Percentage Of Tregs Research Articles

Dysregulation of Regulatory T Cells and Autoimmune Sequelae in DRESS: Insights From Flow Cytometry and NanoString Analysis.

Drug reactions with eosinophilia and systemic symptoms (DRESS) and Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) are severe cutaneous adverse hypersensitivity reactions with distinct clinical manifestations. Regulatory T (Treg) cells may behave differently in these syndromes, contributing to their diverse clinical features and outcomes. This study compared Treg dynamics between DRESS and SJS/TEN patients during the acute and recovery phases. Flow cytometry quantitatively analysed and defined the immunophenotype of CD4+CD25+CD127-FoxP3+ Tregs in blood from DRESS and SJS/TEN patients indicated that Treg percentages were lowest in DRESS patients during the acute phase compared to those in the recovery phase in DRESS patients and the acute phase in SJS/TEN patients. During the acute phase, CTLA-4 expression in Tregs in both DRESS patients with and without autoimmune sequelae was significantly increased, while only DRESS patients without autoimmune sequelae had elevated OX40 expression compared to the healthy controls. High IL-10 expression in Tregs during the acute phase in SJS/TEN patients was also observed. The suppressive function of Tregs was lower in DRESS compared to SJS/TEN, which was determined using a suppression assay by co-culturing autologous Treg and effector T cells. Furthermore, NanoString technology explored mRNA profiles in Tregs. Genes associated with the JAK/STAT pathway were found to be downregulated during the acute phase in DRESS patients who later developed autoimmune sequelae. Our findings evidenced impaired Treg function in DRESS compared to SJS/TEN. The early disturbance of the JAK/STAT pathway may serve as a prognostic marker for autoimmune development in DRESS patients.

Effects of moxibustion in improving synovitis in adjuvant arthritis rats by regulating synovial GPR43 and peripheral blood Th17/Treg balance

To observe the effects of moxibustion on G protein-coupled receptor 43 (GPR43) and helper T cell 17 (Th17)/regulatory T cell (Treg) balance in rats with adjuvant arthritis (AA), so as to investigate the partial mechanism of moxibustion therapy on rheumatoid arthritis (RA). A total of 24 Wistar rats were randomly divided into a normal group, a model group and a moxibustion group, with 8 rats in each group. The AA rat model was replicated using wind, cold and moisture environmental factors + Freund's complete adjuvant (CFA) injection method. In the moxibustion group, moxibustion was performed on bilateral "Zusanli" (ST36) and "Shenshu" (BL23) for 20 min/time, once daily, for 21 d. The changes of joint swelling degree (JSD) and arthritis index (AI) were observed in each group of rats. Transmission electron microscopy and HE staining were used to examine changes in the cellular structure of the ankle joint synovial tissue in each group. Western blot analysis was employed to detect the expression levels of GPR43 in the synovial tissue of the ankle joints. Flow cytometry was used to measure the percentages of Treg and Th17 in peripheral blood. ELISA was used to determine the contents of interleukin-10 (IL-10) and transforming growth factor-β 1 (TGF-β1) in serum. Compared with the normal group, the JSD and AI in the model group increased before and after treatment (P<0.01), with significant synovial pathological and ultrastruct ural injury observed. Additionally, compared with the normal group, the expression of GPR43 in the synovial tissue decreased (P<0.01), the Treg percentage in peripheral blood decreased (P<0.01) and the Th17 percentage increased (P<0.01), serum IL-10 and TGF-β1 contents decreased in the model group (P<0.01). Compared with the model group, the moxibustion group exhibited a significant reduction in JSD and AI after treatment (P<0.01), the degree of pathological and ultrastruct ural damage in the synovium decreased, the expression of GPR43 in the synovial tissue increased (P<0.05), the Treg percentage in peripheral blood increased (P<0.01) and the Th17 percentage decreased (P<0.01), serum IL-10 and TGF-β1 contents increased (P<0.01). The relative expression of GPR43 in synovial tissue was positively correlated with the percentage of Treg in peripheral blood (r=0.967, P<0.01). Moxibustion can significantly improve the inflammatory response in the synovial membrane of AA rats. The mechanism may be related to moxibustion restoring the Th17/Treg balance through regulating GPR43.

Updates on the controversial roles of regulatory lymphoid cells in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most common and severe form of pulmonary fibrosis, characterized by scar formation in the lung interstitium. Transforming growth factor beta (TGF-β) is known as a key mediator in the fibrotic process, acting on fibroblasts and mediating their proliferation and differentiation into myofibroblasts. Although the immune system is not considered responsible for the initiation of IPF, markers of tolerogenic immunity define the pro-fibrotic microenvironment in the lungs. In homeostatic conditions, regulatory T cells (Tregs) constitute the main lymphoid population responsible for maintaining peripheral tolerance. Similar to Tregs, regulatory B cells (Bregs) represent a recently described subset of B lymphocytes with immunosuppressive functions. In the context of IPF, numerous studies have suggested a role for Tregs in enhancing fibrosis, mainly via the secretion of TGF-β. In humans, most studies show increased percentages of Tregs associated with the severity of IPF, although their exact role remains unclear. In mice, the most commonly used model involves triggering acute lung inflammation with bleomycin, leading to a subsequent fibrotic process. Consequently, data are still conflicting, as Tregs may play a protective role during the inflammatory phase and a deleterious role during the fibrotic phase. Bregs have been less studied in the context of IPF, but their role appears to be protective in experimental models of lung fibrosis. This review presents the latest updates on studies exploring the implication of regulatory lymphoid cells in IPF and compares the different approaches to better understand the origins of conflicting findings.

Oral magnesium reduces levels of pathogenic autoantibodies and skin disease in murine lupus

BackgroundSystemic Lupus Erythematosus (SLE) has a strong genetic susceptibility, but little is known about the impact of diet on disease severity. The Western diet is typically deficient in magnesium (Mg), and given the immunomodulatory effects of Mg, we hypothesized that the low Mg intake increases disease risk and that increasing Mg intake would reduce severity of murine lupus. Here, we placed 12-week old MRL/lpr female lupus mice on a normal (Mg500) or a high (Mg2800) Mg diet for 9 weeks. Urine and blood were collected during the study for quantification of urinary albumin, BUN, anti-dsDNA antibodies, and immune phenotyping.ResultsMRL/lpr lupus mice on high Mg2800 diet had significantly fewer skin lesions and less severe skin histology score, and reduced levels of pathogenic anti-dsDNA antibodies, compared with the Mg500 group (143.8±75.0 vs. 47.4±36.2 × 106U/ml; P < 0.05). The high Mg2800 group had a nearly two-fold increase in the percentage of CD4+FOXP3+ Treg cells compared to controls (19.9±5.4 vs. 11.4±5.5%; P < 0.05). Treg percentages inversely correlated with the concentration of anti-dsDNA. None of the mice developed arthritis during the observation period and there were no significant differences in weight, proteinuria, BUN or kidney histology.ConclusionIn conclusion, oral supplementation of Mg has a protective effect in a murine lupus model and may represent an inexpensive and safe adjuvant in the treatment of SLE.

Role of serum cd4+, cd25+, and foxp3+ cells’ segmental and nonsegmental vitiligo

Background Vitiligo is an autoimmune skin disease presented with depigmented macules and patches. Vitiligo has an impact on the quality of life. The etiopathogenesis of vitiligo is multifactorial, including genetic, immune dysregulation, and oxidative stress mechanisms. Lately, several researches have underlined the pivotal function of regulatory T cells (Tregs) in the vitiligo pathogenesis with lack of data regarding their role in segmental vitiligo (SV). Objective To investigate the role of Tregs in SV and nonsegmental vitiligo (NSV) pathogenesis and its correlation with disease activity and severity. Patients and methods This case–control study included 20 cases with NSV and 10 cases with SV in addition to 10 healthy volunteers. Vitiligo Area Scoring Index and Vitiligo Disease Activity scores were estimated for vitiligo cases and all the included participants were assessed for the percentage of serum CD4+, CD25+, and FOXP3+ T cells using flow cytometry staining buffer. Results There was significant reduction of the peripheral Tregs in NSV cases in comparison with healthy participants and negative correlation with their percentage to disease activity. On the other hand, there was insignificant difference between the percentage of peripheral Tregs in SV cases and healthy participants. Also, there was insignificant correlation between peripheral Tregs and both severity and activity of the disease among SV cases. Conclusion NSV cases showed significant reduction of the peripheral Tregs and negative correlation with disease activity, indicating the importance of Tregs in the etiopathogenesis of NSV and hence future targeting therapy. On the other hand, in SV cases, there was insignificant reduction of peripheral Tregs and insignificant correlation between their percentage and both severity and activity indicating mosaic etiopathogenesis.

Prognostic Significance of Regulatory T-Cells and PD-1 + CD8 T-Cells in Chronic Myeloid Leukemia Patients Treated with Generic Imatinib.

The impact of T-regulatory cells (Tregs), PD-1 + CD8 T-cells, and their dynamics during treatment with imatinib mesylate remains poorly understood in patients with chronic myeloid leukemia (CML). We conducted a prospective study on newly diagnosed, treatment-naïve adult (> 18years old) patients with CML in the chronic phase (CP) and age- and sex-matched controls. Peripheral blood samples were collected at diagnosis and after three months of imatinib therapy to assess Tregs and PD-1 + CD8 T-cell levels using flow cytometry. The study comprised 57 patients with a median age of 39years, including 27 males (47%). At baseline, the mean percentage of Tregs was significantly higher in CML patients (3.6 ± 0.32%) compared to controls (1.58 ± 0.21%) (p < 0.0001) but decreased significantly after three months of imatinib treatment (1.73 ± 0.35%) (p < 0.0001). Baseline Treg% exhibited positive correlations with Sokal (r = 0.29), Hasford (r = 0.33), EUTOS (r = 0.28), and ELTS (r = 0.31) risk scores (p < 0.05), as well as with the BCR-ABL transcript levels at three months (p = 0.03). Furthermore, the mean baseline percentage of PD-1 + CD8 T-cells was significantly elevated in CML patients (7.66 ± 0.36%) compared to controls (2.65 ± 0.32%) (p < 0.0001) and also decreased after treatment (3.44 ± 0.37%) (p < 0.0001). The baseline percentage of PD-1 + T-cells demonstrated positive correlations with Sokal (r = 0.26), Hasford (r = 0.27), and ELTS (r = 0.41) risk scores (p < 0.05). Our findings reveal a significantly higher proportion of Tregs and PD-1 + CD8 T-cells in patients with CML-CP compared to healthy controls, notably diminished following imatinib treatment. These observations suggest the potential for immunotherapy as a promising approach to managing immune exhaustion in CML patients.

Activating α7nAChR suppresses systemic inflammation by mitigating neuroinflammation of the medullary visceral zone in sepsis in a rat model.

Our previous studies have shown that activating α7nAChRs suppresses systemic inflammation and immunity through the cholinergic anti-inflammatory pathway (CAP) in early sepsis. Now that the medullary visceral zone (MVZ) is the center of CAP and responsible for regulating systemic inflammation, what changes will occur in MVZ's pathology and function in sepsis, especially when interfering with α7nAChRs? Does activation of MVZ's α7nAChRs contribute to the inhibition of systemic inflammation? To clarify these issues, we explored the systemic inflammation and immunity state by detecting serum levels of TNF-α, IL-6, HMGB1, sCD14, and CD4+CD25+Treg and TH17 lymphocytes percentage, meanwhile, we analyzed the apoptosis of cholinergic and catecholaminergic neurons and the expressions of tyrosine hydroxylase (TH) and choline acetyltransferase (CHAT) in MVZ in sepsis and the interfering effects on α7nAChRs. In this study, we found that in sepsis, serum TNF-α, IL-6, HMGB1, sCD14, CD4+CD25+Treg, and TH17 lymphocytes significantly increased and the ratio of Treg/TH17 significantly decreased, cholinergic and catecholaminergic neurons underwent apoptosis with low expressions of TH and CHAT in MVZ; activation of α7nAChRs not only significantly decreased the levels of septic serum TNF-α, IL-6, HMGB1, sCD14, and TH17 lymphocytes (P ＜ 0.05), but also significantly reduced cholinergic and catecholaminergic neurons' apoptosis, and promoted expressions of TH/CHAT. Our study reveals that sepsis undermines MVZ through neuroinflammation which contributes to the uncontrolled systemic inflammation. Activating central α7nAChRs is not only helpful to restore MVZ's structure and function but also beneficial to subside the inflammatory storm in sepsis. Even if MVZ is damaged in sepsis, cholinergic neurons in MVZ still regulate the systemic inflammation stably.

Decrease of Tregs cells and increase of exhausted Treg cells as the predictors of COVID19 severity

BackgroundT cells and regulatory T cells (Tregs) play a critical role in viral infectious immunity. Exhaustion of T cells during infection and decreased Tregs both contribute to the exacerbation of the disease. In the present study, we assessed T cells and regulatory T cells of COVID-19 patients and a control group according to the expression of the PD-1 molecule. MethodsForty-two COVID-19 patients and 40 controls were enrolled in the study. In COVID-19 patients, blood samples were collected on the first day of their hospitalization. Regulatory T cells (CD4+, CD25+, FOXP3+), CD4+PD-1+, and PD-1+ regulatory T cells were assessed by flow cytometry. ResultsThe percentage of CD4+PD-1 + T cells in COVID-19 patients was significantly higher compared to the control group (P < 0.0001). The percentage of PD-1+ regulatory T cells was significantly increased in the patient group compared to the control group (P < 0.0001). However, the Treg percentage was significantly decreased in the patient group compared to the control group (P < 0.0001). The frequency of CD4+PD-1 + T cells, Tregs, and PD-1+ Tregs had acceptable sensitivity and specificity for assisting in the diagnosis of severe/critical COVID-19. The declined Tregs and enhanced CD4+CD25+, CD4+PD-1+, and PD-1 + T cells were associated with disease severity. ConclusionThe decrease in Tregs and the increase in exhaustion of these cells and T cells play an important role in COVID-19 pathogenesis. These immune parameters could be used as meaningful indicators for assisting in the diagnosis of severe/critical COVID-19.

P-390 The percentage of regulatory T cells is associated with T follicular helper cell quantity in the peripheral blood of pregnant women

Abstract Study question Is there a relationship between the percentage of peripheral regulatory T cells (Treg) and other T cell subtypes in the first trimester of pregnancy? Summary answer The percentage of Treg cells showed a significant positive correlation with the quantity of T follicular helper cells (TFH). What is known already Regulatory T cells (Treg) play an essential role in the process of embryo implantation and maintenance of normal pregnancy. The quantity of these cells in the peripheral blood expands during the first trimester of pregnancy. Several studies report the importance of Treg in the control of the inflammatory process and in the pathophysiology of common adverse pregnancy outcomes. However, there is a scarce data about the quantitative relation between Treg and other important T sub-types during pregnancy. Study design, size, duration This is an observational study of 93 women, 28 to 53 years of age (mean 38 years), who had a blood sample during the first trimester (at 8-10 weeks of pregnancy). The study was conducted between November 2022 and October 2023. The exclusion criteria were history of recent inflammatory or infection disorders, recent antibiotic treatment, autoimmune diseases, oncological diseases, moderate or severe endometriosis, and endometrial polyps. Written informed consent was obtained from each participant. Participants/materials, setting, methods SIx peripheral T cell subtypes were measured by flow cytometry: T helpers % (CD3+CD4+CD8-/CD3+), T killers % (CD3+CD4-CD8+/CD3+), double-positive T cells % (CD3+CD4+CD8+/CD3+), double-negative T cells % (CD3+CD4-CD8-/ CD3+), T follicular helper cells % (CD3+CD4+CD8-CD185+/CD4+), T reg cells (CD4+CD8-CD25+CD127−/CD4+). Spearman’s correlation test was performed with all studied T cell subtypes in SPSS 21 package program. Main results and the role of chance The median percentage (range) of the studied T cell subtypes was: T helpers (51.7%, 12.1%÷91.45%), T killers (39.0%, 4.1%÷72.5%), DPT cells (1.2%, 0÷8.75), DNT cells (8.4%, 1.9%÷46.2%), Treg cells (4.6%, 1.9%÷12.7%), TFH cells (7.6%, 1.4%÷42.6%). To examine the association between the percentage of Treg cells and the other T cell subtypes, the Spearman’s Rho Correlation coefficient for non-paramentric data was used. No correlation was found between the quantity of Treg and T helpers, T killers, Total T cells, double-positive T cells (p &amp;gt; 0.05). In contrast, the percentage of Treg showed a significant positive correlation with the percentage of TFH (R = 0.36; p = 0.01), and a significant negative correlation with double-negative T cells (R= - 0.29; p = 0.04). Limitations, reasons for caution The main limitation of the study is that it was carried out only in a limited period of time during the pregnancy. Thus, it would be beneficial to compare the percentage of Treg cells with other T cell sub-types in the second and third trimester of pregnancy. Wider implications of the findings Our study demonstrated that Treg cells have stronger association with TFH cells than with other T cell subtypes. The strength of this quantitative relationship might be important for the normal progress of pregnancy, and it remains subject of further studies. Trial registration number not applicable

BET Inhibitor JQ1 Selectively Reduce Tregs by Upregulating STAT3 and Suppressing PD-1 Expression in Patients with Multiple Myeloma.

Multiple myeloma (MM) stands as a prevalent hematological malignancy, primarily incurable, originating from plasma cell clones. MM's progression encompasses genetic abnormalities and disruptions in the bone marrow microenvironment, leading to tumor proliferation, immune dysfunction, and compromised treatment outcomes. Emerging evidence highlights the critical role of regulatory T cells (Tregs) in MM progression, suggesting that targeting Tregs could enhance immune functionality and treatment efficacy. In this study, a notable increase in Treg proportions within MM patients' bone marrow (BM) compared to healthy individuals is observed. Additionally, it is found that the bromodomain and extraterminal domain (BET) inhibitor JQ1 selectively diminishes Treg percentages in MM patients' BM and reduces TGF-β1-induced Tregs. This reduction occurs via inhibiting cell viability and promoting apoptosis. RNA sequencing further indicates that JQ1's inhibitory impact on Tregs likely involves upregulating STAT3 and suppressing PD-1 expression. Collectively, these findings suggest JQ1's potential to modulate Tregs, bolstering the immune response in MM and introducing a promising avenue for MM immunotherapy.

