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Monoclonal antibodies against the NK cell-FcR and the T3-complex potentiate normal lymphocyte killing
CELLULAR
IMMUNOLOGY
100,
158-166 (1986)
Monoclonal Antibodies against the NK Cell-FcR and the T3-Complex Potentiate Normal Lymphocyte Killing MIKAEL
JONDAL, CHARLOTTE KULLMAN, MAJ-BRITT AND KRISTINA LJUNGGREN
ALTER,
Department of Immunology, Karolinska Institute, 104 01 Stockholm, Sweden Received December 3, 1985; acceptedFebruary 2, I986 Pretreatment of normal human lymphocytes with monoclonal IgG against the NK cell-FcR (IgG) or the T3 complex was found to potentiate killing of most NK sensitive target cells with the exception of T-cell derived cells. Anti-FcR IgM monoclonals were suppressive for all target cells. IgG anti-FcR mediated potentiation required minute amounts of antibody but was also seenat high anti-FcR concentrations that modulated FcR activity. Potentiated and FcR modulated cells retained anti-FcR IgG on the membrane and conjugated normally to target cells. Anti-FcR potentiation blocked antibody-dependent killing but did not influence lectin-dependent killing, with anti-T3 the opposed effect was seen.Combined anti-FcR and anti-T3 treatment resulted in decreasedpotentiation. The results suggestthat the NK cell-FcR may be activated during normal NK cell killing (without the addition of antibody) as suggestedfor FcR in B cell triggering. 0 1986 Academic
Press, Inc.
INTRODUCTION Saxena and Adler (2) and Brunda et al. (3) have demonstrated that in some in vitro systems NK killing can be enhanced by allo-antisera against the effector population and that only some antisera were reactive. In Brunda’s work anti-H2, anti-Ia, and anti-Thy 1.2 increased killing by anti-asialo GMl-positive NK cells from nu/nu mice (3). More recent work in the human system show that anti-T3 can inhibit specific T-cell killing or increase non-selective killing (4-7). Anti-T3 antibody reacts with almost all post-thymic T cells and is mitogenic for peripheral T cells (8- 12). In addition, anti-T3 also inhibit antigen-specific T-cell proliferation and the generation of cytotoxic T cells and has been used as an immunosuppressant in organ grafting (13). The mitogenic properties of anti-T3 has prompted several studies on the effect of anti-T3 on killer T cells and on NK cells as mitogenic plant lectins readily induce killing. In the present work we have compared anti-T3 effectson human lymphocytes with several other monoclonal antibodies and found that monoclonal IgG specific for the NK cell-Fcr (IgG) potentiate NK killing. We hypothesize that the mechanism behind this potentiation may be similar to the earlier suggested“pro-receptor” function of FcR in B-cell triggering (1) and may depend on the interaction between FcR and Ig-like domains on a NK cell receptor molecule (18-20). Severalcell-surface molecules with receptor functions show a structural relationship with Ig such asCD8, CD4, CDl, the T antigen receptor, MHC, and the poly Ig receptor ( 14-16). 158 0008~8749/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved
FcR AND
T3 INDUCED
KILLING
159
FcR are expressedon most hematopoetic cells and structurally unique FcR (IgG) have been isolated from human monocytes, B, null, and T lymphocytes ( 17). Whether this heterogeneity depends on distinct receptor molecules or on differences in glycosylation is not clear. FcR positive cells have important immunological effector functions such as antibody-dependent killing, phagocytosis of opsonized particles, and regulation of cellular and serological immune responses( 18,2 1). FcR against different Ig isotypes are inducible on T cells and FcR beearing T-cell subpopulations appear in blood when concentrations of the corresponding isotype increase in serum (22). Human NK cells and granulocytes expressa specific FcR (IgG) and several monoclonal antibodies have been produced that selectively react with this FcR (23-27). MATERIAL
AND METHODS
Isolation oflymphocytes. Peripheral blood lymphocytes were obtained from normal healthy donors. Lymphocytes were collected by buoyant density centrifugation of Ficoll-Isopaque and washed three times in phosphate-buffered saline. Cells were then resuspendedin RPMI- 1640 that contained 10%fetal calf serum (FCS). Adherent cells were removed by the scrubbed nylon wool column technique (28). Isolated effector cells consisted of non-adherent T cells and null cells with occasional contaminating non-lymphoid cells. Only cells collected the same day as the experiment were used. Cell lines. Target cell lines were maintained in tissue culture in RPMI-1640 that contained 10% fetal calf serum, 10 mM Hepes and antibiotics. Monoclonal antibodies. MAS 1532 was purchased from Sera-Lab, United Kingdom. NEI-04 was purchased from New England Nuclear, Boston. The TAC monoclonal was a gift from Dr. T. Waldman, (29) and supplied as ascitic fluid. Anti-FcR monoclonal antibodies included 3G8, Leu-1 lB, VEP-13, and B73.1. 3G8 was received as a gift from Dr. Jay C. Unkeless, Rockefeller University, New York in the form of ascitic fluid (23). Leu-l lb, Leu-5, and Leu-7 were purchased from Becton Dickinson. VEP- 13 was a gift from Dr. H. Rumpold, University of Vienna, Austria and supplied as ascitic fluid (24). B73.1 was received from Dr. G. Trinchieri, Wistar Institute of Anatomy and Biology, Philadelphia, as ascitic fluid (25). “Cr cytotoxicity tests. Cytotoxicity assayswere performed with duplicate wells in V-shaped microplates using a total volume of 150 ~1. “Cr labeled target cells ( 104) and 2 X 1O5isolated lymphocytes as effector cells in RPMI- 1640 with 10% fetal calf serum were added in each well (28). Fifty microliters of the supernatant from each well was harvested after 3 hr and counted in a LKB gamma counter. Releasedradioactivity was calculated as described (28). Antibody-dependent killing was tested with rabbit IgG anti-IgM (Dako-Patts, Hagersten, Sweden) against the surface IgM positive target cell line Daudi. Anti-IgM was used as described in Table 4 and Fig. 3. Lectindependent killing was tested against the same target cell line using concanavalin A (Con A) (Pharmacia, Uppsala, Sweden) as inducing agent as described in the legend to Table 4. RESULTS Eflect of D$erent Anti-lymphocyte Monoclonal Antibodies on Killing of Daudi Cells The following monoclonal antibodies were tested for induction of Daudi cell killing 3G8 (anti-NK cell-FcR (IgG)), Leu4 (anti-T3), MAS 1532 (anti-MHC, class I), NEI-
160
JONDAL ET AL.
I
Cont
1
2
Mab
3
4
5
6
7
6
concentration
FIG. 1. Effect of 3G8 on killing of Molt-4 or Daudi target cells. Lymphocytes were pretreated for 60 min at 37°C with 3G8 monoclonal antibodies at a concentration of 1:100of the antibody containing ascitic fluid with eight consecutive 1:10 dilutions. Cells were then washed (filled symbols) or unwashed at 1 (A) or 18 (B) hr and tested against Daudi (triangles) or Molt-4 (circles) target cells. Standard “Cr release assay.
04 (anti-MHC, class II), TAC (anti-IL-2 receptor), Leu-5 (anti-SRBC receptor), and Leu-7 (anti-HNK-1). Anti-FcR and anti-T3 monoclonals induced killing of Daudi (Figs. 1 and 2) whereas no effect was seenwith the other antibodies (data not shown). A. 40 30
L-IL
20 10
P
b;’ u-l
i
501 t-B. .
&
*--
-T
40-
=
30. b
20.
P
10 1
I Cont; ; 4 4 Cont; ; $ i FIG. 2. Effect of Leu-4 on killing of Daudi and K-562 target cells with two different lymphocyte donors. Donor 1 (left part) is high-reactive and donor 2 (right part) low-reactive in NK lysis. Lymphocytes were pretreated for 60 min at 37°C with Leu-4 at a concentration of 2 &ml and four consecutive 15 dilutions. Cells were then washed (filled symbols) or unwashed (open symbols) and tested against Daudi (A) or K-562 (B) target cells. Standard “Cr releaseassay.
161
FcR AND T3 INDUCED KILLING
The non-inductive monoclonals MAS 1532 and Leu-5 were of the same isotype as the inductive 3G8 and Leu4, and reactive with NK cells. Efect ofAnti-FcR and Anti-T3 Monoclonal Antibodies on Normal Lymphocyte Killing Normal, non-adherent peripheral lymphocytes were pretreated with anti-FcR monoclonal antibody 3G8 and tested either in the absenceor presenceof 3G8 against target cells Molt-4 and Daudi (Fig. 1). Treated cells were potentiated against Daudi and suppressed against Molt-4 cells. The potentiation agaisnt Daudi was detectable at low dilutions of 3G8 ( 10w8)and unchanged with cells that were either pretreated or cultured in the presence of antibody for 18 hr (Fig. 1, B). Background killing of Daudi increased somewhat during tissue culture overnight as noted earlier. The potentiation of Daudi killing by 3G8 was compared to the effect of the antiT3 monoclonal Leu-4 on both Daudi and K-562 killing (Fig. 2). Both with antibody present in the test and with pretreated effector cells, both antibodies potentiated killing of both target cell lines. Potentiation was more extensive with the NK low reactive lymphocyte donor (donor 2). Several other target cell lines and anti-FcR monoclonals were tested as summarized in Table 1. The IgG anti-FcR monoclonal B73.1 acted in a similar way as 3G8 by potentiating killing of several different NK sensitive hematopoetic target cell lines (group A) but inhibiting killing of Molt-4 cells and other T-cell lines (group B). With NK resistant cell lines, including the Epstein-Barr virus transformed normal B-cell line LCL-ES-Bl, the Burkitt lymphomaderived Raji line, the myeloma cell line U-266, and the mouse T-cell line YAC there was no induction of killing (group C). The two IgM anti-FcR monoclonals, known to react with the same molecule as 3G8 and B73.1, suppressedkilling of all NK-sensitive target cells at higher concentrations. With Leu-4 there was potentiation of killing with all NK-sensitive target cells but no inhibition of T-cell target killing at the tested concentrations.
TABLE 1 Effect of Anti-NK Cell FcR and Anti-T3 Monoclonal Antibodies on Killing of Different Target Cells Effect of monoclonals on NK killing” Specificity
Mab
NK-FcR NK-FcR NK-FcR NK-FcR T3-complex
3G8
I&l
B73.1 VEP- I3 La-1 lb
If@, IgM kM
La-4
IgGl
kotype
A high-intermediateb
B high-intermediate
Increase as in Fig. 1 Increase as in Fig. 1 Decrease as in Fig. I
Decreaseas in Fig. I Deaease as in Fig. 1 Decreaseas in Fig. 1 Decrease’ No influence’
DeCRaSeC
Increase as in Fig. 2
c low No No No No No
induction induction induction induction induction
Note. The influence of the antibodies was tested in several repeat experiments using a wide range of antibody concentrations in standard “Cr releaseNK assays.The origin of the different monoclonal antibodies are given in the MM section. ’ Different target cell groups include: (A) K-562, U937, HL-60, Daudi, and Namalva, (B) Molt-A Jurkat, and CCRF-CEM, (C) LCL-ES-Bl, Raji, U-266, and YAC. ’ Indicate NK sensitivity of target cells. ’ Jondal, M., Kullman, C., Alter, M-B., and Ljunggren, K. (submitted for publication).
162
JONDAL ET AL.
