a 2,5 IL4 (4h) in Ja18-/- mice b 2,5 IFN-γ (16h) in Ja18-/- mice 2 2 ng/ml 1,5 ng/ml 1,5 1 1,5,5 4ClPh NC PyrC 4ClPh NC PyrC upplemental Figure 1
a b c PyrC-α-GalCer NU-α-GalCer α-galcer CD4 (PE) MFI CD4 (PE) 6 4 2 %CD4+ cells 3 2 1 CD8 (FITC) MFI CD8 (PE) 15 1 5 %CD8+ cells 6 4 2 ICOSL (PE) MFI ICOSL (PE) 4 2 %ICOSL+ cells 15 1 5 OX4L (PE) MFI OX4L (PE) 1 5 %OX4L+ cells 3 2 1 CD86 (FITC) Supplemental Figure 2 MFI CD86 (PE) 8 6 4 2 PyrC NU α-galcer DMSO %CD86+ cells 2 1 PyrC NU α-galcer DMSO
Table I. Data collection and refinement statistics mtcr-pyrc-α-galcermcd1d mtcr-4clphc-α-galcermcd1d Data collection Resolution range (Å) a 59.33-2.8 (2.95-2.8) 5.-3. (3.11-3.) Completeness (%) a 98.1 (98.8) 98. (97.1) Number of unique reflections 28,24 22,997 Redundancy 4.1 4. R sym (%) 1.7 (45.8) 15.4 (71.8) I/σ a 9.9 (3.6) 9.9 (2.) Refinement statistics Number of reflections (F>) 26,546 21,794 Maximum resolution (Å) 2.8 3. R cryst (%) 19. (25.7) 18.6 (25.9) R free (%) 23.8 (32.8) 22.8 (28.1) Number of atoms 6594 6382 Protein 6342 6233 Glycolipid 79 7 N-linked carbohydrate 7 66 Solvent molecules (waters/dmso) 13 13 Ramachandran statistics (%) Favored 96.3 96.2 Outliers.25.13 R.m.s.d. from ideal geometry Bond length (Å).9.9 Bond angles ( ) 1.176 1.189 Average B values (Å 2 ) Protein 4.7 42.6 Glycolipid 35.3 53.9 Water molecules 33.2 45.2 Carbohydrates 53.9 59.5 a Number in parentheses refer to the highest resolution shell.
Glycolipid 4ClPhC-α-GalCer PyrC-α-GalCer source atoms CD1d target atoms Å Å Ala12 CB CAZ 3,97 ------ Ala12 CB CAI 3,98 ------ Asp153 CG O2A 3,29 3,45 Asp153 CG O3A 3,75 3,74 Asp153 OD1 O2A 3.44 * 3.51 * Asp153 OD1 C3A 3,47 3.51 * Asp153 OD1 O3A 2.89 *** 3.51 * Asp153 OD2 O1A 3.86 * 3.99 * Asp153 OD2 C2A 3,74 3,87 Asp153 OD2 O2A 2.69 *** 2.86 *** Asp153 OD2 C3A 3,73 3,77 Asp153 OD2 O3A 3.93 * 3.91 * Asp8 CG O3 3,43 3,41 Asp8 CG O4 3,96 3,79 Asp8 OD1 O3 ------ 3.92 * Asp8 OD2 C5 3,91 3,95 Asp8 OD2 C3 3,41 3,41 Asp8 OD2 O3 2.41 *** 2.44 *** Asp8 OD2 C4 3,54 3,47 Asp8 OD2 O4 2.8 *** 2.64 *** Cys12 CB CAM 3.91 3,65 Cys12 SG CAY 3,65 ------ Cys12 SG CAX ------ 3,82 Cys168 CB CAX 3,89 ------ Gln14 CB CAK 3,98 ------ Gln14 CB CAL 3,95 ------ Gln14 OE1 CAK 3,42 3,81 Gln14 OE1 CAL 3,27 3,55 Gln14 OE1 CAJ ------ 3,74 Gly155 N O3A 3.68 * 3.93 * Gly155 CA C3A 3,97 ------ Gly155 CA O3A 3,6 3,4 Gly155 C C3A 3,87 ------ Gly155 C O3A 3,49 3,89 Gly155 C C3A 3,87 ------ Gly155 C O3A 3,6 3,89 Ile47 CD1 CAQ 3,81 3,7 Ile81 CD1 C14 3,99 3,77 Leu1 CD2 CAJ ------ 3,94
