Monique Coltey

Evidence for a cyclic renewal of lymphocyte precursor cells in the embryonic chick thymus

Experiments involving sequential transplantations of the chick embryonic thymus at E9 to E12 into a first 3-day host quail embryo and then into a second chick host allowed demonstration of the cyclic periodicity of hemopoietic cell seeding of the embryonic thymus. After a first wave of colonization occurring between E6.5 and E8, the thymus becomes refractory to hemopoietic cell entry for about 4 days. It resumes its capacity to be seeded by a second wave of blood-borne stem cells at E12. After a second period of non receptivity starting at E14, a third wave of incoming cells reaches the thymus around E18. Therefore, with a slightly different periodicity, the same cyclic mechanism regulates the renewal of lymphocytes in chick and quail embryos. Quail hemopoietic cells were immunostained in the chimeric thymuses, with a species specific monoclonal antibody (anti-MB1) which recognizes a common surface antigenic determinant on all endothelial and blood cells of the quail (except erythrocytes). Two steps could thus be distinguished in the seeding process. When the thymus becomes receptive for hemopoietic cells, the latter first accumulate in the intrathymic blood vessels before penetrating massively in the thymic parenchyma. The quail chick-chimera system combined with the use of a species- and cell-type-specific antibody provides a unique tool for studying thymic colonization by lymphocyte precursors.

Postnatal development of a demyelinating disease in avian spinal cord chimeras

Xenogeneic spinal cord chimeras were constructed by grafting fragments of quail neural primordium into chick embryos at 2 days of incubation. Hatched birds displayed normal motor behavior for about 5 to 7 weeks, whereupon they developed a neurological syndrome; in the grafted spinal cord the pathological signs of the disease were very similar to those of the active plaques of multiple sclerosis and of the lesions of experimental allergic encephalomyelitis and neuritis, including Ia expression by brain capillary endothelia, rupture of the blood-brain barrier, leukocytic infiltration in the nervous tissue, and demyelination. In the animals at the most advanced stage of the disease an autoimmune attack occurred on the hosts nervous system with the same histopathological signs.

Ontogenic emergence of a quail leukocyte/endothelium cell surface antigen

The ontogenic emergence of MB1, a quail cell surface antigen expressed by endothelial and hemopoietic cells but not erythrocytes, was followed by direct immunofluorescent staining of transverse sections of the developing blastodisc, from the stage of the cephalic fold until 22 pairs of somites. Along the developmental sequence that leads from hemangioblasts, the mesodermal precursors of both endothelium and hemopoietic cells, to vessels containing blood cells, MB1 is first expressed by arising endothelial cells. These first emerge as flattened cells at the periphery of hemangioblastic clusters in the area opaca from the stage of one pair of somites and slightly later as unicellular angioblasts in the area pellucida and in the embryo. MB1 expression is then maintained on endothelium as vessels develop, in contrast with extraembryonic blood islands in which primitive erythroblasts remain MB1-negative. A small proportion of blood island cells and budding of endothelium contribute a population of MB1-positive hemopoietic cells appearing soon after the onset of angiogenesis.

Successful xenogeneic transplantation in embryos: induction of tolerance by extrathymic chick tissue grafted into quail.

In previous experiments, we have demonstrated that limb buds engrafted during embryonic life at E4, between MHC-mismatched chick embryos, are not only tolerated after birth, but induce in the recipient a state of split tolerance toward cells expressing the donor MHC haplotype: donors skin grafts are permanently tolerated while a proliferative response of hosts T cells is generated in MLR by donor–type blood cells. If the same experiment is performed, using quail embryo as a donor and chick as a recipient, acute rejection of the quail limb starts during the first two weeks after birth, thus suggesting that the peripheral type of tolerance induced in these experiments can be obtained only in allogeneic but not in xenogeneic combinations. We report here the unexpected result that when a chick limb bud is grafted into a quail at E4, it is tolerated and, like allogeneic grafts in chickens, induces adult skin-graft tolerance without modifying the MLR response. Similar results were obtained with grafts from another closely related species of bird, the guinea fowl from the Phasianidae family. In contrast, xenogeneic combinations involving more distant species (chick and quail as recipients and duck, an Anatidae, as donor) resulted in strong and early rejection from both recipients. As a whole, quails exhibit a greater ability than the chick to become tolerant to antigens presented peripherally from early developmental stages. In adult quails, however, skin grafts performed in either direction (i.e., quail to chick or the reverse) are rejected according to a similar temporal pattern. Moreover, lymphocytes of both species are able to respond equally well to quail or chick IL-2. Several hypotheses are envisaged to account for these observations. It seems likely that this type of tolerance is directly related to antigenic load because the load in chick to quail wing chimeras is larger than that in quail to chick chimeras. This view is supported by the protracted delay in graft rejection observed when two quail wing buds instead of one are grafted into chickens.

Restoration of T-cell function in nude mice by grafting the epitheliomesenchymal thymic rudiment from 10-day-old euthymic embryos.

The capacity of the uncolonized thymic epithelium to restore immune function in nude mice was demonstrated by grafting the 3rd branchial arch area taken from euthymic 10-day BALB/c embryos into syngeneic newborn nude mice. Twenty-six percent of the operated animals became immunocompetent. T-cell function was tested with skin grafts and the presence of high levels of Thy-1 positive cells plus a variety of in vitro culture assays: Con A stimulation of T lymphocytes, cytotoxicity and alloreactivity in MLR of the recipient toward allogeneic spleen cells. All these tests showed a pattern of response similar to normal euthymic BALB/c mice.

Avian spinal cord chimeras. Further studies on the neurological syndrome affecting the chimeras after birth

These experiments bring new information concerning the immunological status after birth of quail----chick spinal cord chimeras. Such birds have been produced using recipient flocks of chickens different from those in our previous experiments. The breakdown of tolerance after hatching has been recorded and found to vary with the origin of the embryos. Chickens of broiler JA 657 strain and of a white leghorn strain raised in Japan started to exhibit signs of neural graft rejection later in life than the white leghorn chickens from a French breeder used in our previous studies. As previously described, in two animals, long-term tolerance was observed only for allogeneic chick----chick neural tube grafts. In one chimera the neurological syndrome resulting from rejection was reversible, and no signs of immune attack of the grafted central nervous tissue could be detected at sacrifice. This and other observations reported in this article strongly support the contention that the host immune response to foreign neural tissues starts in peripheral nerves and ganglia where no blood-brain barrier exists rather than in the spinal cord. A humoral response of the host against non-polymorphic quail antigens present on fibroblasts was observed in all birds at the time of rejection.

Kinetics and repertoire selection of T cells derived from the early waves of fetal thymus colonization after thymus grafting in allogeneic nude recipients.

The survival of T cells derived from the early waves of thymus colonization by haemopoietic cell precursors was investigated by grafting thymus from B6.Thy1.1 day 14 embryos (E14) (first wave) or E17 or newborn thymus (subsequent waves) into allogeneic athymic BALB/c (Thy1.2) nude recipients. The survival of donor‐derived Thy1.1 cells was longer when they were derived from early thymocytes. Donor B6.Thy1.1 Vβ5 and Vβ11 T cells, although maturing in BALB/c host presenting Mls2a superantigens, were not deleted, in contrast to host Thy1.2 T cells differentiating from endogenous stem cells. These results show that the population of T cells derived from early precursors undergoes particular selection characteristics, which favour the inclusion of potentially autoreactive cells with prolonged survival, even in H‐2 allogeneic conditions which normally do not allow the survival of peripheral T cells.