Graft-versus-host disease following interleukin-2/lymphokine-activated killer (LAK) cell immunotherapy in a patient with acute myelogenous leukaemia in second complete remission: autologous LAK cells following allogeneic bone marrow transplantation are donor-derived

A 48-year-old man was treated by allogeneic bone marrow transplantation (BMT) in first remission of M4 acute myelogenous leukaemia (AML). He experienced no graft-versus-host disease (GVHD) and 7 months later he relapsed. Following further chemotherapy, he entered a second complete remission; however, he refused a further allogeneic or autologous BMT but agreed to immunotherapy with interleukin-2 and autologous lymphokine-activated killer (LAK) cells. He tolerated this treatment well but went on to develop grade II skin GVHD. Polymerase chain reaction studies of DNA microsatellites of the autologous LAK cells showed that they were of donor origin. The patient remained well for 9 months until, immediately following the introduction of prednisolone for his persistent GVHD, he relapsed. He declined further active treatment and died 5 months later. The case shows that IL-2/LAK cells can be safely given to patients who have experienced no GVHD following allo-BMT and are likely to be effective through an ongoing graft-versus-leukaemia effect.     

Amelioration of acute graft vs host disease due to minor histocompatibility antigens by in vivo administration of anti-interleukin 2 receptor antibody

Lethal acute graft vs host disease (GVHD) elicited by minor histocompatibility antigens was studied in a murine model of bone marrow transplantation (B10.BR----CBA). The severity of GVHD was reduced by both clinical and histologic parameters when transplant recipients received injections of a monoclonal antibody directed against the interleukin 2 receptor. This study suggests that anti-interleukin 2 receptor antibodies may be useful in clinical marrow transplantation and provides additional evidence that monoclonal antibodies that block T cell function in vitro may be of therapeutic value in vivo.     

Allogeneic bone marrow transplantation for leukaemia and aplastic anaemia. Results of the Leipzig BMT Group

In acute leukaemias there was a stable plateau in the survival curve at 45% after two years if grafted in first complete remission (n = 20) but only 13% of the patients are disease-free alive if grafted in a more advanced stage of the disease (n = 8). In 16 patients transplanted for chronic myeloid leukaemia the overall survival is 40%, in cases with graft-versus-host disease (GVHD) prevention by cyclosporine survival rate could be improved. Only 8 patients with severe aplastic anaemia, partially in low performance status were able to be transplanted; three died of infections, another by acute GVHD. The fatal complications in our study characterize the international well-known major problems in BMT: GVHD, interstitial pneumonitis, infections, graft failure in aplastic anaemia and recurrence of leukaemia, especially in more advanced leukaemia stage.     

Bone marrow transplantation: current results in leukemia

Bone marrow transplantation offers two potential therapeutic advantages over more conventional therapy of leukemia. It allows more intensive treatment to be given without regard to marrow toxicity and allows in the case of allogeneic marrow an additional immunotherapeutic effect through graft-versus-host disease (GVHD). Initially, allogeneic transplants in HLA matched sibling donors were only employed in end-stage patients. Although there were encouraging results in terms of long-term therapeutic effects, the overall mortality was prohibitive. Subsequently, patients were transplanted in remission with a marked improvement in overall survival in both acute lymphocytic leukemia and acute non-lymphocytic leukemia. The major obstacles to further improvement in the therapeutic effects of this procedure have been identified (i.e., GVHD, viral infection, and relapse in ALL) and are subject to intensive investigations that already show encouraging results. Syngeneic marrow transplantation is limited for obvious reasons, but early results have shown significant therapeutic effects, in particular, in chronic myelogenous leukemia. These results have encouraged others to use autologous bone marrow. Marrow contamination with unseen tumor cells is being approached by pharmacologic and immunologic techniques designed to "purge" marrow of tumor cells. Animal and initial clinical studies have been encouraging.     

