Vol. 11, No. 5
INFECTiON AND IMMUNITY, May 1975, p. 1031-1037 Copyright i 1975 American Society for Microbiology
Printed in U.S.A.
Immunodepression by Rowson-Parr Virus in Mice: Effect of Rowson-Parr Virus and Friend Leukemia Complex Infections on Contact Sensitivity in Susceptible and Resistant Mice M. BENDINELLI,* M. CAMPA, AND A. TONIOLO Institute of Microbiology, University of Pisa, 56100 Pisa, Italy Received for publication 31 December 1974
Contact sensitivity to 2-phenyl-4-ethoxymethilene oxazolone, as a probe for cell-mediated immunity, was investigated in susceptible BALB/c and resistant C57BL/6 mice after infection with Friend leukemia complex (FLC) or with Rowson-Parr virus (RPV). In BALB/c mice, FLC depressed contact sensitivity when given before primary sensitization but had no effect on established contact sensitivity nor on the response elicited by a booster application of the sensitizer. These findings, together with the failure to alter reactivity to an aspecific inflammatory stimulus, indicate that FLC impairs the afferent limb of the response. In the same strain of mice RPV infection did not significantly depress contact sensitivity, as judged by the extent of the reaction 24 h after challenge, but slightly inhibited the early antibody-mediated phase of this reaction. In C57BL/6 mice neither viral preparation affected contact sensitivity. After early reports of depressed contact sensitivity (1, 9), there has been somewhat conflicting evidence on the functionality of cellmediated immunity in mice infected with the Friend leukemia complex (FLC) (18, 38, 40). In one study, skin graft rejection was reported unimpaired in susceptible CBA and in resistant C57BL/6 mice infected before grafting (39), but later investigations revealed that infected susceptible BALB/c mice exhibit increased rejection times to both H-2 and non-H-2 allografts; this deficit was confirmed in vitro by direct and indirect migration inhibition factor assays (24) and by lymphocyte-mediated cytolysis (30). Spleen cells from infected susceptible mice have been shown to mount normal graft-versus-host reactions (12, 39), but in one report, infected C57BL/6 splenocytes caused accelerated mortality in (DBA/2 x C57) F1 (39). FLC infection has also been found to depress the immune elimination of allogeneic lymphoid cells slightly (6) but was without effects on the rejection of bone marrow allografts (12). The rejection of sarcoma 180 tumor cells was found reduced in infected susceptible DBA/2 and resistant C57 mice (S. B. Deodhar and T. Chiang, Fed. Proc. 29:560, 1970). Although suggestive, the findings that co-infection with FLC abolished the regression of tumors induced in BALB/c mice by murine sarcoma virus (17) and that C57BL/6 but not BALB/c mice injected with Friend lymphomas releasing infectious virus developed
transplantation resistance (28) are difficult to interpret. Classical hypersensitivity to tuberculin has repeatedly been investigated and constantly found depressed in susceptible but not in resistant mice infected either before or after sensitization, as assessed by footpad swelling and by the migration inhibition factor and blastogenic response of spleen cells to purified protein derivative (22, 23, 29, 31). In recent studies, FLC infection of susceptible donors has also been shown to suppress the spleen cell response to concanavalin A (29) and to phytohemagglutinin (43). Taken together, these results indicate that FLC does indeed impair cellmediated immunity though certain assays (graft-versus-host reaction, for example) probably do not indicate the extent of impairment. Rowson-Parr virus (RPV), one of the viruses forming the FLC, in singly infected BALB/c mice induces an early depression of the antibody response to sheep erythrocytes (11, 13) that is accompanied by a slight hyperplasia of the spleen, whereas neoplastic changes follow only after several months (13, 14). From these findings the obvious problem arises of the role played by RPV in the immunodepressive activity of FLC (8). It is therefore of interest to compare the effects of RPV and FLC on cellmediated immune responsiveness. We have done so by using contact sensitivity to oxazolone in mice susceptible (BALB/c) and resistant (C57BL/6) to the leukemogenic activity of FLC.
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BENDINELLI, CAMPA, AND TONIOLO
MATERIALS AND METHODS Animals. Inbred BALB/c mice and outbred C57BL/6 mice grown in this Institute were used. Only females were used because fighting between males can cause undesired ear swelling; age at the start of the experiments was 8 to 10 weeks. In each experiment the animals were randomly assigned to different groups. Viruses. The passage histories of the viruses have already been described and the batches used were prepared as previously reported (10). The titers of the FLC batches varied between 1030- and 1035 mean infectious doses per 0.1 ml. The RPV batches, prepared in newborn mice, ranged in titer between 104.0 and 105.° mean infectious doses per 0.1 ml. Both preparations were free of the lactic dehydrogenase virus, which is known to depress contact sensitivity (1). Infection was performed by retro-orbital injection of 0.1 ml of virus. Sensitization. Sensitization was carried out as described (3) with minor modifications. The mice were anesthetized by intraperitoneal administration of tribromoethanol in amylene hidrate (Avertin, Winthrop, N.Y.) and sensitized by the application of 2-phenyl-4-ethoxymethilene oxazolone (referred to as oxazolone; British Drug Houses, Poole, England) in ethanol to the skin of the abdominal wall. The sensitizer was spread within an area of approximately 4 cm2. Unless stated otherwise, 0.2 ml of 2% oxazolone was applied. Challenge. Unless stated otherwise, the challenge was carried out 6 days after sensitization. The animals were anesthetized and the thickness of both ears was measured twice with a micrometer. Both sides of both ears were then painted with a drop of 1% oxazolone in olive oil. This concentration of oxazolone was chosen on the basis of preliminary studies indicating that it was little irritating in unsensitized animals and highly effective in inducing ear swelling in sensitized mice. In these experiments 2% oxazolone, which had been used as challenge in previous studies and found nonirritating (1), produced marked nonspecific swelling in normal mice. The reason for this discrepancy is not known, but it might have been due to differences in the degree of purity of different batches of oxazolone. Unsensitized mice were included at each testing as controls for unspecific swelling. Quantification of sensitivity. Twenty-four hours after sensitization, the animals were again anesthetized and the ear thickness was measured twice. For each mouse the result was expressed by the difference in thickness found between the measurements carried out before and after challenge (mean of the two ears) in units of 10-i cm. Statistics. Unless otherwise specified, six to seven mice were used per group. For each group the geometric mean of ear thickness increase and the 95% confidence limits of the geometric mean are given. The statistical significance of the differences between groups was assessed by Student's t test.
INFECT. IMMUN.
the duration of contact sensitivity, groups of BALB/c mice were painted with 3% oxazolone and challenged at different intervals thereafter. Groups of unimmunized mice served as controls. A significant increase in ear thickness above the control values was first detected in the group of animals challenged 3 days after sensitization (Fig. 1). At this time there were considerable individual variations. Maximum ear swellings and the lowest degree of individual variation were obtained when the challenge was performed between days 4 and 7 from immunization. By day 9 the extent of swelling had already decreased and individual variations had become larger. This level of sensitivity remained little changed throughout the period of observation (10 weeks). It was possible to evoke a secondary response. Groups of seven BALB/c mice sensitized with 3% oxazolone on day -70 were given a similar second dose of sensitizer on day 0 and challenged on days 2, 3, 6, and 9. Groups of animals given the primary stimulus only were used as controls. The boosted mice showed a clear potentiation of ear swelling and a reduction in individual variations 6 days after the booster as compared with animals given the primary dose alone (Fig. 1). However, the rapidity and extent of the response, as well as the duration of maximum responsiveness, were not different from those observed during the primary response. The kinetics of development of ear swelling during the primary response was also investigated. One group of BALB/c mice sensitized on day -6 and one group of normal mice were 75-
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FIG. 1. Contact sensitivity in BALBIc mice at RESULTS various times after sensitization (-, primary reContact sensitivity in normal mice. To sponse; *, secondary response) and kinetics of the determine the time course of appearance and reaction after challenge (0).
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CONTACT SENSITIVITY IN RPV AND FLC INFECTIONS
challenged at time 0, and ear thickness was measured at various intervals thereafter. In normal animals the increase of ear thickness was very low, reaching a peak of 1.18 3 days after testing. In sensitized mice the response was biphasic: 8 h after challenge, there was a first peak with ear swellings averaging about 50%o of the maximum response, after 10 h the response had slightly but definitely declined, and at 12 h the response had started to increase again to reach the second and highest peak at 24 h. At 72 h the extent of ear swelling was somewhat decreased. The effect of varying the dose of oxazolone used to sensitize BALB/c mice was also investigated. Groups of mice were skin painted with 0.2 ml of increasing concentrations of oxazolone and challenged 6 days later. The results are not reported here in detail because they mimicked those obtained in subsequent experiments with normal and RPV-infected animals (see Fig. 3). The lowest concentration of oxazolone giving maximum sensitization was 2%. The same study was performed in C57BL/6 mice. This strain was more prone to sensitization: painting with as little as 0.1% oxazolone gave a mean ear swelling of 7.64 (4.33 to 13.44) against aspecific swelling of 0.66 (0.31 to 1.41) in unsensitized animals. The lowest concentration giving optimum ear swelling was 1%. To test the degree of depression of contact sensitivity that can be achieved by a treatment known to interfere strongly with cellular immune response, the effect of rabbit anti-mouse thymocyte serum administered 1 and 3 days before sensitization on the extent of ear swelling after challenge was studied in BALB/c mice. Anti-thymocyte serum halved the mean ear swelling as compared with the sensitized controls (P < 0.01), whereas rabbit anti-mouse macrophage and normal sera had no significant effects. These results further demonstrate the suitability of contact sensitivity as a probe of cell-mediated immune responsiveness of mice after various manipulations. Contact sensitivity in RPV- and FLC-infected BALB/c mice. The effect of infection with RPV or FLC performed at various times before sensitization is shown in Fig. 2. Groups of unimmunized infected or uninfected mice served as controls for aspecific swelling; their responses did not significantly differ. RPV given as early as 50 days or 1 day before sensitization did not significantly influence the development of contact sensitivity; in contrast, FLC given 15, 6, or 3 days before sensitization produced marked depression of contact sensitivity. The effect of RPV infection 6 days before sensitization with various doses of oxazolone
was also studied. Normal and infected mice showed superimposable dose response curves (Fig. 3). The time course of development of ear swelling in sensitized uninfected and RPV- or FLCinfected mice was compared by measuring the
L-. z
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10
5
15
20
50
DAYS BETWEEN INFECTION AND SENSITIZATION
FIG. 2. Effect of infection with Rowson-Parr virus (0) or Friend leukemia complex (V) on contact sensitivity in BALBIc and C57BL/6 mice.
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05 1 20 10 CONCENTRATION OF SENSITIZER X 3. FIG. Effect of infection with Rowson-Parr virus on contact sensitivity established with various doses of sensitizer in BALBIc mice (0, infected mice; 0,
uninfected controls).
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BENDINELLI, CAMPA, AND TONIOLO
thickness at various times after challenge in mice sensitized 6 days after infection (Fig. 4). Normal mice showed the usual biphasic response. In FLC-infected mice, the magnitude of the response was strongly reduced throughout the testing period and there was no evidence of the first peak of response, which in normal mice was clearly evident at 8 h. In the RPV-infected mice the magnitude of the 24-h response was normal, but the 8-h peak was reduced. The effects of RPV and FLC infections on long-term established contact sensitivity and on the secondary response were also ascertained. Groups of mice were sensitized on day -71, infected on day 12, and challenged on day 0. Neither FLC nor RPV had any detectable effect on the residual low level of contact sensitivity still detectable 71 days after primary stimulation (Table 1). Other groups of animals were treated as above but on day -6 received a second dose of sensitizer. The recall of contact sensitivity in the infected animals was as high as in the uninfected controls. The depression of ear swelling caused by FLC in sensitized animals as well as the apparent lack of effect on it by RPV infection might be due to the ability of these viral infections to reduce (or to potentiate, in the case of RPV) the ability of mice to mount an inflammatory response. To test this possibility the degree of ear
INFECT. IMMUN.
TABLE 1. Effect of infection with RPV or FLC on long-term established contact sensitivity and on the secondary response in BALBIc micea Contact sensitivity
Sensitiz- Infection Booster No. of ation
mice ~Mean
I
-
+ +
+ +
+ +
None None RPV FLC None RPV FLC
-
+
+ +
6 11 12 10 10 9 9
0.32 1.76 2.22 1.88 10.45 11.59 11.09
95d Confidence limits
0.17-0.61 0.85-3.65 1.38-3.59 0.95-3.70 7.10-14.97 8.20-16.37 7.55-16.30
-
a Sensitization was carried out on day -71, infection on day - 12, booster on day -6, and the challenge on day 0.
nonspecific ear swelling was measured, in normal mice and in mice infected with FLC or RPV 15 days earlier, 2 h after the application of the irritant agent origan oil on the ears. The mean increases of ear thickness were 9.66, 9.50, and 9.85 units, respectively. Contact sensitivity in RPV- and FLC-infected C57BL/6 mice. In the first group of experiments mice were infected with RPV or FLC at various times before sensitization with 2% oxazolone and tested 6 days later (Fig. 2). No differences were observed in contact sensitivity Vf) between infected and control mice. -J 100 Since the doses of sensitizer necessary to establish a substantial level of contact sensitivI.z ity in C57BL/6 mice are markedly lower than 0 those used in the previous experiment, in a further experiment the effect of RPV and FLC 75infections given 7 days before sensitization with 0.1% oxazolone was studied. The mean ear swelling was 5.87 (4.08 to 8.45) for RPV-infected mice and 4.70 (2.95 to 7.48) for FLC-infected animals against 5.50 (3.75 to 8.05) in the sensi3:50tized uninfected controls and 0.64 (0.18 to 2.21) in the sensitized controls. DISCUSSION Contact to simple chemicals in sensitivity 7- 25mice has been extensively investigated in recent years (1-5, 20, 26, 34-36, 44) and has proven a very suitable model for exploring basic aspects of the immune response. Z .n Contact sensitivity has also been exploited as a parameter of immune responsiveness of mice HOURS AFTER CHALLENGE during infection with various viruses or in other FIG. 4. Kinetics of ear swelling after challenge in situations (1, 9, 21, 25). Here the morphology of sensitized uninfected (0), Rowson-Parr virus-infected the response of mice to oxazolone was pre(0), and Friend leukemia complex-infected BALBIc liminarily further characterized and a regimen mice (V). of sensitization and testing suited for assaying 0
0 z
w
w
0
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CONTACT SENSITIVITY IN RPV AND FLC INFECTIONS
the functionality of the immune system in mice receiving various manipulations was determined. Contact sensitivity, like other in vivo delayed hypersensitivity reactions (37), is not a pure cell-mediated entity since low levels of sensitivity can be passively transferred with serum or with macrophages coated with serum (4, 5). After challenge the increase of ear thickness in actively sensitized animals shows two peaks. The first one, which has been shown in previous studies (20, 34) to occur as early as 4 h after challenge, occurred in our experiments at 8 to 10 h. This peak is usually low and is mainly a manifestation of an antibody-mediated reaction (20). The second and highest peak, which reaches its maximum at 24 h, is maintained at 48 h and, generally, is still sustained at 72 h, is instead clearly a typical delayed-type reaction (20, 26). In these and in previous studies (1, 10) aimed to ascertain the effects of viral infections on cell-mediated immunoresponsiveness, we were primarily concerned with the late response to challenge, i.e., with the increase of ear thickness at 24 h. The use of contact sensitivity to oxazolone is a very convenient probe for cellular immunity in virus-infected mice, since a high level of sensitivity can be easily and reproducibly induced and quantitated in 1 week. The method is of great advantage when viruses that kill the host rapidly are studied, making it possible to infect the animals before sensitization and to test the sensitivity before the animals are obviously ill. The possibility of evoking a secondary response is also advantageous, because virus infections may preferentially interfere with primary or secondary immune responses (19, 32). Moreover, the kinetics of the response to challenge can be easily followed, providing indications on whether a given infection affects the early antibody-mediated phase, the late cellmediated phase, or both. Contact sensitivity has been extensively used also as a parameter of immune responsiveness in humans, and therefore correlations between findings in animal models and clinical situations can be drawn. Previous investigations have shown that contact sensitivity to oxazolone and to pycril chloride is depressed in susceptible mice infected with FLC (1, 9). Here the effects of FLC and RPV on contact sensitivity to oxazolone in susceptible and resistant mice are compared. In BALB/c mice, as expected, FLC given between 3 and 15 days before sensitization strongly depressed the primary response, whereas RPV had no detectable effects as judged by the increase in ear thickness at 24 h, regardless of
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the dose used for sensitization and the interval between infection and sensitization. A difference between normal and RPV-infected mice could be noticed only when the time course of ear swelling was studied. In the RPV-infected animals the early peak of swelling was reduced, probably reflecting a depression of the humoral response to oxazolone. In FLC-infected mice both the early and late peaks were depressed, in accordance with previous findings that FLC impairs antibody and cellular responses to simple chemicals (1). The inflammatory response is a key factor in contact sensitivity (2). However, the possibility that FLC and RPV might interfere with the ability of mice to mount a normal inflammatory response is ruled out by the observation that oxazolone and the irritant origan oil caused equal levels of nonspecific ear swelling in infected and uninfected unsensitized mice. The findings that the established contact sensitivity and the secondary response were not depressed by FLC also argue against this possibility and indicate that FLC inhibits contact sensitivity not by affecting its expression but by interfering with the afferent limb of the reaction. However, the failure of FLC infection to inhibit established contact sensitivity strongly contrasts with recent results in which FLC profoundly suppressed an established state of hypersensitivity to PPD and to histocompatibility antigens in susceptible mice (29-31). An explanation of these contradictory results may be that a few days after sensitization the cells mediating contact sensitivity to oxazolone in mice are no longer found in the spleen and lymph nodes but appear in the bone marrow and in the peritoneal cavity (5), peritoneal lymphocytes being affected by FLC infection only very late in the course of the disease (7). Previous reports had indicated that C57BL/6 mice, a strain considered "absolutely resistant" (41) to leukemia induction by FLC, are also resistant to the immunodepressive activity of the virus (16, 33, 39, 42). However, recent investigations in our laboratory and elsewhere have revealed that after FLC infection in this strain, the splenic antibody-forming cell response to sheep erythrocytes (15, 27; unpublished data) is transiently but consistently reduced. RPV infection, in contrast, has no effect on the humoral response to sheep erythrocytes in C57BL/6 mice (unpublished data). The present results, showing that neither virus given before sensitization depresses contact sensitivity in C57BL/6 mice, agree with the finding by others that in this strain FLC fails to impair an established tuberculin hypersensitivity (31).
