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Blood, 15 October 2005, Vol. 106, No. 8, pp. 2871-2878. Prepublished online as a Blood First Edition Paper on July 5, 2005; DOI 10.1182/blood-2005-04-1522.
PHAGOCYTES Quiescent and activated mouse granulocytes do not express granzyme A and B or perforin: similarities or differences with human polymorphonuclear leukocytes?From the Max-Planck-Institut für Immunbiologie, Metschnikoff Laboratory, Freiburg, Germany; the Institut für Immunologie, Universitätsklinikum Heidelberg, Heidelberg, Germany; the Medizinische Klinik 5, Universität Heidelberg, Heidelberg, Germany; and the John Curtin School of Medical Research, Australian National University, Canberra, Australia.
Polymorphonuclear leukocytes have been shown to use a multitude of effector functions to combat pathogens and tumors, including enzymes, defensins, and toxic products such as oxygen radicals and nitrogen oxides. Recent studies provided evidence for the expression of granzymes (gzms) and perforin (perf) within the cytotoxic arsenal of human neutrophils, the validity of which was questioned by 2 subsequent studies. We have now used cytology, intracellular flow cytometry, enzymatic assays, immunoelectron microscopy, and quantitative reverse transcriptase-polymerase chain reaction to obtain evidence of the presence of gzms and/or perf in mouse Gr-1+ granulocyte populations. The data obtained clearly demonstrate that neither in vitro- nor in vivo-derived mouse granulocytes synthesize gzmA and gzmB or perf, even following infection/immunization with pathogens or pathogen-derived material. A parallel comparable analysis on the expression of gzmB in human neutrophils from 3 healthy control subjects and 4 patients with diverse diseases failed to detect gzmB expression. The data indicate that polymorphonuclear leukocytes from mice and humans lack the 3 cytotoxic effector molecules, gzmA, gzmB, and perf, generally associated with natural killer and cytotoxic T lymphocytes. (Blood. 2005;106:2871-2878)
Perforin (perf) and granzymes (gzms) are major components of cytoplasmic granules from natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) and are critical cytolytic effector molecules in NK/CTL-mediated apoptosis and are required for the control of intracellular pathogens and tumors.1-10 In mice and humans, gzms, in particular gzmA and gzmB, and perf are produced mainly by the majority of NK cells and CTLs, a fraction of CD4+ T cells,11-13 and to a lesser extent by related cell types, such as metrial gland cells,14 but not by polymorphonuclear leukocytes (granulocytes/neutrophils) and monocytes.15-17 Two recent studies provided evidence that gzms and perf are also produced by human polymorphonuclear neutrophils (PMNs).18,19 These reports were intriguing in light of the known biologic effects of gzms and perf in NK/CTL-mediated immune responses8,10,20,21 and the role of PMNs as a first-line defense against microbial pathogens22,23 and tumors.24,25 However, the assumption that PMNs use the same set of molecules as NK/CTL to combat pathogens and tumors was challenged by 2 subsequent studies.26,27 To date, the discrepancy of the 4 studies, which used similar techniques for the enrichment of PMNs and to analyze the expression of GZM- and PRF1-specific transcripts and/or proteins, has not been resolved. We have now analyzed in detail the expression of gzmA, gzmB, and perf in in vitro-generated and ex vivo-derived mouse granulocytes by combining cytology, correlative cell surface phenotypic analysis, intracellular flow cytometry, enzyme assays, immunoelectron microscopy (IEM), and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. In addition we have investigated the expression of gzmB in human PMNs from healthy individuals and a panel of patients with distinct diseases.
Mouse strains and cell lines Inbred C57BL/6 (B6) and mouse strains deficient for gzmA (gzmA-/-), gzmB (gzmB-/-), and gzmA x B (gzmA x B-/-) bred on the B6 background were maintained at the Max-Planck-Institut für Immunbiologie, Freiburg, Germany, and analyzed for their genotypes as described.4,5 Male mice of 8 to 12 weeks of age were used in all experiments and were conducted in accordance with the ethical guidelines of the Federation of European Laboratory Animal Science Association. The mouse cell lines 1.3E6SN (CTL; perf+, gzmA+, gzmB+28) and EL4.F15 (thymoma; perf-, gzmA-, gzmB-5) were used as control cells for the analysis of perf- and gzm-specific transcripts and proteins.
