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. 2009 Dec 7;4(12):e8159.
doi: 10.1371/journal.pone.0008159.

Original encounter with antigen determines antigen-presenting cell imprinting of the quality of the immune response in mice

Valérie Abadie  1 , Olivia Bonduelle, Darragh Duffy, Christophe Parizot, Bernard Verrier, Béhazine Combadière
Affiliations

Original encounter with antigen determines antigen-presenting cell imprinting of the quality of the immune response in mice

Valérie Abadie et al. PLoS One. .

Abstract

Background: Obtaining a certain multi-functionality of cellular immunity for the control of infectious diseases is a burning question in immunology and in vaccine design. Early events, including antigen shuttling to secondary lymphoid organs and recruitment of innate immune cells for adaptive immune response, determine host responsiveness to antigens. However, the sequence of these events and their impact on the quality of the immune response remain to be elucidated. Here, we chose to study Modified Vaccinia virus Ankara (MVA) which is now replacing live Smallpox vaccines and is proposed as an attenuated vector for vaccination strategies against infectious diseases.

Methodology/principal findings: We analyzed in vivo mechanisms triggered following intradermal (i.d.) and intramuscular (i.m.) Modified Vaccinia virus Ankara (MVA) administration. We demonstrated significant differences in the antigen shuttling to lymphoid organs by macrophages (MPhis), myeloid dendritic cells (DCs), and neutrophils (PMNs). MVA i.d. administration resulted in better antigen distribution and more sustained antigen-presenting cells (APCs) recruitment into draining lymph nodes than with i.m. administration. These APCs, which comprise both DCs and MPhis, were differentially involved in T cell priming and shaped remarkably the quality of cytokine-producing virus-specific T cells according to the entry route of MVA.

