Aplastic anaemia in donor cells 14 years after bone-marrow transplant.
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Anemia, Aplastic↗
Biomedical subjects
J H Falkenburg
Publications and source records attributed to J H Falkenburg.
HLA-B8 and HLA-A3 coexpressed with HLA-B8 are associated with a reduced risk of the development of chronic myeloid leukemia. The Chronic Leukemia Working Party of the EBMT.
Chronic myeloid leukemia (CML) is characterized by the chromosomal translocation t(9;22) resulting in the chimeric bcr-abl oncogene that encodes the P210 fusion protein, which contains a unique amino acid sequence. If peptides derived from the leukemia-specific part of P210 are expressed in HLA molecules on the cell membrane of leukemic cells, an immunological response may occur. Recent studies using synthetic peptides identical to the bcr-abl fusion region showed that some peptides are capable of binding to HLA-A3, -A11, and -B8 molecules. Cytotoxic T-cell responses have been induced against bcr-abl-derived synthetic peptides bound to HLA-A3 and -B8. We hypothesized that if antigen processing of the P210 fusion protein leads to presentation of peptides from the fusion region by major histocompatibility complex (MHC) molecules in vivo, this may be reflected in a diminished incidence of CML in individuals expressing HLA-A3, -A11, or -B8. Consequently, lower frequencies of these antigens would be expected in patients with CML compared with unaffected individuals. A case-control study and a meta-analysis were performed to test this hypothesis. The multicenter case-control study compared patients with CML from the data base of the European Group for Blood and Marrow Transplantation (EBMT) with unaffected individuals from the registry of Bone Marrow Donors Worldwide. Patients and controls were matched per country. The meta-analysis consisted of five studies reported in the literature. The multicenter case-control study consisting of 1,899 patients and 512, 363 bone marrow donors as controls yielded odds ratios (ORs) of 0.90 (95% confidence interval [CI], 0.80 to 1.00) for HLA-A3, 1.16 (95% CI, 1.02 to 1.33) for HLA-A11, and an OR of 0.73 (95% CI, 0.65 to 0. 82) for HLA-B8. Coexpression of HLA-A3 and HLA-B8 gave an OR of 0.51 (95% CI, 0.40 to 0.67). This can be translated in a protective effect of 27% for HLA-B8, 10% for HLA-A3, and 49% protection for the combination of HLA-A3 and HLA-B8. The meta-analysis comprising 463 CML patients and 4,912 controls showed a 29% risk reduction for individuals expressing HLA-B8 (OR of 0.71; 95% CI, 0.52 to 0.97), but an OR of 1.19 (95% CI, 0.90 to 1.56) for HLA-A3 and an OR of 1. 09 (95% CI, 0.80 to 1.50) for HLA-A11. In conclusion, these results indicate that HLA-B8 expression, in particular when HLA-A3 is coexpressed, is associated with a diminished incidence of CML. A biological mechanism may be that presentation of bcr-abl breakpoint peptides in these HLA molecules can induce a protective immune response.
Case-Control Studies↗
Mismatched transplants for refractory lymphoma?
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Bone Marrow Transplantation↗
Hairy cell leukemia-specific recognition by multiple autologous HLA-DQ or DP-restricted T-cell clones.
We studied in patients with hairy cell leukemia (HCL) whether autoreactive T cells could be isolated with specific reactivity to the HCL cells. HCL cells were activated via triggering of CD40 on the cell membrane and used as stimulator cells to generate autologous T-cell clones. Two types of CD4(+)BV2(+) T-cell clones with different CDR3 rearrangements and one type of CD4(+)BV8S3(+) T-cell clone were generated from the spleen or blood. These clones specifically recognized the autologous HCL cells, without reactivity to autologous peripheral blood mononuclear cells (PBMC), phytohemagglutinin blasts, or Epstein-Barr virus-transformed B cells in a primed lymphocyte test. Blocking and panel studies using HCL cells from 11 other patients showed that recognition of the HCL cells by the BV2(+) T cells was restricted by HLA-DQA1*03/DQB1*0301, and the BV8S3(+) T cells were restricted by DPB1*04. The T-cell clones did not recognize DPB1*04(+) or DQ3(+) PBMC from healthy donors or DP/DQ matched malignant cells from patients with other hematologic malignancies, except for one patient with acute lymphoblastic leukemia. These HCL-specific T-cell clones may be used for the detection of an HCL-specific tumor antigen.
