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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

TCR Vβ subset distribution in ET cells.Splenocytes from C57BL/6 mice infected IV with Y. pseudotuberculosis mE for 7 days (A and filled circles in B) or one year (empty circles in B) were stained with YopE69-77 tetramer, a panel of Vβ antibodies and CD8 antibody conjugated with different fluorophores and were analyzed by flow cytometry. Representative histographs of tetramer (YopE-APC) and Vβ8.1 & 8.2 (left) or Vβ8.3 (right) signals from CD8+ T cells from one mouse are shown in (A). Numerical values correspond to percentages of gated cell populations among total CD8+ T cells. A summary of the percentages of each Vβ subset of total Vβ (% total) among ET cells from 3 mice infected for 7 days and 2 mice infected for 1 year is shown in (B). Mean and SEM is shown for each group of 5 mice.
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ppat.1005167.g001: TCR Vβ subset distribution in ET cells.Splenocytes from C57BL/6 mice infected IV with Y. pseudotuberculosis mE for 7 days (A and filled circles in B) or one year (empty circles in B) were stained with YopE69-77 tetramer, a panel of Vβ antibodies and CD8 antibody conjugated with different fluorophores and were analyzed by flow cytometry. Representative histographs of tetramer (YopE-APC) and Vβ8.1 & 8.2 (left) or Vβ8.3 (right) signals from CD8+ T cells from one mouse are shown in (A). Numerical values correspond to percentages of gated cell populations among total CD8+ T cells. A summary of the percentages of each Vβ subset of total Vβ (% total) among ET cells from 3 mice infected for 7 days and 2 mice infected for 1 year is shown in (B). Mean and SEM is shown for each group of 5 mice.

Mentions: The exceptionally large ET cell response in mice infected with Y. pseudotuberculosis is similar to the magnitude of CD4+ T cell responses to superantigens. Superantigens typically induce a CD4+ T cell response that is limited in diversity with respect to the Vβ usage in αβ T-cell receptors (TCRs). To determine the clonal nature of the ET cells produced during Y. pseudotuberculosis infection of C57BL/6 mice, the Vβ repertoire of these cells was investigated. The genes of functional TCRs are assembled from separate V, D, J region segments through recombination (reviewed in [25]). Mice and humans carry about 20–70 germline V segments that encode about 90 amino acid residues of the mature TCR. Therefore, diversity in Vβ composition demonstrates a polyclonal nature of a T cell population, however, T cells containing the same Vβ regions are further diversified through the addition of D and/or J segments and imprecise joining. To obtain uniform infections we used intravenous (IV) challenges, and because it is difficult to achieve sublethal infections via this route with the wild-type bacteria, our experiments were done with the attenuated Y. pseudotuberculosis strain 32777 encoding catalytically inactive YopER144A (mE, Table 1). C57BL/6 mice were infected IV with mE, and the Vβ composition of the ET cells in spleens was determined using a panel of fluorophore-conjugated antibodies recognizing different Vβ regions in conjunction with tetramer staining and flow cytometry (Fig 1A). Results obtained with an uninfected mouse analyzed in parallel as a control are shown in S1 Fig Seven days post infection (dpi), when the number and/or percentage of ET cells were still increasing; or one year after infection, when only ~2% of total splenic CD8+ T cells were specific for YopE69-77, the most prominent population was composed of Vβ8.1 and 8.2, with an average of 25% of all ET cells in this category (Fig 1A, left, and 1B). The 2nd largest population, however, differed between individual mice (Fig 1B). Overall, all of the Vβ subsets tested were represented within the ET cell population in all the mice examined, ranging in average composition from 3% to 25% among all the ET cells (Fig 1B). These results revealed that the Vβ usage in the ET cell composition is polyclonal and highly diverse, indicating that an antigen-presentation process, rather than a superantigen-like mechanism, is responsible for production of these cells.


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)

TCR Vβ subset distribution in ET cells.Splenocytes from C57BL/6 mice infected IV with Y. pseudotuberculosis mE for 7 days (A and filled circles in B) or one year (empty circles in B) were stained with YopE69-77 tetramer, a panel of Vβ antibodies and CD8 antibody conjugated with different fluorophores and were analyzed by flow cytometry. Representative histographs of tetramer (YopE-APC) and Vβ8.1 & 8.2 (left) or Vβ8.3 (right) signals from CD8+ T cells from one mouse are shown in (A). Numerical values correspond to percentages of gated cell populations among total CD8+ T cells. A summary of the percentages of each Vβ subset of total Vβ (% total) among ET cells from 3 mice infected for 7 days and 2 mice infected for 1 year is shown in (B). Mean and SEM is shown for each group of 5 mice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4607306&req=5

ppat.1005167.g001: TCR Vβ subset distribution in ET cells.Splenocytes from C57BL/6 mice infected IV with Y. pseudotuberculosis mE for 7 days (A and filled circles in B) or one year (empty circles in B) were stained with YopE69-77 tetramer, a panel of Vβ antibodies and CD8 antibody conjugated with different fluorophores and were analyzed by flow cytometry. Representative histographs of tetramer (YopE-APC) and Vβ8.1 & 8.2 (left) or Vβ8.3 (right) signals from CD8+ T cells from one mouse are shown in (A). Numerical values correspond to percentages of gated cell populations among total CD8+ T cells. A summary of the percentages of each Vβ subset of total Vβ (% total) among ET cells from 3 mice infected for 7 days and 2 mice infected for 1 year is shown in (B). Mean and SEM is shown for each group of 5 mice.
Mentions: The exceptionally large ET cell response in mice infected with Y. pseudotuberculosis is similar to the magnitude of CD4+ T cell responses to superantigens. Superantigens typically induce a CD4+ T cell response that is limited in diversity with respect to the Vβ usage in αβ T-cell receptors (TCRs). To determine the clonal nature of the ET cells produced during Y. pseudotuberculosis infection of C57BL/6 mice, the Vβ repertoire of these cells was investigated. The genes of functional TCRs are assembled from separate V, D, J region segments through recombination (reviewed in [25]). Mice and humans carry about 20–70 germline V segments that encode about 90 amino acid residues of the mature TCR. Therefore, diversity in Vβ composition demonstrates a polyclonal nature of a T cell population, however, T cells containing the same Vβ regions are further diversified through the addition of D and/or J segments and imprecise joining. To obtain uniform infections we used intravenous (IV) challenges, and because it is difficult to achieve sublethal infections via this route with the wild-type bacteria, our experiments were done with the attenuated Y. pseudotuberculosis strain 32777 encoding catalytically inactive YopER144A (mE, Table 1). C57BL/6 mice were infected IV with mE, and the Vβ composition of the ET cells in spleens was determined using a panel of fluorophore-conjugated antibodies recognizing different Vβ regions in conjunction with tetramer staining and flow cytometry (Fig 1A). Results obtained with an uninfected mouse analyzed in parallel as a control are shown in S1 Fig Seven days post infection (dpi), when the number and/or percentage of ET cells were still increasing; or one year after infection, when only ~2% of total splenic CD8+ T cells were specific for YopE69-77, the most prominent population was composed of Vβ8.1 and 8.2, with an average of 25% of all ET cells in this category (Fig 1A, left, and 1B). The 2nd largest population, however, differed between individual mice (Fig 1B). Overall, all of the Vβ subsets tested were represented within the ET cell population in all the mice examined, ranging in average composition from 3% to 25% among all the ET cells (Fig 1B). These results revealed that the Vβ usage in the ET cell composition is polyclonal and highly diverse, indicating that an antigen-presentation process, rather than a superantigen-like mechanism, is responsible for production of these cells.

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