Rapid establishment of long-term culture-initiating cells of donor origin after nonmyeloablative allogeneic hematopoietic stem-cell transplantation,and significant prognostic impact of donor T-cell chimerism on stable engraftment and progression-free survival

BACKGROUND: Nonmyeloablative allogeneic hematopoietic stem-cell transplantation (NST) allows establishment of donor hematopoiesis without eradication of recipient stem cells by chemoradiotherapy. Quantification of donor chimerism may predict graft failure and relapse. METHODS: We quantified donor long-term culture-initiating cells (LTC-IC) in nine patients during the early phase after NST and lineage-specific donor cells of myeloid (CD33+, CD34+, granulocytes) and lymphoid lineage (CD3+, CD4+, CD8+, CD56+) in 38 patients with a median follow-up of 40 weeks after NST. Conditioning therapy consisted of fludarabine 90 mg/m2 followed by total body irradiation of 2 Gy. RESULTS: Only rapid establishment of donor T-cell chimerism was essential for stable donor engraftment. Patients with less than 90% of donor T cells 4 weeks after NST had a significantly higher risk of relapse, graft rejection, or both (14 of 18 patients) than patients with donor T-cell chimerism of 90% and higher (3 of 20 patients). Although conditioning therapy was nonmyeloablative, a significant decrease of repopulating stem cells defined as LTC-IC was seen after 2 weeks followed by rapid recovery of LTC-IC to pretransplant values. Interestingly, all LTC-IC were from donor origin 2 and 4 weeks after NST, but rapid establishment of donor LTC-IC was not predictive for progression-free survival. CONCLUSIONS: Rapid establishment of lymphoid but not myeloid donor chimerism is a prognostic factor for stable donor engraftment after NST. It seems that an immunologic shield of alloreactive donor T cells is essential for early hematopoietic progenitors.     

Transfusion of peripheral blood stem cells from donor homozygous for a shared HLA-haplotype: avoiding fatal transfusion-associated graft-versus-host disease while preserving anti-leukemic effect

BACKGROUND: Fatal transfusion-associated graft-versus-host disease was observed in immunocompetent patients transfused with blood from donors homozygous for a shared haplotype with the recipient (the P-F1 barrier). We tested whether it was possible to carry out successful transplantation in a patient with relapsed acute myeloid leukemia, using peripheral blood stem cells from his HLA-homozygous brother (HLA A2, B46, DRB1 901) who shared a haplotype with the patient (HLA A2, B46,75, DRB1 901,12). METHODS: A CD34 positively selected cell fraction (5.46x 10(6) CD34 cells/kg) was infused first, followed by subsequent infusion of graded doses of donor T cells (total 7.25x10(7) T cells/kg). Nonmyeloablative chemotherapy with idarubicin and cytarabine was given during the transplantation to reduce the leukemic burden and facilitate engraftment. Polymerase chain reaction with the VNTR primers, D1S80, was used to detect engraftment. RESULTS: Complete remission (>300days) and successful donor engraftment (90%) were achieved. CONCLUSIONS: Peripheral blood stem cells transplantation from a donor with a homozygous shared haplotype is possible with a minimal preparative regimen.     

Transplantation of the bone marrow microenvironment leads to hematopoietic chimerism without cytoreductive conditioning

BACKGROUND: It has been hypothesized that regimens to induce transplantation tolerance and long-term hematopoietic chimerism require recipient conditioning with whole body irradiation or a cytoablative regimen to create space within the marrow microenvironment to permit pluripotent stem cell engraftment. The purpose of this study was to determine if transplantation of an intact bone marrow microenvironment in the form of a bone graft would permit stable hematopoietic stem cell engraftment, shape the repertoire of developing T cells, and induce donor-specific unresponsiveness in the absence of a conditioning regimen. METHODS: Fragments of femur were transplanted under the kidney capsule of recipient mice. At defined time points after bone graft transplantation recipients were assayed for chimerism, bone graft viability, and responses to donor and third party alloantigens in vitro and in vivo. RESULTS: In the absence of an immunological barrier, bone graft transplantation resulted in long-term multi-lineage hematopoietic chimerism in the peripheral blood. Nude bone graft transplantation into SCID recipients resulted in development of donor- derived T cells that underwent negative selection on bone graft derived I-E+ cells within the thymus. Across a fully allogeneic barrier in immunocompetent recipients treated with combined blockade of the CD40 and CD28 pathways bone graft transplantation resulted in long-term donor-specific hyporesponsiveness in vitro and acceptance of donor specific skin grafts. CONCLUSIONS: Transplantation of bone marrow in the form of a bone graft may facilitate the production of hematopoietic chimerism and lead to long-term donor-specific hyporesponsiveness in the absence of a cytoreductive conditioning regimen.     

