Accessory Signals Research Articles

The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4.

Exon enhancers are accessory pre-mRNA splicing signals that stimulate exon splicing. One class of proteins, the serine-arginine-rich (SR) proteins, have been demonstrated to bind enhancers and activate splicing. Here we report that A/C-rich exon enhancers (ACE elements) are recognized by the human YB-1 protein, a non-SR protein. Sequence-specific binding of YB-1 was observed both to an ACE derived from an in vivo iterative selection protocol and to ACE elements in an alternative exon (v4) from the human CD44 gene. The ACE element that was the predominant YB-1 binding site in CD44 exon v4 was required for maximal in vivo splicing and in vitro spliceosome assembly. Expression of wild-type YB-1 increased inclusion of exon v4, whereas a truncated form of YB-1 did not. Stimulation of exon v4 inclusion by wild-type YB-1 required the ACE necessary for YB-1 binding in vitro, suggesting that YB-1 stimulated exon inclusion in vivo by binding to an exonic ACE element. These observations identify a protein in addition to SR proteins that participates in the recognition of exon enhancers.

Advances in transplantation tolerance

Immunosuppressive drugs developed in the past two decades have improved the short-term survival of organ allografts, but tolerance has not been achieved and almost all transplant recipients continue to require drugs throughout life. Graft rejection arises from the cognate interaction of T cells with antigen-presenting cells, the recognition of alloantigen through the T-cell receptor, and the delivery of accessory stimulation signals. Once activated by the specific antigen, replicating T cells die if they are re-exposed to the same antigen. Since depletion of antigen-activated T cells is one critical mechanism of transplantation tolerance, drugs such as ciclosporin that interfere with activation-induced T-cell death could inhibit tolerance, whereas drugs such as mycophenolate mofetil, that induce the death of activated T cells, could facilitate tolerance. Other tolerance mechanisms depend on inactivation rather than elimination of allograft reactive T cells. When antigen recognition occurs without costimulation through the CD28 and CD154 accessory receptors, or in absence of cell division, T cells become unresponsive. Thus, inhibitors of CD28 and CD154, and inhibition of T-cell division by rapamycin promotes transplantation tolerance.

Expression and function of the costimulatory molecules B7-1 (CD80) and B7-2 (CD86) in an in vitro model of the human blood–brain barrier

The interaction of B7 molecules with their ligand provides important accessory signals for optimal T cell activation and proliferation. In this study the in vitro expression of B7-1 and B7-2 by human brain microvessel endothelial cells (HBMEC) was investigated by semiquantitative reverse transcriptase–polymerase chain reaction (RT–PCR) and immunocytochemistry. In addition, the contribution of B7 molecules to T cell proliferation on cerebral endothelial cells was studied by coincubating purified CD4+ T cells with resting or cytokine activated HBMEC. Untreated cultures constitutively expressed B7-2 RNA and surface protein, but lacked B7-1 expression. Treatment with TNF-α and IFN-γ upregulated B7-2 and induced de novo expression of B7-1. Monoclonal blocking antibodies to B7-1 or B7-2 and human CTLA-4Ig chimeric protein significantly reduced the ability of HBMEC to support α-CD3-induced proliferation of CD4+ T lymphocytes. Expression of B7 glycoproteins and the ability to provide secondary signals for T cell proliferation suggest a potential role of the human cerebral endothelium in T cell activation during the early stages of central nervous system inflammation.

Mechanisms of allergen-specific immunotherapy.

Mechanisms of allergen-specific immunotherapy.

Thymic neuroendocrine self-antigens. Role in T-cell development and central T-cell self-tolerance.

The repertoire of thymic neuroendocrine precursors plays a dual role in T-cell differentiation as the source of either cryptocrine accessory signals in T-cell development or neuroendocrine self-antigens presented by the thymic major histocompatibility complex (MHC) machinery. Thymic neuroendocrine self-antigens usually correspond to peptide sequences highly conserved during the evolution of one family. The thymic presentation of some neuroendocrine self-antigens is not restricted by MHC alleles. Oxytocin (OT) is the dominant peptide of the neurohypophysial family. It is expressed by thymic epithelial and nurse cells (TEC/TNCs) of different species. Ontogenetic studies have shown that the thymic expression of the OT gene precedes the hypothalamic one. Both OT and VP stimulate the phosphorylation of p125FAK and other focal adhesion-related proteins in murine immature T cells. These early cell activation events could play a role in the promotion of close interactions between thymic stromal cells and developing T cells. It is established that such interactions are fundamental for the progression of thymic T-cell differentiation. Insulin-like growth factor 2 (IGF-2) is the dominant thymic polypeptide of the insulin family. Using fetal thymic organ cultures (FTOCs), the inhibition of thymic IGF-2-mediated signaling was shown to block the early stages of T-cell differentiation. The treatment of FTOCs with an mAb anti-(pro)insulin had no effect on T-cell development. In an animal model of autoimmune type 1 diabetes (BB rat), thymic levels of (pro)insulin and IGF-1 mRNAs were normal both in diabetes-resistant and diabetes-prone BB rats. IGF-2 transcripts were clearly identified in all thymuses from diabetes-resistant adult (5-week) and young (2- and 5-days) BB rats. In marked contrast, the IGF-2 transcripts were absent and the IGF-2 protein was almost undetectable in +/- 80% of the thymuses from diabetes-prone adult and young BB rats. These data show that a defect of the thymic IGF-2-mediated tolerogenic function might play an important role in the pathophysiology of autoimmune Type 1 diabetes.

Modulation of leukocyte behavior by an inflamed extracellular matrix.

Inflammation is a response of the immune system to foreign insult or physical damage. Various cellular and humoral components of the immune system are recruited from the vascular system and are translocated through endothelium, and into extracellular matrix (ECM) compartments of inflamed tissues. This translocation is orchestrated by various types of accessory signals, in the form of soluble or complexed molecules, which evoke remarkable transitions in leukocyte activities. Recruited inflammatory cells give rise to mechanisms of migration, including the secretion of enzymes and other pro-inflammatory mediators and the alteration of their adhesive contacts with the ECM. Hence, migrating cells secrete enzymes, chemokines, and cytokines which interact with the ECM, and thereby, provide the cells with intrinsic signals for coordinating their responses. Resultant products of enzymatic modifications to the ECM microenvironment, such as cytokine- and ECM-derived molecules, may be also part of a cell-signaling mechanism that provides leukocytes with information about the nature of their inflammatory activity; such a mechanism may give the immune system data that can be cognitively interpreted for consequential activities. This article reviews the findings that support this notion and describe the dynamic interactions between participants of the inflammatory processes.

CD4 T cell traffic control:in vivo evidence that ligation of OX40 on CD4 T cells by OX40-ligand expressed on dendritic cells leads to the accumulation of CD4 T cells in B follicles

We report here that CD40- but not lipopolysaccharide (LPS)-activated murine dendritic cells (DC) express OX40-ligand (OX40L) as has been reported in humans. To understand how OX40 ligation affects differentiation of CD4 T cells at the time of priming, we constitutively expressed OX40L on DC using the DC-specific promoter of CD11c. Transgenic mice showed greatly increased numbers of CD4 but not CD8 T cells in their B cell areas. This effect was to a great extent immunization dependent, as spleen and lymphoid tissue with no germinal center reactions from mice which had not been deliberately immunized did not show marked CD4 T cell accumulation. The increased numbers of CD4+ CD62low cells in transgenic mice suggest that it is activated CD4 T cells that accumulate within B cell follicles. These data are consistent with the notion that physiological engagement of OX40 (CD134) on activated CD4 T cells either initiates their migration into or causes them to be retained in B follicles. In contrast, LPS-treated CD did not up-regulate OX40L expression. This dichotomy provides a molecular explanation of how DC might integrate environmental and accessory signals to control cytokine differentiation and migration in CD4 effector cells.

Anti-CD86 (B7.2) treatment abolishes allergic airway hyperresponsiveness in mice.

Allergic sensitization in asthma develops as a consequence of complex interactions between T cells and antigen-presenting cells. We have developed several in vivo models to study allergen-specific T cell and B cell function and their relevance to allergic airway hyperresponsiveness (AHR), focusing on the role of the costimulatory molecules CD80 and CD86. Treatment of mice with anti-CD86, but not anti-CD80, significantly inhibited increased serum levels of ovalbumin (OA)-specific IgE and IgG1, airway eosinophilia, and AHR both after 10 d of OA aerosol exposure (in the absence of adjuvant) and after intraperitoneal sensitization followed by repeated airway challenges. Inhibition of AHR was associated with decreased IL-4 and IL-5 levels in the BAL fluid of sensitized mice, suggesting impaired Th2 function in anti-CD86-treated animals. This effect was not seen when mice received treatment only before allergen challenge, indicating that anti-CD86 acts through inhibition of allergic sensitization and not simply by inhibiting the influx of inflammatory cells. These data suggest that the CD86 costimulatory ligand plays a major role in the development of allergic inflammation and AHR in allergen-challenged mice. Further, this study demonstrates that T-B cell interactions during allergic sensitization are amenable to therapeutic manipulation and that selective blockade of accessory signals can be an effective means for modulating distinct T cell functions.

T-independent type 2 antigens induce B cell proliferation in multiple splenic sites, but exponential growth is confined to extrafollicular foci

During the primary splenic response to the T-independent type 2 (TI-2) antigen (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll, small numbers of antigen-specific B cells have entered S phase of the cell cycle 24 h after intraperitoneal immunization. These are distributed in all splenic compartments (T zones, marginal zones, follicles, and red pulp), indicating early proliferation induced by NP-Ficoll does not require accessory signals delivered in a particular splenic microenvironment. Subsequently B blasts accumulate selectively in the outer T zone areas, but exponential growth leading to plasma cell production occurs only in extra-follicular foci. This growth peaks after 5 days, but 20 % of peak numbers of antibody-containing cells are still present 3 months after immunization and 9 % of these cells are proliferating. It is unclear if these late plasmablasts are sustained by self-renewal or continued recruitment of virgin cells into the response. Unlike TD and TI-1 responses NP-specific memory cells do not accumulate in the splenic marginal zones. The level of Cγ3 switch transcripts increases during the first 24 h of the response, but does not increase further during exponential plasmablast growth.

T-independent type 2 antigens induce B cell proliferation in multiple splenic sites, but exponential growth is confined to extrafollicular foci.

During the primary splenic response to the T-independent type 2 (TI-2) antigen (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll, small numbers of antigen-specific B cells have entered S phase of the cell cycle 24 h after intraperitoneal immunization. These are distributed in all splenic compartments (T zones, marginal zones, follicles, and red pulp), indicating early proliferation induced by NP-Ficoll does not require accessory signals delivered in a particular splenic microenvironment. Subsequently B blasts accumulate selectively in the outer T zone areas, but exponential growth leading to plasma cell production occurs only in extrafollicular foci. This growth peaks after 5 days, but 20% of peak numbers of antibody-containing cells are still present 3 months after immunization and 9% of these cells are proliferating. It is unclear if these late plasmablasts are sustained by self-renewal or continued recruitment of virgin cells into the response. Unlike TD and TI-1 responses NP-specific memory cells do not accumulate in the splenic marginal zones. The level of Cgamma3 switch transcripts increases during the first 24 h of the response, but does not increase further during exponential plasmablast growth.

EXPRESSION OF IL-17 IN HUMAN MEMORY CD45RO+T LYMPHOCYTES AND ITS REGULATION BY PROTEIN KINASE A PATHWAY

In the present study, the authors compared the interleukin 17 (IL-17 expression of human naive and phenotypically defined memory T cells as well as its regulation by cAMP pathway. Our data showed that IL-17 mRNA was highly expressed in memory human peripheral CD8+45RO+T cells and CD4+45RO+T cells when peripheral blood mononuclear cells were first stimulated with ionomycin/PMA. IL-17 expression in memory CD8+T cells required accessory signals since culture of ionomycin/PMA-activated CD8+45RO+T cells alone did not result to IL-17 expression. In contrast, memory CD4+T cell population seems to be more independent. IL-17 and interferon γ(IFN-γ) mRNA were both inhibited in the presence of PGE2or the cAMP analogue (dibutyryl-cAMP), while the anti-inflammatory cytokine IL-10 was highly increased. In contrast, naive CD45RA+T cells were unable to express IL-17 whatever the culture conditions. Naive CD4+and CD8+T cells were sensitive to the PKA regulatory pathway since they represent a significant source of IL-10 when PBMC were first cultured with ionomycin/PMA in the presence of either PGE2or db-cAMP. The authors showed that naive cells are highly dependent to their microenvironment, since culture of ionomycin/PMA-activated CD45RA+T cells alone did not result in detectable levels of cytokines even in the presence of PGE2. Results also showed that PGE2induced quite the same levels of intracellular cAMP in naive and memory cells suggesting that these cell populations are equally sensitive to PGE2. However, we suggest that PGE2may be more efficient in blocking both IL-17 and IFN-γ expression in already primed memory T cells, rather than in suppressing naive T cells that could represent a significant source of IL-10. Data suggest that PKA activation pathway plays a critical role in the regulation of cytokine profiles and consequently the functional properties of both human naive and memory CD4+and CD8+T cells during the immune and inflammatory processes.

Resistance to Fas-Mediated T Cell Apoptosis in Asthma

Over activation of CD4+ T cells in the peripheral blood and airway tissues is characteristic of asthma; therefore, we investigated whether activated T cells from asthmatic subjects have altered apoptotic potential through the Fas death receptor. We found that mitogen-stimulated peripheral blood T cells of asthmatic subjects expressed cell surface Fas, but failed to undergo the normal degree of apoptosis after Fas receptor ligation. T cells from asthmatics exhibited normal apoptotic responses to gamma-irradiation (dependent on IL-1 converting enzyme family proteases), ceramide, and mitogen challenge, suggesting functional integrity of the apoptotic pathway. Furthermore, the defect in Fas-dependent apoptosis was overcome by prestimulation with allogeneic accessory cells instead of mitogen. Taken together, the findings suggest that selective resistance to Fas-dependent apoptosis reflects altered Ag-driven, accessory cell-dependent signaling and that ineffective activation of Fas signal transduction may contribute to T cell-dependent immunoinflammation in asthma.

Induction of β-R1/I-TAC by Interferon-β Requires Catalytically Active TYK2

The beta-R1/I-TAC (interferon-inducible T-cell alpha-chemoattractant) gene encodes an alpha-chemokine that is a potent chemoattractant for activated T-cells. We previously reported that beta-R1 was selectively induced by interferon (IFN)-beta compared with IFN-alpha and that the canonical type I IFN transcription factor interferon-stimulated gene factor 3 (ISGF3) was necessary but not sufficient for beta-R1 induction by IFN-beta. These findings suggested that beta-R1 induction by IFN-beta required an accessory component. To begin characterizing this signaling pathway, we examined the function of TYK2 protein in the IFN-beta-mediated induction of beta-R1. This study was motivated by the observation that beta-R1 could not be induced in TYK2-deficient U1 cells by IFN-beta (Rani, M. R. S., Foster, G. R., Leung, S., Leaman, D., Stark, G. R., and Ransohoff, R. M. (1996) J. Biol. Chem. 271, 22878-22884), an unexpected result because IFN-beta evokes substantial expression of IFN-stimulated genes (ISGs) in U1 cells through a TYK2-independent pathway. We now report beta-R1 expression patterns in U1 cells complemented with wild-type or mutant TYK2 proteins. Complementation with wild-type TYK2 rescued IFN-beta-inducible expression of beta-R1. Cells expressing kinase-deficient deletion or point mutants of TYK2 were refractory to induction of beta-R1 by IFN-beta despite robust expression of other ISGs. Transient transfection analysis of a beta-R1 promoter-reporter confirmed that transcriptional activation of beta-R1 by IFN-beta required competent TYK2 kinase. These studies indicate that the catalytic function of TYK2 is required for IFN-beta-mediated induction of beta-R1. Catalytic TYK2 is the first identified component in an accessory signaling pathway that supplements ISGF3/interferon-stimulated response element signaling for gene induction by type I IFNs.

Oligoclonal T cells in human cancer.

Many solid tumors are characterised by the infiltration of lymphocytes and their presence has been correlated with a more favourable prognosis. These tumor-infiltrating lymphocytes (TIL), have been shown to possess specific cytolytic reactivity towards autologous tumours, thus suggesting that tumour cells may express antigens capable of eliciting an immune response. Expression of such tumour-associated antigens (TAA) in combination with appropriate accessory signals would lead to the in vivo accumulation of T cells with anti-tumour specificity. Analysis of the composition of the specific T-cell receptor (TCR) of TIL could thus provide information on the nature of the antigen(s) recognised by TIL. In this review, different aspects of the presence of clonal T cells in patients with cancer are discussed.

Sjögren's autoimmunity: how perturbation of recognition in endomembrane traffic may provoke pathological recognition at the cell surface.

CD4 T cell antigen recognition requires presentation by major histocompatibility complex Class II molecules (MHC II). B cell surface immunoglobulins recognize antigens independently of MHC II, but activation typically requires CD4 cell cytokines as accessory signals. Plasma membrane-endomembrane traffic in lacrimal gland acinar cells, targets of autoimmune activity in Sjögren's syndrome, may satisfy both requirements. The Golgi protein galactosyltransferase and the lysosomal proteins cathepsin B and cathepsin D appear at the plasma membranes during sustained secretomotor stimulation. The RNA transcription termination factor La, a frequent target of Sjögren's autoantibodies, appears in the acinar cell cytoplasm and plasma membranes during viral infection and during in vitro exposure to cytokines. MHC II cycle through endomembrane compartments which contain La, galactosyltransferase, cathepsin B and cathepsin D and which are sites of proteolysis. This traffic may permit trilateral interactions in which B cells recognize autoantigens at the surface membranes, CD4 T cells recognize peptides presented by MHC II, B cells provide accessory signals to CD4 T cells, and CD4 T cells provide cytokines that activate B cells. Acinar cells stimulate lymphocyte proliferation in autologous mixed cell reactions, confirming that they are capable of provoking autoimmune responses.

Mitogenic properties of a bispecific single-chain Fv-Ig fusion generated from CD2-specific mAb to distinct epitopes.

The combination of anti-CD2 mAb 9.6 and 9-1, specific for distinct epitopes, induces proliferation of resting human T cells. The mitogenic activity of this mAb mixture depends upon accessory cells and the 9-1 mAb Fc domain. To further study the functional properties of these mAb, their variable regions were cloned and expressed as monospecific single-chain Fv (scFv) proteins fused to the human IgG1 Fc domain (scFvIg). A novel bispecific scFvIg was constructed by cloning the two monospecific scFv binding sites in tandem, with the 9.6 scFv placed N-terminal to the 9-1 scFvIg. Monospecific scFvIg binding to CD2 was comparable to that of the corresponding parental mAb, while the bispecific scFvIg exhibited binding activity similar to that of the 9-1 scFvIg. The combination of 9.6 scFvIg and 9-1 mAb was mitogenic, whereas mixtures including the 9-1 scFvIg were non-stimulatory, confirming the unique properties of the 9-1 IgG3 Fc. Without the IgG3 tail, the bispecific 9.6/9-1 scFvIg was directly mitogenic and was a more potent mitogen than the mAb mixture, but was accessory cell dependent. Unlike the combination of mAb, the bispecific reagent did not directly mobilize calcium in T cells. In comparison to the mAb mixture, bispecific 9.6/9-1 scFvIg-mediated stimulation of a mixed lymphocyte reaction was significantly more resistant to inhibition of the CD28 co-stimulatory pathway by the inhibitor CTLA-4-Ig. These results show that expression of the 9.6 and 9-1 binding sites together on a bispecific scFvIg increased the mitogenic properties of the mAb and altered the degree of accessory cell signals required for T cell activation.

Tolerance induction in rat renal allografts by chemical modulation of accessory signals for lymphocyte activation without immunosuppressants

Tolerance induction in rat renal allografts by chemical modulation of accessory signals for lymphocyte activation without immunosuppressants

Stimulation of CD2-activated mucosal T cells by intestinal epithelial cells (IEC) requires LFA-3 and is independent of MHC class II and B7

Previous studies from our laboratory demonstrated that stimulation of lamina propria T cell (LPT) proliferation and cytokine synthesis by ailogeneic intestinal epithelial cells (IEC) is dependent on signaling via the CD2 receptor (GE 112:A1007, 1997). The purpose of this study is to identify the costimulatory molecules expressed by IEC which mediate LPT activation. Methods LPT were purified (97%) from normal mucosa by negative selection. Allogeneic IEC or irradiated Caco-2 epithelial ceils were cultured with lxl0 s LPT and a sub-optimal concentration of a stimulatory pair of anti-CD2 antibodies (Tl12/Tl13). Neutralizing antibody to LFA-3 and MHC class II (L243) and the B7 antagonist, CTLA-4-Ig, were added at culture initiation. Proliferation was measured on day 5. Results Conditioned media from IEC or Caco-2 cultures did not stimulate LPT proliferation, suggesting that direct cell-cell contact was required. The addition of the MHC mAb L243 or CILA-4-Ig did not inhibit the proliferation of LPT induced by IEC and Tll2/Tll 3, each alone, or in combination. In contrast, antibody to LFA-3 inhibited greater than 85% of the LPT response to co-stimulation with IEC and Tll2/Tll~. As a control, L243 and CTLA-4-Ig blocked the proliferation of both blood T ceils and LPT to allogeneic monocytes. Conclusion IEC stimulation of CD2-activated LPT is not mediated by the co-stimulatory molecule BT. In addition, our findings show that the restricting element for allogeneic IEC activation of LPT is not MHC class II. These results suggest that another surface IEC protein activates LPT in a CD2 dependent manner in collaboration with LFA-3. The use of alternate accessory proteins and signaling pathways to regulate intestinal T ceils may explain the unique properties of mucosal immunity.

TGF-β Promotes Immune Deviation by Altering Accessory Signals of Antigen-Presenting Cells

AbstractMacrophages incubated with OVA in the presence of TGF-β2 induce immune deviation in vivo (impaired delayed hypersensitivity and IgG2a Ab production) when injected into naive, syngeneic mice. OVA-specific TCR transgenic naive T cells (DO11.10 T cells) produce Th1-type cytokines when stimulated in vitro with OVA-pulsed peritoneal exudate cells (PEC), but if PEC are first treated with TGF-β2 and then pulsed with OVA, the T cells secrete Th2-type cytokines instead. In this study, we investigated the mechanisms that are involved in the modified Ag-presenting functions of macrophages by TGF-β2 pretreatment. We have found that: 1) TGF-β2 impaired the capacity of PEC to produce IL-12 and to express CD40; 2) reduced CD40 expression on TGF-β2-treated PEC impaired IL-12 production when the cells were cocultured with DO11.10 T cells; 3) the failure of TGF-β2-treated PEC to stimulate DO11.10 T cells to secrete IFN-γ was due to their impaired IL-12 production. From these results, we conclude that TGF-β2 treatment impairs the ability of macrophages to produce IL-12 and to express CD40. As a consequence, TGF-β2-treated PEC fail to promote development of pT cells toward the Th1 phenotype.

The Fas Antigen and Fas-Mediated Apoptosis in B-Cell Differentiation

In the B-cell lineage, Fas, a type 1 membrane protein belonging to the tumor necrosis factor receptor (TNF) family, is expressed on B-cells at a restricted developmental stage and on activated B-cells, but not on naive mature B-cells. Apoptosis mediated by Fas-Fas ligand interactions may be involved in the peripheral elimination of autoreactive B-cells and in the regulation of the immune response through deletion of B-cells activated by foreign antigens, as for the T-cell lineage. Fas-mediated apoptosis associated with B-cell activation is affected by costimulation through other accessory signaling molecules like CD40, whose ligands are on T-cells.