EXPERIMENTAL MODEL AND SUBJECT DETAILS Human Subjects and Study Design Detailed protocols of the clinical study are reported in Ott et al., 20207. Patients who were 18 years of age or older with histologically or cytologically conﬁrmed unresectable or metastatic melanoma were eligible for enrollment in this multicenter phase I clinical trial (NCT02897765). The gender and age of all patients evaluated in this manuscript can be found in Table S1 . No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocationduring experiments and outcome assessment. This study was conducted in accordance with the Declaration of Helsinki and with approval by Institutional Review Board at each participating site and written informed consent was obtained for all patients. Key eligibility criteria included an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 (on a 5-point scale, withhigher scores indicating greater disability) and life expectancy of > 6 months. Excluded from the study were patients with untreated central nervous system (CNS) metastases, active or history of autoimmune disease, and who had received previous therapy with PD-1, or PD-L1 inhibitors other than CTLA-4 (cytotoxic T lymphocyte-associated antigen 4). NEO-PV-01 is a personalized vaccinecomposed of up to 20 synthesized peptides that are approximately 14 to 35 amino acids in length. The peptides are divided into up tofour individual pools, each pool consisting of 1 to 5 peptides. On the day of vaccination, each pool of NEO-PV-01 was mixed with polyinosinic polycytidylic acid polylysine carboxymethylcellulose (poly ICLC; Hiltonol /C226) for a concentration of 300 mg/mL of each peptide. Each patient was scheduled to receive 5 doses of vaccination during the priming phase, on days 1 and 4 of week 12 andthen weekly for 3 weeks (weeks 13, 14, and 15) followed by 2 booster vaccinations on weeks 19 and 23. Each pool was administered by subcutaneous injection to one of the four limbs or left or right midriff as an alternative anatomical location. The anatomical site of injection for each pool was constant through the vaccination regimen. All patients, regardless of disease status, were allowed to startvaccination with NEO-PV-01 + adjuvant poly-ICLC. Nivolumab was administered on study day 1 at dose 240 mg by IV over 30 min every 2 weeks. All healthy donor (HD) material used for this study was obtained with written informed consent through external vendors identiﬁed in the Key Resources Table . Clinical assessments Adverse events, laboratory values, ECG, and vital signs were assessed regularly and graded according to the National CancerInstitute Common Terminology Criteria for Adverse Events, version 4.0. Response was assessed at weeks 8, 12, 24, 36, 52, 64, 76, 88, and 100 according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, by the investigator. Peripheral Samples Leukaphereses were obtained from patients at pre-treatment, pre-vaccine (week 10-12) and post-vaccine (week 20). Healthy donor leukaphereses were obtained from various external vendors identiﬁed in the Key Resources Table . PBMCs were isolated within 4h after collection using Ficoll-paque (GE Healthcare) density-gradient centrifugation and cryopreserved in Recovery Cell Culture Freezing Medium (Invitrogen) or cell freezing media (90% FBS and 10% DMSO). PBMCs were stored in vapor-phase liquid nitrogen until time of analysis. METHOD DETAILS T Cell and RNA Isolation for TCR Sequencing PBMCs were thawed and subjected to negative selection using the Pan T Cell Isolation Kit (Miltenyi) according to manufacturer’s protocol. T cells were counted, centrifuged for 15 minutes at 300xg then ﬂash frozen and stored as dry pellets at /C080 /C14C. RNA isolation was performed using the RNeasy Plus Micro Kit (QIAGEN) on the QIAcube (QIAGEN; Protocol: Puriﬁcation of total RNA using gDNA Eliminator and RNeasy MinElute spin columns) according to manufacturer’s protocol. RNA concentration was measured using the QubitTMRNA HS Assay Kit (ThermoFischer) according to manufacturer’s protocol. Eluted RNA was stored at /C080/C14C. TCR Library Preparation and Sequencing TCRaand TCR blibraries were prepared from isolated RNA using the Long Read iR-Proﬁle Reagent System (iRepertoire) either in-house or at the iRepertoire headquarters according to manufacturer’s protocol. For in-house sequencing, libraries were pooled,and concentrations were calculated using the KAPA Library Quantiﬁcation Kit (Roche) on the Lightcycler /C226480 II (Roche). Pooled libraries were sequenced using the MiSeq Reagent Kit v2 500-cycles (Illumina), either in-house or at the iRepertoire headquarters according to manufacturer’s protocol. In-house sequencing runs included a 10% Phi-X spike-in. Throughout the study, the numberof samples per pool was designed to maintain equal sequencing depths across samples. e3Cell Reports Medicine 1, 100141, November 17, 2020Reportll OPEN ACCESS Phenotypic analysis of peripheral samples Patient and healthy donor PBMCs were thawed into FBS, followed by a wash with X-Vivo media (Lonza). Cells were then treated with benzonase (Millipore) for 30 minutes at a 1:1000 dilution in media at 37/C14C. Cells were washed with media and counted using the Guava easyCyte Flow cytometer. A total of 2 x10 ^6 cells per sample were plated for antibody staining and washed once with FACS buffer (PBS + 0.5% BSA). Cells were then incubated with cell surface antibody for 30 minutes on ice, followed by a wash with FACS buffer. Cells were ﬁxed and permeabilized for intracellular staining using either the BD cytoﬁx/cytoperm kit (BD Biosci- ences; used for B cell panel) or Foxp3/Transcription Factor Staining Buffer Set (eBioscience; used for T cell panel) for 20 minuteson ice according the manufacturer’s instructions. Cells were washed with the corresponding permeabilization wash buffer accordingto the manufacturer’s instructions. Cells were then incubated with intracellular antibodies in the corresponding permeabilization wash buffer for 30 minutes on ice, washed with the appropriate permeabilization wash buffer, followed by a ﬁnal wash with FACS buffer. Cells were stored in FACS buffer at 4 /C14C until acquisition on a BD LSR Fortessa instrument. TCR Repertoire Generation TCR repertoires were generated by running a licensed copy of MiXCR34on the paired-end raw sequencing fastq ﬁles. The param- eters included the species speciﬁcations (Human, hsa), starting material (RNA), 50and 30primers (v and c primers, respectively) with no adapters, and searching for either TCR a(tra) or TCR bchains (trb). TCRaor TCR bCDR3 clonotypes were ﬁltered by removal of non-functional sequences (out-of-frame sequences or those contain- ing stop codons). Clonal frequency was calculated based on the count for each clone out of the total count. TCR Repertoire Diversity/Clonality Analysis Clone size category ( Figures 1 B and 1C; Figure S1 ) was based on clonal frequency, Fi as follows: rare (Fi < 1e-6), small (1e-6 %Fi < 1e-5), medium (1e-5 %Fi < 1e-4), large (1e-4 %Fi < 1e-3), and hyperexpanded (1e-3 %Fi). The unique number of nucleotide /amino acid TCR aor TCR bCDR3s was calculated per sample. Global diversity/clonality coef- ﬁcients have been calculated as follows: DE50 – AA CDR3 clonotypes were sorted based on their frequency in descending order. The cumulative frequencies of this sorted frequency vector were calculated. The rank of the ﬁrst value that was equal or larger than 0.50 was divided by the total number of unique aa CDR3 clonotypes to obtain the DE50 value. For example, if the 40 most frequent clones (but not 39) of a repertoire covers 50% of the total counts of the clones in that repertoire, consisting of 1000 clones, the DE50 value would be 0.04.Gini Coefﬁcient – ranges between 0 (all clones are equally frequent – repertoire diversity) and 1 (frequency dominated by one clone, repertoire clonality). Calculated using the ‘‘Gini’’ function from the ‘‘DescTools’’ R package 35. Normalized Shannon’s Entropy – higher values represent higher inequality of the frequencies25,26. Lorentz curves – similarly to the DE50 estimate, but a continuous curve between DE0 and DE100. Calculated using the ‘‘Lc’’ func- tion from the ‘‘DescTools’’ R package35. Sum of squares – the sum of squares measurement is calculated as the sum of the frequencies of the aa CDR3 clonotypes, eachsquared. TCR Repertoire Stability Assessment of the 2-way repertoire turnover was performed using the Jensen-Shannon Divergence 28,29. This measurement ranges between 0 (completely overlapping repertoires – no turn-over) and 1 (completely disjoint repertoires – high turn-over). This calculation was performed using the ‘‘JSD’’ function from the R package ‘‘philentropy’’36, either using the pre-treatment time-point as baseline, and comparing pre-vaccine and post-vaccine samples to that baseline ( Figure 2 A), or by comparing consecutive time-points to each other ( Figure S2 A). Calculation was based on the most frequent aa CDR3 clonotypes covering 20% of the total counts in each repertoire. This analysis was performed for varying levels of repertoire coverage, from 20% to 80% ( Figure S2 B). Signiﬁcance was calculated from a repeated-measures analysis for modeling, with AR(1) covariance structure. Assessment of the 3-way repertoire stability was performed using a Venn diagram approach ( Figure 2 B). The aa CDR3 sequences from the 3 time-points in each patient were intersected using the ‘‘venn’’ function from the R package ‘‘gplots’’37; the size of each of the 7 segments (A-G) was deﬁned as the sum of the frequencies of the clones in that segment. For segments shared by 2 (D-F) or 3 (G)segments, each clone has 2 or 3 frequency values, corresponding to each time-point it was detected in. Therefore, segments D-G have 2 or 3 size values. In addition, we also calculated the intersection of the unique aa CDR3 sequences across the 3 time-points, regardless of their frequencies ( Figure 2 D). Principal Component Analysis Select measurements taken at baseline from either the TCR repertoire analysis or the immunophenotyping of the PBMCs wasaggregated in one matrix. The matrix was centered and scaled, and PCA was calculated