PTPN22 and Regulation of Src-Family Kinases
A prelude to autoimmunity is generally an alteration in the balance of the naïve and memory/effector T cell pools. The size of these pools is tightly regulated, although transient, large expansions of the effector/memory pool occur during an immune response, for example, to infection. Once infection resolves in a healthy individual, the homeostatic balance is reinstated. Another perturbation that can affect immune homeostasis is transient lymphopenia which can occur following infection. This control breaks down in ageing where there is an increase in the size of the memory T cell pool which coincides with an increased incidence of autoimmune disease. Understanding how the memory/effector pool is restrained in a healthy individual and why this restraint fails leading to autoimmunity is our primary goal. Genetic analysis in humans has shown that in terms of single gene contribution, point mutations in a haematopoietic phosphatase, PTPN22, are the second highest risk factor for development of rheumatoid arthritis and the third for type 1 diabetes, and other autoimmune diseases. PTPN22 regulates the activity of Src-family kinases (SFKs) and their downstream targets. We have been investigating how changes in expression of the SFKs in GM mice result in alterations in T cell homeostasis leading to expansion of the memory pool either with age and/or following immune challenge, which leads, in some instances, to autoimmunity. We focus on CD8 T cells from mice predisposed towards deregulated expansion and autoimmunity and are using RNA-Seq and proteomic analysis to identify both known and unknown targets of these important signalling molecules. Such a systematic approach will link genotypes and immunopathology and has the potential for identifying novel pathways that could be targeted for immunotherapy.
Regulation of Translation in T cells
The extent of TCR triggering is linked to T cell fate. For example, strong agonist ligands stimulate maximal clonal proliferation, differentiation to effector function and the development of memory cells, whereas weaker ligands drive less proliferation and effector differentiation, yet still allow differentiation to memory cells. While changes in gene transcription influence these cell fates there is also substantial control regulated at the level of translation. Particular groups of proteins, such as effector cytokines, chemokines and chemokine receptors, have been shown to be highly regulated post-transcriptionally in T cells. In order to gain a better understanding of how T cells regulate translation upon stimulation through the TCR, we are investigating primary CD8+ T lymphocytes expressing a TCR of known specificity (OT-1) that are stimulated with defined peptide ligands in vitro. We are looking at specific signalling pathways that play a role in regulating ribosome biogenesis in T cells at the level of rRNA precursor transcription and processing, and at the TCR signalling pathways that regulate this complex process by the translation of specific genes involved in various stages of pre-rRNA production and maturation. We can correlated manipulations in TCR signalling with their impact on ribosome biogenesis and with T cell proliferation, to show that T cells respond to TCR stimulation in a dynamic fashion by regulating the production, and therefore the availability, of functional ribosomes in the cell, with consequent influences on both global and specific protein translation.
We are also looking at how microRNAs influence T cell activation and differentiation. Abnormal regulation of miRNAs can lead to immune pathology and it is important to learn more about their roles in the adaptive immune system. We are attempting to identify miRNAs and their target genes in murine CD8 T cells by using a novel Ago-2 tagged mouse which will allow biochemical and immunological assays to be combined with bioinformatic analysis. Gaining information about miRNA targets will further our understanding of the mechanism of action of miRNAs as well as the signalling pathways surrounding T cell activation.