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
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
8. Results: Western blot
Polyclonal goat
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 cytometry shows 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
• Validate on 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
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”
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.
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.
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.
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?
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Explain the populations....
----- Meeting Notes (8/5/15 08:52) -----
Normalization: by using the total protein amount
140% increase
