NK细胞在小鼠异基因骨髓移植中的作用

目的:研究自然杀伤(NK)细胞在异基因骨髓移植中对移植物抗宿主病(GVHD)、移植排斥、骨髓植入及造血重建的影响。方法:以近交系小鼠C57/6j(H-2^b)为供鼠、BALB/c(H-2^d)为受鼠,在移植物中增加供者的外周T细胞和/或NK细胞进行异基因骨髓移植,用流式细胞仪检测受鼠的CD₃₄^＋细胞计数和H-2K^b＋细胞表达水平,血细胞自动分析仪检测外周血白细胞计数,并结合临床表现和病理检查,比较不同移植组的存活率、GVHD、植入水平及造血重建等。结果:增加NK细胞组的小鼠存活率显著大于不增加NK细胞组,小鼠出现GVHD的数量少、程度轻,外周血白细胞及骨髓CD₃₄^＋细胞恢复快、H-2K^b＋细胞表达水平高。结论:NK细胞抑制小鼠异基因骨髓移植中的GVHD和移植排斥,促进骨髓植入及造血重建。     

同种反应性自然杀伤细胞对小鼠单倍体骨髓移植后T细胞重建的影响

目的探讨同种反应性自然杀伤（NK）细胞对小鼠单倍体骨髓移植（BMT）后T细胞重建的可能影响。方法以（C57BL/6×BALB/c）BCF1（H-2d/b）为供鼠,BALB/c（H-2d）为受鼠,鼠龄6～8周,体质量16～20 g。建立小鼠单倍体BMT模型。根据移植物不同将实验分为单纯BMT组、非同种反应性NK（non-alloNK）细胞组、同种反应性NK（allo-NK）细胞组,移植后不同时间点以外周血CD3e的表达、外周血血清白细胞介素4（IL-4）、干扰素（IFN-γ）浓度等指标评价同种反应性NK细胞对T细胞重建的可能影响。结果移植后＋10 d受鼠外周血CD3e的表达allo-NK组较non-alloNK组显著增高,分别为（16.58±5.77）%、（8.64±3.60）%,差异具有统计学意义（P〈0.05）。＋30 d时allo-NK组IL-4的浓度显著低于单纯BMT组,分别为（9.73±2.08）pg/mL、（12.44±1.57）pg/mL,差异具有统计学意义（P〈0.05）。non-alloNK组IFN-γ的浓度显著高于单纯BMT组,分别为（98.78±33.07）pg/mL、（52.15±18.87）pg/mL,差异具有统计学意义（P〈0.05）。结论同种反应性NK细胞用于单倍体BMT小鼠,可能促进T细胞的重建,体内诱导Th1免疫反应的偏移。     

混合骨髓移植后过继免疫治疗小鼠白血病

背景：急性白血病自体造血干细胞移植后复发率高,异基因造血干细胞移植后移植相关病死率高,混合造血干细胞移植及移植后过继免疫治疗有可能取长补短,提高疗效。 目的：观察自体骨髓混合H-2半相合异体骨髓移植后供体淋巴细胞输注+白细胞介素2治疗对小鼠白血病的疗效。 方法：将Balb/c小鼠经直线加速器照射3 Gy后分为白血病模型组、白血病模型照射组、混合移植组、自体骨髓移植组,均尾静脉注射5×10⁵ K562(GFP+/NeoR+)或K562(GFP-/NeoR-)细胞。7 d后6 Gy照射,自体骨髓移植组移植自体骨髓细胞或联合白细胞介素2治疗;混合移植组移植小鼠自体骨髓细胞混合1/10的H-2半相合异体骨髓细胞后应用白细胞介素2或联合供体淋巴细胞输注治疗。4周后行小鼠外周血及骨髓细胞形态检查,外周血细胞亚群、GFP及NeoR基因测定,肝、脾匀浆细胞GFP和NeoR基因测定。 结果与结论：白血病模型组小鼠因骨髓造血功能衰竭于20 d内全部死亡,白血病模型照射组小鼠因造血功能衰竭于14 d内全部死亡;自体骨髓移植组、混合移植组均有多少不等小鼠无白血病存活超过28 d,且混合骨髓移植后及自体骨髓移植后应用白细胞介素2治疗可提高白血病小鼠长期无病生存率,在此基础上联合供体淋巴细胞输注可更进一步提高白血病小鼠长期无病生存率。     

