MJ Cells Research Articles

New JAK3-INSL3 Fusion Transcript-An Oncogenic Event in Cutaneous T-Cell Lymphoma.

Constitutively activated tyrosine kinase JAK3 is implicated in the pathogenesis of cutaneous T-cell lymphomas (CTCL). The mechanisms of constitutive JAK3 activation are unknown although a JAK3 mutation was reported in a small portion of CTCL patients. In this study, we assessed the oncogenic roles of a newly identified JAK3-INSL3 fusion transcript in CTCL. Total RNA from malignant T-cells in 33 patients with Sézary syndrome (SS), a leukemic form of CTCL, was examined for the new JAK3-INSL3 fusion transcript by RT-PCR followed by Sanger sequencing. The expression levels were assessed by qPCR and correlated with patient survivals. Knockdown and/or knockout assays were conducted in two CTCL cell lines (MJ cells and HH cells) by RNA interference and/or CRISPR/Cas9 gene editing. SS patients expressed heterogeneous levels of a new JAK3-INSL3 fusion transcript. Patients with high-level expression of JAK3-INSL3 showed poorer 5-year survival (n = 19, 42.1%) than patients with low-level expression (n = 14, 78.6%). CTCL cells transduced with specific shRNAs or sgRNAs had decreased new JAK3-INSL3 fusion transcript expression, reduced cell proliferation, and decreased colony formation. In NSG xenograft mice, smaller tumor sizes were observed in MJ cells transduced with specific shRNAs than cells transduced with controls. Our results suggest that the newly identified JAK3-INSL3 fusion transcript confers an oncogenic event in CTCL.

How Should Researchers Measure Perovskite‐Based Monolithic Multijunction Solar Cells’ Performance? A Calibration Lab's Perspective

Perovskite multijunctions (PVSK MJs) have made remarkable progress with monolithic PVSK/PVSK tandems surpassing the efficiency of single‐junction (1 J) PVSK cells and PVSK/Si cells reported to exceed the 30% efficiency mark. These efficiencies are reported at standard test conditions (STC), established by the photovoltaic (PV) community to facilitate comparison between devices and technologies. Herein, it is discussed why an accurate STC performance measurement for a MJ is more complicated than for a 1 J cell and the special aspects to be considered when measuring the current–voltage characteristics and the performance of PVSK‐containing MJ cells are emphasized. It is discussed why a spectrally adjustable solar simulator is needed and the sequence of accurate performance measurement, namely measurement of the spectral response of each junction, adjustment of the spectrum, and appropriate protocols for measuring the power output of the device at STC, is presented. For all these, common errors and pitfalls that sometime lead to misleading interpretation of the results are presented, and the methods to evaluate the accuracy of the data when a spectrally adjustable solar simulator is not available are recommended. Finally, first step is taken toward recommending performance measurement approaches when high throughput is required as will eventually be in a production line.

Chimerized Anti-ICOS 314.8 Monoclonal Antibodies Inhibit Tumor Cells and Regulatory T Cells in Patients with Sézary Syndrome

Background: In a previous study, we reported the strong expression of Inducible T-cell costimulatory (ICOS) by Sézary cells, and presented the excellent preclinical efficacy results of anti-ICOS antibody drug conjugates (ADCs) in both Sézary syndrome (SS) and angioimmunoblastic T-cell lymphoma. Although exerting a potent direct action on the tumor cell, ADCs have the disadvantage of being associated with a cumulative toxicity, related to the conjugated drug. The development of antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC/ADCP)-inducing anti-ICOS monoclonal antibodies (mAbs) is therefore of great importance to ensure long-term maintenance therapy.Methods: We first determined which anti-ICOS clone was the best candidate to induce an ADCC effect on ICOS + cell lines (MyLa, MJ and HUT78), using Mouse FcγRIII ADCC Bioassay (Promega®). The selected mAb was then chimerized and afucosylated (GlymaxX® technology, Evitria®).Secondly, we evaluated in vitro ADCC and ADCP effect of the chimeric anti-ICOS mAb against ICOS + CTCL cell lines, compared to both positive controls (mogamulizumab (moga) and alemtuzumab) and negative controls (IgG1 isotype control, and rituximab). To perform ADCC experiments, we co-incubated target cells with mAbs and allogenic NK lymphocytes from healthy volunteers (HV). Cellular apoptosis was measured by flow cytometry using the Caspase 3/7 assay (Promega®). For ADCP, monocytes were sorted from HV blood samples and treated with M-CSF. Target cells were labeled with PKH67 (Sigma-Aldrich®) and after co-incubation, CD14 +CD11b +PKH67 + monocytes were analyzed by flow cytometry.Finally, we verified the ADCC/ADCP potency of anti-ICOS mAbs on primary Sézary cells isolated from 16 moga-naïve SS patients, and 6 patients who had developed resistance to moga. We also questioned whether anti-ICOS mAbs were able to promote the autologous apoptosis of Sézary cells and T regulatory cells (Tregs) when directly incubated with peripheral blood mononuclear cells (PBMCs) from patients with SS.Results: Among 7 different anti-ICOS clones, 314.8, 92.17 and 293.1 clones had the higher ADCC activity against MyLa, MJ and HUT78. Of these 3 clones, 314.8 had the best affinity to the receptor, and the best ability to inhibit binding between ICOS receptor and a recombinant ICOS ligand protein. Anti-ICOS 314.8 mAb was then chosen to be chimerized and glyco-engineered.ICOS and CCR4 were strong and similarly expressed on MyLa and MJ. HUT78 had only mild expression of ICOS and CD52. Anti-ICOS mediated potent ADCC of cell lines (respectively 39.1±5% and 52.6±2.4% for MyLa and MJ cells), without significant difference when compared to mogamulizumab. In HUT78 cells, anti-ICOS induced a specific apoptosis of 35.7±5% versus 15.6±5.6% with alemtuzumab (p=0.02; CI95%: 4.1-36.1).Moreover, phagocytosis induced by anti-ICOS was significantly increased than that induced by negative controls. On MyLa cells, anti-ICOS had a greater phagocytosis activity than moga (59.4±5.2% vs 39.4±5.1%, p=0.031).Expression of ICOS by circulating tumor cells was found in all the 16 moga-naïve patients. The expression was strong, as 61±6% of tumor cells expressed ICOS vs 20±8% of non-tumoral CD4 + cells. CCR4 was more expressed than ICOS on both Sézary cells and non-tumoral CD4 + cells (91±6%, and 44±9% respectively). Anti-ICOS induced the apoptosis of 57.1±4.7% Sézary cells, compared to 16.9±2.2% with rituximab (p<0.01; CI95%:-53–27). The efficacy was better than with alemtuzumab, but there was no significant difference with moga. The ADCP effect induced by anti-ICOS did not differ with moga. Interestingly, anti-ICOS were effective in 6 moga-resistant patient, as they induced 38.9±5.9% of apoptosis, compared to 12.4±4.7% with moga (p<0.001).Ex vivo, anti-ICOS allowed 39.4±19.9% and 70.1±20.1% lysis of Sézary cells and Tregs respectively, with no difference with moga and alemtuzumab. However, the depletion of non-tumoral CD4 + and total PBMCs was significantly lower with anti-ICOS mAbs than with moga and alemtuzumab.Discussion: In a recent study, we showed that Treg cells of patients with SS have a high expression of ICOS. Here, we demonstrate that anti-ICOS mAbs induce Tregs depletion, which may improve immune profiles and emphasize Sézary cells killing. Our data suggest robust anti-tumor activity of anti-ICOS mAbs in SS, and xenograft experiments are underway to confirm these findings. DisclosuresLopez: Emergence Therapeutics: Current holder of individual stocks in a privately-held company. Bagot: Takeda: Membership on an entity's Board of Directors or advisory committees. Olive: Alderaan Biotechnology: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; ImCheck Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Emergence Therapeutics: Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

