Cutting edge: The transmembrane E3 ligase GRAIL ubiquitinates the costimulatory molecule CD40 ligand during the induction of T cell anergy

Activation of naive T lymphocytes is regulated through a series of discrete checkpoints that maintain unresponsiveness to self. During this multistep process, costimulatory interactions act as inducible signals that allow APCs to selectively mobilize T cells against foreign Ags. In this study, we provide evidence that the anergy-associated E3 ubiquitin ligase GRAIL (gene related to anergy in lymphocytes) regulates expression of the costimulatory molecule CD40L on CD4 T cells. Using its luminal protease-associated domain, GRAIL binds to the luminal/extracellular portion of CD40L and facilitates transfer of ubiquitin molecules from the intracellular GRAIL RING (really interesting new gene) finger to the small cytosolic portion of CD40L. Down-regulation of CD40L occurred following ectopic expression of GRAIL in naive T cells from CD40(-/-) mice, and expression of GRAIL in bone marrow chimeric mice was associated with diminished lymphoid follicle formation. These data provide a model for intrinsic T cell regulation of costimulatory molecules and a molecular framework for the initiation of clonal T cell anergy.     

Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B (RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8(+) T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-gamma-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.     

CD40 ligand-deficient T cells from X-linked hyper-IgM syndrome carriers have intrinsic priming capability.

Deficiency in CD40 ligand (CD40L) expression is associated with impaired T cell immunity in mouse models and in humans. Previous studies have indicated that this is due to the failure of induction of extrinsic costimulatory molecules. However, other studies have suggested that CD40L is an intrinsic costimulatory molecule. The X-linked hyper-IgM syndrome (XHIM) is a primary immunodeficiency caused by mutations in CD40L, resulting in impaired Ab production and T cell immunity. CD4+ T cells from female carriers of XHIM express a variable degree of normal CD40L based on random X chromosome inactivation. We have examined T cells from XHIM carriers to investigate whether CD40L supports T cell function by acting as an intrinsic costimulator or by induction of other costimulatory molecules by examining coexpression of CD40L and markers of T lymphocyte priming. These carriers provide a unique model for comparison of CD40L-expressing and -nonexpressing lymphocytes in that all factors, including immunological experience, are equivalent between the two populations. Our results show that compared with CD40L-deficient T cells, T cells that express CD40L normally have a minimal advantage in becoming primed, as defined by CD45 RO isoform expression and production of IFN-gamma and TNF-alpha. Conversely, CD40L-deficient T lymphocytes clearly were capable of becoming primed as defined by the same parameters. These findings imply that the intrinsic costimulatory activity of CD40L is not required for attaining primed status, and that CD40L primarily supports T cell function by inducing extrinsic factors that can be shared by CD40L-deficient cells.     

T-cell activation induced by anti-CD3 × anti-B-cell lymphoma monoclonal antibody is enhanced by pretreatment of lymphoma cells with soluble CD40 ligand

 T cells play a key role in the control of abnormal B cell proliferation. Factors that play a role in inadequate T cell responses include absence of expression of costimulatory and adhesion molecules by the malignant B cells and lack of cytotoxic T cells specific for tumor-associated antigens. A number of approaches have been used to enhance T cell response against malignant B cells. Agents such as soluble CD40 ligand can enhance expression of costimulatory molecules by the malignant B cells and improve their ability to activate T cells. Anti-CD3-based bispecific antibodies can retarget T cells toward the tumor cells irrespective of T cell specificity. We used the V 38C13 murine lymphoma model to assess whether the combination of soluble CD40 ligand and anti-CD3-based bispecific antibody can enhance T cell activation induced by malignant B cells more effectively than either approach alone. Expression of CD80, CD86, and ICAM-1 on lymphoma cells was up-regulated by soluble CD40 ligand. Syngeneic T cells were activated more extensively by lymphoma cells when the lymphoma cells were pre-treated with soluble CD40 ligand. Bispecific-antibody induced T cell activation was more extensive when lymphoma cells pretreated with soluble CD40 ligand were present. The combination of soluble CD40 ligand plus bispecific antibody enhanced the median survival of mice compared to mice treated with bispecific anibody alone. We conclude that pretreatment of tumor cells with agents capable of inducing costimulatory molecule expression, such as soluble CD40 ligand can enhance the ability of malignant B cells to activate T cells. This effect is enhanced by the addition of bispecific antibody. The combination of enhanced expression of costimulatory molecules and retargeting of T cells by bispecific antibody may allow for a more effective T-cell-based immunotherapy. Accepted: 14 October 1997     

