Valle et al . Figure S1

No Gate Singlets CD4 262144 262144

5 CD4 4 -/+ +/+ 10 10 196608 196608 4 Singlets 10 CD8 131072 131072 3 CD4 FSC-H

FoxP3 3 SSC-A 10 Lymphocyte 10

65536 65536 2 10 1 2 -10 -10 -/- +/- 0 0 1 2 3 4 5 2 2 3 0 65536 131072196608 262144 0 65536 131072196608 262144 -10 10 10 10 10 -10 10 10 FSC-A FSC-A CD8 Ki67

Figure S1. Gating strategy. The gating strategy used to evaluate by flow cytometry the different circulating subsets based on the expression of CD4, CD8, FoxP3 and Ki67 markers is shown. Gating is shown on top of each panel. Valle et al . Figure S2

A CD4 (Lympho) CD8 (Lympho) B CD4 (Lympho) CD8 (Lympho) C FoxP3 (CD4) D FoxP3 (CD4) FoxP3 FoxP3 FoxP3 FoxP3 FoxP3 FoxP3 FoxP3 FoxP3 CD4 CD4 CD4 CD4

CD8 CD8 CD8 CD8 CD8 CD8 CD4 CD4 CD4 CD4 *** *** *** *** *** *** *** ***

DN cells CD8 + T cells CD4 + T cells DN cells CD8 + T cells CD4 + T cells CD4 +FoxP3 - CD4 +FoxP3 + CD4 +FoxP3 - cells CD4 +FoxP3 + cells

CD3 expression level CD3 expression level CD3 expression level CD3 expression level

Figure S2. Expression of CD3 on different circulating T cells in NOD and Balb/C mice. CD3 expression levels were evaluated in additional flow-cytometry-based experiments that used differing reagents (i.e., differing fluorochrome-conjugated antibodies) and likewise differing staining protocols (i.e., with or without cell permeabilization). (A and B) Peripheral blood of NOD (Panel A, n=16) or Balb/C (Panel B) mice was stained with anti -CD 3ε, -CD 8 and -CD 4 mAbs without cell permeabilization . SPADE analysis was performed on total (lympho ). Subsets are visualized in branched -tree two -dimension structures. Branched-tree visualization of a representative sample is depicted for the three given markers. Nodes are colored on the basis of expression of the specified marker (upper panels). Hierarchical clustering was then used for the selection of the various T-cell subsets (i.e., CD8+, CD4+ and CD4-CD8- double negative (DN) T cells), which were then represented in conventional dot plots to prove the results of the clustering (middle panels). Heatmap of CD3 expression on the above mentioned T-cell subsets is depicted (lower panel). Each line represents one mouse. Statistical significance was evaluated by paired t-test (*** P< 0.001 ). (C and D) Peripheral blood of NOD (Panel C, n= 13 ) or Balb /C (Panle D, n= 5) mice was stained with anti -CD 3ε, -CD 4, -FoxP 3 mAbs . SPADE analysis was performed on CD 4+ T cells (CD4). Subsets are visualized in branched-tree two-dimension structures. Branched-tree visualization of a representative sample is depicted for the three given markers. Nodes are colored on the basis of expression of the specified marker (upper panels). Hierarchical clustering was then used for the selection of the various T-cell subsets (i.e., CD4+FoxP3- and CD4+FoxP3+ T cells), which were then represented in conventional dot plots to prove the results of the clustering (middle panels). Heat map of CD3 expression on the abovementioned T-cell subsets is depicted (lower panel). Each line represents one mouse. Statistical significance was evaluated by paired t-test (*** P<0.001). Valle et al . Figure S3 NOD mice Balb/c mice

*** ***

8000 8000 *** *** 6000 6000 *** ***

4000 4000 MFI CD3 CD3 MFI MFI MFI CD3 CD3 MFI MFI 2000 2000

0 0 - - 4 4 D4+ 4 44+ D8+ 4 44+ C D D C D D CD4+ CD8+ C C C C + + + + 4 4 8 8 D D D D C C C C CD4+CD44-CD4+CD44+ CD8+CD44-CD8+CD44+ Figure S3. Expression of CD3 on murine T cells at different maturation state. CD3 expression was quantified by flow cytometry on peripheral CD44 - (i.e., naive and recently activated) and CD44 + (i.e., effector and central memory) CD4+ and CD8+ T cells. Experiments were performed in NOD (left panel) and Balb/c (right panel) mice. (*** P<0.001, paired t-test). Valle et al . Figure S4

A CD8 (CD3) CD4 (CD3) CD25 (CD3) FOXP3 (CD3)

CD8 + CD4 + FOXP3 -CD25 - FOXP3 -CD25 + FOXP3 + FOXP3 +CD25 - FOXP3 hi 25 hi FOXP3 lo CD25 lo

CD4 + FOXP3 + CD8 FOXP3 FOXP3

CD4 CD4 CD25 CD25 CD25 CD25 CD25 CD25

*** B CD4 (CD3) CD8 (CD3) TCR γδ (CD3) *** ***

CD4 CD8 TCR γδ γδ γδ γδ CD4 CD4 CD4 TCR TCR TCR CD8 + T cells CD4 + T cells TCR γδ T cells

CD8 TCR γδ CD8 CD8 CD8 CD4 CD3 expression level

Figure S4. Expression of CD 3 on different circulating T-cell subsets in humans . (A) SPADE analysis on gated CD 3+ T cells (CD 3) processed as in Figure 7A. Branched-tree visualization of a representative sample is depicted for the four markers under consideration. Hierarchical clustering was used for the selection of various T-cell subsets (i.e., CD8+, CD4+ T cells. Within CD4+ T cells: FOXP3-CD25 -, FOXP3-CD25 + and FOXP3+ T cells. Within CD4+FOXP3+ T cells: FOXP3+CD25 -, FoxP3hi CD25 hi and FoxP3lo CD25 lo T cells), which were then represented in conventional dot plots to prove the results of the clustering . Hierarchical clustering discriminates the various population as expected by a classical manual analysis except for few FOXP 3- T cells which cluster together with FOXP3+ T cells (middle-right and lower-right dot plots). (B) To further consolidate the results obtained in Figure 7, CD3 expression levels were evaluated in additional flow-cytometry experiments that used differing reagents (i.e., differing fluorochrome-conjugated antibodies) and likewise differing staining protocols (i.e., without cell permeabilization) . Peripheral blood of healthy donors was stained with anti - CD 3ε, -CD 4, - CD45RA, -CD25 and -CD127 mAbs. SPADE analysis was performed on CD4+ T cells (CD4). Branched-tree visualization of a representative sample is depicted for the three markers under consideration (upper panels). Hierarchical clustering was used for the selection of the various T-cell subsets: CD127 - CD25 +CD45RA + (i.e., naïve Treg cells), CD127 -CD25 ++ CD45RA - (i.e., memory Treg cells), CD127 +/-CD25 -CD45RA + (i.e., naïve T cells), CD127 +/-CD25 - /+ CD 45 RA - (i.e., memory T cells) . Selected populations were then represented in conventional dot plots to prove the result of the clustering (lower panels) . Heat map of CD3 expression on the abovementioned T-cell subsets is depicted. Each line represents one healthy individuals (n=29). Statistical significance was evaluated by paired t-test (*** P<0.001).