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BENDINELLI, CAMPA, AND TONIOLO
After infection with the viruses of FLC there be a dissociation between development of leukemia and depression of the antibody response to unrelated antigens. This has been found to occur in resistant C57BL/6 mice infected with FLC (15, 27; unpublished data) and in susceptible BALB/c mice infected with RPV (11); in both instances infection is followed by a rapid fall in the ability to produce antibody but not by early signs of leukemia. The results presented here indicate that such dissociation with leukemia development does not seem to exist in the case of the depression of cellmediated immunity. However, it is not known whether the impairment of cellular immunity is a primary effect of FLC infection or a consequence of the induced neoplastic changes. may
ACKNOWLEDGMENTS This investigation was supported by the Italian National Research Council (grant no. CT 73.00462.04). We thank G. Falcone for helpful advice and suggestions, L. Montagnani for skillful technical assistance, and G. Greco for typing the manuscript. LITERATURE CITED 1. Asherson, G. L., and M. Bendinelli. 1969. Immunodepression by viruses: effect of Friend and Riley viruses on contact sensitivity. G. Microbiol. 17:179-188. 2. Asherson, G. L., and R. M. R. Barnes. 1973. Contact sensitivity in the mouse. IX. The role of immunological and non-immunological inflammation in the movement of lymphocytes to immunized lymph nodes. Immunology 24:885-894. 3. Asherson, G. L., and W. Ptak. 1968. Contact and delayed hypersensitivity in the mouse. I. Active sensitization and passive transfer. Immunology 15:405-416. 4. Asherson, G. L., and M. Zembala. 1970. Contact sensitivity in the mouse. IV. The role of lymphocytes and macrophage in passive transfer and the mechanism of their interaction. J. Exp. Med. 132:1-15. 5. Asherson, G. L., and M. Zembala. 1973. Anatomical location of cells which mediate contact sensitivity in the lymph nodes and bone marrow. Nature (London) New Biol. 244:171-177. 6. Bainbridge, D. R., and M. Bendinelli. 1972. Circulation of lymphoid cells in mice infected with Friend leukemia virus. J. Natl. Cancer Inst. 49:773-781. 7. Bendinelli, M. 1968. Haemolytic plaque formation by mouse peritoneal cells, and the effect on it of Friend virus infection. Immunology 14:837-850. 8. Bendinelli, M. 1971. Immunodepression by Friend virus, p. 314. In J. L. Melnick (ed.), Proc. 2nd Int. Congr. Virol. S. Karger, Basel. 9. Bendinelli, M., and G. L. Asherson. 1968. Effect of Friend virus infection on contact sensitivity. Rep. Br. Emp. Cancer Campaign 45:137. 10. Bendinelli, M., and L. Nardini. 1973. Immunodepression by Rowson-Parr virus in mice. I. Growth curves of Rowson-Parr virus and immunological relationships with Friend virus. Infect. Immun. 7:152-159. 11. Bendinelli, M., and L. Nardini. 1973. Immunodepression by Rowson-Parr virus in mice. II. Effect of Rowson-Parr virus infection on the antibody response to sheep red cells in vivo and in vitro. Infect. Immun. 7:160-166. 12. Bennett, M., and R. A. Steeves. 1970. Immunocompetent
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cell functions in mice infected with Friend leukemia virus. J. Natl. Cancer Inst. 44:1107-1119. 13. Carter, R. L., F. C. Chesterman, K. E. K. Rowson, M. H. Salaman, and N. Wedderburn. 1970. A new virus of minimal pathogenicity associated with Friend virus. II. Histological changes and immunodepressive effect. Int. J. Cancer 5:103-110. 14. Carter, R. L., F. C. Chesterman, K. E. K. Rowson, M. H. Salaman, and N. Wedderburn. 1970. Induction of lymphoma in BALB/c mice by Rowson-Parr virus. Int. J. Cancer 6:290-303. 15. Ceglowski, W. S., B. P. Campbell, R. F. Mortensen, and H. Friedman. 1974. Humoral and cellular immune responses in susceptible and resistant strains of mice infected with Friend leukemia virus. Proc. Soc. Exp. Biol. Med. 146:619-624. 16. Ceglowski, W. S., and H. Friedman. 1969. Murine virus leukemogenesis. Relationship between susceptibility and immunodepression. Nature (London) 224:1318-1319. 17. Chirigos, M. A., K. Perk, W. Turner, B. Burka, and M. Gomez. 1968. Increased oncogenicity of the murine sarcoma virus (Moloney) by co-infection with murine leukemia viruses. Cancer Res. 28:1055-1063. 18. Dawson, R. J., R. B. Tacke, and A. H. Fieldsteel. 1968. Relationship between Friend virus and an associated lymphatic leukemia virus. Br. J. Cancer 22:569-576. 19. Dent, P. B. 1972. Immunodepression by oncogenic virus. Prog. Med. Virol. 14:1-35. 20. De Sousa, M. A. B., and D. M. V. Parrot. 1969. Induction and recall in contact sensitivity. Changes in skin and draining lymph nodes of intact and thymectomized mice. J. Exp. Med. 130:671-686. 21. Fabris, N. 1973. Immunological reactivity during pregnancy in the mouse. Experientia 29:610-613. 22. Friedman. H., and W. S. Ceglowski. 1971. Defect in cellular immunity of leukemia virus-infected mice assessed by a macrophage migration-inhibition assay. Proc. Soc. Exp. Biol. Med. 136:154-158. 23. Friedman, H., and W. S. Ceglowski. 1973. Cellular immunity and leukemia virus infection, p. 299-320. In W. S. Ceglowski and H. Friedman (ed.), Virus tumorigenesis and immunogenesis. Academic Press Inc., New York. 24. Friedman, H., H. Melnick, L. Mills, and W. S. Ceglowski. 1973. Depressed allograft immunity in leukemia virus infected mice. Transplant. Proc. 5:981-986. 25. Greenwood, B. M., J. H. L. Playfair, and G. Torrigiani. 1971. Immunosuppression in murine malaria. I. General characteristics. Clin. Exp. Immunol. 8:467-478. 26. Grinswold, D. E., J. A. Di Lorenzo, and P. Calabresi. 1974. Quantification and pharmacological dissection of oxazolone-induced contact sensitivity in the mouse. Cell Immunol. 11:198-204. 27. Kumar, V., M. Bennet, and R. J. Eckner. 1974. Mechanisms of genetic resistance to Friend leukemia virus in mice. I. Role of 89 Sr-sensitive effector cells responsible for rejection of bone marrow allografts. J. Exp. Med. 139:1093-1109.
