Immunoliposomal Delivery of Mir-29b By Targeting Tumor Antigen ROR1 Induces Epigenetic Reprograming in Human-ROR1-Expressed Mouse Model of Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1743-1743
Author(s):  
Chi-Ling Chiang ◽  
Frank W Frissora ◽  
Zhiliang Xie ◽  
Xiaomeng Huang ◽  
Rajeswaran Mani ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Mouse Model ◽  
Cell Line ◽  
Tumor Antigen ◽  
Targeted Delivery ◽  
Leukemia Cell ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia

Abstract Chronic lymphocytic leukemia (CLL), characterized by accumulation of CD5+CD19+sIgM+ B lymphocytes in peripheral blood and lymphoid organs, is classified into indolent and aggressive forms. Patients with indolent CLL generally survive 5 to 10 years and do not require treatment until severe symptoms, while those with aggressive CLL show resistant to standard treatment and survive less than 24 months. While emerging B cell antigen receptor directed therapies are promising, resistance to such therapies pose problems warranting novel therapeutic approaches. MicroRNA (miR) profiling revealed lower expression of miR-29b in aggressive CLL associated with survival, drug resistance and poor prognosis via its up-regulation of anti-apoptotic proteins myeloid leukemia cell differentiation protein 1 (Mcl1) and oncogenic T-cell leukemia 1 (Tcl1). Thus, specific overexpression of miR-29b in B-CLL cells could be a potential therapy for aggressive CLL patients. Despite the promise, short circulation half-life, limited cellular uptake and off-target effects on non-desirable tissues pose a challenge for miR-based therapies. To promote efficiency and specificity of miR-29b delivery, we developed neutral immunonanoparticles with selectivity to CLL via targeting tumor antigen ROR1, which is expressed in over 95% of CLL but not normal B cells. We optimized a novel 2A2-immunoliposome (2A2-ILP) recognizing surface ROR1 on primary CLL cell to promote internalization and miR-29b uptake (n=6, p=0.042*). About 20-fold increased uptake of miR-29b was achieved with 2A2-ILP-miR-29b formulation compared to control. Further ROR1 targeted delivery of miR29b resulted in significant downregulation of DNMT1 and DNMT3a mRNA and protein (n=3, DNMT1: p= 0.0115*; DNMT3a: p=0.0231*, SP1; p=0.0031**) in primary CLL cells and a human CLL cell line OSU-CLL. Consistent with the downregulation of DNMTs, decreased global DNA methylation was observed in OSU-CLL cell line one week post- treatment with 2A2-ILP-miR-29b (n=3, p=0.0003***). To further study the in vivo ROR1-targeting efficiency of 2A2-ILP-miR-29b, we used our recently described Eµ-hROR1x Tcl1 CLL mouse model that develops CLL like disease with human ROR1 antigen in leukemic CD19+CD5+ B cells. Using hROR1+CD19+CD5+ leukemic cell engraftment model, we showed significant in-vivo efficacy of ROR1-ILP-miR-29b formulation associated with a) decreased number of circulating leukemic B220+CD5+ cells b) reduced splenomegaly (p=0.0461*, 2A2-29b: n=9; PBS: n=8) c) with extended survival (p=0.0075**, 2A2-29b: n=9; IgG-29b: n=7; 2A2-SC: n=7; PBS: n=8). In summary, 2A2-ILP effectively delivered functional miR-29b, resulting in downregulation of DNMT1 and DNMT3a, reduction of hypermethylation and anti-leukemic activity. Ongoing studies are aimed at understanding miR-29b mediated in-vivo methylome reprograming using our novel hROR1xTcl1 transgenic mouse model and ROR1-targeted miR-29b delivery formulation. Figure 1. Figure 1. Disclosures Byrd: Acerta Pharma BV: Research Funding.

