T cells are the main cell type expressing B7-1 and B7-2 in the central nervous system during acute, relapsing and chronic experimental autoimmune encephalomyelitis.

T cell co-stimulation through the CD28 receptor on T cells is critical to the induction of experimental autoimmune encephalomyelitis (EAE). In this study, expression of the co-stimulatory ligands B7-1 (CD80) and B7-2 (CD86), as well as the receptors CD28 and CTLA-4, were quantitated in central nervous system (CNS) tissues from mice at various stages of EAE. Immunohistochemistry and flow cytometry of CNS-infiltrating cells revealed a high percentage of infiltrating T cells expressing B7-1 and B7-2 during acute, chronic and relapsing EAE. Of the infiltrating cells 10-20% were CTLA-4(+), most of which were CD4(+) T cells. B7-1 and B7-2 expression within the CNS during active EAE might increase the potential for local activation of autoimmune T cells; however, the high level of expression of B7 molecules may also provide a mechanism for the autoregulation of activated CTLA-4(+) T cells.     

Costimulation by CD28 sFv expressed on the tumor cell surface or as a soluble bispecific molecule targeted to the L6 carcinoma antigen

Interaction of the CD80 (B7-1) and CD86 (B7-2) molecules on antigen presenting cells with the receptors CD28 and CTLA-4 on T cells generates signals important in the regulation of immune responses. Because this receptor system involves multiple receptor-ligand interactions, determining the function for individual receptors has been difficult. One approach is the use of antibodies and their derivatives with singular specificity as substitute ligands to explore the activities of these molecules. We have constructed recombinant mono-and bi-specific sFv molecules specific for the CD28 receptor that are capable of binding and generating costimulatory signals to activate T cells. We demonstrate that these soluble molecules are capable of higher levels of costimulation than soluble CD80Ig at equivalent concentrations. We also constructed artificial adhesion receptors on the cell surface using two different CD28-specific sFvIgs fused to the CD80 cytoplasmic and transmembrane domains. In this report, we compared costimulation by a soluble bispecific (αCD28-α6) single chain sFvIg fusion protein to that generated by L6 antigen positive (L6+) H3347 tumor cells transduced with cell surface expressed forms of aCD28 sFv's. We show that the bispecific protein can target potent CD28 costimulatory activity to L6+ tumor cells in vitro . We also show that transfection of the cell surface forms of the two different CD28 sFvIgs into H3347 tumor cells allows them to generate significant costimulatory signals to activated T cells. Finally, we demonstrate that tumor cell presentation of either the soluble bispecific or transduced cell surface sFv generate similar costimulatory effects resulting in T cell activation.     

Expression and functional significance of CTLA-4, a negative regulator of T cell activation

The generation of an effective immune response involves antigen-specific T cell expansion and differentiation of effector function. T cell activation requires at least two distinct signals, including signaling via the antigen-specific T cell receptor (TCR) and a costimulatory pathway. Antigen stimulation of T cells can lead either to a productive immune response, characterized by proliferation, differentiation, clonal expansion and effector function, or, in the absence of an appropriate costimulation, to a state of long-lasting unresponsiveness, termed anergy. Anergic T cells fail to proliferate and secrete cytokines in response to secondary stimulation. The interaction between the costimulatory molecule CD28 on T cells and members of the B7 family on antigen-presenting cell results in upregulation of T cell proliferation and cytokine production and induces the expression of the anti-apoptotic protein Bcl-xl. Based of these findings, the two-signal requirement model for T cell activation is generally accepted today. The negative regulatory mechanisms during T cell activation are not well understood, but they are crucial for the maintainance of lymphocyte homeostasis. For several years the functional role of the enigmatic CD28 homologue cytotoxic T lymphocyte antigen-4 (CTLA-4) in T cell activation has been both obscure and controversial. CTLA-4 was initially supposed to provide a costimulatory signal in conjunction with TCR/CD3 signaling. Today we know that CD28 and CTLA-4 molecules may have diametrically opposed functions: signaling via CD28, in conjunctive with TCR, is required for T cell activation, while signaling via CTLA-4 is a negative signal that inhibits T cell proliferation. How the T cell integrates signals through the TCR/CD3 complex, CD28 and CTLA-4 to initiate, maintain and terminate antigen-specific immune response is in fact not fully clarified. In this review, we will focus on the emerging role of CTLA-4 as a negative regulator of T lymphocyte activation and its role in the dynamic interplay of activatory and inhibitory signals.     

Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses

Pathways in the B7:CD28 family of costimulatory molecules regulate T cell activation and tolerance. B7-dependent responses in Cd28(-/-)Ctla4(-/-) T cells together with reports of stimulatory and inhibitory functions for Programmed Death-1 Ligand 1 or 2 molecules (PD-L1 or PD-L2) have suggested additional receptors for these B7 family members. We show that B7-1 and PD-L1 interacted with affinity intermediate to that of B7-1:CD28 and B7-1:CTLA-4. The PD-L1:B7-1 interface overlapped with the B7-1:CTLA-4 and PD-L1:PD-1 (Programmed Death-1) interfaces. T cell activation and cytokine production were inhibited by the interaction of B7-1 with PD-L1. The responses of PD-1-deficient versus PD-1,B7-1 double-deficient T cells to PD-L1 and of CD28,CTLA-4 double-deficient versus CD28,CTLA-4,PD-L1 triple-deficient T cells to B7-1 demonstrated that PD-L1 and B7-1 interact specifically to inhibit T cell activation. Our findings point to a substantial bidirectional inhibitory interaction between B7-1 and PD-L1 and add an additional dimension to immunoregulatory functions of the B7:CD28 family.     

The role of B7 co-stimulation in activation and differentiation ofCD4+and CD8+T cells

Summary: The functional significance of B7 co-stimulation in T-cell activation was described first in the context of preventing the induction of anergy. The functions of this pathway are far more complex than initially appreciated in view of the existence of two B7 molecules which have specificities for both CD28 and CTLA-4, which serve to amplify and terminate T-cell responses respectively Mice lacking B7 co-stimulators and CD28 and CTLA-4 co-stimulatory receptors are helping to clarify the functions of this key immunoregulatory pathway. In this review we will focus on the role of B7 co-stimulation in the activation and differentiation of CD4⁺ helper cells and CD8⁺ cytotoxic cells. The contribution of B7 co-stimulation to CD⁺ responses depends upon the activation history of the T-cell and the strength of the T-cell antigen receptor signal. B7 co-stimulation contributes to in Cerleukin (IL)-2 production by both naive and previously activated CD4⁺ T cells. B7 co-stimulation is most critical for the differentiation of naive CD4+ T cells to IL-4 producers, but predominately influences IL-2 production by previously activated CD4+ cells. B7 co-stimulation is important in development of cytotoxic T cells through both effects on T-helper cells and by direct co-stimulation of CDS⁺ cells.     

The role of ICOS and other costimulatory molecules in allergy and asthma

Activation and differentiation of T cells play a critical role in the pathogenesis of allergies and asthma. Upon encounter with specific antigen, naïve T helper precursor (ThP) cells become activated, an event that is regulated not only by engagement of the T cell receptor (TCR) with peptide presented in the context of MHC class II molecules, but also by a number of costimulatory signals. CD28 engagement by B7-1 and B7-2 on resting ThP cells provides a critical signal for initial cell cycle progression, interleukin-2 production and clonal expansion. However, in recent years, other related members of the immunoglobulin (Ig) family, such as inducible costimulatory molecules (ICOS) and the TNF receptor family members which include OX40, have also been demonstrated to play an important role in providing unique and complementary signals that regulate the outcome of immune responses. These positive costimulatory signals are counterbalanced by signals that dampen down immune responses and include CTLA-4, PD-1 and the recently described Ig superfamily members BTLA and TIM-3. This review discusses the role of these costimulatory signals and their potential involvement in the pathogenesis of asthma and allergic responses.     

B7-CD28 interaction is a late acting co-stimulatory signal for human T cell responses

The interaction of CD28 with one of the B7 molecules (CD80 and CD86) on professional antigen-presenting cells (APC) is generally considered as the most important co-stimulatory signal for T cell activation. APC in a resting condition express either no or only low levels of B7 molecules. These are up-regulated as a result of interactions with activated T cells, thus suggesting that B7-CD28 interaction is not required at initiation of T cell activation. To study this issue, we blocked B7-CD28 interaction at various time points after in vitro stimulation of peripheral blood T cells with allogeneic monocytes. Epstein-Barr virus-transformed B cells or soluble antigens. We observed that T cell proliferation and IL-2 production were inhibited by B7- blocking agents (CTLA-4-Ig or anti-B7 mAb) almost to the same degree when added either at initiation of culture or 24 h later. B7-blocking agents still resulted in significant inhibition of allogeneic T cell activation when added after 48 h. Furthermore, when CTLA-4-Ig was added at the start of an allogeneic T cell stimulation, addition of anti-CD28 mAb after 24 h of culture nearly fully restored T cell proliferation to control levels. Finally, we demonstrate that delayed addition of B7- blocking agents together with cyclosporin A 1 day after the onset of culture of T cells with allogeneic B cells is highly efficient to induce energy as evaluated by lack of proliferation, cytotoxic T lymphocyte reactivity and IFN-gamma or IL-5 production upon alloantigen rechallenge. Taken together, our data can explain why B7 expression on APC is not required at the time of initial APC-T cell contact, and suggest that the effect of the CD28 signal indeed consists in prolonging IL-2 production and amplifying T cell responses, rather than in providing a critical co-stimulatory signal at the time of initial TCR triggering.      

Cytotoxic T lymphocyte antigen-4 accumulation in the immunological synapse is regulated by TCR signal strength

CD28 and CTLA-4 engagement with B7 expressed by APCs generates critical regulatory signals for T cell activation. CD28 is expressed on the T cell surface and enhances T cell expansion, while CTLA-4 localizes primarily to an intracellular compartment and inhibits T cell proliferation. We demonstrate that CTLA-4 has several unique trafficking properties that may regulate its ability to attenuate a T cell response. Importantly, accumulation of CTLA-4 at the immunological synapse is proportional to the strength of the TCR signal, suggesting that cells receiving stronger stimuli are more susceptible to CTLA-4-mediated inhibition. This may represent a novel feedback control mechanism in which a stimulatory signal regulates the recruitment of an inhibitory receptor to a functionally relevant site on the cell surface.     

The role of CTLA-4 in induction and maintenance of peripheral T cell tolerance

T cell receptor engagement and the B7-CD28 / CTLA-4 signaling pathways play critical roles in T cell activation and regulation. CD28 engagement results in T cell activation, differentiation and survival while CTLA-4 signals block IL-2 production, cell cycle progression and T cell differentiation. We explored the role of CTLA-4 in peripheral tolerance induced by intravenous administration of ethylene carbodiimide-fixed, antigen-coupled splenocytes in the PLP139 - 151-induced relapsing experimental autoimmune encephalomyelitis system. Tolerance induction with PLP139 - 151-coupled splenocytes correlates with low B7 expression on the fixed antigen-presenting cells, conditions that would favor CTLA-4-mediated inhibition. Administration of CTLA-4Ig or anti-CTLA-4 concomitant with the 'tolerogenic' stimulus, however, failed to reverse tolerance induction. In contrast, blocking CTLA-4 at the time of secondary 'immunogenic' encounter with antigen reversed the tolerant state. These findings indicate that CTLA-4 is required to maintain the unresponsive state of the tolerized T cells upon antigenic stimulation under inflammatory conditions and, therefore, have important implications for therapeutic regulation of autoimmune disease.     

Role of B7 signaling in the differentiation of naive CD4+ T cells to effector interleukin-4-producing T helper cells

Signaling through the T cell receptor must be accompanied by costimulatory signals for the differentiation of naive T cells to cytokine-producing effector T helper cells. The costimulatory signal through CD28 is required for T cell activation resulting in increased interleukin (IL)-2 production in vitro, but its role in the production of IL-4 and in the in vivo response is still unclear. We have examined the effects of blocking CTLA-4 (the CD28 homologue) ligand interactions on the in vivo development of IL-4-producing T helper effector cells during a primary mucosal immune response to the nematode parasiteHeligmosomoides polygyrus and during a primary systemic immune response to immunogenic anti-IgD antibodies. Our results demonstrate that CD28 and/or CTLA-4 signaling is required for T cell priming leading to IL-4 cytokine production, B cell activation, and IgE secretion during both immune responses, suggesting that other signaling molecules do not substitute for these molecules in either of these two different immune responses. Furthermore, the CD28 ligands, B7-1 and B7-2, can substitute for each other in providing the required T cell costimulatory ligand interactions during the primary immune response toH. polygyrus. In contrast, memory T cells during the challenge immune response do not require CD28/CTLA-4 ligand interactions for TL-4 production and T helper effector function. *** DIRECT SUPPORT *** A02GS028 00003     

Phenotypic analysis of CTLA-4 and CD28 expression during transient peptide-induced T cell activation in vivo.

The T cell co-stimulatory receptors CD28 and CTLA-4 appear to have opposite effects on T cell activation, mediating augmentation and inhibition of T cell responses respectively. Since these two receptors use the same ligands, CD80 (B7-1) and CD86 (B7-2), the co-ordinate timing of CD28 and CTLA-4 expression has a major impact on the regulation of immune responses. While the kinetics of co-stimulatory molecules have been established for T cell stimulation in vitro, little is known about CD28 and CTLA-4 expression in response to T cell activation in vivo. In this study we have investigated the kinetics of CD28 and CTLA-4 expression upon CD4(+) T cell activation in response to soluble peptide in vivo. Using mice transgenic for a T cell receptor specific for the I-Au-restricted N-terminal peptide of myelin basic protein MBP Ac1-9, we show maximal up-regulation of both CD28 and CTLA-4 2 days after peptide administration. CTLA-4 expression correlated positively with early activation markers on the same cells and was high on blast cells. Administration of peptide analogs with higher affinity for I-Au MHC class II revealed a higher increase in CTLA-4 than in CD28 expression in response to improved TCR ligation. Further, a small population of CD4(+) T cells expressing CTLA-4, CD25 and CD45RBlow was identified in mice that had not been treated with specific peptide. The implications of these observations for immune regulation are discussed.     

Differential requirements for co-stimulatory signals from B7 family members by resting versus recently activated memory T cells towards soluble recall antigens

The interaction between CD28 on T cells with CD80 (B7-1) andCD86 (B7-2) on APCs is considered to be of critical importancefor primary T cell activation both in vivo and in vitro. Therelative importance of this co-stimulatory signal in memoryT cell activation is, however, less clear, and was thereforestudied by in vitro experiments on T cell responses to solublerecall antigens using peripheral blood mononuclear cells orT cell clones. Our data demonstrate that B7-2 represents themajor co-stimulatory signal for the activation of resting peripheralblood memory T cells with recall antigens, as evidenced by theeffects of anti-B7-1 and anti-B7-2 on T cell proliferation aswell as on IL-2 and INF-y production. Since CTLA-4-lg and anti-CD28Fab fragments had similar inhibitory effects to the combinationof anti-B7-1 plus anti-B7-2, the involvement of a third co-stimulatoryCD28/CTLA-4 ligand is unlikely. Despite the strong effects ofB7-blocking agents, a variable fraction of the memory T cellswas resistant to inhibition. Moreover, T cell clones or in vitropreactivated T cells could efficiently be restimulated by solubleantigens on autologous APCs in the absence of B7-1 or B7-2 co-stimulation.These data show that most memory T cells that are freshly isolatedfrom the blood are still dependent on CD28 triggering for theiractivation. However, recently activated T cells can apparentlybypass the requirement for B7 and use other co-stimulatory signalsfor reactivation, a finding with important implications forthe development of immunosuppressive strategies.     

CD28/CTLA-4--CD80/CD86 and ICOS--B7RP-1 costimulatory pathway in bronchial asthma

Costimulatory molecules are cell surface glycoproteins that can direct, modulate and fine-tune T-cell receptor signals. The B7-1/B7-2--CD28/CTLA-4 and ICOS-B7RP-1 pathway provides key second signals that can regulate the activation, inhibition and fine-tuning of T-lymphocyte responses. The expression of B7-1/B7-2--CD28/CTLA-4 molecules on clinical samples from patients with asthma have been well studied, and the results indicate that different extents of these molecules are expressed on the surface of various cells, and that the concentrations of soluble form of these molecules are elevated in the sera of patients with asthma. There is a burst of papers describing an important role for B7-1/B7-2--CD28/CTLA-4 pathway in the Th1/Th2 balance. Similarly, ICOS stimulates both Th1 and Th2 cytokine production but may have a preferential role in Th2 cell development. Moreover, The B7-1/B7-2-CD28/CTLA-4 and ICOS-B7RP-1 pathway has been suggested of being involved in the development of airway inflammation and airway hyperresponsiveness. Further study of the functions of the pathways within the CD28/CTLA-4--CD80/CD86 and ICOS--B7RP-1 superfamily individually and their interplay should provide insights into the pathogenesis of asthma, and has great therapeutic potential for treatment of asthma.     

B7-h2 is a costimulatory ligand for CD28 in human

CD28 and CTLA-4 are cell surface cosignaling molecules essential for the control of T?cell activation upon the engagement of their ligands B7-1 and B7-2 from antigen-presenting cells. By employing a receptor array assay, we have demonstrated that B7-H2, best known as the ligand of inducible costimulator, was a ligand for CD28 and CTLA-4 in human, whereas these interactions were not conserved in mouse. B7-H2 and B7-1 or B7-2 interacted with CD28 through distinctive domains. B7-H2-CD28 interaction was essential for?the costimulation of human T?cells' primary responses to allogeneic antigens and memory recall responses. Similar to B7-1 and B7-2, B7-H2 costimulation via CD28 induced survival factor Bcl-xL, downregulated cell cycle inhibitor p27(kip1), and triggered signaling cascade of ERK and AKT kinase-dependent pathways. Our findings warrant re-evaluation of CD28 and CTLA-4's functions previously attributed exclusively to B7-1 and B7-2 and have important implications in therapeutic interventions against human diseases.     

