Preparation and application of fluorescent monoclonal antibodies recognizing goat CD4+CD25+ regulatory T cells.

Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.

Causal relationship between immune cells and atrial fibrillation: A Mendelian randomization study.

Atrial fibrillation (AF) is a prevalent cardiac arrhythmia, with recent research indicating a correlation between immune system characteristics and the development of AF. However, it remains uncertain whether the immunological response is the primary underlying component or a secondary consequence of AF. Initially, we investigated the effect of immune cells on AF by performing forward Mendelian randomization (MR) analyses with immune cells as the exposure variable and their associated genetic variants as instrumental variables. Subsequently, we performed reverse MR analyses with AF as the exposure variable and immune cells as the outcome variable to exclude the interference of reverse causality, to distinguish between primary and secondary effects, and to further elucidate the causal relationship between the immune system and AF. We discovered that membrane proteins on specific immune cells, such as CD25 on memory B cells-which functions as a part of the interleukin-2 receptor-may be risk factors for AF development, with odds ratios of 1.0233 (95% confidence interval: 1.0012-1.0458, P = .0383). In addition, certain immune cell counts, such as the CD4 regulatory T cell Absolute Count, play a protective factor in the development of AF (odds ratio: 0.9513, 95% confidence interval: 0.9165-0.9874; P = .0086). More detailed results are elaborated in the main text. Our MR study has yielded evidence that substantiates a genetically inferred causal association between the immune system and AF. Identifying the risk factors associated with AF is vital to facilitate the development of innovative pharmaceutical treatments.

Ischemic stroke outcome after promoting CD4+CD25+ Treg cell migration through CCR4 overexpression in a tMCAO animal model.

The importance of neuroinflammation during the ischemic stroke has been extensively studied. The role of CD4+CD25+ regulatory T (Treg) cells during the recovery phase have shown infarct size reduction and functional improvement, possibly through the mitigation of inflammatory immune responses. We aimed to investigate the molecular factors involved in microglia-Treg cell communication that result in Treg trafficking. First, we observed the migration patterns of CD8+ (cytotoxic) T cells and Treg cells and then searched for chemokines released by activated microglia in an oxygen-glucose deprivation (OGD) model. The transwell migration assay showed increased migration into OGD media for both cell types, in agreement with the increase in chemokines involved in immune cell trafficking from the mouse chemokine profiling array. MSCV retrovirus was transduced to overexpress CCR4 in Treg cells. CCR4-overexpressed Treg cells were injected into the mouse transient middle cerebral artery occlusion (tMCAO) model to evaluate the therapeutic potential via the tetrazolium chloride (TTC) assay and behavioral tests. A general improvement in the prognosis of animals after tMCAO was observed. Our results suggest the increased mobility of CCR4-overexpressed Treg cells in response to microglia-derived chemokines in vitro and the therapeutic potential of Treg cells with increased mobility in cellular therapy.

Depletion of regulatory T cells enhancing the anti-tumor effect of in situ vaccination in solid tumors.

The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the clinical treatment for tumor. However, the low response rate of ICIs remains the major obstacle for curing patients and effective approaches for patients with primary or secondary resistance to ICIs remain lacking. In this study, immune stimulating agent unmethylated CG-enriched (CpG) oligodeoxynucleotide (ODN) was locally injected into the tumor to trigger a robust immune response to eradicate cancer cells, while anti-CD25 antibody was applied to remove immunosuppressive regulatory T cells, which further enhanced the host immune activity to attack tumor systematically. The combination of CpG and anti-CD25 antibody obtained notable regression in mouse melanoma model. Furthermore, rechallenge of tumor cells in the xenograft model has resulted in smaller tumor volume, which demonstrated that the combinational treatment enhanced the activity of memory T cells. Remarkably, this combinational therapy presented significant efficacy on multiple types of tumors as well and was able to prevent relapse of tumor partially. Taken together, our combinational immunotherapy provides a new avenue to enhance the clinical outcomes of patients who are insensitive or resistant to ICIs treatments.

[Circulating populations of CD4+ CD25+ CD127- regulatory t lymphocytes in peripheral blood of allergic asthmatic children]. / Determinación de poblaciones circulantes en sangre periférica de linfocitos T, reguladores CD4+ CD25+ CD127-, en niños asmáticos alérgicos.

OBJECTIVE: To carry out a preliminary analysis on the Treg lymphocyte counts present in the peripheral blood of allergic asthmatic children from the city of Cartagena, Colombia, compared to healthy controls. METHODS: We compared cytometry counts of ten asthmatic patients (age 7-16 years) and seven healthy controls (6-12 years), recruited in the city of Cartagena. Peripheral blood samples were stained using Cytek's 14-color cFluor Immunoprofiling kit (Cytek® cFluor® Immunoprofiling Kit 14 Color RUO kit), and analyzed on a Northern Lights™ spectral cytometer (Cytek® Biosciences, Fremont, CA, USA), to read 50.000 events per sample. The data obtained were analyzed in SpectroFlo® and FlowJo. The study was approved by the ethics committee of the University of Cartagena (SGR, Grant BPIN2020000100405). RESULTS: The frequency of CD3+, CD4+, CD25+, CD127- Tregs was 11% of all CD4+ T cells, with a range of minimum 8,1% and maximum 17,7%. There was no significant difference in the proportion of Tregs between allergic asthmatic patients and healthy controls (P = 0,2). CONCLUSIONS: With this preliminary sample size, no significant differences were found in the Treg lymphocyte population between allergic asthmatic patients and healthy controls. The 14-color multiplexed panel is a useful tool not only to count CD3+ and CD4+ populations, but also to obtain the percentage of regulatory T cells using cell surface markers.

OBJETIVO: Realizar un análisis preliminar sobre los conteos de linfocitos Tregs presentes en sangre periférica de niños asmáticos alérgicos de la ciudad de Cartagena, comparado con controles sanos. MÉTODOS: Se compararon los conteos de citometría de diez pacientes asmáticos (entre 7 y16 años) y siete controles sanos (entre 6 y12 años), reclutados en la ciudad de Cartagena. La muestra de sangre periférica fue teñida empleando el kit de inmunofenotipo multiplexado de 14 colores de Cytek (Cytek® cFluor® Immunoprofiling Kit 14 Color), y analizada en un citómetro espectral Northern Lights™ (Cytek® Biosciences, Fremont, CA, USA), a lectura de 50.000 eventos por muestra. Los datos obtenidos fueron analizados en SpectroFlo® y FlowJo. El estudio fue aprobado por el Comité de Ética de la Universidad de Cartagena. RESULTADOS: El panel de tinción funcionó apropiadamente y dentro de los parámetros apropiados. Se obtuvo un promedio de células Tregs CD3+, CD4+, CD25+ y CD127- del 11% de todos los CD4+ en las muestras estudiadas, con un rango de mínimo de 8,1% y un máximo de 17,7%. No hubo diferencias significativas en la proporción de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos (P = 0.2). CONCLUSIONES: Con este tamaño de muestra preliminar, no se encontraron diferencias significativas en la población de linfocitos Tregs entre los pacientes asmáticos alérgicos y los controles sanos. El panel multiplexado de 14 colores es una herramienta útil no solo para derivar las poblaciones CD3+ y CD4+, sino también para obtener el porcentaje de células T reguladoras empleando marcadores de superficie celular.

Interleukin-2-induced skin inflammation.

Recombinant human IL-2 has been used to treat inflammatory diseases and cancer; however, side effects like skin rashes limit the use of this therapeutic. To identify key molecules and cells inducing this side effect, we characterized IL-2-induced cutaneous immune reactions and investigated the relevance of CD25 (IL-2 receptor α) in the process. We injected IL-2 intradermally into WT mice and observed increases in immune cell subsets in the skin with preferential increases in frequencies of IL-4- and IL-13-producing group 2 innate lymphoid cells and IL-17-producing dermal γδ T cells. This overall led to a shift toward type 2/type 17 immune responses. In addition, using a novel topical genetic deletion approach, we reduced CD25 on skin, specifically on all cutaneous cells, and found that IL-2-dependent effects were reduced, hinting that CD25 - at least partly - induces this skin inflammation. Reduction of CD25 specifically on skin Tregs further augmented IL-2-induced immune cell infiltration, hinting that CD25 on skin Tregs is crucial to restrain IL-2-induced inflammation. Overall, our data support that innate lymphoid immune cells are key cells inducing side effects during IL-2 therapy and underline the significance of CD25 in this process.

Expression of CD25, mast cell markers and T-cell markers in eosinophilic esophagitis.

While eosinophilic esophagitis (EOE) is defined by histologic presence of eosinophils, a few studies have established the presence of mast cells in EOE and even shown their correlation with symptom persistence despite resolution of eosinophils. Expression of aberrant mast cell markers CD25 and CD2 have not been studied in EOE. This study quantifies the number of hotspot cells per high power field expressing CKIT/CD117, tryptase, CD25, CD2 and CD3 by immunohistochemical stains in endoscopic esophageal biopsies of the following three cohorts: (1) established and histologically confirmed EOE, (2) suspected EOE with biopsies negative for eosinophils, and (3) no history of or suspicion for EOE with histologically unremarkable biopsies. In this study, mast cells were highlighted by CKIT and tryptase in EOE, and not seen in other clinically mimicking cases. There were also significantly higher densities of CD25 and pan-T-cell marker staining in EOE cases. These findings suggest an inflammatory cellular milieu in EOE, beyond just eosinophils, that can be demonstrated by immunohistochemistry, and that invite further study into the role that these cells may play in EOE.

Graft Protective and Intercellular Immunomodulatory Effects by Adoptive Transfer of an Agonistic Anti-BTLA mAb (3C10) Induced CD4+CD25+ Regulatory T Cells in Murine Cardiac Allograft Transplant Model.

BACKGROUND: We demonstrated that an agonistic anti-B and T lymphocyte attenuator antibody (3C10) prolonged cardiac survival by inducing regulatory T cells (Treg). However, the mechanisms of immune tolerance in the recipients remained unclear. In this study, we investigated the graft-protective and intercellular immunomodulatory effects of adoptive transfer (AT) of 3C10-induced Tregs in a murine cardiac allograft transplant model. METHODS: Thirty days after transplantation of a C57BL/6 heart into the primary 3C10-treated CBA recipients, splenic CD4+CD25+ cells from these recipients (3C10/AT group) or naïve CBA mice (no-treatment group) were adoptively transferred into secondary CBA recipients with a C57BL/6 heart. To confirm the requirement for 3C10-induced Tregs, we administered an anti-interleukin-2 receptor alpha antibody (PC-61) to secondary CBA recipients. Additionally, histologic and fluorescent staining, cell proliferation analysis, flow cytometry, and donor-specific antibody (DSA) measurements were performed. RESULTS: 3C10/AT-treated CBA recipients resulted in significantly prolonged allograft survival (median survival time [MST], >50 days). Allografts displayed prolonged function with preservation of vessel structure by maintaining high numbers of splenic CD4+CD25+Foxp3+ Treg and intramyocardial CD4+Foxp3+ cells. DSA levels were suppressed in 3C10/AT-treated CBA recipients. Moreover, PC-61 administration resulted in a shorter MSTs of cardiac allograft survivals, a detrimental increase in DSA production, and enhanced expression of programmed cell death (PD)-1. CONCLUSION: AT of 3C10-induced Tregs may be a promising graft-protective strategy to prolong allograft survival and suppress DSA production, driven by the promotion of splenic and graft-infiltrating Tregs and collaboration with PD-1+ T cells and Treg.

Controlled WASp activity regulates the proliferative response for Treg cell differentiation in the thymus.

The Wiskott-Aldrich syndrome protein (WASp) regulates actin cytoskeletal dynamics and function of hematopoietic cells. Mutations in the WAS gene lead to two different syndromes; Wiskott-Aldrich syndrome (WAS) caused by loss-of-function mutations, and X-linked neutropenia (XLN) caused by gain-of-function mutations. We previously showed that WASp-deficient mice have a decreased number of regulatory T (Treg) cells in the thymus and the periphery. We here evaluated the impact of WASp mutations on Treg cells in the thymus of WAS and XLN mouse models. Using in vitro Treg differentiation assays, WAS CD4 single-positive thymocytes have decreased differentiation to Treg cells, despite normal early signaling upon IL-2 and TGF-ß stimulation. They failed to proliferate and express CD25 at high levels, leading to poor survival and a lower number of Foxp3+ Treg cells. Conversely, XLN CD4 single-positive thymocytes efficiently differentiate into Foxp3+ Treg cells following a high proliferative response to IL-2 and TGF-ß, associated with high CD25 expression when compared with WT cells. Altogether, these results show that specific mutations of WASp affect Treg cell development differently, demonstrating a critical role of WASp activity in supporting Treg cell development and expansion.

Additional booster doses in patients with chronic lymphocytic leukemia induce humoral and cellular immune responses to SARS-CoV-2 similar to natural infection regardless ongoing treatments: A study by ERIC, the European Research Initiative on CLL.

Profound immune dysregulation and impaired response to the SARS-CoV-2 vaccine put patients with chronic lymphocytic leukemia (CLL) at risk of severe COVID-19. We compared humoral memory and T-cell responses after booster dose vaccination or breakthrough infection. (Green) Quantitative determination of anti-Spike specific antibodies. Booster doses increased seroconversion rate and antibody titers in all patient categories, ultimately generating humoral responses similar to those observed in the postinfection cohort. In detail, humoral response with overscale median antibody titers arose in >80% of patients in watch and wait, off-therapy in remission, or under treatment with venetoclax single-agent. Anti-CD20 antibodies and active treatment with BTK inhibitors (BTKi) represent limiting factors of humoral response, still memory mounted in ~40% of cases following booster doses or infection. (Blue) Evaluation of SARS-CoV-2-specific T-cell responses. Number of T-cell functional activation markers documented in each patient. The vast majority of patients, including those seronegative, developed T-cell responses, qualitatively similar between treatment groups or between vaccination alone and infection cases. These data highlight the efficacy of booster doses in eliciting T-cell immunity independently of treatment status and support the use of additional vaccination boosters to stimulate humoral immunity in patients on active CLL-directed treatments.

Non-IL-2-blocking anti-CD25 antibody inhibits tumor growth by depleting Tregs and has synergistic effects with anti-CTLA-4 therapy.

CD25, also known as the interleukin-2 receptor α chain (IL-2Rα), is highly expressed on regulatory T cells (Tregs), but relatively lower on effector T cells (Teffs). This makes it a potential target for Treg depletion, which can be used in tumor immunotherapy. However, marketed anti-CD25 antibodies (Basiliximab and Daclizumab) were originally developed as immunosuppressive drugs to prevent graft rejection, because these antibodies can block IL-2 binding to CD25 on Teffs, which in turn destroys the function of Teffs. Recent studies have shown that non-IL-2-blocking anti-CD25 antibodies have displayed exciting antitumor effects. Here, we screened out a non-IL-2-blocking anti-CD25 monoclonal antibody (mAb) 7B7 by hybridoma technology, and confirmed its antitumor activity via depleting Tregs in a CD25 humanized mouse model. Subsequently, we verified that the humanized 7B7, named as h7B7-15S, has comparable activities to 7B7, and that its Treg depletion is further increased when combined with anti-CTLA-4, leading to enhanced remodeling of the tumor immune microenvironment. Moreover, our findings reveal that the Fab form of h7B7-15S has the ability to deplete Tregs, independent of the Fc region. Taken together, our studies expand the application of anti-CD25 in tumor immunotherapy and provide insight into the underlying mechanism.

Causal associations of cytokines and growth factors with cholelithiasis: a bidirectional Mendelian randomization study.

BACKGROUND: It has been reported that patients with cholelithiasis may have changes in levels of cytokines and growth factors, while their causal relationships were still unclear. METHODS: This study was a bidirectional Mendelian randomization (MR) study. Datasets of 41 circulation cytokines and growth factors and the data on cholelithiasis were obtained. Six steps of strict instrumental variable filtration were set, and inverse-variance weighted analysis, MR-Egger regression, and weighted median test were used to identify the causal relationships. Benjamini-Hochberg method was used to adjust the P-values. RESULTS: After adjustments of P-values, four cytokines and growth factors were still causally associated with cholelithiasis significantly: interleukin 2 receptor alpha (adjusted P: 4.59E-02), interleukin 8 (adjusted P: 1.09E-02), monocyte-specific chemokine 3 (adjusted P: 2.73E-04), and stem cell factor (adjusted P: 2.73E-04). In the reverse MR analysis, no significant causal relationship was detected after adjustment. CONCLUSIONS: Four cytokines and growth factors, including interleukin 2 receptor alpha, interleukin 8, monocyte-specific chemokine 3, and stem cell factor, were proven to relate to cholelithiasis causally and unidirectionally.

