Minor Histocompatibility Barriers Research Articles

Histological changes of bile duct in experimental graft-versus-host disease across minor histocompatibility barriers. II. Electron microscopic observations.

Electron microscopic features of intrahepatic bile ducts of experimental mouse graft-versus-host disease (GVHD) across minor histocompatibility barriers were studied for 14 months after transplantation. In GVHD mice, the bile duct epithelial layer was consistently infiltrated by lymphoid cells and often accompanied by polymorphonuclear leukocytes, monocytes and rarely by plasma cells. The epithelial cells in close contact with and in the vicinity of these infiltrated cells showed a variety of degenerative changes, including darkness of the cytoplasm and the nucleus with shrunken, irregular contours, increase in the amount of endoplasmic reticulum and number of mitochondria, and formation of intracytoplasmic vesicles and diverticula, cytoplasmic blebs, and apoptopic bodies. Lymphocytes were in close contact with epithelial cells through a number of point-contacts and located in the lateral intercellular spaces and/or between the basement membranes and the epithelial cells. The localization of infiltrating lymphocytes beneath the epithelial cells with conspicuous detachment from the basement membranes strongly suggested a link with subsequent epithelial cell injury and death. The lymphoid cells had irregular cytoplasmic projections which occasionally extended into spaces created by retractions of the epithelial cell membranes, reflecting an activation of the lymphocytes. These findings support the notion that the bile duct lesions in GVHD across minor histocompatibility barriers are mediated by specifically sensitized lymphocytes against epithelial cell membrane antigens. From our previous finding that a large majority of the infiltrating lymphocytes had a phenotype of helper/inducer T cells, a putative role of these lymphocytes in the induction of the bile duct lesions is discussed.

PROLONGATION OF SKIN ALLOGRAFT SURVIVAL IN RATS BY A NOVEL IMMUNOSUPPRESSIVE AGENT, FK 506

FK506, an immunosuppresant, was isolated from Streptomyces tsukubaensis. Intramuscular administration of FK506 (0.32 mg/kg or more) 5 days a week for two weeks after grafting prolonged the acceptance time of F344 skin allograft to WKA rats. Similar results were obtained with cyclosporine at 32 mg/kg or more, but other immunosuppressives (i.e., prednisolone, azathioprine, and bredinin) gave only a marginal prolongation. The prolonging effect of FK506 was obtained in various donor-recipient combinations across a major or minor histocompatibility barrier. The agent also prolonged the acceptance time of mouse skin xenografts to rats. Furthermore, maintenance doses of 3.2 or 0.32 mg/kg twice a week after an initial 14-day treatment with the agent at 3.2 mg/kg gave graft survival as long as the treatment was continued for more than 120 days. Our findings show that FK506 has a potent immunosuppressive effect in rats and suggest that the agent merits further investigation.

Enhanced interferon- [formula omitted] (IFN- [formula omitted]) and defective IFN-γ production in chronic graft versus host disease: A potential mechanism for immunosuppression

Immunosuppression is a well-characterized consequence of chronic graft-versus-host disease (GVHD). We have previously shown that interferon (IFN) is produced in high levels during acute GVHD. Our objective in this study was to determine if IFN, as a cytokine with known immunosuppressive qualities, could be detected in mice experiencing chronic GVHD-induced immunosuppression. Two different experimental models were used to induce chronic GVHD. The first model involved the injection of parental strain spleen cells into adult F 1 hybrids (AJ → B6AF 1), while the second model utilized GVHD induced across minor histocompatibility barriers (B10·D2 → BALB/c). Results indicated that significant levels of serum IFN- α β are present in mice undergoing chronic GVHD. Spleen cells from chronic GVHD mice were also shown to produce significant levels of IFN- α β upon in vitro culture in medium only. This IFN- α β production was greatly increased when GVHD spleen cells were cultured with either concanavalin A (Con A) or IL-2. In contrast, IFN-γ production was undetectable in these Con A- or IL-2-containing cultures. Additionally, these same spleen cells which produced high levels of IFN- α β were immunosuppressed as measured by mitogen-induced cell proliferation. These results suggest that IFN-γ production is defective in GVHD spleen cells, and that the presence of high IFN- α β production by GVHD mice may contribute to the immunosuppression associated with chronic GVHD.

Cutaneous mast cell depletion and recovery in murine graft-vs-host disease.

