Glucocorticoid-induced Tumor Necrosis Factor Receptor Research Articles

Phase 1 study of MK-4166, an anti-human glucocorticoid-induced tumor necrosis factor receptor (GITR) antibody, as monotherapy or with pembrolizumab (pembro) in patients (pts) with advanced solid tumors.

9509 Background: Ligation of GITR on immune cells decreases Treg-mediated suppression and enhances T cell proliferation. MK-4166 is a humanized IgG1 agonist monoclonal antibody targeting GITR. Data from the first-in-human phase 1 study (NCT02132754) of MK-4166 as monotherapy (mono) or in combination with pembro (combo) are presented. Methods: MK-4166 was tested alone (0.0015 mg IV-900 mg Q3W ×4 doses) or with pembro (fixed dose 200 mg IV Q3W up to 35 doses) in the absence of toxicity or progression. Study included a dose escalation/confirmation cohort (metastatic solid tumors) and an expansion cohort (treatment-naive and pretreated melanoma). A T cell–inflamed gene expression profile (GEP) was assessed using RNA from baseline tumor samples. End points – primary: safety/tolerability, maximum tolerated dose (MTD) of MK-4166; secondary: pharmacokinetics (PK), pharmacodynamics (PD); exploratory: objective response rate (ORR) per irRECIST1.1. Results: Of 113 pts, 48 received mono and 65 combo; 20 were in the melanoma expansion. Common AEs ( > 20%) were fatigue, infusion-related reaction, nausea, abdominal pain, and pruritus; 43 pts had grade ≥3 AEs (38.1%); 6 (5.3%) were treatment-related. One dose-limiting toxicity (bladder perforation in a urothelial pt with a neobladder) possibly related to study drug was observed with mono. MTD was not reached. No treatment-related deaths were observed. MK-4166 PK/PD showed target-mediated drug disposition concomitant with decreased GITR availability on T cells in blood with increasing doses. Four objective responses (4/45; ORR, 9%) were seen with combo in dose escalation. For ICI-naive melanoma pts (n = 13) in expansion, ORR was 69% (95% CI, 38-91), including 4 CRs and 5 PRs. No response was observed in 7 pts previously treated with ICI. High ORRs were observed in noninflamed and inflamed ICI-naive melanoma pts. Conclusions: MK-4166 at a dose up to 900 mg as monotherapy and in combination with pembro was well tolerated, with dose-related evidence of target engagement. Responses were observed with MK-4166 900 mg plus pembro, particularly in pts with melanoma naive to ICIs. Clinical trial information: NCT02132754.

Regulatory T cell features in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in any of the genes encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, responsible for the production of reactive oxygen species (ROS). CGD is marked by invasive bacterial and fungal infections and by autoinflammation/autoimmunity, of which the exact pathophysiology remains elusive. Contributing factors include decreased neutrophil apoptosis, impaired apoptotic neutrophil clearance, increased proinflammatory protein expression and reduced ROS-mediated inflammasome dampening. We have explored a fundamentally different potential mechanism: it has been reported that macrophage-mediated induction of regulatory T cells (Tregs ) depends on ROS production. We have investigated whether numerical or functional deficiencies exist in Tregs of CGD patients. As the prevalence of autoinflammation/autoimmunity differs between CGD subtypes, we have also investigated Tregs from gp91phox -, p47phox - and p40phox -deficient CGD patients separately. Results show that Treg numbers and suppressive capacities are not different in CGD patients compared to healthy controls, with the exception that in gp91phox -deficiency effector Treg (eTreg ) numbers are decreased. Expression of Treg markers CD25, inducible T cell co-stimulator (ICOS), Helios, cytotoxic T lymphocyte antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR) did not provide any clue for differences in Treg functionality or activation state. No correlation was seen between eTreg numbers and patients' clinical phenotype. To conclude, the only difference between Tregs from CGD patients and healthy controls is a decrease in circulating eTregs in gp91phox -deficiency. In terms of autoinflammation/autoimmunity, this group is the most affected. However, upon culture, patient-derived Tregs showed a normal phenotype and normal functional suppressor activity. No other findings pointed towards a role for Tregs in CGD-related autoinflammation/autoimmunity.

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.

