Figure 1 EMRA CM 17.4% Naive Nkp44 29.0% 4 10 EM Nkp46

A C 105 NKp30 Figure 1 EMRA CM 17.4% Naive NKp44 29.0% 4 10 EM NKp46

CD45R A Nkp80 103 Central EMRA Memory KIR3DS 0 31.2% Effector Memory KIR3DL 22.5% KIR2DS 3 4 5 0 10 10 10 KIR2DL CD27 CD161 KLRG1 NKG2D NK Receptors B NKG2F NKG2E NKG2A///NKG2C Naive EMRA + + CD94 CD8 T cells CD8 T cells DNAM-1 CD244 IL18RAP IL18R1 FASLG FASLG CD57 PRF1 PRF1 CX3CR1 SYTL3 CD16 NCAM1/CD56 CD244 FCGR3A/B IL2RB CD56 ZBTB16 CX3CR1 SH2D1B/EAT-2 SYK S1PR5 NKG2A/NKG2C TYROBP KLRF1/NKp80 S1PR5 DLG5 Eomes ITGB1 ITGB1 NK Chemokine CD57 CMKLR1 NCR1/NKp46 KIR2DS4 KIR2DS1 CX3CR1 KIR2DS3 Receptors ITGB1 KLRD1/CD94 ITGAV KIR2DS5 KIR2DL5A KIR2DL4 KIR3DS1 KIR3DL3 FAS ligand KIR2DS2 KIR3DL1 KIR2DL3 KIR2DL2 Perforin KIR2DL1 KIR3DL1/KIR3DL2 SYTL3 Cytotoxicity GZMA CMKLR1 Granzyme B CD3zeta CMKLR1 ITGB1 HCST Granzyme A 2GCLP CD161 ITGB1 IL2RG NKG2E ITGB1 ZEB2 KLRG1 EAT-2 Ly49 PARVB RAB27A RAB27A SAP MYO5A SYTL3 NK adaptors CD226 TBX21 GZMH SYK PRDM1 GZMB ICAM1 SYTL3 DAP12 IL15RA IL2RA IL2RA Foxo4 KLRC4 PTCH1 Foxo3/Foxo3B PTCH1 DAP10 NKp30 NKp44 IL12RB2 IL12RB1 ZAP70 RORC ZAP70 CD69 SH2D1A KLRK1 PLCG1 PLCG1 SELL LCK CD28 LAT CD27 TCRA TCRalpha GZMK LCK Foxo1 LAT CD3E TCR complex CD3D CCR7 CCR7 IL7R CD3G SELL and signaling Foxp3 CD274 CCR7 CD28 Downregulated Upregulated CD27 CD3 TCR

-40 -20 0 20 40 60 Gene expression log10 (Fold change relative to naive) D **** **** **** **** * *** **** **** * * **** *** *** ** *** ** **** 100 ** ** *

ll s CD27+CD45RA+ + -

T ce CD27 CD45RA

+ 50 CD27-CD45RA- - + CD 8 CD27 CD45RA %

0 CD16 CD56 CD57 KLRG1 KIR3DL1 KIR2DL1-3 CD244 NKG2A NKG2C NKG2D NKp44 CD161 KIR2DS1/3/5

E KDa PLCγ1 - 155 ** 2.0 * * * Zap70 - 70 ** 1.0 1.5 1.0 1.0 (Fold) - 57 LCK (Fold) 1.0 0.5 0.5 0.5 LAT - 36 0.5 LAT

LCK (Fold) PLC γ1 - 37 Zap70 (Fold) GAPDH 0.0 0.0 0.0 0.0 -- CD28 + + + + - - - CD27 + + - CD27 28 CD27 28 CD27 28 A B C CD8+IL7R+ CD8+IL7R- Figure 2 C0 Clusters C1 Groups C2 CD8+IL7R- n = 37,192 n = 25,151 C3 CD8+IL7R+ 6 samples 6 samples C4 Combined matrix (after normalization) C5 C6 C7 Doublet removal (Srublet) C8 C9

ScanPy pipeline C10 BBKNN batch effect correction C11 C12

Cluster visualisation Data analysis D CD3E CD8A CD4 IL7R 4 1.50 4

0 0 0.00 0 CD27 CD28 CCR7 SELL (CD62L) 4 2.5 3 3

0 0.0 0 0 KLRG1 PRF1 GZMA IFNG 5 4 4 3

0 0 0 0 A B Figure 3 Selected clusters Selected clusters after a second round of clustering C1_Temra

