Absence Of Tregs Research Articles

IL15/IL15Rα complex induces an anti-tumor immune response following radiation therapy only in the absence of Tregs and fails to induce expansion of progenitor TCF1+ CD8 T cells.

This work seeks to understand whether IL15-incorporating treatments improve response to radiotherapy and uncover mechanistic rationale for overcoming resistance to IL15 agonism using novel therapeutic combinations. Orthotopic tumor models of PDAC were used to determine response to treatment. IL15-/- and Rag1-/- mouse models were employed to determine dependence on IL15 and CTLs, respectively. Flow cytometry was used to assess immune cell frequency and activation state. Phospho-proteomic analyses were used to characterize intracellular signaling pathways. We show that the combination of radiation therapy (RT) and an IL15/IL15Ra fusion complex (denoted IL15c) fails to confer anti-tumor efficacy; however, a CD8-driven anti-tumor immune response is elicited with the concurrent administration of an aCD25 Treg-depleting antibody. Using IL15-/- and Rag1-/- mice, we demonstrate that response to RT + IL15c + aCD25 is dependent on both IL15 and CTLs. Furthermore, despite an equivalent survival benefit following treatment with RT + IL15c + aCD25 and combination RT + PD1-IL2v, a novel immunocytokine with PD-1 and IL2Rβγ binding domains, CTL immunophenotyping and phospho-proteomic analysis of intracellular metabolites showed significant upregulation of activation and functionality in CD8 T cells treated with RT + PD1-IL2v. Finally, we show the immunostimulatory response to RT + PD1-IL2v is significantly diminished with a concurrent lack of TCF+ CD8 T cell generation in the absence of functional IL15 signaling. Our results are illustrative of a mechanism wherein unimpeded effector T cell activation through IL2Rβ signaling and Treg inhibition are necessary in mediating an anti-tumor immune response.

Regulatory T cells shield muscle mitochondria from interferon-γ-mediated damage to promote the beneficial effects of exercise.

Exercise enhances physical performance and reduces the risk of many disorders such as cardiovascular disease, type 2 diabetes, dementia, and cancer. Exercise characteristically incites an inflammatory response, notably in skeletal muscles. Although some effector mechanisms have been identified, regulatory elements activated in response to exercise remain obscure. Here, we have addressed the roles of Foxp3+CD4+ regulatory T cells (Tregs) in the healthful activities of exercise via immunologic, transcriptomic, histologic, metabolic, and biochemical analyses of acute and chronic exercise models in mice. Exercise rapidly induced expansion of the muscle Treg compartment, thereby guarding against overexuberant production of interferon-γ and consequent metabolic disruptions, particularly mitochondrial aberrancies. The performance-enhancing effects of exercise training were dampened in the absence of Tregs. Thus, exercise is a natural Treg booster with therapeutic potential in disease and aging contexts.

Targeting glutamine metabolism mitigates Foxp3-deficiency mediated disease

Abstract Regulatory T (Treg) cells play a key role in enforcing peripheral immunological tolerance to self-antigens. Foxp3 has been widely described as a master regulator for Treg differentiation and maintenance of their suppressive functions. Upon Foxp3 loss of function mutations, Foxp3-deficient Tregs (ΔTreg) develop in the thymus, however, these cells present impaired suppressive capacity and an effector T cell (Teff)-like phenotype. Among the Teff-like characteristics is the shift in their metabolism towards glycolysis and enhanced mitochondrial respiration. Metabolomic analysis revealed an increase in glutamine consumption. Here, we used 6-diazo-5-oxo-norleucine (DON), a broad inhibitor of glutamine-utilizing enzymes to treat Foxp3 deficient mice (Foxp3 ΔeGFPiCre), and observed a significant decrease in Teff cell activation, and IFNγ production from both ΔTreg and Teff cells together with a significant increase in survival. This effect was enhanced when we combined DON treatment with a ΔTreg specific Rictor depletion (Foxp3 ΔeGFPiCreRictor Δ/Δ). Given that glutamine is a key amino acid for T cell activation, we evaluated if this was a pan-T cell effect by inducing Treg depletion in Foxp3 DTRmice with concomitant DON treatment. Surprisingly, in the absence of Tregs, DON was not able to prevent autoimmune disease. These results suggest that DON mitigates Foxp3-deficiency mediated disease in a ΔTreg dependent manner, by improving their suppressive capacities. We are currently performing metabolomic studies to better understand the role of glutamine usage in ΔTreg, more specifically if glutamine is directed to glutaminolysis, nucleotide or glutathione synthesis and how this contributes to Treg cell loss of function. NIH grant 1R01AI153174

Abstract 6414: Concomitant anti-tumor immunity and checkpoint blockade responses are restored by depletion of CD62L expressing cells

Abstract Studies have shown the importance of CD62L in leukocyte trafficking. However, the significance of CD62L-expressing cells in concomitant anti-tumor immunity has not been investigated. Using a model of post-surgical tumor immunity, we show that in vivo depletion of CD62L-expressing cells before or immediately after surgical tumor resection enabled the development of anti-tumor concomitant responses against poorly immunogenic B16 (melanoma) or MTE-Ras (head & neck cancer) tumor cells. Anti-CD62L mAb administration depleted the cellular immune components that express CD62L, including NK, NKT, Mo, Ma, B, and T cells. Within the T cell compartment, naïve T cells (CD44lo CD62Lhi), effector (CD44hi CD62Llo), memory (CD44hi CD62Lhi) and regulatory T cells (Treg, CD4 CD25 Foxp3) were also depleted, while effector CD62L-negative T cells were spared in the TDLN. Tumor immunity did not occur in the absence of a primary tumor (sham surgery group) or after CD8 depletion. Whereas anti-CTLA-4 or anti-PD-1 mAbs failed as monotherapies, their administration during CD62L depletion restored their anti-tumor properties. The unified global gene expression analysis of the TDLN shows the significant enrichment of biological processes involved in the CD8 T cell effector phase, IL-2/IL-15, TNF-α, IFN-γ, IFN-α, IL-6, responses, mitotic cell division, glycolysis, and acetylation. Also, genes related to Treg responses were downregulated. These data point to a process in which depletion of CD62L cells in tumor-bearing hosts transforms the TDLN into a highly active immunological site in which tumor-reactive effector CD8 T cells (CD62L-negative) actively divide in the absence of Treg, accompanied by a cytokine response, glucose consumption, and acetylation of transcriptional factors and histones. These results identify an unrecognized role of CD62L-expressing cells in regulating concomitant anti-tumor immunity and restoring anti-tumor responses by checkpoint blockade inhibitors. Our study provides a new framework that could help understand how anti-tumor responses are limited. Citation Format: Brianna Burke, Lourdes Plaza-Rojas, Cynthia Perez, Elena Kostenko, Anna Austin, Justin Boucher, Kushal Prajapati, Michael Delos Reyes, Christine Chung, Jose-Alejandro Guevara-Patino. Concomitant anti-tumor immunity and checkpoint blockade responses are restored by depletion of CD62L expressing cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6414.

Regulatory T cell coordination of the mucosal NK cell response during viral infection.

Abstract In many infections the mucosal barrier is the site of pathogen exposure, yet the regulation of mucosal immunity is not well understood. Herpes simplex virus-2 (HSV-2) is one of the most prevalent mucosal infections, making this disease an ideal model to study mucosal anti-viral immune response. My goal is to define how regulatory T cells (Tregs) shape anti-viral immune responses through modulation of natural killer (NK) cells during mucosal virus infection. To accomplish my goal, I utilized a mouse model of intravaginal HSV-2 infection. NK cell phenotypes were examined using high parameter flow cytometry. To define how transient Treg depletion during HSV-2 infection impacts NK cells, I utilized Foxp3DTR mice. Treg depletion led to increased maturation of NK cells in the vaginal tract (VT). KLRG1 is associated with NK cell maturation and is a marker of terminally mature NK cell subsets. I found a significant increase in the frequency and total number of KLRG1+ NK cells in the VT two days after HSV-2 infection in the absence of Tregs. I further analyzed NK cell maturation using the expression of the two surface markers CD11b and CD27 and found a significant increase in CD11b+CD27+ NK cells after Treg depletion. This subset of NK cells is known to be significant producers of pro-inflammatory cytokines. The cytokine IL-15 is necessary for NK cell development and maturation. I found that in the VT, CD11b+Ly6C+ monocyte-derived DCs display increased IL-15 transpresentation after Treg depletion. My data suggests that Tregs have a modulatory effect on NK cell development in the VT as Treg depletion leads to altered NK cell maturation and development. This demonstrates the role of Tregs in mediating an effective immune response while limiting immune pathology. Supported by grants from NIH (R01 AI141435-01, T32 AI 7509-21)

Research Progress on the Role of Regulatory T Cell in Tumor Microenvironment in the Treatment of Breast Cancer.

The tumor microenvironment (TME) is a complex ecosystem comprised of cancer cells, stromal cells, and immune cells. Analysis of the composition of TME is essential to assess the prognosis of patients with breast cancer (BC) and the efficacy of different regimes. Treg plays a crucial role in the microenvironment of breast cancer subtypes, and its function contributes to the development and progression of BC by suppressing anti-tumor immunity directly or indirectly through multiple mechanisms. In addition, conventional treatments, such as anthracycline-based neoadjuvant chemotherapy, and neo-therapies, such as immune-checkpoint blockades, have a significant impact on the absence of Tregs in BC TME, thus gaining additional anti-tumor effect to some extent. Strikingly, Treg in BC TME revealed the predicted efficacy of some therapeutic strategies. All these results suggest that we can manipulate the abundance of Treg to achieve the ultimate effect of both conventional and novel treatments. In this review, we discuss new insights into the characteristics of Treg in BC TME, the impact of different regiments on Treg, and the possibilities of Treg as a predictive marker of efficacy for certain treatments.

Regulatory T cells reduce endothelial neutral sphingomyelinase 2 to prevent T-cell migration into tumors.

Endothelial cells are key regulators of transendothelial migration and their secretion of chemokines and expression of adhesion molecules facilitates lymphocyte entry into tissues. Previously, we demonstrated that Tregs can reduce transendothelial migration of T cells into tumors by decreasing endothelial CXCL10 secretion, but the mechanism by which this occurs is still not known. In this study, we aimed to define how Tregs decrease transendothelial migration into tumors. mRNA sequencing of intestinal tumor endothelial cells from Treg depleted mice identified neutral sphingomyelinase 2 (nSMase2) as a gene downregulated in the presence of Tregs. nSMase2 is expressed in human umbilical vein endothelial cells (HUVECs) and was decreased after coculture with Tregs. Furthermore, blocking of nSMase2 activity in vitro decreased VCAM1, CX3CL1, and CXCL10 expression in HUVECs, mirroring the same decrease found in Treg cocultures. In the APCmin/+ mouse model of intestinal cancer, nSMase2 is lower in tumor endothelial cells than in unaffected small intestine and chronic treatment with a nSMase2 inhibitor suppressed the increased migration that is otherwise seen in the absence of Tregs. We conclude that nSMase2 is an important mediator in endothelial cells supporting transendothelial migration, which may be targeted by Tregs to reduce T-cell migration into tumors.

Increased Regulatory T Cells and Decreased Myeloid-Derived Suppressor Cells Induced by High CCL17 Levels May Account for Normal Incidence of Cancers among Patients with Atopic Dermatitis.

The incidence of cancers in atopic dermatitis (AD) is not increased, although the Th2-dominant environment is known to downregulate tumor immunity. To gain mechanistic insights regarding tumor immunity in AD, we utilized CCL17 transgenic (TG) mice overexpressing CCL17, which is a key chemokine in AD. Tumor formation and lung metastasis were accelerated in CCL17 TG mice when melanoma cells were injected subcutaneously or intravenously. Flow cytometric analysis showed increases in regulatory T cells (Tregs) in lymph nodes in CCL17 TG mice with high mRNA levels of IL-10 and Foxp3 in tumors, suggesting that Tregs attenuated tumor immunity. The frequency of myeloid-derived suppressor cells (MDSCs), however, was significantly decreased in tumors of CCL17 TG mice, suggesting that decreased MDSCs might promote tumor immunity. Expression of CXCL17, a chemoattractant of MDSCs, was decreased in tumors of CCL17 TG mice. Depletion of Tregs by the anti-CD25 antibody markedly reduced tumor volumes in CCL17 TG mice, suggesting that tumor immunity was accelerated by the decrease in MDSCs in the absence of Tregs. Thus, CCL17 attenuates tumor immunity by increasing Tregs and Th2 cells, while it decreases MDSCs through reductions in CXCL17, which may work as a “safety-net” to reduce the risk of malignant tumors in the Th2-dominant environment.

Elucidating different pattern of immunoregulation in BALB/c and C57BL/6 mice and their F1 progeny

Helminths are large multicellular parasites that infect one quarter of the human population. To prolong their survival, helminths suppress the immune responses of their hosts. Strongyloides ratti delays its expulsion from the gut by induction of regulatory circuits in a mouse strain-specific manner: depletion of Foxp3+ regulatory T cells (Treg) improves the anti-S. ratti immunity in BALB/c but not in C57BL/6 mice. In the current study we compare the hierarchy of immunoregulatory pathways in BALB/c, C57BL/6 mice and their F1 progeny (BALB/c × C57BL/6). Using multicolor flow cytometry, we show that S. ratti induces a distinct pattern of inhibitory checkpoint receptors by Foxp3+ Treg and Foxp3− T cells. Intensity of expression was highest in C57BL/6 and lowest in BALB/c mice, while the F1 cross had an intermediate phenotype or resembled BALB/c mice. Treg subsets expanded during infection in all three mouse strains. Similar to BALB/c mice, depletion of Treg reduced intestinal parasite burden and increased mucosal mast cell activation in S. ratti-infected F1 mice. Our data indicate that Treg dominate the regulation of immune responses in BALB/c and F1 mice, while multiple regulatory layers exist in C57BL/6 mice that may compensate for the absence of Treg.

