A study of peripheral tolerance through embryonic grafts of the bursal epithelial rudiment between MHC-distinct chick embryos.

We report here that different organ grafts are not equally competent to induce tolerance of the host even if they are performed early in development, i.e. before the host's immune system has started to develop. We have grafted the epithelio-mesenchymal rudiment of the bursa of Fabricius between histoincompatible chicken embryos at E5 and found that the transplant is normally colonized by hemopoietic cells from the host and that a normal contingent of B cells is eventually produced. The grafted bursa is tolerated after birth for a few weeks (> or = 4-8) but is in all cases rejected by an immune mechanism which cannot be assimilated to the physiological involution process which occurs at 4-5 months of age. Moreover, even in the first month after birth, when the bursa is still healthy, skin grafts of the same MHC haplotype are promptly rejected. These observations are in contrast with the outcome of allogeneic limb bud grafts which are permanently tolerated after birth although in an unperfect manner. We show in addition that, as was the case in xenogeneic grafts of limb buds and bursas of Fabricius from quail and chick embryos, the allogeneic in situ graft to thymic epithelium of the MHC haplotype of the bursal implant induces tolerance of the bursa. One common point of xenogeneic and allogeneic embryonic grafts of limb bud, bursa and even thymic rudiments is that none of them induced a complete state of tolerance, since proliferation responses were always obtained in vitro in one-way host-donor mixed leucocyte cultures.

Abnormalities associated with the bursal microenvironment in spontaneous dysgammaglobulinemia of UCD line 140 chickens.

UCD line 140 chickens have been previously reported to develop a syndrome of spontaneous 7s immunoglobulin deficiency and the presence of autoantibodies. Earlier studies demonstrated that these inbred birds have normal peripheral blood T and B cell numbers; they also respond normally to allogeneic stimulation. Although the 7s immunodeficiency does not manifest itself until several months of age, line 140 birds have a premature degeneration of bursa. Because of the recent development of monoclonal reagents specific for bursal elements, including surface epithelium, basement membrane associated epithelium, follicle associated epithelium, and lymphoid subpopulations, we have examined line 140 and control birds for the expression of bursal epithelial cell antigens. Line 140 birds, in contrast to control chickens, have a dramatic early alteration in the expression of an epithelial cell marker in the bursa, thymus, and intestine. Moreover, to further address this issue, we transplanted bursa from 10-day embryos onto the chorioallantoic membrane, a privileged site. Bursae from control birds became abnormal when transplanted onto line 140 CAM; they remained normal when transplanted among several control chicken lines. In contrast, line 140 bursa remained abnormal independent of the transplant procedure. Due to the marked bursal abnormality observed specific to the dysgammaglobulinemia chicken line, we propose that the microenvironmental features of line 140 bursa may predispose these birds to the development of humoral immunodeficiency and autoantibodies.

Suppressor cells for antibody production in vivo, induced by bursa cell injection into agammaglobulinemic chickens, belong to A CT4-, CT8+, TCRI- subset of T cells.

The suppressor cells, induced in agammaglobulinemic chickens by injection of bursa cells and capable of inhibiting adoptive antibody production upon cotransfer with bursa cells, were characterized with respect to phenotype. They were found to be CT8+, CT4-, and TCRI- (gamma delta). No evidence could be obtained that histamine type 2 receptor bearing cells were involved in the suppression of antibody formation in vivo.

Tolerance to class I major histocompatibility complex antigens in chicken B cell chimeras. Effect of B cell depletion on transferability of tolerance.

B cells from bursa of Fabricius of newly hatched chickens are able to reconstitute the B cell compartment of chemically bursectomized chickens. The resulting B cell chimerism can be detected with monoclonal antibodies against donor B cell alloantigen. Chimeric chickens accept donor-type skin grafts and are unresponsive to donor major histocompatibility complex (MHC) antigens in graft-vs.-host splenomegaly assay and mixed lymphocyte reaction. To study the capability of B cells to induce tolerance to selected MHC antigens, we transplanted class I or total MHC-incompatible bursa cells into cyclophosphamide-treated recipients. The recipients of class I or total MHC-incompatible bursa cells were equally tolerant of donor-MHC antigens. To further analyze the mechanisms of tolerance to class I antigens vs. total MHC, spleen cells from tolerant chickens were transferred to irradiated, histocompatible secondary hosts. The secondary recipients were also unresponsive to bursa cell donor-strain MHC antigens. However, if the chimeric B cells were depleted before the spleen cell transfer, the transfer of tolerance to total MHC was severely inhibited. Instead, most recipients of B cell-depleted spleen cells tolerant of class I antigens were still tolerant of bursa cell donor MHC. Our results indicate differences in the transferability of tolerance to class I antigens vs. entire MHC, although in primary recipients of bursa cells the tolerance is similar. These data suggest that a mechanism that is not dependent on the presence of donor cell chimerism contributes to the maintenance of tolerance to donor class I antigens. The transfer of tolerance to total MHC disparity requires the presence of chimeric cells indicating that donor alloantigen expression is needed for induction of tolerance in the secondary hosts.

Thymic epithelium tolerizes chickens to embryonic graft of quail bursa of Fabricius.

We demonstrated previously that isotopic and isochronic grafts of the quail bursa of Fabricius rudiment performed at 5 days of incubation (E5) into chick embryos resulted in the development of a chimeric bursa whose chick host B lymphocytes and accessory cells differentiated in a foreign, quail epithelial environment. Such animals reject their grafted bursa by the age of 2-3 weeks post-hatching (1,2). Isotopic embryonic grafts of the thymus epitheliomesenchymal anlagen from the quail donor of the bursal rudiment were carried out at E4.5 (before their colonization by hemopoietic precursor cells), following partial or complete host thymectomy. The quail thymic epithelial stroma was accepted and invaded by chick hemopoietic precursor cells that further differentiated into lymphocytes and dendritic cells. Tolerance of the foreign bursa was induced in such thymobursal chimeras. This demonstrates that the thymic epithelium has the capacity to induce tolerance of xenogeneic rudiments when both grafts are implanted at early stages of embryonic development. We also report on the production of two birds in which removal of the chick host thymus was complete thus generating chimeras in which host T and B lymphocytes differentiated in a completely xenogeneic epithelial environment.

Ontogeny of the GVH-R inducing capacity, in conventional and germ-free chickens.

Three strains of MHC homozygous chickens were used to study the ontogeny of the GVH-R. It was found that this function of the immune system was acquired with a delay in animals raised in germ-free conditions. In our previous work, we had shown that, contrary to expectation, bursal cells were capable of mounting a GVH-R against embryos, and actually were particularly efficient in this regard when compared to spleen cells. The present experiments have disclosed that splenomegaly induction may be dissociated from the toxic effect that bursal cells also exert on recipient embryos.

A comparative study of the GVH-R in the chick embryo: effects of various allogeneic cells and various administration routes.

Allogeneic cells inoculated into immunologically immature chick embryos induce a Graft-Versus-Host Reaction (GVH-R), one of the manifestations of which is splenic enlargement. Splenomegaly varies with the composition of allogeneic cell suspensions, route of inoculation and age of recipient embryos at inoculation and autopsy. We have compared the splenomegaly induced by lymphoid cells from spleen or bursa of Fabricius with that induced by peripheral blood lymphocytes, after these cell preparations were either grafted on the chorioallantoic membrane (CAM) at 9 or 13 days or injected intravenously at 13 days. We confirm our previous findings that bursa cells grafted on the 9-day CAM induced splenomegaly as efficiently as other cell preparations. On the other hand, bursa cells did not mediate such an effect after they were injected at 13 days. On the whole, a hierarchy of decreasing efficiency is observed from PBL to spleen and finally to bursal cells. It appears that, as the recipient embryo ages, this hierarchy becomes more marked.

[Effect of ACTH injection and graft of bursa of Fabricius on the adrenocortical activity of bursectomized chicks at 80 hours of incubation]. / Effets de l'injection d'ACTH et de la greffe de bourse de Fabricius sur l'activité corticosurrénalienne des poussins et poulets bursectomisés à 80 heures d'incubation.

Early embryonic bursectomy (BFX) disturbed the adrenocortical functioning. The stress-unresponsive period that occurred in controls, and lasted for 2-3 weeks after hatching, no longer appeared in BFX chicks. In contrast, the magnitude of the stress-induced hypercorticosteronemia was much lower in BFX than in sham-operated 5 week-old chicken. It was assumed that such adrenocortical dysfunction was due to bursal deprivation, since grafting bursal buds onto the chorio-allantoic membrane of BFX embryos restored all the parameters under study, i.e., the post-hatching stress unresponsive period and the high magnitude of stress-induced responses in adults. Factor(s) involved in such interregulation are not known but do not seem to affect directly adrenocortical cells because intramuscular injection of a moderate dose of ACTH resulted in the same hypercorticosteronemia whether 3 day-old and 5 week-old chicks had been bursectomized or sham-operated.

The follicle-associated epithelium in the bursa of Fabricius cell origin studied by means of quail-chick chimeras and monoclonal antibodies.

The cell origin of the follicle-associated epithelium (FAE) of the bursa of Fabricius was studied by two different technical approaches. Precolonized quail bursal rudiments were grafted into chick embryos and the grafts were recovered 2 wk after hatching of the recipients. By taking advantage of the distinct nuclear characteristics of chick and quail cells, it could be shown that the specialized FAE consists of a mixture of epithelial cells, with special features, among which hemopoietic cells, originating from the host, are dispersed. Staining of chicken bursas with different monoclonal antibodies reacting either with the epithelial component (BEP-1) or with the hemopoietic cells of the bursa (L22, L17) confirmed that hemopoietic cells, presumably macrophages, are mixed with the epithelial cells at the level of FAE.

A novel method to bursectomize avian embryos and obtain quail----chick bursal chimeras. I. Immunocytochemical analysis of such chimeras by using species-specific monoclonal antibodies.