Solute Transporter OCTN1/Slc22a4 Affects Disease Severity and Response to Infliximab in Experimental Colitis: Role of Gut Microbiota and Immune Modulation

Abstract Background Inflammatory bowel diseases are chronic disabling conditions with a complex and multifactorial etiology, still incompletely understood. OCTN1, an organic cation transporter, could have a role in modulating the inflammatory response, and some genetic polymorphisms of this molecule have been associated with increased risk of inflammatory bowel diseases. Until now, limited information exists on its potential in predicting/modulating patient’s response to therapies. The aim of this study was to evaluate the role of OCTN1 in modifying gut microbiota and mucosal immunity in response to infliximab therapy in murine colitis. Methods A dextran sodium sulphate model of colitis was used to assess the clinical efficacy of infliximab administered intravenously in ocnt1 gene knockout mice and their C57BL/6 controls. Stool, colon, and mesenteric lymph node samples were collected to evaluate differences in gut microbiota composition, histology, and T cell populations, respectively. Results Octn1 -/- influences the microbiota profile and is associated with a worse dysbiosis in mice with colitis. Infliximab treatment attenuates colitis-associated dysbiosis, with an increase of bacterial richness and evenness in both strains. In comparison with wild type, octn1-/- mice have milder disease and a higher baseline percentage of Treg, Tmemory, Th2 and Th17 cells. Conclusions Our data support the murine model to study OCTN1 genetic contribution to inflammatory bowel diseases. This could be the first step towards the recognition of this membrane transporter as a biomarker in inflammatory conditions and a predictor of response to therapies.

Pre-Existing Immunity Predicts Response to First-Line Immunotherapy in Non-Small Cell Lung Cancer Patients.

T-cell-mediated anti-tumoral responses may have significant clinical relevance as a biomarker for response to immunotherapy. The value of peripheral blood pre-existing tumor antigen-specific T cells (PreI+) as a predictive immunotherapy biomarker in NSCLC patients was investigated, along with the frequency of various circulating immune cells. Fifty-two treatment-naïve, stage III/IV NSCLC patients, treated with front-line immune checkpoint inhibitors (ICI)-containing regimens were enrolled. PreI was calculated as the percentages of CD3+IFNγ+ cells after in vitro co-cultures of PBMCs with peptides against four different Tumor-Associated Antigens (TAA). Immunophenotyping of peripheral blood immune cells was performed using multicolor flow cytometry. PreI+ T cells were detected in 44% of patients. Median overall survival (OS) was significantly higher in PreI+ patients compared to PreI- patients (not reached vs. 321 days, respectively; p = 0.014). PreI+ patients had significantly higher numbers of possible exhausted CD3+CD8+PD-1+ cells and lower percentages of immunosuppressive Tregs compared to PreI- patients. Additionally, patients with PreI+ and low numbers of peripheral blood M-MDSCs had a significant survival advantage compared to the rest of the patients. Thus, combining pre-existing tumor antigen-specific immunity before initiation of ICI in NSCLC patients with selected immune-suppressive cells could identify patients who have a favorable clinical outcome when treated with ICI-containing regimens.

Overexpression of ubiquitin-conjugating enzyme 2T induces radiotherapy resistance in hepatocellular carcinoma by enriching regulatory T cells in the tumor microenvironment

To investigate the effect of overexpression of ubiquitin-conjugating enzyme 2T (UBE2T) on radiosensitivity of hepatocellular carcinoma (HCC). Hepa1-6 cells were transfected with a UBE2T-overexpressing or a control lentiviral vector, and the changes in their radiotherapy sensitivity and concentrations of glucose and lactate in the supernatant were assessed using colony-forming assay and colorimetric assay. The transfected cells were inoculated subcutaneously in nude mice or C57BL/6 mice, and tumor growth following irradiation were recorded. The xenografts were collected for analyzing infiltration of CD4+ T cells and regulatory T cells (Tregs) using flow cytometry and detecting expressions of HK1 and LDHA using Western blotting. The correlations of UBE2T expression with immune cell infiltration, glycolysis and Tregs in HCC were analyzed using CIBERSORT algorithm and TCGA database, and the results were verified in a co-culture system of Hepa1-6 cells and Tregs. UBE2T overexpression caused radiotherapy resistance in both cultured Hepa1-6 cells and xenografts in the tumor-bearing mouse models (especially in C57BL/6 mice). CIBERSORT analysis suggested that a high expression of UBE2T was associated with increased percentages of dendritic cells, T follicular helper cells, M2 macrophages, monocytes, lymphocytes and Tregs in HCC. The UBE2T-overexpressing xenografts showed an increased percentage of Tregs and enhanced expressions of HK1 and LDHA, and irradiation increased infiltration of CD4+ T cells and Tregs in the tumor microenvironment. Hepa1-6 cells overexpressing UBE2T showed a decreased glucose concentration and an increased lactate concentration. GSEA analysis suggested that a high UBE2T expression was positively correlated with increased glycolysis and Tregs infiltration in HCC. In the cell co-culture system, UBE2T overexpression significantly enhanced lactate production, proliferation and immunosuppressive functions of Tregs. A high UBE2T expression results in radiotherapy resistance of HCC possibly by enhancing glycolysis and cause enrichment of Tregs in the tumor microenvironment.

