Murphy’s interest in tumour transplants in chicken embryos provided early evidence of GVHD. In his experiments, as described earlier in the introduction, he observed that after transplanting the spleen cells of adult chickens onto the chorioallantoic membrane of chicken embryos, the spleens of these chicken embryos became enlarged. He further noticed the development of nodules on the chorioallantoic membrane. However, he suggested that growth factors were responsible for the formation of these nodules.
It was only in 1957 that Simonsen first recorded the enlargement of the spleen in chicken embryos as a consequence of a GVHR (Simonsen, 1957). Simonsen inoculated chicken embryos with adult spleen cells three days before hatching in the hope of tolerizing the chicks to the adult spleen cells. However, soon after hatching the chicks developed severe
haemolytic anemia and died. Simonsen believed that the chicks were tolerant to the donor cells as there was no evidence of antibodies against the donor cells. Further investigation revealed the enlarged spleens of the chicks. He reported that the donor cells had been activated by host antigens and proliferated, destroying and replacing the host lymphocyte population. He postulated that the recipient had to be young enough to be unable to respond to the donor cells and that the donor cells had to be mature enough to form antibodies in order for this phenomenon to occur. A cell-mediated immune response by the donor cells is currently known as the leading cause for a GVHR. A cell-mediated immune response by the donor cells, however, was not considered by Simonsen at the time.
In the same year that Simonsen reported his findings on GVHR in chicken embryos, Billingham and Brent reported similar findings using two inbred strains of mice. In his attempt to induce tolerance to allogeneic skin grafts he injected immunologically competent cells of adult mice into histoincompatible newborn mice. The newborn mice developed runt disease and eventually died two to three weeks later. Exfoliation of the skin and reduced flexibility were marked features of these mice, with considerable weight loss and the eventual enlargement of all lymph nodes as well as the spleen. They further observed that the donor cells persisted in the recipient. Notably the runt disease only arose with cells from adult spleens and not cells from the kidney or the skin. The fact that only spleen cells were effective provided evidence of the role of the lymphocytes on the development of runt disease in mice. The authors initially thought that the presence of a pathogen in the adult inoculum could have resulted in an infection, which led to death. However, they favoured the idea that the immunologically competent cells in the adult spleens mounted an immune response against the host’s cells. In fact, they observed that the tissues that were most affected during runt disease were those to which the lymphocytes migrated. The migration of the lymphocytes through the lymphoid tissue was later demonstrated by Gowans and Knight in 1964 as described later in the introduction.
In a paper by Billingham and Brent in 1959, they provided conclusive evidence that runt disease in newborn mice was due to a GVHR. They inoculated newborn mice with cells from different histoincompatible donors with varying results. Spleen cells from adult F1,
(AxB) mice inoculated into parental (A) newborn mice did not result in runt disease. However, the inoculation of the spleen cells from the parental strain (A) into F1, (AxB) newborn mice induced runt disease. Furthermore, they noted that the inoculation of bone marrow, which contains fewer immunologically competent cells than spleen, resulted in a less severe form of runt disease in the newborn mice. The authors came to two conclusions: the first is that the donor cells that were inoculated were responsible for the runt disease that developed in the newborn mice and secondly that there was a dose effect between the number of immunologically competent cells and the severity of the disease.
GVHD was not only a phenomenon that occurred in mice and chickens but also in non- human primates such as the rhesus monkey. In 1961 Crouch and colleagues reported that monkeys receiving allogeneic bone marrow with critical contaminating T cells after irradiation also developed GVHD. Thus, it was that the phenomenon of GVHR was firmly demonstrated in a number of different animal species. Furthermore, this response of the donor cells against the host cells eventually leads to GVHD and potentially the death of the host.
1.3.2.2 Whole body irradiation and bone marrow transplantations
As early as 1915 Hektoen noted that with an unknown dose of irradiation, administered over several days, rats were unable to produce antibodies to SRBCs (Hektoen, 1915). This provided early evidence of the effects of irradiation on the immunological competent cells of the body. However, it was much later, after the atom bomb was deployed at the end of World War II that active investigation into the effects of radiation on the hematopoietic cells was undertaken at the Radiobiological Research Unit of the Atomic Energy Research Establishment in Harwell (United Kingdom) and the Radiobiologic Institute in Rijswijk (The Netherlands) (Brent, 1997). During these investigations the lethal dose for mice that resulted in the destruction of the bone marrow was determined. In order to counteract the effects of irradiation they transferred bone marrow from mice that differed at their major histocompatibility antigens into the irradiated mice. Although the transplant appeared initially to be protective, the mice developed extreme diarrhoea and weight loss and eventually died. Since allogeneic bone marrow was used in the transplant, the disease
manifestation was referred to as homologous disease. Homologous grafts were originally grafts transplanted from one individual to another of the same species. However, the homologous graft subsequently became known as the allograft.
The work on GVHD resulting in runt disease in mice, prompted Barnes and Loutit to undertake a series of studies, the findings of which were reported in 1958 (Barnes and Loutit, 1958). They explored the potential relationship between the runt disease observed in neonatal mice and the development of the homologous disease after bone marrow transplants in irradiated recipients. The transplanted cells were implicated in the development of runt disease. Barnes and colleagues inoculated irradiated adult mice with either allogeneic neonatal spleen cells or with adult bone marrow. The mortality rate was much higher when adult bone marrow cells were administered. Moreover, they found that bone marrow from an F1 into an irradiated parental strain increased the survival rate compared to the transfer of bone marrow from the parent into irradiated F1. These findings suggested that the inoculated cells were responsible for the development of the homologous disease in irradiated recipients as were they in neonatal mice and chicken embryos. Therefore, homologous disease was considered to be the same as the runt disease observed in mice and the GVHR observed in the chickens.
1.3.3 Factors determining the development of an acute graft versus host reaction and