Immune Tolerance In Patients Research Articles

Elevated level of multibranched complex glycan reveals an allergic tolerance status

BackgroundAllergen immunotherapy (AIT) is the only disease-modifying therapy that can achieve immune tolerance in patients through long-term allergen stimulation. Glycans play crucial roles in allergic disease, but no information on changes in glycosylation related to an allergic tolerance status has been reported.MethodsFifty-seven patients with house dust mite (HDM) allergies were enrolled. Twenty-eight patients were not treated with AIT, 19 patients had just entered the AIT maintenance treatment phase, and 10 patients had been in the AIT maintenance phase for more than 1 year. Serum protein N-glycans were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), which included linkage-specific sialylation information.ResultsEighty-four N-glycans were identified in all three groups. Compared with the patients treated without AIT, the patients treated with AIT for a shorter time showed downregulated expression of high-mannose glycans and upregulated expression of α2,6 sialic acid. The patients treated with AIT in the maintenance phase for over 1 year, which was considered the start of immunological tolerance, showed downregulated expression of biantennary N-glycans and upregulated expression of multibranched and complex N-glycans. Nine N-glycans were changed between allergic and allergic-tolerant patients.ConclusionsThe glycan form changed from mannose to a more complex type as treatment time increased, and multibranched complex glycans have the potential to be used as a monitoring indicator of immune tolerance. This serum N-glycome analysis provided important information for a deeper understanding of AIT treatment at the molecular level.

HBeAg induces neutrophils activation impairing NK cells function in patients with chronic hepatitis B.

The role of neutrophils in hepatitis B virus (HBV) infection has been a subject of debate due to their involvement in antiviral responses and immune regulation. This study aimed to elucidate the neutrophil characteristics in patients with chronic hepatitis B (CHB). Through flow cytometry and ribonucleic acid-sequencing analysis, the phenotypes and counts of neutrophils were analyzed in patients with CHB. Moreover, the effects of HBeAg on neutrophils and the corresponding pattern recognition receptors were identified. Simultaneously, the cross-talk between neutrophils and natural killer (NK) cells was investigated. Neutrophils were activated in patients with CHB, characterized by higher expression levels of programmed death-ligand 1 (PD-L1), cluster of differentiation 86, and interleukin-8, and lower levels of CXC motif chemokine receptor (CXCR) 1 and CXCR2. Hepatitis B e antigen (HBeAg) partially induces neutrophil activation through the Toll-like receptor 2 (TLR2). A consistent upregulation of the TLR2 and HBeAg expression was observed in patients with CHB. Notably, the genes encoding molecules pivotal for NK-cell function upon NK receptor engagement enriched in neutrophils after HBeAg activation. The HBeAg-activated neutrophils demonstrated the ability to decrease the production of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in NK cells, while the PD-1 and PD-L1 pathways partially mediated the immunosuppression. The immunosuppression of neutrophils induced by HBeAg suggests a novel pathogenic mechanism contributing to immune tolerance in patients with CHB.

GNTI-122: an autologous antigen-specific engineered Treg cell therapy for type 1 diabetes.

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving β cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.

Real-world data on the use of emicizumab in patients with haemophilia A with and without inhibitors in Singapore.

Emicizumab is a bispecific monoclonal antibody that mimics the function of factor VIII by binding to factor IXa and factor X to achieve haemostasis in haemophilia A. The long half-life and subcutaneous mode of administration makes emicizumab a compelling treatment option for bleeding prophylaxis. There is still limited real-world data on its use and management considerations, especially during surgical procedures. The objective of the study is to describe the real-world experience of emicizumab in a cohort of adult and paediatric haemophilia A patients in Singapore, including its use in the periprocedural setting. This was an observational study conducted at the 2 main haemophilia treatment centres in Singapore. All haemophilia A patients who commenced treatment with emicizumab before 1 July 2022 were recruited. A total of 18 patients with haemophilia A were included in this study. Ten (55.6%) patients had active inhibitors. The median annual bleeding rate for all patients before emicizumab use was 4.5 events (interquartile range [IQR] 2.8-8.3) compared with 0 events (IQR 0-0) after emicizumab was commenced (P=0). There were no adverse events of venous or arterial thrombosis, thrombotic microangiopathy, or death. A total of 6 procedures in 5 patients were performed during the study period with no major bleeding complications. Emicizumab effectively protects against bleeding in haemophilia A patients with and without inhibitors, including in children less than 12 years old. More studies are required to address clinical nuances, such as periprocedural management and the role of immune tolerance in patients with inhibitors on emicizumab.

Transplantation of Hematopoietic Stem and Progenitor Cells from Cadaveric Organ Donors Leads to Long-Term Multilineage Engraftment in NSG Mice

Background Infusion of Hematopoietic Stem and Progenitor Cells (HSPC) from cadaveric organ donors (CD) has been used anecdotally in the setting of allogeneic Hematopoietic Stem Cell transplants (HSCT) most commonly with the intention for induction of mixed hematopoietic chimerism and subsequent immunological tolerance. No systematic study has addressed the functional capacity of these cells for inducing long-term multi-lineage engraftment. Since the introduction of post-transplant Cyclophosphamide (PTCy) it has been possible to reliably overcome HLA barriers and achieve stable engraftment across extensively HLA-mismatched donor and recipient pairs. Banked HSPC from cadaveric donors may provide a new approach to expand the number of donors for HSCT. Usage of HSPC from bone marrow (BM) of banked cadaveric donors is viewed as no different than live donor aspirated marrow by the FDA but is not yet available on the registry. This emerging source could be of high importance to allow rapid on-demand access to transplants for patients who lack HSPC donors or induce stable mixed chimerism and immune tolerance in patients undergoing combined organ and BM transplantation. The objective of our studies was to investigate whether hCD34⁺HSC selected from cadaveric BM retain their capacity for long-term multi-lineage engraftment. Methods and Results We compared CD34⁺-selected HSPC obtained from CD and living donors (LD) regarding their in vitro and in vivo function and with particular focus on their ability to achieve long-term multi-lineage engraftment following transplantation into NSG mice. Bone marrow was recovered from vertebral bodies of CD and CD34⁺ selected. 5x10⁵ hCD34⁺ cells from 24 different CD were transplanted i.v. in to NSG or SGM3 mice (n=5 per donor) following 1Gy total body irradiation (TBI). Blood was drawn every two weeks, starting week 4 and human CD45⁺ chimerism was analyzed via flow cytometry. All donors engrafted successfully and robust long-term multi-lineage engraftment >16 weeks was seen in 19 out of 22 donors. To further investigate the long-term repopulating capacity, we performed secondary transplants. BM of 15 primary engrafted donors was harvested, hCD45⁺ were selected with immunobeads and transplanted into 1Gy irradiated NSG mice. Successful multi-lineage engraftment after secondary transfer was observed in 12 out of 15 donors in the peripheral blood (PB). Results of tissue engraftment are still outstanding for most experiments. So far they showed mean engraftment of human CD45⁺ cells of 6.16%+/-2.4% for donor 1 and 8.48%+/-0.74% for donor 2 in the BM. To compare CD and LD we set up two side-by-side transplantation experiments and intra-mouse competitive engraftment studies using HLA-mismatched donors. Using side-by-side experiments no significant difference in human CD45⁺ chimerism was detected in blood measured at weeks 6, 12 and 16 pos-transplant. The tissues showed a mean engraftment of human CD45⁺ cells of 87% +/- 9.6% vs 71% +/- 9.5% in spleen and 76% +/- 5.3% vs 78% +/- 4% (NS) in BM upon takedown 20 weeks post-transplant. For our competitive assays, we transplanted 2.5x10⁵ hCD34⁺ i.v. from LD and CD simultaneously into NSG mice (n=5 per donor pair) following 1Gy TBI. In total 8 CD and 3 LD were used, out of whom 4 CD reached higher engraftment than the LD. Conclusion Our data suggest that CD34+HSPC from CD retain their long-term multi-lineage engraftment capacity, reaching at least equipotent levels as HSPC from living donors. Based on our findings cadaveric bone marrow should be considered as HSPC source for HSCT.

