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Potent graft-versus-leukemia (GVL) effects can be mediated by donor-derived T cells recognizing minor histocompatibility antigens (mHags) in patients treated with donor lymphocyte infusion (DLI) for relapsed hematologic malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). Donor-derived T cells, however, may not only induce GVL, but also mediate detrimental graft-versus-host disease (GVHD). Because HLA-class II is under noninflammatory conditions predominantly expressed on hematopoietic cells, CD4+ T cells administered late after alloSCT may selectively confer GVL without GVHD. Although a broad range of different HLA-class I–restricted mHags have been identified, the first 2 autosomal HLA-class II–restricted mHags have only recently been characterized. By screening a recombinant bacteria cDNA expression library, we identified 4 new HLA-class II–restricted mHags recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia who achieved long-term complete remission and experienced only mild GVHD of the skin after DLI. All CD4+ T cells were capable of recognizing the mHags presented by HLA-DR surface molecules on primary hematopoietic cells, but not on skin-derived (cytokine-treated) fibroblasts. The selective recognition of hematopoietic cells as well as the balanced population frequencies and common HLA-DR restriction elements make the novel mHags possible targets for development of immunotherapeutic strategies.

Potent graft-versus-leukemia (GVL) effects can be mediated by donor-derived T cells recognizing minor histocompatibility antigens (mHags) in patients treated with donor lymphocyte infusion (DLI) for relapsed hematologic malignancies after HLA-matched allogeneic stem cell transplantation (alloSCT). Donor-derived T cells, however, may not only induce GVL, but also mediate detrimental graft-versus-host disease (GVHD). Because HLA-class II is under noninflammatory conditions predominantly expressed on hematopoietic cells, CD4+T cells administered late after alloSCT may selectively confer GVL without GVHD. Although a broad range of different HLA-class I–restricted mHags have been identified, the first 2 autosomal HLA-class II–restricted mHags have only recently been characterized. By screening a recombinant bacteria cDNA expression library, we identified 4 new HLA-class II–restricted mHags recognized by CD4+T cells induced in a patient with relapsed chronic myeloid leukemia who achieved long-term complete remission and experienced only mild GVHD of the skin after DLI. All CD4+T cells were capable of recognizing the mHags presented by HLA-DR surface molecules on primary hematopoietic cells, but not on skin-derived (cytokine-treated) fibroblasts. The selective recognition of hematopoietic cells as well as the balanced population frequencies and common HLA-DR restriction elements make the novel mHags possible targets for development of immunotherapeutic strategies.

Reduced-intensity conditioning (RIC) has allowed the use of allogeneic stem cell transplantation (alloSCT) for haematological malignancies in elderly patients. A major problem of this type of transplantation is the high incidence of persisting chronic graft-versus-host disease (GvHD), leading to increased morbidity and mortality. The inclusion of alemtuzumab added to the graft (‘Campath in the bag’) for donor T-cell depletion offers an easy procedure to diminish the incidence of GvHD. Good engraftment is observed in most patients, whereas almost no GvHD is observed after transplantation. Most patients become mixed chimeric after transplantation, requiring donor lymphocyte infusion for conversion to full donor chimerism. Although subsequent acute and chronic GvHD is observed in 50–60% of patients, it is responsive to therapy in many patients, resulting in a low incidence of persisting chronic GvHD. AlloSCT with RIC and alemtuzumab-induced T-cell depletion offers a suitable platform for the investigation of novel cellular immunotherapy.

Allogeneic stem cell transplantation (alloSCT) or autologous SCT (autoSCT) and intensive consolidation/intensification courses plus maintenance chemotherapy for 1 to 2 years are currently the major options for post-remission treatment of adult patients with acute lymphoblastic leukemia (ALL) in first remission. Comparison of their value with respect to relapse prevention, disease-free survival, and overall survival has been impossible until recently when the results of several randomized trials became available. Herein, we try to dissect data from these randomized trials to evaluate the role of autoSCT in patients with ALL in complete remission. Five prospectively randomized trials were found in which patients with a family donor were eligible for an alloSCT and the remaining patients were randomized between autoSCT and continuation chemotherapy. In addition, in two prospectively randomized trials all patients with a donor were eligible for an alloSCT and the remaining patients were eligible for autoSCT. Using intention to treat, in the majority of ALL studies alloSCT is superior to autoSCT or intensive continuation chemotherapy. It still has to be determined which subgroups of ALL benefit most of allogeneic transplantation, since in some trials the advantage of allogeneic transplantation was confined to the standard-risk ALL patients and in other trials to the high-risk patients. With respect to the role of autoSCT compared to continuation chemotherapy, both treatment modalities show equal, although for high-risk ALL inferior, overall survival chances. In one large trial the disease-free survival in the autoSCT arm was inferior to that in the chemotherapy arm. This finding may eventually have an impact on the overall survival rate. Currently, the main benefit of autoSCT may be its short duration compared with the continuation chemotherapy regimen.

Minor histocompatibility antigens (mHags) play an important role in both graft-versus-tumor effects and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. We applied biochemical techniques and mass spectrometry to identify the peptide recognized by a dominant tumor-reactive donor T-cell reactivity isolated from a patient with relapsed multiple myeloma who underwent transplantation and entered complete remission after donor lymphocyte infusion. A frequently occurring single nucleotide polymorphism in the human ATP-dependent interferon-responsive (ADIR) gene was found to encode the epitope we designated LB-ADIR-1F. Although gene expression could be found in cells from hematopoietic as well as nonhematopoietic tissues, the patient suffered from only mild acute GVHD despite high percentages of circulating LB-ADIR-1F–specific T cells. Differential recognition of nonhematopoietic cell types and resting hematopoietic cells as compared with activated B cells, T cells, and tumor cells was demonstrated, illustrating variable LB-ADIR-1F expression depending on the cellular activation state. In conclusion, the novel mHag LB-ADIR-1F may be a suitable target for cellular immunotherapy when applied under controlled circumstances.

Minor histocompatibility antigens (mHags) play an important role in both graft-versus-tumor effects and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. We applied biochemical techniques and mass spectrometry to identify the peptide recognized by a dominant tumor-reactive donor T-cell reactivity isolated from a patient with relapsed multiple myeloma who underwent transplantation and entered complete remission after donor lymphocyte infusion. A frequently occurring single nucleotide polymorphism in the human ATP-dependent interferon-responsive (ADIR)gene was found to encode the epitope we designated LB-ADIR-1F. Although gene expression could be found in cells from hematopoietic as well as nonhematopoietic tissues, the patient suffered from only mild acute GVHD despite high percentages of circulating LB-ADIR-1F–specific T cells. Differential recognition of nonhematopoietic cell types and resting hematopoietic cells as compared with activated B cells, T cells, and tumor cells was demonstrated, illustrating variable LB-ADIR-1F expression depending on the cellular activation state. In conclusion, the novel mHag LB-ADIR-1F may be a suitable target for cellular immunotherapy when applied under controlled circumstances.

Genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T-cell immunity toward viral infections and malignancies. We previously demonstrated redirected antileukemic reactivity of cytomegalovirus (CMV)–specific T cells by transfer of minor histocompatibility antigen HA-2–specific T-cell receptors (TCRs). HA-2–TCR-transferred CMV-specific T cells were potent effectors against HA-2–expressing leukemic cells, as well as CMV-expressing cells. Functional activity of these T cells correlated with TCR cell-surface expression. In the present study we analyzed which properties of transferred and endogenous TCRs are crucial for efficient cell-surface expression. We demonstrate that expression of the introduced TCR is not a random process but is determined by characteristics of both the introduced and the endogenously expressed TCR. The efficiency of TCR cell-surface expression is controlled by the intrinsic quality of the TCR complex. In addition, we demonstrate that chimeric TCRs can be formed and that efficiency of TCR expression is independent of whether TCRs are retrovirally introduced or naturally expressed. In conclusion, introduced, endogenous, and chimeric TCRs compete for cell-surface expression in favor of the TCR-CD3 complex with best-pairing properties.

Genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T-cell immunity toward viral infections and malignancies. We previously demonstrated redirected antileukemic reactivity of cytomegalovirus (CMV)–specific T cells by transfer of minor histocompatibility antigen HA-2–specific T-cell receptors (TCRs). HA-2–TCR-transferred CMV-specific T cells were potent effectors against HA-2–expressing leukemic cells, as well as CMV-expressing cells. Functional activity of these T cells correlated with TCR cell-surface expression. In the present study we analyzed which properties of transferred and endogenous TCRs are crucial for efficient cell-surface expression. We demonstrate that expression of the introduced TCR is not a random process but is determined by characteristics of both the introduced and the endogenously expressed TCR. The efficiency of TCR cell-surface expression is controlled by the intrinsic quality of the TCR complex. In addition, we demonstrate that chimeric TCRs can be formed and that efficiency of TCR expression is independent of whether TCRs are retrovirally introduced or naturally expressed. In conclusion, introduced, endogenous, and chimeric TCRs compete for cell-surface expression in favor of the TCR-CD3 complex with best-pairing properties.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have emerged as a promising tool for clinical application. Further clinical interest has been raised by the observation that MSCs are immunoprivileged and, more important, display immunosuppressive capacities. These properties may be of therapeutic value in allogeneic transplantation to prevent graft rejection and to prevent and treat graft-versus-host disease. In the present study, we examined the in vivo immunomodulatory properties of MSCs in murine models of allogeneic bone marrow (BM) transplantation. Sublethally irradiated recipients received allogeneic BM with or without host or donor MSCs. The addition of host MSCs significantly enhanced the long-term engraftment associated with tolerance to host and donor antigens. However, the infusion of donor MSCs was associated with significantly increased rejection of allogeneic donor BM cells. Moreover, we showed that the injection of merely allogeneic donor MSCs in naive mice was sufficient to induce a memory T-cell response. Although the observed engraftment-promoting effects of host MSCs in vivo support the therapeutic potential of MSCs, our results also indicate that allogeneic MSCs are not intrinsically immunoprivileged and that under appropriate conditions, allogeneic MSCs induce a memory T-cell response resulting in rejection of an allogeneic stem cell graft.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have emerged as a promising tool for clinical application. Further clinical interest has been raised by the observation that MSCs are immunoprivileged and, more important, display immunosuppressive capacities. These properties may be of therapeutic value in allogeneic transplantation to prevent graft rejection and to prevent and treat graft-versus-host disease. In the present study, we examined the in vivo immunomodulatory properties of MSCs in murine models of allogeneic bone marrow (BM) transplantation. Sublethally irradiated recipients received allogeneic BM with or without host or donor MSCs. The addition of host MSCs significantly enhanced the long-term engraftment associated with tolerance to host and donor antigens. However, the infusion of donor MSCs was associated with significantly increased rejection of allogeneic donor BM cells. Moreover, we showed that the injection of merely allogeneic donor MSCs in naive mice was sufficient to induce a memory T-cell response. Although the observed engraftment-promoting effects of host MSCs in vivo support the therapeutic potential of MSCs, our results also indicate that allogeneic MSCs are not intrinsically immunoprivileged and that under appropriate conditions, allogeneic MSCs induce a memory T-cell response resulting in rejection of an allogeneic stem cell graft.

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched hematopoietic stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated tumor-reactive cytotoxic CD8+T-cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma after allogeneic HLA-matched SCT. Using cDNA expression cloning, the target molecule of an HLA-B7–restricted CTL clone was identified. The CTL clone recognized a minor histocompatibility antigen produced by a single nucleotide polymorphism (SNP) in the angiogenic endothelial-cell growth factor-1 (ECGF1)gene also known as thymidine phosphorylase. The SNP leads to an Arg-to-His substitution in an alternatively translated peptide that is recognized by the CTL. The ECGF1gene is predominantly expressed in hematopoietic cells, although low expression can also be detected in other tissues. The patient from whom this CTL clone was isolated had mild graft-versus-host disease despite high numbers of circulating ECGF-1–specific T cells as detected by tetramer staining. Because solid tumors expressing ECGF-1 could also be lysed by the CTL, ECGF-1 is an interesting target for immunotherapy of both hematologic and solid tumors.

Characterization of the antigens recognized by tumor-reactive T cells isolated from patients successfully treated with allogeneic HLA-matched hematopoietic stem cell transplantation (SCT) can lead to the identification of clinically relevant target molecules. We isolated tumor-reactive cytotoxic CD8+ T-cell (CTL) clones from a patient successfully treated with donor lymphocyte infusion for relapsed multiple myeloma after allogeneic HLA-matched SCT. Using cDNA expression cloning, the target molecule of an HLA-B7–restricted CTL clone was identified. The CTL clone recognized a minor histocompatibility antigen produced by a single nucleotide polymorphism (SNP) in the angiogenic endothelial-cell growth factor-1 (ECGF1) gene also known as thymidine phosphorylase. The SNP leads to an Arg-to-His substitution in an alternatively translated peptide that is recognized by the CTL. The ECGF1 gene is predominantly expressed in hematopoietic cells, although low expression can also be detected in other tissues. The patient from whom this CTL clone was isolated had mild graft-versus-host disease despite high numbers of circulating ECGF-1–specific T cells as detected by tetramer staining. Because solid tumors expressing ECGF-1 could also be lysed by the CTL, ECGF-1 is an interesting target for immunotherapy of both hematologic and solid tumors.

T-cell tolerance is mandatory for major histocompatibility complex (MHC)-mismatched stem-cell transplantation without cytoreduction. Here, we used a cytotoxicity assay based on the infusion of differentially carboxyfluorescein succinimidyl ester (CFSE)-labeled syngeneic and donor splenocytes to determine the survival of donor cells in vivo. In vivo cytotoxicity data showed that treatment with anti-CD40 ligand monoclonal antibody in combination with a low dose of MHC-mismatched bone marrow cells was sufficient to induce T-cell tolerance. However, CFSE-labeled donor cells were still eliminated. A similar elimination pattern was observed in T-cell and natural killer T-cell (NKT-cell)-deficient mice, suggesting the involvement of natural killer (NK) cells. Indeed, in vivo NK-cell depletion resulted in a prolonged survival of CFSE-labeled donor cells, confirming the role of NK cells in this process. Transplantation of a megadose of MHC-mismatched bone marrow cells was required for a complete survival of CFSE-labeled donor cells. This NK-cell tolerance was donor specific and was associated with mixed chimerism. Additional NK-cell depletion significantly enhanced engraftment and allowed long-term chimerism after transplantation of a relatively low dose of donor bone marrow cells. These data demonstrate the importance of NK cells in the rejection of MHC-mismatched hematopoietic cells and may explain the high numbers of bone marrow cells required for transplantation over MHC barriers. (Blood. 2005;106:2215-2220)

T-cell tolerance is mandatory for major histocompatibility complex (MHC)-mismatched stem-cell transplantation without cytoreduction. Here, we used a cytotoxicity assay based on the infusion of differentially carboxyfluorescein succinimidyl ester (CFSE)-labeled syngeneic and donor splenocytes to determine the survival of donor cells in vivo. In vivo cytotoxicity data showed that treatment with anti-CD40 ligand monoclonal antibody in combination with a low dose of MHC-mismatched bone marrow cells was sufficient to induce T-cell tolerance. However, CFSE-labeled donor cells were still eliminated. A similar elimination pattern was observed in T-cell and natural killer T-cell (NKT-cell)-deficient mice, suggesting the involvement of natural killer (NK) cells. Indeed, in vivo NK-cell depletion resulted in a prolonged survival of CFSE-labeled donor cells, confirming the role of NK cells in this process. Transplantation of a megadose of MHC-mismatched bone marrow cells was required for a complete survival of CFSE-labeled donor cells. This NK-cell tolerance was donor specific and was associated with mixed chimerism. Additional NK-cell depletion significantly enhanced engraftment and allowed long-term chimerism after transplantation of a relatively low dose of donor bone marrow cells. These data demonstrate the importance of NK cells in the rejection of MHC-mismatched hematopoietic cells and may explain the high numbers of bone marrow cells required for transplantation over MHC barriers. (Blood. 2005;106:2215-2220)