Carboxyamidotriazole-orotate inhibits the growth of imatinib-resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis

The Bcr/Abl kinase has been targeted for the treatment of chronic myelogenous leukaemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML are often resistant. New therapeutic options are therefore needed for this fatal disease. Although more common in solid tumors, increased microvessel density was also reported in chronic myelogenous leukaemia and was associated with a significant increase of angiogenic factors, suggesting that vascularity in hematologic malignancies is a controlled process and may play a role in the leukaemogenic process thus representing an alternative therapeutic target. Carboxyamidotriazole-orotate (CTO) is the orotate salt form of carboxyamidotriazole (CAI), an orally bioavailable signal transduction inhibitor that in vitro has been shown to possess antileukaemic activities. CTO, which has a reduced toxicity, increased oral bioavailability and stronger efficacy when compared to the parental compound, was tested in this study for its ability to affect imatinib-resistant CML tumor growth in a xenograft model. The active cross talk between endothelial cells and leukemic cells in the bone marrow involving exosomes plays an important role in modulating the process of neovascularization in CML. We have thus investigated the effects of CTO on exosome-stimulated angiogenesis. Our results indicate that CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility for CTO in leukaemia patients.     

Advances in the treatment of acute graft‐versus‐host disease

Allogeneic hematopoietic stem cell transplantation (HSCT) has been widely used for the treatment of hematologic malignant and non‐malignant hematologic diseases and other diseases. However, acute graft‐versus‐host disease (GVHD) is a life‐threatening complication of allogeneic transplantation. Acute GVHD may occur in 30% of transplant recipients, which is a syndrome of erythematous skin eruption, cholestatic liver disease and intestinal dysfunction, resulting from the activation of donor T lymphocytes by host antigen‐presenting cells, resulting in an immune‐mediated inflammatory response. Recent scientific advances in the understanding of the pathogenesis involved in the development of acute GVHD and clinical investigation have provided more effective therapeutic strategies for acute GVHD. This review focuses on major scientific and clinical advances in the treatment of acute GVHD.     

Alterations of glycosphingolipid-based blood group antigen expression on erythrocytes and in plasma studied on consecutive samples after a blood group O to A bone marrow transplantation.

A blood group A1Le(a-b+) individual with chronic myeloid leukaemia had received a bone marrow graft from an HLA-identical OLe(a+b-) donor. Twelve months after bone marrow transplantation (BMT), the red blood cells of the patient became agglutinable with anti-A blood group reagents. To elucidate whether the blood group A antigen expression was of plasma or of bone marrow origin, total non-acid glycosphingolipid fractions were prepared from red blood cells and plasma collected 17 months after BMT, and from plasma collected 13, 15 and 19 weeks after BMT. The glycolipid fractions were analysed by thin-layer chromatography and immunostained with monoclonal A-antibodies, and permethylated and permethylated-reduced derivatives of selected plasma samples were analysed by mass spectrometry. The results strongly indicate the presence of host bone marrow-produced blood group A red blood cells. Furthermore, the presence of a blood group H active pentaglycosylceramide type 1 (H-5-1) (Table I), characteristic for an OLe(a-b-) secretor, was seen in plasma 3-4 weeks before clinical chronic graft versus host disease (GVHD). After treatment of chronic GVHD, this expression disappeared. The blood group ALeb (A-7-1) antigen produced by the recipient seems to be present and to increase with time in all plasma samples. This also seems to be the case for the Leb and A-6-1 antigens.     

Acute graft-versus-host disease after allogeneic bone marrow transplantation.

In 25 patients receiving allogeneic bone marrow transplants methotrexate was used to prevent acute graft-versus-host disease (GVHD). Acute GVHD, grades 2 to 4, developed in only 5 (20%) of the patients. The incidence of acute GVHD in other series of recipients of bone marrow transplants has ranged from 5% to 76%. A review of the literature suggests that this variation cannot be completely accounted for by age, type of disease treated by transplantation or type of GVHD prophylaxis. However, transfusion of allogeneic lymphocytes that have not been completely inactivated by irradiation (e.g., in platelet and granulocyte preparations) and inadequate isolation-decontamination procedures may increase the probability of GVHD following bone marrow transplantation.     

Complete Suppression of the Gut Microbiome Prevents Acute Graft-Versus-Host Disease following Allogeneic Bone Marrow Transplantation

The hypothesis that elimination of facultative and strict anaerobic microorganisms from the gastro-intestinal tract by antimicrobial drugs in the period of time around allogeneic bone marrow transplantation (BMT) prevents acute graft-versus-host disease (GVHD), was examined in a cohort of 112 children grafted between 1989 and 2002 for hematological malignancies. All patients received T-cell replete marrow from human leukocyte antigens (HLA) matched sibling donors under identical transplantation conditions. To eliminate microorganisms from the gastro-intestinal tract, total gastro-intestinal decontamination (GID) was applied by high doses of non-absorbable antimicrobial drugs while the graft recipient was maintained in strict protective isolation. About half of the children (51%) proved to be successfully decontaminated, and about half (49%) unsuccessfully. One recipient got acute GVHD in the first group and 8 in the second group (p = 0.013). The degree of success of total GID was decisive for the occurrence of acute GVHD, irrespective of the presence of other risk factors such as higher age of recipient and/or donor, female donor for male recipient and carriership or reactivation of herpesviruses. Our results demonstrate that successful total GID of the graft recipient prevents moderate to severe acute GVHD. We suppose that substantial translocation of gastro-intestinal microorganisms or parts of these, functioning as microbial-associated molecular patterns (MAMP''s), triggering macrophages/dendritic cells via pattern recognizing receptors (PRR''s) is prohibited. As a consequence the initiation and progression of an inflammatory process leading to acute GVHD is inhibited.     

Extracorporeal photopheresis (photochemotherapy) in the treatment of acute and chronic graft versus host disease: immunological mechanisms and the results from clinical studies

Extracorporeal photopheresis (ECP) is an immunomodulatory alternative for treatment of graft versus host disease (GVHD). The blood is then separated into its various components through apheresis; buffy coat cells are thereafter treated with 8-methoxypsoralen before exposure to ultraviolet light and finally reinfused into the patient. There is a general agreement that this treatment has an anti-GVHD effect, but the mechanisms of action behind this effect are only partly understood. However, altered maturation of dendritic cells (DC) and thereby indirect modulation of T-cell reactivity seems to be one important mechanism together with DC-presentation of antigens derived from apoptotic donor T cells and induction of regulatory T cells. The treatment has been best studied in patients with chronic GVHD (both pediatric and adult patients), but most studies are not randomized and it is difficult to know whether the treatment is more effective than the alternatives. The clinical studies of ECP in adults with acute GVHD are few and not randomized; it is not possible to judge whether this treatment should be a preferred second- or third-line treatment. There is no evidence for increased risk of leukemia relapse or suppression of specific graft versus leukemia reactivity by this treatment, so specific antileukemic immunotherapy may still be possible. Thus, even though the treatment seems effective in patients with GVHD, further clinical (especially randomized) as well as biological studies with careful standardization of the treatment are needed before it is possible to conclude how ECP should be used in acute and chronic GVHD.     

Evaluation of in vitro cytotoxic T lymphocyte assays as a predictive test for the occureence of graft vs host disease

The potential value of in vitro cytotoxic T lymphocyte (CTL) assays for predicting the occurrence of graft vs host disease (GVHD) following allogeneic bone marrow transplantation was evaluated in 12 mouse donor-host combinations associated with various degrees of GVHD. These donor-host combinations were selected after evaluation of GVHD triggered by minor histocompatibility antigens (MiHA) in 24 allogeneic strain combinations derived from six strains of H-2 ^b mice. Recipients (n=475), previously submitted to total body irradiation (9.5 Gy), were transplanted with 10⁷ bone marrow cells along with 5 x 10⁷ spleen cells. While lethal GVHD was observed in half of the strain combinations, it was possible to select 12 donor-host combinations characterized by severe, mild, or absent GVHD. When levels of anti-host CTL activity were assessed following in vivo priming and in vitro boosting, strong CTL-mediated cytotoxicity was observed in all combinations wheteer they developed GVHD or not. CTL frequency measured by limiting dilution analysis (LDA) ranged from 1/16880-1/306. The Spearman rank test revealed no positive correlation between GVHD intensity and donor anti-host CTL activity assayed either in bulk culture experiments or in LDA conditions. These results indicate that MiHA capable of triggering potent CTL responses in vitro do not necessarily initiate GVHD, and that in vitro measurement of donor CTL activity against host-type Con A blasts is not a predictive assay for anti-MiHA GVHD. However, the possibility to recruit CTL populations targeting host MiHA expressed specifically on hematopoietic cells suggests a novel therapeutic strategy for the cure of hematopoietic malignancies. Indeed, transplantation of donor hematopoietic stem cells supplemented with T cells aimed at MiHA specifically expressed by host hematopoietic cells, could possibly potentiate the desirable graft vs leukemia effect without increasing the risk of GVHD.     

