scholarly journals CD200 Expression on Multiple Myeloma Cells Induces Attenuation of T Cell-Mediated Cytotoxicity Via DOK2

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1587-1587
Author(s):  
Pooja Shah ◽  
Thorsten Stuehmer ◽  
Daniela Bruennert ◽  
Umair Munawar ◽  
Ellen Leich ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Sleeping Beauty ◽  
Luciferase Assay ◽  
Vector System ◽  
Activated T Cells ◽  
Blocking Antibody ◽  
Sleeping Beauty Transposon

Abstract Introduction The CD200/CD200 receptor (CD200R) axis is known to exert immunoregulatory effects in myeloid-derived cells and constitutes a putative immune checkpoint in hematological malignancies, in which CD200 expression is associated with poor prognosis. In multiple myeloma (MM), CD200 is expressed in the majority of patient-derived primary cells. However, its functional importance as well as the related downstream mechanism upon CD200 ligand binding to its CD200R on T cells are not well understood. In this study, we analyze the functional role of CD200 as a potential immune checkpoint in MM and decipher the mechanism of CD200-mediated immune escape. Methods Primary MM cells and MM cell lines were analyzed for CD200 surface expression by flow cytometry. To overexpress CD200 on non-expressing MM cell lines we used a Sleeping Beauty transposon vector system. CD200+/- MM cell lines L363, U266 and MM.1s were co-cultured with CD3/CD28-activated healthy donor T cells. T cell-mediated cytotoxicity in these co-cultures was assessed with flow cytometry and/or luciferase assay. Moreover, to analyze the effects of CD200R activation on downstream signaling pathways, activated T cells were treated with recombinant human CD200 (rhCD200) and/or anti-CD200 blocking antibody and subsequently, Western blotting was performed. Results Of n=120 primary MM samples (n=120) analyzed, CD200 protein expression could be detected in ca. 75 % of the cases. In contrast, all n=9 MM cell lines tested neither displayed surface nor cytoplasmic CD200 expression. Therefore, using a Sleeping Beauty transposon vector system we stably expressed CD200 on MM cell lines for further analyses. In the presence of CD200-expressing MM cells up to 50% decrease in CD3+ T cell-mediated cytotoxicity against MM cells was observed in flow cytometry and luciferase assay. Proliferation rates of MM cell lines remained unchanged regardless of the level of CD200 overexpression as determined by Alamar blue assays. In myeloid-derived cells, CD200R directly interacts with docking protein-2 (DOK2). In activated T cells, we observed DOK2 phosphorylation upon CD200 binding when treated with rhCD200 in a time- and concentration-dependent manner. Applying an anti-CD200 blocking antibody, this effect could be reversed, thus revealing a possible mechanism for the observed attenuation of T cell function. Conclusion Our study shows that anti-MM cytotoxicity from primary healthy donor CD3+ T cells is attenuated by CD200 expression on MM cells. We also demonstrate that this inhibitory mechanism in CD3+ T cells is mediated via DOK2, providing a potential target for immunotherapeutic approaches in MM. Disclosures Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding.

scholarly journals A bispecific T cell engager targeting Glypican-1 redirects T cell cytolytic activity to kill prostate cancer cells

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Maria E. Lund ◽  
Christopher B. Howard ◽  
Kristofer J. Thurecht ◽  
Douglas H. Campbell ◽  
Stephen M. Mahler ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Surface ◽  
Cell Lines ◽  
Inflammatory Cytokines ◽  
Cytolytic Activity ◽  
Activated T Cells ◽  
Bispecific T Cell Engager

