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Stimuler le système immunitaire est un enjeu majeur dans le traitement des leucémies aigües. Nous avons centré notre étude sur la leucémie aigüe myéloïde (LAM) et la leucémie à cellules dendritiques plasmacytoïdes (LAPDC). Dans une première partie, nous avons étudié les médicaments immunomodulateurs (IMiDs) utilisés dans le traitement du myélome multiple et des syndromes myélodysplasiques à délétion 5q. Les IMiDs présentent des propriétés anti-angiogéniques, anti-prolifératives, pro-apoptotiques, et immunomodulatrices en particulier sur les cellules NK (Natural Killer). Nous avons évalué les effets anti-leucémiques des IMiDs (lenalidomide et pomalidomide) dans le but d’améliorer l’activité cytotoxique des NK dans la LAM. Nous avons mis en évidence une altération de la survie des blastes de LAM par les IMiDs in vitro, et dans un modèle in vivo de greffe dans les souris immunodéficientes NOD/SCID/IL2rg-/- (NSG). Nous avons également montré une sensibilisation par les IMiDs des blastes de LAM à la lyse par les NK allogéniques, indépendamment de la cible moléculaire connue, le cereblon. Le traitement des blastes de LAM par IMiDs stimule les fonctions NK. Enfin, nous avons décrit des modifications phénotypiques induites par les IMiDs sur les récepteurs NK, et une diminution d’expression de HLA-classe I sur les cellules de LAM. Ces résultats encouragent la poursuite du développement des IMiDs dans la LAM, en particulier les associations stimulant les fonctions NK. Dans une seconde partie, nous avons développé un modèle murin de LAPDC dans la souris NSG. Cet outil préclinique est indispensable dans l’élaboration de stratégies d’immunothérapie dans les leucémies aigües. / Boosting the Immune System is a major challenge in the treatment of acute leukemias. We focused our study on acute myeloid leukemia (AML) and plasmacytoid dendritic cell leukemia (BPDCN). In the first part, we studied immunomodulatory drugs (IMiDs) that are currently used in the treatment of patients with myeloma and myelodysplastic syndrome with 5q deletion. IMiDs exhibit anti-angiogenesis, anti-proliferative, pro-apoptotic, and immunomodulatory properties especially on NK cells and T lymphocytes. We investigated the anti-leukemic effects of two IMiDs (lenalidomide and pomalidomide) in order to improve NK cell cytotoxic activity in AML. We have shown that IMiDs impaired survival of AML blasts in vitro, and in vivo in NOD/SCID/IL2rg-/- (NSG) murine model. In addition, IMiDs treatment sensitized AML blasts to allogeneic NK cell mediated lysis, independently of Cereblon, the known molecular target of IMiDs. IMiDs treatment of AML blasts enhanced NK cell functions such as degranulation and cytokine production. Finally, we have described phenotypic changes induced by IMiDs on NK receptors, and a down-regulation of HLA-class I on AML blasts. These results encourage continuing investigation for the use of IMiDs in AML, especially in combination with immunotherapies based on NK cells. In a second part, we have developed a murine model of plasmacytoid dendritic cell leukemia (BPDCN) in NSG mice. Murine model of leukemia are essential preclinical tools in the development of new immunotherapies in acute leukemias.

