Nature Immunology: doi: /ni Supplementary Figure 1. Examples of staining for each antibody used for the mass cytometry analysis.

Transcription

1 Supplementary Figure 1 Examples of staining for each antibody used for the mass cytometry analysis. To illustrate the functionality of each antibody probe, representative plots illustrating the expected staining profiles of each antibody are shown. Cells gated as shown (annotated above each plot) are represented as two parameter dot plots. 1

2 Supplementary Figure 2 DensVM analysis flowchart. This flow chart describes how tsne transformed data are clustered using the DensVM approach. Using the first two dimensions of tsne map coordinates, a peak detection algorithm is applied that results in some cells assigned to clusters as previously described (see methods). These classified events together with their 38-antibody expression profiles are used to train Support Vector Machine (SVM) algorithm. Using remaining unclassified cells are then classified based on the SVM leaving an output with all cells classified. 2

3 Supplementary Figure 3 tsne analysis of an independently collected data set comprising four mice and six tissues (spleen, liver, mesenteric lymph nodes, lung, bone marrow, brain). Two C57Bl6 wild-type mice and two Csf2rb / mice were analyzed as described for the main dataset resulting in a new two-dimensional tsne map of all myeloid cells derived from all tissues of these four mice. (a, b) As in Figure 2 manual traditional gating was applied to cell events to annotate the major clusters of cells derived from (a) lung and (b) bone marrow. For cluster 6-, 8- and 28-like cells, manual gating as illustrated in Figure 3a was used to annotate these cells. Note that the dominant clusters of cells display similar arrangements as in Fig. 2. (c) As in Figure 6a, the distributions and composition of lung cells are compared between two C57Bl6 wildtype mice and two Csf2rb / mice. Arrows point to clusters of cells (corresponding to alveolar macrophages in magenta, cluster 6-like eosinophils in orange, and cluster 28-like innate lymphoid cells in black) where reductions in frequencies were observed in Csf2rb / mice as described and further validated in Figure 6. 3

4 Supplementary Figure 4 tsne analysis objectively delineates myeloid cell subsets of lymph node, thymus and liver. As in figure 2, cells are plotted as tsne composite dimensions 1 vs. 2. In the left panels, cells are colored according to traditional/biased definitions using gating strategies similar to that in Figure 1. gray color indicates cells that are remaining and unaccounted for by these definitions and predominant clusters are indicated with arrows. Cells in the right panels are colored according to their unbiased cluster designation (see results). Notable clusters including those that correspond to subsets not accounted for by traditional gating are labeled with cluster numbers. Frequencies as percentage of total CD45 + CD90 - CD19 - CD3 - are shown for each subset. 4

5 Supplementary Figure 5 Use of fluorescence flow cytometry based cell sorting to analyze the morphology of ambiguous cell subsets delineated by higher-order analysis. (a) For flow cytometric cell sorting of cells from lung tissue, matching characteristics of cluster 8 (eosinophils), cluster 6 (Ly6C - 5

6 monocytes) and cluster 28 (NK cells) were gated and sorted at shown. Representative Geimsa stained images for each are shown in Figure 3. (b) For flow cytometric cell sorting of bone marrow, cells representative of cluster 4 (Ly6G + neutrophils), cluster 11 and 12 (Ly6C + monocytes, clusters 11 and 12 not distinguished by these fluorescent markers), and cluster 5 (Ly6G - Ly6G/C + Ly6C int granulocyte precursors) were gated as shown. Clusters 5 and differ in the expression of CD48 as shown in the histogram on the right. Representative Giemsa stained cells are shown for cluster (Ly6C + monocytes). Images of clusters 4 and 5 (neutrophils and granulocyte precursors) in are shown in Figure 4. (c) For flow cytometric cell sorting of thymus, cells representative of clusters (DCs), cluster (Ly6C + monocytes) and cluster 7 (CD11c + eosinophils) were gated as shown, sorted, mounted and subsequently Giemsa stained. Representative cells from cluster (DCs) and clusters (Ly6C + monocytes) are shown. A representative cell from cluster 7 (eosinophil) is shown in Figure 3. (d) Additional fluorescence flow cytometry analysis of a thymus-derived ambiguous cell subset. Representative plots used to identify cluster (Ly6C + monocytes, red) and cluster 7 (CD11c + eosinophils, green) are shown. Parallel staining of Siglec-F is also shown. Histograms of Siglec-F staining cells derived from red and green gates are also shown, which demonstrate strong Siglec-F staining on cells similar to those identified as cluster 7. 6

