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Cross-presentation of a spread-defective MCMV is sufficient to prime the majority of virus-specific CD8+ T cells.

Snyder CM, Allan JE, Bonnett EL, Doom CM, Hill AB - PLoS ONE (2010)

Bottom Line: However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells.Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector.Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, United States of America.

ABSTRACT
CD8+ T cells can be primed by peptides derived from endogenous proteins (direct presentation), or exogenously acquired protein (cross-presentation). However, the relative ability of these two pathways to prime CD8+ T cells during a viral infection remains controversial. Cytomegaloviruses (CMVs) can infect professional antigen presenting cells (APCs), including dendritic cells, thus providing peptides for direct presentation. However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells. Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector. We have shown previously that deleting the immune evasion genes from murine cytomegalovirus (MCMV) that target class I MHC presentation, has no impact on the size or breadth of the CD8+ T cell response elicited by infection, suggesting that the majority of MCMV-specific CD8+ T cells in vivo are not directly primed by infected professional APCs. Here we use a novel spread-defective mutant of MCMV, lacking the essential glycoprotein gL, to show that cross-presentation alone can account for the majority of MCMV-specific CD8+ T cell responses to the virus. Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection.

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The ΔgL and K181 wild-type viruses induce a similar immunodominance hierarchy after direct i.p. infection.A) There are fewer splenocytes in ΔgL infected mice. C57BL/6 mice were infected i.p. with 1×105 pfu K181 or ΔgL virus. 7 days after infection the total number of splenocytes and CD8+ T cells were quantified. Statisitical significance was determined by a Student's t-test. Data represents an average of 3 separate mice in this experiment. B) Immunodominant CD8+ T cell responses are readily detected in the blood of ΔgL infected mice. C57BL/6 mice were infected with the indicated viruses and the CD8+ T cell responses in the peripheral blood were examined by tetramer staining 7 days later (n = 7 mice per group combined from 2 independent experiments). Data is representative of at least 5 independent experiments. C) The immunodominance hierarchies induced by wild-type and ΔgL infections are remarkably similar. C57BL/6 mice were infected i.p. with 1×105 pfu of the indicated viruses and spleens were harvested 7 days later (n = 3 mice per group). Cells were stimulated with the indicated peptides and the production of IFN-γ was measured as described. The shaded area reflects the background from unstimulated cells in this experiment. Similar data was obtained in an independent experiment that included a more limited subset of MCMV peptides (not shown).
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pone-0009681-g003: The ΔgL and K181 wild-type viruses induce a similar immunodominance hierarchy after direct i.p. infection.A) There are fewer splenocytes in ΔgL infected mice. C57BL/6 mice were infected i.p. with 1×105 pfu K181 or ΔgL virus. 7 days after infection the total number of splenocytes and CD8+ T cells were quantified. Statisitical significance was determined by a Student's t-test. Data represents an average of 3 separate mice in this experiment. B) Immunodominant CD8+ T cell responses are readily detected in the blood of ΔgL infected mice. C57BL/6 mice were infected with the indicated viruses and the CD8+ T cell responses in the peripheral blood were examined by tetramer staining 7 days later (n = 7 mice per group combined from 2 independent experiments). Data is representative of at least 5 independent experiments. C) The immunodominance hierarchies induced by wild-type and ΔgL infections are remarkably similar. C57BL/6 mice were infected i.p. with 1×105 pfu of the indicated viruses and spleens were harvested 7 days later (n = 3 mice per group). Cells were stimulated with the indicated peptides and the production of IFN-γ was measured as described. The shaded area reflects the background from unstimulated cells in this experiment. Similar data was obtained in an independent experiment that included a more limited subset of MCMV peptides (not shown).

