Ultraviolet irradiation of blood prevents transfusion-induced sensitization and marrow graft rejection in dogs

In a canine model using DLA-identical littermate pairs, we have shown that a regimen of three transfusions of donor blood given 24, 17, and 10 days before transplant uniformly leads to marrow graft rejection, presumably due to sensitization to minor (non-DLA) histocompatibility antigens. Untransfused dogs uniformly achieve sustained engraftment. In the present study, we investigated whether the exposure of blood to ultraviolet (UV) light (220-300 nm) prior to transfusion prevented sensitization of the recipient and allowed for successful marrow engraftment. Ten dogs were each given three pretransplant transfusions from the marrow donor. Each transfusion consisted of 50 mL of whole blood exposed in vitro to UV light for a total of 1.35 J/cm2. All ten dogs achieved engraftment. In contrast, all four dogs that had received sham-exposed transfusions rejected their grafts. In vitro studies revealed that although cell viability was not affected, leukocytes contained in UV-exposed blood were unable to function as stimulator cells in mixed leukocyte cultures or as accessory cells in mitogen- stimulated cultures. These data are consistent with the hypothesis that accessory cells are involved in transfusion-induced sensitization. We conclude that in vitro exposure of blood to UV light before transfusion prevents sensitization and allows for subsequent marrow engraftment.     

Prevention of transfusion-associated graft-versus-host disease by photochemical treatment

Photochemical treatment (PCT) with the psoralen S-59 and long wavelength ultraviolet light (UVA) inactivates high titers of contaminating viruses, bacteria, and leukocytes in human platelet concentrates. The present study evaluated the efficacy of PCT to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in vivo using a well-characterized parent to F1 murine transfusion model. Recipient mice in four treatment groups were transfused with 10(8) splenic leukocytes. (1) Control group mice received syngeneic splenic leukocyte transfusions; (2) GVHD group mice received untreated allogeneic splenic leukocytes; (3) gamma radiation group mice received gamma irradiated (2,500 cGy) allogeneic splenic leukocytes; and (4) PCT group mice received allogeneic splenic leukocytes treated with 150 micromol/L S-59 and 2.1 J/cm2 UVA. Multiple biological and clinical parameters were used to monitor the development of TA-GVHD in recipient mice over a 10-week posttransfusion observation period: peripheral blood cell levels, spleen size, engraftment by donor T cells, thymic cellularity, clinical signs of TA-GVHD (weight loss, activity, posture, fur texture, skin integrity), and histologic lesions of liver, spleen, bone marrow, and skin. Mice in the control group remained healthy and free of detectable disease. Mice in the GVHD group developed clinical and histological lesions of TA-GVHD, including pancytopenia, marked splenomegaly, wasting, engraftment with donor derived T cells, and thymic hypoplasia. In contrast, mice transfused with splenic leukocytes treated with (2,500 cGy) gamma radiation or 150 micromol/L S-59 and 2.1 J/cm2 UVA remained healthy and did not develop detectable TA-GVHD. Using an in vitro T-cell proliferation assay, greater than 10(5.1) murine T cells were inactivated by PCT. Therefore, in addition to inactivating high levels of pathogenic viruses and bacteria in PC, these data indicate that PCT is an effective alternative to gamma irradiation for prevention of TA-GVHD.     

Prevention of graft-versus-host disease by induction of immune tolerance with ultraviolet B-irradiated leukocytes in H-2 disparate bone marrow donor

Transfusions (Tx) of Ultraviolet B (UVB)-irradiated peripheral blood mononuclear leukocytes (MNL) have been shown to induce humoral immune tolerance to major histocompatability complex (MHC) antigens (Blood 88:4375, 1996). To determine whether cellular immune tolerance to MHC antigens can be induced by the same approach, transplantation of bone marrow and spleen cells from tolerant donors across the H-2 barrier was conducted to study its effect on prevention of graft-versus-host disease (GVHD). After immune tolerance induction by four weekly Tx of UVB-irradiated BALB/c (H-2(d)) peripheral blood MNL into CBA/HT6 (H-2(k)) mice, bone marrow cells (BMC) and spleen MNL from tolerant or naive CBA mice were transplanted into lethally irradiated BALB/c mice. The transplanted mice were followed by measuring body weight, peripheral leukocyte counts, GVHD, survival, and cytokine response. All BALB/c recipient mice were fully engrafted with H-2(k) CBA donor cells after transplantation. The severity of GVHD was significantly attenuated in BALB/c mice transplanted with BMC and spleen MNL from tolerant CBA donor mice. The recovery of peripheral leukocyte and lymphocyte counts were faster and more complete in mice transplanted with cells from the tolerant donors. The serum cytokine profile after transplantation with tolerant donor cells showed increased interleukin-4 and reduced gamma interferon that are consistent with a polarized Th2 response. The results pooled from three separate experiments showed that BALB/c mice transplanted with 5 x 10(6) BMC and 4 x 10(5) spleen MNL from tolerant CBA donors had better overall survival than the control group (72% v 17%, P =.018). The findings show that transplantation with bone marrow and spleen cells from tolerant H-2 disparate donor mice is associated with significant attenuation of GVHD and better outcomes. The results also support that transfusions of UVB-irradiated leukocytes may induce cellular immune tolerance.     

Prevention of graft-versus-host disease by anti IL-7Ralpha antibody

Graft-versus-host disease (GVHD) continues to be a serious complication that limits the success of allogeneic bone marrow transplantation (BMT). Using IL-7-deficient murine models, we have previously shown that IL-7 is necessary for the pathogenesis of GVHD. In the present study, we determined whether GVHD could be prevented by antibody-mediated blockade of IL-7 receptor alpha (IL-7Ralpha) signaling. C57/BL6 (H2K(b)) recipient mice were lethally irradiated and underwent cotransplantation with T-cell-depleted (TCD) BM and lymph node (LN) cells from allogeneic BALB/c (H2K(d)) donor mice. Following transplantation, the allogeneic BMT recipients were injected weekly with either anti-IL-7Ralpha antibody (100 mug per mouse per week) or PBS for 4 weeks. Anti-IL-7Ralpha antibody treatment significantly decreased GVHD-related morbidity and mortality compared with placebo (30% to 80%). IL-7Ralpha blockade resulted in the reduction of donor CD4(+) or CD8(+) T cells in the periphery by day 30 after transplantation. Paradoxically, the inhibition of GVHD by anti-IL-7Ralpha antibody treatment resulted in improved long-term thymic and immune function. Blockade of IL-7R by anti-IL-7Ralpha antibody resulted in elimination of alloreactive T cells, prevention of GVHD, and improvement of donor T-cell reconstitution.     

Treatment of acute graft-versus-host disease with PUVA (psoralen and ultraviolet irradiation): results of a pilot study

Acute graft-versus-host disease (aGVHD) is a frequent and major complication after allogeneic stem cell transplantation. For many years psoralen and ultraviolet (UV)-A light have been used in the treatment of chronic cutaneous graft-versus-host disease, but few patients have received PUVA therapy for aGVHD. We assessed 20 patients who received PUVA therapy for acute cutaneous GVHD (grade 2-4). Seven patients showed additional organ manifestations (liver, gut). To better quantify the cutaneous lesions, a new scoring system was introduced: intensity of erythema (0-3) x %body surface + size of bullae (4-5) x %body surface affected. All patients received prednisolone and PUVA for treatment of aGVHD. Fifteen patients (75%), 12 with manifestations restricted to the skin, responded by score classification (average time to a 50% score reduction: 39 days) and reduction of the dosage of prednisolone (average time to a 50% prednisolone reduction: 35 days). PUVA treatment was well tolerated and might play a role in the therapy of acute cutaneous GVHD.     

Prevention of graft-versus-host disease by immunosuppressive agents after transplantation of DLA-nonidentical canine marrow

Dogs given 9.2 Gy of total body irradiation (TBI) followed by hematopoietic grafts from DLA-nonidentical unrelated or littermate donors and no postgrafting immunosuppression all develop acute graft-versus host disease (GVHD) and die within 3 weeks of grafting. Over a period of 10 years, the present study evaluated various immunosuppressive drugs either alone or in combination and compared their effectiveness to that of methotrexate (MTX) in preventing acute GVHD and prolonging survival. Among the single agents tested, 6-mercaptopurine and azathioprine resulted in a slight but significant prolongation of survival compared to controls, but they were less effective than MTX. Procarbazine and cytosine arabinoside were ineffective. Drugs tested in combination included MTX, cyclophosphamide, 6-mercaptopurine, azathioprine, cyclosporin, procarbazine, prednisone and antithymocyte globulin. Drug combinations, while effective in delaying the onset of GVHD, were associated with hematopoietic and other toxicities, which made their use impractical. Administration of MTX twice weekly proved to be no better than once weekly administration. Prednisone administered for the first 4 days after grafting in addition to MTX resulted in a modest improvement in survival. High-dose MTX (200 mg/kg) followed by leukovorin rescue resulted in severe early toxicity, both gastrointestinal and hematopoietic; however, some of those dogs which survived the early postgrafting period became stable long-term survivors. Most single agents tested in the present study proved to be either ineffective or were inferior to MTX in their ability to prevent GVHD. None of the drug combinations tested was as effective as the one involving MTX/cyclosporin reported previously.     