Significance of increased IL-17-producing Foxp3+ Treg in peripheral blood of patients with active systemic lupus erythematosus

Objective To investigate the expression of IL-17-producing regulatory T cells (Treg) in patients with systemic lupus erythematosus (SLE) and to analyze their clinical significance. Methods This study recruited 32 patients with SLE (including 14 with active SLE and 18 with inactive SLE) and 13 healthy subjects matched for age and sex. Flow cytometry was performed to detect the expression of Foxp3 and IL-17 in CD4 T lymphocytes and the phenotypic characteristics of IL-17-producing Treg. Correlations between these cells and clinical indicators of SLE were analyzed. Peripheral CD4+ CD25+ T cells were isolated from five healthy subjects and then stimulated with IL-6 and IL-1β alone or in combination. An in vitro T cell polarization assay was performed to investigate the role of cytokines in the polarization and regulation of IL-17-producing Treg. Results Compared with the healthy subjects and patients with inactive SLE, the patients with active SLE had a higher percentage of IL-17-producing Treg in peripheral blood. Moreover, the expression of Foxp3 and CD45RA by IL-17-producing Treg in the active SLE group was down-regulated, while that of IL-2, granzyme B (GramB), programmed cell death protein 1 (PD-1) and glucocorticoid-induced tumor necrosis factor receptor (GITR) was up-regulated. Inflammatory cytokines such as IL-6 and IL-1 could induce Treg to produce IL-17. Conclusions This study suggested that increased inflammatory cytokines might correlate with higher percentages of IL-17-producing Treg in patients with active SLE. These cells were a subset of pathogenic Treg failing to prevent autoimmune. Key words: Regulatory T cell; Interleukin-17; Inflammatory cytokine; Immunophenotype; T cell plasticity

Increased GITRL Impairs the Function of Myeloid-Derived Suppressor Cells and Exacerbates Primary Sjögren Syndrome.

Although the expansion of myeloid-derived suppressor cells (MDSCs) has been reported in autoimmune disorders, it is largely unclear how MDSCs contribute to the development of primary Sjögren syndrome (pSS). In this study, we found significantly increased MDSCs with gradually diminished suppressive capacity during disease development in mice with experimental Sjögren syndrome (ESS). The ligand for glucocorticoid-induced TNFR family-related protein (GITRL) was increased along ESS progression, whereas the increased GITRL was found to attenuate the immunosuppressive function of MDSCs. Moreover, blocking GITR signal in MDSCs significantly restored their immunosuppressive function and alleviated ESS progression in mice. In pSS patients, expanded MDSCs were found to express low levels of arginase. Significantly increased serum GITRL levels were closely correlated with patients with higher Sjögren syndrome disease activity index. Furthermore, treatment with recombinant GITRL markedly reduced the immunosuppressive function of human MDSCs. Together, our studies have demonstrated a critical role of GITRL in modulating the suppressive function of MDSCs, which may facilitate the validation of GITRL as a therapeutic target for the treatment of pSS.

A single administration of human adipose tissue-derived mesenchymal stromal cells (MSC) induces durable and sustained long-term regulation of inflammatory response in experimental colitis

Current therapies for inflammatory bowel diseases (IBD) are aimed at controlling the exacerbated response in the gut, but no treatment is fully effective for many refractory patients. Mesenchymal stromal cells (MSC) are multi-potent cells with regulatory immunosuppressive activity that may control inflammatory diseases. In this study, we investigated the short- and especially the long-term protective effects of MSC on experimental colitis. We show that MSC elicited protection to acute intestinal inflammation with gain of weight, improvement in the clinical disease score and expressive reduction in the mortality rate of treated mice. MSC changed the population of neutrophils, eosinophils and augmented the frequency of CD4 T lymphocytes in the gut-draining lymph nodes, together with reduced accumulation of these cells in the colon intraepithelial compartment. Interestingly, there were increased levels of programmed death 1 (PD-1) and glucocorticoid-induced tumour necrosis factor receptor family-related receptor (GITR) in the spleen regulatory T cells of mice that received MSC treatment, which also presented a reversal in the pattern of immune response in the gut, with diminished inflammatory, T helper type 1 (Th1) and Th17 profile, in contrast to augmented Th2 responses. Most strikingly, this balanced response elicited by a single administration of MSC during the acute colitis persisted long-term, with restored goblet cells, eosinophils and maintenance of elevated gut interleukin (IL)-4, besides increased CD4+ CD25+ PD-1+ cells in the spleen and reduced Th17 response in mesenteric lymph nodes (MLN) of treated mice on day 60. Taken together, our findings provided a significant contribution to translational immunology by pointing human adipose tissue-derived MSC as a novel therapeutic approach with long-term beneficial regulatory effects in experimental colitis.

