Supplementary Figure 1. Immunophenotyping panels used in this study. Seven 13 or 14- color panels were opWmized to enumerate and phenotype different ...
Panel 1: Panel 2: T Cell T Cell Differentiation Activation Viability CD3 CD4 CD8 CD27 CD45RA CCR7 CD28 CD31 CD57 CD95 CD127 CD244
Viability CD3 CD4 CD8 CD25 CD127 CD39 CD73 CD45RA CD38 CCR5 PD1 HLA-DR
Panel 5: Panel 3: Panel 4: Panel 6: Stem cells; T Cell NK Cell B Cell Polarization Differentiation Innaptive Differentiation Differentiation Viability CD3 CD4 CD8 CD45RA CCR7 PD1 CXCR3 CXCR5 CCR4 CCR6 CCR10 CD161
Viability CD2 CD3 CD4 CD16 CD56 CCR7 CD62L CD158a CD158b CD314 CD335 CD337
Viability CD34 CD3 CD4 CD8 CD1d TCR-Vγ9 TCR-Vδ1 TCR-Vδ2 CD27 CD28 CD45RA CCR7 CCR5
Viability CD19 CD20 IgA IgD IgG IgM CD5 CD10 CD21 CD24 CD27 CD95 CD38
Supplementary Figure 1. Immunophenotyping panels used in this study. Seven 13 or 14color panels were op2mized to enumerate and phenotype different leukocyte popula2ons from cryopreserved PBMC. All seven panels were applied to all subjects using staining temperatures op2mized for each panel. Reagents are roughly ordered by: major lineagedefining markers; sub-lineage defining markers; and then markers to assess differen2a2on stages, ac2va2on state, and/or func2onal capacity.
Panel 7: Myeloid Cells Viability CD14 CD11c CD123 HLA-DR CD1c CD141 CD8 CD16 CD32 CD64 CD83 CD274 HLA-DR
Singlets
FSC-A
Live Cells
CD20
SSC-A
FSC-H
PBMC
B Cells
CD19
Live/Dead (Aq. Blue)
IgM
CD5
+
–
CD27
CD95
CD21
IgM
CD10
CD24
B Cell Combination Gates
IgM
IgA
IgG
IgD
+
–
"Naive B": IgM IgD + IgA – IgG – – "Mem. IgM":+IgM –IgD –IgA –IgG "IgG B": IgG+ IgM– IgD– IgA– "IgA B": IgA –IgM –IgD –IgG– "IgE B": IgG IgM IgD IgA
CD38
IgM
Supplementary Figure 2. Ga9ng for B cell subsets. A different ga2ng strategy was used for Panel 6 than originally described8. B cells were broadly iden2fied as CD20+ live lymphocytes (top). Within these cells, gates iden2fying isotype expression were combined to as-purely-aspossible iden2fy naïve and memory B cells; within the laXer, IgM, IgG, IgA, and puta2ve IgEexpressing cells were gated. Within each of these five, all combina2ons of the seven markers (posi2ve, nega2ve, or ignored) were created by Boolean ga2ng as previously described8. This results in 2,187 CSF traits for each of the five isotype-defined subsets.
Complete Set 89,051 CSF: 88,367 SPEL: 684
Out of range 56,927 CSF: 56,278 SPEL: 649
CSF: % < 0.1, or % > 99 SPEL: MFI < 100 CSF: 32,089 SPEL: 35
Poor reproducibility 37,997
Longitudinal covariance < 0.7 CSF: 18,719 SPEL: 211
CSF: 37,559 SPEL: 438
Unknown or poorly described function CSF: 14,554 SPEL: 49
Final Set 23,394 CSF: 23,005 SPEL: 389
Supplementary Figure 3. Trait QC workflow. Structured equa2on modelling was performed on a robust subset of all traits. Three different filters (shown in green) were applied in succession to the original 89,051 traits to result in the final 23,394. The last filter was a subjec2ve, elimina2ng cell popula2ons such as CD4+CD8+ (double posi2ve) or CD4–CD8– (double nega2ve) T cells from considera2on.
