Transient Graft-versus-host Disease Research Articles

P3.60: Native spleen preservation attenuate graft versus host disease in an experimental model of modified multivisceral transplantation.

Introduction: Native spleen used to be removed in patients undergoing modified multivisceral transplantation (MMVT) increasing the risk of sepsis and graft-versus-host disease (GVHD). The mechanistic basis of these effects including mixed chimerism level is poorly understood. Based on a novel experimental procedure of MMVT that triggers GVHD, we aimed to evaluate the effects of native spleen preservation in this model in order to gain insight into the mechanisms that may mediate this phenomenon. Material and Methods: Heterotopic MMVT from Lewis to Brown Norway rats was performed. MMVT graft consisted of stomach, duodenum, pancreas, spleen and the small bowel of the donor. The native spleen was removed (MMVT-S) in half of the MMVT recipients (N=5), while the remaining preserved their own spleen (MMVT+NSP) (N=5). After MMVT mixed hematopoietic chimerism was determined in native spleen and peripheral blood samples by flow cytometry using strain-specific HLA antibodies. Also, histopathological (using Pintar score) and clinical signs of GVHD such as skin rash were evaluated. Results: all animals in the group MMVT-S presented clinical signs of GVHD such as skin rash, weight loss, and diarrhea, among others, between 7–10 days after transplantation. Skin rash was particularly remarkable in the ears and periocular area. Clinical signs of GVHD were less frequent in MMVT+NSP recipients (40%; p<0.05). Also, significant differences between groups were observed in the histopathological study and mixed chimerism in peripheral blood 3 days after MMVT (36.6 and 23.62 % in MMVT-S and MMVT+NSP respectively) (Figure 1). Also, native spleen showed an 18±5 % of chimerism (CD3+ donor cells) 1 week after transplantation. After day 10 post-MMVT, when the signs of transient GVHD disappeared, both groups presented graft rejection. Conclusions: Native spleen preservation in MMVT recipients attenuated the occurrence of GVHD and reduced the level of chimerism compared to recipients that underwent spleen removal during MMVT. Despite more studies are necessary, our preliminary results suggest that native spleen preservation increase the efficacy in removal of anti-recipient reactive clones that would explain the protective effect of native spleen preservation against GVHD.

Immunological Outcome in Haploidentical-HSC Transplanted Patients Treated with IL-10-Anergized Donor T Cells

T-cell therapy after hematopoietic stem cell transplantation (HSCT) has been used alone or in combination with immunosuppression to cure hematologic malignancies and to prevent disease recurrence. Here, we describe the outcome of patients with high-risk/advanced stage hematologic malignancies, who received T-cell depleted (TCD) haploidentical-HSCT (haplo-HSCT) combined with donor T lymphocytes pretreated with IL-10 (ALT-TEN trial). IL-10-anergized donor T cells (IL-10-DLI) contained T regulatory type 1 (Tr1) cells specific for the host alloantigens, limiting donor-vs.-host-reactivity, and memory T cells able to respond to pathogens. IL-10-DLI were infused in 12 patients with the goal of improving immune reconstitution after haplo-HSCT without increasing the risk of graft-versus-host-disease (GvHD). IL-10-DLI led to fast immune reconstitution in five patients. In four out of the five patients, total T-cell counts, TCR-Vβ repertoire and T-cell functions progressively normalized after IL-10-DLI. These four patients are alive, in complete disease remission and immunosuppression-free at 7.2 years (median follow-up) after haplo-HSCT. Transient GvHD was observed in the immune reconstituted (IR) patients, despite persistent host-specific hypo-responsiveness of donor T cells in vitro and enrichment of cells with Tr1-specific biomarkers in vivo. Gene-expression profiles of IR patients showed a common signature of tolerance. This study provides the first indication of the feasibility of Tr1 cell-based therapy and paves way for the use of these Tr1 cells as adjuvant treatment for malignancies and immune-mediated disorders.

