Translational Mini-Review Series on B cell subsets in disease. Reconstitution after haematopoietic stem cell transplantation - revelation of B cell developmental pathways and lineage phenotypes

Haematopoietic stem cell transplantation (HSCT) is an immunological treatment that has been used for more than 40 years to cure a variety of diseases. The procedure is associated with serious side effects, due to the severe impairment of the immune system induced by the treatment. After a conditioning regimen with high-dose chemotherapy, sometimes in combination with total body irradiation, haematopoietic stem cells are transferred from a donor, allowing a donor-derived blood system to form. Here, we discuss the current knowledge of humoral problems and B cell development after HSCT, and relate these to the current understanding of human peripheral B cell development. We describe how these studies have aided the identification of subsets of transitional B cells and also a robust memory B cell phenotype.     

Complications of haematopoietic stem cell transplantation

Allogeneic haematopoietic stem cell transplantation (HSCT) following myeloablative conventional conditioning regimen is associated with a higher incidence of transplant-related morbidity and mortality, limiting its applicability to younger patients without significant co-morbidities. The morbidity and mortality of HSCT are related closely to relapse of malignancy, conditioning-related toxicities, infection, and donor–recipient human leucocyte antigen (HLA) disparity. Disparities at minor histocompatibility antigens are thought to be responsible for acute graft-versus-host disease in patients receiving HSCT from a HLA-matched donor. Serious complications tend to erupt within specified periods of immunologic reconstitution after bone marrow grafting. Because their innate and acquired immune systems are especially vulnerable, HSCT recipients often have infectious and noninfectious complications. This article reviews the major complications of HSCT.     

Mucormycosis after allogeneic haematopoietic stem cell transplantation: a French Multicentre Cohort Study (2003-2008)

We conducted a nationwide retrospective study to evaluate clinical characteristics and outcome of mucormycosis among allogeneic haematopoietic stem cell transplant recipients. Twenty-nine patients were diagnosed between 2003 and 2008. Mucormycosis occurred at a median of 225 days after allogeneic haematopoietic stem cell transplant, and as a breakthrough infection in 23 cases. Twenty-six patients were receiving steroids, mainly for graft-versus-host disease treatment, while ten had experienced a prior post-transplant invasive fungal infection. Twenty-six patients received an antifungal treatment; surgery was performed in 12. Overall survival was 34% at 3 months and 17% at 1 year.     

Defective expression of apoptosis‐related molecules in multiple sclerosis patients is normalized early after autologous haematopoietic stem cell transplantation

Defective apoptosis might be involved in the pathogenesis of multiple sclerosis (MS). We evaluated apoptosis‐related molecules in MS patients before and after autologous haematopoietic stem cell transplantation (AHSCT) using BCNU, Etoposide, AraC and Melphalan (BEAM) or cyclophosphamide (CY)‐based conditioning regimens. Patients were followed for clinical and immunological parameters for 2 years after AHSCT. At baseline, MS patients had decreased proapoptotic BAD, BAX and FASL and increased A1 gene expression when compared with healthy counterparts. In the BEAM group, BAK, BIK, BIM_EL, FAS, FASL, A1, BCL2, BCLX_L, CFLIP_L and CIAP2 genes were up‐regulated after AHSCT. With the exception of BIK, BIM_EL and A1, all genes reached levels similar to controls at day + 720 post‐transplantation. Furthermore, in these patients, we observed increased CD8⁺ Fas⁺ T cell frequencies after AHSCT when compared to baseline. In the CY group, we observed increased BAX, BCLW, CFLIP_L and CIAP1 and decreased BIK and BID gene expressions after transplantation. At day + 720 post‐AHSCT, the expression of BAX, FAS, FASL, BCL2, BCLX_L and CIAP1 was similar to that of controls. Protein analyses showed increased Bcl‐2 expression before transplantation. At 1 year post‐AHSCT, expression of Bak, Bim, Bcl‐2, Bcl‐xL and cFlip‐L was decreased when compared to baseline values. In summary, our findings suggest that normalization of apoptosis‐related molecules is associated with the early therapeutic effects of AHSCT in MS patients. These mechanisms may be involved in the re‐establishment of immune tolerance during the first 2 years post‐transplantation.     

