Supplementary Figure 1
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1 Supplementary Figure 1 Identification of IFN-γ-producing CD8 + and CD4 + T cells with naive phenotype by alternative gating and sample-processing strategies. a. Contour 5% probability plots show definition of T N, T CM, T E+EM and T EMRA subsets based on the expression of
2 pairs of CD45RA and CCR7 or CD95 and CD28 molecules by total CD8 + T cells from a representative donor (age 71). b. Gating strategies for identification of T N (CD45RA + CCR7 + CD95 lo CD28 int )(top row), T CM (middle row), T E+EM and T EMRA (both bottom row) CD8 + T cell subsets, using gating in reverse compared to Fig. 1, by first applying the CD95/28 gate and then the CD45RA/CCR7 gate. c. Age dependent decrease in phenotypically naive CD8+ T cells (shown in black) and IFN-γ producing phenotypically naive (CD45RA + CCR7 + CD95 lo CD28 int CD8 + T cells (in green) shown as cell number (Log 10 )/µl of blood (n=92). d. Graph shows flow cytometric analysis of percentages of IFN-γ + CD4 + T cells with naive phenotype (gated through CD45RA + CCR7 + CD28 int CD95 lo ) with age. Student s t-test was applied to analyze significance in the age-related increase between the group of donors <40 years old (n=27), 40 65y old (n=44) or >65 years old (n=21; *p<0.05). Data are representative of one experiment. e. Intracellular cytokine staining of human PMA+ionoactivated fresh (not cryopreserved) PBMCs from healthy donors. Dot plots show IFN-γ, TNF-α, and GzmB production by phenotypically naïve (CD45RA + CCR7 + CD95 lo CD28 int CD8 + T cells from freshly isolated blood of 7 donors (age as indicated). Unstimulated (US) PBMCs were used as control. f. Intracellular cytokine staining of sorted naïve (CD45RA + CCR7 + CD95 lo CD28 int CD8 + T cells after 3 hr activation with PMA-iono. Bar graphs show percentage of cytokine producing naive CD8 + T cells in healthy subjects age <40 and > 65, with n=6/group (*p<0.05, experiment repeated once with different subjects, n=4/group).
3 Supplementary Figure 2 Expression of phenotypic markers on T MNP cells relative that on other CD8 + T cell subsets. Graphs show flow cytometry-based analysis of diversity (mean gmfi ± SD) in expression of a given molecule between different CD8 + T cell subsets from 12 donors. Repeated measures ANOVA was used to quantify
4 differences in CD45RA, CCR7, CD95 and CD28 expression between T N and T MNP, T CM, T EM or T EMRA CD8 T cells, defined as in Supplemental Fig. 1d (*p<0.05,**p<0.0001, n=12, ages 27-85, 5 males, 7 females).
5 Supplementary Figure 3 T MNP cells do not respond to bystander stimulation by IL-12 and IL-18. a. Flow cytometry-based evaluation IFNγ production in sorted naïve CD45RA + CCR7 + CD95 lo CD28 int CD49d high and CD49d low CD8 + T cells after overnight IL-12 (5ng/mL) + IL-18 (5ng/mL) culture. Data are representatives of 2 subjects, males ages 52, 64 (of n=3). b. IFN-γ production by different PBMC CD8 + T cell subsets (T N and T MNP, T CM, T EM or T EMRA, defined as in Supplemental Fig. 1d) after overnight IL-12+IL-18 culture and 3hr presence of protein transport inhibitors shown as % of IFN-g + cells (repeated measures ANOVA,*P<0.05, n=9, ages 35-77).
6
7 Supplementary Figure 4 Flow FISH analysis and stability of phenotype after stimulation with PMA plus ionomycin. a. Flow cytometry analysis of human PBMCs from a representative donor (male, age 68). Contour 5% probability plots and histogram show gating strategy used to identify different CD8 + T cell populations (T N, T CM, T E+EM and T EMRA. As shown in the final graph, 4n cells were excluded from the analysis. b. Contour 5% probability plots and histograms show expression of naive phenotypic markers and intensity of CD8 and A2 CMV-NLV tetramer staining after 3hr PMA+iono stimulation (in right column or in red) or in the presence of BFA alone in one representative subject (left column or in blue) of n=8 (age 35-83).
