Mechanism for IL-15-Driven B Cell Chronic Lymphocytic Leukemia Cycling: Roles for AKT and STAT5 in Modulating Cyclin D2 and DNA Damage Response Proteins.

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15, a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients, significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues, and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors, we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore, STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM, 53BP1, and MDC1. Furthermore, protein levels of these DNA damage response molecules are reduced by IL-15, as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally, pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients.

Mechanistic Insights into CpG DNA and IL-15 Synergy in Promoting B Cell Chronic Lymphocytic Leukemia Clonal Expansion.

Malignant cell growth within patients with B cell chronic lymphocytic leukemia (B-CLL) is largely restricted to lymphoid tissues, particularly lymph nodes. The recent in vitro finding that TLR-9 ligand (oligodeoxynucleotide [ODN]) and IL-15 exhibit strong synergy in promoting B-CLL growth may be particularly relevant to growth in these sites. This study shows IL-15-producing cells are prevalent within B-CLL-infiltrated lymph nodes and, using purified B-CLL cells from blood, investigates the mechanism for ODN and IL-15 synergy in driving B-CLL growth. ODN boosts baseline levels of phospho-RelA(S529) in B-CLL and promotes NF-κB-driven increases in IL15RA and IL2RB mRNA, followed by elevated IL-15Rα and IL-2/IL-15Rß (CD122) protein. IL-15→CD122 signaling during a critical interval, 20 to 36-48 h following initial ODN exposure, is required for optimal induction of the cycling process. Furthermore, experiments with neutralizing anti-IL-15 and anti-CD122 mAbs indicate that clonal expansion requires continued IL-15/CD122 signaling during cycling. The latter is consistent with evidence of heightened IL2RB mRNA in the fraction of recently proliferated B-CLL cells within patient peripheral blood. Compromised ODN+IL-15 growth with limited cell density is consistent with a role for upregulated IL-15Rα in facilitating homotypic trans IL-15 signaling, although there may be other explanations. Together, the findings show that ODN and IL-15 elicit temporally distinct signals that function in a coordinated manner to drive B-CLL clonal expansion.

TLR-9 and IL-15 Synergy Promotes the In Vitro Clonal Expansion of Chronic Lymphocytic Leukemia B Cells.

Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) reflects the clone's Ag receptor (BCR) and involves stroma-dependent B-CLL growth within lymphoid tissue. Uniformly elevated expression of TLR-9, occasional MYD88 mutations, and BCR specificity for DNA or Ags physically linked to DNA together suggest that TLR-9 signaling is important in driving B-CLL growth in patients. Nevertheless, reports of apoptosis after B-CLL exposure to CpG oligodeoxynucleotide (ODN) raised questions about a central role for TLR-9. Because normal memory B cells proliferate vigorously to ODN+IL-15, a cytokine found in stromal cells of bone marrow, lymph nodes, and spleen, we examined whether this was true for B-CLL cells. Through a CFSE-based assay for quantitatively monitoring in vitro clonal proliferation/survival, we show that IL-15 precludes TLR-9-induced apoptosis and permits significant B-CLL clonal expansion regardless of the clone's BCR mutation status. A robust response to ODN+IL-15 was positively linked to presence of chromosomal anomalies (trisomy-12 or ataxia telangiectasia mutated anomaly + del13q14) and negatively linked to a very high proportion of CD38(+) cells within the blood-derived B-CLL population. Furthermore, a clone's intrinsic potential for in vitro growth correlated directly with doubling time in blood, in the case of B-CLL with Ig H chain V region-unmutated BCR and <30% CD38(+) cells in blood. Finally, in vitro high-proliferator status was statistically linked to diminished patient survival. These findings, together with immunohistochemical evidence of apoptotic cells and IL-15-producing cells proximal to B-CLL pseudofollicles in patient spleens, suggest that collaborative ODN and IL-15 signaling may promote in vivo B-CLL growth.

Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion.

Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up-regulated p53 following T-cell-independent (TI) stimulation. We here examine p53 gene transcription in such TI clones, with a focus on modulatory effects of prostaglandin E2 (PGE2), and evaluate progeny for p53 mutations. Resting IgM(+)IgD(+)CD27(-) B cells from human tonsils were labeled with CFSE and stimulated in vitro with complement-coated antigen surrogate, IL-4, and BAFF ± exogenous PGE2 (50 nM) or an analog specific for the EP2 PGE2 receptor. We use flow cytometry to measure p53 and AID protein within variably divided blasts, qRT-PCR of p53 mRNA from cultures with or without actinomycin D to monitor mRNA transcription/stability, and single-cell p53 RT-PCR/sequencing to assess progeny for p53 mutations. We report that EP2 signaling triggers increased p53 gene transcriptional activity in AID(+) cycling blasts (P<0.01). Progeny exhibit p53 mutations at a frequency (8.5 × 10(-4)) greater than the baseline error rate (<0.8 × 10(-4)). We conclude that, devoid of the repressive influences of Bcl-6, dividing B lymphoblasts in inflamed tissues should display heightened p53 transcription and increased risk of p53 mutagenesis.

Candidate chromosome 1 disease susceptibility genes for Sjogren's syndrome xerostomia are narrowed by novel NOD.B10 congenic mice.

Sjogren's syndrome (SS) is characterized by salivary gland leukocytic infiltrates and impaired salivation (xerostomia). Cox-2 (Ptgs2) is located on chromosome 1 within the span of the Aec2 region. In an attempt to demonstrate that COX-2 drives antibody-dependent hyposalivation, NOD.B10 congenic mice bearing a Cox-2flox gene were generated. A congenic line with non-NOD alleles in Cox-2-flanking genes failed manifest xerostomia. Further backcrossing yielded disease-susceptible NOD.B10 Cox-2flox lines; fine genetic mapping determined that critical Aec2 genes lie within a 1.56 to 2.17Mb span of DNA downstream of Cox-2. Bioinformatics analysis revealed that susceptible and non-susceptible lines exhibit non-synonymous coding SNPs in 8 protein-encoding genes of this region, thereby better delineating candidate Aec2 alleles needed for SS xerostomia.

T-cell independent, B-cell receptor-mediated induction of telomerase activity differs among IGHV mutation-based subgroups of chronic lymphocytic leukemia patients.

Although B-cell chronic lymphocytic leukemia (B-CLL) clones with unmutated IGHV genes (U-CLL) exhibit greater telomerase activity than those with mutated IGHV genes (M-CLL), the extent to which B-cell receptor (BCR) triggering contributes to telomerase up-regulation is not known. Therefore, we studied the effect of BCR stimulation on modulating telomerase activity. The multivalent BCR ligand, dextran conjugated anti-µ mAb HB57 (HB57-dex), increased telomerase activity and promoted cell survival and proliferation preferentially in U-CLL cases, whereas the PI3K/Akt inhibitor LY294002 blocked HB57-dex induced telomerase activation. Although both U-CLL and M-CLL clones exhibited similar membrane proximal signaling responses to HB57-dex, telomerase activity and cell proliferation, when inducible in M-CLL, differed. B-CLL cells stimulated using bivalent F(ab')(2) -goat anti-µ antibody (goat anti-µ) exhibited higher membrane proximal response in U-CLL than M-CLL cells, whereas telomerase activity, cell survival, and proliferation were induced to lower levels than those induced by HB57-dex. In normal B lymphocytes, HB57-dex induced less protein phosphorylation but more cell proliferation and survival than goat anti-µ. Although both anti-BCR stimuli induced comparable telomerase activity, normal CD5(+) B cells preferentially exhibited higher hTERT positivity than their CD5(-) counterparts. These findings provide an understanding of how BCR-mediated signals impact telomerase modulation in IGHV mutation-based subgroups of B-CLL and normal B cells.

IGHV-unmutated and IGHV-mutated chronic lymphocytic leukemia cells produce activation-induced deaminase protein with a full range of biologic functions.

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.

A p53 axis regulates B cell receptor-triggered, innate immune system-driven B cell clonal expansion.

