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19 Sprent, J., yon Boehmer, H. and Nabholz, M. (1975) J. Exp. Med. 142, 321 20 Yunis, E.J., Ferrandes, G., Smith, J. and Good, R. A. (1976) TransplanZ Proc. 8, 521 21 yon Boehmer, H., Hudson, L. and Sprent, J. (1975) J. Exp. ivied. 142, 989 22 Zinkernagel, R. M., Callahan, G. N., Althage, A., Cooper, S., Klein, P. A. and Klein, J, (I978) J. Exp. Med. 147, 882 23 Kappler, J. W. and Marrack, P. (1978)J. Exp. Med. 148, 1510 24 Bevan, M.J. (1978) Nature (London) 269, 417 25 Miller, J. F. A. P. (1978) Adv. CancerRes. 29, 1 26 Singer, A., Hatchcock, K. S. and Hodes, R.J. (1979) J. Exp. Med. 149, 1208 27 Zinkernagel, R. M. and Doherty, P. C. (1979) Adv. hnmunol. 27, 51 28 Bevan, M.J. (1975)J. Exp. Med. 142, 1349 29 Sprent, J. (1978)j. Exp. Med. 148, 478 30 Korngotd, R. and Sprent, J. (1980) j . Fxp. Med. 151, 1114 3I Cantor, H. and Boyse, E. A. (1975).7. Exp. Med. 141, 1376 and 1390

32 Schendel, D. J., Alter, B. J. and Bach, F. H. (1973) Transplant. Proc. 5, 1651 33 Alter, B.J. and Bach, F. H. (1974)J. Exp. Med. 140, 1410 34 yon Boehmer, H. and Haas, W. (1979)J. Exp. Med. 150, 1134 35 Bennink, J. R., Schwartz, D. tI. and Doherty, P. C. (1981) Cell Imrnunol. (in press) 36 Wagner, H., Hardt, C., Heeg, K., Pfizenmaier, K., Solbach, W., Bartlett, R., Stockinger, H. and R/511inghot'l',M. (1980) Immunol. Rev. 51,215 37 Watson, J., Mochizuki, D. and Gillis, S. (1980) lmrnunol. To@ 1,113 38 Pollard, M., Chang, L. F. and Srivastava, K. K. (1976) Transplanl. Proc. 8, 533 39 Klein, J., Chiang, C. L. and Wakeland, E. K. (I977) Iramunogenel. 5, 445 40 Ledbetter, J. A., Rouse, R. V., Micklem, H. S. and Herzenberg, L. A. (1980);,7. Exp. Med. 152, 280 41 Klein, J. ( 1975) Biology of the Mouse Histocompalibility-2Complex, Springer-Verlag, New York 42 Zinkernagel, R. M. (1978) hnmunol. Rev. 42, 224

The avian immune system Beimhard Fleischer Institute of Virology and Immunobiology, University of Wfirzburg, 8700 Wiirzburg, F.R.G. Some of the mosl pioneering discoveries irl immunology, e.g. lhe dichotomy of the lymphoid system and the extrinsic origin of stem cells in primary lymphoid organs, have been made using the avian immune system. A considerable amount of information has been accumulated but the immune system in birds is still poorly understood compared with the murine or human immune systems, and experimental studies have been confined almost exclusively to chickens. This review discusses some of the major findings concerning the avian immune system. The phylogenetic distance between avian and m a m m a l i a n species is reflected by a n u m b e r of peculiarities in their immune systems. For example, the anatomical organization shows profound differences, the most remarkable being the presence of the bursa of Fabricius as the central organ of B-lymphocyte differentiation. F u r t h e r differences can be seen in the extent of the gut-associated lymphoid tissue, including Peyer's patches and two cecal tonsils, the presence of accessory spleens and the division of the t h y m u s which can form up to 12 isolated lobes. Also noteworthy is the extensive infiltration of the adult thymus with B cells a n d of the bursa with T cells. T h o u g h l y m p h a t i c nodules are present, they do not show t h e typical o r g a n i z a t i o n of m a m m a l i a n l y m p h nodes. T h e physicochemical differences between m a m m a l i a n IgG and avian 7s immunoglobulin are such that the designation IgY has been proposed for the latter 1. Avian antibodies do not activate m a m m a l i a n complement, whereas avian complement is less discriminating and can be activated by m a m m a l i a n antibodies. In spite of all these differences m a n y striking similarities between avian and m a m m a l i a n i m m u n e systems exist. Therefore, comparative studies in birds have often revealed certain aspects of immunology more clearly than have studies in the corresponding

m a m m a l i a n systems.

