Specific Macrophage Immunity to Vaccinia Virus: Macrophage-Virus Interaction

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1 INFECTION AND IMMUNITY, JUlY 1972, p Copyright 1972 American Society for Microbiology Vol. 6, No. 1 Printed in U.S.A. Specific Macrophage Immunity to Vaccinia Virus: Macrophage-Virus Interaction F. R. AVILA, R. M. SCHULTZ, AND W. A. F. TOMPKINS Center for Zoonioses and Comparative Medicine, University of Illinois College of Veterinary Medicine, Urbana, Illinois Received for publication 15 February 1972 Macrophage immunity to vaccinia virus was examined by measuring replication of vaccinia virus in immune and nonimmune rabbit peritoneal macrophages. Vaccinia virus multiplied in macrophages from normal rabbits but failed to replicate in macrophages from immune rabbits. The specificity of vaccinia restriction was demonstrated by the ability of myxoma virus to replicate in nonimmune and vaccinia-immune macrophages. Vaccinia virus absorbed equally well to immune and nonimmune macrophages, but only a small fraction of immune cells supported synthesis of virus antigen. Virus eclipse and infective center neutralization studies suggested that much of the virus remained at the plasma membrane of immune macrophages, even after prolonged incubation in culture. These data support our previous suggestions that macrophages may contribute to specific immunity against vaccinia virus. There is much evidence to suggest that the cellular elements of the immune response act as a major factor in the control of a number of virus infections (1, 3, 8, 13). Macrophages, by virtue of their ability to interact with and phagocytose viruses, can in some instances restrict replication and spread of the virus (6, 13, 21). It is generally believed that this macrophage function does not require prior sensitization of the animal but reflects a nonimmunological resistance on the part of the macrophage. Macrophages may also be instrumental in cell-mediated immunological resistance to virus infection. Reports on in vitro studies of resistance of macrophages to infection by poxviruses have been inconclusive. Whereas Roberts (11) reported an apparent increase in susceptibility of immune macrophages to ectromelia virus, other investigators have found no difference in susceptibility of macrophages from immune and nonimmune animals to poxviruses (13). Others have, on the other hand, demonstrated that macrophages from immune animals are more resistant to virus replication than macrophages from normal animals (14, 18). These differences may reflect individual variations in virus-host systems, methods of immunization, and assays of immunity. Previous studies by Tompkins et al. (18) demonstrated that purified peritoneal macrophages from rabbits sensitized to vaccinia virus were specifically inhibited by vaccinia virus in 9 the migration-inhibition test. Further attempts to investigate this macrophage-associated immunity showed that macrophages from vacciniaimmune rabbits suppressed replication of vaccinia virus, whereas normal macrophages did not. The present study was designed to extend these observations and explore the basic interaction in tissue culture between vaccinia virus and immune macrophages. MATERIALS AND METHODS Tissue culture cells and media. Primary rabbit kidney cells were prepared by trypsinization of kidneys from 2- to 4-week-old New Zealand White rabbits. RK, cells, an established line of rabbit kidney cells, were obtained from Grand Island Biological Co. All cells were grown in Eagle's medium supplemented with 10% newborn calf serum, penicillin (100 units/ml), streptomycin (100,g/ml), and sodium bicarbonate. Preparation of virus stocks. The WR strain of vaccinia virus was grown in monolayers of primary rabbit kidney (PRK) cells in 16-oz (473-ml) prescription bottles. Cell monolayers were inoculated with 107 plaque-forming units (PFU) of virus and allowed to absorb for 2 hr at room temperature, after which 20 ml of medium was added and the cultures were incubated at 37 C for 48 hr. The virus was harvested by three cycles of freeze-thawing and was assayed on monolayers of RK1 cells as previously described (18). Stocks of the Moses strain of myxoma virus were prepared in PRK cells and assayed by the plaque-