using the R function ‘‘prcomp’’ from the ‘‘stats’’ R package 38. The loadings, or contributions of the different measurements to PC1, were retrieved from the rotation matrix (Figure 4 B). Cell Reports Medicine 1, 100141, November 17, 2020 e4Reportll OPEN ACCESS Kaplan-Meier analysis was performed based on categorizing patients as belonging to PC1 < 0 or PC1 > 0. Calculation was per- formed using the ‘‘survﬁt’’ function from the ‘‘survival’’ R package39and plotted using the ‘‘ggsurvplot’’ function from the ‘‘survminer’’ R package40. P value was calculated using the log-ratio test and hazard-ration calculated using a univariate Cox proportional hazards regression model. Public TCR Sequence Analysis The 11 HDs were used to represent healthy population peripheral TCR repertoires. For each patient, each of the top 1000 mostfrequent TCR baa CDR3 sequence found in the pre-treatment time-point was scored by the number of HD repertoires in which it was detected (ranging from 0- no one, to 11 - all). For each score, the fraction of clones with that score in each patient was calculated (Figure S5 B). scTCRseq of neoantigen-speciﬁc TCRs Single-cell TCR sequencing (scTCRseq) was performed as previously described 7. Brieﬂy, patient PBMCs were sorted into 96-well plates either based on tetramer staining or staining with T cell activation markers (i.e., CD69, CD107a, CD25, PD-1) either immediatelyafter overnight rest ( ex vivo ) or after neoantigen peptide exposure for 5-6 days and recall for 6-24h (stim). Cells were stained with T cell activation markers as described in the peripheral immunophenotyping section, with anti-CD107a antibody added 6h prior to the end of co-culture during peptide recall. Final sort gates for neoantigen-reactive subpopulations were determined based on controltetramer staining or control co-cultures with DMSO. Plates were submitted to iRepertoire for scTCRseq (iPair-TCR). Pairedsequencing information was used to determine clonal composition of sorted cell populations and candidate alpha/beta chain pairs were tested for neoantigen-reactivity after lentivirus-based T cell transductions 41. For the transductions, second generation lentivirus vectors were generated on 293FT producer cell lines using shuttle plasmids (GenScript) containing TCR beta and alpha chains under the control of a SFFV promoter and separated by a furin cleavage site and a P2A ribosomal skip sequence. Human TCR variable regions were fused to cysteine-modiﬁed mouse constant regions for both chains resulting in recombinant mTCR42,43. Neoantigen-reactivity of mTCR Jurkat cells was determined by IL-2 secretion measured by electrochemiluminescence (MSD), according to the manufacturer’s instructions, in 24h co-culture assays of mTCR transduced Jurkat cells with neoantigen peptide and allele matched antigen presenting cell lines or CD3+T cell depleted autologous patient PBMCs using CD3 microbeads (Miltenyi). IFN-gELISpot assay for CEF Reactivity IFN-gELISpot assays to assess reactivity to CMV, EBV, or inﬂuenza (CEF) were performed using 96-well MultiScreen Filter Plates (Millipore) and the Ready-Set-Go! Human IFN- gELISpot Kit (Invitrogen) according to manufacturer’s instructions and as previously described7. Plates were coated overnight at 4/C14C with anti-IFN gCapture Antibody diluted in 1X Coating Buffer, washed with 1X Coating Buffer and blocked with X-Vivo media (Lonza) containing 1% penicillin/streptomycin (GIBCO) for 1h. PBMCs plated in triplicate with 1 3106cells per well after they were rested overnight in X-Vivo media (Lonza) supplemented with 1% penicillin/ streptomycin (GIBCO) at 37/C14C. CEF viral peptide pools were added to ELISpot wells at a 5 mM ﬁnal concentration. Each plate included a healthy donor positive and negative control with the CEF viral peptide pool (JPT) and Cell Stimulation Cocktail (Life Technologies) to conﬁrm reagent performance. Plates were incubated overnight at 37/C14C. Plates were washed 3 times using PBS with 0.05% Tween-20 and detection antibody diluted in 1X ELISpot diluent was added to wells for 2h. After washing 3 times with PBS with 0.05% Tween-20, Avidin-HRP was diluted in 1X ELISpot diluent and added to wells for 45 min. Plates were washed with both PBS with Tween-20 and PBS 3 times. AEC substrate-chromogen (BD Biosciences) was then added for 20 min. Plateswere rinsed with deionized water 3 times and allowed to dry at room temperature overnight. Spots were imaged and enumeratedusing an Immunospot analyzer (Cellular Technology Limited). QUANTIFICATION AND STATISTICAL ANALYSISStatistical Analysis All analyses were performed in the R language and environment for statistical computing (version 3.5.1). Likewise, all ﬁgures werecreated using an R script relying on the ‘‘ggplot2’’ package 44. Unless otherwise speciﬁed, P values are derived from a two-tailed Stu- dent’s t test. ADDITIONAL RESOURCES All patient data presented in this manuscript is from the multicenter phase I clinical trial NCT02897765. https://clinicaltrials.gov/ e5Cell Reports Medicine 1, 100141, November 17, 2020Reportll OPEN ACCESS