FcR Activity in Relation to Presenceof FcR Molecules in Cell Membrane Four different anti-FcR monoclonals were tested for inhibition of FcR activity expressed as rosette formation with ox RBC sensitized with rabbit anti-OxRBC IgG (Table 2). All antibodies suppressed FcR activity after short-term preincubation of lymphocytes at room temperature. The same antibody concentrations that gave complete FcR activity suppression, induced NK killing. The results indicate that strong NK cell activity can occur in cells without any detectable FcR activity and raised the question whether the FcR molecules were cleared from the cell surface or still present, although in an inactive state. This was analyzed by labeling 3G8 treated cells with FITC anti-mouse IgG antibodies and subsequent testing by flow cytometry in a FACS system. No major difference between 3G8 treated and control cells were seen, even after prolonged incubation ( 18 hr) at 37°C suggestingthat although FcR activity was inhibited, FcR molecules remained in the membrane (data not shown). In addition, immunosorbent purified rabbit anti-mouse IgG added to 3G8 potentiated effector cells inhibited the 3G8 induced killing and did so even after overnight tissue culture demonstrating the presence of 3G8 (and FcR) (Fig. 3). Eflect of 3G8 and Leu-4 on Conjugation of Effector Cells to Target Cells 3G8 or Leu-4 were included in single cell cytotoxicity assaysin agaroseand compared with “Cr release under identical conditions (Table 3). Concentrations of 3G8 that suppressedMolt-4 killing or potentiated Daudi killing did dot change the number of target binding lymphocytes when treasured at the initiation of the 5’Cr releaseassay. Leu-4 induced Daudi killing did not influence target binding demonstrating that both anti-FcR and anti-T3 antibodies exert their influence during the triggering phaserather than the binding phase. Efect of 3G8 and Leu-4 on Antibody and Lectin-Dependent Killing Normal lymphocyte effector cells were pretreated with either 3G8 or Leu-4 antibodies and tested in antibody or lectin-dependent systems (Table 4). Antibody-dependent killing was induced by a rabbit heteroantisera against human IgM expressed on the TABLE 2 Effect of Anti-NK Cell FcR-Monoclonal Antibodies on EA (IgG) Rosette Formation EA (IgG) rosette formation at mab titer 10-l
10-S lo-* (% of lymphocytes)
lo-4
0.2 1.9 0 0
0 1.5 0.8 2.1
1.9 3.6 9.1 8.3
Mab 3G8 B73.1 VEP- 13 Leu-1 lb Control
0.6 2.6 3.4 7.2
(15.3)
Note. FcR activity was measured as rosette formation between rabbit IgG sensitized ox RBC and nonadherent peripheral lymphocytes after preincubation for 30 min at room temperature with lymphocytes and the different anti-FcR monoclonals.
163
FcR AND T3 INDUCED KILLING
R anti-mtg
concentration
FIG. 3. Suppression of 3G8 induced killing by rabbit anti-mouse Ig at Days 0 and 1. Lymphocytes were pretreated for 30 min at 37°C with 3G8 monoclonal antibodies at dilutions of IO-’ (0) and IO-’ (0) or untreated (*) and washed. Cells were then tested for killing of Daudi cells either directly or after a day of tissue culture in the presence of immunosorbent purified rabbit IgG anti-mouse Ig at concentrations from 1:10m3and six consecutive I:3 dilutions.
surface of Daudi cells and lectin-dependent killing by Con A with the same target cell. Con A induced strong killing of Daudi cells and the presence of 3G8 antibodies did not influence lectin-dependent killing. Antibody-dependent killing was completely inhibited at high 3G8 concentrations in accordancewith the dependenceof this activity on an intact FcR expression. Leu-4 antibodies left antibody-dependent killing undisturbed but suppressed Con A-induced killing demonstrating the dependence of the latter activity on the T3 complex. Eflect of Combined Treatments with 3G8 and Leu-4 Normal lymphocytes were potentiated with either 3G8 and Leu-4 alone or with combinations of both monoclonals (Fig. 4). When a titration of 3G8 was tested in the presence of a fixed Leu-4 concentration (1: 100) the dose-response curve was shifted downward demonstrating a negative combined effect. When the reversedcombination was used, i.e., a titration of Leu-4 and a fixed 3G8 concentration a clear negative effect of anti-T3 on anti-FcR induced killing was seen. TABLE 3 Effect of 3G8 and Leu-4 Monoclonal Antibodies on Conjugation of Effector Cells to Target Cells
NK killing (% Yr release)
Target binding (% of total lymphocyte population)
Mab
Titer of mab
Molt-4
Daudi
Molt-4
Daudi
3G8 3G8 Leu-4 Control
10-2 10-6 10-2 -
10.4 33.5 37.1 38.6
40.2 54.4 39.2 3.4
10.1 9.4 9.9 10.8
11.1 10.1 9.8 12.0
Note. NK killing was tested after preincubation of effector cells and mab for 30 min at room temperature. Target binding and 5’Cr release was tested under identical conditions using the agarose single cell immobilization assay(35). Target binding was scored at initiation of the NK test.
164
JONDAL ET AL. TABLE 4 Effect of 3G8 and Anti-T3 Monoclonal Antibodies on Antibody and Lectin-Dependent Killing ADCC and LDCC killing (% “0
Expt Expt 1
Mab 3G8 3G8 3G8
Control Expt 2
Titer of mab
Medium
Con A
10-3
37.6 42. I 40.2 8.2
71.7 73.5 70.6 73.6
36.4 45.4 58.1 70.4
35.4 28.9
33.4 32.3 46.1 55.4
59.6 58.6 58.1 57.7
10-4 10-5
-
La-4
1O-2
till-4
10-3 1o-4 -
Leu-4
Control
release)
16.7 12.4
Anti-&I
Note. Antibody-dependent killing (ADCC) was tested with rabbit IgG anti&M against the IgM positive target cell Daudi and &tin-dependent killing (LDCC) against the same target cell using concanavalin A. The experiments were done with titration curves for anti-IgM and Con A, the optimal values are given. Effector cells were pretreated with the indicated concentrations of mab for 60 min at 37°C and washedtwice before used in assays.
DISCUSSION The present data demonstrate that IgG monoclonal antibodies against an NK cell FcR potentiate killing of most NK sensitive target cells with the exception of some T-cell lines. Anti-Fc antibodies are active at low concentrations, have prolonged effects on NK cell killing, and do not influence target cell binding. Similar results were obtained with anti-T3 in our work and earlier (6, 7). Anti-FcR and anti-T3 either react with distinct NK cell subpopulations, as suggestedby recent distribution studies of these markers (26, 32) or with NK cells expressing both FcR and T3. The demonstration of FcR and T3 on large fractions of active NK cells in single cell assays(33) support the latter possibility. The combination of 3G8 and Leu-4 antibodies also resulted in inhibition of FcR-dependent induction indicating the coexpression in some NK cells. Roberts and Moore cloned B73.1 positive and negative cells and analyzed the different clones for killing and expression of phenotypic markers (34). They found that
Cant123456 Mab
123456 concentration
FIG. 4. Effect of combined 3G8 and Leu-4 pretreatment on killing of Daudi target cells. (A) Lymphocytes were pretreated for 30 min at 37°C with either 3G8 (A) or Let14 (0) at starting concentrations of 1:100 of ascitic fluid and 2 pg/ml, respectively and six consecutive I:10 dilutions. (B) Combined treatments were done with 3G8 at 1:100and a similar dilution seriesof Let14 (A) or with Leu-4 at 2 pgjml and corresponding 3G8 dilutions (0).
FcR AND T3 INDUCED KILLING
165
although clones derived from B73.1 positive cells retained an LGL morphology only some expressedNK activity and most of these were T3 positive. No correlation between any surface marker and NK cell killing was found and one of their lytic clones, derived from B73.1 positive cells, did not expresseither B73.1 or T3. These combined data indicate that NK cells may use alternating membrane interactions for triggering of lysis. The addition of anti-T3 directly into short-term radioisotope release assaysresult in inhibition of T-cell lysis (4). Long-term (46-64 hr) pretreatment of T-memory cells cause the specific activation of effector cells if tested in the absenceof anti-T3 but in inhibition with antibody present in the test system (7). Long-term (16 hr) treatment of isolated large granular lymphocytes augmented killing of both Molt-4 and Daudi target cells caused by lymphokine secreting T cells present in the LGL fraction (7). In contrast, Leeuvenberg et al. did not detect any anti-T3 induction of killing with fresh peripheral cells but found induction of NK like killing in MLC generated effector cells in combination with inhibition of specific killing (6). Further analysis of the mechanism behind anti-T3 induced NK killing suggestedcrosslinking between IgG anti-T3 and Fc receptors for mouse IgG present on the target cell population (6). In our present work it is clear that anti-T3 indeed potentiate, normal unstimulated peripheral lymphocytes but that the specificity of the induced activity is slightly different from that reported by Leeuvenberg et al. We found no killing of the target cell line Raji which is reported to be positive for mouse Ig FcR (6). The induction of anti-T3dependent killing may thus depend on an alternative mechanism, i.e., the polymerization of the T3 complex to receptor structures involved in NK cell recognition such as the Ti-like, clonotypic heterodimer described by Ritz et al. (36-38). The stronger effect of anti-FcR IgG for the induction of NK killing supports the concept that induction primarily depends on events on the effector cell surface as these antibodies can interact both at the antigen specific and at the Fc end during polymerization of FcR. The regulatory influence of anti-FcR and anti-T3 include both down-regulation and up-regulation of activity. IgM anti-NK cell FcR (and IgG anti-FcR against some target cells) inhibit killing by FcR positive cells and IgG anti-FcR potentiate killing of most NK sensitive target cell lines. These effectsare important becausethey suggest novel modes of selectiveimmunomodulation in vivo asproposed (5,7). Conventionally, antibody effects have been studied in relation to target cell specific antibodies that either induce or inhibit immune reactivity, but our present data suggest that antieffector cell antibodies may be used to selectively activate or suppressdifferent effector functions. In the present work we have studied killer cells but the same approach may be used for modulation of other cell-mediated functions such as suppression or helper activity. In conclusion, IgG monoclonal antibodies against a specific NK cell-FcR potentiate killing and IgM antibodies of the same specificity suppress NK activity. Anti-T3 antibodies have similar effects as earlier reported (7). This result suggeststhat FcR may be involved in normal NK cell killing (without added antibody) and that anti-FcR, anti-T3, and other relevant antibodies should be systematically investigated for their potential capacity as immunological response modifiers. ACKNOWLEDGMENT This work was supported by grants from the Swedish Cancer Association, The Karolinska Institute, and the Swedish Medical Association.
JONDAL ET AL.
