Natural history of mixed chimerism after bone marrow transplantation with CD6-depleted allogeneic marrow: a stable equilibrium

Mixed hematopoietic chimerism (MC) is a common finding after allogeneic bone marrow transplantation (BMT), but the natural history of this phenomenon remains unclear. To understand the evolution and the implications of this finding, we performed a prospective analysis of the development of mixed chimerism in 43 patients with hematologic malignancies who received bone marrow (BM) from human leukocyte antigen (HLA)-identical sibling donors. T-cell depletion in vitro with anti-T12 (CD6) monoclonal antibody and rabbit complement was used as the only method of graft-versus-host disease (GVHD) prophylaxis. Overall, MC was identified in peripheral blood (PB) and BM in 22 of 43 (51%) patients evaluated. MC was found by restriction fragment length polymorphism (RFLP) analysis in 21 of 40 (53%) patients, by cytogenetic analysis in 6 of 29 (21%) patients, and by red blood cell phenotyping in 4 of 9 (44%) patients. RFLP studies were performed at 0.5, 1, 3, 6, 9, and 12 months post-BMT and then every 6 months, and showed a high probability of developing MC in the first 6 months after BMT followed by stabilization after 12 months. Cytogenetic analysis was less sensitive in detecting MC. Once MC was detected after BMT, the percentage of recipient cells increased very slowly over more than 3 years of follow-up, and no patient reverted to complete donor hematopoiesis (CDH). Thus, recipient and donor cells remained in a relative state of equilibrium for prolonged periods that seemed to favor recipient cells over donor cells. Patient's disease, remission status, or intensity of the transplant preparative regimen did not influence the subsequent development of mixed chimerism. Early immunologic reconstitution was the only factor that correlated with the subsequent chimeric status of the patients. The percentage and absolute number of T3 (CD3) and T4 (CD4) positive cells at day 14 after BMT were significantly higher in the patients who maintained CDH but NK cell reconstitution was similar in both groups, suggesting that early reconstitution with T cells may play a role in preventing recovery of recipient cells after BMT. GVHD was also associated with maintenance of CDH, but the probability of relapse, survival, and disease-free survival was identical in patients with MC and CDH.     

Surface antigen expression of human neoplastic plasma cells includes molecules associated with lymphocyte recirculation and adhesion

Adherent lymphokine activated killer (ALAK) cells are a subpopulation of activated natural killer (NK) cells with MHC unrestricted antitumour activity distinguished by their propensity to adhere to plastic in the presence of interleukin-2 (IL-2). We generated ALAK cells from seven patients with chronic myeloid leukaemia (CML) following Campath-1-depleted bone marrow transplantation (BMT). Five had relapsed and were in chronic phase, one had cytogenetic evidence of relapse and one had prior evidence of cytogenetic relapse but was in complete remission at time of study. Phenotypically the ALAK cells included both CD56+/CD3- NK cells and CD56-/CD3+ T cells. The CD3- subpopulation were studied cytogenetically and their functional activity tested in a 4 h 51Cr release cytotoxicity assay using the pretransplant leukaemia cells as targets. Cytogenetic studies showed that the ALAK cells from six patients were Ph negative, and where donor and recipient were sex mismatched, ALAK cells were exclusively of donor origin. In one patient ALAK cells were Ph positive and of recipient origin in eight of nine metaphases. In the 51Cr release assay the ALAK cells showed significant lysis of the pretransplant leukaemia in five of the seven patients tested. These data indicate that in CML patients who relapse post-BMT the NK cells are usually of donor origin but may be recipient-derived. In most patients these ALAK cells have antileukaemic activity in vitro.     

