Viral Cross-Class Serpin Inhibits Vascular Inflammation and T Lymphocyte Fratricide; A Study in Rodent Models In Vivo and Human Cell Lines In Vitro

Poxviruses express highly active inhibitors, including serine proteinase inhibitors (serpins), designed to target host immune defense pathways. Recent work has demonstrated clinical efficacy for a secreted, myxomaviral serpin, Serp-1, which targets the thrombotic and thrombolytic proteases, suggesting that other viral serpins may have therapeutic application. Serp-2 and CrmA are intracellular cross-class poxviral serpins, with entirely distinct functions from the Serp-1 protein. Serp-2 and CrmA block the serine protease granzyme B (GzmB) and cysteine proteases, caspases 1 and 8, in apoptotic pathways, but have not been examined for extracellular anti-inflammatory activity. We examined the ability of these cross-class serpins to inhibit plaque growth after arterial damage or transplant and to reduce leukocyte apoptosis. We observed that purified Serp-2, but not CrmA, given as a systemic infusion after angioplasty, transplant, or cuff-compression injury markedly reduced plaque growth in mouse and rat models in vivo. Plaque growth was inhibited both locally at sites of surgical trauma, angioplasty or transplant, and systemically at non-injured sites in ApoE-deficient hyperlipidemic mice. With analysis in vitro of human cells in culture, Serp-2 selectively inhibited T cell caspase activity and blocked cytotoxic T cell (CTL) mediated killing of T lymphocytes (termed fratricide). Conversely, both Serp-2 and CrmA inhibited monocyte apoptosis. Serp-2 inhibitory activity was significantly compromised either in vitro with GzmB antibody or in vivo in ApoE/GzmB double knockout mice. Conclusions The viral cross-class serpin, Serp-2, that targets both apoptotic and inflammatory pathways, reduces vascular inflammation in a GzmB-dependent fashion in vivo, and inhibits human T cell apoptosis in vitro. These findings indicate that therapies targeting Granzyme B and/or T cell apoptosis may be used to inhibit T lymphocyte apoptosis and inflammation in response to arterial injury.


Introduction
Serine protease inhibitors or serpins have extensive regulatory actions, moderating thrombotic and immune responses [1,2]. Poxviruses encode highly active serpins, including a secreted serpin Serp-1, which inhibits extracellular thrombolytic and thrombotic proteases and markedly reduces arterial inflammation and plaque growth in animal models [2][3][4]. This serpin, when injected as a purified protein, also significantly reduces markers of myocardial damage after stent implant in patients with unstable coronary syndromes [5]. These studies suggest that other viral serpins may have therapeutic potential. With the studies reported herein we explore a second class of viral cross-class serpins with different protease targets that block apoptosis and inflammation.
Serp-2 treatment also reduced plaque in rat aortic transplant models (ACI donor to Lewis recipient, p,0.05, data not shown) and reduced mononuclear cell invasion, when compared to CrmA, in both intimal (p,0.0001, data not shown) and adventitial layers.
These initial studies demonstrate an arterial anti-inflammatory effect for an intracellular viral serpin, Serp-2, in both angioplasty injury as well as aortic allograft transplant models in rodents, when infused into the circulating blood immediately after injury. This effect was specific to Serp-2 and was neither reproduced by another intracellular serpin, CrmA nor by two active site mutants of Serp-2.
