Cytotoxic Lymphocyte Functions Granzymes C And/Or F Are Important for Granzyme B and the Downstream

Granzyme B and the Downstream Granzymes C and/or F Are Important for Cytotoxic Lymphocyte Functions

This information is current as Paula A. Revell, William J. Grossman, Dori A. Thomas, of October 1, 2021. Xuefang Cao, Rajesh Behl, Jane A. Ratner, Zhi Hong Lu and Timothy J. Ley J Immunol 2005; 174:2124-2131; ; doi: 10.4049/jimmunol.174.4.2124 http://www.jimmunol.org/content/174/4/2124 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Granzyme B and the Downstream Granzymes C and/or F Are Important for Cytotoxic Lymphocyte Functions1

Paula A. Revell,2* William J. Grossman,2,3† Dori A. Thomas,2,4* Xuefang Cao,2* Rajesh Behl,2,5* Jane A. Ratner,* Zhi Hong Lu,* and Timothy J. Ley6*

Although the functions of granzyme A (GzmA) and GzmB are well-defined, a number of orphan granzymes of unknown function are also expressed in cytotoxic lymphocytes. Previously, we showed that a targeted loss-of-function mutation for GzmB was associated with reduced expression of several downstream orphan granzyme genes in the lymphokine-activated killer cell compartment. To determine whether this was caused by the retained phosphoglycerate kinase I gene promoter (PGK-neo) cassette in the GzmB gene, we retargeted the GzmB gene with a LoxP-flanked PGK-neo cassette, then removed the cassette in embryonic stem cells by transiently expressing Cre recombinase. Mice homozygous for the GzmB null mutation containing the PGK-neo cassette GzmB؊/؊/؉PGK-neo) displayed reduced expression of the closely linked GzmC and F genes in their MLR-derived CTLs and) Downloaded from .lymphokine-activated killer cells; removal of the PGK-neo cassette (GzmB؊/؊/⌬PGK-neo) restored the expression of both genes Cytotoxic lymphocytes derived from mice with the retained PGK-neo cassette (GzmB؊/؊/؉PGK-neo) had a more severe cytotoxic defect than those deficient for GzmB only (GzmB؊/؊/⌬PGK-neo). Similarly, GzmB؊/؊/؉PGK-neo mice displayed a defect in the allogeneic clearance of P815 tumor cells, whereas GzmB؊/؊/⌬PGK-neo mice did not. These results suggest that the retained PGK-neo cassette in the GzmB gene causes a knockdown of GzmC and F expression, and also suggest that these granzymes are relevant for the function of cytotoxic lymphocytes in vitro and in vivo. The Journal of Immunology, 2005, 174: 2124–2131. http://www.jimmunol.org/

ytotoxic lymphocytes can use multiple pathways to in- perturbations that may be relevant for the entry of other granule duce target cell death, including secretory mechanisms proteins, including granzymes. Perforin also appears to play a critical C (e.g., TNF-␣ and IFN-␥) and contact-dependent mecha- role in the trafﬁcking of granzymes once they enter the target cell. nisms, including the granule exocytosis pathway and the Fas path- Granzymes, a class of neutral serine proteases, are thought to induce way (1–3). In several studies the killing of tumor cells has been target cell death by cleaving critical intracellular substrates (1, 2). shown to be dependent on the granule exocytosis pathway, but Granzyme A (GzmA)7 and B have very different substrate speciﬁci- variable results have been obtained with different tumor targets in ties, and appear to cleave nonoverlapping sets of target cell proteins

different laboratories, suggesting that more than one mechanism is (1, 2, 12, 13). Although GzmB induces classical apoptosis associated by guest on October 1, 2021 relevant, and that different mechanisms may be important for dif- with mitochondrial depolarization, nuclear collapse and fragmenta- ferent tumors (1, 4–11). tion, and activation of caspase-activated DNase, GzmA induces cell The secretory granules of cytotoxic lymphocytes (CTL, NK, and death that has many of the features of apoptosis, but is clearly different LAK cells) migrate along microtubules to the site of contact be- from death induced by GzmB (1, 2, 14–16). The apparent nonredun- tween the effector and target cells, where a tight immunologic dancy of these two granzymes might be explained in part by the fact synapse is formed (1–3). There, the contents of the cytotoxic gran- that viruses and tumor cells sometimes express inhibitors of granules are secreted into the small intercellular space, where perforin zymes, presumably to thwart the ability of cytotoxic lymphocytes to can polymerize in the presence of calcium to form membrane kill these cells (1). Under these circumstances, alternative granzymes with different speciﬁcities would be required to induce cell death.

