Purified hematopoietic stem cell allografts reconstitute immunity superior to bone marrow

Antigen-specific immune responses are impaired after allogeneic hematopoietic cell transplantation (HCT). The events contributing to this impairment include host hematolymphoid ablation and donor cell regeneration, which is altered by pharmacologic immune suppression to prevent graft-versus-host disease (GVHD). A generally accepted concept is that graft T cell depletion performed to avoid GVHD yields poorer immune recovery because mature donor T cells are thought to be the major mediators of protective immunity early post-HCT. Our findings contradict the idea that removal of mature donor cells worsens immune recovery post-HCT. By transplantation of purified hematopoietic stem cells (HSC) compared with bone marrow (BM) across donor and recipient pairs of increasing genetic disparity, we show that grafts composed of the purified progenitor population give uniformly superior lymphoid reconstitution, both qualitatively and quantitatively. Subclinical GVHD by T cells in donor BM likely caused this lympho-depleting GVHD. We further determined in the major histocompatibility complex (MHC)-mismatched pairs, that T cell restricted proliferative responses were dictated by donor rather than host elements. We interpret these latter findings to show the importance of peripheral antigen presentation in the selection and maintenance of the T cell repertoire.     

Cytokine requirement for the development of T-lymphoid lineage potential in clonal lymphohaematopoietic progenitors in vitro

The early process of T-cell development prior to thymic colonization has been poorly investigated because of the lack of a sensitive assay. We have developed a two-step in vitro culture system by combining a clonal culture with a fetal thymus organ culture (FTOC) and analysed the early development of T cells from lymphohaematopoietic progenitors. Cells of immature colonies derived from bone marrow cells of 5-fluorouracil (5FU)-treated mice using various combinations of early acting cytokines were transferred into a FTOC. All the combinations of stem cell factor (SCF), interleukin (IL)-3 and IL-6 capable of inducing colony formation supported T-cell generation. IL-11 and the Flt3 ligand possessed T-lineage promotional effects similar to IL-6 and SCF respectively. However, there were some quantitative differences in the final T-cell yield among cytokine combinations. Thus, the commitment towards T lineage in lymphohaematopoietic progenitors may be an event determined intrinsically rather than induced by specific stimuli, but there may be a hierarchy between the activity of cytokines in further development. Furthermore, we examined the T-lineage potential of individual colonies derived from Lin(-)c-Kit(+)Sca-1(+) cells clone-sorted from post-5FU marrow cells. No colonies that contained only myelocytic progenitors showed T-lineage potential, but 23.3% of colonies with a haematopoietic multipotentiality did. Therefore, the divergence of the T lineage from other lineages such as myeloid potential may occur at an early stage of the hierarchy of haematopoiesis. The proposed method should prove valuable for exploring the molecular and cellular changes that occur during early T-cell development before thymic colonization.     

Quantification of progenitors capable of generating T cells in human cord blood

Objective:  For transplantation of cord blood (CB) cells, it is important to select a CB sample that can reconstitute not only myelo-erythropoiesis but also lymphopoiesis in recipients. However, until now the reconstitution ability of CB samples has been assessed by colony forming unit-culture (CFU-C) assay or by simply counting CD34⁺ cells. The present study aims at establishing a method capable of assessing the potential of T lymphopoieses of CB samples.
Methods:  CD34⁺CD38⁻ cells sorted from CB were cultured on a monolayer of murine stromal cell line TSt-4, transduced with the human Delta-like 1 gene.
Results:  Immature T cells expressing CD5 and/or CD7 were generated in the culture. As these immature T cells can easily be discriminated from mature T cells that are included in the mononuclear cell population (MNCs), we can use the MNCs as starting material for quantification of progenitors capable of generating T cells (TGP). By applying a limiting dilution analysis, we succeeded in determining the frequency of TGP in MNCs. It was found that the ratios for the number of TGP vs. that of CFU-C differ among CB samples maximally by 3.5 times.
Conclusion:  The present assay system provides a novel tool for the evaluation of CB samples, especially for their T-cell-generating potential.     