2 10 AVILA, SCHULTZ, AND TOMPKINS INFECT. IMMUNITY counting method on monolayers of RK1 cells without an overlay. Immunization of rabbits. Young-adult, male New Zealand White rabbits, 12 to 18 weeks of age, were immunized by inoculating 0.1 ml (106 PFU) of vaccinia virus intradermally at four sites on their shaved backs. The rabbits were given a second identical inoculation 3 to 4 weeks later. A delayed cutaneous reaction at the site of inoculation was taken as evidence that the rabbits had been sensitized. Rabbits were used in experiments 1 to 2 weeks after the second inoculation of virus. Preparation of peritoneal macrophages. Peritoneal exudate cells were obtained from rabbits after injection of paraffin oil as previously described (16, 18). Purified macrophages were obtained by placing the exudate cells in 16-oz prescription bottles in 10 ml of Eagle's medium and incubating for 10 to 15 min at 37 C. The unattached cells were removed, and the adhering macrophages were washed three times with cold Hanks balanced salt solution (HBSS) at ph 7.2 to 7.4. The adhering cells were then gently scraped from the glass surface in Eagle's medium, washed, and counted in a hemacytometer. Macrophages were kept in an ice bath prior to use to prevent adherence to the glass surface. This procedure usually resulted in a yield of 2 X 107 to 8 X 107 macrophages for each rabbit, with a viability of 90% or greater. Inoculation of macrophages with virus. Virus was adsorbed to macrophages in a 1-ml suspension for 90 min at 4 C with frequent shaking. Nonadsorbed virus was removed by two washes in medium, and the cells were resuspended to a concentration of 2.5 X 101 cells/ml. For growth curve experiments, 1-ml portions were placed in loose-capped culture tubes and incubated at 37 C in the presence of 5% CO2 in air. Duplicate samples were frozen at the beginning of the experiment and at the intervals to be tested. Virus to be assayed was released from the cells by three cycles of freeze-thawing, the duplicate tubes were pooled, and the virus was titered by plaquing on monolayers of RK1 cells. Assay of infective centers. Macrophages free in the medium and macrophaages removed from the glass surface by trypsin (0.25%) were pooled from duplicate tubes, washed three times with HBSS, resuspended in Eagle's medium, and counted. Appropriate dilutions of each sample were prepared, and 100 or 1,000 cells were plated onto monolayers of RK1 cells in 1-oz (ca. 30-ml) prescription bottles. After 30 min at 37 C had been allowed for macrophages to adhere to the monolayers, the cultures were covered with 1% agar overlay and incubated at 37 C. Plaques were counted after 72 hr. Neutralization of infective centers. To neutralize virus adherent to the cell membrane, macrophages were prepared as above. However, before plating, the washed cells were resuspended in 1 ml of a 1:4 dilution of antivaccinia serum or normal serum in HBSS and were incubated for 1 hr at 4 C with frequent shaking. After incubation, the cells were washed twice in cold HBSS and plated for infective center assay as described above. Vaccinia immune serum prepared in rabbits (50% virus neutralizing titer, 1:1,024) and in guinea pigs (50% virus neutralizing titer, 1:512) were used in this test. Immnunofluorescence test. Infected and noninfected macrophages were seeded onto cover slips in shell vials in 2 ml of Eagle's medium and were incubated at 37 C in a 5% CO2 in air atmosphere. At intervals after seeding, duplicate cover slips were prepared for the indirect immunofluorescence test as described (17). Briefly, the monolayers were washed twice in ph 7.6 tris(hydroxymethyl)aminomethane-buffered saline (TBS), allowed to air-dry, and fixed in acetone for 3 min at room temperature. Standard heat-inactivated (30 min at 56 C) vaccinia-immune and normal rabbit sera diluted 1:4 in TBS were used throughout the study. Fixed cell monolayers were treated with antiserum for 30 min at 37 C, washed three times in TBS (5 min/wash), and counterstained for 30 min at 37 C with fluorescein-conjugated goat anti-rabbit gamma globulin. The cells were washed three times, mounted in TBS-glycerol, and observed with a Zeiss fluorescence microscope. RESULTS Replication of vaccinia virus and myxoma virus in vaccinia-immune and nonimmune macrophages. Inoculation of nonimmune macrophages with an input of 0.1 PFU of vaccinia virus per cell resulted in an increase from 5.2 X 103 to 6.5 X 105 PFU of virus/ml between 0 and 48 hr after infection (Fig. 1). In contrast, cultures of vaccinia-immune macrophages showed only an lo' 105-F// a-,, Days After Infection FIG. 1. Replication of vaccinia virus (solid lines) and myxoma virus (dashed lines) in peritoneal macrophages from vaccinia-immune (0) and nonimmune (A) rabbits.

