Suppressed Immune Responsiveness of Chickens Exposed to Antibody as Embryos1

Suppressed Immune Responsiveness of Chickens Exposed to Antibody as Embryos ! FRANK SETO

Zoology Department, University of Oklahoma, Norman, Oklahoma 73069 (Received for publication October 30, 1973) ABSTRACT In the adaptation of the in vivo culture method for the analysis of the chicken immune system, immunocompetent cells (ICC) from antigen-primed allogeneic donors are grown in chick embryo hosts and their antibody-forming capacity assessed. When the surviving in vivo hosts were tested later as juvenile and young adult chickens for their immune capacity, the hemagglutinin output and ICC levels in the blood in response to the specific antigen mouse erythrocytes (MRBC) were significantly less than normal. The extent of the immunosuppression was related in part to (1) the B-antigen disparity between donor and host and (2) the degree of immunologic maturity of the donor. The depressed immune responsiveness in hosts inoculated with ICC from B-mismatched donors could be explained as the consequence of the graft-vmushost reaction, but the explanation is inadequate to account for the impaired development of immunity of hosts that received blood cells from B-antigen matched donors. The immunosuppression was observed in hosts inoculated with a mixture of ICC and MRBC but not apparent in those receiving ICC only. It appears that the activation of the grafted ICC and subsequent antibody formation in some way suppressed the development of the immune response to MRBC in the young hosts. POULTRY SCIENCE 53: 1408-1414, 1974

INTRODUCTION

T

HE in vivo culture model (Albright et al., 1964) was adapted for the kinetic analysis of immunocyte formation and antibody production in the chicken (Seto, 1970a). Immunologically immature chick embryos, which have little adverse effect on the grafted cells and are incapable of responding immunologically to mouse erythrocytes (MRBC) used as antigen, provide an ideal culture medium for the growth of donor immunocompetent cells (ICC) (Seto, 1968, 1971). Within a week after the inoculation of the donor blood-antigen mixture, the activated ICC in the donor blood produce high levels of specific hemagglutinating antibodies in the host embryos. The in vivo antibody response profile is much like that of the intact bird (Seto, 1973). The magnitude of the antibody formation in in vivo culture is dependent on several experimental factors as reported in earlier studies (Seto, 1970a, b, 1971) and

1. Research aided in part with a grant-in-aid from the Executive Committee of the Oklahoma University Research Institute

under proper conditions high antibody titers are obtained consistently. It was observed in exceptional experiments, however, that the in vivo immune antibody titers obtained with donor cells from apparently healthy adult birds were considerably lower than normal. Our records revealed that the donors were used previously as embryo hosts in earlier in vivo experiments. It was suspected that their use as hosts had in some way impaired the normal development of the immune response to MRBC. Experiments were conducted to confirm our suspicions and to ascertain the cause of the immune impairment induced in the in vivo hosts. MATERIALS AND METHODS Materials. Embryonated eggs, baby chicks and adults were obtained from a flock of White Leghorn chickens maintained at the Oklahoma University animal facilities. The inbred chickens have been maintained for several years by closed flock matings within blood group lines ( B ' B 1 C 4 C 4 and B 2 B 2 C 2 C 2 ) . Eggs were incubated in a Sears elec-

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SUPPRESSED IMMUNE RESPONSIVENESS