Factors influencing delayed type graft-versus-host disease after allogeneic bone marrow transplantation

Effects of the number of transplanted bone marrow cells and splenectomy performed before the transplantation on delayed type graft-versus-host disease (GVHD) were investigated in mice. The relation between delayed type GVHD and suppressor cells was also studied. Bone marrow cells (1 x 10(5)-1 x 10(7) cells) from CBA/N mice were transplanted to lethally irradiated (10 Gy) C57BL/6 mice, and the same type of transplantation was performed in other mice that underwent splenectomy. Suppressive effects of spleen cells from chimera mouse were measured by the percentage of suppression against alloantigen specific mixed lymphocyte reaction (MLR). The survival rate decreased with the number of grafted cells. However, survival rate was high in the splenectomy group even when the number of grafted cells was small. The incidence of delayed type GVHD was 0% in the 1 x 10(7) cell transplanted group, but that was more than 50% in the 1 x 10(5) cell transplanted group. There was no significant difference in the incidence of delayed type GVHD between the splenectomy group and non-splenectomy group. Spleen cells from chimera mouse without delayed type GVHD had greater suppressive effect against MLR than those with GVHD. These results suggest that alloantigen specific suppressor cells in the spleen of chimera mice inhibit delayed type GVHD.     

Alleviation of chronic GVHD in mice by oral immuneregulation toward recipient pretransplant splenocytes does not jeopardize the graft versus leukemia effect

Chronic graft versus host disease (cGVHD) is the result of an immune-mediated attack by transplanted donor lymphocytes, entailing inflammatory damage to host target organs. Clinically, the post-bone marrow transplantation (BMT) graft versus leukemia (GVL) effect may be associated with GVHD. Immune hyporesponsiveness induced by oral antigen administration has recently been shown to prevent the development of cGVHD in a murine model. To evaluate whether amelioration of cGVHD in mice by induction of oral immune regulation in donors toward recipient pretransplant lymphocyte antigens is associated with attenuation of the GVL effect donor B10.D2 mice were fed with Balb/c splenocytes, B10.D2 splenocytes, bovine serum albumin (BSA), or regular chow, every other day for 10 days. Subsequently, transplantation of 2 x 10(7) splenocytes from donor B10.D2 mice to recipient Balb/c mice was undertaken, followed by inoculation of 3 x 10(3) BCL-1 leukemia on the day of BMT. Control groups were fed identically without leukemia inoculation. Mice were followed for survival and leukemia progression. Induction of tolerance was assessed by a mixed lymphocyte reaction (MLR). Cutaneous GVHD was assessed macroscopically. To elucidate the mechanism of any observed effect, serum interferon (IFN), interleukin (IL-2), IL-12, IL-4, and IL-10 were determined by enzyme-linked immunosorbent assay and flow cytometry analysis for CD4+, CD8+, and NK1.1+ lymphocyte subpopulations was performed. There was no significant difference in leukemia progression manifested by survival or white blood cell counts of orally immune-regulated mice compared with control animals. Cutaneous cGVHD was significantly ameliorated in Balb/c mice transplanted from tolerized B10.D2 mice. This effect was associated with a significant reduction in the mixed lymphocyte response of effector splenocytes from tolerized B10.D2 mice against Balb/c target splenocytes; significantly decreased serum IFN-gamma and IL-2; increased serum IL-12 levels; increased peripheral NK1.1+ cells; and CD4+/CD8+ lymphocyte ratio. Oral tolerization of BMT donors toward recipient antigens ameliorates cGVHD without hampering the GVL effect.     