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Ramasamy, R., Munro, A., and Milstein, C., Nature (London) 249, 573, 1974. Saxena, R. K., and Adler, W. H., J. Immunol. 123, 846, 1979. Brunda, M. J., Herbeman, R. H., and Holden, H. T., Int. J. Cancer 27, 205, 1981. Landegren, U., Ramstedt, U., Axberg, I., Ullberg, M., Jondal, M., and Wigzell, H., J. Exp. Med. 155, 1579, 1982. 5. Spits, H., Yssel, H., Leeuwenberg, J., and de Vries J. E., Eur. J. Immunol. 15, 88, 1985. 6. Leeuwenberg, J. F. M., Spits, H., Tax, W. J. M., and Cape], P. J. A., J. Immunol. 6, 3770, 1985. 7. Suthanthiran, M., Williams, P. S., Salomon, S. D., Rubin, A. L., and Stenzel, K. H., J. Clin. Invest. 74, 2263, 1984. 8. Reinherz, E. L., Meuer, S. C., and Schlossman,S. F., Immunol. Today4, 5, 1983. 9. Moller, G. (ed.) Immunol. Rev. 81, 1984. 10. Reinherz, E. L., Immunol. Today6,75, 1985. 11. Weiss, M. J., Daley, J. F., Hodgon, J. C., and Reinherz, E. L., PNAS 81, 6836, 1984. 12. van Wauwe, J. P., de Mey, J. R., and Goossens,J. G., J. Immunol. 124, 2708, 1980. 13. Cosimi, A. B., Burton, R. C., Calvin, R. B., Goldstein, G., Delmonico, F. L., LaQuaglia, M. P., Tolkolf, N., Rubin, R. H., Henin, J. T., and Russel, P. S., Transplantation 32, 535, 1981. 14. Cushley W., and Owen, M. J., Immunol. Today 4,88, 1983. 15. Williams, F. (ed.) Nature (London) 308, 12, 1984. 16. Williams, F. (ed.) Nature (London) 314, 579, 1985. 17. Cohen, L., Sharp, S., and Kulczycki, A., J. Immunol. 131, 378, 1983. 18. Dickler, H. B., Adv. Immunol. 24, 167, 1976. 19. Dickler, H. B., and Kubieck, M. T., J. Exp. Med. 153, 1329, 198 1. 20. Dickler, H. B., Kubieck, M. T., and Finkelman, F. D., J. Immunol. 128, 1271, 1982. 2 1. Unkeless, J. C., Fleit, H., and Mellman, I. S., Adv. Immunol. 31, 247, 198 1. 22. MSller, G. (ed.) Immunol. Rev. 56, 1981. 23. Rumpold, H., Kraft, D., Obexer, G., Bock, G., and Gebhart, W., J. Immunol. 129, 1458, 1982. 24. Pert&a, B., Starr, S., Abraham, S., Fanning, V., and Trinchieri, G., J. Immunol. 130,2133, 1983. 25. Fleit, H. B., Wright, S. D., and Unkeless, J. C., PNAS 79, 3275, 1982. 26. Peru&a, B., Trinchieri, G., Jackson, A., Warner, N. L., Faust, J., Rumpold, H., Kraft, D., and Lanier, L. L., J. Immunol. 133, 180, 1984. 27. Perussia,B., and Trinchieri, G., J. Immunol. 132, 1410, 1984. 28. Merrill, J. E., Ullberg, M., and Jondal, M., Eur. J. Immunol. 11, 536, 1981. 29. Uchiyama, T., Broder, S., and Waldmann, T. A., J. Immunol. 126, 1393, 1981. 30. Minowada, J., Ohnuma, T., and More, G. E., J. Natl. Cancer Inst. 49, 891, 1972. 31. Lozzio, C. B., and Lozzio, B. B., J. Natl. Cancer Inst. 50, 535, 1973. 32. Lanier, L. L., Le, A. M., Phillips, J. H., Warner, N. L., and Babcock, G. F., J. Immunol. 131, 1789, 1983.
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IMMUNOLOGY
100,
158-166 (1986)
Monoclonal Antibodies against the NK Cell-FcR and the T3-Complex Potentiate Normal Lymphocyte Killing MIKAEL
JONDAL, CHARLOTTE KULLMAN, MAJ-BRITT AND KRISTINA LJUNGGREN
ALTER,
Department of Immunology, Karolinska Institute, 104 01 Stockholm, Sweden Received December 3, 1985; acceptedFebruary 2, I986 Pretreatment of normal human lymphocytes with monoclonal IgG against the NK cell-FcR (IgG) or the T3 complex was found to potentiate killing of most NK sensitive target cells with the exception of T-cell derived cells. Anti-FcR IgM monoclonals were suppressive for all target cells. IgG anti-FcR mediated potentiation required minute amounts of antibody but was also seenat high anti-FcR concentrations that modulated FcR activity. Potentiated and FcR modulated cells retained anti-FcR IgG on the membrane and conjugated normally to target cells. Anti-FcR potentiation blocked antibody-dependent killing but did not influence lectin-dependent killing, with anti-T3 the opposed effect was seen.Combined anti-FcR and anti-T3 treatment resulted in decreasedpotentiation. The results suggestthat the NK cell-FcR may be activated during normal NK cell killing (without the addition of antibody) as suggestedfor FcR in B cell triggering. 0 1986 Academic
Press, Inc.
INTRODUCTION Saxena and Adler (2) and Brunda et al. (3) have demonstrated that in some in vitro systems NK killing can be enhanced by allo-antisera against the effector population and that only some antisera were reactive. In Brunda’s work anti-H2, anti-Ia, and anti-Thy 1.2 increased killing by anti-asialo GMl-positive NK cells from nu/nu mice (3). More recent work in the human system show that anti-T3 can inhibit specific T-cell killing or increase non-selective killing (4-7). Anti-T3 antibody reacts with almost all post-thymic T cells and is mitogenic for peripheral T cells (8- 12). In addition, anti-T3 also inhibit antigen-specific T-cell proliferation and the generation of cytotoxic T cells and has been used as an immunosuppressant in organ grafting (13). The mitogenic properties of anti-T3 has prompted several studies on the effect of anti-T3 on killer T cells and on NK cells as mitogenic plant lectins readily induce killing. In the present work we have compared anti-T3 effectson human lymphocytes with several other monoclonal antibodies and found that monoclonal IgG specific for the NK cell-Fcr (IgG) potentiate NK killing. We hypothesize that the mechanism behind this potentiation may be similar to the earlier suggested“pro-receptor” function of FcR in B-cell triggering (1) and may depend on the interaction between FcR and Ig-like domains on a NK cell receptor molecule (18-20). Severalcell-surface molecules with receptor functions show a structural relationship with Ig such asCD8, CD4, CDl, the T antigen receptor, MHC, and the poly Ig receptor ( 14-16). 158 0008~8749/86 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved
FcR AND
T3 INDUCED
KILLING
159
FcR are expressedon most hematopoetic cells and structurally unique FcR (IgG) have been isolated from human monocytes, B, null, and T lymphocytes ( 17). Whether this heterogeneity depends on distinct receptor molecules or on differences in glycosylation is not clear. FcR positive cells have important immunological effector functions such as antibody-dependent killing, phagocytosis of opsonized particles, and regulation of cellular and serological immune responses( 18,2 1). FcR against different Ig isotypes are inducible on T cells and FcR beearing T-cell subpopulations appear in blood when concentrations of the corresponding isotype increase in serum (22). Human NK cells and granulocytes expressa specific FcR (IgG) and several monoclonal antibodies have been produced that selectively react with this FcR (23-27). MATERIAL