Targeting of Chronic Lymphocytic Leukemia B Cells with a Novel Monoclonal Antibody to ROR1

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 984-984
Author(s):  
Bing CUi ◽  
George F. Widhopf ◽  
Jian Yu ◽  
Daniel Martinez ◽  
Esther Avery ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Adoptive Transfer ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Phosphorylated Akt

Abstract Abstract 984 ROR1 is an orphan receptor tyrosine kinase that is expressed on leukemia cells of patients with chronic lymphocytic leukemia (CLL), but not on most adult tissues of healthy adults, including CD5+ B cells. To generate anti-ROR1 antibodies, we immunized mice using different strategies employing vaccines comprised of recombinant ROR1 protein, polynucleotide-ROR1 vaccines and CD154 genetic adjuvants, or replication-defective adenovirus vectors encoding ROR1 and CD154. We extirpated the spleens of animals that developed high-titer serum anti-ROR1 antibodies and used these to generate monoclonal-antibody-(mAb)-producing hybridomas or antibody phage-display libraries that subsequently were screened for ROR1-binding. Over 70 unique mAbs were generated that each bound the extra-cellular domain of native ROR1. Most mAbs recognized an epitope(s) within the ROR1 Ig-like domain, which appears to represent the immune dominant epitope. Other mAb recognized epitopes within the conserved ROR1 Kringle domain. One mAb (UC D10-001) had distinctive binding to an intradomain epitope of human ROR1 (hROR1). UC D10-001 was the only mAb we found directly cytotoxic for hROR1-expressing leukemia cells cultured in media without complement for 6 hours. We found that UC D10-001 could induce significant reductions in basal levels of phosphorylated AKT in hROR1-expressing leukemia cells. Moreover, UC D10-001 significantly decreased the basal levels of phosphorylated AKT in freshly isolated human CLL cells (N=4) to levels comparable to that observed in co-cultures containing 10 mM LY294002, a broad-spectrum inhibitor of PI3K. We examined whether this mAb had cytotoxic activity for leukemia cell in vivo. For this we examined whether we could inhibit the adoptive transfer of human-ROR1-expressing leukemia cells to young, syngeneic recipient mice made transgenic for human ROR1 under control of a B-cell specific promoter. Cohorts of 5 animals per group were each given intravenous injections of antibody at a dose of at 10 mg/kg. Each cohort was treated with UC D10-001, control IgG, or 4A5, an anti-ROR1 mAb specific for a non-cross-reactive epitope located in the Ig-like domain of ROR1. Each animal received an intravenous injection of 5 × 105 ROR1-expressing leukemia cells and then was assessed weekly for circulating leukemia cells by flow cytometry. UC D10-001, but not control IgG or 4A5, significantly inhibited engraftment of the ROR1+ leukemia. Four weeks after adoptive transfer, animals treated with UC D10-001 had a 10-fold lower median number of leukemia B cells in the blood than animals treated with control IgG or 4A5. We also tested UC D10-001 for its capacity to induce clearance of human ROR1+ CLL cells engrafted into the peritoneal cavity of Rag-2−/−/γc−/− immune deficient mice. Each of these mice received intraperitoneal injections of equal numbers of human ROR1+ CLL cells prior to receiving D10-001, control IgG, or 4A5, each at 10 mg/kg. These animals were sacrificed seven days later and the human leukemia cells were harvested via peritoneal lavage. In mice treated with UC D10-001 we harvested an average of only 6 × 104 ± 3 × 104 CLL cells. This number of cells was significantly less than the average number of CLL cells harvested from control IgG or 4A5-treated mice (8 × 105 ± 4 × 105 or 7 × 105 ± 2 × 105, respectively, p <0.01). These studies indicate that the anti-ROR1 mAb UC D10-001 can be directly cytotoxic for ROR1-expressing leukemia cells in vitro and in vivo, a property that apparently is unique to this mAb among other anti-ROR1 mAbs. Because of the restricted expression of ROR1 on leukemia cells and the distinctive properties of this mAb, we propose that UC D10-001 might have potential utility in the treatment of patients with CLL. Disclosures: No relevant conflicts of interest to declare.

