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1 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: Phone:

2 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 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 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 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 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 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.

3 % HLA-DRB1 Allele Expression MGH & SC OPE C ohort (n =1085) In dependent C ohort 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.

4 a 6 P=0.25 b 1500 P=0.37 Lo g Vira l Load 4 2 CD4 T cell c ount 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).

5 Gag Nef Env Pol Acc Total Number of peptides in each protein Number of peptides eliciting a CD4 T cell response* 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 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).

6 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* Gag GKKKYKLKHIVWASREL 10 DRB1*0101, *0701, *0801, *1101, *1302, *1303, DRB3*0101, DRB3*0301, DRB4*0101, DRB5* Gag KHIVWASRELERFAV 6 DRB1*0301, *1301, *1302, *1303, DRB3*0101, DRB5* Gag SGGELDRWEKIRLRPGGK 5 DRB1*0701, *1101, *1301, *1302, DRB3* Gag WIILGLNKIVRMYSPTSI 5 DRB1*0302, *1101, *1301, *1501, * Nef LWVYHTQGYFPDWQNY 5 DRB1*0701, *1301, *1401, DRB3*0101, DRB4* Gag ERFAVNPGLLETSEGCR 4 DRB1*1302, *1303, *1501, DRB3* Gag AFSPEVIPMFSALSEGA 4 DRB1*0401, *0701, *1501, DRB4* Gag STLQEQIGWMTNNPPIPV 4 DRB1*0101, *1303, *DRB3*0101, DRB3* Nef KFDSRLAFHHMARELH 4 DRB1*0302, *1302, *1303, DRB3* Tat TKGLGISYGRKKRRQRRR 4 DRB1*1302, *1303, DRB3*0101, DRB3* Gag ASRELERFAVNPGLL 3 DRB1*1301, *1302, DRB4* Gag RQANFLGKIWPSHKGR 3 DRB1*1301, *1501, DRB5* Gag GKIWPSHKGRPGNFLQSR 3 DRB1*0701, *1301, * Nef KFDSRLAFHHMARELH 3 DRB1*1302, *1303, DRB3* Env NVTENFNMWKNNMVEQMH 3 DRB1*1101, *1501, * Env YALFYKLDVVPIDNDNTSY 3 DRB1*0101, *0701, * Env KVSFEPIPIHYCAPAGFA 3 DRB1*0101, *1101, DRB3* Gag TGSEELRSLYNTVATLY 2 DRB1*0405, * Gag SLYNTVATLYCVHQRIEV 2 DRB1*0701, * Gag PRTLNAWVKVVEEKAF 2 DRB1*1301, * Gag SDIAGTTSTLQEQIGWM 2 DRB1*0404, DRB1* Gag WMTNNPPIPVGEIYKRWI 2 DRB3*0101, DRB3* Gag CFNCGKEGHIAKNCRAPR 2 DRB1*0901, *1301, 55 Gag HIAKNCRAPRKKGCWK 2 DRB1*0701, * Nef YKAAVDLSHFLKEKGGL 2 DRB1*0701, * Rev PSPEGTRQARRNRRRRW 2 DRB1*1301, DRB3* Pol VIWGKTPKFKLPIQKETW 2 DRB1*1301, DRB3* Env AAEQLWVTVYYGVPVWK 2 DRB1*1101, * Gag IVQNLQGQMVHQAISPR 1 DRB1* Gag WVKVVEEKAFSPEVIPMF 1 DRB1* Gag AAEWDRLHPVHAGPIA 1 DRB1* Gag LHPVHAGPIAPGQMREPR 1 DRB1*1101

7 40 Gag GPKEPFRDYVDRFYKTLR 1 DRB1* Gag EVKNWMTETLLVQNA 1 DRB1* Gag AATLEEMMTACQGVGGPGH 1 DRB1* Gag TNSATIMMQRGNFRNQRK 1 DRB1* Gag RAPRKKGCWKCGKEGHQM 1 DRB1* Gag FLQSRPEPTAPPEESFRF 1 DRB1* Gag QKQEPIDKELYPLASLR 1 DRB1* Gag KELYPLASLRSLFGNDPSSQ 1 DRB1* Nef RSVVGWPAVRERMRRA 1 DRB1* Nef PAVRERMRRAEPAADGV 1 DRB1* Nef GLEGLIYSQKRQDILDLW 1 DRB1* Nef QKRQDILDLWVYHTQGYF 1 DRB1* Nef FGWCFKLVPVEPEKVEEA 1 DRB1* Vpu IVFIEYRKILRQKIDRL 1 DRB1* Pol EEKIKALVEICTEMEK 1 DRB1* Pol QKTELQAIHLALQDSGL 1 DRB1* Pol TIHTDNGSNFTSTTVKAA 1 DRB1* Pol PYNPQSQGVVESMNKELK 1 DRB1* Pol LKTAVQMAVFIHNFKRK 1 DRB1* Vpr ILQQLFIHFRIGCQHSR 1 DRB1* Env LGMLMICSAAEQLWVTVY 1 DRB1* Env TVYYGVPVWKEATTTLF 1 DRB1* Env LFCASDACAYDTEVHNVW 1 DRB3* Env NPQEVVLENVTENFNMWK 1 DRB1* Env CSFNITTSIRDKVQKEYA 1 DRB1* Env IRDKVQKEYALFYKLDVV 1 DRB1* Env NCTRPNNNTRKSIHI 1 DRB1* Env SFNCGGEFFYCNTTQLF 1 DRB1* Env ELYKYKVVKIEPLGVA 1 DRB1* Env LWNWFDITNWLWYIKIFI 1 DRB1* Env IFIMIVGGLVGLRIVFAV 1 DRB1* Env RLVDGFLALIWVDLRSL 1 DRB1* Vif HIPLGDARLVITTYWGL 1 DRB1* 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.

8 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.

9 HLA-DRB1 Allele Expressed (n=1085) % Allele Frequency (n=1085) DRB1*01: DRB1*01: DRB1*01: DRB1*03: DRB1*03: DRB1*04: DRB1*04: DRB1*04: DRB1*04: DRB1*07: DRB1*08: DRB1*08: DRB1*08: DRB1*09: DRB1*10: DRB1*11: DRB1*11: DRB1*11: DRB1*12: DRB1*13: DRB1*13: DRB1*13: DRB1*13: DRB1*14: DRB1*15: DRB1*15: DRB1*15: DRB1*16: DRB1*16: Supplementary Table 4: HLA-DRB1 allele expression frequencies in our MGH & SCOPE cohort of HIVinfected treatment naive individuals (n=1085).

10 HLA-DRB1 P-value OR 95% CI 15: : : : : : : 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= Fishers exact test, Fig. 3b P= Mann-Whitney Test).

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