Time-course analysis of antigen-loaded APCs localization.

<p>C57BL/6 mice were immunized by the nasal route with OVA-Alexa fluor 647 conjugate (25 µg/mouse) and CpG ODN (20 µg/mouse). At different time points (0, 12, 24 and 72 h), DCs and B cells were isolated from draining (mediastinal [MedLN]) and distal (iliac [ILN], mesenteric [MLN]) lymph nodes and spleen (SPL). The percentages of DCs (A) and B cells (B) positive for fluorescent OVA were analysed by flow cytometry. Shown is the percentage of OVA-Alexa fluor 647 positive cells among total live CD11c⁺ MHC class II⁺ cells purified using anti CD11c-conjugated magnetic beads (A) or B220⁺ cells (B).</p

Effect of treatment with FTY720 on dissemination of primed T cells after nasal immunization with OVA plus CpG ODN.

<p>CD4⁺ and CD8⁺ T cells, isolated from OT-II and OT-I mice, were labelled with CFSE and adoptively transferred into recipient C57BL/6 Ly5.1 mice. Twenty-four hours later, recipient mice were immunized with OVA (25 µg/mouse) and CpG ODN (20 µg/mouse) by the nasal route. FTY720 was administered i.p. 18 h after immunization and every 24 h until harvest 5 days after immunization. <b>A–B</b>. CD4⁺ (A) and CD8⁺ (B) TgN T- cell proliferation assessed in cervical (CLN), mediastinal (MedLN), iliac (ILN), and mesenteric (MLN) lymph nodes, and spleen (SPL), by CFSE dilution. Histograms are gated on CD4⁺ CD45.2⁺ (A) or CD8⁺ CD45.2⁺ (B) population, with light scatter properties of lymphocytes. Results are representative of three independent experiments performed with three mice per group. Reported values indicate the percentage of dividing OVA-specific TgN T cells. <b>C.</b> Ratio between the absolute number of OVA-specific CD4⁺ and CD8⁺ TgN T cells detected in FTY720- treated <i>versus</i> untreated mice. Values of ratios for each organ are reported.</p

Antigen-specific T-cell distribution in draining, distal lymph nodes and spleen following vaginal or nasal immunization.

<p>CFSE-labelled OT-II CD4⁺ T cells were transferred into recipient C57BL/6J mice. Twenty-four hours later, recipient mice were immunized with OVA (25 µg/mouse) and CpG ODN 1826 (20 µg/mouse) by the vaginal or nasal route. The average number of OVA-specific CD4⁺ T cells was calculated in draining, distal lymph nodes and spleen of mice 57 hours following vaginal or nasal immunization or in naïve mice. Here the ratio between the absolute number of OVA-specific CD4⁺ T cells detected in lymphoid organs of mice immunized by the vaginal or nasal route <i>versus</i> naïve mice is reported.</p

Time course analysis of the OVA-specific CD4⁺ T-cell clonal expansion following vaginal or nasal immunization.

<p>The clonal expansion of adoptively transferred CD4⁺ T cells following vaginal or nasal immunization with OVA plus CpG ODN 1826 was analyzed on days 2.5, 3, 5, and 7 post immunization in draining and distal lymph nodes and spleen. Values reported in y axis indicate the percentage of proliferating antigen-specific T cells. OVA-specific T cells were identified as CD4⁺ CFSE-positive populations, with light scatter properties of lymphocytes. The percentages of proliferating cells are shown on the <i>y</i> axis. Bars represent the means ± standard errors of the means (SEM) of values from nine mice assessed in three separate experiments.</p

A Galton Watson branching process in the discrete time.

<p>A) Schematic representation of the stochastic behavior of the branching process. The <i>i</i> in the circle denotes the cell generation tracked with CFSE. Between two subsequent time-steps a single cell in <i>i</i> generation could take independently by the other cells, three biological decisions: entering in division with probability γ<i>_i</i> and creating two cells of <i>i</i>+1 generation, resting undivided in generation <i>i</i> with probability δ<i>_i</i>, dying with probability α<i>_i</i> = 1−δ<i>_i</i>−γ<i>_i</i>. The formalism allows us to calculate the moments of cell counts. B) Proliferation of antigen-specific CD4⁺ T cells following treatment with isotype control or anti-CD62L antibody. Clonal division was assessed in draining lymph nodes 57 hours following immunization and analyzed as CFSE dilution (x axis) on the gated CFSE⁺ CD4⁺ populations with light scatter properties of lymphocytes. C) Comparison of the experimental relative frequencies obtained from 5 mice at the time-point <i>t</i> = 57 h and the predicted values by the branching process for each cell generation following vaginal immunization.</p

Clonal expansion of OVA-specific CD4⁺ T cells following vaginal or nasal immunization.

<p>OVA-specific proliferation of CD4⁺ T cells was assessed 57 hours following immunization in draining lymph nodes (iliac for vaginally and cervical for nasally immunized mice, respectively) and analyzed as CFSE dilution (x axis) on the gated CFSE⁺ CD4⁺ populations with light scatter properties of lymphocytes. The number of respective cell generation is reported for each peak. Results are representative of two experiments performed with six animals per group.</p

Antigen uptake by DCs following vaginal or nasal immunization.

