Phytohemagglutinin-induced leukocyte blastogenesis in normal and avian leukosis virus-infected chickens

CELLUL.%R

IMML’NOLOGY

27,

140-146

(1976)

Phytohemagglutinin-Induced and Avian Leukosis PAUL

MEYERS,

Department

GRAHAM

Leukocyte Blastogenesis in Normal Virus-Infected Chickens1 D.

of Microbiology, Rorhester,

RITTS,

AND

DANIEL

Mayo Clinic and Mayo Minnesota 55901

Received

July

R. Mcdiral

JOHNSON School,

2, 1976

The effects of congenital avian leukosis virus (ALV) infection on cell-mediated immunity in the chicken have been investigated. We assessed the capabilities of leukocytes from ALV-infected birds to respond to phytohemagglutinin (PHA), a T-cell mitogen, using an in vitro blastogenic transformation assay. There were no significant differences in PHA responsiveness between ALV congenitally infected and normal control birds as measured at times from several weeks posthatching to adulthood. However, there was a quantitative difference between the two groups in the amount of PHA needed to stimulate their leukocytes maximally. This latter phenomenon may be a consequence of virus infe,tion of the lymp.,o-ytes or altered R- or T-cell ratios.

INTRODUCTION It is now well established that infection of a host with certain oncogenic or nononcogenic viruses may induce alterations in the host’s immunological capacity, affecting cellular or humoral responsesor both (1, 2). Of those oncogenic viruses which affect chickens, the effects of Marek’s diseasevirus (MDV) on immunological responsiveness have been well studied, and apparently both cellular and humoral responses are adversely affected (1, 3-12). E-ar less information is available on possible immunosuppression caused by infection of chickens with another group of oncogenic agents, the avian leukosis viruses (ALV). Impairment of antibody responsesby ALV has been reported (3, 13-16) as well as both increases (1.5) or normal function (14-16), the reported effects apparently depending in part on the challenge antigen response measured, the particular strain of ALV used, the age of the animals, and other factors. Data on cell-mediated immunity (CMI) in ALV-infected birds are sparse and also equivocal and have been largely derived from in z&o assessmentsof CM1 (3, 4, 16). There is only one brief report (17) on the effects of congenital ALV infection, which produces a chronic lifelong viremia (18)) on CM1 in chickens. The purpose of the work presented here was to assessthe capabilities of lymphocytes from ALV congenitally infected chickens to respond in vitro to phytohemagglutinin (PHA), a specific T-cell mitogen in the chicken ( 19-23). We have, therefore, developed a sensitive microtechnique for chicken lymphocyte blastogenesis,using lymphocytes obtained from peripheral blood, and examined the 1. Supported

by Public

Health

Service

Grant

CA15494 110

Copyright All

rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

from

the National

Cancer

Institute.

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ontogeny of T-cell responsiveness to PHA in ALV congenitally infected and control birds. To our knowledge, this is the first report utilizing in vitro blastogenesis to assessCM1 capabilities directly in ALV-infected birds. MATERIALS

