No Improvement In Long-Term Overall Survival After The Introduction Of Chemo(Immuno)Therapy For Chronic Lymphocytic Leukemia Patients Belonging To Stereotyped Subset #2

HIGHER-ORDER IMMUNOGLOBULIN SEQUENCE RELATIONS FOR MAJOR SUBSETS OF CHRONIC LYMPHOCYTIC LEUKEMIA : UNIQUENESS VERSUS EQUIVALENCE

Human B-cell chronic lymphocytic leukemia (CLL) is a clonal CD5(+) CD19(+) B-lymphocyte whose B-cell receptor may be classified as unmutated (U-CLL) or mutated (M-CLL) depending on the level of IGHV mutations. Aggressive CLL associates with acquisition of new gene mutations and cytogenetic aberrations, not necessarily in the IGHV or IGLV loci and perhaps caused by activation-induced deaminase (AID). To test if CLL cells can produce functional AID, CLL cells were activated in vitro with CD32-transfected murine L cells, anti-CD40 and interleukin-4 (7 and 14 days U-CLL1278, 0.0% mutated IGHV3-30; 14 days M-CLL1299, 4.9% mutated IGHV3-23), plus irradiated T lymphocytes (10 or 14 days M-CLL1299). CLL cells in these cultures produced detectable AID protein. To evaluate mutational activity, CLL IGHV cDNA was ultra-deep sequenced using the 454 FLX system (Roche) prior to (day 0) or after activation. The resulting 458,124 sequence reads were processed to generate fixed sequence length datasets. Individual subclone sequences occurring at least twice were extracted and unique de novo subclones not shared between day 0 and activation were analyzed for new mutations. All culture conditions showed increases in IGHV mutation frequencies relative to the IGHM constant region. U-CLL1278 showed increased mutation at AID hotspots and a lower transition mutation frequency. M-CLL1299 showed an overall high frequency of transitions and an increase in mutation at AID hotspots in T cell cultures. Thus, de novo mutations consistent with AID activity were found, with some differences between U-CLL and M-CLL. Mutationally-active AID in CLL could lead to adverse consequences.

Not All IGHV3-21 CLL Are Equal : Subset #2 Displays a Distinctive Clinicobiological Profile with Remarkable Similarities to Subset #169, its Close Immunogenetic Relative

Observations of B-cell chronic lymphocytic leukemia (B-CLL) antibody or immunoglobulin (Ig) sequences suggest some common B-CLL antigen reactivity, such as an infectious agent, for which human endogenous retrovirus (HERV) is a candidate. However, we were unable to detect HERV-K particle nucleic acid in plasma obtained from B-CLL patients. Rather than search for individual infectious agents, we developed a method to isolate and identify infectious particle nucleic acids from plasma. A large amount of plasma was obtained following therapeutic phlebotomy from an untreated B-CLL patient with polycythemia vera. Amplified DNA from plasma particles revealed unique restriction enzyme digestible fragments, which were cloned and sequenced. We identified 31 unique sequences from 49 clones. Over 20% of the clones represented a new virus previously not described in B-CLL. The remaining clones represented various new bacterial sequences, of which about 15% represented one species. These sequences may identify new infectious agents that react with the B-CLL antibody.

Interleukin-10 (IL-10), a cytokine that regulates inflammation, may play an important role in B-cell chronic lymphocytic leukemia (B-CLL), because of the reported association of high serum IL-10 levels with a lower prognosis of survival. Using the Bio-Plex protein array system, we confirmed that B-CLL patients exhibit higher median IL-10 levels (3.54 pg/ml, n=50) as compared to controls (1.28 pg/ml, n=33) (p<0.0001). We are in the midst of determining B-CLL VH gene mutation status and IL-10 levels. To determine if elevated IL-10 levels are due to inherent genetic polymorphisms, we examined three single nucleotide polymorphisms (SNPs) in the proximal end of the promoter region of the IL-10 gene (-1082 A/G, −819 T/C, and −592 A/C) that may affect IL-10 transcription levels. DNA from an overlapping set of 54 B-CLL patients and 48 normals was genotyped using the Transgenomic WAVE system. The difference in allele frequencies at a single locus showed no trend towards significance between groups using the Pearsons chi-square test and Odds Ratios (OR) with 95% Confidence Intervals (CI) for each SNP (-1082 (p = 0.686, OR = 1.122, CI = 0.641–1.966), −819 (p = 0.844, OR = 1.062, CI = 0.585–1.926) and −592 (p = 0.720, OR = 1.116, CI = 0.613–2.031)). B-CLL cases and normal subjects showed no significant departure from Hardy-Weinberg equilibrium in single locus genotype frequencies. Differences in single locus genotype frequencies between B-CLL cases and controls showed no significant differences for each SNP (−1082 (p = 0.600, OR = 1.366, CI = 0.425–4.389), −819 (p = 0.638, OR = 0.727, CI = 0.192–2.749), and −592 (p = 0.638, OR = 0.727, CI = 0.192–2.749)). The haplotype frequencies were calculated using maximum likelihood method based on the observed genotypes. The differences in maximum likelihood haplotype frequencies, between the B-CLL cases and controls, were insignificant (p = 0.754). The haplotype pair genotype frequency differences between the B-CLL cases and controls were also insignificant (p = 0.921). In conclusion, the allele, genotype, and haplotype frequencies of IL-10 SNPs −1082, −819, and −592 show no trend towards significance overall. This suggests that these promoter SNPs do not contribute to elevated serum IL-10 in B-CLL. However, a suggestion of association with VH gene mutation status is being further investigated with a larger sample size.

