Enhanced allostimulatory activity of host antigen-presenting cells in old mice intensifies acute graft-versus-host disease

Older bone marrow transplantation (BMT) recipients are at heightened risk for acute graft-versus-host disease (GVHD) after allogeneic BMT, but the causes of this association are poorly understood. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older mice. GVHD mortality, morbidity, and pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice, which also secreted more TNF-alpha and IL-12 after LPS stimulation. In a B6 --> B6D2F1 model, CD4(+) donor T cells but not CD8(+) T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using B6D2F1 BM chimeras created with either old or young BM. Four months after chimera creation, allogeneic BMT from B6 donors caused significantly worse GVHD in old BM chimeras. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. These data demonstrate a hitherto unsuspected mechanism of amplified donor T cell responses by aged allogeneic host APCs that increases acute GVHD in aged recipients in this BMT model.     

复制缺陷性慢病毒介导的双自杀基因在小鼠异基因骨髓移植中的应用

目的　探讨复制缺陷性慢病毒介导的胞嘧啶脱氨酶 (cytosinedeaminase,CD)和胸苷激酶(thymidinekinase ,TK)双自杀基因在异基因骨髓移植 (allo BMT)时控制移植物抗宿主病 (GVHD)的可行性及其有效性。方法　将携带双自杀基因的复制缺陷性慢病毒通过脂质体转染入T细胞中。将经6 0　Coγ射线照射后的荷瘤小鼠分组进行骨髓移植 ,观察骨髓移植后荷瘤鼠CFU S、CFU GM、Y染色体整合、T细胞中CD TK基因整合、生存率和生存期、体重及病理组织学等指标。结果　复制缺陷性慢病毒载体可将双自杀基因高效稳定转染入T细胞。在allo BMT中 ,除空白对照组外 ,各组小鼠的CFU S的数目和CFU GM产率差异无显著性 ,且均有Y染色体整合。使用前体药物治疗的小鼠未发生明显的GVHD ,其生存率和生存期明显高于其它各组 ;双自杀基因的效果优于单自杀基因。结论　复制缺陷性慢病毒介导的双自杀基因系统可在不影响移植物存活的情况下有效控制GVHD的发生 ,显著提高allo BMT的成功率。     

Reduced graft-versus-host disease-inducing capacity of T cells after activation, culturing, and magnetic cell sorting selection in an allogeneic bone marrow transplantation model in rats.

Graft-versus-host disease (GvHD), a major complication of allogeneic bone marrow transplantation, has been ascribed to mature T cells in the graft. Because T cells play an important role in engraftment of the bone marrow and decrease the probability of relapse of leukemia, a treatment strategy was developed to preserve the benefits of T cells in the graft and to control the severe complications of GvHD. This can be accomplished by the genetic modification of donor T cells with a suicide gene that allows their selective in vivo elimination and subsequently the abrogation of GvHD. For clinical benefit the alloreactivity of herpes simplex virus thymidine kinase (HSV-TK) gene-transduced T cells should be retained. Therefore, we investigated the influence of gene transduction and the selection procedure on T cells. We demonstrated that activation and culturing of T cells reduce their capacity to induce lethal GvHD in an allogeneic rat bone marrow transplantation model. Furthermore, positive immunomagnetic selection of gene-transduced T cells resulted in loss of the GvHD-inducing capacity of HSV-TK(+) T cells directly after MACS (magnetic cell sorting) selection; this loss could be recovered by a 1-day expansion of the selected T cells. No effect on alloreactivity was observed to be caused by the gene transduction procedure. Our study resulted in the development of an optimized culture and gene transduction protocol with preservation of T cell alloreactivity. Treatment of transplanted rats with ganciclovir resulted in a rapid reduction in the number of HSV-TK(+) T cells in the peripheral blood and in increased survival of the animals.     

PKCθ is required for alloreactivity and GVHD but not for immune responses toward leukemia and infection in mice

When used as therapy for hematopoietic malignancies, allogeneic BM transplantation (BMT) relies on the graft-versus-leukemia (GVL) effect to eradicate residual tumor cells through immunologic mechanisms. However, graft-versus-host disease (GVHD), which is initiated by alloreactive donor T cells that recognize mismatched major and/or minor histocompatibility antigens and cause severe damage to hematopoietic and epithelial tissues, is a potentially lethal complication of allogeneic BMT. To enhance the therapeutic potential of BMT, we sought to find therapeutic targets that could inhibit GVHD while preserving GVL and immune responses to infectious agents. We show here that T cell responses triggered in mice by either Listeria monocytogenes or administration of antigen and adjuvant were relatively well preserved in the absence of PKC isoform θ (PKCθ), a key regulator of TCR signaling. In contrast, PKCθ was required for alloreactivity and GVHD induction. Furthermore, absence of PKCθ raised the threshold for T cell activation, which selectively affected alloresponses. Most importantly, PKCθ-deficient T cells retained the ability to respond to virus infection and to induce GVL effect after BMT. These findings suggest PKCθ is a potentially unique therapeutic target required for GVHD induction but not for GVL or protective responses to infectious agents.     

IL-11 separates graft-versus-leukemia effects from graft-versus-host disease after bone marrow transplantation

We recently showed that IL-11 prevents lethal graft-versus-host disease (GVHD) in a murine bone marrow transplantation (BMT) model of GVHD directed against MHC and minor antigens. In this study, we have investigated whether IL-11 can maintain a graft-versus-leukemia (GVL) effect. Lethally irradiated B6D2F1 mice were transplanted with either T cell-depleted (TCD) bone marrow (BM) alone or with BM and splenic T cells from allogeneic B6 donors. Animals also received host-type P815 mastocytoma cells at the time of BMT. Recipients were injected subcutaneously with recombinant human IL-11 or control diluent twice daily, from 2 days before BMT to 7 days after BMT. TCD recipients all died from leukemia by day 23. All control- and IL-11-treated allogeneic animals effectively rejected their leukemia, but IL-11 also reduced GVHD-related mortality. Examination of the cellular mechanisms of GVL and GVHD in this system showed that IL-11 selectively inhibited CD4-mediated GVHD, while retaining both CD4- and CD8-mediated GVL. In addition, IL-11 treatment did not affect cytolytic effector functions of T cells after BMT either in vivo or in vitro. Studies with perforin-deficient donor T cells demonstrated that the GVL effect was perforin dependent. These data demonstrated that IL-11 can significantly reduce CD4-dependent GVHD without impairing cytolytic function or subsequent GVL activity of CD8(+) T cells. Brief treatment with IL-11 shortly after BMT may therefore represent a novel strategy for separating GVHD and GVL.     

GITR activation induces an opposite effect on alloreactive CD4(+) and CD8(+) T cells in graft-versus-host disease

Glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) is a member of the tumor necrosis factor receptor (TNFR) family that is expressed at low levels on unstimulated T cells, B cells, and macrophages. Upon activation, CD4(+) and CD8(+) T cells up-regulate GITR expression, whereas immunoregulatory T cells constitutively express high levels of GITR. Here, we show that GITR may regulate alloreactive responses during graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). Using a BMT model with major histocompatibility complex class I and class II disparity, we demonstrate that GITR stimulation in vitro and in vivo enhances alloreactive CD8(+)CD25(-) T cell proliferation, whereas it decreases alloreactive CD4(+)CD25(-) proliferation. Allo-stimulated CD4(+)CD25(-) cells show increased apoptosis upon GITR stimulation that is dependent on the Fas-FasL pathway. Recipients of an allograft containing CD8(+)CD25(-) donor T cells had increased GVHD morbidity and mortality in the presence of GITR-activating antibody (Ab). Conversely, recipients of an allograft with CD4(+)CD25(-) T cells showed a significant decrease in GVHD when treated with a GITR-activating Ab. Our findings indicate that GITR has opposite effects on the regulation of alloreactive CD4(+) and CD8(+) T cells.     

CD4(+)CD25(+) immunoregulatory T Cells: new therapeutics for graft-versus-host disease

CD4(+)CD25(+) immunoregulatory T cells play a pivotal role in preventing organ-specific autoimmune diseases and in tolerance induction to allogeneic organ transplants. We investigated whether these cells could also control graft-versus-host disease (GVHD), the main complication after allogeneic hematopoietic stem cell transplantation (HSCT). Here, we show that the few CD4(+)CD25(+) T cells naturally present in the transplant regulate GVHD because their removal from the graft dramatically accelerates this disease. Furthermore, the addition of freshly isolated CD4(+)CD25(+) T cells at time of grafting significantly delays or even prevents GVHD. Ex vivo-expanded CD4(+)CD25(+) regulatory T cells obtained after stimulation by allogeneic recipient-type antigen-presenting cells can also modulate GVHD. Thus, CD4(+)CD25(+) regulatory T cells represent a new therapeutic tool for controlling GVHD in allogeneic HSCT. More generally, these results outline the tremendous potential of regulatory T cells as therapeutics.     

Increased risk of lethal graft-versus-host disease-like syndrome after transplantation into NOD/SCID mice of human mobilized peripheral blood stem cells, as compared to bone marrow or cord blood

We tested the ability of human cells from different hematopoietic tissues to generate graft versus host disease-like syndrome (GVHD) in sublethally irradiated non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Tissue sources of human hematopoietic cells were: (1) bone marrow (BM), (2) nonmobilized peripheral blood (PB), (3) mobilized peripheral blood stem-progenitor cells (PBSC), and (4) cord blood (CB). To avoid interindividual donor variation, part of this study was done using BM, PB, and PBSC donated by a single healthy adult volunteer. A total of 179 NOD/SCID mice received graded human hematopoietic cell doses [5-500 x 10(6) mononuclear cells (MNC), containing 2-325 x 10(6) CD3(+) T cells, per mouse] from individual donors. Mice were observed for the development of GVHD and sacrificed 60 days after transplantation (earlier if ill). Mice were analyzed quantitatively by flow cytometry for human hematopoietic cell types and histologically, especially for human T lymphocytes infiltrating BM. No mouse transplanted with the tested doses of human CB or BM cells developed GVHD (experimentally defined as >10% human T lymphocytes infiltrating the mouse BM). For PB and PBSC, the frequencies of death, death with GVHD, and GVHD were directly related to the dose and source of human cells. Because PB cells contaminate harvested BM, the results from infused BM and PB were next combined for further analysis (BM/PB). The relative risks (hazard ratios estimated from the proportional hazards model) for death with GVHD, for each 10 human T cell dose increase, were 1.15 for BM/PB (p < 0.0001) and 1.47 for PBSC (p < 0.0001). In this in vivo xenogeneic model, the average T cell from human PBSC generated GVHD more potently than did the average T cell from human BM/PB, and the average CB T cell had a much lower GVHD potential. These results suggest that the potential for clinical GVHD from an HLA-disparate donor graft is likely to be quantitatively dependent both on the total number of T lymphocytes in the donor graft and the tissue source of the graft. Quantitative criteria for optimal T cell content of allogeneic donor hematopoietic grafts from different sources are discussed.     

人骨髓间充质干细胞体外对异基因T淋巴细胞表型的影响

为了研究间充质干细胞 (mesenchymalstemcell,MSC)的免疫调节作用 ,探讨防治异基因骨髓移植中移植物抗宿主病 (GVHD)和移植排斥反应 (HVGR)的可能途径 ,从人骨髓中分离培养间充质干细胞 ,并通过其形态的均一性及流式细胞术检测表面标志以鉴定其纯度 ,将MSC分别以 8× 10 4 (A组 )、4× 10 4 (B组 )和 2× 10 4 (C组 )个细胞 孔分别接种于 6孔板 ,并与经分离的外周血T淋巴细胞共培养 7天 ,以单独培养的外周血T淋巴细胞为对照组 ,用流式细胞仪分别在培养 0、2 4、72小时和 7天测定各组T细胞上CD3、CD4、CD8、CD2 5表达的变化。结果表明 :T细胞和MSC共培养组与T细胞单独培养组相比 ,A组和B组CD4 CD2 5 免疫调节性T细胞和CD8 T细胞明显增多 ,A组和B组之间无明显差别。实验组和对照组相比CD3、CD4、CD2 5表达无明显差别。结论 :骨髓MSCs在体外可使外周血T细胞表型发生改变 ,CD4 CD2 5 免疫调节性T细胞和CD8 T细胞明显增多 ,本研究结果为临床异基因造血干细胞移植预防GVHD时MSCs的输注数量提供了参考。     

Donor-derived interferon gamma is required for inhibition of acute graft-versus-host disease by interleukin 12.

We have demonstrated that a single injection of interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits acute graft-versus-host disease (GVHD) in mice. This effect of IL-12 can be diminished by anti-interferon (IFN)-gamma mAb. To determine the mechanism by which IFN-gamma affects IL-12-mediated GVHD protection, we have compared the effect of IL-12 on GVHD in C57BL/6 wild-type (WT) or IFN-gamma gene knockout (GKO) recipients of fully major histocompatibility complex plus minor antigen-mismatched allogeneic BMT from WT or GKO BALB/c mice. Lethal acute GVHD was readily induced in the absence of IFN-gamma. IL-12 inhibited GVHD mortality to a similar extent in WT and GKO recipients of WT allogeneic BMT. However, neither WT nor GKO recipients were protected by IL-12 from GVHD induced by GKO allogeneic BMT. Moreover, the effective inhibition of host-reactive donor T cell activation and expansion that is associated with IL-12-mediated GVHD protection was dependent on the ability of BALB/c donors to produce IFN-gamma. These results demonstrate that (a) acute GVHD can be induced in the absence of IFN-gamma, (b) host IFN-gamma does not play a critical role in IL-12-induced GVHD protection, and (c) the protective effect of IL-12 against GVHD is dependent on the ability of the donor to produce IFN-gamma.     

免疫磁珠选择性清除人异基因反应性T细胞的实验研究

本研究用磁性细胞分离系统清除CD25^＋和CD69^＋人同种异体反应性T细胞，为减轻异基因骨髓移植后的移植物抗宿主病探索新的手段、对健康供者外周血单个核细胞与白血病缓解病人的骨髓单个核细胞进行单向混合淋巴细胞培养。3天后，通过磁性细胞分离系统清除CD25^＋和CD69^＋的同种异体反应性T细胞；通过^3H-TdR掺入实验，观察处理后的供者外周血单个核细胞对同一病人的骨髓单个核细胞及无关者外周血单个核细胞的反应性．结果表明：与未清除组相比，清除后供者细胞对同一病人抗原的反应性降低50％，但保留了大部分对无关者抗原的反应性。结论：在体外反应中清除同种异体反应性T细胞可降低对刺激抗原反应，同时保留大部分对无关者抗原的反应。     

Bone marrow NK1.1(-) and NK1.1(+) T cells reciprocally regulate acute graft versus host disease

Sorted CD4(+) and CD8(+) T cells from the peripheral blood or bone marrow of donor C57BL/6 (H-2(b)) mice were tested for their capacity to induce graft-versus-host disease (GVHD) by injecting the cells, along with stringently T cell-depleted donor marrow cells, into lethally irradiated BALB/c (H-2(d)) host mice. The peripheral blood T cells were at least 30 times more potent than the marrow T cells in inducing lethal GVHD. As NK1.1(+) T cells represented <1% of all T cells in the blood and approximately 30% of T cells in the marrow, the capacity of sorted marrow NK1.1(-) CD4(+) and CD8(+) T cells to induce GVHD was tested. The latter cells had markedly increased potency, and adding back marrow NK1.1(+) T cells suppressed GVHD. The marrow NK1.1(+) T cells secreted high levels of both interferon gamma (IFN-gamma) and interleukin 4 (IL-4), and the NK1.1(-) T cells secreted high levels of IFN-gamma with little IL-4. Marrow NK1.1(+) T cells obtained from IL-4(-/-) rather than wild-type C57BL/6 donors not only failed to prevent GVHD but actually increased its severity. Together, these results demonstrate that GVHD is reciprocally regulated by the NK1.1(-) and NK1.1(+) T cell subsets via their differential production of cytokines.     

Donor T cell DNMT3a regulates alloreactivity in mouse models of hematopoietic stem cell transplantation

DNA methyltransferase 3a (DNMT3a) is an important part of the epigenetic machinery that stabilizes patterns of activated T cell responses. We hypothesized that donor T cell DNMT3a regulates alloreactivity after allogeneic blood and marrow transplantation (allo-BMT). T cell conditional Dnmt3a KO mice were used as donors in allo-BMT models. Mice receiving allo-BMT from KO donors developed severe acute graft-versus-host disease (aGVHD), with increases in inflammatory cytokine levels and organ histopathology scores. KO T cells migrated and proliferated in secondary lymphoid organs earlier and demonstrated an advantage in trafficking to the small intestine. Donor T cell subsets were purified after BMT for whole-genome bisulfite sequencing (WGBS) and RNA-Seq. KO T cells had global methylation similar to that of WT cells, with distinct, localized areas of hypomethylation. Using a highly sensitive computational method, we produced a comprehensive profile of the altered epigenome landscape. Hypomethylation corresponded with changes in gene expression in several pathways of T cell signaling and differentiation. Additionally, Dnmt3a-KO T cells resulted in superior graft-versus-tumor activity. Our findings demonstrate a critical role for DNMT3a in regulating T cell alloreactivity and reveal pathways that control T cell tolerance. These results also provide a platform for deciphering clinical data that associate donor DNMT3a mutations with increased GVHD, decreased relapse, and improved survival.     

In Vivo Selection of CD4(+) T Cells Transduced with a Gamma-Retroviral Vector Expressing a Single-Chain Intrabody Targeting HIV-1 Tat

Abstract We evaluated the potential of an anti-human immunodeficiency virus (HIV) Tat intrabody (intracellular antibody) to promote the survival of CD4(+) cells after chimeric simian immunodeficiency virus (SIV)/HIV (SHIV) infection in rhesus macaques. Following optimization of stimulation and transduction conditions, purified CD4(+) T cells were transduced with GaLV-pseudotyped retroviral vectors expressing either an anti-HIV-1 Tat or a control single-chain intrabody. Ex vivo intrabody-gene marking was highly efficient, averaging four copies per CD4(+) cell. Upon reinfusion of engineered autologous CD4(+) cells into two macaques, high levels of gene marking (peak of 0.6% and 6.8% of peripheral blood mononuclear cells (PBMCs) and 0.3% or 2.2% of the lymph node cells) were detected in vivo. One week post cell infusion, animals were challenged with SHIV 89.6p and the ability of the anti-HIV Tat intrabody to promote cell survival was evaluated. The frequency of genetically modified CD4(+) T cells progressively decreased, concurrent with loss of CD4(+) cells and elevated viral loads in both animals. However, CD4(+) T cells expressing the therapeutic anti-Tat intrabody exhibited a relative survival advantage over an 8- and 21-week period compared with CD4(+) cells expressing a control intrabody. In one animal, this survival benefit of anti-Tat transduced cells was associated with a reduction in viral load. Overall, these results indicate that a retrovirus-mediated anti-Tat intrabody provided significant levels of gene marking in PBMCs and peripheral tissues and increased relative survival of transduced cells in vivo.     

Effect of combined cytostatic cyclosporin A and cytolytic suicide gene therapy on the prevention of experimental graft-versus-host disease

The immunosuppressive drug cyclosporin A (CsA) represents the standard preventive treatment of graft-versus-host disease (GVHD), the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). However, its efficacy is only partial and many patients develop lethal GVHD despite CsA. A strategy of genetic immunosuppression based on conditional elimination of donor T cells expressing the Herpes simplex type 1 thymidine kinase (TK) suicide gene was recently developed. In this system, ganciclovir (GCV) selectively kills dividing but not quiescent TK T cells. Since CsA is known to have a cytostatic effect on T cells, it could negatively interfere with the division-dependent TK gene therapy. We thus tested whether administration of CsA would antagonize elimination of alloreactive donor TK T cells mediated by GCV in a murine model of GVHD. In vivo experiments revealed that, contrary to GCV, CsA only transiently controlled alloactivation-induced T cell proliferation, and likewise could not prevent lethal GVHD. When T cells resumed proliferation under CsA, they were however still sensitive to GCV. Survival, as well as immune reconstitution, was excellent in mice treated with GCV alone or in combination with CsA. These observations should help to design improved suicide gene therapy trials in the field of allogeneic HSCT.     