Relationship between Thy-1 expression and cell-cycle distribution in human bone marrow hematopoietic progenitors

Analysis of the relationship between Thy-1 expression and cell-cycle distribution of hematopoietic stem cells (HSCs) showed that freshly isolated Thy-1+ and Thy-1- subsets of the CD34highCD38-flt-3-Lin- population were predominantly in G0/G1 phase and remained essentially quiescent, whereas after 6 days of cytokine stimulation, the Thy-1+ subset of the population entered the cycling state while the Thy-1- subset again remained quiescent. Expression of Thy-1 antigen resulted in a drastic increase in the percentage of cycling cells in CD34highCD38-flt-3-Lin-Thy-1+- as well as CD34highCD38-flt-3-Lin- Thy-1(-)-cell-initiated cultures. The Thy-1+ subset of the CD34highCD38-flt-3-Lin- population exists in the freshly isolated CD34highCD38-flt-3-Lin- Thy-1+ fraction, loses Thy-1 expression during 6 days, and re-expresses Thy-1 for an additional 2 days. Cell-cycle analysis demonstrated that this unique subset contains abundant S/G2M cells. Thus, Thy-1 expression appears to be an indicator of cell-cycle phase in targeting HSC, which might serve in the cell subset best suited for gene transfer.     

小鼠骨髓干细胞诱导分化为肝细胞的实验研究

目的探讨成年小鼠骨髓干细胞在肝细胞生长因子(HGF)作用下分化为肝细胞的可能性及其分化特性，为肝细胞移植提供实验基础。方法HGF100ng／ml体外诱导小鼠骨髓干细胞向肝细胞分化，于诱导的第0、7、l4、21、28天，观察细胞形态特征；半定量逆转录聚合酶链反应(RT-PCR)法检测细胞白蛋白(ALB)、甲胎蛋白(AFP)mRNA水平的表达；免疫细胞化学法检测ALB、AFP和细胞角蛋白l9(CK19)蛋白水平的表达。结果新鲜分离的骨髓干细胞ALB及AFP mRNA呈弱阳性。在非诱导组，培养7d时ALB mRNA已检测不到，AFP mRNA明显减弱，14d时消失。在诱导组，7d时ALB mRNA检测不到，14d时再次出现阳性条带，21d时表达量最大，而AFP mRNA在诱导组始终呈阳性，14d时表达量最大，以后逐渐减少。免疫细胞化学结果与RT-PCR结果基本一致，但CK19蛋白始终阴性。结论小鼠骨髓干细胞在HGF单独作用下能诱导分化为肝细胞样细胞，诱导分化的最佳时期是2～3周。     

Incubation of murine bone marrow cells in hypoxia ensures the maintenance of marrow-repopulating ability together with the expansion of committed progenitors

We developed previously a hypoxic culture system in which progenitors endowed with marrow-repopulating ability (MRA), unlike committed progenitors, were selected and maintained better than in air. We report here an improvement to this system targeted at combining the maintenance of progenitors sustaining MRA with the numerical expansion of multipotent and committed progenitors. Murine bone marrow cells were incubated at 1% oxygen in liquid medium supplemented with stem cell factor, granulocyte colony-stimulating factor, interleukin-6 and interleukin-3. In day 8 hypoxic cultures, the numbers of high proliferative potential and granulocyte/macrophage colony-forming cells (HPP-CFC and CFU-GM) were increased with respect to time zero. Colonies generated by HPP-CFC derived from hypoxic cultures exhibited a high replating ability, whereas colonies generated by HPP-CFC derived from control cultures exhibited a low replating ability. MRA was fully maintained in hypoxia and markedly reduced in air. Thus, severe hypoxia is able to ensure a full maintenance of progenitors sustaining MRA, together with a significant expansion of in vitro-detectable clonogenic progenitors, including those endowed with replating ability. This system could contribute to the improvement of current techniques for the in vitro treatment of human haematopoietic cell populations before transplantation.     

Immune reconstitution to cytomegalovirus following partially matched‐related donor transplantation: impact of in vivo T‐cell depletion and granulocyte colony‐stimulating factor‐primed peripheral blood/bone marrow mixed grafts

Cytomegalovirus (CMV) infection and delayed immune reconstitution remains a serious obstacle for successful partially matched‐related donor transplantation (PMRD). We evaluated 42 patients for the development of CMV‐specific CD8+ T lymphocytes (CTL_CMV) following granulocyte colony‐stimulating factor‐primed peripheral blood (PB) and bone marrow (BM) with anti‐thymocyte globulin (ATG)‐based PMRD. PMRD recipients achieved a high frequency, proliferation capacity, and interferon‐γ response of CTL_CMV at 1 year post transplantation. CTL_CMV with the central memory CD45RO+CD62L+ cell phenotype expanded in PB and BM‐resident CTL_CMV displayed distinct phenotypes when CMV was reactivated. Although the incidence of CMV reactivation was high in PMRD patients (87.67%), only 11.90% of them developed CMV disease. In conclusion, after PMRD using mixed grafts with ATG‐based conditioning, immune recovery to CMV seems to be early and fast, thereby reducing the incidence of CMV disease.     

