1. Volume 25, Issue 10, Pages 2309-2322 (October 2017)
Intradermal Immunization with rAAV1 Vector Induces Robust Memory CD8+ T Cell Responses Independently of Transgene Expression in DCs 
Alexandre Ghenassia, David-Alexandre Gross, Stéphanie Lorain, Fabiola Tros, Dominique Urbain, Sofia Benkhelifa-Ziyyat, Alain Charbit, Jean Davoust, Pascal Chappert 
Molecular Therapy 
Volume 25, Issue 10, Pages (October 2017)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Figure 1
Intradermal Immunization with rAAV2/1 Vector Induces Robust CD4+ T Cell Help-Dependent Primary CD8+ T Cell Responses
(A) Representation of the mOVA-HY construct. SS, signal sequence; TM, transmembrane domain. (B) Female or male mice were immunized in the ear dermis (i.d.) with 1010 viral genomes (vg) rAAV2/1-mOVA-HY (mOVA-HY) or 1× PBS (Ctrl). Representative dot plots for IFNγ and CD4 (left) and frequencies of IFNγ+ cells (right) in DBY608-stimulated live CD4+ T cells at day 14 in the spleen of the indicated individuals are shown. (C) Female or male mice were immunized in the tibialis anterior (i.m.) or the ear dermis (i.d.) with 3 × 1010 vg rAAV2/1-mOVA-HY (mOVA-HY). Representative dot plots (left) and frequencies of CD44hi Kb/OVA257 Tetramer+ (OVA257+) (right) in CD8+ T cells at day 14 in the blood of immunized individuals are shown. Mean ± SEM (n = 8–10 mice per group, pooled from at least three independent experiments). *p < 0.05 and ****p < (two-way ANOVA/Sidak’s test). (D) Female mice were immunized in the ear dermis (i.d.) with 5 × 1010 vg rAAV2/1-mOVA-HY (mOVA-HY). Injected ear was collected at day 14 after immunization, and 20-μm ear sections were stained with anti-CD8 (red) and anti-MHC class II (blue) antibodies. Left image shows the Z-project (maximum intensity) of an original 1,345 × 852 × 8-μm mosaic image acquired on a LEICA SP8 confocal microscope using a 40×/1.30 Oil CS2 PL APO objective. Right image shows an enlargement of the region of interest depicted in the top right corner of the left image. Images analysis was performed using ImageJ software.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 2
Route of Immunization and CD4+ T Cell Help Synergize in Regulating the Generation of Effector/Memory CD8+ T Cells
Anti-OVA memory CD8+ T cell responses were analyzed between day 60 and day 80 post-immunization in the spleen or draining lymph nodes (dLNs) of male or female mice immunized in the tibialis anterior (i.m.) or ear dermis (i.d.) with 3 × 1010 vg rAAV2/1-mOVA-HY (mOVA-HY) (see also Figure S1 for mice immunized with a control rAAV2/1 vector). (A) Frequencies of CD44hi Kb/OVA257 Tetramer+ (OVA257+) (bottom) in CD8+ T cells in the blood of immunized mice. (B) Representative dot plots for CD44 and Kb/OVA257 tetramer staining in live splenic CD8+ T cells (top) and for CD62L and KLRG1 staining in live splenic CD44hiOVA257+CD8+ T cells (bottom). (C) Frequencies of CD44hiOVA257+ CD62L+KLRG1− (OVA257+ Tcm), CD62L−KLRG1− (OVA257+ Tem), or CD62L−KLRG1+ (OVA257+ Teff) in total live CD8+ T cells isolated from the spleen (top) or draining lymph nodes (bottom) of the indicated individuals. (D) Representative histograms for IL-7Rα (CD127) expression in live splenic OVA257+ Tem (bold) versus endogenous naive CD8+ T cells (filled gray) (left) and relative IL-7Rα expression in live splenic OVA257+ Tem from the indicated individuals (right). (E) Relative IL-7Rα expression in live splenic OVA257+ Teff (dark gray), OVA257+ Tem (light gray), or OVA257+ Tcm (white) from i.d. immunized female mice. Relative IL-7Rα expression is represented as the ratio of the indicated cell population MedianFI over the MedianFI of the endogenous naive CD8+ T cell population of the same individual to account for potential inter-experimental variations in the CD127 staining. Only mice with significant levels of each OVA257+ T memory subset of interest (above mean + 3 SD of control vector) were included in the analysis. Mean ± SEM (A–E, n = 5–11 mice per group, pooled from at least three independent experiments). n.s., non-significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < (A–D, two-way ANOVA/Sidak’s test; E, repeated-measure (RM) one-way ANOVA/Sidak’s test).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 3