AND

METHODS

Al&&s. Fertile white Leghorn eggs were obtained from flocks free of exogenous leukosis viruses, MDV, and other common avian pathogens (SPAFAS, Inc., Sorwich, Conn.). Chicks were hatched in a standard commercial incubator. Chicks congenitally infected with ALV were obtained as previously described (14) by injecting 7-day-old embryonated SPAFAS eggs in the yolk sac with 105-10” infectious units of ALV-F42, a subgroup A leukosis virus, After hatching, these chicks were tested for viremia by a radioimmunoassay for avian group-specific (gs) antigen (24, 25) and for the absence of neutralizing antibody to the homologous pseudotype sarcoma virus, RSV( F42) ( 14). Uninfected control chicks were hatched and housed separately from AI,V-infected animals. Cell preparation. Blood was drawn by cardiac puncture or from the wing vein into a syringe containing heparin (Panheparin, Abbott Laboratories, North Chicago? Ill.). The blood was then transferred to 16 x 125-mm screw-cap tubes and centrifuged at 50g for 12-15 min, and the plasma and buffy coat were aspirated into another 16 X 125mm tube. Care was taken to avoid erythrocytes. The leukocyte-plasma suspension was then centrifuged at 400~ for 10 min, the supernatant was removed and discarded, and the cell pellet was resuspended in RPM1 1640 medium (Grand Island Biological Co., Grand Island, N.Y.) containing 10% fetal calf serum (Grand Island Biological Co.), 100 units/ml of penicillin, and 100 pg/ml of streptomycin. A single lot of calf serum was used for most of the experiments. A portion of the cell suspensionwas counted using Natt-Herrick’s stain (26)) and the cell concentration was adjusted to lo7 mononuclear cells/ml. Leztkocyte cultwe. The cells were distributed into No. 3040 Falcon microtiter plates (Microtest II, Falcon Plastics, Oxnard, Calif.,) using a 200-~1 MLA pipet (Medical Laboratory Automation, Inc., Mt. Vernon, N.Y.) so that each well contained 2 x 10G cells in 200 ~1. We should note that this cell number was determined experimentally to give optimum results under our assay conditions (unpublished). Phytohemagglutinin (PHA-M, Difco, Detroit, Mich.) was reconstituted to 5 ml according to manufacturer’s instructions, and further dilutions were based on this original stock nominally being considered “undiluted” PHA. The dilutions of PHA (in medium 1640) were made so that when added to the microtiter wells the final mitogen dilutions were 1 : 10, 1 : 20, 1: 40, 1 : 80, and 1 : 160. A single lot of PHA was used for most of the work described. When a second lot was needed it was selected on the basis of comparisonsof its blastogenic capability to the first lot. All dilutions were assayed in triplicate or quadruplicate, except for occasional samplesfrom very young birds when limited cell numbers provided only enough cells for duplicate samples.Nonstimulated leukocyte cultures (medium only) were always run in parallel with the PHA-stimulated cultures. After 56 hr of incubation at 37°C in a 5% CO2 atmosphere, 1.0 &i of tritiated thymidine (6.7 Ci/mmol, New England Nuclear Corp., Boston, Mass.) was added to each well. After an additional 16 hr of incubation, the cells were harvested by means of a multiple automated sample harvester onto filter paper (934AH, Whatman, Inc., Clifton, N.J.) and washed with physiological saline and 5% TCA. After

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drying, those portions of the filter paper containing the precipitated material were cut out, transferred to scintillation vials containing 10 ml of Fluoralloy TLA (Beckman Instruments, Inc., Irvine, Calif.,) in toluene, and counted for 5 min or to 1% error in a Beckman LS-330 liquid scintillation spectrometer. RESULTS Since we wished to compare the PHA responsiveness of the leukocytes of normal control birds with those of ALV congenitally infected animals, it was first necessary to establish the normal ontogeny of in vitro blastogenesis. Figure 1 shows the maximum PHA-induced incorporation of [ 3H] thymidine in individual control birds ranging in age from 11 days to adults, presented as log, (T-N) where T is the mean counts per minute in the PHA-stimulated cultures and N is the mean counts per minute in the nonstimulated cultures. It may be seen that after about 5 weeks of age most birds responded well, with some tendency for hens to be poor responders at earlier ages. There was, however, considerable scatter in the PHA response of individuals except at later ages. The same comments apply to the lymphocyte responses of individual ALV congenitally infected birds presented in Fig. 2. The individual variation in these animals appears to be even more pronounced. When the means of the data from all of the birds in each age group are plotted (Fig. 3) it may be seen that these means have overlapping 95% confidence limits and therefore there appeared to be no difference in the ability of leukocytes from normal or ALV congenitally infected birds to respond to PHA. However, as described above, the data presented in Figs. l-3 represent maximum stimulation of [SH]thymidine incorporation by PHA. When the responses were analyzed in terms of the dilution of PHA required to obtain maximal stimulation there are clear-cut differences in ALV-infected and control birds. Figure 4 presents the percentage frequency of occurrence of maximal leukocyte stimulation of normal and ALV-infected birds at dilutions of PHA ranging from 1: 10 to 1: 160. It may be seen that nearly twice as many ALV-infected birds’ leukocytes required a 1: 10 concentration of PHA for maximal [3H] thymidine uptake as did the leukocytes of normal control birds (40 vs 22%, respectively). Since we did not test any

FIG. 1. Maximum [*HI thymidine incorporation by control chickens expressed as log, of the total counts Closed circles, males; crosses, females ; open circles, mean of two to four replicate cultures (see text) from

PHA-stimulated leukocytes from normal per minute (T) minus background (N). sex unknown; each point represents the a single bird.

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FIG. 2. Maximum [8H] thymidine incorporation by PHA-stimulated leukocytes from ALV congenitally infected chickens expressed as log2 of the total counts per minute (T) minus background (N). Closed circles, males ; crosses, females ; open circles, sex unknown ; each point represents the mean of two to four replicate cultures (see text) from a single bird.