CD80 and CD86 C domains play an important role in receptor binding and co-stimulatory properties

CD80 and CD86 expressed on the surface of antigen-presenting cells interact with cytotoxic T lymphocyte antigen-4 [CTLA-4 (CD152)] expressed on activated T cells and mediate critical T cell inhibitory signals. CD80 and CD86 are type I glycoproteins, and are made up of two extracellular (EC) Ig-like domains-a transmembrane region and a cytoplasmic tail. The N-terminal (V domain) and membrane-proximal (C) domains share homology with the variable region (V) and the constant region (C) of Ig respectively. Co-crystallographic structures of both CD80 and CD86 bound to CTLA-4 indicate that there is no direct interaction of the C domain of either CD80 or CD86 with the CTLA-4. In contrast, previous mutagenesis studies have identified specific amino acids within the C domain of CD80 that are critical for CTLA-4 binding. To further understand the importance of C domains in the functioning of CD80 and CD86, we constructed chimeric human CD80 and CD86 molecules by swapping their respective C domains, and tested their ability to stimulate T cells. A Chinese hamster ovary (CHO) cell line expressing CD86 activated murine T cells. In contrast, CHO cells expressing either CD80 or a chimeric construct of the CD86 V domain and the CD80 C domain showed a significantly reduced activation. Our studies further demonstrated that the decreased activation by cells expressing the CD80 or a chimera containing CD80 C domain is most likely due to enhanced CTLA-4 binding. From these results we conclude that C domains play a critical, albeit indirect, role in determining CTLA-4 binding affinities and co-stimulatory properties.     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.     

CTLA-4 and autoimmunity: New insights into the dual regulator of tolerance

Cytotoxic T-Lymphocye Antigen 4 (CTLA-4) or CD152 is an inhibitory molecule that plays a critical role in maintenance of tolerance to self-antigens. CTLA-4 is structurally as well as functionally related to CD28, since it shares 31% of homology and binds the B7 family molecules CD80 and CD86 with higher affinity. Nevertheless, CTLA-4 has opposing effects on T cell activation and current evidence shows that its inhibitory role goes beyond the ligand-binding interaction. CTLA-4 competes with CD28 in binding to B7, interacts within the immunological synapsis elements and with clathrin adaptor proteins and tyrosine phosphatases through its cytoplasmic domain to regulate cell trafficking and to set the activation threshold within T cells. Moreover, we have learned from the knock out model that CTLA-4 plays a key role in regulatory T cells and in central tolerance. Because of its importance in maintenance of peripheral tolerance, CTLA-4 has been implicated in several autoimmune diseases, such as systemic lupus erythematosus. Multiple single-nucleotide polymorphisms have been located to human Ctla-4 gene, and their association with autoimmune disease is still a matter of controversy. Despite the promising results of abatacept or CTLA-4-Ig in rheumatoid arthritis and murine lupus nephritis, more clinical randomized trials and standardization of outcomes are needed to prove its efficacy and safety in human lupus nephritis.     

Regulation of CD28 expression on CD8+ T cells by CTLA-4

CD28 and CTLA-4 are the critical costimulatory receptors that predominantly determine the outcome of T cell stimulation, with CD28 promoting positive costimulation and CTLA-4 inducing inhibitory signals. Blockage of the B7-CD28/CTLA-4 pathway leads to transplantation tolerance. However, the exact mechanism of the inhibitory function of CTLA-4 remains elusive. Here, we investigated the influence of CTLA-4 expression on CD28 using CTLA-4-transfected Jurkat T cells as well as primary T cells. Up-regulation of CTLA-4 induced abrogation of IL-2 production, indicating an anergic phenotype of CTLA-4(high) T cells. Besides the negative signaling function of CTLA-4, we show for the first time that CTLA-4 expression promotes the down-regulation of CD28 on the T cell surface as a result of enhanced internalization and degradation of CD28. These data suggest that apart from the established competition for B7.1 and B7.2 by CTLA-4, inhibition of T cells by CTLA-4 might be additionally explained by reduction of CD28 on the cell surface, which might impede T cell response to stimulation. Our data provide a previously unrecognized mechanism for T cell regulation.