Aldehyde dehydrogenases inhibition eradicates leukemia stem cells while sparing normal progenitors.

The vast majority of patients with acute myeloid leukemia (AML) achieve complete remission (CR) after standard induction chemotherapy. However, the majority subsequently relapse and die of the disease. A leukemia stem cell (LSC) paradigm has been invoked to explain this failure of CR to reliably translate into cure. Indeed, LSCs are highly enriched in CD34+CD38- leukemic cells that exhibit positive aldehyde dehydrogenase activity (ALDH+) on flow cytometry, these LSCs are resistant to currently existing treatments in AML such as cytarabine and anthracycline that, at the cost of great toxicity on normal cells, are highly active against the leukemic bulk, but spare the LSCs responsible for relapse. To try to combat the LSC population selectively, a well-characterized ALDH inhibitor by the trivial name of dimethyl ampal thiolester (DIMATE) was assessed on sorted CD34+CD38- subpopulations from AML patients and healthy patients. ALDH activity and cell viability were monitored by flow cytometry. From enzyme kinetic studies DIMATE is an active enzyme-dependent, competitive, irreversible inhibitor of ALDH1. On cells in culture, DIMATE is a powerful inhibitor of ALDHs 1 and 3, has a major cytotoxic activity on human AML cell lines. Moreover, DIMATE is highly active against leukemic populations enriched in LSCs, but, unlike conventional chemotherapy, DIMATE is not toxic for healthy hematopoietic stem cells which retained, after treatment, their self-renewing and multi-lineage differentiation capacity in immunodeficient mice, xenografted with human leukemic cells. DIMATE eradicates specifically human AML cells and spares healthy mouse hematologic cells.

Natural killer cells and malignant haemopathies: a model for the interaction of cancer with innate immunity.

Despite recent progress in the therapeutic approach of malignant haemopathies, their prognoses remain frequently poor. Immunotherapy offers an alternative of great interest in this context but defect or abnormal expression of human leukocyte antigens (HLA), frequently observed in cancer cells, limits its efficiency. Natural killer (NK) cells, which are able to kill target cells in a HLA-independent way, represent a novel tool in the treatment of haematological malignancies. Abnormal NK cytolytic function is observed in all the haematological malignancies studied, such as acute leukaemia, myelodysplastic syndromes or chronic myeloid/lymphoid leukaemia. Several mechanisms are involved in the alterations of NK cytotoxicity: decreased expression of activating receptors, increased expression of inhibitory receptors or defective expression of NK ligands on target cells. Further studies are needed to identify how each type of haematological malignancy escapes from the innate immune response. Attempts to increase the expression of activating receptors, to counteract inhibitory receptors expression, or to increase NK cell cytotoxic capacities could overcome tumour escape from innate immunity. These therapies are based on monoclonal antibodies or culture of NK cells in presence of cytokines or dendritic cells. Moreover, many novel drugs used in haematological malignancies [tyrosine kinase inhibitors, IMIDs(®), proteasome inhibitors, demethylating agents, histone deacetylase inhibitors (HDACis), histamine dihydrochloride] display interesting immunomodulatory properties that affect NK cells. These data suggest that combined modalities associating cytotoxic drugs with innate immunity modulators may represent a major breakthrough in tumour eradication.

Stimulation of non-Hodgkin's lymphoma via HVEM: an alternate and safe way to increase Fas-induced apoptosis and improve tumor immunogenicity.

Stimulation by CD40 ligand (L) improves B-cell malignancy immunogenicity, and also induces proliferative signals. To avoid these tumorigenic effects, we studied an alternate way of tumor-cell stimulation by homologous to lymphotoxin, inducible expression, competing for GpD of herpesvirus, which binds to the herpesvirus entry mediator (HVEM), and is expressed on T-lymphocytes (LIGHT), the ligand for HVEM, a new member of the tumor necrosis factor (TNF)/TNF-receptor (-R) family. HVEM is constitutively expressed on the surface of tumor B cells. We focused our attention on mantle cell lymphoma, a subtype of B-cell malignancy of poor prognosis. Triggering by LIGHT, in contrast to CD40L stimulation, did not increase lymphoma proliferation nor decrease chemotherapy entrance. We observed an upregulation of the TNFR apoptosis-inducing ligand Fas, and in contrast to CD40L-induced protection, an enhancement of lymphoma sensitivity to Fas-induced apoptosis. LIGHT triggering increased lymphoma cell recognition in a mixed lymphocyte response. In conclusion, LIGHT-mediated triggering renders B-cell lymphomas more immunogenic and sensitive to apoptosis, without inducing proliferation. Since LIGHT triggering also enhances the functions of T-lymphocytes and dendritic cells, it could be a unique way to restore an efficient cancer control by its pleiotropic effects on immune effectors and tumor cells.

A novel mechanism of antitumor response involving the expansion of CD3+/CD56+ large granular lymphocytes triggered by a tumor-expressed activating ligand.

We describe a patient with acute myeloid leukemia (AML) who developed polyclonal large granular lymphocyte (LGL) proliferation. The reciprocal evolution of AML and LGLs suggested that these LGLs had an anti-tumor activity. The patient's LGLs killed autologous leukemia cells in a different way to classical T lymphocyte-mediated cytotoxicity since it did not rely on the recognition of target antigens presented by major histocompatibility complex (MHC) class I molecules by the CD3/TcRalphabeta complex. This killing was also different from natural killer (NK)-mediated cytotoxicity, which depends on the absence of MHC class I molecule recognition by NK inhibitory receptors. The LGLs were polyclonal, had a CD3+/CD8+/CD56+ phenotype, and did not express the natural killer cell receptors (NKRs) for MHC class I molecules. The LGLs did not express the NK-specific activating natural cytotoxicity receptors but expressed the 2B4 non-MHC restricted triggering receptor, whose ligand CD48 was expressed by leukemic cells and normal bone marrow cells. The 2B4 receptor participated in the ability of LGLs to lyse patient's leukemia. This represents a novel function for 2B4 in man, since this molecule, at variance with the murine system, was considered not to have direct effects on CD8+ T cell-mediated cytotoxicity. This case report allowed us to describe a novel T lymphocyte-mediated anti-tumor mechanism which relied on (1) the abnormal expansion of the rare 2B4-positive CD3+/CD8+/CD56+ T lymphocyte subset, (2) an as yet undescribed cytotoxicity mechanism in man which depended on 2B4 molecule. The relevance of this observation in human cancer immunotherapy has to be further investigated.

The TNF superfamily members LIGHT and CD154 (CD40 ligand) costimulate induction of dendritic cell maturation and elicit specific CTL activity.

LIGHT is a recently identified member of the TNF superfamily that is up-regulated upon activation of T cells. Herpesvirus entry mediator, one of its receptors, is constitutively expressed on immature dendritic cells (DCs). In this report, we demonstrate that LIGHT induces partial DC maturation as demonstrated by Ag presentation and up-regulation of adhesion and costimulatory molecules. LIGHT-stimulated DCs show reduced macropinocytosis and enhanced allogeneic stimulatory capacity but fail to produce significant amounts of IL-12, IL-6, IL-1beta, or TNF-alpha compared with unstimulated DCs. However, LIGHT cooperates with CD154 (CD40 ligand) in DC maturation, with particular potentiation of allogeneic T cell proliferation and cytokine secretion of IL-12, IL-6, and TNF-alpha. Moreover, LIGHT costimulation allows DCs to prime in vitro-enhanced specific CTL responses. Our results suggest that LIGHT plays an important role in DC-mediated immune responses by regulating CD154 signals and represents a potential tool for DC-based cancer immunotherapy.

Reciprocal expression of the TNF family receptor herpes virus entry mediator and its ligand LIGHT on activated T cells: LIGHT down-regulates its own receptor.

The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8(+) than CD4(+) T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.

Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities.

The destruction of cells capable of initiating and maintaining leukemia challenges the treatment of human acute myeloid leukemia. Recently, CD34+/CD38- leukemia progenitors have been defined as new leukemia-initiating cells less mature than colony-forming cells. Here we show that CD34+/CD38- leukemia precursors have reduced in vitro sensitivity to daunorubicin, a major drug used in leukemia treatment, in comparison with the CD34+/CD38+ counterpart, and increased expression of multidrug resistance genes (mrp/lrp). These precursors show lower expression of Fas/Fas-L and Fas-induced apoptosis than CD34+/CD38+ blasts. Moreover, the CD34+/CD38- leukemic subpopulation induces a weaker mixed leukocyte reaction of responding T-lymphocytes than the CD34+/CD38+ leukemic counterpart, either in a MHC-unmatched or MHC-matched settings. This weaker immunogenicity could be linked to lower expression on CD34+/CD38- leukemia precursors of major immune response molecules (MHC-DR, LFA-3, B7-1, or B7-2) than CD34+/CD38+ leukemic cells. Nonetheless, the susceptibility of the immature CD38- precursors to cytotoxicity was not different from the sensitivity of the CD38+ counterpart. Finally, CD34+/CD38- leukemia precursors, in contrast with CD38+ precursors, failed, under appropriate conditions, to differentiate into dendritic cells, a central step for antigen recognition. This is to our knowledge the first demonstration that the very immature phenotype of CD34+/CD38- leukemic progenitors confers both chemotherapy resistance and decreased capacities to induce an immune response. Because the susceptibility of the immature leukemia cells as cytotoxic targets is maintained, our data underline the importance of improving the initial steps of leukemia recognition, more particularly by defining optimal conditions of dendritic cell transformation of the very immature hematopoietic precursors.

Acute myeloid leukaemia triggering via CD40 induces leukocyte chemoattraction and cytotoxicity against allogenic or autologous leukemic targets.

The CD40 antigen is a member of the tumor necrosis factor receptor superfamily which interacts with its ligand and regulates the immune response via a dialogue between T-lymphocytes and antigen-presenting or tumor cells. Tumor triggering via CD40 exerts direct effects on cancer cells, which have mainly been investigated in terminally differentiated hematological malignancies such as low-grade lymphoma. We focused our attention on minimally differentiated acute myeloid leukemia (AML-M0), an aggressive hematological malignancy in which severe prognosis suggests the requirement for innovative therapeutic strategies. Here we demonstrate, for the first time to our knowledge, a CD40-triggered IL-8, RANTES and IL-12 secretion by leukemic cells. Supernatants from CD40-stimulated leukemia cells had chemoattractant effects on T-lymphocytes, natural killer cells and monocytes. Moreover, these supernatants, when complemented with low-dose IL-2, induced significant lymphokine-activated and natural killer cytotoxicity, leading to leukemia lysis both in allogenic HLA-matched and autologous settings. Stimulation of leukemia cells via CD40 could participate significantly to the anti-leukemia immune response by contributing to the development of an inflammatory response and to in situ cytotoxicity. Leukemia(2000) 14, 123-128.

[Mechanisms of tumor escape from immunologic response]. / Mécanismes d'échappement tumoral à la réponse immunitaire.

INTRODUCTION: The rationale for immune control of cancer is now better defined via the immunovirology of transforming viruses, definition of human tumor antigens recognized by T-lymphocytes, and cellular and humoral components of the anticancer response. Nonetheless tumors can escape from immune surveillance. To better define immunomodulation strategies, we describe some of the various strategies developed by transformed cells to evade the immune response. CURRENT KNOWLEDGE AND KEY POINTS: Both the lack of specific tumor antigen and down-regulation of major histocompatibility complex (MHC) molecule expression hamper recognition of neoplastic cells by T-lymphocytes. In presence of defective expression of ligands for the T-cell co-stimulatory receptors, tumor recognition may lead to the development of tolerance instead of specific cytotoxic activity. Tumor cell counter-attack against effector T-cells has also been described, using either inhibitory cytokines (IL-10), apoptosis induction (via Fas signalling), functional inactivation (disruption of normal CD40/CD40 ligand interactions), or induction of anergy. FUTURE PROSPECTS AND PROJECTS: Despite the many different mechanisms of tumor escape, the immune system has developed efficient counter-attacks. For instance, natural killer cells may detect and destroy tumor cells that lack class 1 MHC molecules and thus escape from specific T-lymphocyte cytolysis. Moreover, immunogenicity can be restored, at least in vitro, by different means such as tumor cell stimulation by cytokines or CD40, suggesting that therapeutic strategies will soon be developed in order to stimulate an efficient antitumoral immune response.