Animals↗
Impaired expression of CD28 on T cells in hairy cell leukemia.
T cells from patients with active hairy cell leukemia (HCL), a chronic B cell malignancy, show poor proliferation in response to allogeneic peripheral blood mononuclear cells (PBMC). In order to study the T cell dysfunction, the expression of several adhesion and costimulatory molecules was analyzed by flow cytometry. Circulating T cells from HCL patients showed increased percentages of CD28(-) in all T cell subsets. In some patients the percentage of CD28(-) T cells within the CD4(+) subset was increased up to 80%. These CD4(+)CD28(-) T cells did not proliferate in a mixed lymphocyte culture (MLC) against allogeneic PBMC. After enrichment for CD4(+)CD28(+) T cells, the proliferative response in the MLC was recovered, but this response was still lower than the proliferative response from control T cells. In conclusion, lack of CD28 on T cells and a restricted T cell repertoire may contribute to immune deficiency in patients with HCL.
Blood Donors↗
Tetrameric HLA class I-minor histocompatibility antigen peptide complexes demonstrate minor histocompatibility antigen-specific cytotoxic T lymphocytes in patients with graft-versus-host disease.
Graft-versus-host disease (GvHD) is a chief complication of allogeneic bone marrow transplantation. In HLA-identical bone marrow transplantation, GvHD may be induced by disparities in minor histocompatibility antigens (mHags) between the donor and the recipient, with the antigen being present in the recipient and not in the donor. Cytotoxic T lymphocytes (CTLs) specific for mHags of the recipients can be isolated from the blood of recipients with severe GvHD (ref. 3). A retrospective study demonstrated an association between mismatch for mHags HA-1, -2, -4 and -5 and the occurrence of GvHD in adult recipients of bone marrow from HLA genotypically identical donors. Tetrameric HLA-peptide complexes have been used to visualize and quantitate antigen-specific CTLs in HIV-infected individuals and during Epstein-Barr virus and lymphocytic choriomeningitis virus infections. Here we show the direct ex vivo visualization of mHag-specific CTLs during GvHD using tetrameric HLA-class and I-mHag HA-1 and HY peptide complexes. In the peripheral blood of 17 HA-1 or HY mismatched marrow recipients, HA-1- and HY-specific CTLs were detected as early as 14 days after bone marrow transplantation. The tetrameric complexes demonstrated a significant increase in HA-1- and HY-specific CTLs during acute and chronic GvHD, which decreased after successful GvHD treatment. HLA class I-mHag peptide tetramers may serve as clinical tools for the diagnosis and monitoring of GvHD patients.
Adult↗
T cells recognizing leukemic CD34(+) progenitor cells mediate the antileukemic effect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation.
Adoptive immunotherapy with donor lymphocyte infusions (DLI) is an effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. To identify the effector and target cell populations responsible for the elimination of the leukemic cells in vivo we developed an assay to measure the frequency of T lymphocyte precursor cells capable of suppressing leukemic progenitor cells. Target cells in this assay were CML cells that were cultured in the presence of stem cell factor, interleukin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. [3H]thymidine incorporation at day 7 represented the proliferation of the progeny of the CD34(+) CML progenitor cells, and not of the more mature CD34(-) CML cells. Effector cells were mononuclear cells, which were used in a limiting dilution analysis to measure the frequencies of CML progenitor cell-inhibitory lymphocyte precursors (PCILp) in peripheral blood of seven patients before and after DLI for relapsed CML. In the six patients who entered complete remission, a 5- to 100-fold increase of PCILp was found during the clinical response. In the patient with resistant relapse the frequency of PCILp was <10 per ml before and after DLI. Leukemia-reactive helper T lymphocyte precursor frequencies remained unchanged after DLI. A significant increase in cytotoxic T lymphocyte precursor frequency against more mature leukemic cells was found in only two responding patients. These results indicate that T cells specifically directed against CD34(+) CML progenitor cells mediate the antileukemic effect of DLI.
