Proc. Nati. Acad. Sci. USA Vol. 78, No. 5, pp. 3160-3164, May 1981 Immunology Dissection of distinct human immunoregulatory T-cell subsets by a monoclonal antibody recognizing a cell surface antigen with wide tissue distribution (T- and B-cell lines/T- and B-cell leukemias/helper and suppressor T cells) OSCAR H. IRIGOYEN*, PHILIP V. RIZZOLO*, YOLENE THOMAS*, MARTIN E. HEMLERt, HARRY H-L SHEN* STEVEN M. FRIEDMAN*, JACK L. STROMINGERt, AND LEONARD CHESS* *Department of Medicine, Division of Rheumatology, Columbia University, College of Physicians and Surgeons, New York, New York 10032; and tSidney Farber Cancer Institute, Boston, Massachusetts 02115 Contributed by Jack L. Strominger, December 31, 1980 ABSTRACT A monoclonal antibody, PVR-1, was obtained wide tissue distribution but is preferentially expressed on pre- after hybridization of X63Ag8.653 murine myeloma cells with cursors of helper T lymphocytes. spleen cells from a mouse immunized with human lymphocytes. It recognizes a 175,000- to 185,000-dalton surface antigen present on -"80% ofnormal human peripheral T lymphocytes, 50% ofnon- MATERIALS AND METHODS T non-B cells, and <10% ofB cells as determined by complement- dependent microcytotoxicity. It is also present on various leukemia Lymphocytes, Cell Lines, and Leukemic Cells. Fresh pe- T cells, on some but not all T lymphoblastoid cell lines, and on a ripheral blood mononuclear cells were isolated from healthy small fraction of some B lymphoblastoid cell lines. Some B-cell human volunteers by Ficoll/diatrizoate density gradient cen- chronic lymphocytic leukemia cells also express the PVR-11 anti- trifugation. Highly enriched populations of T and B cells were gen. Functional analysis of normal human T lymphocytes dem- obtained by rosetting with sheep erythrocytes and passage onstrated that the PVR-11-depleted T-cell subset contains the pre- through a column ofrabbit anti-human F(ab')2 coupled to Seph- cursors ofboth cytotoxic and suppressor cells but lacks helper cells. adex G-200 as described (21). The non-T non-B cell population On the other hand, cytotoxic effector T cells express the PVR-11 was further separated into adherent (designated "macro- antigen. These results demonstrate that antigenic determinants phages") and nonadherent (designated "null") cell populations with relatively wide tissue distribution can dissect functionally by adherence to plastic. distinct human immunoregulatory T-cell subsets. The Epstein-Barr virus-transformed B lymphoblastoid cell lines were established in collaboration with Frank Nakamura During the last several years, a number of antibodies that rec- and Arthur Bloom (Cancer Center, College of Physicians and ognize distinct surface membrane antigens on human T lym- Surgeons, Columbia University). CJ is a new T-LCL generated phocytes have been described. These antibodies have different in this institution. The leukemic T cells (E+ SmIg-) and B cells specificities that can be grouped into at least three distinct cat- (E- SmIg+) were obtained after informed consent from patients egories. The first group includes antibodies reacting with all or being followed at the Presbyterian Hospital (New York). nearly all normal peripheral T lymphocytes. Elimination ofthe Production of the Monoclonal Antibody PVR-11. BALB/c cells reactive with these antibodies abrogates all T-cell depen- mice were immunized with acute lymphocytic leukemia T cells dent functions (1-4). The second group consists of antibodies (T-ALL) and reimmunized 8 weeks later with normal allogeneic that recognize determinants restricted to cells ofT-cell lineage peripheral T lymphocytes. Spleen cells were hybridized and and dissect subsets of functionally distinct T lymphocytes monoclonal antibodies were produced according to standard (5-13). Antibodies in these first two groups, which have been techniques (22) with X63Ag8.653 myeloma cells, a gift from developed either by conventional means or by somatic cell hy- Matthew Scharff (Albert Einstein College of Medicine). The bridization techniques (14), react minimally or not at all with complement-binding antibody obtained, designated "PVR-11" normal B lymphocytes, B lymphoblastoid cell lines (B-LCL), was found to be an IgG2a K immunoglobulin by using standard or B cell chronic lymphocytic leukemia (B-CLL) cells. immunodiffusion techniques. The third group includes antibodies recognizing antigens Analysis of Cell Populations by Complement (C)-Mediated common to both normal peripheral T lymphocytes and B-CLL Microcytotoxicity Assay. Ten thousand 51Cr-labeled cells in 10 cells. Those initially reported were heteroantibodies generated ,ul of final medium (RPMI-1640 medium containing 25 mM by immunization of rabbits with leukemic T cells (15), thymus Hepes, 0.15% Na bicarbonate, 12% fetal calf serum, and pen- (15, 16), or brain (17). More recently, monoclonal antibodies icillin/streptomycin) were incubated with 10 ,ul of monoclonal recognizing single antigenic determinants of69,000-71,000 and