HDAC3 single-nucleotide polymorphism rs2530223 is associated with increased susceptibility and severity of primary immune thrombocytopenia.

Primary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disorder characterized by a low platelet count and increased risk of bleeding. We previously reported that low-dose chidamide, a histone deacetylase (HDAC) inhibitor, restores immune tolerance in patients with ITP. This study aimed to evaluate the association of a single-nucleotide polymorphism (SNP) rs2530223 in the HDAC3 gene with susceptibility to ITP and its clinical features. Patients with ITP and age-matched healthy participants were recruited for this case-control study. Genotyping of the HDAC3 rs2530223 polymorphism was performed using MassARRAY platform. Individuals with T allele of HDAC3 rs2530223 exhibited a 1.472-fold increased risk of ITP susceptibility (OR 1.472; 95% CI 1.100-1.969; p=.009), while ones with the TT genotype under the codominant and recessive models, and the TC/TT genotypes under the dominant model all revealed increased risk of ITP susceptibility (dominant odds ratio[OR] 1.965; 95% CI: 1.046-3.656; p=.036; codominant OR 2.264; 95% CI 1.175-4.360; p=.015; and recessive OR 1.512; 95% CI 1.028-2.224; p=.036, respectively). Regarding platelet counts in ITP patients, we observed that the TC/TT genotypes exhibited a 3.932-fold increased risk for platelet (PLT) <30 × 109 /L (OR 3.932; 95% CI 1.426-10.842; p=.008). This study indicates that HDAC3 rs2530223 may be an important genetic factor related to ITP susceptibility and platelet count in ITP patients, providing new perspectives on disease progression, new therapeutic targets, and severity prediction.

Clinical Features and T Cell Immune Characteristics of Postpartum Hepatitis Flare in Pregnant Women With HBeAg-Positive Chronic HBV Infection.

BackgroundThe extent of the increase in postpartum alanine transaminase (ALT) varies significantly among pregnant women in the immune tolerance stage of nucleoside analogue (NA) intervention, so this study is an attempt to analyze the clinical features of patients with and without postpartum hepatitis flare and preliminarily explore the differences in their immune functions.MethodsPregnant women with a gestational age of 24–28 w and in the immune tolerance stage of NA intervention for hepatitis B virus (HBV) infection were included and divided into a hepatitis group (Group 1) and a nonhepatitis group (Group 2) according to the ALT level at 6–12 w after childbirth. The clinical features were analyzed, and the phenotypes, functions, and cytokines of clusters of differentiation CD8+ T cells in the two groups of patients were detected using flow cytometry before and after childbirth.ResultsA total of 15 patients with postpartum hepatitis flare were enrolled in Group 1, and 10 matched patients were selected as controls for Group 2. Compared with the individuals in Group 2, the postpartum clinical features in Group 1 included a remarkable elevation of the ALT level on the basis of a relatively low HBV DNA level, usually accompanied by a decline in hepatitis B virus surface antigen levels as well as HBeAg levels. In addition, CD8+ T cell activation was enhanced after childbirth in Group 1. In particular, there was a notable difference in the activation of TEMRA subsets, and the frequency of CD8+ T cells expressing perforin and granzyme B increased.ConclusionThe changes in the immune characteristics of CD8+ T cells may play a certain role in breaking down immune tolerance in patients with postpartum hepatitis flare, and the indexes related to activating and killing functions may help to indicate the population with hepatitis flare after childbirth.

Chinese Guideline on Allergen Immunotherapy for Allergic Rhinitis: The 2022 Update.

