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Ontiveros_Sharon_Presentation
- 1. Sharon Ontiveros Cuevas,Laurence Pellerin, Maria Grazia Roncarolo, Rosa Bacchetta Department of Pediatrics, Stanford University School of Medicine CD25 expression in subjects with CD25 gene mutations
- 2. Background: IL-2 signaling •IL-2: produced mainly by activated CD4+ T lymphocytes • IL-2 signals through IL-2R (IL-2Rβ and γ or IL-2Rα, β and γ) • IL-2Rα/CD25 required for proliferation of Teff & survival of Treg CD25 CD122 CD132
- 3. Background: CD25 deficiency •Primary immunodeficiency with immune dysregulation • Autoimmune enteropathy, eczema, and viral infections • Few cases in the world Diffuse eczema Neutropenia Thrombocytopenia Inflammatory bowel disease Alopecia universalis Hypothyroidism Hepatosplenomegaly
- 4. Preliminary Studies &hypothesis
- 5. Preliminary Studies &hypothesis Healthy donor CD25 CD25 Heterozygous (mutation 1) Are the patients able to express CD25? If so, which isoform is expressed? Heterozygous (mutation 2) Compound Heterozygous
- 6. Experimental Design +PHA +IL-2 unstimulated stimulated 2weeks Flow Cytometry Western BlotWestern Blot Flow Cytometry • staining • protein extraction • protein extraction• staining • anti-CD3/anti- CD28 beads • IL-7 • IL-15
- 7. Results: Flow Cytometry Unstained Unstimulated,stained Stimulated, stained CD25 %ofMax Conclusion: Our experimental design is appropriate to induce and detect a high expression of CD25
- 8. Results: Western blot Polyclonalgoat anti-CD25 antibody Monoclonal mouse anti-CD25 antibody Conclusion: The monoclonal mouse anti-CD25 antibody has a stronger signal than the polyclonal goat anti-CD25 antibody 0 0.1 0.2 0.3 0.4 0.5 Unstimulated PHA blasts Stimulated PHA blasts ArbitraryUnits
- 9. Conclusions • Flow cytometryshows that our experimental design is appropriate to induce and detect a high expression of CD25 • WB: the monoclonal mouse anti-CD25 antibody works better • WB detected higher expression of CD25 protein after T cell activation
- 10. Future Directions • Validateon activated primary T cells • Test CD25 expression on mutated T cells from patients by WB
- 11. Acknowledgements • Laurence Pellerin •Rosa Bacchetta • Members of the Roncarolo Laboratory • California Institute of Regenerative Medicine (CIRM) • Stanford Institutes of Medicine Research Program (SIMR)
Editor's Notes
- #2 Hi I’m Sharon Ontiveros, I have been working with Laurence Pellerin, Maria Grazia Roncarolo, and Rosa Bacchetta in the Roncarolo Lab, and my project is “CD25/IL-2 receptor alpha expression in subjects with CD25 gene mutations”
- #3 Here’s the background: Interleukin 2 signaling is “mainly produced by activated CD4+ T lymphocytes”. IL-2 signals IL-2 Receptor. IL-2 Receptor is composed out of three subunits: IL-2 Receptor alpha, IL-2 Receptor beta, and IL-2 gamma chain. These subunits are also called Cluster of Differentiation CD25, CD122, and CD132. CD122 and CD32 create a low affinity receptor for IL-2, however when CD25 is added the IL-2 receptor becomes a stronger affinity receptor for IL-2 by the hundred fold. CD25 is required for the proliferation of T effector cells and the survival of T regulatory cells.
- #4 CD25 deficiency causes a primary immunodeficiency with immune dysregulation. Which is characterized by autoimmune enteropathy, eczema, and viral infections. There have been few cases in the world making it difficult to study CD25 deficiency. *Points to baby* Here are also common symptoms associated with CD25 deficiency: alopecia universalis: balding, Inflammatory bowel disease: constant and severe diarrhea, neutropenia: the deficiency of neutrophils, and thrombocytopenia: the deficiency of platelets in the blood.
- #5 My study focuses on three related patients that present two different CD25 gene mutations. From preliminary studies, we predicted the structure of the two different CD25 proteins. The first CD25 protein is the wildtype/healthy donor. The first mutation as you may notice is shorter than the wildtype. This is because the first mutation results in the formation of a premature STOP codon. The mutation 2 is a frameshift mutation that results in the formation of an elongated protein.
- #6 Preliminary studies from the lab have assessed the expression of CD25 by flow cytometry on fresh CD3+CD4+ T lymphocytes using two different monoclonal CD25 antibodies. The 1st plot is HD, the second a patient that presents the mutation 1 heterozygous,… The healthy donor presents 17.2% of CD25+ T lymphocytes. The patient that carries the heterozygous mutation 1 shows 3.39% of CD25+ T lymphocytes. The patient that carries the heterozygous mutation 2 shows 2.18% of CD25+ T lymphocytes. The compound heterozygous that carries both mutations one and mutation two shows a near absence of CD25 since it has a 0.471% of CD25+ T lymphocytes. From this data we formulated our hypothesis: Whether the patients are able to express CD25? And if so, which isoform is expressed?
- #8 ----- Meeting Notes (8/5/15 08:52) ----- Explain the populations....
- #9 ----- Meeting Notes (8/5/15 08:52) ----- Normalization: by using the total protein amount 140% increase