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Examination of Thymic Positive and Negative Selection by Flow Cytometry
Published on: October 8, 2012
Phenotypical and functional characterization of double-negative (CD4-CD8-) alpha beta T-cell receptor positive cells
N Illum1, E Ralfkiaer, G Pallesen
1Department of Paediatrics, University of Copenhagen, Denmark.
Insights
This study identifies unusual CD4-CD8- double-negative T cells in an immunodeficiency patient. These cells show impaired signaling, suggesting a defect in transmembrane signal transduction.
Area of Science:
- Immunology
- T-cell biology
- Cellular signaling
Background:
- Characterization of CD4-CD8- double-negative (DN) alpha beta TCR+ T cells in a patient presenting with immunodeficiency, lymphocytosis, lymphadenopathy, and hepatosplenomegaly.
- The majority of peripheral blood lymphocytes were identified as DN alpha beta TCR+ T cells via flow cytometry and biochemical analysis.
Observation:
- The DN T cells exhibited a specific phenotype (alpha beta TCR+, CD4-, CD8-, CD2+, CD3+, CD5+, CD28+, CD45RA+, CD57+).
- Southern blot and flow cytometry analyses indicated a polyclonal T-cell expansion.
- Lymph node biopsies revealed expanded paracortical areas infiltrated by these DN T cells.
Findings:
- DN T cells demonstrated a significantly reduced proliferative response to mitogens and TCR/CD3, CD2, and CD28 stimulation.
- Exogenous interleukin-2 (IL-2) minimally enhanced proliferation.
- Combined calcium ionophore and phorbol 12-myristate 13-acetate (PMA) restored proliferative capacity, indicating intact protein kinase C activity.
Implications:
- The findings suggest a defect in transmembrane signal transduction pathways within these atypical DN T cells.
- This cellular defect may contribute to the observed immunodeficiency and lymphoproliferative disorder.
- Further research into T-cell signaling defects could offer insights into novel therapeutic targets for related immune disorders.
Abstract:
We have characterized CD4-CD8- double-negative (DN) alpha beta TCR+ T cells from a patient with immunodeficiency, lymphocytosis, lymphadenopathy, and hepatosplenomegaly. The majority of peripheral blood lymphocytes were DN alpha beta TCR+ T cells as evaluated by FACS and biochemical analysis. The DN T cells showed the following phenotype: alpha beta TCR+, gamma delta TCR-, CD2+, CD3+, CD4-, CD5+, CD7-, CD8-, CD16-, CD25-, CD26-, CD28+, CD45RO-, CD45RA+, CD57+, and HLA-DR+. Both southern blot analysis of TCR genes and FACS analysis applying a panel of V beta and V alpha monoclonal antibodies (MoAbs) indicated a polyclonal T-cell expansion. Thymic biopsy showed normal histology, whereas lymph node biopsy samples showed altered histological and immunohistological patterns with markedly expanded paracortical areas containing the DN T cells of the same phenotype as found in peripheral blood T cells. In functional studies, the DN T cells showed a profoundly reduced proliferative response upon stimulation with mitogens as well as MoAbs against the TCR/CD3 complex, CD2, and CD28, respectively. Addition of exogenous interleukin-2 (IL-2) only minimally augmented the proliferative response. In contrast, the addition of a combination of Ca2+ ionophore and phorbol 12-myristate 13-acetate (PMA) restored the proliferative response of the DN T cells to almost normal levels. This observation strongly suggests that the protein kinase C activity of the DN T cells was intact, but that the normal mechanism for transmembrane signal transduction was impaired in these unusual DN T cells.
