SOM 1 *** *** *** * * n.s GFP + (NK 10 4 ) Naive DNFB OXA. Donor sensitization: Naive OXA DNFB 1,200 6,000 4,000

Transcription

1 SOM 1 a n.s GFP + (NK 1 4 ) Naive DNFB OXA splenic NK hepatic NK b Donor sensitization: Naive OXA DNFB NK cells (% of CD ) 1, NK cells (% of CD ) 6, 4, 2,

2 Supplementary Figure. 1. Long-term survival of adoptively transferred NK-cells is not affected by sensitization or lymphopenia. (a) 1 5 Thy1 + NK1.1 + CD3 NKcells expressing GFP under the actin promoter were transferred into naïve C57BL/6 mice, and recipients were challenged 6 weeks later. Two weeks later, recipient mice were analyzed by FACS and total numbers of splenic and hepatic GFP + NK1.1 + CD3 cells were determined. No GFP + CD3 + cells were present in recipient mice (not shown). (b) Recruitment of memory NK-cells to sites of challenge is Ag-specific. Hepatic Thy1 + NK1.1 + NK-cells from naïve CD WT donors (C57BL/6) and from CD DNFB or OXA sensitized WT or actin- GFP transgenic donors were mixed (1 5 each) and adoptively transferred into naïve Rag2 / Il2rg / recipients. One month after adoptive transfer, recipient ears were challenged with either DNFB or OXA. s and ears were harvested at 24, 48 and 72 hrs, and analyzed for the presence of NK-cells whose origin was distinguished by congenic/fluorescent markers using FACS. No NK-cells were found in acetone challenged control ears (not shown). Symbols and error bars depict mean and range of n=2 ears for each day. p<1-2 ; p<1-3 ; p<1-11.

3 SOM 2 a b c

4 Supplementary Figure. 2. Characterizaon of influenza A virus- like parcles (VLPs). Transmission electron micrographs of influenza VLPs containing (a) HA and M1 or (b) M1 only. (c) Blots for HA (top) and M1 (bohom) were probed using mouse an- PR8 sera and purified mouse an- M1 IgG anbody (Serotec), respecvely. Lane 1, influenza VLPs containing PR8 HA and M1; lane 2, HA negave M1 VLPs; lane 3, negave control (human immunodeficiency virus- like parcles containing gag/env).

5 NK1.1 SOM 3 a b Ear swelling (µm) Phenotype GFP + GFP + GFP GFP Donor organ Spleen Spleen NK cells ( 1 5 ) n.s. n.s. n.s Phenotype Donor organ GFP + GFP GFP + GFP Spleen Spleen c Donor organ Phenotype Spleen 1x x1 5 95% 93% 1 GFP x x1 5 Spleen GFP + 1x x1 5 95% 75% x x1 5 1x x1 5 22% 4% 1 GFP x x1 5 Spleen GFP 1x x1 5 4% 1% x x1 5 GFP

6 Supplementary Figure. 3. NK cell- expressed CXCR6 is required for NK- cell mediated adapve immunity to haptens. (a) CXCR6 + NK- cells mediate OXA specific CHS responses. 8, GFP + or GFP - hepac or splenic NK- cells from Cxcr6 +/ mice were sorted from OXA sensized donor mice and transferred into Rag2 / Il2rg / recipients, recipients challenged one month post adopve transfer with OXA on one ear and solvent on the other, and ear swelling determined every 24 hrs using a micrometer. Background swelling was determined using naïve mice and subtracted from experimental groups mice/group. p<1-2 ; p<1-3. (b) Mice were analyzed six weeks post adopve transfer for the presence and locaon of adopvely transferred NK- cells using FACS analysis (c) and their GFP expression determined.

7 SOM 4 a 5 C57BL/6 cxcr6 +/ Cxcr6 / 18 Rag1 / (C57BL/6) cxcr6 +/ Cxcr6 / 4 15 Ear swelling (µm) Ear swelling (µm) b Rag1 / C57BL/6 cxcr6 +/ C57BL/6 cxcr6 +/ % CXCR6 + NK 8 4 p>.62 p>.12 p>.66 p>.2 p>.22 p>.37 8 p>.57 p>.6 p>.11 p>.41 p>.7 p>.23 % CXCR6 + NK LN BM Spleen Lung Blood LN BM Spleen Lung Blood isotype anti-cxcr6 anti-cxcl16 4 c C57BL/6 Rag1 / C57BL/6 Ear swelling (µm) Isotype Anti-CXCR6 Ear swelling (µm) Istoype Anti-CXCR