Route of Immunization and CD4+ T Cell Help Synergize in Regulating the Quality of Effector/Memory CD8+ T Cells
The cytokine production potential of anti-OVA memory CD8+ T cell responses was analyzed between day 60 and day 80 post-immunization in the spleen or dLNs of male or female mice immunized in the tibialis anterior (i.m.) or ear dermis (i.d.) with 3 × 1010 vg rAAV2/1-mOVA-HY (mOVA-HY). The production of IFNγ, TNF-α, and IL-2 was measured after OVA257 peptide (1 μg/mL) in vitro re-stimulation of splenic cell suspensions. (A) Representative dot plots for IFNγ and TNF-α expression in OVA257-stimulated (top) or unstimulated (middle) live splenic CD8+ T cells. Representative dot plots for IL-2 and TNF-α expression in OVA257-stimulated live splenic CD44hiIFNγ+CD8+ T cells (bottom) are shown. (B) Relative proportion of single- (white), double- (light gray), and triple- (dark gray) cytokine-producing OVA257-stimulated live CD8+ T cells isolated from spleen (top) or dLNs (bottom). Only mice with significant levels of OVA257+ memory T cells in the spleen (above mean + 3 SD of control vector) were included in this analysis (n = 5–8 mice per group, pooled from at least three independent experiments). Analysis and presentation of distributions were performed using SPICE version 5.1 on background- (unstimulated) corrected data. Pairwise comparison of all distributions inside each panel was performed using a Student’s t test and a partial permutation test as described;56 only significant (p < 0.05) differences are displayed (*p < 0.05 and **p < 0.01).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 4
Potent Intradermally Induced Primary CD8+ T Cell Responses Are Generated in the Absence of Transgene Expression in Hematopoietic Cells
(A) Representation of the mOVA-HY-miR construct. SS, signal sequence; TM, transmembrane domain; miR142-3pT, miR142-3p target sequence. (B and C) To analyze the role of direct presentation in rAAV-induced CD8+ T cell responses, male (B) or female (C) mice were immunized in the tibialis anterior (i.m.) or ear dermis (i.d.) with 3 × 1010 vg rAAV2/1-mOVA-HY (mOVA-HY), rAAV2/1-mOVA-HY-miR142-3pT (mOVA-HY-miR), or a control rAAV2/1 vector (Ctrl). (B) Representative dot plots at day 14 (top) and frequencies of OVA-specific CD44hiOVA257+ in CD8+ T cells at the indicated time points in the blood of immunized male mice (bottom) are shown. (C) Frequencies of OVA-specific CD44hiOVA257+ in CD8+ T cells at the indicated time points in the blood of immunized female mice are shown. Mean ± SEM (n = 4–11 mice per group and per time point, pooled from at least two independent experiments). **p < 0.01 and ****p < (two-way ANOVA/Sidak’s test; p < and p = 0.46, respectively, for the overall impact of the miR142-3p-mediated regulation on the rAAV-induced CD8+ T cell response following i.m. or i.d. immunization).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 5
Cross-Presentation of Skin-Targeted Antigen Induces Bona Fide Protective Memory Responses
(A–D) Anti-OVA memory CD8+ T cell responses were analyzed between day 60 and day 80 post-immunization in the spleen or dLNs of male or female mice immunized in the tibialis anterior (i.m.) or ear dermis (i.d.) with 3 × 1010 vg rAAV2/1-mOVA-HY-miR142-3pT (mOVA-HY-miR). (A) Frequencies of CD44hiOVA257+ CD62L+KLRG1− (OVA257+ Tcm), CD62L−KLRG1− (OVA257+ Tem), or CD62L−KLRG1+ (OVA257+ Teff) in total live CD8+ T cells isolated from the spleen (left) or dLNs (right) of the indicated individuals. (B and C) Relative IL-7Rα expression in live splenic OVA257+ Tem from the indicated individuals (B) and in live splenic OVA257+ Teff (dark gray), OVA257+ Tem (light gray), or OVA257+ Tcm (white) from i.d. immunized female mice (C), as described in Figure 2. (D) The production of IFNγ, TNF-α, and IL-2 was measured after OVA257 peptide (1 μg/mL) in vitro re-stimulation of splenic cell suspensions. Relative proportion of single- (white), double- (light gray), and triple- (dark gray) cytokine-producing OVA257-stimulated live CD8+ T cells isolated from spleen or dLNs is displayed as described in Figure 3. Mean ± SEM (A–D, n = 5–11 mice per group, pooled from at least three independent experiments). n.s., non-significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < (A and B, two-way ANOVA/Sidak’s test; C, RM one-way ANOVA/Sidak’s test). (E) The protective potential of anti-OVA memory CD8+ T cell responses induced by the sole cross-presentation of tissue-expressed rAAV transgene was analyzed at day 60 after primary immunization by challenging i.p. with 106 CFUs OVA-expressing recombinant Listeria monocytogenes (Lm-OVA) male or female mice previously immunized in the tibialis anterior (i.m.) or ear dermis (i.d.) with 3 × 1010 vg rAAV2/1-mOVA-HY-miR142-3pT (mOVA-HY-miR) vector. Weight loss over time (left) and Lm-OVA titer at day 3 after challenge are expressed as CFUs/spleen for individual mice (right). Mean ± SEM (n = 9 mice for the “male mOVA-HY-miR i.d.” group, n = 10 mice per group for all other groups, pooled from two independent experiments). **p < 0.01 and ****p < (left, two-way ANOVA/Sidak’s test; right, Kruskal-Wallis/Dunn’s test).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

7. Figure 6
Transgene Products from Both Hematopoietic and Non-hematopoietic Origin Are Actively Cross-Presented by Multiple DC Subsets
To analyze antigen presentation by individual DC subsets following rAAV immunization, male mice were immunized in the tibialis anterior (i.m.) (A, D, and E) or in the ear dermis (i.d.) (B–E) with 1010 vg rAAV2/1-mOVA-HY (A–E) or rAAV2/1-mOVA-HY-miR142-3pT vector (C and E). Then 5 days after rAAV2/1 immunization, individual DC subsets were sorted from draining lymph nodes or injected tissues (see Figure S5 for sort strategies and DC subset frequencies). (A–C) OVA-mRNA expression in indicated DC subset, injected muscle (TA), or skin tissue (ear), at day 5 post-immunization. (D and E) Indicated number of sorted DCs were put in culture with 5 × 103 VPD-labeled naive OVA-specific TCR transgenic OT-1 T cells. (D) Representative histograms show VPD fluorescence in live CD8+VPD+OT-1 after 64-hr culture with 2 × 103 DCs, and (E) frequencies of divided cells in live CD8+VPD+OT-1 after 64-hr culture with the indicated number of DCs are shown. Mean ± SEM. (A–C) Data are pooled from three independent experiments for CD11b+ DC subsets and two for all others. Dots in graphs correspond to one experiment. (D and E) Data are pooled from three independent experiments for all DC subsets. For each experiment, organs from n = 3 immunized mice per group were pooled prior to DC sort. **p < 0.01 and ****p < (two-way ANOVA).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

8. Figure 7
Target Tissue Dictates the Efficiency of Tissue-Expressed Transgene Cross-Presentation
To analyze transgene cross-presentation following rAAV immunization, male or female mice were immunized in the tibialis anterior (i.m.) with 1010 vg rAAV2/1-mOVA-HY or rAAV2/1-mOVA-HY-miR142-3pT (A–C), in the ear dermis (i.d.) with 1010 vg rAAV2/1-mOVA-HY-miR142-3pT (C), or in the ear dermis (i.d.) with the indicated dose of rAAV2/1-mOVA-HY or rAAV2/1-mOVA-HY-miR142-3pT (D). 2 × 106 VPD-labeled naive OVA-specific TCR transgenic OT-1 T cells were infused at the indicated time point following rAAV immunization, and their propensity to upregulate overnight CD69 and CD25, two early T cell activation markers, was assessed. (A–C) Representative dot plots for CD69 and CD25 expression (left) and frequencies of CD69+CD25+ (right) in live VPD+OT-1 T cells recovered from muscle or ear draining lymph nodes 18 hr after a day 5 (dot plots) or indicated time point (graph) OT-1 transfer. (D) Frequencies of CD69+CD25+ (right) in live VPD+OT-1 T cells recovered from ear draining lymph nodes 18 hr after a day 5 OT-1 transfer. Mean ± SEM (A, n = 3–6 mice per group and per time point, pooled from at least two independent experiments, except for day 7.5 and 9, n = 3 mice per group from one experiment only [see also Figure S4 for data on transgene presentation in the spleen and contralateral non-draining lymph nodes]; B, n = 3–6 mice per group and per time point, pooled from at least two independent experiments; C, n = 5 mice per group and per time point, pooled from two independent experiments; and D, n = 3 mice per group, pooled from two independent experiments). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < (two-way ANOVA/Sidak’s test).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions