Glucocorticoid-induced TNFR Family-related Protein Research Articles

GITRL impairs the immunosuppressive function of MDSCs via PTEN-mediated signaling pathway in experimental Sjögren syndrome.

Recent studies have revealed a role of the ligand for glucocorticoid-induced TNFR family-related protein (GITRL) in mediating functional dysregulations of myeloid-derived suppressor cells (MDSCs) in the pathogenesis of primary Sjögren syndrome (pSS), but the underlying molecular mechanism is largely unclear. In this study, we aimed to elucidate GITRL-mediated signaling pathways in MDSCs during the development of experimental SS (ESS). MDSCs were stimulated with recombinant GITRL, the activation of PTEN, AKT and STAT3 in MDSCs was analyzed by Western blot. MDSCs with different treatment were adoptively transferred to ESS mice. ELISA was used to detect the level of autoantibodies. Proportions of Th1 and Th17 cells were examined by flow cytometry. Histological evaluation of glandular destruction was analyzed by hematoxylin and eosin (HE) staining. The interaction of GITR, TRAF3 and PP2A was detected by CoIP. Upon the engagement of GITR on MDSCs, PTEN was activated and led to the inhibition of downstream AKT/STAT3 signaling pathway, therefore, resulting in the impaired immunosuppressive function of MDSCs. In ESS mice, blocking the activity of PTEN could efficiently restore the immunomodulatory effect of MDSCs and alleviate the progression of ESS. Furthermore, TRAF3 was found to bind to GITR, and then recruited PP2A to dephosphorylate PTEN, thus enhancing the activity of PTEN. This study elucidated the molecular mechanism underlying the effect of GITRL in regulating the function of MDSCs, which may provide a new therapeutic target for the treatment of pSS.

Abstract P057: Targeting Treg cells with GITR activation alleviates resistance to immunotherapy in murine glioblastomas

Abstract Glioblastoma (GBM) shows high level of resistance to currently available treatments including the standard of care and immunotherapy, representing the most fatal cancer type. Our study revealed that immune suppression by regulatory T cells (Treg) secondary to therapy with immune checkpoint blocker (anti-PD1) confers this resistance. In the GBM tumor microenvironment, Treg cells with increased suppressive phenotype were found of which frequency and anergic phenotype increase after ICB therapy, potentially contributing to the resistance. Targeting Treg has a dual-barreled effect on enhancing anti-tumor immunity: GBM is highly infiltrated with Treg while CD8 T cells are excluded. In view of Treg's intrinsic reactivity to self-antigens, mobilizing converted Treg as effector T cells can be an effective strategy to a tumor type that expresses a low level of neoantigens including GBM. Our study revealed that GITR (glucocorticoid induced TNFR family related protein) is a desirable therapeutic target based on its increased expression on GBM Tregs as compared to peripheral Tregs. Engagement of GITR with agonistic antibody led to conversion of Treg to Th1-like effector T cells, which is accompanied with downregulation of Helios and IL-10 expression that are associated with Treg suppressive function. Through combining anti-GITR with anti-PD1 therapy, tumor recognition by converted Treg and CD8 T cells could be enhanced via IFNg induced promotion of MHC class I and II expression by GBM cells, which also resulted in T cell memory formation in the long-term survivors. To obtain clinically relevant information, we established a standard of care regimen consisting of surgery, radiation, and chemotherapy for orthotopic mouse GBM. We found that the anti-GITR +anti-PD1 therapy tailored to the GBM specific TME synergizes with the standard of care, suggesting a translational potential in patients. Citation Format: Zohreh Amoozgar, Jonas Kloepper, Jun Ren, Rong En Tay, Samuel W. Kazer, Evgeny Kiner, Shanmugarajan Krishnan, Jessica M. Posada, Mitrajit Ghosh, Emilie Mamessier, Christina Wong, Gino B. Ferraro, Ana Batista, Nancy Wang, Mark Badeaux, Sylvie Roberge, Lei Xu, Peigen Huang, Alex K. Shalek, Dai Fukumura, Hye-Jung Kim, Rakesh K. Jain. Targeting Treg cells with GITR activation alleviates resistance to immunotherapy in murine glioblastomas [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P057.

Ptpn22 Modifies Regulatory T Cell Homeostasis via GITR Upregulation.