8 Supplementary Figure. 4. Primed CXCR6 + NK- cells preferenally transfer sensivity to OXA into naïve hosts. (a) Cxcr6 +/ and Rag1 / Cxcr6 +/ mice were sensized with OXA on days and 1, and challenged with OXA on day four on one ear, and solvent on the other. Ear swelling was determined every 24 hrs post challenge using a micrometer. Background swelling was determined using naïve animals, and subtracted from experimental groups. n= 8-1 recipients/group., p<1-2 ;, p<1-3 ;, p<1-4. (b) An- CXCR6 or an- CXCL16 mabs do not deplete or CXCR6- expressing NK cells. The percentage of CXCR6- expressing (i.e. GFP + ) NK- cells from CXCR6 +/gfp mice were analyzed by flow cytometry. NK- cells were idenfied as CD45 + NK1.1 + cells, and CXCR6 expression monitored using GFP in mice treated intravenously with 1µg isotype control, or an- CXCR6 mab or an- CXCL16 mab 24 hrs before cell isolaon. (c) Blocking mab to CXCR6 abrogates CHS responses in Rag1 / mice. WT and Rag1 / mice were sensized with OXA day and 1, intravenously injected with 1µg an- CXCR6 or isotype control on day four, and challenged with OXA on day five on one ear, and solvent on the other. Ear swelling was determined every 24 hrs post challenge using a micrometer. Background swelling was determined using naïve animals, and subtracted from experimental groups mice/group., p<1-2 ;, p<1-3 ;, p<1-4.

9 SOM 5 a Rag1 / C57BL/6 Rag1 / C57BL/ n.s. n.s. NK ( 1 4 ) Spleen NK ( 1 5 ) Genotype cxcr6 +/+ cxcr6 +/ cxcr6 /. Genotype cxcr6 +/+ cxcr6 +/ cxcr6 / CXCR6 + or GFP + CXCR6 + or GFP + CXCR6 or GFP CXCR6 or GFP b 2. NK cells ( 1 5 per recipient) Donor genotype cxcr6 +/ cxcr6 / Spleen

10 Supplementary Figure. 5. CXCR6 regulates hepac, but not splenic NK- cell homeostasis. (a) Numbers of CXCR6- posive NK- cells were compared in spleens and livers of Rag1 /, Rag1 / Cxcr6 +/ and Rag1 / Cxcr6 / mice. WT CXCR6 was visualized using anbody staining, while CXCR6 was visualized in Rag1 / Cxcr6 +/ mice and Rag1 / Cxcr6 / mice using GFP mice/group. p = (b) Recipient mice shown in Figure 6e were analyzed by FACS six weeks post adopve transfer and number of donor NK- cells determined (n=8 mice/group). p =.16.

11 SOM 6 Gated on CD45 + NK1.1 + cells Sensitization Ab Hepatic NK Splenic NK 25K 25K 2K 2K Acetone Isotype 15K 1K 15K 1.58% 1.4% 1K 5K 5K x x1 5 25K 25K 2K 2K DNFB Isotype 15K 1K 15K 12.6% 1.71% 1K 5K 5K x x1 5 25K 25K 2K 2K DNFB Anti-CXCR6 in vitro FSC A 15K 1K 12.7% 15K 1.28% 1K 5K 5K x x1 5 25K 25K DNFB Anti-CXCR6 in vivo 2K 15K 1K 2K 1.1% 1.27% 15K 1K 5K 5K x x1 5 25K 25K 2K 2K OXA Isotype 15K 1K 15K 2.71% 1.47% 1K 5K 5K x x1 5 Lamp-1

12 Supplementary Figure. 6. DNFB primed NK- cells degranulate upon exposure to DNBS- conjugated B- cells in a sensizaon- dependent, organ- specific, and Ag- specific manner. NK1.1 + cells were analyzed for an- Lamp- 1 incorporaon by FACS. Rag1 / donor mice were sensized with acetone, DNFB or OXA on days and 1, and NK- cells were sorted from livers or spleens. Indicated groups of donor mice were injected with 1µg an- CXCR6 or isotype control mab 12 hrs pre- NK isolaon. NK- cells were cocultured with DNBS labeled B- cells and 1µg/ml mab specific for Lamp- 1, in the presence of an- CXCR6 1 µg/ml or isotype control. NK- cells were FACS analyzed for an- Lamp- 1 incorporaon aler three hrs. The same gang strategy was used to obtain the data shown in Figure 7d. Data is pooled from 3-5 independent experiments; 1-18 donor mice total; 12-2 individual wells/group.