Genetically modified hematopoietic stem/progenitor cells that produce IL-10–secreting regulatory T cells

Random amino acid copolymers used in the treatment of multiple sclerosis in man or experimental autoimmune encephalomyelitis (EAE) in mice [poly(Y,E,A,K)n, known as Copaxone, and poly(Y,F,A,K)n] function at least in part by generation of IL-10-secreting regulatory T cells that mediate bystander immunosuppression. The mechanism through which these copolymers induce Tregs is unknown. To investigate this question, four previously described Vα3.2 Vβ14 T cell receptor (TCR) cDNAs, the dominant clonotype generated in splenocytes after immunization of SJL mice, that differed only in their CDR3 sequences were utilized to generate retrogenic mice. The high-level production of IL-10 as well as IL-5 and small amounts of the related cytokines IL-4 and IL-13 by CD4+ T cells isolated from the splenocytes of these mice strongly suggests that the TCR itself encodes information for specific cytokine secretion. The proliferation and production of IL-10 by these Tregs was costimulated by activation of glucocorticoid-induced TNF receptor (GITR) (expressed at high levels by these cells) through its ligand GITRL. A mechanism for generation of cells with this specificity is proposed. Moreover, retrogenic mice expressing these Tregs were protected from induction of EAE by the appropriate autoantigen.

Mature IgDlow/- B cells maintain tolerance by promoting regulatory T cell homeostasis

A number of different B cell subsets have been shown to exhibit regulatory activity using a variety of mechanisms to attenuate inflammatory diseases. Here we show, using anti-CD20-mediated partial B cell depletion in mice, that a population of mature B cells distinguishable by IgDlow/- expression maintains tolerance by, at least in part, promoting CD4+Foxp3+ regulatory T cell homeostatic expansion via glucocorticoid-induced tumor necrosis factor receptor ligand, or GITRL. Cell surface phenotyping, transcriptome analysis and developmental study data show that B cells expressing IgD at a low level (BDL) are a novel population of mature B cells that emerge in the spleen from the transitional-2 stage paralleling the differentiation of follicular B cells. The cell surface phenotype and regulatory function of BDL are highly suggestive that they are a new B cell subset. Human splenic and peripheral blood IgDlow/- B cells also exhibit BDL regulatory activity, rendering them of therapeutic interest.

Tandem immunotherapy achieves synergy

Cancer Immunology Immune checkpoint–inhibitor therapies bolster the antitumor activity of CD8+ T lymphocytes. Wang et al. performed single-cell analysis of tumor-infiltrating lymphocytes in mouse cancer models in which inhibitory anti–PD-1 (programmed cell death protein 1) and stimulatory anti–GITR (glucocorticoid-induced tumor necrosis factor receptor–related protein) antibodies together enhanced tumor control. This combination immunotherapy led to a synergistic increase in tumor antigen–specific memory precursor effector T cells dependent on the CD226 costimulatory pathway. Biochemical studies in liposomes identified CD226 as an additional target of dephosphorylation mediated by the PD-1–SHP2 (Src homology region 2) complex. Thus, further clinical trials could usefully test the efficacy of combined anti-GITR and anti–PD-1 immunotherapy in human cancer. Sci. Immunol. 3 , eaat7061 (2018).

Dose escalation results from a first-in-human, phase 1 study of glucocorticoid-induced TNF receptor\u2013related protein agonist AMG 228 in patients with advanced solid tumors