Human bone marrow stromal cells simultaneously support B and T/NK lineage development from human haematopoietic progenitors: a principal role for flt3 ligand in lymphopoiesis

The regulation of human early lymphopoiesis remains unclear. B- and T-lineage cells cannot develop simultaneously with conventional stromal cultures. Here we show that telomerized human bone marrow stromal cells supported simultaneous generation of CD19(+) CD34(lo/-) CD10(+) cyCD79a(+) CD20(+/-) VpreB(-) pro-B cells and CD7(+) CD34(+) CD45RA(+) CD56(-) cyCD3(-) early T/Natural Killer (NK) cell precursors from human haematopoietic progenitors, and the generation of both lymphoid precursors was promoted by flt3 ligand (flt3L). On the other hand, stem cell factor or thrombopoietin had little or no effect when used alone. However, both acted synergistically with flt3L to augment the generation of both lymphoid precursors. Characteristics of these lymphoid precursors were evaluated by gene expression profiles, rearrangements of IgH genes, or replating assays. Similar findings were observed with primary human bone marrow stromal cells. Notably, these two lymphoid-lineage precursors were generated without direct contact with stromal cells, indicating that early B and T/NK development can occur, at least in part, by stromal cell-derived humoral factors. In serum-free cultures, flt3L elicited similar effects and appeared particularly important for B cell development. The findings of this study identified the potential of human bone marrow stromal cells to support human early B and T lymphopoiesis and a principal role for flt3L during early lymphopoiesis.     

Selective secretion of chemoattractants for haemopoietic progenitor cells by bone marrow endothelial cells: a possible role in homing of haemopoietic progenitor cells to bone marrow

To elucidate the mechanisms by which haemopoietic progenitor cells lodge in the bone marrow, we examined the secretion of chemoattractants for haemopoietic progenitor cells by bone marrow and lung endothelial cells. The bone marrow endothelial cells, but not lung endothelial cells, secreted chemoattractants for the haemopoietic progenitor cell line, FDCP-2, and normal haemopoietic progenitor cells. Checkerboard analysis demonstrated that the conditioned medium of the bone marrow endothelial cells had chemotactic activity and random motility-stimulating activity. The bone marrow endothelial cells expressed stromal-cell-derived factor-1 (SDF-1) mRNA and produced SDF-1 protein, whereas the lung endothelial cells did not. Adhesion of FDCP-2 cells to the bone marrow endothelial cells was partially inhibited by anti-SDF-1 antibody. These findings suggest that the chemoattractants for haemopoietic progenitor cells including SDF-1 and random motility-stimulating factor(s) selectively secreted by the bone marrow endothelial cells may contribute to the homing of haemopoietic progenitor cells to bone marrow.     

Early suppressive effects of chemotherapy and cytokine treatment on committed versus primitive haemopoietic progenitors in patient bone marrow

These studies investigated the effectiveness of in vivo administration of cytokines in ameliorating potential marrow damage induced by chemotherapy. Breast cancer patients received 5-fluorouracil, leucovorin, doxorubicin and cyclophosphamide (FLAC) followed by either GM-CSF, PIXY321, or no cytokine. Marrow was obtained before and after one or two cycles of FLAC once blood cell counts had recovered. Colony-forming units for granulocytes and macrophages (CFU-GM) were used to indicate the effect of therapy on recovery of committed progenitor cells responsible for early blood cell recovery. The frequency and number of CFU-GM in marrow obtained after FLAC + PIXY321 were significantly lower than in marrow obtained after FLAC + GM-CSF or FLAC without cytokine. CD34⁺ cell numbers were also reduced after FLAC + PIXY321. CFU-GM production in marrow long-term cultures (LTC) was used to assess the effect of therapy on primitive progenitors. After 5 weeks the number of CFU-GM in LTC of post-therapy marrow from all three treatment arms was < 15% of the number in pre-therapy LTC. Suppressive effects of FLAC on primitive progenitors were observed even when committed progenitors and CD34⁺ cells had recovered to pre-therapy levels. These results demonstrate that cytokine treatment did not ameliorate suppressive or toxic effects of FLAC on the functional integrity of the marrow.     