Anti-recipient cytotoxic T lymphocyte precursors are present in the spleens of mice with acute graft versus host disease due to minor histocompatibility antigens

A model for bone marrow transplantation across minor histocompatibility barriers was developed by using mouse strains that were H-2 identical and mutually non-reactive in MLC. Acute graft-vs-host disease was induced only when donor lymphoid cells were included in the marrow inoculum, in both C57BL/6 recipients of LP cells and BALB/c recipients of B10.D2/nSN cells. GVHD was prevented by treating the lymphoid cells with anti-Thy 1.2 and C before transplantation. Spleen cells from mice with acute GVHD were not directly cytotoxic to recipient strain target cells. However, when spleen cells from mice with GVHD were boosted in vitro to recipient strain stimulator cells they generated a specific anti-recipient cytotoxic response. Spleen cells from mice without GVHD did not generate a cytotoxic response in vitro. The cytotoxic effector cells and their precursors were shown to be T lymphocytes. This model and the in vitro method described may be useful in further studies of the immunobiology of GVHD due to minor histocompatibility antigens and of transplantation tolerance.     

Lethal graft-vs-host disease across major histocompatibility barriers: requirement for leucyl-leucine methyl ester sensitive cytotoxic T cells

L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) is selectively toxic for human natural killer (NK) cells and cytotoxic T lymphocytes (CTL) at both the precursor and effector stage of differentiation. The present studies explored the effects of Leu-Leu-OMe on murine spleen cell function. Leu-Leu-OMe exposure removed NK function from murine spleen cells but spared their capacity to proliferate in response to lipopolysaccharide and Con A. The capacity to generate CTL from both L3T4 (+) and Lyt-2 (+) precursors was lost after Leu-Leu-OMe treatment, whereas alloantigen-induced proliferation and interleukin 2 (IL 2) production by L3T4 (+) T helper cells remained intact. Lethal graft vs host disease (GVHD), which developed in irradiated (C57BL/6 X DBA/2)F1 recipients of C57BL/6 bone marrow and spleen cells was completely prevented by Leu-Leu-OMe treatment of donor cells. In contrast depletion of Lyt-2 positive cells from the donor inoculum did not prevent acute GVHD in this fully major histo-compatibility complex (MHC) incompatible strain combination. However, Leu-Leu-OMe treatment of the Lyt-2 depleted inoculum completely prevented lethal GVHD, although the treated cells retained the capacity to proliferate and secrete IL 2 normally after in vitro stimulation with (C57BL/6 X DFA/2)F1 spleen cells. These findings indicate that L3T4 (+) T helper cells alone are unable to initiate lethal GVHD in this H-2 incompatible strain combination. Rather, lethal GVHD requires the transfer of a Leu-Leu-OMe sensitive T cell subset, likely to be thymus educated pre-CTL. Leu-Leu-OMe treatment should provide a useful way to delineate subpopulations of cells involved in the production of lethal GVHD and an approach to preventing this complication of bone marrow transplantation.     

