GITR pathway activation abrogates tumor immune suppression through loss of regulatory T cell lineage stability.
ABSTRACT: Ligation of GITR (glucocorticoid-induced tumor necrosis factor (TNF) receptor-related gene, or TNFRSF18) by agonist antibody has recently entered into early phase clinical trials for the treatment of advanced malignancies. Although the ability of GITR modulation to induce tumor regression is well-documented in preclinical studies, the underlying mechanisms of action, particularly its effects on CD4(+)foxp3(+) regulatory T cells (Treg), have not been fully elucidated. We have previously demonstrated that GITR ligation in vivo by agonist antibody DTA-1 causes a >50% reduction of intra-tumor Treg with down modulation of Foxp3 expression. Here we show that the loss of Foxp3 is tumor-dependent. Adoptively-transferred Foxp3(+)Treg from tumor-bearing animals lose Foxp3 expression in the host when treated with DTA-1, whereas Treg from naïve mice maintain Foxp3 expression. GITR ligation also alters the expression of various transcription factors and cytokines important for Treg function. Complete Foxp3 loss in intra-tumor Treg correlates with a dramatic decrease in Helios expression and is associated with the upregulation of transcription factors T-Bet and Eomes. Changes in Helios correspond with a reduction in IL-10 and an increase in IFN? expression in DTA-1-treated Treg. Together, these data show that GITR agonist antibody alters Treg lineage stability inducing an inflammatory effector T cell phenotype. The resultant loss of lineage stability causes Treg to lose their intra-tumor immune suppressive function, making the tumor susceptible to killing by tumor-specific effector CD8(+) T cells.
Project description:In vivo GITR ligation has previously been shown to augment T-cell-mediated anti-tumor immunity, yet the underlying mechanisms of this activity, particularly its in vivo effects on CD4+ foxp3+ regulatory T cells (Tregs), have not been fully elucidated. In order to translate this immunotherapeutic approach to the clinic it is important gain better understanding of its mechanism(s) of action. Utilizing the agonist anti-GITR monoclonal antibody DTA-1, we found that in vivo GITR ligation modulates regulatory T cells (Tregs) directly during induction of melanoma tumor immunity. As a monotherapy, DTA-1 induced regression of small established B16 melanoma tumors. Although DTA-1 did not alter systemic Treg frequencies nor abrogate the intrinsic suppressive activity of Tregs within the tumor-draining lymph node, intra-tumor Treg accumulation was significantly impaired. This resulted in a greater Teff:Treg ratio and enhanced tumor-specific CD8+ T-cell activity. The decreased intra-tumor Treg accumulation was due both to impaired infiltration, coupled with DTA-1-induced loss of foxp3 expression in intra-tumor Tregs. Histological analysis of B16 tumors grown in Foxp3-GFP mice showed that the majority of GFP+ cells had lost Foxp3 expression. These "unstable" Tregs were absent in IgG-treated tumors and in DTA-1 treated TDLN, demonstrating a tumor-specific effect. Impairment of Treg infiltration was lost if Tregs were GITR(-/-), and the protective effects of DTA-1 were reduced in reconstituted RAG1(-/-) mice if either the Treg or Teff subset were GITR-negative and absent if both were negative. Our results demonstrate that DTA-1 modulates both Teffs and Tregs during effective tumor treatment. The data suggest that DTA-1 prevents intra-tumor Treg accumulation by altering their stability, and as a result of the loss of foxp3 expression, may modify their intra-tumor suppressive capacity. These findings provide further support for the continued development of agonist anti-GITR mAbs as an immunotherapeutic strategy for cancer.
Project description:Helios, an Ikaros family transcription factor, is preferentially expressed at the mRNA and protein level in regulatory T cells. Helios expression previously appeared to be restricted to thymic-derived Treg. Consistent with recent data, we show here that Helios expression is inducible in vitro under certain conditions. To understand phenotypic and functional differences between Helios(+) and Helios(-) Treg, we profiled cell-surface markers of FoxP3(+) Treg using unmanipulated splenocytes. We found that CD103 and GITR are expressed at high levels on a subset of Helios(+) Treg and that a Helios(+) Treg population could be significantly enriched by FACS sorting using these two markers. Quantitative real-time PCR (qPCR) analysis revealed increased TGF-? message in Helios(+) Treg, consistent with the possibility that this population possesses enhanced regulatory potential. In tumor-bearing mice, we found that Helios(+) Treg were relatively over-represented in the tumor-mass, and BrdU studies showed that, in vivo, Helios(+) Treg proliferated more than Helios(-) Treg. We hypothesized that Helios-enriched Treg might exert increased suppressive effects. Using in vitro suppression assays, we show that Treg function correlates with the absolute number of Helios(+) cells in culture. Taken together, these data show that Helios(+) Treg represent a functional subset with associated CD103 and GITR expression.
