DOCSLIB.ORG

Neonatal and Adult CD4 CD3 Cells Share Similar Gene Expression

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Neonatal and Adult CD4 CD3 Cells Share Similar Gene Expression The Journal of Immunology Neonatal and Adult CD4؉CD3؊ Cells Share Similar Gene Expression Profile, and Neonatal Cells Up-Regulate OX40 Ligand in Response to TL1A (TNFSF15)1 Mi-Yeon Kim, Kai-Michael Toellner, Andrea White, Fiona M. McConnell, Fabrina M. C. Gaspal, Sonia M. Parnell, Eric Jenkinson, Graham Anderson, and Peter J. L. Lane2 We report here the quantitative expression of a set of immunity-related genes, including TNF family members, chemokine -receptors, and transcription factors, in a CD4؉CD3؊ accessory cell. By correlating gene expression between cell-sorted popula -tions of defined phenotype, we show that the genetic fingerprint of these CD4؉CD3؊ cells is distinct from dendritic cells, plas macytoid dendritic cells, T cells, B cells, and NK cells. In contrast, it is highly similar to CD4؉CD3؊ cells isolated from embryonic and neonatal tissues, with the exception that only adult populations express OX40L and CD30L. We have previously reported that IL-7 signals regulate CD30L expression. In the present study, we show that both neonatal and adult CD4؉CD3؊ cells express the TNF family member, death receptor 3 (TNFRSF25), and that addition of TL1A (TNFSF15), the ligand for death receptor 3, up-regulates OX40L on neonatal CD4؉CD3؊ cells. Finally, we demonstrate that this differentiation occurs in vivo: neonatal -CD4؉CD3؊ cells up-regulate both CD30L and OX40L after adoptive transfer into an adult recipient. The Journal of Immu nology, 2006, 177: 3074–3081. e have previously reported that CD4ϩCD3Ϫ CD30L but not OX40L expression (3). Our failure to induce CD11cϪB220Ϫ cells (CD4ϩCD3Ϫ cells) provide sur- OX40L on neonatal CD4ϩCD3Ϫ cells raised the possibility that W vival signals to activated CD4 T cells via their con- they were different cells from those that we found in adult mice. In stitutive expression of OX40L (CD252 and TNFSF4) and CD30L the present study, we report three independent pieces of evidence (CD153 and TNFSF8) in adult mouse (1, 2). These cells are that further support a relationship. We show that cells of related located in B follicles but also T cell areas, especially the outer T lineage show strong correlations in the quantitative mRNA expres- zone. In B follicles they are attached to the T cells that select sion of a 96-gene set of immunity related genes: for example, germinal center (GC)3 B cells, and in the absence of OX40 and subsets of dendritic cells (DCs), T and B cells, are closely corre- CD30 signals, GC T cells fail to survive, with consequent failure lated. This relationship also holds for neonatal and adult popula- of affinity maturation of the Ab responses (2). Even more signif- tions of CD4ϩCD3Ϫ cells. Of particular interest was the shared icantly, T cell memory for Ab responses is abrogated in OX40 and high levels of mRNA for the TNF ligands, lymphotoxin (LT) ␣, CD30 double-deficient mice (2), and we have speculated that LT␤, TNF-␣, and TNF-related activation-induced cytokine memory T cells are normally maintained by their associations with (TRANCE) (TNFSF11). Like OX40L and CD30L on adult these cells in the outer T zone. CD4ϩCD3Ϫ cells, these cells appear to express high levels of When we investigated the presence of these cells in neonatal these ligands constitutively. They also express receptor activator tissues, we found a similar population: however, OX40L and of NF-␬B (RANK) (TNFRSF11A), death receptor 3 (DR3) CD30L, the T cell survival molecules, were lacking (3). These (TNFRSF25), IL-2R␣ (CD25), IL-7R␣ (CD127), common cyto- ␥ ␥ studies demonstrated that the expression of OX40L and CD30L kine receptor -chain ( c) (CD132), CCR7, and CXCR5. was regulated independently: IL-7 signals were important for Because we found that both neonatal and adult CD4ϩCD3Ϫ cells expressed high levels of DR3, we added recombinant TL1A (TNFSF15) to both neonatal and adult populations. This signal Medical Research Council Centre for Immune Regulation, Institute for Biomedical induced high levels of OX40L expression on embryonic/neonatal Research, Birmingham Medical School, Birmingham, United Kingdom populations, and the expression of OX40L was further augmented ϩ Ϫ Received for publication April 4, 2006. Accepted for publication June 16, 2006. on adult cells. Finally, we show that embryonic CD4 CD3 cells The costs of publication of this article were defrayed in part by the payment of page following transfer into an adult recipient up-regulate OX40L and charges. This article must therefore be hereby marked advertisement in accordance CD30L expression to comparable levels to the adult host CD4ϩ with 18 U.S.C. Section 1734 solely to indicate this fact. CD3Ϫ cells in vivo. 1 This work was supported by a Wellcome Programme Grant (to P.J.L.L.). 2 Address correspondence and reprint requests to Dr. Peter J. L. Lane, Medical Re- search Council Centre for Immune Regulation, Institute for Biomedical Research, Materials and Methods Birmingham Medical School, Birmingham B15 2TT, U.K. E-mail address: Mice [email protected] All experiments were performed in accordance with the U.K. laws and with 3 ␤ ␤ Abbreviations used in this paper: GC, germinal center; 2m, 2-microglobulin; CC, Ϫ/Ϫ ␥ the approval of the local ethics committee. Normal, RAG1 , and T cell- correlation coefficient; DC, dendritic cell; DR3, death receptor 3; c, common ␥ deficient mice were bred and maintained in our animal facility. Neonatal -chain; HVEM, herpes virus entry mediator; LT, lymphotoxin; LIGHT, LT-related ϩ Ϫ inducible ligand that competes for glycoprotein D binding to HVEM on T cell; pDC, lymph node or spleen CD4 CD3 cells were isolated from 1- to 2-day-old Ϫ/Ϫ plasmacytoid DC; TRANCE, TNF-related activation-induced cytokine; RANK, re- normal BALB/c litters or RAG1 mice. Spleens from normal C57BL/6 ϩ Ϫ ceptor activator of NF-␬B. or BALB/c E15 embryos were used to isolate E15 CD4 CD3 cells. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 3075 Preparation of CD4ϩCD3Ϫ cells, plasmacytoid DCs (pDCs), DCs, and other cells Cell suspensions for isolation of CD4ϩCD3Ϫ cells, DCs, and pDCs were made from the spleens of adult RAGϪ/Ϫ mice as described pre- viously (1, 3). Neonatal CD4ϩCD3Ϫ cells were isolated from either BALB/c or C57BL/6 mice that were 1 or 2 days old. Briefly, CD11cϩ cells were positively enriched by using CD11c-coated magnetic beads (Miltenyi Biotec) and then FACS sorted into CD8ϩ and CD8Ϫ popu- lations. CD4ϩ cells were enriched from CD11cϩ-depleted populations using CD4-coated magnetic beads, and the resulting CD4ϩ-enriched populations sorted into CD4ϩCD3ϪB220ϪCD11cϪ (CD4ϩCD3Ϫ) and CD4ϩCD3ϪB220ϩCD11clow (pDC) populations. CD45Ϫ stromal cells were FACS sorted from BALB/c mice. For the preparation of E15 CD4ϩCD3Ϫ cells, embryos from normal pregnant mice of gestation day 15 were obtained and the spleens removed. The spleens were placed in culture medium with 100 ng/ml IL-7 (Pepro- Tech) on a 0.8-␮m sterile Nuclepore filter (Millipore) on a sterile arti wrap sponge. The petri dish was then cultured in a contained humid environment in a 10% CO2 incubator for 5 days. On day 5, cultured spleens were teased apart with fine forceps and CD4 cells enriched as above. Follicular B (CD21lowCD23ϩIgMint) cells, marginal zone B (CD21highCD23ϪIgMϩ) cells, and NK cells from normal mice were sorted to make cDNA. Th1 and Th2 cells were prepared under Th1 conditions (10 ng/ml IL-12 and 10 ␮g/ml anti-IL-4) and Th2 conditions (10 