Antigens, CD34↗
Correction of abnormal T-cell receptor repertoire during interferon-alpha therapy in patients with hairy cell leukemia.
Patients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-alpha (IFN-alpha) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-alpha in the past, at initiation, and at several intervals up to 6 years of IFN-alpha treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers. In all seven patients improvement of the skewed T-cell repertoire was not seen until 2 years of treatment. It consisted of disappearance of oligoclonal subpopulations and (polyclonal) reappearance of absent TCRBV gene families. The RT-PCR results were correlated with the TCRBV protein expression using TCRBV-specific monoclonal antibodies. T lymphocytes from four patients with active HCL contained large expansions of particular TCRBV-expressing cells (up to 25% of the CD3+ cells; 600 to 700/microL whole blood), which decreased during IFN-alpha therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as measured by proliferative, helper, and cytotoxic T-lymphocyte precursor frequencies against major histocompatibility complex-unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T lymphocytes expressing the same TCRBV family. Moreover, IFN-alpha can restore the skewed T-cell repertoire and suppress persistent T-cell clones upon treatment of the accompanying malignancy.
Adult↗
Selective response of CD5+ B cell malignancies to activation of the CD72 antigen.
The function of the simultaneous expression of CD5 and its ligand CD72 on B cell malignancies like chronic lymphocytic leukemia and mantle cell lymphoma was assessed. It is unknown if reciprocal interactions between CD72 and CD5 exert an autocrine growth-promoting or -inhibiting effect. CD5+ (n = 13) and CD5- (n = 9) B cell malignancies were cultured with the anti-CD72 mAb WL225. For comparison, five other anti-CD72 mAbs were tested. Only CD5+ B cell malignancies proliferated upon CD72 activation (9 out of 13 cases). A strong suppressive effect of IL-4 on the anti-CD72-induced [3H]thymidine incorporation, partially caused by downmodulation of the CD72 expression, was seen. Stimulation of the CD5 antigen by L cells transfected with human CD72 (LhCD72) and the anti-CD5 mAb 1C12 exerted no (n = 9) or a minor effect (2 out of 8 cases), respectively. Finally, the results of CD72 stimulation were compared with CD40 stimulation, as this "CD40 system" is an effective method for stimulating B cell malignancies. In 4 of the 7 anti-CD72 responsive cases a costimulatory effect was seen.
Antigens, CD↗
Generation of leukemia-reactive cytotoxic T lymphocytes from HLA-identical donors of patients with chronic myeloid leukemia using modifications of a limiting dilution assay.
Donor leukocyte transfusions (DLT) have an anti-leukemic effect in most patients with a relapse of chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. However, DLT are often complicated by graft-versus-host disease. Selection of donor lymphocytes with a relative specificity for leukemic cells is desirable. The generation of leukemia-reactive cytotoxic T lymphocyte (CTL) responses between HLA-identical donors and patients in bulk cultures showed major variations, and false negative results were observed. In a modification of a limiting dilution analysis (LDA) two-fold serial dilutions of HLA-identical donor mononuclear cells (MNC) were cultured in the presence of CML cells. The anti-leukemic CTL precursor frequencies in these donors varied between <1 and 9 per 106 MNC. HLA-restricted CD4+ or CD8+ lymphocytes as well as MHC non-restricted gammadelta T cells were responsible for the anti-leukemic responses. A positive correlation between cytotoxicity in the various wells after 3, 4 and 5 weeks of culture could be found. The LDA may be superior to bulk cultures in selecting stable immune responses and in separating multiple different anti-leukemic T cell responses in each donor-patient combination.
Cell Survival↗
Unconventional rapid Erk1,2 activation is indispensable for proliferation of the growth factor-independent myeloid leukemic cell line KG1.
