Cytomegalovirus and tumor stress-surveillance by human γδ T cell receptor binding to Endothelial Protein C Receptor

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1 Cytomegalovirus and tumor stress-surveillane y human γδ T ell reeptor inding to Endothelial Protein C Reeptor Carrie R. Willox, Vinent Pitard, Sonia Netzer, Lionel Couzi, Mahoo Salim, Toias Silerzahn, Jean-François Moreau, Adrian C. Hayday, Benjamin E. Willox, and Julie Déhanet-Merville Nature Immunology: doi:1.138/ni.2394

2 Supplementary Tale 1. Illumina expression profiling of 2E9L + vs 2E9L (-) ell lines. Expression profiling omparisons highlighted 231 genes expressed in two 2E9L + ell lines (K562 and U937) ut not in two 2E9L (-) ell lines (Hutu8 and Huh7), of whih 66 were plasma-memrane loalized (shown in order of dereasing fold indution). Fold hange in expression was alulated as average expression in K562 and U937 divided y average expression in Hutu8 and Huh7. The tale summarizes dataase analyses for eah of the 66 plasma memrane expressed gene produts, assessing funtion, expression and loalization. ( Nature Immunology: doi:1.138/ni.2394

3 kda s kda R d g δ γ LES stcr NR anti-γδ TCR ma LES stcr MAU stcr Control E9 injetion HLA-A2 4 5 Supplementary Figure 1. Conformational validation and speifiity of reognition y 2E9. (a) SDS-PAGE analysis of solule (left) and solule LES TCR (right, under reduing (R) and non-reduing (NR) onditions). (, left) Injetion of anti-γδ TCR onformational ma over immoilized reominant LES and MAU TCR, showing unequivoal inding. Negligile responses were oserved over ontrol flow ells. (, right) Injetion of Ativated Protein C (at 1µM) over immoilized, indiating speifi inding. () Surfae plasmon resonane (SPR) sensorgram of 2E9 injetion over immoilized or ontrol HLA-A2. Results are representative of at least two experiments. Nature Immunology: doi:1.138/ni.2394

4 αvδ1 LES Clone αvδ5 JRT3-TCR Vγ9Vδ2 Vγ4Vδ1 Vγ9Vδ3 Vγ8Vδ1 Vγ4Vδ5=LES Vδ5 Vδ5 Vγ9 Vδ2 Donor 1 Pan-δδ Pan-δ Pan-δ Vδ5 Vγ9 Donor 2 Pan-δ Pan-δ Nature Immunology: doi:1.138/ni.2394 Supplementary Figure 2: Charaterization of the anti-vδ5 ma. (a) Speifi staining of Les lone y anti-vδ5 ma versus anti-vδ1 ma (left). Speifi staining of JRT3-LES versus JRT3 transdued with other indiated γδ TCRs y the anti-vδ5 ma (right). () Staining of PBMC from an healthy lood donor showing high rate of Vγ9Vδ2 ells within γδ T ells and asene of staining with anti-vδ5 ma. () Staining of PBMC from a reast aner patient with a signifiant proportion of Vδ5+ ells within γδ T ells. Results are representative of at least three experiments.

5 2E9 staining (MFI) Ctrl 2E9 wt HT29 wt U373 wt U251 CD69 expression (MFI) JRT3-MAU Ctrl 2E9 wt wt wt HT29 U373 U251 CD69 expression (MFI) JRT3-MAU wt CMV (MOI) Supplementary Figure 3. overexpression and its effet on JRT3-MAU ativation. (a) 2E9 staining of -transdued versus wild-type (WT) tumor ells. () No effet of overexpression on JRT3-MAU ativation y tumor ells. () No effet of overexpression and CMV-infetion on JRT3-MAU ativation y U251 ells. Results are representative of three independent experiments. Nature Immunology: doi:1.138/ni.2394

6 (U251) LES TCR Ctrl TCR (U251+CMV) LES TCR MAU TCR 3 (U373) 2 (HeLa) Supplementary Figure 4. LES TCR inds similarly to produed from ativatory and nonativatory ell lines. (a) Solule produed from non-infeted (left panel) or CMV-infeted U251 ells (middle panel) was injeted at equivalent onentrations over immoilised LES TCR and over an irrelevant ontrol TCR. The purity of solule produed from U251 ells in the asene (lane 1) or presene (lane 2) of CMV infetion was verified y SDS-PAGE (right panel). () Similar experiment using solule derived from the ativatory ell line U373 (left panel) and non-ativatory HeLaEV2 ell line (right panel). Results are representative of one experiment (a) and three experiments (). Nature Immunology: doi:1.138/ni.2394

7 TNFα seretion (pg/ml) Ctrl mas αcd58+αcd2 2E9 TNFα seretion (pg/ml) Ctrl mas αcd58+αcd2 + αcd58+αcd2 αcd3 TNFα seretion (pg/m ml) FSF HT29 Infeted -FSF EC Ctrl ma αcd58+αcd2 +αcd58+αcd2 αcd3 l) TNFα seretion (pg/ml Infeted -EC K U937 FSF Ctrl mas αcd11a αcd54 αcd54 + αcd11a Infeted-FSF Supplementary Figure 5. Ativation of LES lone y CMV-infeted ells and tumour ells depends on o-stimulatory moleules. TNFα prodution y LES lone ultured 4h with uninfeted or CMV-infeted firolasts (a,) or EC (a) or with tumour ells () in the presene or asene of the indiated mas. Results are representative of at least five experiments for FSF and tumuor ells, and of two experiments for EC. Nature Immunology: doi:1.138/ni.2394