by SCOTT P. BARTLETT AND ROBERT C. BURTON*. (FrotTt the Transplantation Unit. General Surgical Services, and the Department of Surgery, Harvard ...
AJEBAK 60 (Pt. 6) 571-579 (1982)
©STUDIES ON NATURAL KILLER (NK) CELLS. HETEROGENEITY OF NK CELLS IN BEIGE MUTANT MICE by SCOTT P. BARTLETT AND ROBERT C. BURTON* (FrotTt the Transplantation Unit. General Surgical Services, and the Department of Surgery, Harvard Medical School at the Massachusetts General Hospital, Boston, Massachusetts, U.S.A., 02114.) (Accepted for puhlieation June 22, 1982.) Summary. Mice homozygous for the recessive beige mutation show grossly impaired natural killer ( N K ) activity against lymphoma targets in short term assays. When spleen cells from homozygous C57 Bt,/6 and B6C3Fi beige mutant mice were examined in assays of up lo 30 h tjuration and tesled with ;inti-NK-l -2 antiserum prior to assay, it was found ( ! ) thai the impaired NK-1 2+ ( N K A ) aclivity aguinst lymphoma targets could be partiiilly or wholly overcome by prolonging the assay time to > 1 2 h, {2) lhe NKi activity as revealed by prolonged assay times showed considerable variability in homozygous beige mutant mice (from ^4-fold less than to 2-fold greater than Ihal of normal controls). (3) that NK-I 2" (NKii) activity against two non-lymphoma targets was normal in these mutant mice and (4) thai two independently derived beige mutations had the same effects on NKA and NKn cell activity.
INTRODUCTION Roder and co-workers (Roder, 1979; Roder and Duwe, 1979; Roder, et al., 1979) have drawn attention to a deficiency of NK and antibody-dependent, cell-mediated cytolysis (ADCC) of tumour cells in C57BL/6 mice homozygous for the recessive beige mutation. They showed that the NK defect was not due to altered organ distribution or frequency of target binding NK cells or to a change in target selectivity, and concluded that the NK deficiency seen in these mice involved the final lethal hit event of the NK cell against its target. Recent observations from thi.s laboratory and others (Stutman. Paige and Figarella, 1978; Paige et al., 1978; Kumar, Ben-Ezra and Sonnenfeld,^979; Burton, 1980; Burton. Bartlett and Winn. 1980; Burton et al.. 1981; Lust et al... 1981; Stutman and Cuttito, 1981 ) have shown that there is a heterogeneity amongst murine NK cells. One class of NK cells (NK.\ cells) displays ^' Present address: Faculty of Medicine, The University of Newcastle, New South Wales 2308, Australia. Abbreviations u.sed in this paper: ADCC. antibody-dependent, cell-mediated cytolysis; TCM, tissue culture medium; C, complement; NK, natural killer; E:T, effector:target ratio.
572
SCOTT P. BARTLETT AND ROBERT C. BURTON
preferential cytotoxicity towards lymphoid tumour targets, is sensitive to treatment with specific anti-NK anti-serum and complement (C) and its activity is absent or reduced in ""''Sr-trcated mice (Kumar et al.. 1979; Burton. 1980; Burton et al., 1980; Burton et al, 1981). A second class of NK cells preferentially lyses adherent solid tumour targets and is resistant to treatment with specific anti-NK alloantisera and C. Normal levels of activity of these latter cells have been reported in ^"Sr-treated mice and these cells have been termed natural cytotoxic (NC) or NKu ceils (Kumar et al, 1979; Burton et al, 1981; Lust et al, 1981). Recently, we and others have provided data which indicate that NC or NKK cell activity is normal in C57BL/6 mice homo/ygous for the beige mutation (Burton et al, 1981; Lust et al. 1981; Stutman and Cuttito, 198!). In this report we provide data on the deficiency of NK-1-2+ ( N K A ) cell activity in mice bearing the beige gene derived from mutations which occurred independently in two strains of mice, and show that this deficiency can often be partially and sometimes wholly overcome by prolonging the in vitro assay time. In addition, we have included more detailed data than has heretofore been published