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And B-Cell Lymphomas in Patients Following Treatment with Anti-Thymocyte Globulin

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And B-Cell Lymphomas in Patients Following Treatment with Anti-Thymocyte Globulin [CANCER RESEARCH 42, 2465-2469, June 1982] Objective Regressions of T- and B-Cell Lymphomas in Patients following Treatment with Anti-Thymocyte Globulin Richard I. Fisher,1 Bruce A. Silver, Christian P. Vanhaelen, Elaine S. Jaffe, and Jeffrey Cossman Medicine Branch, Clinical Oncology Program, Division of Cancer Treatment [R. l. F., B. A. S., C. P. V.I, and the Section of Hematopathology, Laboratory of Pathology [E. S. J., J. C.J, National Cancer Institute, Bethesda, Maryland 20205 ABSTRACT tempts have been made to classify the specificity and mecha nism of action of the ATG towards normal lymphocyte subpop- We have conducted a clinical trial utilizing anti-thymocyte ulations as well as the patients' own tumor cells. globulin (ATG) for the treatment of patients with non-Hodgkin's lymphomas. Six patients were treated; 50% reductions in tumor mass of short duration were observed in one patient with a T- MATERIALS AND METHODS cell lymphoma and two patients with B-cell lymphomas. In vitro assays have been performed in an attempt to study the reac ATG. ATG (ATGAM) was kindly provided by the Upjohn Co., Kala- tivity and potential mechanism of antitumor action of the ATG. mazoo, Mich. ATG is the IgG fraction isolated from the plasma of horses that have been immunized with human thymocytes. The ATG The ATG bound to essentially all normal blood mononuclear leukocytes as well as tumor cells from patients with T-, B-, or has been absorbed with human plasma and RBC stroma. Preparation of Cell Suspensions. Peripheral blood MNL were iso null cell lymphomas demonstrating its lack of specificity. Fur lated from heparinized blood by centrifugation over Ficoll-Hypaque thermore, complement-mediated lysis of normal mononuclear (Lymphoprep; Litton Bionetics, Kensington, Md.) as described previ leukocytes, normal T- or B-cells, and tumor cells from two ously (3). Single-cell suspensions from lymph nodes involved by lym unresponsive patients were all comparable; moreover, since phoma were prepared immediately following surgery. Cell surface this lysis occurred only at concentrations of ATG that are not markers were determined immediately, or the cells were cryopreserved attainable in vivo, it is unlikely that complement-mediated cy- over liquid nitrogen. Immunological markers were not altered by the totoxicity accounts for the responses observed. Peripheral freezing or thawing process (4). Immunotyping of Neoplastic Cells. Surface membrane immuno- blood lymphocyte counts and total erythrocyte rosettes did decrease during ATG treatment. Thus, objective tumor re globulin was detected by direct immunofluorescence using individual sponses in both B- and T-cell non-Hodgkin's lymphomas can heavy- and light-chain specific antibodies as well as polyvalent anti- immunoglobulin antibodies as described previously (6). The B-cell be achieved with a very nonspecific antiserum although sig lymphomas expressed only one light-chain isotype, or the neoplastic nificant toxicity resulted. Whether the magnitude or duration of cells were uniformly stained by the polyvalent antibody. response can be increased with monoclonal antibodies remains T-Cells were identified by their ability to form rosettes with sheep to be determined. Future success with serotherapy might re erythrocytes which had been treated with aminoethylisothiouronium quire use of either a battery of different monoclonal antibodies (9). The percentage of the cell suspension forming rosettes was initially or a single monoclonal antibody that can deliver radioisotopes, determined. The cell suspension was then cytocentrifuged, placed on slides, and stained with a modified Wright's stain. Lymphomas were chemotherapy, or toxins to the tumor cells. considered to be composed of T-cells when the morphologically ab normal lymphoid cells on the cytocentrifuge preparations demonstrated INTRODUCTION erythrocyte rosette formation. The cell suspensions also contained variable numbers of surface immunoglobulin-positive normal-appearing Several investigators have documented the occurrence of lymphocytes that expressed both K and A light chains. All cases were objective tumor regressions in patients with Sézarysyndrome also studied for intranuclear terminal deoxynucleotidyl transferase by who were treated with ATG2 (1, 7, 8). The ATG used in the indirect immunofluorescence using an anti-terminal deoxynucleotidyl studies from this country has been utilized extensively in the transferase antibody (provided by Dr. Frederick Bollum, Uniformed prevention of allograft rejections following renal transplantation Services University for the Health Sciences, Bethesda, Md.) (2). Ter (5). However, the specificity and mechanism of action of this minal deoxynucleotidyl transferase activity was identified only in lym antiserum have not been well characterized. To date, there are phomas of the lymphoblastic type. Separation of T- and B-Cells. T- and B-cells were separated from no reports of ATG