Supplementary Methods Antibodies for flow cytometry Cytotoxicity assay

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1 Supplementary Methods Antibodies for flow cytometry HLA-Cw3 and CD2 expression: LCL cell lines were stained with anti-hla-abc-apc (clone G46-2.6, BD) or migg-apc (clone MOPC-2, BD) antibody, or with unconjugated rituximab and a secondary anti-higg-pe antibody and analyzed by flow cytometry. KIR saturation in vitro: splenocytes used in the in vitro cytotoxicity assay were incubated with the same doses of lirilumab or IC mab as for cytotoxicity assay for 3 min. at 4 C and mab binding was revealed using a secondary monoclonal antibody coupled to PE (clone HP625, Southern Biotechnology). KIR saturation in vivo: RagKO-Tg KIR mice (n=3 per dose) were injected i.v. with the indicated doses of lirilumab. Blood lymphocytes were isolated using Lympholyte - Mammal (Cedarlane) and stained with lirilumab-pe and NK.-APC mabs for the detection of free KIR2DL3 on NK cells. Quantum PE MESF (Molecules of Equivalent Soluble Fluorochrome) beads (Bangs Laboratories, Inc.) were used to allow the comparison of MFI values across several time points. Percentages of KIR2DL3 occupancy on NK cells were calculated for each mouse with the following formula: - [x(mesf time-point - basal MESF)/(MESF controls - basal MESF)], where MESF time-point is the converted MFI of lirilumab-pe into MESF units on the mouse of interest, MESF controls is the MESF value of lirilumab-pe on 3 control mice (injected with diluent only) and basal MESF the background value on NK cells. Cytometry data acquisition was performed on FACS Canto II (BD) and analysis was done using FACSDiva software. Cytotoxicity assay For cytotoxicity assay against 22 cell lines, RagKO-Tg KIR mice (n=3/group) were injected with 5µg of poly-ic (pic, Invivogen) i.v. 8h before the experiment. A pic-treated group was also depleted in NK cells by injecting µg of anti-nk. mab i.p. (PK36). The day of the experiment, splenocytes from all mice were isolated by manual disruption and red blood cells were lysed using.8% NH 4 Cl solution. Splenocytes were then co-cultured with target cell lines (22, 22 HLA-Cw3) pre-loaded with 5 Cr for 4 hours at 37 C at 3: ratio. Anti-KIR or higg4 isotype control (IC) antibodies were added at different concentrations, ranging from µg/ml to.ng/ml. Chromium released in co-culture supernatants was measured as described in M&M.

2 Supplementary Results KIR occupancy in vivo is antibody dose-dependent We wanted to determine the relation between KIR occupancy on NK cells and lirilumab dose injected into RagKO-Tg KIR mice. In order to monitor in vivo KIR occupancy, we have set up a flow cytometry-based assay allowing the detection of free receptors at NK cell surface with fluorochrome-conjugated lirilumab. After a single i.v. injection of different doses of lirilumab, ranging from.5mg/kg and 5mg/kg, all KIR were saturated at a level >95% at early time points (5 min.) following injection. (Supplementary Figure 2). KIR occupancy duration depended on the dose of mab injected, with complete saturation of KIR (> 95%) on peripheral NK cells for respectively 24h and 48h at.5mg/kg and.5mg/kg doses of lirilumab. Doses corresponding to 5mg/kg and 5 mg/kg induced complete KIR saturation for 8 days and 4 days respectively (Supplementary Figure 2). Lirilumab therapeutic efficacy is dose-dependent Anti-KIR treatment was delayed 5 days after HLA+ tumor challenge in KIR Tg mice (Supplementary Figure 3A). Several doses of anti-kir mab were tested in single injections, ranging from.5mg/kg to 5mg/kg. At the lowest dose, corresponding to -day complete KIR saturation on NK cells (Supplementary Figure 3A), a significant prolongation in survival was observed (median 45.5 days, range 3-69) as compared to mice who did not receive anti-kir mab (median 26.5 days, range 23-37) (Supplementary Figure 3B). The highest dose tested, 5mg/kg, allowed the survival of one third of the mice days after tumoral challenge, with a median survival of mice of 64.5 days. In vivo KIR-HLA interaction blockade, even five days after HLA+ tumor cell injection, significantly improved the outcome of the disease in KIR Tg mice.