Tumor escape from immune surveillance.

The bases for an efficient anti-tumor immune response begin to be better defined. Nonetheless, neoplastic cells develop various strategies to escape immune surveillance, which are discussed here in order to better design the therapeutic possibilities of immune manipulation. The absence of specific tumor antigen as well as the weak expression of major histocompatibility complex (MHC) molecules hinder the recognition of the neoplastic cells by T lymphocytes. The defect of expression by the tumor of the ligands for the T cell activation costimulatory molecules is particularly harmful for the immune response since it induces tolerance. Finally, tumor cells can inactivate effector T lymphocytes through the secretion of inhibitory cytokines, induction of apoptosis or functional inactivation. The multiplicity of the means to oppose an effective anti-tumor response challenges the adaptative mechanisms of the immune system. For example, the natural killer cells target tumor cells not expressing MHC class I molecules. Numerous possibilities of tumor immunogenicity restoration have been demonstrated at least in vitro, such as stimulation of the cancerous cells by CD40 or cytokine treatment, which could lead to several promising therapeutical approaches.

Differential modulation of immune recognition molecules by interleukin-7 in human acute leukaemias.

Clinical animal models and in vitro data afford evidence for anti-leukaemia immunity. Many reports have underlined the interest of interleukin-7 (IL-7) use in cancer and its pivotal role in immune recognition. This cytokine, initially identified as a B cell growth factor, enhances the anti-tumour properties of immune effector cells via T lymphocyte activation, increased specific cytotoxicity and cytokine secretion. Nonetheless, few data are available regarding the effect of IL-7 on the expression at the leukaemia cell surface of molecules involved in the immune response, which defective expression could induce tolerance or anergy. This prompted us to study the effects of IL-7 on 20 cases of acute myeloid leukaemia (AML) and 9 cases of lymphoid leukaemia (ALL), in comparison with gamma-interferon, a potent inducer of immune regulation molecule expression. In AML and ALL, IL-7 increased MHC class I molecule expression, while class II molecules were weakly modified. The expression of the tumour necrosis factor family members CD40 and Fas/CD95, together with the adhesion molecules ICAM-1/CD54 and CD58/LFA-3, was also increased in both types of leukaemia. The IL-7 was an efficient inducer of B7-2/CD86 expression in AML and ALL, while increased expression of B7-1/CD80 was only observed in AML. In the corresponding, co-cultured T lymphocyte population, IL-7 more particularly increased B7-1/CD80 and CD58/LFA-3 expression. Finally, pre-incubation of leukaemic cells with IL-7 increased the proliferation of responding, normal allogenic T lymphocytes and their secretion of gamma-IFN and IL-2 in mixed the lymphocyte-tumour reaction. We concluded that IL-7 is efficient at increasing the membrane expression of molecules which are central for the development of the immune response, and at improving allogenic immune recognition. The clinical implications of such data require further in vivo investigation.

Regulation of CD80/B7-1 and CD86/B7-2 molecule expression in human primary acute myeloid leukemia and their role in allogenic immune recognition.

Clinical data and animal models afford evidence for anti-leukemia immunity in humans, but the interactions critical for blast cell recognition are unresolved. Expression of B7 molecules by antigen-presenting cells (APC) provides co-stimulatory signals to T lymphocytes via CD28 and CTLA-4 which prevent the induction of alloantigen-specific tolerance. Conversely, expression of CD40 ligand by stimulated T cells activates APC via CD40. In human hematological B cell malignancies (follicular lymphoma and chronic lymphocytic leukemia), the defect in alloantigen presentation of tumoral cells can be repaired by up-regulation of B7 and other co-stimulatory molecules via CD40. We studied the role of B7 molecules in alloimmune recognition and the various ways to improve the antitumoral response on peripheral blood leukemic cells from 20 patients with a diagnosis of primary acute myeloid leukemia (AML). We focused on myelo/monocytic M4/M5 French-American-British classification subtypes which are considered as the neoplastic counterpart of normal monocytes, a prototypic APC. In one-way mixed lymphocyte reaction of CD4+ T cells against leukemic cells, differences in B7-1, B7-2 or CD40 expression by AML cells did not induce specific cytokine secretion; interleukin (IL)-2 and interferon (IFN)-gamma were detected but not IL-4, corresponding to a Th1 pattern. Blockade experiments showed that proliferation and IFN-gamma secretion only partially depended on B7 molecules, which in contrast had a pivotal role in IL-2 synthesis. In contrast with murine models which suggest a pivotal role for CD80/B7-1 in the immune response against AML, our data support a greater role for CD86/B7-2, in line with the baseline expression of CD86/B7-2 and lack of CD80/B7-1 on most M4/M5 AML cells. AML cell stimulation via CD40: (1) significantly improved IL-2 secretion but not proliferation of responding T lymphocytes, (2) increased CD54/ICAM-1 expression in three quarters of cases, (3) failed in most cases to induce CD40-specific CD80/B7-1 up-regulation, and (4) had a weak effect on CD86/B7-2 expression. These data contrast with the very efficient up-regulation of both B7 co-stimulatory molecule expression and tumoral cell alloimmune recognition following CD40 stimulation in B cell malignancy models. The role of the defective B7 molecule up-regulation by the CD40 pathway in inefficient tumor immunogenicity of primary AML cells has to be further investigated, in particular using transfection experiments of CD80/B7-1-deficient AML cell lines. From our in vitro data we conclude that B7 molecules play an important role in the alloimmune surveillance of AML as suggested by the high B7 molecule dependency of IL-2 secretion. Nonetheless, the contribution of B7 molecules to alloimmune T cell proliferation against primary AML cells in human and the way to improve it--regulation via CD40 in particular--differ from B cell malignancies and murine models, suggesting the requirement for specific strategies in the development of antitumor immunity.

Value of PCR analysis for long term survivors after allogeneic bone marrow transplant for chronic myelogenous leukemia: a comparative study.

At present, allogeneic bone marrow transplantation (BMT) seems to be the only curative treatment for patients with chronic myeloid leukemia (CML). The mechanisms of this therapeutic approach probably include anti-leukemic immune response or "graft-versus-leukemia" (GVL) reaction against leukemic cell clones, since even aggressive chemotherapy is not sufficient per se to cure this type of disease. In many patients, allogeneic BMT results in the disappearance of the Ph chromosome; this negativity does not exclude the presence of residual leukemic cells since sensitivity of cytogenetic analysis is low. The chimeric BCR-ABL gene in CML is a tumor-specific marker that is well suited to allowing detection of low numbers of residual leukemic cells through the extremely sensitive detection method of polymerase chain reaction (PCR). Using this method, 1 leukemic cell in 10(5) or 10(6) normal cells can be detected. Many studies have evaluated the clinical significance and predictive value of BCR-ABL gene rearrangement detected by PCR assay after BMT, most often on heterogeneous populations. Results from such studies have been conflicting. We have conducted a review of the literature, focussing on long-term survivors (> 3 years from BMT) of CML to determine the value of PCR in such patients (n = 183). With the consideration that such a population is obviously heterogenous, long-term PCR negativity strongly correlated with achieving a cure since no patient relapsed in this population (n = 117). Among the PCR positive patients (n = 66), only 5 (8%) relapsed, suggesting a poor prognostic value for a PCR+ test > 36 months post-transplant. Besides the potential clinical value of these data, they contribute to the understanding of the biology of the disease and may suggest a crucial role for the long term GVL effect of marrow transplantation.

Minor breakpoint cluster region (m-BCR) positive chronic myeloid leukaemia with an acute lymphoblastic leukaemia onset: a case report.

m-BCR chronic myeloid leukaemia (CML) is a rare entity. We report a patient presenting with Philadelphia (Ph)-positive, m-BCR-positive acute lymphoblastic leukaemia (ALL) who achieved complete remission after induction chemotherapy, but showed a majority of Ph-positive mitoses during this remission. A diagnosis of m-BCR CML was established and the patient was given interferon alpha therapy. This is the first m-BCR CML presenting as ab initio ALL. This report emphasizes the importance of karyotyping Ph-positive ALL during remission so as not to misdiagnose CML patients who can benefit from Interferon therapy.