T-cell regulation by CD28 and CTLA-4

Activation of T lymphocytes is thought to require at least two signals, one delivered by the T-cell receptor complex after antigen recognition, and one provided on engagement of co-stimulatory receptors, such as CD28. Recent studies are providing clues as to the specific signalling roles of co-stimulatory receptors. Furthermore, superimposition of inhibitory signals, such as those delivered by cytotoxic T-lymphocyte antigen 4 (CTLA-4), leads to a complex network of positive and negative co-stimulatory signals, the integration of which modulates immune responses.     

Migration of Th1 lymphocytes is regulated by CD152 (CTLA-4)-mediated signaling via PI3 kinase-dependent Akt activation

Efficient adaptive immune responses require the localization of T lymphocytes in secondary lymphoid organs and inflamed tissues. To achieve correct localization of T lymphocytes, the migration of these cells is initiated and directed by adhesion molecules and chemokines. It has recently been shown that the inhibitory surface molecule CD152 (CTLA-4) initiates Th cell migration, but the molecular mechanism underlying this effect remains to be elucidated. Using CD4 T lymphocytes derived from OVA-specific TCR transgenic CD152-deficient and CD152-competent mice, we demonstrate that chemokine-triggered signal transduction is differentially regulated by CD152 via phosphoinositide 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt). In the presence of CD152 signaling, the chemoattractant CCL4 selectively induces the full activation of Akt via phosphorylation at threonine 308 and serine 473 in pro-inflammatory Th lymphocytes expressing the cognate chemokine receptor CCR5. Akt signals lead to cytoskeleton rearrangements, which are indispensable for migration. Therefore, this novel Akt-modulating function of CD152 signals affecting T cell migration demonstrates that boosting CD152 or its down-stream signal transduction could aid therapies aimed at sensitizing T lymphocytes for optimal migration, thus contributing to a precise and effective immune response.     

CTLA-4 overexpression inhibits T cell responses through a CD28-B7-dependent mechanism

CTLA-4 has been shown to be an important negative regulator of T cell activation. To better understand its inhibitory action, we constructed CTLA-4 transgenic mice that display constitutive cell surface expression of CTLA-4 on CD4 and CD8 T cells. In both in vivo and in vitro T cell responses, CTLA-4 overexpression inhibits T cell activation. This inhibition is dependent on B7 and CD28, suggesting that overexpressed CTLA-4 inhibits responses by competing with CD28 for B7 binding or by interfering with CD28 signaling. In addition, expression of the transgene decreases the number of CD25+Foxp3+ T cells in these mice, but does not affect their suppressive ability. Our data confirm the activity of CTLA-4 as a negative regulator of T cell activation and that its action may be by multiple mechanisms.     

CTLA-4 and CD28 mRNA are coexpressed in most T cells after activation. Expression of CTLA-4 and CD28 mRNA does not correlate with the pattern of lymphokine production.

Ag-presenting cells provide at least two distinct signals for T cell activation. T cell receptor-dependent stimulation is provided by presentation of a specific peptide Ag in association with MHC molecules. In addition, APC also supply costimulatory signals required for T cell activation that are neither Ag- nor MHC restricted. One such costimulatory signal is mediated via the interaction of B7 on APC with the CD28 receptor on T cells. Recently, CTLA-4 has been shown to be a second B7 receptor on T cells. In the present report, we have examined the expression of CD28 and CTLA-4 on a panel of resting and activated normal T cell subsets and T cell clones by RNA blot analysis in an attempt to determine whether their expression defines reciprocal or overlapping subsets. CD28 was detected in resting T cells, whereas CTLA-4 was not. After stimulation with PHA and PMA for 24 h, CTLA-4 mRNA was expressed in both the CD4+ and CD8+ subsets as well as in CD28+ T cells. We examined 37 human and six murine T cell clones that had been previously characterized for their cytokine production. After activation, CTLA-4 and CD28 mRNA were coexpressed in 36 of 37 human T cell clones and all six murine T cell clones. These included T cells of CD4+8-, CD4-8+, and CD4-8- phenotypes as well as clones with Th1 and Th2 cytokine profiles. In contrast, CD28 but not CTLA-4 mRNA was detected in leukemic T cell lines and myelomas. CTLA-4 and B7 mRNA but not CD28 mRNA was detected in two long term HTLV-I-transformed T cell lines. These data demonstrate that CD28 and CTLA-4 mRNA are coexpressed in most activated T cells and T cell clones, providing evidence that they do not define reciprocal subsets. Moreover, they are consistent with the hypothesis that B7 transmits its signal through a single receptor, CD28, on resting T cells, and multiple receptors, CD28 and CTLA-4, on activated T cells.     

