Proportion Of Tregs Research Articles

Research on Mechanism of Sevoflurane Carried with Superparamagnetic Iron Oxide Nanoparticles in Regulating Metabolism and Function of Anterior Cervical Lymphocytes Through Induction of PI3K/AKT Signal Pathway

We in this study assessed mechanism of sevoflurane carried with superparamagnetic iron oxide nanoparticles in regulating metabolism and function of anterior cervical lymphocytes through induction of PI3K/AKT signal pathway. 50 of SDF-grade SD rats were divided into 5 groups, including blank group, nanometer group, sevoflurane group, positive control group and pathway agonist group. The expressions ofβ-ACTIN, HIF1A, GLUT1 and HK2 were tested. Proportions of CD3 and T-cells, CD4/CD8 ratio, change of B220 and B-lymphocytes, Th1/Th2, Treg proportion, change of Tc1/Tc2 ratio, and presentation of factors in PI3K/AKT signal pathway were also observed. The expressions of factors related with metabolism of lymphocytes in blank group were found to be highest. = The proportions of CD3 and T-cells in the blank and nanometer groups were lowest while the CD/CD8 ratio was highest. The proportion of CD3 and T-cells in the sevoflurane and positive groups was highest while the CD/CD8 ratio was lowest. The proportion of lymphocytes was regulated by sevoflurane through restraining the activity of PI3K/AKT signal pathway so as to affect the body immunity function. The proportion of lymphocytes could return to normal through activating the activity of PI3K.AKT signal pathway so as to improve the immunological function. The PI3K/AKT signal pathway could be adopted as therapeutic target point for improving the action of anesthetic drugs on body immunity.

Evaluation of the effects of 1,25VitD3 on Th17 cells and Tregs in HTLV-1 infected cell lines

Human T-lymphotropic virus type 1 (HTLV-1) causes chronic infections of human T lymphocytes. The present study aimed to evaluate the effects of 1,25VitD3 on the proportion of Tregs and Th17 cells, the expression of related transcription factors (ROR-γt and FOXP3) and cytokines (IL-10, TGF-β, IL-6, and IL-17 A) in the HTLV-1infected cell lines MT-2 and MT-4. MT-2 and MT-4 cells and control PBMCs were treated with 1,25VitD3 and percentages of Tregs and Th17 cells was determined by flow cytometry. Gene expression and cytokine levels were analyzed by real-time PCR and ELISA, respectively. Treatment with-1,25VitD3 increased the percentage of Tregs in MT-2 and MT-4 cells, while it decreased the percentage of Th17 cells among MT-2 cells. 1,25VitD3 treatment also significantly improved FOXP3 gene expression in MT-2 cells, while reducing ROR-γt-gene expression in MT-2 and MT-4 cells comparing to untreated cells. Treatment with 1,25VitD3 significantly improved IL-10 levels in MT-2 cells, as well as TGF-β levels in both cell lines culture supernatants. 1,25VitD3 treatment diminished IL-6 levels in cell culture supernatants of MT-2 and MT-4 as well as IL-17 A levels in MT-2. Here we showed, that 1,25VitD3 modulated immune responses by enhancing Tregs differentiation and functions as well as inhibiting Th17 differentiation and actions in HTLV-1 infected cell lines. This suggests that VitD3 may have therapeutic effects in HTLV-1-related diseases by suppressing adverse inflammatory responses. Keywords: Tregs; Th17 cells; HTLV-1; 1, 25VitD3.

Various phenotypes of LRBA gene with compound heterozygous variation: A case series report of pediatric cytopenia patients.

Objective:LPS-responsive beige-like anchor (LRBA) deficiency is one of the most common monogenic disorders causing common variable immunodeficiency (CVID) and CVID-like disorders. However, the clinical spectrum of compound heterozygous (CHZ) LRBA variation should be extended. In this study, we presented five cases of compound heterozygous LRBA with various refractory cytopenias.Materials and Methods:Retrospective analysis of the clinical manifestations, management, and outcomes of five cases (from five pedigrees) with LRBA gene CHZ variants which initially manifested as single/multilineage immune cytopenias was performed. Results: 1. Gene variations: All five patients inherited the compound heterozygous LRBA variations from their parents which were thought to be pathogenic. BEACH, DUF4704, and LamG were the main affected domains of LRBA gene in this case series. 2. Immune dysregulation of clinic: (1) Hypogammaglobulinemia were recorded in four patients, and the proportion of Treg was decreased in two patients. Only one patient had been with increased TCRαβ+CD4/CD8 double-negative T cells (DNT). (2) Lymphoproliferative manifestations were seen in three patients. (3) All five patients were complained with cytopenia, although they showed different clinical manifestations. None of the parents was asymptomatic. (4) Other immune disorders: P5 also had relapsed infections and autoimmune endocrinopathy. 3. Management and outcomes: P1 and P5 responded well to immunomodulatory therapy and P3 was effectively treated with hemophagocytic lymphohistiocytosis (HLH) first-line regimen chemotherapy. P4 showed no responses to steroids and IVIG. However, TPO-R agonist was effective.Conclusion:Unlike homozygous mutations, compound heterozygous LRBA variation should always be kept in mind for the various phenotypes and different treatment responses.

SCM-198 Alleviates Endometriosis by Suppressing Estrogen-ERα mediated Differentiation and Function of CD4+CD25+ Regulatory T Cells.

Background: Endometriosis (EMS), a typical endocrine immune disorder, associates with dramatically increased estrogen production and disorganized immune response in ectopic focus. Peritoneal regulatory T cells (Tregs) expansion in women with EMS and their pathogenic role attributable to endometriotic immunotolerance has been reported. Whether local high estrogen promotes EMS by discipling Tregs needs to be further explored. Up to date, there is no effective medicine for the treatment of EMS. SCM-198 is a synthetic leonurine with multiple physiological activities. Whether SCM-198 could regulate Tregs via estrogen and facilitate the radical cure of EMS has not yet been reported.Methods: Proportion of Tregs in peritoneal fluid of patients with EMS was firstly analyzed via flow cytometry. Peritoneal estrogen concentration and the mRNA levels of estrogen receptor α (ERα) and estrogen receptor β (ERβ) of Tregs were detected by ELISA and RT-PCR, respectively. Grouped in vitro induction assays were performed to explore the effects of SCM-198 and estrogen signaling on Tregs. Cell invasion and viability assays were utilized to detect the crosstalk between Tregs and ectopic endometrial stromal cells (eESCs), with or without SCM-198 treatment. Furthermore, EMS mice models were established to verify the therapeutic effects of SCM-198.Results: Increased Tregs were found in peritoneal fluid of EMS patients, accompanied with estrogen-ERα overactivation. Estrogen-ERα triggered the expansion of Tregs and their cytokine production (IL-10 and TGF-β1), which could be reversed by SCM-198 treatment. Moreover, SCM-198 abated the invasion and viability of eESCs enhanced by Tregs. In vivo experiments confirmed that SCM-198 obviously retarded the growth of ectopic lesions and downregulated the functions of Tregs via estrogen-ERα inactivation.Conclusions: These data suggest that SCM-198 attenuates Tregs expansion via the inhibition of estrogen-ERα signaling in EMS and offer a promising therapy for such a refractory disease.

Regulatory T and B cells in transient hypogammaglobulinemia of infancy.

Transient hypogammaglobulinemia of infancy (THI) is a heterogeneous disorder caused by an abnormal delay in reaching normal IgG levels in the first three years of life. Although THI is a common primary immune deficiency, its pathogenesis has not been fully elucidated. We aimed to investigate the role of regulatory T cells (Tregs) and B cells (Bregs) in the pathogenesis of THI. T and B cell subsets were evaluated in 40 patients with THI aged 6-41 months and 23 healthy controls aged 6-51 months using flow cytometry. CD4 and interleukin-2 receptor-α alpha (CD25) expression and a lack of interleukin-7 receptor-α (CD127) were used for Treg identification. FoxP3 expression in Tregs was determined as a percentage and mean fluorescence intensity. B cell subsets (plasmablast, mature naive, primarily memory, new memory) and Bregs were defined according to CD19, CD38, and CD24 expressions. Patients with THI (15 females and 25 males; mean age: 18.8 ± 8.6 months) and controls (10 females and 13 males; mean age: 22.6 ± 13.1 months) participated in this study. While the proportion of Tregs of children with THI were significantly increased compared to the controls, primarily memory B cells were reduced. Additionally, the proportions of CD127 in CD3+ and CD3+CD4+ T cells were significantly reduced in the patients with THI compared to the control. No significant difference was detected in the FoxP3 expression of Tregs and the frequency of Bregs in the children with THI. Increased Tregs and decreased primarily memory B cells may cause antibody production delay in children with THI. Changes in the T and B cell compartments may be related to chronic immune activation and affected cellular immunity in THI. Further studies are needed to use T and B cell subsets in the prediction of IgG level recovery.

Abstract P062: PI3Kγδ inhibitor plus radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer and humanized patient-derived xenograft model

Abstract Purpose: Breast cancer is generally viewed as immunologically ‘cold', imposing an immune-suppressive tumor microenvironment (TME) and responding poorly to lone immune checkpoint blockade (ICB). As an adjunct to ICB, radiation therapy (RT) holds promise in terms of in situ tumor vaccination effect, although it is known to promote immune suppression, increasing regulatory T cells (Treg), myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages (TAMs). It was our contention that combined use of RT and a PI3Kγδ inhibitor to combat immune suppression might enhance the efficacy of ICB. Methods: Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated by 3 fractions of 24 Gy. A PD-1 blockade and a PI3Kγδ inhibitor were then administered every other day for 2 weeks. Tumors from humanized patient-derived breast cancer xenograft (PDX) model was sequenced to identify immune-related pathways and to profile infiltrated immune cells. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas (TCGA) pan-cancer cohort, and the deconvolution algorithm was used to profile immune cell repertoire. Results: In the immune-competent syngenic 4T1 murine tumor model, PD-1 blockade alone led to tumor hyperprogression, whereas a three-pronged strategy of PI3Kγδ inhibitor, RT, and PD-1 blockade significantly delayed primary tumor growth, boosted abscopal effect, and improved animal survival by comparison. The immune-deficient syngenic 4T1 murine tumor model failed to show this synergism in delaying tumor growth and the abscopal effect. According to FACS analysis, RT significantly increased not only CD8+cytotoxic T-cell fractions but also immune-suppressive Tregcells, MDSCs, and M2 TAMs. However, PI3Kγδ inhibitor significantly lowered proportions of Treg, MDSCs, and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumor CD8+ T-cell populations after triple combination therapy. There were significantly decreased tumor expressions of p-AKT, PD-L1, and HIF1α by PI3Kγδ inhibition. Triple combination therapy significantly delayed primary tumor growth in humanized PDX model as well and analyses of RNA sequencing data of humanized PDX samples showed decreased immune suppressive pathways with decreased and M2 macrophage and increased CD8+ T-cell by triple combination therapy. In the TCGA pan-cancer cohort, high Treg/CD8+T-cell and M2/M1 TAM ratios and poor overall patient survival was associated with high PIK3CG (PI3Kγ) or PIK3CD (PI3Kδ) gene expression. Conclusion: These findings collectively indicate that PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME. Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may thus be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors such as breast cancer. Citation Format: In Ah Kim, Min Guk Han, Bum Sup Jang, Mi Hyun Kang. PI3Kγδ inhibitor plus radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer and humanized patient-derived xenograft model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P062.

Effect of Donor Lymphocyte Infusion From Two Types of Donors on Mixed Chimerism With Secondary Graft Failure After Allogeneic Hematopoietic Stem Cell Transplantation

Mixed chimerism (MC) and secondary graft failure (SGF) with recipient- or donor-type chimerism is a major obstacle in allogeneic hematopoietic stem cell transplantation (HSCT). Donor lymphocyte infusion (DLI) can eradicate minimal residual disease or be used to rescue a hematologic relapse, being able to induce durable remissions after HSCT. This study aimed to analyze the efficacy and immune mechanism of DLI from the original and alternative donor for patients of mixed donor chimerism with SGF. The alternative donor refers to the candidate relative donor who did not initially provide stem cells and includes HLA-matched sibling donor or HLA-haploidentical donor. We conducted a retrospective study of 246 patients with a median age of 37 (9-58) years who had regularly detected MC, complete donor chimera (CC), and regulatory T cells (Treg). The median diagnosis time of SGF was 69 (39-141) days after transplantation. Sixteen patients with SGF received DLI from the alternative donor, including 3 patients who chose DLI from the original donor with no initial response and 13 patients who directly chose DLI from the alternative donor. Sixteen patients with SGF existed mixed chimerism synchronously and the rate calculated overall chimerism of MC was 63% (range 42%-85%) after transplantation. The proportion of Treg decreased significantly in SGF patients from a median of 2.66% ± 0.80% to 0.93% ± 0.57% at a time point after transplantation (P = .02). The DLI of the alternative donor in 14 patients achieved complete response and MC gradually convert to CC state, simultaneously there was significant increase in the Treg fraction (SGF versus complete response: 0.93% ± 0.57% versus 3.61% ± 0.82% [P = .01]). For the clinical nonresponders from 2 types of donors, there was no significant change in MC and Treg cells. The OS and disease-free survival at 2 years after DLI were 69.7% ± 3.19% and 61.3% ± 4.80%, respectively. DLI from the alternative donor may be an effective treatment for MC with SGF, and the mechanism is closely related to the activation of Treg cells level.

Mechanism of miR-181a-5p in Regulatory T/T-Helper 17 Immune Imbalance and Asthma Development in Mice with Allergic Rhinitis

Introduction: Allergic rhinitis (AR) is an immune disorder and also a risk factor of asthma. microRNAs (miRNAs) are implicated in autoimmune diseases, including RA. This study investigated effect of miR-181a-5p on regulatory T (Treg)/ T-helper (Th) 17 immune imbalance in AR. Methods: A murine model of AR was established and treated with lentivirus modified miR-181a-5p. The allergic symptoms of mice were examined. The contents of Th17-related cytokines (interferon [IFN]-γ and interleukin [IL]-6), Treg-related cytokine (IL-10), and Treg-specific nuclear transcription factor (Foxp3) in nasal mucosa and lung tissues were determined. The proportion of Treg and Th17 cells was analyzed by flow cytometry. The level of ovalbumin-specific immunoglobulin E in the serum, and the contents of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid and IFN-γ, IL-6, and IL-10 in nasal lavage fluid were measured. The targeting relationship between miR-181a-5p and high mobility group box chromosomal protein 1 (HMGB1) was verified. HMGB1 and receptor for advanced glycation end products (RAGE) expression in RA were determined, and the interaction between HMGB1 and RAGE was detected. Results: miR-181a-5p expression was reduced in AR mice. miR-181a-5p overexpression attenuated allergic behaviors, alleviated Treg/Th17 imbalance, and delayed asthma development. HMGB1 and RAGE were elevated in AR mice. miR-181a-5p targeted HMGB1, and HMGB1 bound to RAGE, while miR-181a-5p overexpression reduced the binding between them. Activating HMGB1/RAGE reversed the protective effect of miR-181a-5p overexpression on AR and induced the development of asthma. Conclusion: miR-181a-5p overexpression reduced the binding of HMGB1 and RAGE by inhibiting HMGB1, thus alleviating Treg/Th17 immune imbalance and blocking AR from developing into asthma.

Acupuncture Can Regulate the Distribution of Lymphocyte Subsets and the Levels of Inflammatory Cytokines in Patients With Mild to Moderate Vascular Dementia.

Background: Vascular dementia (VD) is the second most common type of dementia after Alzheimer’s disease, but there is a lack of definitive treatment for VD. Acupuncture treatment is effective in improving the cognitive impairment and behavioral capacity of patients with VD. In recent years, more studies indicated that peripheral inflammation and abnormal peripheral immune function may aggravate neuroinflammation and cognitive dysfunction. However, there are few studies about the acupuncture and the abnormal peripheral immune function of VD. Also, few studies concern the regulating effect of acupuncture on peripheral immunity of patients with VD.Objective: The aim of this study was to explore the effect of the “sanjiao” acupuncture method on peripheral immunity of patients with mild to moderate VD.Methods: A total of 30 patients with VD were involved in the acupuncture group (AG), which was treated with the “sanjiao” acupuncture method once a day for six times a week and lasted for 12 weeks, and 30 healthy elderly people were assigned to the normal group (NG), which had no treatment. The distribution of lymphocyte subsets and the levels of some inflammatory cytokines in the peripheral blood of subjects were evaluated using the flow cytometry (FCM) and the enzyme-linked immunosorbent assay (ELISA).Results: A total of 60 subjects were involved in this study, while 58 subjects completed the entire trial. Before treatment, the levels of CD3+ T, CD4+ T cells, CD4+/CD8+, Tregs, B cells, IFN-γ, and IL-10 in patients with VD were significantly decreased compared with the normal group (all P < 0.05 or P < 0.01). The level of TNF-α in peripheral blood of patients with VD was significantly increased (P < 0.01). After acupuncture treatment, the levels of CD3+ T, CD4+ T cells, and IFN-γ were significantly increased (all P < 0.05 or P < 0.01). The level of TNF-α was significantly decreased (P < 0.01). The proportion of Tregs was increased (P < 0.01), but it was still lower than that of the normal group (P < 0.05).Conclusion: The acupuncture method can increase the proportion of CD3+, CD4+ T cells, and Tregs in peripheral blood of patients with VD. And, it reduces the levels of pro-inflammatory factor TNF-α, which achieves the anti-inflammatory effects and immunostimulation. It suggests that acupuncture can improve the peripheral immune dysfunction of patients with VD by regulating the distribution of lymphocyte subsets and the levels of inflammatory cytokines.Clinical Trial Registration: [www.chictr.org.cn], identifier [ChiCTR-IOR-17012052].

Single-Cell Sequencing Technology Reveals the Heterogeneity of the Immune Microenvironment and Key Molecules of Drug Resistance in Angioimmunoblastic T-Cell Lymphoma

Objective:Angioimmunoblastic T-cell lymphoma (AITL) is a common subtype of peripheral T-cell lymphoma (PTCL). AITL is an aggressive malignancy with a poor prognosis, and its clinical manifestations vary greatly among individuals. The current chemotherapy regimens based on anthracycline show limited efficacy, and there is no best rescue treatment for patients with relapsed and refractory (RR) AITL. In addition, the lack of optimal AITL models in vitro greatly limits the basic research on the mechanism of disease occurrence and progression, and also hinders the development of new drugs and preclinical trials. Our study aims to deeply analyze the tumor heterogeneity and clonal evolution of AITL, discovering key molecules of drug resistance and potential theraputic targets.Methods:We detected fresh lymph node samples from newly diagnosed and relapsed/ refractory AITL patients using single-cell RNA sequencing, combined with imaging mass cytometry (IMC) and whole exome sequencing. IMC was performed to analyze the spatial position relationship and protein expression characteristics of different subgroups in the tumor microenvironment of AITL. In addition, AITL patient-derived organoid model was established to study the regulatory role of YY1 and its inhibitors in relapsed and refractory AITL.Results:ScRNA-seq revealed the significant differences in the tumor microenvironment of newly diagnosed and RR-AITL patients (Fig A,B). B cells and myeloid subgroups may play important roles in the development of AITL (Fig C). Transcription factor YY1, highly expressed in follicular helper T cell (Tfh) of RR-AITL patients, promoted the proliferation and drug resistance of AITL cells (Fig E,F). The proportion of CD8+ T cells in the RR-AITL sample was reduced, while the proportion of Treg was increased, as well as the depletion of T cells (Fig G,H). Furthermore, the stemness of B cells in RR-AITL was enhanced and exhibits significant malignant characteristics (Fig C,I-K). We also found decreased interaction in RR-AITL samples (Fig L,M). Moreover, for the first time, we established AITL patient-derived organoid models that can be stablely cultured in vitro (Fig N). On this basis, we could further clarify the important roles of transcription factor YY1 in the drug resistance of AITL, evaluate the cytotoxic effect of YY1 inhibitor NP-001 on AITL tumor cells.Conclusion:In conclusion, our study revealed the differences between newly diagnosed and relapsed /refractory AITL in terms of immune microenvironment, single-cell transcriptomes, and signal pathway activation. YY1 may serve as an novel target for drug resistance for RR-AITL patients. These findings may provide a theoretical foundation for improving the clinical treatment of AITL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Endothelial Cell Dysfunction Is Involved in the Progression of Myelodysplastic Syndromes

