(A) Schematic comparison of the regions encoding RHVP pQa-1 and mouse H2-Qa-1. The genomic sequence is represented by a black line (solid for mouse and dotted for virus), and genome coordinates are …
(A) Schematic depiction of RHVP ORFs and the RNA splicing events. The genomic sequence is represented by a dotted black line. Previously annotated ORFs are represented by pattern-filled block …
The pQa-1 ectodomain amino acid sequences are compared to closely related MHC-I proteins using ESPript3 (Robert and Gouet, 2014). The secondary structure of mouse Qa-1 is depicted above the …
(A) Schematic depiction of the pQa-1 expression constructs used in the study. The C-terminal 26aa containing predicted GPI attachment site (marked by red star) is shown under the C-terminus of the …
(A) Following incubation of MEF-pQa-1cells with 100 μM Qdm (AMAPRTLLL) or OVA257-264 (SIINFEKL) peptide at 37°C for 4 hr, surface pQa-1 and H2-Kb were analyzed by flow cytometry for comparison. (B) …
(A) MEF-pQa-1 (upper panel) or MEF-Qa-1 (lower panel) cells were transduced with vector, pK3 or pK3 RING mutant (pK3 RM). Surface pQa-1/Qa-1 levels were determined by anti-HA/anti-Qa-1 staining. …
(A) Spleen lymphocytes isolated from wildtype C57BL/6 or NKG2A-/- knockout mice were stained with PE-labeled pQa-1-dtSCT tetramer (PE-pQa-1 Tet), or PE-labeled streptavidin (PE-SA) as negative …
(A) Schematic depiction of pQa-1-SCT. A disulfide-trapped version (dtSCT) was made by substitutions of Linker1 G2C and pQa-1 Y84C. Surface expression of pQa-1-SCT on CHO cells was compared with …
(A) Schematic depiction of the constructs for expression of CD94, CD94/NKG2C or CD94/NKG2A. (B) Mouse T lymphoma cell line BWZ.36 expressing CD94 alone indicated by GFP could not be detected by …
C57BL/6 splenocytes were co-cultured with CHO cells expressing the indicated constructs and the NK cells were subsequently analyzed by flow cytometry (A–F). The ratio of IFNγ production between …
(A) Cells used in Figure 6A–F. Surface pQa-1 and pQa-1-SCT but not Qa-1 molecules were susceptible to PI-PLC treatment (0.5 U/ml for 1 hr). (B) Cells used in Figure 6G. (C) Cells used in Figure 6H. …
(A) The GFP+ target cells (RMA cells expressing pK3 or pK3 RM by IRES-GFP vector or co-expressing pK3 and pQa-1) were mixed with the effectors (NK cells isolated from wildtype C57BL/6 or NKG2A-/- …
Because pK3 or pK3 RM was transduced into RMA cells using an IRES.GFP vector, GFP signal in the cells served as indicator of pK3 or pK3 RM expression. These cells were used as target of syngeneic NK …
NK cytotoxicity assay was conducted as described in Figure 7 after treatment of RMA cells co-expressing pK3 and pQa-1 (RMA-pK3 +pQa-1) with 0.5 U/ml PI-PLC at 37°C for 1 hr. PI-PLC treatment led to …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (M. musculus) | Klrc1-/- | PMID: 26680205 | Dr. Marco Colonna laboratory (Washington University School of Medicine) | |
Strain, strain background (M. musculus) | C57BL/6NCr | Charles River Laboratories | Strain code: 556 | |
Cell line (M. musculus) | B6/WT-3 | Dr. Stephen R Jennings (Louisiana State University Health Sciences Center); PMID: 6316651 | an SV-40 transformed, C57BL/6 murine embryo fibroblasts line | |
Cell line (M. musculus) | BWZ.36 | Dr. Nilabh Shastri (University of California, Berkeley); PMID:8186188 | a mouse T lymphoma line | |
Cell line (M. musculus) | FT1- (Tap1-/-) | Dr. Michael Edidin (Johns Hopkin University); PMID:10485658 | a fibroblast line derived from Tap1 knockout mice | |
Cell line (M. musculus) | 3KO (H2-K-/-, H2-D-/-, B2m-/- murine embryo fibroblast line) | Dr. Ted H. Hansen laboratory; PMID:12530981 | 3KO mice were derived by breeding H2-Kb/-Db doubly deficient mice (PMID:10229092) with B2m-/- mice (Jackson Laboratories) | |
Cell line (Homo sapiens) | T2 (174 x CEM.T2) | American Type Culture Collection | ATCC CRL-1992, RRID:CVCL_2211 | a TAP deficient T-B lymphoblast hybrid (PMID: 3522223) |
Cell line (M. musculus) | RMA | PMID: 3877776 | RRID:CVCL_J385 | |
Cell line (M. musculus) | B16-F10 | American Type Culture Collection | ATCC CRL-6475, RRID:CVCL_0159 | |
Cell line (Cricetulus griseus) | CHO | Dr. Pamela Stanley laboratory (Albert Einstein College of Medicine) | ||
Cell line (Homo sapiens) | 293F (FreeStyle 293 F Cells) | Thermo Fisher Scientific | R79007, RRID:CVCL_D603 | |
Cell line (Homo sapiens) | 293T (HEK 293T) | American Type Culture Collection | ATCC CRL-3216, RRID:CVCL_0063 | |
Antibody | FITC labeled anti-mouse NKG2A/C/E | eBioscience | cat# 11-5896-82 | clone: 20D5 (1:100) |
Antibody | FITC labeled isotype control (RatIgG2a) | eBioscience | cat# 11-4321-85 | clone: eBR2a (1:100) |
Antibody | eFluor 660 labeled anti-mouse CD107a | eBioscience | cat# 50-1071-82 | clone: eBio1D4B (1:1000 in culture) |
Antibody | APC-eFluor labeled anti-mouse CD19 | eBioscience | cat# 47-0193-82 | clone: eBio 1D3 (1:100) |
Antibody | PE-Cy7 labeled anti-mouse CD19 | eBioscience | cat# 25-0193-82 | clone: eBio1D3 (1:200) |
Antibody | APC-eFluor 780 labled anti-mouse CD3e | eBioscience | cat# 47-0031-82 | clone: 145–2 C11 (1:100) |
Antibody | Pacific Blue labeled anti-mouse CD3e | Biolegend | cat# 100214 | clone: 17A2 (1:100) |
Antibody | PE-eFluor 610 labeled anti-mouse CD8a | eBioscience | cat# 61-0081-80 | clone: 53–6.7 (1:200) |
Antibody | PE labeled anti-mouse CD94 | eBioscience | cat# 12-0941-82 | clone: 18D3 (1:100) |
Antibody | mouse anti-HA tag | Covance | cat# MMS-101P | clone: 16B12 (1:500) |
Antibody | eFluor 450 labeled anti-mouse IFNg | eBioscience | cat# 48-7311-82 | clone: XMG1.2 (1:100) |
Antibody | PE-Cy7 labeled anti-mouse NK1.1 | eBioscience | cat# 25-5941-82 | clone: PK136 (1:100) |
Antibody | PerCp Cy5.5 labeled anti-mouse NK1.1 | eBioscience | cat# 45-5941-82 | clone: PK136 (1:200) |
Antibody | PE-labeled anti-mouse Thy1.1 | BD PharMingen | cat# 551401 | clone: OX-7 (1:150) |
Antibody | biotin labeled anti-mouse Qa-1 | BD PharMingen | cat# 559829 | clone: 6A8.6F10.1A6 (1:200) |
Recombinant DNA reagent | pMIG_pQa-1-HA | current study | Schematic depiction is shown in Figure 2A | |
Recombinant DNA reagent | pMIN_pQa-1-HA | current study | pMSCV.IRES.neo (pMIN) was described previously (PMID:15280476) | |
Recombinant DNA reagent | pMIG_pK3 | PMID:22403403 | Dr. Ted H Hansen laboratory | |
Recombinant DNA reagent | pMIN_Qa-1 | current study | DNA sequence encoding Qa-1b (NM_034528) was subcloned into pMIN vector | |
Recombinant DNA reagent | pMIG_pQa-1-SCT | current study | Schematic depiction is shown in Figure 5—figure supplement 1 | |
Recombinant DNA reagent | pFM1.2R_pQa- 1-SCT-BirA | current study | Described in the DAN constructs section in Materials and methods | |
Peptide, recombinant protein | pQa-1-SCT | current study | Described in the Production of pQa-1-SCT Proteins and Tetramers section in Materials and methods | |
Commercial assay or kit | EasySep mouse NK cell isolation kit | StemCell Tecknologies | cat# 19815 | |
Chemical compound, drug | PI-PLC (phosphatidylinositol -specific phospholipase C) | Sigma | P8804 | |
Software, algorithm | PHYLIP Neighbor Joining algorithm | http://evolution.genetics.washington.edu/phylip/phylipweb.html | ||
Others | rodent herpesvirus Peru (RHVP) | PMID: 21209105 | Dr. Herbert W. Virgin laboratory in Washington University School of Medicine |
RHVP encodes a protein with high identity to Qa-1.
Peptides used for pQa-1 surface stabilization assay.