A herpesvirus encoded Qa-1 mimic inhibits natural killer cell cytotoxicity through CD94/NKG2A receptor engagement

  1. Xiaoli Wang
  2. Sytse J Piersma
  3. Christopher A Nelson
  4. Ya-Nan Dai
  5. Ted Christensen
  6. Eric Lazear
  7. Liping Yang
  8. Marjolein Sluijter
  9. Thorbald van Hall
  10. Ted H Hansen
  11. Wayne M Yokoyama
  12. Daved H Fremont  Is a corresponding author
  1. Washington University School of Medicine, United States
  2. Leiden University Medical Center (LUMC), The Netherlands
7 figures, 1 table and 2 additional files

Figures

Figure 1 with 2 supplements
RHVP encodes a Qa-1-like protein.

(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 …

https://doi.org/10.7554/eLife.38667.002
Figure 1—figure supplement 1
Three previously annotated RHVP ORFs are spliced to form mRNA encoding an MHC-I-like protein.

(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 …

https://doi.org/10.7554/eLife.38667.003
Figure 1—figure supplement 2
Sequence alignment of pQa-1 with selected classical and non-classical MHC-I proteins.

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 …

https://doi.org/10.7554/eLife.38667.004
RHVP pQa-1 is GPI anchored, cell surface expressed and assembles with β2m.

(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 …

https://doi.org/10.7554/eLife.38667.005
Qdm and Qdm-like peptides stabilize pQa-1 at the cell surface.

(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) …

https://doi.org/10.7554/eLife.38667.006
Qa-1 is sensitive to pK3-induced downregulation while pQa-1 is resistant.

(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. …

https://doi.org/10.7554/eLife.38667.007
Figure 5 with 2 supplements
The Qdm-β2m-pQa-1 single chain trimer (SCT) specifically recognizes the CD94/NKG2A receptor.

(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 …

https://doi.org/10.7554/eLife.38667.008
Figure 5—figure supplement 1
pQa-1 single chain trimer design and increased thermal stability.

(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 …

https://doi.org/10.7554/eLife.38667.009
Figure 5—figure supplement 2
pQa-1-dtSCT tetramer stains CD94/NKG2A expressing cells stronger than CD94/NKG2C-expressing cells.

(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 …

https://doi.org/10.7554/eLife.38667.010
Figure 6 with 1 supplement
Expression of pQa-1 bound with Qdm/Qdm-like peptide inhibits NKG2ANK cell activation and prevents tumor rejection in vivo.

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 …

https://doi.org/10.7554/eLife.38667.011
Figure 6—figure supplement 1
Surface level of pQa-1 or Qa-1 on cells used in the assays in Figure 6.

(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. …

https://doi.org/10.7554/eLife.38667.012
Figure 7 with 2 supplements
Downregulation of MHC-I by pK3 leads to NK killing susceptibility, while pQa-1 co-expression provides protection in an NKG2A-dependent manner.

(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-/-

https://doi.org/10.7554/eLife.38667.013
Figure 7—figure supplement 1
Surface level of endogenous Qa-1, MHC-I and transduced pQa-1 on RMA cells expressing pK3 or pK3 RING mutant (pK3 RM).

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 …

https://doi.org/10.7554/eLife.38667.014
Figure 7—figure supplement 2
PI-PLC treatment diminishes pQa-1 mediated protection of RMA target cells expressing pK3.