The role of glycosylated recombinant human granulocyte colony-stimulating factor (G-CSF) in the induction treatment of older adults with acute myeloid leukemia (AML) is still uncertain. In this trial, a total of 722 patients with newly diagnosed AML, median age 68 years, were randomized into 4 treatment arms: (A) no G-CSF; (B) G-CSF during chemotherapy; (C) G-CSF after chemotherapy until day 28 or recovery of polymorphonuclear leukocytes; and (D) G-CSF during and after chemotherapy. The complete remission (CR) rate was 48.9% in group A, 52.2% in group B, 48.3% in group C, and 64.4% in group D. Analysis according to the 2 × 2 factorial design indicated that the CR rate was significantly higher in patients who received G-CSF during chemotherapy (58.3% for groups B + D vs 48.6% for groups A + C; P = .009), whereas no significant difference was observed between groups A + B and C + D (50.6% vs 56.4%, P = .12). In terms of overall survival, no significant differences were observed between the various groups. Patients who received G-CSF after chemotherapy had a shorter time to neutrophil recovery (median, 20 vs 25 days; P < .001) and a shorter hospitalization (mean, 27.2 vs 29.7 days; P < .001). We conclude that although priming with G-CSF can improve the CR rate, the use of G-CSF during and/or after chemotherapy has no effect on the long-term outcome of AML in older patients.

The role of glycosylated recombinant human granulocyte colony-stimulating factor (G-CSF) in the induction treatment of older adults with acute myeloid leukemia (AML) is still uncertain. In this trial, a total of 722 patients with newly diagnosed AML, median age 68 years, were randomized into 4 treatment arms: (A) no G-CSF; (B) G-CSF during chemotherapy; (C) G-CSF after chemotherapy until day 28 or recovery of polymorphonuclear leukocytes; and (D) G-CSF during and after chemotherapy. The complete remission (CR) rate was 48.9% in group A, 52.2% in group B, 48.3% in group C, and 64.4% in group D. Analysis according to the 2 × 2 factorial design indicated that the CR rate was significantly higher in patients who received G-CSF during chemotherapy (58.3% for groups B + D vs 48.6% for groups A + C; P= .009), whereas no significant difference was observed between groups A + B and C + D (50.6% vs 56.4%, P= .12). In terms of overall survival, no significant differences were observed between the various groups. Patients who received G-CSF after chemotherapy had a shorter time to neutrophil recovery (median, 20 vs 25 days; P< .001) and a shorter hospitalization (mean, 27.2 vs 29.7 days; P< .001). We conclude that although priming with G-CSF can improve the CR rate, the use of G-CSF during and/or after chemotherapy has no effect on the long-term outcome of AML in older patients.

T cells directed against minor histocompatibility antigens (mHags) might be responsible for eradication of hematological malignancies after allogeneic stem cell transplantation. We investigated whether transfer of T cell receptors (TCRs) directed against mHags, exclusively expressed on hematopoietic cells, could redirect virus-specific T cells toward antileukemic reactivity, without the loss of their original specificity. Generation of T cells with dual specificity may lead to survival of these TCR-transferred T cells for prolonged periods of time in vivo due to transactivation of the endogenous TCR of the tumor-reactive T cells by the latent presence of viral antigens. Furthermore, TCR transfer into restricted T cell populations, which are nonself reactive, will minimize the risk of autoimmunity. We demonstrate that cytomegalovirus (CMV)-specific T cells can be efficiently reprogrammed into leukemia-reactive T cells by transfer of TCRs directed against the mHag HA-2. HA-2-TCR–transferred CMV-specific T cells derived from human histocompatibility leukocyte antigen (HLA)-A2+ or HLA-A2− individuals exerted potent antileukemic as well as CMV reactivity, without signs of anti–HLA-A2 alloreactivity. The dual specificity of these mHag-specific, TCR-redirected virus-specific T cells opens new possibilities for the treatment of hematological malignancies of HLA-A2+ HA-2–expressing patients transplanted with HLA-A2–matched or –mismatched donors.

For the clinical evaluation of the efficacy of cellular immunotherapy it is necessary to analyze the effector functions of T cells against primary leukemic target cell populations which are usually considerably heterogeneous caused by differential maturation stages of the leukemic cells. An appropriate assay should not only allow the quantitative analysis of rapid cell death induction as measured by the conventional 51Cr release assay but also of the more slowly executing pathways of T-cell-induced apoptosis occurring within days instead of hours which cannot be measured using this method. Furthermore, it should dissect the differential susceptibility to T-cell-induced cell death of various target cell subpopulations and characterize the malignant precursor cells capable of producing malignant progeny. To fulfill these requirements we developed a new assay based on carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the target cell population combined with antibody staining of specific cell populations and addition of fluorescent microbeads to quantitatively monitor target cell death occurring within a longer time frame up to at least 5 days. This new assay facilitates the analysis of differential recognition of distinct cell types within a heterogeneous target cell population and allows simultaneously evaluation of the proliferative status of surviving target cells in response to relevant cytokines. (Blood. 2004;103: 2677-2682)

For the clinical evaluation of the efficacy of cellular immunotherapy it is necessary to analyze the effector functions of T cells against primary leukemic target cell populations which are usually considerably heterogeneous caused by differential maturation stages of the leukemic cells. An appropriate assay should not only allow the quantitative analysis of rapid cell death induction as measured by the conventional51Cr release assay but also of the more slowly executing pathways of T-cell-induced apoptosis occurring within days instead of hours which cannot be measured using this method. Furthermore, it should dissect the differential susceptibility to T-cell-induced cell death of various target cell subpopulations and characterize the malignant precursor cells capable of producing malignant progeny. To fulfill these requirements we developed a new assay based on carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the target cell population combined with antibody staining of specific cell populations and addition of fluorescent microbeads to quantitatively monitor target cell death occurring within a longer time frame up to at least 5 days. This new assay facilitates the analysis of differential recognition of distinct cell types within a heterogeneous target cell population and allows simultaneously evaluation of the proliferative status of surviving target cells in response to relevant cytokines. (Blood. 2004;103: 2677-2682)

Since endotoxins are potent inducers of stem cell mobilization, we hypothesized that their presence in the gut may play a role in cytokine-induced mobilization. To address this possibility we added ciprofloxacin and polymyxin B to the drinking water of Balb/c mice mobilized with either interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), or flt3 ligand (FL). The yield of colony-forming units (CFUs) was significantly reduced in all mice treated with these antibiotics when compared with controls (IL-8: 192 ± 61 vs 290 ± 64, P < .05; G-CSF: 1925 ± 1216 vs 3371 ± 1214, P < .05; FL: 562 ± 213 vs 1068 ± 528, P < .05). Treatment with ciprofloxacin eliminated only aerobic Gramnegative bacteria from the feces without effect on mobilization. Polymyxin B treatment did not result in decontamination but significantly reduced the number of mobilized hematopoietic progenitor cells (HPCs) most likely due to the endotoxin binding capacity of polymyxin B. More than 90% of the gastrointestinal flora consists of anaerobic bacteria. Elimination of the anaerobic flora by metronidazol led to a significantly reduced number of mobilized HPCs when compared with controls (IL-8: 55 ± 66 vs 538 ± 216, P < .05). Germ-free OF1 mice showed a significantly reduced mobilization compared with their wild-type controls (IL-8 controls: 378 ± 182, IL-8 germ free: 157 ± 53, P < .05). Finally, we performed reconstitution experiments adding Escherichia coli–derived endotoxins to the drinking water of decontaminated mice. This resulted in partial restoration of the IL-8–induced mobilization (67 ± 28 vs 190 ± 98.1, P <.01). Our results indicate that endotoxins serve as cofactors in cytokine-induced mobilization. Modification of the endotoxin content by antibiotic treatment may affect the yield of cytokine-induced mobilization.

Since endotoxins are potent inducers of stem cell mobilization, we hypothesized that their presence in the gut may play a role in cytokine-induced mobilization. To address this possibility we added ciprofloxacin and polymyxin B to the drinking water of Balb/c mice mobilized with either interleukin-8 (IL-8), granulocyte colony-stimulating factor (G-CSF), or flt3 ligand (FL). The yield of colony-forming units (CFUs) was significantly reduced in all mice treated with these antibiotics when compared with controls (IL-8: 192 ± 61 vs 290 ± 64, P< .05; G-CSF: 1925 ± 1216 vs 3371 ± 1214, P< .05; FL: 562 ± 213 vs 1068 ± 528, P< .05). Treatment with ciprofloxacin eliminated only aerobic Gramnegative bacteria from the feces without effect on mobilization. Polymyxin B treatment did not result in decontamination but significantly reduced the number of mobilized hematopoietic progenitor cells (HPCs) most likely due to the endotoxin binding capacity of polymyxin B. More than 90% of the gastrointestinal flora consists of anaerobic bacteria. Elimination of the anaerobic flora by metronidazol led to a significantly reduced number of mobilized HPCs when compared with controls (IL-8: 55 ± 66 vs 538 ± 216, P< .05). Germ-free OF1 mice showed a significantly reduced mobilization compared with their wild-type controls (IL-8 controls: 378 ± 182, IL-8 germ free: 157 ± 53, P< .05). Finally, we performed reconstitution experiments adding Escherichia coli–derived endotoxins to the drinking water of decontaminated mice. This resulted in partial restoration of the IL-8–induced mobilization (67 ± 28 vs 190 ± 98.1, P<.01). Our results indicate that endotoxins serve as cofactors in cytokine-induced mobilization. Modification of the endotoxin content by antibiotic treatment may affect the yield of cytokine-induced mobilization.

Donor-derived T lymphocytes directed against minor histocompatibility antigens (mHags) exclusively expressed on cells of the hematopoietic lineages can eliminate hematologic malignancies. Transfer of T-cell receptors (TCRs) directed against these mHags into T lymphocytes may provide a strategy to generate antileukemic T cells. To investigate the feasibility of this strategy the TCR usage of mHag HA-2-specific T-cell clones was characterized. Thirteen different types of HA-2-specific T-cell clones were detected, expressing TCRs with diversity in TCR α- and β-chain usage, however, containing in the TCR α chain a single conserved gene segment Jα42, indicating that Jα42 is involved in HA-2-specific recognition. We transferred various HA-2 TCRs into T lymphocytes from HLA-A2-positive HA-2-negative individuals resulting in T cells with redirected cytolytic activity against HA-2-expressing target cells. Transfer of chimeric TCRs demonstrated that the HA-2 specificity is not only determined by the Jα42 region but also by the N-region of the α chain and the CDR3 region of the β chain. Finally, when HA-2 TCRs were transferred into T cells from HLA-A2-negative donors, the HA-2 TCR-modified T cells exerted potent antileukemic reactivity without signs of anti-HLA-A2 alloreactivity. These results indicate that HA-2 TCR transfer may be used as an alternative strategy to generate HA-2-specific T cells to treat hematologic malignancies of HLA-A2-positive, HA-2-expressing patients that received transplants from HLA-A2-matched or -mismatched donors. (Blood. 2003;102:3530-3540)

Donor-derived T lymphocytes directed against minor histocompatibility antigens (mHags) exclusively expressed on cells of the hematopoietic lineages can eliminate hematologic malignancies. Transfer of T-cell receptors (TCRs) directed against these mHags into T lymphocytes may provide a strategy to generate antileukemic T cells. To investigate the feasibility of this strategy the TCR usage of mHag HA-2-specific T-cell clones was characterized. Thirteen different types of HA-2-specific T-cell clones were detected, expressing TCRs with diversity in TCR α- and β-chain usage, however, containing in the TCR α chain a single conserved gene segment Jα42, indicating that Jα42 is involved in HA-2-specific recognition. We transferred various HA-2 TCRs into T lymphocytes from HLA-A2-positive HA-2-negative individuals resulting in T cells with redirected cytolytic activity against HA-2-expressing target cells. Transfer of chimeric TCRs demonstrated that the HA-2 specificity is not only determined by the Jα42 region but also by the N-region of the α chain and the CDR3 region of the β chain. Finally, when HA-2 TCRs were transferred into T cells from HLA-A2-negative donors, the HA-2 TCR-modified T cells exerted potent antileukemic reactivity without signs of anti-HLA-A2 alloreactivity. These results indicate that HA-2 TCR transfer may be used as an alternative strategy to generate HA-2-specific T cells to treat hematologic malignancies of HLA-A2-positive, HA-2-expressing patients that received transplants from HLA-A2-matched or -mismatched donors. (Blood. 2003;102:3530-3540)

In the European Organization for Research and Treatment of Cancer Leukemia Group and Gruppo Italiano Malattie Ematologiche dell' Adulto (EORTC-LG/GIMEMA) acute myeloid leukemia (AML)–10 trial, patients in first complete remission (CR1) received a single intensive consolidation (IC) course. Subsequently, those patients younger than 46 years with an HLA-identical sibling donor were assigned to undergo allogeneic (allo) stem cell transplantation (SCT), and patients without such a donor were planned for autologous (auto) SCT. Between November 1993 and December 1999, of 1198 patients aged younger than 46 years, 822 achieved CR. The study group constituted 734 patients who received IC: 293 had a sibling donor and 441 did not. Allo-SCT and auto-SCT were performed in 68.9% and 55.8%, respectively. Cytogenetic determination was successfully performed in 446 patients. Risk groups were good (t(8;21), inv16), intermediate (NN or –Y only), and bad/very bad (all others). Median follow-up was 4 years; 289 patients relapsed, 66 died in CR1, and 293 died. Intention-to-treat analysis revealed that the 4-year disease-free survival (DFS) rate of patients with a donor versus those without a donor was 52.2% versus 42.2%, P = .044; hazard ratio = 0.80, 95% confidence interval (0.64, 0.995), the relapse incidence was 30.4% versus 52.5%, death in CR1 was 17.4% versus 5.3%, and the survival rate was 58.3% versus 50.8% (P = .18). The DFS rates in patients with and without a sibling donor were similar in patients with good/intermediate risk but were 43.4% and 18.4%, respectively, in patients with bad/very bad risk cytogenetics. In younger patients (15-35 years), the difference was more pronounced. The strategy to perform early allo-SCT led to better overall results than auto-SCT, especially for younger patients or those with bad/very bad risk cytogenetics.

In the European Organization for Research and Treatment of Cancer Leukemia Group and Gruppo Italiano Malattie Ematologiche dell’ Adulto (EORTC-LG/GIMEMA) acute myeloid leukemia (AML)–10 trial, patients in first complete remission (CR1) received a single intensive consolidation (IC) course. Subsequently, those patients younger than 46 years with an HLA-identical sibling donor were assigned to undergo allogeneic (allo) stem cell transplantation (SCT), and patients without such a donor were planned for autologous (auto) SCT. Between November 1993 and December 1999, of 1198 patients aged younger than 46 years, 822 achieved CR. The study group constituted 734 patients who received IC: 293 had a sibling donor and 441 did not. Allo-SCT and auto-SCT were performed in 68.9% and 55.8%, respectively. Cytogenetic determination was successfully performed in 446 patients. Risk groups were good (t(8;21), inv16), intermediate (NN or –Y only), and bad/very bad (all others). Median follow-up was 4 years; 289 patients relapsed, 66 died in CR1, and 293 died. Intention-to-treat analysis revealed that the 4-year disease-free survival (DFS) rate of patients with a donor versus those without a donor was 52.2% versus 42.2%,P= .044; hazard ratio = 0.80, 95% confidence interval (0.64, 0.995), the relapse incidence was 30.4% versus 52.5%, death in CR1 was 17.4% versus 5.3%, and the survival rate was 58.3% versus 50.8% (P= .18). The DFS rates in patients with and without a sibling donor were similar in patients with good/intermediate risk but were 43.4% and 18.4%, respectively, in patients with bad/very bad risk cytogenetics. In younger patients (15-35 years), the difference was more pronounced. The strategy to perform early allo-SCT led to better overall results than auto-SCT, especially for younger patients or those with bad/very bad risk cytogenetics.

Hematopoietic reconstitution after stem cell transplantation requires excessive replicative activity because of the limited number of stem cells that are used for transplantation. Telomere shortening has been detected in hematopoietic cells after bone marrow transplantation. This has been thought to result from excessive replication of the stem cells, with putative concomitant reduction of their replicative potential. Hematopoietic stem cells from cytokine-mobilized peripheral blood are increasingly used for stem cell transplantation. These grafts contain higher numbers of hematopoietic stem cells, resulting in a faster hematopoietic reconstitution. We have performed a combined prospective and cross-sectional study of hematologic recovery and telomere length dynamics in the immediate reconstitution period after allogeneic T-cell–depleted blood stem cell transplantation. We analyzed hematologic recovery and telomere length of granulocytes, monocytes, B cells, and T-cell subsets in 30 donor/recipient combinations. We found fast recovery in combination with transient telomere shortening in the myeloid lineages. This initial reduction of telomere length was followed by an increase in telomere length to such an extent that 1 year after transplantation the telomere length in recipient cells was similar to the telomere length in donor-derived cells. Therefore, our data indicate telomere length homeostasis after peripheral blood stem cell transplantation, implying no loss of replicative capacity of the stem cells. Our data indicate that fast expansion is accompanied by a reduction of telomere length and that telomere length homeostasis is achieved by de novo generation of hematopoietic cells from stem cells without transplantation-related telomere loss.

Hematopoietic reconstitution after stem cell transplantation requires excessive replicative activity because of the limited number of stem cells that are used for transplantation. Telomere shortening has been detected in hematopoietic cells after bone marrow transplantation. This has been thought to result from excessive replication of the stem cells, with putative concomitant reduction of their replicative potential. Hematopoietic stem cells from cytokine-mobilized peripheral blood are increasingly used for stem cell transplantation. These grafts contain higher numbers of hematopoietic stem cells, resulting in a faster hematopoietic reconstitution. We have performed a combined prospective and cross-sectional study of hematologic recovery and telomere length dynamics in the immediate reconstitution period after allogeneic T-cell–depleted blood stem cell transplantation. We analyzed hematologic recovery and telomere length of granulocytes, monocytes, B cells, and T-cell subsets in 30 donor/recipient combinations. We found fast recovery in combination with transient telomere shortening in the myeloid lineages. This initial reduction of telomere length was followed by an increase in telomere length to such an extent that 1 year after transplantation the telomere length in recipient cells was similar to the telomere length in donor-derived cells. Therefore, our data indicate telomere length homeostasis after peripheral blood stem cell transplantation, implying no loss of replicative capacity of the stem cells. Our data indicate that fast expansion is accompanied by a reduction of telomere length and that telomere length homeostasis is achieved by de novo generation of hematopoietic cells from stem cells without transplantation-related telomere loss.

De novo erythroleukemia (EL) is a rare disease. Reported median survival are poor and vary from 4 to 14 months. The value of hematopoietic stem cell transplantation (HSCT) for EL is unknown. This EBMT registry study reports on the largest series of patients with EL treated with HSCT in first complete remission—103 autologous and 104 HLA identical sibling allogeneic HSCT. Outcome and identification of prognostic factors for each type of transplantation were evaluated. For autologous HSCT, outcome at 5 years showed a leukemia-free survival (LFS) of 26% ± 5%, a relapse incidence (RI) of 70% ± 6%, and a transplant-related mortality (TRM) of 13% ± 4%. By multivariate analysis, the only prognostic factor was age. For allogeneic HSCT, outcome at 5 years showed an LFS of 57% ± 5%, an RI of 21% ± 5%, and a TRM of 27% ± 5%. By multivariate analysis, prognostic factors were graft-versus-host disease and age. This study represents the largest series of de novo EL treated with HSCT and shows that allogeneic HSCT is by far the most effective treatment.

De novo erythroleukemia (EL) is a rare disease. Reported median survival are poor and vary from 4 to 14 months. The value of hematopoietic stem cell transplantation (HSCT) for EL is unknown. This EBMT registry study reports on the largest series of patients with EL treated with HSCT in first complete remission—103 autologous and 104 HLA identical sibling allogeneic HSCT. Outcome and identification of prognostic factors for each type of transplantation were evaluated. For autologous HSCT, outcome at 5 years showed a leukemia-free survival (LFS) of 26% ± 5%, a relapse incidence (RI) of 70% ± 6%, and a transplant-related mortality (TRM) of 13% ± 4%. By multivariate analysis, the only prognostic factor was age. For allogeneic HSCT, outcome at 5 years showed an LFS of 57% ± 5%, an RI of 21% ± 5%, and a TRM of 27% ± 5%. By multivariate analysis, prognostic factors were graft-versus-host disease and age. This study represents the largest series of de novo EL treated with HSCT and shows that allogeneic HSCT is by far the most effective treatment.

Adoptive immunotherapy using in vitro–generated donor-derived cytotoxic T lymphocytes (CTLs) can be effective in the treatment of relapsed leukemia after allogeneic transplantation. To determine effector cell characteristics that result in optimal in vivo antileukemic efficacy, we developed an animal model for human CTL therapy. Nonobese diabetic/severe combined immunodeficiency (NOD/scid) mice were inoculated with either of 2 primary human acute lymphoblastic leukemia (ALL), denoted as SK and OF. Anti-SK and anti-OF CTLs were generated in vitro by repeated stimulation of donor peripheral blood mononuclear cells with either SK or OF cells. Both CTL lines displayed HLA-restricted reactivity against the original targets and non-major histocompatibility class (MHC)–restricted cross-reactivity in vitro. The CTLs were administered intravenously weekly for 3 consecutive weeks to mice engrafted with either SK or OF leukemia. In 3 of 8 SK-engrafted and anti-SK–treated mice, complete remissions were achieved in blood, spleen, and bone marrow. In the remaining 5 animals partial remissions were observed. In 4 of 4 OF-engrafted anti-OF–treated mice partial remissions were observed. The antileukemic effect of specific CTLs was exerted immediately after administration and correlated with the degree of HLA disparity of the donor-patient combination. In cross-combination–treated animals, no effect on leukemic progression was observed indicating that in vivo antileukemic reactivity is mediated by MHC-restricted effector cells. The CTLs, however, displayed an impaired in vivo proliferative capacity. Ex vivo analysis showed decreased reactivity as compared to the moment of infusion. We therefore conclude that the model can be used to explore the requirements for optimal in vivo efficacy of in vitro– generated CTLs.

Magnetic resonance imaging (MRI) can be used to distinguish bone marrow (BM) from cartilage and may therefore be used to measure BM volume in intact bones. We used MRI to measure the total human fetal BM volume in intact fetuses during the second trimester of pregnancy and determined the contribution of the individual bones to the total compartment. The total BM volume ranged from 934 μL at 17 to 18 weeks to 4563 μL at 22 to 23 weeks of gestation. The largest contributor to the total BM volume was the spine, constituting 26.4% ± 2.7% of the total volume. By analyzing leukocyte content and percentages of CD34+ cells, lymphocytes, granulocytes, and monocytes of determined volumes, absolute numbers of these cell populations in BM could be measured. The cellular composition of the BM compartment did not significantly change throughout the second trimester of gestation. Absolute white blood cell counts per fetus increased from 111 × 106 at 16 to 17 weeks to 1229 × 106 at 21 to 22 weeks. The absolute numbers of CD34+ cells increased from 25 × 106 at 16 to 17 weeks to 256 × 106 at 21 to 22 weeks. Similar analysis of liver and spleen revealed comparable absolute numbers of CD34+ cells in BM and liver throughout the second trimester of gestation. In fetal liver, CD34+ cells differentiate into red cells, myeloid cells, and platelets, while lymphopoiesis mainly occurs in BM or spleen. Combining MRI and cell counts provides a method to quantify specific cell populations in fetal compartments. This study may enable better evaluation of fetal diagnostics and therapies.

Adoptive immunotherapy using in vitro–generated donor-derived cytotoxic T lymphocytes (CTLs) can be effective in the treatment of relapsed leukemia after allogeneic transplantation. To determine effector cell characteristics that result in optimal in vivo antileukemic efficacy, we developed an animal model for human CTL therapy. Nonobese diabetic/severe combined immunodeficiency (NOD/scid) mice were inoculated with either of 2 primary human acute lymphoblastic leukemia (ALL), denoted as SK and OF. Anti-SK and anti-OF CTLs were generated in vitro by repeated stimulation of donor peripheral blood mononuclear cells with either SK or OF cells. Both CTL lines displayed HLA-restricted reactivity against the original targets and non-major histocompatibility class (MHC)–restricted cross-reactivity in vitro. The CTLs were administered intravenously weekly for 3 consecutive weeks to mice engrafted with either SK or OF leukemia. In 3 of 8 SK-engrafted and anti-SK–treated mice, complete remissions were achieved in blood, spleen, and bone marrow. In the remaining 5 animals partial remissions were observed. In 4 of 4 OF-engrafted anti-OF–treated mice partial remissions were observed. The antileukemic effect of specific CTLs was exerted immediately after administration and correlated with the degree of HLA disparity of the donor-patient combination. In cross-combination–treated animals, no effect on leukemic progression was observed indicating that in vivo antileukemic reactivity is mediated by MHC-restricted effector cells. The CTLs, however, displayed an impaired in vivo proliferative capacity. Ex vivo analysis showed decreased reactivity as compared to the moment of infusion. We therefore conclude that the model can be used to explore the requirements for optimal in vivo efficacy of in vitro– generated CTLs.

Magnetic resonance imaging (MRI) can be used to distinguish bone marrow (BM) from cartilage and may therefore be used to measure BM volume in intact bones. We used MRI to measure the total human fetal BM volume in intact fetuses during the second trimester of pregnancy and determined the contribution of the individual bones to the total compartment. The total BM volume ranged from 934 μL at 17 to 18 weeks to 4563 μL at 22 to 23 weeks of gestation. The largest contributor to the total BM volume was the spine, constituting 26.4% ± 2.7% of the total volume. By analyzing leukocyte content and percentages of CD34+cells, lymphocytes, granulocytes, and monocytes of determined volumes, absolute numbers of these cell populations in BM could be measured. The cellular composition of the BM compartment did not significantly change throughout the second trimester of gestation. Absolute white blood cell counts per fetus increased from 111 × 106at 16 to 17 weeks to 1229 × 106at 21 to 22 weeks. The absolute numbers of CD34+cells increased from 25 × 106at 16 to 17 weeks to 256 × 106at 21 to 22 weeks. Similar analysis of liver and spleen revealed comparable absolute numbers of CD34+cells in BM and liver throughout the second trimester of gestation. In fetal liver, CD34+cells differentiate into red cells, myeloid cells, and platelets, while lymphopoiesis mainly occurs in BM or spleen. Combining MRI and cell counts provides a method to quantify specific cell populations in fetal compartments. This study may enable better evaluation of fetal diagnostics and therapies.

The β2 integrins leukocyte function antigen-1 (LFA-1, CD11a) and macrophage antigen-1 (Mac-1, CD11b) have been reported to play a role in the attachment of CD34+ cells to stromal cells in the bone marrow. When administered prior to interleukin-8 (IL-8), anti–LFA-1 antibodies completely prevent the IL-8–induced mobilization of hematopoietic stem cells in mice. Here, we studied the role of anti-β2 integrin antibodies in granulocyte colony-stimulating factor (G-CSF)–induced mobilization of hematopoietic progenitor cells. Administration of antibodies against the α chain of LFA-1 or against the α chain of Mac-1 followed by daily injections of G-CSF for more than 1 day resulted in a significant enhancement of mobilization of hematopoietic progenitor cells when compared with mobilization induced by G-CSF alone. Also, the number of late (day 28) cobblestone area–forming cells in vitro was significantly higher after mobilization with anti–LFA-1 antibodies followed by 5 μg G-CSF for 5 days than with G-CSF alone (119 ± 34 days vs 17 ± 14 days), indicating mobilization of repopulating stem cells. Pretreatment with blocking antibodies to intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of LFA-1 and Mac-1, did not result in an effect on G-CSF–induced mobilization, suggesting that the enhancing effect required an interaction of the β2 integrins and one of their other ligands. Enhancement of mobilization was not observed in LFA-1–deficient (CD11a) mice, indicating that activated cells expressing LFA-1 mediate the synergistic effect, rather than LFA-1–mediated adhesion.

The β2 integrins leukocyte function antigen-1 (LFA-1, CD11a) and macrophage antigen-1 (Mac-1, CD11b) have been reported to play a role in the attachment of CD34+cells to stromal cells in the bone marrow. When administered prior to interleukin-8 (IL-8), anti–LFA-1 antibodies completely prevent the IL-8–induced mobilization of hematopoietic stem cells in mice. Here, we studied the role of anti-β2 integrin antibodies in granulocyte colony-stimulating factor (G-CSF)–induced mobilization of hematopoietic progenitor cells. Administration of antibodies against the α chain of LFA-1 or against the α chain of Mac-1 followed by daily injections of G-CSF for more than 1 day resulted in a significant enhancement of mobilization of hematopoietic progenitor cells when compared with mobilization induced by G-CSF alone. Also, the number of late (day 28) cobblestone area–forming cells in vitro was significantly higher after mobilization with anti–LFA-1 antibodies followed by 5 μg G-CSF for 5 days than with G-CSF alone (119 ± 34 days vs 17 ± 14 days), indicating mobilization of repopulating stem cells. Pretreatment with blocking antibodies to intercellular adhesion molecule-1 (ICAM-1; CD54), a ligand of LFA-1 and Mac-1, did not result in an effect on G-CSF–induced mobilization, suggesting that the enhancing effect required an interaction of the β2 integrins and one of their other ligands. Enhancement of mobilization was not observed in LFA-1–deficient (CD11a) mice, indicating that activated cells expressing LFA-1 mediate the synergistic effect, rather than LFA-1–mediated adhesion.