Bi-directional activation between human airway smooth muscle cells and T lymphocytes: role in induction of altered airway responsiveness

Because both T lymphocyte and airway smooth muscle (ASM) cell activation are events fundamentally implicated in the pathobiology of asthma, this study tested the hypothesis that cooperative intercellular signaling between activated T cells and ASM cells mediates proasthmatic changes in ASM responsiveness. Contrasting the lack of effect of resting human T cells, anti-CD3-activated T cells were found to adhere to the surface of naive human ASM cells, increase ASM CD25 cell surface expression, and induce increased constrictor responsiveness to acetylcholine and impaired relaxation responsiveness to isoproterenol in isolated rabbit ASM tissues. Comparably, exposure of resting T cells to ASM cells prestimulated with IgE immune complexes reciprocally elicited T cell adhesion to ASM cells and up-regulated T cell expression of CD25. Extended studies demonstrated that: 1) ASM cells express mRNAs and proteins for the cell adhesion molecules (CAMs)/costimulatory molecules, CD40, CD40L, CD80, CD86, ICAM-1 (CD54), and LFA-1 (CD11a/CD18); 2) apart from LFA-1, ASM cell surface expression of the latter molecules is up-regulated in the presence of activated T cells; and 3) pretreatment of ASM cells and tissues with mAbs directed either against CD11a or the combination of CD40 and CD86 completely abrogated both the activated T cell-induced changes in expression of the above CAMs/costimulatory molecules in ASM cells and altered ASM tissue responsiveness. Collectively, these observations identify the presence of bi-directional cross-talk between activated T cells and ASM cells that involves coligation of specific CAMs/costimulatory molecules, and this cooperative intercellular signaling mediates the induction of proasthmatic-like changes in ASM responsiveness.     

SVR angiosarcomas can be rejected by CD4 costimulation dependent and CD8 costimulation independent pathways

PURPOSE: We wished to determine whether virally- induced endothelial tumors are rejected by CD4 and CD8 lymphocytes, and whether there are differences in requirements for costimulation in the rejection of these tumors by lymphocyte subsets. EXPERIMENTAL DESIGN: We have developed a model of endothelial tumorigenesis through the sequential introduction of SV40 large T antigen and oncogenic H-ras into endothelial cells. These cells (SVR cells) form highly aggressive angiosarcomas in immunocompromised mice, but do not grow in syngeneic C57BL/6 mice. Using both acute blockade with systemic administration of antibodies and mice genetically deficient in the costimulatory molecules CD28, CD40, and CD40L, we have delineated the requirements of costimulation required to reject this virally-induced endothelial tumor. RESULTS: Control of SVR angiosarcoma is mediated through T lymphocytes, and both CD4 and CD8 lymphocytes are capable of controlling SVR angiosarcoma growth in vivo. Mice genetically deficient in CD28, CD40, and CD40L were able to reject SVR tumors, but depletion of these mice of CD8, but not CD4 cells led to rapid tumor growth. This data suggests that CD4 mediated rejection has a greater dependence of costimulation than CD8 mediated rejection. Surprisingly, acute depletion of costimulatory molecules in immunocompetent C57BL/6 mice led to rapid tumor growth. CONCLUSIONS: Significant differences exist in the immune status of mice acutely depleted of costimulatory molecules versus genetically deficient mice. Our results suggest that acute depletion is more immunosuppressive than genetic depletion. Humans who undergo costimulatory blockade may require periodic surveillance for virally-induced tumors.     

Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts

While CD28 functions as the major T cell costimulatory receptor, a number of other T cell molecules have also been described to induce T cell costimulation. Here, we investigated the mechanisms by which costimulatory molecules other than CD28 contribute to T cell activation. Non-CD28 costimulatory molecules such as CD5, CD9, CD2, and CD44 were present in the detergent-insoluble glycolipid-enriched (DIG) fraction/raft of the T cell surface, which is rich in TCR signaling molecules and generates a TCR signal upon recruitment of the TCR complex. Compared with CD3 ligation, coligation of CD3 and CD5 as an example of DIG-resident costimulatory molecules led to an enhanced association of CD3 and DIG. Such a DIG redistribution markedly up-regulated TCR signaling as observed by ZAP-70/LAT activation and Ca2+ influx. Disruption of DIG structure using an agent capable of altering cholesterol organization potently diminished Ca2+ mobilization induced by the coligation of CD3 and CD5. This was associated with the inhibition of the redistribution of DIG although the association of CD3 and CD5 was not affected. Thus, the DIG-resident costimulatory molecules exert their costimulatory effects by contributing to an enhanced association of TCR/CD3 and DIG.     

A recombinant vector expressing transgenes for four T-cell costimulatory molecules (OX40L,B7-1, ICAM-1, LFA-3) induces sustained CD4+ and CD8+ T-cell activation,protection from apoptosis,and enhanced cytokine production

The role of OX40L on the activation of T cells was investigated using poxvirus vectors expressing OX40L alone or in combination with three other T-cell costimulatory molecules: B7-1, ICAM-1, and LFA-3. Poxvirus vector-infected cells were used to stimulate nai;ve or activated CD4(+) and CD8(+) T cells. These studies demonstrate that (a) OX40L plays a role in sustaining the long-term proliferation of CD8(+) T cells in addition to the known effect on CD4(+) T cells following activation, (b) OX40L enhances the production of Th1 cytokines (IL-2, IFN-gamma, and TNF-alpha) from both CD4(+) and CD8(+) while no change in IL-4 expression was observed, and (c) the anti-apoptotic effect of OX40L on T cells is likely the result of sustained expression of anti-apoptotic genes while genes involved in apoptosis are inhibited. In addition, these are the first studies to demonstrate that the combined use of a vector driving the expression of OX40L with three other costimulatory molecules (B7-1, ICAM-1, and LFA-3) both enhances initial activation and then further potentiates sustained activation of nai;ve and effector T cells.     

Lipopolysaccharide modulation of dendritic cells is insufficient to mature dendritic cells to generate CTLs from naive polyclonal CD8+ T cells in vitro, whereas CD40 ligation is essential.

Many cytotoxic CD8+ T cell responses are dependent on the interactions between CD40 ligand on the helper CD4+ T cell and CD40 on the APC. Although CD40 triggering of dendritic cells (DC) has been shown to mature the DC by increasing the level of expression of costimulatory molecules and inducing IL-12 secretion, the precise mechanisms by which CD40-CD40 ligand interactions allow DC to drive CTL responses remain unknown. We have used an in vitro model in which naive polyclonal CD8+ T cells can be activated by bone marrow-derived DC to investigate factor(s) that are responsible for this CD40-dependent generation of CTLs. DC modulated with agonistic anti-CD40 mAb (aCD40) are able to generate Ag-specific CTL responses while DC modulated with the microbial stimulus LPS alone do not. We compared the Ag-presenting capacity, levels of costimulatory molecules, and release of cytokines and chemokines of DC modulated with aCD40 to that of DC modulated by LPS. None of the factors assayed account for the unique capacity of anti-CD40-matured DC to drive CTL but this model provides a simplified system for further investigation. Although we attempted to use an LPS-free system for these studies, we are unable to rule out the possibility that very low levels of endotoxin (<20 pg/ml) may synergize with CD40 ligation in the generation of CTLs.     

IFN-alpha beta promote priming of antigen-specific CD8+ and CD4+ T lymphocytes by immunostimulatory DNA-based vaccines

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-alphabeta and -gamma, TNF-alpha, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8(+) and CD4(+) lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-alphabeta pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8(+) CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4(+) Th cells by ISS-based vaccines.     

Expression of CD86 on human islet endothelial cells facilitates T cell adhesion and migration

Pancreatic islet endothelial cells (ECs) form the barrier across which autoreactive T cells transmigrate during the development of islet inflammation in type 1 diabetes. Little is known about the immune phenotype of islet ECs that might shape their molecular interaction with autoreactive T cells before and during the development of islet inflammation. In this study we examined the expression and functional significance of costimulatory molecules by human islet ECs. Freshly isolated human islet ECs constitutively expressed CD86 (B7-2) and ICOS ligand but not CD80 (B7-1) or CD40 costimulatory molecules. The functional activity of islet EC-expressed CD86 was examined by coculture of resting islet ECs with CD4 T cells stimulated by CD3 ligation alone. Marked T cell proliferation in the coculture was completely abrogated by mAb blockade of CD86, confirming that costimulatory properties are conferred on ECs by CD86 expression. In view of its location on the vasculature, we hypothesized a role for CD86 in T cell adhesion/transmigration. In keeping with this, adhesion/transmigration of activated (CD3 ligated) memory (CD45R0(+)) CD4 T cells across islet ECs was completely inhibited in the presence of CD86 blocking mAb. Identical results were obtained for T cell adhesion using either CTLA-4 blocking mAb or CTLA-4Ig (abatacept), indicating CTLA-4 as the T cell ligand for these CD86-mediated effects. These data suggest a novel role for CD86 expression on the microvasculature, whereby ligation of CTLA-4 on CD4 T cells by CD86 on islet ECs is key to the adhesion of recently activated T cells.     

