异基因造血干细胞移植中的树突状细胞

树突状细胞(DC)是目前已知的启动免疫反应最强大的抗原呈递细胞(APC),也是惟一能激活初始T细胞的APC。近年来,DC在移植免疫中的作用已成为研究的焦点。简要综述了DC在异基因造血干细胞移植中的研究进展。     

A Murine Model of Hepatic Veno-occlusive Disease Induced by Allogeneic Hematopoietic Stem Cell Transplantation

Hepatic veno-occlusive disease (HVOD) is a life-threatening complication of bone marrow stem cell transplantation. The understanding of this clinical condition is hampered by the lack of suitable animal models. Here, we present a murine (BALB/c-based) model of HVOD induced by allogeneic hematopoietic stem cell transplantation (allo-HSCT). The chimerism rate of bone marrow was measured on days 5 and 10, while the chimerism rate of peripheral blood was measured on day 15 after allo-HSCT. Percentages of peripheral reticulocytes and serum levels of bilirubin and alanine aminotransferase (as liver function tests) were measured on days 5, 10, 15, 20, and 30. Livers were obtained on days 5, 10, 15, 20, and 30, and fixed in formaldehyde or glutaric dialdehyde. Liver slices were processed using the hematoxylin–eosin, Masson’s trichrome, or immunohistochemistry staining, and examined by light or transmission electron microscopy. Sinusoidal damages were the earliest pathological changes occurring in the allo-HSCT-induced HVOD, followed by coagulative necrosis of liver cells. The liver cell necrosis was later attenuated and sinusoidal endothelial cell morphology improved. However, on day 30, the edema and necrosis of liver cells became aggravated again. Furthermore, sinusoidal lining cell regeneration and partly attenuated liver cell necrosis were followed by the moderate to severe central vein fibrosis. In conclusion, we have successfully established a murine model of HSCT-HVOD. This model develops moderate to severe HVOD which cannot heal without intervention.     

Herpesvirus-Associated Central Nervous System Diseases after Allogeneic Hematopoietic Stem Cell Transplantation

Herpesvirus infections of the central nervous system (CNS) are associated with encephalitis/myelitis and lymphoproliferative diseases in immunocompromised individuals. As of now, data of herpesvirus-associated CNS diseases in transplant recipients is limited. Hence, in this prospective study, we investigated the incidence of herpesvirus-associated CNS diseases and explored the diagnosis of these diseases in 281 allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients. Herpesvirus-DNA and cerebrospinal fluid (CSF) cells were sampled from 58 recipients with herpesvirus-associated diseases or with unexplainable CNS manifestations. Results showed that 23 patients were diagnosed as herpesvirus-associated CNS diseases, including 15 Epstein-Barr virus (EBV)-associated diseases (4 encephalitis and 11 lymphoproliferative diseases), 5 herpes simplex virus type 1 encephalitis, 2 cytomegalovirus encephalitis/myelitis and 1 varicella zoster virus encephalitis. The median time of diseases onset was 65 (range 22-542) days post-transplantation. The 3-year cumulative incidence of herpesvirus-associated encephalitis/myelitis and post-transplant lymphoproliferative disorder (PTLD) was 6.3% ±1.9% and 4.1% ±1.2%, respectively. Of the evaluable cases, CSF cells mainly consisted of CD19⁺CD20⁺ B cells (7/11) and had clonal rearrangement of immunoglobulin genes (3/11) in patients with CNS-PTLD. On the contrary, in patients with encephalitis/myelitis, CSF cells were comprised of different cell populations and none of the gene rearrangement was detected. Herpesvirus-associated CNS diseases are common in the early stages of allo-HSCT, wherein EBV is the most frequent causative virus. The immunophenotypic and clonal analysis of CSF cells might be helpful in the differential diagnosis between encephalitis and lymphoproliferative diseases.     

Efficacy of Mesenchymal Stem Cell Therapy for Steroid-Refractory Acute Graft-Versus-Host Disease following Allogeneic Hematopoietic Stem Cell Transplantation: A Systematic Review and Meta-Analysis

Background

Mesenchymal stem cells (MSCs) have been broadly used experimentally in various clinical contexts. The addition of MSCs to initial steroid therapy for acute graft-versus-host disease (aGVHD) may improve patient outcomes. However, investigations regarding prognostic factors affecting the efficacy of MSC therapy for steroid-refractory aGVHD remain controversial. We thus conducted a systematic review and meta-analysis of published clinical trials to determine possible prognostic factors affecting the efficacy of MSCs in treating steroid-refractory aGVHD.

