Pharmacologic marrow purging in murine T cell leukemia

Abstract Deoxycoformycin in combination with deoxyadenosine was used to purge 6C3HED malignant T cells from murine marrow in vitro. Adenosine deaminase activity of 6C3HED cells was ablated by incubation with 10(- 6) mol/L deoxycoformycin (dCF). During a 12-hour incubation with 10(-6) mol/L dCF and 10(-4) mol/L deoxyadenosine, tumor cells sequentially accumulated dATP, became depleted of NAD followed by ATP, then died. More than 5 logs of 6C3HED cells were killed as measured by survival of mice injected with treated tumor cells. Identical incubation of 5 x 10(6) marrow cells did not interfere with rescue of syngeneic lethally irradiated mice. Long-term survival was demonstrated in 12 of 14 mice that received marrow that had been contaminated with 5% 6C3HED cells, incubated with deoxycoformycin and deoxyadenosine, then used to rescue lethally irradiated mice. This murine model provides information not available from in vitro assays and may be useful in the development of strategies to purge malignant T cells from marrow.

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Pharmacologic marrow purging in murine T cell leukemia

Blood ◽

10.1182/blood.v71.6.1656.bloodjournal7161656 ◽

1988 ◽

Vol 71 (6) ◽

pp. 1656-1661

Author(s):

EA Copelan ◽

SC Johnson ◽

MR Grever ◽

JF Sheridan ◽

PJ Tutschka

Keyword(s):

T Cells ◽

Tumor Cells ◽

In Vitro Assays ◽

Cell Leukemia ◽

Term Survival ◽

Adenosine Deaminase Activity ◽

Marrow Purging ◽

Malignant T Cells

Deoxycoformycin in combination with deoxyadenosine was used to purge 6C3HED malignant T cells from murine marrow in vitro. Adenosine deaminase activity of 6C3HED cells was ablated by incubation with 10(- 6) mol/L deoxycoformycin (dCF). During a 12-hour incubation with 10(-6) mol/L dCF and 10(-4) mol/L deoxyadenosine, tumor cells sequentially accumulated dATP, became depleted of NAD followed by ATP, then died. More than 5 logs of 6C3HED cells were killed as measured by survival of mice injected with treated tumor cells. Identical incubation of 5 x 10(6) marrow cells did not interfere with rescue of syngeneic lethally irradiated mice. Long-term survival was demonstrated in 12 of 14 mice that received marrow that had been contaminated with 5% 6C3HED cells, incubated with deoxycoformycin and deoxyadenosine, then used to rescue lethally irradiated mice. This murine model provides information not available from in vitro assays and may be useful in the development of strategies to purge malignant T cells from marrow.

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108 MCY-M11, a CAR-PBMC cell product transiently expressing a mesothelin targeted mRNA CAR, exhibits desirable functional and immune phenotype attributed to sustained antitumor immunity in vitro

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0108 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A120-A120

Author(s):

Sashi Kasimsetty ◽

Himavanth Gatla ◽

Dhana Chinnasamy

Keyword(s):

T Cells ◽

Tumor Cells ◽

Antitumor Immunity ◽

Memory T Cells ◽

Cell Populations ◽

Epitope Spreading ◽

Antitumor Response ◽

Cell Product

BackgroundMCY-M11, an anti-mesothelin CAR (Meso-CAR) mRNA transfected PBMC cell product manufactured through <1 day-process is under clinical evaluation for the treatment of advanced ovarian cancer and peritoneal mesothelioma. In this in-vitro study, we characterized the phenotypic and functional status of immune cell populations in MCY-M11 and their possible role in antitumor immunity.MethodsMCY-M11 cell product were generated using unmanipulated healthy donor PBMCs (n=5) by transfection of Meso-CAR mRNA using MaxCyte’s proprietary Flow Electroporation® system. Frozen MCY-M11 cell product was thawed and cultured for 18 hours, then co-cultured with hMSLNneg or hMSLNpos human mesothelioma cell line, MSTO-211H, or stimulated with anti-CD3/anti-CD28 antibodies in vitro for 8 days. Distinct cell populations in MCY-M11 were evaluated for kinetics and duration of CAR expression, differentiation, activation, exhaustion, and their ability to secrete various immunomodulatory molecules during in vitro stimulation. Antigen-specific proliferation and cytotoxicity of MCY-M11 against hMSLNpos tumor cells as well as their ability to mount long-term antitumor immunity through epitope spreading mechanisms were studied.ResultsIndividual cell populations in MCY-M11 exhibited a consistent but transient Meso-CAR expression persisting for about 7 days. Cell subsets in MCY-M11 acquired early signs of activation and differentiation within 18–24 hours post-culture, but only attained full activation and lineage-specific differentiation upon specific response to hMSLNpos tumor cells. hMSLN antigen experienced MCY-M11 retained significant fractions of Naïve and Central Memory T cells and increased percentage of Effector Memory T cells along with increased expression of CD62L, CD27, and chemokine receptors (CCR5, CCR7, and CXCR3). MCY-M11 exhibited strong antigen-specific cytotoxicity against hMSLNpos tumor cells with corresponding increase in activation and proliferation of CD4+ and CD8+ T cell subsets and displayed low or no acquisition of known exhaustion markers. NK cells also exhibited a functionally superior molecular signature exhibiting increased levels of NKG2D, NKp44, NKp46, FAS, and TRAIL. The Monocytes and B cells in MCY-M11 also acquired an activated, differentiated, and mature phenotype, expressing molecules required for antigen presentation (HLA-DR, HLA-ABC, and CD205) and T cell co-stimulation (CD80 and CD86) to mount a strong antitumor response. These phenotypic changes in cell subsets of MCY-M11 transpired with simultaneous secretion of potent immunostimulatory molecules and chemokines facilitating an extended antitumor response through epitope spreading.ConclusionsWe demonstrated that MCY-M11 is a unique cell product possessing a complete built-in immune cellular machinery with favorable phenotype and enhanced functions specialized in mediating an effective and long-term antitumor response.Trial RegistrationNCT03608618

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IL15, but Not IL2, Supports Long-Term Survival and Function of Human and Macaque Antigen-Specific CD8+ T Cell Clones.

Blood ◽

10.1182/blood.v104.11.3237.3237 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3237-3237

Author(s):

Carolina S. Berger ◽

Michael Jensen ◽

Stanley R. Riddell

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cell ◽

Term Survival ◽

Long Term Survival ◽

Cd8 T Cell Memory ◽

And Function

Abstract The adoptive transfer of antigen-specific CD8+ cytotoxic T lymphocyte (CTL) clones that have been isolated and expanded in vitro is a promising treatment modality for both human malignancies and infections. However, establishing immunity of sufficient magnitude and persistence for sustained efficacy is a limitation of this approach. Recent studies have identified a critical role for cytokine signaling including that mediated by IL15 in the establishment and maintenance of CD8+ T cell memory, suggesting that protocols for generating and transferring antigen-specific T cells might be improved. Interleukin-2 (IL2) is the T cell growth factor that has been widely used in vitro and in vivo for promoting T cell proliferation and persistence, but prolonged exposure of T cells to IL2 can enhance susceptibility to cell death and limit CD8+ memory T cell survival. IL15 is a novel cytokine that shares some activities with IL2 such as the induction of T cell proliferation, but exerts contrasting effects on the homeostasis of CD8+ T cell memory in experimental models. Here, we study the utility of IL15 to enhance the long-term survival and function of human and macaque antigen-specific CD8+ CTL clones in vitro. Human and macaque CD8+ CTL clones reactive against CMV were isolated by limiting dilution, expanded over 14 days in the presence of IL2 or IL15 (1–10 ng/ml), and then rested for >4 weeks in media alone and with IL2 or IL15 at 0.01–10 ng/ml. Surviving T cells were enumerated at intervals, monitored for cell surface phenotype, and assayed for cytotoxicity by chromium release assay. CTL expanded in IL2 or IL15 proliferated equivalently over 14 days with a median of 1100 and 1400 fold increase in number, displayed surface markers consistent with an effector memory phenotype (CD45RA−CD62L−CCR7−CD28−), and showed comparable cytotoxicity (n=4). However, exposure after 14 days to IL15 at doses as little as 0.05-0.1 ng/ml greatly enhanced the survival of the CD8+ CTL as determined by Annexin V staining. By contrast, cells cultured without cytokines or with IL2 declined >80% in number over 3 or 11 days, respectively. Of note, IL15 at higher doses (>0.5 ng/ml), but not IL2, efficiently promoted sustained cell growth illustrated by labeling cells with CFSE. Cells cultured with IL15 displayed 1.5-fold increased expression of antiapoptotic molecules such as Bcl-xL and Bcl-2 over those plated in IL2 (n=4), indicating IL15 mediated its effects at least in part by preventing apoptosis. Of note, the cytotoxicity of CTL rested in IL2 was markedly reduced (>60%, n=3), while the presence of IL15 permitted for sustained CTL function and expansion after restimulation. The responses of human and macaque CTL clones to IL15 were equivalent suggesting in vivo studies of T cell transfer in macaques may be predictive of results in humans. We have constructed retroviral vectors encoding intracytoplasmic truncated macaque CD34 or CD19 genes that could serve as nonimmunogenic selectable marker to track macaque T cells after transfer. Macaque T cells were efficiently transduced to express CD34t and CD19t (>50%), and enriched to high purity by immunomagnetic selection. Studies to examine the safety and utility of IL15 on the survival of adoptively transferred CTL in macaques are in progress. Collectively, our data support that novel cytokines such as IL15 may prove useful to augment the long-term survival and effector function of ex vivo expanded antigen-specific CD8+ CTL clones after transfer.

