Discordant Regulation of Granzyme H and Granzyme B Expression in Human Lymphocytes

We analyzed the expression of granzyme H in human blood leukocytes, using a novel monoclonal antibody raised against recombinant granzyme H. 33-kDa granzyme H was easily detected in unfractionated peripheral blood mononuclear cells, due to its high constitutive expression in CD3-CD56+ natural killer (NK) cells, whereas granzyme B was less abundant. The NK lymphoma cell lines, YT and Lopez, also expressed high granzyme H levels. Unstimulated CD4+ and particularly CD8+ T cells expressed far lower levels of granzyme H than NK cells, and various agents that classically induce T cell activation, proliferation, and enhanced granzyme B expression failed to induce granzyme H expression in T cells. Also, granzyme H was not detected in NK T cells, monocytes, or neutrophils. There was a good correlation between mRNA and protein expression in cells that synthesize both granzymes B and H, suggesting that gzmH gene transcription is regulated similarly to gzmB. Overall, our data indicate that although the gzmB and gzmH genes are tightly linked, expression of the proteins is quite discordant in T and NK cells. The finding that granzyme H is frequently more abundant than granzyme B in NK cells is consistent with a role for granzyme H in complementing the pro-apoptotic function of granzyme B in human NK cells. We analyzed the expression of granzyme H in human blood leukocytes, using a novel monoclonal antibody raised against recombinant granzyme H. 33-kDa granzyme H was easily detected in unfractionated peripheral blood mononuclear cells, due to its high constitutive expression in CD3-CD56+ natural killer (NK) cells, whereas granzyme B was less abundant. The NK lymphoma cell lines, YT and Lopez, also expressed high granzyme H levels. Unstimulated CD4+ and particularly CD8+ T cells expressed far lower levels of granzyme H than NK cells, and various agents that classically induce T cell activation, proliferation, and enhanced granzyme B expression failed to induce granzyme H expression in T cells. Also, granzyme H was not detected in NK T cells, monocytes, or neutrophils. There was a good correlation between mRNA and protein expression in cells that synthesize both granzymes B and H, suggesting that gzmH gene transcription is regulated similarly to gzmB. Overall, our data indicate that although the gzmB and gzmH genes are tightly linked, expression of the proteins is quite discordant in T and NK cells. The finding that granzyme H is frequently more abundant than granzyme B in NK cells is consistent with a role for granzyme H in complementing the pro-apoptotic function of granzyme B in human NK cells. Cytotoxic T lymphocytes and natural killer (NK) 1The abbreviations used are: NK, natural killer; NKT, NK T; gzm, granzyme; PBMC, peripheral blood mononuclear cell; mAb, monoclonal antibody; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; PHA, phytohemagglutinin.1The abbreviations used are: NK, natural killer; NKT, NK T; gzm, granzyme; PBMC, peripheral blood mononuclear cell; mAb, monoclonal antibody; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; PHA, phytohemagglutinin. cells are cytotoxic lymphocytes that are responsible for inducing rapid apoptosis of virus-infected or transformed cells (1Barry M. Bleackley R.C. Nat. Rev. Immunol. 2002; 2: 401-409Crossref PubMed Google Scholar, 2Trapani J.A. Smyth M.J. Nat. Rev. Immunol. 2002; 2: 735-747Crossref PubMed Scopus (859) Google Scholar). Cytotoxic lymphocytes utilize two pathways for killing target cells, both of which require direct cell contact. The first pathway involves exocytosis of potent toxins from secretory granules stored in the effector cell cytoplasm, whereas the second is triggered by clustering of death receptors on the target cell membrane following interaction with their respective ligands (tumor necrosis factor superfamily members) expressed on the killer cell (1Barry M. Bleackley R.C. Nat. Rev. Immunol. 2002; 2: 401-409Crossref PubMed Google Scholar, 2Trapani J.A. Smyth M.J. Nat. Rev. Immunol. 