Expression of ZAP-70 Does Not Accelerate Leukemia Development and Progression in the Eμ-TCL1 Transgenic Mouse Model of Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 925-925
Author(s):  
Stefania Gobessi ◽  
Francesca Belfiore ◽  
Sara Bennardo ◽  
Brendan Doe ◽  
Luca Laurenti ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Mouse Model ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Leukemia Development ◽  
Low Levels ◽  
And Behavior

Abstract Abstract 925 One of the most relevant prognostic factors in chronic lymphocytic leukemia (CLL) is expression of the protein tyrosine kinase ZAP-70. Typically, patients whose leukemic cells express ZAP-70 at 30–100% of the levels in normal T cells have aggressive disease, whereas patients whose leukemic cells do not express ZAP-70 or express only low levels of this protein have indolent disease. Previously, we and others demonstrated that ZAP-70 modulates B-cell receptor signaling and thus affects the capacity of the leukemic cells to respond to antigen stimulation. However, a direct link between an altered antigen response and CLL pathogenesis has still not been established and, more importantly, the question whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of aggressive CLL still remains to be answered. We have now addressed these issues by analyzing in vivo the impact of forced expression of ZAP-70 on the development and behavior of leukemias that arise in the Eμ-TCL1 transgenic (tg) mouse model of CLL. This model is characterized by the development of antigen-driven leukemias that resemble human CLL in many aspects but are always ZAP-70-negative. To force the expression of ZAP-70 in TCL1 leukemias, we generated two tg lines with targeted expression of ZAP-70 in the B cell compartment (ZAP70high and ZAP70low) and crossed them with Eμ-TCL1 tg mice. B cells in ZAP70high tg mice express similar levels of ZAP-70 as normal mouse T cells, whereas the levels of ZAP-70 in B cells of ZAP70lowtg mice are approximately 10 times lower. Both cohorts of Eμ-TCL1/ZAP70 double tg mice developed characteristic TCL1 leukemias. Eμ-TCL1/ZAP70low tg mice developed leukemias with onset and rate of progression similar to their ZAP-70-negative littermates, indicating that low levels of ZAP-70 do not alter the development and behavior of the disease. Surprisingly, Eμ-TCL1/ZAP70high tg mice developed leukemias with an approximately 2 month delay compared to their ZAP-70-negative Eμ-TCL1 tg littermates, which was contrary to the expectation that high ZAP-70 expression will accelerate leukemia development. The delay in leukemia development was especially evident at 6 months of age, when leukemic cells could be detected in the PB of 77% (10/13) of Eμ-TCL1 tg mice and only 24% (4/17) of Eμ-TCL1/ZAP70hightg mice (P=0.011). Since ZAP-70 expression can affect the migratory and adhesion capacity of human CLL cells in vitro, we first investigated if the delayed appearance of leukemic cells in the PB of Eμ-TCL1/ZAP70high tg mice could be due to increased retention of the leukemic cells in the lymphoid tissues. Assessment of tumor burden in the spleen, peritoneal cavity (PC), bone marrow and PB of 7 months old mice showed that the number of tumor cells in each compartment was significantly lower in Eμ-TCL1/ZAP70hightg mice than their Eμ-TCL1 littermates, suggesting that the delay in leukemia appearance is not caused by increased tissue retention but rather by reduced tumor growth. To investigate if ZAP-70 impairs tumor growth by affecting proliferation, we performed in vivo BrdU incorporation analysis of leukemic cells from spleen and PC of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice. Spleen and PC samples were analyzed because they are the major sites of leukemia proliferation in Eμ-TCL1 tg mice. Interestingly, while the percentage of proliferating leukemic cells in the spleens of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice was similar (mean % of BrdU+ cells ±SD: 6.81 ±1.67 and 6.15 ±2.92, respectively; P=n.s.), the percentage of proliferating leukemic cells in the PC of Eμ-TCL1/ZAP70high tg mice was significantly lower (mean % of BrdU+cells ±SD: 1.74 ±1.05 and 0.56 ±0.39, respectively; P=0.024). In summary, this study shows that ZAP-70 expression, per se, is unable to accelerate leukemia development and progression in an established in vivo model of CLL and suggests that ZAP-70 is not directly responsible for the greater disease severity in the poor prognosis subset of CLL. In addition, this study reveals that ZAP-70 in certain tissue environments can function as a negative regulator of leukemic cell proliferation, contrary to the widespread perception of ZAP-70 as a positive regulator of leukemic cell responses. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The novel plant-derived agent silvestrol has B-cell selective activity in chronic lymphocytic leukemia and acute lymphoblastic leukemia in vitro and in vivo

Blood ◽  
2009 ◽  
Vol 113 (19) ◽  
pp. 4656-4666 ◽  
Author(s):  
David M. Lucas ◽  
Ryan B. Edwards ◽  
Gerard Lozanski ◽  
Derek A. West ◽  
Jungook D. Shin ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Translational Inhibition

Abstract Therapeutic options for advanced B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL) are limited. Available treatments can also deplete T lymphocytes, leaving patients at risk of life-threatening infections. In the National Cancer Institute cell line screen, the structurally unique natural product silvestrol produces an unusual pattern of cytotoxicity that suggests activity in leukemia and selectivity for B cells. We investigated silvestrol efficacy using primary human B-leukemia cells, established B-leukemia cell lines, and animal models. In CLL cells, silvestrol LC50 (concentration lethal to 50%) is 6.9 nM at 72 hours. At this concentration, there is no difference in sensitivity of cells from patients with or without the del(17p13.1) abnormality. In isolated cells and whole blood, silvestrol is more cytotoxic toward B cells than T cells. Silvestrol causes early reduction in Mcl-1 expression due to translational inhibition with subsequent mitochondrial damage, as evidenced by reactive oxygen species generation and membrane depolarization. In vivo, silvestrol causes significant B-cell reduction in Eμ-Tcl-1 transgenic mice and significantly extends survival of 697 xenograft severe combined immunodeficient (SCID) mice without discernible toxicity. These data indicate silvestrol has efficacy against B cells in vitro and in vivo and identify translational inhibition as a potential therapeutic target in B-cell leukemias.

scholarly journals P66Shc: A Pleiotropic Regulator of B Cell Trafficking and a Gatekeeper in Chronic Lymphocytic Leukemia

Cancers ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 1006 ◽  
Author(s):  
Laura Patrussi ◽  
Nagaja Capitani ◽  
Cosima T. Baldari
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Chemokine Receptors ◽  
Adaptor Protein ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Cell Trafficking ◽  
Gene Encoding ◽  
Cell Invasiveness

Neoplastic B cells from chronic lymphocytic leukemia patients (CLL) have a profound deficiency in the expression of p66Shc, an adaptor protein with pro-apoptotic and pro-oxidant activities. This defect results in leukemic B cell resistance to apoptosis and additionally impinges on the balance between chemokine receptors that control B cell homing to secondary lymphoid organs and the sphingosine phosphate receptor S1PR1 that controls their egress therefrom, thereby favoring leukemic B cell accumulation in the pro-survival lymphoid niche. Ablation of the gene encoding p66Shc in the Eµ-TCL1 mouse model of human CLL enhances leukemogenesis and promotes leukemic cell invasiveness in both nodal and extranodal organs, providing in vivo evidence of the pathogenic role of the p66Shc defect in CLL pathogenesis. Here we present an overview of the functions of p66Shc in B lymphocytes, with a specific focus on the multiple mechanisms exploited by p66Shc to control B cell trafficking and the abnormalities in this process caused by p66Shc deficiency in CLL.

Aberrant expression of B-lymphocyte stimulator by B chronic lymphocytic leukemia cells: a mechanism for survival

Blood ◽  
2002 ◽  
Vol 100 (8) ◽  
pp. 2973-2979 ◽  
Author(s):  
Anne J. Novak ◽  
Richard J. Bram ◽  
Neil E. Kay ◽  
Diane F. Jelinek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
B Lymphocyte ◽  
Leukemic Cell ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Aberrant Expression ◽  
B Lymphocyte Stimulator

