Our long-term goal is to understand the role of T cells in the pathogenesis of systemic sclerosis (SSc). We have recently identified oligoclonal subpopulations of gamma/delta and CD8+ T cells in the lungs of SSc patients with alveolitis. The hypothesis of this proposal is that these T cells play a causal role in the development of pulmonary fibrosis in SSc. Our general strategy is to analyze T cells within the lungs of SSc patients before, during, and after the development of interstitial inflammation. Analyses of T cells will include the T cell antigen (TCR) repertoire, response to self antigens, and effector functions. SSc patients will be followed with serial bronchoalveolar lavage (BAL). We will compare results in SSc patients who develop alveolitis and restrictive lung disease to those in patients who do not develop pulmonary involvement and to healthy controls. The first specific aim is to analyze the TCR repertoire expressed by CD4+alpha/beta, CD8+alpha/beta, and gamma/delta T cells isolated serially from the blood and BAL of SSc patients and controls. We will use two approaches to analyze TCR diversity. First, we will test the level of expression of different variable (V)alpha, Vbeta, Vgamma, and Vdelta gene families, using a RT-PCR technique. Results will be confirmed with anti-V gene monoclonal antibodies. Second, we will test the diversity of expressed TCR junctional regions, using analyses of nucleotide lengths and DNA sequences. These studies will determine whether oligoclonal T cells infiltrate the lungs of SSc patients before other inflammatory cells, whether their presence predicts the development of restrictive lung disease, and whether they disappear with resolution of lung inflammation. The second specific aim is to characterize the functional capacities of T cells that have undergone oligoclonal expansion in the lungs of SSc patients. We will develop long-term clones of these T cells. The T cell clones will be tested for their capacity to proliferate in response to a panel of self antigens. Self antigens will include B cell autoantigens, autologous peripheral blood mononuclear cells, autologous B cells, autologous fibroblasts, and homogenates of human lung, fat, and skin. We will determine the pattern of cytokines made by these T cell clones. These studies will focus on T cell-derived cytokines that will identify a Th1, Th2, or ThO phenotype, promote fibrosis, and recruit or stimulate other inflammatory cells within the lungs. The ability of these T cell clones to kill autologous cells will be determined.