Identification of an early T cell progenitor for a pathway of T cell maturation in the bone marrow

We have identified a rare ( approximately 0.05-0.1%) population of cells (Thy-1(hi)CD16(+)CD44(hi)CD2(-)TCRalphabeta(-)B220(-)M ac-1(-)NK1. 1(-)) in the adult mouse bone marrow that generates CD4(+) and CD8(+) TCRalphabeta(+) T cells after tissue culture for 48 hr in the presence of Ly5 congenic marrow cells. The essential stages in the maturation of the progenitors were determined; the stages included an early transition from CD2(-)CD16(+)CD44(hi)TCRalphabeta(-) to CD2(+)CD16(int/-)CD44(int/-)TCRalphabeta(-) cells, and a later transition to CD4(+)CD8(+)TCRalphabeta(+) double-positive T cells that rapidly generate the CD4(+) and CD8(+) single-positive T cells. The maturation of the progenitors is almost completely arrested at the CD2(+)TCRalphabeta(-) stage by the presence of mature T cells at the initiation of cultures. This alternate pathway is supported by the marrow microenvironment; it recapitulates critical intermediary steps in intrathymic T cell maturation.     

Analysis of the Expression of the TRBC1 in T lymphocyte tumors

T cell therapy represents a new class of immunotherapies garnering considerable attention. T cell receptor beta chain constant region 1 (TRBC1) is partially expressed in subsets of normal T cells. However, the immunotherapy of T lymphocyte tumors is rarely validated in clinical trials. Here, we aim to explore whether TRBC1 is a promising target for the immunotherapy of T lymphocyte tumors. This study examined TRBC1 expression in 25 healthy bone marrow samples, 39 patients with T-lineage acute lymphocytic leukemia (T-ALL), 4 patients with mature T cell neoplasms, and 5 patients suspected with mature T cell neoplasms with evidence of T cell neoplasia. Moreover, the expression of TRBC1 was evaluated by flow cytometry and through PCR detection of TCR gene rearrangements. The expression of monophasic TRBC1 was identified in all 25 normal bone marrows (23.83% ± 2.74% positive rate). The expression of TRBC1 was positive in 5 patients (12.8%) among the 39 T-ALL patients. TRBC1 was partially expressed in 1 patient (25%) with T cell non-Hodgkin''s lymphoma (T-NHL) and in 1 patient (20%) suspected to have T-NHL. Healthy donors showed a pattern of partial expression and patients with T-lymphocyte tumors showed a polytypic TRBC1 expression pattern. Thus, TRBC1 may be a diagnostic and therapeutic marker for T lymphocyte tumors.     

Megakaryocyte progenitors in the bone marrow and peripheral blood of patients with myeloproliferative diseases

We studied the behavior in culture of megakaryocyte progenitor cells (CFU-Mk) from peripheral blood (PB) and bone marrow (BM) cells in eight patients with myeloproliferative diseases (MPD). In seven patients we observed megakaryocyte (Mk) colony formation from PB cells, which were generated in the absence of any added stimulator and which did not increase after the addition of a source of Mk-colony stimulating activity (CSA-Mk). The number of BM CFU-Mk was significantly higher in patients than in controls, and in seven out of eight patients the responsiveness to added CSA-Mk was retained. Plasma obtained from six patients did not stimulate normal donors' BM target cells to form Mk colonies. These data demonstrate an expansion of the CFU-Mk pool in MPD patients without increased plasma levels of CSA-Mk, and suggest that PB and BM CFU-Mk of MPD patients might have different kinetic properties.     