Analysis of the in vivo dendritic cell response to the bacterial superantigen staphylococcal enterotoxin B in the mouse spleen

To investigate the in vivo effects of Staphylococcal enterotoxin B (SEB) on dendritic cells (DCs) in the spleen, a single dose of SEB (50 microg/kg) was administered to BALB/c mice by intraperitoneal injection. Afterwards, the mice were sacrificed at 2, 6 and 24 hr, 2, 4, 7 and 15 days, and the spleens were removed. The immunocytochemical characterization of the cells was carried out using various monoclonal antibodies in cryostat-cut sections. The distribution patterns of DCs and their major costimulatory molecules, CD80, CD86 and CD40 in the spleen were identified, and the evidence for maturation of DCs in vivo in response to SEB was obtained. It was found that systemic administration of SEB induced the migration of most of the immature, splenic DCs from the marginal zone to the periarterial lymphatic sheath within 6 hr. This movement paralleled a maturation process, as assessed by upregulation of CD40, CD80 and CD86 expression in the interdigitating dendritic cells (IDCs). The upregulation of costimulatory molecule expression was conspicuous only in DCs in contrast to other antigen-presenting cells (APCs) such as macrophages and B cells which did not show any significant alterations in their costimulatory molecule expression. We also demonstrated the temporal expression pattern of these costimulatory molecules on the activated DCs. The upregulation of costimulatory molecules on DCs reached a peak level 6 hr after SEB injection, while the increase in number of T cells expressing T cell receptor V138 reached a peak level on day 2 after SEB treatment. In conclusion, we demonstrated the in vivo DC response to SEB in the mouse spleen, especially a potent stimulative effect of SEB on DCs in vivo, a temporal distribution pattern of DCs as well as T cells including TCR Vbeta8+ T cells, and a differential expression pattern of costimulatory molecules on the activated DCs. The results of the present study indicate that DCs are the principal type of APCs which mediate T cell activation by SAg in vivo, and that each costimulatory molecule may have different role in the activation of DCs by SAg. Thus, it is plausible to speculate that DCs play a critical role in the T cell clonal expansion by SAgs and other SAg-induced immune responses in vivo.     

The net effect of costimulatory blockers is dependent on the subset and activation status of the autoreactive T cells

In this study, we investigated whether CD4 and CD8 autoreactive T cells have different costimulatory requirements for their activation in vitro by testing the effect of a panel of Abs specific for various costimulatory molecules. Our results showed that CD8 interphotoreceptor retinoid-binding protein-specific T cells are more dependent on costimulatory molecules for activation than their CD4 counterparts. Interphotoreceptor retinoid-binding protein-specific T cells are less dependent on costimulatory molecules in the secondary response than the primary response. We also showed that blockade of costimulatory molecules can either promote or inhibit the proliferation of autoreactive T cells, depending on the degree of activation of the cells. Our results show that anti-costimulatory molecule treatment can have diverse actions on autoreactive T cell subsets, the net effect being determined by the subset of immune cells affected and the type and dose of treatment used.     

Naive CD8+ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells

Most current models of T cell activation postulate a requirement for two distinct signals. One signal is delivered through the TCR by engagement with peptide/MHC complexes, and the second is delivered by interaction between costimulatory molecules such as CD28 and its ligands CD80 and CD86. Soluble peptide/MHC tetramers provide an opportunity to test whether naive CD8+ T cells can be activated via the signal generated through the TCR-alphabeta in the absence of any potential costimulatory molecules. Using T cells from two different TCR transgenic mice in vitro, we find that TCR engagement by peptide/MHC tetramers is sufficient for the activation of naive CD8+ T cells. Furthermore, these T cells proliferate, produce cytokines, and differentiate into cytolytic effectors. Under the conditions where anti-CD28 is able to enhance proliferation of normal B6 CD4+, CD8+, and TCR transgenic CD8+ T cells with anti-CD3, we see no effect of anti-CD28 on proliferation induced by tetramers. The results of this experiment argue that given a strong signal delivered through the TCR by an authentic ligand, no costimulation is required.     

