Gregorio Aversa

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.

SLAM and its role in T cell activation and Th cell responses.

Following the initial events of T cell activation, triggered by binding of specific peptide‐MHC complex to the TCR for antigen and engagement of costimulatory molecules, a number of activation molecules are expressed on the cell surface. Many of these molecules regulate T cell function. T‐T cell interactions and the interaction of T cells with other cells. One such molecule is SLAM, a multifunctional 70 kDa glycoprotein member of the Ig superfamily with multiple isoforms. SLAM is rapidly induced on natve T cells and B cells following activation. Engagement of SLAM by a specific antibody (mAb A12) results in IL‐2‐independent T cell expansion and induction/up‐regulation of IFN‐γ by activated T cells, including Th2 cells. SLAM was found to be a high‐affinity self‐ligand mediating molecular and cellular homophilic interactions. In this review we discuss SLAM as a receptor involved in T cell expansion and in directing immune responses to a Th0‐Th 1 pathway.

Regulation of IgE synthesis by cytokines

Considerable progress has been made in our understanding of the mechanisms underlying regulation of human IgE synthesis. Interleukin-4 induces IgE production specifically, but costimulatory signals provided by T cells are required. Other cytokines modulate interleukin-4-induced IgE synthesis. The roles of T cells and cytokines in regulating IgE switching are discussed.

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.

Gene transfer with IL‐4 and IL‐13 improves survival in lethal endotoxemia in the mouse and ameliorates peritoneal macrophages immune competence

Systemic anti‐cytokine therapies have been unsuccessful in preventing mortality from gram‐negative bacteremia in humans partly because of the failure to neutralize pro‐inflammatory cytokines at sites of exaggerated production. In an attempt to deliver anti‐inflammatory cytokines to organs directly, gene transfer was employed. Thirty‐six BALB/c mice were injected intraperitoneally with cationic liposomes containing plasmids encoding the human interleukin‐4 (hIL‐4) or IL‐13 gene. Both, hIL‐4 and hIL‐13 mRNA were detected by reverse transcription‐polymerase chain reaction analysis in the liver and the spleen of the animals. Fourty‐eight hours after the in vivo gene transfer, these 36 mice and 18 mock‐transfected mice, were challenged with a lethal dose of E. coli lipopolysaccharide with D‐galactosamine (D‐GalN). Gene transfer with hIL‐4 reduced the serum tumor necrosis factor (TNF)‐α production in response to endotoxin/D‐GalN by 80 % from 113.1 pg/ml in mock‐transfected animals to 22.2 pg/ml ( p < 0.05); human IL‐13 gene transfer reduced serum TNF‐α levels by 90 % (113.1 pg/ml to 11.6 pg/ml; p < 0.05). Survival was improved from 20 % to over 83 % in both treatment groups ( p < 0.001). Our data demonstrate a potent in vivo anti‐inflammatory action of both IL‐4 and IL‐13. In addition, the immune functions of peritoneal macrophages are significantly ameliorated in both treatment groups, with IL‐13 demonstrating better macrophage immune modulation than IL‐4 ( p < 0.05).

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.

Interleukin 10 regulates cell surface and soluble LIR-2 (CD85d) expression on dendritic cells resulting in T cell hyporesponsiveness in vitro.

Dendritic cells (DC) are unique in their ability to stimulate naive T cells to proliferate and to differentiate into effector T cells. DC, however, can also inhibit T cell activation and play a role in central and peripheral tolerance. IL‐10 has been shown to render DC tolerogenic by unknown mechanisms. Using a combined monoclonal antibody/retroviral expression cloning approach, we show here that the inhibitory receptor LIR‐2 (leukocyte immunoglobulin‐like receptor‐2, CD85d) is specifically up‐regulated by IL‐10 on maturing human DC. LPS‐stimulated, LIR‐2‐transfected DC inhibited the proliferation of T cells in autologous, as well as allogeneic culture systems in vitro. In addition, overexpression of LIR‐2 on resting T cells, which lack LIR‐2 expression, inhibited T cell proliferation induced by TCR activation. A novel soluble form of LIR‐2 was detected in culture supernatants of maturing DC. IL‐10 treatment of DC potently inhibited the production of soluble LIR‐2. Recombinant soluble LIR‐2 was able to completely restore the proliferation of T cells activated with LPS‐plus IL‐10‐treated DC. Thus, IL‐10 renders DC hypostimulatory by up‐regulating cell surface LIR‐2 and by inhibiting soluble LIR‐2 in vitro.

Signaling lymphocytic activation molecule (SLAM) regulates T cellular cytotoxicity

Signaling lymphocytic activation molecule (SLAM) is a CD2‐related surface receptor expressed by activated T cells and B cells. SLAM is a self ligand and enhances T cellular proliferation and IFN‐γ  production. A defective SLAM associated protein (SAP) causes X‐linked lymphoproliferative syndrome (XLP), a frequently lethal mononucleosis based on the inability to control EBV. We reportthat SLAM augments TCR‐mediated cytotoxicity. In normal CD4+ and CD8+ T cells, SLAM enhanced TCR‐mediated cytotoxicity. In CD4+ and CD8+ Herpesvirus saimiri (H.saimiri) infected T cells, SLAM engagement alone triggered cytotoxicity. Using H.saimiri‐transformed T cells as a model system we found that SLAM‐engagement promotes the release of lytic granules and a CD95‐independent killing that requires extracellular Ca2+, cytoskeletal rearrangements, and signaling mediated by mitogen‐activated protein kinase kinases MEK1/2. SLAM‐enhanced cytotoxicity implies an immunoregulatory function by facilitating the elimination of APC and a role in overcoming infections with pathogens requiring a cytotoxic immune response.

A novel role for p21-activated protein kinase 2 in T cell activation.

To identify novel components of the TCR signaling pathway, a large-scale retroviral-based functional screen was performed using CD69 expression as a marker for T cell activation. In addition to known regulators, two truncated forms of p21-activated kinase 2 (PAK2), PAK2ΔL1–224 and PAK2ΔS1–113, both lacking the kinase domain, were isolated in the T cell screen. The PAK2 truncation, PAK2ΔL, blocked Ag receptor-induced NFAT activation and TCR-mediated calcium flux in Jurkat T cells. However, it had minimal effect on PMA/ionomycin-induced CD69 up-regulation in Jurkat cells, on anti-IgM-mediated CD69 up-regulation in B cells, or on the migratory responses of resting T cells to chemoattractants. We show that PAK2 kinase activity is increased in response to TCR stimulation. Furthermore, a full-length kinase-inactive form of PAK2 blocked both TCR-induced CD69 up-regulation and NFAT activity in Jurkat cells, demonstrating that kinase activity is required for PAK2 function downstream of the TCR. We also generated a GFP-fused PAK2 truncation lacking the Cdc42/Rac interactive binding region domain, GFP-PAK283–149. We show that this construct binds directly to the kinase domain of PAK2 and inhibits anti-TCR-stimulated T cell activation. Finally, we demonstrate that, in primary T cells, dominant-negative PAK2 prevented anti-CD3/CD28-induced IL-2 production, and TCR-induced CD40 ligand expression, both key functions of activated T cells. Taken together, these results suggest a novel role for PAK2 as a positive regulator of T cell activation.

Antagonistic peptides specifically inhibit proliferation, cytokine production, CD40L expression, and help for IgE synthesis by Der p 1–specific human T-cell clones

BACKGROUND Allergic disorders are characterized by IgE antibody responses to a multitude of allergens as a result of the ability of these antibodies to specifically bind to high-affinity IgE receptors on mast cells and basophils. This interaction results in receptor activation and release of soluble mediators such as histamine and leukotrienes, which cause allergic reactions in various target organs. Because the synthesis of IgE is tightly regulated by cytokines and CD40 ligand (L) interactions, CD4+ helper T cells are obvious targets, with the aim to modulate allergen-induced IgE responses. OBJECTIVES Because of the central role of allergen-specific T-helper type 2 (TH2) cells in the pathway leading to IgE synthesis in vitro and in vivo, we have evaluated the possibility of inhibiting allergen-induced activation of these cells by using allergen-derived peptides that have been modified by single amino acid substitutions. METHODS Three cloned human TH2-like CD4+ T-cell lines, specific for Der p 1, the major allergen in house dust, were used in this study. Upon activation with Der p 1 or specific Der p 1-derived wild-type peptides, these T-cell clones produce high levels of IL-4 and IL-5 and low levels of interferon-gamma and IL-2, respectively, and furthermore give help to B cells for the production of IgE in vitro. Modified synthetic peptides were generated by the introduction of single amino acid substitutions into two different T-cell activation-inducing epitopes on Der p 1. The effects of these modified peptides were studied in Der p 1-induced proliferation, cytokine production, and in vitro IgE production assays. RESULTS Several substituted Der p 1-derived peptides failed to induce T-cell proliferation, in contrast to the native peptides. In addition, some of these peptides acted as antagonists by strongly inhibiting wild-type peptide-induced proliferation as well as the production of interferon-gamma, IL-2, IL-4, and IL-5, although the production of the latter two cytokines was less affected than that of interferon-gamma, even at a 100-fold molar excess of the antagonistic peptides. In addition, the presence of an excess of each of the antagonistic peptides during the activation of Der p I-specific T-cell clones prevented induction of CD40L expression, resulting in a failure of these cells to give help to B cells for the production of IgE in vitro, even in the presence of exogenous IL-4. CONCLUSIONS Substitution of certain amino acid residues in immunogenic Der p 1-derived peptides results in the generation of peptides that fail to induce proliferation of Der p 1-specific T-cell clones. In addition, these modified peptides have strong antagonistic activities on Der p 1-induced proliferation, cytokine production, and CD40L expression by allergen-specific T-cell clones as well as on T cell-mediated IgE production by B cells. These findings suggest that modified peptides interfere with allergen-induced activation of T cells, including the production of cytokines and the expression of surface molecules important for successful T cell-B cell interactions, and may therefore have therapeutic potential by inhibiting the expansion and function of allergen-specific TH2 cells.