Elizabeth S. Raveche

Overview of monoclonal B‐cell lymphocytosis

Monoclonal B‐cell lymphocytosis (MBL) has been the subject of more intensive investigation for the last 10u2003years. The increased presence of MBL in unaffected, first‐degree relatives with familial chronic lymphocytic leukaemia (CLL) suggest that it is surrogate marker for early disease. In normal population studies, MBL is found to be increased in ageing subjects. Consensus criteria for the diagnosis of MBL have been proposed. The differential diagnosis has been further clarified and the prevalence of MBL is most prominent in the elderly. The aetiology of MBL is unknown but probably involves immune mechanism of senescence or altered response. Environmental health studies suggest that exposure to certain toxins may lead to MBL but further work is needed. MBL is a precursor to CLL but may also regress, remain stable or progress to clinical CLL.

Possible immunoregulatory role for CD5 + B cells.

CD5 + B cells represent a subpopulation of B cells which have the characteristic of employing unmutated immunoglobulin variable region genes. These cells are found to be increased early in ontogeny. The percentage of CD5 + B cells is highest in the fetus and decreases after birth. The antibodies produced by CD5 + B cells are polyreactive and are the natural autoantibodies. These autoantibodies may not be pathogenic. CD5 + B cells are elevated in certain autoimmune disease states and are the malignant cell type in B-CLL, with a strong genetic component involved in determining elevated CD5 + B cell states. Elevated CD5 + B cells are found in immunodeficient states (young, aged, and autoimmune). CD5 + B cells may normally act as a first-line defense against invading foreign pathogens but are not involved in the specific immune response. There is some evidence, at least in newborns, that CD5 + B cells may affect the emerging B cell repertoire of conventional B cells via idiotype cascade. However, the action of CD5 + B cells in the newborn may be quite different than their activity in the adult. Nonimmunoglobulin-producing CD5 + B cells may be immunosuppressors. In this report, a unique subpopulation of CD5 + B cells was investigated. These cells were found only in the spleens of aged NZB mice. The CD5 + B cells were clonal and possessed extra chromosomes and did not appear to be producing antibodies. These cells were capable of rapid proliferation in unirradiated recipients. By taking advantage of this proliferative capability, the effect of exogenous clonal CD5 + B cells on recipient immune system was evaluated. Clonal CD5 + B cells from NZB mice were immunosuppressive and decreased the numbers of conventional B cells as well as the level of natural antibodies. In summary, CD5 + B cells may play different roles in the immune system depending upon environment, age, and their differentiation state (i.e., proliferation versus antibody secretion). The natural antibody produced by CD5 + B cells may be involved in maintenance functions such as removal of dead cells and first-line defense mechanisms. In addition, CD5 + B cells may themselves regulate the immune system and produce a factor which is immunosuppressive. An understanding of the various functions of CD5 + B cells may elucidate fundamental immunoregulatory circuits.

Antitumor activity of Type I and Type III interferons in BNL hepatoma model

Hepatocellular carcinoma (HCC) occurs most commonly secondary to cirrhosis due to chronic hepatitis C or B virus (HCV/HBV) infections. Type I interferon (IFN-α) treatment of chronic HCV/HBV infections reduces the incidence of HCC in cirrhotic patients. However, IFN-α toxicity limits its tolerability and efficacy highlighting a need for better therapeutic treatments. A recently discovered type III IFN (IFN-λ) has been shown to possess antiviral properties against HCV and HBV in vitro. In phase I clinical trials, IFN-λ treatment did not cause significant adverse reactions. Using a gene therapy approach, we compared the antitumor properties of IFN-α and IFN-λ in a transplantable hepatoma model of HCC. BALB/c mice were inoculated with syngeneic BNL hepatoma cells, or BNL cells expressing IFN-λ (BNL.IFN-λ cells) or IFN-α (BNL.IFN-α cells). Despite the lack of antiproliferative activity of IFNs on BNL cells, both BNL.IFN-λ and BNL.IFN-α cells displayed retarded growth kinetics in vivo. Depletion of NK cells from splenocytes inhibited splenocyte-mediated cytotoxicity, demonstrating that NK cells play a role in IFN-induced antitumor responses. However, isolated NK cells did not respond directly to IFN-λ. There was also a marked NK cell infiltration in IFN-λ producing tumors. In addition, IFN-λ and, to a lesser extent, IFN-α enhanced immunocytotoxicity of splenocytes primed with irradiated BNL cells. Splenocyte cytotoxicity against BNL cells was dependent on IL-12 and IFN-γ, and mediated by dendritic cells. In contrast to NK cells, isolated from spleen CD11c+ and mPDCA+ dendritic cells responded directly to IFN-λ. The antitumor activities of IFN-λ against hepatoma, in combination with HCV and HBV antiviral activities warrant further investigation into the clinical use of IFN-λ to prevent HCC in HCV/HBV-infected cirrhotic patients, as well as to treat liver cancer.

Murine models of chronic lymphocytic leukaemia: role of microRNA-16 in the New Zealand Black mouse model.

Mouse models are valuable tools in the study of human chronic lymphocytic leukaemia (CLL). The New Zealand Black (NZB) strain is a naturally occurring model of late‐onset CLL characterized by B‐cell hyperproliferation and autoimmunity early in life, followed by progression to CLL. Other genetically engineered models of CLL that have been developed include (NZBu2003×u2003NZW) F1 mice engineered to express IL5, mice expressing human TCL1A, and mice overexpressing both BCL2 and a tumour necrosis factor receptor‐associated factor. The applicability to human CLL varies with each model, suggesting that CLL is a multifactorial disease. Our work with the de novo NZB model has revealed many similarities to the human situation, particularly familial CLL. In NZB, the malignant clones express CD5, zap‐70, and have chromosomal instability and germline Ig sequence. We also identified a point mutation in the 3′‐flanking sequence of Mirn16‐1, which resulted in decreased levels of the microRNA, miR‐16 in lymphoid tissue. Exogenous restoration of miR‐16 to an NZB malignant B‐1 cell line resulted in cell cycle alterations, suggesting that the altered expression of Mirn15a/16‐1 is an important molecular lesion in CLL. Future studies utilizing the NZB mouse could ascertain the role of environmental triggers, such as low dose radiation and organic chemicals in the augmentation of a pre‐existing propensity to develop CLL.

Prevalence of monoclonal B-cell lymphocytosis: A systematic review†‡§

Individuals with monoclonal B‐cell lymphocytosis (MBL) have been identified in clinic outpatients, in unaffected relatives of patients with chronic lymphocytic leukemia (CLL), and in general populations. MBL and its relationship with CLL have been actively investigated over the last decade. This report systematically reviews the prevalence of MBL in the context of the populations studied and the evolution of laboratory methods used to define MBL.

Correlation of ZAP-70 expression in B cell leukemias to the ex vivo response to a combination of fludarabine/genistein

The role of ZAP-70 expression on the ex vivo response of blood cells from CLL and PLL patients to a combination of fludarabine, a purine analog, and genistein, a tyrosine kinase inhibitor was studied. Patient cells were studied for the expression of ZAP-70 mRNA and its relation to the induction of apoptosis in response to treatment with genistein 15–60xa0μM and/or fludarabine 3xa0μM. The combination of genistein and fludarabine resulted in a significantly increased induction of apoptosis relative to the fludarabine alone. The ex vivo patient cells with a high ZAP-70 expression underwent more apoptosis in response to genistein than did patient cells with a low ZAP-70 mRNA expression. In contrast, basal IL-10 mRNA expression correlated negatively with apoptosis induction in response to genistein (Pxa0<xa00.01). These studies suggest that, in malignant B cells that express elevated levels of the ZAP-70 signaling molecule, genistein may inhibit the ZAP-70 tyrosine kinase activity, resulting in cell death. The ZAP-70 may serve as a target for therapy. In addition, these studies suggest that the IL-10 expression by malignant B cells may not only suppress anti-tumor T cell responses in vivo, but also promote the survival of malignant B cells despite treatment with chemotherapeutic agents.

B-cell repertoire and clonal analysis in unaffected first degree relatives in familial chronic lymphocytic leukaemia kindred.

Monoclonal B cell lymphocytosis (MBL) was detected in four unaffected first‐degree relatives (FDR) in a familial chronic lymphocytic leukaemia (CLL) kindred. The proband remains untreated and two male siblings have died. The four unaffected siblings have been followed for a five‐year period. All four FDR developed a kappa+CD5+ MBL detected by flow cytometry. Poymerase chain reaction (PCR) for IGHV rearrangement showed evidence of oligoclonality in three of these individuals. Single cell PCR of flow cytometric sorted kappa+ cells combined with Ig kappa light chain gene sequencing revealed further evidence of monoclonality in two of these individuals. Three of these individuals all showed evidence of hyper‐somatic mutations. The B‐cell repertoire in unaffected FDR in familial CLL offers a new area to investigate the interface between the immune system and lymphoid neoplasm.

Three dimensional structure of a natural auto antibody — A predicted model of the antigen binding site

In the complex network of the immune system, antibodies play a major role in binding antigen, thereby activating the immune humoral response and aiding in elimination of the foreign particle. There are millions of antibodies in the body ready to encounter the myriad of different antigens. Only when a proper fit occurs between the antibody and the antigen, a strong immune response is elicited and the body gets rid of the antigen. Unfortunately, by some unknown mechanism autoantibodies are also produced in the body of some individuals. These autoantibodies bind specifically to self-antigens, thus leading to an unfavorable pathogenic situation of autoimmunity. In addition, natural autoantibodies are produced by a subset of B cells (B-1) which are polyreactive and may bind several self antigens. In this paper, we modeled the 3-D structure of a natural autoantibody whose deduced amino acid sequence is known. This antibody was produced by malignant, hyperdiploid B-1 clones. We compared the antigen binding site (Fab) of this natural autoantibody to a normal antibody and found that the tertiary structure of this autoantibody resulted in an antibody(Ig) with a shallow binding groove relative to the original antibody. It is possible that alterations in the binding groove are responsible for polyreactivity.

Abstract 4045: Restoring miR-15a/16 in the NZB mouse model of chronic lymphocytic leukemia reduces disease and enhances drug sensitivity

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnAlterations in the human 13q14 genomic region containing the microRNAS, mir-15a and mir-16-1 are present in a majority of human chronic lymphocytic leukemia (CLL). We have previously found the development of CLL in the New Zealand Black (NZB) murine model to be associated with a point mutation in the primary mir-15a/16-1 region, as well as a decrease in both mature miR-16 and miR-15a levels. Addition of exogenous miR-15a and miR-16 led to an accumulation of cells in G1 in both normal B cell and NZB-derived malignant B-1 cell lines. However, the NZB line had a significantly greater G1 accumulation, suggesting a restoration of cell cycle control upon exogenous miR-15a/16 addition. Our experiments demonstrated a reduction in protein levels of cyclin D1, a miR-15a/16 target and cell cycle regulator of G1/S transition, in the NZB cell line following miR-15a/16 addition. These miRNAs were shown to directly target the cyclin D1 3′UTR using a GFP lentiviral sensor. In vivo restoration of NZB with a miR-16-producing lentiviral vector resulted in the depletion of endogenous-transduced malignant aneuploid B-1 cells. In addition, experimentally increasing miR-16 in either a drug-resistant malignant NZB B-1 cell line or ex vivo splenic NZB B-1 cells augmented apoptosis induction by the following agents: genistein, a soy isoflavone tyrosine kinase inhibitor; nutlin, an MDM2 antagonist; and 2-fluoroadenine-9-B-D-arabinofuranoside, a purine nucleotide analog. Our data support a role for miR-16 in apoptosis induction, either alone, or in synergy with chemotherapeutic agents to increase their efficacy in murine CLL cells.nnThis research was funded in part by the NJCCR pre-doctoral fellowship awarded to E.S.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4045.