T‐cell responses after haematopoietic stem cell transplantation for aggressive relapsing–remitting multiple sclerosis

Autologous haematopoietic stem cell transplantation (HSCT) for relapsing–remitting multiple sclerosis is a potentially curative treatment, which can give rise to long‐term disease remission. However, the mode of action is not yet fully understood. The aim of the study was to evaluate similarities and differences of the CD4⁺ T‐cell populations between HSCT‐treated patients (n = 12) and healthy controls (n = 9). Phenotyping of memory T cells, regulatory T (Treg) cells and T helper type 1 (Th1) and type 17 (Th17) cells was performed. Further, T‐cell reactivity to a tentative antigen, myelin oligodendrocyte glycoprotein, was investigated in these patient populations. Patients treated with natalizumab (n = 15) were included as a comparative group. White blood cells were analysed with flow cytometry and T‐cell culture supernatants were analysed with magnetic bead panel immunoassays. HSCT‐treated patients had similar levels of Treg cells and of Th1 and Th17 cells as healthy subjects, whereas natalizumab‐treated patients had lower frequencies of Treg cells, and higher frequencies of Th1 and Th17 cells. Cells from HSCT‐treated patients cultured with overlapping peptides from myelin oligodendrocyte glycoprotein produced more transforming growth factor‐β₁ than natalizumab‐treated patients, which suggests a suppressive response. Conversely, T cells from natalizumab‐treated patients cultured with those peptides produced more interleukin‐17 (IL‐17), IL‐1 and IL‐10, indicating a Th17 response. In conclusion, we demonstrate circumstantial evidence for the removal of autoreactive T‐cell clones as well as development of tolerance after HSCT. These results parallel the long‐term disease remission seen after HSCT.     

Skewed T cell receptor repertoire of Vdelta1(+) gammadelta T lymphocytes after human allogeneic haematopoietic stem cell transplantation and the potential role for Epstein-Barr virus-infected B cells in clonal restriction

The proliferation of Vdelta1(+) gammadelta T lymphocytes has been described in various infections including human immunodeficiency virus (HIV), cytomegalovirus (CMV) and malaria. However, the antigen specificity and functions of the human Vdelta1(+) T cells remain obscure. We sought to explore the biological role for this T cell subset by investigating the reconstitution of T cell receptor (TCR) repertoires of Vdelta1(+) gammadelta T lymphocytes after human allogeneic haematopoietic stem cell transplantation (HSCT). We observed skewed TCR repertoires of the Vdelta1(+) T cells in 27 of 44 post-transplant patients. Only one patient developed EBV-associated post-transplant lymphoproliferative disorder in the present patient cohort. The -WGI- amino acid motif was observed in CDR3 of clonally expanded Vdelta1(+) T cells in half the patients. A skew was also detected in certain healthy donors, and the Vdelta1(+) T cell clone derived from the donor mature T cell pool persisted in the recipient's blood even 10 years after transplant. This T cell clone expanded in vitro against stimulation with autologous EBV-lymphoblastoid cell lines (LCL), and the Vdelta1(+) T cell line expanded in vitro from the same patient showed cytotoxicity against autologous EBV-LCL. EBV-infected cells could also induce in vitro oligoclonal expansions of autologous Vdelta1(+) T cells from healthy EBV-seropositive individuals. These results suggest that human Vdelta1(+) T cells have a TCR repertoire against EBV-infected B cells and may play a role in protecting recipients of allogeneic HSCT from EBV-associated disease.     

造血干细胞移植后免疫重建

造血干细胞移植（HSCT）已广泛应用于临床,是治疗血液系统疾患、实体瘤、基因缺陷及自身免疫性疾病的重要手段干细胞移植伴随着严重的免疫缺陷,这一时期,患者有合并严重感染的风险固有免疫恢复迅速．NK细胞、树突状细胞移植后数周内即可恢复。而适应性免疫系统恢复缓慢,T细胞和B细胞移植后数月才可恢复正常,而T细胞功能恢复需要数年的时间：研究造血系统各细胞存移植后数量、功能上的恢复规律,对如何加快免疫重建,降低移植死亡率有重要意义.     

