Proc. Nat. Acad. Sci. USAVol. 72, No. 5, pp. 1671-1675, May 1975

Augmentation of Lymphocyte Cytotoxicity by Antibody to Herpesvirus saimiriAssociated Antigens

(membrane immunoflui e/lymphoma/disease pattern/multiple serum factors)

JEAN-MARIE PREVOST*, THOMAS W. ORR, AND GARY R. PEARSONf

Viral Biology Branch, National Cancer Institute, NIH, Bethesda, Maryland 20014

Communicated by George Klein, January 27, 1975

ABSTRACT Sera from owl monkeys infected withHerpesvirus saimiri (HVS) mediated antibody-dependentlymphocyte cytotoxicity (ADLC) against virus-infectedowl monkey kidney cells. Peripheral blood lymphocytesfrom rhesus monkeys served as effector cells in this cyto-toxic assay. ADLC titers increased along with membraneimmunofluorescence (MF) titers but among some sera,the ADLC titers were much higher than expected from theMF titers, suggesting that multiple serum factors wereinvolved in mediating ADLC in this system. Absorption ofboth low and high titered sera with HVS-infected owlmonkey kidney cells removed all ADLC activity. Pre-liminary results from serial serum samples from two in-fected monkeys that developed leukemia and/or lym-phoma demonstrated that ADLC but not MF titers in-creased to high titers with progression of disease andfollowed essentially a different kinetic pattern than thatnoted by MF. The possible significance of these findings inrelation to malignant disease induced by this virus isdiscussed.

Herpesvirus saimiri, isolated in 1968 from degeneratingsquirrel monkey kidney cultures by Mel6ndez et al. (1),produces malignant lymphoma and leukemia when inoculatedinto different species of New World primates, includingmarmosets and owl monkeys (2). The target cell for trans-formation by this virus has been shown to be a thymus-derived lymphocyte (T-cell) so that the malignant diseaseinduced by HVS has the characteristics of a T-cell lymphoma(3). Disease has not been recognized, however, in its naturalhost, the squirrel monkey, even though the majority ofanimals in this species are infected by the virus as determinedfrom virus isolation experiments and serological analyses (2).Furthermore, capuchin monkeys, common marmosets, andsome owl monkeys also failed to develop lymphoma followingHVS inoculation even though they developed a persistingvirus infection similar to that noted in squirrel monkeys(4-7).A previous study provided evidence, based on the kinetics

of the development of antibody responses to virus-associatedantigens in resistant and susceptible species, which indicatedthat immunological factors might be responsible for the re-

sistance of the natural host to lymphoma induction (8).

Abbreviations: HVS, Herpesvirus saimiri; ADLC, antibody-dependent lymphocyte cytotoxicity; MF, membrane immuno-fluorescence; OMK, owl monkey kidney; PFU, plaque-formingunits; T-cell, thymus-derived lymphocyte.* Visiting scientist from Institut J. Bordet, Brussels, Belgium.t Send reprint requests to: Dr. Gary R. Pearson, National CancerInstitute, NIH, Building 37, Room lB-05, Bethesda, Md. 20014.

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The nature of the active immune factors, however, is stillunknown. Preliminary attempts to identify an active T-cellimmunity in chronically infected monkeys from differentspecies have, for the most part, been unsuccessful (unpub-lished results). Experiments were therefore undertaken todetermine the possible mechanism by which antibody directedagainst HVS-associated cell membrane antigens, previouslyidentified on the membranes of virus-infected cells (9), couldparticipate in immunity to cells infected or transformed byHVS. A question of particular interest was whether antibodydirected against HVS-associated antigens could mediate thedestruction of cells expressing these antigens through normallymphoid elements. This augmentation of in vitro lymphocytecytotoxicity by immune serum, originally described by Perl-mann and coworkers (10, 11), involves an interaction betweenantibody or antigen-antibody complexes with a non-thymusderived lymphoid cell bearing the receptor for the Fc fragmentof the immunoglobulin molecule (11). Antibody-dependentlymphocyte cytotoxicity (ADLC) has now been demonstratedwith a variety of cellular antigens including those associatedwith viruses (12-17). Evidence has also been reported whichsuggested that this cytotoxicity mechanism might indeed beactive in vivo in immunity against virus-induced malignancies(13).The results presented in this paper demonstrate that anti-

body directed against HVS-associated cell membrane antigensexpressed in productively infected cells is active in mediatingthe in vitro destruction of these cells by nonsensitized lympho-cytes. The preliminary data also indicate that the antibodytiters determined by the ADLC assay but not by membraneimmunofluorescence (MF) increase markedly with the de-velopment of malignant disease and suggest that multiplehumoral factors might be responsible for mediating ADLC inthe diseased animal.

