Amplification of Rabies Virus-Induced Stimulation of Human T-Cell Lines and Clones by Antigen-Specific Antibodies

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1 JOURNAL OF VIROLOGY, Nov. 1985, p Vol. 56, No X/85/ $2./ Copyright C) 1985, American Society for Microbiology Amplification of Rabies Virus-Induced Stimulation of Human T-Cell Lines and Clones by Antigen-Specific Antibodies ESTEBAN CELIS,lt* TADEUSZ J. WIKTOR,2 BERNHARD DIETZSCHOLD,2 AND HILARY KOPROWSKI2 Centocor, Malvern, Pennsylvania 19355,1 and The Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania Received 8 April 1985/Accepted 3 July 1985 The effect of antigen-specific antibodies on the response of human T-cell lines and clones to rabies virus was studied. Plasmas from rabies-immune vaccine recipients, but not those from nonimmune individuals, enhanced the proliferative response of rabies-reactive T cells to whole inactivated virus or to the purified glycoprotein and nucleocapsid from the rabies virion. Rabies-immune plasma also increased the antigen-induced production of gamma interferon by the rabies-specific T-cell lines. Experiments performed on T-cell clones specific for either rabies glycoprotein or nucleocapsid showed that immune plasma as well as antiglycoprotein and antinucleoprotein murine monoclonal antibodies possessed the capacity to increase significantly the antigeninduced proliferative responses of these clones. The overall results indicate that this in vitro effect of antigen-specific antibodies on the response of regulatory T lymphocytes to rabies virus could be an important factor in the development of effective immune responses in vivo to rabies virus. Antibodies are one of the major end products of immune responses and possess a large variety of effector functions all directed towards one main goal, the elimination of an antigenic threat. When an effective immune response is triggered by an antigen, the resultant antibodies will attempt to destroy the infectious agents that carry those antigenic determinants that were present in the antigen that originated the response. The elimination of an infectious organism by antibodies is usually achieved by several mechanisms, which include neutralization, complement-mediated lysis, opsonization, and antibody-dependent cellular cytotoxicity (5). The synthesis of antibodies is a direct function of B lymphocytes, and in most cases this synthesis takes place only with the direct intervention of regulatory T cells. Helper T lymphocytes, which are also triggered by the antigenic stimulus, deliver specific signals to the B cells, inducing them to proliferate and differentiate into antibody-producing plasma cells (1, 12). However, helper T cells, unlike antibodies, do not bind soluble antigens directly but will do so when the antigen is displayed on the surface of accessory cells. Macrophages and monocytes are some of the cells known to capture, ingest, process, and present the antigen on their surface (2). It is only then that the helper T cells will interact with the antigen in combination with class II major histocompatibility molecules (DR or Ta) expressed on the accessory cell surface (11, 13, 17). Aside from the effector functions of antibodies, these molecules are also capable of regulating the response of T cells to antigens and thus indirectly their own synthesis (2, 4, 15, 19, 21). Both effector and regulatory properties of antibodies are restricted to certain classes and subclasses of immunoglobulins, and most of these properties seem to be mediated through the Fc portion of their molecules (3, 5, 19). We recently described the isolation of human T-cell lines * Corresponding author. t Present address: E. I. Du Pont de Nemours & Co., Biomedical Products Department, Glasgow Research Laboratory, Wilmington, DE and clones reactive with rabies virus (E. Celis, R. W. Miller, T. J. Wiktor, B. Dietzchold, and H. Koprowski, submitted for publication). Peripheral blood mononuclear cells (PBM) from five vaccine recipients were stimulated in vitro with inactivated rabies virus in the presence of accessory cells and interleukin-2 (IL2). After two to three stimulation cycles, long-term-growth T-cell lines were obtained that specifically proliferated to whole rabies virus and to purified preparations of rabies glycoprotein (G) and nucleocapsid (NC). Significant amounts of gamma interferon (IFN--y) were synthesized by these T-cell lines when they were stimulated by the rabies antigens. Most of the rabies-reactive T-cells from all five vaccine recipients belonged to the T4+-T8- (helper-inducer) subclass of T lymphocytes. Clones selected from two of the T-cell lines reacted specifically to the rabies G or NC. The present work deals with the regulatory effect of antibodies on the response of antigen-specific T cells to rabies antigens. The results point to the important role that antirabies antibodies play in potentiating the antigen-induced proliferative response and production of IFN--y of T lymphocytes specific for rabies virus. The present observations could be of help in understanding some of the mechanisms involved in the protective active immune response to rabies virus when both the vaccine (antigen) and rabies immune globulin are used together in the postexposure treatment of rabies (1). MATERIALS AND METHODS Antigens and antibodies. Inactivated rabies virus strain PM, for cell culture (rabies vaccine), was obtained from the Institut Merieux, Lyon, France. The contents of each vial were suspended in 1 ml of culture medium and assigned a concentration of 1, arbitrary units per ml. For one experiment, rabies vaccine was suspended in 7% (vol/vol) formic acid at a concentration of 1, U/ml to completely denature the proteins. After 48 h at 4 C, the formic acid was evaporated by vacuum centrifugation, and the dried antigen preparation was suspended in culture medium. Radioimmunoassay with human antirabies immunoglobulin G (IgG) Downloaded from on August 27, 218 by guest

2 VOL. 56, 1985 ANTIBODY REGULATION OF RABIES-SPECIFIC T CELLS 427 revealed that the formic acid-treated rabies vaccine preparation did not react with the antibodies and thus appeared to have been irreversibly denatured by the organic acid. Purified rabies G and NC were prepared as described elsewhere (6, 16) Ṗlasmas from rabies vaccine recipients and from normal, nonimniune individuals were used as sources of human polyclonal antirabies antibodies and controls, respectively. The plasmas from the rabies vaccinees were found to have antibodies specific for both rabies G and rabies NC. Antirabies-specific human IgG was purified by chromatography on protein A-Sepharose (Sigma Chemical Co., St. Louis, Mo.). The mouse monoclonal antirabies antibodies 52-2 and 59-6 were prepared by somatic cell hybridization techniques as described elsewhere (7, 8). Antibodies 52-2 and 59-6 were found to be specific for rabies nucleoprotein (N) from the NC (7) and for rabies G (8), respectively. Ascitic fluids containing 3 to 5 mg of monoclonal antibodies per ml were used as a source of antirabies antibodies. Hepatitis B surface antigen (HBsAg) was purified from the plasma of a chronic carrier as described elsewhere (3). The mouse monoclonal anti-hbsag antibody A5C3 (IgG2a) was obtained and purified as reported elsewhere (3). Immune complexes were prepared by mixing A5C3 and HBsAg at a molar ratio of 1:1 (antibodies to antigen) and incubating the mixture at 4 C for at least 48 h. Rabies-specific T-celi lines and clones. The isolation of the IL2-dependent T-cell lines (RBL-1, RBL-2, RBL-3, RBL-4, and RBL-5) and clones (RBC-9G and RBC-4NC) was described in detail elsewhere (Celis et al., submitted). Briefly, PBM from five rabies vaccine recipients were incubated in vitro in the presence of 4 U of rabies vaccine per ml for 1 week. After this time, the antigen-reactive cells were expanded with a commercial source of IL2 (T Cell Growth Supplement, Meloy Laboratories, Springfield, Va.). Clones of these T-cell lines were selected by a limiting dilution in 96-flat-well plates (Costar, Cambridge, Mass.) by placing 1 cell per well. Clones RBC-9G and RBC-4NC were derived from RBL-1 and were shown to react specifically with rabies G and NC, respectively. The T-cell lines and clones were maintained in culture by weekly stimulation with 4 U of rabies vaccine per ml, IL2, and autologous-irradiated (2, rads) PBM as a source of accessory cells. The culture medium consisted of RPMI 164 (KC Biologicals, Lenexa, Kans.) with 25 mm L-glutamine, 25 mm HEPES (N-2- hydroxyethylpiperazine-n'-2-ethanesulfonic acid), 5 jig of gentamicin per ml, 5 x 1-5 M 2-mercaptoethanol, and 1% heat-inactivated fetal calf serum (HyClone, Sterile Systems Inc., Logan, Utah). Cells were maintained in culture at 37 C in a 5% C2-humid air incubator. Cell proliferation assays. For every well of a 96-flat-well plate, 2 x 14 rabies-reactive T cells were incubated with 15 autologous irradiated PBM and different concentrations of antigens and antibodies for 3 days. In one experiment, the autologous irradiated PBM or antigen-presenting cells (APC) were pulsed with antigen (rabies vaccine, 5 U/ml) at 16 cells per ml in the presence or absence of HBsAg immune complexes (1,ug of ASC3 plus 1,g of HBsAg per ml) for 1 h at 37 C in culture medium. After the APC were washed three times by centrifugation, they were incubated at different numbers with 2 x 14 rabies-specific RBL-1 cells for 3 days. [3H]thymidine (1,uCi) was added to each well 18 h before the assays were terminated. The cultures were harvested and washed onto glass fiber filters, and the amount of radioactivity incorporated into DNA was measured by scintillation spectroscopy. Results of the cell proliferation assay were calculated by the mean counts per minute of [3H]thymidine incorporated into DNA by duplicate cultures, and the standard error of the mean was below 1% of the