Supporting Information

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1 Supporting Information Chapuis et al /pnas SI Methods Selection of Patients, Targets, Isolation, and Expansion of Melanoma- Specific CTL Clones. Patients were HLA-typed, and their tumors were tested for the expression of melanoma-associated antigens (MART-1, tyrosinase, and gp100). Only patients with previously identified HLA-restricted, melanoma-specific peptides were selected for enrollment in the study (1 3). Between March 2007 and December 2010, 22 patients were enrolled on protocol Fred Hutchinson Cancer Research Center (FHCRC) 2140 and their PBMCs were collected by way of leukapheresis. Because a period of 3 mo was required for the generation of antigen-specific T-cell clones, patients were encouraged to receive alternate treatments in the interval. One patient received melanomaspecific T cells on a different clinical protocol, 8 patients were ineligible to receive T cells (death or development of brain metastasis), 2 patients responded to the alternate treatment deferring T-cell infusion, and 11 patients received melanomaspecific T-cell clones. Antigen-specific T-cell clones were obtained for all 18 enrolled patients for whom manufacturing was initiated. All ex vivo manipulations involving processing of products destined for reinfusion were performed as previously described (4, 5). Before infusions, CTL clones were tested for surface expression of CD3, CD8, CD4, CD45RO, CD28, CD62L, binding to the corresponding MHC-peptide specific multimer, lysis of HLA-matched B-lymphoblastoid cell lines (B-LCL) pulsed with titrating amounts of the relevant peptide, and monoclonality by analysis of TCR Vβ use (6) (Table S1). Treatment Plan. Patients were scheduled to receive 4,000 mg/m 2 of CY administered over 2 d, 72 h, and 48 h before the infusion of melanoma-specific CTL clones/m 2 [determined safe from previous studies (4)]. Eight patients (cohort 1) received LD s.c. IL-2 (500,000 U/m 2 twice daily) within 6 h of the T-cell infusion for 14 d, and three patients (cohort 2) received HD IL-2 i.v. (600,000 IU/kg) every 8 h for a target of 14 total doses. Patients were monitored for toxicities based on Common Toxicity Criteria v4.0 (7), as well as for persistence and function of transferred cells for at least 8 wk after infusion. Staging studies were obtained at 4 and 8 wk as well as at later time points 12 and/or 16 wk after the T-cell infusion. Radiological responses were evaluated after infusion according to RECIST 1.1 criteria (8, 9) (Fig. S2). Cytotoxicity Assays. Cytotoxic responses of HIV antigen-specifict cells were examined as previously described (10). Telomere Lengths. Telomere lengths were measured as previously described (11). T-Cell Tracking in Paraffin-Embedded Blocks. For patient 1, genomic DNA was purified using PureGene or a QIAxtractor System (Qiagen) on both the skin biopsy paraffin-embedded block and infused clone. TCR-γ rearrangements were amplified by multiplex PCR and then size-fractionated on an Applied Biosystems 3130 Sequence Analyzer as described (12). T-Cell Tracking by MHC-Peptide Multimers. Where possible, melanoma-specific MHC-peptide multimers [FHCRC in-house production (13)] were preferentially used to detect transferred CTL clones in PBMCs collected after infusions (Table S1). The sensitivity of multimer staining was fixed at 0.1% of total CD8 + T cells, below which the capacity to distinguish between transferred cells and background was greatly diminished. Persistence was calculated as the last time point at which multimer-positive T cells were twofold background levels or >0.1%. T-Cell Tracking by Quantitative PCR. Primers flanking the CDR3 region of infused melanoma-specific CTL clones were designed as described (14), and genomic DNA isolated from PBMCs was used as a template for quantitative real-time PCR. Clone-specific TCR copies were normalized to β-actin copies assuming each live cell in each sample analyzed contained two copies of β-actin. Total CD8 + T cells were determined by flow cytometry for each sample, and an equation (100/[% total CD8 + T cells in each sample] [TCR copies/{β-actin copies/2} 100]) was used to determine the number of TCR copies per 100 CD8 + T cells. This method can detect as low as 1 TCR copy per 100,000 DNA cell equivalents (0.001%) as determined by serial dilutions of clonal cells in autologous preinfusion PBMCs. Flow Cytometry. Infused autologous CTL clones in PBMCs obtained after transfer were identified by binding to specific multimer constructs (Table S1) and analyzed by flow cytometry after staining with fluorochrome-conjugated mabs to CD3, CD4, CD16, CD19, CD8, CD28, CD27, CD62L, CCR7, CD45RA, CD45RO, CD137 (4-1BB), CD132 [IL-2 receptor (R) γ], CD127 (IL-7Rα), CD57, and PD-1 (BD Pharmingen). Intracellular cytokine expression of IFN-γ, TNF-α, and IL-2 by responding CTLs pulsed for 4 5 h with relevant peptide was assessed as described (15). Cells were analyzed on an LSRII (Becton Dickinson) using FACS-Diva software. ELISpots. Human IFN-γ ELISpot assays were performed as previously described (16) using capture and detection antibodies D1K and 7-B6-1 (10 μg/ml; Mabtech and Nacka), respectively. Results are presented as the mean number of spot-forming cells per 10 5 PBMCs. Plasma Cytokine Levels. Plasma samples were drawn at a preinfusion time point and at days 0, 7, 14, and 21 of the second T-cell infusion. Plasma samples were frozen, thawed, and analyzed simultaneously by the Cytokine Analysis Facility at the FHCRC for IL-7, IL-15, and, in selected cases, IL-2 by sandwich ELISA. Statistical Analysis. Statistical tests were performed with GraphPad Prism software version 3.0 or with the R-package for statistical analysis. Two-tailed signed-rank tests were used to determine P values. 1. Brichard V, et al. (1993) The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 178: Brichard VG, et al. (1996) A tyrosinase nonapeptide presented by HLA-B44 is recognized on a human melanoma by autologous cytolytic T lymphocytes. Eur J Immunol 26: Kawakami Y, et al. (1994) Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes. J Exp Med 180: Wallen H, et al. (2009) Fludarabine modulates immune response and extends in vivo survival of adoptively transferred CD8 T cells in patients with metastatic melanoma. PLoS ONE 4:e Yee C, et al. (2002) Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: In vivo persistence, migration, and antitumor effect of transferred T cells. Proc Natl Acad Sci USA 99: Monteiro J, et al. (1995) Oligoclonality in the human CD8+ T cell repertoire in normal subjects and monozygotic twins: Implications for studies of infectious and autoimmune diseases. Mol Med 1: of5