C6_Temra C0_naive C0_Naive C1_Temra C4_naive C2_Temra

C8_naive C2_Temra C4_Naive C6_Temra C8_Naive

n = 39,634

C D

NKG7 GNLY FCGR3A (CD16) FCRL6 KLRD1 (CD94) **** 3

KLRG1 TYROBP (DAP12) KLRK1 (NKG2D) KLRC1 (NKG2A) KLRC2 (NKG2C) 1 NK scores 0

Naive Temra

E F

B3GAT1 (CD57) CDKN1A (p21) CDKN2A (p16) SESN2 PRKAA1 ****

MAPKAPK2 MAPK1 MAPK8 MAPK14 TAB1 (MAP3K7IP1) 1

0 Senescence scores Naive Temra Figure 4 B C1R Cells C1R-MICA

A 0.21% 98.5% D C1R C1R MICA 30 *** FCS-A 40 NS 25 ** ** ** NS 0 103 104 105 0 103 104 105 30 20 * MICA/B (% cells) 15 20

CD107a (% ) 10 10

C CD107a (% ) 5 0 0 % Max CD28+27+ CD28+27- CD28-27- NK cells siCTR siNKG2D

0 103 104 105 NKG2D (MFI) siCTR 2486 siNKG2D 688 G E F IP: NKG2D 40 CD28 + -- *** CD28 + – CD27 30 + + - KDa * DAP12 - 12 20 DAP12 IgGL 0.18 1.37 12.39 10

GAPDH - 37 AP 12 (%) WCL D 0 DAP12

CD27+28+ CD27+28- CD27-28- NK cells

WB in CD28-27- T cells NKG2D stimulation H I J 40 ** ** KDa 100 *** 8 * 30 80 p-Zap70/Syk - 70 *** 6 60 0.20 1.02 1.62 20 ce ll s) 4 GAPDH 40 - 37 2 10 ( % 20 CD107a (% ) IFN γ (% cells) Granzyme B anti-CD3 – + – 0 0 0 anti-NKG2D – – + siCTR siDAP12 CD27+´28+ CD27+ 28- C1R CD27- 28- C1R MICA A B Figure 5

** *** 1.0 CD27-28- 764 1.0 1495

(Fold) CD27+28- 1028 1579 0.5 0.5 CD27+28+ 2161 3656 (Fold)

Unstim 394 219 p-Zap70/Syk

p-CD3ζ 0.0 0.0 3 4 5 % Max 3 4 5 % Max 0 10 10 10 0 10 10 10 p-CD3ζ (pY142) p-Zap70 (pY319)/Syk(Y352)

C 10000 *** D 5000 **** ** *** 8000 4000 CD27-28- 5459 CD27-28- 4047 6000 3000 CD27+28- 4956 CD27+28- 1506 4000 2000 CD27+28+ 4563 CD27+28+ 1815 n2 (MFI ) Ses Sesn1 (MFI) FMO 1775 2000 FMO 1540 1000 0 103 104 105 0 103 104 105 Sesn1 0 Sesn2 0 E CD8 CD28 + - - ** CD27 + + - 0.8 1.0 * * Sesn2 0.8 * 0.6 0.30 0.48 0.72 0.6 0.4 P-Jnk 0.4 0.45 0.20 0.60 0.2 0.2 GAPDH

n2 /GAPDHSes (AU) 0.0 0.0 P-Jnk/ GA PDH (A U) BF DAPI Sesn2 DAP12 P-Jnk Overlay

Sesn2 Only Figure 6 A NKG2D IP WCL B CD28 + – + – DAP12 Only DAP12

Sesn2 P-Jnk Only

P-Jnk CD27+CD28+ IgGL

CD27-CD28-

C 4 6 DE shCtrl 11.2 1 .5 * shCtrl shSesn

3 Sesn2 *

4 0.073 0.036 cells 1 .0 + 2 DAP12 250K shSesn 4.52 2 0.143 0.027 200K 0 .5

1 150K

GAPDH 100K %NKG2D

50K P-Jnk/Sesn2 BDS > 2

DAP12/Sesn2 BDS > 2 0 0 0 .0 0 SSC 3 4 5 + + - - 0 10 10 10 CD27 28 CD27 28 NKG2D shCtrl shSesn