antibody in triplicate at 22°C. After 45 min, 20 Aul of a 1:6 di- 65,000 daltons on T cells and B-CLL cells have been reported lution ofrabbit serum as a source ofC was added and incubation (18-20). was continued for 1 hr at 37°C. The plates were centrifuged at The functional significance of these cell surface antigens ex- 1000 rpm for 10 min, and the supernatants were collected and pressed on both T cells assayed for radioactivity in a gamma counter. Incubation of tar- and B-CLL cells is currently unknown. cells with C alone was used to In the present report we describe the characterization of a get measure spontaneous release. monoclonal antibody which recognizes a surface antigen with Abbreviations: LCL, lymphoblastoid cell lines; CLL, chronic lympho- cytic leukemia; ALL, acute lymphocytic leukemia; SmIg, surface im- The publication costs ofthis article were defrayed in part by page charge munoglobulin; E, rosettes with sheep erythrocytes; C, complement; payment. This article must therefore be hereby marked "advertise- PHA, phytohemagglutinin; PFC, plaque-forming cells; PWM, poke- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. weed mitogen. 3160 Downloaded by guest on September 27, 2021 Immunology: Irigoyen et al. Proc. Natl. Acad. Sci. USA 78 (1981) 3161 Percentage lysis was calculated as: Table 1. PVR-11 expression on OKT4' and OKT8' T-cell subsets % lysis by monoclonal (experimental release - spontaneous release x 100 antibodyt maximum release - spontaneous release T-cell subset* PVR-11 OKT3 OKT4 OKT8 OKT4+ 100 100 45 -3 Maximal release was the 5"Cr released from cells incubated with 56 100 Triton X-100. 0KT4- 5 70 Isolation of Human T-Cell Subsets. The isolation of T-cell OKT8+ 61 100 11 100 subpopulations by C-mediated lysis with monoclonal antibodies OKT8- 79 100 73 2 has been described (13). Peripheral T lymphocytes were also separated into OKT4+ and OKT4- cells by positive selection * T-cell subsets were obtained by positive selection with antibody- coated erythrocytes. with antibody-coated erythrocytes as described (23). t Assessed Labeling and Immunoprecipitation ofCell Surface Proteins. by C-dependent microcytotoxicity. Purified T cells were cultured for 4-6 days in phytohemagglu- tinin [PHA (Difco)] at 10 ,ug/ml to obtain PHA-activated T cells. RESULTS The surface proteins were externally labeled with "2I by using PVR-11 Distribution on Normal Peripheral Blood Mono- 1,3,4,6-tetrachloro-3,6-diphenylglycoluril (IODO-GEN, Pierce), nuclear Cells, Continuous Cell Lines, and Malignant Cells. immunoprecipitated, and then electrophoretically analyzed as PVR-11 reacted by C-mediated cytotoxicity with 75-80% of described (10). After labeling, cells were extracted with 1% ,ormal T lymphocytes, 50-60% of both "null", cells and mac- Nonidet P-40 in 2 mM phenylmethanesulfonyl fluoride/10 mM rophages and =10% of B lymphocytes (Fig. 1). Furthermore, iodoacetamide/10 mM Tris'HCl, pH 8.0. All extracts were PVR-11 recognized >90% ofOKT4' lymphocytes and 60-80% cleared with 3-4 ,ul of normal mouse serum prior to specific ofOKT8' lymphocytes (Table 1). Conversely, PVR-11-depleted immunoprecipitation with 2-3 ,ul of purified PVR-11 (2 mg/ T lymphocytes were largely depleted of OKT4' cells and en- ml). riched in OKT8' cells (data not shown). Thus, PVR-11 reacts Functional Assays. Polyclonal induction and suppression of with the majority ofOKT4' cells and a fraction ofOKT8' cells. antibody-secreting cells. The culture conditions and the reverse Results ofT-LCL surface antigen analysis with several mono- hemolytic plaque assay for the assessment ofinduction and reg- clonal antibodies are shown in Table 2. All the T-cell lines re- ulation of plaque-forming cell (PFC) generation has been de- acted weakly or not at all with OKT3 and were unreactive with scribed in detail (13). In brief, 2 X 106 B cells were cultured the anti-monocyte antibody OKM1. With the exception of in 2 ml offinal medium containing pokeweed mitogen (PWM). YT4E, they also were unreactive with the anti-human la anti- To these cultures were added various numbers of the relevant body OKIL. Only three ofthe seven T-LCL were highly reactive T-cell subsets. Control cultures consisted of B cells cultured with PVR-11. In contrast, four ofthem reacted with OKT8 and alone or in the presence of PWM without added T cells. After only one of them reacted significantly with OKT4. Thus, si- 5-6 days ofincubation, plaques were counted in triplicate; the multaneous analysis with other monoclonal antibodies against results are expressed as the mean PFC per 106 B cells in the T cells indicated that the expression of PVR-11 is independent original culture. SEM always was <20%. of OKT3, OKT4, and OKT8. Cell-mediated lympholysis. Lympholysis by putative killer In addition, we analyzed leukemic T cells from six patients. cells generated during mixed lymphocyte culture was assessed Two patients had T-CLL, one had T-ALL, and three had the on 51Cr-labeled target cells in a 5 hr assay as described (21).