Dynamics of early histopathological changes in GVHD after busulphan/cyclophosphamide conditioning regimen

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for otherwise incurable diseases. Conditioning regimen is an important part of HSCT and consists of chemotherapy with or without irradiation. Conditioning exerts myelosuppressive, immunosuppressive and antitumor effects, but also contributes to HSCT-related complications including graft-versus-host disease (GVHD). Since almost 50% of the transplanted patients are conditioned with cytostatics without irradiation, we developed and characterized a GVHD mouse model following conditioning with busulphan and cyclophosphamide. Recipient Balb/c female mice were treated with busulphan (20 mg/kg/day for 4 days) and cyclophosphamide (100 mg/kg/day for two days). After one day of rest, recipient mice were transplanted with 2×10(7) bone marrow and 3×10(7) spleen cells from male C57BL/6 (allogeneic group) or female Balb/c (syngeneic/control group) mice. The allogeneic, but not syngeneic transplanted mice developed GVHD. Histopathology of the major internal organs (liver, pancreas, spleen, lungs, heart and kidney) was examined before conditioning start, after conditioning's end and 5, 7 and 21 days after transplantation using hematoxylin-eosin staining. Decreased spleen cellularity and diminished glycogen content in the liver were observed after conditioning regimen. Histopathological changes such as vasculitis, inflammation and apoptotic cell forms in liver, spleen, pancreas, lungs and heart were observed in allogeneic transplanted mice, however, only hypocellular spleen and extramedullar hematopoiesis were detected in syngeneic transplanted animals. No morphological changes were observed in kidney in either HSCT setting. This is the first study describing early histopathological changes after conditioning regimen with busulphan/cyclophosphamide and dynamics of GVHD development in several major internal organs.     

Effects of intrabone marrow-bone marrow transplantation plus adult thymus transplantation on survival of mice bearing leukemia

We recently found that allogeneic intrabone marrow-bone marrow transplantation (IBM-BMT) plus adult thymus transplantation (ATT) from the same donor is effective in mice bearing solid tumors. In the current study, we examined the effects of this strategy on the survival of mice with leukemia. One week after intravenous injection of 1×10(6) leukemic cells (EL-4, H-2(b)) into 8-week-old B6 (H-2(b)) mice, the mice were 8 Gy irradiated and transplanted with 1×10(7) bone marrow cells (BMCs) from 8-week-old BALB/c mice (H-2(d)) by IBM-BMT with or without donor lymphocyte infusion (DLI) or ATT. All the mice without treatment died within 70 days after injection of EL-4. About 40% of those treated with IBM-BMT alone died within 100 days due to tumor relapse. In contrast, those treated with IBM-BMT+DLI or ATT showed the longest survival rate without relapse of leukemia. In addition, the former showed less graft versus host disease (GVHD) than the latter. The mice treated with IBM-BMT+ATT also showed an intermediate percentage of effector memory (EM) and central memory (CM) cells between those treated with BMT alone and those treated with IBM-BMT+DLI. The numbers and functions of T cells increased in those treated with IBM-BMT+ATT with interleukin-2 and interferon-γ production. These results suggest that IBM-BMT+ATT is effective in the treatment of leukemia with strong graft versus leukemia without increased risk of GVHD.     

Graft-facilitating doses of ex vivo activated gammadelta T cells do not cause lethal murine graft-vs.-host disease.