administration in an attempt to treat patients with other more common forms of non-Hodgkin's lymphomas. peripheral blood MNL by modification of the method of Dean ef al. (6). In brief, unfractionated peripheral blood MNL were incubated with Indeed, the therapeutic usefulness of this form of treatment still neuraminidase-treated sheep RBC at 4°.The erythrocyte-rosetting T- remains undefined. cell fraction was separated from the non-T-cell fraction by centrifuga In this report, we present the results of a study of the tion over Ficoll-Hypaque. Both T- and non-T-cell fractions were then therapeutic value of ATG for widespread non-Hodgkin's lym subjected to a second erythrocyte rosette procedure with separation phomas that are refractory to combination chemotherapy. At- over Ficoll-Hypaque. Sheep RBC were lysed by brief exposure to a hypotonie lysing buffer. Lymphocytes that twice formed rosettes with ' To whom requests for reprints should be addressed, at National Cancer sheep RBC comprise our T-cell population; cells that were erythrocyte Institute, NIH, Building 10, Room 12N226, 9000 Rockville Pike, Bethesda, Md. rosette negative in both separations, i.e., non-T-cells, are referred to 20205. as our B-cell population even though they contain B-cells, monocytes, 2 The abbreviations used are: ATG, anti-thymocyte globulin; MNL, mononu and null cells. In our laboratory, the T-cell fractions obtained in this clear leukocytes; FACS II, Fluorescence Activated Cell Sorter II; i.d., intradermal; i.e., intracranial. manner contain 94.3 ±0.74% (S.E.) erythrocyte rosette-forming cells Received November 18, 1981; accepted March 3, 1982. and 1.0 ±0.4% B-cells as determined by immunofluorescence with a JUNE 1982 2465 Downloaded from cancerres.aacrjournals.org on October 4, 2021. © 1982 American Association for Cancer Research. R. I. Fisher et al. F(ab'>2 fragment of goat anti-human immunoglobulin (Cappel Labora lesions was considered a partial responder, while any patient with a tories, Cochranville, Pa.). The non-T-cell fraction contains 2.2 ±0.5% response of lesser magnitude was considered a nonresponder. Each T-cells and 57.8 ±4.8% surface immunoglobulin-positive cells. patient received 14 doses of ATG unless rapidly progressive lymphoma Cellular Binding of ATG. Binding of ATG to normal human peripheral developed. Patients who did not develop progressive disease were blood MNL and tumor cells from the patients was determined by an retreated with ATG for 7 consecutive days at monthly intervals. indirect immunofluorescence assay. MNL or tumor cells were sus pended in phosphate-buffered saline (containing in g/liter: 0.2 KCI; 0.2 KH2PO„;8NaCI; and 2.1 Na2HPO«-7H2O)with 2% fetal calf serum RESULTS and incubated with or without the desired concentration of ATG for 30 min at 37°.The cells were washed 3 times in the previous medium plus Binding of ATG to Normal MNL. The ability of the ATG to 0.02% sodium azide at 4°. An appropriate dilution of a fluorescein- bind to normal peripheral blood MNL was assayed using indi conjugated IgG fraction goat anti-horse IgG (heavy- and light-chain) rect immunofluorescence and FACS II. Preliminary experiments antibody (Cappel Laboratories) was added for 30 min at 4°.The cells demonstrated that a 1:100 dilution of ATG and a 1:16 dilution were washed 3 additional times and analyzed for fluorescent staining of the fluorescein-conjugated goat anti-horse immunoglobulin with FACS II (Becton Dickinson Electronics Laboratory, Sunnyvale, gave optimal staining. As shown in Chart 1, ATG bound to 96% Calif.). of normal MNL. Background staining was less than 2%. Sub Complement-dependent Antibody Cytotoxicity. Complement-de sequent studies revealed that the percentage of cells bound pendent cytotoxicity of the ATG against normal peripheral blood MNL, with ATG did not differ among the various batch preparations T-cells, B-cells, and patients' tumor cells was assayed by modification of ATG that were used in this trial. of the method of Sachs (13). Briefly, 2 x 107 lymphocytes or tumor Complement-dependent Antibody Cytotoxicity of Normal cells were suspended in Roswell Park Memorial Institute Tissue Culture Lymphocytes. The ATG was also able to lyse normal peripheral Medium 1640 with 10% heat inactivated AB serum and 0.3 ml of blood MNL in a complement-dependent antibody cytotoxicity radioactive sodium chromate (Amersham Corp., Arlington Heights, II.) (specific activity, 1 mCi/ml) for 1 hr at 37° and then washed twice. assay. As shown in Table 1, the cytotoxicity was maximal During this time, ATG at the desired maximal concentration was added between a 1:10 and a 1:20 dilution of ATG but diminished to the appropriate wells of a 96-well U-bottom microtiter plate (Cooke rapidly with subsequent dilutions of the ATG. The highest Laboratory Products, Alexandria, Va.); serial 1:2 dilutions filled the percentage of cytotoxicity observed in 5 normal individuals remainder of the experimental wells. No antibody was added to wells was 68.2 ± 4.6% with background lysis from complement that would be used to determine background or maximum chromium alone ranging from 11 to 15%. When T-cells were separated release.
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