Residual Cytotoxicity and Granzyme K Expression in Granzyme A-deficient Cytotoxic Lymphocytes

Cytotoxic lymphocytes contain granules that have the ability to induce apoptosis in susceptible target cells. The granule contents include perforin, a pore-forming molecule, and several granzymes, including A and B, which are the most abundant serine proteases in these granules. Granzyme B-deficient cytotoxic T lymphocytes (CTL) have a severe defect in their ability to rapidly induce apoptosis in their targets, but have an intact late cytotoxicity pathway that is in part perforin-dependent. In this report, we have created mice that are deficient for granzyme A and characterized their phenotype. These mice have normal growth and development and normal lymphocyte development, activation, and proliferation. Granzyme A-deficient CTL have a small but reproducible defect in their ability to induce 51Cr and125I-UdR release from susceptible allogeneic target cells. Since other granzyme A-like tryptases could potentially account for the residual cytotoxicity in granzyme A-deficient CTL, we cloned the murine granzyme K gene, which is linked to granzyme A in humans, and proved that it is also tightly linked with murine granzyme A. The murine granzyme K gene (which encodes a tryptase similar to granzyme A) is expressed at much lower levels than granzyme A in CTL and LAK cells, but its expression is unaltered in granzyme A−/− mice. The minimal cytotoxic defect in granzyme A−/− CTL could be due to the existence of an intact, functional early killing pathway (granzyme B dependent), or to the persistent expression of additional granzyme tryptases like granzyme K. Cytotoxic lymphocytes contain granules that have the ability to induce apoptosis in susceptible target cells. The granule contents include perforin, a pore-forming molecule, and several granzymes, including A and B, which are the most abundant serine proteases in these granules. Granzyme B-deficient cytotoxic T lymphocytes (CTL) have a severe defect in their ability to rapidly induce apoptosis in their targets, but have an intact late cytotoxicity pathway that is in part perforin-dependent. In this report, we have created mice that are deficient for granzyme A and characterized their phenotype. These mice have normal growth and development and normal lymphocyte development, activation, and proliferation. Granzyme A-deficient CTL have a small but reproducible defect in their ability to induce 51Cr and125I-UdR release from susceptible allogeneic target cells. Since other granzyme A-like tryptases could potentially account for the residual cytotoxicity in granzyme A-deficient CTL, we cloned the murine granzyme K gene, which is linked to granzyme A in humans, and proved that it is also tightly linked with murine granzyme A. The murine granzyme K gene (which encodes a tryptase similar to granzyme A) is expressed at much lower levels than granzyme A in CTL and LAK cells, but its expression is unaltered in granzyme A−/− mice. The minimal cytotoxic defect in granzyme A−/− CTL could be due to the existence of an intact, functional early killing pathway (granzyme B dependent), or to the persistent expression of additional granzyme tryptases like granzyme K. The cytolytic granules of cytotoxic T lymphocytes (CTL) 1The abbreviations used are: CTL, cytotoxic T lymphocytes; NK, natural killer; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); BLT,N α-benzyloxycarbonyl-l-lysine thiobenzyl ester; MLR, mixed lymphocyte reaction; IL, interleukin;125I-UdR, 125Iiododeoxyuridine. 1The abbreviations used are: CTL, cytotoxic T lymphocytes; NK, natural killer; PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s); BLT,N α-benzyloxycarbonyl-l-lysine thiobenzyl ester; MLR, mixed lymphocyte reaction; IL, interleukin;125I-UdR, 125Iiododeoxyuridine. and natural killer (NK) cells contain several different neutral serine proteases called granzymes, the pore-forming protein perforin, and other proteins of unknown function. The granule exocytosis model of cellular cytotoxicity postulates that granzymes, in concert with perforin, induce target cell apoptosis after cytotoxic lymphocytes specifically recognize the target cell, and release their granule contents into the intercellular space between the effector and target cells (1Henkart P.A. Annu. Rev. Immunol. 