III‐V//CuxIn1−yGaySe2 multijunction solar cells with 27.2% efficiency fabricated using modified smart stack technology with Pd nanoparticle array and adhesive material

AbstractMultijunction (MJ) solar cells achieve high efficiencies by effectively utilizing the solar spectrum. Previously, we have developed III‐V MJ solar cells using smart stack technology, a mechanical stacking technology that uses a Pd nanoparticle array. In this study, we fabricated an InGaP/AlGaAs//CuxIn1−yGaySe2 three‐junction solar cell by applying modified smart stack technology with a Pd nanoparticle array and adhesive material. Using adhesive material (silicone adhesive), the bonding stability was improved conspicuously. The total efficiency achieved was 27.2% under AM 1.5 G solar spectrum illumination, which is a better performance compared to our previous result (24.2%) for a two‐terminal solar cell. The performance was achieved by optimizing the structure of the upper GaAs‐based cell and by using a CuxIn1−yGaySe2 solar cell with a specialized performance for an MJ configuration. In addition, we assessed the reliability of the InGaP/AlGaAs//CuxIn1−yGaySe2 three‐junction solar cell through a heat cycle test (from −40°C to +85°C; 50 cycles) and were able to confirm that our solar cells show high resistivity under severe conditions. The results demonstrate the potential of III‐V//CuxIn1−yGaySe2 MJ solar cells as next‐generation photovoltaic cells for applications such as vehicle‐integrated photovoltaics; they also demonstrate the effectiveness of modified smart stack technology in fabricating MJ cells.

Kinome profiling analysis identified Src pathway as a novel therapeutic target in combination with histone deacetylase inhibitors for cutaneous T-cell lymphoma

BackgroundHistone deacetylase inhibitors (HDACi) are used to treat patients with cutaneous T-cell lymphoma (CTCL), but they show limited efficacy. Hence, combination therapies should be explored to enhance the effectiveness of HDACis. ObjectiveThis study was conducted to identify novel therapeutic targets that can be combined with HDACis for treating CTCL. MethodsWe performed a global kinome profiling assay of three CTCL cell lines (HH, MJ, and Hut78) with three HDACis (romidepsin, vorinostat, and belinostat) using the PamChip® microarray. The three cell lines were co-treated with romidepsin and an inhibitor against the tyrosine kinase pathway. ResultsPrincipal component analysis revealed that kinome expression patterns were mainly related to the cell origin and were not affected by the drugs. Few kinases were commonly activated by the HDACis. Most identified kinases were Src-associated molecules, such as annexin A2, embryonal Fyn-associated substrate, and progesterone receptor. Phosphorylated Src was not observed in any untreated cell lines, whereas Src phosphorylation was detected in two of the three cell lines after HDACi treatment. Ponatinib, a Src inhibitor, significantly enhanced romidepsin-induced apoptosis not only in HH, MJ, and Hut78 cells, but also in Myla and SeAx CTCL cell lines. ConclusionThe Src pathway is a possible target for combination therapy involving HDACis for CTCL.

589 Effects of CCR4 antagonists in cutaneous T-cell lymphoma cells

CC chemokine receptor 4 (CCR4) is responsible for T-cell skin homing. It is overexpressed in type 2 helper T-cells (Th2) as well as cutaneous T-cell lymphoma (CTCL) cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are two most common types of CTCL. Currently, Mogamulizumab, a humanized anti-CCR4 antibody, has been FDA-approved to treat MF and SS. However, the clinical response rates for SS patients was 30% and for MF patients was 22% and severe drug eruptions following treatment were reported. Thus, a more effective and less toxic strategy is needed for CCR4-targeted therapy. CCR4 antagonists have been studied in diseases where Th2 cells participate, such as asthma and atopic dermatitis, but little has been done for CTCL. Here, we tested two CCR4 antagonists, C021 (class I antagonist) and AZD2098 (class II antagonist), in MF derived cell line (MJ) and SS derived cell line (Hut78). The effects of two compounds on cell chemotaxis, proliferation, apoptosis, and Th2 cytokine secretion were assessed. As expected, CCR4 was highly expressed on MJ and Hut78 cells. MJ and Hut78 cells showed chemotactic responses to TARC/CCL17 and MDC/CCL22. Both C021 and AZD2098 inhibited chemotactic responses to TARC/CCL17 and MDC/CCL22 in MJ and Hut78 cells. Of note, C021 inhibited chemotaxis to CCL17 at a much lower concentration (IC50: 0.186 μM) than to CCL22 (1.3 μM) in MJ cells. AZD2098 inhibited chemotaxis to CCL17 at a much lower concentration (IC50: 0. 12 μM) than to CCL22 (0.866 μM) in Hut78 cells. Interestingly, only C021 downregulated CCR4 expression on cell surfaces. Only C021 inhibited cell proliferation, at a higher concentration (IC50: 3.21 μM in MJ cells; 5.98 μM in Hut78 cells), and induced cell apoptosis. AZD2098 had no such effect at any concentrations tested. In addition, the expressions of IL-5 and IL-13 proteins were decreased after treatment with C021 but not with AZD2098. Our results suggest that both CO21 and AZD2098 have inhibitory effects on chemotaxis of CTCL cells, but only C021 exerts other effects on CTCL cells. The in vivo study is on the way.