Leu1 CD2 CAI 3,51 ------ Leu1 CD2 CAJ ------ 3,94 Leu1 CD2 CAI ------ 3,74 Leu1 CD2 CAH 3,82 ------ Leu116 CD1 CAG ------ 3,76 Leu143 CD2 C15 ------ 3,79 Leu15 CD1 C11 3,77 3,79 Leu15 CD1 C13 ------ 3,98 Leu15 CD2 O4 3,98 3,78 Leu163 CD1 CAE 3,83 4 Leu66 CD2 CAU 3,84 3,86 Leu84 CB C18 3,85 ------ Met162 SD CL1 3,84 ------ Met69 CG CCL 3,76 ------ Phe1 CZ CAT 3,89 3,85 Phe1 CE2 CAV 3,83 ------ Phe12 CE1 C17 3,94 3,7 Phe171 CE2 CAT 3,92 3,69 Phe171 CD2 CAV 3,91 ------ Phe171 CE2 CAT ------ 3,69 Phe171 CD2 CAV 3,91 ------ Phe171 CD2 CAU 3,85 ------ Phe171 CD2 CAT ------ 3,97 Phe7 CD1 CAN 3,93 ------ Phe7 CE1 CAN 3,92 ------ Phe7 CE2 CAS ------ 3,99 Phe77 CD2 C6 ------ 3,97 Ser28 OG CAM 4 ------ Ser28 CB CAN ------ 3,78 Ser28 OG CAN ------ 3,85 Ser76 CB C2 3,78 3,76 Ser76 CB O3 3,18 3,45 Ser76 CB C1 3,8 3,64 Ser76 OG C1 3,87 3,6 Ser76 C O3 3,93 ------ Ser76 O O3 3.97 * ------ Thr156 N C3A 3,73 3,78 Thr156 N O3A 3.56 * 3.7 * Thr156 CB N2 3,9 3,85 Thr156 OG1 CAB 3,64 3,71 Thr156 OG1 CAA 3,63 3,66 Thr156 OG1 N2 2.75 *** 2.75 *** Thr156 OG1 C2 3,59 3,58
Thr156 OG1 C3 3,58 3,49 Thr156 OG1 C4 3,73 3,82 Thr156 OG1 O1A 3.41 * 3.33 * Thr156 OG1 C3A 3,65 3,72 Thr156 CG2 CAB 3,84 3,86 Thr156 CG2 N2 3,9 3,85 Thr159 OG1 NCJ 3.7 * ------ Thr159 OG1 CCU 3,73 ------ Thr159 OG1 CCT 3,29 ------ Thr159 CG2 CAC 3,99 ------ Trp133 CH2 C9 3,78 3,65 Trp133 CH2 C1 3,89 3,99 Trp142 CZ3 C18 3,97 ------ Trp63 CZ3 CAS 3,98 Tyr73 CA OAA 3,62 3,65 Tyr73 CB OAA 3,54 3,68 Tyr73 CD1 CAE 3,71 ------ Tyr73 CD1 CAF ------ 3,82 Tyr73 CD1 C5 3,99 3,96 Tyr73 CD1 CAD 3,68 3,72 Tyr73 CE1 C7 3,91 3,83 Tyr73 CE1 CAF ------ 3,66 Tyr73 CE1 C6 ------ 3,92 Tyr73 CZ CAH 3,66 ------ Tyr73 CZ CAJ ------ 3,89 Tyr73 OH CAH 3,79 ------ Tyr73 OH CAJ ------ 3,81 Tyr73 CE2 CAH 3,86 ------ Tyr73 CE2 CAJ ------ 3,69 Val118 CG1 C12 3,79 3,92 Val118 CG2 C1 3,99 Val16 CG2 CAD 3,87 3,78 Val16 CG2 CAE ------ 3,85 Val3 CG2 CAP ------ 3,7
atoms Glycolipid 4ClPhC-α-GalCer PyrC-α-GalCer TCR target atoms Å Å D94 O C1A 3,76 3,95 G96 N C2A 3,71 3,88 G96 N O2A 2.8*** 2.96*** G96 N O3A ----- 3.89* G96 CA O2A 3,85 ----- N3 CA O4A ----- 3,97 N3 CG O3A 3,89 3,66 N3 ND2 C2A 3,77 3,79 N3 ND2 C3A 3,73 3,61 N3 ND2 O3A 2.7 *** 2.48*** N3 ND2 O4A 3.77* 3.67* P28 CB O6A 3,36 3,3 P28 CG C1A 3,81 3,48 P28 CG O6A 3,31 3,24 Q52 CG N ----- 3,87 Q52 CD CCQ ----- 3,94 Q52 CD N ----- 3,44 Q52 CD CCS ----- 3,39 Q52 CD CCT ----- 3,84 Q52 OE1 CCU ----- 3,65 Q52 OE1 CCL ----- 3,55 Q52 OE1 CCQ ----- 3,42 Q52 OE1 N ----- 3.38* Q52 OE1 CCS ----- 3,47 Q52 OE1 CCT ----- 3,6 Q52 NE2 N ----- 3.89* Q52 NE2 CCS ----- 3,35 Q52 NE2 CCT ----- 3,69 R95 CA C2A 3,96 ----- R95 CA O2A 3,35 3,26 R95 CB O2A 3,85 3,89 R95 CD O3 3,61 3,39 R95 CD O1A 3,76 ----- R95 CD O2A 3,76 3,54 R95 CD C1A 3,95 ----- R95 NE O3 3.3*** 2.87*** R95 CZ O3 3,74 3,57 R95 NH1 O3 3.97* 3.88* R95 C O2A 3,52 3,58
Atomic contacts determined using the CCP4i implementation of Contact and cut-off 4. Å. *** indicates the strong possibly of a hydrogen bond at this contact (distance < 3.3 Å). * indicates a weak possibility of a hydrogen bond at this contact (distance > 3.3 Å). Blank indicates that the CCP4i implementation of Contact program considers there is no possibility of a hydrogen bond. Van der waals interactions defined as non-hydrogen bond contact distances of 4. Å or less.
TCR CD1d agalcer NU-aGalCerNP255 NP263 Arg11 NH1 Glu83 OE1 3,96 * 3,47 * Arg11 NH1 Glu83 OE2 3,18 *** Arg11 NH2 Glu83 OE2 3,92 * Arg11 NH2 Glu83 OE1 3,13 *** 3,4 * Arg11 N Arg79 NH2 3,42 * 3,57 * 3,58 * Arg11 NH2 Lys86 NZ 3,99 * Arg95 NE Asp8 OD1 2,9 *** 2,69 *** 2,78 *** 2,71 *** Arg95 NE Asp8 OD2 3,47 * 3,1 *** 3,3 *** Arg95 NH1 Asp8 OD1 3,1 *** 2,96 *** Arg95 NH1 Asp8 OD2 3,79 * Arg95 NH1 Asp8 OD1 2,81 *** Arg95 NH1 Ser76 O 3,9 * 3,32 * 3,2 *** 3,1 *** Arg95 NH1 Ser76 OG 3,18 *** 3,59 * Arg95 NH2 Ser76 OG 3,85 * Arg95 NH2 Arg79 NH1 3,6 * 3,58 * Arg95 NH1 Arg79 NH1 3,45 * Arg95 NH1 Arg79 NE 3,89 * 3,65 * 3,81 * Arg95 NH1 Arg79 NH2 3,97 * 3,97 * Arg95 NH2 Arg79 NH2 3,38 * 3,48 * Asp94 OD1 Arg79 NH1 3,66 * 3,8 *** 3,9 * Asp94 OD1 Arg79 NH2 3,38 * 3,49 * 3,26 *** 2,77 *** Asp94 OD2 Arg79 NH1 2,79 *** 3,84 * 2,75 *** 2,98 *** Asp94 OD2 Arg79 NH2 3,86 * 2,76 *** 3,32 * 3,36 * Gly96 N Asp153 OD2 3,19 *** Gly96 O Asp153 N 3,6 * 3,91 * 3,74 * 3,59 * Gly96 N Asp153 OD1 3,68 * 3,96 * Gly96 O Ala152 O 2,85 *** 3,19 *** 3,31 * 3,36 * Leu99 O Arg79 NH2 2,85 *** 3,3 * 3,39 * Leu99 O Arg79 NH1 3,25 *** Ser97 OG Val149 O 3,97 * 3,93 *
Supplemental Figure 1 Additional cytokine data. (a+b) No detectable cytokine secretion at the expected peak time points (4 hours for IL-4 and 16 hours for IFN-γ) after injection of 5 µg glycolipid (i.p.) in mice lacking inkt (Jα18-/-) cells. Experiment was performed once, 3-4 mice per group. Supplemental Figure 2 6 -OH altered glycolipids induce a different costimulatory landscape on spleen dendritic cells. Dendritic cells from spleens of C57/BL6 mice that were injected with 6 glycolipid pulsed BMDCs were stained with antibodies against CD4, OX4L, ICOSL, CD8 and CD86. (a) Costimulatory molecule expression was analyzed by gating on CD11c + CD11b + splenocytes. Filled histograms represent the DMSO control. (b) Graphs indicate the mean with s.e.m. of MFI for each costimulatory molecule. (c) Graphs indicate the mean with s.e.m. of the percentage of CD11c + CD11b + double positive splenocytes that are positive for each costimulatory molecule. Supplemental Table 1 Data collection and refinement statistics Supplemental Table 2 Molecular contacts