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Nuclear events after activation of CD4+8+ thymocytes
J S Riegel1, E R Richie, J P Allison
1Department of Molecular and Cellular Biology, University of California, Berkeley 94720.
Insights
Immature thymocytes fail to produce IL-2 due to a lack of inducible NFAT-1 activity. Functional T cell maturation involves acquiring the ability to induce NFAT-1, crucial for IL-2 gene expression.
Area of Science:
- Immunology
- Molecular Biology
- Cellular Signaling
Background:
- Mature T cells secrete IL-2 and proliferate upon Ag receptor stimulation.
- Immature CD4+8+ thymocytes lack IL-2 secretion and proliferation responses.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the differential IL-2 gene expression in T cell populations.
- To compare IL-2 mRNA expression and transcription factor activity in mature vs. immature T cells.
Main Methods:
- Analysis of mRNA expression (c-myc, IL-2) in murine thymocytes and lymphoma cell lines.
- Assessment of nuclear DNA-binding factors NFAT-1 and NFIL2-A activity via electrophoretic mobility shift assays.
Main Results:
- All cell types accumulated c-myc mRNA upon stimulation.
- CD4+ thymocytes and C6VL-B lymphoma expressed IL-2 mRNA, while CD4+8+ thymocytes and 1010 lymphoma did not.
- NFIL2-A binding activity was constitutive in all cells.
- NFAT-1 binding activity was inducible in CD4+ cells but minimally induced in CD4+8+ cells upon activation.
Conclusions:
- The inability of CD4+8+ thymocytes to express IL-2 mRNA is partly due to the lack of inducible NFAT-1 binding activity.
- Functional T cell maturation correlates with the acquisition of inducible NFAT-1 activity, essential for IL-2 gene expression.
Abstract:
Functionally mature T cells respond to stimulation via the Ag receptor by secretion of IL-2 and/or other lymphokines and by proliferation. However, immature CD4+8+ thymocytes do not secrete IL-2 or proliferate in response to stimulation. We have analyzed murine thymocyte populations enriched for CD4+ and CD4+8+ cells as well as the functionally mature CD4+ lymphoma C6VL-B and the CD4+8+ lymphoma 1010 for their ability to express mRNA related to early products of T cell activation signals. When stimulated with the calcium ionophore (Ionomycin) plus PMA, all the cells, regardless of their phenotype, accumulated abundant levels of c-myc mRNA. However, in contrast to the CD4+ thymocytes and C6VL-B, which accumulated abundant levels of IL-2 transcripts, neither the normal CD4+8+ thymocytes nor 1010 expressed IL-2 mRNA before or after stimulation. We have also examined these cells for the presence of the murine equivalents of two nuclear DNA-binding factors, NFAT-1 and NFIL2-A, which have been shown to be involved in IL-2 gene expression in human T cells. Our results indicate: 1) NFIL-2A binding activity is constitutively expressed in both CD4+ and CD4+8+ thymocytes and lymphomas and 2) NFAT-1 binding activity is readily detected in CD4+ thymocytes and C6VL-B, but is detected in very minimal amounts in populations enriched for CD4+8+ thymocytes and in 1010 upon activation. These results suggest that the failure of CD4+8+ thymocytes to express IL-2 mRNA upon stimulation may be in part due to the lack of inducibility of NFAT-1 binding activity, and that functional maturation of this population might be associated with acquisition of the ability to induce NFAT-1 activity.