trie incubator. Baby chicks were housed for the first few weeks in heated battery brooders and later transferred to small cages. Older chickens, especially the breeding stocks were maintained as blood group lines in individual cages or in small pens. All chicks were dye-marked and later wing-banded for identification. METHODS Immunization Procedure. Saline-washed erythrocyte suspension from a Swiss albino strain of mice was used as antigen to immunized donor chickens and to activate the grafted blood immunocytes (ICC) in the in vivo culture assay. Baby chicks, two weeks of age or less were immunized by cardiac injection with 1/2 ml. of a 1% mouse erythrocyte (MRBC) suspension; three to four week old birds received an ml. of a 1% MRBC suspension, and older birds were injected intravenously with higher immunization doses. Serum samples from baby chicks and from embryos in in vivo culture were collected six days after immunization and stored in a freezer at least two days prior to titration. Cyclophosphamide Treatment. Some host embryos were pretreated with cyclophosphamide (CY) a day before the inoculation of cells. CY was obtained through the generosity of Mead Johnson and Co. (Evansville, Indiana). A solution of the drug in Hank's balanced salt solution was prepared immediately before its administration intravenously into host embryos. A sublethal dose of 1.5 to 2.0 mg. per embryo is sufficient to suppress the host system and thereby enhanced the immune activity of the grafted cells (Seto, 1970a, 1971). Them vivo Culture Method. The procedure has been reported in detail earlier (Seto, 1970a) and will be briefly described. Donor chickens were immunized with an intravenous or cardiac injection of MRBC as antigen

and four days later whole blood was procured by cardiac puncture in Alsever's anticoagulant solution. An equal volume of 4% MRBC suspension as antigen was added to the donor blood and the mixture was administered intravenously into 14-day host embryos. The ICC in the donor blood is activated by the antigen to produce antibody in the embryo host. The embryos were bled six days later and the serum tested for antibody content. The antibody titer provides an indirect measure of the ICC content of the donor blood inoculum. The antiserum titration was carried out in Microtiter plates (Cooke Engineering, Alexandria, Va.) with a Wo MRBC suspension as antigen. RESULTS In the initial experiments four apparently healthy young adult donors, three B ' B ' and one B 2 B 2 birds, all former hosts of earlier in vivo experiments, were immunized with an ml. of 10% MRBC suspension and four days later their blood was tested for the ICC content with the in vivo antibody assay. These birds had been inoculated as embryos with a mixture of antigen and ICC-containing blood from B-antigen matched adult donors. Three groups of 14-day embryo hosts were used to assay the ICC-forming capacity of donor # 2 in experiment 1. Group A hosts were inoculated with a mixture of antigen and donor blood; group B hosts received only donor blood, and group C hosts, pretreated with Cy a day before, received the antigendonor blood mixture. In experiment 2, a mixture of antigen and blood from donor # 3 was prepared. Three different concentrations of the mixture was administered into three groups of 14-day embryo hosts. A similar procedure was followed in experiment 3 with donor # 4 . In the last experiment host embryos of different ages were injected with an inoculation mixture prepared with blood from donor # 5 . Group D in experiment 4 was pretreated with Cy a day before the

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F. SETO

TABLE 1.—In vivo antibody assay for ICC-forming capacity of four donor chickens previously used as hosts in in vivo culture experiments. These received ICC-containing blood from B-antigen matched donors

1

Blood type B'B'

A

No. of hosts 14

2

B'B1

A B C

5 5 9

14 days 14 days 14 days

Blood + ag Blood + ag Blood + ag

4.0 ± 2.7 b 4.2 ± 0.5 5.4 ± 0.7=

3

B'B1

A B C

7 8 6

14 days 14 days 14 days

Blood + ag Blood/4 + ag Blood/16 + ag

4.8 ± 2.0 2.4 ± 1.5 1.1 ± 0.4

4

B'B'

A B C

6 5 5

14 days 14 days 14 days

Blood + ag Blood/4 + ag Blood/16 + ag

5.7 ± 1.1 5.2 ± 2.7 1.6 ± 2.5

5

B2B2

A B C D

6 6 8 6

15 17 18 18

Blood Blood Blood Blood

4.0 4.4 3.9 6.6

Donor

Group

Host age

Inoculum

Mean Ab titer ± SD

14 days

Blood + ag

10.3 ± 1.0"

days days days days

+ + + +

ag ag ag ag

± ± ± ±

1.5 1.8 0.9 2.1 c

a Normal control bird. b All experimental groups c

significandy reduced (P less than 0.01). Hosts pretreated with CY; antibody titers higher than the corresponding experimental group.

