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Selective lysis of target cells by interleukin-2-expanded peripheral blood mononuclear leukocyte clones
CELLULAR
IMMUNOLOGY
118,458-469 ( 1989)
Selective Lysis of Target Cells by Interleukin-2-Expanded Blood Mononuclear Leukocyte Clones’
Peripheral
BENJAMIN P. CHEN,*JACQUELYN A.HANK,* ERICE.KRAUS,* AND PAUL M. S~NDEL*~~X~Y~ Departments of *Human Oncology, TPediatrics, and $Genetics, University of Wisconsin, Madison, Wisconsin 53792 Received August 23, 1988; accepted October IO, I988 The presence ofdistinct cytolytic subsets within interleukin-2-expanded peripheral blood leukocytes (IEL) cultures was demonstrated by clonal analysis. Thirty-seven IEL clones were isolated from two healthy blood donors; 2 1 destroyed both Daudi and K562 cell lines. Of those 2 1 clones, 1 clone could destroy autologous PBM, 7 clones could destroy fresh allogeneic ovarian carcinoma (OVA-CA) cells, and 6 clones could destroy normal autologous PBM and fresh OVACA cells. Twelve of the 37 clones destroyed only one of the four targets tested: 8 clones destroyed K562,2 clones destroyed Daudi, and 1 clone each was selective for autologous PBM or OVACA. Of the remaining 4 clones, 1 destroyed OVA-CA and Daudi cells, 1 destroyed PBM and K562, 1 destroyed PBM and Daudi cells, and 1 destroyed PBM, Daudi, and OVA-CA. These results suggest that these functionally heterogeneous cytolytic clones may use different cell recognition or cytolytic mechanisms to enable these distinct and, at times, reciprocal patterns of target cell selectivity. 0 I989 Academic Press, Inc.
INTRODUCTION Activation of human peripheral blood mononuclear leukocytes (PBM) with IL-2 generates phenotypically heterogeneous populations of effector cells which mediate non-MHC-restricted cytotoxicity of various tumor cell lines, fresh tumor cells, and to a lesser extent, normal vascular endothelial cells and PBM ( 1- 13). This non-MHCrestricted cytotoxicity has been designated the lymphokine-activated killer (LAK) phenomenon (1, 2). The heterogeneity of cell phenotypes mediating the LAK phenomenon could suggest that IL-2 induces diverse populations of lymphoid cells to nonspecifically lyse both tumor and normal cells, or that IL-2 expands subsets of IEL with different target cell “selectivities.” Data are consistent with both of these two mutually nonexclusive possibilities (5- 12). Skinner et al. (5) and Brooks et al. (6, 7) have shown that Sendai virus-specific and alloantigen-specific cytotoxic T cell clones (CTL), upon exposure to a high concentration of IL-2, become nonspecific killer cells capable of destroying both natural killer (NK)-sensitive and NK-resistant target cells. While Philips and Lanier (8) Ortaldo et al. (9), and Ferrini et al. (10) have reported ’ This study was supported by Grants NIH-CA 32685 and American Cancer Society CH-237. ’ To whom correspondence should be addressed at K4/448 UWCCC, 600 Highland Ave., Madison, WI
53792. 458 0008-8749/89$3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
CLONAL
ANALYSIS
OF IL-2-EXPANDED
LEUKOCYTES
459
that the majority of tumoricidal activity of IEL cultures is mediated by a population of CD3-, CD 16+, NM 1’ killer cells, Malkovsky et al. ( 1 1), Damle et al. ( 12), and Tilden et al. (13) have further documented that IL2 could induce other lymphoid cell populations to mediate non-MHC-restricted cytotoxicity of tumor cells. Our previous study with bulk populations of IEL show that unlabeled tumor cell lines could competitively inhibit much of the non-MHC-restricted killing of autologous normal PBM target cells, suggesting that some of the IEL which kill tumor cell lines also “recognize” the PBM target cells ( 14). In this study, we have used split well analysis of limiting dilution cultures and clonal analysis to document that IL-2 expands IEL subsets with different target cell selectivities. These experiments suggest that different non-MHC-restricted effector clones may utilize different recognition (or lytic) mechanisms to mediate quantitatively distinct levels of target cell selectivity. MATERIALS
AND METHODS
Leukocytes Fresh PBM from healthy volunteer donors were obtained by Ficoll-Hypaque sedimentation of anticoagulated whole blood. Viability of PBM as determined by eosin dye exclusion was >98%. Blood donors signed consent forms approved by the University of Wisconsin committee for the protection of human subjects. Frequency Analysis of IEL Precursors by Limiting
Dilution
Cultures
Fresh PBM were diluted serially to yield 2000, 1500, 1000,750,500, and 375 cells/ well (48 replicate wells of each) in tissue culture media HS-RPM1 [RPM1 1640 supplemented with 25 mM Hepes buffer, 2 mM L-glutamine, penicillin, streptomycin (all purchased from Flow Lab., McLean, VA), and 10% pooled heat-inactivated nontransfused human male serum (HS)]. Each microwell also received 5 X lo4 irradiated (4000 cGy) autologous PBM as feeder cells and 100 units/ml of human recombinant IL-2 (a gift from the Cetus Corp., Emeryville, CA). These cultures were incubated at 37°C in a 5% CO2 humidified incubator for 14 days prior to analysis. Estimates of the frequencies of IEL cytolytic for different target cell types were obtained by the minimum x2 method from the Poisson distribution relationship between the number of PBM initially plated per well and the negative logarithm of the percentage of noncytolytic (negative) cultures ( 15). Cell Lines and Fresh Tumor Cells The NK-sensitive erythroleukemia cell line K562 and the HLA class I-deficient Burkitt’s lymphoma Daudi cell line were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). These cell lines were maintained in HS-RPMI. Fresh ovarian carcinoma cells were obtained by Ficoll-Hypaque sedimentation of ascites fluids from patients and were cryopreserved in HS-RPM1 containing 10% dimethyl sulfoxide. These tumor cells were thawed for use as target cells on the day of assay.
460
CHEN ET AL. TABLE 1 Frequencies of IEL Precursors Cytolytic for Autologous PBM, OVA-CA, and Tumor Cell Lines Precursor frequency estimations 0T Target cells
Donor EK
Donor BC
Donor PS
Autologous PBM OVA-CA Daudi K562
1f5950b l/973 l/386 l/442
I/5556 l/5654 l/615 l/535
l/8172 l/9062 l/856 l/410
a Estimations based on Poisson’s distribution, concentration. b Average of two independent estimations.
using six cell concentrations with 48 wells at each cell
Derivation of IEL Clones Three approaches were taken to generate IEL clones. (A) Cloning by limiting dilution. Fresh PBM from healthy donors were plated at 1000,500, 100,50, 10, and I cell per well in the presence of 100 units/ml of IL-2 and 5 X 1O4irradiated autologous PBM as feeder cells. Microcultures with visible growth within lo- 14 days were selected and tested for cytotoxicity. The cloning efficiency at the highest cell concentration is usually less than 10% and regression analysis demonstrated a linear correlation of the logarithm of the percentage of negative cultures with the number of PBM initially plated per well indicating that these clones most likely were derived from a single cell. IEL clones generated by this approach are identified in the text with the prefix LD. (B) Cloning by polyclonal activation of PBM with anti-CD3 monoclonal antibody (MoAb). Fresh PBM were plated at one cell every two to three wells in the presence of 5 x 1O4irradiated (4000 cGy) autologous PBM and a predetermined optimal concentration of anti-CD3 MoAb and IL2 at 100 units/ml. Cytotoxic cultures were selected within lo- 14 days and expanded in HS-RPM1 media containing 100 units/ml of IL-2. These clones were restimulated every 2 weeks with anti-CD3 MoAb and irradiated autologous PBM as feeder cells. All cytotoxic clones generated by this method were washed and cultured in medium with IL-2 and without anti-CD3 MoAb for at least 7 days prior to their being tested in the cytotoxicity assay. IEL clones derived by this approach are designated with the prefix CD. (C) Cloning by serial limiting dilution. Fresh PBM at 2 X lo4 cells/ml were plated in 48 replicate wells and serially diluted to yield six cell concentrations ranging from 150 to 2000 cells/well. Each culture well also received 5 X lo4 irradiated (4000 cGy) autologous PBM feeder cells and IL2 at 100 units/ml. Between Days 10 and 14, cultures with visible growth and flanked by two or more negative wells of the same dilution series were picked and screened for cytotoxicity. Clones generated with this approach are identified by the prefix SD. All cytotoxic clones generated by these three cloning procedures were propagated in multiple microwells and expanded, when confluent, into macrowells and then flasks using standard cell growth technique (10).
CLONAL
ANALYSIS
OF IL2-EXPANDED
LEUKOCYTES
461
Cell-Mediated Lysis (CML) For precursor frequency estimation, each individual limiting dilution microculture was equally split into four replicate microwells (Costar, Cambridge, MA) and assayed separately for cytotoxicity against autologous PBM, Daudi, K562, and freshly thawed OVA-CA target cells. When clones were assayed, aliquots of the cells from each clone were split into replicate wells and also tested simultaneously on these same four target cell populations. Cytotoxicity was measured in an 8-hr 5’Cr release assay. Target cells were labeled with 0.25 mCi of 5’Cr (New England Nuclear, Boston, MA) in 0.3 ml of HS-RPM1 for 2 hr, washed three times in media, and diluted and 5 X lo3 cells were added in 0.1 ml to the microwells containing the effector cells. The plates were centrifuged at 200g for 5 min before culturing at 37°C. After 8 hr of incubation, the plates were centrifuged at 5OOg for 5 min and supernatants were collected with Skatron harvesting frames (Stering, VA) for measuring 5’Cr release in a gamma counter. Percentage cytotoxicity is calculated using the formula cytotoxicity
=
experimental cpm - spontaneous cpm x 100%. maximum cpm - spontaneous cpm
Spontaneous release values were obtained by incubating target cells with medium alone. Maximum release values were determined by incubating target cells in cetrimide detergent (Sigma Chemicals, St. Louis, MO). For each population of target cells, the mean spontaneous release value plus three times the standard deviation was chosen as the cutoff value to separate negative cultures from positive cultures for frequency analysis.
Monoclonal Antibodies Anti-CD3 (CRL-800 I), -CD4 (CRL 8002), and -CD8 (CRL 80 14) used for inhibition studies were ammonium sulfate-precipitated hybridoma supernatants; the hybridomas for these MoAb were obtained from ATCC.
Phenotyping of Leukocytes Indirect immunofluorescence was done with anti-CD3, -CD4, and -CD8 (BectonDickinson MoAb center, Mountain View, CA) and NKI-I-1 (Coulter, Hialeah, FL) and developed with a fluorescein-labeled anti-mouse immunoglobulin. (Coulter). Five thousand cells were analyzed for each marker on an Ortho Cytofluorograph Systems Model 50 flow cytometer (Ortho, Raritan, NJ). RESULTS
Diflerential Distribution of IEL Precursors Cytolytic for Tumor Cells and PBM To estimate the precursor frequencies of IEL cytolytic for Daudi, K562, autologous PBM, and OVA-CA target cells, limiting dilution cultures of IEL were split into four equal aliquots and tested against each of these four target cells. The IEL precursor frequencies, averages of two separate determinations for each of the three unrelated healthy blood donors, are shown in Table 1. The frequencies of IEL capable of lysing Daudi and K562 are much higher than those cytolytic for autologous PBM and OVACA for all three donors. Of all the estimates, greatest variations are seen in the fre-
462 I 60 am b 40 ” 20 .=
a
.o ;: ‘0 O z0 -20
o\"
0
20
40
60
80
100
0
20
40
K562 %
60
Daudi Cytotoxicity
80
100
0
20
40
Ovarian
60
80
100
Tumor
1. Split well analysis of limiting dilution cultures. IEL activated in microwells were split into replicates and assayed simultaneously for killing against Dam%, K562, OVA-CA, and autologous PBM in a 8hr “Cr release assay. Paired comparison of cytotoxicity of autologous PBM vs tumor cell targets used IEL microcultures that were initially plated at 2000, 1500, 1000, 750, 500, and 375 cells/well. Thus each individual well was tested (by replicate plating) on all four targets. These data are presented here in three plots, showing for each well (plotted as a single point) the destruction of the PBM targets versus the destruction of K562, Daudi, and OVA-CA targets in (a), (b), and (c). The lines represent percentage cytotoxicity equivalent to the mean plus three times the standard deviation of 24 spontaneous release values measured on each target. The multiple cultures that did not kill either target cells on a given graph are included in the shaded area. FIG.
quencies of IEL tumoricidal for OVA-CA. Cytotoxicity results of limiting dilution cultures from donor EK are further analyzed in split culture plots, Fig. 1. Each plot compares the cytotoxicity on autologous PBM target cells by cells from an individual well with cytotoxicity by replicates of that same well on K562, Daudi, or OVA-CA target cells. A total of 144 wells, 24 wells each initially seeded at 6 cell concentrations ranging from 375 to 2000 cells/well, were analyzed. As shown in Fig. 1, 14, 69, 123, and 110 wells mediated significant destruction of PBM, OVA-CA, K562, and Daudi target cells, respectively. The differential distribution of IEL precursors capable of lysing the four target cells is due to the presence of IEL subpopulations which mediate different patterns of selective target cell destruction and not explicable solely by differential lysability of the target cells, as shown below with IEL clones. IEL Clones Display Selective Cytotoxicity The cytotoxic activities of 4 IEL clones are shown in Fig. 2. These 4 IEL clones each gave a different pattern of cytotoxicity for the four target cells. Although the magnitude of killing of susceptible target cells increased with greater numbers of effector cells, the pattern of cytotoxicity unique to each of these four clones remains the same at all the effector to target cell ratios tested. These results suggest that the “selectivity” observed with IEL clones is not the sole result of differential lysability of the target cells used. An additional 33 cytotoxic IEL clones were isolated from two healthy donors PS and BC. All 37 clones can be segregated into six major groups (Table 2). Six of these clones were cytotoxic for Daudi, K562, autologous PBM, and OVA-CA target cells (group I). Seven clones lysed the two tumor cell lines and fresh tumor cells but not autologous PBM (group II). Seventeen clones destroyed tumor cell lines but not fresh tumor cells or PBM (groups III, IV, and V). Of these 17 clones, 7 clones were cytotoxic for both Daudi and K562 (group III), 2 clones were cytolytic for Daudi only (group IV), and 8 clones only lysed K562 cells (group V). Other IEL
CLONAL
ANALYSIS
OF IL-Z-EXPANDED
LEUKOCYTES
463
CLONE
SD-CD2 80 3 :g 6o
.-.
Autologous PBM Ovor~on Tumor
B 2 G 40
0 25
5
IO
20
25
5
IO
Effector
20
-
to Torget
25
Cell
5
IO
20
Ratkos
FIG. 2. IEL clones with differential cytotoxic activities. Cytotoxicity of IEL clones SD-CDlc, SD-CD2, LD-10, and LD-3 were measured in an S-hr “Cr release assay.Various numbers ofeffector cells were added to 5 X 1O3 of the indicated target cells in microwells to achieve the specific effector to target cell ratios. Results represent the mean + standard deviation of triplicate cultures.
clones that do not fall in any of the five groups described are represented in group VI. Within this group, IEL clone CD-BA 10 lysed only the OVA-CA cells, clone SD-CD 1c lysed autologous PBM only, clones SD-2C, SD-CD2, and LD-3 lysed PBM and one or both tumor cell lines but not fresh tumor cells. Clone LD-BC2 lysed OVA-CA and Daudi, and clone SD-2D lysed OVA-CA, PBM, and Daudi cells but not K562. All these clones gave reproducible results in two to four independent experiments. Phenotypes of 18 IEL clones, representatives from all six groups, are shown in Table 3. All of these clones express the CD3 marker, 11 of these clones are CDS+/ CD4-, 4 clones are the CD4+/CD8-, 2 clones are CD4+/CDS+, and 1 clone, clone LD-BC2, does not express the CD4 or CD8 marker. All clones, except clone SDCD2, also express the NKH-1 marker. Although there is no distinct correlation of IEL phenotypes with target cell selectivity, these phenotypically heterogeneous IEL clones may be modulated by different MoAb directed at these markers.