In the last few decades, there has been a progressive increase in the prevalence of allergic rhinitis (AR) in China, where it now affects approximately 250 million people. AR prevention and treatment include allergen avoidance, pharmacotherapy, allergen immunotherapy (AIT), and patient education, among which AIT is the only curative intervention. AIT targets the disease etiology and may potentially modify the immune system as well as induce allergen-specific immune tolerance in patients with AR. In 2017, a team of experts from the Chinese Society of Allergy (CSA) and the Chinese Allergic Rhinitis Collaborative Research Group (C2AR2G) produced the first English version of Chinese AIT guidelines for AR. Since then, there has been considerable progress in basic research of and clinical practice for AIT, especially regarding the role of follicular regulatory T (TFR) cells in the pathogenesis of AR and the use of allergen-specific immunoglobulin E (sIgE) in nasal secretions for the diagnosis of AR. Additionally, potential biomarkers, including TFR cells, sIgG4, and sIgE, have been used to monitor the incidence and progression of AR. Moreover, there has been a novel understanding of AIT during the coronavirus disease 2019 pandemic. Hence, there was an urgent need to update the AIT guideline for AR by a team of experts from CSA and C2AR2G. This document aims to serve as professional reference material on AIT for AR treatment in China, thus improving the development of AIT across the world.

Engineering a safe monoclonal anti‐human IL‐2 that is effective in a murine model of food allergy and asthma

Regulatory T cells (Tregs) are known to protect against allergies. Moreover, the decrease in the frequency and efficiency of Tregs amplifies allergic symptoms. This study investigated whether expanding Tregs in vivo with an IL-2/IL-2 antibody complex could be safe, well tolerated and efficient in a therapeutic setting in allergies. We produced an anti-IL-2 antibody (1C6) and demonstrated that when it is complexed to human IL-2, it increases IL-2 efficiency to induce Tregs in vivo without any detectable side effects. Furthermore, the IL-2/1C6 complex induces an increase in Helios expression by Tregs, suggesting that it not only elevated Treg numbers but also boosted their functions. Using mouse models of house-dust-mite-induced airway inflammation and wheat-gliadin-induced food allergies, we investigated the therapeutic potential of the IL-2/1C6 complex in allergies. IL-2/1C6 treatment significantly reduced allergic symptoms, specific IgE production, the adaptive immune response and tissue damage. Interestingly, IL-2/1C6 treatment modulated innate lymphoid cells by increasing ILC2s in asthma and decreasing ILC3s in food allergies. In conclusion,complexed IL-2/anti-IL-2may restore Treg numbers and function in respiratory and food allergies, thereby improving allergic markers and symptoms. Our IL-2/anti-IL-2 complex offers new hope for reestablishing immune tolerance in patients with allergies.

The skin microbiome-useful for diagnosis and therapy?

Our skin is avery important and complex organ of the body. The microorganisms of the skin, the so-called microbiome, represent an important part of the healthy skin barrier and are influenced by various external and internal factors. The question to what extent the skin microbiome represents adiagnostic or even therapeutic target in the context of skin diseases is discussed. Aliterature search was performed. Several diseases are associated with negative alterations of the skin microbiome. In atopic dermatitis, acorrelation between severity and increased availability of Staphylococcus aureus is known, with aloss of bacterial diversity on the skin. In the future, S.aureus will not only be used as adiagnostic marker in atopic dermatitis, but also represents apromising target as apredictive marker for therapeutic success. The role of the skin microbiome in psoriasis has not yet been researched in depth. However, there is evidence that dysbiosis of the skin microbiome contributes to the course of psoriasis and that there is adisturbance in immune tolerance in patients. In the case of acne, the involvement of Cutibacterium acnes in the clinical picture is well known; however, recent findings show that it is not sufficient to identify the species, but certain characteristics of C.acnes strains are associated. Microbial biomarkers are currently only established in atopic dermatitis. For other diseases, this might be the case in the future; however combinations of microorganisms, single species and also strains with specific characteristics must be considered.

Oral Administration of Brain Protein Combined With Probiotics Induces Immune Tolerance Through the Tryptophan Pathway.

