1. Significant functional heterogeneity among KIR2DL1 alleles and a pivotal role of arginine245
by Rafijul Bari, Teresa Bell, Wai-Hang Leung, Queenie P. Vong, Wing Keung Chan, Neha Das Gupta, Martha Holladay, Barbara Rooney, and Wing Leung
Blood
Volume 114(25):
December 10, 2009
©2009 by American Society of Hematology

2. Generation of stable YT-Indy cell line expressing KIR2DL1 alleles.
Generation of stable YT-Indy cell line expressing KIR2DL1 alleles. YT-Indy cells were stably transfected with pcDNA3 vector containing various alleles of KIR2DL1 (*00301, *00401, *006, and *010) or with pcDNA3 vector alone as control. Expression of alleles of KIR2DL1 in sorted and stably transfected YT-Indy cells was confirmed to be similar as assessed by flow cytometry.
Rafijul Bari et al. Blood 2009;114:
©2009 by American Society of Hematology

3. KIR2DL1 alleles differentially inhibit the cytotoxicity, degranulation, and cytokine production of YT-Indy cells.
KIR2DL1 alleles differentially inhibit the cytotoxicity, degranulation, and cytokine production of YT-Indy cells. YT-Indy cells transfected with empty vector were used as control. (A) Specific lysis by YT-Indy cells expressing various alleles of KIR2DL1 (*00301, *00401, *006, and *010) were assessed against Cw6 at various ratios of effector to target cells using a BADTA release assay. The E/T ratios were 40:1, 20:1, 10:1, and 5:1. Data shown are average of 3 independent experiments. (B) Specific lysis of target Cw6 cells by YT-Indy transfectants was assessed at E/T = 20:1. Data shown are average of 9 independent experiments. ***P < .01. (C) Relative expression of CD107 at the surface of YT-Indy cells expressing various alleles of KIR2DL1 was detected by flow cytometry after challenge with target Cw6 cells. The results represent the mean of 3 independent experiments; *P = .22, **P < .05. Error bars represent SD. (D) Production of IFN-γ in YT-Indy transfectants was assessed after stimulation with target Cw6 cells. To separate effector from target cells, YT-Indy transfectants were first stained with CD45 antibody, followed by incubation with target cells. Cell mixtures were fixed, permeabilized, and stained with IFN-γ antibody. YT-Indy transfectants were gated based on CD45 (left panel), and IFN-γ production was assessed (right panels). Data shown are representative of 3 independent experiments.
Rafijul Bari et al. Blood 2009;114:
©2009 by American Society of Hematology

4. Functional differences among KIR2DL1 alleles observed in vitro are confirmed in vivo.
Functional differences among KIR2DL1 alleles observed in vitro are confirmed in vivo. YT-Indy cells expressing different alleles of KIR2DL1 were mixed with Cw6 at a 10:1 (E/T) ratio and injected subcutaneously into NOD-scid IL2Rγnull mice. A total of 30 mice, 5 mice for each group, were given injections. After the tumors reached 20% of body mass in the positive control group (mice injected with only target cells), the mice were humanely killed, dissected, and photographed. The image of a representative tumor from each group is shown. (A) Vector, *00301, *00401, *006, and *010 are tumors from mice given injections of YT-Indy transfected with empty vector, KIR2DL1*00301, KIR2DL1*00401, KIR2DL1*006, or KIR2DL1*010, together with expressing HLA-Cw6. Cw6 was a tumor from a mouse given only expressing HLA-Cw6. Tumor mass (B) and tumor volume (C) were also measured. The experiment was repeated 3 times. *P = .08, **P < .05, ***P < .01. Error bars represent SD.
Rafijul Bari et al. Blood 2009;114:
©2009 by American Society of Hematology