*Division of Oncology, Departments of Internal Medicine and Genetics, Siteman In both humans and mice, a variety of additional granzymes of Cancer Center, Washington University School of Medicine, and †Division of Hema- unknown function (orphans) are expressed in cytotoxic lympho- tology/Oncology, Department of Pediatrics, St. Louis Children’s Hospital, St. Louis, cytes (17). In the mouse a large number of functional granzyme MO 63110 genes are found downstream from GzmB in a locus commonly Received for publication September 3, 2004. Accepted for publication October ϳ 24, 2004. known as the GzmB gene cluster. GzmC lies 24 kb downstream from GzmB and is most closely related to human GzmH, which is the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance only orphan granzyme gene found downstream from human GzmB. with 18 U.S.C. Section 1734 solely to indicate this fact. In the mouse, however, several additional granzyme genes lie down- 1 This work was supported by grants from the National Institutes of Health stream from GzmC, including (5Ј-Ͼ3Ј) GzmF, G, L, N, D, and E, (5T32AI07163 (to P.A.R.) and DK49786 (to T.J.L.)) and the Hope Street Kids Foun- followed by cathepsin G (a promyelocyte-speciﬁc serine proteinase of dation (to W.J.G.). similar structure) and several mast cell chymases (17). 2 P.A.R., W.J.G., D.A.T., X.C., and R.B. contributed equally to this work. In our initial GzmB knockout mouse, we removed the coding 3 Current address: Division of Hematology/Oncology, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226. sequences from exon 1 and part of intron 1 and replaced these 4 Current address: Department of Cancer Biology and Genetics, Memorial Sloan Ket- tering Cancer Center, New York, NY 10021. 7 Abbreviations used in this paper: Gzm, granzyme; 7-AAD, 7-aminoactinomycin; 5 Current address: Center for Human Genetics, Bangalore 560066, India. ES, embryonic stem cell; FloKA, ﬂow-based killing assay; LAK, lymphokine-acti- 6 Address correspondence and reprint requests to Dr. Timothy J. Ley, Washington vated killer; PGK-neo, phosphoglycerate kinase I gene promoter; qRT-PCR, quanti- University School of Medicine, 660 South Euclid Avenue, Campus Box 8007, St. tative RT-PCR; rh, recombinant human; [125I]UdR, [125I]5-iodo-2[prime]-deoxyuri- Louis, MO 63110. E-mail address: [email protected] dine; WT, wild type; LCR, locus control region.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 2125 sequences with a functional phosphoglycerate kinase I gene pro- suspensions were made by gently crushing spleens between frosted micro- moter (PGK)-neo cassette (18). We later learned that mixed strain scope slides. Red cells were lysed in ACK buffer (154 mM NH4Cl, 10 mM KHCO , and 0.1 mM EDTA; sterile-filtered) and washed twice in PBS. mice containing this mutation display significantly reduced the ex- 3 The cell suspension from each BALB/c spleen was resuspended in 5 ml of pression of several downstream granzyme genes (C, F, and D) in complete K10 medium (RPMI 1640, 10% heat-inactivated FCS, 10 mM the lymphokine-activated killer (LAK) cell compartment (19). To HEPES, 1% nonessential amino acids, 1% sodium pyruvate, 1% L-glu- determine whether this was a neighborhood knockdown effect tamine, 1ϫ penicillin/streptomycin, 0.57 ␮M ␤-ME) and 50 U/ml recom- caused by the retained PGK-neo cassette, we retargeted the GzmB binant human IL-2 (rhIL-2) and irradiated (2000 cGy). The cell suspensions from WT, GzmBϪ/Ϫ/⌬PGK-neo, and GzmBϪ/Ϫ/ϩPGK-neo spleens gene with a LoxP-flanked PGK-neo cassette, then removed it from were each resuspended in 40 ml of complete K10 medium and 50 U/ml the targeted ES cells with Cre recombinase. Removal of the PGK- rhIL-2. Irradiated BALB/c stimulators and 129/SvJ, GzmBϪ/Ϫ/⌬PGK-neo, neo cassette restored the expression of the downstream granzymes, or GzmBϪ/Ϫ/ϩPGK-neo responders were combined into 45 ml of com- which allowed us to compare the functions of cytotoxic lympho- plete K10 medium and 50 U/ml rhIL-2. Samples were mixed, plated in cytes deficient for GzmB only (GzmBϪ/Ϫ/⌬PGK-neo) or GzmB three 100-mm non-tissue culture-treated plates, and incubated for 4 days. Before FloKA, the mononuclear cells were purified over Ficoll (Sigma- Ϫ/Ϫ ϩ plus reduced levels of GzmC and F (GzmB / PGK-neo). To Aldrich), washed with PBS, and resuspended in complete K10 medium and our surprise, cytotoxic lymphocytes deficient for GzmB, C, and F 50 U/ml rhIL-2. displayed a more striking defect in killing than cytotoxic lymphocytes deficient for GzmB only; this was true not only for LAK Quantitative real-time RT-PCR (qRT-PCR) cells, but also for CTL derived from MLR. To validate these re- Total RNA was isolated from 10 ϫ 106 LAK cells or Ficoll-purified cells sults in vivo, we assessed the ability of these mice to clear an from MLRs using the RNeasy Mini Kit (Qiagen). One microgram of total allogeneic tumor (P815, H-2Kd) and again found that mice defi- RNA from each sample was reverse-transcribed into cDNA using the Taq- Man reverse transcriptase (ABI; Applied Biosystems) and analyzed by Downloaded from cient for GzmB, C, and F have a more significant clearance defect PCR. The relative mRNA abundance of each granzyme mRNA was mea- than those deficient for GzmB only. In sum, these results have sured by the 7300 real-time PCR system from Applied Biosystems, using shown that the retained PGK-neo cassette in the GzmB gene does primer sets that were proven to amplify only the self granzyme cDNA cause a neighborhood effect, and the reduced expression of GzmC using the conditions described below. The primer sets used in these studies Ј and F is relevant for the functions of CTLs and LAK cells in vitro were as follows: GzmA-specific primer set: forward, 5 -AGA CCG TAT ATG GCT CTA CT-3Ј; and reverse, 5Ј-CCC TCA CGT GTA TAT TCA and in vivo. TC-3Ј; GzmB-specific primer set: forward, 5Ј-ATC AAG GAT CAG CAG CCT GA-3Ј; and reverse, 5Ј-TGA TGT CAT TGG AGA ATG TCT-3Ј; http://www.jimmunol.org/ Materials and Methods GzmC-specific primer set: forward, 5Ј-ATG AGT TTC TGA AAG TTG Construction of the GzmB targeting vector GTG-3Ј; and reverse, 5Ј-TCA TTA GAA CGG TCA TCA GG-3Ј; GzmF- specific primer set: forward, 5Ј-TGA GGT TTG TGA AAG ATA ATG-3Ј; A 4.16-kb EcoRI fragment containing the entire GzmB locus was sub- and reverse, 5Ј-TCA CTG GTG TTG TCC TTA TC-3Ј; GzmG-specific cloned into pUC19 to generate the backbone of the GzmB targeting vector, primer set: forward, 5Ј-GTT CAT TAA GTC TGT GGA TAT CG-3Ј; and similar to that previously described (18). A 350-bp AvrII fragment con- reverse, 5Ј-GTC TTG GAA TAG GTG TAC CAG-3Ј; GzmD-specific taining the initiation and start sites and the entire first exon (and part of the primer set: forward, 5Ј-AAA CAG CTC TGT CCA AAG CTC-3Ј; and first intron) of the GzmB gene was replaced with a 1.6-kb NotI fragment reverse, 5Ј-CAA ATC TCT GTG GTC TCA GTG-3Ј; and GzmE-specific containing a LoxP-flanked neomycin phosphotransferase gene driven by primer set: forward, 5Ј-CTG CTC ACT GCA GGA ACA GGA-3Ј; and

PGK-neo (20). The resulting targeting vector contained 1.03 kb of genomic reverse, 5Ј-CAA ATC TCT GTG GTC TCA GTG-3Ј. The qRT-PCRs were by guest on October 1, 2021 DNA from the GzmB 5Ј flank upstream from the PGK-neo insertion and conducted in the presence of the DNA intercalating dye SYBR Green. PCR 2.75 kb of genomic DNA containing exons 2–5 downstream from PGK- conditions used for all primer sets were as follows: 95°C hot start for 10 neo. The targeting vector was linearized with SalI and gel-purified before min, followed by 40 amplification cycles of 95°C for 30 s (denaturing), electroporation into Rw4 embryonic stem cells (ES; 129/SvJ). 59°C for 40 s (annealing), and 72°C for 40 s (extension). A PCR ampli-