TGF-beta-mediated suppression by CD4+CD25+ T cells is facilitated by CTLA-4 signaling

CD4+CD25+ T cells play a pivotal role in immunological homeostasis by their capacity to exert immunosuppressive activity. However, the mechanism by which these cells function is still a subject for debate. We previously reported that surface (membrane) TGF-beta produced by CD4+CD25+ T cells was an effector molecule mediating suppressor function. We now support this finding by imaging surface TGF-beta on Foxp3+CD4+CD25+ T cells in confocal fluorescence microscopy. Then, using a TGF-beta-sensitive mink lung epithelial cell (luciferase) reporter system, we show that surface TGF-beta can be activated to signal upon cell-cell contact. Moreover, if such TGF-beta signaling is blocked in an in vitro assay of CD4+CD25+ T cell suppression by a specific inhibitor of TGF-betaRI, suppressor function is also blocked. Finally, we address the role of CTLA-4 in CD4+CD25+ T cell suppression, showing first that whereas anti-CTLA-4 does not block in vitro suppressor function, it does complement the blocking activity of anti-TGF-beta. We then show with confocal fluorescence microscopy that incubation of CD4+CD25+ T cells with anti-CTLA-4- and rB7-1/Fc-coated beads results in accumulation of TGF-beta at the cell-bead contact site. This suggests that CTLA-4 signaling facilitates TGF-beta-mediated suppression by intensifying the TGF-beta signal at the point of suppressor cell-target cell interaction.     

Differential requirement for CD28/CTLA-4-CD80/CD86 interactions in drug-induced type 1 and type 2 immune responses to trinitrophenyl-ovalbumin

The use of mAbs to abrogate costimulatory interactions has attracted much attention with regard to prevention and modulation of adverse (auto)immune-like reactions. However, the role of costimulatory molecules and possible therapeutic use of Ab-treatment in drug-induced immunostimulation is poorly elucidated. In the present studies, we show that CD28/CTLA-4-CD80/CD86 costimulatory interactions differently regulate drug-induced type 1 and type 2 responses to an identical bystander Ag, TNP-OVA, in BALB/c mice using the reporter Ag popliteal lymph node assay. The antirheumatic drug D-Penicillamine, which may induce lupus-like side-effects, stimulated type 2 responses against TNP-OVA, characterized by the production of IL-4 and TNP-specific IgG1 and IgE. These responses were abrogated in CD80/CD86-deficient mice and in wild-type mice that were treated with anti-CD80 and anti-CD86, or CTLA-4-Ig. Anti-CTLA-4 intensively enhanced the D-Penicillamine-induced effects. In contrast, the type 1 response (IFN-gamma, TNF-alpha, IgG2a) to TNP-OVA induced by the diabetogen streptozotocin still developed in the absence of CD80/CD86 costimulatory signaling. In addition, it was demonstrated that coadministration of anti-CD80 and anti-CD86 mAbs slightly enhanced streptozotocin-induced type 1 responses, whereas the CTLA-4-Ig fusion protein completely abrogated this response. In conclusion, different drugs may stimulate distinct types of immune responses against an identical bystander Ag, which are completely dependent on (type 2) or independent of (type 1) the CD28/CTLA-4-CD80/CD86 pathway. Importantly, the effects of treatment with anti-CD80/CD86 mAbs and CTLA-4-Ig may be considerably different in responses induced by distinct drugs.     

Polarization of naive CD4+ T cells toward the Th1 subset by CTLA-4 costimulation

In this study, we examined in vitro the role of CTLA-4 costimulation in the polarization of naive CD4+ T cells toward the Th1 subset. When CTLA-4 costimulation was blocked by the inclusion of anti-CTLA-4 Fab in cultures during priming of naive CD4+ T cells with anti-CD3 in the presence of splenic adherent cells, they were polarized toward the Th2 subset. Conversely, the engagement of CTLA-4 with immobilized anti-CTLA-4 or with CD80-P815 cells polarized naive CD4+ T cells costimulated with anti-CD3 and anti-CD28 toward the Th1 subset. The CTLA-4 costimulation during priming augmented TGF-beta1 mRNA accumulation in naive CD4+ T cells, and the inclusion of anti-TGF-beta in cultures for priming suppressed the effect of CTLA-4 costimulation on the Th1 polarization. The addition of low doses of TGF-beta1 in cultures for priming of naive CD4+ T cells enhanced the production of Th1 cytokines upon secondary stimulation, although Th2 cytokine production was not affected by the doses of TGF-beta1. The CTLA-4 costimulation was also shown to suppress IL-4 production of naive CD4+ T cells upon priming. These results indicate that the costimulation against CTLA-4 drives polarization of naive CD4+ T cells toward the Th1 subset independent of IL-12 through, at least in part, the enhancement of TGF-beta1 production, and it also hampers Th2 subset differentiation by affecting IL-4 production of naive CD4+ T cells.     

Regulation of cell surface expression of CTLA-4 by secretion of CTLA-4-containing lysosomes upon activation of CD4+ T cells

CTLA-4 is expressed on the surface of activated T cells and negatively regulates T cell activation. Because a low-level expression of CTLA-4 on the cell surface is sufficient to induce negative signals in T cells, the surface expression of CTLA-4 is strictly regulated. We previously demonstrated that the association of CTLA-4 with the clathrin-associated adaptor complex AP-2 induces internalization of CTLA-4 and keeps the surface expression low. However, the mechanism to induce high expression on the cell surface upon stimulation has not yet been clarified. To address this, we investigated the intracellular dynamics of CTLA-4 by analyzing its localization and trafficking in wild-type and mutant CTLA-4-transfected Th1 clones. CTLA-4 is accumulated in intracellular granules, which we identified as lysosomes. CTLA-4 is degraded in lysosomes in a short period, and the degradation process may serve as one of the mechanisms to regulate CTLA-4 expression. Upon TCR stimulation, CTLA-4-containing lysosomes are secreted as proven by the secretion of cathepsin D and beta-hexosaminidase in parallel with the increase of surface expression of CTLA-4 and lysosomal glycoprotein 85, a lysosomal marker. These results suggest that the cell surface expression of CTLA-4 is up-regulated upon stimulation by utilizing a mechanism of secretory lysosomes in CD4(+)T cells.     

Coordinate regulation of T cell activation by CD2 and CD28

T cell activation requires co-engagement of the TCR with accessory and costimulatory molecules. However, the exact mechanism of costimulatory function is unknown. Mice lacking CD2 or CD28 show only mild deficits, demonstrating that neither protein is essential for T cell activation. In this paper we have generated mice lacking both CD2 and CD28. T cells from the double-deficient mice have a profound defect in activation by soluble anti-CD3 Ab and Ag, yet remain responsive to immobilized anti-CD3. This suggests that CD2 and CD28 may function together to facilitate interactions of the T cell and APC, allowing for efficient signal transduction through the TCR.     

The role of CTLA-4 in regulating Th2 differentiation.

To examine the role of CTLA-4 in Th cell differentiation, we used two newly generated CTLA-4-deficient (CTLA-4-/-) mouse strains: DO11. 10 CTLA-4-/- mice carrying a class II restricted transgenic TCR specific for OVA, and mice lacking CTLA-4, B7.1 and B7.2 (CTLA-4-/- B7.1/B7.2-/- ). When purified naive CD4+ DO11.10 T cells from CTLA-4-/- and wild-type mice were primed and restimulated in vitro with peptide Ag, CTLA-4-/- DO11.10 T cells developed into Th2 cells, whereas wild-type DO11.10 T cells developed into Th1 cells. Similarly, when CTLA-4-/- CD4+ T cells from mice lacking CTLA-4, B7. 1, and B7.2 were stimulated in vitro with anti-CD3 Ab and wild-type APC, these CTLA-4-/- CD4+ T cells produced IL-4 even during the primary stimulation, whereas CD4+ cells from B7.1/B7.2-/- mice did not produce IL-4. Upon secondary stimulation, CD4+ T cells from CTLA-4-/- B7.1/B7.2-/- mice secreted high levels of IL-4, whereas CD4+ T cells from B7.1/B7.2-/- mice produced IFN-gamma. In contrast to the effects on CD4+ Th differentiation, the absence of CTLA-4 resulted in only a modest effect on T cell proliferation, and increased proliferation of CTLA-4-/- CD4+ T cells was seen only during secondary stimulation in vitro. Administration of a stimulatory anti-CD28 Ab in vivo induced IL-4 production in CTLA-4-/- B7.1/B7.2-/- but not wild-type mice. These studies demonstrate that CTLA-4 is a critical and potent inhibitor of Th2 differentiation. Thus, the B7-CD28/CTLA-4 pathway plays a critical role in regulating Th2 differentiation in two ways: CD28 promotes Th2 differentiation while CTLA-4 limits Th2 differentiation.     

Established T cell-driven germinal center B cell proliferation is independent of CD28 signaling but is tightly regulated through CTLA-4

CD4 T cell activation is positively (CD28) and negatively (CTLA-4) regulated by the costimulatory ligands CD80 and CD86. A central question is how the balance between these two opposing forces is controlled as T cells differentiate. We have previously shown that CD28 signaling is absolutely required to prime naive CD4 T cells to differentiate into effectors that provide help for germinal centers and class-switched Ab responses. In this study, we show that the requirement for CD28 signaling is transient and effector CD4 T cells do not require CD28 signals to sustain their function. The CD28 independence of effector T cells within germinal centers suggested that a key function for CD80/CD86 under these circumstances might be to provide negative regulatory signals via the CD28 homologue CTLA-4. By examining germinal center responses in mice where the ability to signal through T cell CTLA-4 was compromised, we provide data that supports a critical role for CTLA-4 in down-regulating T cell help for germinal center B cells.     

CD28, Ox-40, LFA-1, and CD4 modulation of Th1/Th2 differentiation is directly dependent on the dose of antigen

The involvement of specific accessory/costimulatory molecules in differentiation to Th1 and Th2 phenotypes is controversial. Reports suggest that molecules such as CD4, CD28, and Ox-40 support Th2 differentiation and suppress Th1 differentiation, whereas others such as LFA-1 support Th1 responses and suppress Th2 responses. We have previously defined an in vitro model of differentiation that is absolutely dependent on the initial dose and affinity of peptide presented to a naive CD4 cell. The dose and affinity of Ag regulate autocrine production of IL-2, IL-4, and IFN-gamma, which in turn govern differentiation to Th1 and Th2 phenotypes. We have used this system to confirm that CD4, CD28, and Ox-40 interactions can promote, and LFA-1 interactions can suppress, differentiation of cells secreting the Th2 cytokines IL-5 and IL-13. However, for CD4 and LFA-1, this is only seen over a certain range of peptide doses. In addition, CD28 and Ox-40 interactions also promote Th1 differentiation. In general, agonist Abs to accessory molecules shifted the response curves for IFN-gamma, IL-5, and IL-13 to lower doses, whereas antagonist reagents resulted in similar curves shifted toward the higher doses. We conclude that ligation of cell surface accessory receptors enables low doses of Ag to promote responses normally induced only by higher doses. Individual receptors do not intrinsically regulate one cytokine phenotype or another, suggesting that differentiation is controlled by the level of expression of multiple accessory molecule pairs integrated with the number and affinity of peptide/MHC complexes.     

Differences in allergen-induced T cell activation between allergic asthma and rhinitis: Role of CD28, ICOS and CTLA-4

Background

Th2 cell activation and T regulatory cell (Treg) deficiency are key features of allergy. This applies for asthma and rhinitis. However with a same atopic background, some patients will develop rhinitis and asthma, whereas others will display rhinitis only. Co-receptors are pivotal in determining the type of T cell activation, but their role in allergic asthma and rhinitis has not been explored. Our objective was to assess whether allergen-induced T cell activation differs from allergic rhinitis to allergic rhinitis with asthma, and explore the role of ICOS, CD28 and CTLA-4.

Methods

T cell co-receptor and cytokine expressions were assessed by flow cytometry in PBMC from 18 house dust mite (HDM) allergic rhinitics (R), 18 HDM allergic rhinitics and asthmatics (AR), 13 non allergic asthmatics (A) and 20 controls, with or without anti-co-receptors antibodies.

Results

In asthmatics (A+AR), a constitutive decrease of CTLA-4+ and of CD4+CD25+Foxp3+ cells was found, with an increase of IFN-γ+ cells. In allergic subjects (R + AR), allergen stimulation induced CD28 together with IL-4 and IL-13, and decreased the proportion of CTLA-4+, IL-10+ and CD4+CD25+Foxp3+ cells. Anti-ICOS and anti-CD28 antibodies blocked allergen-induced IL-4 and IL-13. IL-13 production also involved CTLA-4.

Conclusions

T cell activation differs between allergic rhinitis and asthma. In asthma, a constitutive, co-receptor independent, Th1 activation and Treg deficiency is found. In allergic rhinitis, an allergen-induced Treg cell deficiency is seen, as well as an ICOS-, CD28- and CTLA-4-dependent Th2 activation. Allergic asthmatics display both characteristics.     

Differential regulation of Th1 and Th2 functions of NKT cells by CD28 and CD40 costimulatory pathways

Valpha14 NKT cells produce large amounts of IFN-gamma and IL-4 upon recognition of their specific ligand alpha-galactosylceramide (alpha-GalCer) by their invariant TCR. We show here that NKT cells constitutively express CD28, and that blockade of CD28-CD80/CD86 interactions by anti-CD80 and anti-CD86 mAbs inhibits the alpha-GalCer-induced IFN-gamma and IL-4 production by splenic Valpha14 NKT cells. On the other, the blockade of CD40-CD154 interactions by anti-CD154 mAb inhibited alpha-GalCer-induced IFN-gamma production, but not IL-4 production. Consistent with these findings, CD28-deficient mice showed impaired IFN-gamma and IL-4 production in response to alpha-GalCer stimulation in vitro and in vivo, whereas production of IFN-gamma but not IL-4 was impaired in CD40-deficient mice. Moreover, alpha-GalCer-induced Th1-type responses, represented by enhanced cytotoxic activity of splenic or hepatic mononuclear cells and antimetastatic effect, were impaired in both CD28-deficient mice and CD40-deficient mice. In contrast, alpha-GalCer-induced Th2-type responses, represented by serum IgE and IgG1 elevation, were impaired in the absence of the CD28 costimulatory pathway but not in the absence of the CD40 costimulatory pathway. These results indicate that CD28-CD80/CD86 and CD40-CD154 costimulatory pathways differentially contribute to the regulation of Th1 and Th2 functions of Valpha14 NKT cells in vivo.     

Costimulation light: activation of CD4+ T cells with CD80 or CD86 rather than anti-CD28 leads to a Th2 cytokine profile

To examine the role of CD28 and CTLA-4 in Th cell differentiation, we used a novel microsphere-based system to compare the effects of CD28 ligation by Ab or CD80/CD86. One set of beads was prepared by coating with anti-CD3 and anti-CD28 Ab. Another set of beads was prepared by immobilizing anti-CD3 and murine CD80-Ig fusion protein or murine CD86-Ig fusion protein on the beads. The three sets of beads were compared in their effects on the ability to activate and differentiate splenic CD4 T cells. When purified naive CD4(+) cells were stimulated in vitro, robust proliferation of similar magnitude was induced by all three sets of beads. When cytokine secretion was examined, all bead preparations induced an equivalent accumulation of IL-2. In contrast, there was a marked difference in the cytokine secretion pattern of the Th2 cytokines IL-4, IL-10, and IL-13. The B7-Ig-stimulated cultures had high concentrations of Th2 cytokines, whereas there were low or undetectable concentrations in the anti-CD28-stimulated cultures. Addition of anti-CTLA-4 Fab augmented B7-mediated IL-4 secretion. These studies demonstrate that B7 is a critical and potent stimulator of Th2 differentiation, and that anti-CD28 prevents this effect.     

CD28 costimulation accelerates IL-4 receptor sensitivity and IL-4-mediated Th2 differentiation.

The development of Th1 and Th2 cells is determined by the type of antigenic stimulation involved in the initial cell activation step. Evidence indicates that costimulatory signals, such as those delivered by CD28, play an important role in Th2 development, but little is known about how CD28 costimulation contributes to Th2 development. In this study, TCR cross-linking was insufficient for Th2 development, while the addition of CD28 costimulation drastically increased Th2 generation through the IL-4-mediated pathway. Th2 generation following CD28 costimulation was not simply explained by the enhancement of IL-4 production in naive T cells. To generate Th2 cells after TCR cross-linking only, it was necessary to add a 20- to 200-fold excess of IL-4 generated after TCR and CD28 stimulation. TCR cross-linking increased the expression level and binding property of the IL-4R, but enhanced the sensitivity to IL-4 only slightly. In contrast, as evidenced by the enhanced phosphorylation of Jak3, the IL-4Ralpha-chain, and STAT6 following IL-4 stimulation, CD28 costimulation increased IL-4R sensitivity without affecting its expression and binding property. This evidence of the enhancement of IL-4R sensitivity increases our understanding of how CD28 costimulation accelerates Th2 development.