Excessive inflammation leads to secondary immune damage after traumatic brain injury (TBI). The intestinal mucosa is a key component of immune tolerance due to gut-brain axis regulation, but the curative effect is not optimal. Intestinal dysfunction impairs the establishment of immune tolerance in patients with TBI. Therefore, we orally administered brain protein (BP) combined with probiotics to induce immune tolerance and explored the mechanism by which the homeostasis of the microbiota contributes to the enhancement of curative effects by BPs. Herein, we demonstrated that patients with TBI and surgical brain injury (SBI) models of rats had obvious dysbiosis. Notably, the intestinal barrier, proinflammatory cytokines, and activation of microglia demonstrated that excessive inflammatory damage was better controlled in the combined group (oral administration of BP combined with probiotics) than in the BP group (oral administration of BP). Fundamentally, tandem mass tag (TMT)-based quantitative proteomics analysis revealed that BP and probiotics preferentially affect Try-related pathways. A series of experiments further confirmed that Indoleamine 2,3 dioxygenase (IDO)/Kynurenine (Kyn)/Aryl hydrocarbon receptor (AhR) expression was high in the BP group, while Tryptophan hydroxylase 1(TpH1)/5-hydroxytryptamine (5-HT) only changed in the combined group. This study suggests that probiotics can enhance the efficacy of oral BP-induced immune tolerance through the Try pathway.

Mycophenolate mofetil enhances the effects of tacrolimus on the inhibitory function of regulatory T cells in patients after liver transplantation via PD-1 and TIGIT receptors

Objective Regulatory T cells (Tregs) induce immune tolerance in patients after organ transplantation. Various immunosuppressors can affect Tregs function through different mechanisms. PD-1 and TIGIT are important receptors on Tregs surface. Here, we investigated the effects of Tacrolimus and mycophenolate mofetil (MMF) on the inhibitory function of Tregs and explored the regulatory mechanism in patients after liver transplantation. Methods Thirty patients that underwent a liver transplant and 15 healthy people were enrolled. Fifteen patients received Tacrolimus only, and 15 received a combination of Tacrolimus and MMF. Tregs and effector T cells (Teffs) were isolated using magnetic beads and were mixed at different ratios of 0:1, 1:4, 1:2 and 1:1. An inhibition assay was performed by adding anti-PD-1 and anti-TIGIT when the mixture ratio was 1:1. The Tregs inhibition rate was determined and the levels of IFN-γ and TNF-α were measured. Results As the ratios of Tregs to Teffs in the mixture increased, the Tregs inhibition rate increased and the levels of IFN-γ and TNF-α decreased. At each mixture ratio, Tacrolimus + MMF group had the highest Tregs inhibition rate compared to Tacrolimus and control group. At the specific mixture ratio of 1:1, the addition of both anti- PD-1 and anti-TIGIT led to lower Tregs inhibition rate and higher IFN-γ and TNF-α levels in all three groups as opposed to the addition of each antibody separately. Additionally, both the decrease in the Tregs inhibition rate and the increase in the IFN-γ and TNF-α levels were the most for Tacrolimus + MMF group among all cases, either adding antibodies alone or mixed. Conclusion Tacrolimus and MMF enhanced the function of Tregs by synergistically affecting PD-1 and TIGIT in liver transplant patients.

Expansion of CD45RA−FOXP3++ regulatory T cells is associated with immune tolerance in patients with combined kidney and bone marrow transplantation

ObjectivesSimultaneous transplantation of a solid organ and bone marrow from the same donor is a possible means of achieving transplant tolerance. Here, we attempted to identify biomarkers that indicate transplant tolerance for discontinuation of immunosuppressants in combined kidney and bone marrow transplantation (CKBMT).MethodsConventional kidney transplant (KT) recipients (n = 20) and CKBMT recipients (n = 6) were included in this study. We examined various immunological parameters by flow cytometry using peripheral blood mononuclear cells (PBMCs), including the frequency and phenotype of regulatory T (Treg) cell subpopulations. We also examined the suppressive activity of the Treg cell population in the setting of mixed lymphocyte reaction (MLR) with or without Treg cell depletion.ResultsAmong six CKBMT recipients, three successfully discontinued immunosuppressants (tolerant group) and three could not (non‐tolerant group). The CD45RA−FOXP3++ Treg cell subpopulation was expanded in CKBMT recipients compared to conventional kidney transplant patients, and this was more obvious in the tolerant group than the non‐tolerant group. In addition, high suppressive activity of the Treg cell population was observed in the tolerant group. The ratio of CD45RA−FOXP3++ Treg cells to CD45RA−FOXP3+ cells indicated good discrimination between the tolerant and non‐tolerant groups.ConclusionThus, our findings propose a biomarker that can distinguish CKBMT patients who achieve transplant tolerance and are eligible for discontinuation of immunosuppressants and may provide insight into tolerance mechanisms in CKBMT.

