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Neonatal and Adult CD4 CD3 Cells Share Similar Gene Expression

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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.
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  • Tumor Necrosis Factor Receptor Superfamily 12 May Destabilize Atherosclerotic Plaques by Inducing Matrix Metalloproteinases

    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.