3 Supplementary Figure KIR2DL3 saturation on transgenic NK cells in vitro is dosedependent. (A) shows staining of KIR2DL3 molecules or IC binding on RagKO-Tg KIR NK cells analyzed by flow cytometry. To demonstrate anti-kir mab recognition of all KIR Tg NK cells in a dosedependent manner, freshly isolated splenocytes from RagKO-Tg KIR mice were incubated with increasing doses of anti-kir mab ( ) or isotype control (IC, )(B). Three groups of mice (n=3/group) were tested: untreated mice, pic-injected mice (5 µg i.v. the day before the experiment) and pic-activated and NK cell-depleted ( µg anti-nk. i.v. a few hours before pic injection) mice. Dose-dependent binding of anti-kir mab or IC to KIR Tg NK cells (defined as NKp46+ cells among splenocytes) was measured by flow cytometry with PE-coupled secondary anti-higg4 monoclonal Ab. Blockade of KIR2DL3/HLA-C interaction induces tumor cell killing by NK cells in vitro. To demonstrate inhibition of lysis of 22 cells by KIR Tg NK cells due to over-expression of HLA-Cw3, expression of HLA-ABC molecules (open histogram) or isotypic control binding (migg, grey-filled histogram) at 22 and 22 HLA-Cw3 cell surface was analyzed by flow cytometry (C). Freshly isolated splenocytes from RagKO-Tg KIR mice were co-incubated with 22 or 22 HLA-Cw3 tumor cells at 3: ratio for 4h, with increasing doses of anti-kir (22- Cw3, ; 22, ) or IC mab (22-Cw3, )(D). Supplementary Figure 2 Duration of KIR saturation in vivo is lirilumab dose-dependent. Four dose levels of anti-kir mab (lirilumab, higg4) were injected i.v. into RagKO-Tg KIR mice (.5 mg/kg,.5 mg/kg, 5 mg/kg, or 5 mg/kg Δ; n=3 to 8 mice/group). (A) shows KIR occupancy assessed by flow cytometry on PBMC, by detecting free KIR on NK cells (defined as NKp46+). Supplementary Figure 3 Lirilumab therapy improves survival in a therapeutic HLA + tumor model in a dose-dependent manner (A) shows the schema of lirilumab administration. To determine therapeutic efficacy of lirilumab therapy, 22 HLA-Cw3 tumor cells ( 7 cells) were injected i.v. into RagKO-Tg KIR mice. Starting five days after tumor challenge, groups received either isotype control (o) or lirilumab at.5 mg/kg ( ),.5 mg/kg (Δ),5 mg/kg ( ) or 5 mg/kg ( )(B). Mice (n=6 per group except for group.5 mg/kg, only 5 mice) were then monitored for overall survival.

4 SUPPLEMENTARY FIGURE A B unstimulated NK cells pic-activated NK cells MFI II mab-pe MFI II mab-pe IC lirilumab C D % of Specific Lysis unstimulated splenocytes.... % of Specific Lysis pic-activated splenocytes.... % of Specific Lysis pic-activated NK cell-depleted Cw3 + IC 22-Cw3 + lirilumab 22 + lirilumab

5 SUPPLEMENTARY FIGURE 2 2 % KIR occupancy mg/kg.5mg/kg 5mg/kg 5 mg/kg Time (days)

6 SUPPLEMENTARY FIGURE 3 A 7 22 HLA- Cw3 RagKO- Tg KIR mice lirilumab Days B Percent survival Vehicle lirilumab.5mg/kg lirilumab.5mg/kg lirilumab 5mg/kg lirilumab 5mg/kg Days after tumor inoculation