Mentions: Typically, MCMV infection develops progressively, infecting a limited subset of cells in the first round before spreading to other cells, including splenic DCs [18]. Since the ΔgL virus is only capable of a single round of infection, the types of cells infected in vivo should be limited. As viral protein expression is influenced by the cell types and organs that are infected [19]–[21], we might expect some differences in the immunodominance hierarchy elicited by ΔgL and replication-competent K181 MCMV. Moreover, the immunodominance hierarchy was characterized previously using the Smith strain of MCMV [6], [22], which could elicit a different pattern of CD8+ T cell responses. Thus, in order to characterize the CD8+ T cell response to K181 (wild-type) and ΔgL viruses, C57BL/6 (B6) mice were infected via the intraperitoneal route with either virus. T cell responses were measured at the peak of expansion, 7 days later. It was noted that the intensity of the immune response was lower after ΔgL infection in comparison to wild-type K181 infection. Relative to K181, there were approximately 40% fewer total splenocytes after ΔgL infection and half the number of CD8+ T cells (Figure 3A). Nevertheless, the immunodominance hierarchy was similar after the two infections. In the peripheral blood, M45-, m139- and M57-specific CD8+ T cells dominated the immune response, although the relative intensity of the M45-specific T cell response was significantly reduced compared to m139- and M57-specific responses after ΔgL infection (Figure 3B). When splenoctyes were tested with an expanded panel of 18 epitopes, the overall hierarchy was very similar for K181 and ΔgL infections (Figure 3C) and closely resembled, though did not precisely match, the hierarchy elicited by Smith MCMV [6], [22]. Again, the relative intensity of the M45-specific T cell response was significantly reduced in ΔgL infected mice. Moreover, the responses to three epitopes, m141, M78 and M33, appeared reduced relative to M57-, M86- and M38316-specific responses in ΔgL infected mice, although these differences were not statistically significant. MCMV infects many cell types in vivo, including epithelial cells, endothelial cells, macrophages and dendrtic cells [18], [23]. We speculate that the slight differences in immunodominance could reflect the intensity of viral gene expression in the cells infected immediately after injection, which is all that is available to the ΔgL virus.


Cross-presentation of a spread-defective MCMV is sufficient to prime the majority of virus-specific CD8+ T cells.

Snyder CM, Allan JE, Bonnett EL, Doom CM, Hill AB - PLoS ONE (2010)

The ΔgL and K181 wild-type viruses induce a similar immunodominance hierarchy after direct i.p. infection.A) There are fewer splenocytes in ΔgL infected mice. C57BL/6 mice were infected i.p. with 1×105 pfu K181 or ΔgL virus. 7 days after infection the total number of splenocytes and CD8+ T cells were quantified. Statisitical significance was determined by a Student's t-test. Data represents an average of 3 separate mice in this experiment. B) Immunodominant CD8+ T cell responses are readily detected in the blood of ΔgL infected mice. C57BL/6 mice were infected with the indicated viruses and the CD8+ T cell responses in the peripheral blood were examined by tetramer staining 7 days later (n = 7 mice per group combined from 2 independent experiments). Data is representative of at least 5 independent experiments. C) The immunodominance hierarchies induced by wild-type and ΔgL infections are remarkably similar. C57BL/6 mice were infected i.p. with 1×105 pfu of the indicated viruses and spleens were harvested 7 days later (n = 3 mice per group). Cells were stimulated with the indicated peptides and the production of IFN-γ was measured as described. The shaded area reflects the background from unstimulated cells in this experiment. Similar data was obtained in an independent experiment that included a more limited subset of MCMV peptides (not shown).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2837378&req=5