Ectonucleotidase CD39 is highly expressed on ATLL cells and is responsible for their immunosuppressive function

Adult T-cell leukemia/lymphoma (ATLL) patients have an extremely poor prognosis, partly due to their immunosuppressive state. The majority of ATLL patients have leukemic cells with phenotype similar to Tregs, prompting suggestions that ATLL cells themselves have immunosuppressive functions. In this study, we detected CD39 expression on ATLL cells, particularly frequent on aggressive subtypes. CD39 and CD73 convert extracellular adenosine triphosphate (ATP) into adenosine, a key player in Tregs’ immunosuppression. In vitro culture, both CD39+ ATLL cells and normal Tregs converted rapidly extracellular ATP to AMP, which was disturbed by CD39 inhibitors, and was negated in the CD39 knockout MJ cell line. The proliferation of cocultured CD4+/CD8+ normal T cells was suppressed by CD39+ MJ cells, but not by CD39 knockout MJ cells. Supplemented ATP was exhausted by an EG7-OVA T-cell line with stable CD39 induction, but not by mock. When these cell lines were subcutaneously transplanted into murine flanks, Poly(I:C) peritoneal administration reduced tumor size to 1/3 in mock-transplanted tumors, but not in CD39 induced tumors. Overall, we found that ATLL cells express CD39 at a high rate, and our results suggest that this helps ATLL cells escape antitumor immunity through the extracellular ATPDase-Adenosine cascade. These findings will guide future clinical strategies for ATLL treatment.

The retinoid derivant ECPIRM selectively exhibited anti-proliferation effects in cutaneous T-Cell lymphoma via ITK-mediated signaling pathway

BackgroundThe treatment of Cutaneous T-cell lymphoma (CTCL) met huge challenges because of the heterogeneity and the scarcity of targeted drugs. ECPIRM derived from isotretinoin exhibited strong anti-proliferation effects in Hut78 and MJ cells rather than Myla cells. However, there was no data regarding the potential target of ECPIRM for its selective activity. ObjectivesTo investigate the potential target of ECPIRM for its selective anti-proliferation activity. MethodsWe evaluated the cell viability of CTCL cells after ECPIRM treatment, and detected the effects of ECPIRM on the biomarker genes of CTCL. Subsequently, the mRNA and protein level of Interleukin-2-inducible T-cell kinase (ITK) was determined. Then the induction of apoptosis triggered by ITK inhibitor BMS-509744 and ITK siRNAs were detected, and the docking of ECPIRM interacted with ITK and the effects of ECPIRM on ITK-mediated signaling pathway were analyzed. Finally, we evaluated the anti-growth activity of ECPIRM in Hut78-xenografted nude mice, and the relative expression of cleaved caspase-3, ITK, p-ERK and p-Akt were determined. ResultsITK was highly expressed in Hut78 and MJ cells rather than Myla cells, and targeted inhibition of ITK triggered cell apoptosis. ECPIRM efficiently bound the hydrophobic active pocket of ITK, and significantly inhibited ITK-mediated signaling pathway. In addition, ECPIRM suppressed tumor growth in Hut78-xenografted model, and upregulated the expression of cleaved caspase 3 and inhibited the expression of ITK, p-ERK and p-Akt in tumor tissues, which was consistent with in vitro study. ConclusionECPIRM might provide a novel strategy for CTCL by inhibiting ITK-mediated signaling pathway.

DT2216, a BCL-XL Proteolysis Targeting Chimera (PROTAC), Is a Potent Anti T-Cell Lymphoma Agent That Does Not Induce Significant Thrombocytopenia

There is an urgent need for new therapeutic agents to treat patients with T-cell lymphoma (TCL). Multiple hematologic malignancies evade apoptosis through overexpression of anti-apoptotic proteins in the BCL-2 family, including BCL-2, BCL-XL, and MCL-1. We and others recently showed that a large fraction of cutaneous and peripheral TCL cell lines, patient-derived xenografts and primary patient samples depend on BCL-XL for survival (Koch et al, Blood. 2019; 133:566-575). These findings suggest that targeted inhibition of BCL-XL could offer therapeutic benefit for some TCL patients. Currently available small molecule BCL-XL inhibitors have failed during clinical development due to on-target and dose-limiting thrombocytopenia, as platelets depend on BCL-XL for survival. To overcome this toxicity, we developed DT2216, a novel proteolysis-targeting chimera (PROTAC) that targets BCL-XL to the Von Hippel Lindau (VHL) E3 ligase for proteasomal degradation. We selected the VHL ligase because platelets express very low levels of VHL, suggesting that they would be spared from the pro-apoptotic effects of DT2216. Here we examined the therapeutic potential of DT2216 against different TCL cell lines in vitro and in TCL xenograft mouse models. We first profiled the expression of different anti-apoptotic BCL-2 family proteins in multiple TCL cell lines (Fig. 1a) and tested their sensitivity to selective inhibitors for different BCL-2 family proteins as well as commonly-used chemotherapeutic agents (Table 1). The results showed that the TCL cells with higher levels of BCL-XL were more resistant to doxorubicin, etoposide and vincristine. DT2216 was more effective in reducing the viability of BCL-XL-dependent TCLs such as MyLa cells than ABT263 (a dual BCL-2/XL inhibitor). The EC50 value of DT2216 for MyLa TCL cells was less than 10 nM and DT2216 killed MyLa cells through induction of BCL-XL degradation and cellular apoptosis (Fig. 1b-f). Moreover, DT2216 was less toxic to human platelets than ABT263 in vitro with an EC50 &amp;gt; 3 μM. We further validated the effect and specificity of DT2216 in MJ cells, another BCL-XL-dependent TCL cell line, and demonstrated that its antitumor activity was dependent on proteasome activity. In vivo, when DT2216 was given to mice with MyLa TCL xenografts by i.p. injections at 10 mpk/q4d, it significantly inhibited tumor growth, whereas ABT263 at the same dose had no significant effect (Fig. 1g). More importantly, after MyLa xenografted mice failed to respond to ABT263 treatment, we subjected the mice to DT2216 (10 mpk/q4d), which induced rapid tumor regression and increased the survival of the mice without causing significant reduction of blood platelets (Fig. 1h). These effects were associated with a significant reduction in BCL-XL expression and activation of caspase cascade in tumor xenografts. However, some TCLs depend on not only BCL-XL but also BCL-2 or MCL-1 for evasion of apoptosis. Therefore, we next assessed the therapeutic potential of DT2216 in combination with a selective BCL-2 inhibitor using the TCL PDX DFTL-28776, which depends on both BCL-XL and BCL-2 for survival. We found that the combination of DT2216 with ABT199 (a selective BCL-2 inhibitor) could more effectively kill DFTL-28776 TCL cells than either agent alone in cell culture. The effect of the combination treatment on the growth of DFTL-28776 PDX in vivo is under examination. Collectively, our findings suggest that targeting BCL-XL using DT2216 can selectively kill BCL-XL-dependent TCL cells without causing significant platelet toxicity. Moreover, the combination of DT2216 with an inhibitor targeting other anti-apoptotic BCL-2 family proteins may have broad therapeutic utility against multiple TCL types and other cancers dependent on BCL-XL. Disclosures He: University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Khan:University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Zhang:University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer agents. Zheng:Dialectic Therapeutics: Equity Ownership, Other: Co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents. Weinstock:Celgene: Research Funding. Zhou:Dialectic Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: co-founders of Dialectic Therapeutics that develops Bcl-xl PROTACs as anti-cancer agents; Unity Biotechnology: Equity Ownership, Other: Co-founder of Unity Biotechnology which develops small-molecule senolytic drugs; University of Arkansas for Medical Sciences: Patents &amp; Royalties: inventor of a pending patent application for use of Bcl-xl PROTACs as anti-cancer and anti-aging agents.

Abstract 3132: Inhibition of the integrin αVβ3 improves the effect of bexarotene in the treatment of cutaneous T-cell lymphoma (CTCL)

Abstract CTCL are exposed to a complex paracrine and endocrine environment that influence their progression from skin to visceral disease. One of the most common treatments for CTCL is RXR agonists or “rexinoids” such as bexarotene (BEX). The most prevalent side effect of rexinoids (~95% of patients) is the development of hypothyroidism hence these patients are prophylactically treated with thyroid hormone (TH). Paradoxically, we recently found (PMID: 25488971) that TH can activate transcriptional programs required for proliferation of CTCL. We also found that the effects of TH on CTCL are mediated through the activation of two different receptors, the classical nuclear TR and a membrane receptor integrin αVβ3. Our aim was to study how the anti-lymphoma activity of BEX is modified by the activation of TR and αVβ3 during the TH replacement therapy, and unknown topic with clinical implications. We first evaluate the cell viability of CTCL cells HuT78 and MJ treated with BEX in presence and absence of physiological levels of TH. As expected, BEX decreased the viability of CTCL cells, however in presence of TH both effects decreased by 25-60% and 20-50% on HuT78 and MJ cells. However, hypothyroidism is associated with a higher dissemination of TCL cells in syngeneic mice. Thus, to evaluate the impact of TH replacement therapy in BEX-treated mice, we implanted TCL EL-4 cells subcutaneously in C57BL/6 mice. When tumors reached ~75 mm3, mice were randomized in three groups: vehicle, bexarotene alone (BEX) and bexarotene with T4 replacement (to reach euthyroidism) (BEX-T4). Tumor growth significantly decreased in both bexarotene arms (vs. vehicle). However, in contrast to mice receiving BEX-T4, mice receiving BEX alone showed a decreased infiltration of tumor-suppressive immune cells. Specifically, these tumors had decreased NK and CD3+CD8+ cells due to BEX-induced hypothyroidism. This indicates that although TH could decrease the effect of BEX in CTCL cells, the hypothyroidism would impact in the local antitumor response. Thus determine if the inhibition of the non-canonical TH membrane receptor integrin αVβ3 would decrease the pro-survival effect of TH on CTCL treated with BEX. We found that either, αVβ3 silencing or the pharmacological inhibitor Cilengitide, not only avoided the pro-survival effect of TH but increased the anti-lymphoma activity of BEX-T4 by 15-35% in CTCL cells. To evaluate the molecular program underpinning these effects, we conducted RNA-sequencing in HuT78 cells treated with BEX-T4 in presence of siRNA for TR, αVβ3 or controls. Among the top pathways we identified genes involved in “apoptosis”, “angiogenesis” and “IL6-JAK-STAT3 signaling” as differentially expressed in si-αVβ3 vs. the others. These genes were independently validated by using Cilengitide. Our data suggest that inhibition of the non-canonical TH receptor, the integrin αVβ3, could be an effective strategy to improve BEX treatment in CTCL Citation Format: Florencia Cayrol, Victoria Revueta, Alejandra Paulazo, Mercedes Debernardi, Maria Celeste Diaz Flaque, Helena Sterle, Leandro Cerchietti, Graciela Cremaschi. Inhibition of the integrin αVβ3 improves the effect of bexarotene in the treatment of cutaneous T-cell lymphoma (CTCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3132. doi:10.1158/1538-7445.AM2017-3132

Oncogenic role of microRNA-155 in mycosis fungoides: an in vitro and xenograft mouse model study.

MicroRNA (miR)-155 contributes to the proliferation of mycosis fungoides (MF) in vitro and is upregulated in tumours of MF compared with early MF lesions. To investigate the contribution of miR-155 to the cancerous phenotype and drug resistance of MF/Sézary cell lines. miR-155 was inhibited in MF cell lines (MyLa and MJ) by transduction of miRZip anti-miR-155, and overexpressed in Hut78 cells by transduction of miRVec-miR-155; empty plasmids served as controls. Cells were analysed for response to inducers of apoptosis and cell-cycle arrest, using fluorescence-activated cell sorting. Transduced MyLa cells were subcutaneously injected into severe combined immunodeficient mice, and tumours were analysed immunohistochemically and for final size. MyLa and MJ cells expressed a high level of miR-155; Hut78 cells expressed a low level. MF cell lines stably expressing miR-155 inhibitor showed increased G2/M arrest in response to N-p-tolyl-2-(3,4,5-trimethoxyphenyl quinazolin-4-amine) (SL111), an inducer of cell-cycle arrest, followed by increased apoptosis. Additionally, they showed increased apoptosis in response to suberoylanilide hydroxamic acid (SAHA). Tumours formed in mice from injected anti-miR-155-expressing MyLa cells had a significantly lower volume and higher occurrence of apoptosis than controls. Stable overexpression of miR-155 in Hut78 cells had no effect. Oncogenic miR-155 appears to contribute to the cancerous phenotype of MyLa and MJ cells, but not of Hut78 cells, by interrupting activation of the G2/M checkpoint in response to SL111, and decreasing apoptosis in response to SL111 and SAHA, thereby facilitating tumour growth. These findings have implications for the potential development of novel therapeutic modalities for MF incorporating miR-155 inhibitors.

Apoptosis Induction in Cutaneous T-Cell Lymphoma Cells after Treatment with Lenalidomide

Introduction: Cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of extranodal non-Hodgkin lymphomas, derived from skin-homing mature T-cells. The two most common types of CTCL are Mycosis fungoides (MF) (50%-72%), which is generally indolent in behavior, and Sézary syndrome (SS) (1%-3%), an aggressive leukemic form of the disease. Currently, there is no effective treatment for CTCL patients. Research on new therapies for CTCL is largely centered on defining novel therapeutic targets, but in most cases the response is short and the survival rate remains the same. Therefore, the CTCL’s resistance to chemotherapy and the lack of full understanding of their pathogenesis request further investigation.Aims: With the view of a more targeted therapy, we evaluated in vitro the effectiveness of lenalidomide, an immunomodulatory agent (IMID) with clinical efficacy, among others, in plasma cell dysplasias and lymphoproliferative disorders.Methods: Four CTCL cell lines were used: MJ and MyLa (derived from MF), SeAx and Hut-78 (both derived from peripheral blood of patients with Sézary syndrome). SeAx and Hut-78 cells were cultured in RPMI 1640, supplemented with 10% FBS and 2 mM L-glutamine. MJ and MyLa cells were cultured in RPMI 1640 and IMDM, respectively, supplemented with 20% FBS. All cell lines were maintained at 37°C in a humid atmosphere of 5% CO2. Cells were treated with various concentrations of lenalidomide (1μΜ, 10μΜ and 100μΜ) for 24, 48 and 72h. Apoptosis was determined by flow cytometry using the Annexin V/PI method. The proliferative capacity of untreated and lenalidomide- treated cells was measured using the BrdU assay.One-way Anova and LSD/ Bonferroni methods were applied for the statistical analysis of the results.Results: Our data indicate that all cell lines responded with enhanced apoptosis at various lenalidomide treatment conditions. Between the two MF lines tested, MyLa cells were affected the most. Specifically, MyLa cells exhibited a statistically significant augmentation on their apoptosis compared to untreated cells after treatment with 10μΜ and 100μΜ lenalidomide for 24h (9.7 and 8.66 vs 4.83, p=0.000 and p<0,001, respectively) and 48h (6.1 and 4.46 vs 3,36, p=0.000 and p<0,007, respectively), as well as after treatment with 10μΜ Lenalidomide for 72h (5.3 vs 4.7, p=0.000). Similarly, MJ cells also responded with enhanced apoptosis after 24h and 48h of treatment with 100μΜ lenalidomide (8.8 vs 7.43 and 11.13 vs 8.66, p=0.000, respectively) and 72h of treatment with 1 μM lenalidomide (10.63 vs 7.96, p=0.000). Regarding the response of the two SS cell lines to lenalidomide treatment, SeAx cells were significantly affected, exhibiting high apoptotic rates compared to untreated cells after treatment with 1μΜ lenalidomide for 48h (13.76 vs 5.1, p=0.000) and 1μΜ, 10μΜ and 100 μΜ lenalidomide for 72h (1.5, 2.4 and 7.03 vs 0.13, p=0.000, respectively). Lenalidomide had a rather moderate effect on the apoptosis of Hut-78 cells, which presented with statistically significant enhanced apoptosis only after treatment with 1μΜ lenalidomide for 72h (1.33 vs 0.8, p<0,009). The proliferation capacity of the CTCL cell lines tested presented no statistically significant changes under any of the treatment conditions.Conclusions: Our observations demonstrate that lenalidomide doesn't appear to have any significant effect on the proliferation of the CTCL cell lines. On the contrary, it seems to lead both MF and SS cell lines to significantly enhanced apoptosis. Interestingly, the MF cell lines seem to be more sensitive to lenalidomide treatment in terms of apoptosis induction compared to SS cell lines. Moreover, in all CTCL cell lines tested, high concentrations of lenalidomide contributed to enhanced apoptosis after a shorter period of treatment, compared to lower concentrations, which seemed to be more effective after a longer period, with the exception of SeAx cells. Although these initial results need to be further confirmed both in vitro and in vivo, they appear very encouraging for the integration of lenalidomide treatment, alone or in combination, in CTCL therapy. DisclosuresNo relevant conflicts of interest to declare.

Thymic Stromal Chemokine TSLP Acts through Th2 Cytokine Production to Induce Cutaneous T-cell Lymphoma.

