CELLULAR
IMMUNOLOGY
E-Rosette
4,
179-185 (1979)
Receptors Induced K Cells Expressing
EDWIN
W. ADES,‘.*
by Phytohemagglutinin on Human T-Cell Surface Antigens’
PATRICIA DOUGHERTY,* STEVEN AND CHARLES M. BALCH”,~,*
L. SHORE,?
*Cc~llular Immunobiology Unit of the Tumor Institute, Depurtments of Microbiology und Surgery, the Comprehensive Cancer Center, University ofAlabamu Medicul Center, Birmingham, Alabama 35294, und tClinicu1 Immunology Luborutory, Center for DiseuscJ Control, Public Health Service, U.S. Depurtment of Health. Education and Welfure, Atlanta. Georgia 30333.’ Received
October
24. 1978
Killer cells (K cells) enriched from human blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) were examined for surface markers. Sixty-seven percent of the E-rosette-negative, sIg-negative cells reacted with anti-T cell serum (AMT) previously shown to react with immunochemically defined T-cell antigens. Phytohemagglutinin induced 25% of K cells to express an E-rosette receptor. When these induced cells were isolated, greater than 98% reacted with AMT and 17% expressed the Fc receptor for IgG. Furthermore, they retained their functional capacity in ADCC. These findings demonstrate that an E-rosette receptor can be induced on human K cells. The data suggest the K-cell fraction included a population of thymus-dependent lymphocytes which can function as effector cells in ADCC.
INTRODUCTION A major portion of the mononuclear effector cells or killer cells (K cells) in human blood which mediate antibody-dependent cellular cytotoxicity (ADCC) lack the surface marker profile characteristic of either B cells or T cells (1,2). These K cells, bearing surface receptors for the Fc fragment of IgG, are enriched in a minor fraction of cells comprising about 4% of the peripheral blood mononuclear cells (MC; 2, 3). It is unclear whether K cells are a distinct third population of lymphoid cells or whether they are lymphocytes of T-cell or B-cell lineage (4). In this study, evidence is presented that a subpopulation of T cells can function as effector cells in ADCC. MATERIALS
AND METHODS
Zsolation oflv cells. Cellular fractions enriched in K cells were isolated from the
peripheral
blood MC of healthy donors by sequential passage through nylon wool
r This investigation was supported by grants from the Medical Research Service of the Veteran’s Administration and from the National Institute of Health (CA13148 CA16673). 2 Recipient of a Special Fellowship from the Leukemia Society of America. 3 Recipient of a Faculty Fellowship in Oncology from the American Cancer Society. 4 To whom requests for reprints should be sent: Department of Surgery, 750 Lyons-Harrison Research, University of Alabama Medical Center, Birmingham, Ala. 35294. 179 0008-8749/79/050179-07$02.00/O Copyright 0 1979by Academic Press, Inc. All rights of reproduction in any form reserved.
180
ADES ET AL.
columns and E-rosette depletion has been described (2) except that neuraminidase-treated sheep erythrocytes (SRBC) and a 4°C incubation temperature were employed for the E-rosette depletion step. This latter system has provec more efficient and sensitive than our previously described system for E-rosette depletions. Erythrocyte-rosette-forming cells (E-RFC) were separated from nonrosetting cells in the K-cell fraction following phytohemagglutinin (PHA) incubation (see below), rosetting suspensions were layered over a Ficoll-Hypaque cushion as described previously (5) and centrifuged for 40 min at 4OOg. Non-rosetting cells at the interface were harvested. Contaminating SRBC were removed by resuspending the E-RFC pellet into 2-3 ml of distilled water for 15 set and then rapidly adding an equal volume of 0.30 M NaCl. Both rosetting and nonrosetting cell populations were then washed twice with RPM1 1640 before cell function in ADCC was examined. Analysis of cellpopulations. A double-label immunofluorescence assay was used to simultaneously detect T and B lymphocytes in human blood (6). T cells were identified by using a purified anti-T-cell reagent (AMT) conjugated with fluorescein isothiocyanate (FITC). The preparation, characterization, and specificity of this same lot of AMT for human T cells have been described (7,8). This reagent has been shown to react with two differentiation antigens on T cells with molecular weights of 25,000 and 16,000 (8). Surface immunoglobulin (sIg)-bearing B lymphocytes were detected simultaneously by using the F(ab’), fragment of rhodamine-conjugated (RITC) goat antiserum raised against purified human IgG, IgM, and IgA (Cappell Laboratories5 Downington, Pa.). In enumeration of E-RFC, cell fractions were washed twice in Hanks’ balanced salt solution with 10% heat-inactivated fetal calf serum (FCS) and adjusted to a concentration of 7.5 x 10Vml. Equal volumes of cell suspensions and 1% neuraminidase-treated SRBC (9) were mixed, incubated at 37°C for 10 min, centrifuged at 2Og for 10 min at 4°C and incubated on ice for 60 min. Cells binding three or more SRBC were scored as positive. Purified populations of normal blood B cells were prepared as previously described (13). These cells and cultured B-lymphoblastoid cell line (RAJI) served as control cells. Rhodamine-conjugated PHA was prepared according to our previous methods for conjugation of antisera (7). Ox erythrocytes (ORBC) coated with either rabbit IgG or IgM antiORBC (EAw or EAc) were prepared and used for Fc receptor assays and isolation of EAc-positive K cells according to the method of Moretta and colleagues (9). Cells were also examined for the presence of cytoplasmic IgM (10). Induction of E-rosette receptor expression. For PHA-induced expression of E-rosette receptors, K cells were resuspended to a concentration of 5 x IO5 cells/ml in RPM1 1640 with 20% FCS and gentamicin (0.05 mg/ml), and then seeded in sterile plastic tubes in 1 ml volumes. Test cells were incubated with PHA (Difco, 5 ~1 of a 1:5 dilution of a stock solution/l x lo5 cells) for 72 hr in a 37°C incubator with 5% CO, in air. Control cells were treated similarly, but were not stimulated with mitogen. 5 Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education, and Welfare.
HUMAN
T LYMPHOCYTES
AS ADCC EFFECTOR
181
CELLS
Antibody-dependent cellular cytotoxicity. ADCC effector-cell activity was assessed against 51Cr-labeled Chang liver cells acutely infected with type 1 Herpes simplex virus (HSV) as previously described (2). The human serum antibody to HSV which mediates the cytotoxic reaction in this system is of the IgG class (11). Cytotoxicity was calculated according to the following equation:
Percentage
specific release = ___ c-D M-B
x 100
’
where C is the percentage isotope release from target cells in the presence of MC and a 1:200 dilution of pooled heat-inactivated human serum HSV antibodies, D is the percentage isotope release from target cells in the presence of MC and a 1:200 dilution of pooled human serum lacking HSV antibodies, M is the maximal isotope release (85%) from target cells after three freeze-thawed cycles, and B is the background release from target cells alone (5- 15%). RESULTS We first examined surface markers on populations of MC at different stages of K-cell isolation (Table 1). The MC remaining after nylon wool adsorption and E-RFC depletion represented 4% of the unfractionated cells. In this K-enriched fraction, 66% of the cells reacted with AMT, 47% expressed Fc receptors for IgG (EA, positive) and 4% had Fc receptors for IgM (EAIII positive). Comparative analysis using two different methods for isolation or enumeration of spontaneous E rosettes in the K-enriched fraction (1% or 5% neuraminidase-treated SRBC) showed no significant differences in the numbers of E rosettes. Virtually none of the K cells formed spontaneous E rosettes. To insure that K cells were not reacting with AMT via Fc receptors, we also used fluoresceinated staphylococcal protein A (FITC-SPA) according to the method of Ades et al. (12). There was no significant difference in the number of K cells detected by AMT with either the direct immunofluorescence assays or with AMT plus FITC-SPA. A small percentage (13%) of cells expressed sIg and also were reactive with a specific B-cell antiserum (6, 13) in a double-label direct immunofluorescent assay. No cells were doubly stained when incubated with RITC anti-Ig and FITC-AMT. None contained cytoplasmic IgM. TABLE
1
Surface Markers on Blood Lymphocytes during K-Cell Fractionation Cell fraction Unfractionated MC Nylon wool-nonadherent Nylon wool-nonadherent, RFC-depleted MC (K-enriched)
MC
sIg”
E
AMT
EA,
EA,
12 * qll)b 1 ” l(14)
70 2 7(10) n.t.d
79 k 6(9) 95 k 3(8)
11 2 4(35) nt.
58 + 9(35)’ n.t.
13 ?I 5(14)
o(lO)
66 + 9(14)
47 2 9(5)
4 k 4(3)
’ Surface immunoglobulin positive. * Number of individual samples tested are given in parentheses. c Normal values established at the Cellular Immunobiology Unit, University of Alabama Medical Center. d Not tested.
182
ADES ET AL.
Since the cells participating in the ADCC reaction possess surface IgG-Fc receptors (14), the K-cell-enriched fraction was further subdivided into populations positive or negative for IgG-Fc receptors by Ficoll-Hypaque centrifugation. In the EA,-positive subset, 85 k 4% (N = 3) expressed a T-cell differentiation antigen(s) reactive with AMT. Induction experiments were then conducted to determine whether Erosette-negative cells in the K-cell-enriched fraction would express E-rosette receptors after incubation for 72 hr with different doses of PHA. In three experiments with PBL from different normal subjects, an average of 25% of the mitogen-activated cells became E-rosette positive, whereas unstimulated cells remained E-rosette negative (Table 2). To confirm that the E-rosette-positive cells did not result from proliferation of any contaminating E-RFC, we first irradiated aliquots of cells with 2000 R before incubation with PHA. Irradiation only slightly reduced the percentage of E-RFC (Table 2). Preincubation of the K-cell-enriched fractions with mitomycin C (50 &3-4 x lo6 cells) also did not appreciably reduce the percentage of PHA-induced E-RFC. As a further control to be sure that PHA was not acting as a nonspecific ligand joining SRBC to lymphocytes, we incubated B-lymphoblastoid cells (RAJI) or purified normal blood B lymphocytes for 72 hr with an optimal stimulating dose of PHA (Table 2). No E-RFC were induced with the B-lymphoblastoid cells despite the observation that rhodamine-conjugated PHA bound to the RAJI cell membranes. Furthermore, blood B cells from PHA cultures showed clumping indicative of the agglutinating property of this mitogen, but less than 1% of the cells formed E rosettes. In addition, the percentage of E-RFC in K-cell-enriched fractions incubated with PHA did not reach maximum levels until the last day of the 72-hr culture period. In a typical experiment, for example, there were 0.75% E-RFC at 24 hr, 4.5% E-RFC at 48 hr, and 19.5% E-RFC at 72 hr. Similar values were obtained in parallel cultures by using mitomycin C TABLE
2
Induction of E-Rosette Receptor Expression in Cell Culture
Cell fraction
Stimulus
RFC after 72-hr incubation (So)
RFC before to incubation m
None PHA”
0 0
0 25 f 9(10)b
(irradiated)
PHA
0
20( 1)C
K-enriched (mitomycin C-treated)
PHA
0
RAJId
PHA
0
Purified B cells
None PHA
K-enriched K-enriched
16 f 6(3) 0
IMMUNOLOGY
E-Rosette
4,
179-185 (1979)
Receptors Induced K Cells Expressing
EDWIN
W. ADES,‘.*
by Phytohemagglutinin on Human T-Cell Surface Antigens’
PATRICIA DOUGHERTY,* STEVEN AND CHARLES M. BALCH”,~,*
L. SHORE,?
*Cc~llular Immunobiology Unit of the Tumor Institute, Depurtments of Microbiology und Surgery, the Comprehensive Cancer Center, University ofAlabamu Medicul Center, Birmingham, Alabama 35294, und tClinicu1 Immunology Luborutory, Center for DiseuscJ Control, Public Health Service, U.S. Depurtment of Health. Education and Welfure, Atlanta. Georgia 30333.’ Received
October
24. 1978
Killer cells (K cells) enriched from human blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) were examined for surface markers. Sixty-seven percent of the E-rosette-negative, sIg-negative cells reacted with anti-T cell serum (AMT) previously shown to react with immunochemically defined T-cell antigens. Phytohemagglutinin induced 25% of K cells to express an E-rosette receptor. When these induced cells were isolated, greater than 98% reacted with AMT and 17% expressed the Fc receptor for IgG. Furthermore, they retained their functional capacity in ADCC. These findings demonstrate that an E-rosette receptor can be induced on human K cells. The data suggest the K-cell fraction included a population of thymus-dependent lymphocytes which can function as effector cells in ADCC.
INTRODUCTION A major portion of the mononuclear effector cells or killer cells (K cells) in human blood which mediate antibody-dependent cellular cytotoxicity (ADCC) lack the surface marker profile characteristic of either B cells or T cells (1,2). These K cells, bearing surface receptors for the Fc fragment of IgG, are enriched in a minor fraction of cells comprising about 4% of the peripheral blood mononuclear cells (MC; 2, 3). It is unclear whether K cells are a distinct third population of lymphoid cells or whether they are lymphocytes of T-cell or B-cell lineage (4). In this study, evidence is presented that a subpopulation of T cells can function as effector cells in ADCC. MATERIALS
AND METHODS
Zsolation oflv cells. Cellular fractions enriched in K cells were isolated from the
peripheral
blood MC of healthy donors by sequential passage through nylon wool
r This investigation was supported by grants from the Medical Research Service of the Veteran’s Administration and from the National Institute of Health (CA13148 CA16673). 2 Recipient of a Special Fellowship from the Leukemia Society of America. 3 Recipient of a Faculty Fellowship in Oncology from the American Cancer Society. 4 To whom requests for reprints should be sent: Department of Surgery, 750 Lyons-Harrison Research, University of Alabama Medical Center, Birmingham, Ala. 35294. 179 0008-8749/79/050179-07$02.00/O Copyright 0 1979by Academic Press, Inc. All rights of reproduction in any form reserved.
180
ADES ET AL.
columns and E-rosette depletion has been described (2) except that neuraminidase-treated sheep erythrocytes (SRBC) and a 4°C incubation temperature were employed for the E-rosette depletion step. This latter system has provec more efficient and sensitive than our previously described system for E-rosette depletions. Erythrocyte-rosette-forming cells (E-RFC) were separated from nonrosetting cells in the K-cell fraction following phytohemagglutinin (PHA) incubation (see below), rosetting suspensions were layered over a Ficoll-Hypaque cushion as described previously (5) and centrifuged for 40 min at 4OOg. Non-rosetting cells at the interface were harvested. Contaminating SRBC were removed by resuspending the E-RFC pellet into 2-3 ml of distilled water for 15 set and then rapidly adding an equal volume of 0.30 M NaCl. Both rosetting and nonrosetting cell populations were then washed twice with RPM1 1640 before cell function in ADCC was examined. Analysis of cellpopulations. A double-label immunofluorescence assay was used to simultaneously detect T and B lymphocytes in human blood (6). T cells were identified by using a purified anti-T-cell reagent (AMT) conjugated with fluorescein isothiocyanate (FITC). The preparation, characterization, and specificity of this same lot of AMT for human T cells have been described (7,8). This reagent has been shown to react with two differentiation antigens on T cells with molecular weights of 25,000 and 16,000 (8). Surface immunoglobulin (sIg)-bearing B lymphocytes were detected simultaneously by using the F(ab’), fragment of rhodamine-conjugated (RITC) goat antiserum raised against purified human IgG, IgM, and IgA (Cappell Laboratories5 Downington, Pa.). In enumeration of E-RFC, cell fractions were washed twice in Hanks’ balanced salt solution with 10% heat-inactivated fetal calf serum (FCS) and adjusted to a concentration of 7.5 x 10Vml. Equal volumes of cell suspensions and 1% neuraminidase-treated SRBC (9) were mixed, incubated at 37°C for 10 min, centrifuged at 2Og for 10 min at 4°C and incubated on ice for 60 min. Cells binding three or more SRBC were scored as positive. Purified populations of normal blood B cells were prepared as previously described (13). These cells and cultured B-lymphoblastoid cell line (RAJI) served as control cells. Rhodamine-conjugated PHA was prepared according to our previous methods for conjugation of antisera (7). Ox erythrocytes (ORBC) coated with either rabbit IgG or IgM antiORBC (EAw or EAc) were prepared and used for Fc receptor assays and isolation of EAc-positive K cells according to the method of Moretta and colleagues (9). Cells were also examined for the presence of cytoplasmic IgM (10). Induction of E-rosette receptor expression. For PHA-induced expression of E-rosette receptors, K cells were resuspended to a concentration of 5 x IO5 cells/ml in RPM1 1640 with 20% FCS and gentamicin (0.05 mg/ml), and then seeded in sterile plastic tubes in 1 ml volumes. Test cells were incubated with PHA (Difco, 5 ~1 of a 1:5 dilution of a stock solution/l x lo5 cells) for 72 hr in a 37°C incubator with 5% CO, in air. Control cells were treated similarly, but were not stimulated with mitogen. 5 Use of trade names is for identification only and does not constitute endorsement by the Public Health Service or by the U.S. Department of Health, Education, and Welfare.
HUMAN
T LYMPHOCYTES
AS ADCC EFFECTOR
181
CELLS
Antibody-dependent cellular cytotoxicity. ADCC effector-cell activity was assessed against 51Cr-labeled Chang liver cells acutely infected with type 1 Herpes simplex virus (HSV) as previously described (2). The human serum antibody to HSV which mediates the cytotoxic reaction in this system is of the IgG class (11). Cytotoxicity was calculated according to the following equation:
Percentage
specific release = ___ c-D M-B
x 100
’
where C is the percentage isotope release from target cells in the presence of MC and a 1:200 dilution of pooled heat-inactivated human serum HSV antibodies, D is the percentage isotope release from target cells in the presence of MC and a 1:200 dilution of pooled human serum lacking HSV antibodies, M is the maximal isotope release (85%) from target cells after three freeze-thawed cycles, and B is the background release from target cells alone (5- 15%). RESULTS We first examined surface markers on populations of MC at different stages of K-cell isolation (Table 1). The MC remaining after nylon wool adsorption and E-RFC depletion represented 4% of the unfractionated cells. In this K-enriched fraction, 66% of the cells reacted with AMT, 47% expressed Fc receptors for IgG (EA, positive) and 4% had Fc receptors for IgM (EAIII positive). Comparative analysis using two different methods for isolation or enumeration of spontaneous E rosettes in the K-enriched fraction (1% or 5% neuraminidase-treated SRBC) showed no significant differences in the numbers of E rosettes. Virtually none of the K cells formed spontaneous E rosettes. To insure that K cells were not reacting with AMT via Fc receptors, we also used fluoresceinated staphylococcal protein A (FITC-SPA) according to the method of Ades et al. (12). There was no significant difference in the number of K cells detected by AMT with either the direct immunofluorescence assays or with AMT plus FITC-SPA. A small percentage (13%) of cells expressed sIg and also were reactive with a specific B-cell antiserum (6, 13) in a double-label direct immunofluorescent assay. No cells were doubly stained when incubated with RITC anti-Ig and FITC-AMT. None contained cytoplasmic IgM. TABLE
1
Surface Markers on Blood Lymphocytes during K-Cell Fractionation Cell fraction Unfractionated MC Nylon wool-nonadherent Nylon wool-nonadherent, RFC-depleted MC (K-enriched)
MC
sIg”
E
AMT
EA,
EA,
12 * qll)b 1 ” l(14)
70 2 7(10) n.t.d
79 k 6(9) 95 k 3(8)
11 2 4(35) nt.
58 + 9(35)’ n.t.
13 ?I 5(14)
o(lO)
66 + 9(14)
47 2 9(5)
4 k 4(3)
’ Surface immunoglobulin positive. * Number of individual samples tested are given in parentheses. c Normal values established at the Cellular Immunobiology Unit, University of Alabama Medical Center. d Not tested.
182
ADES ET AL.
Since the cells participating in the ADCC reaction possess surface IgG-Fc receptors (14), the K-cell-enriched fraction was further subdivided into populations positive or negative for IgG-Fc receptors by Ficoll-Hypaque centrifugation. In the EA,-positive subset, 85 k 4% (N = 3) expressed a T-cell differentiation antigen(s) reactive with AMT. Induction experiments were then conducted to determine whether Erosette-negative cells in the K-cell-enriched fraction would express E-rosette receptors after incubation for 72 hr with different doses of PHA. In three experiments with PBL from different normal subjects, an average of 25% of the mitogen-activated cells became E-rosette positive, whereas unstimulated cells remained E-rosette negative (Table 2). To confirm that the E-rosette-positive cells did not result from proliferation of any contaminating E-RFC, we first irradiated aliquots of cells with 2000 R before incubation with PHA. Irradiation only slightly reduced the percentage of E-RFC (Table 2). Preincubation of the K-cell-enriched fractions with mitomycin C (50 &3-4 x lo6 cells) also did not appreciably reduce the percentage of PHA-induced E-RFC. As a further control to be sure that PHA was not acting as a nonspecific ligand joining SRBC to lymphocytes, we incubated B-lymphoblastoid cells (RAJI) or purified normal blood B lymphocytes for 72 hr with an optimal stimulating dose of PHA (Table 2). No E-RFC were induced with the B-lymphoblastoid cells despite the observation that rhodamine-conjugated PHA bound to the RAJI cell membranes. Furthermore, blood B cells from PHA cultures showed clumping indicative of the agglutinating property of this mitogen, but less than 1% of the cells formed E rosettes. In addition, the percentage of E-RFC in K-cell-enriched fractions incubated with PHA did not reach maximum levels until the last day of the 72-hr culture period. In a typical experiment, for example, there were 0.75% E-RFC at 24 hr, 4.5% E-RFC at 48 hr, and 19.5% E-RFC at 72 hr. Similar values were obtained in parallel cultures by using mitomycin C TABLE
2
Induction of E-Rosette Receptor Expression in Cell Culture
Cell fraction
Stimulus
RFC after 72-hr incubation (So)
RFC before to incubation m
None PHA”
0 0
0 25 f 9(10)b
(irradiated)
PHA
0
20( 1)C
K-enriched (mitomycin C-treated)
PHA
0
RAJId
PHA
0
Purified B cells
None PHA
K-enriched K-enriched
16 f 6(3) 0