2002; 2: 735-747Crossref PubMed Scopus (859) Google Scholar). Cytotoxic granules contain two major types of toxin that co-operatively induce target cell apoptosis: granzymes (gzms), a family of serine proteases, and a pore-forming protein, perforin. The precise mechanism of gzm/perforin synergy has not been clarified; however, it is likely that gzms enter the target cell by endocytosis (3Motyka B. Korbutt G. Pinkoski M.J. Heibein J.A. Caputo A. Hobman M. Barry M. Shostak I. Sawchuk T. Holmes C.F. Gauldie J. Bleackley R.C. Cell. 2000; 103: 491-500Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 4Trapani J.A. Sutton V.R. Thia K.Y. Li Y.Q. Froelich C.J. Jans D.A. Sandrin M.S. Browne K.A. J. Cell Biol. 2003; 160: 223-233Crossref PubMed Scopus (92) Google Scholar), whereas perforin enables pro-apoptotic gzms access to their substrates in the target cell cytosol by destabilizing endosomes (5Froelich C.J. Orth K. Turbov J. Seth P. Gottlieb R. Babior B. Shah G.M. Bleackley R.C. Dixit V.M. Hanna W. J. Biol. Chem. 1996; 271: 29073-29079Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 6Browne K.A. Blink E. Sutton V.R. Froelich C.J. Jans D.A. Trapani J.A. Mol. Cell Biol. 1999; 19: 8604-8615Crossref PubMed Scopus (176) Google Scholar). The more traditionally held notion of perforin permitting access to the cytosol through plasma membrane pores may also apply in some circumstances (6Browne K.A. Blink E. Sutton V.R. Froelich C.J. Jans D.A. Trapani J.A. Mol. Cell Biol. 1999; 19: 8604-8615Crossref PubMed Scopus (176) Google Scholar). Gzms are closely related to one another structurally, and their genes are clustered in several loci on distinct chromosomes (7Smyth M.J. O'Connor M.D. Trapani J.A. J. Leukocyte Biol. 1996; 60: 555-562Crossref PubMed Scopus (70) Google Scholar). In both humans and rodents, each locus encodes proteases with a single broad type of substrate cleavage. Gzms A and K are trypsin-like proteases (“tryptases”) that cleave proteins at basic residues, and their genes are linked on human chromosome 5 (8Baker E. Sayers T.J. Sutherland G.R. Smyth M.J. Immunogenetics. 1994; 40: 235-237Crossref PubMed Scopus (37) Google Scholar). Gzm M preferentially cleaves at residues with long, uncharged side chains (Met, Leu), and its gene is closely linked to the neutrophil elastase gene on human chromosome 19 (9Smyth M.J. Sayers T.J. Wiltrout T. Powers J.C. Trapani J.A. J. Immunol. 1993; 151: 6195-6205PubMed Google Scholar). The genes for gzms B and H are located on chromosome 14, tightly linked with the gene encoding cathepsin G, another chymotrypsin-like protease (“chymase”) that, unlike the gzms, is expressed in cells of the myeloid lineage but not in lymphocytes (7Smyth M.J. O'Connor M.D. Trapani J.A. J. Leukocyte Biol. 1996; 60: 555-562Crossref PubMed Scopus (70) Google Scholar, 10Haddad P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google Scholar). These three genes all map to within 60 kb of one another. Gzm B cleaves its substrates adjacent to acidic residues, particularly Asp (11Odake S. Kam C.M. Narasimhan L. Poe M. Blake J.T. Krahenbuhl O. Tschopp J. Powers J.C. Biochemistry. 1991; 30: 2217-2227Crossref PubMed Scopus (225) Google Scholar), and plays a central role in eliciting death of the target cell by perturbing mitochondria and activating caspases (12Sutton 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 (298) Google Scholar, 13Sutton V.R. Wowk M.E. Cancilla M. Trapani J.A. Immunity. 2003; 18: 319-329Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Its absence from the granules of lymphocytes from gene knockout mice slows the rate of target cell DNA damage (14Heusel J.W. Wesselschmidt R.L. Shresta S. Russell J.H. Ley T.J. Cell. 1994; 76: 977-987Abstract Full Text PDF PubMed Scopus (743) Google Scholar). In the mouse, the gene for gzm B is tightly linked to at least four other granzyme genes predicted to code for functional chymases (gzms C-F); however, only one such chymase, gzm H, performs this function in humans (15Edwards K.M. Kam C.M. Powers J.C. Trapani J.A. J. Biol. Chem. 1999; 274: 30468-30473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Gzm C is now known to possess an unusual type of pro-apoptotic activity characterized by mitochondrial swelling and disruption that does not require prior caspase activity (16Johnson H. Scorrano L. Korsmeyer S.J. Ley T.J. Blood. 