NK Cells Mediate Costimulation Blockade-Resistant Rejection of Allogeneic Stem Cells During Nonmyeloablative Transplantation

Although T-cell CD28/CD40 costimulation blockade represents a powerful mechanism to promote immune tolerance during murine allotransplantation, it has not yet been successfully translated to clinical transplantation. We determined the impact of natural killer (NK) cells on costimulation blockade-resistant rejection of donor bone marrow. We found that NK cells represent a potent barrier to engraftment: host NK depletion led to increased donor stem cell survival, increased mixed hematopoietic chimerism and to engraftment of low doses of donor marrow (1 x 10(8)/kg) that were otherwise rejected. To understand the mechanisms of NK alloreactivity, we employed an in vivo NK-specific cytotoxicity assay. We found that an increased proportion of target cells were killed between days 2 and 8 after cell transfer, and that NK killing of parental targets was inducible: NK cells preprimed with allotargets were more efficient at their elimination upon reexposure. Finally, both transplant and in vivo NK-killing models were used to determine the contribution of LFA-1 to NK alloreactivity. Blockade of LFA-1 led to decreased NK-mediated killing, and increased alloengraftment. These results identify NK alloreactivity as an integral component to costimulation blockade-resistant rejection, and suggest that its inhibition may represent an important target in the clinical translation of tolerance-induction transplantation.     

Transplantation of human peripheral blood stem cells into fetal rhesus monkeys (Macaca mulatta)

BACKGROUND: Methods for assessing engraftment efficiency have been explored in a primate xenogeneic model of in utero hematopoietic stem cell transplantation. METHODS: Human peripheral blood stem cells (PBSC) were obtained by leukapheresis from a human male donor after 4 days of administration of recombinant human granulocyte-colony stimulating factor (5 microg/kg/ day). PBSC were enriched for the CD34+ population with and without T-cell depletion. The resulting mononuclear cells consisted of two cell populations, one that was stem cell enriched (0.83% CD3+ cells, 95% CD34+; group 1) and one that was stem cell enriched and T-cell depleted (<0.03% CD3+ cells, 98% CD34+; group 2). Four fetal monkeys (two per group) received either two or four i.p. injections (approximately 5x10(6) cells/injection) via ultrasound guidance every other day over a 7-day period (gestational days 50, 52, 54, and 56). One fetus in each group also received i.p. recombinant human stem cell factor (25 microg/kg) and recombinant human granulocyte-colony stimulating factor (10 microg/kg) posttransplant every 10 days from gestational day 60-150. RESULTS: Four healthy newborns were delivered at term, and specimens were analyzed by polymerase chain reaction for the human Y chromosome (birth, monthly to 6 months; blood, marrow, progenitor assays). Polymerase chain reaction results were positive for all four newborns in all specimens assessed, and flow cytometric analysis for human CD45 in marrow showed engraftment ranging from 0.1-1.7%. There was no evidence of graft-versus-host disease in any of the animals. CONCLUSION: These studies show that (1) multilineage engraftment of human PBSC can be achieved in the fetal rhesus recipient, (2) the rhesus fetus appears to tolerate relatively high numbers of human CD3+ cells, and (3) healthy chimeric rhesus infants can be delivered at term after multiple in utero procedures.     

Increased engraftment and GVHD after in utero transplantation of MHC-mismatched bone marrow cells and CD80low, CD86(-) dendritic cells in a fetal mouse model.

BACKGROUND: Bone marrow transplantation (BMT) is the only known cure for a variety of inherited diseases and requires the administration of high doses of immunosuppressive and myeloablative therapy. Because the fetus is immunoincompetent early in gestation, in utero stem cell transplantation (IUT) could avoid the need for this toxic conditioning. A major limitation to date of IUT is the low level of engraftment and failure to induce tolerance. Dendritic cells (DC) are considered very potent antigen-presenting cells, but DC progenitors (pDC) are strongly tolerogenic. METHODS: We examined the effect of donor pDC on the degree of engraftment and tolerance induction after IUT. Bone marrow-derived pDC (CD80low, CD86-) from male C57BL/6 mice (H2b) were injected with and without donor bone marrow (BM) intraperitoneally into 13 to 15-day BALB/c (H2d) fetuses. Engraftment was determined by flow cytometry and quantitative polymerase chain reaction and tolerance by skin grafts and the mixed lymphocyte reaction. RESULTS: At 1-month posttransplant, mice that received BM+pDC showed a higher degree of engraftment (29+/-46%) than mice that received pDC-enriched cells or BM cells alone (0.11+/-0.70% and 1.71+/-1.66%, respectively, P<0.001). However, 5/19 recipients of BM+pDC died within 6 weeks; 4/5 had significant donor cell engraftment in blood and/or tissues. Also, these mice had evidence of graft-versus-host disease (GVHD). Two mice out of 15 long-term survivors in the BM+pDC group had virtually complete replacement of host with donor hematopoietic cells. Skin grafts and mixed lymphocyte reaction studies showed no durable tolerance induction other than in the two fully engrafted recipients of BM+pDC. CONCLUSIONS: These results suggest that donor pDC, along with donor BM, can have a significant impact on engraftment of MHC-mismatched donor cells associated with an increased incidence of GVHD. However, marrow-derived pDC do not result in an increase in tolerance induction in utero even when microchimerism is present.     

A decrease in graft-vs.-host disease without loss of graft-vs.-leukemia reactivity after MHC-matched bone marrow transplantation by selective depletion of donor NK cells in vivo.

It is thought that natural killer cells may play a role in graft-vs.-host reactions after allogeneic bone marrow transplantation, but the use of NK cell-specific reagents has been limited. In this report, an NK allele-specific monoclonal antibody, anti-NK 1.1, was used to study the impact of in vivo donor NK cell depletion on GVH disease, graft-vs.-leukemia (GVL) reactivity and donor T cell chimerism after allogeneic murine BMT. AKR/J (H-2k) recipient mice were preconditioned with suboptimal irradiation (9 Gy = LD50) and transplanted with major histocompatibility complex-matched B10.BR (H-2k) BM cells with or without added spleen cells as a source of T cells. The addition of increasing numbers of spleen cells to the BM inoculum produced GVHD of varying intensities. The beneficial effect of NK depletion on GVHD was dependent on the intensity of the GVH reaction. Donor NK cell depletion had no effect on the survival of mice with severe GVHD after MHC-matched BMT (B10.BR into AKR) or after MHC-mismatched BMT (B10.BR into DBA/2; H-2k into H-2d). However, donor NK depletion increased survival of AKR hosts given sufficient B10.BR splenic T cells to induce mild-to-moderate GVHD. Ex vivo depletion of donor CD8+ T cells also reduced GVH-associated mortality, but the use of both CD8 and NK depletion offered no improvement over either alone, suggesting an interaction between CD8+ and NK 1.1+ cells. In contrast to CD8 depletion, donor NK depletion did not compromise the rapid and complete establishment of donor T cell chimerism nor the ability of chimeras to mount an effective GVL reaction. Thus, elimination of donor NK cells provides an alternate strategy for reducing GVHD without loss of GVL reactivity following MHC-matched allogeneic BMT.     

Immune modulation to prevent antibody-mediated rejection after allogeneic hematopoietic stem cell transplantation

It has been shown that antibodies to donor CD34+/VEGFR-2+ stem cells or antibodies against mismatched HLA are associated with graft rejection after hematopoietic stem cell transplantation (HSCT). CD34/VEGFR-2 positive stem cells have been implicated to play a major role in engraftment after HSCT. In this study we treated four patients with an imminent risk of antibody-mediated rejection with immune modulation, i.e. plasma exchange, intravenous immunoglobulin (IVIG), and rituximab before HSCT. Three of the patients had been previously transplanted and rejected their initial grafts after 12 months, 1 month, and less than 1 month, respectively. The fourth patient was not transplanted previously but had HLA directed antibodies present against the graft. During the immune modulatory treatment we followed the pattern of antibodies in sera using FACS and microcytotoxicity assay. We could show that two patients had antibodies against donor CD34+/VEGFR-2+ cells while the other two had antibodies directed against HLA. All four patients tolerated the immune modulatory regimen without any side effects. In this preliminary study we show that immune modulatory treatment may be used to reduce antibody levels and to prevent rejection after HSCT. In two of the three patients which experienced previous rejections and had detectable anti-HLA or anti-CD34+/VEGFR-2+ antibodies, immune modulation resulted in engraftment. In the fourth patient with known anti-HLA-class I antibodies, the treatment also resulted in engraftment. Our results encourage further studies regarding this treatment regimen.     

A role for natural killer cells in the rapid death of cultured donor myoblasts after transplantation

BACKGROUND: The rapid and massive death of cultured donor myoblasts after injection in vivo is a major problem for clinical myoblast transfer therapy (MTT). This study shows blood-borne factors are responsible and that ablating the host natural killer (NK) cell response greatly enhances the survival of such donor myoblasts. METHODS: Cultured male donor myoblasts were injected into muscles of female host mice and surviving donor male DNA (myoblasts) quantified using a Y-chromosome specific (Y1) probe. Survival of donor myoblasts transfected with m144, a murine major histocompatibility complex (MHC) class I homologue that protects against NK attack, was quantified. In addition, donor myoblast survival was investigated in host mice following initial (before MTT) and sustained (repeatedly for 3 weeks after MTT) depletion of host NK1.1+ and CD4+/CD8+ cells using specific monoclonal antibodies (either alone or in combination) for up to 3 weeks after MTT, as well as in beige (deficient in NK activity) and in perforin-deficient mdx host mice. RESULTS: A major role for blood-borne factors (especially cells) was confirmed by MTT experiments in irradiated and perfused host mice. Substantially enhanced myoblast survival was seen with donor myoblasts modified by transfection with the m144 molecule or following antibody depletion of host NK1.1+ cells and in beige host mice. Other studies support some role for CD8+ but not CD4+ cells. CONCLUSIONS: These combined data support a central role for host NK cells in the rapid initial death of donor myoblasts. The demonstrated role of NK cells provides strategies to enhance the efficacy of clinical myoblast transplantation.     

MiHA Reactive CD4 and CD8 T-Cells Effect Resistance to Hematopoietic Engraftment Following Reduced Intensity Conditioning

Reduced intensity conditioning (RIC) prior to allogeneic hematopoietic cell transplantation (HCT) has shown promise in lowering the incidence of post-transplant complications including infection and graft-versus-host disease. T-cell-mediated graft rejection, however, remains a crucial factor in determining how 'mild' a level of immunosuppression can be administered. Understanding the kinetics of resistance responses as well as the role of CD4+ and CD8+ T cells underlies the development of protocols to circumvent resistance and support hematopoietic engraftment. In these studies, a major histocompatibility complex (MHC)-matched/minor histocompatibility antigen (MiHA) disparate RIC HCT model was developed in which resistance against donor hematopoietic progenitors as well as mature peripheral blood cells could be assessed. Interestingly, resistance was diminished in the absence of either host CD4+ or CD8+ T cells. However, its impairment was more severe in CD4-/- mice where resistance was not detected. Host CD4+ T cells were required for optimal expansion of specific (H60) T-cell receptor (TCR) expressing host anti-donor MiHA reactive CD8+ T cells following HCT. These observations demonstrate a critical role for host CD4+ T cells in resistance against MiHA disparate HCT. This RIC HCT resistance model will be useful for the analysis of the barrier to engraftment mediated by host T cells and the development of strategies to support engraftment.     

Relevance of Chimerism Analysis After Allogeneic Stem Cell Transplantation

Hematopoietic stem cell transplantation is a potentially curative therapy for a range of malignant and non-malignant hematological diseases. Analysis of chimerism following allogeneic stem cell transplantation has been a routine method for the assessment of engraftment and early detection of graft failure. Lineage-specific chimerism monitoring is progressively used to specifically detect chimerism in one or more cell subsets, which may be undetected in assessment of the whole leukocyte population. The chimerism study in different leukocyte subpopulations increases sensitivity and specificity in the monitoring after transplantation, especially the analysis of T lymphocytes. All peripheral blood samples were separated into mononuclear cells and granulocytes by Ficoll density gradient centrifugation and T, B, and CD34+ was separated by immunomagnetic automatic cell separator. After DNA extraction, chimerism monitoring was performed using short tandem repeat by multiplex polymerase chain reaction followed by capillary electrophoresis. Quantification of chimerism was performed by determining the ratio of peak areas from donor and recipient informative short tandem repeat. Donor-recipient chimerism analysis in patients after allogeneic stem cell transplantation is a practical, feasible, and useful tool that predicts clinical outcomes and provides a guide for suitable therapeutic interventions.     

Flt3-L augments the engraftment of donor-derived bone marrow cells when combined with sublethal irradiation and costimulatory (CD28/B7 and CD40/CD40L) blockade

T-cell costimulatory blockade as a constituent for recipient conditioning prior to bone marrow transplantation has led to the development of less toxic protocols for the establishment of donor cell chimerism. We therefore hypothesized that the addition of the hematopoietic growth factor, Flt3-ligand (Flt3-L), to the perioperative inhibition of the CD28/B7 and CD40/CD40 ligand costimulatory pathways would enhance the engraftment of allogeneic bone marrow. Recipient BALB/c ByJ (H-2(d), Mls(c), Vbeta6+/Vbeta8+ TCR) received a single sublethal dose of total body irradiation (300 rad) 6 h prior to transplantation IV with unfractionated donor CBA/J (H-2(k), Mls(d), Vbeta6-/Vbeta8+ TCR) bone marrow cells. CTLA4-Ig and/or MRI were administered at 500 microg IP on days 0, 2, 4, and 6 posttransplantation. Flt3-L was administered at 10 microg IP on days 0-6. Donor cell chimerism was determined on days 30-90 by flow cytometric analysis. Donor-specific tolerance was assessed by skin grafting. In vitro TCR cross-linking assays and flow cytometry were utilized to explore the deletion of donor-reactive T cells. Recipients receiving CTLA4-Ig and MRI engrafted allogeneic bone marrow cells in the peripheral blood (3/6; 50%) with chimerism being detected at 2-31%. Addition of Flt3-L to this preconditioning regimen enhanced the incidence of engraftment of donor bone marrow cells (10/13; 3-70%). Long-term survival of donor but not third-party-specific skin grafts demonstrated that donor-specific tolerance had been achieved in the chimeric recipients. Deletion of the donor-reactive T cells within the chimeric recipients was also observed. The addition of hematopoietic growth factors and cytokines to the nonmyeloablative regimen of sublethal irradiation and T-cell costimulatory blockade provides a novel strategy for the establishment of donor cell chimerism and for the induction of stable and robust donor-specific tolerance. The deletion of donor-reactive T cells using this protocol suggests the reliability and feasibility of this protocol for clinical transplantation.     

Absence of a facilitory role for NK 1.1-positive donor cells in engraftment across a major histocompatibility barrier in mice

Ex vivo T cell depletion of donor marrow grafts in humans and mice has virtually eliminated severe graft-versus-host disease (GVHD). However, as a consequence of T cell depletion, sustained donor cell engraftment is likely compromised. Since the majority of T cell depletion techniques also deplete natural killer (NK) cells, we investigated the role of donor NK cells in engraftment and GVHD in a murine model. Using a monoclonal antibody directed against an NK-specific epitope, we have selectively depleted NK cells while preserving donor marrow T cells. In an established model of engraftment, selective NK depletion demonstrated that removal of donor NK cells did not impair the engraftment process under conditions in which donors and recipients are major histocompatibility complex-disparate. In contrast, recipients of anti-Thy 1.2 plus complement (C')-treated marrow grafts had a significantly higher incidence of either partial engraftment or graft rejection as compared with recipients of selective NK-depleted donor grafts or control grafts. In addition, we have observed that NK-specific depletion of donor marrow/splenocyte inocula does not alter the incidence of GVHD. Recipients of NK-depleted donor grafts developed lethal acute GVHD, whereas recipients of anti-Thy 1.2-depleted donor grafts did not (P less than 0.0001). Interestingly, NK 1.1-depleted donor graft recipients had a significantly increased mortality in comparison with control groups receiving C'-treated grafts (P = 0.04) or anti-Thy 1.2 plus C'-treated grafts (P less than 0.05). Thus, NK depletion may reduce immunosurveillance, thereby increasing the risk of posttransplant infection. We conclude from these results that donor NK cells play an insignificant role in engraftment as well as in the afferent phase of GVHD, but may be important in immunosurveillance when murine bone marrow is transplanted across the major histocompatibility barrier.     

The effect of human fetal liver-derived mesenchymal stem cells on CD34+ hematopoietic stem cell repopulation in NOD/Shi-scid/IL-2Rã(null) mice

Mesenchymal stem cells (MSCs) are progenitors that are capable of differentiating into mesenchymal tissues. They are known to support allogeneic hematopoietic stem cell transplantation by facilitating engraftment without increasing the risk of graft-versus-host disease. We optimized culture conditions for human fetal liver-derived MSCs (hFL-MSCs) to investigate the role of hFL-MSCs on repopulation of hematopoietic stem cells in NOD/Shi-scid/IL-2Rγ(null) (NOG) mice using CD34(+) hematopoietic stem cells (HSCs) derived from umbilical cord blood (UCB). FL-MSCs and CD34(+) HSCs were prepared from fetal liver and UCB, respectively. Twenty-four hours after irradiation, CD34(+) HSCs and hFL-MSCs were injected intravenously and intratibially into NOG mice. During 24 weeks posttransplantation, engraftment levels of human cells were analyzed in bone marrow, peripheral blood, and spleen of transplanted mice by flow cytometry. hFL-MSCs showed a fibroblast-like morphology and immunophenotypic characteristics appropriate for MSCs. hFL-MSCs prolonged the survival of NOG mice that had been cotransplanted with UCB CD34(+) cells. Fluorescence-activated cell-sorting analysis showed that engraftment of human cells was increased by cotransplantation of hFL-MSCs. However, significant enhancement of human cell engraftment was not detected in NOG mice regardless of the number of cotransplanted MSCs. Although survival of repopulating NOG mice and engraftment of human cells were prolonged by cotransplantation of hFL-MSCs, 8.0 × 10(6) MSCs were not sufficient to increase HSC engraftment in irradiated NOG mice in vivo.     

Donor bone marrow cells play a role in the prevention of accelerated graft rejection induced by semi-allogeneic spleen cells in transplantation

Spleen or spleen plus bone marrow cells from (BALB/cxC57Bl/6)F1 donors were transferred into BALB/c recipients 21 days before skin or cardiac transplantation. Prolonged graft survival was observed on recipients treated with the mixture of donor-derived cells as compared to those treated with spleen cells alone. We evaluated the expression of CD45RB and CD44 by splenic CD4+ and CD8+ T cells 7 and 21 days after donor cell transfer. The populations of CD8+CD45RBlow and CD8+CD44high cells were significantly decreased in mice pre-treated with donor spleen and bone marrow cells as compared to animals treated with spleen cells only, although these cells expanded in both groups when compared to an earlier time-point. No differences were observed regarding CD4+ T cell population when recipients of donor-derived cells were compared. An enhanced production of IL-10 was observed seven days after transplantation in the supernatants of spleen cell cultures of mice treated with spleen and bone marrow cells. Taken together these data suggest that donor-derived bone marrow cells modulate the sensitization of the recipient by semi-allogeneic spleen cells in part by delaying the generation of activated/memory CD8+ T cells leading to enhanced graft survival.     

由脐血单个核细胞和富集的CD34+细胞扩增的造血干/祖细胞的生理功能比较

目的 探讨由脐血单个核细胞(MNC)和富集的CD34+细胞起始扩增所得的造血干/祖细胞在体内植入及造血重建的能力.方法 从人脐血中分离出MNC和CD34+细胞,在体外扩增7 d.将非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠分为四组,在接受亚致死剂量铯源照射后,进行细胞移植,实验A组接受由MNC培养得到的CD34+细胞和CD34-细胞;实验B组接受由富集的CD34+细胞培养得到的CD34+细胞和CD34-细胞;阳性对照组接受从脐血新鲜分离的CD34+细胞和CD34-细胞;阴性对照组不接受细胞移植,仅输注相同体积的IMDM培养基.6周后处死存活的小鼠,取其骨髓、脾脏和外周血细胞,分别进行细胞表型分析、集落和人特异性基因的检测.结果 经过体外扩增,以富集的CD34+细胞起始培养者的细胞总扩增倍数为39.8倍,远高于以MNC为起始细胞者的1.88倍.移植6周后,所有接受细胞移植的小鼠均存活,存活小鼠的骨髓和脾脏细胞中均能检测到人源细胞(CD45+细胞)及人源的各系血细胞,实验A组各类细胞的含量稍高于实验B组,且接受细胞移植小鼠的骨髓和脾脏细胞中可检测出人特异的Alu序列.结论 与从脐血中新鲜分离的细胞相比,扩增后的造血干/祖细胞的体内植入能力有所下降,以MNC起始扩增的造血干/祖细胞体内植入能力优于以富集的CD34+细胞起始扩增者,但二者体内造血重建能力的差异不显著.     

