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Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL. Key Points: • In a series of 29 856 CLL patients, the incidence of BcR stereotypy peaked at 41%. • Higher-order relations exist between stereotyped subsets, particularly for those from U-CLL, for which satellite subsets were identified.

International audience; Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease exemplary of this process, and a model for neoplasms in general, we created transgenic mice overexpressing the enzyme, activation-induced deaminase (AID), whose normal function is to induce DNA mutations in B lymphocytes. AID allows normal B lymphocytes to develop more effective immunoglobulin (Ig)-mediated immunity, but also is able to mutate non-Ig genes, predisposing to cancer. In chronic lymphocytic leukemia (CLL), AID expression correlates with poor prognosis suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Em-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL-cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in non-Igs genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically-similar amino acid substitutions as in human CLL and lymphoma.Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

Cancer pathogenesis involves the interplay of tumor- and microenvironment-derived stimuli. Here we focused on the influence of an immunomodulatory cell type, myeloid-derived suppressor cells (MDSCs), and their lineage-related subtypes on autologous T lymphocytes. Although MDSCs as a group correlated with an immunosuppressive Th repertoire and worse clinical course, MDSC subtypes (polymorphonuclear, PMN-MDSC, and monocytic, M-MDSCs) were often functionally discordant. In vivo, PMN-MDSCs existed in higher numbers, correlated with different Th-subsets, and more strongly associated with poor clinical course than M-MDSCs. In vitro, PMN-MDSCs were more efficient at blocking T-cell growth and promoted Th17 differentiation. Conversely, in vitro M-MDSCs varied in their ability to suppress T-cell proliferation, due to the action of TNFα, and promoted a more immunostimulatory Th compartment. Ibrutinib therapy impacted MDSCs differentially as well, since after initiating therapy, PMN-MDSC numbers progressively declined, whereas M-MDSC numbers were unaffected, leading to a set of less immunosuppressive Th cells. Consistent with this, clinical improvement based on decreasing CLL-cell numbers correlated with the decrease in PMN-MDSCs. Collectively, the data support a balance between PMN-MDSC and M-MDSC numbers and function influencing CLL disease course.

Progression of chronic lymphocytic leukemia (CLL) results from the expansion of a small fraction of proliferating leukemic B cells. When comparing the global gene expression of recently divided CLL cells with that of previously divided cells, we found higher levels of genes involved in regulating gene expression. One of these was the oncogene Musashi 2 (MSI2), an RNA-binding protein that induces or represses translation. While there is an established role for MSI2 in normal and malignant stem cells, much less is known about its expression and role in CLL. Here we report for the first time ex vivo and in vitro experiments that MSI2 protein levels are higher in dividing and recently divided leukemic cells and that downregulating MSI2 expression or blocking its function eliminates primary human and murine CLL and mature myeloid cells. Notably, mature T cells and hematopoietic stem and progenitor cells are not affected. We also confirm that higher MSI2 levels correlate with poor outcome markers, shorter time-to-first-treatment, and overall survival. Thus, our data highlight an important role for MSI2 in CLL-cell survival and proliferation and associate MSI2 with poor prognosis in CLL patients. Collectively, these findings pinpoint MSI2 as a potentially valuable therapeutic target in CLL.

Craniosynostosis (CRS) is a congenital abnormality deformity with a heterogenous genetic contribution. Previously, there are two attempts to collect genes that are genetically associated with craniosynostosis and some related syndromes with 57 (Twigg and Wilkie, 2015) and 39 (Goos and Mathijssen, 2019) genes identified, respectively. We expanded this list of craniosynostosis genes by adding another 17 genes with an updated literature search. These genes are shown to be more likely to be intolerant to functional mutations. Of these 113 craniosynostosis genes, 21 (19% vs. 1.5% baseline frequency) are cancer driver genes, a 14-fold enrichment. The cancer-craniosynostosis connection is further validated by an over-representation analysis of craniosynostosis genes in KEGG cancer pathway and several cancer related gene-sets. Many cancer-craniosynostosis overlapping genes participate in intracellular signaling pathways, which play a role in both development and cancer. This connection can be viewed from the oncogenesis recapitulates ontogenesis framework. Nineteen craniosynostosis genes are transcription factor genes (16.8% vs. 8.2% baseline), and craniosynostosis genes are also enriched in targets of certain transcription factors or micro RNAs.

Amino acid replacement mutations in certain chronic lymphocytic leukemia (CLL) stereotyped B cell receptor (BCR) immunoglobulins (IGs) at defined positions within antigen-binding sites strongly imply antigen selection. Prime examples of this are CLL subset 4 BCR IGs using IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements. Conspicuously, and unlike most CLL IGs, subset 4 IGs do not bind apoptotic cells. By testing the (auto)antigenic reactivities of subset 4 IGs toward viable lymphoid-lineage cells and specific autoantigens typically bound by IGHV4-34+ IGs, we found that IGs from both subset 4 and non-subset 4 IGHV4-34-expressing CLL cases bound naïve B cells. However, only subset 4 IGs reacted with memory B cells. Furthermore, subset 4 IGs did not bind DNA nor i or I carbohydrate antigens that are common targets of IGHV4-34-utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively. Notably, we found that subset 4 IG binding to memory B lymphocytes depends on an aspartic acid at position 66 of FR3 in the rearranged IGKV2-30 gene; this amino acid residue is acquired by somatic mutation. Our findings illustrate the importance of positive and negative selection criteria for structural elements in CLL IGs and suggest that autoantigens driving normal B cells to become subset 4 CLL cells differ from those driving IGHV4-34+ B cells in other diseases.

Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication. Experimental Design: In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34–expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes. Results: We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly (P < 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). Conclusions: Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Clin Cancer Res; 23(17); 5292–301. ©2017 AACR.

Intraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.

Six new limonoids, munropins A−F (1–6), were isolated from the aerial parts of a Chinese medicinal plant, Munronia pinnata (Meliaceae). The structures of 1–6 were assigned by detailed analyses of their spectroscopic data. Munropins A (1) and B (2) are limonoids possessing a prieurianin skeleton with α,β-unsaturated γ-lactam moieties at C-17. Munropins C (3), D (4), and E (5) are also prieurianin type limonoids with an α,β-unsaturated γ-lactone moiety, an acetyl group, and an acetoxyacetyl group at C-17, respectively. Munropin F (6) was assigned as a nimbolinin type limonoid with a γ-hydroxy-α,β-unsaturated γ-lactone moiety.

Background Although the etiology of chronic lymphocytic leukemia (CLL), the most common type of adult leukemia, is still unclear, strong evidence implicates antigen involvement in disease ontogeny and evolution. Primary and 3D structure analysis has been utilised in order to discover indications of antigenic pressure. The latter has been mostly based on the 3D models of the clonotypic B cell receptor immunoglobulin (BcR IG) amino acid sequences. Therefore, their accuracy is directly dependent on the quality of the model construction algorithms and the specific methods used to compare the ensuing models. Thus far, reliable and robust methods that can group the IG 3D models based on their structural characteristics are missing. Results Here we propose a novel method for clustering a set of proteins based on their 3D structure focusing on 3D structures of BcR IG from a large series of patients with CLL. The method combines techniques from the areas of bioinformatics, 3D object recognition and machine learning. The clustering procedure is based on the extraction of 3D descriptors, encoding various properties of the local and global geometrical structure of the proteins. The descriptors are extracted from aligned pairs of proteins. A combination of individual 3D descriptors is also used as an additional method. The comparison of the automatically generated clusters to manual annotation by experts shows an increased accuracy when using the 3D descriptors compared to plain bioinformatics-based comparison. The accuracy is increased even more when using the combination of 3D descriptors. Conclusions The experimental results verify that the use of 3D descriptors commonly used for 3D object recognition can be effectively applied to distinguishing structural differences of proteins. The proposed approach can be applied to provide hints for the existence of structural groups in a large set of unannotated BcR IG protein files in both CLL and, by logical extension, other contexts where it is relevant to characterize BcR IG structural similarity. The method does not present any limitations in application and can be extended to other types of proteins.

Chronic lymphocytic leukemia (CLL) is a clonal disease of B lymphocytes manifesting as an absolute lymphocytosis in the blood. However, not all lymphocytoses are leukemic. In addition, first-degree relatives of CLL patients have an ~15 % chance of developing a precursor condition to CLL termed monoclonal B cell lymphocytosis (MBL), and distinguishing CLL and MBL B lymphocytes from normal B cell expansions can be a challenge. Therefore, we selected FMOD, CKAP4, PIK3C2B, LEF1, PFTK1, BCL-2, and GPM6a from a set of genes significantly differentially expressed in microarray analyses that compared CLL cells with normal B lymphocytes and used these to determine whether we could discriminate CLL and MBL cells from B cells of healthy controls. Analysis with receiver operating characteristics and Bayesian relevance determination demonstrated good concordance with all panel genes. Using a random forest classifier, the seven-gene panel reliably distinguished normal polyclonal B cell populations from expression patterns occurring in pre-CLL and CLL B cell populations with an error rate of 2 %. Using Bayesian learning, the expression levels of only two genes, FMOD and PIK3C2B, correctly distinguished 100 % of CLL and MBL cases from normal polyclonal and mono/oligoclonal B lymphocytes. Thus, this study sets forth effective computational approaches that distinguish MBL/CLL from normal B lymphocytes. The findings also support the concept that MBL is a CLL precursor.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset # 2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset # 2. Within subset # 2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset # 2/IGHV3-21 was enriched for IGHV-unmutated cases (P =.002). Subset # 2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset # 2/IGHV3-21 (22 vs 60 months, P =.001). No such difference was observed between non-subset # 2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset # 2 emerges as uniformly aggressive, contrasting non-subset # 2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

Tumor-specific metabolic changes can reveal new therapeutic targets. Our findings implicate a supporting role for fatty acid metabolism in chronic lymphocytic leukemia (CLL) cell survival. Peroxisome proliferator–activated receptor (PPAR)-α, a major transcriptional regulator of fatty acid oxidation, was recently shown to be upregulated in CLL. To evaluate PPARα as a potential therapeutic target, we developed a highly selective, potent small molecule antagonist of PPARα, NXT629. NXT629 inhibited agonist-induced transcription of PPARα-regulated genes, demonstrating target engagement in CLL cells. Furthermore, NXT629 induced apoptosis of CLL cells even in the presence of a protective microenvironment. To mimic the proliferative lymphoid compartment of CLL, we examined the activity of NXT629 on CLL cells that were stimulated to proliferate in vitro. NXT629 reduced the number of leukemia cells undergoing cell division. In addition, in two xenograft mouse models of CLL (one a model for nondividing and one for dividing CLL), NXT629 reduced the number of viable CLL cells in vivo. Overall, these results suggest that fatty acid metabolism promotes survival and proliferation of primary CLL cells and that inhibiting PPARα gene regulation could be a new therapeutic approach to treating CLL.