AND METHODS
Isolation oflymphocytes. Peripheral blood lymphocytes were obtained from normal healthy donors. Lymphocytes were collected by buoyant density centrifugation of Ficoll-Isopaque and washed three times in phosphate-buffered saline. Cells were then resuspendedin RPMI- 1640 that contained 10%fetal calf serum (FCS). Adherent cells were removed by the scrubbed nylon wool column technique (28). Isolated effector cells consisted of non-adherent T cells and null cells with occasional contaminating non-lymphoid cells. Only cells collected the same day as the experiment were used. Cell lines. Target cell lines were maintained in tissue culture in RPMI-1640 that contained 10% fetal calf serum, 10 mM Hepes and antibiotics. Monoclonal antibodies. MAS 1532 was purchased from Sera-Lab, United Kingdom. NEI-04 was purchased from New England Nuclear, Boston. The TAC monoclonal was a gift from Dr. T. Waldman, (29) and supplied as ascitic fluid. Anti-FcR monoclonal antibodies included 3G8, Leu-1 lB, VEP-13, and B73.1. 3G8 was received as a gift from Dr. Jay C. Unkeless, Rockefeller University, New York in the form of ascitic fluid (23). Leu-l lb, Leu-5, and Leu-7 were purchased from Becton Dickinson. VEP- 13 was a gift from Dr. H. Rumpold, University of Vienna, Austria and supplied as ascitic fluid (24). B73.1 was received from Dr. G. Trinchieri, Wistar Institute of Anatomy and Biology, Philadelphia, as ascitic fluid (25). “Cr cytotoxicity tests. Cytotoxicity assayswere performed with duplicate wells in V-shaped microplates using a total volume of 150 ~1. “Cr labeled target cells ( 104) and 2 X 1O5isolated lymphocytes as effector cells in RPMI- 1640 with 10% fetal calf serum were added in each well (28). Fifty microliters of the supernatant from each well was harvested after 3 hr and counted in a LKB gamma counter. Releasedradioactivity was calculated as described (28). Antibody-dependent killing was tested with rabbit IgG anti-IgM (Dako-Patts, Hagersten, Sweden) against the surface IgM positive target cell line Daudi. Anti-IgM was used as described in Table 4 and Fig. 3. Lectindependent killing was tested against the same target cell line using concanavalin A (Con A) (Pharmacia, Uppsala, Sweden) as inducing agent as described in the legend to Table 4. RESULTS Eflect of D$erent Anti-lymphocyte Monoclonal Antibodies on Killing of Daudi Cells The following monoclonal antibodies were tested for induction of Daudi cell killing 3G8 (anti-NK cell-FcR (IgG)), Leu4 (anti-T3), MAS 1532 (anti-MHC, class I), NEI-
160
JONDAL ET AL.
I
Cont
1
2
Mab
3
4
5
6
7
6
concentration
FIG. 1. Effect of 3G8 on killing of Molt-4 or Daudi target cells. Lymphocytes were pretreated for 60 min at 37°C with 3G8 monoclonal antibodies at a concentration of 1:100of the antibody containing ascitic fluid with eight consecutive 1:10 dilutions. Cells were then washed (filled symbols) or unwashed at 1 (A) or 18 (B) hr and tested against Daudi (triangles) or Molt-4 (circles) target cells. Standard “Cr release assay.
04 (anti-MHC, class II), TAC (anti-IL-2 receptor), Leu-5 (anti-SRBC receptor), and Leu-7 (anti-HNK-1). Anti-FcR and anti-T3 monoclonals induced killing of Daudi (Figs. 1 and 2) whereas no effect was seenwith the other antibodies (data not shown). A. 40 30
L-IL
20 10
P
b;’ u-l
i
501 t-B. .
&
*--
-T
40-
=
30. b
20.
P
10 1
I Cont; ; 4 4 Cont; ; $ i FIG. 2. Effect of Leu-4 on killing of Daudi and K-562 target cells with two different lymphocyte donors. Donor 1 (left part) is high-reactive and donor 2 (right part) low-reactive in NK lysis. Lymphocytes were pretreated for 60 min at 37°C with Leu-4 at a concentration of 2 &ml and four consecutive 15 dilutions. Cells were then washed (filled symbols) or unwashed (open symbols) and tested against Daudi (A) or K-562 (B) target cells. Standard “Cr releaseassay.
161
FcR AND T3 INDUCED KILLING
The non-inductive monoclonals MAS 1532 and Leu-5 were of the same isotype as the inductive 3G8 and Leu4, and reactive with NK cells. Efect ofAnti-FcR and Anti-T3 Monoclonal Antibodies on Normal Lymphocyte Killing Normal, non-adherent peripheral lymphocytes were pretreated with anti-FcR monoclonal antibody 3G8 and tested either in the absenceor presenceof 3G8 against target cells Molt-4 and Daudi (Fig. 1). Treated cells were potentiated against Daudi and suppressed against Molt-4 cells. The potentiation agaisnt Daudi was detectable at low dilutions of 3G8 ( 10w8)and unchanged with cells that were either pretreated or cultured in the presence of antibody for 18 hr (Fig. 1, B). Background killing of Daudi increased somewhat during tissue culture overnight as noted earlier. The potentiation of Daudi killing by 3G8 was compared to the effect of the antiT3 monoclonal Leu-4 on both Daudi and K-562 killing (Fig. 2). Both with antibody present in the test and with pretreated effector cells, both antibodies potentiated killing of both target cell lines. Potentiation was more extensive with the NK low reactive lymphocyte donor (donor 2). Several other target cell lines and anti-FcR monoclonals were tested as summarized in Table 1. The IgG anti-FcR monoclonal B73.1 acted in a similar way as 3G8 by potentiating killing of several different NK sensitive hematopoetic target cell lines (group A) but inhibiting killing of Molt-4 cells and other T-cell lines (group B). With NK resistant cell lines, including the Epstein-Barr virus transformed normal B-cell line LCL-ES-Bl, the Burkitt lymphomaderived Raji line, the myeloma cell line U-266, and the mouse T-cell line YAC there was no induction of killing (group C). The two IgM anti-FcR monoclonals, known to react with the same molecule as 3G8 and B73.1, suppressedkilling of all NK-sensitive target cells at higher concentrations. With Leu-4 there was potentiation of killing with all NK-sensitive target cells but no inhibition of T-cell target killing at the tested concentrations.
TABLE 1 Effect of Anti-NK Cell FcR and Anti-T3 Monoclonal Antibodies on Killing of Different Target Cells Effect of monoclonals on NK killing” Specificity
Mab
NK-FcR NK-FcR NK-FcR NK-FcR T3-complex
3G8
I&l
B73.1 VEP- I3 La-1 lb
If@, IgM kM
La-4
IgGl
kotype
A high-intermediateb
B high-intermediate
Increase as in Fig. 1 Increase as in Fig. 1 Decrease as in Fig. I
Decreaseas in Fig. I Deaease as in Fig. 1 Decreaseas in Fig. 1 Decrease’ No influence’
DeCRaSeC
Increase as in Fig. 2
c low No No No No No
induction induction induction induction induction
Note. The influence of the antibodies was tested in several repeat experiments using a wide range of antibody concentrations in standard “Cr releaseNK assays.The origin of the different monoclonal antibodies are given in the MM section. ’ Different target cell groups include: (A) K-562, U937, HL-60, Daudi, and Namalva, (B) Molt-A Jurkat, and CCRF-CEM, (C) LCL-ES-Bl, Raji, U-266, and YAC. ’ Indicate NK sensitivity of target cells. ’ Jondal, M., Kullman, C., Alter, M-B., and Ljunggren, K. (submitted for publication).
162
JONDAL ET AL.
FcR Activity in Relation to Presenceof FcR Molecules in Cell Membrane Four different anti-FcR monoclonals were tested for inhibition of FcR activity expressed as rosette formation with ox RBC sensitized with rabbit anti-OxRBC IgG (Table 2). All antibodies suppressed FcR activity after short-term preincubation of lymphocytes at room temperature. The same antibody concentrations that gave complete FcR activity suppression, induced NK killing. The results indicate that strong NK cell activity can occur in cells without any detectable FcR activity and raised the question whether the FcR molecules were cleared from the cell surface or still present, although in an inactive state. This was analyzed by labeling 3G8 treated cells with FITC anti-mouse IgG antibodies and subsequent testing by flow cytometry in a FACS system. No major difference between 3G8 treated and control cells were seen, even after prolonged incubation ( 18 hr) at 37°C suggestingthat although FcR activity was inhibited, FcR molecules remained in the membrane (data not shown). In addition, immunosorbent purified rabbit anti-mouse IgG added to 3G8 potentiated effector cells inhibited the 3G8 induced killing and did so even after overnight tissue culture demonstrating the presence of 3G8 (and FcR) (Fig. 3). Eflect of 3G8 and Leu-4 on Conjugation of Effector Cells to Target Cells 3G8 or Leu-4 were included in single cell cytotoxicity assaysin agaroseand compared with “Cr release under identical conditions (Table 3). Concentrations of 3G8 that suppressedMolt-4 killing or potentiated Daudi killing did dot change the number of target binding lymphocytes when treasured at the initiation of the 5’Cr releaseassay. Leu-4 induced Daudi killing did not influence target binding demonstrating that both anti-FcR and anti-T3 antibodies exert their influence during the triggering phaserather than the binding phase. Efect of 3G8 and Leu-4 on Antibody and Lectin-Dependent Killing Normal lymphocyte effector cells were pretreated with either 3G8 or Leu-4 antibodies and tested in antibody or lectin-dependent systems (Table 4). Antibody-dependent killing was induced by a rabbit heteroantisera against human IgM expressed on the TABLE 2 Effect of Anti-NK Cell FcR-Monoclonal Antibodies on EA (IgG) Rosette Formation EA (IgG) rosette formation at mab titer 10-l
10-S lo-* (% of lymphocytes)
lo-4
0.2 1.9 0 0
0 1.5 0.8 2.1
1.9 3.6 9.1 8.3
Mab 3G8 B73.1 VEP- 13 Leu-1 lb Control
0.6 2.6 3.4 7.2
(15.3)
Note. FcR activity was measured as rosette formation between rabbit IgG sensitized ox RBC and nonadherent peripheral lymphocytes after preincubation for 30 min at room temperature with lymphocytes and the different anti-FcR monoclonals.
163
FcR AND T3 INDUCED KILLING
R anti-mtg
concentration
FIG. 3. Suppression of 3G8 induced killing by rabbit anti-mouse Ig at Days 0 and 1. Lymphocytes were pretreated for 30 min at 37°C with 3G8 monoclonal antibodies at dilutions of IO-’ (0) and IO-’ (0) or untreated (*) and washed. Cells were then tested for killing of Daudi cells either directly or after a day of tissue culture in the presence of immunosorbent purified rabbit IgG anti-mouse Ig at concentrations from 1:10m3and six consecutive I:3 dilutions.
surface of Daudi cells and lectin-dependent killing by Con A with the same target cell. Con A induced strong killing of Daudi cells and the presence of 3G8 antibodies did not influence lectin-dependent killing. Antibody-dependent killing was completely inhibited at high 3G8 concentrations in accordancewith the dependenceof this activity on an intact FcR expression. Leu-4 antibodies left antibody-dependent killing undisturbed but suppressed Con A-induced killing demonstrating the dependence of the latter activity on the T3 complex. Eflect of Combined Treatments with 3G8 and Leu-4 Normal lymphocytes were potentiated with either 3G8 and Leu-4 alone or with combinations of both monoclonals (Fig. 4). When a titration of 3G8 was tested in the presence of a fixed Leu-4 concentration (1: 100) the dose-response curve was shifted downward demonstrating a negative combined effect. When the reversedcombination was used, i.e., a titration of Leu-4 and a fixed 3G8 concentration a clear negative effect of anti-T3 on anti-FcR induced killing was seen. TABLE 3 Effect of 3G8 and Leu-4 Monoclonal Antibodies on Conjugation of Effector Cells to Target Cells
NK killing (% Yr release)
Target binding (% of total lymphocyte population)
Mab
Titer of mab
Molt-4
Daudi
Molt-4
Daudi
3G8 3G8 Leu-4 Control
10-2 10-6 10-2 -
10.4 33.5 37.1 38.6
40.2 54.4 39.2 3.4
10.1 9.4 9.9 10.8
11.1 10.1 9.8 12.0
Note. NK killing was tested after preincubation of effector cells and mab for 30 min at room temperature. Target binding and 5’Cr release was tested under identical conditions using the agarose single cell immobilization assay(35). Target binding was scored at initiation of the NK test.
164
JONDAL ET AL. TABLE 4 Effect of 3G8 and Anti-T3 Monoclonal Antibodies on Antibody and Lectin-Dependent Killing ADCC and LDCC killing (% “0
Expt Expt 1
Mab 3G8 3G8 3G8
Control Expt 2
Titer of mab
Medium
Con A
10-3
37.6 42. I 40.2 8.2
71.7 73.5 70.6 73.6
36.4 45.4 58.1 70.4
35.4 28.9
33.4 32.3 46.1 55.4
59.6 58.6 58.1 57.7
10-4 10-5
-
La-4
1O-2
till-4
10-3 1o-4 -
Leu-4
Control
release)
16.7 12.4
Anti-&I
Note. Antibody-dependent killing (ADCC) was tested with rabbit IgG anti&M against the IgM positive target cell Daudi and &tin-dependent killing (LDCC) against the same target cell using concanavalin A. The experiments were done with titration curves for anti-IgM and Con A, the optimal values are given. Effector cells were pretreated with the indicated concentrations of mab for 60 min at 37°C and washedtwice before used in assays.
DISCUSSION The present data demonstrate that IgG monoclonal antibodies against an NK cell FcR potentiate killing of most NK sensitive target cells with the exception of some T-cell lines. Anti-Fc antibodies are active at low concentrations, have prolonged effects on NK cell killing, and do not influence target cell binding. Similar results were obtained with anti-T3 in our work and earlier (6, 7). Anti-FcR and anti-T3 either react with distinct NK cell subpopulations, as suggestedby recent distribution studies of these markers (26, 32) or with NK cells expressing both FcR and T3. The demonstration of FcR and T3 on large fractions of active NK cells in single cell assays(33) support the latter possibility. The combination of 3G8 and Leu-4 antibodies also resulted in inhibition of FcR-dependent induction indicating the coexpression in some NK cells. Roberts and Moore cloned B73.1 positive and negative cells and analyzed the different clones for killing and expression of phenotypic markers (34). They found that
Cant123456 Mab
123456 concentration
FIG. 4. Effect of combined 3G8 and Leu-4 pretreatment on killing of Daudi target cells. (A) Lymphocytes were pretreated for 30 min at 37°C with either 3G8 (A) or Let14 (0) at starting concentrations of 1:100 of ascitic fluid and 2 pg/ml, respectively and six consecutive I:10 dilutions. (B) Combined treatments were done with 3G8 at 1:100and a similar dilution seriesof Let14 (A) or with Leu-4 at 2 pgjml and corresponding 3G8 dilutions (0).
FcR AND T3 INDUCED KILLING
165
although clones derived from B73.1 positive cells retained an LGL morphology only some expressedNK activity and most of these were T3 positive. No correlation between any surface marker and NK cell killing was found and one of their lytic clones, derived from B73.1 positive cells, did not expresseither B73.1 or T3. These combined data indicate that NK cells may use alternating membrane interactions for triggering of lysis. The addition of anti-T3 directly into short-term radioisotope release assaysresult in inhibition of T-cell lysis (4). Long-term (46-64 hr) pretreatment of T-memory cells cause the specific activation of effector cells if tested in the absenceof anti-T3 but in inhibition with antibody present in the test system (7). Long-term (16 hr) treatment of isolated large granular lymphocytes augmented killing of both Molt-4 and Daudi target cells caused by lymphokine secreting T cells present in the LGL fraction (7). In contrast, Leeuvenberg et al. did not detect any anti-T3 induction of killing with fresh peripheral cells but found induction of NK like killing in MLC generated effector cells in combination with inhibition of specific killing (6). Further analysis of the mechanism behind anti-T3 induced NK killing suggestedcrosslinking between IgG anti-T3 and Fc receptors for mouse IgG present on the target cell population (6). In our present work it is clear that anti-T3 indeed potentiate, normal unstimulated peripheral lymphocytes but that the specificity of the induced activity is slightly different from that reported by Leeuvenberg et al. We found no killing of the target cell line Raji which is reported to be positive for mouse Ig FcR (6). The induction of anti-T3dependent killing may thus depend on an alternative mechanism, i.e., the polymerization of the T3 complex to receptor structures involved in NK cell recognition such as the Ti-like, clonotypic heterodimer described by Ritz et al. (36-38). The stronger effect of anti-FcR IgG for the induction of NK killing supports the concept that induction primarily depends on events on the effector cell surface as these antibodies can interact both at the antigen specific and at the Fc end during polymerization of FcR. The regulatory influence of anti-FcR and anti-T3 include both down-regulation and up-regulation of activity. IgM anti-NK cell FcR (and IgG anti-FcR against some target cells) inhibit killing by FcR positive cells and IgG anti-FcR potentiate killing of most NK sensitive target cell lines. These effectsare important becausethey suggest novel modes of selectiveimmunomodulation in vivo asproposed (5,7). Conventionally, antibody effects have been studied in relation to target cell specific antibodies that either induce or inhibit immune reactivity, but our present data suggest that antieffector cell antibodies may be used to selectively activate or suppressdifferent effector functions. In the present work we have studied killer cells but the same approach may be used for modulation of other cell-mediated functions such as suppression or helper activity. In conclusion, IgG monoclonal antibodies against a specific NK cell-FcR potentiate killing and IgM antibodies of the same specificity suppress NK activity. Anti-T3 antibodies have similar effects as earlier reported (7). This result suggeststhat FcR may be involved in normal NK cell killing (without added antibody) and that anti-FcR, anti-T3, and other relevant antibodies should be systematically investigated for their potential capacity as immunological response modifiers. ACKNOWLEDGMENT This work was supported by grants from the Swedish Cancer Association, The Karolinska Institute, and the Swedish Medical Association.
JONDAL ET AL.
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