1. Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells
by Susanne M. Schmidt, Kerstin Schag, Martin R. Müller, Markus M. Weck, Silke Appel, Lothar Kanz, Frank Grünebach, and Peter Brossart
Blood
Volume 102(2):
July 15, 2003
©2003 by American Society of Hematology

2. Survivin expression in human tumor cell lines.
Survivin expression in human tumor cell lines. (A) RT-PCR using survivin-specific primers was performed to analyze the RNA expression of survivin in human tumor cell lines. β2-Microglobulin–specific primers were used as controls. (B) Western blot analysis using a monoclonal antibody against survivin protein. Peripheral blood mononuclear cells (PBMNCs) and purified B and T lymphocytes were included in the analysis of protein expression as controls (C).
Susanne M. Schmidt et al. Blood 2003;102:
©2003 by American Society of Hematology

3. Induction of survivin-specific CTL responses in vitro using peptide-pulsed mature DCs as antigen-presenting cells.
Induction of survivin-specific CTL responses in vitro using peptide-pulsed mature DCs as antigen-presenting cells. DCs generated from adherent PBMNCs in the presence of GM-CSF, IL-4, and TNF-α were pulsed with the synthetic peptide derived from the survivin protein and used to induce a CTL response in vitro. Cytotoxic activity of induced CTLs was analyzed in a standard 51Cr-release assay using T2 cells pulsed with the cognate survivin peptide (T2 + Sv, ⋄) or an irrelevant (adipophilin) peptide (T2 + Ad, □) as targets. E:T indicates effector-to-target ratio.
Susanne M. Schmidt et al. Blood 2003;102:
©2003 by American Society of Hematology

4. Antigen-specific lysis of human tumor cell lines endogenously expressing survivin.
Antigen-specific lysis of human tumor cell lines endogenously expressing survivin. Human renal cell carcinoma (MZ 1774, MZ 1257, A 498), melanoma (Mel 1479), breast cancer (MCF-7), multiple myeloma (U 266), and the EBV-immortalized Croft cells expressing survivin and HLA-A2 as well as the ovarian cancer cell line SK-OV-3 (HLA-A2-/survivin+) were used as targets in a standard 51Cr-release assay. K 562 cells were included to determine the NK cell activity (A-C). The HLA-A2 restriction of the CTL lines was analyzed using a HLA-A2–specific monoclonal antibody (U anti–HLA-A2, 3C; A498 + anti–HLA-A2, 3D). An isotype antibody was used as control (control immunoglobulin). A cold target inhibition assay was performed to analyze the antigen specificity of the CTL lines (D). Unlabeled T2 cells pulsed with the cognate survivin peptide were used to block lysis of A 498 cells (A T2 + Sv). No inhibition of lysis was detected when using T2 cells pulsed with the irrelevant adipophilin peptide (A T2 + Ad). The ratio of inhibitor to target cells was 20:1.
Susanne M. Schmidt et al. Blood 2003;102:
©2003 by American Society of Hematology

5. Lysis of primary leukemia cells by survivin-specific CTLs
Lysis of primary leukemia cells by survivin-specific CTLs. Primary malignant cells from HLA-A2–positive (CLL-K, AML-Ah, AML-Bu, AML-Na, ALL-Sc) or HLA-A2–negative (CLL-F, AML-Bd, AML-Ra, AML-Fi) patients with acute myelogenous leukemia (AML), acute lymph...
Lysis of primary leukemia cells by survivin-specific CTLs. Primary malignant cells from HLA-A2–positive (CLL-K, AML-Ah, AML-Bu, AML-Na, ALL-Sc) or HLA-A2–negative (CLL-F, AML-Bd, AML-Ra, AML-Fi) patients with acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL) were used as targets in a standard 51Cr-release assay.
Susanne M. Schmidt et al. Blood 2003;102:
©2003 by American Society of Hematology

6. Survivin-specific CTLs recognize autologous dendritic cells transfected with RNA isolated from A 498 tumor cells.
Survivin-specific CTLs recognize autologous dendritic cells transfected with RNA isolated from A 498 tumor cells. Autologous DCs from a healthy HLA-A2+ donor generated from peripheral blood monocytes were pulsed with the cognate survivin peptide (Sv) or electroporated with tumor RNA isolated from the survivin-expressing A 498 tumor cell line and were used as target cells in a standard 51Cr-release assay (▪, •). DCs pulsed with an irrelevant HIV peptide (HIV) or electroporated with EGFP in vitro transcript (IVT) were used as controls in the assay (⋄, ▵).
Susanne M. Schmidt et al. Blood 2003;102:
©2003 by American Society of Hematology

7. Survivin expression in dendritic cells.
Survivin expression in dendritic cells. (A) Survivin expression in human monocyte-derived dendritic cells was analyzed by Western blotting. (B) Standard 51Cr-release assay was performed using autologous immature DCs and autologous DCs activated with TNF-α from a healthy HLA-A2–positive as well as the HLA-A2– and survivin–positive colon carcinoma cell line HCT 116 as targets for survivin-specific CTLs.
Susanne M. Schmidt et al. Blood 2003;102:
©2003 by American Society of Hematology

8. Survivin-specific CTLs do not recognize activated B and T lymphocytes.
Survivin-specific CTLs do not recognize activated B and T lymphocytes. Survivin-specific CTLs were assayed against autologous-activated B and T lymphocytes either pulsed with the cognate survivin (Sv) peptide (filled symbols) or left unpulsed (open symbols).
Susanne M. Schmidt et al. Blood 2003;102:
©2003 by American Society of Hematology

9. Survivin-specific CTLs can lyse autologous malignant chronic lymphatic leukemia cells.
Survivin-specific CTLs can lyse autologous malignant chronic lymphatic leukemia cells. Malignant autologous CLL cells from an HLA-A2+ patient with CLL and autologous immature DCs were pulsed with the cognate survivin peptide (Sv) or an irrelevant adipophilin peptide (Ad) and used as targets. Autologous nonmalignant B cells were included as controls. The antigen specificity of the CTL lines was analyzed in cold target inhibition assays using unlabeled T2 cells pulsed with the cognate survivin (Sv) peptide or the irrelevant adipophilin (Ad) at an inhibitor-to-target ratio of 20:1.
Susanne M. Schmidt et al. Blood 2003;102:
©2003 by American Society of Hematology