Julie Stockis

Membrane protein GARP is a receptor for latent TGF-beta on the surface of activated human Treg.

Human Treg and Th clones secrete the latent form of TGF‐β, in which the mature TGF‐β protein is bound to the latency‐associated peptide (LAP), and is thereby prevented from binding to the TGF‐β receptor. We previously showed that upon TCR stimulation, human Treg clones but not Th clones produce active TGF‐β and bear LAP on their surface. Here, we show that latent TGF‐β, i.e. both LAP and mature TGF‐β, binds to glycoprotein A repetitions predominant (GARP), a transmembrane protein containing leucine rich repeats, which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF‐β mediated by binding to GARP may be necessary for the ability of Treg to activate TGF‐β upon TCR stimulation. However, it is not sufficient as lentiviral‐mediated expression of GARP in human Th cells induces binding of latent TGF‐β to the cell surface, but does not result in the production of active TGF‐β upon stimulation of these Th cells.

The CD4+ T-Cell Response of Melanoma Patients to a MAGE-A3 Peptide Vaccine Involves Potential Regulatory T Cells

Melanoma patients were injected with various vaccines containing a MAGE-A3 peptide presented by HLA-DP4. Anti-MAGE-A3.DP4 T cells were not detectable in the blood before vaccination, but their frequencies after vaccination ranged from 2 x 10(-6) to 2 x 10(-3) among the CD4(+) blood T lymphocytes of the patients. The CD4(+) blood T lymphocytes that stained ex vivo with HLA-DP4 tetramers folded with the MAGE-A3 peptide were selected by flow cytometry and amplified under clonal conditions. About 5% of the CD4(+) T-cell clones that recognized the MAGE-A3.DP4 antigen had a CD25(+) phenotype in the resting state. These CD25(+) clones had a high capacity to suppress the proliferation of another T-cell clone after peptide stimulation in vitro. Most of them had high FOXP3 expression in the resting state and an unmethylated FOXP3 intron 1. They produced active transforming growth factor-beta but none of cytokines IFN-gamma, interleukin-2 (IL-2), IL-4, IL-5, and IL-10. About 20% of CD25(-) clones had a significant but lower suppressive activity. Most of the CD25(-) clonal populations contained cells that expressed FOXP3 in the resting state, but FOXP3 demethylation was not observed. We conclude that MAGE-A3.DP4 vaccination can produce CD4(+) T cells that may exert regulatory T-cell function in vivo.

Frequency of Circulating Tregs with Demethylated FOXP3 Intron 1 in Melanoma Patients Receiving Tumor Vaccines and Potentially Treg-Depleting Agents

Purpose: Regulatory T cells (Tregs) are thought to inhibit antitumor immune responses, and their depletion could therefore have a synergistic effect with therapeutic cancer vaccines. We investigated the impact of three medications on blood Treg frequency in vaccinated cancer patients. Experimental Design: To date, the most specific marker for human Tregs is demethylation in the DNA that encodes the transcription factor FOXP3. Thus, we used a FOXP3 methylation-specific quantitative PCR assay (MS-qPCR) to measure Treg frequencies in the peripheral blood mononuclear cells (PBMCs) of melanoma patients. The patients participated in three clinical trials that combined tumor vaccines with potential Treg-depleting agents: low-dose cyclophosphamide, anti-CD25 monoclonal antibody daclizumab, and the IL-2/diphtheria toxin fusion protein denileukin diftitox. Results: In the nine control patients, blood Treg frequencies varied over time; there was a 46% reduction in one patient. In treated patients, a more than 2-fold decrease in Tregs was observed in one out of 11 patients receiving cyclophosphamide and in four out of 13 receiving daclizumab, but there was no such Treg decrease in any of the six patients who received denileukin diftitox. As a positive control, a more than 2-fold increase in blood Tregs was detected in four out of nine patients who were treated with interleukin-2. Conclusions: We used a MS-qPCR method that detects Tregs but not other activated T lymphocytes; however, none of the Treg-depleting strategies that we tested led, in the majority of patients, to a conservative 50% reduction in blood Tregs. Clin Cancer Res; 17(4); 1–8. ©2010 AACR.

