Volume 19, Issue 11, Pages 2021-2030 (November 2011) Capsid-specific T-cell Responses to Natural Infections With Adeno-associated Viruses in Humans Differ From Those of Nonhuman Primates  Hua Li, Marcio O Lasaro, Bei Jia, Shih Wen Lin, Larissa H Haut, Katherine A High, Hildegund CJ Ertl  Molecular Therapy  Volume 19, Issue 11, Pages 2021-2030 (November 2011) DOI: 10.1038/mt.2011.81 Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Gating for the human cells. Cells were first gated onto (a) single cells and then on (b) lymphocytes and (c) AmCyan negative live cells. (d) APC-Cy7 was used as a dump gate for CD14 and CD19. (e) Cells were then gated onto CD8+ or CD4+ cells, which were then gated further according to (f) expression of CD45RO and CD27. (g) CD45ROhiCD27hi cells (central memory cells, TCM), CD45RO hiCD27low cells (effector memory cells, TEM) and CD45RO lowCD27low cells (effector cells, TEff) were then gated onto interferon-γ (IFN-γ), or interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α), perforin and macrophage inflammatory protein-1α (MIP-1α) as shown. Molecular Therapy 2011 19, 2021-2030DOI: (10.1038/mt.2011.81) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Frequencies and cell numbers of adeno-associated virus serotype 2 (AAV2) capsid-specific CD8+ or CD4+ peripheral blood mononuclear cells (PBMCs) in humans. Lymphocytes isolated from 17 human blood samples were tested by intracellular cytokine staining for the production of interferon-γ, interleukin-2, tumor necrosis factor-α, perforin, and macrophage inflammatory protein-1α upon stimulation with the AAV2 capsid peptide pool. (a) The sum of the responses in total CD8+ and CD4+ T cells in different people are shown. (b) The numbers of AAV2 capsid-specific cells of each subset per 107 live PBMCs are shown. The average relative distributions of the (c) AAV2 capsid-specific different CD8+ and (d) CD4+ T-cell subsets were calculated. Background responses (responses without peptides) were subtracted prior to plotting of responses. TCM, central memory cells; TEff, effector cells; TEM, effector memory cells. Molecular Therapy 2011 19, 2021-2030DOI: (10.1038/mt.2011.81) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 ELISPOTs for interferon-γ (IFN-γ) and interleukin-2 (IL-2) responses and neutralizing antibodies to adeno-associated virus serotype 8 (AAV8) in nonhuman primates (NHPs). Freshly isolated peripheral blood mononuclear cells (PBMCs) from naive rhesus macaques were test by an ELISpot assay for IFN-γ and IL-2 secretion upon stimulation with four different peptide pools containing overlapping peptides of the AAV8 capsid. The graphs showed responses of 21 animals. Responses to medium (negative control) were subtracted from responses to each pool. (a,b) Our cut-off for positive responses was 55 spots/106 PBMCs. Error bar showed the SD of two different wells. (c) Plasmas from these rhesus macaques were also collected and titers of neutralizing antibodies to AAV8 were tested. Molecular Therapy 2011 19, 2021-2030DOI: (10.1038/mt.2011.81) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Frequencies and cell numbers of adeno-associated virus serotype 8 (AAV8) capsid-specific CD8+ or CD4+ peripheral blood mononuclear cells (PBMCs) in nonhuman primates (NHPs). PBMCs from six naive rhesus macaques were tested by intracellular cytokine staining for production of interferon-γ, interleukin-2 and tumor necrosis factor-α upon stimulation with the AAV8 capsid peptide pool. (a) The sum of the responses in total CD8+ and CD4+ T cells in different NHP samples are shown. (b) The numbers of AAV8 capsid-specific cells of each subset per 107 live PBMCs are shown. The average relative distributions of the (c) AAV8 capsid-specific different CD8+ and (d) CD4+ T-cell subsets were calculated. Background responses (responses without peptides) were subtracted before plotting of responses. TCM, central memory cells; TEff, effector cells; TEM, effector memory cells. Molecular Therapy 2011 19, 2021-2030DOI: (10.1038/mt.2011.81) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Functional properties of CD8+ or CD4+ T cells from human blood. The same 17 human peripheral blood mononuclear cells (PBMCs) shown in Figure 2 were tested for secretion of interferon-γ (IFN-γ) (g), interleukin-2 (IL-2) (l), tumor necrosis factor-α (TNF-α) (T), perforin (p) and macrophage inflammatory protein-1α (MIP-1α) (M) by intracellular cytokine staining of CD8+ or CD4+ T cells that had been stimulated with the adeno-associated virus serotype 2 (AAV2) peptide pool. (a,b)The bars show the frequencies of different subsets of cells that stained for the different function either alone or in combinations. Frequencies <0.005% over all cells of the subset and frequencies of T cells producing perforin only are not shown. The error bar shows the standard error of mean of 17 human PBMCs. (c,d)The average relative distribution of IFN-γ, IL-2 or TNF-α secreting AAV2 capsid-specific CD8+ and CD4+ T cells are shown. Background responses (responses without peptides) were subtracted. Molecular Therapy 2011 19, 2021-2030DOI: (10.1038/mt.2011.81) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Functional properties of CD8+ or CD4+ T cells from nonhuman primates (NHPs). The same NHP peripheral blood mononuclear cells (PBMCs) shown in Figure 4 were tested for secretion of interferon-γ (IFN-γ) (g), interleukin-2 (IL-2) (l) and tumor necrosis factor-α (TNF-α) (T) by intracellular cytokine staining of CD8+ or CD4+ T cells that had been stimulated with adeno-associated virus serotype 8 (AAV8) peptide pool. (a,b) The bars showed the frequencies of different subsets of cells that secreted one, two or three cytokines alone or in combinations. The error bar showed the standard error of mean of six NHPs. (c,d) The average relative distribution of IFN-γ, IL-2 or TNF-α secreting AAV8 capsid-specific CD8+ and CD4+ T cells are shown. Background responses (responses without peptides) were subtracted. Molecular Therapy 2011 19, 2021-2030DOI: (10.1038/mt.2011.81) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Phenotypes of adeno-associated virus (AAV) capsid-specific different CD8+ and CD4+ T-cell subsets. The average expression levels of PD1, Ki67 were determined on interferon-γ (IFN-γ) and/or interleukin-2 (IL-2) secreting (TCM+,TEM+,TEff+) and IFN-γ and IL-2 double negative (TCM−,TEM−,TEff−) CD8+ and CD4+ T-cell subsets in both nonhuman primate (NHP) and human peripheral blood mononuclear cells (PBMCs). (a) The expression levels in naive cell population are shown for comparison. (b–e) The geometric mean fluorescence intensity (GMFI) of PD1, Ki67 expression in different cell subsets are shown. The error bars show the SD of three to four different samples which had sufficient numbers of cells for the analysis in each group. Molecular Therapy 2011 19, 2021-2030DOI: (10.1038/mt.2011.81) Copyright © 2011 The American Society of Gene & Cell Therapy Terms and Conditions