Progress and prospects for the use and the understanding of the mode of action of autologous hematopoietic stem cell transplantation in the treatment of multiple sclerosis

Introduction: A substantial proportion of patients with multiple sclerosis (MS) do not respond to pharmacological treatments and no currently approved therapy has been convincingly demonstrated to prevent or stop disease progression. With MS widely believed to be an auto-immune disease, immunoablative therapy followed by autologous haematopoietic stem cell transplantation (I/AHSCT) is being investigated as an alternative therapeutic option.

Areas covered: With the results of phase III comparative trials only a few years away, this article reviews animal and clinical trials of I/AHSCT in the treatment of MS and discusses possible immunological mechanisms behind its action.

Expert commentary: I/AHSCT can induce long-term suppression of inflammatory disease activity and can halt or reverse neurological deterioration even in progressive stages of the disease, altering the fundamental disease course. However, toxicity of the therapy remains a problem and longer term follow up is required. Immunological investigations of the reconstituting immune system have discovered that qualitative changes take place at the cellular and molecular levels, which support the hypothesis of a ‘resetting’ of the immune system towards a tolerant and anti-inflammatory state.     

Immunological characteristics and T‐cell receptor clonal diversity in children with systemic juvenile idiopathic arthritis undergoing T‐cell‐depleted autologous stem cell transplantation

Children with systemic Juvenile Idiopathic Arthritis (sJIA), the most severe subtype of JIA, are at risk from destructive polyarthritis and growth failure, and corticosteroids as part of conventional treatment can result in osteoporosis and growth delay. In children where there is failure or toxicity from drug therapies, disease has been successfully controlled by T‐cell‐depleted autologous stem cell transplantation (ASCT). At present, the immunological basis underlying remission after ASCT is unknown. Immune reconstitution of T cells, B cells, natural killer cells, natural killer T cells and monocytes, in parallel with T‐cell receptor (TCR) diversity by analysis of the β variable region (TCRVb) complementarity determining region‐3 (CDR3) using spectratyping and sequencing, were studied in five children with sJIA before and after ASCT. At time of follow up (mean 11·5 years), four patients remain in complete remission, while one child relapsed within 1 month of transplant. The CD8⁺ TCRVb repertoire was highly oligoclonal early in immune reconstitution and re‐emergence of pre‐transplant TCRVb CDR3 dominant peaks was observed after transplant in certain TCRVb families. Further, re‐emergence of pre‐ASCT clonal sequences in addition to new sequences was identified after transplant. These results suggest that a chimeric TCR repertoire, comprising T‐cell clones developed before and after transplant, can be associated with clinical remission from severe arthritis.     

Diagnostic potential of tetramer-based monitoring of cytomegalovirus-specific CD8+ T lymphocytes in allogeneic stem cell transplantation

Cytomegalovirus (CMV) infection remains a significant problem in allogeneic stem cell transplant (SCT) recipients despite the availability of effective antiviral drugs. This problem concerns patients which are unable to mount an effective T-lymphocyte response against CMV. Therefore, the development and use of tetramer technology to enumerate CMV-specific T cells will be valuable to identify these patients as early as possible. Here, we review clinical studies in which CMV-specific CD8(+) T cells have been monitored in allogeneic SCT recipients using tetramers in the context of similar studies that are based on functional assays of CMV-specific T cells. The results thus far warrant the further development of tetramer technology as a diagnostic tool to monitor CMV-specific T cells in SCT recipients and other groups of immunocompromised patients threatened by CMV.     

自体外周血造血干细胞移植后淋巴细胞亚群的恢复

目的探讨自体外周血造血干细胞移植(auto-PBSCT)后免疫重建的规律。方法测定血液肿瘤患者auto-PBSCT前后淋巴细胞各亚群的绝对值。结果部分亚群在移植前低于正常。移植后各细胞亚群恢复时间不同,其中NK细胞在6个月恢复;B细胞在9个月恢复正常。T细胞总数在9个月恢复正常,其中主要为CD3 CD8 细胞。CD3 CD4 细胞在18个月恢复,而CD4 CD28 细胞在2年内仍未恢复。CD3 CD8 细胞在移植后早期有所下降,在3个月时恢复并高于正常,至9个月时逐渐下降至正常。CD3 CD8 细胞的主要部分是免疫表型呈活化的T细胞,即CD8 HLA-DR 细胞和CD8 CD38 细胞。两者分别在1个月和3个月时高于正常,在9个月时恢复正常。结论血液肿瘤患者移植前即存在淋巴细胞亚群异常的前提下,移植后NK细胞最先恢复,其次为B细胞,T细胞各亚群中CD3 CD8 细胞恢复较早,其中主要为活化T细胞。     

Cellular immune function monitoring after allogeneic haematopoietic cell transplantation: evaluation of a new assay

Managing the patient's immune system after haematopoietic cell transplantation (HCT) is a challenge, mainly in the unstable period immediately after the transplant. Currently there is no standardized non‐invasive diagnostic tool for the evaluation of immunological complications such as graft‐versus‐host disease (GVHD) and for managing the cellular immune function of the transplant recipient. The ImmuKnow assay for cellular immune function monitoring has been incorporated successfully into the clinical follow‐up routine of solid organ transplant recipients. This study aims to explore the relevance and potential contribution of immune monitoring using the assay in the setting of HCT. We found that ImmuKnow‐level measurement can distinguish between states of immune function quiescence and between events of acute GVHD. ImmuKnow levels were significantly higher in patients going through GVHD than the levels measured for the same patients during immunological stability. Moreover, we demonstrate a patient case where longitudinal monitoring using the ImmuKnow assay provided a trustworthy depiction of the patient's cellular immune function post‐HCT. In conclusion, we provide evidence for the potential contribution of the ImmuKnow assay for longitudinal individualized cellular immune function monitoring of patients following HCT. Further studies are necessary in order to establish the optimal practice for utilizing the assay for this purpose.     

Differential diagnosis of skin lesions after allogeneic haematopoietic stem cell transplantation

Allogeneic haematopoietic stem cell transplantation (i.e. bone marrow or peripheral blood stem cell transplantation) is a common procedure in the treatment of various haematological disorders such as aplastic anaemia, (pre)leukaemias, some malignant lymphomas, multiple myeloma and immunodeficiency states. Many of these patients develop erythematous skin lesions following transplantation. Although graft- versus-host disease is the major differential diagnosis in these situations, many other causes of erythema are encountered. The large number of transplant patients means that more and more pathologists are confronted with the challenging problem of making a correct diagnosis in these situations. In this review article we therefore describe the different causes of erythema and their differential diagnoses. In most cases the clinical presentation is related to the microscopical features. Besides acute and chronic graft-versus-host disease, we discuss the (common) drug reactions and non-specific features such as Sweet's syndrome, erythema nodosum and eosinophilic folliculitis. In addition, we deal with the recurrence of original diseases and infections. With this knowledge every pathologist should feel comfortable when looking at skin biopsies of patients after haematological stem cell transplantation.     

Tweaking the immune system: Gene therapy-assisted autologous haematopoietic stem cell transplantation as a treatment for autoimmune disease

Autoimmune diseases represent a major challenge for medical research. The aberrant self-recognition by the immune system leads to a range of pathologies for which cures have not been forthcoming. Treatments are commonly non-specific and often lead to unwanted side-effects. A number of strategies are currently being explored to tackle autoimmunity; aimed at eliminating existing pathogenic clones and the induction of immune tolerance through resetting or regulating the immune system. Autologous haematopoietic stem cell transplantation (HSCT) is one such strategy and is being trailed in a number of autoimmune diseases. However, a common feature of this strategy is disease relapse and may indicate incomplete tolerance mechanisms. It is well known that bone marrow derived cells have a major influence on immune tolerance. It is also well documented that ectopic expression of antigens within the immune system can promote robust tolerance. This review considers these observations in the context of promoting a strategy involving genetic manipulation of haematopoietic stem cells together with HSCT to induce immune tolerance and tackle autoimmunity.     

Incidence of invasive fungal disease after unmanipulated haploidentical stem cell transplantation was significantly higher than that after HLA-matched sibling transplantation

The aim of this study was to determine the incidence, clinical features and outcome of invasive fungal disease (IFD) after either unmanipulated haploidentical haematopoietic stem cell transplantation (HSCT) or human leukocyte antigen (HLA)-matched sibling HSCT. This was a head-to-head comparative study performed at a single centre. Patients were admitted between 2007 and 2010, and IFD was evaluated according to the revised EORTC/MSG criteria, with only proven and probable cases included. Of the 1042 consecutive patients enrolled, 390 received the HLA-matched HSCT and 652 received unmanipulated haploidentical HSCT. A total of 61 (5.8%) patients had IFD, including 15 proven cases and 46 probable cases. The incidence of IFD after unmanipulated haploidentical HSCT was significantly higher than that after HLA-matched transplantation (7.1% vs. 3.3%, respectively; p 0.007). IFD occurred later in patients receiving HLA-matched transplantation compared with patients receiving unmanipulated haploidentical HSCT (141.5 vs. 23 days, respectively; p 0.04). In multivariate analysis, acute graft-versus-host disease (GVHD) grades III to IV (HR = 2.214, 95% CI, 1.139–4.304; p 0.019), extensive chronic GVHD (HR = 2.413, 95% CI, 1.377–4.228; p 0.002) and haploidentical transplantation (HR = 2.648, 95% CI, 1.111–6.310; p 0.028) were identified as significant risk factors associated with IFD. The response to antifungal therapy and the IFD-attributable mortality were similar between the two types of transplantation. In conclusion, patients who received unmanipulated haploidentical HSCT had a higher risk of IFD than those patients who received HLA-matched HSCT, but the prognosis of IFD was not associated with the HLA type.     

Up-regulation of fas and fasL pro-apoptotic genes expression in type 1 diabetes patients after autologous haematopoietic stem cell transplantation

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T cell-mediated destruction of pancreatic β cells, resulting in insulin deficiency and hyperglycaemia. Recent studies have described that apoptosis impairment during central and peripheral tolerance is involved in T1D pathogenesis. In this study, the apoptosis-related gene expression in T1D patients was evaluated before and after treatment with high-dose immunosuppression followed by autologous haematopoietic stem cell transplantation (HDI-AHSCT). We also correlated gene expression results with clinical response to HDI-AHSCT. We observed a decreased expression of bad, bax and fasL pro-apoptotic genes and an increased expression of a1, bcl-x(L) and cIAP-2 anti-apoptotic genes in patients' peripheral blood mononuclear cells (PBMCs) compared to controls. After HDI-AHSCT, we found an up-regulation of fas and fasL and a down-regulation of anti-apoptotic bcl-x(L) genes expression in post-HDI-AHSCT periods compared to pre-transplantation. Additionally, the levels of bad, bax, bok, fasL, bcl-x(L) and cIAP-1 genes expression were found similar to controls 2 years after HDI-AHSCT. Furthermore, over-expression of pro-apoptotic noxa at 540 days post-HDI-AHSCT correlated positively with insulin-free patients and conversely with glutamic acid decarboxylase autoantibodies (GAD65) autoantibody levels. Taken together, the results suggest that apoptosis-related genes deregulation in patients' PBMCs might be involved in breakdown of immune tolerance and consequently contribute to T1D pathogenesis. Furthermore, HDI-AHSCT modulated the expression of some apoptotic genes towards the levels similar to controls. Possibly, the expression of these apoptotic molecules could be applied as biomarkers of clinical remission of T1D patients treated with HDI-AHSCT therapy.     

乙型肝炎患者骨髓造血干细胞来源的树突状细胞表型分析

目的 检测乙型肝炎患者骨髓造血干细胞（HSC）诱导的树突状细胞（DC）的表面分子的表达,评价其相关因素及临床意义.方法 收集慢性乙型肝炎患者的骨髓液9例,健康者7例后用磁珠分离仪分离纯化骨髓液CD34＋细胞,在含有干细胞生长因子（SCF）、酪氨酸激酶受体家族Ⅲ的配体（FLT3）、促血小板生成素（TPO）、IL-3和10%FBS的IMDM培养基中孵育并进行扩增,在干细胞扩增基础上,在GM-CSF和IL-4作用下诱生DC,通过流式细胞仪分析其表面分子的表达并对DC进行形态学观察.结果 乙型肝炎患者HSC经诱导为DC后,其免疫表型CD80、CD86、CD1a的表达均低于正常对照组,差异有统计学意义：CD1a（t=3.94,P＜0.05）,CD80（t=7.08,P＜0.01）,CD86（t=3.65,P＜0.05）,而HLA-DR表达与正常组比较差异无统计学意义（t=0.34,P＞0.05）.结论 慢性乙型肝炎患者HSC来源的部分DC的表面分子表达低下可能与HBV感染HSC后出现病态的免疫细胞分化有关.     

Allogeneic haematopoietic stem cell transplantation in patients with human immunodeficiency virus: the experiences of more than 25 years

For treatment of several malignancies, transplantation of allogeneic haematopoietic stem cells (HSCT) derived from bone marrow or peripheral blood has been used as a therapeutic procedure for decades. In the past, HSCT has been suggested as a treatment option for infection with the human immunodeficiency virus type 1 (HIV-1), but these attempts were mostly unsuccessful. Today, after the introduction of an active anti-retroviral therapy, the lifetime expectancy of HIV-infected patients has improved substantially, but nevertheless the incidence rate of malignancies in these patients has increased considerably. Therefore, it can be assumed that there will be a rising necessity for HIV-1-infected patients with malignancies for allogeneic HSCT. At the same time, there is increasing interest in treatment methods which might target the HIV-1 reservoir more effectively, and the question has been raised as to whether allogeneic HSCT could be linked to such strategies. In this paper the data of more than 25 years experience with allogeneic HSCT in patients with HIV-1 are reviewed and analysed.     

Morphology of the bone marrow after stem cell transplantation

In many haematological conditions the only curative option is stem cell (SCT) or bone marrow (BM) transplantation. Little information exists about BM morphology following non-ablative engraftment. During the pretransplantation period and depending on the kind of pretreatment, there may be hypoplasia, residual disease and varying degrees of fibrosis. In the post-transplantation period, after 1-3 weeks of transfusion-dependent pancytopenia, the first signs of successful engraftment are indicated by the recurrence of neutrophils, monocytes and erythrocytes in the peripheral blood. In the BM there is slow regeneration of erythropoiesis, followed by the other lineages of haematopoiesis and increase in reticulin fibres or even a resolution of fibrosis. Diagnostic problems arise when neoplastic lympho- or haematopoiesis are maintained following transplantation. Moreover, there may be a significant graft versus tumour response reaction or an already relapsing disease needing aggressive treatment. On the other hand, a conspicuous dyshaematopoiesis should not be mistaken as representing a myelodysplastic syndrome. The presence of granulomas being treatment-related or a manifestation of intercurrent granulomatous disease has to be considered. More advanced knowledge of the histological features of regenerating BM will certainly aid the recognition of relapsing disease and is needed for the adequate reporting of post-transplant alterations associated with a successful or failing engraftment.     

Gonadal status in reproductive age women after haematopoietic stem cell transplantation for haematological malignancies

BACKGROUND: Ovarian failure is a frequent complication occurring after haematopoietic stem cell transplantion (SCT), which is generally ascribed to radiation treatment and antiblastic alkylating agents. METHODS: Ovarian morphology and function were studied in reproductive age women 12-24 months after allogeneic SCT (n = 23) received from an HLA identical sibling, or autologous SCT (n = 22). Thirteen allo-transplanted women were suffering from chronic graft-versus-host disease (cGVHD). RESULTS: Menstrual cycles recovered in two and four women in the allo- and auto-SCT groups respectively, being associated with younger age and longer period elapsed from transplant. There was no difference in previous use of alkylating agents between allo- and auto-transplantation, while corticosteroid treatment was longer and more recent in the allo-SCT group. Significantly higher gonadotrophin levels and lower estradiol were seen in the combined group of patients than in controls. In allo-transplanted women, androgens were also significantly lower than in controls. Ovarian and uterine volumes were lower in patients than in controls, and in the allo- than in the auto-transplanted women. Within the allo-SCT group, endocrine function and ovarian and uterine volumes were significantly lower in the patients suffering from cGVHD. CONCLUSIONS: Ovarian failure in SCT recipients is likely to be caused principally by myelo-ablative treatments, but the condition of gonadal and androgen insufficiency can be worsened by an altered immunomodulation in allogeneic setting.