Cell lines were cultured in minimal essential medium (MEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 2-mercapto ethanol (10-5 M) at 7% CO2, as described29; for 1.3E6SN cells, medium was supplemented with 10% Concanavalin A supernatant (ConA SN). ConA SN was used as cytokine source for expansion of mouse T cells. To produce ConA SN the SN of rat spleen cells was previously stimulated with ConA (5 µg/mL, 2 days) and subsequently treated with 20 mg/mL
In vitro-propagated alloreactive T-cell lines (H-2b anti-H-2d) were generated by weekly restimulation with irradiated spleen cells and ConA SN, as described.30 For analysis of intracellular gzmA/gzmB expression, alloreactive T-cell lines ( Isolation of PBMCs and PMNs from human blood Blood was taken from 4 healthy donors (2 men, 2 women) and 4 patients. The patients were from the University Hospital Heidelberg, Germany. Patient 1 was a 28-year-old woman with Still syndrome, rheumatoid arthritis, acute inflammatory episode with fever, joint pains, and high C-reactive protein (CRP; 150 mg/L). Patient 2 was a 70-year-old woman with chronic lymphatic leukemia, fever, and tumor progression (CRP, 30 mg/L). Patient 3 was a 81-year-old man with B-non-Hodgkin-lymphoma with fever (no CRP increase, no infection). Patient 4 was a 56-year-old woman with acute progressive myeloid leukemia with fever, pneumonia, and sepsis (CRP, 86 mg/L). Cells from one healthy donor were used for cytospin analysis, cells of the other 3 donors were used for fluorescence-activated cell sorting (FACS) staining. Blood was taken by venous puncture using EDTA (ethylenediaminetetraacetic acid)-coated tubes (Sarstedt, Nürnbrecht, Germany), and cells were separated on Ficoll Paque gradient (Amersham Pharmacia Biotech, Freiburg, Germany) according to established protocols. The interphase containing the peripheral blood mononuclear cells (PBMCs) was removed and stored on ice. The pellet containing the PMNs and the erythrocytes were mixed with phosphate-buffered saline (PBS) and 3% Dextran (Amersham Pharmacia Biotech; diluted in PBS) was added 1:1 and mixed. After sedimentation of erythrocytes at room temperature for 20 minutes the supernatant (SN) was removed, cells were resuspended in buffer containing 155 mM NH4Cl/10 mM KH2CO3/0.1 mM EDTA in H2O, and incubated for 15 minutes on ice to lyse resting erythrocytes. After centrifugation the SN was discarded, and the pellet containing neutrophils was resuspended in RPMI. Approval was obtained from the Medizinische Klinki 5, University Heidelberg institutional review board for these studies. Informed consent was provided according to the Declaration of Helsinki. In vitro generation of mouse granulocytes Mouse granulocyte populations were generated from bone marrow (BM). BM cells were rinsed from femur and tibia (needle 23G; Terumo, Roma, Italy) with MEM/1 x nonessential amino acids/1 mM sodium pyruvate/1 mM L-glutamine/10-5 M 2-mercapto ethanol (MEM-complete [MEM-comp]), adjusted to 1 x 106 cells/mL, and cultivated in a 24-well plate (MEM-comp + 20% horse serum [HS]; Cell Concepts, Umkirch, Germany; 1 mL/well; 37°C, 7% CO2) in the presence of 5 ng/mL granulocyte colony-stimulating factor (G-CSF; PeproTech, Rocky Hill, NJ) and 5 µM hydrocortisone (Sigma-Aldrich, Taufkirchen, Germany). After 5 days, 500 µL medium was replaced by 500 µL fresh MEM-comp + 20% HS for a further 4 days. Infection of mice and activation of leukocytes and granulocytes in vivo and in