Volume 18, Issue 9, Pages 1714-1723 (September 2010) IL-28B/IFN-λ3 Drives Granzyme B Loading and Significantly Increases CTL Killing Activity in Macaques  Matthew P Morrow, Jian Yan, Panyupa Pankhong, Devon J Shedlock, Mark G Lewis, Kendra Talbott, Roberta Toporovski, Amir S Khan, Niranjan Y Sardesai, David B Weiner  Molecular Therapy  Volume 18, Issue 9, Pages 1714-1723 (September 2010) DOI: 10.1038/mt.2010.118 Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Group design, plasmid construction, and immunization schedule. (a) Rhesus macaques were divided into four groups of varying immunizations consisting of (b) HIV antigens with or without IL-12 or IL-28B/IFN-λ3 constructs. (c) IL-12 and IL-28B/IFN-λ3 expression and secretion were verified after in vitro transfection. (d) Animals were immunized and analysis was performed at indicated time points. HIV, human immunodeficiency virus; IFN, interferon; IL, interleukin; PBMC, peripheral blood mononuclear cells. Molecular Therapy 2010 18, 1714-1723DOI: (10.1038/mt.2010.118) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Interferon-γ ELISpot responses. HIV-specific interferon-γ secretion induced by immunization with or without IL-12 or IL-28B/IFN-λ3 was measured via ELISpot over the course of the immunization period and analyzed on per (a) animal basis as well as on (b) a group-wide basis. ELISpot, enzyme-linked immunospot assay; HIV, human immunodeficiency virus; IFN, interferon; IL, interleukin; SFU, spot-forming units. Molecular Therapy 2010 18, 1714-1723DOI: (10.1038/mt.2010.118) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Analysis of T-cell function in the periphery (PBMC) and mesenteric lymph nodes (MLN) 3 months after immunization. (a) Interferon γ and CD107a responses were measured from peripheral (PBMC—top) and mesenteric (MLN—bottom) CD8+ T cells 3 months after vaccination. *P < 0.05 for CD107a responses between IL-28B/IFN-λ3 group and any other group. **P < 0.05 for summed responses between IL-28B/IFN-λ3 and IL-12. (b) Analysis of CD8+ T-cell responses are shown by group in the periphery (PBMC) and MLN. (c) Long-lived CD8+ T cells were analyzed for multiple functions including coexpression of CD107a and interferon γ (PBMC and MLN), CD107a and granzyme B (PBMC and MLN) or perforin release (PBMC). The P value shown is for comparison of CD107a/GrzB+ CD8+ T cells in the IL-28B/IFN-λ3 group compared with each of the other vaccination groups when looking in the PBMC subset. (d) Long-lived CD4+ T-cell function was measured in both the periphery (PBMC) and lymph nodes (MLN). HIV, human immunodeficiency virus; IFN, interferon; IL, interleukin; PBMC, peripheral blood mononuclear cells. Molecular Therapy 2010 18, 1714-1723DOI: (10.1038/mt.2010.118) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Lytic granule loading assay of CD8+ T cells from mesenteric lymph nodes. Cells isolated from MLN of animals 3 months postimmunization were incubated with HIV Gag and Pol peptides for 6 days, at the end of which time cells were stained for activation markers CD38, HLA-DR, and Ki67. Representative staining is shown in a. (b) After the 6 day stimulation, CD8+ T cells from MLN exhibiting activation as determined from two-marker positivity were assayed for granzyme B content. (c) A more restrictive triple-positive activation requirement was applied to the stimulated CD8+ MLN T cells, and granzyme B content was again determined. HIV, human immunodeficiency virus; HLA, human leucocyte antigen; IFN, interferon; IL, interleukin; MLN, mesenteric lymph nodes; PBMC, peripheral blood mononuclear cells. Molecular Therapy 2010 18, 1714-1723DOI: (10.1038/mt.2010.118) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Activated granzyme B delivery from antigen-specific CTLs to targets. CD8+ T cells (CTLs) from MLN left unstimulated (day 0) or stimulated for 6 days (day 6) with HIV Gag and Pol in the same fashion as the lytic granule loading assay were incubated with autologous targets for 1 hour. The presence of active granzyme B was determined by assaying for fluorescent granzyme B substrate in target cells. Representative staining is shown in a. (b) Overlays of CTL-based killing via delivery of active granzyme B from day 0 and day 6 cells, as well graphical representations of killing on day 0, day 6, or the average increase in killing from day 0 to day 6. CTL, cytotoxic T lymphocytes; GrB, granzyme B; HIV, human immunodeficiency virus; HLA, human leucocyte antigen; IFN, interferon; IL, interleukin; MLN, mesenteric lymph nodes;PBMC, peripheral blood mononuclear cells. Molecular Therapy 2010 18, 1714-1723DOI: (10.1038/mt.2010.118) Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions