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1 Supplementary information Intrahepatic myeloid cell-aggregates enable local CD8 + T cell expansion and successful immunotherapy against chronic viral liver infection Li- Rung Huang, Dirk Wohlleber, Florian Reisinger, Craig N. Jenne, Ru- Lin Cheng, Zeinab Abdullah, Frank A. Schildberg, Margarete Odenthal, Hans- Peter Dienes, Nico van Rooijen, Edgar Schmitt, Natalio Garbi, Michael Croft, Christian Kurts, Paul Kubes, Ulrike Protzer, Mathias Heikenwalder & Percy A. Knolle

2 Supplementary Figure 1

3 Supplementary Figure 1: T cell expansion in the liver upon TLR signaling. (a) Numbers CD CTLs and BrdU + CD CTLs in liver and spleen at d3 after transfer. CTLs were in vitro- stimulated with α- CD3 and α- CD28- coated microbeads in the presence or absence of IL- 12 p70 (5 ng/ml) before transfer (n=3 per group). (b) Fold of increase of hepatic CD CTLs in livers d3 after transfer and application of different TLR agonists compared to saline- treated mice (n=3 per group). (c) Total numbers of CD CTLs in spleens and livers at d3 after application of different doses of TLR9- L (n=3 per group). (d) Serum ALT values at different time points after CTL transfer and TLR9- L application (n=3 per group). (e) CFSE- profile of CD CTLs in spleen, liver and lung at 4 or 72hrs after transfer and TLR9- L application. Data are representative of histograms of three different mice. (f) Number of endogenous CD8 + cells in spleen and liver of mice as in (c) at d3 after application of different doses of TLR9- L. (g i) Flow sorted spleen- derived naïve CD44 low KLRG1 neg (N) or memory CD44 hi KLRG1 neg CD127 + CD8 + T cells (M) (0.6x10 6 per mouse) from CD mice were transferred into Ctrl- or TLR9- L- treated CD CD recipients fed with BrdU drinking water for 4 days. Transfer of equal number of in vitro- activated CD CTL (CTL) served as positive control. (g) At d4 after TLR9- L challenge, BrdU- incorporation by CD or CD cells in the liver of TLR9- L- treated mice. (h) Fold increase of T cells derived from transferred naïve or memory T cells. (i) Absolute number of T cells from experiments in (h). (j) Fold of change of the endogenous CD8 + T cells in the organs at d4 after FTY720 treatment (n=3 per group). NS, not significant. *P < 0.05, **P < 0.01, ***P < (unpaired Student s t- test). Data are representative of 2 independent experiments. (a d, f, h j; error bars, s.d.)

4 Supplementary Figure 2

5 Supplementary Figure 2: Characteristics of imates. (a) Immunohistochemistry for MHCII and CD11b in the liver at d3 after application of TLR agonists. (b) Number of CD CTLs in different organs of TLR9- L- treated mice at d3 after daily application of LTβR Ig (100µg per mouse i.p.) or control isotype Ig. (c) Quantification of the numbers of imates at indicated time points after TLR9- L treatment. (n=3 per group per time point). (d) Immunohistochemistry for CD11b in the liver at indicated time points after TLR9- L treatment. (e) Staining for cleaved caspase 3 in liver tissue of mice at d3 after TLR9- L challenge. (f) Immunohistochemistry for NKp46, CD19 or Ly6C + Ly6G (Gr- 1) in liver tissue at d3 after TLR9- L challenge. Arrows indicate imate and arrowheads point out CD19 + cells. (g) Number of CD CTLs in spleen or liver of TLR9- L- treated mice at d3 after daily application of anti- Ly6G Ig (1A8) or isotype Ig (2A3) (d- 1- d2; 100ug per mouse i.v.) (n=3 per group). Scale bar, 100 µm. NS, not significant. *P < 0.05, **P < 0.01, ***P < (unpaired Student s t- test). Data are representative of two independent experiments with 3 mice per group. (b, c, g; error bars, s.d.)

6 Supplementary Figure 3

7 Supplementary Figure 3: Preferential CTL expansion in imates. (a) Immunohistochemistry for CD11b and CD90.1 in the liver of splenectomized FTY720- treated mice at indicated time points after transfer of CD CTLs and TLR9- L challenge. Mice received BrdU in drinking water (0.8 mg/ml) during the entire time of the experiment (n=3 per group per time point). Arrowheads indicate imates. (b) BrdU- incorporation in transferred CD cells from liver or lymph node (LN) of TLR9- L- treated mice as described in (a). Data are representative of two independent experiments with 3 mice per group. Scale bar, 100 µm.

