The lysosomal protease cathepsin B has been proposed to protect cytotoxic T lymphocytes from the membrane-disruptive effects of perforin secreted during the execution phase of target cell death. Accordingly, cathepsin B that translocates to the lymphocyte surface upon degranulation has been postulated to cleave and inactivate perforin molecules that diffuse back to the killer cell. We have found that recombinant perforin is cleaved inefficiently by cathepsin B and shows no significant reduction in its lytic activity following co-incubation. Furthermore, purified CD8+ cytotoxic T lymphocytes of cathepsin B-null gene-targeted mice were able to induce normal death of target cells both in vitro and in vivo and to survive the encounter with target cells as efficiently as cathepsin B-expressing killer cells. We conclude that cathepsin B is not essential for protection of cytotoxic lymphocytes from the toxic effects of their secreted perforin. The lysosomal protease cathepsin B has been proposed to protect cytotoxic T lymphocytes from the membrane-disruptive effects of perforin secreted during the execution phase of target cell death. Accordingly, cathepsin B that translocates to the lymphocyte surface upon degranulation has been postulated to cleave and inactivate perforin molecules that diffuse back to the killer cell. We have found that recombinant perforin is cleaved inefficiently by cathepsin B and shows no significant reduction in its lytic activity following co-incubation. Furthermore, purified CD8+ cytotoxic T lymphocytes of cathepsin B-null gene-targeted mice were able to induce normal death of target cells both in vitro and in vivo and to survive the encounter with target cells as efficiently as cathepsin B-expressing killer cells. We conclude that cathepsin B is not essential for protection of cytotoxic lymphocytes from the toxic effects of their secreted perforin. Cytotoxic T lymphocytes (CTL) 3The abbreviations used are: CTL, cytotoxic T lymphocytes; CatB, cathepsin B; CFSE, carboxyfluorescein diacetate, succinimidyl ester; NK, natural killer. recognize virus-infected or transformed cells and induce their apoptotic death through two independent mechanisms. The first involves death receptor triggering to bring about classic caspase-dependent apoptosis of the target cell (1Nagata S. Golstein P. Science. 1995; 267: 1449-1456Crossref PubMed Scopus (3991) Google Scholar). The granule exocytosis pathway constitutes the second mechanism and involves the secretion of cytotoxic proteins from lysosome-like granules toward the target cell following conjugate formation (2Jenne D.E. Tschopp J. Immunol. Rev. 1988; 103: 53-71Crossref PubMed Scopus (239) Google Scholar). The granule constituents cooperate to induce both caspase-dependent and -independent death of the target cell (3Trapani J.A. Smyth M.J. Nat. Rev. Immunol. 2002; 2: 735-747Crossref PubMed Scopus (918) Google Scholar). Perforin is a key component of cytotoxic granules, and its crucial role in the clearance of intracellular pathogens and immune surveillance is evident from studies on gene-engineered perforin-deficient mice. These mice are unable to clear a variety of viruses despite having a functional death receptor pathway, and the majority develop spontaneous B cell lymphoma as they age (4Kagi D. Ledermann B. Burki K. Seiler P. Odermatt B. Olsen K.J. Podack E.R. Zinkernagel R.M. Hengartner H. Nature. 1994; 369: 31-37Crossref PubMed Scopus (1542) Google Scholar, 5Smyth M.J. Thia K.Y. Street S.E. MacGregor D. Godfrey D.I. Trapani J.A. J. Exp. Med. 2000; 192: 755-760Crossref PubMed Scopus (448) Google Scholar). Perforin's precise mechanism of action is unclear. It is commonly believed that following target cell recognition, perforin and other components of the cytotoxic granules are released into the immunological synapse where exposure to millimolar calcium levels and neutral pH results in perforin monomers binding and inserting into the target membrane, forming transmembrane channels that can lead to loss of plasma membrane homeostasis and osmotic lysis (6Trapani J.A. Int. Rev. Cytol. 