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Identification of targets of CD8⁺ T cell responses to malaria liver stages by genome-wide epitope profiling.

Hafalla JC, Bauza K, Friesen J, Gonzalez-Aseguinolaza G, Hill AV, Matuschewski K - PLoS Pathog. (2013)

Bottom Line: While both PbS20₃₁₈ and PbTRAP₁₃₀ elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice, only PbTRAP₁₃₀-specific CD8⁺ T cells exhibit in vivo cytotoxicity.We conclude that PbTRAP is an immunodominant antigen during liver-stage infection.Together, our results underscore the presence of CD8⁺ T cells with divergent potencies against distinct Plasmodium liver-stage epitopes.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom. Julius.Hafalla@lshtm.ac.uk

ABSTRACT
CD8⁺ T cells mediate immunity against Plasmodium liver stages. However, the paucity of parasite-specific epitopes of CD8⁺ T cells has limited our current understanding of the mechanisms influencing the generation, maintenance and efficiency of these responses. To identify antigenic epitopes in a stringent murine malaria immunisation model, we performed a systematic profiling of H(2b)-restricted peptides predicted from genome-wide analysis. We describe the identification of Plasmodium berghei (Pb) sporozoite-specific gene 20 (S20)- and thrombospondin-related adhesive protein (TRAP)-derived peptides, termed PbS20₃₁₈ and PbTRAP₁₃₀ respectively, as targets of CD8⁺ T cells from C57BL/6 mice vaccinated by whole parasite strategies known to protect against sporozoite challenge. While both PbS20₃₁₈ and PbTRAP₁₃₀ elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice, only PbTRAP₁₃₀-specific CD8⁺ T cells exhibit in vivo cytotoxicity. Moreover, PbTRAP₁₃₀-specific, but not PbS20₃₁₈-specific, CD8⁺ T cells significantly contribute to inhibition of parasite development. Prime/boost vaccination with PbTRAP demonstrates CD8⁺ T cell-dependent efficacy against sporozoite challenge. We conclude that PbTRAP is an immunodominant antigen during liver-stage infection. Together, our results underscore the presence of CD8⁺ T cells with divergent potencies against distinct Plasmodium liver-stage epitopes. Our identification of antigen-specific CD8⁺ T cells will allow interrogation of the development of immune responses against malaria liver stages.

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Vaccination with PbTRAP elicits high levels of PbTRAP130-specific CD8+ T cell responses.(A) Schematic diagram of methodology. B6 mice were immunised with Ad PbTRAP (or Ad vector control) and boosted eight weeks later with M PbTRAP (or M vector control). Two weeks later, the frequencies and quality of PbTRAP-specific CD8+ T cells on peripheral blood leukocytes were quantified by ICS. (B) Flow cytometry plots of IFN-γ co-staining with markers of effector and effector memory phenotypes (CD62Llo and CD11ahi). Cells were either not stimulated (no peptide) or stimulated with PbTRAP130 (5 µg/ml) or with a pool of 20-mer peptides overlapping by 10 amino acids spanning PbTRAP (PbTRAPpool: final concentration is 5 µg/mL for each peptide). (C) Flow cytometry plots of IFN-γ co-staining with other effector cytokines, TNF and IL-2. Figures are representative data from one of 4 experiments with 5 mice/group/experiment. (D) Polyfunctional analysis of cytokine secretion based on (C). Bars represent the mean value of the % of PbTRAP130-specific CD8+ cells. Individual data are also shown (blue diamond for PbTRAP130 and red diamond for PbS20318). Figures B, C and D are representative data from one of at least 4 experiments with 4–5 mice/experiment.
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ppat-1003303-g006: Vaccination with PbTRAP elicits high levels of PbTRAP130-specific CD8+ T cell responses.(A) Schematic diagram of methodology. B6 mice were immunised with Ad PbTRAP (or Ad vector control) and boosted eight weeks later with M PbTRAP (or M vector control). Two weeks later, the frequencies and quality of PbTRAP-specific CD8+ T cells on peripheral blood leukocytes were quantified by ICS. (B) Flow cytometry plots of IFN-γ co-staining with markers of effector and effector memory phenotypes (CD62Llo and CD11ahi). Cells were either not stimulated (no peptide) or stimulated with PbTRAP130 (5 µg/ml) or with a pool of 20-mer peptides overlapping by 10 amino acids spanning PbTRAP (PbTRAPpool: final concentration is 5 µg/mL for each peptide). (C) Flow cytometry plots of IFN-γ co-staining with other effector cytokines, TNF and IL-2. Figures are representative data from one of 4 experiments with 5 mice/group/experiment. (D) Polyfunctional analysis of cytokine secretion based on (C). Bars represent the mean value of the % of PbTRAP130-specific CD8+ cells. Individual data are also shown (blue diamond for PbTRAP130 and red diamond for PbS20318). Figures B, C and D are representative data from one of at least 4 experiments with 4–5 mice/experiment.

Mentions: Adenovirus chimpanzee serotype 63 (Ad) and Modified Vaccinia Ankara (M) vaccines expressing a mammalian codon-optimised fragment of PbTRAP were generated. Referred to as Ad-M PbTRAP combination vaccine, they were used to vaccinate B6 mice with an 8-week resting period between priming and boosting (Figure 6A). Since the recombinant PbTRAP vaccines contained sequences in addition to PbTRAP130, we used both PbTRAP130 and a pool of overlapping peptides to PbTRAP (PbTRAPpool) in stimulation assays to verify if other PbTRAP-derived sequences were able to induce T cell responses. As shown in Figure 6B–D, the frequencies of IFN-γ secreting CD8+ T cells were indistinguishable between the two stimulations; approximately ∼17% (range: 14%–50%) of the total CD8+ T cells produce IFN-γ specific for PbTRAP130 or PbTRAPpool. Consistent with the induction of effector responses, these activated IFN-γ-producing cells coincided with the modulation of the corresponding expression markers CD62Llo and CD11ahi (Figure 6B). Polyfunctional analysis revealed that the responses were predominantly IFN-γ positive cells and IFN-γ/TNF double positive cells (Figures 6C,D). This intracellular cytokine pattern of PbTRAP130-specific CD8+ T cells were similar to that measured by immunizations with irradiated sporozoites (Figure S2). Cells stimulated with no peptide did not respond to either PbTRAP130 or PbTRAPpool. No cytokine-producing CD4+ T cells were detected following PbTRAP130 or PbTRAPpool stimulation. These results demonstrated the induction PbTRAP130-specific CD8+ T cells by vaccination and confirmed that PbTRAP130 is the only T cell epitope in PbTRAP in this infection model.


Identification of targets of CD8⁺ T cell responses to malaria liver stages by genome-wide epitope profiling.

Hafalla JC, Bauza K, Friesen J, Gonzalez-Aseguinolaza G, Hill AV, Matuschewski K - PLoS Pathog. (2013)

Vaccination with PbTRAP elicits high levels of PbTRAP130-specific CD8+ T cell responses.(A) Schematic diagram of methodology. B6 mice were immunised with Ad PbTRAP (or Ad vector control) and boosted eight weeks later with M PbTRAP (or M vector control). Two weeks later, the frequencies and quality of PbTRAP-specific CD8+ T cells on peripheral blood leukocytes were quantified by ICS. (B) Flow cytometry plots of IFN-γ co-staining with markers of effector and effector memory phenotypes (CD62Llo and CD11ahi). Cells were either not stimulated (no peptide) or stimulated with PbTRAP130 (5 µg/ml) or with a pool of 20-mer peptides overlapping by 10 amino acids spanning PbTRAP (PbTRAPpool: final concentration is 5 µg/mL for each peptide). (C) Flow cytometry plots of IFN-γ co-staining with other effector cytokines, TNF and IL-2. Figures are representative data from one of 4 experiments with 5 mice/group/experiment. (D) Polyfunctional analysis of cytokine secretion based on (C). Bars represent the mean value of the % of PbTRAP130-specific CD8+ cells. Individual data are also shown (blue diamond for PbTRAP130 and red diamond for PbS20318). Figures B, C and D are representative data from one of at least 4 experiments with 4–5 mice/experiment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649980&req=5

