American Journal of Pathology, Vol. 140, No. 5, May> 1992 Copyright C American Association of Pathologists

Rapid Communication Immunohistochemical Identification of Cytotoxic Lymphocytes Using Human Perforin Monoclonal Antibody Arif Hameed,* Kristin J. Olsen, Lirong Cheng,t William M. Fox, II,* Ralph H. Hruban,* and Eckhard R. Podackt From the Department of Pathology, The Johns Hopkins

Hospital, Baltimore, Maryland, and the Department of Mirobiology & Immunology,J University of Miami School of Medicine, Miami, Florida

Perforin is a potent cytolytic pore-forming protein expressed in cytoplasmic granules of cytotoxic T lymphocytes and natural killer cells. A new monoclonal antibody raised against human perforin was used to detect both in vitro and in vivo perforin expression in cytotoxic cells. Immunohistochemical analysis of human peripheral blood mononuclear cells cultured in recombinant interleukin-2 (rIL-2) showed strong granular cytoplasmic staining of the IL-2 activated cytotoxic cells. Fresh-frozen tissue sections from patients with heart allograft rejection were also stained. Strong granular cytoplasmic staining of the mononuclear inflammatory infiltrate characteristic for perforn in cardiac allograft rejection was observed. The detection and quantitative analysis ofperforin-associated cytotoxic cells by the human anti-perfortn monoclonal antibody will help to evaluate the significance of these functionally distinct cytotoxic cells in human tissue. (Am J Pathol 1992, 140:1025-1030)

Human cytotoxic T cells and natural killer (NK) cells express multicomponent cytoplasmic granules containing cytolytic effecter molecules such as perforin,12 proteoglycans,3 and serine proteinases45 (alternately called human granzymes). In the presence of calcium, purified perforin nonspecifically lyses a variety of target cells (for review, 1823).6 Human cDNA clones have been isolated encoding human perforin.7 Human perforin mRNA is ex-

pressed specifically in cytolytic lymphocytes regardless of whether they were generated in vivo or in vitro. There is constitutive expression of perforin in human CD3-, 56+ NK cells, CD3+ large granular lymphocyte (LGL), and gamma/delta T cells. This expression is significantly induced in CD8+ T cells, but to lesser extent in gamma/ delta T cells and in natural killer cells. Strong correlation exists between cytolytic potential and perforin mRNA expression in LGL, NK cells, gamma/delta T cells, and CD8+ T cells."10 The induction of perforin mRNA is partially blocked by the immunosuppressive drug cyclosporin A." A mouse monoclonal antibody against a rat perforin that crossreacts with human perforin has been used to stain cytotoxic lymphocytes by immunoperoxidase techniques. 10 12,13 We have generated a new monoclonal antibody against native human perforin and subsequently used it to detect cytotoxic cells by the immunoperoxidase technique. The study demonstrates that the human antiperforin monoclonal antibody is a useful immunohistochemical marker for detection of perforin-associated cytotoxic cells in vitro and in fresh-frozen human tissue.

Materials and Methods

Production of Human Perforin Monoclonal Antibody Briefly, mouse anti-human perforin was generated by immunizing BalbC mice with purified granules from the human cytotoxic large granular lymphocytes lymphoma line YT.14 Hybridoma supernatants, generated by convenSupported by funds from the NIH and the American Cancer Society. Dr. Hameed is a recipient of the Individual National Research Service Award from the National Institutes of Health, Bethesda, Maryland. Accepted for publication March 10, 1992. Address reprint requests to Dr. Arif Hameed, Department of Pathology, Division of Gynecologic Pathology, The Johns Hopkins Hospital, 600 N. Wolfe Street, Baltimore, MD 21205.

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tional procedures, were then screened by indirect immunofluorescence techniques, for binding to permeabilized YT and for lack of reactivity with K562, Raji, or Daudi cells. The anti-perforin antibody used in the current study was further characterized by the following criteria. In immunoprecipitation assays, a 70 kd protein (perforin) is precipitated from YT cells but not from other cell lines; anti-perforin-Sepharose, on preincubation with hemolytically active granules, depletes hemolytic activity. Finally, transfection of perforin negative cells with human perforin cDNA renders previously negative cells reactive with the anti-perforin antibody. The anti-perforin is a murine IgG2b specific for human perforin and unreactive with mouse

Briefly, the granules were extracted with an equal volume of 1 mol/l sodium-potassium-phosphate buffer (pH 6.5), containing 1 mmol/l ethyleneglycol bis (P-aminoethyl ether) N,N,N' tetraacetic acid (EGTA), 0.2% azide, and 1 mmol/l MgCI2. After 30 minutes of centifugation at 20,000 rpm in a Sorvall centrifuge, the soluble extract was applied to gel-filtration column (Protein PAK 300 sw) using an HPLC System (Waters, Millipore, Milford, MA). The eluted fractions were tested for hemolytic activity as described.'0 The hemolytically active fractions were pooled and stored at - 700C.

perforin (data not shown).

Immunostaining was performed on cytocentrifuge slides and paraffin-embedded cell blocks of LAK cells as well as on the fresh-frozen tissue sections. The staining was performed according to the manufacturer's instructions using the Histoscan Kit (Biomedia Corp., Foster City, CA). Briefly, the deparaffinized or acetone-fixed frozen tissue sections and cytocentrifuge slides of LAK cells were hydrated in PBS for 3 minutes followed by addition of tissue conditioner reagent for 10 minutes at room temperature. After rinsing in PBS, the slides were covered with the blocking reagent for 5 minutes. The perforin antibody was diluted to a 1:1000 dilution, and the slides were incubated with the primary antibody for 20 minutes. The slides were rinsed with PBS for 5 minutes and incubated with the secondary antibody for 20 minutes. After PBS wash, the peroxidase reagent was added for 20 minutes. The immunostaining was developed using 3-amino-9ethylcarbazole. The slides were counterstained with Mayer's hematoxylin. In absorption experiments, the antiperforin antibody was preincubated with various concentrations of the partially purified, hemolytically active human perforin, homogenates of LAK cells, and the human erythroleukemic cell line, K562. The preincubation was carried out at room temperature for 60 minutes. After pre-incubation, the binding of antiperforin antibody to inflammatory cells in tissue sections with cardiac allograft rejection, as well as in vitro IL-2 activated cytotoxic cells, was analyzed by the immunoperoxidase technique.

Human Tissue Fresh-frozen tissue from 17 heart transplant recipients with heart allograft rejection was used for the study. The selected fresh tissue sections containing mononuclear inflammatory infiltrates were fixed in acetone. The degree of rejection present in each of the tissue sections was graded according to the new working formulation proposed by the International Society for Heart Transplantation.15 In all cases, the degree of rejection in the material frozen was representative of that present in the myocardium. The fresh normal tissue sections of various organs were obtained from surgical specimens.

Generation of Human Cytotoxic Cells Human peripheral blood mononuclear cells were obtained from healthy volunteers by Ficoll-Hypaque density gradient centrifugation. After washing, the cells were placed at a density of 1.5 x 106 per ml in Iscove's modified Dulbecco's minimum essential medium (DMEM) (GIBCO, Grand Island, NY) containing 10% blood group compatible human serum and 1000 pJml recombinant interleukin-2 (rlL-2) (Hoffman La Roche, Nutley, NJ). After approximately 5 days, the cells began to proliferate, and the culture was divided every 2 days in the same medium. Cells were harvested after 20 days and were washed in phosphate-buffered saline (PBS). The cytocentrifuge preparations of these cells, as well as resting peripheral blood mononuclear cells, were prepared and fixed in either acetone, 4% paraformaldehyde, or 10% formalin, and were kept at 4°C.

Partial Purification of Human Perforin Cytotoxic granules from human lymphokine-activated (LAK) cells were prepared as described previously.16,17

Immunohistochemical Staining

Results The antiperforin monoclonal antibody used in this study stained the cytotoxic cells in both cytocentrifuge slides of IL-2 activated cytotoxic cells (positive control) as well as those in fresh-frozen tissue sections of cardiac allograft rejection. Figure 1 a shows LAK cells with approximately 80% of the cells staining with the antiperforin antibody at a 1:1000 dilution. The staining pattern generally was granular and cytoplasmic. Most LAK cells reacted with antibodies to CD3, CD4, or CD8 and CD16, whereas

Perforin-positive Cytotoxic Cells

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Figure 1. Immunohistocbemical staining with anti-human perforin. A: LAK cells (positive control) using human perforin antibody. B: LAK cells stained with normal nonimmunized mouse serum (negative control). C: Immunostaining by the anti-perforin antibody of a tissue section of a human cardiac biopsy during allograft rejection. D: Enlarged view ofperforin positive cells delineated by the rectangle in (C), (original magnification A,B,C x 63, D x 100).

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Table 1. Lack of Reactivity of Perforin Antibody with Various Normal (Noninflammatory) Tissues* Perforin monoclonal Tissues antibody reactivity

Gastrointestinal tract Intestinal epithelium Kidney Tubular epithelium Glomeruli Interstitium Liver Hepatocytes Biliary tract epithelium Pancreas Pancreatic acini Pancreatic ductal epithelium Lung Respiratory epithelium Lung parenchyma Endocrine glands Thyroid Adrenal Pituitary Skin Epidermis Dermis Muscles Cardiac muscle Skeletal muscle Smooth muscle Nervous System Cerebrum Cerebellum Spinal cord Peripheral nerve *

Five specimens of each type of tissue were stained.