1998; 182: 111-192Crossref PubMed Google Scholar). Perforin is also essential for the induction of apoptotic death in cooperation with granule proteases called granzymes, especially granzyme B, which (like caspases) can cleave its substrates adjacent to key aspartate residues (3Trapani J.A. Smyth M.J. Nat. Rev. Immunol. 2002; 2: 735-747Crossref PubMed Scopus (918) Google Scholar, 7Sutton V.R. Davis J.E. Cancilla M. Johnstone R.W. Ruefli A.A. Sedelies K. Browne K.A. Trapani J.A. J. Exp. Med. 2000; 192: 1403-1414Crossref PubMed Scopus (312) Google Scholar, 8Sutton V.R. Wowk M.E. Cancilla M. Trapani J.A. Immunity. 2003; 18: 319-329Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). It has been proposed that granzyme uptake occurs in response to “repair” of plasma membrane perturbations brought about by perforin (9Keefe D. Shi L. Feske S. Massol R. Navarro F. Kirchhausen T. Lieberman J. Immunity. 2005; 23: 249-262Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Therefore, target cells exposed to high concentrations of purified granzyme B remain healthy unless perforin is also present in low concentrations (10Waterhouse N.J. Sedelies K.A. Browne K.A. Wowk M.E. Newbold A. Sutton V.R. Clarke C.J. Oliaro J. Lindemann R.K. Bird P.I. Johnstone R.W. Trapani J.A. J. Biol. Chem. 2005; 280: 4476-4482Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). CTL are relatively resistant to their granule toxins, allowing them to consecutively kill several target cells in vitro (11Martz E. Transplantation. 1976; 21: 5-11Crossref PubMed Scopus (48) Google Scholar, 12Isaaz S. Baetz K. Olsen K. Podack E. Griffiths G.M. Eur. J. Immunol. 1995; 25: 1071-1079Crossref PubMed Scopus (194) Google Scholar). To date, it remains unclear how CTL are protected from the toxic effects of perforin, particularly following its exocytosis into the immunological synapse. Perforin biosynthesis and trafficking are closely regulated to minimize the possibility of inadvertent damage to the CTL organelles. It is thought that perforin is synthesized as an inactive precursor and that its lytic activity is achieved only when a heavily glycosylated C-terminal oligopeptide is cleaved after packaging within the secretory granules (13Uellner R. Zvelebil M.J. Hopkins J. Jones J. MacDougall L.K. Morgan B.P. Podack E. Waterfield M.D. Griffiths G.M. EMBO J. 1997; 16: 7287-7296Crossref PubMed Scopus (165) Google Scholar). Thereafter, the acidic pH of this compartment prevents perforin from binding calcium ions, an obligate requirement for lipid binding and insertion (14Voskoboinik I. Thia M.C. Fletcher J. Ciccone A. Browne K. Smyth M.J. Trapani J.A. J. Biol. Chem. 2005; 280: 8426-8434Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). But how perforin is able to selectively damage the target cell and spare the CTL after its release remains unclear. One hypothesis suggests that a CTL granule component may act as a specific inhibitor of perforin following degranulation. Recently, a role for the lysosomal protease Cathepsin B (CatB) was proposed (15Balaji K.N. Schaschke N. Machleidt W. Catalfamo M. Henkart P.A. J. Exp. Med. 2002; 196: 493-503Crossref PubMed Scopus (181) Google Scholar). CatB is a papain-like cysteine protease that has been reported to participate in various biological processes such as apoptosis induction, enzyme activation, and tissue remodeling (16Turk B. Turk D. Turk V. Biochim. Biophys. Acta. 2000; 1477: 98-111Crossref PubMed Scopus (705) Google Scholar). Following granule exocytosis, enzymatically active CatB was found to be expressed transiently on the surface of degranulating T cells, and inhibition of CatB in vitro resulted in the death of CD8+ T cells, resulting in reduced target cell killing. The authors concluded that CatB may cleave and inactivate perforin molecules that diffuse