ppat-1003303-g006: Vaccination with PbTRAP elicits high levels of PbTRAP130-specific CD8+ T cell responses.(A) Schematic diagram of methodology. B6 mice were immunised with Ad PbTRAP (or Ad vector control) and boosted eight weeks later with M PbTRAP (or M vector control). Two weeks later, the frequencies and quality of PbTRAP-specific CD8+ T cells on peripheral blood leukocytes were quantified by ICS. (B) Flow cytometry plots of IFN-γ co-staining with markers of effector and effector memory phenotypes (CD62Llo and CD11ahi). Cells were either not stimulated (no peptide) or stimulated with PbTRAP130 (5 µg/ml) or with a pool of 20-mer peptides overlapping by 10 amino acids spanning PbTRAP (PbTRAPpool: final concentration is 5 µg/mL for each peptide). (C) Flow cytometry plots of IFN-γ co-staining with other effector cytokines, TNF and IL-2. Figures are representative data from one of 4 experiments with 5 mice/group/experiment. (D) Polyfunctional analysis of cytokine secretion based on (C). Bars represent the mean value of the % of PbTRAP130-specific CD8+ cells. Individual data are also shown (blue diamond for PbTRAP130 and red diamond for PbS20318). Figures B, C and D are representative data from one of at least 4 experiments with 4–5 mice/experiment.
Mentions: Adenovirus chimpanzee serotype 63 (Ad) and Modified Vaccinia Ankara (M) vaccines expressing a mammalian codon-optimised fragment of PbTRAP were generated. Referred to as Ad-M PbTRAP combination vaccine, they were used to vaccinate B6 mice with an 8-week resting period between priming and boosting (Figure 6A). Since the recombinant PbTRAP vaccines contained sequences in addition to PbTRAP130, we used both PbTRAP130 and a pool of overlapping peptides to PbTRAP (PbTRAPpool) in stimulation assays to verify if other PbTRAP-derived sequences were able to induce T cell responses. As shown in Figure 6B–D, the frequencies of IFN-γ secreting CD8+ T cells were indistinguishable between the two stimulations; approximately ∼17% (range: 14%–50%) of the total CD8+ T cells produce IFN-γ specific for PbTRAP130 or PbTRAPpool. Consistent with the induction of effector responses, these activated IFN-γ-producing cells coincided with the modulation of the corresponding expression markers CD62Llo and CD11ahi (Figure 6B). Polyfunctional analysis revealed that the responses were predominantly IFN-γ positive cells and IFN-γ/TNF double positive cells (Figures 6C,D). This intracellular cytokine pattern of PbTRAP130-specific CD8+ T cells were similar to that measured by immunizations with irradiated sporozoites (Figure S2). Cells stimulated with no peptide did not respond to either PbTRAP130 or PbTRAPpool. No cytokine-producing CD4+ T cells were detected following PbTRAP130 or PbTRAPpool stimulation. These results demonstrated the induction PbTRAP130-specific CD8+ T cells by vaccination and confirmed that PbTRAP130 is the only T cell epitope in PbTRAP in this infection model.

Bottom Line: While both PbS20₃₁₈ and PbTRAP₁₃₀ elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice, only PbTRAP₁₃₀-specific CD8⁺ T cells exhibit in vivo cytotoxicity.We conclude that PbTRAP is an immunodominant antigen during liver-stage infection.Together, our results underscore the presence of CD8⁺ T cells with divergent potencies against distinct Plasmodium liver-stage epitopes.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom. Julius.Hafalla@lshtm.ac.uk

ABSTRACT
CD8⁺ T cells mediate immunity against Plasmodium liver stages. However, the paucity of parasite-specific epitopes of CD8⁺ T cells has limited our current understanding of the mechanisms influencing the generation, maintenance and efficiency of these responses. To identify antigenic epitopes in a stringent murine malaria immunisation model, we performed a systematic profiling of H(2b)-restricted peptides predicted from genome-wide analysis. We describe the identification of Plasmodium berghei (Pb) sporozoite-specific gene 20 (S20)- and thrombospondin-related adhesive protein (TRAP)-derived peptides, termed PbS20₃₁₈ and PbTRAP₁₃₀ respectively, as targets of CD8⁺ T cells from C57BL/6 mice vaccinated by whole parasite strategies known to protect against sporozoite challenge. While both PbS20₃₁₈ and PbTRAP₁₃₀ elicit effector and effector memory phenotypes in both the spleens and livers of immunised mice, only PbTRAP₁₃₀-specific CD8⁺ T cells exhibit in vivo cytotoxicity. Moreover, PbTRAP₁₃₀-specific, but not PbS20₃₁₈-specific, CD8⁺ T cells significantly contribute to inhibition of parasite development. Prime/boost vaccination with PbTRAP demonstrates CD8⁺ T cell-dependent efficacy against sporozoite challenge. We conclude that PbTRAP is an immunodominant antigen during liver-stage infection. Together, our results underscore the presence of CD8⁺ T cells with divergent potencies against distinct Plasmodium liver-stage epitopes. Our identification of antigen-specific CD8⁺ T cells will allow interrogation of the development of immune responses against malaria liver stages.

Show MeSH
Related in: MedlinePlus