Patterns of antigenic expression on human monocytes as defined by monoclonal antibodies

CELLULAR

IMMUNOLOGY

78, 83-99 (1983)

Patterns of Antigenic Expression on Human Monocytes as Defined by Monoclonal Antibodies M. A. TALLE, P. E. RAO, E. WESTBERG,N. ALLEGAR, M. MAKOWSKI, R. S. MITTLER, AND G. GOLDSTEIN Immunobiology

Division,

Ortho Pharmaceutical

Corporation,

Raritan, New Jersey 08869

Received November 18, 1982; accepted January 25, 1983

A seriesof seven monoclonal antibodies directed at determinants on human peripheral blood monocytes were produced and characterized. The antibodies were separated into three groups basedon cell distribution and percentagesof monocytes bearing antigen. Hybridoma antibodies, termed OKM l,OKM9, and OKM 10,recognized antigen(s) expressedon the majority of adherent monocytes, null cells, and granulocytes. The second group, comprising OKMS and OKM8, reacted with most adherent monocytes and platelets. OKM3 and OKM6, comprising a third group of antibodies, reacted with a subpopulation of adherent monocytes and platelets. OKM antibodies were not expressed on lymphocytes, thymocytes, and lymphoblastoid cells, with the exception of OKM3 which reacted with three B-cell lines. SDS gels of immunoprecipitates formed with OKM antibodies yielded the following tentative molecular weight results: OKMl and OKM9 antigens appeared to be 160,000(nonreduced) and 170,000(reduced); OKM 10 precipitated two polypeptides of 170,000 and 115,ooO(reduced); OKMS and OKM8 precipitated a single polypeptide of 88,@YJ(reduced, nonreduced); OKM6 antigen appeared to be 116,000 (nonreduced) and 130,000 (reduced). INTRODUCTION

Cells of myelomonocytic lineage perform diverse immunologic functions (1). In vitro, macrophages are necessaryfor the optimal response of T lymphocytes to plant lectins, allogeneic lymphocytes, and various soluble antigens (2-4). Macrophages, by virtue of target-specific Fc receptor-bound Ig, may be indirectly cytotoxic (ADCC) (5) or directly cytotoxic as well as phagocytic (6, 7). Their role in processing and displaying antigen for appropriate T lymphocyte responseshas been extensively documented ( 1) and a suppressor role has recently been demonstrated (8). Functional surface molecules displayed by macrophages, as well as by peripheral blood monocytes capable of differentiating into macrophages, include HLA-DR antigens and receptors for various components of complement and the Fc region of IgG. These markers are not, however, restricted to macrophages or even to cells of myelomonocytic lineage (9, 10). The development of hybridoma technology by Kohler and Milstein (11) has permitted us ( 12, 13) and others ( 14-16) to discriminate functional subsets of T lymphocytes by production of monoclonal antibodies to distinctive surface molecules. 83 ooo8-8749183$3.00 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

84

TALLE ET AL.

Application of this technology has likewise resulted in the identification of molecules restricted to myelomonocytic cells (17-27) or molecules shared by these and cells of other origins (28-32). Previously we have described a monoclonal antibody, termed OKM 1, directed to an antigenic marker common to monocytes, granulocytes, and null cells (17). The wide variety of functions associatedwith cells displaying the OKMI antigen prompted us to searchfor more restricted monocyte markers. In this communication we describe six new monoclonal antibodies reactive with peripheral blood monocytes, one of which, OKM5, in conjunction with OKMl, defines the stimulator population in autologous mixed lymphocyte culture (33). We anticipate that these reagents will aid in the delineation and isolation of additional functional populations of monocytes. MATERIALS

AND METHODS

Production of monoclonal antibodies. Production of OKMl has been described (17). The remaining OKM series are products of a fusion between spleen cells from a CAFl mouse that had been immunized three times intraperitoneally with 2 X 10’ peripheral blood sheep erythrocyte negative (E-) mononuclear cells at 2-week intervals and Sp 2/O-Ag 14 myeloma cells according to the method of Kohler and Milstein (11) and as previously described (12). Hybrids surviving culture in hypoxanthine, aminopterin, thymidine (HAT) selective media, and secreting antibodies reactive with E- but not sheep erythrocyte rosette positive (E+) mononuclear cells by indirect immunofluorescence were cloned twice by limiting dilution. Ascites were prepared by injecting l-2 X IO’ cells from selected clones into pristane primed CAF, mice. Zmmunofluorescenceanalysis. The percentagesof cells expressing antigen reactive with an OKM series antibody were generally determined by indirect immunofluorescence. In brief, saturating levels of OKM ascites (usually a 1:100 dilution) were incubated with IO6cells for 30 min at 4°C in medium containing 0.1% sodium azide, 3 mMethylenediaminetetraacetic acid (EDTA), and 0.1 mg human immunoglobulin/ ml. Cells were washed free of unbound antibody and incubated for 30 min at 4°C with a mixture of fluorescein-conjugated goat anti-mouse IgG and IgM (Meloy Laboratories, Springfield, Va.) diluted in the same medium. After two additional washes, cells (defined as lymphocytes, monocytes, or granulocytes by light scattering properties) were analyzed for fluorescence on a Cytofluorograf FC 200/48OOA (Ortho Diagnostic Systems, Westwood, Mass.). Cells displaying fluorescence intensity above those stained with P3 X 63 Ag 8 ascites (a myeloma producing nonspecific light and heavy chains) were considered positive. To ascertain the reactivity of OKM monoclonal antibodies with peripheral blood B lymphocytes, two color immunofluorescence was employed. Adherent celi depleted E- mononuclear cells were incubated (see above) with a saturating level of a pan B cell reagent, OKB7,’ conjugated to biotin (36). Cells were washed free of unbound antibody, incubated with 25 pg of rhodaminem-avidin-D (Vector Laboratories, Burlingame, Calif.) for 30 min, and washed. Saturating levels of OKM monoclonal ’ Mittler, R. S., Talle,M. A., Carpenter,K., Rao,P. E., and Goldstein,G. 1983.Generationand characterization of monoclonal antibodies reactive with human B lymphocytes. J. Immunol. (submitted for publication).