Differential roles of IL-1 and TNF-α on graft-versus-host disease and graft versus leukemia

We demonstrate an increase in graft-versus-host disease (GVHD) after experimental bone marrow transplant (BMT) when cyclophosphamide (Cy) is added to an otherwise well-tolerated dose (900 cGy) of total body irradiation (TBI). Donor T cell expansion on day +13 was increased after conditioning with Cy/TBI compared with Cy or TBI alone, although cytotoxic T lymphocyte (CTL) function was not altered. Histological analysis of the gastrointestinal tract demonstrated synergistic damage by Cy/TBI and allogeneic donor cells, which permitted increased translocation of LPS into the systemic circulation. TNF-alpha and IL-1 production in response to LPS was increased in BMT recipients after Cy/TBI conditioning. Neutralization of IL-1 significantly reduced serum LPS levels and GVHD mortality, but it did not affect donor CTL activity. By contrast, neutralization of TNF-alpha did not prevent GVHD mortality but did impair CTL activity after BMT. When P815 leukemia cells were added to the bone marrow inoculum, allogeneic BMT recipients given the TNF-alpha inhibitor relapsed at a significantly faster rate than those given the IL-1 inhibitor. To confirm that the role of TNF-alpha in graft versus leukemia (GVL) was due to effects on donor T cells, cohorts of animals were transplanted with T cells from either wild-type mice or p55 TNF-alpha receptor-deficient mice. Recipients of TNF-alpha p55 receptor-deficient T cells demonstrated a significant impairment in donor CTL activity after BMT and an increased rate of leukemic relapse compared with recipients of wild-type T cells. These data highlight the importance of conditioning in GVHD pathophysiology, and demonstrate that TNF-alpha is critical to GVL mediated by donor T cells, whereas IL-1 is not.     

供鼠的活化自然杀伤细胞对小鼠异基因骨髓移植的影响

目的　研究供鼠的活化自然杀伤细胞（ Ｎ Ｋ 细胞） 对小鼠异基因骨髓移植的影响。方法　将荷瘤鼠照射后,于输注骨髓细胞（ 含 Ｔ细胞） 之前４ 小时或之后７２ 小时,注射供鼠的活化 Ｎ Ｋ 细胞,观察荷瘤鼠 Ｃ Ｆ Ｕ Ｓ、 Ｃ Ｆ Ｕ Ｇ Ｍ、生存期、体重、病理组织学和双肺瘤结节等指标。结果　输注供鼠的活化 Ｎ Ｋ细胞能显著促进荷瘤鼠 Ｃ Ｆ Ｕ Ｓ和 Ｃ Ｆ Ｕ Ｇ Ｍ 增殖。输注骨髓细胞前４ 小时输注供鼠的活化 Ｎ Ｋ 细胞能显著提高荷瘤鼠生存率,减轻移植物抗宿主病（ Ｇ Ｖ Ｈ Ｄ） 的病理损伤程度,并使肺瘤结节数量减少;而输注骨髓细胞后７２ 小时输注供鼠的活化 Ｎ Ｋ 细胞能显著加重 Ｇ Ｖ Ｈ Ｄ 病理损伤程度。结论　①在小鼠异基因骨髓移植的初始阶段,输注供鼠的活化 Ｎ Ｋ 细胞能促进造血重建,预防 Ｇ Ｖ Ｈ Ｄ 并同时增强移植物抗肿瘤（ Ｇ Ｖ Ｔ） 效应;输注骨髓细胞之后,输注供鼠的活化 Ｎ Ｋ 细胞则起到加重 Ｇ Ｖ Ｈ Ｄ 的不良作用;② Ｇ Ｖ Ｔ和 Ｇ Ｖ Ｈ Ｄ 是能够被分开的。     

LPS antagonism reduces graft-versus-host disease and preserves graft-versus-leukemia activity after experimental bone marrow transplantation.