PTPN22 gene variation associates with multiple autoimmune diseases, including type 1 diabetes and rheumatoid arthritis. Loss of function studies have demonstrated that PTPN22 impinges on the homeostatic behavior of regulatory T (Treg) cells, a lineage critical for immune tolerance. The frequency and absolute number of Treg cells is increased in Ptpn22-deficient mice, but the mechanism driving this increase is unknown. In this study, we show that Ptpn22 knockdown (KD) promoted the expansion of the Treg cell compartment by upregulating the glucocorticoid-induced TNFR family-related protein (GITR) and increasing GITR signaling. Ptpn22 KD did not accelerate cell division but instead prolonged Treg cell survival, as measured by a decrease in the frequency of apoptotic Treg cells. Loss of Ptpn22 caused a concomitant increase in the proportion of CD44(hi)CD62L(lo) effector Treg cells, at the expense of CD44(lo)CD62L(hi) central Treg cells. The increase in Treg cell numbers, but not their differentiation toward an effector phenotype, was dependent on GITR signaling, because blockade of GITR ligand prevented Treg cell expansion caused by Ptpn22 KD. These findings indicate that GITR plays a key role in regulating the overall size of the Treg cell pool. Our results suggest that the size and composition of the Treg cell compartment are independently controlled and have implications for the design of immunotherapies that seek to improve Treg cell function.

Potential regulatory role of in vitro-expanded Vδ1 T cells from human peripheral blood

γδ T cells represent a relevant proportion of T lymphocytes that express T cell receptors (TCRs) encoded by the γ and δ T cell receptor genes. Whereas the most circulating γδ T cell type, Vδ2 T cells, has been described and studied intensively, the potential role of Vδ1 T cells remains largely unclear. Here, we identified that expanded peripheral blood Vδ1 T cells stimulated with anti-human TCR Vδ1 monoclonal antibody (mAb) in vitro predominantly expressed forkhead box p3 (Foxp3). In addition, these cells also expressed other regulatory T cell-related molecules, such as CD25, glucocorticoid-induced TNFR family-related protein and cytotoxic T lymphocyte-associated protein-4 (CTLA-4), and held the potent capacity for the production of transforming growth factor beta 1 (TGF-β1). These autocrine and/or paracrine TGF-β1 could bind TGF-β1 receptors on Vδ1 T cells and induce sustained Foxp3 expression. Moreover, Foxp3 expression coincided with high CD25 expression. CD25(high) Vδ1 T cells exhibited stronger suppression on CD4(+) T cell proliferation compared with TGF-β1-induced CD25(high) CD4(+) regulatory T cells. Therefore, our phenotypic and functional analyses first demonstrate the potential regulatory property of anti-human TCR Vδ1 mAb-activated Vδ1 T cells. These results will broaden our understanding about the role of peripheral blood Vδ1 T cells under physical and pathological conditions.

A Simple Detection Method for Low-Affinity Membrane Protein Interactions by Baculoviral Display

BackgroundMembrane protein interactions play an important role in cell-to-cell recognition in various biological activities such as in the immune or neural system. Nevertheless, there has remained the major obstacle of expression of the membrane proteins in their active form. Recently, we and other investigators found that functional membrane proteins express on baculovirus particles (budded virus, BV). In this study, we applied this BV display system to detect interaction between membrane proteins important for cell-to-cell interaction in immune system.Methodology/Principal FindingsWe infected Sf9 cells with recombinant baculovirus encoding the T cell membrane protein CD2 or its ligand CD58 and recovered the BV. We detected specific interaction between CD2-displaying BV and CD58-displaying BV by an enzyme-linked immunosorbent assay (ELISA). Using this system, we also detected specific interaction between two other membrane receptor-ligand pairs, CD40-CD40 ligand (CD40L), and glucocorticoid-induced TNFR family-related protein (GITR)-GITR ligand (GITRL). Furthermore, we observed specific binding of BV displaying CD58, CD40L, or GITRL to cells naturally expressing their respective receptors by flowcytometric analysis using anti-baculoviral gp64 antibody. Finally we isolated CD2 cDNA from a cDNA expression library by magnetic separation using CD58-displayng BV and anti-gp64 antibody.ConclusionsWe found the BV display system worked effectively in the detection of the interaction of membrane proteins. Since various membrane proteins and their oligomeric complexes can be displayed on BV in the native form, this BV display system should prove highly useful in the search for natural ligands or to develop screening systems for therapeutic antibodies and/or compounds.

