Tresp Cells Research Articles

FRI616 Combined Elf5a And Notch Inhibition Reprograms The Effector T Cell Fate Decisions Towards Treg Phenotype Without Any Off Target Effect

Abstract Disclosure: S. Rafiqi: None. A. Aldasouqi: None. S. faisal: None. S. Qureshi: None. A. Mahmood: None. J.C. Jaume: None. S. Imam: None. We explored immune resetting and plasticity of T effector cells towards T regulatory cells (CD4+CD25+FOXP3+) by elF5A and Notch inhibition. We validated our approach for plasticizing human T effector cells into Treg cells conducted on recent onset latent autoimmune diabetes of adult (LADA) patients (n=3), and nondiabetic patients (n=4) with simultaneous use of GC7 (100 μM) and anti-DLL4 (10 μg/ml). We also investigated the functional stability of plasticized Tregs and compared it with freshly isolated naïve Tregs from the same patient. Tregs were co-cultured with T effector cells and suppression/proliferation was accessed after 5 days. To create an ex-vivo Treg deficient environment, we sorted out CD4+ cells from the PBMCs. CD4+ deficient PBMCs and sorted single positive CD4+CD25- cells were treated with anti-DLL4+GC7+rhGAD65 with and without IL-2 supplementation and were compared with conventional anti-(CD3+CD28) stimulation and absolute control (20%FCS+RPMI). The presence of Treg cells was confirmed by flow cytometry. 30.5±5.1% of CD4 cells were plasticized into Tregs in the treatment group supplemented with IL-2 while only 18.8±4.6 % were plasticized in treatment group without IL-2 supplementation. Also, 11.2±3.1% plasticized to Tregs in treatment group in presence of CD3/CD28 microbeads compared to 6.19±2.4% in absolute control group. Similarly, 13±4.3% of CD8 cells had plasticized into Tregs in treatment group supplemented with IL-2 while only 11.5±6.7% plasticized in treatment group without IL-2 supplementation. 14.3±5.8% plasticized to Tregs in treatment group in presence of CD3/CD28 microbeads compared to 12.4±6.3% in absolute control group. Interestingly, the plasticized Tregs from LADA patients were more robust than those of non-diabetic patients at a 1:2 ratio (Treg:Tresp). Furthermore, the plasticized Tregs had comparable suppressive capacity over Tresp (CD4+CD25-) cells as that of freshly isolated naïve human Tregs. To investigate any off-target effects of (GC7 and anti-DLL4), live, dead, and apoptotic cell count at 24, 48, 96 hours, and 7 days post-treatment using dead cell apoptosis kit (Invitrogen). In our study, there was no significant difference between treatment and control groups in terms of live, dead, and apoptotic cells at different time intervals. On the 7th day of culture 64.06±8.66% and 68.88±6.15% of cells were live in treatment and absolute control groups respectively while 7.24±1.59% and 10.0±2.02% cells were found apoptotic respectively. Therefore, we define the reprogramming of T effector cells towards Treg cells with functional suppressive capacity using GC7 and anti-DLL4 without any off-target effect (toxicity). The present study expands the concept of T cell plasticity and establishes the functional stability of plasticized T cells that can accelerate pre-clinical trials for therapeutic use in T1D and as an adjunct to immunotherapies. Presentation: Friday, June 16, 2023

A dendritic cell receptor-targeted chimeric immunotherapeutic protein (C-HBV) for the treatment of chronic hepatitis B

ABSTRACT In chronic Hepatitis B Virus (HBV) infections HBV-specific T cells are functionally impaired. Immunotherapy may restore HBV-specific T cell responses essential for sustained disease remission off-treatment and induction of a functional cure. Chimigen® Molecules are fusion proteins of antigen(s) with the Fc fragment of a xenotypic antibody designed to target specific receptors on dendritic cells (DCs). Here we describe the production and pre-clinical evaluation of Chimigen® HBV (C-HBV), containing HBV PreS1 and PreS2 peptide fragments, HBV core and murine Fc, produced in insect cells. C-HBV binding to immature DCs and internalization by endocytosis was FcγRII (CD32) and mannose receptor (CD206) dependent and led to increased MHC I and MHC II surface expression. Upon exposure of human T cells isolated from HBV un-infected healthy and chronically HBV-infected donors to C-HBV-pulsed mature DCs ex vivo, C-HBV induced vigorous T cell proliferation and enhanced expression of IFN-γ, TNF-α, perforin and granzyme B in both CD4+ and CD8+ T cell subsets. Re-stimulation of C-HBV-activated T cells from chronically infected donors with HBV PreS1/PreS2 and core overlapping peptides induced IFN-γ production in both CD4+ and CD8+ populations. C-HBV-activation of peripheral blood mononuclear cells (PBMCs) from chronically HBV-infected patients stimulated granzyme B production by CD4+CD25− T responder (Tresp) cells, accompanied by an increase in Annexin V staining on CD4+CD25+ T regulatory (Treg) cell phenotype, consistent with apoptosis. The observed HBV-specific cellular responses induced by C-HBV ex vivo suggest that C-HBV is a promising immunotherapeutic candidate for the treatment of chronic HBV infections.

