The goal of this research project is to understand the process of T cell differentiation that underlies the switch from a naive T cell to a memory T cell. Upon stimulation of the T cell antigen receptor/CD3 complex, naive T cells are incapable of secreting lymphokines other than interleukin 2 (IL-2), whereas memory T cells secrete many different lymphokines almost immediately. Individual lymphokines are responsible for the recruitment and activation of the cells that actually mediate the immune reaction. Therefore, the capacity of memory T cells to immediately direct the participation of all Immune/inflammatory cells accounts in large part for the increased Intensity and accelerated nature of the anamnestic immune response. The total absence or loss of memory results in immunodeficiency and susceptibility to a broad range of Infectious organisms, as in AIDS. By comparison, the absence or loss of antigen-specific memory provides an explanation for tolerance or anergy to specific infectious agents. Accordingly, as a result of this research project the information gained is expected to have immediate relevance for the regulatory mechanisms in AIDS, infectious diseases, autoimmunity, and organ transplantation. Having developed and established cell separation procedures and the polymerase chain reaction (PCR) to amplify lymphokine mRNA transcripts, naive T cells derived from neonates will be examined to determine what proportion of cells actually differentiate. In addition, individual cell clones will be examined to determine whether each primed T cell responds to a secondary stimulation by expressing the entire lymphokine repertoire, or Whether different cells express a more restricted lymphokine repertoire. The phenotype and differentiative stage of T cells present in adults will also be examined to determine whether naive T cells uncontaminated by memory T cells can be detected and isolated. Furthermore, the expression of genes encoding cytolytic molecules will be examined to determine whether a similar differentiative event occurs. The mechanisms that could account for the absence of detectable lymphokine mRNA transcripts after priming stimulation of neonatal T cells focus on one of two possibilities; the lack of transcription vs. rapid mRNA degradation, with the former being the most likely. To identify directly gene regulatory features that could preclude transcription, such as cytosine methylation and/or transcriptional silencing proteins, ligation-mediated PCR-based genomic sequencing will be used. IL-2 is critical to the differentiative step(s) that allow naive cells to switch to the expression of the full lymphokine repertoire upon a secondary stimulation. Therefore, experiments to determine whether the IL-2 effect is direct or indirect, requiring new gene expression will be performed. Together, these experiments will provide a molecular dissection of the pathways responsible for the T cell differentiative switch that gives rise to a functional, memory T cell.
| Chiocchia, G; Smith, K A (1997) Highly sensitive method to detect mRNAs in individual cells by direct RT-PCR using Tth DNA polymerase. Biotechniques 22:312-4, 316, 318 |