value of the mean in all determinations. Production and measurements of IFN--y. For the production of IFN--y, cells were stimulated with antigen in the presence and absence of antibodies for 2 days in the same way as for the proliferation assays. Cell-free supernatants were collected and assayed for amounts of IFN--y by a radioimmunoassay specific for biologically active human IFN--y (Centocor Inc., Malvern, Pa.). RESULTS The antigen-induced proliferative response of an IL2- dependent T-cell line (RBL-1) to different concentrations of inactivated rabies virus (vaccine) was tested in the presence and absence of rabies-immune plasma (final dilution, 1:5). The results (Fig. 1) show that T-cell proliferation to rabies vaccine was significantly higher in the presence of immune plasma containing antibodies to rabies antigens. Similar results were obtained with T-cell lines derived from four other vaccine recipients (RBL-2, RBL-3, RBL-4, and RBL-5). The results shown in Fig. 2 indicate that the rabies-immune plasma potentiated the proliferative response of the four rabies-specific T-cell lines derived from different vaccine recipients to whole inactivated virus. Three samples of rabies-immune plasma from separate individuals and two plasma samples from normal, nonimmune individuals were tested for their capacity to increase the T-cell proliferative response to various concentrations of rabies vaccine. All three immune plasmas increased to a great extent the reactivity of the T cells from RBL-1 to rabies virus, specifically at low concentrations of antigen (Fig. 3). On the other hand, the nonimmune plasmas did not have the same effect. To determine if the enhancing effect of the immune plasma on the proliferative response of T cells to rabies vaccine was related to antibodies reacting with the antigens, the following experiment was performed. The effects of the IgG fraction of *4.-4 P- -C.) C,, I-I Rabies Vaccine (units/ml) 1 FIG. 1. Effect of rabies-immune plasma in the proliferative response of the antigen-specific T-cell line RBL-1 to whole inactivated rabies virus (vaccine). Proliferation to different concentrations of antigen was determined in the absence () and presence (O) of rabies-immune plasma from a vaccine recipient at a final dilution of 1:5. Downloaded from on August 27, 218 by guest

3 428 CELIS ET AL. J. VIROL B.- U Rabies Vaccine (units/ml) D FIG. 2. Potentiation of the proliferative response of rabies-specific T-cell lines to whole inactivated rabies virus. Antigen-induced proliferation was measured as for Fig. 1, with the T cells from four different rabies vaccine recipients. (A) RBL-2; (B) RBL-3; (C) RBL-4; (D) RBL-5. The assays were done in both the absence (open symbols) and presence (closed symbols) of a 1:5 final dilution of rabies-immune plasma. rabies-immune plasma (purified on protein A-Sepharose) on the proliferation of antigen-specific T cells to rabies vaccine and formic acid-denatured rabies vaccine were examined. The results of this experiment (Fig. 4) indicate that the rabies-immune IgG increased to a great degree the proliferative response of RBL-1 cells to the untreated rabies vaccine preparation. In contrast, no effect of the immune IgG on the T-cell proliferation was observed when the formic aciddenatured rabies antigens were used. Both the untreated and the formic acid-denatured rabies vaccine preparations were equally effective in stimulating the proliferative response of the RBL-1 cells in the absence of immune IgG. Protein G#) 3 5 ;.) A-purified IgG from normal, nonimmune plasma did not show this effect (data not shown). The role of rabies-immune IgG in the capacity of accessory cells (autologous irradiated PBM) to capture and present antigen to the T cells was studied. APC pulsed with 5 U of rabies vaccine per ml in the presence of immune IgG were '4-4 _ ' co6. _-4 4 Downloaded from on August 27, 218 by guest 1 Rabies Vaccine (units/ml) FIG. 3. Comparison of normal and rabies-immune human plasmas for their effects in the antigen-induced proliferation of a rabies-specific T-cell line. Three rabies-immune plasmas from different vaccine recipients (A, O, ) and two plasmas from nonimmune individuals (+, *) were tested at a final dilution of 1:5 for their capacity to enhance the proliferative response of a T-cell line to various concentrations of rabies vaccine. Proliferation to rabies vaccine was also determined in the absence of human plasma (). Rabies Vaccine (units/ml) FIG. 4. Effect of rabies-immune IgG in the proliferative response of T cells to native and denatured rabies vaccine antigens. Protein A-purified IgG from rabies-immune plasma was tested for its ability to enhance the proliferative response of RBL-1 cells to various concentrations of untreated (closed symbols) or formic aciddenatured (open symbols) rabies vaccine. Cell proliferation was determined either in the presence of 1,ug of rabies immune IgG (, ) or in its absence (A, A).

