T cell repertoire in patients with stable scleroderma

Abstract

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.

Fig. 1

Qualitative T cell repertoire analysis and method for quantifying the extent of T cell repertoire perturbations. (a) Examples of immunoscope profiles of unsorted PBMC, and CD4 and CD8 T cells from dSSC, lSSc, RP patients and Ctrl. Only the first 7 BV (out of the 14 BV families analysed) are displayed. RFI, relative fluorescence intensity. (b) To quantify repertoire perturbations, the CDR3 profiles for each BV family is translated into a probability distribution P^BV(i) for each peak i (i = 1–8). The extent of perturbation D^BV(i) for each peak i is computed by the distance between P^BV(i) values from the sample and P_ref^BV(i) values from a reference distribution (mean ± SEM). The perturbation D^BV is then calculated as Σ_i |D^BV(i)|/2. The mean of D^BV perturbations for all 14 BV families yields the average perturbation D = Σ_k D^BVk/14. An example of calculation is given for BV5 in CD8 T cells from Ctrl-01. All D^BV results for this sample are displayed in (a). Average D-values (computed on 14 BV families) are also given in (a) for all displayed samples.

Fig. 2

Quantitative T cell repertoire analysis. Perturbation indices D of unsorted (a) PBMC, (b) CD8 and (c) CD4 sorted T cells, and (d) IL-2-cultured CD4 T cells are given for dSSc, lSSc, RP and Ctrl groups. Mean ± SD [range] of D indices for the different cell subsets are displayed for each group. Horizontal bar indicates mean. Inter-group differences were not statistically significant (Mann–Whitney test).

Fig. 3

Repertoire analysis of CD4 T cells after culture with IL-2. Example of immunoscope profiles of CD4 T cells (before culture and d7 postculture) from dSSC, lSSc, RP and Ctrl. Only the last 7 BV (out of the 14 BV families analysed) are displayed. Perturbation indices D are given for all samples.

Fig. 4

Histological analysis of skin sections. (a) Anti-CD3 immunohistochemical analysis of skin section from patient dSSc-03 shows a discrete T cell infiltrate. Original magnification ×200. (b) Hematoxin and eosin staining of skin section from patient dSSc-04 shows thickened collagen bundles characteristic of SSc. Original magnification ×200.