Immunization with radiation-attenuated Plasmodium berghei sporozoites induces liver cCD8alpha+DC that activate CD8+T cells against liver-stage malaria

Abstract

Immunization with radiation (gamma)-attenuated Plasmodia sporozoites (gamma-spz) confers sterile and long-lasting immunity against malaria liver-stage infection. In the P. berghei gamma-spz model, protection is linked to liver CD8+ T cells that express an effector/memory (T(EM)) phenotype, (CD44(hi)CD45RB(lo)CD62L(lo)), and produce IFN-gamma. However, neither the antigen presenting cells (APC) that activate these CD8+ T(EM) cells nor the site of their induction have been fully investigated. Because conventional (c)CD8alpha+ DC (a subset of CD11c+ DC) are considered the major inducers of CD8+ T cells, in this study we focused primarily on cCD8alpha+ DC from livers of mice immunized with Pb gamma-spz and asked whether the cCD8alpha+ DC might be involved in the activation of CD8+ T(EM) cells. We demonstrate that multiple exposures of mice to Pb gamma-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+ T(EM) cells. Upon adoptive transfer, liver CD11c+NK1.1(-) DC from Pb gamma-spz-immunized mice induced protective immunity against sporozoite challenge. Moreover, in an in vitro system, liver cCD8alpha(+) DC induced naïve CD8+ T cells to express the CD8+ T(EM) phenotype and to secrete IFN-gamma. The in vitro induction of functional CD8+ T(EM) cells by cCD8alpha+ DC was inhibited by anti-MHC class I and anti-IL-12 mAbs. These data suggest that liver cCD8alpha+ DC present liver-stage antigens to activate CD8+ T(EM) cells, the pre-eminent effectors against pre-erythrocytic malaria. These results provide important implications towards a design of anti-malaria vaccines.

Figure 1. Both CD11c⁺NK1.1⁻ DC and CD8⁺ T_EM cells (CD44^hiCD45RB^lo) accumulate in the livers of Pb γ−spz-immunized mice.

(A) HMNC were identified by FSC/SSC and after exclusion of CD3/CD8β⁺ T cells, CD11c⁺ cells are segregated into CD11c⁺NK1.1⁺ and CD11c⁺NK1.1⁻ populations. (B) Dot plots show the level or absence of B cells, T cells and macrophages within the gated CD11c⁺NK1.1⁻ population. (C) HMNC were isolated from individual C57BL/6 mice before and after prime and boost immunizations with Pb γ−spz or uninfected mosquito debris (sham). Cells were stained with a cocktail of mAbs and analyzed by flow cytometry for identification of CD11c⁺NK1.1⁻ DC and CD8⁺ T_EM. Panels show representative contour plots of CD11c⁺NK1.1⁻ DC in the livers of naïve mice and livers of mice 6 days after the primary (1°), secondary (2°) and tertiary (3°) immunizations. The percentages of the CD11c⁺NK1.1⁻ DC in relation to the total HMNC/liver for each representative mouse are indicated in each panel. (D) The results show the mean %±SD of CD11c⁺NK1.1⁻ DC in total HMNC of naïve, Pb γ−spz-immunized and sham-immunized mice. (E) Panels show representative contour plots of CD8⁺ T_CM cells (CD44^hiCD45RB^hi ) and CD8⁺ T_EM cells (CD44^hiCD45RB^lo) in the livers of naïve, Pb γ−spz-immunized and sham-immunized mice at the time-points indicated in (C). The percentages of the CD8+ T_EM and CD8+ T_CM cells in relation to the gated liver CD3⁺CD8⁺ T cells are indicated in the panels. (F) The results show the mean %±SD of CD8⁺ T_EM in the gated liver CD3⁺CD8⁺ T cells in naïve, Pb γ−spz-immunized and sham-immunized mice. The results are representative of three individual mice per group from three independent experiments.

Figure 2. CD11c⁺NK1.1⁻ DC are constitutively present in the spleens of naïve mice but do not substantially increase following immunization.

(A) CD11c⁺NK1.1⁻DC in Pbγ−spz-immune splenic MNC were identified according to the procedure described for HMNC in Fig 1. 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 Pbγ−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, Pb 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 Pb 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.

Figure 3. cCD8α⁺ DC, relatively absent in the livers of naïve mice, are induced after prime-boost immunizations with Pbγ-spz.

(A) Hepatic CD11c⁺NK1.1⁻ DC (see Fig. 1) were stained with Abs specific for B220 and CD8α and analyzed by flow cytometry to reveal 3 subpopulations of DC . (B) HMNC isolated from livers of Pbγ-spz-fully immunized mice 6 days after the 3° immunization were incubated with a cocktail of biotinylated microbeads to deplete T cells, B cells, NK cells, granulocytes and macrophages as described in Materials and Methods. 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 Materials and Methods. Panels show photographs at 100× of Giemsa stained cytospins of cCD8α⁺DC, cCD8α⁻DC and pDC. (C and E) HMNC and (D and F) splenic MNC were isolated from individual naïve mice and from individual mice at 6 days after 1°, 2° and 3° immunizations with Pb γ−spz and after 3° immunizations with uninfected mosquito debris (sham). Cells were stained with a cocktail of mAbs for identification of DC subpopulations as described in (A). Bar graphs show the mean % ± SD of the cCD8α⁺DC (C and D) and cCD8α⁻DC (E and F) in the gated CD11c⁺NK1.1⁻ cells. Data are representative of three individual mice per group at each time-point in two independent experiments.

Figure 4. CD8β mRNA is absent in purified liver cCD8α⁺DC.

HMNC were pooled from livers of Pbγ-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 Materials and Methods. cCD8α⁺DC were further isolated from the enriched CD11c⁺NK1.1⁻ population by positive magnetic selection as described in Materials and Methods. 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.

Figure 5. Hepatic cCD8α⁺ DC from Pbγ-spz-immunized and Pbγ-spz-immunized and challenged mice mediate in vitro activation of naïve CD8⁺ T cells.

HMNC were pooled from livers of Pbγ-spz-fully immunized mice (n = 20) 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 Materials and Methods. 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 Materials and Methods. (A) Dot plots show the relative % of T cells and NK1.1 cells within the gated CD11c⁺CD8α⁺DC. (B) cCD8α⁺ DC, cCD8α⁻ DC and pDC were purified from CD11c⁺NK1.1⁻ DC isolated from pooled livers either 6 days after 3° immunization or 3 days after the challenge of Pbγ-spz-immunized as well as naïve mice . CD8⁺ T cells were isolated from the spleens of naïve mice using magnetic beads. Liver DC subpopulations and splenic CD8⁺ T cells were co-cultured at a ratio of 1 DC : 2 CD8⁺T cells for 4 days. Culture supernatants were harvested and analyzed for IL-10 and IL-12p40 by ELISA. 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 representative contour plots of CD8⁺T cells co-cultured with cDC and pDC subpopulations from Pb γ-spz-immunized-challenged mice. Bar graphs show the mean percentage±SD of CD8⁺ T_EM in the gated CD3⁺CD8⁺ T cell population after co-culture with (C) cCD8α⁺ DC, (D) cCD8α⁻ DC and (E) pDC each isolated from Pbγ-spz-fully-immunized, Pbγ-spz-immunized-challenged and naïve-challenged mice. The experiments were performed twice yielding similar results. (F) IL-12p40 production in the culture supernatants of CD8⁺ T cells with the liver DC populations from the Pbγ-spz-immunized mice. The results expressed as pg/ml (mean±SD) represent data from two representative experiments. (G) cCD8α⁺DC were isolated from pooled livers of Pbγ-spz-fully-immunized mice, Pbγ-spz-immunized and challenged mice or naïve-challenged mice and co-cultured with purified naïve splenic CD8⁺ T cells for 4 days. Supernatants were harvested and analyzed for IL-12p40 and IL-10 by ELISA.

Figure 6. Hepatic cCD8α⁺ DC from Pbγ-spz-immunized and challenged mice mediate in vitro activation of naïve CD8⁺ T cells.

HMNC were pooled from livers of Pbγ-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 Materials and Methods. 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 Materials and Methods. (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 Pbγ-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 Pbγ-spz-immunized-challenged mice.

Figure 7. Liver cCD8α⁺ DC are more efficient than splenic cCD8α⁺ DC in inducing differentiation of and IFN-γ production by CD8⁺ T cells.

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 Fig. 3. CD8α⁺ DC were purified as described in Figs 1 and 3. 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.

Figure 8. Liver CD11c⁺NK1.1⁻ cells from Pbγ-spz-immunized mice induce protection against infectious sporozoites.

(A) 1×10⁶ CD11c⁺NK1.1⁻ cells, isolated from the livers of Pbγ-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 Pbγ-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). Pb 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.