3 VOL. 6, 1972 [*. t... Ja _w w -: iv -, ^ t t t..,.ak s - ss.uj IMMUNITY TO VACCINIA VIRUS 11 %! *.; # r % % % a IS. G _ ' * W * v b s [,; * _e t,::-^, T.1m V w:ti~~~ _.,I!W ạ... ll Ji '3! o -w~ Ik- "I- VA_k FIG. 2. Hematoxylin and eosin stain of immune (a) and nonimmune (b) peritoneal macrophages at 48 hr after infection in culture with vaccinia virus. increase from 4.1 x 103 to 5.3 X 103 PFU of virus/ml between 0 and 48 hr after infection. Some experiments with immune macrophages actually resulted in a significant decrease in infectious virus during this incubation period. The specificity of suppression of vaccinia virus by vaccinia-immune macrophages was shown by the observation that myxoma virus replicated equally well in vaccinia-immune and normal macrophages. Increases of 2.1 and 2.7 log10 of infectious myxoma virus at 48 hr after infection were recorded in normal and vaccinia-immune macrophages, respectively (Fig. 1). Cytopathogenic effect of vaccinia virus on immune and nonimmune peritoneal macrophage cultures. Immune and nonimmune macrophages were inoculated with 0.1 PFU of virus per cell as described, seeded onto cover slips in shell vials, and stained at intervals for evidence of cytopathogenic changes. Normal macrophages demonstrated large eosinophilic cytoplasmic inclusions by 24 hr after infection, with the cells showing signs of rounding up and aggregation. By 48 hr after infection, the cytopathogenic changes in the culture were extensive, with many I_ TABLE 1. Adsorption oj vaccinia virus to immune and nonimmune peritoneal macrophagesa Source of Total PFU of Cells adsorbing peritoneal virus adsorbed virus (%)b macrophages Immune rabbits (i0. 6) X (±-1.3) Nonimmune rabbits (±0. 7) X (4 1.8) a Macrophages (2.5 X 105) were inoculated with an input of 0.1 PFU of vaccinia virus per cell at 4 C. The results represent the mean (and standard deviation) of three separate experiments with each type of macrophage. b Percentage of cells adsorbing virus measured by infective center assay immediately after adsorption at 4 C. cells coming off the cover glass; the attached cells were enlarged and clumped and were showing cytolytic effects of the virus. Characteristic nuclear changes were also evident, with many nuclei demonstrating fragmentation and pyknosis at 48 hr after infection (Fig. 2b). In contrast, 'O.T. 6VA-

4 12 AVILA, SCHULTZ, AND TOMPKINS INFECT. IMMUNITY r,ultures of immune macrophages showed no evidence of cytopathogenic changes. Macrophages remained adherent to the cover glass, retaining their characteristic cellular and nuclear morphology with no viral inclusions or cyto- TABLE 2. Percentage ofmacrophages with infectious virus and virus antigen at intervals after infection in culture with vaccinia virus Infective centers Cells showing Incu- Infective centers cytoplasmicfluorescence bation at 37 C (hr)a Immune Nonimmnune Immune Nonimmune macro- macro- macro- macrophages phages phages phages < < <1 _ a Macrophages inoculated in suspension with 0.1 PFU/cell at 4 C. bestimates determined by counting the number of positive and negative cells in several areas of the slides. plasmic granulation (Fig. 2a). Furthermore, it was evident from these studies that immune cells had a tendency to spread on the glass surface with distinct pseudopod formations, whereas normal macrophages tended to remain rounded with few visible pseudopods. Adsorption of vaccinia virus to immune and nonimmune macrophages. In an attempt to explain the differences in susceptibility of immune and nonimmune macrophages to vaccinia virus, experiments were designed to test for differences in adsorption. Macrophages were inoculated with 0.1 PFU of virus per cell and were washed to remove residual virus; the total adsorbed virus and the fraction of cells adsorbing virus were then measured. The data recorded in Table 1 demonstrated that there was no significant difference in the total amount of virus adsorbed by immune and normal cells and that the number of cells adsorbing virus was essentially the same in the two populations. At 4 C, 4.2% of the cells in the immune macrophage population adsorbed a total of 6.6 X 103 PFU of virus, as compared with 3.7% of the normal macrophages adsorbing 6.2 X 103 PFU of virus. Downloaded from on January 17, 2019 by guest FIG. 3. Immunofluorescence stain for vaccinia virus antigens at 48 hr after infection of immune (a) and nonimmune (b) peritoneal macrophages in culture.