8 Supplementary Figure 5 T MNP cells undergo robust proliferation in response to TCR stimulation. a,b. Overlaid histograms show proliferation (dilution of CTV proliferation dye) of a. sorted T MNP (FN-γ + CD45RA + CCR7 + CD95 lo CD28 int ) cells (red line), isolated using IFN-γ capture assay (as in methods) and T N (naive IFN-γ - CD45RA + CCR7 + CD95 lo CD28 int ) CD8 + T cells(blue line), 3 days after activation with anti-cd3+anti- CD2+anti-CD28 Ab-coated beads (two donors of n=3, 2 females, 1 male, ages 65-73, are shown, t-test, NS) and b. sorted T MNP (CD45RA + CCR7 + CD95 lo CD28 int CD49d hi ), T N (CD45RA + CCR7 + CD95 lo CD28 int CD49d lo ), T CM (CD45RA - CCR7 + CD95 lo CD28 hi ) and T EM+TEMRA (CD45RA -or+ CCR7 + CD95 hi CD28 int or lo ) cells at 6 days after activation with anti-cd3+ anti-cd2+anti-cd28 Ab-coated beads, two representative donors, age 32 and 35 are shown. Proliferation and division indices are shown by scatter graphs (shown as mean ±SEM; repeated measures oneway ANOVA,*P<0.05, n=5, 4 females, 1 male, age 32-69).
9 Supplementary Figure 6 TCR V β distribution in various subsets of CD8 + T cells from a healthy donor (female, 65 years of age), isolated 24 months apart. 3D line graphs showing TCR Vβ distribution in different CD8 + T cells subsets (indicated in the legend). Percentages of each of the 24 tested TCR Vβ (marked on the x axis) are shown. Total analyzed were n=2 (1 female, 1 male, age 65,81). Data are representative of one experiment.
10 Supplementary Figure 7 Flow cytometry analyzing IFN-γ production by naive IAV-specific CD8 + T cells before and 14 d after vaccination against IAV shows no bystander or nonspecific activation of T MNP cells. PBMCs were activated briefly with PMA+Iono. Contour 5% probability plots show gating strategy to evaluate the naive phenotype (CD45RA + CCR7 + CD95 lo CD28 int ) and IFN-γ production by IAV matrix GIL peptide:hla- A*0201 or CMV pp65 NLV: HLA-A*0201 tetramer + CD8 + T cells. Numbers represent percentages of the population within each gate after subsequent gating. Data from two representative donors (one CMV seropositive and one seronegative (of n=7, ages 65-80).
11 Supplementary Figure 8 Major CD8 + T cell subsets do not change in number regardless of the severity of symptoms in WNV-exposed subjects. Quantification of CD8 + T cell subsets in WNV asymptomatic and symptomatic subjects is represented as absolute number of T N, T E+EM, T EMRA and T CM subset/10 6 CD8 + T cells within each subject. Repeated measures one-way ANOVA was used to quantify the subsets differences between asymptomatic (n=11,,39-92) and symptomatic (n=16, ages 43-84).