Serp-2 reduces plaque growth in Apolipoprotein E deficient (ApoE 2/2 ) mice Serpin treatment in hyperlipidemic ApoE 2/2 mice after carotid cuff compression injury was examined, both at the site of cuff injury and at the aortic root where no surgical injury occurs (Fig. 2) [32]. This model provides a means to assess both effects of serpin treatment after arterial surgical injury and also at a site of de novo growth of plaque induced by genetic hyperlipidemia with no arterial surgical injury (e.g. apolipoprotein E deficiency). Serp-2 significantly reduced plaque area in the aortic root (N = 11, p,0.001, Fig. 2B, 2D) where there was no surgical injury, but with borderline significance at sites of carotid cuff compression injury (N = 11, p = 0.06, Fig. 2E), when compared to saline ( Fig. 2A, 2D). Plaque reductions with Serp-2 treatment were comparable at both sites, 42% for aortic root versus 44% for the carotid (Fig. 2D, 2E), while CrmA treatment (N = 11) had no effect (Fig. 2C, 2D, 2E). Compared to saline, plaque lipid content was also significantly reduced on Oil-red O stained sections with Serp-2 treatment, but not with CrmA (Fig. 2F). This reduction in lipid-laden cells is similar to the reduction in inflammatory cell invasion seen with Serp-2 treatment in the aortic angioplasty and allograft models.

Cross-class serpin treatments modify apoptotic responses in vitro
Effects of serpin treatments on cellular apoptotic responses were assessed both at early times after angioplasty injury in vivo and in To test for potential effects on individual cell types associated with arterial inflammation and plaque growth, inhibition of apoptotic responses were measured in HUVEC (human umbilical vein endothelial cells), THP-1 monocytes, and Jurkat T lymphocytes in vitro, with and without serpin treatment. Apoptotic responses were induced through three pathways using staurosporine (STS; intrinsic pathway), and camptothecin (CPT; double strand break initiation).
In HUVEC cultures treated with serum deprivation, staurosporine, or camptothecin caspase 3, 7, 8 and GzmB were increased (p,0.001, not shown). Serp-2 and CrmA both significantly reduced caspase 8 after CPT treatment (p,0.0001) in HUVEC, but had no effect on caspase 3 and 7 (not shown). STS-induced apoptosis was not altered by Serp-2, CrmA, D294A, or D294E in HUVEC (not shown). In all cell lines tested after FasL treatment Serp-2, CrmA, and D294A had no inhibitory activity (not shown). The Serp-2 RCL mutant, D294E did, however, reduce caspase 3, 7, 8 and GzmB activity after FasL treatment of T cells (p,0.0005, not shown). Cathepsin K, S, L, V activity in T cells was not affected by serpin treatment (p = 0.386, not shown).
In summary, Serp-2, but not CrmA, inhibited camptothecinand staurosporine-induced caspase activity in T cells in vitro. Both Serp-2 and CrmA inhibited camptothecin-induced caspase activity in monocytes with CrmA displaying greater inhibitory activity in monocytes, suggesting T lymphocytes as a primary target for Serp-2. No differential effects were produced by Serp-2 and CrmA in vivo in arterial sections isolated early after angioplasty injury, which may be due to the fact that multiple cell types are assessed in arterial sections.

Serp-2 reduces T cell apoptotic responses to cytotoxic T lymphocyte (CTL) granzyme B
Activated T cells (TH1) and cytotoxic T lymphocytes/ natural killer (CTL/NK) cells release GzmB into the surrounding medium, initiating death responses in other cells, and also in T lymphocytes [12,14,15]. The role of GzmB in Serp-2 mediated anti-apoptotic activity was assessed using medium from T cells activated to a CTL-like state with phorbol myristic acid (PMA) and ionophore [12,14]. These activated CTL-like cells express and secrete increased GzmB. Naive HUVEC, THP-1, and Jurkat cells were then treated with medium from the CTL-like T cells (CTLm) with and without serpin treatments (Fig. 4, Fig. S1).