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31. Mortensen, R. F., W. S. Ceglowski, and H. Friedman. 1974. Susceptibility and resistance to Friend leukemia virus: effect on production of migration-inhibition factor. J. Natl. Cancer Inst. 52:409-505. 32. Notkins, A. L., S. E. Mergenhagen, and R. J. Howard. 1970. Effect of virus infections on the function of the immune system. Annu. Rev. Microbiol. 24:525-538. 33. Odaka, T., H. Ishii, K. Yamaura, and T. Yamamoto. 1966. Inhibitory effect of Friend leukemia virus infection on the antibody formation to sheep erythrocytes in mice. Jpn. J. Exp. Med. 36:277-290. 34. Parrot, D. M. V., M. A. B. De Sousa, and J. Fachet. 1970. The response of normal, thymectomized and reconstituted mice in contact sensitivity. Clin. Exp. Immunol. 7:387-393. 35. Phanuphak, P., J. W. Moorhead, and H. N. Claman. 1974. Tolerance and contact sensitivity to DNFB in mice. I. In vivo detection by ear swelling and correlation with in vitro cell stimulation. J. Immunol. 112:115-123. 36. Pritchard, H., and H. S. Micklem. 1972. Immune responses in congenitally thymusless mice. I. Absence of response to oxazolone. Clin. Exp. Immunol. 10:151-161. 37. Revillard, J. P. 1971. Introduction, p. 1-5. In J. P. Revillard (ed.), Cell-mediated immunity. In vitro correlates. University Park Press, Baltimore. 38. Rowson, K. E. K., and I. Parr. 1970. A new virus of
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minimal pathogenicity associated with Friend virus. I. Isolation by end-point dilution. Int. J. Cancer 5:96-102. Schneider, M., and J. F. Dore. 1969. Effet de l'injection d'un virus leucemogene murin (Friend) sur les reactions immunitaires des souris sensible et resistantes a ce virus. Rev. Fr. Etud. Clin. Biol. 10:1010-1014. Steeves, R. A., R. J. Eckner, M. Bennett, E. A. Mirand, and P. J. Trudel. 1971. Isolation and characterization of a lymphatic leukemia virus in the Friend virus complex. J. Natl. Cancer. Inst. 46:1209-1217. Stutman, O., and J. M. Dupuy. 1972. Resistance to Friend leukemia virus in mice: effect of immunosuppression. J. Natl. Cancer Inst. 49:1283-1293. Wedderburn, N., and M. H. Salaman. 1968. The immunodepressive effect of Friend virus. II. Reduction of splenic hemolysin-producing cells in primary and secondary responses. Immunology 15:439-454. Wheelock, E. F., S. T. Toy, 0. S. Weislow, and M. H. Levy. 1974. Restored immune and nonimmune functions in Friend virus leukemia mice treated with statolon. Prog. Exp. Tumor Res. 19:369-389. Zembala, M., and G. L. Asherson. 1970. Contact sensitivity in the mouse. V. The role of macrophage cytophilic antibody in passive transfer and the effect of trypsin and anti-gamma globulin serum. Cell. Immunol. 1:276-289.
INFECTiON AND IMMUNITY, May 1975, p. 1031-1037 Copyright i 1975 American Society for Microbiology
Printed in U.S.A.
Immunodepression by Rowson-Parr Virus in Mice: Effect of Rowson-Parr Virus and Friend Leukemia Complex Infections on Contact Sensitivity in Susceptible and Resistant Mice M. BENDINELLI,* M. CAMPA, AND A. TONIOLO Institute of Microbiology, University of Pisa, 56100 Pisa, Italy Received for publication 31 December 1974
Contact sensitivity to 2-phenyl-4-ethoxymethilene oxazolone, as a probe for cell-mediated immunity, was investigated in susceptible BALB/c and resistant C57BL/6 mice after infection with Friend leukemia complex (FLC) or with Rowson-Parr virus (RPV). In BALB/c mice, FLC depressed contact sensitivity when given before primary sensitization but had no effect on established contact sensitivity nor on the response elicited by a booster application of the sensitizer. These findings, together with the failure to alter reactivity to an aspecific inflammatory stimulus, indicate that FLC impairs the afferent limb of the response. In the same strain of mice RPV infection did not significantly depress contact sensitivity, as judged by the extent of the reaction 24 h after challenge, but slightly inhibited the early antibody-mediated phase of this reaction. In C57BL/6 mice neither viral preparation affected contact sensitivity. After early reports of depressed contact sensitivity (1, 9), there has been somewhat conflicting evidence on the functionality of cellmediated immunity in mice infected with the Friend leukemia complex (FLC) (18, 38, 40). In one study, skin graft rejection was reported unimpaired in susceptible CBA and in resistant C57BL/6 mice infected before grafting (39), but later investigations revealed that infected susceptible BALB/c mice exhibit increased rejection times to both H-2 and non-H-2 allografts; this deficit was confirmed in vitro by direct and indirect migration inhibition factor assays (24) and by lymphocyte-mediated cytolysis (30). Spleen cells from infected susceptible mice have been shown to mount normal graft-versus-host reactions (12, 39), but in one report, infected C57BL/6 splenocytes caused accelerated mortality in (DBA/2 x C57) F1 (39). FLC infection has also been found to depress the immune elimination of allogeneic lymphoid cells slightly (6) but was without effects on the rejection of bone marrow allografts (12). The rejection of sarcoma 180 tumor cells was found reduced in infected susceptible DBA/2 and resistant C57 mice (S. B. Deodhar and T. Chiang, Fed. Proc. 29:560, 1970). Although suggestive, the findings that co-infection with FLC abolished the regression of tumors induced in BALB/c mice by murine sarcoma virus (17) and that C57BL/6 but not BALB/c mice injected with Friend lymphomas releasing infectious virus developed
transplantation resistance (28) are difficult to interpret. Classical hypersensitivity to tuberculin has repeatedly been investigated and constantly found depressed in susceptible but not in resistant mice infected either before or after sensitization, as assessed by footpad swelling and by the migration inhibition factor and blastogenic response of spleen cells to purified protein derivative (22, 23, 29, 31). In recent studies, FLC infection of susceptible donors has also been shown to suppress the spleen cell response to concanavalin A (29) and to phytohemagglutinin (43). Taken together, these results indicate that FLC does indeed impair cellmediated immunity though certain assays (graft-versus-host reaction, for example) probably do not indicate the extent of impairment. Rowson-Parr virus (RPV), one of the viruses forming the FLC, in singly infected BALB/c mice induces an early depression of the antibody response to sheep erythrocytes (11, 13) that is accompanied by a slight hyperplasia of the spleen, whereas neoplastic changes follow only after several months (13, 14). From these findings the obvious problem arises of the role played by RPV in the immunodepressive activity of FLC (8). It is therefore of interest to compare the effects of RPV and FLC on cellmediated immune responsiveness. We have done so by using contact sensitivity to oxazolone in mice susceptible (BALB/c) and resistant (C57BL/6) to the leukemogenic activity of FLC.