F 80 150 1.5 *** ** ** 60

cells 100 1.0 cells + + DMSO 40 JnkI/siRNA

50 0.5

20 %CD28 P-Lck (MFI fold) %NKG2D

0 0 0 Figure 7 A Cells Singlets

65.4 SSC 2.79 33.8 5.39 NK cells FSC-H

FSC FSC-A 24.2 iNKT cells CD4

Live Lymphocytes NK1.1 CD4 T cells

CD1d Tetramer 91.3 CD8 T cells SSC SSC TCRβ CD3 CD8a Live/Dead FSC C D B 20 Y WT O WT 7.91 26.2 50 ** * 15 40 * 30 10 O Sesn1-/- O Sesn2-/- 5 %CD8 T cells 10 15.9 19.0 + 20 5 4 %Total cells 10 10 103

0 0 0 NKG2D -/-

NK iNKT CD8 TCD4 T CD44 0 103 104 105 Y WT O Sesn1 -/- EF NKG2D O WT O Sesn2 30 100 100 G H * Sesn2-/- 255 80 80 Sesn1-/- 212 20 60 60 O WT 310 %NK cells + %iNKT cells

40 + 40 Y WT 231 %CD8 T cells 10 +

20 20 FMO 183 0 102 103 104

NKG2D 0 0 0 NKG2D

DAP12 DAP12 Old WT * 100 95 I 107:107 JK * + Rae-1 90

50 85

Rae-1- 80 % specific lysis specific % 0 75 WT KO WT KO %Rae-1+cells retrieved Old Sesn-/- Figure S1

A iNKT.0.32%

Singlets Live cells NKR iNKT FSC-H FSC-H SSC-A Lymphocytes FSC-A FSC-A Live/dead TCRαβ CD3

42.0 17.6 Naive CM EM EMRA Gated on TCRαβ+CD3+CD8+ cells 0.59% 1.38% 6.04% 15.3% CD45RA 31.7 8.67 NKR+ (%) CD27 B Naive CM EM EMRA 105 18.6 11.5 0 77.9 0 29.3 37.8 3.14 1.07 0.12

104

103 CD28 CD45RA 0

15.3 54.6 0 22.1 0 70.7 56.6 2.40 95.3 3.54 0 103 104 105 CD27 CD27

CM NAIVE CM NAIVE CD57+ CD57+ KLRG1+ KLRG1+ 9.27 1.61 CD244+ CD244+ 982 39.7 14.4 46.7 25.2 EMRA EMRA 777 EM 1283 EMRA 696 EM 4 CM 0 10 CM EM EM EMRA 404 NAIVE 962 CM CD57+ CD57+ NAIVE EM EMRA KLRG1+ KLRG1+ CD244+ CD244+ 28.4 34.7 259 NAIVE 39.7 81.0 68.5 93.0 267 641 151 167 411 4 0 10 4 4 3 4 5 4 3 4 5 0 10 0 10 0 10 10 10 0 10 4 CD57 (%) CD57 MFI 0 10 0 10 10 10 KLRG1 (%) KLRG1 MFI CD244 (%) CD244 MFI

NKG2D+ NKG2D+ 44.0 90.1 NKG2C+ NKG2C+ KIR2DL KIR2DL EMRA 2.45 7.64 1.25 1.62 EM 730 521 EMRA 285 CM EMRA EM 501 NAIVE 185 EM 224 CM NKG2D+ NKG2D+ CM NKG2C+ NKG2C+ NAIVE KIR2DL 73.5 93.2 KIR2DL NAIVE 305 11.3 40.8 168 4.53 8.95 115

701 189 109 4 4 3 4 5 4 4 3 4 5 0 10 0 10 0 10 10 10 0 10 0 10 0 10 10 10 NKG2C (%) KIR2DL (%) KIR2DL MFI NKG2D (%) NKG2D MFI NKG2C MFI A Figure S2 CD27+ 28+ CD27 + 28- CD27- 28-

