Phosphorylated S6 ribosomal protein: a novel biomarker of antibody-mediated rejection in heart allografts.

We tested the hypothesis that phosphorylation of S6 ribosomal protein (S6RP), a downstream target of the PI3K/Akt/mTOR pathway, is a biomarker of antibody-mediated rejection (AMR) in heart allografts. Primary cultures of human aortic and microvascular endothelial cells (EC) were treated with anti-HLA class I and class II antibodies (Ab) and cell lysates were studied for phosphorylation of S6 ribosmal protein at Serine235/236 (p-S6RP). Treatment of cultured EC with anti-class I and class II Ab stimulated S6RP phosphorylation. Immunohistochemical techniques were used to detect the level of p-S6RP in endomyocardial biopsies (n = 131) from 46 heart transplant recipients and the results were correlated with histopathological diagnosis of rejection, C4d staining, production of posttransplant anti-HLA Ab and clinical outcome. Increased phosphorylation of S6RP in endomyocardial biopsies was significantly associated with the diagnosis of AMR (p < 0.0001). No significant association between acute cellular rejection (ACR) and p-S6RP was observed. C4d staining was positively associated with both AMR and p-S6RP. Posttransplant anti-HLA class II Ab production was also significantly associated with a positive p-S6RP status in cardiac biopsies. These results indicate that p-S6RP is a useful biomarker for the diagnosis of AMR.

Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors.

HLA-F is a human non-classical MHC molecule. Recombinant HLA-F heavy chain was refolded with 2-microglobulin to form a stable complex. This complex was used as an immunogen to produce a highly specific, high-affinity monoclonal antibody (FG1) that was used to study directly the cellular biology and tissue distribution of HLA-F. HLA-F has a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA-F could be immunoprecipitated from B cell lines and from HUT-78, a T cell line. HLA-F binds TAP, but unlike the classical human class I molecules, was undetected at the cell surface. HLA-F tetramers stain peripheral blood monocytes and B cells. HLA-F tetramer binding could be conferred on non-binding cells by transfection with the inhibitory receptors ILT2 and ILT4. Surface plasmon resonance studies demonstrated a direct molecular interaction of HLA-F with ILT2 and ILT4. These results, together with structural predictions based on the sequence of HLA-F, suggest that HLA-F may be a peptide binding molecule and may reach the cell surface under favorable conditions, which may include the presence of specific peptide or peptides. At the cell surface it would be capable of interacting with LIR1 (ILT2) and LIR2 (ILT4) receptors and so altering the activation threshold of immune effector cells.