inoculation of the mixture. Blood from a normal adult chicken, # 1 served as the control. The results of the in vivo assay of the four experiments are summarized in Table 1. The antibody production in all experimental groups were considerably lower than the control. The antibody titer of group B hosts of experiment 1 represents the background

antibody level as these embryos received only donor blood. Since the mean antibody titers of hosts in the group A's of all four experiment are not distinguishable from group B, the ICC-forming capacity of the four donors is minimal. Still lower titers were obtained, as might be expected, when diluted antigendonor blood mixtures were inoculated into other groups of hosts i.e. groups 2B, 2C and

TABLE 2.—In vivo antibody assay for ICC-forming capacity of 3- to 4-week chicks used as embryo hosts in earlier in vivo experiments Donor

Blood

Previous in vivo treatment of donors

Age when assayed

No. of hosts

Mean Ab titers ± SD

3 weeks 4 weeks

3 6

7.7 ± 1.5 9.2 ± 0.8

1 2

B'B' B'B'

None None

3 4 5 6 7 8

B'B' B'B' B'B1 B'B' B2B2 B2B2

B'B'blood B'B'blood B ' B ' blood B ' B ' blood B 2 B 2 blood B 2 B 2 blood

+ + + + + +

ag ag ag ag ag ag

3 3 3 4 3 3

weeks weeks weeks weeks weeks weeks

6 3 7 4 4 2

4.3 2.5 1.8 4.8 1.6 2.7

± ± ± ± ± ±

2.9 a 2.3 1.4 3.1 2.3 3.2

9 10 11 12 13

B'B' B'B1 B'B' B'B' B'B1

B2B2 B2B2 B2B2 B2B2 B2B2

+ ag + ag only only + ag

11/2 3 3 3 11/2 3

weeks weeks weeks weeks weeks weeks

6 4 4 4 5 4

2.0 0.5 1.6 0.5 0.5 0.5

± ± ± ± ± ±

2.2 0.0 2.3 0.0 0.0 0.0

blood blood blood blood blood

a NS compared to 3 week control; all other experimental groups were significantly reduced (P less than 0.05).

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SUPPRESSED IMMUNE RESPONSIVENESS

3C. The mean titers of hosts pretreated with Cy as in groups IC and 4D are significantly enhanced compared to the corresponding experimental group but still obviously lower than the normal control. The data clearly reveal the underdeveloped condition of the ICC-forming potential in the four former in vivo hosts. In a second experimental series, 11 survivors of in vivo experiments were tested as juvenile chicks for their ICC-forming capacity. Six of these received ICC-containing blood from B-antigen matched donors and five received cells from B-mismatched young donors. Two untreated B ' B 1 chicks of comparable ages were used as controls. The results of the in vivo assay of the ICC content of blood following the immunization of the 13 birds are summarized in Table 2. Five of the six birds exposed previously to activated immunocytes from B-matched donors (3 to 8) were significantly depressed in their capacity to produce ICC in response to MRBC immunization as compared to the 3 and 4 week control birds. The depression appears even greater among the donors (9 to 13) that received blood from mismatched donors in earlier in vivo treatment. This appears to be the case whether a mixture of donor cells and antigen (as in donors 9 and 10) or donor cells only (as in donors 11 and 12) were used. The immunosuppres-

sion was practically complete in B ' B ' donor 13 which had been exposed as an embryo host to an antigen-blood cell mixture of B 2 B 2 donor origin. The in vivo antibody assay was negative when the birds was tested at 10 and at 21 days of age. Suppression of the ICCforming capacity was clearly observed in 10 of 11 former in vivo hosts when tested as juvenile chicks. Donors with depressed ICC-forming capacity also appeared to be deficient in hemagglutinin formation in response to MRBC immunization. Experiments were conducted to ascertain this reduction and to determine further the role of immunologic maturity of the donors in the impaired immunologic development of host embryos exposed to their cells. Three groups of donors, (1) 2-3 day baby chicks, (2) 3-week chicks and (3) young chickens 6-10 weeks of age, were immunized with MRBC and four days later bled, the donor blood mixed with antigen and inoculated into host embryos. Other host embryos received an equivalent amount of blood from unprimed young chicken donors. The host eggs were inculated and allowed to hatch. Over half of the hosts that received donor cells from older birds died within two weeks of age. The surviving hosts were immunized with MRBC at 2, 3, or 4 weeks of age and bled six days later for serum hemagglutinin determination. Untreated chicks 2 and 3

TABLE 3.—Hemagglutinin-forming capacity of 2 and 3 week control chicks (1) and 2 to 4 week chicks inoculated as embryos with blood from unprimed donors (2) or a mixture of antigen and blood from antigen primed donors (3). All hosts received blood from B-antigen matched donors Group 1

a b

Source of donor blood for in vivo treatment None None

Age at immunization 2 weeks 3 weeks

No. of birds 8 8

Mean Ab titer ± SD 11.1 ± 1.8 13.4 ± 1.0

2

Young chicken Young chicken

2 weeks 4 weeks

14 9

13.1 ± 1.4" 14.4 ± 1.0"

3

B' B' B' B' B' B' B2B2

2 3 4 4

11 9 7 7

11.8 9.4 9.0 10.1

baby chicks 3-week chicks young chickens young chickens

weeks weeks weeks weeks

Significantly different with t-test; P less than 0.05. NS.

±2.6" ± 3.2" ± 3.0 a ± 1.4"

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F. SETO

weeks of age served as controls. The results of the experiments are summarized in Table 3. Impairment in the development of the hemagglutinin response to MRBC was observed only in those hosts that received a mixture of antigen and blood from MRBC-primed young chicken donors (group 3). The other experimental groups, those inoculated with blood from baby chicks, 3 week chicks and from unprimed young chickens (group 2), did not exhibit immunosuppression. In fact the two week donors of group 2 showed as a group an enhanced response. When 11 birds of group 2 were tested for their ICC-forming capacity, it was normal in all birds. DISCUSSION When immunologically mature allogeneic donors are used in the in vivo culture system in chickens, the hosts rarely survive beyond three weeks after inoculation and mortality is presumed to be due to a graft-versus-host (GVH) reaction. The host survival rate is considerably higher when the donor and host are matched with respect to the B-antigen. The mortality is this case is attributable to other minor histocompatibility factors. Whether the donors were matched or mismatched in B-antigen, the surviving in vivo hosts were immunologically impaired as revealed by their significantly lower hemagglutinin response and ICC-forming capacity. Greater inhibition of immune responsiveness occurred in the allogeneic combinations in which there was B-antigen disparity and is believed to have resulted from the GVH reaction that occurs concurrently with the in vivo antibody production by the donor cells. Immunosuppression concomitant with the GVH reaction has been reported in experiment with mammals (Moller, 1971; Zaleski and Milgrom, 1973; Medziharadsky and Novotna, 1972). The interaction of donor immunocompetent cells with the allogeneic host antigens initiates a complex counter

response by the host that is manifested in hepatosplenomegaly, runting, hemopoietic depression and mortality (Simonsen, 1962; Seto and Albright, 1965; Elkins, 1971). The GVH reaction is triggered by disparity in the histocompatibility antigen, especially the B and C systems in chickens (Schierman and Nordskog, 1961, 1965) and by other minor antigen systems. A number of mechanisms have been offered to explain the GVH reaction related immunosuppression. Immune inhibition as the consequence of antigen competition (Lawrence and Simonsen, 1967) or the exhaustion of the macrophage antigen processing capacity (Zaleski and Milgrom, 1973) emphasize the complexity and excessive quantities of antigens involved in the complex interactions. The role of thymusderived suppressor cells (Bennett et al., 1973) and the participation of a lymphocyte secreted proliferation inhibiting chalone (Mathe, 1972) present new possible mechanisms. The more conventional interpretation, however, proposes an initial cytotoxic destruction of host cells by donor cells which is followed by the release of lymphotoxins and other humoral reactants that further damage other cells nonspecifically (Elkins, 1971; Singh et al., 1972). A comparable situation has been described in chick embryos undergoing GVH reactions. The interaction of donor lymphocytes and host target cells stimulates a proliferative response involving both donor and host cells (Seto and Albright, 1965). The pathologic proliferation of the host hemopoietic elements and the depletion of multipotential yolk sac derived precursors are characteristic changes (Killby et al., 1972; Walker et al., 1973). Mammals suffering from the GVH reaction are deficient in plaque forming cells and bone marrow stem cells (Petrov et al., 1969; Lengerova et al., 1971; Medzihradsky and Novotna, 1972). The massive destruction and/or abnormal differentiative diversion of common hemopoietic precursors deplete the stem cells available

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for immunodifferentiation and subsequently cause impairment in immune development. Although uniformity with respect to the B and C antigens was maintained among the experimental materials, the effect of other minor histocompatibility factors cannot be ruled out since the chickens were not syngeneic. In our material the GVH reaction was minor when donor and host were B-antigen matched (Seto, 1973). The observation that chicks inoculated as embryos with blood from B-matched unprimed donors were not immune-depressed also suggest that these minor factors are insignificant in the effects described in this paper. Impairment in immune responsiveness to the test antigen was clearly evident in the recipients of a mixture of antigen and blood from B-matched donors. The activation of the ICC of donor origin, their proliferation and formation of antibody in the in vivo hosts in some way affected the development of the anti-MRBC response of the hosts. Antibody-mediated immunosuppression or regulation of antibody output is well known (Uhr, 1968) but the mechanism is still unclear (Safford and Tokuda, 1971). Any explanation at the cellular level must take into account, moreover, the interaction of several cell types (Mosier, 1967; Claman et al., 1966; Weigle, 1973) as well as the molecular events in which antibodies, with its twin binding propensities for antigen and cell receptors, moderate antibody production (Siskind and Benacerraf, 1969). A promising model to explain the regulatory function of antibody in which specific immune unresponsiveness is mediated by the formation of an anti-antibody, has been recently proposed and has some intriguing possibilities (Rowley et al, 1973). The impaired immune status of chicks resulting from their earlier experience as in vivo hosts may be explained in part as the consequence of the GVH reaction in those combinations involving B-antigen mismatched donor-host combinations, but it is

insufficient to explain the impairment observed in chicks used as in vivo hosts for the culture of immunocytes from B-antigen matched donors. Our earlier studies indicate that exposure of embryos to MRBC neither elicits an antibody response nor causes tolerance (Seto and Henderson, 1968). The suppression described in this paper is causally related to the activation of the donor immunocytes in the embryo hosts and is probably antibody-mediated. The suppressive effect is somewhat lasting and resembles in some respect immunologic tolerance. It may be that the embryonic immunocyte progenitor cells are more susceptible to the inhibiting effects of antibody. Experiments are in progress to elucidate the nature of the susceptibility. REFERENCES Albright, J. F., T. Makinodan and E. E. Capalbo, 1964. Factors regulating antibody production by spleen cells cultured in vivo. Proc. 9th Congr. Intern. Blood Transf. Mexico: 301-318. Bennett, M., M. Sturgeon and J. P. Engler, 1973. Graft-versus-host reactions in mice. IV. Thymus cell suppression of antibody formation. Am. J. Pathol. 71: 135-150. Claman, H. N., E. A. Chaperon and R. F. Triplett, 1966. Immunocompetence of transferred thymusmarrow cell combinations. J. Immunol. 97: 828-832. Elkins, W. L., 1971. Cellular immunology and the pathogenesis of the graft-versus-host reactions. Progr. Allergy, 15: 78-187. Killby, J. A. A., K. J. Lafferty and M. A. Ryan, 1972. Interaction of embryonic chicken spleen cells and adult allogeneic leucocytes. Aust. J. Exp. Biol. Med. 50: 309-321. Lawrence, W. Jr., and M. Simonsen, 1967. The property of "strength" of histocompatibility antigens and their ability to produce antigenic competition. Transplantation, 5: 1304-1322. Lengerova, A., J. Matousek and J. Zeleny, 1971. Quantitative analysis of allogeneic inhibition of colony-forming performance of bone marrow cells. The possibilities of the method for detecting minor differences in the H-2 structure of the cell membrane. Folia Biol. 17: 145-155. Mathe, G., 1972. Lymphocyte inhibitors fulfilling the definition of chalone and immunosuppression. Europ. J. Clin. Biol. Res. 17: 548-551.