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 …

https://doi.org/10.7554/eLife.38667.015

Tables

Key resources table
Reagent
type (species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Strain, strain
background
(M. musculus)
Klrc1-/-PMID: 26680205Dr. Marco Colonna
laboratory
(Washington University
School of Medicine)
Strain, strain
background
(M. musculus)
C57BL/6NCrCharles River
Laboratories
Strain code: 556
Cell line
(M. musculus)
B6/WT-3Dr. 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.36Dr. 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)
RMAPMID: 3877776RRID:CVCL_J385
Cell line
(M. musculus)
B16-F10American Type
Culture Collection
ATCC
CRL-6475,
RRID:CVCL_0159
Cell line
(Cricetulus griseus)
CHODr. Pamela Stanley
laboratory (Albert
Einstein
College of Medicine)
Cell line
(Homo sapiens)
293F (FreeStyle
293 F Cells)
Thermo Fisher ScientificR79007,
RRID:CVCL_D603
Cell line
(Homo sapiens)
293T (HEK 293T)American Type
Culture Collection
ATCC
CRL-3216,
RRID:CVCL_0063
AntibodyFITC labeled
anti-mouse
NKG2A/C/E
eBiosciencecat# 11-5896-82clone: 20D5
(1:100)
AntibodyFITC labeled isotype
control (RatIgG2a)
eBiosciencecat# 11-4321-85clone: eBR2a
(1:100)
AntibodyeFluor 660 labeled
anti-mouse CD107a
eBiosciencecat# 50-1071-82clone: eBio1D4B
(1:1000 in culture)
AntibodyAPC-eFluor labeled
anti-mouse CD19
eBiosciencecat# 47-0193-82clone: eBio
1D3 (1:100)
AntibodyPE-Cy7 labeled
anti-mouse CD19
eBiosciencecat# 25-0193-82clone: eBio1D3
(1:200)
AntibodyAPC-eFluor 780
labled anti-mouse
CD3e
eBiosciencecat# 47-0031-82clone: 145–2 C11
(1:100)
AntibodyPacific Blue labeled
anti-mouse CD3e
Biolegendcat# 100214clone: 17A2
(1:100)
AntibodyPE-eFluor 610
labeled anti-mouse
CD8a
eBiosciencecat# 61-0081-80clone: 53–6.7
(1:200)
AntibodyPE labeled
anti-mouse CD94
eBiosciencecat# 12-0941-82clone: 18D3
(1:100)
Antibodymouse anti-HA tagCovancecat# MMS-101Pclone: 16B12
(1:500)
AntibodyeFluor 450 labeled
anti-mouse IFNg
eBiosciencecat# 48-7311-82clone:
XMG1.2 (1:100)
AntibodyPE-Cy7 labeled
anti-mouse NK1.1
eBiosciencecat# 25-5941-82clone:
PK136 (1:100)
AntibodyPerCp Cy5.5 labeled
anti-mouse NK1.1
eBiosciencecat# 45-5941-82clone: PK136
(1:200)
AntibodyPE-labeled
anti-mouse Thy1.1
BD PharMingencat# 551401clone: OX-7
(1:150)
Antibodybiotin labeled
anti-mouse Qa-1
BD PharMingencat# 559829clone:
6A8.6F10.1A6
(1:200)
Recombinant
DNA reagent
pMIG_pQa-1-HAcurrent studySchematic
depiction
is shown in
Figure 2A
Recombinant
DNA reagent
pMIN_pQa-1-HAcurrent studypMSCV.IRES.neo (pMIN)
was described
previously
(PMID:15280476)
Recombinant
DNA reagent
pMIG_pK3PMID:22403403Dr. Ted H Hansen
laboratory
Recombinant
DNA reagent
pMIN_Qa-1current studyDNA sequence
encoding
Qa-1b (NM_034528)
was subcloned
into pMIN vector
Recombinant
DNA reagent
pMIG_pQa-1-SCTcurrent studySchematic depiction
is shown in

Figure 5—figure supplement 1
Recombinant
DNA reagent
pFM1.2R_pQa-
1-SCT-BirA
current studyDescribed in
the DAN constructs
section in
Materials and methods
Peptide,
recombinant
protein
pQa-1-SCTcurrent studyDescribed 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)
SigmaP8804
Software,
algorithm
PHYLIP Neighbor
Joining algorithm
http://evolution.genetics.washington.edu/phylip/phylipweb.html
Othersrodent herpesvirus
Peru (RHVP)
PMID: 21209105Dr. Herbert
W. Virgin laboratory
in Washington
University School
of Medicine

Additional files

Supplementary file 1

RHVP encodes a protein with high identity to Qa-1.

https://doi.org/10.7554/eLife.38667.017
Supplementary file 2

Peptides used for pQa-1 surface stabilization assay.

https://doi.org/10.7554/eLife.38667.016

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