Methods and Findings

Clinical trials using MSC therapy for steroid-refractory aGVHD were identified by searching PubMed and EMBASE databases. A total of 6,963 citations were reviewed, and 13 studies met the inclusion criteria. A total of 301 patients from thirteen studies were included. Of these, 136 patients showed a complete response (CR), and 69 patients displayed a partial (PR) or mixed response (MR). In total, 205 patients exhibited overall response (ORR). Patients with skin steroid-refractory aGVHD showed a better clinical response than gastrointestinal (CR: odds ratio [OR] = 1.93, 95% confidence interval [95%CI]: 1.05–3.57, p < 0.05) and liver (CR: OR = 2.30, 95%CI: 1.12–4.69, p < 0.05, and ORR: OR = 2.93, 95%CI: 1.06–8.08, p < 0.05) steroid-refractory aGVHD. Those with grade II steroid-refractory aGVHD exhibited a better clinical response following MSC therapy than recipients with grade III–IV (CR: OR = 3.22, 95%CI: 1.24–8.34, p < 0.05). Completion therapy may improve the CR but reduce ORR compared with induction therapy (CR: OR = 0.20, 95%CI: 0.09–0.44, p < 0.05; ORR: OR = 2.18, 95%CI: 1.17–4.05, p = 0.01). There was also a trend towards a better clinical response in children compared with adults (CR: OR = 2.41, 95%CI: 1.01–5.73, p = 0.05).

Conclusions

Age, skin involvement, lower aGVHD grade, and the number of infusions are the main prognostic factors affecting the efficacy of MSC therapy for steroid-refractory aGVHD.     

Ceruloplasmin Is a Potential Biomarker for aGvHD following Allogeneic Hematopoietic Stem Cell Transplantation

Acute graft-versus-host-disease (aGvHD) is the major cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recently, diagnostic biomarkers for aGvHD have been shown to play important roles in evaluating disease status and mortality risk after allo-HSCT. To identify plasma biomarkers for aGvHD with high sensitivity and specificity, a quantitative proteomic approach using 8-plex isobaric tags for relative and absolute quantitation (8-plex iTRAQ) was employed to screen differentially expressed proteins in peripheral blood before and after the onset of aGvHD. Four target proteins, ceruloplasmin (CP), myeloperoxidase (MPO), complement factor H (CFH), and alpha-1-acid glycoprotein (AGP), were chosen for preliminary validation with enzyme linked immunosorbent assay (ELISA) in 20 paired samples at both the time of diagnosis of aGvHD and the time of complete response. The most promising candidate, ceruloplasmin, was further validated at fixed time points after allo-HSCT and during aGvHD. The plasma ceruloplasmin levels were significantly increased during the period of aGvHD onset and were markedly decreased as aGvHD resolved. The plasma ceruloplasmin levels at different time points post-transplant in the aGvHD (+) group were significantly higher than those in the aGvHD (−) group (p<0.001). The elevation of ceruloplasmin level in patients with active aGvHD was independent of infection status. Patients whose ceruloplasmin levels were elevated above 670 μg/ml at 7, 14 and 21 days after allo-HSCT had a remarkably increased probability of subsequently developing aGvHD. In conclusion, our results suggest that plasma ceruloplasmin is a potential plasma biomarker of aGvHD, and it also has prognostic value for risk-adapted prophylaxis during the consecutive time points monitored in the first month after allo-HSCT.     

Differential Effect of MyD88 Signal in Donor T Cells on Graft-versus-Leukemia Effect and Graft-versus-Host Disease after Experimental Allogeneic Stem Cell Transplantation

Despite the presence of toll like receptor (TLR) expression in conventional TCRαβ T cells, the direct role of TLR signaling via myeloid differentiation factor 88 (MyD88) within T lymphocytes on graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect after allogeneic stem cell transplantation (allo-SCT) remains unknown. In the allo-SCT model of C57BL/6 (H-2^b) → B6D2F1 (H-2^b/d), recipients received transplants of wild type (WT) T-cell-depleted (TCD) bone marrow (BM) and splenic T cells from either WT or MyD88 deficient (MyD88KO) donors. Host-type (H-2^d) P815 mastocytoma or L1210 leukemia cells were injected either subcutaneously or intravenously to generate a GVHD/GVL model. Allogeneic recipients of MyD88KO T cells demonstrated a greater tumor growth without attenuation of GVHD severity. Moreover, GVHD-induced GVL effect, caused by increasing the conditioning intensity was also not observed in the recipients of MyD88KO T cells. In vitro, the absence of MyD88 in T cells resulted in defective cytolytic activity to tumor targets with reduced ability to produce IFN-γ or granzyme B, which are known to critical for the GVL effect. However, donor T cell expansion with effector and memory T-cell differentiation were more enhanced in GVHD hosts of MyD88KO T cells. Recipients of MyD88KO T cells experienced greater expansion of Foxp3- and IL4-expressing T cells with reduced INF-γ producing T cells in the spleen and tumor-draining lymph nodes early after transplantation. Taken together, these results highlight a differential role for MyD88 deficiency on donor T-cells, with decreased GVL effect without attenuation of the GVHD severity after experimental allo-SCT.     

Diabetes Mellitus after Hematopoietic Stem Cell Transplantation

ObjectiveTo review the current literature on posttransplant diabetes mellitus after hematopoietic stem cell transplantation, including its epidemiologic features, transplant-related risk factors, and treatment.MethodsA literature search was conducted in PubMed for articles on diabetes mellitus after hematopoietic stem cell transplantation and effects of immunosuppressants on glucose metabolism.ResultsWithin 2 years after hematopoietic stem cell transplantation, up to 30% of patients may have diabetes. Although some of these cases resolve, the rates of diabetes and metabolic syndrome remain elevated in comparison with those in the nontransplant patient population during long-term follow-up. Traditional risk factors for diabetes as well as features related to the transplantation process, including immunosuppressive medications, are associated with posttransplant diabetes. Cardiovascular risk also appears to be increased in this population. Limited data are available on hypoglycemic agents for posttransplant diabetes; thus, treatment decisions must be based on safety, efficacy, and tolerability, with consideration of each patient’s transplant-related medications and comorbidities.ConclusionTreatment of diabetes mellitus in patients who have undergone hematopoietic stem cell transplantation necessitates attention to the posttransplant medication regimen and clinical course. Although no guidelines specific to treatment of posttransplant diabetes in this patient population currently exist, treatment to goals similar to those for nontransplant patients with diabetes should be considered in an attempt to help reduce long-term morbidity and mortality. (Endocr Pract. 2010;16:699-706)     

Tolerance Associated Gene Expression following Allogeneic Hematopoietic Cell Transplantation

Biologic markers of immune tolerance may facilitate tailoring of immune suppression duration after allogeneic hematopoietic cell transplantation (HCT). In a cross-sectional study, peripheral blood samples were obtained from tolerant (n = 15, median 38.5 months post-HCT) and non-tolerant (n = 17, median 39.5 post-HCT) HCT recipients and healthy control subjects (n = 10) for analysis of immune cell subsets and differential gene expression. There were no significant differences in immune subsets across groups. We identified 281 probe sets unique to the tolerant (TOL) group and 122 for non-tolerant (non-TOL). These were enriched for process networks including NK cell cytotoxicity, antigen presentation, lymphocyte proliferation, and cell cycle and apoptosis. Differential gene expression was enriched for CD56, CD66, and CD14 human lineage-specific gene expression. Differential expression of 20 probe sets between groups was sufficient to develop a classifier with > 90% accuracy, correctly classifying 14/15 TOL cases and 15/17 non-TOL cases. These data suggest that differential gene expression can be utilized to accurately classify tolerant patients following HCT. Prospective investigation of immune tolerance biologic markers is warranted.     

Decreased Levels of Circulating IL17-Producing CD161+CCR6+ T Cells Are Associated with Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

The C-type lectin-like receptor CD161 is a well-established marker for human IL17-producing T cells, which have been implicated to contribute to the development of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT). In this study, we analyzed CD161⁺ T cell recovery, their functional properties and association with GVHD occurrence in allo-SCT recipients. While CD161⁺CD4⁺ T cells steadily recovered, CD161^hiCD8⁺ T cell numbers declined during tapering of Cyclosporine A (CsA), which can be explained by their initial growth advantage over CD161^neg/lowCD8⁺ T cells due to ABCB1-mediated CsA efflux. Interestingly, occurrence of acute and chronic GVHD was significantly correlated with decreased levels of circulating CD161⁺CD4⁺ as well as CD161^hiCD8⁺ T cells. In addition, these subsets from transplanted patients secreted high levels of IFNγ and IL17. Moreover, we found that CCR6 co-expression by CD161⁺ T cells mediated specific migration towards CCL20, which was expressed in GVHD biopsies. Finally, we demonstrated that CCR6⁺ T cells indeed were present in these CCL20⁺ GVHD-affected tissues. In conclusion, we showed that functional CD161⁺CCR6⁺ co-expressing T cells disappear from the circulation and home to GVHD-affected tissue sites. These findings support the hypothesis that CCR6⁺CD161-expressing T cells may be involved in the immune pathology of GVHD following their CCL20-dependent recruitment into affected tissues.     

Edema and Tetraparesis in a Miniature Pig after Allogeneic Hematopoietic Cell Transplantation

A 3-mo-old, 12-kg, intact, miniature pig presented with severe neurologic signs on day 8 after hematopoietic cell transplantation. This pig had received an immunosuppressive regimen before transplantation that included an antiCD3 immunotoxin for T-cell depletion, 100 cGy of total-body irradiation, and cyclosporine for 45 d. The pig began exhibiting erythematous lesions on posttransplantation day 7. He also demonstrated increased conscious proprioceptive deficits and recumbency but normal mentation. Neurologic signs worsened over several days; the pig became lethargic but remained afebrile. Conjunctival swelling developed on posttransplantation day 9, which subsequently spread to the animal''s head, ears and hocks by day 10. Analgesics were given for pain, and cyclosporine levels were decreased. Despite the measures taken, neurologic signs progressed. Given the worsening subcutaneous edema and neurologic status, Escherichia coli infection was suspected, and treatment with a third-generation cephalosporin was instituted. The clinical signs resolved within 12 h after the start of antibiotics. ‘Shiga-like’ toxin from E. coli can cause peracute toxemia and induce ataxia, paralysis, and recumbency. Other common and pathognomonic findings include periocular edema and variable edema in other subcutaneous regions. A fecal sample demonstrated an overgrowth of gram-negative, lactose-fermenting colonies. On the basis of the clinical presentation, exclusion of other potential conditions compatible with edema and neurologic diseases, physical exam findings, microbiology and the resolution of signs after therapy, the pig was diagnosed with edema disease.Abbreviations: HCT, hematopoietic cell transplantation; PTD, posttransplantation dayHematopoietic cell transplantation (HCT) is a common therapy for many hematologic malignancies including leukemia and lymphoma.⁵ Patients undergoing HCT are maintained under immunosuppression and are at increased risk of acquiring viral, bacterial, and fungal infections.In our laboratory we use miniature pigs of defined major histocompatibility complex as a large animal model for the study of HCT.^22,23,27,28 The pig is an excellent and well-accepted large animal model in biomedical research^15,19 Pigs that undergo HCT develop transplantation-related complications that are very similar to what is reported in the human clinic, such as posttransplantation lymphoproliferative disease^7,17 and thrombotic microangiopathy.¹⁸ However, swine pathogens can often be overlooked in lieu of typical transplantation-related complications. Vigilance for veterinary pathogens is important, and their identification is crucial. Failure to identify these pathogens may have a detrimental impact on experimental outcomes. Here we describe a case of edema disease in an immunosuppressed miniature pig after HCT. Edema disease due to Escherichia coli is a common condition in swine^11,13,35 and has been used as a model in microbiologic studies.⁶ The symptomatology resembled and overlapped with known and relatively common posttransplantation complications. For these reasons, diagnosis was particularly challenging in this case. To our knowledge, this is the first report of edema disease in an animal undergoing hematopoietic cell transplantation.     

造血干细胞移植条件对小鼠造血重建的影响

通过同种基因型小鼠构建造血干细胞移植模型,将预处理的全骨髓单个核细胞或c-Kit⁺造血干细胞移植至致死剂量照射的受体小鼠体内,动态监测移植2～16周后受体小鼠体内供体来源细胞造血重建以及嵌合情况,以期揭示不同群体的供体细胞以及预处理等因素对小鼠造血干细胞移植后造血重建的影响。实验结果显示,移植后早期(2周)全骨髓单个核细胞组髓系比例要高于c-Kit⁺细胞移植组,但全骨髓移植组受体小鼠呈现出较大的移植后不良反应,出现脱毛、食欲不振以及体重减轻的症状。c-Kit⁺细胞移植组在淋系重建上要早于全骨髓移植组,供体细胞的嵌合植入也早于全骨髓移植组,但两组实验组最终均能完成造血重建过程。实验结果表明c-Kit⁺细胞移植组在移植后能够较快地实现供体细胞植入,进而开始造血重建,且c-Kit⁺细胞移植组的不良反应要低于全骨髓移植组。结果说明在整体造血重建效果上c-Kit⁺细胞移植组要优于全骨髓移植组。     

Successful Fertility Restoration after Allogenic Hematopoietic Stem Cell Transplantation