Graft rejection or graft-versus-host (GVH) disease after HLA-identical stem cell transplantation is the result of recognition of minor histocompatibility antigens (mHags) by immunocompetent T lymphocytes from recipient or donor origin, respectively. Cytolytic T lymphocyte (CTL) clones can be isolated during graft rejection and GVH disease to identify mHags and their corresponding genes. Thus far, all human mHags identified appeared to be HLA class I–restricted. Here, we report the characterization of the first human HLA class II–restricted sex-linked mHag involved in GVH disease. Previously, we isolated an HLA-DQ5–restricted CD4+ CTL clone from a male patient with chronic myeloid leukemia who developed acute GVH disease grade III-IV after transplantation of HLA genotypically identical female stem cells. Using a panel of female HLA-DQ5+ EBV cells that we stably transfected with Y chromosome–specific genes, we determined that the HLA class II male-specific mHag (H-Y) was encoded by the Y chromosome–specific gene DBY. The H-Y epitope was localized in the DBY protein using female HLA-DQ5+peripheral blood mononuclear cells loaded with DBY protein fragments. The minimal peptide sequence leading to maximal recognition by the specific HLA-DQ5–restricted CTL clone was characterized as the 12–amino acid sequence HIENFSDIDMGE. Although the epitope differed by 3 amino acids from its X-homolog DBX, only 2 polymorphisms were shown to be essential for recognition by the CTL clone.

Graft rejection or graft-versus-host (GVH) disease after HLA-identical stem cell transplantation is the result of recognition of minor histocompatibility antigens (mHags) by immunocompetent T lymphocytes from recipient or donor origin, respectively. Cytolytic T lymphocyte (CTL) clones can be isolated during graft rejection and GVH disease to identify mHags and their corresponding genes. Thus far, all human mHags identified appeared to be HLA class I–restricted. Here, we report the characterization of the first human HLA class II–restricted sex-linked mHag involved in GVH disease. Previously, we isolated an HLA-DQ5–restricted CD4+CTL clone from a male patient with chronic myeloid leukemia who developed acute GVH disease grade III-IV after transplantation of HLA genotypically identical female stem cells. Using a panel of female HLA-DQ5+EBV cells that we stably transfected with Y chromosome–specific genes, we determined that the HLA class II male-specific mHag (H-Y) was encoded by the Y chromosome–specific gene DBY.The H-Y epitope was localized in the DBY protein using female HLA-DQ5+peripheral blood mononuclear cells loaded with DBY protein fragments. The minimal peptide sequence leading to maximal recognition by the specific HLA-DQ5–restricted CTL clone was characterized as the 12–amino acid sequence HIENFSDIDMGE. Although the epitope differed by 3 amino acids from its X-homolog DBX, only 2 polymorphisms were shown to be essential for recognition by the CTL clone.

Development of resistance to cytarabine (AraC) is a major problem in the treatment of patients with acute myeloid leukemia (AML). Inactivation of deoxycytidine kinase (dCK) plays an important role in AraC resistance in vitro. We have identified inactive, alternatively spliced dCK forms in leukemic blasts from patients with resistant AML. Because these dCK-spliced variants were only detectable in resistant AML, it was hypothesized that they might play a role in AraC resistance in vivo. In the current study, the biologic role of the alternatively spliced dCK forms in AraC resistance was further investigated by retroviral transductions in rat leukemic cells. Introduction of inactive, alternatively spliced dCK forms into AraC-resistant K7 cells, with no endogenous wild-type (wt) dCK activity, could not restore AraC sensitivity, whereas wt dCK fully restored the AraC-sensitive phenotype. Transfection of alternatively spliced dCK forms into AraC-sensitive KA cells, as well as in human leukemic U937 cells and in phytohemagglutinin-stimulated T cells, did not significantly change sensitivity toward AraC. In addition, cotransduction of wt dCK with alternatively spliced dCK in K7 cells did not result in altered sensitivity to AraC compared with K7 cells only transduced with wt dCK. These data indicate that the alternatively spliced dCK forms cannot act as a dominant-negative inhibitor on dCK wt activity when they are coexpressed in a single cell. However, a cell expressing alternatively spliced dCK forms that has lost wt dCK expression is resistant to the cytotoxic effects of AraC.

Development of resistance to cytarabine (AraC) is a major problem in the treatment of patients with acute myeloid leukemia (AML). Inactivation of deoxycytidine kinase (dCK) plays an important role in AraC resistance in vitro. We have identified inactive, alternatively spliced dCK forms in leukemic blasts from patients with resistant AML. Because these dCK-spliced variants were only detectable in resistant AML, it was hypothesized that they might play a role in AraC resistance in vivo. In the current study, the biologic role of the alternatively spliced dCK forms in AraC resistance was further investigated by retroviral transductions in rat leukemic cells. Introduction of inactive, alternatively spliced dCK forms into AraC-resistant K7 cells, with no endogenous wild-type (wt) dCK activity, could not restore AraC sensitivity, whereas wt dCK fully restored the AraC-sensitive phenotype. Transfection of alternatively spliced dCK forms into AraC-sensitive KA cells, as well as in human leukemic U937 cells and in phytohemagglutinin-stimulated T cells, did not significantly change sensitivity toward AraC. In addition, cotransduction of wt dCK with alternatively spliced dCK in K7 cells did not result in altered sensitivity to AraC compared with K7 cells only transduced with wt dCK. These data indicate that the alternatively spliced dCK forms cannot act as a dominant-negative inhibitor on dCK wt activity when they are coexpressed in a single cell. However, a cell expressing alternatively spliced dCK forms that has lost wt dCK expression is resistant to the cytotoxic effects of AraC.

This study investigated the feasibility of allogeneic (alloSCT) and autologous stem cell transplantation (ASCT) as postconsolidation therapy for patients with myelodysplastic syndromes (MDSs) or acute myeloid leukemia after MDS. Patients with a histocompatible sibling were candidates for alloSCT and the remaining patients for ASCT. Remission-induction therapy consisted of 1 or 2 courses with idarubicin, cytarabine, and etoposide, followed by one intensive consolidation course with cytarabine and mitoxantrone. Initially, bone marrow cells were used for ASCT. Subsequently, mobilized blood stem cells were used in an attempt to shorten posttransplantation hypoplasia. With a median follow-up of 3.6 years the 184 evaluable patients showed a 4-year survival rate of 26% and a median survival of 13 months. The remission-induction chemotherapy induced complete remission (CR) in 100 patients (54%). The 4-year disease-free survival (DFS) rate was 29% and the median DFS was 12 months. Twenty-eight of 39 patients (72%) with a donor were allografted in CR-1, including 2 patients who underwent transplantation in CR-1 without a consolidation course. Thirty-six of 59 patients (61%) without a donor received ASCT in CR-1. The 4-year DFS rates in the group of patients with or without a donor were 31% and 27%, respectively. The 4-year survival rates from CR were 36% and 33%, respectively. This large prospective study shows the feasibility of both alloSCT and ASCT. This treatment approach leads to a relatively high remission rate, and the majority of patients in remission received the SCT in CR-1. The ongoing study investigates whether this approach is better than treatment with chemotherapy only.

This study investigated the feasibility of allogeneic (alloSCT) and autologous stem cell transplantation (ASCT) as postconsolidation therapy for patients with myelodysplastic syndromes (MDSs) or acute myeloid leukemia after MDS. Patients with a histocompatible sibling were candidates for alloSCT and the remaining patients for ASCT. Remission-induction therapy consisted of 1 or 2 courses with idarubicin, cytarabine, and etoposide, followed by one intensive consolidation course with cytarabine and mitoxantrone. Initially, bone marrow cells were used for ASCT. Subsequently, mobilized blood stem cells were used in an attempt to shorten posttransplantation hypoplasia. With a median follow-up of 3.6 years the 184 evaluable patients showed a 4-year survival rate of 26% and a median survival of 13 months. The remission-induction chemotherapy induced complete remission (CR) in 100 patients (54%). The 4-year disease-free survival (DFS) rate was 29% and the median DFS was 12 months. Twenty-eight of 39 patients (72%) with a donor were allografted in CR-1, including 2 patients who underwent transplantation in CR-1 without a consolidation course. Thirty-six of 59 patients (61%) without a donor received ASCT in CR-1. The 4-year DFS rates in the group of patients with or without a donor were 31% and 27%, respectively. The 4-year survival rates from CR were 36% and 33%, respectively. This large prospective study shows the feasibility of both alloSCT and ASCT. This treatment approach leads to a relatively high remission rate, and the majority of patients in remission received the SCT in CR-1. The ongoing study investigates whether this approach is better than treatment with chemotherapy only.

For more than 10 years umbilical cord blood (UCB) has been used as an alternative source of hematopoietic stem cells for allogeneic transplantation. Although the clinical results are encouraging, UCB is associated with delayed engraftment. To address these issues we have used the NODSCID mouse model to study the engraftment potential of cord blood cells in more detail, to assess the engraftment potential of expanded megakaryocytic progenitor cells, and to study the effect of co-transplantation of mesenchymal stem cells on engraftment.

The treatment of posttransplant lymphoproliferative disorder (PTLD) remains empirical. We review our treatment of seven cases of PTLD consisting of five interventions 1) reduction of immunosuppression; 2) antiviral drugs; 3) interferon-; 4) gamma-globulins; and 5) anti-CD19 monoclonal antibodies.

Rejection of a graft after human leukocyte antigen (HLA)-identical stem cell transplantation (SCT) can be caused by recipient's immunocompetent T lymphocytes recognizing minor histocompatibility antigens on donor stem cells. During rejection of a male stem cell graft by a female recipient, 2 male (H-Y)–specific cytotoxic T lymphocyte (CTL) clones were isolated from peripheral blood. One CTL clone recognized an HLA-A2–restricted H-Y antigen, encoded by the SMCY gene. Another CTL clone recognized an HLA-B60–restricted H-Y antigen. In this study UTY was identified as the gene coding for the HLA-B60–restricted H-Y antigen. The UTY-derived H-Y antigen was characterized as a 10-amino acid residue peptide, RESEEESVSL. Although the epitope differed by 3 amino acids from its X-homologue, UTX, only 2 polymorphisms were essential for recognition by the CTL clone HLA-B60 HY. These results illustrate that CTLs against several H-Y antigens derived from different proteins can contribute simultaneously to graft rejection after HLA-identical, sex-mismatched SCT. Moreover, RESEEESVSL-specific T cells could be isolated from a female HLA-B60+ patient with myelodysplastic syndrome who has been treated with multiple blood transfusions, but not from control healthy HLA-B60+ female donors. This may indicate that RESEEESVSL-reactive T cells are more common in sensitized patients.

Rejection of a graft after human leukocyte antigen (HLA)-identical stem cell transplantation (SCT) can be caused by recipient's immunocompetent T lymphocytes recognizing minor histocompatibility antigens on donor stem cells. During rejection of a male stem cell graft by a female recipient, 2 male (H-Y)–specific cytotoxic T lymphocyte (CTL) clones were isolated from peripheral blood. One CTL clone recognized an HLA-A2–restricted H-Y antigen, encoded by the SMCYgene. Another CTL clone recognized an HLA-B60–restricted H-Y antigen. In this study UTYwas identified as the gene coding for the HLA-B60–restricted H-Y antigen. The UTY-derived H-Y antigen was characterized as a 10-amino acid residue peptide, RESEEESVSL. Although the epitope differed by 3 amino acids from its X-homologue, UTX, only 2 polymorphisms were essential for recognition by the CTL clone HLA-B60 HY. These results illustrate that CTLs against several H-Y antigens derived from different proteins can contribute simultaneously to graft rejection after HLA-identical, sex-mismatched SCT. Moreover, RESEEESVSL-specific T cells could be isolated from a female HLA-B60+ patient with myelodysplastic syndrome who has been treated with multiple blood transfusions, but not from control healthy HLA-B60+ female donors. This may indicate that RESEEESVSL-reactive T cells are more common in sensitized patients.

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.

Relapse of chronic myeloid leukemia (CML) in chronic phase after allogeneic stem cell transplantation (SCT) can be successfully treated by donor lymphocyte infusion (DLI). However, relapse of accelerated phase CML, blast crisis, or acute leukemia after allogeneic SCT are resistant to DLI in the majority of cases. In vitro-selected and expanded leukemia-reactive T-cell lines may be more effective in inducing an antileukemic response in vivo. To treat a patient with accelerated phase CML after allogeneic SCT, leukemia-reactive cytotoxic T-lymphocyte (CTL) lines were generated from her HLA-identical donor. Using a modification of a limiting dilution assay, T cells were isolated from the donor, selected based on their ability to inhibit the in vitro growth of CML progenitor cells, and subsequently expanded in vitro to generate CTL lines. Three CTL lines were generated that lysed the leukemic cells from the patient and inhibited the growth of leukemic progenitor cells. The CTL did not react with lymphocytes from donor or recipient and did not affect donor hematopoietic progenitor cells. The 3 leukemia-reactive CTL lines were infused at 5-week intervals at a cumulative dose of 3.2 × 109 CTL. Shortly after the third infusion, complete eradication of the leukemic cells was observed, as shown by cytogenetic analysis, fluorescence in situ hybridization, molecular analysis of BCR/ABL-mRNA, and chimerism studies. These results show that in vitro cultured leukemia-reactive CTL lines selected on their ability to inhibit the proliferation of leukemic progenitor cells in vitro can be successfully applied to treat accelerated phase CML after allogeneic SCT.

Recently, we have demonstrated that antibodies that block the function of the ß2-integrin leukocyte function-associated antigen-1 (LFA-1) completely abrogate the rapid mobilization of hematopoietic progenitor cells (HPC) with colony-forming and radioprotective capacity induced by interleukin-8 (IL-8) in mice. These findings suggested a direct inhibitory effect of these antibodies on LFA-1–mediated transmigration of stem cells through the bone marrow endothelium. Therefore, we studied the expression and functional role of LFA-1 on murine HPC in vitro and in vivo. In steady state bone marrow ± 50% of the mononuclear cells (MNC) were LFA-1neg. Cultures of sorted cells, supplemented with granulocyte colony-stimulating factor (G-CSF)/granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-1/IL-3/IL-6/stem cell factor (SCF) and erythropoietin (EPO) indicated that the LFA-1neg fraction contained the majority of the colony-forming cells (CFCs) (LFA-1neg 183 ± 62/7,500 cells v LFA-1pos 29 ± 17/7,500 cells,P < .001). We found that the radioprotective capacity resided almost exclusively in the LFA-1neg cell fraction, the radioprotection rate after transplantation of 103, 3 × 103, 104, and 3 × 104 cells being 63%, 90%, 100%, and 100% respectively. Hardly any radioprotection was obtained from LFA-1pos cells. Similarly, in cytokine (IL-8 and G-CSF)–mobilized blood, the LFA-1neg fraction, which comprised 5% to 10% of the MNC, contained the majority of the colony-forming cells, as well as almost all cells with radioprotective capacity. Subsequently, primitive bone marrow-derived HPC, represented by Wheat-germ-agglutinin (WGA)+/Lineage (Lin)-/Rhodamine (Rho)- sorted cells, were examined. More than 95% of the Rho- cells were LFA-1neg. Cultures of sorted cells showed that the LFA-1neg fraction contained all CFU. Transplantation of 150 Rho- LFA-1neg or up to 600 Rho-LFA-1pos cells protected 100% and 0% of lethally irradiated recipient mice, respectively. These results show that primitive murine HPC in steady-state bone marrow and of cytokine-mobilized blood do not express LFA-1.