F4/8 % in the peritoneal lavage 6 4 2 p=.15 n.s p=.76 CD115 F4/8 hi CD115 + F4/8 + CD115 + F4/8 hi CD115 + F4/8 + CD115 + MHCII MHCII Supplementary Figure S1. CD11b deficiency affects the cellular responses to thigolycolate. Peritoneal cells were harvested from or mice 4 days after thioglycolate injection. Representative flow cytometry profiles of F4/8 and CD115 staining on peritoneal cells. Histogram showing the MHCII expression on different MF subpopulations (F4/8 hi /CD115 + and F4/8 + /CD115 + ) and their respective percentage (mean ±S.E.M, n=3) in the peritoneal lavage from and mice. p<.5 (t-test).
RANTES (ng/ml) RANTES (ng/ml) RANTES (ng/ml) IL-6 (pg/ml) IL-6 (pg/ml) IL-6 (pg/ml) a 2 MHCII 75 5 CD8 2 CD86 1 1 25 GM-A GM-F GM/IL-4 GM-A GM-F GM/IL-4 GM-A GM-F GM/IL-4 4 3 CD4 15 1 TLR4 3 2 CD14 2 1 5 1 GM-A GM-F GM/IL-4 GM-A GM-F GM/IL-4 GM-A GM-F GM/IL-4 b GM-CSF derived adherent cells (GM-A) 5 4 3 2 p=.2 p=.2 5 4 3 2 GM-CSF derived floating cells (GM-F) p=.392 p=.193 p=.83 2 1 GM-CSF/IL-4 derived (GM/IL-4) p=.7 p=.8 p=.87 1 1 3 2 1 1 1 1 (ng/ml) 6 4 2 1 1 1 (ng/ml) p=.36 p=.44 p=.29 5 4 3 2 1 1 1 (ng/ml) p=.11 p=.438 p=.163 1 c 4 3 1 1 1 (ng/ml) TLR4 11 1 9 8 7 1 1 1 (ng/ml) CD14 1 1 1 (ng/ml) 2 3 1 2 1 BM-MFs BM-MFs Supplementary Figure S2. CD11b is required for -induced cytokine production by myeloid cells differentiated with GM-CSF alone. Bone marrow cells from or mice were cultured in 16.67ng/ml of GM-CSF. Floating cells (GM-F) and adherent cells (GM-A) were collected at day 6. BM- DCs (GM/IL-4) were also generated. (a) Surface expression of MHC class II, CD8, CD86, CD4, TLR4 and CD14 on unstimulated and BM-derived cells were examined by flow cytometry (=test-isotype control) (mean ±S.E.M, n=3). (b) Cells were then treated with a range of concentrations from 1ng/ml to 1ng/ml. IL-6 and RANTES were measured by ELISA 24hrs later. Pooled data from three independent experiments are presented (mean ±S.E.M, n=9). (c) Surface expression of TLR4 and CD14 on unstimulated and BM-MFs and measured by flow cytometry. Data are expressed as delta mean fluorescent intensity (=test-isotype control) (mean ±S.E.M, n=3). p<.5, p<.1, p<.5 (t-test).
ng/ml ng/ml a Day 5 Day 6 BM-MFs Bone marrow cells + M-CSF + GM-CSF MF GM MF GM/IL4 Stimulate with 1mg/ml Bone marrow cells + GM-CSF + IL-4 b 3 2 F4/8 1 CD11c 75 4 3 MHCII 1 5 25 2 1 c IL-6 p=. 75 3 RANTES p=.121 5 2 25 1 1 2 3 4 1 2 3 4 Supplementary Figure S3. CD11b dependency in responses is restricted to and is a lineage/differentiation effect. (a) Schematic outline of the experimental design: bone marrow cells from and mice were differentiated with M-CSF for 5 days to obtain BM-MFs. The M-CSFcontaining medium was removed at day 5, the wells were washed with PBS and fed with media containing either GM-CSF or GM-CSF plus IL-4. BM cells conventionally differentiated into or BM-MFs were used as controls. On day 6 cells were stimulated with 1mg/ml of for 24hrs. (b) Surface expression of F4/8, CD11c and MHCII on unstimulated and BM-derived cells described in (a). Data are expressed as delta mean fluorescent intensity (=test-isotype control) (mean ±S.E.M, n=3). (c) Production of IL-6 and RANTES by the BM-derived cells described in (a) were measured by ELISA 24hrs after the stimulation. Data represent mean ± S.E.M (n=3) p<.5, p<.1 (t-test).
Counts TLR4 (Sa15-21) Counts TLR4(Sa15-21) Counts TLR4(MTS51) Counts TLR4 (MTS51) a MFs 3 min 9 min MFs 3 min 9 min 4 3 BM- MFs p=.2 p=.5 p=.1 p=.3 2 1 3 9 3 9 TLR4 ( MTS51) time (min) after stimulation DCs 3 min 9 min DCs 3 min 9 min 3 n.s p=.6 p=. n.s p=.46 n.s p=.6 2 1 TLR4 ( MTS51) 3 9 3 9 time (min) after stimulation b MFs 3 min 9 min MFs 3 min 9 min 4 3 p=.8 p=.65 BM- MFs p=.37 p=.13 2 1 3 9 3 9 TLR4 (Sa15-21) time (min) after stimulation DCs 3 min 9 min DCs 3 min 9 min 1 75 p=.17 p=.17 n.s p=.31 n.s p=.53 5 25 3 9 3 9 TLR4 (Sa15-21) time (min) after stimulation Supplementary Figure S4. CD11b is required for -TLR4 binding and the subsequent TLR4 internalization in. or BM-MFs and were treated with (1mg/ml) for the times indicated. (a) Cell were stained with the MTS51 mab, which recognizes only -free TLR4-MD-2 complex, to measure binding. (b) Cells were stained with the Sa15-21 mab that detects the TLR4-MD-2 complex irrespective of the presence of. The loss of surface Sa15-21 staining indicates receptor internalization. Representative histograms are shown. Data are expressed as delta mean fluorescent intensity ( = test isotype control ) (mean ±S.E.M, n=3). p<.5, p<.1,p<.5 (t-test).
Fold of phospho-syk induction Fold of phospho-akt induction MHCII surface staining (fold induction) Counts % of surface CD14 % of surface CD11c Counts % of surface CD14 a MFs MFs b 3 min 1 8 6 9 min 4 2 MFs MFs 3 6 9 CD14 DCs DCs time (min) after stimulation p=.49 1 8 6 p=.14 b 1 8 6 4 2 DCs DCs 4 2 CD14 3 6 9 time (min) after stimulation 3 6 9 time (min) after stimulation c 72 72 3 min 9 min p-syk (tyr-525/526) Total SyK d 4 3 2 p=.145 p=.94 6 p-akt 1 6 3 3 Total Akt 3 min 9 min 3 min 9 min BM-MFs 2 2 1 1 3 45 9 Time (min) 3 45 9 Time (min) Supplementary Figure S5. CD11b contributes to CD14 internalization but not to Akt or Syk phosphorylation in. or BM-MFs and were treated with (1mg/ml) for the times indicated. Surface expression of (a) CD14 or (b) CD11c was measured by flow cytometry. (a,b) Displayed are the of specific receptor staining at each time point ( at time as 1%) (mean ±S.E.M, n=3). Representative histograms are shown. (c) or were left untreated () or treated with (1mg/ml) for the time indicated. The presence of phosphorylated (p-)syk and Akt was examined by western blot. Quantification of band intensity showing the fold increase in phospho-protein levels relative to untreated cells and normalized to total protein levels (mean ±S.E.M, n=3). (d) or BM-MFs and were treated with (1mg/ml) for the times indicated. The fold increase in MHC class II expression was compared to untreated cells (mean ±S.E.M, n=3). p<.5, p<.1 (t-test).