CD80 (B7-1) and CD86 (B7-2) are functionally equivalent in the initiation and maintenance of CD4+ T-cell proliferation after activation with suboptimal doses of PHA

Effective activation of T cells requires engagement of two separate T-cell receptors. The antigen-specific T-cell receptor (TCR) binds foreign peptide antigen-MHC complexes, and the CD28 receptor binds to the B7 (CD80/CD86) costimulatory molecules expressed on the surface of antigen-presenting cells (APC). The simultaneous triggering of these T-cell surface receptors with their specific ligands results in an activation of this cell. In contrast, CTLA-4 (CD152) is a distinct T-cell receptor that, upon binding to B7 molecules, sends an inhibitory signal to T cell activation. Many in vitro and in vivo studies demonstrated that both CD80 and CD86 ligands have an identical role in the activation of T cells. Recently, functions of B7 costimulatory molecules in vivo have been investigated in B7-1 and/or B7-2 knockout mice, and the authors concluded that CD86 could be more important for initiating T-cell responses, while CD80 could be more significant for maintaining these immune responses. In this study, we directly compared the role of CD80 and CD86 in initiating and maintaining proliferation of resting CD4(+) T cells in an in vitro mode system that allowed to provide the first signal-to-effector cells through the use of suboptimal doses of PHA and the second costimulatory signal through cells expressing CD80 or CD86, but not any other costimulatory molecules. Using this experimental system we demonstrate that the CD80 and CD86 molecules can substitute for each other in the initial activation of resting CD4(+) T cells and in the maintenance of their proliferative response.     

Therapeutic peptidomimetic strategies for autoimmune diseases: costimulation blockade.

Cognate interactions between immune effector cells and antigen-presenting cells (APCs) govern immune responses. Specific signals occur between the T-cell receptor peptide and APCs and nonspecific signals between pairs of costimulatory molecules. Costimulation signals are required for full T-cell activation and are assumed to regulate T-cell responses as well as other aspects of the immune system. As new discoveries are made, it is becoming clear how important these costimulation interactions are for immune responses. Costimulation requirements for T-cell regulation have been extensively studied as a way to control many autoimmune diseases and downregulate inflammatory reactions. The CD28:B7 and the CD40:CD40L families of molecules are considered to be critical costimulatory molecules and have been studied extensively. Blocking the interaction between these molecules results in a state of immune unresponsiveness termed 'anergy'. Several different strategies for blockade of these interactions are explored including monoclonal antibodies (mAbs), Fab fragments, chimeric, and/or fusion proteins. We developed novel, immune-specific approaches that interfere with these interactions. Using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis mediated by central nervous system (CNS)-specific T-cells, we developed a multi-targeted approach that utilizes peptides for blockade of costimulatory molecules. We designed blocking peptide mimics that retain the functional binding area of the parent protein while reducing the overall size and are thus capable of blocking signal transduction. In this paper, we review the role of costimulatory molecules in autoimmune diseases, two of the most well-studied costimulatory pathways (CD28/CTLA-4:B7 and CD40:CD40L), and the advantages of peptidomimetic approaches. We present data showing the ability of peptide mimics of costimulatory molecules to suppress autoimmune disease and propose a mechanism for disease suppression.     

Essential role for both CD80 and CD86 costimulation, but not CD40 interactions, in allergen-induced Th2 cytokine production from asthmatic bronchial tissue: role for alphabeta, but not gammadelta, T cells

CD80 and CD86 interact with CD28 and deliver costimulatory signals required for T cell activation. We demonstrate that ex vivo allergen stimulation of bronchial biopsy tissue from mild atopic asthmatic, but not atopic nonasthmatic, subjects induced production of IL-5, IL-4, and IL-13. Explants from both study groups did not produce IFN-gamma, but secreted the chemokine RANTES without any overt stimulation. In addition to allergen, stimulation of asthmatic explants with mAbs to CD3 and TCR-alphabeta but not TCR-gammadelta induced IL-5 secretion. Allergen-induced IL-5 and IL-13 production by the asthmatic tissue was inhibited by anti-CD80 and, to a lesser extent, by anti-CD86 mAbs. In contrast, the production of these cytokines by PBMCs was not affected by mAbs to CD80, was inhibited by anti-CD86, and was strongly attenuated in the presence of both Abs. FACS analysis revealed that stimulated asthmatic bronchial tissue was comprised of CD4+ T cells that expressed surface CD28 (75. 3%) but little CTLA-4 (4.0%). Neutralizing mAbs to CD40 ligand had no effect on the cytokine levels produced by asthmatic tissue or PBMCs. Collectively, these findings suggest that allergen-specific alphabeta T cells are resident in asthmatic bronchial tissue and demonstrate that costimulation by both CD80 and CD86 is essential for allergen-induced cytokine production. In contrast, CD86 appears to be the principal costimulatory molecule required in PBMC responses. Attenuation of type 2 alphabeta T cell responses in the bronchial mucosa by blocking these costimulatory molecules may be of therapeutic potential in asthma.