ObjectiveMyeloablative conditioning regimens given prior to hematopoietic stem cell transplantation (HSCT) frequently cause permanent sterility in men. In patients with sickle cell disease (SCD) we use a nonmyeloablative regimen with sirolimus, alemtuzumab, and low-dose total-body irradiation (300 centigrays) with gonadal shielding preceding allogeneic HSCT. We report here the restoration of azoospermia in a patient with SCD after allogeneic HSCT. We discuss the impact of our patient’s underlying chronic medical conditions and the therapies he had received (frequent blood transfusions, iron chelating drugs, ribavirin, hydroxyurea, opioids), as well as the impact of the nonmyeloablative conditioning regimen on male gonadal function, and we review the literature on this topic.MethodsWe determined the patient’s reproductive hormonal values and his semen parameters before, during, and after HSCT and infertility treatment. In addition, we routinely measured his serum laboratory parameters pertinent to SCD and infertility, such as iron and ferritin levels. A karyotype analysis was performed to assess the potential presence of Klinefelter syndrome. Finally, imaging studies of the patient’s brain and testes were done to rule out further underlying pathology.ResultsA 42-year-old man with SCD, transfusional iron overload, and hepatitis C underwent a nonmyeloablative allogeneic HSCT. One year later he desired to father a child but was found to be azoospermic in the context of hypogonadotropic hypogonadism. Restoration of fertility was attempted with human chorionic gonadotropin (2,000 IU) plus human menopausal gonadotropin (75 IU follicle-stimulating hormone) injected subcutaneously 3 times weekly. Within 6 months of treatment, the patient’s serum calculated free testosterone value normalized, and his sperm count and sperm motility improved. After 10 months, he successfully initiated a pregnancy through intercourse. The pregnancy was uncomplicated, and a healthy daughter was delivered naturally at term.ConclusionDespite exposure to several gonadotoxins, transfusional iron overload and nonmyeloablative conditioning with radiation causing severe testicular atrophy suggesting extensive damage to seminiferous tubules and possibly Leydig cells, gonadotropins were efficacious in restoring our patient’s reproductive capability. (Endocr Pract. 2014;20:e157-e161)     

Immune Deficits in Allogeneic Hematopoietic Stem Cell Transplant (HSCT) Recipients

Immune deficits account for the high frequency of life threatening bacterial, viral, and fungal opportunistic infections seen in allogeneic HSCT recipients. Despite advances in infectious disease management, the integrity of host defenses remains the mainstay of defense. The intensity of the preparative regimen, degree of HLA matching, source of stem cells (marrow, blood, or cord), extent of T-cell depletion, and immunosuppressive therapy are some of the factors that impact the kinetics, characteristics, and quality of immune reconstitution. Graft-versus-host disease and its prophylaxis or treatment produce a host environment that is particularly vulnerable to infections. Mucosal disruption and prolonged severe neutropenia usually confine their impact to the early course of transplant. After initial engraftment, HSCT recipients remain at great risk for opportunistic infections and this is related to prolonged and severe T-lymphocyte dysfunction of a complex multifactorial nature. B cell dysfunction is less problematic clinically, but includes deficiencies of immunoglobulin subclasses and impaired ability to mount a vaccine response. Advances in understanding of these immune deficits have resulted in successful strategies including revaccination, growth factors, thymic protection, and adoptive cellular therapy with antigen-specific cells.     

Identification of Varicella-Zoster Virus-Specific CD8 T Cells in Patients after T-Cell-Depleted Allogeneic Stem Cell Transplantation

To study the role of CD8 T cells in the control of varicella-zoster virus (VZV) reactivation, we developed multimeric major histocompatibility complexes to identify VZV-specific CD8 T cells. Potential HLA-A2 binding peptides from the putative immediate-early 62 protein (IE62) of VZV were tested for binding, and peptides with sufficient binding capacity were used to generate pentamers. Patients with VZV reactivation following stem cell transplantation were screened with these pentamers, leading to the identification of the first validated class I-restricted epitope of VZV. In 42% of HLA-A2 patients following VZV reactivation, these IE62-ALW-A2 T cells could be detected ex vivo.Varicella-zoster virus (VZV) infects about 95% of the population, persists throughout life, and may lead to herpes zoster when the virus reactivates. After T-cell-depleted allogeneic stem cell transplantation (TCD alloSCT), reactivation of the virus leads to considerable morbidity (10). Primary infection elicits both humoral and cellular responses, but cellular immunity is essential for preventing herpes zoster. The VZV genome comprises more than 70 unique open reading frames that encode proteins that are coordinately expressed during replication. The product of open reading frame 62, the immediate-early 62 (IE62) protein, is required for the initiation of VZV replication (9) and is expressed at high levels before viral replication has occurred (8). Previous research has demonstrated that IE62-specific T cells were detected after primary VZV infection and in immune subjects (2, 4). In addition, T cells recognizing various other IE proteins and glycoproteins of VZV, as demonstrated by gamma interferon (IFN-γ) production upon stimulation with peptides or lysate derived from these proteins, have been described (1, 6, 13). The VZV-specific memory T cells found in these studies were predominantly CD4 T cells, while no VZV-specific CD8 T cells were demonstrated without prior in vitro expansion, possibly due to the low frequency of VZV-specific CD8 T cells or to the low sensitivity of the screening methods used to detect CD8 T cells by IFN-γ production upon stimulation. Frey et al. described CD8 epitopes of IE62 detected following in vitro restimulation. However, the HLA restriction and specificity of these T cells were not confirmed (4). Due to the lack of validated VZV-derived immunodominant peptides for major histocompatibility complex (MHC) class I, the analysis of VZV-specific CD8 T-cell responses is hampered (14). To be able to analyze the role of CD8 T cells in VZV reactivation, we therefore set out to identify epitopes for VZV by using VZV-IE62-specific MHC class I peptide complexes.The predictive algorithms BIMAS (11) and SYFPEITHI (12) were used to select potential HLA-A2 binding peptides from the IE62 protein. Peptides with a score of ≥3 (BIMAS) or ≥20 (SYFPEITHI) were considered to have potentially significant binding affinity. The 81 resulting 9-mer peptides were synthesized and tested for binding affinity with the REVEAL MHC-peptide binding assay (ProImmune, Oxford, United Kingdom). HLA-A2 binding affinity was determined by the ability of the peptides to stabilize the HLA-peptide complex. Based on the binding affinity measurements, 34 high- to medium-affinity HLA-A2 binding peptides were selected and used to generate ProVE MHC pentamers (ProImmune, Oxford, United Kingdom). To enable screening of this large number of pentamers, the pentamers were divided into five pools, each containing six or seven pentamers. In the initial screening with pooled pentamers, four HLA-A2-positive patients were screened after a clinical diagnosis of VZV reactivation after TCD alloSCT. The presence of viral DNA in plasma at the time of clinical observations of VZV reactivation was confirmed by real-time PCR on plasma samples as previously described (7). After informed consent was obtained, peripheral blood mononuclear cells (PBMCs) were cryopreserved and thawed and 0.5 × 10⁶ cells were incubated with pentamers at a concentration of 0.03 mg/ml for 10 min at room temperature in RPMI medium supplemented with 2% fetal bovine serum. After the cells were washed twice, 8 μl of FluoroTag-phycoerythrin (PE) was added for 20 min of incubation at 4°C and the cells were counterstained with CD4, CD40, and CD19-fluorescein isothiocyanate (FITC). Flow cytometric analysis was performed on a FACScalibur fluorescence-activated cell sorter (FACS; Becton-Dickinson [BD], San Jose, CA). In one of four patients, pentamer pool 6, containing pentamers 61, 62, 64, 65, 66, and 67, was positive (0.06% of CD8 T cells); no other positive signals were observed. Staining with the individual pentamers revealed that pentamer 66, containing the epitope ALWALPHAA derived from the IE62 protein of VZV (IE62-ALW-A2) was responsible for the positive signal (0.06% of CD8 T cells, Fig. ​Fig.1B1B).Open in a separate windowFIG. 1.Screening with pentamers containing VZV-derived immunogenic epitopes. PBMCs of a patient after VZV reactivation following TCD alloSCT were incubated with pentamers and then stained with FluoroTag-PE to detect the pentamer-positive cells (A and B) and counterstained with CD4-, CD40-, and CD19-FITC. Pentamer staining of the CD4-, CD40-, and CD19-negative cells is shown. (A) PBMCs stained with pentamer 67 containing the epitope ALPHAAAAV, showing no specific staining. (B) PBMCs stained with pentamer 66 containing the epitope ALWALPHAA, showing specific staining. IE62-ALW-A2-specific T-cell clones were sorted into a single cell per well and expanded nonspecifically. The clones were stained with an irrelevant tetramer (C) and the IE62-ALW-A2 tetramer (D) in combination with CD8-FITC. Clones 1 and 2 were stained with a Vβ kit (BD) to demonstrate that clone 1 (E) and clone 2 (F) express different T-cell receptors. The results demonstrate that we isolated different T-cell clones that specifically stain with the IE62-ALW-A2 tetramer.To confirm the specificity of the IE62-ALW-A2-specific T cells, the pentamer-positive T cells were sorted into a single cell per well with a FACSDiva (BD) and expanded as previously described (5). The expanded T-cell clones were labeled specifically with the IE62-ALW-A2 PE-conjugated tetramer that was constructed as previously described (3) (Fig. ​(Fig.1D),1D), and Vβ analysis with the T-cell receptor Vβ repertoire kit (BD) showed that at least two different T-cell clones were isolated, demonstrating the oligoclonal origin of IE62-ALW-A2-positive T cells (Fig. 1E and F). To assess the cytolytic capacity of IE62-ALW-A2 T cells, chromium release assays were performed as described earlier (5). ⁵¹Cr-labeled Epstein-Barr virus (EBV) lymphoblastoid cell lines (LCLs) loaded with the IE62-ALW peptide were incubated with IE62-ALW-A2 T cells for 4 h. As demonstrated in Fig. ​Fig.2A,2A, HLA-A2-positive EBV LCLs loaded with the IE62-ALW-A2 peptide were lysed by both T-cell clones, whereas unloaded EBV LCLs were not lysed. To determine the avidity of the T-cell clones, the IE62-ALW-A2 peptide was titrated on EBV LCLs, and after 24 h of coculture, supernatants were harvested and used to determine the IFN-γ production of the stimulated T cells by standard enzyme-linked immunosorbent assay. Half-maximum IFN-γ production of the T-cell clones was observed when the stimulator cells were loaded with 10 ng/ml peptide, indicative of high-avidity T-cell clones (Fig. ​(Fig.2B).2B). To determine whether the T cells recognized cells endogenously expressing the IE-62-encoding gene, COS-A2 cells were transfected with Lipofectamine (Invitrogen, Carlsbad, CA) by using pcDNA vectors coding for different VZV genes, which were kindly provided by E. Wiertz (Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands). The transfected COS-A2 cells were used 24 h after transfection as stimulator cells in this assay. After 24 h of coculture, supernatants were harvested and used to determine the IFN-γ production of the stimulated T cells. IE62-ALW-A2 T-cell clones produced IFN-γ in response to COS-A2 cells endogenously expressing the IE62 protein, as well as COS-A2 cells pulsed with the IE62-ALW-A2 peptide. No IFN-γ was produced when the COS-A2 cells were transfected with the IE63-encoding gene of VZV or pulsed with an irrelevant peptide (Fig. ​(Fig.2C2C).Open in a separate windowFIG. 2.IE62-ALW-A2 T cells recognize IE62-ALW-A2 peptide-loaded target cells and target cells endogenously expressing IE62. (A) The cytolytic activity of IE62-ALW-A2-positive T-cell clones 1 and 2 was analyzed with the ⁵¹Cr release assay. T cells were incubated for 4 h with IE62-ALW-A2 peptide (pep)-loaded or unloaded, HLA-A2-positive EBV LCLs at an effector-to-target ratio of 10:1. (B) IE62-ALW-A2 T-cell clone 1 was stimulated with HLA-A2-positive EBV LCLs loaded with different concentrations of the IE62-ALW-A2 peptide. Release of IFN-γ (pg/ml) after 24 h of stimulation is shown. (C) IE62-ALW-A2 T-cell clones 1 and 2 were stimulated with HLA-A2-positive COS-A2 cells, left untreated, or loaded with the IE62-ALW-A2 peptide or with the IE4-ALR-B8 peptide as an irrelevant peptide or transfected with the IE63-encoding gene (COS-A2-IE63) or the IE62-encoding gene (COS-A2-IE62). Release of IFN-γ (picograms per milliliter) after 24 h of stimulation is shown.To determine whether IE62-ALW-A2-specific T cells were present in healthy individuals, cryopreserved PBMCs from 18 healthy, VZV-seropositive, HLA-A2-positive individuals were screened with the PE-conjugated VZV tetramer. PBMCs were labeled with tetramers for 15 min at 37°C in RPMI medium without phenol supplemented with 2% fetal bovine serum, washed, and analyzed with a FACScalibur. In 3 of these 18 serologically VZV-positive individuals, IE62-ALW-A2 tetramer-positive T cells could be detected (range, 0.01 to 0.02% of CD8 T cells). These data demonstrate that IE62-ALW-A2-specific T cells can be observed and that the frequency of these T cells is low under steady-state conditions in immunocompetent persons.To assess the frequency of IE62-ALW-A2-specific T cells in a cohort of patient who suffered from VZV reactivation following TCD alloSCT, 19 HLA-A2-positive patients after VZV reactivation following TCD alloSCT were screened by using the IE62-ALW-A2 tetramer. We screened these patients at a median of 47 days after the clinical diagnosis of VZV reactivation. In 8 of these 19 patients, IE62-ALW-A2-specific T cells could be directly detected ex vivo (mean, 0.04% [range, 0.01 to 0.11%] of CD8 T cells), indicating that this epitope is recognized in 42% of the HLA-A2-positive patients during VZV reactivation (Table ​(Table1).1). In VZV-seronegative patients (six screened), no IE62-ALW-A2 tetramer-positive cells could be detected.

TABLE 1.

Presence of IE62-ALW-A2-specific T cells in HLA-A2 patients after VZV reactivation following TCD alloSCT

大鼠异基因骨髓移植急性移植物抗宿主病模型的建立

目的建立较稳定的异基因骨髓移植急性移植物抗宿主病动物模型,为异基因骨髓移植后的急性移植物抗宿主病（aGVHD）的相关研究提供实验参照。方法以雄性SD大鼠为供鼠,雌性Wistar大鼠为受鼠,受体大鼠随机分成A、B、C、D、E 5组,移植当天所有受鼠均接受8.5 GY的全身照射（TBI）,于照射后4～6 h内,A组回输等量培养液,B组经尾静脉输注供鼠骨髓细胞（2×10^8个/kg）,C、D、E组分别回输供鼠骨髓细胞（2×10^8个/kg）＋不同比例的脾细胞。观察各组大鼠生存期、外周白细胞计数、及有无aGVHD的临床及病理表现。结果A组大鼠于15d内全部死亡,外周血白细胞计数明显减低,骨髓病理示造血组织减少,提示死于造血衰竭。B、C、D、E组大鼠外周血白细胞计数均有明显恢复,B组大鼠8只存活超过50 d,C、D、E组大鼠均于50 d观察期内死亡,并有aGVHD的临床表现及病理表现,但C组大鼠aGVHD的程度较轻且时间不集中,其中D、E组大鼠可于相对集中的时间内观察到典型aGVHD临床及病理。结论TBI预处理的方式是可行的,单纯输入异基因骨髓细胞不能引起明显的aGVHD,骨髓细胞与脾细胞1∶1及1∶1.5混合组均可作为异基因骨髓移植后理想的aGVHD动物模型。     

胚胎干细胞移植治疗帕金森病

胚胎干细胞起源于植入前胚胎的内细胞团(inner cell mass,ICM),能够在体外进行增殖并能分化成三个胚层的所有细胞类型.如果能有效地将胚胎干细胞(embryonic stem cell,ES细胞)诱导分化为特定的细胞类型,ES细胞将成为细胞替代治疗中供体细胞的一个理想来源,从而可以应用于帕金森病(Parkinson's disease,PD)、糖尿病、心脓死等的退行性病变疾病的治疗.近年来,将ES细胞来源的多巴胺能神经元用于PD治疗的研究越来越广泛.现对这些研究中有关基因表达调控、实验研究进展、临床应用以及所遇到的问题作一综述.虽然这些研究还没能最终大面积应用于临床.但为PD的治疗提供了一个广阔的应用前景.     

Infusion of Endothelial Progenitor Cells Accelerates Hematopoietic and Immune Reconstitution, and Ameliorates the Graft-Versus-Host Disease After Hematopoietic Stem Cell Transplantation

Hematopoietic stem cells transplantation (HSCT) causes endothelial cell damage, disrupting hematopoietic microenviroment and leading to various complications. We hypothesized that infusion of endothelial progenitor cells (EPCs) may improve endothelium repair, facilitate hematopoietic reconstitution, and alleviate complications associated with HSCT. C57Bl6, and BALB/c mice received total body irradiation followed by infusion of C57Bl6-derived bone marrow (BM) cells, with or without concomitant infusion of C57Bl6-derived EPCs. The time course of hematopoietic and immune reconstitution and the severity of the graft-versus-host disease (GVHD) were monitored. Further, to confirm that EPCs promote endothelial cell recovery, HSCT mice were treated with anti-VE-cadherin antibody targeting the endothelium. The EPCs-treated mice exhibited accelerated recovery of BM vasculature, cellularity, hematopoietic stem and progenitor cell recovery, improved counts of lymphocyte subsets in peripheral blood, and facilitated spleen structure reconstruction. EPCs infusion also ameliorated the GVHD in the C57Bl6????BALB/c allo-HSCT model. Systemic administration of anti-VE-cadherin antibody significantly delayed hematological and immune reconstitution in the EPCs-infused mice. In conclusion, our data demonstrate that infusion of EPCs augments the hematopoietic and immune reconstitution, and alleviates the GVHD. These findings further highlight the relationship between the microvascular recovery, hematopoietic and immune reconstitution, and the GVHD.     

Assessing the Influence of Different Comorbidities Indexes on the Outcomes of Allogeneic Hematopoietic Stem Cell Transplantation in a Developing Country

Although the application of Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) has enabled better prediction of transplant-related mortality (TRM) in allogeneic hematopoietic stem cell transplants (AHSCT), data from developing countries are scarce. This study prospectively evaluated the HCT-CI and the Adult Comorbidity Evaluation (ACE-27), in its original and in a modified version, as predictors of post-transplant complications in adults undergoing a first related or unrelated AHSCT in Brazil. Both bone marrow (BM) and peripheral blood stem cells (PBSC) as graft sources were included. We analyzed the cumulative incidence of granulocyte and platelet recovery, sinusoidal obstructive syndrome, acute and chronic graft-versus-host disease, relapse and transplant-related mortality, and rates of event-free survival and overall survival. Ninety-nine patients were assessed. Median age was 38 years (18–65 years); HCT-CI ≥ 3 accounted for only 8% of cases; hematologic malignancies comprised 75.8% of the indications for AHSCT. There was no association between the HCT-CI or the original or modified ACE-27 with TRM or any other studied outcomes after AHSCT. These results show that, in the population studied, none of the comorbidity indexes seem to be associated with AHSCT outcomes. A significantly low frequency of high-risk (HCT-CI ≥ 3) in this Brazilian population might justify these results.