Associated Antigens (membrane immunoflui

Transcription

1 Proc. Nat. Acad. Sci. USA Vol. 72, No. 5, pp , May 1975 Augmentation of Lymphocyte Cytotoxicity by Antibody to Herpesvirus saimiri Associated 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 214 Communicated by George Klein, January 27, 1975 ABSTRACT Sera from owl monkeys infected with Herpesvirus saimiri (HVS) mediated antibody-dependent lymphocyte cytotoxicity (ADLC) against virus-infected owl monkey kidney cells. Peripheral blood lymphocytes from rhesus monkeys served as effector cells in this cytotoxic assay. ADLC titers increased along with membrane immunofluorescence (MF) titers but among some sera, the ADLC titers were much higher than expected from the MF titers, suggesting that multiple serum factors were involved in mediating ADLC in this system. Absorption of both low and high titered sera with HVS-infected owl monkey kidney cells removed all ADLC activity. Preliminary results from serial serum samples from two infected monkeys that developed leukemia and/or lymphoma demonstrated that ADLC but not MF titers increased to high titers with progression of disease and followed essentially a different kinetic pattern than that noted by MF. The possible significance of these findings in relation to malignant disease induced by this virus is discussed. Herpesvirus saimiri, isolated in 1968 from degenerating squirrel monkey kidney cultures by Mel6ndez et al. (1), produces malignant lymphoma and leukemia when inoculated into different species of New World primates, including marmosets and owl monkeys (2). The target cell for transformation by this virus has been shown to be a thymusderived lymphocyte (T-cell) so that the malignant disease induced by HVS has the characteristics of a T-cell lymphoma (3). Disease has not been recognized, however, in its natural host, the squirrel monkey, even though the majority of animals in this species are infected by the virus as determined from virus isolation experiments and serological analyses (2). Furthermore, capuchin monkeys, common marmosets, and some owl monkeys also failed to develop lymphoma following HVS inoculation even though they developed a persisting virus 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-associated antigens in resistant and susceptible species, which indicated that immunological factors might be responsible for the resistance of the natural host to lymphoma induction (8). Abbreviations: HVS, Herpesvirus saimiri; ADLC, antibodydependent lymphocyte cytotoxicity; MF, membrane immunofluorescence; OMK, owl monkey kidney; PFU, plaque-forming units; T-cell, thymus-derived lymphocyte. * Visiting scientist from Institut J. Bordet, Brussels, Belgium. t Send reprint requests to: Dr. Gary R. Pearson, National Cancer Institute, NIH, Building 37, Room lb-5, Bethesda, Md The nature of the active immune factors, however, is still unknown. Preliminary attempts to identify an active T-cell immunity in chronically infected monkeys from different species have, for the most part, been unsuccessful (unpublished results). Experiments were therefore undertaken to determine the possible mechanism by which antibody directed against HVS-associated cell membrane antigens, previously identified on the membranes of virus-infected cells (9), could participate in immunity to cells infected or transformed by HVS. A question of particular interest was whether antibody directed against HVS-associated antigens could mediate the destruction of cells expressing these antigens through normal lymphoid elements. This augmentation of in vitro lymphocyte cytotoxicity by immune serum, originally described by Perlmann and coworkers (1, 11), involves an interaction between antibody or antigen-antibody complexes with a non-thymus derived lymphoid cell bearing the receptor for the Fc fragment of the immunoglobulin molecule (11). Antibody-dependent lymphocyte cytotoxicity (ADLC) has now been demonstrated with a variety of cellular antigens including those associated with viruses (12-17). Evidence has also been reported which suggested that this cytotoxicity mechanism might indeed be active in vivo in immunity against virus-induced malignancies (13). The results presented in this paper demonstrate that antibody directed against HVS-associated cell membrane antigens expressed in productively infected cells is active in mediating the in vitro destruction of these cells by nonsensitized lymphocytes. The preliminary data also indicate that the antibody titers determined by the ADLC assay but not by membrane immunofluorescence (MF) increase markedly with the development of malignant disease and suggest that multiple humoral factors might be responsible for mediating ADLC in the diseased animal. MATERIALS AND METHODS Virus and Cell Culture. Herpesvirus saimiri (HVS) passaged in owl monkey kidney cells (OMK) served as the virus inoculum for these experiments (4). The OMK cell line was maintained in this laboratory for virus studies as previously described (9). Membrane Antigen Production. HVS with a titer of approximately 1 X 15 plaque-forming units (PFU) was adsorbed for 1 hr at 37 on confluent OMK monolayers and then the cultures were incubated at 37 for 3-5 days. Cultures harvested at this time by trypsinization usually contained a high

2 1672 Immunology: Prevost et al. Proc. Nat. Acad. Sci. USA 72 (1975) 8 7F 6 - ~ X 5 -)4 percentage of viable membrane antigen-positive cells as determined by MF as previously reported (9). Preparation of Rhesus Monkey Peripheral Blood Leukocytes. Heparinized blood was collected from three to four adult rhesus monkeys by venipuncture. The blood was pooled and diluted 1: 3 in growth media and then the leukocyte fraction separated on Ficoll-Hypaque gradients (18) (Litton Bionetics, Inc., Kensington, Md.). The lymphocytes were washed 3 X in RPMI plus 1% fetal calf serum and then diluted to the appropriate concentration with the same media. Different rhesus monkeys served as blood donors in different experiments. Sera. Sera were collected from owl monkeys at different times after HVS infection. The serum fractions were heatinactivated at 56 and stored at -7. Serum dilutions were made in phosphate-buffered saline without calcium and magnesium. Membrane Immunofluorescence Assay. The indirect MF assay was performed as previously described (9) using a fluorescein-conjugated goat anti-human IgG (H and L-chain specific) reagent (Hyland Laboratories, Los Angeles, Calif.). The percentage of membrane antigen-positive cells in each preparation was determined on a cytofluorograf (Biophysics Systems, Inc.). Antibody-Dependent Lymphocyte Cytotoxicity (ADLC) Assay. Sera were assayed for ADLC activity against 61Cr- 31-2F 1 1 C ' RATIO OF LYMPHOID TO TARGET CELLS FIG. 1. Cytotoxicity of different numbers of rhesus monkey lymphocytes in the presence of two different dilutions of a negative and anti-hvs positive serum on HVS-infected OMK cells. (@) Anti-HVS positive serum at final dilution 1:6, () positive serum diluted at 1: 24, (a) negative sample at 1: 6 and () at 1: 24 dilutions, (A) Cytotoxic figures for lymphoid cells alone. Enhancement of cytotoxicity is statistically significant (P <.1) at all lymphoid to target cells ratios for the positive serum and insignificant (P >.5) for the negative sample RATIO OF LYMPHOID TO TARGET CELLS FIG. 2. Cytotoxicity of different numbers of rhesus monkey lymphocytes in the presence of two different dilutions of a normal and anti-hvs positive serum against uninfected OMK cells. Same symbols as Fig. 1. No statistically significant (P >.5) enhancement is observed for either serum at the different lymphoid to target cell ratios. labeled infected or uninfected OMK cells using a microassay previously described in detail (13). The microplates, however, were incubated at 37 for 4 hr instead of 18 hr as originally performed (13). Cytotoxicity for the lymphocyte-serum mixtures or lymphocytes alone was calculated as previously described (13). ADLC was calculated by subtracting the cytotoxicity figure for lymphocytes alone from the cytotoxicity figure in the presence of the test sera. Statistically significant ADLC was determined by Student's t test (19). The final serum dilution mediating an increase in lymphocyte cytotoxicity significant at P <.5 in comparison with the cytotoxic figure for lymphoid cells alone was chosen as the serum titer. Absorption of ADLC Activity. HVS-infected or uninfected OMK cells harvested by trypsinization were washed 3 X in phosphate buffered saline without calcium and magnesium and containing 3% heat-inactivated fetal calf serum. Ten million cells were then resuspended in 1. ml of a test serum which was diluted in the buffer so that the dilution to be absorbed 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 suspension. The cells were removed by centrifugation at 41 X g for 1 min and the absorbed serum samples tested for residual ADLC activity against HVS-infected OMK cells. RESULTS Demonstration of ADLC Activity Against HVS Antigens with Rhesus Monkey Lymphocytes. Peripheral blood lymphocytes from a number of different species of non-human primates, including rhesus, capuchin, and owl monkeys, were tested as possible effector cells in ADLC against target cells