1985; 3: 31-58Crossref PubMed Scopus (510) Google Scholar). Perforin is thought to produce channels in the target cell membrane through which the granzymes pass; in the target cell, granzymes are thought to cleave and activate critical substrates, thereby initiating the apoptotic program of the cell. Several distinct granzymes from mice, rats, and humans have been cloned and characterized. To date, granzymes A through G and metase-1 have been identified in mice (2Pasternack M.S. Verret C.R. Liu M.A. Eisen H.N. Nature. 1986; 322: 740-743Crossref PubMed Scopus (183) Google Scholar, 3Gershenfeld H.K. Weissman I.L. 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Except for granzyme B, which is required for the rapid induction of target cell apoptosis during CTL attack (30Heusel J.W. Wesselschmidt R.L. Shresta S. T.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, S. J.W. T.J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, T.A. T.J. 1996; PubMed Google Scholar), the of the other granzymes are A and B are the most expressed granzymes in CTL with a of Granzyme A is a tryptase (which after or at the and granzyme B is an (which after at the The these proteases have been to different murine and granzyme A are and a of H.K. Hershberger R.J. Shows T.B. Weissman I.L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1184-1188Crossref PubMed Scopus (101) Google Scholar, K. Denizot F. Mattei Golstein P. F. 1987; Scholar, T.M. P. H. Zimmer M. Jenne D.E. 1993; PubMed Scopus Google Scholar), and murine and granzyme B are of K. Mattei M.-G. Simon D. M. P. M. Golstein P. Immunogenetics. 1988; 28: PubMed Scopus Google Scholar, P.A. T.J. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus (47) Google Scholar, J.W. T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The granzyme B gene is part of a of tightly linked granzymes and other serine that are specifically and expressed in distinct cell The murine granzyme B granzymes and other cell the granzyme B is to contain granzymes B and H, and cell that the of in the murine granzyme B gene the expression of granzymes and G in cells, potentially of of the from these mice J.W. T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). the defect in the ability of granzyme LAK cells to induce apoptosis of susceptible could be due in part to the of granzyme or G. CTL from granzymes A and B. in of granzyme CTL, the defect in the rapid induction of apoptosis is most due to the of granzyme B of granzyme mice have that CTL are with a granzyme of cytotoxicity (30Heusel J.W. Wesselschmidt R.L. Shresta S. T.J. Cell. 1994; Full Text PDF PubMed Scopus Google S. J.W. T.J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). Several of granzyme A a for this granzyme A from CTL or granules apoptosis of or target cells L. Kam C.-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (420) Google P.A. J. Exp. Med. 1989; PubMed Scopus Google cells with and granzyme induce in target cells and granzyme A or B J.W. L. P.A. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, H. P.A. J. Exp. Med. 1995; PubMed Scopus Google and granzyme A is a granzyme A apoptosis after of with of for the of granzyme apoptosis L. Kam C.-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (420) Google Scholar). tryptase called granzyme K is also a for granzyme The murine of granzyme K M. Yoast S. Schmidt B.F. FEBS Lett. 1995; 364: 268-271Crossref PubMed Scopus (20) Google Scholar, 29Sayers T.J. Lloyd A.R. McVicar D.W. O'Connor M.D. Kelly J.M. Carter C.R.D. Wiltrout T.A. Wiltrout R.H. Smyth M.J. J. Leukocyte Biol. 1996; 59: 763-768Crossref PubMed Scopus (27) Google been but granzyme K been and its been cloned L. Kam C.-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (420) Google Scholar, 18Sayers T.J. Wiltrout T. Smyth M.J. Ottaway K.S. Pilaro A.M. Sowder R. Henderson L.E. Sprenger H. Lloyd A.R. J. Immunol. 1994; 152: 2289-2297PubMed Google Scholar). to granzyme A, granzyme K from a cell apoptosis of target cells in a late cytotoxicity L. Kam C.-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (420) Google Scholar). granzymes A and K to the E. Sayers T.J. Smyth M.J. Immunogenetics. 