Cutting edge: regulation of T cell activation threshold by CD28 costimulation through targeting Cbl-b for ubiquitination

Optimal T cell activation requires signaling through the TCR and CD28 costimulatory receptor. CD28 costimulation is believed to set the threshold for T cell activation. Recently, Cbl-b, a ubiquitin ligase, has been shown to negatively regulate CD28-dependent T cell activation. In this report, we show that CD28 costimulation selectively induces greater ubiquitination and degradation of Cbl-b in wild-type T cells than CD3 stimulation alone, and TCR-induced Cbl-b ubiquitination and degradation are significantly reduced in CD28-deficient T cells. Stimulation of CD28-deficient T cells with higher doses of anti-CD3 results in increased ubiquitination of Cbl-b, which correlates with enhanced T cell responses. Our results demonstrate that CD28 costimulation regulates the threshold for T cell activation, at least in part, by promoting Cbl-b ubiquitination and degradation.     

The roles of CD28 and CD40 ligand in T cell activation and tolerance

Costimulation of T cell activation involves both the B7:CD28 as well as the CD40 ligand (CD40L):CD40 pathway. To determine the importance of these pathways to in vitro and in vivo T cell activation, a direct comparison was made of the responses of TCR transgenic T cells lacking either CD28 or CD40L. In vitro, CD28-/- T cells showed a greater reduction in proliferative responses to Ag than did CD40L-/- T cells. The absence of CD28 resulted in defective Th2 responses, whereas CD40L-/- T cells were defective in Th1 development. In vivo, CD28-/- T cells failed to expand upon immunization, whereas CD40L-/- T cells could not sustain a response. These results suggest that CD28 is critical for initiating T cell responses, whereas CD40L is required for sustained Th1 responses. The different functional roles of these costimulatory pathways may explain why blocking B7:CD28 and CD40L:CD40 interactions has an additive effect in inhibiting T cell responses.     

Mechanism of peripheral T cell activation by coengagement of CD44 and CD2.

A number of CD44 antibodies are known to augment peripheral T cell proliferation stimulated with suboptimal concentrations of activating pairs of CD2 mAb. These findings have implicated the CD44 adhesion receptor in the activation of peripheral T cells via CD2. We have investigated early events after CD44 and CD2 coengagement on peripheral T cells. CD44 and CD2 coengagement resulted in enhanced [Ca2+]i mobilization. However, the increase in [Ca2+]i mobilization did not occur until at least 3 min after CD2 and CD44 coengagement, suggesting that other events precede the elevation in [Ca2+]i. Using a T cell/fibroblast adhesion assay, we could demonstrate a dramatic increase in T cell adhesiveness after about 1 min after CD44 and CD2 coengagement. The increase in T cell adhesiveness was comparable to that induced by PMA. In the absence of antibodies or treatment with mAb directed to other T cell surface Ag, there was little if any adhesion between unstimulated peripheral T cells and fibroblasts. Enhancement of T cell adhesiveness through CD44 engagement was not mediated by a direct effect on lymphocyte-function associated Ag-3, the known ligand of CD2. However, cross-linking of CD44 resulted in epitopic modulation of CD2 as demonstrated by the increased expression of the T11(3) activation epitope. Furthermore, anti-CD44 could substitute for anti-T11(2) in the activation of peripheral T cells via CD2. These results suggest that CD44 ligation has profound effects on CD2-mediated events by inducing epitopic modulation of CD2.     

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.     

Negative regulation of T cell homeostasis by lymphocyte activation gene-3 (CD223)

Lymphocyte homeostasis is a central biological process that is tightly regulated. However, its molecular and cellular control is poorly understood. We show that aged mice deficient in lymphocyte activation gene 3 (LAG-3), an MHC class II binding CD4 homologue, have twice as many T cells as wild-type controls. CD4(+) and CD8(+) LAG-3-deficient T cells showed enhanced homeostatic expansion in lymphopenic hosts, which was abrogated by ectopic expression of wild-type LAG-3, but not by a signaling-defective mutant. In addition, in vivo treatment with anti-LAG-3 mAb resulted in enhanced T cell expansion to a level comparable to that in LAG-3-deficient cells. This deregulation of T cell homeostasis also resulted in the expansion of multiple cell types, including B cells, macrophages, granulocytes, and dendritic cells. Lastly, regulatory T cells were dependent on LAG-3 for their optimal control of T cell homeostasis. Our data suggest that LAG-3 negatively regulates T cell homeostasis by regulatory T cell-dependent and independent mechanisms.     

CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation.

We investigate, here, the mechanism of the costimulatory signals for CD8 T cell activation and confirm that costimulation signals via CD28 do not appear to be required to initiate proliferation, but provide survival signals for CD8 T cells activated by TCR ligation. We show also that IL-6 and TNF-alpha can provide alternative costimulatory survival signals. IL-6 and TNF-alpha costimulate naive CD8 T cells cultured on plate-bound anti-CD3 in the absence of CD28 ligation. They act directly on sorted CD8-positive T cells. They also costimulate naive CD8 T cells from Rag-2-deficient mice, bearing transgenic TCRs for HY, which lack memory cells, a potential source of IL-2 secretion upon activation. IL-6 and TNF-alpha provide costimulation to naive CD8 T cells from CD28, IL-2, or IL-2Ralpha-deficient mice, and thus function in the absence of the B7-CD28 and IL-2 costimulatory pathways. The CD8 T cell generated via the anti-CD3 plus IL-6 and TNF-alpha pathway have effector function in that they express strong cytolytic activity on Ag-specific targets. They secrete only very small amounts of any of the cytokines tested upon restimulation with peptide-loaded APC. The ability of the naive CD8 T cells to respond to TCR ligation and costimulatory signals from IL-6 and TNF-alpha provides a novel pathway that can substitute for signals from CD4 helper cells or professional APC. This may be significant in the response to viral Ags, which can be potentially expressed on the surface of any class I MHC-expressing cell.     

Dynamic regulation of T cell immunity by CD43

During a viral response, Ag-specific effector T cells show dramatically increased binding by the mAb 1B11 and the lectin peanut agglutinin (PNA). We investigated the contribution of CD43 expression to 1B11 and PNA binding as well as its role in generation and maintenance of a CD8 T cell response. Analysis of CD43(-/-) mice revealed no increased 1B11 binding and reduced PNA binding on virus-specific CD8 T cells from -/- mice compared with +/+ mice. Furthermore, we examined the role of CD43 in the kinetics of an immune response. We show that CD43 expression modestly effects generation of a primary virus-specific CD8 T cell response in vivo but plays a more significant role in trafficking of CD8 T cells to tissues such as the brain. More interestingly, CD43 plays a role in the contraction of the immune response, with CD43(-/-) mice showing increased numbers of Ag-specific CD8 T cells following initial expansion. Following the peak of expansion, Ag-specific CD8 T cells from -/- mice show similar proliferation but demonstrate increased Bcl-2 levels and decreased apoptosis of Ag-specific effector CD8 T cells in vitro. Consistent with a delay in the down-modulation of the immune response, following chronic viral infection CD43(-/-) mice show increased morbidity. These data suggest a dynamic role of CD43 during an immune response: a positive regulatory role in costimulation and trafficking of T cells to the CNS and a negative regulatory role in the down-modulation of an immune response.     