Prevention of acute graft-versus-host disease by blocking T-cell entry to secondary lymphoid organs

In acute graft-versus-host disease (aGVHD), donor T cells attack the recipient's gastrointestinal tract, liver, and skin. We hypothesized that blocking access to distinct lymphoid priming sites may alter the specific organ tropism and prevent aGVHD development. In support of this initial hypothesis, we found that different secondary lymphoid organs (SLOs) imprint distinct homing receptor phenotypes on evolving alloreactive effector T cells in vivo. Yet preventing T-cell entry to specific SLOs through blocking monoclonal antibodies, or SLO ablation, did not alter aGVHD pathophysiology. Moreover, transfer of alloreactive effector T cells into conditioned secondary recipients targeted the intestines and liver, irrespective of their initial priming site. Thus, we demonstrate redundancy of SLOs at different anatomical sites in aGVHD initiation. Only prevention of T-cell entry to all SLOs could completely abrogate the onset of aGVHD.     

Prevention of the acute graft-versus-host disease (GVHD) in rats by the immunomodulating drug leflunomide

Summary The grafting of immunocompetent allogeneic cells into MHC-discordant, genetically nonresponsive F₁ hybrids of inbred rat strains consistently leads to an acute, lethal graft-versus-host disease (GVHD). The novel immunomodulating drug leflunomide, which has been shown to be efficacious in animal models of autoimmunity and adverse transplantation reactions, was studied in a rat model of GVHD. It was found that this drug not only was a powerful agent to prevent this otherwise terminal disorder, but was also proficient when used as a therapy of an established GVHD. Since leflunomide has been shown to be efficacious and safe in patients with chronic rheumatoid arthritis, it would also be reasonable to investigate this drug in clinical trials for bone marrow transplantation and GVHD in human beings.This work was supported by the Wilhelm Sander-Stiftung Neuburg a. d. Donau, Number 90.059.1     

Ultraviolet irradiation modulates MHC-alloreactive cytotoxic T-cell precursors involved in the onset of graft-versus-host disease

Ultraviolet B (UVB) irradiation of cellular blood components has been proposed as a new technology to prevent HLA sensitization in recipients. Earlier studies have shown that a dose of 2 J/cm2 abrogates the ability of lymphocytes to serve as stimulators in mixed lymphocyte cultures (MLC). In this study we have evaluated the effect of UV energy on T-lymphocytes for the prevention of transfusion-associated graft-versus-host disease (TA-GvHD). The response of cytotoxic T-lymphocyte precursors against host alloantigens was almost undetectable at a dose of 0.5 J/cm2. T-cell proliferation in MLC or in response to phytohaemagglutinin was inhibited by more than 95% at doses of 1 J/cm2 or higher. The data suggest that UV irradiation can be used to prevent both HLA sensitization and TA-GvHD in recipients.     

Recipient CD4+ T cells that survive irradiation regulate chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is an increasingly common cause of morbidity and mortality in allogeneic stem cell transplantation (alloSCT). Relative to acute GVHD (aGVHD), much less is understood about cGVHD. Using the B10.D2 --> BALB/c murine cGVHD model, which shares critical pathologic features with human cGVHD, we find that radiation-resistant host T cells regulate cGVHD. We initially observed that recipients lacking all lymphocytes developed accelerated and more severe cGVHD. Using genetically deficient recipients, we determined that alphabeta+CD4+ T cells were required to regulate cGVHD. Increased cGVHD severity was not due to the absence of T cells per se. Rather, the potency of regulation was proportional to host T-cell receptor (TCR) diversity. Only CD4+CD25+, and not CD4+CD25-, host T cells ameliorated cGVHD when added back, indicating that host T cells acted not via host-versus-graft activity or by reducing homeostatic proliferation but by an undefined regulatory mechanism. Thus, preparative regimens that spare host CD4+CD25+ T cells may reduce cGVHD. Donor CD4+CD25+ T cells also reduced cGVHD. Depletion of CD4+CD25+ cells from the inoculum exacerbated disease, whereas transplantation of additional CD4+CD25+ cells protected against severe cGVHD. Additional CD4+CD25+ cells also promoted healing of established lesions, suggesting that their effects persist during the evolution of cGVHD.