Deficiency in the frequency and function of Tr1 cells in IgAV and the possible role of IL-27.

OBJECTIVE: Type 1 regulatory T (Tr1) cells are involved in the pathogenesis of numerous immune-mediated diseases. However, little is known about whether and how Tr1 cells affect the development of IgA vasculitis (IgAV). We aimed to investigate this question in IgAV patients. METHODS: . Tr1 cells in peripheral blood and kidney tissue of IgAV patients were analysed by multi-parametric flow cytometry and immunofluorescence techniques. An in vitro assay of suppression of T cell proliferation and cytokine release was performed to evaluate the function of Tr1 cells. Real-time PCR and cell stimulation in vitro were used to explore the roles of IL-27 and early growth response gene 2 (EGR2). RESULTS: The frequency of Tr1 cells was decreased in peripheral blood but increased in kidney tissue from IgAV patients. A defective suppressive function of Tr1 cells in IgAV was observed. The frequency of Tr1 cells and the cytokines secreted by them were up-regulated in the presence of recombinant IL-27 in vitro. Moreover, IL-27 also increased the expression of EGR2. Furthermore, lower frequency of Tr1 cells during remission had a higher recurrence rate. CONCLUSION: Tr1 cells are involved in the pathogenesis of IgAV. The low IL-27 in IgAV is responsible for impaired frequency and function of Tr1 cells, and EGR2 may be the specific transcription factor involved in the progression. Tr1 may be a risk factor for IgAV recurrence.

Quantifying immunoregulation by autoantigen-specific T-regulatory type 1 cells in mice with simultaneous hepatic and extra-hepatic autoimmune disorders.

Nanoparticles (NPs) displaying autoimmune disease-relevant peptide-major histocompatibility complex class II molecules (pMHCII-NPs) trigger cognate T-regulatory type 1 (Tr1)-cell formation and expansion, capable of reversing organ-specific autoimmune responses. These pMHCII-NPs that display epitopes from mitochondrial protein can blunt the progression of both autoimmune hepatitis (AIH) and experimental autoimmune encephalomyelitis (EAE) in mice carrying either disease. However, with co-morbid mice having both diseases, these pMHCII-NPs selectively treat AIH. In contrast, pMHCII-NPs displaying central nervous system (CNS)-specific epitopes can efficiently treat CNS autoimmunity, both in the absence and presence of AIH, without having any effects on the progression of the latter. Here, we develop a compartmentalized population model of T-cells in co-morbid mice to identify the mechanisms by which Tr1 cells mediate organ-specific immunoregulation. We perform time-series simulations and bifurcation analyses to study how varying physiological parameters, including local cognate antigenic load and rates of Tr1-cell recruitment and retention, affect T-cell allocation and Tr1-mediated immunoregulation. Various regimes of behaviour, including 'competitive autoimmunity' where pMHCII-NP-treatment fails against both diseases, are identified and compared with experimental observations. Our results reveal that a transient delay in Tr1-cell recruitment to the CNS, resulting from inflammation-dependent Tr1-cell allocation, accounts for the liver-centric effects of AIH-specific pMHCII-NPs in co-morbid mice as compared with mice exclusively having EAE. They also suggest that cognate autoantigen expression and local Tr1-cell retention are key determinants of effective regulatory-cell function. These results thus provide new insights into the rules that govern Tr1-cell recruitment and their autoregulatory function.

Galectin-1 is essential for the induction of MOG35-55 -based intravenous tolerance in experimental autoimmune encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE), intravenous (i.v.) injection of the antigen, myelin oligodendrocyte glycoprotein-derived peptide, MOG35-55 , suppresses disease development, a phenomenon called i.v. tolerance. Galectin-1, an endogenous glycan-binding protein, is upregulated during autoimmune neuroinflammation and plays immunoregulatory roles by inducing tolerogenic dendritic cells (DCs) and IL-10 producing regulatory type 1 T (Tr1) cells. To examine the role of galectin-1 in i.v. tolerance, we administered MOG35-55 -i.v. to wild-type (WT) and galectin-1 deficient (Lgals1(-/-) ) mice with ongoing EAE. MOG35-55 suppressed disease in the WT, but not in the Lgals1(-/-) mice. The numbers of Tr1 cells and Treg cells were increased in the CNS and periphery of tolerized WT mice. In contrast, Lgals1(-/-) MOG-i.v. mice had reduced numbers of Tr1 cells and Treg cells in the CNS and periphery, and reduced IL-27, IL-10, and TGF-ß1 expression in DCs in the periphery. DCs derived from i.v.-tolerized WT mice suppressed disease when adoptively transferred into mice with ongoing EAE, whereas DCs from Lgals1(-/-) MOG-i.v. mice were not suppressive. These findings demonstrate that galectin-1 is required for i.v. tolerance induction, likely via induction of tolerogenic DCs leading to enhanced development of Tr1 cells, Treg cells, and downregulation of proinflammatory responses.

Primary Tr1 cells from metastatic melanoma eliminate tumor-promoting macrophages through granzyme B- and perforin-dependent mechanisms.

In malignant melanoma, tumor-associated macrophages play multiple roles in promoting tumor growth, such as inducing the transformation of melanocytes under ultraviolet irradiation, increasing angiogenesis in melanomas, and suppressing antitumor immunity. Because granzyme B- and perforin-expressing Tr1 cells could specifically eliminate antigen-presenting cells of myeloid origin, we examined whether Tr1 cells in melanoma could eliminate tumor-promoting macrophages and how the interaction between Tr1 cells and macrophages could affect the growth of melanoma cells. Tr1 cells were characterized by high interleukin 10 secretion and low Foxp3 expression and were enriched in the CD4+CD49b+LAG-3+ T-cell fraction. Macrophages derived from peripheral blood monocytes in the presence of modified melanoma-conditioned media demonstrated tumor-promoting capacity, exemplified by improving the proliferation of cocultured A375 malignant melanoma cells. But when primary Tr1 cells were present in the macrophage-A375 coculture, the growth of A375 cells was abrogated. The conventional CD25+ Treg cells, however, were unable to inhibit macrophage-mediated increase in tumor cell growth. Further analyses showed that Tr1 cells did not directly eliminate A375 cells, but mediated the killing of tumor-promoting macrophages through the secretion of granzyme B and perforin. The tumor-infiltrating interleukin 10+Foxp3-CD4+ T cells expressed very low levels of granzyme B and perforin, possibly suggested the downregulation of Tr1 cytotoxic capacity in melanoma tumors. Together, these data demonstrated an antitumor function of Tr1 cells through the elimination of tumor-promoting macrophages, which was not shared by conventional Tregs.

Inhibition of CD8+ T cells and elimination of myeloid cells by CD4+ Foxp3- T regulatory type 1 cells in acute respiratory distress syndrome.

Acute lung injury and acute respiratory distress syndrome (ARDS) are caused by rapid-onset bilateral pulmonary inflammation. We therefore investigated the potential role of interleukin (IL)-10+ CD4+ Tr1 cells, a regulatory T cell subset with previously identified immunosuppressive functions, in ARDS patients. We first showed that circulating Tr1 cells were upregulated in active and resolved ARDS patients compared to healthy controls and pneumonia patient controls. A significant fraction of these Tr1 cells expressed granzyme B and perforin, while most Tr1 cells did not express interferon gamma (IFN-γ), IL-4, IL-17 or FOXP3, suggesting that the effector functions of these Tr1 cells were primarily mediated by IL-10, granzyme B, and perforin. Indeed, Tr1 cells effectively suppressed CD8+ T cell IFN-γ production and induced lysis of monocytes and dendritic cells in vitro. The elimination of myeloid antigen-presenting cells depended on granzyme B production. We also discovered that Tr1 cells could be identified in the bronchoalveolar lavage fluid collected from ARDS patients. All these results suggested that Tr1 cells possessed the capacity to downregulate inflammation in ARDS. In support of this, we found that ARDS patients who resolved the inflammation and survived the syndrome contained significantly higher levels of Tr1 cells than ARDS patients who succumbed to the syndrome. Overall, this report added a novel piece of evidence that ARDS could be intervened by regulatory T cell-mediated suppressive mechanisms.

Cytotoxic CD4 T cells in the mucosa and in cancer.

CD4 T cells were initially described as helper cells that promote either the cellular immune response (Th1 cells) or the humoral immune response (Th2 cells). Since then, a plethora of functionally distinct helper and regulatory CD4 T cell subsets have been described. CD4 T cells with cytotoxic function were first described in the setting of viral infections and autoimmunity, and more recently in cancer and gut dysbiosis. Regulatory CD4 T cell subsets such as Tregs and T-regulatory type 1 (Tr1) cells have also been shown to have cytotoxic potential. Indeed, Tr1 cells have been shown to be important for maintenance of stem cell niches in the bone marrow and the gut. This review will provide an overview of cytotoxic CD4 T cell development, and discuss the role of inflammatory and Tr1-like cytotoxic CD4 T cells in maintenance of intestinal stem cells and in anti-cancer immune responses.

Substance P promotes immunotherapy efficacy for airway allergy.

Background: Allergen-specific immunotherapy (AIT) has been employed in the treatment of allergic diseases for many years. However, the effectiveness of AIT requires improvement. Substance P (SP) can interact with immune cells, modulate immune cell activity, and regulate immune reaction. The purpose of this study is to use SP as an immune regulator to enhance the therapeutic efficacy of AIT. Methods: An established mouse model of the airway allergy disorder (AAD) was employed with ovalbumin as a specific antigen. The AAD response was evaluated through established procedures. AAD mice were treated with AIT employing SP as an immune regulator. Dendritic cells were isolated from the airway tissues by magnetic cell sorting, and were analyzed by RNA sequencing (RNAseq). Results: We observed that after sensitization with ovalbumin, mice exhibited AAD-like symptoms, serum specific IgE, and Th2 polarization. The presence of SP in the course of sensitization prevented the development of AAD. Treating mice with SP by nasal instillations induced IL-10, but not TGF-ß, in dendritic cells of the airway tissues. The most differentially expressed genes (DEG) in the dendritic cells were those related to the IL-10 expression, including Il10, Tac1r, and Mtor. The gene ontology analysis showed that these DEGs mainly mapped to the tachykinin-PI3K-AKT-mTOR pathway. The addition of SP substantially enhanced the therapeutic efficacy of AIT for AAD by inducing antigen specific type 1 regulatory T cells (Tr1 cells). Conclusion: Acting as an immune regulator, SP promotes the therapeutic efficacy for AAD by inducing antigen specific Tr1 cells in the airway tissues.

Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation.

Intestinal colonization by antigenically foreign microbes necessitates expanded peripheral immune tolerance. Here we show commensal microbiota prime expansion of CD4 T cells unified by the Kruppel-like factor 2 (KLF2) transcriptional regulator and an essential role for KLF2+ CD4 cells in averting microbiota-driven intestinal inflammation. CD4 cells with commensal specificity in secondary lymphoid organs and intestinal tissues are enriched for KLF2 expression, and distinct from FOXP3+ regulatory T cells or other differentiation lineages. Mice with conditional KLF2 deficiency in T cells develop spontaneous rectal prolapse and intestinal inflammation, phenotypes overturned by eliminating microbiota or reconstituting with donor KLF2+ cells. Activated KLF2+ cells selectively produce IL-10, and eliminating IL-10 overrides their suppressive function in vitro and protection against intestinal inflammation in vivo. Together with reduced KLF2+ CD4 cell accumulation in Crohn's disease, a necessity for the KLF2+ subpopulation of T regulatory type 1 (Tr1) cells in sustaining commensal tolerance is demonstrated.

Expression of CD226 is upregulated on Tr1 cells from neuromyelitis optica spectrum disorder patients.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is a central and acute demyelinating disease of the central nervous system with unusual clinical course. The development of novel biomarkers for NMOSD is critical for implementing effective clinical treatment. CD226 is known to be expressed on many types of peripheral lymphoid cells. However, the expression level and function of CD226 on type 1 T regulatory (Tr1) cells during NMOSD is unknown. METHODS: Eighteen patients with NMOSD and 10 healthy volunteers were enrolled in the test group to probe the difference of CD226 expression on Tr1 cells using flow cytometric analysis. RESULTS: The expression of CD226 on Tr1 cells exhibited significantly increased tendency in NMOSD patients. Additionally, methylprednisolone and rituximab treatment decreased the expression of CD226 on Tr1 cells. Furthermore, the expression of CD226 on Tr1 cells was correlated with disease severity. CONCLUSION: This study provides a new basic insight into CD226 expression pattern on Tr1 cells, which have great potential to be biomarkers for monitoring the development and treatment of NMOSD.

The Mechanism of Action of Antigen Processing Independent T Cell Epitopes Designed for Immunotherapy of Autoimmune Diseases.

Immunotherapy with antigen-processing independent T cell epitopes (apitopes) targeting autoreactive CD4+ T cells has translated to the clinic and been shown to modulate progression of both Graves' disease and multiple sclerosis. The model apitope (Ac1-9[4Y]) renders antigen-specific T cells anergic while repeated administration induces both Tr1 and Foxp3+ regulatory cells. Here we address why CD4+ T cell epitopes should be designed as apitopes to induce tolerance and define the antigen presenting cells that they target in vivo. Furthermore, we reveal the impact of treatment with apitopes on CD4+ T cell signaling, the generation of IL-10-secreting regulatory cells and the systemic migration of these cells. Taken together these findings reveal how apitopes induce tolerance and thereby mediate antigen-specific immunotherapy of autoimmune diseases.

Induction of Tolerance to Therapeutic Proteins With Antigen-Processing Independent T Cell Epitopes: Controlling Immune Responses to Biologics.

The immune response to exogenous proteins can overcome the therapeutic benefits of immunotherapies and hamper the treatment of protein replacement therapies. One clear example of this is haemophilia A resulting from deleterious mutations in the FVIII gene. Replacement with serum derived or recombinant FVIII protein can cause anti-drug antibodies in 20-50% of individuals treated. The resulting inhibitor antibodies override the benefit of treatment and, at best, make life unpredictable for those treated. The only way to overcome the inhibitor issue is to reinstate immunological tolerance to the administered protein. Here we compare the various approaches that have been tested and focus on the use of antigen-processing independent T cell epitopes (apitopes) for tolerance induction. Apitopes are readily designed from any protein whether this is derived from a clotting factor, enzyme replacement therapy, gene therapy or therapeutic antibody.

Function of T regulatory type 1 cells is down-regulated and is associated with the clinical presentation of coronary artery disease.

T regulatory type 1 (Tr1) cells can promote tolerance and suppress inflammation. Atherosclerosis may be induced by the proinflammatory activation of cells in the vasculature and the immune system. Hence, we wondered whether defects in Tr1 function were a contributing factor to coronary artery disease (CAD). Data showed that the frequency of IL-10+ Tr1 cells was significantly lower in CAD patients than in controls. Compared to healthy controls, Tr1 cells from CAD patients presented lower CTLA-4 but higher PD-1 expression, in addition to lower IL-10 secretion. When co-incubated with Tconv cells, the CD4+CD49b+LAG-3+CD45RO+ Tr1 cells presented IL-10-dependent inhibitory effects, and those from CAD patients presented significantly lower suppression capacity than those from healthy controls. Interestingly, the characteristics of Tr1 cells were associated with clinical features of CAD patients. The frequency of Tr1 cells and the IL-10 and LAG-3 expression by Tr1 cells were negatively correlated with the BMI of the CAD patients. In addition, the Tr1 frequency and the LAG-3 and CTLA-4 expression on Tr1 cells were lower in CAD patients with higher numbers of narrowed vessels. Together, these results suggest that in CAD, Tr1 cells present multiple defects, which are associated with the clinical presentation of the disease.

Regulation of allergic airway inflammation by adoptive transfer of CD4+ T cells preferentially producing IL-10.

Anti-inflammatory pharmacotherapy for asthma has mainly depended on the inhalation of glucocorticoids, which non-specifically suppress immune responses. If the anti-inflammatory cytokine interleukin (IL)-10 can be induced by a specific antigen, asthmatic airway inflammation could be suppressed when individuals are exposed to the antigen. The purpose of this study was to develop cellular immunotherapeutics for atopic diseases using IL-10-producing CD4+ T cells. Spleen cells isolated from ovalbumin (OVA)-sensitized mice were cultured with the antigen, OVA and growth factors, IL-21, IL-27 and TGF-ß for 7 days. After the 7-day culture, the CD4+ T cells were purified using a murine CD4 magnetic beads system. When the induced CD4+ T cells were stimulated by OVA in the presence of antigen-presenting cells, IL-10 was preferentially produced in vitro. When CD4+ T cells were adoptively transferred to OVA-sensitized mice followed by intratracheal OVA challenges, IL-10 was preferentially produced in the serum and bronchoalveolar lavage fluid in vivo. IL-10 production coincided with the inhibition of eosinophilic airway inflammation and epithelial mucus plugging. Most of the IL-10-producing CD4+ T cells were negative for Foxp3 and GATA-3, transcription factors of naturally occurring regulatory T cells and Th2 cells, respectively, but double positive for LAG-3 and CD49b, surface markers of inducible regulatory T cells, Tr1 cells. Collectively, most of the induced IL-10-producing CD4+ T cells could be Tr1 cells, which respond to the antigen to produce IL-10, and effectively suppressed allergic airway inflammation. The induced Tr1 cells may be useful for antigen-specific cellular immunotherapy for atopic diseases.