Abstract Background Glypican-1 is a heparan sulfate proteoglycan that is overexpressed in prostate cancer (PCa), and a variety of solid tumors. Importantly, expression is restricted in normal tissue, making it an ideal tumor targeting antigen. Since there is clinical and preclinical evidence of the efficacy of Bispecific T cell Engager (BiTE) therapy in PCa, we sought to produce and test the efficacy of a GPC-1 targeted BiTE construct based on the Miltuximab® sequence. Miltuximab® is a clinical stage anti-GPC-1 antibody that has proven safe in first in human trials. Methods The single chain variable fragment (scFv) of Miltuximab® and the CD3 binding sequence of Blinatumomab were combined in a standard BiTE format. Binding of the construct to immobilised recombinant CD3 and GPC-1 antigens was assessed by ELISA and BiaCore, and binding to cell surface-expressed antigens was measured by flow cytometry. The ability of MIL-38-CD3 to activate T cells was assessed using in vitro co-culture assays with tumour cell lines of varying GPC-1 expression by measurement of CD69 and CD25 expression, before cytolytic activity was assessed in a similar co-culture. The release of inflammatory cytokines from T cells was measured by ELISA and expression of PD-1 on the T cell surface was measured by flow cytometry. Results Binding activity of MIL-38-CD3 to both cell surface-expressed and immobilised recombinant GPC-1 and CD3 was retained. MIL-38-CD3 was able to mediate the activation of peripheral blood T cells from healthy individuals, resulting in the release of inflammatory cytokines TNF and IFN-g. Activation was reliant on GPC-1 expression as MIL-38-CD3 mediated only low level T cell activation in the presence of C3 cells (constitutively low GPC-1 expression). Activated T cells were redirected to lyse PCa cell lines PC3 and DU-145 (GPC-1 moderate or high expression, respectively) but could not kill GPC-1 negative Raji cells. The expression of PD-1 was up-regulated on the surface of MIL-38-CD3 activated T cells, suggesting potential for synergy with checkpoint inhibition. Conclusions This study reports preclinical findings into the efficacy of targeting GPC-1 in PCa with BiTE construct MIL-38-CD3. We show the specificity and efficacy of the construct, supporting its further preclinical development.

scholarly journals Sunitinib Exerts In Vitro Immunomodulatory Activity on Sarcomas via Dendritic Cells and Synergizes With PD-1 Blockade

2021 ◽  
Vol 12 ◽  
Author(s):  
Darina Ocadlikova ◽  
Mariangela Lecciso ◽  
Javier Martin Broto ◽  
Katia Scotlandi ◽  
Michele Cavo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Cell Lines ◽  
Effector T Cells ◽  
Sarcoma Cell ◽  
Ifn Γ ◽  
Sarcoma Cells

BackgroundHigh-grade sarcomas are a heterogeneous group of aggressive tumors arising in bone and soft tissues. After relapse, treatment options are limited. The multi-targeted receptor tyrosine kinase inhibitors (TKIs) sunitinib and inhibitor of PD-1 (anti-PD-1) nivolumab have shown antitumor activity in selected subtypes. In this study, we examine the role of TKIs and PD-1 based therapy in in vitro cocultures of sarcoma.MethodsThe human osteosarcoma (SaOS-2) and synovial sarcoma (SYO-1) cell lines were treated with sunitinib. After cell death and proliferation assessment, expression of PD-L1 was analyzed by flow cytometry. Sunitinib-treated sarcoma cells were cocultured with dendritic cells (DCs), and the phenotype of mature DCs was determined by flow cytometry. Mature DCs were cultured with autologous T cells. PD-1 expression on T cells, their proliferation, T regulatory cell (Tregs) induction and IFN-γ production, before and after nivolumab exposure, were analyzed.ResultsAlong with its anti-proliferative and direct pro-apoptotic effect on sarcoma cell lines, sunitinib prompted PD-L1 upregulation on sarcoma cells. Interestingly, sunitinib-treated sarcoma cells drive DCs to full maturation and increase their capacity to induce sarcoma-reactive T cells to produce IFN-γ. Conversely, no effect on T cell proliferation and T cell subpopulation composition was observed. Moreover, both bone and synovial sarcoma cell lines induced Tregs through DCs but sunitinib treatment completely abrogated Treg induction. Finally, sarcoma cell lines induced PD-1 upregulation on both effector T cells and Tregs when loaded into DCs, providing a rationale for using PD-1 blockade. Indeed, PD-1 blockade by nivolumab synergized with sunitinib in inducing IFN-γ-producing effector T cells.ConclusionsTaken together, our in vitro data indicate that the treatment of sarcoma cells with sunitinib can exert significant changes on immune cell subsets toward immune activation, leading to DC-based cross-priming of IFN-γ-producing effector T cells and reduced Treg induction. PD-1 blockade with nivolumab has a synergistic effect with sunitinib, supporting the use of TKI and anti-PD-1 approach in sarcomas, and perhaps in other cancers. DC-targeted drugs, including toll-like receptor 3 inhibitors and CD47 inhibitors, are under development and our preclinical model might help to better design their clinical application.

scholarly journals Rapid Flow Cytometry Method for Quantitation of LFA-1-Adhesive T Cells

2006 ◽  
Vol 13 (3) ◽  
pp. 403-408 ◽  
Author(s):  
Brian Crucian ◽  
Mayra Nelman-Gonzalez ◽  
Clarence Sams
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
T Cell Subsets ◽  
Activated T Cells ◽  
Peripheral Blood Mononuclear ◽  
Simple Method ◽  
Rapid Flow

ABSTRACT Adhesion molecules are important for leukocyte endothelial attachment and migration to sites of inflammation. The LFA-1 (CD11a and CD18) integrin molecule is constitutively expressed on the T-cell surface. Following T-cell activation, a rapid conformational change of LFA-1 to an “adhesive” state occurs, allowing LFA-1 binding to intracellular cell adhesion molecule type 1 (ICAM-1)-expressing targets, such as antigen-presenting cells. For this study, a rapid flow cytometry method for the quantitation of LFA-1-adhesive T cells following activation was developed. Purified ICAM-1 was bound to 4.5-μm-diameter beads. Following peripheral blood mononuclear cell activation culture (phorbol myristate acetate and ionomycin), the cells were incubated with the ICAM-1 beads, which allowed attachment to occur. The T cell-bead complexes were then resolved from unbound T cells by flow cytometry. Multicolor analysis allowed a complete phenotypic analysis of the adhesive T-cell subsets. Experimental controls indicated that the T cell-bead attachment was LFA-1 and ICAM-1 specific. Very little binding between unactivated T cells and ICAM beads or between activated T cells and plain beads was observed. The kinetics of the response was extremely rapid, with nearly maximal numbers of adhesive T cells observed following 5 min of activation. Scanning electron microscopy analysis was used to characterize legitimate bead-cell binding. By using multicolor cytometry, the responding adhesive T-cell population was usually identified as a distinct subset of T cells with the following phenotype: CD3+ CD4+ or CD8+ CD19− CD16− CD45RO+ CD62L+ CD27+ CD57−. A rapid and simple method for the scoring of LFA-1-adhesive T cells was developed and may have significant utility for immune function studies.

624 IFNγ secreted by tebentafusp (IMCgp100)-redirected T cells inhibits expression of melanin synthesis pathway genes in healthy melanocytes

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A660-A660
Author(s):  
Mariantonella Vardeu ◽  
David Depoil ◽  
Camille Britton-Rivet ◽  
Jane Houghton ◽  
Jane Harper ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adverse Events ◽  
Cancer Cells ◽  
Cell Lines ◽  
Uveal Melanoma ◽  
Melanin Synthesis ◽  
Activated T Cells ◽  
Melanoma Cancer ◽  
Melanoma Cell Lines

BackgroundTebentafusp (IMCgp100) is a bispecific T cell redirector comprised of an affinity-enhanced TCR recognising melanocyte lineage antigen gp100 and a T cell engaging anti-CD3 scFv domain. Tebentafusp has shown activity as monotherapy in advanced cutaneous and uveal melanoma (Middleton et al., ASCO 2019), and we have previously reported that over half of uveal melanoma patients treated with tebentafusp display melanocyte-related adverse events (MRAE). These include vitiligo/skin hypopigmentation, leukotrichia, and hyperpigmentation and, collectively, are associated with better overall survival in uveal patients receiving tebentafusp (Orloff et al, AACR 2020). In this study, we dissected the mechanisms by which tebentafusp may induce MRAE and highlight the potential clinical significance.MethodsIn vitro studies were conducted to assess the direct and indirect effects of tebentafusp on epidermal melanocytes from healthy donors. Expression of gp100 and the gp100:HLA*02:01 target complex by melanocytes were quantified at the mRNA level and on the cell surface by confocal microscopy, respectively. Melanocytes co-cultured with PBMC and increasing concentrations of tebentafusp were assessed for their susceptibility to lysis and/or ability to stimulate cytokine production. These readouts were compared to gp100-positive and negative melanoma cancer cell lines. Melanin production by melanocytes was quantified and the melanin synthesis pathway interrogated at the mRNA and protein level following exposure to secretomes from tebentafusp-redirected PBMC against melanoma cancer cells.ResultsHealthy melanocytes expressed 2 to 3-fold lower levels of gp100 peptide-HLA complexes on their surface compared to gp100-positive melanoma cell lines. In the presence of tebentafusp, this lower target expression translated into 3–6 fold lower levels of IFNγ and more than 100 fold lower granzyme B production by redirected T cells and these melanocytes were resistant to direct tebentafusp-induced killing (EC50 for melanocytes greater than 1nM vs EC50 melanoma cell lines of 23–50 pM). Supernatants from T cells activated in response to melanoma cancer cells by tebentafusp downregulated the melanin content of healthy melanocytes (20–30% reduction). Western blotting revealed 30–40% inhibition of two key components of the melanin synthesis pathway; the tyrosinase-related protein (TRP)-1 and TRP-2. This inhibition was reversed by blocking IFNγ in supernatants from activated T cells.ConclusionsMRAEs, especially vitiligo, associated with response to tebentafusp, may be explained, at least in part, by the downregulation of melanin biosynthesis pathway genes by IFNγ secreted by tebentafusp-activated T cells.Ethics ApprovalThe study was approved by the South Central - Oxford A Research Ethics Committee (UK), REC reference 13/SC/0226ReferencesMiddleton, et al., Relationship between clinical efficacy and AEs of IMCgp100, a novel bispecific TCR–anti-CD3, in patients with advanced melanoma. Journal of Clinical Oncology. 2019.Orloff, et al., Vitiligo and other clinical melanocyte-related adverse events following tebentafusp (IMCgp100) exposure in patients with uveal melanoma. AACR (American Association for Cancer Research), 2020.