ng/ml IL-4 and 10 ␮g/ml anti-IL-12) for 6 days in vitro culture. Stimulation of E15 or neonatal or adult CD4ϩCD3Ϫ cells Prepared cells were cultured with a wide range (0.1–100 ng/ml) of recom- binant mouse TL1A (R&D Systems) for 2 or 6 days of culture and then stained for flow cytometry analysis or for MoFlo cell sorting. FACS staining CD4ϩCD3Ϫ cells were stained with anti-CD4 PE, anti-CD3 FITC, anti- CD11c FITC, and anti-B220 FITC mAbs or anti-B220 allophycocyanin mAbs (BD Biosciences) and then stained with biotinylated mAbs against OX40L, CD30L, and CXCR4 (BD Biosciences) or TRANCE (R&D sys- tems) in conjunction with streptavidin CyChrome (BD Biosciences) as the second-step staining reagent. TaqMan low-density array analysis TaqMan primer sets are designed to work with an efficiency approaching 100%, enabling the quantitative comparison of mRNA expression for dif- ferent genes not only within a cell type, but also between cells of different ␤ ␤ ␤ lineages. Housekeeping genes ( -actin, 2-microglobulin ( 2m), or 18S rRNA) were used to correct for total mRNA. TaqMan low-density real-time PCR arrays (Applied Biosystems) were designed with a 96-gene format. A list of all of the genes measured is as follows: chemokines (CCL19, CXCL12, and CXCL13), chemokine recep- tors (CCR7, CXCR3, and CXCR5), cytokines (IL-1␣, IL-1␤, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p35, IL-12p40, IL-13, IL-15, IL-18, TSLP, IFN-␣1, IFN-␤, IFN-␥, and TGF-␤1), cytokine receptors (IL-2R␣, IL-2R␤, IL-2R␥, IL-4R␣, IL-7R␣, IL-10R␣, IL-10R␤, IL-12R␤1, IL-12R␤2, IFN-␥R1, and IFN-␥R2), costimulatory molecules (CD80, CD86, CTLA4, ICOS, and ICOSL), DC marker (DC-SIGN, ca- ␣ ␤ thepsin S, and integrin x), housekeeping (CD4, -actin, 18S RNA, and ␤ 2m), MHC class II (CD74), TLR (MyD-88, TLR2, TLR3, TLR4, TLR5, spleens. E15 embryonic, day 2 neonatal CD4ϩCD3Ϫ cells, follicular B (fol B), marginal zone (MZ) B, and NK cells were from normal mice. Th1 and Th2 cells were differentiated in vitro for 6 days. A, Correlation of gene expressions between two cell types.
Load more

Recommended publications
  • Human Angiogenin Fused to Human CD30 Ligand (Ang-CD30L) Exhibits Specific Cytotoxicity Against CD30-Positive Lymphoma1
    [CANCER RESEARCH 61, 8737–8742, December 15, 2001] Human Angiogenin Fused to Human CD30 Ligand (Ang-CD30L) Exhibits Specific Cytotoxicity against CD30-positive Lymphoma1 Michael Huhn, Stephanie Sasse, Mehmet K. Tur, Ba¨rbel Matthey, Timo Schinko¨the, Susanna M. Rybak, Stefan Barth,2 and Andreas Engert Fraunhofer IME, Department of Pharmaceutical Product Development, 52074 Aachen, Germany [M. K. T., M. H., S. B.]; Laboratory of Immunotherapy, Department I of Internal Medicine, University Hospital Cologne, 50931 Cologne, Germany [S. S., B. M., T. S., A. E.]; Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702 [S. M. R.] ABSTRACT first and then to administer ITs to kill residual H-RS cells. One of the most promising target antigens for immunotherapy of malignant lym- A number of different immunotoxins composed of cell-specific target- phoma such as Hodgkin’s lymphoma or anaplastic large cell lym- ing structures coupled to plant or bacterial toxins have increasingly been phoma is the CD30 receptor. This antigen was originally discovered evaluated for immunotherapy. Because these foreign proteins are highly immunogenic in humans, we have developed a new CD30 ligand-based on cultured H-RS cells using the moab Ki-1 (1). The gene encoding fusion toxin (Ang-CD30L) using the human RNase angiogenin. The com- the CD30 receptor molecule (2) is located on chromosome 1p36. The pletely human fusion gene was inserted into a pET-based expression naturally occurring CD30 ligand has also been identified and cloned plasmid. Transformed Escherichia coli BL21(DE3) were grown under (3).