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MATERIALS AND METHODS Animals. Inbred BALB/c mice and outbred C57BL/6 mice grown in this Institute were used. Only females were used because fighting between males can cause undesired ear swelling; age at the start of the experiments was 8 to 10 weeks. In each experiment the animals were randomly assigned to different groups. Viruses. The passage histories of the viruses have already been described and the batches used were prepared as previously reported (10). The titers of the FLC batches varied between 1030- and 1035 mean infectious doses per 0.1 ml. The RPV batches, prepared in newborn mice, ranged in titer between 104.0 and 105.° mean infectious doses per 0.1 ml. Both preparations were free of the lactic dehydrogenase virus, which is known to depress contact sensitivity (1). Infection was performed by retro-orbital injection of 0.1 ml of virus. Sensitization. Sensitization was carried out as described (3) with minor modifications. The mice were anesthetized by intraperitoneal administration of tribromoethanol in amylene hidrate (Avertin, Winthrop, N.Y.) and sensitized by the application of 2-phenyl-4-ethoxymethilene oxazolone (referred to as oxazolone; British Drug Houses, Poole, England) in ethanol to the skin of the abdominal wall. The sensitizer was spread within an area of approximately 4 cm2. Unless stated otherwise, 0.2 ml of 2% oxazolone was applied. Challenge. Unless stated otherwise, the challenge was carried out 6 days after sensitization. The animals were anesthetized and the thickness of both ears was measured twice with a micrometer. Both sides of both ears were then painted with a drop of 1% oxazolone in olive oil. This concentration of oxazolone was chosen on the basis of preliminary studies indicating that it was little irritating in unsensitized animals and highly effective in inducing ear swelling in sensitized mice. In these experiments 2% oxazolone, which had been used as challenge in previous studies and found nonirritating (1), produced marked nonspecific swelling in normal mice. The reason for this discrepancy is not known, but it might have been due to differences in the degree of purity of different batches of oxazolone. Unsensitized mice were included at each testing as controls for unspecific swelling. Quantification of sensitivity. Twenty-four hours after sensitization, the animals were again anesthetized and the ear thickness was measured twice. For each mouse the result was expressed by the difference in thickness found between the measurements carried out before and after challenge (mean of the two ears) in units of 10-i cm. Statistics. Unless otherwise specified, six to seven mice were used per group. For each group the geometric mean of ear thickness increase and the 95% confidence limits of the geometric mean are given. The statistical significance of the differences between groups was assessed by Student's t test.
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the duration of contact sensitivity, groups of BALB/c mice were painted with 3% oxazolone and challenged at different intervals thereafter. Groups of unimmunized mice served as controls. A significant increase in ear thickness above the control values was first detected in the group of animals challenged 3 days after sensitization (Fig. 1). At this time there were considerable individual variations. Maximum ear swellings and the lowest degree of individual variation were obtained when the challenge was performed between days 4 and 7 from immunization. By day 9 the extent of swelling had already decreased and individual variations had become larger. This level of sensitivity remained little changed throughout the period of observation (10 weeks). It was possible to evoke a secondary response. Groups of seven BALB/c mice sensitized with 3% oxazolone on day -70 were given a similar second dose of sensitizer on day 0 and challenged on days 2, 3, 6, and 9. Groups of animals given the primary stimulus only were used as controls. The boosted mice showed a clear potentiation of ear swelling and a reduction in individual variations 6 days after the booster as compared with animals given the primary dose alone (Fig. 1). However, the rapidity and extent of the response, as well as the duration of maximum responsiveness, were not different from those observed during the primary response. The kinetics of development of ear swelling during the primary response was also investigated. One group of BALB/c mice sensitized on day -6 and one group of normal mice were 75-
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FIG. 1. Contact sensitivity in BALBIc mice at RESULTS various times after sensitization (-, primary reContact sensitivity in normal mice. To sponse; *, secondary response) and kinetics of the determine the time course of appearance and reaction after challenge (0).
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CONTACT SENSITIVITY IN RPV AND FLC INFECTIONS
challenged at time 0, and ear thickness was measured at various intervals thereafter. In normal animals the increase of ear thickness was very low, reaching a peak of 1.18 3 days after testing. In sensitized mice the response was biphasic: 8 h after challenge, there was a first peak with ear swellings averaging about 50%o of the maximum response, after 10 h the response had slightly but definitely declined, and at 12 h the response had started to increase again to reach the second and highest peak at 24 h. At 72 h the extent of ear swelling was somewhat decreased. The effect of varying the dose of oxazolone used to sensitize BALB/c mice was also investigated. Groups of mice were skin painted with 0.2 ml of increasing concentrations of oxazolone and challenged 6 days later. The results are not reported here in detail because they mimicked those obtained in subsequent experiments with normal and RPV-infected animals (see Fig. 3). The lowest concentration of oxazolone giving maximum sensitization was 2%. The same study was performed in C57BL/6 mice. This strain was more prone to sensitization: painting with as little as 0.1% oxazolone gave a mean ear swelling of 7.64 (4.33 to 13.44) against aspecific swelling of 0.66 (0.31 to 1.41) in unsensitized animals. The lowest concentration giving optimum ear swelling was 1%. To test the degree of depression of contact sensitivity that can be achieved by a treatment known to interfere strongly with cellular immune response, the effect of rabbit anti-mouse thymocyte serum administered 1 and 3 days before sensitization on the extent of ear swelling after challenge was studied in BALB/c mice. Anti-thymocyte serum halved the mean ear swelling as compared with the sensitized controls (P < 0.01), whereas rabbit anti-mouse macrophage and normal sera had no significant effects. These results further demonstrate the suitability of contact sensitivity as a probe of cell-mediated immune responsiveness of mice after various manipulations. Contact sensitivity in RPV- and FLC-infected BALB/c mice. The effect of infection with RPV or FLC performed at various times before sensitization is shown in Fig. 2. Groups of unimmunized infected or uninfected mice served as controls for aspecific swelling; their responses did not significantly differ. RPV given as early as 50 days or 1 day before sensitization did not significantly influence the development of contact sensitivity; in contrast, FLC given 15, 6, or 3 days before sensitization produced marked depression of contact sensitivity. The effect of RPV infection 6 days before sensitization with various doses of oxazolone
was also studied. Normal and infected mice showed superimposable dose response curves (Fig. 3). The time course of development of ear swelling in sensitized uninfected and RPV- or FLCinfected mice was compared by measuring the
L-. z
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10
5
15
20
50
DAYS BETWEEN INFECTION AND SENSITIZATION
FIG. 2. Effect of infection with Rowson-Parr virus (0) or Friend leukemia complex (V) on contact sensitivity in BALBIc and C57BL/6 mice.