Isolation and Characterization of CD4+ T Cells Specific for the HPA 1a Epitope Associated with Neonatal Alloimmune Thrombocytopenia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2091-2091
Author(s):  
Maria T. Ahlen ◽  
Mette K. Killie ◽  
Bjorn Skogen ◽  
Anne Husebekk ◽  
Tor B. Stuge
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cell Detection ◽  
Severe Thrombocytopenia ◽  
Isolation And Characterization ◽  
T Cell Lines ◽  
Alloimmune Thrombocytopenia

Abstract Neonatal alloimmune thrombocytopenia (NAIT) can cause severe complications such as intrauterine death or intracranial hemorrhage (ICH) in the newborn, and is caused by the transfer of platelet-depleting antibodies from the mother to the fetus during pregnancy. These antibodies react with allogeneic epitopes, most commonly human platelet antigen (HPA) 1a, when present on fetal platelets. Although these responses are thought to be a result of a T cell-dependent immune response, HPA 1a specific T cells have not yet been isolated. To examine whether HPA 1a specific T cells could be detected and isolated, we collected PBMC post delivery from an HPA 1a negative mother who gave birth to an HPA 1a positive neonate suffering from severe thrombocytopenia (platelet count <50×109/L). The cells were stimulated with HPA 1a peptides (20aa) in long term cultures supplemented with IL-7 and IL-2, and subsequently, IL-15. After 4 weeks in culture these cells were labeled with CFSE dye and restimulated with HPA 1a or control peptides. After additional 2 weeks in culture supplemented with IL-2 and IL-15, specific proliferative responses were detectable by CFSE dye dilution by flow cytometry. The cells were cloned by fluorescent-activated cell sorting (FACS) and expanded in numbers with anti-CD3 stimulation in the presence of irradiated allogeneic PBMC and IL-2. The resulting clonal T cell lines were characterized in proliferation assays, ELISPOT assays and phenotyped by flow cytometry. All clones were CD3+, CD4+ and CD19−, and the majority of the clones proliferated and secreted cytokines in response to stimulation with HPA 1a peptides, but not control peptides. In ELISPOT assays, peptide-pulsed antigen-presenting cells were required for T cell detection. These clonal HPA 1a specific CD4+ T cell lines represent formal evidence of the existence of HPA 1a specific T cell responses related to NAIT and will serve as important tools for further characterization of maternal immune responses associated with NAIT.

PD-1/PD-1-Ligand Interaction Contributes to Immunosuppressive Microenvironment of Hodgkin Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 379-379
Author(s):  
Ryo Yamamoto ◽  
Momoko Nishikori ◽  
Toshio Kitawaki ◽  
Tomomi Sakai ◽  
Masakatsu Hishizawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Signaling System ◽  
Peripheral T Cells ◽  
Ifn Γ ◽  
Immunosuppressive Microenvironment

Abstract Programmed death-1 (PD-1), a member of the CD28 costimulatory receptor superfamily, inhibits T cell activity by providing a second signal to T cells in conjunction with signaling through the T-cell receptor. PD-1/PD-1 ligand (PD-L) signaling system is indicated to be involved in the functional impairment of T cells such as in chronic viral infection or tumor immune evasion. We hypothesized that this signaling system is also involved in the pathogenesis of Hodgkin lymphoma (HL). We examined expression of B7-H1 and B7-DC, two known PD-Ls, in lymphoid cell lines using RT-PCR and flow cytometry. They were expressed in HL and several T-cell lines, whereas most B-NHL lines lacked their expression. Immunohistochemical staining of HL tissues demonstrated that PD-Ls were also expressed in primary H/RS cells. As gene expression of B7-H1 and B7-DC was increased in Epstein-Barr virus (EBV)-transformed lymphoblastoid B-cell lines, we examined the effect of EBV latent membrane proteins on their gene regulation. By luciferase reporter assay, both LMP1 and LMP2A were shown to enhance promoter activity of B7-H1 and B7-DC genes. This finding implies that in cases of EBV-positive HL, latent membrane proteins may help H/RS cells escape from host immune surveillance by upregulating PD-L gene expression. We next analyzed PD-1 expression of tumor-infiltrating T cells of HL tissue samples by flow cytometry, and found that PD-1+ cells were elevated markedly in these cells. As HL patients are well recognized as having defective cellular immunity, we compared PD-1 expression level in peripheral blood T cells of HL patients with those of healthy volunteers and B-NHL patients. PD-1 was significantly elevated in peripheral T cells of HL patients compared to the other two groups. PD-1+ T cells were highest in patients with active disease, and tended to decline along with treatment. Although regulatory T cells are reported to play a part in the pathogenesis of HL, FOXP3+ T cells were not significantly elevated in peripheral T cells of HL patients, and PD-1+ T cells did not overlap with these regulatory population. To elucidate whether the PD-1/PD-L signaling pathway is functional in the immunosuppressive microenvironment of HL, we finally examined the effect of blockade of this pathway. After culturing bulk HL tumor cells with anti-PD-L blocking antibodies, IFN-γ production was measured by ELISA. Blockade of PD-Ls augmented IFN-γ production of HL-infiltrating T cells. We concluded that anti-tumor activity of HL-infiltrating T cells was inhibited via the PD-1/PD-L pathway, and this inhibition could be successfully relieved by PD-L blockade. Taken together, our observations indicate that “T-cell exhaustion” is essential to the pathogenesis of HL, and tumor-infiltrating T cells around H/RS cells seem to be kept in balance by this inhibitory signaling. Our findings provide a potentially effective and clinically applicable strategy for the immunotherapy of HL.

scholarly journals T cell growth factor receptors. Quantitation, specificity, and biological relevance

1981 ◽  
Vol 154 (5) ◽  
pp. 1455-1474 ◽  
Author(s):  
RJ Robb ◽  
A Munck ◽  
KA Smith
Keyword(s):  
T Cells ◽  
T Cell ◽  
Growth Factor ◽  
Cell Lines ◽  
Binding Sites ◽  
Activated T Cells ◽  
Receptor Sites ◽  
Polypeptide Hormones ◽  
T Cell Lines ◽  
T Cell Growth Factor

