Clin. exp. Immunol. (1978) 34, 281-287.
Receptors for IgG (Fc receptors) on human lymphocytes: re-evaluation by multiple techniques P. J. CLEMENTS & J. LEVY Department of Medicine, UCLA School of Medicine, Los Angeles, California, USA
(Received 24 April 1978)
SUMMARY
This study examines whether receptors for IgG (Fc receptors), as identified by different methods, are found on identical human lymphocyte subpopulations, and the relationship of Fc receptors to surface immunoglobulin (SIg) and receptor for complement (C'). Fc receptors were identified by two rosetting techniques (antibody-sensitized human erythrocytes, HuEA, or sheep erythrocytes, ShEA) and two immunofluorescent techniques (heat-aggregated IgG of human origin, HuAgg, or of guinea-pig origin, GPAgg). When lymphocytes depleted of cells resetting with ShEA were compared to HuEA-depleted lymphocytes, the two subpopulations appeared to be significantly different. However, when lymphocytes were depleted of cells rosetting with ShEA which had been sensitized with lower concentrations of antibody, the subpopulation so depleted appeared to be virtually identical to HuEAdepleted lymphocytes. These studies suggest that more than one lymphocyte subpopulation has Fc receptors and that each subpopulation can, in part, be identified and distinguished from the other by the capacity to bind IgG at differing concentrations. In particular, these experiments may serve to resolve the controversy concerning the presence of Fc receptors on lymphocytes bearing surface immunoglobulin (SIg). Depletion of lymphocytes rosetting with ShEA removed most of the SIg-bearing lymphocytes; depletion of cells resetting with ShEA which had been sensitized with lower concentrations of IgG antibody, however, failed to deplete SIg-bearing lymphocytes even though other Fc-bearing populations were completely depleted. These results suggest that SIg-bearing lymphocytes (B lymphocytes) do have Fc receptors but that high concentrations of IgG are needed to demonstrate them.
INTRODUCTION Human thymus-derived (T) lymphocytes are commonly recognized by surface receptors for sheep red blood cells (SRBC), whereas bone-marrow derived (B) lymphocytes are recognized by surface immunoglobulin (SIg) and receptors for complement (WHO/IARC Workshop, 1974). Another group of cells are distinguished by having surface receptors for the Fc portion of IgG immunoglobulins (Fc receptors). Depending upon the situations, these Fc receptors have putatively been detected on B lymphocytes, T lymphocytes, and lymphocytes without the common T and B markers (reviewed by Dickler, 1976). Since Fc receptors in these various reports were assayed by different methods, many of the discrepancies may be artifacts of the techniques used to detect them. Because of this, this investigation examined the question of whether Fc receptors, as identified by four different methods, were found on identical lymphocyte subpopulations and the relation of these Fc receptors to the conventional T and B markers. MATERIALS AND METHODS Preparation ofreagents. The preparation of all immunoglobulin reagents is outlined in Table 1. Trypsin (twice-crystallized, salt-free from bovine pancreas, Worthington Laboratories, Freehold, New Jersey) was used at a concentration of 5 mg/ml. Sheep red blood cells in Alsever's solution were obtained from Mission Laboratories, Rosemead, Correspondence: Dr P. J. Clements, UCLA/Department of Medicine, 1000 Veteran Avenue, Los Angeles, California 90024, USA. 0099-9104/78/0110-281$02.00 (0 1978 Blackwell Scientific Publications
281
282
P. J. Clements & J. Levy TABLE 1. Preparation of immunoglobulin reagents
Protein and source
Preparation prior to use
IgG fraction of goat antiserum to human Protein was conjugated with IgG, heavy chain specific. Miles fluorescein isothiocyanate (FITC, Laboratories, Kankakee, Illinois isomer I, Baltimore Biologicals Laboratories, Cockeysville, Maryland) by an adaptation of dialysis method of Shepard & Clark (1963) IgG fraction of goat antiserum to Protein FITC conjugated as above. guinea pig gamma globulin, Cappel Laboratories, Downington, Pennsylvania Guinea-pig gamma globulin, Cappel Protein was FITC-conjugated as above. Laboratories. It was aggregated at 630C for 20 min at a protein concentration of 10 mg/ml and diluted to 5 mg/ml prior to use. IgG fractions of rabbit antiserum to Since further purification by DEAE human red blood cells or to sheep red cellulose chromatography did not change blood cells. Cappel Laboratories. the performance characteristics of either antiserum they were used as received from Cappel Laboratories. Human gamma globulins (Fraction II). It was aggregated at 630C for 20 min at a Nutritional Biochemicals Division, ICN, protein concentration of 50 mg/ml and Cleveland, Ohio. diluted to 2 mg/ml prior to use. FITC-conjugated goat polyvalent Antiserum was centrifuged at 50,000 g antiserum to human immunoglobulins. for 30 min prior to use. Meloy Laboratories, Springfield, Virginia
Reagent as finally used FITC-conjugated goat antiserum to human IgG.
FITC-conjugated goat antiserum to guinea-pig gamma-globulin. Heat-aggregated FITCconjugated guinea-pig gammaglobulin. IgG fractions of rabbit antiserum to human red blood cells or to sheep red blood cells.
Heat-aggregated human gammaglobulin. FITC-conjugated goat polyvalent antiserum to human immunoglobulins
California. Human red blood cells (blood type 0+) were obtained from the same normal volunteer throughout the study. Foetal bovine serum (Rehatuin) was obtained from Reheis, Los Angeles, California. Zymosan particles were obtained from Sigma Chemical Company, St Louis, Missouri. Blood and lymphocyte separation. Subjects for these experiments were adult laboratory personnel and normal volunteers. Total lymphocyte counts were determined by routine methods from total white blood cell counts and Wright stained differential blood smears. Mononuclear blood cells were separated from peripheral blood by Ficoll-Hypaque sedimentation (Yu & Clements, 1976). Mononuclear blood cells were incubated for 45 min at 370C with carbonyl iron filings (40 mg carbonyl iron, GAF, for each 1 ml of cell suspension containing 5 x 106 cells) in the presence of 20% foetal bovine serum, to facilitate phagocytosis as well as shedding of cytophilic IgG (Horwitz & Lobo, 1975; Yu, Ramer & Kacena, 1977). Phagocytic cells were removed with the use of a magnet. Purified lymphocytes were washed twice with 37°C media and divided into aliquots for identification of cell surface markers. Identification of cell surface markers. Immunofluorescent detection of surface immunoglobulin of all classes. Using a method similar to that of Horwitz & Lobo (1975), lymphocytes were incubated for 30 min at 4°C in the presence of an FITC-conjugated goat anti-human immunoglobulin antisera and washed. For this and all immunofluorescent experiments to follow, a minimum of 200 viable lymphocytes were examined under phase contrast and epifluorescent microscopy and the percentage of fluorescent cells was determined. Immunofluorescent detection of surface immunoglobulin of the IgG class. A separate aliquot of cells was incubated directly with FITC- conjugated goat antisera to human IgG (FGx-HiIgG) to determine the number of cells with surface immunoglobulin of the IgG class. Immunofluorescent detection of binding of aggregated human IgG (HuAgg). Using an adaptation of previously described methods (Dickler, 1974; Yu & Clements, 1976), lymphocytes were incubated with heat-aggregated human gamma-globulin for 30 min at 4°C. Cells were washed and incubated with fluorescein-conjugated goat anti-human IgG (FGx-HuIgG) for 30 min at 4°C. Immunofluorescent detection of binding of aggregated guinea-pig IgG (GPAgg).A similar technique, adapted from Horwitz & Lobo (1975), was used as in demonstrating receptors for HuAgg. Cells were incubated with heat-aggregated FITCconjugated guinea-pig gamma-globulin at 4°C for 30 min. Cells were then washed and incubated with FITC-conjugated goat antisera to guinea-pig gamma-globulin (FGx-GPIgG) at 4°C for 30 min.
Re-evaluation of Fc receptors on lymphocytes
283
Rosetting with complement-zymosan particles (ZyC) and with sheep red blood cells (E or SRBC). The method for the simultaneous identification of ZyC rosettes and E rosettes has been described previously (Yu & Clements, 1976). SRBC were suspended as a 0 5%y solution in Hanks' balanced salt solution (HBSS) supplemented with 20% foetal calf serum which had previously been absorbed with SRBC. Zymosan granules (Zy) were suspended in HBSS and sonicated. Zy were washed and incubated 30 min at 370C in HBSS supplemented 20% with fresh or deep frozen human serum as a source of complement. Complement-coated zymosan (ZyC) were washed and suspended in HBSS to give an approximate concentration of 1 x 108 ZyC per ml. To form rosettes, 0-1 ml of ZyC, 0-1 ml of the 0.50% solution of SRBC, and 0-1 ml of lymphocytes (5 x 106 per ml) were mixed, incubated at 370C for 5 min, and centrifuged at 50 g for 5 min. After an overnight incubation at 4VC, the mixture was resuspended in the presence of crystal violet (to identify lymphocytes). Resuspension of all rosette assays were done on a vertical rotator for 24 min, and a minimum of 400 lymphocytes were examined afterwards. Lymphocytes with two or more adherent ZyC, or with three or more adherent SRBC's were counted as rosettes. Rosettiug with antibody-sensitized red blood cells. Fc receptors were also evaluated by two rosette methods: (1) The first method adapted from Zighelboim et al. (1974) employed antibody-sensitized SRBC (ShEA). Washed SRBC (suspended in 2%, concentration in HBSS) were incubated for 30 min at 370C with 5 mg/ml of trypsin. After the trypsinized SRBC (T-SRBC) were washed, aliquots were incubated with serial doubling dilutions of the IgG fraction of rabbit antiSRBC antisera for 30 min at 370C. The antibody-sensitized SRBC (ShEA) were washed and resuspended at a 0.5% concentration. 0-1 ml of lymphocytes (5x 106) were mixed with 0-1 ml of ShEA (sensitized at a subagglutinating titre) and centrifuged at 50 g for 5 min. After a 60-min incubation at room temperature, the cells were resuspended by rotation. Cells with four or more adherent ShEA were counted as rosettes. Control experiments in which trypsinized SRBC's (without sensitizing antibody) were used routinely gave less than 2% rosetting lymphocytes. (2) The second method, adapted from Froland, Wisloff & Michaelsen (1974), employed sensitized human red blood cells (HuEA). Washed human red blood cells (HRBC) were incubated at a 2%4 concentration in serial doubling dilutions of a heat-inactivated IgG fraction of rabbit anti-HRBC antisera for 30 min at 37°C. The HRBC sensitized at subagglutinating titre were washed and resuspended at a 1% concentration. The rosetting procedure was identical to that used for ShEA rosettes. Depletion of lymphocyte subpopulations by buoyant density gradients. Depletion of ShEA and of HuEA rosette-forming lymphocytes was accomplished by Ficoll-Hypaque gradient centrifugation. Rosettes of lymphocytes with HuEA or ShEA were formed as described above. 4-6 ml of the rosette suspensions were pooled and layered onto Ficoll-Hypaque solution. The tubes were centrifuged for 20 min at 400 g. Cells at the interface were collected for evaluation of their surface markers. Statistical analysis. All data given in the tables, figures and text are expressed as mean+ standard error of the mean. Statistical comparisons utilized Student's t-test or paired t-test when appropriate.
RESULTS
Comparison of different methods for detecting Fc receptors on lymphocytes Four techniques for demonstrating Fc receptors were compared: immunofluorescence for binding of aggregated human gamma-globulin (HuAgg) and aggregated guinea-pig gamma-globulin (GPAgg), and rosetting with antibody-sensitized sheep red blood cells (ShEA) and with sensitized human red blood cells (HuEA). TABLE 2. Percentages of lymphocytes bearing SIg or binding GPAgg or HuAgg as evaluated before and after depletion of lymphocytes rosetting with ShEA or with HuEA
Percentages of lymphocytes positive for surface receptor
I. Before rosette depletion (10) II. After depletion of: A. HuEA-rosetting lymphocytes (2) B. ShEA-rosetting lymphocytes (2)
SIg
HuAGG
GPAgg
7-2+0-3
18-0+ 1-4*
18-2+ 1-7*
6-8+ 1-5
61+ 1-3
6-9+ 10
2-3±0-5
0-5±0-5t
) = Number of experiments. * P< 0-01 HuAgg or GPAgg as compared to SIg. t P< 0 025 ShEA-depleted compared to HuEA-depleted.
1-0+±00t
284
P. 9. Clements & 5. Levy (d)
0 .0
(0
Q0
15
E
@':iR~ ~ ~ ~ ~ ~ ~ ~M
FIG. 1. Percentages of lymphocyte
subpopulations as identified byvarious surface receptors (a) before and subpopulations: (a) Iymphocytes before depletion, fourteen subjects; (b) lymphocytes remaining after depletion of lymphocytes rosettingFaith SRIBC sensitized with the maximal subagglutinating concentration of antisera (ShEA), ten subjects; (c) Iymphocytes remaining after depletion of lymphoc tes rosettingwsithHlRBC sensitized with the maximal subagglutinating concentration of antisera (HuEA), sevensubjects; and (d) lymphocytes remaining after depletion oflymphocytes resetting with SRBC sensitizedwith a four-fold dilution of the subagglutinating concentration of antisera (ShEAsub), ten subjects. The average total lymphocyte follossing depletion experiments aere (expressed as a percentage of the recovery of control gradients): (b) 72+22",, (±s.d.); (c) 72+25",,; and (d) 62+ 18",' HuAgg-binding, ShEA-rosetting, H) HuEA-rosetting, (U) SIg-bearing,(a)Z, C-bearing, and E-rosetting. Other abbre'iaitionis: I luAgg-binding lymphocytes binding aggregated human IgG. SIgbearing = lymphocytes bearing surface immunoglobulin. ZyC-rosetting lymphocytes rosetting with zymosan complement particles. E-rosetting lymphocytes rosettingwith SRBC. f P< 0001 lymphocytes depleted of ShEA compared to cells depleted of HuEA. P< 005 lymphocytes depleted of ShEA compared to cells depleted of HuEA. § P< 0001 lymphocytes depleted of HuEA compared to cells at baseline. 11 P< 005 lymphocytes depleted of HuEA compared to cells at baseline.
(bhd) after depletion of differentlImphocxte
reco-cries
(E)
=
=
=
+
As shown in Fig. and Table 2, the percentages of cells in the first three categories similar (i.e. 18 2+ 17/,, and 17-0+ 1±%, respectively). These significantly higher (P< 0001) than the 0+ 110% of HuEA-rosetting cells. Following depletion of ShEA rosettes HuEA rosettes, lymphocytes binding HuAgg, GPAgg or ShEA behaved in parallel fashion (Fig. 1). Depletion of ShEA rosettes (Fig.lb) resulted in almost complete removal of lymphocytes positive for HuEA, GPAgg or HuAgg, whereas removal of HuEA rosettes (Fig. ic) only partially removed lymphocytes positive for ShEA, GPAgg, or HuAgg. were
17-9+2-6%,
were
or
F c receptors to other surface receptors Relation of Depletion of ShEA rosettes, but not of HuEA rosettes, removed almost all lymphocytes bearing SIg. Similarly, depletion of ShEA rosettes depleted 70- 7500 of complem ent-rosetting lymphocytes, but
depletion of HuEA rosettes depleted only 25-30% HuEA or ShEA there was a rise in the percentage
of them. In all depletion of E-rosetting lymphocytes.
experiments
employing
Effect of suboptimal sensitization of SRBC In all the experiments described above, the indicator RBC sensitized with the agglutinating titre of antibodies. Additional experiments done in which T-SRBC with a four-fold dilution of that subagglutinating titre. The rosetting lymphocytes were
were
maximum sub-
Ficoll-Hypaque gradient. When surface receptors
were
evaluated
on
lymphocytes remaining
face, the percentages of SIg, complement rosettes, E-rosettes, and Fc receptors results which occurred following depletion of HuEA rosettes (Fig. ic, d).
were
sensitized by at the inter-
were
were
depleted
similar
to
the
Re-evaluation of Fc receptors on lymphocytes
285
Additional control experiments Control gradients were formed for the depletion experiments using HRBC in place of HuEA (six subjects) or T-SRBC in place of ShEA (five subjects). The average total cell recovery at the interfaces of HRBC and T-SRBC control gradients was 72+ 18% (± s.d.) and 59++ 8% respectively. Results of the examination of surface receptors on lymphocytes remaining at the interface were not significantly different from those at baseline. When the FGx-GPIgG antisera was incubated directly with human lymphocytes, less than 1% of the lymphocytes were noted to fluoresce. The indirect method used to identify HuAgg-binding cells also identified cells with SIg of the IgG class; therefore, a separate aliquot of cells was stained directly with FGx-HuIgG (ten subjects): 2-3+ 0 3% (mean+ s.e.) were fluorescent prior to gradient depletion; 1 6+ 0.40 after depletion, of HuEA rosettes and 0 9+ 0.2% after depletion of ShEA rosettes. DISCUSSION The present investigators compared cells with several non-SRBC rosette markers: SIg, ZyC rosettes, and binding of HuAgg, GPAgg, sensitized SRBC (ShEA) and sensitized HRBC (HuEA). The last four are commonly regarded as markers for Fc receptors. The study was carried out by examining cells remaining after selective depletion of ShEA or HuEA. These experiments were based on the assumption that if a group of cells possessed more than one marker simultaneously, depletion of this group should lead to decreases in the percentages of all cells found positive when assayed for those markers. Conversely if two markers were present on two different types of cells, depletion of one type should lead to selective enrichment of the other. Our results showed that depletion of HuEA rosettes failed to deplete cells with SIg but did lead to a partial reduction (about 60%) in the percentages of cells which bound ShEA, GPAgg or HuAgg. This was then compared to results obtained by depletion of ShEA rosettes. Depletion of the latter removed 7000 of cells with SIg and 95% of all lymphocytes with Fc receptors demonstrable by the other three techniques. Based upon these experiments a diagram was constructed (Fig. 2) to illustrate the content and the overlap of these subpopulations. One possibility to account for this difference between ShEA and HuEA was that the HRBC was more easily agglutinable by the sensitizing antibodies than was the SRBC and so carried less immunoglobulin on the surface to which the lymphocytes could bind. To test this hypothesis a submaximal titre of antisera was used to sensitize the SRBC (ShEASUb) and gradient depletion was repeated. The percentages of surface receptors on lymphocytes depleted of ShEASUb then equalled those of cells depleted of HuEA rosettes. This would appear to confirm our hypothesis. These rosette experiments (using particulate complexes) complement work by Grey et al. (1975) who showed that multiple-cell subpopulations could bind soluble aggregated IgG, but to widely varying extents depending upon the cell type. These observations together suggest that more than one lymphocyte subpopulation has Fc receptors (each with its own avidity or affinity) and that each subpopulation can be identified and distinguished from the others by its capacity to bind IgG at different concentrations. In particular it may have a bearing on the controversy concerning the presence of Fc receptors on SIgbearing lymphocytes: some investigators have observed Fc receptors on most (Jondal, 1974; Forni & Pernis, 1975; Dickler, 1976), on some (Samarut, Brochier & Revillard, 1976; Gergely et al., 1977), or on few (Froland et al., 1974; Kurnick & Grey, 1975) SIg-bearing lymphocytes. In the present experiments, depletion of ShEA rosettes was found to remove most of the SIg-bearing cells; conversely depletion of ShEASUb rosettes (which are in fact ShEA which have been sensitized with a reduced amount of IgG) failed to deplete SIg-bearing cells even though other Fc-bearing populations were completely depleted. Had we performed experiments with ShEASub only or with HuEA only, we too would have concluded that SIg-bearing cells did not have Fc receptors. But having performed experiments with both ShEA and ShEASub) we are forced to conclude that SIg-bearing cells do have Fc receptors but that high concentrations of IgG are needed to demonstrate them. It is likely too, that had higher concentrations of
P. J. Clements & J.
286
Levy
rI
|
ShEAsub-rosetting*
|ShEA- rosetting 17.0 + 1. 1
A HuAgg -bindi ng
|-
17¢ 9 +2- 6l
I Sig - bearing 72!O -33 ZyC-rosetting 12*8 0'9
0
5
10
15
20
25
Lymphocyte subpopulations (/)
FIG. 2. Diagrammatic representation of the content and overlap of lymphocyte subpopulations (as identified by a variety of surface receptors). Results are based upon gradient depletion experiment outlined in the text. *Although the ShEASlib-rosetting population was not itself directly measured, depletion experiments outlined in the text suggest its placement in relation to the other subpopulations. tAlthough GPAgg-binding lymphocytes are not shown, the experiments outlined in the text suggest that they were essentially identical to HuAgg-binding lymphocytes.
IgG been used in previous experiments, more reports would have found Fc receptors on SIg-bearing cells. The results here showed that when assaying Fc-receptor cells, ShEA and HuEA rosettes are not identical. The former encompasses most of the other non-SRBC markers, but not so theHuEA rosettes. These data have to be interpreted with two reservations in mind. First, we cannot exclude the possibility that rosette depletion of ShEA did not non-specifically deplete other cell types through a mechanism other than resetting, and that this non-specific mechanism was absent when we depleted HuEA. Since, in the unseparated preparation, the number of ShEA exceeded the number of HuEA and the percentages of ShEA were indistinguishable from those of HuAgg and GPAgg, we feel this would be an unlikely mechanism to account for our results. The second reservation is that differences in surface markers do not necessarily reflect functional differences. This will have to be determined before one can conclude whether ShEA and HuEA constitute two functionally different groups. This work was presented in part at the Eleventh Leukocyte Culture Conference, Tucson, Arizona, 20 September, 1976 and was supported in part by USPHS Grant GM 15759. We would like to thank R. Weisbart, M.D. and D. T. Y. Yu, M.D. for their comments during the preparation of this manuscript and to acknowledge Zmira Timor for her assistance in the execution of this work.
REFERENCES CLARK, H.F. & SHEPAmw, C.C. (1963) A dialysis technique DICKLER, H.B. (1976) Lymphocyte receptors for immunoglobulin. Advances in Immunology (eds. F.J. Dixon & for preparing fluorescent antibody. Virology, 20, 642. H.G. Kunkel), p. 167, Academic Press. DICKLER, H.B. (1974) Studies of the human lymphocyte receptor for heat-aggregated or antigen-complexes FoRNI, L. & PERNIS, B. (1975) Interactions between Fc receptors and membrane immunoglobulins on B lymphoimmunoglobulin. 3. exp. Med. 140, 508.
Re-evaluation ofFc receptors on lymphocytes cytes. Membrane Receptors of Lymphocytes (eds. M. Seligmann, J.L. Preudhomme & F.M. Kourilsky), p. 193, North Holland Publishing Co. FROLAND, S.S., WISLOFF, F. & MICHAELSEN, T.E. (1974) Human lymphocytes with receptors for IgG. A population of cells distinct from T and B lymphocytes. Int. Arch. Allergy, Appl. Immunol. 47, 124. GERGELY, P., BAKACS, T., CORNAIN, S. & KLEIN, E. (1977) Fc receptors on human blood B lymphocytes. Clin. exp. Immunol. 28, 99. GREY, H.M., ANDERSON, C.L., HEUSSER, C.H. & KURNICK, J.T. (1975) Fc receptors on lymphocytes other than B cells in mouse and man. Membrane Receptors of-Lymphocytes. (eds. M. Seligmann, J.L. Preud'homme & F.M. Kourilsky), p. 185, North Holland Publishing Co. HORWITZ, D.A. & LOBO, P.I. (1975) Characterization of two populations of human lymphocytes bearing easily detectable surface immunoglobulin. 3. cdin. Invest. 56, 1464. JONDAL, M. (1974) Surface markers on human B and T lymphocytes. IV. Distribution of surface markers on resting and blast-transformed lymphocytes. Scand. 3. Immunol. 3, 739.
287
KURNICK, J.T. & GREY, H.M. (1975) Relationship between immunoglobulin-bearing lymphocytes and cells reactive with sensitized human erythrocytes. A. Immunol. 115, 305. SAMARUT, C., BROCHIER, J. & REVILLARD, J.P. (1976) Distribution of cells binding erythrocyte-antibody (EA) complexes in human lymphoid populations. Scand. 7. Immunol. 5, 221. WHO/IARC WORKSHOP SPECIAL TECHNICAL RESPOT (1974) Identification, enumeration, and isolation of B and T lymphocytes from human peripheral blood. Scand. 7. Immunol. 3, 521. Yu, D.T.Y. & CLEMENTS, P.J. (1976) Human lymphocyte subpopulations. Effect of epinephrine. Clin. exp. Immunol. 25, 472. Yu, D.T.Y., RAMER, S. & KACENA, A. (1977) Effect of corticosteroids on the response of lymphocytes to stimulation by galactose oxidase modified lymphocytes. Immunology, 33, 247. ZIGHELBOIM, J., GALE, R.P., CHIN, A., BONAVIDA, B., OSSORIO, R.C. & FAHEY, J.L. (1974) Antibody dependent cellular cytotoxicity: Cytotoxicity mediated by non T lymphocytes. Clin. Immunol. Immunopathol. 3, 193.
Receptors for IgG (Fc receptors) on human lymphocytes: re-evaluation by multiple techniques P. J. CLEMENTS & J. LEVY Department of Medicine, UCLA School of Medicine, Los Angeles, California, USA
(Received 24 April 1978)
SUMMARY
This study examines whether receptors for IgG (Fc receptors), as identified by different methods, are found on identical human lymphocyte subpopulations, and the relationship of Fc receptors to surface immunoglobulin (SIg) and receptor for complement (C'). Fc receptors were identified by two rosetting techniques (antibody-sensitized human erythrocytes, HuEA, or sheep erythrocytes, ShEA) and two immunofluorescent techniques (heat-aggregated IgG of human origin, HuAgg, or of guinea-pig origin, GPAgg). When lymphocytes depleted of cells resetting with ShEA were compared to HuEA-depleted lymphocytes, the two subpopulations appeared to be significantly different. However, when lymphocytes were depleted of cells rosetting with ShEA which had been sensitized with lower concentrations of antibody, the subpopulation so depleted appeared to be virtually identical to HuEAdepleted lymphocytes. These studies suggest that more than one lymphocyte subpopulation has Fc receptors and that each subpopulation can, in part, be identified and distinguished from the other by the capacity to bind IgG at differing concentrations. In particular, these experiments may serve to resolve the controversy concerning the presence of Fc receptors on lymphocytes bearing surface immunoglobulin (SIg). Depletion of lymphocytes rosetting with ShEA removed most of the SIg-bearing lymphocytes; depletion of cells resetting with ShEA which had been sensitized with lower concentrations of IgG antibody, however, failed to deplete SIg-bearing lymphocytes even though other Fc-bearing populations were completely depleted. These results suggest that SIg-bearing lymphocytes (B lymphocytes) do have Fc receptors but that high concentrations of IgG are needed to demonstrate them.
INTRODUCTION Human thymus-derived (T) lymphocytes are commonly recognized by surface receptors for sheep red blood cells (SRBC), whereas bone-marrow derived (B) lymphocytes are recognized by surface immunoglobulin (SIg) and receptors for complement (WHO/IARC Workshop, 1974). Another group of cells are distinguished by having surface receptors for the Fc portion of IgG immunoglobulins (Fc receptors). Depending upon the situations, these Fc receptors have putatively been detected on B lymphocytes, T lymphocytes, and lymphocytes without the common T and B markers (reviewed by Dickler, 1976). Since Fc receptors in these various reports were assayed by different methods, many of the discrepancies may be artifacts of the techniques used to detect them. Because of this, this investigation examined the question of whether Fc receptors, as identified by four different methods, were found on identical lymphocyte subpopulations and the relation of these Fc receptors to the conventional T and B markers. MATERIALS AND METHODS Preparation ofreagents. The preparation of all immunoglobulin reagents is outlined in Table 1. Trypsin (twice-crystallized, salt-free from bovine pancreas, Worthington Laboratories, Freehold, New Jersey) was used at a concentration of 5 mg/ml. Sheep red blood cells in Alsever's solution were obtained from Mission Laboratories, Rosemead, Correspondence: Dr P. J. Clements, UCLA/Department of Medicine, 1000 Veteran Avenue, Los Angeles, California 90024, USA. 0099-9104/78/0110-281$02.00 (0 1978 Blackwell Scientific Publications
281
282
P. J. Clements & J. Levy TABLE 1. Preparation of immunoglobulin reagents
Protein and source
Preparation prior to use
IgG fraction of goat antiserum to human Protein was conjugated with IgG, heavy chain specific. Miles fluorescein isothiocyanate (FITC, Laboratories, Kankakee, Illinois isomer I, Baltimore Biologicals Laboratories, Cockeysville, Maryland) by an adaptation of dialysis method of Shepard & Clark (1963) IgG fraction of goat antiserum to Protein FITC conjugated as above. guinea pig gamma globulin, Cappel Laboratories, Downington, Pennsylvania Guinea-pig gamma globulin, Cappel Protein was FITC-conjugated as above. Laboratories. It was aggregated at 630C for 20 min at a protein concentration of 10 mg/ml and diluted to 5 mg/ml prior to use. IgG fractions of rabbit antiserum to Since further purification by DEAE human red blood cells or to sheep red cellulose chromatography did not change blood cells. Cappel Laboratories. the performance characteristics of either antiserum they were used as received from Cappel Laboratories. Human gamma globulins (Fraction II). It was aggregated at 630C for 20 min at a Nutritional Biochemicals Division, ICN, protein concentration of 50 mg/ml and Cleveland, Ohio. diluted to 2 mg/ml prior to use. FITC-conjugated goat polyvalent Antiserum was centrifuged at 50,000 g antiserum to human immunoglobulins. for 30 min prior to use. Meloy Laboratories, Springfield, Virginia
Reagent as finally used FITC-conjugated goat antiserum to human IgG.
FITC-conjugated goat antiserum to guinea-pig gamma-globulin. Heat-aggregated FITCconjugated guinea-pig gammaglobulin. IgG fractions of rabbit antiserum to human red blood cells or to sheep red blood cells.
Heat-aggregated human gammaglobulin. FITC-conjugated goat polyvalent antiserum to human immunoglobulins
California. Human red blood cells (blood type 0+) were obtained from the same normal volunteer throughout the study. Foetal bovine serum (Rehatuin) was obtained from Reheis, Los Angeles, California. Zymosan particles were obtained from Sigma Chemical Company, St Louis, Missouri. Blood and lymphocyte separation. Subjects for these experiments were adult laboratory personnel and normal volunteers. Total lymphocyte counts were determined by routine methods from total white blood cell counts and Wright stained differential blood smears. Mononuclear blood cells were separated from peripheral blood by Ficoll-Hypaque sedimentation (Yu & Clements, 1976). Mononuclear blood cells were incubated for 45 min at 370C with carbonyl iron filings (40 mg carbonyl iron, GAF, for each 1 ml of cell suspension containing 5 x 106 cells) in the presence of 20% foetal bovine serum, to facilitate phagocytosis as well as shedding of cytophilic IgG (Horwitz & Lobo, 1975; Yu, Ramer & Kacena, 1977). Phagocytic cells were removed with the use of a magnet. Purified lymphocytes were washed twice with 37°C media and divided into aliquots for identification of cell surface markers. Identification of cell surface markers. Immunofluorescent detection of surface immunoglobulin of all classes. Using a method similar to that of Horwitz & Lobo (1975), lymphocytes were incubated for 30 min at 4°C in the presence of an FITC-conjugated goat anti-human immunoglobulin antisera and washed. For this and all immunofluorescent experiments to follow, a minimum of 200 viable lymphocytes were examined under phase contrast and epifluorescent microscopy and the percentage of fluorescent cells was determined. Immunofluorescent detection of surface immunoglobulin of the IgG class. A separate aliquot of cells was incubated directly with FITC- conjugated goat antisera to human IgG (FGx-HiIgG) to determine the number of cells with surface immunoglobulin of the IgG class. Immunofluorescent detection of binding of aggregated human IgG (HuAgg). Using an adaptation of previously described methods (Dickler, 1974; Yu & Clements, 1976), lymphocytes were incubated with heat-aggregated human gamma-globulin for 30 min at 4°C. Cells were washed and incubated with fluorescein-conjugated goat anti-human IgG (FGx-HuIgG) for 30 min at 4°C. Immunofluorescent detection of binding of aggregated guinea-pig IgG (GPAgg).A similar technique, adapted from Horwitz & Lobo (1975), was used as in demonstrating receptors for HuAgg. Cells were incubated with heat-aggregated FITCconjugated guinea-pig gamma-globulin at 4°C for 30 min. Cells were then washed and incubated with FITC-conjugated goat antisera to guinea-pig gamma-globulin (FGx-GPIgG) at 4°C for 30 min.
Re-evaluation of Fc receptors on lymphocytes
283
Rosetting with complement-zymosan particles (ZyC) and with sheep red blood cells (E or SRBC). The method for the simultaneous identification of ZyC rosettes and E rosettes has been described previously (Yu & Clements, 1976). SRBC were suspended as a 0 5%y solution in Hanks' balanced salt solution (HBSS) supplemented with 20% foetal calf serum which had previously been absorbed with SRBC. Zymosan granules (Zy) were suspended in HBSS and sonicated. Zy were washed and incubated 30 min at 370C in HBSS supplemented 20% with fresh or deep frozen human serum as a source of complement. Complement-coated zymosan (ZyC) were washed and suspended in HBSS to give an approximate concentration of 1 x 108 ZyC per ml. To form rosettes, 0-1 ml of ZyC, 0-1 ml of the 0.50% solution of SRBC, and 0-1 ml of lymphocytes (5 x 106 per ml) were mixed, incubated at 370C for 5 min, and centrifuged at 50 g for 5 min. After an overnight incubation at 4VC, the mixture was resuspended in the presence of crystal violet (to identify lymphocytes). Resuspension of all rosette assays were done on a vertical rotator for 24 min, and a minimum of 400 lymphocytes were examined afterwards. Lymphocytes with two or more adherent ZyC, or with three or more adherent SRBC's were counted as rosettes. Rosettiug with antibody-sensitized red blood cells. Fc receptors were also evaluated by two rosette methods: (1) The first method adapted from Zighelboim et al. (1974) employed antibody-sensitized SRBC (ShEA). Washed SRBC (suspended in 2%, concentration in HBSS) were incubated for 30 min at 370C with 5 mg/ml of trypsin. After the trypsinized SRBC (T-SRBC) were washed, aliquots were incubated with serial doubling dilutions of the IgG fraction of rabbit antiSRBC antisera for 30 min at 370C. The antibody-sensitized SRBC (ShEA) were washed and resuspended at a 0.5% concentration. 0-1 ml of lymphocytes (5x 106) were mixed with 0-1 ml of ShEA (sensitized at a subagglutinating titre) and centrifuged at 50 g for 5 min. After a 60-min incubation at room temperature, the cells were resuspended by rotation. Cells with four or more adherent ShEA were counted as rosettes. Control experiments in which trypsinized SRBC's (without sensitizing antibody) were used routinely gave less than 2% rosetting lymphocytes. (2) The second method, adapted from Froland, Wisloff & Michaelsen (1974), employed sensitized human red blood cells (HuEA). Washed human red blood cells (HRBC) were incubated at a 2%4 concentration in serial doubling dilutions of a heat-inactivated IgG fraction of rabbit anti-HRBC antisera for 30 min at 37°C. The HRBC sensitized at subagglutinating titre were washed and resuspended at a 1% concentration. The rosetting procedure was identical to that used for ShEA rosettes. Depletion of lymphocyte subpopulations by buoyant density gradients. Depletion of ShEA and of HuEA rosette-forming lymphocytes was accomplished by Ficoll-Hypaque gradient centrifugation. Rosettes of lymphocytes with HuEA or ShEA were formed as described above. 4-6 ml of the rosette suspensions were pooled and layered onto Ficoll-Hypaque solution. The tubes were centrifuged for 20 min at 400 g. Cells at the interface were collected for evaluation of their surface markers. Statistical analysis. All data given in the tables, figures and text are expressed as mean+ standard error of the mean. Statistical comparisons utilized Student's t-test or paired t-test when appropriate.
RESULTS
Comparison of different methods for detecting Fc receptors on lymphocytes Four techniques for demonstrating Fc receptors were compared: immunofluorescence for binding of aggregated human gamma-globulin (HuAgg) and aggregated guinea-pig gamma-globulin (GPAgg), and rosetting with antibody-sensitized sheep red blood cells (ShEA) and with sensitized human red blood cells (HuEA). TABLE 2. Percentages of lymphocytes bearing SIg or binding GPAgg or HuAgg as evaluated before and after depletion of lymphocytes rosetting with ShEA or with HuEA
Percentages of lymphocytes positive for surface receptor
I. Before rosette depletion (10) II. After depletion of: A. HuEA-rosetting lymphocytes (2) B. ShEA-rosetting lymphocytes (2)
SIg
HuAGG
GPAgg
7-2+0-3
18-0+ 1-4*
18-2+ 1-7*
6-8+ 1-5
61+ 1-3
6-9+ 10
2-3±0-5
0-5±0-5t
) = Number of experiments. * P< 0-01 HuAgg or GPAgg as compared to SIg. t P< 0 025 ShEA-depleted compared to HuEA-depleted.
1-0+±00t
284
P. 9. Clements & 5. Levy (d)
0 .0
(0
Q0
15
E
@':iR~ ~ ~ ~ ~ ~ ~ ~M
FIG. 1. Percentages of lymphocyte
subpopulations as identified byvarious surface receptors (a) before and subpopulations: (a) Iymphocytes before depletion, fourteen subjects; (b) lymphocytes remaining after depletion of lymphocytes rosettingFaith SRIBC sensitized with the maximal subagglutinating concentration of antisera (ShEA), ten subjects; (c) Iymphocytes remaining after depletion of lymphoc tes rosettingwsithHlRBC sensitized with the maximal subagglutinating concentration of antisera (HuEA), sevensubjects; and (d) lymphocytes remaining after depletion oflymphocytes resetting with SRBC sensitizedwith a four-fold dilution of the subagglutinating concentration of antisera (ShEAsub), ten subjects. The average total lymphocyte follossing depletion experiments aere (expressed as a percentage of the recovery of control gradients): (b) 72+22",, (±s.d.); (c) 72+25",,; and (d) 62+ 18",' HuAgg-binding, ShEA-rosetting, H) HuEA-rosetting, (U) SIg-bearing,(a)Z, C-bearing, and E-rosetting. Other abbre'iaitionis: I luAgg-binding lymphocytes binding aggregated human IgG. SIgbearing = lymphocytes bearing surface immunoglobulin. ZyC-rosetting lymphocytes rosetting with zymosan complement particles. E-rosetting lymphocytes rosettingwith SRBC. f P< 0001 lymphocytes depleted of ShEA compared to cells depleted of HuEA. P< 005 lymphocytes depleted of ShEA compared to cells depleted of HuEA. § P< 0001 lymphocytes depleted of HuEA compared to cells at baseline. 11 P< 005 lymphocytes depleted of HuEA compared to cells at baseline.
(bhd) after depletion of differentlImphocxte
reco-cries
(E)
=
=
=
+
As shown in Fig. and Table 2, the percentages of cells in the first three categories similar (i.e. 18 2+ 17/,, and 17-0+ 1±%, respectively). These significantly higher (P< 0001) than the 0+ 110% of HuEA-rosetting cells. Following depletion of ShEA rosettes HuEA rosettes, lymphocytes binding HuAgg, GPAgg or ShEA behaved in parallel fashion (Fig. 1). Depletion of ShEA rosettes (Fig.lb) resulted in almost complete removal of lymphocytes positive for HuEA, GPAgg or HuAgg, whereas removal of HuEA rosettes (Fig. ic) only partially removed lymphocytes positive for ShEA, GPAgg, or HuAgg. were
17-9+2-6%,
were
or
F c receptors to other surface receptors Relation of Depletion of ShEA rosettes, but not of HuEA rosettes, removed almost all lymphocytes bearing SIg. Similarly, depletion of ShEA rosettes depleted 70- 7500 of complem ent-rosetting lymphocytes, but
depletion of HuEA rosettes depleted only 25-30% HuEA or ShEA there was a rise in the percentage
of them. In all depletion of E-rosetting lymphocytes.
experiments
employing
Effect of suboptimal sensitization of SRBC In all the experiments described above, the indicator RBC sensitized with the agglutinating titre of antibodies. Additional experiments done in which T-SRBC with a four-fold dilution of that subagglutinating titre. The rosetting lymphocytes were
were
maximum sub-
Ficoll-Hypaque gradient. When surface receptors
were
evaluated
on
lymphocytes remaining
face, the percentages of SIg, complement rosettes, E-rosettes, and Fc receptors results which occurred following depletion of HuEA rosettes (Fig. ic, d).
were
sensitized by at the inter-
were
were
depleted
similar
to
the
Re-evaluation of Fc receptors on lymphocytes
285
Additional control experiments Control gradients were formed for the depletion experiments using HRBC in place of HuEA (six subjects) or T-SRBC in place of ShEA (five subjects). The average total cell recovery at the interfaces of HRBC and T-SRBC control gradients was 72+ 18% (± s.d.) and 59++ 8% respectively. Results of the examination of surface receptors on lymphocytes remaining at the interface were not significantly different from those at baseline. When the FGx-GPIgG antisera was incubated directly with human lymphocytes, less than 1% of the lymphocytes were noted to fluoresce. The indirect method used to identify HuAgg-binding cells also identified cells with SIg of the IgG class; therefore, a separate aliquot of cells was stained directly with FGx-HuIgG (ten subjects): 2-3+ 0 3% (mean+ s.e.) were fluorescent prior to gradient depletion; 1 6+ 0.40 after depletion, of HuEA rosettes and 0 9+ 0.2% after depletion of ShEA rosettes. DISCUSSION The present investigators compared cells with several non-SRBC rosette markers: SIg, ZyC rosettes, and binding of HuAgg, GPAgg, sensitized SRBC (ShEA) and sensitized HRBC (HuEA). The last four are commonly regarded as markers for Fc receptors. The study was carried out by examining cells remaining after selective depletion of ShEA or HuEA. These experiments were based on the assumption that if a group of cells possessed more than one marker simultaneously, depletion of this group should lead to decreases in the percentages of all cells found positive when assayed for those markers. Conversely if two markers were present on two different types of cells, depletion of one type should lead to selective enrichment of the other. Our results showed that depletion of HuEA rosettes failed to deplete cells with SIg but did lead to a partial reduction (about 60%) in the percentages of cells which bound ShEA, GPAgg or HuAgg. This was then compared to results obtained by depletion of ShEA rosettes. Depletion of the latter removed 7000 of cells with SIg and 95% of all lymphocytes with Fc receptors demonstrable by the other three techniques. Based upon these experiments a diagram was constructed (Fig. 2) to illustrate the content and the overlap of these subpopulations. One possibility to account for this difference between ShEA and HuEA was that the HRBC was more easily agglutinable by the sensitizing antibodies than was the SRBC and so carried less immunoglobulin on the surface to which the lymphocytes could bind. To test this hypothesis a submaximal titre of antisera was used to sensitize the SRBC (ShEASUb) and gradient depletion was repeated. The percentages of surface receptors on lymphocytes depleted of ShEASUb then equalled those of cells depleted of HuEA rosettes. This would appear to confirm our hypothesis. These rosette experiments (using particulate complexes) complement work by Grey et al. (1975) who showed that multiple-cell subpopulations could bind soluble aggregated IgG, but to widely varying extents depending upon the cell type. These observations together suggest that more than one lymphocyte subpopulation has Fc receptors (each with its own avidity or affinity) and that each subpopulation can be identified and distinguished from the others by its capacity to bind IgG at different concentrations. In particular it may have a bearing on the controversy concerning the presence of Fc receptors on SIgbearing lymphocytes: some investigators have observed Fc receptors on most (Jondal, 1974; Forni & Pernis, 1975; Dickler, 1976), on some (Samarut, Brochier & Revillard, 1976; Gergely et al., 1977), or on few (Froland et al., 1974; Kurnick & Grey, 1975) SIg-bearing lymphocytes. In the present experiments, depletion of ShEA rosettes was found to remove most of the SIg-bearing cells; conversely depletion of ShEASUb rosettes (which are in fact ShEA which have been sensitized with a reduced amount of IgG) failed to deplete SIg-bearing cells even though other Fc-bearing populations were completely depleted. Had we performed experiments with ShEASub only or with HuEA only, we too would have concluded that SIg-bearing cells did not have Fc receptors. But having performed experiments with both ShEA and ShEASub) we are forced to conclude that SIg-bearing cells do have Fc receptors but that high concentrations of IgG are needed to demonstrate them. It is likely too, that had higher concentrations of
P. J. Clements & J.
286
Levy
rI
|
ShEAsub-rosetting*
|ShEA- rosetting 17.0 + 1. 1
A HuAgg -bindi ng
|-
17¢ 9 +2- 6l
I Sig - bearing 72!O -33 ZyC-rosetting 12*8 0'9
0
5
10
15
20
25
Lymphocyte subpopulations (/)
FIG. 2. Diagrammatic representation of the content and overlap of lymphocyte subpopulations (as identified by a variety of surface receptors). Results are based upon gradient depletion experiment outlined in the text. *Although the ShEASlib-rosetting population was not itself directly measured, depletion experiments outlined in the text suggest its placement in relation to the other subpopulations. tAlthough GPAgg-binding lymphocytes are not shown, the experiments outlined in the text suggest that they were essentially identical to HuAgg-binding lymphocytes.
IgG been used in previous experiments, more reports would have found Fc receptors on SIg-bearing cells. The results here showed that when assaying Fc-receptor cells, ShEA and HuEA rosettes are not identical. The former encompasses most of the other non-SRBC markers, but not so theHuEA rosettes. These data have to be interpreted with two reservations in mind. First, we cannot exclude the possibility that rosette depletion of ShEA did not non-specifically deplete other cell types through a mechanism other than resetting, and that this non-specific mechanism was absent when we depleted HuEA. Since, in the unseparated preparation, the number of ShEA exceeded the number of HuEA and the percentages of ShEA were indistinguishable from those of HuAgg and GPAgg, we feel this would be an unlikely mechanism to account for our results. The second reservation is that differences in surface markers do not necessarily reflect functional differences. This will have to be determined before one can conclude whether ShEA and HuEA constitute two functionally different groups. This work was presented in part at the Eleventh Leukocyte Culture Conference, Tucson, Arizona, 20 September, 1976 and was supported in part by USPHS Grant GM 15759. We would like to thank R. Weisbart, M.D. and D. T. Y. Yu, M.D. for their comments during the preparation of this manuscript and to acknowledge Zmira Timor for her assistance in the execution of this work.
REFERENCES CLARK, H.F. & SHEPAmw, C.C. (1963) A dialysis technique DICKLER, H.B. (1976) Lymphocyte receptors for immunoglobulin. Advances in Immunology (eds. F.J. Dixon & for preparing fluorescent antibody. Virology, 20, 642. H.G. Kunkel), p. 167, Academic Press. DICKLER, H.B. (1974) Studies of the human lymphocyte receptor for heat-aggregated or antigen-complexes FoRNI, L. & PERNIS, B. (1975) Interactions between Fc receptors and membrane immunoglobulins on B lymphoimmunoglobulin. 3. exp. Med. 140, 508.
Re-evaluation ofFc receptors on lymphocytes cytes. Membrane Receptors of Lymphocytes (eds. M. Seligmann, J.L. Preudhomme & F.M. Kourilsky), p. 193, North Holland Publishing Co. FROLAND, S.S., WISLOFF, F. & MICHAELSEN, T.E. (1974) Human lymphocytes with receptors for IgG. A population of cells distinct from T and B lymphocytes. Int. Arch. Allergy, Appl. Immunol. 47, 124. GERGELY, P., BAKACS, T., CORNAIN, S. & KLEIN, E. (1977) Fc receptors on human blood B lymphocytes. Clin. exp. Immunol. 28, 99. GREY, H.M., ANDERSON, C.L., HEUSSER, C.H. & KURNICK, J.T. (1975) Fc receptors on lymphocytes other than B cells in mouse and man. Membrane Receptors of-Lymphocytes. (eds. M. Seligmann, J.L. Preud'homme & F.M. Kourilsky), p. 185, North Holland Publishing Co. HORWITZ, D.A. & LOBO, P.I. (1975) Characterization of two populations of human lymphocytes bearing easily detectable surface immunoglobulin. 3. cdin. Invest. 56, 1464. JONDAL, M. (1974) Surface markers on human B and T lymphocytes. IV. Distribution of surface markers on resting and blast-transformed lymphocytes. Scand. 3. Immunol. 3, 739.
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KURNICK, J.T. & GREY, H.M. (1975) Relationship between immunoglobulin-bearing lymphocytes and cells reactive with sensitized human erythrocytes. A. Immunol. 115, 305. SAMARUT, C., BROCHIER, J. & REVILLARD, J.P. (1976) Distribution of cells binding erythrocyte-antibody (EA) complexes in human lymphoid populations. Scand. 7. Immunol. 5, 221. WHO/IARC WORKSHOP SPECIAL TECHNICAL RESPOT (1974) Identification, enumeration, and isolation of B and T lymphocytes from human peripheral blood. Scand. 7. Immunol. 3, 521. Yu, D.T.Y. & CLEMENTS, P.J. (1976) Human lymphocyte subpopulations. Effect of epinephrine. Clin. exp. Immunol. 25, 472. Yu, D.T.Y., RAMER, S. & KACENA, A. (1977) Effect of corticosteroids on the response of lymphocytes to stimulation by galactose oxidase modified lymphocytes. Immunology, 33, 247. ZIGHELBOIM, J., GALE, R.P., CHIN, A., BONAVIDA, B., OSSORIO, R.C. & FAHEY, J.L. (1974) Antibody dependent cellular cytotoxicity: Cytotoxicity mediated by non T lymphocytes. Clin. Immunol. Immunopathol. 3, 193.