CD4+Foxp3-IL-10+ Tr1 Cells Promote Relapse of Diffuse Large B Cell Lymphoma by Enhancing the Survival of Malignant B Cells and Suppressing Antitumor T Cell Immunity.

Diffuse large B cell lymphoma (DLBCL) is a common B cell malignancy. Complete remission can be achieved in most patients by conventional treatment with rituximab and chemotherapy. However, a subset of remission individuals will develop a relapsed disease for obscure reasons. CD4+Foxp3-IL-10+ cell (Tr1) is a novel cell subtype with the capacity to suppress pro-inflammatory responses, but has not been extensively studied in most tumors. In this study, we investigated the potential role of Tr1 cells in DLBCL. We found that compared to that in healthy controls, the frequency of Tr1 cells was significantly increased in DLBCL patients, even during complete remission. Further study showed that these Tr1 cells were enriched in the CD25low/-Foxp3-CD49b+LAG-3+ fraction and could be developed in vitro from naive CD45RA+ CD4+ T cells. To examine the effect of Tr1 upregulation, we cocultured the enriched in vitro-induced Tr1 cells (iTr1) with autologous primary DLBCL cells and CD3+ T cells and found that iTr1 cells both enhanced the survival of CD20+ DLBCL tumor cells and suppressed the antitumor response of CD3+ T cells through the production of IL-10. Furthermore, the frequency of CD4+Foxp3-IL-10+ Tr1 cells in DLBCL patients during complete remission is directly associated with the risk of relapse. Together, these results suggested that Tr1 cells contributed to tumor cell maintenance and may serve as a prognostic marker and therapeutic target.

CD4+Foxp3- type 1 regulatory T cells in glioblastoma multiforme suppress T cell responses through multiple pathways and are regulated by tumor-associated macrophages.

CD4+Foxp3- type 1 regulatory T (Tr1) cells are potent producers of interleukin 10 (IL-10) and transforming growth factor beta (TGF-ß), through which they suppress pathogenic inflammation and autoimmune responses. The role of Tr1 response in glioblastoma multiforme (GBM) is still unclear. Here, we examined the frequency, phenotype, induction mechanism, and function of Tr1 cells in GBM patients. Compared to healthy controls, GBM patients presented significantly higher frequency of Tr1 cells in peripheral blood. The Tr1 frequency was further elevated in the tumor. By surface marker expression, the Tr1 cells were enriched in the antigen-experienced effector/memory cell compartment. A minority of Tr1 cells presented IL-10+TGF-ß+ double expression. Interestingly, naive CD4+CD45RA+ T cells could differentiate into IL-10- and TGF-ß-expressing cells, if incubated with tumor-associated macrophages (TAMs) or with macrophages conditioned with primary glioma cells, suggesting that tumor cells and TAMs had a role in inducing Tr1 cells in GBM patients. Coculture of Tr1 cells with proinflammatory CD4+ T cells resulted in TGF-ß-dependent reduction of interferon gamma (IFN-γ) and IL-10-dependent reduction of tumor necrosis factor alpha (TNF-α), while coculture of Tr1 cells with CD8+ T cells resulted in lower tumor-specific cytotoxicity. Together, these results demonstrated an upregulation of Tr1 cells in GBM with anti-inflammatory functions.

Studies on the immunotherapy of EAE by adoptive transfer of immunoregulatory T cells specific for the spreading determinant / 中国免疫学杂志

Objective:To explore the therapeutic potential of preemptive targeting of the epitope spreading cascade by adoptive transfer after EAE onset of immunoregulatory T cells specific for the MBP87-99 spreading determinant.Methods:EAE was induced by PLP139-151 in SWXJ mice, all mice were weighed and examined daily for neurologic signs. On days 6 after EAE onset, mice were injected with IL-2prom→IL-10cDNA-transfected Th2/Tr1-like T cells specific for MBP87-99,PLP104-117,or BSA. Finally demyelination was quantified by analysis of digitized images of PLP immunostained spinal cord sections.Results:Mice received Th2/Tr1-like T cells specific for MBP87-99 had a significantly improved clinical outcome(P=0.02) compared with control mice, reduction in the relapse rate and delay in mean time to onset of first relapse were also observed(P