Immunization with Radiation-Attenuated Plasmodium berghei Sporozoites Induces Liver cCD8α+DC that Activate CD8+T Cells against Liver-Stage Malaria

Immunization with radiation (γ)-attenuated Plasmodia sporozoites (γ-spz) confers sterile and long-lasting immunity against malaria liver-stage infection. In the P. berghei γ-spz model, protection is linked to liver CD8+ T cells that express an effector/memory (TEM) phenotype, (CD44hiCD45RBloCD62Llo ), and produce IFN-γ. However, neither the antigen presenting cells (APC) that activate these CD8+ TEM cells nor the site of their induction have been fully investigated. Because conventional (c)CD8α+ DC (a subset of CD11c+ DC) are considered the major inducers of CD8+ T cells, in this study we focused primarily on cCD8α+ DC from livers of mice immunized with Pb γ-spz and asked whether the cCD8α+ DC might be involved in the activation of CD8+ TEM cells. We demonstrate that multiple exposures of mice to Pb γ-spz lead to a progressive and nearly concurrent accumulation in the liver but not the spleen of both the CD11c+NK1.1− DC and CD8+ TEM cells. Upon adoptive transfer, liver CD11c+NK1.1− DC from Pb γ-spz-immunized mice induced protective immunity against sporozoite challenge. Moreover, in an in vitro system, liver cCD8α+ DC induced naïve CD8+ T cells to express the CD8+ TEM phenotype and to secrete IFN-γ. The in vitro induction of functional CD8+ TEM cells by cCD8α+ DC was inhibited by anti-MHC class I and anti-IL-12 mAbs. These data suggest that liver cCD8α+ DC present liver-stage antigens to activate CD8+ TEM cells, the pre-eminent effectors against pre-erythrocytic malaria. These results provide important implications towards a design of anti-malaria vaccines

Fine epitope signature of antibody neutralization breadth at the HIV-1 envelope CD4-binding site

Major advances in donor identification, antigen probe design, and experimental methods to clone pathogen-specific antibodies have led to an exponential growth in the number of newly characterized broadly neutralizing antibodies (bnAbs) that recognize the HIV-1 envelope glycoprotein. Characterization of these bnAbs has defined new epitopes and novel modes of recognition that can result in potent neutralization of HIV-1. However, the translation of envelope recognition profiles in biophysical assays into an understanding of in vivo activity has lagged behind, and identification of subjects and mAbs with potent antiviral activity has remained reliant on empirical evaluation of neutralization potency and breadth. To begin to address this discrepancy between recombinant protein recognition and virus neutralization, we studied the fine epitope specificity of a panel of CD4-binding site (CD4bs) antibodies to define the molecular recognition features of functionally potent humoral responses targeting the HIV-1 envelope site bound by CD4. Whereas previous studies have used neutralization data and machine-learning methods to provide epitope maps, here, this approach was reversed, demonstrating that simple binding assays of fine epitope specificity can prospectively identify broadly neutralizing CD4bs–specific mAbs. Building on this result, we show that epitope mapping and prediction of neutralization breadth can also be accomplished in the assessment of polyclonal serum responses. Thus, this study identifies a set of CD4bs bnAb signature amino acid residues and demonstrates that sensitivity to mutations at signature positions is sufficient to predict neutralization breadth of polyclonal sera with a high degree of accuracy across cohorts and across clades

Cell Type Specificity and Host Genetic Polymorphisms Influence Antibody-Dependent Enhancement of Dengue Virus Infection ▿

Antibody-dependent enhancement (ADE) is implicated in severe, usually secondary, dengue virus (DV) infections. Preexisting heterotypic antibodies, via their Fc-gamma receptor (FcγR) interactions, may increase disease severity through enhanced target cell infection. Greater numbers of infected target cells may contribute to higher viremia and excess cytokine levels often observed in severe disease. Monocytes, macrophages, and immature and mature dendritic cells (DC) are considered major cellular targets of DV. Apheresis of multiple donors allowed isolation of autologous primary myeloid target cell types for head-to-head comparison of infection rates, viral output, and cytokine production under direct infection (without antibody) or ADE conditions (with antibody). All studied cell types except immature DC supported ADE. All cells undergoing ADE secreted proinflammatory cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) at enhancement titers, but distinct cell-type-specific patterns were observed for other relevant proteins (alpha/beta interferon [IFN-α/β] and IL-10). Macrophages produced type I interferons (IFN-α/β) that were modulated by ADE. Mature DC mainly secreted IFN-β. Interestingly, only monocytes secreted IL-10, and only upon antibody-enhanced infection. While ADE infection rates were remarkably consistent in monocytes (10 to 15%) across donors, IL-10 protein levels varied according to previously described regulatory single nucleotide polymorphisms (SNPs) in the IL-10 promoter region. The homozygous GCC haplotype was associated with high-level IL-10 secretion, while the ACC and ATA haplotypes produced intermediate and low levels of IL-10, respectively. Our data suggest that ADE effects are cell type specific, are influenced by host genetics, and, depending on relative infection rates, may further contribute to the complexity of DV pathogenesis

Liver CD11c⁺NK1.1⁻ cells from <i>Pb</i>γ-spz-immunized mice induce protection against infectious sporozoites.