Mast cells as studied by light microscopy with metachromatic staining, have been noted to "disappear" from the skin of mice with chronic graft-vs-host disease (GVHD) produced across minor histocompatibility barriers. This mast cell disappearance is accompanied by ultrastructural evidence of loss of granule contents. In this study, we followed cutaneous mast cells in chronic GVHD over 9 mo by three methods: Light microscopy of toluidine blue-stained sections showed that mast cells not seen at day 42 reappeared between days 94 and 125, were supramaximal at days 146 and 164, and returned to normal levels at days 195 and 280. Double immunofluorescent staining of mast cells for the presence of surface IgE receptors and cytoplasmic granules (avidin) revealed IgE receptor-bearing cells that lacked avidin-binding granules at the time when mast cells were not apparent on light microscopy. By electron microscopy, reappearing mast cells have the morphology of immature dermal mast cells. Ultrastructural abnormalities of mast cells persist some 150 days after GVHD induction. The possible relationship of these mast cell changes to the development of dermal fibrosis in chronic GVHD is discussed.

BONE MARROW TRANSPLANTATION WITH T-CELL-DEPLETED GRAFTS

Graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (BMT), can be prevented by in vitro depletion of T cells from the bone marrow (BM) prior to transplantation. The purpose of this study was to assess the role of BMT cells in the reconstitution of various immune functions following BMT across minor histocompatibility barriers. Lethally irradiated CBA/J (H-2k) mice were grafted with either 10(7) unseparated or T-cell-depleted BM cells from B10.BR (H-2k, minor-histoincompatible) mice. Blood counts, BM colonies in agar, and various immune functions of spleen cells from the recipient mice were tested 2-12 weeks post-BMT and compared with those of normal donors. The following observations were made: (A) Peripheral blood lymphocyte counts decreased to 30% of normal 2 weeks post-BMT with almost normal recovery at 8 weeks. (B) The percentage of Thy1.2+ splenocytes reached normal levels at 8 weeks post-BMT. (C) The number of BM colonies (GM-CFU) was reduced to 10% at 2 weeks and fully recovered at 12 weeks. (D) Proliferative response to the B-cell mitogen LPS was fully reconstituted after 4 weeks; however, anti-SRBC PFC (following Mishell-Dutton cultures) was restored 50% at 8-12 weeks. (E) Reconstitution of T cell functions including proliferative responses to concanavalin A, phytohemagglutinin, and allogeneic leukocytes, and allocytotoxicity, did not exceed 50% even 12 weeks post-BMT. Overall, depletion of T cells from donor BM allografts incompatible at minor histocompatibility loci, did not seem to significantly alter the rate of immunohematopoietic reconstitution in the lethally irradiated BM recipients.

BONE MARROW TRANSPLANTATION WITH T-CELL-DEPLETED GRAFTS

Graft-versus-host disease (GVHD), a serious complication of allogeneic bone marrow transplantation (BMT), can be prevented by in vitro depletion of T cells from the bone marrow (BM) prior to transplantation. The purpose of this study was to assess the role of BM T cells in the reconstitution of various immune functions following BMT across minor histocompatibility barriers. Lethally irradiated CBA/J (H-2k) mice were grafted with either 107 unseparated or T-cell-depleted BM cells from B10.BR (H-2k, minor-histoincompatible) mice. Blood counts, BM colonies in agar, and various immune functions of spleen cells from the recipient mice were tested 2-12 weeks post-BMT and compared with those of normal donors. The following observations were made: (A) Peripheral blood lymphocyte counts decreased to 30% of normal 2 weeks post-BMT with almost normal recovery at 8 weeks. (B) The percentage of Thy 1.2+ splenocytes reached normal levels at 8 weeks post-BMT. (C) The number of BM colonies (GM-CFU) was reduced to 10% at 2 weeks and fully recovered at 12 weeks. (D) Proliferative response to the B-cell mitogen LPS was fully reconstituted after 4 weeks; however, anti-SRBC PFC (following Mishell-Dutton cultures) was restored 50% at 8-12 weeks. (E) Reconstitution of T cell functions including proliferative responses to concanavalin A, phytohemagglutinin, and allogeneic leukocytes, and allocytotoxicity, did not exceed 50% even 12 weeks post-BMT. Overall, depletion of T cells from donor BM allografts incompatible at minor histocompatibility loci, did not seem to significantly after the rate of immunohematopoietic reconstitution in the lethally irradiated BM recipients.

Variable capacity of L3T4+ T cells to cause lethal graft-versus-host disease across minor histocompatibility barriers in mice.

Highly purified populations of L3T4+ and Lyt-2+ T cell subsets were compared for their capacity to cause lethal GVHD in six different H-2-compatible, multiple minor histocompatibility antigen-different murine strain combinations. In four of these combinations (C3H.SW----B6, DBA/2----B10.D2, B10.BR----CBA, and B10.S----SJL), lethal GVHD appeared to be caused almost entirely by Lyt-2+ cells; the injection of L3T4+ cells resulted in low mortality even when these cells were presensitized to the recipient antigens. In the remaining two combinations (B10.D2----DBA/2 and B10.D2----BALB/c), L3T4+ T cells were able to cause a high incidence of GVHD and were more potent than the Lyt-2+ cells. The implications of these findings are discussed.

Monoclonal antibody analysis of skin in chronic murine graft vs host disease produced across minor histocompatibility barriers

Chronic graft vs host disease (GVHD) across minor histocompatibility barriers was produced in BALB/c mice by the injection of spleen cells from B10.D2 mice. Changes in the skin were analyzed in frozen sections using a panel of monoclonal antibodies detected by immunoperoxidase methods. Compared to control animals, a number of changes occurred in the skin of animals with chronic GVHD. In the epidermis, there were increased numbers of Thy-1-positive dendritic cells; keratinocytes expressed Thy-1 and Ia antigens. T lymphocytes appeared in both dermis and epidermis. In the early stages, cells with “helper” and “suppressor” phenotypes were present, while at later times “helper” cells remained in the epidermis and “suppressor” cells remained in the dermis. Cells bearing markers of macrophages were prominent in both dermis and epidermis after the second week. Of great interest was the appearance of spindle-shaped cells in the dermis which expressed Thy-1 and Ia. These cells resembled fibroblasts which may be activated to produce the excess collagen seen in the skin of chronic GVHD.

Mast cell "disappearance" in chronic murine graft-vs-host disease (GVHD)-ultrastructural demonstration of "phantom mast cells".

Mast cells were studied during the induction of chronic graft-vs-host disease (GVHD) induced in mice across minor histocompatibility barriers. B10.D2 spleen cells (or control BALB/c cells) were injected into irradiated (600 rad) BALB/c recipients. Serial skin biopsies were taken over 26 days, during which time changes occurred resembling scleroderma, namely, dermal fibrosis, a mononuclear cell infiltrate, and loss of fat and appendages. Mast cells, when stained with toluidine blue, "disappeared" from GVHD, but not from control skin. Ultrastructural analysis showed that mast cells in GVHD skin were indeed present but underwent degranulation. Some mast cells showed only pale expanded sacs, indicating granule depletion. Because these cells could not be seen by toluidine blue staining but were plainly present, we have called them "phantom mast cells." Cellular activation occurred in many GVHD mast cells as shown by increased cytoplasmic activity, with numerous Golgi complexes, ribosomes, granular endoplasmic reticulum, and small vesicles. No identifiable mast cells were seen after day 19. No significant changes were seen in the mast cells of syngeneic control mice. We believe that immunologic processes in chronic GVHD cause a slow release of mast cell granule contents, which is different from anaphylactic degranulation. The depleted mast cells (invisible by toluidine blue staining) are also activated, perhaps in an attempt to replete their stores of granule contents. We discuss the relation of mast cell changes to fibrosis.

Limb Allotransplantation in the Rat

We report the variability of the rejection process among the several tissues of a limb allograft. We used a rat hind limb allograft model transplanting across a well-defined minor histocompatibility barrier (Fischer RT-1(1v1)), donor animals, and Lewis (RT-1(1)) recipient animals. Continuous cyclosporin and prednisone immunosuppression was used. Four immunosuppressive regimens all produced extended limb survival. The rejection process was most severe and difficult to control in the skin. Nonskin tissues reverted to a nearly normal appearance after a period of cellular infiltration 2 to 3 weeks posttransplantation. Clinical and electromyographic evidence of nerve regeneration and end-organ reinnervation was demonstrated in long-term surviving animals.

Pituitary transplantation: cyclosporine enables transplantation across a minor histocompatibility barrier.

Pituitary glands from neonatal donors were transplanted to the median eminence of hypophysectomized adult rats. Rats with transplants were then treated for 2 weeks with the immunosuppressive drug cyclosporine. For 5 weeks thereafter, blood was drawn at regular intervals for determination of serum thyroxine, prolactin, and luteinizing hormone. Cyclosporine-treated recipients of grafts with minor histocompatibility differences had normal levels of thyroxine and prolactin, whereas untreated animals did not. In addition, the treated animals responded to oophorectomy with a marked elevation in serum luteinizing hormone. This evidence indicates that cyclosporine enables successful transplantation across a minor histocompatibility barrier. It also suggests that these grafts interact with the hypothalamus. Transplantation across a major histocompatibility barrier was unsuccessful even in the presence of cyclosporine.

