Interaction of granzyme B and mouse embryonic fibroblasts deficient for Sulf1 and Sulf2

The textbook model of the uptake of granzymes into target cells during CTL-mediated cytotoxicity describes that perforin forms pores in the target cell membrane through which granzymes can enter the cell. This model was challenged by the finding that granzymes are sequestered in a complexed form bound to the 250 kDa chondroitin sulphate serglycin, which is then way too large to enter through those pores (Metkar et al. 2002; Raja et al.

2002). Furthermore, sublytic concentrations of perforin, which do not form pores, are suffi-cient to release granzymes from endosomes and to induce apoptosis (Froelich et al. 1996a).

Several other pathways for the uptake were suggested including dynamin-dependent and independent pathways (Tran et al. 2003; Veugelers et al. 2004) and pathways for which the MPR300 is essential (Motyka et al. 2000; Veugelers et al. 2006) or not (Dressel et al.

2004a, 2004b; Kurschus et al. 2005).

Raja and colleagues (Raja et al. 2005) could demonstrate that the anionic granzymes exchange from the cationic serglycin to more cationic sites on the cell surface (sulphated PGs). By using cell lines either lacking xylosyl transferase or HS polymerase, mutants with decreased levels of PGs or HSPGs, respectively, were gained. Both of them showed a diminished uptake of GrB indicating that those cell surface structures are involved in the uptake of GrB. The findings suggest that sulphation of cell surface HS plays a role in the uptake of GrB into target cells and led us to analyse the effect of the knock-out of HS 6-O-endosulphatases 1 and 2 on GrB uptkake. The avian QSULF1 and QSULF2

6 Discussion

desulphate cell surface HS (Ai et al. 2006, 2003). A knock-out of mouse Sulf1and Sulf2 genes leads to higher sulphated cell surface HS on MEFs (Lamanna et al. 2006).

It was tested, whether the increased sulphation of MEFs deficient for Sulf1or Sulf2 or both, would lead to elevated uptake of GrB and therefore elevated levels of apoptosis.

The level of GrB-uptake did not differ between the single Sulf clones as determined by the addition of Alexa488-labelled GrB for 60 min. Raja and colleagues (Raja et al. 2005) and also Kurschus and colleagues (Kurschus et al. 2005) detected a correlation between the density of sulphation of HS and the uptake of GrB. After treatment with sodium chlorate, which generally inhibits the sulphation of HS-chains, both groups could show a concentration-dependent reduction in GrB binding but also a 2.6-fold decrease in up-take of monomeric or complexed GrB in Jurkat and CHO-K1 cells. According to our results an increase in sulphation of HS-chains does not augment the levels of GrB binding and uptake. This does not necessarily indicate a discrepancy between our findings and literature. Sodium chlorate is a selective inhibitor of ATP sulphurylase (ATP sulphate adenylyltransferase), the first enzyme in the sulphate activation pathway, to activate sul-phation. ATP sulphurylase does not only activate sulphate synthesis but also catalyses GTP-hydrolysis, both of which reactions are coupled, implying the possibility that more than just the sulphation of HSs is abrogated (Sukal and Leyh 2001). On the other hand, avian QSULF1 and QSULF2 were not only shown to specifically desulphate cell surface HS but also cell matrix HS (Ai et al. 2006). The enzyme specificity of SULF1 and SULF2 is not restricted to di- and tri-sulphated 6S disaccharide units within the HS chain, but additionally the Sulf1 and Sulf2 genes also have an impact on the 6O-sulphotransferase activity (Lamanna et al. 2008). Therefore, the knock-out ofSulf1and Sulf2 leads to spe-cific patterns of sulphation and not to a general suppression of sulphation as in a sodium chlorate treatment. Furthermore, it might be that a higher level of sulphation does not increase binding and uptake of GrB into cells, because a saturation could occur maybe caused by steric hindrance.

To analyse whether CTL-mediated lysis is affected by the deficiency of the Sulf1 and Sulf2 genes, ⁵¹Chromium-release assays were performed with the MEFs. ⁵¹ Chromium-release assays are considered to show perforin-dependent lysis as the Chromium-release of⁵¹Chromium just requires the disruption of the cell membrane. This disruption can be achieved by the formation of pores by perforin but also in the final stages of apoptosis induced by granzymes after about 2 hrs and 20 min of incubation with killer cells as determined by time-lapse microscopy (Waterhouse et al. 2006b).

The increased sulphation of the HSPG on the cell surface of cells double-deficient for Sulf1and Sulf2 led to a slightly but significantly higher lysis by antigen-specific CTLs in comparison to Wt cells with a p-value of less than 0.001 as determined with ANOVA. Most

6 Discussion

of the phenotype of the Sulf Dko cell line could be accredited to the deficiency of Sulf1, as comparisons of Wt andSulf1^−/− cells showed (p < 0.01). Although, the uptake of GrB was not increased by elevated levels of sulphation of HS chains on MEFs deficient forSulf1 and Sulf2, the perforin-dependent lysis seemed to be increased especially in the double-deficient cell line. In all cytotoxic experiments with the cell lines double-deficient for Sulf1 and Sulf2 a control with EGTA was performed. EGTA is a chelating agent of divalent cations such as Ca²⁺ and can remove those cations so that granule secretion is blocked and no calcium-dependent killing can take place. The killing was always very low in this control meaning that hardly any killing not being calcium-dependent takes place and therefore the granule-exocytosis pathway is the pathway mainly used to lyse target cells. A second negative control was performed in which no SIINFEKL peptide was added during the 4 hrs of co-incubation of CTLs and target cells. The absence of killing in these controls indicated that no peptide-independent killing took place, meaning that only peptide-specific CTLs caused the lysis of target cells.

Next, the susceptibility of theSulf clones towards apoptosis induced by peptide-specific CTLs was determined in [³H]-Thymidine assays, which are considered to be GrB-depen-dent. [³H]-Thymidine-release assays indicate apoptotic DNA fragmentation. No difference between theSulf double-deficient and the Wt clones could be detected here (p = 0.167).

Also for the cell lines deficient for eitherSulf1(p = 0.10) orSulf2(p = 0.15) no difference in killing could be detected in comparison to Wt cells. In general, the percentage of apoptosis was comparibly low in those tests and even the stimulation of target cells with IFN-γ to increase H2K^b expression did not yield better results. The results of the [³H]-Thymidine assays are therefore not fully conclusive. For all cytotoxic assays withSulf-deficient cells, a Wt was chosen having a nearly identical H2K^b expression. Thus a difference in lysis between the cell lines caused by differential expression of the SIINFEKL restriction element H2K^b can be excluded.

Therefore, a specific GrB assay was performed, where GrB was delivered into the Sulf MEFs by using AdV type 5 as an endosomolytic agent to induce GrB-dependent apoptosis, which was analysed by sub G1-peak analysis in flow cytometry. No significant difference in GrB-induced apoptosis was detected between the 4 differentially sulphated MEFs clones.

Thus, no difference in apopototic killing was found in [³H]-Thymidine release assays and in GrB-induced apoptosis between theSulf clones. This is in accordance with the findings that the uptake of GrB did not differ between the single Sulf clones. The difference in lysis between the Sulf double-deficient and the Sulf1-deficient clone in comparison to the Wt in ⁵¹Chromium-release assays is therefore assumed to be perforin-dependent. This is in accordance with literature. It was recently shown that CHO mutant cells A745 (lacking xylosyl transferase and therefore HSPGs) showed less membrane permeabilisation

6 Discussion

by perforin than Wt cells did, implying that perforin might bind to HS as well (Metkar et al. 2005). Thus, perforin might bind with different affinity to higher sulphated HSs and a higher sulphation leads to a higher lysis. Sulf1-deficiency led to increased sulphation on target cells and might cause increased perforin-dependent lysis. In contrast to granzymes, perforin is not redundant but instead essential for CTLs and NK cells as mentioned in the introduction.

Thus, an alteration of perforin binding by an alteration of HS sulphation patterns might be functionally important.

Generally, one can say that the question of how GrB is taken-up into cells is still not ultimately solved, whereby it is very likely that more than one pathway exists. For ex-ample, mutants of GrB reduced in their ability to bind to HS were not endocytosed but still successfully delivered into the cytosol of the target cells. Probably translocation into the cytosol of target cells is achieved by a process involving repairable membrane pores (Kurschus et al. 2008).

6.2.2 Uptake of adenovirus type 5 into mouse embryonic fibroblasts deficient