Frequency of clonal intraepithelial T lymphocyte proliferations in enteropathy-type intestinal T cell lymphoma, coeliac disease, and refractory sprue

BACKGROUND: Clonal T cell receptor (TCR) gene rearrangements and loss of T cell antigens such as CD8 and TCR-beta in intraepithelial lymphocytes (IELs) may indicate the development of an enteropathy-type intestinal T cell lymphoma (EITCL) in patients with refractory sprue. AIMS: To define the diagnostic value of these markers in duodenal biopsies from patients with villous atrophy as a result of various underlying disorders. PATIENTS AND METHODS: Duodenal biopsies from eight patients with coeliac disease and five patients with villous atrophy caused by defined disorders were compared with three patients with refractory sprue evolving into overt EITCL, two patients with ulcerative jejunitis, and with eight patients with overt EITCL, for expression of CD3, CD4, CD8, and TCR-beta in IELs using immunohistochemistry and for clonal TCR-gamma gene rearrangements using polymerase chain reaction. In addition, biopsies from six consecutive patients with refractory sprue of uncertain cause were examined. RESULTS: Clonal TCR-gamma gene rearrangements were found in all resected tumours of patients with EITCL, in 3/8 duodenal biopsies of patients with EITCL, in 2/2 patients with ulcerative jejunitis, in 2/3 patients with refractory sprue evolving into overt EITCL, and in 1/6 patients with refractory sprue. No rearrangements were found in biopsies from patients with refractory sprue caused by defined disorders or those with coeliac disease. Clonality in duodenal biopsies was associated with an abnormal phenotype of IELs in all cases and in all but one case in patients with evidence of underlying coeliac disease. Specificity for detection of an EITCL using immunohistology was 77% for CD8 and for TCR-beta staining, and 100% for detection of a clonal TCR-gamma gene rearrangement. Sensitivity was 62% for staining with CD8 and clonality investigation, while sensitivity reached 100% for TCR-beta staining in all investigated patients with EITCL. CONCLUSIONS: Clonal proliferations of phenotypically abnormal IELs in refractory sprue represent an early manifestation of EITCL, for which the term "sprue-like intestinal T cell lymphoma" is proposed. This constellation is also found in duodenal biopsies from patients with an overt EITCL and is not related to other sprue syndromes, resulting in a high specificity for detection of an EITCL or refractory sprue evolving into EITCL. Overt EITCL may develop directly from coeliac disease without a precursor lesion (refractory sprue with clonal IELs) being demonstrable in duodenal biopsies or via a "sprue-like intestinal T cell lymphoma". This latter entity is a complication of coeliac disease.     

Exclusive expression of G-CSF receptor on myeloid progenitors in bone marrow CD34+ cells

Although granulocyte colony-stimulating factor (G-CSF) has been reported to act on cells of neutrophilic lineage, the administration of G-CSF to induce the mobilization of various haematopoietic progenitors into the circulation. We analysed the expression of receptors for G-CSF (G-CSFR) on human bone marrow and G-CSF-mobilized peripheral blood CD34+ cells, and examined the proliferation and differentiation capabilities of sorted CD34+G-CSFR+ and CD34+G-CSFR- cells using methylcellulose clonal culture. Flow cytometric analysis showed that G-CSFR was expressed on 14.9 +/- 4.9% of bone marrow CD34+ cells, most of which were included in CD34+CD33+ and CD34+CD38+ cell fractions. In clonal cultures, CD34+G-CSFR+ cells produced only myeloid colonies, whereas CD34+G-CSFR- cells produced erythroid bursts, megakaryocyte and multilineage colonies. When incubated with the cytokine cocktail for 5 d, CD34+G-CSFR- cells generated CD34+G-CSFR+ myeloid progenitors. In G-CSF-mobilized peripheral blood, CD34+ cells contained 10.8 +/- 5.8% of G-CSFR+ cells, most of which were also myeloid progenitors, although CD34+G-CSFR- cells contained a substantial number of myeloid progenitors. These results indicated that the expression of G-CSFR on CD34+ cells is restricted to myeloid progenitors, suggesting that the specific activity of G-CSF on myelopoiesis depends on the exclusive expression of its receptor on myeloid progenitors, and that the mobilization of various haematopoietic progenitors is not a direct effect of G-CSF in humans.     

The impact of early CD4+ lymphocyte recovery on the outcome of patients who undergo allogeneic bone marrow or peripheral blood stem cell transplantation

Background

Different factors influence the clinical outcome of allogeneic transplants, the foremost being good immune recovery.

Materials and methods

The purpose of this study was to evaluate the influence of different factors, such as stem cell source, type of donor, conditioning regimen and acute graft-versus-host disease, on early lymphocyte recovery after transplantation. We then analyzed the impact of early CD4+ cell count on overall survival, transplant-related mortality and disease-related mortality.