Comparison of stable human Treg and Th clones by transcriptional profiling

From cancerous and non‐cancerous patients, we derived stable clones of CD4+ Treg, defined as clones that expressed high CD25 at rest, were anergic in vitro, and suppressed the proliferation of co‐cultured CD4+ cells. A conserved region of FOXP3 intron 1 was demethylated in all Treg clones, whereas it was methylated in non‐regulatory Th and CTL clones. In our panel of human clones, this stable epigenetic mark correlated better with suppressive activity than did FOXP3 mRNA or protein expression. We used expression microarrays to compare Treg and Th clones after activation, which is required for suppressive function. The transcriptional profile that is specific of activated Treg clones includes a TGF‐β signature. Both activated Treg and Th clones produced the latent form of TGF‐β. However, SMAD2 phosphorylation was observed after activation in the Treg but not in the Th clones, indicating that only activated Treg clones produced the bioactive form of TGF‐β. A TGF‐β signature was also displayed by a Th clone “suppressed” by a Treg clone. In conclusion, the hallmark of our panel of activated human Treg clones is to produce bioactive TGF‐β which has autocrine actions on Tregs and can have paracrine actions on other T cells.

Arsenic Trioxide Treatment Decreases the Oxygen Consumption Rate of Tumor Cells and Radiosensitizes Solid Tumors

Arsenic trioxide (As(2)O(3)) is an effective therapeutic against acute promyelocytic leukemia and certain solid tumors. Because As(2)O(3) inhibits mitochondrial respiration in leukemia cells, we hypothesized that As(2)O(3) might enhance the radiosensitivity of solid tumors by increasing tumor oxygenation [partial pressure of oxygen (pO(2))] via a decrease in oxygen consumption. Two murine models of radioresistant hypoxic cancer were used to study the effects of As(2)O(3). We measured pO(2) and the oxygen consumption rate in vivo by electron paramagnetic resonance oximetry and (19)fluorine-MRI relaxometry. Tumor perfusion was assessed by Patent blue staining. In both models, As(2)O(3) inhibited mitochondrial respiration, leading to a rapid increase in pO(2). The decrease in oxygen consumption could be explained by an observed decrease in glutathione in As(2)O(3)-treated cells, as this could increase intracellular reactive oxygen species that can disrupt mitochondrial membrane potential. When tumors were irradiated during periods of As(2)O(3)-induced augmented oxygenation, radiosensitivity increased by 2.2-fold compared with control mice. Notably, this effect was abolished when temporarily clamped tumors were irradiated. Together, our findings show that As(2)O(3) acutely increases oxygen consumption and radiosensitizes tumors, providing a new rationale for clinical investigations of As(2)O(3) in irradiation protocols to treat solid tumors.

Monoclonal antibodies against GARP/TGF-β1 complexes inhibit the immunosuppressive activity of human regulatory T cells in vivo

Monoclonal antibodies that inhibit human Treg function in vivo may be used therapeutically in cancer or infections. Immunotherapy according to GARP Regulatory T cells (Tregs) play a critical role in preventing autoimmunity but can be co-opted by cancer cells to block immune surveillance of tumors. Cuende et al. report that a membrane protein, GARP, which binds transforming growth factor–β1 (TGF-β1) on the cell surface of Tregs, is involved in Treg-mediated inhibition of immune responses. What’s more, the authors develop anti-GARP monoclonal antibodies that block TGF-β1 production by Tregs and inhibit the activity of these cells in a xenogeneic mouse model of graft-versus-host disease. Thus, blocking GARP, either alone or in combination with other checkpoint inhibitors, could add to our arsenal for cancer immunotherapy. Regulatory T cells (Tregs) are essential to prevent autoimmunity, but excessive Treg function contributes to cancer progression by inhibiting antitumor immune responses. Tregs exert contact-dependent inhibition of immune cells through the production of active transforming growth factor–β1 (TGF-β1). On the Treg cell surface, TGF-β1 is in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. We demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-β1 by human Tregs. These antibodies recognize a conformational epitope that requires amino acids GARP137–139 within GARP/TGF-β1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-β1 production by Tregs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human Tregs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer via a mechanism of action distinct from that of currently available immunomodulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy.

Role of glycolysis inhibition and poly(ADP-ribose) polymerase activation in necrotic-like cell death caused by ascorbate/menadione-induced oxidative stress in K562 human chronic myelogenous leukemic cells.

Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K3) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD+ is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP‐ribose) polymerase (PARP) activation, as shown by the high amount of poly‐ADP‐ribosylated proteins. The addition of N‐acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells.