Myeloid mixed chimerism is associated with relapse in bcr-abl positive patients after unmanipulated allogeneic bone marrow transplantation for chronic myelogenous leukemia

BACKGROUND AND OBJECTIVE: Although bcr-abl polymerase chain reaction (PCR) positivity after bone marrow transplantation (BMT) for chronic myelogenous leukemia (CML) is significantly related to relapse, the predictive value of the assay is not very high and therefore most investigators consider that qualitative RT-PCR data alone are too imprecise to enable clinical decisions to be taken in individual cases. To define the clinical outcome of bcr-abl positive patients after unmanipulated BMT better, we sought the origin of hematopoiesis and traced its evolution over time. DESIGN AND METHODS: Forty-nine patients received allogeneic BMT for CML (39 in chronic phase and 10 in accelerated phase/blast crisis). Median follow-up was 61 months (range 4-92). mRNA and DNA were used to assess bcr-abl and chimerism status respectively. Quantitative VNTR-PCR on total cells and lymphoid or myeloid population allowed us to assign and measure the origin of hematopoiesis. RESULTS: Both bcr-abl positivity and the presence of mixed chimerism (MC) were significantly associated with relapse (p = 0.0009 and p < 0.0001 respectively). Relapse was observed in one of 39 patients with complete donor chimerism and in 6 of 9 patients with MC. These six cases showed increasing levels of host hemopoiesis and bcr-abl positivity in the CD15-positive population prior to relapse. The other three cases had decreasing or stable low-level MC which was restricted to the T-cells as well as bcr-abl negativity. INTERPRETATION AND CONCLUSIONS: Whereas the simple detection of bcr-abl fails to identify patients who will relapse with certainty, the assessment of MC by VNTR-PCR does identify patients headed to relapse. Confirmation of myeloid involvement and increasing levels over time further elucidates the clinical outcome of bcr-abl positive patients after BMT.     

Graft-versus-leukaemia following allogeneic bone marrow transplantation: emergence of cytotoxic T lymphocytes reacting to host leukaemia cells.

Cytotoxic T lymphocyte precursor (CTLp) frequency assays were examined in patients with chronic myeloid leukaemia (CML) following bone marrow transplantation (BMT) using recipient lymphocytes or CML cells as targets in a 51Cr release cytotoxicity assay. Eighteen patients were studied; 11 received marrow from a fully HLA A, B and DR matched sibling donor, and six from matched unrelated donors or a partially matched sibling (one patient). Two of the unrelated donor transplant recipients received marrow depleted of T lymphocytes, and the remainder received unmanipulated marrow and cyclosporin with or without methotrexate as prophylaxis against graft-versus-host disease (GVHD). Donor cells tested before BMT did not generate CTL against the patients' leukaemia, but up to 9 months after BMT a low frequency of CTLp directed against the patients' CML cells (Lk-CTLp) was detected in all patients. The Lk-CTLp frequency was significantly lower than the frequency of CTLp directed against the recipients' PHA transformed pretransplant lymphocytes (Ly-CTLp) (p less than 0.05). Lk-CTLp showed MHC restricted cytotoxicity and did not demonstrate cytotoxicity in an NK assay. The Lk-CTLp frequency correlated with both GVHD severity and relapse: severe GVHD was only seen with Lk-CTLp frequencies greater than 1:400,000, while leukaemic relapse was only observed in two patients with Lk-CTLp frequencies less than 1:400,000. These results show that a low frequency of alloreactive cells of presumed donor origin with cytotoxic potential against residual leukaemia normally circulate after BMT. Their relationship with the graft-versus-leukaemia phenomenon and their cross-reaction with GVHD reacting cells remain to be determined.     

Adoptive immunotherapy of BCR-ABL-induced chronic myeloid leukemia-like myeloproliferative disease in a murine model

Donor leukocyte infusion (DLI) can induce graft-versus-leukemia (GvL) reactions in patients with chronic myeloid leukemia (CML) relapsing after allogeneic bone marrow transplantation (BMT), but the mechanisms of the antileukemic effect of DLI are unknown, and the procedure is complicated by graft-versus-host disease (GvHD) and graft failure. Here, we adapted a murine retroviral BMT model of Philadelphia(+) leukemia by combining allogeneic bone marrow (BM) from C57Bl/6 (H-2(b)) mice with BCR-ABL-transduced Balb/c (H-2(d)) BM, inducing mixed chimerism and myeloproliferative disease in recipients resembling relapse of CML following allogeneic BMT. Infusions of allogeneic splenocytes eliminated BCR-ABL-induced CML-like disease in the majority of mixed chimeras, with significant GvL effects mediated by both CD4(+) and CD4(-) cells. BCR-ABL-induced acute B-lymphoblastic leukemia was also eradicated by DLI in major histocompatibility complex (MHC)-mismatched chimeras. Most DLI-treated mice converted to full allogeneic chimerism but succumbed frequently to GvHD or graft failure. When MHC-matched B10.D2 (H-2(d)) mice were the allogeneic donors, CML-like disease was more resistant to DLI. These results suggest that depletion of CD8(+) cells from DLI could impair GvL against CML, while increased MHC disparity between donor and recipient may improve the responsiveness of Philadelphia(+) B-lymphoblastic leukemia to DLI.     

Anti-asialo GM1 antiserum treatment of lethally irradiated recipients before bone marrow transplantation: evidence that recipient natural killer depletion enhances survival, engraftment, and hematopoietic recovery

Natural killer (NK) cells are reported to have an important role in the resistance of lethally irradiated recipients to bone marrow transplantation (BMT). Therefore, we investigated the effects of recipient NK depletion on survival, chimerism, and hematopoietic reconstitution after lethal irradiation and the transplantation of limiting amounts of T-cell-deficient bone marrow (BM). When administered before BMT, anti-asialo GM1 (ASGM1) antiserum treatment, effective in depleting in vivo NK activity, was associated with a marked increase in survival in 3 of 3 allogeneic combinations (BALB/c into C3H/HeN, C57B1/6, or C3B6F1). This enhanced survival was independent of the susceptibility of each recipient strain to accept BALB/c BM. Moreover, recipient anti-ASGM1 treatment was also effective in increasing survival in recipients of syngeneic BM, suggesting that NK cells can adversely affect engraftment independent of genetically controlled polymorphic cell surface determinants. Analysis of chimerism in surviving animals 2 months post-BMT showed that recipient NK depletion significantly increased the level of donor engraftment when high doses of BM were transplanted. These studies also demonstrated that anti-ASGM1 pretreatment mainly resulted in an increase in extramedullary hematopoiesis in the second and third week after irradiation. Anti-ASGM1 treatment also dramatically accelerated the rate of appearance of donor-derived cells with a higher level of donor-cell engraftment apparent at a time when the differences in survival between NK-depleted and control BMT recipients became significant. Peripheral cell counts were also affected by NK depletion, with significantly enhanced platelet and red blood cell recovery and a moderate increase in granulocyte recovery. The overall favorable influence of anti-ASGM1 recipient treatment on hematopoietic events post-BMT suggests that, in humans, pretransplant regimens aimed toward NK depletion should be evaluated.     

Evidence for a graft-versus-mast-cell effect after allogeneic bone marrow transplantation

Mast cell leukemia (MCL) is a rare form of aggressive mastocytosis with a reported median survival below 6 months. Casuistic reports suggest the effectiveness of allogeneic bone marrow transplantation (BMT) for MCL. However, these reports lack clear evidence for a graft-versus-mast-cell (GvMC) effect. We prospectively investigated the GvMC at different time points after allogeneic BMT and donor-lymphocyte infusions (DLI). Samples were gathered from a patient with MCL treated with allogeneic BMT from an unrelated HLA identical donor. Parameters for detection of a GvMC effect included flow cytometrical analysis of mast cell (MC) populations in peripheral blood and BM, BM smear and histology, chimerism analysis of flow cytometrically sorted BM CD117+/CD34- MC and testing for anti-mast cell reactivity of donor lymphocytes by interferon (IFN)-gamma ELISPOT. DLIs reduced MC from 5 to 0.5%. MC chimerism analysis demonstrated a complete recipient genotype after BMT, suggesting that the persistent mastocytosis was part of residual neoplastic disease. At 3.7 years after BMT, there is some evidence for relapse. In summary, BMT and DLIs attenuated the mastocytosis from an aggressive to an indolent form and may have improved the patients' prognosis. The in vitro data of our study indicate for the first time the existence of a GvMC effect.     

Mixed chimerism of erythro- and megakaryopoiesis following allogeneic bone marrow transplantation

Until now, studies on mixed chimerism (MCh) after allogeneic bone marrow transplantation (BMT) have predominantly focused on the B- and T-lymphocyte population, but not on distinct myeloid cell lineages like nucleated erythroid precursors and megakaryocytes. To evaluate the lineage-restricted MCh more explicitly in 10 patients with chronic myelogenous leukemia (CML), a quantitative analysis was performed on bone marrow biopsies following a sex-mismatched host/donor constellation. Techniques included immunophenotyping (antiglycophorin C, CD61) for the identification of erythro- and megakaryopoiesis and a simultaneously conducted genotyping with x- and y-chromosome-specific DNA probes. Normal bone marrow and specimens taken before BMT served as controls. Contrasting a total gender-dependent sex-typing in the latter samples in the early and late posttransplant period (up to 586 days), 3-9% erythroid precursors and about 16% megakaryocytes revealed a host-type origin. This significantly higher number of host megakaryocytes is explained by their polyploidy generating an increased probability to detect positive signals at a certain section level of the corresponding biopsies. A striking conversion of MCh to a recipient cell type was found in leukemic relapse with a more than 90% host-derived erythroid and megakaryocytic cell population in 4 patients approximately 643 days after BMT.     

Donor cell-derived acute myeloid leukemia developing 14 months after matched unrelated bone marrow transplantation for chronic myeloid leukemia.

We report a patient with Ph-positive CML who developed a Ph-negative AML in donor cells 14 months after BMT from an HLA-identical male unrelated donor. The Ph translocation could not be detected by either conventional cytogenetics, FISH or RT-PCR analysis excluding relapse of CML in myeloid blast crisis. Chimerism studies were performed by variable number of tandem repeats (VNTR) analysis. These revealed donor-type hematopoiesis in both unseparated mononuclear cells and CD34+ selected blasts proving the leukemia to be of donor origin. The patient received three cycles of polychemotherapy with mitoxantrone, topotecan and ara-c resulting in CR after the first treatment cycle and reconstitution with donor hematopoiesis. A second transplant from a female alternative matched unrelated donor was performed after conditioning with fludarabine and 200 cGy TBI and was well tolerated. Nine months after the second transplant the patient is at home and in CR. T cell chimerism was studied by sex chromosome analysis and revealed complete female donor chimerism.     

Combined immunocytochemistry and FISH: an improved method to study engraftment of accessory bone marrow stromal cells

In aplastic anaemia (AA), correction of bone marrow (BM) stromal function may contribute to the outcome of bone marrow transplantation (BMT). Engraftment of BM stromal cells is rarely observed, but engraftment of accessory cells (macrophages and T cells) may be important. We have improved a method of combined immunocytochemistry and FISH described by van Tol et al. (1998) to define the cellular origin and time course of engraftment of BM stromal accessory cells after sex-mismatched BMT. Long-term bone marrow cultures were trypsinized and cytospin preparations stained by immunocytochemistry using monoclonal antibodies against specific cell lineages followed by FISH for X and Y chromosomes. Low level phase contrast microscopy was used to study staining of individual cells simultaneously with fluorescence microscopy to define chromosomal pattern. In controls, the combined procedure did not affect the intensity of APAAP staining or the accuracy of sex chromosome determination. In cultures from AA patients after sex-mismatched BMT, cell lineages could be identified and donor or recipient origin determined unequivocally. This procedure enabled us to examine the origin (host/donor) of different cell lineages with high confidence, in addition to producing images of the combined staining.     

Patterns of hematopoietic chimerism following bone marrow transplantation for childhood acute lymphoblastic leukemia from volunteer unrelated donors

Hematopoietic chimerism was analyzed in serial bone marrow samples taken from 28 children following T-cell depleted unrelated donor bone marrow transplants (UD BMT) for acute lymphoblastic leukemia (ALL). Chimeric status was determined by polymerase chain reaction (PCR) of simple tandem repeat (STR) sequences (maximal sensitivity, 0.1%). At least two serial samples were examined in 23 patients. Of these, two had evidence of complete donor engraftment at all times and eight showed stable low level mixed chimerism (MC) (<1% recipient hematopoiesis). All 10 of these patients remain in remission with a minimum follow-up of 24 months. By contrast, 13 patients demonstrated a progressive return of recipient hematopoiesis. Five of these relapsed (4 to 9 months post BMT), one died of cytomegalovirus pneumonitis and seven remain in remission with a minimum follow-up of 24 months. Five children were excluded from serial analysis as two serial samples were not collected before either relapse (3) or graft rejection (2). We conclude that as with sibling transplants, ex vivo T depleted UD BMT in children with ALL is associated with a high incidence of MC. Stable donor engraftment and low level MC always correlated with continued remission. However, detection of a progressive return of recipient cells did not universally correlate with relapse, but highlighted those patients at greatest risk. Serial chimerism analysis by PCR of STRs provides a rapid and simple screening technique for the detection of relapse and the identification of patients with progressive MC who might benefit from detailed molecular analysis for minimal residual disease following matched volunteer UD BMT for childhood ALL.     