While normal hematopoietic progenitor cells are dependent on colony-stimulating factors for in vitro proliferation, myeloid leukemic cells are frequently factor-independent. In this study we investigated several signalling intermediates of the Ras-Er1,2 pathway which may be involved in the development of growth factor independence. In the growth factor independent cell line KG1, an extremely short activation pattern of Erk1,2 with a maximum at 30 s was observed in response to FBS. In contrast, stimulation of the IL-3 receptor in AML193 cells resulted in a transient Erk activation peaking at 5 min and returning to base levels after 15 min. Although the Erk activation in KG1 cells is short-lived, using the MEK inhibitor PD98059, we demonstrated that Erk phosphorylation is essential for proliferation of these cell lines. We also detected major differences in Shc phosphorylation between factor-dependent and -independent cells. These data suggest that Erk activation is essential for proliferation of growth factor-dependent and -independent leukemic cells. The minimal Erk activation observed in KG1 cells in response to serum is sufficient for mitogenesis of these cells.
Adaptor Proteins, Signal Transducing↗
T-cell dysfunction in hairy cell leukemia: an updated review.
Hairy cell leukemia (HCL) is clinically associated with severe T-cell dysfunction. Several new observations have given more insight into the abnormal T-cell responses seen in this disease. T-lymphocytes in the spleen of patients with HCL seem to be abnormally activated. On the other hand, they are non-responsive, possibly as a result of monocytopenia which may lead to inadequate antigen presentation. This, together with the lack of CD28 on T-cells, may cause T-cell dysfunction. Furthermore, there is a very restricted repertoire of the T-cell receptor-beta family, which may also result in non-responsiveness. Otherwise, T-cell clonal excess may be indicative for activated, possibly autoreactive T-cells.
Cell Adhesion↗
Umbilical cord blood banking in The Netherlands.
Cord blood banks are being developed in the United States and Europe. In The Netherlands, the EuroCord Nederland Foundation (ECN) has been established to coordinate the development of a national cord blood bank for unrelated transplants. The aim of ECN is to collect at least 5000 transplants for patients who lack an HLA-identical related or unrelated bone marrow donor. Four blood banks in Leiden, Groningen, Nijmegen and Amsterdam, Europdonor Foundation and the Department of Hematology of the Leiden University Medical Center participate in this organization. From March 1995 to November 1997, 720 units have been collected, processed, HLA typed, tested for transmissible diseases and cryopreserved in the Dutch cord blood bank.
Blood Banks↗
Comparison of repopulating ability of hematopoietic progenitor cells isolated from human umbilical cord blood or bone marrow cells in NOD/SCID mice.
The repopulating ability and homing potential of CD34+ cells isolated from either bone marrow (BM) or umbilical cord blood (UCB) was compared in NOD/SCID mice. Mice were sublethally irradiated (3.5 Gy) and within 24 h transplanted i.v. with 10(5)-10(6) CD34+ cells. Four weeks post-transplantation blood was collected from the tail vein for detection of human cells and after 6-7 weeks the mice were sacrificed and blood, BM, thymus, lymph nodes, spleen, liver, lung and kidney were harvested and single cells suspensions were made. Human cells were detected by flow cytometry, and staining was performed for CD34, CD45, and markers of the myeloid, and lymphoid lineages. CD34+ cells from UCB successfully engrafted into the NOD/SCID mice. Eighty-five percent of cells in BM of the mice were of human origin, depending on the dose of cells injected. In all other organs these percentages were always lower, and maximally 63, 13, 3 and 2% in spleen, liver, lung and kidney, respectively. Transplantation of CD34+ cells isolated from human BM, on the other hand, resulted in a very low percentage of human cells after 6-7 weeks of transplantation, not exceeding 3% of the cell suspension. Whether this difference in repopulating ability can be explained by an intrinsic qualitative difference or by differences in quantity of stem cells within the CD34+ compartment from UCB or BM remains to be determined.
Animals↗
HLA class II restricted T-cell reactivity to a developmentally regulated antigen shared by leukemic cells and CD34+ early progenitor cells.