on the NK-1 2~ (NKn) activity in mice homozygous for the beige mutation. These mice have levels of NKn activity equal to those of their heterozygous Iittermates and normal mice of the same strain. MATERIALS AND METHODS Animals C3H/HeJ. C57BL/6J (B6) and C57BL/6J-bgJ (homozygous bciye mutant) mice were purchased from the Jackson Laboratory. Bar Harbor. Maine. (C57/BL6I-bgVbg^ X C3H/ HcJ-bg-J/+)Fi. hereafter referred to as B6C3Fi heterozygous beige or B6C3F| homozygous beige mice, were kindly supplied by Dr. G. Cudkowicz, Slate University of New York, Buffalo. New York. Mice of either sex were used at 6 to 12 weeks of age. Tumour cell lines The tumour cell lines YAC (A strain T-Iymphoma). EK-4 1B6 l-lymphoma) WEHII64-! (BALB/c fibrosarcoma) and FLD-3 (BALB/c crythrolcukemia) used in this study were mainlained in stationary suspension culture (Burton. Thompson and Warner, 1975) and have been described in detail elsewhere (Kiessling. Klein and Wigzell. 1975: Burton, Chism and Warner. 1977; Burton et til. 1980). We have previously shown that YAC and EL-4 are killed exclusively by NK* cells and that WEHM64.1 and FLD-3 are targets for NKB celis (Burton et al. 1981). Tissue culture media Tissue culture medium {TCM) used for all studies was RPMI-1640 supplemented with 2 tnM glutamine. \^c non-esseniial amino acids, and 20 mM HEPES buffer (Grand Island Biological Co. (GIBCO), Grand Island. N.Y.). Foetal calf serum (FCS-GIBCO) was added to a final concentration of 10% immediately prior to use. Spleen cell preparations Red cell free spleen cell preparations were made by mechanically dissociating spleens in buffered isotonic NR|CI and resuspending the cells in TCM after one wash as previously described (Burton et ai. 1975).
NK CELLS IN BEIGE MUTANT MICE
573
Serological reagents and complement Anti-NK-1 2 was made by immunizing CE mice with CBA spleen and lymph node preparations. The specificity and full characterization of this alloantiserum has been described previously (Burton, 1980; Burton and Winn, 1981), Batches of rabbit complement (Pelfreeze Biologicals, Inc., Rogers, ARK.) were screened for high C activity and low non-specific cytotoxicity againsi murine spleen using anti-NK-! 2 antiserum. Treatment of efjector cells Fresh spleen cells were trented as 10' viable cells per ml of RPMI-1640 plus 20 niM HF.PES buffer (RPMI-H) in conical 15 ml centrifuge tubes (Corning Glass Works. Corning, New York). Anti-NK-1 2 antiserum was added to the cell suspension to a final serum concentration of 1:50 and the mixture was then incubated at 37" for 30 min. The cells were then washed with 5 ml of RPMl-H, resuspended in pre-tesled rabbit C diluted with RPMI-H (1:6-1:15 depending upon the batch) and incubated for an additional 30 min al 37". The cells were then washed twice in RPMI-H and resuspended to the initial pre treatment volume in TCM before testing as described below. Target cell labelling Tumour cell targets were labelled with '''Cr (Na'-'CrOi, 1 mCi/ml. New England Nuclear. Boston, MA) by adding 5-10 ^Ci per ml of the i.sotope to dilute concentrations of the cell line during its logarithmic phase of growth and incubating overnight at 37" in 5% COj as previously described f Btirton et al. 1975). Labelling of tumour cells wiLh "Mn (i^Mn oxine in 100% ethanol 20 MCi/ml, New England Nuclear) was as described by Wiltrcut and colleagues (Wiltrout, Taramelli and Holden. 1982). Briefly, tumour cells were admixed with 5-10 y«Ci of ^ I n and TCM to a final volume of 0-5 ml and incubated at room temperature for 15 min. After labelling with either '''Cr or "Un ihe cells were washed thrice in RPMI-H and resuspended in TCM. Only those experiments in which spontaneous release, as a percentage of maximal uptake, was i-hgJ Experiment No. 2 36 26 12 C57BL/6J 10 C51BL/6i-hg^ 8 3 Experiment No. 3 Ifi 12 7 C57BL/6J 0 0 0 C57BL/6J-/.J,'-' * mean of 4 replicates (S.E.M. — 3%).-'i Cr release assay.
lysis* 100:1
WEHI-Ifi4 I 50:1
25:1
11 8
10 7
6 2
12
7
1
7
0
0
15
11 8
4
13
0
the results of three such experiments. These data are in agreement with those of Roder and eo-workers (Roder and Duwe, 1979; Roder, 1979). as they show a marked deficiency of NKA function in the mutant mice. NKH function, however, was not signifieantly different in homozygous beige mutant a.s compared to normal B6 mice (Students' t test, p > 0 05; mean ± S.E.M. of all observations for each E:T ratio), although there was a trend in all three experiments for NKn activity to be lower in the homozygous beige mutant mice. Since previous observations (Stutman et al, 1978; Burton. 1980) had shown that the detection of high levels of NKn activity required a prolonged assay time, several 16 h assays were subsequently performed (Table 2). In these long-tertn assays, NKi, activity against WEHI-164 1 was not significantly different in B6 or homozygous beige mutant mice. Furthermore, when these prolonged assay times were used, a majority of experiments revealed significant NK aetivity against YAC in the homozygous beige mutant mice. In three experiments (No. I, No. 3. No. 6) this activity was 4-foId lower in the mutant miee, as judged by comparisons of the E:T ratios for equivalent levels of lysis with normal B6 controls. However, in two experiments the levels of lysis observed were comparable to those of the controls (No. 4,
575
NK CELLS IN BEIGE MUTANT MICE
EfTectur spleen cells Experiment No. I C57BL/6J CMHUM-hgi Experiment No. 2 C57HL/6I C57BL/6J.M Experiment No. 3 C57BL''6I C57HI /f^i-hgJ
Experiment No. 4 C57HI./6J C57BL/61-/.,W Experiment No. 5 C57BL/6J C57HlJ(yl-bsJ
TABLE 2 Natural killer activity of fresh murine spleen—16 h assay. Percent specific lysis YAC WI H I IM 1 25: 1 5H: 1 100:1 50:1 100:1 :vi
12 5:1
25 0
12 I
4 0
76 78
70 70
52 47
33 28
45 72
20 35
11 14
60 55
43 45
27 33
14 16
45 20
36 N
21 7
35 36
28 32
21 27
14 12
21 15
8 8
2 0
79 89
74 83
77 80
59 58
27 23
17 10
8 3
100 100
ino 100
97 100
74 81
Percent specific lysis 100:1 Experiment No. 6 C.^7BI M C.'i7Bl /6J-/'i,-'
^9 12
YAC
50:1
25: 1
100:1
29 8
9 1
31 27
FLD-3 50: 1 25:1 18 27
16 18
12 5:1 15 16
No. 5). and in one experiment the NKA aetivit>' of homozygous beige mutant spleen cells was twice that of the controls. Also shown in Table 2 are the detailed data from one experiment (No. 6). in whieh the NKu target FLD-3 (Burton et al, 1981) was also used. Comparable levels of NKn activity against this target were obtained with both homozygous beige mutant and normal B6 spleen cells.
DURATION
OF ASSAY
(HOURS)
Fid. 1. Lytic activity of fresh C57BL/6J (*» and C57BL/6J-/'i''' ( • ) spleen as a funeiion of assay lime. In Figs. lA and IB Ihe same cell suspensions were used. Fig. IC is a separate experiment. ,^. N K , sensilivc largei VAC. E:T. 100:1; '''Cr rcle:isc assay. B. NK» sensitive target YAC. H; 1". 100:1; ' " I n release assay. C. N K B sensitive target WEHI-164 1. E:T. 100:1; i i ' I n release assay.
576
SCOTT P. BARTLETT AND ROBERT C. BURTON
NKA and NKu activity of beige mutant mice—prolonged assay times The ^'Un and "'^Cr release assays were compared using normal B6 and homozygous beige mutant spleen cells (Fig. 1). In the ''^Cr release assay {Fig. lA), NKA activity against YAC increased throughout the assay period for both the B6 and homozygous beige mutant spleen cells, with the former manifesting signiikantly higher levels of lysis by 24 h. In the ^^^In assay (Fig. IB) the homozygous beige mutant effectors manifested a greater rate of killing during the last 12 h of incubation, such that by 30 h lysis of YAC by both effector cell types was comparable. By contrast, killing of ^' Un labelled WEHI-164 1 (Fig. IC) was maximal by 18 h of assay and B6 and homozygous beige mutant mice showed similar levels of NKH activity throughout the assay period. The aetivity of spleen cells from mice bearing two independently derived beige mutant genes was tested against four different tumour targets. Table 3 shows the activity of normal mice and C57BL/6J and B6C3Fi homozygous TABLE 3
\'K \ and NKH activity of jri'sh mtirinc .spleen againstvoritni.s tumour targets. Percent specific lysis* EL-4 WEHI-164-1 FLD-3 YAC Effector spleen cells C57BL/'6] C3H/HeJ C57BL/6J-hf,'J B6C3Fi homozygous beige
281 33 10 II
24 24 8 3
89 75
85
m
47 64 3» 57
* mciin of 4 rcpliciilcs (S.E.M. < 3^^^ ). \(^ h -'Kr rclciise iissay. TiT. 100:1. t single figures taken from ihc corresponding linear parts of Ihe E:T versus specific lysis curves.
beige mutant mice against two NK.K sensitive targets (YAC, EL-4) and two NKn sensitive targets (WEHI-164 I, FLD-3). NKA activity was present in both strains of beige mutant mice, but was reduced as compared to normal controls. In contrast, NKu activity was comparable in the two groups of animals. Effect of anti-NK-1 -2 antiserum and C treatment on NKK and NK\K activity in homozygous beige mutant mice In order to show that the cytotoxic activity of spleen cells from homozygous beige mutant mice was due to NK-1 2 ' NKA cells, fresh spleen cell suspensions were treated with anti-NK-1 2 alloantiserum plus C before being tested in a 16 h ''^Cr release assay. The experimental results are shown in Table 4. NK activity against YAC of spleen eells from normal mice and two strains of beige mutant mice was fully sensitive to this treatment, indicating that the killing of YAC was due to NK-1-2^ NKA cells. By contrast, NKi, activity of both normal and homozygous beige mutant mice against WEHI164 1 and FLD-3 was not affected by this treatment, indicating that killing of these target cells was not due to NK-1 2 ' NKA cells.
577
NK CELLS IN BEIGE MUTANT MICE TABLE 4 Serological reactivity of NKA and NK» cells. Effector spleen cells Experiment No. 1 C57BL/6J Experiment No. 2 C57BL/6I
C57BL/6J C57BL/6J-M
24 15
12 5
Percent specific lysis Y A C Anti-NK-l 2 25:1 100:1 50:1 5 2
1 0
100:1
21 9 6 7 15 28 3 12 Percent specific lysis WEHI-K>4 1 * C Anti-NK-l 50:1 25:1 12 5:1 100:1 50:1
100 95
100 92
86 87
68
71
88 84
24 36
19 33
17 22
M 17
37 38
B6C3F| heterozygous beige B6C3Fi homozygous beige
Experiment No. 1 C57BL/6J C57BL/6J hgJ Experiment No. 2 C57BL/(SJ C57BL/6J heJ
100:1
C 50:1
45 20 54 26
36 14 41 15
100: 1 31 31
C 50:1
25:1
1 0
0 0
5 1 7 4
5 0 5 0
2+ C 25:1
12 5:1
84 78
76 79
64 72
32 34
22 25
23 21
Percent specific lysis FLD-3* Anti-NK-1 2 + C 25:1 100:1 50:1 25:1
25 27
23 20
29 27
28 20
20 18
• Mean of 4 replicates (S.E.M. ^ 3% ). 16 h
DISCUSSION The NK deficiency in beige mutant mice was originally defined using an NKA sensitive target, YAC, and a 4 h -^''Cr release assay (Roder and Duwe, 1979). In that system homozygous beige mutant mice were profoundly deficient in NK.\ activity. By employing both 4-24 h '''Cr release assays and 16-30 h '^Mn release assays, we have demonstrated that spleen ceils from homozygous beige mutant mice can mediate substantial levels of lysis of NKA sensitive targets as the assay times are prolonged (Table 2, Fig. IB). In the *"!n assay system against YAC. more lysis by spleen cells from homozygous beige mutant mice as compared to the B6 effectors was observed during the latter hours of the assay, a phenomenon not observed in the '''Cr assay of shorter duration (Fig. 1). We have shown that killing of YAC by the homozygous beige NKA cells is generally less efficient than that of the B6 NKA cells (Tables 1 and 2). In the ' " I n assay this inefiiciency tends to be obscured, perhaps due only to the prolonged assay period. An alternate hypothesis is that the ' " I n assay is a more sensitive indicator of target cell damage. Further studies to compare these aspects of the ''^Cr and the ' " I n assay systems are in order. It was also observed that even with prolonged assay times there was considerable variability of NK.v mediated killing in different experiments. In some experiments little or no NKA activity was observed in homozygous beige
578
SCOTT P. BARTLETT AND ROBERT C. BURTON
