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Notch signaling is involved in tumorigenesis, but its role in B–chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor–kappa B (NF-κB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the γ-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-κB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

Notch signaling is involved in tumorigenesis, but its role in B–chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor–kappa B (NF-κB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the γ-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-κB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

Patients with primary plasma-cell leukemia (PCL) are usually treated with regimens used for multiple myeloma because no official guidelines exist for the treatment of PCL. Ballantiet al. discuss the case of a 68-year-old man with primary PCL who developed sustained ventricular tachycardia that was thought to be caused by thalidomide. The authors emphasize the need for complete cardiac evaluation and clinical monitoring of patients undergoing thalidomide treatment.

The proteasome inhibitor bortezomib may increase osteoblast-related markers in multiple myeloma (MM) patients; however, its potential osteoblastic stimulatory effect is not known. In this study, we show that bortezomib significantly induced a stimulatory effect on osteoblast markers in human mesenchymal cells without affecting the number of osteoblast progenitors in bone marrow cultures or the viability of mature osteoblasts. Consistently we found that bortezomib significantly increased the transcription factor Runx2/Cbfa1 activity in human osteoblast progenitors and osteoblasts without affecting nuclear and cytoplasmatic active β-catenin levels. Consequently a stimulatory effect of bortezomib on bone nodule formation was also demonstrated in osteoblast progenitors. These in vitro observations were confirmed in vivo by the finding of a significant increase in the number of osteoblastic cells × mm2 of bone tissue and in the number of Runx2/Cbfa1-positive osteoblastic cells that was observed in MM patients who responded to bortezomib. Our in vitro and in vivo observations support the hypothesis that a direct stimulatory effect on bone formation process could occur during bortezomib treatment.

The proteasome inhibitor bortezomib may increase osteoblast-related markers in multiple myeloma (MM) patients; however, its potential osteoblastic stimulatory effect is not known. In this study, we show that bortezomib significantly induced a stimulatory effect on osteoblast markers in human mesenchymal cells without affecting the number of osteoblast progenitors in bone marrow cultures or the viability of mature osteoblasts. Consistently we found that bortezomib significantly increased the transcription factor Runx2/Cbfa1 activity in human osteoblast progenitors and osteoblasts without affecting nuclear and cytoplasmatic active β-catenin levels. Consequently a stimulatory effect of bortezomib on bone nodule formation was also demonstrated in osteoblast progenitors. These in vitro observations were confirmed in vivo by the finding of a significant increase in the number of osteoblastic cells × mm2of bone tissue and in the number of Runx2/Cbfa1-positive osteoblastic cells that was observed in MM patients who responded to bortezomib. Our in vitro and in vivo observations support the hypothesis that a direct stimulatory effect on bone formation process could occur during bortezomib treatment.

Much progress has been made in the clinical, biological and technical aspects of the T-cell-depleted full-haplotype mismatched transplants for acute leukemia. Our experience demonstrates that infusing a megadose of extensively T-cell-depleted hematopoietic peripheral blood stem cells after an immunomyeloablative conditioning regimen in acute leukemia patients ensures sustained engraftment with minimal graft-vs-host disease (GvHD) without the need of any post-transplant immunosuppressive treatment. Since our first successful pilot study, our efforts have concentrated on developing new conditioning regimens, optimizing the graft processing and improving the post-transplant immunological recovery. The results we have so far achieved in more than 200 high-risk acute leukemia patients show that haploidentical transplantation is now a clinical reality. Because virtually all patients in need of a hematopoietic stem cell transplant have a full-haplotype mismatched donor, who is immediately available, a T-cell depleted mismatched transplant should be offered, not as a last resort, but as a viable option to high risk acute leukemia patients who do not have, or cannot find, a matched donor.

We found that MEK1 inhibitor PD184352 strikingly increased apoptosis induced by arsenic trioxide (ATO) in 21 of 25 patients with primary acute myelogenous leukemia (AML). Isobologram analysis confirmed the synergistic (13 of 25 patients) or additive (8 of 25 patients) nature of this interaction. Moreover, we demonstrated that the p53-related gene p73is a molecular target of the combined treatment in AML blasts. Indeed, ATO modulates the expression of the p73gene by inducing the proapoptotic and antiproliferative TAp73 and the antiapoptotic and proproliferative ΔNp73 isoforms, thereby failing to elevate the TA/ΔNp73 ratio. Conversely, treatment with PD184352 reduces the level of ΔNp73 and blunts the arsenic-mediated up-regulation of ΔNp73, thus causing an increase in the TA/ΔNp73 ratio of dual-treated cells. High doses of ATO induced p53 accumulation in 11 of 21 patients. Combined treatment resulted in the induction of the proapoptotic p53/p73 target gene p53AIP1(p53-regulated apoptosis-inducing protein 1) and greatly enhanced the apoptosis of treated cells.

We found that MEK1 inhibitor PD184352 strikingly increased apoptosis induced by arsenic trioxide (ATO) in 21 of 25 patients with primary acute myelogenous leukemia (AML). Isobologram analysis confirmed the synergistic (13 of 25 patients) or additive (8 of 25 patients) nature of this interaction. Moreover, we demonstrated that the p53-related gene p73 is a molecular target of the combined treatment in AML blasts. Indeed, ATO modulates the expression of the p73 gene by inducing the proapoptotic and antiproliferative TAp73 and the antiapoptotic and proproliferative ΔNp73 isoforms, thereby failing to elevate the TA/ΔNp73 ratio. Conversely, treatment with PD184352 reduces the level of ΔNp73 and blunts the arsenic-mediated up-regulation of ΔNp73, thus causing an increase in the TA/ΔNp73 ratio of dual-treated cells. High doses of ATO induced p53 accumulation in 11 of 21 patients. Combined treatment resulted in the induction of the proapoptotic p53/p73 target gene p53AIP1 (p53-regulated apoptosis-inducing protein 1) and greatly enhanced the apoptosis of treated cells.

In this study we determined whether human stromal cells could be engineered with a retroviral vector carrying the interleukin 7 (IL-7) gene and investigated the effects on T cells in vitro and in vivo in a murine model. Transduced mesenchymal cells strongly express CD90 (98.15%), CD105 (87.6%), and STRO-1 (86.7%). IL-7 production was 16.37 (±2 SD) pg/ml, which remained stable for 60 days. In vitro-immunoselected naive T cells maintained the CD45RA+CD45RO− naive phenotype (4.2 times more than controls) after 7 days of culture with IL-7-engineered stromal cells. The apoptosis rate (4.7%) of the naive T cells cultured with transduced stromal cells overlapped with that of freshly isolated cells. Immunohistological analysis detected stromal cells in bone marrow, spleen, and thymus. Cotransplantation of IL-7-engineered stromal cells with CD34+ cells improved engraftment in terms of CD45+ cells and significantly increased the CD3+ cell count in peripheral blood, bone marrow, and spleen. These data demonstrate the following: (1) human stromal cells can be transduced, generating a normal layer; (2) transduced stromal cells in vitro maintain the naive T cell phenotype; and (3) IL-7-transduced stromal cells in vivo home to lymphoid organs and produce sufficient IL-7 in loco, supporting T cell development in a cotransplantation model. Because of their efficient cytokine production and homing, IL-7-engineered stromal cells might be an ideal vehicle to hasten immunological reconstitution in T cell-depleted hosts.

The Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) 0496 protocol, through the central handling of bone marrow samples at presentation, allowed us to combine cytogenetic and molecular information on a large series of adults with acute lymphoblastic leukemia (ALL) treated homogeneously, enabling us to define as broadly as possible their genetic profile and to determine the impact on outcome of the cytogenetic-molecular signature. Of 414 patients centrally processed, 325 were considered for the categorization into the following cytogenetic-molecular subgroups: normal, t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1;19)/E2A-PBX1, 9p/p15-p16deletions, 6q deletions, miscellaneous structural abnormalities, and hyperdiploid. The inclusion into each subgroup was based on a hierarchical approach: molecular abnormalities with adverse prognosis had precedence over karyotypic changes with less-defined prognosis and the latter over ploidy. Patients without abnormalities and those with isolated 9p/p15-p16deletions showed a relatively favorable outcome (median disease-free survival [DFS], > 3 years). The t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1; 19)/E2A-PBX1defined a group with dismal prognosis (median DFS, 7 months), whereas 6q deletions, miscellaneous aberrations, and hyperdiploidy predicted an intermediate prognosis (median DFS, 19 months). This study highlights the importance of a combined cytogenetic-molecular profiling of adult ALL at presentation as a critical independent determinant of their outcome, providing further evidence of the necessity of a risk-adapted therapeutic algorithm for an optimal management of these patients.

