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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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Kinetics of PbS20318 and PbTRAP130-specific CD8+ T cell responses following immunisation with Pb γ-Spz.(A) B6 mice were immunised either once or twice with Pb γ-Spz as shown in the schematic diagram. On days 7, 14 and 180 after the last immunisation, PbS20318 and PbTRAP130-specific CD8+ T cell responses were quantified in the spleens and the livers by peptide stimulation followed by ICS. Representative flow cytometry plots showing IFN-γ-secretion by CD8+ T cells in the spleens and the livers of Pb γ-Spz-immunised mice. (B) Data in (A) presented as bar graphs: white squares = 1° immunisation, black squares = 2° immunisation (blue for PbS20318 and red for PbTRAP130), differences between 1° vs 2° immunisations: **p<0.01 and *p<0.05, Mann-Whitney test. Dotted lines represent baseline responses based on peptide stimulation of spleens from naïve mice. Experiments were performed at least 5 times with 3–5 mice per group. Frequencies of epitope-specific CD8+ T cells were also compared among the different time points and were found to be statistically different (p<0.05) using the Kruskal Wallis test.
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ppat-1003303-g002: Kinetics of PbS20318 and PbTRAP130-specific CD8+ T cell responses following immunisation with Pb γ-Spz.(A) B6 mice were immunised either once or twice with Pb γ-Spz as shown in the schematic diagram. On days 7, 14 and 180 after the last immunisation, PbS20318 and PbTRAP130-specific CD8+ T cell responses were quantified in the spleens and the livers by peptide stimulation followed by ICS. Representative flow cytometry plots showing IFN-γ-secretion by CD8+ T cells in the spleens and the livers of Pb γ-Spz-immunised mice. (B) Data in (A) presented as bar graphs: white squares = 1° immunisation, black squares = 2° immunisation (blue for PbS20318 and red for PbTRAP130), differences between 1° vs 2° immunisations: **p<0.01 and *p<0.05, Mann-Whitney test. Dotted lines represent baseline responses based on peptide stimulation of spleens from naïve mice. Experiments were performed at least 5 times with 3–5 mice per group. Frequencies of epitope-specific CD8+ T cells were also compared among the different time points and were found to be statistically different (p<0.05) using the Kruskal Wallis test.

Mentions: PbS20318 and PbTRAP130 represent the first reported endogenously processed CD8+ T cell epitopes of malaria liver stages in the B6 model. To determine expansion and contraction of PbS20318- and PbTRAP130-specific CD8+ T cells over time, we quantified the responses in the spleen and the liver after one or two immunisations with Pb γ-Spz (Figure 2A). After a single immunisation, CD8+ T cell responses in both the spleen and the liver reach the highest magnitude on day 7 (Figure 2A,B). The responses were slightly decreased on day 14 as contraction of the response occurs but they remained quantifiable for up to 180 days after immunisation. The percentages of antigen-specific CD8+ T cells were generally higher in the liver that in the spleen. More robust responses were observed after two immunisations with Pb γ-Spz (Figure 2A,B) Polyfunctional analysis of PbS20318- and PbTRAP130-specific CD8+ T cells revealed the induction of IFN-γ positive cells and IFN-γ/tumour necrosis factor (TNF) double positive CD8+ T cells (Figure S2). Consistent with the generation of effector and effector memory responses, PbS20318 and PbTRAP130-specific IFN-γ-producing CD8+ T cells were immunophenotyped as CD62Llo, CD44hi, CD11ahi, and CD49dhi (Figure 3, S3). Cells stimulated with no peptide or cells from naïve mice stimulated with either peptide did not respond to either PbS20318 or PbTRAP130 (data not shown). Together, these results indicate that immunisation with Pb γ-Spz recruits antigen-specific CD8+ T cells to undergo differentiation, proliferation, and long-term persistence.


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)

Kinetics of PbS20318 and PbTRAP130-specific CD8+ T cell responses following immunisation with Pb γ-Spz.(A) B6 mice were immunised either once or twice with Pb γ-Spz as shown in the schematic diagram. On days 7, 14 and 180 after the last immunisation, PbS20318 and PbTRAP130-specific CD8+ T cell responses were quantified in the spleens and the livers by peptide stimulation followed by ICS. Representative flow cytometry plots showing IFN-γ-secretion by CD8+ T cells in the spleens and the livers of Pb γ-Spz-immunised mice. (B) Data in (A) presented as bar graphs: white squares = 1° immunisation, black squares = 2° immunisation (blue for PbS20318 and red for PbTRAP130), differences between 1° vs 2° immunisations: **p<0.01 and *p<0.05, Mann-Whitney test. Dotted lines represent baseline responses based on peptide stimulation of spleens from naïve mice. Experiments were performed at least 5 times with 3–5 mice per group. Frequencies of epitope-specific CD8+ T cells were also compared among the different time points and were found to be statistically different (p<0.05) using the Kruskal Wallis test.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649980&req=5

ppat-1003303-g002: Kinetics of PbS20318 and PbTRAP130-specific CD8+ T cell responses following immunisation with Pb γ-Spz.(A) B6 mice were immunised either once or twice with Pb γ-Spz as shown in the schematic diagram. On days 7, 14 and 180 after the last immunisation, PbS20318 and PbTRAP130-specific CD8+ T cell responses were quantified in the spleens and the livers by peptide stimulation followed by ICS. Representative flow cytometry plots showing IFN-γ-secretion by CD8+ T cells in the spleens and the livers of Pb γ-Spz-immunised mice. (B) Data in (A) presented as bar graphs: white squares = 1° immunisation, black squares = 2° immunisation (blue for PbS20318 and red for PbTRAP130), differences between 1° vs 2° immunisations: **p<0.01 and *p<0.05, Mann-Whitney test. Dotted lines represent baseline responses based on peptide stimulation of spleens from naïve mice. Experiments were performed at least 5 times with 3–5 mice per group. Frequencies of epitope-specific CD8+ T cells were also compared among the different time points and were found to be statistically different (p<0.05) using the Kruskal Wallis test.
Mentions: PbS20318 and PbTRAP130 represent the first reported endogenously processed CD8+ T cell epitopes of malaria liver stages in the B6 model. To determine expansion and contraction of PbS20318- and PbTRAP130-specific CD8+ T cells over time, we quantified the responses in the spleen and the liver after one or two immunisations with Pb γ-Spz (Figure 2A). After a single immunisation, CD8+ T cell responses in both the spleen and the liver reach the highest magnitude on day 7 (Figure 2A,B). The responses were slightly decreased on day 14 as contraction of the response occurs but they remained quantifiable for up to 180 days after immunisation. The percentages of antigen-specific CD8+ T cells were generally higher in the liver that in the spleen. More robust responses were observed after two immunisations with Pb γ-Spz (Figure 2A,B) Polyfunctional analysis of PbS20318- and PbTRAP130-specific CD8+ T cells revealed the induction of IFN-γ positive cells and IFN-γ/tumour necrosis factor (TNF) double positive CD8+ T cells (Figure S2). Consistent with the generation of effector and effector memory responses, PbS20318 and PbTRAP130-specific IFN-γ-producing CD8+ T cells were immunophenotyped as CD62Llo, CD44hi, CD11ahi, and CD49dhi (Figure 3, S3). Cells stimulated with no peptide or cells from naïve mice stimulated with either peptide did not respond to either PbS20318 or PbTRAP130 (data not shown). Together, these results indicate that immunisation with Pb γ-Spz recruits antigen-specific CD8+ T cells to undergo differentiation, proliferation, and long-term persistence.

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