Ϫ/Ϫ fication profile was derived by recording the SYBR Green fluorescence Generation of 129/SvJ mice containing the GzmB /ϩPGK- intensity, which was in linear relation to the amount of formed PCR prod- Ϫ Ϫ neo and GzmB / /⌬PGK-neo mutations uct (as a function of PCR cycle number). Standard curves were generated by plotting the PCR cycle number at which a reaction entered exponential ϩ/Ϫ ϩ GzmB / PGK-neo ES were generated by electroporation of the GzmB amplification vs the amount of input DNA of plasmids containing the targeting vector in Rw4 ES cells, neomycin-resistant clones were ex- cDNA of each granzyme gene. The standard curves were then used for a panded, and homologous recombination events were identified by Southern determination of sample template concentration. blot analysis using an external probe (data not shown) (18). Correct targeting was confirmed by Southern blot analysis using an internal probe Western blot analysis after EcoRI digestion (Fig. 1). ES clones with specific homologous recombination were injected into C57BL/6 blastocysts that were implanted into Ficoll-purified MLR and LAK cell lysates were prepared and analyzed pseudopregnant Swiss-Webster mice. Male chimeras were bred with 129/ using standard Western blotting techniques as previously described (16, SvJ female mice, and germline transmission of the mutant alleles was 22). Primary Abs included rabbit anti-mouse GzmA antiserum (MA2A) confirmed by Southern blot analysis. Heterozygous animals were inter- (16), rabbit anti-mouse GzmB antiserum (526B) (22), rabbit anti-mouse crossed to generate homozygous GzmBϪ/Ϫ/ϩPGK-neo deficient mice. GzmC antiserum (428A) (23), and goat anti-mouse ␤-actin-HRP antiserum GzmBϩ/Ϫ/⌬PGK-neo ES clones were generated by transiently transfecting (Santa Cruz Biotechnology). Primary Abs were detected using secondary GzmBϩ/Ϫ/ϩPGK-neo ES clones with a Turbo-Cre-expressing plasmid as goat anti-rabbit HRP or horse anti-goat HRP with standard chemilumines- previously described (20, 21), resulting in the recombination of LoxP sites cence procedures (Amersham Biosciences). Recombinant murine GzmA, and the deletion of the internal PGK-neo cassette. Resultant GzmBϩ/Ϫ/ B, and C were purified as previously described (16, 23, 24). One hundred ⌬PGK-neo clones were screened for neomycin sensitivity, and deletion of nanograms of each recombinant granzyme was used to demonstrate anti- the PGK-neo cassette was confirmed by Southern blot analysis (Fig. 1). serum specificity. Twenty-five to 50 ␮g of MLR or LAK cell protein was Mutant mice were generated exactly as described above. Heterozygous used in each lane. animals were intercrossed to generate homozygous GzmBϪ/Ϫ/⌬PGK-neo deficient mice. Both mutant strains were maintained as homozygous breed- Animals ing colonies in the 129/SvJ background. WT C57BL/6J, BALB/c, and 129/SvJ mice were obtained from The Jack- MLR and LAK cell preparations for in vitro cytotoxicity and son Laboratory. GzmBϪ/Ϫ/ϩPGK-neo- and GzmBϪ/Ϫ/⌬PGK-neo mice in flow cytometry assays the 129/SvJ background were generated as described above. The gld/gld and perforinϪ/Ϫ mice in C57BL/6 backgrounds were previously described MLR and LAK cell preparations were performed as previously described (25). GzmBϪ/Ϫ/ϩPGK-neo mice in the C57BL/6J background were gen- (18, 22). For MLR preparations, stimulator (BALB/c wild-type (WT); erated by backcrossing our previously described mutant mice (18) to H-2Kd) and effector (129/SvJ WT, GzmBϪ/Ϫ/ϩPGK-neo, and GzmBϪ/Ϫ/ C57BL/6J mice for 10 generations. All mice were bred and kept in patho- ⌬PGK-neo; all H-2Kb) spleens were harvested into PBS, and single cell gen-free housing in accordance with Washington University School of 2126 ORPHAN GRANZYME AND CTL

Medicine animal care guidelines, using protocols approved by the animal studies committee. Intracellular granzyme staining and flow cytometry Unstimulated splenocytes or MLR cells were stained for surface markers and intracellular GzmB expression using a GzmB-specific Ab as previously described (26). Briefly, 1 ϫ 106 cells were first labeled with fluo- rochrome-conjugated Abs against cell surface markers (anti-mouse CD4, CD8, and DX5; BD Pharmingen). Samples were fixed and permeabilized (Cytofix/Cytoperm; BD Pharmingen), then stained with primary conjugated anti-GzmB Ab (GB12; Caltag Laboratories) diluted at 1/400 in staining buffer. During all steps of staining, permeabilization, and washing, anti-murine Fc␥RII/III-blocking Ab (0.1 ␮g/␮l, final concentration; BD Pharmingen) and human albumin (1%; Aventis Behring) were used to block nonspecific FcR Ab binding. Samples were analyzed on a FACScan (BD Biosciences). All FACScans depicted are representative of four or more independent experiments. Flow-based killing assay (FloKA) and [125I]5-iodo-2[prime]- deoxyuridine ([125I]UdR) release assay CTL and LAK cells were assessed by FloKA as previously described (26). d