Protective low-avidity anti-tumour CD8+ T cells are selectively attenuated by regulatory T cells.

SummaryObjectivesRegulatory T cells (Treg) play a major role in the suppression of protective anti-tumour T cell responses. In the CT26 BALB/c murine model of colorectal carcinoma, Tregs differentially suppress responses to two characterised CD8+ T epitopes, AH1 and GSW11, which results in an absence of detectable IFN-γ-producing GSW11-specific T cells in the spleen and lymph nodes of tumour challenged mice. Activation of GSW11-specific T cells correlates with protection against tumour progression. We wanted to examine the presence of non-functional GSW11-specific T cells in Treg replete and depleted mice, assess their phenotype and their affinity compared to AH1-specific T cells.MethodsWe used peptide-specific tetramers to identify tumour-specific CD8+ T cells and assessed the cell surface expression of markers associated with exhaustion (PD-1, Tim3 and Lag-3) and their function by IFN-g production using flow cytometry. We also assessed the T cell receptor (TcR) clonality of tumour-specific T cells. Tetramer competition assays were performed to determine the relative affinity of identified TcR.ResultsHere, we show that GSW11-specific T cells are in fact induced in Treg-replete, CT26-bearing mice, where they make up the majority of tumour-infiltrating CD8+ lymphocytes, but exhibit an ‘exhausted’ phenotype. This dysfunctional phenotype is induced early in the anti-tumour response in tumours. Depletion of Tregs prior to tumour challenge correlates with an altered T cell receptor (TcR) repertoire. Moreover, the avidity of GSW11-specific TcRs that expanded in the absence of Tregs was significantly lower compared with TcRs of CD8+populations that were diminished in protective anti-tumour responses.ConclusionOur results indicate that Tregs suppress the induction of protective anti-tumour T cell responses and may signify that low-avidity T cells play an important role in this protection.

Abstract 1031: Liver metastasis mediated control of distant tumor-specific immunity and response to checkpoint immunotherapy

Abstract Background: PD-1 blockade achieves objective responses in 30-55% of melanoma patients, but there remain 45-70% of patients where it is ineffective, either as a single agent or in combination with anti-CTLA-4. We lack salvage strategies for this population and a mechanistic understanding of checkpoint inhibitor refractory melanoma. Previously, we reported that patients with liver metastasis have worse response rate and survival than those without liver metastasis despite treatment with checkpoint immunotherapy and cutaneous biopsy samples from patients with liver metastasis have significantly less activated tumor-infiltrating lymphocytes (TILs) than those with metastasis elsewhere. However, the mechanism of how liver metastasis influences the systemic antitumor immune response in this context is unclear. We hypothesize that tumor antigen-specific tolerance can be induced when metastatic cells invade the liver, resulting in reduced systemic antitumor immunity and immunotherapy resistance. Results: In this study, we developed a syngeneic two-site tumor animal model in which tumor cells are injected simultaneously into a subcutaneous (SQ) site and into the liver or other organ sites of wild-type C57BL/6 mice to investigate the unique effects of the intrahepatic tumor on antitumor immunity at a distant site. We found that the presence of tumor or tumor antigen within the liver, as opposed to other organs, reduced systemic antitumor immunity and effector T cell function that is evident at the distant SQ site. Tetramer-based flow cytometry and single cell-RNAseq analysis were done on the TILs within the SQ site and the results suggest that tumor-specific CD8 T cells were preferentially dysfunctional but not sequestered by the liver or clonally deleted, with reduced expression of activation markers and effector cytokines (ICOS, CTLA-4, IFNγ, and TNFα). Collectively, the data suggest a liver and antigen-specific immunoregulatory process mediated by the coordinated activation of Foxp3+ regulatory T cells (with increased expression of CTLA-4 and ICOS) and significant changes in the non-T cell immune composition at the distant SQ site in the liver-tumor bearing mice. Genetic studies using a mouse strain expressing the diphtheria toxin receptor under the control of Foxp3 (Foxp3-DTR) with or without the systemic depletion of Tregs revealed that in the absence of Tregs, there was no difference in the distal immunity against the SQ tumor between mice with and without experimental liver metastasis. Finally, we found that this form of systemic antitumor effector T cell dysfunction was not reversed by anti-PD-1 monotherapy but rather by combination therapy with the deletion (anti-CTLA-4) or destabilization (EZH2 inhibitor) of Tregs, suggesting a distinct regulatory mechanism contributing to anti-PD-1 resistance in the setting of liver metastasis that could be overcome to achieve complete tumor regression at both sites. Conclusion: Our results reveal a novel mechanism of checkpoint inhibitor resistance and suggest a strategy to improve the outcome of immunotherapy treatments for stage IV cancer patients with liver metastasis. Citation Format: James C. Lee, Sadaf Mehdizadeh, Arabella Young, Jennifer Bridge, Ilgiz A. Mufazalov, Adil Daud, Jeffrey A. Bluestone. Liver metastasis mediated control of distant tumor-specific immunity and response to checkpoint immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1031.

