The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades

The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades. of type I NKT cells in real time binding assays with Asiaticoside high affinity, only a few activate type I NKT cells in or experiments. The differences in biological responses are likely a result of different pharmacokinetic properties of each lipid, which carry modifications at different parts of the molecule. Our results indicate a need to perform a variety of assays to ascertain the therapeutic potential of type I NKT cell GSL activators. within 90 min. Since this initial discovery, many glycolipids have been studied that sway the response of the immune system predominantly toward either a Th1 or a Th2 response (12). One of the earliest Th1 skewing lipids studied to date is ? and ? difference electron density maps using COOT (33). The GSLs were built into 2? map and refined using REFMAC (34). Refmac geometric libraries for the glycolipids were obtained using the PRODRG server (35). Data collection and refinement statistics are summarized in Table 1. TABLE 1 Refinement statistics for the CD1d-GSL-TCR complexes NA means not available. (?)78.6, 149.7, 101.479.4, 150.4, 102.579.6, 191.9, 151.979.1, 191.4, 151.379.4, 150.3, 100.8????, , ()90, 96.5,9090, 96.4, 9090, 90, 9090, 90, 9090, 96.2, 90????Resolution range (?) (outer shell)40C3.2 (3.31C3.2)40C3.1 (3.15C3.1)66.21C2.60 (2.71C2.60)95.7C2.9 (3.06C2.9)500C3.05 (3.12C3.05)????No. of reflections38,28642,69434,68125,09244,418????(37), and is related to the previously crystallized SMC124 lipid (16). The sugar headgroup and fatty acid chain; = 247 86 nm) and GCK152 (= 197 22 nm) show the lowest V14V8.2 TCR affinity. This is similar to the affinity reported for the parent -C-GalCer (= Asiaticoside 247 nm) (36) but is 10-fold weaker than GalCer, which in our hands ranges in affinity from 11 to 25 nm (18, 29). Of note, the binding affinity is still high compared with mouse TCR affinities for MHC-presented peptides, which most often are in the micromolar range (39, 40). The higher affinity group is composed of the NC-GC (= 37.1 14.10 nm), similar to NU-GC (36), EF77 (44.7 0.4 nm), and 7DW8-5 (94 2.8 nm). The division into lower and higher affinity groups was not maintained in the SPR evaluation using the human being V24V11 TCR and human being Compact disc1d (Fig. 2values of 6.85 2.6 and 3.4 2.71 m, respectively. The additional lipids had identical affinity to GalCer, which inside our hands runs from 1 to 3 m. GCK127 (1.45 0.05 m) and NC-GC (1.45 0.35 m) were virtually identical, and 7DW8-5 led to the best TCR affinity (1.13 0.9 m). We mentioned that in the mouse studies the off-rate for the type I NKT cell TCR for both GCK127 and GCK152 (= 1.28 0.0014 10?2 and 1.66 0.0016 10?2 s?1, respectively) is 10 times faster than the other ligands, including GalCer (= 2.2 0.52 10?3 s?1) (data not shown), but is similar to -C-GalCer (36). Therefore, we assume that the GCK glycolipids were not able to induce the closure of the roof over the CD1d F pocket. As reported previously, some GSLs like GalCer induce RHOC the formation of the F roof closure prior to TCR docking by orienting CD1d side chains at Leu-84, Val-149, and Leu-150 to an optimal conformation for engagement by the TCR CDR3 residue Leu-99. The pre-formed F roof closure has been correlated with a slower off-rate of the type I NKT cell TCR (41). In the human SPR studies, we noted that the off-rates for all the GSLs were similar, likely due to the inability of human CD1d to pre-form the closed F roof, as the Leu-84 of mouse CD1d is altered to Phe-84 in human CD1d, and a fully closed F roof has not been observed in the hCD1d-GalCer structure (42). Open in a separate window FIGURE 2. Real time TCR binding kinetics. Binding of refolded mouse V14V8.2 TCR (showing the binding response of increasing concentrations of TCR (GCK152; GCK127; NC-GC; 7DW8-5; EF77. CD1d is shown in and 2M in and TCR chain in NC-GC; EF77; 7DW8-5; GCK152; GCK127. dual binding motif for acyl chain of 7DW8-5. 2electron density is drawn as a around the glycolipid (GCK127; GCK152; NC-GC; 7DW8-5; EF77; overlay of NC-GC (and cell-based assay in which an A20 B cell lymphoma cell line transfected with CD1d was pulsed with Asiaticoside 100 ng of the indicated GSL and co-cultured with a V14V8.2 NKT cell hybridoma (1.2). TCR engagement was measured after 24 h through IL-2 production in serum measured with a sandwich ELISA. Data represent samples in triplicate and are representative of three independent experiments. human type I NKT cells were activated by human.