1994; PubMed Scopus Google Scholar). These that murine granzyme K is to and that it be the granzyme A gene In this we the of murine granzyme K and the between murine granzymes A and K. have created a of granzyme A and a of with a have that granzyme A−/− mice are and have normal development, and similar to the mice K. M. F. A. M. M. Simon M.M. EMBO J. 1995; PubMed Scopus Google Scholar), which a different in the granzyme A of the granzyme K gene is the of the in the granzyme A Granzyme A−/− CTL have a ability to attack and allogeneic target cells, have an intact granzyme B persistent expression of granzyme K which is also a from with for or from an cell from mice T the granzyme and T. J. a P. N. 1987; Google Scholar). The of with and with the of that from and to the most between the and granzyme K and A into the The with an A murine granzyme A the of granzyme A of The and in to its ability to granzyme A used to a murine that with the granzyme A and for the of granzyme K gene in the murine granzyme K the with the granzyme K from or and and into and from with a and with the murine granzyme A or a murine granzyme K and The and of these J.W. T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with or with mixed lymphocyte (30Heusel J.W. Wesselschmidt R.L. Shresta S. T.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). cells also with for to LAK cells, S. J.W. T.J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). cellular from lymphocytes and an T.J. J. 1989; PubMed Scopus Google Scholar). for murine granzyme A, murine granzyme B, and murine (30Heusel J.W. Wesselschmidt R.L. Shresta S. T.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). A that specifically of murine granzyme K created a from the the of granzyme K. granzyme K a of from to the The murine granzyme A that the used to a A an most of the murine granzyme A gene The into and and that this the and through of granzyme A. used to the granzyme A gene A of the granzyme A gene with a the gene the gene M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google in an in the granzyme A The in the from granzyme A. The of a and from and a including and from cells from with the granzyme A and (30Heusel J.W. Wesselschmidt R.L. Shresta S. T.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). The a that with granzyme A R.J. H.K. Weissman I.L. L. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, K. Kramer M.D. Simon M.M. 1992; PubMed Scopus Google Scholar). The a and a of of and and these in a with a with cells from of the and into (30Heusel J.W. Wesselschmidt R.L. Shresta S. T.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). with a of with and their for of the granzyme A the to produce mice. and from with or cell and a and (30Heusel J.W. Wesselschmidt R.L. Shresta S. T.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). CTL in of and and of and for and at for at to the from cellular of this for tryptase the α-benzyloxycarbonyl-l-lysine thiobenzyl E. PubMed Scopus Google Scholar). the mixed with of with and the at for of to the The a the and with and or the 51Cr release T. G. J. Immunol. Google Scholar). To the of and granzyme K, we that to the most between the and between and granzyme K a from and a cell that the from a and most of the of this The but the of through and a identified in the murine granzyme K A, of the murine granzyme K The is the are and their are A B, of murine granzymes A and K and are but these are that the granzyme A gene the The granzyme K which is to is to the and the of the is at the in The of murine granzyme K with the and In the of the and granzyme K, the and and of that are to and are The and a The granzyme K contain for the murine granzymes, granzyme K is most similar to granzyme A A, K, and other tryptases have an at to the that is thought to for or in the in to granzyme A (which a due to an the granzyme K a it contain the additional the murine granzyme K protein is to be a in with the of L. Kam C.-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (420) Google Scholar, 18Sayers T.J. Wiltrout T. Smyth M.J. Ottaway K.S. Pilaro A.M. Sowder R. Henderson L.E. Sprenger H. Lloyd A.R. J. Immunol. 1994; 152: 2289-2297PubMed Google and granzyme K Immunol. 