Background T cells (TC) expressing Vδ1+ TC receptors recognize lipids presented by CD1d (1). Oligoclonal Vδ1+ TC infiltrate skin and internal organs and are activated in the peripheral blood (PB) in systemic sclerosis (SSc) suggesting lipids as pathogenic immunogens (2,3).
Objectives To determine effects and molecular pathways of the auto antigenic lipid – cardiolipin- in SSc and healthy controls (HC).
Methods Mononuclear cells (MC) were isolated from PB of 12 SSc patients and 8 HC by density centrifugation and cultured in IL2C (RPMI-1640 with 10% fetal bovine serum + 100 international units of interleukin-2), CARDC or OCHC [IL2C+cardiolipin 2.5μg/ml or OCH 50ng/ml (analogue of α-galactosyl ceramide) respectively]. Zoledronate (zol, 2μM), a Vγ9+ γδTC stimulant that induces their CD1d expression, was added to parallel cultures (labeled ILCzol etc.) (4,5). Flow-cytometry (FC) of 4–5 day cultures after staining with monoclonal antibodies (mAb) or CD1d tetramers to detect %CD25+ cells (%CD25+) or tetramer+ cells, was performed.Means were compared by two tailed student t-test; p<0.05 was considered significant.
Results Vδ1+ and Vγ9+TC consisted 11.85±2.0% and 14.7±3.5% vs 10.42±2.4% and 7.08±1.2% of the CD3+ TC in SSc and HC IL-2C respectively (values not significantly different). In HC cultures, %CD25+ was non significantly different in IL2C, OCHC and CARDC for all TC subsets.Furthermore, IL2Czol increased %CD25+ on all TC subsets relative to IL2C, which was further amplified on Vδ1+ TC in CARDCzol (33.16±7.0% to 56.93±6.3% p<0.007). In SSc, in contrast OCHC and CARDC enhanced %CD25+Vδ1+TC whereas CARDC %CD25+Vγ9+TC was suppressed (76.06±8.7% vs 66.9±10.9%, p<0.036) relative to IL2C. In IL2Czol, however, %CD25+Vγ9+TC increased to 73.93±8.12%. In contrast, IL2Czol decreased SSc %CD25+Vδ1+TC (69.58±8.29% to 55.02±10.54%, p<0.028), but this decrease was partially abrogated in CARDCzol. Both SSc and HC IL2C Vδ1+TC were highly reactive with native and OCH-bound CD1d tetramers (25–70%, n=5 and 50–54% n=2 respectively), but binding was suppressed in SSc samples by zol. In addition, blocking mAb against CD1d abolished the increased expression of CD25 in SSc CARDCzol Vδ1+ TC (from +27±3% to -8.9±2.3%, n=5 p<0.0251) but not in HC (n=2).
Conclusions Cardiolipin induced suppression of CD25 expression on Vγ9+ TC in SSc, which could be reversed by zol. On the other hand cardiolipin reversed SSc- specific zol induced suppression of CD25 expression on Vδ1+TC in a CD1d dependent-manner. These results suggest that cardiolipin may suppress Vγ9+ TC while augmenting Vδ1+TC activation in SSc, at least in part via CD1d mediated interactions.
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Acknowledgement Supported by a donation to I.B. via the Sheba Research Authority
Disclosure of Interest None declared