Figure 1.
GzmBW and GzmBP have distinct substrate preferences.
(A) Predicted amino acid sequences of the p (common inbred) and w (wild) alleles of mouse GzmB. A comparison with the sequences of human (RAH allele) and rat GzmB is shown, commencing with the 18-residue leader peptide and the activation peptide GE which is trimmed in the cytotoxic granules by cathepsin C or H. The mature protein commences with the tetrapeptide IIGG. Single amino acid code is used, with dots denoting identical amino acid at the same position. (B) Progress curves of recombinant granzymes at indicated concentrations cleaving the peptide thioester substrate Boc-Ala-Ala-Asp (AAD)-SBzl. (C) Interaction of recombinant granzymes with Serpinb9. One µg of granzyme was incubated with 10 µg serpin and complex formation assessed by 10% SDS-PAGE with Coomassie Blue staining. Note that all the granzyme shifts into complex (arrowed), indicating fully active preparations.
Table 1.
GzmBW has a substrate preference similar to human GzmB.
Figure 2.
GzmBW cleaves Bid more efficiently and procaspases less efficiently than GzmBP.
In vitro translated 35S-labeled mouse procaspase 3, mouse procaspase 7, or mouse Bid were incubated with the indicated amounts of granzymes at 37°C for 30 min. Products were separated by 15% SDS-PAGE and visualized by fluorography.
Figure 3.
GzmBW and GzmBP have equivalent cytotoxic potential in vitro.
(A) Mouse P815, (B) mouse EL-4, (C) human Jurkat and (D) HeLa cells were labeled with 51Cr, and exposed to sub-lytic concentrations of recombinant perforin in combination with either mouse p (black bars) or w (white bars) recombinant GzmB. Specific 51Cr release is shown as the mean of individual experiments (each performed in triplicate) ± SEM. The number of experiments performed were: P815 (n = 5), EL-4 (n = 2), Jurkat (n = 3) and HeLa (n = 4).
Table 2.
Non-synonymous polymorphisms in Gzm genes linked to GzmB on Chr 141.
Figure 4.
Granule enzyme activity and GzmB expression in T cell lysates from GzmBW/W and GzmBP/P mice.
(A) GzmB (ASPase) activity was measured as the maximum rate of cleavage of the peptide thioester substrate Boc-Ala-Ala-Asp (AAD)-SBzl or (B) the activity detected through cleavage of Ac-IEPD-pNA. (C) Chymotrypsin-like (chymase) activity (cleavage of Suc-Phe-Leu-Phe SBzl) was used as an independent measure of granule enzyme activity. The data points show the mean ± SEM of triplicate readings. The data is representative of 3 individual experiments. Equivalent results were obtained with lysates generated from IL-2 activated NK cells. (D) Western blot analysis for GzmB expression in OT1 T cells from outbred w/w mice, B6 and control GKO (B6.Pfp−/− and B6.GzmAB−/−) mice. The blot was re-probed for actin expression.
Figure 5.
GzmBW/W mice are sensitive to infection with Δm157 MCMV.
(A) B6 mice (black square) or GzmBW/W mice (white square) were infected with 2×104 pfu of MCMV Δm157 and survival monitored over the indicated time course (n = 5 for each group). ***P<0.0001. (B) The indicated mouse strains were infected with 2×104 pfu of MCMV Δm157, the indicated organs were removed at day 7 post-infection, and viral load quantified by plaque assay. Data are pooled from 2 independent experiments, mean ± SEM are plotted, where n≥8. *P<0.05. (C) Livers from uninfected or MCMV-infected mice were isolated at day 6 post-infection, fixed and tissue sections stained with haematoxylin and eosin. The results are representative of two independent experiments. (D) Liver enzymes in the serum of B6 mice (black bar) or GzmBW/W mice (white bar) were measured at day 6 post-infection. Data are pooled from two independent experiments where n≥5. *P<0.05.
Figure 6.
Increased viral load in mice lacking GzmB.
The indicated mouse strains were infected with 2×104 pfu of MCMV Δm157. At days 4 and 6 post-infection viral loads in the (A) spleen, (B) liver and (C) lungs were quantified by plaque assay. Data are pooled from 3 independent experiments, mean ± SEM are plotted, where n≥6. *P<0.05.**P<0.005.
Figure 7.
Liver damage in GzmBW/W mice is not the result of immune-mediated pathology.
(A) B6 mice (black bar) or GzmBW/W mice (white bar) were infected with Δm157 MCMV and total liver leukocytes, inflammatory monocytes, and granulocytes enumerated at the indicated times. Data are pooled from 2 independent experiments, mean ± SEM are plotted, where n≥5. *P<0.05. **P<0.01. (B) Levels of TNF-α and IFN-γ in the liver at the indicated times post-infection were quantified by ELISA. (C) Livers from uninfected or MCMV-infected mice at day 6 post-infection were stained with the Chroma 101 anti-IE1 antibody followed by detection with a DAB substrate. Sections were counterstained with haematoxylin. The results are representative of two independent experiments.
Figure 8.
GzmBW/W CD8 T cells are unable to lyse MCMV infected cells.
(A) The numbers of CD8 and CD4 T cells localizing to the liver of B6 mice (black bar) or GzmBw/w mice (white bar) after infection with MCMV Δm157 are shown. (B) At the indicated times post-infection, splenocytes were stained with anti-CD8, anti-TCRβ, and M45 tetramers. Representative FACS plots showing the percentage of M45-specific CD8 T cells are shown, and (C) the total numbers of M45-specific CD8 T cells are plotted. Data are pooled from 2 independent experiments, where n≥5. (D) Splenocytes were isolated from MCMV Δm157 infected B6 (black square) and GzmBw/w mice (white circle), or from uninfected B6 mice (white square) and GzmBw/w mice (black circle). Splenocytes were cultured with 51Cr-labeled M45 pulsed EL4 cells for 4 h and specific lysis determined. n = 6 for each data point. (E) CD8 T cells were purified from B6 mice (black square) and GzmBw/w mice (open circle) and co-cultured with MCMV infected IC-21 macrophages for 18 h at the indicated E∶T ratios. n = 5 for each data point.