141, 3142–3147Hameed. A., Lowrey, D. M., Lichtenheld, M., and Podack, E. R.J. Immunol. , 141, 3142–3147.Google Scholar). a from mice a the of granzyme A, and that with this of from the of murine granzyme A the of the granzyme A in These a murine granzyme K through with this A or of with the granzyme K and and that into the and the The the and and the and most of and the and and The of granzyme K is in and are in the The gene of and a for this of Granzyme A and R.J. H.K. Weissman I.L. L. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, K. Kramer M.D. Simon M.M. 1992; PubMed Scopus Google the granzyme K gene of and to have Since the of granzymes A and K to that the are we to the between from with and with granzyme A or The granzyme of a that the The granzyme K the and and and granzyme K in of the the of kb, which a of to the granzyme A and K that the are at most of the with that or the the a the between the is the expression of murine granzyme K a granzyme in granzyme K an of from A granzyme K in murine small or granzyme K levels in CTL, and in LAK cells in with for In to the granzyme K for granzyme A and used for lymphocyte activation, and for and The in the of granzyme K with the expression of granzyme A and B in CTL and LAK cells. of these that granzyme K levels are that of granzyme A in cells, and that of granzyme A in LAK cells. granzyme A−/− mice the in A The granzyme A a of (which contain the of the a that of the used for A, of into to that the granzyme A in the In B, which a of the granzyme A the and granzyme A the mice with of mice. Granzyme and mice have normal development and and are from their and with CTL LAK cells from granzyme or a granzyme A (which of the a of granzyme A in granzyme A−/− CTL Granzyme A−/− cytotoxic effector cells a normal ability to activate in to allogeneic or to in LAK cell levels of granzyme B in granzyme and levels of granzyme K in the A−/− the of the in the granzyme A gene the expression of the linked granzyme K the ability of granzyme or CTL to cleave the B that a of is from granzyme A-deficient a small of residual in these the of lymphocytes in the and of granzyme A−/− mice. is a of granzyme or lymphocyte with murine and cell Granzyme or the of cells and small of or cells and mice of of cells in their granzyme and CTL of and cells, and the of these CTL cell similar of and CTL in that granzyme A−/− the cell and that granzyme A-deficient T lymphocytes and allogeneic in The normal and of granzyme A−/− in to to specifically the ability of the CTL to attack allogeneic of 51Cr release and release at effector to target at an early or a late after the of effector and target cells In the and CTL cytotoxic the at and Granzyme A−/− CTL to induce normal levels or release from the allogeneic or target cells at early and late The levels of 51Cr and release the CTL in a of target cells, including cells with a or the are are and In this report, we have the of granzyme A−/− mice gene to granzyme A−/− mice created and K. M. F. A. M. M. Simon M.M. EMBO J. 1995; PubMed Scopus Google Scholar), granzyme A-deficient have normal development and Cytotoxic lymphocytes from these mice have normal of and and normal levels of the tightly linked granzyme K In with CTL from these mice a small defect in their ability to induce 51Cr and release from allogeneic target cells at and The of granzyme A−/− mice the in cytotoxic effector cells from granzyme A-deficient mice and K. M. F. A. M. M. Simon M.M. EMBO J. 1995; PubMed Scopus Google Scholar). The small defect in cytotoxicity in granzyme A-deficient mice is granzyme A is to be an that is of apoptosis in target cells. the similar in the mice K. M. F. A. M. M. Simon M.M. EMBO J. 1995; PubMed Scopus Google and in this that the minimal in The mice K. M. F. A. M. M. Simon M.M. EMBO J. 1995; PubMed Scopus Google contain an of in this gene could potentially be to produce granzyme A that contain and granzyme A protein could be in that and minimal residual tryptase defect in the cytotoxicity of granzyme A−/− CTL but these mice to the in normal A. K. R. T. C. Simon M.M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). In of the that part of the and the of the A small of residual tryptase in the CTL from a small in cytotoxicity in to the normal cytotoxicity in the K. M. F. A. M. M. Simon M.M. EMBO J. 1995; PubMed Scopus Google mice. The mice K. M. F. A. M. M. Simon M.M. EMBO J. 1995; PubMed Scopus Google are in an and mice are in a mixed different in this gene in different have similar it that these an of the granzyme A are several granzyme A−/− CTL have cytotoxicity in granzyme A−/− mice have a granzyme B In from we have that in the granzyme B are required for the rapid induction of apoptosis in susceptible target cells (30Heusel J.W. Wesselschmidt R.L. Shresta S. T.