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1 Supplementary Information Titles Journal: Nature Medicine Article Title: Corresponding Author: Supplementary Item & Number Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Table 1 Supplementary Table 2 Protective HIV-specific CD8 + T cells evade T reg suppression. Helen Horton Title or Caption Correlation of suppression of proliferation by Treg cells and the precursor frequency or magnitude of the effector CD8+ T cells. Knockdown of Gal-9 in Treg cells using sirna and knockdown of GzmB in CD8+ T cells using sirna. Examples of CFSE dilution data for GzmB inhibitor and GzmB sirna studies. Examples of killing of non-treg CD4+ T cells by B27/57-restricted CTL from LTNP and examples of flow data. Proliferation of CFSE labeled Treg-depleted PBMC stimulated with HIV epitopes alone or together with Treg cells, and also with a GzmB peptide inhibitor. Treg suppression of HSV- and EBV-specific CD8+ T cells in HIV seronegative individuals. Participants clinical data and HLA genotypes. Measured CD8+ T cells response.
2 Supplementary Figure 1. Correlation of suppression of proliferation by T reg cells and the precursor frequency or magnitude of the effector CD8 + T cells. (a) Initial precursor frequency of the epitope-specific CD8 + T cells (as measured by IFN-g ELISpot) correlated to the magnitude of T reg suppression. (b) Magnitude of CD8 + CTL proliferation correlated to the magnitude of T reg suppression. (c) Magnitude of CD8 + CTL proliferation correlated to the magnitude of T reg suppression for a subset of low frequency T cell responses whose proliferation is below 3% CFSE lo CD8 + T cells. (a-c) Spearman rank tests were used. Percentage T reg suppression Percentage T reg suppression Percentage T reg suppression a b c 1,000 2,000 3,000 SFC/10 6 PBMC p = 0.01 r 2 = 0.10 p = r 2 = Percentage CFSE lo CD8 + T cells Percentage CFSE lo CD8 + T cells p = r 2 = 0.21
3 Supplementary Figure 2. (a) Knockdown of Gal-9 in T reg cells (CD4 + CD25 + CD127 /lo ) using sirna. Expression of Gal-9, 24 h post transfection was analyzed by RT-PCR. GAPDH was used as a house-keeping gene. This experiment is representative of three repeat experiments using cells from different LTNPs. (b-d) Knockdown of GzmB in CD8 + T cells using sirna. (b) CD8 + T cells were analyzed for the GzmB expression by ICS prior to stimulation with their corresponding epitopes. (c) CD8 + T cells were electroporated with nonhybridizing negative control (sicontrol) or (d) GzmB specific (sigzmb) sirna oligonucleotides and were stimulated with their cognate epitopes to determine GzmB expression by ICS. This experiment is representative of three repeat experiments using cells from different LTNPs. GAPDH Galectin-9 Neg Ctrl RNA b c d a + sirna CD8 GranzymeB
4 Supplementary Figure 3. (a) Examples of CFSE dilution data for GzmB inhibitor studies. Proliferation of CFSE labeled CD8 + T cells stimulated with their corresponding epitopes alone or together with Treg cells (at 1:0.25 ratio), and also in the presence or absence of a GzmB peptide inhibitor (z-aad-cmk). The measures of coculture suppression by Treg cells in the presence or absence of GzmB inhibitor are shown for a representative experiment from four repeat experiments. (b) Examples of CFSE dilution data for GzmB sirna studies. Proliferation of CFSE labeled CD8+ T cells electroporated with GzmB specific (sigzmb) or non-hybridizing negative control (sicontrol) sirna oligonucleotides and stimulated with their cognate epitopes alone or with Tregs (at 1:0.25 ratio). This experiment is representative of three experiments using cells from different LTNPs. a CD8 b CD8 B57 B27 A03 A CFSE B57 B27 A03 B CFSE CD8 + T reg + GzmB Inh CD8 + GzmB Inh CD8 + T reg cells CD8 alone CD8 + T reg + sir-gzmb CD8 + sir-gzmb CD8 + T reg CD8 alone
5 Supplementary Figure 4. Examples of killing of non-t reg CD4 + T cells by B27/57-restricted CTL from LTNP. Percentage Annexin V + non-treg CD4 + T cells vs. T reg cells, when PBMC were stimulated with epitopes restricted by (a) HLA-B27 or B57, (b) non-hlab57/27, (c) no peptide controls, and (d) examples of flow data. Data are from 18 experiments using cells from different LTNPs. Wilcoxon Signed Rank (WSR) tests were used. a b c B57/B27 Non-B57/B27 No peptide B57 B27 B39 No peptide d CD4+ PBMC alone PBMC + GzmB Inh Annexin V
6 Supplementary Figure 5. T reg co-culture at a ratio of 1:16 T reg :PBMC. Proliferation of CFSE labeled T reg -depleted PBMC (TrØPBMC) stimulated with HIV epitopes alone or together with T reg cells (at 1:16 T reg :PBMC ratio; i.e. approx 6% T reg frequency), and also in the presence or absence of a GzmB peptide inhibitor (z-aad-cmk). B27 B27 A03 A03 TrØPBMC + T reg + GzmB Inh (1:16 T reg :PBMC) CD8 TrØPBMC + T reg (1:16 T reg :PBMC) TrØPBMC CFSE
7 Supplementary Figure 6. T reg suppression of HSV- and EBV-specific CD8 + T cells in HIV seronegative individuals. Percentage suppression of proliferation by T reg cells of (a) HSV-specific CD8 + CTL responses restricted by HLA-B27, B07, and A02, and (b) EBV-specific CD8 + CTL responses restricted by HLA-B27 and A02 in HIV seronegative individuals. 125 a HSV 125 b EBV Percentage T reg suppression Percentage T reg suppression B27 WMW p = Non-B27-25 B27 WMW p = Non-B27
