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CD8+ T Cell Cross-Priming via Transfer of Proteasome Substrates

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Science  28 May 2004:
Vol. 304, Issue 5675, pp. 1318-1321
DOI: 10.1126/science.1096378
  • Fig. 1.

    Schematic of expression vector–encoded gene products used to study cross-priming. The gene constructs utilized in this study are schematically diagrammed.

  • Fig. 2.

    Cross-priming does not require proteasome activity in virus-infected cells. (A) Uninfected β2mneg cells (UN) or cells infected with VV-ovalbumin (OVA) were injected 4 hours after infection into mice that had received OT-I TCR CD8+ T cells 3 hours previously. “OVA/LC” cells were treated with the irreversible proteasome inhibitor lactacystin, which was started 1 hour before infection. Sixteen hours after immunization, activation of CD8+, Vα2+ OT-1 T cells was determined via measurement of increased expression of CD69 by flow cytometry. Data are expressed as the percentage of CD69 highly expressing cells. These data are representative of nine consecutive experiments in which immunogenicity was either enhanced (3 times) or remained unaffected by lactacystin treatment (6 times). (B) Mice were immunized intraperitoneally (i.p.) with IAV in the presence or absence of a neutralizing monoclonal antibody (mAb) against hemagglutinin (α-HA). Six days later, influenza virus–specific CD8+ T cells present in spleens were enumerated by flow cytometry using Db-NP366-374 molecules tetramerized by binding to fluorescent strepavidin. The percentage of CD8+ T cells that specifically bind tetramers is shown. (C) Uninfected β2mneg cells or β2mneg cells infected with IAV for 5 hours were injected i.p. in the presence of a neutralizing mAb against HA, and CD8+ T cells were enumerated as in (B). Where indicated, cells were treated with lactacystin (LC) from 1 hour before infection. The data shown represent the mean ±SEM of duplicate mice and are representative of four similar experiments.

  • Fig. 3.

    Cross-priming is not mediated by exogenous or endogenous peptides. (A) We measured binding of Ova257-264 to splenocytes derived from B6 or Kbm1 mice by flow cytometry using a fluorescent version of the peptide that mimics the binding of the unmodified peptide. An additional experiment yielded similar results. MCF, mean channel flourescence. (B) Splenocytes from B6 or Kbm1 mice pulsed with synthetic Ova257-264 peptide at the indicated concentrations were injected into B6 mice into which fluorescent OT-I TCR transgenic CD8+ T cells were adoptively transferred 3 hours previously. The percentage of dividing OT-I cells was determined flow cytometrically by decreased cellular fluorescence. This experiment was repeated twice with similar results. (C and D). B6 mice were immunized with the rVV expressing the indicated gene product (C) or with P815 cells infected for 12 hours with rVVs expressing the gene product indicated (D). Six days later, numbers of responding Ova257-264-specific CD8+ T cells present in spleens was determined by their expression of IFN-γ. Data represent averages from three mice for each group; errors bars show ±SEM. The figure is representative of nine additional experiments performed with P815 or HeLa cells. (E) After 293 cells were transfected 24 hours earlier with plasmids encoding the indicted gene product, they were introduced into B6 mice, and the responding CD8+ T cells enumerated by expression of IFN-γ. Averages from four mice for each group with SEM are shown. An additional experiment yielded similar results.

  • Fig. 4.

    Rapidly degraded proteins are not substrates for cross-priming. Mice were immunized with rVV expressing the protein indicated (A) or P815 cells infected for 12 hours with rVVs expressing the gene product indicated (B). Six days after immunization, numbers of responding splenic Ova257-264-specific CD8+ T cells were determined by their expression of IFN-γ. Where indicated, 20 μM lactacystin was added to cells 1 hour after addition of virus and was maintained throughout the 12-hour infection. Averages from three mice for each group. This experiment was repeated three times with similar results.

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  • Abstract
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    CD8+ T Cell Cross-Priming via Transfer of Proteasome Substrates
    C. C. Norbury, S. Basta, K. B. Donohue, D. C. Tscharke, M. F. Princiotta, P. Berglund, J. Gibbs, J. R. Bennink, J. W. Yewdell

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