7 Supplementary Figure 6 tsne analysis used to compare cellular composition of tissues derived from individual Csf2rb / and Csf2rb +/+ mice. (a-c) As in figure 6a, bone marrow-, spleen-, and liver-derived cells color-coded by DensVM cluster number are plotted by tsne scores. For each panel, the three upper plots represent three replicate C57Bl6 wild-type tissue cells and the three lower plots represent three Csf2rb / tissue cells run in parallel. 7

8 Supplementary Figure 7 Biaxial plots comparing Csf2rb / and Csf2rb +/+ lung myeloid subsets. (a) Standard biaxial plots to were used to delineate lung alveolar macrophages (CD11c+MHC II -/low ) and their quantities were compared between Csf2rb / vs. Csf2rb +/+ mice (Average ± S.E.M. frequencies are provided, n=3 mice each). (b) Based on the gate in (a) to exclude AM and DCs, frequencies of Ly6C-Ly6G- (non-monocyte, non-neutrophils) were quantified. These cells are composed of both eosinophils (cluster 6) and NK cells (cluster 28), both found to be reduced in Csf2rb / mice and here their frequencies are compared between Csf2rb / vs. Csf2rb +/+ mice (Average ± S.E.M. frequencies are provided, n=3 mice each). 8

9 Supplementary Figure 8 Comparison of granulocyte composition in various tissues derived from Csf2rb / and Csf2rb +/+ mice. (a) As in figure 4, the five different populations of neutrophil-like cells and two different populations of eosinophil-like cells were used for ISOMAP dimensionality reduction analysis to compare their overall phenotypic relatedness. For each cell cluster (from Csf2rb +/+ tissues, left and Csf2rb / tissues, right), the median values in ISOMAP dimension 1 are plotted against the relative frequencies of each cluster for each tissue (expressed as percentage of total granulocytes for each tissue). 9

10 Supplementary Figure 9 Florescence flow cytometric gating strategy to quantify and validate changes in composition observed in tissues from Csf2rb / (bottom) and Csf2rb +/+ mice (top). (a) Representative plots are shown as they were used to identify cells corresponding to cluster 16 (MHCII lo /CD11c + : AM), cluster 28 was interrogated by further gating on CD45 + MHCII Ly6C low/ Ly6G CD11b low (NK cells) and clusters 6 and 8 were further gated on by F4/80 + CD11b + and specifically interrogated for CD11c (Cluster 8: Ly6C low monocytes) and CD24 (Cluster 6: eosinophils). The same gating strategy was used to analyze (b) liver and (c) spleen. In panels b and c, for cluster 28, we further excluded more abundant CD3 + and B220 + cells. 10

11 Supplementary Figure 10 Comparison of DC composition in lung tissue derived from Csf2rb / and Csf2rb +/+ mice. (a) To compare CD103 expression in the DC compartment of Csf2rb / vs. Csf2rb +/+ CD11c + MHCII + cells are shown. Note, reduction of CD103 expression on CD11b - cells in Csf2rb -/- lung tissue. (b) Gating strategy to identify CD11b /CD24 + (also referred to as CD103 + DCs red), CD11b + CD24 + CD301 + DCs (green) and CD11b + CD24 CD301 interstitial macrophages (orange). (c) To specifically focus on DC phenotypic profiles, tsne analysis was repeated using CD11c+MHCII+ cells derived from spleen, liver, bone marrow and lung. From this analysis two new tsne dimensions were used to segregate lung DCs with better resolution. To annotate this representation, cells gated in (a) were color coded and overlaid on tsne contour plots (CD11b - CD24 + DCs in red, CD11b + CD24 - CD301 + DCs in green and CD11b + CD24 - CD301 - interstitial macrophages in orange). Note the prominence of the two DC subsets in Csf2rb +/+ mostly lost in Csf2rb / replaced by a prominent population of interstitial macrophage-like cells and an additional unknown population. (d) To compare the phenotypes of the four cell populations defined by zoomed-in tsne analysis in (c), the median intensities of each marker for each population are summarized as a heat-plot. 11

12 Supplementary Table 1. Table of antibodies used for mass cytometry analysis listing metal conjugate, antibody clone name, supplier and description of each marker. Supplementary Table 2. Table of defining markers for major types of traditionally defined mouse myeloid cells. Supplementary Table 3. Table of descriptions, defining markers for clusters to of mouse myeloid cells delineated by tsne/densvm in this study.

13 Supplementary Table 1. Isotope tag Sequence Clone/Company Purpose Qdot CD19 6D5 (Invitrogen) B cell marker (Cd112/114) Rh- 103 Barcode Barcode Pd- 104 Barcode Barcode Pd- 106 Barcode Barcode Pd- 108 Barcode Barcode Pd- 110 Barcode Barcode Ln- 113 Barcode Barcode Pr- 141 CD F11 (Biolegend) Hematopoetic cell marker Nd- 142 MHC class II Y3P (BioXcell) Antigen presenting cells Nd- 143 B220 RA3.341/61 (BioXCell) B cells and pdc Nd- 144 CD11a FD441.8 (BD) Integrin, important for leukocyte homing Nd- 145 Ly6G/C RB6-8C5 (BD) Granulocytes/monocytes Nd- 146 CD (BD) T cell and DC Sm- 147 Ly6G IA8 (BD) Granulocyte Nd- 148 Ly6c HK1.4 (Biolegend) Monocyte Sm- 149 CD4 H (BD) T cell and low levels on DC Nd- 150 CD54 YNI.7.4 (BioXCell) ICAM- 1, adhesion molecule. Eu- 151 CD62L MEL14 (Biolegend) L- Selectin, adhesion molecule Sm- 152 PDL2 Ty25 (Biolegend) Co- inhibitory molecule expressed by APCs Eu- 153 CD11b M1/70 (BD) Most myeloid cells, subset of NK cells Sm- 154 CD C11 (BD) T cell marker, T cell receptor Gd- 155* CD86 GL7 (BD) Costimulatory molecule expressed by APCs Gd- 156 BST2 PDCA- 1 (Purified in house) Key marker for plasmacytoid dendritic cells Gd- 157 Mertk (SAv) Mertk- biotin- accession Key marker for macrophages number (R&D Systems) Recombinant 6xCys- Strepatvin (see Newell, et al., 2013) Gd- 158 Ter119 TER119 (Biolegend) Marks erythrocytes and cells of the erythrocyte lineage Tb- 159 F4/80 C1:A3-1 (Serotec) Expressed many cells of mononuclear phagocyte system Gd- 160 FcER1 MAR- 1 (ebioscience) Fc- receptor, Marks macrophages as well as inflammatory monocyte derived cells Dy- 161 CD25 PC61 (Biolegend) IL2Ra subunit, expressed by numerous IL- 2 responsive cell types Dy- 162 PDL- 1 10F.9G2 (Biolegend) Co- inhibitory molecule expressed by APCs Dy- 163 CD11c N418 (Biolegend) Marks most antigen- presenting cells Dy- 164 CD103 2e7 (Biolegend) Expressed by a subpopulation of DCs, Binds epcam, localizes expressing cells to the epithelial region Ho- 165 CD64 X54-5/7.1 (Biolegend) Fcgr1a on macrophages Er- 166 CD48 HM48-1 (Biolegend) Expressed at varying levels by different

14 Er- 167 CCR2 (a- APC) CCR2 APC (R&D systems) a- APC - APC003 (Biolegend) leukocyte subsets Chemokine receptor, important for monocyte migration Er- 168 CD44 IM7 (Biolegend) Adhesion molecule Tm- 169 CD115 AF5 (purified in- house) CSF- 1R, receptor for M- CSF, expressed by most mononucleated myeloid cells Er- 170 CD38 90 (Biolegend) cyclic ADP ribose hydrolase, expressed by many leukocytes Yb- 171 SiglecH 551 (Biolegend) Key marker for plasmacytoid dendritic cells and microglia Yb- 172 CD301 (a- PE) CD301 PE - LOM- 14 (ebioscience) ape - PE001 (Biolegend) subpopulation of mononuclear phagocyte lineage Yb- 173 SIRPa (CD172a) P84 (BD) Marks subpopulation of mononuclear phagocyte lineage Yb- 174 CD24 M1/69 (Biolegend) Marks subpopulation of mononuclear phagocyte lineage Lu- 175 ESAM 1G8 (ebioscience) Marks subpopulation of DCs Yb- 176 CD90 T24/31 (BioXcell) Pan T cell marker lr- 191/193 DNA DVS Sciences Cell identifier 195 CisPlatin Sigma Aldrich Viability stain live/dead Cell length Cell length N/A Number of TOF pushes collected for the cell event