2 7. Trotti A, et al. (2003) CTCAE v3.0: Development of a comprehensive grading system for the adverse effects of cancer treatment. Semin Radiat Oncol 13(3): Eisenhauer EA, et al. (2009) New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur J Cancer 45: Schwartz LH, et al. (2009) Evaluation of lymph nodes with RECIST 1.1. Eur J Cancer 45: Riddell SR, et al. (1992) Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 257: Lansdorp PM (2006) Stress, social rank and leukocyte telomere length. Aging Cell 5: Juarez T, et al. (2005) Analysis of T-cell receptor gene rearrangement for predicting clinical outcome in patients with cutaneous T-cell lymphoma: A comparison of Southern blot and polymerase chain reaction methods. Arch Dermatol 141: Altman JD, et al. (1996) Phenotypic analysis of antigen-specific T lymphocytes. Science 274: Hunder NN, et al. (2008) Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N Engl J Med 358: Papagno L, Almeida JR, Nemes E, Autran B, Appay V (2007) Cell permeabilization for the assessment of T lymphocyte polyfunctional capacity. J Immunol Methods 328(1-2): Scheibenbogen C, et al. (2000) Quantitation of antigen-reactive T cells in peripheral blood by IFNgamma-ELISPOT assay and chromium-release assay: A four-centre comparative trial. J Immunol Methods 244(1-2): of5

3 Fig. S1. Phenotypic and functional characteristics of melanoma-specific CD8 + T-cell clones isolated and expanded for infusion. (A) Lytic activity of each of the melanoma-specific CD8 + T-cell clones to established melanoma cell lines (A375 [HLA-B* ] and/or Mel-526 [HLA-A* ], effector/target ratio = 10:1) and HLA-matched B-lymphoblastoid cell lines (B-LCL) pulsed with decreasing concentrations of specific peptide in a 51 Cr-release assay. No HLA A* melanoma cell line was available for patient (Pt) 4. (B) Expression of CD45RO (Left), CD27 (Center), and CD28 (Right) on melanoma-specific CD8 + T-cell clones (bold line) compared with isotype control (gray area). Inset values represent percentages of CD45RO +, CD27 +, and CD28 + CD8 + T cells, respectively. (C) Expression of CD127, CD62L, and CCR7 (bold line) on a representative CTL clone used for infusion (patient 7, HLA B*4402/Tyr). All clones were otherwise negative for CD45RA, CD62L, CCR7, CD127, CD137, CD132 (IL-2Rγ), CD57, and PD-1. 3of5

