Categorical analysis of human T cell heterogeneity with One-SENSE

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1 2 3 4 Supplementary Information Categorical analysis of human T cell heterogeneity with One-SENSE 5 6 Running title: T cell Analysis by One-SENSE 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 Yang Cheng*, Michael T. Wong*, Laurens van der Maaten, Evan W. Newell* *Singapore Immunology Network, Agency of Science, Technology and Research, Singapore, Singapore Pattern Recognition Laboratory, Delft University of Technology, Delft, The Netherlands To whom correspondence should be addressed: Evan W. Newell 8A Biomedical Grove Immunos, #3-6 Singapore 138648 Email: evan_newell@immunol.a-star.edu.sg Tel: (+65) 647-641 1

1a Rh13Di: DNA 1 3 1 2 1 1 1 7.8 Pt195Di: Cisplatin 1 3 1 2 1 1 1 61 Cd112Di: CD14_1 1 3 1 2 1 1 1 96.3 2 4 6 8 1 Length 2 4 6 8 1 Length Cd114Di: CD14_2 Y89Di: CD45 1 3 1 2 1 1 1 82 EQ beads Dy162Di: CD4CD19CD56 1 3 1 2 1 1 1 3.1 Palladium-based dual mass tag cell debarcoding system Dimentionality Reduction: t-sne and One-SENSE Ce14Di: CD3 Nd142Di: CD8 See Figure 1 & Materials and Methods 1b Gated on s 1c Gated on CD4 T cells 1 3 7.76 3.4 1 3 1.4 3.35 1 3 3.26.43 1 3 4.3 2.76 1 3 4.49 2.39 Pm147Di: TNF-a 1 2 1 1 1 59 2.82 1 2 1 1 1 79.3 Ho165Di: CD4L 6.89 Gd157Di: IL-8 1 2 1 1 1 83 13.4 1 2 1 1 1 92.7.244 Sm152Di: CD25 1 2 1 1 1 9.4 2.77 Ho165Di: CD39 1 3 32.9 6.57 1 3 15.6 1.78 1 3 6.62 1.14 1 3 6.8.715 1 3 6.81.255 Nd143Di: GrzB 1 2 1 1 1 59.2 Er167Di: GrzA 1.31 Gd158Di: CD17a 1 2 1 1 1 77.3 Sm152Di: Perforin 5.28 Gd155Di: CTLA-4 1 2 1 1 1 9.9 Gd156Di: CD39 1.37 1 2 1 1 1 92.9.189 Er17Di: IL-1 1 2 1 1 1 91.6 1.34 Dy164Di: LAG-3 1 3 1 2 1 1 1 26 7.84 63.3 2.87 Pm147Di: TNF-a 1 3 1 2 1 1 1 21.9 16.3 6.2 1.63 Dy163Di: IL-1 1 3 1 2 1 1 1.124.1 89.7 1.2 1 3 1 2 1 1 1 1.8 6.1 1 3 1 2 1 1 1 5.99.969 Nd146Di: MIP1b Dy161Di: MIP1a Yb172Di: GM-CSF 46.7 46.1 Nd15Di: CTLA-4 29.6 63.4 Lu176Di: CD127 Functional Tregness Supplementary Figure 1. Validation of mass cytometry staining and T cell subgating scheme. (A) Shown are biaxial plots of subgating strategy to identify CD8 + T cells and the subsequent dimensionality reduction analysis workflow, including One-SENSE (B) Shown are representative plots of 15 different functional markers that were used as category Function. Numbers indicate frequency of parent cells. Plots show combined cells from three donors. (C) Representative biaxial dotplots show the staining quality of Treg-associated markers on gated CD4 + T cells. Numbers indicate frequency of parent (total CD4 + ) cells. FoxP3, CD25, CD39, CTLA-4, IL-1 and LAG-3 were markers used for Tregness dimension of One-SENSE in Figure 6.