Background:Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective hematopoiesis, refractory anemia, and a tendency to transform to acute myeloid leukemia (AML). Ineffective hematopoiesis progression and immune deregulation are dominating pathophysiological process of MDS. Emerging evidences showed the role of bone marrow (BM) microenvironment in MDS. In MDS murine model, integral BM microenvironment contributes to inferior hematopoietic function and disease progression. As an important component of BM microenvironment, the relationship between endothelial cells (ECs) and MDS progression remains largely unknown. Although ECs from MDS patients have been identified to have decreased supporting ability to normal hematopoietic stem cells (HSCs), the supporting ability of ECs in different clinical stages of MDS remains to be elucidated. In addition, the role of BM ECs from MDS patients in supporting leukemia cells and their immunomodulatory ability remains unclear.Aims:To determine the number and functions of BM ECs in different subtypes of MDS patients. Moreover, to explore the correlation between BM ECs and MDS progression, which may represent a potential therapeutic target for MDS patients.Methods:In the prospective cohort study, patients with multilineage dysplasia (MDS-MLD, N=15), MDS with excess blasts (MDS-EB, N=15), or AML(N=15) and healthy donors (HD, N=15) were enrolled. BM ECs were analyzed in HD and patients by flow cytometry and in situ histological analyses. The functions of BM ECs were analyzed by migration, angiogenesis capacities, levels of apoptosis and reactive oxygen species (ROS). To evaluate the supporting abilities of BM ECs on HSCs, leukemia cells and T cells, in vitro co-culture strategies were used. The levels of apoptosis, ROS and colony-forming unit-plating (CFU) efficiency of CD34+ and HL-60 cells were investigated. T cell subsets were analyzed by flow cytometry as previously reported. To further investigate the underlying mechanism of dysfunctional ECs, RNA sequencing (RNA-Seq) analyses and real time-PCR (qRT-PCR) were performed in BM ECs from HD and MDS patients with different subtypes.Results:In the current study, gradually increased BM ECs were observed from MDS-MLD, MDS-EB to AML patients. Furthermore, dysfunctional BM ECs were found with MDS progression, characterized by increased levels of migration, angiogenesis capacities, apoptosis and ROS. More importantly, BM ECs from MDS patients exhibited decreased supporting ability of HSCs whereas increased supporting ability of leukemia cells in vitro with MDS progression. After coculture with ECs, levels of apoptosis and ROS in CD34+ cells were increased whereas their CFU efficiency reduced. On the other hand, levels of apoptosis and ROS of HL-60 cells were decreased. The proliferation capacity and leukemia CFU efficiency of HL-60 cells after co-cultured with ECs were enhanced with MDS progression. Furthermore, following coculture with BM ECs, deregulated differentiation was demonstrated in T cell subsets, characterized by elevating proportion of Th2 and Treg and decreasing proportion of Th1 and Th17 with MDS progression. RNA-Seq showed that the expression profile of BM ECs from MDS-EB was closer to MDS-MLD, whereas that of MDS-EB was closer to AML. Different gene expression profiles indicated the expression of hematopoiesis and immune related genes increased in BM ECs with MDS progression. Mechanistically, the mRNA levels of CX CL12, SCF and NFKB of ECs were increased with MDS progression.Summary/Conclusion:In summary, the number of BM ECs gradually increased, BM EC dysfunction more and more severe, and the supporting abilities of BM ECs on HSCs decreased, whereas on leukemia cells increased with MDS progression. Moreover, ECs regulated the differentiation of T cells into immune tolerant cells with MDS progression. Although further validation is required, these findings indicated that the improvement of BM ECs may represent a potential therapeutic approach for MDS patients.Keywords: Myelodysplastic syndromes, endothelial cells, disease progression, ineffective hematopoiesis, immune deregulation DisclosuresNo relevant conflicts of interest to declare.

Combinatorial Analysis of AT-Rich Interaction Domain 1A and CD47 in Gastric Cancer Patients Reveals Markers of Prognosis.

Background: The AT-rich interaction domain 1A (ARID1A) is thought to be a tumor suppressive gene, and most of its mutations result in loss of expression of ARID1A protein. Combined with SIRPα on the surface of macrophages, CD47 on the surface of cancer cells can send an antiphagocytic “Don’t eat me” signal to the immune system that helps to avoid immune surveillance. However, the relationship between ARID1A and CD47 expression and their prognostic value in gastric cancer (GC) are still unknown.Methods: In this study, we evaluated ARID1A and CD47 expression in 154 GC patients’ tissues using tissue microarray. Expressions of ARID1A and CD47 in GC cell lines were determined by western blot and quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) techniques, and cell membranous CD47 expression was quantified by flow cytometry. In addition, chromatin immunoprecipitation (ChIP)–qPCR was used to determine the aspects of regulation of CD47 by ARID1A. The proportions of tumor-infiltrating immune cells were estimated on The Cancer Genome Atlas (TCGA) data set by using quanTIseq and EPIC algorithms. The infiltration of M1-polarized macrophages, M2-polarized macrophages, and regulatory T cells (Tregs) in GC tissues was determined by multispectral immunofluorescence.Results: A significant correlation was found between loss of ARID1A and high expression of CD47 at protein level in GC. By integrating 375 bulk RNA sequencing samples from TCGA data set, we found that mutated ARID1A correlated with high CD47 expression. In GC cell lines, knockdown of ARID1A significantly increased CD47 expression both at protein and mRNA levels as measured by western blot, qRT-PCR, and flow cytometry. Moreover, ChIP-qPCR revealed that CD47 was a direct downstream target gene of ARID1A in GC. Utilizing univariate and multivariate survival analyses, we found that patients with ARID1AlossCD47high expression had a worse prognosis. Estimation of infiltrating immune cells on TCGA data set showed that a higher infiltration proportion of M2 macrophages and Tregs was found in ARID1AmutatedCD47high expression subgroup. Furthermore, application of multispectral immunofluorescence revealed a higher infiltration proportion of M2 macrophages and Tregs in ARID1AlossCD47high GC tissues.Conclusion: Loss of ARID1A is strongly correlated with high CD47 expression in GC, and combination of ARID1A and CD47 is a promising prognosis factor in GC.

Circulating Immunosuppressive Regulatory T Cells Predict Risk of Incident Cutaneous Squamous Cell Carcinoma.

Ultraviolet radiation exposure (UVR) is a risk factor for cutaneous squamous cell carcinoma (cuSCC) and has been shown to be positively associated with circulating immunosuppressive regulatory T cells (“Tregs”). However, the risk of cuSCC in association with circulating Tregs has not been studied. The aim of this study was to determine whether circulating Treg levels are associated with cuSCC development, particularly in the context of high UVR. Blood and spectrophotometer-based UVR measurements were obtained on 327 immunocompetent individuals undergoing routine skin cancer screenings at baseline and followed for up to 4 years for incident cuSCC development within a prospective cohort study. Proportions of phenotypically distinct Tregs, especially CCR4hi and CLA+ cells which are associated with activation and homing, respectively, were measured by flow cytometry. Tregs in cuSCC tumors were assessed using immunohistochemistry and graded for solar elastosis, a measure of cumulative UVR damage. Of several Treg phenotypes examined, higher levels of circulating CCR4hi Tregs at baseline were significantly associated with increased risk of subsequent cuSCC; those with higher levels of both CCR4hi and UVR were four times more likely to develop cuSCC compared to those with lower levels of both (Hazard Ratio = 4.11, 95% CI = 1.22–13.90). Within cuSCC tumors, CCR4hi Tregs were positively associated with solar elastosis. Results show that a higher proportion of CCR4hi peripheral Tregs predicts incident cuSCC up to 4 years, especially among highly UV-exposed individuals. Research of the underpinning biology of Tregs in UVR-associated skin damage may possibly reveal novel opportunities for screening, prevention, and treatment.

Biological Effects of Prostagrandin E2-EP4 Antagonist (AAT-008): Enhancement of Immunoresponse to Radiotherapy and a Potential as a Radiosensitizer

The cyclooxygenase-2 product prostaglandin E2 (PGE2) promotes tumor growth and metastasis by acting on a family of four G protein-coupled receptors (EP1-4). Recently, an antagonist of PGE2-EP4 (AAT-008) has been newly developed for clinical use. The purpose of this study is to investigate the biological effects of AAT-008 as a radiosensitizer for the treatment of murine colon cancer (CT26WT) in vivo and explore the mechanism using flow cytometry.CT26WT tumor cells transplanted into the hind legs of BALB/c mice were used. Treatments were carried out when the mean diameter of the tumors reached approximately 10 mm for tumor growth delay assay and 5 mm for flow cytometry (day 0). AAT-008 (0, 3, 10, and 30 mg/kg/day) was administered orally twice a day from day 0 to day 18. The tumor was irradiated at 9 Gy on day 3 for the radiation treatment (RT) group. In the tumor growth assay, twelve mice were used for each group. The three dimensions of each tumor were measured every other day using a caliper and the tumor doubling time was calculated. For the first flow cytometry, the dose of AAT-008 was fixed to 10 mg/kg/day. Administration of AAT-008 and RT dose were the same as in the tumor growth assay. The population of cytotoxic T-cells (CD45+CD8+CD69+: CTL) was investigated using the tumor cells on day 19. For the second flow cytometry, the dose of AAT was fixed to 30 mg/kg/day. AAT-008 was administered from day 0 to day 12. The population of CTL and the ratio of CTL / regulatory T-cells (Foxp3+: Treg) were investigated using the tumor cells on day 13.In the tumor growth delay assay, the median tumor doubling times in the 0-, 3-, 10-, and 30-mg group without RT were 3.2, 4.4, 4.4, and 5.7 days, respectively. The times in those dose groups with RT were 6.1, 6.6, 17.3, and 19.8 days, respectively (P < 0.001 between the 0- and 10-mg/30-mg groups with RT). There was no significant difference between the 10- and 30-mg groups with RT (P = 0.56). In the first flow cytometry, the mean CTL proportion of the irradiated tumors in the 10 mg + RT (n = 4) and 0 mg + RT groups (n = 5) was 43% and 31%, respectively. Of note, 67% of CTL were observed in responded mice (n = 2) in the 10 mg + RT group. In the second flow cytometry, the mean Treg proportion and the CTL/ Treg ratio of the irradiated tumors in the 30 mg + RT (n = 5) and 0 mg + RT groups (n = 5) were 4.0% and 1.5%, respectively (P = 0.02) and 10 and 22, respectively (P = 0.02).AAT-008 had a potential of enhancing radiosensitivity in the treatment of colon cancer cells. It was assumed that the optimal dose of AAT-008 with RT was 10 mg/kg/day. It was suggested that AAT-008 stimulated the immune system against the cancer cells.

The Responsiveness of Bee Venom Phospholipase A2 on Regulatory T Cells Correlates with the CD11c+CD206+Population in Human Peripheral Blood Mononuclear Cells.

Bee venom phospholipase A2 (bvPLA2) has been reported to have therapeutic effects such as neuroprotection, anti-inflammation, anti-nociception, anti-cancer properties, caused by increasing regulatory T cells (Tregs). The mechanism of Tregs modulation by bvPLA2 has been demonstrated by binding with the mannose receptor, CD206 in experimental models of several diseases. However, it remains unknown whether this mechanism can also be applied in human blood. In this study, we collected peripheral blood samples from healthy donors and analyzed the percentages of monocyte-derived dendritic cells with CD206 (CD206+ DCs) before expansion, the proportion of Tregs, and the subpopulations after expansion treated with bvPLA2 or PBS using flow cytometry and the correlations among them. The percentage of Tregs tended to be higher in the bvPLA2 group than in the control group. There were significant positive correlations between the CD206 population in hPBMC and the proportions of Tregs treated with bvPLA2, especially in the Treg fold change comparing the increase ratio of Tregs in bvPLA2 and in PBS. These findings indicate that bvPLA2 increased the proportion of Tregs in healthy human peripheral blood and the number of CD206+ DCs could be a predictor of the bvPLA2 response of different individuals.

SJMHE1 Peptide from Schistosoma japonicum Inhibits Asthma in Mice by Regulating Th17/Treg Cell Balance via miR-155.

PurposeHelminths and their products can regulate immune response and offer new strategies to control and alleviate inflammation, including asthma. We previously found that a peptide named as SJMHE1 from Schistosoma japonicum can suppress asthma in mice. This study mainly investigated the molecular mechanism of SJMHE1 in inhibiting asthma inflammation.MethodsSJMHE1 was administered to mice with OVA-induced asthma via subcutaneous injection, and its effects were detected by testing the airway inflammation of mice. The Th cell distribution was analyzed by flow cytometry. Th-related transcription factor and cytokine expression in the lungs of mice were analyzed using quantitative real-time PCR (qRT-PCR). The expression of miR-155 and levels of phosphorylated STAT3 and STAT5 were also determined after SJMHE1 treatment in mice by qRT-PCR and Western blot analysis. The in vitro mouse CD4+ T cells were transfected with lentivirus containing overexpressed or inhibited miR-155, and the proportion of Th17, Treg cells, CD4+p-STAT3+, and CD4+p-STAT5+ cells were analyzed by flow cytometry.ResultsSJMHE1 ameliorated the airway inflammation of asthmatic mice, upregulated the proportion of Th1 and Treg cells, and the expression of Th1 and Treg-related transcription factor and cytokines. Simultaneously, SJMHE1 treatment reduced the percentage of Th2 and Th17 cells and the expression of Th2 and Th17-related transcription factor and cytokines. SJMHE1 treatment decreased the expression of miR-155 and p-STAT3 but increased p-STAT5 expression. In vitro, the percentage of Th17 and CD4+p-STAT3+ cells increased in CD4+ T cells transfected over-expression of miR-155, but SJMHE1 inhibited the miR-155-mediated increase of Th17 cells. Furthermore, SJMHE1 increased the proportion of Treg and CD4+p-STAT5+ cells after transfected over-expression or inhibition of miR-155.ConclusionSJMHE1 regulated the balance of Th17 and Treg cells by modulating the activation of STAT3 and STAT5 via miR-155 in asthma. SJMHE1 might be a promising treatment for asthma.

PI3Kγδ inhibitor plus radiation enhances the antitumour immune effect of PD-1 blockade in syngenic murine breast cancer and humanised patient-derived xenograft model

IntroductionWe hypothesised that the combined use of radiation therapy and a phosphoinositide 3-kinaseγδ inhibitor to reduce immune suppression would enhance the efficacy of an immune checkpoint inhibitor. MethodsMurine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumours were irradiated by 3 fractions of 24 Gy. A PD-1 blockade and a phosphoinositide 3-kinase (PI3K)γδ inhibitor were then administered every other day for 2 weeks. The same experiments were performed in humanised patient-derived breast cancer xenograft model and its tumour was sequenced to identify immune-related pathways and profile infiltrated immune cells. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas pan-cancer cohort, and the deconvolution algorithm was used to profile immune cell repertoire. ResultsUsing a PI3Kγδ inhibitor, radiation therapy (RT) and PD-1 blockade significantly delayed primary tumour growth, boosted the abscopal effect and improved animal survival. RT significantly increased CD8+cytotoxic T-cell fractions, immune-suppressive regulatory T cells (Tregs), myeloid-derived suppressor cells and M2 tumour-associated macrophages (TAMs). However, the PI3Kγδ inhibitor significantly lowered the proportions of Tregs, myeloid-derived suppressor cells and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumour CD8+ T-cell populations after triple combination therapy. In a humanised patient-derived breast cancer xenograft model, triple combination therapy significantly delayed tumour growth and decreased immune-suppressive pathways. In The Cancer Genome Atlas cohort, high Treg/CD8+ T cell and M2/M1 TAM ratios were associated with poor overall patient survival. ConclusionThese findings indicate PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME, and combining PI3Kγδ inhibitor, RT and PD-1 blockade may overcome the therapeutic resistance of immunologically cold tumours. SynopsisCombining PI3Kγδ inhibitor, RT, and PD-1 blockade may be a viable clinical approach, helping to overcome the therapeutic resistance of immunologically cold tumours such as breast cancer.

Immunomodulatory Effects of Lenvatinib Plus Anti-Programmed Cell Death Protein 1 in Mice and Rationale for Patient Enrichment in Hepatocellular Carcinoma.

Lenvatinib is an effective drug in advanced HCC. Its combination with the anti-PD1 (programmed cell death protein 1) immune checkpoint inhibitor, pembrolizumab, has generated encouraging results in phase Ib and is currently being tested in phase III trials. Here, we aimed to explore the molecular and immunomodulatory effects of lenvatinib alone or in combination with anti-PD1. We generated three syngeneic models of HCC in C57BL/6J mice (subcutaneous and orthotopic) and randomized animals to receive placebo, lenvatinib, anti-PD1, or combination treatment. Flow cytometry, transcriptomic, and immunohistochemistry analyses were performed in tumor and blood samples. A gene signature, capturing molecular features associated with the combination therapy, was used to identify a subset of candidates in a cohort of 228 HCC patients who might respond beyond what is expected for monotherapies. In mice, the combination treatment resulted in tumor regression and shorter time to response compared to monotherapies (P<0.001). Single-agent anti-PD1 induced dendritic and T-cell infiltrates, and lenvatinib reduced the regulatory T cell (Treg) proportion. However, only the combination treatment significantly inhibited immune suppressive signaling, which was associated with the TGFß pathway and induced an immune-active microenvironment (P<0.05 vs. other therapies). Based on immune-related genomic profiles in human HCC, 22% of patients were identified as potential responders beyond single-agent therapies, with tumors characterized by Treg cell infiltrates, low inflammatory signaling, and VEGFR pathway activation. Lenvatinib plus anti-PD1 exerted unique immunomodulatory effects through activation of immune pathways, reduction of Treg cell infiltrate, and inhibition of TGFß signaling. A gene signature enabled the identification of ~20% of human HCCs that, although nonresponding to single agents, could benefit from the proposed combination.

Endocervical Regulatory T Cells Are Associated With Decreased Genital Inflammation and Lower HIV Target Cell Abundance.

Regulatory T cells (Tregs) play important roles in tissue homeostasis, but few studies have investigated tissue Tregs in the context of genital inflammation, HIV target cell density, and vaginal microbiota in humans. In women from Nairobi (n=64), the proportion of CD4+ CD25+ CD127low Tregs in the endocervix correlated with those in blood (r=0.31, p=0.01), with a higher Treg frequency observed in the endocervix (median 3.8 vs 2.0%, p<0.0001). Most Tregs expressed FOXP3 in both compartments, and CTLA-4 expression was higher on endocervical Tregs compared to blood (median 50.8 vs 6.0%, p<0.0001). More than half (34/62, 55%) of participants displayed a non-Lactobacillus dominant vaginal microbiota, which was not associated with endocervical Tregs or CD4+ T cell abundance. In a multivariable linear regression, endocervical Treg proportions were inversely associated with the number of elevated pro-inflammatory cytokines (p=0.03). Inverse Treg associations were also observed for specific cytokines including IL-1β, G-CSF, Eotaxin, IL-1RA, IL-8, and MIP-1 β. Higher endocervical Treg proportions were associated with lower abundance of endocervical CD4+ T cells (0.30 log10 CD4+ T cells per log10 Treg, p=0.00028), with a similar trend for Th17 cells (p=0.09). Selectively increasing endocervical Tregs may represent a pathway to reduce genital tract inflammation in women.

Immunological Differences in Human Peripheral Blood Mononuclear Cells Treated with Traditional Japanese Herbal Medicines Hochuekkito, Juzentaihoto, and Ninjin'yoeito from Different Pharmaceutical Companies.

Hochuekkito (HET), Juzentaihoto (JTT), and Ninjin'yoeito (NYT) have been used as Hozai, a group of traditional Japanese herbal medicines, to treat physically and mentally weak cancer patients. Their compositions are quite different, and Japanese pharmaceutical companies have been using different types or quantities of herbs for formulations with the same name. Here, we compared the immunological differences between HET, JTT, and NYT with respect to the induced T cell subsets and cytokines. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and treated with 0 (control), 25, 50, 100, 200, or 400 μg/mL HET, JTT, or NYT (manufactured by Tsumura [TJ], Kracie [KR], and Kotaro [KO]). PBMC proliferation, CD4+ T cell, CD8+ T cell, and regulatory T cell (Treg) proportions and interleukin (IL) concentrations (IL-6, IL-10, IL-17A, interferon-γ, tumor necrosis factor-α, and transforming growth factor (TGF)-β) secreted by PBMCs were measured using Cell Counting Kit-8 or flow cytometry bead analysis. PBMC proliferation and CD4+ T cell percentages were similar in the HET, JTT, NYT, and control groups; however, the percentage of CD8+ T cells tended to increase after treatments. Tregs were suppressed by HET, JTT, and NYT, and TJ-JTT significantly decreased Treg numbers (compared with control). The concentrations of all cytokines except TGF-β were increased in a concentration-dependent manner (p < 0.05); particularly, KR-HET induced IL-6 secretion (compared with the control, TJ-HET, and KO-HET; 37-, 7-, and 17-fold, respectively; p < 0.05). The TGF-β concentration was decreased in a concentration-dependent manner by HET, JTT, and NYT (compared with the control). These results suggest that, compared with TJ-HET and KO-HET, KR-HET should be administered with caution. Although HET, JTT, and NYT belong to the same Hozai group and have the same names among companies, their differing effects on immune activity must be considered and they must be administered with caution.