CD28 is required for T cell activation and IFN-gamma production by CD4+ and CD8+ T cells in response to Trypanosoma cruzi infection

In the present study we evaluated the mechanisms behind the implication of the costimulatory molecule CD28 for the immune response against the intracellular protozoan parasite Trypanosma cruzi. Our results reveal a critical role for CD28 in the activation of both CD4+ and CD8+ T cells and induction of the effector mechanisms that ultimately mediate the control of parasite growth and pathogenesis in infected mice. CD28-deficient (CD28-/-) mice are highly susceptible to T. cruzi infection, presenting higher parasitemia and tissue parasitism, but less inflammatory cell infiltrate in the heart than C57Bl/6 wild-type (WT) mice. All the infected WT mice survived acute infection, whereas 100% of CD28-/- mice succumbed to it. The increased susceptibility of the CD28-/- mice was associated with a dramatic decrease in the production of IFN-gamma by both CD4+ and CD8+ T cells resulting in a diminished capacity to produce nitric oxide (NO) and mediate parasite killing. T cell activation was also profoundly impaired in CD28-/- mice, which presented decreased lymphoproliferative response after the infection compared to WT mice. Together, these data represent the first evidence that CD28 is critical for efficient CD4+ T cell activation in response to T. cruzi infection in mice.     

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.     

CD2 can mediate TCR/CD3-independent T cell activation.

T lymphocytes can be activated clonotypically through TCR/CD3 complex or polyclonally via the CD2 molecule. Whether CD2-mediated activation is dependent on TCR/CD3 expression or signaling is controversial. We have re-explored this issue by using a series of CD2-transfected, TCR/CD3 surface membrane-negative human and mouse T cells. Our results clearly show that such T cells can be triggered for IL-2 secretion and increases in intracellular Ca2+ through the CD2 molecule in the absence of surface expression of TCR/CD3 complexes. These responses are only observed when cells express high levels of CD2 and there is a critical threshold of CD2 expression necessary for such activation in the absence of CD3. Concomitant expression of TCR/CD3 complex markedly lowers the level of CD2 required for activation via the latter pathway. These results provide a clear resolution of the controversy concerning the requirement for surface CD3 expression in T cell activation through CD2 and further suggest a possible role for CD2 in activation of TCR/CD3-negative cells.     

Glucose uptake is limiting in T cell activation and requires CD28-mediated Akt-dependent and independent pathways

T cell activation potently stimulates cellular metabolism to support the elevated energetic and biosynthetic demands of growth, proliferation, and effector function. We show that glucose uptake is limiting in T cell activation and that CD28 costimulation is required to allow maximal glucose uptake following TCR stimulation by up-regulating expression and promoting the cell surface trafficking of the glucose transporter Glut1. Regulation of T cell glucose uptake and Glut1 was critical, as low glucose prevented appropriate T cell responses. Additionally, transgenic expression of Glut1 augmented T cell activation, and led to accumulation of readily activated memory-phenotype T cells with signs of autoimmunity in aged mice. To further examine the regulation of glucose uptake, we analyzed CD28 activation of Akt, which appeared necessary for maximal glucose uptake of stimulated cells and which we have shown can promote Glut1 cell surface trafficking. Consistent with a role for Akt in Glut1 trafficking, transgenic expression of constitutively active myristoylated Akt increased glucose uptake of resting T cells, but did not alter Glut1 protein levels. Therefore, CD28 appeared to promote Akt-independent up-regulation of Glut1 and Akt-dependent Glut1 cell surface trafficking. In support of this model, coexpression of Glut1 and myristoylated Akt transgenes resulted in a synergistic increase in glucose uptake and accumulation of activated T cells in vivo that were largely independent of CD28. Induction of Glut1 protein and Akt regulation of Glut1 trafficking are therefore separable functions of CD28 costimulation that cooperate to promote glucose metabolism for T cell activation and proliferation.     

Soluble CD8 during T cell activation

The CD8 Ag has long been used as a surface marker for the identification of cytotoxic and suppressor cells. Recently CD8-positive cells have been shown to release a soluble form of the CD8 Ag. We have devised a sandwich monoclonal enzyme immunoassay for the quantitation of this released CD8. Soluble CD8 was released in response to lymphocyte activation. In vitro, PHA or anti-CD3 mAb-mediated T cell activation led to release of CD8 into the culture supernatant. In vivo, serum from patients with EBV-induced infectious mononucleosis (IM), a disease associated with intense CD8+ T cell activation, demonstrated elevations in soluble CD8 (7939 U/ml, day 0) compared to serum from normal controls (289 U/ml). Levels of soluble CD8 correlated (r = 0.82, p less than 0.001) with the increased percentage of CD8+/HLA-DR+ (activated CD8+ T cells) observed in acute IM. Sequential analysis of serum during the course of IM shows that soluble CD8 levels parallel the decline in CD8+/HLA-DR+ cells that occurs with the resolution of the disease. These data suggest that released CD8 may be of value in monitoring the involvement of CD8+ T cells in response to a pathologic event. The functional role of the released CD8 molecule will require further investigation.     

CD44 contributes to T cell activation

We demonstrate here that the CD44 molecule, which mediates lymphocyte adhesion to high endothelial venules (HEV), is also involved in the delivery of an activation signal to the T cell. We have produced a CD44 mAb (H90) which is able to block the binding of lymphocytes to high endothelial venules. H90 had no effect on [3H]TdR incorporation of whole PBL stimulated by lectins, allogeneic cells, or CD3 mAb in the soluble phase; in contrast, it strongly increased [3H]TdR incorporation of PBL stimulated by CD2 pairs of mAb or by CD3 mAb linked to the plastic culture plates, when purified T cells were used, H90 mAb could efficiently induce them to proliferate after a primary signal of activation delivered via cross-linked CD3 or via CD2, an effect mediated by Il-2 synthesis and Il-2R expression. Thus, the effect of H90 mAb resembles the mitogenic effect of CD28 "9.3" mAb. However, several results show that CD28 and CD44 mediate different signals to the T cells: i) in contrast to CD28 mAb, CD44 mAb cannot complement the signal delivered by a soluble CD3 mAb, lectins, or PMA; ii) CD44 mAb, at the difference of CD28 mAb, cannot induce CD3+ thymocytes to proliferate in conjunction with a first signal provided via cross-linked CD3 or via CD2; iii) F(ab) fragments of H90 were efficient, whereas divalent fragments of CD29 9.3 mAb are required to produce activation signals; and iv) CD44 and CD28 mAb produce a very strong synergistic effect on T cell proliferation. These results fit with previous ones showing that endothelial cells can play the role of accessory cell in T cell activation and that a hierarchy of signaling can be delivered to T cells via CD3 and CD2.     

Negative regulation of T cell activation by placental protein 14 is mediated by the tyrosine phosphatase receptor CD45

CD45 is the major protein tyrosine phosphatase receptor on T cell surfaces that functions as both a positive and a negative regulator of T cell receptor (TCR) signaling. Although CD45 is required for the activation of TCR-associated Src family kinases, it also dephosphorylates phosphoproteins involved in the TCR-signaling cascade. This study links CD45 to the inhibitory activity of placental protein 14 (PP14), a major soluble protein of pregnancy that is now known to be a direct modulator of T cells and to function by desensitizing TCR signaling. PP14 and CD45 co-capped with each other, pointing to a physical linkage between the two. Interestingly, however, the binding of PP14 to T cell surfaces was not restricted to CD45 alone, with evidence showing that PP14 binds to other surface molecules in a carbohydrate-dependent fashion. Notwithstanding the broader molecular binding potential of PP14, its interaction with CD45 appeared to have special functional significance. Using transfected derivatives of the HPB.ALL mutant T cell line that differ in CD45 expression, we established that the inhibitory effects of PP14 are dependent upon the expression of intact CD45 on T cell surfaces. Based upon these findings, we propose a new immunoregulatory model for PP14, wherein one of its surface molecular targets, CD45, mediates its T cell inhibitory activity, accounting for the intriguing capacity of PP14 to elevate TCR activation thresholds and thereby down-regulate T cell activation.