Skin-Resident Effector Memory CD8+CD28- T Cells Exhibit a Profibrotic Phenotype in Patients with Systemic Sclerosis

Abstract

Loss of CD28 expression by CD8⁺ T cells occurs with age and during chronic inflammatory conditions. CD8⁺CD28^- T cells are a heterogeneous cell subpopulation whose function ranges from immunosuppressive to effector. Here we analyzed the role of CD8⁺CD28^- T cells in the pathogenesis of systemic sclerosis (SSc), a connective tissue disorder characterized by autoimmunity, vasculopathy, and extensive cutaneous and visceral fibrosis. We show that the frequency of CD8⁺CD28^- T cells is increased in the blood and affected skin of SSc patients, independent of patient age, and correlates with the extent of skin fibrosis. We found that most skin-tropic CD8⁺CD28^- T cells are resident in the skin lesions of patients in the early stage of the disease, exhibit an effector memory phenotype, and present a strong cytolytic activity ex vivo. Skin-resident and circulating SSc CD8⁺CD28^- T cells produce high levels of the profibrotic cytokine IL-13, which induces collagen production by normal and SSc dermal fibroblasts. Thus, our findings indicate that CD8⁺CD28^- T cells represent a pathogenic T-cell subset in SSc and likely play a critical role in the early stage of SSc skin disease.

Figure 1. Age-independent accumulation of CD8+CD28− T cells in the blood and skin of SSc patients correlates with the extent of skin fibrosis

Representative CD28 expression profiles by peripheral blood CD8+ T cells in controls or patients (a). Lymphocyte population was gated according to light scatter characteristics (FSC/SSC). CD8+ T cells were identified as CD8+CD3+ cells. Proportion of circulating CD8+CD28− T cells in patients (n=65) and controls (n=32) (b) or in dcSSc (n=28) and lcSSc (n=37) patients (c). Groups in (b) and (c) are shown as boxplots with the median indicated as horizontal line and whiskers from minimum to maximum. P values were determined by Wilcoxon rank sum test. (d) Representative cell surface phenotype of skin-resident CD8+ T cells in normal (NS, n=5) or SSc (n=5) skin. Freshly isolated skin cell suspensions were gated on lymphocyte scatter and CD8 positivity. Comparison of skin-homing receptor expression (E) and skin-resident marker expression (f) by CD8+CD28− T cells in blood and skin of SSc patients. Statistics by ANOVA followed by post hoc Tukey’s test.

Figure 2. Blood and skin-resident SSc CD8⁺CD28⁻ T cells exhibit effector and effector/memory phenotypes

(a–b) Cell surface CD28 expression by circulating CD8+ T-cell subsets. (a) Data are gated on lymphocyte scatter and CD8 positivity. CD8+-gated cells were separated into naïve (CD45RA+CD27+), CM (CM, CD45RA−CD27+), effector/memory (EM, CD45RA− CD27−), and effector (E, CD45RA+CD27−) based on CD45RA and CD27 expression. Each of these subsets was analyzed for CD28 co-expression. A representative example is depicted. (b) Frequency of CD8+CD28− T cells in total, effector, naïve, CM and EM CD8+ T cells from SSc patients (n=29) and NDs (n=18). Each symbol represents one patient or a control. The mean response is shown as a horizontal line. (c) Expression of CD45RA and CD27 by SSc skin-resident CD8+CD28− T cells. CD8+CD28− T cells were gated as described in Figure 1d. A representative example is shown (upper panel). Phenotype of skin-resident CD8+CD28− T cells from 5 patients. In panels (b and d), statistics by ANOVA followed by post-hoc Tukey’s test.

Figure 3. SSc CD8⁺CD28⁻ T cells in blood and skin express markers of cytotoxicity

(a) Perforin and GraB expression in total CD8+ and CD8+CD28+/− circulating T cells in a representative SSc patient. (b) Frequency of cells expressing perforin and GraB in each CD8 subset from 8 SSc patients. Each symbol represents one patient or a control. Statistics by ANOVA and post-hoc Tukey’s test. (c) Surface expression of CD107a and intracellular content of perforin were analyzed by flow cytometry in stimulated and unstimulated SSc CD8+CD28+/− T cells. A representative example is shown. (d) Expression of GraB by CD8+ T cells in the affected skin of patients with early dcSSc. Representative examples of H&E staining of NS and SSc skin (upper panel – scale bar = 40μm). Double-color immunohistochemistry for CD8 (red) and GraB (black) in normal (NS, n=4) and SSc skin (n=5), (200×, Inset 500×) (lower panel – scale bar = 20μm). A representative example is shown.

Figure 4. SSc CD8+CD28− T cells produce high levels of IL-13

Purified CD8+CD28+ and CD8+CD28− T-cell subsets from SSc patients and NDs were cultured in vitro for 5 days and then stimulated for 6 hours with PMA and ionomycin. Intracellular cytokine staining was used to determine the proportion of IL-13+ (n=9), IL-10+ (n=8), and IFNγ+ (n=8) cells. (a) A representative example from one SSc patient is shown. (b) Comparison of the mean percentage of cytokine-positive cells in each CD8+ T-cell subset from SSc patients and age-matched NDs. Each symbol represents one patient or a control. Statistics by ANOVA followed by post hoc Tukey’s test. (c) Cytokine production by skin-resident CD8+CD28− T cells was determined as indicated above. A representative example is shown (upper panels). Comparison of cytokine production by CD8+CD28− T cells in the blood and skin of (n=5) (lower panel). Statistics by ANOVA followed by post hoc Tukey’s test. (d) Representative examples of H&E staining of NS and SSc skin (upper panel – scale bar = 40μm). Representative example of double color immunofluorescence staining for CD8 and IL-13, 1000× (lower panels – scale bar = 1μm). Skin samples from 5 early dcSSc patients and 4 NDs were analyzed giving similar results. DAPI stains nuclei.

Figure 5. SSc CD8+CD28− T cells are pro-fibrotic and cytotoxic to dermal fibroblasts in vitro

(a–b) Normal dermal fibroblasts were stimulated for 24 hours with CD8+CD28− and CD8+CD28− T-cell subset supernatants from ND (n=7) or SSc patients (n=8) with or without pre-incubation with a neutralizing anti-IL-13 antibody. COL1A1 production was determined in culture supernatants (sp) or cell lysates (lys) by Western blot. A representative experiment out of seven independent experiments from different SSc patients is shown. SSc dermal fibroblasts were obtained from two early dcSSc patients (pt 1 and 2) and were cultured with ND (n=8) and SSc (n=10) CD8+CD28− T-cell supernatants. (c) Representative Western blot for COL1A1 protein expression in fibroblast culture media (sp) and densitometric quantification of secreted COL1A1 protein (d). SSc CD8+CD28− cells (ei) or fibroblasts (eii) alone as well as mixed at a 5:1 ratio (eiii, eiv) were incubated in media for 0 or 3 hours. Lymphocyte and fibroblast populations were gated according to light scatter characteristics (FSC/SSC). The horizontal lines in panels ei–eiv designate the CD8-negative target cell gate used for analysis of Annexin V positivity in panels ev–eviii. This experiment is representative of three similar experiments. Percent Annexin V-positive cells was determined by population gating (f). Statistics in b,d, and f by ANOVA followed by post hoc Tukey’s test.