Significance A pathogenetic role of NOTCH1 in chronic lymphocytic leukemia (CLL) has been implied by the presence of deregulating mutations in a relatively small fraction of cases. Our results now indicate that ∼50% of CLL cases devoid of mutations express the active form of NOTCH1 ICN1 (intracellular portion of NOTCH1), thus implicating a much broader role of this transcription factor in the disease. ICN1 + CLL cases display equivalent NOTCH1-dependent transcriptional responses regardless of the gene mutation status, indicating that the detection of ICN1 represents a reliable biomarker of NOTCH1 activation for diagnostic and therapeutic targeting. Finally, our results identify the NOTCH1-dependent transcriptional program in CLL cells, thus providing direct insights into the pathogenesis of a large fraction of CLL cases.

BACKGROUND. Ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL) that inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. METHODS. We used stable isotopic labeling with deuterated water (2H2O) to measure directly the effects of ibrutinib on leukemia cell proliferation and death in 30 patients with CLL. RESULTS. The measured average CLL cell proliferation ("birth") rate before ibrutinib therapy was 0.39% of the clone per day (range 0.17%-1.04%); this decreased to 0.05% per day (range 0%-0.36%) with treatment. Death rates of blood CLL cells increased from 0.18% per day (average, range 0%-0.7%) prior to treatment to 1.5% per day (range 0%-3.0%) during ibrutinib therapy, and they were even higher in tissue compartments. CONCLUSIONS. This study provides the first direct in vivo measurements to our knowledge of ibrutinib's antileukemia actions, demonstrating profound and immediate inhibition of CLL cell proliferation and promotion of high rates of CLL cell death. TRIAL REGISTRATION. This trial was registered at clinicaltrials.gov (NCT01752426). FUNDING. This study was supported by a Cancer Center Support Grant (National Cancer Institute grant P30 CA016672), an NIH grant (CA081554) from the National Cancer Institute, MD Anderson's Moon Shots Program in CLL, and Pharmacyclics, an AbbVie company.

Array comparative genomic hybridization (aCGH) has yet to be fully leveraged in a prognostic setting in chronic lymphocytic leukemia (CLL). Genomic imbalance was assessed in 288 CLL specimens using a targeted array. Based on 20 aberrations in a hierarchical manner, all 228 treatment-naive specimens were classified into a group with poor outcome (20.6%) exhibiting at least one aberration that was univariately associated with adverse outcome (gain: 2p, 3q, 8q, 17q, loss: 7q, 8p, 11q, 17p, 18p), good outcome (32.5%) showing 13q14 loss without any of the other 10 aberrations (gain: 1p, 7p, 12, 18p, 18q, 19, loss: 4p, 5p, 6q, 7p) or intermediate outcome (remainder). The three groups were significantly separated with respect to time to first treatment and overall survival (p < 0.001), and validation of the stratification scheme was performed in two independent datasets. Gain of 3q and 8q, and 17p loss were determined to be independent unfavorable prognostic biomarkers. TP53, NOTCH1 and SF3B1 mutations correlated with the presence of one poor outcome aCGH marker, at a considerably higher frequency than when only considering poor risk aberrations routinely detected by fluorescence in situ hybridization (FISH). These data support genomic imbalance evaluation in CLL by aCGH to assist in risk stratification.

CLL cases display heterogeneous responses to B cell receptor (BCR) engagement which correlate with clinical course and survival. Typically, cases with more aggressive disease show greater activation of downstream signaling molecules, such as the kinases SYK, AKT and ERK, upon BCR engagement. The heterogeneous BCR signaling responses have been attributed to varying levels of anergy occurring as a consequence of chronic antigen engagement in the absence of T cell help. However, an alternative explanation for the anergic phenotype is that it is induced by binding of serum IgM to the high-affinity Fcμ receptor (FCMR), which is highly overexpressed on CLL cells and has been reported to physically interact with the BCR in normal murine B cells. To investigate whether binding of soluble IgM to the FCMR can affect BCR signaling, we investigated activation of downstream BCR signaling molecules in primary CLL B cells after FCMR prestimulation with monoclonal IgM or Fcμ fragment followed by BCR stimulation with polyclonal anti-light chain antibody. For 10 of the 18 cases tested, substantial reduction in the levels of phospho-SYK, phospho-AKT and phospho-ERK was observed in IgM- or Fcμ-prestimulated CLL cells consistent with FCMR stimulation inhibiting BCR signaling. Stimulation with IgM or Fcμ did not cause any appreciable change in surface IgM expression, suggesting that inhibition of BCR signaling is not caused by receptor downmodulation but rather by recruitment of negative regulatory mechanism(s). Investigation of these mechanisms is still ongoing, but preliminary data suggest involvement of the phosphatases SHIP and SHP1. To investigate the impact of serum IgM/FCMR interactions on the clinical course, we correlated FCMR expression and serum IgM levels with treatment-free survival (TFS) in a large series of CLL patients (Figure 1). Cases with FCMR expression levels below median value (n=67) had a median TFS of 41 months, which was significantly shorter compared to cases with FCMR levels equal to or above median value (n=71, median TFS=132 months, P Collectively, these data show that serum IgM/FCMR interactions inhibit BCR signaling, which could at least in part explain the anergic phenotype of peripheral blood CLL cells. Along with our finding that the FCMR is downregulated by IL4 and expressed at lower levels on LN compared to PB CLL cells (Gobessi S et al, Blood 2016 128:2015), these data suggest that serum IgM/FCMR interactions are involved in regulating the intensity of the BCR signal in different compartments and consequently can influence the clinical behavior of the disease. Disclosures Chen: Janssen: Research Funding; Pharmacyclics: Research Funding; ArgenX: Research Funding; Beigene: Research Funding; Verastem: Research Funding. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

The growth of CLL cells stems from a small fraction of dividing CD5+B cells. The size and rate of growth of this proliferative fraction (PF) correlates directly with poor outcome prognostic markers and inversely with time-to-first-treatment. Furthermore, since dividing cells upregulate DNA mutators (AID and APOBEC), the PF can acquire new DNA abnormalities that can lead to more lethal disease. Hence, cells of the PF are important targets for therapy. By gene expression profile analysis, we found that Musashi 2 (MSI2) is highly expressed in the PF (CXCR4DimCD5Bright) compared with the resting fraction (RF) that expresses the reciprocal phenotype (CXCR4BrightCD5Dim). In normal cells, MSI2 binds to mRNA and blocks or enhances protein translation. MSI2 levels are higher in proliferating normal and malignant stem cells and during tumorigenesis. In CLL, high MSI2 mRNA levels associate with poor outcome. Nevertheless, nothing is known about the function of MSI2 in CLL cells. Therefore, we studied the biological role of MSI2 in CLL B cells. We found that CLL B cells express higher levels of MSI2 protein than those of healthy donors (HD). MSI2 levels were higher in U-CLL than M-CLL, and M-CLL B cells expressed more than HD cells, consistent with MSI2 associating with poor prognosis. Within a CLL clone, MSI2 was higher in PF than RF. Also, microenvironment signals that induce B cell proliferation also increased MSI2 protein levels. Consistent with these observations in patients, in vitro experiments showed that dividing cells contain more MSI2 protein than undivided cells. Next, we investigated the importance of MSI2 on CLL survival. In primary CLL cells, we knocked down MSI2 levels using MSI2 siRNA and co-cultured CLL cells with stromal cells (HS5) or stimulated them with CpG ODN + IL15, with or without HS5 cells. In the three conditions tested, knock-down of MSI2 resulted in a significant decrease in tumor-cell survival compared with the control (P = 0.0342, 0.0079, and 0.0274, respectively). In addition, down regulation of MSI2 led to an upregulation of cleaved caspase3 (1.3 fold change;P= 0.029), p27kip1 (1.10 fold change; P= 0.0026) and phospho p53 (1.16 fold change; P= 0.017) compared with siRNA control (siCTR). Collectively, the results highlight the importance of MSI2 in primary CLL-cell survival and suggest that these are mediated by caspase 3, p27kip1 and p53 signaling pathways. Reported comparisons of the RNAs that MSI2 binds in different cell types suggest that MSI2 regulates both common and unique mRNA targets in a cell type-specific manner. Hence to study the role of MSI2 in CLL, we analyzed the proteomes of MSI2 knockdown B cells from 12 CLL patients and controls. This showed that MSI2 knock down significantly increased the levels of 12 proteins (P ≤ 0.01 and fold change ≤ -1.2 ≥ 1.2). Since Ingenuity Pathway Analysis of these proteins suggested an increase cellular movement, we confirmed protein levels by flow cytometry of proteins involved in migration: Fer (non-tyrosine-protein kinase receptor), VAV1 (guanine nucleotide exchange factors) and its active form (phosphorY174 VAV, pVAV). Since Fer and VAV1 play a role in actin cytoskeleton regulation, we next knocked down MSI2 in B cells from the same 12 CLL patients and evaluated by microscopy cytoskeleton rearrangement of cells bound to fibronectin-coated slides. We used an established method to classify round cells as having a non-modified cytoskeleton and elongated cells as having undergone cytoskeleton rearrangement. Also, we counted the number of cells with an accumulation of polarized actin and others with multiple protrusions. MSI2 downregulation increased the number of elongated, polarized and cells with multi protrusions. Thus, MSI2 levels are higher in B cells from poor outcome patients and also in the dividing/divided cells of the PF before and after stimulation. Also, MSI's downregulation induces apoptosis of CLL cells and increases adhesion and migration. Therefore, we propose that MSI2 is a valuable target for therapeutic intervention since inhibiting its functions will likely abort clonal evolution and disease progression, making CLL an even more chronic and manageable condition. Disclosures Barrientos: Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Kolitz:Magellan Health: Consultancy, Honoraria. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