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108 Fast and accurate prediction of optimal CAR T-cell function using z-Movi® Cell Avidity Analyzer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.108 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A119-A119

Author(s):

Will Singleterry ◽

Andrea Candelli ◽

Rogier Reijmers ◽

Jens Eberlein

Keyword(s):

T Cells ◽

Tumor Cells ◽

Cell Function ◽

New Technology ◽

Interaction Strength ◽

Cell Interaction ◽

In Vitro Assays ◽

Antigen Receptors ◽

Car T

BackgroundAffinity between himeric antigen receptors (CARs) to their target has shown to be a poor predictor of functional capacity of T cells. Recent studies have revealed that the overall binding strength (or avidity) between T cells and tumor cells represents a crucial parameter for identifying and developing potent cancer immunotherapies. Compared with affinity, cell avidity provides a more complete and physiologically relevant picture that reflects the bona fide interaction between T cells and tumor cells. Therefore, this interaction could better predict cellular responses in vitro, drive better, more informed decisions and potentially improve clinical outcome. However, one of the main obstacles in the process of measuring avidity is the lack of fast, specific, and accurate technologies to assess cellular avidity.MethodsThe z-Movi® Cell Avidity Analyzer is a novel and unique instrument for direct measurement of cell–cell interaction strength using acoustic forces. This new technology provides predictive, reproducible, and fast high-throughput results at a single-cell level.ResultsIn this poster we will demonstrate that data obtained with the z-Movi Cell Avidity Analyzer correlates strongly with standard in vitro assays, such as cytokine release and cytotoxicity measurements. We will review the simple principles behind the z-Movi, the workflow to set up an experiment consisting of multiple runs, and how the intuitive software package will assist in getting precise cell avidity information of hundreds of cells simultaneously. Finally, we will demonstrate the great potential of the z-Movi for accelerating the development of CAR T immunotherapy against cancer.ConclusionsFinally, we will demonstrate the great potential of the z-Movi for accelerating the development of CAR T immunotherapy against cancer.

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Improving efficacy of PD-1 blockade in unresponsive lymphoma tumors with in situ vaccination through induction of a highly efficient cross-presenting dendritic cell subset.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.5_suppl.76 ◽

2018 ◽

Vol 36 (5_suppl) ◽

pp. 76-76

Author(s):

Linda Hammerich ◽

Maxime Dhainaut ◽

Thomas A. Davis ◽

Tibor Keler ◽

Andres M. Salazar ◽

...

Keyword(s):

