Volume 23, Issue 5, Pages 964-973 (May 2015) T-cell Subsets in Peripheral Blood and Tumors of Patients Treated With Oncolytic Adenoviruses  Taipale Kristian, Liikanen Ilkka, Juhila Juuso, Karioja-Kallio Aila, Oksanen Minna, Turkki Riku, Linder Nina, Lundin Johan, Ristimäki Ari, Kanerva Anna, Koski Anniina, Joensuu Timo, Vähä-Koskela Markus, Hemminki Akseli  Molecular Therapy  Volume 23, Issue 5, Pages 964-973 (May 2015) DOI: 10.1038/mt.2015.17 Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 The balance of T lymphocytes in patients receiving oncolytic virotherapy is shifted towards cytotoxic T cells after the first viral treatment. (a–d) T-cell levels were measured from peripheral blood mononuclear cell samples with flow cytometry. T-cell levels are presented as percentage of immediate parent population (CD3+ and CD3+CD4+ for CD4/CD8 and Th1/Treg respectively). Panel a includes the average differences in T-cells for all patients. In the panels b through d, the differences in average T-cell levels are shown for different virus treatment groups. The P values measured by paired Student's t-test for the pre-postdifference in all patients (n = 50) were <0.0001 for CD4 cells and <0.0001 for CD8 cells. The P values for different virus treatment groups were 0.0011 (CD4) and 0.0075 (CD8) for CGTG-102 (n = 16), 0.0370 (CD4), and 0.0491 (CD8) for CGTG-401 (n = 12) and 0.0026 (CD4) and 0.0224 (CD8) for CGTG-602 (n = 22). No significant differences in the Th1 or Treg populations were seen. Error bars are shown as mean + SEM. Molecular Therapy 2015 23, 964-973DOI: (10.1038/mt.2015.17) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Virus design and concomitant treatments influence the change in T cell subpopulations. (a) Effects of different virus constructs on CD8 and CD4 changes. Differences in T-cell levels are shown as change in percentage points between pre- and post-treatment percentages from parent population (CD3+) in all panels. The light (CD8) and dark gray (CD4) bars represent the median change in different treatment groups. Difference between CGTG-102 and CGTG-602 patients was borderline significant (P = 0.0745) for the CD8 change. (b) The T-cell level changes are smaller in patients receiving metronomic cyclophosphamide and temozolomide as concomitant treatments. Patient groups are based on concomitant treatments that were given with the oncolytic adenovirus treatment. CP, cyclophosphamide; TMZ, temozolomide. The light (CD8) and dark gray (CD4) bars indicate the median change in different concomitant treatment groups. The differences in CD4 level changes between cyclophosphamide and cyclophosphamide + temozolomide groups were considered significant (P = 0.041). (c,d) Reduction in anti-survivin and anti-Ad5 T-cell activity (by IFN-γ ELISPOT) associates with smaller increase in cytotoxic T-cells after the first viral treatment. Patients were grouped according to the change between pretreatment and post-treatment ELISPOT result. Panel c and d display patients grouped by anti-Ad5 and anti-survivin ELISPOT results respectively. The light (CD8) and dark gray (CD4) bars represent the median change in different ELISPOT change groups. The P values were not considered significant. Error bars are shown as mean + SEM in all panels.ELISPOT, enzyme-linked immunospot assay. Molecular Therapy 2015 23, 964-973DOI: (10.1038/mt.2015.17) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Changes of CD4 and CD8 T cells in tumor biopsies are correlated with changes in blood. (a,b) Percentual changes in tumor biopsy IHC scores of CD4 cells are inversely correlated with changes in blood CD4 levels measured by flow cytometry, while changes in CD8 biopsy scores are positively correlated with changes in blood CD8 levels. Light gray dots represent the percentual change of the CD4 or CD8 staining in tumor biopsy samples between pre- and post-treatment biopsies. Dark gray dots indicate the change in blood CD4 or CD8 cells between pre- and post-treatment samples measured by flow cytometry. Patients are sorted based on the tumor biopsy change from largest to smallest change. Pearson's correlation coefficients were −0.828 (P = 0.084) and 0.829 (P = 0.083) for CD4 and CD8 cells, respectively. Molecular Therapy 2015 23, 964-973DOI: (10.1038/mt.2015.17) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Percentual changes of B cell marker CD19, T cell activation marker CD25 and monocyte/macrophage markers CD68 and CD163 in tumor biopsy IHC staining scores. (a–d). Light gray dots represent the percentual changes of the staining scores in tumor biopsy samples for individual patients between pre- and post-treatment biopsies. Patients are sorted as in panel a of Figure 3. Molecular Therapy 2015 23, 964-973DOI: (10.1038/mt.2015.17) Copyright © 2015 American Society of Gene & Cell Therapy Terms and Conditions