2a 1 3 1 3 2.93 1 3 Differentiation 1 2 1 1 1 Function 1 2 1 1 1 Ho165Di: CD4L Gd156Di: CD39 1 2 1 1 1 2.63 Gd155Di: CTLA-4 Functional profiles of subsets 1 1 1 1 8 8 8 8 6 4 6 4 6 4 6 4 2 2 2 2 Pm147Di: TNF-a Gd158Di: CD17a Sm152Di: Perforin 1 1 1 1 8 8 8 8 6 4 6 4 6 4 6 4 2 2 2 2 Nd143Di: GrzB Er167Di: GrzA Ho165Di: CD4L 1 1 1 1 8 8 8 8 6 4 6 4 6 4 6 4 2 2 2 2 Dy161Di: MIP1a Nd146Di: MIP1b Yb172Di: GM-CSF Gd157Di: IL-8 1 8 6 4 2 Dy163Di: IL-1 1 8 6 4 2 Gd156Di: CD39 1 8 6 4 2 Gd155Di: CTLA-4 Naive Supplementary Figure 2. Differential multi-functional profiles of CD8 + T cells. (A) Different subsets of T cells on the column of Function axis are color coded and annotated as shown in Figure 4a. Two rare subsets are defined. CD8 + T cells exclusively co-express the highest IL-2 and CD4L. CD8 + T cells are identified by Treg functional markers CD39 and CTLA-4. The divergent expression of functional profiles shows the enormous heterogeneity between different subsets.

2b Differentiation and Trafficking profiles 1 1 1 1 8 8 8 8 6 4 6 4 6 4 6 4 2 2 2 2 In115Di: CD57 Nd144Di: CD45RA Eu151Di: CCR7 Nd15Di: KLRG1 1 1 1 1 8 8 8 8 6 4 6 4 6 4 6 4 2 2 2 2 Nd148Di: CD28 Nd145Di: CD27 Er166Di: CD38 Lu176Di: CD127 1 8 6 4 1 8 6 4 1 8 6 4 Naive 2 2 2 Dy164Di: CD62L Tm169Di: CXCR1 Yb173Di: CCR4 2c % of functional CD8 + T cells 15 1 5 Donor 1 Donor 2 Donor 3 Supplementary Figure 2. Differential multi-functional profiles of CD8 + T cells, continued. (B) Expression of differentiation and trafficking markers show is the most cytotoxic-activated (with Supplementary Figure 2a) CD8 + T cell subset. (C) The frequency of different functional-profiled CD8 + T cell subsets in each donor.

3a Pm147Di: CD45RO Gd157Di: CD27 Gd16Di: CD28 Sm154Di: CD4L Naive 1 2 3 4 5 6 7 Tm169Di: IL-4 Dy163Di: IL-22 Eu151Di: IL-2 Nd142Di: TNF-a Er168Di: CCR2 Yb171Di: CCR6 Lu175Di: CCR5 Yb173Di: CCR4 Er166Di: CXCR5 Gd155Di: CXCR6 Nd148Di: CLA 3b 4_1 4_2 4_3 4_4 Eu151Di: IL-2 Nd142Di: TNF-a Nd148Di: CLA Yb173Di: CCR4 5_1 5_2 3c % of regulatory CD4 + T cells 25 2 15 1 5 1 2 3 4_1 4_2 4_3 4_4 5_1 5_2 6 7 Nd148Di: CLA Donor 1 Donor 2 Donor 3 Er168Di: CCR2 t-sne_1 Lu175Di: CCR5 Supplementary Figure 3. Differential profiles of regulatory CD4 + T cells (Treg) subsets. (A) The diverse differentiation, function and trafficking profiles of seven regulatory CD4 + T cells subsets are shown as histogram. (B) Multiple subpopulations of 4 (blue series) and 5 (brown series) were further segregated by distinct functional and trafficking status. n=3. (C) The frequency of different regulatory-like CD4 + T cell subsets in each healthy individual. (D) t-sne presentation of subpopulation of 4 subset. 3d t-sne_2

Supplementary Table 1: CyTOF staining panel for One-SENSE analysis CD8 panel (Functionality) CD4 panel (Tregness) Metal-Isotope Antibody (or label) Clone Company t-sne Antibody (or label) Clone Company t-sne Y-89 CD45 HI3 Fluidigm (DVS) CD45 HI3 Fluidigm (DVS) Cd-112/114 Qdot8-CD14 TuK4 Molecular Probes Qdot8-CD14 TuK4 Molecular Probes Rh-13 DNA Intercalator Fluidigm (DVS) DNA Intercalator Fluidigm (DVS) Pd-14 Barcode Barcode Pd-15 Barcode Barcode Pd-16 Barcode Barcode Pd-18 Barcode Barcode Pd-11 Barcode Barcode Ln-113 Barcode Barcode In-115 CD57 HCD57 Biolegend CD57 HCD57 Biolegend La-139 Vα7.2 3C1 Biolegend γδtcr-pe (1 Ab) SA6.E9 Invitrogen anti-pe (2 Ab) PE1 Biolegend Ce-14 CD3 UCHT1 BioXcell CD3 UCHT1 BioXcell Pr-141 IFN-γ 4S.B3 ebioscience IFN-γ 4S.B3 ebioscience Nd-142 CD8α SK1 Biolegend TNF-α Mab11 ebioscience Nd-143 Granzyme B CLB-GB11 Abcam IL-17A BL168 Biolegend Nd-144 CD45RA HI1 BD Bioscience CCR7 1553 R&D Systems Nd-145 CD27 LG.7F9 ebioscience CD62L DREG56 BD Bioscience Nd-146 MIP1-β D21-1351 BD Bioscience CD8α SK1 Biolegend Sm-147 TNF-α Mab11 ebioscience CD45RO UCHL1 Biolegend Nd-148 CD28 CD28.2 Biolegend CLA HECA-452 Biolegend Sm-149 IL-2 MQ1-17H12 ebioscience CD4 SK3 Biolegend Nd-15 KLRG-1 Hanspeter Pircher Granzyme B CLB-GB11 Abcam Eu-151 CCR7 1553 R&D Systems IL-2 MQ1-17H12 ebioscience Sm-152 Perforin B-D48 Abcam CD25 M-A251 BD Bioscience Eu-153 CXCR5 RF8B2 BD Bioscience CD17a H4A3 BD Bioscience Sm-154 CD45RO UCHL1 Biolegend CD4L 24-31 ebioscience Gd-155 CTLA-4 BNI3 BD Bioscience CTLA-4 BNI3 BD Bioscience Gd-156 CD39 A1 Biolegend CD19 HIB19 Biolegend Gd-157 IL-8 H8A5 Biolegend CD27 LG.7F9 ebioscience Gd-158 CD17a H4A3 BD Bioscience CD56 NCAM16.2 BD Bioscience Tb-159 ICOS C398.4A Biolegend CXCR3 1C6 BD Bioscience Gd-16 PD-1 ebioj15 ebioscience CD28 CD28.2 Biolegend Dy-161 MIP1 α 11A3 BD Bioscience Foxp3-Biotin (1 Ab) PCH11 ebioscience Strepavadin-Dy161 (2 Ab) In-House Dy-162 CD4 SK3 Biolegend CD38 HIT2 Biolegend Dy-162 CD19 HIB19 Biolegend Dy-162 CD56 NCAM16.2 BD Bioscience Dy-163 IL-1 JES3-9D7 ebioscience IL-22 Poly5161 Biolegend Dy-164 CD62L DREG56 BD Bioscience LAG-3 87451 R&D Systems Ho-165 CD4L 24-31 ebioscience CD39 A1 Biolegend Er-166 CD38 HIT2 Biolegend CXCR5 RF8B2 BD Bioscience Er-167 Granzyme A CB9 Biolegend PD-1 ebioj15 ebioscience Er-168 CXCR3 1C6 BD Bioscience CCR2 K36C2 Biolegend Tm-169 CXCR1 4275 R&D Systems IL-4 MP4-25D2 Biolegend Er-17 CXCR4 12G5 Biolegend IL-1 JES3-9D7 ebioscience Yb-171 CXCR6 K41E5 Biolegend CCR6 G34E3 Biolegend Yb-172 GM-CSF BVD2-21C11 Biolegend GM-CSF BVD2-21C11 Biolegend Yb-173 CCR4 2541 R&D Systems CCR4 2541 R&D Systems Yb-174 CCR6 G34E3 Biolegend CD45RA HI1 BD Bioscience Lu-175 CCR5 ab7346 Abcam CCR5 ab7346 Abcam Yb-176 CD127 A19D5 Biolegend CD127 A19D5 Biolegend Ir-191/193 CD161 HP-3G1 Biolegend CD161 HP-3G1 Biolegend Pt-195 Cisplatin Live/Dead Fluidigm (DVS) Cisplatin Live/Dead Fluidigm (DVS) One-SENSE category Function Differentiation Trafficking Tregness

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