BackgroundThis open-label, first-in-human, phase 1 study evaluated the safety, pharmacokinetics, pharmacodynamics, and maximum tolerated dose (MTD) of AMG 228, an agonistic human IgG1 monoclonal antibody targeting glucocorticoid-induced tumor necrosis factor receptor−related protein (GITR), in patients with refractory advanced solid tumors.MethodsAMG 228 was administered intravenously every 3 weeks (Q3W). Dose escalation was in two stages: single-patient cohorts (3, 9, 30, and 90 mg), followed by “rolling six” design (n = 2–6; 180, 360, 600, 900, and 1200 mg). Primary endpoints included incidence of dose-limiting toxicities (DLTs), AEs, and pharmacokinetics. Additional endpoints were objective response and pharmacodynamic response.ResultsThirty patients received AMG 228, which was well tolerated up to the maximum planned dose (1200 mg). No DLTs occurred; the MTD was not reached. The most common treatment-related AEs were fatigue (13%), infusion-related reaction (7%), pyrexia (7%), decreased appetite (7%), and hypophosphatemia (7%). Two patients had binding anti−AMG 228 antibodies (one at baseline); no neutralizing antibodies were detected. AMG 228 exhibited target-mediated drug disposition, and serum exposure was approximately dose proportional at 180–1200 mg and greater than dose proportional at 3–1200 mg. Doses > 360 mg Q3W achieved serum trough coverage for 95% in vitro GITR occupancy. Despite GITR coverage in peripheral blood and tumor biopsies, there was no evidence of T-cell activation or anti-tumor activity.ConclusionsIn patients with advanced solid tumors, AMG 228 Q3W was tolerable up to the highest tested dose (1200 mg), exhibited favorable pharmacokinetics, and provided target coverage indicating a pharmacokinetic profile appropriate for longer intervals. However, there was no evidence of T-cell activation or anti-tumor activity with AMG 228 monotherapy.Trial registrationClinicalTrials.gov, NCT02437916.

Abstract LB-152: Local selivery of GITR agonistic antibody induces a stronger antitumor immunity than systemic administration

Abstract The field of cancer immunotherapy is expanding rapidly with the success of an antagonistic antibody against CTLA-4 and PD-1/PD-L1. However, since approximately half of patients do not respond to the therapies even the combination regimen, the development of novel checkpoint inhibitors is desired for the recurrent or refractory patients. Recently, newer targets including select members of the tumor necrosis factor receptor (TNFR) family, including 4-1BB, OX40 and glucocorticoid-induced tumor necrosis factor receptor (GITR), are gathering attention. GITR is expressed at a low basal level on naïve murine T cells and at a very low level on human T cells, whereas a GITR ligand (GITRL) was abundantly expressed in murine dendritic cells and macrophages. Multiple studies have shown that GITR-GITRL interaction can provide a co-stimulatory signal to both CD4+ and CD8+ naïve T cells, enhancing proliferation and effector function, particularly in the setting of suboptimal T cell receptor stimulation. In contrast, murine and human Tregs constitutively express GITR, and it had been shown that activation of GITR signaling by GITR ligand or agonistic antibody inhibit the suppressive activity of Tregs. To enhance the antitumor effect of immune stimulatory reagents, we have been focusing on the intratumoral administration route. Since the GITR agonistic Ab directly activates effector T cells and suppresses Tregs, the increase of Ab concentration in tumors and surrounding tissues including lymph nodes by the intratumoral route may enhance only the tumor-infiltrating T cells and break the tumor-specific immune-tolerant microenvironment, sparing the unexpected effect on the systemic immune system. In this study, first, in a murine colon cancer model, we showed that the intratumoral delivery of Ab significantly increased the number of effector T cells infiltrated into tumors, and suppressed tumor growth more effectively than the intraperitoneal injection did. Then, we found that the injection of Ab into the peritumoral area induced a systemic antitumor immunity at a similar level to the intratumoral injection. Therefore, we hypothesized that the transfer of locally administrated Ab into tumor-draining lymph nodes (TDLNs) plays an important role in inducing an effective immunity. In fact, intratumorally or peritumorally injected Ab was detected in TDLNs, and resection of Ab-injected TDLNs significantly reduced GITR Ab-mediated systemic tumor immunity. Intratumoral injection showed less number of auto-reactive T cells in the spleen than the intraperitoneal injection did. Furthermore, the AH-1 tetramer assay and ELISpot assay showed that the combination of GITR Ab with anti-PD-1 Ab synergistically enhanced an induction of antitumor immunity, leading to eradication of the tumor. Intratumoral delivery of GITR Ab is a promising approach to induce an effective immunity than the systemic delivery. Citation Format: Kazunori Aoki, Kenta Narumi, Chihiro Shibasaki, Reina Miyakawa. Local selivery of GITR agonistic antibody induces a stronger antitumor immunity than systemic administration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-152.

Abstract 1699: Treatment with agonist anti-GITR antibody after chemotherapy enhances tumor immunity

Abstract Stimulation of glucocorticoid-induced tumor necrosis factor receptor (GITR) has been shown to enhance antitumor immunity by stimulating effector CD4+ and CD8+ T cells and attenuating suppression and depleting by CD4+Foxp3+ regulatory T cells (Treg). However, GITR monotherapy does not effectively control tumor growth. We hypothesize that cyclophosphamide (CTX), a cytotoxic chemotherapeutic agent with key immunomodulatory properties, can enhance the potency of GITR engagement anti-tumor effects. Further, we hypothesize that CTX can induce neoantigen formation by increasing tumor mutation burden. In this study, we administered high-dose CTX (250 mg/kg) followed the next day by an agonist anti-GITR antibody (DTA-1) to C56BL/6 mice bearing poorly immunogenic B16 melanoma tumors. We found that the combination therapy can cause regression of established tumors whereas the single agents did not. Mechanistically, we found that CTX administration results in tumor antigen release, which enhances the antitumor T cell response after treatment with DTA-1. This effect is lost when lower doses of CTX are given or CTX is administered prior to tumor inoculation, suggesting that tumor cell death and cross-presentation of tumor antigens to T cells is necessary. Furthermore, Treg cells are an important target of combination therapy. Intratumoral Treg levels were substantially reduced in treated mice, which greatly increased the ratio of T effector/Treg in the tumor. In conclusion, CTX and agonist anti-GITR antibody slows tumor progression and can induce regression of palpable, poorly immunogenic B16 melanoma tumors, likely by enhancing cross-priming and increasing the T effector/Treg ratio in the tumor. Thus, this combination warrants further investigation as a novel immunotherapy approach and provides rational for exploring combination of GITR agonist antibodies with chemotherapeutic treatments in clinical settings. Citation Format: Allison Betof Warner, Daniel Hirschhorn, Adam Cohen, Cailian Liu, Walter Newman, Cyndi Sirard, Jedd Wolchok, Taha Merghoub. Treatment with agonist anti-GITR antibody after chemotherapy enhances tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1699.

Abstract 3826: GITRL-Fc biomarker and mechanism study: GITRL-Fc reduces Treg frequency in tumors and requires effector function for inhibition of tumor growth

Abstract Activation of the co-stimulatory receptor GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor) by GITR-Ligand (GITRL) promotes proliferation and activation of effector T cells (T eff) and inhibits suppressive activity of regulatory T cells (Treg). Here, we have further characterized the mechanism of action of a single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc, 336B3) by examining pharmacodynamic (PD) biomarkers in time course studies. Mice bearing CT26.WT colon tumors were treated with weekly GITRL-Fc and sacrificed 24 hours, 7 and 14 days after the first dose. Immuno-phenotyping of tumor-associated immune cells revealed a reduction in Treg frequency in tumor by 24 hours post-dose that was maintained at 7 and 14 days. Furthermore, GITRL-Fc treatment increased activation markers on tumor-associated CD4+ and CD8+ T cells, suggesting an increased cytotoxic environment within the tumor. This was supported by significant and sustained increase in CD8+ T cell:Treg ratio in the tumor after GITRL-Fc treatment. To determine whether intratumoral (IT) injection of GITRL-Fc is an effective route of administration, we compared efficacy, pharmacodynamic (PD) markers and pharmacokinetics in IT- and intraperitoneal (IP)-injected mice bearing bilateral CT26.WT tumors. Both routes of administration showed similar tumor growth inhibition (TGI) and PD markers in both the treated and the abscopal tumors, but IT injection resulted in a significantly lower serum GITRL-Fc concentration, suggesting that IT administration may be an alternative route of administration to IP with similar efficacy. The GITRL-Fc molecule 336B3 is effector function competent and able to induce cell-mediated cytotoxicity upon binding. To determine whether this effector function is required for GITRL-Fc-induced TGI, we treated CT26.WT tumor-bearing mice with 336B3 and 336B22, a GITRL-Fc molecule deficient in effector function. The effector function-competent 336B3 induced significant TGI and a more robust activation of Teff cells and reduction in Treg frequency, when compared to 336B22, suggesting that effector function is important for efficacy. To identify biomarkers for GITRL-Fc, we performed microarray analyses on multiple syngeneic mouse models treated with GITRL-Fc and developed GITRL gene signatures from blood and from tumors. We also developed multiplexed immunohistochemistry panels designed to quantify frequency of GITR and GITRL expression (GITR+CD8, GITR+FOXP3) in tumors. In conclusion, we have examined the effects of GITRL-Fc on preclinical mouse models. Biomarker analysis showed that loss of Tregs, activation of T cells and Fc-mediated effector function are key elements in the mechanism of action of the molecule. We have identified potential biomarkers to be used for PD and potential predictive analysis in clinical trial patient samples. Citation Format: Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Reyhaneh Lahmy, Erwan Le Scolan, Rose Harris, Alayne Brunner, Min Wang, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Earth Light Lowe, Gilbert O'Young, Austin Gurney, Ann M. Kapoun. GITRL-Fc biomarker and mechanism study: GITRL-Fc reduces Treg frequency in tumors and requires effector function for inhibition of tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3826.