Diferential Eflects of Anti-CD3, -CD4, and -CD8 MoAb on Cytotoxicity Mediated by IEL Clones We have previously observed that the nonspecific killing of tumor cell lines by bulk IEL cultures can be potentiated by anti-CD3 MoAb, and at times weakly inhibited by anti-CD8 MoAb (( 14) and unpublished results). The weak inhibition of bulk IEL cultures could result from selective inhibition by anti-CD8 MoAb of phenotypically different cell subsets within the bulk IEL populations. We tested the effect of antiCD3, -CD4, and -CDS MoAb on two CD3+/CD4-/CDS+ IEL clones (clones CDBCl and SD-2D), one CD3+/CD4+/CDSf IEL clone (clone CD-AH2), and a CD3-/ CD4-/CDS-, Fc+ killer clone initially described as a natural killer-like clone (NK77) ( 16). IEL clones CD-BCl and CD-AH2 and NK77 killed all PBM, OVA-CA, Daudi, and K562 target cells tested, while clone SD-2D did not kill K562 cells (Fig. 3). The addition of anti-CD3 MoAb enhanced the killing of PBM and OVA-CA target cells by all three CD3+ clones, but not by the CD3- NK77 clone. Anti-CD3 also enhanced the killing of Daudi target cells by clone CD-BC 1 and SD-2D, but inhibited the killing
464
CHEN ET AL. TABLE 2 Differential Cytotoxic Activities of IEL Clones % Cyotoxicity of target”
Group I
II
III
IV V
VI
Bulk culture
IEL clone
E:Tb
SD-36~11~ SD-4D LD-10 LD-17 CD-BC 1 CD-AB3
10 10 30 30 30 10
19 (3)d 22 (4)
CD-AC 1 CD-BH 12 CD-AE6 CD-AE7 CD-AH2 SDdC SD-6A
30 30 30 10 10 20 10
6(l)
CD-BClO CD-BGl2 CD-AA7 LD-13 SD-52GII SDdB SD-CD 11
30 30 30 20 10 20 20
CD-AG 1 SD-5A
30 20
l(3) 8 (3)
CD-BB9 SD-l 3GII SD-32PII SD54PII SD-1C SD-ID SD-CD3 SD-CD9
30 10 10 10 12 10 10 20 20
l(l) 5 (3) 3 (3) 3 (3) 4 (5) -10(4) -13 (5) -l(l)
30 20 10 20 20 30 10
28 (4) 27 (8) 31(7) 16 (3)
2(l)
5 (2) 29 (4)
80(2)
-3 (1) 3 (2) 79 (5) 56 (1) 81(4) 7(l)
10 (2)
12(l)
11(l)
-2 (2)
20
32 (2)
71(10)
82 (9)
92 (3)
CD-BA 10 SD-CDlc SD-2C SD-CD2 LD-3 LD-BC2 SD-2D
Auto]. PBLC
Ova’
Daudi
K562
20(6)
21(2) 26(l)
12 (3) 19(2) 44 (2) 25 (1)
13 (2) 14(l)
12(1)
21(2)
20 (3)
39 (9)
57 48 77 47 85 85
14(l) 14 (2)
93 (3) 49 (2) 14(l) 30 (2) 78 (5) 97(10) 80(11)
7 (2) 4 (3)
6(l) 5 (1) -3 (5) 8 (4) 3(l) -l(l) -l(3)
8(l) -2 (3) -7 (3) 7(l)
-
12(1) 21(3) 15 (4) 19(8) 25 (5) 3 (2) 7 (2) 5 (2) 10 (3) l(2)
48 (6) 91(5) 61(3)
8 (2)
30(l) 38 (1) 27 (2) 31(l) 51 (8) 69 (8)
4 (2)
18(l)
8 (3) 3 (3)
43 (5)
6 (2) 9 (3) 3 (3)
6 t-3 3 (2) 2 (3) l(2) 4(l)
6(l)
8(l)
6 (2)
15 (2)
8(l) 2 (3)
8(2)
16(l) l(1) l(3) -4 (5) 4 (8)
-8 (6) -2 (4) 0 (3) l(l) 4(l) 3 (2) 3 (2) 77 (6) 9(l) 7(l)
(7) (16) (4) (2) (1) (5)
88 (4) 75 (3)
80(l) 85 (3) 85 (4)
81@) 77 (5) 88 (3) 86 (3) 72 (5) 39 (2) 19 (2) 55 (4) 66 (3) 0 (2) 10 (4) 46 (2) 39 (2) 36 (2) 37 (2) 28 (3) 23 (9)
26(8) 23 (1) 29 (2)
’ Cytotoxicity was measured in an 8-hr “Cr release assay. b Cytotoxicity was tested at two to four effector to target cell ratios. Only cytotoxicity at the highest E:T ratio is shown. ‘Autoi. PBL = autologous PBL cultured in medium for 3 days; OVA = freshly thawed ovarian carcinoma cells. d Mean cytotoxicity ? standard deviation of triplicate cultures. ’ IEL SD and LD clones were cloned in IL-2 alone; CD clones were cloned in the presence of anti-CD3 monoclonal antibody.
CLONAL
ANALYSIS
OF IGZ-EXPANDED
LEUKOCYTES
465
TABLE 3 Phenotypes of IEL Clones’ IEL clones
CD3
CD4
CD8
NKH-1
I
LD-IO LD-17 CD-BC 1
+ + +
+ -
+ + +
+ + -t
II
CD-AC 1 CD-BH12 CD-AE6 CD-AH2
+ + + +
+ +
+ + +
+ + + +
III
CD-BClO CD-BGl2 LD-13
+ + +
-
+ + +
+ + +
IV
CD-AG 1 SD-5A
+ +
+ +
-
+ +
V
CD-BB9 SD-1C
+ +
-
+ +
+ +
VI
SD-2C SD-CD2 LD-BC2 SD-2D
+ + + +
+ -
+ +
+ + +
Groups
a The phenotypes of each IEL clone was assessedby indirect immunofluorescence of 5000 cells for each marker.
of the same target cells by clone CD-AH2. The very high level (plateau) killing of K562 mediated by clones CD-BCl and CD-AH2 was not affected by anti-CD3 MoAb; this MoAb did enhance the killing of K562 cells by these two clones in a separate experiment performed at a lower E:T ratio (data not shown). Anti-CD3 MoAb also induced significant killing of K562 cells by clone SD-2D, although this clone normally did not kill K562 target cells. MoAb anti-CD8 only inhibited the killing of Daudi cells by clone CD-BC I and CD-AH2, and anti-CD4 MoAb did not affect the lytic activity of all four clones tested. DISCUSSION A variety of cell types are lysed by bulk IEL populations with greatest lysis of tumor cell lines, intermediate lysis of fresh tumor cells, and minimal, yet significant, lysis of autologous PBM targets (3). The present studies evaluated this lysis by limiting dilution and clonal analysis. If all lytic clones mediated this same pattern of lysis (i.e., greatest killing on the tumor cell lines, and least killing on PBM), it would have suggested that all cells mediating the LAK phenomenon use the same mechanism for recognizing susceptible target cells, and that different levels of lysis observed resulted from quantitative (rather than qualitative) differences in recognition structures or lysability of the different target cells tested. However, these studies showed that different subpopulations of IEL mediate cytolysis with different patterns of “selectiv-
466
CHEN ET AL.
CLONE
0 ‘15.0 km0
‘/IO00 0 ho ‘/ZOO ‘/IO00 0 ‘150 ‘/ZOO ‘/IO00 0 ‘/SO c200 ~/loo0
Dilution
of Monoclonol
Antibodies
FIG. 3. Potentiation of IEL-mediated cytotoxicity by anti-CD3 monoclonal antibodies. IEL clones were preincubated with media (0) or three dilutions of MoAb (l/50, l/200, and l/1000) for 1 hr before the addition of target cells. Monoclonal antibodies were ammonium sulfate-precipitated hybridoma supernatants added to the culture for the duration of the CML assay.Cytotoxicity was measured in an S-hr 5’Cr release assayat an effector to target ratio of 10: 1. Each data point represents the mean of triplicate cultures.