非清髓性干细胞移植患者供体细胞嵌合率的动态定量研究

目的　对非清髓性干细胞移植 (NST)后供受嵌合体形成动力学 ,嵌合体的转归 ,以及供体细胞嵌合率 (DC)对评价植入、复发、移植物抗宿主病 (GVHD)和长期生存的作用进行研究。方法　18例接受HLA全相合NST的患者进行DC的定量分析。术前采集供者和受者外周血 ,术后不同时间段采集受者外周血或骨髓。用QIAamp全血DNA抽提试剂盒提取样本DNA ,用AmpF/STRprofilerplus试剂盒进行 9个STR位点复合扩增后 ,产物用ABI 310遗传分析仪进行毛细管电泳 ,由Genescane和Genotype软件确定基因位点及峰面积 ,根据供受体基因型的差异计算供体细胞嵌合率。结果　 (1)移植早期植入表明 ,移植后 8d供体细胞开始占优势 (DC >6 0 % ) ,比造血重建提早 4d ;此外 ,由于移植前免疫状态不同 ,慢性髓细胞性白血病 (CML)患者供体细胞植入滞后于急性白血病和其他非恶性血液病患者 ;(2 )NST后嵌合状态有一个由混合嵌合 (MC)向完全供体细胞嵌合 (FDC)状态转化的过程 ;(3)FDC组GVHD的发生率高于MC组 (90 0 %、6 2 5 % ) ,而且从FDC状态的建立到急性GVHD发生的中位时间仅为 9d ;(4)移植后能获得无白血病生存的患者均有FDC或供体细胞高比例MC稳定嵌合的特点 ,而复发或排斥患者均在发生临床症状之前 ,出现供体细胞嵌合率的进行性下降。结论　DC     

Genetic fingerprinting in mouthwashes of patients after allogeneic bone marrow transplantation.

Detection of chimerism by PCR analysis of short tandem repeats (STR) in blood samples of patients who received allogeneic bone marrow transplantation (BMT) has proved to be an important method for early detection of relapse. The prerequisite for this type of analysis is knowledge of donor and recipient pretransplantation genotypes. In some cases, recipient cells from time points prior to BMT are not available and the pretransplant fingerprint cannot be determined. As BM recipients only alter their genotype in blood cells, we attempted to identify patient's pretransplantation genotypes after transplantation in mouthwash samples that contain easily accessible epithelial cells. Of 17 patients who had undergone BMT between one week and 45 months prior to analysis, DNA was isolated from mouthwash cell pellets or from epithelial cells obtained from mouthwashes. PCR analysis of STR loci in the von Willebrand and the tyrosine hydroxylase genes were performed. Even though the mouthwash cell pellets contained about 75% epithelial cells (presumably of recipient origin) and only about 25% leukocytes (presumably of donor origin), three of five patients showed donor genotype and only two patients exhibited chimeric DNA patterns, when cellular DNA was obtained by boiling of mouthwash cell pellets. Following phenol/chloroform extraction, eight of 10 DNA samples exhibited a chimeric pattern, while two of 10 DNAs showed only donor genotype. Of three patients, epithelial cells were attached to magnetic beads prior to DNA isolation. Even this DNA contained donor and recipient material. From our results it appears that blood cells serve as preferential DNA source in mouthwash samples and cannot be removed by epithelial cell separation.     

Predominant or complete recipient T-cell chimerism following alemtuzumab-based allogeneic transplantation is reversed by donor lymphocytes and not associated with graft failure

The clinical significance of mixed chimerism following allogeneic haematopoietic stem cell transplantation (HSCT) remains controversial. Its relevance and incidence are probably influenced by the conditioning regimen and incorporation of T‐cell depletion. The presence of recipient chimerism levels >40–50% following T‐cell replete reduced intensity transplantation correlates with a high risk of graft rejection, regardless of donor‐lymphocyte infusions, but it is unclear whether this finding translates to T‐cell depleted transplants. We conducted a retrospective single‐institution analysis of patients receiving alemtuzumab‐based HSCT. 27/152 (18%) evaluable cases had predominantly recipient T‐cell chimerism at 3 months or beyond. By contrast, coincident chimerism in the granulocyte lineage was predominantly of donor origin (median 100%) in all but one patient. Donor lymphocyte infusion effectively converted predominantly recipient T‐cell chimerism to ful donor chimerism in all evaluable cases including three cases with no detectable donor T cells. The only graft failure occurred in the patient with predominantly recipient myeloid chimerism in whom rejection occurred rapidly before donor lymphocytes could be administered. We conclude that predominant or complete recipient T‐cell chimerism following alemtuzumab‐based regimens does not have the same clinical implications as that following T‐cell replete transplants and can be effectively converted with donor lymphocytes without the need for lympho‐depleting agents or re‐conditioning.     

Monitoring of residual disease and guided donor leucocyte infusion after allogeneic bone marrow transplantation by chimaerism analysis with short tandem repeats

In this study, we analysed the chimaeric status of peripheral blood leucocytes (PBLs) in recipients of allogeneic bone marrow transplantation (BMT) with the use of short tandem repeat (STR) microsatellite markers for monitoring the efficacy of BMT and donor leucocyte infusions (DLIs). A set of four STR markers was used with a highly discrimative capacity between individuals. STRs were detected by polymerase chain reaction (PCR) and were analysed by gene scanning (STR-GS). Between June 1990 and December 1998, 52 patients treated with BMT for chronic myeloid leukaemia (CML) were analysed. Seventeen patients relapsed after BMT and two patients never achieved remission after BMT. Fourteen of the 17 patients achieved a complete donor chimaerism after BMT, as detected by the presence of only donor STR-GS fragments, and in three cases a weak recipient STR-GS signal remained persistently detectable after BMT. A reappearance or increase of recipient STR-GS signals was indicative of relapse, which was mostly detected by STR-GS several months before relapse was diagnosed clinically. Nineteen patients were treated with DLI for reappearance of CML after BMT which resulted in complete remission in 17 patients, concordant with the disappearance of recipient STR-GS signals. More importantly, DLI treatment could be guided based upon the STR-GS data, which prevented unnecessary extra DLI courses that could cause toxicity. This study indicates that STR-GS is an effective and reliable method for monitoring BMT recipients.     

Minimal residual disease is more common in patients who have mixed T- cell chimerism after bone marrow transplantation for chronic myelogenous leukemia

Determining both lymphoid chimerism and the presence of minimal residual disease after allogeneic bone marrow transplantation (BMT) for chronic myelogenous leukemia (CML) could be helpful to the understanding of the biology of leukemic relapse in this disease. We prospectively investigated 32 patients with CML post-BMT by assessing T- cell chimerism and minimal residual disease using sensitive polymerase chain reaction (PCR) methodologies. Patients were studied between 1 and 24 months post-BMT. Thirty patients received a T-cell-depleted marrow grafts and 2 received unmanipulated marrow. All but 1 patient were conditioned with total body irradiation (TBI)+thiotepa+cyclophosphamide (Cy). The other patient received TBI+Cy as conditioning. The T cells were exclusively of donor origin in 12 of 16 patients who were tested at 1 month post-BMT, but were mixed chimeric in 11 of these patients by > or = 3 months. Once mixed T-cell chimerism was documented, no patient returned to having all donor T-cells. At a median follow-up of 12 months, minimal residual disease was present in 18 of 22 patients with mixed T-cell chimerism and in 3 of 10 patients with full donor chimerism. The actuarial molecular relapse rate at 24 months for the two groups is 91% and 33%, respectively (P < .02). The finding of BCR- ABL mRNA within the first 6 months of transplant or on two consecutive assays was highly predictive of subsequent cytogenetic or hematologic relapse (P = .032 and P < .02, respectively). Ten patients, 9 with mixed T-cell chimerism, have relapsed (4 clinical, 6 cytogenetic) at a median of 12 months post-BMT. These data suggest that mixed T-cell chimerism may be a marker for abrogation of graft-versus-leukemia activity that is thought to be pivotal in eradicating minimal residual disease after BMT for CML.     