15 Supplementary Table 2. Subset label Neutrophils Eosinophils Ly6C + Monocytes Ly6C low Monocytes Microglia Red pulp macrophages Alveolar macrophages CD8α/CD103 + DCs CD11b + DCs pdcs Defining markers CD45+, Ly6G+, CD11b+, F480-, CD115+ CD45+, CD11b+, F480+, CD115+, Siglec- F+ CD45+, CD115+, CD11b+, Ly6C+, CD11c-, CD43- CD45+, CD115+, CD11b+, Ly6Clow, CD11c+, CD43+ CD45+, CD11b+, F4/80+ CD45+, CD11c+, CD11b+, F4/80+ SSChigh, CD45+, CD11c+, MHC II- /+, CD11b-, F4/80+ CD45+, CD11c+, MHC II+, CD103/CD8a+, CD24+ CD45+, CD11c+, MHC II+, CD11b+, CD24+, Sirpa+ CD45+, CD11c int, MHC II- /+, B220+, Ly6C+, Siglec- H+, BST2+

16 Supplementary Table 3 C57BL6 average frequency per tissue Cluster# Subset label Defining markers BM Brain Kidney Liver LN Lung Spleen Thymus 1 Neutrophils CD45+, Ly6G+, CD11b+, F480-, CD115+, CD62L low, CD25-2 Neutrophils CD45+, Ly6G+, CD11b+, F480-, CD115+, CD62L+, CD25+ 3 Neutrophils CD45+, Ly6G+, CD11b+, F480-, CD115+, CD62L+, CD25+, MerTK+, PDL2+ 4 Neutrophils CD45+, Ly6G+, CD11b+, F480+, CD115+, CD62L+, CD25+, MerTK+, PDL2+, FceR+ 5 Neutrophils CD45+, Ly6G+, CD11b+, F480+, CD115+, CD62L+, CD25-, MerTK+, PDL2+, FceR+ 6 Eosiniophils CD45+, CD11b+, F480+, CD115+, CD62L-, CD11c- 7 Eosiniophils CD45+, CD11b+, F480+, CD115+, CD62L+, CD11c+ 8 Monocytes CD45+, CD115+, CD11b+, Ly6C low, CD11c+, MerTK+, CCR2+, PDL1+ 9 Monocytes CD45+, CD115+, CD11b+, Ly6C+, CD11c-, MerTK-, CCR2+, Ly6g/c- 10 Monocytes CD45+, CD115+, CD11b+, Ly6C+, CD11c-, MerTK+, CCR2+, Ly6g/c-, CD4+ 11 Monocytes CD45+, CD115+, CD11b+, Ly6C+, CD11c-, MerTK+, CCR2+, Ly6g/c+, CD4+, CD64+, FceR+, MHC II+ 12 Monocytes CD45+, CD115+, CD11b+, Ly6C+, CD11c-, MerTK+, CCR2+, Ly6g/c+, CD4+, CD64-, FceR-, MHC II+ 13 Microglia CD45+, CD11b+, F4/80+, CD64+, MerTK+, CD24+, Sirpa+ 1.5% 2.0% 0.4% 11.6% 0.4% 2.3% 2.1% 0.1% 2.1% 0.0% 1.0% 2.1% 0.2% 11.6% 7.3% 0.2% 2.7% 0.0% 0.7% 3.4% 0.2% 12.5% 6.1% 1.5% 57.7% 0.0% 0.0% 0.5% 0.1% 1.5% 1.1% 0.4% 13.2% 0.3% 0.7% 0.9% 0.2% 0.6% 1.3% 0.2% 2.9% 0.4% 1.8% 7.9% 1.3% 3.9% 4.5% 2.4% 0.0% 0.0% 0.6% 2.2% 0.8% 0.7% 0.6% 27.8% 1.3% 0.3% 1.8% 9.5% 1.5% 11.6% 5.7% 0.4% 0.0% 0.5% 0.1% 1.0% 0.0% 0.0% 0.1% 0.0% 0.0% 0.0% 0.0% 0.1% 0.0% 0.0% 0.0% 0.0% 2.8% 0.4% 7.7% 14.1% 4.3% 20.3% 12.6% 2.0% 9.5% 1.0% 2.6% 6.5% 0.8% 2.2% 3.4% 0.3% 0.1% 92.9% 0.9% 0.0% 0.0% 0.0% 0.0% 0.0%

