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CCR2+ Inflammatory Dendritic Cells and Translocation of Antigen by Type III Secretion Are Required for the Exceptionally Large CD8+ T Cell Response to the Protective YopE69-77 Epitope during Yersinia Infection.

Zhang Y, Tam JW, Mena P, van der Velden AW, Bliska JB - PLoS Pathog. (2015)

Bottom Line: The features of the interaction between pathogen and host that result in this large CD8+ T cell response are unknown.Additionally, a smaller YopE69-77-specific CD8+ T cell response (~10% of the large expansion) can be generated in a "translocation-independent" pathway in which CD8α+ DCs cross present secreted YopE.A requirement for inflammatory DCs in producing a protective CD8+ T cell response to a bacterial antigen has not previously been demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Diseases and Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, United States of America.

ABSTRACT
During Yersinia pseudotuberculosis infection of C57BL/6 mice, an exceptionally large CD8+ T cell response to a protective epitope in the type III secretion system effector YopE is produced. At the peak of the response, up to 50% of splenic CD8+ T cells recognize the epitope YopE69-77. The features of the interaction between pathogen and host that result in this large CD8+ T cell response are unknown. Here, we used Y. pseudotuberculosis strains defective for production, secretion and/or translocation of YopE to infect wild-type or mutant mice deficient in specific dendritic cells (DCs). Bacterial colonization of organs and translocation of YopE into spleen cells was measured, and flow cytometry and tetramer staining were used to characterize the cellular immune response. We show that the splenic YopE69-77-specific CD8+ T cells generated during the large response are polyclonal and are produced by a "translocation-dependent" pathway that requires injection of YopE into host cell cytosol. Additionally, a smaller YopE69-77-specific CD8+ T cell response (~10% of the large expansion) can be generated in a "translocation-independent" pathway in which CD8α+ DCs cross present secreted YopE. CCR2-expressing inflammatory DCs were required for the large YopE69-77-specific CD8+ T cell expansion because this response was significantly reduced in Ccr2-/- mice, YopE was translocated into inflammatory DCs in vivo, inflammatory DCs purified from infected spleens activated YopE69-77-specific CD8+ T cells ex vivo and promoted the expansion of YopE69-77-specific CD8+ T cells in infected Ccr2-/- mice after adoptive transfer. A requirement for inflammatory DCs in producing a protective CD8+ T cell response to a bacterial antigen has not previously been demonstrated. Therefore, the production of YopE69-77-specific CD8+ T cells by inflammatory DCs that are injected with YopE during Y. pseudotuberculosis infection represents a novel mechanism for generating a massive and protective adaptive immune response.

No MeSH data available.


Related in: MedlinePlus

Detection of YopE-TEM1 translocation into different splenocyte populations.C57BL/6 mice were IV infected with 105 CFU of YopE-Bla or 5X106 CFU of ΔB YopE-Bla for 6 days. Splenocytes isolated from surviving mice were incubated with CCF4-AM to detect the presence of translocated YopE-TEM1, and subsequently stained with a panel of antibodies and analyzed with flow cytometry. (A) A representative histograph of splenocytes from a mouse infected with YopE-Bla showing the gate for translocation positive cells (Blue). (B) Spleen and liver colonization levels were determined by CFU assay for YopE-Bla, and ΔB YopE-Bla and the combined value of spleen and liver colonization was plotted against the percentage of blue splenocytes in the same mice. Data shown are the summary of four independent experiments. (C) Splenocytes from mice infected with YopE-Bla for 6 days were analyzed by flow cytometry and the percentage of splenocytes positive for the indicated markers (top) and blue cells among these cells (bottom) were determined. (D) Percentage of Ly6Chi infDCs or Ly6Cmed PMNs among the CD11b+ splenocytes from mice infected with YopE-Bla for 6 days were determined by flow cytometry (top), and the percentage of blue cells among these were plotted at bottom. Representative histograph of overlaid blue signal strength of PMNs (E) or infDC (F) from an individual mouse infected for 6 days with YopE-Bla (black lines) or ΔB YopE-Bla (light gray lines). The percentage of blue cells from the YopE-Bla-infected sample was indicated in the gate. In B-D, each dot represents the value obtained from one mouse, and wherever applicable, the bar indicated the average.
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ppat.1005167.g006: Detection of YopE-TEM1 translocation into different splenocyte populations.C57BL/6 mice were IV infected with 105 CFU of YopE-Bla or 5X106 CFU of ΔB YopE-Bla for 6 days. Splenocytes isolated from surviving mice were incubated with CCF4-AM to detect the presence of translocated YopE-TEM1, and subsequently stained with a panel of antibodies and analyzed with flow cytometry. (A) A representative histograph of splenocytes from a mouse infected with YopE-Bla showing the gate for translocation positive cells (Blue). (B) Spleen and liver colonization levels were determined by CFU assay for YopE-Bla, and ΔB YopE-Bla and the combined value of spleen and liver colonization was plotted against the percentage of blue splenocytes in the same mice. Data shown are the summary of four independent experiments. (C) Splenocytes from mice infected with YopE-Bla for 6 days were analyzed by flow cytometry and the percentage of splenocytes positive for the indicated markers (top) and blue cells among these cells (bottom) were determined. (D) Percentage of Ly6Chi infDCs or Ly6Cmed PMNs among the CD11b+ splenocytes from mice infected with YopE-Bla for 6 days were determined by flow cytometry (top), and the percentage of blue cells among these were plotted at bottom. Representative histograph of overlaid blue signal strength of PMNs (E) or infDC (F) from an individual mouse infected for 6 days with YopE-Bla (black lines) or ΔB YopE-Bla (light gray lines). The percentage of blue cells from the YopE-Bla-infected sample was indicated in the gate. In B-D, each dot represents the value obtained from one mouse, and wherever applicable, the bar indicated the average.

