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Blood, Vol. 105, Issue 1, 233-240, January 1, 2005
NKG2D receptor–mediated NK cell function is regulated by inhibitory Ly49 receptors
Blood Regunathan et al.
105: 233
Supplemental materials for: Regunathan et al, Vol 105, Issue 1, 233-240
Files in this Data Supplement:
- Figure S1. Expression levels of ligands for NKG2D on LPS induced splenocytes and tumor cells (JPG, 44 KB)
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(A) Splenocytes from BALB.B and BALB/c mice express significant quantities of ligands for NKG2D, compared with cells from B6 or B10.D2 mice. Blast cells from the indicated strains were generated by inducing splenocytes with LPS (10µg/mL) for 48 hours. Live cells were separated by density gradient, and expression levels of ligands on blast cells were quantified using sNKG2D-Ig fusion protein. Soluble immunoglobulin Fc (Ig) was used to determine background staining levels. (B) Pro B line, C3 derived from (B10×129J)F1 or lymphoblast line, YAC-1 (A/Sn), were stained as described in panel A. (C) Levels of H60 expression in EL4, ST61, and ST63 are shown for comparison with splenocytes and tumor cells.
- Figure S2. Presentation of antigenic epitopes by H-2b MHC class I molecules are comparable between ST61 and ST63 sublines and parental EL4 cells (JPG, 21 KB)
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The functionality of H-2b MHC on EL4, ST61, and ST63 was characterized by analyzing their ability to present antigenic epitopes and to stimulate CD8+ T-cell hybridomas. BCZ103 is specific for the LTFNYRNL peptide derived from H60 and presented on H2-Kb. Since H60 was electroporated into EL4 and expressed at high (ST63) or low (ST61) levels, the activation of BCZ103 T cells by these target cells also varied depending on the H60 protein levels. EL4 endogenously processes and presents several viral epitopes. KZ30.6 is specific for the ANYDFITC peptide derived from the envelop protein of MMTV and presented on H2-Kb. BEZ48.3 is specific for SSWDFITV, which is derived from the envelope protein of MMLV and presented on H2-Kb (S.M., unpublished). Comparable levels of activation of either KZ30.6 or BEZ48.3 by EL4, ST61, and ST63 were observed, indicating that expression of H2-Kb was not altered. To analyze the expression levels and functionality of the H2-Db molecule, we exogenously added the KAPDNRDTL peptide derived from minor histocompatibility antigen H7 to target cells, and tested their ability to stimulate BCZ1644 T cell hybridoma (S.M., unpublished). Results indicated all the targets were able to activate BCZ1644 T cells, demonstrating that H2-Db function is also unaltered. T-cell responses specific for peptide/MHC were measured by the production of  -galactosidase (LacZ) activity in the T-cell hybrids (10×104) after overnight co-culturing with APC (5×104) in 96-well plates. LacZ activity was quantified using the substrate chlorophenol red -D-galactopyrannoside (CPRG). The conversion of CPRG to chlorophenol red was measured at 595 nm, using 655 nm as reference wavelength, in a 96-well microplate reader (BioRad, Richmond, CA). Data shown are the mean absorbance of triplicate cultures and are representative of at least 3 independent experiments.
- Figure S3: Expression pattern of inhibitory Ly49 receptors on NK cells derived from B6 and B10.D2 mice (JPG, 35 KB)
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IL2-activated NK cells were used on day 7 to quantify the percentage of Ly49 subsets using appropriate mAbs as described in "Materials and methods." Ly49 subsets from (A) B6- and (B) B10.D2-derived NK cells are shown. The percentages of respective subsets are indicated. The purity of the NK cell preparation was analyzed with anti-NK1.1 mAb (bottom panels).
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