B-cell chronic lymphocytic leukemia (B-CLL) is defined by the accumulation of CD5+ B cells in the periphery and bone marrow. This disease is not characterized by highly proliferative cells but rather by the presence of leukemic cells with significant resistance to apoptosis and, therefore, prolonged survival. B-lymphocyte stimulator (BLyS) is a newly identified tumor necrosis factor (TNF) family member shown to be critical for maintenance of normal B-cell development and homeostasis and it shares significant homology with another TNF superfamily member, APRIL. The striking effects of BLyS on normal B-cell maintenance and survival raises the possibility that it may be involved in pathogenesis and maintenance of hematologic malignancies, including B-CLL. In this study, we investigated the status of APRIL and BLyS expression, as well as their receptors, in this disease. All B-CLL patient cells studied expressed one or more of 3 known receptors for BLyS; however, the pattern of expression was variable. In addition, we demonstrate for the first time that B-CLL cells from a subset of patients aberrantly express BLyS and APRIL mRNA, whereas these molecules were not detectable in normal B cells. Furthermore, we provide in vitro evidence that BLyS protects B-CLL cells from apoptosis and enhances cell survival. Because these molecules are key regulators of B-cell homeostasis and tumor progression, leukemic cell autocrine expression of BLyS and APRIL may be playing an important role in the pathogenesis of this disease.

scholarly journals Chronic Lymphocytic Leukemia B Cells Can Undergo Somatic Hypermutation and Intraclonal Immunoglobulin VHDJH Gene Diversification

2002 ◽  
Vol 196 (5) ◽  
pp. 629-639 ◽  
Author(s):  
Carmela Gurrieri ◽  
Peter McGuire ◽  
Hong Zan ◽  
Xiao-Jie Yan ◽  
Andrea Cerutti ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Lymphocyte ◽  
Somatic Hypermutation ◽  
Point Mutations ◽  
Single Strand Conformation Polymorphism ◽  
Lymphocytic Leukemia ◽  
Gene Diversification

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.

Liposomal Targeted Delivery Overcomes Immunostimulatory Effects of Oligonucleotide Based Therapy In Chronic Lymphocytic Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1475-1475
Author(s):  
Bo Yu ◽  
Yicheng Mao ◽  
Li-Yuan Bai ◽  
Sarah E.M. Herman ◽  
Asha Ramanunni ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Targeted Delivery ◽  
Therapeutic Efficacy ◽  
Hind Limb ◽  
Conflicts Of Interest ◽  
Day Treatment ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Hind Limb Paralysis

Abstract Abstract 1475 Therapeutic application of several CpG-containing anti-sense oligodeoxyribonucleotides (ODN) and siRNAs are limited due to their potent immune stimulatory properties. To resurrect these promising agents for clinical application, we have developed an antibody based liposomal delivery strategy that overcomes the immunostimulatory properties without compromising the target down modulation. We show here G3139, an archetypical anti-sense ODN for Bcl-2, induced activation of NF-kB, which results in the up-regulation of its downstream anti-apoptotic targets such as Bcl-2 (22.5%, n=10, p=0.04) and Mcl-1 (35.0%, n=4, p<0.005) and co-stimulatory markers such as CD86 (47.5%, n=10, p<0.05) and HLA-DR (175%, n=10, p<0.005) as well as cytokine flare in B chronic lymphocytic leukemia (CLL) cells. These adverse immunostimulatory responses are abrogated by Rituximab mediated immunoliposomal delivery of G3139, resulting in significant Bcl-2 down-regulation and ∼60% enhanced sensitivity to fludarabine induced cytotoxicity (n=4, p<0.001). Consistent with the in-vitro observations, significant in-vivo therapeutic efficacy was demonstrated in a SCID mouse xenograft leukemia/lymphoma model. A total of 74 female 6- to 8-week-old SCID mice were engrafted with 2×106 Raji cells through tail vein injection on Day 0. The 10-day treatment was initiated via i.p. injection on Day 4. Fludarabine and G3139 were injected into the mice at the dose of 100mg/kg/day and 5mg/kg on alternative days respectively. Hind limb paralysis was considered as the key endpoint while other early removal criteria were also considered. The median survival time for placebo controls was 15 to 18 days without liposomal G3139 treatment. Although the in-vivo experiment is still in progress, CD20-targeting ILP-G3139 has already shown significant therapeutic efficacy with >80% survival rate (n=14) at day 58 of the study as a single agent or in combination with fludarabine treatment, compared to the mice treated with the control Her-ILP-G3139 that showed hind-limb paralysis before Day 45 in all mice. These results indicate potential use of targeted delivery vehicles to resurrect and improve the clinical efficacy of immunostimulatory oligonucleotide therapeutics for B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