Mentions: YopE-Bla was attenuated in comparison to mE in our IV mouse infection model, and therefore an infection dose of 105 CFU was used. At 6 dpi of C57BL/6 mice with YopE-Bla, approximately 16% of splenocytes were blue as a result of translocation of the YopE-TEM1 fusion protein (Fig 6A). Translocation of YopE-TEM1 increased with increasing colonization levels of YopE-Bla in spleen and liver (Fig 6B). Translocation largely depended on YopB because an average of only 0.5% of the total splenocytes were blue after infection with the control ΔB YopE-Bla strain (Table 1) (Fig 6B).


CCR2+ Inflammatory Dendritic Cells and Translocation of Antigen by Type III Secretion Are Required for the Exceptionally Large CD8+ T Cell Response to the Protective YopE69-77 Epitope during Yersinia Infection.

Zhang Y, Tam JW, Mena P, van der Velden AW, Bliska JB - PLoS Pathog. (2015)

Detection of YopE-TEM1 translocation into different splenocyte populations.C57BL/6 mice were IV infected with 105 CFU of YopE-Bla or 5X106 CFU of ΔB YopE-Bla for 6 days. Splenocytes isolated from surviving mice were incubated with CCF4-AM to detect the presence of translocated YopE-TEM1, and subsequently stained with a panel of antibodies and analyzed with flow cytometry. (A) A representative histograph of splenocytes from a mouse infected with YopE-Bla showing the gate for translocation positive cells (Blue). (B) Spleen and liver colonization levels were determined by CFU assay for YopE-Bla, and ΔB YopE-Bla and the combined value of spleen and liver colonization was plotted against the percentage of blue splenocytes in the same mice. Data shown are the summary of four independent experiments. (C) Splenocytes from mice infected with YopE-Bla for 6 days were analyzed by flow cytometry and the percentage of splenocytes positive for the indicated markers (top) and blue cells among these cells (bottom) were determined. (D) Percentage of Ly6Chi infDCs or Ly6Cmed PMNs among the CD11b+ splenocytes from mice infected with YopE-Bla for 6 days were determined by flow cytometry (top), and the percentage of blue cells among these were plotted at bottom. Representative histograph of overlaid blue signal strength of PMNs (E) or infDC (F) from an individual mouse infected for 6 days with YopE-Bla (black lines) or ΔB YopE-Bla (light gray lines). The percentage of blue cells from the YopE-Bla-infected sample was indicated in the gate. In B-D, each dot represents the value obtained from one mouse, and wherever applicable, the bar indicated the average.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4607306&req=5

ppat.1005167.g006: Detection of YopE-TEM1 translocation into different splenocyte populations.C57BL/6 mice were IV infected with 105 CFU of YopE-Bla or 5X106 CFU of ΔB YopE-Bla for 6 days. Splenocytes isolated from surviving mice were incubated with CCF4-AM to detect the presence of translocated YopE-TEM1, and subsequently stained with a panel of antibodies and analyzed with flow cytometry. (A) A representative histograph of splenocytes from a mouse infected with YopE-Bla showing the gate for translocation positive cells (Blue). (B) Spleen and liver colonization levels were determined by CFU assay for YopE-Bla, and ΔB YopE-Bla and the combined value of spleen and liver colonization was plotted against the percentage of blue splenocytes in the same mice. Data shown are the summary of four independent experiments. (C) Splenocytes from mice infected with YopE-Bla for 6 days were analyzed by flow cytometry and the percentage of splenocytes positive for the indicated markers (top) and blue cells among these cells (bottom) were determined. (D) Percentage of Ly6Chi infDCs or Ly6Cmed PMNs among the CD11b+ splenocytes from mice infected with YopE-Bla for 6 days were determined by flow cytometry (top), and the percentage of blue cells among these were plotted at bottom. Representative histograph of overlaid blue signal strength of PMNs (E) or infDC (F) from an individual mouse infected for 6 days with YopE-Bla (black lines) or ΔB YopE-Bla (light gray lines). The percentage of blue cells from the YopE-Bla-infected sample was indicated in the gate. In B-D, each dot represents the value obtained from one mouse, and wherever applicable, the bar indicated the average.
Mentions: YopE-Bla was attenuated in comparison to mE in our IV mouse infection model, and therefore an infection dose of 105 CFU was used. At 6 dpi of C57BL/6 mice with YopE-Bla, approximately 16% of splenocytes were blue as a result of translocation of the YopE-TEM1 fusion protein (Fig 6A). Translocation of YopE-TEM1 increased with increasing colonization levels of YopE-Bla in spleen and liver (Fig 6B). Translocation largely depended on YopB because an average of only 0.5% of the total splenocytes were blue after infection with the control ΔB YopE-Bla strain (Table 1) (Fig 6B).

Bottom Line: The features of the interaction between pathogen and host that result in this large CD8+ T cell response are unknown.Additionally, a smaller YopE69-77-specific CD8+ T cell response (~10% of the large expansion) can be generated in a "translocation-independent" pathway in which CD8α+ DCs cross present secreted YopE.A requirement for inflammatory DCs in producing a protective CD8+ T cell response to a bacterial antigen has not previously been demonstrated.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Diseases and Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, United States of America.

ABSTRACT
During Yersinia pseudotuberculosis infection of C57BL/6 mice, an exceptionally large CD8+ T cell response to a protective epitope in the type III secretion system effector YopE is produced. At the peak of the response, up to 50% of splenic CD8+ T cells recognize the epitope YopE69-77. The features of the interaction between pathogen and host that result in this large CD8+ T cell response are unknown. Here, we used Y. pseudotuberculosis strains defective for production, secretion and/or translocation of YopE to infect wild-type or mutant mice deficient in specific dendritic cells (DCs). Bacterial colonization of organs and translocation of YopE into spleen cells was measured, and flow cytometry and tetramer staining were used to characterize the cellular immune response. We show that the splenic YopE69-77-specific CD8+ T cells generated during the large response are polyclonal and are produced by a "translocation-dependent" pathway that requires injection of YopE into host cell cytosol. Additionally, a smaller YopE69-77-specific CD8+ T cell response (~10% of the large expansion) can be generated in a "translocation-independent" pathway in which CD8α+ DCs cross present secreted YopE. CCR2-expressing inflammatory DCs were required for the large YopE69-77-specific CD8+ T cell expansion because this response was significantly reduced in Ccr2-/- mice, YopE was translocated into inflammatory DCs in vivo, inflammatory DCs purified from infected spleens activated YopE69-77-specific CD8+ T cells ex vivo and promoted the expansion of YopE69-77-specific CD8+ T cells in infected Ccr2-/- mice after adoptive transfer. A requirement for inflammatory DCs in producing a protective CD8+ T cell response to a bacterial antigen has not previously been demonstrated. Therefore, the production of YopE69-77-specific CD8+ T cells by inflammatory DCs that are injected with YopE during Y. pseudotuberculosis infection represents a novel mechanism for generating a massive and protective adaptive immune response.

No MeSH data available.


Related in: MedlinePlus