T-cells are lymphocytes that make up part of the adaptive arm of the immune system, and are essential for efficient protection from and eradication of viruses and pathogens. T-cells not only play an important role in protection from external agents, but also regulate and prevent activation towards self-peptides and detect and remove erratically growing cells. Alterations in T-cell activation and suppression contribute to auto-immunity, immunocompromised disorders, and cancer progression. The immune system, and T-cells in particular, provides daily surveillance, recognition and destruction of aberrant cells. Although the immune system is proficient at suppressing malignant progression, tumor cells acquire various methods of immune evasion. Myelodysplastic Syndrome (MDS) is a pre-malignant dysplastic disorder of the bone marrow characterized by ineffective hematopoiesis and clonality in the myeloid lineage, where lack of immune response has been implicated in the propensity for progression to acute myeloid leukemia (AML). Leukemia progression is associated with the acquisition of complex genetic abnormalities. Alterations in immune system regulation have been implicated in various stages of the disease process, although the role of the immune system in response to several therapies in MDS has not been fully discovered. Lenalidomide is a small molecule therapeutic preferentially effective in MDS patients with an interstitial chromosome 5q deletion (del(5q)). Improved erythropoiesis has also been reported to occur in 20-30% of low-risk, non-del(5q) patients. Although lenalidomide is a potent immunomodulatory drug that potentiates T-cell and NK-cell responses, the T-cell compartment in MDS is highly deregulated by aberrant repertoire skewing, decreased function and abnormal naïve and memory cell homeostasis. The presence of lymphoid infiltrates in the bone marrow of lenalidomide-responsive patients suggests that T-cells may participate in the hematopoietic response, but it is unclear if lenalidomide is capable of reversing these functional T-cell defects. We therefore assessed immunological changes in low-risk MDS patients before and after 16-weeks of lenalidomide therapy, and assessed the relationship to erythroid response. Although MDS T-cells were anergic prior to treatment, we have shown that T-cells in responders have a significant increase in antigen-induced proliferative response and T helper type-1 (Th1) cytokine production (IL-2, IFN-γ, TNF-α) compared to non-responders. The change in function positively correlated with an increase in naïve T-cells and a decrease in memory cells, indicating that lenalidomide has immunomodulatory activity to reverse anergy in MDS. Although it is known that lenalidomide may increase T-cell activation and proliferation in the absence of co-stimulatory signals, a direct mechanism of action has yet to be elucidated. Since CD28 is one of the most important co-stimulatory molecules deregulated in cancer, we therefore set out to determine if the expression of CD28 was essential for lenalidomide's mechanism in T-cells. We knocked out CD28 expression in healthy donor T-cells, and sorted on inherently deficient, CD28null, T-cells that accumulate in older healthy donors and found that lenalidomide-induced proliferation and function were completely ablated within the CD28null subset. These data indicate the immunotyrosine-based activation motifs (ITAMs) on the intracellular domain of the CD28 receptor are necessary for lenalidomide action. Interestingly, during the natural aging process, repeated exposure to antigens results in the accumulation of CD28null T-cells that are phenotypically distinct and functionally deficient due to excessive proliferative history in vivo. We therefore examined whether CD28 expression on MDS patient T-cells affected responses to lenalidomide, and if this could be used as a predictive biomarker of responsiveness. We found that patients who fail lenalidomide therapy had higher CD8+ Terminal Effector Memory (TEM), which are inherently CD28null, and that non-responders had an overall increase in CD4+ and CD8+CD28null T-cells, as well as an increase in CD28null cells within the TEM compartment compared to hematologic responders. We then sought to determine the particular protein target of lenalidomide responsible for increased CD28 receptor signaling in T-cells. Several targets in a variety of cell types have been postulated, although the direct mechanism in T-cells is unclear. Our group has previously shown that lenalidomide inhibits the activity of two haplodeficient phosphatases located within the commonly deleted region (CDR) on chromosome 5q in the MDS myeloid clone, Protein Phosphatase 2A (PP2A) and Cdc25c. PP2Ac is known to bind CD28 and is hypothesized to inhibit T-cell co-stimulation. Therefore, it is plausible that lenalidomide and other IMiDs inhibit the phosphatase activity of PP2A which leads to increased activation of T-cell proximal signals dependent on CD28 expression. We examined this hypothesis using molecular modeling and virtual screening and found that all of the IMiDs (lenalidomide, pomalidomide, and thalidomide) can theoretically interact with the catalytic pocket of the PP2A heterotrimer, potentially inhibiting PP2Ac activity. In vitro phosphatase activity assays supported these findings as lenalidomide-inhibition of PP2Ac activity was seen in both ad293 and Jurkat cell lines, and in primary T-cells. Mutations of theorized lenalidomide hydrogen-bond sites within the catalytic pocket of PP2A rendered the enzyme catalytically dead, indicating that these are important residues for enzymatic activity, but unfortunately could not be used to determine if lenalidomide activity was disrupted by mutation of those sites. These data together suggest that the ability of lenalidomide to augment immune activation in vivo in MDS patients, and potentially other diseases, is extremely important to patient response. Also, that CD28 expression on T-cells is essential for lenalidomide immune-mediated tumor eradication through CD28 downstream signaling, and potentially through inhibition of PP2A. These results are useful in designing future lenalidomide-combination therapy trials in other hematologic and solid malignancies, and could be used to help stratify patients for future therapeutic decisions in MDS and other malignancies.