Serp-2 binding to T cells is reduced with granzyme B inhibition
To determine whether Serp-2 or CrmA binds to T cells, FITClabeled Serp-2 and CrmA binding was measured by flow cytometry (Fig. 5A) and fluorescence microscopy (Fig. 5B). In these studies Serp-2 displayed a greater binding affinity than CrmA (Fig. 5A, 5B) for T cells in vitro and mouse peripheral circulating lymphocytes in vivo (not shown). Serp-2 significantly reduced caspases 3/7 activity in CTLm treated T cells (Fig. 5C, p,0.001) and treatment with the GzmB inhibitor ZAAD-CMK, which predominately inhibits intracellular granzyme B, further increased Serp-2-mediated inhibition of caspases, indicating that Serp-2 actions may be predominately extra-cellular (Fig. 5C, p,0.005). The uptake of FITC-labeled Serp-2 into Jurkat T cells, as measured by soluble lysate content, was also reduced after treatment with antibody to GzmB (Fig. 5D, p,0.003) or perforin (Fig. 5D, p,0.012), further supporting a role for Serp-2 binding to GzmB and inhibiting of T cell responses.
These studies support GzmB as one of the central targets for Serp-2 mediated anti-inflammatory and anti-atherogenic activity. The CTLm was also applied to naive T cells in culture to induce apoptosis and increased levels of caspase 3 and granzyme B were observed as IEPDase activity (C, p,0.0001) and DEVDase activity (D, p,0.0001) respectively. Treatment with Serp-2 reduced both granzyme B (C, p,0.01) and caspase 3 (D, p,0.0009) activities significantly. Antibody to granzyme B (GzmB) blocked Serp-2 mediated reductions in CTLm induced granzyme B (C) when compared to Serp-2 treatment alone (p,0.0004), but with a still significant decrease (p,0.005) when compared to CTLm treatment alone. Antibody to granzyme B also blocked the Serp-2 mediated decrease in caspase 3 (D, p,0.149) when compared to CTLm activation. This Serp-2 mediated inhibition of CTLm induced granzyme B activity was also blocked by incubation of cells with antibody to perforin (C, p = 0.412) but not the caspase 3 activity (D, p,0.0193). The results shown here represent mean 6 SE from 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance (ANOVA) with secondary Fishers least significant difference and Mann Whitney analysis.

Serp-2 reduces early apoptosis in invading inflammatory cells after aortic allograft transplant
Early changes in T cell and macrophage responses can initiate inflammatory responses that drive plaque development and plaque growth at later times in arterial plaque growth and disease. To assess effects of Serp-2 and CrmA on inflammatory T cell responses in vivo in a mouse model, we examined markers for T cell invasion and apoptosis in aortic allograft transplants in mice at early 72 hour follow up (Fig. 7). Serp-2 treatment (Fig. 7) induced no significant T lymphocyte responses at 72 hrs follow up, although there is a minor trend toward an increase in CD3 positive T cells. On immunohistochemical staining, however, Serp-2 but not CrmA, markedly reduced inflammatory cell apoptosis (Fig. 7G, p,0.0002).

Discussion
Many cell types are associated with atherosclerotic plaque growth. Injury to the arterial wall is believed to cause endothelial cell dysfunction and activation of inflammatory cells, specifically monocytes that transform into macrophages, T lymphocytes as well as smooth muscle cells, and other cells types such as mast cells, neutrophils and even B lymphocytes. Damage to the arterial wall and loss of supporting connective tissue can additionally cause programmed cell death or apoptosis, which can lead to release of increased levels of inflammatory cytokines. Activated or dysfunctional T cells can also induce transformation of other cells to a suicidal or apoptotic state. These initial changes in inflammatory cell responses are believed to then drive further damage to the arterial wall and cause intimal plaque growth and arterial narrowing.
It is evident that there are multiple factors that drive plaque growth with some known shared or common pathways. We elected to assess the effect of an anti-apoptotic serpin, as the apoptotic pathways are becoming recognized as a driving force in arterial injury responses and inflammation. Apoptosis in endothelial and in macrophage cells has been reported, as has apoptosis in SMC and T cells in plaque development. However, apoptosis altering the many T cell sub-populations remains poorly defined. It is not known whether interruption of apoptotic responses will alter plaque development and whether this applies to the wide range of arterial injury states that can cause plaque formation.