Briefly, allogeneic (P815 and TA3-H-2K ; American Type Culture Col- Downloaded from lection) or MHC class I-deficient (RMAS and YAC-1; American Type Culture Collection) murine target cells were washed with PBS and labeled with 125 nM (final concentration) CFSE (Molecular Probes). Labeling re- actions were stopped with complete K10 medium. Labeled cells were added to 96-well, V-bottom, tissue culture-treated plates (Corning Glass) along with the indicated effector cells (CTLs from MLRs, or LAK cells; see above) in complete medium containing 50 U/ml rhIL-2. E:T cell ratios varied from 5:1 to 20:1 in different experiments. Immediately before anal- http://www.jimmunol.org/ ysis, 1 ␮g/ml (final concentration) of 7-aminoactinomycin D (7-AAD; Cal- FIGURE 1. Targeting strategy and screening. A, Targeting vector used for biochem) was added to each sample. Control samples (targets only) had homologous recombination containing the PGK-neo selection cassette flanked effectors added immediately before analysis at the indicated E:T cell ratio. 125 by two LoxP recombination sites. Recombination of the targeting vector with [ I]UdR release assays were performed as previously described (18, 27). ϩ Ϫ the endogenous allele results in formation of GzmB / /ϩPGK-neo ES cells. All experiments examining WT vs Gzm-deficient mice were performed in ϩ/Ϫ ϩ parallel. All samples were analyzed in duplicate. Data presented in graphic Transfection of a Cre-expressing plasmid in GzmB / PGK-neo ES cells form are the combination of three or more individual experiments. The data results in excision of the PGK-Neo selection cassette through recombination of ϩ Ϫ shown in individual FACS scans are representative of three or more indi- the flanking LoxP sites and formation of GzmB / /⌬PGK-neo ES cells. E, vidual experiments. Statistical analyses were performed by one-way-ANOVA EcoRI. Probe A was used for the Southern blot shown in B. B, Southern blot with Bonferroni post-test analysis or Student’s t test, using PRISM version analysis of EcoRI-digested tail DNA derived from WT mice or heterozygous by guest on October 1, 2021 3.0a for Macintosh (GraphPad). mice containing the GzmBϩ/Ϫ/ϩPGK-neo or GzmBϩ/Ϫ/⌬PGK-neo mutations. The expected fragment sizes are shown. Allogeneic tumor cell clearance in vivo All mice were sublethally irradiated (400 cGy) on day Ϫ1 and subse- quently injected i.v. in the lateral tail vein with 1 ϫ 107 P815 mastocytoma Ϫ Ϫ Ϫ Ϫ 129/SvJ mice, and the mutations were transmitted through the germ- cells on day 0. WT, GzmB / /ϩPGK-neo, perforin / , and gld/gld mice (all on the C57BL/6J background) were monitored over a 60-day period. line. The expected EcoRI fragments were detected in the tail DNA of ϩ Ϫ WT, GzmBϪ/Ϫ/⌬PGK-neo, and GzmBϪ/Ϫ/ϩPGK-neo mice (on the 129/ mice heterozygous for the mutations (4.4 kb GzmB / /ϩPGK-neo, SvJ background) were similarly injected and monitored. 2.8 kb GzmBϩ/Ϫ/⌬PGK-neo, and 4.2 kb WT) with Southern blot analysis using an internal probe (Fig. 1, A and B) and also an external Results probe (data not shown). Heterozygous matings yielded offspring with Creation of mice with targeted mutations in the GzmB gene the expected Mendelian frequencies. Homozygous GzmBϪ/Ϫ/ Ϫ Ϫ We generated GzmBϪ/Ϫ/ϩPGK-neo mice using the targeting vec- ϩPGK-neo and GzmB / /⌬PGK-neo mice were found to have nor- tor shown in Fig. 1A (top panel). The targeted GzmB allele con- mal development and fertility. tained a 350-bp deletion that starts in the 5Ј untranslated region of the gene, and removes all the coding sequences of exon 1, and part of Neighborhood effect on downstream granzyme gene expression intron 1 (18). Targeted ES cells containing the mutant GzmBϩ/Ϫ/ caused by the retained PGK-neo cassette ϩPGK-neo allele contained the PGK-neo selection cassette flanked We measured the expression of multiple granzyme genes in cells by two LoxP sites. We generated GzmBϩ/Ϫ/⌬PGK-neo ES cells derived from MLRs and LAK preparations from both GzmBϪ/Ϫ/ by transiently transfecting the GzmBϩ/Ϫ/ϩPGK-neo ES clones ϩPGK-neo and GzmBϪ/Ϫ/⌬PGK-neo mice using qRT-PCR with with pTurbo-Cre, which resulted in recombination of the two LoxP primer pairs specific for each granzyme gene (Fig. 2). All primer sites and deletion of the internal PGK-neo selection cassette (Fig. pairs were shown to amplify only the cloned self target cDNA in 1A, top panel). Using Southern blot analysis with internal and qRT-PCRs (data not shown). However, annealing efficiencies var- external probes along with new restriction sites introduced by the ied among the primer pairs, so comparisons of the absolute abun- targeting vector, we demonstrated correct targeting of the vector in dances of different granzyme mRNAs are not valid. We therefore two independent Rw4 (129/SvJ) ES clones; precise deletion of the normalized the abundance of each granzyme mRNA to the level PGK-neo cassette in the targeted clones was likewise demon- detected in WT LAK cells. In all experiments activations were strated with Southern blot analysis. The correctly targeted ES performed concurrently with all three genotypes. Neither GzmB clones were injected into C57BL/6 blastocysts, which were then mutant strain had detectable GzmB mRNA expression in LAK implanted into pseudopregnant female recipients. All ES clones cells or CTLs (Fig. 2). There was no significant difference in the gave rise to chimeric male mice that were then directly mated to expression levels of GzmA in any of the mutant mice, because The Journal of Immunology 2127

FIGURE 2. Quantitative RT-PCR analysis of granzyme mRNA abundance in MLR and LAK cells. Quan- titative RT-PCR was performed on three independent MLR and three independent LAK cell preparations from mice of the described genotypes. All data for each speciﬁc granzyme mRNA was normalized to the level of expression detected in WT LAK cells. Because primer pairs an- nealed to their targets with different efﬁciencies, compar- ison of absolute mRNA abundance among different granzymes is not valid. Values shown are the mean Ϯ SD. .p Ͻ 0.001 ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء

GzmA is located on a different chromosome than GzmB (17). The similar to those observed with mRNA analysis. GzmA protein ex- qRT-PCR analysis of GzmC and F mRNAs in LAK cells derived pression was equivalent in LAK cell lysates from WT and the two from GzmBϪ/Ϫ/ϩPGK-neo mice demonstrated that expression GzmB knockout strains (Fig. 3, middle panels); GzmA protein could was reduced 5- to 6-fold compared with that in WT LAK cells, but not be detected in day 4 MLR-derived lysates, probably because this Downloaded from was normal in LAK cells derived from GzmBϪ/Ϫ/⌬PGK-neo mice gene is activated later than GzmB and C (28). Recombinant murine (Fig. 2). GzmC and F expression in MLR-derived cells from granzymes were used for each Western blot as a control for antiserum GzmBϪ/Ϫ/ϩPGK-neo mice was reduced compared with that in specificity (Fig. 3, right panels), and each blot was analyzed for ␤-ac- WT cells, but expression levels were very low, and the difference tin protein levels as an internal control for protein loading. Specific was not statistically significant. GzmC and F expression in Abs for GzmD, E, F, and G are not yet available. Ϫ/Ϫ GzmB /⌬PGK-neo CTLs was reproducibly increased compared http://www.jimmunol.org/ with that in WT animals (Fig. 2). mRNAs for GzmD, E, and G Normal lymphocyte subsets in GzmB-deficient mice were not detectably expressed in MLR-derived CTLs, but all were To ensure that the observed differences in granzyme mRNA and Ϫ/Ϫ detected in LAK cells and were not altered in GzmB /ϩPGK- protein levels between GzmBϪ/Ϫ/ϩPGK-neo and GzmBϪ/Ϫ/ neo derived LAKs. ⌬PGK-neo mice were not due to skewed granzyme-expressing Examination of granzyme protein levels from MLR and LAK populations, we examined these mice for the main lymphocyte cell lysates yielded a similar expression profile (Fig. 3). MLR and populations known to express granzymes, namely CD4ϩ and Ϫ/Ϫ Ϫ/ LAK cell lysates from both GzmB /ϩPGK-neo and GzmB CD8ϩ T cells and NK cells (DX5-positive cells). As shown in Ϫ/⌬PGK-neo mice lacked a detectable GzmB signal using West- Fig. 4A, no differences were found in the percentages of these ern blot analysis (Fig. 3, left and middle panels). LAK cells from lymphocyte subpopulations between WT mice and the two by guest on October 1, 2021 Ϫ/Ϫ GzmB /ϩPGK-neo mice had an ϳ80% reduction in GzmC pro- GzmB-deficient mouse strains. Resting WT splenocytes do not tein expression compared with WT LAKs (Fig. 3, middle panels). contain detectable amounts of GzmB (Fig. 4B), in contrast to Ϫ/Ϫ In contrast, LAK cell lysates from GzmB /⌬PGK-neo mice had resting human PBMCs, where 15–25% of resting CD8ϩ T cells restoration of GzmC protein expression to WT levels. Analysis of contain detectable amounts of this enzyme (26). Ϫ/Ϫ MLR-derived protein lysates from GzmB /⌬PGK-neo mice We next examined the intracellular GzmB expression profile demonstrated GzmC protein expression levels that were elevated over from WT MLR-derived cells using flow cytometry and an intra- the GzmC protein levels found in wild-type CTLs (Fig. 3, left panels), cellular staining assay for GzmB (26). These studies demonstrated that the majority of GzmB-expressing cells in MLRs are CD8ϩ T cells (Fig. 4C); however, a small percentage of CD4ϩ T cells also express GzmB, a finding consistent with that of human PBMC (26). As expected, no GzmB intracellular expression was detected in either CD4ϩ or CD8ϩ T cells in MLRs from GzmBϪ/Ϫ/ϩPGK- neo and GzmBϪ/Ϫ/⌬PGK-neo mice (Fig. 4C). The polyclonal Ab against GzmC used in the Western blots in Fig. 3 was not useful in flow-based studies; mAbs specific for murine GzmA or C are not yet available. A small amount of nonspecific staining (presumably representing activated macrophages) was observed in MLR- derived cells from all mouse strains (Fig. 4C), which was not pre- vented by Fc␥RII-III-blocking Abs.

Defects in cytotoxicity in vitro To determine whether the two GzmB knockout strains have defects in cytotoxicity, we first examined CTLs generated from MLR for their ability to kill allogeneic P815 or TA3 tumor cells (both d FIGURE 3. Western blot analysis of cell lysates made from LAK cells from an H-2K background) using a flow-based killing assay Ϫ/Ϫ ϩ and MLR-derived CTLs. Purified murine recombinant granzymes (rGzms) (FloKA). We found that GzmB / PGK-neo CTLs were signif- were used for specificity controls of Abs in each Western blot (top, middle, icantly less efficient at inducing target cell death than WT CTLs at and bottom panels). Data shown are representative of three or more inde- all time points tested (representative data are shown in Fig. 5A,R1 pendent experiments. and R2 gates). The same cytotoxic defects were detected with both 2128 ORPHAN GRANZYME AND CTL

cytotoxic defect than GzmBϪ/Ϫ/ϩPGK-neo CTLs (Figs. 5A and 6A, R1 gates). A similar defect in the production of late stage apoptotic events was seen with LAK cells generated from both Gzm-deficient strains against two different NK-sensitive target cell lines (RMAS and YAC-1 cells; Fig. 6B). The most severe cytotoxic defect was observed with GzmBϪ/Ϫ/ϩPGK-neo LAK cells, with GzmBϪ/Ϫ/⌬PGK-neo LAKs again demonstrating an inter- mediate defect (Fig. 6B). Although the severity of the cytotoxic phenotype was partly dependent upon the target cells used, the trends were the same for all target cells tested. When we examined GzmBϪ/Ϫ/ϩPGK-neo and GzmBϪ/Ϫ/ ⌬PGK-neo LAKs for their ability to release dsDNA fragments using classic [125I]UdR release assays, we detected no difference between the two Gzm-deficient strains regardless of time or E:T cell ratio (Fig. 5B). CTLs from both Gzm-deficient strains displayed a severe defect in [125I]UdR clearance at 2 h, but this defect was not apparent after 8 h (Fig. 5B), similar to results previously described (18). No abnormalities were detected in 51Cr release with GzmB-deficient LAK cells, as expected (data not shown). Downloaded from Defects in tumor clearance in vivo To determine whether the cytotoxic defects detected in vitro were relevant in vivo, we examined the clearance of P815 cells in sublethally irradiated (400 cGy) C57BL/6 mice deficient for perforin, GzmBϪ/Ϫ/ϩPGK-neo, or FasL (gld). The GzmBϪ/Ϫ/ϩPGK-neo and perforin-deficient mice were equivalently impaired in their http://www.jimmunol.org/ ability to survive a P815 challenge, compared with WT mice (Fig. 7A). In contrast, gld/gld mice were able to clear P815 cells as efficiently as WT mice (Fig. 7A). To examine the role of orphan granzymes in allogeneic tumor clearance, we compared the clearance of P815 cells in sublethally irradiated (400 cGy) 129/SvJ mice (WT vs GzmBϪ/Ϫ/ϩPGK-neo vs GzmBϪ/Ϫ/⌬PGK-neo). GzmBϪ/Ϫ/ϩPGK-neo mice were sig-