T Cells ◽

Tumor Cells ◽

Cd8 T Cells ◽

Tumor Regression ◽

Low Grade ◽

Dendritic Cell Subset ◽

Term Survival ◽

Intratumoral Injections

76 Background: Low-grade non-Hodgkin’s B-cell lymphomas are generally incurable; preliminary results with anti-PD-1 therapy have yielded low response rates. Tumoral DC infiltration correlates with efficacy of checkpoint blockade and tumor-targeted vaccines represent promising, novel treatment strategies to induce anti-tumor T cells. Methods: A20 lymphoma-bearing mice were treated with a PD-1 blocking antibody with an in situ vaccine (ISV) consisting of intratumoral injections of FMS-like tyrosine kinase-3 ligand (Flt3L), local irradiation (XRT) of the tumor and intratumoral injections of the TLR3 agonist poly-ICLC (pIC). Results: Untreated lymphoma tumors contained very low numbers of DC and treatment with anti-PD1 alone did not induce tumor regression or increase survival. Flt3L treatment resulted in a dramatic increase of IRF8+TLR3+ DC at the tumor site, the draining lymph node and the spleen. XRT of A20 cells induced activation of Flt3L-treated splenic DC in vitro and local XRT of the tumor in vivo induced expression of CD103 on infiltrating TLR3+ DC. Local XRT also enhanced uptake of dying tumor cells by DC. Interestingly, tumor antigens were taken up mainly by CD103+ DC and not CD103- subtypes. CD103+ expression distinguishes a subset of migratory cross-presenting DC. Accordingly, CD103+ DC isolated from the tumor induced proliferation of tumor-specific CD8+ T cells more efficiently than CD103- subsets. The combination of Flt3L with XRT and pIC induced tumor-reactive, IFNg-producing T cells, but delayed tumor growth and improved survival only in 40% of mice. ISV also increased expression of PD-L1 on tumor cells and tumor infiltrating DC. Consistently, combination of ISV with PD-1 blockade led to complete tumor regression and increased long-term survival in the majority of mice. PD-1 blockade also increased the number of tumor-reactive T cells and depletion of CD8+ T cells abrogated the anti-tumor effect. Conclusions: In situ vaccination can improve efficacy of anti-PD-1 in checkpoint-unresponsive lymphoma tumors through induction of a cross-presenting DC subset leading to long-term regression of established lymphoma tumors.

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Donor treatment with pegylated G-CSF augments the generation of IL-10-producing regulatory T cells and promotes transplantation tolerance

Blood ◽

10.1182/blood-2003-08-2864 ◽

2004 ◽

Vol 103 (9) ◽

pp. 3573-3581 ◽

Cited By ~ 94

Author(s):

Edward S. Morris ◽

Kelli P. A. MacDonald ◽

Vanessa Rowe ◽

Diana H. Johnson ◽

Tatjana Banovic ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Interleukin 10 ◽

Term Survival ◽

Donor T Cells ◽

Dose Dependent Fashion ◽

Dose Dependent ◽

Donor Pretreatment

Abstract We investigated whether the protection from graft-versus-host disease (GVHD) afforded by donor treatment with granulocyte colony-stimulating factor (G-CSF) could be enhanced by dose escalation. Donor treatment with human G-CSF prevented GVHD in the B6 → B6D2F1 murine model in a dose-dependent fashion, and murine G-CSF provided equivalent protection from GVHD at 10-fold lower doses. Donor pretreatment with a single dose of pegylated G-CSF (peg-G-CSF) prevented GVHD to a significantly greater extent than standard G-CSF (survival, 75% versus 11%, P < .001). Donor T cells from peg-G-CSF-treated donors failed to proliferate to alloantigen and inhibited the responses of control T cells in an interleukin 10 (IL-10)-dependent fashion in vitro. T cells from peg-G-CSF-treated IL-10-/- donors induced lethal GVHD; T cells from peg-G-CSF-treated wild-type (wt) donors promoted long-term survival. Whereas T cells from peg-G-CSF wt donors were able to regulate GVHD induced by T cells from control-treated donors, T cells from G-CSF-treated wt donors and peg-G-CSF-treated IL-10-/- donors did not prevent mortality. Thus, peg-G-CSF is markedly superior to standard G-CSF for the prevention of GVHD following allogeneic stem cell transplantation (SCT), due to the generation of IL-10-producing regulatory T cells. These data support prospective clinical trials of peg-G-CSF-mobilized allogeneic blood SCT. (Blood. 2004;103:3573-3581)

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Differential Effects of Microenvironmental Elements On Tumor Cells Survival in Chronic Lymphocytic Leukemia Patient Subsets with Good or Poor Prognosis.

Blood ◽

10.1182/blood.v114.22.2333.2333 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2333-2333

Author(s):

Marta Coscia ◽

Francesca Pantaleoni ◽

Chiara Riganti ◽

Micol Rigoni ◽

Candida Vitale ◽

...