mutant mice (Table 2, Experiments No. 1 and No. 6 ) , while at other times activity of these spleen cells was as great or greater than that observed in normal controls (Table 2. Experiments No. 2 and No. 5). These experiments were performed over a priod of 1 year with different lots of mice. Exogenous influences such as infection can alter the NK activity seen in normal laboratory animals (Orn et al, 1980; Welsh and Kiessling. 1980), and these results indicate that homozygous beige mutant mice may be similarly affected by the environment. It might also be argued that the activity we observed against YAC in these mutant mice using prolonged assay times was due to a different effector than the NKA cell. However, it is evident from the data in Table 4 that this is not so. There it is clearly shown that al! the homozygous beige mutant NK activity was mediated by NK-1 2"' cells: NKA cells. Furthermore, these findings were not restricted to mice homozygous for the B6 beige mutant gene bg^. Similar findings were obtained when Fi homozygous and heterozygous Httermatcs bearing recessive beige tnutant genes which occurred independently in the two parental strains (C57BL/6J and C3H/ HeJ) were compared (Table 4 ) . In each of the experiments reported herein. NK activity against WEHI164-1 or FLD-3 in both C57BL/'6 and B 6 C 3 F T homozygous beige mutant mice was equal to that in normal controls. Further, this activity was not significantly afl'ected by treatment with anti-NK-1 2 alloantisera plus C (Table 4 ) . We conclude, therefore, that NKM activity is normal in mice homozygous for the beige mutation. It must be noted that C57BL/6 and C3H beige mutant mice are not the only strains to show a marked deficiency in NK coll activity against the YAC target. SJL mice also show such a defect (Kaminsky and Cudkowicz, 1980; Cudkowicz, personal communication), and therefore tests of spleen cells from this strain against NKu targets such as WEHI-164 1 or FLD-3 would be of interest. Acknowledgements. The aulhors wish to acknowledge Ihe cxperl counsel of Dr. Henry I. Winn find the excellent tcchniciil assistance ol" Mrs. Joanne Forlin. This study w:is supporieii by gr;mis CA-17800 jind CA-20()44 awjinied hy Ihe NalioriLi! Cancer Insliliile. Belhcsda. Maryland and by Grants AM-n7055 and HL-I864fi from Ihc Nalional Insliiules of Health. Bethesiia. M;iryl;inil. In Ihc course of this study Dr Robert C. Burton was supported first by a Nalional Hcalih and Medical Research Council of Auslralia Fellowship in Applied Keallh Sciences am! subsequently by John Mitchell Crouch Fellowship of Royal Australasian College of Surgeons. REFERENCES BuRixjN. R. C. (1980): 'Alloanlisera seiecBURTXJN. R. C and WINN. H . J. (1981): lively reactive with NK celts: characleri'Studies of natural killer (NK) cells. I. zation and use in defining NK cell classes.' N.K. cell specific antihodies in CE anliIn "Natural Cell-Mediated Immunity CBA serum." J. Immunol. 126. 1985Against Tumors" (R. Herberman. ed.). 1989. Acadamic Press. Inc.. New York, p. 19-
35.
BI'RTON. K. C , BARTIETT, S. P.. and WINN.
H. I. (1980): 'Heterogeneity of natural
NK CELLS IN BEIGE MUTANT MICE killer cells: A serological study with specific anti-NK ailoantisera,' In "Genetic Control of Natural Resistance to Infection and Malignancy" (R. Skamcne and P. A. L. Kongshavn. eds.), Academic Press. Inc.. New York, p. 413-418. BURTON. R. C:.. BARTIKTT, S. P., KUMAR,
V., and WINN. H . I. (1981): 'Heterogeneity of niitural killer cells in ihe mouse' Transplant Proc.. 13, 783-786. BURTON. R. C . CHtsM. S. E.. and WARNER.
N. L. (1977): 'In vitro Induction of tumour-specific immutiity. III. Lack of requirement for H-2 compatability in lysis of tumour targets by T cells activated in vitro to oncofetal and plasmacytoma antigens." J. Immunol. 118, 971-980. BLRTON. R. C . THOMPMVN, I., and WARNER.
N. L. ( 1975): 7« vitro induction of tumour specific immunity. I. Development of optimal conditions for induction and assay of cytotoxic lymphocytes.' / . Immunol Methods. 8. 133-150. KAMINSKY. S.. and CiUKowicy, G. (1980): 'Natural killing and resistance to marrow grafts: correlations in four "beige" mutant mouse lities." I'vd. l'r