8 Supplementary Figure 4

9 Supplementary Figure 4: Typical forms of imates. (a) Immunohistochemistry for GFP and Ki- 67 in the liver at d3 after TLR9- L- challenge and transfer of GFP + CTLs (3x10 6 ). Distinct forms of imates are shown: a condensed form with either few or many proliferating CTLs, a dispersed form with many proliferating CTLs and one form that was almost dissolved with hardly any Ki67 + CTLs. (b) 3D structures of imates derived from serial liver sections with immunohistochemistry for CD11b and GFP from mice treated as in (a). 3D reconstruction of imates from 30 serial sections was generated using the novel software µcore (microdimensions GmbH, München, Germany) using a Mirax Midi Slide Scanner (Carl Zeiss microimaging GmbH, München, Germany). The resultant virtual slide data was then loaded into µcore where individual sections were automatically detected and separated. A 3D reconstruction was created using image- based registration, a technique to compute a common coordinate system for two images based on the images content. Consecutive sections were registered to each other and care was taken to avoid error propagation and bad results due to micro- artifacts like folding or tears. To achieve high- quality reconstruction also on high magnification levels an initial reconstruction result on low- resolution images was propagated from low to high magnification levels inherently using the pyramidal structure of virtual slides. Volumetric information for imates was computed by using an ellipsoidal shape as an approximation. Principal axes were measured four times each in µcore resulting in means (a, b, c) and corresponding standard deviations (σ!, σ!, σ! ). Volume: V!""#$%&#' =!! πabc Total variance: σ =!! π bcσ! + acσ! + abσ!

10 Supplementary Figure 5

11 Supplementary Figure 5: Phenotypic and functional analysis of imate- forming cells. (a) Gating strategies for analysis of CD11b + cells in the liver at d3 after TLR9- L application. CD146 + (LSECs), NKp46 + (NK cells) and Ly6G + (neutrophils) cells were precluded from further analysis of CD11b + cells. The NKp46 - Ly6G + CD11b+ cells are further divided into Kupffer cells (F4/80 hi CD11b + ), non- KC F4/80 + CD11b + and F4/80 - CD11b + cells based on their expression levels of F4/80 and CD11b. (b) Kinetics of increase in numbers of Kupffer cells (F4/80 hi CD11b + ), non- KC F4/80 + CD11b + cells and F4/80 - CD11b + cells in liver after TLR9- L- challenge. (n=3 per time point). (c) BrdU- incorporation into the different hepatic cell populations at d3 after TLR9- L challenge. (d) Flow cytometric analysis of Ly6C expression by Kupffer cell or non- KC F4/80 + CD11b + cells from the liver of mice at d3 after TLR9- L treatment. (e) Flow cytometric analysis of Ly6C, MHC II or CD11c expression on non- KC F4/80 + CD11b + cells from the liver at d3 after TLR9- L treatment. (f) Immunohistochemistry for CD11b in the liver of wild- type (WT) or CD11c.DOG mice at d3 after TLR9- L challenge and daily treatment with diphtheria toxin (DT) (30ng/g body weight, i.p.). (g) Numbers of cells in subpopulations distinguished by different expression level of CD11c in non- KC F4/80 + CD11b + cells in the liver at d3 after TLR9- L challenge ± DT application (n=2 per group). (h) CTL expansion in the liver of wild- type or CD11c- DOG mice (C57BL/6N) at d3 after transfer of CFSE- labeled CTLs (C57BL/6N), TLR9- L challenge and daily DT treatment (n=3 per group). All the data are representative of two independent experiments. NS, not significant. *P < 0.05, **P < 0.01, ***P < (unpaired Student s t- test). (b, g, h; error bars, s.d.)