<p>C57BL/6 mice were immunized by the vaginal or nasal route with OVA Alexa Fluor 647-conjugate (25 µg/mouse) and CpG ODN (20 µg/mouse). Groups of animals were sacrificed 0, 12, 24 and 72 hours after mucosal immunization and iliac lymph nodes (considered draining after vaginal and distal after nasal immunization respectively), cervical lymph nodes (distal after vaginal and draining after nasal immunization respectively) and spleen were collected. DCs positive for fluorescent OVA were analysed by flow cytometry. Shown is the percentage of OVA-Alexa Fluor 647 positive cells among total CD11c⁺ MHC class II⁺ cells.</p

Phenotypic analysis of proliferating antigen-specific CD4⁺ T cells.

<p>The expression of CD45RB, CD69, CD44 and CD62L was analysed as a function of cell divisions on OVA-specific CD4⁺ T cells from draining lymph nodes 72 hours following vaginal or nasal immunization with OVA and CpG ODN 1826. A). Dot plot analysis of CFSE dilution (x axis) <i>versus</i> CD45RB, CD69, CD44 and CD62L expression (y axis) on OVA-specific CD4⁺ T cells in naïve and vaginally or nasally immunised mice. B). Mean fluorescence intensity of CD45RB, CD69, CD44 and CD62L expression per each cell generation of OVA-specific CD4⁺ T cells from naïve mice (open histogram) and vaginally or nasally immunised mice (dark and light gray respectively). Cell divisions (from 0 to 6) are identified by CFSE intensity dilutions. Phenotypic analysis was performed on single mice. Results are representative of two experiments with six animals per group.</p

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<p>The induction and modulation of the immune response to vaccination can be rationally designed by combining different vaccine formulations for priming and boosting. Here, we investigated the impact of heterologous prime-boost approaches on the vaccine-specific cellular and humoral responses specific for a mycobacterial vaccine antigen. C57BL/6 mice were primed with the chimeric vaccine antigen H56 administered alone or with the CAF01 adjuvant, and boosted with H56 alone, or combined with CAF01 or with the squalene-based oil-in-water emulsion adjuvant (o/w squalene). A strong secondary H56-specific CD4⁺ T cell response was recalled by all the booster vaccine formulations when mice had been primed with H56 and CAF01, but not with H56 alone. The polyfunctional nature of T helper cells was analyzed and visualized with the multidimensional flow cytometry FlowSOM software, implemented as a package of the R environment. A similar cytokine profile was detected in groups primed with H56 + CAF01 and boosted with or without adjuvant, except for some clusters of cells expressing high level of IL-17 together with TNF-α, IL-2, and IFN-γ, that were significantly upregulated only in groups boosted with the adjuvants. On the contrary, the comparison between groups primed with or without the adjuvant showed a completely different clusterization of cells, strengthening the impact of the formulation used for primary immunization on the profiling of responding cells. The presence of the CAF01 adjuvant in the priming formulation deeply affected also the secondary humoral response, especially in groups boosted with H56 alone or o/w squalene. In conclusion, the presence of CAF01 adjuvant in the primary immunization is crucial for promoting primary T and B cell responses that can be efficiently reactivated by booster immunization also performed with antigen alone.</p

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<p>The induction and modulation of the immune response to vaccination can be rationally designed by combining different vaccine formulations for priming and boosting. Here, we investigated the impact of heterologous prime-boost approaches on the vaccine-specific cellular and humoral responses specific for a mycobacterial vaccine antigen. C57BL/6 mice were primed with the chimeric vaccine antigen H56 administered alone or with the CAF01 adjuvant, and boosted with H56 alone, or combined with CAF01 or with the squalene-based oil-in-water emulsion adjuvant (o/w squalene). A strong secondary H56-specific CD4⁺ T cell response was recalled by all the booster vaccine formulations when mice had been primed with H56 and CAF01, but not with H56 alone. The polyfunctional nature of T helper cells was analyzed and visualized with the multidimensional flow cytometry FlowSOM software, implemented as a package of the R environment. A similar cytokine profile was detected in groups primed with H56 + CAF01 and boosted with or without adjuvant, except for some clusters of cells expressing high level of IL-17 together with TNF-α, IL-2, and IFN-γ, that were significantly upregulated only in groups boosted with the adjuvants. On the contrary, the comparison between groups primed with or without the adjuvant showed a completely different clusterization of cells, strengthening the impact of the formulation used for primary immunization on the profiling of responding cells. The presence of the CAF01 adjuvant in the priming formulation deeply affected also the secondary humoral response, especially in groups boosted with H56 alone or o/w squalene. In conclusion, the presence of CAF01 adjuvant in the primary immunization is crucial for promoting primary T and B cell responses that can be efficiently reactivated by booster immunization also performed with antigen alone.</p