Lentivectors induce gradual increase in antigen load in expressor cells.

<p>(<b>A</b>) Mice were immunized with Lv-Luc vector (encoding also eGFP) and luciferase expression was tracked <i>in vivo</i>. (<b>B</b>) Part of the Lv-Luc primed mice were euthanized on days 1, 5, 7 and 14 and their ears were collected and processed to obtain genomic DNA. DNA samples were subjected to quantitative real-time PCR analysis in order to calculate the relative amount of lentiviral DNA in each day tested. Lentiviral DNA was quantified using eGFP specific primers and was standardized according to the levels of endogenous 18S DNA. (<b>C</b>) The ears of Lv-Luc immunized mice were removed on days 1 and 11 post priming and were then subjected to immunofluorescence analysis. Images of confocal microscopy of the ear pinna are shown with a 5-µm-thick section using a 10×0.6 objective and 25× zoom. Control image represents staining with secondary antibody only. (Blue, nuclei stained with the DNA intercalating dye DAPI; green, anti-GFP antibody). Arrow heads indicate GFP-expressing cells. Dotted line was added to define the auricular cartilage (AC), D-dermis, E-epidermis. One representative out of two independent experiments is depicted. *, <i>P</i><0.005, compared to all immunized groups. #, <i>P</i><0.001, compared to DNA copies measured on days 1, 5 and 7 post-immunization.</p

Kinetics of antigen expression control lentivector-induced secondary CD8⁺ T-cell response.

<p>(<b>A</b>) B6 mice were primed, or primed and boosted intradermally with 5×10⁶ TU of Lv-OVA/Luc, and luciferase expression was determined <i>in vivo</i> using whole body imagining. The mean relative light unit (RLU) values expressed by a group of 4 mice ± SE are presented. (<b>B</b>) Immunization site of Lv-OVA (5×10⁶ TU) homologously boosted mice was removed on days 2, 4 or 6 following immunization, and the magnitude of OVA-specific CD8⁺ T cells was analyzed. Data are showed as the mean percentage ± SE of CD8⁺ tetramer⁺ T cells. n  = 3 mice per group for each time point. (<b>C</b>) Mice were primed with Lv-OVA and 7 weeks later were boosted with Lv-OVA (Lv-Lv) or Ad-OVA (Lv-Ad). In parallel, other groups of naïve mice were primed with Lv-OVA (Lv prime) or Ad-OVA (Ad prime) using the same viral vector employed for boosting to allow adequate comparison. Two weeks after immunization tetramer analysis was performed on blood samples obtained from the mice (n  = 4 to 5 mice for each group). The graph represents the fold increase in the magnitude of OVA-specific CD8⁺ T cells, Lv-Lv versus Lv prime, and Lv-Ad versus Ad prime. Results described in this figure are representative of two independent experiments. *, <i>P</i><0.01, prime versus boost response at the indicated time points. #, <i>P</i><0.05, compared to control uncut boosted group.</p

Shortened kinetics of antigen presentation <i>in vivo</i> in lentivector-boosted mice.

<p>B6 mice were primed, or primed and boosted with Lv-OVA, and then were adoptively transferred i.v. with 2×10⁶ CFSE-labeled OT-I (<b>A</b>) or OT-II (<b>B</b>) splenocytes at the indicated days. Three days later the LNs were harvested and the CFSE dilution was assessed by flow cytometry to analyze the proliferation of the transferred CD8⁺ or CD4⁺ T cells, respectively. Results are shown as representative flow plots gating on dividing CD8⁺ or CD4⁺ lymphocytes; numbers indicate the percentages of dividing cells and represent the mean of three mice per group for each time point ± SE. One representative out of 2 independent experiments is depicted. *, <i>P</i><0.01, primed mice versus boosted mice at the time points indicated.</p

image_1_Multiple Regulatory Levels of Growth Arrest-Specific 6 in Mucosal Immunity Against an Oral Pathogen.jpeg

<p>Growth arrest-specific 6 (GAS6) expressed by oral epithelial cells and dendritic cells (DCs) was shown to play a critical role in the maintenance of oral mucosal homeostasis. In this study, we demonstrate that the induction of pathogen-specific oral adaptive immune responses is abrogated in Gas6^−/− mice. Further analysis revealed that GAS6 induces simultaneously both pro- and anti-inflammatory regulatory pathways upon infection. On one hand, GAS6 upregulates expression of adhesion molecules on blood vessels, facilitating extravasation of innate inflammatory cells to the oral mucosa. GAS6 also elevates expression of CCL19 and CCL21 chemokines and enhances migration of oral DCs to the lymph nodes. On the other hand, expression of pro-inflammatory molecules in the oral mucosa are downregulated by GAS6. Moreover, GAS6 inhibits DC maturation and reduces antigen presentation to T cells by DCs. These data suggest that GAS6 facilitates bi-directional trans-endothelial migration of inflammatory cells and DCs, whereas inhibiting mucosal activation and T-cell stimulation. Thus, the orchestrated complex activity of GAS6 enables the development of a rapid and yet restrained mucosal immunity to oral pathogens.</p

image_2_Multiple Regulatory Levels of Growth Arrest-Specific 6 in Mucosal Immunity Against an Oral Pathogen.jpeg

<p>Growth arrest-specific 6 (GAS6) expressed by oral epithelial cells and dendritic cells (DCs) was shown to play a critical role in the maintenance of oral mucosal homeostasis. In this study, we demonstrate that the induction of pathogen-specific oral adaptive immune responses is abrogated in Gas6^−/− mice. Further analysis revealed that GAS6 induces simultaneously both pro- and anti-inflammatory regulatory pathways upon infection. On one hand, GAS6 upregulates expression of adhesion molecules on blood vessels, facilitating extravasation of innate inflammatory cells to the oral mucosa. GAS6 also elevates expression of CCL19 and CCL21 chemokines and enhances migration of oral DCs to the lymph nodes. On the other hand, expression of pro-inflammatory molecules in the oral mucosa are downregulated by GAS6. Moreover, GAS6 inhibits DC maturation and reduces antigen presentation to T cells by DCs. These data suggest that GAS6 facilitates bi-directional trans-endothelial migration of inflammatory cells and DCs, whereas inhibiting mucosal activation and T-cell stimulation. Thus, the orchestrated complex activity of GAS6 enables the development of a rapid and yet restrained mucosal immunity to oral pathogens.</p

T regulatory cells migrate toward R5 HIV gp120.

<p>CD4+CD25+T regulatory cells were purified from naive human PBMC and exposed to various concentrations of gp120 in a Boyden chamber migration assay. A) Normalized transmigration index of Tregs that migrate towards various gp120 concentrations (500 pg/ml, 5 ng/ml, 500 ng/ml) in presence of CCR5 antagonist, TAK-779, or pretreated with pertussis toxin (Ptx 100 ng/ml) (*p<0.05 vs TAK-779). B) Normalized transmigration index of Tregs that migrate away from gp120 concentrations (500 pg/ml, 5 ng/ml, 500 ng/ml) in presence of CCR5 antagonist, TAK-779 (40 nM), or pretreated with pertussis toxin (Ptx, 100 ng/ml).</p

Increased numbers of CD4+CD25+CD127low T cells in the LN of R5-SHIV infected RM.

<p>RM were sampled at 5 and 12 weeks post-inoculation. PB and LN samples were examined by multi-colour flow cytometry for the proportion of Treg cells present. A) Representative dotplots from PB and LN gated on size and granularity as well as CD3 and CD4, and subsequently on CD25 vs CD127 as shown (% frequency of parent). B) Gating strategy demonstrating the fluorescent minus one scheme utilized as above and CD25 vs CD127 as shown. C & D) Individual RM Treg cell frequencies of parental gate at 5 weeks post-infection *p<0.05 LN compared to PB (C), and 12 weeks <u>(non-significant)</u> (D) post-infection. E) Time course of Treg accumulation at 5 and 12 weeks post infection in PB and LN, mean ±SEM. F) LN samples depleted of CD4+CD25+ T cells and stimulated with overlapping SIVmac239 Gag peptide pools. Data depicted as relative change of Gag- and gp120 specific CD8 responses in CD4+CD25+ depleted compared to non-depleted samples, (p = 0.06 vs change in gp120 specific CD8 T cells.) G) Correlation of Tregs with gp120 in the LN of intravaginally challenged RM (r² = 0.7, two-tailed p = 0.035).</p

Enhanced basal T cell apoptosis from PB, but not LN of R5-SHIV-infected RM.

<p>Naïve and R5-SHIV challenged RM were sampled at 5 and 12 weeks post-inoculation. PB and LN samples from either naïve or 5 and 12 weeks post-inoculation samples were either non-stimulated (NS) or stimulated with plate-bound anti-CD3 (CD3) for 24 hours. A) Percent of apoptotic CD4 T cells from naïve RM or infected RM at 5 or 12 weeks post inoculation comparing non-stimulated to CD3 stimulated is shown (naïve PBMC non-stimulated vs naïve PBMC stimulated p<0.005; naïve PBMC non-stimulated vs 5 weeks infected PBMC non-stimulated p<0.005; naïve PBMC non-stimulated vs 12 weeks infected non-stimulated p<0.05). B) Percent of apoptotic T cells from naïve RM or infected RM at 5 or 12 weeks post inoculation comparing non-stimulated to CD3 stimulated, (naïve PBMC non-stimulated vs naïve PBMC stimulated p<0.005; naïve PBMC, non-stimulated vs 5 weeks infected PBMC, non-stimulated p<0.05).</p

Reduced antigen-specific CD4 and CD8 T cell responses in LN RM during early R5-SHIV infection.

<p>RM were inoculated intravaginally (n = 4) or intravenously (n = 2) with SHIV-1157ipd3N4 (R5-SHIV). Blood samples were drawn for viral load analysis at 0, 1, 2, 3, 8 and 12 weeks post infection (A). Paired peripheral blood samples (PB) and lymph nodes (LN) from RM during early infection were sampled at 5 and 12 weeks post-inoculation (B-G). PB and LN lymphocytes were stimulated with overlapping clade C gp120 peptide pools at 5 and 12 weeks post inoculation and IFN-γ⁺ gp120-specific CD4 (B,C) and CD8 (D,E) T cell were assayed. F & G) PB and LN derived cells were stimulated with overlapping SIVmac239 Gag peptide pools at 5 and 12 weeks post inoculation and IFN-γ⁺ Gag-specific CD4 (F) and CD8 (G) T cell responses were assayed. Data representative of frequency of total (C,E-G) or frequency of parent (B,D) are shown. Error bars ±SEM, *p<0.05, ** p<0.01.</p

Levels of gp120 and p27 in the Plasma, Lymph Node and Spleen of RM 12 weeks post-inoculation with R5-SHIV.

<p>LN: Lymph Node.</p