A repertoire-independent and cell-intrinsic defect in murine GVHD induction by effector memory T cells

AbstractEffector memory T cells (TEM) do not cause graft-versus-host disease (GVHD), though why this is has not been elucidated. To compare the fates of alloreactive naive (TN) or memory (TM) T cells, we developed a model of GVHD in which donor T cells express a transgene-encoded TCR specific for an antigenic peptide that is ubiquitously expressed in the recipient. Small numbers of naive TCR transgenic (Tg) T cells induced a robust syndrome of GVHD in transplanted recipients. We then used an established method to convert TCR Tg cells to TM and tested these for GVHD induction. This allowed us to control for the potentially different frequencies of alloreactive T cells among TN and TM, and to track fates of alloreactive T cells after transplantation. TEM caused minimal, transient GVHD whereas central memory T cells (TCM) caused potent GVHD. Surprisingly, TEM were not inert: they, engrafted, homed to target tissues, and proliferated extensively, but they produced less IFN-γ and their expansion in target tissues was limited at later time points, and local proliferation was reduced. Thus, cell-intrinsic properties independent of repertoire explain the impairment of TEM, which can initiate but cannot sustain expansion and tissue damage.

Graft-versus-host-disease-associated thymic damage results in the appearance of T cell clones with anti-host reactivity.

We studied whether T-cell clones, which appear in the periphery as a result of the failure of thymic negative selection during graft-versus-host disease (GVHD), have any in vivo anti-host reactivity and can cause GVHD in an adoptive transfer model. We performed our studies in a murine model (B10.BR into CBA/J) for allogeneic bone marrow transplantation with major histocompatibility complex-matched and minor histocompatibility antigen-mismatched unrelated donors and unique Vbeta T-cell deletion patterns in donors and recipients. GVHD resulted in the appearance of Vbeta6+ T cells as a result of a loss of negative selection. We found that Vbeta6+ T cells from normal donors proliferated in vitro and in vivo. Depletion of Vbeta6+ T cells from the donor T-cell inoculum resulted in less GVHD morbidity and a decrease in the loss of thymic cellularity. To test the anti-host reactivity of de novo generated Vbeta6+ T cells in animals with GVHD, we developed an adoptive transfer model of splenic T cells from CBA/J host animals with GVHD into sublethally irradiated CBA/J recipients Depletion of Vbeta6+ T cells from the splenic T cells before adoptive transfer could significantly decrease the transient GVHD morbidity in the sublethally irradiated hosts. Our data indicate that GVHD-associated thymic damage results in a loss of thymic negative selection, which leads to the appearance of T-cell clones with anti-host reactivity in vitro and in vivo.

Allogeneic Transplants of Canine Peripheral Blood Stem Cells Mobilized by Recombinant Canine Hematopoietic Growth Factors

We have studied graft-versus-host disease (GVHD) after transplantation of allogeneic peripheral blood stem cells (PBSC) mobilized by either recombinant canine granulocyte colony-stimulating factor (rcG-CSF) alone or combined with stem cell factor (rcSCF). These studies were prompted by the observation of extremely rapid and sustained engraftment of growth factor-mobilized PBSC in the autologous setting using genetically marked cells and changes in function of T lymphocytes from donors that had undergone mobilization. Specifically, lymphocytes from growth factor-treated donors were hyporesponsive in mixed leukocyte culture and in response to Con A, raising hopes that GVHD in dogs given growth factor mobilized allogenic PBSC might be altered in a beneficial way. Eighteen dogs were given a median of 17.1 x 10(8) PBSC/kg from littermate donors after 920 cGy of total body irradiation without postgrafting immunosuppression. Donors were either genotypically DLA-identical (n = 9) or DLA-haploidentical (n = 9). The median number of colony-forming unit-granulocyte macrophage (CFU-GM) infused was 27 x 10(4)/kg, and the number of CD34+ cells in the transplant was on the order of 4.6 x 10(6)/kg. The dogs received a median of 52.8 x 10(7) CD4 cells/kg and 13.7 X 10(7) CD8 cells/kg. All 18 dogs had prompt hematopoietic engraftment of donor cells as assessed by chimerism studies using variable number tandem repeat, as well as cytogenetic markers. Three of the nine dogs given grafts from DLA-identical littermates had fatal GVHD, five had transient GVHD, and one had no GVHD. All nine DLA-haploidentical recipients of PBSC developed fatal hyperacute GVHD. In conclusion, the expectation about rapid engraftment was fulfilled. However, incidence and severity of acute GVHD after transplantation of mobilized PBSC were not different than previously reported for nonmobilized PBSC or marrow. This model will allow for further studies, including T-cell depletion to minimize GVHD without increasing graft rejection.