2003; 101: 3093-3101Crossref PubMed Scopus (91) Google Scholar). However, no such evidence has yet come forward for human gzm H. We recently showed that recombinant gzm H expressed in baculovirus-infected insect cells preferentially cleaves substrates with Phe or Tyr at the P1 position (15Edwards K.M. Kam C.M. Powers J.C. Trapani J.A. J. Biol. Chem. 1999; 274: 30468-30473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Despite sharing this substrate specificity with mouse gzms C-F, gzm H has no direct structural equivalent in the mouse and appears to be a uniquely human protein. Gzm H has a very high amino acid identity (>90%) with many portions of the gzm B sequence, particularly near the amino terminus of the molecule (10Haddad P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google Scholar) despite performing a distinct enzymic function. It has been inferred from the high degree of sequence similarity that the gzmH gene arose by duplication of part of the 5′ end of the gzmB gene (particularly exons 3 and 4) and fusion with the 3′ end of an another primordial serine protease gene (10Haddad P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google Scholar). The function of gzm H is unknown, and its study has been greatly hindered by a lack of reagents able to distinguish it from gzm B at the protein or mRNA levels. The cellular expression of gzm H has been reported in just one study that assessed gzm H mRNA expression in human T cells (10Haddad P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref PubMed Scopus (51) Google Scholar). This study concluded that like gzm B, gzm H is expressed at high levels in activated T cells. However, in another study, a portion of the gzmH promoter was used to preferentially direct the transgenic expression of SV-40 T antigen to the mouse lymphoid compartment. The transgene was not activated in allo-stimulated cytotoxic T lymphocytes that expressed high levels of gzm B but was highly expressed in activated NK cells and in NK and NK/T cell tumors that arose in these animals (17MacIvor D.M. Pham C.T. Ley T.J. Blood. 1999; 93: 963-973Crossref PubMed Google Scholar). Although the genes encoding cathepsin G and gzms B and H are very tightly linked, cathepsin G and gzm B have markedly different cellular expression profiles, as cathepsin G is expressed exclusively by cells of the myeloid lineage, particularly neutrophils and monocytes (18Hohn P.A. Popescu N.C. Hanson R.D. Salvesen G. Ley T.J. J. Biol. Chem. 1989; 264: 13412-13419Abstract Full Text PDF PubMed Google Scholar). It is therefore quite feasible that gzms B and H may be differentially expressed and regulated among leukocyte subsets. The availability of correctly folded, proteolytically active gzm H enabled us to attempt raising monospecific reagents for this protease. Having produced a novel gzm H-specific mAb, we now demonstrate that gzm H is expressed solely in lymphocytes, but its constitutive expression pattern is quite distinct from that of gzm B, as is its expression in response to a variety of stimuli that strongly induce gzm B expression. Antibodies—Antibodies used in Western blotting and immunohistochemistry included several mAbs made by our laboratory: 2C5 (antigzm B) (19Trapani J.A. Browne K.A. Dawson M. Smyth M.J. Biochem. Biophys. Res. Commun. 1993; 195: 910-920Crossref PubMed Scopus (60) Google Scholar), 4H10 (anti-gzm M) (20Smyth M.J. O'Connor M.D. Kelly J.M. Ganesvaran P. Thia K.Y. Trapani J.A. Biochem. Biophys. Res. Commun. 1995; 217: 675-683Crossref PubMed Scopus (22) Google Scholar), PB2 (anti-perforin) (21Geisberg M. Trapani J.A. Dupont B. Tissue Antigens. 1990; 35: 229-233Crossref PubMed Scopus (12) Google Scholar), and 7D8 (anti-PI-9) (22Hirst C.E. Buzza M.S. Sutton V.R. Trapani J.A. Loveland K.L. Bird P.I. Mol. Hum. Reprod. 