Counts % CD25 + Foxp3 - T cells (gated CD4 + cells) % of CD8 + Uty + cells (gated CD8 + cells) CD4 % IFNg % a TGF-b TGF-b + IL-6 TGF-b + DC TGF-b + DC 14 % of Th17 cells p=.171 11 8 5 IL-17 2 15 % of Foxp3 + cells p=.329 1 Foxp3 b HY peptide HY peptide/ c 75 5 25 ex vivo after in vitro re-stimulation 5 4 p=.262 3 2 CFSE (DC:T ratio = 2:1) 1 ex vivo after in vitro re-stimulation Supplementary Figure S6. CD11b regulates the T cell responses elicited by -primed DCs in vitro and in vivo. (a) Naïve CD4 + T cells were stimulated with plate-bound anti-cd3/cd28 antibodies in the presence of TGF-b (with/without IL-6) or conditioned medium () from -treated or DCs. The percentage of Foxp3 + cells was assessed at day 3. Cells were re-stimulated with PMA and ionomycin in the presence of GolgiSTOP for 5hrs and the percentage of Th17 cells was determined by intracellular staining. Data represent mean ±S.E.M from four independent experiments (p<.5,t-test). (b) Female or mice (n=3) were given twice i.n. 1mg HYDby peptide or peptide plus 3mg. Splenic CD11c + cells, isolated 24hrs later, were co-cultured with Marilyn T cells for 72hrs. Representative flow cytometry histograms of CFSE-labelled Marilyn T cells. (c) HY peptide/ treated and mice that had rejected the male grafts were boosted intraperitoneally with male spleen cells. The mice were sacrificed seven days later and the splenocytes were re-stimulated in vitro with irradiated male splenocytes for 1 week. The percentage of antigen specific CD8 + Uty + and CD4 + CD25 + Foxp3 - T cells were analysed by flow cytometry before (ex vivo) and after the in vitro restimulation. Data represent mean ± S.E.M (n=7) (p<.5,mann-whitney test).
RANTES (ng/ml) RANTES (ng/ml) IL-6 (pg/ml) IL-6 (pg/ml) BM- MFs BM- DCs 4 p=.243 1 3 75 2 5 1 25 + ic3b-coated RBC + ic3b-coated RBC 5 4 p=.42 5 4 3 3 2 2 1 1 + ic3b-coated RBC + ic3b-coated RBC Supplementary Figure S7. ic3b-coated red blood cells (RBC) downregulate -induced cytokine production in BM-MFs but not. Guinea pig RBCs were opsonised with mouse C5-deficient serum to avoid complement lysis and were added to BM-MFs and at a 1:1 ratio (RBC:cells). Cells were then stimulated with 1ng/ml of. The amounts of IL-6 and RANTES secreted were measured by ELISA at 24hr. Data represent mean ± S.E.M (n=3) p<.5, p<.1 (ttest).
a min 3 min 9 min p-p38 13 13 Total p38 13 13 3 min 3 min 13 13 p-erk1/2 p-jnk Total Erl1/2 13 Total JNK 13 3 min 13 p-ikba 13 GAPDH
b min 3 min 9 min 18 min p-p38 13 13 Total p38 13 13 3 min 9 min p-erk1/2 13 13 Total Erl1/2 13 13 3 min 15min 3 min 13 13 p-jnk p-ikba 13 13 Total JNK GAPDH upplementary Figure S8 : (a) BM-MFs (b), full Blots relating to Figure 2.
BM-MFs 3 min 9 min 3 min 9 min P-TBK1 225 15 12 76 225 15 12 76 Total TBK1 225 15 12 76 225 15 12 76 3 min 9 min 3 min 9 min 13 13 P-IRF-3 13 13 Total RF-3 Supplementary Figure S9 : full Blots relating to Figure 5.
P-IRF-3 13 13 Total IRF-3 13 13 9 min Supplementary Figure S1 : full Blots relating to Figure 6.
3 min 9 min p-syk (tyr-525/526) 13 Total Syk 13 3 min 9 min 13 p-akt 13 Total Akt Supplementary Figure S11 : full Blots relating to Supplementary Figure S5.