back to the CTL surface following their release; however, the efficiency of this interaction was not studied (15Balaji K.N. Schaschke N. Machleidt W. Catalfamo M. Henkart P.A. J. Exp. Med. 2002; 196: 493-503Crossref PubMed Scopus (181) Google Scholar). In the current study, we further investigated the possible role for CatB in CTL protection from perforin in a more physiological setting by studying the lymphocytes of CatB-deficient mice. We found that the CTL are able to induce normal target cell death both in short- and long-term killing assays and are not compromised in inducing target cell death in vivo. We therefore conclude that CatB is dispensable for the protection of CTL from their secreted perforin. Antibodies and Other Reagents—Rat anti-perforin monoclonal antibody P1–8 was used as described (17Kawasaki A. Shinkai Y. Kuwana Y. Furuya A. Iigo Y. Hanai N. Itoh S. Yagita H. Okumura K. Int. Immunol. 1990; 2: 677-684Crossref PubMed Scopus (96) Google Scholar). A polyclonal antiserum detecting human fibronectin was purchased from Sigma. Anti-mouse CD107a (lamp-1) was purchased from BD Biosciences. Anti-mouse CD3 antibody (clone 2C11) was used to induce degranulation through T cell receptor ligation. T cell markers CD8, TCR Vα2, CD25, CD44, and CD69 were purchased from BD Biosciences. Propidium iodide was purchased from Sigma. Recombinant mouse perforin was purified from baculovirus-infected cells as described (18Voskoboinik I. Thia M.C. De Bono A. Browne K. Cretney E. Jackson J.T. Darcy P.K. Jane S.M. Smyth M.J. Trapani J.A. J. Exp. Med. 2004; 200: 811-816Crossref PubMed Scopus (60) Google Scholar). CatB purified from human liver and fibronectin from human plasma were purchased from Calbiochem. The CatB inhibitor CA074 and general cysteine protease inhibitor E64 were purchased from Sigma, dissolved as a 50-mm stock solution in dimethyl sulfoxide, and stored in aliquots at –70 °C. The fluorogenic substrate Z-Arg-Arg-aminomethylcoumarin was purchased from Sigma. The peptide SIINFEKL (OVA, amino acids 257–264) was purchased from Auspep. 51Cr (as sodium dichromate) was from Amersham Biosciences. CFSE (carboxyfluorescein diacetate, succinimidyl ester) was purchased from Invitrogen and dissolved as a 5-mm stock solution in dimethyl sulfoxide and stored in aliquots at –20 °C. Precast 4–20% Tris/glycine SDS gradient gels were purchased from Invitrogen. Mice—C57BL/6 mice were purchased from the Walter and Eliza Hall Animal Facility (Parkville, Australia). CatB–/– mice were generated on a BL/6 background (19Deussing J. Roth W. Saftig P. Peters C. Ploegh H.L. Villadangos J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4516-4521Crossref PubMed Scopus (234) Google Scholar, 20Halangk W. Lerch M.M. Brandt-Nedelev B. Roth W. Ruthenbuerger M. Reinheckel T. Domschke W. Lippert H. Peters C. Deussing J. J. Clin. Investig. 2000; 106: 773-781Crossref PubMed Scopus (475) Google Scholar). CatB–/– mice were crossed with OT-I (OVA-specific, H-2Kb-restricted T cell receptor transgenic) mice to generate OT-I transgenic CatB+/+, CatB+/–, and CatB–/– mice (21Hogquist K.A. Jameson S.C. Heath W.R. Howard J.L. Bevan M.J. Carbone F.R. Cell. 1994; 76: 17-27Abstract Full Text PDF PubMed Scopus (2320) Google Scholar). Genotyping of the mice for CatB status was carried out by PCR using the following primers: 5′-GGTTGCGTTCGGTGAGG-3′ and 5′-AACAAGAGCCGCAGGAGC-3′. Expression of the OT-I transgenic T cell receptor was assessed by co-staining peripheral blood lymphocytes with anti-CD8 and anti-Vα2 antibodies and followed by flow cytometry. Cells and Cell Culture—All cells used in this study were cultured at 37 °C in a humidified CO2 incubator in medium supplemented with 2 mm glutamine and 10% fetal bovine serum. The mouse lymphoma cell line EL4 was maintained in Dulbecco's modified Eagle's medium and Jurkat human lymphoma cells in RPMI. For the generation of mouse CTLs, splenocytes from naïve C57BL/6 mice were pulsed with the cognate