Background/Purpose: Systemic sclerosis (SSc) is an auto-immune disorder  leading to destructive tissue fibrosis. Abnormal responses of  SSc T cells to lipid antigens and low molecular weight phospho-antigens have been observed but not extensively explored.  We determined  how exposure to cardiolipin, a lipid auto antigen, and  zoledronate (zol),  an  inducer of intracellular phospho-antigens (e.g isopentenyl pyrophosphate), affect  cytokine secretion and activation of SSc T cells.   We focused on   non-conventional innate gd T cells   since these:  1. Are   oligoclonally expanded in SSc  2. infiltrate damaged organs,  and 3. include  lipid reactive Vdelta (d)1⁺ and phospho-antigen reactive Vgamma (g)9⁺  gammadelta (gd) T cells.

Methods: Peripheral blood mononuclear cells (PBMC) were isolated by ficoll hypaque density centrifugation from 12 SSc patients (SScp) and 13 healthy controls (HC). Expression of CD25, a marker of activation, on Vg9⁺,  Vd1⁺  and  total CD3⁺ T cells in PBMC cultured for at 2X10⁶ cells/ml 96 hour in RPMI-1640  tissue culture medium supplemented with 10% fetal bovine serum, penicillin streptomycin, and glutamine, (TCM) containing 100 international units of  interleukin (IL)-2 and added reagents as indicated, was measured by flow cytometry – as were cell surface binding  of  CD1d tetramers,  and  effects of monoclonal antibody (mAb) mediated blockade of CD1d. Intracellular production of  anti fibrotic [interferon (IFN) g]  and pro fibrotic [IL- 4] cytokines was measured by flow cytometry after overnight incubation of PBMC in medium with added reagents.

Results: Secretion of IFNg (but not  IL-4) was suppressed in SSc relative to HC CD3⁺   and Vg9⁺ , but not Vd1⁺ T cells cultured in  TCM alone.  However, both Vd1⁺  and Vg9⁺  T cell IFNg  secretion in SSc were relatively reduced by adding cardiolipin (2.5 mg/ml),  which, by contrast, augmented IL-4⁺  SSc Vg9⁺  T cells. Moreover,   IFNg⁺  SSC Vg9⁺  T cells were relatively suppressed by zol (2mMol),  consistent with subset restricted specific effects of  cognate antigens.  Conversely,  %CD25⁺ CD3⁺ and  Vd1⁺ ,  and to a lesser degree Vg9+ T cells, were elevated significantly  in 96 h cultured SSc   PBMC compared to  HC.  Moreover, cardiolipin and zol respectively, significantly suppressed   SSc but not HC  %CD25⁺ Vg9⁺ and Vd1⁺  (but not   total %CD25⁺CD3⁺ )  T cells   suggesting   antigen driven cross inhibitory effects by these gd T cell subsets. These effects could be  partially reversed  by culture with  zol + cardiolipin , whereas this combination more strongly amplified  both CD25⁺ gd T cell subsets of HC.  Importantly,  SSc and HC Vd1⁺ T cells > Vd1^– T cells were highly reactive with lipid presenting CD1d – tetramers. Furthermore, a CD1d – blocking mAb decreased the zol + cardiolipin   enhancement of %CD25⁺Vd1⁺ T cells in SSc PBMC cultures

Conclusion:     SSc patient Vd1⁺ and Vg9⁺  T cell subsets exhibit disturbed and interactive responses to cardiolipin and   zol in vitro,  mediated in part by CD1d. Thus, the abnormal perception of cardiolipin and phospho-antigens by gd T cells may play a role  in the immune pathology in SSc,  notably that of pathological fibrosis, warranting further study.