Selective Survival of Naturally Occurring Human CD4+CD25+Foxp3+ Regulatory T Cells Cultured with Rapamycin

Naturally occurring CD4(+)CD25(+) regulatory T (nTreg) cells are essential for maintaining T cell tolerance to self Ags. We show that discrimination of human Treg from effector CD4(+)CD25(+) non-nTreg cells and their selective survival and proliferation can now be achieved using rapamycin (sirolimus). Human purified CD4(+)CD25(high) T cell subsets stimulated via TCR and CD28 or by IL-2 survived and expanded up to 40-fold in the presence of 1 nM rapamycin, while CD4(+)CD25(low) or CD4(+)CD25(-) T cells did not. The expanding pure populations of CD4(+)CD25(high) T cells were resistant to rapamycin-accelerated apoptosis. In contrast, proliferation of CD4(+)CD25(-) T cells was blocked by rapamycin, which induced their apoptosis. The rapamycin-expanded CD4(+)CD25(high) T cell populations retained a broad TCR repertoire and, like CD4(+) CD25(+) T cells freshly obtained from the peripheral circulation, constitutively expressed CD25, Foxp3, CD62L, glucocorticoid-induced TNFR family related protein, CTLA-4, and CCR-7. The rapamycin-expanded T cells suppressed proliferation and effector functions of allogeneic or autologous CD4(+) and CD8(+) T cells in vitro. They equally suppressed Ag-specific and nonspecific responses. Our studies have defined ex vivo conditions for robust expansion of pure populations of human nTreg cells with potent suppressive activity. It is expected that the availability of this otherwise rare T cell subset for further studies will help define the molecular basis of Treg-mediated suppression in humans.

Glucocorticoid‐induced TNFR family‐related protein (GITR) activation exacerbates murine asthma and collagen‐induced arthritis

Glucocorticoid-induced TNFR family-related protein (GITR) is expressed at low levels on resting T cells, B cells and macrophages but at high levels on regulatory T cells (Treg). Although GITR expression is up-regulated on CD4+ effector cells upon activation, the role of GITR in Th1 and Th2 cell development is unclear. We report here that activation of GITR signalling by anti-GITR antibody markedly enhanced the induction of both Th1 and Th2 cytokine production by naive CD4+CD25- T cells. Consistent with this observation, anti-GITR antibody significantly enhanced the expression of the key Th1 (T-bet) and Th2 (GATA3) transcription factors in vitro. Administration of anti-GITR mAb in a murine model of arthritis significantly exacerbated the severity and onset of joint inflammation with elevated production of TNF-alpha, IFN-gamma, IL-5, and collagen-specific IgG1. Administration of anti-GITR mAb also significantly exacerbated murine allergic airways inflammation with elevated production of OVA-specific IFN-gamma, IL-2, IL-4, IL-5, and IgE. Finally, we demonstrated that adoptive transfer of CD4+GITR+ T cells effectively abolished airway inflammation induced in SCID mice reconstituted with CD4+GITR- T cells. Our results therefore provide direct evidence that GITR can modulate both Th1- and Th2-mediated inflammatory diseases, and may be a potential target for therapeutic intervention.

Correction: Resistance of regulatory T cells to glucocorticoid‐induced TNFR family‐related protein (GITR) during Plasmodium yoelii infection

Correction: Resistance of regulatory T cells to glucocorticoid‐induced TNFR family‐related protein (GITR) during <i>Plasmodium yoelii</i> infection

The IL-4 Receptor α-Chain-Binding Cytokines, IL-4 and IL-13, Induce Forkhead Box P3-Expressing CD25+CD4+ Regulatory T Cells from CD25−CD4+ Precursors

The mechanisms underlying the extrathymic generation of CD25+CD4 regulatory T cells (Tregs) are largely unknown. In this study the IL-4R alpha-chain-binding cytokines, IL-4 and IL-13, were identified as inducers of CD25+ Tregs from peripheral CD25-CD4 naive T cells. IL-4-induced CD25+ Tregs phenotypically and functionally resemble naturally occurring Tregs in that they are anergic to mitogenic stimulation, inhibit the proliferation of autologous responder T cells, express high levels of the Forkhead box P3 and the surface receptors glucocorticoid-induced TNFR family-related protein and CTLA-4, and inhibit effector T cells in a contact-dependent, but cytokine-independent, manner. The IL-4-induced generation of peripheral Tregs was independent of the presence of TGF-beta or IL-10, but was dependent on Ag-specific stimulation and B7 costimulation. The significance of the IL-4Ralpha-binding cytokines in the generation of Ag-specific Tregs was emphasized in a mouse model of oral tolerance, in which neutralization of IL-4 and IL-13 in mice transgenic for the TCR specific for OVA completely inhibited the expansion of OVA-specific Tregs that can be induced in untreated mice by feeding the nominal Ag. Together, our results demonstrate that IL-4 and IL-13 play an important role in generating Forkhead box P3-expressing CD25+ Tregs extrathymically in an Ag-dependent manner and therefore provide an intriguing link between the well-established immunoregulatory capacity of Th2 cells and the powerful CD25+ Treg population. Moreover, our findings might provide the basis for the design of novel therapeutic approaches for targeted immunotherapy with Tregs to known Ags in autoimmune diseases or graft-vs-host reactions.

Adult T Cell Leukemia/Lymphoma (ATLL) Is a Malignant Counterpart of Regulatory T Cells (Treg).

Adult T cell leukemia/lymphoma (ATLL) is a lymphoproliferative disorder caused by a retrovirus, human T-lymphotropic virus type 1 (HTLV-1). ATLL is subclassified into four subtypes, smoldering, chronic, acute, and lymphoma. The acute type progresses rapidly and is usually resistant to conventional chemotherapy. In contrast, the chronic type shows an indolent clinical course and the patients survive for several years, even without chemotherapy. Irrespective of the subtypes, however, ATLL patients are in a severely immune-suppressed condition and can easily acquire opportunistic infections such as Pneumocystis Carinii pneumonitis. Suppression of cell-mediated immunity has also been reported in HTLV-1 carriers. Although ATLL cells show the activated helper/inducer T-cell phenotypes, CD4+ and CD25+, they exhibit strong immune-suppressive activity in vitro. The recent notion of CD4+ CD25+ regulatory T cells (Treg) prompted us to investigate the origin of ATLL cells from the standpoint of Treg. Forkhead/winged helix transcription factor (Foxp3) is a functional marker of Treg, which plays a central role in their generation. There are other marker molecules for Treg, including glucocorticoid-induced TNFR family-related protein (GITR) and the chemokine receptors CCR4 and CCR8.In the present study, we examined primary ATLL cells from 48 patients: 36 patients with acute type and 12 patients with chronic type. We also examined ATLL cell lines, HTLV-1-infected T-cell lines and peripheral blood mononuclear cells (PBMC) from healthy adults as control cells. We used RT-PCR for detection of Foxp3, GITR, CCR4, and CCR8 mRNA expression. Foxp3 and/or GITR mRNA were detected in over 90% of the patients, and 50% of the patients expressed both. There was no difference between subtypes. In contrast, Foxp3 and GITR mRNA were scarcely detected in the PBMC from healthy adults. Furthermore, we confirmed GITR expression at the protein level by flow cytometry. CCR4 and CCR8 mRNA were also detected in almost all ATLL samples, at significantly higher levels than in the normal PBMC. Corresponding to the results of the primary cells, ATLL cell lines and HTLV-1-infected T-cell lines also expressed GITR mRNA, although HTLV-1-negative cell lines, Jurkat and Molt4, completely lack it. Next, we examined whether GITR affects ATLL cell proliferation using a GITR- expressing IL-2-dependent ATLL cell line, KK1. We found that GITR ligand induced proliferation of KK1 cells in an IL-2-negative condition. Thus, these results indicate the Treg origin of ATLL cells and show that GITR expression is possibly involved in the development of ATLL.

CD4+CD25bright T cells in human intestinal lamina propria as regulatory cells.

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only active suppression by regulatory T cells plays an important role in the normal intestinal homeostasis, but also its dysregulation leads to the development of inflammatory bowel disease. In this study, we demonstrate that the CD4(+)CD25(bright) T cells reside in the human intestinal lamina propria (LP) and functionally retain regulatory activities. All human LP CD4(+) T cells regardless of CD25 expression constitutively expressed CTLA-4, glucocorticoid-induced TNFR family-related protein, and Foxp3 and proliferate poorly. Although LP CD4(+)CD25(-) T cells showed an activated and anergic/memory phenotype, they did not retain regulatory activity. In LP CD4(+)CD25(+) T cells, however, cells expressing CD25 at high levels (CD4(+)CD25(bright)) suppressed the proliferation and various cytokine productions of CD4(+)CD25(-) T cells. LP CD4(+)CD25(bright) T cells by themselves produced fewer amounts of IL-2, IFN-gamma, and IL-10. Interestingly, LP CD4(+)CD25(bright) T cells with regulatory T activity were significantly increased in patients with active inflammatory bowel disease. These results suggest that CD4(+)CD25(bright) T cells found in the normal and inflamed intestinal mucosa selectively inhibit the host immune response and therefore may contribute to the intestinal immune homeostasis.