peptide SIINFEKL (presented on Kb, 1 μg/ml) for 60 min at 37 °C, lethally irradiated, and cultured at a 1:1 ratio with responder splenocytes harvested from CatB-sufficient or -deficient mice. CD8+ cells were purified by using and the cell CTL were generated by lymphocyte as described V. E. L. M. H. T. Trapani J. Transplantation. PubMed Scopus Google Scholar). of CTL was by for and the markers CD25, CD44, and CD69 and by flow using a of Cathepsin Cathepsin B, by its to be active was in found to be active as by an active with the inhibitor E64 as described A.A. H. M. K. J. PubMed Scopus Google Scholar). In substrate various of CatB were with Z-Arg-Arg-aminomethylcoumarin substrate for min at 37 °C in sodium or 1 mm In the specific CatB inhibitor CA074 was also was on a at For of mouse perforin, substrate and protease were at a ratio of the for For of the were used as for perforin the CatB was on a of perforin and fibronectin were on 10% and 4–20% gradient to and with anti-perforin or The lytic activity of perforin following was by perforin with CatB with 2 Jurkat cells for 2 at followed by of 51Cr release into the using a assays CD8+ CTL were at various with EL4 target cells pulsed with SIINFEKL for 1 at 37 or the was harvested and 51Cr release was on a For the spontaneous release of the of the was not 10% of To CTL during the purified CTL were with 51Cr in the and with EL4 or with CTL in mice. in vivo was assessed by the of or target cells from the of CatB-deficient or mice following into the The was to that described R.M. Heath W.R. Carbone F.R. J. Immunol. 2002; PubMed Scopus Google Scholar). In the cells were with high CFSE and the cells with low CFSE, in other the CFSE concentrations were The following was used to specific was as CFSE CFSE lysis was 1 ratio of peptide R.M. Heath W.R. Carbone F.R. J. Immunol. 2002; PubMed Scopus Google Scholar). of CTL to were to by with antibody as described (15Balaji K.N. Schaschke N. Machleidt W. Catalfamo M. Henkart P.A. J. Exp. Med. 2002; 196: 493-503Crossref PubMed Scopus (181) Google Scholar). CD8+ CTL were with various of recombinant mouse perforin the or of the general cathepsin inhibitor for 2 at 37 °C. was by of the lysosomal CD107a on the cell surface as by flow cytometry. Recombinant Perforin a for CatB in has been proposed that CatB from the lysosomal compartment to the surface of degranulating CTL can cleave and inactivate perforin molecules that diffuse back to the CTL following exocytosis (15Balaji K.N. Schaschke N. Machleidt W. Catalfamo M. Henkart P.A. J. Exp. Med. 2002; 196: 493-503Crossref PubMed Scopus (181) Google Scholar). mechanism the of CTL to however, the of CatB to inactivate purified perforin has not been To further this we the of CatB to cleave recombinant mouse perforin in CatB specific activity of as by active was able to cleave the oligopeptide substrate Z-Arg-Arg-aminomethylcoumarin at granule or neutral pH and cleaved the substrate fibronectin to and The of the was by the inhibition of when the specific CatB inhibitor CA074 was in CA074 has been on the of the general protease inhibitor E64 and is to to the only in CatB A. T. K. T. T. Y. M. K. J. 1997; PubMed Scopus Google Scholar). recombinant mouse perforin and purified CatB were for min at 37 °C a variety of and a ratio was used with of the perforin a of was also by at both acidic and neutral the generation of which was by CA074 To perforin reduced its lytic we the perforin with Jurkat cells, which are to perforin at pH (14Voskoboinik I. Thia M.C. Fletcher J. Ciccone A. Browne K. Smyth M.J. Trapani J.A. J. Biol. Chem. 2005; 280: 8426-8434Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). We found that perforin activity was not reduced following exposure to CatB at acidic or neutral pH with and Perforin that been with CatB at pH for min lysis acidic perforin is unable to calcium (14Voskoboinik I. Thia M.C. Fletcher J. Ciccone A. Browne K. Smyth M.J. Trapani J.A. J. Biol. Chem. 2005; 280: 8426-8434Abstract Full Text Full Text PDF PubMed Scopus (126) Google and remains of the pH is to In perforin to at pH in the of calcium can and was to activity (6Trapani J.A. Int. Rev. Cytol. 