CD57 antigen expression was rare (data not shown). The normal nonimmunized mouse serum, used as negative control, showed no staining of the LAK cells (Figure 1 B). In addition, the unstimulated peripheral blood mononuclear cells showed rare perforin positive mononuclear cells probably representing NK cells or in vivo activated T lymphocytes. The polymorphonuclear leukocytes, monocytes, and macrophages of the peripheral blood as well as tissue infiltrates did not stain with the perforin antibody. Figure 1C and D show granular cytoplasmic staining of the mononuclear inflammatory cells infiltrating the myocardium of a patient with cardiac allograft rejection. The staining was observed in approximately 1 to 10% of the infiltrating (activated) mononulcear cells found in the perivascular, intravascular, and interstitial areas. There was no staining of the mononuclear inflammatory infiltrate of cardiac allograft rejection by the nonimmune mouse serum used as a negative control (data not shown). Of the 17 hearts examined, 2 had grade 0 rejection, 6 had grade 1 A rejection, 4 had grade 3A rejection, and 5 had grade 3B rejection. The two (0%) hearts with grade 0 rejection did not contain lymphocytes that stained for perforin, four of the six (66%) hearts with grade 1 A rejection contained lymphocytes that stained for perforin, three of the four (74%) hearts with grade 3A rejection contained

perforin-positive lymphocytes, and four of the five (80%) hearts with grade 3B rejection contained perforin-positive lymphocytes. All patients had received triple-drug immunosuppression, which included cyclosporin. Cyclosporin is known to inhibit perforin expression during T-cell activation.11 The specificity of the antiperforin antibody staining is summarized in Table 1. Normal tissue sections from multiple organs showed no reactivity with the antibody. Furthermore, the specific staining noted previously was blocked when the antiperforin antibody was preincubated with the LAK cells extract or partially purified human perforin (Table 2). The fixation of cytocentrifuge slides as well as cell blocks of LAK cells in 10% buffered formalin caused complete loss of staining by the antiperforin antibody (data no shown).

Discussion The granule exocytosis model of NK cell and cytotoxic T-lymphocyte (CTL) mediated cytolysis proposes that cytotoxic cells, on recognition of their target, release the contents of their cytotoxic granules.1"21 Perforin, one of the apparently constant components of granules of human cytotoxic lymphocytes, is a cytolytic mediator and forms transmembrane channels (pores) in the target membrane in the presence of calcium.2223 Polyclonal antibodies raised against rat perforin have been shown to block rat NK-cell-mediated killing but not rat CTL killing.24 However, inhibition of perforin expression by antisense perforin oligonucleotides in T lymphocytes activated in vitro results in a proportional decrease of CTL cytotoxicity in mice.25 The role of human perforin in in vivo cytolytic reactions has not been extensively studied. One study, using a rabbit polyclonal antiserum raised against recombinant human perforin and against N-terminal peptides of human perforin, has described the staining of CTL and NK cells among inflammatory infiltrates during acute myocarditis by the immunoperoxidase technique.26 The understanding of perforin-mediated reactions in in vivo, in part, requires detection of perforin Table 2. Absorption Experiments with Anti-perforin MonoclonalAntibody Cardiac Perforin monoclonal allograft LAK antibody rejection cells* + Unabsorbed + Absorbed with K562 cells extract Absorbed with LAK cells extract Absorbed with human perforint * Lymphokine-activated killer cells. t Partially purified perforin.

+

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-

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positive cells in various human tissues. Our study, using mouse monclonal antibody raised against native human perforin, localizes cytotoxic cells in human tissue by the immunoperoxidase technique. The percentage of perforin positive mononuclear inflammatory cells in the tissue sections with heart allograft rejection ranges approximately 1 to 10%. The lack of staining in the remaining mononuclear infiltrates may be explained by one or more of the following alternatives: 1) the unstained mononuclear cells may not be cytotoxic cells; 2) it may imply in vivo degranulation of the cytotoxic cells after lethal hit delivery; 3) the cyclosporin immunosuppression of those patients may inhibit perforin expression; 4) because inflammatory reaction takes place within days, the cellular infiltrate in different areas may represent different stages of activation. As a result, the cells with poor and/or no staining may represent an infiltrate at a relatively earlier time, and as yet incomplete activation and synthesis of perforin. However, all these considerations are speculative and require further analysis. We emphasize that our study with the antiperforin antibody was directed towards immunohistochemical localization of perforin antigen by the monoclonal antibody, both in vitro as well as in vivo. The presence of perforinassociated cytotoxic cells in cardiac allograft rejection suggests their role in the disease process. However, careful quantitative analysis of these cytotoxic cells in human tissues in conjunction with the patient's clinical course is required to understand their role in disease processes. The production of monoclonal antiperforin antibody with high specificity, high titer, and unlimited supply could make it an invaluable tool for characterizing subsets of human perforin-positive cytotoxic cells among inflammatory infiltrate in multiple organs during various immune reactions.

Acknowledgment The authors thank Dr. Frederic B. Askin for review of this manuscript, and Sue Skierkowski for excellent secretarial assistance.

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