5 VOL. 6, 1972 IMMUNITY TO VACCINIA VIRUS 13 Percentage of macrophages associated with virus at intervals after infection. The previous experiment indicated that immune and nonimmune macrophages adsorbed vaccinia virus with equal facility, and that the virus was apparently not neutralized after adsorption to immune macrophages. The next experiment was designed to determine whether there is a spread of virus in the culture after inoculation of immune macrophages. This was done by measuring the fraction of macrophages with infectious virus, virus antigen, or both at intervals after infection. Macrophages from normal rabbits inoculated with 0.1 PFU of virus/cell showed a progressive increase in infective centers with time in culture such that 89.1% of the cells were infected after 72 hr (Table 2). Immune macrophages, in contrast, did not show an increase in infective centers with time in culture, but rather were subject to a constant decrease. The percentage of immune macrophages that registered infectious virus dropped from 3.8 to 1.8% between 0 and 72 hr after infection (Table 2). Immunofluorescence studies on fixed macrophage cultures on cover slips demonstrated that essentially all of the cells in the normal macrophage population had cytoplasmic antigen by 48 hr after infection (Table 2). These cells showed large well-localized fluorescing cytoplasmic inclusions between 18 and 24 hr after infection. Between 24 and 48 hr after infection, the cells rounded up, showing massive amounts of antigen throughout the cytoplasm (Fig. 3b). Immunofluorescence studies on immune macrophages clearly demonstrated that only a small fraction (less than 1 %) of cells were capable of supporting the synthesis of virus antigen and that there was no increase in antigen-positive cells by 72 hr after infection (Table 2, Fig. 3a). Inoculation of immune macrophages at a higher multiplicity of infection (input of 1 PFU of virus per cell) did not result in an increase in macrophage susceptibility. Infective center assay showed 17.3% of cells with infectious virus at 0 hr after infection, as compared to 9.2% after 48 hr. Furthermore, under these conditions, there was no increase in total virus in the cultures, and again less than 1 %/ of the macrophages stained for virus-specific antigen. Also, it was evident by comparing the results of the infective center and immunofluorescence tests that only a fraction of immune cells adsorbing virus supported synthesis of virus antigen. These data support the hypothesis that the majority of the cells in the immune macrophage population are refractory to virus replication and that the block is at an early stage in the replication cycle. Neutralization of vaccinia virus after adsorption to immune and nonimmune macrophages. The previous experiment suggested that the mechanism of suppression of virus replication by immune macrophages functions at some stage between virus adsorption and synthesis of new virus antigens. To test for a possible defect in penetrations of vaccinia virus into immune macrophages, cells were inoculated with virus at 4 C as described before, and were seeded into tubes for incubation at 37 C. A portion of the cells (0-hr samples) were kept at 4 C and resuspended in 1 ml of a 1:4 dilution of vacciniaimmune rabbit serum to neutralize virus adherent to the cell membrane; this preparation was seeded onto monolayers of RK1 cells for infective center assay. As controls, cells were incubated in a 1:4 dilution of normal rabbit serum before plating. This procedure was repeated at 24 and 48 hr after incubation at 37 C. Treatment of immune macrophages with virus-immune serum immediately after adsorption of virus reduced the infective center titer by 78.3% (Table 3). Similar reductions of 67.2 and 69.9% were seen with cells treated at 24 and 48 hr after TABLE 3. Neutralizationi of virus-macrophage inzfective centers at intervals after inifectionz with vaccinia virus Source of peritoneal macrophages Source of seruma antivaccinia Per cent reduction in infective centers for time of infectionb (immune serum/nonimmune serum) Ohr 24hr 48 hr Nonimmune rabbits Rabbit 76.2 i i 5.7 Vaccinia-immune rabbits Rabbit Vaccinia-immune rabbits Guinea pig 87. lc 77. 3c 72.8c a Antivaccinia serum with a 50%0 virus neutralizing titer of 1:1,024 (rabbit) and 1:512 (guinea pig) used at 1:4 dilution. b Calculated from the number of cells registering as infective centers per 100 or 1,000 cells plated, after treatment with vaccinia-immune or normal serum. Mean of three experiments. c Mean of two experiments only.

6 14 AVILA, SCHULTZ, AND TOMPKINS INFECT. IMMUNITY infection, suggesting that much of the virus re- DISCUSSION mains on the surface of the cells even alter pro- Tissue culture studies abortive and prolonged incubation at 37 C. In the case c)f normal ductive virus infections of mononuclear cells macrophages, vaccinia-immune seruim neuhave suggested that macrophages may play a tralized 76.2C of the infective centerss at hr after infection but only 22.6%ro at 24 significant role in the control of virus diseases. * hr after Much of this evidence has come from the study infection. Vaccinia-immune serum pre2pared guinea pigs showed an almost identica in of the interaction between macrophages and il pattern poxviruses. Nishmi and Niecikowski (10) showed of neutralization of infective centers, with rethat vaccinia virus fails to multiply in mouse ductions of 87.1, 77.3, and 72.8%f at 0 24, and macrophages, although the virus replicates in 48 hr after infection of immune macirophages. renal epithelium and lung fibroblasts from mice Virus eclipse in immune and nonimmurie macro- of the same strain. In contrast, Beard and Rous phages. In an attempt to substantiate the data (2) and Florman and Enders (4) demonstrated from the previous experiment suggesting much of the input virus remained boua that replication of vaccinia virus in rabbit macrond to the phages. Roberts (11) reported that macrophages plasma membrane of immune macirophages, from ectromelia-sensitive mice were more susvirus eclipse studies were performed Macro- * ceptible to infection with the virulent virus than phages were inoculated with an input af 1 PFU with the attenuated virus. A somewhat similar of virus cell, washed twice to removie nonad- abortive infection results after infection of mouse sorbed virus, and incubated at 37 C. IDuplicate macrophages with herpesvirus, in which virus samples were frozen at intervals after aldsorption antigens accumulate in the cells but no complete and assayed for infectious viruses. infectious virus is produced (15). These infectn nonlm- tions are characterized by inherently resistant The growth curve of vaccinia virus i mune macrophages showed only a sligi between 0 and 6 hr, with a continuousst decline cells in which the virus may enter the cell cyto- plasm but is unable to in virus titers thereafter, suggesting asn eclipse produce infectious progeny phase of approximately 6 hr (Fig. 4). Ir the case virus. n the case There is evidence for second category of immune macrophages, no virus eclipse was of abortive macrophage infections, which deobserved at any point between 0 and 244 hr a.fter pends on the immune status of the host. Although infection. Although the difference in vir us eclipse cultured spleen cells from normal rabbits sup- macro- port vaccinia virus replication, these same cells curves between immune and nonimmur phages was not marked, it was reprocle repeated experiments and supports tucible from animals sensitized to the virus are rethet sthgt fractory to infection (14). Similarly, Tompkins gestions from the previous experimr et al. (18) demonstrated that purified peritoneal tent ata vaccinia virus does not enter the cell after ad- macrophages from vaccinia-immune rabbits sorption to immune macrophages. suppressed replication of the virus, whereas normal peritoneal macrophages did not. The IL _ -~~~~~~~~~~~~~01 i4ic- /~// / / Hours After Infection FIG. 4. Eclipse of vaccinia virus after to vaccinia-immuw1e (solid line) and nonimmi line) peritoneal macrophtages.,' data presented in this paper confirm and extend these findings. Inoculation of peritoneal macrophages from nonimmune rabbits with a low input of vaccinia virus resulted in a 2 to 3 log1o increase in infectious virus after 72 hr of infection. Infective centers and immunofluorescence studies demonstrated that, after the first cycle of infection, progeny virus quickly spread to infect essentially all of the cells in the macrophage population. This infection resulted in a characteristic vacciniainduced cytopathogenic effect on the macrophage culture. In contrast, cultures of macrophages from vaccinia-immune rabbits did not support replication of the virus. No increase in infectious virus was observed between 0 and 72 hr after 20 2 infection, and immune macrophages did not demonstrate the virus-induced degenerative adsorption changes observed with infected normal macrolne (dashed phages. Inoculation of vaccinia-immune macrophages with myxoma virus resulted in virus repli-

