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Supplemental Material 1 Supplemental Figure 1 A CD8+ è Tet+ è B CD8+ è Tet+ è Gang strategy for defining subpopulaons of CD8+ T cells in terms of expression of combinaons of transcrip#on factors, T-bet (TB) and Eomesodermin (EO), and differen#aon markers, CCR7 (R7), CD27 (27), and CD45RA (RA). A. Sample in AIM, labeled with pMHC1 tetramers to detect EBV-specific CD8+ T cells with specificity for HLA-A*0201 restricted immunodominant epitope in BMRF1 (TLD) is shown. Top row, leY to right shows gates used to define CD8+ T cells. Middle row, leY to right shows gates used to define populaons of CD8+ T cells expressing each marker, and tetramer- posi#ve cells. Bo[om row, leY to right shows marker gates applied to the tetramer- posi#ve populaon. All marker and tetramer gates were checked against a “fluorescence minus one” control. B. Sample in CONV that is paired with sample represented in top panel of this figure with same gang strategy applied. Supplemental Figure 2 A p < 0.05 p < 0.05 B p < 0.05 p < 0.05 p < 0.05 100 8000 80 6000 60 4000 40 (median; IQR) 2000 20 Relative Percentage 0 Relative Signal Intensity 0 CD27 IL7R CCR7 CD57 CD27 IL7R CCR7 CD57 Surface Marker Gene Expressed C D Tcm/naive: CD27+ CD127+ CCR7+ CD57-CD127 Tem/early:Phenotype CD27+ CD127+ or CCR7+CD27 CD57- CCR7 CD57 Tem/int_late: CD27+ CD127- CCR7- CD57+/-(IL-7R) Teff: CD27- CD127- CCR7- CD57+/- Tcm/naïve (central memory or naïve) + + + - Tem/early (effector memory, early differen#aon) + +/- +/- - Tem/late (effector memory, late differen#aon) + - - +/- Teff (effector) - - - +/- A. Relave percentages of CD8+ T-cells expressing individual surface markers of differen#aon in AIM (black bars) and CONV (white bars). Brackets indicate significant differences (Mann-Whitney test). B. Gene expression of selected surface markers in CD8+ T cells in AIM and CONV. Mean relave microarray signal intensi#es with SEM are shown. Brackets indicate significant differences (Mann- Whitney test). C. and D. Phenotypes of EBV-specific CD8+ T cells in AIM and CONV.Tcm/naive: CD27+ CD127+ CCR7+ CD57- Tem/early: CD27+ CD127+ or CCR7+ CD57- Relave percentages of subpopulaons, defined by expression of CD27, IL-7R, CCR7, Tem/int_late: CD27+ CD127- CCR7- CD57+/- and CD57. C. EBV Ly#c (A2-BMLF1-GLC and A2-BRLF1-YVL), and Teff: CD27- CD127-D. Latent (B7- CCR7- CD57+/- EBNA3a-RPP, A2-EBNA3c-LLD, and A2-LMP2-CLG) an#gen specific CD8+ T-cells are represented similarly. Time frame: AIM (Week 1 or 2) to CONV (Week 26 or 52). Supplemental Table I: Gene expression in total CD8+ T cells in AIM and CONV (highlighting differentially expressed genes related to T cell differentiation, activation, functionality, and exhaustion) Gene AIM CONV Fold Change AIM CONV Fold Change Category* P-value Gene Symbol Category* P-value Symbol Signal Signal AIM to CONV Signal Signal AIM to CONV CD38 1 3282 836 -3.9 3.50E-08 GZMH 4 2752 1038 -2.6 3.70E-08 CD27 1 4281 3408 -1.3 2.10E-02 GZMA 4 2831 1141 -2.5 9.30E-08 CD28 1 635 796 1.3 2.60E-03 GZMB 4 1961 1072 -1.8 2.10E-05 IL7R 1 1775 5877 3.3 1.20E-06 SPON2 4 1626 911 -1.8 4.80E-05 STAT1 2 4295 2048 -2.1 1.70E-05 PRF1 4 6682 4504 -1.5 3.10E-04 GFI1 2 946 541 -1.7 8.80E-07 CTLA4 5 522 269 -1.9 6.90E-07 ZNF683 2 792 481 -1.6 4.00E-05 LAG3 5 867 494 -1.8 1.00E-05 BATF 2 484 315 -1.5 1.30E-06 FASLG 5 428 253 -1.7 1.90E-07 NFKBIB 2 1128 741 -1.5 2.50E-06 FAS 5 886 530 -1.7 2.10E-06 EOMES 2 1827 1206 -1.5 5.80E-06 CD160 5 2200 1287 -1.7 1.40E-03 TBX21 2 1150 902 -1.3 7.80E-05 KLRD1 5 2670 1777 -1.5 3.00E-03 NFATC2 2 3406 2594 -1.3 1.80E-04 HAVCR2 (TIM3) 5 428 316 -1.4 4.00E-02 IRF4 2 943 712 -1.3 2.10E-02 PDCD1 5 584 500 -1.2 8.20E-03 PRDM1 2 2283 1808 -1.3 3.30E-02 KLRG1 5 2131 1728 -1.2 4.10E-02 STAT2 2 890 723 -1.2 6.70E-03 PTGER2 5 992 1376 1.4 2.10E-02 RUNX3 2 5580 4788 -1.2 3.00E-02 CD70 1 205 189 -1.1 2.20E-01 GATA3 2 1114 916 -1.2 4.70E-02 PTPRC 1 5339 5060 -1.1 4.10E-01 STAT4 2 1744 1549 -1.1 2.50E-02 SELL 1 7906 7722 -1 6.90E-01 ID2 2 5405 4978 -1.1 4.60E-02 CD69 1 3277 3720 1.1 3.00E-01 NFKB2 2 435 529 1.2 2.00E-03 NFIL3 2 648 533 -1.2 2.80E-01 ID3 2 158 186 1.2 7.30E-03 RORA 2 2178 1920 -1.1 6.10E-02 XBP1 2 1632 1921 1.2 1.00E-02 BCOR 2 536 472 -1.1 1.10E-01 RORC 2 266 308 1.2 1.90E-02 MBD2 2 1673 1574 -1.1 1.90E-01 PBX3 2 411 532 1.3 9.40E-04 STAT3 2 3158 3013 -1 3.80E-01 NFATC1 2 793 1015 1.3 9.80E-04 BCL6B 2 222 230 1 4.50E-01 BCL11B 2 1740 2424 1.4 3.30E-05 STAT6 2 1706 1763 1 6.90E-01 RUNX2 2 1056 1429 1.4 1.80E-02 BCL6 2 740 769 1 7.40E-01 BMI1 2 741 1126 1.5 1.30E-03 NOTCH1 2 684 687 1 9.50E-01 NFKBIZ 2 917 1476 1.6 1.70E-04 NOTCH2 2 1191 1332 1.1 2.00E-01 TCF7 2 2221 3900 1.8 3.60E-05 AHR 2 969 1434 1.5 6.10E-02 NFKBIA 2 2952 5433 1.8 9.80E-05 FOS 2 3621 5715 1.6 1.00E-01 TCF7L2 2 324 659 2 8.00E-05 CCR1 3 304 225 -1.4 1.90E-01 LEF1 2 2997 6187 2.1 1.80E-06 IL2RB 3 5927 4912 -1.2 5.90E-02 SCML1 2 60 143 2.4 1.80E-04 ITGB1 3 1339 1151 -1.2 1.20E-01 BACH2 2 351 1019 2.9 6.10E-08 ITGAD 3 290 284 -1 6.00E-01 NR3C2 2 207 664 3.2 6.20E-08 IL2 3 25 26 1 4.30E-01 IFNG 3 819 220 -3.7 1.10E-06 CXCL2 3 108 111 1 8.70E-01 CX3CR1 3 1430 623 -2.3 8.30E-05 IL21 3 40 44 1.1 8.90E-02 IL12RB1 3 1367 808 -1.7 3.60E-10 XCL1 3 291 309 1.1 7.50E-01 IL32 3 4719 2946 -1.6 2.70E-05 GZMK 4 5537 4767 -1.2 1.60E-01 IL28RA 3 416 294 -1.4 9.10E-07 GZMM 4 1788 1637 -1.1 1.60E-01 ITGAL 3 5732 4144 -1.4 7.30E-05 GNLY 4 3660 3208 -1.1 3.20E-01 CCL5 3 12098 9296 -1.3 7.30E-06 PROK2 4 420 388 -1.1 4.10E-01 IL2RG 3 10768 8351 -1.3 3.40E-04 LAMP1 4 1328 1292 -1 5.80E-01 ADAM8 3 768 616 -1.2 5.20E-06 KLRC1 5 173 242 1.4 8.20E-02 ITGB2 3 5145 4353 -1.2 2.60E-04 CXCR6 3 195 167 -1.2 3.60E-02 *Categories: CMTM8 3 184 218 1.2 3.60E-02 1 Markers of differentiation and activation IL18R1 3 124 157 1.3 4.50E-02 2 Transcription Factors IL2RA 3 138 202 1.5 5.60E-04 3 Cytokines, chemokines, and their receptors IL4R 3 686 1186 1.7 3.90E-05 4 Effector/immune function IFNGR2 3 261 521 2 2.30E-03 5 Negative regulators and exhaustion markers CCR7 3 2073 5297 2.6 2.60E-06 ITGA6 3 621 1664 2.7 1.40E-06 Genes with significant differential expression have p-values highlighted in bold and are listed in the top section of the table. The sections of the table are sorted by gene category. Supplemental Table II: Gene expression in EBV-specific CD8+ T cells in AIM and CONV (highlighting differentially expressed genes in the Nanostring codeset) Gene Fold Change Fold Change Category* Description P-value Gene Symbol Category* Description