MATERIALS AND METHODS

Virus and Cell Culture. Herpesvirus saimiri (HVS) passagedin owl monkey kidney cells (OMK) served as the virus inocu-lum for these experiments (4). The OMK cell line was main-tained in this laboratory for virus studies as previouslydescribed (9).

Membrane Antigen Production. HVS with a titer of approxi-mately 1 X 105 plaque-forming units (PFU) was adsorbed for1 hr at 370 on confluent OMK monolayers and then thecultures were incubated at 370 for 3-5 days. Cultures har-vested at this time by trypsinization usually contained a high

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RATIO OF LYMPHOID TO TARGET CELLS

FIG. 1. Cytotoxicity of different numbers of rhesus monkeylymphocytes in the presence of two different dilutions of anegative and anti-HVS positive serum on HVS-infected OMKcells. (@) Anti-HVS positive serum at final dilution 1:60, (0)positive serum diluted at 1: 240, (a) negative sample at 1: 60 and(0) at 1: 240 dilutions, (A) Cytotoxic figures for lymphoid cellsalone. Enhancement of cytotoxicity is statistically significant(P < 0.01) at all lymphoid to target cells ratios for the positiveserum and insignificant (P > 0.05) for the negative sample.

percentage of viable membrane antigen-positive cells asdetermined by MF as previously reported (9).

Preparation of Rhesus Monkey Peripheral Blood Leukocytes.Heparinized blood was collected from three to four adultrhesus monkeys by venipuncture. The blood was pooled anddiluted 1: 3 in growth media and then the leukocyte fractionseparated on Ficoll-Hypaque gradients (18) (Litton Bionetics,Inc., Kensington, Md.). The lymphocytes were washed 3 Xin RPMI plus 10% fetal calf serum and then diluted to theappropriate concentration with the same media. Differentrhesus monkeys served as blood donors in different experi-ments.

Sera. Sera were collected from owl monkeys at differenttimes after HVS infection. The serum fractions were heat-inactivated at 560 and stored at -70°. Serum dilutions weremade in phosphate-buffered saline without calcium andmagnesium.

Membrane Immunofluorescence Assay. The indirect MFassay was performed as previously described (9) using afluorescein-conjugated goat anti-human IgG (H and L-chainspecific) reagent (Hyland Laboratories, Los Angeles, Calif.).The percentage of membrane antigen-positive cells in eachpreparation was determined on a cytofluorograf (BiophysicsSystems, Inc.).

Antibody-Dependent Lymphocyte Cytotoxicity (ADLC) As-say. Sera were assayed for ADLC activity against 61Cr-

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RATIO OF LYMPHOID TO TARGET CELLS

FIG. 2. Cytotoxicity of different numbers of rhesus monkeylymphocytes in the presence of two different dilutions of a normaland anti-HVS positive serum against uninfected OMK cells.Same symbols as Fig. 1. No statistically significant (P > 0.05)enhancement is observed for either serum at the different lym-phoid to target cell ratios.

labeled infected or uninfected OMK cells using a microassaypreviously described in detail (13). The microplates, however,were incubated at 370 for 4 hr instead of 18 hr as originallyperformed (13). Cytotoxicity for the lymphocyte-serum mix-tures or lymphocytes alone was calculated as previouslydescribed (13). ADLC was calculated by subtracting the cyto-toxicity figure for lymphocytes alone from the cytotoxicityfigure in the presence of the test sera. Statistically significantADLC was determined by Student's t test (19). The finalserum dilution mediating an increase in lymphocyte cyto-toxicity significant at P < 0.05 in comparison with the cyto-toxic figure for lymphoid cells alone was chosen as the serumtiter.

Absorption of ADLC Activity. HVS-infected or uninfectedOMK cells harvested by trypsinization were washed 3 Xin phosphate buffered saline without calcium and magnesiumand containing 3% heat-inactivated fetal calf serum. Tenmillion cells were then resuspended in 1.0 ml of a test serumwhich was diluted in the buffer so that the dilution to be ab-sorbed was 8 to 16 times below its previously determined AD-LC titer. The mixtures were then incubated for 3 hr in a 37°water bath with intermittent shaking to keep the cells in sus-pension. The cells were removed by centrifugation at 4100 Xg for 1 min and the absorbed serum samples tested for residualADLC activity against HVS-infectedOMK cells.

RESULTS

Demonstration of ADLC Activity Against HVS Antigenswith Rhesus Monkey Lymphocytes. Peripheral blood lympho-cytes from a number of different species of non-human pri-mates, including rhesus, capuchin, and owl monkeys, weretested as possible effector cells in ADLC against target cells

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TABLE 1. Specificity of antibody-dependent lymphocytecytotoxicity to HVS antigens

Antibody-dependent lymphocytecytotoxicity (%)

Serum OMK HVS-OMK*

1 1.6 11.62 0.3 7.53 1.9 19.64 2.9 23.95 4.3 23.46 2.6. 20.97 1.0 16.58 0 21.89 0 26.710 0 28.511 1.1 27.212 0 36.7

* This culture contained 40-50% MF-positive cells as deter-mined with an anti-HVS reference serum.

from HVS-infected OMK cultures containing at least 50%MF-positive cells. Although lymphocytes from all the speciestested were active in this assay, it was decided to use lympho-cytes from rhesus monkeys since it was easier to get largenumbers of lymphocytes for each assay from this species thanfrom the other species available at Litton Bionetics PrimateFacility due to their larger size.The cytotoxicity of varying numbers of rhesus monkey

lymphocytes against HVS-infected OMK cells in the presenceof two different dilutions (1:60, 1:240) of a normal and ananti-HVS positive serum (MF titer of 320-640) is shown inFig. 1. The percent cytotoxicity induced by the lymphocytesfollowing incubation in the presence of the two different dilu-tions of normal serum was approximately the same at thedifferent lymphocyte to target cell ratios as that induced bylymphocytes alone. This background cytotoxicity decreasedwith decreasing numbers of lymphocytes from a high of ap-proximately 35% at 400: 1 to about 5% at a ratio of 20 to 1.In contrast to these findings, the cytotoxic activity of thelymphocyte preparation was markedly enhanced when in-cubated in the presence of a serum containing antibodies toHVS. The percent cytotoxicity induced by both serum dilu-tions at the 400 to 1 ratio was approximately 75%. The cyto-toxic activity decreased with decreasing numbers of lympho-cytes but was significantly higher than control values at everyratio tested including 20 to 1. ADLC calculated as describedin Materials and Methods was optimal (37-44%) at the threehighest ratios. Therefore, a lymphocyte to target cell ratio of100 to 1 was used in subsequent experiments.

Fig. 2 presents the cytotoxicity values for lymphocytesincubated in the presence of these same dilutions of normaland immune serum against uninfected OMK. It is apparentfrom these results that the immune serum did not enhancethe lymphocyte cytotoxicity significantly against these targetcells at any of the ratios tested.The ADLC values for 12 different sera from owl monkeys

infected with HVS against OMK and HVS-OMK are shownin Table 1. All sera were screened at a dilution of 1:60.Significant ADLC against HVS-infected OMK ranged from7.5% to 36.7%. No significant ADLC was detected against

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FIG. 3. Representative titration curves with five differentantibody positive serum samples, in ADLC cytotoxicity test, onHVS-infected OMK cells. (X) Cytotoxicity of lymphoid cellsalone.

uninfected OMK with any of the sera, demonstrating againthe HVS specificity of the reaction.

Representative titration curves for five different sera testedin the ADLC test are shown in Fig. 3. A number of differentpatterns were apparent. The cytotoxicity induced by thelower titered sera in this assay decreased with serum dilutionto the level of cytotoxicity induced by lymphocytes alone.As the titer of the test serum increased, a plateau was notedat the low serum dilutions with a subsequent decrease incytotoxicity with higher dilutions. With the very high titeredsera, a "prozone" phenomenon was observed where cyto-toxicity figures initially increased with serum dilution beforedropping. This phenomenon has been observed in other sys-tems with the ADLC assay (20).

