Because of their particular composition, “lipid rafts” found within the plasma membrane of T lymphocytes selectively associate with certain membrane proteins and as such, play a critical role in activation and signaling. Lipid rafts have also been shown to translocate to the immune synapse (IS) following ligation of the T cell receptor (TCR) and co-stimulatory receptors (such as CD28) by antigen presenting cells. We have observed that several proteins which play key roles in T cell activation, such as LAT, Lck, CD3-zeta, ZAP70 and CD28 are more abundant in lipid rafts isolated from cells maintained for several hours under mild thermal stress (MTS) when compared to rafts from normothermic cells. Since co-stimulation is thought to help cluster essential signaling molecules in the lipid raft, we hypothesize that MTS may function as a co-stimulatory signal for T cell activation.
We have observed that exposure of CD4+ T lymphocytes to a fever-range thermal stress (39.5°C) prior to T cell receptor mediated activation reduces the need for costimulation via CD28 for the production of IL-2 compared to cells maintained at 37°C. This data reveals that the current paradigm regarding the requirements for co-stimulation of CD4 + T lymphocytes, which have been entirely defined at 37°C, may not apply under conditions of fever or local inflammation. Similar effects were seen using human peripheral blood CD4+ cells, Jurkat T cells, CD4+ T cells isolated from CD28 knock-out mice and ova-specific CD4+ T cells isolated from DO11.10 transgenic mice.
We have further studied changes in the protein composition of lipid rafts isolated from Jurkat T cells given TCR stimulation with either a classical co-stimulatory signal or exposed to 39.5°C. We observed similar changes in the relative expression of the proteins LAT, Lck, CD3-zeta, ZAP70 and CD28 in lipid rafts from T cells given either standard activation with TCR and CD28 ligation, and T cells given TCR ligation and MTS.
For a more global analysis of the protein changes which occur in lipid rafts in response to MTS, we used Ettan-DIGE followed by MALDI-TOF mass spectrometry. We identified several cytoskeletal proteins, such as spectrin and actin, whose association with lipid rafts increased when T cells were maintained under MTS. This has led us to hypothesize the involvement of the cytoskeleton in MTS induced raft movement. Of particular interest we identified the stomatin-like protein 2 (STML2) to have higher expression in lipid rafts from MTS treated T cells. STML2 has been shown to link mitochondria to the TCR in the immunological synapse during T cell activation. Since we have also identified several mitochondrial proteins to be more highly expressed in lipid rafts of MTS T cells, we have hypothesized that MTS not only results in a clustering of lipid rafts, but also promotes the association of the mitochondria with these clusters via STML2.
Determining how physiologically relevant temperatures affect the composition and function of lipid rafts will lead to a better understanding of how CD4 + T cell activation is regulated by the thermal microenvironment and hopefully to new therapeutic approaches using thermal therapy.