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, S. J.W. T.J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, J.W. T.J. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). Since the rapid killing pathway is intact in granzyme A-deficient the granzyme A defect be to that are deficient for granzymes A and B, we be to the of granzyme A for the granzyme cytotoxicity that we have (30Heusel J.W. Wesselschmidt R.L. Shresta S. T.J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, S. J.W. T.J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, S. T.J. PubMed Google Scholar). of the cytotoxic of these also to other in cytolytic granules to granzyme A B have the that are it that additional granule proteins to to these are in for the minimal cytotoxic defect of granzyme A deficient mice be the of granzyme A other granzymes with a similar granzyme K is a its with granzyme A, the granzyme K expression in granzyme A−/− In to granzyme K, granzyme is CTL tryptase D. Tschopp J. Cell. 1987; 49: 679-685Abstract Full Text PDF PubMed Scopus (338) Google Scholar), and its expression is unaltered in granzyme A-deficient mice it in the granzyme B granzymes and K to the tryptase in CTL, than in granzyme A-deficient mice. the that the of residual tryptase granzymes K, and other granzymes is to the mice from the of granzyme A an the of these other granzymes for CTL we have cloned the murine granzyme K gene which encodes a tryptase that is similar to granzyme A. Granzyme K that are to serine proteases the the the and are at and of a of are in and are to the granzymes with these granzyme K is most similar to granzyme A. granzymes A and K have the to the including at and are also in in granzyme K. these tryptase the (which are of serine are in in granzymes A and K. These that these proteases cleave similar and have similar granzyme K contain the that granzyme A to are several of that are in granzyme K but in granzyme A, that granzyme K could have different from granzyme A. serine to in the and the with the and granzyme B gene have that this are expressed specifically in the lymphocyte K. Mattei M.-G. Simon D. M. P. M. Golstein P. Immunogenetics. 1988; 28: PubMed Scopus Google Scholar, P.A. T.J. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus (47) Google Scholar, J.W. T.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). with from the that granzymes A and K are linked In the and T.M. P. H. Zimmer M. Jenne D.E. 1993; PubMed Scopus Google have that the granzyme A gene to a that is between and the granzyme A gene is in E. Sayers T.J. Smyth M.J. Immunogenetics. 1994; PubMed Scopus Google have that the granzyme K also to a of the tryptase of serine In we these murine granzymes A and K in a from a The of the of these is but it that these that the expression of granzymes A and K is to T cells and cells. that CTL and LAK cells much lower levels of granzyme K than granzyme A and Immunol. 141, 3142–3147Hameed. A., Lowrey, D. M., Lichtenheld, M., and Podack, E. R.J. Immunol. , 141, 3142–3147.Google Scholar), have granzymes A and K from LAK that granzyme K protein is much abundant than granzyme A. in that granzyme K the of granzyme A. granzyme K been to be a expressed granzyme that be from the lymphocyte cell L. Kam C.-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (420) Google Scholar, 18Sayers T.J. Wiltrout T. Smyth M.J. Ottaway K.S. Pilaro A.M. Sowder R. Henderson L.E. Sprenger H. Lloyd A.R. J. Immunol. 1994; 152: 2289-2297PubMed Google and L. Kam C.-M. Powers J.C. Aebersold R. Greenberg A.H. J. Exp. Med. 1992; 176: 1521-1529Crossref PubMed Scopus (420) Google have that granzyme K is abundant than granzyme A in cells. of cytotoxic lymphocyte is to the of granzyme A K in cytotoxic and to the of these granzymes in cell for and T. N. and A. for their critical of this