Relation Between MF and ADLC Titers. Since the ADLCactivity in the sera from monkeys infected with HVS pre-sumably was mediated by antibody directed against HVS-associated membrane antigens, sera were titrated for anti-bodies by both the MF and ADLC assays. The comparativefindings for 42 sera are shown in Fig. 4. None of these seramediated complement-dependent cytotoxicity against HVS-infected OMK cells (H. Rabin, personal communication).MF antibody was only detected in the IgG fraction of the seraas determined with a variety of fluorescent reagents (un-published results). There was a good correlation between theantibody titers determined by both assays in that ADLCtiters tended to increase with increasing MF titers. Titersdetermined by the ADLC assay, however, were generallymuch higher than those determined by MF. Sera from mon-keys infected with Epstein-Barr virus did not contain anti-bodies cross-reactive with HVS antigens by either assay. Theexperimental findings presented in this figure also suggestthat at least with the high-titered sera, multiple serum factorsmight be active in mediating theADLC activity. Sera negativefor MF were also negative for ADLC. In sera with MF titersranging from 40 to 640, ADLC titers were comparable tothose determined by MF with some sera, but with others,titers were much higher than expected based on their MFtiters. Interestingly, one serum with an MF titer of 640 hadno detectable ADLC activity. Almost all the sera testedwith MF titers greater than 640 had extremely high ADLC

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983040 r TABLE 2. MF and ADLC titers in two owl monkeysthat developed lymphoma following HVS inoculation

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1280 5120

FIG. 4. Correlation between antibody titers detected bymembrane immunofluorescence (MF) and by ADLC cyto-toxicity tests. (0) Serum samples from HVS-infected monkeys,(0) serum samples from EBV-infected animals. The regressionline was calculated according to the least square method-correlation coefficient: r = 0.86, P < 0.01.

titers ranging up to 400,000. These findings show that not allsera were concordant for these two activities suggesting that,at least with some of the high-titered sera, factors other thanthe antibody species detected by the MF assay were par-ticipating in the ADLC reaction.To determine if the factor(s) mediating ADLC activity in

sera with low, moderate, or high titers were reacting withantigens expressed on infected OMK cells, a number of dif-ferent sera were absorbed with infected and uninfected OMKcells. Serum 1 had an ADLC titer of 640, serum 2 of 80, serum

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0SERUM 1 SERUM 2 SERUM 3 SERUM 4

FiG. 5. Absorption of ADLC activity with HVS-infectedOMK cells from four sera with different ADLC titers. (0) Un-absorbed serum, (1X) absorbed with uninfected OMK, (-)absorbed with HVS-infected OMK. Unabsorbed ADLC titers:serum (1) 640; (2) 80; (3) 80,000; and (4) 40,000. (- -) cytotox-icity of lymphocytes in the absence of serum. Absorption issignificant at P < 0.001 for all four samples after exposure toHVS-infected OMK cells only.

Months Antibody titers

Owl monkey post-inoculation MF ADLC ADLC/MF

447* 2 80 <60 <0. 754 320 3,840 12.06 640 40,960 64.0

481t 2 640 <60 <0. 104 5,120 7,680 1.56 5,120 480,000 93.8

* This monkey died approximately 6 months post-HVS infecttion from generalized malignant lymphoma.

t This animal was initially diagnosed to be leukemic at 3months post-HVS infection and survived approximately 8months before dying from leukemia and malignant lymphoma.

3 of 80,000, and serum 4 of 40,000. The results are shown inFig. 5. Absorption of all of these sera with infected but notuninfected OMK cells abolished the ADLC activity. Thus, ifmultiple factors are present in high-titered sera which arecapable of mediating ADLC, they are all directed againstHVS-associated antigens expressed in productively infectedcells.