Studies on transient graft-versus-host disease in BALB/c nude mice injected with allogeneic C57BL/6 splenocytes

Graft-versus-host disease (GVHD) is known to cause profound dysregulation of the immune system, although its effector mechanisms are not fully understood. In this study, we investigated what factors influenced the development of GVHD. BALB/c nude mice (H-2 d) injected with MHC-disparate B6(H-2 b) spleen cells exhibited transient GVHD such as hunched back, diarrhea, loss of body weight and splenomegaly. No animals died during the period of observation. BALB/c nude mice produced alloantibodies to the donor cells. The injection of the serum from GVHD nude mice into naive nude mice can protect from GVHD. Donor derived H-2 b+ cells were recognized in the recipient lymph nodes and skin. Prevention of GVHD was achieved by the pretreatment of spleen cells with anti-Thy-1.2 antibody or anti-CD4 antibody and complement, while it was not done by the pretreatment of spleen cells with anti-CD8 antibody and complement. These data demonstrate that Thy-1.2 + CD4 + CD8 − lymphocytes are important effector cells and alloantibodies to the donor cells prevent GVHD in this model.

Evidence that the elevation of soluble MHC class I antigens in the serum precedes the onset of graft-versus-host disease and is correlated with the severity of the disease in rats

We examined the changes in the levels of soluble major histocompatibility antigen complex (MHC) class I antigens in the serum under a lethal or nonlethal state of graft-versus-host-disease (GVHD) induced by injecting various doses of PVG rat splenic lymphocytes into (DA × PVG)F 1 rats. All rats receiving 4 × 10 8 lymphocytes (lethal dose) died on day 20–36 showing typical features of GVHD, while the injection of 4 × 10 7 cells (nonlethal dose) induced no sign of GVHD. When rats were inoculated with a nonlethal dose of lymphocytes prior to the injection of a lethal dose, all rats survived with or without showing transient GVHD. Preceding the onset of GVHD the levels of soluble class I antigens increased significantly to 1094 ± 487 ng/ml (mean ± SD, n = 4) from 3 days after the injection of a lethal dose to the time of death, whilst the levels in the nonlethal dose group remained unchanged. Rats with transient GVHD in the preinoculated group showed the increase of soluble class I antigens to the same extent as rats with lethal GVHD, suggesting that GVHD was systematically ongoing. The levels of soluble class I antigens also correlated with the severity of GVHD as judged by daily observation and histological studies. Rats receiving a lethal dose showed destructive alteration of spleen structure and cellular infiltration in the portal area of the liver before the animals started to show signs of GVHD, whereas rats in the nonlethal dose group exhibited no marked change. These data suggest the possibility of serum soluble class I antigens being not only a diagnostic but also a prognostic marker for GVHD.

Graft-versus-host reaction enhanced by ultraviolet radiation.

Ultraviolet (UV) radiation to the whole body is known to cause selective systemic immunosuppression. Delayedtype hypersensitivity and contact hypersensitivity reactions are suppressed by UV radiation [8, 12, 13], although other general immune reactions, such as the mitogenic response, antibody formation, cytolytic T-cell induction and allograft rejection, are not affected [6, 9, 11]. Graft-versus-host disease (GVHD) is a complex process involving many different cell types and soluble mediators in immunoincompetent recipients after transfusion of allogeneic immunocompetent cells [5]. When normal F 1 hybrid mice, which are immunologically unresponsive to parental cells, are injected with parental strain lymphoid cells they develop transient GVHD, for example splenomegaly, but not lethal GVHD [2, 4]. In the present study, using this classical parent to F 1 GVHD model, we examined how in vivo UV irradiation, which is usually associated with suppression of immune responses, affects GVHD. Contrary to our expectation that UV would suppress GVHD, we found that UV enhanced GVHD. Female C57BL/6 (B6) (H-2 b) and (BALB/c x C57BL/6)F 1 (CB6F1) (H-2 b/d) mice were bred and maintained under pathogen-free conditions in the Experimental Animal Center, Kyoto University. All mice were used at 6-8 weeks of age. Systemic GVHD was induced in the CB6F 1 mice by intravenous injection of B6 spleen cells (5 x 107). As controls, CB6F1 mice were injected with syngeneic spleen cells. The dorsal hair of all CB6F 1 mice was shaved. Within 1 h of donor spleen cell injection, the recipient CB6F 1 mice were exposed for 1 h to UVB (280-320 nm) radiation emitted from a bank of five sunlamps (FL20S.E-30, Toshiba Electric, Tokyo, Japan). During irradiation, the

Effects of donor pretreatment with antilymphocyte serum and cyclosporin on rejection and graft-versus-host disease after small bowel transplantation in immunosuppressed and nonimmunosuppressed rats

After fully allogeneic small bowel transplantation, both graft-versus-host disease (GVHD) and rejection may occur. Donor pretreatment may prevent GVHD, but this sometimes leads to accelerated graft rejection. To study a possible balance between GVHD and rejection, fully allogeneic total orthotopic small bowel transplantation was performed in rats using the WAG-to-BN donor-host combination. Untreated control grafts were rejected in 16.6 +/- 2.7 days (mean +/- SEM), and 35% of the animals had mild, transient GVHD. Pretreatment of the donor with antilymphocyte serum on days -2 and -1 before grafting, with antilymphocyte serum on days -2 and -1 before grafting, with antilymphocyte serum on days -2 and -1 before grafting, either intravenously or intraperitoneally, completely eliminated the occurrence of clinical GVHD but led to significantly shortened survival times (12.3 +/- 0.8 and 10.3 +/- 0.9 days, respectively). Donor pretreatment with 50 mg/kg cyclosporin (CyA) on days -2 and -1 prolonged graft survival significantly to 22.1 days but had no significant effect on the incidence of GVHD. Administration of 25 mg/kg CyA on days 0, 1, 2, 4, and 6 after grafting prolonged survival to 38.3 days with no evidence of GVHD. Pretreatment of the donor with antilymphocyte serum (ALS), combined with the same postoperative, short-term CyA regimen, increased survival to more than 50 days, again with no evidence of GVHD. When CyA was used as both donor pretreatment and postoperative therapy, there was no survival advantage compared to the use of postoperative CyA alone. These results show that an in vivo balance between GVHD and rejection exists and that abrogation of GVHD leads to accelerated rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of donor pretreatment with antilymphocyte serum and cyclosporin on rejection and graft-versus-host disease after small bowel transplantation in immunosuppressed and nonimmunosuppressed rats

Abstract. After fully allogeneic small bowel transplantation, both graft-versus-host disease (GVHD) and rejection may occur. Donor pretreatment may prevent GVHD, but this sometimes leads to accelerated graft rejection. To study a possible balance between GVHD and rejection, fully allogeneic total orthotopic small bowel transplantation was performed in rats using the WAG-to-BN donor-host combination. Untreated control grafts were rejected in 16.6 ± 2.7 days (mean ± SEM), and 35 % of the animals had mild, transient GVHD. Pretreatment of the donor with antilymphocyte serum on days -2 and -1 before grafting, either intravenously or intraperitoneally, completely eliminated the occurrence of clinical GVHD but led to significantly shortened survival times (12.3 ±0.8 and 10.3 ± 0.9 days, respectively). Donor pretreatment with 50 mg/kg cyclosporin (CyA) on days -2 and -1 prolonged graft survival significantly to 22.1 days but had no significant effect on the incidence of GVHD. Administration of 25 mg/kg CyA on days 0,1, 2,4, and 6 after grafting prolonged survival to 38.3 days with no evidence of GVHD. Pretreatment of the donor with antilymphocyte serum (ALS), combined with the same postoperative, short-term CyA regimen, increased survival to more than 50 days, again with no evidence of GVHD. When CyA was used as both donor pretreatment and postoperative therapy, there was no survival advantage compared to the use of postoperative CyA alone. These results show that an in vivo balance between GVHD and rejection exists and that abrogation of GVHD leads to accelerated rejection. Immunosuppression of the recipient may overrule this accelerated rejection while preserving the beneficial effect of donor pretreatment: elimination of clinical GVHD.

Severe combined immunodeficiency: treatment by bone marrow transplantation in 15 infants using HLA-haploidentical donors.

