The mechanisms by which CD28 and CTLA-4 transmit their respective signals are not well understood. Despite their opposing roles in T cell function, both molecules share some basic features. It has been shown that not only do CD28 and CTLA-4 compete for the ligands CD80 and CD86 [22, 23] but both also initiate signalling pathways. However, both molecules lack intrinsic catalytic activity in their cytoplasmic tails and they therefore require association with further signalling molecules. Despite their opposing functions during T cell responses, CD28 and CTLA-4 interact with identical signalling molecules: the phosphoinositide 3-kinase (PI-3K) and the protein phosphatase 2A (PP2A) (Fig. 1) [31–34]. However, the functional relevance and consequences of these shared properties are not well understood.
It is still controversial whether CD28 transmits a unique signal or only amplifies TCR signals. After engagement of CD28 by its ligand, tyrosine residues in the cytoplasmic tail of CD28 become phosphorylated by Src-family kinases , leading to the binding of PI-3K to CD28 [31, 32]. Additionally, CD28 triggering induces the phosphorylation and activation of the kinases Tec and Itk [36, 37] as well as other signalling molecules such as the guanine-nucleotide-exchange factor Vav-1 or phospholipase Cγ1 . All of these molecules are also activated by TCR signalling, so CD28 might only be an amplifier. A unique signal could arise from the dependence of full phospholipase Cγ1 activation on a signal provided by CD28 that involves PI-3K, Vav-1, and the adapter molecule SLP-76 .
In another model, CD28 sets the threshold for T cell activation and amplifies the TCR signal by enhancing the recruitment of lipid rafts to the plasma membrane [40, 41]. In resting/naive cells, lipid rafts are stored in intracellular vesicles and are redistributed to the plasma membrane after stimulation. This redistribution is strongly enhanced by CD28 and facilitates the full signal leading to T cell activation. However, the signal required for raft relocalization is unknown at present.
Indications for both the quantitative and the qualitative signal mediated by CD28 can be derived from the analysis of gene expression after stimulation with TCR alone, CD28 alone, or a combination of TCR and CD28 . This study shows that CD28 acts primarily as a signal amplifier of TCR signalling but also leads to the activation of a few, though important, distinct genes (such as CD69 and tumor necrosis factor).
Like CD28, CTLA-4 becomes phosphorylated on tyrosine residues after stimulation, which is mediated by Src-family kinases, JAK-2 or Rlk [43–45]. The tyrosine residue is located within a YVKM motif and this has been shown to serve as the binding site for several molecules (Fig. 1). In its unphosphorylated state this motif is bound to the medium-chain subunit AP-50 of the AP-2 clathrin adapter [12, 13], leading to the rapid endocytosis of CTLA-4. In contrast, tyrosine phosphorylation results in the surface retention of CTLA-4 and the binding of PI-3K to the YVKM motif . It has been also described that CTLA-4 can be found in a complex together with CD3ζ and the tyrosine phosphatase SHP-2 [46–48]. The direct interaction between SHP-2 and the signalling molecule CD3ζ is thought to be a mechanism by which CTLA-4 downregulates TCR signalling. This could also explain the observation that CD3ζ is hyperphosphorylated in CTLA-4 knockout mice . However, the crosslinking of CTLA-4 in combination with TCR and CD28 did not lead to a decreased phosphorylation of CD3ζ . In addition, our own results, gained by the retroviral transduction of SHP-2 mutants into primary T cells, do not support the idea of a prominent contribution of SHP-2 in CTLA-4 signalling (H Hoff and MC Brunner-Weinzierl, unpublished observation).
A second phosphatase that has been shown to interact with CTLA-4 is the serine/threonine phosphatase PP2A [34, 50]. Because PP2A has been described as a negative regulator for the mitogen-activated protein kinases extracellular signal-related kinase and c-Jun N-terminal kinase, and these molecules are downregulated after CTLA-4 engagement , PP2A might serve as the mediator for these downstream effects of CTLA-4. However, so far only the opposite role for PP2A as a negative regulator for CTLA-4 function has been described .
CTLA-4 is also able to interfere with raft recruitment to the plasma membrane. It has been shown that CTLA-4 can be found in lipid rafts  and is able to suppress raft aggregation mediated by TCR and CD28 . This mechanism would account for a general downregulation of early T cell activation events by CTLA-4, such as a lack of NFAT translocation to the nucleus and IL-2 gene transcription but would dismiss further downstream specific CTLA-4 signals [22, 42]. The nature of this specific signal is still unknown. Further studies should seek to analyze the integration of the CTLA-4 signal into the cell signalling machinery  on cells that have already formed rafts. We have recently reported that already upregulated molecules such as the α-chain of the IL-2 receptor cannot be downregulated by CTLA-4 on activated T cells , suggesting that the gene transcription of activated T cells, rather than the regulation of proteins, is altered by CTLA-4.
It is not yet clear whether CTLA-4 interferes with CD28 costimulation or with TCR stimulation. Most probably it interferes with both via the inhibition of raft accumulation, because it inhibits TCR-mediated effects such as the upregulation of cyclin-dependent kinases and CD28-mediated effects such as enhanced accumulation of NFAT in the nucleus . However, the engagement of CTLA-4 does not interfere with the CD28-mediated stabilization of IL-2 mRNA .