Results

Univariate analysis with Spearman’s rho showed a significant correlation between early CD4+ cell recovery and overall survival, transplant-related mortality, stem cell source and type of donor. In multivariate analysis CD4+ cell count was significantly associated with (i) stem cell source, being higher in patients whose haematopoietic progenitor cells were obtained by apheresis than in those whose source of grafted cells was bone marrow, and (ii) type of donor, being higher in patients transplanted from sibling donors than in those whose graft was from an alternative donor. The ROC curve of CD4+ cell count indicated that a cut-off of 115 CD4+ cells/mL could differentiate groups with different outcomes. At 2 years follow-up, patients achieving this CD4+ cell count had significantly lower cumulative transplant-related mortality compared to patients who did not have this count (10%±4% versus 40%±8%, p=0.0026). At the 5-year follow-up, the overall survival rates were 77.5%±0.6% and 36%±7% (p=0.000) in patients with a CD4+ cell count ≥115/mL and in patients with CD4+ cell count ≤ 115/mL, respectively.

Conclusion

Early CD4+ cell recovery after allogeneic transplantation has a relevant impact on overall survival and transplant-related mortality and is influenced by two factors: stem cell source and type of donor.     

Functional reconstitution of HBV-specific CD8 T cells by in vitro polyphenol treatment in chronic hepatitis B

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Impact of whole body irradiation and vascular endothelial growth factor-A on increased beta cell mass after bone marrow transplantation in a mouse model of diabetes induced by streptozotocin

Aims/hypothesis  Recent studies have shown that bone marrow transplantation reduces hyperglycaemia in a mouse model of diabetes induced by streptozotocin. However, the essential factors for the improvement of hyperglycaemia by bone marrow transplantation have not been fully elucidated. The aim of this study was to search for such factors. Methods  We investigated the effect of irradiation to whole body, to abdomen alone or to whole body excluding abdomen, followed by infusion or no infusion of bone marrow cells. We also investigated the effect of bone marrow transplantation on beta cell-specific vascular endothelial growth factor-A gene (Vegfa) knockout mice. Results  Bone marrow transplantation improved streptozotocin-induced hyperglycaemia and partially restored islet mass. This change was associated with increased islet vascularisation. Among the other methods investigated, low-dose irradiation of the whole body without infusion of bone marrow cells also improved blood glucose level. In streptozotocin-treated beta cell-specific Vegfa knockout mice, which exhibit impaired islet vascularisation, bone marrow transplantation neither improved hyperglycaemia, relative beta cell mass nor islet vascularisation. Conclusion/interpretation  Our results indicate that whole body irradiation is essential and sufficient for restoration of beta cell mass after streptozotocin treatment independent of infusion of bone marrow cells. Vascular endothelial growth factor-A produced in beta cells is also essential for this phenomenon. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

HLA同胞全相合异基因造血干细胞移植后T细胞重建的规律

目的通过胸腺近输出功能和T细胞受体克隆谱型来研究同胞全相合骨髓移植后T细胞免疫重建。方法应用实时定量PCR的方法定量检测23例同胞全相合骨髓移植后不同时间及70例正常供者T细胞受体重排删除环（TRECs）水平;应用RT-PCR扩增其中10例患者骨髓移植后及5例正常供者外周血的T细胞受体β链可变区（TCRBV）24个家族的基因序列,通过基因扫描的方法判断TCRBV家族的克隆表达、互补决定区3（CDR3）克隆性质及计算TCRBV家族的利用率。结果70例正常人外周血单个核细胞中TRECs为（3351．1±3711．1）拷贝／10^5细胞,年龄与TRECs含量呈负相关。移植前患者TRECs定量检测结果为（307．9±433．3）拷贝／10^5细胞,明显低于正常人。移植后1～5个月患者TRECs水平均明显下降,部分患者未能检测到TRECs。移植后6个月TRECs明显升高并持续1年;移植后24个月TRECs水平有较大提高并接近移植前状态。移植后2～15个月,10例患者在7～16个BV家族有表达,且多克隆表达率为48％,单克隆及寡克隆分布在24个BV家族。结论在移植后早期TRECs为低水平并持续一段时间。移植后6个月,胸腺产生一定数量的初始T细胞并且TCRBV CDR3谱型显示BV家族的利用及多克隆的表达均在增加。