T cell repertoire in patients with stable scleroderma.

At onset of systemic sclerosis (SSc), T cells have been found to oligoclonally expand in the skin, presumably in response to auto-antigens, but the T cell repertoire has not been evaluated at a later stage. To determine whether a perpetuating immune response contributes to the pathogenesis of stable SSc, the T cell repertoire was analysed in patients with diffuse (d) or limited (l) SSc, and compared to patients with primary Raynaud's phenomenon (RP) or healthy volunteers (Ctrl). The T cell repertoire (total, CD4 or CD8 sorted blood T cells) was analysed by qualitative and quantitative immunoscope (14 BV families analysed) in 11 untreated dSSc and 11 untreated lSSc, 10 RP and 11 Ctrl. To better detect in vivo activated cells, repertoire analysis was also performed on sorted CD4 T cells after in vitro culture with IL-2. In parallel, 6 skin biopsies from SSc patients were analysed. After 7-8 years of disease evolution, SSc patients did not show detectable clonal T cell expansions in the skin, even after tentative expansion from the biopsy with IL-2. Total T cell, sorted CD4 and CD8 T cell repertoires from the blood of patients with SSc did not show significant perturbation as compared to patients with RP and Ctrl. After IL-2 culture for 7 days, blood CD4 T cells from the patients did not preferentially expand as compared to RP and Ctrl. These findings suggest that antigen-driven immune responses may play a lesser role in established SSc than at disease onset.

Severe perturbations of the blood T cell repertoire in polymyositis, but not dermatomyositis patients.

Polymyositis and dermatomyositis are diseases characterized by muscle weakness and muscle inflammatory infiltrates. Their pathogenesis remains unclear. A central role for endomysial autoaggressive CD8(+) T cells is suspected in polymyositis and for perivascular B cells in dermatomyositis. We compared the T cell repertoire of 10 polymyositis and 10 dermatomyositis patients by immunoscope, a method providing a global assessment of the T cell repertoire and a sensitive detection of clonal T cell expansions. Samples were analyzed qualitatively and quantitatively in the blood (unsorted cells and CD4(+) and CD8(+) cells) and in muscle infiltrates. Dramatic perturbations of the T cell repertoire were observed in the blood of polymyositis but not dermatomyositis patients (p < 0.0005), the latter being undistinguishable from controls. These perturbations were due to oligoclonal expansions of CD8(+) T cells and most blood clonal expansions were also found in muscle. These results indicate that the pathogenesis of polymyositis and dermatomyositis is different and reinforce the view that polymyositis but not dermatomyositis is an autoimmune CD8(+) T cell-mediated disease. Moreover, this method may be helpful for the differential diagnosis of polymyositis and dermatomyositis and for noninvasive follow-up of polymyositis patients.

Retrovirus-mediated gene transfer into T cells: 95% transduction efficiency without further in vitro selection.

This study was designed to retrovirally transduce T cells by a protocol that would be simple, short, cost effective, applicable for clinical use, and efficient enough to avoid further selection of transduced T cells. Because retrovirally mediated infection is depending on the cell cycle, we first optimized the conditions for activating T cells in the presence of immobilized CD3 monoclonal antibodies and recombinant interleukin 2. Cell cycle analysis indicated that CD8+ and total T cells reach a maximum of cycling within 4 days whereas CD4+ T cells attain their maximum of cycling only by day 6. Taking into account these data, CD4+, CD8+, and total T cells were preactivated for 5 and 3 days, respectively, and then infected for 24 hr with supernatant containing retrovirus pseudotyped with gibbon-ape leukemia virus envelope, using a cell centrifugation protocol. Results show that approximately 95% of CD4+, CD8+, and total T cells can be transduced, this transduction efficiency being significantly higher than that obtained with amphotropic retrovirus vectors. Furthermore, under permanent growth stimulation, transduced T cells can be expanded approximately 1,000-fold in 4 weeks of culture with maintenance of transgene expression. However, Immunoscope analysis revealed alterations of T cell repertoire diversity after 2-3 weeks in culture that was not due to retroviral transduction per se. Overall, these data provide evidence that T cells can be transduced at levels that may alleviate the need for both further selection of transduced cells and in vitro expansion, thereby preserving the repertoire diversity of the transduced T cells to be reinfused.