At-Risk and Recent-Onset Type 1 Diabetic Subjects Have Increased Apoptosis in the CD4+CD25+(high) T-Cell Fraction

BACKGROUND: In experimental models, Type 1 diabetes T1D can be prevented by adoptive transfer of CD4+CD25+ FoxP3+ suppressor or regulatory T cells. Recent studies have found a suppression defect of CD4+CD25+(high) T cells in human disease. In this study we measure apoptosis of CD4+CD25+(high) T cells to see if it could contribute to reduced suppressive activity of these cells. METHODS AND FINDINGS: T-cell apoptosis was evaluated in children and adolescent 35 females/40 males subjects comprising recent-onset and long-standing T1D subjects and their first-degree relatives, who are at variable risk to develop T1D. YOPRO1/7AAD and intracellular staining of the active form of caspase 3 were used to evaluate apoptosis. Isolated CD4+CD25+(high) and CD4+CD25− T cells were co-cultured in a suppression assay to assess the function of the former cells. We found that recent-onset T1D subjects show increased apoptosis of CD4+CD25+(high) T cells when compared to both control and long-standing T1D subjects p<0.0001 for both groups. Subjects at high risk for developing T1D 2–3Ab+ve show a similar trend p<0.02 and p<0.01, respectively. On the contrary, in long-standing T1D and T2D subjects, CD4+CD25+(high) T cell apoptosis is at the same level as in control subjects p = NS. Simultaneous intracellular staining of the active form of caspase 3 and FoxP3 confirmed recent-onset FoxP3+ve CD4+CD25+(high) T cells committed to apoptosis at a higher percentage 15.3±2.2 compared to FoxP3+ve CD4+CD25+(high) T cells in control subjects 6.1±1.7 p<0.002. Compared to control subjects, both recent-onset T1D and high at-risk subjects had significantly decreased function of CD4+CD25+(high) T cells p = 0.0007 and p = 0.007, respectively. CONCLUSIONS: There is a higher level of ongoing apoptosis in CD4+CD25+(high) T cells in recent-onset T1D subjects and in subjects at high risk for the disease. This high level of CD4+CD25+(high) T-cell apoptosis could be a contributing factor to markedly decreased suppressive potential of these cells in recent-onset T1D subjects

T Regulatory Cells in Cord Blood—FOXP3 Demethylation as Reliable Quantitative Marker

Regulatory T-cells (Tregs), characterized as CD4+CD25(hi) T-cells expressing FOXP3, play a crucial role in controlling healthy immune development during early immune maturation. Recently, FOXP3 demethylation was suggested to be a novel marker for natural Tregs in adults. In cord blood, the role and function of Tregs and its demethylation is poorly understood. We assessed FOXP3 demethylation in cord blood in relation to previously used Treg markers such as CD4+CD25(hi), FOXP3 mRNA, protein expression, and suppressive Treg function

Identification, frequency, activation and function of CD4+ CD25highFoxP3+ regulatory T cells in children with juvenile idiopathic arthritis

The aim of the study was to test the frequency of CD4+ CD25highFoxP3 regulatory T cells in JIA patients and to assess their activation status and functional activity. The study involved 12 children with JIA and 35 healthy control subjects. PBMC were stained with monoclonal antibodies (anti-CD25, anti-CD4, anti-CD127, anti-CD69, anti-CD71, and anti-FoxP3). The samples were evaluated using flow cytometer. CD4+ CD25− and CD4+ CD25+ cells were isolated by negative and positive selection with magnetic microbeads. CD4+ CD25+ and CD4+ CD25− cells were cultured separately and co-cultured (1:1) with or without PHA. The percentage of Tregs in JIA patients was significantly decreased in comparison with controls (median, 3.2 vs. 4.6; P = 0.042). Relative fluorescence intensities of FoxP3 were higher in JIA patients than in controls (median, 9.1 vs. 6.8). The percentage of activated Tregs (CD71+) was significantly higher in JIA patients in comparison with controls (median, 6.5 vs. 2.8; P = 0.00043). CD4+ CD25+ cells derived from JIA patients and controls were anergic upon PHA stimulation, while CD4+ CD25− cells showed intensive proliferative response. The proliferation rate of CD4+ CD25− cells stimulated by PHA was decreased in co-cultures. In JIA patients, the inhibition of proliferation of CD4+ CD25− cells by CD4+ CD25+ cells was 37.9%, whereas in controls it was significantly lower (55.7%, P = 0.046). JIA patients had statistically lower percentage of Tregs in peripheral blood compared to controls. CD4+ CD25+ cells sorted from peripheral blood of JIA patients had statistically lower ability to suppress CD4+ CD25− cell proliferation in comparison with cells obtained from controls