8 Supplementary Table 1 Participants clinical data and HLA 1 genotypes. PTID 2 HLA-A HLA-B HLA-C LTNP or DP HIV Plasma Viral load Virus CD4 Count NP G LTNP HIV < NP02 02G1 24G LTNP HIV NP04 03G G LTNP HIV NP LTNP HIV NP G LTNP HIV < NP G DP HIV NP G1 LTNP HIV < NP11 02G G LTNP HIV < NP13 03G LTNP HIV < NP14 03G1 68G G DP HIV NP LTNP HIV < NP LTNP HIV < NP26 02G1 03G G1 07G1 DP HIV NP29 24G LTNP HIV NP G G1 DP HIV NP DP HIV NP G LTNP HIV NP G LTNP HIV < PIC / LTNP HIV < PIC LTNP HIV PIC DP HIV PIC LTNP HIV PIC DP HIV SAC LTNP HIV AG NA HSV ND ND ND EYPI 3
9 AG NA HSV ND ND ND JW NA HSV ND ND ND TM NA HSV ND ND ND MD NA HSV ND ND ND M NA HSV ND ND ND KL NA HSV ND ND ND RW NA HSV ND ND ND EB NA HSV ND ND ND MG NA HSV ND ND ND NA HSV ND ND ND NA HSV ND ND ND NA HSV ND ND ND NA HSV ND ND ND AG NA EBV ND ND ND AG NA EBV ND ND ND JW NA EBV ND ND ND TM NA EBV ND ND ND MG NA EBV ND ND ND NA EBV ND ND ND NA EBV ND ND ND NA EBV ND ND ND 1 Human Leukocyte Antigen, 2 Patient Identification Number, 3 Estimated years post infection Not detected (ND), Not applicable (NA)
10 Supplementary Table 2 Measured CD8+ T cells response. Virus ELISpot with Treg ELISpot without Treg CFSE with Treg CFSE without Treg PTID 1 Allele Sequence HLA 2 NP02 A02 ILKEPVHGV HIV NP02 A02 LKEPVHGV HIV NP02 A24 RYLKDQQLL HIV NP02 B27 GLNKIVRMY HIV NP02 B27 IRLRPGGKK HIV NP02 B27 KRWIILGLNK HIV NP04 A03 QVPLRPMTYK HIV NP04 B07 FPVTPQVPLR HIV NP04 B07 TPGPGVRYPL HIV NP10 A26 EVIPMFSAL HIV NP10 B57 YFPDWQNYT HIV NP10 B57 ISPRTLNAW HIV NP10 B57 KAFSPEVIPMF HIV NP10 B57 IVLPEKDSW HIV NP13 A03 RLRPGGKKK HIV NP13 B27 IRLRPGGKK HIV NP13 B27 KRWIILGLNK HIV NP14 A03 AIFQSSMTK HIV NP14 A03 QVPLRPMTYK HIV NP14 A03 IRLRPGGKKK HIV NP14 B53 QASQEVKNW HIV NP14 B57 ISPRTLNAW HIV NP14 B57 KAFSPEVIPMF HIV NP14 B57 IVLPEKDSW HIV NP26 A02 SLYNTVATL HIV NP26 A03 RLRPGGKKKY HIV NP26 A03 AIFQSSMTK HIV NP26 A03 TVYYGVPVWK HIV NP26 B08 GGKKKYKL HIV NP26 B08 YLKDQQLL HIV NP26 B08 GPKVKQWPL HIV NP29 A24 KYKLKHIVW HIV NP29 A25 QAISPRTLNAW HIV NP29 A25 ETINEEAAEW HIV NP29 B39 GHQAAMQML HIV NP29 B57 AVRHFPRIW HIV NP29 B57 QASQEVKNW HIV NP29 B57 ISPRTLNAW HIV NP29 B57 KAFSPEVIPMF HIV NP29 B57 TSTLQEQIGW HIV NP31 A25 ETINEEAAEW HIV NP31 B08 GGKKKYKL HIV
11 NP40 A32 PIQKETWETW HIV NP40 B44 TWEAWWTEYW HIV NP41 A03 RLRPGGKKK HIV NP41 A03 MVHQAISPR HIV NP41 A03 LVWASRELERF HIV NP41 A03 KIRLRPGGK HIV NP41 B57 ISPRTLNAW HIV NP41 B57 KAFSPEVIPMF HIV NP41 B57 TSTLQEQIGW HIV NP41 B57 IVLPEKDSW HIV PIC1458 A24 RYPLTFGWCF HIV PIC1458 B27 RRGWEALKY HIV PIC1458 B27 VRYPLTFGW HIV SAC628 A02 SLYNTVATL HIV SAC628 A02 TAFTIPSI HIV SAC628 A32 PIQKETWETW HIV SAC628 B08 FLKEKGGL HIV SAC628 B08 NPDCKTIL HIV SAC628 B08 ANPDCKTIL HIV SAC628 B08 GEIYKRWII HIV SAC628 B14 ERYLKDQQL HIV SAC628 B14 DRFYKTLRA HIV NP01 B52/57 KAAVDLSHF