Jill Waukau

Apoptosis of CD4+CD25high T Cells in Type 1 Diabetes May Be Partially Mediated by IL-2 Deprivation

Background Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease targeting the insulin-producing pancreatic β cells. Naturally occurring FOXP3+CD4+CD25high regulatory T cells (Tregs) play an important role in dominant tolerance, suppressing autoreactive CD4+ effector T cell activity. Previously, in both recent-onset T1D patients and β cell antibody-positive at-risk individuals, we observed increased apoptosis and decreased function of polyclonal Tregs in the periphery. Our objective here was to elucidate the genes and signaling pathways triggering apoptosis in Tregs from T1D subjects. Principal Findings Gene expression profiles of unstimulated Tregs from recent-onset T1D (n = 12) and healthy control subjects (n = 15) were generated. Statistical analysis was performed using a Bayesian approach that is highly efficient in determining differentially expressed genes with low number of replicate samples in each of the two phenotypic groups. Microarray analysis showed that several cytokine/chemokine receptor genes, HLA genes, GIMAP family genes and cell adhesion genes were downregulated in Tregs from T1D subjects, relative to control subjects. Several downstream target genes of the AKT and p53 pathways were also upregulated in T1D subjects, relative to controls. Further, expression signatures and increased apoptosis in Tregs from T1D subjects partially mirrored the response of healthy Tregs under conditions of IL-2 deprivation. CD4+ effector T-cells from T1D subjects showed a marked reduction in IL-2 secretion. This could indicate that prior to and during the onset of disease, Tregs in T1D may be caught up in a relatively deficient cytokine milieu. Conclusions In summary, expression signatures in Tregs from T1D subjects reflect a cellular response that leads to increased sensitivity to apoptosis, partially due to cytokine deprivation. Further characterization of these signaling cascades should enable the detection of genes that can be targeted for restoring Treg function in subjects predisposed to T1D.

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.

The role of NF‐κB and Smad3 in TGF‐β‐mediated Foxp3 expression

The transcription factor Foxp3 is essential for the development of functional, natural Treg (nTreg), which plays a prominent role in self‐tolerance. Suppressive Foxp3+ Treg cells can be generated from naïve T cells ex vivo, following TCR and TGF‐β1 stimulations. However, the molecular contributions from the different arms of these pathways leading to Foxp3 expression are not fully understood. TGF‐β1‐activated Smad3 plays a major role in the expression of Foxp3, since TGF‐β1‐induced‐Treg generation from Smad3−/− mice is markedly reduced and abolished by inactivating Smad2. In the TCR pathway, deletion of Bcl10, which activates NF‐κB, markedly reduces both IL‐2 and Foxp3 production. However, partial rescue of Foxp3 expression occurs on addition of exogenous IL‐2. TGF‐β1 significantly attenuates NF‐κB binding to the Foxp3 promoter, while inducing Foxp3 expression. Furthermore, deletion of p50, a NF‐κB subunit, results in increased Foxp3 expression despite a decline in the IL‐2 production. We posit several TCR‐NF‐κB pathways, some increasing (Bcl10‐IL‐2‐Foxp3) while others decreasing (p50‐Foxp3) Foxp3 expression, with the former predominating. A better understanding of Foxp3 regulation could be useful in dissecting the cause of Treg dysfunction in several autoimmune diseases and for generating more potent TGF‐β1‐induced‐Treg cells for therapeutic purposes.

Comparison of apoptosis and mortality measurements in peripheral blood mononuclear cells (PBMCs) using multiple methods