120 *** *** * *** * * *** *** * *** ** 100 ** ***

s Tce ll s CD8+ 20

% 0 CD16 CD56 CD57 KLRG1 KIR3DL1 KIR2DL1-3 CD244 NKG2A NKG2C NKG2D NKp44 CD161 KIR2DS1-5 Inhibitory receptors Stimulatory receptors

B CD27+ 28+ CD27 - 28+ CD27- 28- ** *** ** *** ** ** ns **

100 ** ** ** * ** * **** * s Tce ll s

+ 50 CD4 0 % CD16 CD56 CD57 KLRG1 KIR2DL1-3CD244 NKG2A NKG2C NKG2D NKp44 CD161 KIR2DS1/3/5 Figure S3 A B IL7R_Neg IL7R_pos 10000 8000

7500 4000 6000 Groups Groups 4000 IL7R_Neg 5000 IL7R_Neg

count IL7R_pos IL7R_pos 2000 2000 2500 Number of cells 0 0 0 1000 2000 3000 1000 2000 3000 Number of genes IL7R_pos IL7R_Neg Groups C Number of cells per individual before/after Doublet removal & filtration 1 2 3 4 5 6 6000 8000 6000 7500 6000 6000 4000 6000 4000 4000 5000 4000 4000 2000 2000 2000 2500 2000 2000 0 0 0 0 0 0 Condition 7 8 9 10 11 12 Before 4000 8000 After 4000 3000 3000 6000 6000 6000 2000 4000 4000 4000 Number of cells 2000 2000 1000 1000 2000 2000 2000 0 0 0 0 0 0

D E Individuals 1.00 Individuals 10X Batches: after batch correction 1 1.00 2 0.75 3 4 0.75 5 Batches 0.50 6 7 0.50 B1 8 B2 0.25 9 0.25 Proportion of cells 10 11 12

Proportion of cells 0.00 0.00 0 1 2 3 4 5 6 7 8 9 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 All Clusters F Groups: after batch correction 10000 9263 1.00 8390 8133 7131 0.75 7500 6221 Groups 0.50 IL7R_Neg 5000 4121 4003 3674 3624 3337 IL7R_pos 2122 0.25 2500 1409 915 Number of cells Proportion of cells 0.00 0 0 1 2 3 4 5 6 7 8 9 All 10 11 12 0 1 2 3 4 5 6 7 8 9 10 11 12 Clusters Clusters (n=13) G IL7R Expression

Clusters (n=13) Figure S4 A B

C CD3E CD8A IL7R CD4

CD27 CD28 CCR7 SELL (CD62L)

KLRG1 PRF1 GZMA IFNG Figure S5 A B C

100 80 5000 E:T 20:1 NK 80 4000 DN 60 60 ys is (%) 3000 40 40 2000

pe cific L 20 S 20 DAP10 (MFI) DAP10 1000

0 Specific Lysis (%) 0 0 20 510 12 0.5 DP SP DN NK

NK cells E:T ratio CD27+28- CD27-28- CD27+28+ D 104

✱

103 Sesn2 (gMFI RFU) Sesn2 (gMFI

102 Young Old Figure S6 A Vaccination Challenge Figure S7 mBSA + CFA 1 mg/ml mBSA s.c. intraplantar

Acclimation

4 h 24 h 36 h 48 h 72 h 96 h 120 h 144 h 168 h Paw measurements Sacrifice Harvest spleens and inguinal LN Stain for T cell subsets and NKRs B C D 400 **** 0.20 2.5 **** * *** 350 0.15 2.0

300 0.10 1.5 Paw swelling

-/- Y WT O Sesn1 250 0.05 1.0 O WT O Sesn2-/- Spleen weight (g) Total response (AUC)

200 0.00 0 50 100 150 -/- Time (h) Y WT O Sesn1 O WT O Sesn2-/-

E Y WT O WT O Sesn1-/- O Sesn2-/- 6 31 21 49 10 34 13 44

5 58 3 27 4 51 5 39 CD44 CD62L

F 80

40 Y WT

%Total cells %Total O WT 20 O Sesn1-/- O Sesn2-/- 0 Naive Effector Central Figure S8 ** 40 ** * * 30 * Y WT 20 * O WT

%Total cells %Total -/- 10 O Sesn1 O Sesn2-/- 0 NKG2D NKG2A/C/E KLRG1 Ly49 NKp46