The role of H-2 and non-H-2 antigens and genes in the rejection of murine cardiac allografts.

Genes of the major histocompatibility complex (MHC) in the mouse (H-2 complex) have been shown to be an important factor in determining the immune responsiveness of various strains of mice to isolated antigens (e.g., lysozyme). The role of MHC genes in controlling the responsiveness of mice to multiple alloantigens is less well-defined, and although non-MHC genes have been shown to be important in determining responsiveness in some systems (e.g., haptens), they have not been demonstrated as yet to influence the rejection of vascularized organ allografts. In this study, the responsiveness of mice to vascularized cardiac allografts transplanted across well-defined major (H-2) and minor (non-H-2) histocompatibility barriers was investigated using congenic mice in 32 different donor/recipient combinations. The results show that both H-2 and non H-2 gene products can act as target alloantigens for rejection. At the responder level, they may interact to effect responsiveness of a recipient strain to multiple alloantigens. In no case in this study has any one gene or group of genes been found to confer universal high or low responder status.

Graft-vs-host reactions (GVHR) across minor murine histocompatibility barriers. II. Development of natural suppressor cell activity.

We explored the immunoincompetence of mice undergoing a chronic graft-vs-host reaction (GVHR) across minor histocompatibility barriers. BALB/c and B10.D2 mice are H-2d and mls b, and differ only with regard to minor histocompatibility antigens (MiHA). A large number of BALB/c mice were unirradiated or were irradiated with 300, 600, or 900 R. They then were injected with 5 X 10(7) spleen cells from either allogeneic B10.D2 or syngeneic BALB/c mice. The spleen cells from these recipient mice were assayed at various times post-irradiation/injection for their proliferative response to Con A and LPS, their ability to suppress the mitogen responses of normal spleen cells, and for the genetic specificity of this suppression. Spleen cells from BALB/c mice that had received 600 or 900 R (but not 0 or 300 R), and allogeneic B10.D2 lymphocytes, became very hyporesponsive to mitogens and became suppressive in vitro by days 7 to 10 post-irradiation/injection. These phenomena persisted for the entire 49 days of the experiment. After an initial period of splenomegaly, the spleens of these mice gradually became depleted of viable lymphocytes. Initial characterization of suppressor cells found in the spleens of GVH mice showed that they were not removed by treatment with anti-Thy-1.2 plus complement. GVH suppressors also were not adherent to plates coated with antiserum directed towards murine Ig. In addition, these cells did not adhere to plastic plates. Thus, we believe that the suppressor cells found in mice undergoing GVHD across MiHA are not mature T cells, B cells, or macrophages, but belong to a class of suppressor cells termed natural suppressor (NS). Genetic analysis of NS cell activity showed that as early as 10 days post-irradiation/injection, NS cells inhibited mitogen responses of all mouse strains tested, the exception being the relative difficulty in suppressing the LPS response of B10.D2 (syngeneic with donor cells). By day 42, this had developed into an almost complete inability to suppress a B10.D2 LPS response, although at this time NS cells were still capable of inhibiting all the other mitogen responses of all strains tested, including the Con A response of B10.D2 spleen cells. Moderate amounts of mitogen unresponsiveness and suppressor activity were seen in the syngeneic groups (BALB/c----BALB/c) but only if recipients received 600 or 900 R. This was a transient phenomenon that was maximal at day 14, and which we believe to be a similar but less severe degree of immunoincompetence when compared with that seen with allogeneic stimulation in the B10.D2----BALB/c GVH model.(ABSTRACT TRUNCATED AT 400 WORDS)

Synergism between T and non-T cells in the in vivo induction and in vitro expression of graft-vs.-host disease-induced natural suppressor cells.

We have been studying the mitogen hyporesponsiveness and immunosuppression induced in chronic murine graft-vs.-host disease (GVHD) induced across minor histocompatibility (MiHA) barriers. In this system, donor and recipient mice are major histocompatibility complex- and mls-identical, and are nonreactive in primary mixed leukocyte reactions. Spleen cells from B10.D2 (H-2d, mls b) mice were injected into irradiated (600 rad) BALB/c (H-2d, mls b) recipients. Recipient spleen cells are hyporesponsive to mitogens, and contain natural suppressor (NS) cells. We investigated the cellular requirements for both the in vivo induction and the in vitro expression of this GVH suppression. T cells are required in the graft, but they are not sufficient to induce suppression, and a non-T cell population is also required for maximum induction in vivo. T cells are also required for the maximum expression of NS cell suppressive ability in vitro. Early in the course of GVH, the suppressor cells are able to suppress the Con A and LPS response of all mouse strains tested (except for the relative difficulty in suppressing the B10.D2 LPS response). Later, they become almost completely unable to suppress the B10.D2 LPS response; while still being able to suppress the Con A and LPS response of all other strains tested (including the B10.D2 Con A response). This inability to suppress a B10.D2 LPS response can be brought back to almost complete suppression by the addition of concanavalin A supernatant (CAS). We present a hypothesis to explain what may be a common mechanism for GVH-induced suppression, total lymphoid irradiation-induced suppression, and neonatal tolerance. These situations all include rapidly proliferating lymphohematopoietic stem cell populations, and also have large numbers of NS cells. NS cells can suppress proliferating lymphoid populations, and their development and activity are greatly enhanced by T cell signals such as are supplied by donor T cells in chronic GVHD. Thus, NS cells may feed back on and downregulate self-reactive T cells or T cells responding to introduced foreign antigens.

Chronic graft-versus-host disease as a model for scleroderma: II. Mast cell depletion with deposition of immunoglobulins in the skin and fibrosis

We explored the pathologic changes in the skin of mice undergoing a chronic graft-versushost (GVH) reaction. In rodents and in man, chronic GVH includes the deposition of excess collagen in the skin—a reaction which resembles idiopathic scleroderma. GVH disease across minor histocompatibility barriers was produced by injecting B10.D2 cells into irradiated BALB/c mice. These strains are identical at the H-2 and Mls loci but differ in minor histocompatibility antigens. Control BALB/c mice received irradiation and BALB/c cells. Serial skin biopsies were taken and studied for histological changes characteristic of chronic GVHD, for mast cell density, and for the deposition of immunoreactants. GVHD was produced in B10.D2 → BALB/c mice as measured by body weight loss and the production of skin changes including dermal fibrosis, loss of fat and appendages, and a mononuclear cell infiltrate. Dermal mast cells, assessed by toluidine blue staining, were normal at Day 11, but had disappeared by Days 21–63 and returned to normal by Day 104. Immunoglobulins IgG, IgA, and IgM appeared at the dermo-epidermal junction and along the basement membrane zone of hair follicles. This deposition was maximal at Day 42 and waned thereafter. Thus the appearance of immunoglobulins in the skin was maximal when mast cell staining was minimal. The changes in this GVHD model leading to a scleroderma-like picture in the skin are compatible with an immune etiology for the fibrosis. Vasodilation following liberation of mast cell mediators would facilitate the deposition of immunoglobulins. The disappearance of mast cell staining may be caused by extensive degranulation. We postulate an interaction between GVHD-activated T cells, mast cell stimulation, fibroblast activation, and fibrosis.

The Long-term Fate of Fresh and Frozen Orthotopic Bone Allografts in Genetically Defined Rats

Fresh and frozen orthotopic iliac crest bone grafts in rats were studied histologically for determination of the long-term effects of histocompatibility matching and the freezing process on orthotopic bone graft incorporation. Grafts exchanged between groups of inbred rats, syngeneic or differing with respect to major or minor histocompatibility loci, were studied histologically at 20, 30, 40, 50, and 150 days after bone transplantation. A numerical histologic scoring system was developed and used by three observers for evaluation of coded hematoxylin and eosin sections. All frozen graft groups had the same fate regardless of histocompatibility relations between donors and recipients, and all grafts were inferior to fresh syngeneic grafts. Both fresh allograft groups received similar scores and initially at 20 and 30 days had scores similar to those of the fresh syngeneic groups. In the later intervals, however, the fresh allografts were inferior to the fresh syngeneic grafts and similar to the frozen groups. This is consistent with an older model describing two distinct phases of osteogenesis. In the long term, frozen syngeneic and fresh and frozen allografts across major and minor histocompatibility barriers were comparable, but all were significantly inferior to fresh syngeneic bone grafts.

Mast Cell Depletion in Murine Chronic Graft-versus-Host Disease

A murine model of chronic graft-versus-host disease (GVHD) was induced across minor histocompatibility barriers. This was done by injecting B10.D2 (H-2d) spleen cells into irradiated BALB/c (H-2d) mice. Chronic GVHD in this model includes features common to human idiopathic scleroderma, such as dermal fibrosis, loss of dermal fat and appendages, and a mononuclear cell filtrate. Serial skin biopsies showed a progressive loss of stainable mast cells in GVHD but not in irradiated controls. Mast cell depletion was noted as well in the tongue and kidney capsule of GVHD mice. Mast cell depletion was noted as early as 11 days after GVHD induction and persisted for at least 56 days. A hypothesis is put forth linking the T-cell activation of GVHD, mast cell degranulation, and increased fibrosis. The pertinence of this hypothesis to idiopathic scleroderma is pointed out.

Graft-vs-host reactions (GVHR) across minor murine histocompatibility barriers. I. Impairment of mitogen responses and suppressor phenomena.

In our laboratory, we have developed a murine model to examine GVHD across minor histocompatibility antigens. In our model, GVHD is induced by injecting B10.D2 spleen cells into irradiated BALB/c recipients. Seven to 10 days after irradiation and injection of cells, there are significant changes in cell function in the recipient spleens. In the B10.D2----BALB/c (600 rad) model, recipient spleen cells are profoundly unresponsive to Con A and LPS stimulation but show increased B cell activity measured by Staphylococcus aureus protein A plaque-forming activity. Spleen cells from such GVH mice profoundly suppress the mitogenic responses of normal BALB/c or B10.D2 spleen cells to Con A and LPS. The degree of impairment of the mitogenic response and the ability to suppress normal cells is proportional to the dose of cells used to induce GVH reactions. Both the inability to respond to mitogens and the capacity to suppress are also related to the dose of irradiation given to the recipients. In addition, immunosuppression across minor histocompatibility antigens shows an unevenhandedness. If we inject parental B10.D2 or BALB/c cells into F1 recipients (P----F1), there is greater inhibition of mitogenic responses when B10.D2 parental cells are given than when BALB/c cells are given to the irradiated F1 recipients. These experiments show that significant immunosuppression occurs during GVH reactions across minor histocompatibility barriers. The degree of suppression varies according to the dose of cells used to induce GVH, the dose of irradiation to the recipient and the "strength" of the GVH recognition system. Such experiments provide models for GVH disease seen in humans who receive treatment for leukemia or other diseases that involves recipient irradiation and infusion of HLA-identical bone marrow.

Vascularized bone allograft transplantation in a genetically defined rat model.

A heterotopic subcutaneous model for experimental vascularized bone allograft transplantation has been presented. This model uses genetically defined rats and allows serial assessment of graft viability. The reliability of this model has been proven by successful isograft transplantation. This model was used to study the effect of matching at the major histocompatibility complex on vascularized bone allograft survival. Whereas grafts transplanted across a minor histocompatibility barrier survived until sacrifice, grafts transplanted across a major histocompatibility barrier were victims of an acute rejection process. This study, therefore, showed genetic disparity to be a critical determinant of vascularized bone allograft survival. It indicates that primary vascularized bone allografts are as susceptible to rejection as heart and kidney allografts. For these reasons, it can be anticipated that genetic matching will be important in clinical vascularized bone allograft transplantation. The model used in this study should be useful for obtaining further fundamental immunologic information concerning vascularized bone allograft transplantation.

Minor antigen graft-versus-host reactions revealed in irradiated spleen and popliteal lymph node assays.

The graft-versus-hot (GVH) reaction across minor (non-H-2) histocompatibility barriers was studied in mice, in vivo. To increase GVH potential and to mimic clinical bone marrow transplantation protocols, we modified the popliteal lymph node (PLN) and the splenomegaly assays by irradiating the recipients before they received allogeneic lymphoid cell suspensions. In several combinations across major (H-2), minor (non-H-2) and multiple minor (non-H-2 plus minor lymphocyte stimulation) barriers, increased recipient organ weight (a measure of GVH activity) was seen with irradiated F1 recipients of parental cells. The irradiated splenomegaly (x-splenomegaly) assay was more sensitive than the (x-PLN) assay, but both correlated with in vivo GVH experiments of the P----F1 variety. The x-splenomegaly test indicated histoincompatibility in a system (B10.D2----BALB/c) in which the primary in vitro mixed leukocyte reactions is nonreactive, but in which systemic GVH can be induced. The x-splenomegaly test should be useful in analyzing complex reactions involving minor histocompatibility antigens in vivo.