4 VOL. 56, 1985 ANTIBODY REGULATION OF RABIES-SPECIFIC T CELLS 429 Xo P' ~~~4 ~ APC/Culture FIG. 5. Enhancement of the proliferative response of T cells by accessory cells pulsed with antigen in the presence of rabiesimmune IgG. Autologous irradiated PBM were incubated with 5 U of rabies vaccine per ml for 1 h in the presence or absence of immune IgG (1,ug/ml) and HBsAg immune complexes (1,g/ml) as explained in more detail in Materials and Methods. After the APC were washed, they were mixed at various cell numbers with RBL-3 cells, and proliferation was determined on day 3. The different conditions were rabies vaccine alone (A), rabies vaccine plus immune IgG (A), rabies vaccine plus immune IgG and HBsAg immune complexes (U), rabies vaccine and HBsAg immune complexes (O), and immune IgG plus immune complexes without rabies vaccine (O). Results are the mean determiniations of triplicate samples for which the standard errors of the means were below 1% of the value of the means. significantly more effective than APC pulsed with rabies vaccine alone in stimulating the proliferation of RBL-3 cells (Fig. 5). The results (Fig. 5) also indicate that the enhancing effect of the immune IgG co4ld be inhibited to a great degree by the addition of immune complexes (HBsAg-anti-HBsAg) during the time that the APC were pulsed with rabies vaccine and immune IgG. Similar results were obtained with tetanus toxoid-antitetanus immune complexes (data not shown). The immune complexes, however, did not inhibit the T-cell proliferation to rabies vaccine alone (in the absence of immune IgG). Lastly, no significant proliferation of the RBL-3 cells was observed at all numbers of APC in the absence of rabies vaccine and in the presence of immune complexes and immune IgG. The potentiating effect of rabies-immune plasma was tested in the proliferative response of the T-cell lines to purified rabies antigens. The results shown in Fig. 6 demonstrate that the immune plasma increased the proliferative response of the T cells from RBL-1 to both rabies G and rabies NC. However, the potentiating action of immune plasma was more prominent in the response to pure NC than to pure G. Similar results were observed with the rabiesspecific T-cell lines RBL-2, RBL-3, and RBL-5, which were obtained from three other vaccine recipients (Fig. 7 and 8). The effect of rabies-immune plasma was studied in the antigen-induced production of IFN--y by T-cell lines from four vaccine recipients (RBL-1, RBL-2, RBL-4, and RBL-5). The results (Table 1) indicate that significantly higher amounts of IFN--y were produced by all T-cell lines in the presence of immune plasma than in its absence in response to whole rabies virus, purified G, or NC. The proliferative response of T-cell clone RBC-9G (derived from RBL-1), which recognizes rabies G specifically, was studied with whole rabies virus or purified G and in the presence and absence of immune plasma or mouse monoclonal antirabies antibodies. The results (Fig. 9A) show that both human immune plasma and monoclonal anti-g antibodies increased the response of the clone to whole virus. On the other hand, monoclonal anti-n antibodies had no effect in the response of this clone to the same antigen. When the reactivity of T-cell clone RBC-9G to purified G protein was determined under the same conditions, it was found that only the rabies-immune plasma increased to a small degree the proliferative response (Fig. 9B). Similar experiments were performed with a T-cell clone specific for rabies NC proteins. The results indicate that the proliferative response to whole rabies virus of clone RBC- 4NC (also derived from RBL-1) was significantly higher iti the presence of immune plasma or monoclonal anti-g antibodies (Fig. 1A). The effect, however, was more dramatic with the human immune plasma than with the monoclonal antibody. As with clone RBC-9G, no significant effect of monoclonal anti-n in the response of RBC-4NC to rabies vaccine was observed. On the other hand, different results were observed when the response of clone RBC-4NC to purified NC proteins was tested. The data show that the rabies-immune plasma and, in particular, the monoclonal anti-n antibodies potentiated the proliferative response of this clone to NC proteins (Fig. 1B). DISCUSSION The regulatory effect of antigen-specific antibodies in the response of human T cells to rabies viral antigens was studied. The results presented here indicate that antibodies were capable of magnifying the reactivity of T-cell lines and clones to some of the antigens of rabies virus. In general, it was observed that rabies-immune plasma potentiated the proliferative responses to whole virus (vaccine) better than the responses to NC proteins and G (Fig. 2, 9, and 1). In turn, the immune plasma was more effective in increasing the proliferative response of the T cells to NC than to G (Fig. 6, 7, and 8). The antigen-induced production of IFN--y by four rabies-specific T-cell lines were also much greater in the presence of rabies-immune plasma than in its absence (Table 1). The potentiating effect of antibodies in the production of IFN--y by rabies-specific T cells appeared to be larger and more consistent in response to stimulation with NC than with G (Table 1). The elements in the rabies-immune plasmas that are responsible for increasing the antigen-induced proliferative response of T cells are most probably antirabies antibodies, since no significant effect was apparent when plasma from nonimmune individuals was used (Fig. 3). The enhancing effect of rabies-immune plasma appears to be specific to T cells that react with rabies antigens, since no potentiation of the response of HBsAg-specific T cells to HBsAg (or rabies vaccine) has been observed with rabies-immune, HBsAgnonimmune plasmas from human donors (data not shown). Furthermore, a preparation of human rabies immune globulin was shown to potentiate in a similar fashion the response of the T cells to rabies virus (Fig. 4 and 5). The enhancing effect of immune plasma (and IgG) appeared to require the binding reaction of antibodies with the rabies antigens, since no effect was observed-when rabies-immune IgG was tested Downloaded from on August 27, 218 by guest

5 43 CELIS ET AL. J. VIROL. 6-4 Q 4-4Q Rabies Antigens (ug/ml) FIG. 6. Enhancement of the proliferation of rabies-specific T cells to purified rabies antigens by immune plasma. The proliferative response of T-cell line RBL-1 to various concentrations of rabies G (, ) or NC proteins (i\, A) was determined in the absence (open symbols) and presence (closed symbols) of rabies-immune plasma. Proliferation in the presence of immune plasma and the absence of antigens was also determined (*). with a preparation of irreversibly denatured rabies vaccine antigens (Fig. 4). However, the denatured rabies vaccine, which was no longer recognized by antibodies (data not shown), induced the T-cell proliferative response as effectively as the untreated vaccine preparation, indicating that the rabies-immune T lymphocytes appear to recognize a different type of antigenic determinant than do antibodies. Studies in the HBsAg system showed that human polyclonal and mouse monoclonal anti-hbsag antibodies potentiated the proliferative response of human T-cell clones to the same antigen (2, 3). Anti-HBsAg antibodies of the IgG ) C.) class were significantly more effective than those of the IgM class in increasing the reactivity of the T-cell clones to HBsAg, and this effect was mediated through the Fc portion of the antibody molecules (3). It was recently reported that anti-hbsag antibodies of the IgG class increased by at least 1 times the amount of HBsAg that was captured and ingested by monocytes (Celis et al., submitted). Since these accessory cells are known to express surface Fc receptors for antibodies of the IgG class, this fact would explain their enhanced efficiency in binding immune-complexed antigen compared with free antigen. Therefore, it seems reasonable to assume that the amount of antigen that is captured by 4-) ~4- Downloaded from on August 27, 218 by guest 2 1 C) -4 2) G Protein (ng/ml) FIG. 7. Effect of rabies-immune plasma in the proliferative response of T-cell lines from different individuals to purified G. Three rabies-specific T-cell lines (RBL-2, RBL-4, and RBL-5) were tested for their proliferation to various concentrations of pure G both in the absence (open symbols) and presence (closed symbols) of immune plasma at a final dilution of 1:5. Cell lines: RBL-2 (, *), RBL-4 (A, A), and RBL-5 (O, *) NC Protein (ng/ml) FIG. 8. Effect of immune plasma in the proliferation of rabiesreactive T-cell lines to pure rabies NC proteins. The same rabiesspecific T-cell lines as are shown in Fig. 7 were tested for their reactivity to rabies NC proteins in the absence (open symbols) and presence (closed symbols) of human immune plasma at a final dilution of 1:5.