2001; 7: 1133-1142Crossref PubMed Scopus (75) Google Scholar). In addition, mAb GB7 (anti-gzm B) was purchased from Serotec. Cell lines, Tissue Samples, and Immunohistochemistry—The human tumor cell lines used in this study were all cultured in RPMI medium supplemented with 10% fetal bovine serum, 2 mm l-glutamine, penicillin, and streptomycin in a CO2 incubator at 37°C. These included YT (human NK), HL-60 and U937 (human myeloid), Daudi and Raji (human B cell), Jurkat and CEM (human T cell), HeLa (cervical carcinoma), Lovo and COLO205 (colon adenocarcinoma), and MDA-MB-435 (human breast adenocarcinoma) tumor cell lines. Lysates of homogeneous populations of human T cell lymphoma samples of two unrelated individuals (derived from lymph node biopsies) were also analyzed. Immunohistochemical studies were performed as described previously (23Sayers T.J. Brooks A.D. Ward J.M. Hoshino T. Bere W.E. Wiegand G.W. Kelly J.M. Smyth M.J. Kelley J.M. J. Immunol. 2001; 166: 765-771Crossref PubMed Scopus (82) Google Scholar). Recombinant gzm Expression—Recombinant human gzm B and gzm H were purified from the culture supernatants of baculovirus-infected Sf9 cells, 3-5 days after infection, as described (15Edwards K.M. Kam C.M. Powers J.C. Trapani J.A. J. Biol. Chem. 1999; 274: 30468-30473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The poly-histidine-tagged gzm proteins were purified by nickel affinity chromatography and dialyzed against PBS. The gzms were tested for their proteolytic activity by assaying the cleavage of tripeptide substrates Ala-Ala-Asp-S-benzyl (for gzm B, a kind gift from Drs. Chih-Min Kam and Jim Powers, Georgia Institute of Technology, Atlanta, GA) or Phe-Leu-Phe-S-benzyl (for gzm H, purchased from Sigma). mAb Production and Screening—Five- to -seven-week-old female BALB/c mice were immunized into the peritoneal cavity with purified, recombinant gzm H (50 μg) mixed with Freund's complete adjuvant. Two to four weeks later, the mice were boosted with the same antigen mixed with incomplete adjuvant. Three days following a further similar immunization, a mouse was sacrificed, and its spleen cells were fused with NS-1 mouse myeloma cells, using polyethylene glycol. Ten days later, hybridomas were screened for secretion of anti-gzm H mAbs using a solid phase ELISA assay in which gzm H (or gzm B as a control) was coated onto the wells of a 96-well polyvinyl chloride plate. Positive hybridomas were cloned by limiting dilution, and the clones were expanded and cryopreserved in liquid nitrogen. Production of a Rabbit Polyclonal Antiserum—Polyclonal antiserum was raised in New Zealand White rabbits by immunization with purified recombinant human gzm B (200 μg) mixed with Freund's complete adjuvant and injected into multiple sites subcutaneously in volumes of <0.1 ml. Three booster immunizations were given intramuscularly with incomplete adjuvant at 3-4-week intervals. Isolation of Leukocyte Subsets—For isolation of neutrophils, cell pellets collected after Ficoll-Hypaque centrifugation were dispersed in PBS containing 3% dextran and left to stand at ambient temperature for 30 min to rouleau the red cells. Neutrophils were then pelleted from the supernatants and resuspended in hypotonic buffer containing 0.2% NaCl for 1.5-2.0 min to lyse the remaining red cells. The tonicity was then adjusted to 0.9% NaCl, and the cells were again pelleted and washed. Cells were >95% neutrophils, by Giemsa staining. Monocytes were sorted from peripheral blood mononuclear cells (PBMC) with anti-CD14 fluorescein isothiocyanate and were at least 82% pure. CD3+CD56- T cells and CD3-CD56+ NK cells were purified from PBMC by fluorescence-activated cell sorter sorting and were reproducibly >96% pure. For isolation of NKT cells, PBMC were cultured in medium containing IL-2, IL-7, and α-galactosylceramide (generously provided by Kirin Breweries, Gunma, Japan) for 7 days. Cells that stained positive with phytohemagglutinin-conjugated CD1d/a-galactosylceramide tetramer (generously provided by Drs Stephané Sidobre and Mitchell Kronenberg, La Jolla Institute for Allergy and Immunology, La Jolla, CA) and CD3-fluorescein isothiocyanate were collected by fluorescence-activated cell sorter sorting, and purity was found to be 95%. T and NK Cell Stimulation—Human PBMC and purified populations of T and NK cells were incubated in medium supplemented with recombinant human interleukin-2 (IL-2, 100-500 units/ml; Chiron Corp.) and/or phytohemagglutinin (PHA) (2 μg/ml) or anti-CD3 mAb HIT3A (10 ng/ml; Pharmingen) for up to 10 days prior to harvest and analysis for gzm expression. Protein Immunoblotting—Cell lysates were fractionated on 12.5% SDS-polyacrylamide gels, and the proteins were electroblotted onto polyvinylidene difluoride membranes. Nonspecific protein binding was blocked by preincubation with 5% nonfat skim milk in PBS at 4°C for 2 h. Membranes were incubated with mAb (hybridoma culture supernatant, typically diluted 1/5 to 1/20 in PBS) at 4°C overnight and washed, and then bound Ig was detected with goat anti-mouse Ig conjugated to horseradish peroxidase. Generation of gzm-specific cDNA Probes Northern Blotting, and Real-time PCR Analysis—cDNA probes specific for the 3′-untranslated regions of gzm B and H mRNA/cDNA were produced by PCR amplification of each respective cDNA clone, using synthetic oligonucleotide primers. The gzm B probe was amplified using the oligonucleotides 5′-GAAACGCTACTAACTACAGG-3′ and 5′-CCACTCAGCTAAGAGGT-3′ and was 129 nucleotides in length. The gzm H probe was similarly amplified using the oligonucleotides 5′-AGAACAATGAAGCGCCTC-3′ and 5 ′-ACACGCGGCCGCGATCCATTTATTACAGTCC-3′ and was 138 nucleotides in length. Each probe was purified from 2% agarose gels and labeled with [α-32P]dATP by nick translation or random priming with synthetic hexanucleotides. The radiolabeled probes were used to identify gzm-specific mRNA in Northern blot experiments. Total cellular RNA was isolated from various populations of human cells and cell lines (10 μg/lane) and separated by electrophoresis on 1% formaldehyde/agarose gels and then transferred to nylon membranes by capillary transfer. The membrane was allowed to dry at room temperature and then baked at 80°C for 2 h. Prehybridization was in buffer containing 50% formamide, 1% SDS, and 5× SSC (pH 6.8) solution at 42°C. Following hybridization with preboiled radiolabeled cDNA probe for 16 h, the filters were washed at low stringency and then under high stringency conditions (0.1× SSC, 1% SDS, 60°C for 2 × 20 min), air-dried, and exposed to x-ray film (Kodak) for 1-3 days at -80°C. In some experiments, plasmid DNA was spotted onto nylon membranes (50 ng/spot) and then denatured in NaOH. The membranes were baked at 80°C for 2 h and then prehybridized and hybridized as described above. For real-time-PCR, 2 μg of DNase-treated RNA was reverse transcribed using M-MLV reverse transcriptase Rnase H minus, point mutant (Promega). PCR primers for each gene were designed using Primer Express software (Applied Biosystems, Foster City, CA) with a melting temperature at 58-60°C and a resulting product of 75-150 bp. Triplicate 20-μl PCR reactions were carried out using SYBR green master mix (Applied Biosystems), initially for 15 min at 95°C followed by 39 cycles of 95°C for 15 s and 60°C for 60 s in the ABI Prism 7700 sequence detection system. The levels of gzm H and B mRNA were normalized to that of ribosomal protein L32. Primer pairs were as follows: L32, forward, 5′-TTCCTGGTCCACAACGTCAAG-3′ and reverse, 5′-TGTGAGCGATCTCGGCAC-3′; gzm B, forward, 5′-GCGGTGGCTTCCTGATACAAG-3′, and reverse, 5′-CCCCCAAGGTGACATTTATGG-3′; gzm H, forward, 5′-TGGCGGCATCCTAGTGAGAA-3′, and reverse 5′-GCCCCCAAGGTGACATTTATG-3′. Primer efficiency for gzms B and H was demonstrated by the overlapping amplification profiles of equal quantities of gzm B and H cDNA. Granule Fractionation—For cell fractionation experiments, YT human NK tumor cells (3 × 108) were harvested, washed several times in PBS, and lysed by nitrogen cavitation, as described (24S. V., Davis J.E. Browne K.A. Trapani J.A. J. Immunol. Methods. 2003; Google Scholar). The cell lysate was fractionated on a continuous Percoll density gradient as described, and 1-ml fractions were collected, starting with the dense (bottom) end of the gradient. Following the removal of Percoll by ultracentrifugation, aliquots of each fraction were analyzed by SDS-PAGE and Western blot. To determine the cellular and subcellular expression of gzm H, it was first necessary to produce an antiserum that could distinguish it from other granzymes, particularly its closest structural relative gzm B, with which it shares ∼75% amino acid identity (25Klein J.L. Selvakumar A. Trapani J.A. Dupont B. Tissue Antigens. 