nificantly impaired in their ability to clear P815 cells (Fig. 7B). In by guest on October 1, 2021 contrast, GzmBϪ/Ϫ/⌬PGK-neo mice cleared P815 cells as efficiently as WT 129/SvJ mice. Discussion In this report we describe two strains of mice that are deficient for GzmB. In one, a retained PGK-neo cassette in the GzmB gene causes knockdown of the most closely linked genes, GzmC and F. Removal of the PGK-neo cassette from the GzmB gene by Cre- FIGURE 4. Flow cytometric analysis of lymphocyte subpopulations mediated recombination restored the expression of GzmC and F to and GzmB expression. A, CD4ϩ, CD8ϩ, and NK (DX5) cell subpopulation normal levels in LAK cells; however, in MLR-derived CTLs, Ϫ Ϫ analysis in unstimulated splenocytes from WT, GzmB / /ϩPGK-neo, and GzmC and F were expressed at higher than normal levels. The GzmBϪ/Ϫ/⌬PGK-neo mice, showing similar cellular compositions. B, ϩ ϩ cytotoxic potential of MLR-generated CTLs from both GzmB-de- Lack of GzmB expression in unstimulated CD4 , CD8 , and NK (DX5) ficient strains was reduced, but the defect was more severe in mice cell populations in WT spleens. C, Intracellular GzmB detection in WT ϩ ϩ with the retained PGK-neo cassette, implying that GzmC and/or F CD4 and CD8 cells from 4-day MLRs. The lack of detectable GzmB expression in GzmBϪ/Ϫ/ϩPGK-neo and GzmBϪ/Ϫ/⌬PGK-neo MLR cells may be relevant for in vitro cytotoxicity. Finally, these results were is demonstrated, with the exception of nonspecific background macrophage corroborated in vivo, where the allogeneic clearance of P815 tu- Ϫ/Ϫ staining, which is demonstrated in the top panels. mor cells was significantly impaired in GzmB /ϩPGK-neo mice, but was normal in mice deficient for GzmB only. These data suggest that the orphan GzmC and/or F are relevant for cytotoxic P815 and TA3 targets, and the findings were consistent and highly lymphocyte functions in vitro and in vivo. reproducible. The most striking defect was the inability of these Retained PGK-neo cassettes in a targeted locus can cause the effectors to generate the small, 7-AADlowϩhigh cells that are lo- expression of tightly linked genes to be reduced (19, 20, 29–31). cated in the R1 gate, which probably represent cells in the late Although many examples of this neighborhood effect have been stages of apoptosis. CTLs derived from both Gzm-deficient strains reported, the mechanism and rules that govern the effect are not yet induced an increased number of target cells in the R2 gate (large clear. The interposition of an active selectable marker cassette be- cells that are 7-AADhigh, probably representing cells with early tween a locus control region (LCR) and its functional targets can apoptotic events), suggesting that the induction of target cell significantly reduce the expression of those linked genes over tens death is delayed with GzmB-deficient effectors, a finding that cor- of kilobases. This effect can often be eliminated by removing the roborates earlier studies of CTL function with these mice (18). selectable marker cassette, using LoxP/Cre (or Frt/Flp) recombi- GzmBϪ/Ϫ/⌬PGK-neo generated CTLs demonstrated a less severe nation technology (20, 29–31). Indeed, removal of the PGK-neo The Journal of Immunology 2129

FIGURE 5. Functional assays of CTLs. A, Represen- tative FloKA data of allogeneic P815 and TA3 target cells coincubated with MLR-derived CTL at an E:T cell ratio of 20:1 for the indicated times. CFSE-positive target cells were gated and analyzed for forward scatter and 7-AAD incorporation. The R1 gate contains small 7-AADlowϩhigh cells that are in the late stages of apoptotic cell death. The R2 gate contains large 7-AADhigh cells that are in the early stages of cell death. Both Gzm- deficient strains have a defect in the killing of P815 and TA3 allogeneic tumor cells, specifically in the percentages of late apoptotic cells (R1 gate), with GzmBϪ/Ϫ/ ϩPGK-neo CTLs demonstrating the most severe defect. Downloaded from Similar trends were obtained using E:T cell ratios of 5:1 and 10:1. B,[125I]UdR release assay using LAK cell effectors coincubated with [125I]UdR-labeled YAC-1 target cells for 2 h (left panel)or8h(right panel). LAK cells from both GzmB-deficient strains demonstrated a similar severe defect in the release of [125I]UdR at2hat http://www.jimmunol.org/ all E:T cell ratios tested. This defect had resolved at 8 h, as previously described (18). by guest on October 1, 2021

cassette from the GzmB gene restored expression of GzmC and F, were performed, using primers absolutely specific for each granzyme which are located 24.7 and 44.8 kb downstream from GzmB, re- mRNA. Regardless, it is clear that the retained PGK-neo cassette spectively. The expression of the granzyme genes located further caused a knockdown of GzmC and F expression in both mutant downstream (G, D, and E) was not affected by the retained PGK- mouse strains, and that this expression was restored upon removal of neo cassette, which suggests that the effect is limited to the 5Ј end the cassette. of this multigene cluster. We previously examined the effect of a We do not yet understand why expression of the GzmC and F retained PGK-neo cassette in the murine cathepsin G gene, which genes is higher than normal in MLR-derived CTLs from GzmBϪ/Ϫ/ lies just downstream from the GzmE gene (32); this retained cas- ⌬PGK-neo mice. The targeted mutation of the GzmB gene creates sette knocked down the expression of the mast cell chymase gene a deletion between two naturally occurring AvrII sites. The 5Ј end just downstream from it, but did not affect the expression of the of the deletion is in the very short 5Ј-untranslated region of GzmB. granzymes found upstream, suggesting a unidirectional component The deletion extends 350 bp downstream and includes all coding of the neighborhood effect. sequences within the first exon and part of the first intron of the In our previously described mouse model of GzmB deficiency gene. The transcription start site of the gene is therefore removed. (18), the GzmB gene was targeted with a standard PGK-neo cas- It is possible that removal of the transcriptional start site disrupts sette, and expression studies were performed in mice on a mixed a functional interaction between a putative LCR and the 5Ј end of strain background (129/SvOlaϫC57BL/6). In that mouse model, the GzmB gene, allowing the LCR to scan downstream for the next the expressions of GzmC, F, and D were all significantly reduced available promoter (i.e., GzmC). Alternatively, it is possible that in LAK cells (19). In the current study both targeted mutations high level transcription of the GzmB gene normally causes inter- were made in a 129/SvJ ES line (Rw4). Chimeric males made from ference with the transcription of GzmC and F; this transcriptional these ES cells were bred to 129/SvJ females, so that the mutations interference would be relieved by the start site deletion. However, were immediately anchored in a pure 129/SvJ background. In the the expression levels of GzmC and F are normal in LAK cells from current model we detected a striking reduction in GzmC and F ex- GzmBϪ/Ϫ/⌬PGK-neo mice, making it less likely that these mech- pression in LAK cells, but detected no reduction in GzmD expression. anisms are involved. Alternatively, it is possible that a specific This may reflect strain-specific differences in the mouse models, or it subpopulation of cells within an MLR preferentially expresses could explain differences in the measurement technology. In the pre- GzmC and F in this setting; we cannot address this issue experi- vious study experiments were performed using S1 nuclease protection mentally as yet, because mAbs that permit flow-based detection of assays (19, 32); in the current study, quantitative RT-PCR studies these granzymes are not yet available. 2130 ORPHAN GRANZYME AND CTL

