KEVIN L. HOLMES*, ROGER G. E. PALFREEt, ULRICH HAMMERLINGt, AND HERBERT C. MORSE, III*. *Laboratory of Immunopathology, National Institute of ...
Proc. Natl. Acad. Sci. USA Vol. 82, pp. 7706-7710, November 1985 Immunology
Alleles of the Ly-17 alloantigen define polymorphisms of the murine IgG Fc receptor (flow microfluorometry/immunoprecipitation)
KEVIN L. HOLMES*, ROGER G. E. PALFREEt, ULRICH HAMMERLINGt, AND HERBERT C. MORSE, III* *Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20205; and tMemorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021
Communicated by Edward A. Boyse, July 18, 1985
ABSTRACT Antibodies specific for allelic determinants of the cell membrane alloantigen Ly-17 were found to react with genetically determined polymorphic sites on the murine IgG Fc receptor (FcyR). Monoclonal antibodies specific for Ly-17.2 and the FcyR detected identical populations of cells in thymus, spleen, lymph node, and bone marrow and had nearly identical reactivity with a large number of hematopoietic neoplasms. Antibodies to the FcyR and either allele of Ly-17 blocked binding of rabbit IgG dimers to the FcyR on spleen and bone marrow cells. Antibodies to the FcyR also blocked binding of antibodies to, either allele of Ly-17, whereas anti-Ly-17 antibodies only partially blocked the binding of antibodies to the FcyR. Biochemical studies showed that antibodies to Ly-17 and the FcyR precipitated proteins of Mr 55,000-60,000. The identity of the proteins recognized by these antibodies was confirmed by sequential immunoprecipitation. The Ly-J 7 locus on the distal end of mouse chromosome 1 has two alleles that determine expression of the cell membrane alloantigens, Ly-17.1 and Ly-17.2,f recognized by allo- and monoclonal antibodies, respectively (1-4). This locus is part of a cluster of genes that control the expression of cell-surface antigens [Ly-9, Ly-17, Ly-22, H-25, Mls, and Ly M (2-9)] or that are involved in the transmission of murine retroviruses [Bxv-J, Rmc-J, Mtv-7, Mtv-10 (4, 10-13)]. Two other loci mapped to this region [gid and Ctl-J (4, 14-16)] control the expression of autoimmune disorders in several strains of
mice. Although many of these loci have been found to be genetically discrete and functionally unrelated (2-16), Ly-17 is genetically inseparable from the Mls locus (2, 8) which controls the expression of cell surface antigens that stimulate mixed lymphocyte reactions in H-2-compatible strains of mice (8, 9). The possibility that Ly-17.1 and Ly-17.2 are cell surface antigens related to Mls has been examined by several approaches. Recently, Ly-17.2 has been characterized biochemically as a monomeric glycoprotein ofMr 55,000-60,000 with no detectable intramolecular disulfide bonds (unpublished results). We now report that antibodies to Ly-17.1 and Ly-17.2 detect genetically determined polymorphisms of the murine IgG Fc receptor (FcyR).
K9.361; ref. 2), to the FcyR (clone 2.4G2; ref. 19), and to 3-nitro-4-hydroxy-5-iodophenyl (clone J-1.2); with mouse alloantisera to Ly-17.1 (1); and with a FITC-labeled goat anti-mouse K light chain (Southern Biotechnology Associates, Birmingham, AL). Cells were also reacted with FITClabeled monoclonal antibodies to Thy-1.2, Mac-i, Lyb-2.1, ThB, Ly-5, Ly-5(B220), Ly-i, and Ly-2 or with unlabeled rat antibodies to Ly-24 and L3T4 or mouse antibodies to Tla as described (17, 20-22). FITC-labeled goat anti-mouse IgG (Southern Biotechnology Associates) and FITC-labeled rabbit anti-rat IgG (Zymed Laboratories, San Francisco, CA) were used for detection of binding of unlabeled antibodies. Cells were analyzed on a fluorescence-activated cell sorter (FACS II, Becton Dickinson) equipped with argon and krypton lasers, in which nonviable cells were electronically gated from analysis by both forward light-scattering and uptake of propidium iodide (23). Two-color analysis was performed using FITC-labeled anti-Ly-17.2 (K9.361) and biotin-labeled anti-Ly-5(B220) (clone RA3-6B2; refs. 17 and 24) or anti-Mac-1 (clone Mi/70; ref. 25), prepared in our laboratory, and Texas red-labeled (26) avidin (Cappel Laboratories, Malvern, PA). Nonviable cells were electronically gated from analysis by forward light-scattering alone. Blocking antibodies, used at concentrations twice that required for saturation, were incubated with cells for 20 min at 40C, in the presence of 0. 1% sodium azide. Cells then were washed twice before incubation with antibodies for specific staining. Cell Surface Radioiodination. Mouse spleen cells or tissue
culture cells were washed twice in cold Earle's balanced salts solution and once with cold phosphate-buffered saline (PBS: 150 mM NaCl/2.8 mM NaH2PO4/7.2 mM Na2HPO4, pH 7.2). A cell count was then performed, and only those cell preparations with >90% viability (trypan blue exclusion) were used. In general, 5 x 107 tumor cells in 0.5 ml of PBS were radiolabeled with 1 mCi (1 Ci = 37 GBq) of Na'25i
(Amersham).
The solid-phase reagent, Iodobeads (Pierce), was used to radiolabel cells according to the procedure described by the manufacturer. Briefly, prewashed Iodobeads (2 or 3 per reaction mixture) were added to the cell suspension with Na125I and the reaction was allowed to proceed at room temperature for 20 min. After this incubation, the beads were removed, and radiolabeled cells were harvested and washed twice with cold PBS. Cell Lysate Preparation. Radioiodinated cells were solubilized in 1 ml of lysis buffer (0.02 M Tris HCl, pH 7.4/0.1 M NaCl, 0.5% Nonidet P-40/0.5% deoxycholate (Sigma)/1% (vol/vol) Trasylol (FBA Pharmaceuticals, New York)/10
MATERIALS AND METHODS Flow Microfluorometry. Analyses were performed on single-cell suspensions of spleen, lymph node, and bone marrow from 3-month-old C57BL/6N or NFS/N mice (obtained from the small animal section, National Institutes of Health) or on primary tumors and continuous cell lines as described (17, 18). Cells were reacted with fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies to Ly-17.2 (clone
Abbreviations: sIg, surface immunoglobulin; FcyR, IgG Fc receptor;
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
FITC, fluorescein isothiocyanate. *Note that the provisional designation Lym-20.2 for the alloantigen described by Kimura et al. (2) has been changed to Ly-17.
7706
Immunology: Holmes et A mM iodoacetamide/1 mM phenylmethylsulfonyl fluoride (Sigma) by incubation at 40C for 20 min, after which cell nuclei and debris were removed by centrifugation and the supernatant was clarified further by spinning in a Microfuge (15.000 x g, 4 min). Immunoprecipitation. Before immunoprecipitation, cell lysates were incubated with rabbit anti-mouse IgM in the presence of Pansorbin (Calbiochem-Behring). The lysates were cleared by centrifugation at 12,000 x g for 5 min, and the supernatants were cleared again after addition of rabbit anti-rat IgG-coated, protein A-Sepharose beads. Precipitation was done by incubating 0.1 ml of lysate either with 2-5 pkl of monoclonal antibody or without antibody for 30 min at 40C. To collect the antibody-antigen complexes, washed protein A-Sepharose beads (Pharmacia) were added and incubation was continued for 40 min at 40C. (When the rat antibody 2.4G2 was used for immunoprecipitation, the protein A-Sepharose beads were precoated with rabbit antirat IgG.) The protein A-Sepharose beads with the bound immunoglobulins then were collected by centrifugation and washed 4-6 times with 0.05 M Tris-HCl, pH 7.4/0.15 M NaCl/5 mM EDTA/0.1% Nonidet P-40/fat-free bovine serum albumin (1 mg/ml; Sigma)/i% Trasylol/1 mM phenylmethylsulfonyl fluoride and then twice with the same buffer but without albumin. Polyacrylamide Gel Electrophoresis. NaDodSO4/PAGE was performed according to the procedure of Laemmli (27) using a 12 cm long, 10% acrylamide slab as separation gel. Radiolabeled antigen was released from protein A-Sepharose beads by heating for 3-5 min at 90°C in 50 ,u1 of sample buffer [0.06 M Tris-HCl, pH 6.8/3% NaDodSO4 (Bio-Rad)/4% (vol/vol) glycerol; note that disulfides were not reducpd]. After electrophoresis, gels were stained with Coomassie blue R-250 (Bio-Rad), destained, dried, and exposed to x-ray film in the presence of a Cronex intensifying screen (Dupont). Affinity Purification of Lyl7.2. K36 tumor cell lysate§ were prepared as for immunoprepipitation and passed through Affi-Gel (Bio-Rad) couplep with purified K9.361 antibody. After thorough washing of the column with solubilization buffer followed by 50 mM Tris-HCl (pH 8.2) containing 0.1% taurodeoxycholate, bound antigen was eluted with 0.1 M diethylamine-HCl (pH 11.5) and then dialyzed against the Tris buffer containing 0.1% taurpdeoxycholate. Radioimmunoassay. 3H-labeled K9.361 antibody was obtained from hybridoma cultures containing a mixture of 3H-labeled amino acids (New England Nuclear) and was purified on protein A-Sepharose. Affinity-purified Ly-17.2 was applied to nitrocellulose in a Bio-Dot apparatus (BioRad). After nonspecific binding sites were blocked by incubation with 3% bovine serum albumin, radiolabeled K9.361 was added to each well of the apparatus in combination with various amounts of unlabeled antibody. After 30 min at room temperature, suction was applied and the nitrocellulose was washed thoroughly with PBS containing 0.04% Tween 20. After the final wash, the nitrocellulose sheet was removed and dried, and the spots were cut out and transferred to vials for liquid scintillation counting. RESULTS AND DISCUSSION Ly-17 was described initially as a B-cell-specific alloantigen (1). Previous studies of normal cells showed that Ly-17.1 and Ly-17.2 specificities are expressed preferentially on B cells from lymph node and spleen, as well as on 20-60% of bone marrow cells and rarely, if at all, on thymocytes (1-3). Additional investigations of primary hematopoietic tumors and tumor cell lines showed that >90% of B-lineage lymphomas and myelogenous leukemias, 16% of T-cell lymphomas, and 33% of erythroleukemias were Ly-17'. This suggested that expression of this antigen among hematopoi-
Proc. Natl. Acad. Sci. USA 82 (1985)
7707
Table 1. Expression of Ly-17 and FcyR on cells of hematopoietic neoplasms Tumor or cell line Ly-17-/FcyRLy-17'/FcyR' Type K36 NFS-970*, -972*, T-cell -975*, -981*, -982*, -983*, -984*, -1082*, -1161*, -1176*, -1177*, -1178*, -1181*, -1182*, -1183*, -1207*, -1208*, -1209*, -1259* BCL 1, NFS-1.0, 2PK3, B-cell BALB-20Y*, WEHI-5, T27A, CWD-1297* Pre-B-cell B3; D4; BAC14; HRC3; BASC6C2; HS1C5; JP1; JP; JP5; JP4-1C2; JP4-2; HAFTL1; HAFTL2; Y1C1; AAC6; KFFTL1; E-1; NFS-1164, -112, -1135*, -5, -25, -971 Myeloid P388D1, ABPL1-11, ABPL2-12, ABPL3-1, ABPL4-6 Erythroid DS-19 Single-cell suspensions of primary tumors (*) or continuous cell lines were typed by flow microfluorometry. All B-cell lymphomas were sIg+. All pre-B-cell lymphomas were sIg- Lyb-2+ and most were Ly-5(B220)+. All myeloid tumors were Mac-i+. All T-cell lymphomas were Thy-l+Ly-1+. The origins and other characteristics of many of these iumors will be described elsewhere.
etic lineages was more extensive than was previously appreciated (2, 17). To evaluate this possibility, we performed two different studies. First, a large series of primary spontaneous and virus-induced lymphomas and leukemias and some cultured cell lines were examined by flow microfluorometry for expression of Ly-17 as well as expression of Thy-1, Ly-1, Ly-2, L3T4, Tla, surface immunoglobulin (slg), Ly-5(B220), ThB, Lyb-2, Ia, Ly-5, Ly-24, and the FcyR, using a panel of monoclonal a'nd alloantibodies. The results of these studies (Table 1) showed that Ly-17 was expressed by all the B-lineage and myeloid tumors and the single erythroid tumor but that only one of the T-cell lymphomas was Ly-17+. The data also revealed complete concordance for expression of Ly-17 and expression of the FcyR recognized by the rat monoclonal antibody 2.4G2. By comparison, there were multiple dissociations between expression of Ly-17 and all the other cell surface antigens examined (data not shown). These results strengthened the suggestion that Ly-17 might be expressed on cells of various hematopoietic lineages and indicated a possible relationship between expression of Ly-17 and the FcyR during hematopoietic differentiation. Second, spleen, lymph node, and bone marrow cells were examined by simultaneous two-color flow microfluorometry for expression of Ly-17 in relation to the B-lineage marker Ly-5(B220) or to the myeloid marker Mac-i. In some experiments, cells were pretreated with unlabeled 2.4G2 to prevent binding of IgG antibodies to cells via the FcyR. Assays performed without FcyR blockade showed that all Ly5(B220)' and Mac-l cells were Ly-17', confirming the suggestion that normal myeloid as well as B-lineage cells express this antigen (data not shown). However, for cells pretreated with 2.4G2, no Ly-17' cells were detected even though normal frequencies of Ly-5(B220)' and Mac-l+ cells
7708
Immunology: Holmes et al.
Proc. Natl. Acad. Sci. USA 82 (1985)
were observed in each tissue (data not shown). The failure of anti-Ly-17 antibodies to bind to cells in the presence of 2.4G2 suggested either that these antibodies bind to cells via their Fc portions or that the antigenic determinants recognized by these antibodies are not accessible on cells with bound 2.4G2. The former possibility was deemed unlikely because antiLy-17.2 antibodies reacted with cells of mice possessing the b but not the a allele of the Ly-17 locus and anti-Ly-17.1 antibodies showed the reciprocal pattern of reactivity. It therefore seemed likely that antibodies to allelic specificities of Ly-17 might react with genetically determined polymorphisms of the FcyR or with a molecule almost always associated with the FcyR in the cell membrane. Such serQlogically detectable polymorphisms associated with the 1.0
FcyR have been described by Dickler et al. (28). It is likely that the antigen defined by their antisera, especially C3H/HeJ anti-CBA/J, is identical to LyM and Ly-17. The view that Ly-17 might be identical to FcyR was also supported by the observations that monoclonal antibodies to Ly-17.2 and the FcyR detected the same frequencies of antigen-positive cells in thymus, spleen, lymph node, and bone marrow (Table 2). These data also suggested that few, if any, T cells in spleen or lymph node expressed Ly-17 at levels comparable to 13 cells or myeloid cells, since the sum of K+ and Mac-1+ cells in these tissues closely approximated the frequencies of Ly-17' and FcyR+ cells (Table 2). However, in bone marrow, Ly-17+ and FcyR+ cells were represented at higher frequencies than the sum of K+ and Mac-X+
A C57BL/6N spleen IgG dimer ----
0.6
2.4G2 blockade
C57BL/6N spleen B _____IgG dimer
I%I'
%I
----
II
Anti-Ly-17.2 blockade
0.2
1.0
0.6
0.2
I
D NFS/N spleen Anti-Ly-17.1 2aG2 blok blockade
C57BL/ON spleen
C Anti-Ly-17.2 - _.-_ 2.4G2 blockade
I
I
CIS v. 1.0
NFS/N spleen ----
0.6
NFS/N bone marrow
E
Anti-Ly-17.1 blockade
F
,,__Anti-Ly-17.1 blockade 'I I
0.2 ~%*
1.0
0.6. 'I
0.2
C57BL/6N bone
G C57BL/6N spleen -2.4G2 .- Anti-Ly-17.2 blockade
'I
I
marrow
----
:, %
I I
H
2.4G2 Anti-Ly-17.2 blockade
I'
II
I
I~ .,, 10
100
1000 Fluorescence intensity
10
100
1000
FIG. 1. (A and B) C57BL/6N spleen cells treated with medium alone (solid lines) or with either unlabeled 2.4G2 anti-Fc'yR (A, broken line) anti-Ly-17.2 (B, broken line) and incubated with rabbit IgG dimers, followed by staining with FITC-labeled F(ab')2 goat anti-rabbit IgG. (C) C57BL/6N spleen cells treated with medium alone (solid line) or with unlabeled 2.4G2 anti-FcyR (broken line) and stained with FITC-labeled anti-Ly-17.2. (0) NFS/N spleen cells treated with medium alone (solid line) or with unlabeled 2.402 anti-Fcy4 (broken line) and incubated with anti-Ly-17.1 alloantiserum, followed by staining with FITC-labeled goat anti-mouse IgG2a. (E and NFS/N spleen (E) and bone marrow (F) cells treated with medium alone (solid lines) or with anti-Ly-17.1 alloantiserum (broken lines) and stained with FITC-labeled 2.4G2 anti-FcyR. (G and H) C57BL/6N spleen (G) and bone marrow (H) cells treated with medium alone (solid lines) or with unlabeled anti-Ly-17.2 and incubated with FITC-labeled 2.4G2 anti-FcyR.
or unlabeled
Table 2. Flow microfluorometric assays of thynus, spleen, lymph node, and bone marrow cells of C57BL/6N mice % positive cells Ly-17.2 FcyR KAPPA Mac-1 Thy-1.2 Tissue 99 3