The purpose of this study was to examine the ability of gamma(delta) T cells to cause graft-vs.-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) and to determine whether these cells offered any therapeutic advantages relative to alphabeta T cells. Due to the paucity of naive gamma(delta) T cells in mice and humans, gamma(delta), T cells (obtained from alpha(beta) T cell-deficient murine donors) were ex vivo activated and expanded in interleukin (IL)-2 so as to achieve sufficient cell numbers and to serve as a more clinically feasible strategy. After transplantation into lethally irradiated hosts, donor gamma(delta) T cells were detected in target organs of GVHD such as the spleen and intestines 2 weeks after BMT and constituted the primary T cell subpopulation. Large doses (150 x 10(6)) of activated gamma(delta) T cells, which we have previously shown capable of facilitating engraftment in MHC-disparate recipients, failed to cause fatal GVHD in lethally irradiated recipients of MHC-incompatible donor marrow grafts (C57BL/6 [H-2b]-->B10.BR [H-2k] and C57BL/6 [H-2b]-B6D2F1[H-2b/d]). The absence of GVHD was confirmed by histologic analysis of target organs, splenic B cell reconstitution, and appropriate negative selection in the thymus, that were all comparable to those observed in mice transplanted with T cell-depleted BM only. While early splenic reconstitution was attributable to donor gamma(delta) T cells, analysis of durably engrafted chimeras 2 months posttransplant revealed that the vast majority of donor splenic T cells expressed the alpha(beta) T cell receptor. The results of secondary adoptive transfer assays showed that these cells were tolerant of recipient alloantigens in vivo, demonstrating that gamma(delta) T cells did not prevent the subsequent development of donor anti-host tolerance in BM-derived alpha(beta) T cells. When comparatively evaluated, the minimal number of naive alpha(beta) T cells necessary for donor engraftment caused significantly more fatal GVHD than the corresponding minimal dose of activated gamma(delta) T cells and thus had a superior therapeutic index. These studies indicate that doses of activated gamma(delta) T cells that are able to promote alloengraftment do not cause lethal GVHD in mice transplanted with MHC-incompatible marrow grafts.     

类人慢性粒细胞性白血病小鼠模型的建立

 目的：为了进一步阐明慢性粒细胞性白血病(chronic myeloid leukemia,CML) 的发病机制、寻找新的治疗靶点,有必要建立CML动物模型。方法：首先改进逆转录病毒包装技术,在相同实验条件下,改变以下条件之一,如质粒的质量、包装细胞的状态、转染当天包装细胞的密度等,进行病毒滴度的测定,优化实验条件后进行后续实验。实验组为BCR/ABL(含BCR/ABL融合基因的逆转录病毒载体)组小鼠。供体小鼠经5-氟尿嘧啶（5-FU）预处理后,骨髓细胞经p210 BCR/ABL逆转录病毒上清感染,感染的骨髓细胞经尾静脉注射给经致死剂量X光照射的受体小鼠。观察移植后BCR/ABL组小鼠的活动情况、发病鼠外周血和骨髓的细胞形态、肝脾病理改变等。同时观察MigR1(逆转录病毒空载体)组小鼠。结果：通过提高质粒的质量、改善包装细胞的状态、优化转染当天包装细胞的密度等,提高了病毒的滴度,用于小鼠移植模型实验。BCR/ABL组小鼠均于移植后19～25 d发病死亡。发病时外周血及骨髓均见中晚幼及成熟粒细胞大量增生,肝脾肿大伴正常结构破坏及大量粒细胞浸润。MigR1组小鼠均无发病。结论: 通过改进逆转录病毒包装技术,提高包装病毒的滴度,我们在体外将BCR/ABL转入小鼠骨髓细胞并移植入受体小鼠,成功建立了类人CML小鼠模型。     

EL9611红白血病小鼠急性GVHD动物模型的建立

目的: 建立EL9611红白血病小鼠急性移植物抗宿主病(GVHD)的动物模型。方法: 同种异基因骨髓移植(allo-BMT)以C57BL/6(H-2^b)鼠为供鼠,BALB/c(H-2^d)为受鼠。设白血病组(n=10)、照射对照组(白血病鼠照射后不进行allo-BMT,n=4)、GVHD组(白血病鼠照射+allo-BMT,n=10)及正常对照组(n=4)。白血病组采用每只BALB/c鼠尾静脉输注2×10⁶个EL9611红白血病细胞建立红白血病动物模型;GVHD组于接种白血病细胞7 d后行总剂量为8.0 Gy的1次性 γ射线全身照射(TBI),照射后5 h内每只小鼠尾静脉输注C57BL/6鼠骨髓细胞2×10⁶个＋脾细胞1×10⁷个,建立EL9611红白血病小鼠的急性GVHD动物模型。观察小鼠体位、皮毛、大便等临床表现,病理检查肝脾、皮肤、小肠、外周血和骨髓,计算生存率。结果: 白血病组生存时间(14.5±2.1) d ,生存时间与GVHD组相比P<0.01,死亡率100％,无自发缓解,死亡时肝脾肿大(肝重2.40 g±0.48 g,脾重0.84 g±0.20 g,与正常对照组比P<0.01),外周血WBC升高 ,病理检查示组织正常结构破坏,白血病细胞浸润。照射对照组生存时间为(9.0±0.7) d,生存时间与GVHD组和正常对照组相比差异显著(P<0.01),死亡率100％,病理检查显示造血衰竭。GVHD组生存时间为(32.0±3.2)d,生存时间与其它各组相比P<0.01,死亡率100％,allo-BMT后第10-13 d出现症状,临床表现和病理检查符合Ⅰ到Ⅱ度GVHD的改变。结论: 采用EL9611红白血病细胞(2×10⁶/鼠)静脉输注的方式可成功建立EL9611红白血病动物模型;接种EL9611红白血病细胞第7 d行TBI +allo-BMT可成功建立EL9611红白血病小鼠的急性GVHD动物模型。     

Selective elimination of alloreactive donor T cells attenuates graft-versus-host disease and enhances T-cell reconstitution.

Impaired T-cell immune reconstitution is a major complication after allogeneic bone marrow transplantation (BMT) and is particularly exacerbated in the setting of graft-versus-host disease (GVHD). Conventional approaches to reduce GVHD, such as T-cell depletion or pharmacologic immunosuppression, typically fail to enhance T-cell immunity and often further exacerbate this problem. An alternative strategy to mitigate GVHD severity is the selective elimination of graft-versus-host-reactive donor T cells by using an incorporated thymidine kinase suicide gene. This approach has been shown to effectively reduce GVHD, although the effect of this strategy on T-cell reconstitution is unresolved. We addressed this question in a murine BMT model (C57BL/6 [H-2(b)] --> AKR/J [H-2(k)]) in which donor and recipient differ at major and minor histocompatibility antigens. Lethally irradiated AKR recipients transplanted with T cell-depleted bone marrow plus thymidine kinase-positive T cells followed by post-BMT ganciclovir (GCV) administration had more prompt and complete normalization of the T-cell repertoire than phosphate-buffered saline-treated GVHD control animals. By 60 days after transplantation, mice administered GCV had T-cell repertoires that were virtually indistinguishable from those of mice that underwent transplantation with T cell-depleted bone marrow alone (no GVHD controls) when assayed by T-cell receptor (TCR) spectratyping. In contrast, phosphate-buffered saline-treated animals had persistent skewing in most Vbeta families. T cells obtained from GCV-treated mice also had significantly higher in vitro proliferative responses after posttransplantation inoculation with ovalbumin than GVHD animals, indicating that CD4(+) T-cell responses against a nominal antigen were better preserved in these chimeras. Finally, GCV-treated mice had augmented immune reconstitution in response to exogenous interleukin-7 administration, as evidenced by increased overall spleen cellularity and absolute numbers of T and B cells. This was in contrast to GVHD control animals, which had a blunted response to interleukin-7 administration. These data indicate that GVHD severity can be significantly reduced by selective elimination of alloreactive donor T cells without compromise of T-cell immunity. Moreover, in light of previous studies demonstrating that this strategy can reduce GVHD without loss of alloengraftment and antileukemia reactivity, further examination of this approach in humans seems warranted.     

Inhibition of natural killer cell-mediated lysis of tumor cells by normal and regenerating bone marrow

The natural killer (NK) cells which can lyse certain tumor cells during brief incubation in vitro have also been postulated to be the cells responsible for natural resistance to transplanted hemopoietic cells in vivo. To test this hypothesis, we have now measured: 1) the ability of bone marrow cells to compete with tumor cells as targets for spleen NK cells and 2) the effect of a brief incubation with spleen cells on the hemopoietic grafting potential of bone marrow cells. Firstly, when CBA/J mouse spleen cells were incubated with 51Cr-labelled YAC tumor cells together with DBA/2 mouse bone marrow cells, tumor cell lysis was reduced compared with incubation of spleen cells with tumor cells alone. Tumor cell lysis was even less when post-irradiation regenerating bone marrow was used. Secondly, C57B1/6 mouse bone marrow cells incubated with an excess of DBA/2 mouse spleen cells showed a reduced ability to produce hemopoietic spleen colonies in irradiated 129/J mice, whereas incubation with either thymus cells or fewer spleen cells produced no such effect. The results show that, when incubated with spleen cells under the conditions of a standard NK cell assay, regenerating bone marrow cells competitively inhibit the killing of YAC tumor cells and bone marrow progenitor cells are rendered ineffective in their hemopoietic colony-forming potential (CFU-s). These findings suggest that certain hemopoietic progenitor cells and YAC tumor cells can both serve as targets for NK cells, consistent with the view that the spontaneous cytolysis of tumor cells in vitro and natural resistance to bone marrow transplantation in vivo are mediated by cells of a common lineage.     

Reduction of graft-versus-host disease in neonatal F1 hybrid mice.

Pre-immunization of BALB/c (H-2d) mothers with C57BL/10 (H-2b) or CBA/H (H-2k) spleen cells partially protected the F1 hybrid offspring of (BALB/c x C57BL/10) or (BALB/c x CBA/H) matings from graft-versus-host-disease (GVHD) induced by neonatal intraperitoneal inoculation with spleen cells of the paternal strain. The effects achieved were manifest as a reduction in mortality. Experiments to establish whether the phenomenon was antibody mediated were performed by passive pre-immunization of BALB/c mothers with alloantisera obtained from BALB/c previously immunized with C57BL/10 spleen cells. Alloantisera produced an equivalent reduction in GVHD mortality. Some of the F1 mice that survived challenge with paternal strain spleen cells were proven to be haemopoietic chimaeras using immunofluorescence with anti-MHC monoclonal antibodies and polymorphism of the enzyme glucose-phosphate-isomerase present in the strains used. The possible mechanisms of protection from GVHD in our mouse model are discussed.     

Differences in MHC-class I presented minor histocompatibility antigens extracted from normal and graft-versus-host disease (GVHD) mice

Graft-versus-host disease (GVHD) may develop after allogeneic bone marrow transplantation (BMT) between donors and recipients incompatible for minor histocompatibility antigens (mHAg). Here, we examined the possible relationship between tissue-specific distribution of dominant mHAg peptides and specific organ destruction caused by GVHD. In the B6 anti-Balb/b (H-2b) strain combination, a GVHD developed against Balb/b mHAgs. Despite the high number of incompatible mHAgs between these two strains, both cytotoxic T lymphocyte (CTL) response and GVHD could be attributed to a limited number of dominant mHAgs. We studied CTL-defined expression of dominant mHAgs in normal tissues and their GVHD-related modifications. mHAg peptides were prepared by acid elution and reversed-phase high pressure liquid chromatography fractionation from the spleen, liver, gut and skin as GVHD target tissues and from the heart and kidney as control tissues. Peptidic fractions extracted from normal and GVHD tissues were incubated with RMA-S targets and analysed using bulk B6 anti-Balb/b CTL. In each tissue several fractions were recognized with a given pattern of mHAg expression. GVHD induced qualitative and quantitative changes in antigenic peptide expression. Modifications in mHAg presentation during GVHD concerned preferentially GVHD target organs as opposed to non-GVHD target organs. In addition, when immunizing tissues were derived from GVHD mice instead of normal mice, the profile of CTL recognition was different. In conclusion, these data indicate that broad differences could exist in peptide presentation between various normal and GVHD-target organs.     