FIGURE 7. In vivo allogeneic tumor cell clearance. A, Sublethally ir- Downloaded from radiated mice (H-2Kb, C57BL/6J background) were injected with 1 ϫ 107 allogeneic P815 tumor cells and monitored for survival. GzmBϪ/Ϫ/ϩPGK- neo and perforinϪ/Ϫ mice had significantly diminished survival compared with WT and gld/gld mice. B, Sublethally irradiated mice (H-2Kb,129/SvJ FIGURE 6. Summary of CTL and LAK cytotoxicity studies. A, FloKA background) were injected with 1 ϫ 107 allogeneic P815 tumor cells and monitored for survival. GzmBϪ/Ϫ/ϩPGK-neo deficient mice had a signif- analysis of CTL cytotoxicity against allogeneic targets (P815 and TA3 http://www.jimmunol.org/ Ϫ Ϫ tumor cells) from WT, GzmBϪ/Ϫ/ϩPGK-neo, and GzmBϪ/Ϫ/⌬PGK-neo icantly diminished survival compared with WT and GzmB / /⌬PGK-neo mice at a 20:1 E:T cell ratio after3hofincubation. GzmBϪ/Ϫ/ϩPGK-neo mice. and GzmBϪ/Ϫ/⌬PGK-neo CTLs demonstrate significantly decreased ability to induce late stage apoptotic cells (R1 gated cells) vs WT in P815 and TA3 target cells. Both GzmBϪ/Ϫ/ϩPGK-neo and GzmBϪ/Ϫ/⌬PGK-neo CTLs had a significantly higher fraction of large 7-AADhigh cells (P815 sistent with previous results (18). Alternatively, the differences in and TA3) in the early stages of cell death (R2 gated cells) compared with WT CTLs. B, FloKA analysis of LAK cytotoxicity against NK-sensitive the target cell FLoKA patterns between the two strains may rep- targets (RMAS and YAC-1 tumor cells) from WT, GzmBϪ/Ϫ/ϩPGK-neo, resent specific, biochemically unique apoptotic events induced by and GzmBϪ/Ϫ/⌬PGK-neo mice at a 10:1 E:T cell ratio after4hofcoin- GzmC and/or F. Indeed, data from our laboratory have demon- by guest on October 1, 2021 cubation. All effectors demonstrated significant target cell killing compared strated that GzmC can directly induce mitochondrial depolariza- with targets only (p Ͻ 0.0001). Data shown are the combination of four tion and nuclear collapse in a noncaspase-dependent manner (23). independent experiments, each performed in duplicate. Values shown are Regardless, these findings suggest that the FloKA assay can detect Ͻ ءءء Ͻ ءء Ͻ ء Ϯ the mean SD. , p 0.05; , p 0.01; , p 0.001. subtle changes in target cells undergoing cell death that are not detected by traditional cytotoxicity assays (that rely on the release of radiolabeled proteins and/or DNA from target cells). The strong The generation of these two GzmB-deficient mouse strains has correlation between the cytotoxic defects detected by this method allowed us to assess the importance of granzyme genes downstream from GzmB in cytotoxic killing assays in vitro and in vivo. and the tumor clearance defect detected in vivo suggests that these Traditional [125I]UdR and 51Cr release assays did not reveal sig- changes may indeed be physiologically relevant. nificant differences between the cytotoxic profiles of LAK cells We detected a strong defect in the clearance of P815 tumor cells in perforin-deficient mice and in mice with our original with and without the PGK-neo cassette. However, FloKA revealed Ϫ Ϫ Ϫ Ϫ / ϩ that the CTLs derived from GzmB / /ϩPGK-neo mice had a GzmB / PGK-neo mutation on the C57BL/6 background. more significant killing defect than those from GzmBϪ/Ϫ/⌬PGK- These data strongly suggest that GzmB and/or the orphan gran- neo mice. Previous work by Lecoeur et al. (33, 34) revealed that zymes affected by this mutation (i.e., GzmC, F, and D) are relevant target cells with the FSClow/7-AADhigh phenotype represent late for the perforin-dependent clearance of this tumor cell line in vivo. apoptotic target cells (as defined by increased activation of These data are similar to those reported by Pardo et al. (4), who caspases-3 and -8, as well as increased mitochondrial depolariza- showed that GzmA and B cluster enzymes are important for per- tion, DNA fragmentation, and annexin V staining). In contrast, forin-dependent clearance of RMAS cells in vivo. However, our FSChigh/7-AADhigh target cells (R2 gate) were shown to represent data are different from those reported by Smyth, Trapani, and col- cells in the early stages of apoptosis (defined by lower levels of leagues (5, 6), who detected perforin-dependent, but granzyme- mitochondrial depolarization, DNA fragmentation, and annexin V independent, clearance of several tumor cell lines in vivo. The staining) (33, 34). In this study we noted a significant decrease in reasons for these discrepancies are not yet clear. Differences in the percentage of late apoptotic target cells (e.g., FSClow/7- laboratory stocks of tumor cell lines could be relevant, and subtle AADlowϩhigh; R1 gate) induced by MLR-generated CTLs and differences in the strain backgrounds of the mutant mice could also Ϫ Ϫ LAK cells from both GzmB-deficient strains, but the defect was be important, because GzmB / /ϩPGK-neo mice on the more severe with CTLs from the GzmBϪ/Ϫ/ϩPGK-neo mice. C57BL/6J background are clearly more susceptible to death than CTLs from both strains caused excess target cells to appear in the the same mice on the 129/SvJ background (see Fig. 7). Clearly, early apoptotic gate (R2), suggesting that the full induction of tar- additional studies will be required to understand the causes of get cell death requires additional time if GzmB is missing, con- these laboratory-to-laboratory differences. The Journal of Immunology 2131