Regulatory T Cells in the Pathogenesis and Healing of Chronic Human Dermal Leishmaniasis Caused by Leishmania (Viannia) Species

The immune inflammatory response is a double edged sword. During infectious diseases, regulatory T cells can prevent eradication of the pathogen but can also limit inflammation and tissue damage. We investigated the role of regulatory T cells in chronic dermal leishmaniasis caused by species of the parasite Leishmania that are endemic in South and Central America. We found that although individuals with chronic lesions have increased regulatory T cells in their blood and at skin sites where immune responses to Leishmania were taking place compared to infected individuals who do not develop disease, their capacity to control the inflammatory response to Leishmania was inferior. However, healing of chronic lesions at the end of treatment was accompanied by an increase in the number and capacity of regulatory T cells to inhibit the function of effector T cells that mediate the inflammatory response. Different subsets of regulatory T cells, defined by the expression of molecular markers, were identified during chronic disease and healing, supporting the participation of distinct regulatory T cells in the development of disease and the control of inflammation during the healing response. Immunotherapeutic strategies may allow these regulatory T cell subsets to be mobilized or mitigated to achieve healing

DRhigh+CD45RA−-Tregs Potentially Affect the Suppressive Activity of the Total Treg Pool in Renal Transplant Patients

Recent studies show that regulatory T cells (Tregs) play an essential role in tolerance induction after organ transplantation. In order to examine whether there are differences in the composition of the total CD4+CD127low+/−FoxP3+- Treg cell pool between stable transplant patients and patients with biopsy proven rejection (BPR), we compared the percentages and the functional activity of the different Treg cell subsets (DRhigh+CD45RA−-Tregs, DRlow+CD45RA−-Tregs, DR−CD45RA−-Tregs, DR−CD45RA+-Tregs). All parameters were determined during the three different periods of time after transplantation (0–30 days, 31–1,000 days, >1,000 days). Among 156 transplant patients, 37 patients suffered from BPR. The most prominent differences between rejecting and non-rejecting patients were observed regarding the DRhigh+CD45RA−-Treg cell subset. Our data demonstrate that the suppressive activity of the total Treg pool strongly depends on the presence of these Treg cells. Their percentage within the total Treg pool strongly decreased after transplantation and remained relatively low during the first year after transplantation in all patients. Subsequently, the proportion of this Treg subset increased again in patients who accepted the transplant and reached a value of healthy non-transplanted subjects. By contrast, in patients with acute kidney rejection, the DRhigh+CD45RA−-Treg subset disappeared excessively, causing a reduction in the suppressive activity of the total Treg pool. Therefore, both the monitoring of its percentage within the total Treg pool and the monitoring of the HLA-DR MFI of the DR+CD45RA−-Treg subset may be useful tools for the prediction of graft rejection

Treatment with Natalizumab in Relapsing–Remitting Multiple Sclerosis Patients Induces Changes in Inflammatory Mechanism

Natalizumab is a widely accepted drug for the relapsing–remitting subtype of multiple sclerosis (RRMS). The present longitudinal exploratory study in RRMS patients analyzes the effects of natalizumab treatment on the levels of pro-inflammatory and anti-inflammatory cytokine protein levels and also the frequency and suppressor function of regulatory T cells. Flow cytometry was used to determine cytokines and regulatory T cell frequency while regulatory T cell suppressor function was assayed in vitro at different time-points after starting with natalizumab. Results showed serum levels of pro-inflammatory interferon gamma and interleukin (IL)-12p70, as well as anti-inflammatory IL-4 and IL-10, were elevated just a few hours or days after first IV infusion of natalizumab. Interestingly, other cytokines like IL-5 or IL-13 were also elevated while pro-inflammatory IL-17, IL-2, and IL-1β increased only after a long-term treatment, suggesting different immune mechanisms. In contrast, we did not observe any effect of natalizumab treatment on regulatory T cell frequency or activity. In conclusion, these results suggest natalizumab has other immunological effects beyond VLA-4 interaction and inhibition of CNS extravasation, the relevance of which is as yet unknown and warrants further investigation