1990; 35: 220-228Crossref PubMed Scopus (23) Google Scholar). We have previously expressed and purified catalytically active gzm H secreted by baculovirus-infected insect cells (15Edwards K.M. Kam C.M. Powers J.C. Trapani J.A. J. Biol. Chem. 1999; 274: 30468-30473Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), so this protein was used as an immunogen for mAb production in BALB/c mice. Of 800 hybridoma culture supernatants screened for mAb secretion in a solid phase ELISA assay, 48 were found to secrete an Ig that bound to gzm H. All but one of these mAbs produced an equivalent signal when tested on purified gzm B, indicating they detected an epitope common to both granzymes (data not shown). However, the mAb produced by hybridoma 4G5 specifically recognized gzm H and failed to react with gzm B in the ELISA assay (Fig. 1A). Conversely, mAb 2C5, which we have used extensively in studies on gzm B (12Sutton 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 (298) Google Scholar, 13Sutton V.R. Wowk M.E. Cancilla M. Trapani J.A. Immunity. 2003; 18: 319-329Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 19Trapani J.A. Browne K.A. Dawson M. Smyth M.J. Biochem. Biophys. Res. Commun. 1993; 195: 910-920Crossref PubMed Scopus (60) Google Scholar, 26Sun J. Bird C.H. Sutton V. McDonald L. Coughlin P.B. De Jong T.A. Trapani J.A. Bird P.I. J. Biol. Chem. 1996; 271: 27802-27809Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 27Sutton V.R. Vaux D.L. Trapani J.A. J. Immunol. 1997; 158: 5783-5790PubMed Google Scholar), was specific for gzm B and failed to recognize gzm H. MAb 4G5 detected as little as 3 ng of gzm H but did not react at all with gzm B in Western blots (Fig. 1B). In the reciprocal experiment, mAb 2C5 reacted strongly with gzm B in Western blots but did not react with gzm H (data not shown). By contrast with both mAbs, a polyclonal rabbit antiserum raised against gzm B showed strong cross-reactivity with gzm H, once again demonstrating the close structural similarity of the two granzymes (Fig. 1B). Interestingly, mAb GB7, a commercially available reagent purported to specifically detect gzmB, produced an equivalent signal with gzms B and H (Fig. 1C). Similar analyses using other recombinant granzymes also demonstrated that both 4G5 and 2C5 did not react with recombinant human gzm M, a more distant relative of gzms B and H (∼35% amino acid identity; data not shown) (7Smyth M.J. O'Connor M.D. Trapani J.A. J. Leukocyte Biol. 1996; 60: 555-562Crossref PubMed Scopus (70) Google Scholar). Having shown that 4G5 specifically detects gzm H, we next used the mAb in Western blotting on whole cell lysates made from a series of hemopoietic and non-hemopoietic cells lines. The human NK leukemia cell lines YT (Fig. 2) and Lopez (data not shown) both demonstrated a 33-kDa protein that co-migrated with recombinant gzmH. The predicted molecular mass on reduced SDS-PAGE was consistent with a gzm H polypeptide backbone of 26 kDa (deduced from the cDNA sequence) and ∼7 kDa of added carbohydrate. Gzm H was not expressed by the acute T cell leukemia lines Jurkat and CEM, the B lymphoma cell lines Daudi and Raji, the myeloid cell lines HL-60 and U937, or cells from two lymph node biopsies excised from patients with disseminated T cell lymphoma. Similarly, no signal was detectable in the human carcinoma cell lines 293 (embryonal kidney), HeLa (cervical), or the adenocarcinomas Lovo, COLO 205, and MDA-MB435. As Lopez and YT cells both contain large numbers of cytolytic granules and also express perforin and gzms B and M (24S. V., Davis J.E. Browne K.A. Trapani J.A. J. Immunol. Methods. 2003; Google Scholar), the co-expression of gzm H by these cells lines was consistent with a possible role for gzm H in cytotoxic granule-mediated cell death. To determine the subcellular localization of gzm H, we initially performed immunohistochemistry on pelleted YT cells, using mAbs 2C5 (anti-gzmB) and 4H10 (anti-gzmM) as controls (Fig. 3A). As was shown previously, gzms B and M were localized to granules in the cytoplasm of YT cells (20Smyth M.J. O'Connor M.D. Kelly J.M. Ganesvaran P. Thia K.Y. Trapani J.A. Biochem. Biophys. Res. Commun. 