For antigen recognition, lampreys use leucine-rich repeats (LRR) instead of immunoglobulin V-(D)-J domains to generate variable lymphocyte receptors (VLR) of three types: VLRA, VLRB, and VLRC. VLRB-bearing lymphocytes respond to immunization with proliferation and differentiation into plasmacytes that secrete multivalent VLRB antibodies. Here, we immunized lampreys with B cells from patients with chronic lymphocytic leukemia (CLL) to generate recombinant monoclonal VLRB antibodies, one of which, VLR39, was specific for the donor CLL cells. The target epitope of VLR39 was shown to be the complementarity determining region 3 (CDR3) of the heavy chain variable region (VH) of the B-cell receptor. Using this antibody to monitor the CLL donor after chemoimmunotherapy-induced remission, we detected VLR39+ B cells in the patient 51 months later, before significant increase in lymphocyte count or CD5+ B cells. This indication of reemergence of the leukemic clone was verified by VH sequencing. Lamprey antibodies can exhibit exquisite specificity for a protein epitope, a CLL signature VH CDR3 sequence in this case, and offer a rapid strategy for generating anti-idiotype antibodies for early detection of leukemia recurrence. Cancer Immunol Res; 1(4); 223–8. ©2013 AACR.

Clonal evolution occurs during the course of chronic lymphocytic leukemia (CLL) and activation-induced deaminase (AID) could influence this process. However, this possibility has been questioned in CLL because the number of circulating AID mRNA+ cells is exceedingly low; synthesis of AID protein by blood CLL cells has not been demonstrated; the full range of AID functions is lacking in unmutated CLL (U-CLL), and no prospective analysis linking AID expression and disease severity has been reported. The results of the present study show that circulating CLL cells and those within secondary lymphoid tissues can make AID mRNA and protein. This production is related to cell division because more AID mRNA was detected in recently divided cells and AID protein was limited to the dividing fraction and was up-regulated on induction of cell division. AID protein was functional because AID+ dividing cells exhibited more double-stranded DNA breaks, IGH class switching, and new IGHV-D-J mutations. Each of these actions was documented in U-CLL and mutated CLL (M-CLL). Furthermore, AID protein was associated with worse patient outcome and adverse cytogenetics. We conclude that the production of fully functional AID protein by U-CLL and M-CLL cells could be involved in clonal evolution of the disease.

on behalf of ERIC, the European Research Initiative on CLL; International audience; We report on markedly different frequencies of genetic lesions within subsets of chronic lymphocytic leukemia patients carrying mutated or unmutated stereotyped B-cell receptor immunoglobu-lins in the largest cohort (n=565) studied for this purpose. By combining data on recurrent gene mutations (BIRC3, MYD88, NOTCH1, SF3B1 and TP53) and cytogenetic aberrations, we reveal a subset-biased acquisition of gene mutations. More specifically, the frequency of NOTCH1 mutations was found to be enriched in subsets expressing unmutated immunoglobu-lin genes, i.e. #1, #6, #8 and #59 (22-34%), often in association with trisomy 12, and was significantly different (P

A high-performance liquid chromatographic method (HPLC) with evaporative light scattering detection (ELSD) has been developed for fingerprint analysis of Flos Lonicerae. The samples were separated with an Agilent C18 column using acetonitrile (A) and 0.1% formic acid solution under gradient conditions (0→10 min：11.5%→15% A, 10→12 min：15% →22% A, 12→18min：22%→33% A; 18→30min: 33%→45% A) as the mobile phase at a flow rate of 0.8ml min−1 within 30 min. The ELSD conditions were optimized at nebulizer-gas flow rate 2.7 L min−1 and drift tube temperature 105°C. Precision experiments showed relative standard deviation (R.S.D.) of peak area and retention time were better than 2.5%, inter-day and intra-day variabilities showed that R.S.D. was ranged from 0.66% to 4.17%. Accuracy validation showed that average recovery was between 95.33% and 104.10%. The method was validated to achieve the satisfactory precision and recovery. Relative retention time and relative peak area were used to identify the common peaks for fingerprint analysis. There are nine common peaks in the fingerprint. The quality of sixteen batches of Flos Lonicerae samples was evaluated by hierarchical clustering analysis, which classfied all bacthes into two clusters. Furthermore, the contents of important medicinal compounds (chlorogenic acid, macranthoidin B and dipsacoside B) in different batches of Flos Lonicerae samples were determined simultaneously using the developed HPLC-ELSD method. The developed analytical procedure was proved to be a reliable and rapid method for the quality control of Flos Lonicerae.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes that produce mAbs often reactive with microbial or autoantigens. Long-term culture of B-CLL clones would permit the collection and characterization of B-CLL mAbs to study antigen specificity and of B-CLL DNA to investigate molecular mechanisms promoting the disease. However, the derivation of long-term cell lines (eg, by EBV), has not been efficient. We have improved the efficiency of EBV B-CLL transformation of CpG oligonucleotide-stimulated cells by incubating patient peripheral blood mononuclear cells in the presence of an irradiated mouse macrophage cell line, J774A.1. Using this approach, peripheral blood mononuclear cells isolated from 13 of 21 B-CLL patients were transformed as documented by IGHV-D-J sequencing. Four clones grew and retained CD5 expression in culture for 2 to 4 months. However, despite documentation of EBV infection by expression of EBNA2 and LMP1, B-CLL cells died after removal of macrophage feeder cells. Nevertheless, using electrofusion technology, we generated 6 stable hetero-hybridoma cell lines from EBV-transformed B-CLL cells, and these hetero-hybridomas produced immunoglobulin. Thus, we have established enhanced methods of B-CLL culture that will enable broader interrogation of B-CLL cells at the genetic and protein levels.

Individual cytokines and groups of cytokines that might represent networks in chronic lymphocytic leukemia (CLL) were analyzed and their prognostic values determined. Serum levels of 23 cytokines were measured in 84 patients and 49 age-matched controls; 17 levels were significantly elevated in patients. Unsupervised hierarchical bicluster analysis identified 3 clusters (CLs) of highly correlated but differentially expressed cytokines: CL1 (CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL19, IL-5, IL-12, and IFNγ), CL2 (TNFα, IL-6, IL-8, and GM-CSF), and CL3 (IL-1β, IL-2, IL-4, IL-15, IL-17, and IFNα). Combination scores integrating expression of CL1/CL2 or CL1/CL3 strongly correlated (P < .005) with time-tofirst-treatment and overall survival (OS), respectively. Patients with the worst course had high CL1 and low CL2 or CL3 levels. Multivariate analysis revealed that CL1/CL2 combination score and immunoglobulin heavy chain variable region mutation status were independent prognostic indicators for time-to-first-treatment, whereas CL1/CL3 combination score and immunoglobulin heavy chain variable region mutation status were independent markers for OS. Thus, we identified groups of cytokines differentially expressed in CLL that are independent prognostic indicators of aggressive disease and OS. These findings indicate the value of multicytokine analyses for prognosis and suggest therapeutic strategies in CLL aimed at reducing CL1 and increasing CL2/CL3 cytokines.

An infectious etiology has been proposed for many human cancers, but rarely have specific agents been identified. One difficulty has been the need to propagate cancer cells in vitro to produce the infectious agent in detectable quantity. We hypothesized that genome amplification from small numbers of cells could be adapted to circumvent this difficulty. A patient with concomitant chronic lymphocytic leukemia (CLL) and polycythemia vera (PV) requiring therapeutic phlebotomy donated a large amount of phlebotomized blood to test this possibility. Using genome amplification methods, we identified a new isolate (BIS8-17) of torque teno virus (TTV) 10. The presence of blood isolate sequence 8-17 (BIS8-17) in the original plasma was confirmed by polymerase chain reaction (PCR), validating the approach, since TTV is a known plasma virus. Subsequent PCR testing of plasmas from additional patients showed that BIS8-17 had a similar incidence (~20%) in CLL (n = 48) or PV (n = 10) compared with healthy controls (n = 52). CLL cells do not harbor BIS8-17; PCR did not detect it in CLL peripheral blood genomic deoxyribonucleic acid (DNA) (n = 20). CLL patient clinical outcome or prognostic markers (immunoglobulin heavy chain variable region [IGHV ] mutation, CD38 or zeta-chain associated protein kinase 70 kDa [ZAP-70]) did not correlate with BIS8-17 infection. Although not causative to our knowledge, this is the first reported isolation and detection of TTV in either CLL or PV. TTV could serve as a covirus with another infectious agent or TTV variant with rearranged genetic components that contribute to disease pathogenesis. These results prove that this method identifies infectious agents and provides an experimental methodology to test correlation with disease.