After allogeneic bone marrow transplantation (BMT), the beneficial graft-versus-leukemia (GVL) effect but also the life-threatening graft-versus-host disease (GVHD) are mediated by T cells of the grafted marrow. The identification of leukemia cell-reactive T cells and their ligands are, therefore, crucial for the development of new anti leukemia strategies. Here we describe a leukemia-reactive allo-HLA class II restricted CD4+ T-cell clone, 6.2, isolated from a healthy individual after stimulation with allogeneic leukemic cells. Clone 6.2 recognizes leukemic cells from several AML patients without showing reactivity to unfractioned peripheral blood mononuclear cells, monocytes, B cells, T-cell blasts, and proximal tubulus epithelial cells. Interestingly, clone 6.2 also recognizes BM cells derived from healthy individuals and inhibits the colony formation of myeloid and erythroid cell lineages. In the BM, clone 6.2 recognizes only CD34+ early precursor cells but not CD34-, more differentiated cells. Thus, the target antigen of clone 6.2 is developmentally regulated and expressed only by leukemic cells and CD34+ early progenitor cells in the hematopoietic system. We suggest that targeting the T-cell immune response to leukemia-associated, developmentally regulated antigens of the hematopoietic system can provide a basis for the separation of GVL from GVHD, and may lead to new therapeutic approaches for residual and relapsed leukemia.
Acute Disease↗
Apoptin induces apoptosis in human transformed and malignant cells but not in normal cells.
The chicken anemia virus protein apoptin induces a p53-independent, Bcl-2-insensitive type of apoptosis in various human tumor cells. Here, we show that, in vitro, apoptin fails to induce programmed cell death in normal lymphoid, dermal, epidermal, endothelial, and smooth-muscle cells. However, when normal cells are transformed they become susceptible to apoptosis by apoptin. Long-term expression of apoptin in normal human fibroblasts revealed that apoptin has no toxic or transforming activity in these cells. In normal cells, apoptin was found predominantly in the cytoplasm, whereas in transformed and malignant cells it was located in the nucleus, suggesting that the localization of apoptin is related to its activity. These properties make apoptin a potential agent for the treatment of a large number of tumors, also those lacking p53 and/or overexpressing Bcl-2.
Apoptosis↗
Generation of dendritic cells expressing bcr-abl from CD34-positive chronic myeloid leukemia precursor cells.
Patients with a relapse of chronic myeloid leukemia (CML) after allogeneic bone marrow transplantation can be successfully treated with blood mononuclear cells from the original bone marrow donor. However, the antileukemic effect of this treatment is often accompanied by graft-versus-host disease (GVHD). Treatment with cytotoxic T-lymphocyte (CTL) lines or clones that are specifically generated against leukemic antigen-presenting cells from the patient, may separate antileukemic effects from GVHD. In this report we demonstrate that after culturing CD34-positive cells purified from bone marrow of patients with chronic phase CML in medium containing human serum, GM-CSF, TNF alpha, and IL-4 up to 28% of the cultured cells were dendritic cells, characterized by morphology, phenotypic analysis, and their efficient capacity to stimulate allogeneic T lymphocytes. The expression of HLA and costimulatory molecules and the stimulatory capacity of the dendritic cell-enriched cell suspensions were optimal between days 7 and 10 after onset of the cultures. Fluorescence in situ hybridization revealed that all cultured dendritic cells contained the CML specific t(9;22) translocation. PCR analysis showed expression of the translocation specific bcr-abl mRNA. These leukemic dendritic cells may enhance the induction and proliferation of CTL lines and clones with more specificity for the leukemic cells.
Antigens, CD34↗
8-Bromo-cAMP induces a proliferative response in an IL-3 dependent leukemic cell line and activates Erk 1,2 via a Shc-independent pathway.
In a number of cell types, elevation of intracellular cAMP concentrations antagonizes growth factor-induced mitogenesis by abrogating the downstream signaling of RasGTP to extracellular-signal-regulated kinases (Erk 1,2). We studied the effect of elevation of cAMP concentrations on the IL-3-induced mitogenic response in the leukemic cell line AML193. We observed that 8-bromo-cAMP (8-Br-cAMP) had no inhibitory effect on the magnitude of this response. On the contrary. 8-Br-cAMP alone induced a proliferative response in these cells. 8-Br-cAMP activated Erk 1,2 in these cells without involvement of Shc phosphorylation. These findings suggest the presence of a novel cAMP-dependent signaling pathway in AML193 cells, which activates Erk 1,2 via a Shc-independent pathway and leads to the generation of a mitogenic response.
8-Bromo Cyclic Adenosine Monophosphate↗