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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.

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.

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.

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.

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)

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)

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.

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.

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.

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.

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.