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. 2011 Apr;41(4):926-35.
doi: 10.1002/eji.201041040. Epub 2011 Mar 1.

PA28 and the proteasome immunosubunits play a central and independent role in the production of MHC class I-binding peptides in vivo

Natascha de Graaf  1 , Mary J G van Helden, Kathrin Textoris-Taube, Tomoki Chiba, David J Topham, Peter-Michael Kloetzel, Dietmar M W Zaiss, Alice J A M Sijts
Affiliations

PA28 and the proteasome immunosubunits play a central and independent role in the production of MHC class I-binding peptides in vivo

Natascha de Graaf et al. Eur J Immunol. 2011 Apr.

Abstract

Proteasomes play a fundamental role in the processing of intracellular antigens into peptides that bind to MHC class I molecules for the presentation of CD8(+) T cells. Three IFN-γ-inducible catalytic proteasome (immuno)subunits as well as the IFN-γ-inducible proteasome activator PA28 dramatically accelerate the generation of a subset of MHC class I-presented antigenic peptides. To determine whether these IFN-γ-inducible proteasome components play a compounded role in antigen processing, we generated mice lacking both PA28 and immunosubunits β5i/LMP7 and β2i/MECL-1. Analyses of MHC class I cell-surface levels ex vivo demonstrated that PA28 deficiency reduced the production of MHC class I-binding peptides both in cells with and without immunosubunits, in the latter cells further decreasing an already diminished production of MHC ligands in the absence of immunoproteasomes. In contrast, the immunosubunits but not PA28 appeared to be of critical importance for the induction of CD8(+) T-cell responses to multiple dominant Influenza and Listeria-derived epitopes. Taken together, our data demonstrate that PA28 and the proteasome immunosubunits use fundamentally different mechanisms to enhance the supply of MHC class I-binding peptides; however, only the immunosubunit-imposed effects on proteolytic epitope processing appear to have substantial influence on the specificity of pathogen-specific CD8(+) T-cell responses.

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Figures

Figure 1
Figure 1. Kinetic analysis of the effects of PA28 and immunosubunits on generation of HBV cAg 131-162 cleavage products
(A) Amino acid sequence of the HBV cAg131-162 polypeptide substrate. Arrows indicate dominant cleavage sites of constitutive or immunoproteasomes (32). (B) HBV cAg131-162 was incubated with constitutive or immunoproteasomes of T2 or T2 + β1i/LMP2+β5i/LMP7 cells, in the absence or presence of a four-molar excess of PA28, purified from induced MEC-PA28 cells [6], as indicated. Digestion products were separated by RP-HPLC and identified by mass spectrometry. Accumulation of different peptide products over time of digestion is shown. HBV cAg131-162 incubated with PA28 without proteasomes was not degraded.
Figure 2
Figure 2. Absence of PA28 and immunosubunit expression in gene-deficient mouse strains
Splenocytes of indicated mouse strains were extracted, the lysates were separated by SDS/PAGE, and β5i/LMP7, β2i/MECL-1, PA28α and PA28β were detected by Western blotting.
Figure 3
Figure 3. Effects of PA28 and immunosubunits on CD4:CD8 T cell ratios in lymphoid tissues of uninfected mice
(A) CD8 T cells were detected in the spleens of B6 wt, PA28-/-, β5i/LMP7+β2i/MECL-1-/- and PA28+β5i/LMP7+β2i/MECL-1-/- mice by staining with anti-CD8α mAb and FACS analysis. Frequencies of CD8 T cells as percentage of total lymphocytes are shown (means plus SEM, n=3). (B) CD4:CD8 T cell ratios in spleen and thymus of B6 wt, PA28-/-, β5i/LMP7+β2i/MECL-1-/- and PA28+β5i/LMP7+β2i/MECL-1-/- mice. Data are representative for two independent experiments (means plus SEM; n=3).
Figure 4
Figure 4. Role of immunosubunits and PA28 in the production of MHC class I ligands
(A) Splenocytes of uninfected mice and mice, infected 8 days earlier with rLM, were stained with anti-B220 and a conformation-dependent anti-H-2Kb mAb. (B) Unstimulated and LPS-stimulated splenocytes of uninfected mice were stained with anti-CD19 and anti-H-2Kb mAbs. MFI of H-2Kb staining on B cells are shown (means plus SEM; n=3). (C) Upregulation of MHC class I H-2Kb cell surface levels on B220+ splenocytes of rLM-infected mice compared to uninfected mice (left panel) or on LPS-stimulated compared to unstimulated B220+ splenocytes (right panel). MFI are shown (means plus SD; n=2-4).
Figure 5
Figure 5. Effects of PA28 and immunosubunits on T cell responses to rLM- and Influenza-derived antigens
B6 wt, PA28-/-, β5i/LMP7+β2i/MECL-1-/- and PA28+β5i/LMP7+β2i/MECL-1-/- mice were infected with 5×103 rLm-E1 (A), 5×104 U Influenza/HKx31 (B) or 1×105 rLM-ova (C) or immunized i.v. with PA224-233-pulsed BM DC (D). (A, C) Eight days after rLM-infection, frequencies of E1B192-200- or ova257-264 -specific CD8 T cells or LLO189-201-specific CD4 T cells were determined in the spleen by staining for CD8 or CD4 and intracellular IFNγ. B, Percentages of NP366-374 and PA224-233 specific CD8 T cells in the BAL, seven days after Influenza infection. D, Percentages of PA224-233-specific CD8 T cells in the spleen, seven days after injection of peptide-pulsed DC. Background responses measured in the absence of stimulating peptides were subtracted. Data are representative for 2-4 experiments. Means plus SEM (A-C) or values for individual mice (D) are shown.
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