Supplemental Data Cytomegalovirus Evasion of Innate Immunity by ...

Immunity, Volume 26

Supplemental Data Cytomegalovirus Evasion of Innate Immunity by Subversion of the NKR-P1B:Clr-b Missing-Self Axis Sebastian Voigt, Aruz Mesci, Jakob Ettinger, Jason H. Fine, Peter Chen, Wayne Chou, and James R. Carlyle

Figure S1. Quantitative Real-Time RT-PCR Analysis of Gene Expression during RCMV Infection over an Extended Time Course (A) REF cells were infected with the indicated viruses (WT, ∆RCTL, REV), then RNA was extracted and cDNA prepared. Samples were normalized according to input cDNA, then subjected to real-time PCR analysis using primers and probes specific for the indicated target sequences (RCTL, rClr-b, rCLEC2D5; see Supplementary Table 2). *Values are plotted for the indicated target gene products normalized relative to host c-myc expression. Values plotted on the x-axis represent undetectable quantities; Ø, mock infections. (B) As in (A) except that WTinfected REF cells were pre-treated with cycloheximide (CHX) or phosphonoacetic acid (PAA) prior to infection. CHX results, 6h post-infection; PAA results, 9h post-infection.

Immunity, Volume 26

Figure S2. In Vivo Depletion of Rat NK Cells Splenocytes derived from undepleted (UND) or NK-depleted (NKD) rats (WAG or SD strain) were analyzed by flow cytometry 3 days after a single intravenous administration of rabbit polyclonal antiasialo-GM-1 antibody plus 3.2.3 mAb (150 µg of each). Cells were stained using biotinylated 10/78 mAb (anti-NKR-P1A/B) plus secondary streptavidin-PE. Percent positive cells are indicated for the gate shown at the right. RCN, relative cell number. Table S1. Relatedness of RCMV and Rodent Clr Sequences Percent Nucleotide Identity

Divergence

rClrb

RCTL

mClrb

mClrg

65.9

85.1

77.5

60.4

58.7

RCTL

47.4

mClrb

16.7

59

mClrg

27.1

64.9

83.6 18.6

rClrb rClrb Divergence

rClrb

Percent Amino Acid Identity RCTL

mClrb

mClrg

60.2

69.7

61.7

49.7

48.1

RCTL

56.1

mClrb

38.7

80.6

mClrg

53.3

85.2

67.5 42.5

Abbreviations: rClrb, rat Clr-b (rCLEC2D11); RCTL, rat cytomegalovirus C-type lectin; mClrb, mouse Clr-b (mCLEC2D8); mClrg, mouse Clr-g (mCLEC2D3).

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Table S2. Gene-Specific Primers and Probes used in RT-PCR and qPCR Analysis Gene Product

Forward Primer (5′ → 3′)

CD3Z/P1B/RCTL a 5’ fusion CD3Z/P1B/RCTL 3’ fusiona rNKR-P1Aa

CTC GAG ATG AGA GCA AAA TTC AGC AGG AGT G GTC TTG GTG CTA TCA GTA CAA CGA TCG ATA GAT CCG CTC GAG ATG GAC ACA GCA CGT GTC TAC

rNKR-P1Ba

CTC GAG ATG GAC ACG GCA GTG GTC TAT G

rNKR-P1Fa

CTC GAG ATG GAC ACA TCA AGA GTC TAT G

rNKR-P1Ga

CTC GAG ATG GAT GCA CCA GTG CTC TAT G

RCTLa

CTC GAG ATG CTG GCC CAT GAG GAT TAC A

RCTLb

CCC ATG AGG ATT ACA TCA GAT TTG C

RCTL-probe

b,c

CGG ATC TAT CGA TCG TTG TAC TGA TAG CAC CAA GAC AGC GGC CGC CTA TGT AGA ACA AAG CTG GCA ACG GAG AGC GGC CGC TCA GGA GTC ATT ACA CAT GCA TTC AGC GGC CGC TCA GGA GTC ATT ACA CAT GCA TTC AGC GGC CGC TCA GAC ATG TTT CAG GGT CTT TTG AGC GGC CGC TCA GAC ACG TTT CAG GGT CTT TTG AGC GGC CGC CTA TGT AGA ACA AAG CTG GCA ACG GAG GTA GAA CAA AGC TGG CAA CGG