Ontogeny of p r i m a r y l y m p h o i d organs Because of its accessibility during all stages of development the avian e m b r y o provides unique opportunities to observe the migration and homing of stem cells during the ontogeny of T and B lymphoid lineages. Using chromosomally marked embryos Moore and O w e n d e m o n s t r a t e d that the lymphoid cell populations in thymus and bursa were (at least in part) derived from extrinsic i m m i g r a n t precursors 2. In a series of elegant experiments, Le D o u a r i n el al. investigated the colonization of thymus and bursa in detail using chick-quail embryonic chimeras (reviewed in Ref. 3). This model system relies on the complete compatibility of quail and chick embryonic tissues and the fact that the cells of each species can clearly be identified, by the structure of their interphase nuclei, within the chimeric tissue. Grafting lymphoid organ rudiments of various developmental stages into hosts at different stages of development revealed definite receptive periods during which the lymphoid primordia allow the influx of stem cells (present in the embryo several days before the receptive period). T h e thymic cortex and medulla are colonized simultaneously by three waves of immigraElsevier/North-Holland

Biomedical Press 1981

0167 4919/81/0001)-0000/$02.75

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tion, each lasting 24 h in the quail and 36 h in the chicken (Fig. 1). The population arriving during the first wave reaches a maximum after one week and dies out within 2 weeks. Therefore, the lymphoid cells present in the young hatched bird seem to derive exclusively from the second and third influx. No such cyclic renewal of the bursal population has been found. Here, the influx lasts much longer (days 8-14 in the chick and days 7-11 in the quail). Since the receptivity of an unpopulated organ can be maintained if the influx of stem cells is experimentally prevented and since the duration of influx seems to be dependent on the number of invading cells, it was suggested that homing was regulated by a chemotactic factor produced by the epithelium. The production of this attractant would then be subject to negative feedback regulation by the invaded cells. Though yolk sac cells are capable of homing to the lymphoid organ rudiments 4, it seems that during normal development the lymphoid precursor cells are derived exclusively from the intra-embryonic mesenchyme, whereas stem cells present in the yolk sac are secondary immigrants originating from intra-embryonic sites 5. The use of the IgG allotype marker as well as a chromosome marker in these studies has demonstrated that cells from intra-embryonic hemopoietic sites can develop into mature |gG producing cells. An important question is whether pluripotent or at least bipotent stem cells migrate to the lymphoid rudiments or whether stem cells precommitted to the T or B lymphoid lineage home to the respective organs. By in-vitro and in-vivo co-cultivation of a quail bursa in its postreceptive period with a receptive chick thymus it was found that the cells present in the bursa could home to the thymus and differentiate to form thymocytes since thymus antigen bearing quail cells were found in the chick thymus 6. It has also been reported that embryonic bursa cells at day 18 restore thymus as well as spleen and bursa morphology in cyclophosphamide treated recipients 7. It should be noted in this respect that homing to thymus and bursa occur as Age (days)

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successive events to prevent competition (Fig. 1). Other authors, however, failed to detect a significant influx of transferred bursa cells into the thymus of irradiated recipients and suggested that functional, mitogen-responsive T cells could not be generated from recently bursa-derived cells 8. Acquisition of B l y m p h o c y t e functions Because of the unique availability of a central B lymphocyte organ the majority of studies concerning the avian immune system have been centered on the maturation of B lymphocytes in the absence or presence of the bursa v. Several events in normal B-cell development, in the chicken, are summarized in Table I. The first IgM-bearing cells are detected on the 12th day of embryogenesis in the bursa. 4 days later they appear in the spleen 1°. In contrast to mammalian preB cells in bone marrow and fetal liver they display cytoplasmic and surface IgM simultaneously 1°. Specific antigen binding cells have been found to appear in predetermined sequence scattered among bursal follicles I~. Since each follicle is believed to contain only a few stem cells, one stem cell probably gives rise to several different clones of B lymphocytes. From the sequential generation of B-cell clones i{ has been proposed that the diversity of B cells is generated in this period of embryogenesis. This is substantiated by the limited immunoglobulin diversity found in chickens bursectomized at this time 12. Controversy exists as to whether antigenic stimulation has an influence on B cell development. In fact, within the mature bursa plaque-forming cells can be generated by intracloacal stimulation 13. An active uptake of environmental antigens into the bursal lumen has been demonstrated 14 and after intravenous immunization enhanced migration of bursal lymphocytes into the spleen can be detected is. However, the developmental pattern of generation of diversity is not influenced by stimulation with specific antigen I1. Therefore, no more than an antigen-driven recruitment of mature B lymphocytes may be envisaged and 9 I

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Fig. 1. Colonization of lymphoid primordia in the quail embryo. Bars indicate receptive periods during wl~iehlymphoid stern cells enter the primordia (adapted from Ref. 3).

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TABLE I. Developmental events in the ontogeny of chicken B lymphocytes d8-d 14 Receptive period of bursal primordium, stem cells entering dl2 Cells with cytoplasmic and surface IgM detected in bursa d14 FcR-bearing cells reaching highest percentage in bursa d14 First lgM cells appear in the spleen d16 Cells binding ~2SI-TGALor ~2SI-KLHdetected in bursa Cells binding SRBC detected, IgM secreting cells d18 demonstrated in bursa d 21 (hatch) IgG producing cells detected in bursa 3 da.h. IgG cells detected in the spleen d 18-2 w a.h. Bursal stem cells predominating in bursa Postbursal stern cells demonstrated in bursa 4wa.h. 7wa.h. Postbursal stem cells found in peripheral lymphoid organs d: day ofembryogenesis d (w) a.h.: days (weeks) after hatching it seems that the mature bursa acts as a peripheral l y m p h o i d organ, taking advantage of the natural routes of antigen entry. T h e developmental switch from I g M to and then to IgA producing cells is thought to take place within the bursa and is reflected by the sequential a p p e a r a n c e of these cells in peripheral l y m p h o i d organs 1c',17. Alternatively, it has been suggested that IgA-forming cells are derived directly from I g M forming precursors ~8. Contradictory views have been presented as to a possible extrabursal site of B cell maturation. Arguments for this possibility came from observations of the consequences of bursectomy. Chickens bursectomized early after hatching usually show IgMh y p e r g a m m a g l o b u l i n e m i a , depressed levels of serum IgG a n d a r e d u c e d n u m b e r of c i r c u l a t i n g B lymphocytes. T h o u g h they are deficient in their p r i m a r y responses, they respond to h y p e r i m m u n i z a tion, usually with an I g M response. T h e same observations were made in chickens subjected to bursectomy during embryogenesis by application of high doses of androgens. Due to a specific deleterious effect of the hormone on the cloacal endoderm, the development of bursal follicles and with it the generation of B lymphocytes at this site are aborted. Since no b u r s a l r e m n a n t s were found u p o n h i s t o l o g i c a l examination it was suggested that other sites in the chicken embryo are capable of conditioning or supplying cells competent for I g M production whereas the switch to IgG would be dependent on the bursal environment 9. T h e elevated I g M levels could be the consequence of a deficient regulatory feedback by IgG ~9. This notion was further supported by the finding that surgical removal of the bursal primordium prior to 70 h of incubation did not abrogate the formation of I g M antibodies 2°. O n the other hand, treatment of the embryos with xenogeneic anti-g antibodies on day 13 and again

igG

following bursectomy at hatching resulted in most cases in complete and persistent a g a m m a g l o b u l i n e m i a due to the susceptibility of avian pre-B cells to arrest by sIgM modulation ~°. T r e a t m e n t with anti-g alone resulted only in transient h y p o g a m m a g l o b u l i n e m i a . This supported the conclusion that sites other than the bursa are unable to induce B-cell differentiation and that the degree of a g a m m a g l o b u l i n e m i a after bursectomy is dependent on the time allowed for the bursa to function before removal 21. In an attempt to reconcile all these findings it has been suggested that during normal development, the bursa m a y be the only site of B-cell differentiation, and that other sites may influence B-cell m a t u r a t i o n if the bursa is removed early enough ~°. Recently, other authors have claimed that in the 3 day embryo immunoglobulinpositive lymphocytes can already be detected 22. Further insights into the m a t u r a t i o n of bursal cells came from reconstitution experiments. Chickens treated with cyclophosphamide (CY) as embryos or shortly after hatching provide a useful model of B-cell deficiency due to a preferential toxicity of the drug to B lymphoid cells. Several m a t u r a t i o n steps of bursal cells could be defined based on the capacity to induce morphological and functional restoration of the B cell system in CY-treated recipients 23. 'Bursal stem cells' are the p r e d o m i n a n t cell type in the bursa in late embryogenesis and in the first weeks after hatching. These cells are unable to produce antibodies and need the influence of the bursal epithelium to differentiate further. After transplantation into syngeneic recipients, they induce complete functional and morphological reconstitution of the recipients including the morphology of the bursa. 'Postbursal stem cells' a p p e a r four weeks after hatching in the bursa and later in bone marrow, spleen and thymus. T h e y are independent of' the bursal environment and restore humoral immunity without normalization of the bursal structure. Their t r a n s p l a n t a t i o n even into surgically bursectomized, CY-treated syngeneic recipients leads to complete reconstitution of antibody production. Bursal stem cells can acquire postbursal maturity equally well in an allogeneic or syngeneic environment 23. However, in an allogeneic recipient only the response to T-cell-independent antigens is restored, since a p p a r e n t l y the t r a n s p l a n t e d B cells or their progeny cannot co-operate with the allogeneic T cells provided by the host 24. After secondary transplantation into syngeneic or semi-allogeneic recipients full reconstitution was found 24. Apparently, the restriction in the co-operation with T cells is imposed on the B cells, not by the developmental or by the priming environment, but exclusively by the genotype.

Fc-receptor bearing cells T h e percentage of cells bearing the Fc-receptor for lgG (FcR) as determined by rosette-formation with

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antibody-coated SRBC reaches a maximum in the bursa at day 14 and then declines. Afterwards these cells appear in the spleen and in other peripheral lymphoid organs 2s, This finding has led to the conclusion that FcR cells are seeded by the bursa and are therefore B cells. Since they were found in bursectomized chickens to the same extent as in normal chickens, it was assumed that the rosette-formation was a B cell function independent of the bursa 26. This view has been challenged by the finding that in bursectomized chickens 95°70 of FcR cells in the spleen are monocytes or macrophages and that the remaining 5% were T cells 27. Furthermore, the majority of FcR cells in the pre-hatch bursa of normal chickens exhibit macrophage-like characteristics. Therefore, the question as to the nature and ontogeny of FcR cells is still open. More recently, it has been reported that FcR cells are present in the intraembryonic mesenchyme of the 3 day embryo and morphologically resemble promyelocytes, embryonic macrophages and fetal lymphocytes 2s. Because of this development of FcR cells long before ontogeny of thymus and bursa it has been suggested that FcR cells may have important functions during embryogenesis.

ents 39. The mechanism underlying this effect is still obscure and divergent findings may be due to different treatments of the donors. Suppressed recipients still possess peripheral B lymphocytes but lack plasma cells and germinal centers. Thymus-dependent and -independent humoral responses are equally affected thus excluding an effect on T helper cells, it has been shown that 'immunization' of ay donors with syngeneic or allogeneic bursa cells enhances and accelerates the development of suppressor cells 4°. Therefore, these birds may lack tolerance to a B-cell differentiation antigen, a concept supported by the finding that presensitization with day 17, but not day 14, embryonic bursa cells is effective 4°. In the system originally described 36 suppressor T cells with a specificity for individual immunoglobulin isotypes were found 39 in limiting dilution transfers. A naturally occurring analogue of this experimental system is the inherited 7s immunoglobulin deficiency found in U C D line 140 chickens < . Besides other immunological abnormalities these chickens have suppressor T cells suppressing IgG but not IgM synthesis in syngeneic or allogeneic recipients. This disease is quite similar to the common variable hypogammaglobulinemia in Inan.

Thymus-dependent cell interactions Most lymphocyte interactions previously described in the mouse have - with some delay - also been found in the chicken 29, These studies were almost always performed by adoptive transfers of the respective cell populations into CY-treated irradiated recipients. Agammaglobulinemic (ay) chickens (see below) served in most cases as a source of T cells free of B cells. The synergism between T cells, B cells and macrophages in the primary response against SRBC has been demonstrated in vivo 3° and in vilro 3z as well as the carrier-specific T cell help in the humoral anti-hapten response 32. The T helper cell in this latter system has been demonstrated to be a non-adherent T cell lacking the Fc-receptor for IgG 33. Immunoregulatory cells have been found in the young chicken thymus 3