CLL Microenvironment Funded Projects

The goal of this study is to define the communications between CLL B cells, T-cell subsets (in particular Th17 cells), and myeloid-derived suppressor cells (MDSCs), and thereby subsequently devise new therapies that take advantage or modulate these interactions. We have chosen to focus on these three cell types because of the novel initial findings we have made about them: [1] CLL B cells affect Th17 cells, with resting CLL B cells augmenting and activated B cells inhibiting the production of Th17 in vitro; [2] patients with higher numbers of circulating Th17 cells have better clinical outcomes; and [3] the granulocyte-type of CLL-derived MDSCs suppress T cell proliferation, whereas the monocyte-type do not. Therefore, over the next two years, we will investigate: [1] the effects that CLL B lymphocytes and their products have on the polarization of T lymphocytes to various T-cell subsets, including Th17 cells, and on the polarization of autologous monocytes to MDSCs; [2] the effects of Th17 cells and Th17 cytokines on the growth and survival of CLL B cells and the differentiation and function of MDSCs; and [3] the interactions that occur between MDSCs and CLL cells and between MDSCs and T cells. In each instance, these studies will be carried out in vitro and in vivo. In the final year, we will define the key mechanisms that mediate these communications and develop approaches to interrupt or support these for clinical benefit, thereby facilitating development of new immunomodulatory therapies for this still incurable disease.

CLL B lymphocytes require inputs from other normal white blood cells to survive and grow. In this study we are analyzing the inputs received from T lymphocytes, in particular the T helper cell 17 subtype (“Th17 cells”), and from myeloid-derived suppressor cells on the survival and growth of CLL B lymphocytes. Over the past year, we have made two observations that impact on our understanding of CLL disease development and progression. First, we have found that CLL B cells alter the ability of a patient’s bone marrow to make Th17 cells. This is important since our second finding is that products of Th17 cells can limit the survival of CLL cells. This is consistent with our earlier findings that patients with high numbers of Th17 cells have a better clinical course and longer survival. We will investigate these two issues in more depth in 2018 in hopes of identifying ways to increase the apparent benefit to patients by increasing the numbers of Th17 cells.

The role of the monocyte/macrophage lineage in CLL has been extensively studied in vitro but only recently has been investigated in in vivo models. The hypothesis of the present proposal is that tumor-associated macrophages (TAMs) and malignant cells cross-talk during leukemia progression and dissemination and that the targeting of this interdependence may be exploited to remodel the therapeutic scenario of CLL.

In this project we will exploit in vivo and in vitro systems of CLL to: a) investigate, at molecular level to what extent CLL malignant lymphocytes depend on the support of TAMs; b) elucidate how the manipulation of leukemic cell/TAM interactions impacts on other immune cells of the microenvironment; c) finalize the in vitro and mouse systems results to design novel therapeutic strategies to be translated into clinical practice.

CLL is the most frequent adult leukemia in the western world, due to the accumulation of mature neoplastic B lymphocytes. Despite the use of intensive immuno-chemotherapeutic treatments, CLL is still an incurable disease. Given that extensive studies demonstrate how leukemic development and expansion correlate with microenvironmental stimuli delivered by nonmalignant cells, a way to modify the present therapeutic perspective could derive from a better understanding of the biological mechanisms underlying the microenvironment-based molecular and cellular interactions. The role of the monocyte/macrophage lineage in the leukemic progression and dissemination is poorly understood. Therapeutic approaches aimed at targeting the monocyte/macrophage-CLL cell interaction are under evaluation. We integrated different approaches shuttling between human primary cells and animal models to investigate the molecular mechanisms that regulate cellular cross talk between leukemic and monocyte/macrophage lineage cells during leukemia progression. Overall these approaches allowed: a) to understand the cellular and molecular dynamics of leukemic cellmonocyte/macrophage interactions that occur during leukemia progression and b) to validate trabectedin, an innovative therapeutic approach aimed at targeting the monocyte/macrophage lineage.

The aim of this proposal is to identify novel BCR ligands expressed on nurse-like cells (NLC) to gain better understanding about the mechanism regulating BCR activation and survival in the leukemia microenvironment. Supported by our preliminary results, we expect common pattern of BCR ligands in NLC samples. We also expect to find patterns of proteins that are CLL-BCR stereotype-specific. In the era of new BCR signaling inhibitors, it is evident that disruption of BCR signaling and other interactions between CLL cells and their microenvironment is a highly successful therapeutic strategy. An alternative strategy aimed at targeting NLC derived antigens, as proposed here as the ultimate goal of this work, would allow direct inhibition of the BCR antigen interaction, even before signaling initiation. Our project therefore could then provide the framework for an entirely novel therapeutic approach. This is the first systematic approach to identify antigens on NLC for CLL BCR. These experiments will allow for discovery of new and may confirm previously identified CLL antigens. These antigens could be responsible for BCR signaling activation in lymphatic tissues, where CLL cells proliferate in a BCR signaling-dependent fashion. NLC are a validated model to recapitulate conditions in the CLL lymph node microenvironment. Findings will be validated on patient samples. Once these aims have been accomplished we will have gained knowledge about mechanism of CLL cell activation that are central for CLL pathogenesis and may open avenues for alternative therapy approaches.