Chronic inflammation promotes the formation of ectopic lymphoid tissue morphologically resembling secondary lymphoid tissues, though it is unclear whether this is a location where Ag-specific immune responses develop or merely a site of lymphocyte accumulation. Ectopic lymphoid tissue formation is associated with many humoral autoimmune diseases, including lupus induced by tetramethylpecadentane in mice. We examined whether an immune response to 4-hydroxy-3-nitrophenyl acetyl-keyhole limpet hemocyanin (NP-KLH) and NP-OVA develops within ectopic lymphoid tissue (“lipogranulomas”) induced by tetramethylpecadentane in C57BL/6 mice. Following primary immunization, NP-specific B cells bearing V186.2 and related heavy chains as well as λ-light chains accumulated within ectopic lymphoid tissue. The number of anti-NP-secreting B cells in the ectopic lymphoid tissue was greatly enhanced by immunization with NP-KLH. Remarkably, the H chain sequences isolated from individual lipogranulomas from these mice were diverse before immunization, whereas individual lipogranulomas from single immunized mice had unique oligo- or monoclonal populations of presumptive NP-specific B cells. H chain CDR sequences bore numerous replacement mutations, consistent with an Ag-driven and T cell-mediated response. In mice adoptively transferred with OT-II or DO11 T cells, there was a striking accumulation of OVA-specific T cells in lipogranulomas after s.c. immunization with NP-OVA. The selective colocalization of proliferating, Ag-specific T and B lymphocytes in lipogranulomas from tetramethylpecadentane-treated mice undergoing primary immunization implicates ectopic lymphoid tissue as a site where Ag-specific humoral immune responses can develop. This has implications for understanding the strong association of humoral autoimmunity with lymphoid neogenesis, which may be associated with deficient censoring of autoreactive cells.

Bispecific antibodies and antibody fragments in various formats have been explored as a means to recruit cytolytic T cells to kill tumor cells. Encouraging clinical data have been reported with molecules such as the anti-CD19/CD3 bispecific T cell engager (BiTE) blinatumomab. However, the clinical use of many reported T cell-recruiting bispecific modalities is limited by liabilities including unfavorable pharmacokinetics, potential immunogenicity, and manufacturing challenges. We describe a B cell-targeting anti-CD20/CD3 T cell-dependent bispecific antibody (CD20-TDB), which is a full-length, humanized immunoglobulin G1 molecule with near-native antibody architecture constructed using "knobs-into-holes" technology. CD20-TDB is highly active in killing CD20-expressing B cells, including primary patient leukemia and lymphoma cells both in vitro and in vivo. In cynomolgus monkeys, CD20-TDB potently depletes B cells in peripheral blood and lymphoid tissues at a single dose of 1 mg/kg while demonstrating pharmacokinetic properties similar to those of conventional monoclonal antibodies. CD20-TDB also exhibits activity in vitro and in vivo in the presence of competing CD20-targeting antibodies. These data provide rationale for the clinical testing of CD20-TDB for the treatment of CD20-expressing B cell malignancies.

Mansouri et al. applied targeted deep sequencing to identify mutations within NF-κB core complex genes in CLL. NFKBIE, the gene encoding the inhibitory IκBε molecule, was most frequently mutated, especially in poor-prognostic subgroups of CLL. The authors show that NFKBIE mutations were associated with significantly reduced IkBε expression and p65 inhibition, ultimately leading to NF-κB activation and a more aggressive disease., NF-κB is constitutively activated in chronic lymphocytic leukemia (CLL); however, the implicated molecular mechanisms remain largely unknown. Thus, we performed targeted deep sequencing of 18 core complex genes within the NF-κB pathway in a discovery and validation CLL cohort totaling 315 cases. The most frequently mutated gene was NFKBIE (21/315 cases; 7%), which encodes IκBε, a negative regulator of NF-κB in normal B cells. Strikingly, 13 of these cases carried an identical 4-bp frameshift deletion, resulting in a truncated protein. Screening of an additional 377 CLL cases revealed that NFKBIE aberrations predominated in poor-prognostic patients and were associated with inferior outcome. Minor subclones and/or clonal evolution were also observed, thus potentially linking this recurrent event to disease progression. Compared with wild-type patients, NFKBIE-deleted cases showed reduced IκBε protein levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65. Considering the central role of B cell receptor (BcR) signaling in CLL pathobiology, it is notable that IκBε loss was enriched in aggressive cases with distinctive stereotyped BcR, likely contributing to their poor prognosis, and leading to an altered response to BcR inhibitors. Because NFKBIE deletions were observed in several other B cell lymphomas, our findings suggest a novel common mechanism of NF-κB deregulation during lymphomagenesis.

B cell chronic lymphocytic leukemia (B-CLL) is a clonal overgrowth of CD5+B lymphocytes. In this disease, the B cell antigen receptor (BCR) is intimately linked to disease severity, because patients with BCRs, comprised of unmutated VHgenes, follow a much more aggressive course. This and related observations suggest that B-CLL derives from a B cell subset comprised of restricted BCR structural diversity and that antigen-selection and drive are major factors promoting the disease. Nevertheless, the initiating event(s) that lead to the development of B-CLL are still unclear, in part because of the lack of an animal model that spontaneously evolves the molecular abnormalities that occur in the human disease. Because overexpression of the TCL1 gene in murine B cells leads to a CD5+B cell lymphoproliferative disorder with many of the features of human B-CLL, we studied leukemias emerging in these mice to examine the extent to which their BCRs resemble those in B-CLL. Our data indicate that the immunoglobulin heavy and light chain rearrangements in TCL1 mice display minimal levels of somatic mutations and exhibit several molecular features found in the human disease. Like human B-CLL, TCL1 leukemic rearrangements from different mice can be very similar structurally and closely resemble autoantibodies and antibodies reactive with microbial antigens. Antigen-binding analyses confirm that selected TCL1 clones react with glycerophospholipid, lipoprotein, and polysaccharides that can be autoantigens and be expressed by microbes. This (auto)antigen-driven mouse model reliably captures the BCR characteristics of aggressive, treatment-resistant human B-CLL.

Adventitious shoots were successfully regenerated from hypocotyl explants of in vitro cultures of Euonymus japonicus Cu zhi. Hypocotyl slices were cultured on Murashige and Skoog (MS) and B 5 basal medium supplemented with varied concentration of different plant growth-regulators, e.g., α-naphthaleneacetic acid (NAA) and indole-3-butyric acid (IBA) in combination with 6-benzylaminopurine (6-BA) and kinetin. The study showed that shoots could be directly regenerated from hypocotyl explants without the intervening callus phase; MS medium was more suitable for adventitious shoots regeneration. The ability of hypocotyls segments to produce shoots varied depending upon their position on the seedlings. The highest regeneration rate was obtained with hypocotyl segments near to the cotyledon cultured on MS basal medium supplemented with 1.5 mg L −1 6-BA and 0.05 mg L −1 NAA (63.64%). The regenerated shoots were readily elongated on the same medium as used for multiplication and rooted on half-strength MS basal medium supplemented with 1.0 mg L −1 IBA and 100 mg L −1 activated carbon. After being transferred to greenhouse conditions, 96% of the plantlets were successfully acclimatized. This regeneration system is applied for genetic transformation now.

Chronic lymphocytic leukemia (CLL) arises from the clonal expansion of a CD5+ B lymphocyte that is thought not to undergo intraclonal diversification. Using VHDJH cDNA single strand conformation polymorphism analyses, we detected intraclonal mobility variants in 11 of 18 CLL cases. cDNA sequence analyses indicated that these variants represented unique point-mutations (1–35/patient). In nine cases, these mutations were unique to individual submembers of the CLL clone, although in two cases they occurred in a large percentage of the clonal submembers and genealogical trees could be identified. The diversification process responsible for these changes led to single nucleotide changes that favored transitions over transversions, but did not target A nucleotides and did not have the replacement/silent nucleotide change characteristics of antigen-selected B cells. Intraclonal diversification did not correlate with the original mutational load of an individual CLL case in that diversification was as frequent in CLL cells with little or no somatic mutations as in those with considerable mutations. Finally, CLL B cells that did not exhibit intraclonal diversification in vivo could be induced to mutate their VHDJH genes in vitro after stimulation. These data indicate that a somatic mutation mechanism remains functional in CLL cells and could play a role in the evolution of the clone.

The targeting of mutations by Activation-Induced Deaminase (AID) is a key step in generating antibody diversity at the Immunoglobulin (Ig) loci but is also implicated in B-cell malignancies such as chronic lymphocytic leukemia (CLL). AID has previously been shown to preferentially deaminate WRC (W = A/T, R = A/G) hotspots. WGCW sites, which contain an overlapping WRC hotspot on both DNA strands, mutate at much higher frequency than single hotspots. Human Ig heavy chain (IGHV) genes differ in terms of WGCW numbers, ranging from 4 for IGHV3-48*03 to as many as 12 in IGHV1-69*01. An absence of V-region mutations in CLL patients ("IGHV unmutated", or U-CLL) is associated with a poorer prognosis compared to "IGHV mutated" (M-CLL) patients. The reasons for this difference are still unclear, but it has been noted that particular IGHV genes associate with U-CLL vs M-CLL. For example, patients with IGHV1-69 clones tend to be U-CLL with a poor prognosis, whereas patients with IGHV3-30 tend to be M-CLL and have a better prognosis. Another distinctive feature of CLL is that ~30% of (mostly poor prognosis) patients can be classified into "stereotyped" subsets, each defined by HCDR3 similarity, suggesting selection, possibly for a self-antigen. We analyzed >1000 IGHV genes from CLL patients and found a highly significant statistical relationship between the number of WGCW hotspots in the germline V-region and the observed mutation frequency in patients. However, paradoxically, this correlation was inverse, with V-regions with more WGCW hotspots being less likely to be mutated, i.e., more likely to be U-CLL. The number of WGCW hotspots in particular, are more strongly correlated with mutation frequency than either non-overlapping (WRC) hotspots or more general models of mutability derived from somatic hypermutation data. Furthermore, this correlation is not observed in sequences from the B cell repertoires of normal individuals and those with autoimmune diseases.

B cell receptor signaling is a key factor in chronic lymphocytic leukemia (CLL), evinced by inhibitory drug ibrutinib's efficacy. Studies of normal and CLL B cells indicate surface membrane IgM or IgD engagement has diverse signaling consequences. But little is known about relative amounts of sIg/co-receptor components within intraclonal fractions and how their compositions affect signaling. We studied relative densities of IgM and IgD and Ig-associated stimulatory and inhibitory co-receptors in two subsets based on: [1] relative densities of IgM and IgD and [2] reciprocal expression of CXCR4/CD5 indicating activation state. Samples from 5 U and 5 M-CLL patients, pre and during (4-6 weeks) ibrutinib treatment, were tested by conventional and by imaging flow cytometry using an ISX Mark II providing multiple spectral images of individual cells in a flow setting. After subfractionating clones for IgM (IgMDim, IgMInt, IgMBright) or IgD (IgDDim, IgDInt, IgDBright) densities, we quantified relative densities of stimulatory/inhibitory molecules on the subsets. A directly proportional change was observed for CD5, CD19, CD20, Siglec10, CD25, HLA-DR, and CD38 as IgM moved from Dim to Bright. Exceptions were CXCR4, which dramatically decreased as IgM density increased, and CD22, that had a constant density in all fractions. Similar changes were seen for IgD except for CD22 which increased in IgDBright density. Again CXCR4 showed the opposite pattern. CD25 and HLADR remained constant within IgD increments. Stimulatory markers CD25 and HLADR changed upward only in the transition to IgMBright. Together these imply signaling through IgM heightens as IgM density increases, but does not through IgD, likely due to increased Siglec 10 and CD22. The reduced amounts of smCXCR4 suggest impaired ability of cells with high smIgM or smIgD to traffic. We examined subpopulations based on CXCR4 and CD5. IgD and IgM densities increased from resting (RF) CXCR4Bright/CD5Dim to intermediate (INT) CXCR4Int/CD5Int to proliferative (PF) CXCR4Dim/CD5Bright fractions, although the degree of upregulation was more marked for IgD than IgM. All stimulatory/inhibitory coreceptors also increased in density from RF to PF. Interestingly, CD22 and IgD retained a constantdensity from RF to INT but increased considerably at the PF. Although stimulatory molecules CD25, CD38 and HLADR had upward trends, peaking occurred in the PF. Collectively, this implies signaling capacity through smIgM amplifies toward the PF due to higher smIgM density and IgD upregulation. Finally, we evaluated CLL clones as a whole as well as based on the subsets above for ibrutinib treatment induced changes. Clonally, a density decrement occurred for smIgD and an increment for smIgM. Data for the other molecules fell into 3 categories based on relative density changes: Decreasing: CD5, CD20, CD38, CD25, HLA-DR; Increasing: CXCR4: Invariant: CD22, CD19, Siglec10. For subfractionating based on smIgs density, ibrutinib drastically reduced density differences for co-receptors and activation markers among IgM or IgD density subpopulations with IgMBright and IgDBright fractions more affected than the Dim and Intermediate density counterparts. Similarly for the CXCR4/CD5 subpopulations, lower differences in relative expression of coreceptors from RF to PF were found. As for IgM and IgD levels, the PF was the most reduced. The composite effect was a reduced slope of change among the original sets of density change categories (IgMDim -> IgMBright; IgDDim -> IgDBright; RF to PF), leading to more phenotypically homogenous subpopulations. In summary, prior to ibrutinib therapy, both the Ig and most of the associated molecules increase density in subsets marked by increased IgM and IgD densities and marked by decreasing CXCR4/CD5 densities. However, there was a major difference in density correlations for CXCR4 and CD22. CXCR4, levels were lowest on IgMBright and IgDBright cells which differs from published data using anti-IgM beads. For CD22 the highest levels were in IgDBright but not in IgMBright and in CXCR4/CD5 subset with the highest IgD and IgM (CXCR4Dim/CD5Bright). In contrast, ibrutinib treatment led to an overall change in coreceptor molecules altering considerably the density relationships with IgM and IgD. Concomitantly, these culminate in a downregulatory membrane stimulatory environment. Disclosures No relevant conflicts of interest to declare.

CLL results from the accumulation of monoclonal B lymphocytes that derive from a small fraction of cells with proliferative activity. Because expression of the DNA mutator, activation-induced cytidine deaminase (AID) is restricted to these dividing cells, they can develop new DNA abnormalities leading to more lethal disease. Hence, such cells are important targets for therapy. Our previous study indicated that the B-cell subset with low levels of CXCR4 and high levels of CD5 (CXCR4DimCD5Bright) is enriched in these cells ("proliferative fraction", PF), whereas the less vital, resting cells exhibit a CXCR4BrightCD5Dim phenotype ("resting fraction", RF). In this study, we focused on analyzing the significantly differentially expressed genes (DEGs) between PF and RF. PF and RF were isolated from 26 CLL (13 U-CLL and 13 M-CLL) and microarrays (llumina HT12) were performed. Selected DEGs between PF and RF were confirmed by rtQ-PCR and/or by flow cytometry. Array data were interpreted using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). First, focusing on the Immunologic Signature of B lymphocytes with GSEA, we found the PF was enriched in gene sets shared with IgM memory cells and pre-germinal center B cells, whereas RF was enriched in gene sets in common with naïve, IgD+ B cells. Notably, the PF also shared gene sets with myeloid dendritic cells and monocytes. Protein expression of 10 myeloid markers (CD68, CD1c, CD11a, CD11b, CD11c, CD13, CD31, CD205, CXCR3 and CLECL1) was documented in PF B cells from 11 U-CLL and 11 M-CLL patients, and each was more highly expressed in the PF than RF. No difference in myeloid markers was observed between U-CLL and M-CLL, suggesting that this expression is independent of IGHV mutation status. DEGs were also determined based on expression ratios for PF and RF for each patient; t tests were performed using R. With a fold change cutoff of > 1.5 or < -1.5 and a level of significance of P < 0.01, we identified 198 genes significantly upregulated in PF and 88 in RF. The top biological-function categories identified using IPA indicated that these genes related to cellular development (n=37), cellular growth and proliferation (n=49), cellular movement (n=42) and cell survival and death (n=50). In addition, these DEGs mapped to 8 canonical pathways with Z-scores ≥2, the highest being the integrin signaling pathway (Z-score = 2.887). Twelve of 13 genes were upregulated in the PF and correlated significantly with the integrin pathway: ACTG, ARPC5, ARPC1B, BCAR3, CAPNS1, ITGAX, ITGB1, ITGB2, ITGB7, PFN1, RAC2 and RHOC. Upregulation of integrin subunits was confirmed by Q-PCR and cell surface staining by flow cytometry. Preliminary cellular adhesion experiments suggest PF bind to fibronectin coated plates, and those cells that bind survive better. Next, comparing PF and RF expression ratios for U-CLL vs. M-CLL revealed 502 DEGs in U-CLL and 179 in M-CLL; 144 genes were shared by both U-CLL and M-CLL. IPA analysis of the latter genes correlated best with integrin signaling, and the potential upstream regulators of these were IFNg, IL1, F2 and IFNa. The Rho Family GTPases signaling pathway was significant (10 genes) for DEGs unique to U-CLL. No significant pathway or bio-function relationship was observed in DEGs only in M-CLL. Finally, IPA analysis showed IL4 as an upstream regulator of DEGs unique to the PF of U-CLL based on upregulation of 12 IL4 target genes (APRT, CCL5, CDKN1A, DECR1, IL17RB, ITGAL, ITGB1, LTA, MAPKAPK3, PIGR, SAMSN1, TIMP1). Three other IL-4 targeted genes were paradoxically under-represented in the U-CLL PF (BCL2, CCR7 and VIPR1). The upregulated findings are consistent with PF B cells inducing T cells to produce IL-4 via co-receptors on B/myeloid cells that foster a Th2 response (e.g., CLECL1). In conclusion, gene expression profiling indicates that cells of the PF display a dual activated B cell/myeloid cell phenotype suggesting enhanced antigen-presentation capacities. Integrin signaling appears to be a key pathway for these cells which could foster cell proliferation, survival, and migration, especially in U-CLL clones where integrin activation can lead to Rho GTPases activation. Finally, genes regulated by IL-4 in the PF could be induced by interactions of autologous T cells with CLL B cells. These findings suggest antigen-presentation and integrin and IL-4 signaling pathways as therapeutic targets in CLL, particularly for U-CLL. Disclosures Barrientos: AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Janssen: Consultancy.

In chronic lymphocytic leukemia (CLL) monoclonal B cells expand and progressively accumulate in the bone marrow, eventually migrating to secondary lymphoid organs for even greater proliferation. At both sites, CLL cells engage in complex, incompletely defined cellular and molecular interactions involving multiple cell types such as T cells, myeloid cells, mesenchymal stromal cells, and matrix, collectively referred to as the "tumor microenvironment". This microenvironment is critical for the survival and proliferation of CLL cells, and data indicate that T cells and myeloid cells have an important role in these processes. In this study, we focus on two cells types: CD4+ T lymphocytes and myeloid-derived suppressor cells (MDSCs). In CLL patients, these populations are altered and impact on clinical outcome. CD4+ T cells comprise several subtypes, and CLL patients often have expanded Th2 and Tregs populations, consistent with the immunosuppressive status of these patients. Moreover, patients with higher numbers of another CD4+ subset, Th17 cells that produce IL-17 and other pro-inflammatory cytokines, can have longer survival times. Although studied minimally in CLL, MDSCs are known suppressors of T cell proliferation in vitro, and expand along with malignant cells in several cancers. However, no information is available about their effects on CD4+ T cell differentiation or on B-cell biology in CLL. In a cohort of 56 untreated CLL patients, we first explored correlation of the numbers of MDSCs and autologous T cells, using flow cytometry. CD3+ cell numbers significantly paralleled total MDSCs and monocyte-like MDSCs (mMDSCs) (P = 0.002, Spearman r = 0.44; P = 0.004, Spearman r = 0.41, respectively). Interestingly, MDSCs correlated with CD4+ and CD8+ T-cells (P < 0.001, Spearman r = 0.646; P < 0.001, Spearman r = 0.61, respectively). However, the correlation of MDSC subpopulations with CD4+ and CD8+ cells differed: mMDSCs associated significantly with CD4+ cells (P < 0.001, Spearman r = 0.73) and granulocyte-like MDSCs (gMDSCs) with CD8+ cells (P= 0.008; Spearman r = 0.45). Furthermore, although gMDSCs did not correlate with the numbers of CD4+ T-cells, we observed that they positively paralleled Tregs defined as CD3+/CD4+/CD25+/CD127-/FoxP3+ cells (P = 0.020, Spearman r = 0.44). Other subpopulations are currently under study. To address the effect of MDSCs on CD4+ cell differentiation, we FACS sorted CD3+/CD45RO- naïve CD4+ lymphocytes and stimulated them in vitro with anti-CD3/CD28 beads and IL2 in the presence or absence of mMDSCs (HLA-DRlo/CD11b+/CD33+/CD14+), gMDSCs (HLA-DRlo/CD11b+/CD33+/CD15+) or monocytes (HLA-DRhi/CD11b+/CD14+); these studies involved samples from 3 CLLs and 3 healthy controls (HCs). On day 7, cells were harvested and cytokine production was quantified by intracellular flow cytometry as the percentages of the following populations: Th1 (INFγ), Th2 (IL-4), Tregs (FoxP3), Th17 (IL-17A and IL-17F), Th9 (IL-9) and Th22 (IL-22). Culturing CLL or HC T cells in the absence of MDSCs revealed a lower percentage of cytokine-producing cells (24% vs. 55%; P = 0.017) in CLL, which was mainly due to a reduction in IL-4+ cells (P = 0.066). However, when analyzing the effects of MDSC subsets on the polarization of CLL or HC T cell, gMDSCs promoted significantly more FoxP3+ and less IL-22+ cells in CLL than in HC (P = 0.025 and P = 0.048, respectively). When analyzing only CLL T cells, supplementation with mMDSCs induced a reduction in IL-22+ cells (P = 0.027) and an insignificant increase of IL-4+ and IL-17+ cells. Conversely monocytes supported an expansion of INFγ+ T-cells (P=0.066), and gMDSCS promoted an increase of IL-9+ cells (P = 0.046) and a reduction of FoxP3+ cells (P = 0.019). In summary, in CLL the absolute numbers of total MDSCs and T cells are tightly linked. There is a significant correlation between CD4+ T cells and mMDSCs, and between CD8+ T cells and gMDSCs. Additionally, in CLL naïve CD4+ differentiation appears reduced compared to HC, in concordance with lower T-cell responses previously reported. Moreover, the preliminary aspects of the study suggest that CLL mMDSCs promote an expansion of Th2, Th17 cells and a reduction of Th22 cells, and monocytes enhance Th1s. Unexpectedly, since we observed a significant positive correlation in the PBMCs, gMDSCs may reduce Tregs and augment Th9. These findings depict differential consequences of CLL T cell - MDSC / mMDSC / gMDSC interactions. Disclosures Stamatopoulos: Abbvie: Honoraria, Other: Travel expenses; Gilead: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Ag selection has been suggested to play a role in chronic lymphocytic leukemia (CLL) pathogenesis, but no large-scale analysis has been performed so far on the structure of the Ag-binding sites (ABSs) of leukemic cell Igs. We sequenced both H and L chain V(D)J rearrangements from 366 CLL patients and modeled their three-dimensional structures. The resulting ABS structures were clustered into a small number of discrete sets, each containing ABSs with similar shapes and physicochemical properties. This structural classification correlates well with other known prognostic factors such as Ig mutation status and recurrent (stereotyped) receptors, but it shows a better prognostic value, at least in the case of one structural cluster for which clinical data were available. These findings suggest, for the first time, to our knowledge, on the basis of a structural analysis of the Ab-binding sites, that selection by a finite quota of antigenic structures operates on most CLL cases, whether mutated or unmutated.

(Auto)antigen engagement by the B-cell receptor (BCR) and possibly the sites where this occurs influence the outcome of chronic lymphocytic leukemia (CLL). To test if selection for autoreactivity leads to increased aggressiveness and if this selection plays out equally in primary and secondary tissues, we used T-cell leukemia (TCL)1 cells reactive with the autoantigen phosphatidylcholine (PtC). After repeated transfers of splenic lymphocytes from a single mouse with oligoclonal PtC-reactive cells, outgrowth of cells expressing a single IGHV-D-J rearrangement and superior PtC-binding and disease virulence occurred. In secondary tissues, increased PtC-binding correlated with enhanced BCR signaling and cell proliferation, whereas reduced signaling and division of cells from the same clone was documented in cells residing in the bone marrow, blood, and peritoneum, even though cells from the last site had highest surface membrane IgM density. Gene-expression analyses revealed reciprocal changes of genes involved in BCR-, CD40-, and PI3K-signaling between splenic and peritoneal cells. Our results suggest autoantigen-stimulated BCR signaling in secondary tissues promotes selection, expansion, and disease progression by activating pro-oncogenic signaling pathways, and that--outside secondary lymphoid tissues--clonal evolution is retarded by diminished BCR-signaling. This transferrable, antigenic-specific murine B-cell clone (TCL1-192) provides a platform to study the types and sites of antigen-BCR interactions and genetic alterations that result and may have relevance to patients.

While many prognostic markers in B-cell chronic lymphocytic leukemia provide insight into the biology of the disease, few have been demonstrated to be useful in the daily management of patients. B-cell receptor signaling is a driving event in the progression of B-cell chronic lymphocytic leukemia and markers of B-cell receptor responsiveness have been shown to be of prognostic value. Single cell network profiling, a multiparametric flow cytometry-based assay, allows functional signaling analysis at the level of the single cell. B-cell receptor signaling proteins (i.e. p-SYK, p-NF-κB p65, p-ERK, p-p38, p-JNK) were functionally characterized by single cell network profiling in samples from patients with B-cell chronic lymphocytic leukemia in an exploratory study (n=27) after stimulation with anti-IgM. Significant associations of single cell network profiling data with clinical outcome (i.e. time to first treatment), as assessed by Cox regression models, were then confirmed in patients' samples in two other sequential independent studies, i.e. test study 1 (n=30), and test study 2 (n=37). In the exploratory study, higher responsiveness of the B-cell receptor signaling proteins to anti-IgM was associated with poor clinical outcomes. Patients' clustering based on signaling response was at least as powerful in discriminating different disease courses as traditional prognostic markers. In an unselected subgroup of patients with Binet stage A disease (n=21), increased anti-IgM-modulated p-ERK signaling was shown to be a significant, independent predictor of shorter time to first treatment. This result was independently confirmed in two test cohorts from distinct populations of patients. In conclusion, these findings support the utility of the single cell network profiling assay in elucidating signaling perturbations with the potential for the development of a clinically useful prognostic test in patients with early stage B-cell chronic lymphocytic leukemia. These data support the clinical relevance of B-cell receptor signaling in B-cell chronic lymphocytic leukemia, and suggest a key role of ERK activation in the physiopathology of this leukemia.

Streptococcus suis, one of the most important and prevalent pathogens in swine, presents a major challenge to global public health. HP0197 is an S. suis surface antigen that was previously identified by immunoproteomics and can bind to the host cell surface. Here, we investigated the interaction between HP0197 and the host cell surface glycosaminoglycans (GAGs) using indirect immunofluorescence and cell adhesion inhibition assays. In addition, we determined that a novel 18-kDa domain in the N-terminal region of HP0197 functions as the GAG-binding domain. We then solved the three-dimensional structures of the N-terminal 18-kDa and C-terminal G5 domains using x-ray crystallography. Based on this structural information, the GAG-binding sites in HP0197 were predicted and subsequently verified using site-directed mutagenesis and indirect immunofluorescence. The results indicate that the positively charged residues on the exposed surface of the 18-kDa domain, which are primarily lysines, likely play a critical role in the HP0197-heparin interaction that mediates bacterium-host cell adhesion. Understanding this molecular mechanism may provide a basis for the development of effective drugs and therapeutic strategies for treating streptococcal infections. Background: The function of the Streptococcus suis antigen HP0197 is unknown, but it may be potentially involved in pathogenesis. Results: A novel GAG-binding domain on HP0197 was identified, and its structure was determined. Conclusion: Three clusters of basic residues on an 18-kDa domain of HP0197 are critical for heparin binding and cell adhesion. Significance: Understanding this mechanism will contribute to the design of drugs to treat streptococcal infections.

Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of “CLL-biased” features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.

