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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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PbTRAP130-specific CD8+ T cell responses are cytolytic in vivo.(A) Schematic diagram of methodology. Target cells were prepared by pulsing syngeneic spleen cells with PbS20318, PbTRAP130, or no peptides prior to labelling with CFSE. Target cells were transferred into naïve or immunised mice 14 days from last Pb γ-Spz immunisation. Spleens of recipient mice were harvested 24 hours later and analysed for CFSE fluorescence. (B) Representative histogram plots showing the fates of transferred cells in naïve (left) or immune (right) mice. The disappearance of a fluorescent peak signifies cytolysis of labelled splenocytes. (C) Quantification of in vivo cytolytic activity (**p<0.01, Mann-Whitney test). Figures are representative data from one of 3 experiments with 4 mice/group/experiment.
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ppat-1003303-g004: PbTRAP130-specific CD8+ T cell responses are cytolytic in vivo.(A) Schematic diagram of methodology. Target cells were prepared by pulsing syngeneic spleen cells with PbS20318, PbTRAP130, or no peptides prior to labelling with CFSE. Target cells were transferred into naïve or immunised mice 14 days from last Pb γ-Spz immunisation. Spleens of recipient mice were harvested 24 hours later and analysed for CFSE fluorescence. (B) Representative histogram plots showing the fates of transferred cells in naïve (left) or immune (right) mice. The disappearance of a fluorescent peak signifies cytolysis of labelled splenocytes. (C) Quantification of in vivo cytolytic activity (**p<0.01, Mann-Whitney test). Figures are representative data from one of 3 experiments with 4 mice/group/experiment.

Mentions: To determine the in vivo cytotoxic potential of PbS20318- and PbTRAP130-specific CD8+ T cells, we utilised an assay that allows the quantification of rapid killing of adoptively transferred target cells by activated CD8+ T cells in vivo[47]. CFSE-labelled and peptide-pulsed syngeneic targets were transferred to Pb γ-Spz-immunised mice 14 days after the last immunisation (Figure 4A). We observed considerable (∼90%) disappearance of PbTRAP130,-pulsed (Figure 4B,C), but not PbS20318-pulsed, target cells when transferred to mice that were immunised twice with Pb γ-Spz. To corroborate our finding that PbTRAP130-specific CD8+ T cells exhibit significant cytotoxic activity, we repeated the cell transfer to mice that were immunised only once and to naïve controls (Figure S4). Cytotoxicity against cells presenting PbTRAP130, but not PbS20318, was already apparent after a single immunisation.


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)

PbTRAP130-specific CD8+ T cell responses are cytolytic in vivo.(A) Schematic diagram of methodology. Target cells were prepared by pulsing syngeneic spleen cells with PbS20318, PbTRAP130, or no peptides prior to labelling with CFSE. Target cells were transferred into naïve or immunised mice 14 days from last Pb γ-Spz immunisation. Spleens of recipient mice were harvested 24 hours later and analysed for CFSE fluorescence. (B) Representative histogram plots showing the fates of transferred cells in naïve (left) or immune (right) mice. The disappearance of a fluorescent peak signifies cytolysis of labelled splenocytes. (C) Quantification of in vivo cytolytic activity (**p<0.01, Mann-Whitney test). Figures are representative data from one of 3 experiments with 4 mice/group/experiment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649980&req=5

ppat-1003303-g004: PbTRAP130-specific CD8+ T cell responses are cytolytic in vivo.(A) Schematic diagram of methodology. Target cells were prepared by pulsing syngeneic spleen cells with PbS20318, PbTRAP130, or no peptides prior to labelling with CFSE. Target cells were transferred into naïve or immunised mice 14 days from last Pb γ-Spz immunisation. Spleens of recipient mice were harvested 24 hours later and analysed for CFSE fluorescence. (B) Representative histogram plots showing the fates of transferred cells in naïve (left) or immune (right) mice. The disappearance of a fluorescent peak signifies cytolysis of labelled splenocytes. (C) Quantification of in vivo cytolytic activity (**p<0.01, Mann-Whitney test). Figures are representative data from one of 3 experiments with 4 mice/group/experiment.
Mentions: To determine the in vivo cytotoxic potential of PbS20318- and PbTRAP130-specific CD8+ T cells, we utilised an assay that allows the quantification of rapid killing of adoptively transferred target cells by activated CD8+ T cells in vivo[47]. CFSE-labelled and peptide-pulsed syngeneic targets were transferred to Pb γ-Spz-immunised mice 14 days after the last immunisation (Figure 4A). We observed considerable (∼90%) disappearance of PbTRAP130,-pulsed (Figure 4B,C), but not PbS20318-pulsed, target cells when transferred to mice that were immunised twice with Pb γ-Spz. To corroborate our finding that PbTRAP130-specific CD8+ T cells exhibit significant cytotoxic activity, we repeated the cell transfer to mice that were immunised only once and to naïve controls (Figure S4). Cytotoxicity against cells presenting PbTRAP130, but not PbS20318, was already apparent after a single immunisation.

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