High IL2RA/CD25 expression is a prognostic stem cell biomarker for pediatric acute myeloid leukemia without a core-binding factor.

CD25 is an aberrant marker expressed on the leukemic stem cell (LSC) surface and an immunotherapy target in acute myeloid leukemia (AML). However, the clinical prevalence and significance of CD25 expression in pediatric AML are unknown. High IL2RA/CD25 expression in pediatric AML showed a stem cell-like phenotype, and elevated CD25 expression was associated with lower overall survival (p < .001) and event-free survival (p < .001) in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 study. This finding was reproduced in AML without a core-binding factor in the Children's Oncology Group study cohort. High CD25 expression has prognostic significance in pediatric AML.

Celastrol, which targets IL-2/CD25 binding inhibition, induces T cell-mediated antitumor activity in melanoma.

Interleukin-2 (IL-2) induces contrasting immune responses depending on its binding receptor subunit; thus, selective receptor binding is considered a key challenge in cancer therapeutic strategies. In this study, we aimed to investigate the inhibition of IL-2 action and antitumor activity of celastrol (CEL), a compound identified in a screen for IL-2/CD25 binding inhibitors, and to elucidate the underlying role of CEL in immune cells. We found that CEL selectively impairs the binding of IL-2 and CD25 and directly binds to IL-2 but not to CD25. CEL significantly suppressed the proliferation and signaling of IL-2-dependent murine T cells and interfered with IL-2-responsive STAT5 phosphorylation in IL-2 reporter cells and human PBMCs. After confirming the impact of CEL on IL-2, we evaluated its antitumor activity in C57BL/6 mice bearing B16F10 tumors and found that CEL significantly inhibited tumor growth by increasing CD8+ T cells. We also found that CEL did not inhibit tumor growth in T cell-deficient BALB/c nude mice, suggesting that its activity was mediated by the T-cell response. Moreover, combination therapy with low-dose CEL and a TNFR2 antagonist synergistically improved the therapeutic efficacy of the individual monotherapies by increasing the ratio of intratumoral CD8/Treg cells and suppressing Foxp3 expression. These findings suggest that CEL, which inhibits CD25 binding by targeting IL-2, exerts antitumor activity by mediating the T-cell response and could be a promising candidate for combination therapy in cancer immunotherapy against melanoma.

The soluble IL-2 receptor α/CD25 as a modulator of IL-2 function.

The pleiotropic cytokine interleukin-2 (IL-2) is an integral regulator of healthy and pathological immune responses, with the most important role in regulating the homeostasis of regulatory T cells. IL-2 signalling involves three distinct receptors: The IL-2 receptor α (IL-2Rα/CD25), IL-2Rß, and IL-2Rγ/γc . While IL-2Rß and γc are essential for signal transduction, IL-2Rα regulates the affinity of the receptor complex towards IL-2. A soluble form of the IL-2Rα (sIL-2Rα) is present in the blood of healthy individuals and increased under various pathological conditions. Although it is known that the sIL-2Rα retains its ability to bind IL-2, it is not fully understood how this molecule affects IL-2 function and thus immune responses. Here, we summarize the current knowledge on the generation and function of the sIL-2Rα. We describe the molecular mechanisms leading to sIL-2Rα generation and discuss the different IL-2 modulating functions that have been attributed to the sIL-2Rα. Finally, we describe attempts to utilize the sIL-2Rα as a therapeutic tool.

IL2RA rs2104286 and IL2 rs2069762 polymorphisms may be associated with bipolar disorder and its clinical findings.