HD1 (FLU) HD2 (EBV) HD2 (FLU)
ramer staining + anti-pe beads ramer staining a HD1 (FLU) HD2 (EBV) HD2 (FLU).73.11.56.46.24 1.12 b CD127 + c CD127 + d CD127 - e CD127 - PD1 - PD1 + PD1 + PD1-1 1 1 1 %CD127 + PD1-8 6 4 2 + anti-pe %CD127
Low Avidity CMV + T Cells accumulate in Old Humans
Supplementary Figure Legends Supplementary Figure 1. CD45RA expressing CMVpp65-specific T cell populations accumulate within HLA-A*0201 and HLA-B*0701 individuals Pooled data showing the size of the NLV/HLA-A*0201-specific
Supplementary Table 1 Clinicopathological characteristics of 35 patients with CRCs
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ILC1 and ILC3 isolation and culture Following cell sorting, we confirmed that the recovered cells belonged to the ILC1, ILC2 and
Supplementary Methods and isolation and culture Following cell sorting, we confirmed that the recovered cells belonged to the, ILC2 and subsets. For this purpose we performed intracellular flow cytometry
Supplementary Figure 1. IL-12 serum levels and frequency of subsets in FL patients. (A) IL-12
1 Supplementary Data Figure legends Supplementary Figure 1. IL-12 serum levels and frequency of subsets in FL patients. (A) IL-12 serum levels measured by multiplex ELISA (Luminex) in FL patients before
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Supplementary Figure 1. Characterization of basophils after reconstitution of SCID mice
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<10. IL-1β IL-6 TNF + _ TGF-β + IL-23
3 ns 25 ns 2 IL-17 (pg/ml) 15 1 ns ns 5 IL-1β IL-6 TNF
Supplementary Figure 1. Example of gating strategy
Supplementary Figure 1. Example of gating strategy Legend Supplementary Figure 1: First, gating is performed to include only single cells (singlets) (A) and CD3+ cells (B). After gating on the lymphocyte
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Supplemental Figure 1. Signature gene expression in in vitro differentiated Th0, Th1, Th2, Th17 and Treg cells. (A) Naïve CD4 + T cells were cultured
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Supplemental Figure 1
Supplemental Figure 1 1a 1c PD-1 MFI fold change 6 5 4 3 2 1 IL-1α IL-2 IL-4 IL-6 IL-1 IL-12 IL-13 IL-15 IL-17 IL-18 IL-21 IL-23 IFN-α Mut Human PD-1 promoter SBE-D 5 -GTCTG- -1.2kb SBE-P -CAGAC- -1.kb
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x Lymphocyte count /µl CD8+ count/µl 800 Calculated
% Lymphocyte in CBC A. 50 40 30 20 10 Lymphocyte count /µl B. x10 3 2.5 1.5 C. 50 D. 1000 % CD3+CD8+ Cells 40 30 20 Calculated CD8+ count/µl 800 600 400 200 10 0 #61 #63 #64 #65 #68 #71 #72 #75 Figure
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SDC (Supplemental Digital Content) Figure S1. Percentages of Granzyme B expressing CD8 + T lymphocytes
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% of Cells A B C. Proliferation Index. T cell count (10 6 ) Division Index. % of Max CFSE. %Ki67+ cells. Supplementary Figure 1.
A B C T cell count (1 6 ) 3. 2. 1.. * % of Max CFSE ormoxia ypoxia o Stim. Proliferation Index 2.5 2. 1.5 1. * Division Index 2. 1.5 1..5. D E %Ki67+ cells 1 8 6 4 2 % of Cells 8 ormoxia 6 ypoxia 4 2 *
Supplementary Figure 1
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Supplementary appendix
Supplementary appendix This appendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Supplement to: Chandran SS, Somerville RPT, Yang JC, et al.
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Supplemental Figure 1. Furin is efficiently deleted in CD4 + and CD8 + T cells. a, Western blot for furin and actin proteins in CD4cre-fur f/f and fur f/f Th1 cells. Wild-type and furin-deficient CD4 +
of whole cell cultures in U-bottomed wells of a 96-well plate are shown. 2
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d f a IL7 b IL GATA RORγt h HDM IL IL7 PBS Ilra R7 PBS HDM Ilra R7 HDM Foxp Foxp Ilra R7 HDM HDM Ilra R7 HDM. 9..79. CD + FOXP + T reg cell CD + FOXP T conv cell PBS Ilra R7 PBS HDM Ilra R7 HDM CD + FOXP
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