To examine directly the hypothesis that T cell growth factor (TCGF) interacts with target cells in a fashion similar to polypeptide hormones, the binding of radiolabeled TCGF to various cell populations was investigated. The results indicate that TCGF interacts with activated T cells via a receptor through which it initiates the T cell proliferative response. Internally radiolabeled TCGF, prepared from a human T leukemia cell line and purified by gel filtration and isoelectric focusing, retained biological activity and was uniform with respect to size and charge. Binding of radiolabeled TCGF to TCGF-dependent cytolytic T cells occurred rapidly (within 15 rain at 37 degrees C) and was both saturable and largely reversible. In addition, at 37 degrees C, a receptor- and lysosome-dependent degradation of TCGF occurred. Radiolabeled TCGF binding was specific for activated, TCGF-responsive T cells. Whereas unstimulated lymphocytes of human or murine origin and lipopolysaccharide-activated B cell blasts expressed few if any detectable binding sites, lectin- or alloantigen-activated cells had easily detectable binding sites. Moreover, compared with lectin- or alloantigen-activated T cells, long-term TCGF-dependent cytolytic and helper T cell lines and TCGF-dependent neo-plastic T cell lines bound TCGF with a similar affinity (dissociation constant of 5-25 pM) and expressed a similar number of receptor sites per cell (5,000-15,000). In contrast, a number of TCGF-independent cell lines of T cell, B cell, or myeloid origin did not bind detectable quantities of radiolabeled TCGF. Binding of radiolabeled TCGF to TCGF-responsive cells was specific, in that among several growth factors and polypeptide hormones tested, only TCGF competed for binding. Finally, the relative magnitude of T cell proliferation induced by a given concentration of TCGF closely paralleled the fraction of occupied receptor sites. As the extent of T cell clonal expansion depends on TCGF and on the TCGF receptor, the dissection of the molecular events surrounding the interaction of TCGF and its receptor that these studies permit, should provide new insight into the hormonelike regulation of the immune response by this lymphokine.

Aberrant Expression and Activation of Nuclear Factor of Activated T cells (NFAT1) in Human T-Cell Acute Lymphocytic Leukemia (T-ALL) May Contribute to Leukemogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4474-4474
Author(s):  
Peter Haviernik ◽  
Mathew L Lesniewski ◽  
Richard Patrick Weitzel ◽  
Mary J Laughlin
Keyword(s):  
Cell Cycle ◽  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Jurkat Cells ◽  
Nuclear Factor ◽  
Flow Cytometry Analysis ◽  
Wild Type ◽  
Activated T Cells ◽  
Aberrant Expression

Abstract Nuclear Factor of Activated T-cells 1 (NFAT1) is a member of the NFAT family of transcription factors (NFAT1-NFAT5) that has been shown to play an important role in regulating genes related to T-cell expansion, differentiation, and apoptosis. As murine NFAT1 knockout mice exhibit lymphoproliferative disease, we hypothesize that aberrant expression of NFAT1 may impact cell cycle dysregulation underlying T-ALL pathogenesis. Four T-ALL cell lines – including 3 mature T-cell lines: CCRF-CEM (no clear chromosomal abnormalities), Jurkat (karyotype 46, XY, -2, -18, del(2) (p21p23), del(18) (p11.2)), Loucy (translocation t(16;20)(p12;q13), and p53 overexpression), and one immature T-cell line MOLT-4 (hypertetraploid chromosomes and 6q-, t(7;7)) (ATCC Manassas, VA), established from peripheral blood of T-ALL patients were analyzed and compared to normal resting adult CD4+ T-cells. Flow cytometry analysis was performed as previously described including CD34, CD38, HLA-DR, CD3, CD4, CD8, CD2, and CD7 to determine maturation stage; MOLT-4 being the most primitive and Jurkat the most mature. Methods: Growth curves were determined and proliferation potential was determined using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. The cell cycle phase distribution was assessed by flow cytometry analysis of Hoechst 33342-stained cells. mRNA expression was examined by quantitative rtPCR using TaqMan probes (ABI) on cDNA derived from TRIzol purified total RNA. For stimulation, we used 2 μM ionomycin treatment for 3 hours. Proteins were prepared as whole cell extract from radioimmunoprecipitation assay (RIPA) buffer lysed cells. Protein expression was evaluated by Western blotting analysis of 20 μg of proteins using anti-NFAT1 antibody (BD Biosciences) and anti-γ-tubulin antibody (Sigma) as a loading control. Transient transfection of GFP vector or plasmid containing the wild-type or constitutively active NFAT1 gene along with the GFP gene was performed by either electroporation (Amaxa) or Lipofectamine 2000 (Invitrogen). GFP positive cells were sorted by FACSAria sorter. Results: Despite their different maturation states, all cell lines (except Loucy) have similar high growth rates. However, the cell cycle distribution analysis of Hoechst-stained cells revealed a lower proportion of Loucy cells in the G0/G1 phase (35% vs. 48–51% for the other 3 cell lines) and a higher proportion in the G2/M phase (37% vs. 19–24% for the other 3 cell lines). In addition, Loucy cells have a higher rate of apoptosis as measured by Annexin V staining. Analysis of NFAT1 expression demonstrated decreased levels of NFAT1 mRNA (30-70-fold) and protein (2-10-fold) in these cell lines compared to resting adult peripheral blood CD4+ T lymphocytes. Figure Figure Moreover, ionomycin stimulation of the calcium-calcineurin pathway in these cells revealed aberrant activation of NFAT1. There was no dephosphorylated or activated NFAT1 in MOLT-4 and Jurkat cells; in contrast, dephosphorylated or activated NFAT1 was degraded in CCRF-CEM and Loucy. As NFAT1 is implicated in the regulation of cell cycle and apoptosis, the expression of cell cycle and apoptosis genes was measured by qrtPCR. Consistent with the negative regulatory role of NFAT1 in cell cycle, T-ALL cells expressing low level of NFAT1 showed upregulation of cyclin A2 (20-80-fold), cyclin E2 (5-8-fold) and CDK4 (3-7-fold) as well as downregulation of p21Cip1 (20-470-fold) and p27Kip1 (20-180-fold). In addition, these cells also demonstrated downregulation of the expression of the pro-apoptotic gene Nur77 (2.5-10-fold). Introduction of exogenous NFAT1 gene into Jurkat cells resulted in decreased proliferation rate to 64% and 42%, for wild-type and constitutively active form of NFAT1 gene, respectively, compare to the cells transfected with the empty GFP vector. Conclusion: Aberrant expression of NFAT1 contributes to leukemogenesis via dysregulation of proliferation and apoptosis. Targeted NFAT1 expression may be an effective therapeutic strategy in T-ALL.