Foxp3 and IL-10 Expression Correlates with Parasite Burden in Lesional Tissues of Post Kala Azar Dermal Leishmaniasis (PKDL) Patients

Post kala azar dermal leishamniasis (PKDL), an unusual dermatosis develops in 5–15% of apparently cured visceral leishmaniasis cases in India and in about 60% of cases in Sudan. PKDL cases assume importance since they constitute a major human reservoir for the parasite. Inadequate treatment of VL, genetics, nutrition and immunological mechanisms that allow renewed multiplication of latent parasites or reinfection predispose to PKDL. Immunopathogenesis of PKDL is poorly understood. IL-10 is widely accepted as an immuno-suppressive cytokine and produced by diverse cell populations including, B cells, macrophages and CD4+ T cells. Natural T regulatory (nTreg) cells are subpopulation of CD4+ T cells that inhibit the response of other T cells. In this study we reported the accumulation of nTreg cells in lesion tissues of PKDL patients. Further correlation of Treg markers and IL-10 with parasite load in lesion tissues suggested a role of IL-10 and Treg in parasite establishment or persistence. Further studies are warranted to explore antigen specific IL-10 source in lesion tissues and unravel the concerted induction or accumulation of Treg in PKDL

Characterization of regulatory T cells in urban newborns

© 2009 Ly et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Graft-vs-tumor effect in patients with advanced nasopharyngeal cancer treated with nonmyeloablative allogeneic PBSC transplantation

While nonmyeloablative peripheral blood stem cell transplantation (NST) has shown efficacy against several solid tumors, it is untested in nasopharyngeal cancer (NPC). In a phase II clinical trial, 21 patients with pretreated metastatic NPC underwent NST with sibling PBSC allografts, using CY conditioning, thymic irradiation and in vivo T-cell depletion with thymoglobulin. Stable lymphohematopoietic chimerism was achieved in most patients and prophylactic CYA was tapered at a median of day +30. Seven patients (33%) showed partial response and three (14%) achieved stable disease. Four patients were alive at 2 years and three showed prolonged disease control of 344, 525 and 550 days. With a median follow-up of 209 (4–1147) days, the median PFS was 100 days (95% confidence interval (CI), 66–128 days), and median OS was 209 days (95% CI, 128–236 days). Patients with chronic GVHD had better survival—median OS 426 days (95% CI, 194–NE days) vs 143 days (95% CI, 114–226 days) (P=0.010). Thus, NST may induce meaningful clinical responses in patients with advanced NPC

CD4(+)CD25(+)FOXP3(+) Regulatory T Cells Suppress Anti-Tumor Immune Responses in Patients with Colorectal Cancer

BACKGROUND: A wealth of evidence obtained using mouse models indicates that CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) maintain peripheral tolerance to self-antigens and also inhibit anti-tumor immune responses. To date there is limited information about CD4(+) T cell responses in patients with colorectal cancer (CRC). We set out to measure T cell responses to a tumor-associated antigen and examine whether Treg impinge on those anti-tumor immune responses in CRC patients. METHODOLOGY AND PRINCIPAL FINDINGS: Treg were identified and characterized as CD4(+)CD25(+)FOXP3(+) using flow cytometry. An increased frequency of Treg was demonstrated in both peripheral blood and mesenteric lymph nodes of patients with colorectal cancer (CRC) compared with either healthy controls or patients with inflammatory bowel disease (IBD). Depletion of Treg from peripheral blood mononuclear cells (PBMC) of CRC patients unmasked CD4(+) T cell responses, as observed by IFNγ release, to the tumor associated antigen 5T4, whereas no effect was observed in a healthy age-matched control group. CONCLUSIONS/SIGNIFICANCE: Collectively, these data demonstrate that Treg capable of inhibiting tumor associated antigen-specific immune responses are enriched in patients with CRC. These results support a rationale for manipulating Treg to enhance cancer immunotherapy