Keyword(s):

T Cells ◽

Chronic Lymphocytic Leukemia ◽

Tumor Cells ◽

Peripheral Blood ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Spontaneous Apoptosis ◽

Survival Effect

Abstract Abstract 2333 Poster Board II-310 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IgVH): patients with unmutated (UM) IgVH have a worse prognosis than patients with mutated (M) IgVH. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Leukemic cells purified by negative selection from the PB of UM CLL patients showed significantly higher rates of spontaneous apoptosis after long-term in vitro culture as compared to CLL cells isolated from M patients. Both M and UM CLL cells showed high basal level of Bcl-2 expression and NF-kB activity soon after purification. In vitro spontaneous apoptosis of purified UM CLL cells was associated with a progressive downregulation of the intracellular expression of Bcl-2 and with a complete loss of the active nuclear form of NF-kB. On the contrary, the higher long term viability of M CLL cells was paralleled by a maintained Bcl-2 and NF-kB expression. IL-4 and CD40L, used alone or in combination, as well as murine and human BMSC were capable of rescuing UM tumor cells from apoptosis. The pro-survival effect of these stimuli was exerted through the upregulation of Bcl-2 and was totally independent from the recovery of NF-kB nuclear translocation. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (PBMC) as compared to purified leukemic cells. This higher cell viability was associated with a retained expression of Bcl-2 and of the nuclear form of NF-kB, thus suggesting the presence of a pro-survival element in the peripheral blood of these patients. The extremely low numbers of NLCs generated from PMBC of UM patients ruled out a role for these cells in supporting the survival of unpurified leukemic cells. Conversely, a pro-survival effect on UM CLL cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM cells were cultured in the presence of autologous peripheral blood T cells (PBT). The prosurvival effect of circulating T cells was particularly evident at high T:B ratio, did not require a cell-cell contact and was mediated by the upregulation of Bcl-2 and the activation of NF-kB in leukemic cells. These data indicate that the survival of UM tumor cells is highly dependent on the action of multiple microenviromental stimuli. Conversely, M cells are intrinsically more resistant to apoptosis and minimally influenced by the local microenviroment. The higher dependency of UM CLL cells from extrinsic signals might be exploited to develop new therapies targeting the tumor microenvironment and to improve the outcome of more aggressive CLL. Disclosures: No relevant conflicts of interest to declare.

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Concomitant Administration of Gene-Modified T Cells Expressing a Chimeric CD16-CD3ζ Receptor with Mogamulizmab Synergistically Suppresses Adult T Cell Leukemia Cells in Vivo

Blood ◽

10.1182/blood.v124.21.307.307 ◽

2014 ◽

Vol 124 (21) ◽

pp. 307-307

Author(s):

Kazushi Tanimoto ◽

Hiroshi Fujiwara ◽

Hiroki Tanaka ◽

Fumihiro Ochi ◽

Hiroaki Asai ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Tumor Cells ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Adcc Activity ◽