Preliminary investigation on the abnormal mechanism of CD4+FOXP3+CD25high regulatory T cells in pediatric B-cell acute lymphoblastic leukemia.

The current study aimed to investigate the changes and regulatory mechanism of cluster of differentiation (CD)4+CD25high forkhead box protein 3 (Foxp3+) regulatory T cells (Tregs) in childhood B-cell acute lymphocytic leukemia (B-ALL). A total of 18 children with B-ALL and 15 age-matched healthy children were included. Reverse-transcription quantitative polymerase chain reaction was used to evaluate the mRNA levels of Foxp3, cytotoxic T-lymphocyte associated protein 4 (CTLA4), glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte activation gene 3 (LAG3), interleukin (IL)-2 receptor (R)β/γ, IL-6Rα/β, mothers against decapentaplegic homolog (Smad)3/4 and runt-related transcription factor (RUNX)1/3 in CD4-positive cells. The concentration of cytokines in plasma were measured using a cytometric bead array. Additionally, the proportion of CD4+CD25highFoxp3+ Tregs and levels of associated proteins was analyzed using flow cytometry. The results demonstrated that the proportion of CD4+CD25highFoxp3+ and expression of Foxp3 in children with B-ALL was significantly higher compared with healthy controls (P<0.05) and that transcription levels of CTLA4, GITR and LAG3 were also significantly elevated (P<0.05). Compared with healthy controls, the expression of IL-2Rα/β and its downstream molecule phosphorylated signal transducer and activator of transcription 5 (pSTAT5) in CD4-positive cells significantly increased (P<0.05); however, no significant difference of IL-2Rγ levels was identified between the two groups. Correlation analysis demonstrated a significant positive correlation between the expression of phosphorylated (p) signal transducer and activator of transcription factor (STAT)5 and CD4+CD25highFoxp3+ Tregs in children with B-ALL (r=0.17; P<0.05). The plasma concentration of TGF-β, the expression of its receptor TGF-βRI/II and downstream molecules Smad3/4 were significantly upregulated in children with B-ALL (P<0.05), whereas the expression of RUNX1/3 was lower compared with healthy controls (P<0.05). Furthermore, the expression of Smad3 and RUNX1 was positively correlated with CD4+CD25highFoxp3+ Tregs in children with B-ALL (r=0.87 and 0.60, respectively; P<0.05). Additionally, the expression of pSTAT3 in CD4-positive cells decreased significantly in pediatric patients with B-ALL when compared with healthy controls; however, plasma concentrations of IL-6 was significantly higher (P<0.05). Furthermore, a negative correlation was identified between pSTAT3 and CD4+CD25highFoxp3+ Tregs in pediatric patients with B-ALL (r=-0.39; P<0.05). However, no significant differences in IL-6Rα/β expression were identified between the two groups. The results demonstrated that the excessive activation of IL-2/pSTAT5 and TGF-β/Smad signaling, and insufficiency of pSTAT3 may be correlated with increased CD4+CD25highFoxp3+ Tregs in pediatric B-ALL.

First-in-human phase 1 study of MK-1248, an anti-human glucocorticoid-induced tumor necrosis factor receptor (GITR) monoclonal antibody, as monotherapy or in combination with pembrolizumab in patients with advanced solid tumors.