12 Supplementary Figure 6

13 Supplementary Figure 6: Relevance of OX40L for CTL expansion in the liver. (a) Flow cytometric analysis of CD28 and OX40 expression by freshly isolated naïve CD8 + T cells or CTLs after in vitro stimulation with anti- CD3 + anti- CD28 microbeads in the presence IL- 12p70 (5ng/mL). (b) 1:1 Ratio of CFSE- labeled wild- type CTLs (WT, CD ) and OX40 / (KO, CD ) CTLs before adoptive transfer into WT or Cd80- Cd86- DKO recipients. Before transfer, CTLs were CFSE- labeled (2µM) to allow in vivo tracking and detection of proliferation. (c) Dot plots showing percentage of WT or OX40 / CTLs among total CD8 + T cells in spleen or liver in WT mice at d3 after transfer and TLR9- L application. CFSE dilution profile of transferred CTLs shown in histograms. (d) Dot plots showing percentage of WT or OX40 / CTLs among total CD8 + T cells in spleen or liver of Cd80- Cd86- DKO mice at d3 after transfer and TLR9- L application. CFSE dilution profile of transferred CTLs shown in histograms. Data are representative of two independent experiments with 3 mice per group.

14 Supplementary Figure 7 Supplementary Figure 7: Relevance of CCR2 + cells for control of acute LCMV infection. (a) Immunohistochemistry for CD11b + and CD8 + or Ki and CD8 + cells in the liver at d9 after acute LCMV infection (2x10 4 pfu per mouse). (b) Time kinetics of LCMV titers in blood of CCR2- CFP- DTR mice (TG) or non- transgenic littermates (Non- TG) after LCMV infection ± DT treatment (n=3-4 per group). Data are representative of 2 experiments. (b; error bars, s.d.)

15 Legends to supplementary video files Supplementary Video 1. & 2: Hepatic microvasculature and uptake of TLR9- L by sinusoidal cells. Liver intravital spinning- disc confocal microscopy in C57BL/6 mice at directly after TLR9- L treatment (10 µg per mouse i.v.). Mice were treated with anti- Ly6G 24h before imaging to eliminate CD11b + neutrophils from the circulation. TLR9- L was taken up by sinusoidal cells within 10 minutes after application (Video 1). There are few round CD11b + cells crawling or floating in sinusoids (Video 2). CD11b + cells are labeled with FITC- conjugated anti- CD11b (green); Kupffer cells labeled with PerCp- Cy5.5- conjugated anti- F4/80 (pink); TLR9- L labeled with Cy5 (blue); neutrophils (if remaining) labeled with PE- conjugated anti- Ly6G (red). Scale bars: 80 µm (video 1); 40 µm (video 2). Supplementary Video 3. & 4: Circulating CD11b + cells adhere in sinusoids after TLR9- L application. Liver imaging at 4h after receiving the same treatment as mentioned above. Increase in F4/80 - CD11b + cells appearing in sinusoids at 4h after TLR9- L application (Video 3). F4/80 - CD11b + cells firmly adhere to hepatic sinusoids but sinusoidal perfusion remains intact (Video 4). Scale bars: 80 µm (video 3); 40 µm (video 4). Supplementary Video 5 to 8: CD11b + cells form non- perfused structures, imates. At 24h after TLR9- L injection, F4/80 - CD11b + cells recruited from the blood but not F4/80 hi Kupffer cells form a non- perfused cell cluster (Video 5). At high magnification it is apparent that F4/80 - CD11b + cells firmly adhere within sinusoids and block sinusoidal blood perfusion; non- perfused areas can be identified by focusing at sinusoidal cells that have taken up fluorochrome- labeled TLR9- L (in blue), i.e. previously perfused areas (Video 6). At 48h, imates have grown in size and are still not perfused (Video 7 & 8). Scale bars: 80 µm (video 5 & 7); 40 µm (video 6 & 8). Supplementary Video 9: CD8 + T cells migrate slowly within imates. Transferred CD CD8 + T cells as well as endogenous CD8 + T cells were retained in imates and

16 migrated slowly within imates. CD11b + cells labeled with FITC- conjugated anti- CD11b (green); CD8 + T cells (both endogenous and transferred CD8 + T) labeled with PE- conjugated anti- CD8α (red); transferred CD8 + T cells labeled with allophycocyanin- conjugated anti- CD45.1 (blue); Kupffer cells labeled with Cy7- conjugated anti- F4/80 (pink). Scale bar: 80 µm. Supplementary Video 10-12: 3D structures of imates. 3D reconstruction of serial liver sections subjected to immunohistochemistry for detection of CD11b + cells (pink) and GFP + CTLs (brown) showing the different forms of imates.

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