Expansion Of Treg Cells Research Articles

Gut microbiome, parathyroid hormone, and bone.

Microorganisms in the gut (the 'microbiome') and the metabolites they produce (the 'metabolome') regulate bone mass through interactions between parathyroid hormone (PTH), the immune system, and bone. This review summarizes these data and details how this physiology may relate to CKD-mediated bone disease. The actions of PTH on bone require microbial metabolite activation of immune cells. Butyrate is necessary for CD4+ T-cell differentiation, T-reg cell expansion and CD8+ T-cell secretion of the bone-forming factor Wnt10b ligand. By contrast, mice colonized with segmented filamentous bacteria exhibit an expansion of gut Th17 cells and continuous PTH infusion increases the migration of Th17 cells to the bone marrow, contributing to bone resorption. In the context of CKD, a modified diet, frequent antibiotic therapy, altered intestinal mobility, and exposure to multiple medications together contribute to dysbiosis; the implications for an altered microbiome and metabolome on the pathogenesis of renal osteodystrophy and its treatment have not been explored. As dysregulated interactions between PTH and bone ('skeletal resistance') characterize CKD, the time is ripe for detailed, mechanistic studies into the role that gut metabolites may play in the pathogenesis of CKD-mediated bone disease.

MHC Class II Antigen Presentation by Lymphatic Endothelial Cells in Tumors Promotes Intratumoral Regulatory T cell-Suppressive Functions.

Several solid malignancies trigger lymphangiogenesis, facilitating metastasis. Tumor-associated lymphatic vessels significantly contribute to the generation of an immunosuppressive tumor microenvironment (TME). Here, we have investigated the ability of tumoral lymphatic endothelial cells (LEC) to function as MHC class II-restricted antigen-presenting cells in the regulation of antitumor immunity. Using murine models of lymphangiogenic tumors engrafted under the skin, we have shown that tumoral LECs upregulate MHC class II and the MHC class II antigen-processing machinery, and that they promote regulatory T-cell (Treg) expansion ex vivo. In mice with LEC-restricted lack of MHC class II expression, tumor growth was severely impaired, whereas tumor-infiltrating effector T cells were increased. Reduction of tumor growth and reinvigoration of tumor-specific T-cell responses both resulted from alterations of the tumor-infiltrating Treg transcriptome and phenotype. Treg-suppressive functions were profoundly altered in tumors lacking MHC class II in LECs. No difference in effector T-cell responses or Treg phenotype and functions was observed in tumor-draining lymph nodes, indicating that MHC class II-restricted antigen presentation by LECs was required locally in the TME to confer potent suppressive functions to Tregs. Altogether, our study suggests that MHC class II-restricted antigen-presenting tumoral LECs function as a local brake, dampening T cell-mediated antitumor immunity and promoting intratumoral Treg-suppressive functions.

Donor Treg expansion by liposomal α‐galactosylceramide modulates Tfh cells and prevents sclerodermatous chronic graft‐versus‐host disease

Background and AimChronic graft‐versus‐host disease (cGVHD) is a major cause of nonrelapse morbidity and mortality following hematopoietic stem cell transplantation (HSCT). α‐Galactosylceramide (α‐GC) is a synthetic glycolipid that is recognized by the invariant T‐cell receptor of invariant natural killer T (iNKT) cells in a CD1d‐restricted manner. Stimulation of iNKT cells by α‐GC leads to the production of not only immune‐stimulatory cytokines but also immune‐regulatory cytokines followed by regulatory T‐cell (Treg) expansion in vivo.MethodsWe investigated the effect of iNKT stimulation by liposomal α‐GC just after transplant on the subsequent immune reconstitution and the development of sclerodermatous cGVHD.ResultsOur study showed that multiple administrations of liposomal α‐GC modulated both host‐ and donor‐derived iNKT cell homeostasis and induced an early expansion of donor Tregs. We also demonstrated that the immune modulation of the acute phase was followed by the decreased levels of CXCL13 in plasma and follicular helper T cells in lymph nodes, which inhibited germinal center formation, resulting in the efficient prevention of sclerodermatous cGVHD.ConclusionsThese data demonstrated an important coordination of T‐ and B‐cell immunity in the pathogenesis of cGVHD and may provide a novel clinical strategy for the induction of immune tolerance after allogeneic HSCT.