FAM-CAA CTA CTC GCT TTC TAT-MGB NFQ

rCLEC2D3a

CTC GAG ATG CCG TCC AGT GCA CAC CTA C

rCLEC2D5a

CTC GAG ATG CCG TCC AGT GCA CAC CTA CAG

rCLEC2D5b

AGA CCC TCT GTA AGG AAC AAG G

rCLEC2D5probeb rCLEC2D11a

NED-TCA GGT TAC TGG TTC G-MGB NFQ

rCLEC2D11b

Reverse Primer (5′ → 3′)

AGC GGC CGC TCA CTG GGA CTG TGA GAT GTA TTT TTT G AGC GGC CGC CTA CAG GCA CTG TGA GGT GTA GC AGG TGT AGC TGT TGG GCT TC

CTC GAG ATG AGT GCC AAA AAG GCT TCC CAA C AGC GGC CGC CTA CAT AGG AGA AAA AGG AGT TTT G GCA GAT ACA AA GGG AGT TTT GAT T CTA CAG CTC CAT CTT TTG TCT GC

rCLEC2D11probeb β-actina

VIC-CCT ACC TGA ACG ACA TC-MGB NFQ AGC TGC GTG TGG CCC C

AGG TCT CAA ACA TGA TCT GGG TCA

RCMVgBb

FAM-CAA GGC CAT CAG AAC GGA TC-TAMRA

TCA CTC CCC GAT GCG TTA TA

RCMVgB-probeb CAC AAA CGT TGC GGT CAG ACT TTT CGA C c-mycb c-myc-probe a

GCC AGA GGA GGA ACG AGC T b

GGG CCT TTT CAT TGT TTT CCA

FAM-TGC CCT GCG TGA CCA GAT CC-TAMRA

RT-PCR primers. Real-time qPCR primers and probes. c cDNA-specific probe, spanning exons 4-5. Underlined sequences indicate restriction enzyme sites used for subcloning; bold text indicates probe modifications. Abbreviations are as follows: FAM, 6-carboxyfluorescein; MGB, minor-groove binding; NFQ, non-fluorescent quencher; TAMRA, 6-carboxytetramethylrhodamine; NED & VIC, proprietary dyes (Applied Biosystems, Foster City, CA). b

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Table S3. GenBank Accession Numbers and Sequences Gene Producta

GenBank Accession Numberb

Referencec,d

RCTL

AF302184

(Voigt et al., 2001)

rClr-b/rOcil/rCLEC2D11

DQ168419.1, EF100690

This study; (Hao et al., 2006)

mClr-b/mOcil/mCLEC2D8

AY320031

(Carlyle et al., 2004; Iizuka et al., 2003)

mClr-g/mOcilrp2/mCLEC2D3

NM_020257

(Iizuka et al., 2003; Plougastel et al., 2001a)

rCLEC2D2

EF100686


rCLEC2D3

EF100687


rCLEC2D5

EF100688


rCLEC2D6

EF100689


rNKR-P1AWAG

EF100677

This study

EF100683

This study

rNKR-P1ARNK

EF100681

This study; (Li et al., 2003)

rNKR-P1AF344

M62891

(Giorda et al., 1990; Li et al., 2003)

rNKR-P1BWAG

EF100678

This study

AF525533.1

This study

NM_001040189.2

This study; (Kveberg et al., 2006)

rNKR-P1BSD

EF100684

This study

rNKR-P1BRNK

EF100682

This study; (Li et al., 2003)

rNKR-P1BF344

rNKR-P1A

SD

BS

rNKR-P1B PVG

rNKR-P1B

PVG

/*C

NM_173292.1, U56936.1

(Li et al., 2003)

TO

AF541943.1

This study

WAG

EF100679

This study

rNKR-P1FSD

EF100685

This study

rNKR-P1FF344

X97477.1

This study; (Carlyle et al., 2006)

rNKR-P1GWAG

EF100680

rNKR-P1B rNKR-P1F

rNKR-P1G

BN

This study; (Carlyle et al., 2006)

mNKR-P1AB6

M77676

(Giorda and Trucco, 1991)

mNKR-P1BB6/*DB6

NM_030599, AF338321

(Carlyle et al., 2006)

mNKR-P1BSw/SJL

M77677, AF354259

(Carlyle et al., 1999; Kung et al., 1999)

B6

M77678

(Giorda and Trucco, 1991; Ryan et al., 1992)

B6

NM_153094

(Iizuka et al., 2003; Plougastel et al., 2001b)

mNKR-P1GB6

XM_355818

(Carlyle et al., 2006)

hNKR-P1A

NM_002258

(Lanier et al., 1994)

mNKR-P1C mNKR-P1F

a

ENSRNOT00000035766

This study b

Abbreviations: R, RCMV; r, rat; m, mouse; h, human; *, indicates an alternate designation given to an allele derived from the Klrb1b locus. b Ensembl gene ID given where no GenBank sequence available.

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See also references contained therein. All other sequences used in this study were derived from (Hao et al., 2006).

d

Supplemental References Carlyle, J.R., Martin, A., Mehra, A., Attisano, L., Tsui, F.W., and Zuniga-Pflucker, J.C. (1999). Mouse NKR-P1B, a novel NK1.1 antigen with inhibitory function. J. Immunol. 162, 5917–5923. Carlyle, J.R., Mesci, A., Ljutic, B., Belanger, S., Tai, L.H., Rousselle, E., Troke, A.D., Proteau, M.F., and Makrigiannis, A.P. (2006). Molecular and genetic basis for strain-dependent NK1.1 alloreactivity of mouse NK cells. J. Immunol. 176, 7511–7524. Giorda, R., and Trucco, M. (1991). Mouse NKR-P1. A family of genes selectively coexpressed in adherent lymphokineactivated killer cells. J. Immunol. 147, 1701–1708. Giorda, R., Rudert, W.A., Vavassori, C., Chambers, W.H., Hiserodt, J.C., and Trucco, M. (1990). NKR-P1, a signal transduction molecule on natural killer cells. Science 249, 1298–1300. Hao, L., Klein, J., and Nei, M. (2006). Heterogeneous but conserved natural killer receptor gene complexes in four major orders of mammals. Proc. Natl. Acad. Sci. USA 103, 3192–3197. Iizuka, K., Naidenko, O.V., Plougastel, B.F., Fremont, D.H., and Yokoyama, W.M. (2003). Genetically linked C-type lectin-related ligands for the NKRP1 family of natural killer cell receptors. Nat. Immunol. 4, 801–807. Kung, S.K., Su, R.C., Shannon, J., and Miller, R.G. (1999). The NKR-P1B gene product is an inhibitory receptor on SJL/J NK cells. J. Immunol. 162, 5876–5887. Kveberg, L., Back, C.J., Dai, K.Z., Inngjerdingen, M., Rolstad, B., Ryan, J.C., Vaage, J.T., and Naper, C. (2006). The novel inhibitory NKR-P1C receptor and Ly49s3 identify two complementary, functionally distinct NK cell subsets in rats. J. Immunol. 176, 4133–4140. Lanier, L.L., Chang, C., and Phillips, J.H. (1994). Human NKR-P1A. A disulfide-linked homodimer of the C-type lectin superfamily expressed by a subset of NK and T lymphocytes. J. Immunol. 153, 2417–2428. Li, J., Rabinovich, B.A., Hurren, R., Shannon, J., and Miller, R.G. (2003). Expression cloning and function of the rat NK activating and inhibitory receptors NKR-P1A and -P1B. Int. Immunol. 15, 411–416. Plougastel, B., Dubbelde, C., and Yokoyama, W.M. (2001a). Cloning of Clr, a new family of lectin-like genes localized between mouse Nkrp1a and Cd69. Immunogenetics 53, 209–214. Ryan, J.C., Turck, J., Niemi, E.C., Yokoyama, W.M., and Seaman, W.E. (1992). Molecular cloning of the NK1.1 antigen, a member of the NKR-P1 family of natural killer cell activation molecules. J. Immunol. 149, 1631–1635. Voigt, S., Sandford, G.R., Ding, L., and Burns, W.H. (2001). Identification and characterization of a spliced C-type lectinlike gene encoded by rat cytomegalovirus. J. Virol. 75, 603–611.