SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Association of HLA-DRB1-restricted CD4 + T cell responses with HIV immune control Srinika Ranasinghe 1, Sam Cutler 1, Isaiah Davis 1, Richard Lu 2, Damien Z. Soghoian 1, Ying Qi 3, John Sidney 4, Gregory Kranias 2, Michael Flanders 1, Madelene Lindqvist 1, Bjorn Kuhl 1, Galit Alter 1, Steven G. Deeks 5, Bruce D. Walker 1, 6, Xiaojiang Gao 3, Alessandro Sette 4, Mary Carrington 1, 3 and Hendrik Streeck 2 1 Ragon Institute of MGH, MIT, and Harvard, Massachusetts General Hospital and Harvard Medical School, Cambridge, Massachusetts, USA. 2 new affiliation: U.S. Military HIV Research Program (MHRP), Henry M. Jackson Foundation, Walter Reed Army Institute of Research (WRAIR), Silver Spring, Maryland, USA. 3 Cancer and Inflammation Program, Laboratory of Experimental Immunology, SAIC-Frederick, NCI Frederick, Frederick, Maryland, USA. 4 La Jolla Institute for Allergy and Immunology, La Jolla, California, USA. 5 UCSF Department of Medicine at San Francisco General Hospital, San Francisco, California, USA. 6 Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. * Corresponding author Hendrik Streeck, MD/PhD Mail: hstreeck@hivresearch.org Phone: 301.319.9704

DRB1 *0 4:0 1 FQ 1 2 ( FYKTLR AEQASQ) or FS1 1 ( FYKTLR AEQAS) DRB1 *07 :0 1 RA1 1 ( RFYKT LRAE QA) or RQ 10 (RF YKT L RAE Q ) O LP-41 (YVDRFYKTLRAEQ ASQ EV) YVDRFYKTLRAEQ ASQ YVDRFYKTLRAEQ A YVDRFYKTLRAE YVDRFYKTLRA YVDRFYKTLR YVDRFYKTL YVDRFKT DRFYKTLRAEQ ASQ EV RFYKTLRAEQ ASQ EV FYKTLRAEQ ASQ EV KTLRAEQ ASQ EV LRAEQ ASQ EV RAEQ ASQ EV AEQ ASQ EV 0 50 100 150 IFN y r es pons e ( % m ax im um ) O LP-41 (YVDRFYKTLRAEQ ASQ EV) YVDRFYKTLRAEQ ASQ YVDRFYKTLRAEQ A YVDRFYKTLRAE YVDRFYKTLRA YVDRFYKTLR YVDRFYKTL YVDRFKT DRFYKTLRAEQ ASQ EV RFYKTLRAEQ ASQ EV FYKTLRAEQ ASQ EV KTLRAEQ ASQ EV LRAEQ ASQ EV RAEQ ASQ EV AEQ ASQ EV 0 50 100 150 IFN y r es pons e ( % m ax im um ) DRB1 *1 1:0 1 FA 10 (FY KTLR AEQ A) or FQ 9 (FY KTLR AEQ) DRB1 *13: 01 YE1 2 ( YVD RF YKT L RAE) o r VE1 1 ( VDRF YK TL RAE) O LP-41 (YVDRFYKTLRAEQ ASQ EV) YVDRFYKTLRAEQ ASQ YVDRFYKTLRAEQ A YVDRFYKTLRAE YVDRFYKTLRA YVDRFYKTLR YVDRFYKTL YVDRFKT DRFYKTLRAEQ ASQ EV RFYKTLRAEQ ASQ EV FYKTLRAEQ ASQ EV KTLRAEQ ASQ EV LRAEQ ASQ EV RAEQ ASQ EV AEQ ASQ EV 0 50 100 150 IFN y r es pons e ( % m ax im um ) O LP-41 (YVDRFYKTLRAEQ ASQ EV) YVDRFYKTLRAEQ ASQ YVDRFYKTLRAEQ A YVDRFYKTLRAE YVDRFYKTLRA YVDRFYKTLR YVDRFYKTL YVDRFKT DRFYKTLRAEQ ASQ EV RFYKTLRAEQ ASQ EV FYKTLRAEQ ASQ EV KTLRAEQ ASQ EV LRAEQ ASQ EV RAEQ ASQ EV AEQ ASQ EV 0 50 100 150 IFN y r espo nse ( % m axim um ) DRB1 *1 3: 02 YE1 2 ( YVD RF YKT L RAE) o r VE1 1 ( VDRF YK TL RAE) O LP-41 (YVDRFYKTLRAEQ ASQ EV) YVDRFYKTLRAEQ ASQ YVDRFYKTLRAEQ A YVDRFYKTLRAE YVDRFYKTLRA YVDRFYKTLR YVDRFYKTL YVDRFKT DRFYKTLRAEQ ASQ EV RFYKTLRAEQ ASQ EV FYKTLRAEQ ASQ EV KTLRAEQ ASQ EV LRAEQ ASQ EV RAEQ ASQ EV AEQ ASQ EV 0 50 100 150 IFN y r es pons e ( % m ax im um ) Supplementary Figure S1: OLP-41 is promiscuously presented by multiple HLA-DRB1 alleles that share largely overlapping epitope-binding repertoires. The minimal stimulatory epitope(s) were delineated for each of the five HLA-DRB1 alleles. In fine-mapping analysis, each OLP-41 specific CD4 T cell line was tested in triplicate against 20 um of the original 18-mer OLP-41 (YVDRFYKTLRAEQASQEV) and 14 serial truncations from the N- and C-terminus presented by the restricting HLA-DRB1 expressing LCL. Bars denote standard error. The IFNγ SFU response to OLP-41 was normalized to 100%. Concordant with other CD4 T cell studies (15) the "minimal stimulatory epitope" was defined as the shortest peptide sequence triggering an IFNy response 50% of the original OLP-41 response.

% HLA-DRB1 Allele Expression 25 20 15 10 5 MGH & SC OPE C ohort (n =1085) In dependent C ohort 0 1 01 1 02 1 03 3 01 3 02 4 01 4 03 4 04 4 05 7 01 8 01 8 04 8 11 9 01 10 01 11 01 11 02 11 04 12 01 13 01 13 02 13 03 13 04 14 01 15 01 15 02 15 03 16 01 16 02 DRB1 alle le s Supplementary Figure S2: The MGH & SCOPE cohort (n=1085) and our independent cohort (n=42) of treatment naive chronically HIV-infected subjects demonstrated similar HLA-DRB1 allele expression frequencies.

a 6 P=0.25 b 1500 P=0.37 Lo g Vira l Load 4 2 CD4 T cell c ount 1000 500 0 Low OR High OR 0 Low OR High OR as sociated HL A-DR associated HL A-DR as sociated HL A-DR assoc iated HLA -DR Supplementary Figure S3: HIV-infected subjects expressing 1 DRB1 alleles associated with low OR vs. high OR demonstrated no significant differences in their mean viral load (log HIV RNA copies/ml) (a) or CD4 count (b) (P>0.05 Mann-Whitney). The 43 treatment-naive chronically HIV-infected subjects from MGH included 15 subjects expressing 1 DRB1 alleles linked with low OR and 28 subjects expressing 1 DRB1 alleles linked to higher OR, that were assayed for their plasma HIV viral load and CD4 T cell count at MGH, as previously described (8).

Gag Nef Env Pol Acc Total Number of peptides in each protein 66 27 113 133 71 410 Number of peptides eliciting a CD4 T cell response* 59 19 63 57 41 239 Number of peptides tested for HLA-DRB1 restriction Number of peptides with successfully defined HLA-DRB1 restriction % Peptides restricted by HLA-DRB1 after adjustment for CD4 T cell targeting 53 16 43 24 19 155 30 9 16 6 6 67 63 80 67 58 55 74 Supplementary Table 1: A similar percentage of peptide restrictions were identified for each protein after adjustment for protein size and HIV-specific CD4 T cell targeting. The item highlighted with an asterisk (*) denotes data gathered from the solid black bars in Fig 1A, as previously described (8).

Peptide (OLP) Protein Peptide Sequence Number of restrictions HLA-DR defined restrictions 41 Gag YVDRFYKTLRAEQASQEV 12 DRB1*0401, *0701, *0801, *1001, *1101, *1301, *1302, *1303, *1501, *1502, DRB4*0101, DRB5*0101 4 Gag GKKKYKLKHIVWASREL 10 DRB1*0101, *0701, *0801, *1101, *1302, *1303, DRB3*0101, DRB3*0301, DRB4*0101, DRB5*0101 5 Gag KHIVWASRELERFAV 6 DRB1*0301, *1301, *1302, *1303, DRB3*0101, DRB5*0101 2 Gag SGGELDRWEKIRLRPGGK 5 DRB1*0701, *1101, *1301, *1302, DRB3*0101 37 Gag WIILGLNKIVRMYSPTSI 5 DRB1*0302, *1101, *1301, *1501, *1502 82 Nef LWVYHTQGYFPDWQNY 5 DRB1*0701, *1301, *1401, DRB3*0101, DRB4*0101 7 Gag ERFAVNPGLLETSEGCR 4 DRB1*1302, *1303, *1501, DRB3*0301 23 Gag AFSPEVIPMFSALSEGA 4 DRB1*0401, *0701, *1501, DRB4*0101 34 Gag STLQEQIGWMTNNPPIPV 4 DRB1*0101, *1303, *DRB3*0101, DRB3*0301 92 Nef KFDSRLAFHHMARELH 4 DRB1*0302, *1302, *1303, DRB3*0301 116 Tat TKGLGISYGRKKRRQRRR 4 DRB1*1302, *1303, DRB3*0101, DRB3*0301 6 Gag ASRELERFAVNPGLL 3 DRB1*1301, *1302, DRB4*0101 59 Gag RQANFLGKIWPSHKGR 3 DRB1*1301, *1501, DRB5*0101 60 Gag GKIWPSHKGRPGNFLQSR 3 DRB1*0701, *1301, *1501 92 Nef KFDSRLAFHHMARELH 3 DRB1*1302, *1303, DRB3*0301 301 Env NVTENFNMWKNNMVEQMH 3 DRB1*1101, *1501, *1502 312 Env YALFYKLDVVPIDNDNTSY 3 DRB1*0101, *0701, *1001 316 Env KVSFEPIPIHYCAPAGFA 3 DRB1*0101, *1101, DRB3*0101 11 Gag TGSEELRSLYNTVATLY 2 DRB1*0405, *0701 12 Gag SLYNTVATLYCVHQRIEV 2 DRB1*0701, *1401 21 Gag PRTLNAWVKVVEEKAF 2 DRB1*1301, *1304 33 Gag SDIAGTTSTLQEQIGWM 2 DRB1*0404, DRB1*1502 35 Gag WMTNNPPIPVGEIYKRWI 2 DRB3*0101, DRB3*0303 54 Gag CFNCGKEGHIAKNCRAPR 2 DRB1*0901, *1301, 55 Gag HIAKNCRAPRKKGCWK 2 DRB1*0701, *1301 78 Nef YKAAVDLSHFLKEKGGL 2 DRB1*0701, *0804 98 Rev PSPEGTRQARRNRRRRW 2 DRB1*1301, DRB3*0101 217 Pol VIWGKTPKFKLPIQKETW 2 DRB1*1301, DRB3*0101 293 Env AAEQLWVTVYYGVPVWK 2 DRB1*1101, *1501 19 Gag IVQNLQGQMVHQAISPR 1 DRB1*0901 22 Gag WVKVVEEKAFSPEVIPMF 1 DRB1*1101 29 Gag AAEWDRLHPVHAGPIA 1 DRB1*0701 30 Gag LHPVHAGPIAPGQMREPR 1 DRB1*1101

40 Gag GPKEPFRDYVDRFYKTLR 1 DRB1*1301 43 Gag EVKNWMTETLLVQNA 1 DRB1*1401 47 Gag AATLEEMMTACQGVGGPGH 1 DRB1*0404 51 Gag TNSATIMMQRGNFRNQRK 1 DRB1*1502 56 Gag RAPRKKGCWKCGKEGHQM 1 DRB1*0901 62 Gag FLQSRPEPTAPPEESFRF 1 DRB1*0701 65 Gag QKQEPIDKELYPLASLR 1 DRB1*1502 66 Gag KELYPLASLRSLFGNDPSSQ 1 DRB1*1502 68 Nef RSVVGWPAVRERMRRA 1 DRB1*1302 69 Nef PAVRERMRRAEPAADGV 1 DRB1*1302 80 Nef GLEGLIYSQKRQDILDLW 1 DRB1*1301 81 Nef QKRQDILDLWVYHTQGYF 1 DRB1*1301 86 Nef FGWCFKLVPVEPEKVEEA 1 DRB1*0701 126 Vpu IVFIEYRKILRQKIDRL 1 DRB1*0405 170 Pol EEKIKALVEICTEMEK 1 DRB1*0405 229 Pol QKTELQAIHLALQDSGL 1 DRB1*1301 256 Pol TIHTDNGSNFTSTTVKAA 1 DRB1*0701 260 Pol PYNPQSQGVVESMNKELK 1 DRB1*0405 264 Pol LKTAVQMAVFIHNFKRK 1 DRB1*1101 286 Vpr ILQQLFIHFRIGCQHSR 1 DRB1*0401 292 Env LGMLMICSAAEQLWVTVY 1 DRB1*1304 294 Env TVYYGVPVWKEATTTLF 1 DRB1*0901 296 Env LFCASDACAYDTEVHNVW 1 DRB3*0101 300 Env NPQEVVLENVTENFNMWK 1 DRB1*0804 310 Env CSFNITTSIRDKVQKEYA 1 DRB1*0701 311 Env IRDKVQKEYALFYKLDVV 1 DRB1*0701 328 Env NCTRPNNNTRKSIHI 1 DRB1*1101 339 Env SFNCGGEFFYCNTTQLF 1 DRB1*0901 353 Env ELYKYKVVKIEPLGVA 1 DRB1*0101 378 Env LWNWFDITNWLWYIKIFI 1 DRB1*1501 380 Env IFIMIVGGLVGLRIVFAV 1 DRB1*1501 388 Env RLVDGFLALIWVDLRSL 1 DRB1*0701 409 Vif HIPLGDARLVITTYWGL 1 DRB1*1301 418 Vif IRNAILGHIVSPRCEYQA 1 DRB1*1501 Supplementary Table 2: Peptide-DRB restrictions were identified across the HIV proteome in our IFNγ ELISpot HLA-DR restriction assay. HIV-specific CD4 T cell peptides are here defined as 'promiscuous' if a peptide is recognized in the context of two or more DRB alleles expressed by different individuals. Yet, please note that the list of potential HLA-DRB restrictions is most likely far greater than determined here.

Supplementary Table 3: Five immunodominant HIV-specific CD4 T cell peptides demonstrated high levels of promiscuity in a well-characterized radioactively labeled competition assay to determine the peptide-drb1 binding capacity. Each peptide was tested against a standardized panel of 13 HLA-DRB1 alleles, as previously described (13). IC 50 nm values were generated for each peptide-drb1 interaction, with IC 50 values less than the threshold value of 1000 nm denoting 'binders', while non-binders (>1000 nm ) are highlighted in grey with bold font.

HLA-DRB1 Allele Expressed (n=1085) % Allele Frequency (n=1085) DRB1*01:01 183 9.4 DRB1*01:02 37 1.9 DRB1*01:03 28 1.4 DRB1*03:01 162 8.3 DRB1*03:02 15 0.8 DRB1*04:01 167 8.5 DRB1*04:03 19 1 DRB1*04:04 68 3.5 DRB1*04:05 20 1 DRB1*07:01 305 15.6 DRB1*08:01 37 1.9 DRB1*08:04 9 0.5 DRB1*08:11 0 0 DRB1*09:01 21 1.1 DRB1*10:01 13 0.7 DRB1*11:01 133 6.8 DRB1*11:02 7 0.4 DRB1*11:04 58 3 DRB1*12:01 43 2.2 DRB1*13:01 144 7.4 DRB1*13:02 105 5.4 DRB1*13:03 35 1.8 DRB1*13:04 0 0 DRB1*14:01 58 3 DRB1*15:01 229 11.7 DRB1*15:02 26 1.3 DRB1*15:03 2 0.1 DRB1*16:01 28 1.4 DRB1*16:02 4 0.2 Supplementary Table 4: HLA-DRB1 allele expression frequencies in our MGH & SCOPE cohort of HIVinfected treatment naive individuals (n=1085).

HLA-DRB1 P-value OR 95% CI 15:02 0.003 0.22 0.08 0.61 10:01 0.07 0.29 0.08 1.09 13:03 0.06 0.49 0.24 1.02 13:02 0.03 0.61 0.40 0.94 15:01 0.07 1.33 0.98 1.81 01:01 0.06 1.39 0.99 1.96 03:01 0.004 1.68 1.18 2.39 Supplementary Table 5: The P-values, odds ratio (OR) and 95% confidence intervals (95% CI) are shown for DRB1 alleles trending to lower OR (DRB1*15:02, *10:01, *13:03, *13:02) and DRB1 alleles trending to higher OR (DRB1 *15:01, *01:01 *03:01) at 10% significance prior to multiple comparisons. The statistical differences observed in our functional analysis remained significant when HLA-DRB1 alleles were more stringently selected based on 10% significance prior to multiple comparisons (Fig. 3a P=0.0148 Fishers exact test, Fig. 3b P= 0.045 Mann-Whitney Test).