1998; 182: 111-192Crossref PubMed Google it was to for the of CatB Accordingly, perforin activity the however, exposure to active CatB not further lysis the CTL from CatB-deficient Cell in CatB CTL from after a possible of CatB be the of CTL following with target cells. To this possibility in an we crossed CatB-deficient mice with mice the transgenic T cell receptor which can recognize the peptide SIINFEKL on target cells (21Hogquist K.A. Jameson S.C. Heath W.R. Howard J.L. Bevan M.J. Carbone F.R. Cell. 1994; 76: 17-27Abstract Full Text PDF PubMed Scopus (2320) Google Scholar). of CD8+ T cells with a of markers were generated from CatB-deficient and as by co-staining with antibodies and the markers CD25, CD44, and CD69 not by the purified CD8+ CTL was through as it was when was with at a high ratio EL4 mouse cells used as only low levels of functional and are not to cell death. We found no in the killing of EL4 cells or upon for of the CD8+ CTL were from mice for CatB or or two no target cell death was in the of the cognate To more the of CTL during the a was in which the purified CD8+ T cells were with 51Cr the EL4 significant CTL death was at of the To an of CTL to the perforin pathway, we exposed the CTL to CTL in mice and found that both CatB-sufficient and -deficient C57BL/6 target cells were efficiently and to a CTL of CatB-deficient and to Perforin in the of purified CTL from and mice to lysis by recombinant mouse perforin purified from baculovirus-infected cells (18Voskoboinik I. Thia M.C. De Bono A. Browne K. Cretney E. Jackson J.T. Darcy P.K. Jane S.M. Smyth M.J. Trapani J.A. J. Exp. Med. 2004; 200: 811-816Crossref PubMed Scopus (60) Google and found them to be active CatB is proposed to be expressed on the cell surface following degranulation (15Balaji K.N. Schaschke N. Machleidt W. Catalfamo M. Henkart P.A. J. Exp. Med. 2002; 196: 493-503Crossref PubMed Scopus (181) Google Scholar). To that CatB was from the lysosomal compartment to the plasma membrane, the CTL were exposed to antibody or not which degranulation as by the in cell surface of the lysosomal CD107a CTL following degranulation was as described (15Balaji K.N. Schaschke N. Machleidt W. Catalfamo M. Henkart P.A. J. Exp. Med. 2002; 196: 493-503Crossref PubMed Scopus (181) Google Scholar). was no in the of and CTL to granule components not or to recombinant perforin To a cysteine protease other CatB may be in the CTL following a general protease inhibitor was to and CTL exposed to recombinant perforin. we found no in of the CTL to recombinant perforin, of CatB in CTL in CatB-deficient to the in vivo efficiency of target cell killing in CatB-deficient and OT-I transgenic in vivo assays were as described R.M. Heath W.R. Carbone F.R. J. Immunol. 2002; PubMed Scopus Google Scholar). target splenocytes with a high of CFSE were with a of lymphocytes with SIINFEKL and a low of CFSE and into the of mice. target cells. the mice were and the death of target cells assessed within the In the the death of target cells was in that the ratio of high to low CFSE lymphocytes in the of mice was when SIINFEKL was on with a mouse lymphocytes not of the death of target cells and CatB–/– mice that the of CatB not the of the mice to clear target cells and cell have been by the of how lymphocytes that perforin remain selectively resistant to its membrane-disruptive studies have of several independent through which the CTL or natural killer cell remains protected from perforin during its and Perforin of lytic activity only at the of its packaging into the cytotoxic granules when a glycosylated C-terminal oligopeptide is (13Uellner R. Zvelebil M.J. Hopkins J. Jones J. MacDougall L.K. Morgan B.P. Podack E. Waterfield M.D. Griffiths G.M. EMBO J. 1997; 16: 7287-7296Crossref PubMed Scopus (165) Google Scholar). Thereafter, within the acidic of the lysosome-like compartment which aspartate residues are prevents perforin from binding with cells and target cells, both CTL and killer lymphocytes are resistant to purified perforin other such as and induce lysis of CTL and A.A. J. Exp. Med. PubMed Scopus Google Scholar, C. A.A. M. J. Immunol. 