7 VOL. 6, 1972 IMMUNITY TO VACCINIA VIRUS 15 cation and cytopathogenic changes in the cell cultures, indicating a specific immunological suppression of vaccinia virus. Virus adsorption studies showed the fraction of cells adsorbing virus and the total virus adsorbed to be the same for immune and nonimmune macrophages. In agreement with this, Steinberger and Rights (14) showed no difference in uptake of vaccinia virus by spleen cells from immune and nonimmune rabbits, and Glasgow (5) demonstrated that chikungunya virus adsorbed to peritoneal leukocytes from immune and nonimmune mice at identical rates. These results argue against any alteration in capacity of macrophages to adsorb virus, after immunization. Furthermore, since the number of cells registering infectious virus was as large and the total amount of virus adsorbed to immune macrophages was not less than that adsorbed to normal macrophages, the virus apparently is not neutralized after adsorption to immune macrophages. Although there is no difference in adsorption of virus by immune and nonimmune macrophages, subsequent events in the virus replication cycle are different. Infective center studies revealed that essentially all of the macrophages in the normal cell population were susceptible to virus infection. Similarly, immunofluorescence studies showed accumulation of virus antigen in the cytoplasm of most of the normal macrophages by 48 hr after infection. In contrast, the infection of cultures of immune macrophages did not appear to spread from the cells originally adsorbing virus. Indeed, there was a decrease in the fraction of cells associated with infectious virus between 0 and 72 hr after infection. Decrease in infective centers with time could represent thermal inactivation of virus, gradual elution of virus from the surface of the cell, or active neutralization or degradation on the part of the immune macrophage. At the present time, we have no information on which of these or other mechanisms may be operable. Immunofluorescence studies also demonstrated that only a small fraction of cells (< 1%) in the immune macrophage population supported the synthesis of virus antigens. The number of cells with virus antigens appeared to remain relatively constant throughout the experiment and was always significantly less than the number of cells shown to carry infectious virus by the infective center assay. It is possible that these cells represent a small number of susceptible contaminating fibroblasts which are always present in our macrophage preparations. In any event, we can conclude from these data that only a small fraction of cells in the immune macrophage population are susceptible to virus infection and that, for the most part, adsorption of virus to immune cells does not lead to production of virus antigen or infectious virus. This is in contrast to observations made on nonimmune abortive infections of macrophages with herpesvirus, which result in the production of virus antigens and incomplete virus particles but no infectious virus (15). Although vaccinia virus can be shown to eclipse in normal macrophages, no evidence of uncoating was found after adsorption of virus to immune macrophages. Furthermore, it was observed that a high percentage of immune macrophage infective centers at 24 and 48 hr after infection were rendered noninfectious by treatment with vaccinia-immune serum. In the case of nonimmune macrophages, immune serum was significantly less effective in neutralizing infective centers after 24 hr of incubation at 37 C, suggesting that much of the infectious virus was intracellular at that time. We have no explanation for the apparent fixation of vaccinia virus at the surface of immune macrophages. Unanue and Cerottini (19) observed that a small fraction of soluble antigens such as hemocyanin remains bound to the plasma membrane of macrophages even after prolonged incubation in vitro. Enhancement of pinocytosis with heparin or dextrose sulfate did not result in endocytosis of these molecules. However, there has been no previous evidence to suggest retention of particulate antigens