In 15 infants with severe combined immunodeficiency (SCID), immunological reconstitution was attempted by bone marrow transplantation (BMT) from HLA-haplo-identical parents. To prevent graft versus host disease (GvHD), marrow grafts were depleted of contaminating T-lymphocytes using lectin agglutination and rosette formation with sheep red blood cells. Thirteen patients received transplants without undergoing prior cytoreductive conditioning. Eleven of these developed donor-dependent T-cell functions, two failed to do this. One of these two as well as two further patients received cytoreductive treatment prior to repeat and to first transplants and in two, complete lymphohemopoietic reconstitution was observed. Of the 15 patients who received transplants, 11 are currently alive. Two recently treated patients remain in the hospital, nine are at home with stable T-cell functions. Normal humoral immune functions have developed upto now in three patients. In the others, gamma globulins are regularly substituted. Complications of acute or chronic GvHD were not observed with the exception of one case who developed transient GvHD of the skin. These results suggest that in a majority of patients with SCID, T-cell functions can develop without GvHD following haploidentical, T-cell-depleted BMT. Exceptional patients require preconditioning to allow donor cell engraftment, an approach that also appears to facilitate reconstitution of humoral immune functions.

Bone marrow transplantation in two patients with aplastic anaemia

Bone marrow transplantation is now established as a significant advance in the management of patients with severe aplastic anaemia. Two such patients, a male of 19 yr and a female aged 14, received bone marrow transplants from female sibling donors at Royal Perth Hospital in January 1979. In each case, donor and recipient were HLA identical and mutually non-stimulatory in mixed lymphocyte culture. The patients were isolated in laminar flow (Clemco) units for 2 wk prior to transplantation and for 4 wk subsequently. They received cyclophosphamide 50 mg/kg daily from day –5 to day –2 as preconditioning, and methotrexate 15 mg weekly post-transplant as prophylaxis against graft versus host disease (GVHD). The girl's transplant was ABO incompatible, the donor being group AB and the recipient group O. A Haemonetics-30 cell separator was used to reduce the anti-A and anti-B titres of the recipient prior to the marrow infusion. Plasmaphereses, each of 4 1, were performed on days – 2, – 1, and 0. No haemolysis was encountered during marrow transfusion. She received 3.0×10⁸ marrow cells/kg. Engraftment was observed on day 15 and the peripheral count rose rapidly to normal levels. Her blood group gradually converted to AB. and the anti-A and anti-B titres disappeared. The pre-engraftment phase was complicated by severe oral herpes simplex ulceration and adenovirus cystitis. On day 39 GVHD became evident. Prednisolone in a dose of 2 mg/kg/d improved the skin rash, but diarrhoea and vomiting persisted and hepatic function deteriorated. Antithymocyte globulin in a dose of 10 mg/kg on alternate days was commenced on day 57, and continued for 3 wk without effect. She eventually became thrombocytopenic with severe megakaryocyte hypoplasia and died from gastrointestinal haemorrhage on day 114. The boy received 2.15×10⁸ marrow cells/kg. Engraftment was first evident on day 18. The peripheral blood count improved slowly, not reaching normal levels until day 110. Apart from transient GVHD on day 70, a mild chest infection around day 100, and a photosensitivity rash attributed to Septrin, he has remained well throughout.

BONE MARROW TRANSPLANTATION IN THE BUSULFAN-TREATED RAT

It was previously shown that lethal doses of busulfan (BU) were not immunosuppressive in the rat. In the present studies the effect of rabbit antirat thymocyte serum (RARTS) and cyclophosphamide (CY) as immunosuppressive preparation for marrow allografts in the rat given supralethal doses of BU was investigated. Lewis or LBNF rats were given RARTS for 4-12 consecutive daily doses. They were given a supralethal dose of BU in addition on the last day of RARTS administration. Ag-B-incompatible (ACI). Ag-B-compatible (F344), or syngeneic marrow was infused 24 hr later. In general, RARTS was not toxic to the marrow graft and permitted engraftment of Ag-B-incompatible and compatible marrow. All animals given Ag-B-incompatible marrow and Ag-B-compatible marrow that survived to day 21 were shown to be chimeric, but all those with incompatible marrow subsequently died from severe graft-versus-host disease (GVHD). In other experiments Lewis or LBNF1 rats were given Ag-B-incompatible (ACI) and compatible (F344) marrow after a supralethal dose of BU and single graded doses of CY. Doses of CY as low as 25-50 mg/kg provided sufficient immunosuppression to allow allogeneic engraftment. Transient chimerism was seen up to doses of 100 mg/kg of CY. Permanent chimerism was seen with doses of CY from 150 to 250 mg/kg. The incidence of lethal GVHD increased in parallel with the percentage of animals that were demonstrated to be chimeric. Mild but transient GVHD was seen with Ag-B-compatible marrow given to Lewis rats.