Recently, we determined that IL4I1 is an L-amino acid oxidase (LAAO) located in lysosomes, suggesting it has an antigen presentation role. Indeed, by quantitative PCR, IL4I1 RNA expression is highest in antigen presenting cells (APCs), including macrophages and immature/mature dendritic cells (DCs). Furthermore, DCs have detectable IL4I1 LAAO enzyme activity. Transfected cells overexpressing IL4I1 have increased MHC class II surface levels, suggesting an APC functional role. IL4I1 overexpression enhanced antigen presentation of whole KLH protein to KLH-specific T cells. Thus, IL4I1 may affect APC function perhaps by modification of the peptide repertoire leading to increased MHC class II expression.

IL-4-induced gene-1 (Il4i1or Fig1) initially isolated as a gene of unknown function from mouse B lymphocytes, is limited in expression to primarily immune tissues and genetically maps to a region of susceptibility to autoimmune disease. The predicted Il4i1 protein (IL4I1) sequence is most similar to apoptosis-inducing protein and Apoxin I, both L-amino acid oxidases (LAAO; Enzyme Commission 1.4.3.2). We demonstrate that IL4I1 has unique LAAO properties. IL4I1 has preference for aromatic amino acid substrates, having highest specific activity with phenylalanine. In support of this selectivity, IL4I1 is inhibited by aromatic competitors (benzoic acid and para-aminobenzoic acid), but not by nonaromatic LAAO inhibitors. Il4i1 protein and enzyme activity is found in the insoluble fraction of transient transfections, implying an association with cell membrane and possibly intracellular organelles. Indeed, IL4I1 has the unique property of being most active at acidic pH (pH 4), suggesting it may reside preferentially in lysosomes. IL4I1 is N-linked glycosylated, a requirement for lysosomal localization. Confocal microscopy of cells expressing IL4I1 translationally fused to red fluorescent protein demonstrated that IL4I1 colocalized with GFP targeted to lysosomes and with acriflavine, a green fluorescent dye that is taken up into lysosomes. Thus, IL4I1 is a unique mammalian LAAO targeted to lysosomes, an important subcellular compartment involved in Ag processing.

Mouse interleukin-four induced gene-1 (mFig1) maps to a region of susceptibility for systemic lupus erythematosus (SLE) that includes the Sle3 locus. To begin examining this relationship in humans, we have isolated and characterized the human homolog of mFig1. Human Fig1 (hFig1) has the same eight exon genomic structure as mFig1. The predicted 63-kDa protein, like mFig1, contains a signal peptide, a large internal sequence that is most similar (43% identical over 484 amino acids) to l-amino acid oxidase (LAAO), and a carboxy terminal domain with no similarity to known genes. When compared to the LAAO crystal structure, hFig1 conserves key residues thought to be involved in catalysis and binding of the flavin adenine dinucleotide cofactor. Surprisingly, the carboxy terminal domains of hFig1 and mFig1 have little similarity (<11% identity), different lengths and amino acid composition. Like mFig1, hFig1 RNA is induced by interleukin-4 (IL-4) in B lymphocytes, and is primarily found in immune tissues. Finally, hFig1 maps to the predicted mFig1 syntenic region on human chromosome 19q13.3–19q13.4, a hot spot for susceptibility to several autoimmune diseases, including SLE.