Abstract. Death through apoptosis is the main process by which aged cells that have lost their function are eliminated. Apoptotic cells are usually detected microscopically by changes in their morphology. However, determination of early apoptotic events is important for in vitro (and ex vivo) studies. The main objective of the present study is to find the most sensitive method for apoptosis detection in human peripheral blood mononuclear cells (PBMCs) by comparing six different methods following five different means of immunological stimulation at 3 and 5 days. Each of six apoptosis quantification methods, except the trypan blue exclusion test, is a combination of two stains, one for the specific detection of apoptotic cells and the other for the unspecific detection of dead cells. Values for apoptosis and mortality were compared with a reference method. The choice of apoptosis detection method is more important following 3 days of stimulation than after 5 days of stimulation (P = 2 × 10−6 versus P = 1 × 10−2). In contrast, we find mortality measurements following the different means of stimulation highly significant at both 3 and 5 days (F2.28 = 7.9, P = 1.4 × 10−6 at 3 days and F2.28 = 8.5, P = 4.5 × 10−7 at 5 days). Variation as a result of the combination of specific PBMC stimulation and the method used to detect apoptosis is reduced considerably with time (F1.58 + 3.7, P + 3 × 10−7 at 3 days to F = (1.58) = 0.97, P = 0.5 at 5 days). Based on Tukeys test, YO‐PRO‐1 is the most sensitive stain for apoptosis and, when combined with 7‐AAD, provides an accurate measure of apoptosis and mortality. In conclusion, we propose YO‐PRO‐1/7‐AAD as a new combination and low‐cost alternative for the sensitive detection of early apoptosis.

Dynamic changes in CD4+ CD25+ high T cell apoptosis after the diagnosis of type 1 diabetes

Because type 1 diabetes (T1D) is a chronic, autoimmune, T cell‐mediated disease, interventions affecting T cells are expected to modulate the immune cascade and lead to disease remission. We propose that increased CD4+ CD25+high T cell apoptosis, a trait we discovered in recent‐onset T1D subjects, reflects T1D partial remission within the first 6 months after diagnosis. Apoptosis of forkhead box P3 (FoxP3)+ CD4+ CD25+high T cells, in addition to total daily doses of insulin (TDD), blood glucose, HbA1c and age, were measured in 45 subjects with T1D at various times after diagnosis. Sixteen healthy control subjects were also recruited to the study. Higher CD4+ CD25+high T cell apoptosis levels were detected within the first 6 months of diagnosis (odds ratio = 1·39, P = 0·009), after adjustment for age, TDD and HbA1c. A proportional hazards model confirmed that the decline of apoptosis after diagnosis of T1D was related significantly to survival time (hazards ratio = 1·08, P = 0·014), with TDD and age also contributing to survival. During this time there was an inverse relationship between CD4+ CD25+high T cell apoptosis with TDD (r = −0·39, P = 0·008). The CD4+ CD25+high T cell apoptosis levels decline significantly after the first 6 months from diagnosis of T1D and may help in the close monitoring of autoimmunity. In parallel, there is an increase in TDD during this time. We also propose that CD4+ CD25+high T cell apoptosis assay can be used to gauge the efficacy of the several immune tolerance induction protocols, now under way.

Inducible regulatory T cells (iTregs) from recent-onset type 1 diabetes subjects show increased in vitro suppression and higher ITCH levels compared with controls

CD4+CD25+high regulatory T cells (Tregs) play a pivotal role in the control of the immune response. A growing body of evidence suggests the reduced function of these cells in autoimmune diseases, including type 1 diabetes (T1D). Restoration of their function can potentially delay further disease development. In the present study, we have converted conventional effector T cells into induced Tregs (iTregs) in recent-onset (RO) T1D (n=9) and compared them with the same cells generated in controls (n=12) and in long-standing (LS) T1D subjects (n=9). The functional potential of in-vitro-generated Tregs was measured by using an in vitro proliferation assay. We noted that the suppressive potential of iTregs exceeded that of natural regulatory T cells (nTregs) only in the RO T1D subjects. We showed that iTregs from RO T1D subjects had increased expression of Foxp3, E3 ubiquitin ligase (ITCH) and TGF-β-inducible early gene 1 (TIEG1) compared with control and LS T1D subjects. We also expanded natural, thymically derived Tregs (nTregs) and compared the functional ability of these cells between subject groups. Expanded cells from all three subject groups were suppressive. RO T1D subjects were the only group in which both iTregs and expanded Tregs were functional, suggesting that the inflammatory milieu impacts in vitro Treg generation. Future longitudinal studies should delineate the actual contribution of the stage of disease to the quality of in-vitro-generated Tregs.