1995; 217: 675-683Crossref PubMed Scopus (22) Google Scholar, 26Sun J. Bird C.H. Sutton V. McDonald L. Coughlin P.B. De Jong T.A. Trapani J.A. Bird P.I. J. Biol. Chem. 1996; 271: 27802-27809Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Cytoplasmic staining in YT cells was also observed with the gzmH mAb; however, the strength of the staining varied considerably from cell to cell: some YT cells (about 50%) stained strongly, a minority stained weakly, and the remainder (about 25%) showed no staining. The heterogeneity of staining was not due to limiting Ig, as similar numbers of strongly and weakly staining cells were observed over a range of antiserum concentrations (data not shown). None of the granzyme mAbs bound to Jurkat cells, which were used as a negative control in this experiment (Fig. 2). To definitively demonstrate gzmH localization to cytotoxic granules, a lysate of mechanically disrupted YT cells was fractionated on a Percoll density gradient (Fig. 3B). The fractions were analyzed by Western blotting with antibodies specific for gzms B and H, perforin, and the serpin, PI-9, which is localized to the cytosol of cytotoxic lymphocytes rather than granules (26Sun J. Bird C.H. Sutton V. McDonald L. Coughlin P.B. De Jong T.A. Trapani J.A. Bird P.I. J. Biol. Chem. 1996; 271: 27802-27809Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Gzm H was co-localized with the granule proteins perforin and gzmB in fractions 6-8 at the dense end of the gradient (specific gravity 1.10-1.13 g/ml, data not shown). Fractions 6-8 were free of PI-9, which is abundant in the cytosol of YT cells (represented by the least dense fractions, 14-18) but is not a significant constituent of the granules (26Sun J. Bird C.H. Sutton V. McDonald L. Coughlin P.B. De Jong T.A. Trapani J.A. Bird P.I. J. Biol. Chem. 1996; 271: 27802-27809Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Almost all of the human and rodent granzymes described to date are expressed constitutively by NK cells and in an inducible manner by CD8+ (and some CD4+) T cells, when they become activated and adopt an effector phenotype (28Trapani J.A. Int. Rev. Cytol. 1998; 182: 111-192Crossref PubMed Google Scholar). The two granzymes known to play a pivotal role in target cell apoptosis, gzm B, which is encoded by a gene that is very tightly linked to that of gzm H, and gzm A, encoded on a different chromosome, Chr 5 (8Baker E. Sayers T.J. Sutherland G.R. Smyth M.J. Immunogenetics. 1994; 40: 235-237Crossref PubMed Scopus (37) Google Scholar) are both regulated in this way. One exception to this general “rule” is gzm M, which is constitutively expressed by NK cells but not by human or rodent T cells when they become activated (20Smyth M.J. O'Connor M.D. Kelly J.M. Ganesvaran P. Thia K.Y. Trapani J.A. Biochem. Biophys. Res. Commun. 1995; 217: 675-683Crossref PubMed Scopus (22) Google Scholar, 23Sayers T.J. Brooks A.D. Ward J.M. Hoshino T. Bere W.E. Wiegand G.W. Kelly J.M. Smyth M.J. Kelley J.M. J. Immunol. 2001; 166: 765-771Crossref PubMed Scopus (82) Google Scholar). We therefore wished to determine which leukocyte subsets (if any) in normal human peripheral blood express gzmH, both in their quiescent state or following exposure to activating stimuli. Initially, we subjected unfractionated PBMC from normal individuals to Western blotting, either prior to or after exposure to agents that cause T cell activation and proliferation in vitro (Fig. 4A). Surprisingly, freshly isolated, unstimulated PBMC from unrelated donors showed strong constitutive gzm H expression, whereas the expression of gzm B by the same cells was very low or absent (Fig. 4A). From past studies, gzmB is known to be expressed at low levels in unstimulated NK cells (23Sayers T.J. Brooks A.D. Ward J.M. Hoshino T. Bere W.E. Wiegand G.W. Kelly J.M. Smyth M.J. Kelley J.M. J. Immunol. 2001; 166: 765-771Crossref PubMed Scopus (82) Google Scholar) and is induced from very low levels in stimulated CD4+ and CD8+ T cells (10Haddad P. Jenne D. Tschopp J. Clement M.V. Mathieu-Mahul D. Sasportes M. Int. Immunol. 1991; 3: 57-66Crossref