Molecular analysis of relapse in chronic myeloid leukemia after allogeneic bone marrow transplantation

Four patients with Philadelphia (Ph') positive chronic myeloid leukemia (CML) were studied before, after, and on relapse following allogeneic bone marrow transplantation (BMT). Southern analysis of DNA from cells collected before and at relapse after BMT was performed in order to investigate the origin of the leukemia at relapse. Using minisatellite probes we showed that the relapse occurred in cells of host origin in all four patients and this was confirmed with a Y chromosome specific probe in two male patients who had a female donor. Furthermore, using two probes for the breakpoint cluster region (bcr) on chromosome 22, we showed that leukemic cells at relapse bore identical rearrangements to those in the disease at time of presentation of each patient. We conclude that relapse in all four patients is due to re-emergence of the original leukemic clone.     

Engraftment kinetics after nonmyeloablative allogeneic peripheral blood stem cell transplantation: full donor T-cell chimerism precedes alloimmune responses.

Nonmyeloablative allogeneic stem cell transplantation has recently been explored as a safer alternative to conventional high-dose transplant regimens. Although a high incidence of mixed chimerism after nonmyeloablative procedures has been reported, the exact kinetics of engrafting donor cells in specific cellular lineages has yet to be defined. We investigated lineage-specific chimerism in 15 patients receiving an allogeneic peripheral blood stem cell (PBSC) transplant from an HLA-identical (n = 14) or a 5/6 antigen-matched sibling donor after a preparative regimen of cyclophosphamide and fludarabine. Donor chimerism was assessed weekly in T lymphocytes and myeloid cells by polymerase chain reaction (PCR) of minisatellite regions. Eight patients survived between 121 to 409 days after transplant. Ten of 14 patients surviving more than 30 days (71.4%) had delayed disease regression consistent with a graft-versus-malignancy (GVM) effect. One patient rejected the transplant with subsequent recovery of autologous hematopoiesis. Hematological recovery was rapid (median, 11 days to >/=500 neutrophils/microL) and was initially predominantly recipient in origin. Donor myeloid chimerism gradually supplanted recipient hematopoiesis and became fully donor in all survivors by 200 days after transplantation. In contrast, T-cell engraftment was more rapid, with full chimerism in 7 patients by day 30 and in 6 further patients by day 200 after cyclosporine withdrawal and donor lymphocyte infusion. Full donor T-cell engraftment preceded donor myeloid engraftment, acute graft-versus-host disease, and disease regression, consistent with a requirement for 100% donor T-cell chimerism for full expression of the alloresponse. These results emphasize the importance of lineage-specific chimerism analysis to successfully manipulate engraftment after nonmyeloablative allogeneic PBSC transplantation.     

Long-term chimerism and B-cell function after bone marrow transplantation in patients with severe combined immunodeficiency with B cells: A single-center study of 22 patients.

We retrospectively analyzed the B-cell function and leukocyte chimerism of 22 patients with severe combined immunodeficiency with B cells (B(+) SCID) who survived more than 2 years after bone marrow transplantation (BMT) to determine the possible consequences of BMT procedures, leukocyte chimerism, and SCID molecular deficit on B-cell function outcome. Circulating T cells were of donor origin in all patients. In recipients of HLA-identical BMT (n = 5), monocytes were of host origin in 5 and B cells were of host origin in 4 and of mixed origin in 1. In recipients of HLA haploidentical T-cell-depleted BMT (n = 17), B cells and monocytes were of host origin in 14 and of donor origin in 3. Engraftment of B cells was found to be associated with normal B-cell function. In contrast, 10 of 18 patients with host B cells still require Ig substitution. Conditioning regimen (ie, 8 mg/kg busulfan and 200 mg/kg cyclophosphamide) was shown neither to promote B-cell and monocyte engraftment nor to affect B-cell function. Eight patients with B cells of host origin had normal B-cell function. Evidence for functional host B cells was further provided in 3 informative cases by Ig allotype determination and by the detection, in 5 studied cases, of host CD27(+) memory B cells as in age-matched controls. These results strongly suggest that, in some transplanted patients, host B cells can cooperate with donor T cells to fully mature in Ig-producing cells.