Monoclonal antibodies for equine CD25 improve detection of regulatory T cells in horses

CD25, the interleukin-2 receptor α-chain, is expressed on cell surfaces of different immune cells and is commonly used for phenotyping of regulatory T cells (Tregs). CD25 has essential roles in the maintenance of hemostasis and immune tolerance and Treg cell involvement has been shown in human diseases and murine models for allergy, autoimmunity, cancer, chronic inflammation, and many others. In horses, a cross-reactive anti-human CD25 antibody has previously been used for characterizing Tregs. Here, we developed monoclonal antibodies (mAbs) to equine CD25 and compared their staining pattern with the anti-human CD25 antibody by flow cytometry. The comparison of the two reagents was performed by two separate analyses in independent laboratories. Overall, similar staining patterns for equine peripheral blood lymphocytes were obtained with the anti-human CD25 antibody and equine CD25 mAb 15–1 in both laboratories. Both reagents identified comparable CD4+CD25+ and CD4+CD25+FOXP3+ percentages after stimulation of peripheral blood mononuclear cells (PBMC) with pokeweed mitogen. However, when compared to the anti-human CD25 antibody, the equine CD25 mAb 15–1 resulted in a better staining intensity of the equine CD25+ cells and increased the percentages of Tregs and other CD25+ cells ex vivo and after culturing of PBMC without stimulation. In summary, the equine CD25 mAbs provide new, improved reagents for Tregs and CD25+ cell phenotyping in horses.

#1715 Role of T cell profile (Th1/Th2/Th17/Treg) and podocyturia in the peripheral blood of patients with preeclampsia versus normal pregnancy

Abstract Background and Aims The concept of the balance between Th17/Treg cell immunity plays important role in determining pregnancy outcomes. Does Treg/TH17 play a role in pathogenesis of preeclampsia associated proteinuria and its implication on irreversible renal damage was the research question. Method This hospital based prospective study conducted in the Department of obstetrics and gynecology at a tertiary care medical college between October 2018 till October 2020 and assessment of T cell profiling and urinary podocyturia among all pregnancies with clinical evidence of preeclampsia and compared it with normal pregnancy monitoring for maternal and foetal outcomes. Repeat assessment of Th profiling and podocyturia was done after 3 months post partum to look for any residual effect. Results A total of 40 patients of preeclampsia and 40 control were enrolled in the study with median age group (24.58 ± 1.22 vs 25.16 ± 1.01). The percentage of Th1 and Th2 were higher for the preeclampsia group as compared to the control group with no change in the percentage of Th17 subsets while Th17 1 were found higher and percentage of Treg (8.1 ± 3.19 vs 2.3 ± 1,05, p = 0.001) lower in the peripheral blood of the preeclampsia group at time of delivery however the ratio of Th1/Treg, Th2/Treg, Th17/Treg and Th17.1/Treg were found significantly lowered after 6 weeks of delivery among those with preeclampsia nearly comparable to the normal pregnancy group The quantitative assessment of podocyte in urine using antinephrin showed increase numbers among preeclamptic pregnancies compared to normal yet it settled within 6 weeks of delivery. Conclusion Women with preeclampsia per se does not increase the risk to exacerbation of kidney disease however greater research mandates to assess the women at greatest risk of kidney disease after pre-eclampsia to have good maternal and foetal outcomes.

#2751 CD14++CD16+ monocytes and CD8+ T cells are independently associated with annual change in eGFR and proteinuria in kidney transplant recipients

Abstract Background and Aims Immune cellular responses are implicated in all clinical aspects of kidney transplantation. There is little data regarding the additional value of regular monitoring of immune cells subsets expression in kidney transplant recipients (KTRs). The aim of our prospective study was a longitudinal follow-up analysis of monocytes subpopulations, natural killer (NK) cells and lymphocytes subsets including regulatory T cells (Tregs) in the circulation of KTRs and potential clinical correlations. Method 48 stable KTRs were initially enrolled. All KTRs were under immunosuppressive regimen with corticosteroids, mycophenolate mofetil or mycophenolate acid and calcineurin inhibitors (CNIs). Exclusion criteria were history or development during follow-up of acute rejection, cardiovascular disease, malignancy and active or chronic infections. Patients were followed for 12 months. The peripheral blood immune cell subsets CD14++CD16-, CD14++CD16+ and CD14+CD16++ absolute values and percentages out of total monocytes and NK cells (CD3+CD16+56+), CD3-CD19+ B lymphocytes, CD3+ CD4+ T cells, CD3+CD8+ T cells and Tregs (CD4+CD25+ FoxP3+) absolute values and percentages out of total lymphocytes were measured by flow cytometry at baseline (T0) and after 12 months (T1). Clinical and laboratory parameters were recorded at T0 and T1. Delta (Δ) eGFR and Δ spot urine protein to creatinine ratio (ΔUPCR) were defined as their respective differences between T1 and T0. Likewise, Delta (Δ) of immune cells subtypes or laboratory indices was defined as their respective difference between T1 and T0. Results 35 KTRs (mean age 53 ± 9.28 years, 71% males, mean transplant vintage 96 ± 66 months, 63% on Tacrolimus and 37% on Cyclosporine) were included in the final analysis. Significant differences were observed between T0 and T1 in monocytes number (653 ± 244 and 538 ± 197/μL respectively, p = 0.001), monocytes percentage (7.6 ± 2.9 and 6.6 ± 2.2% respectively, p = 0.006) as well as in the number of classical CD14++CD16- monocytes (534 ± 225 and 452 ± 185/μL respectively, p = 0.04). The rest immune cells subsets did not show any significant differences between T0 and T1. Mean eGFR declined from 58 ± 17 at T0 to 53 ± 18 ml/min/1.73 m2 at T1 (p = 0.004). No significant changes were observed between T0 and T1 in median UPCR [0.16 (IRQ, 0.09-0.56) at T0 and 0.16 (IQR, 0.10-0.70) gr protein/gr creatine at T1, p = 0.489], in median CRP [4.0 (IQR, 3-7) at T0 and 5 (IQR, 2.5-7.5) mg/L) at T1, p = 0.919], in mean ESR (20 ± 14 at T0 and 22 ± 19 mm/hour at T1, p = 0.381) or other parameters, including CNIs blood levels. ΔeGFR was correlated with the T0 percentage of monocytes (rho = 0.359, p = 0.037), the T0 number and T0 percentage of CD14++CD16+ monocytes (rho= 0.502, p = 0.003 and rho = 0.438, p = 0.008 respectively). On the other hand, a borderline inverse correlation was observed between ΔeGFR and ΔCD14++CD16+ monocytes number (rho = −0.339, p = 0.05). Additional correlates of ΔeGFR included serum albumin at T0 (rho = 0.395, p = 0.021) and Δuric acid (rho = 0.567, p = 0.000). At stepwise linear regression analysis, CD14++CD16+ monocytes (β = 0.338, p = 0.04) and Δuric acid (β = 0.477, p = 0.006) remained independent significant correlates of ΔeGFR. ΔUPCR was significantly correlated with the percentage of B-lymphocytes (rho = 0.385, p = 0.027) and CD4+ T-cells (rho = 0.352, p = 0.044) at T0, and inversely correlated with the T0 percentage of T-lymphocytes (rho = −0.402, p = 0.02) and CD8+ T cells (rho = −0.603, p = 0.000) as well as with Δhemoglobin (rho = −0.385, p = 0.027). At stepwise linear regression analysis, only the CD8+ T cells percentage at T0 remained independently correlated to ΔUPCR (β = –0.379, p = 0.03). Conclusion The results of our study indicate that intermediate CD14++CD16+ monocytes are associated with annual eGFR change whereas CD8+ T cells negatively correlate with the annual trend of proteinuria change in KTRs. Further research is needed to clarify underlying pathophysiological mechanisms for these findings. Finally, future studies including larger cohorts and with longer follow-up are required to specify the potential utility of immune subpopulations monitoring as potential prognostic biomarkers in KTRs.

Majie Cataplasm Alleviates Asthma by Regulating Th1/Th2/Treg/Th17 Balance

Introduction: T cells play a critical role in inflammatory diseases. The aim of the present study was to investigate the effects of Majie cataplasm (MJC) on asthma and to propose a possible mechanism involved in this process. Methods: Airway inflammation, infiltration of inflammatory cells, levels of interleukin (IL)-4, IL-10, IL-17, and interferon (IFN)-γ, levels of Th2, Treg, Th17, and Th1 cells, and the expressions of IL-4, IL-10, IL-17, IFN-γ, GATA binding protein 3 (GATA-3), Foxp3, RAR-related orphan receptor gamma (RORγt), and T-bet were detected. Result: MJC treatment reduced lung airway resistance and inflammatory infiltration in lung tissues. MJC treatment also reduced the numbers of eosinophils and neutrophils in the blood and bronchoalveolar lavage fluid (BALF). The levels of IL-4 and IL-17 in the blood, BALF, and lungs were suppressed by MJC, and IFN-γ and IL-10 were increased. Furthermore, MJC suppressed the percentage of Th2 and Th17 and increased the percentage of Th1 and Treg in spleen cells. In addition, MJC can inhibit asthma by increasing expressions of IFN-γ, IL-10, T-bet, and Foxp3, as well as decreasing expressions of IL-4, IL-17, GATA-3, and RORγt. Conclusion: MJC may improve airway hyperresponsiveness and inflammation by regulating Th1/Th2/Treg/Th17 balance in ovalbumin-induced rats. And MJC may be a new source of anti-asthma drugs.