In addition to the defect in allogeneic tumor cell clearance in 9. Tsukada, N., T. Kobata, Y. Aizawa, H. Yagita, and K. Okumura. 1999. Graft- GzmBϪ/Ϫ/ϩPGK-neo mice, we have detected a defect in the con- versus-leukemia effect and graft-versus-host disease can be differentiated by cytotoxic mechanisms in a murine model of allogeneic bone marrow transplanta- trol of gammaherpesvirus (␥HV68) latency and reactivation in tion. Blood 93:2738. these mice (35); this defect is more severe than that in mice defi- 10. Pan, L., T. Teshima, G. R. Hill, D. Bungard, Y. S. Brinson, V. S. Reddy, K. R. Cooke, and J. L. Ferrara. 1999. Granulocyte colony-stimulating factor- cient for GzmB only. These data support the observations reported mobilized allogeneic stem cell transplantation maintains graft-versus-leukemia in this study and implicate GzmC and/or F in the control of gam- effects through a perforin-dependent pathway while preventing graft-versus-host maherpesviruses in vivo. disease. Blood 93:4071. Ϫ/Ϫ 11. Schmaltz, C., O. Alpdogan, K. J. Horndasch, S. J. Muriglan, B. J. Kappel, The neighborhood effect in LAK cells derived from GzmB / T. Teshima, J. L. Ferrara, S. J. Burakoff, and M. R. van den Brink. 2001. Dif- ϩPGK-neo mice predominantly affects GzmC and F. Recent stud- ferential use of Fas ligand and perforin cytotoxic pathways by donor T cells in ies in our laboratory have demonstrated that GzmC can induce graft-versus-host disease and graft-versus-leukemia effect. Blood 97:2886. 12. Kam, C. M., D. Hudig, and J. C. Powers. 2000. Granzymes (lymphocyte serine target cell death that has many of the features of apoptosis, includ- proteases): characterization with natural and synthetic substrates and inhibitors. ing nuclear collapse, annexin V positivity, and mitochondrial de- Biochim. Biophys. Acta. 1477:307. 13. Bredemeyer, A. J., R. M. Lewis, J. P. Malone, A. E. Davis, A. E. Gross, polarization. However, GzmC does not cause nuclear fragmenta- R. R. Townsend, and T. J. Ley. 2004. A proteomic approach for the discovery of tion or double-stranded intranucleosomal DNA cleavage, and it protease substrates. Proc. Natl. Acad. Sci. USA 101:11785. does not cleave any of the recognized substrates of GzmB (23). 14. Ebnet, K., M. Hausmann, F. Lehmann-Grube, A. Mullbacher, M. Kopf, M. Lamers, and M. M. Simon. 1995. Granzyme A-deficient mice retain potent The ability of GzmC to cause cell death is related to its protease cell-mediated cytotoxicity. EMBO J. 14:4230. activity, because a mutation in its active site serine significantly 15. Beresford, P. J., Z. Xia, A. H. Greenberg, and J. Lieberman. 1999. Granzyme A reduces its ability to kill target cells in vitro; however, the speci- loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation. Immunity 10:585. ficity of this protease and its relevant substrates are currently un- 16. Shresta, S., T. A. Graubert, D. A. Thomas, S. Z. Raptis, and T. J. Ley. 1999. known. Because GzmC can induce target cell death, its reduced Granzyme A initiates an alternative pathway for granule-mediated apoptosis. Im- Downloaded from Ϫ/Ϫ ϩ munity 10:595. expression in GzmB / PGK-neo-derived CTLs may well ex- 17. Grossman, W. J., P. A. Revell, Z. H. Lu, H. J. Johnson, A. J. Bredemeyer, and T. J. Ley. plain the reduced cytotoxicity of these cells, at least in part. The 2003. The orphan granzymes of humans and mice. Curr. Opin. Immunol. 15:544. ability of GzmF to induce cell death has not yet been examined; 18. Heusel, J. W., R. L. Wesselschmidt, S. Shresta, J. H. Russell, and T. J. Ley. 1994. Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA frag- likewise, its substrates are not yet known. Regardless, it is impor- mentation and apoptosis in allogeneic target cells. Cell 76:977. tant to note that the overexpression of GzmC and F in MLR-de- 19. Pham, C. T., D. M. MacIvor, B. A. Hug, J. W. Heusel, and T. J. Ley. 1996. rived CTLs does not rescue the cytotoxicity defect caused by the Long-range disruption of gene expression by a selectable marker cassette. Proc. http://www.jimmunol.org/ Natl. Acad. Sci. USA 93:13090. loss of GzmB, suggesting that the enzymes are not functionally 20. Hug, B., R. Wesselschmidt, S. Fiering, M. Bender, E. Epner, M. Groudine, and redundant. Additional studies and knockout mice will be required T. J. Ley. 1996. Analysis of mice containing a targeted deletion of ␤-globin locus control region 5Ј hypersensitive site 3. Mol. Cell. Biol. 16:2906. to dissect the relative contributions of GzmC and F to the pheno- 21. Westervelt, P., A. A. Lane, J. L. Pollock, K. Oldfather, M. S. Holt, types observed in this report. D. B. Zimonjic, N. C. Popescu, J. F. DiPersio, and T. J. Ley. 2003. High pen- In summary, these studies have shown that a retained PGK-neo etrance mouse model of acute promyelocytic leukemia with very low levels of PML/RAR␣ expression. Blood 102:1857. cassette in the GzmB gene causes a knockdown of the expression 22. Shresta, S., D. M. MacIvor, J. W. Heusel, J. H. Russell, and T. J. Ley. 1995. levels of GzmC and F just downstream from the GzmB gene. Natural killer and lymphokine-activated killer cells require granzyme B for the CTLs derived from these mice have a more severe cytotoxic defect rapid induction of apoptosis in susceptible target cells. Proc. Natl. Acad. Sci. USA 92:5679. by guest on October 1, 2021 than those from mice deficient for GzmB only. These data clearly 23. Johnson, H., L. Scorrano, S. J. Korsmeyer, and T. J. Ley. 2003. Cell death in- implicate GzmC and F in CTL functions and suggest the need for duced by granzyme C. Blood 101:3093. 24. Pham, C. T., D. A. Thomas, J. D. Mercer, and T. J. Ley. 1998. Production of fully additional studies of their substrate specificities and killing activities. active recombinant murine granzyme B in yeast. J. Biol. Chem. 273:1629. Furthermore, because GzmC is the probable orthologue of human 25. Graubert, T. A., J. F. DiPersio, J. H. Russell, and T. J. Ley. 1997. Perforin/ GzmH (17), it will be important to learn whether human GzmH is granzyme-dependent and independent mechanisms are both important for the development of graft-versus-host disease after murine bone marrow transplanta- important for the ability of human cytotoxic lymphocytes to kill their tion. J. Clin. Invest. 100:904. targets. 26. Grossman, W. J., J. W. Verbsky, B. L. Tollefson, C. Kemper, J. P. Atkinson, and T. J. Ley. 2004. Differential expression of granzymes A and B in human cytotoxic Acknowledgments lymphocyte subsets and T regulatory cells. Blood 10:1182. 27. Russell, J. H., V. Masakowski, T. Rucinsky, and G. Phillips. 1982. Mechanisms We thank the Siteman Cancer Center High Speed Cell Sorter Core and the of immune lysis. III. 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