It is for this reason we have elected to assess plaque growth and responses to the viral anti-inflammatory serpin, Serp-2, in a range of models to determine whether the effects of this protein will be evident in different animal models of arterial disease in order to assess whether this will be of more widespread potential interest. Furthermore, in order to better isolate the contributions of individual cellular subpopulations, individual serpins were tested on human cell lines. Activated or dysfunctional T cells can also induce apoptosis of endothelial cells and monocytes/ macrophages, among other cell types; in the plaque, this leads to an increased release of cytokines and activate thrombolytic serine proteases tissue-and urokinase-type plasminogen activators (tPA and uPA, respectively) and the matrix metalloproteases (MMPs), which breakdown collagen and elastin, weakening the plaque's fibrotic cap [45][46][47]. In addition to these activated and apoptotic cells, the newly exposed necrotic core and eroded cap structure also activate leukocytes and may initiate plaque rupture, subsequent thrombus formation, leading to heart attacks and strokes. Through a series of complex cross-talk and feedback mechanisms, the serine proteases in the coagulation and fibrinolytic pathways interact on many levels with the inflammatory and apoptotic responses and vice versa.
It is evident that there are multiple factors that drive plaque growth with some known shared or common pathways. We elected to assess the effect of an anti-apoptotic serpin, as the apoptotic pathways are now becoming recognized as a driving force in arterial injury responses and inflammation [48]. Apoptosis in endothelial cells and in macrophage has been reported as has  (Fig 6C, p,0.024) in PAI-1 2/2 mice 4 weeks post-aortic transplant. Immunostained sections for caspase 3 illustrate reduced staining in mononuclear cells in Serp-2 treated PAI-1 2/2 aortic transplants when compared toD294E treatment (Fig 6D, Mag 400X). doi:10.1371/journal.pone.0044694.g006 Viral Serpin Reduces Plaque and T Cell Apoptosis PLOS ONE | www.plosone.org apoptosis in SMC and T cells in plaque development. However, apoptosis altering the many T cell sub-populations remains poorly defined. It is not known whether interruption of apoptotic responses will alter plaque development and whether this applies to the wide range of arterial injury states that can cause plaque formation. For this reason, we have elected to assess plaque growth and responses to the viral anti-inflammatory serpin, Serp-2, in a range of models to determine whether the effects of this protein will be evident in different animal models of arterial disease. Furthermore, in order to better tease apart the contributions of individual cellular subpopulations, individual serpins were tested on human cell lines.
Intravenous infusion of Serp-2, a reputed intracellular myxomaviral cross-class serpin, effectively inhibited plaque growth in a series of animal models of vascular disease (Figs. 1, 2) irrespective of the model being vascular surgery based or hyperlipidemic mice. These studies demonstrate marked extracellular, GzmB-dependent inhibitory actions for Serp-2, previously thought to function in a predominantly intracellular capacity. This inhibitory activity was unique to Serp-2; the cowpox viral serpin, CrmA and two Serp-2 active site mutants were inactive in these models.
Serp-2 blockade of plaque growth in donor aortic allografts was absent when the transplanted tissue was from GzmB 2/2 donors.
Local deficiency of GzmB was not sufficient to reduce plaque growth in donor allografts treated with saline, but did block increased plaque in ApoE 2/2 mice treated with CrmA. Thus, granzyme B may have greater effects on vascular disease when active locally rather than systemically during inflammatory cell responses. Although Serp-2 was infused systemically, the loss of activity in donor allografts from knockout mice suggests that Serp-2 acts locally on donor aorta after infusion (Fig. 6). In vitro studies suggest that Serp-2 specifically inhibits T cell apoptosis. Further work using isografts, GzmB 2/2 transplant recipients, and caspase 1 deficient transplants is needed to assess and contrast the roles of systemic GzmB and caspase 1 in allograft vasculopathy and as a target for Serp-2.