Study results supported that immuno-inflammatory pathways in the brain and environment contribute to the etiopathogenesis of bipolar disorder (BD), a chronic affective disease. Our study aimed to assess the relationship between BD risk and interleukin 2 (IL2) and interleukin 2 receptor subunit alpha (IL2RA) variants in a Turkish population. Genomic DNA from 86 diagnosed BD patients and 100 healthy blood donors was extracted. IL2RA rs2104286, IL2 rs2069762, and IL2 rs2069763 variants were genotyped using the polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method. It was compared to the relationship between the genotype distributions of these variants and clinical characteristics. Results were evaluated statistically. A statistically significant difference in the genotype distribution of the IL2RA rs2104286 variant was found between patients and controls. There was no GG genotype in the patient group. The IL2RA rs2104286 AA genotype was more common in the patient group than the controls, and the AG genotype was higher in the controls compared to the patients (p = 0.001, p = 0.001, respectively). The IL2 rs2069762 and IL2 rs2069763 genotype distributions did not differ between the patient and control groups (p > 0.05). We found that the clinical global impression severity (CGI-S) score was higher in those with IL2 rs2069762 TG and GG genotypes. In this study, we showed for the first time that the genotype distribution of IL2RA rs2104286 and IL2 rs2069762 is associated with BD susceptibility and CGI-S score in a Turkish population.

Serum sCD25 is an indicator for rheumatoid arthritis-associated interstitial lung disease.

OBJECTIVES: CD25 (IL-2Rα) is one of IL-2 receptor's polypeptide subunits, and its soluble form is increased in patients with various inflammatory or autoimmune diseases. This study aimed to evaluate the clinical correlation of serum soluble CD25 (sCD25) with interstitial lung disease (ILD) in rheumatoid arthritis (RA) patients. METHODS: 294 RA patients, including 72 in the discovery cohort (15 patients with ILD, 57 patients without ILD), 222 in the validation cohort (41 patients with ILD and 181 patients without ILD), and 58 healthy controls (HCs) were recruited. High-resolution computed tomography (HRCT) scan provided evidence and patterns of RA-ILD. Serum sCD25 concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Clinical and laboratory data were recorded and the association with sCD25 was also analysed. RESULTS: In the discovery cohort, 16 RA-related molecules including cytokines, chemokines and functional soluble cell surface proteins were investigated. The results showed that sCD25 was significantly higher in RA-ILD than in RA-no-ILD group (p=0.004). ROC analysis also showed RA-ILD was discriminated with RA-no-ILD by sCD25 (AUC=0.695, 95% CI=0.541-0.849). Logistics regression demonstrated that sCD25 was one of the risk factors of RA-ILD. This result was further confirmed in validation cohort (p<0.001). According to the cut-off value in the discovery cohort, the sensitivity and specificity of sCD25 in RA-ILD were 51.2%, 77.3%, respectively. Compared with RA-no-ILD, serum level of sCD25 was also higher in different HRCT patterns including UIP, NSIP and RA-ILA. The ROC curves revealed sCD25 as diagnostic marker in UIP, NSIP and RA-ILA (with AUCs of 0.730, 0.761, and 0. 694, respectively, p<0.05). The result indicated that sCD25 was a biomarker for RA-ILD subtypes. Although sCD25 was not correlated with HRCT scores, it was significantly higher in consolidation pattern by HRCT. CONCLUSIONS: sCD25 was significantly elevated in RA-ILD (including UIP, NSIP and RA-ILA) compared to RA-no-ILD and HCs, which supports their value as a potential biomarker in RA-ILD screening and assessment.

Plasma neuron specific enolase (NSE), tumour necrosis factor-alpha (TNF-α) and soluble IL-2 receptor alpha (sIL-2Rα) levels in children with developmental delay (DD): Use of combined ROC curves to increase their diagnostic value.

BACKGROUND: Developmental delay (DD) occurs when children fail to reach developmental milestones in comparison to peers of the same age range. However, there are no valuable biomarkers for the early diagnosis of DD. Since there is no specific marker for screening the disease, we evaluated plasma NSE, TNF-α and sIL2-Rα as potential markers for this purpose. METHODS: In this cross-sectional randomized case-control study, a total of 174 DD patients and 49 matched elderly controls aged between 2 months and 60 months were recruited. A sensitive enzyme-linked immunosorbent assay and an immunoradiometric assay were used to evaluate the levels of plasma IL-1, IL-6, IL-8, IL-10, sIL2-Rα, TNF-α, and NSE. Statistical analyses using t test, χ2, ANOVA, ROC curves and binary logistic regression models were performed. RESULTS: In comparison to the control group, the DD group had greater levels of NSE, TNF-α, and sIL2-Rα(p < 0.05). In the binary logistic regression analysis of DD, NSE had an odds ratio (OR) of 1.783 (95 % CI 1.297 to 2.451, p = 0.000), indicating that NSE was an independent risk factor for DD. The plasma TNF-α level was positively correlated with plasma NSE and sIL2-Rα levels in the DD group (r = 0.366 and 0.433, respectively), and the DQ score and plasma sIL2-Rα level in the DD group were positively correlated. The ROC curve revealed that the respective areas under the NSE, TNF-α, and sIL2-Rα ROC curves were 0.9797, 0.9365, and 0.8533, respectively. Moreover, a significant increase in AUC was observed using combined ROC curve analysis. CONCLUSIONS: Children with DD have significantly altered plasma concentrations of sIL2-Rα, NSE, and TNF-α. NSE, TNF-α and sIL2-Rα can be used as DD blood biomarkers. This information may be helpful in early diagnosis and intervention.

Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4+CD25+FOXP3+ T Cells.

Staphylococcal superantigens induce massive activation of T cells and inflammation, leading to toxic shock syndrome. Paradoxically, increasing evidence indicates that superantigens can also induce immunosuppression by promoting regulatory T cell (Treg) development. In this study, we demonstrate that stimulation strength plays a critical role in superantigen-mediated induction of immunosuppressive human CD4+CD25+FOXP3+ T cells. Suboptimal stimulation by a low dose (1 ng/ml) of staphylococcal enterotoxin C1 (SEC1) led to de novo generation of Treg-like CD4+CD25+FOXP3+ T cells with strong suppressive activity. In contrast, CD4+CD25+ T cells induced by optimal stimulation with high-dose SEC1 (1 µg/ml) were not immunosuppressive, despite high FOXP3 expression. Signal transduction pathway analysis revealed differential activation of the PI3K signaling pathway and expression of PTEN in optimal and suboptimal stimulation with SEC1. Additionally, we identified that FOXP3 isoforms in Treg-like cells from the suboptimal condition were located in the nucleus, whereas FOXP3 in nonsuppressive cells from the optimal condition localized in cytoplasm. Sequencing analysis of FOXP3 isoform transcripts identified five isoforms, including a FOXP3 isoform lacking partial exon 3. Overexpression of FOXP3 isoforms confirmed that both an exon 2-lacking isoform and a partial exon 3-lacking isoform confer suppressive activity. Furthermore, blockade of PI3K in optimal stimulation conditions led to induction of suppressive Treg-like cells with nuclear translocation of FOXP3, suggesting that PI3K signaling impairs induction of Tregs in a SEC1 dose-dependent manner. Taken together, these data demonstrate that the strength of activation signals determined by superantigen dose regulates subcellular localization of FOXP3 isoforms, which confers suppressive functionality.

Effector-Phase IL-2 Signals Drive Th1 Effector and Memory Responses Dependently and Independently of TCF-1.

Following viral infection, CD4+ T cell differentiation is tightly regulated by cytokines and TCR signals. Although most activated CD4+ T cells express IL-2Rα after lymphocytic choriomeningtis virus infection, by day 3 postinfection, only half of activated T cells maintain expression. IL-2Rα at this time point distinguishes precursors for terminally differentiated Th1 cells (IL-2Rαhi) from precursors for Tfh cells and memory T cells (IL-2Rαlo) and is linked to strong TCR signals. In this study, we test whether TCR-dependent IL-2 links the TCR to CD4+ T cell differentiation. We employ a mixture of anti-IL-2 Abs to neutralize IL-2 throughout the primary CD4+ T cell response to lymphocytic choriomeningitis virus infection in mice or only after the establishment of lineage-committed effector cells (day 3 postinfection). We report that IL-2 signals drive the formation of Th1 precursor cells in the early stages of the immune response and sustain Th1 responses during its later stages (after day 3). Effector-stage IL-2 also shapes the composition and function of resulting CD4+ memory T cells. Although IL-2 has been shown previously to drive Th1 differentiation by reducing the activity of the transcriptional repressor TCF-1, we found that sustained IL-2 signals were still required to drive optimal Th1 differentiation even in the absence of TCF-1. Therefore, we concluded that IL-2 plays a central role throughout the effector phase in regulating the balance between Th1 and Tfh effector and memory cells via mechanisms that are both dependent and independent of its role in modulating TCF-1 activity.