Acute graft-versus-host disease (GVHD) and leukemic relapse remain the two major obstacles to successful outcomes after allogeneic bone marrow transplantation (BMT). Recent studies have demonstrated that the loss of gastrointestinal tract integrity, and specifically the translocation of LPS into the systemic circulation, is critical to the induction of cytokine dysregulation that contributes to GVHD. Using a mouse BMT model, we studied the effects of direct LPS antagonism on GVHD severity and graft-versus-leukemia (GVL) activity. Administration of B975, a synthetic lipid-A analogue from day 0 to day +6, reduced serum TNF-alpha levels, decreased intestinal histopathology, and resulted in significantly improved survival and a reduction in clinical GVHD, compared with control-treated animals. Importantly, B975 had no effect on donor T cell responses to host antigens in vivo or in vitro. When mice received lethal doses of P815 tumor cells at the time of BMT, administration of B975 did not impair GVL activity and resulted in significantly improved leukemia-free survival. These findings reveal a critical role for LPS in the early inflammatory events contributing to GVHD and suggest that a new class of pharmacologic agents, LPS antagonists, may help to prevent GVHD while preserving T cell responses to host antigens and GVL activity.     

Application of peripheral blood stem cells (PBSC) mobilized by recombinant human granulocyte colony stimulating factor for allogeneic PBSC transplantation and the comparison of allogeneic PBSC transplantation and bone marrow transplantation.

Recombinant human granulocyte colony stimulating factor (rhG-CSF)-mobilized peripheral blood stem cells (PBSC) are now widely used for allogeneic PBSC transplantation (alloPBSCT). Large numbers of hematopoietic progenitor cells mobilized by rhG-CSF would be considered equivalent or better than bone marrow (BM) cells and would be used as an alternative to BM for allogeneic hematopoietic stem cell transplantation. The complications associated with the administration of rhG-CSF and apheresis in PBSC collection in formal donors are well tolerated and usually acceptable in the short term but some hazardous adverse events such as splenic rupture and cardiac arrest are reported although the incidence is very low. Protective means and stopping rules for safe donation in the collection of PBSC are established. The characteristics of PBSC were clarified; the expression of some adhesion molecules such as CD49d on CD34 positive cells of PBSC have been shown to be low compared to BM stem cells. In alloPBSCT compared with allogeneic BM transplantation (alloBMT), the incidence and frequency of graft versus host disease (GVHD) is of concern because high number of T lymphocytes are infused in alloPBSCT. The incidence and severity of acute GVHD are not increased but chronic GVHD is higher in alloPBSCT compared with alloBMT. The outcome of alloPBSCT and BMT are almost equivalent and conclusive results regarding survival are not yet available.     

非清除性异基因骨髓移植治疗小鼠白血病的实验研究

目的　研究非清除性异基因骨髓移植 (BMT)及其加供者淋巴细胞输注 (DLI)治疗小鼠白血病是否在保留移植物抗白血病效应 (GVL)的同时 ,又能减轻移植物抗宿主病 (GVHD)及移植相关并发症。方法　采用L6 15淋巴细胞白血病小鼠模型 ,非清除性预处理后移植BALB c小鼠骨髓细胞和脾细胞。分 4组 :清除性BMT对照组 (A组 )、非清除性预处理组 (B组 )、非清除性BMT组 (C组 )、非清除性BMT +DLI组 (D组 )。通过受鼠生存期、外周血白细胞和L6 15细胞计数、死亡鼠尸解观察GVL效应 ;通过体重改变、弓背、翘毛、腹泻等表现和肝脏、小肠、皮肤病理学检查观察GVHD ;通过染色体检查和PCR检测嵌合状态。结果　A、B、C、D组生存时间分别为 (2 0 .3± 13.4 )d、(15 .9± 1.1)d、(2 1.6± 1.7)d和 (37.8± 2 .0 )d ,C组和A组生存时间无统计学意义 (P >0 .0 5 ) ,C组生存时间长于B组 (P <0 .0 1) ,D组长于C组 (P <0 .0 1) ;A组有 6 0 %出现GVHD表现及病理学改变 ,而C组和D组均无典型GVHD表现和病理学改变 ;A组有 4 0 %在 2周内死于移植相关并发症 ,C组和D组均无移植相关死亡发生 ;A组一直保持异基因嵌合 ,C组渐被排斥。结论　非清除性BMT有一定的GVL效应 ,加DLI能增强GVL效应 ;非清除性BMT能减轻GVHD及移植相关并发症。