at macrophage membranes (20). These studies for the most part differ from the present study in that they were not dealing with immune macrophages or infectious virus which may bind to specific cellular receptors. Under these conditions, the normal phagocytic or pinocytic events may not occur, or these virus-binding areas may not participate in endocytosis. Although it has been reported that macrophages activated by bacterial infections have enhanced phagocytic capabilities (7), recent in vitro studies have demonstrated that peritoneal macrophages from Candida albicans-sensitized guinea pigs had a decreased ability to phagocytose C. albicans (9, 12). This unexpected observation led the authors to propose that cellular immunity to C. albicans is associated with reduced activity of phagocytic macrophages, thereby promoting localization of the lesions (12). A similar mechanism may be operative in the case of vaccinia virus and rabbit macrophages. It is possible that immune macrophages play a major role in virus infections, particularly poxvirus infections, in this manner. Binding of vaccinia virus to immune macrophages might not only prevent replication and spread of the virus but might also hold it in such a fashion that it is easily accessible to neutralizing antibody. Ad-

8 16 AVILA, SCHULTZ, AND TOMPKINS INFECT. IMMUNITY ditional experiments are necessary before any conclusions can be drawn about this type of cellular immunity to vaccinia virus. ACKNOWLEDGMENTS This study was supported by research grant from the Illiniois Division of the American Cancer Society. LITERATURE CITED 1. Allison, A. C Cell mediated immune responses to virus infections and virus-induced tumors. Brit. Med. Bull. 23: Beard, J. W., and P. J. Rous The fate of vaccinia virus on cultivation in vitro with Kupfer cells (R E cells). J. Exp. Med. 67: Blanden, R. V Mechanisms of recovery from generalized viral infections: mousepox. J. Exp. Med. 132: Florman, A. C., and J. F. Enders The effect of homologous antiserum and complement on the multiplication of vaccinia virus in roller tube cultures of blood mononuclear cells. J. Immunol. 43: Glasgow, L. A Leukocytes and interferon in the host response to viral infections. II. Enhanced interferon response of leukocytes from immune animals. J. Bacteriol. 91: Hirsch, M. S., B. Zisman, and A. C. Allison Macrophages and age-dependent resistance to herpes simplex virus in mice. J. Immunol. 104: MacKaness, G. B The monocyte in cellular immunity. Seminars Hematol. 7: Mims, C. A Aspects of the pathogenesis of virus diseases. Bacteriol. Rev. 28: immunity in vitro: Infect. Immunity Neta, R., and migration S. B. Salvin. inhibition Cellular and phagocytosis. 4: Nishmi, M., and H. Niecikowski Interaction of vaccinia virus and cells in primary and continuous culture. Nature (London) 199: Roberts, J. A Growth of virulent and attenuated ectromelia virus in cultured macrophages from normal and ectromedia immune mice. J. Immunol. 92: Salvin, S. B., and S. L. Cheng Lymphoid cells in delayed hypersensitivity. IH. In vitro phagocytosis and cellular immunity. Infect. Immunity 3: Silverstein, S Macrophages and viral immunity. Seminars Hematol. 7: Steinberger, A., and I. L. Rights Effect of immunization on tissue susceptibility to vaccinia in vitio. Virology 21: Stevens, J. G., and M. L. Cook Restriction of herpes simplex virus by macrophages. An analysis of cell-virus interaction. J. Exp. Med. 133: Tompkins, W. A. F., C. Adams, and W. E. Rawls An in vitro measure of cellular immunity to fibroma virus. J. Immunol. 104: Tompkins, W. A. F., N. A. Crough, S. S. Tevethia, and W. E. Rawls Characterization of surface antigen on cells infected by fibroma virus. J. Immunol. 105: Tompkins, W. A. F., J. M. Zarling, and W. E. Rawls In vitro assessment of cellular immunity to vaccinia virus: contribution of lymphocytes and macrophages. Infect. Immunity 2: Unanue, E. R., and J. C. Cerottini Persistence of antigen on the surface of macrophages. Nature (London) 222: Unanue, E. R., and J. C. Cerottini The function of macrophage in the immune response. Seminars Hematol. 7: Zisman, B., M. S. Hirsch, and A. C. Allison Selective effects of anti-macrophage serum, silica and anti-lymphocyte serum on pathogenesis of herpes virus infections of young adult mice. J. Immunol. 104: