Immunology Letters, 28 (1991) 31-38 Elsevier IMLET 01558
Interleukin-2 activated T cells (T-LAK) express CD16 antigen and are triggered to target cell lysis by bispecific antibody Taizo Nitta, M o t o m i N a k a t a , H i d e o Yagita a n d Ko O k u m u r a Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan (Received 10 October 1990; revision received 28 November 1990; accepted 6 December 1990)
Human PBMCs from healthy donors were cultured with 100 U/ml rlL-2 for up to 5 weeks and tested at short and long activation times for the ability to mediate CD3 and CD16 targeted cytotoxicity using chemically cross-linked bispecific antibodies. At each period, LAK activity was augmented with the use of bispecific antibodies (BA), whereas interestingly enough, at later periods ( 4 - 5 weeks) when CD16 positive lymphocytes are not present by flow cytometry, CD16 targeted cytotoxicity was induced. We suspected the possibility of CD16 expression on activated T cells and have purified the T cell subpopulations to see the targeted cytotoxicity. Populatio~as enriched for T cells by Percoll density centrifugation, treatment with anti-CD16 plus complement or sorting for CD5 ÷ cells, were all able to mediate CD16 targeted cytotoxicity following activation with rlL-2. These data suggest that IL-2 activated T cells express CD16 in addition to CD3.
cells with bispecific antibody (BA) could form the basis of a new form of cancer immunotherapy by augmenting cytolytic activity of effector cells against target cells [1, 2]. It was shown that CD3 antigen can serve as a trigger molecule of cytolysis in T cells [3] and also CD16 (Fc'y receptor III) antigen can trigger ADCC in NK cells [4, 5]. In this paper, we explore the targetable cytotoxicity of LAK cells after short and long-term culture. LAK cells have been conventionally generated by stimulating peripheral lymphocytes with IL-2. Such cells gain the ability to lyse NK-resistant and fresh tumor cells in an MHC unrestricted fashion [6]. The actual phenotype of the LAK cell remains to be known, but they are considered to be derived from NK and some populations of T ceils [7- 9]. Phenotypically, some express CD16 or CD3 and a few populations express both. We have then attempted to target LAK ceils with anti-CD3 and anti-CD16 containing BA to study the cellular characterization of short- and long-term LAK cells with BA.
2. Introduction
3. Materials and Methods
1. Summary
Specific targeting of cytotoxic cells against tumor Key words: CD16; cytotoxicity
Lymphokine-activated killer; Targeted
Correspondence to: Taizo Nitta, M.D., Dept. of Neurology and Neurological Science, Stanford University School of Medicine, Stanford CA 94305-5235, U.S.A. Abbreviations." LAK, lyrnphokine-activated killer; LGL, large granular lymphocyte; ADCC, antibody dependent cellular cytotoxicity; PBMCs, peripheral blood mononuclear cells; MHC, major histocompatibility complex; rlL-2, recombinant interleukin-2. 0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V.
Human PBMCs from normal healthy donors were separated by centrifugation on a Ficoll-Hypaque gradient and depleted of monocytes by adherence to plastic to save a purified lymphocyte preparation. PBL were fractionated on a five step discontinuous Percoll gradient (Pharmacia Fine Chem.) as described [10]. LGL were collected from the low density fractions (fraction 1), whereas T cells were found in the higher density bottom fraction. Such LGLenriched preparations contained an average of 9 0 - 9 5 % LGL, which were 85% CD16 +, 5-10070 CD3 ÷ NKH-1 (Leu 19) + T cells. The T-enriched 31
fraction contained < 2°7oLGL and there were > 95°70 CD3 + cells. PBL were sorted into NKH-1 + T cells (CD5 +, Leu 19 +) and NKH-1 T cells (CD5 +, Leu 19-) using a FACstar (Becton-Dickinson). Reanalysis indicated 98°70 purity in both populations. CTL clones, 6D5 (CD3+4 8+16 ) and 1G4 (CD3+4+8-16 - ) were obtained by limiting dilution technique from primary and secondary mixed lymphocyte culture with normal PBMCs and irradiated allogenic B cell line (YM) [11]. The clones were maintained in complete culture medium with 100 U/ml of rIL-2 and irradiated allogenic lymphocytes were added as stimulators and feeder once a week. They do not have any cytotoxic potential against autologous tumor cells. PBMCs were incubated for 45 min at 0°C with 1 #g/106 cells of anti-Leu llb (anti-CD16 mAb; IgM). After washing, rabbit complement (Low Tox-H; Cedarlane, Hornby, Ontario) was added at a final dilution of 1/10 and cells were incubated for 1.5 h at 37 °C. Examination by flow cytometry revealed no contamination by Leu lla + NK cells and their purity was confirmed at each period (day 3 and 7 after culture). Culture medium was RPMI 1640 supplemented with 5°70 pooled AB serum, 5×10 -5 M 2-ME, 100 U/ml penicillin, 50/~g/ml steptomycin (Sigma Co.). This medium will be referred to hereafter as complete medium. Lymphocytes or lymphocyte subsets were cultured at 5× 105 cells/ml in complete medium with 100 U/ml of rIL-2 (Shinogi). K562, an erythroleukemic cell line; P815, a mastocytoma and EL 4, a mouse T cell lymphoma were used for in vitro cytotoxicity assays and it was confirmed that EL 4 did not express any Fc-yRs by FCM using antiFcTR mAbs. All were purchased from the American Type Culture Collection (ATCC). EL 4 was modified with nitrophenyl (NP) groups as described [12]. All anti-Leu 1, anti-Leu 2a, anti-Leu 3a, anti-Leu 4 and anti-Leu 19 recognize CD5, CD8, CD4, CD3 and NKH-1 respectively [8]. Anti-CD16 mAbs were anti-Leu lla (IgG1), anti-Leu llb (IgM), anti-Leu llc (IgG1) and 3G8 (IgG1) [13], which were all kind gifts from J. C. Unkeless. FC2 (IgG2a) was from J. Ledbetter and MG12 (IgG1) from M. Yokoyama [14]. These anti-CD16 mAbs recognize different epitopes of Fc-yRIII, except for anti-Leu lla and anti-Leu llb which recognize the same epitope [15]. OKT3, purchased from ATCC and anti-Leu 4 recognize different epitopes on CD3. Anti-NP mAb (C6-8-2; IgG1) 32