Dynamics of the early immune cellular reactions after myogenic cell transplantation

The role of immune cells in the early donor cell death/survival following myoblast transplantation is confusing, one of the reasons being the lack of data about the immune reactions following cell transplantation. We used outbred mice as hosts for transplantation of primary cultured muscle cells and T-antigen-immortalized myoblasts. The host muscles were analyzed 1 h to 7 days after cell injection. No net loss of the donor primary cultured cell population was observed in this period. The immune cellular reaction in this case was: 1) a brief (<48 h) neutrophil invasion; 2) macrophage infiltration from days 1 to 7; 3) a specific response involving CTL and few NK cells (days 6 and 7), preceded by a low CD4+ cell infiltration starting at day 3. In contrast, donor-immortalized myoblasts completely disappeared during the 7-day follow-up. In this case, an intense infiltration of CTL and macrophages, with moderate CD4+ infiltration and lower amounts of NK cells, was observed starting at day 2. The nonspecific immune response at days 0 and 1 was similar for both types of donor cells. The present observations set a basis to interpret the role of immune cells on the early death/survival of donor cells following myoblast transplantation.     

Allogeneic transplantation of selected peripheral CD34+ cells with controlled CD3+ cells add-back in high-risk patients

We evaluated the feasibility of allogeneic transplantation of CliniMACS-selected peripheral CD34+ cells from siblings (four patients: AML-M4, M2, CLL, MDS); nonoptimal related donors (two patients: AML-M4, CML); and unrelated donors (two patients: CML, ALL, both without engraftment after preceding URDBMT). All patients had high-risk of aGVHD and/or graft failure due to multiple transplantation risk factors. Conditioning treatment was myeloablative (n=7) or nonmyeloablative (n=1). Immunosuppression consisted of CsA (n=8), Mtx (n=5), ATG (n=4). Selected CD34+ cells were transplanted (average 3.91 x 10(6)/kg, range 1.29 to 7.27 x 10(6)/kg) together with 0.01 to 0.5 x 10(7) CD3+ cells/kg to assure proper engraftment. The remaining CD34-negative fraction was cryopreserved for further CD3+ cell add-back. Average recovery and purity of CD34+ cells following CliniMACS selection were 74% and 97%. No severe complications were observed in the first 100 days. Regeneration times were satisfactory in seven of eight patients (87.5%) with ANO > 0.5 g/L and Plt > 50 g/L reached on average on days +26 and +32 (range 15 to 29 and 15 to 67), respectively. In three patients (37.5%) T-lymphocytes were added-back one to three times (due to low numbers of initially transfused CD3+ cells in two patients, in one patient with PRCA caused by ABO incompatibility). One to four additional transplantations of nonselected peripheral cells were performed on days +28 to +270 in consequence of infections (CMV-two patients; parvovirus-one patient), poor regeneration and residual disease (one patient) and prolonged transfusion dependency (one patient). Severe aGVHD grade III or IV developed in three patients (37.5%) following the nonselected cells transplantation. Finally, five patients (62.5%) are alive and in remission (median follow-up 815 days). We conclude that allogeneic transplantation of selected peripheral CD34+ cells (CliniMACS) with controlled add-back of CD3+ cells is an effective, well, tolerated procedure in high-risk patients.     

Expression of stem cell markers on mononuclear cells derived from heparinized cadaveric organ donors before and after disconnection from the respirator

We have attempted to evaluate the level of the earliest human hematopoietic cell marker expression (CD34, CD117, CD133, CD184) on cells obtained from heparinized cadaveric organ donors before and after disconnection from the respirator. Moreover, we compared various cell populations: (1) coexpressing CD34/CD117; (2) CD34/CD133; (3) highly enriched hematopoietic stem cells (CD34+CXCR4+CD45+); and (4) highly enriched tissue-committed stem cells (CD34+CXCR4+CD45-). Finally, we analyzed whether the level of hematopoietic stem cell marker expression depended on the age of the donor. The expression of the membrane receptors (CD34, CD45, CD117, CD133, CD184) was studied by flow cytometry. We observed that the proportion of mononuclear cells expressing these markers slightly decreased in bone marrow harvested after disconnection from the respirator compared with the samples obtained before disconnection. Moreover, the proportion of cells expressing CD117 antigen depended on age of the donor.