Treg-specific IL-2 therapy can reestablish intrahepatic immune regulation in autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic autoimmune inflammatory disease that usually requires life-long immunosuppression. Frequent relapses after discontinuation of therapy indicate that intrahepatic immune regulation is not restored by current therapies. As steroid therapy preferentially depletes intrahepatic regulatory T cell (Tregs), immune regulation might be re-established by increasing and functionally strengthening intrahepatic Tregs. In recent clinical trials with low dose IL-2, the Treg compartment was strengthened in autoimmune diseases. Therefore, we tested complexed IL-2/anti-IL-2 to increase the selectivity for Tregs. We used our model of experimental murine AIH (emAIH) and treated the mice with complexed IL-2/anti-Il-2 in the late course of the disease. The mice showed increased intrahepatic and systemic Treg numbers after treatment and a reduction in activated, intrahepatic effector T cells (Teffs). This resulted in a reduction in liver-specific ALT levels and a molecular pattern similar to that of healthy individuals. In conclusion, complexed IL-2/anti-IL-2 restored the balance between Tregs and Teffs within the liver, thereby improving the course of emAIH. Treg-specific IL-2 augmentation offers new hope for reestablishing immune tolerance in patients with AIH.

Persistent IL-2 Receptor Signaling by IL-2/CD25 Fusion Protein Controls Diabetes in NOD Mice by Multiple Mechanisms.

Low-dose interleukin-2 (IL-2) represents a new therapeutic approach to regulate immune homeostasis to promote immune tolerance in patients with autoimmune diseases, including type 1 diabetes. We have developed a new IL-2-based biologic, an IL-2/CD25 fusion protein, with greatly improved pharmacokinetics and pharmacodynamics when compared with recombinant IL-2 to enhance this type of immunotherapy. In this study, we show that low-dose mouse IL-2/CD25 (mIL-2/CD25), but not an equivalent amount of IL-2, prevents the onset of diabetes in NOD mice and controls diabetes in hyperglycemic mice. mIL-2/CD25 acts not only to expand regulatory T cells (Tregs) but also to increase their activation and migration into lymphoid tissues and the pancreas. Lower incidence of diabetes is associated with increased serum levels of IL-10, a cytokine readily produced by activated Tregs. These effects likely act in concert to lower islet inflammation while increasing Tregs in the remaining inflamed islets. mIL-2/CD25 treatment is also associated with lower anti-insulin autoantibody levels in part by inhibition of T follicular helper cells. Thus, long-acting mIL-2/CD25 represents an improved IL-2 analog that persistently elevates Tregs to maintain a favorable Treg/effector T cell ratio that limits diabetes by expansion of activated Tregs that readily migrate into lymphoid tissues and the pancreas while inhibiting autoantibodies.

