearance in LCMV-infected GITRL tg mice was entirely dependent on CD4 T cells. We found no major differences for NK cell and regulatory T cell responses, whereas the humoral response to the virus was increased in GITRL tg mice, but only in the late phase of the infection when the virus was almost eradicated. Based on these findings, we conclude that enhanced GITR-triggering mediates its protective, anti-viral effect on the CD8 T cell compartment by boosting CD4 T cell help. As such, increasing costimulation through GITR may be an attractive strategy to increase anti-viral CTL responses without exacerbating pathology, in particular to persistent viruses such as HIV and HCV.

Combined PD-1 blockade and GITR triggering induce a potent antitumor immunity in murine cancer models and synergizes with chemotherapeutic drugs

BackgroundThe coinhibitory receptor Programmed Death-1 (PD-1) inhibits effector functions of activated T cells and prevents autoimmunity, however, cancer hijack this pathway to escape from immune attack. The costimulatory receptor glucocorticoid-induced TNFR related protein (GITR) is up-regulated on activated T cells and increases their proliferation, activation and cytokine production. We hypothesize that concomitant PD-1 blockade and GITR triggering would synergistically improve the effector functions of tumor-infiltrating T cells and increase the antitumor immunity. In present study, we evaluated the antitumor effects and mechanisms of combined PD-1 blockade and GITR triggering in a clinically highly relevant murine ID8 ovarian cancer model.MethodsMice with 7 days-established peritoneal ID8 ovarian cancer were treated intraperitoneally (i.p.) with either control, anti-PD-1, anti-GITR or anti-PD-1/GITR monoclonal antibody (mAb) and their survival was evaluated; the phenotype and function of tumor-associated immune cells in peritoneal cavity of treated mice was analyzed by flow cytometry, and systemic antigen-specific immune response was evaluated by ELISA and cytotoxicity assay.ResultsCombined anti-PD-1/GITR mAb treatment remarkably inhibited peritoneal ID8 tumor growth with 20% of mice tumor free 90 days after tumor challenge while treatment with either anti-PD-1 or anti-GITR mAb alone exhibited little antitumor effect. The durable antitumor effect was associated with a memory immune response and conferred by CD4+ cells and CD8+ T cells. The treatment of anti-PD-1/GITR mAb increased the frequencies of interferon-γ-producing effector T cells and decreased immunosuppressive regulatory T cells and myeloid-derived suppressor cells, shifting an immunosuppressive tumor milieu to an immunostimulatory state in peritoneal cavity. In addition, combined treatment of anti-PD-1/GITR mAb mounted an antigen-specific immune response as evidenced by antigen-specific IFN-γ production and cytolytic activity of spleen cells from treated mice. More importantly, combined treatment of anti-PD-1/GITR mAb and chemotherapeutic drugs (cisplatin or paclitaxel) further increased the antitumor efficacy with 80% of mice obtaining tumor-free long-term survival in murine ID8 ovarian cancer and 4 T1 breast cancer models.ConclusionsCombined anti-PD-1/GITR mAb treatment induces a potent antitumor immunity, which can be further promoted by chemotherapeutic drugs. A combined strategy of anti-PD-1/GITR mAb plus cisplatin or paclitaxel should be considered translation into clinic.