间充质干细胞诱导的CD8α~+ Jagged2~(high) CD11b~(high)调节性树突状细胞防治小鼠aGVHD

目的:观察间充质干细胞诱导的CD8α+Jagged2highCD11bhigh调节性树突状细胞(MSC-DCregs),对小鼠急性移植物抗宿主病(a GVHD)的影响。方法:体外诱导MSC-DCregs。以C57BL/6(H-2b)小鼠为供鼠、接受清髓性全身照射(TBI)预处理的BALB/c(H-2d)小鼠为受鼠进行移植。分为以下5组:为正常对照组;TBI组;移植组:移植物为1×107骨髓细胞(BMCs);a GVHD组:移植物为1×107BMCs+1×107脾细胞(Sp Cs);MSC-DCregs组:移植物为1×107BMCs+1×107Sp Cs+1×106MSC-DCregs。监测植入情况、H2-Kb嵌合率、a GVHD评分、生存和病理学改变。结果:处理1 0 d后所有小鼠造血恢复,30 d后嵌合率检测转为供鼠型。a GVHD组和MSC-DCregs组中位生存期分别为27 d和33 d,MSC-DCregs组生存期较a GVHD组延长(P0.01),临床评分比a GVHD组低(P0.01),33 d的皮肤、肝脏病理学改变均优于a GVHD组。结论:MSC-DCregs具有防治a GVHD的作用,其相关作用机理待进一步研究。     

Control of mouse graft-versus-host disease following allogeneic bone marrow transplantation by blocking the CD28/B7 signaling pathway with lentiviral vector-mediated RNA interference

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective way to cure hematological malignancies. However, graft-versus-host disease (GVHD) following transplantation limits the clinical application to some extent. The donor T lymphocytes play a central role in the occurrence and development of GVHD. Control of GVHD by inhibition of T cell proliferation by blocking the CD28/B7 signaling pathway with RNA interference has not been examined. In this study, we constructed a lentiviral vector carrying CD28 shRNA and generated genetically engineered splenocytes through transduction in a murine allogeneic bone marrow transplantation model. The survival and the occurrence of GVHD in transplanted mice were monitored every day. Liver, intestine, skin, and other tissues from the mice in each group were used for histological examination. We also determined plasma concentrations of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-13, and interferon gamma (IFN-γ). Recipient bone marrow from mice that had survived for an extended period was examined to detect chimerism. We succeeded in suppressing the expression of CD28 gene and controlling mouse GVHD following allogeneic bone marrow transplantation in the engineered spleen cell group. These suggest that blocking the CD28/B7 signal transduction pathway with lentiviral vector-mediated RNA interference effectively controlled the occurrence of mouse GVHD following allogeneic bone marrow transplantation. Its mechanism could be due to the inhibition of T cell proliferation and, simultaneously, the promotion of the differentiation of TH0 to TH2 cells, thereby reducing GVHD in the mouse transplantation model.     

移植耐受诱导的异基因胎肝移植小鼠的免疫功能重建

本文用门静脉注射异型脾细胞加腹腔注射环磷酰胺方法，成功地诱导了成年Ｂａｌｂ／ｃ小鼠（Ｈ－２ｄ）对Ｃ５７ＢＬ／６（Ｈ－２ｂ）小鼠的免疫耐受。致死照射的耐受Ｂａｌｂ／ｃ小鼠用Ｃ５７ＢＬ／６（Ｂ６）小鼠的胎肝细胞移植后，无移植排斥产生。嵌合状态分析的结果表明，在胎肝移植后９０ｄ和２４０ｄ，重建的Ｂａｌｂ／ｃ小鼠的脾细胞分别有７４．４％和８３．７％来自于供体Ｂ６小鼠．证明Ｂ６小鼠胎肝造血干细胞已经在致死照射的Ｂａｌｂ／ｃ小鼠体内稳定植入。免疫功能检测的结果表明，在胎肝移植后９０ｄ，照射Ｂａｌｂ／ｃ小鼠的免疫功能已经重建。     

Delaying acute graft-versus-host disease in mouse bone marrow transplantation by treating donor cells with antibodies directed at l-selectin and alpha4-integrin prior to infusion

