Supplementary Information

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1 Supplementary Information High throughput epitope discovery reveals frequent recognition of neo-antigens by CD4 + T-cells in human melanoma Carsten Linnemann, Marit M. van Buuren, Laura Bies, Els M.E.Verdegaal, Remko Schotte, Jorg J.A. Calis, Sam Behjati, Arno Velds, Henk Hilkmann, Dris el Atmioui, Marten Visser, Michael R. Stratton, John B.A.G. Haanen, Hergen Spits, Sjoerd H. van der Burg and Ton N.M. Schumacher Supplementary Item Title Supplementary Figure 1 Experimental setup for the identification of neo-antigen reactive CD4 + T-cells in tumor lesions. Supplementary Figure 2 Detection of neo-antigen reactive CD4 + T- cells in a melanoma lesion using BCL- 6/BCL-XL immortalized but not Epstein- Barr Virus (EBV) immortalized autologous B-cells. Supplementary Figure 3 Cytokine production by intratumoral CD4 + T-cells in response to melanoma mutanomes. Supplementary Figure 4 Intratumoral CD4 + T-cells only react against peptides within the autologous mutatome. Supplementary Figure 5 Isolation of neo-antigen reactive CD4 + T- cells from human melanoma lesions. Supplementary Figure 6 Recognition of mutant and wildtype peptide by neo-antigen reactive T-cell clones. Supplementary Figure 7 Idnetification of neo-antigen reactive CD4 + T-cells in the melanoma lesion of NKIRTIL27. Supplementary Table 1 Characteristics of the mutational landscape in the analyzed melanoma lesions. Nature Medicine: doi:1.138/nm.3773

2 Supplementary Figure 1 1b: Tumor excision 1a: Isolation & immortalization of B cells 2b: Isolation of TIL 2a: Identification of tumor-specific mutations 3: Sorting & expansion of CD4 + T cells 4: Synthesis of mutated long peptides IRRSSTSGDTEEEEEVVPFFSSDEQKRRSEA 5: Loading of autologous B cells with long peptides 6: Culture with autologous CD4 + T cells 7: Measurement of cytokine levels in culture supernatant 8: Confirmation of putative hit-peptides by intracellular cytokine staining Supplementary Figure 1. Experimental setup for the identification of neo-antigen reactive CD4 + T- cells in tumor lesions. Whole exome-sequencing of tumor and healthy material in combination with RNAsequencing identifies tumor-specific, non-synonymous mutations within genes with confirmed RNAexpression. This information is used to create a library of putative neo-epitopes comprised of 31 aminoacid long peptides, extending each identified mutation by 15 amino acids on either side. Autologous B- cells immortalized by retroviral gene transfer of BCL-6 and BCL-XL provide an infinite source of autologous antigen-presenting cells, allowing the profiling of CD4 + T-cell reactivity against all MHC-class II haplotypes of the subject. Stimulation of CD4 + T-cells obtained from a tumor lesion or peripheral blood of the same subject by neo-antigen peptide-loaded B-cells enables the detection of neo-antigen specific CD4 + T-cell reactivity through the analysis of cytokine levels within culture supernatant. After initial screens against complete melanoma mutanomes, the small set of putative neo-epitopes (peptides inducing substantial cytokine production over background, generally comprising some.5% of the total mutanome) is subsequently used for independent confirmation by intracellular cytokine staining. Nature Medicine: doi:1.138/nm.3773

3 Supplementary Figure 2 1, IFN- 5 CIRH1A P>L GART V>A ASAP1 P>L Supplementary Figure 2. Detection of neo-antigen reactive CD4 + T-cells in a melanoma lesion using BCL-6/BCL-XL immortalized but not Epstein-Barr Virus (EBV) immortalized autologous B- cells. IFN-γ in culture supernatant after a 48 h co-culture of in vitro expanded intratumoral CD4 + T-cells of patient NKIRTIL18 with peptide loaded autologous B-cells immortalized by stable transduction with BCL-6/BCL-XL (red) or by EBV infection (black). Red and black dotted lines indicate cytokine s after co-culture of CD4 + T-cells with unloaded BCL-6/BCL-XL and EBV immortalized B-cells, respectively. Nature Medicine: doi:1.138/nm.3773

4 Supplementary Figure 3 a NKIRTIL18 15 IFN- 2 IL-4 IL-6 TNF IL-1 IL-17a IL-2 b NKIRTIL34 75 IFN- IL-4 IL-6 TNF IL-1 IL-17a IL-2 # Supplementary Figure 3. Cytokine production by intratumoral CD4 + T-cells in response to melanoma mutanomes. Cytokine in culture supernatant after a 48 h co-culture of peptide loaded, autologous B-cells with in vitro expanded intratumoral CD4 + T-cells from (a) NKIRTIL18 and (b) NKIRTIL34. Red dotted line indicates cytokine s after co-culture of CD4 + T-cells with unloaded B-cells. IL-2 production upon incubation with peptides marked # was above background in this initial screen but could not be confirmed in an independent experiment. Nature Medicine: doi:1.138/nm.3773

5 Supplementary Figure 4 a 8 A P>L H1 CIR NKIRTIL18 IFN- (pg ml 1) >A RT V GA P1 P>L A AS 1 b NKIRTIL34 8 D3 P RN >S IFN- (pg ml 1) Supplementary Figure 4. Intratumoral CD4 + T-cells only react against peptides within the autologous mutanome. IFN-γ in culture supernatant after a 48 h co-culture of in vitro + expanded intratumoral CD4 T-cells obtained from (a) NKIRTIL18 and (b) NKIRTIL34 with autologous B-cells loaded with peptide libraries of the respective other patient. Red dotted line indicates IFN-γ + production of CD4 T-cells after co-culture with unloaded B-cells. Identified neo-epitopes of NKIRTIL18 and NKIRTIL34 were used as positive control samples (light grey). Black bars indicate analyses of reactivity towards individual non-autologous mutanome peptides. Nature Medicine: doi:1.138/nm.3773

6 Supplementary Figure 5 6, CIRH1A P>L IFN- 4, 2, , GART V>A IFN- 4, 2, , ASAP1 P>L IFN- 4, 2, Supplementary Figure 5. Isolation of neo-antigen reactive CD4 + T-cells from human melanoma lesions. IFN-γ in culture supernatant after a 48h co-culture of GART V>A, CIRH1A P>L and ASAP1 P>L specific CD4 + T-cell clones derived from NKIRTIL18 with unloaded (white bars) or peptide loaded (black bars) autologous B-cells. Nature Medicine: doi:1.138/nm.3773

7 Supplementary Figure 6 GART V>A TCR I GART V>A TCR II GART V>A TCR III GART V>A TCR IV 1 4 Clone 5 Clone 21 Clone 1 Clone 6 Clone 12 IFN Peptide ( g ml 1 ) Supplementary Figure 6. Recognition of mutant and wildtype peptide by neo-antigen reactive T-cell clones. IFN-γ in culture supernatant after a 48 h co-culture of GART V>A reactive CD4 + T-cell clones derived from NKIRTIL18 with autologous B-cells loaded with the indicated s of mutant (open symbols) or wild-type (black symbols) peptide. TCR clonotypes are indicated as determined in (Fig. 2b). Nature Medicine: doi:1.138/nm.3773

8 Supplementary Figure 7 IFN- 1, LEMD2 P>L Supplementary Figure 7. Identification of neo-antigen reactive CD4 + T-cells in the melanoma lesion of NKIRTIL27. IFN-γ in culture supernatant after a 48 h co-culture of peptide loaded, autologous B-cells with in vitro expanded intratumoral CD4 + T-cells. Red dotted line indicates IFN-γ production of CD4 + T-cells after co-culture with unloaded B-cells. Nature Medicine: doi:1.138/nm.3773

9 Supplementary Table 1 Sex Mutational load # somatic coding mutations UV associated mutations % of somatic mutations Synonymous mutations Non-synonymous mutations RNA expressed, non-synonymous NKIRTIL18 NKIRTIL45 NKIRTIL34 NKIRTIL27 BO F M M F M Supplementary Table 1. Characteristics of the mutational landscape in the analyzed melanoma lesions. Nature Medicine: doi:1.138/nm.3773