Monoclonal antibodies against human granulocytes and myeloid differentiation antigens

Monoclonal Antibodies Against Human Granulocytes and Myeloid Differentiation Antigens

Patrice Mannoni, Anna Janowska-Wieczorek, A. Robert Turner, Locksley McGann, and Jean-Michel Turc

ABSTRACT: Monoclonal antibodies eMCA~ were obtained b3 immunizing BALBk mfie with 9 9 ~ pure granMoo'tes from normal donors or u'ith a u'hok kukoo'te smp~nsion obtained ~'om a chronh" mydogenous kakemia ¢CML~ patient, and then fusing the mouse spleen cdZ~ u'itb a 315- 43 myeloma cell clone. Four MCA we~x, selected and stmlied using ELISA. intmanofluorescence. ,ytotoxicity assays, and FACS analysZ,'. Antibodies 8OH. 1.80H.3. and 80H.5 ¢ fi'om norntal~ and S I H. ! ¢ from CMLI detected antigens expressed on neatrophils. Antibodies 8OH. 1 and SOH.3 ¢lgGI ako reacted u'ith monoo, tes but not u'ith other bkmd cell subsets. Antibodies 80H.5 and 81H. 1 ¢lgM ~ u'e~v cytotoxic and reacted stnmgl3 u'ith most of the celL*" ¢~the neutrophil maturation .wquence. i.e.. no'e/oblasts, prono,eloo, tes. myeloo'tes, and matm~ granuloo'tes. Antibodies 80H.5 and 81H. ! alto inhibited CFU-GM gmu'th stimulated ~' kukocyte feeder layers or placental conditioned media, but dM not inhibit BFU-E and CFU-E. Antigens recognized ~r 80H.3. 8OH. 5. and 8 ! H. 1 u'ere expressed both on a proportion of celfi ~ ~'om HL. 60. KG. I. M L. 1. and K562 no'doid cell lines, and on a proportion of blast cell, is'olated ~'om patknts with acute mydogenous leukemia. They u'em not found on ~,mphoid cd/ lines or (rmphoid leukemia cell~. These MCA nav~gnize either late differentiation antigens e.xpressed on mature neutrophi~* and nmnoo'tes ¢801t. ! and 80H.31 or ea~' differentiation antigens ¢80H. 5 and 81H. I ~ specific to the granaloo'th" lineage. They ntco' be useful for a better definition of those antigens specific to hematopoietic stem cells and thdr relationsh@ wOh normal or neoplastk" hematopoiesis.

ABBREVIATIONS ALL AML BFU-E

BSA CFU-GM

CFU-E

CML

FCS

acute lymphocytic leukemia ELISA enzyme-linked immunosorbent acute myelocytic leukemia assay burst fi~rming unit, FACS fluorescent activated cell

Erythroid sorter bovine serum albumin FITC fluorescein isothiocyanate colony forming unit, granu- GAT granuloagglutination test

Iocyte/macrophage GCT granulocytotoxicity test colony forming unit, HAT hypoxanthine~aminopterine-

erythroid thymidine chronic myelocytic IFT immunofluorescence test

leukemia MCA monoclonal antibodies fetal calf serum PBS phosphate buffer saline

From the Canadian Red Cross. Blood Transfnsion Servh'e and Unizvr.dO' o/" Alberta. D@artmcnt ,,f Pathology. Edmonton eP.M.. L.M.. J.-M.T,I and the Department of Medicine. Cross Cancer iustit~te. Edmonton tA.J.-W.. A.R.T.I Alberta. Canada.

Address r~qnests for reprints to Dr. Patrice Mannoni. Departmrnt of Patholo.g~. Unirer~io ¢,l Al& .rta. Edmonton. Al&rta. T6G 2G3 Canada.

R~dred May 24. 1982: accepted August 6. 1982.

H ~ Immucmlragy 5, 309-323 (19821 © ~ ~/~v.g¢ i~blishing Co., Inc., 1982 52 V ~ I ¢ A~¢.. New York, N~ 10017 0108-8859t821080~09-15$2.7 5

310 p. Mannoni et al.

INTRODUCTION Neutrophil or granulocyte antigens have been studied by using alloantisera developed in patients immunized by pregn~ndes or transfusions or by heteroantisera raised in animals [ 1 ]. Such sera often contain makiple specificities corresponding to different ant/body populations.

Somatic cell hybridization of immune spleen lymphocytes and myeloma cells has been used to create cell lines secreting MCA% highly specific to one antigenic determinant [2]. These MCA have been used to define specific markers on a wide variety of cells. Extensive studies on normal lymphoid cells using MCA have led to the definition of different subsets of lymph~gytes with specific func- tions, such as inducer-helper or suppressor cytotoxk cells as well as lymphoid precursor cells [3]. In comparison, little work has been done with MCA against mature human neutrophils or monocytes [4-6]. Such MCA would facilitate the study ofgranulocyte membrane antigens, whkh change during the different/ation of hematopoietic precursors to mature cells [7]. in a~]dition, the study of the expression or lack of expression of specific antigens on neoplastic cells should lead to an increased understanding of the leukemic process [8,9].

This report describes the production a ~ chasacterization of flint MCA against mature human neutrophils and monocytes that recognize determinants specific to the myeloid lineage.

MATERIALS AND METHODS

Prad~ction of ~o~odolml anti&Mi¢~, Hurl~n ~iphcr~l b ~ g~nuk~ytcs were obtained from defibrin~ted b h ~ col~cttd f~m normM donors. B i ~ was diluted in PBS*/E~A gO.5 mM E ~ A in 0~15 M PBS~ at a r~tio of 1:0.4. Two milliliters of a 6~. ~ x t r a n T S ~ solutMn {Fi~ Chemicals Pharm~i& Uppsala) were ~ded to 10 ml of diluted M ~ . After mixing, t ~ b l ~ was Mlowcd to ~diment for 30 rain ~n a sili¢onized t u ~ ~ 4~C. ~ e rich leuki~yte fr~don was removed ~nd centrifugal, ~ad t ~ ~lMt w~Md once with PB~EDTA solution.

Cells were sus~nded in ~ ~ i u ( ~ ~ le~ ~ BSA" in PBS at a ¢on~entot/on of 2.5 x 10 ~ celi~m~ ~ l~yered on t ~ top ~ , di~ondnuous gradient of Percoll []0]. Five concentrat~on~ of Percoll ~l~tMns in PDS {36~.. 45%. 58.5~. 67~.. and 72~.) corre~nding to den , i t~ r ~ n g from IA~4 to t.089 wet~ prepared and layered on top of e~h other. After ,¢n,rifu~i~m fi~r 20 rain at 8(R} x g at 4°C, five ¢eli bands we~¢ rtgovtf¢-d ~ o ~ i ~ g from tM n~p to the ~t tom: cell debris a ~ p~e~ t , , ~ ~ y t e ~ # g ' h ~ . lympM~yt¢~, ;++~d two b~nds of granui~ytes, res~ctively. ~ h ~ a ~ w~ ¢~efully rcmoYed ,~nd w~hed twice in PB~BSA. Only t ~ ~an~k~yt¢ ~ wer~ u~d to immunize mice, but M! were u~d to ~rform ~ t i ~ y ~ r ~ n i ~ ~ Mentific~m ~tud~es. ~uk~y te s from the b l ~ of a p ~ n t with CML ~ ~¢~ ~f i f i td by ~dimcntation on ~ x - tran, w~hed with PD~BSA ~flutMn. ~ inC,'ted inu~ mi6e. BAL~c mice {Uni- versity of A I ~ m ~ S ~ t A n ~ l P r ~ were in~ected intravenously with either 10 ~ ~ M ~ y t e s ~ CML M u ~ y t ~ s ~ d in 0~5 nfl of Ca 2 ~- ~ d Mg 2 *-free PDS. ~tice were ~ t e d i n t ~ . e ~ l y ~'ice with the ~ e do~ of cells, at l¢mst 1 ~ n t h d ~ r tM p r~ in~ ~ ~ ~ ~ t I week ~ffdelay ~ , e e n the first ~ d t ~ ~ o n d ~ t . ~ d~y~ ~ r t ~ ~ t ~ s t , the ~pteen w~ removed a~pdc~y ~ l y m ~ t ~ ~ ~ . In order to i m r ¢ ~ the humor of re~tive cells, spl~n , e ~ wcr¢ ~ ~ wieh ~ i ~ ? ~ ¢ ~ f i d c s {E. Cdi strain 026 ~ , 20 mMml~ in RPMi wkh I ~ FCS" f t , 18 hr at }7~C {11L

F~.~ion prot~,~. ~ m y ¢ ~ ¢¢~ ~ M ~ ~ 1%43, a ~ c r e t i n g variant of HAT~-~nsit/ve, o u ~ n ~ r ~ t ~ t D ~ ¢ ~ o ~ ~ ¢~d for (~ion ~ de-

MCAs to Human Myeloid Antigens ~! !

scribed elsewhere [ 11,12]. Briefly, cells were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum and 2.0 × 10 ~ ' M mercapu~ethano| (RPMI/FCS). While in the logarithmal phase of growth, myeloma cells were fused with spleen cells at a ratio of 5:1, by using a solution of 30% polyethylene glycol 1500. After fusion, the cell suspension was diluted in RPMI/FCS medium with HAT. Mouse blood cells were used as feeders and added to the HAT medium to a final concentration of I × 10 ~ mouse blood cells/ml. In 96-well culture plates (Limbro or Costar) 200/zl of suspension were plated. After 7 days, !(g.} /zl of supernatant were removed and replaced with new HAT medium. Super- natant screening took place 10-14 days after the fusion day when clones were distinct in wells. Expansion of positive chines, subcloning, and production of large amount of supernatants and ascites fluids were performed using standard techniques.

Screening pr~'edure. The MCA described in this report were selected using EL[SA *. Target cells (granulocytes) isolated on discontinuous gradients of Percoll were coated on 96-well microtitration plates (Dynatech). Each well of the flat-bottom microplates had been pretreated for at least 2 hr at room temperature with l ~ l ~l of 0.1% glutaraldehyde in carbonate buffer (pH 8.2). Plates were then washed with PBS and 100,000 cells were added in each well. After slow centrifugation (200 × g) and removal of supernatant, 100/zl of 0 .05~ glutaraldehyde diluted in PBS (0.15 M) were added for a period of 3 rain. The glutaraldehyde was removed and cells were washed again with PBS. In order to saturate the free glutaraldehyde radicals, 200/A of 1% BSA fin PBS) were added to each well and left for 2 hr at room temperature. Plates were kept in this medium for up to 2 weeks at 4°C, or were frozen at 70°C. Before using, plates were washed with PBS/BSA.

Twenty-five to 50/.tl of culture supernatant, diluted l:2 in PBS/TWEEN 20 ~0.05% final concentration) were dispensed in each well of granulocyte-coated plates. Incubation was performed either overnight at 4°C or for 2 hr at room temperature. Supernatants were removed and plates were washed six times with cold (4°C) PBS/TWEEN. The screening reagent was sheep Flab'L, anti-mouse lgG (heavy and light chain specific) coupled to ~-galactosidase ~BRL reagent, Bethesda, MD) and was used at a dilution of 1:250 to 1:300 in PBS. After incubation for 90 rain at room temperature and further washings with cold PBS¢ TWEEN, 50/zl of substrate (p-/~-D-galactoside, Sigma; 1 mg/ml in PBS + 0.0i mM MgCI,) were added to each well. After 1 hr the reaction was stopped by adding 25 /zl of 0.5 M sodium carbonate, and the change in optical densi~" recorded by an ELISA reader using a filter of 405 nm. Negative and positive controls were present in each plate. Only changes in optical density, above two standard deviations of the mean value of the negative controls were scored as positive results.

Selected hybridoma cultures were recloned by limiting dilutions. Specific clones were cultured until a sufficient number of cells could be obtained for freezing and fo," injection into BALB/c mice primed with 2,6,10,14-tetramethylpenta- decane (Aldrich Chemical, Milwaukee). The ascites from these mice were col- lected and clarified by centrifugation.

Specifi'city tests. Normal cells isolated from peripheral blood were prepared by centrifugation on a Percoll gradient as described above. To obtain B cells, de- pletion of T cells was achieved by either rosetting them with trypsinized sheep red blood cells or by adhering B cells to nylon wool. Bone marrow cells were obtained from volunteers or from patients undergoing cardiac surgery and sep-

312 P. Mannoni et al.

arated on a discontinuous gradient of Percoil (density 1.(~$4-1.089L Leukemic cells were obtained from the peripheral blood of patients with CML, AML', and ALL*, and from patients with hypereosinophili:L Sedimentation on Dextran alone or followed by centrifugation on Percoll gradients was used to obtain such cells, depending on their cotmentrat/on, their tendency to sediment, and the contamination by nonleukemic ceils.

Peripheral blood and bone marrow cell populations were assessed fi)r puritg, by the following meth~ds. Cytospi~l slides were staineti with Ma},-Griinwald- Giemsa. Volume distributions were performed fi)r all cell |a)pulations. and nonspecific esterase and latex or yeast phggocytosis were used fi~r monocyte and granulocyte identification, Surface immunoglobulins were determined in populations of mononuclear ceils by using fluorescein coniugated anti-human im- munogiobulins gheavy and light chain specific, Cappel Laboratories, Cochranville. PA) in order to assess the percentage of B cells. Monoclonal anti-B (anti-la) anti anti-T antibody iOKT3 and our local MCA) were Mso used .~s controls. Cells were obtained from normal donors ~n~| were typed tbr HLA (ABC, DR) and neutrophil antigens ~NA 1. NA2, NBI L

tl/mta~2 Cell Libra. Myeioid cell lines HL-(~O [13] ~kindl~' pn)vid~d b~' Dr. C. Cass, Univ,:rsity of Aiherta?, KG-I [13] (kindly provided b~' Dr. H.P. Koefiler. U.C.LA.L K-562 [ 13] gkindly provMed by Dr. M. Longenecker, University, of Alberta). ML. 1. and U.937, a monocytk cell line [ l,t] (kind|y pn~vided by Dr. R. Painter, University of TorontoL were gr~nvn in our |abor~n~ry. Lymphoid cell lines of B, T and null types (Daudi; R~ii; RPM18392; CCRLSB; BALL i: I~ALM 2; MOLT 3, 4; CCRF-CEM; NALM-I. 6, 12; REH; NALLL~ were also tesfed b~' using ELISA and immunofluorescence assays.

SerMog&'a/A.(sa.~. Two binding ~,~ys. the ELISA and an |tel TM [15,1g~L were developed for the identification of s~c/fi~itk.~. ~ e ELISA assay was the s~me as used for supernat~nt ~mening with minor ~ i f i¢~ t~ns . Different dilutions of culture su~rnatants t 1:2 to 1:20) and ~ i t e s ~1:25~) to I:HL~R)) ~¢r¢ umd. In mldition, ~m¢ c¢11 iine~ ~MALM~! or K562. fbr instance} r¢quired a higher concentotion of g!~taraMeh~'d¢ ~0,1 to 0.5~) n~ Mlow ~ r ~ n ~ ¢ to the ~ t tom of the well.

in the direct immu~flu,~m~ene¢ g,~, , ¢¢~ were fir, t tr¢~ted wiih 1~ paraformaldehyde in PBS ~ I ml for 3 n~ 5 ~ l~g' geB~) f t , _~ rain in onl~r ~o decrea~ the nons~cific a n t i ~ y M~ing b~' Fc ~ c ~ o r , [ 16L After w~snil,. ~2.5-5) x 10 ~ cells were ingu~ed with different d iMt~s ~ff MCA fi~r 3 ~ rain at r ~ m tem~ratur¢ in a *~weJJ culture p h ~ g ~ o or Cost~. After three washings with cold PB~BSA l~., l (~ # of ~ r c g e i n I~J~d V~b')2 anti- mouse 1 ~ ~heavy ~ d l i~ t c ~ n s ~ i ~ ) w ~ i m u ~ e d fi~r 4~ rain at 4~C. Several commercM FITC ~ ~ n t i ~ were t ¢ ~ ~ d F ~ ' ~ : f~n~.n~s of ~heep or rabbit ~ t l -mou~ ig ~ C ~ ! ~ m r ~ C~hr~nvil~. PA~ were ~l~gted ~ giving the most r e p r ~ i ~ ~ t s ~ ~ ~ t ~ n ~ i f i ¢ fluorescence. Preparations were w ~ M ~ ~ o d r ~ ~ ¢ ~ u t ~ m ~ 3 ~ # ~ ¢ r d gin PBS) were ~ded to t ~ ~lMt f ~ e ~ i ~ b)' ~ ~n E P i - f l ~ e ~ e ~ e mkro- sco~. The ~ rcen t~e of f l ~ e ~ e n t ¢ ~ w~ ¢ ~ t e d ~ t ~ M ~ t m s s of t ~ staining w~ ~ o ~ d ~ f ~ l ~ s : ~ v¢~' M ~ t ; b, M ~ t ; ¢, int~t~dia~; d, ~*¢~. For cell ~ e r ~ y ~ s , t ~ cells were ~ ¢ ~ to a m ~ ~ resus~nd¢~i in 0.5-1 m l d PBS with le6 BSA ~ ~ d ~ ~PIC~V cell ~)~¢r ~C~mlter ElectronksL C o m p l e ~ n t ~ n * e y ~ w~., ~ M d ~ differen~ cell ~pulations b7 i ~ u ~ i ~ MCA ~ r ~ t ~ u r ¢ f ~ (~ rain ~ith fl~e t ~ t cells. MCA were Mw~ys dilu~d in ~ h u ~ AB ~r~m. &f,~t wa.~i~,

MCAs to Human Myeloid Antigens .~l.~

rabbit complement was added, and incubation was performed at room temperature for 45 min. Cell death was determined either by staining with eosin (fi~r T-lymphocyte or B-lymphocyte cytotoxicity tests) or by using the technique of double fluorochromasia using two fluorescent compounds: fluorescein diacetate (FDA) and ethidium bromide (Sigma) [17]. Microtechniques were developed using 72-well Terasaki trays.' Five thousand cells preincubated with FDA were pipetted in each well under mineral oil, and 1 ~tl of different dilutions of MCA were added to each well. After 1 hr of incubation at room temperature, one washing was performed with 5 /xl PBS or Hanks balanced salt solution (HBSS) and then ,~omplement added. Low Tox Complement (Cedarlane) or normal rabbit complement were selected. Each batch has been tested for nonspecific cytotoxicity, and eventually absorbed twice at 4°C with human red blood cells. Lysis of target cells by MCA and complement was determined by the percentage of either eosin-positive cells or ethidium-bromide-positive cells (red coloration;. A fluorescent microscope tZeiss EP! IV) with two filters was used for the latter test. Reading was performed in the microplates and a score from 1 to 4 was determined according to the percentage of dead cells. Living cells retaining FDA were identified by their strong green fluorescence.

Granuloagglutination test. A specific GAT* was performed according to Lalezari [15]. In brief, MCA at different dilutions in PBS (1:2 fi~r culture supernatants and 1:20 to 1:100 for ascites) were mixed with 1 p.l of a suspension of 5 × 10"/ mi granulocytes in the well of a microplate, under oil. Readings of the tests were performed after 2, 5, and 24 hr of incubation at room temperature. Strongly agglutinating human anti-NA l and anti-NA2 serums served as positive controls. The class and subclass of each MCA was determined by using anti-mo~se lgM. lgGl , and lgG2 coupled with fluorescein.

Fluor~'scence actirated ,dl sorter analysis. Quantitative FACS* analysis was performed with the EPICS-V system (Coulter Electronics). For each histogram (1 - 3) × IO * cells were counted by light scattering and analyzed for fluorescent intensity by using the same reagents used in the microscopic technique. Single- and two-parameter histograms were acquired and displayed by the MDADS (muitiparameter data acquisition and display system) in order to identify different cell populations and to define the number of fluorescent cells in each population.

Colony a~says. After sedimentation on Dextran TSO0, a nonadherent bone marrow cell population was prepared by sequential adherence on plastic tissue culture dishes for CFU-GM * assays [ 18]. Complement-dependent antibody cytotoxicity was assessed by incubating the nonadherent fraction of bone marrow cells at the concentration of 10 ~ cells/ml with several dilutions of MCA for 30 min at room temperature, followed by the addition of rabbit noncytotoxic complement iCe- darlane Laboratories) at a final dilution of 1:6, for 1 hr at 22°C. After incubation, cells were washed three times with ~ medium plus 5% FCS and plated in the appropriate assays.

Cytotoxicity was assessed by comparison to the controls incubated in medium and in complement or antibody alone. Three separate experiments were performed with each MCA. The complement had been previously tested an8 found to have no inhibitory activity against myeloid progenitors. In the assay fi~r granulocyte/macrophage progenitors (CFU-GM), bone marrow cells were cultured according to a modification of the technique described by Pike and Robinson [19]. Briefly, nonadherent cells were added to a mixture of 2% methylcellulose (Fluka) in minimum essential alpha media (Gibco) containing 15% FCS (Flow)


at a final concemradon of I0 ~ cellcml. One millil/ter of this mixture was overlaid onto 10 x 35-ram tissue culture dishes (Coming) containing I0 c' normal human leukocytes suspended in a 0.5% agar gGibco) solution. These peripheral ieu- kocytes acted as a source of colony-stimulating activity. To exclude any effect of the antibodies on coMny-sdmula¢ing-activiq~-pcoducing cells in leukocyte feeder layers, MCA were also ~zsg~l using ceil-free placental conditioned medium as the source of colony-stimulating activity. Placem~l conditioned medium was prepared by the method of Burghs et al. [20] and comparative studies were performed by placing 10 ~ cdl/ml in 10% cell-free pl~en~l conditioned medium in methykellulose plus 15% FCS in alpha medium. Triplicate experiments of each method were incubated a~ 37°C in a fully humidified atmosphere and 7~, CO2 for 14 days.

Aggregates of greater than 50 cells were scored as colonies with an inverted microscope ~ day 14. The nature of the cells in colonies was ~ssessed by preparing cytospins of pooled cobnies aspirated with pipettes aM staining with Wright- G'emsa stain.

Eq, throid progenitors (BFU-E,* CFU-E*~ were ~ssayed in mcthykellulose cultures containing in 30q~ FCS, 1% BSA ~Frgt/on V, SigmaL !0- ~ 2-mercapto- ethanol alpha medium with pen/¢iilin ~ strep¢omycin and leuk~gyte conditioned medium ~LCM) at a final concentr;~ion of 9% lob21]. Cells were plated to give 2 x 10 ~ cells/dish. Effthropoiedn g h u ~ u r i p ~ , gift of Dr. C.A. Eaves) was added at a final corgena, afion of 2.5 U/mL Cul~io were incubated at 37°C in a humidified atmosphere of 5% CO~ in ~ir. CFff-E were c~mnted ~n day 7; BFU-E, large colonies compris/~g ~hree or m~e s~!xolonics, were counted on da~ 14.

MCA Nomendature, Each MCA w~ ~amcd by ~ number, the letter H. and a number. The first number indic~te~ the fus~n number. The H means that human cells were used for immunization, ~ d ~h¢ l~t nuraber is arb~¢rary, assigned to each positive hybridoma ~nd i~s MCA.

R E S U L T S

General Properties of 8OH.I, 80H.3, 8OH.5, and 81HA

Antiixxlies 80H. 1, 3, ~nd 5 were ~ d f r ~ • ~ u ~ [ ~ n [ z e d whh ~ormal g r a n u I ~ e s a~d 81H, 1 from a ~ u ~ ~ e d wi~h CML I¢~k~y~es. Th¢~ four a n t i ~ i e s r e . t ed with ~ r i ~ r ~ ~ ~ g ~'tcs from ~! donors tested, but not with lymp~ytcs , phte~ts, o~ red celh ~T~lc ! L I~n~no~obulin

TABLE ~ General proper~k~ of SOH.i, 3, 5, ~ d 81H.| m~aodonal ~atibedics

T ~ o~ ~ ~ g y ~ c s ~

Antibodies (I~ chs~s) ELISA G i f t GCT GAT

80H. I ( lgG2 ) 1: | ~.~ |: ~ ~ ¢ ~g~dvc ~H,3(I~I) I : ~ ~:~ ~ " I:~ ~H.5~IgM) ~ : 1 ~ ~ : 1 ~ ~ : ~ !:20 81H.HigM) I : ~ 1 : ~ l : 1 6 ~ 1:20

~ELISA, c ~ y ~ y ; G|i~T, ~ ~ ~ ; ~ T ~ ~ ~ ; GAT~ ~ ~ . ~¢~m i~d ~ ~ ~ ~ ~ s ~ ~ ~ ~ ~ G~T. ~ r ~ .

M C A s to H u m a n Mye lo id Ant igens 3 1 5

subclasses and activity titers determined by binding and cytotoxic assays are presented in Table 2. Antibodies 80H.3, 80H.5 and 81H.I induced a strong direct agglutination of normal granulocytes. Cells from 24 donors have been tested in order to rule out the possibility that this MCA detected some alkan- tigens. Antibodies 80H.I and 80H.3 have also been found to react with more than 90°~. of peripheral blood monocytes (Table 2). All were completely negative with all subsets of lymphoid cells tested by cytotoxicity or binding assays. When tested on eosinophils obtained from one patient with hypereosinophilia, 8OH. I and 80H.3 were negative and 80H. 5 and 81H. 1 were weakly positive, and 8OH. 1, lgG2 and 80H.3, IgG1 were not cytotoxic to target cells in the presence of rabbit complement. Antibody 80H. 1 was strongly positive by using ELISA assay and weakly by IFT; 80H.3 was strongly positive in both the ELlSA and IFT exclusively to monocytes and granulocytes. Antibodies 80H.5 and 81H. 1, both of the IsVM class, were strongly cytotoxic to granulocytes, and to all myeloid cells or ce|l lines recognized by these two MCA. All of these data obtained using manual techniques like ELISA, IFT, and GCT* were confirmed by FACS analysis as reported below. ;

Controls, including mouse lgG1, IgG2, and IgM produced as ascites by r~y- eloma tumors implanted in BALB/c mice, failed to react by immunofluorescevce and cytotoxicity. Even though they occasionally induced a high background in the ELISA assay, the values obtained were far below the values obtained with the specific antibodies.

TABLE 2 lmmunofluorescence tests of monoclonal antibodies on normal blood, bone marrow, and CML cells (microscope examination)

Number ,)f fluorescent cells

Cell type 8 0 H . 1 8 0 H . 3 8 0 H . 5 8 1 H . I

B l o o d cells Red blood cells . . . . .

Platelets . . . . .

B L y m p h o c y t c s . . . .

T L y m p h o c y t e s . . . .

M o n o c y t e s 90 ' 90 ' - - - -

Neutrophils 90 '~ 100 t' IO(Y l()t~

Marrow cells Monocy tes/Macrophages 90 ' 9() ~ - - - -

Myeloblasts/Promyeh)cytes/Lymphocytes - - I 0 - 3 ( ) ~' 5 0 - 8 ( Y 50-~;tV

M yelocytes/Metamyelocy tes - - 60 ~' IO(F I|X~ ~

Bands/Granulocytes 90 a 1 ()0 t' 1 O0 ~ 1 (XV

C M L cells Myeloblasts/Promyelocytes - - 1 0 - 2 0 ' 90" 9W

Myeloc),tes/Metamyelocytes - - 6 0 - 9 0 ~ 10t~ I¢~*

Bands/Neutrophils 90 't 100 ~ I 0 0 ~ IOiV

No,':: Immunofluoreseent tests were perh)rmed on peripheral blood cells obtained from 2,t blood d o m e . ~m bone marrow cells obtained from 3 different normal donors, and on [c'ukocytes isolated from 3 patients in chn)mc pl)~se of chronic myelo~ytic leukemia (CML). ~'Vety bright fluorescence. ~Br/ght. 'Intermediate. '~eak.


Reactivity with Normal Bone Marrow Cells

Fractionation of bone marrow on a discontinuous gradient of Percoll resulted in the separation of different cell populations according to their density. The first band on the top of the gradient mainly comprised monocytes, and approximately 80~ of these cells were labeled with 80H.I and 80H.3. Antib~xties 80H.5 and 81H.1 were negative. Band 2 contained myeloblasts (5-15~), promyek)cytes, and lO-20e/~ Iymphocyms. Antibodies 8OH.5 and 81H.I induced a bright fluorescence on myeloid cells; 80H. ! was negative, and 80H.3 reacted with a smaller amount of cel|s in comparison to 80H.5 or 81H.l (Table 2). Bands 3, 4, and 5, which included populations of myel~Kytes and mature granulocytes, were stained strongly by fluorescent anti-mouse antibody after incubation with 80H.5 and 81H. 1 antibodies. No difference in intensity was observed in the different cell populations when tested with these two antibodies. To the contrary, the binding of 80H.3 was correlated with the degree of cell maturation, and 8OH. ! was only weakly positive on mature granulocytes.

Reactivity with Leukocytes Isolated from Patients with CML

Since the percentage of myeloblasts in b~me marrow is low. even after Percoll separation, peripheral blcmd from CML patients in the chronic phase was sepa- rated in a discontinuous Perco|i gradient. By using this approach, it has [yeen possible to obtain a myeloblast enrichment as high as 2(F'/f, the remaining cells found in the same gradient being pn~myel(u:ytes and myelocytes. Under these conditions, 80H. l and 80H.3 were clearly negative, and 80H.5 and 81H. i were strongly positive with more than 9~F*/~: of the cells ~Table 2L

Reactivity with Human Leukemic Cell Lines

The results of l i t performed on different human cell lines arc- shown m Table 3. None of the I~mphoid ce~l l i~s ~es~ed ~ m ~ any of the~ MCA. Results obtained with m~eloid cell l i ~ HL(~L KG.I, ML~ ~nd with K562 cell~ demonstrated that 80H. ! did not u~ re~t with ~a~ ~ff t ~ ce~l line~. An t i~ ty 8OH. 3 was ne~tive when te~ted on KG. l, ML 1, ~mt K562 but ~tained only 9~ of cells of the most different~ted mye~fid eell line, HLX~L

A n t i , l i e s 80H.5 ~ 81H.I re . ted ~ t n ~ y with HL~} and KG.i. Al- though the pattern c h ~ d s~ightly from o ~ t¢~ to ~nother, dewnding on the maturation st~¢ of the culture, ~ m t 4 ~ 7 ~ of the ¢¢~1s were ~trong!y fluore~ent wkh HL.60. In all tests, 3 ~ 4 ~ . ~.~f t ~ cells remained u~,stMned by M)th anti~iies. Thi~ w~ ~ e d wi,h ~ h cell l i ~ a ~ w~ m~t de~'ndent on a n t i ~ y concentr~ion. Moreover, t ~ i ~ t ~ ~ gr~nuhKVte differentiathm by the ~dition of redm~ic ~M [22] did mn i ~ r ~ the ~ r c e n t ~ of ~:~:,l~ ~ l e to bind the~ myeloM MCA ~d~a tv2t $ ~ n L A ~ t ~ fluore~ent ~ pulations, it h~ ~ e n ~s f ib~ ~o Mentif~" different sub~ts de~mii~g ~n the fluore~ent inmnsicy. ~ m ~ ~ i d v e eelh were t ~ more differenthted tells, and cells with weber intensity ~ F * ~ to ~ l~s mature ~ judg~t by their morphology in # a ~ contr~t m k r ~ , . H ~ e L u ~ l~ge cell~ with the m o r p h o l ~ ~ leukemk m y e ~ * s were ~ " gM~d by 80H.5 and 81H. I.

When 80H.5 ~ 81H.I ~ i ~ d ~ h K ~ 2 ceB$, avery hete~ogenous pattern w~ ~ 6 M . From ~ ~ ~ ~ ceih ~ r e s ~ b~ tke FITC anti- mou~ 1 ~ . S u ~ b n i ~ ~ t ~ ~ K ~ 2 ceR ~ d ~ r ~ e d t ~ ~s~Mlity of obtaini~ s u ~ b ~ s , expre~i~ ~ v ~ ~ n t f ~ t ~ different ~ k e r s (manu~fipt in p r ~ m M n L W ~ n t~t~{ ~ U.937, ~ tell l i ~ from hi~d~Kytk

MCAs to Human Myehfid Antigens 317

TABLE 3 Immunofluorescence assays of monocional antibodies on human cell lines (microscope examination)

':; Pos i t ive cells

Cell line 80H. I 80H ~, 80H.5 s I H. l

K 5 6 2 " o 0 5 0 - 8 0 ~ 5 0 - 8 W

H L . 6 0 " 0 5 ~ 5 0 - - 0 ~ i ~ - 5 ~ Y

K G . l ' O 0 20- '~O ~ ~W

ML. 1 0 0 I00 ~ I(~

U937 ')0 '~ 9(F 88 '~ 88 ~ B Cell

~RAJI, CCRFSB, RPMI 8392, BALL 1, BALM 2, DAUDb 0 0 0 (I

T Cell ~MOLT ~. MOLT 4, CCRF-CEM~ o o o o

Null (NALM l, 6A2; NALL I, REH~ 0 0 0 (]

FACS

Not~: Cell line K562, HL.60. and KG.I values obtained m three diflZ.rcm cxpcnmcn[s. ~Vcry brigh~ lluorcstcm l l l tc t ls i fy. ~Brtghr. ' Intermediate. ~Wcak.

origin which expresses monocytic/macrophagic features [ 14], 80t-I. 1 and 80H.3 reacted weakly on about 90% of the cells. Antibodies 80H. 5 and 81 I-|. 1 induced a bright staining of about 80% of the cells. These different results are summarized in Table 3. Titration of MCA on positive cell lines gave similar results when compared with values observed with normal cells.

Analysis

Results obtained by measuring the fluorescence activity and by comparing it to the light scattering of the cell population confirmed the observations made by microscope examination. The strongest fluorescence was again observed with 80H.5 and 81H. l, and the specificity of the binding fi)r myeloid cells only was. confirmed. The fluorometry analysis ITable 4) confirmed that 8OH. 1 and 80H.3 were positive with peripheral blood monocytes and neutrophils. Reactivity of 80H. I to granulocytes was weaker than 80H.3. Both antibodies did not bind eosinophils; whereas, 80H.5 was strongly positive and 81H. 1 recognized alx~ut 50% of the cell preparation. The weak positivity observed with 80H. 1 and 80H.3 was due to the neutrophil contamination of the cell suspension. When tested on a bone marrow cell fraction containing 20% lymphocytes and 80% cells belonging to the myeloid lineage, the FACS analysis confirmed that (a) 80H. 1 was negative on the myeloid fraction, (b) 80H.3 stained only 40°~ of the cells, and ~c) 80H.5 and 81H.1 bound to the majority of the cells. These results suggest that the majority and possibly the totality of the myeloid cells are recognized by these two MCA.

No binding was observed in the different lymphoid populations tested what- ever the technique of separation used (gradient density on albumin or Percoll, E Rosetting, adhesion on nylon wool). By using the two-parameter analysis tMDADS program) it has also been possible to demonstrate that the antibody binding was

318

TABLE 4

P. Mannoni et al.

Microfluorometry analysis: Percentage of fluorescent cells after treatment with MCA plus FITC anti-mouse lg

Cells

M~m~ghmal antibodies t MCA}" Purity of the ce!! Negative suspension {~ ~ controls ~ 8~)H. 1 80H. 3 80H.5 81H. I

Mom~cytes Granulocyees Eosinophils T lymphocytes B lymphocytes Bone marrow ~

ceils CML leakocytes

8 9 - 9 8 5 ~ 4 ) 8 ~ ± 13) 95~-+41 ~ - * 3~ 3(--21 95-¢)8 21~ 1~ ¢M~ ~ 9} ~)~ ~ i ) 85~ ~8~ 92~ ~ 6 )