Resting mature human B cells undergo a dynamic process of clonal expansion, followed by clonal contraction, during an in vitro response to surrogate C3d-coated Ag and innate immune system cytokines, IL-4 and BAFF. In this study, we explore the mechanism for clonal contraction through following the time- and division-influenced expression of several pro- and anti-apoptotic proteins within CFSE-labeled cultures. Several findings, involving both human and mouse B cells, show that a mitochondria-dependent apoptotic pathway involving p53 contributes to the high activation-induced cell death (AICD) susceptibility of replicating blasts. Activated B cell clones exhibit elevated p53 protein and elevated mRNA/protein of proapoptotic molecules known to be under direct p53 transcriptional control, Bax, Bad, Puma, Bid, and procaspase 6, accompanied by reduced anti-apoptotic Bcl-2. Under these conditions, Bim levels were not increased. The finding that full-length Bid protein significantly declines in AICD-susceptible replicating blasts, whereas Bid mRNA does not, suggests that Bid is actively cleaved to short-lived, proapoptotic truncated Bid. AICD was diminished, albeit not eliminated, by p53 small interfering RNA transfection, genetic deletion of p53, or Bcl-2 overexpression. DNA damage is a likely trigger for p53-dependent AICD because susceptible lymphoblasts expressed significantly elevated levels of both phosphorylated ataxia telangiectasia mutated-Ser(1980) and phospho-H2AX-Ser(139). Deficiency in activation-induced cytosine deaminase diminishes but does not ablate murine B cell AICD, indicating that activation-induced cytosine deaminase-induced DNA damage is only in part responsible. Evidence for p53-influenced AICD during this route of T cell-independent clonal expansion raises the possibility that progeny bearing p53 mutations might undergo positive selection in peripherally inflamed tissues with elevated levels of IL-4 and BAFF.

B Cell Chronic Lymphocytic Leukemia Development in Mice with Chronic Lung Exposure to Coccidioides Fungal Arthroconidia.

Links between repeated microbial infections and B cell chronic lymphocytic leukemia (B-CLL) have been proposed but not tested directly. This study examines how prolonged exposure to a human fungal pathogen impacts B-CLL development in Eµ-hTCL1-transgenic mice. Monthly lung exposure to inactivated Coccidioides arthroconidia, agents of Valley fever, altered leukemia development in a species-specific manner, with Coccidioides posadasii hastening B-CLL diagnosis/progression in a fraction of mice and Coccidioides immitis delaying aggressive B-CLL development, despite fostering more rapid monoclonal B cell lymphocytosis. Overall survival did not differ significantly between control and C. posadasii-treated cohorts but was significantly extended in C. immitis-exposed mice. In vivo doubling time analyses of pooled B-CLL showed no difference in growth rates of early and late leukemias. However, within C. immitis-treated mice, B-CLL manifests longer doubling times, as compared with B-CLL in control or C. posadasii-treated mice, and/or evidence of clonal contraction over time. Through linear regression, positive relationships were noted between circulating levels of CD5+/B220low B cells and hematopoietic cells previously linked to B-CLL growth, albeit in a cohort-specific manner. Neutrophils were positively linked to accelerated growth in mice exposed to either Coccidioides species, but not in control mice. Conversely, only C. posadasii-exposed and control cohorts displayed positive links between CD5+/B220low B cell frequency and abundance of M2 anti-inflammatory monocytes and T cells. The current study provides evidence that chronic lung exposure to fungal arthroconidia affects B-CLL development in a manner dependent on fungal genotype. Correlative studies suggest that fungal species differences in the modulation of nonleukemic hematopoietic cells are involved.

A cyclooxygenase-2/prostaglandin E2 pathway augments activation-induced cytosine deaminase expression within replicating human B cells.

Within inflammatory environments, B cells encountering foreign or self-Ag can develop tertiary lymphoid tissue expressing activation-induced cytosine deaminase (AID). Recently, this DNA-modifying enzyme was detected in nonlymphoid cells within several inflamed tissues and strongly implicated in malignant transformation. This study examines whether a cyclooxygenase 2 (COX-2) pathway, often linked to inflammation, influences AID expression in activated B lymphocytes. In this paper, we report that dividing human B cells responding to surrogate C3d-coated Ag, IL-4, and BAFF express AID, as well as COX-2. A progressive increase in AID with each division was paralleled by a division-related increase in a COX-2-linked enzyme, microsomal PGE(2) synthase-1, and the PGE(2)R, EP2. Cells with the greatest expression of AID expressed the highest levels of EP2. Although COX-2 inhibitors diminished both AID expression and IgG class switching, exogenous PGE(2) and butaprost, a selective EP2 agonist, augmented AID mRNA/protein and increased the numbers of IgG(+) progeny. Despite the latter, the proportion of IgG(+) cells within viable progeny generally declined with PGE(2) supplementation. This was not due to PGE(2)-promoted differentiation to plasma cells or to greater downstream switching. Rather, because phosphorylated ataxia telangiectasia mutated levels were increased in progeny of PGE(2)-supplemented cultures, it appears more likely that PGE(2) facilitates AID-dependent DNA double-strand breaks that block B cell cycle progression or promote activation-induced cell death, or both. Taken together, the results suggest that a PGE(2) feed-forward mechanism for augmenting COX-2 pathway proteins promotes progressively increased levels of AID mRNA, protein, and function.

Structural requirements for the interaction of human IgM and IgA with the human Fcalpha/mu receptor.

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fcalpha/mu receptor (hFcalpha/muR). Ligand polymerization status was crucial for the interaction, because hFcalpha/muR binding did not occur with monomeric Ab of either class. hFcalpha/muR bound IgM with an affinity in the nanomolar range, whereas the affinity for dimeric IgA (dIgA) was tenfold lower. Panels of mutant IgM and dIgA were used to identify regions critical for hFcalpha/muR binding. IgM binding required contributions from both Cmu3 and Cmu4 Fc domains, whereas for dIgA, an exposed loop in the Calpha3 domain was crucial. This loop, comprising residues Pro440-Phe443, lies at the Fc domain interface and has been implicated in the binding of host receptors FcalphaRI and polymeric Ig receptor (pIgR), as well as IgA-binding proteins produced by certain pathogenic bacteria. Substitutions within the Pro440-Phe443 loop resulted in loss of hFcalpha/muR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA-binding proteins were shown to inhibit the dIgA-hFcalpha/muR interaction. Therefore, we have identified a motif in the IgA-Fc inter-domain region critical for hFcalpha/muR interaction, and highlighted the multi-functional nature of a key site for protein-protein interaction at the IgA Fc domain interface.

Identification of residues in the Cmu4 domain of polymeric IgM essential for interaction with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1).

The binding of nonspecific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Herein we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4beta domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H chain and requires the IgM Cmu4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4beta domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cmu4 domain are equivalent to those regions of IgG and IgA recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.

APRIL and BAFF promote increased viability of replicating human B2 cells via mechanism involving cyclooxygenase 2.

Of relevance to both protective and pathogenic responses to Ag is the recent finding that soluble molecules of the innate immune system, i.e., IL-4, B cell-activation factor of the TNF family (BAFF), and C3, exhibit significant synergy in promoting the clonal expansion of human B2 cells following low-level BCR ligation. Although IL-4, BAFF, and C3dg each contribute to early cell cycle entry and progression to S phase, only BAFF promotes later sustained viability of progeny needed for continued cycling. The present study sought to further clarify the mechanisms for BAFF's multiple functions. By comparing BAFF and a proliferation-inducing ligand (APRIL) efficacy at different stages in the response (only BAFF binds BR3; both bind transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation Ag, the early role was attributed to BR3, while the later role was attributed to TACI/B cell maturation Ag. Importantly, BAFF- and APRIL-promoted viability of cycling lymphoblasts was associated with sustained expression of cyclooxygenase 2 (COX-2), the rate-limiting enzyme for PGE2 synthesis, within replicating cells. Supernatants of cultures with BAFF and APRIL contained elevated PGE2. Although COX-2 inhibitors diminished daughter cell viability, exogenous PGE2 (1-1000 nM) increased the viability and recovery of lymphoblasts. Increased yield of viable progeny was associated with elevated Mcl-1, suggesting that a BAFF/APRIL --> TACI --> COX-2 --> PGE2--> Mcl-1 pathway reduces activation-related, mitochondrial apoptosis in replicating human B2 cell clones.

Innate immunity and human B cell clonal expansion: effects on the recirculating B2 subpopulation.

