Fcγ-receptor-bearing, non-B lymphocytes in human peripheral blood: Cytophilic immunoglobulin binds almost exclusively to large granular lymphocytes

CELLULAR

IMMUNOLOGY

90,

196-207 (1985)

Fey-Receptor-Bearing, Non-B Lymphocytes in Human Peripheral Blood: Cytophilic lmmunoglobulin Binds Almost Exclusively to Large Granular Lymphocytes ANNE B. WILSON AND R. R. A. CGGMBS Division of Immunology,Departmentof Pathology, University of Cambridge, Cambridge, England Received July 9, 1984; accepted August 6, 1984 Cytophilic IgG (CYT-Ig) has previously been reported to bind to both the “To” (E+, FcrR+) and “L” (E-, FcyR+) subsets of non-B lymphocytes in human peripheral blood. Present investigations show that IgG-binding cells, as detected by a sensitive antiglobulin resetting reaction, are contained almost entimly within the large gtanular lymphocyte (LGL) subpopulation, and that fewer than 5% of other non-B lymhocytes acquire IgG from serum. Cell membranebound IgG sterically blocks the reaction of LGL with sheep red blood cells and therefore influences the proportions of these cells characterized as To (E+) or L (E-) lymphocytes. Although the majority of To lymphocytes are LGL, a further subpopulation of E+, FcyR+ cells are detectable under particular test conditions. Unlike LGL, these lymphocytes do not react with rabbit &G-coated ox RBC (EAo) in saline, but will form EAo rosettes when the reaction is enhanced in the presence of Ficoll. These FcyR+ cells are mostly of typical smalllymphocyte morphology and do not bind detectable amounts of CYT-Ig, nor do they express the monoclonal antibody-defined VEP 13 determinant associated with FcyR on LGL. o 1985 Academic Press,Inc.

INTRODUCTION The binding of cytophilic immunoglobulin IgG (CYT-Ig) to a non-T, non-B (null) cell subpopulation of human peripheral blood lymphocytes was demonstrated by Horwitz and Lobo (1) and by Kumagai et al. (2). CYT-Ig is released from cellmembrane receptors in serum-free medium at 37’C and, because of its labile binding, lymphocytes acquiring CYT-Ig were termed “L” cells (3). These cells bear high avidity (or density) receptors (FcyR) for the Fc portion of IgG which is reported to bind not only as aggregatebut also in the monomeric form (4). A proportion of E-rosette-forming (E+) human lymphocytes also have high avidity FcyR, indistinguishable from those of L cells (5), and will bind monomeric IgG (6). These cells belong to the To lymphocyte subpopulation, characterized as E+, FcyR+ cells. Like L cells, To lymphocytes will react with EAo complexes formed either as human red blood cells (RBC) coated with Ripley anti-CD Rh antibodies (7) or as rabbit IgG-sensitized bovine (ox) RBC (8). A proportion of L cells and To lymphocytes also share a similar morphology and cytochemistry (9), and belong to the large granular lymphocyte (LGL) subset which includes effector cells for antibody-dependent (K) and natural (NK) cytotoxic responses (10). Both the E+ and E- effector cells for K-cell killing have high avidity FcyR (1 l), and the E+ 196 OOOS-8749/85$3.00 Copyright 6 1985 by Academic FVess,Inc. AU rights of reproduction in any form reserved.

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fraction of NK effector lymphocytes are reported to bind monomeric IgG (12). In addition, IgG has been shown to inhibit both antibody-dependent (13) and NK ( 14) target cell killing. In the present report, these observations are drawn together in a study on the human peripheral blood lymphocytes which bind CYT-Ig, as detected by the direct antiglobulin rosette-forming reaction (DARR). When this reaction is carried out using ox RBC as a carrier for anti-immunoglobulin antibody, it is greatly enhanced by pretreating the RBC with trypsin (15-17). The DARR is then more sensitive than direct immunofluorescent antiglobulin staining (DIF) for the detection of lymphocyte cell-membrane Ig ( 18). Recent investigations ( 17) have shown that the DIF-, DARR+ lymphocytes become negative in the DARR after prolonged incubation at 37°C and that they carry passively acquired CYT-Ig which may be abnormally firmly bound to FcyR by exposure to Ficoll during lymphocyte purification. The lymphocytes binding CYT-Ig have been characterized, as described below, using the enhanced DARR in combination with lymphocyte subpopulation depletion and morphological examination of Ig-bearing cells. These investigations show that CYT-Ig binds to over 95% of cells with LGL morphology, and that few (~5%) other non-B lymphocytes acquire surface Ig from serum in vitro. A further subset of Ef lymphocytes with lower avidity for complexed IgG has been demonstrated by reacting with EAo in the presence of Ficoll which enhances cell-cell interactions, including EAo-rosette formation (19). These To lymphocytes do not bind detectable amounts of CYT-Ig. MATERIALS

AND METHODS

Preparation of Peripheral Blood Lymphocytes Lymphocytes were isolated by different procedures depending on the requirement for high or low retention of CYT-Ig. Full details of these procedures have been described previously (17). Briefly, blood from normal donors was defibrinated and lymphocytes were isolated on Ficoll-Hypaque at either 20°C (Ficoll-Hypaque 61.077, 4OOg,40 min), to give lymphocytes carrying relatively large amounts of CYT-Ig, or at 37°C (Ficoll-Hypaque 61.09, 4OOg,40 min), to provide lymphocytes free of surface-bound Ig. Phagocytes were removed with carbonyl iron either before or after gradient centrifugation. Purified lymphocytes were washed three times at room temperature (RT) or 37°C as appropriate, and twice more at 4”C, using phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA, crystalline Armour). Lymphocyte preparations contained 0 to 3% monocytes by morphology and esterase positivity and approximately 1% basophils; both cell types were excluded from counts of reacting lymphocytes. In Vitro Passive Uptake and Release of Lymphocyte-Cytophilic Ig Maximum binding of CYT-Ig was achieved by placing lymphocytes in 10% fresh autologous serum, diluted in RPM1 1640 medium, at 4°C for 1 hr or longer. The lymphocytes were then washed three times with cold PBS/BSA before testing by the DARR. To induce the release of FcyR-bound CYT-Ig, lymphocytes were incubated at 37°C in RPM1 1640 medium, pH 7.2, buffered with 20 mA4 Hepes and containing

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10% heat-inactivated FCS with penicillin (100 IU ml-‘), and streptomycin (100 pg ml-‘). AFter 2-3 hr, lymphocytes were washed three times with PBS/BSA at RT, and tested for surface-Ig.

Antiglobulin and Other Antibodies for Coupling to Ox RBC IgG and IgM fractions of the following reagents were prepared as described previously (17) and were dialysed against 0.9% sodium chloride. Sheep anti-human Fab antiserum (T379/4) was obtained from Mr. J. Herbert (Institute of Animal Physiology, Babraham), and sheep antibodies specific for human Ig isotypes (236 1G anti-IgD, 263 1V anti-IgM, Z5 11G anti-IgG, Z649U anti-&A, and 285 1G anti+@) were gifts from Dr. A. R. Bradwell (Immunodiagnostics Unit, University of Birmingham). Mouse monoclonal antibodies to human Ig light chains (clone 6el antkK and clone C4 anti-X) were provided by Dr. N. R. Ling (University of Birmingham), who also supplied mouse IgGl hybridoma antibodies (JL5 12, JG4, and RJ4) to human IgG subclasses 1, 3, and 4, respectively. A mouse IgM monoclonal antibody (VEP 13) to a determinant associated with FcrR on LGL and polymorphonuclear neutrophils was obtained from Dr. D. Kraft (University of Vienna), and a rat IgG2a monoclonal antibody to an MHC class II, Ia-like, determinant (YCL 6.8) antibody was provided by Dr. H. Waldmann of this department. Control IgG and IgM reagents were prepared from normal sera of sheep, mouse, and rat or from ascitic fluids containing monoclonal antibodies unreactive with human leucocytes. All reagents were coupled to ox RBC at protein concentrations between 0.75 and 2 mg ml-‘.

Preparationsof Antibody-CoupledOx RBC and RosetteFormation with Lymphocytes Relatively “inagglutinable” (Inagg) and “agglutinable” (Agg) ox RBC were obtained from animals (A40 and E16) at the Institute of Animal Physiology, Babraham. Sheep anti-human Fab was coupled by chromic chloride to both untreated Inagg and Agg Ox RBC and to trypsin-treated (TT) Inagg ox RBC. Other antibodies were chromic chloride-linked to TT Inagg ox RBC only. Details of the trypsinization of RBC and antibody-coupling procedure, together with the results of haemagglutination assayscomparing the performance of the three types of anti-Fab-coupled RBC are given elsewhere ( 17). Antibody-coupled RBC were used as a 1% suspension in PBS and were stored for up to 2 weeks at 4°C. For rosette formation, equal volumes (25 ~1) of lymphocytes (2-3 X lo6 ml-‘) and RBC were mixed in 5 X 50-mm tubes and centrifuged (35Og, 2 min, 4°C). Rosettes were resuspended after 30 min at 4°C and transferred with toluidine blue stain to siliconed microscope slides. Between 200 and 1000 lymphocytes were examined per slide. Although included in every test, control RBC carrying nonantibody IgG or IgM were consistently negative, and results are not shown.

SheepRBC and EAo Sheep RBC were well washed (X5) and treated with papain (20). E rosettes were formed by mixing lymphocytes and 1% pap-SRBC for 15 min at RT, and then centrifuging as above. Rosettes were left 1 hr or longer at 4°C before being resuspended. EAo complexes were produced by sensitizing “agglutinable” (E 16) ox RBC (2% v/v) with an equal volume of DEAE-cellulose-purified rabbit IgG anti-ox RBC for

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45 min at RT, then washing three times with PBS. The IgG antibody was used either at the maximum subagglutinating dilution (1:40) or at a suboptimal dose ( 1:160). EAo rosettes were usually prepared in PBS as described above for antibodycoupled ox RBC. In some tests, EAo binding was enhanced by rosetting in the presence of 14% Ficoll using the method of Binns and Licence (19). For this, Agg ox RBC were sensitized with 1:160 diluted rabbit IgG antibody, and the rosettes were examined after 1 hr at 4°C. Depletion of Lymphocyte Subpopulations

B lymphocytes were removed from cell suspensions prepared free of CYT-Ig. Up to 1S-ml aliquots of lymphocytes (3 X lo6 ml-‘) were mixed with equal volumes of 1% untreated Agg ox RBC coupled to sheep anti-human Fab antibody and centrifuged at 35Og for 5 min at 4°C. After 30 min at 4”C, the rosettes were resuspended and layered over Percoll (Pharmaciaj which had been made isotonic and adjusted to 61.077 with PBS. Following centrifugation at 400g for 10 min at RT, interface lymphocytes, free of rosettes, were washed twice with PBS/BSA at RT and twice at 4°C. FcyR-bearing and VEP 13+ lymphocytes were similarly depleted following interaction with EAo in PBS or with TTox RBC linked to VEP 13 monoclonal IgM, respectively. Giemsa-Stained Cytocentrifuge Preparations

Unreacted lymphocytes or aliquots of rosetted cells were diluted in 10% FCS and spun (500 rpm, 5 min) onto microscope slides using a “Cytospin” cytocentrifuge (Shandon). The slides were dried, fixed 15 min in methanol, and stained 10 min with 10% Giemsa (Gurr) in PBS. RESULTS Identification

of Cytophilic Ig-Bearing LGL in Fresh Lymphocyte Suspensions

Previous investigations (17) showed that the binding of CYT-Ig to fresh human blood lymphocytes is artefactuahy increased by separating the cells on FicollHypaque at 20°C or below, although lymphocytes isolated on the same medium at 37°C lack detectable labile Ig. The typical pattern of reactivity given by lymphocytes separated at 20°C or at 37°C when tested with anti-Fab-linked ox RBC and with other markers is shown in Table 1. These lymphocyte suspensions contained similar percentages of B cells, as shown by their expression of IgD and IgM and by close agreement in the percentages of Ia+ lymphocytes, the majority of which are B cells. The presence or absence of CYT-Ig on non-B lymphocytes, however, was clearly demonstrated by differences at the two temperatures in the percentagesof lymphocytes reacting with ox RBC coupled to sheep anti-Fab or mouse monoclonal anti-light chain antibodies. Polyclonal anti-Fab antibody on TTox RBC provided the most sensitive test for CYT-Ig on lymphocytes isolated at 20°C. These antiglobulin-linked RBC reacted with all B cells plus a mean of about 20% non-B lymphocytes carrying surface-bound CYT-Ig. Untreated Agg ox RBC coupled to anti-Fab were less sensitive, reacting with all B cells and a smaller proportion (approx 10%) of CYTIg-bearing lymphocytes, while untreated Inagg ox RBC-anti-Fab indicator cells

200

WILSON AND COOMBS TABLE 1

Detection of Surface lmmunoglobulin (SIg) by the Direct Antiglobulin Resetting Reaction, and Tests for Other Phenotype Markers, on Peripheral Blood Lymphocytes Isolated on Ficoll-Hypaque at 20°C and 37°C Percentage of reacting lymphocytes Cells isolated at 20°C

Cells isolated at 37°C

Inagg ox RBC-sheep anti-Fab Agg ox RBC-sheep anti-Fab TTox RBC-sheep anti-Fab

10.7 + 3.1 21.0 + 5.9 27.9 f 6.5

7.0 * 1.1 9.0 +- 1.3 9.6 + 1.5

Light chains

TTox RBC-mcl 6el anti-r TTox RBC-mcl C4 anti-X

14.9 + 3.3 9.7 + 2.3

6.2 f 0.8 2.9 + 0.7

Isotypes

TTox RBC-sheep anti-IgD TTox RBC-sheep anti-IgM

6.0 + 1.5 4.2 f I.0

5.4 f 1.0 3.5 f 0.6

Ia+

TTox RBC-mcl YCL 6.8 anti-la-like determinant

11.8 + 1.2

10.4 f 1.2

FcyR+

EA, (W,

26.4 f 5.1’

17.4 f 3.7”

E-RFC

Pap-SRBC

78.6 f 4Sb

86.7 f 2.26

Lymphocyte subpopulation

Marker

Fab SIg+

l/40)

Note. Results are expressed as means f SEM (6 experiments). Retention of cytophilic immunoglobulin on non-B lymphocytes at 20°C is shown by high reactions with anti-Fab and anti-Ig light chain antibodies (figures in bold type). 0 As reported previously (17), the proportions of EA,-binding lymphocytes are consistently higher when cells are separated at 20°C compared to 37°C. This may result from Ficollenhanced binding of EA, to FcyR on lymphocytes prepared at 20°C, and/or from spontaneous shedding of FcyR in serum-free medium at 37°C. bThe percentages of E-RFC vary inversely with the amount of cytophilic immunoglobulin bound to lymphocytes. This is discussedin the text relating to in vitro-binding experiments.

detected only 70-80% of B cells and were extremely insensitive for the detection of CYT-Ig. Granular lymphocytes, containing cytoplasmic granules of variable size and number, were identified in Giemsa-stained cytocentrifuge preparations of unrosetted peripheral blood lymphocytes isolated at either 20 or 37°C. These cells usually represented between 10 and 25% of total lymphocytes, and their number was unaffected by the method of isolation. Although most granular lymphocytes were of typical LGL morphology, a minor population appeared smaller, with a lower cytoplasm:nucleus ratio. These cells were similar to LGL, however, in regard to IgG-binding and reactivity with VEP 13 monoclonal antibody, which identifies an FcyR-associated determinant found only on the granular subpopulation of lymphocytes (2 1, 22), and they were included in counts of LGL. Examination of cytocentrifuge spreads of lymphocyte rosettes formed in the DARR showed that the proportions of LGL which reacted with the most sensitive indicator, TTox RBC-sheep anti-Fab antibody, varied inversely with the temperature of lymphocyte preparation. Thus, in cell suspensions isolated from the same blood sample at 37, 20, and 4”C, the percentages of LGL carrying surface-Ig were 3, 75, and 98%, respectively. In a further four preparations of lymphocytes separated at

HUMAN

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20°C between 55 and 88% of LGL reacted with TTox RBC-anti-Fab. By contrast, only O-4% of LGL in the same (2O”Cprepared) lymphocyte suspensions bound sheep anti-Fab coupled to the least sensitive carrier, untreated Inagg ox RBC. When lymphocytes from three donors were separated at 37°C no Ig-bearing LGL were observed in tests with TTox RBC-anti-Fab. These results, therefore, showed a close correlation between percentages of Ig+ LGL and the degree of retention of CYT-Ig on freshly prepared peripheral blood lymphocytes, and they provided the basis for more detailed in vitro studies. In Vitro Uptake and Release of Lymphocyte-Cytophilic Ig, as Monitored by the DARR Initial tests showed, as expected, that CYT-Ig was slowly releasedfrom lymphocytes in FCS at 37°C but not at 4°C (Table 2). Incubation of lymphocytes in autologous serum at 4°C led to an increased uptake of Ig, so that it was detectable by the most sensitive DARR on up to 30% of total lymphocytes. Giemsa-stained preparations of lymphocyte rosettes formed with TTox RBC-anti-Fab again showed a direct correlation between the binding of CYT-Ig and the numbers of Ig+ LGL, the proportion of reacting LGL varying from over 90% following exposure to serum at 4°C to 5% or fewer after 37°C incubation in FCS. The binding of CYT-Ig at 4°C led to a reduction in lymphocyte reactivity with both EAo and SRBC (Tables 2 and 4). Partial blocking of lymphocyte FcrR by cell membrane-bound CYT-Ig was reversible, and was most evident in tests using suboptimally sensitized EAo . With regard to E rosetting, it has been noted previously (17) and shown in Table 1 that the percentage of SRBC rosettes formed by freshly isolated lymphocytes varies inversely with the number of lymphocytes carrying CYT-Ig. In vitro IgG-binding experiments further showed that the retention of

TABLE 2 Effect of Incubating Lymphocytes” in Foetal Calf Serum or Autologous Human Serum at 37°C and at 4°C on the Retention (Figures in Bold Type) or Release of Cytophilic Immunoglobulin, and Its Influence on Measurement of Fey Receptors and E Rosetting Percentage of reacting lymphocytes

Incubated in FCS Lymphocyte subpopulation

Incubated in autologous serum

Marker

Prior to incubation

4°C

37°C

4°C

s1g+

Inagg ox RBC-sheep anti-Fab TTox RBC-sheep anti-Fab

3 20

4 20

3 6

8 30

FcyR+

EAo (IgG l/160)

27

29

20

12

E-RFC

Pap-SRBC

81

81

83

70

a Lymphocytes were isolated from whole blood on Ficoll-Hypaque (20”(J), washed, and then treated with carbonyl iron in RPM1 1640/10% FCS (37°C 30 min.). They were incubated for 2 hr in either 10% FCS or 10% fresh, autologous serum and subsequently washed 3X before testing.

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CYT-Ig in sufficient amounts sterically blocks, again in a reversible manner, the interaction of SRBC with approximately 10% of total lymphocytes. In agreement with other reports (lo), it was found that at least 50% of LGL will bind SRBC, and subpopulation depletion experiments confirmed that the blocking of rosette formation by IgG occurs on these cells. Characterization of Ig-Binding Lymphocytes as LGL Plus a Minor Subset of Other Non-B Cells In order to determine precisely the proportion of CYT-Ig-binding cells represented by LGL it was first necessary to remove B lymphocytes carrying innate surface Ig. Lymphocytes prepared free of detectable CYT-Ig were therefore reacted in the DARR, and rosette-forming B cells were separated out by centrifugation on Percoll (Tables 3 and 4). The percentagesof EAo-reacting and CYT-Ig-binding lymphocytes in these B cell-depleted suspensions were similar to each other and to those of LGL, whether the latter formed a low percentage (8%) of total lymphocytes, as in Table 3, or a higher percentage (32%), as in Table 4. Removal of the non-B, EAo-binding cells by rosette depletion (step 2, Table 3) also removed the majority of LGL and abrogated subsequent uptake of CYT-Ig. Finally, the selective removal of LGL from other non-B lymphocytes using VEP 13 monoclonal antibody (21, 22) left fewer than 5% lymphocytes capable of binding Ig from serum in vitro (data not shown). By examining cytocentrifuge preparations of rosettes formed by the DARR in experiments such as that described in Table 4, it was found that over 95% of LGL can acquire surface-bound Ig, and that these cells represent at least 95% of the lymphocytes carrying detectable CYT-Ig. Furthermore, CYT-Ig can be repeatedly adsorbed by and released from FcyR on LGL without the loss of receptor activity which follows “modulation” by complexed IgG (8), suggesting that CYT-Ig from serum is bound mainly as monomer. As found previously with freshly prepared lymphocyte suspensions (17), CYT-Ig attached to cell-surface FcyR did not react in the DARR with anti-Ig isotypespecific antibodies coupled to TTox RBC. This was clearly demonstrated with nonB lymphocytes which had acquired CYT-Ig from serum in vitro (step 2, Table 4). These cells, unlike IgG-coated monocytes, reacted very weakly, if at all, with antiIgG Fc-specific reagents, and no reactions were seen with antibodies specific to other Ig isotypes. Attempts to characterize lymphocyte-bound CYT-Ig using TTox RBC coupled to monoclonal anti-IgG subclass-specific antibodies (JL5 12 anti-IgGl-Fc, ZG4 anti-IgG3 F(ab’)* hinge region, and RJ4 anti-IgG4-Fc) were unsuccessful. These reagents also failed to react with CYT-Ig-bearing non-B lymphocytes (results not shown), although they bound to B cells expressing IgG. The latter were enumerated as percentages of total lymphocytes (mean f SEM) in the peripheral blood of nine normal adults, and gave results showing 1.4 f 0.4% IgGl+, 1.1 + 0.2% IgG3+, and 0.2 + 0.2% IgG4+, which are similar to those recently reported in a separate study using the same monoclonal anti-&G subclass antibodies (23). Additional Non-LGL, Fe-y-Receptor-Bearing(Td Lymphocytes Detected by Enhanced EAo Binding in the Presence of Ficoll From the above results, it was apparent that all human peripheral blood lymphocytes which bind CYT-Ig carry FcrR reactive in PBS with EA, complexes

Which Acquire Cytophilic

EAo (IgG. l/40)

Pap-SRBC

FcyR+

E-RFC

8 7 96

7.5 9 92

92

5

8

1

3

5 I

1

Treated autologous serum (4°C)

99

0

1

0

0

0 0 0

Untreated

97

0

1

0

0

0 0

0

Treated autologous serum (4’C)

Nole. Results are given as percentagesof reacting lymphocytes. ’ Lymphocytes were isolated free of cytophilic Ig at 37’C. In step I. B cells were removed by sedimenting DARR rosettes on Percoll. In step 2, B-cell-depleted lymphocytes were rosetted with EAo, and the FcyR+ lymphocytes were removed on Percoll. The two depleted lymphocyte populations were treated with 10% fresh autologous serum at 4°C for I hr. then washed 3X (PBS/I% BSA) at 4’C before testing. b LGL were enumerated in Giemsa-stained cytocentrifuge preparation of undepleted and depleted lymphocyte suspensions.

(Giemsa stain)

I.5

0

0

4.5

TTox RBC-mcl 6el anti-n

TTox RBC-mcl C4 anti-h

Light chains

0

4 0 0

Untreated

Freshly isolated lymphocytes”

B cells and EA& lymphocytes removed

LGL)

B cells removed

Non-B Subset (Mainly

step 2

to the EA,-Binding,

step I

Ig in Vifro (Figures in Bold Type) Correspond

3

6 6

Agg ox RBC-sheep anti-Fab TTox RBC-sheep anti-Fab

Fab

lnagg ox RBC-sheep anti-Fab

Marker

Showing That Lymphocytes

LGLb

s1g+

Lymphocyte subpopulation

Experiment

TABLE

5

s D

2 z

$ &I

k B 4 “0

3

,‘I

204

WILSON

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TABLE 4 Experiment

Demonstrating Sequential Adsorption (Step 2, Figures in Bold Type) and Elution (Step 3) of Cytophilic Ig from Lymphocytes in a B-Cell-Depleted Preparation

Lymphocyte subpopulation

Ig+

Marker

Fresh lymphocytes lacking CYT-I@

step I B removed

step 2 Autologous serum (4°C)

step 3 = (37OC)

Fab

Inagg ox RBC-sheep anti-Fab Agg ox RBC-sheep anti-Fab TTox RBC-sheep anti-Fab

6.5 7 8.5

0 0 0

16 33 35’

0 0
Light chains

TTox RBC-mcl 6el anti-x TTox RBC-mcl C4 antid

7

0

26

1.5

0

9.5

0 0

1sotypes

TTox TTox TTox TTox TTox

5 1.5 <0.5 I
0 0 0 0 0

0 0 2.5 0 0

RBC-sheep anti-IgD RBC-sheep anti-IgM RBC-sheep anti-IgG RBC-sheep anti-@ RBC-sheep anti-IgE

LGL

(Giemsa stain)b

24 (3%)

32 (0)

32 (96%)

32 (I%)

FcyR+

EA, UgG, l/40)

28

33

22

29

13

19

10

22

82

81

17

87

EA, UgG. E-RFC

PapSRBC

W60)

Nom. Results are shown as percentagesof reacting lymphocytes. “Lymphocytes were isolated at 20°C. then incubated in 10% FCS at 37°C to release cytophilic Ig. (Prior to 37°C incubation 19% of lymphocytes carried cytophilic Ig, including 65% of the LGL subset;data not shown.) Following the removal of B cells as DARR rosettes on Percoll (step l), non-B lymphocytes were first exposed to 10%autologous serum (1 hr, 4’C) in order to adsorb cytophilic Ig (step 2) then incubated Ii/z hr at 37°C in 10%FCS for subsequentreleaseof acquired Ig (step 3). b LGL were enumerated in Giemsa-stained slides of undepleted and B-cell-depleted lymphocyte preparations. Figures in parenthesesrepresent percentagesof the LGL which carried surface-Ig, enumerated from stained cytocentrifuge spreads of TTox RBC-anti-Fab rosettes. ’ LGL represented 95% of the lymphocytes which passively acquired Ig from serum (see text).

formed by optimally sensitized, rabbit IgG-coated “agglutinable” ox RBC. Observation of fixed, stained EAo rosettes given by lymphocytes under the usual test conditions (i.e., in PBS/BSA) showed that, in addition to reacting with a proportion of B cells, the EAo also bound to between 95 and 100% of LGL and to a small number (~5%) of other non-B lymphocytes. Additional lymphocytes bearing FcyR of lower avidity (or density) were detected, however, by rosetting with EAo in the presence of 14% Ficoll as described by Binns and Licence (19). In 14 tests on the lymphocytes of 11 normal persons (cells separatedon Ficoll-Hypaque at 2O”C), the proportion of E& rosettes was increased from 26.8 + 2.5% (mean -t SEM) to 34.0 zk 2.8% in Ficoll. Lymphocyte subpopulation-depletion experiments showed that the majority (approximately 80%) of the cells with low avidity FcyR also formed E rosettes. From cytocentrifuge preparations of EAo rosettes given by non-B lymphocytes

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(two preparations), it was found that the proportion of rosettescontaining nongranular lymphocytes had increased from around 5% in PBS to between 25 and 40% in Ficoll. Most of the additional E+, EAo-binding cells were of typical small lymphocyte morphology, and it was concluded that they represent a non-LGL subset of To lymphocytes which carry relatively low avidity FcyR and do not bind CYT-Ig in detectable amounts. DISCUSSION A variable proportion of the Ig-bearing cells measured by the DARR in human peripheral blood lymphocyte suspensions are FcyR+, non-B lymphocytes which carry passively acquired, not innate, surface-Ig. Although lymphocytes binding CYT-Ig have previously been shown to occuf in both the null, L (1, 24) and To (5, 6) subpopulations, they have not previously been examined as a whole in B celldepleted suspensions. The current investigation has shown that over 95% of lymphocytes carrying CYT-Ig have LGL morphology, and that between 95 and 100% of LGL, carrying FcyR detectable by VEP 13 monoclonal antibody, can acquire surface-bound Ig from autologous serum. Since 50% or more of the LGL have an affinity for SRBC, it is apparent that the E- subset of LGL comprise the null, L cells, while the E+ LGL belong to the To lymphocyte subpopulation. These subsets, therefore, share a similar morphology and an ability to bind CYT-Ig by means of high avidity FcyR. They differ, among other things, in their capacity to form rosettes with SRBC, but as this reaction is influenced by the amount of CYTIg retained on the LGL, the relative proportions of LGL identified as To or L cells will depend, at least in part, on this variable factor. Although it is clear that most, if not all, L cells are LGL, there is little doubt that a proportion of To cells are of a different morphology. Human To lymphocytes separated from peripheral blood as E+, EAo-rosetting cells by Ferrarini et al. (9) contained only 80% of cells with LGL morphology, while Timonen et af. (10) reported that approximately 40% of the lymphocytes binding to monolayers of immune complexes were non-LGL which lacked NK and K cytotoxic functions and, unlike LGL, had a high a5nity for SRBC. Also, a monoclonal antibody (OKT3) to a determinant on mature T lymphocytes which reacts with few LGL (25) or NK or K effector cells in normal human peripheral blood (6, 26, 27), has been reported to bind to between 14 and 72% of purified human To lymphocytes (28, 29). Although LGL can be made to express the OKT3 antigen when cultured for several days in suitably conditioned medium (30) it would appear that the OKT3+ cells isolated in To fractions of normal blood lymphocytes are predominantly nongranular lymphocytes. A similar subset of E+, FcyR+ lymphocytes, morphologically distinct from LGL and unreactive with VEP 13 monoclonal antibody, has been demonstrated in the present investigations. These cells represented up to 40% of FcyR+ non-B lymphocytes, but compared to LGL they were found to have a much lower avidity for IgGFc. Thus, they were shown to bind EAq complexes only when the reaction was enhanced by the presence of Ficoll, and they did not acquire detectable amounts of CYT-Ig from serum. Becauseof their relatively weak binding to IgG, the proportion of nongranular lymphocytes isolated in purified To preparations will vary according to the sensitivity of the test for FcyR activity. Further investigations are required,

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therefore, to determine the part played by non-LGL T cells in the mitogen-induced and other responses which have been observed with To (8) but not with L cell preparations (3 1). Earlier reports that both K and NK effector cells in human blood were Ig-positive in DARR tests using anti-Fab antibodies linked to trypsinized ox RBC (32, 33) were apparently contradictory to others claiming that the cytotoxic cells lacked surface Ig when tested by immunofluorescent antiglobulin and other techniques. Since all NK and most K effector lymphocytes are contained within the LGL subpopulation (lo), this discrepancy can be explained by the present finding that these lymphocytes carry passively acquired Ig in amounts which are usually detectable only by very sensitive tests such as the DARR. The lack of reaction observed with polyclonal and monoclonal antibodies specific to human IgG in DARR tests on LGL carrying CYT-Ig idicates that the corresponding determinants of lymphocyte-bound IgG are inaccessible for interaction with these RBC-coupled antibodies. Using other antibodies, however, the DARR-type of assay is proving especially suitable for examining non-Ig cell-surface components of LGL. A particular advantage of these rosetting reactions is that they carry little risk of nonspecific binding of IgG antibodies to cell-membrane FcyR, apparently as a result of the blocking of IgG-Fc sites during chromic chloride-coupling of antibody to the carrier RBC (34). Although LGL form a heterogeneous population with regard to cell-surface markers and functions, their characteristic morphology makes them readily distinguishable from other lymphocytes, and the reactivity of LGL with monoclonal anti-T cell and other antibodies is currently being investigated by examination of stained cytocenttifuge slides of rosetted preparations. This method allows a study to be made of LGL subsets as a whole, and, for some tests, it has advantages over procedures utilizing LGL-enriched preparations. ACKNOWLEDGMENTS This work was supported by grants from the Wellcome Trust and the Sir Halley Stewart Trust. We thank Mrs. V. Woods for excellent technical assistanceand Mrs. A. Hancock for typing this manuscript. We are also grateful to Dr. A. R. Bradwell, Dr. N. R. Ling, Mr. J. Herbert, Dr. D. Kraft, and Dr. H. Waldmann for generously supplying monoclonal and polyclonal antibodies.

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