Ovalbumin-Induced Airway Inflammation Is Ameliorated in Dectin-1-Deficient Mice, in Which Pulmonary Regulatory T Cells Are Expanded through Modification of Intestinal Commensal Bacteria.

Asthma is an allergic chronic respiratory disease that affects more than 300 million people around the world. Dysbiosis of intestinal commensal microbiota influences the development of asthma. Dectin-1 (gene symbol: Clec7a), a C-type lectin receptor, plays an important role in the intestinal immune homeostasis by controlling regulatory T (Treg) cell differentiation through regulation of intestinal microbiota. However, it is not clear whether intestinal immune conditions affect immune responses in other organs. In this study, we examined the effects of Dectin-1 deficiency on allergic airway inflammation (AAI). OVA-induced AAI was attenuated in Clec7a -/- mice. Treg cells were more abundant in colonic lamina propria, mesenteric lymph nodes, and bronchoalveolar lavage fluid of Clec7a -/- mice after AAI induction. Treatment with antibiotics, but not an antifungal agent, decreased the abundance of intestinal Treg cells and aggravated the symptoms of AAI in Clec7a -/- mice. Transplantation of gut microbiota from Clec7a -/- mice into antibiotic-treated hosts increased the abundance of intestinal Treg cells and ameliorated AAI. Overcolonization by Lactobacillus murinus, a Dectin-1 signaling-regulated commensal bacterium, also promoted expansion of Treg cells in the colon and suppressed lung inflammation. Depletion of Treg cells with anti-CD25 Ab eliminated the phenotypic differences between wild-type and Clec7a -/- mice in OVA-induced AAI. These observations suggest that inhibition of Dectin-1 signaling ameliorates AAI by increasing the abundance of Treg cells in lungs through modification of intestinal commensal bacteria, suggesting a role for commensal microbiota in regulating inflammation in organs other than the intestine.

Microbial metabolite Urolithin A induces expansion of Regulatory T Cells in aryl hydrocarbon receptor (AhR)-dependent manner

Abstract Intestinal inflammation and loss of gut barrier integrity contribute to severity of inflammatory bowel diseases (IBD). Gut microbiota and their metabolites play an important role in maintenance of immunological balance within the gut. Previously, our group demonstrated that treatment with Urolithin A enhanced gut barrier function by inducing junctional proteins and blocked LPS-induced inflammation. Moreover, treatment with this microbial metabolite mitigated colitis in pre-clinical models. In the current study, we identified novel function for Urolithin A, where treatment with Urolithin A significantly expanded FoxP3+ T regulatory cells (Tregs) both ex vivo and in vivo models. Urolithin A failed to induce Treg expansion in AhR−/− mice suggesting Urolithin A mediates its activities through AhR. Moreover, treatment with Urolithin A mitigated murine model of dextran sulfate sodium (DSS)-colitis and enhanced Treg cell population compared to vehicle treatment. These results suggest that Urolithin A offers multi-pronged beneficial activities on gut epithelial layer (enhanced barrier function), macrophages (down regulation of inflammatory cytokines) and expansion of Treg cells to protect against colitis.

Immune Monitoring during Therapy Reveals Activitory and Regulatory Immune Responses in High-Risk Neuroblastoma.

Simple SummaryNeuroblastoma is a type of childhood cancer accounting for approximately 15% of childhood cancer deaths. Despite intensive treatment, including immunotherapy, prognosis of high-risk neuroblastoma is poor. Increasing amounts of research show that the fighting capacity of the immune system is very important for the outcome of neuroblastoma patients. Therefore, we investigated the fighting capacity of immune cells in blood at diagnosis and during the different phases of therapy. In this study, we observed both processes that stimulate and processes that decrease fighting capacity of immune cells in neuroblastoma patients during therapy. Despite this, we show that overall fighting capacity of the immune system of neuroblastoma patients is impaired at diagnosis as well as during therapy. In addition, we observed a lot of variation between patients, which might explain differences in therapy efficacy between patients. This study provides insight for improvement of therapy timing as well as new therapy strategies enhancing immune cell fighting capacity.Despite intensive treatment, including consolidation immunotherapy (IT), prognosis of high-risk neuroblastoma (HR-NBL) is poor. Immune status of patients over the course of treatment, and thus immunological features potentially explaining therapy efficacy, are largely unknown. In this study, the dynamics of immune cell subsets and their function were explored in 25 HR-NBL patients at diagnosis, during induction chemotherapy, before high-dose chemotherapy, and during IT. The dynamics of immune cells varied largely between patients. IL-2- and GM-CSF-containing IT cycles resulted in significant expansion of effector cells (NK-cells in IL-2 cycles, neutrophils and monocytes in GM-CSF cycles). Nonetheless, the cytotoxic phenotype of NK-cells was majorly disturbed at the start of IT, and both IL-2 and GM-CSF IT cycles induced preferential expansion of suppressive regulatory T-cells. Interestingly, proliferative capacity of purified patient T-cells was impaired at diagnosis as well as during therapy. This study indicates the presence of both immune-enhancing as well as regulatory responses in HR-NBL patients during (immuno)therapy. Especially the double-edged effects observed in IL-2-containing IT cycles are interesting, as this potentially explains the absence of clinical benefit of IL-2 addition to IT cycles. This suggests that there is a need to combine anti-GD2 with more specific immune-enhancing strategies to improve IT outcome in HR-NBL.