Conclusions/significance: This study improves our understanding of the mechanisms of antigen delivery and their consequences on the quality of immune responses and provides new insights for vaccine development.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Magnitude and quality of MVA–specific T cell responses following i.d. and i.m. antigen delivery.
(A) C57BL/6 mice were immunized i.d. or i.m. with 5.106 PFU of MVA or saline buffer as a control. Seven days after inoculation, IFN-γ–producing DLNs T cells were quantified by ELISPOT assay. (B) Mice received 5.106 PFU of MVA i.d. or i.m.. MVA–specific T cell responses in the DLNs on day 7 were measured with an intracellular cytokine staining assay. The magnitude of MVA–specific CD4+ T cells producing cytokines is shown. (C) The functional composition of the CD4+ T cell response is presented. The pie charts present the mean frequencies of the CD4+ T cells positive for the indicated cytokines. TP, triple producers; DP, double producers; and SP, single producers. (D) The magnitude of the total CD8+ T cell response is presented. (E) The cytokine coexpression profile of MVA–specific CD8+ T cells is shown (as in C). * p<0.05, *** p<0.0001. Data are representative of two (B–E) or three (A) independent experiments (n = 10 individual mice for each group).
Figure 2
Figure 2. Persistence of rMVA–egfp particles and phenotype of cells infiltrating the inoculation sites after i.d. and i.m. antigen delivery.
(A) Fibered confocal fluorescence microscopy of skin and muscle from 4 to 48 h after inoculation of 5.106 PFU of rMVA–egfp or saline buffer as a control by the i.d. and i.m. routes. (B) Flow cytometric analyses of skin-infiltrating cells. Ly-6C+Ly-6G+ PMNs were gated on CD11bhi cells. Mo/MΦ (F4/80+CD11c−) and DC (CD11c+) subpopulations were gated on CD11b+ cells. (C) Results are expressed as the percentage of CD11b+ cells isolated from ears at 4, 24, and 48 h after injection of 5.106 PFU of MVA. PMNs are expressed as the percentage of CD11bhi. Values at point 0 correspond to the percentage of positive cells extracted from ears of control mice. Each percentage represents the mean values obtained for three mice per group. *P<0.05, **P<0.01. This experiment was performed twice with similar results. (D) Similar representative flow cytometric analyses of muscle infiltrating-cells. The percentages of cells for each subpopulation are indicated in the quadrant. (E) CD11b+ cells present in muscle were analyzed for expression of both Ly-6C and Ly-6G, and for expression of CD11c and F4/80 by flow cytometry. Results are expressed as the percentage of CD11b+ cells (except PMNs that are all CD11bhi) isolated from muscle at 4, 24, and 48 h after injection of 5.106 PFU of MVA. Values at point 0 correspond to the percentage of positive cells extracted from control mice. Each percentage represents the mean values obtained for three mice per group. *P<0.05, **P<0.01, ***P<0.0001. This experiment was performed twice with similar results.
Figure 3
Figure 3. Differential biodistribution of rMVA–egfp particles after i.d. and i.m. antigen delivery.
(A) At 4 to 48 h after i.d. and i.m. injection of 5.106 PFU of rMVA–egfp, DLN suspensions were analyzed for the presence of eGFP+ cells by flow cytometry. Each histogram bar represents the mean percentage for three independent experiments (n = 3 mice per group and per time point for each experiment). (B) Four h after rMVA–egfp inoculation, eGFP+ ADLN cells were analyzed by flow cytometry and identified as CD11b+F4/80+ M√s, CD11b+CD11c+ mDCs, and Ly-6G+Ly-6C+ PMNs. Pie chart represents the proportion of each eGFP+ cell subset for three independent experiments (n = 3 mice per group and per time point for each experiment). (C) Immunohistochemistry of DLN samples stained with anti-B220 (green) and anti-vaccinia (red) to localize MVA–infected cells in the node. Slides were analyzed under an Olympus fluorescence microscope with a UPlanFL N 10×/0.30 lens.
Figure 4
Figure 4. Cell recruitment into DLNs according to route of MVA administration.
At 4 to 48 h after i.d. (black bars) or i.m. (grey bars) MVA inoculation, leukocyte subpopulations present in DLN suspensions were characterized by flow cytometry (A, PMNs; B MΦs; C mDCs; D lymphoid DCs; E pDCs). Each histogram bar represents the mean percentage for three independent experiments (n = 3 mice per group and per time point for each experiment). *P<0.05, **P<0.01, ***P<0.0001.
Figure 5
Figure 5. Differential involvement of myeloid cells in T cell priming according to antigen delivery route.
(A) Number of IFN-γ positive spots produced by CD4+ (open symbols) and CD8+ (black symbols) T cells after incubation with the indicated number of DCs (left panel) or MΦs (right panel) isolated from Mock (⋄, ♦), ID (○, •), and IM (□, ▪) MVA–inoculated DLNs on day 2. Each data point represents triplicates from three independent experiments. (B) 48 h after MVA i.d. administration, a section cut from frozen ADLN was immunostained with anti-F4/80 (blue), anti-CD3 (red), and anti-vaccinia (green) to localize interactions between MVA–infected F4/80+cells and T cells in the node. Slides were analyzed under a Leica Sp2 AOBS confocal microscope with a 63x/1.4 NA lens. (C) CD11c+ DCs from DLNs on day 2 were sorted into CD8α+ DCs and CD11b+ DCs. DC subsets were cocultured with CD4+ (open symbols) and CD8+ (black symbols) naïve T cells, and tested in duplicate wells for their ability to induce IFN-γ production by T cells. Data are representative of three independent experiments.
Figure 6
Figure 6. Myeloid APCs qualitatively shape MVA–specific T cell responses.
(A) F4/80+ cells were isolated from MVA i.d. inoculated mice on day 2 or from control mice, and transferred intravenously into i.m. MVA–inoculated mice the same day. DLNs were isolated from recipient mice on day 7 to determine the cytokine coexpression profile of T cells. (B) Mice were inoculated with MVA i.d. or i.m. and received intraveneously F4/80+ cells isolated from MVA i.d. inoculated mice on day 2 or from control mice. Seven days after inoculation, IFN-γ–producing DLNs T cells were quantified by ELISPOT assay. Representative data from two independent experiments are presented (n = 10 individual mice for each group). * p<0.05, *** p<0.0001, NS Non Significant. (C) DLNs from mice inoculated with MVA i.d. or i.m. and from MVA i.m. immunized mice that received F4/80+ cells were analyzed on day 7 for their MVA–specific T cell response. DLN cells were stimulated in vitro for 12 h with MVA antigen. Cell surfaces were stained with anti-CD8α and anti-CD4, and then cells were intracellularly stained with anti-IFN-γ, anti-TNF-α, and anti-IL-2. Cytokine co-expression patterns are represented as the mean frequencies of the T cell subsets positive for the indicated cytokines. Representative data from two independent experiments are presented (n = 10 individual mice for each group).
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