Immunosuppressive therapy influences the accelerated age-dependent T-helper cell differentiation in systemic lupus erythematosus remission patients

BackgroundCD4+ T cells are of great importance in the pathogenesis of systemic lupus erythematosus (SLE), as an imbalance between CD4+ regulatory T cells (Tregs) and CD4+ responder T cells (Tresps) causes flares of active disease in SLE patients. In this study, we aimed to find the role of aberrant Treg/Tresp cell differentiation for maintaining Treg/Tresp cell balance and Treg functionality.MethodsTo determine differences in the differentiation of Tregs/Tresps we calculated the percentages of CD45RA+CD31+ recent thymic emigrant (RTE) Tregs/Tresps and CD45RA+CD31− mature naive (MN) Tregs/Tresps, as well as CD45RA−CD31+ and CD45RA−CD31− memory Tregs/Tresps (CD31+ and CD31− memory Tregs/Tresps) within the total Treg/Tresp pool of 78 SLE remission patients compared with 94 healthy controls of different ages. The proliferation capacity of each Treg/Tresp subset was determined by staining the cells with anti-Ki67 monoclonal antibodies. Differences in the autologous or allogeneic Treg function between SLE remission patients and healthy controls were determined using suppression assays.ResultsWith age, we found an increased differentiation of RTE Tregs via CD31+ memory Tregs and of RTE Tresps via MN Tresps into CD31− memory Tregs/Tresp in healthy volunteers. This opposite differentiation of RTE Tregs and Tresps was associated with an age-dependent increase in the suppressive activity of both naive and memory Tregs. SLE patients showed similar age-dependent Treg cell differentiation. However, in these patients RTE Tresps differentiated increasingly via CD31+ memory Tresps, whereby CD31− memory Tresps arose that were much more difficult to inhibit for Tregs than those that emerged through differentiation via MN Tresps. Consequently, the increase in the suppressive activity of Tregs with age could not be maintained in SLE patients. Testing the Tregs of healthy volunteers and SLE patients with autologous and nonautologous Tresps revealed that the significantly decreased Treg function in SLE patients was not exclusively attributed to an age-dependent diminished sensitivity of the Tresps for Treg suppression. The immunosuppressive therapy reduced the accelerated age-dependent Tresp cell proliferation to normal levels, but simultaneously inhibited Treg cell proliferation below normal levels.ConclusionsOur data reveal that the currently used immunosuppressive therapy has a favorable effect on the differentiation and proliferation of Tresps but has a rather unfavorable effect on the proliferation of Tregs. Newer substances with more specific effects on the immune system would be desirable.

Decreased regulatory function of CD4+CD25+CD45RA+ T cells and impaired IL-2 signalling pathway in patients with type 2 diabetes mellitus.

In this study, the frequency and function of CD4+CD25+CD45RA+ regulatory T cells (Treg) and intracellular IL-2 signalling molecules in patients with type 2 diabetes mellitus (T2DM) were investigated. Tregs and responder T cells (Tresp, CD4+CD25- T cells) were sorted and suppression assays were performed using flow cytometry. Phosphorylation of signal transducer and activator of transcription-5 (pSTAT5) were assessed using flow cytometry. Gene expression of FOXP3 was performed with the SYBR green Real Time PCR method. Production of IL-2 from cultured cells was assessed using ELISA. We observed a functional defect of CD4+CD25+CD45RA+ Tregs in T2DM patients with higher proliferation of Tresp cells, in response to anti-CD3 and anti CD28 stimulation in the presence of Tregs invitro. The results showed that the proliferation of Tresps in the absence of Treg cells was higher in T2DM patients than in healthy controls. Decreased FOXP3 mRNA expression and pSTAT5 were observed within the Tregs of the patients, whereas the level of secreted IL-2 from PBMCs culture was not statically different between T2DM patients and healthy individuals. Changes in intracellular IL-2 pathways and FOXP3 gene expression may contribute to the defect of Tregs in T2DM patients. These findings indicating that the purified CD4+CD25+CD45RA+ Treg cells have reduced functional capacity together with impaired IL-2 pathway in T2DM, and the Tregs could be used for a potential novel therapeutic target.

End‐stage renal disease, dialysis, kidney transplantation and their impact on CD4+ T‐cell differentiation

Premature aging of both CD4+ regulatory T (Treg) and CD4+ responder-T (Tresp) cells in patients with end-stage renal disease (ESRD) is expected to affect the success of later kidney transplantation. Both T-cell populations are released from the thymus as inducible T-cell co-stimulator-positive (ICOS+ ) and ICOS- recent thymic emigrant (RTE) Treg/Tresp cells, which differ primarily in their proliferative capacities. In this study, we analysed the effect of ESRD and subsequent renal replacement therapies on the differentiation of ICOS+ and ICOS- RTE Treg/Tresp cells into ICOS+ CD31- or ICOS- CD31- memory Treg/Tresp cells and examined whether diverging pathways affected the suppressive activity of ICOS+ and ICOS- Treg cells in co-culture with autologous Tresp cells. Compared with healthy controls, we found an increased differentiation of ICOS+ RTE Treg/Tresp cells and ICOS- RTE Treg cells through CD31+ memory Treg/Tresp cells into CD31- memory Treg/Tresp cells in ESRD and dialysis patients. In contrast, ICOS- RTE Tresp cells showed an increased differentiation via ICOS- mature naive (MN) Tresp cells into CD31- memory Tresp cells. Thereby, the ratio of ICOS+ Treg/ICOS+ Tresp cells was not changed, whereas that of ICOS- Treg/ICOS- Tresp cells was significantly increased. This differentiation preserved the suppressive activity of both Treg populations in ESRD and partly in dialysis patients. After transplantation, the increased differentiation of ICOS+ and ICOS- RTE Tresp cells proceeded, whereas that of ICOS+ RTE Treg cells ceased and that of ICOS- RTE Treg cells switched to an increased differentiation via ICOS- MN Treg cells. Consequently, the ratios of ICOS+ Treg/ICOS+ Tresp cells and of ICOS- Treg/ICOS- Tresp cells decreased significantly, reducing the suppressive activity of Treg cells markedly. Our data reveal that an increased tolerance-inducing differentiation of ICOS+ and ICOS- Treg cells preserves the functional activity of Treg cells in ESRD patients, but this cannot be maintained during long-term renal replacement therapy.

The number and function of T regulatory cells in obese atopic female asthmatics

ABSTRACTBackground: Mechanisms underlying the association between asthma and obesity remain poorly understood. Obesity appears to be a risk factor for asthma, and obese asthmatics fare poorly compared to lean asthmatics. Objectives: To explore the possibility that reduced regulatory T cell (Treg) number and function contribute to the obesity-asthma association. We concentrated on obese females with childhood-onset asthma, since Treg may be involved in this phenotype. Methods: We recruited 64 women (ages 18–50) into four groups: lean (BMI 18–25 kg/m2) controls (n = 17) and asthmatics (n = 13), and obese (BMI ≥ 35 kg/m2) controls (n = 17) and asthmatics (n = 17). Asthmatics had atopy and childhood-diagnosed asthma. We assessed lung function, asthma control and quality of life. Peripheral blood CD4+/CD25+/FoxP3+ Treg cells were identified and counted by flow cytometry and expressed as % total CD4+ T cells. We assessed Treg cell function by the ability of CD4+/CD25+ Treg cells to suppress autologous CD4+/CD25- responder T cell (Tresp) proliferation and measured as % suppression of Tresp cell proliferation. Results: Obese asthmatics had worse lung function, asthma control, and quality of life compared to lean asthmatics. Compared to lean or obese control groups, the number of Treg cells in the obese asthmatics was approximately 1.58- or 1.73-fold higher. The ability of Treg cells from obese-asthmatics to suppress Tresp cell proliferation was reduced. Conclusions: Obese, atopic women with childhood diagnosed asthma demonstrate increased Treg cell number and mildly decreased Treg cell function. Our data do not support the view that reduced Treg cell number contributes to this obese-asthma phenotype.

The effect of interleukin (IL)-21 and CD4+ CD25++ T cells on cytokine production of CD4+ responder T cells in patients with myasthenia gravis.

Impairment of the suppressive function of regulatory T (Treg ) cells has been reported in myasthenia gravis (MG). In this study, cytokine-related mechanisms that may lead to the defect of Treg were investigated in patients with anti-acetylcholine receptor antibody-positive MG (AChR + MG). Proliferation and cytokine production of responder T (Tresp ) cells in response to polyclonal activation were measured in a suppression assay. The effect of interleukin (IL)-21 on suppression was evaluated in vitro in co-culture. IL-21 increased the proliferation of Tresp cells in Tresp /Treg co-cultures. Tresp cells from patients with MG secreted significantly lower levels of IL-2. In patients with MG, IL-2 levels did not change with the addition of Treg to cultures, whereas it decreased significantly in controls. In Tresp /Treg co-cultures, IL-4, IL-6 and IL-10 production increased in the presence of Treg in patients. Interferon (IFN)-γ was decreased, whereas IL-17A was increased in both patient and control groups. IL-21 inhibited the secretion of IL-4 in MG and healthy controls (HC), and IL-17A in HC only. The results demonstrated that IL-21 enhances the proliferation of Tresp cells in the presence of Treg . An effect of IL-21 mainly on Tresp cells through IL-2 is implicated.

The characteristic profiles of PD-1 and PD-L1 expressions and dynamic changes during treatment in active tuberculosis

PD-1 is a cell surface receptor of activated T and B lymphocytes and it's role in tuberculosis is controversial because of lack of congruence between clinical study and animal model. To investigate the immunological pathogenesis mechanisms of tuberculosis and to develop the immune therapy target essential for controlling tuberculosis, here we explored the expression characteristics and dynamic changes of PD-1/PD-L1 pathway in different CD4+T cell subsets. We enrolled 24 human subjects including 15 active tuberculosis (ATB) patients and 9 healthy donors (HD). The expressions of PD-1 and PD-L1 on CD4+T cells increased significantly in ATB patients than HD. ATB patients had a higher proportion of regulatory T cells (Treg, CD4+CD25 + Foxp3+) than HD. The expressions of PD-1 and PD-L1 increased remarkably on CD4+T cell subsets, including Treg cells, Tresp (CD4+CD25−) cells and Teff (CD4+CD25 + Foxp3-) cells. Finally, clinical improvement following effective anti-TB therapy is correlated with significantly decreased expression of PD-1 in Tresp and Teff cells, but not in Treg cells. Thus, expression profiles of PD-1 in T cell subpopulations may be used as a candidate to predict the clinical efficacy of anti-tuberculosis therapy. Modulation of PD-1/PD-L1 pathway in CD4 subsets may offer an immunotherapy target for the control of tuberculosis.

Dysfunction of regulatory T cells in patients with psoriasis and related mechanisms of regulation by the STAT3 signaling pathway

Objective To evaluate the function of regulatory T (Treg) cells in peripheral blood from patients with psoriasis, and to explore the possible role of the STAT3 signaling pathway in Treg cell dysfunction. Methods Totally, 81 patients with psoriasis vulgaris, who all presented with chronic plaques and had a psoriasis area and severity index (PASI) score of 10-30, were enrolled into this study. Forty-six healthy blood donors served as the control group. Venous blood samples were collected from these subjects followed by isolation of Treg cells and responder T (Tresp) cells. Flow cytometry was performed to determine the proportion of Treg cells in peripheral blood as well as that of cells secreting phosphorylated-STAT3 (p-STAT3), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and interleukin 17 (IL-17) in Treg cells, and quantitative real-time PCR (qRT-PCR) to measure the expression levels of IFN-γ, TNF-α and IL-17 mRNAs in Treg cells. Some Treg cells and Tresp cells were cultured in vitro alone or in combination, and flow cytometry was conducted to estimate cellular proliferative activity on day 7 after stimulation with IL-2. Some patient-derived Treg cells were classified into several groups to be cultured alone or in combination with Tresp cells with or without the presence of the STAT3 pathway inhibitor, Stattic V (10 or 50 μg/L), for 7 days. Subsequently, flow cytometry was performed to evaluate the proliferative activity of Tresp cells, and qRT-PCR to measure the expression levels of IFN-γ, TNF-α and IL-17 mRNAs in Treg cells. Results No significant differences were observed in the proportion of Treg cells in peripheral blood between the patient group and control group (6.437% ± 0.186% vs. 6.812% ± 0.241%, t = 1.224, P> 0.05). Compared with control-derived Treg cells, the patient-derived Treg cells showed significantly decreased proliferative activity and inhibitory effects on Tresp cells, but increased proportion of cells secreting p-STAT3, IFN-γ, TNF-α and IL-17 (all P< 0.05). After the treatment with 50 μg/L Stattic V, a significant increase was observed in the inhibitory effect of patient-derived Treg cells on Tresp cells (inhibition rate: 61.670% ± 4.640% vs. 28.820% ± 11.490%, P< 0.05), but a significant decrease in the mRNA expressions of IFN-γ (2-△△Ct: 1.654 ± 0.879 vs. 23.350 ± 6.721, P< 0.05), TNF-α (0.850 ± 0.705 vs. 4.847 ± 1.525, P< 0.05) and IL-17 (0.572 ± 0.135 vs. 3.095 ± 0.650, all P< 0.05) in patient-derived Treg cells compared with untreated patient-derived Treg cells. Conclusions The negative regulatory effect of Treg cells on Tresp cells is decreased in patients with psoriasis, which may be associated with abnormal activation of the STAT3 signaling pathway, and inhibition of the pathway may restore the function of Treg cells to a certain extent. Key words: Psoriasis; T-lymphocytes, regulatory; STAT3 transcription factor; Interferon-gamma; Tumor necrosis factor-alpha; Interleukin-17; Stattic V

Aberrant GITR expression on different T cell subsets and the regulation by glucocorticoid in systemic lupus erythematosus.

The dysfunction of T regulatory cells is important for the pathogenesis of systemic lupus erythematosus (SLE). Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is expressed at low levels on resting responder T lymphocytes (Tresps) and is up-regulated on T regulatory cells (Tregs) and activated T cells, diminishing suppressive activity of Tregs and/or leading to resistance to suppression of Tregs by activated effector T cells. We aimed to explore whether SLE patients had an aberrant expression of GITR on Tregs and responder T cells (Tresps) and the regulation by glucocorticoids. The surface GITR expression on Tregs and Tresps cells were analyzed by flow cytometry in 32 patients and 15 normal controls. Purified Tregs or Tresps were cultured with glucocorticoid. Apoptosis of the cells were determined by the staining of Annexin V. Systemic lupus erythematosus patients had higher levels of GITR expressed on CD4(+) CD25(+) , CD4(+) CD25(high) and CD4(+) CD25(+) CD127(low/-) Tregs as well as on CD4(+) CD25(-) Tresps compared to healthy controls. The expression of GITR on Tregs and Tresps were positively correlated with score of SLE disease activity index (SLEDAI). In vitro glucocorticoid induced GITR expression on purified Tresp cells, but not on Tregs, and almost all of the GITR positive cells induced by glucocorticoid encountered apoptosis. Aberrant expression of GITR may contribute to SLE pathogenesis. Glucocorticoid may achieve its therapeutic effect partly by inducing GITR expression on Tresps rather than Tregs, which initiates the apoptosis of Tresp cells in SLE patients.

Critical stoichiometric ratio of CD4+ CD25+ FoxP3+ regulatory T cells and CD4+ CD25− responder T cells influence immunosuppression in patients with B‐cell acute lymphoblastic leukaemia

Regulatory T (Treg) cells act to suppress activation of the immune system and thereby maintain immunological homeostasis and tolerance to self-antigens. The frequency and suppressing activity of Treg cells in general are high in different malignancies. We wanted to identify the role and regulation of CD4(+) CD25(+) FoxP3(+) Treg cells in B-cell acute lymphoblastic leukaemia (B-ALL). We have included patients at diagnosis (n=54), patients in clinical remission (n=32) and normal healthy individuals (n=35). These diagnosed patients demonstrated a lower number of CD4(+) CD25(+) cells co-expressing a higher level of FoxP3, interleukin-10, transforming growth factor-β and CD152/CTLA-4 than the normal population. Treg cells from patients showed a higher suppressive capability on CD4(+) CD25(-) responder T (Tresp) cells than normal. The frequency and immunosuppressive potential of CD4(+) CD25(+) FoxP3(+) Treg cells became high with the progression of malignancy in B-ALL. Relative distribution of Tresp and Treg cells was only ~5:1 in B-ALL but ~35:1 in normal healthy individuals, further confirming the elevated immunosuppression in patients. A co-culture study at these definite ex vivo ratios, indicated that Treg cells from B-ALL patients exhibited higher immunosuppression than Treg cells from normal healthy individuals. After chemotherapy using the MCP841 protocol, the frequency of CD4(+) CD25(+) cells was gradually enhanced with the reduction of FoxP3, interleukin-10 positivity corresponded with disease presentation, indicating reduced immunosuppression. Taken together, our study indicated that the CD4(+) CD25(+) FoxP3(+) Treg cells played an important role in immunosuppression, resulting in a positive disease-correlation in these patients. To the best of our knowledge, this is the first detailed report on the frequency, regulation and functionality of Treg cells in B-ALL.

