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Chapter 4 Assessment of promoter methylation Genomic DNA was isolated from relapsed (TCR T cell treated) and progressed (Mock T cell treated) tumors as described in the Materials and Methods section and treated with sodium bisulphite (EZ DNA Methylation, Zymo research; Irvine, CA) according to the manufacturer’s protocol. Methylation-specific PCR was performed on treated DNA using primers specific for either unmethylated (U) or methylated (M) DNA sequences of the 5’ LTR promoter of the pLXSN provirus, upstream of the antigen gene. Primer sequences were: U forward primer, 5’-TGTGTTTTATTTGAATTAATTAATTAGTTT-3’; U reverse primer, 5’-CACAATCTATCAAAAAAC- TAACACC-3’; M forward primer, 5’- TTTTATTTGAATTAATTAATTAGTTCGT-3’; and M reverse primer, 5’- GCAATCTATCGAAAAACTAACGC-3’. PCR products analyzed by gel electrophoresis (see Supplementary Figure 5a). Antigen mRNA and genomic DNA Quantitative PCRs were performed to assess levels of antigen mRNA and genomic DNA. Levels were calculated using the formula 2-ΔCT , in which C is defined as the cycle number at T which the amplified fluorescence signal crosses a pre-set threshold and ΔC is defined as the T difference between the C value of the A2-YLEP gene and that of an endogenous reference T gene, namely TRP2. Sequences of primers and probes used to quantify levels of A2-YLEP and A2 DNAs are provided in Figure 5a. Sequences of primers and probe to quantify TRP2 DNA were: (forward primer) 5‘-TAATTGTGGAGGCTGCAAGTTC-3’; (reverse primer) 5’-AGGATGGCC- GGCTTCTTC-3’; and (probe) FAM-5’-CTGGACCGGCCCCGACTGTAATC-3’ (Applied Biosystems, Foster City, CA). Detection was monitored with an MX3000P real-time PCR System using MX Pro data analysis (Stratagene, La Jolla, CA). Ex vivo treatments of tumor cells to up-regulate antigen expression Relapsed tumors were isolated, single cell suspensions were prepared and cells were cultured ex vivo for 5-7 days (see Materials and Methods section). Tumor cells were subjected to 3 different treatments. First, Azacitidine (AZA) (Sigma-Aldrich) was added to the culture media at day 4 for 3 consecutive days (final concentration 2-10mM). Second, mouse IFNg (100 Units/ ml) (Sanquin) was added at day 5 and tumor cells were left untreated for 48. Third, tumor cells were cultured for extended periods of 14-21 days. Following treatment, tumor cells were analyzed for HLA-A2 expression by flow cytometry (see Supplementary Figure 5c). 130
A CD8 T cells (%) C B16 target cells 100 90 80 70 60 50 40 30 20 10 0 B16:A2-YLEP B16:A2/gp100AV B16:A2/gp100 B16WT none *** *** pB:TCRαβ pB:opt TCRαβ pMP71: opt β-T2A-α T cells with gp100/A2 TCR transgenes ** * * * TCR-Vβ27 gp100/A2 pMHC Mock T cells pB:TCR α+β pB:opt TCR α+β pMP71:opt TCR β-T2A-α Mock 0 5000 10000 15000 20000 25000 30000 IFNγ (pg/ml) TCR T cells do not induce loss of tumor immunogenicity figure S1. Validation of TCR and antigen gene constructs a. Murine splenocytes gene-engineered to express gp100/A2 TCRab. Murine splenocytes were transduced with the following gp100/A2-specific TCR constructs: pB:TCRa+b (two separate pB vectors with wild type sequences for TCRa and TCRb); pB:optTCRa+b (two separate pB vectors with codon optimized sequences for TCRa and TCRb); and pMP71:optTCR b-T2A-a (single pMP71 vector with codon optimized TCRab in a b-T2A-a cassette). See Materials and Methods section and Supplementary text for details on the construction of TCR constructs. At day 5 following T cell activation CD8 T cells were analyzed for TCR-Vb27 expression (white bars) and gp100/A2 pMHC binding (black bars) by flow cytometry. Data represent mean % ± SEM, n=5 separate transductions. B. B16 melanoma cells gene-engineered to express HLA-A2 and human gp100. B16 cells were transduced with the following gp100/A2 antigen constructs: pLXSN:A2+pLXSH:gp100 (B16:A2/gp100 cells, dashed histogram); pLXSN:A2+pLXSH:gp100AV (B16:A2/ gp100AV cells, black histogram) and pLXSN:A2-YLEP+pLXSH:gp100 (B16:A2-YLEP cells, bold black histogram). See Materials and Methods section and Supplementary text for details on the construction of antigen constructs. Tumor cells were stained with a FITC-labeled anti-HLA-A2 or anti-hgp100 mAb followed by a PE-labeled anti-mouse Ig and analyzed by flow cytometry. Non-transduced B16WT cells were used as a negative control (grey filled histogram). C. IFNg production by TCR-engineered T cells in response to gp100/A2-positive B16 cells in vitro. TCR-transduced splenocytes (from S1a) were incubated with different populations of B16 target cells (from S1b) and supernatants were harvested after 20 hours and analyzed for murine IFNg. Data are presented as mean pg/ml IFNg + SEM, n=5. D. B16 cells that express the A2-YLEP antigen are not immunogenic in vivo. B16:A2-YLEP or B16WT cells (0.5x10 6 ) were injected s.c. into HLA-A2 transgenic mice. Tumor volumes were measured 3 times a week with a caliper and data are presented as mean mm 3 ± SEM, n=5. No significant differences were observed between the in vivo growth kinetics of B16 cell lines with or without A2-YLEP. Statistical differences were calculated with Student’s t tests: *p
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Towa r d s c l i n i c a l Tc r g e
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Studies described in this thesis we
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PROMOTIECOMMISSIE Promotor: Prof.dr
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6.2.3 Tumor relapse 168 6.2.3.1 Pot
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1.1 IMMunE ThERaPy fOR ThE TREaTMEn
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1. Isolation and activation of peri
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General Introduction synovial carci
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Table 3. Challenges of TCR gene the
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General Introduction Table 4. Stren
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1.3.2 Environmentally induced tumor
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General Introduction adoptive T cel
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General Introduction would require
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General Introduction different plat
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REfEREnCES General Introduction 1.
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General Introduction metastatic syn
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General Introduction 51. Schambach
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General Introduction 80. Leach DR,
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General Introduction 112. Umansky V
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General Introduction 142. Hanson HL
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General Introduction 172. Liu W, Ch
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Chapter 2 aBSTRaCT Importance of th
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Chapter 2 receptors, CARs) to treat
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Chapter 2 Table 2. advantages of Ma
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Chapter 2 within a vector is of imp
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Chapter 2 expression and pMHC multi
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Chapter 2 potential in eradicating
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Chapter 2 differentiation state and
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Chapter 2 recognize such a self-ant
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Chapter 2 Table 3. Strategies to im
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Chapter 2 60 3. Lymphodepletion Iso
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Chapter 2 CC, Rudy SF, VanWaes C, D
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Chapter 2 42. Schultz ES, Schuler-T
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Chapter 2 73. Sadelain M, Brentjens
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Chapter 2 105. Moroz A, Eppolito C,
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Chapter 2 134. Willemsen RA, Sebest
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Chapter 3 T cell receptor gene ther
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InTRODuCTIOn TCR-engineered T cells
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OV-CAR3 (MUC-CD) cells OC SCID Beig
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Page 112 and 113: Chapter 4 RESulTS TCR T cells preve
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Page 118 and 119: Chapter 4 116 B genomic DNA (arbitr
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Page 130 and 131: Chapter 4 42. Zou W (2005) Immunosu
Page 134 and 135: Chapter 4 132 lymphocytes (number /
Page 136 and 137: Chapter 4 HLA-A2 transgenic mice be
Page 138 and 139: Chapter 5 aBSTRaCT Adoptive therapy
Page 140 and 141: Chapter 5 in up to 50% of non-small
Page 142 and 143: Chapter 5 140 MC2/A2 peptide MHC (p
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Page 150 and 151: Chapter 5 figure 7. Ma3/DP4 TCR CD4
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Page 156 and 157: Chapter 5 12. Parkhurst MR, Yang JC
Page 158 and 159: Chapter 5 40. Carrasco J, Van Pel A
Page 160 and 161: Chapter 5 68. Kuball J, Schmitz FW,
Page 162 and 163: Chapter 5 melanoma cells; MC2 pos /
Page 164 and 165: Chapter 6 Currently, TCR gene thera
Page 166 and 167: Chapter 6 murine T cells with solub
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Chapter 6 F, Bordignon C & Bonini C
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Chapter 6 58. Spiotto MT, Rowley DA
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Chapter 6 CD8+ T cells expressing i
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Chapter 6 118. Kantarjian HM, O’B
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SuMMaRy Summary To date, adoptive T
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Summary 20% of mice durable remissi
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SaMEnVaTTIng Samenvatting Momenteel
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Samenvatting experimenten laten wij
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Curriculum Vitae List of publicatio
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lIST Of PuBlICaTIOnS List of public
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PhD portfolio 1.4b Poster presentat
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Samenvatting In het bijzonder wil i
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