Volume 13, Issue 2, Pages 270-279 (February 2006) Recombinant Adenovirus Vaccines Can Successfully Elicit CD8+ T Cell Immunity under Conditions of Extreme Leukopenia  Natalie Grinshtein, Teng-Chih Yang, Robin Parsons, James Millar, Galina Denisova, Dilan Dissanayake, Jaina Leitch, Yonghong Wan, Jonathan Bramson  Molecular Therapy  Volume 13, Issue 2, Pages 270-279 (February 2006) DOI: 10.1016/j.ymthe.2005.09.018 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Effects of the myeloablative treatment on total cellularity. C57BL/6 (CD45.2) mice (three mice per group) were lethally irradiated and supplemented with 5 × 106 bone marrow cells from a congenic, B6.SJL (CD45.1+) donor. (A) Bone marrow, draining lymph nodes, and spleen were retrieved at different time points postirradiation and the total cell yield was enumerated. The results are presented as percentage from nonirradiated mice ± SEM. Representative data from one of two independent experiments are shown. (B) Relative representation of host versus donor cells in the irradiated mice 6 weeks posttreatment. Spleens were harvested, processed, and stained for the expression of various surface markers. Numbers in parentheses indicate the percentage of CD45.2+ cells from each gated population ± SEM for a group of three mice. The histograms are representative of one mouse and the data are representative of two independent experiments with three mice each. Molecular Therapy 2006 13, 270-279DOI: (10.1016/j.ymthe.2005.09.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Characterization of the CD8+ effector T cells primed by rAd vaccine under conditions of extreme leukopenia. For all the experiments represented, C57BL/6 mice (five mice per group) were lethally irradiated and reconstituted with 5 × 106 bone marrow cells from a congenic B6.SJL (CD45.1+) donor. (A) Examples of intracellular IFN-γ staining assay. Splenocytes were obtained from irradiated and nonirradiated mice immunized 42 days earlier with AdSIINFEKL-Luc-004 and restimulated with peptide for measurement of intracellular IFN-γ. The top graphs are representative of samples that were stimulated with SIINFEKL and the lower graphs are representative of cells stimulated with an irrelevant peptide. (B) Examples of tetramer staining assay. Lymphocytes were obtained from the blood of irradiated and nonirradiated mice immunized 42 days earlier with AdSIINFEKL-Luc-004 and stained with Kb/SIINFEKL tetramer. Results using lymphocytes from a naïve mouse are included to demonstrate the specificity of the tetramer. (C) The irradiated and reconstituted mice were immunized with Ad-SIINFEKL-Luc-004 either 1 or 14 days following irradiation. SIINFEKL-specific CD8+ T cells were enumerated by IFN-γ staining of spleen populations harvested 28, 35, and 42 days after irradiation. (D) The irradiated and reconstituted mice were immunized the next day with AdSIINFEKL-Luc-004 (closed bars). Animals were bled weekly and SIINFEKL-specific CD8+ T cells were identified by tetramer staining. Immunized, but nonirradiated, mice were used as controls (open bars). Each point represents the mean ± SEM for a group of five mice. (E) SIINFEKL-specific CD8+ T cells were enumerated in spleen, draining lymph nodes, and lungs 6 weeks after immunization with Ad-SIINFEKL-Luc-004 using the IFN-γ staining method. Each bar represents the mean ± SEM for a group of five mice and the data represent one of three independent experiments. (F) C57BL/6 mice (five mice per group) were lethally irradiated and reconstituted with 5 × 106 bone marrow cells from a congenic, B6.SJL (CD45.1+) donor. The next day mice were immunized with Ad-SIINFEKL-Luc-004. SIINFEKL-specific CD8+ T cells were enumerated by IFN-γ staining Total numbers of IFN-γ-producing cells of total CD8+ cells from either host (CD45.2+) or donor (CD45.1+) origin are shown (means ± SEM; n = 5) and the data represent one of three independent experiments. Unless otherwise noted, the differences between the groups were not found to be significant. Asterisks denote significant differences (i.e., P < 0.05). Molecular Therapy 2006 13, 270-279DOI: (10.1016/j.ymthe.2005.09.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Induction of protective immunity to melanoma challenge. C57BL/6 mice (six mice per group) were lethally irradiated and reconstituted with bone marrow cells from a syngeneic donor. The next day mice were immunized with either AdSIINFEKL-Luc-004 or AdLacZ. Nonirradiated mice were similarly immunized with AdSIINFEKL-Luc-004 and AdLacZ. Mice were challenged 6 weeks postimmunization with B16M05 cells and tumor size was monitored daily. The percentage of tumor-free mice over the course of the experiment is shown. Molecular Therapy 2006 13, 270-279DOI: (10.1016/j.ymthe.2005.09.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Gene expression in vivo following rAd vaccination. C57BL/6 mice (n = 3) were lethally irradiated and reconstituted with 5 × 106 bone marrow cells from a syngeneic donor. Mice were immunized with AdSIINFEKL-Luc-004 the day after irradiation. Nonirradiated mice were similarly immunized (n = 3). Muscles (bottom) and draining lymph nodes (top) were retrieved from the animals on various days postimmunization, homogenized, and assayed for luciferase expression. Luciferase activity was normalized to the amount of protein in the homogenate and the results are shown as means ± SEM. Molecular Therapy 2006 13, 270-279DOI: (10.1016/j.ymthe.2005.09.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Antigen presentation following rAd vaccination. C57BL/6 mice (n = 4) were lethally irradiated and reconstituted with 5 × 106 bone marrow cells from a syngeneic donor. The following day, mice were immunized with either AdSIINFEKL-Luc-004 or AdLuc (108 pfu). Nonirradiated mice were similarly immunized (n = 3). CFSE-labeled OT-I CD8+ T cells were injected into the immunized mice on days 3, 26, or 44 postimmunization, as indicated above each column. The draining lymph nodes were retrieved 3 days post-OT-1 cell transfer and proliferation was visualized as dilution of the CFSE signal. One representative mouse of four is shown from each group. Molecular Therapy 2006 13, 270-279DOI: (10.1016/j.ymthe.2005.09.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Generation of SIINFEKL-specific CD8+ T cells in allogeneic bone marrow chimeras. C57BL/6 mice were lethally irradiated and reconstituted with 5 × 106 bone marrow cells from either a syngeneic (Bl/6 → Bl/6) or an allogeneic (bm1 → Bl/6) donor. Similarly, bm1 mice were lethally irradiated and reconstituted with 5 × 106 bone marrow cells from an allogeneic donor (Bl/6 × bm1). The next day, mice were immunized with AdSIINFEKL-Luc-004. Immunized, but nonirradiated, mice were used as controls for this experiment (Bl/6). The frequency of SIINFEKL-specific CD8+ T cells in the spleen was determined 6 weeks later by tetramer staining. Pooled data from two independent experiments are shown. Molecular Therapy 2006 13, 270-279DOI: (10.1016/j.ymthe.2005.09.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Antigen presentation in allogeneic bone marrow chimeras. C57BL/6 mice were lethally irradiated and reconstituted with 5 × 106 bone marrow cells from either a syngeneic (Bl/6 → Bl/6) or an allogeneic (bm1 → Bl/6) donor. Similarly, bm1 mice were lethally irradiated and reconstituted with 5 × 106 bone marrow cells from an allogeneic donor (Bl/6 → bm1). The next day, mice were immunized with AdSIINFEKL-Luc-004 or AdLuc. Mice were injected with CFSE-labeled OT-I CD8+ T cells 3 days postimmunization. The draining lymph nodes were retrieved 3 days following OT-1 cell transfer and proliferation was visualized as dilution of the CFSE signal. One representative mouse of four is shown from each group. Molecular Therapy 2006 13, 270-279DOI: (10.1016/j.ymthe.2005.09.018) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions