gms | German Medical Science

Introduction: CD8+ cytotoxic T cells are involved in the pathogenesis of systemic sclerosis (SSc) [1]. Cytomegalovirus (CMV)-specific CD8+ T cells that adhere to the endothelium by means of activated integrins and drive inflammation, may contribute to vasculopathy in SSc. CMV-specific CD8+ T cells can be assessed by flow cytometric detection of integrin activation using fluorochrome-conjugated integrin ligands, called multimeric intercellular adhesion molecule-1 (mICAM-1)-conjugates [2]. Here, we used this rapid method to detect and compare functionally activated T cells and, in particular, CMV-specific CD8+ T cells in SSc-patients and healthy controls (HC).

Methods: Frequencies and phenotype (CD27, CD28) of CD3+CD8+ total and CMV-specific T cells were examined in whole blood of SSc patients (n=19) compared to HC (n=9). Whole blood was stimulated with staphylococcal enterotoxin B (SEB, 4 µg/ml, 8 min, positive control), CMV-NLV peptide (4 µg/ml, 8 min) or CMV-A2/NLV fluorochrome-conjugated dextramers (directly before mICAM-1 staining). Thereafter, activation and enumeration of total or HLA-typed CMV-specific CD3+ CD8+ T cell subsets was assessed by flow cytometry using mICAM-1 conjugates.

Results: Following stimulation with SEB, a significant increase of mICAM-1 binding and thus in the number of activated T cells was observed in comparison to no stimulation for both SSc patients (p<0.0001) and HC (p< 0.001). Overall, the activation of CD8+ T cells was more pronounced in SSc. Following short-term stimulation with the A2/NLV fluorochrome-conjugated dextramer, the number of activated CMV-specific CD8+ T cells was elevated in SSc compared to no stimulation (p<0.01). The subsets of CMV-specific CD8+ T cells displayed predominantly a late differentiated pattern of CD27 and CD28 expression. In terms of short-term peptide stimulation, our findings showed that both the unconjugated and the fluorochrome-conjugated dextramer CMV peptide are sufficient to induce activation of CMV peptide-specific human CD8+ T cells.

Conclusion: The mICAM-1 assay is suitable to detect functional, CMV-specific T cells in peripheral blood of SSc patients and HC. Compared to other activation assays, the mICAM-1 assay has the advantage that initiation of relevant signals by the cell requires only a short incubation time of minutes and low amounts of peripheral blood.

Disclosures: The authors have nothing to declare.