6 VOL. 56, 1985 ANTIBODY REGULATION OF RABIES-SPECIFIC T CELLS 431 TABLE 1. Effects of antibodies in the antigen-induced production of IFN--y by rabies-specific human T-cell lines Response to: IFN-y (U/ml) produced by cell linea: Group Rabies Immune RBL-l RBL-2 RBL-4 RBL-5 vaccineb GI NCc plasmad < < < <.2 <.2 <.2 < <.2 <.2 <.2 <.2 a Determined by radioimmunoassay as described in Materials and Methods. b Used at 1.25 U/ml. c Used at 12 ng/ml. d Used at a final dilution of 1:5. monocytes and macrophages is likely to be a direct function of the efficiency of these cells to present antigen to T lymphocytes. Our present results also agree with the abovementioned hypothesis, since antigen-pulsed APC in the presence of rabies-immune IgG were clearly more effective in triggering the T-cell proliferative response than were APC pulsed with antigen alone (Fig. 5). Furthermore, immune complexes prepared with HBsAg and an IgG suppressed to a great degree the potentiating effect of rabies-immune IgG on the antigen-induced proliferation of T cells (Fig. 5), suggesting that the surface Fc receptors of APC play an important role in this phenomenon. Monoclonal antibodies specific for rabies G increased the proliferative response of anti-g and anti-nc T-cell clones to whole rabies virus (Fig. 9A and 1A). On the other hand, monoclonal antibodies that react with rabies NC did not significantly modify the response of the T-cell clones to whole viru's (Fig. 9A and 1A). When the T-cell clones were tested against their purified specific antigens, it became evident that the monoclonal anti-n antibody increased the '4-4 p response of clone RBC-4NC to NC proteins, and as expected, the monoclonal anti-g had no effect (Fig. 1B). However, results with clone RBC-9G showed that none of the monoclonal antibodies increased the response of this clone to pure G (Fig. 9B). The differences observed between the sources of antibodies (immune plasma versus monoclonal antibodies) and between the various antigens used (whole rabies virus versus pure G and NC) could well be explained by differences in antibody concentration and affinity, accessibility of the antigenic determinants in the rabies molecules, and capacity of the rabies immune complexes to interact with Fc receptors on the surface of accessory cells. For example, antibodies specific for rabies G (monoclonal antibody 59-6) potentiated the response of NC-specific T cells (clone RBC-4NC) to whole virus (Fig. 1A), but not to pure NC (Fig. 1B). It seems reasonable to assume that the anti-g antibodies react with the G present on the external portion of the virus and that these immune complexes are then bound to Fc receptors on monocytes. Phagocytosis of the virus takes place, and once inside of the Downloaded from on August 27, 218 by guest Q). -) Rabies Vaccine G Protein (ng/ml) (units/ml) FIG. 9. Enhancement of the antigen-induced proliferative response of a T-cell clone specific for rabies G by immune plasma and mouse monoclonal antibodies. Proliferation of clone RBC-9G to different concentrations of whole rabies virus (A) or to pure G (B) was determined in the absence () or presence (A) of human immune plasma, monoclonal anti-rabies G antibody 59-6 (O), or monoclonal anti-rabies N antibody 52-2 (O). The final dilutions of plasma and monoclonal antibodies from ascites were 1:5 in all cases.