ity” for tumor cell lines, fresh tumor cells, and normal PBM target cells (Table 2). Some clones kill one tumor line and not the other, others kill PBM and not fresh tumor, some kill all target cells, etc. Since these individual experiments tested replicate well lysis on all four target cells simultaneously in parallel to one another, the identification of any clone which kills PBM greater than the tumor lines (i.e., clone SD-CDlc kills PBM but not Daudi or K562; or clone SD-CD2 which kills PBM greater than Daudi but less than K562) proves that for at least some effector populations, PBM targets are not merely less susceptible to lysis than the tumor cell lines. As such, the clones from the six distinct groups identified in Table 2 must either recognize these target populations using different surface molecule recognition structures or use different molecules to mediate lysis, or both, to account for the different patterns of selective target cell destruction. The methods used to generate the clones reported here indicate that many are likely to be “true clones” of single cell origin. However, definitive subcloning of these to prove clonal origin was not performed, leaving open the possibility that some of these populations represent the progeny of more than a single cell. However, these popula-
CLONAL
ANALYSIS
OF
IL-ZEXPANDED
LEUKOCYTES
467
tions (particularly those in Table 2, groups II-VI) show patterns of target cell lysis that are not seen with bulk cultures activated with the same stimuli (i.e., IL-2 with or without anti-CD3 MoAb). These data, and the cell surface marker phenotype data, are consistent with the clonal origin of these populations which we refer to here as clones. Nevertheless, the biological conclusions to be made from these studies do not hinge on proof of clonal origin. Rather, they are derived from the distinct specificity patterns (selectivity) mediated by subpopulations derived from bulk populations without specificity. With respect to surface recognition molecules, both bulk culture and clonal analyses would suggest that most cells mediating the LAK phenomenon may not use the CD3/Ti receptor molecule to recognize target cells, nor does recognition usually involve MHC molecules ( 17). In contrast, the family of cell surface adhesion molecules, designated integrins (18), appear to play a prominent role in the approximation of IEL and target cells. Antibody to laminin ( 19) and LFA- 1 molecules ( 14,20) can each inhibit non-MHC-restricted cytotoxicity by IL-Zactivated bulk cultures and clones. The different selectivity patterns seen here could result from a different proportion or density of these (or other) adhesion molecules on different IEL clones. As such, IEL clones with a broad target spectrum (group I of Table 2) may have adhesion structures that allow effective binding to virtually all tissues; candidates for such ubiquitous adhesion structures include the LFA- 1 and the I-CAM- 1 molecules to which they bind (20,2 1). Some IEL may also use other adhesion/accessory molecules such as the CD4 or CD8 molecules. Recent data suggest that CD4 or CD8 null cells transfected with CD4 or CD8 genes express the molecule and can bind respectively to class II or class I MHC molecule bearing cells, without involving antigen-specific receptors ((22) and Salter et al., personal communication). Conjugates formed via these molecules, however, may be of low affinity and play a secondary role to other adhesion molecules. The selective killing of target cells by IEL clones could also be due to the use of different lytic molecules by individual IEL clones. Cytolytic molecules such as the lymphotoxins (23) and the perform class of lytic molecules (24,25) have been shown to be used by different effector cell types to destroy susceptible targets (26). Different IEL clones may use various amounts of these (or other) lytic molecules and different target cells may show differential susceptibility to the lytic molecules released by a given IEL clone. Combinations of these. recognition and lytic control mechanisms may determine the degree to which a given target cell is lysed by a given IEL clone. In this regard, IEL clones can show distinct patterns of inhibition by different MoAb, when lysis is tested on different target populations (Fig. 3 and P. Fisch, J. A. Hank, and P. M. Sondel, unpublished). These MoAb could either differentially affect the binding of target cells by individual IEL or they may affect different lytic mechanisms (14,20,27). The precise mechanism whereby IEL are activated is not known. Data from several laboratories suggest that IEL represent activated natural killer cells (8-lo), while other studies suggest that some IEL killers are antigen-specific cytotoxic T cells, reversibly “converted” by high concentration of rIL-2 to nonspecific killer cells (5-7). The “nonspecific” IEL clones described in this study (group I) could represent killer cells generated via the latter mechanism. The demonstration of IEL clones with target cell selectivity, however, suggests that not all killer cells can be driven by rIL-2 to kill nonspecifically. This may again suggest that all IEL clones do not possess the same
468
CHEN ET AL.
repertoire of IL-2 inducible @tic mechanisms, or that some effector cells can maintain specificity despite the exposure to high concentrations ofIL-2. The clones which lysed autologous PBM or tumor cells alone (i.e., clones SD-CDIc and CD-BAlO) may indeed be antigen-specific clones that maintained specificity. Clone SD-CDlc may resemble class II-restricted murine autolytic clones with immunoregulatory function (28), while clone SD-BA 10, which lysed the fresh tumor cells and not PBM or tumor Cell lines, may represent an alloreactive clone recognizing ahoantigens on the OVACA. More extensive studies would be required to confirm these possibilities. Nevertheless, these clones were obtained, without antigen-specific priming, using in vitro stimuli shown to induce the LAK phenomenon in bulk cultures ( 1, 3, 14,29). Phenotypicalfy, all the IEL clones generated in this study are CD3+ and, with the exception of clone LD- 17, express either the CD4 or the CD8 marker. The expression of the CD3+ marker by all our IEL clones is consistent with the majority of the cells in the bulk culture expressing the CD3 marker (3, 29). By limiting dilution cloning of Day 4 LAK cultures in the presence of PHA and IL-2 containing supernatant, Rayner et al. (30) have also generated exclusively CD3+ LAK clones, similar to our clones shown in group II of Table 2; those clones killed all tumor target cells tested and were not cytolytic for normal PBM. Similar to our results, Allevena and Ortaldo (3 1) and Hercend et al. (32, 33) have also generated CD3+ killer clones by cloning Percoll gradient-enriched large granular leukocytes; their CD3+ killer clones also demonstrated a heterogeneous pattern of reactivity against a panel of tumor cell lines. However, it is not known if those clones could destroy fresh tumor cells or normal PBM, since those target cells were not included in their studies. LAK cells that are CD3- can be generated by culturing Leul l+ PBM in IL-2 as shown by Ferrini et al. (IO) and Moretta et al. (34); in their studies, the majority of the killer clones were shown to be nonspecific killers, killing both fresh tumor cells and tumor cell lines. One of our IEL clones, clone LD-BC2, is CD3+, CD4-, CD8-, and NKH- 1+. Previous reports have documented nonspecific cytotoxicity by CD3+, CD4-, CD8 cells (16, 17); uniquely, clone LD-BC2 with this phenotype described in this study showed some target cell selectivity, Table 2. We do not know if this clone expresses an antigen-specific T cell receptor. The physiological role of the LAK phenomenon remains undetermined; much of the activity may reflect that of activated NK cells. The precise regulation of the degree of lysis a given IEL clone will mediate on a single target cell population has not been clarified. The present experiments indicate this regulation is a multifactorial process which cannot be explained merely by quantitative differences in “lysability.” By clarifying these mechanisms and learning to control them experimentally, it may someday be feasible to use this information to protect normal tissues from the LAK phenomenon, or more selectively activate those effector cells (or their products) which preferentially mediate lysis of the autologous tumor. ACKNOWLEDGMENTS We thank Dr. Paul Fisch for helpful discussions, Drs. R. Bolhuis and Greg Litton for the generous gift of clone NK77, and Ms. Jean Surfus for her excellent technical assistance.
REFERENCES 1. Rosenberg, S. A., and Lotze, M. T., In “Annual Review ofImmunology” (W. E. Paul, C. G. Fathman, and H. Metzger, Eds.), pp. 68 l-709. Annual Reviews, Palo Alto, CA, 1986.
CLONAL
ANALYSIS
OF IL-ZEXPANDED
469
LEUKOCYTES
2. Grimm, E. A., and Wilson, D. J., Cell. Immunol. 94,568, 1985. 3. Sondel, p. M., Hank, J. A., Kohler, P. C., Chen, B. C., Minkoff, D. Z., and Molenda, J. A., J. ~~~uno~ 137,502,1986.
4. 5. 6. 7. 8. 9. 10.
Damle, N. K., Doyle, L., Bender, J., and Bradley, E., J. Zmmunof. 138, 1779, 1987. Skinner, M., Ertl, H. C. J., and Finberg, R. W., Cell. Zmmunol. 18% 159, 1987. Brooks, C. G., Holscher, M., andurdal, D., J. Zmmunol. 135,1145,1985. Brooks, C., and Holscher, M., J. Irnmunol. 138, 1331, 1987. Philips, J., and Lanier, L., J. Exp. Med. 164,814, 1986. Ormldo, J. R., Mason, A., and Overton, R., J. Exp. Med. 164,1193,1986. Ferrini, S., Miescher, S., Zocchi, M. R., von Fliedner, V., and Moretta, A., J. Immunol. 138, 1297, 1987.
11. Malkovsky, M., Loveland, B., North, M., Asherson, G. L., Gao, L., Ward, P., and Fiers, W., Nature (London) 325,262,1987. 12. Damle, N. K., Doyle, L., and Bradley, E., J. Immunol. 137,2814, 1987. 13. Tilden, A. B., Itoh, K., and Balch, C. M., J. Zmmunol. 138,1068, 1987. 14. Chen, B. P., Malkovsky, M., Hank, 3. A., and Sondel, P. M., Cell. Immunol. 110,282,1987. 15. Taswell,C.,J. Immunol. 126,1614, 1981. 16. Griend, R. J., Tax, W., Krimpen, B., Vreugenhil, R., Ronteltap, C., and Bolhuis, R., J. Immunol. 138, 1627,1987. 17. Hersey, P., and Bolhuis, R., Immunol. Today8,233, 1987. 18. Rouslahti, E., and Pierschbacher, M. D., Science 238,49 I, 1987. 19. Hiserodt, J. C., Vujanovic, N. L., Reynolds, C. W., Herberman, R. B., and Cramer, D. W ., In “Cellular Immunotherapy ofCancer” (R. L. Truitt, R. P. Gale, and M. M. Bortin, Eds.), pp. 137-146. A. R. Liss, New York, 1987. 20. Schmidt, R. E., Caulfield, J. P., Michar, J., Hein, A., Kamada, M. M., MacDermott, R. P., Stevens, R. L., and Ritz, J., J. Immunol. 140,990,1988. 2 1. Springer, T. A., Dustin, M. L., Hishmoto, T. K., and Marlin, S. D., In “Annual Review of Immunology” (W. E. Paul, C. G. Fathman, and H. Metzger, Eds.), pp. 621-669. Annual Reviews, Palo Alto, California, 1987. 22. Doyle, C., and Strominger, J. L., Nature (London) 330,256, 1987. 23. Granger, G. A., On; S. L., and Yamamoto, R. S., J. C/in. Immunol. 5,2 17, i985. 24. Young, J. D. E., Leong, L. G., Liu, C. C., Damiano, A., and Cohn, Z. A., Proc. Natl. Acad. Sci. US,4 83,5668,1986.
Liu, C. C., Steffen, M., King, F., and Young, J. D. E., Ce// 51,393, 1987. 26. Green, L. M., Stem, M. T., Haviland, D. L., Mills, B. J., and Ware, C. F., J. Immunol, 135, 4034, 25.
1985.
Blue, M. L., Hafer, D. A., Daley, J. F., Levine, H., Craig, K. A., Bretmeyer, J. B., and &hlosman, S. F., J. Immunol. 140,376, 1988. 28. Shioshara, T., Ruddle, N. H., Horowitz, M., Moellmann, G. E., and Lemer, A. B., J. Immunol. 138, 1971,1987. 29. Ochoa, A. C., Gromo, G., Alter, B. J., Sondel, P. M., and Bach, F. H., J. Immunol. 138,2728, 1987. 30. Rayner, A. A., Grimm, E. A., Lotze, M. T., Wilson, D. J., and Rosenberg, S. A., J. Natl. Cancer Inst. 27.
75,67,
1985.
3 1. Allavena, P., and Ortaldo, J. R., J. Immunof. 1322363, 1984. 32. Hercend, T., Reinherz, E. L., Meuer, S., Schlossman, S. F., and Ritz,
J., Nature
(Lon&~)
~1,
158,
1983.
33. Hercend, T., Meuer, S., Brennan, A., Edson, M. A., Acute, O., Reinherz, E. L., Schlossman, S. F., and Ritz, J., J. Exp. Med. 158, 1547, 1983. 34. Moretut, L., Pende, D., Cozzani, R., Merli, A., Bagnasco, M., and Mingari, M. C., Eur. J. Immunol. 16,1623,1986.
IMMUNOLOGY
118,458-469 ( 1989)
Selective Lysis of Target Cells by Interleukin-2-Expanded Blood Mononuclear Leukocyte Clones’
Peripheral
BENJAMIN P. CHEN,*JACQUELYN A.HANK,* ERICE.KRAUS,* AND PAUL M. S~NDEL*~~X~Y~ Departments of *Human Oncology, TPediatrics, and $Genetics, University of Wisconsin, Madison, Wisconsin 53792 Received August 23, 1988; accepted October IO, I988 The presence ofdistinct cytolytic subsets within interleukin-2-expanded peripheral blood leukocytes (IEL) cultures was demonstrated by clonal analysis. Thirty-seven IEL clones were isolated from two healthy blood donors; 2 1 destroyed both Daudi and K562 cell lines. Of those 2 1 clones, 1 clone could destroy autologous PBM, 7 clones could destroy fresh allogeneic ovarian carcinoma (OVA-CA) cells, and 6 clones could destroy normal autologous PBM and fresh OVACA cells. Twelve of the 37 clones destroyed only one of the four targets tested: 8 clones destroyed K562,2 clones destroyed Daudi, and 1 clone each was selective for autologous PBM or OVACA. Of the remaining 4 clones, 1 destroyed OVA-CA and Daudi cells, 1 destroyed PBM and K562, 1 destroyed PBM and Daudi cells, and 1 destroyed PBM, Daudi, and OVA-CA. These results suggest that these functionally heterogeneous cytolytic clones may use different cell recognition or cytolytic mechanisms to enable these distinct and, at times, reciprocal patterns of target cell selectivity. 0 I989 Academic Press, Inc.
INTRODUCTION Activation of human peripheral blood mononuclear leukocytes (PBM) with IL-2 generates phenotypically heterogeneous populations of effector cells which mediate non-MHC-restricted cytotoxicity of various tumor cell lines, fresh tumor cells, and to a lesser extent, normal vascular endothelial cells and PBM ( 1- 13). This non-MHCrestricted cytotoxicity has been designated the lymphokine-activated killer (LAK) phenomenon (1, 2). The heterogeneity of cell phenotypes mediating the LAK phenomenon could suggest that IL-2 induces diverse populations of lymphoid cells to nonspecifically lyse both tumor and normal cells, or that IL-2 expands subsets of IEL with different target cell “selectivities.” Data are consistent with both of these two mutually nonexclusive possibilities (5- 12). Skinner et al. (5) and Brooks et al. (6, 7) have shown that Sendai virus-specific and alloantigen-specific cytotoxic T cell clones (CTL), upon exposure to a high concentration of IL-2, become nonspecific killer cells capable of destroying both natural killer (NK)-sensitive and NK-resistant target cells. While Philips and Lanier (8) Ortaldo et al. (9), and Ferrini et al. (10) have reported ’ This study was supported by Grants NIH-CA 32685 and American Cancer Society CH-237. ’ To whom correspondence should be addressed at K4/448 UWCCC, 600 Highland Ave., Madison, WI
53792. 458 0008-8749/89$3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
CLONAL
ANALYSIS
OF IL-2-EXPANDED
LEUKOCYTES
459
that the majority of tumoricidal activity of IEL cultures is mediated by a population of CD3-, CD 16+, NM 1’ killer cells, Malkovsky et al. ( 1 1), Damle et al. ( 12), and Tilden et al. (13) have further documented that IL2 could induce other lymphoid cell populations to mediate non-MHC-restricted cytotoxicity of tumor cells. Our previous study with bulk populations of IEL show that unlabeled tumor cell lines could competitively inhibit much of the non-MHC-restricted killing of autologous normal PBM target cells, suggesting that some of the IEL which kill tumor cell lines also “recognize” the PBM target cells ( 14). In this study, we have used split well analysis of limiting dilution cultures and clonal analysis to document that IL-2 expands IEL subsets with different target cell selectivities. These experiments suggest that different non-MHC-restricted effector clones may utilize different recognition (or lytic) mechanisms to mediate quantitatively distinct levels of target cell selectivity. MATERIALS
AND METHODS
Leukocytes Fresh PBM from healthy volunteer donors were obtained by Ficoll-Hypaque sedimentation of anticoagulated whole blood. Viability of PBM as determined by eosin dye exclusion was >98%. Blood donors signed consent forms approved by the University of Wisconsin committee for the protection of human subjects. Frequency Analysis of IEL Precursors by Limiting
Dilution
Cultures
Fresh PBM were diluted serially to yield 2000, 1500, 1000,750,500, and 375 cells/ well (48 replicate wells of each) in tissue culture media HS-RPM1 [RPM1 1640 supplemented with 25 mM Hepes buffer, 2 mM L-glutamine, penicillin, streptomycin (all purchased from Flow Lab., McLean, VA), and 10% pooled heat-inactivated nontransfused human male serum (HS)]. Each microwell also received 5 X lo4 irradiated (4000 cGy) autologous PBM as feeder cells and 100 units/ml of human recombinant IL-2 (a gift from the Cetus Corp., Emeryville, CA). These cultures were incubated at 37°C in a 5% CO2 humidified incubator for 14 days prior to analysis. Estimates of the frequencies of IEL cytolytic for different target cell types were obtained by the minimum x2 method from the Poisson distribution relationship between the number of PBM initially plated per well and the negative logarithm of the percentage of noncytolytic (negative) cultures ( 15). Cell Lines and Fresh Tumor Cells The NK-sensitive erythroleukemia cell line K562 and the HLA class I-deficient Burkitt’s lymphoma Daudi cell line were obtained from the American Type Culture Collection (ATCC) (Rockville, MD). These cell lines were maintained in HS-RPMI. Fresh ovarian carcinoma cells were obtained by Ficoll-Hypaque sedimentation of ascites fluids from patients and were cryopreserved in HS-RPM1 containing 10% dimethyl sulfoxide. These tumor cells were thawed for use as target cells on the day of assay.
460
CHEN ET AL. TABLE 1 Frequencies of IEL Precursors Cytolytic for Autologous PBM, OVA-CA, and Tumor Cell Lines Precursor frequency estimations 0T Target cells
Donor EK
Donor BC
Donor PS
Autologous PBM OVA-CA Daudi K562
1f5950b l/973 l/386 l/442
I/5556 l/5654 l/615 l/535
l/8172 l/9062 l/856 l/410
a Estimations based on Poisson’s distribution, concentration. b Average of two independent estimations.
using six cell concentrations with 48 wells at each cell
Derivation of IEL Clones Three approaches were taken to generate IEL clones. (A) Cloning by limiting dilution. Fresh PBM from healthy donors were plated at 1000,500, 100,50, 10, and I cell per well in the presence of 100 units/ml of IL-2 and 5 X 1O4irradiated autologous PBM as feeder cells. Microcultures with visible growth within lo- 14 days were selected and tested for cytotoxicity. The cloning efficiency at the highest cell concentration is usually less than 10% and regression analysis demonstrated a linear correlation of the logarithm of the percentage of negative cultures with the number of PBM initially plated per well indicating that these clones most likely were derived from a single cell. IEL clones generated by this approach are identified in the text with the prefix LD. (B) Cloning by polyclonal activation of PBM with anti-CD3 monoclonal antibody (MoAb). Fresh PBM were plated at one cell every two to three wells in the presence of 5 x 1O4irradiated (4000 cGy) autologous PBM and a predetermined optimal concentration of anti-CD3 MoAb and IL2 at 100 units/ml. Cytotoxic cultures were selected within lo- 14 days and expanded in HS-RPM1 media containing 100 units/ml of IL-2. These clones were restimulated every 2 weeks with anti-CD3 MoAb and irradiated autologous PBM as feeder cells. All cytotoxic clones generated by this method were washed and cultured in medium with IL-2 and without anti-CD3 MoAb for at least 7 days prior to their being tested in the cytotoxicity assay. IEL clones derived by this approach are designated with the prefix CD. (C) Cloning by serial limiting dilution. Fresh PBM at 2 X lo4 cells/ml were plated in 48 replicate wells and serially diluted to yield six cell concentrations ranging from 150 to 2000 cells/well. Each culture well also received 5 X lo4 irradiated (4000 cGy) autologous PBM feeder cells and IL2 at 100 units/ml. Between Days 10 and 14, cultures with visible growth and flanked by two or more negative wells of the same dilution series were picked and screened for cytotoxicity. Clones generated with this approach are identified by the prefix SD. All cytotoxic clones generated by these three cloning procedures were propagated in multiple microwells and expanded, when confluent, into macrowells and then flasks using standard cell growth technique (10).
CLONAL
ANALYSIS
OF IL2-EXPANDED
LEUKOCYTES
461
Cell-Mediated Lysis (CML) For precursor frequency estimation, each individual limiting dilution microculture was equally split into four replicate microwells (Costar, Cambridge, MA) and assayed separately for cytotoxicity against autologous PBM, Daudi, K562, and freshly thawed OVA-CA target cells. When clones were assayed, aliquots of the cells from each clone were split into replicate wells and also tested simultaneously on these same four target cell populations. Cytotoxicity was measured in an 8-hr 5’Cr release assay. Target cells were labeled with 0.25 mCi of 5’Cr (New England Nuclear, Boston, MA) in 0.3 ml of HS-RPM1 for 2 hr, washed three times in media, and diluted and 5 X lo3 cells were added in 0.1 ml to the microwells containing the effector cells. The plates were centrifuged at 200g for 5 min before culturing at 37°C. After 8 hr of incubation, the plates were centrifuged at 5OOg for 5 min and supernatants were collected with Skatron harvesting frames (Stering, VA) for measuring 5’Cr release in a gamma counter. Percentage cytotoxicity is calculated using the formula cytotoxicity
=
experimental cpm - spontaneous cpm x 100%. maximum cpm - spontaneous cpm
Spontaneous release values were obtained by incubating target cells with medium alone. Maximum release values were determined by incubating target cells in cetrimide detergent (Sigma Chemicals, St. Louis, MO). For each population of target cells, the mean spontaneous release value plus three times the standard deviation was chosen as the cutoff value to separate negative cultures from positive cultures for frequency analysis.
Monoclonal Antibodies Anti-CD3 (CRL-800 I), -CD4 (CRL 8002), and -CD8 (CRL 80 14) used for inhibition studies were ammonium sulfate-precipitated hybridoma supernatants; the hybridomas for these MoAb were obtained from ATCC.
Phenotyping of Leukocytes Indirect immunofluorescence was done with anti-CD3, -CD4, and -CD8 (BectonDickinson MoAb center, Mountain View, CA) and NKI-I-1 (Coulter, Hialeah, FL) and developed with a fluorescein-labeled anti-mouse immunoglobulin. (Coulter). Five thousand cells were analyzed for each marker on an Ortho Cytofluorograph Systems Model 50 flow cytometer (Ortho, Raritan, NJ). RESULTS
Diflerential Distribution of IEL Precursors Cytolytic for Tumor Cells and PBM To estimate the precursor frequencies of IEL cytolytic for Daudi, K562, autologous PBM, and OVA-CA target cells, limiting dilution cultures of IEL were split into four equal aliquots and tested against each of these four target cells. The IEL precursor frequencies, averages of two separate determinations for each of the three unrelated healthy blood donors, are shown in Table 1. The frequencies of IEL capable of lysing Daudi and K562 are much higher than those cytolytic for autologous PBM and OVACA for all three donors. Of all the estimates, greatest variations are seen in the fre-
462 I 60 am b 40 ” 20 .=
a
.o ;: ‘0 O z0 -20
o\"
0
20
40
60
80
100
0
20
40
K562 %
60
Daudi Cytotoxicity
80
100
0
20
40
Ovarian
60
80
100
Tumor
1. Split well analysis of limiting dilution cultures. IEL activated in microwells were split into replicates and assayed simultaneously for killing against Dam%, K562, OVA-CA, and autologous PBM in a 8hr “Cr release assay. Paired comparison of cytotoxicity of autologous PBM vs tumor cell targets used IEL microcultures that were initially plated at 2000, 1500, 1000, 750, 500, and 375 cells/well. Thus each individual well was tested (by replicate plating) on all four targets. These data are presented here in three plots, showing for each well (plotted as a single point) the destruction of the PBM targets versus the destruction of K562, Daudi, and OVA-CA targets in (a), (b), and (c). The lines represent percentage cytotoxicity equivalent to the mean plus three times the standard deviation of 24 spontaneous release values measured on each target. The multiple cultures that did not kill either target cells on a given graph are included in the shaded area. FIG.
quencies of IEL tumoricidal for OVA-CA. Cytotoxicity results of limiting dilution cultures from donor EK are further analyzed in split culture plots, Fig. 1. Each plot compares the cytotoxicity on autologous PBM target cells by cells from an individual well with cytotoxicity by replicates of that same well on K562, Daudi, or OVA-CA target cells. A total of 144 wells, 24 wells each initially seeded at 6 cell concentrations ranging from 375 to 2000 cells/well, were analyzed. As shown in Fig. 1, 14, 69, 123, and 110 wells mediated significant destruction of PBM, OVA-CA, K562, and Daudi target cells, respectively. The differential distribution of IEL precursors capable of lysing the four target cells is due to the presence of IEL subpopulations which mediate different patterns of selective target cell destruction and not explicable solely by differential lysability of the target cells, as shown below with IEL clones. IEL Clones Display Selective Cytotoxicity The cytotoxic activities of 4 IEL clones are shown in Fig. 2. These 4 IEL clones each gave a different pattern of cytotoxicity for the four target cells. Although the magnitude of killing of susceptible target cells increased with greater numbers of effector cells, the pattern of cytotoxicity unique to each of these four clones remains the same at all the effector to target cell ratios tested. These results suggest that the “selectivity” observed with IEL clones is not the sole result of differential lysability of the target cells used. An additional 33 cytotoxic IEL clones were isolated from two healthy donors PS and BC. All 37 clones can be segregated into six major groups (Table 2). Six of these clones were cytotoxic for Daudi, K562, autologous PBM, and OVA-CA target cells (group I). Seven clones lysed the two tumor cell lines and fresh tumor cells but not autologous PBM (group II). Seventeen clones destroyed tumor cell lines but not fresh tumor cells or PBM (groups III, IV, and V). Of these 17 clones, 7 clones were cytotoxic for both Daudi and K562 (group III), 2 clones were cytolytic for Daudi only (group IV), and 8 clones only lysed K562 cells (group V). Other IEL
CLONAL
ANALYSIS
OF IL-Z-EXPANDED
LEUKOCYTES
463
CLONE
SD-CD2 80 3 :g 6o
.-.
Autologous PBM Ovor~on Tumor
B 2 G 40
0 25
5
IO
20
25
5
IO
Effector
20
-
to Torget
25
Cell
5
IO
20
Ratkos
FIG. 2. IEL clones with differential cytotoxic activities. Cytotoxicity of IEL clones SD-CDlc, SD-CD2, LD-10, and LD-3 were measured in an S-hr “Cr release assay.Various numbers ofeffector cells were added to 5 X 1O3 of the indicated target cells in microwells to achieve the specific effector to target cell ratios. Results represent the mean + standard deviation of triplicate cultures.
clones that do not fall in any of the five groups described are represented in group VI. Within this group, IEL clone CD-BA 10 lysed only the OVA-CA cells, clone SD-CD 1c lysed autologous PBM only, clones SD-2C, SD-CD2, and LD-3 lysed PBM and one or both tumor cell lines but not fresh tumor cells. Clone LD-BC2 lysed OVA-CA and Daudi, and clone SD-2D lysed OVA-CA, PBM, and Daudi cells but not K562. All these clones gave reproducible results in two to four independent experiments. Phenotypes of 18 IEL clones, representatives from all six groups, are shown in Table 3. All of these clones express the CD3 marker, 11 of these clones are CDS+/ CD4-, 4 clones are the CD4+/CD8-, 2 clones are CD4+/CDS+, and 1 clone, clone LD-BC2, does not express the CD4 or CD8 marker. All clones, except clone SDCD2, also express the NKH-1 marker. Although there is no distinct correlation of IEL phenotypes with target cell selectivity, these phenotypically heterogeneous IEL clones may be modulated by different MoAb directed at these markers.
Diferential Eflects of Anti-CD3, -CD4, and -CD8 MoAb on Cytotoxicity Mediated by IEL Clones We have previously observed that the nonspecific killing of tumor cell lines by bulk IEL cultures can be potentiated by anti-CD3 MoAb, and at times weakly inhibited by anti-CD8 MoAb (( 14) and unpublished results). The weak inhibition of bulk IEL cultures could result from selective inhibition by anti-CD8 MoAb of phenotypically different cell subsets within the bulk IEL populations. We tested the effect of antiCD3, -CD4, and -CDS MoAb on two CD3+/CD4-/CDS+ IEL clones (clones CDBCl and SD-2D), one CD3+/CD4+/CDSf IEL clone (clone CD-AH2), and a CD3-/ CD4-/CDS-, Fc+ killer clone initially described as a natural killer-like clone (NK77) ( 16). IEL clones CD-BCl and CD-AH2 and NK77 killed all PBM, OVA-CA, Daudi, and K562 target cells tested, while clone SD-2D did not kill K562 cells (Fig. 3). The addition of anti-CD3 MoAb enhanced the killing of PBM and OVA-CA target cells by all three CD3+ clones, but not by the CD3- NK77 clone. Anti-CD3 also enhanced the killing of Daudi target cells by clone CD-BC 1 and SD-2D, but inhibited the killing
464
CHEN ET AL. TABLE 2 Differential Cytotoxic Activities of IEL Clones % Cyotoxicity of target”
Group I
II
III
IV V
VI
Bulk culture
IEL clone
E:Tb
SD-36~11~ SD-4D LD-10 LD-17 CD-BC 1 CD-AB3
10 10 30 30 30 10
19 (3)d 22 (4)
CD-AC 1 CD-BH 12 CD-AE6 CD-AE7 CD-AH2 SDdC SD-6A
30 30 30 10 10 20 10
6(l)
CD-BClO CD-BGl2 CD-AA7 LD-13 SD-52GII SDdB SD-CD 11
30 30 30 20 10 20 20
CD-AG 1 SD-5A
30 20
l(3) 8 (3)
CD-BB9 SD-l 3GII SD-32PII SD54PII SD-1C SD-ID SD-CD3 SD-CD9
30 10 10 10 12 10 10 20 20
l(l) 5 (3) 3 (3) 3 (3) 4 (5) -10(4) -13 (5) -l(l)
30 20 10 20 20 30 10
28 (4) 27 (8) 31(7) 16 (3)
2(l)
5 (2) 29 (4)
80(2)
-3 (1) 3 (2) 79 (5) 56 (1) 81(4) 7(l)
10 (2)
12(l)
11(l)
-2 (2)
20
32 (2)
71(10)
82 (9)
92 (3)
CD-BA 10 SD-CDlc SD-2C SD-CD2 LD-3 LD-BC2 SD-2D
Auto]. PBLC
Ova’
Daudi
K562
20(6)
21(2) 26(l)
12 (3) 19(2) 44 (2) 25 (1)
13 (2) 14(l)
12(1)
21(2)
20 (3)
39 (9)
57 48 77 47 85 85
14(l) 14 (2)
93 (3) 49 (2) 14(l) 30 (2) 78 (5) 97(10) 80(11)
7 (2) 4 (3)
6(l) 5 (1) -3 (5) 8 (4) 3(l) -l(l) -l(3)
8(l) -2 (3) -7 (3) 7(l)
-
12(1) 21(3) 15 (4) 19(8) 25 (5) 3 (2) 7 (2) 5 (2) 10 (3) l(2)
48 (6) 91(5) 61(3)
8 (2)
30(l) 38 (1) 27 (2) 31(l) 51 (8) 69 (8)
4 (2)
18(l)
8 (3) 3 (3)
43 (5)
6 (2) 9 (3) 3 (3)
6 t-3 3 (2) 2 (3) l(2) 4(l)
6(l)
8(l)
6 (2)
15 (2)
8(l) 2 (3)
8(2)
16(l) l(1) l(3) -4 (5) 4 (8)
-8 (6) -2 (4) 0 (3) l(l) 4(l) 3 (2) 3 (2) 77 (6) 9(l) 7(l)
(7) (16) (4) (2) (1) (5)
88 (4) 75 (3)
80(l) 85 (3) 85 (4)
81@) 77 (5) 88 (3) 86 (3) 72 (5) 39 (2) 19 (2) 55 (4) 66 (3) 0 (2) 10 (4) 46 (2) 39 (2) 36 (2) 37 (2) 28 (3) 23 (9)
26(8) 23 (1) 29 (2)
’ Cytotoxicity was measured in an 8-hr “Cr release assay. b Cytotoxicity was tested at two to four effector to target cell ratios. Only cytotoxicity at the highest E:T ratio is shown. ‘Autoi. PBL = autologous PBL cultured in medium for 3 days; OVA = freshly thawed ovarian carcinoma cells. d Mean cytotoxicity ? standard deviation of triplicate cultures. ’ IEL SD and LD clones were cloned in IL-2 alone; CD clones were cloned in the presence of anti-CD3 monoclonal antibody.
CLONAL
ANALYSIS
OF IGZ-EXPANDED
LEUKOCYTES
465
TABLE 3 Phenotypes of IEL Clones’ IEL clones
CD3
CD4
CD8
NKH-1
I
LD-IO LD-17 CD-BC 1
+ + +
+ -
+ + +
+ + -t
II
CD-AC 1 CD-BH12 CD-AE6 CD-AH2
+ + + +
+ +
+ + +
+ + + +
III
CD-BClO CD-BGl2 LD-13
+ + +
-
+ + +
+ + +
IV
CD-AG 1 SD-5A
+ +
+ +
-
+ +
V
CD-BB9 SD-1C
+ +
-
+ +
+ +
VI
SD-2C SD-CD2 LD-BC2 SD-2D
+ + + +
+ -
+ +
+ + +
Groups
a The phenotypes of each IEL clone was assessedby indirect immunofluorescence of 5000 cells for each marker.
