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1 gbt-i. GFP+ Resident memory cells: gbt-i.gfp+ Recruited memory cells: gbt-i.cd gbt-i. flu.gb sc. CD45.1+ Graft with gbt-i.gfp+ 1 Recipient 1 re Graft with gbt-i.gfp+ and gbt-i.cd45.1+ Distriution of gbt-i.gfp+ and gbt-i.cd45-1+ in HSV i.v. spleen and graft? 2 Recipient 2 28 Supplementary Figure 1. Experimental strategy for the analysis of migrational differences etween tissue resident (green) and recruited (red) gbt-i CD8 + T cells as descried in the text and in the legend for Figure 1.

2 a gbt-i. GFP+ Resident memory cells: gbt-i.gfp+ Recruited memory cells: gbt-i.cd gbt-i. HSV sc. CD45.1+ Graft with gbt-i.gfp+ 1 Recipient 1 re Graft with gbt-i.gfp+ and gbt-i.cd45.1+ Distriution of gbt-i.gfp+ and gbt-i.cd45-1+ in HSV i.v. spleen and graft? 2 Recipient 2 28 CD45.1 Graft Rec. 16. Res Control.2 c d P <.1 P >.3 GFP Supplementary Figure 2. Tissue resident memory T cells fail to migrate following systemic viral. (a-d) Antiody-deficient µmt mice were seeded with naïve gbt-i.cd45.1 (recruited) CD8 + T cells (5 x 1 4 each) one day prior to sucutaneous foot hock with HSV-1 KOS (1 x 1 6 pfu) and left to rest for 3 at which time latently infected dorsal root ganglia containing gbt-i.gfp memory CD8 + T cells (resident) were transplanted under the kidney capsule. On day 5 after transplantation the grafts were recovered and re-transplanted into naïve recipient µmt mice. On day 2 after re-transplantation mice were intravenously challenged with HSV (1 x 1 5 pfu) and recruited (gbt-i.cd45.1) and tissue-resident (gbt-i.gfp) memory CD8 + T cells in grafts and spleens were analysed y flow cytometry. () Representative flow cytometry plots gated on PI - CD8 + events with rectangles indicating the two memory populations. Inserted values indicate percentages of memory gbt-i populations among all CD8 + cells for the depicted experiment. (c) Percentage of oth memory gbt-i T cell populations among all CD8 + cells in the spleen. (d) Numers of gbt-i.gfp and gbt-i.cd45.1 CD8 + memory T cells recovered from grafts. Data represent means ± SEM derived from two independent experiments with 6-8 mice per group. Statistical analysis was performed using the paired student s t-test.

3 gbt-i. GFP+ Resident memory cells: gbt-i.gfp+ Recruited memory cells: gbt-i.cd gbt-i. flu-gb sc. Graft with CD45.1+ gbt-i.gfp+ 1 Recipient 1 re Graft with gbt-i.gfp+ and gbt-i.cd45.1+ Distriution of gbt-i.gfp+ and gbt-i.cd45-1+ in oth grafts? 2 Recipient 9 Graft without gbt-i 2 Supplementary Figure 3. Experimental strategy for the analysis of migrational differences etween tissue resident (green) and recruited (red) gbt-i CD8 + T cells as descried in the text and in the legend for figure 1.

4 a Acute Memory Mock P <.5 P <.5 P >.5 Acute Memory Control HSV L 4.5 L 13.4 C 2. c Acute P <.1 Memory P <.1 R 32.4 R 8.4 P <.1 P <.1 K gb-tet P >.5 P >.5 CD44 HSV HSV Supplementary Figure 4. Retention of endogenous, HSV-specific CD8 + T cells at the site of previous viral. C57BL/6 mice were sujected to mock or HSV-1 KOS (1 x 1 6 pfu) of the left flank and virus-specific endogenous CD8 + T cells in the spleen and skin samples from oth flanks were analysed 9-1 (acute) or 3-35 (memory) later. (a) Representative flow cytometry plots from spleen and skin preparations gated on PI - CD8 + CD events. Numers indicate the percentage of virus-specific (CD8 + CD44 hi K gb-tetramer + ) T cells (rectangles) among all events shown. () Numers of HSV-specific (CD8 + CD44 hi K gb-tetramer + ) T cells in the spleen at acute and early memory time points. Controls represent mock-infected mice. (c) Numers of HSV-specific CD8 + CD44 hi K gb-tetramer + T cells in the skin at similar time points. Data represent means ± SEM derived from three independent experiments. Statistical analyses were performed using one-way ANOVA followed y Tukey s post test comparison.