4 Fig. S2. Treatment plan. All patients received MART-1 or tyrosinase-specific CD8 + T-cell clones on day 0 ( cells), preceded by 2 g/m 2 of CY on days 3 and 2. Infusions for patients 1 8 were followed by LD IL-2 for 14 d ( IU twice daily). Infusions for patients 9 11 were followed by HD IL-2 (600,000 IU/ kg i.v.) administered every 8 h for a target total of 14 doses. Patients were evaluated for responses 4, 8, and 12/16 wk after infusions. Fig. S3. Increased plasma levels of IL-2, IFN-γ, IL-18, and MCP-1 evaluated after infusions. Plasma samples were obtained before CY, immediately before T-cell infusions, and up to 56 d after T-cell infusions. (A) Plasma levels of IL-2 in patients who received LD IL-2 (Left) and HD IL-2 (Right) increased in a dose-dependent manner. Plasma levels of proinflammatory cytokines IFN-γ (B) and IL-18 (C) measured after CTL infusions. (D) Plasma levels of the proinflammatory cytokine MCP-1 in patients who received LD IL-2 (Left) and HD IL-2 (Right). Note the difference in the y-axis scale: maximum of 2,000 pg/ml (Left) and maximum of 20,000 pg/ml (Right). The lower limit of detection was 3.6 pg/ml for plasma levels of IL-2, IL-18, and Monocyte chemotactic protein-1 (MCP-1), and it was 0.3 pg/ml for IFN-γ. Plasma levels of IFN-α, IL-1α, IL-1β, IL-10, IL-12p70, IL-17, IL-1Rα, IL-21, IL-2Rα, IL-4, IL-5, IL-6, IL-7, IL-8, macrophage inflammatory protein-1α (MIP-1α), TNF-α, and VEGF were evaluated after infusions, and no changes were detected throughout the evaluation period. 4of5

5 Fig. S4. Dynamics of CD4 + T-regs after CY lymphodepletion followed by exogenous IL-2. (A) Percentages of CD4 +, CD127 lo, CD25 hi, and FOXP3 + cells (T-regs) (y axis) plotted over time (x axis) for all patients. Gray lines show individual patients, and filled circles show mean and SD. (B) Absolute numbers of T-regs (y axis) plotted over time (x axis) for all patients. Gray lines show individual patients, and filled circles show mean and SD. (C) Box and whisker plots of percentages of K i -67 expressing cells expressed by (Left to Right) T-regs before treatment (CY and T cells), CD4 + T-effector cells (defined as CD4 + CD25 lo, CD127 hi, FOXP3 cells) before treatment, T-regs 14 d after T-cell infusion, and CD4 + T-effector cells 14 d after T-cell infusion. Bars represent medians. A two-tailed paired signed-rank test was used for statistical analysis. Table S1. Characteristics of infused melanoma-specific CD8 + T-cell clones Patient no. T-cell specificity Class I restriction Epitope Corresponding tetramer TCR Vβ use LD IL-2 1 MART1 HLA-A*0201 AAGIGILTV A*0201/AAGIGILTV-Mart1 Vβ3 2 Tyrosinase HLA-B*4402 SEIWRDIDF NA Vβ8 3 gp100 HLA-A*0201 KTWGQYWQV A*0201/KTWGQYWQV-gp100 Vβ17 4 gp100 HLA-A*0301 ALLAVGATK A*0301/ALLAVGATK-gp100 Vβ22 5 Tyrosinase HLA-A*0201 YMDGTMSQV A*0201/YMDGTMSQV-Tyr Vβ7 6 Tyrosinase HLA-A*0201 YMDGTMSQV *0201/YMDGTMSQV-Tyr Vβ14 7 Tyrosinase HLA-B*4402 SEIWRDIDF NA Vβ7 8 MART1 HLA-A*0201 AAGIGILTV A*0201/AAGIGILTV-Mart1 Vβ7 HD IL-2 9 MART1 HLA-A*0201 AAGIGILTV A*0201/AAGIGILTV-Mart1 Vβ2 10 MART1 HLA-A*0201 AAGIGILTV A*0201/AAGIGILTV-Mart1 Vβ14 11 MART1 HLA-A*0201 AAGIGILTV A*0201/AAGIGILTV-Mart1 Vβ3 NA, not available. Table S2. Telomere lengths of infused melanoma-specific CD8 + T-cell clones Patient no. Age at time of infusion, y Infused CD8 + T-cell clone MTL Estimated normal lymphocyte MTL at age * MTL, median telomere length. *Consistent with previous studies (1, 2), telomere lengths were shorter than in vivo age-matched lymphocyte subpopulations. 1. Shen X, et al. (2007) Persistence of tumor infiltrating lymphocytes in adoptive immunotherapy correlates with telomere length. J Immunother 30: Zhou J, et al. (2005) Telomere length of transferred lymphocytes correlates with in vivo persistence and tumor regression in melanoma patients receiving cell transfer therapy. J Immunol 175: of5