7 432 CELIS ET AL. J. VIROL. 12 B.- - C' v Rabies Vaccine (units/ml) NC Protein (ng/ml) FIG. 1. Effects of immune plasma and monoclonal antirabies antibodies in the proliferative response of a rabies NC protein-specific T-cell clone. Proliferation of clone RBC-4NC to various concentrations of whole rabies virus (A) or purified rabies NC proteins (B) was determined in the absence () or presence (A) of rabies-immune plasma and monoclonal anti-g (O) or anti-n (O) antibody, all at a final dilution of 1:5. monocyte, all of the antigenic components including NC are processed and transported back onto the cell surface for presentation to the T cells. On the other hand, the failure of the monoclonal anti-n antibodies (52-2) to potentiate T-cell responses to whole virus (Fig. 9A and IOA) or to G (Fig. 9B) is probably due to the inability of these antibodies to bind directly to these antigens. Howfver, the rabies-immune plasma and the monoclonal anti-n antibodies were capable of increasing the response of NC-specific T cells to purified NC (Fig. lob), indicating that antibody-mediated potentiation of T-cell responses to this antigen can take place when this antigen is acces'sible to antibodies. Unexpectedly, the proliferative responses to pure rabies G were not increased by monoclonal anti-g monoclonal antibodies, and a very small potentiating effect by antibodies from the rabiesimmune plasma was evident in these responses (Fig. 7 and 9B). Since the same antibodies were capable of significantly increasing the T-cell responses to whole virus (Fig. 9A and 1OA), it seems possible that during the purification procedure, the antigenic determinants recognized by the antibodits have been somehow modified, and thus the formation of stable immnune complexes is affected. Similar conclusions can be made from the observations of the effect of antibodies in the antigen-induced production of IFN--y by the rabiesreactive T cells (Table 1). The regulation by antibody of T-cell reactivity with antigen is a phenomenon that probably also takes place in in vivo immune responses. Immunization of animals with antigen complexed to antibodies, and especially when low doses of antigen are used, has been reported by several investigators working in different systems to induce stronger and faster antibody primary immune responses (9, 14, 18). At present, we are examining the effect of antibodies in rabies vaccination in mice. It could prove to be beneficial in both humans and animals if more effective pre-exposure immune responses to rabies virus were to be achieved by immunization with immune complexes. The most effective and recommended postexposure treatment for rabies virus infection is the administration of both the vaccine and antigen-specific antibodies (1). It is possible that some of the mechanisms involved in the success of this treatment are related to the capacity of the antirabies antibodies to potentiate the response of T lymphocytes to the rabies virus in vivo. LITERATURE CITED 1. Anderson, L. J., K. G. Nicholson, R. V. Tauxe, and W. G. Winkler Human rabies in the United States, 196 to 1979: epidemiology, diagnosis, and prevention. Anq. Intern. Med. 1: Celis, E., and T. W. Chang Antibodies to hepatitis B surface antigen potentiate the response of human T lymphocyte clones to the same antigen. Science 224: Celis, E., V, R. Zurawski, Jr., and T. W. Chang Regulation of T cell function by antibodies: enhancement of the response of human T cell clones to hepatitis B surface antigen by antigen-specific monoclonal antibodies. Proc. Natl. Acad. Sci. USA 81: Cohen, B. E., A. S. Rosenthal, and W. E. Paul Antigenmacrophage interaction. II. Relative roles of cytophilic antibody and other membrane sites. J. Immunol. 111: Davies, D. R., and H. Metzger Structural basis of antibody function. Annu. Rev. Immunol. 1: Dietzschold, B., T. J. Wiktor; W. H. Wunner, and A. Varrichio Chemical and immunological analysis of the rabies glycoprotein. Virology 124: Flamand, A., T. J. Wiktor, and H. Koprowski Use of hybridoma monoclonal antibodies in the detection of antigenic differences between rabies and rabies-related virus proteins. I. The nucleocapsid protein. J. Gen. Virol. 48: Flamand, A., T. J. Wiktor, and H. Koprowski Use of hybridoma monoclonal antibodies in the' detection of antigenic differences between rabies and rabies-related virus proteins. II. The glycoprotein. J. Gen. Virol. 48: Houston, W. E., k. J. Kremer, C. L. Crabbs, and R.. Spertzel Inactivated Venezuelan equine encephalomyelitis virus vaccine complexed with specific antibody: enhanced primary immune response and altered antibody class elicited. J. Infect. Dis. 135: Howard, M., and W. E. Paul Regulation of B-cell growth and differentiation by soluble factors. Annu. Rev. Immunol. 1: Kappler, J. W., and P. C. Marrack Helper T cells recognize antigen and macrophage surface components simultaneously. Nature (London) 262: Downloaded from on August 27, 218 by guest

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