<p>(A) 1×10⁶ CD11c⁺NK1.1⁻ cells, isolated from the livers of <i>Pb</i>γ-spz-fully-immunized mice 6 days after the 3° immunization, were adoptively transferred (i.v.) into naïve recipients. Seven days later the adoptively transferred recipients (n = 3) as well as naïve infectivity control mice (n = 3) were challenged with 10K infectious sporozoites. The results show the level of parasitemia assessed in each individual mouse and expressed as the mean of parasitemia per mice/group at 4, 7 and 10 days following challenge. (B) 1×10⁶ hepatic CD11c⁺NK1.1⁻ cells, purified as described in (A) were isolated from <i>Pb</i>γ-spz-immunized-challenged mice and from naïve-challenged mice and adoptively transferred into naïve syngeneic recipients (n = 13) that were challenged 7 days later with either 250 (n = 6) or 1000 (n = 7) infectious sporozoites. The protected group (250 sporozoites) were re-challenged 60 days later along with another group of naïve infectivity control mice (n = 3). <i>Pb</i> γ-spz-immunized mice (n = 3), used as positive controls, were sterily protected at challenge and re-challenge. Parasitemia and survival were evaluated from day 2 post-challenge.</p

Liver cCD8α⁺ DC are more efficient than splenic cCD8α⁺ DC in inducing differentiation of and IFN-γ production by CD8⁺ T cells.

<p>Differentiation and induction of CD8⁺ T cell function is MHC class I- and IL-12 dependent. HMNC or splenic MNC were prepared after the 3° immunization as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#pone-0005075-g003" target="_blank">Fig. 3</a>. CD8α⁺ DC were purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#pone-0005075-g001" target="_blank">Figs 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#pone-0005075-g003" target="_blank">3</a>. Splenic (A and C) or hepatic (B and D) cCD8α⁺ DC were co-cultured for 4 days either alone or with purified CD8⁺ T cells from the livers (open bars) or spleen (filled bars) of naïve mice. Cells were harvested, stained with the appropriate mAb and analyzed by flow cytometry. Culture supernatants were analyzed for IFNγ by ELISA. (A and B) Results show the mean % of CD8⁺ T_EM in the gated CD3⁺CD8⁺ T cell population and (C and D) the amount of IFNγ in the culture supernatant. Data are representative of two individual experiments. (E and F) Liver cCD8α⁺ DC were co-cultured with naïve splenic CD8⁺ T cells in the presence or absence of anti-IL-12 (clone C17.1) and/or anti-MHC class I (clone 28-8-6) mAbs for 4 days. (E) Results show the mean % of CD8⁺ T_EM in the gated CD3⁺CD8⁺ T cell population and (F).the amount of IFNγ. Data is representative of two individual experiments.</p

Numbers of splenic CD11c⁺NK1.1⁻ DC and cCD8α⁺DC in naïve and <i>Pb</i>γ-spz-immunized mice^a

a<p>SMNC were isolated from individual C57BL/6 mice before and after prime and boost immunizations with <i>Pb</i> γ−spz. Cells were stained with a cocktail of mAbs for identification of CD11c⁺NK1.1⁻ DC and cCD8α⁺DC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#s4" target="_blank">Materials and Methods</a> and analyzed by flow cytometry. Data represent the mean±SD of the number of cells of three mice per group and are representative of three independent experiments.</p

CD8β mRNA is absent in purified liver cCD8α⁺DC.

<p>HMNC were pooled from livers of <i>Pb</i>γ-spz-fully immunized mice 6 days after the 3° immunization and were incubated with a cocktail of biotinylated microbeads to deplete T cells, B cells, NK cells, granulocytes and macrophages as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#s4" target="_blank">Materials and Methods</a>. cCD8α⁺DC were further isolated from the enriched CD11c⁺NK1.1⁻ population by positive magnetic selection as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#s4" target="_blank">Materials and Methods</a>. Staining with anti-CD8α, CD3, CD8b and CD11c was performed on permeabilized cells to reveal both the surface and the intracellular presence of these markers. (A) Dot plot show the relative % of T cells (red) and cCD8α⁺DC (blue) within the purified cCD8α⁺DC population. (B) Two-step quantitative real-time PCR was performed on RNA isolated from magnetic-bead purified liver cCD8α⁺DC (described in A). Ratio of CD8β/CD8α gene expression was calculated using standard curves for each gene. Measurements were done in duplicates in wells containing 1000, 1, 0.1 cells/well, or non-template control (NTC). Representative results of one out of two experiments are shown. (C) Histogram plots show expression of DEC 205, I-A^b and costimulatory molecules on the cCD8α⁺DC population (black lines). Grey lines represent staining of the isotype controls.</p