3 Proc. Nat. Acad. Sci. USA 72 (1975) TABLE 1. Specificity of antibody-dependent lymphocyte cytotoxicity to HVS antigens Antibody-dependent lymphocyte cytotoxicity (%) Serum OMK HVS-OMK* * This culture contained 4-5% MF-positive cells as determined with an anti-hvs reference serum. from HVS-infected OMK cultures containing at least 5% MF-positive cells. Although lymphocytes from all the species tested were active in this assay, it was decided to use lymphocytes from rhesus monkeys since it was easier to get large numbers of lymphocytes for each assay from this species than from the other species available at Litton Bionetics Primate Facility due to their larger size. The cytotoxicity of varying numbers of rhesus monkey lymphocytes against HVS-infected OMK cells in the presence of two different dilutions (1:6, 1:24) of a normal and an anti-hvs positive serum (MF titer of 32-64) is shown in Fig. 1. The percent cytotoxicity induced by the lymphocytes following incubation in the presence of the two different dilutions of normal serum was approximately the same at the different lymphocyte to target cell ratios as that induced by lymphocytes alone. This background cytotoxicity decreased with decreasing numbers of lymphocytes from a high of approximately 35% at 4: 1 to about 5% at a ratio of 2 to 1. In contrast to these findings, the cytotoxic activity of the lymphocyte preparation was markedly enhanced when incubated in the presence of a serum containing antibodies to HVS. The percent cytotoxicity induced by both serum dilutions at the 4 to 1 ratio was approximately 75%. The cytotoxic activity decreased with decreasing numbers of lymphocytes but was significantly higher than control values at every ratio tested including 2 to 1. ADLC calculated as described in Materials and Methods was optimal (37-44%) at the three highest ratios. Therefore, a lymphocyte to target cell ratio of 1 to 1 was used in subsequent experiments. Fig. 2 presents the cytotoxicity values for lymphocytes incubated in the presence of these same dilutions of normal and immune serum against uninfected OMK. It is apparent from these results that the immune serum did not enhance the lymphocyte cytotoxicity significantly against these target cells 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 shown in Table 1. All sera were screened at a dilution of 1:6. Significant ADLC against HVS-infected OMK ranged from 7.5% to 36.7%. No significant ADLC was detected against Antibody-Dependent Lymphocyte Cytotoxicity 1673 x -2 3r SERUM DILUTION FIG. 3. Representative titration curves with five different antibody positive serum samples, in ADLC cytotoxicity test, on HVS-infected OMK cells. (X) Cytotoxicity of lymphoid cells alone. uninfected OMK with any of the sera, demonstrating again the HVS specificity of the reaction. Representative titration curves for five different sera tested in the ADLC test are shown in Fig. 3. A number of different patterns were apparent. The cytotoxicity induced by the lower titered sera in this assay decreased with serum dilution to the level of cytotoxicity induced by lymphocytes alone. As the titer of the test serum increased, a plateau was noted at the low serum dilutions with a subsequent decrease in cytotoxicity with higher dilutions. With the very high titered sera, a "prozone" phenomenon was observed where cytotoxicity figures initially increased with serum dilution before dropping. This phenomenon has been observed in other systems with the ADLC assay (2). Relation Between MF and ADLC Titers. Since the ADLC activity in the sera from monkeys infected with HVS presumably was mediated by antibody directed against HVSassociated membrane antigens, sera were titrated for antibodies by both the MF and ADLC assays. The comparative findings for 42 sera are shown in Fig. 4. None of these sera mediated complement-dependent cytotoxicity against HVSinfected OMK cells (H. Rabin, personal communication). MF antibody was only detected in the IgG fraction of the sera as determined with a variety of fluorescent reagents (unpublished results). There was a good correlation between the antibody titers determined by both assays in that ADLC titers tended to increase with increasing MF titers. Titers determined by the ADLC assay, however, were generally much higher than those determined by MF. Sera from monkeys infected with Epstein-Barr virus did not contain antibodies cross-reactive with HVS antigens by either assay. The experimental findings presented in this figure also suggest that at least with the high-titered sera, multiple serum factors might be active in mediating the ADLC activity. Sera negative for MF were also negative for ADLC. In sera with MF titers ranging from 4 to 64, ADLC titers were comparable to those determined by MF with some sera, but with others, titers were much higher than expected based on their MF titers. Interestingly, one serum with an MF titer of 64 had no detectable ADLC activity. Almost all the sera tested with MF titers greater than 64 had extremely high ADLC

4 1674 Immunology: Prevost et al. Proc. Nat. Acad. Sci. USA 72 (1975) Crx 9834 r TABLE 2. MF and ADLC titers in two owl monkeys that developed lymphoma following HVS inoculation F /. a a Months Antibody titers 6144 Owl monkey post-inoculation MF ADLC ADLC/MF <6 / S 447* 2 8 <6 < , , t 2 64 <6 < ,12 7, ,12 48, 93.8 * This monkey died approximately 6 months post-hvs infect tion from generalized malignant lymphoma. t This animal was initially diagnosed to be leukemic at 3 months post-hvs infection and survived approximately 8 months before dying from leukemia and malignant lymphoma. < MF TITER FIG. 4. Correlation between antibody titers detected by membrane immunofluorescence (MF) and by ADLC cytotoxicity tests. () Serum samples from HVS-infected monkeys, () serum samples from EBV-infected animals. The regression line was calculated according to the least square methodcorrelation coefficient: r =.86, P <.1. titers ranging up to 4,. These findings show that not all sera were concordant for these two activities suggesting that, at least with some of the high-titered sera, factors other than the antibody species detected by the MF assay were participating in the ADLC reaction. To determine if the factor(s) mediating ADLC activity in sera with low, moderate, or high titers were reacting with antigens expressed on infected OMK cells, a number of different sera were absorbed with infected and uninfected OMK cells. Serum 1 had an ADLC titer of 64, serum 2 of 8, serum 6, SERUM 1 SERUM 2 SERUM 3 SERUM 4 FiG. 5. Absorption of ADLC activity with HVS-infected OMK cells from four sera with different ADLC titers. () Unabsorbed serum, (1X) absorbed with uninfected OMK, (-) absorbed with HVS-infected OMK. Unabsorbed ADLC titers: serum (1) 64; (2) 8; (3) 8,; and (4) 4,. ( - -) cytotoxicity of lymphocytes in the absence of serum. Absorption is significant at P <.1 for all four samples after exposure to HVS-infected OMK cells only. 3 of 8,, and serum 4 of 4,. The results are shown in Fig. 5. Absorption of all of these sera with infected but not uninfected OMK cells abolished the ADLC activity. Thus, if multiple factors are present in high-titered sera which are capable of mediating ADLC, they are all directed against HVS-associated antigens expressed in productively infected cells. Relation of ADLC Titers to Disease Course. To determine the development of ADLC titers to disease course in HVS-infected owl monkeys, we performed preliminary experiments with serum samples collected at bimonthly intervals from two monkeys that developed malignant disease following HVS infection. MF antibody titers were also determined for comparison purposes. These findings are shown in Table 2. In both monkeys, MF antibodies were detected earlier than ADLC activity and the titers increased between 2 and 4 months post-inoculation but did not change significantly between 4 and 6 months when both animals were diseased. ADLC titers, however, showed a definite disease related pattern. In monkey 447, which developed malignant lymphoma, ADLC titers increased from less than 6 at 2 months post-inoculation to almost 41, at 6 months. Owl monkey 481 died of malignant lymphoma and leukemia. The ADLC titers in serum samples from this monkey increased from less than 6 to almost 5, over this 6-month period. This progressive increase in ADLC but not MF titers with disease is also reflected in the ADLC/MF figures presented for both monkeys in this Table. These findings indicated that the progression of ADLC titers in the HVS system was related to disease course and again suggested the participation of multiple humoral factors in the mediation of this activity. DISCUSSION The findings reported here demonstate that antibody directed against HVS-associated antigens can mediate ADLC against productively infected cells expressing the appropriate viral antigens. These results confirm reports by others on this antibody-dependent cytotoxic reaction and extend the number of virus systems where this cytotoxic reaction has been demonstrated (12-17). Although these findings demonstrate this cytotoxic reaction against acutely infected cells, the question of whether the ADLC assay might also detect antigens in the membranes of nonproducer transformed cells still remains to be answered. Initial attempts to demonstrate ADLC against

5 Proc. Nat. Acad. Sci. USA 72 (1975) a HVS-transformed marmoset cell line (21) not expressing membrane antigens detectable by MF (22) have so far yielded equivocal results (unpublished findings). Further studies are needed with nonproducer cell lines and fresh biopsy cells before it can be determined whether this in vitro assay might be useful for demonstrating new antigens on cells showing minimum virus expression. ADLC titers in general tended to increase along with MF titers indicating that antibody detected by the MF assay could also be involved in the ADLC reaction. However, there were a number of discordant sera based on titer determinations in the two assays which suggested that other factors might also be active in mediating ADLC. In the group of sera with MF titers between 4 and 64, ADLC titers showed a wide variation. Some sera had ADLC titers comparable to titers determined by the MF assay while, in other sera, the ADLC titers were much higher than expected, ranging as high as 1,. Interestingly, there was also one serum in this group with an MF titer of 64 but no detectable ADLC. As shown in Table 2, this serum sample was collected two months postinfection with HVS at a time when the monkey was free of clinical signs of disease. Sera with MF titers greater than 64 usually had very high ADLC titers ranging from 3, to almost 5,. These findings suggest the involvement of multiple serum factors in the mediation of ADLC. The exact nature of these mediators is still unknown. However, the following possibilities are under consideration: (1) an antibody species different from the 7S antibody detected in the MF test but directed at the same antigen; (2) multiple antibodies directed against a variety of HVS-associated membrane antigen specificities, not all of which can also be detected by the MF assay; and (3) antigen-antibody complexes since it has been reported that complexes can mediate ADLC in some systems (11). The results from the absorption experiments reported here suggest that complexes may not be involved in the reaction in this system since all ADLC activity was removed even from the high titered serum (ADLC titer of 8,) by HVS-infected OMK cells. It is difficult to visualize why complexes would be removed by this absorption procedure although this possibility can still not be completely ruled out. Preliminary attempts to absorb ADLC activity with rhesus monkey lymphocytes have been unsuccessful. Absorption studies with a larger number of sera collected at different times during the disease process are still needed. These preliminary findings, however, would tend to favor the first two possibilities discussed above. Fractionation studies should provide more definitive information on this question. Unexpectedly, results from serial serum samples collected at different time intervals following HVS infection from two animals that developed lymphoma showed that the increases in ADLC titers correlated with progressing disease. This was unexpected since published findings from experiments in other virus systems showed that the highest titers of ADLC activity were usually detected in the sera of immune and not tumor-bearing animals (13, 14). The reason for this difference is unclear. However, these preliminary findings reported here indicate that in the HVS system the development of ADLC activity correlated with malignant disease progression and thereby raises questions on the in vivo significance of this Antibody-Dependent Lymphocyte Cytotoxicity 1675 activity against lymphomas induced by this virus. In checking the records of other sera shown in Fig. 4, it was found that the majority of the high-titered sera were also collected from monkeys with active disease, supporting this relationship. Results from five HVS-infected owl monkeys now under study suggest a similar trend in the development of ADLC titers (to be published). Therefore, the ADLC assay may be another useful parameter in addition to the ones already described (4, 5) for monitoring the induction and development of malignant disease in non-human primates infected with HVS. The potential usefulness of this assay for monitoring humans with herpesvirus-associated malignancies has yet to be determined. J.-M.P; is a Fulbright Hays Scholar and is supported by a grant from the R. and J. Hoguet Foundation, Brussels, Belgium. Rhesus monkey blood was provided through Contract no. NO1-CP from the Virus Cancer Program to Litton Bionetics, Inc., Kensington, Md. 1. Mel6ndez, L. V., Daniel, M. D., Hunt, R. D. & Garcia, F. G. (1968) Lab. Anim. Care 18, Deinhardt, F. (1973) in The Herpesviruses, ed. Kaplan, A. S. (Academic Press, Inc., New York), pp Wallen, W. C., Neubauer, R. H., Rabin, H. & Cicmanec, J. (1973) J. Nat. Cancer Inst. 51, Pearson, G. R., Orr, T. W., Rabin, H., Cicmanec, J., Ablashi, D. & Armstrong, G. (1973) J. Nat. Cancer Inst. 51, Pearson, G. R., Rabin, H., Wallen, W. C., Neubauer, R. H., Orr, T. W. & Cicmanec, J. L. (1974) J. Med. Primatol. 3, 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. 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