Figure 4.
Figure 4. AAV capsid is MHC I presented by cDCs, which do not require intrinsic TLR9-MyD88 signaling. (A) Transgenic mice expressing Cre recombinase under the control of a CD11c promoter were crossed with mice containing a MyD88 gene flanked by loxP sites. The resulting animals were termed DC-MyD88−/− mice. Genomic DNA was isolated and polymerase chain reaction analysis of MyD88 was performed (100 ng DNA/sample), along with genomic DNA from a global MyD88−/− mouse (that had been generated by others by Cre excision from the same MyD88fl/fl strain) to confirm that detection is lost after Cre-mediated excision. (B) Spleens were harvested from WT or DC-MyD88−/− mice (n = 4-5/group) and analyzed for Cre expression using the GFP transgene in B cells (CD19+), cDCs (CD11c+ PDCA-1−), and pDCs (CD11cint PDCA-1+). Percent GFP+ was calculated via Overton subtraction using WT mice as a control, and GFP mean fluorescence intensity in each cell population was quantified. (C) WT or DC-MyD88−/− mice were IM injected with AAV2-SIINFEKL and tetramer-positive cells were quantified over time (n = 4/group). The dotted line at 0.1% represents the limit of detection of capsid-specific CD8+ T cells using the tetramer. (D) Experimental outline of TLR9−/− cDC adoptive transfers. CD11c-DTR mice received 100 ng DT in order to deplete endogenous cDC. Mice then either received both AAV2-SIINFEKL (IM) and 3 to 4 × 106 cDC from TLR9−/− mice or AAV2-SIINFEKL only. Another group of mice that received AAV2-SIINFEKL but no DT served as positive control for the immune response, and is labeled as “Control.” Mice were injected with 5 × 106 CTV-labeled OT-1 cells 2 days later. (E) Representative plots of proliferation of adoptively transferred OT-1 cells. (F) Quantification of the percent proliferation, percent divided (percent of original cells that divided at least once), and the division index (the average number of cells that a dividing cell became). (G) Detection of capsid antigen presentation in inguinal lymph node by staining with a phycoerythrin-labeled antibody specific to SIINFEKL/H-2Kb as a function of time after IM injection of 1 × 1011 vg of AAV2-SIINFEKL vector in WT C57BL/6 mice. Shown are average mean fluorescence intensity ± SEM for cDCs (CD11chi, PDCA-1−) and pDCs (CD11cmid, PDCA-1+) 1, 2, or 3 days after vector injection (n = 4, 4, or 2, respectively). Cells from PBS-injected mice, analyzed in parallel, serve for baseline level of staining (n = 4). Also shown is a representative histogram for a PBS- vs vector-injected (2-day time point) mouse. P values: (B) ***P < .0001 for differences in % GFP, **P =.003 for MFI; (F) percent proliferation, *P =.025 for control vs depleted and *P =.048 for depleted vs “+ TLR9−/− cDCs”; percent divided, *P =.025 for control vs depleted and **P =.003 for depleted vs “+ TLR9−/− cDCs”; division index, **P =.0025 for control vs depleted and ***P < .0001 for depleted vs “+ TLR9−/− cDCs”; (G) **P < .01 by ANOVA. GFP, green fluorescent protein.

AAV capsid is MHC I presented by cDCs, which do not require intrinsic TLR9-MyD88 signaling. (A) Transgenic mice expressing Cre recombinase under the control of a CD11c promoter were crossed with mice containing a MyD88 gene flanked by loxP sites. The resulting animals were termed DC-MyD88−/− mice. Genomic DNA was isolated and polymerase chain reaction analysis of MyD88 was performed (100 ng DNA/sample), along with genomic DNA from a global MyD88−/− mouse (that had been generated by others by Cre excision from the same MyD88fl/fl strain) to confirm that detection is lost after Cre-mediated excision. (B) Spleens were harvested from WT or DC-MyD88−/− mice (n = 4-5/group) and analyzed for Cre expression using the GFP transgene in B cells (CD19+), cDCs (CD11c+ PDCA-1−), and pDCs (CD11cint PDCA-1+). Percent GFP+ was calculated via Overton subtraction using WT mice as a control, and GFP mean fluorescence intensity in each cell population was quantified. (C) WT or DC-MyD88−/− mice were IM injected with AAV2-SIINFEKL and tetramer-positive cells were quantified over time (n = 4/group). The dotted line at 0.1% represents the limit of detection of capsid-specific CD8+ T cells using the tetramer. (D) Experimental outline of TLR9−/− cDC adoptive transfers. CD11c-DTR mice received 100 ng DT in order to deplete endogenous cDC. Mice then either received both AAV2-SIINFEKL (IM) and 3 to 4 × 106 cDC from TLR9−/− mice or AAV2-SIINFEKL only. Another group of mice that received AAV2-SIINFEKL but no DT served as positive control for the immune response, and is labeled as “Control.” Mice were injected with 5 × 106 CTV-labeled OT-1 cells 2 days later. (E) Representative plots of proliferation of adoptively transferred OT-1 cells. (F) Quantification of the percent proliferation, percent divided (percent of original cells that divided at least once), and the division index (the average number of cells that a dividing cell became). (G) Detection of capsid antigen presentation in inguinal lymph node by staining with a phycoerythrin-labeled antibody specific to SIINFEKL/H-2Kb as a function of time after IM injection of 1 × 1011 vg of AAV2-SIINFEKL vector in WT C57BL/6 mice. Shown are average mean fluorescence intensity ± SEM for cDCs (CD11chi, PDCA-1−) and pDCs (CD11cmid, PDCA-1+) 1, 2, or 3 days after vector injection (n = 4, 4, or 2, respectively). Cells from PBS-injected mice, analyzed in parallel, serve for baseline level of staining (n = 4). Also shown is a representative histogram for a PBS- vs vector-injected (2-day time point) mouse. P values: (B) ***P < .0001 for differences in % GFP, **P =.003 for MFI; (F) percent proliferation, *P =.025 for control vs depleted and *P =.048 for depleted vs “+ TLR9−/− cDCs”; percent divided, *P =.025 for control vs depleted and **P =.003 for depleted vs “+ TLR9−/− cDCs”; division index, **P =.0025 for control vs depleted and ***P < .0001 for depleted vs “+ TLR9−/− cDCs”; (G) **P < .01 by ANOVA. GFP, green fluorescent protein.

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