High frequency but impaired function of Tregs in active CD patients

Objectives: Coeliac disease (CD) is an immune-mediated systemic disorder elicited by gluten and characterized by a small intestinal enteropathy. Besides active CD, we recognize an early stage of CD characterized by a normal small intestinal mucosa and positive CD serology. The aim of the study was to investigate the immunoregulatory mechanisms acting in the gut of active and potential CD patients. We evaluated the expression of IFN-γ and IL-10 in CD4+ T cells and the frequency of Foxp3+ regulatory T cells (Tregs) in duodenal biopsies. Finally, we investigated the functional properties of intestinal Foxp3+ Tregs from both groups of patients and the effects of IL-15 on their suppressive activity. Methods: CD4+ T cells were isolated from duodenal biopsies of 11 controls, 13 active CD and 15 potential CD patients (7 Marsh 0 (M0) and 8 Marsh 1 (M1)). IFN-γ and IL-10 expression were assessed by flow cytometry. The frequency of CD4+ CD25+ Foxp3+ Tregs frequency and function were investigated. Intestinal Tregs suppressive function was assessed on autologous peripheral blood responder CD4+ CD25− T cells (Tresp), in the presence of a polyclonal stimulus and with or without IL-15. Results: CD4+ IFN-γ+ T cell frequency was higher in duodenal biopsies of active (mean ± sd: 12.9 ± 3.2) and M1 potential CD (17.8 ± 5.9) than controls (4.9 ± 0.7; p < 0.05 and p < 0.05 respectively). On the contrary the highest number of CD4+ IL10+ T cells was observed in M0 potential CD patients (17 ± 5.8) compared to active (6.7 ± 1.7, p < 0.05) and control patients (6.1 ± 0.6; p < 0.05). The percentage of CD4+ CD25+ Foxp3+ T cells was significantly increased in active (14.1 ± 2.7%) and potential CD (9.9 ± 3.2%) if compared to controls (6.5 ± 3.1%; p < 0.01 and p < 0.05 respectively). In particular, in potential M1 CD it was significantly higher (10.9 ± 3.1%; p < 0.05). Intestinal Tregs from both active and potential CD exerted significant suppressive effects on Tresp cells (p < 0.05) in in vitro suppressive assay. Interestingly IL-15 was able to counteract their suppressive activity in active CD but not in potential CD. Conclusions: IFN-γ expression in CD4+ T cells correlates with CD stage, as it is increased in M1 potential and active patients; on the contrary in early CD (M0) a high frequency of IL-10 producing CD4+ T cells was found. This might prevent the progression towards mucosal damage down-regulating IFN-γ production and promoting Foxp3+ Tregs. These cells are significantly increased in the small intestinal mucosa of CD patients and are functionally active; however their suppressive activity is impaired by IL-15 only in active CD patients, suggesting a relation between high IL-15 expression, an impaired Tregs function and villous atrophy in active CD.

Impaired regulatory function in circulating CD4+CD25highCD127low/− T cells in patients with myasthenia gravis

Previous studies have reported alterations in numbers or function of regulatory T (Treg) cells in myasthenia gravis (MG) patients, but published results have been inconsistent, likely due to the isolation of heterogenous "Treg" populations. In this study, we used surface CD4, CD25(high), and CD127(low/-) expression to isolate a relatively pure population of Tregs, and established that there was no alteration in the relative numbers of Tregs within the peripheral T cell pool in MG patients. In vitro proliferation assays, however, demonstrated that Treg-mediated suppression of responder T (Tresp) cells was impaired in MG patients and was associated with a reduced expression of FOXP3 in isolated Tregs. Suppression of both polyclonal and AChR-activated Tresp cells from MG patients could be restored using Tregs isolated from healthy controls, indicating that the defect in immune regulation in MG is primarily localized to isolated Treg cells, and revealing a potential novel therapeutic target.

Dysfunctional Regulatory T Cells in Patients with Autoimmune Myasthenia Gravis: Therapeutic Targets? (PD6.006)

Objective: To investigate circulating regulatory T cell (Treg) numbers and suppressive function in patients with autoimmune myasthenia gravis (MG). Background Published studies examining the role of Tregs in the pathogenesis of MG have reported conflicting results. In these investigations, isolation of Tregs was based on expression of CD25 alone, which is also expressed on a subset of recently activated and memory T cells. More recently, low expression of the IL-7Rα chain (CD127) combined with high expression of CD25 has allowed for isolation of pure Treg populations with high suppressive functionality. Design/Methods: The expression of surface CD4, CD25, and CD127, and intracellular Foxp3 was analyzed by flow cytometry in 16 MG patients vs. 14 normal control subjects. The function of Tregs (CD4+CD25highCD127low/- cells) was examined by co-culture experiments with CD25-negative autologous responder T cells (Tresp), and their suppressive capacity was determined in T cell proliferation assays. Expression of intracellular Foxp3 was assessed by flow cytometry, and cytokine profiles from the T cell culture supernatants were analyzed. GM-CSF was added to T cell co-cultures and the effects on Tregs investigated. Results: No alteration in the relative numbers of Tregs within the peripheral CD4+ T cell pool was seen in MG patients. A reduced expression of Foxp3 within Tregs, and a defect in the ability of these cells to suppress Tresp cells was demonstrated for both polyclonal and AChR-induced T cell proliferation. Cytokine profiles revealed altered levels of IL-6, IL-17, IFN-γ, and IL-10 in MG patients. The demonstrated defects in Treg function/phenotype were partially reversed by in vitro exposure to GM-CSF. Conclusions: The suppressive function of Tregs is impaired in MG patients, likely playing a role in the immunopathogenesis of the disease. Enhancement of Treg function in vitro is feasible, and may represent a novel therapeutic strategy in MG. Supported by: The NIH (National Institute of Neurologic Disorders and Stroke, K08NS058800, and the Muscular Dystrophy Association (MDA) MNM and JRS; and National Institute of Allergy and Infectious Diseases, RO1 AI 058190, BSP. This project was also supported by the University of Illinois at Chicago (UIC) Center for Clinical and Translational Science (CCTS), Award Number UL1RR029879 from the National Center For Research Resources. Disclosure: Dr. Meriggioli has received personal compensation for activities with Athena Diagnostics, Talecris Biotherapeutics, Gliknik, and Celegene Corporation. Dr. Meriggioli receives patent payments for a patent on bispecific antibody coated dendritic cells. Dr. Meriggioli has received research support from the NIH/NINDS and the Muscular Dystrophy Association. Dr. Muthusamy has nothing to disclose. Dr. Rowin has nothing to disclose. Dr. Sheng has nothing to disclose. Dr. Prabhakar has received personal compensation for activities with Gliknik Inc. as a consultant.Dr. Prabhakar has received research support from NIH.

Isolation strategies of regulatory T cells for clinical trials: Phenotype, function, stability, and expansion capacity

Recent clinical results demonstrate the highly effective potency of regulatory T cells (Tregs) to control graft-versus-host disease (GvHD). In this presented study, we directly compared different Treg subpopulations in order to define the most promising Treg target cell population for cellular intervention studies with respect to their phenotype, functional properties, stability, and expansion capacity. Different Treg cell populations have been isolated from healthy donors and characterized by fluorescence activated cell sorting (FACS) analysis for their phenotypic marker and purity, functional properties by suppression assay, stability by Treg-specific demethylated region (TSDR) of the Foxp3 promoter and their invitro expansion capacity. The direct comparison of the respective Treg target cell populations identified CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs as the most promising Treg population for fresh cell infusions in clinical trials with respect to cell yield, phenotype, function, and stability. The CD4(+)CD25(hi) Tregs qualified as the best candidate for invitro expansion combining a highly stable phenotype with strong suppressive potential and attractive cell yield after repetitive stimulation. The suppressive capacity of freshly isolated CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs is comparable to freshly isolated CD4(+)CD25(hi), but inferior to CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs. Invitro expansion of CD4(+)CD25(hi)CD45RA(+) and CD49d(-)CD127(-) Tregs resulted in cell populations with less suppressive potency compared with CD4(+)CD25(hi) expanded Tregs correlating well with a higher TSDR demethylation level. In conclusion, future clinical trials should favor CD4(+)CD25(hi)CD127(-) and CD4(+)CD25(hi)ICOS(+) Tregs for direct Treg cell transfer, whereas CD4(+)CD25(hi) Tregs qualify as best candidate for invitro expansion.

Transfer of cell membrane components via trogocytosis occurs in CD4+Foxp3+CD25+ regulatory T-cell contact-dependent suppression

A key component of the immune system is its ability to establish and maintain peripheral tolerance. Naturally occurring CD4+CD25+Foxp3+ regulatory T (nTreg) cells represent an important means by which this is accomplished, through their potent ability to suppress the actions of both CD4+ and CD8+ effector (Teff) cells in vitro and in vivo. We hypothesized that direct contact between nTreg and Teff cells is sufficient for nTreg cell-contact suppression. We first show that nTreg cell suppression is independent of APCs and their derived co-stimulatory signals. We then used a two-colour, lipid dye labelling and quantification approach to formally demonstrate that nTreg cells specifically form cell conjugates with responding T (Tresp) cells only under TCR activating conditions. Strikingly, activated CD4+ nTreg cells undergo progressive trogocytosis, a process by which membrane fragments are transferred from one cell subset to another, with Tresp cells more readily than Teff cells. These results are the first to show that nTreg cell cognate interactions with Tresp cells leads to trogocytosis between the cells, and the first to relate the degree of trogocytosis with the level of nTreg-mediated suppression.

CD4+CD25+Foxp3+ Regulatory T Cells Promote Th17 Cells In Vitro and Enhance Host Resistance in Mouse Candida albicans Th17 Cell Infection Model

Th17 cells and CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells are thought to promote and suppress inflammatory responses, respectively. Here we explore why under Th17 cell polarizing conditions, Treg cells did not suppress, but rather upregulated, the expression of interleukin-17A (IL-17A), IL-17F, and IL-22 from responding CD4(+) Tcells (Tresp cells). Upregulation of IL-17 cytokines in Tresp cells was dependent onconsumption of IL-2 by Treg cells, especially at early time points both invitro and invivo. During an oral Candida albicans infection in mice, Treg cells induced IL-17 cytokines in Tresp cells, which markedly enhanced fungal clearance and recovery from infection. These findings show how Treg cells can promote acute Th17 cell responses to suppress mucosal fungus infections and reveal that Treg cellshave a powerful capability to fight infections besides their role in maintaining tolerance or immune homeostasis.

T-cell specific defect in expression of the NTPDase CD39 as a biomarker for lupus

Regulatory T cells (Tregs) are critical for maintenance of peripheral tolerance via suppression of T-cell responses, and absence of Tregs results in autoimmunity. The role of aberrations in the Treg pool for the development of systemic lupus erythematosus (SLE, lupus) remains uncertain. Treg-mediated generation of adenosine, dependent on the ectonucleotidase CD39, is an important mechanism for suppression of T-cell responses. We tested whether decreases in numbers of Tregs, and specifically CD39-expressing Tregs, are associated with human lupus. We studied 15 SLE patients, six patients with rheumatoid arthritis (RA) and 24 healthy controls. Treg phenotypic markers, including CD39 expression, were studied by flow cytometry. Varying numbers of sorted Tregs cells were co-cultured with responder T (Tresp) cells, with proliferation assessed by 3H-thymidine incorporation. The proportion of Tregs as defined by Foxp3+ CD25+high CD127−/low was similar in lupus and control populations. CD39-expressing Tregs comprised 37±13% of the Treg population in healthy controls and 36±21% in lupus subjects using nonsteroidal immunosuppressants to control active disease, but was nearly absent in five of six lupus subjects with minimally active disease. In contrast to healthy controls and lupus subjects without the CD39 defect, in SLE subjects with the CD39 defect, adenosine-dependent Treg-mediated suppression was nearly absent. These results suggest that functional defects in Tregs, rather than reduced Treg numbers, are important for the loss of peripheral tolerance in lupus. Presentation of this defect may serve as a biomarker for untreated disease.

Low responder T cell susceptibility to the suppressive function of regulatory T cells in patients with dilated cardiomyopathy

ObjectiveThe pathogenesis of dilated cardiomyopathy (DCM) is closely connected with dysfunction of the autoimmune system, and CD4+CD25highCD127low/− regulatory T (Treg) cells have a vital role in maintaining self-tolerance. In this...

A study on the function of CD4+CD25+ regulatory T cells in peripheral blood of patients with psoriasis vulgaris.

Objective To analyse the function of CD4~+CD25~+ regulatory T cells in patients with psoriasis vulgaris(PV), and try to explore their role in the pathogenesis of PV.Methods Twelve patients with chronic plaque PV, who had a psoriasis area and severity score(PASI)of 15 to 34.5, were included in this study together with 6 healthy human controls.By using immunomagnetic beads, CD4~+CD25~+ regulatory T (Treg)cells and CD4~+CD25~- responder T(Tresp)cells were isolated from peripheral blood of these subjects.Then, these Treg and Tresp cells were subjected to monoculture or coculture under the stimulation with CD3 and CD28.IL-2 was also used to stimulate the proliferation of the Treg cells.[~3H]-thymidine incorporation assay was performed to analyze the proliferation of cells, enzyme linked immunosorbent assay(ELISA)to detect the levels of interferon-gamma(IFN-γ), interleukin-10(IL-10)and transforming growth factor-beta(TGF-β)in the supernatant of monoculture or cocuhure system, and RT-PCR to measure the expression of Foxp3 mRNA.Results After stimulation with CD3 and CD28, neither normal nor psoriatic Treg cells showed obvious proliferation, while this condition was reversed by the addition of IL-2.Cocultured with Tresp cells at a ratio of 1:1,Treg cells inhibited the proliferation of Tresp cells by 60% in the control group and 19.5% in the psoriasis group(P ＜ 0.01).The level of supernatant IFN-γ was 179.66 ± 48.97 ng/L and 109.55± 48.0 ng/L in the monoculture and coculture system of psoriatic Tresp cells, respectively, significantly different from that of control Tresp cells(87.36 ± 33.36 ng/L, 32.55 ± 15.69 ng/L, both P＜ 0.05); the secretion of IFN-γ by Tresp cells was suppressed by 40% in psoriatic group and 63% in control group, by Treg cells(P ＜ 0.05).Treg cells secreted more TGF-β than Tresp cells in the normal control group(3025 ± 769 ng/L vs 2450 ± 431 ng/L, P ＜0.05).Between the patient and control group, there was no statistical difference in the secretion of IL-10 or TGF-β by Treg cells or Tresp cells in monoculture or coculture system(all P ＞ 0.05).In the case of mRNA expression of Foxp3, Treg cells was significantly higher than Tresp cells, whereas no significant difference was observed between psoriatic and control Treg or Tresp cells.Conclusions The impairment of inhibitory function of CD4~+CD25~+ Treg cells leads to an incontrollable proliferation of CD4~+CD25~- Tresp cells in psoriatic patients, which may take part in the pathogenesis of psoriasis. Key words: Psoriasis; CD4~+CD25~+Treg; Cd4~+CD25~-Tresp; Foxp3