Regulatory T Cells Modulate CD4 Proliferation after Severe Trauma via IL-10.

Objective: Severely injured patients frequently develop an immunological imbalance following the traumatic insult, which might result in infectious complications evoked by a persisting immunosuppression. Regulatory T cells (Tregs) maintain the immune homeostasis by suppressing proinflammatory responses, however, their functionality after trauma is unclear. Here, we characterized the role of Tregs in regulating the proliferation of CD4+ lymphocytes in traumatized patients (TP). Methods: Peripheral blood was obtained daily from 29 severely injured TP (Injury Severity Score, ISS ≥16) for ten days following admission to the emergency department (ED). Ten healthy volunteers (HV) served as controls. The frequency and activity of Tregs were assessed by flow cytometry. Proliferation of CD4+ cells was analyzed either in presence or absence of Tregs, or after blocking of either IL-10 or IL-10R1. Results: The frequencies of CD4+CD25high and CD4+CD25+CD127− Tregs were significantly decreased immediately upon admission of TP to the ED and during the following 10 post-injury days. Compared with HV CD4+ T cell proliferation in TP increased significantly upon their admission and on the following days. As expected, CD4+CD25+CD127− Tregs reduced the proliferation of CD4+ cells in HV, nevertheless, CD4+ proliferation in TP was increased by Tregs. Neutralization of IL-10 as well as blocking the IL-10R1 increased further CD4+ T cell proliferation in Tregs-depleted cultures, thereby confirming an IL-10-mediated mechanism of IL-10-regulated CD4+ T cell proliferation. Neutralization of IL-10 in TP decreased CD4+ T cell proliferation in Tregs-depleted cultures, whereas blocking of the IL-10R1 receptor had no significant effects. Conclusions: The frequency of Tregs in the CD4+ T lymphocyte population is reduced after trauma; however, their inductiveness is increased. The mechanisms of deregulated influence of Tregs on CD4+ T cell proliferation are mediated via IL-10 but not via the IL-10R1.

Adoptive Immunotherapy with Regulatory and Conventional T Cells in Haploidentical Transplantation Primes Dendritic Cells to Promote T Cell Alloreactivity in the Bone Marrow and Tolerance in the Periphery

In high-risk acute leukemia patients undergoing HLA haploidentical T cell-depleted tranplantation, we demonstrated that adoptive immunotherapy with donor T regulatory cells (Tregs; 2x106/kg) co-infused with conventional T cells (Tcon; 1x106/kg ) provided significant protection from acute graft-versus-host disease (aGvHD) and was associated with an almost complete control of leukemia relapse (graft versus leukemia effect, GvL) (Di Ianni et al., Blood 2011; Martelli et al., Blood 2014; Ruggeri et al., ASH 2018). In the present study we investigated whether Tregs interact with bone marrow (BM) and peripheral blood (PB) dendritic cells (DCs) and whether such interaction is responsible for GvHD protection and GvL effect. Twenty six patients (median age 54 ; 20 AML; 4 ALL; 2 MDS) transplanted between July 2016 and April 2019 were evaluated up to one year after the transplant. BM and PB DCs (using CD123 for plasmocitoid DC-pDC; CD11c for myeloid DC-mDC; CD80/CD86 for costimulatory molecules) and T cells (CD3/CD4/CD8; CD4/CD25/CD127; CD28/PD-1/TIM3) were analysed by flow-cytometry. DCs were also sorted and analysed by RT-PCR for a panel of genes involved in activation (IL-6; TNF-a; IL-12; CCR7; NOTCH ligands) vs tolerigenic (TGF-beta; PD-1/PDL1; IDO; IL-10; ICOS) pathways. To study the effects of DCs on T cell proliferation, pre-activated (with GM-CSF at 50 ng/ml, IL-4 at 800 U/ml and TNF-a at 50 ng/ml for 18 hrs) BM and PB CD1c+ DCs were co-cultured for 96 hrs with autologous CFSE labelled BM and PB CD3+ cells at a DC:CD3 ratio of 1:10. mDC numbers were significantly higher in BM than PB during the first 6 months after transplant. BM-derived mDCs expressed higher levels of the co-stimulatory receptor CD86. No differences emerged in pDCs. RT-PCR showed an activation signature in BM-DCs (significantly higher IL-6 level) and a tolerigenic signature in PB-DCs (significantly higher TGF-beta and PDL-1 levels). BM-derived CD8+ T cells displayed a higher expression of the co-stimulatory receptor CD28 than PB-derived CD8+ T cells (30.3±18.8 vs 9.2±4.9; p<0.05 ). In contrast, the expression of the immune checkpoint inhibitor PD-1 was significantly higher in both PB-derived CD4 (69%±29 vs 24±11) and CD8 (65±25 vs 4±3; p<0.05) T cells than BM-derived T lymphocytes. T cells from both BM and PB did not express the T cell exhaustion marker TIM-3. CD3/CFSE+-DCs co-cultures showed a T cell proliferation rate that was significantly higher in BM than in PB (25±7.2 vs 6.7±8.7; p<0.05). These data show that haploidentical transplantation with Treg/Tcon immunotherapy promotes the reconstitution of DCs with an activating signature in the BM and a tolerigenic signature in the PB. Human peripheral blood Tregs that are used for adoptive immunotherapy are largely CD45RO+ and express low level of CxCR4 bone marrow homing receptor. When infused in immunodeficient mice they migrate to the periphery (spleen, gut, liver) but are unable to home to the bone marrow (Ruggeri et al., ASH 2018). In conclusion, Tregs/DC interaction induce tolerance in the periphery (and may protect from GvHD). In the BM, in the absence of Tregs, DCs activate alloreactive Tcon and may favour killing of the leukemic targets. Therefore, Tregs/DC interactions may contribute to the separation between GvL effect and GvHD in the Treg based haploidentical transplantation. Disclosures No relevant conflicts of interest to declare.