1988; Google Scholar, C. Tschopp J. J. Immunol. 1994; Google Scholar). that the to lysis by perforin in CTL is perforin have been to the of CTL to perforin during target cell the of the CTL membrane may be from that of a target cell and more resistant to from CTL were to be resistant to and this by a in and cells P.A. Immunol. PubMed Scopus Google Scholar, W. J. Cell Biol. 1990; PubMed Scopus Google Scholar). A in lipid or of the plasma membrane of CTL with target cells has been and is believed to the of perforin to or to the target membrane C. Tschopp J. J. Immunol. 1994; Google Scholar, R. P. Immunol. PubMed Scopus Google Scholar, J. Immunol. 1990; Google Scholar). a CTL granule component may act as a specific inhibitor of perforin following degranulation. perforin is released into the immunological synapse and of cell damage are this it has been proposed that lysosomal CatB expressed on the surface of killer lymphocytes upon of the granule membrane with the plasma membrane may inactivate perforin molecules that diffuse back to the cell surface (15Balaji K.N. Schaschke N. Machleidt W. Catalfamo M. Henkart P.A. J. Exp. Med. 2002; 196: 493-503Crossref PubMed Scopus (181) Google Scholar). study has the of purified CatB to cleave and inactivate perforin and also in the of an CTL in vitro and in vivo. that CatB has to cleave perforin, we found that CatB is not for CTL during and following killing of target cells. We have using in vitro assays that recombinant perforin is a relatively substrate for CatB in which fibronectin is cleaved to Furthermore, perforin active and maintained their lytic with this is the that lytic activity has been for several from the perforin especially at the S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, N. J. Chem. Biol. Full Text PDF PubMed Scopus Google Scholar). The reported of perforin by purified CatB were not with purified perforin used perforin within a CTL cell as the It is possible that the CTL proteases of perforin to or that CatB a second protease for perforin and purified CD8+ CTL from CatB-sufficient or we also that the of CatB not the of target cell death or the of CTL for to following encounter with target cells. The of CatB-deficient mice to study the of the protease in a and the requirement for CatB which the study heavily upon to induce a of CatB inhibition (15Balaji K.N. Schaschke N. Machleidt W. Catalfamo M. Henkart P.A. J. Exp. Med. 2002; 196: 493-503Crossref PubMed Scopus (181) Google Scholar). studies with CatB-deficient lymphocytes that CatB is dispensable for perforin and CatB role in perforin proteases also be of this the human B, and are expressed by cytotoxic only is expressed by CTL C. D. 1997; PubMed Scopus Google Scholar). Recently, studies using mice have that not the of cytotoxic lymphocytes to or following degranulation J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, CatB, not have a role in cytotoxic cell a role in CTL it is that a high of may the proteases lysis of CTL from or mice no in to death with or the general cysteine protease inhibitor These results that cysteine proteases are not in the CTL from death following degranulation. The used by CTL to from perforin following degranulation therefore remains Other studies have the possibility that lipid of may CTL by the to perforin and is that may be more in the CTL membrane, the for perforin to or the lipid R. P. Immunol. PubMed Scopus Google Scholar). The of purified recombinant perforin studies that to We and for and We for with the CatB
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