Targeting Enclysis in Liver Autoimmunity, Transplantation, Viral Infection and Cancer.

Persistent liver inflammation can lead to cirrhosis, which associates with significant morbidity and mortality worldwide. There are no curative treatments beyond transplantation, followed by long-term immunosuppression. The global burden of end stage liver disease has been increasing and there is a shortage of donor organs, therefore new therapies are desperately needed. Harnessing the power of the immune system has shown promise in certain autoimmunity and cancer settings. In the context of the liver, regulatory T cell (Treg) therapies are in development. The hypothesis is that these specialized lymphocytes that dampen inflammation may reduce liver injury in patients with chronic, progressive diseases, and promote transplant tolerance. Various strategies including intrinsic and extracorporeal expansion of Treg cells, aim to increase their abundance to suppress immune responses. We recently discovered that hepatocytes engulf and delete Treg cells by enclysis. Herein, we propose that inhibition of enclysis may potentiate existing regulatory T cell therapeutic approaches in patients with autoimmune liver diseases and in patients receiving a transplant. Moreover, in settings where the abundance of Treg cells could hinder beneficial immunity, such us in chronic viral infection or liver cancer, enhancement of enclysis could result in transient, localized reduction of Treg cell numbers and tip the balance towards antiviral and anti-tumor immunity. We describe enclysis as is a natural process of liver immune regulation that lends itself to therapeutic targeting, particularly in combination with current Treg cell approaches.

AhR Ligands Modulate the Differentiation of Innate Lymphoid Cells and T Helper Cell Subsets That Control the Severity of a Pulmonary Fungal Infection.

In agreement with other fungal infections, immunoprotection in pulmonary paracoccidioidomycosis (PCM) is mediated by Th1/Th17 cells whereas disease progression by prevalent Th2/Th9 immunity. Treg cells play a dual role, suppressing immunity but also controlling excessive tissue inflammation. Our recent studies have demonstrated that the enzyme indoleamine 2,3 dioxygenase (IDO) and the transcription factor aryl hydrocarbon receptor (AhR) play an important role in the immunoregulation of PCM. To further evaluate the immunomodulatory activity of AhR in this fungal infection, Paracoccidioides brasiliensis infected mice were treated with two different AhR agonists, L-Kynurenin (L-Kyn) or 6-formylindole [3,2-b] carbazole (FICZ), and one AhR specific antagonist (CH223191). The disease severity and immune response of treated and untreated mice were assessed 96 hours and 2 weeks after infection. Some similar effects on host response were shared by FICZ and L-Kyn, such as the reduced fungal loads, decreased numbers of CD11c+ lung myeloid cells expressing activation markers (IA, CD40, CD80, CD86), and early increased expression of IDO and AhR. In contrast, the AhR antagonist CH223191 induced increased fungal loads, increased number of pulmonary CD11c+ leukocytes expressing activation markers, and a reduction in AhR and IDO production. While FICZ treatment promoted large increases in ILC3, L-Kyn and CH223191 significantly reduced this cell population. Each of these AhR ligands induced a characteristic adaptive immunity. The large expansion of FICZ-induced myeloid, lymphoid, and plasmacytoid dendritic cells (DCs) led to the increased expansion of all CD4+ T cell subpopulations (Th1, Th2, Th17, Th22, and Treg), but with a clear predominance of Th17 and Th22 subsets. On the other hand, L-Kyn, that preferentially activated plasmacytoid DCs, reduced Th1/Th22 development but caused a robust expansion of Treg cells. The AhR antagonist CH223191 induced a preferential expansion of myeloid DCs, reduced the number of Th1, Th22, and Treg cells, but increased Th17 differentiation. In conclusion, the present study showed that the pathogen loads and the immune response in pulmonary PCM can be modulated by AhR ligands. However, further studies are needed to define the possible use of these compounds as adjuvant therapy for this fungal infection.