P-value Symbol AIM to CONV AIM to CONV cytotoxic T-lymphocyte-associated TYMS 7 thymidylate synthetase -13.73 0 CTLA4 5 -2.21 0.0769 protein 4 MKI67 7 marker of proliferation Ki-67 -8.15 0.001 CCNA2 7 cyclin A2 -2.18 0.1321 CDCA7 7 cell division cycle associated 7 -8.03 0.0006 CCNB2 7 cyclin B2 -2.03 0.071 CD38 1 CD38 molecule -7.4 0.0009 CPT1A 7 carnitine palmitoyltransferase 1A (liver) -1.58 0.1683 glyceraldehyde-3-phosphate GAPDH 7 -3.75 0 FAS 5 Fas cell surface death receptor -1.58 0.1316 dehydrogenase granzyme A (granzyme 1, GZMA 4 cytotoxic T-lymphocyte- -3.61 0.0031 CD160 5 CD160 molecule -1.48 0.1637 associated serine esterase 3) hypoxanthine zinc finger and BTB domain containing HPRT 7 -3.16 0.0036 ZBTB7B 2 -1.47 0.3157 phosphoribosyltransferase 1 7B IRF4 2 interferon regulatory factor 4 -3.12 0 NOTCH1 2 notch 1 -1.46 0.1444 TUBB 7 tubulin, beta class I -3.1 0.0002 RUNX2 2 runt-related transcription factor 2 -1.37 0.2439 signal transducer and activator of STAT1 2 -2.68 0 FASLG 5 Fas ligand (TNF superfamily, member 6) -1.36 0.2602 transcription 1, 91kDa v-myc avian myelocytomatosis hepatitis A virus cellular receptor 2 (syn: MYC 2 -2.67 0.0139 HAVCR2 5 -1.35 0.4495 viral oncogene homolog TIM3) topoisomerase (DNA) II alpha growth factor independent 1 TOP2A 7 -2.62 0.0019 GFI1 2 -1.34 0.2179 170kDa transcription repressor SH2D1A 6 SH2 domain containing 1A -2.58 0.0103 ZEB1 2 zinc finger E-box binding homeobox 1 -1.34 0.3338 SELL 1 selectin L; CD62 -2.54 0.0117 LAG3 5 lymphocyte-activation gene 3 -1.31 0.0884 granzyme B (granzyme 2, GZMB 4 cytotoxic T-lymphocyte- -2.28 0.02 IFNG 3 interferon, gamma -1.27 0.3581 associated serine esterase 1) T cell immunoreceptor with Ig and inhibitor of DNA binding 2, dominant TIGIT 4 -2.18 0.0465 ID2 2 -1.22 0.0545 ITIM domains negative helix-loop-helix protein signal transducer and activator of STAT2 2 -2.15 0.0001 CD44 1 CD44 molecule (Indian blood group) -1.14 0.2451 transcription 2, 113kDa CD244 molecule, natural killer cell EGR2 2 early growth response 2 -1.99 0.0041 CD244 5 -1.1 0.7556 receptor 2B4 signal transducer and activator of GATA3 2 GATA binding protein 3 -1.99 0.0001 STAT3 2 transcription 3 (acute-phase response -1.1 0.5861 factor) CCL5 3 chemokine (C-C motif) ligand 5 -1.96 0 LEF1 2 lymphoid enhancer-binding factor 1 -1.05 0.8965 chemokine (C-X-C motif) receptor basic helix-loop-helix family, member CXCR3 3 -1.96 0.0003 BHLHE40 2 -1.04 0.7471 3 e40 GUSB 7 glucuronidase, beta -1.93 0.0366 ATM 7 ataxia telangiectasia mutated -1.01 0.9859 lipase A, lysosomal acid, cholesterol ZBP1 2 Z-DNA binding protein 1 -1.92 0.0061 LIPA 7 1.01 0.9724 esterase PRF1 4 perforin 1 (pore forming protein) -1.58 0 XBP1 2 X-box binding protein 1 1.05 0.8853 RORA 2 RAR-related orphan receptor A -1.29 0.0229 BCL6 2 B-cell CLL/lymphoma 6 1.08 0.7677 signal transducer and activator of STAT4 2 1.39 0.017 AHR 2 aryl hydrocarbon receptor 1.16 0.5843 transcription 4 lysine (K)-specific denticleless E3 ubiquitin protein ligase KMT2E 2 methyltransferase 2E (synonym: 1.4 0.0037 DTL 7 1.19 0.6406 homolog (Drosophila) MLL5) KLF2 2 Kruppel-like factor 2 1.55 0.0098 CCL4 3 chemokine (C-C motif) ligand 4 1.2 0.3746
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