The Gruppo Italiano Malattie Ematologiche dell'Adulto (GIMEMA) 0496 protocol, through the central handling of bone marrow samples at presentation, allowed us to combine cytogenetic and molecular information on a large series of adults with acute lymphoblastic leukemia (ALL) treated homogeneously, enabling us to define as broadly as possible their genetic profile and to determine the impact on outcome of the cytogenetic-molecular signature. Of 414 patients centrally processed, 325 were considered for the categorization into the following cytogenetic-molecular subgroups: normal, t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1;19)/E2A-PBX1, 9p/p15-p16 deletions, 6q deletions, miscellaneous structural abnormalities, and hyperdiploid. The inclusion into each subgroup was based on a hierarchical approach: molecular abnormalities with adverse prognosis had precedence over karyotypic changes with less-defined prognosis and the latter over ploidy. Patients without abnormalities and those with isolated 9p/p15-p16 deletions showed a relatively favorable outcome (median disease-free survival [DFS], > 3 years). The t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1; 19)/E2A-PBX1 defined a group with dismal prognosis (median DFS, 7 months), whereas 6q deletions, miscellaneous aberrations, and hyperdiploidy predicted an intermediate prognosis (median DFS, 19 months). This study highlights the importance of a combined cytogenetic-molecular profiling of adult ALL at presentation as a critical independent determinant of their outcome, providing further evidence of the necessity of a risk-adapted therapeutic algorithm for an optimal management of these patients.

Establishment of cell lines capable of killing leukemia cells, in the absence of alloreactivity against normal host cells, represents a most desirable goal in bone marrow transplantation (BMT) and cancer immunotherapy. By using a human → mouse chimeric model, we demonstrate that allogeneic anti-third-party cytotoxic T lymphocytes (CTLs) depleted of alloreactivity are endowed with a potent anti-B-cell chronic lymphocytic leukemia (B-CLL) reactivity. Likewise, CTL preparations generated from autologous T cells of the same patients with B-CLL exhibited comparable leukemia eradication, suggesting that the reactivity of allogeneic anti-third-party CTLs is not mediated by residual antihost clones. This specificity was also exhibited in vitro, and annexin staining revealed that B-CLL killing is mediated by apoptosis. While the CTLs killing of third-party cells could be blocked by anti-CD3 antibody, the lysis of the B-CLL cells was not inhibited by this antibody, suggesting a T-cell receptor (TCR)-independent cytotoxicity. The role of cell contact leading to apoptosis of B-CLL cells is shown in transwell plates and by anti-lymphocyte function-associated antigen-1 (LFA-1)-blocking antibody. Up-regulation of CD54 and the subsequent apoptosis of B-CLL cells depend on the initial LFA-1/ICAM-1 (intercellular adhesion molecule 1) interaction. Taken together, these results suggest that allogeneic or autologous host nonreactive anti-third-party CTLs may represent a new therapeutic approach for patients with B-CLL. (Blood. 2005;105:3365-3371)

Establishment of cell lines capable of killing leukemia cells, in the absence of alloreactivity against normal host cells, represents a most desirable goal in bone marrow transplantation (BMT) and cancer immunotherapy. By using a human → mouse chimeric model, we demonstrate that allogeneic anti-third-party cytotoxic T lymphocytes (CTLs) depleted of alloreactivity are endowed with a potent anti-B-cell chronic lymphocytic leukemia (B-CLL) reactivity. Likewise, CTL preparations generated from autologous T cells of the same patients with B-CLL exhibited comparable leukemia eradication, suggesting that the reactivity of allogeneic anti-third-party CTLs is not mediated by residual antihost clones. This specificity was also exhibited in vitro, and annexin staining revealed that B-CLL killing is mediated by apoptosis. While the CTLs killing of third-party cells could be blocked by anti-CD3 antibody, the lysis of the B-CLL cells was not inhibited by this antibody, suggesting a T-cell receptor (TCR)-independent cytotoxicity. The role of cell contact leading to apoptosis of B-CLL cells is shown in transwell plates and by anti-lymphocyte function-associated antigen-1 (LFA-1)-blocking antibody. Up-regulation of CD54 and the subsequent apoptosis of B-CLL cells depend on the initial LFA-1/ICAM-1 (intercellular adhesion molecule 1) interaction. Taken together, these results suggest that allogeneic or autologous host nonreactive anti-third-party CTLs may represent a new therapeutic approach for patients with B-CLL. (Blood. 2005;105:3365-3371)

Previous studies suggest that cells within the CD34+ hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34+ cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti–IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34+ cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34+ cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34+ cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti–tumor necrosis factor-α (anti–TNF-α) and not by anti–transforming growth factor-β (anti–TGF-β), suggesting a potential role for TNF-α in the regulatory activity of CD34+ cells.

Previous studies suggest that cells within the CD34+hematopoietic stem cell compartment are endowed with immune regulatory activity. Furthermore, it is possible to expand the human regulatory cells upon short-term culture of purified CD34+cells with an early-acting cytokine cocktail. We now show that addition of anti-CD28, anti-CD2, interleukin-2 (IL-2), anti–IL-10, or IL-12 to the bulk mixed lymphocyte reaction (MLR) cannot reverse the inhibitory activity of the CD34+cells, ruling out anergy-based mechanisms or mechanisms involving Th1-Th2 skewing. Furthermore, phenotyping of cells present after addition of CD34+cells to the bulk MLR ruled out potential induction of plasmacytoid dendritic precursors, known to be endowed with regulatory activity. In contrast, the inhibitory activity of CD34+cells could be reversed by adding the caspase inhibitor BD-FMK to the bulk MLR, indicating a deletion-based mechanism. The deletion can be inhibited by anti–tumor necrosis factor-α (anti–TNF-α) and not by anti–transforming growth factor-β (anti–TGF-β), suggesting a potential role for TNF-α in the regulatory activity of CD34+cells.

Thiols are important molecules to control apoptosis. This study examined the effect of N‐acetyl‐L‐cysteine (NAC) on in vitro spontaneous apoptosis of human tonsillar B lymphocytes (TBL). Results show that NAC inhibits TBL apoptosis and maintains their survival in vitro. The antiapoptotic action of NAC is progressively reduced when its addition to culture is delayed, is reversible, and is not blocked by cycloheximide. The antiapoptotic activity of NAC is associated with its ability to inhibit caspase‐3 and ‐7 proteolytic processing, DNA‐fragmentation factor 45 cleavage, and DNA fragmentation. Furthermore, NAC inhibits BID cleavage and cytochrome c release from mitochondria and increases the expression of Bcl‐2 and BclXLsurvival proteins. However, it has no effect on caspase‐9 cleavage and increases that of caspase‐8 and poly(adenosine 5′‐diphosphate‐ribose)polymerase. We conclude that NAC‐induced inhibition of TBL apoptosis is associated with inhibition of caspase‐3 and ‐7 processing and is accompanied by changes in several regulatory components of the apoptotic process. These results pose the question of whether microenvironment thiols may in part contribute to in vivo B cell survival.

Endothelial cells (EC) were extracted through a lectin-based method from bone marrow of 57 patients with active multiple myeloma (MM) and compared with their healthy quiescent counterpart, human umbilical vein EC (HUVEC). MMECs exhibit specific antigens that indicate ongoing angiogenesis and embryo vasculogenesis; solid intercellular connections, hence stability of MM neovessels; and frequent interactions with plasma cells, hence tumor dissemination. They show heterogeneous antigen expression, hence existence of subsets. Their main genetic markers are indicative of a vascular phase. They show intrinsic angiogenic ability, because they rapidly form a capillary network in vitro, and extrinsic ability, because they generate numerous new vessels in vivo. They vividly secrete growth and invasive factors for plasma cells. They signal through kinases mandatory for development of neovascularization. Ultrastructurally, they are abnormal and show metabolic activation, like tumor ECs. Thalidomide heavily interferes with their functions. Vasculogenesis and angiogenesis might contribute to the MM vascular tree and progression, in the form of growth, invasion, and dissemination. In view of the heterogeneity of the antigenic phenotype of MMECs, a mixture (or a sequence) of antiangiogenic agents coupled with thalidomide would seem plausible for the biologic management of MM.

Endothelial cells (EC) were extracted through a lectin-based method from bone marrow of 57 patients with active multiple myeloma (MM) and compared with their healthy quiescent counterpart, human umbilical vein EC (HUVEC). MMECs exhibit specific antigens that indicate ongoing angiogenesis and embryo vasculogenesis; solid intercellular connections, hence stability of MM neovessels; and frequent interactions with plasma cells, hence tumor dissemination. They show heterogeneous antigen expression, hence existence of subsets. Their main genetic markers are indicative of a vascular phase. They show intrinsic angiogenic ability, because they rapidly form a capillary network in vitro, and extrinsic ability, because they generate numerous new vessels in vivo. They vividly secrete growth and invasive factors for plasma cells. They signal through kinases mandatory for development of neovascularization. Ultrastructurally, they are abnormal and show metabolic activation, like tumor ECs. Thalidomide heavily interferes with their functions. Vasculogenesis and angiogenesis might contribute to the MM vascular tree and progression, in the form of growth, invasion, and dissemination. In view of the heterogeneity of the antigenic phenotype of MMECs, a mixture (or a sequence) of antiangiogenic agents coupled with thalidomide would seem plausible for the biologic management of MM.

To develop a trial with lymphocyte suicide gene therapy in patients with hematological malignancies, we transduced human T lymphocytes with a retroviral vector (LSN-tk) encoding the herpes simplex virus thymidine kinase (tk) and the neomycin resistance (NeoR) genes. Precise quantification of gene transfer is crucial for any gene therapy protocol, but methods using semiquantitative PCR are inaccurate and subject to variations. Real-time quantitative PCR could be a valid alternative. A TaqMan probe was designed to hybridize with the NeoR gene. The PCR product is 64 nucleotides long and readily quantified by TaqMan probe binding. The analysis was performed soon after transduction and repeated after the selection procedure. This method was more accurate, reproducible, and sensitive than the semiquantitative PCR assay. Accuracy was the same whether the analysis was performed at the highest rate or at the lowest rate of transduction. Additionally we used real-time PCR to monitor the kinetics of enrichment of the transduced cells over the selection time and showed how 7 days of selection are needed. This study precisely quantified the percentages of cells transduced by the retroviral vector and could have major implications in gene therapy studies.

Studies in mice and humans demonstrate that transplantation of hematopoietic progenitors in numbers larger than commonly used (“megadose” transplants) overcomes major genetic barriers without induction of graft-versus-host disease. Therefore, transplantation from a human leukocyte antigen mismatched family member is a viable option for patients with acute leukemia at high risk of relapse who urgently need a transplant and who do not have a well matched unrelated donor. In vitro studies suggest that veto cells, contained in the population of hematopoietic progenitors, facilitate this favorable outcome.

Stem cell–dose escalation is one way to overcome immune rejection of incompatible stem cells. However, the number of hematopoietic precursors required for overcoming the immune barrier in recipients pretreated with sublethal regimens cannot be attained with the state-of-the-art technology for stem cell mobilization. This issue was addressed by the observation that cells within the human CD34+ population are endowed with veto activity. In the current study, we demonstrated that it is possible to harvest about 28- to 80-fold more veto cells on culturing of purified CD34+cells for 7 to 12 days with an early-acting cytokine mixture including Flt3-ligand, stem cell factor, and thrombopoietin. Analysis of the expanded cells with fluorescence-activated cell-sorter scanning revealed that the predominant phenotype of CD34+CD33− cells used at the initiation of the culture was replaced at the end of the culture by cells expressing early myeloid phenotypes such as CD34+CD33+ and CD34−CD33+. These maturation events were associated with a significant gain in veto activity as exemplified by the minimal ratio of veto to effector cells at which significant veto activity was detected. Thus, whereas purified unexpanded CD34+ cells exhibited veto activity at a veto-to-effector cell ratio of 0.5, the expanded cells attained an equivalent activity at a ratio of 0.125. The availability of novel sources of veto cells such as those in this study might contribute to the realization of immunologic tolerance in “minitransplants,” without any risk of graft-versus-host disease.

Stem cell–dose escalation is one way to overcome immune rejection of incompatible stem cells. However, the number of hematopoietic precursors required for overcoming the immune barrier in recipients pretreated with sublethal regimens cannot be attained with the state-of-the-art technology for stem cell mobilization. This issue was addressed by the observation that cells within the human CD34+population are endowed with veto activity. In the current study, we demonstrated that it is possible to harvest about 28- to 80-fold more veto cells on culturing of purified CD34+cells for 7 to 12 days with an early-acting cytokine mixture including Flt3-ligand, stem cell factor, and thrombopoietin. Analysis of the expanded cells with fluorescence-activated cell-sorter scanning revealed that the predominant phenotype of CD34+CD33−cells used at the initiation of the culture was replaced at the end of the culture by cells expressing early myeloid phenotypes such as CD34+CD33+and CD34−CD33+. These maturation events were associated with a significant gain in veto activity as exemplified by the minimal ratio of veto to effector cells at which significant veto activity was detected. Thus, whereas purified unexpanded CD34+cells exhibited veto activity at a veto-to-effector cell ratio of 0.5, the expanded cells attained an equivalent activity at a ratio of 0.125. The availability of novel sources of veto cells such as those in this study might contribute to the realization of immunologic tolerance in “minitransplants,” without any risk of graft-versus-host disease.

The GIMEMA ALL 0288 trial was designed to evaluate the impact of a 7-day prednisone (PDN) pretreatment on complete remission (CR) achievement and length, the influence of the addition of cyclophosphamide (random I) to a conventional 4-drug induction on CR rate and duration, and whether an early post-CR intensification (random II) by an 8-drug consolidation could improve CR duration. Median follow-up of this study was 7.3 years. From January 1988 to April 1994, among 794 adult (> 12 but < 60 years) patients registered, 778 were eligible. Their median age was 27.5 years; 73% had B-lineage acute lymphoblastic leukemia (ALL) and 22% had T-lineage disease; 18% showed associated myeloid markers; 47 of 216 analyzed patients (22%) had Philadelphia chromosome–positive ALL. Response to PDN pretreatment was observed in 65% of cases. CR was achieved in 627 patients (82%). Resistant patients and induction death rates were 11% and 7%, respectively. Random II was applied to 388 patients with CR; 201 had maintenance alone and 187 had consolidation followed by maintenance. The relapse rate was 60%; isolated central nervous system relapses were 8% of all CRs and 13% of all relapses. Median survival (overall survival [OS]), continuous complete remission (CCR), and disease-free survival (DFS) were 2.2, 2.4, and 2 years, respectively. PDN pretreatment response resulted the main independent factor influencing CR achievement, OS, CCR, and DFS; the addition of cyclophosphamide in induction significantly influenced CR achievement in a multivariate analysis. Neither induction intensification nor early consolidation appeared to influence CCR and DFS duration. For the first time PDN pretreatment response proved to be a powerful factor predicting disease outcome in adult ALL patients.

The GIMEMA ALL 0288 trial was designed to evaluate the impact of a 7-day prednisone (PDN) pretreatment on complete remission (CR) achievement and length, the influence of the addition of cyclophosphamide (random I) to a conventional 4-drug induction on CR rate and duration, and whether an early post-CR intensification (random II) by an 8-drug consolidation could improve CR duration. Median follow-up of this study was 7.3 years. From January 1988 to April 1994, among 794 adult (> 12 but < 60 years) patients registered, 778 were eligible. Their median age was 27.5 years; 73% had B-lineage acute lymphoblastic leukemia (ALL) and 22% had T-lineage disease; 18% showed associated myeloid markers; 47 of 216 analyzed patients (22%) had Philadelphia chromosome–positive ALL. Response to PDN pretreatment was observed in 65% of cases. CR was achieved in 627 patients (82%). Resistant patients and induction death rates were 11% and 7%, respectively. Random II was applied to 388 patients with CR; 201 had maintenance alone and 187 had consolidation followed by maintenance. The relapse rate was 60%; isolated central nervous system relapses were 8% of all CRs and 13% of all relapses. Median survival (overall survival [OS]), continuous complete remission (CCR), and disease-free survival (DFS) were 2.2, 2.4, and 2 years, respectively. PDN pretreatment response resulted the main independent factor influencing CR achievement, OS, CCR, and DFS; the addition of cyclophosphamide in induction significantly influenced CR achievement in a multivariate analysis. Neither induction intensification nor early consolidation appeared to influence CCR and DFS duration. For the first time PDN pretreatment response proved to be a powerful factor predicting disease outcome in adult ALL patients.