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05 1 20 10 CONCENTRATION OF SENSITIZER X 3. FIG. Effect of infection with Rowson-Parr virus on contact sensitivity established with various doses of sensitizer in BALBIc mice (0, infected mice; 0,
uninfected controls).
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BENDINELLI, CAMPA, AND TONIOLO
thickness at various times after challenge in mice sensitized 6 days after infection (Fig. 4). Normal mice showed the usual biphasic response. In FLC-infected mice, the magnitude of the response was strongly reduced throughout the testing period and there was no evidence of the first peak of response, which in normal mice was clearly evident at 8 h. In the RPV-infected mice the magnitude of the 24-h response was normal, but the 8-h peak was reduced. The effects of RPV and FLC infections on long-term established contact sensitivity and on the secondary response were also ascertained. Groups of mice were sensitized on day -71, infected on day 12, and challenged on day 0. Neither FLC nor RPV had any detectable effect on the residual low level of contact sensitivity still detectable 71 days after primary stimulation (Table 1). Other groups of animals were treated as above but on day -6 received a second dose of sensitizer. The recall of contact sensitivity in the infected animals was as high as in the uninfected controls. The depression of ear swelling caused by FLC in sensitized animals as well as the apparent lack of effect on it by RPV infection might be due to the ability of these viral infections to reduce (or to potentiate, in the case of RPV) the ability of mice to mount an inflammatory response. To test this possibility the degree of ear
INFECT. IMMUN.
TABLE 1. Effect of infection with RPV or FLC on long-term established contact sensitivity and on the secondary response in BALBIc micea Contact sensitivity
Sensitiz- Infection Booster No. of ation
mice ~Mean
I
-
+ +
+ +
+ +
None None RPV FLC None RPV FLC
-
+
+ +
6 11 12 10 10 9 9
0.32 1.76 2.22 1.88 10.45 11.59 11.09
95d Confidence limits
0.17-0.61 0.85-3.65 1.38-3.59 0.95-3.70 7.10-14.97 8.20-16.37 7.55-16.30
-
a Sensitization was carried out on day -71, infection on day - 12, booster on day -6, and the challenge on day 0.
nonspecific ear swelling was measured, in normal mice and in mice infected with FLC or RPV 15 days earlier, 2 h after the application of the irritant agent origan oil on the ears. The mean increases of ear thickness were 9.66, 9.50, and 9.85 units, respectively. Contact sensitivity in RPV- and FLC-infected C57BL/6 mice. In the first group of experiments mice were infected with RPV or FLC at various times before sensitization with 2% oxazolone and tested 6 days later (Fig. 2). No differences were observed in contact sensitivity Vf) between infected and control mice. -J 100 Since the doses of sensitizer necessary to establish a substantial level of contact sensitivI.z ity in C57BL/6 mice are markedly lower than 0 those used in the previous experiment, in a further experiment the effect of RPV and FLC 75infections given 7 days before sensitization with 0.1% oxazolone was studied. The mean ear swelling was 5.87 (4.08 to 8.45) for RPV-infected mice and 4.70 (2.95 to 7.48) for FLC-infected animals against 5.50 (3.75 to 8.05) in the sensi3:50tized uninfected controls and 0.64 (0.18 to 2.21) in the sensitized controls. DISCUSSION Contact to simple chemicals in sensitivity 7- 25mice has been extensively investigated in recent years (1-5, 20, 26, 34-36, 44) and has proven a very suitable model for exploring basic aspects of the immune response. Z .n Contact sensitivity has also been exploited as a parameter of immune responsiveness of mice HOURS AFTER CHALLENGE during infection with various viruses or in other FIG. 4. Kinetics of ear swelling after challenge in situations (1, 9, 21, 25). Here the morphology of sensitized uninfected (0), Rowson-Parr virus-infected the response of mice to oxazolone was pre(0), and Friend leukemia complex-infected BALBIc liminarily further characterized and a regimen mice (V). of sensitization and testing suited for assaying 0
0 z
w
w
0
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CONTACT SENSITIVITY IN RPV AND FLC INFECTIONS
the functionality of the immune system in mice receiving various manipulations was determined. Contact sensitivity, like other in vivo delayed hypersensitivity reactions (37), is not a pure cell-mediated entity since low levels of sensitivity can be passively transferred with serum or with macrophages coated with serum (4, 5). After challenge the increase of ear thickness in actively sensitized animals shows two peaks. The first one, which has been shown in previous studies (20, 34) to occur as early as 4 h after challenge, occurred in our experiments at 8 to 10 h. This peak is usually low and is mainly a manifestation of an antibody-mediated reaction (20). The second and highest peak, which reaches its maximum at 24 h, is maintained at 48 h and, generally, is still sustained at 72 h, is instead clearly a typical delayed-type reaction (20, 26). In these and in previous studies (1, 10) aimed to ascertain the effects of viral infections on cell-mediated immunoresponsiveness, we were primarily concerned with the late response to challenge, i.e., with the increase of ear thickness at 24 h. The use of contact sensitivity to oxazolone is a very convenient probe for cellular immunity in virus-infected mice, since a high level of sensitivity can be easily and reproducibly induced and quantitated in 1 week. The method is of great advantage when viruses that kill the host rapidly are studied, making it possible to infect the animals before sensitization and to test the sensitivity before the animals are obviously ill. The possibility of evoking a secondary response is also advantageous, because virus infections may preferentially interfere with primary or secondary immune responses (19, 32). Moreover, the kinetics of the response to challenge can be easily followed, providing indications on whether a given infection affects the early antibody-mediated phase, the late cellmediated phase, or both. Contact sensitivity has been extensively used also as a parameter of immune responsiveness in humans, and therefore correlations between findings in animal models and clinical situations can be drawn. Previous investigations have shown that contact sensitivity to oxazolone and to pycril chloride is depressed in susceptible mice infected with FLC (1, 9). Here the effects of FLC and RPV on contact sensitivity to oxazolone in susceptible and resistant mice are compared. In BALB/c mice, as expected, FLC given between 3 and 15 days before sensitization strongly depressed the primary response, whereas RPV had no detectable effects as judged by the increase in ear thickness at 24 h, regardless of