80 6 13 21 92 52 ~-95 0 0 0 o 0 ~5 0 0 0 0 0

10 (lymph~;'tes) 5 6 40 85 8~ 90 ~myeloM cells~ I(R) ~mydoid ceils) 3 0 53 95 98

Notr: Tests were performed on six differem ~Fc~mcns ~ff ~ r i ~ * r M ~ M momgyt,,s ~nd gr~nuhgytes, three samples of T and B ~ells, one su~ns~*m ~f ¢'*~i~u~i~ f g ~ a p~t~nt ~.ith ehnmk eo~inophi~m. ~nd t.te ~ l l e marrow and CML cell sus~nsion. ~Mcan ~ standard deviat~m, ~Xhis fr~tion of ~me m~rrow cells c~m~ined I ~ ~ 'mp~y~¢s. ' ~ m ? e ~ s ~ s . ~5% p~-mVehgy~es ~ d m~c- hgytus, and 4 ( ~ metamyehg~'tes ~nd ~ u r c ~n~g~ '~¢s .

restricted to the granulocyte popu|ation for 80H.5 and 81H.I and to the monocytes and granulocyte populations for 80H.I and 80H.3. All other lymphoid cell populations contaminating the preparation were negative.

Effect of MCA on Colony Assays

In order to better define the bone m~rrow cells regcting with theg~ myehfid- specific MCA, these antibodies were tested for their potential effect on the growth of granulocytic/monocydc colonies {CFUoGM) and, ~s controls, on the growth of erythroid colonies ~BFU-E, CFU-EL Because different bone m~rrows might contain different popolations of colony-stimuhting ceils as well as different per- centares of the cells involved in the CFU growth, M! e~periments were performed in triplicate by using three different bone marrow donors. No inhibition Of CFU- GM was observed with 8OH. 1 ~nd 80H.3, which ~re n~t cytologic and recogni~ mainly mature forms of granuMcytes. Antibodies 80H.5 and 8 i l l . 1, hJth lgM, strongly inhibited the growth of CFU-GM stlmuhted with feeder laye~ or with cell-free placental conditioned medium ~Table 5L This inhibition occu~':ed with dilution of MCA as high as 1:4000 for 80H.5 ~Tahle 6). No inhibhion was obtained by incubating MCA or complement alone with the bone marrow cells. This effect was specific to the myelomonocytic line~.ae since no inhibition was observed with BFU-E or CFU-E cobnies. As positive contro|s, an anti-HLA and an anti-in MCA (9H. 1, 7H.3, provided by Dr. B.M. Longenecker) were tested under the same conditions. Both of them were able to inhibit m~,ebid ,ohmy growth.

React ivi ty on Leukemic Cells

Blast cells were obtained from the peripheral blood of patients with acute lymphocytic leukemia (6 patients), acute myek/Mastic leukemia { 14 patients), and chronic myelocydc leukemia ~5 patients). All l m d ~ i e s were negative when tested on leukemic cells from lymphoid o ¢ i ~ . A ~ n g the acute m~'e|ocvtk

MCAs to Human Myeloid Antigens

TABLE 5 Effect of cytotoxic MCA on granulocytic and erythropoietic colony formation by bone marrow cells

319

Colonies

Treatment o f b o n e C F U - G M BFU-E C F U - E marrow cells [pe r 10 s m l t ( +-SE)] [pe r 2 x In * m l * ~ ±SE~]

Medium ,16 0 ± 2.6 -35.7 _-z- 7.2 108 ~ 5 .6 C o m p l e m e n t 51.7 _+ 2.9 26.0 ± 3.5 128 ± I ' L l 8 0 H . 5 + c o m p l e m e n t 5.3 x 1.3 25.5 ± 6.1 142 ± 9.~ 8 1 H . I + c o m p l e m e n t 11.4 ± 3.6 3~.0 ± 9.1 I V ~ ± 5.5 an t i - h " + c o m p l e m e n t 2.8 ± 1.5 0.7 ± 0.5 6-~." z 5 .6 anti H L A t" + c o m p l e m e n t 1.0 +- 0 .6 N D ' N D lgb l ~ + c o m p l e m e n t 40.0 ~ 19 N D N D

Not,: Monoc~onal antibodies (MCA) were diluted 1:20 in alpha minimal essential medium plus 2 0 ~ t~:¢M *.Mr serum, and after incubation cells were plated with 10cA placental conditioned medium. Mean of three diffi:~cm experiments arc given. ~Aw:i-la and ar, ti-HLA MCA were kindly pro,dried by Dr. M. h~ngencckt:r ~Departmcnt of Imn'mnolog.v. t ;m*.-*.-r.,*~: of AlbertaL qgM MCA without any reactivity on human cells was selected as control. 'ND, not done.

leukemias, 80H. 1 did not show reactivity against any of the leukemic myeloblasts tested.

The reactivity of 80H.3 was more polymorphic. This antibody did not react with the leukemic myeloblasts obtained from less differentiated forms of AML (type M 1 and M2) [23] and reacted weakly with 10-20% of cells from patients with more differentiated forms (AML type M3 and M4).

Antibodies 80H.5 and 81H.1 reacted with 10-50% of the myeloblasts obtained from differentiated AML (M3 and M4). In the M1 and M2 forms, the percentage of positive cells varied from 0% to 30% of the leukemic cell population, with three patients being completely negative when tested with these MCA (unpublished observations). A different pattern of reaction was observed wir.h the different myeioid populations obtained from CML patients during the chronic phase of the disease. Thi:~ pattern was the same as those observed ~ith

TABLE 6 Percentage of colony inhibition by cytotoxic monoclonal antibodies (MCA) ,

C F U - G M

Dilutions 8 0 H . 5 8 | H . ~

1:3 95 .0 92 .4 1:20 88 .6 86 .9 1:400 76.1) 5 | .0 1:4,000 67.8 O 1:40 ,000 9.3 ~

Notv: MCA was diluted in alpha minimal essetMal medbim plus 20% fetal calf serum, and after incubati*m, ,:ei~s were plated on feeder layers. The percentage of inhibition was calculated by comparing the number tff ¢ ~ r ~ s nb~etved after treatment with MCA plus compl~ment to the number of cohmies obtained with MCA al*~m.- ~m,¢~"i of three experiments).


normal myeloid bone marrow cells; for example, 80H. I and 80H.3 recognized mainly mature forms of myeloid lineage, whereas 80H.5 and 81H.I stained all the myeloid lineage cells gTable 2).

DISCUSSION This study describes two kinds of MCA raised against ce!!s from the granulocyte/ monocyte lineage. These MCA appear specific for this lineage since they did not recognize other blood cell subsets. Antibodies 8OH. i and 80H.3 defined mature or late antigens common to neutrophils and monocytes and those not expressed on the early stages of the myeloid pathway. This pattern was confirmed when testing the reactivity of these MCA on leukemic cells and myeloid cell lines. The 8OH. 1 antigen was not expressed and the presence of 80H.3 was detectable only on the more differentiated forms of myeloid cell lines {HL(~) or leukemic cells. When tested against bone marrow celL's, 80H.3 reacted strongly with metamyelocytes and granulocytes and weakly with promyel~gytes and myelocytes, thus suggesting the progressive appearance of this antigen during the maturation process. The serologic patterns of these two antibodies, when tested on different cell subsets, were different suggesting ~hat they recognized different antigens or different constituents of the same antigen. It is unlikely that these antibodies bound nonspccifically to cells by the receptor for the Fc fragment of IgG. The few Fc receptors expressed on granulocytes are ofh~w affinity [24] and treatment of cells by paraformaldehyde would inhibit the nonspecific binding of proteins [ 16]. Moreover, these antibodies did not bind B lymphocytes or B-lymphocyte cell lines.

In contrast, 80H.5 and 81H. 1 recognized early myebid differentiadtm antigens expressed on committed progenit~r cells and their progeny. They inhibited CFU-GM growth but not BFU-£ and CFU-E, thus demonstrating that these stem cells have distinct antigens. It has been demonstrated in the mouse that the antigenic pattern of pluripotential cells is different from CFU-E. The cyt~noxic effect of 80H.5 and 81H.1 on cobny growth ~ppe~rs specific and is different to the inhibition obtained by mon¢glonal or ~lo~til~u3dies against HLA or la antigens, which suppress equMly the growth ofervthroid ~ mye|oid colonies {25- 27, and data presented here). The inhibitMn of CFU-GM growth by 80H.5 and 81 f t . 1 would appear to be due m the s~ifk killing d myebid pr~genit~r cells and not to some effect on lymphoM or access~gy cell populations since 80H.5 and 81H. 1 did not react with such cells. The s~if ic i ty of the inhibition ¢~f CFU- GM growth was suplx~rted by the inhibitMn ob~t~ed w ~ n pl~ental condi'ioned medium was used as a source of cobny-stimuhtit~ ~ctivity. This is the first example of specific inhibition of the CFU~3M by mom~hmal ant/bodies. Similar results have been reported by using heteroantibod~ developed in rabbits immunized with human neutrophils [28]. However, the reactivity of the~ antibodies is relatively weak compared to the killit~g ind~ed by the MCA reported here, and this heteroantiserum re~ts strong|y with human monocytes. T~e binding and the cytotoxic activity of 801t.5 and 8 |Hoi on mature gr~nulocytes, metamyelocytes, myelocytes, and promyelocytes were readily assessed. The binding to myeloblasts was more difficult to pro~e since there is no avaihble technique to obtain a pure population of m y e l o ~ s . The FACS analysis and the microscopic examination showed that more than ~)Oe~ o of the m~,e|oid eel|s, obtained from normal bone marrow or from patients ~ t h CML bound these MCA, suggesting that myeloblasts were also recogrfized by them. However, these antibodies did not react with poorly differentiated myelobhsts fro~ p~dents with AML type M 1 cells and very infrequently with blasts from ~ n t s with AML t y ~ M2 cells;

MCAs to Human Myeioid Antigens 321

whereas, the reactivity on M3 and M~t blast cells was stronger. This was also observed with leukemic cell lines; only differentiated cell lines ~HL.60, ML. |~ express these antigens on a large number of cells. The nonexpression o f such antigens on myeloid leukemic cells probably results from the loss o r absence ,~f synthesis of some membrane structures, as repor ted in patients with chroni~ granulocytic leukemia [29]. The absence of such antigens from the cell surface might: result from genetic dysfunction, an antigenic modulat ion induced by ex- ternal factors, or cell cycle related events.

The antigens recognized by these fi)ur MCA differ from the neutr,~phil alloantigens (NA, NB) since they do not appear to have allotypic variar~ts and since the neutrophi l alloantigens are not t,xpressed on monocytes or on myeloid progeni tor cells [ 15].

It is difficult to assess whether 80H.5 and 81H.1 recognize the same determinant or two distinct antigens. One ~80H.5) has been raised after immunizat ion with normal granulocytes and has reacted with a larger populat ion of eosinophils and leukemic cells than 81H. 1. The second one (81H. 1) has been obtained by immunizing with CML cells. Reactivity on normal neutrophils is s t ronger than for 80H.5 . To the contrary, the C F U - G M inhibition is weaker. Although this could be due to a different affinity, this may suggest that these two MCA recognize distinct determinants . The MCA repor ted here could be useful to positively select normal myeloid progeni tor cells and to study the myel~)id pathway and the re- lat ionship between mononuclear and polymorphonuclear phagocytic cells.

ACKNOWLEDGMENTS This study was supported by a grant from the Alberta Heritage Fund for Medical Research.

We wish to acknowledge Mrs. A. de Boer and Mrs. S. Shamim for excellent technical assistance in these studies and Mrs. S. Fownes fi~r preparing the manuscript. We thank Dr. T. Mossman and Dr. B.M. Longenecker for their continuous support and their helpful discussions during this study and in the preparation of the manuscript. Thanks also to Drs. J.C. Cailaghan and E.T. Gelfand who provided us with bone marrow cells obtained during cardiac surgery. The FACS analysis was perfi~rmed in The Naval Blood Re~, rch (Dr. Valeri) in Boston with the help of Dr. Horland and B. Weiblen.

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MCAs to Human Myeloid Antigens ~2~

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Monoclonal antibodies against human granulocytes and myeloid differentiation antigens

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