CCR4:CCL17 Interaction Influences TLR-9 Mediated Cell Survival and Proliferation In Chronic Lymphocytic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3593-3593
Author(s):  
Sonal C. Temburni ◽  
Ryon M. Andersen ◽  
Luke Janson ◽  
Xiao-Jie Yan ◽  
Barbara Sherry ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Dose Range ◽  
Serum Levels ◽  
Lymphocytic Leukemia ◽  
Novel Therapy

Abstract Abstract 3593 Unlike other hematologic disorders, chronic lymphocytic leukemia(CLL) exhibits remarkable heterogeneity in the rates of disease progression among cases. CLL cells survive by receiving signals from the microenvironment via various receptors: B-cell antigen receptor (BCR), Toll-like receptors (TLRs) and cytokine and chemokine receptors. We previously reported that CLL clones with somatically mutated IGHVs and high (≥30%) percentage of CD38 expressing cells have the highest percentage of CCR4-expressing cells. To further explore the functional contribution of the CCR4:CCL17 axis in CLL, we studied CCL17-induced chemotactic behavior in 16 CLL cases. In transwell cultures we observed a bimodal migratory response to CCL17 at 2 doses in a dose range of 0.78– 25ng/ml, in ~60% of cases; the remaining cases showed maximal migration at a single dose (1.56 or 3.12ng/ml). A comparison of phenotypes of the migrated and non-migrated cell populations was undertaken in 10 cases, analyzing CXCR3, CXCR4, CCR4 and CCR7 that are involved in homing of cells to sites favoring growth, and CD31, CD38 and CD69, activation related molecules. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells suggesting a role for CD38 in the CCR4-mediated downstream pathway. CCR4 ligand, CCL17, is constitutively expressed in the thymus and is produced by dendritic cells, endothelial cells, keratinocytes and fibroblasts, whereas CCL22 is produced by tumor cells and the tumor microenvironment. Serum levels of both these ligands in untreated patients were quantified by ELISA. CCL17 levels ranged between 45-1, 229 pg/ml in U-CLL cases (n=23) and between 43-1, 418 pg/ml in M-CLL cases (n=30). CCL22 levels ranged between 121-5, 497 pg/ml in U-CLL cases (n=23) and 409-5, 502 pg/ml in M-CLL cases (n=30). The percentages of CCR4- expressing B cells directly correlated with percentages of T cells expressing CCR4 in individual cases, whereas they inversely correlated with both, serum levels of CCL17 (p< 0.01) and CCL22 (p< 0.05). CCL17 produced by DCs in peripheral organs may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells. We therefore tested if CCL17 supported BCR- and TLR-mediated proliferative responses in a cohort of 31 (16 U-CLL and 15M-CLL) CLL cases. CCL17 augmented BCR-mediated B-cell proliferation in 9/16 (56%) U-CLL cases, but only in 3/15 (20%) M-CLL cases. On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 (87%) M-CLL cases at a dose of 2ng/nl (approximating that detected in serum); it also augmented TLR-9 mediated B cell proliferation in 6/16 U-CLL cases but at a 5-fold or higher dose (10-25 ng/ml). In a subset of this cohort (8 cases) CCL17-induced modulation of molecules involved in the apoptotic process was studied. We found upregulation of anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulation of pro-apoptotic molecules Bim, PUMA, and Bid in 5 of these cases. The pro-survival effects of CCL17 were partially abrogated by the blocking anti-CCR4 mAb (1G1). Taken together, these findings suggest that CCL17 plays a role in modulating TLR-9-mediated signaling and migration in CLL. Therefore, inhibition of CCR4:CCL17 interaction in vivo represents a novel therapy by preventing migration of CLL cells towards an environment that promotes their survival. Disclosures: No relevant conflicts of interest to declare.

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