Supplementary Figure Legends
Suppl.Fig.1. Lx bead distribution and uptake by macrophages in liver. A Distribution of Lx
particles without (control) or with macrophage depletion (+CloLip) in relation to Cx3cr1+
myeloid cells in different organs. Representative FACS plots and percentage of body-wide
distribution is depicted. B Kupffer cell susceptibility to depletion by liposomal clodronate.
Clodronate-loaded liposomes (CloLip) were injected i.v., and liver leukocytes were analyzed 12h
later. Cells were pregated on CD45+ and Ly6G-. C Analysis of liver sinusoidal endothelial cells
(LSEC) as potential phagocytes. Liver tissue was isolated 12hrs after Lx bead injection. Cells
were isolated by collagenase digestion and stained for the LSEC marker CD146. Cells were gated
as CD45- to eliminate possible LSEC-Kupffer cell aggregates. D Morphological analysis by
cytospin following FACS sorting according to the gating strategy depicted in Figure 1C. Please
note that the red particles are primarily located in KC. All experiments were performed in groups
of 3 animals and results were confirmed in two independent experiments. *P<0.05; **P<0.01;
***P<0.001
Suppl.Fig.2. Antigen presentation of particle associated OVA to CD4 but not CD8 T cells. A
Regulatory T cells were isolated from OTII donor mice by MACS isolation (Treg isolation kit).
Cells were stained with CFSE, and 2x106 cells were adoptively transferred into wt recipients.
Mice were challenged with latex particles and proliferation was measured in the blood 10d after
challenge. Optional depletion of phagocytes was performed by injection of clodronate-loaded
liposomes (CloLip) 12hrs before injection of Latex particles. B CFSE proliferation of OVAspecific CD8 and CD4 T cells. OTI and OTII cells were CFSE labeled, and 1x106 cells were
coinjected with 0.04% (v/v) latex (Lx) particles that were either plain or coupled to OVA using
10µl/g body weight. Phagocytic cells were depleted by injection of 200µl clodronate-loaded
liposomes (0.5mg/ml clodronate). Subcutaneous immunization with 50µl OVA-CFA (10mg/ml
OVA mixed 1:1 with CFA) was used as a positive control for T cell activation. Activation of T
cells was assessed by determining the amount of proliferating T cells in the blood 12d after
injection. Cells were pregated on CD45, Vα2 and CD4 or CD8 respectively. C Sorting of OTII
Foxp3.gfp cells. T cells were gated on expression of CD4 and CD45 and then sorted for GFP+
and GFP- cells.
Suppl.Fig.3. Models of tolerance induction in vivo. A Experimental design of in vivo
tolerance and cytotoxicity assay. To assess the tolerogenic effect of particle associated antigens,
animals were challenged i.v. with Lx particles, injecting 200µl with a concentration of 0.04%
(v/v) solid spheres. Optional depletion of phagocytes was performed by i.v. injection of 200µl
clodronate-loaded liposomes (0.5mg/ml clodronate) one day before Lx bead injection. 10 days
later, mice were immunized s.c. using IFA with or without OVA. In the next step, target cells
were injected i.v. 10 days after immunization. To this end, splenocytes were incubated with
OVA257-264 (SIINFEKL) peptide and labeled with 5µM CFSE, while non-pulsed splenocytes were
labeled with 0.5µM CFSE. Both target cell populations were mixed in a 1:1 ratio, and 2x106 of
each cell type were injected i.v. into recipient mice. Lysis of OVA257-26 target cells was measured
by flow cytometry 3 days later, assessing the ratio change between the peptide loaded and the
peptide free cells. B Flow cytometry analysis (representative plots) of target cell deletion during
in vivo cytotoxicity assay. Negative controls were treated by s.c. injection of PBS-IFA, positive
controls by plain Lx injection following s.c. injection of OVA-IFA. Depletion of macrophages
was performed by injection of clodronate-loaded liposomes (0.5mg/ml clodronate).