Glucocorticoids control allergic inflammation via Foxp3+regulatory T cells

Abstract Asthma is a chronic inflammatory disease in the lung characterized by airway inflammation and airway hyper-responsiveness (AHR). Synthetic glucocorticoids such as Dexamethasone (Dex) are a mainstay treatment option for the clinical management of asthma. However, the target cells that mediate their therapeutic effects and the underlying mechanisms remain unclear. Utilizing a cockroach antigen-induced allergic inflammation model in mice, we sought to test the roles of Foxp3+ regulatory T cells (Tregs) during Dex-mediated regulation of allergic inflammation. Administration of Dex significantly reduced the development of airway inflammation and AHR. Accumulation of eosinophils and effector CD4 T cells expressing Th2 cytokines in the lung were dramatically reduced following Dex administration. To our surprise, the Dex-induced reduction was completely abolished in the absence of Tregs. Inflammatory cell accumulation, cytokine production, and AHR development remained unchanged in this condition, suggesting that Tregs may be an important target cell of Dex in vivo. Indeed, adoptive transfer of in vitro generated Tregs into Treg-depleted mice fully restored Dex effects. We performed an RNA-sequencing analysis comparing gene expression profiles in lung infiltrating effector CD4 T cells from mice treated with control, Dex, and Dex with Treg depletion. Genes upregulated or downregulated by Dex treatment remained unchanged by Treg depletion. Taken together, our results uncover a novel function of Tregs during Dex-mediated treatment of allergic inflammation.

052 Regulatory T cell-mediated, OX40-dependent peripheral tolerance to autoantigen, desmoglein 3

Peripheral immunological tolerance is critical to prevent autoimmunity but detail of the mechanism has not been exactly understood. The purpose of this study is to unveil the mechanisms of peripheral tolerance to a pemphigus autoantigen, desmoglein 3 (Dsg3). To achieve this, Dsg3-specific TCR transgenic (Dsg3H1) CD4+ T cell was used. This T cell was deleted by central tolerance, but developed into the periphery in the absence of Dsg3 as in Dsg3-/- mice. When Dsg3H1 T cells were adoptively transferred, the peripheral Dsg3H1 T cells disappeared in skin-draining LNs in WT mice by Day14, but survived in Dsg3-/- mice, indicating antigen-specific elimination. To identify crucial cell subsets and molecules involved in this process, various gene-engineered or antibody-treated mice were used as recipients. The elimination was affected in MHC II-/- (n=6), but not in Aire-/- (n=3) or anti-PD-1 treated mice (n=3), indicating an essential role of MHC II+ antigen presenting cells, but not of Aire or PD-1. Moreover, under in vivo ablation of Foxp3+ Treg in DEREG mice, Dsg3H1 T cells were not eliminated, but expanded and induced dermatitis (n=12). Furthermore, Dsg3H1 T cells were eliminated even in three different Foxp3 mutant knock-in mice which have functionally-affected Treg to various degrees. Therefore, Treg-specific molecules that remain functional in those mutants can be responsible for the elimination. Transcriptome analysis of the mutant Treg identified OX40, co-stimulatory molecule involved in lymphocyte survival, as a candidate molecule. In fact, anti-OX40L blocking antibody partially restored the elimination of Dsg3H1 T cells in the absence of Treg in DEREG mice (n=3). Thus, these findings indicated Treg-mediated, OX40-dependent elimination of autoreactive CD4+ T cells as a novel skin peripheral tolerance mechanism.