Relation ofADLC Titers to Disease Course. To determine thedevelopment of ADLC titers to disease course in HVS-infectedowl monkeys, we performed preliminary experiments withserum samples collected at bimonthly intervals from twomonkeys that developed malignant disease following HVSinfection. MF antibody titers were also determined for com-parison purposes. These findings are shown in Table 2. Inboth monkeys, MF antibodies were detected earlier thanADLC activity and the titers increased between 2 and 4months post-inoculation but did not change significantlybetween 4 and 6 months when both animals were diseased.ADLC titers, however, showed a definite disease relatedpattern. In monkey 447, which developed malignant lym-phoma, ADLC titers increased from less than 60 at 2 monthspost-inoculation to almost 41,000 at 6 months. Owl monkey481 died of malignant lymphoma and leukemia. The ADLCtiters in serum samples from this monkey increased from lessthan 60 to almost 500,000 over this 6-month period. Thisprogressive increase in ADLC but not MF titers with diseaseis also reflected in the ADLC/MF figures presented for bothmonkeys in this Table. These findings indicated that theprogression of ADLC titers in the HVS system was related todisease course and again suggested the participation of mul-tiple humoral factors in the mediation of this activity.

DISCUSSIONThe findings reported here demonstate that antibody directedagainst HVS-associated antigens can mediate ADLC againstproductively infected cells expressing the appropriate viralantigens. These results confirm reports by others on thisantibody-dependent cytotoxic reaction and extend the numberof virus systems where this cytotoxic reaction has been dem-onstrated (12-17). Although these findings demonstrate thiscytotoxic reaction against acutely infected cells, the questionof whether the ADLC assay might also detect antigens in themembranes of nonproducer transformed cells still remains tobe answered. Initial attempts to demonstrate ADLC against

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Antibody-Dependent Lymphocyte Cytotoxicity 1675

a HVS-transformed marmoset cell line (21) not expressingmembrane antigens detectable by MF (22) have so far yieldedequivocal results (unpublished findings). Further studies areneeded with nonproducer cell lines and fresh biopsy cellsbefore it can be determined whether this in vitro assay mightbe useful for demonstrating new antigens on cells showingminimum virus expression.ADLC titers in general tended to increase along with MF

titers indicating that antibody detected by the MF assaycould also be involved in the ADLC reaction. However, therewere a number of discordant sera based on titer determina-tions in the two assays which suggested that other factorsmight also be active in mediating ADLC. In the group ofsera with MF titers between 40 and 640, ADLC titers showeda wide variation. Some sera had ADLC titers comparable totiters determined by the MF assay while, in other sera, theADLC titers were much higher than expected, ranging as highas 100,000. Interestingly, there was also one serum in this groupwith an MF titer of 640 but no detectable ADLC. As shownin Table 2, this serum sample was collected two monthspostinfection with HVS at a time when the monkey was freeof clinical signs of disease. Sera with MF titers greater than640 usually had very high ADLC titers ranging from 30,000to almost 500,000. These findings suggest the involvement ofmultiple serum factors in the mediation of ADLC. The exactnature of these mediators is still unknown. However, thefollowing possibilities are under consideration: (1) an antibodyspecies different from the 7S antibody detected in the MFtest but directed at the same antigen; (2) multiple antibodiesdirected against a variety of HVS-associated membraneantigen specificities, not all of which can also be detected bythe MF assay; and (3) antigen-antibody complexes since ithas been reported that complexes can mediate ADLC in somesystems (11). The results from the absorption experimentsreported here suggest that complexes may not be involved inthe reaction in this system since all ADLC activity wasremoved even from the high titered serum (ADLC titer of80,000) by HVS-infected OMK cells. It is difficult to visualizewhy complexes would be removed by this absorption proce-dure although this possibility can still not be completelyruled out. Preliminary attempts to absorb ADLC activitywith rhesus monkey lymphocytes have been unsuccessful.Absorption studies with a larger number of sera collected atdifferent times during the disease process are still needed.These preliminary findings, however, would tend to favor thefirst two possibilities discussed above. Fractionation studiesshould provide more definitive information on this question.

