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. 2010 May 1;184(9):5193-9.
doi: 10.4049/jimmunol.1000050. Epub 2010 Mar 26.

Caspases in virus-infected cells contribute to recognition by CD8+ T lymphocytes

Daniel López  1 , Margarita García-Calvo, Geoffrey L Smith, Margarita Del Val
Affiliations

Caspases in virus-infected cells contribute to recognition by CD8+ T lymphocytes

Daniel López et al. J Immunol. .

Abstract

CD8(+) cytotoxic T lymphocytes recognize infected cells in which MHC class I molecules present pathogen-derived peptides that have been processed mainly by proteasomes. Many infections induce a set of proteases, the caspases involved in apoptosis or inflammation. In this study, we report that processing and presentation of a short vaccinia virus-encoded Ag can take place also by a nonproteasomal pathway, which was blocked in infected cells with chemical inhibitors of caspases. By cleaving at noncanonical sites, at least two caspases generated antigenic peptides recognized by T lymphocytes. The sites and the peptidic products were partially overlapping but different to those used and produced by proteasomes in vitro. Antigenic natural peptides produced in infected cells by either pathway were quantitatively and qualitatively similar. Finally, coexpression of the natural vaccinia virus protein B13, which is an inhibitor of caspases and apoptosis, impaired Ag presentation by the caspase pathway in infected cells. These data support the hypothesis that numerous cellular proteolytic systems, including those induced during infection, such as caspases involved in apoptosis or in inflammation, contribute to the repertoire of presented peptides, thereby facilitating immunosurveillance.

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Figures

FIGURE 1
FIGURE 1. Diagram showing nature of the rVACV.
The rVACV-m19 and all viruses derived from vaccinia virus strain Copenhagen have the B13R gene disrupted. In contrast, this gene is functional in vaccinia virus strain WR.
FIGURE 2
FIGURE 2. Ag-presentation assays with proteasome and caspases inhibitors.
L/Ld cells were infected overnight with 10 PFU/cell of rVACV-m19 or rVACV-m17 in the presence of LC, z-VAD-fmk (z-VAD), or z-FA-fmk (z-FA) and tested with 9pp89-specific CTLs (A, B). Similarly, L/Dd cells were infected with rVACV-sA (C) or rVACV-sI (D) and tested with HIV ENV-specific CTLs. Inhibitors were present throughout infection and during [51Cr] labeling until addition of specific CTLs to target cells. The percentage specific lysis in the absence of LC was 45%, 50%, 45%, and 39% for m19, m17, sA, and sI, respectively, and was used as a reference to calculate percentage of specific inhibition, which is expressed as the mean ± SD (n = 6).
FIGURE 3
FIGURE 3. In vitro digestions of m19 synthetic peptide with different caspases (Casp) or proteasomes (Prot).
The m19 synthetic peptide was digested with the indicated purified proteases. Peptides generated in each digestion were separated by RP-HPLC. Fractions were tested with 9pp89-specific CTLs to analyze the presence of antigenic peptides. The results obtained with each protease in the cytotoxicity assay are depicted. The arrows indicate antigenic peaks other than the m19 substrate peptide.
FIGURE 4
FIGURE 4. Digestion pattern of m19 synthetic peptide with purified proteasomes or different caspases analyzed by MS.
The m19 synthetic peptide was digested with the indicated purified proteases (A) or negative controls (B) and analyzed by MS/MS. The sequence of the CMV epitope is boxed. Horizontal lines show peptide products identified by MS/MS analysis. Line thickness indicates approximate amount detected. The arrows indicate cleavages. Their thickness indicates frequency of each cleavage, deduced from the approximate amounts of identified peptide products. Amounts are depicted relative to the most abundant product within each digestion.
FIGURE 5
FIGURE 5. Natural endogenous peptides generated after processing of m19 in infected cells.
Naturally processed peptides were acid-extracted from L/Ld cells infected overnight with 10 PFU/cell of rVACV-m19 in the presence of LC (■), z-VAD-fmk (▲), or nothing (○) and separated by RP-HPLC. Fractions were tested with 9pp89-specific CTLs to analyze the presence of antigenic peptides.
FIGURE 6
FIGURE 6. Activation of caspases after vaccinia virus infection.
Inhibition by the vaccinia virus protein B13. L/Ld cells were infected for 4 h with the PFU/cell shown in brackets with the indicated vaccinia viruses or combinations of two viruses (A–D). Refer to Fig. 1 for presence of B13R in each virus. The percentage of apoptotic cells was determined by flow cytometry after staining with CaspACE FITC-VAD-fmk. CTL-L2 cells in A were incubated for 4 h with 1 μM staurosporine (ST).
FIGURE 7
FIGURE 7. Inhibition of Ag presentation to CTLs by a combination of LC and the vaccinia virus protein B13.
L/Ld cells were singly infected overnight in the presence of LC with 10 PFU/cell of rVACV-m19 (■) or a negative control (●) or doubly infected with 10 PFU/cell of either vΔB13R (▲) or vB13R+rev (lines). LC was kept throughout infection and [51Cr] labeling until the addition of CTLs to target cells. The percentage specific lysis at each E:T ratio is shown on the vertical axis (A). The percentage specific inhibition caused by the coinfection is plotted as the mean ± SD (n = 4) of all E:T ratios (B).
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