CD11c⁺NK1.1⁻ DC are constitutively present in the spleens of naïve mice but do not substantially increase following immunization.

<p>(A) CD11c⁺NK1.1⁻DC in <i>Pb</i>γ−spz-immune splenic MNC were identified according to the procedure described for HMNC in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#pone-0005075-g001" target="_blank">Fig 1</a>. After exclusion of T cells, CD11c⁺ cells were segregated into CD11c⁺NK1.1⁺ and CD11c⁺NK1.1⁻ populations. Splenic mononuclear cells were isolated from individual mice before and after prime and boost immunizations with <i>Pb</i>γ−spz or uninfected mosquito debris (sham). Cells were stained with a cocktail of mAbs and NK1.1⁻ DC and CD8⁺ T_EM cells were identified by flow cytometry. (B) Panels show representative contour plots of DC in the spleens of naïve mice and in spleens of mice 6 days after either the 1°, 2° and 3° immunizations and in sham-immunized mice 6 days after the 3° immunization. The percentages of the CD11c⁺NK1.1⁻ DC in relation to the total SMNC/spleen for each representative mouse are indicated in each panel. (C) The results show the mean percentage ±SD of CD11c⁺NK1.1⁻ DC in total spleens of naïve mice, <i>Pb</i> γ−spz-immunized mice at day 6 after each immunization and in sham-immunized mice at day 6 after the 3° immunization. (D) Panels show representative contour plots of CD8⁺ T_CM cells and CD8⁺ T_EM cells in the spleens of naïve mice as well as of <i>Pb</i> γ−spz-immunized mice and sham-immunized mice at the same time-points described in (B). The numbers indicate the percentages of the T_CM and T_EM cells in the gated splenic CD3⁺CD8⁺ T cell population. (E) The results show the mean percentage ±SD of CD8⁺T_EM in the gated splenic CD3⁺CD8⁺ T cell population of naïve and immunized mice at day 6 after each immunization. Contour plots and bar graphs are representative of three individual mice per group in three independent experiments.</p

Hepatic cCD8α⁺ DC from <i>Pb</i>γ-spz-immunized and challenged mice mediate <i>in vitro</i> activation of naïve CD8⁺ T cells.

<p>HMNC were pooled from livers of <i>Pb</i>γ-spz-fully immunized and challenged mice (n = 18) and were incubated with a cocktail of biotinylated microbeads to deplete T cells, B cells, NK cells, granulocytes and macrophages as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#s4" target="_blank">Materials and Methods</a>. DC subpopulations were further isolated from the enriched CD11c⁺NK1.1⁻ population by positive magnetic selection for cCD8α⁺DC and pDC and by negative selection for CD8α⁻DC, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005075#s4" target="_blank">Materials and Methods</a>. (A) Dot plots show the relative % of T cells and NK1.1 cells within the purified cCD8α⁺DC population. (B) CD8⁺ T cells were isolated from the spleens of naïve mice using magnetic beads and labeled with 2 µM CFSE. Dot plots show the gating scheme for the analysis of CFSE-labeled CD3⁺CD8⁺T cells for expression of the CD45RB^loCD44^hi phenotype. (C) cCD8α⁺ DC, cCD8α⁻ DC and pDC were purified from CD11c⁺NK1.1⁻ DC isolated from pooled livers 3 days after the challenge of <i>Pb</i>γ-spz-immunized mice. Liver DC subpopulations and CFSE-labeled splenic CD8⁺ T cells were co-cultured at a ratio of 1 DC : 2 CD8⁺T cells for 4 days. Cells were harvested, stained with a cocktail of mAbs and the % of CD3⁺CD8⁺CD45RB^loCD44^hi cells (CD8⁺ T_EM) was analyzed by flow cytometry. Results show contour plots of CD8⁺T cells co-cultured with cDC and pDC subpopulations. (D) Bar graphs show the percentage of CD8⁺ T_EM in the gated CD3⁺CD8⁺ T cell population after co-culture with cCD8α⁺ DC, cCD8α⁻ DC and pDC each isolated from <i>Pb</i>γ-spz-immunized-challenged mice.</p