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antibodies conjugated to fluorescein (37) were added to the cells, incubated as above, and washed free of unbound antibody. Two color flUOre%mCe andySiS was performed on a Cytofluorograf 50-H (Ortho Diagnostic Systems, Westwood, Mass.) interfaced to a model 2150 computer (Ortho Diagnostic Systems, Westwood, Mass.). Immunoglobulin isotype of OKM monoclonals was ascertained by using fluorescein-conjugated goat anti-mouse isotype specific sera (Meloy Laboratories, Springfield, Va.). Competitive immunofluorescence. Fluoresce&conjugated OKMl or OKM8 was titrated against mononuclear cells, and the level staining approximately 75% of the cells expressing antigen was used in competition experiments. Increasing amounts of the competing monoclonal antibodies were incubated with constant levels of tluorescein isothiocyanate (FITC)-OKMl or FITC-OKM8 and mononuclear cells; the cells were washed and analzyed for percentages of stained cells, as above. The antibodies used in these experiments were partially purified by column chromatography on Sephacryl S-200 (Pharmacia, Piscataway, N.J.). Isolation ofperipheral blood components. Fresh peripheral blood collected in heparin was spun on Ficoll-Hypaque density gradients (d = 1.077). The interface mononuclear cells and pelleted granulocytes plus red cells were recovered separately. Lysis of red cells by T&buffered ammonium chloride resulted in a highly enriched granulocyte population (>95% by Wright-Giemsa stain). Mononuclear cells were fractionated into T lymphocytes, null cells, and adherent monocytes. Highly purified T cells (>95% OKT 11A positive) (38) were obtained by rosetting with sheep red blood cells as described by Mendes et al. (39). Surface Ig positive (sIg+) cells were removed from the nonrosetted population by passageover a Sephadex G-200 anti-F(abfi column (35). Column nonadherent cells were treated with OKT3 and complement to eliminate residual T cells (12), and adherent monocytes were removed by incubation on plastic resulting in a highly enriched null cell population. Adherent cells were isolated by incubating 3 X 10’ mononuclear cells or 2 X 10’ E- cells on lOO-mm plastic culture dishes (Coming, Coming, N.Y.) in RPM1 1640 medium (Gibco Laboratories, Grand Island, N.Y.) containing either 10% fetal calf serum (FCS) or 15% heat-inactivated autologous plasma. Plates were stored at 37°C for 60 min and washed free of nonadherent cells with four volumes of serum-sup plemented medium. Adherent cells were treated with serum-free RPM1 at 4°C containing 3 n&f EDTA for 3 min prior to gentle scraping with a rubber-tipped syringe plunger. More than 90% of the recovered cells displayed light scattering properties characteristic of monocytes in the 90” versus forward angle scattergram and less than 5% of the C&S gave positive staining with OKTl 1A (34), rabbit anti-human IgG, and OKB2.’ This population was therefore considered to be highly enriched for adherent monocytes. Viability was >90%. Purified platelets were isolated from blood collected in citrate according to the method of McKean et al. (40). Preparation of lymphoid tissue cells. Thymus was obtained from pediatric patients undergoing cardiac surgery; normal spleen was obtained from patients re&ving kidney tmnspknts; and tonsil was obtained from patients undergoing therapeutic tonsillectomy. OKM antibody reactivity was measured with single cell suspensions prepared from tissues not more than 24 hr after surgery.

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Culture Ofcell lines. T-cell lymphoblastoid lines (HSB-2, CEM, RPMI-8402, 1301, MOLT-3, and MOLT4), B-cell lines (EB3, U266, Raji, B88, and B89), and myeloid lines (HL-60, K-562, and U-937) were maintained in RPM1 medium supplemented with 10% FCS, 2 m&f glutamine, and 50 pg gentamycin/ml (Gibco Laboratories, Grand Island, N.Y.). U-937 was provided by Dr. Yolene Thomas, Columbia University, and the remaining cell lines by Dr. Jun Minowada, Roswell Park Memorial Institute. Proteolytic digestions. Nonrosetting mononuclear cells were treated with 1 mg nonspecific bacterial proteases (Type-XIV) (6 U/mg; Sigma, St. Louis, MO.) per 5 X IO6cells in 1 ml of serum-free RPMI for 30 min at 37OC.Treated and untreated cells were stored at either 4 or 37°C in RPM1 with platelet-free FCS or plateletenriched autologous plasma. Viability of treated and untreated cells was >95% as judged by trypan blue exclusion. Induction of Ok34 antigens on myeloid cell lines. HL-60 and U-937 cells were treated with 1.5 X lob8 12-O-tetradecanoylphorbol 13-acetate(TPA) (P. L. Biochemicals, Milwaukee, Wise.) at lo6 cells/ml for 48 hr or with 1.25% dimethyl sulfoxide (DMSO) (Mallinckrodt, St. Louis, MO.) for 7 days at 5 X lo5 cells/ml. At the end of culture, adherent cells were recovered by scraping with a rubber-tipped syringe plunger in cold RPM13 nut4 EDTA. Preparation of monocytesfor surface iodination. Monocytes for surface iodination were prepared as described by DeBoer et al. (41). Briefly, peripheral blood mononuclear cells (prepared as above) were washed in Hanks’ balanced salt solution (HBSS) and suspended in phosphate-buffered saline (PBS) with 0.38% trisodium citrate and 0.5% human albumin and pelleted. The supematant containing platelets was discarded; however, this did not eliminate platelets completely. Pelleted cells were resuspended in RPM1 supplemented with 0.38% t&odium citrate, 25 n&f Tris-HCl (pH 7.4), and 10% heat-inactivated human AB serum and incubated for 30 min at 37°C. Cells were pelleted again and resuspendedin 1.2 ml of ice cold Ficoll-Hypaque (d = 1.062; 70,000 MW) (Pharmacia, Piscataway, N.J.) in RPM1 with 0.38% trisodium citrate, 25 mM Tris-HCl (pH 7.4) and 1% human albumin and were overlaid with 0.5 ml RPMI-trisodium citrate-Tris-HCl. A monocyte-enriched polulation was recovered from the interface following immediate centrifugation at 2500 t-pm for 10 min at 4°C in a Sorvall RC-3 centrifuge with HL-8 rotor (DuPont Instruments, New-town, Conn.). Cell surface iodination and extraction. Monocytes, granulocytes, or E- mononuclear cells were suspendedin PBS at 10scells/200 ~1and iodinated with 1 mCi Na12’I (New England Nuclear, Boston, Mass.) using enzymobeads (Bio-Rad, Richmond, Calif.) as described by the manufacturer. Iodinated cells were freed of unincorporated iodine by three washes of PBS with 5 mM NaI and were then extracted for 15 min on ice in 10 mM Tris acetate (pH 8.0), 0.5% nonidet P40 (NP40), 2 mh4 phenylmethylsufonylfluoride (PMSF), 50 ti iodoacetamide, and pepstatin A (1 rg/ml) at 2.5 X 10’ cells/ml. Extracts were cleared of nuclei and cell debris by spinning at 40,000 rpm for 45 min in a 50.3 Ti rotor using an LS-80 ultracentrifuge (Beckman Instruments, Palo Alto, Calif). Immunoprecipitation. For the determination of reduced molecular weights, aggregates of sheep anti-mouse (SHAM) immunoglobulin (Ig) and mouse monoclonal

MONOCLONAL

ANTIBODIES TO HUMAN

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87

antibody were used, whereas SHAM-conjugated Sepharose 4B beads reacted with mouse monoclonal antibody were used in determination of nonreduced molecular weights. Prior to immunoprecipitation with hybridoma antibodies, cell extracts were cleared of nonspecifically adhering materials by three 1 hr adsorptions to either aggregatesof SHAM and normal mouse IgG (Cappel Laboratories, West Chester, Penn.) or to SHAM-conjugated Sepharose4B beads previously reacted with normal mouse IgG. Cleared cell extracts were reacted with aggregatesor beads prepared with hybridoma antibodies for 2 hr at 4°C with constant mixing. Aggregatesor beads were pelleted in an Eppendorf centrifuge (Brinkman Instruments, Westbury, N.Y.) and the supernatants removed. Nonspecifically adsorbed material was removed by four washesof the pellet in 10 mJ4Tris acetatebuffer (pH 8.0), 0.2% NP40, 5 mMEDTA, and 0.15 M NaCI. Immunoprecipitates were stored at -20°C until sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis. SDS-PAGE. Immunoprecipitates were dissociated in 25-50 ~1of sample buffer as described by Laemmli (42). In brief, samples were immersed in boiling water for 23 min prior to being loaded onto SDS-polyacrylamide gels, Gels were stained and dried, and autoradiography was performed at -70°C using Kodak XAR film (Eastman Kodak, Rochester, N.Y.) and Cronex lightning plus enhancing screens(DuPont, Wilmington, Del.) (43) to detect precipitated ‘*%ontaining protein bands. RESULTS Antigen Distribution on Peripheral Blood Components A series of seven monoclonal antibodies reactive with human peripheral blood adherent monocytes has been developed. Basedon percentagesof adherent monocytes stained and fluorescence profiles of antigen intensity, the antibodies were divided into three groups for study (Fig. 1 and Table 1). The distribution of antigens recognized by the monoclonal antibodies designated OKM9 and OKM 10 in populations of periphera1 blood cells cIosely resembled that of antigens bound by OKM 1, a monoclonal antibody previously shown to react with

FLUORESCENCE

INTENSITY

I%. I. Immunofluorescence profiles of peripheral blood adherent monocytes with OKM series monoclonal antibodies. Background fluorescencestaining (solid line) was obtained by incubating c&s with as&es (1: 100) from the Ig producing myeloma P3X63Ag8. (A) - - - OKM9; - 0 - OK&I1 and OKMIO; (B)---OKMS; -O---KM&(C)---OKM6;-•-OKM3.

88

TALLE ET AL. TABLE 1 Percentagesof Peripheral Blood Cells Stained by Indirect Immunofluorescence” Adherent monoeytes (n = 7)

OKMI

oKM9 OKMlO

OKMS

87k 83k 87k

7 3 4

(“E,‘3)

oKB7+* (n = 3)

10 + 8

9+2

8+4 6_+4

10 + 8 14 2 3

121

OKM8

70-+ 18 74+ 18

3+2 323

2+1

oKM3 OKhS6

36k 17 27 + 12

2+2 422

I k 1c I+1

Null (n = 4) 72+ 63+ 75*

6

8 4

GWUdOCytes (n = 4) 96k 3 89 f 12 91 f 13

5-+ 4

Platelets (n = 2) 5f

I

7f 4 7_+ 3

12* II 12 * 12

4*

2

63k 78f

7_+ 7 6+ I

5*

4+

3 2

85k 4 82 f 11

4 3

k subclass w2 WI Mb

W, kG w kG

a Mean z!zSD. b E- adherent cell depleted mononuckar cells were stained with biotinylated OKB7, a pan B cell reagent followed by rhodamine-avidin as described under Materials and Methods. Cells were counterstained with fluoresceinated OKM monoclonal antibodies. The percentagesof cells bearing OKB7 antigen and an OKM antigen are reported. ’ Cells were sequentially incubated with OKM3, FITC-goat anti-mouse IgM, biotinylated OKB7, and rhodamine-avidin. This procedure was necessarysince attempts to fluoresozinate OKM3 (IgM &type) were unsuccessfuland cells precoated with rhodamine-avidin bound OKM3 nonspecifically.

most adherent and some nonadherent mononuclear cells (17). The OKMl positive population is known to contain cells necessary for presentation in soluble antigeninduced proliferation as well as a large fraction of the Ty cell population (17, 44). OKM9 and OKMlO, like OKMl, recognize antigen(s) expressed on almost all adherent mononuclear cells, most null cells and granulocytes, but not expressed on most T or B lymphocytes or platelets (Table 1). The density of OKM9 antigen on adherent monocytes is lower than that of OKMl and OKMlO as judged by fluorescence intensity (Fig. 1). Two monoclonal antibodies, termed OKM5 and OKMS, react with most adherent monocytes but not with T cells or B cells. They are clearly distinguished from the members of the OKMl group on the basis of their reactivity with platelets and lack of reactivity with granulocytes or most null cells. The intensity with which OKM8 stains adherent monocytes is consistently higher than OKM5, suggestinga difference in the antigenic molecule or epitope. A third group of monoclonal antibodies, OKM3 and OKM6, are reactive with only a fraction of adherent monocytes and nonreactive with all T and B lymphocytes, null cells, and granulocytes. Both OKM3 and OKM6 stain platelets equally well, but OKM3 appears to react with a population of adherent monocytes somewhat larger than that recognized by OKM6 (36% + 17 vs 27% + 12); in addition, the two antibodies yield slightly different fluorescence profiles. In view of the striking similarities in the distributions of antigens in peripheral blood recognized by these three groups of antibodies (Table I), additional cellular distribution studies with lymphoid tissues and established cell lines were performed in an attempt to distinguish antigens recognized by the antibodies of each group.

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Antigen Expression on Cells from Lymphoid Tissues Single-cell suspensions of thymus, spleen, and tonsil were prepared and the entire mononuclear cell population was examined for the presence of OKM antigens by indirect immunofluorescence. As illustrated in Table 2, the OKM antibodies reacted with very few thymus cells. OKMl, OKM9, and OKMlO labeled only 5-20% of the mononuclear cells from spleen and tonsil, whereas OKM5, OKM8, OKM3, and OKM6 reacted with even fewer cells from these organs. Most of these OKM reactive cells were restricted to the monocyte cluster as defined by the forward angle versus 90” angle light scatter cytogram (data not shown). Antigen Expression on Myeioid and Lymphoid Cell Lines Several myeloid and lymphoid cell lines were examined for OKM antigen expression. OKM 1, OKM9, and OKMlO were slightly reactive with K-562, a possible erythroid precursor line derived from a patient with chronic myelogenous leukemia (45), were less reactive with U-937, a monoblastic cell line derived from a histiocytic lymphoma (46, 47), and were not reactive with HL-60, a promyelocyte line (48) established from a patient with acute promyelomonocytic leukemia (49) (Table 3). OKM5,OKM8, and OKM6 antigens were not expressedon any of the myeloid lines tested, however, a low level of OKM3 antigen was present on K-562. Six T-cell lines were uniformly unreactive with the OKM series, and five B-cell lines were negative for all but OKM3 antibody. OKM3, the only IgM subclass antibody of the OKM series (Table 1) stained 15-20% of B-cell lines EB3, B88, and B89, further distinguishing OKM3 from OKM6. It is unlikely that the OKM3 reactivity with B-cell lines is due to Fc receptor binding since aggregatedhuman immunoglobulin is present throughout the staining procedure, and OKM3 reactivity did not correlate with expression of Fc receptors by the cell lines (50).

TABLE 2 Antigen Expression on Cells from Lymphoid Tissues” Thymus (n = 3)

Spleen (n = 9)

Tonsil (n = 4)

OKMI ow9 OKMIO

422 6+5 5r2

13 k 6 10 + 5 14 + 7

11 +4 11+3 12 2 4

oKM5 OKM8

4-c3 321

7k5 8-t7

6+2 721

oKM3 OKM6

Sk-3 2-cl

91+4 Sk5

722 8t_2

a Percentagepositive cells by indirect immunofluorescence (mean f SD); Cytofluorograf gated on lymphocyte + monocyte cluster.

90

TALLE ET AL. TABLE 3 Reactivity with Myeloid and Lymphoid Cell Lines Myeloid lines” K562

HL-60

u-937

T-cell lines*

B-cell lines’

oKM9 OKMlO

25 11 17

2 2 2

9 3 11

NEG NEG NEG

NEG NEG NEG

oKM5 OKM8

2 2

1 1

1 1

NEG NEG

NEG NEG

oKM3 OKM6

6 2

1 1

1 1

NEG NEG

&3/Y NEG

OKMI

’ Percentage positive cells by indirect immunofluorescence. * MOLT-3, MOLT-4, 1301, 8402, CEM, HSB-2. ‘EB3, U266, B88, B89, Raji. d Twenty percent positive or less on EB3, B88, and B89.

Efect of Proteases on Ok34 Antigen Expression A monocyte-enriched cell population was treated with nonspecific bacterial proteases (Sigma-Type XIV) to detect differences in antigen sensitivity to proteolytic enzymes and to determine if OKM5, OKM8, OKM3, and OKM6 reactivity with monocytes was due exclusively to the presence of contaminating adherent platelets. Protease-treatedcells were stored overnight at either 4 or 37°C to respectively prevent or permit antigen re-expression; untreated control cells were also incubated at these temperatures. The results in Table 4 (representing four similar experiments) demTABLE 4 Antigen Expression following Protease Treatment” Treated*

Untreated 37°C

4OC

4°C

37°C

OKMI oKM9 OKMIO

67 67 84

87 84 91

92 57 95

75 45 84

oKM5 OKM8

71 80

84 85

6 12

40 71

oKM3 OKM6

55 32

18 14

3 2

59 24

e Percentageswere determined by flow cytometric analysis (monocyte cluster) of cells labeled by indirect immunofluorescence. * E- peripheral blood leukocytes were treated with sigma-Type XIV nonspecific bacterial protease ( 1 mg/ ml/5 X lo6 cells) for 30 min at 37°C and stored in platelet-free medium at either 37 or 4°C for 18 hr prior to analysis.

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onstrate complete loss of OKM5, OKM8, OKM3, and OKM6 and partial loss of OKM9 antigen expression upon protease treatment. After 18 hr at 37°C expression of OKM5, OKM8, OKM3, and OKM6 antigens returned to levels approximating those of untreated cells. The data presented in Table 4 were obtained from cells stored in medium supplemented with 10% FCS which had been passedthrough a 0.20~pm filter and then examined microscopically to verify the absence of platelets. In a separate experiment protease-treated monocytes were stored at 37°C in medium containing either 10% platelet-free fetal calf serum or 15% platelet-enriched autologous plasma. Cells stored in platelet-enriched medium were at most 5% more positive with platelet-reactive antibodies than those stored in platelet-free medium (data not shown). In addition, purified platelets were unable to regenerate OKhQOKM8, OKM3, and OKM6 following protease treatment. Induction of Antigen Expression on Myeloid Cell Lines Further evidence for the monocyte nature of the markers recognized by the OKM series was provided by experiments employing the differentiation-inducing agents DMSO and the phorbol diester TPA on myeloid cell lines. HL-60, the promyelocyte line, acquired either monocyte or granulocyte characteristics when treated with TPA or DMSO, respectively (48, 51). Incubation of HL-60 and U-937 with either 1.5 X 10e8M TPA for 48 hr or 1.25% DMSO for 7 days resulted in a population containing greater than 75% adherent cells. Nonadherent cells were discarded and the remaining cells were recovered in cold RPMI-3 mit4 EDTA by gentle scraping with a rubber-tipped syringe plunger. Viability was greater than 80%. As shown in Table 5, both TPA and DMSO were capable of inducing expression of the OKM markers shared by monocytes and granulocytes (i.e., OKMl, OKM9, and OKMlO) on both cell lines. The remaining OKM antigens did not appear on HL-60 even after treatment with TPA. This may suggest that TPA-induced differentiation of HL-60 to monocytes is incomplete. OKM5, OKM8, and OKM3 were,

TABLE 5 Induction of Antigen Expression on HL-60 and U-937 Cells by Phorbol Diester and Dimethyl Sulfoxide” HL-60

u-937

Control

TPA

DMSO

Control

TPA

DMSO

OKMl oKM9 OKMlO

3 2 3

69 57 68

22 17 20

10 4 10

36 26 37

71 47 74

OKM5 0KM8

1 1

2 2

0 0

3 4

23 32

10 11

oKM3 OKM6

I 0

7 0

0 0

0 0

18 5

4 5

’ Percentagepositive cells by indirect immunotluorescence.

92

TALLE ET AL.

however, induced on U-937, a line thought to be committed exclusively to the monocyte/macrophage differentiation pathway (46, 47).

Molecular Weight Determinations Molecular sizesof antigens recognized by OICM seriesantibodies were determined by SDS-PAGE of immunoprecipitates obtained from nonionic detergent extracts of radioiodinated monocytes, granulocytes, or E rosette negative cells. Prior to the addition of OKM antibodies, the cell extracts were freed of nonspecifically precipitating material by incubating with SHAM-normal mouse IgG aggregates.The washed pellet obtained by three cycles of nonspecific adsorption was reduced and electrophoresed in parallel with material specifically precipitated from the cleared cell extract by hybridoma antibodies complexed to SHAM. SHAM coupled to Sepharose 4B was substituted for SHAM in preparation of both nonspecific and specific immunoprecipitates used in determining nonreduced molecular weights. As illustrated in Figs. 2 and 3, both OKMl and OKM9 appeared to precipitate proteins of 170,000 da (reduced) and 160,000 da (nonreduced), thus distinguishing them from OKMIO, which precipitated polypeptides of 115,000 and 170,000 da (reduced) and 125,000 and 160,000 da (nonreduced). If either OKM5 or OKM8 was the immunoprecipitating agent, a band appeared at 88,000 da under reducing and nonreducing conditions suggestingthat theseOKM antibodies, like OKMl and OKM9,

12

45

3 200

7

6

8

9

10

200

116.3 92.5 67

116.3 92.5

30

20.1 14.4

FIG. 2. Reduced molecular weights of antigensrecognized by OKM antibodies.‘251-labeledsurface proteinsfromhumanperipheralbloodmonocytes, granulocytes, or E- mononuclear cells were extracted with NP40 asdescribed underMaterialsandMethods. Extracts were cleared of nonspecifically adhering material with preformed aggregatesof sheep anti-mouse IgG and normal mouse IgG (lanes 1, 4, 7, 9). Specific antigens were then precipitated using aggregatesof sheep anti-mouse IgG and the monoclonal antibodies OKMI (lane 2) and OKM9 (lane 3) from granulocyte extract, OKM5 (lane 5), 0-8 (lane 6), and OKM6 (lane 8) from monocyte extract and OKMIO (lane 10) from E- mononuclear cell extract. Aggregateswere dissolved in Laemmli sample buffer and 2-mercaptoethanol was added to a concentration of 10%. Samples were loaded onto 8-158 (lanes 1-6, 9, 10) or 8% (lanes 7, 8) polyacrylamide gels. Autoradiographs from dried gels were exposed for 1-3 weeks at -70°C using an enhancing screen.

MONOCLONAL

ANTIBODIES TO HUMAN

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93

266

ll6.3

116.3 92.1

92.6 67 67 46 45

FIG. 3. Nomeduced molecular weights of antigens recognized by OKM antibodies. ‘2sI-labeled surface proteins from human peripheral blood monocytes, granulocytes, or E- mononuclear cells were extracted with NP40 as described under Materials and Methods Extracts were cleared of nonspecifically adhering material by absorptions with aggregatesof SHAM-conjugated Se&rose 4B beads previously reacted with normal mouse IgG (lanes 2,4, 7, 9). Specific antigens were then precipitated using aggregatesof SHAMconjugated sepharose4B beads and the monoclonal antibodies OKMl (lane 1) and OKM9 (lane 3) from gmnulocyte extract, OKM5 (lane 5). OKM8 (lane 6), and OKM6 (lane 8) from monocyte extract, and OKM 10 (lane 10) from E- mononuclear cell extract. Precipitated material was dissolved in sample buffer and iodacetamide was added to 20 fl. Umeduced samples were loaded onto 7% (lanes l-6), 8% (lanes 7, 8), or 8- 15% gradient polyacrylamide gels (Lanes9, 10). Autoradiographs of dried gels were produced by exposing to Kodak X-OMAT-AR film at -70°C with an enhancing screen from 1 to 3 weeks.

may recognize common molecules (Figs. 2 and 3). An apparent molecular weight of 116,000 (nonreduced) or 130,000 (reduced) was determined for antigen recognized by OKM6. At present we have not successfully precipitated the OKM3 antigen. Cross-Immunoprecipitates Because antigen size and cellular distribution profiles were unable to distinguish OKMl from OKM9 and OKM5 from OKM8, cross-immunoprecipitations were performed to determine if antigens of similar size were in fact the same molecule. Radioiodinated granulocyte extracts cleared with SHAM-normal mouse IgG complexes were subjected to four sequential precipitations with OKM9 before reacting the residual material with OKM 1. As shown in Fig. 4A, no OKM 1 reactive antigen was precipitated (lane 7) after the fourth precipitation with OKM9, indicating that OKMl and OKM9 recognize the same molecule. In the converse experiment however, some OKM 1 reactive material was still present (lanes 2, 3) and a small amount of OKM9 binding material remained (lane 4) by the fourth precipitation with OKM 1. The inability of OKMl to remove all OKM9 precipitable material was probably due to an affinity difference as has been suggestedfor the two directional cross-precipitates of OKT3 with Leu 4 (52). In a similar experiment, OKMS antigen from a radioiodinated monocyte extract was exhaustively precipitated prior to cross-precipitation with OKM5. The bands

94

TALLE ET AL.

A 1234567

200

12

3

A

-

116.392.5-

45 -

FIG.4. Cross-precipitation of OKM 1 and OKM9 antigens and OKM5 and OKM8 antigens. ‘251-labeled granulocyte extracts (OKMI and OKM9) or monocyte extracts (OKM5 and OKM8) were precipitated as described using normal mouse IgC (A lane 1, B lane 4). Antigen was precipitated repeatedly with one monoclonal antibody and a precipitation was then made with a second monoclonal antibody. Precipitates were dissolved in sample buffer and run on 7% (A) or 8% (B) polyacrylamide gels. Dried gels were exposed to Kodak X-OMAT-AR film at -70°C with an enhancing screen from 1 to 3 weeks. (A) Lane 2, first precipitation with OKM 1; lane 3, fourth precipitation with OKM 1; lane 4, precipitation with OKM9 from same aliquot; lane 5, first precipitation with OKM9; lane 6, fourth precipitation with OKM9; lane 7, precipitation with OKMI from same aliquot. (B) Lane 3, first precipitation with OKM8; lane 2, fourth precipitation with OKM8; lane l,OKM5 antigen precipitated from same aliquot. OKM6 antigen was then precipitated from the same aliquot as a control for the condition of antigens in the extracts after handling (data not shown).

appearing at 88,000 da (Fig. 4B) represent the first and fourth immunoprecipitates with OKM8 (lanes 3 and 2, respectively). OKM5 did not precipitate any additional material (lane l), demonstrating recognition of a common molecule by OKM5 and OISM8. Epitope Competition Competition experiments were performed to determine if the reactivities of OKM 1 and OKM9 were directed toward distinct epitopes on a common molecule. A fixed level of FITC-OKMl was analyzed for its ability to stain monocytes in the presence of increasing amounts of competing monoclonal antibody (Fig. 5a). OKM 1 was able to compete with FITC-OKMI, whereas there was no displacement of FITC-OKMI by up to 10 rg of OKM9 or OISM5. It was concluded, therefore, that OKMl and OKM9 recognize discrete epitopes on the same molecule. In a similar experiment comparing OKM5 and OKM8 (Fig. 5b), OKM5 was shown to displace OKM8 as effectively as OKM8 competed with itself. This suggeststhat OKM5 recognizes an epitope identical to or in close proximity to the epitope reactive with OKM8.

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lmmunotluorescent Competition for OKMl (a) and OKMBIb) 100 J

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+ OKMS

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.-.

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x 5 f .A = Y

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FIG. 5. (a) Ability of OKM monoclonal antibodies to compete for FITC-OKMl (0.25 g, approximately 75% saturation). 0 - - - 0, OKMl; 0 - - - 0, OKM9; A - - - A, OKM5. (Details are provided under Materials and Methods.) (b) Ability of OKM monoclonals to compete for FITC-OKMB (0.05 ~g, approximately 75% saturation). 0 - - - 0, OKMS; 0 - - l ,OKM5; A - - - A, OKM9.

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TALLE ET AL.

DISCUSSION We have obtained seven monoclonal antibodies which identify at least six separate determinants present on human peripheral blood monocytes. Three antigens, defined by indirect immunofluorescent staining with OKMl, OKM9, and OKMlO were expressed on granulocytes and null cells in addition to monocytes but were absent from platelets and the majority of T and B lymphocytes. Reactivity of OKMl, OKMg, and OKM 10 with single cell suspensionsof thymus, spleen, and tonsil were primarily restricted to cells possessingmonocyte light scattering properties, and the percentages of cells displaying antigen within these populations were consistent with the expected number of monocytes in these tissues. Polyacrylamide gel electrophoresis of OKM 1 and OKM9 immunoprecipitable material revealed a single protein band of 160,000 da (nonreduced) or 170,000 da (reduced) (Figs. 2 and 3). OKMl did not precipitate any additional protein from granulocyte extracts which had been exhaustively reacted with OKM9, indicating that OKMl and OKM9 recognize the same molecule (Fig. 4A). In the converse experiment, however, OKMl was unable to remove all OKM9 precipitable material suggesting perhaps the OKM9 interaction with antigen is of higher affinity. In competition experiments, unlabeled OKM9 was unable to displace fluorescein-conjugated OKM 1 (Fig. Sa). We concluded, therefore, that OKMl and OKM9 discriminate epitopes present on a common molecule. OKMlO, although recognizing an antigenic determinant with cellular distribution patterns identical to those of OKMl and OKM9, differs from the latter antibodies with respect to the size of the antigen it binds since OKMlO precipitates two ‘*$ labeled polypeptides of 115,000 and 170,000 da (reducing conditions) (Fig. 2). The relationship of the 170,000-da protein precipitated by OKM 10 and to the 170,000da protein precipitated by OKMl and OKM9 has not been determined, however, no 115,000-da precipitable material was ever obtained with OKMI or OKM9. Material precipitated by OKMl and OKM9 in the 120,000 molecular weight range was also present in the nonimmune precipitates, and, therefore, does not represent antigen. Other monoclonal antibodies defining antigens with cellular distribution patterns similar to OKM 1, OKM9, and OKM 10 have been described. B43.4, described by Perussia et al. (2 l), was reported not to compete with OKM 1, however, similar studies with OKM9 and OKMlO are necessary. The molecular weight(s) of the antigen(s) recognized by B43.4 have not been published. Anti-MO1 and OKMl (Todd et al.) (22, 53) were reported to recognize glycoproteins on monocytes consisting of two subunits of 94,000 and 155,000 da. We were unable to detect any specifically precipitated protein in the 94,000-da range with OKMl or OKM9, however the nonimmune precipitate (Fig. 2, lane 1) contained 94,OOO-damaterial which may be masking specific antigen. In addition, granulocytes rather than monocytes were the antigen source for our immunoprecipitations with OKMl and OKM9. Two additional monoclonal antibodies described herein, OKM5 and OKMS, define antigenic determinant(s) that were more restricted in their distribution. Within the peripheral blood, only monocytes and platelets expressed the OKM5 and OKM8 reactive antigen, and within lymphoid tissues lessthan 15%of single cell suspensions were stained with OKM5 or OKM8 (Tables 1 and 2).

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TO HUMAN

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Although similar percentages of cells were recognized by OKM5 and OKM8, the latter consistently stained adherent monocytes more intensely than the former at srtturing levels of antibody (Fig. 1), suggesting that the antibodies were identifying either distinct molecules or epitopes. Polyacrylamide gel electrophoresis of O&%5 and 0~~8 immunoprecipitates revealed, however, reactivity wtih a common 88,OOOda molecule (Pi. 4B), and competition experiments showed that unlabeled OKMS competed as effectively for fluorescein-conjugated 0048 reactive antigen as unlabeled OKM8 (Fig. 5b). OKM5 and OKM8, therefore, recognize epitopes that are either identical or in close proximity. One possible explanation to account for the difference in fluorescence intensity could be a higher affinity of the fluorescein-conjugated goat anti-mouse sera for OKM8. Previously described monoclonal antibodies MY3 (23) and B44.1 (21) differed from OKM5 and OKMS in that they do not react with platelets. 63D3 ( 18) recognizes a 200,OOOdamolecule and weakly stains granulocytes, thus distinguishing it from OKM5 and OKM8. Anderson et al. (26) reported a monocyte-specific monoclonal antibody that precipitates two polypeptides with apparent molecular weights of 90,000 and 100,000 (major and minor bands, respectively). Although we have not detected any lOO,OWda precipitable material with OKMS and OKMS, these three antibodies may recognize a common molecule. Reactivity of the antibody described by Anderson et al. (26) with platelets was not reported. Anti-Mo4 described by Todd et al. (22) was indistinguishable from OKM5 and OKM8 from the published data; however, biochemical characterization was not presented for anti-Mo4. OKM3 and OICM6 appeared to identify monocyte subsets. Fluorescence profiles of adherent monocytes stained with OKM3 and OKM6 revealed a discrete population of cells expressing high-density antigen; however, it is possible that the remaining population of cells possessesa small amount of antigen. OKM3 can be distinguished from OKM6 becauseit identified a slightly larger population of monoqtes, and the antigen it recognized was weakly expressed on three out of five B lymphoblastoid cell lines tested. OKM6 precipitated a single polypeptide of 130,000 da under reducing conditions and 116,OOO da nonreduced (Figs. 2 and 3). Raff et al. (54) have described a monoclonal antibody, termed Mac-120, which identified a subset of human macrophages necessary for soluble antigen induced proliferation of peripheral blood mononuclear cells. In addition, the Mac-120+ cell can function as a stimulator cell in autologous mixed lymphocyte cultures. Cellular distribution and molecular weight of the Mac- 120 reactive antigen ( 120,000) appeared similar to that of OKM6. Because several of the OKM antibodies bound to platelets, it was necessary to determine if their reactivity with monocytes was an artifact due to platelet adherence (55). Data presented in Table 4 demonstrated that OKM5, OKM8, OKM3, and OKM6 antigens were sensitive to nonspecific bacterial proteases and could be regenerated by monocytes in the presence of platelet-free medium. Interestingly, the degree of expression of various antigens on untreated cells (Table 4) differed depending on the storage temperature. Induction experiments using the promyelocytic line HL-60 and the monoblastic line U-937 were performed to establish if the OKM reactive antigens could be detected on those lines following activation. TPA induced expression ofOKM5,0~8, and OKM3 antigens on U-937 (Table 5), providing additional evidence that these antigens were products of mono&c lineage cells.

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While the functions of the OICM reactive antigens themselves have not been established, a small subset of monocytes identified as OKMl-5+ has been shown to contain the autologous mixed lymphocyte culture stimulator cell (56). PerhapsOKM6 or OKM3 will distinguish additional functional subsetsas Mac- 120 has been reported to do. These various monoclonal antibodies reacting with cell surface molecules of monocytes and other cells should provide valuable reagents for furthering our knowledge of the functions of these cells; furthermore, they may be of clinical utility in following changes of the cells they enumerate in health and disease. ACKNOWLEDGMENTS The authors would like to thank Mr. Rich Look for performing the two color immunofluorescence analysis, Ms. Marilyn Sanders for her help in editing the manuscript, and Ms. Nancy Geshner in typing the manuscript.

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