17 14 Red pulp CD45+, CD11c+, CD11b+, F4/80+, Ly6c+, 0.3% 0.1% 0.2% 2.0% 0.1% 0.0% 5.7% 0.8% macrophages MHC II+, Sirpa+ 15 Spleen/Kidney CD45+, CD11c-, CD11b+, F4/80+, Ly6c+, 0.3% 0.0% 3.4% 0.5% 0.6% 0.6% 1.8% 0.3% macrophages MHC II+, Esam+, Sirpa- 16 Alveolar CD45+, CD11c+, MHC II- /+, CD11b-, F4/80+, 0.0% 0.0% 0.0% 0.0% 0.0% 12.2% 0.0% 0.0% macrophages Sirpa+ 17 DCs CD45+, CD11c+, MHC II+, CD11b+, CD24+, 0.1% 1.0% 39.2% 1.2% 1.2% 0.4% 0.1% 0.6% Sirpa+, F4/ DCs CD45+, CD11c+, MHC II+, CD11b+, CD24+, 0.5% 0.2% 8.7% 8.1% 2.5% 3.5% 2.1% 4.1% Sirpa+, CD301+, CD DCs CD45+, CD11c+, MHC II+, CD11b+, CD24+, 0.1% 0.0% 0.4% 1.0% 8.2% 0.1% 16.5% 1.3% Sirpa+, Esam+, CD4+ 20 DCs CD45+, CD11c+, MHC II+, CD103+, CD24+, 0.0% 0.0% 1.4% 2.0% 26.0% 0.7% 1.2% 7.1% Sirpa- 21 DCs CD45+, CD11c+, MHC II+, CD8+, CD24+, 0.0% 0.0% 0.5% 1.8% 8.5% 0.8% 5.5% 6.9% Sirpa- 22 pdcs CD45+, CD11c+, MHC II+, B220+, Ly6C+, 2.0% 0.0% 10.7% 1.9% 9.6% 1.2% 3.2% 11.9% Siglec- H+, BST2+ 23 Innate CD11c-, CD11a+, MHC II+, CD4+, F4/80+, 0.0% 0.0% 0.1% 0.0% 7.0% 0.1% 0.1% 1.5% lymphoid cells CD25+, CD62L- 24 Innate CD11c-, CD11a+, MHC II-, CD4+, F4/80-, 0.0% 0.0% 1.3% 0.8% 14.0% 0.2% 0.5% 1.7% lymphoid cells CD25 low, CD62L+ 25 Innate CD11c-, CD11a+, MHC II-, CD4-, F4/80+, 2.4% 0.5% 5.9% 6.4% 3.0% 1.1% 2.7% 2.6% lymphoid cells CD25-, CD62L- 26 Innate CD11c-, CD11a+, MHC II-, CD4+, F4/80+, 0.0% 0.0% 0.7% 0.4% 0.9% 0.1% 0.2% 24.0% lymphoid cells CD25-, CD62L- 27 Innate CD11c+, CD11a+, MHC II-, CD4-, F4/80-, 0.0% 0.1% 1.6% 7.3% 2.8% 0.4% 1.5% 0.6% lymphoid cells CD25-, CD62L- 28 Innate CD11c+, CD11a+, MHC II-, CD4-, F4/80-, 0.3% 0.1% 7.3% 6.9% 5.6% 11.7% 14.4% 1.2% lymphoid cells CD25-, CD62L+ Total Non- T/Non- B/CD45+ CD19- CD90- CD % 100% 100% 100% 100% 100% 100% 100%