pone-0009681-g003: The ΔgL and K181 wild-type viruses induce a similar immunodominance hierarchy after direct i.p. infection.A) There are fewer splenocytes in ΔgL infected mice. C57BL/6 mice were infected i.p. with 1×105 pfu K181 or ΔgL virus. 7 days after infection the total number of splenocytes and CD8+ T cells were quantified. Statisitical significance was determined by a Student's t-test. Data represents an average of 3 separate mice in this experiment. B) Immunodominant CD8+ T cell responses are readily detected in the blood of ΔgL infected mice. C57BL/6 mice were infected with the indicated viruses and the CD8+ T cell responses in the peripheral blood were examined by tetramer staining 7 days later (n = 7 mice per group combined from 2 independent experiments). Data is representative of at least 5 independent experiments. C) The immunodominance hierarchies induced by wild-type and ΔgL infections are remarkably similar. C57BL/6 mice were infected i.p. with 1×105 pfu of the indicated viruses and spleens were harvested 7 days later (n = 3 mice per group). Cells were stimulated with the indicated peptides and the production of IFN-γ was measured as described. The shaded area reflects the background from unstimulated cells in this experiment. Similar data was obtained in an independent experiment that included a more limited subset of MCMV peptides (not shown).
Mentions: Typically, MCMV infection develops progressively, infecting a limited subset of cells in the first round before spreading to other cells, including splenic DCs [18]. Since the ΔgL virus is only capable of a single round of infection, the types of cells infected in vivo should be limited. As viral protein expression is influenced by the cell types and organs that are infected [19]–[21], we might expect some differences in the immunodominance hierarchy elicited by ΔgL and replication-competent K181 MCMV. Moreover, the immunodominance hierarchy was characterized previously using the Smith strain of MCMV [6], [22], which could elicit a different pattern of CD8+ T cell responses. Thus, in order to characterize the CD8+ T cell response to K181 (wild-type) and ΔgL viruses, C57BL/6 (B6) mice were infected via the intraperitoneal route with either virus. T cell responses were measured at the peak of expansion, 7 days later. It was noted that the intensity of the immune response was lower after ΔgL infection in comparison to wild-type K181 infection. Relative to K181, there were approximately 40% fewer total splenocytes after ΔgL infection and half the number of CD8+ T cells (Figure 3A). Nevertheless, the immunodominance hierarchy was similar after the two infections. In the peripheral blood, M45-, m139- and M57-specific CD8+ T cells dominated the immune response, although the relative intensity of the M45-specific T cell response was significantly reduced compared to m139- and M57-specific responses after ΔgL infection (Figure 3B). When splenoctyes were tested with an expanded panel of 18 epitopes, the overall hierarchy was very similar for K181 and ΔgL infections (Figure 3C) and closely resembled, though did not precisely match, the hierarchy elicited by Smith MCMV [6], [22]. Again, the relative intensity of the M45-specific T cell response was significantly reduced in ΔgL infected mice. Moreover, the responses to three epitopes, m141, M78 and M33, appeared reduced relative to M57-, M86- and M38316-specific responses in ΔgL infected mice, although these differences were not statistically significant. MCMV infects many cell types in vivo, including epithelial cells, endothelial cells, macrophages and dendrtic cells [18], [23]. We speculate that the slight differences in immunodominance could reflect the intensity of viral gene expression in the cells infected immediately after injection, which is all that is available to the ΔgL virus.

Bottom Line: However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells.Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector.Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, Oregon, United States of America.

ABSTRACT
CD8+ T cells can be primed by peptides derived from endogenous proteins (direct presentation), or exogenously acquired protein (cross-presentation). However, the relative ability of these two pathways to prime CD8+ T cells during a viral infection remains controversial. Cytomegaloviruses (CMVs) can infect professional antigen presenting cells (APCs), including dendritic cells, thus providing peptides for direct presentation. However, the viral immune evasion genes profoundly impair recognition of infected cells by CD8+ T cells. Nevertheless, CMV infection elicits a very strong CD8+ T cell response, prompting its recent use as a vaccine vector. We have shown previously that deleting the immune evasion genes from murine cytomegalovirus (MCMV) that target class I MHC presentation, has no impact on the size or breadth of the CD8+ T cell response elicited by infection, suggesting that the majority of MCMV-specific CD8+ T cells in vivo are not directly primed by infected professional APCs. Here we use a novel spread-defective mutant of MCMV, lacking the essential glycoprotein gL, to show that cross-presentation alone can account for the majority of MCMV-specific CD8+ T cell responses to the virus. Our data support the conclusion that cross-presentation is the primary mode of antigen presentation by which CD8+ T cells are primed during MCMV infection.

Show MeSH
Related in: MedlinePlus