LFA-1 interaction with ICAM-1 and ICAM-2 regulates Th2 cytokine production

The role of CD28/B7 and LFA-1/ICAM costimulation in proliferation and Th1/Th2 differentiation of naive CD4+ T cells was addressed using T cells from DO11.10 TCR transgenic mice stimulated by dendritic cells. The blockade of either CD28/B7 or LFA-1/ICAM interactions partially inhibited T cell proliferation. By comparison, blocking CD28/B7 costimulation inhibited IL-4 and IL-5 (Th2 cytokine) production, whereas blocking LFA-1/ICAM-1 or LFA-1/ICAM-2 led to a significant increase (15- to 40-fold) of Th2 cytokines. The combination of anti-ICAM-1 and anti-ICAM-2 mAbs had a synergistic effect with a 100- to 1000-fold increase of Th2 cytokine production. Thus, these two costimulatory pathways have opposing roles in the regulation of Th2 development.     

Critical role of CD28/B7 costimulation in the development of human Th2 cytokine-producing cells

CD28 is a major costimulatory signal receptor for T cells. We have used human naive CD4+ cells from cord blood to analyze the effect of the CD28/B7 costimulatory pathway on development of T helper (Th) subsets. We show that CD28 costimulation is critical for development of the Th2 cytokine-producing cells and that in the absence of CD28 costimulation, cells are not primed to produce Th2 cytokines and consequently "default" to the Th1 subset, independent of the presence of exogenous cytokines. After CD28 costimulation, cells differentiate into a subset that produces Th2 cytokines. However, further CD28 costimulation is not required to maintain Th2 cytokine production. We conclude that D28 costimulation is critical for the development of Th0 and Th2 subsets, but not for the maintenance of cytokine production.     

Studies using antigen-presenting cells lacking expression of both B7-1 (CD80) and B7-2 (CD86) show distinct requirements for B7 molecules during priming versus restimulation of Th2 but not Th1 cytokine production

The differentiation of CD4+ T cells into a Th1 vs Th2 phenotype profoundly influences the outcome of autoimmune and infectious diseases. B7 costimulation has been shown to affect the production of both Th1 and Th2 cytokines, depending on the system studied. There is, consequently, great interest in manipulating the B7 costimulatory signal for therapeutic purposes. To optimally manipulate this key immunoregulatory pathway, the contribution of B7 costimulation to cytokine production requires further clarification. We have compared the B7 requirement for cytokine production by naive vs previously activated T cells using DO11.10 TCR transgenic CD4+ T cells and splenic APCs from mice lacking B7 expression. Our data indicate that induction of IL-4 production and Th2 differentiation by naive T cells is highly dependent on B7 molecules, whereas IL-4 production by previously activated T cells is B7 independent. The predominant contribution of B7-mediated signals to Th1 cytokine production by both naive and primed T cells is upon IL-2 production (and expansion) rather than IFN-gamma (effector cytokine) production. Thus, our studies demonstrate that the antigenic experience of a T cell at the time of B7 blockade may determine whether blockade predominantly affects T cell expansion, differentiation, or effector cytokine production. These differential effects of B7 costimulation on IL-2 vs IFN-gamma production and on IL-4 production by naive vs primed T cells have important implications for understanding how B7:CD28/CTLA4 blockade can be effectively used to manipulate cytokine production in vivo.     

Costimulation through B7-2 (CD86) is required for the induction of a lung mucosal T helper cell 2 (TH2) immune response and altered airway responsiveness

The recruitment of eosinophils into the airways after allergen exposure is dependent on interleukin (IL) 5 secreted from antigen-specific CD4+ T cells of the T helper cell (Th) 2 subset. However, while it is established that costimulation through CD28 is required for TCR-mediated activation and IL-2 production, the importance of this mechanism for the induction of a Th2 immune response is less clear. In the present study, we administered the fusion protein CTLA-4 immunoglobulin (Ig) into the lungs before allergen provocation to determine whether CD28/CTLA-4 ligands are required for allergen-induced eosinophil accumulation and the production of Th2 cytokines. Administration of CTLA-4 Ig inhibited the recruitment of eosinophils into the lungs by 75% and suppressed IgE in the bronchoalveolar lavage fluid. CTLA-4 Ig also inhibited the production of IL-4, IL-5, and IL-10 by 70-80% and enhanced interferon-gamma production from CD3-T cell receptor-activated lung Thy1.2+ cells. Allergen exposure upregulated expression of B7-2, but not B7-1, on B cells from the lung within 24 h. Moreover, airway administration of an anti-B7-2 monoclonal antibody (mAb) inhibited eosinophil infiltration, IgE production, and Th2 cytokine secretion comparable in magnitude to that observed with CTLA-4 Ig. Treatment with an anti-B7-1 mAb had a small, but significant effect on eosinophil accumulation, although was less effective in inhibiting Th2 cytokine production. The anti-B7-2, but not anti-B7-1, mAb also inhibited antigen-induced airway hyperresponsiveness in vivo. In all of the parameters assessed, the combination of both the anti-B7-1 and anti-B7-2 mAb was no more effective than anti-B7-2 mAb treatment alone. We propose that strategies aimed at inhibition of CD28 interactions with B7-2 molecules may represent a novel therapeutic target for the treatment of lung mucosal allergic inflammation.     

Human 4-1BB regulates CD28 co-stimulation to promote Th1 cell responses

Our present study provides evidence that the 4-1BB signal is critical to CD28 co-stimulation in maintaining T cell activation when CD28 has been down-regulated because of repeated stimulation. The 4-1BB signal synergized with CD28 co-stimulation by lowering the threshold of anti-CD28 required to sustain proliferation and IL-2 production. The 4-1BB signal also modulated CD28-mediated cytokine profiles by markedly enhancing Th1 but suppressing Th2-type cytokine production. The 4-1BB signal generated Th1-type cells, as identified by intracellular IFN-gamma production. IFN-gamma induction was detected preferentially in 4-1BB-expressing cells, but not in those expressing CD30. 4-1BB and CD30 were induced in both CD4+ and CD8+ cells, but the location of the two molecules was mutually exclusive in each T cell subset. Our study suggests that the 4-1BB signal regulates CD28 co-stimulation in the targeted subset cells to favor Th1 development and maintain long-term cell growth.     

Sucrase-isomaltase and other brush border glycosidases in colorectal tumors

CD28 ligation delivers a costimulatory signal important in T cell activation. This study demonstrates that the disruption of the CD28/B7 pathway early in the nonobese diabetic mouse strain, using CD28-/- and CTLA41g transgenic mice, promoted the development and progression of spontaneous autoimmune diabetes. Functional analyses of T cells isolated from CD28-deficient mice demonstrated that the GAD-specific T cells produced enhanced Th1-type cytokines (IL-2 and IFN gamma) and diminished Th2-type cytokine, IL-4. Moreover, there was a significant decrease in serum levels of anti-GAD antibodies of the IgG1 isotype consistent with a profound suppression of Th2-type responses in these animals. Thus, the early differentiation of naive diabetogenic T cells into the Th2 subset is dependent upon CD28 signaling and extends our understanding of the importance of Th1/Th2 balance in the regulation of this spontaneous autoimmune disease.     

Maturation of human neonatal CD4+ and CD8+ T lymphocytes into Th1/Th2 effectors

The increased susceptibility of neonates to infections has been ascribed to the immaturity of their immune system. More particularly, T cell-dependent responses were shown to be biased towards a Th2 phenotype. Our studies on the in vitro maturation of umbilical cord blood T cells suggest that the Th2 bias of neonatal response cannot be simply ascribed to intrinsic properties of neonatal T cells. Phenotypically, neonatal CD4+ T cells are more immature than their adult CD45RO-/RA+ naive counterparts and they contain a subset (10-20%) of CD45RO-/RA+ CD31- cells which is very low in adults and displays some unique functional features. The activation and maturation of neonatal CD4+ T cells is particularly dependent upon the strength of CD28-mediated cosignal which dictates not only the cytokine profile released upon primary activation but also the response to IL-12. Activation of adult as well as neonatal CD4+ T cells in the context of low CD28 costimulation yields to the production of low levels of only one cytokine, i.e. IL-2. In contrast, strong CD28 costimulation supports the production of high levels of type 1 (IL-2, IFN gamma and TNF beta) and low levels of type 2 (IL-4 and IL-13) cytokines by neonatal T cells. The low levels of naive T cell-derived IL-4 are sufficient to support their development into high IL-4/IL-5 producers by an autocrine pathway. The ability of IL-12 to prime neonatal CD4+ T cells for increased production of IL-4 (in addition to IFN gamma) is observed only when CD28 cosignal is minimal. Under optimal activation conditions (i.e. with anti-CD3/B7.1 or allogenic dendritic cells) the response and the maturation of neonatal and adult naive T cells are similar. Thus the Th2 bias of neonatal immune response cannot be simply ascribed to obvious intrinsic T cell defect but rather to particular conditions of Ag presentation at priming. Unlike CD4+ T cells, neonatal CD8+ T cells strictly require exogenous IL-4 to develop into IL-4/IL-5 producers. Most importantly, anti-CD3/B7-activated neonatal CD8 T cells coexpress CD4 as well as CCR5 and CXCR4 and are susceptible to HIV-1 infection in vitro.     

Neonatal activation of CD28 signaling overcomes T cell anergy and prevents autoimmune diabetes by an IL-4-dependent mechanism

Optimal T cell responsiveness requires signaling through the T cell receptor (TCR) and CD28 costimulatory receptors. Previously, we showed that T cells from autoimmune nonobese diabetic (NOD) mice display proliferative hyporesponsiveness to TCR stimulation, which may be causal to the development of insulin-dependent diabetes mellitus (IDDM). Here, we demonstrate that anti-CD28 mAb stimulation restores complete NOD T cell proliferative responsiveness by augmentation of IL-4 production. Whereas neonatal treatment of NOD mice with anti-CD28 beginning at 2 wk of age inhibits destructive insulitis and protects against IDDM by enhancement of IL-4 production by islet-infiltrating T cells, administration of anti-CD28 beginning at 5-6 wk of age does not prevent IDDM. Simultaneous anti-IL-4 treatment abrogates the preventative effect of anti-CD28 treatment. Thus, neonatal CD28 costimulation during 2-4 wk of age is required to prevent IDDM, and is mediated by the generation of a Th2 cell-enriched nondestructive environment in the pancreatic islets of treated NOD mice. Our data support the hypothesis that a CD28 signal is requisite for activation of IL-4-producing cells and protection from IDDM.     

Signaling via CD28 costimulates lymphokine production, but does not reverse unresponsiveness to interleukin-2 in anti-CD3 triggered Th1 cells

Previously, it has been described that the ability of murine Th1 cells to proliferate in response to exogenous interleukin (IL)-2 is blocked when these cells are exposed to immobilized anti-CD3 antibodies. In the present study we examined whether simultaneous triggering of the T cell antigen CD28 can prevent the induction of unresponsiveness to IL-2 in Th1 cells. We report that costimulation of Th1 cells with anti-CD28 monoclonal antibodies (mAb) did not overcome unresponsiveness to IL-2 induced by various amounts of immobilized anti-CD3 antibodies. However, stimulation with anti-CD28 mAb strongly augmented IL-2 and interferon-gamma production in anti-CD3-exposed Th1 cells. Thus, despite the fact that anti-CD28 mAb is a potent costimulus for lymphokine production, signaling through CD28 does not seem to be sufficient to trigger proliferation in Th1 cells activated via the T cell receptor. These data suggest the existence of at least three signals to trigger Th1 cell activation. The first is mediated by ligation of the T cell receptor. One cosignal, delivered by the CD28 molecule, leads to IL-2 production. A third, still undefined, signal is required for proliferation in response to IL-2.     

T cell-derived IL-4 and dendritic cell-derived IL-12 regulate the lymphokine-producing phenotype of alloantigen-primed naive human CD4 T cells

Previous studies on human Th subset development were restricted to the analysis of naive T cells activated with anti-CD3 mAb in the absence of physiologic APC. In this study, we have analyzed the role of cytokines and physiologic APC on T cell maturation in an Ag-specific system, in which naive neonatal CD4 T cells were primed with allogeneic dendritic cells (DC). We found that the cytokine profile of primed cells was dependent upon 1) the ratio between T cells and allogeneic DC and 2) the endogenous production of IL-4 and IL-12. Neutralization of IL-4 during primary MLR increased IFN-gamma production at priming and shifted the phenotype of primed cells from Th0 to Th1. These effects were IL-12 dependent, in that they were suppressed by anti-IL-12 Abs. The production of IL-12 in primary MLR was further evidenced by the presence of IL-12 p40 in the culture supernatant fluids. IL-12 production was suppressed by exogenous IL-4 and increased by anti-IL-4 blocking mAbs, indicating that endogenous IL-4 down-regulated IL-12 production by DC. Finally, IL-12 was produced as a result of T cell/DC interaction involving the CD40/CD40 ligand and CD28/B7 costimulation pathways, as revealed by the inhibitory effect of anti-CD40 ligand mAb and CTLA-4Ig. These observations suggest that in neutral conditions, Ag presentation by DC results in the coordinate production of naive T cell-derived IL-4 and DC-derived IL-12 that in concert shape the cytokine profile of Th cells.     

Temporal and subunit-specific modulations of the Rel/NF-kappaB transcription factors through CD28 costimulation

Stimulation of highly purified primary T lymphocytes through CD2 and CD28 adhesion molecules induces a long-term proliferation, dependent on persistent autocrine secretion of interleukin 2 (IL-2), high and prolonged expression of inducible CD25/IL-2 receptor alpha chain (IL-2Ralpha), and secretion of growth factors such as the granulocyte-macrophage colony-stimulating factor (GM-CSF). CD28 costimulation appears to activate cytokine gene expression through conserved kappaB-related CD28 response (CD28RE) or cytokine 1 (CK-1) elements in addition to canonical NF-kappaB-binding sites. In this report, we assess: 1) the evolution of the expression, over an 8-day time period, of the Rel/NF-kappaB family of proteins in costimulated versus TcR/CD3-stimulated primary T cells; 2) the impact of changes on the in vitro occupancy of GM-CSF kappaB and CK-1, as well as IL-2Ralpha kappaB sites; and 3) the differential regulation of newly synthesized p65 and c-Rel by IkappaB proteins. We show that CD2+CD28 stimulation specifically induces, at maximal T cell proliferation phase, sustained nuclear overexpression of NFKB2 p52 and c-Rel subunits which might rely on long-lasting processing of p100 precursor for p52 and increased neosynthesis of c-Rel. This up-regulation correlates with sustained occupancy of GM-CSF kappaB and CK-1 elements by both proteins. Conversely, these subunits do not appear to bind to the IL-2Ralpha kappaB site. Costimulation, but not TcR/CD3 stimulation, appears supported by sustained down-regulation of both IkappaBalpha and -beta regulators. Furthermore, contrary to p65, c-Rel appears to display little affinity for p105, p100 and IkappaBalpha regulators.     

Costimulation through CD86 is involved in airway antigen-presenting cell and T cell responses to allergen in atopic asthmatics

Atopic allergic asthma is characterized by activation of Th2-type T cells in the bronchial mucosa. Previous reports have suggested an important role for costimulation through the CD28/CTLA4-CD80/CD86 pathway in allergen activation of T cells in animal models of inhaled allergen challenge. However, human allergen-specific lines and clones were reported to be costimulation independent. We therefore examined CD80 and CD86 dependence of allergen-induced T cell proliferation and cytokine production in peripheral blood and bronchoalveolar lavage from atopic asthmatic subjects and controls. Both allergen-induced proliferation and IL-5 production from PBMC were inhibited by CTLA4-Ig fusion protein and anti-CD86, but not anti-CD80 mAbs. When allergen-specific CD4+ T cell lines from peripheral blood were examined, proliferation and cytokine production were found to be independent of CD80 or CD86 costimulation. However, when cells obtained directly from the airways were examined, allergen-induced proliferation of bronchoalveolar lavage T cells from atopic asthmatic subjects was inhibited by anti-CD86 but not anti-CD80. In addition, bronchoalveolar lavage-adherent cells from asthmatic, but not control subjects showed APC activity to autologous T cells. This was also inhibited by anti-CD86 but not anti-CD80. Thus allergen-induced T cell activation and IL-5 production in the airway in asthmatic subjects is susceptible to blockade by agents interfering with costimulation via CD86, and this may hold therapeutic potential in asthma.     

IL-10 is required for development of protective Th1 responses in IL-12-deficient mice upon Candida albicans infection

IL-12 is both required and prognostic for Th1 development in mice with Candida albicans infection. To delineate further the physiologic role of IL-12 in antifungal immunity, mice deficient for this cytokine were assessed for susceptibility to C. albicans infections, and for parameters of innate and adaptive immunity. IL-12-deficient mice were highly susceptible to gastrointestinal infection or to reinfection and showed elevated production of Candida-specific IgE and IL-4 and defective production of IFN-gamma. The failure to mount protective Th1 responses occurred despite the presence of an unimpaired innate antifungal immune response, which correlated with unaltered IFN-gamma production, but defective production of, and responsiveness to, inhibitory IL-10. IL-10 or IL-12 neutralization increased the innate antifungal resistance in wild-type mice. However, in IL-12-deficient mice, treatment with exogenous IL-12 or IL-10 impaired IL-4 production and increased resistance to infection, through a negative effect on the CTLA-4/B7-2 costimulatory pathway. These results confirm the obligatory role of IL-12 in the induction of anticandidal Th1 responses, and indicate the existence of a positive regulatory loop between IL-12 and IL-10 that may adversely affect the innate antifungal response, but is required for optimal costimulation of IL-12-dependent CD4+ Th1 cells.     

Differential requirements for CD28 and CD40 ligand in the induction of experimental autoimmune myasthenia gravis

The interactions of CD28-B7 and CD40-CD40 ligand (CD40L) pathways in T cell costimulation and autoimmune disease are incompletely understood. We sought to address this issue by investigation of the genesis of acetylcholine receptor (AChR)-induced antibody-mediated experimental autoimmune myasthenia gravis (EAMG) in CD28- and CD40L-deficient mice (CD28-/-, CD40L-/-). Compared to wild-type mice, the CD28-/- mice became less susceptible, and CD40L-/- mice were completely resistant to EAMG induction. Analysis of T helper functions, reflected by cytokine responses, revealed a switch to a Th1 profile in CD28-/- mice. Consistently, levels of serum AChR-specific antibodies of the IgG1 isotype were decreased in CD28-/- mice. In the CD40L-/- mice, both Th1 and Th2 cytokine responses were diminished, and T cell-dependent AChR-reactive B cell responses were more severely impaired than in the CD28-/- mice. Thus, CD28 and CD40L are differentially required for induction of EAMG.