Jan E. de Vries

A CD4 + T-cell subset inhibits antigen-specific T-cell responses and prevents colitis

Induction and maintenance of peripheral tolerance are important mechanisms to maintain the balance of the immune system. In addition to the deletion of T cells and their failure to respond in certain circumstances, active suppression mediated by T cells or T-cell factors has been proposed as a mechanism for maintaining peripheral tolerance. However, the inability to isolate and clone regulatory T cells involved in antigen-specific inhibition of immune responses has made it difficult to understand the mechanisms underlying such active suppression. Here we show that chronic activation of both human and murine CD4+T cells in the presence of interleukin (IL)-10 gives rise to CD4+T-cell clones with low proliferative capacity, producing high levels of IL-10, low levels of IL-2 and no IL-4. These antigen-specific T-cell clones suppress the proliferation of CD4+T cells in response to antigen, and prevent colitis induced in SCID mice by pathogenic CD4+CD45RBhighsplenic T cells. Thus IL-10 drives the generation of a CD4+T-cell subset, designated T regulatory cells 1 (Tr1), which suppresses antigen-specific immune responses and actively downregulates a pathological immune response in vivo .

Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones

A serum-free lymphocyte culture medium is described in which serum is replaced by bovine serum albumin, transferrin, insulin, ethanolamine and a mixture of saturated and unsaturated fatty acids (linoleic acid, oleic acid and palmitic acid). In this serum-free medium proliferative and cytotoxic responses induced in mixed lymphocyte culture were comparable with those obtained in medium containing serum. Antigen-specific cytotoxic and helper T cells were isolated and could be propagated in serum-free medium without loss of function.

Interleukin-10 Down-Regulates MHC Class II αβ Peptide Complexes at the Plasma Membrane of Monocytes by Affecting Arrival and Recycling

Interleukin-10 (IL-10) inhibits antigen-specific T cell responses when human monocytes are used as antigen-presenting cells. This is correlated with a down-regulation of MHC class II molecules on the surface of the monocyte. Here we show that IL-10 does not affect MHC class II transcription, polypeptide synthesis, subunit assembly, or antigenic peptide loading. Instead, newly synthesized mature MHC class II molecules are localized to the MHC class II loading compartment but are prevented from reaching the plasma membrane. In addition, treatment of monocytes with IL-10 leads to an accumulation of internalized MHC class II complexes in intracellular vesicles. These results indicate that IL-10 affects antigen presentation by regulating MHC exocytosis and recycling.

Molecular Biology of Interleukin 4 and Interleukin 5 Genes and Biology of their Products that Stimulate B Cells, T Cells and Hemopoietic Cells

Lymphokines produced by helper T cells activated by antigen mediate numerous efTector functions of T cells. Unlike immunoglobulin or the T cell receptor, lymphokines do not bear antigen specificity and stimulate proliferation and differentiation of lymphocytes and hemopoietic cells (Arai et al. 1986). Since many lymphokines are composed of single polypeptide chains, their coding sequences can be isolated by functional expression in appropriate host cells. Using a pcD cDNA expression vector (Okayama & Berg 1983), we have developed a screening procedure employing transfection of plasmid DNAs into mammalian cells followed by assay of transfected cell supernatants for lymphokine activities of interest (Yokota et al. 1984. 1985, 1987c). Based on this expression cloning protocol, many T cell lymphokine genes have been isolated and their primary structures determined. These studies have revealed a regulatory network formed between lymphoid cells and hemopoietic cells through the action of multiple lymphokines produced by activated T cells. For example, interleukin 2 (IL-2) stimulates predominantly T cells whereas interleukin 3 (lL-3) and granulocytemacrophage colony stimulating factor (GM-CSF) stimulate hemopoietic cells. A number of molecules stimulate B cells. Among them, interleukin 4 (IL-4), interleukin 5 (IL-5) and B cell stimulatory factor 2 (BSF-2) have been chemically defined by molecular cloning (Lee et al. 1986, Noma et al. 1986, Yokota et al. !986,

IL-10 alters DC function via modulation of cell surface molecules resulting in impaired T-cell responses.

IL-10 is a potent inhibitor of T-cell activation and has tolerizing effects on these cells. These effects are primarily mediated via modulation of antigen presenting cell function. Here, it is demonstrated that IL-10 completely inhibits LPS-induced DC maturation, resulting in altered DC-T-cell interactions and reduced T-cell responses. IL-10 inhibited LPS-induced upregulation of costimulatory molecules, MHC Class II, and the secretion of IL-12, TNF-alpha, IL-6, and IL-1beta by DCs, although it upregulated the SLAM (CD150) expression at both the mRNA and protein levels. IL-10 pre-treated DC did not respond to subsequent LPS activation and its stimulatory ability for allogeneic and antigen-specific T-cells was severely impaired. Importantly, T-cells derived from co-cultures with Ag-pulsed, IL-10-treated DC were impaired in their responses to subsequent Ag-specific restimulation. Transwell and DC-derived plasma membrane experiments indicated that the capacity of IL-10-treated DC to induce T-cell unresponsiveness results from alterations in the cell surface molecules rather than modulation of cytokine secretion.

Targeting CLA/E‐selectin interactions prevents CCR4‐mediated recruitment of human Th2 memory cells to human skin in vivo

Naive Th cells, bearing receptors for cutaneous antigens, become activated in skin‐draining lymph nodes and express cutaneous lymphocyte antigen (CLA), which confers to these cells the capacity to migrate into the skin to exert their normal effector functions. In the case of atopic dermatitis (AD), allergen‐specific Th2 cells generate exacerbated responses and induce skin inflammation. In such a situation, interfering with the specific mechanism of skin homing would provide a therapeutic benefit. Here we report that CLA+ Th2 memory cells, derived from skin lesions of AD patients, selectively migrate to human skin grafts transplanted onto SCID mice in response to CCR4 but not CCR3, CCR8 or CXCR3 ligands. Skin homing of human CCR4+ Th2 memory cells was Pertussis toxin sensitive and restricted to the CLA+ subset. Furthermore, treatment of these mice with anti‐E‐selectin monoclonal antibody was sufficient to prevent CCL22‐mediated Th2 cell migration to human skin, which both, validates the model and highlights the importance of CLA/E‐selectin interactions in the homing process of Th2 cells to the skin. Using this mechanistic model we demonstrate that skin homing of human Th2 memory cells can be efficiently suppressed using a low molecular weight E‐selectin antagonist, which is of clinical relevance for the treatment of inflammatory skin diseases, including AD.

Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1

Increased expression of vascular cell adhesion molecule 1 (VCAM1) is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention. We have recently identified CAM741, a derivative of a fungus-derived cyclopeptolide that acts as a selective inhibitor of VCAM1 synthesis in endothelial cells. Here we show that the compound represses the biosynthesis of VCAM1 in cells by blocking the process of cotranslational translocation, which is dependent on the signal peptide of VCAM1. CAM741 does not inhibit targeting of the VCAM1 nascent chains to the translocon channel but prevents translocation to the luminal side of the endoplasmic reticulum (ER), through a process that involves the translocon component Sec61β. Consequently, the VCAM1 precursor protein is synthesized towards the cytosolic compartment of the cells, where it is degraded. Our results indicate that the inhibition of cotranslational translocation with low-molecular-mass compounds, using specificity conferred by signal peptides, can modulate the biosynthesis of certain secreted and/or membrane proteins. In addition, they highlight cotranslational translocation at the ER membrane as a potential target for drug discovery.

Receptors and cytokines involved in allergic TH2 cell responses.

Cytokines produced by allergen-specific TH2 cells play a central role in the induction and maintenance of allergic responses. Therefore antagonizing TH2 differentiation and TH2 effector functions will provide an effective way to intervene in allergic diseases. This article discusses that antagonizing the effects of IL-4 and IL-13 by IL-4Ralpha antagonists inhibits human TH2 development and IgE synthesis. In addition, it is shown that the activation of allergen-specific TH2 cells with an agonistic anti-CDw150 mAb redirects the cytokine production profile of these TH2 cells to a TH0 phenotype.

Isolation of large numbers of highly purified lymphocytes and monocytes with a modified centrifugal elutriation technique

A modified centrifugal elutriation technique is described for the isolation of large numbers of lymphocytes and monocytes. Elutriation was carried out by lowering the rotor speed at a constant flow rate which was generated by hydrostatic pressure. The flow rate could be kept constant if the separation procedure was performed at high pressure and high systemic resistance. Up to 2.3 X 10(9) mononuclear cells derived from 2000 ml blood were separated in one single experiment in approximately 1 h. The lymphocytes and monocytes were isolated at purities of 98 +/- 1% and 94 +/- 1% respectively. The purity of the lymphocytes was increased to 99.8 +/- 0.1% by a second elutriation run. Additional advantages of the elutriation procedures are that the choice of medium is free, and that relatively large numbers of cells may be separated with high recoveries.

Systematic identification of regulatory proteins critical for T-cell activation

Background The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. Results In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10Rα and integrin α2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. Conclusions This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.