    [Show full text]
  • The TNF and TNF Receptor Review Superfamilies: Integrating Mammalian Biology
    Cell, Vol. 104, 487±501, February 23, 2001, Copyright 2001 by Cell Press The TNF and TNF Receptor Review Superfamilies: Integrating Mammalian Biology Richard M. Locksley,*²³k Nigel Killeen,²k The receptors and ligands in this superfamily have and Michael J. Lenardo§k unique structural attributes that couple them directly to *Department of Medicine signaling pathways for cell proliferation, survival, and ² Department of Microbiology and Immunology differentiation. Thus, they have assumed prominent ³ Howard Hughes Medical Institute roles in the generation of tissues and transient microen- University of California, San Francisco vironments. Most TNF/TNFR SFPs are expressed in the San Francisco, California 94143 immune system, where their rapid and potent signaling § Laboratory of Immunology capabilities are crucial in coordinating the proliferation National Institute of Allergy and Infectious Diseases and protective functions of pathogen-reactive cells. National Institutes of Health Here, we review the organization of the TNF/TNFR SF Bethesda, Maryland 20892 and how these proteins have been adapted for pro- cesses as seemingly disparate as host defense and or- ganogenesis. In interpreting this large and highly active Introduction area of research, we have focused on common themes that unite the actions of these genes in different tissues. Three decades ago, lymphotoxin (LT) and tumor necro- We also discuss the evolutionary success of this super- sis factor (TNF) were identified as products of lympho- familyÐsuccess that we infer from its expansion across cytes and macrophages that caused the lysis of certain the mammalian genome and from its many indispens- types of cells, especially tumor cells (Granger et al., able roles in mammalian biology.
    [Show full text]
  • Tools for Cell Therapy and Immunoregulation
    RnDSy-lu-2945 Tools for Cell Therapy and Immunoregulation Target Cell TIM-4 SLAM/CD150 BTNL8 PD-L2/B7-DC B7-H1/PD-L1 (Human) Unknown PD-1 B7-1/CD80 TIM-1 SLAM/CD150 Receptor TIM Family SLAM Family Butyrophilins B7/CD28 Families T Cell Multiple Co-Signaling Molecules Co-stimulatory Co-inhibitory Ig Superfamily Regulate T Cell Activation Target Cell T Cell Target Cell T Cell B7-1/CD80 B7-H1/PD-L1 T cell activation requires two signals: 1) recognition of the antigenic peptide/ B7-1/CD80 B7-2/CD86 CTLA-4 major histocompatibility complex (MHC) by the T cell receptor (TCR) and 2) CD28 antigen-independent co-stimulation induced by interactions between B7-2/CD86 B7-H1/PD-L1 B7-1/CD80 co-signaling molecules expressed on target cells, such as antigen-presenting PD-L2/B7-DC PD-1 ICOS cells (APCs), and their T cell-expressed receptors. Engagement of the TCR in B7-H2/ICOS L 2Ig B7-H3 (Mouse) the absence of this second co-stimulatory signal typically results in T cell B7-H1/PD-L1 B7/CD28 Families 4Ig B7-H3 (Human) anergy or apoptosis. In addition, T cell activation can be negatively regulated Unknown Receptors by co-inhibitory molecules present on APCs. Therefore, integration of the 2Ig B7-H3 Unknown B7-H4 (Mouse) Receptors signals transduced by co-stimulatory and co-inhibitory molecules following TCR B7-H5 4Ig B7-H3 engagement directs the outcome and magnitude of a T cell response Unknown Ligand (Human) B7-H5 including the enhancement or suppression of T cell proliferation, B7-H7 Unknown Receptor differentiation, and/or cytokine secretion.