HIV ND ND NP01 B57 ISPRTLNAW HIV ND ND NP01 B57 KAFSPEVIPMF HIV ND ND NP01 B57 TSTLQEQIGW HIV ND ND NP01 B57 IVLPEKDSW HIV ND ND NP01 B57 HTQGYFPDWQ HIV ND ND NP05 B57 QASQEVKNW HIV ND ND NP05 B57 KAFSPEVIPMF HIV ND ND NP05 B52 RMYSPTSI HIV ND ND NP08 3 B57 ISPRTLNAW HIV ND ND NP08 3 B57 TSTLQEQIGW HIV ND ND NP08 3 B57 IVLPEKDSW HIV ND ND NP08 3 A24 KYKLKHIVW HIV ND ND NP11 B57 ISPRTLNAW HIV ND ND NP11 B57 KAFSPEVIPMF HIV ND ND NP11 B57 TSTLQEQIGW HIV ND ND NP22 B57 KAFSPEVIPMF HIV ND ND NP22 B57 IVLPEKDSW HIV ND ND NP22 B57 QASQEVKNW HIV ND ND 0 0 NP22 A03 RLRPGGKKK HIV ND ND NP22 A03 RLRPGGKKKY HIV ND ND NP22 A03 RLRDLLLIVTR HIV ND ND NP24 B57 QASQEVKNW HIV ND ND NP24 B57 ISPRTLNAW HIV ND ND
12 NP24 B57 IVLPEKDSW HIV ND ND NP24 B57 HTQGYFPDWQ HIV ND ND NP24 B18 YPLTFGWCY HIV ND ND NP24 B35 NSSKVSQNY HIV ND ND NP33 B57 ISPRTLNAW HIV ND ND NP33 B57 KAFSPEVIPMF HIV ND ND NP33 B57 KTAVQMAVF HIV ND ND NP33 B40 IEIKDTKEAL HIV ND ND NP33 B57 YFPDWQNYT HIV ND ND NP33 A02 ILKEPVHGV HIV ND ND PIC B57 ISPRTLNAW HIV ND ND PIC B57 KAFSPEVIPMF HIV ND ND PIC B57 IVLPEKDSW HIV ND ND PIC B40 KELYPLTSL HIV ND ND PIC B40 KELYPLASL HIV ND ND PIC A68 ELYPLASLK HIV ND ND PIC B57 ISPRTLNAW HIV ND ND PIC B57 KAFSPEVIPMF HIV ND ND PIC B57 IVLPEKDSW HIV ND ND PIC A02 FLGKIWPSYK HIV ND ND PIC A02 PIVLPEKDSW HIV ND ND NP08 4 B57 ISPRTLNAW HIV ND ND NP08 4 B57 TSTLQEQIGW HIV ND ND NP08 4 B57 IVLPEKDSW HIV ND ND NP08 4 A24 KYKLKHIVW HIV ND ND PIC B57 ISPRTLNAW HIV ND ND PIC B57 KAFSPEVIPMF HIV ND ND PIC B57 IVLPEKDSW HIV ND ND PIC B40 KELYPLTSL HIV ND ND PIC B40 KELYPLASL HIV ND ND PIC A68 ELYPLASLK HIV ND ND PIC B57 ISPRTLNAW HIV ND ND PIC B57 KAFSPEVIPMF HIV ND ND PIC B57 IVLPEKDSW HIV ND ND PIC A02 FLGKIWPSYK HIV ND ND PIC A02 PIVLPEKDSW HIV ND ND AG11220 B27 RRAQMAPKR HSV ND ND AG10569 B27 RRAQMAPKR HSV ND ND JW10352 B27 RRAQMAPKR HSV ND ND JW10352 B07 RPRGEVRFL HSV ND ND MD12719 B07 RPRGEVRFL HSV ND ND M11563 B07 RPRGEVRFL HSV ND ND KL7466 B07 RPRGEVRFL HSV ND ND RW4196 B07 RPRGEVRFL HSV ND ND EB5491 B07 RPRGEVRFL HSV ND ND A02 FLWEDQTLL HSV ND ND
13 8770 A02 FLWEDQTLL HSV ND ND A02 FLWEDQTLL HSV ND ND A02 FLWEDQTLL HSV ND ND AC11220 B27 RRARSLSAERY EBV ND ND AC11220 B27 RRIYDLIEL EBV ND ND AG10569 B27 RRIYDLIEL EBV ND ND JW10352 B27 RRARSLSAERY EBV ND ND JW10352 B27 RRIYDLIEL EBV ND ND TM10062 B27 RRIYDLIEL EBV ND ND MG11331 B27 RRIYDLIEL EBV ND ND MG11331 B27 RRARSLSAERY EBV ND ND A02 CLGGLLTMV EBV ND ND A02 GLCTLVAML EBV ND ND A02 CLGGLLTMV EBV ND ND A02 GLCTLVAML EBV ND ND A02 GLCTLVAML EBV ND ND Patient Identification Number 2 Human Leukocyte Antigen 3 Prior to progression 4 Post progression Not detected (ND)
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