3029Background: MK-1248 is a humanized IgG4 agonist monoclonal antibody (mAb) that targets GITR. GITR is expressed on regulatory T cells (Treg), resting CD4+ and CD8+ T cells, NK (natural killer) c...

Combination of NIR therapy and regulatory T cell modulation using layer-by-layer hybrid nanoparticles for effective cancer photoimmunotherapy.

The efficacy of combined near-infrared (NIR) and immune therapies for inhibiting tumor growth and recurrence has gained increasing research attention. Regulatory T cells in the tumor microenvironment constitute a major obstacle in achieving robust CD8+ T cell antitumor immunotherapy. In the present study, we designed a photoimmunotherapy-based strategy involving a combination of photothermal and photodynamic therapies, followed by Treg cell suppression, for eliciting an immune response with IR-780- and imatinib-loaded layer-by-layer hybrid nanoparticles.Methods: The layer-by-layer hybrid nanoparticles were prepared through electrostatic interactions. Their photothermal effect, photodynamic effect as well as their effect on inhibiting Treg cells' suppressive function were investigated in vitro and in vivo. Their antitumor effect was evaluated using B16/BL6 and MC-38 tumor-bearing mice.Results: The layer-by-layer hybrid nanoparticles, which were pH-sensitive, enabled the release of IR-780 dye for NIR-induced photothermal and photodynamic effects, and the release of imatinib-loaded glucocorticoid-induced TNF receptor family-related protein/poly(lactic-co-glycolic acid) (GITR-PLGA) nanoparticles to initiate antitumor immunotherapy. The photothermal and photodynamic effects caused by IR-780 under NIR exposure resulted in direct tumor apoptosis/necrosis and the production of tumor-associated antigen, promoted dendritic cell maturation, and enhanced the presentation of tumor-associated antigen to T cells, while the imatinib-loaded GITR-PLGA cores reduced the suppressive function of Treg cells, and consequently activated effective CD8+ T cells towards tumors.Conclusion: With the significant photothermal, photodynamic and immunotherapies, the system successfully eradicated tumor growth, diminished tumor recurrence, and improved survival in vivo. The proposed nanoparticles provide a novel and versatile approach to boost antitumor photoimmunotherapy.

IL-17 producing CD4+CD45RO+ T-cells in atherosclerosis express GITR molecule

BackgroundAtherosclerosis (AS) is a chronic inflammatory disease of vessel walls associated with infiltration of immune cells which their function is controlled by different co-stimulatory and co-inhibitory receptors. We investigated the expression of co-inhibitory molecules on the memory and effector T-cells in patients with Atherosclerosis. MethodsPatients included 9 hypertensive, dyslipidemic, non-diabetic, non-smoker individuals with the diagnosis of coronary artery disease and controls were 8 normotensive, normolipemic, non-diabetic, non-smoker individuals with normal coronary angiography/insignificant coronary artery disease. PBMCs were separated from the blood and memory T-cell subsets as well as the expression of Glucocorticoid-induced tumor necrosis factor receptor (GITR), Programmed Death-1 (PD-1), IL-17A and IFN-γ were quantified by flowcytometry. ResultsCD4+CD45RO+ memory T-cells and CD4+CD45RO− effector T-cells in patients expressed the highest level of GITR molecule. The IL-17 producing memory CD4+CD45RO+ T-cells were enriched in GITR molecule in the patients group (P = 0.03). The increased population of GITR+effector CD4+CD45RO− T-cells in patients, however, did not produce IL-17 (P = 0.03). PD-1 expression on memory T-cells of the patients was higher than the controls and was concomitant with the lack of IFN-γ expression (P = 0.05). IFN-γ production by effector T-cells was only seen in the PD-1− population in both groups. ConclusionsWe provide data on the expression of GITR molecule on IL-17 producing memory T-cells in patients with CAD. A population of memory T-cells, which expressed PD-1 and were not producing IFN-γ, also increased in patients' blood. These data suggest the modified phenotype/function of T-cell subsets in the atherosclerotic inflammation.

Emerging targets in cancer immunotherapy.