of the same target cells by clone CD-AH2. The very high level (plateau) killing of K562 mediated by clones CD-BCl and CD-AH2 was not affected by anti-CD3 MoAb; this MoAb did enhance the killing of K562 cells by these two clones in a separate experiment performed at a lower E:T ratio (data not shown). Anti-CD3 MoAb also induced significant killing of K562 cells by clone SD-2D, although this clone normally did not kill K562 target cells. MoAb anti-CD8 only inhibited the killing of Daudi cells by clone CD-BC I and CD-AH2, and anti-CD4 MoAb did not affect the lytic activity of all four clones tested. DISCUSSION A variety of cell types are lysed by bulk IEL populations with greatest lysis of tumor cell lines, intermediate lysis of fresh tumor cells, and minimal, yet significant, lysis of autologous PBM targets (3). The present studies evaluated this lysis by limiting dilution and clonal analysis. If all lytic clones mediated this same pattern of lysis (i.e., greatest killing on the tumor cell lines, and least killing on PBM), it would have suggested that all cells mediating the LAK phenomenon use the same mechanism for recognizing susceptible target cells, and that different levels of lysis observed resulted from quantitative (rather than qualitative) differences in recognition structures or lysability of the different target cells tested. However, these studies showed that different subpopulations of IEL mediate cytolysis with different patterns of “selectiv-
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CLONE
0 ‘15.0 km0
‘/IO00 0 ho ‘/ZOO ‘/IO00 0 ‘150 ‘/ZOO ‘/IO00 0 ‘/SO c200 ~/loo0
Dilution
of Monoclonol
Antibodies
FIG. 3. Potentiation of IEL-mediated cytotoxicity by anti-CD3 monoclonal antibodies. IEL clones were preincubated with media (0) or three dilutions of MoAb (l/50, l/200, and l/1000) for 1 hr before the addition of target cells. Monoclonal antibodies were ammonium sulfate-precipitated hybridoma supernatants added to the culture for the duration of the CML assay.Cytotoxicity was measured in an S-hr 5’Cr release assayat an effector to target ratio of 10: 1. Each data point represents the mean of triplicate cultures.
ity” for tumor cell lines, fresh tumor cells, and normal PBM target cells (Table 2). Some clones kill one tumor line and not the other, others kill PBM and not fresh tumor, some kill all target cells, etc. Since these individual experiments tested replicate well lysis on all four target cells simultaneously in parallel to one another, the identification of any clone which kills PBM greater than the tumor lines (i.e., clone SD-CDlc kills PBM but not Daudi or K562; or clone SD-CD2 which kills PBM greater than Daudi but less than K562) proves that for at least some effector populations, PBM targets are not merely less susceptible to lysis than the tumor cell lines. As such, the clones from the six distinct groups identified in Table 2 must either recognize these target populations using different surface molecule recognition structures or use different molecules to mediate lysis, or both, to account for the different patterns of selective target cell destruction. The methods used to generate the clones reported here indicate that many are likely to be “true clones” of single cell origin. However, definitive subcloning of these to prove clonal origin was not performed, leaving open the possibility that some of these populations represent the progeny of more than a single cell. However, these popula-
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tions (particularly those in Table 2, groups II-VI) show patterns of target cell lysis that are not seen with bulk cultures activated with the same stimuli (i.e., IL-2 with or without anti-CD3 MoAb). These data, and the cell surface marker phenotype data, are consistent with the clonal origin of these populations which we refer to here as clones. Nevertheless, the biological conclusions to be made from these studies do not hinge on proof of clonal origin. Rather, they are derived from the distinct specificity patterns (selectivity) mediated by subpopulations derived from bulk populations without specificity. With respect to surface recognition molecules, both bulk culture and clonal analyses would suggest that most cells mediating the LAK phenomenon may not use the CD3/Ti receptor molecule to recognize target cells, nor does recognition usually involve MHC molecules ( 17). In contrast, the family of cell surface adhesion molecules, designated integrins (18), appear to play a prominent role in the approximation of IEL and target cells. Antibody to laminin ( 19) and LFA- 1 molecules ( 14,20) can each inhibit non-MHC-restricted cytotoxicity by IL-Zactivated bulk cultures and clones. The different selectivity patterns seen here could result from a different proportion or density of these (or other) adhesion molecules on different IEL clones. As such, IEL clones with a broad target spectrum (group I of Table 2) may have adhesion structures that allow effective binding to virtually all tissues; candidates for such ubiquitous adhesion structures include the LFA- 1 and the I-CAM- 1 molecules to which they bind (20,2 1). Some IEL may also use other adhesion/accessory molecules such as the CD4 or CD8 molecules. Recent data suggest that CD4 or CD8 null cells transfected with CD4 or CD8 genes express the molecule and can bind respectively to class II or class I MHC molecule bearing cells, without involving antigen-specific receptors ((22) and Salter et al., personal communication). Conjugates formed via these molecules, however, may be of low affinity and play a secondary role to other adhesion molecules. The selective killing of target cells by IEL clones could also be due to the use of different lytic molecules by individual IEL clones. Cytolytic molecules such as the lymphotoxins (23) and the perform class of lytic molecules (24,25) have been shown to be used by different effector cell types to destroy susceptible targets (26). Different IEL clones may use various amounts of these (or other) lytic molecules and different target cells may show differential susceptibility to the lytic molecules released by a given IEL clone. Combinations of these. recognition and lytic control mechanisms may determine the degree to which a given target cell is lysed by a given IEL clone. In this regard, IEL clones can show distinct patterns of inhibition by different MoAb, when lysis is tested on different target populations (Fig. 3 and P. Fisch, J. A. Hank, and P. M. Sondel, unpublished). These MoAb could either differentially affect the binding of target cells by individual IEL or they may affect different lytic mechanisms (14,20,27). The precise mechanism whereby IEL are activated is not known. Data from several laboratories suggest that IEL represent activated natural killer cells (8-lo), while other studies suggest that some IEL killers are antigen-specific cytotoxic T cells, reversibly “converted” by high concentration of rIL-2 to nonspecific killer cells (5-7). The “nonspecific” IEL clones described in this study (group I) could represent killer cells generated via the latter mechanism. The demonstration of IEL clones with target cell selectivity, however, suggests that not all killer cells can be driven by rIL-2 to kill nonspecifically. This may again suggest that all IEL clones do not possess the same
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repertoire of IL-2 inducible @tic mechanisms, or that some effector cells can maintain specificity despite the exposure to high concentrations ofIL-2. The clones which lysed autologous PBM or tumor cells alone (i.e., clones SD-CDIc and CD-BAlO) may indeed be antigen-specific clones that maintained specificity. Clone SD-CDlc may resemble class II-restricted murine autolytic clones with immunoregulatory function (28), while clone SD-BA 10, which lysed the fresh tumor cells and not PBM or tumor Cell lines, may represent an alloreactive clone recognizing ahoantigens on the OVACA. More extensive studies would be required to confirm these possibilities. Nevertheless, these clones were obtained, without antigen-specific priming, using in vitro stimuli shown to induce the LAK phenomenon in bulk cultures ( 1, 3, 14,29). Phenotypicalfy, all the IEL clones generated in this study are CD3+ and, with the exception of clone LD- 17, express either the CD4 or the CD8 marker. The expression of the CD3+ marker by all our IEL clones is consistent with the majority of the cells in the bulk culture expressing the CD3 marker (3, 29). By limiting dilution cloning of Day 4 LAK cultures in the presence of PHA and IL-2 containing supernatant, Rayner et al. (30) have also generated exclusively CD3+ LAK clones, similar to our clones shown in group II of Table 2; those clones killed all tumor target cells tested and were not cytolytic for normal PBM. Similar to our results, Allevena and Ortaldo (3 1) and Hercend et al. (32, 33) have also generated CD3+ killer clones by cloning Percoll gradient-enriched large granular leukocytes; their CD3+ killer clones also demonstrated a heterogeneous pattern of reactivity against a panel of tumor cell lines. However, it is not known if those clones could destroy fresh tumor cells or normal PBM, since those target cells were not included in their studies. LAK cells that are CD3- can be generated by culturing Leul l+ PBM in IL-2 as shown by Ferrini et al. (IO) and Moretta et al. (34); in their studies, the majority of the killer clones were shown to be nonspecific killers, killing both fresh tumor cells and tumor cell lines. One of our IEL clones, clone LD-BC2, is CD3+, CD4-, CD8-, and NKH- 1+. Previous reports have documented nonspecific cytotoxicity by CD3+, CD4-, CD8 cells (16, 17); uniquely, clone LD-BC2 with this phenotype described in this study showed some target cell selectivity, Table 2. We do not know if this clone expresses an antigen-specific T cell receptor. The physiological role of the LAK phenomenon remains undetermined; much of the activity may reflect that of activated NK cells. The precise regulation of the degree of lysis a given IEL clone will mediate on a single target cell population has not been clarified. The present experiments indicate this regulation is a multifactorial process which cannot be explained merely by quantitative differences in “lysability.” By clarifying these mechanisms and learning to control them experimentally, it may someday be feasible to use this information to protect normal tissues from the LAK phenomenon, or more selectively activate those effector cells (or their products) which preferentially mediate lysis of the autologous tumor. ACKNOWLEDGMENTS We thank Dr. Paul Fisch for helpful discussions, Drs. R. Bolhuis and Greg Litton for the generous gift of clone NK77, and Ms. Jean Surfus for her excellent technical assistance.
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