Acute graft-versus-host disease (GVHD) is still a major hurdle for successful bone marrow transplantation (BMT). Although many immunosuppressive drugs are available, none of them alone or in combination are able to completely abolish acute GVHD. The lifelong immunosuppression profoundly reduces the quality of life of BMT recipients. Therefore, new therapeutic approaches are needed. We previously reported that, in an acute GVHD model using SCID mice as recipient, incubating donor spleen cells with antibodies directed at CD49d and CD62L could significantly delay the occurrence of acute GVHD. To test the potential usefulness of this treatment in BMT, we examined this therapeutic protocol in a mouse BMT model. The present mouse BMT study confirmed our previous results that incubation of donor cells with antibodies directed at CD49d and CD62L prior to infusion into the recipient can effectively delay acute GVHD, allowing the recipients to recover from the side effects of total body irradiation. This one-time treatment is easy and simple and may be modified for clinical usage.     

Repifermin (keratinocyte growth factor-2) reduces the severity of graft-versus-host disease while preserving a graft-versus-leukemia effect.

Graft-versus-host disease (GVHD) is the principal complication after allogeneic bone marrow transplantation (BMT). Reductions in systemic GVHD are frequently associated with a corresponding diminishment of the graft-versus-leukemia (GVL) response. In this study, we tested the effects of a novel recombinant human keratinocyte growth factor, repifermin (keratinocyte growth factor-2), on the induction of GVHD in a well-defined murine BMT model (B6 --> B6D2F1). Administration of repifermin (5 mg/kg/d) to allogeneic BMT recipients resulted in a significant decrease in both systemic GVHD and target organ histopathology. Repifermin treatment also reduced serum levels of tumor necrosis factor alpha and lipopolysaccharide compared with control mice. In contrast, repifermin did not affect T-cell proliferation, cytokine production, or cytotoxic responses to host antigens. When 2000 host-derived P815 (H-2(d)) leukemia cells were added to the bone marrow inoculum, repifermin preserved GVL effects and resulted in significantly delayed mortality compared with control-treated allogeneic BMT recipients. Collectively, these data suggest that repifermin administration may represent a novel strategy to separate the toxicity of GVHD from the beneficial GVL effects after allogeneic BMT.     

Recipient-Derived Allo-iTregs Induced by Donor DCs Effectively Inhibit the Proliferation of Donor T Cells and Reduce GVHD

To compare the potency of recipient-derived, antigen-specific regulatory T cells induced by different dendritic cells (DCs; iTregs) and freshly isolated natural regulatory T cells (nTregs) in preventing mouse graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). CD4⁺ T cells from recipient BALB/c mice were stimulated with DCs from recipient BALB/c (syn-DCs), donor B6 (allo-DCs), and third-party C3H (third-party-DCs) mice to induce different iTregs. In parallel, nTregs were isolated from spleen cells of recipient BALB/c (syn-nTregs) and donor B6 (allo-nTregs) mice using magnetic-activated cell sorting. Mixed lymphocyte reaction (MLR) assays were performed to evaluate the suppressive ability of these various regulatory T cells (Tregs). Both the iTregs and nTregs were transfused to GVHD mice on Days 0, 1, 3, and 5. Body weight, GVHD score, and survival time were monitored. Peripheral Tregs were subsequently examined on Days 7, 14, 21, and 28 after BMT, while chimerism was evaluated on Days 14 and 60. Histopathology of colon, liver, and spleen were also performed. DCs markedly induced CD25⁺ and Foxp3⁺ expression on CD4⁺ T cells. The allo-DC-induced Tregs (allo-iTregs) suppressed the proliferation of alloreactive T cells better than the other iTregs/nTregs in MLR assays (P < 0.05). Meanwhile, transfusion of the allo-iTregs reduced the severity of GVHD (P < 0.05), increased survival time compared with the GVHD group (P < 0.05), and enhanced the chimerism proportion. On Day 28 after BMT, the allo-iTregs group had the highest frequency of peripheral Tregs (P < 0.05). Recipient-derived allo-iTregs induced by donor DCs included predominant clones that specifically recognized donor antigens. These allo-iTregs not only prevented GVHD by suppressing the proliferation of donor-alloreactive T cells, but also promoted engraftment, and prolonged the survival of GVHD mice. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:825–836, 2019. © 2018 Wiley Periodicals, Inc.