5. Arginine residue in the transmembrane domain of KIR2DL1 plays an important role in its inhibitory function.
Arginine residue in the transmembrane domain of KIR2DL1 plays an important role in its inhibitory function. Arginine in the transmembrane domain of KIR2DL1*010 was replaced with alanine. A stable YT-Indy cell line expressing the KIR2DL1*010 mutant was generated. YT-Indy transfected with vector was used as control. (A) Specific killing of YT-Indy expressing wild-type KIR2DL1*010 (*010), KIR2DL1*010 mutants (*010 R245A), and KIR2DL1*00401 (*00401) was assessed against Cw6 by BADTA release assay. *P = .62, **P < .05. (B) Relative expression of CD107 at the surface of YT-Indy cells expressing wild-type (*010) and mutated (*010 R245A) KIR2DL1*010 was assessed by CD107 mobilization assay. **P < .05. (C) Production of IFN-γ was assessed after YT-Indy cells expressing wild-type (top) and mutated (bottom) KIR2DL1*010 were stimulated with target Cw6 cells. Results are representative of 3 independent experiments. (D) YT-Indy cells expressing wild-type and mutated KIR2DL1*010 alleles were mixed with target Cw6 cells at a 10:1 (E/T) ratio and injected into mice subcutaneously. A total of 10 mice, 5 for each group, were given injections. After tumors reached 20% of the body mass in some mice, the mice were humanely killed, dissected, and photographed. The image of a representative tumor from each group is shown. The tumor shown at left is from a mouse that was given YT-Indy expressing wild-type KIR2DL1*010 (*010), and the tumor shown at right is from a mouse that was given YT-Indy cells expressing the mutated KIR2DL1*010 (*010 R245A) allele. Mass and volume of these tumors are shown at bottom. **P < .05, ***P < .01. The experiments were repeated 3 times. Error bars represent SD.
Rafijul Bari et al. Blood 2009;114:
©2009 by American Society of Hematology

6. KIR2DL1 alleles *00401 and *010 showed different signaling intensity and inhibition of lipid raft polarization at immune synapse upon stimulation with Cw6 cells.
KIR2DL1 alleles *00401 and *010 showed different signaling intensity and inhibition of lipid raft polarization at immune synapse upon stimulation with Cw6 cells. YT-Indy cells were retrovirally transduced with KIR2DL1 alleles *00401 and *010 fused with FLAG tag. Similarly expressing cells were collected by flow cytometry cell sorting. YT-Indy cells expressing FLAG-tagged KIR2DL1 alleles *00401 and *010 were stimulated with target Cw6 cells at a 1:2 (E/T) ratio for the indicated times. The stimulated cells were lysed and immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblotting (IB) with antibodies to (A) SHP-2 and (B) β-arrestin 2. Membranes were stripped and reprobed with FLAG antibody for loading control. (C) Positive and negative are representatives of lipid raft polarization and inhibition of lipid raft polarization, respectively. 2DL1 indicates cells stained with KIR2DL1 antibody, GM1 for lipid raft, and BF for conjugate formation. Calculated percentages of lipid raft polarization in YT-Indy/KIR2DL1* and YT-Indy/KIR2DL1* in the presence of Cw6 are shown in the lower panel. Images were obtained by a Nikon Eclipse E800 fluorescence microscope. The experiment was repeated 3 times. **P < .05. Error bars represent SD.
Rafijul Bari et al. Blood 2009;114:
©2009 by American Society of Hematology

7. Transmembrane arginine prevents down-regulation of cell-surface expression of KIR2DL1 after interaction with its ligand.
Transmembrane arginine prevents down-regulation of cell-surface expression of KIR2DL1 after interaction with its ligand. YT-Indy cells expressing KIR2DL1 alleles *00401 and *010 were coincubated with target Cw6 cells for the indicated times. Cell-surface expression of KIR2DL1 was detected using KIR2DL1 mAb. Left panel shows cell-surface expression of KIR2DL1*00401, and right panel shows KIR2DL1*010. Data shown are representative of 3 independent experiments.
Rafijul Bari et al. Blood 2009;114:
©2009 by American Society of Hematology