    [Show full text]
  • Autologous T Cells Expressing CD30 Chimeric Antigen Receptors For
    Published OnlineFirst August 31, 2016; DOI: 10.1158/1078-0432.CCR-16-1365 Cancer Therapy: Clinical Clinical Cancer Research Autologous T Cells Expressing CD30 Chimeric Antigen Receptors for Relapsed or Refractory Hodgkin Lymphoma: An Open-Label Phase I Trial Chun-Meng Wang1, Zhi-Qiang Wu2,YaoWang3, Ye-Lei Guo3,Han-RenDai3, Xiao-Hui Wang2, Xiang Li2, Ya-Jing Zhang1,Wen-YingZhang1, Mei-Xia Chen1, Yan Zhang1, Kai-Chao Feng1,YangLiu4,Su-XiaLi4, Qing-Ming Yang1, and Wei-Dong Han3 Abstract Purpose: Relapsed or refractory Hodgkin lymphoma is a chal- tolerated, with grade 3 toxicities occurring only in two of 18 lenge for medical oncologists because of poor overall survival. We patients. Of 18 patients, seven achieved partial remission and six aimed to assess the feasibility, safety, and efficacy of CD30- achieved stable disease. An inconsistent response of lymphoma targeting CAR T cells in patients with progressive relapsed or was observed: lymph nodes presented a better response than refractory Hodgkin lymphoma. extranodal lesions and the response of lung lesions seemed to Experimental Design: Patients with relapsed or refractory be relatively poor. Lymphocyte recovery accompanied by an Hodgkin lymphoma received a conditioning chemotherapy fol- increase of circulating CAR T cells (peaking between 3 and 9 days lowed by the CART-30 cell infusion. The level of CAR transgenes after infusion) is a probable indictor of clinical response. Analysis in peripheral blood and biopsied tumor tissues was measured of biopsied tissues by qPCR and immunohistochemistry revealed periodically according to an assigned protocol by quantitative the trafficking of CAR T cells into the targeted sites and reduction PCR (qPCR).
    [Show full text]
  • Human TNFRSF18 ELISA Kit (ARG81453)
    Product datasheet [email protected] ARG81453 Package: 96 wells Human TNFRSF18 ELISA Kit Store at: 4°C Component Cat. No. Component Name Package Temp ARG81453-001 Antibody-coated 8 X 12 strips 4°C. Unused strips microplate should be sealed tightly in the air-tight pouch. ARG81453-002 Standard 2 X 10 ng/vial 4°C ARG81453-003 Standard/Sample 30 ml (Ready to use) 4°C diluent ARG81453-004 Antibody conjugate 1 vial (100 µl) 4°C concentrate (100X) ARG81453-005 Antibody diluent 12 ml (Ready to use) 4°C buffer ARG81453-006 HRP-Streptavidin 1 vial (100 µl) 4°C concentrate (100X) ARG81453-007 HRP-Streptavidin 12 ml (Ready to use) 4°C diluent buffer ARG81453-008 25X Wash buffer 20 ml 4°C ARG81453-009 TMB substrate 10 ml (Ready to use) 4°C (Protect from light) ARG81453-010 STOP solution 10 ml (Ready to use) 4°C ARG81453-011 Plate sealer 4 strips Room temperature Summary Product Description ARG81453 Human TNFRSF18 ELISA Kit is an Enzyme Immunoassay kit for the quantification of Human TNFRSF18 in serum, plasma (heparin, EDTA) and cell culture supernatants. Tested Reactivity Hu Tested Application ELISA Specificity There is no detectable cross-reactivity with other relevant proteins. Target Name TNFRSF18 Conjugation HRP Conjugation Note Substrate: TMB and read at 450 nm. Sensitivity 31.25 pg/ml Sample Type Serum, plasma (heparin, EDTA) and cell culture supernatants. Standard Range 62.5 - 4000 pg/ml Sample Volume 100 µl www.arigobio.com 1/3 Precision Intra-Assay CV: 6.3% Inter-Assay CV: 7.0% Alternate Names Tumor necrosis factor receptor superfamily member 18; AITR; CD357; CD antigen CD357; Activation- inducible TNFR family receptor; GITR-D; GITR; Glucocorticoid-induced TNFR-related protein Application Instructions Assay Time ~ 5 hours Properties Form 96 well Storage instruction Store the kit at 2-8°C.