Interleukin-33-Dependent Accumulation of Regulatory T Cells Mediates Pulmonary Epithelial Regeneration During Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) triggered mostly by infection, is a syndrome that involves respiratory failure. ARDS induces strong local infiltration of regulatory T cells (Treg cells) in the lungs, and Treg cells were recently highlighted as being related to the repair of various tissue. However, at present, there is still a lack of adequate evidence showing the impact of Treg cells on pulmonary regeneration during ARDS. Here, we verified that Treg cells are strongly induced in ARDS mice and Treg depletion results in impaired lung repair. Moreover, Treg cells show high expression of ST2, a cellular receptor for the tissue alarmin IL-33, which is strongly upregulated in the lung during ARDS. In addition, we demonstrated that IL-33 signaling is crucial for Treg cell accumulation, and ST2-blocked mice show a decrease in the Treg cell population. Critically, transfer of exogenous IL-33 into Treg depleted mice restored Treg cells and facilitated lung regeneration by promoting alveolar type II cell (AEC2) recovery in ARDS, with elevated neutrophils infiltration and upregulated TGF-β1 release. These results emphasized the importance of IL-33 in accelerating the expansion of pulmonary Treg cells and promoting their activity to mediate pulmonary epithelial regeneration during ARDS in a TGF-β1-dependent manner.

Decreased Treg Cell and TCR Expansion Are Involved in Long-Lasting Graves’ Disease

Graves’ disease (GD) is a T cell-mediated organ-specific autoimmune disorder. GD patients who have taken anti-thyroid drugs (ATDs) for more than 5 years with positive anti-thyroid stimulating hormone receptor autoantibodies value were defined as persistent GD (pGD). To develop novel immunotherapies for pGD, we investigated the role of T cells in the long-lasting phase of GD. Clinical characteristics were compared between the pGD and newly diagnosed GD (nGD) (N = 20 respectively). Flow cytometric analysis was utilized to determine the proportions of Treg and Th17 cells (pGD, N = 12; nGD, N = 14). T cell receptor sequencing (TCR-seq) and RNA sequencing (RNA-seq) were also performed (pGD, N = 13; nGD, N = 20). Flow cytometric analysis identified lower proportions of Th17 and Treg cells in pGD than in nGD (P = 0.0306 and P = 0.0223). TCR-seq analysis revealed a lower diversity (P = 0.0025) in pGD. Specifically, marked clonal expansion, represented by an increased percentage of top V-J recombination, was observed in pGD patients. Interestingly, pGD patients showed more public T cell clonotypes than nGD patients (2,741 versus 966). Meanwhile, RNA-seq analysis revealed upregulation of the inflammation and chemotaxis pathways in pGD. Specifically, the expression of pro-inflammatory and chemotactic genes (IL1B, IL13, IL8, and CCL4) was increased in pGD, whereas Th17 and Treg cells associated genes (RORC, CARD9, STAT5A, and SATB1) decreased in pGD. Additionally, TCR diversity was negatively correlated with the expression of pro-inflammatory or chemotactic genes (FASLG, IL18R1, CCL24, and CCL14). These results indicated that Treg dysregulation and the expansion of pathogenic T cell clones might be involved in the long-lasting phase of GD via upregulating chemotaxis or inflammation response. To improve the treatment of pGD patients, ATDs combined therapies, especially those aimed at improving Treg cell frequencies or targeting specific expanded pathogenic TCR clones, are worth exploring in the future.

The role of decidual regulatory T cells in the induction and maintenance of fetal antigen-specific tolerance: Imbalance between regulatory and cytotoxic T cells in pregnancy complications

Fetal antigen-specific tolerance is important for maintaining allogeneic pregnancies. Maternal conventional T cells recognize fetal antigens; however, regulatory T (Treg) cells suppress immune reactions against the fetus. Fetal antigen-specific Treg cells are induced in the decidua upon contact with antigen-presenting cells and extravillous trophoblasts (EVTs). Functional alteration of cytotoxic T cells (CTLs) in the decidua also contributes to maintaining the pregnancy. Reduced, dysfunctional, and imbalanced Treg cell distribution likely contributes to the pathogenesis of pregnancy complications, such as miscarriage and preeclampsia. Recent studies have revealed differences in Treg cell characteristics during preeclampsia and miscarriage. Treg cell reduction in the decidua is likely associated with miscarriage. Insufficient expansion of fetal antigen-specific Treg cells in the decidua probably plays a role in preeclampsia pathogenesis. In addition, the balance between Treg cell-mediated tolerance and functional alteration of CTLs is important. Further investigations of functional molecules in Treg cells will contribute to the development of immunotherapy for pregnancy complications.

SND1 promotes Th1/17 immunity against chlamydial lung infection through enhancing dendritic cell function.

To date, no reports have linked the multifunctional protein, staphylococcal nuclease domain-containing protein 1 (SND1), to host defense against intracellular infections. In this study, we investigated the role and mechanisms of SND1, by using SND1 knockout (SND1-/-) mice, in host defense against the lung infection of Chlamydia muridarum, an obligate intracellular bacterium. Our data showed that SND1-/- mice exhibited significantly greater body weight loss, higher organism growth, and more severe pathological changes compared with wild-type mice following the infection. Further analysis showed significantly reduced Chlamydia-specific Th1/17 immune responses in SND1-/- mice after infection. Interestingly, the dendritic cells (DCs) isolated from SND1-/- mice showed lower costimulatory molecules expression and IL-12 production, but higher IL-10 production compared with those from wild-type control mice. In the DC-T cell co-culture system, DCs isolated from SND1-/- infected mice showed significantly reduced ability to promote Chlamydia-specific IFN-γ producing Th1 cells but enhanced capacity to induce CD4+T cells into Foxp3+ Treg cells. Adoptive transfer of DCs isolated from SND1-/- mice, unlike those from wild-type control mice, failed to protect the recipients against challenge infection. These findings provide in vivo evidence that SND1 plays an important role in host defense against intracellular bacterial infection, and suggest that SND1 can promote Th1/17 immunity and inhibit the expansion of Treg cells through modulation of the function of DCs.

D-mannose ameliorates autoimmune phenotypes in mouse models of lupus

BackgroundSystemic lupus erythematosus is an autoimmune disease characterized by an overproduction of autoantibodies resulting from dysregulation in multiple immune cell types. D-mannose is a C− 2 epimer of glucose that exhibits immunoregulatory effects in models of autoimmune diseases, such as type 1 diabetes, induced rheumatoid arthritis, and airway inflammation. This study was conducted to evaluate the efficacy of D-mannose treatment in mouse models of lupus.ResultsFirstly, the effect of D-Mannose was evaluated by flow cytometry on the in vitro activation of non-autoimmune C57BL/6 (B6) bone marrow-derived dendritic cells (BMDCs) and their ability to induce antigen-specific CD4+ T cell proliferation and activation. D-mannose inhibited the maturation of BMDCs and their induction of antigen-specific T cell proliferation and activation. In vivo, D-mannose increased the frequency of Foxp3+ regulatory T cells in unmanipulated B6 mice. To assess the effect of D-mannose in mouse models of lupus, we used the graft-versus-host disease (cGVHD) induced model and the B6.lpr spontaneous model. In the cGVHD model, D-mannose treatment decreased autoantibody production, with a concomitant reduction of the frequency of effector memory and follicular helper T cells as well as germinal center B cells and plasma cells. These results were partially validated in the B6.lpr model of spontaneous lupus.ConclusionOverall, our results suggest that D-mannose ameliorates autoimmune activation in models of lupus, at least partially due to its expansion of Treg cells, the induction of immature conventional dendritic cells and the downregulation of effector T cells activation. D-Mannose showed however a weaker immunomodulatory effect in lupus than in other autoimmune diseases.

Immunomodulatory Activity of Human Bone Marrow and Adipose-Derived Mesenchymal Stem Cells Prolongs Allogenic Skin Graft Survival in Nonhuman Primates.

Objective In the present study, we examined the tolerance-inducing effects of human adipose-derived mesenchymal stem cells (hAD-MSCs) and bone marrow-derived MSCs (hBM-MSCs) on a nonhuman primate model of skin transplantation. Materials and Methods In this experimental study, allogenic and xenogeneic of immunomodulatory properties of human AD-MSCs and BM-MSCs were evaluated by mixed lymphocyte reaction (MLR) assays. Human MSCs were obtained from BM or AD tissues (from individuals of either sex with an age range of 35 to 65 years) and intravenously injected (2×106 MSCs/kg) after allogeneic skin grafting in a nonhuman primate model. The skin sections were evaluated by H&E staining for histopathological evaluations, particularly inflammation and rejection reaction of grafts after 96 hours of cell injection. At the mRNA and protein levels, cellular mediators of inflammation, such as CD4+IL-17+ (T helper 17; Th17) and CD4+INF-γ+ (T helper 1, Th1) cells, along with CD4+FoxP3+ cells (Treg), as the mediators of immunomodulation, were measured by RT-PCR and flow cytometry analyses. Results A significant Treg cells expansion was observed in MSCs-treated animals which reached the zenith at 24 hours and remained at a high concentration for 96 hours; however, Th1 and Th17 cells were significantly decreased. Our results showed that human MSCs significantly decrease Th1 and Th17 cell proliferation by decreasing interleukin-17 (IL-17) and interferon-γ (INF-γ) production and significantly increase Treg cell proliferation by increasing FoxP3 production. They also extend the allogenic skin graft survival in nonhuman primates. Histological evaluations showed no obvious presence of inflammatory cells or skin redness or even bulging after MSCs injection up to 96 hours, compared to the group without MSCs. There were no significant differences between hBM-MSCs and hAD-MSCs in terms of histopathological scores and inflammatory responses (P<0.05). ConclusionIt seems that MSCs could be regarded as a valuable immunomodulatory tool to reduce the use of immunosuppressive agents.

Dialogue between gastrointestinal tract and skin: New insights into the Helicobacter pylori and atopic dermatitis.

Although many studies have focused on the protective function of H pylori in some allergic diseases, it remains unknown as whether H pylori infection exerts a similar protective effect on atopic dermatitis(AD). Thus, the aim of this study was to evaluate the association between H pylori infection and AD. An animal model of H pylori infection-AD was established by epicutaneous sensitization with calcipotriol after infection with H pylori by gavage. The Treg cells were analyzed by flow cytometry and immunohistochemistry. The expression of key inflammatory cytokines in dermal tissues was investigated at the mRNA level by real-time PCR. Compared with that in the H pylori-negative AD group, the severity of skin lesions, such as hyperemia, erythema, and swelling, was lower in the H pylori-positive AD group, while the serum IgE level decreased significantly in the H pylori-positive AD group. The percentage of CD4+ CD25+ Foxp3+ Treg cells in the peripheral blood and the number of Foxp3+ cells in dermal tissues increased significantly in the H pylori-positive AD group. The expression of IL-10 and TGF-β was upregulated, while the expression of IL-4 mRNA was downregulated in dermal tissues in the H pylori-positive AD group. The adoptive transfer assay showed that the number of CFSE+ Treg cells in the cervical lymph nodes of AD mice was significantly higher than that in normal mice, indicating the Tregs in H pylori-positive mice had a tendency to migrate to the skin tissue. It was also found that H pylori infection induced CCR4+ Treg cells expansion synchronously in gastric lymph nodes, spleen, blood, mesenteric lymph node (MLN), and cervical lymph nodes by the time of H pylori infection. H pylori infection alleviated calcipotriol-inducing AD manifestations by inducing the amplification of CD4+ CD25+ Foxp3+ Treg cells in the peripheral blood. H pylori showed possible protection against atopic dermatitis, suggesting an immune dialogue between gastrointestinal tract and skin.

Induction of Antigen-Independent Proliferation of Regulatory T-Cells by TNF Superfamily Ligands OX40L and GITRL.

TNF receptor superfamily comprises many T-cell costimulatory receptors, including TNFRSF1, TNFRSF2, TNFRSF4 (OX40), TNFRSF9 (4-1BB), TNFRSF18 (GITR), and TNFRSF7 (CD27). Signaling through these costimulatory stimulatory receptors can promote conventional T-cell (Tconv) proliferation, and effector functions in an antigen-dependent manner. Thus, agonistic antibodies and ligands for OX40, 4-1BB, GITR, and CD27 have been tested for inducing T-cell-mediated antitumor responses in several cancers. However, recently emerging reports show critical role for TNFR signaling in regulatory T-cell (Treg) differentiation and expansion, which might suppress effector T-cell proliferation and functions. Here, we show preferential over expression of TNFR2, OX40, 4-1BB, and GITR in Treg cells over Tconv cells, and the ability of OX40L and GITRL to induce selective proliferation of Treg cells, but not Tconv cells, in an antigen-independent manner. We describe the standard protocols used for Affymetrix gene expression profiling, T-cell isolation, and Cell Trace Violet-based cell proliferation assay.

Platelet-Targeted Gene Therapy Induces Robust Immune Tolerance Even in a Primed Model Via Peripheral Clonal Deletion of CD4 and CD8 T Cells and Expansion of Treg Cells

Our previous studies have demonstrated that platelet-specific gene transfer under control of the αIIb promoter (2b) results in effective immune tolerance induction to FVIII and FIX as well as the non-coagulant protein ovalbumin (OVA) in the unprimed model. Here we explored how the reactivity of immune responses affects the efficacy of transgene expression and the potential immune tolerance mechanisms in platelet-specific gene transfer in the primed model. We used OVA as a surrogate protein for this study. Recipient wild-type (WT) CD45.1 mice were primed with OVA protein. Platelet-OVA expression was introduced by 2bOVA lentivirus transduction of Sca-1+ cells from either WT/CD45.2 or OTII/CD45.2 donors followed by transplantation into OVA-primed WT/CD45.1 recipients preconditioned with 6.6Gy irradiation. We found that pre-existing high-reactive immune responses devastate platelet-OVA expression in recipients in both the WT/WT and OTII/WT models. Using the WT/WT mouse model, 6 of 7 2bOVA-transduced recipients had sustained platelet-OVA expression with levels ranging from 4.53-8.84 ng/108 platelets. Anti-OVA total IgG titers declined with time in 2bOVA-transduced OVA-primed recipients even when rechallenged with OVA. In contrast, recall memory immune responses were elicited in 2bGFP and untransduced transplanted controls. Furthermore, full-thickness tail skin grafts from CAG-OVATg mice were successfully engrafted onto 2bOVA-transduced recipients and survived throughout the remaining lifetimes of the animals. In contrast, skin grafts were completely rejected in control 2bGFP-transduced and untransduced transplanted recipients. To ensure that the immune system was not inactive in 2bOVA-transduced recipients, animals were immunized with unrelated antigen recombinant human FVIII (rhF8) using a protocol known to induce anti-F8 immune responses even in WT mice. All 2bOVA-transduced recipients developed anti-F8 inhibitory antibodies after rhF8 immunization with no differences between the 2bOVA group and the 2bGFP and untransduced control groups. Using the OVA-specific CD4 TCR transgenic model (OTII/WT), we showed that sustained platelet-OVA expression was achieved in 90% of 2bOVA-transduced recipients. OVA-specific CD4 T cells were deleted and Treg cells were expanded in peripheral lymphoid organs in 2bOVA-transduced OVA-primed recipients. The levels of platelet-OVA expression negatively correlates with the percentages of OVA-specific CD4 T cells, but positively correlates with Treg cells. Immune tolerance was also achieved in the OTII/WT model even though all donor-derived CD4 T cells are OVA-specific. However, our studies showed that high-reactive immune responses could influence the neoprotein expression in platelet-targeted gene therapy. Using the OVA-specific CD8 TCR transgenic model (OTI/WT), in which all donor-derived CD8 T cells are OVA-specific, we found that low levels of platelet-OVA expression were obtained at week 5 after transplantation (0.72±0.65 ng/108 platelets) and then dropped to barely detectable at subsequent time points. Flow cytometry analysis showed that there were 33.9±12.9% platelets positive with GFP in 2bGFP LV-transduced recipients, demonstrating that high transduction efficiency and successful transplantation were achieved. Of note, OVA-specific CD8 T cells in the 2bOVA group were 48% lower than in the 2bGFP control group, demonstrating that antigen-specific CD8 T cells were partially deleted in the OTI/WT model after platelet-targeted OVA gene transfer. Interestingly, we found that the percentage of the antigen-specific CD8+Foxp3+ cells in the 2bOVA group was significantly higher than in the 2bGFP group (0.19±0.14% vs. 0.05±0.01%, respectively). These results suggest that there is a counterpace process between immune reaction and immune tolerance after platelet-targeted gene therapy. In conclusion, our studies demonstrate that platelet-targeted gene therapy can induce antigen-specific immune tolerance even when the immune response has been mounted via peripheral clonal deletion of antigen-specific CD4 and CD8 T cells and expansion of Tregs, suggesting that platelet-targeted gene therapy is a promising approach to induce immune tolerance for treatment of diseases with undesired immune responses or even with pre-existing immune responses, such as hemophilia A with inhibitors or autoimmune diseases. Disclosures No relevant conflicts of interest to declare.

An All-in-One Nanomedicine Consisting of CRISPR-Cas9 and an Autoantigen Peptide for Restoring Specific Immune Tolerance.

Nanotechnology has shown great promise in treating diverse diseases. However, developing nanomedicines that can cure autoimmune diseases without causing systemic immunosuppression is still quite challenging. Herein, we propose an all-in-one nanomedicine comprising an autoantigen peptide and CRISPR-Cas9 to restore specific immune tolerance by engineering dendritic cells (DCs) into a tolerogenic phenotype, which can expand autoantigen-specific regulatory T (Treg) cells. In brief, we utilized cationic lipid-assisted poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA) nanoparticles to simultaneously encapsulate an autoimmune diabetes-relevant peptide (2.5mi), a CRISPR-Cas9 plasmid (pCas9), and three guide RNAs (gRNAs) targeting costimulatory molecules (CD80, CD86, and CD40). We demonstrated that the all-in-one nanomedicine was able to effectively codeliver these components into DCs, followed by simultaneous disruption of the three costimulatory molecules and presentation of the 2.5mi peptide on the genome-edited DCs. The resulting tolerogenic DCs triggered the generation and expansion of autoantigen-specific Treg cells by presenting the 2.5mi peptide to CD4+ T cells in the absence of costimulatory signals. Using autoimmune type 1 diabetes (T1D) as a typical disease model, we demonstrated that our nanomedicine prevented autoimmunity to islet components and inhibited T1D development. Our all-in-one nanomedicine achieved codelivery of CRISPR-Cas9 and the peptide to DCs and could be easily applied to other autoimmune diseases by substitution of different autoantigen peptides.

Foxp3+ Regulatory T Cells Inhibit CCl4-Induced Liver Inflammation and Fibrosis by Regulating Tissue Cellular Immunity.

Foxp3+ regulatory T (Treg) cells are pivotal in maintaining immunological self-tolerance and tissue homeostasis; however, it remains unclear how tissue Treg cells respond to liver injury and regulate chronic inflammation, which can cause liver fibrosis. We report here that hepatic Treg cells play a critical role in preventing liver pathology by suppressing inflammatory cellular immunity that can promote liver damage and fibrosis. Chronic liver inflammation induced by injections of carbon tetrachloride (CCl4) led to preferential expansion of hepatic Treg cells that prevented liver fibrosis. In contrast, depletion of Treg cells in the CCl4-induced liver fibrosis model exacerbated the severity of liver pathology. Treg depletion unleashed tissue cellular immunity and drove the activation and expansion of the pro-fibrotic IL-4-producing T helper 2 cells, as well as CCR2high Ly-6Chigh inflammatory monocytes/macrophages in the inflamed liver. Although Treg expression of amphiregulin plays a key role in tissue remodeling and repair in various inflammation models, amphiregulin from hepatic Treg cells, the largest producer among liver immune cells, was dispensable for maintaining liver homeostasis and preventing liver fibrosis during CCl4-induced chronic inflammation. Our results indicate that Treg cells control chronic liver inflammation and fibrosis by regulating the aberrant activation and functions of immune effector cells. Harnessing Treg functions, which effectively regulate tissue cellular immunity, may be a therapeutic strategy for preventing and treating liver fibrosis.

An Adaptive Control Scheme for Interleukin-2 Therapy.

SummaryRegulatory T cells (Treg) are suppressor cells that control self-reactive and excessive effector conventional T helper cell (Tconv) responses. Breakdown of the balance between Tregs and Tconvs is a hallmark of autoimmune and inflammatory diseases. Interleukin-2 (IL-2) is a growth factor for both populations and subtle leverage to restore the healthy immune balance in IL-2 therapy. By using a mechanistic mathematical model, we introduced an adaptive control strategy to design the minimal therapeutic IL-2 dosage required to increase and stabilize Treg population and restrict inflammatory response. This adaptive protocol allows for dose adjustments based on the feedback of the immune kinetics of the patient. Our simulation results showed that a minimal Treg population was required to restrict the transient side effect of IL-2 injections on the effector Tconv response. In silico results suggested that a combination of IL-2 and adoptive Treg transfer therapies can limit this side effect.