A mutational signature consistent with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) activity has been identified in somatic mutations found in large-scale surveys of ultra-deep sequencing data from many human cancers including chronic lymphocytic leukemia (CLL). APOBEC is a cytidine deaminase family made up of eleven genes, including AID (activation-induced cytidine deaminase) and APOBEC3B, both of which have been implicated in somatic mutation in various cancers, including CLL. These observations have led to the hypothesis that APOBEC cytidine deaminases may be driving somatic mutations leading to the development of more aggressive cancers. Therefore, we examined APOBEC gene family member RNA expression levels in CLL to test for correlations with expression levels and patient outcome. We further examined if CLL cells generated de novo APOBEC family member mutational patterns in the immunoglobulin variable region gene (IGHV) after implantation in a mouse xenograft model of CLL. CLL peripheral blood mononuclear cells (PBMCs) and associated clinical data were collected from patients after informed consent as approved by the Institutional Review Board at the North Shore-Long Island Jewish Health System and in accordance with the Helsinki Declaration. CLL samples were chosen based on availability with no pre-established inclusion/exclusion criteria. CLL RNA expression levels were examined by microarray or quantitative real-time PCR (qPCR). For microarray studies, CLL B cells were purified prior to RNA isolation and acquisition of microarray expression data using Illumina Human WG6 and HT12 bead chips, followed by quantile normalization using GenomeStudio software (Illumina). For qPCR, RNA expression from CLL PBMCs was measured relative to glyceraldehyde 3-phosphate dehydrogenase gene expression by Taqman assay with Roche UPL probes and LightCycler 480. To examine de novo mutations in CLL, the IGHV region was ultra-deep sequenced (Roche 454 FLX system) from human CLL cells recovered from the NOD/Shi-scid,γcnull (NSG) xenograft mouse model of CLL as approved by the Institutional Animal Care and Use Committee at the North Shore-Long Island Jewish Health System. CLL patient (N = 65) RNA expression by microarray showed very low levels of APOBEC1, 2, 3A, 3B, 3D, 4, and AID, modest levels of APOBEC3C and 3H, and high levels of APOBEC3F and 3G. Higher AID expression levels significantly correlated (P To test if CLL cells can acquire de novo mutations indicative of APOBEC gene family member activity, human CLL cells were transferred into NSG mice. After CLL cells proliferated for 4-14 weeks in this xenograft model, the IGHV region was amplified, ultra-deep sequenced, and analyzed for specific mutational characteristics of various APOBEC gene family members. The results of these ongoing analyses will be presented. In conclusion, the expression levels of the APOBEC gene family members AID, APOBEC3B, and potentially APOBEC3F and 3H, correlate with worse patient outcome. These data are consistent with the hypothesis that APOBEC gene family member activity may promote new mutations at sites outside the IG gene loci leading to the evolution of aggressive CLL. Disclosures Barrientos: Pharmacyclics, Celgene, and Genentech: Membership on an entity's Board of Directors or advisory committees; Gilead, Pharmacyclics, and AbbVie: Research Funding.

Introduction. Somatic hypermutation (SHM) and class-switch recombination (CSR) are critical physiologic events in an effective normal B-cell immune response. Notably, both SHM and CSR are initiated by activation-induced cytidine deaminase (AID). In CLL, IGHV clonal mutations correlate strongly with better clinical outcomes. Eμ-T-cell leukemia-1 (TCL1) transgenic (Tg) mice are a valuable model of CLL. However because SHM and CSR occur rarely in the leukemic clones from these animals, TCL1 Tg mice mimic only IGHV -unmutated CLL and hence may not provide an understanding of the roles of SHM and CSR in disease evolution. To address these issues, we developed two new TCL1 strains by interbreeding mice over-expressing AID in all cells (Em-TCL1xActin-AID) or only in B lymphocytes (Em-TCL1xVκ-AID). Methods. B-cell clonal expansions were identified in spleen cells from 22 TCL1 and 33 TCL1xAID Tg (10 Em-TCL1xActin-AID plus 23 Em-TCL1xVκ-AID) mice at 10-20 months of age. This was done by amplifying cDNAs by PCR using consensus IGHV FR and IgM-, IgG-, and IgA-specific primers for IgH chains and Vκ FR and Cκ for IgL chain genes. DNA sequences of these amplicons were compared to murine germline IGHV s and IGKV s, and IGHV-D-JH and IGKV-JK rearrangements by IMGT V-Quest. Because there were no major differences in the parameters listed below for the two TCL1xAID Tg mouse strains, data were combined. Results. Clonal expansions: Monoclonal/oligoclonal expansions were detected in all TCL1 mice; these used only µ H and κ L chains. Similar expansions were detected in 31 of 33 TCL1xAID mice; each animal bore an IgMκ+ clone and 7 also exhibited an IgGκ+ clone. IGHV gene use in IgMκ+ clones: IGHV use did not differ significantly between IgMκ+ TCL1 and IgMκ+ TCL1xAID clones. Approximately 50% used VH1-55, VH11-2, or VH12-3, some of which encoded stereotyped anti-phosphatidylcholine antibodies. SHM: Among the IgM+ TCL1 clones, there was a mutation frequency of 0.05% for IGHV and 0.09% IGKV. In contrast, the IGHV and IGKV mutation frequencies were considerably higher in TCL1xAID mice. Specifically, for IGHV, the TCL1xAID animals displayed a frequency of 0.47% in IgM+ and 3.0% in IgG+ clones; for IGKV, the frequency was 0.9% for IgM+ and IgG+ combined. Consistent with these mutations being mediated by AID, mutations localized more frequently in AID hotspots than coldspots at a ratio of ≥7:1. However, SHM did not affect all clones equally. Although the mutation frequency in VH12-3 and VH11-2 clones was higher (0.38%, range: 0-1.9%) than the TCL1 level (0.05%), it was considerably less than that found in the TCL1xAID clones using other IGHV s (0.80%, range: 0-3.8%). In addition, no mutations were detected in VH1-55 clones. Consistent with a reduced AID mutational activity in clones expressing these specific genes, none of the genes were found in the isotype-switched, IgG-expressing clones. IGHV gene use and SHM in clones that underwent CSR: Notably, in only 2 of 9 instances was the same IGHV-D-J rearrangement found in IgMκ+ and IgGκ+ clones from the same mice; these IgM+ and IgG+ shared clones used VH5 genes. For the remaining 7, only the IgG+ version was detected; all but one of these used a VH1 gene. Also, within the IgG-only group, IGHV1-47 was used by 2 different clones and these were highly mutated (8.9%). Stereotyped IGHV-D-J and IGκ V-Jκ rearrangements: Among 43 IgM clones from TCL1xAID mice, we found 8 clones expressing VH11-2 and Vκ14-126 and 10 clones expressing VH12-3 and Vκ4-91; these IGHV-D-J and IGκV-Jκ rearrangements were very similar to canonical anti-phosphatidylcholine-producing clones. Summary and conclusions.Over-expression of AID in TCL1 mice leads to markedly increased SHM and CSR. However, SHM is not equivalent for all IGHV genes since despite AID over-expression certain IGHVs and IGKVs appeared less sensitive to major increases in the extent of SHM and the occurrence of CSR. This property resembles some human CLL IGHV s that rarely develop SHMs or undergo CSR despite the B-cell's ability to synthesize AID (e.g., many IGHV1-69+ clones). AID overexpression also led to IgG+ clones for which an IgM precursor was not found. This resembles those human stereotyped CLL clones that are only found as IgGs (e.g., stereotyped subsets 4 and 8). Finally, the two new TCL1xAID mouse strains described provide new models to study IGHV -mutated and IGHV -unmutated CLL and represent novel tools to evaluate the role of AID in leukemic progression. Disclosures No relevant conflicts of interest to declare.

Immune imbalance is a common characteristic of patients with chronic lymphocytic leukemia (CLL). This feature is shared with Eμ-TCL1 transgenic mice that, like CLL patients, exhibit an expansion of CD5+ B cells with associated non-B-cell defects. In patients and in mice, T-cell responses are often ineffective. This alteration is generally considered due to the direct effects of the leukemic cells. The expansion of myeloid derived suppressor cells (MDSCs), which play a major role in helping tumor cells escape immune surveillance by inhibiting T-cell responses, is promoted by many cancers. MDSCs are a heterogeneous population of cells that are subdivided into monocyte-like (m-MDSC) and granulocyte-like (g-MDSC) subsets, both in humans and mice. There we have investigated the extent that patients with CLL have expansions of MDSCs, what are their types and functions, and how these correlate with the Eμ-TCL1 mice model. Using flow cytometry on cryopreserved PBMCs, we found that the absolute numbers of MDSCs (HLA-DRlo/CD11b+/CD33+) in 49 untreated CLL patients were significantly higher than 15 healthy controls (HCs) (966 446 vs. 163 578 cells/ml, P When we evaluated the ability of MDSCs to inhibit autologous T-cell proliferation in CLL patients (n=7), we observed a consistent reduction of proliferation only when co-culturing with g-MDSCs(P=0.034). In contrast, the effects of m-MDSCs on T-cell expansion were varied and insignificant statistically. In 5 CLL samples, we induced m-MDSCs (im-MDSCs) from purified CD33+ cells in vitro with GM-CSF, IL10, and IL6; the im-MDSCs effectively suppressed T-cell proliferation in 4 of 5 cases at an average inhibition of 33% (range: 10-79%). Thus, dysfunctional m-MDSC suppression was not inherent and functional suppression could be achieved by stimulation of CLL precursor cells. Similarly in 3 independent experiments performed with MDSCs from Eμ-TCL1 mice (12-14 months of age), we observed a reduction of in vitro proliferation with g-MDSCs (P=0.049) and not with m-MDSCs. In addition, for those Eμ-TCL1 animals for which sufficient sample was available, we subdivided the g-MDSC population into the two subpopulations based on CD11b density; the CD11blo subset present less nuclear segmentation and higher suppressive activity. In summary, absolute numbers of MDSCs in the blood of CLL patients and Eμ-TCL1 mice are elevated and correlate with the levels of expansion of the leukemia. The major subtype in both situations was g-MDSCs.These g-MDSCs were functionally competent suppressors, whereas m-MDSCs were impaired in this function. In CLL patients, this m-MDSC suppressor defect could be corrected by in vitro stimulation with growth factors that support monocyte differentiation. The high similarity between CLL patients and Eμ-TCL1 mice in relation to MDSC number and function suggest that an imbalance in g-MDC vs. m-MDSC function may affect CLL development and expansion, altering interactions with members of the microenvironment such as T cells. Disclosures No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia (CLL) is an incurable disease in which most of the tumor cells in the blood are arrested in G0/G1 stages of the cell cycle with only a minimal number displaying proliferative activity. In this regard, our group has found by gene expression profiling (GEP) that the proliferative fraction (PF) of CLL cells is enriched in the intraclonal subset marked by CXCR4dim CD5brite expression. Indeed, this subset differs by more than 1000 genes from the CXCR4brite CD5dim resting fraction (RF). The genes over-expressed in the PF relate to replication and migration as well as regulation of gene expression. One of these genes is Musashi 2 (MSI2). Of note, MSI2 is expressed at the highest levels in IGHV unmutated CLL (U-CLL) clones and their PFs. Normally, MSI2 binds mRNA and blocks translation of proteins, playing an important role in post-transcriptional regulation. In addition, MSI2 has been linked to proliferation of normal and malignant stem cells, tumorigenesis, and poor prognosis. In CLL, high MSI2 mRNA expression has been identified in patients with worse prognosis. Nevertheless, nothing is known about the function of MSI2 in CLL cells. Therefore, we have begun to study the biological role of MSI2 in B-CLL cells and its possible association with B-cell proliferation and CLL disease progression. Fist, we studied MSI2 protein expression by flow cytometry in CD19+ B cells from healthy donors (HD) and CD5+CD19+ cells from CLL patients, observing an up-regulation in CLL compared to HD. Also, we documented higher MSI2 expression in U-CLL compared to IGHV-mutated (M-CLL) CLL as well as HD. Within the leukemic clone, we observed that MSI2 expression was highest in the PF, lower in the intermediate (INT) fraction (defined as CXCR4int CD5int), and much lower in the RF (PF>INT>RF). The PF expressed 40% more MSI2 than the RF, suggesting that the highest amounts of MSI2 protein is in dividing and recently-divided cells of the clone. Since CLL B-cell proliferation occurs in the microenvironment of lymphoid organs, presumably delivered by external signals, we tested whether such signals could stimulate MSI2 expression. After stimulating CLL cells with TLR9 agonist + IL15 + IL2 in vitro MSI2 protein was up-regulated form 0.3 to 2.5 fold. In addition, the increase in MSI2 protein was associated with an enhancement in Ki-67+ cells and in phosphorylation of MAPK/ERK and AKT signaling components, measured by flow cytometry. These results suggest that signals from the microenvironment that induce cell growth and proliferation lead to MSI2 synthesis in CLL B cells. In order to study a possible association between MSI2 expression and cell division, we labeled CLL PBMCs with a dilutable cell tracer, CFSE, and then stimulated them in vitro with TLR9 agonist + IL15 + IL2. These studies indicated that MSI2 protein synthesis was increased in the activated cells and that MSI2 protein levels increased with each cell division. However, it was also clear that this increase was not directly associated to the extent of cell replication as CLL B cells from only 10% of the patients underwent 4 cycles of cell division. Since we observed an increase in MSI2 and Ki-67 expression after stimulation in all patients' clones but did not detect replication of CLL cells in all patients, we studied the effects of in vitro stimulation on cell cycle entry and completion and how this related to MSI2 expression. Experiments using propidium iodide to evaluate DNA content of PBMCs showed that in vitro stimulation increased the percentage of cells in S phase (5-25%) compared to control cells without activation ( These results, and the facts that MSI2 plays an important role in post-transcriptional regulation and is associated with cell proliferation and poor prognosis in cancer, suggest that a better understanding of the role of MSI2 in CLL patients will provide clues to understanding the birth and growth of CLL B cells and to identifying and designing new therapeutic strategies for the disease. Disclosures No relevant conflicts of interest to declare.

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.

The failure of chemotherapeutic regimens to eradicate cancers often results from the outgrowth of minor subclones with more dangerous genomic abnormalities or with self-renewing capacity. To explore such intratumor complexities in B-cell chronic lymphocytic leukemia (CLL), we measured B-cell kinetics in vivo by quantifying deuterium (2H)-labeled cells as an indicator of a cell that had divided. Separating CLL clones on the basis of reciprocal densities of chemokine (C-X-C motif) receptor 4 (CXCR4) and cluster designation 5 (CD5) revealed that the CXCR4dimCD5bright (proliferative) fraction contained more 2H-labeled DNA and hence divided cells than the CXCR4brightCD5dim (resting) fraction. This enrichment was confirmed by the relative expression of two cell cycle-associated molecules in the same fractions, Ki-67 and minichromosome maintenance protein 6 (MCM6). Comparisons of global gene expression between the CXCR4dimCD5bright and CXCR4brightCD5dim fractions indicated higher levels of pro-proliferation and antiapoptotic genes and genes involved in oxidative injury in the proliferative fraction. An extended immunophenotype was also defined, providing a wider range of surface molecules characteristic of each fraction. These intraclonal analyses suggest a model of CLL cell biology in which the leukemic clone contains a spectrum of cells from the proliferative fraction, enriched in recently divided robust cells that are lymphoid tissue emigrants, to the resting fraction enriched in older, less vital cells that need to immigrate to lymphoid tissue or die. The model also suggests several targets preferentially expressed in the two populations amenable for therapeutic attack. Finally, the study lays the groundwork for future analyses that might provide a more robust understanding of the development and clonal evolution of this currently incurable disease.

We investigated a mechanism leading to activation of murine T cells by staphylococcal enterotoxin E (SEE). L cells transfected with I-Ab genes but not control L cells supported IL-2 production by SEE-induced C57BL/6 T lymphoblasts upon restimulation with SEE. mAb to I-Ab markedly inhibited the above response. Flow cytometric analyses showed that SEE-induced C57BL/6 T lymphoblasts are composed of both CD4+ T cells and CD8+ T cells, and that larger parts of them bore V beta 11 (40-75%). mAb to V beta 11 markedly inhibited the SEE-induced proliferative response and IL-2 production by T cells. Analysis of SEE-induced IL-2 production in spleen cells from various mouse strains showed that C57BL/6 and B10.A(4R) mice (I-E, not expressed; V beta 11+ T cells, normally generated) are highly responsive to SEE. In contrast, BALB/c, C3H/HeN, (C57BL/6 x BALB/c or C3H/HeN) F1 mice (I-E, normally expressed and V beta 11+ T cells, deleted), and SJL and C57L mice (V beta 11 genes, deleted) are weakly responsive to SEE. The results indicate that SEE activates mainly T cells bearing V beta 11 in physical association with MHC class II molecules expressed on AC. In addition, the results indicate that SEE activates both CD4+ T cells and CD8+ T cells.

Human umbilical vascular endothelial cells (HUVEC) stimulated with recombinant human IFN-γ were investigated for expression of HLA class II molecules, toxic shock syndrome toxin-1 (TSST-l)-binding activity and accessory cell (AC) activity in TSST-1-induced T cell activation. HUVEC stimulated with recombinant human IFN-γ ranging from 10 to 1,000 units/ml for 72 h express HLA class II molecules. Intensity of the expression was dependent on the concentration of IFN-γ. HUVEC stimulated with 1,000 units/ml of IFN-γ for 72 h exhibited 125I-TSST-l-binding that was blocked by the unlabeled toxin and monoclonal antibodies (mAb) to DR/DP. The activity was not removed by paraformaldehyde fixation. The IFN-γ-stimulated HUVEC exhibited AC activity in TSST-1-induced IL-2 production by T cells from human peripheral blood mononuclear cells. The activity was blocked by mAb to DR. The above two activities were also observed in L cells transfected with DR2 genes but not in the unstimulated HUVEC and control L cells. In view of the fact that TSST-1 binds directly to HLA class II molecules and activates human T cells in association with HLA class II molecules on AC, it is likely that the acquisition of TSST-1-binding activity and AC activity in the toxin-induced T cell activation is mediated by the expression of HLA class II molecules. Vascular endothelial cells may play an important role in the development of pathological changes in TSS patients.

Leukemic B lymphocytes of a large group of unrelated chronic lymphocytic leukemia (CLL) patients express an unmutated heavy chain immunoglobulin variable (V) region encoded by IGHV1-69, IGHD3-16, and IGHJ3 with nearly identical heavy and light chain complementarity-determining region 3 sequences. The likelihood that these patients developed CLL clones with identical antibody V regions randomly is highly improbable and suggests selection by a common antigen. Monoclonal antibodies (mAbs) from this stereotypic subset strongly bind cytoplasmic structures in HEp-2 cells. Therefore, HEp-2 cell extracts were immunoprecipitated with recombinant stereotypic subset-specific CLL mAbs, revealing a major protein band at approximately 225 kDa that was identified by mass spectrometry as nonmuscle myosin heavy chain IIA (MYHIIA). Reactivity of the stereotypic mAbs with MYHIIA was confirmed by Western blot and immunofluorescence colocalization with anti-MYHIIA antibody. Treatments that alter MYHIIA amounts and cytoplasmic localization resulted in a corresponding change in binding to these mAbs. The appearance of MYHIIA on the surface of cells undergoing stress or apoptosis suggests that CLL mAb may generally bind molecules exposed as a consequence of these events. Binding of CLL mAb to MYHIIA could promote the development, survival, and expansion of these leukemic cells.

Since its discovery in follicular lymphoma cells at the breakpoint t(14;18), Bcl-2 has been studied extensively in many basic and clinical science settings. Bcl-2 can locate as an integral mitochondrial membrane component, where its primary role is to block apoptosis by maintaining membrane integrity. Here we show that Bcl-2 also can position on the outer cell surface membrane of B cells from patients with chronic lymphocytic leukemia (B-CLL) and certain other leukemias that do not classically possess the chromosomal breakpoint t(14;18). Although low levels of Bcl-2 can be detected on the surface membrane of apparently healthy leukemic and normal B cells, expression of Bcl-2 correlates best with spontaneous or induced apoptosis. Notably, upon induction of apoptosis, B-CLL cells were much more efficient in upregulating surface Bcl-2 than normal B cells. It is not clear if this surface membrane expression is a passive consequence of the apoptotic process or an active attempt by the B cell to abort cell death by stabilizing the plasma membrane.