Chick embryos were bursectomized at 5 days of incubation according to a novel surgical technique described in this article. This method yields birds that are able to hatch and are devoid of the physiologic deficiencies resulting from the previously used method, which involved resection of the cloacal and posterior embryonic region. The bursectomized embryos were grafted in situ with a quail bursa of the same age, which thereafter became chimeric through chick host hemopoietic cell invasion. By means of species-specific antibodies, the chimeric condition revealed 1) that the bursal epithelium expresses a unique antigenic determinant (MB1 determinant), until now considered to be an exclusive feature of blood vessel endothelium and hemopoietic cells, and 2) that this determinant appears in bursal epithelium at the time and site of hemopoietic cell invasion. The other point arising from this work concerns the apparent constitutive Ia expression by perifollicular blood capillary endothelial cells in normal and chimeric bursas.

Lymphoid cell chimerism and transplantation tolerance induced by bursal and postbursal cells.

Transplantation of allogeneic bursal cells into cyclophosphamide-treated, immunodeficient chickens is a useful experimental model for analyzing the mechanisms of transplantation tolerance, especially because transplanted bursal cells do not produce graft-versus-host disease. In this study we have determined B-lymphoid chimerism in various lymphoid organs after transplantation of allogeneic bursal stem cells or postbursal cells, and used a variety of tests to determine presence of immunological tolerance. Transplanted bursal stem cells induced a state of stable chimerism that could easily be detected in peripheral blood and other lymphoid organs. Chimerism induced by postbursal cells was low in peripheral blood, but clearly observable in other lymphoid organs, especially in spleen and thymus. Both bursal and postbursal cells induced specific unresponsiveness to donor-line alloantigens. Bursal cell recipients accepted donor line skin grafts--and their graft-versus-host reactivity, as assayed by embryonal splenomegaly, and mixed lymphocyte reactivity against donor line alloantigens were significantly decreased. Despite differences in chimerism, a strong transplantation tolerance was readily induced with bursal stem cells and with postbursal cells.

A retroviral myc gene induces preneoplastic transformation of lymphocytes in a bursal transplantation assay.

Treatment of chicken embryos with cyclophosphamide results in ablation of bursal lymphocytes. Bursal follicles can be reconstructed by infusion of embryonic bursal cells. Histologic examination of reconstituting bursal follicles showed that the first lymphocytes to appear were large pyrinophilic lymphoblasts that lined up adjacent to the bursal basement membrane and appeared to serve as progenitors for the differentiation of bursal medullary lymphocytes. When these cells were infected with the avian myelocytomatosis virus HB1 bearing a v-myc oncogene they appeared to home to the region of the bursal basement membrane but failed to differentiate. Instead, they formed structures indistinguishable from the preneoplastic transformed follicles that develop during bursal lymphomagenesis induced by lymphoid leukosis viruses. The DNA from these transformed follicles contained the HB1 v-myc gene but lacked the ability to transform NIH/3T3 mouse cells. Therefore these preneoplastic lesions were induced directly by HB1 myc and did not require the expression of Blym-1 or similar oncogenes. Exploitation of this transplantation technique with the chicken bursa will provide a useful method for assessing the stage-specific activity of oncogenes in vivo.

Clones of B lymphocytes in individual follicles of the bursa of Fabricius.

To discover whether individual bursal follicles can contain clones of B lymphocytes, we estimated the numbers of lymphoid cell precursors populating single follicles in two types of chicken chimera. The first type was produced by establishing parabiotic connections between blood vessels of embryo chorioallantoic membranes. Under these conditions, and most likely during normal development, most follicles are populated by more than one, but less than ten, precursor cells. However, in a second type of chimera, a cyclophosphamide-treated chick reconstituted with normal bursal cells, most follicles in the reconstituted bursa are clonal (their lymphocytes are derived from a single precursor cell). Individual follicles can readily be isolated from bursae of reconstituted birds and should be useful in studies of B cell development.

Lymphocyte requirement for the functional development of follicle-associated epithelium.

Follicular development in the bursa of Fabricius was disrupted by testosterone treatment and chorioallantoic membrane grafting. Analysis by a quantitative histological technique demonstrated that testosterone treatment causes a dose dependent delay in development by inhibiting lymphoid proliferation. Inhibition of lymphoid development prevented the development of carbon transport ability by follicle associated epithelium. Reconstitution of follicular development by grafting treated bursae to the chorioallantoic membrane of untreated hosts results in the development of functional follicle associated epithelium. These results establish the lymphoid requirement for the development of transport ability by the follicle-associated epithelium.

Ontogenic restriction of colonization of the bursa of Fabricius.

The capacity of hemopoietic precursor cells (HPC) to home to embryonic bursal and thymic grafts was investigated in embryonic and newly hatched chickens. Whereas thymic grafts developed normal histogenesis in both types of recipients, the bursal rudiment was colonized and developed in embryonic, but not in newly hatched hosts. In the latter, noncolonized bursal grafts developed neither lymphoid follicles nor granulopoiesis in the mesenchyme. These results are interpreted in terms of ontogenic "maturation" of the HPC which lose their homing potential towards the bursa while they preserve their thymic seeding capacity. This hypothesis is consistent with previously reported data which indicated cyclic continuous recruitment of the thymic lymphoid population, but restricted bursal colonization to a relatively brief period of embryonic life.