Thymic stromal lymphopoietin (TSLP) activates dendritic cells to induce Th2-mediated inflammation. Periostin, an extracellular matrix protein produced by fibroblasts, induces chronic inflammation by stimulating TSLP production. Recently, a reinforcing cycle linking Th2-type immune responses with periostin-induced keratinocyte activation has been proposed in atopic dermatitis pathogenesis. In this study, we investigated the role of TSLP and periostin in the development of cutaneous T-cell lymphoma (CTCL), where Th2 cytokines and chemokines are also dominant. TSLP and periostin mRNA expression levels were elevated in CTCL lesional skin, both of which correlated with IL4 expression levels. In vitro and ex vivo, IL4 or IL13 stimulated periostin expression by dermal fibroblasts, and fibroblasts from CTCL lesional skin expressed higher levels of periostin than those from control skin. Serum periostin levels of CTCL patients were also significantly higher than those of healthy individuals. Hut78 and MJ, CTCL cell lines, and peripheral blood mononuclear cells from leukemic CTCL patients expressed the TSLP receptor. TSLP induced production of IL4 and IL13 by Hut78 and MJ cells through the activation of STAT5. Moreover, TSLP induced proliferation of CTCL cells both in vitro and in vivo These data suggest that periostin-mediated TSLP production by keratinocytes directly stimulates CTCL tumor cell growth in addition to inducing a Th2-dominant tumor environment in CTCL. Cancer Res; 76(21); 6241-52. ©2016 AACR.

ONC201 Induces Apoptosis in Cutaneous T-Cell Lymphoma Cells through a Mechanism That Involves the Integrated Stress Response and Inactivation of Jak/Stat Signaling

Background: ONC201 (also called TIC10) is an orally active, first-in-class small molecule that is in phase II clinical trials for advanced cancers based on its ability to activate apoptosis in tumor cells, but not normal cells, in a p53-independent manner. Recent studies have implicated the integrated stress response as an early stage mechanism of ONC201 that may lead to the previously observed downstream anti-cancer effects. This study was conducted to evaluate the anti-tumor activity of ONC201 on Cutaneous T-Cell Lymphoma (CTCL) cell lines and Sézary cells.Methods: We treated 8 CTCL cell lines (H9, HH, Hut78, Mac2A, MJ, MyLA, Pb2b and SeAx), peripheral blood mononuclear cells (PBMC) from 5 Sézary syndrome (SS) patients and 6 healthy donors with ONC201. MTS viability Assay was used to assess the anti-proliferation effect. Apoptosis was assessed using Annexin V FITC/PI staining and sub-G1 analysis by flow cytometry. Protein expression by western blotting was analyzed in 3 CTCL cell lines (HH, Hut78, MJ) and in PBMCs from 2 SS patients treated for 72 hours with ONC201including Activating Transcription Factor 4 (ATF4), interferon regulatory factor 7 (IRF7/pIRF7), Signal transducer and activator of transcription 3 (STAT3), Janus Kinase 3 (JAK3/pJAK3) and NF-κB family members (p65, RelB, c-Rel and p105).Results: MTS viability assay showed pronounced cell growth inhibition, significantly increasing in a time-dependent manner in H9, HH, Hut78, Mac2A, MJ, MyLa, Pb2b, and SeAx lines after a 96-hour incubation within a very narrow efficacy threshold ranging from 1.25 to 10µM (n = 8, p < 0.05). To determine whether growth inhibition of ONC201 is due to cell-cycle arrest and/or to apoptosis in CTCL cell lines, sub-G1 analysis by flow cytometry was measured in 3 CTCL cell lines (HH, Hut78 and MJ). The percentages of HH, Hut78 and MJ cells with sub-G1 population increased, suggesting that cells are undergoing apoptosis (n = 3, p < 0.05). Induction of apoptosis was further confirmed by Annexin V FITC/PI staining, revealing dose and time dependent apoptosis induction in all 8 cell lines (Figure 1). To confirm cell line results in refractory ex vivo samples, we tested the pro-apoptotic effects of ONC201 on PBMCs from 5 SS patients who had high circulating CD4+CD26- malignant T-cells compared with PBMCs from 6 healthy donors. ONC201 induced significant levels of apoptosis in PBMCs from SS patients, but not in normal PBMCs (Figure 1, p < 0.001). Western blot analysis revealed that ONC201 increased the expression of ATF4, a hallmark of the integrated stress response and a negative regulator of IRF7. ONC201 decreased expression of IRF7/pIRF7 and down-regulated JAK3/pJAK3 and STAT3 expression in CTCL cell lines and PBMCs from SS patients. ONC201 also decreased the protein expression of NF-κB family members ( p65, RelB, c-Rel and p105) that can cause resistance to apoptosis in CTCL cells.Conclusions: ONC201 appears to be active as a single oral agent. It may impact key signaling pathways in CTCL preclinical models by inhibiting proliferation and inducing apoptosis through a mechanism that involves the integrated stress response, leading to inactivation of JAK/STAT signaling and down-regulation of the NFκB pathway. [Display omitted] DisclosuresDuvic:Eisai: Research Funding; MiRagen Therapeutics: Consultancy; Cell Medica Ltd: Consultancy; Tetralogics SHAPE: Research Funding; Soligenics: Research Funding; Array Biopharma: Consultancy; Innate Pharma: Research Funding; Allos (spectrum): Research Funding; Rhizen Pharma: Research Funding; Spatz Foundation: Research Funding; Oncoceutics: Research Funding; Huya Bioscience Int'l: Consultancy; Therakos: Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kyowa Hakko Kirin, Co: Membership on an entity's Board of Directors or advisory committees, Research Funding. Off Label Use: Most drugs for ctcl are non approved. This drug is a small molecule and is not approved for any cancer. Tarapore:Oncoceutics, Inc: Employment, Equity Ownership. Allen:Oncoceutics, Inc: Employment, Equity Ownership.

Abstract 2096: High sensitivity of cutaneous T-cell lymphoma (CTCL) to CABE, a component of propolis

Abstract Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of diseases characterized by clonal expansion of malignant T-cells in the skin. In the course of our quest to screen and identify a potent, selective anti-proliferative drug for CTCL, we discovered that caffeic acid benzyl ester (CABE), a natural component of edible plant species found in high concentrations in propolis, showed increased potency for CTCL (IC50 4 μM) in vitro compared to other tumor types such as melanoma (IC50 25 μM), glioma (IC50 25 μM), multiple myeloma (MM) (IC50 70 μM), head and neck squamous cell carcinoma (HNSCC) (IC50 25 μM), ovarian cancer (OC) (IC50 17 μM), breast carcinoma (IC50 43 μM), and Hodgkin's B-cell lymphoma (IC50 33 μM). Cell death in CTCL MJ and HH cell lines was accompanied by increased caspase-3 and poly (ADP-ribose) (PARP) cleavage and annexin V staining. There is growing evidence that CABE and its analogs exert their anticancer activity by modulating expression level of key transcription factors. Here, we evaluated the effects of CABE on the activity of different transcription factors and the expression of total and phosphorylated proteins in MJ cells by use of the transcription factor activation profiling array (TFAPA) and the reverese-phase protein array profiling (RPPA) assays. The activity of 48 transcription factors (TFs) and expression of 225 unique total and phosphorylated proteins could be monitored simultaneously by using a collection of biotin-labeled DNA probes and 225 unique antibodies. TFAPA analysis revealed that 18 TFs (including STAT1, STAT4, and RXR) were up-regulated and 3 TFs (STAT3, STAT5, and SATB1) were down-regulated to a variable extent in cells treated with 10 μM CABE for 5 h as compared to vehicle-treated control. RPPA analysis revealed 41 differentially expressed proteins between vehicle control and cells treated with 10 μM CABE. These can be functionally categorized into functional groups: DNA repair, cell cycle, apoptosis, phosphoinositide 3-kinase (PI3K)/AKT/mTOR, and receptor kinase pathways. Our results suggest that CABE targets multiple signaling pathways in CTCL in vitro, warranting the development of a pre-clinical animal model for the testing of CABE efficacy in vivo. Acknowledgement: These studies were supported in part by the grant from Moleculin, LLC Citation Format: Arumugam Jayakumar, Radjendirane Venugopal, Rafal Zielinski, Aleksandra Rusin, Izabela Fokt, Stanislaw Skora, Xiao Ni, Madeleine Duvic, Waldemar Priebe. High sensitivity of cutaneous T-cell lymphoma (CTCL) to CABE, a component of propolis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2096. doi:10.1158/1538-7445.AM2015-2096

Optimization of virus detection in cells using massively parallel sequencing

Massively parallel sequencing (MPS)-based virus detection has potential regulatory applications. We studied the ability of one of these approaches, based on degenerate oligonucleotide primer (DOP)-polymerase chain reaction (PCR), to detect viral sequences in cell lines known to express viral genes or particles. DOP-PCR was highly sensitive for the detection of small quantities of isolated viral sequences. Detected viral sequences included nodavirus, bracovirus, and endogenous retroviruses in High Five cells, porcine circovirus type 1 and porcine endogenous retrovirus in PK15 cells, human T-cell leukemia virus 1 in MJ cells, human papillomavirus 18 in HeLa cells, human herpesvirus 8 in BCBL-1 cells, and Epstein–Barr Virus in Raji cells. Illumina sequencing (for which primers were most efficiently added using PCR) provided greater sensitivity for virus detection than Roche 454 sequencing. Analyzing nucleic acids extracted both directly from samples and from capsid-enriched preparations provided useful information. Although there are limitations of these methods, these results indicate significant promise for the combination of nonspecific PCR and MPS in identifying contaminants in clinical and biological samples, including cell lines and reagents used to produce vaccines and therapeutic products.

Thyroid Hormones Maintain The CTCL Malignant Phenotype Through Membrane- and Nuclear-Initiated Transcriptional Programs

Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of T-cell non-Hodgkin lymphomas (T-NHL) derived from skin-homing mature T cells. Thyroid hormones (THs) are crucial regulators of cellular differentiation, growth and metabolism. We recently found that THs stimulate the proliferation and metabolism of T cells and that this pathway is co-opted by T-NHL cells to maintain their malignant phenotype. Accordingly, a decrease in TH levels affects the proliferation of T-NHL cells. We also found that THs modulate these functions by activating both canonical nuclear (TR) and membrane receptors (mTR, that for most cells is represented by integrin αvβ3). We now hypothesized that the characterization of the relative contribution of these receptors to the malignant phenotype of T-NHLs will allow for a more precise therapeutic intervention. The study presented here was conducted to determine (i) whether THs are pro-survival factors in CTCL and (ii) the relative contribution of TR and mTR to the effect of THs in CTCLs.To test this hypothesis, we first analyzed the expression of TR and mTR in CTLC cells (HuT 78, MJ and HuT102) vs. normal T cells (from peripheral blood) by qRT-PCR and immunoblotting. We found that CTCL expressed 2 to 10 fold more THRA, ITGAV and ITGB3 mRNA and protein than their normal counterpart (p<0.05, T test). Moreover, in a tissue microarray of 52 CTCLs, the integrin avβ3 was overexpressed in 92% of the cases. To differentiate between nuclear vs. membrane-initiated effects, CTCL cells were treated with physiological concentrations of free and cell impermeable agarose-bound T3/T4 (TH-ag). After 24h of treatment, cell proliferation increased 20-38% in HuT-78 cells and 18-32% in MJ cells with both forms of THs (p<0.05 for both comparisons vs. control). Long-term exposure (96h) to free TH and TH-ag accelerated HuT-78 doubling time by 17-25% and 16-22%, respectively (p<0.05 for both comparisons). By time course analysis of mRNA abundance (1, 2, 3, 6, 12 and 24 h post TH), we found that TH-induced phenotypic changes were associated with 1.5 to 3 fold up-regulation of proliferation markers such as PCNA, CCND1, CCND2, CCND3, CCNE2 and CCNB1 in addition to a concomitant decrease of the tumor suppressor genes TP53 and CDKN1A.To determine the specific transcriptional programs regulated by mTR and TR, T-NHLs were treated with free T3/T4 (for total TR activity) and agarose-coupled T3/T4 (for mTR activity) for 24h and analyzed by mRNA-sequencing. Differentially regulated genes were further analyzed using Ingenuity Pathway Analysis. We found the TNFR and interleukin-2 pathways (e.g. TNFRSF19, TNFRSF9, IL2RA) among the top programs regulated by free THs through canonical nuclear TR activation whereas mTR activation upregulated genes involved in angiogenesis induction (e.g. PDGFR, VEGFA, VEFGB), lymphocyte proliferation/differentiation and DNA replication (e.g. IL4, DOK2). We independently validated these genes in HuT-78 and MJ cell lines by qRT-PCR. Taken together, these data suggest that TR and mTR regulate distinct transcriptional programs that are complimentary towards CTCL proliferation.Considering that (i) mTR are over-expressed in CTCL vs. normal T cells (in contrast to canonical nuclear TR that are broadly expressed) and (ii) mTR regulate pro-survival pathways in CTLC, we wished to determine whether mTR inhibition will be sufficient to decrease CTCL proliferation. We therefore tested the effect of mTR abrogation by using siRNA to ITGAV and ITGB3 as well as the effect of pharmacological mTR inhibitors (i.e: RGD peptide and Tetrac) in CTCL proliferation. We found that mTR inhibition with siRNA or compounds affected mTR-induced transcriptional programs. In fact, the TH-ag mediated upregulation of VEGFB, VEGFA, DOK2 and IL4 mRNA levels were totally abrogated in CTCL cells transfected with the siRNA to ITGAV and ITGB3. Moreover, almost 100% of the TH-ag-induced and 80% of the free-TH-induced CTCL proliferative effect was abrogated by ITGAV- and/or ITGB3-targeting siRNA.Taken together these data suggest that THs are important inducers of CTCL proliferation by transcriptionally regulating major survival pathways, and inhibition of mTR could constitute a selective chemotherapy-free treatment for CTCLs. Disclosures:No relevant conflicts of interest to declare.

Alkylphosphocholines and curcumin induce programmed cell death in cutaneous T-cell lymphoma cell lines

While most patients with early-stage cutaneous T-cell lymphomas (CTCL) have a very good prognosis, the survival of patients with extensive tumour stage and visceral involvement remains extremely poor and necessitates the development of more effective treatment modalities. In this study, we evaluated the in vitro effects of two alkylphosphocholines (APCs, miltefosine and erufosine) and the polyphenolic compound curcumin on 5 human CTCL cell lines (Hut-78, HH, MJ, My-La CD4+ and My-La CD8+). All tested drugs showed considerable cytotoxic activity, as determined by the MTT dye reduction assay. The IC50 values of both APCs ranged from the low micromolar level (Hut-78 cells) to 60-80μM (HH cells). The IC50 values of curcumin ranged from 12 to 24μM. All tested drugs induced apoptosis, as ascertained by morphological changes, DNA fragmentation and activation of caspase cascades. Miltefosine and erufosine induced dephosphorylation of Akt in My-La CD8+ cells and phosphorylation of JNK in Hut-78 and My-La CD8+ cells. APCs increased the level of the autophagic marker LC3B in Hut-78 and MJ cells. Results from co-treatment with autophagy modulators suggested that the cytotoxicity of APCs in CTCL cells is mediated, at least in part, by induction of autophagy.

Production and characterization of a CD25-specific scFv-Fc antibody secreted from Pichia pastoris

Antibodies against CD25 would be novel tools for the diagnosis and treatment of adult T cell leukemia lymphoma (ATLL) and many other immune disorders. In our previous work, we successfully produced the single-chain fragment of a variable antibody against CD25, the Dmab(scFv) antibody, using Pichia pastoris. Here, we describe a novel form of an antibody against CD25, the Dmab(scFv)-Fc antibody, also produced by P. pastoris. To construct the Dmab(scFv)-Fc antibody, the Dmab(scFv) antibody was genetically fused to the Fc fragment of a human IgG1 antibody. A fusion gene encoding Dmab(scFv)-Fc antibody was cloned into the pPIC9K plasmid and expressed at high levels, 60-70 mg/l, by P. pastoris under optimized conditions. The Dmab(scFv)-Fc antibody was similar to the Dmab(scFv) antibody in its binding specificity but different in its molecular form and Fc-mediated effector functions. The Dmab(scFv)-Fc antibody and the Dmab(scFv) antibody both bound to CD25-positive MJ cells but not to CD25-negative K562 cells. The Dmab(scFv)-Fc antibody existed as a dimer whereas the Dmab(scFv) antibody was a monomer because it lacks the Fc fragment. The Dmab(scFv)-Fc antibody enhanced the antibody-dependent cellular cytotoxicity of CD25-positive cancer cells, whereas the Dmab(scFv) antibody was inactive in the antibody-dependent cellular cytotoxicity assays. In addition, compared to the Dmab(scFv) antibody, the Dmab(scFv)-Fc antibody showed stronger immunosuppressive activity in the Con A-stimulated lymphocyte proliferation system and in the mixed lymphocyte reaction system. These results demonstrate that the Dmab(scFv)-Fc antibody produced in P. pastoris is functional, and therefore it might be developed as a novel diagnostic and therapeutic tool for ATLL and other immune disorders.

Differential CCR4 Expression And Function in Cutaneous T-Cell Lymphoma Cell Lines

Cutaneous T cell lymphoma (CTCL) is a clonal epidermotropic malignancy of memory T cells primarily involving the skin. However, the mechanisms governing migration of CTCL cells have not been fully clarified. It has been shown that certain chemokine receptors are upregulated in CTCL cells, but it remains unanswered whether these chemokine receptors play a critical role in the migration dynamics of CTCL. Using cell lines originally derived from patients with different subtypes of CTCL, we have shown higher CCR4 expression in the line derived from the mycosis fungoides (MJ), compared with the line derived from Sezary syndrome (Hut78). In specific responses to CCL22 (a CCR4 ligand) treatments, MJ cells showed significant chemotactic migration, enhanced activation and adhesion of certain integrins (CD49d and CD29) in vitro, while the control cells (Hut78, CD4+CD45RO+ memory T cells, and Jurkat cells) did not. Furthermore, compared with Hut78 cells, MJ cells manifested significantly more transendothelial migration in responses to treatments with either CCL22 or conditioned medium from dendritic cells in vitro. These results provide further dynamic evidence, in line with the multistep cascade paradigm for leukocyte transendothelial migration, to support a critical role for CCR4 in CTCL migration.