016 Deficiency of the G protein-coupled receptor HCA2 alters the phenotype and function of dendritic cells

Short chain fatty acids (SCFA), bacterial products from fermentation of fiber, exert anti-inflammatory effects in the colon and the skin via induction of regulatory T cells (Treg). One of the most relevant receptors for SCFA is the G protein-coupled receptor 109a/HCA2. To understand the mechanism by which Treg are induced via HCA2 signaling, bone marrow derived-dendritic cells (BMDC) from HCA2-/- mice were characterized. IL-6 and IL-17, cytokines which promote Th17 cells and suppress the development of Treg, were upregulated in HCA2-/- BMDC in comparison to wild type (WT) BMDC. Aldehyde dehydrogenase Aldh1a1 which converts naïve T cells into Treg was weaker expressed in HCA2-/- BMDC than in WT BMDC. FACS analysis of CD4+CD25- T cells which were coincubated with WT or HCA2-/- BMDC revealed that WT BMDC were more potent in inducing Treg markers such as Foxp3, Garp, CTLA-4. This may be due to soluble factors, since the same was observed upon incubation of CD4+CD25- cells with BMDC-derived supernatants. Accordingly, IL-10, a relevant promotor of Treg, was downregulated in HCA2-/- mice. To prove the functional relevance, HCA2-/- and WT BMDC were pulsed with the hapten TNBS and injected subcutaneously into naïve mice which were challenged 5 days later with TNCB. To our surprise upon injection of HCA2-/- BMDC the sensitization response was not enhanced (as supposed due to the reduced number of Treg) but reduced in comparison to WT BMDC. As initiation of an immune response is dependent on antigen uptake and presentation to T cells, we studied antigen uptake of HCA2-/-BMDC in an antigen uptake assay using FITC-conjugated OVA. HCA2-/- BMDC were less potent in taking up FITC-OVA compared to WT BMDC. The reduced antigen uptake might explain why the sensitizing capacity of DC lacking HCA2 is reduced despite the absence of Treg. Taken together, these findings indicate HCA2 as a crucial receptor during antigen presentation which might essentially influence the outcome of an immune response.

Regulatory T Cell-Dependent and -Independent Mechanisms of Immune Suppression by CD28/B7 and CD40/CD40L Costimulation Blockade.

Blocking of costimulatory CD28/B7 and CD40/CD40L interactions is an experimental approach to immune suppression and tolerance induction. We previously reported that administration of a combination of CTLA-4Ig and MR1 (anti-CD40L mAb) for blockade of these interactions induces tolerance in a fully mismatched allogeneic splenocyte transfer model in mice. We now used this model to study whether regulatory T cells (Tregs) contribute to immune suppression and why both pathways have to be blocked simultaneously. Mice were injected with allogeneic splenocytes, CD4(+) T cells, or CD8(+) T cells and treated with MR1 mAb and different doses of CTLA-4Ig. The graft-versus-host reaction of CD4(+) T cells, but not of CD8(+) T cells, was inhibited by MR1. CTLA-4Ig was needed to cover CD8(+) T cells but had only a weak effect on CD4(+) T cells. Consequently, only the combination provided full protection when splenocytes were transferred. Importantly, MR1 and low-dose CTLA-4Ig treatment resulted in a relative increase in Tregs, and immune suppressive efficacy was abolished in the absence of Tregs. High-dose CTLA-4Ig treatment, in contrast, prevented Treg expansion and activity, and in combination with MR1 completely inhibited CD4(+) and CD8(+) T cell activation in a Treg-independent manner. In conclusion, MR1 and CTLA-4Ig act synergistically as they target different T cell populations. The contribution of Tregs to immune suppression by costimulation blockade depends on the concentration of CTLA-4Ig and thus on the degree of available CD28 costimulation.