Unexpectedly, results from serial serum samples collectedat different time intervals following HVS infection from twoanimals that developed lymphoma showed that the increasesin ADLC titers correlated with progressing disease. This wasunexpected since published findings from experiments in othervirus systems showed that the highest titers of ADLC ac-tivity were usually detected in the sera of immune and nottumor-bearing animals (13, 14). The reason for this differenceis unclear. However, these preliminary findings reported hereindicate that in the HVS system the development of ADLCactivity correlated with malignant disease progression andthereby raises questions on the in vivo significance of this

activity against lymphomas induced by this virus. In checkingthe records of other sera shown in Fig. 4, it was found that themajority of the high-titered sera were also collected frommonkeys with active disease, supporting this relationship.Results from five HVS-infected owl monkeys now under studysuggest a similar trend in the development of ADLC titers(to be published). Therefore, the ADLC assay may be anotheruseful parameter in addition to the ones already described(4, 5) for monitoring the induction and development ofmalignant disease in non-human primates infected with HVS.The potential usefulness of this assay for monitoring humanswith herpesvirus-associated malignancies has yet to be de-termined.

J.-M.P; is a Fulbright Hays Scholar and is supported by agrant from the R. and J. Hoguet Foundation, Brussels, Belgium.Rhesus monkey blood was provided through Contract no.NO1-CP4-3224 from the Virus Cancer Program to LittonBionetics, Inc., Kensington, Md.

1. Mel6ndez, L. V., Daniel, M. D., Hunt, R. D. & Garcia, F. G.(1968) Lab. Anim. Care 18, 374-381.

2. Deinhardt, F. (1973) in The Herpesviruses, ed. Kaplan, A. S.(Academic Press, Inc., New York), pp. 595-625.

3. Wallen, W. C., Neubauer, R. H., Rabin, H. & Cicmanec, J.(1973) J. Nat. Cancer Inst. 51, 967-975.

4. Pearson, G. R., Orr, T. W., Rabin, H., Cicmanec, J.,Ablashi, D. & Armstrong, G. (1973) J. Nat. Cancer Inst. 51,1939-1943.

5. Pearson, G. R., Rabin, H., Wallen, W. C., Neubauer, R. H.,Orr, T. W. & Cicmanec, J. L. (1974) J. Med. Primatol. 3,54-67.

6. Rabin, H., Pearson, G. R., Wallen, W. C., Neubauer, R. H.,Ciemanec, J. L. & Orr, T. W. (1975) J. Nat. Cancer Inst.,in press.

7. Laufs, R., Steinke, H., Steinke, G. & Petzold, D. (1974) J.Nat. CancerInst. 53, 195-200.

8. Klein, G., Pearson, G., Rabson, A., Ablashi, D. V., Falk, L.,Wolfe, L., Deinhardt, F. & Rabin, H. (1973) Int. J. Cancer12, 270-289.

9. Pearson, G., Ablashi, D., Orr, T., Rabin, H. & Armstrong, G.(1972) J. Nat. Cancer Inst. 49, 1417-1424.

10. Perlmann, P. & Holm, G. (1969) Advan. Immunol. 11, 117-193.

11. Perlmann, P., Perlmann, H. & Wigzell, H. (1972) Trans-plant. Rev. 13, 91-114.

12. Pollack, S., Heppner, G., Brawn, R. J. & Nelson, K. (1972)Int. J. Cancer 9, 316-323.

13. Harada, M., Pearson, G., Pettigrew, H., Redmon, L. &Orr, T. (1973) Cancer Res. 33, 2886-2893.

14. de Landazuri, M. O., Kedar, E. & Fahey, J. L. (1974) J.Nat. Cancer Inst. 52, 147-152.

15. Skurzak, H. M., Klein, E., Yoshida, T. 0. & Lamon, E. W.(1972) J. Exp. Med. 135, 997-1002.

16. Shore, S. L., Nahmias, A. J., Starr, S. E., Wood, P. A. &McFarlin, D. E. (1974) Nature 251, 350-352.

17. Rager-Zisman, B. & Bloom, B. R. (1974) Nature 251, 542-543.

18. Boyum, A. (1962) Scand. J. Clin. Lab. Invest. 21, 1.19. Snedecor, G. W. & Cochran, W. G. (1967) Statistical

Methods (The Iowa State University Press, Ames, Iowa),6th ed.

20. Perlmann, P. & Perlmann, H. (1970) Cell. Immunol. 1,300-315.

21. Rabson, A. S., O'Conor, G. T., Lorenz, D. E., Kirschstein,R. L., Legallais, F. Y. & Tralka, T. S. (1971) J. Nat. CaneerInst. 46, 1099-1109.

22. Neubauer, R. H., Wallen, W. C. & Rabin, H. (1974) J.Virol. 14, 745-750.

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