PHA dilutions at less than 1 : 10, these differences could possibly have been even greater. At a PHA dilution of 1 : 40, 34% of the control birds’ leukocytes showed maximum stimulation while only 20% of the ALV-infected birds responded. The percentage of normal birds whose leukocytes required a PHA dilution of 1: 20 or less for maximal stimulation was 47%, whereas fully 61% of congenitally infected birds required this amount of PHA. These data are based on 204 normal animals and 75 ALV-infected birds and were consistent even within a given experiment where leukocytes of control and infected birds were cultured under exactly identical conditions. This held true especially in the case of older birds where the maximum PHA responsesof individuals tended to be less scattered. Thus, taken as an overall pattern, the leukocytes of normal birds tended to require less PHA for maximum stimulation than did the cells of ALV-infected birds.

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FIG. 3. Mean maximum [8H] thymidine incorporation of PHA-stimulated leukocytes by normal control (-•-) and ALV congenitally infected (- -O- -) chickens. Each point represents the mean of S-23 chickens of each age tested. The vertical bars are 95% confidence limits.

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I:20

I:40

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FIG. 4. Percentage frequency of occurrence of maximum PHA-induced leukocyte blastogenesis at a given dilution of PHA. Open bars represent controls; hatched bars are ALV congenitally infected birds.

DISCUSSION Cell-mediated immunity in the chicken develops as early as the second week or so of embryonic life (27-31) as judged mostly by in z&o allograft reactivity (27-29, 31). Thus, it is not surprising that high levels of PHA-induced leukocyte blastogenesis are detectable at 10 days posthatching in both normal and ALV congenitally infected chickens. Although some birds’ leukocytes responded poorly at this early age, others showed high levels of stimulation. Fluctuations in the level of peripheral blood leukocytes and a “plateau level” of reactivity such as we observed have also been reported (27). We are not aware of any previous rqlorts measuring CM1 in vitro over the course of a large age span in chickens. In contrast to the situation with Marek’s disease (5, 7-lo), infection of chickens with ALV-F42 does not appear to have a dramatic suppressive effect on CM1 as judged by PHA-induced leukocyte blastogenesis. However the lymphoproliferative diseasecaused by MDV primarily involves thymus-derived (T) cells (6, 32-34) whereas ALV causes lesions involving bursa-derived (B) cells (15, 35, 36). It is therefore not unexpected that previous reports on the effect of ALV on CM1 have been equivocal or negative (3, 4, 16) especially since they employed less sensitive in zrivo measures of CMI. In one case there were slight differences in CM1 in ALV-infected chickens, as compared to controls, when birds were tested at 10 weeks of age, but no differences when birds were tested at a later age (3). We should also point out that birds congenitally infected with the strain of ALVI;42 we employed develop clinical diseaselate in their lifespan and even then often only a fairly small number is affected (37)) so that we have measured responses in birds not clinically ill. One brief report (17) examined eight RAV-1 congenitally infected birds. Of four birds which had clinical leukosis and four which had “inqpparent” leukosis only the former group had a depressed CM1 as judged by a cytotoxicity assay. The rather subtle effects of congenital ALV infection on the optimum dilution ot PHA necessary for maximal leukocyte stimulation, which we describe, may be