The first generation of immune checkpoint inhibitors (anti-CTLA-4 and anti-PD-1/PD-L1) targeted natural immune homeostasis pathways, co-opted by cancers, to drive anti-tumor immune responses. These agents led to unprecedented results in patients with previously incurable metastatic disease and may become first-line therapies for some advanced cancers. However, these agents are efficacious in only a minority of patients. Newer strategies are becoming available that target additional immunomodulatory mechanisms to activate patients' own anti-tumor immune responses. Herein, we present a succinct summary of emerging immune targets with reported pre-clinical efficacy that have progressed to active investigation in clinical trials. These emerging targets include co-inhibitory and co-stimulatory markers of the innate and adaptive immune system. In this review, we discuss: 1) T lymphocyte markers: Lymphocyte Activation Gene 3 [LAG-3], T-cell Immunoglobulin- and Mucin-domain-containing molecule 3 [TIM-3], V-domain containing Ig Suppressor of T cell Activation [VISTA], T cell ImmunoGlobulin and ITIM domain [TIGIT], B7-H3, Inducible T-cell Co-stimulator [ICOS/ICOS-L], CD27/CD70, and Glucocorticoid-Induced TNF Receptor [GITR]; 2) macrophage markers: CD47/Signal-Regulatory Protein alpha [SIRPα] and Indoleamine-2,3-Dioxygenase [IDO]; and 3) natural killer cell markers: CD94/NKG2A and the Killer Immunoglobulin-like receptor [KIR] family. Finally, we briefly highlight combination strategies and potential biomarkers of response and resistance to these cancer immunotherapies.

Changes in DNA Methylation of Glucocorticoid-Induced Tumor Necrosis Factor Receptor and Its Ligand in Liver Transplantation

Liver transplantation (LT) is the criterion standard of care in patients with end-stage liver disease and those with tumors of hepatic origin in the setting of liver dysfunction. Chronic immune rejection of the liver transplant can lead to bad prognosis for patients. Glucocorticoid-induced tumor necrosis factor receptor (GITR) play a key role in dominant immunologic self-tolerance maintained by CD25+/CD4+ regulatory T cells. Here, we investigated the DNA methylation variations of GITR and GITR ligand (GITRL) using pyrosequencing by analyzing blood DNA samples of patients after LT. Our results showed that the methylation level of certain CpGs, such as CpG_13, in GITRL was significantly reduced after LT. Furthermore, we found that the GITRL methylation statuses of cohorts with no chronic immune rejection were significantly lower compared with cohorts with chronic immune rejection after LT treatment. However, the methylation statuses of GITR were less varied. Using linear regression analysis, we further found that factors such as upper gastrointestinal hemorrhage, splenectomy, and creatinine might affect DNA methylation patterns in chronic immune rejection cohorts. These findings provide novel insights into the pivotal role of GITRL as a potential molecular biomarker for the triage of liver transplantation.

The Proinflammatory Cytokine GITRL Contributes to TRAIL-mediated Neurotoxicity in the HCN-2 Human Neuronal Cell Line.

Cytokines belonging to the TNF superfamily play a relevant role in neurodegenerative processes. Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), released during neuronal injury, has proven to potently mediate and sustain neurotoxic processes leading to neuronal death. Similarly to TRAIL, the cytokine Glucocorticoid-induced TNF receptor ligand (GITRL) is able to transduce proapoptotic signals. In spite of the array of reports suggesting relationships between TRAIL and other cytokines, scanty data are, so far, available about a GITRL/TRAIL crosstalk. Here, we investigated possible interactions between TRAIL and the GITRL system in an in vitro model of neurodegeneration, using the human cortical neuronal cell line HCN-2. Cultured HCN-2 neurons were incubated at different times with GITRL and/or TRAIL, and thereafter nucleic acid and protein expression were measured. Real-time PCR analysis showed that the human cortical neuronal cell line HCN-2 does not express GITRL mRNA, but the latter is induced after treatment with TRAIL. In addition, HCN-2 cells did not express the GITRL receptor GITR mRNA, neither in control cultures, nor after treatment with TRAIL. All mRNA data were confirmed by western blot analysis of proteins. Cell viability assay showed that TRAIL, when associated to GITRL, was able to exert additive toxic effects. A counterproof was provided in experiments performed blocking GITRL, in which TRAIL-mediated toxicity appeared significantly reduced. Results suggest that GITRL/TRAIL redundancy during neurodegenerative processes implies extended potentiation of detrimental effects of both cytokines on neurons, eventually leading to larger cell damage and death. Finally, characterization of novel molecular targets within the TRAIL/GITRL interplay may represent a platform for innovative therapy of neurodegenerative disorders.