SAT0278 LOW-DOSE IL-2 RESTORES TREG-MEDIATED IMMUNE TOLERANCE IN PATIENTS WITH ANCA-ASSOCIATED VASCULITIS

Background:Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune disease that can cause systemic organ damage, including granulomatosis with polyangiitis(GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis(EGPA)[1]. Several observations have showed that the breakdown of immune tolerance was involved in the pathogenesis of AAV [2], furthermore, a single, open and clinical trial demonstrates that IL-2 can be used to treat patients with GPA [3]. But there is still a lack of understanding of the relationship between Th17 / Treg and AAV and evidence for the therapeutic effect of IL-2 on AAV, which needs further exploration.Objectives:We first measured the absolute number of CD4+T subsets in peripheral blood of patients to explore the pathogenesis of AAV, and then investigated the effects of short-term and low-dose recombinant human IL-2 (rhIL-2) on CD4+T subsets of patients to analyze the regulatory effect of IL-2 on AAV.Methods:49 patients with AAV, hospitalized at the Second Hospital of Shanxi Medical University from the May 2016 to the November 2019 were enrolled, including 36 patients who were only received conventional glucocorticoids and DMARDs, and other 13 patients who were not only received these treatments but were also injected subcutaneously rhIL-2(50WIU/day for a 5-day course). 31 age and gender-matched healthy adults were selected as controls. The absolute number of Th17 and Treg cells in peripheral blood of health controls and the patients before and after treatment was detected by flow cytometry.Results:There was significant decreased level of Treg cells in the patients with AAV compared with healthy controls (P&lt;0.001) leading to a higher Th17/Treg ratio in the patients with AAV, but there was no statistically significant in the absolute number of Th17 cells between the patients and healthy controls. After the treatment of short-term and low-dose IL-2, there was a significant increase in the absolute number of Treg cells (P&lt;0.01) leading to a decrease in the ratio of Th17 and Treg (p&lt;0.05).The absolute number of Th17 had a trend towards higher values but was not statistical significance.Conclusion:The difference of Treg cells between the patients and healthy controls suggested that the decreased number of Treg cells failed to control autoimmune inflammatory response contributing to the pathogenesis of AAV. After the treatment of short-term and low-dose rhIL-2, there was a more significant increase in the absolute number of Treg cells showing that IL-2 could selectively stimulate the growth of Treg cells and restore the Treg-mediated immune tolerance in patients with AAV to achieve disease remission.

Microencapsulated G3C Hybridoma Cell Graft Delays the Onset of Spontaneous Diabetes in NOD Mice by an Expansion of Gitr+ Treg Cells.

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet β-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3+ Treg-cell expansion in the spleen of wild-type mice but not in Gitr-/- mice. G3C/cps also induced the expansion of nonconventional Cd4+Cd25-/lowFoxp3lowGitrint/high (GITR single-positive [sp]) Tregs. Both Cd4+Cd25+GitrhighFoxp3+ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.

Synthetically glycosylated antigens induce antigen-specific tolerance and prevent the onset of diabetes.

Homeostatic antigen presentation by hepatic antigen-presenting cells, which results in tolerogenic T-cell education, could be exploited to induce antigen-specific immunological tolerance. Here we show that antigens modified with polymeric forms of either N-acetylgalactosamine or N-acetylglucosamine target hepatic antigen-presenting cells, increase their antigen presentation and induce antigen-specific tolerance, as indicated by CD4+ and CD8+ T-cell deletion and anergy. These synthetically glycosylated antigens also expanded functional regulatory T cells, which are necessary for the durable suppression of antigen-specific immune responses. In an adoptive-transfer mouse model of type-1 diabetes, treatment with the glycosylated autoantigens prevented T-cell-mediated diabetes, expanded antigen-specific regulatory T cells and resulted in lasting tolerance to a subsequent challenge with activated diabetogenic T cells. Glycosylated autoantigens targeted to hepatic antigen-presenting cells might enable therapies that promote immune tolerance in patients with autoimmune diseases.

Abstract 1337: Circulating angiogenic factors predict metastatic liver progression in rectal cancer patients given curative-intent oxaliplatin-containing neoadjuvant therapy