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the dose used for sensitization and the interval between infection and sensitization. A difference between normal and RPV-infected mice could be noticed only when the time course of ear swelling was studied. In the RPV-infected animals the early peak of swelling was reduced, probably reflecting a depression of the humoral response to oxazolone. In FLC-infected mice both the early and late peaks were depressed, in accordance with previous findings that FLC impairs antibody and cellular responses to simple chemicals (1). The inflammatory response is a key factor in contact sensitivity (2). However, the possibility that FLC and RPV might interfere with the ability of mice to mount a normal inflammatory response is ruled out by the observation that oxazolone and the irritant origan oil caused equal levels of nonspecific ear swelling in infected and uninfected unsensitized mice. The findings that the established contact sensitivity and the secondary response were not depressed by FLC also argue against this possibility and indicate that FLC inhibits contact sensitivity not by affecting its expression but by interfering with the afferent limb of the reaction. However, the failure of FLC infection to inhibit established contact sensitivity strongly contrasts with recent results in which FLC profoundly suppressed an established state of hypersensitivity to PPD and to histocompatibility antigens in susceptible mice (29-31). An explanation of these contradictory results may be that a few days after sensitization the cells mediating contact sensitivity to oxazolone in mice are no longer found in the spleen and lymph nodes but appear in the bone marrow and in the peritoneal cavity (5), peritoneal lymphocytes being affected by FLC infection only very late in the course of the disease (7). Previous reports had indicated that C57BL/6 mice, a strain considered "absolutely resistant" (41) to leukemia induction by FLC, are also resistant to the immunodepressive activity of the virus (16, 33, 39, 42). However, recent investigations in our laboratory and elsewhere have revealed that after FLC infection in this strain, the splenic antibody-forming cell response to sheep erythrocytes (15, 27; unpublished data) is transiently but consistently reduced. RPV infection, in contrast, has no effect on the humoral response to sheep erythrocytes in C57BL/6 mice (unpublished data). The present results, showing that neither virus given before sensitization depresses contact sensitivity in C57BL/6 mice, agree with the finding by others that in this strain FLC fails to impair an established tuberculin hypersensitivity (31).
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BENDINELLI, CAMPA, AND TONIOLO
After infection with the viruses of FLC there be a dissociation between development of leukemia and depression of the antibody response to unrelated antigens. This has been found to occur in resistant C57BL/6 mice infected with FLC (15, 27; unpublished data) and in susceptible BALB/c mice infected with RPV (11); in both instances infection is followed by a rapid fall in the ability to produce antibody but not by early signs of leukemia. The results presented here indicate that such dissociation with leukemia development does not seem to exist in the case of the depression of cellmediated immunity. However, it is not known whether the impairment of cellular immunity is a primary effect of FLC infection or a consequence of the induced neoplastic changes. may
ACKNOWLEDGMENTS This investigation was supported by the Italian National Research Council (grant no. CT 73.00462.04). We thank G. Falcone for helpful advice and suggestions, L. Montagnani for skillful technical assistance, and G. Greco for typing the manuscript. LITERATURE CITED 1. Asherson, G. L., and M. Bendinelli. 1969. Immunodepression by viruses: effect of Friend and Riley viruses on contact sensitivity. G. Microbiol. 17:179-188. 2. Asherson, G. L., and R. M. R. Barnes. 1973. Contact sensitivity in the mouse. IX. The role of immunological and non-immunological inflammation in the movement of lymphocytes to immunized lymph nodes. Immunology 24:885-894. 3. Asherson, G. L., and W. Ptak. 1968. Contact and delayed hypersensitivity in the mouse. I. Active sensitization and passive transfer. Immunology 15:405-416. 4. Asherson, G. L., and M. Zembala. 1970. Contact sensitivity in the mouse. IV. The role of lymphocytes and macrophage in passive transfer and the mechanism of their interaction. J. Exp. Med. 132:1-15. 5. Asherson, G. L., and M. Zembala. 1973. Anatomical location of cells which mediate contact sensitivity in the lymph nodes and bone marrow. Nature (London) New Biol. 244:171-177. 6. Bainbridge, D. R., and M. Bendinelli. 1972. Circulation of lymphoid cells in mice infected with Friend leukemia virus. J. Natl. Cancer Inst. 49:773-781. 7. Bendinelli, M. 1968. Haemolytic plaque formation by mouse peritoneal cells, and the effect on it of Friend virus infection. Immunology 14:837-850. 8. Bendinelli, M. 1971. Immunodepression by Friend virus, p. 314. In J. L. Melnick (ed.), Proc. 2nd Int. Congr. Virol. S. Karger, Basel. 9. Bendinelli, M., and G. L. Asherson. 1968. Effect of Friend virus infection on contact sensitivity. Rep. Br. Emp. Cancer Campaign 45:137. 10. Bendinelli, M., and L. Nardini. 1973. Immunodepression by Rowson-Parr virus in mice. I. Growth curves of Rowson-Parr virus and immunological relationships with Friend virus. Infect. Immun. 7:152-159. 11. Bendinelli, M., and L. Nardini. 1973. Immunodepression by Rowson-Parr virus in mice. II. Effect of Rowson-Parr virus infection on the antibody response to sheep red cells in vivo and in vitro. Infect. Immun. 7:160-166. 12. Bennett, M., and R. A. Steeves. 1970. Immunocompetent
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