were a generous gift from T. Azuma. Phenotypic analysis of effector lymphocytes was performed as described previously by FACscan (Becton-Dickinson) using fluorescent isothiocyanate (FITC) or phycoerythrin (PE)-conjugated mAbs. For three-color analysis, some mAbs were biotinylated and followed by reaction with streptoavidin Duochrome TM (Becton-Dickinson). Bispecific hetero F(ab')2 fragments were performed using a thiol activating reagent DTNB(5,5" dithiobis-(2-nitrobenzoic acid); Sigma) according to the published procedure [16]. Briefly, F(ab')2 fragments of OKT3 or 3G8 were reduced with 0.5 mM DTT (dithiothreitol) for 30 min at pH 7.5. The reduction was stopped by addition of DTNB at a final concentration of 5 mM. The nitrobenzoic acid derivatives of OKT3 and 3G8 Fab fragments (Fab-SNB) were separated by gel chromatography (TSK 3000 SWXL; TOSO, Japan). F(ab')2 fragments of anti-NP mAb were also reduced to Fab-SH with 0.5 mM DTT, and excess reducing agents were removed by gel filtration. The two were mixed in a 1:1 ratio and incubated for 4 h at 37 °C. Coupled hetero F(ab')2 fragments were purified by gel filtration. These coupled F(ab')2 fragments were found to be pure heterodimers from the target absorption assay [171. Cytotoxicity was measured using a standard 51Cr release assay as described [16]. Antibodies were added simultaneously with effector and target cells. The value represents the mean of triplicate determinations. Standard deviation was within 5°70. 4. Results
PBMCs collected from healthy donors were cultured with 100 U/ml of rIL-2 up to 5 weeks. Their cytotoxicity with and without BA (anti-CD3 × antitarget, anti-CD16 × anti-target) was examined against NP haptenized EL 4 (NP-EL 4). As Fig. 1 clearly shows, LAK activity of PBMCs without any BA was at a maximum during week one and then it declined, increasing gradually thereafter. Killer activity in the presence of BA was higher than LAK activity irrespective of the trigger molecule (CD3 or CD16) at each period. LAK activity against "native" EL 4 and NP-EL 4 was almost the same, but only the cytotoxicity against NP-EL 4 was greatly augmented after LAK cells were precoated with BA (data not
x~x
shown). From this result, we concluded that strong targeted or untargeted cytotoxic activity could be generated by culturing PBL with rIL-2 for one week. LAK cells were also analyzed for the expression of each marker at various periods. As Fig. 2 shows, CD16 expression was high at week one, but decreased at later time periods. Both T (CD3 ÷) and NK (CD16 +) cells expressed NKH-1 (Leu 19) after one week of stimulation with rIL-2, but expressed little of this marker prior to stimulation with rIL-2. Similar results (not shown) were obtained using two other anti-CD16 mAbs (3G8 and FC 2). It has been reported that precursor cells of late-LAK activity are derived from CD3 ÷ T cells [18]. However, our results using BA suggested that early on, both NK and T cells are able to lyse target cells. Paradoxically, this was true of cells targeted through CD16, as well as through CD3, even though CD16 expression was barely detectable at later periods. Targetable cytotoxicity was measured in PBMCs after fractionation on a Percoll density gradient.
none
:
~ Anti-CD3 XAnti-NP Anti-CD16 X A n t i - N P
50
u~
O
x
1
2 culture
3
4
periods
5
of PBMC
Fig. 1. Nonadherent PBMCs from a healthy donor were cultured with 100 U / m l of rIL-2 for 5 weeks. Cytotoxicity was assayed at weekly intervals by a standard 4-h ~ C r release assay with and without BA at a final concentration of 1 #g/ml. Effector to target ratio was 5:1. NP-EL 4 was used as target cells.
Fresh
1 week
2 week
3 week
I
-~1....... .~ ..
..
~- - % ? : . . .
5 week
IJ_ ..~::.~ . •: , ~ , - , . ,:.. •.,,'~.~ ~ -.. , .
i. FL3
FL3
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I J
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....
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h ..,:~;~:...:,,E;:=. :.%;~.Vi ~,~,,'.,~;-,.. • .,~,~:~..,.!;;~! ~..
FL3
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•
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FL1
h
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h
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.
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./:.
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.
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FLI (Leul
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: Anti-NKH-1
FLI (l_eu19)
(OKT3)
Fig. 2. Flow cytometric analysis o f the co-expression o f Leu llc, Leu 19 and OKT3 in nonadherent P B M C s cultured for various times with 100 U / m l of rIL-2. The donor was the same as in Fig. 1. Cells were stained with FITC-coniugated anti-Leu 1lc, PE-conjugated anti-Leu 19 and biotinylated OKT3 followed by streptoavidin Duochrome TM.
33
BA enhanced the lytic activity of both NK-enriched and T-enriched populations. Next, PBMCs were treated with anti-Leu lib and rabbit complement. Flow cytometric analysis was due to rule out contamination by CD16 ÷ NK cells. After stimulation with 100 U/ml o f rlL-2 on days 0, 1, 3, 7, killer activity with and without T cells could be triggered to lyse target cells in the presence of antiCD16x anti-target BA. Fig. 3 shows that on day one, killer activity triggered by anti-CD16 was depressed compared to whole PBMCs, but the activity approached that of whole PBMCs after 3 days. Nonadherent PBMCs from healthy donors were separated by flow cytometry into NKH-1 ÷ T cells (CD5 ÷ , Leu 19 ÷ , 7o7o) and NKH-1 - T cells (CD5 ÷ , Leu 19-, 50°7o). Each fraction was cultured with 100 U/ml r I ~ 2 for 7 days. Anti-CD5 was used to isolate T cells, since anti-CD3 has been shown to either inhibit or induce T cell-mediated cytotoxicity. These fractions were tested for n o n - M H C restricted cytotoxicity against K562 and NP-EL 4 cells and then tested for BA-dependent cytotoxicity against NP-EL 4. Table 2 shows that activity of CD5 ÷ NKH-1 ÷ and N K H - 1 - cells was much less than that of whole PBMCs. However, cytolysis was greatly enhanced in both CD5 ÷ subsets by not only anti-
PBMCs from healthy donors were fractionated on a 5-step discontinuous Percoll gradient and cultured with 100 U/ml rlL-2 for 7 days. Table 1 shows that at one week most LAK activity against NP-EL 4 was in the low density, LGL fraction. Enriched T cells (high buoyant density) had less cytotoxic activity than the LGL. In the presence of anti-CD3 x antitarget BA, most of the cytotoxicity was in the Tenriched populations, while anti-CD16x anti-target TABLE 1 Killer activity of subpopulations of Percoll-separated lymphocytes. Cell type a
PBL LGL T
Cells staining positively
on day 3 (°70)
L A K activity (LU/107 cells) and triggered with: b
CD3
CD16
(-)
anti-CD3
anti-CD16
64 7 95
15 85 1
101 300 75
415 344 850
433 790 275
aLGL are low buoyant cells isolated as Percoll, the T ceils represent the high buoyant density fraction, bCytotoxicity using NPEL 4 as target cells and either no antibody, anti-CD3 x anti-NP or a n t i - C D 1 6 x anti-NP.
o
o non-
:-
=
treated
treated with A n U - L e u - 1 1 b and complement
¢J
r,,)
50-
50-
no Ab
Anti-Target
0
1
3
7
0
i
i
1
1
3
7
nti-Target
culture periods (days) Effector : Target=10 : 1 Fig. 3. PBMCs were depleted of NK cells with anti-CD16 mAb (anti-Leu lib) plus complement and cultured with 100 U/ml of rlL-2 for 7 days. At 0, 1, 3, 7 days, cytotoxicityagainst NP-EL 4 was assayedwith and without BA at a final concentrationof 1 /zg/ml. Effector to target ratio was 10.'1. 34
TABLE 2 Inductionof bispecificantibody dependent cytolysisin NKH-I + and in NKH-I- T cells. Lytic a c t i v i t y a g a i n s t t a r g e t cells b
E f f e c t o r cells a
K562
NP-EL 4
(-)
PBL CD5 ÷ NKH-1 ÷ CD5 ÷ NKH-I -
97.0 18.2 7.7
anti-NP
OKT8x anti-NPc
71.8 41.8 31.3
68.6 53.7 62.1
3G8x
44.5 12.9 7.7
apBL from a healthy donor were sorted into CD5 + (Leu-1), NKH-1 ÷ and CD5 + NKH-1 - fractions by FACS and were cultured for 7 days with 100 U/ml of rlL-2, bLytic activity was expressed as percent of 4-h 51Cr release assay. CBispecificantibody was added to the effector ceils30 min before addition of radiolabeled target cells at a final concentration of 1 /~g/ml.
CD3 x anti-target b u t also anti-CD16 x anti-target. F r o m this result, it was suspected that T cellm e d i a t e d n o n - M H C restricted cytotoxicity resides
m a i n l y in the NKH-1 + subset, b u t a n t i - C D 3 a n d anti-CD16 triggered activity were mediated by b o t h p o p u l a t i o n s . There was some discrepancy o f targeted T cell cytotoxicity against N P - E L 4 by the T cell p u r i f i c a t i o n procedure. Two h u m a n C T L clones 6D5 ( C D 3 + 4 - 8 + 1 6 - ) a n d 1G4 ( C D 3 + 4 + 8 - 1 6 - ) were tested with BA dep e n d e n t cytotoxicity against N P - E L 4 target cells. Fig. 4 shows that either clone by itself was u n a b l e to lyse N P - E L 4 target cells a n d neither clone mediated A D C C when target cells were precoated with antiN P mAb. However, lysis was i n d u c e d by antiCD16 x a n t i - N P a n d a n t i - C D 3 x anti-NP. It is conceivable that the C T L clones might co-express CD3 a n d CD16 antigens, b u t CD16 expression was so faint that it could n o t be detected by flow cytometry. CD16 a n t i g e n expression in C T L clones were further investigated by b l o c k i n g studies. I n the experiment s h o w n in Fig. 5, 6D5 cells were p r e i n c u b a t e d with a n t i - C D 3 F ( a b ' ) 2 fragments. Several anti-CD16 m A b s were able to redirect cytolysis o f 6D5 against % specific lysis 50
)
0D16 PERCENT SPECIFIC LYSIS
6D5
50
Ab (-I 3G8 F(ab')2 3G8 XAnti-NP
Ab(_
7/'///////////////////////////]
+ 3 G 8 F(ab')2 h 3G8 XAnti-NP +OKT3 F(ab )2
3G8 XAnti-N~ IlllllllJlllll]rllil]rllil/rllll[FIIiitrllllrllJrlpJ[ipl
I CD3 I Effector
to Target
1G4
ratio=2
' I
5O i
/
Ab (-I OKT3 F(ab')2
Effector to Target ratio = 5 50 '
D
E
I~
0 K T 3 X A nt i - N P ~ ' / ' / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / ~ 1
A b (-) + 3 G 8 F(ab )2 X Anti'-NP
OKT3
A n t i - N P mAb
•
lllilllllllllllllllJlJllljlllillllllllllll[lllllillll[lllilllillllilllll
I Effector
: Human CTL
Anti-CD1fXAnti-NP
Target
; NP-EL4
TARGET NP-EL4
Bispecific Abs : OKT3 XAnti-NP; F(ab')2 3 G 8 X A n t i - N P ; F(ab')2
I Fig. 4. Two human CTL clones, 6D5 (CD3+4-8 + 16-) and 1G4 (CD3+4+8-16 -) were assayed for ADCC activity and BAdependent cytotoxicity against NP-EL 4. Anti-NP mAb (C6- 82) and BA (OKT3x anti-NP, 3G8 x anti-NP) were added at a final concentration of 1/~g/ml. Effector to target ratio was 2:1.
6D5
Fig. 5. Inhibition of BA dependent cytolysis against/~P-EL 4, was tested by pretreating 6D5 cells with 10/~g/ml of anti-CD3 (OKT3) F(ab')2 or anti-CD16 (3G8) F(ab')2 for 30 min before adding 1/xg/ml BA and NP-EL 4 target cells. Effector to target ratio was 5:1. 35
_~
FC 2
o/'-'
[]
m
X~ I
x I
x ~ X ~
Ab{-) J
1 2 5 10 EFFECTOR TO TARGET RATIO EFFECTOR TARGET rn Abs
6 D 5 (CD 3+ 4 - 8+ 16-) NP-EL 4 ANTi-CD 3 OKT3 ANTI-CD16 3G8, FC 2, MG12
Fig. 6. Redirected cytolysis of 6D5 by using anti-CD3 mAb (OKT3) and anti-CD16 mAbs (3G8, FC2, MG12) was examined against Fc'rR bearing P815 mastocytoma. Each mAb was added at a final concentration of 1 tzg/ml.
P815, a murine target cell expressing Fc-yR but not the CD16 epitope found in human Fc3,RIII. Fig. 6 shows that 3G8 and FC2 could render 6D5 cytolytic against P815 while MG12 could not. 5. Discussion
While discussions so far focus only upon how many effector LAK cells would be required for adoptive immunotherapy, a current issue is to obtain more cytotoxic killer cells on a per cell basis. Many attempts have been done to increase the efficiency of effector cells and among them, specific targeting of effector LAK cells using BA may be one of the promising approaches. With these effector LAK cells coated with BA as one would expect, favorable clinical results have been obtained against malignant gliomas [19]. In this paper, we explored the diversity of targeted cytotoxicity in regard to culture periods of LAK cells with rIL-2. To explore killer activity and precursor ceils, we simply divided the culture period into short-term LAK (early LAK; until one week) and long-term LAK (late LAK; over 2 weeks). Few papers have been published in regard to the effector cell populations and cytolytic activity of long36
term LAK. Some studies have shown that LAK activity is maximal at 5 - 1 0 days and then decreases weekly [20]. Also in this study~ optimal killing was apparent at the first week after it declined; it gradually recovered 3 weeks later. BA dependent cytolytic activity, regardless of the trigger molecules (CD3 or CD16), showed maximum activity at one week. Therefore, we think that LAK cells cultured with rIL-2 for one week would be suitable for specific targeting therapy. In the earlier periods, both T and NK cells were triggered by BA containing relevant mAb and both populations were detectable by flow cytometry. However, in the late stage of culture, although CD16 + cells were scarcely detected by flow cytometry using various mAbs, BA containing anti-CD16 could render them cytotoxic against target cells. This result may raise some questions about the phenotype of effector cells targeted by anti-CD16 mAb. As the other report shows, NK cells have gradually lost expression of Leu llc after they were stimulated with rIL-2 for more than one week [8]. In this study, we explored whether CD16 antigen epitopes expressed on late LAK cells may be different from Leu llc or not, by using other anti-CD16 mAbs. These mAbs (FC 2, 3G8, MG12) other than anti-keu llc, failed to react with late LAK effector cells (data not shown). On the other hand, a subpopulation of normal CD3 + T cells, bearing Fc3,R (CD16), which lacks CD4 and CD8, but shows NK-like, non-MHC restricted cytotoxicity might be expanded for longer periods [5, 21]. But from Fig. 2, no CD3+16 + T cells were shown in fresh PBL and late LAK cells. These findings encouraged us to suspect that T-LAK cells cultured for a long time may coexpress CD3 and CD16, which is undetectable by flow cytometry. This was confirmed by using various T cell purification methods. PBMCs depleted of CD16 + NK cells by Cmediated lysis demonstrate depressed anti-CD16 triggered cytolysis on the first day, but 3 days later, show killer activity equal to that of whole lymphocytes. This supports the result that anti-CD16 triggered cytolysis was induced from Percoll-separated cells (high buoyant cells) and FACS sorted cells (CD5 +). In contrast to the assumption that T-LAK cells derived from NKH-1 + T cells (LGL-T), antiCD3 triggered cytolysis was induced from "pan T cells" from our data, too [11]. With one week of stimulation with rIL-2, T cells express CD16, where-
as NK-depleted PBLs with b r i e f exposure to IL-2 for 24 h, showed d i m i n i s h e d lytic activity triggered by anti-CD16. However, a 3-day s t i m u l a t i o n or more with rIL-2 would be required for T cells to express CD16. Recent reports showed that C D 4 + T cells m a y be infected, n o t t h r o u g h CD4 protein b u t t h r o u g h any Fc3,R expressed o n T cells [22]. The expression of CD16 o n activated T cells was also clarified from the study of C T L clones. I n this study, BA (anti-CD16 × anti-target) d e p e n d e n t cytolysis was i n d u c e d b u t showed n o A D C C activity. We consider the i n t e r a c t i o n o f the anti-target m A b ' s Fc d o m a i n with the Fc3,R o f effector cells is p r o b a b l y less t h a n the i n t e r a c t i o n o f anti-CD16 m A b with Fc3'R in terms o f i n d u c i n g the signal for cytolytic activity in effector cells [1]. Otherwise, sideway A D C C [4] which differs from classical A D C C using polyclonal antisera, m a y n o t be i n d u c e d by IgG~ isotype mAb. A m o n g the same anti-CD16 m A b s o f the same class (3G8 a n d MG12; IgGl), redirected cytolysis by a n t i b o d y was different (Fig. 6). T h e epitope o n CD16 which MG12 recognizes m a y be different from the epitope reactive with 3G8 [14]. By the way, the expression o f the CD16 molecule o n activated T cells m a y throw some insight into the T cell o n t o g e n y as well as p r o m o t e specific targeting therapy.
References [1] Karpovsky, B., Titus, J.A., Stephany, D.A. and Segal, D. M. (1984) J. Exp. Med. 160, 1686. [2] Perez, P., Hoffman, R. W., Titus, J. A. and Segal, D. M. (1986) J. Immunol. 139, 3153. [3] Leeuwenberg,J. E M., Spits, H., Tax, W. J. M. and Capel,
P. J. A. (1985) J. Immunol. 134, 3770. [4] Unkeless, J. C., Scigliano, E. and Freedom, V. H. (1988) Annu. Rev. Immunol. 6, 251. [5] Van de Griend, R. J., Bolhuis, R. L. H., Stoter, R. L. G. and Roozenmond, R. C. (1987) J. Immunol. 138, 3137. [6] Ortaldo, J. R., Mason, A. and Overton, R. (1986) J. Exp. Med. 164, 1193. [7] Philips, J. H. and Lanier, L. L. (1986) J. Exp. Med. 164, 814. [8] Lanier, L. L., Le, A. M., Civin, C. I., Loken, M. R. and Philips, J. H. (1986) J. Immunol. 136, 4480. [9] ltoh, K., Tiden, A. B., Kumagai, K. and Balch, C. M. (1985) J. Immunol. 134, 802. [10] Timonen, T. and Saksela, E. (1980) J. Immunol. Methods 36, 285. [ll] Burns, G. F., Triglia, T. and Wermeister, J. A. (1984) J. Immunol. 133, 1656. [12] Weinberger, J. Z., Germain, R. N., Benacerraf, B. J. and Dorf, M. F. (1980) J. Exp. Med. 152, 161. [13] Fleit, H. B., Wright, S. D. and Unkeless, J. C. (1982) Proc. Natl. Acad. Sci. USA 79, 3275. [14] Tettero, P. A. T., Van der Schoot, C. E., Visser, E J., Bos, M. J. E. and von dem Borne, A. E. G. (1987)Leukocytetyping III, p. 702. Oxford University Press, Oxford. [15] Perrusia, B., Trinchieri, G., Jackson, A., Warner, N. L., Faust, J., Rumpold, H., Kraft, D. and Lanier, L. L. (1984) J. Immunol. 133, 180. [16] Nitta, T., Yagita,H., Azuma, T. and Okumura, K. (1989)Eur. J. Immunol. 19, 1437. [17] Nitta, T., Sato, K., Okumura, K. and Ishii, S. (1990) J. Neurosurg. 72, 476. [18] Sawada, H., Abo, T., Sugamura, S. and Kumagai, K. (1988) J. Immunol. 140, 3688. [19] Nitta, T., Sato, K., Yagita, H., Okumura, K. and Ishii, S. (1990) Lancet 335, 368. [20] Ochoa, A. C., Gromo, G., Alter, B. J., Sondel, P. M. and Bach, E H. (1987) J. Immunol. 138, 2728. [21l Lanier, L. L., Ruitenberg, J. J. and Philips, J. H. (1986) J. Exp. Med. 164, 339. [22] Homsy, J., Meyer, M., Tateno, M., Clarkson, S. and Levy, J. A. (1989) Science 244, 1357.
37
Interleukin-2 activated T cells (T-LAK) express CD16 antigen and are triggered to target cell lysis by bispecific antibody Taizo Nitta, M o t o m i N a k a t a , H i d e o Yagita a n d Ko O k u m u r a Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan (Received 10 October 1990; revision received 28 November 1990; accepted 6 December 1990)
Human PBMCs from healthy donors were cultured with 100 U/ml rlL-2 for up to 5 weeks and tested at short and long activation times for the ability to mediate CD3 and CD16 targeted cytotoxicity using chemically cross-linked bispecific antibodies. At each period, LAK activity was augmented with the use of bispecific antibodies (BA), whereas interestingly enough, at later periods ( 4 - 5 weeks) when CD16 positive lymphocytes are not present by flow cytometry, CD16 targeted cytotoxicity was induced. We suspected the possibility of CD16 expression on activated T cells and have purified the T cell subpopulations to see the targeted cytotoxicity. Populatio~as enriched for T cells by Percoll density centrifugation, treatment with anti-CD16 plus complement or sorting for CD5 ÷ cells, were all able to mediate CD16 targeted cytotoxicity following activation with rlL-2. These data suggest that IL-2 activated T cells express CD16 in addition to CD3.
cells with bispecific antibody (BA) could form the basis of a new form of cancer immunotherapy by augmenting cytolytic activity of effector cells against target cells [1, 2]. It was shown that CD3 antigen can serve as a trigger molecule of cytolysis in T cells [3] and also CD16 (Fc'y receptor III) antigen can trigger ADCC in NK cells [4, 5]. In this paper, we explore the targetable cytotoxicity of LAK cells after short and long-term culture. LAK cells have been conventionally generated by stimulating peripheral lymphocytes with IL-2. Such cells gain the ability to lyse NK-resistant and fresh tumor cells in an MHC unrestricted fashion [6]. The actual phenotype of the LAK cell remains to be known, but they are considered to be derived from NK and some populations of T ceils [7- 9]. Phenotypically, some express CD16 or CD3 and a few populations express both. We have then attempted to target LAK ceils with anti-CD3 and anti-CD16 containing BA to study the cellular characterization of short- and long-term LAK cells with BA.
2. Introduction
3. Materials and Methods
1. Summary
Specific targeting of cytotoxic cells against tumor Key words: CD16; cytotoxicity
Lymphokine-activated killer; Targeted
Correspondence to: Taizo Nitta, M.D., Dept. of Neurology and Neurological Science, Stanford University School of Medicine, Stanford CA 94305-5235, U.S.A. Abbreviations." LAK, lyrnphokine-activated killer; LGL, large granular lymphocyte; ADCC, antibody dependent cellular cytotoxicity; PBMCs, peripheral blood mononuclear cells; MHC, major histocompatibility complex; rlL-2, recombinant interleukin-2. 0165-2478 / 91 / $ 3.50 © 1991 Elsevier Science Publishers B.V.
Human PBMCs from normal healthy donors were separated by centrifugation on a Ficoll-Hypaque gradient and depleted of monocytes by adherence to plastic to save a purified lymphocyte preparation. PBL were fractionated on a five step discontinuous Percoll gradient (Pharmacia Fine Chem.) as described [10]. LGL were collected from the low density fractions (fraction 1), whereas T cells were found in the higher density bottom fraction. Such LGLenriched preparations contained an average of 9 0 - 9 5 % LGL, which were 85% CD16 +, 5-10070 CD3 ÷ NKH-1 (Leu 19) + T cells. The T-enriched 31
fraction contained < 2°7oLGL and there were > 95°70 CD3 + cells. PBL were sorted into NKH-1 + T cells (CD5 +, Leu 19 +) and NKH-1 T cells (CD5 +, Leu 19-) using a FACstar (Becton-Dickinson). Reanalysis indicated 98°70 purity in both populations. CTL clones, 6D5 (CD3+4 8+16 ) and 1G4 (CD3+4+8-16 - ) were obtained by limiting dilution technique from primary and secondary mixed lymphocyte culture with normal PBMCs and irradiated allogenic B cell line (YM) [11]. The clones were maintained in complete culture medium with 100 U/ml of rIL-2 and irradiated allogenic lymphocytes were added as stimulators and feeder once a week. They do not have any cytotoxic potential against autologous tumor cells. PBMCs were incubated for 45 min at 0°C with 1 #g/106 cells of anti-Leu llb (anti-CD16 mAb; IgM). After washing, rabbit complement (Low Tox-H; Cedarlane, Hornby, Ontario) was added at a final dilution of 1/10 and cells were incubated for 1.5 h at 37 °C. Examination by flow cytometry revealed no contamination by Leu lla + NK cells and their purity was confirmed at each period (day 3 and 7 after culture). Culture medium was RPMI 1640 supplemented with 5°70 pooled AB serum, 5×10 -5 M 2-ME, 100 U/ml penicillin, 50/~g/ml steptomycin (Sigma Co.). This medium will be referred to hereafter as complete medium. Lymphocytes or lymphocyte subsets were cultured at 5× 105 cells/ml in complete medium with 100 U/ml of rIL-2 (Shinogi). K562, an erythroleukemic cell line; P815, a mastocytoma and EL 4, a mouse T cell lymphoma were used for in vitro cytotoxicity assays and it was confirmed that EL 4 did not express any Fc-yRs by FCM using antiFcTR mAbs. All were purchased from the American Type Culture Collection (ATCC). EL 4 was modified with nitrophenyl (NP) groups as described [12]. All anti-Leu 1, anti-Leu 2a, anti-Leu 3a, anti-Leu 4 and anti-Leu 19 recognize CD5, CD8, CD4, CD3 and NKH-1 respectively [8]. Anti-CD16 mAbs were anti-Leu lla (IgG1), anti-Leu llb (IgM), anti-Leu llc (IgG1) and 3G8 (IgG1) [13], which were all kind gifts from J. C. Unkeless. FC2 (IgG2a) was from J. Ledbetter and MG12 (IgG1) from M. Yokoyama [14]. These anti-CD16 mAbs recognize different epitopes of Fc-yRIII, except for anti-Leu lla and anti-Leu llb which recognize the same epitope [15]. OKT3, purchased from ATCC and anti-Leu 4 recognize different epitopes on CD3. Anti-NP mAb (C6-8-2; IgG1) 32
were a generous gift from T. Azuma. Phenotypic analysis of effector lymphocytes was performed as described previously by FACscan (Becton-Dickinson) using fluorescent isothiocyanate (FITC) or phycoerythrin (PE)-conjugated mAbs. For three-color analysis, some mAbs were biotinylated and followed by reaction with streptoavidin Duochrome TM (Becton-Dickinson). Bispecific hetero F(ab')2 fragments were performed using a thiol activating reagent DTNB(5,5" dithiobis-(2-nitrobenzoic acid); Sigma) according to the published procedure [16]. Briefly, F(ab')2 fragments of OKT3 or 3G8 were reduced with 0.5 mM DTT (dithiothreitol) for 30 min at pH 7.5. The reduction was stopped by addition of DTNB at a final concentration of 5 mM. The nitrobenzoic acid derivatives of OKT3 and 3G8 Fab fragments (Fab-SNB) were separated by gel chromatography (TSK 3000 SWXL; TOSO, Japan). F(ab')2 fragments of anti-NP mAb were also reduced to Fab-SH with 0.5 mM DTT, and excess reducing agents were removed by gel filtration. The two were mixed in a 1:1 ratio and incubated for 4 h at 37 °C. Coupled hetero F(ab')2 fragments were purified by gel filtration. These coupled F(ab')2 fragments were found to be pure heterodimers from the target absorption assay [171. Cytotoxicity was measured using a standard 51Cr release assay as described [16]. Antibodies were added simultaneously with effector and target cells. The value represents the mean of triplicate determinations. Standard deviation was within 5°70. 4. Results
PBMCs collected from healthy donors were cultured with 100 U/ml of rIL-2 up to 5 weeks. Their cytotoxicity with and without BA (anti-CD3 × antitarget, anti-CD16 × anti-target) was examined against NP haptenized EL 4 (NP-EL 4). As Fig. 1 clearly shows, LAK activity of PBMCs without any BA was at a maximum during week one and then it declined, increasing gradually thereafter. Killer activity in the presence of BA was higher than LAK activity irrespective of the trigger molecule (CD3 or CD16) at each period. LAK activity against "native" EL 4 and NP-EL 4 was almost the same, but only the cytotoxicity against NP-EL 4 was greatly augmented after LAK cells were precoated with BA (data not
x~x
shown). From this result, we concluded that strong targeted or untargeted cytotoxic activity could be generated by culturing PBL with rIL-2 for one week. LAK cells were also analyzed for the expression of each marker at various periods. As Fig. 2 shows, CD16 expression was high at week one, but decreased at later time periods. Both T (CD3 ÷) and NK (CD16 +) cells expressed NKH-1 (Leu 19) after one week of stimulation with rIL-2, but expressed little of this marker prior to stimulation with rIL-2. Similar results (not shown) were obtained using two other anti-CD16 mAbs (3G8 and FC 2). It has been reported that precursor cells of late-LAK activity are derived from CD3 ÷ T cells [18]. However, our results using BA suggested that early on, both NK and T cells are able to lyse target cells. Paradoxically, this was true of cells targeted through CD16, as well as through CD3, even though CD16 expression was barely detectable at later periods. Targetable cytotoxicity was measured in PBMCs after fractionation on a Percoll density gradient.
none
:
~ Anti-CD3 XAnti-NP Anti-CD16 X A n t i - N P
50
u~
O
x
1
2 culture
3
4
periods
5
of PBMC
Fig. 1. Nonadherent PBMCs from a healthy donor were cultured with 100 U / m l of rIL-2 for 5 weeks. Cytotoxicity was assayed at weekly intervals by a standard 4-h ~ C r release assay with and without BA at a final concentration of 1 #g/ml. Effector to target ratio was 5:1. NP-EL 4 was used as target cells.
Fresh
1 week
2 week
3 week
I
-~1....... .~ ..
..
~- - % ? : . . .
5 week
IJ_ ..~::.~ . •: , ~ , - , . ,:.. •.,,'~.~ ~ -.. , .
i. FL3
FL3
FL3
FL3
FL3
I J
.:~;.......
....
~ f;~. ,;~..
h ..,:~;~:...:,,E;:=. :.%;~.Vi ~,~,,'.,~;-,.. • .,~,~:~..,.!;;~! ~..
FL3
FL3
."i "..
•
~ . ~ .~'":'4/"
FL1
h
..=,.
~.~,.;:.:~:;.
h
:Anti-CD16
FL3
:Anti-CD3
.
&":!:' i
.FE3
FL3
J
./:.
lc)
.
.
LL .:,~:,!'...
FLI (Leul
C~
' ::..
FL3
FL1 FLI
[L
FLI FL2
: Anti-NKH-1
FLI (l_eu19)
(OKT3)
Fig. 2. Flow cytometric analysis o f the co-expression o f Leu llc, Leu 19 and OKT3 in nonadherent P B M C s cultured for various times with 100 U / m l of rIL-2. The donor was the same as in Fig. 1. Cells were stained with FITC-coniugated anti-Leu 1lc, PE-conjugated anti-Leu 19 and biotinylated OKT3 followed by streptoavidin Duochrome TM.
33
BA enhanced the lytic activity of both NK-enriched and T-enriched populations. Next, PBMCs were treated with anti-Leu lib and rabbit complement. Flow cytometric analysis was due to rule out contamination by CD16 ÷ NK cells. After stimulation with 100 U/ml o f rlL-2 on days 0, 1, 3, 7, killer activity with and without T cells could be triggered to lyse target cells in the presence of antiCD16x anti-target BA. Fig. 3 shows that on day one, killer activity triggered by anti-CD16 was depressed compared to whole PBMCs, but the activity approached that of whole PBMCs after 3 days. Nonadherent PBMCs from healthy donors were separated by flow cytometry into NKH-1 ÷ T cells (CD5 ÷ , Leu 19 ÷ , 7o7o) and NKH-1 - T cells (CD5 ÷ , Leu 19-, 50°7o). Each fraction was cultured with 100 U/ml r I ~ 2 for 7 days. Anti-CD5 was used to isolate T cells, since anti-CD3 has been shown to either inhibit or induce T cell-mediated cytotoxicity. These fractions were tested for n o n - M H C restricted cytotoxicity against K562 and NP-EL 4 cells and then tested for BA-dependent cytotoxicity against NP-EL 4. Table 2 shows that activity of CD5 ÷ NKH-1 ÷ and N K H - 1 - cells was much less than that of whole PBMCs. However, cytolysis was greatly enhanced in both CD5 ÷ subsets by not only anti-
PBMCs from healthy donors were fractionated on a 5-step discontinuous Percoll gradient and cultured with 100 U/ml rlL-2 for 7 days. Table 1 shows that at one week most LAK activity against NP-EL 4 was in the low density, LGL fraction. Enriched T cells (high buoyant density) had less cytotoxic activity than the LGL. In the presence of anti-CD3 x antitarget BA, most of the cytotoxicity was in the Tenriched populations, while anti-CD16x anti-target TABLE 1 Killer activity of subpopulations of Percoll-separated lymphocytes. Cell type a
PBL LGL T
Cells staining positively
on day 3 (°70)
L A K activity (LU/107 cells) and triggered with: b
CD3
CD16
(-)
anti-CD3
anti-CD16
64 7 95
15 85 1
101 300 75
415 344 850
433 790 275
aLGL are low buoyant cells isolated as Percoll, the T ceils represent the high buoyant density fraction, bCytotoxicity using NPEL 4 as target cells and either no antibody, anti-CD3 x anti-NP or a n t i - C D 1 6 x anti-NP.
o
o non-
:-
=
treated
treated with A n U - L e u - 1 1 b and complement
¢J
r,,)
50-
50-
no Ab
Anti-Target
0
1
3
7
0
i
i
1
1
3
7
nti-Target
culture periods (days) Effector : Target=10 : 1 Fig. 3. PBMCs were depleted of NK cells with anti-CD16 mAb (anti-Leu lib) plus complement and cultured with 100 U/ml of rlL-2 for 7 days. At 0, 1, 3, 7 days, cytotoxicityagainst NP-EL 4 was assayedwith and without BA at a final concentrationof 1 /zg/ml. Effector to target ratio was 10.'1. 34
TABLE 2 Inductionof bispecificantibody dependent cytolysisin NKH-I + and in NKH-I- T cells. Lytic a c t i v i t y a g a i n s t t a r g e t cells b
E f f e c t o r cells a
K562
NP-EL 4
(-)
PBL CD5 ÷ NKH-1 ÷ CD5 ÷ NKH-I -
97.0 18.2 7.7
anti-NP
OKT8x anti-NPc
71.8 41.8 31.3
68.6 53.7 62.1
3G8x
44.5 12.9 7.7
apBL from a healthy donor were sorted into CD5 + (Leu-1), NKH-1 ÷ and CD5 + NKH-1 - fractions by FACS and were cultured for 7 days with 100 U/ml of rlL-2, bLytic activity was expressed as percent of 4-h 51Cr release assay. CBispecificantibody was added to the effector ceils30 min before addition of radiolabeled target cells at a final concentration of 1 /~g/ml.
CD3 x anti-target b u t also anti-CD16 x anti-target. F r o m this result, it was suspected that T cellm e d i a t e d n o n - M H C restricted cytotoxicity resides
m a i n l y in the NKH-1 + subset, b u t a n t i - C D 3 a n d anti-CD16 triggered activity were mediated by b o t h p o p u l a t i o n s . There was some discrepancy o f targeted T cell cytotoxicity against N P - E L 4 by the T cell p u r i f i c a t i o n procedure. Two h u m a n C T L clones 6D5 ( C D 3 + 4 - 8 + 1 6 - ) a n d 1G4 ( C D 3 + 4 + 8 - 1 6 - ) were tested with BA dep e n d e n t cytotoxicity against N P - E L 4 target cells. Fig. 4 shows that either clone by itself was u n a b l e to lyse N P - E L 4 target cells a n d neither clone mediated A D C C when target cells were precoated with antiN P mAb. However, lysis was i n d u c e d by antiCD16 x a n t i - N P a n d a n t i - C D 3 x anti-NP. It is conceivable that the C T L clones might co-express CD3 a n d CD16 antigens, b u t CD16 expression was so faint that it could n o t be detected by flow cytometry. CD16 a n t i g e n expression in C T L clones were further investigated by b l o c k i n g studies. I n the experiment s h o w n in Fig. 5, 6D5 cells were p r e i n c u b a t e d with a n t i - C D 3 F ( a b ' ) 2 fragments. Several anti-CD16 m A b s were able to redirect cytolysis o f 6D5 against % specific lysis 50
)
0D16 PERCENT SPECIFIC LYSIS
6D5
50
Ab (-I 3G8 F(ab')2 3G8 XAnti-NP
Ab(_
7/'///////////////////////////]
+ 3 G 8 F(ab')2 h 3G8 XAnti-NP +OKT3 F(ab )2
3G8 XAnti-N~ IlllllllJlllll]rllil]rllil/rllll[FIIiitrllllrllJrlpJ[ipl
I CD3 I Effector
to Target
1G4
ratio=2
' I
5O i
/
Ab (-I OKT3 F(ab')2
Effector to Target ratio = 5 50 '
D
E
I~
0 K T 3 X A nt i - N P ~ ' / ' / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / / ~ 1
A b (-) + 3 G 8 F(ab )2 X Anti'-NP
OKT3
A n t i - N P mAb
•
lllilllllllllllllllJlJllljlllillllllllllll[lllllillll[lllilllillllilllll
I Effector
: Human CTL
Anti-CD1fXAnti-NP
Target
; NP-EL4
TARGET NP-EL4
Bispecific Abs : OKT3 XAnti-NP; F(ab')2 3 G 8 X A n t i - N P ; F(ab')2
I Fig. 4. Two human CTL clones, 6D5 (CD3+4-8 + 16-) and 1G4 (CD3+4+8-16 -) were assayed for ADCC activity and BAdependent cytotoxicity against NP-EL 4. Anti-NP mAb (C6- 82) and BA (OKT3x anti-NP, 3G8 x anti-NP) were added at a final concentration of 1/~g/ml. Effector to target ratio was 2:1.
6D5
Fig. 5. Inhibition of BA dependent cytolysis against/~P-EL 4, was tested by pretreating 6D5 cells with 10/~g/ml of anti-CD3 (OKT3) F(ab')2 or anti-CD16 (3G8) F(ab')2 for 30 min before adding 1/xg/ml BA and NP-EL 4 target cells. Effector to target ratio was 5:1. 35
_~
FC 2
o/'-'
[]
m
X~ I
x I
x ~ X ~
Ab{-) J
1 2 5 10 EFFECTOR TO TARGET RATIO EFFECTOR TARGET rn Abs
6 D 5 (CD 3+ 4 - 8+ 16-) NP-EL 4 ANTi-CD 3 OKT3 ANTI-CD16 3G8, FC 2, MG12
Fig. 6. Redirected cytolysis of 6D5 by using anti-CD3 mAb (OKT3) and anti-CD16 mAbs (3G8, FC2, MG12) was examined against Fc'rR bearing P815 mastocytoma. Each mAb was added at a final concentration of 1 tzg/ml.
P815, a murine target cell expressing Fc-yR but not the CD16 epitope found in human Fc3,RIII. Fig. 6 shows that 3G8 and FC2 could render 6D5 cytolytic against P815 while MG12 could not. 5. Discussion
While discussions so far focus only upon how many effector LAK cells would be required for adoptive immunotherapy, a current issue is to obtain more cytotoxic killer cells on a per cell basis. Many attempts have been done to increase the efficiency of effector cells and among them, specific targeting of effector LAK cells using BA may be one of the promising approaches. With these effector LAK cells coated with BA as one would expect, favorable clinical results have been obtained against malignant gliomas [19]. In this paper, we explored the diversity of targeted cytotoxicity in regard to culture periods of LAK cells with rIL-2. To explore killer activity and precursor ceils, we simply divided the culture period into short-term LAK (early LAK; until one week) and long-term LAK (late LAK; over 2 weeks). Few papers have been published in regard to the effector cell populations and cytolytic activity of long36
term LAK. Some studies have shown that LAK activity is maximal at 5 - 1 0 days and then decreases weekly [20]. Also in this study~ optimal killing was apparent at the first week after it declined; it gradually recovered 3 weeks later. BA dependent cytolytic activity, regardless of the trigger molecules (CD3 or CD16), showed maximum activity at one week. Therefore, we think that LAK cells cultured with rIL-2 for one week would be suitable for specific targeting therapy. In the earlier periods, both T and NK cells were triggered by BA containing relevant mAb and both populations were detectable by flow cytometry. However, in the late stage of culture, although CD16 + cells were scarcely detected by flow cytometry using various mAbs, BA containing anti-CD16 could render them cytotoxic against target cells. This result may raise some questions about the phenotype of effector cells targeted by anti-CD16 mAb. As the other report shows, NK cells have gradually lost expression of Leu llc after they were stimulated with rIL-2 for more than one week [8]. In this study, we explored whether CD16 antigen epitopes expressed on late LAK cells may be different from Leu llc or not, by using other anti-CD16 mAbs. These mAbs (FC 2, 3G8, MG12) other than anti-keu llc, failed to react with late LAK effector cells (data not shown). On the other hand, a subpopulation of normal CD3 + T cells, bearing Fc3,R (CD16), which lacks CD4 and CD8, but shows NK-like, non-MHC restricted cytotoxicity might be expanded for longer periods [5, 21]. But from Fig. 2, no CD3+16 + T cells were shown in fresh PBL and late LAK cells. These findings encouraged us to suspect that T-LAK cells cultured for a long time may coexpress CD3 and CD16, which is undetectable by flow cytometry. This was confirmed by using various T cell purification methods. PBMCs depleted of CD16 + NK cells by Cmediated lysis demonstrate depressed anti-CD16 triggered cytolysis on the first day, but 3 days later, show killer activity equal to that of whole lymphocytes. This supports the result that anti-CD16 triggered cytolysis was induced from Percoll-separated cells (high buoyant cells) and FACS sorted cells (CD5 +). In contrast to the assumption that T-LAK cells derived from NKH-1 + T cells (LGL-T), antiCD3 triggered cytolysis was induced from "pan T cells" from our data, too [11]. With one week of stimulation with rIL-2, T cells express CD16, where-
as NK-depleted PBLs with b r i e f exposure to IL-2 for 24 h, showed d i m i n i s h e d lytic activity triggered by anti-CD16. However, a 3-day s t i m u l a t i o n or more with rIL-2 would be required for T cells to express CD16. Recent reports showed that C D 4 + T cells m a y be infected, n o t t h r o u g h CD4 protein b u t t h r o u g h any Fc3,R expressed o n T cells [22]. The expression of CD16 o n activated T cells was also clarified from the study of C T L clones. I n this study, BA (anti-CD16 × anti-target) d e p e n d e n t cytolysis was i n d u c e d b u t showed n o A D C C activity. We consider the i n t e r a c t i o n o f the anti-target m A b ' s Fc d o m a i n with the Fc3,R o f effector cells is p r o b a b l y less t h a n the i n t e r a c t i o n o f anti-CD16 m A b with Fc3'R in terms o f i n d u c i n g the signal for cytolytic activity in effector cells [1]. Otherwise, sideway A D C C [4] which differs from classical A D C C using polyclonal antisera, m a y n o t be i n d u c e d by IgG~ isotype mAb. A m o n g the same anti-CD16 m A b s o f the same class (3G8 a n d MG12; IgGl), redirected cytolysis by a n t i b o d y was different (Fig. 6). T h e epitope o n CD16 which MG12 recognizes m a y be different from the epitope reactive with 3G8 [14]. By the way, the expression o f the CD16 molecule o n activated T cells m a y throw some insight into the T cell o n t o g e n y as well as p r o m o t e specific targeting therapy.
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P. J. A. (1985) J. Immunol. 134, 3770. [4] Unkeless, J. C., Scigliano, E. and Freedom, V. H. (1988) Annu. Rev. Immunol. 6, 251. [5] Van de Griend, R. J., Bolhuis, R. L. H., Stoter, R. L. G. and Roozenmond, R. C. (1987) J. Immunol. 138, 3137. [6] Ortaldo, J. R., Mason, A. and Overton, R. (1986) J. Exp. Med. 164, 1193. [7] Philips, J. H. and Lanier, L. L. (1986) J. Exp. Med. 164, 814. [8] Lanier, L. L., Le, A. M., Civin, C. I., Loken, M. R. and Philips, J. H. (1986) J. Immunol. 136, 4480. [9] ltoh, K., Tiden, A. B., Kumagai, K. and Balch, C. M. (1985) J. Immunol. 134, 802. [10] Timonen, T. and Saksela, E. (1980) J. Immunol. Methods 36, 285. [ll] Burns, G. F., Triglia, T. and Wermeister, J. A. (1984) J. Immunol. 133, 1656. [12] Weinberger, J. Z., Germain, R. N., Benacerraf, B. J. and Dorf, M. F. (1980) J. Exp. Med. 152, 161. [13] Fleit, H. B., Wright, S. D. and Unkeless, J. C. (1982) Proc. Natl. Acad. Sci. USA 79, 3275. [14] Tettero, P. A. T., Van der Schoot, C. E., Visser, E J., Bos, M. J. E. and von dem Borne, A. E. G. (1987)Leukocytetyping III, p. 702. Oxford University Press, Oxford. [15] Perrusia, B., Trinchieri, G., Jackson, A., Warner, N. L., Faust, J., Rumpold, H., Kraft, D. and Lanier, L. L. (1984) J. Immunol. 133, 180. [16] Nitta, T., Yagita,H., Azuma, T. and Okumura, K. (1989)Eur. J. Immunol. 19, 1437. [17] Nitta, T., Sato, K., Okumura, K. and Ishii, S. (1990) J. Neurosurg. 72, 476. [18] Sawada, H., Abo, T., Sugamura, S. and Kumagai, K. (1988) J. Immunol. 140, 3688. [19] Nitta, T., Sato, K., Yagita, H., Okumura, K. and Ishii, S. (1990) Lancet 335, 368. [20] Ochoa, A. C., Gromo, G., Alter, B. J., Sondel, P. M. and Bach, E H. (1987) J. Immunol. 138, 2728. [21l Lanier, L. L., Ruitenberg, J. J. and Philips, J. H. (1986) J. Exp. Med. 164, 339. [22] Homsy, J., Meyer, M., Tateno, M., Clarkson, S. and Levy, J. A. (1989) Science 244, 1357.
37