Abstract Purpose: Systemic failure remains a challenge in rectal cancer. Our recent data from a prospective study of intensified treatment in locally advanced rectal cancer indicated that neoadjuvant chemotherapy (NACT) and sequential long-course chemoradiotherapy (CRT), both modalities containing oxaliplatin, may have eradicated hypoxic tumor components and promoted an immune response that favored survival without metastatic progression. Because a tumor microenvironment with aberrant vasculature and hypoxia provokes immune tolerance, which is frequently connected to systemic inflammation, we hypothesized that circulating angiogenic factors may predict development of metastatic disease in rectal cancer patients given curative-intent treatment. Recognizing that angiopoietin 2 (ANGPT2) disrupts vascular remodeling by inducing endothelial cell apoptosis, we analyzed the soluble ANGPT2 and its endothelial receptor TEK at diagnosis and their linkage to inflammatory factors and progression-free survival (PFS). Experimental procedures: We used clinical data from 275 rectal cancer patients prospectively enrolled onto three studies. In two of the studies, patients were referred either directly to pelvic surgery or to standard CRT (without oxaliplatin) or short-course radiotherapy before surgery. In the third, introduced above, 80 patients received oxaliplatin-based therapy (two cycles of NACT followed by CRT) before resection. PFS was defined as time from study enrolment until the first metastatic event or up to five years after surgery in the case of no event. Serum ANGPT2 and TEK were measured by immunoassays. The measures (range of 1.60-8.16 ng/ml for ANGPT2 and 1.42-2.97 ng/ml for TEK) were normalized according to each intracohort mean value to enable analysis of pooled samples. Statistical methods were Pearson product correlation and Cox regression. Results: A correlation was seen between ANGPT2 and TEK levels (r = 0.34, p &amp;lt; 0.001), and each factor correlated with C-reactive protein (for ANGPT2: r = 0.29, p &amp;lt; 0.01; for TEK: r = 0.19, p &amp;lt; 0.01). The higher ANGPT2 or TEK, the higher risk of developing liver metastasis (for ANGPT2: hazard ratio 1.01, p = 0.002; for TEK: hazard ratio 1.01, p = 0.039) in the oxaliplatin-exposed cohort. No such association was found for patients given treatment devoid of oxaliplatin. Conclusions: High circulating ANGPT2 and TEK at diagnosis were associated with systemic inflammation and metastatic progression to the liver in rectal cancer patients receiving oxaliplatin-containing neoadjuvant therapy. Since we have previously shown that oxaliplatin may have breached the immune tolerance in patients with favorable outcome, there is a rationale for adding an anti-angiogenic agent in patients with hypoxic tumors as reflected by elevated circulating angiogenic factors. Citation Format: Sebastian Meltzer, Kathrine Røe Redalen, Svein Dueland, Annette Torgunrud Kristensen, Arne Mide Solbakken, Kjersti Flatmark, Anne Hansen Ree. Circulating angiogenic factors predict metastatic liver progression in rectal cancer patients given curative-intent oxaliplatin-containing neoadjuvant therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1337.

Haptoglobin improves acute phase response and endotoxin tolerance in response to bacterial LPS

Sepsis is characterized by delayed acute phase response and lowered immune tolerance in patients. Acute phase serum proteins, like Haptoglobin (Hp), have been associated with increased mortality in bacteria mediated acute lung inflammation and sepsis in neonates. However, it’s direct role in modulating the immune response by regulating pro-inflammatory mediators leading to immune tolerant state and if gender affects its expression levels during bacterial infection, especially in blood has not been fully explored. To understand its specific role in endotoxin-mediated immune response, we investigated the correlation between the rise in Hp levels on bacterial infection and its influence on the expression of pro-inflammatory mediators in male and female Whole blood (WHB) and PBMCs. Here, we observed pathogen-specific and gender-specific expression of Hp. Gonadal steroid hormones differentially influenced the Hp expression in LPS-induced WHB, where the addition of Estrogen increased Hp expression, with suppression of TNFα, in both genders. Further on evaluating, the influence of Hp on TNFα expression in endotoxin tolerance (ET), we show that increased Hp levels directly reduced TNFα expression in ET models. Interestingly, blockade of secreted Hp significantly reversed the (ET) state, confirmed by a significant rise in TNFα expression in both ex vivo and in vitro ET models, indicating a possible feedback inhibition by Hp on inflammatory mediators like TNFα. We also investigated the role of PKCδ in the regulation of LPS induced secretion of acute phase proteins (Hp) in serum, where inhibition of PKCδ, reduced secretion of anti-microbial proteins in response to LPS shown by restored bacterial growth. These findings clearly highlight the crucial role of Hp in maintaining immune tolerance via suppressing the pro-inflammatory mediators and also in preventing bacterial proliferation in blood during infection.