vitro Mice were infected with 5 x 103 colony-forming units (CFUs) Listeria monocytogenes (2.5 x 104 CFU/mL; wild-type, strain EGD; PBS) or injected with 2 µg lipopolysaccharide (LPS; Salmonella abortus equi, smooth form; provided by C. Galanos, Max-Planck-Institut für Immunbiologie, Freiburg, Germany; 10 µg/mL in PBS) intraperitoneally. At indicated time points, the peritoneal cells were recovered by rinsing with PBS and stored on ice for further analysis. Mice were infected with 1 x 105 plaque-forming units lymphocytic choriomeningitis virus (LCMV)-WE intraperitoneally according to established protocols.5,31 At day 8 after infection, spleens were removed for further analysis. In vitro-enriched granulocytes (BM cells, day 9, G-CSF) were incubated in the presence of either LPS (Salmonella minnesota, rough form; provided by C. Galanos; 1 µg/mL) and recombinant lipoprotein outer surface protein A (Lip-OspA; 10 µg/mL; GlaxoSmithKline Beecham, Rixensart, Belgium) in MEM-comp + 20% HS for 8 hours or with 50 µg/L x 105 cells zymosan A (Sigma-Aldrich) in MEM-comp + 20% HS for 90 minutes. Mouse splenocytes were activated with 5 µg/mL ConA (Amersham Pharmacia Biotech) with or without 10% IL-2 (ConA SN) in MEM (10% FCS/10-5 M 2-mercaptoethanol) for 2 to 3 days. Morphologic analysis of cells by cytospin Cells were washed with PBS, and 1 x 105 cells/150 µL PBS were centrifuged on microscope slides in a Cytospin 3 (Shandon Thermo Electron, Pittsburgh, PA). The cells were incubated with 150 µL May-Grünwald (Merck, Darmstadt, Germany) for 2 minutes, the same volume of H2O was added for a further 3 minutes. After washing the slides twice with H2O, cells were stained with Giemsa (diluted 1:20 in H2O; Sigma-Aldrich) for 20 minutes, washed, and embedded with Entellan (Merck). The images were taken using a Zeiss Axioskop 10 microscope, a Zeiss Axiocam as analysis camera, and Zeiss Vision 3.0 as software (Carl Zeiss, Jena, Germany). The objective used was a Zeiss PlanNeofluor, with a magnification of 40 x and a numeric aperture of 0.75. Flow cytometry
All monoclonal antibodies (mAbs) for surface staining, except anti-(
For analysis of intracellular gzmA and gzmB, 1.3E6SN, EL4.F15, splenocytes from LCMV-immune mice (day 8 after infection), mouse granulocytes, and human PBMCs or PMNs were stained with surface markers as described in "Flow cytometry" and then fixed with 100 µL PBS containing 2.5% PFA for 15 minutes at 4°C. Subsequently, cells were incubated in 100 µL permeabilizing buffer (PBS, 5% FCS, 0.1% NaN3, 0,1% Saponin; Roth, Karlsruhe, Germany) for 10 minutes at 4°C, then stained for 45 minutes at 4°C with 40 µL rabbit
For cell sorting, the enriched granulocytes (BM, day 9, G-CSF) were washed with FACS-washing buffer and incubated with 50 to 100 µL FITC/PE-labeled Enzymatic assays
Cell lysates from 1.3E6SN, EL4.F15, positively sorted ( Electron microscopy
In vitro-enriched granulocytes and 1.3E6SN cells were fixed in 4% PFA in 0.2 M PHEM buffer (240 mM PIPES [Piperazine-1,4-bis(2-ethanesulfonic acid)]/100 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]/40 mM EGTA [ethyleneglycoltetraacetic acid]/8 mM MgCl2, pH 6.), stored in 0.5% PFA in 0.1 M PHEM buffer, and then processed for ultrathin cryosectioning as previously described.32 Cryosections (45 nm) cut at 125°C using diamond knives (Drukker, Cuijk, The Netherlands) in an ultracryomicrotome (Leica Aktiengesellschaft, Vienna, Austria) were transferred with a mixture of sucrose and methylcellulose onto formvar-coated copper grids.33 The grids were placed on 35-mm Petri dishes containing 2% gelatin. For double immunolabeling, a published procedure34 was followed with 10- and 15-nm protein-A-conjugated colloidal gold probes, using Western blot analysis
Intracellular gzmA was determined in cell lysates from magnetic-activated cell sorting (MACS)-sorted LCMV-immune CD8+ cells of B6, gzmA-/-, and gzmB-/- mice by Western blotting under nonreducing conditions. The Probing for mRNA transcription Total RNA was extracted from up to 5 x 106 cells, using the QIAshredder spin columns, the RNeasy Mini Kit, and the RNase-free DNase Kit (all from Qiagen, Hilden, Germany) according to the manufacturer's instructions. mRNA was transcribed by incubating total RNA with random hexamer primer (660 ng/µL; Pharmacia, Freiburg, Germany), RNasin inhibitor (20 U; Promega, Madison, WI), deoxyribonucleoside triphosphate (0.5 mM; Qbiogene, Heidelberg, Germany), and Omniscript reverse transcriptase (4 U; Qiagen) as advised by the manufacturer. Reverse transcription was performed in a Thermocycler PTC-200 DNA-Engine (MJ Research, Waltham, MA). The transcription reaction profile was as follows: 37°C for 60 minutes and 70°C for 10 minutes. The resulting cDNAwas used as a template for RT-PCR hypoxanthin:guanine phosphoribosyl-transferase (Hprt1), Prf1, Gzma, and Gzmb amplification. Polymerase chain reaction (PCR) for Hprt1, Prf1, Gzma, and Gzmb was carried out in a Thermocycler PTC-200 as described in "Probing for mRNA transcription." The PCR reaction profile for Hprt1, Prf1, Gzma, and Gzmb was as follows: 1 cycle at 94°C for 2.5 minutes as an initial denaturation step; then denaturation at 94°C for 20 seconds; annealing at 56°C for 20 seconds for Hprt1 or 55°C for 20 seconds for Prf1, Gzma, and Gzmb; extension at 72°C for 20 seconds for Hprt1 or 72°C for 30 seconds for Prf1, Gzma, and Gzmb (35 cycles); followed by further incubation for 3 minutes at 72°C (1 cycle). The PCR reaction profile for the cathepsin G gene (Ctsg) was as follows: 1 cycle at 95°C for 1 minute as an initial denaturation step, then denaturation at 94°C for 30 seconds, annealing at 48°C for 30 seconds, extension at 70°C for 1 minute (35 cycles), followed by further incubation for 8 minutes at 70°C. The following primers were used for amplification: Hprt1-specific primers, sense (5'-GCT GGT GAAAAG GAC CTC T-3') and antisense (5'-CAC AGG ACT AGA ACA CCT GC-3' amplifying a 249-base pair (bp) segment; Prf1-specific primers, sense (5'-GAG CCC CTG CACACATTACTG GAA-3') and antisense (5'-ACA TTC TCA AAG TCC ATC T-3') amplifying a 380-bp segment; Gzma-specific primers, sense (5'-GGG GAT CTA CAA CTT GTA CGG-3') and antisense (5'-ATT GCA GGA GTC CTT TCC ACC AC-3') amplifying a 291-bp segment; Gzmb-specific primers sense (5'-TCA GGC TGC TGA TCC TTG ATC G-3') and antisense (5'-ATG AAG ATC CTC CTG CTA CTG C-3') amplifying a 135-bp segment; Ctsg-specific primers, sense (5'-CAT CCA AAT GCG AGA AAG GAC-3') and antisense (5'-CAT CTG CAC TCT TAG CTG CAC-3') amplifying a 272-bp segment. The primers for Hprt1, Gzma, Gzmb, and Prf1 were synthesized by Hermann (Freiburg, Germany), for Ctsg by Thermo Electron (Ulm, Germany). The PCR products were analyzed by gel electrophoresis (2% agarose), stained with ethidium bromide, and analyzed using an UV-transluminator (Vilber Lourmat, New Ark, NJ). As size markers, 1-kilobase (kb) DNA marker was used (BioLabs, Frankfurt/Main, Germany).
Phenotype of BM-derived and in vitro-propagated mouse granulocytes
Granulocytes were generated from mouse BM by in vitro cultivation with G-CSF (day 9) and further enriched by FACS sorting with mAb to Gr-1. This protocol leads to a highly purified granulocyte population (Figure 1A): freshly isolated BM cells consisted of approximately 50% Gr-1+, 3% NK1.1+, 44% B220+, 6% Thy1.2+, and 48% Mac-1+ cells (Figure 1Ai), with approximately 94% of Gr-1+ cells staining in addition for Mac-1 and approximately 2% to 3% for NK1.1, B220, or Thy1.2. The cell population propagated in G-CSF consisted of greater than 93% Gr-1+Mac-1+ (Figure 1Aii) and on further enrichment by FACS of greater than 99% Gr-1+Mac-1+ with only a minority (
In vitro-derived mouse granulocytes do not express gzmA or gzmB intracellularly
In vitro-enriched BM-derived granulocytes were analyzed by FACS for intracellular expression of gzmA and gzmB by using
The data obtained by FACS analysis were verified by IEM (Figure 4, only shown for gzmA; Cell lysates from in vitro-enriched mouse granulocytes as well as from 1.3E6SN and EL4.F15 cell lines were tested for proteolytic activities of gzmB and cathepsin G, a granulocyte-specific protease,39 by using appropriate chromogenic substrates. 1.3E6SN lysates contained gzmB- (19 U/106 cells) but not cathepsin G-related activity (0 U/106 cells), whereas EL4.F15 lysates did not harbor either of the 2 enzymatic activities (0 U/106 cells). In addition, lysates from ex vivo-derived and enriched LCMV-immune CD8+ T cells from B6 (3 U/106 cells) but not gzmAxB-/- mice (0 U/106 cells) expressed gzmB activity, although at much lower levels (3 U versus 19 U/106 cells, as expected from previous studies; M.M.S. unpublished data, February 2004) when compared with the long-term cultured T-cell line, 1.3E6SN. Both ex vivo-derived T-cell populations expressed low but significant levels of cathepsin G or cathepsin G-like activity (1.3 U/106 cells). In contrast, lysates from in vitro-enriched granulocytes, unstimulated or LPS-stimulated, expressed only cathepsin G-related (17 U/106 cells), but not gzmB-related (0 U/106 cells), enzymatic activities.
Ex vivo-derived mouse granulocytes do not express gzmB
B6 mice were injected intraperitoneally with LPS or infected intraperitoneally with L monocytogenes. Twenty hours and 48 hours after infection, respectively, cells were recovered from the peritoneal cavity and analyzed by FACS for surface phenotype and the expression of intracellular gzmB. Approximately 45% of cells from LPS-challenged mice stained Gr-1+, although with differential intensity. Only approximately 3% to 4% of cells expressing Gr-1 were also positive for NK1.1 and Thy1.2, and mainly in the Gr-1low cell population. When tested for the expression of gzmB, only few Gr-1hi but significantly more Gr-1low cells stained for gzmB. In contrast to Gr-1+ cells, a much higher proportion from Thy1.2+ cells (
The peritoneal cell population from L monocytogenes-infected mice contained approximately 34% Gr-1+ cells, with approximately 74% and approximately 6% being also positive for NK1.1 and Thy1.2, respectively (Figure 5B). However, none of the Gr-1+ cells expressed gzmB, whereas approximately 4% of the NK1.1+ cells ( Mouse BM-derived granulocytes do not express transcripts for gzmA, gzmB, and perf In vitro-enriched and FACS-sorted Gr-1+ BM-derived granulocyte populations from B6 and gzmB-/- mice were assayed by RT-PCR for the expression of Gzma, Gzmb, and Prf1-specific transcripts. None of the 2 cell populations expressed Gzma, Gzmb, or Prf1-specific mRNA, irrespective of whether they were stimulated with LPS or left untreated (Figure 6A). As expected, mRNA from 1.3E6SN, but not EL4.F15, cells expressed transcripts for Gzma, Gzmb, and Prf1. In contrast, when in vitro-enriched but unsorted granulocyte populations were used, Gzmb-specific transcripts were detectable, the level of which was enhanced on pretreatment with LPS (Figure 6B; lanes 2 and 3). No Gzm- or Prf1-specific transcripts were seen in in vitro-generated and FACS-enriched Gr-1+ cells (Figure 6B; lane 1). In contrast, specific bands for the granulocyte-specific Ctsg were only detected in samples of granulocytes, but not in 1.3E6SN and EL4.F15 (Figure 6B). Similar results were obtained when mRNA preparations were subjected to quantitative RT-PCR using the light cycler system (data not shown). In summary, these results clearly demonstrate that mouse granulocytes, even after in vitro activation or on encounter with pathogens in vivo, do not express gzmA, gzmB, or perf. The present data obtained by cytologic, intracellular flow cytometric, enzymatic, IEM, and semiquantitative RT-PCR analyses, support earlier studies, indicating that in mice the 3 cytolytic effector molecules, gzmA, gzmB, and perf, are produced mainly by NK and CTL,12,15,28 and to some extend also by metrial gland cells,14 but not by other cells of the lymphoid or myeloid lineages. Human granulocytes from patients with various disease symptoms and healthy individuals do not express gzmB
In light of the published conflicting results on the expression of gzms and perf in human neutrophils,18,19,26,27 we tested a panel of PBMC and PMN populations from 4 healthy individuals (cells of 1 individual were used for cytospin, cells of the other 3 individuals were stained intracellularly) and 4 patients with either an autoimmune disorder or B-cell chronic lymphatic leukemia, B-cell non-Hodgkin lymphoma, or acute myelogenous leukemia. PBMCs and PMNs were separated by standard methods and subsequently applied to cytospin and FACS analysis. Both cell populations were stained for surface markers CD4, CD8, and CD66b as well as intracellularly for gzmB using
Our findings that only human PBMCs, in particular fractions of CD4+ and CD8+ T cells, but none of the enriched PMN populations from the healthy individuals and 4 patients with various disease symptoms expressed gzmB are in line with 2 recent studies,26,27 but challenge 2 others.18,19 Final proof whether human PMNs are able to produce gzms and/or perf under certain conditions is still pending. A cautionary note regarding proof of principle needs to be added to avoid erroneous conclusions. This pertains, in particular, to enrichment of granulocytes, via mAb: We found that the majority of cytologically defined mouse granulocytes are Gr-1+, but that they may differentially express NK- and T-cell-specific markers, depending on their activation state. However, fractions of ex vivo-derived spleen cells or spleen cells, previously activated in vitro with ConA or ConA plus ConA SN were double positive for either NK1.1 and Gr-1 or Thy1.2 and Gr-1 (Figure 1C). Phenotype analysis of human PBMCs and PMNs led to similar observations. Thus, highly enriched PBMC populations contained small fractions of CD56+ or CD3+ cells, which also expressed CD66b and, vice versa, highly enriched PMNs (> 95% CD66b) contained a small fraction of cells, which expressed either CD56 or CD3 in addition to CD66b. This also holds true when another marker, CD15, mainly associated with human PMNs,18,26 was applied (data not shown). Thus, granulocyte populations/PMNs selected solely by Gr-1 (mouse) or CD66b/CD15 (humans) may either contain granulocyte/neutrophil subpopulations expressing NK/T-cell markers or may be contaminated with cells from other lineages, in particular NK, NKT (natural killer T), and T cells, which are the main producers of gzms and perf.
The data presented here provide strong supportive evidence that mouse granulocytes do not express gzmA, gzmB, and perf under physiologic or pathophysiologic conditions. This is supported by the lack of Prf1- and Gzma-/Gzmb-specific mRNA and the respective proteins in in vitro-generated or ex vivo-derived granulocyte populations, as revealed by cytologic, intracellular flow cytometry, enzymatic, IEM, and RT-PCR analyses. Gzms and perf could not be detected in granulocytes previously activated either in vitro and/or in vivo, by L monocytogenes or by pathogen-derived material, such as LPS, Lip-OspA, or the yeast product, zymosan. This indicates that mouse granulocytes do not use the known NK/CTL-associated cytotoxic effector molecules, gzmA, gzmB, and perf, as defense against pathogens and tumors. Our finding that fractions of CD4+ and CD8+ T cells, but not highly purified PMNs from 4 patients and 3 healthy individuals, expressed gzmB is in support of recent work from 2 independent groups,26,27 but contrasts with other related studies.18,19 Thus, it remains to be verified whether the discrepant results obtained using human PMNs are merely due to technical differences and whether mouse and/or human PMNs or fractions thereof are able to produce gzms and/or perf, but only under stringent conditions unlike the expression in NK/CTLs.
We thank Jero Calafat from The Netherlands Cancer Institute, Division of Cell Biology, Amsterdam, for performing the IEM experiments and for her critical comments, and Verena Weber, Claudia Luckner, Christiane Brenner, and Aynur Ekiciler for expert technical assistance.
Submitted April 14, 2005; accepted June 10, 2005.
Prepublished online as Blood First Edition Paper, July 5, 2005; DOI 10.1182/blood-2005-04-1522.
Supported in part by an Alexander von Humboldt Foundation Fellowship (7000188979) (J.P.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Markus M. Simon, Max-Planck-Institut für Immunbiologie, Stübeweg 51, Freiburg, Germany; e-mail: simon{at}immunbio.mpg.de.
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Abstract
Introduction
-methyl-D-mannopyranosid to block residual ConA activity. 
85%-90% CD8+) of B6, gzmA-/-, and gzmB-/- mice obtained after the third in vitro stimulation were used. 
-actin mAb (clone C-11) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Then the blot was stained with peroxidase-conjugated secondary antibodies, goat 
