Immune deficiency and autoimmunity in patients with CTLA-4 (CD152) mutations.

Immune deficiency disorders are a heterogeneous group of diseases of variable genetic aetiology. While the hallmark of immunodeficiency is susceptibility to infection, it is increasingly clear that autoimmunity is prevalent, suggestive of a more general immune dysregulation in some cases. With the increasing use of genetic technologies, the underlying causes of immune dysregulation are beginning to emerge. Here we provide a review of the heterozygous mutations found in the immune checkpoint protein CTLA-4, identified in cases of common variable immunodeficiency disorders (CVID) with accompanying autoimmunity. Study of these mutations provides insights into the biology of CTLA-4 as well as suggesting approaches for rational treatment of these patients.

Understanding the CD28/CTLA-4 (CD152) pathway and its implications for costimulatory blockade.

T cell activation is a key event in the adaptive immune system and vital in the generation of protective cellular and humoral immunity. Activation is required to generate CD4 effector T cell responses and provide help for B cell and cytotoxic T cell responses. While defective T responses to foreign antigen result in infectious pathology, over-reactive T cell responses against self-antigens result in autoimmunity and, in a transplantation setting, tissue rejection. Understanding how T cell activation is normally regulated is critical to therapeutic intervention and the CD28/CTLA-4 (CD152) pathway represents the initial activation checkpoint in molecular terms. In particular, while the CTLA-4 pathway is well established as an essential regulator of self-reactivity, its mechanism of action is still uncertain. Such mechanistic issues are important given its central position in T cell activation and the increasing number of therapeutic modalities aimed at manipulating the CD28/CTLA-4 pathway. Here, we provide an updated view of CTLA-4 biology, reviewing the established features of the system and highlighting its interplay with CD28. We then discuss how recent progress in our understanding of this pathway affects our interpretations following intervention.

Expression and function of the autoimmune regulator (Aire) gene in non-thymic tissue.

Educational immune tolerance to self-antigens is induced primarily in the thymus where tissue-restricted antigens (TRAs) are presented to T lymphocytes by cells of the thymic stroma - a process known as central tolerance. The expression of these TRAs is controlled in part by a transcription factor encoded by the autoimmune regulatory (Aire) gene. Patients with a mutation of this gene develop a condition known as autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED), characterized by autoimmune destruction of endocrine organs, fungal infection and dental abnormalities. There is now evidence for TRA expression and for mechanisms of functional tolerance outside the thymus. This has led to a number of studies examining Aire expression and function at these extra-thymic sites. These investigations have been conducted across different animal models using different techniques and have often shown discrepant results. Here we review the studies of extra thymic Aire and discuss the evidence for its expression and function in both human and murine systems.

Lack of activation induced cell death in human T blasts despite CD95L up-regulation: protection from apoptosis by MEK signalling.

The generation of effective immunity requires that antigen-specific T cells are activated, clonally expanded and ultimately eliminated by apoptosis. The involvement of CD95-mediated apoptosis in T-cell elimination is well established, but the conditions which regulate the death pathway under normal circumstances are still emerging. Using superantigen-activated human T cells, we found that whilst T-cell receptor (TCR) signalling triggered up-regulation of CD95 ligand (CD95L), the majority of T cells were resistant to apoptosis induction, despite co-expressing high levels of CD95. Resistance was maintained following direct antibody-mediated cross-linking of CD95 and was not confined to early time periods following activation. Our data implicate TCR-derived signals in protection from apoptosis and reveal a role for the mitogen-activated protein (MAP) kinase pathway by use of a MAP kinase kinase (MEK) inhibitor. Collectively these data demonstrate that resistance to activation-induced cell death in human T cells is prolonged rather than transient, is not attributable to a lack of CD95L up-regulation and is due, at least in part, to signalling via the MEK pathway.

IL-2-independent activation and proliferation in human T cells induced by CD28.

Although the role of CD28 in T cell costimulation is firmly established, the mechanisms by which it exerts its costimulatory actions are less clear. In many circumstances it is difficult to distinguish the effects of CD28 from subsequent actions of cytokines, such as IL-2, on T cell proliferation. Here, we report a model of CD28 costimulation using PMA plus the natural ligand CD80 that resulted in very limited stimulation of IL-2, as evidenced by both cytokine production and IL-2 promoter stimulation. Promoter assays revealed CD28-dependent effects on both NF-kappaB and AP-1, but not on NF-AT or the intact IL-2 promoter. In addition, T cell proliferation was completely resistant to the actions of the immunosuppressant cyclosporin A (CsA). Moreover T cell proliferation was unaffected by the addition of blocking Abs to both IL-2 and the IL-2 receptor, demonstrating that this form of costimulation by CD28 was independent of IL-2. We also investigated the effects of stimulating T cell blasts with CD80 alone and found that there was a limited requirement for IL-2 in this system. We conclude that CD28 costimulation can cause substantial T cell proliferation in the absence of IL-2, which is driven by a soluble factor independent of NF-AT transactivation.

Positive and negative regulation of human T cell activation mediated by the CTLA-4/CD28 ligand CD80.

CD28 and CTLA-4 are related receptors that differentially regulate T cell activation. Despite the fact that they bind the same ligands, CD28 is a classical costimulator enhancing proliferation whereas CTLA-4 appears to perform negative regulatory functions. In this study, we have utilized the natural ligand for CD28 and CTLA-4 (CD80) to determine under what circumstances positive and negative effects are operative. We show here that the stimulation of purified human T cells with phorbol ester and ionomycin is inhibited in the presence of Chinese hamster ovary (CHO) cells expressing CD80. This inhibition is reversed by blocking with both anti-CD80 or Fab fragments of anti-CTLA-4 but also requires CD28 engagement. Furthermore, we show that the inhibitory function of CD80 requires elevated intracellular calcium since inhibition was observed only in the presence of ionomycin. In the absence of intracellular calcium elevation, CTLA-4 was not expressed at the cell surface, and CD80 acted positively as a costimulator of T cells, via CD28. These results demonstrate that the natural ligand CD80 can either costimulate or inhibit T cell responses depending on the conditions of T cell stimulation.

Down-regulation of CD28 via Fas (CD95): influence of CD28 on T-cell apoptosis.

Following antigen engagement of the T-cell receptor (TCR), T-cell survival is largely dictated by the provision of additional signals, such as those from costimulatory receptors and cytokine receptors. Whilst CD28-mediated signalling is increasingly associated with survival, ligation of alternative T-cell antigens, such as Fas (CD95), can trigger apoptosis. The T-cell response following antigen engagement may therefore be influenced by the relative expression levels of these coreceptors as well as by the availability of their ligands (CD80/86 and Fas-L). In this study we demonstrate functional interplay between the death receptor Fas and the costimulatory receptor CD28 in human T cells. In Jurkat T cells, we show that Fas signalling leads to rapid and selective CD28 down-regulation, and that this is associated with a specific decrease in mRNA for CD28, indicating that mechanisms exist which target CD28 at a transcriptional level. Moreover, cells that down-regulate CD28 also undergo apoptosis. Studies on activated human peripheral blood T cells demonstrate that cells expressing high levels of CD28 are resistant to Fas-mediated apoptosis whereas cells expressing low levels are more susceptible, implicating CD28 in the provision of anti-apoptotic signals. Consistent with this hypothesis, direct ligation of CD28 using B7 transfectants concomitant with anti-Fas challenge protects from apoptosis. Since antigen-presenting cells may express Fas-L under certain circumstances, the maintenance of T-cell CD28 expression may be crucial for the prevention of Fas-mediated apoptosis during the course of antigen engagement.

Activation of human T cells with superantigen (staphylococcal enterotoxin B) and CD28 confers resistance to apoptosis via CD95.

Ag recognition is an essential component for an effective T cell response. However, T cell activation is also subject to additional regulation by accessory molecules. CD28 provides essential costimulatory signals that allow T cells to proliferate, whereas molecules such as CTLA-4 and CD95 (Fas) appear to be negative regulators. Currently, which outcome predominates under conditions of antigenic challenge is poorly understood. In particular it has been suggested that one consequence of antigenic activation of T cells is the up-regulation of both CD95 and CD95 ligand, thereby exposing activated T cells to apoptotic death. We have investigated this possibility in normal human peripheral blood T cells triggered by the superantigen SEB either in the presence of endogenous APCs or transfectants expressing DR4 and CD80. In either case, we find that such activation does not expose the majority of T cells to anti-CD95-induced apoptosis as detected by annexin V externalization and DNA fragmentation. Furthermore, by phenotypically identifying, by flow cytometry, those cells that received both antigenic and costimulatory signals from those cells that did not, we observed that CD95-induced apoptosis was not seen in activated T cells receiving Ag and costimulatory signals via CD28. However, while not all T cells were stimulated by superantigen, CD95 expression was found to be homogeneously up-regulated, suggesting a mechanism whereby bystander cells might be made susceptible to CD95-induced death. We conclude that antigenic activation of T cells via the TCR and CD28 engagement provides protection from CD95-induced apoptosis.

Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B.

The intracellular signaling pathways activated upon ligation of the co-stimulatory receptor CD28 remain relatively ill-defined, although CD28 ligation does result in the strong association with, and activation of, phosphatidylinositol (PI) 3-kinase. The downstream effector targets of the CD28-activated PI 3-kinase-dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3-kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of CD28 to PI 3-kinase and the known protective effects of both CD28 and PI 3-kinase against apoptosis in different cell models, we investigated the effects of CD28 on PKB activation. We demonstrate that ligation of CD28 by either anti-CD28 monoclonal antibodies or the natural ligand B7.1, results in the marked activation of PKB in both the leukemic T cell line Jurkat and freshly isolated human peripheral blood-derived normal T lymphocytes. Our data suggest therefore, that PKB may be an important intracellular signal involved in CD28 signal transduction and demonstrate CD28 coupling to downstream elements of a signaling cascade known to promote cell survival.

Evidence that a kinase distinct from protein kinase C and phosphatidylinositol 3-kinase mediates ligation-dependent serine/threonine phosphorylation of the T-lymphocyte co-stimulatory molecule CD28.

The CD28 cytoplasmic tail contains several potential phosphorylation sites for the serine/threonine kinase protein kinase C (PKC) and/or proline-directed serine/threonine kinases, such as extracellular signal-regulated kinases. We demonstrate that ligation of CD28 by B7.1 results in strong serine/threonine phosphorylation of CD28. It is unlikely that ligation-stimulated phosphorylation of CD28 is mediated via activation of PKC, since it was not prevented by pre-treatment of Jurkat cells with inhibitors of PKC, and it was not mimicked by treatment with PKC activators such as PMA. Nevertheless, despite for lack of detectable effects of PMA treatment on CD28 phosphorylation, PMA did partially inhibit the association of CD28 with the putative signalling molecule phosphatidylinositol 3-kinase (PI 3-kinase) and the subsequent accumulation of PtdIns(3,4,5)P3. PI 3-kinase exhibits dual specificity as both a lipid kinase and a protein serine kinase, and site-specific mutagenesis of the Tyr173 residue in the CD28 cytoplasmic tail, which abolishes CD28 coupling to PI 3-kinase [Pages, Ragueneau, Rottapel, Truneh, Nunes, Imbert and Olive (1994) Nature (London) 369, 327-329], also prevents ligation-stimulated phosphorylation of CD28. However, the two PI 3-kinase inhibitors wortmannin and LY294002 had no effect on phosphorylation of CD28 after ligation by B7.1. This study therefore demonstrates that (1) a CD28-activated serine/threonine kinase distinct from both PKC and PI 3-kinase mediates ligation-stimulated CD28 phosphorylation, and (2) the PMA-stimulated down-regulation of the coupling of CD28 to PI 3-kinase is not due to PMA-stimulated phosphorylation of CD28.

Induction of activator protein (AP)-1 and nuclear factor-kappaB by CD28 stimulation involves both phosphatidylinositol 3-kinase and acidic sphingomyelinase signals.

A major obstacle in understanding the signaling events that follow CD28 receptor ligation arises from the fact that CD28 acts as a costimulus to TCR engagement, making it difficult to assess the relative contribution of CD28 signals as distinct from those of the TCR. To overcome this problem, we have exploited the observation that activated human T cell blasts can be stimulated via the CD28 surface molecule in the absence of antigenic challenge; thus, we have been able to observe the response of normal T cells to CD28 activation in isolation. Using this system, we observed that CD28 stimulation by B7-transfected CHO cells induced a proliferative response in T cells that was not accompanied by measurable IL-2 production. However, subsequent analysis of transcription factor generation revealed that B7 stimulation induced both activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) complexes, but not NF-AT. In contrast, engagement of the TCR by class II MHC/superantigen, either with or without CD28 ligation, resulted in the induction of NF-AT, AP-1, and NF-kappaB as well as IL-2 production. Using selective inhibitors, we investigated the signaling pathways involved in the CD28-mediated induction of AP-1 and NF-kappaB. This revealed that NF-kappaB generation was sensitive to chloroquine, an inhibitor of acidic sphingomyelinase, but not to the phosphatidylinositol 3-kinase inhibitor, wortmannin. In contrast, AP-1 generation was inhibited by wortmannin and was also variably sensitive to chloroquine. These data suggest that in activated normal T cells, CD28-derived signals can stimulate proliferation at least in part via NF-kappaB and AP-1 generation, and that this response uses both acidic sphingomyelinase and phosphatidylinositol 3-kinase-linked pathways.

Differential expression of the costimulatory molecules B7.1 (CD80) and B7.2 (CD86) in rheumatoid synovial tissue.

CD4+ T-lymphocytes require two signals to become activated--antigen receptor (TcR) occupancy and an antigen-presenting cell (APC)-derived costimulus. The latter may be provided by B7.1 (CD80) or B7.2 (CD86) on APC interacting with CD28 on T-cells. We have studied the expression of these costimulatory molecules in rheumatoid and osteoarthritic synovial membrane. Very few B7.1-positive cells were seen in synovial tissue from either established or early rheumatoid disease, or in rheumatoid arthritis (RA) or osteoarthritis (OA) synovia at arthroplasty. In contrast, B7.2 was readily detected in rheumatoid synovia, predominantly in the lining layer, in a pattern of expression that corresponded to the presence of CD68-positive macrophages. Only occasional B7.2-positive cells were seen in OA synovia. The presence of B7.2 but the relative lack of expression of B7.1 may be partly responsible for the observations of 'frustrated' T-cell activation or T-cell hyporesponsiveness in the rheumatoid synovium.

Human vascular endothelial cells process and present autoantigen to human T cell lines.

The effectiveness of cultured human umbilical vein endothelial cells as accessory cells for T cell activation has been investigated using T cell clones and lines derived from patients with myasthenia gravis which were specific for different epitopes on the alpha subunit of the human acetylcholine receptor. The endothelial cells were induced with IFN-gamma to express HLA-DR and -DQ at high and low levels respectively. They could then efficiently present specific peptides of the alpha subunit to an HLA-DR- and an HLA-DQw5-restricted T cell line. They could also process epitopes for both T cell lines from the full-length recombinant alpha subunit (r1-437) of the human acetylcholine receptor, where the known epitopes are 80 amino acid residues apart. The endothelial presentation of r1-437, but not of the peptides, was sensitive to chloroquine inhibition. Presentation appeared slightly less efficient (by 1.5- to 3.0-fold) with endothelial cells than with presenting cells from peripheral blood. This may reflect differences in accessory signalling since mAb blocking studies suggested that ligands for CD28 provided important accessory signalling by peripheral blood presenting cells while LFA-3 was used by endothelial cells.

Inhibition of CD28-mediated T cell costimulation by the phosphoinositide 3-kinase inhibitor wortmannin.

T lymphocyte activation requires at least two signals, one via the antigen-specific T cell receptor and a second via the surface molecule CD28 which provides signals critical to interleukin-2 (IL-2) production and T cell proliferation. We have previously shown (Ward S. G., Westwick, J., Hall N. and Sansom D. M. Eur. J. Immunol. 1993. 23: 2572) that CD28 stimulates phosphoinositide (PI) 3-kinase activity, indicating that D-3 phosphoinositides may act as mediators of CD28-induced T cell costimulation. Here, we report that immunoprecipitation of CD28 molecules from Jurkat cells stimulated with the CD28-ligand B7, results in a ligand-dependent association of CD28 with PI 3-kinase. This association correlates with the appearance of PI 3-kinase enzymatic activity in CD28 immunoprecipitates and the formation of D-3 phosphoinositides. Consistent with the hypothesis that D-3 phosphoinositides are important mediators of CD28 signaling, treatment of T cells with the PI 3-kinase inhibitor wortmannin, inhibited both T cell proliferation and production of IL-2, but not the response of T cells to exogenous IL-2. Hence, abrogation of PI 3-kinase activity by wortmannin, appears sufficient to disrupt the costimulatory pathway utilized by CD28, indicating a central role for this enzyme in the CD28 signaling pathway.

Antibody ligation of CD7 leads to association with phosphoinositide 3-kinase and phosphatidylinositol 3,4,5-trisphosphate formation in T lymphocytes.

The CD7 40-kDa glycoprotein is present on a major subset of human T cells and in the presence of phorbol esters mediates an accessory pathway of T cell activation. Hitherto, the intracellular events elicited by CD7 have been ill-defined. This report demonstrates that cross-linking of CD7 results in the formation of phosphatidic acid in the absence of phosphatidylinositol-4,5-bisphosphate metabolism and also the formation of D-3 phosphoinositides lipids which have been postulated to act as intracellular regulatory molecules. The magnitude of D-3 phosphoinositide formation was similar to that induced by CD3. Both the CD7- and CD3-induced elevation of phosphatidylinositol 3,4,5-trisphosphate approximately 5-10 fold less than that elicited by ligation of the costimulatory molecule CD28 by its counter receptor CD80. The formation of D-3 phosphoinositides following ligation of CD7 coincided with the co-association of CD7 with phosphoinositide 3-kinase, the enzyme which mediates the formation of D-3 phosphoinositide lipids. In contrast, ligation of another reported T cell accessory molecule CD5, failed to elicit formation of D-3 phosphoinositides, implying that phosphoinositide 3-kinase is not coupled to all T cell molecules with accessory functions. Since D-3 phosphoinositides have been suggested to play a pivotal role in T cell costimulatory signals induced by CD28, the results presented in this study suggest that CD7 may also influence T cell activation via this pathway.

Mutually exclusive binding of peptide and invariant chain to major histocompatibility complex class II antigens.

The invariant chain is a membrane protein associated with the major histocompatibility complex class II antigens both intra- and extracellularly. The extracellular portion of the human invariant chain (Ii) was expressed in Escherichia coli as a fusion protein with a polyhistidine tail and purified by metal affinity chromatography. The recombinant Ii was used as a ligand to probe binding to the cell surface of Chinese hamster ovary cells stably transfected with human class II alpha and beta genes of the DR4 isotype. We show that recombinant Ii inhibits peptide loading on class II polypeptides and also the converse; the presence of peptide in the antigen groove prevents binding of fluorescein-conjugated Ii. Moreover, blocking of Ii binding by peptide did not require a transition of the class II dimers to an SDS-stable state. A monoclonal antibody, L243, known to bind to (or close to) the peptide pocket of the class II molecule likewise blocked Ii-fluorescein binding. Further, we investigated whether or not the Ii, a variety of bacterial superantigens or the CD4 molecule, have overlapping binding sites on the class II heterodimer. Of the class II ligands tested, reduced binding was detected for the Staphylococcus superantigen type SEB on cells precincubated with soluble Ii while the binding of the other ligands was either unchanged or marginally changed. These data clarify by a direct biochemical approach the binding characteristics of Ii in comparison with other class II ligands.