GARP Is Regulated by miRNAs and Controls Latent TGF-β1 Production by Human Regulatory T Cells

GARP is a transmembrane protein present on stimulated human regulatory T lymphocytes (Tregs), but not on other T lymphocytes (Th cells). It presents the latent form of TGF-β1 on the Treg surface. We report here that GARP favors the cleavage of the pro-TGF-β1 precursor and increases the amount of secreted latent TGF-β1. Stimulated Tregs, which naturally express GARP, and Th cells transfected with GARP secrete a previously unknown form of latent TGF-β1 that is disulfide-linked to GARP. These GARP/TGF-β1 complexes are possibly shed from the T cell surface. Secretion of GARP/TGF-β1 complexes was not observed with transfected 293 cells and may thus be restricted to the T cell lineage. We conclude that in stimulated human Tregs, GARP not only displays latent TGF-β1 at the cell surface, but also increases its secretion by forming soluble disulfide-linked complexes. Moreover, we identified six microRNAs (miRNAs) that are expressed at lower levels in Treg than in Th clones and that target a short region of the GARP 3’ UTR. In transfected Th cells, the presence of this region decreased GARP levels, cleavage of pro-TGF-β1, and secretion of latent TGF-β1.

Retinoic acid synergizes ATO-mediated cytotoxicity by precluding Nrf2 activity in AML cells

Background:Standard therapy for acute promyelocytic leukaemia (APL) includes retinoic acid (all-trans retinoic acid (ATRA)), which promotes differentiation of promyelocytic blasts. Although co-administration of arsenic trioxide (ATO) with ATRA has emerged as an effective option to treat APL, the molecular basis of this effect remains unclear.Methods:Four leukaemia cancer human models (HL60, THP-1, NBR4 and NBR4-R2 cells) were treated either with ATO alone or ATO plus ATRA. Cancer cell survival was monitored by trypan blue exclusion and DEVDase activity assays. Gene and protein expression changes were assessed by RT-PCR and western blot.Results:ATO induced an antioxidant response characterised by Nrf2 nuclear translocation and enhanced transcription of downstream target genes (that is, HO-1, NQO1, GCLM, ferritin). In cells exposed to ATO plus ATRA, the Nrf2 nuclear translocation was prevented and cytotoxicity was enhanced. HO-1 overexpression reversed partially the cytotoxicity by ATRA-ATO in HL60 cells. The inhibitory effects of ATRA on ATO-mediated responses were not observed in either the ATRA-resistant NB4-R2 cells or in NB4 cells pre-incubated with the RARα antagonist Ro-41-52-53.Conclusions:The augmented cytotoxicity observed in leukaemia cells following combined ATO-ATRA treatment is likely due to inhibition of Nrf2 activity, thus explaining the efficacy of combined ATO-ATRA treatment in the APL therapy.

Blocking immunosuppression by human Tregs in vivo with antibodies targeting integrin αVβ8.

Significance Immunosuppression by regulatory T cells (Tregs) is essential for the maintenance of self-tolerance, but it is detrimental in cancer because Tregs inhibit antitumor immunity. Development of therapeutic tools to block Tregs in patients with cancer requires a precise understanding of how human Tregs suppress immune responses. We recently identified an important mechanism implicating release of the active form of TGF-β1, a potently immunosuppressive cytokine, from GARP/latent TGF-β1 complexes on the surface of human Tregs. Here we unravel the molecular process leading to this release. We identify integrin αVβ8 as indispensable for TGF-β1 activation from GARP/latent TGF-β1 complexes. We show that anti-β8 monoclonals block immunosuppression by human Tregs in vivo and could thus serve in cancer immunotherapy. Human regulatory T cells (Tregs) suppress other T cells by converting the latent, inactive form of TGF-β1 into active TGF-β1. In Tregs, TGF-β1 activation requires GARP, a transmembrane protein that binds and presents latent TGF-β1 on the surface of Tregs stimulated through their T cell receptor. However, GARP is not sufficient because transduction of GARP in non-Treg T cells does not induce active TGF-β1 production. RGD-binding integrins were shown to activate TGF-β1 in several non-T cell types. Here we show that αVβ8 dimers are present on stimulated human Tregs but not in other T cells, and that antibodies against αV or β8 subunits block TGF-β1 activation in vitro. We also show that αV and β8 interact with GARP/latent TGF-β1 complexes in human Tregs. Finally, a blocking antibody against β8 inhibited immunosuppression by human Tregs in a model of xenogeneic graft-vs.-host disease induced by the transfer of human T cells in immunodeficient mice. These results show that TGF-β1 activation on the surface of human Tregs implies an interaction between the integrin αVβ8 and GARP/latent TGF-β1 complexes. Immunosuppression by human Tregs can be inhibited by antibodies against GARP or against the integrin β8 subunit. Such antibodies may prove beneficial against cancer or chronic infections.