To try to separate out the effects of different cell lineages found in plaques, the effects of the serpins on apoptosis-induced cell lines were examined. Serp-2 bound to T cells in vitro in culture and selectively inhibited caspase 3/7 in camptothecin (CPT)-treated Jurkat T cells (Figs. 3 and 4) and was dependent upon GzmB and perforin (Figs. 5 and 6). Additionally, Serp-2 but not Serp-2 D294E was able to reduce levels of active caspase 3 in ApoE and ApoE/GzmB knockout mouse aortic cross-sections after transplant (Fig. 6). This Serp-2 mediated reduction for apoptosis was substantiated when comparing Serp-2 to CrmA and saline 72hrs after C57Bl/6 aortic transplant into Balb/C mice (Fig. 7A-C,  7H). Based upon these studies we postulate that Serp-2 decreases T cell mediated apoptosis, inducing a generalized reduction in vascular inflammation.
The extracellular activity for this viral serpin is predicted to begin with binding to GzmB. GzmB mediates apoptosis upon release from T cells and can also induce apoptosis in other T cells. Many viral proteins have multiple functions [2,8,31] and expanded actions of these cross-class serpins upon release from infected cells is predicted [2][3][4][5][6][7][8][9][31][32][33]. This inhibitory activity is present either with camptothecin treatment of T cells or after treatment with CTL medium from PMA and ionophore treated T cells, containing granzyme B. Serp-2 may thus either bind GzmB outside the cell or may be internalized via perforin pathways. The GzmB inhibitor ZAAD-CMK, which is cell membrane permeable and inhibits GzmB inside the cell, further increased Serp-2 inhibition of caspase activity indicating that Serp-2 actions are extra-cellular. Serp-2 inhibition of camptothecin-mediated apoptosis is consistent with GzmB inhibition.
Many viral proteins are also reported to derive functions through mimicking mammalian genes, as well as the converse. Two mammalian serpins, murine serine protease inhibitor 6 (SPI-6) [36] and human protease inhibitor 9 (PI-9) [37] target GzmB and protect cells from CTL-induced apoptosis. Serp-2 protein may thus mimic this mammalian serpin pathway, hindering T cell apoptosis and inflammation, e.g. T cell fratricide. We have not yet, however, determined whether selected T cell subsets are targeted by Serp-2 protection. It is undeniable that Serp-2 is protecting both T cells and other lineages from GzmB mediated apoptosis (Fig. 4) in vitro.
GzmB mediates DNA degradation, interfering with DNA repair responses. Topoisomerase, iCAD, and PARP are involved in DNA damage repair and are GzmB substrates. Camptothecin binds topoisomerase I, an enzyme class that alters DNA topography, interfering with DNA re-ligation [38] and creating persistent DNA breaks. Inhibition of topoisomerase also leads to caspase activation [37]. Poly (ADP ribosylation, PAR) is a post-translational modification driven by the PAR polymerase-1 (PARP-1) that reactivates topoisomerase complexes, preventing further damage [39] and is also a transcription initiation factor for NFkB [40]. Once cleaved, iCAD no longer inhibits caspase activated DNAse permitting DNA degradation [41]. Serp-2 mediated inhibition of GzmB or inhibition of secondary induction of caspase 3 may alter the balance between DNA repair and damage in T cells [42][43][44].
We conclude that Serp-2, a viral anti-apoptotic cross-class serpin, has the potential to inhibit arterial vascular disease progression in animal models through inhibition of GzmBdependent T cell apoptosis. Granzyme B-inhibition of T cell fratricide may represent a potential new target for intervention in inflammation-based disease. Serp-2 inhibition is generalized, with expanded inhibitory function for plaque growth in hyperlipidemic ApoE 2/2 mice and after arterial surgery, indicative of blockade of central regulatory pathways.
