Volume 11, Issue 6, Pages 950-959 (June 2005) Induction of leukemia-specific CD8+ cytotoxic t cells with autologous myeloid leukemic cells maturated with a fiber-modified adenovirus encoding TNF-α  Aurore Saudemont, Selim Corm, Thomas Wickham, Dominique Hetuin, Bruno Quesnel  Molecular Therapy  Volume 11, Issue 6, Pages 950-959 (June 2005) DOI: 10.1016/j.ymthe.2004.12.016 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 1 TNF-α production by transduced leukemic cells. (A) Time-course analysis of TNF-α production by K562 leukemic cells transduced with the AdTNF.F(pK7) vector at various m.o.i. (B) TNF-α production by AML cells 72 h after transduction with AdZ.F(pK7), AdTNF, or AdTNF.F(pK7) vector. (C) Time-course analysis of TNF-α production (top) by 30 samples of AML cells transduced with the AdTNF.F(pK7) vector and viability of these cells measured by annexin V/PI (bottom). The horizontal mark indicates the median. (D) Detailed results of (C), top, for the seven AML samples that could be cultured for up to 3 weeks. Molecular Therapy 2005 11, 950-959DOI: (10.1016/j.ymthe.2004.12.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 2 Immunophenotypic analysis of maturated AML cells. (A) CD1a, (B) CD80, (C) CD86, (D) MHC I, and (E) MHC II expression in AML cells maturated with recombinant TNF-α, AdZ.F(pK7), or AdTNF.F(pK7). Data represent means and standard deviations of experiments performed in 20 AML samples, each one analyzed in triplicate. Molecular Therapy 2005 11, 950-959DOI: (10.1016/j.ymthe.2004.12.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 3 IL12 production of maturated AML cells and proportion of leukemic cells after maturation. (A) IL12 production by AML cells maturated with recombinant TNF-α, AdZ.F(pK7), or AdTNF.F(pK7). Data represent means and standard deviation of experiments performed in quadruplicate. (B) Ratio of AML1-ETO-positive leukemic cells in AML cells maturated with recombinant TNF-α, AdZ.F(pK7), or AdTNF.F(pK7) compared to AML cells cultured in control medium. Data represent means and standard deviation of experiments performed in triplicate with AML cells isolated from one patient with AML1-ETO-positive AML. Molecular Therapy 2005 11, 950-959DOI: (10.1016/j.ymthe.2004.12.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Blocking experiments with anti-TNF-α in maturated AML cells. Immunophenotypic analysis of one AML sample representative of three maturated with AdZ.F(pK7) or AdTNF.F(pK7) and incubated with anti-TNF-α blocking antibody. Data represent means and standard deviation of experiments performed in quadruplicate. Molecular Therapy 2005 11, 950-959DOI: (10.1016/j.ymthe.2004.12.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 5 Immunophenotypic modification induced by signal transduction inhibitors. Incubation of one AML sample representative of three maturated with AdTNF.F(pK7), with PD980509 and U0126 MAP/ERK inhibitor, SB203580 p38SAP kinase inhibitor, rapamycin, wortmannin, and LY294002. Data represent means and standard deviation of experiments performed in triplicate. Molecular Therapy 2005 11, 950-959DOI: (10.1016/j.ymthe.2004.12.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Cell expansion and activation of T cells cultured with maturated AML cells. (A) T cell expansion and (B) CD69 expression of T cells isolated from the peripheral blood of four patients with AML and cultured for 2 weeks with autologous AML cells maturated with recombinant TNF-α, AdZ.F(pK7), or AdTNF.F(pK7). Data represent means and standard deviation of experiments performed in triplicate. Molecular Therapy 2005 11, 950-959DOI: (10.1016/j.ymthe.2004.12.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Cytotoxicity experiments. (A and B) Results from patients 3 and 4, respectively. Cytotoxicity of CD8+ T cells, sorted from T cells expanded with autologous AML cells maturated by recombinant TNF-α, AdZ.F(pK7), or AdTNF.F(pK7), against autologous AML cells or K562 cells. CD8+ T cells from patient 3 were tested against AML cells from patient 4 and vice versa. Data represent means and standard deviation of experiments performed in quadruplicate. Molecular Therapy 2005 11, 950-959DOI: (10.1016/j.ymthe.2004.12.016) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions