2 ± 0.5), compared to preconversion (0.86 ± 0.2; P = 0.02) or those with rejection (0.9 ± 0.1; P = 0.01). For the liver biopsy cultures, there was significant variability in cell growth, precluding an appropriate pre- and postconversion statistical analysis (Supporting Table 3). Of the biopsies that had growth pre- and postconversion, two had decreases, six had no change, and three had increases in Treg percentages. In the others, two lost growth, but seven had new Treg growth after conversion, perhaps suggesting a trend toward increased intragraft Tregs after culture. However, given the variability of culture growth, these data are not fully conclusive. There has been recent interest in functional assays (Cylex ImmuKnow; Cylex,

Inc.) assessing nonspecific CD4 responses to distinguish alloreactive from immunosuppressed states.34 In the present study, mean ATP values did not change after SRL conversion (266 ± 132 to 274 ± 149 ng/mL; P = 0.15), suggesting that SRL conversion and Treg generation did not appear to lead to nonspecific over-immunosuppression. We have also recently reported on a novel in vitro immune monitoring assay in humans (the Treg MLR) demonstrating favorable immunoregulatory effects of SRL versus TAC when added directly to MLR cultures.21,

22 As another functional measure, we therefore questioned whether the addition of patient sera containing TAC versus SRL and, possibly, other buy JNK inhibitor resulting regulatory molecules might suppress lymphoproliferation and enhance Treg generation.21, 22 Both pre- and postconversion sera equally suppressed MLR lymphoproliferation (stimulation indices) below media controls (n = 13; P < 0.05) (Fig. 3A). However, TAC sera also suppressed CD4+CD25highFOXP3+ cell generation (n = 13; P < 0.01) (Fig. 3B), whereas SRL sera did not. Genomic, proteomic, and cytokine signatures may have the potential to predict tolerance.9, 35 In previous reports, transcripts for cell-proliferation arrest proteins and T- and NK-cell receptors have been identified Bupivacaine as putative LT tolerance signatures, correlating with increased circulating Tregs. We examined whether similar signatures of immunoregulation might be also observed after

SRL conversion. In the present study, several gene transcripts (n = 288; Supporting Table 4) and plasma proteins (n = 22; Table 3), many involved in immunoregulatory pathways, were found to be significantly different after SRL conversion (P < 0.005). Within the heat map displayed in Fig. 4 were up-regulated transcripts of FOXP3, CD25, and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), transforming growth factor beta (TGF-β), and CD4 and down-regulated transcripts of chemokine (C-C motif) receptor 3, apolipoprotein C4 (ApoC-IV) and collagen type IV (Supporting Table 4). Also, a number of proteins known to be involved in lymphocyte and DC activation (e.g., IL-3, IL-7, IL-13, macrophage inflammatory protein 1-alpha [MIP-1α], and CD40), lymphocyte trafficking (e.g.