Leukemia cells from CLL patients are activated and they grow because of signals that the leukemia cells receive from the microenvironment, especially a surface receptor called the B cell receptor (BCR). This receptor now is targeted by treatments such as ibrutinib, that shut down the signals from the BCR. Here, we analyzed the mechanism how the BCR is activated in patients, where this BCR activation occurs exclusively in the lymph nodes. We found a molecule called Calreticulin located on the surface of nurse-like cells that binds CLL BCRs. Nurse-like cells are macrophages from CLL patients that are key players for BCR activation in CLL. BCR from CLL patients bind to calreticulin, and in turn presumably activate the BCR. We dissected out which subgroup of CLL patients have BCR that bind to Calreticulin, and this strategy now allows us to better understand disease mechanism that result in BCR activation and may open avenues for new treatment approaches.

The intent behind this study is to: a) Determine how CLL-derived exosomes “educate” monocytes; b) Isolate, expand and study the characteristics of CLL patients’ fibrocytes; c) Investigate the effects of CLL-derived fibrocytes on hematopoietic cells; d) Determine whether fibrocytes play a role in CLL bone marrow failure and whether serum amyloid P (SAP) could reverse it.

Much attention has been paid to the role of T-lymphocytes in the CLL microenvironment whereas little is known about the role of monocytes or monocyte-derive cells. A few studies suggested that monocytes and monocyte-derived cells play a role in the pathogenesis of CLL: a) A high monocyte count was found to correlate with CLL patients’ poor prognosis; b) Increased frequency of CD14+ /HLA-DRlo/neg monocytes was associated with decreased time to progression; c) Monocytes were shown to increase the survival of CLL cells by secreting soluble CD14, which induces nuclear factor-κB (NF-κB) activation in CLL cells; d) Monocyte-derived nurse-like cells were shown to provide CLL cells with survival advantage; and e) A recent study demonstrated that the survival of CLL cells, in a mouse model, is macrophage-dependent. This data suggests that a cross-talk between CLL cells and monocytes exists and that monocyte-derived cells support, protect and/or stimulate the CLL clone.

Cell-derived nano-particles capable of affecting cell function have been identified in the circulating blood and various tissues of CLL patients and healthy individuals. We found that CLL cell-derived nano-particles named exosomes, may suppress the generation of normal blood elements and induce a low degree of scarring of the bone marrow of patients with CLL. In the upcoming year we intend to investigate how do exosomes induce this effect and how could we reverse it.

Chronic lymphocytic leukemia (CLL) is characterized by generalized immune suppression and susceptibility to infectious complications and secondary malignancies. A number of studies have reported both quantitative and qualitative defects in T-cell function in CLL. However, the mechanisms underlying CLL-induced immunosuppression have been difficult to dissect, as they involve complex bidirectional interactions among leukemic cells, components of the tumor microenvironment and immune effectors. Regulatory B cells (Bregs) are a recently defined subset of B cells with potent immunoregulatory function. A subset of Bregs, known as B10 cells, suppress effector T-cell function through STAT3-mediated production of IL-10 and have been implicated in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus, immune thrombocytopenia, active chronic sarcoidosis, and multiple sclerosis, as well as alloimmune disorders such as graft-versus-host disease. Tedder et al recently reported that CLL B cells are capable of secreting IL-10 and possess regulatory functions comparable to those of normal B10 cells. These intriguing observations suggest a means by which CLL cells could induce immunosuppression in patients, but a mechanistic basis for IL-10 production by CLL cells is lacking.

The CLL microenvironment supports tumor cell survival via a number of soluble and surface–bound factors, such as the CXC chemokine ligand 12 (CXCL12), BAFF, APRIL and CD40 ligand (CD154). CXCL12 binds its receptor CXCR4 on the surface of CLL cells and directs chemotaxis, supports tumor survival and activates various signaling pathways, including STAT3. We hypothesize that CXCL12-CXCR4 interaction results in activation of the STAT3 pathway and IL-10 production by CLL cells, which may in turn contribute to immune suppression in patients. We will test this hypothesis in the following specific aims:

Aim 1. Determine if the CXCL12-CXCR4 chemokine ligand/receptor axis activates the STAT3 pathway and CLL B10 function
Aim 2. Determine if CLL induces T cell suppression through phosphorylation of Y705-STAT3
Aim 3. Investigate if lenalidomide modulates T cell function in CLL by inhibiting the STAT3 pathway

Recent data support an important role for the immune system in the control of cancer. However, defects of the immune system have been described in CLL. Indeed, progressive dysfunction of the immune system often parallels disease progression. The aim of our study was to determine the mechanism by which CLL cells induce dysfunction in T cells. We have shown that CLL cells produce an immunosuppressive protein, interlukin-10 (IL-10), through which they suppress T- cell function. A detailed understanding of the underlying mechanism for the defects in T cell function in CLL will help us develop strategies to boost the immune response in CLL. One such strategy is treatment with an immunomodulatory drug, lenalidomide.