Adult T Cell Leukemia

Abstract [Background and purpose] Mogamulizumab, a newly developed monoclonal antibody (mAb) targeting the receptor for C-C chemokine 4 (CCR4) has initially demonstrated a promising clinical outcome for the treatment of therapy-resistant Adult T cell leukemia (ATL) of which tumor cells broadly express CCR4. However, in practice, we sometimes encounter unsuccessful cases of ATL treated with mogmulizumab, especially in the setting of combination therapy with multiple anticancer agents. In those cases, ATL tumor cells were still positive for CCR4, but the chemotherapy-induced lymphocytopenia was reproducibly noticeable. Considering the pharmacologic action of mogamulizumab, the defucosylation-enhanced antibody-dependent cellular cytotoxicity (ADCC) exerted by FcγRIIIa (CD16) expressing effector cells in vivo, as observed for Natural Killer (NK) cells, we hypothesized that adoptive transfer of ADCC effector cells might be able to regain the anti-ATL efficacy of mogamulizumab. Accordingly, we examined in vivo the feasibility of T cells gene-modified to express a newly generated chimeric CD16-CD3ζ receptor as a transferable alternative effector cell for mogamulizumab. [Methods] A novel affinity-matured chimeric CD16 with a 158V/V-CD3ζ (cCD16ζ ) gene construct was lentivirally introduced into CD3+ T cells from both healthy individuals (n=4) and patients with adult T cell leukemia (ATL) (n=3) (cCD16ζ-T cells). Using ATL or HTLV-1 infected cell lines variably expressing CCR4 on the cell surface (n=7), and primary ATL tumor cells (n=3), in the context of ADCC activity, functional properties of cCD16ζ-T cells were extensively examined both in vitro and in vivo, compared with those of NK cells from healthy donors (n=3). Next, we examined the in vivo therapeutic efficacy of concomitantly infused cCD16ζ-T cells with mogamulizumab via tail vein using a xenografted mouse model. Finally, we examined the feasibility of double gene-modified T cells to express human telomerase reverse transcriptase (hTERT)-specific TCR and cCD16ζ receptor, because we recently demonstrated that HLA-A*24:02-restricted and hTERT461-469 (VYGFVRACL)-specific TCR gene-modified CD8+ T cells displayed the cytocidal activity against ATL tumor cells (Blood, 2014). [Results] cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human (rh)IL-2, and they successfully displayed ADCC-mediated tumoricidal activity against CCR4+ MT-4 and ATN-1 (ATL cell lines) cells, but not CCR4- K562 cells with mogamulizumab, in a effector cell number and an antibody dose dependent manner. Pharmacological dose of 0.1μg/ml mogamulizumab could bind to ATL cell line cells expressing variable extent of CCR4 ranging from MFI 1.01 for HUT102 cell line to 12.3 for MT-2 (0.57 for K562 as a negative control), and could mediate the similar degree of ADCC activity exerted by cCD16ζ-T cells. The magnitude of ADCC activity mediated by cCD16ζ-T cells against opsonized ATN-1 with mogamulizumab was almost similar to that by activated NK cells using rhIL-2. This cytotoxicity was inhibited by anti-CD16 mAb. During ADCC, ligation of opsonized cancer cells with mogamulizumab to cCD16ζ receptor stimulated cCD16ζ-T cells to release toxic granules shown by CD107a expression. cCD16ζ-T cells generated from patients with ATL (n=3) successfully displayed ADCC activity against autologous tumor cells in vitro. Human cCD16ζ-T cells infused concomitantly with mogamulizumab synergistically inhibited the growth of disseminated luciferase gene-modified ATN-1 cells in immunodeficient mice, demonstrated using an in vivo bioluminescence assay. Additionally, this tumor suppressive effect contributed to the longer survival of treated mice. Finally, the double-gene modified CD3+ T cells could successfully recognize ATN-1 through both the mogamulizumab-opsonized CCR4 and hTERT epitope/HLA-A24*02 complex on the cell surface. Disclosures Okamoto: CDM Center, Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Employment. Shiku:Takara Bio Inc.: Research Funding.

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The Ciliary Rootlet Maintains Long-Term Stability of Sensory Cilia

Molecular and Cellular Biology ◽

10.1128/mcb.25.10.4129-4137.2005 ◽

2005 ◽

Vol 25 (10) ◽

pp. 4129-4137 ◽

Cited By ~ 95

Author(s):

Jun Yang ◽

Jiangang Gao ◽

Michael Adamian ◽

Xiao-Hong Wen ◽

Basil Pawlyk ◽

...

Keyword(s):

Cell Biology ◽

Structural Integrity ◽

In Vitro Assays ◽

Ciliated Cells ◽

Term Survival ◽

Functional Deficits ◽

Structural Support ◽

Long Term Stability

ABSTRACT The striated ciliary rootlet is a prominent cytoskeleton originating from basal bodies of ciliated cells. Although a familiar structure in cell biology, its function has remained unresolved. In this study, we carried out targeted disruption in mice of the gene for rootletin, a component of the rootlet. In the mutant, ciliated cells are devoid of rootlets. Phototransduction and ciliary beating in sensory and motile cilia initially exhibit no apparent functional deficits. However, photoreceptors degenerate over time, and mutant lungs appear prone to pathological changes consistent with insufficient mucociliary clearance. Further analyses revealed a striking fragility at the ciliary base in photoreceptors lacking rootlets. In vitro assays suggest that the rootlet is among the least dynamic of all cytoskeletons and interacts with actin filaments. Thus, a primary function of the rootlet is to provide structural support for the cilium. Inasmuch as photoreceptors elaborate an exceptionally enlarged sensory cilium, they are especially dependent on the rootlet for structural integrity and long-term survival.

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