Foci of autoantigen-specific B lymphocytes in nonlymphoid tissues have been associated with development of autoimmune disease. To better understand the genesis of such ectopic lymphoid tissue, this study investigated whether several B cell-tropic innate immune system molecules, known to be elevated in response to inflammatory stimuli, can cooperate in fostering the T cell-independent clonal expansion of mature human B2 cells under conditions of limiting BCR engagement. Notable synergy was observed between BCR coligation with the C3dg-binding CD21/CD19 costimulatory complex, B cell-activating factor belonging to the TNF family (BAFF), and IL-4 in generating B cell progeny with sustained CD86 and DR expression. The synergy was observed over a wide range of BCR:ligand affinities and involved: 1) cooperative effects at promoting early cell cycle progression and viability; 2) BCR:CD21 coligation-promoted increases in BAFF receptors that were highly regulated by IL-4; 3) reciprocal effects of IL-4 and BAFF at dampening daughter cell apoptosis typical of stimulation by BCR:CD21 and either cytokine alone; and 4) BAFF-sustained expression of antiapoptotic Mcl-1 within replicating lymphoblasts. The results suggest that significant clonal proliferation of recirculating B2 cells occurs upon limited binding to C3dg-coated Ag in an inflammatory in vivo milieu containing both BAFF and IL-4. When rare autoantigen-presenting B cells undergo such expansions, both B cell and T cell autoimmunity may be promoted.

Antigen receptor triggered upregulation of CD86 and CD80 in human B cells: augmenting role of the CD21/CD19 co-stimulatory complex and IL-4.

The impact of BCR:CD21 co-engagement on B cell expression of molecules critical for T cell activation was investigated with receptor-specific mAbs conjugated to high MW dextran as stimulatory ligands. In the absence of IL-4, BCR:CD21 co-ligation augmented BCR-triggered CD86 only under conditions of very low BCR ligand dose or affinity, and CD80 was minimally induced by BCR and/or CD21 crosslinking. In the presence of IL-4, BCR:CD21 co-ligation augmented CD86 and CD80 expression under conditions of greater BCR engagement. However, with very high level BCR engagement, no bonus effect of BCR:CD21 crosslinking was observed. Co-ligation-promoted CD86 and CD80 expression was associated with heightened B cell activation of resting allogeneic T cells. The data suggest that co-clustering of BCR and the CD21/CD19 co-stimulatory complex following B cell engagement with C3d-bound microbial or self-antigens will enhance B cell recruitment of T cell help only when IL-4 is present and/or BCR engagement is very limiting.

Role of complement-binding CD21/CD19/CD81 in enhancing human B cell protection from Fas-mediated apoptosis.

Defective expression of Fas leads to B cell autoimmunity, indicating the importance of this apoptotic pathway in eliminating autoreactive B cells. However, B cells with anti-self specificities occasionally escape such regulation in individuals with intact Fas, suggesting ways of precluding this apoptosis. Here, we examine whether coligation of the B cell Ag receptor (BCR) with the complement (C3)-binding CD21/CD19/CD81 costimulatory complex can enhance the escape of human B cells from Fas-induced death. This was warranted given that BCR-initiated signals induce resistance to Fas apoptosis, some (albeit not all) BCR-triggered events are amplified by coligation of BCR and the co-stimulatory complex, and several self Ags targeted in autoimmune diseases effectively activate complement. Using a set of affinity-diverse surrogate Ags (receptor-specific mAb:dextran conjugates) with varying capacity to engage CD21, it was established that BCR:CD21 coligation lowers the BCR engagement necessary for inducing protection from Fas apoptosis. Enhanced protection was associated with altered expression of several molecules known to regulate Fas apoptosis, suggesting a unique molecular model for how BCR:CD21 coligation augments protection. BCR:CD21 coligation impairs the generation of active fragments of caspase-8 via dampened expression of membrane Fas and augmented expression of FLIP(L). This, in turn, diminishes the generation of cells that would be directly triggered to apoptosis via caspase-8 cleavage of caspase 3 (type I cells). Any attempt to use the mitochondrial apoptotic protease-activating factor 1 (Apaf-1)-dependent pathway for apoptosis (as type II cells) is further blocked because BCR:CD21 coligation promotes up-regulation of the mitochondrial antiapoptotic molecule, Bcl-2.