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caused by a number of factors. These include a simple increase in the number of B-cells (35) and/or a reduction in the number of T-cells in ALV-infected birds. In this case, however, one would expect the effects to have been more dramatic. Another possibility may involve the fact that chickens congenitally infected with ALV-F42 are known to have lymphocytes which actively replicate virus (37) _ Although there is no direct evidence that these are specifically T-cells, there is no reason to doubt that T-cells are infected, since virtually every cell type in the body of an ALV congenitally infected bird produces virus (37). Depressed lymphocyte reactivity to PHA has been noted in other chronic viral animal infections (e.g., 38) and a phenomenon directly analogous to that which we observed, i.e., depressed lymphocyte responses at “suboptimal” concentrations of PHA, was reported to occur in humans with measles virus infection (39). One further consideration is that plant lectins such as PHA may bind directly to avian tumor virus glycoprotein molecules (40). Such binding of PHA to viral glycoproteins on the surface of virus-producing, infected leukocytes might also account for the present results if PHA were bound to budding or free virus particles, thus making less lectin available to activate the leukocytes. REFERENCES 1. Dent, P. B., Progr. Med. Viral. 14, 1, 1972. 2. Friedman, H., In “Viruses and Immunity” (C. Koprowski and H. Koprowski, Eds.), p. 17. Academic Press, New York, 1975. 3. Purchase, H. G., Chubb, R. C., and Biggs, P. M., J. Xat. Cancrv Zusf. ‘40, 583, 1968. 4. Payne, L. N., Proc. Roy. Sot. Med. 63, 16, 1970. 5. Lu, Y.-S., and Lapen, R. F., A~lzer. J. Vet. Res. 35, 977, 1974. 6. Payne, L. N., Powell, P. C., and Rennic, M., Cold Spring Harbor Sptp. Qzrant. Biol. 39, 817, 1974. 7. Kermani-Arab, V., Mall, T., Cho, B. R., Davis, W. C., and Lu, Y.-S., Infect. Zm?urzl~. 12, 1058, 1975. 8. Theis, G. A., McBride, R. A., and Schierman, L. W., J. 1~rnlultol. 115, 848, 1975. 9. Aim, G. V., Siccardi, F. J., and Peterson, R. D. A., Acta Pathol. Micvobiol. Stand. Sect. A 80, 109, 1972. 10. Burg, R. W., Feldbush, T., Morris, C. A., and Maag, T. A., Asian Dis. 15, 662, 1971. 11. Evans, D. L., and Patterson, L. T., Infect. Znmztn. 4, 567, 1971. 12. Jakowski, R. M., Fredrickson, T. N., and Luginbuhl, R. E., 1. 1+mm~~zol. 111, 238, 1973. 13. Peterson, R. I)., Purchase, H. G., Burrnester, B. R., Cooper, M. D., and Good, R. A., J. Nat. Cancer Inst. 36, 585, 1966. 14. Meyers, P., and Dougherty, R. M., J. Nat. Cam-m Inst. 46, 701, 1971. 15. Cooper, M. D., Purchase, H. G., Bockman, D. E., and Gathings, W. E., J. 1~t+m~101. 113, 1210, 1974. 16. Dent, P. B., Cooper, M. D., Payne, L. N., Solomon, J. J., Burnlester, R. R., and Good, R. A., J. Naf. Caxrer Inst. 41, 391, 1968. 17. Granlund, D. J., and Loan, R. W., J. Nat. Carxer Imt. 52, 1373, 1974. 18. Rubin, H., Bartcriol. Rev. 26, 1, 1962. 19. Greaves, M. F., Roitt, I. M., and Rose, hI. E., Nature (Lortdon) 220, 293, 196s. 20. Alm, G. V., Acta Pathol. dficrobiol. Scaud. Sect. B 78, 632, 1970. 21. Kirchner, H., Oppenheim, J. J., and Blaese, R. M., In “Proceedings of the Seventh Leukocyte Culture Conference,” (F. Daguillard, Ed.), p. 501. Academic Press, New York, 1973. 22. Weber. W. T., J. Reticuloendothel. Sot. 14, 538, 1973. 23. Toivanen, P., and Toivanen, A., 1. Z?nmztnol. 111, 1602, 1973. 24. Fritz, R. B., and Qualtiere, L. F., J. Vivol. 11, 736, 1973. 25. Meyers, P., J. Nat. Cancer Ircst. 56, 381, 1976. 26. Natt, M. P., and Herrick, C. A., Poult. Sci. 3, 735, 1952.

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and Lafferty, K. J., Amt. J. Exp. BioZ. Med. Sci. 49 1971. Seto, F., J. Exp. 2001. 177, 343, 1971. Sillstrijm, J. F., and Aim, G. V., Int. Arch. Allergy 47, 388, 1974. JankovZ, B. D., IsakoviC, K., Lukie, M. L., VujanoviC, N. L., PetroviE, S., and Marl B. M., Immunology 29, 497, 197.5. Payne, L. N., and Rennie, M., J. Nat. Cancer Inst. 45, 387, 1970. Rouse, B. T., Wells, R. J. H., and Warner, N. L., J. ZmmunoE. 110, 534, 1973. Nazerian, K., and Sharma, J. M., J. Nut. Cancer mst. 54, 277, 1975. Payne, L. N., and Rennie, M., Vet. Rec. 96, 454, 1975. Qualtiere, L. F., and Meyers, P., J. Immunol., in press. Dougherty, R. M., and DiStefano, H. S., Progr. Med. Viral. 11, 154, 1969. Perryman, L. E., Banks, K. L., and McGuire, T. C., J. Immunol. 115, 22, 1975. Finkel, A., and Dent, P. B., CelE. Iwzmunol. 6, 41, 1973. Ishizaki, R., and Bolognesi, D. P., J. ViroE. 17, 132, 1976. G. I.,