Dissection of Distinct Human Immunoregulatory T-Cell Subsets by A

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 lymphocytic 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 phytohemagglutinin [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). Sezary syndrome. The T cells from all of them were highly re- Percentage cytotoxicity was calculated as in the microcytotox- active with PVR-11 (Fig. 2). In contrast, of 11 B-LCL tested, icity assay. Spontaneous release was the 51Cr release from target the majority expressed low or no reactivity with PVR-11. The cells incubated with medium alone. B-CLL studied were heterogeneous with respect to reactivity with PVR-11: two sets of cells were only marginally reactive (10%), three had an intermediate reactivity (30-40%), and four were highly reactive (70-80%). 100 Characterization ofthe PVR-11 Antigen. In the presence of a reducing agent, PVR-1l specifically immunoprecipitated '25I- 80 labeled proteins ofapproximately 175,000185,000 daltons from PHA-activated T cells (Fig. 3A, lane a). Other faint bands of lower molecular weight were also present, but these were non- x 60 0 specific contaminants because they also appeared in the control

:Y 40 Table 2. PVR-11 expression on human T-cell lines % lysis by monoclonal antibody* 20 F T-cell line PVR-11 OKT3 OKT4 OKT8 OKM1 OKI1 CEM 100 15 92 83 0 0 CJ 100 0 11 50 0 0 1 2 4 8 16 32 Molt 3 87 0 26 50 0 0 Molt 4 28 0 0 100 0 0 (1/antibody dilution) x 10-3 HSB-2 8 0 0 0 0 0 RPMI-8402 0 0 0 0 0 0 FIG. 1. Reactivity ofPVR-11 on peripheral blood mononuclear cell YT4E 8 8 8 6 0 100 subpopulations as assessed by C-dependent microcytotoxicity. 0, T cells [erythrocyte-rosette positive (E+ cells)]; o, B cells (SmIg+ cells); *~sessed by C-dependent mic-ocytotoxicity. Antibodies OKT3, OKT4, *, null cells (E-, SmIg-, plastic nonadherent cells); A, macrophages OKT8, OKM1, and OKI1 were a gift from P. Kung and G. Goldstein, (E-, SmIg-, plastic adherent cells). Ortho (4, 9, 13, 24-26). Downloaded by guest on September 27, 2021 3162 Immunology: Irigoyen et al. Proc. Natl. Acad. Sci. USA 78 (1981)

100 0@ A H . L-200- . 0 80 _ so 0

._x 0 60 _ 0 92.5 C.) -0 401 0- 69 0 0 20 - 0 *. 0 0 0 46' I I_ 0 Tcell Tcell B cell B cell lines leukemia lines CLL

FIG. 2. Reactivity of PVR-11 on T and B leukemia and lymphoblastoid cell lines as assessed by C-dependent microcytotoxicity. Each point represents an individual cell population.

immunoprecipitation (lane b). This control experiment was car-

ried out with another mouse monoclonal antibody that recog- .i nizes a 240,000-dalton protein. Analysis of a PVR-11 immunoprecipitation in the absence of a reducing agent (Fig. 3B) again FIG. 4. Immunoprecipitation of the PVR-11 antigen on T and B showed a broad protein band at 175,000-185,000 daltons, and lymphoblastoid cell lines. Extracts of '25I-labeled HSB (lane a), JY the number of background bands was decreased. Thus, the (lane b), and peripheral blood T cells (lane c) were immunoprecipitated PVR-11 antigen does not appear to contain disulfide-bonded with PVR-11. The peripheral blood T-cell extract was also immuno- subunits. precipitated with the anti-HLA monoclonal antibody W6/32 (lane d). The B lymphoblastoid cell line JY (Fig. 4, lane b) did not Standards (sizes in kilodalton indicated) were as in Fig. 3. express the PVR-11 antigen (175,000-185,000 daltons) as detected by immunoprecipitation. However, the PVR-11 antibody did precipitate bands (175,000-185,000 daltons) from the T-cell line HSB-2 (Fig. 4, lane a) and from normal peripheral blood T cells (Fig. 4, lane c). A control experiment using the B monoclonal antibody W6/32 (27) to precipitate the 44,000- and A 12,000-dalton bands ofHLA from peripheral blood T cells (Fig. 4, lane d) did not precipitate any bands in the 175,000- to 185,000-dalton region. -200 -24o0 Functional Analysis of T-Cell Subsets Defined by PVR-11. The evidence obtained by using normal T lymphocytes and T cell lines suggests that the PVR-11 antigen may be expressed only at certain stages ofT-cell differentiation. To provide further evidence to support this idea, detailed functional studies of un- selected and PVR-11-depleted T-cell populations were 92.5 undertaken.

-92 .5 Regulation of B lymphocyte function by PVR-11-depleted T lymphocytes. (i) Help. It has been demonstrated that PWM- -69

20,000 FIG. 3. Immunoprecipitation of the PVR-11 antigen from 1251-la- up 15,000 beled PHA-activated T cells. Gels were 7-15% linear gradients of to acrylamide, and samples were 4 10,000 boiled without mercaptoethanol (B) or with 5% 0 5 2-mercaptoethanol (A). 5,000 --30o Protein standards (shown in lane c ofA; not shown for B) were myo- sin (200,000), phosphorylase b (92,500), albumin (68,000), oval- 0.5 1 2 bumin (46,000), and carbonic T cells added, no. x 10- anhydrase (30,000) (all from New England Nuclear). Immunoprecip- FIG. 5. OKT4+-enriched but not PVR-11-depleted T-cell popula- was out x were -b-= itation carried with PVR- tions induce B-cell differentiation. B cells (2 106) cultured in 11 (A, lane a; B) and an unrelated the presence of PWM and either OKT4' (e) or PVR-11-depleted (o) T control mouse monoclonal antibody cells. After 5-6 days, cultures were harvested and assayed for PFC (A, lane b). activity. Downloaded by guest on September 27, 2021 Immunology: Irigoyen et al. Proc. Natl. Acad. Sci. USA 78 (1981) 3163

Table 3. PVR-11-depleted T cells can suppress B-cell antibody production Addition of PVR-11- cells Addition of OKT8' cells T cells added, PFC/106 Suppression,t PFC/106 Suppression, Exp. no. x 10-6 per culture* B cellst % B cells S 1 0 18,600 - 18,000 - 1.0 6,000 68 9,700 48 2.0 5,350 72 8,850 52 4.0 4,600 76 5,100 73 2 0 13,150 - 13,150 - 1.0 9,250 30 7,600 43 2.0 1,150 92 ND - 4.0 1,000 95 ND - 3 0 16,250 - 16,250 - 0.2 5,800 65 5,750 65 1.0 2,525 85 3,475 79 2.0 2,275 86 2,350 86 4.0 1,750 90 1,350 92 * PVR-11- or OKT8' cells were added, prior to incubation, to cultures containing 2 x 106 B lymphocytes, 0.2 x 106 OKT4' T cells, and 10 Ag of PWM. ND, not done. t PFC generation was assessed by the reverse hemolytic plaque assay after 5-6 days in culture. t Suppression was calculated as follows: % suppression = [1 - PFC (etd culture) x 100. Standard culture consisted of B cells, OKT4' cells, and PWM without putative suppressor cells added.

triggered B-cell differentiation is dependent on OKT4' T lym- triggered polyclonal induction of B-cell differentiation. In con- phocytes (13). Therefore, we compared the helper capacity of trast, the T-cell subset remaining after depletion ofPVR-11-re- PVR-11-depleted T lymphocytes to that of OKT4' T lympho- active T cells contains the suppressor cells that negatively reg- cytes. To 2 X 106 B cells were added various numbers ofeither ulate the induction ofB-cell differentiation. Thus, viewed from OKT4' or PVR-11 depleted T cells, and the cell mixture was the universe of cell surface molecules present on T cells, the cultured for 5-6 days with PWM. Antibody production was antigen recognized by PVR-11 represents a differentiation an- measured by the reverse hemolytic plaque assay. The data tigen which can distinguish functionally distinct immunoregu- clearly indicate that PVR-11-depleted T cells do not have helper latory T-cell subsets. Further support for this concept came capacity (Fig. 5). Thus, helper T cells are included in the PVR- from studies demonstrating that depletion of PVR-11-reactive 11+ subset. T cells does not alter the generation of alloreactive cytotoxic (ii) Suppression. The OKT4' T-cell-induced antibody pro- effector cells. However, the PVR-11 determinant can be de- duction by B cells can be suppressed by addition ofOKT8 lym- tected on already committed cytotoxic effector T cells. This lat- phocytes (13). Various numbers of either OKT8' or PVR-11- ter finding suggests that either the PVR-11 antigen becomes depleted T cells were added to cultures containing 2 x 106 B expressed after allo-activation, or it is present on the precursor lymphocytes, 5% OKT4' T cells, and PWM (Table 3). PVR-11- cytotoxic cells but at an antigenic density or distribution not depleted lymphocytes were able to suppress antibody produc- detectable by C-mediated lysis. tion to the same extent that OKT8+ lymphocytes did. Thus, Although this distribution ofthe PVR-11 determinant is dis- *suppressor cells are contained within the PVR-11- T-cell tinct on functionally different T-cell subsets, the overall tissue subset. distribution of this antigen is not restricted to T lymphocytes. Cell-mediated cytotoxicity by PVR-11-depleted T lympho- For example, PVR-11 recognizes approximately 10% ofnormal cytes. Equal numbers of either normal or PVR-11-depleted T lymphocytes were cultured for 6 days with irradiated allogeneic E- cells and tested for the generation ofcytotoxic T cells. PVR- A B 11-depleted T cells were able to generate cytotoxic T cells (Fig. 40 40 6A). In contrast, when-untreated T lymphocytes were cultured >, 0 for 6 days in mixed lymphocyte culture and then depleted of x'x 30 30 PVR-11 reactive cells, the already committed cytotoxic effector 0 T cells were eliminated (Fig. 6B). Taken together, these data 2 20 o Ns.J 20 demonstrate that precursors of both suppressor and cytotoxic & T cells are contained within the PVR-1ll- subset. On the other 10 10 hand, helper cells and cytotoxic effectorTcells express the PVR- 11 determinant. 40:1 20:110:1 5:1 40:1 20:110:1 5:1 Killer-to-target ratio DISCUSSION FIG. 6. Cytotoxic effector but not precursor cells are PVR-11'. The monoclonal antibody described in this report, designated Responder T cells, either before (A) or after (B) in vitro sensitization PVR-11, recognizes a cell surface determinant present on a sub- to irradiated [1250 rad (12.5grays)] allogenic stimulators, were treated with C alone (s) orPVR-11 plus C(). Viable cells from each treatment set of normal peripheral T lymphocytes and on some but not group were assayed, at the killer-to-target ratios indicated, in a 5-hr all established human T-cell lines. The T-cell subset recognized cellular cytotoxicity assay against 51Cr-labeled cells autologous to the by PVR-11 contains the helper cells necessary for the PWM- stimulators. Downloaded by guest on September 27, 2021 3164 Immunology: Irigoyen et al. Proc. Natl. Acad. Sci. USA 78 (1981)

peripheral B lymphocytes and approximately 50% of adherent 5. Woody, J. N., Ahmed, A., Knudsen, R. C., Strong, D. M. & and nonadherent non-B non-T cells. At the present time it is Sell, J. W. (1975)J. Clin. Invest. 55, 956-966. unknown whether the normal non-T-cell populations bearing 6. Brouet, J. C. & Toben, H. (1976)J. Immunol. 116, 1041-1044. 7. Evans, R. L., Breard, J. M., Lazarus, A., Schlossman, S. F. & this antigen represent functionally different subsets. Prelimi- Chess, L. (1977)J. Exp. Med. 145, 221-233. nary studies have shown that PVR-11 is not present on the B 8. Friedman, S. M., Irigoyen, 0. H., Thomas, Y. & Chess, L. cells effecting the PFC response and that PVR-11-depleted E- (1980) in Regulatory T. Lymphocytes, eds. Pernis, B. & Vogel, cells are enriched in natural killing activity. H. J. (Academic, New York), p. 251. The concept that certain antigenic markers with relatively 9. Reinherz, E. L., Kung, P. C., Goldstein, G. & Schlossman, S. wide tissue distribution can dissect subsets ofT cells is not with- F. (1979) Proc. Natl. Acad. Sci. USA 76, 4061-4065. 10. Haynes, B. F., Mann, D. L., Hemler, M. E., Schroer, J. A., out precedent. Analogous findings have been reported in mu- Shelhamer, J. H., Eisenbarth, G. S., Strominger, J. L., Thomas, rine studies on both the Ly and Qa alloantigenic systems. For C. A., Mostowski, H. S. & Fauci, A. S. (1980) Proc. Natl. Acad. example, Ly6 and Ly7 antigens are expressed on peripheral T Sci. USA 77, 2914-2918. cells and B cells. Functional analysis demonstrated that Ly6 is 11. Engleman, E. G., Benike, C., Osborne, B. & Goldsby, R. (1980) recognized on a fraction ofcytotoxic effector cells but not on the Proc. Natl. Acad. Sci. USA 77, 1607-1611. precursor cells, whereas Ly7 is selectively expressed on effector 12. Hausman, P. B., Raff, H. V., Gilbert, R. C., Picker, L. J. & Stobo, J. D. (1980)J. Immunol. 125, 1374-1379. helper T cells (28-30). The alloantigens Qa2 and Qa3 are found 13. Thomas, Y., Sosman, J., Irigoyen, 0. H., Friedman, S. M., predominantly on T cells but are also expressed on non-T cells. Kung, P. C., Goldstein, G. & Chess, L. (1980)J. Immunol. 125, Selective elimination of Qa2' or Qa3' cells inhibits mitogen 2402-2408. proliferation in response to concanavalin A or PHA. Further- 14. Kohler, G. & Milstein, C. (1975) Nature (London) 256, 495-497. more, elimination of Qa2' cells reduces proliferation in mixed 15. Boumsell, L., Bernard, A., Lepage, V., Degos, L., Lemerle, J. lymphocyte culture and inhibits the generation ofcytotoxic cells & Dausset, J. (1978) Eur. J. Immunol. 8, 900-904. 16. Foon, K. A., Billing, R. J. & Terasaki, P. I. (1980) Blood 55, but does not affect the cytotoxic effector cells (31, 32). 16-20. It is of considerable interest that PVR-11 is also expressed 17. Whiteside, T. L., Winkelstein, A. & Rabin, B. S. (1977) Cancer on some but not all B-CLL cells. Comparison ofPVR-11 to other 39, 1109-1118. monoclonal antibodies recognizing antigens on normal T cells 18. Wang, C. Y., Good, R. A., Ammirati, P., Dymbort, G. & Evans, and B-CLL cells reveals distinct differences. First, all of the R. L. (1980)J. Exp. Med. 151, 1539-1544. previously reported antibodies have been found to be nonreac- 19. Boumsell, L., Coppin, H., Pham, D., Raynal, B., Lemerle, J., Dausset, J. & Bernard, A. (1980) J. Exp. Med. 152, 229-234. tive with normal peripheral B cells or monocytes. Second, un- 20. Royston, I., Majda, J. A., Baird, S. M., Meserve, B. L. & Grif- like the other antibodies, PVR-11 reacts with a small but definite fiths, J. C. (1980)J. Immunol. 125, 725-731. proportion of some B-LCL. Finally, and perhaps most impor- 21. Sondel, P. M., Chess, L., MacDermott, R. P. & Schlossman, S. tantly, PVR-11 precipitates an antigen of 175,000-185,000 dal- F. (1975)J. Immunol. 114, 982-987. tons, whereas the previously described antibodies detect an- 22. Kwan, S. P., Yelton, D. E. & Scharff, M. D. (1980) in Genetic tigens of much smaller size (i.e., 65,000-71,000 daltons). Engineering, eds. Setlow, J. K. & Hollaender, A. (Plenum, New York), Vol. 2, p. 31. This protein band precipitated by PVR-11 is broad enough 23. Strelkauskas, A. J., Teodorescu, M. & Dray, S. (1975) Clin. Exp. to be a doublet, perhaps with more than one protein sharing Immunol. 22, 62-71. the same antigenic determinant or alternatively, there may be 24. Breard, J., Reinherz, E. L., Kung, P. C., Goldstein, G. & some heterogeneity in glycosylation. It is of interest that the Schlossman, S. F. (1980)J. Immunol. 124, 1943-1948. PVR-11 antigen is strikingly similar in band shape and molecular 25. Reinherz, E. L., Kung, P. C., Pesando, J. M., Ritz, J., Gold- stein, G. & Schlossman, S. F. (1979) J. Exp. Med. 150, weight to some of the bands recognized by the rabbit heter- 1472-1482. oantisera C-51 and anti-JAN on variousT-cell lines and activated 26. Friedman, S. M., Hunter, S. B., Irigoyen, 0. H., Kung, P. C., T cells (33). However, sequential immunoprecipitation exper- Goldstein, G. & Chess, L. (1981)J. Immunol., in press. iments (not shown) suggest that those heteroantisera recognize 27. Barnstable, C. J., Bodmer, W. J., Brown, G., Galfre, G., Mil- antigens unrelated to the PVR-11 antigen. Other investigators stein, C., Williams, A. J. & Ziegler, A. (1978) Cell 14, 9-20. 28. Potter, T. A., McKenzie, I.F.C., Morgan, G. M. & Cherry, M. studying mouse (34-36) and human (37) resting and activated (1980)J. Immunol. 125, 541-545. T cells have detected multiple proteins in the 170,000- to 29. Potter, T. A., Morgan, G. M. & McKenzie, I.F.C. (1980)1. Im- 190,000-dalton region. PVR-11 might be useful in sorting out munol. 125, 546-550. these previous results and also might provide further insight 30. Pilarski, L. M., Al-Adra, A. R. & McKenzie, I.F.C. (1980)J. Im- into the relationship between specific surface determinants and munol. 125, 365-369. 31. Flaherty, L., Zimmerman, D. & Sullivan, K. A. (1978) J. Im- cell activation and function. munol. 121, 1640-1643. We gratefully acknowledge the expert technical assistance of Gary 32. Sullivan, K. & Flaherty, L. (1979)J. Immunol. 123, 2920-2924. 33. Judd, W., Poodry, C. A., Broder, S., Friedman, S. M., Chess, Thompson. This work was supported in part by National Institutes of L. & Strominger, J. L. (1980) Proc. Natl. Acad. Sci. USA 77, Health Grants 14969 and 15524, the Robert Wood Johnson, Jr., Char- 6805-6809. itable Trust, and the Arthritis Foundation. M. E. H. was supported by 34. Sarmiento, M., Glasebrook, A. L. & Fitch, F. W. (1980) Proc. the Damon Runyon-Walter Winchell Cancer fund; J. L. S. was sup- Natl. Acad. Sci. USA 77, 1111-1115. ported by National Institutes of Health Grant A 1-1566Q. 35. Dunlap, B., Bach, F. H. & Bach, M. L. (1978) Nature (London) 1. Aiuti, F. & Wigzell, H. (1973) Clin. Exp. Immunol. 13, 171-181. 271, 253-255. 2. Brown, G. & Greaves, M. F. (1974) Eur.J. Immunol. 4, 302-310. 36. Michaelson, J., Scheid, M. & Boyse, E. A. (1979) Immunogenet- 3. Owen, F. L. & Fanger, M. W. (1974) J. Immunol. 113, ics 9, 193-197. 1128-1144. 37. Anderson, L. C., Karhi, K. K., Gahmberg, C. G. & Rodt, H. 4. Kung, P. C., Goldstein, G., Reinherz, E. L. & Schlossman, S. (1980) Eur.J. Immunol. 10, 359-362. F. (1979) Science 206, 347-349. Downloaded by guest on September 27, 2021