Project description:Crosslinking of glucocorticoid-induced TNF family-related receptor (GITR) with agonist antibodies restores cancer immunity by enhancing effector T cell (Teff) responses while interfering with intra-tumor regulatory T cell (Treg) stability and/or accumulation. However, how anti-GITR antibody infusion changes T cell receptor (TCR) repertoire of Teffs and Tregs engaged in anti-tumor immune response is unclear. Here, we used a transgenic mouse model (TCRmini) where T cells express naturally generated but limited TCR repertoire to trace the fate of individual T cells recognizing B16 melanoma in tumor-bearing mice, treated or non-treated with an anti-GITR monoclonal antibody DTA-1. Analysis of TCRs of CD4+ T cells from these mice revealed that the TCR repertoire of dominant tumor-reactive Teff clones remained rather similar in treated and non-treated mice. In contrast, both tumor-associated and peripheral TCR repertoire of Tregs, which were mostly distinct from that of Teffs, underwent DTA-1 mediated remodeling characterized by depletion of dominant clones and an emergence of more diverse, low-frequency clones bearing increased numbers of TCRs shared with Teffs. We conclude that the DTA-1 infusion eliminates activated Tregs engaged in the initial maintenance of tolerogenic niche for tumor growth, but over time, it favors tumor replenishment by Tregs expressing an array of TCRs able to compete with Teffs for recognition of the same tumor antigens which may prevent its complete eradication.
Project description:The transcription factor Helios is expressed in a large subset of Foxp3+ Tregs. We previously proposed that Helios is a marker of thymic derived Treg (tTreg), while Helios- Treg were induced from Foxp3- T conventional (Tconv) cells in the periphery (pTreg). To compare the two Treg subpopulations, we generated Helios-GFP reporter mice and crossed them to Foxp3-RFP reporter mice. The Helios+ Treg population expressed a more activated phenotype, had a slightly higher suppressive capacity in vitro and expressed a more highly demethylated TSDR but were equivalent in their ability to suppress inflammatory bowel disease in vivo. However, Helios+ Treg more effectively inhibited the proliferation of activated, autoreactive splenocytes from scurfy mice. When Helios+ and Helios- Treg were transferred to lymphoreplete mice, both populations maintained comparable Foxp3 expression, but Foxp3 expression was less stable in Helios- Treg when transferred to lymphopenic mice. Gene expression profiling demonstrated a large number of differentially expressed genes and showed that Helios- Treg expressed certain genes normally expressed in CD4+ Foxp3- T cells. TCR repertoire analysis indicated very little overlap between Helios+ and Helios- Treg. Thus, Helios+ and Helios- Treg subpopulations are phenotypically and functionally distinct and express dissimilar TCR repertoires.
Project description:Immunotherapy for cancer using antibodies to enhance T-cell function has been successful in recent clinical trials. Many molecules that improve activation and effector function of T cells have been investigated as potential new targets for immunomodulatory antibodies, including the tumor necrosis factor receptor superfamily members GITR and OX40. Antibodies engaging GITR or OX40 result in significant tumor protection in preclinical models. In this study, we observed that the GITR agonist antibody DTA-1 causes anaphylaxis in mice upon repeated intraperitoneal dosing. DTA-1-induced anaphylaxis requires GITR, CD4(+) T cells, B cells, and interleukin-4. Transfer of serum antibodies from DTA-1-treated mice, which contain high levels of DTA-1-specific immunoglobulin G1 (IgG1), can induce anaphylaxis in naive mice upon administration of an additional dose of DTA-1, suggesting that anaphylaxis results from anti-DTA-1 antibodies. Depletion of basophils and blockade of platelet-activating factor, the key components of the IgG1 pathway of anaphylaxis, rescues the mice from DTA-1-induced anaphylaxis. These results demonstrate a previously undescribed lethal side effect of repetitive doses of an agonist immunomodulatory antibody as well as insight into the mechanism of toxicity, which may offer a means of preventing adverse effects in future clinical trials using anti-GITR or other agonist antibodies as immunotherapies.
Project description:Two distinct subsets of CD4(+)Foxp3(+) regulatory T (Treg) cells have been described based on the differential expression of Helios, a transcription factor of the Ikaros family. Efforts to understand the origin and biological roles of these Treg populations in regulating immune responses have, however, been hindered by the lack of reliable surface markers to distinguish and isolate them for subsequent functional studies. Using a single-cell cloning strategy coupled with microarray analysis of different Treg functional subsets in humans, we identify the mRNA and protein expression of TIGIT and FCRL3 as a novel surface marker combination that distinguishes Helios(+)FOXP3(+) from Helios(-)FOXP3(+) memory cells. Unlike conventional markers that are modulated on conventional T cells upon activation, we show that the TIGIT/FCRL3 combination allows reliable identification of Helios(+) Treg cells even in highly activated conditions in vitro as well as in PBMCs of autoimmune patients. We also demonstrate that the Helios(-)FOXP3(+) Treg subpopulation harbors a larger proportion of nonsuppressive clones compared with the Helios(+)FOXP3(+) cell subset, which is highly enriched for suppressive clones. Moreover, we find that Helios(-) cells are exclusively responsible for the productions of the inflammatory cytokines IFN-?, IL-2, and IL-17 in FOXP3(+) cells ex vivo, highlighting important functional differences between Helios(+) and Helios(-) Treg cells. Thus, we identify novel surface markers for the consistent identification and isolation of Helios(+) and Helios(-) memory Treg cells in health and disease, and we further reveal functional differences between these two populations. These new markers should facilitate further elucidation of the functional roles of Helios-based Treg heterogeneity.
Project description:No curative treatment options are available for advanced hepatocellular carcinoma (HCC). Anti-PD1 antibody therapy can induce tumor regression in 20% of advanced HCC patients, demonstrating that co-inhibitory immune checkpoint blockade has therapeutic potential for this type of cancer. However, whether agonistic targeting of co-stimulatory receptors might be able to stimulate anti-tumor immunity in HCC is as yet unknown. We investigated whether agonistic targeting of the co-stimulatory receptor GITR could reinvigorate ex vivo functional responses of tumor-infiltrating lymphocytes (TIL) freshly isolated from resected tumors of HCC patients. In addition, we compared GITR expression between TIL and paired samples of leukocytes isolated from blood and tumor-free liver tissues, and studied the effects of combined GITR and PD1 targeting on ex vivo TIL responses. In all three tissue compartments, CD4+ FoxP3+ regulatory T cells (Treg) showed higher GITR- expression than effector T-cell subsets. The highest expression of GITR was found on CD4+ FoxP3hi CD45RA- activated Treg in tumors. Recombinant GITR-ligand as well as a humanized agonistic anti-GITR antibody enhanced ex vivo proliferative responses of CD4+ and CD8+ TIL to tumor antigens presented by mRNA-transfected autologous B-cell blasts, and also reinforced proliferation, IFN-γ secretion and granzyme B production in stimulations of TIL with CD3/CD28 antibodies. Combining GITR ligation with anti-PD1 antibody nivolumab further enhanced tumor antigen-specific responses of TIL in some, but not all, HCC patients, compared to either single treatment. In conclusion, agonistic targeting of GITR can enhance functionality of HCC TIL, and may therefore be a promising strategy for single or combinatorial immunotherapy in HCC.
Project description:OBJECTIVE:To assess the use of Helios in combination with FoxP3 as a superior method for identifying non-cytokine-producing human Treg cells in patients with systemic lupus erythematosus (SLE) and to determine if FoxP3+Helios+ Treg cells are maintained at normal levels in patients with clinically active disease. METHODS:Peripheral blood mononuclear cells (PBMCs) were purified from the blood of healthy volunteer donors and from 52 consecutive patients with SLE of varying clinical activity (Systemic Lupus Erythematosus Disease Activity Index scores of 0, 2-4, and ≥ 5). PBMCs (either fresh or after 4 hours of stimulation for cytokine production) were then analyzed by flow cytometry for the expression of cell surface markers (CD4, CD25, CD127, and CD45RA) and transcription factors (FoxP3 and Helios), as well as for the production of cytokines (interleukin-2 and interferon-γ). RESULTS:FoxP3+Helios+ Treg cells were found to be non-cytokine producing in both SLE patients and healthy controls. Patients with clinically active SLE had higher percentages of FoxP3+Helios+ Treg cells than did patients with inactive SLE or healthy controls. When corrected for the total CD4 cell count, the absolute numbers of FoxP3+Helios+ Treg cells in patients with moderately-to-highly active SLE were normal. CONCLUSION:Previous reports of a deficiency in Treg cell number or function in SLE are limited by their use of CD25, either alone or in combination with other markers, to identify human Treg cells. Helios in combination with FoxP3 is a superior method for detecting all non-cytokine-producing Treg cells, irrespective of CD25 or CD45RA expression. Using this method, we showed that FoxP3+Helios+ Treg cell numbers are not reduced in patients with clinically active SLE.
Project description:Glucocorticoid-induced tumor necrosis factor receptor family-related protein (TNFRSF18, CD357) is constitutively expressed on regulatory T cells (Tregs) and is inducible on effector T cells. In this report, we examine the role of glucocorticoid-induced TNF receptor family-related protein ligand (GITR-L), which is expressed by antigen presenting cells, on the development and expansion of Tregs. We found that GITR-L is dispensable for the development of naturally occurring FoxP3(+) Treg cells in the thymus. However, the expansion of Treg in GITR-L (-/-) mice is impaired after injection of the dendritic cells (DCs) inducing factor Flt3 ligand. Furthermore, DCs from the liver of GITR-L (-/-) mice were less efficient in inducing proliferation of antigen-specific Treg cells in vitro than the same cells from WT littermates. Upon gene transfer of ovalbumin into hepatocytes of GITR-L (-/-)FoxP3(GFP) reporter mice using adeno-associated virus (AAV8-OVA) the number of antigen-specific Treg in liver and spleen is reduced. The reduced number of Tregs resulted in an increase in the number of ovalbumin specific CD8(+) T effector cells. This is highly significant because proliferation of antigen-specific CD8(+) cells itself is dependent on the presence of GITR-L, as shown by in vitro experiments and by adoptive transfers into GITR-L (-/-) Rag (-/-) and Rag (-/-) mice that had received AAV8-OVA. Surprisingly, administering ?CD3 significantly reduced the numbers of FoxP3(+) Treg cells in the liver and spleen of GITR-L (-/-) but not WT mice. Because soluble Fc-GITR-L partially rescues ?CD3 induced in vitro depletion of the CD103(+) subset of FoxP3(+)CD4(+) Treg cells, we conclude that expression of GITR-L by antigen presenting cells is requisite for optimal Treg-mediated regulation of immune responses including those in response during gene transfer.
Project description:Foxp3(+) regulatory T cells (Tregs) play a pivotal role in control of autoimmunity and pathological immune responses. Helios, the Ikarus family transcription factor, binds to the Foxp3 promoter, stabilizing its expression, and is expressed in 70% of peripheral Tregs of healthy individuals. This frequency is altered during malignancy, infection, and autoimmunity, although the mechanisms that control proliferation and relative numbers of Helios(+/-) Tregs remain largely unknown. Using a T-cell-monocyte in vitro stimulation assay, we now show that proliferation of Helios(+) Tregs is inhibited by CD16(+) monocyte subset. Antibody blocking with anti-interleukin (IL)-12 reversed this inhibition, whereas addition of IL-12 suppressed Helios(+) Treg expansion, indicating that CD16(+) monocyte control of Helios(+) Treg numbers is mediated through IL-12. In contrast, proliferation of Helios(-) Tregs, which express higher levels of tumor necrosis factor receptor II (TNFRII), was suppressed by TNF-?, whereas anti-TNF-? and anti-TNFRII reversed the inhibition. CD16(-) monocyte subset was mainly responsible for TNF-?-mediated control of Helios(-) Treg expansion. Altogether, these data suggest a differential role for monocyte subsets in control of Helios(+/-) Treg development that is mediated by distinct inflammatory cytokines. These data may have important implications for understanding the pathogenesis as well as control of chronic inflammatory and autoimmune diseases.