scholarly journals The human OX40/gp34 system directly mediates adhesion of activated T cells to vascular endothelial cells.

1996 ◽  
Vol 183 (5) ◽  
pp. 2185-2195 ◽  
Author(s):  
A Imura ◽  
T Hori ◽  
K Imada ◽  
T Ishikawa ◽  
Y Tanaka ◽  
...  
Keyword(s):  
T Cells ◽  
Endothelial Cells ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Cell Leukemia ◽  
Activated T Cells ◽  
T Cell Lines

Fresh leukemic cells from patients with adult T cell leukemia (ATL) and some ATL-derived T cell lines show adhesion to human umbilical vein endothelial cells (HUVECs) mainly through E-selectin, but a proportion of this binding remains unaffected by the addition of combinations of antibodies against known adhesion molecules. By immunizing mice with one of such cell lines, we established monoclonal antibodies (mAbs), termed 131 and 315, that recognize a single cell surface antigen (Ag) and inhibit the remaining pathway of the adhesion. These mAbs did not react with normal resting peripheral blood mononuclear cells (PBMC) or most of the cell lines tested except for two other human T cell leukemia virus type I (HTLV-I)-infected T cell lines. After stimulation with phytohemagglutinin (PHA), PBMC expressed Ag 131/315 transiently, indicating that these mAbs define a T cell activation Ag. Western blotting and immunoprecipitation revealed that Ag 131/315 has an apparent molecular mass of 50 kD. Expression cloning was done by transient expression in COS-7 cells and immunological selection to isolate a cDNA clone encoding Ag 131/315. Sequence analysis of the cDNA indicated that it is identical to human OX40, a member of the tumor necrosis factor/nerve growth factor receptor family. We then found that gp34, the ligand of OX40, was expressed on HUVECs and other types of vascular endothelial cells. Furthermore, it was shown that the adhesion of CD4+ cells of PHA-stimulated PBMC to unstimulated HUVECs was considerably inhibited by either 131 or 315. Finally, OX40 transfectants of Kit 225, a human interleukin 2-dependent T cell line, were bound specifically to gp34 transfectants of MMCE, a mouse epithelial cell line, and this binding was blocked by either 315 or 5A8, an anti-gp34 mAb. These results indicate that the OX40/gp34 system directly mediates adhesion of activated T cells or OX40+-transformed T cells to vascular endothelial cells.

scholarly journals A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes

1998 ◽  
Vol 188 (9) ◽  
pp. 1593-1602 ◽  
Author(s):  
George R. Ehring ◽  
Hubert H. Kerschbaum ◽  
Claudia Eder ◽  
Amber L. Neben ◽  
Christopher M. Fanger ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Lines ◽  
K Channel ◽  
Na Channel ◽  
Activated T Cells ◽  
K Channels ◽  
Voltage Gated

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin–resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl− channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.

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