Immunology 1991 73 444-449
ADONIS 0019280591001798
NK cells inhibit T-cell
responses:
responses are
LFA3 + but not LFA3 suppressed
T-cell
P. D. MASON*, G. LOMBARDIt & R. I. LECHLERt Departments of *Medicine and tImmunology, Royal Postgraduate Medical School, Hammersmith Hospital, London Acceptedfor publication 24 April 1991
SUMMARY There is increasing evidence that natural killer (NK) cells have immunoregulatory effects in addition to their ability to lyse tumour and virus-infected target cells. However, much of the evidence to date is based on the reported effect of adding relatively impure NK cell populations to various in vitro cultures, and the effect on T cells has been contradictory. Here we report the inhibitory effect of highly purified CD16+ NK cells on mixed lymphocyte reaction (MLR), using unseparated peripheral blood mononuclear cells (PBMC) and purified T cells as responders. Marked inhibition was observed (up to 75%) which was proportional to the number of CD16+ cells present, and was abrogated by ultraviolet irradiation. In contrast, the addition of CD 16+ cells had no effect on the proliferative responses of five CD4+ anti-DR I alloreactive T-cell clones. To test the relative sensitivity of previously primed versus virgin T cells to NK cell-mediated inhibition, freshly isolated T cells from PBMC were separated into LFA3+ (memory) and LFA3- (virgin) populations. CD16+ cells caused inhibition of proliferation of LFA3 + but not LFA3 - cells in an M LR. In addition, the recall response of T cells to influenza was inhibited. These results further illustrate the regulatory potential of CD16+ NK cells, and suggest that previously primed cells are more susceptible to NK-mediated inhibition. However, activated (rather than resting) cells may escape regulation.
INTRODUCTION
however, another report suggested that highly purified CD 16+ cells actually enhance MLR-induced proliferation of T cells.9 In most of the above reports the source of NK cells has been either LGL or Leu-7a+ cells, although it is now known that anti-LeuI I b (CD 16) more completely defines the population of NK cells. Earlier work, demonstrating that enrichment of peripheral blood mononuclear cells (PBMC) with CD16+ cells suppressed and depletion enhanced, PWM-induced immunoglobulin synthesis, confirmed that CD16+ cells could play an immunoregulatory role." Subsequently, preliminary experiments suggested that enrichment with CD16+ cells had similar effects on phytohaemagglutinin (PHA)- and MLR-induced proliferation of PBMC, but CD16+ cells did not suppress the proliferation of alloreactive T-cell clones.'2 The experiments described here extend the previous studies, and report the effect of CD 16 + cells on MLR-induced proliferation by PBMC, purified T cells, CD 16 + -depleted cells, LFA3 + and LFA3 - cells and allospecific CD4+ T-cell clones. The results suggest that CDI6+ cells suppress MLR- and antigen-induced proliferation by resting LFA3+ but not LFA3- cells.
Natural killer (NK) cells are now accepted as being CD3- and CD16+ or CD56+ cells which are capable of non-major histocompatibility complex (MHC)-restricted cytotoxicity.' In addition to their ability to lyse various tumour or virus-infected cells, it has been found more recently that NK cells may mediate immunoregulatory effects. Cell populations enriched for NK activity have been demonstrated to suppress B-cell2 6 and T-cell functions.3 7 9 Some of the effects on B cells have been suggested to be mediated via direct regulation of T cells. Large granular lymphocytes (LGL) suppressed in vitro immunogloblin synthesis by B cells in the presence of T cells (but not T-cell supernatant) after pokeweed mitogen (PWM) stimulation,4 and more recently these results were confirmed in experiments in which CD 1 6 + cells suppressed PWM-induced plaque-forming cells. These authors also claimed that their data provided evidence that co-culture of NK cells with T cells induced 'suppressor' activity.7 Other data have been published suggesting that NK cells inhibit mixed lymphocyte reactions (MLR) in man3 and autologous MLR (AMLR) in man' and in mice.'0 In contrast,
MATERIALS AND METHODS Lymphocyte preparation PBMC were prepared from blood obtained from healthy volunteers (aged 18-45 years) on a Ficoll-Metrizoate gradient (Lymphoprep Nygaard, Denmark).'3
Correspondence: Dr P. D. Mason, Dept. of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Ducane Road, London W12 ONN, U.K.
444
NK cell inhibition of T-cell responses
445
(a)
(a)
20x
LFA3 37-8%
CD16+
E15
23-3%
E~ 10CL -N
c
10
-
5
(b) 0
CD16+ 98-9% Control
(b)
3
x
2
1:1 1:2 1:4 1:8 CD16+: PBMC ratio
1:16
LFA3+
LGFL
Figure 1. Flow cytometer histograms during cell sorting. (a) PBMC stained with anti-CD16 anti-LFA3 and control antibody. (b) Reanalysis of positively sorted cells using the same control.
a)
.'C C
0
E
For isolation of T cells, PBMC were suspended in RPMI1640 (Flow Laboratories, Irvine, Ayrshire, U.K.) supplemented with 10% foetal calf serum (FCS; Imperial Laboratories, Andover, U.K.), 2 mM L-glutamine (Gibco, Paisley, Renfrewshire, U.K.), penicillin 100,000 IU/litre and streptomycin 100 mg/litre (Imperial Laboratories) at 2 x 106 per ml, and incubated for 1 hr in a tissue culture grade Petri dish (Sterilin, Teddington, Middlesex, U.K.) at 370 in 5% CO2. Non-adherent cells were carefully removed, washing the dish gently with prewarmed medium. These cells, resuspended in 5 ml medium, were carefully added to a 1 5-ml nylon-wool column which had previously been flushed through with 50 ml of warm medium (with FCS). After 1 hr at 370, non-adherent cells were eluted from the column with 30 ml of warm medium. These cells were < 1% esterase positive and proliferated very poorly in response to PHA, suggesting that they were effectively depleted of accessory cells. The cells adherent to the Petri dish were removed with the gentle use of a rubber policeman.
Complement-mediated depletion Two-hundred microlitres of rabbit complement (Buxted Rabbit Co., Buxted, Surrey, U.K.; preadsorbed with 4 x 107 human PBMC per ml for 30 min on ice) were added to 107 cells in 200 ,l of RPMI-10, and the mixture incubated at 370 for 45 min. Two ,g/ml of sterile DNAse (Sigma, Poole, Dorset, U.K.) were added during the final 10 min to prevent clumping. Cells were then washed three times and recounted. Control antibodytreated cells were always > 99% viable. T-cell clones A series of anti-DRI and anti-DQwl alloreactive clones was used.'4 These were generated from PBMC by culture with yirradiated DR1-positive PBMC stimulators and cloned in limiting dilution, and were maintained in culture by weekly stimulation with irradiated PBMC and 10 U/ml of recombinant interleukin-2 (IL-2) (Boehringer-Mannheim, Lewes, Sussex,
U.K.). Cells used as feeder or stimulator cells, or for experimental controls, were y-irradiated (4000 rads). For control experiments
0
2
Responder Figure 2. Effect of enriching and depleting CD16+ cells on MLCinduced proliferation of PBMC. (a) 5 x 104 responder PBMC were cultured with 5 x 104 irradiated stimulator PBMC alone (A), with the indicated ratio of CD16+ cells untreated (0), or after UV-irradiation (-). (b) 5 x 104 control or depleted responder PBMC were cultured with 5 x 104 irradiated stimulator PBMC from subjects 1(o), 2 (E) or 3 (s), and the data expressed as stimulation index compared with undepleted control cultures. The relative increase in CD3 + cells in depleted responder populations as determined by flow cytometry was 150% and 190%, for responders 1 and 2, respectively. Actual counts per minute in the allogenic-MLR ranged from 2 x 104 to 105, and 2-5 x 103 for autologous MLR (A). All cultures were pulsed with [3H]thymidine for 16 hr and harvested on Day 7. Error bars represent standard deviations.
CD16+ cells were UV-irradiated in I ml of RPMI with 10% FCS in a 60-mm Petri dish for 5 min, 12 cm from a 15W UV-tube (Phillips TOU- 1 5W, delivering 25 mW/M2 at 12 cm and 253 7 nm). Monoclonal antibodies The following monoclonal antibodies were used at optimal concentrations: anti-Leu- 1 b (anti-CD 16; Becton-Dickinson Co, Mountain View, CA) TS3/2 (anti-LFA3; ATCC, Rockville, MD), OKT3 (anti-CD3, ATCC) and L243 (anti-DR; ATCC).
Fluorescence-activated cell sorting (FA CS) PBMC were incubated with optimum concentrations of different monoclonal antibodies for 30 min on ice, folowed by a FITC-labelled rabbit anti-mouse F(ab)2 (Dako, High Wycombe, Bucks, U.K.). The cells were then analysed or sorted
P. D. Mason, G. Lombardi & R.
446
I.
Lechler
Table 1. CD16+ cells suppress MLR by PBMC, T cells and CD 16-depleted cells
Exp. I (PBMC)
Exp. 2 (PBMC) °0
So
C.p.m. MLR alone MLR+NK 1:1 1:2 1:4 1:8 1:16 M LR + uvNK 1:1 Irr. MLR+NK 1:1
suppression*
C.p.m.
suppression*
%
C.p.m.
3742 3121 5143 5867 6480
74 78 64 59 55
8890 11,364 19,994 12,855 15,786
40 24 0 13 0
10,617
26
12,025
19
supression*
C.p.m.
O/o
47 41 39 27 NA
C.p.m.
suppression *
96,868
119,828
5000 5634 5776 6929 NA
Exp. 5 (CD 16-)
o
suppression*
9501
14,919
14,415
Exp. 4 (T cells)
Exp. 3 (T cells)
74,780 93,516 118,561 118,877 125,270
38 22 1 1 0
115,958
3
58,257 74,690 77,732 100,716 102,189
40 23 20 0 0
1144
358
Cultures were set up as described in the text with 5 x 104 responder cells (PBMC, T cells or CD 16 - cells) and 5 x 104 irradiated stimulator allogeneic PBMC. Different numbers of CD16+ cells were added and the cultures harvested on Day 7, 16 hr after pulsing with [3H]thymidine. control c.p.m. -NK-enriched c.p.m. 100%. *Suppression= coto ~~.x control c.p.m. NA, not available.
to o
Table 2. CD16+ cells do not affect allospecific proliferation of T-cell clones
120
x
E -6
100
Exp.
80
6
I
S 60C
40-
E
20-
D
APC CD16+ No clone
2
0
1:1.4 1:3 CD16+: T-cell clone ratio 3:1
15:1
1:6
Figure 3. CD16+ cells do not inhibit allospecific responses of T-cell clones. 104 DRI-reactive clones were cultured with 3 x 104 DRI + BCL [(O) G8; (0) G12] alone or with the indicated number of untreated CD16+ cells [(0) G8, (-) G12] or UV-treated CD16+ cells [(O) G8; (0) G 12]. Cultures were harvested at 48 hr, 16 hr after pulsing with [3H]thymidine.
into positive and negative populations
on an
Epics CSTM flow
cytometer (Coulter Electronics, Luton, Beds, U.K.).
3
+ + + + + + + + +
+ +UV
+ +UV + +UV
378
1581 4887 1768 436 1990 2670 1658 1602 896
G3
G12
498 56,252 76,512 61,267 481 52,612 54,491 48,991 369 64,373 53,111 53,469
870 73,911 100,506 74,093 415 62,548 62,662
66,002 590 19,316 21,242 17,407
DAF 7
G68
349 34,242 52,697 35,285 822 294 2181 2833 1664 7686 320 430 41,894 42,111 44,545
Four DRI-specific CD4+ human T-cell clones (G3, G12, G68 and DAF7) were used. 104 cells of each clone were cultured alone or with 3 x 104 APC (irradiated DR I + LCL). Doubling dilutions of CD 16+ cells were added to replicate cultures, but the values shown in the table are from cultures with the largest number (CD16+: T cell ratio of 34: 1). Othercontrols included UV-irradiated CDl6+ cells as described in the Materials and Methods. All values are the mean of triplicate
cultures.
Cell cultures Exact conditions varied with particular experiments, details of which are given in the Results. In general, cells were cultured in round-bottomed 96-well plates with RPMI supplemented with 0% AB serum pulsed with I ,uCi [3H]thymidine at 48 hr (T-cell clones) or 5 days (MLC and mitogen-stimulated cultures), harvested 16 hr later onto glass fibre filters using an automatic cell harvester (Titertek, Flow Laboratories), and counted in a scintillation counter (Beckman, High Wycombe, Bucks, U.K.).
RESULTS Effect of CD16+ cells on MLR-induced proliferation CD16+ sorted cells were always >95% (and usually >98%/,) pure and LFA3+ sorted cells were always >90% (usually >950/)) pure by re-analysis (Fig. 1). CD16+ cells suppressed
NK cell inhibition of T-cell responses Exp. 1
2*0 -
2-0
447 Exp. 2
-
1-5 -
9.5
190
-
9*0
0-5 -
0-5
-
0*0
-
2*0
-
9.5
-
0 C 0.0
05cnE
-
I
I
I
I
I
I
I
l
Exp. 3
5*0 -
2 *0 -
Exp. 4
I*0 -
O*0
-
0-0
-
0
1:1
1:4
1:16
0-5
-
O*0
-
1:654
I
I
0
1:1
I
I
I
t-
1:16
1:4
1:64
CD16 : responder ratio
Figure 4. CD16+ cells inhibit MLR-induced proliferation by LFA3 + but not LFA3 - cells. The results of four experiments are presented, expressed as a stimulation ratio of proliferation of cultures with and without added CD 16 + cells. 5 x 104 responder LFA3 + (U) or LFA3 (0) cells were cultured with 5 x I04 irradiated stimulator PBMC alone (0), or with the indicated ratio of CD16+ cells. Cultures were pulsed with [3H]thymidine for 16 hr and harvested on Day 7.
Table 3. Effect of CD16+ cells on MLR by LFA3 + and LFA3- responder T cells
Exp. 1
C.p.m. LFA3+ MLR +CD16+:LFA3+ +1:1 + 1:2 + 1:4 + 1:8 + 1:16 + 1:32 + 1:64 LFA3+MLR+ UV CD16+t
12,600 1246 2145 3600 2884
NT
Exp. 2
% suppression*
90 82 71 63
NT
LFA3- MLR +CD16+:LFA3- +1:1 + 1:2 + 1:4 +1:8 + 1:16 + 1:32 +1:64
909 1312 2145 2526 1784
-
LFA3-MLR+UVCD16+
NT
NT
C.p.m. 27,000 10,499 15,150 24,574 34,564 20,203 31,361 29,109 8738
16,503 22,760 26,367 34,442 30,676 27,079 22,102 23,602 16,394
Exp. 3
% suppression*
Exp. 4
C.p.m.
OX, suppression*
-
24,000 9697 13,235 17,043 18,000 20,388 21,577 23,861
60 45 29 25 15 10 1
42
60,985
-
14,798 13,768 19,361 19,188 19,221
1
16,572
62 46 12 -
27
Cultures were set up as described in the test and the results of four experiments are presented. * Suppression = c.p.m. (MLR)-c.p.m. (MLR+CD16+) I000/ c.p.m. (MLR) t Effect of UV-treated CDl16+ cells in equal numbers to responder cells; NT, not tested.
7 -
C.p.m.
OX/ suppression*
78,913 36,209 46,457 47,339 70,295 72,434 79,359
64 41 40 11 8 1
115,958
3
101,001 113,203 127,882 141,370 140,029 102,949 150,116 138,743 139,659
-
P. D. Mason, G. Lombardi & R. I. Lechler
448 x
20
E
A.
Q. 150 -6
.50.I 10 0
.5c 5.0
1:1
1:2 1:4 1:8 1:16
CD16+: PBMC ratio Figure 5. Effect of CD l 6 + cells on proliferation of T cells in response to influenza antigen. Cultures contained T cells, macrophages and influenza antigen alone (A) or with the indicated ratio of CDl6+ cells (El).
5
x
104 responder T cells
were
cultured with 3 x 104 autologous
irradiated macrophages and influenza antigen in round-bottomed wells for 5 days pulsed with [3H]thymidine for the final 16 hr.
MLR-induced proliferation of autologous PBMC. The effects abrogated by UV-irradiation (Fig. 2a). Suppression ranged between 40% and 75%, and was marked even at relatively low ratios of CD 16+ cells: responder PBMC (Fig. 2a, Table 1). The effect was observed when PBMC, T or CD16cells were used as responder cells (Table 1). Furthermore, the reciprocal experiment gave the same result, in that depletion of CD16+ cells from PBMC resulted in an enhanced MLRinduced proliferation, and the results of two representative experiments are shown in Fig. 2b. The enhancement was often greatest in autologous MLR-induced proliferation. were
CD16+ cells do not affect the allospecific-specific responses of Tcell clones Five different alloreactive clones were used as the responders. 104 cells of each T-cell clone were cultured with optimal numbers (3 x 104) of DRI + irradiated EBV-transformed lymphoblastoid cell lines, with or without CD16+ cells (and UV-irradiated CD 1 6 + cells were used as controls). Figure 3 represents a typical experiment, and Table 2 summarizes the data from several experiments. CD16+ cells did not suppress the allospecific proliferation of the T-cell clones used, even with a CDI 6+: responder ratio of 4: 1. In a further series of experiments using (i) PBMC and L cells transfected with DRl as alternative stimulator cells, and (ii) different ratios of responder to stimulator cells, no suppression was observed (data not shown). Low level proliferation of CD16+ cells was seen in the presence of added IL-2 (data not shown) and also when co-cultured with irradiated T-cell clones and their stimulator cells (data not shown), probably due to the IL-2 generated by the clones, but this was generally small in comparison to the proliferation of T-cell clones. Effect of CD16+ cells on MLR by LFA3+ and LFA3- cells In view of the observed suppression of MLR but not of alloreactive T-cell clones by CD16+ cells, experiments were designed to determine whether NK cells had a differential effect on 'naive' rather than 'memory' T cells. For this purpose, peripheral blood T cells were separated by cell sorting into
LFA3+ ('memory') or LFA3- ('naive') cells for use as responders in the MLR. CD16+ cells were obtained, as before, by flow cytometry on the first day and kept overnight in RPMI supplemented with AB serum at 37" (which preserved NK cytotoxicity; data not shown). T cells and macrophages were isolated from the same donor on the following day, and the T cells were stained with TS/32 (anti-LFA3) and sorted into LFA3+ and LFA3- cells (Fig. 1). MLR cultures were set up using whole PBMC, T cells, LFA3+ and LFA3- cells as responders, and third-party yirradiated PBMC as stimulator cells. CD16+ cells were added in different numbers to MLR cultures, which were pulsed with [3H]thymidine on Day 5 and harvested 16 hr later. CD16+ cells consistently inhibited MLR responses by unseparated T cells and LFA3+ but not by LFA3- cells (Fig. 4, Table 3). CD16+ cells also suppressed an antigen-specific recall response, as measured by the proliferation of autologous T cells in resonse to influenza antigen in the presence of macrophages (Fig. 5). The effect was abrogated by irradiation of CD16+ cells with UV light.
DISCUSSION The experiments reported here were designed to determine, in more detail, the phenotype of the T cells susceptible to regulation by CD16+ NK cells. Previous reports have given conflicting results, which may have been due in part to the use of relatively impure populations of NK cells. Data have been published that rather impure NK populations could inhibit Tcell proliferation in MLR3 and autologous MLR8 in man, and in mice, although some of the latter data suggested the NK effect acted via dendritic cells.'0 A recent report concluded that purified CD1 6+ cells actually enhanced allogeneic T-cell stimu-
lation.9 In the studies reported here, NK cells were prepared by positive selection by FACS, and so all cells are stained with antiCD16. Binding of antibody to NK cells does result in calcium flux and increased phosphoinositol turnover,'5 and may induce transcription of various genes including CD25, IFN-y and TNF.'6 However, anti-CD16 antibodies have no short-term effect on NK cytotoxicity of target cells, which have no Fcy receptors, but enhance cytotoxicity of those bearing Fcy receptors when the anti-CD 16 monoclonal is of the IgG class.'7 The anti-CD 1 6 used in these experiments (anti-Leu- II b) is an IgM antibody and does not enhance cytotoxicity, although it may have an effect on NK immunoregulation since previous studies demonstrated that addition of anti-Leu-l lb to PBMC reduced PWM-induced immunoglobulin synthesis." Enrichment of PBMC, purified T cells or CD16- cells with sorted CD16+ cells resulted in a marked suppression (30-750%) of MLR-induced proliferation. The effect was almost completely abrogated by UV-irradiation of NK cells. This suggests that the observed suppression was not simply due to an effect of additional cells in the cultures. It implies that the UV-irradiation inhibited some function of metabolically active, live cells, the nature of which is undefined. In some experiments, y-irradiated CD16+ cells had an intermediate effect on suppression (data not shown), suggesting that proliferation of CD16+ cells is not essential. It is also notable that the effects reported here were observed when the ratio of CD16+ cells to responding T cells was similar to that present in PBMC and, conversely, that
NK cell inhibition of T-cell responses depletion of CD16+ cells from PBMC enhanced MLC responses. However, CD16+ cells had no effect on the allospecific proliferation of five alloreactive CD4+ clones, stimulated under a variety of conditions, and with a NK: T cell ratio of up to 4: 1. Indeed, in some experiments, NK-enriched cultures showed enhanced proliferation, although some of this was undoubtedly due to the proliferation of the CD 16 + cells in response to the IL2 generated by the T-cell clones, and disappeared following UVirradiation of CD16+ cells. One of the possible reasons why CD16+ cells were able to inhibit an MLR but were unable to inhibit allospecific proliferation by T-cell clones is that CD16+ cells act on 'naive' but not 'memory' T cells, since the MLR seems to be due to proliferation of both types of T cells,'8 '9 while T-cell clones are, by definition, 'memory' cells. In an attempt to address this possibility, the effect of CD16+ cells on MLR by unseparated T cells, LFA3+ and LFA3- cells (representing, respectively, 'memory' and 'naive' populations) was examined. CD16+ cells suppressed MLR responses by LFA3+ (60-90%) but not by LFA3- cells. CD16+ cells also suppressed the proliferative response of peripheral blood T cells to influenza antigen. In vitro responses to recall antigens are dependent on previously primed cells. These results rule out this explanation for the failure of NK cells to inhibit proliferation of T-cell clones. Three other explanations can be offered to account for the differential susceptibility of PBMC versus T-cell clones to NKinduced inhibition. First, it may be that NK cells can only inhibit resting and not activated T cells. Due to the way in which T-cell clones are maintained in vitro, they are permanently in an activated state. In contrast T cells derived from peripheral blood are usually 'resting'. Second, the effector: target ratio may have been too low. Although NK: T ratios of up to 4: 1 were used, this is likely to be far lower than the ratio of NK: responding T cells in a primary MLR. The published frequencies of alloreactive CD4+ T cells stimulated by HLA-mismatched stimulator cells vary between 1:1000 and 1:40,000. The third possible explanation is that in order to exert their suppressive effect CD16+ cells might require another cell type which is present in PBMC but not in the T-cell clone cultures. There is published data suggesting that NK cells mediate their effect by interaction with accessory cells in mice2 and humans.9 The differential susceptibility of LFA3+ versus LFA3- T cells to NK cell-mediated inhibition may be explained by accessory molecular interactions. One of the ligands for LFA3 is CD2 which, of course, is present on virtually all CD16+ NK cells. ' If LFA3 - T cells were not susceptible to regulation by NK cells, it is possible that the NK effect might be mediated via CD2-LFA3 interaction, and so the effect of CD 16 + on mitogenstimulated proliferation of LFA3- cells would be of interest. The effect of a variety of monoclonal antibodies (including antiCD2 and anti-LFA3) on NK-mediated suppression should also be investigated. In conclusion, the series of experiments described here provides clear support for the ability of cells with 'NK' phenotype and activity to exert important immunoregulatory functions. CD16+ cells are able to inhibit alloreactive and antigen-specific responses of LFA3 + cells but not the alloreactive responses of LFA3- cells. The lack of susceptibility of established alloreactive T-cell clones may suggest that once activated, T cells lose susceptibility to NK-mediated inhibition.
449
ACKNOWLEDGMENT We wish to thank Dr Ann Rees for critical comments on the manuscript.
REFERENCES 1. HERCEND T. & SCHMIDT R.E. (1988) Characteristics and uses of natural killer cells. Immunol. Today, 9, 291. 2. ABRUZZO L.V. & ROWLEY D.A. (1983) Homeostasis of the antibody response: immunoregulation by NK cells. Science, 222, 581. 3. TILDEN A.B., Aso T. & BALCH C.M. (1983) Suppressor cell function of human granular lymphocytes identified by the HNK-l (Leu7) monoclonal antibody. J. Immunol. 130, 1171. 4. ARAI S., YAMAMOTO H., ITOH K. & KUMAGAI K. (1983) Suppressive effect of human natural killer cells on pokeweed mitogen-induced B cell differentiation. J. Immunol. 131, 651. 5. BRIEVA J.A., TARGAN S. & STEVENS R.H. (1984) NK and T cell subsets regulate antibody production by human in tito antigeninduced lymphoblastoid B cells. J. Immunol. 132, 611. 6. KATZ P., WHALEN G., MITCHELL S.R., Cupps T.R. & EVANS M. (1990) Modulation of suppression of mitogen-induced T celldependent B cell responses by natural killer cells. Clin. Immunol. Immunopathol. 55, 148. 7. KATZ P., MITCHELL S.R., Cupps T.R., EVANS M. & WHALEN G. (1989) Suppression of B cell responses by natural killer cells is mediated through direct effects on T cells. Cell. Immunol. 119, 130. 8. POPE R.M., MCCHESNEY L., STEBBING N., GOLDSTEIN L. & TALAL N. (1985) Regulation of T cell proliferation by cloned interferon-a mediated by Leu 1 lb-positive cells. J. Immunol. 135, 4048. 9. WEISSLER J.C., YARBROUGH W.C., TOEws G.B. & NICOD L.P. (1989) Human natural killer cells enhance a mixed leukocyte reaction. J. Leuk. Biol. 43, 291. 10. SHAH P.D., GILBERTSON S.M. & ROWLEY D.A. (1985) Dendritic cells that have interacted with antigen are targets for natural killer cells. J. exp. Med. 162, 625. 11. MASON P.D., WEETMAN A.P., SISSONS J.G.P. & BORYSIEWICZ L.K. (1988) Suppressive role of NK cells in pokeweed mitogen-induced immunoglobulin synthesis: effect of depletion/enrichment of Leu 1 l b I cells. Immunology, 65, 113. 12. MASON P.D., LOMBARDI G., SISSONS J.G.P., BORYSIEWICZ L.K. & LECHLER R.I. (1989) Target specificity of immunoregulation by natural killer cells. Transpl. Proc. 21, 201. 13. BOYUM A. (1968) Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, (Supple. 97) 77. 14. LOMBARDI G., SIDHU S., LAMB J.R., BATCHELOR J.R. & LECHLER R.I. (1989) Co-regulation of endogenous antigens with HLA-DRI by alloreactive human T cell clones. J. Immunol. 142, 753. 15. CASSATELLA M.A., ANEGON I., CUTURI M.C., GRISKEY P., TRINCHIERI G. & PERUSSIA B. (1989) FcyR (CD16) interaction with ligand induces Ca2+ mobilization and phosphoinositide turnover in human natural killer cells. J. Immunol. 169, 549. 16. ANEGON I., CUTURI M.C., TRINCHIERI G. & PERUSSIA B. (1988) Interaction of Fc receptor (CD 16) ligands induces transcription of interleukin 2 receptor (CD25) and lymphokine genes and expression of their products in human natural killer cells. J. exp. Med. 167, 452. 17. MORETTA A., TAMBUSSI G., CICCONE E., PENDE D., MELIOLI G. & MORETTA L. (1989) CD16 surface molecules regulate the cytolytic function of CD3- CD16+ human natural killer cells. Int. J. Cancer, 44, 727. 18. MERKENSCHLAGER M., TERRY L., EDWARDS R. & BEVERLEY P.C.L. (1988) Limiting dilution analysis of proliferative responses in human lymphocyte populations defined by the monoclonal antibody UCHLI: implications for differential CD45 expression in T cell memory formation. Eur. J. Immunol. 18, 1653. 19. LOMBARDI G., SIDHU S., DALY M., BATCHELOR J.R., MAKGOBA W. & LECHLER R.I. (1990) Are primary allo responses truely primary? Int. Immunol. 2, 9.
ADONIS 0019280591001798
NK cells inhibit T-cell
responses:
responses are
LFA3 + but not LFA3 suppressed
T-cell
P. D. MASON*, G. LOMBARDIt & R. I. LECHLERt Departments of *Medicine and tImmunology, Royal Postgraduate Medical School, Hammersmith Hospital, London Acceptedfor publication 24 April 1991
SUMMARY There is increasing evidence that natural killer (NK) cells have immunoregulatory effects in addition to their ability to lyse tumour and virus-infected target cells. However, much of the evidence to date is based on the reported effect of adding relatively impure NK cell populations to various in vitro cultures, and the effect on T cells has been contradictory. Here we report the inhibitory effect of highly purified CD16+ NK cells on mixed lymphocyte reaction (MLR), using unseparated peripheral blood mononuclear cells (PBMC) and purified T cells as responders. Marked inhibition was observed (up to 75%) which was proportional to the number of CD16+ cells present, and was abrogated by ultraviolet irradiation. In contrast, the addition of CD 16+ cells had no effect on the proliferative responses of five CD4+ anti-DR I alloreactive T-cell clones. To test the relative sensitivity of previously primed versus virgin T cells to NK cell-mediated inhibition, freshly isolated T cells from PBMC were separated into LFA3+ (memory) and LFA3- (virgin) populations. CD16+ cells caused inhibition of proliferation of LFA3 + but not LFA3 - cells in an M LR. In addition, the recall response of T cells to influenza was inhibited. These results further illustrate the regulatory potential of CD16+ NK cells, and suggest that previously primed cells are more susceptible to NK-mediated inhibition. However, activated (rather than resting) cells may escape regulation.
INTRODUCTION
however, another report suggested that highly purified CD 16+ cells actually enhance MLR-induced proliferation of T cells.9 In most of the above reports the source of NK cells has been either LGL or Leu-7a+ cells, although it is now known that anti-LeuI I b (CD 16) more completely defines the population of NK cells. Earlier work, demonstrating that enrichment of peripheral blood mononuclear cells (PBMC) with CD16+ cells suppressed and depletion enhanced, PWM-induced immunoglobulin synthesis, confirmed that CD16+ cells could play an immunoregulatory role." Subsequently, preliminary experiments suggested that enrichment with CD16+ cells had similar effects on phytohaemagglutinin (PHA)- and MLR-induced proliferation of PBMC, but CD16+ cells did not suppress the proliferation of alloreactive T-cell clones.'2 The experiments described here extend the previous studies, and report the effect of CD 16 + cells on MLR-induced proliferation by PBMC, purified T cells, CD 16 + -depleted cells, LFA3 + and LFA3 - cells and allospecific CD4+ T-cell clones. The results suggest that CDI6+ cells suppress MLR- and antigen-induced proliferation by resting LFA3+ but not LFA3- cells.
Natural killer (NK) cells are now accepted as being CD3- and CD16+ or CD56+ cells which are capable of non-major histocompatibility complex (MHC)-restricted cytotoxicity.' In addition to their ability to lyse various tumour or virus-infected cells, it has been found more recently that NK cells may mediate immunoregulatory effects. Cell populations enriched for NK activity have been demonstrated to suppress B-cell2 6 and T-cell functions.3 7 9 Some of the effects on B cells have been suggested to be mediated via direct regulation of T cells. Large granular lymphocytes (LGL) suppressed in vitro immunogloblin synthesis by B cells in the presence of T cells (but not T-cell supernatant) after pokeweed mitogen (PWM) stimulation,4 and more recently these results were confirmed in experiments in which CD 1 6 + cells suppressed PWM-induced plaque-forming cells. These authors also claimed that their data provided evidence that co-culture of NK cells with T cells induced 'suppressor' activity.7 Other data have been published suggesting that NK cells inhibit mixed lymphocyte reactions (MLR) in man3 and autologous MLR (AMLR) in man' and in mice.'0 In contrast,
MATERIALS AND METHODS Lymphocyte preparation PBMC were prepared from blood obtained from healthy volunteers (aged 18-45 years) on a Ficoll-Metrizoate gradient (Lymphoprep Nygaard, Denmark).'3
Correspondence: Dr P. D. Mason, Dept. of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, Ducane Road, London W12 ONN, U.K.
444
NK cell inhibition of T-cell responses
445
(a)
(a)
20x
LFA3 37-8%
CD16+
E15
23-3%
E~ 10CL -N
c
10
-
5
(b) 0
CD16+ 98-9% Control
(b)
3
x
2
1:1 1:2 1:4 1:8 CD16+: PBMC ratio
1:16
LFA3+
LGFL
Figure 1. Flow cytometer histograms during cell sorting. (a) PBMC stained with anti-CD16 anti-LFA3 and control antibody. (b) Reanalysis of positively sorted cells using the same control.
a)
.'C C
0
E
For isolation of T cells, PBMC were suspended in RPMI1640 (Flow Laboratories, Irvine, Ayrshire, U.K.) supplemented with 10% foetal calf serum (FCS; Imperial Laboratories, Andover, U.K.), 2 mM L-glutamine (Gibco, Paisley, Renfrewshire, U.K.), penicillin 100,000 IU/litre and streptomycin 100 mg/litre (Imperial Laboratories) at 2 x 106 per ml, and incubated for 1 hr in a tissue culture grade Petri dish (Sterilin, Teddington, Middlesex, U.K.) at 370 in 5% CO2. Non-adherent cells were carefully removed, washing the dish gently with prewarmed medium. These cells, resuspended in 5 ml medium, were carefully added to a 1 5-ml nylon-wool column which had previously been flushed through with 50 ml of warm medium (with FCS). After 1 hr at 370, non-adherent cells were eluted from the column with 30 ml of warm medium. These cells were < 1% esterase positive and proliferated very poorly in response to PHA, suggesting that they were effectively depleted of accessory cells. The cells adherent to the Petri dish were removed with the gentle use of a rubber policeman.
Complement-mediated depletion Two-hundred microlitres of rabbit complement (Buxted Rabbit Co., Buxted, Surrey, U.K.; preadsorbed with 4 x 107 human PBMC per ml for 30 min on ice) were added to 107 cells in 200 ,l of RPMI-10, and the mixture incubated at 370 for 45 min. Two ,g/ml of sterile DNAse (Sigma, Poole, Dorset, U.K.) were added during the final 10 min to prevent clumping. Cells were then washed three times and recounted. Control antibodytreated cells were always > 99% viable. T-cell clones A series of anti-DRI and anti-DQwl alloreactive clones was used.'4 These were generated from PBMC by culture with yirradiated DR1-positive PBMC stimulators and cloned in limiting dilution, and were maintained in culture by weekly stimulation with irradiated PBMC and 10 U/ml of recombinant interleukin-2 (IL-2) (Boehringer-Mannheim, Lewes, Sussex,
U.K.). Cells used as feeder or stimulator cells, or for experimental controls, were y-irradiated (4000 rads). For control experiments
0
2
Responder Figure 2. Effect of enriching and depleting CD16+ cells on MLCinduced proliferation of PBMC. (a) 5 x 104 responder PBMC were cultured with 5 x 104 irradiated stimulator PBMC alone (A), with the indicated ratio of CD16+ cells untreated (0), or after UV-irradiation (-). (b) 5 x 104 control or depleted responder PBMC were cultured with 5 x 104 irradiated stimulator PBMC from subjects 1(o), 2 (E) or 3 (s), and the data expressed as stimulation index compared with undepleted control cultures. The relative increase in CD3 + cells in depleted responder populations as determined by flow cytometry was 150% and 190%, for responders 1 and 2, respectively. Actual counts per minute in the allogenic-MLR ranged from 2 x 104 to 105, and 2-5 x 103 for autologous MLR (A). All cultures were pulsed with [3H]thymidine for 16 hr and harvested on Day 7. Error bars represent standard deviations.
CD16+ cells were UV-irradiated in I ml of RPMI with 10% FCS in a 60-mm Petri dish for 5 min, 12 cm from a 15W UV-tube (Phillips TOU- 1 5W, delivering 25 mW/M2 at 12 cm and 253 7 nm). Monoclonal antibodies The following monoclonal antibodies were used at optimal concentrations: anti-Leu- 1 b (anti-CD 16; Becton-Dickinson Co, Mountain View, CA) TS3/2 (anti-LFA3; ATCC, Rockville, MD), OKT3 (anti-CD3, ATCC) and L243 (anti-DR; ATCC).
Fluorescence-activated cell sorting (FA CS) PBMC were incubated with optimum concentrations of different monoclonal antibodies for 30 min on ice, folowed by a FITC-labelled rabbit anti-mouse F(ab)2 (Dako, High Wycombe, Bucks, U.K.). The cells were then analysed or sorted
P. D. Mason, G. Lombardi & R.
446
I.
Lechler
Table 1. CD16+ cells suppress MLR by PBMC, T cells and CD 16-depleted cells
Exp. I (PBMC)
Exp. 2 (PBMC) °0
So
C.p.m. MLR alone MLR+NK 1:1 1:2 1:4 1:8 1:16 M LR + uvNK 1:1 Irr. MLR+NK 1:1
suppression*
C.p.m.
suppression*
%
C.p.m.
3742 3121 5143 5867 6480
74 78 64 59 55
8890 11,364 19,994 12,855 15,786
40 24 0 13 0
10,617
26
12,025
19
supression*
C.p.m.
O/o
47 41 39 27 NA
C.p.m.
suppression *
96,868
119,828
5000 5634 5776 6929 NA
Exp. 5 (CD 16-)
o
suppression*
9501
14,919
14,415
Exp. 4 (T cells)
Exp. 3 (T cells)
74,780 93,516 118,561 118,877 125,270
38 22 1 1 0
115,958
3
58,257 74,690 77,732 100,716 102,189
40 23 20 0 0
1144
358
Cultures were set up as described in the text with 5 x 104 responder cells (PBMC, T cells or CD 16 - cells) and 5 x 104 irradiated stimulator allogeneic PBMC. Different numbers of CD16+ cells were added and the cultures harvested on Day 7, 16 hr after pulsing with [3H]thymidine. control c.p.m. -NK-enriched c.p.m. 100%. *Suppression= coto ~~.x control c.p.m. NA, not available.
to o
Table 2. CD16+ cells do not affect allospecific proliferation of T-cell clones
120
x
E -6
100
Exp.
80
6
I
S 60C
40-
E
20-
D
APC CD16+ No clone
2
0
1:1.4 1:3 CD16+: T-cell clone ratio 3:1
15:1
1:6
Figure 3. CD16+ cells do not inhibit allospecific responses of T-cell clones. 104 DRI-reactive clones were cultured with 3 x 104 DRI + BCL [(O) G8; (0) G12] alone or with the indicated number of untreated CD16+ cells [(0) G8, (-) G12] or UV-treated CD16+ cells [(O) G8; (0) G 12]. Cultures were harvested at 48 hr, 16 hr after pulsing with [3H]thymidine.
into positive and negative populations
on an
Epics CSTM flow
cytometer (Coulter Electronics, Luton, Beds, U.K.).
3
+ + + + + + + + +
+ +UV
+ +UV + +UV
378
1581 4887 1768 436 1990 2670 1658 1602 896
G3
G12
498 56,252 76,512 61,267 481 52,612 54,491 48,991 369 64,373 53,111 53,469
870 73,911 100,506 74,093 415 62,548 62,662
66,002 590 19,316 21,242 17,407
DAF 7
G68
349 34,242 52,697 35,285 822 294 2181 2833 1664 7686 320 430 41,894 42,111 44,545
Four DRI-specific CD4+ human T-cell clones (G3, G12, G68 and DAF7) were used. 104 cells of each clone were cultured alone or with 3 x 104 APC (irradiated DR I + LCL). Doubling dilutions of CD 16+ cells were added to replicate cultures, but the values shown in the table are from cultures with the largest number (CD16+: T cell ratio of 34: 1). Othercontrols included UV-irradiated CDl6+ cells as described in the Materials and Methods. All values are the mean of triplicate
cultures.
Cell cultures Exact conditions varied with particular experiments, details of which are given in the Results. In general, cells were cultured in round-bottomed 96-well plates with RPMI supplemented with 0% AB serum pulsed with I ,uCi [3H]thymidine at 48 hr (T-cell clones) or 5 days (MLC and mitogen-stimulated cultures), harvested 16 hr later onto glass fibre filters using an automatic cell harvester (Titertek, Flow Laboratories), and counted in a scintillation counter (Beckman, High Wycombe, Bucks, U.K.).
RESULTS Effect of CD16+ cells on MLR-induced proliferation CD16+ sorted cells were always >95% (and usually >98%/,) pure and LFA3+ sorted cells were always >90% (usually >950/)) pure by re-analysis (Fig. 1). CD16+ cells suppressed
NK cell inhibition of T-cell responses Exp. 1
2*0 -
2-0
447 Exp. 2
-
1-5 -
9.5
190
-
9*0
0-5 -
0-5
-
0*0
-
2*0
-
9.5
-
0 C 0.0
05cnE
-
I
I
I
I
I
I
I
l
Exp. 3
5*0 -
2 *0 -
Exp. 4
I*0 -
O*0
-
0-0
-
0
1:1
1:4
1:16
0-5
-
O*0
-
1:654
I
I
0
1:1
I
I
I
t-
1:16
1:4
1:64
CD16 : responder ratio
Figure 4. CD16+ cells inhibit MLR-induced proliferation by LFA3 + but not LFA3 - cells. The results of four experiments are presented, expressed as a stimulation ratio of proliferation of cultures with and without added CD 16 + cells. 5 x 104 responder LFA3 + (U) or LFA3 (0) cells were cultured with 5 x I04 irradiated stimulator PBMC alone (0), or with the indicated ratio of CD16+ cells. Cultures were pulsed with [3H]thymidine for 16 hr and harvested on Day 7.
Table 3. Effect of CD16+ cells on MLR by LFA3 + and LFA3- responder T cells
Exp. 1
C.p.m. LFA3+ MLR +CD16+:LFA3+ +1:1 + 1:2 + 1:4 + 1:8 + 1:16 + 1:32 + 1:64 LFA3+MLR+ UV CD16+t
12,600 1246 2145 3600 2884
NT
Exp. 2
% suppression*
90 82 71 63
NT
LFA3- MLR +CD16+:LFA3- +1:1 + 1:2 + 1:4 +1:8 + 1:16 + 1:32 +1:64
909 1312 2145 2526 1784
-
LFA3-MLR+UVCD16+
NT
NT
C.p.m. 27,000 10,499 15,150 24,574 34,564 20,203 31,361 29,109 8738
16,503 22,760 26,367 34,442 30,676 27,079 22,102 23,602 16,394
Exp. 3
% suppression*
Exp. 4
C.p.m.
OX, suppression*
-
24,000 9697 13,235 17,043 18,000 20,388 21,577 23,861
60 45 29 25 15 10 1
42
60,985
-
14,798 13,768 19,361 19,188 19,221
1
16,572
62 46 12 -
27
Cultures were set up as described in the test and the results of four experiments are presented. * Suppression = c.p.m. (MLR)-c.p.m. (MLR+CD16+) I000/ c.p.m. (MLR) t Effect of UV-treated CDl16+ cells in equal numbers to responder cells; NT, not tested.
7 -
C.p.m.
OX/ suppression*
78,913 36,209 46,457 47,339 70,295 72,434 79,359
64 41 40 11 8 1
115,958
3
101,001 113,203 127,882 141,370 140,029 102,949 150,116 138,743 139,659
-
P. D. Mason, G. Lombardi & R. I. Lechler
448 x
20
E
A.
Q. 150 -6
.50.I 10 0
.5c 5.0
1:1
1:2 1:4 1:8 1:16
CD16+: PBMC ratio Figure 5. Effect of CD l 6 + cells on proliferation of T cells in response to influenza antigen. Cultures contained T cells, macrophages and influenza antigen alone (A) or with the indicated ratio of CDl6+ cells (El).
5
x
104 responder T cells
were
cultured with 3 x 104 autologous
irradiated macrophages and influenza antigen in round-bottomed wells for 5 days pulsed with [3H]thymidine for the final 16 hr.
MLR-induced proliferation of autologous PBMC. The effects abrogated by UV-irradiation (Fig. 2a). Suppression ranged between 40% and 75%, and was marked even at relatively low ratios of CD 16+ cells: responder PBMC (Fig. 2a, Table 1). The effect was observed when PBMC, T or CD16cells were used as responder cells (Table 1). Furthermore, the reciprocal experiment gave the same result, in that depletion of CD16+ cells from PBMC resulted in an enhanced MLRinduced proliferation, and the results of two representative experiments are shown in Fig. 2b. The enhancement was often greatest in autologous MLR-induced proliferation. were
CD16+ cells do not affect the allospecific-specific responses of Tcell clones Five different alloreactive clones were used as the responders. 104 cells of each T-cell clone were cultured with optimal numbers (3 x 104) of DRI + irradiated EBV-transformed lymphoblastoid cell lines, with or without CD16+ cells (and UV-irradiated CD 1 6 + cells were used as controls). Figure 3 represents a typical experiment, and Table 2 summarizes the data from several experiments. CD16+ cells did not suppress the allospecific proliferation of the T-cell clones used, even with a CDI 6+: responder ratio of 4: 1. In a further series of experiments using (i) PBMC and L cells transfected with DRl as alternative stimulator cells, and (ii) different ratios of responder to stimulator cells, no suppression was observed (data not shown). Low level proliferation of CD16+ cells was seen in the presence of added IL-2 (data not shown) and also when co-cultured with irradiated T-cell clones and their stimulator cells (data not shown), probably due to the IL-2 generated by the clones, but this was generally small in comparison to the proliferation of T-cell clones. Effect of CD16+ cells on MLR by LFA3+ and LFA3- cells In view of the observed suppression of MLR but not of alloreactive T-cell clones by CD16+ cells, experiments were designed to determine whether NK cells had a differential effect on 'naive' rather than 'memory' T cells. For this purpose, peripheral blood T cells were separated by cell sorting into
LFA3+ ('memory') or LFA3- ('naive') cells for use as responders in the MLR. CD16+ cells were obtained, as before, by flow cytometry on the first day and kept overnight in RPMI supplemented with AB serum at 37" (which preserved NK cytotoxicity; data not shown). T cells and macrophages were isolated from the same donor on the following day, and the T cells were stained with TS/32 (anti-LFA3) and sorted into LFA3+ and LFA3- cells (Fig. 1). MLR cultures were set up using whole PBMC, T cells, LFA3+ and LFA3- cells as responders, and third-party yirradiated PBMC as stimulator cells. CD16+ cells were added in different numbers to MLR cultures, which were pulsed with [3H]thymidine on Day 5 and harvested 16 hr later. CD16+ cells consistently inhibited MLR responses by unseparated T cells and LFA3+ but not by LFA3- cells (Fig. 4, Table 3). CD16+ cells also suppressed an antigen-specific recall response, as measured by the proliferation of autologous T cells in resonse to influenza antigen in the presence of macrophages (Fig. 5). The effect was abrogated by irradiation of CD16+ cells with UV light.
DISCUSSION The experiments reported here were designed to determine, in more detail, the phenotype of the T cells susceptible to regulation by CD16+ NK cells. Previous reports have given conflicting results, which may have been due in part to the use of relatively impure populations of NK cells. Data have been published that rather impure NK populations could inhibit Tcell proliferation in MLR3 and autologous MLR8 in man, and in mice, although some of the latter data suggested the NK effect acted via dendritic cells.'0 A recent report concluded that purified CD1 6+ cells actually enhanced allogeneic T-cell stimu-
lation.9 In the studies reported here, NK cells were prepared by positive selection by FACS, and so all cells are stained with antiCD16. Binding of antibody to NK cells does result in calcium flux and increased phosphoinositol turnover,'5 and may induce transcription of various genes including CD25, IFN-y and TNF.'6 However, anti-CD16 antibodies have no short-term effect on NK cytotoxicity of target cells, which have no Fcy receptors, but enhance cytotoxicity of those bearing Fcy receptors when the anti-CD 16 monoclonal is of the IgG class.'7 The anti-CD 1 6 used in these experiments (anti-Leu- II b) is an IgM antibody and does not enhance cytotoxicity, although it may have an effect on NK immunoregulation since previous studies demonstrated that addition of anti-Leu-l lb to PBMC reduced PWM-induced immunoglobulin synthesis." Enrichment of PBMC, purified T cells or CD16- cells with sorted CD16+ cells resulted in a marked suppression (30-750%) of MLR-induced proliferation. The effect was almost completely abrogated by UV-irradiation of NK cells. This suggests that the observed suppression was not simply due to an effect of additional cells in the cultures. It implies that the UV-irradiation inhibited some function of metabolically active, live cells, the nature of which is undefined. In some experiments, y-irradiated CD16+ cells had an intermediate effect on suppression (data not shown), suggesting that proliferation of CD16+ cells is not essential. It is also notable that the effects reported here were observed when the ratio of CD16+ cells to responding T cells was similar to that present in PBMC and, conversely, that
NK cell inhibition of T-cell responses depletion of CD16+ cells from PBMC enhanced MLC responses. However, CD16+ cells had no effect on the allospecific proliferation of five alloreactive CD4+ clones, stimulated under a variety of conditions, and with a NK: T cell ratio of up to 4: 1. Indeed, in some experiments, NK-enriched cultures showed enhanced proliferation, although some of this was undoubtedly due to the proliferation of the CD 16 + cells in response to the IL2 generated by the T-cell clones, and disappeared following UVirradiation of CD16+ cells. One of the possible reasons why CD16+ cells were able to inhibit an MLR but were unable to inhibit allospecific proliferation by T-cell clones is that CD16+ cells act on 'naive' but not 'memory' T cells, since the MLR seems to be due to proliferation of both types of T cells,'8 '9 while T-cell clones are, by definition, 'memory' cells. In an attempt to address this possibility, the effect of CD16+ cells on MLR by unseparated T cells, LFA3+ and LFA3- cells (representing, respectively, 'memory' and 'naive' populations) was examined. CD16+ cells suppressed MLR responses by LFA3+ (60-90%) but not by LFA3- cells. CD16+ cells also suppressed the proliferative response of peripheral blood T cells to influenza antigen. In vitro responses to recall antigens are dependent on previously primed cells. These results rule out this explanation for the failure of NK cells to inhibit proliferation of T-cell clones. Three other explanations can be offered to account for the differential susceptibility of PBMC versus T-cell clones to NKinduced inhibition. First, it may be that NK cells can only inhibit resting and not activated T cells. Due to the way in which T-cell clones are maintained in vitro, they are permanently in an activated state. In contrast T cells derived from peripheral blood are usually 'resting'. Second, the effector: target ratio may have been too low. Although NK: T ratios of up to 4: 1 were used, this is likely to be far lower than the ratio of NK: responding T cells in a primary MLR. The published frequencies of alloreactive CD4+ T cells stimulated by HLA-mismatched stimulator cells vary between 1:1000 and 1:40,000. The third possible explanation is that in order to exert their suppressive effect CD16+ cells might require another cell type which is present in PBMC but not in the T-cell clone cultures. There is published data suggesting that NK cells mediate their effect by interaction with accessory cells in mice2 and humans.9 The differential susceptibility of LFA3+ versus LFA3- T cells to NK cell-mediated inhibition may be explained by accessory molecular interactions. One of the ligands for LFA3 is CD2 which, of course, is present on virtually all CD16+ NK cells. ' If LFA3 - T cells were not susceptible to regulation by NK cells, it is possible that the NK effect might be mediated via CD2-LFA3 interaction, and so the effect of CD 16 + on mitogenstimulated proliferation of LFA3- cells would be of interest. The effect of a variety of monoclonal antibodies (including antiCD2 and anti-LFA3) on NK-mediated suppression should also be investigated. In conclusion, the series of experiments described here provides clear support for the ability of cells with 'NK' phenotype and activity to exert important immunoregulatory functions. CD16+ cells are able to inhibit alloreactive and antigen-specific responses of LFA3 + cells but not the alloreactive responses of LFA3- cells. The lack of susceptibility of established alloreactive T-cell clones may suggest that once activated, T cells lose susceptibility to NK-mediated inhibition.
449
ACKNOWLEDGMENT We wish to thank Dr Ann Rees for critical comments on the manuscript.
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