异基因免疫治疗中控制GVHD发生的研究进展

目前,异基因造血干细胞移植(allo-HSCT)已成为治疗某些恶性血液病的有效手段之一,然而伴随其而来的移植物抗宿主病(GVHD)是导致移植后患者死亡的重要并发症之一。因此,如何诱导移植后免疫耐受来控制GVHD,尤其是控制急性移植物抗宿主病(a GVHD)的发生及已成为研究的重要内容。GVHD的发生机制非常复杂,但最终为供者骨髓内的成熟T淋巴细胞识别受者体内的细胞表面的MHC-I和MHC-II及所递呈的多肽而导致供者的T细胞的激活、增殖并浸润到GVHD的靶器官如皮肤、小肠及肝脏并导致靶器官的损伤[1]。临床移植学和移植免疫学主要攻克的内容之一就是如何控制GVHD的发生发展。其预防和治疗是决定着同种异基因造血干细胞移植(allo-HSCT)是否成功的关键所在,移植个体是否长期存活的主要因素之一。本文章就导致GVHD现象的原因及最新进展做一总结。     

IgG anti-tetanus toxoid antibody production induced by Epstein-Barr virus from B cells of human marrow transplant recipients

This investigation shows that Epstein-Barr virus (EBV)-activated human B cells from marrow transplant recipients can produce in vitro IgG anti-tetanus toxoid antibody (anti-TT) without booster immunizations with tetanus toxoid (TT). Purified B cells (E-rosette negative) from 8 normal subjects, 6 healthy long-term marrow graft recipients, and 15 long-term marrow graft recipients with chronic graft-vs-host disease (GVHD), were stimulated for 12 days with EBV to induce anti-TT production in culture supernatants. The amount of anti-TT in culture supernatants was quantitated using a enzyme-linked immunosorbent assay. B cells from all 8 normal controls produced in vitro IgG anti-TT after EBV stimulation. Five of 6 healthy recipients had B cells that produced anti-TT after EBV stimulation. Four of 15 recipients with chronic GVHD had B cells capable of producing anti-TT after EBV stimulation. The number of cultures making anti-TT responses was less in those with chronic GVHD than in those without chronic GVHD or normal individuals (P less than 0.001). B cells from patients with chronic GVHD had fewer responses exceeding the overall median of 0.7 ng/ml when compared with the other two groups (P less than 0.03). These data show that B cells of donor origin can produce in vitro IgG anti-TT antibody to tetanus toxoid antigen in a T-independent fashion.     

Cellular interactions in marrow-grafted patients. I. Impairment of cell-mediated lympholysis associated with graft-vs-host disease and the effect of interleukin 2

Forty patients with hematologic malignancy or aplastic anemia were given allogeneic marrow after conditioning with high-dose cyclophosphamide alone or in combination with total body irradiation. Between 28 and 3857 days after transplantation, their peripheral blood mononuclear leukocytes were tested for reactivity in indirect cell-mediated lympholysis against normal leukocytes from unrelated individuals, and the results were compared to those with cells from their healthy marrow donors. An impairment of cell-mediated lympholysis was found with cells from most patients with acute and chronic graft-vs-host disease (GVHD) whereas cells from most short-term and long-term patients without GVHD had cell-mediated lympholysis reactivity comparable to that of cells from the marrow donors. When interleukin 2 was added to the mixed leukocyte cultures during the sensitization phase, the impaired cell-mediated immunity of cells from most short-term patients with acute GVHD, but not that of cells from most patients with chronic GHVD, could be restored to normal levels. These results suggest the impairment of cell-mediated immunity seen in cells of short-term patients with acute GVHD is attributable to helper cell defects or to ineffective communication between antigen-presenting cells and helper T cells. The impairment in cell-mediated immunity seen in patients with chronic GVHD, however, may reside on the effector cells (or their precursors) or may be due to the presence of suppressor cell activity.     

A Thymic Hormone Protects Mice from Enteropathy during Acute Graft-versus-Host Disease

We have previously reported that a nonapeptide thymic hormone, facteur thymique serique (FTS), is involved in the differentiation and activation of intestinal intraepithelial lymphocytes (i-IEL) in mice. In this study, we examined the effect of FTS treatment on enteropathy in a murine model for acute graft-vs.-host disease (GVHD) induced by injection of parental C57BL/6 splenocytes into unirradiated (C57BL/6XDBA/2) F₁ hybrids. FTS treatment significantly protected mice from developing acute GVHD as assessed by mortality rate, splenomegaly and enteropathy. The infiltration of donor-derived TCRαβ i-IEL bearing CD8αβ was significantly inhibited in the small intestine of FTS-treated mice, and the frequencies of apoptosis of crypt cells in the intestinal mucosa were decreased in these mice during acute GVHD. These results suggest that FTS treatment contributes to protection against enteropathy of acute GVHD. Thus, FTS may provide a useful approach to control acute GVHD after blood transfusion or bone marrow transplantation.     

Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease.

Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by discontinuous Percoll density gradients. The fractionated low density (1.050-1.060 g/ml) C57BL/Ka spleen cells completely suppressed acute lethal graft vs host disease (GVHD) when coinjected with unfractionated C57BL/Ka spleen cells into sublethally irradiated (400 rad) BALB/c mice. In dose response experiments, as few as 0.5 x 10(6) low density cells from the spleen fractions suppressed acute GVHD induced by 2.5 x 10(6) unfractionated allogeneic spleen cells. Although the low density spleen fractions inhibited acute GVHD, the high density (1.075-1.090 g/ml) spleen fractions induced acute GVHD in sublethally irradiated BALB/c recipients. Fractionation of C57BL/Ka bone marrow cells showed that none of the high or low density fractions or unfractionated cells induced lethal GVHD. When these fractions were tested for their capacity to suppress GVHD by coinjection with C57BL/Ka unfractionated spleen cells, all fractions protected the BALB/c recipients. Unfractionated bone marrow cells showed modest protection. Evaluation of the dose response characteristics of the suppressive activity of the low and middle density (1.060-1.068 g/ml) bone marrow cell fraction showed that reproducible protection could be achieved at a 5:1 ratio of inducing to suppressing cells. The low density fractions of both bone marrow and spleen cells had a marked depletion of typical TCR(+)-alpha beta CD4+ or CD8+ T cells, and a predominant population of TCR(+)-alpha beta CD4- CD8- T cells. Purified populations of the latter cells suppressed GVHD. Recipients given unfractionated C57BL/Ka spleen cells and protected with low-density bone marrow or spleen cells were chimeras.     

Laminar air flow versus barrier nursing in marrow transplant recipients

Forty-eight patients with acute leukaemia in relapse (n = 14), acute leukaemia in complete remission (n = 19), chronic myeloid leukaemia (n = 8) or severe aplastic anaemia (n = 7) received a marrow transplant. The first 26 patients were nursed in laminar-air-flow plastic isolators while the next 22 patients were treated in barrier nursing rooms. Gnotobiotic parameters and morbidity in the 2 groups are compared. Good decontamination of the gastro-intestinal tract was obtained using either of the 2 isolation techniques. The incidence of bacterial and mycotic infections, as well as the supportive care required by the patients was almost equal in both groups. Our results also suggest that the incidence of graft versus host disease may decrease with efficient decontamination of the patients.     

Cell-mediated immunity to non-HLA antigens of the host by donor lymphocytes in patients with chronic graft-vs-host disease

Forty-four patients with aplastic anemia or leukemia were given marrow grafts from siblings selected on the basis of HLA-A and -B identity and mutual nonreactivity of their lymphocytes in mixed leukocyte culture (MLC). Twenty-two to 1089 days after grafting, their lymphocytes (of donor origin) were tested for reactivity in MLC to lymphocytes from the host (cryopreserved before grafting), the marrow donor, and unrelated individuals. Lymphocytes from 14 of 22 long-term survivors with chronic graft-vs-host disease (GVHD) showed unidirectional reactivity in response to host lymphocytes manifested as high stimulation indices (SI) and high relative responses (RR). Lymphocytes from only 1 of 12 long-term survivors without chronic GVHD showed unidirectional reactivity to host lymphocytes. Statistical analysis showed that lymphocytes from patients with chronic GVHD displayed anti-host responses that were significantly higher than those of lymphocytes from either marrow donors (p < 0.001) or patients without GVHD (p = 0.03). Lymphocytes from 5 patients with and 5 without acute GVHD, tested shortly after marrow grafting, failed to show responses to host cells. The results are consistent with a participation of cell-mediated immunity of graft against host in chronic GVHD.