Prostaglandin I2 signaling in Treg restrains ST2 expression and associated pathogenicity

Abstract We reported Prostaglandin I2 (PGI2) signaling within Treg is critical for Treg suppressive function in allergic airway inflammation (AAI). Interestingly, pathogenic Treg that have impaired suppressive capacity and promote AAI have greater ST2 expression. We hypothesized that Treg PGI2 signaling limits ST2 expression via restraint of β-Catenin signaling in AAI to prevent Treg pathogenicity. The percentage of ST2+ Tregs was elevated at baseline in IP deficient (IP KO) Treg, which lack the receptor for PGI2, compared to WT Treg. To further test our hypothesis, we utilized an Alternaria alternata (Alt) extract sensitization and challenge protocol. Alt-challenged mice that lacked IP signaling only on Treg (Foxp3YFPcrexIPflox) had elevated BAL IL-13, BAL eosinophils, lung vascular remodeling, and lung ST2+ Treg percentages compared to Foxp3YFPcre controls. Moreover, there were elevated total lung ST2+IL13+ Treg and ex-Treg in Alt-challenged fate-mapping IP KO mice compared to fate-mapping controls. Interestingly, BAL IL-13, BAL eosinophils, lung ST2+ Treg percentages were significantly decreased in Alt-challenged mice that had Treg specific deficiency for both IP and β-Catenin (Foxp3YFPcrexIPfloxxCtnnb1flox) compared to Foxp3YFPcrexIPflox mice. In summary, we demonstrate an association of Treg ST2 expression with enhanced Treg pathogenicity in a mouse model of AAI in IP deficient Treg mediated by β-Catenin. These data support PGI2 as a therapeutic for asthma.

Deletion of RANKL in regulatory T cells ameliorates bone erosion in arthritis

Abstract Rheumatoid arthritis (RA) is an autoimmune disease that leads to bone erosion mediated through osteoclasts.Osteoclast differentiation is promoted through engagement of RANK by its ligand (RANKL) expressed by multiple lineages. Whereas regulatory T cells (Tregs) typically suppress osteoclastogenesis, Tregs developing in a pro-inflammatory environment can express RANKL and promote osteoclast function (Levescot et al.2021).To test the importance of osteoclastogenic Tregs in vivo, we developed a mouse selectively lacking RANKL expression in Tregs (FoxP3YFP/creRANKLflox/flox, hereafter RANKLΔTreg). Arthritis was induced by administration of K/BxN serum containing arthritogenic IgG antibodies. Joint inflammation and synovial neutrophil infiltration in RANKLΔTreg mice were similar to WT animals. Compared with Tregs from lymph nodes, Tregs from inflamed joints exhibited increased expression of activation markers including CD25,Foxp3,CD44, and CTLA4, without a difference between RANKLΔTreg and WT animals; RANKLΔTreg mice exhibited a trend for higher Treg PD-1 expression. In WT mice, the percentage of Tregs expressing RANKL was elevated in synovial fluid compared to lymph nodes. Correspondingly, microCT showed a significant reduction in bone erosion in RANKLΔTreg compared to WT mice (p=0.003). These findings are consistent with a pathogenic role of RANKL-expressing Treg for RA bone erosion in arthritis; whether RANKL impacts other Treg functions remains to be established.

Deficiency in CD103DC function leading to decreased pTregs in female lupus-prone mice is due to defects in intestinal lamina propria stromal cell function that can be restored by male microbiota.

Abstract Active lupus in humans is associated with low Treg percentages, but not function. We have found that lupus-prone female BWF1 have decreased peripherally-derived Tregs (pTregs), but not function vs lupus-resistant male BWF1 mice. Gut CD103+ DC (CD103DC), the major pTreg inducer, function is decreased in female mice due to defects in the retinoic acid (RA) synthesis pathway, i.e., lower RALDH2 (critical enzyme) and higher Zfp36 expression (degrades RALDH2 RNA). Both normal CD103DC development/function and RALDH2 expression require exogenous RA. Treating female CD103DC with RA restored function and increased RALDH2 and decreased Zfp36 expression indicating CD103DC are not defective themselves. Lamina propria stromal cells (LPSC) are required for CD103DC development via RA and GM-CSF production. Female BWF1 LPSC had decreased RALDH2 expression/activity and GM-CSF suggesting lower CD103DC function is due to decreased LPSC function. We have found differences in female and male gut microbiota in BWF1 mice, and transfer of male microbiota to female mice protects against disease and increases survival, increases percentages/numbers of total Tregs/pTregs, and restores CD103DC and LPSC function that appears to be mediated by metabolites, phytol/phytanic acid, produced by male microbiota. Lower pTregs in female BWF1 mice may be due to defects in LPSC support of CD103DC development and can be restored by metabolites produced by male microbiota that contribute to protection from disease.