Viral serpins were infused intravenously (i.v.) immediately after surgery in rats at doses of 0.3 ng -3000 ng/rat (0.001-10 ng/g), with follow up at 4 weeks (Table 1). Daily subcutaneous injections of saline, CrmA (240 ng/mouse/day, 12 ng/g/day) and Serp2 (1800 ng/mouse/day, 90 ng/g/day) were started two weeks after collar placement in ApoE 2/2 mice and continued for 4 weeks until sacrifice. 125 I labeled CrmA and Serp-2 were injected on the first day and the last two days in two ApoE 2/2 mice detecting serum concentrations of 0.16 nM for CrmA and 1.72 nM for Serp-2. For the mouse aortic transplant studies a single i.v. injection (15 mg/ mouse; 500 ng/g) of Serp-2, CrmA, or D294A or D294E mutants was administered immediately after allograft surgery. A separate group of 100 rats had angioplasty injury with 300 ng of each serpin by i.v. injection for early follow up at 0, 12, or 72 hours to assess early apoptotic pathway enzyme activity (6 animals/treatment group; Table 1). There were no deaths after angioplasty or aortic transplant in the rats, 14 mice died after aortic transplant, and one mouse died during placement of the carotid cuff. In the mouse aortic transplant model two mice died after treatment with Serp-2, 5 after CrmA, 2 after D294A, 2 after D294E, and 2 after saline treatment with overall survival of 85.4% (p = NS). Body weight was measured weekly and mice were checked daily for signs of distress and necessity for analgesic.

Histological, Morphometric, and Fluorescence Analysis
At the designated study end (4 weeks for plaque analysis and 0-72 hours for protease activity or apoptosis) rats and mice were sacrificed with Euthanyl (Bimenda MTC Animal Health Company, Cambridge, Ontario, Canada). For mouse and rat angioplasty and allograft transplant models, arterial sections were fixed, processed, paraffin embedded, and cut into 5 mm sections (2-3 sections per site) for histological analysis, as previously described [2][3][4][31][32][33][34]. For the ApoE 2/2 mice with carotid cuff compression, the aortic valve area (10 mm sections throughout the valve area) and the carotid artery from the bifurcation through the site of cuff compression (5 mm sections at 25 mm intervals) were assessed. Sections were stained with Haematoxylin/ Eosin, Trichrome and Oil Red O for analysis of plaque area, thickness, and invading mononuclear cells, as previously described [2][3][4][31][32][33]. Plaque area as well as intimal thickness and medial thickness were measured by morphometric analysis via the Empix Northern Eclipse trace application program (Mississauga, ON, Canada) on images captured by a video camera (Olympus, Orangeburg, NY, USA) attached to and calibrated to the Olympus microscope objective. The mean total cross-sectional area of the intima as well as diameter of the intima and media were calculated for each arterial section. For immunohistochemistry, T cells were labeled with rabbit anti-mouse CD3 antibody, both then labeled with secondary goat anti-rabbit antibody (CD3; Cat # AB5690, Secondary anti-rabbit; Cat# AB80437. Abcam, Cambridge, MA, USA). For Caspase 3 staining, sections were incubated with anti-caspase 3 polyclonal antibody (Cat# AB3623 1:20) as primary antibody with secondary rabbit specific-HRP conjugated antibody (Cat # AB80437). Either numbers of positively stained cells in three high power fields in the intimal, medial, and adventitial layers were counted and the mean calculated for each specimen or, when fewer cells were detected, (displayed in earlier follow up times) cell counts for positive staining in all three layers were averaged.