Regulatory T-Cell Plasticity

During the past 2 decades, human and animals studies have clearly documented the crucial role of inflammation in the development and complications of atherosclerosis.1 Both innate immunity and adaptive immunity are involved in this process. The first evidence that suggested a role of adaptive immunity in atherosclerosis was the widespread detection of the major histocompatibility class II in human atherosclerotic plaques and the presence of a large amount of CD3+ lymphocytes in human and mouse atherosclerotic lesions. Most of the T cells in mouse and human atherosclerotic plaques are CD4+ T-helper (Th) cells expressing the αβ T-cell antigen receptor. Among CD4+ T cells, Th1 cells have been shown to exert proatherogenic effects, whereas regulatory T cells (Tregs) display atheroprotective properties. The role of Th2 and Th17 cells is still debated.1 Helper T-cell subsets are defined by the production of cytokines or the expression of characteristic lineage-defining transcription factors. Th1 cells are generated on priming in the presence of interleukin (IL)-12 that promotes the expression of the transcription factor T-bet and stimulates the production of the Th1 prototypical cytokine interferon-γ (IFN-γ).2 Tregs have a central role in the dominant control of immunologic tolerance and maintenance of immune homeostasis. They were first identified in mice and later in humans.3 The transcription factor FoxP3 is essential for the generation and the functions of Tregs.4 FoxP3 deficiency leads to a multiorgan autoimmune disease as can be observed in the scurfy mouse and in human immune dysregulation, polyendocrinopathy, enteropathy X–linked syndrome patients.5 Natural or thymic FoxP3+ Tregs acquire regulatory lineage commitment already on maturation in the thymus,3 whereas adaptive or peripheral FoxP3+ Tregs can be induced from mature CD4+ Th cells in the periphery under the influence of different stimulations, especially transforming …

Post-transplant bendamustine reduces GvHD while preserving GvL in experimental haploidentical bone marrow transplantation.

Advances in haploidentical bone marrow transplantation (h-BMT) have drastically broadened the treatment options for patients requiring BMT. The possibility of significantly reducing the complications resulting from graft-versus-host disease (GvHD) with the administration of post-transplant cyclophosphamide (PT-CY) has substantially improved the efficacy and applicability of T cell-replete h-BMT. However, higher frequency of disease recurrence remains a major challenge in h-BMT with PT-CY. There is a critical need to identify novel strategies to prevent GvHD while sparing the graft-versus-leukaemia (GvL) effect in h-BMT. To this end, we evaluated the impact of bendamustine (BEN), given post-transplant, on GvHD and GvL using clinically relevant murine h-BMT models. We provide results indicating that post-transplant bendamustine (PT-BEN) alleviates GvHD, significantly improving survival, while preserving engraftment and GvL effects. We further document that PT-BEN can mitigate GvHD even in the absence of Treg. Our results also indicate that PT-BEN is less myelosuppressive than PT-CY, significantly increasing the number and proportion of CD11b(+) Gr-1(hi) cells, while decreasing lymphoid cells. In vitro we observed that BEN enhances the suppressive function of myeloid-derived suppressor cells (MDSCs) while impairing the proliferation of T- and B-cells. These results advocate for the consideration of PT-BEN as a new therapeutic platform for clinical implementation in h-BMT.

Depletion of regulatory T cells leads to an exacerbation of delayed-type hypersensitivity arthritis in C57BL/6 mice that can be counteracted by IL-17 blockade.

ABSTRACTRodent models of arthritis have been extensively used in the elucidation of rheumatoid arthritis (RA) pathogenesis and are instrumental in the development of therapeutic strategies. Here we utilise delayed-type hypersensitivity arthritis (DTHA), a model in C57BL/6 mice affecting one paw with synchronised onset, 100% penetrance and low variation. We investigate the role of regulatory T cells (Tregs) in DTHA through selective depletion of Tregs and the role of IL-17 in connection with Treg depletion. Given the relevance of Tregs in RA, and the possibility of developing Treg-directed therapies, this approach could be relevant for advancing the understanding of Tregs in inflammatory arthritis. Selective depletion of Tregs was achieved using a Foxp3-DTR-eGFP mouse, which expresses the diphtheria toxin receptor (DTR) and enhanced green fluorescent protein (eGFP) under control of the Foxp3 gene. Anti-IL-17 monoclonal antibody (mAb) was used for IL-17 blockade. Numbers and activation of Tregs increased in the paw and its draining lymph node in DTHA, and depletion of Tregs resulted in exacerbation of disease as shown by increased paw swelling, increased infiltration of inflammatory cells, increased bone remodelling and increased production of inflammatory mediators, as well as increased production of anti-citrullinated protein antibodies. Anti-IL-17 mAb treatment demonstrated that IL-17 is important for disease severity in both the presence and absence of Tregs, and that IL-17 blockade is able to rescue mice from the exacerbated disease caused by Treg depletion and caused a reduction in RANKL, IL-6 and the number of neutrophils. We show that Tregs are important for the containment of inflammation and bone remodelling in DTHA. To our knowledge, this is the first study using the Foxp3-DTR-eGFP mouse on a C57BL/6 background for Treg depletion in an arthritis model, and we here demonstrate the usefulness of the approach to study the role of Tregs and IL-17 in arthritis.