5 a group HSV-1 Infection left right A rgb-l8a scr B scr rgb C rgb-l8a rgb D scr scr Acute c A B Memory C D A B C D Supplementary Figure 5. Cognate antigenic encounter in the skin is not a prerequisite for accumulation and persistence of CD8 + T ce lls. Groups of C57BL/6 mice were infected on oth the l eft and the r ight flanks using two different strains of rec ominant HSV-1 (8.5 x 1 5 each). One stra in was a control virus expressing the wild type gb epitope (HSV-1 KOS.rgB) whereas the other strain expressed a mutated epitope variant (HSV-1 KOS.gB-L8A) not e ing recognized y endogenous K gb-tetramer inding CD8 + T ce lls. Note that oth viruses were strongly attenuated for skin replication and did not cause zosteriform flank lesions. Four groups of mice were infected according to the tale shown in (a). Mock s included control arasion of the epidermal skin layer. Numers of HSV-specific (CD44 hi K gb-tetramer + CD8 + ) T cells in the skin () 8-1 (acute) and (c) 28-3 (memory) after. Data are derived from three independent experiments.

6 a rachial LN ctrl CD62L c CD122 CD69 Supplementary Figure 6. -resident virus-specific CD8+ T cells phenotypically differ from their circulating counterparts. (a-c) Naïve gbti.cd45.1 CD8+ T cells (5 x 14 ) were transferred into C57BL/6 mice one day prior to HSV-1 KOS (1 x 16 pfu) skin. Flow cytometric analysis of (a) CD62L, () CD122, and (c) CD69 surface expression on memory gbti.cd45.1 CD8+ T cells isolated from rachial lymph node, spleen and skin 3 5 after. Control staining (ctrl) was performed with CD8+ splenocytes from naïve mice.

7 a gbt-i gbt-i in flank skin? Graft with gbt-i gbt-i in graft and ctrl skin? Recipient Recipient G.2 G 1 C <.1 C.4 99 CD45.1 Vα2 CD45.1 Vα2 < CD45.1 Vα2 CD8 c d CD8 P <.5 Recipient Supplementary Figure 7. resident virus-specific T cells persist in situ for at least 5 weeks after transplantation of previously infected skin onto naïve recipients. (a-d) Naïve gbt-i.cd45.1 CD8 + T cells (5 x 1 4 ) were transferred into C57BL/6 mice one day prior to HSV-1 KOS (1 x 1 6 pfu) skin. Two to four weeks after, one group of mice was sacrificed and previously infected skin was transplanted onto naïve C57BL/6 recipients. Two to five weeks after transplantation, distriution of gbt-i.cd45.1 CD8 + T cells in grafts, control skin tissue, and spleens from recipient and donor mice was analysed y flow cytometry. () Representative flow cytometry plots derived from pooled recipient tissues (graft, control skin and spleen) isolated 35 after transplantation as well as individual donor tissues (previously HSVinfected skin and spleen). HSV-specific T cells were identified as PI - CD8 + CD CD Va2 + events. Numers indicate the percentage of gbt- I.CD45.1 CD8 + T cells among all events in the plots. (c+d) Numers of gbt- I.CD45.1 CD8 + T cells isolated from (c) skin tissues and (d) spleens. Symols Nature Immunology: depict individual doi:1.138/ni.1718 mice derived from two independent experiments. Statistical analyses were performed using the paired student s t-test.

8 Supplementary Figure 8. Local protection y skin-resident memory T cells is virus-specific. C57BL/6 mice were infected with HSV-1 KOS (1 x 1 6 pfu) on the left flank and left to rest. After 2-3 months, oth, the left and the right flanks of these mice were re-challenged with vaccinia-np (5 x 1 7 pfu) and viral titres at the inoculation sites were determined two later. Symols depict individual mice derived from two independent experiments.

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