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In JoVE (1)
Other Publications (6)
Articles by Jill Waukau in JoVE
JoVE Immunology and Infection
Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance
1Department of Pediatrics/Allergy, Medical College of Wisconsin, 2Flow Cytometry Core Facility, Medical College of Wisconsin, 3Max McGee National Research Center for Juvenile Diabetes and Human Molecular Genetics Center, Medical College of Wisconsin
Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.
Other articles by Jill Waukau on PubMed
The Design of a Gene Chip for Functional Immunological Studies on a High-quality Control Platform
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.
At-risk and Recent-onset Type 1 Diabetic Subjects Have Increased Apoptosis in the CD4+CD25+ T-cell Fraction
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.
Apoptosis of CD4+ CD25(high) T Cells in Type 1 Diabetes May Be Partially Mediated by IL-2 Deprivation
Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease targeting the insulin-producing pancreatic beta cells. Naturally occurring FOXP3(+)CD4(+)CD25(high) regulatory T cells (T(regs)) play an important role in dominant tolerance, suppressing autoreactive CD4(+) effector T cell activity. Previously, in both recent-onset T1D patients and beta cell antibody-positive at-risk individuals, we observed increased apoptosis and decreased function of polyclonal T(regs) in the periphery. Our objective here was to elucidate the genes and signaling pathways triggering apoptosis in T(regs) from T1D subjects.
The Role of NF-kappaB and Smad3 in TGF-beta-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-beta1 stimulations. However, the molecular contributions from the different arms of these pathways leading to Foxp3 expression are not fully understood. TGF-beta1-activated Smad3 plays a major role in the expression of Foxp3, since TGF-beta1-induced-Treg generation from Smad3(-/-) mice is markedly reduced and abolished by inactivating Smad2. In the TCR pathway, deletion of Bcl10, which activates NF-kappaB, markedly reduces both IL-2 and Foxp3 production. However, partial rescue of Foxp3 expression occurs on addition of exogenous IL-2. TGF-beta1 significantly attenuates NF-kappaB binding to the Foxp3 promoter, while inducing Foxp3 expression. Furthermore, deletion of p50, a NF-kappaB subunit, results in increased Foxp3 expression despite a decline in the IL-2 production. We posit several TCR-NF-kappaB 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-beta1-induced-Treg cells for therapeutic purposes.
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-beta-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.
The Type of Responder T-cell Has a Significant Impact in a Human in Vitro Suppression Assay
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(+)CD25(low) T cells is important for disease development. However, both Treg dysfunction and hyperactive responder T-cell proliferation contribute to disease.
