Supplemental Information. Granulocyte-Monocyte Progenitors and. Monocyte-Dendritic Cell Progenitors Independently

Transcription

1 Immunity, Volume 47 Supplemental Information Granulocyte-Monocyte Progenitors and Monocyte-endritic ell Progenitors Independently Produce Functionally istinct Monocytes lberto Yáñez, Simon G. oetzee, ndre Olsson, avid E. Muench, enjamin P. erman, ennis J. Hazelett, Nathan Salomonis, H. Leighton Grimes, and Helen S. Goodridge

2 c-kit 34 X3R %Lin - cells c-kit Figure S LKS 34 + cells Lin cells 34 G GP iii ii i iv v vi i - ii - + lo iii iv G v GP vi + i ii iii iv v vi X3R-GFP i + + lo - - lo ii + lo + + lo + - lo iii + + lo + - hi hi 0.9 iv G + + hi - - lo lo 3.5 v GP + + hi - + lo lo 3.8 vi hi - + hi hi 2.9 Figure S. haracterization of myeloid progenitors from mouse bone marrow. (related to Figure ) () MS-sorted lineage-negative (Lin ) mouse bone marrow cells were evaluated by flow cytometry (c-kit [7], Sca-, 34, [6 and 32],,, ). c-kit + Sca cells were gated and visne analysis was performed using ytobank.,, and expression was visualized in a stacked dot plot of and tsne2 (left panels) to facilitate identification of progenitor populations (right panel). () Flow cytometry gating of bone marrow Lin cells for progenitor identification and sorting. lear separation of the lo and hi gates is very important for optimal separation of the progenitor fractions. () X3R expression by progenitor subsets from X3R-GFP reporter mice was assessed by flow cytometry. () Summary of surface marker expression by myeloid progenitor subsets.

3 + G FU Figure S2 post-sort progenitor analysis methylcellulose cultures c-kit c-kit c-kit lo hi hi lo G G-derived cells Lin c-kit M G GM GEMM -derived cells Lin c-kit G- or derived cells Lin c-kit E differentiation trajectory G Figure S2. haracterization of myeloid progenitors from mouse bone marrow. (related to Figure ) () Post-sort analysis of progenitor subsets after FS sorting from mouse bone marrow. () 000 Gs or s were seeded in methylcellulose medium, and colonies were evaluated week later (M monocyte, G granulocyte, GM granulocyte-monocyte, GEMM granulocyte-erythrocytemegakaryocyte-monocyte). (-) Gs and s were cultured in vitro with M-SF for 48 hours, and or production was assessed by flow cytometry (overlaid in ). (E) Trajectory of monocytecommitted progenitor ( or ) production by Gs and s.

4 day 0 day 0 day 7 day 7 day 4 day 4 day 7 day 7 FU day 4 day 4 Figure S3 G-derived cells - bone marrow -derived cells - bone marrow + + mp mp mp mp neutrophils monocytes cs ps - + lo -derived cells - bone marrow - -derived cells - bone marrow methylcellulose cultures lo - M GM E GEMM G EMk Mk - + lo mp- mp- G GP P mp- mp- neutrophils hi monocytes cs and ps mp- mp- neutrophils monocytes ps + monocytes Figure S3. Myeloid cell production by subsets. (related to Figure ) (-) The progeny of adoptively transferred Gs and s () and - + lo and - cells () from donor (45.2) mice were identified by flow cytometry of recipient (45.) bone marrow cells on the days indicated. () lo and - cells per well were plated in methylcellulose media and colonies were counted after week of culture. G granulocyte, M monocyte, E erythrocyte, Mk megakaryocyte, GM granulocyte + monocyte, EMk erythrocyte + megakaryocyte, GEMM granulocyte + erythrocyte + monocyte + megakaryocyte. () Model of neutrophil, monocyte and production by - + lo cells, with 2 independent pathways of monocyte differentiation via i) - cells, Gs and s, and ii) s and s.

5 8a MH II WT Figure S4 ay 7 ay 4 bone marrow blood spleen bone marrow blood spleen hi X3R-GFP X3R-GFP X3R-GFP hi + Irf8 / WT G-derived Irf8 / G-derived hi hi hi + E bone marrow blood spleen F spleen MH II MH II MH II Figure S4. Profiling of G- and -derived monocyte subsets. (related to Figure 2) () Flow cytometry gating strategy for characterization of + + cells in the bone marrow, blood and spleen. () + + cells from the bone marrow, blood and spleen of X3R-GFP reporter mice were analyzed by flow cytometry to assess X3R expression by the hi, + and subsets. () For assessment of fully differentiated hi monocytes in Irf8-deficient (plus WT and Nur77 (Nr4a)-deficient) mice, monocytes were also gated on + cells. () Flow cytometry gating strategy for assessing production of the hi (and + ), + and subsets of + + cells by adoptively transferred Gs, s and s+s from 45.2 donor mice. Gating of G-derived cells is shown. (E) MH II and expression by the hi, + and subsets of + + cells from the bone marrow, blood and spleen was assessed by flow cytometry. (F) Expression of Zbtb46 by splenic cs and ps from Zbtb46-GFP reporter mice was assessed by flow cytometry. hi ps Zbtb46-GFP + cs 8a + cs

6 -derived G-derived -log0 FR Figure S5 M-SF M-SF c-kit + c-kit + + G (ex vivo) (ex vivo) M-SF G-derived hi monocyte (G-mono) M-SF -derived hi monocyte (M-mono) G-mono M-mono log2 (G/). G UP 932 (6.75%). UP 407 (2.95%) G UP G UP G UP G UP UP G-mono UP UP M-mono UP G-mono UP UP UP M-mono UP UP UP UP UP UP M-mono UP UP G-mono UP UP M-mono UP UP G-mono UP G pathway-enriched: G UP = G>, UP = >, G-mono UP = G-mono>M-mono pathway-enriched: UP = G<, UP = <, M-mono UP = G-mono<M-mono Figure S5. Isolation of G- and -derived cells for RN sequencing. (related to Figure 3) (-) Gs and s isolated from the pooled bone marrow of 20 mice were cultured in vitro with M-SF to derive monocyte-committed progenitors (s and s) and hi monocytes. Monocyte progenitors and hi monocytes derived from Gs (s and G-mono) and s (s and M-mono) were harvested from the cultures on days 2, 3 and 4, and the three timepoint samples were pooled for RN extraction. RN was also extracted from the ex vivo G and fractions from which the monocyte progenitors and monocytes were derived. The whole process was repeated using 20 additional mice to obtain a replicate set of samples. () Volcano plot of genes enriched (2-fold, adjusted p value < 0.05) in Gs (G UP ) versus s ( UP ). The number of enriched genes and percentage of the total genes expressed by Gs and is indicated. () Venn diagrams (plotted to scale) showing overlapping sets of enriched genes ( UP = enriched in the specified subset compared to its counterpart in the other lineage).

7 Figure S6 G monocyte progenitors (Olsson et al., Nature 206) s s Figure S6. Profiling of G- and -derived monocyte progenitor subsets. (related to Figure 3) () Gene expression by the progenitors (Gs, s, s and s) was viewed through the lens of progenitor population-specific genes and selected markers previously defined in a single-cell RN sequencing study (Olsson et al., 206). ll genes with an RPKM> are shown. HSP/P hematopoietic stem cell progenitor, NK natural killer cell, Meg megakaryocyte, Eryth erythrocyte, dendritic cell, Mono monocyte, Gran granulocyte, Myelocyte myelocyte and metamyelocyte. () Heatmap of differential gene expression (2-fold, p<0.05) by the presumed s and s ( s and s ) among the 58 previously identified monocyte progenitors profiled by single-cell RN sequencing (Olsson et al., 206).

8 tsne2 tsne2 tsne2 tsne2 tsne2 SS- SS- E 73 Figure S MHII G-derived hi monocytes -derived hi monocytes + cs cs lusters ells M-monoderived G-monoderived MHII MHII lustering: total hi monocytes 209a H2-b H2-a Figure S7. Single-cell RN sequencing of hi monocytes. (related to Figures 4-6) () (surface) and MHII (surface and intracellular) expression by splenic hi monocytes derived from adoptively transferred Gs and s, as well as endogenous s, was assessed by flow cytometry. MFI values are indicated. (-) hi monocytes were sorted from 3-day in vitro cultures of Gs and s with M-SF, and then cultured with GM-SF for a further 4 days prior to assessment of and MHII expression by flow cytometry (). 86 and expression by subsets of + cells was also assessed by flow cytometry (). () ack-gating of hi monocyte subsets from IRF8- GFP Gfi-tdTomato reporter mouse bone marrow. (E-G) Gene expression by hi monocytes from the bone marrow of wild-type and IRF8-GFP Gfi-tdTomato reporter mice (78 and 4 monocytes, respectively) was assessed by single-cell RN sequencing. (E) Unsupervised clustering of hi monocytes using IGS in ltnalyze. (F) omb plots of transcription factor expression by individual hi monocytes and monocyte progenitors. (G) Total wild-type hi monocytes were also clustered using ytobank (tsne plots are shown). lustering was performed using 252 genes that were differentially expressed in Gand -derived hi monocytes (G-mono UP and M-mono UP genes identified by bulk sequencing) and also detected by single-cell RN sequencing. Expression intensities (log0 RPKM) of mo-producing monocyte markers (209a, H2-b, H2-a and ) and the c transcription factor Zbtb46 are shown. 86 G-mono-derived + MHII /lo cells M-mono-derived + MHII /lo cells M-mono-derived + MHII hi cells ells: total hi monocytes IRF8-GFP hi Gfi-Tomato hi hi monocytes G F FS- IRF8-GFP hi Gfi-Tomato hi hi monocytes total hi monocytes Zbtb46 total bone marrow cells hi monocytes: IRF8-GFP hi Gfi-Tomato hi IRF8-GFP lo Gfi-Tomato lo Irf8 Gfi