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Medziharadsky, J., and L. Novotna, 1972. Direct plaque-forming cell response to sheep red blood cells in rat spleen undergoing graft-versus-host reaction. Neoplasma, 19: 19-25. Moller, G., 1971. Suppressive effect of graft versus host reactions on the immune response to heterologous red cells. Immunology, 20: 597-609. Mosier, D. E., 1967. A requirement for two cell types for antibody formation in vitro. Science, 158: 15731575. Petrov, R. V., V. M. Manyko, E. I. Panteleyev and L. S. Seslavina, 1969. Inactivation of stem cells after transplantation of mixtures of haemopoietic or lymphoid cells of different genotypes. Transplantation, 7: 165-175. Rowley, D. A., F. W. Fitch, F. R. Stuart, H. Kohler and H. Cosenza. 1973. Specific suppression of immune responses. Science, 181: 113-1141. Safford, J. W. Jr., and S. Tokuda. 1971. Antibodymediated suppression of the immune response: effect on the development of immunologic memory. J. Immunol. 107: 1213-1225. Schierman, L. W., and A. W. Nordskog, 1961. Relationship of blood type to histocompatibility in chickens. Science, 134: 1008-1009. Schierman, L. W., and A. W. Nordskog, 1965. Evidence for a second blood group histocompatibility system in chickens. Transplantation, 3: 44-48. Seto, F., 1970a. Antibody production in chick embryo hosts by allogeneic donor cells. Proc. Oklahoma Acad. Sci. 50: 45-48. Seto, F., 1970b. Immunocompetent cells in the blood of immunized chickens. Poultry Sci. 49: 1673-1680. Seto, F., 1971. Allograft reactivity in chick embryos. J. Exp. Zool. 177: 343-352. Seto, F., 1973a. B-antigen disparity as a factor in suppression of antibody formation in the allogeneic

in vivo system of chickens. Proc. Oklahoma Acad. Sci. 53: 61-64. Seto, F., 1973b. Kinetic analysis of chicken immunocytes with the allogeneic in vivo antibody assay. Poultry Sci. 52: 1714-1721. Seto, F., and J. F. Albright, 1965. An analysis of host and donor contributions to splenic enlargement in chick embryos inoculated with adult chicken spleen cells. Develop. Biol. 11: 1-24. Seto, F., and W. G. Henderson, 1968. Natural and immune hemagglutinin forming capacity of immature chickens. J. Exp. Zool. 169: 501-511. Simonsen, M., 1962. Graft versus host reactions. Their natural history and applicability as tools of research. Progr. Allergy, 6: 349-467. Singh, J. N., E. Sabbadini and A. H. Sehon. 1972. Cytotoxicity in graft-versus-host reaction. I. Role of donor and host spleen cells. J. Exp. Med. 136: 36-48. Siskind,G. W.,andB. Benacerraf, 1968. Cell selection by antigen in the immune response. Adv. Immunol. 10: 1-50. Uhr, J. W., 1968. Inhibition of antibody formation by serum antibody. In: Regulation of the Antibody Response, C. C. Thomas, Springfield, Illinois, pp. 114-126. Walker, K. Z., G. I. Schoefl, K. J. Lafferty and F. P. Adams, 1972. Pathogenesis of the graft-versushost reaction in chick embryos. Pathological changes in the yolk sac, thymus bone marrow and bursa of Fabricius. Aust. J. Exp. Biol. Med. 50: 675-688. Weigle, W. O., 1973. Immunological unresponsiveness. Adv. Immunol. 16: 61-122. Zaleski, M., and F. Milgrom, 1973. Immunosuppressive effect of graft-versus-host reactions. Cell. Immunol. 7: 268-274.

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