    [Show full text]
  • Clinical Trials of CAR-T Cells in China Bingshan Liu1,2, Yongping Song2* and Delong Liu2*
    Liu et al. Journal of Hematology & Oncology (2017) 10:166 DOI 10.1186/s13045-017-0535-7 RAPID COMMUNICATION Open Access Clinical trials of CAR-T cells in China Bingshan Liu1,2, Yongping Song2* and Delong Liu2* Abstract Novel immunotherapeutic agents targeting tumor-site microenvironment are revolutionizing cancer therapy. Chimeric antigen receptor (CAR)-engineered T cells are widely studied for cancer immunotherapy. CD19-specific CAR-T cells, tisagenlecleucel, have been recently approved for clinical application. Ongoing clinical trials are testing CAR designs directed at novel targets involved in hematological and solid malignancies. In addition to trials of single-target CAR-T cells, simultaneous and sequential CAR-T cells are being studied for clinical applications. Multi-target CAR-engineered T cells are also entering clinical trials. T cell receptor-engineered CAR-T and universal CAR-T cells represent new frontiers in CAR-T cell development. In this study, we analyzed the characteristics of CAR constructs and registered clinical trials of CAR-T cells in China and provided a quick glimpse of the landscape of CAR-T studies in China. Background “third generation chimeric,” and “fourth generation chimeric”; Novel immunotherapeutic agents targeting CTLA-4, country: China. All relevant trials registered at the Clinical- programmed cell death-1 protein receptor (PD-1), and the Trials.gov prior to July 18, 2017, were included in the analysis. ligand PD-L1 are revolutionizing cancer therapy [1–7]. One trial was excluded (NCT03121625) because the target Cancer immunotherapy by re-igniting T cells through antigen was not disclosed. A search of the PubMed blocking PD-1 and PD-L1 is highly potent in a variety of databasewasalsodonetoincludethosetrialsand malignancies [8–12].
    [Show full text]
  • CD134/OX40 Code No
    D125-3 For Research Use Only. Page 1 of 2 Not for use in diagnostic procedures. MONOCLONAL ANTIBODY CD134/OX40 Code No. Clone Subclass Quantity Concentration D125-3 W4-3 Rat IgG2a 100 L 1 mg/mL BACKGROUND: OX40 (CD134/TNFRSF4/ACT35) is SPECIES CROSS REACTIVITY: a 50 kDa of cell surface glycoprotein. OX40, a member of Species Human Mouse Rat the tumor necrosis factor (TNF) superfamily, is expressed primarily on activated CD4+ T cells. OX40 interacts with Cells MT2, HPB-MLT Not Tested Not Tested OX40 ligand antigen (OX40L, also known as CD252/gp34/CD134L) expressed on activated B cells and Reactivity on FCM + antigen presenting cells results in enhanced T cell proliferation and induction of IL-2 production. REFERENCES: OX40/OX40L interaction provides a costimulatory signal, 1) Kawamata, S., et al., J. Biol. Chem. 273, 5808-5814 (1998) resulting in enhanced T cell proliferation and cytokine 2) Latza, U., et al., Europ. J. Immun. 24, 677-683 (1994) production. Then, cell proliferation and immunoglobulin secretion in activated B cells are enhanced. OX40/OX40L system mediates the adhesion of activated or HTLV-I-transformed T cells to vascular endothelial cells. TRAF2 and TRAF5 binding to OX40 led to NF-B activation, and TRAF3 binding appeared to inhibit NF-B activation SOURCE: This antibody was purified from C.B-17 SCID mice ascites fluid using protein A agarose. This hybridoma (clone W4-3) was established by fusion of mouse myeloma cell SP2/0 with WKA/H rat splenocyte immunized with the human OX40 transfectant. Flow cytometric analysis of CD134 expression on HPB-MLT (left) and PM1 FORMULATION: 100 g IgG in 100 L volume of (right).
    [Show full text]
  • TNFRSF18 (Human) ELISA Kit
    TNFRSF18 (Human) ELISA Kit Catalog Number KA1720 96 assays Version: 01 Intended for research use only www.abnova.com I. INTRODUCTION GITR (glucocorticoid induced tumor necrosis factor receptor family related gene) is a type-1 transmembrane protein of 228 amino acids belonging to the TNF and NGF receptor family of proteins. GITR is expressed in normal T-lymphocytes from thymus, spleen. Constitutive expression of a transfected GITR gene induces resistance to apoptosis induced by anti CD3 monoclonal antibodies. The human homolog of GITR is expressed in lymph node and peripheral blood leukocytes. Its expression is up-regulated in human peripheral mononuclear cells mainly after stimulation with antibodies against CD3 and CD28. TNFRSF18 (Human) ELISA Kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human GITR in cell lysate and tissue lysate. This assay employs an antibody specific for human GITR coated on a 96-well plate. Standards and samples are pipetted into the wells and GITR present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-human GITR antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of GITR bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. II. REAGENTS 1. GITR Microplate (Item A): 96 wells (12 strips x 8 wells) coated with anti-human GITR.