Genetic association of HLA DQB1 with CD4+CD25+ high T-cell apoptosis in type 1 diabetes

Type 1 diabetes (T1D) has a strong genetic component and the major locus lies in the HLA DQB1 region. We found earlier an increased apoptosis with decreased viability and function of the CD4+CD25+high T-cell subset (Treg) in human subjects with recent-onset T1D and in multiple autoantibody-positive, high at-risk individuals. Tregs normally inhibit or delay onset of T1D in animal models and increased Treg apoptosis could bring on or accelerate disease from effector T-cell-mediated destruction of insulin-producing beta cells. In this study, we test the hypothesis that HLA DQB1 genotypes are associated with increased CD4+CD25+high T-cell apoptosis. HLA DQ-based genetic risk status was significantly associated with CD4+CD25+high T-cell apoptosis, after adjustment for age, gender and phenotypic status (n=83, F=4.04 (d.f.=3), P=0.01). Unaffected, autoantibody-negative high risk HLA DQB1 control subjects showed increased CD4+CD25+high apoptosis levels compared with low risk HLA DQB1 control subjects (n=26, P=0.002), confirming that the association precedes disease. The association of specific HLA DQB1 genotypes with Treg apoptosis was also tested, showing significance for HLA DQB1*0302, DQB1*0201 and HLA DQB1*0602 alleles. Our study shows an association of HLA DQB1 genotypes with CD4+CD25+high T-cell apoptosis, which implicates CD4+CD25+high T-cell apoptosis as a new intermediate trait for T1D.

The type of responder T-cell has a significant impact in a human in vitro suppression assay.

Background In type 1 diabetes (T1D), a prototypic autoimmune disease, effector T cells destroy beta cells. Normally, CD4+CD25+high, or natural regulatory T cells (Tregs), counter this assault. In autoimmunity, the failure to suppress CD4+CD25low T cells is important for disease development. However, both Treg dysfunction and hyperactive responder T-cell proliferation contribute to disease. Methods/Principal Findings We investigated human CD4+CD25low T cells and compared them to CD4+CD25- T cells in otherwise equivalent in vitro proliferative conditions. We then asked whether these differences in suppression are exacerbated in T1D. In both single and co-culture with Tregs, the CD4+CD25low T cells divided more rapidly than CD4+CD25- T cells, which manifests as increased proliferation/reduced suppression. Time-course experiments showed that this difference could be explained by higher IL-2 production from CD4+CD25low compared to CD4+CD25- T cells. There was also a significant increase in CD4+CD25low T-cell proliferation compared to CD4+CD25- T cells during suppression assays from RO T1D and at-risk subjects (n = 28, p = 0.015 and p = 0.024 respectively). Conclusions/Significance The in vitro dual suppression assays proposed here could highlight the impaired sensitivity of certain responder T cells to the suppressive effect of Tregs in human autoimmune diseases.

The design of a gene chip for functional immunological studies on a high-quality control platform.

Abstract: We have created an immunology‐related microarray chip containing primarily known genes with well‐studied functional properties. By looking at known genes rather than expressed sequence tags, we hope to gain a better understanding of immunological pathways and how they work. The immunology gene chip contains genes from the following functional categories: T cell genes; B cell genes; dendritic cell genes; chemokine and cytokine genes; apoptosis genes; cell cycle genes; cell interaction genes; general hematology and immunology genes; and adhesion genes. We have also developed a novel three‐color cDNA array platform in which arrays are directly visualized before hybridization, which allows us to select only high‐quality chips for our experiments. In an effort to provide quantitative quality control for each array element as well as the entire chip, we have developed Matarray, a software package for image processing and data acquisition. With Matarray, we have built a quantitative data filtering and normalization scheme that has proved to be more efficient than the existing methods. The list of immunology chip genes is available from the authors.

Human in vitro suppression as screening tool for the recognition of an early state of immune imbalance.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic β-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.