Natural Killer cells (NK) are critical components of the innate immune system. Often referred to by their morphology, these large granular lymphocytes (LGLs) are bone marrow-derived lymphocytes and can be found throughout the body. NK cells reside in the liver, lymph nodes, spleen, thymus, and mucosal-associated lymphoid tissues (MALT). Importantly, NK cells also circulate throughout the blood where they function as surveyors of the body and are armed to eliminate malignant, infected, damaged, or foreign cells. NK cells function by a dual receptor system. That is, NK receptors are broadly categorized as inhibitory or activating. It is a fine balance, or lack thereof, that dictates the function of an NK cell. Unlike their T and B cell adaptive counterparts, NK cell receptors (NKR) are germline encoded and do not undergo gene rearrangement. NKRs are expressed in a variegated but overlapping fashion such that different cell subsets in the NK compartment elaborate different combinations of activating and inhibitory NKR. Varying the array of NKRs used by each subset increases the potential specificities of the NK compartment, while retaining tolerance to self. Thus, a diverse and balanced NK cell receptor repertoire (NKRR) is extremely important in order for this lineage to respond to various immunologic challenges and to do so in a normal, effective manner. As we have previously shown, aberrations in the expression of NKRs or downstream signaling can lead to severe immune deficiency, as observed in SHIP-deficient mice. We also showed that in the absence of SHIP-1, 2B4 becomes highly upregulated, functioning as a dominant inhibitory receptor and rendering the SHIP-1-deficient NK cell unresponsive to complex tumor targets. Traditionally MHC-I inhibitory ligands are largely responsible for the regulation of NK function. However, we show here that 2B4, which mediates MHC-I-independent inhibition, is required for formation of a normal NKRR, NK homeostasis, and effector functions. Moreover, in the absence of 2B4 and SHIP-1, NK cells have improper licensing, or education. In addition to SHIP-1 and 2B4 we show that the nature of the MHC-I ligands also play a significant role in repertoire formation, NK effector functions, and NK cell education. As described above, NK cells are critical components of the immune system. Understanding how NK cell biology and function are regulated, or affected in the context of pathology is of high significance. NK function is often severely impaired in a diseased state, and more importantly, NK cells are frequently adversely affected by the treatments themselves. Here we sought out to determine the effects of an immunomodulating drug, lenalidomide, on the biology and function of healthy NK cells. Lenalidomide is a unique drug that displays immune enhancing functions yet can be cytotoxic to tumor cells. However, lenalidomide treatment can result in immune suppression and severe cytopenia, and has the ability to impair NK viability. We show here that if used in combination with cytokine treatment (e.g. IL-2 or IL-15), many of these negative affects can be overcome. Furthermore, we show that lenalidomide treatment results in what appears to be an NK activating phenotype with a down-modulation of inhibitory KIRs and upregulation of CD16. Lenalidomide also leads to a sustained and robust activation of STAT5 and consequential increase in perforin and granzyme B. Finally, we find that treatment with lenalidomide in combination with IL-2 or IL-15 enhances the expression of IL-Rβ and IL-2Rγ chains, a presumed mechanism of action, which may provide a positive feedback loop. These findings have important clinical application. We propose that using lenalidomide in combination with IL-15 can augment its immune activating effects, while minimizing unwanted cytopenias.