    [Show full text]
  • Engineering Strategies to Enhance TCR-Based Adoptive T Cell Therapy
    cells Review Engineering Strategies to Enhance TCR-Based Adoptive T Cell Therapy Jan A. Rath and Caroline Arber * Department of oncology UNIL CHUV, Ludwig Institute for Cancer Research Lausanne, Lausanne University Hospital and University of Lausanne, 1015 Lausanne, Switzerland * Correspondence: [email protected] Received: 18 May 2020; Accepted: 16 June 2020; Published: 18 June 2020 Abstract: T cell receptor (TCR)-based adoptive T cell therapies (ACT) hold great promise for the treatment of cancer, as TCRs can cover a broad range of target antigens. Here we summarize basic, translational and clinical results that provide insight into the challenges and opportunities of TCR-based ACT. We review the characteristics of target antigens and conventional αβ-TCRs, and provide a summary of published clinical trials with TCR-transgenic T cell therapies. We discuss how synthetic biology and innovative engineering strategies are poised to provide solutions for overcoming current limitations, that include functional avidity, MHC restriction, and most importantly, the tumor microenvironment. We also highlight the impact of precision genome editing on the next iteration of TCR-transgenic T cell therapies, and the discovery of novel immune engineering targets. We are convinced that some of these innovations will enable the field to move TCR gene therapy to the next level. Keywords: adoptive T cell therapy; transgenic TCR; engineered T cells; avidity; chimeric receptors; chimeric antigen receptor; cancer immunotherapy; CRISPR; gene editing; tumor microenvironment 1. Introduction Adoptive T cell therapy (ACT) with T cells expressing native or transgenic αβ-T cell receptors (TCRs) is a promising treatment for cancer, as TCRs cover a wide range of potential target antigens [1].
    [Show full text]
  • CD30-Redirected Chimeric Antigen Receptor T Cells Target CD30 And
    Published OnlineFirst August 7, 2018; DOI: 10.1158/2326-6066.CIR-18-0065 Research Article Cancer Immunology Research CD30-Redirected Chimeric Antigen Receptor T Cells Target CD30þ and CD30– Embryonal Carcinoma via Antigen-Dependent and Fas/FasL Interactions Lee K. Hong1, Yuhui Chen2, Christof C. Smith1, Stephanie A. Montgomery3, Benjamin G. Vincent2, Gianpietro Dotti1,2, and Barbara Savoldo2,4 Abstract Tumor antigen heterogeneity limits success of chimeric (NSG) mouse model of metastatic EC. We observed that CD30. þ antigen receptor (CAR) T-cell therapies. Embryonal carcino- CAR T cells, while targeting CD30 EC tumor cells through mas (EC) and mixed testicular germ cell tumors (TGCT) the CAR (i.e., antigen-dependent targeting), also eliminated – containing EC, which are the most aggressive TGCT subtypes, surrounding CD30 EC cells in an antigen-independent man- are useful for dissecting this issue as ECs express the CD30 ner, via a cell–cell contact-dependent Fas/FasL interaction. In – þ – antigen but also contain CD30 /dim cells. We found that CD30- addition, ectopic Fas (CD95) expression in CD30 Fas EC was redirected CAR T cells (CD30.CAR T cells) exhibit antitumor sufficient to improve CD30.CAR T-cell antitumor activity. activity in vitro against the human EC cell lines Tera-1, Tera-2, Overall, these data suggest that CD30.CAR T cells might be and NCCIT and putative EC stem cells identified by Hoechst useful as an immunotherapy for ECs. Additionally, Fas/FasL dye staining. Cytolytic activity of CD30.CAR T cells was com- interaction between tumor cells and CAR T cells can be plemented by their sustained proliferation and proinflamma- exploited to reduce tumor escape due to heterogeneous antigen tory cytokine production.