For spectroscopic analysis of Serp-2 and CrmA binding, 1610 6 cells/mL were treated with 1ug/mL of FITC-labeled protein for two hours, lysed with cell lysis buffer, and fluorescence emission at 525 nm quantified during excitation at 485 nm. For fluorescence microscopy, cells treated with Serp-2 FITC for 2 hrs at 4uC, then fixed with 2% formaldehyde, mounted with 10% glycerol mounting solution and viewed with a Zeiss fluorescent microscope as previously described [4]. For FACS analysis, 1610 6 cells/mL were treated with 1 mg/mL of FITC labeled Serp-2 or CrmA for two hours and run on FACS (FACS Calibur, BD Falcon) acquiring data for 20,000 events with three replicates (Cell Quest data analysis program). To measure uptake of Serp-2, Jurkat T cells were treated with PMA and ionophore to stimulate granzyme B production, then FITC-labeled Serp-2 was added to the activated cells and incubated. Cells were lysed and the membrane and insoluble fractions were separated by centrifugation. FITC-Serp-2 presence was quantitated by absorbance at 525 nm (Fluorscan) measurements for both fractions. Source and Purification of Serp-2, CrmA, D294A, and D294E All serpins were His-tagged at the amino-terminus, expressed in vaccinia/T7 vector in HeLa cells (Dr Richard Moyer, University of Florida, Gainesville, USA) [6], and purified by immobilized metal affinity using His-Bind resin (Novagen) [2][3][4][5][6][7][8]31,33]. The D294A protein is a site-directed mutant of Serp-2 with P1 Aspartate 294 changed to Alanine to inactivate the serpin, while the D294E protein has P1 Aspartate 294 replaced by Glutamic acid to alter the inhibition spectrum [6][7][8]. Eluted proteins were judged .90% pure by SDS-12% PAGE, silver staining and immunoblotting. Serp-2 and CrmA were tested for Casp 1 and GzmB inhibitory activity, CrmA displaying greater (.5-6 fold) Casp 1 inhibition than Serp-2 (data not shown), as previously reported [6][7][8][9].
Serp-2 and CrmA were labeled with Fluorescein Isothionate (Fluorotag FITC conjugation kit, Sigma-Aldrich Canada Ltd., Mississauga, Ontario) and passed through G-25M gel filtration column to separate unbound FITC. The F/P (FITC/protein) ratio was 2.2 and 2.1, for Serp-2 and CrmA respectively. The caspase 1 inhibitory activity of FITC labeled proteins was assayed, displaying normal activity. BSA was labeled in parallel with FITC and used as control in entry assays.
A subset of T cell cultures were treated with phorbol myristic acid (PMA, 1 ug/mL) and Ionophore A23187 (1 mg/mL) to induce a CTL-like (cytotoxic T lymphocyte) state. Medium from treated T cell cultures containing GzmB and perforin was removed after 2 hours incubation and applied to fresh, untreated T cell cultures together with Serp-2, CrmA, or D294A or E, with and without antibody to GzmB or perforin (Sigma) or the cell permeable small molecule inhibitor, ZAAD-CMK (ZAAD-chloromethylketone, Calbiochem, CedarLane, Hornby, ON), incubation for 12 or 24 hours [44].

Statistical Analysis
The apoptotic enzyme activity measurement results unless otherwise mentioned represents mean 6 SE from 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance (ANOVA) with secondary Fishers least significant difference and Mann Whitney; p values ,0.05 considered significant.

Supporting Information
Figure S1 Viral cross-class serpins alter Staurosporineinduced apoptotic responses in T cells and monocytes, in vitro. Apoptotic responses were induced in T cells and monocytes using staurosporine. The ability of Serp-2, CrmA, or Serp-2 mutants to counteract this induction was measured by granzyme B and caspase 8 activity by IEPDase activity. Serp-2, but not CrmA nor D294A and D294E treatment of Jurkat T cells reduced caspase 8 and Granzyme B activity after staurosporine (STS) (A, p#0.001) apoptosis actuator treatment. In THP-1 human monocytes, no cross-class serpins significantly reduced granzyme B or caspase 8 activity (B). The results shown here represent mean 6 SE from 3 to 5 replicates for each experiment. Significance was assessed by analysis of variance (ANOVA) with secondary Fishers least significant difference and Mann Whitney analysis. (TIFF)