Aims and Background To investigate the safety and efficacy of a high-dose chemotherapy regimen with etoposide, carboplatin and thiotepa in high-risk stage II-IIIA breast cancer and in responsive metastatic patients.Study Design From April 1992 to December 1998, 24 patients with high-risk stage II-IIIA breast cancer (≥9 positive nodes) and 9 responsive metastatic patients were enrolled in the trial. After induction chemotherapy with an anthracycline-based regimen, peripheral blood stem cells were mobilized with cyclophosphamide (7 g/m2) and G-CSF (5-16 μg/kg/sc/day). The high-dose chemotherapy regimen consisted of etoposide (1000 mg/m2), carboplatin (800 mg/m2) and thiotepa (500 mg/m2). At the end of the high-dose chemotherapy, all stage II-IIIA patients received radiotherapy to the breast or chest wall and draining nodes; stage IV patients were irradiated to sites of disease, if feasible. All ER+ and/or PgR+ patients were treated with hormone therapy.Results For stage II-IIIA high-risk patients, the median follow-up was 4.36 years (range, 1.93-6.94), and the Kaplan-Meier estimate at 5 years of disease-free survival and overall survival was 54.8 ± 11% SE and 76.73 ± 9.4% SE, respectively. For metastatic patients, the median follow-up was 4.93 years (range, 4.15-7.95), and the Kaplan-Meier estimate at 5 years of progression-free survival and overall survival was 22.2 ± 13.9% SE and 76.2 ± 14.8% SE, respectively. No treatment-related deaths were observed.Conclusions Our results are comparable to those obtained in other high-dose chemotherapy trials but do not seem to be superior to conventional-dose therapy given to similar patients.

In human leukocyte antigen haplotype–mismatched transplantation, extensive T-cell depletion prevents graft-versus-host disease (GVHD) but delays immune recovery. Granulocyte colony-stimulating factor (G-CSF) is given to donors to mobilize stem cells and to recipients to ensure engraftment. Studies have shown that G-CSF promotes T-helper (Th)-2 immune deviation which, unlike Th1 responses, does not protect against intracellular pathogens and fungi. The effect of administration of G-CSF to recipients of mismatched hematopoietic transplants with respect to transplantation outcome and functional immune recovery was investigated. In 43 patients with acute leukemia who received G-CSF after transplantation, the engraftment rate was 95%. However, the patients had a long-lasting type 2 immune reactivity, ie, Th2-inducing dendritic cells not producing interleukin 12 (IL-12) and high frequencies of IL-4– and IL-10–producing CD4+cells not expressing the IL-12 receptor β2chain. Similar immune reactivity patterns were observed on exposure of donor cells to G-CSF. Elimination of postgrafting administration of G-CSF in a subsequent series of 36 patients with acute leukemia, while not adversely affecting engraftment rate (93%), resulted in the anticipated appearance of IL-12–producing dendritic cells (1-3 months after transplantation versus > 12 months in transplant recipients given G-CSF), of CD4+cells of a mixed Th0/Th1 phenotype, and of antifungal T-cell reactivity in vitro. Moreover, CD4+cell counts increased in significantly less time. Finally, elimination of G-CSF–mediated immune suppression did not significantly increase the incidence of GVHD (< 15%). Thus, this study found that administration of G-CSF to recipients of T-cell–depleted hematopoietic transplants was associated with abnormal antigen-presenting cell functions and T-cell reactivity. Elimination of postgrafting administration of G-CSF prevented immune dysregulation and accelerated functional immune recovery.

In human leukocyte antigen haplotype–mismatched transplantation, extensive T-cell depletion prevents graft-versus-host disease (GVHD) but delays immune recovery. Granulocyte colony-stimulating factor (G-CSF) is given to donors to mobilize stem cells and to recipients to ensure engraftment. Studies have shown that G-CSF promotes T-helper (Th)-2 immune deviation which, unlike Th1 responses, does not protect against intracellular pathogens and fungi. The effect of administration of G-CSF to recipients of mismatched hematopoietic transplants with respect to transplantation outcome and functional immune recovery was investigated. In 43 patients with acute leukemia who received G-CSF after transplantation, the engraftment rate was 95%. However, the patients had a long-lasting type 2 immune reactivity, ie, Th2-inducing dendritic cells not producing interleukin 12 (IL-12) and high frequencies of IL-4– and IL-10–producing CD4+ cells not expressing the IL-12 receptor β2 chain. Similar immune reactivity patterns were observed on exposure of donor cells to G-CSF. Elimination of postgrafting administration of G-CSF in a subsequent series of 36 patients with acute leukemia, while not adversely affecting engraftment rate (93%), resulted in the anticipated appearance of IL-12–producing dendritic cells (1-3 months after transplantation versus > 12 months in transplant recipients given G-CSF), of CD4+ cells of a mixed Th0/Th1 phenotype, and of antifungal T-cell reactivity in vitro. Moreover, CD4+ cell counts increased in significantly less time. Finally, elimination of G-CSF–mediated immune suppression did not significantly increase the incidence of GVHD (< 15%). Thus, this study found that administration of G-CSF to recipients of T-cell–depleted hematopoietic transplants was associated with abnormal antigen-presenting cell functions and T-cell reactivity. Elimination of postgrafting administration of G-CSF prevented immune dysregulation and accelerated functional immune recovery.

The engraftment capacity of bone marrow–derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell–depleted allograft from human leukocyte antigen (HLA)–matched or –mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit–F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18?±?0.13?×?104/kg vs 1.19?± 0.19?×?104/kg; P?=?.97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell–depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.

The engraftment capacity of bone marrow–derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell–depleted allograft from human leukocyte antigen (HLA)–matched or –mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit–F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18 ± 0.13 × 104/kg vs 1.19 ± 0.19 × 104/kg; P≥ .97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell–depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.

We developed a combined methodological approach to enrich and to proliferate in vitro fetal CD34+ stem progenitor cells. Using a magnetic cell-sorting technique, CD34+ cells from pregnant women at the early-second trimester were isolated and enriched and compared to those isolated from blood of nonpregnant women. The number and frequency of CD34+ cells were significantly higher (p < 0.001) in the pregnant women. Unenriched peripheral blood mononuclear cells (PBMC) and enriched CD34+ cells were cultured in a methylcellulose system to evaluate the cloning potential of progenitor cells. After culture, the numbers of burst-forming units erythroid/colony-forming units erythroid (BFU-E/CFU-E) and colony-forming units granulocyte-macrophage (CFU-GM) colonies were increased by 33 and 16 times, respectively. Finally, to distinguish between fetal and maternal cells, four cases of cultured cells were hybridized with specific probes for X and Y chromosomes and two cases with a specific probe for chromosome 21. In normal pregnancies, we identified a high number of male fetal cells and an elevated fetal/maternal ratio. When we analyzed blood samples from pregnancies with trisomic fetuses, we scored a high ratio of trisomic cells respect to maternal cells that was significantly different from the ratio of pregnancies with normal fetuses. Our results demonstrate fetal progenitor cells may be cultured and detected successfully with an appropriate combined methodological approach, which may significantly increase the feasibility of noninvasive prenatal diagnosis.

In this study, we assessed the efficiency of T lymphocyte transduction with a retroviral vector carrying the herpes simplex virus thymidine kinase (HSV-tk) and neomycin phosphotransferase (neo) genes by four different protocols: standard supernatant infection, supernatant infection plus centrifugation steps, supernatant infection on fibronectin fragment-coated wells, and cocultivation. After retrovirus-mediated gene transfer of tk-neo in PHA/IL-2-stimulated primary T lymphocytes and G418 selection, T cells retained their proliferative activity, alloresponsiveness, ability to produce and to respond to IL-2, and ganciclovir (gcv)-specific sensitivity. When the four different transduction techniques were compared, no significant differences were seen in terms of cellular viability, proliferation capacity, and immunophenotyping. tk gene expression was the same in all transduced selected populations, as indicated by gcv sensitivity. Transduction efficiency was evaluated by semiquantitative PCR. Using the standard supernatant infection method, the rate of infection was extremely low (<5%). After adding the centrifugation step or performing supernatant infection on fibronectin fragment-coated wells, PCR analysis showed a 30%-40% rate of transduced cells. After infection by cocultivation, the rate of transduced cells was 30%-40%. These results demonstrate that supernatant infection plus centrifugation, supernatant infection on fibronectin fragment-coated wells, and cocultivation methods provide equivalent rates of transduced cells. The lack of reproducibility and safety indicates that cocultivation is not suitable for clinical studies. In our view, supernatant infection plus centrifugation is easier to perform than using fibronectin fragments, and it is currently the optimal method for clinical studies when large quantities of T lymphocytes are being processed.

In this study, we assessed the efficiency of T lymphocyte transduction with a retroviral vector carrying the herpes simplex virus thymidine kinase (HSV-tk) and neomycin phosphotransferase (neo) genes by four different protocols: standard supernatant infection, supernatant infection plus centrifugation steps, supernatant infection on fibronectin fragment-coated wells, and cocultivation. After retrovirus-mediated gene transfer of tk-neo in PHA/IL-2-stimulated primary T lymphocytes and G418 selection, T cells retained their proliferative activity, alloresponsiveness, ability to produce and to respond to IL-2, and ganciclovir (gcv)-specific sensitivity. When the four different transduction techniques were compared, no significant differences were seen in terms of cellular viability, proliferation capacity, and immunophenotyping. tk gene expression was the same in all transduced selected populations, as indicated by gcv sensitivity. Transduction efficiency was evaluated by semiquantitative PCR. Using the standard supernatant infection method, the rate of infection was extremely low (&lt;5%). After adding the centrifugation step or performing supernatant infection on fibronectin fragment-coated wells, PCR analysis showed a 30%-40% rate of transduced cells. After infection by cocultivation, the rate of transduced cells was 30%-40%. These results demonstrate that supernatant infection plus centrifugation, supernatant infection on fibronectin fragment-coated wells, and cocultivation methods provide equivalent rates of transduced cells. The lack of reproducibility and safety indicates that cocultivation is not suitable for clinical studies. In our view, supernatant infection plus centrifugation is easier to perform than using fibronectin fragments, and it is currently the optimal method for clinical studies when large quantities of T lymphocytes are being processed.

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR gene on chromosome 22 with c-ABL sequences translocated from chromosome 9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase (PTK) activity exerting a key role in malignant transformation. Targeting the tyrosine kinase activity of BCR/ABL or using agents capable of triggering apoptosis might represent attractive therapeutic approaches for ex vivo purging. AG957, a member of the tyrphostin compounds, exerts a selective inhibition of p210BCR/ABLtyrosine phosphorylation. We report here that preincubation of CML or normal CD34+ cells with graded concentration of AG957 (1 to 100 µmol/L) resulted in a statistically significant, dose-dependent suppression of colony growth from multipotent, erythroid, and granulocyte-macrophage progenitors as well as the more primitive long-term culture-initiating cells (LTC-IC). However, AG957 doses causing 50% inhibition (ID50) of CML and normal progenitors were significantly different for multilineage colony-forming units (CFU-Mix; 12 v 64 µmol/L; P = .008), burst-forming unit-erythroid (BFU-E; 29 v 89 µmol/L;P = .004), colony-forming unit–granulocyte-macrophage (CFU-GM; 34 v 85 µmol/L; P = .004), and LTC-IC (43 v 181 µmol/L; P = .004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by reverse transcription-polymerase chain reaction showed that AG957 at 50 µmol/L significantly reduced the mean (±SD) percentage of BCR/ABL-positive progenitors (92% ± 10% v 33 ± 5%;P = .001). Because AG957 treatment resulted in significantly higher percentages of apoptotic cells (30% v9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93 cells (BCR/ABL-negative), we investigated the combined effects of AG957 with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that triggers apoptosis. As compared with AG957 alone, the sequential treatment of CML CD34+ cells with AG957 (1 µmol/L) and CH11 (1 µg/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the treatment of normal CD34+ cells with AG957 and CH11 failed to enhance AG957-induced colony growth inhibition. We conclude that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived colony formation by both primitive LTC-IC as well as committed CFU-Mix, BFU-E, and CFU-GM; (2) this growth inhibition is associated with the selection of a substantial amount of BCR/ABL-negative progenitors; and (3) the antiproliferative effect of AG957 is dramatically increased by combining this compound with the anti–Fas-R antibody CH11. These data may have significant therapeutic applications.

ABSTRACTWe have studied the in vivo tropism of human herpesvirus 6 (HHV-6) for hemopoietic cells in patients with latent HHV-6 infection. Having used a variety of cell purification, molecular, cytogenetic, and immunocytochemical procedures, we report the first evidence that HHV-6 latently infects early bone marrow progenitor cells and that HHV-6 may be transmitted longitudinally to cells which differentiate along the committed pathways.

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR gene on chromosome 22 with c-ABL sequences translocated from chromosome 9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase (PTK) activity exerting a key role in malignant transformation. Targeting the tyrosine kinase activity of BCR/ABL or using agents capable of triggering apoptosis might represent attractive therapeutic approaches for ex vivo purging. AG957, a member of the tyrphostin compounds, exerts a selective inhibition of p210BCR/ABLtyrosine phosphorylation. We report here that preincubation of CML or normal CD34+cells with graded concentration of AG957 (1 to 100 μmol/L) resulted in a statistically significant, dose-dependent suppression of colony growth from multipotent, erythroid, and granulocyte-macrophage progenitors as well as the more primitive long-term culture-initiating cells (LTC-IC). However, AG957 doses causing 50% inhibition (ID50) of CML and normal progenitors were significantly different for multilineage colony-forming units (CFU-Mix; 12 v64 μmol/L; P= .008), burst-forming unit-erythroid (BFU-E; 29 v89 μmol/L;P= .004), colony-forming unit–granulocyte-macrophage (CFU-GM; 34 v85 μmol/L; P= .004), and LTC-IC (43 v181 μmol/L; P= .004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by reverse transcription-polymerase chain reaction showed that AG957 at 50 μmol/L significantly reduced the mean (±SD) percentage of BCR/ABL-positive progenitors (92% ± 10% v33 ± 5%;P= .001). Because AG957 treatment resulted in significantly higher percentages of apoptotic cells (30% v9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93 cells (BCR/ABL-negative), we investigated the combined effects of AG957 with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that triggers apoptosis. As compared with AG957 alone, the sequential treatment of CML CD34+cells with AG957 (1 μmol/L) and CH11 (1 μg/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the treatment of normal CD34+cells with AG957 and CH11 failed to enhance AG957-induced colony growth inhibition. We conclude that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived colony formation by both primitive LTC-IC as well as committed CFU-Mix, BFU-E, and CFU-GM; (2) this growth inhibition is associated with the selection of a substantial amount of BCR/ABL-negative progenitors; and (3) the antiproliferative effect of AG957 is dramatically increased by combining this compound with the anti–Fas-R antibody CH11. These data may have significant therapeutic applications.

We review the rationale for, and the results of, clinical trials on chemoradiotherapy‐based pretransplant regimens for non‐Hodgkin's lymphomas, Hodgkin's disease and multiple myeloma. What clearly emerges from this review is the lack of any conclusive evidence that total‐body irradiation (TBI)‐containing regimens are better than chemotherapy alone in diseases which are considered to be radiosensitive. Due to the variety of pretransplant regimens adopted, the relatively low number of patients enrolled in each trial, and the lack of randomized studies, no one conditioning scheme, with or without TBI, could be identified as superior to another. Only randomized clinical studies will indicate whether TBI‐containing regimens are superior to chemotherapy‐only regimens and whether TBI and/or involved field radiation therapy have a place in autologous stem cell transplantation programs for lymphoproliferative disorders. And finally, the best TBI dose, schedule, and technique should be defined.

V pre-B and λ-like genes are selectively expressed in human pre-B cells and encode polypeptide chains that associate in a μ-pseudolight chain complex that may regulate some crucial steps of early B-cell differentiation. We have followed by polymerase chain reaction and Northern blot analysis the expression of these “pre-B-specific” genes in correlation with the status (rearranged v germline) of Ig gene loci (H, κ, λ) in a panel of 32 leukemias pertaining mostly to the B lineage and including a number of ambiguously characterized samples. All cells that had rearranged the H locus only expressed V pre-B and λ-like transcripts, in agreement with a pre-B status. In this group, some biphenotypic leukemias (mostly My/B) might, in fact, be already engaged in the B lineage. Rearrangement of Vκ or Vλ loci correlated with the disappearance of the pre-B gene products. In a pre-B acute lymphoblastic leukemia cell line that was induced to mature to the B-cell stage in culture upon κ gene rearrangement, the μ-pseudolight chain complex was actually replaced by the classical μ-Κ molecule. Finally, V pre-B and λ-like genes were found expressed in two leukemic cells that had retained all Ig loci in germline configuration. This finding raises the possibility of having an early pro-B progenitor in which V pre-B and λ-like products associate with a H chain surrogate in a complex that would trigger an early event of B-cell differentiation such as the H locus rearrangements. © 1991 by The American Society of Hematology.

The immunophenotype of 304 adult lymphoblastic leukemias (> 18 years) diagnosed on the basis of the FAB criteria was determined at the time of diagnosis using a panel of monoclonal antibodies. The series comprised cases diagnosed and immunophenotyped in 43 Italian centers (GIMEMA Cooperative Group) between April 1988 and June 1991. The immunophenotypic characterization consisted of two consecutive steps. The initial screening was based on the reactivity for Tdt, HLA-Dr, CD7, CD10, CD13, CD19, CD24, CD33 and CD41. According to the results obtained, the second level of investigation assessed the positivity for intra cytoplasmic (Cy) Ig, CD la, CD2, CD3, CD4, CDS, CDS and CD20. Based on the hierarchial expression of the different B- and T-cell related antigens, each case was assigned to a given differentiation stage. B-lineage ALL were classified in five subgroups (B0-B4) and T-lineage ALL in four subgroups (T0-T3). Cases in which the blasts were lymphoid according to the FAB criteria, but expressed myeloid antigens in association with B- and T-lymphoid markers were defined as hybrid leukemias.As expected, CD10 + cases (B2-B3) were the most frequent within the B-lineage ALL (83.2% of cases). Cylg+ (B3) accounted for about 20% of CD10+ ALL. Twenty eight cases (13.4%) were at a pre-cALL stage (B0-B1) and of these, 8 (3.8% of the total series) were positive only for TdT and HLA-Dr (BO). Intermediate and mature thymic phenotypes (T2-T3) were predominant within the T-ALL (67.2%) groups. Five cases, were positive only for TdT and CD7 (CD5 +), and classified as T0. 9.2% of cases fulfilled the definition of hybrid leukemia, largely in view of the co-expression of B-lymphoid and myeloid markers. In most cases the cells were at a B2 stage of differentiation. Analysis at the DNA level, carried out in 6 cases, showed that all had a monoclonal rearrangement of the Ig heavy chain gene, sometimes coupled with concomitant TCR gene rearrangement.Mediastinal enlargement was found in 38.9% of the T-ALL cases, but was undetectable in the other subgroups. Anemia was more pronounced in B-lineage ALL while high WBC counts were more frequent in T-ALL., where a greater tumor load was documented. Overall, only 7 cases (2.3%) diagnosed as ALL on the basis of the FAB criteria were re-classified as AML after immunologic characterization. The diagnostic role of phenotypic and genotypic analyses is discussed.

Patients who undergo transplantation with haploidentical “three-loci” mismatched T-cell-depleted bone marrow (BM) are at high risk for graft failure. To overcome the host-versus-graft barrier, we increased the size of the graft inoculum, which has been shown to be a major factor in controlling both immune rejection and stem cell competition in murine models. Seventeen patients (mean age, 23.2 years; range, 6 to 51 years) with end-stage chemoresistant leukemia were received transplants of a combination of BM with recombinant human granulocyte colony-stimulating factor-mobilized peripheral blood progenitor cells from HLA-haploidentical “three-loci” incompatible family members. The average concentration of colony-forming unit-granulocyte-macrophage in the final inoculum was sevenfold to 10-fold greater than that found in BM alone. The sole graft-versus-host disease (GVHD) prophylaxis consisted of T-cell depletion of the graft by the soybean agglutination and E-rosetting technique. The conditioning regimen included total body irradiation in a single fraction at a fast dose rate, antithymocyte globulin, cyclophosphamide and thiotepa to provide both immunosuppression and myeloablation. One patient rejected the graft and the other 16 had early and sustained full donor-type engraftment. One patient who received a much greater quantity of T lymphocytes than any other patient died from grade IV acute GVHD. There were no other cases of GVHD ≥grade II. Nine patients died from transplant-related toxicity, 2 relapsed, and 6 patients are alive and event-free at a median follow-up of 230 days (range, 100 to 485 days). Our results show that a highly immunosuppressive and myeloablative conditioning followed by transplantation of a large number of stem cells depleted of T lymphocytes by soybean agglutination and E-rosetting technique has made transplantation of three HLA-antigen disparate grafts possible, with only rare cases of GVHD.

Sixty-one adults aged <55 years with acute lymphoblastic leukaemia (ALL) in first bone marrow relapse were enrolled in an Italian cooperative study (ALL R-87 protocol) from 12 GIMEMA Institutions. The treatment programme consisted of: (1) an induction phase with intermediate-dose cytarabine (IDARA-C 1 g/m2, 6 h daily infusion ×6 d) plus idarubicin (IDA; 5 mg/m2/d × 6 d) and prednisone (40 mg/m2/d × 21 d), (2) a consolidation phase followed by (3) bone marrow transplant (BMT). Median first complete remission (CR) duration was 8.5 months (range 1–54 months). 34/61 patients achieved CR (56%); 24 (39%) failed to respond and three (5%) died during induction. Most responders (24 patients) could not enter the BMT programme; 15 relapsed early (median time to relapse 2 months); nine were withdrawn due to toxicity and one died in CR of infection. Nine of the 34 CRs underwent BMT (five autologous and four allogeneic). Three of the four allotransplanted patients are alive in continuous CR at 22, 43 and 63 months; only one of the five who underwent an autologous BMT is alive in CR at 46 months.

In this study, we investigated the impact of recombinant interleukin-2 (rIL-2) after high dose chemotherapy and autologous bone marrow transplantation (ABMT) in 25 patients with refractory or relapsed Hodgkin's disease (HD) (11 patients) and non Hodgkin's lymphoma (NHL) (14 patients). 48% of patients had resistant disease, 84% achieved complete remission after ABMT. rIL-2 was started at a median of 54 days post-transplant and consisted of a first cycle of 5 days followed by 4 cycles of 2 days every other week. Patients received a mean of 160 ×× 106IU/m2rIL-2 and hematological toxicity was moderate and transient. None of the 5 evaluable patients with measurable disease responded to rIL-2. After a 5 year median follow-up, the probability of survival and DFS is 72% (HD: 73% and NHL: 70%, p = NS) and 45% (HD: 36% and NHL: 48%, p = NS) respectively. These somewhat encouraging results warrant further evaluation of rIL-2 after ABMT in controlled studies, especially in NHL patients stratified for previous chemosensitivity.

Intracytoplasmic lysozynie was studied by the peroxidase antiperoxidase (PAP) and protein A-peroxidase methods in 130 cases of various myeloproliferative and lymphoproliferative disorders and 21 lymph nodes and bone marrow metastases from solid primary tumors. This marker, which can be identified in formalin or Zenker-fixed tissues, as well as in peripheral blood and bone marrow smears, proved useful to distinguish malignant myeloid and histiocytic tumors from malignant lymphoid and undifferentiated epithelial metastases. The diagnostic application of these findings are discussed.

The cytogenetic follow-up of a case of refractory anemia with excess of blasts (RAEB) that rapidly evolved to acute myeloblastic leukemia (Ml-FAB type) is described. Bone marrow analysis at presentation revealed two chromosomally abnormal clones that shared an interstitial deletion of the long arm of chromosome 5 (5q−) and a terminal deletion of the short arm of chromosome 12 (12p-), but that differed from one another in the localization of a very similar segment of chromosome 17 (i.e. 17q11−12qter) on two clearly distinct karyotypic sites: 2q37 and 17q25. Fourteen percent of the metaphases examined bore the 2q+ marker and 38% the 17q+ marker; the remaining cells had a normal karyotype. A second study carried out 4 months later, at onset of the acute phase, revealed that the clone with normal karyotype had almost completely disappeared and that there had been an inversion in the ratio of the two abnormal cell populations. In the final study, made 1 month before death, the cells with t(2;17) had totally effaced the other clone.

Since vimentin intermediate filament (IF) expression in hemopoietic cells varies with the cell lineage as well as the state of differentiation of the cells, we studied the vimentin cytoskeleton by direct immunofluorescence and electron microscopy in 50 cases of acute nonlymphocytic leukemias. We found that malignant cells tend to reproduce the vimentin organization characteristic of their normal cellular counterpart. Thus, in M2 and M3 leukemias (French-American-British classification), vimentin was often reduced to a juxtanuclear bundle of filaments contrasting with the rich filamentous network expressed by M4or M5 leukemias. In erythroblastic leukemias (M6) and mega- karyoblastic leukemias, both identified by the expression of lineage-specific antigens, the absence of vimentin IFs could be correlated with the level of differentiation reached by the blasts. M1 leukemias displayed an abnormal pattern of vimentin organization with aggregated filaments giving a ring-like structure. However, no abnormality of the vimentin polypeptide could be detected by two-dimen-sional electrophoresis. These results show that the expression of the vimentin IF cytoskeleton may be a useful marker of differentiation in the study of leukemic cells. © 1985 by Grune & Stratton, Inc.

Cord blood samples from healthy full-term newborns were tested with antimature and antiimmature lymphoid-cell monoclonal antibodies, as well as more traditional markers, in order to identify the phenotype of circulating precursor cells. The results demonstrated that human cord blood contains a lower number of OKT3+, E-rosetting mature T cells than adult blood, very high levels of OKT10+ cells, and few OKT9+, OKT8+OKT3-, and OKT4+OKT3- cells. Although the finding of OKT9+ and OKT10+ cord circulating cells could be indicative of cell activation, double marker studies in newborn blood pointed to phenotypically immature lymphoid subsets at different stages of maturation, according to Reinherz's hypothesis. In addition, the absence of nuclear Tdt-positive and hot-rosetting cells, together with the fact that most of these are OKT3+, OKT10+, OKT4+, or OKT8+ cells, suggests that the surface phenotype of newborn lymphocytes is similar to that of mature thymocytes.

We previously demonstrated that when human bone marrow-derived mesenchymal cells (hMSCs) were co-cultured with CD4+/CD25+ T regulatory cells (Tregs) they maintained the T regulatory phenotype and function over time. Here we studied the TGF-beta/Smad signalling and mitogen-activated protein kinase (MAPK) pathways after Tregs/hMSCs co-culture. After 7 days co-culture of highly purified, immuno-selected Tregs and hMSCs from healthy donors, TGF-beta and IL-10 concentrations were dosed and mRNA of a panel of genes (NFAT, Smad4, Smad7, AP-1, Foxp3 and CD127) was quantified on harvested lymphocytes. Western blotting was performed by incubation with antiSMAD2, anti-pSMAD2, anti-ERK1/2 and anti-pERK1/2 polyclonal antibodies. After immuno-magnetic cell separation the final Treg fraction showed a mean purity of 93.6%±1. The CD4+/CD25+bright constituted 29%±7 of Tregs, FoxP3 cells constituted 51.9%±15.1 and CD127+ cells 19%±11.5. CD4+/CD25+ cells inhibited CD4+/CD25− cells (mean inhibition percentage: 52.1±29.6% (ratio 1:1). Tregs produced no TGF-beta and only a small quantity of IL-10 (8.67±4 pg/ml). hMSCs produced high quantity of TGF-beta (229.3 ± 54.8 pg/ml) associated with little IL-10 (2.7 ± 1.2 pg/ml). After co-culture, the TGF-beta concentration was 91.5±48.5 pg/ml while the IL-10 concentration was no different to baseline. To identify TGF-beta signaling pathways, we focused on Smad2, a central element in the cascade. In Tregs the strongly expressed pSmad2 signal after selection rapidly decreased after 7 days culture. When Tregs were co-cultured in presence of hMSCs the phosphorylated form of Smad2 did not change, indicating TGF-beta signaling was up-regulated. After co-culture mRNA quantification showed hMSCs down-regulated expression of SMAD7 (−8.9± 4.4 times vs Tregs without hMSCs), a negative regulator of TGF-beta signaling. After co-culture with hMSCs non-regulatory CD4+/CD25− control cells did not show any differences in SMAD7 expression. To study the MAPK kinase pathways, harvested Tregs were assayed for ERK1/2 kinase activity by determining their phosphorylated forms. pERK1/2 was almost absent in selected Tregs; it rapidly increased after 7 days in vitro culture without hMSCs but remained low after co-culture with hMSCs, indicating impaired ERK1/2 activation. AP-1 activation was also reduced (AP-1 mRNA −32±29 times less than controls). In conclusion, co-cultures of hMSCs and Tregs have the potential to set up a positive feedback loop that heightens responsiveness to TFG-beta in Treg cells. In fact, hMSCs produce TGF-beta which is consumed in co-culture with Tregs; TGF-beta1 signaling in Tregs is maintained through pSMAD2 up-regulation and SMAD7 down-regulation; hMSCs also inhibit ERK phosphorylation which consequently down-regulates AP-1 mRNA. Thus Tregs signalling is maintained through culture on a layer of hMSCs. Manipulation of Tregs signaling through culture on a layer of hMSCs may represent a novel strategy for maintenance of Treg phenotype and function.

We previously demonstrated that when human bone marrow-derived mesenchymal cells (hMSCs) were co-cultured with CD4+/CD25+T regulatory cells (Tregs) they maintained the T regulatory phenotype and function over time. Here we studied the TGF-beta/Smad signalling and mitogen-activated protein kinase (MAPK) pathways after Tregs/hMSCs co-culture. After 7 days co-culture of highly purified, immuno-selected Tregs and hMSCs from healthy donors, TGF-beta and IL-10 concentrations were dosed and mRNA of a panel of genes (NFAT, Smad4, Smad7, AP-1, Foxp3 and CD127) was quantified on harvested lymphocytes. Western blotting was performed by incubation with antiSMAD2, anti-pSMAD2, anti-ERK1/2 and anti-pERK1/2 polyclonal antibodies. After immuno-magnetic cell separation the final Treg fraction showed a mean purity of 93.6%±1. The CD4+/CD25+brightconstituted 29%±7 of Tregs, FoxP3 cells constituted 51.9%±15.1 and CD127+ cells 19%±11.5. CD4+/CD25+cells inhibited CD4+/CD25−cells (mean inhibition percentage: 52.1±29.6% (ratio 1:1). Tregs produced no TGF-beta and only a small quantity of IL-10 (8.67±4 pg/ml). hMSCs produced high quantity of TGF-beta (229.3 ± 54.8 pg/ml) associated with little IL-10 (2.7 ± 1.2 pg/ml). After co-culture, the TGF-beta concentration was 91.5±48.5 pg/ml while the IL-10 concentration was no different to baseline. To identify TGF-beta signaling pathways, we focused on Smad2, a central element in the cascade. In Tregs the strongly expressed pSmad2 signal after selection rapidly decreased after 7 days culture. When Tregs were co-cultured in presence of hMSCs the phosphorylated form of Smad2 did not change, indicating TGF-beta signaling was up-regulated. After co-culture mRNA quantification showed hMSCs down-regulated expression of SMAD7 (−8.9± 4.4 times vs Tregs without hMSCs), a negative regulator of TGF-beta signaling. After co-culture with hMSCs non-regulatory CD4+/CD25−control cells did not show any differences in SMAD7 expression. To study the MAPK kinase pathways, harvested Tregs were assayed for ERK1/2 kinase activity by determining their phosphorylated forms. pERK1/2 was almost absent in selected Tregs; it rapidly increased after 7 days in vitro culture without hMSCs but remained low after co-culture with hMSCs, indicating impaired ERK1/2 activation. AP-1 activation was also reduced (AP-1 mRNA −32±29 times less than controls). In conclusion, co-cultures of hMSCs and Tregs have the potential to set up a positive feedback loop that heightens responsiveness to TFG-beta in Treg cells. In fact, hMSCs produce TGF-beta which is consumed in co-culture with Tregs; TGF-beta1 signaling in Tregs is maintained through pSMAD2 up-regulation and SMAD7 down-regulation; hMSCs also inhibit ERK phosphorylation which consequently down-regulates AP-1 mRNA. Thus Tregs signalling is maintained through culture on a layer of hMSCs. Manipulation of Tregs signaling through culture on a layer of hMSCs may represent a novel strategy for maintenance of Treg phenotype and function.

As Chronic Lymphocytic Leukemia (CLL) is associated with several defects in the T cell compartment, the impact of tumour burden on the autologous immune system was studied. Gene expression profiles (using Applied Biosystem Human Genome Microarray) identified 237 genes with significantly increased expression and 221 genes with significantly decreased expression (p<0.05) in CD3+ cells from CLL patients compared with healthy donors. Panther software analysis identified 34/237 upregulated genes and 26/221 downregulated genes that were involved in specific pathways, mainly cell differentiation and proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T cell activation. The 26 dowregulated genes included Zap70, a member of the syk family protein tyrosine kinase, which is involved in T-cell activation. Zap-70 results were validated by mRNA quantification by RT-PCR (−1.77 fold in comparison with healthy controls) and by flow-cytometric analysis (Mean Intensity Fluorescence=33±12 vs 80±23.62 in controls, p<0.05). To test the hypothesis that activation with OKT3 /IL-2 could bypass these T cell deficiencies, activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity (using the 51chromium release assay) against mutated and unmutated (according to IgVH mutational status) autologous B cells, DAUDI, K562 and P815 cell lines. After 10 days’ culture, the T cell count remained unchanged; CD8 cells expanded more than CD4; TCR spectratyping analysis indicated no differences in TCR repertoires. Activation restored the ZAP-70 mRNA (+1.67 fold). The 51chromium release cytotoxicity assay showed an index > 30% in 5/20 patients. The other 15 were partially cytotoxic against P815, K562 and Daudi. Cell line analysis in all 20 confirmed prevalently T cell-mediated cytotoxicity and poor NK/LAK activity. Cytotoxicity did not correlate with B cell mutational status. We tested the cytotoxic activity of autologous activated T cells in NOD/SCID mice co-transplanted with leukaemic B cells. Only activated T cells exerting cytotoxicity vs autologous B-cell CLL prevent CLL in human-mouse chimera, as confirmed by PCR and FACS analysis which visualised only CD3+ cells. In conclusion, in patients with CLL, activating autologous T cells with OKT3 /IL-2 bypasses, at least in part, the T cell immunological deficiencies. These in vitro and in vivo findings might serve to throw light on new mechanisms that could be exploited in immunotherapy designed to exert disease control.

Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Since mesenchymal cells (MSCs) exert an immune regulatory function and suppress T cell proliferation, this in vitro study investigated their role in Treg recruitment and function. hMSCs and different T cell populations (CD3+, CD3+/CD45RA+, CD3+/CD45RO+, CD4+/CD25+, CD4+/CD25+/CD45RO+, CD4+/CD25+/CD45RA+) from healthy donors were co-cultured for up to 15 days. Harvested lymphocytes were analysed by flow-cytometry; FoxP3 and CD127 expression were measured by real time PCR; regulatory activity was assessed. Within CD3+ fraction, percentages of CD4+/CD25bright and CD4+/CD25bright/Foxp3+ cells were higher in the presence of hMSCs (42% vs 34% for the CD4+/CD25bright and 20.5 vs 3.5 for the CD4+/CD25bright/Foxp3+). Within CD3+/CD45RA+ and CD3+/CD45RO+ fractions, the T reg starting fraction of the naïve population rose from 0.05% ± 0.01 CD4/CD25 positive cells to 0.2% ± 0.14 and the T reg starting fraction of the memory population rose from 0.3% ± 0.05 CD4/CD25 positive cells to 1.5% ± 0.9. Within CD4+/CD25+, CD4+/CD25+/CD45RA+, CD4+/CD25+/CD45RO+, FoxP3 expression did not change in CD4+/CD25+ cells. It increased 5.38±2.3 fold in CD4+/CD25+/CD45RA+ and 7.98±1.9 fold in CD4+/CD25+/CD45RO+ cells. CD127 expression decreased by -582±29.2 fold in CD4+/CD25+ cells, by -216±17.5 fold in CD4+/CD25+/CD45RA+ and by -71.95±12.6 fold in CD4+/CD25+/CD45RO+ cells. Cytofluorimetric analysis showed CD4+/CD25+/FoxP3+ was up-regulated to 14%±4 and CD127 down-regulated to 4.4%±1.5 vs respectively 0.2%±0.28 and 21.9%±3.5 in controls without MSCs (CD4+/CD25+ alone). FoxP3 up-regulation was more marked in CD4+/CD25bright cells (51%±10 vs 0.1%±0.7 in controls). Sorted Tregs exert potent immunosuppressive activity on CD4+/CD25- cell populations. Inhibitory activity was lost within 15 days when sorted T regs were cultured without hMSC. On day +15 proliferation using CD4+/CD25+ cells as suppressor cells was 1.7±0.8 vs 45.2±12 in controls; proliferation using CD4/CD25+/CD45RA+ as suppressor cells was 5.4±3.1, vs 21.3±11.2 in controls, and proliferation with CD4+/CD25+/RO+ as suppressor cells was 2.3±2 vs 33.5±8.7 in controls. We demonstrate MSC recruit Tregs from a fraction of CD3+ and from immunoselected CD3+/CD45RA+ and CD3+/CD45RO+ fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4+/CD25bright/FoxP3 subset and CD127 expression was down regulated. When purified Treg populations (CD4/CD25+, CD4/CD25+/CD45RA+ and CD4/CD25+/CD45RO+) were used as fraction for hMSC co-cultures, they maintain FoxP3 expression and CD127 expression is down-regulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. In conclusion, our study demonstrates that MSCs recruit, regulate and maintain T regulatory phenotype and function over time.

Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Since mesenchymal cells (MSCs) exert an immune regulatory function and suppress T cell proliferation, this in vitro study investigated their role in Treg recruitment and function. hMSCs and different T cell populations (CD3+, CD3+/CD45RA+, CD3+/CD45RO+, CD4+/CD25+, CD4+/CD25+/CD45RO+, CD4+/CD25+/CD45RA+) from healthy donors were co-cultured for up to 15 days. Harvested lymphocytes were analysed by flow-cytometry; FoxP3 and CD127 expression were measured by real time PCR; regulatory activity was assessed. Within CD3+ fraction, percentages of CD4+/CD25brightand CD4+/CD25bright/Foxp3+ cells were higher in the presence of hMSCs (42% vs 34% for the CD4+/CD25brightand 20.5 vs 3.5 for the CD4+/CD25bright/Foxp3+). Within CD3+/CD45RA+ and CD3+/CD45RO+ fractions, the T reg starting fraction of the naïve population rose from 0.05% ± 0.01 CD4/CD25 positive cells to 0.2% ± 0.14 and the T reg starting fraction of the memory population rose from 0.3% ± 0.05 CD4/CD25 positive cells to 1.5% ± 0.9. Within CD4+/CD25+, CD4+/CD25+/CD45RA+, CD4+/CD25+/CD45RO+, FoxP3 expression did not change in CD4+/CD25+ cells. It increased 5.38±2.3 fold in CD4+/CD25+/CD45RA+ and 7.98±1.9 fold in CD4+/CD25+/CD45RO+ cells. CD127 expression decreased by -582±29.2 fold in CD4+/CD25+ cells, by -216±17.5 fold in CD4+/CD25+/CD45RA+ and by -71.95±12.6 fold in CD4+/CD25+/CD45RO+ cells. Cytofluorimetric analysis showed CD4+/CD25+/FoxP3+ was up-regulated to 14%±4 and CD127 down-regulated to 4.4%±1.5 vs respectively 0.2%±0.28 and 21.9%±3.5 in controls without MSCs (CD4+/CD25+ alone). FoxP3 up-regulation was more marked in CD4+/CD25brightcells (51%±10 vs 0.1%±0.7 in controls). Sorted Tregs exert potent immunosuppressive activity on CD4+/CD25- cell populations. Inhibitory activity was lost within 15 days when sorted T regs were cultured without hMSC. On day +15 proliferation using CD4+/CD25+ cells as suppressor cells was 1.7±0.8 vs 45.2±12 in controls; proliferation using CD4/CD25+/CD45RA+ as suppressor cells was 5.4±3.1, vs 21.3±11.2 in controls, and proliferation with CD4+/CD25+/RO+ as suppressor cells was 2.3±2 vs 33.5±8.7 in controls. We demonstrate MSC recruit Tregs from a fraction of CD3+ and from immunoselected CD3+/CD45RA+ and CD3+/CD45RO+ fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4+/CD25bright/FoxP3 subset and CD127 expression was down regulated. When purified Treg populations (CD4/CD25+, CD4/CD25+/CD45RA+ and CD4/CD25+/CD45RO+) were used as fraction for hMSC co-cultures, they maintain FoxP3 expression and CD127 expression is down-regulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. In conclusion, our study demonstrates that MSCs recruit, regulate and maintain T regulatory phenotype and function over time.