    [Show full text]
  • Supplementary Tables
    Supplementary Table 1 Lymphocyte activation Activation of immune response Cell adhesion Rank Gene p-value Fold change Rank Gene p-value Fold change Rank Gene p-value Fold change 1 BATF 0.00001 6.31564 1 DUSP6 0.00002 6.39359 1 IL21 0.00014 8.20000 2 MIF 0.00007 2.68925 2 TANK 0.00020 1.69235 2 IL2RA 0.00037 7.70668 3 NFATC2 0.00008 0.40405 3 IRAK1 0.00057 1.63217 3 C1QBP 0.00039 3.79258 4 INPP5D 0.00028 0.31780 4 CHUK 0.00080 1.59486 4 CCL28 0.00010 2.43756 5 TNFSF12 0.00002 0.30456 5 HRAS 0.00004 1.58111 5 BCL10 0.00036 0.62896 6 CASP8 0.00001 0.23611 6 CREB1 0.00037 0.58528 6 IRF1 0.00027 0.62762 7 SMAD3 0.00002 0.23043 7 CD3E 0.00023 0.49823 7 CD46 0.00033 0.52342 8 RORA 0.00001 0.14902 8 ZAP70 0.00068 0.46392 8 SPN 0.00022 0.48806 9 CTSS 0.00024 0.30270 9 JAK2 0.00086 0.45576 10 PLA2G6 0.00002 0.22338 10 TGFB1 0.00006 0.44888 11 TLR3 0.00045 0.13067 11 IL12RB1 0.00021 0.39881 12 ITGA4 0.00098 0.37128 13 ITGAL 0.00013 0.32985 14 CCR2 0.00049 0.29760 15 CD44 0.00097 0.21387 Supplementary Table 1. Differentially expressed genes (p<0.05) in CAR compared to delta CAR T cells. The table displays the p-values and fold changes of genes that are differentially regulated in CAR T cells compared to the truncated CAR (ΔCAR) control T cells collected 24 hours post-stimulation with PSCA.
    [Show full text]
  • Tumor Necrosis Factor Receptor Superfamily 12 May Destabilize Atherosclerotic Plaques by Inducing Matrix Metalloproteinases
    RAPID COMMUNICATION Jpn Circ J 2001; 65: 136–138 Tumor Necrosis Factor Receptor Superfamily 12 may Destabilize Atherosclerotic Plaques by Inducing Matrix Metalloproteinases Se-Hwa Kim, BS; Won-Ha Lee, PhD; Byoung S. Kwon, PhD*; Goo Taeg Oh, PhD**; Yoon-Ho Choi, MD; Jeong-Euy Park, MD, PhD Immunohistochemical staining of human atherosclerotic plaques revealed expression of the tumor necrosis factor receptor superfamily (TNFRSF) 12 in regions rich in macrophage/foam cells. The role of TNFRSF12 in the functioning of monocytes in relation to atherogenesis was investigated by analysis of cellular events after stimulation of TNFRSF12 in a human macrophage-like cell line, THP-1. Activation of the THP-1 cells on plates coated with monoclonal antibody against TNFRSF12 induced the expression of matrix metalloproteinases (MMPs) -1, -9, and -13. Furthermore, the expression patterns of TNFRSF12 and the MMPs overlapped in atherosclerotic plaques. Signaling of TNFRSF12 may thus contribute to the induction of extracellular matrix degrading enzymes in macrophages. (Jpn Circ J 2001; 65: 136–138) Key Words: Atherosclerosis; Matrix metalloproteinase; Tumor necrosis factor receptor superfamily he stability of atherosclerotic plaques depends greatly sion in atherosclerotic plaques. Subsequently, we looked on the integrity of the fibrous cap, which in turn into the expression of MMPs in THP-1 cells (American T depends on its extracellular matrix protein content. Type Culture Collection, Rockville, MD, USA) after stim- Dysregulation of the interplay between matrix degrading ulation of the TNFRSF12. enzymes, such as matrix metalloproteinases (MMPs), and The immunohistochemical analysis of human athero- their inhibitors, such as tissue inhibitor of metalloproteinases sclerotic plaques obtained from carotid endoarterectomies (TIMPs), is believed to be responsible for the rupture of was approved by an institutional review committee and the atherosclerotic plaques.1 MMP-1 and MMP-13, which subjects gave their informed consent.
    [Show full text]