In this study, we illustrated the dynamic aspects of the mechanisms of CD28-mediated co-stimulation in relation to TCR MCs. CD28 is co-localized with TCR to form TCR-CD28 MCs together with signaling molecules as signalsomes, where CD28 plays a critical role in recruiting PKCθ to MCs at initial activation, and then to the outer region of c-SMAC possibly for sustained activation to exhibit effector functions. Previous studies have indicated that TCR MCs generated at the periphery of IS induce activation signals while c-SMAC may not be responsible for transducing activation signals, on the basis of our observation that phosphorylated proteins are localized almost in peripheral MCs and not obviously detected in c-SMAC (Yokosuka et al., 2005
), and that Ca2+
signaling is not sustained in the absence of peripheral MCs (Varma et al., 2006
). However, the present result of CD28-PKCθ signaling at the outer region of c-SMAC revises it to the idea that c-SMAC also provides signal competency for co-stimulation and that different signal clusters are responsible for TCR-mediated signals and co-stimulatory signals for the maintenance of T cell activation.
The translocation of CD28 to the T cell—APC interface before c-SMAC formation was previously reported (Andres et al., 2004a
), but we demonstrated here the co-localization of CD28 with TCR and its downstream molecules as a TCR-CD28 MC using a planar bilayer system. This co-localization of TCR with CD28 in the same MC coincides with the findings that Lck phosphorylates both TCR and CD28 after TCR engagement, and that CD28 shares most of the downstream molecules with TCR. We searched for a signaling molecule contributing specifically to CD28 signals that initially co-localizes with TCR MCs and then translocates to CD28 clusters at the outer region of c-SMAC. We demonstrated that CD28 enhanced the co-localization of PI3K with CD28, but PI3K clusters were generated onlytransiently and did not accumulate at c-SMAC. We finally observed that PKCθ met the criteria for the specific mediator of CD28 signals; initial accumulation at TCR-CD28 MCs as well as at c-SMAC later.
PKCθ is widely known as a downstream molecule of CD28. PKCθ- and CD28-deficient animals demonstrated similar functional defects (Pfeifhofer et al., 2003
; Sun et al., 2000
), although the physical interaction has not been shown. We provided evidence of the functional assembly between CD28 and PKCθ in three aspects. 1) PKCθ is contained within early activation signalsomes as TCR-CD28 MCs and translocated to c-SMAC with CD28. 2) Physical association of PKCθ with CD28 is demonstrated by co-immunoprecipitation upon PKCθ activation. 3)Functional importance of the early and later localization of PKCθ at TCR-CD28 MCs and at c-SMAC was evidenced in IL-2 production. Recently, CD19 was reported to enhance B cell functions through its co-localization with B cell receptors (BCRs) and Syk as BCR MCs (Depoil et al., 2008
). These and our results suggest that MCs are initially generated as signalsomes with TCRs and BCRs and that their own co-stimulatory molecules are critical for the consequent signals.
In the last decade, PKCθ has been considered as a marker for c-SMAC during T cell activation (Monks et al., 1998
), while CD28 was suggested to segregate PKCθ in IS (Huang et al., 2002
; Tseng et al., 2005
). In this study, in addition to the finding of co-localization of CD28-PKCθ in TCR-MC, we unveiled a new functional domain of c-SMAC by identification of a sub-region into which CD28 and PKCθ accumulated. The generation of this sub-region within cSMAC appears to depends on both localizations and densities of CD28 since PKCθ was co-localized with CD28high
but not TCR/CD3high
. Furthermore, the formation of CD28 clusters is subjected to the density of CD28 and CD80/CD86 on T cells and APCs, respectively, and to the avidity of TCR to MHCp, substituting to the competition between CD28—CD80/CD86 and TCR—MHCp (unpublished observation). We propose here a functional region of c-SMAC, CD3dim
, as a co-stimulatory signalsome for T cell activation. We demonstrated that no signaling molecule was translocated to CD3high
, and that CD3dim
was more flexible and dynamically regulated than CD3high
by FRAP analysis (unpublished observation). Together with our previous report that lysobisphosphatidic acid (LBPA), a marker for protein degradation, was localized in c-SMAC (Varma et al., 2006
), c-SMAC is suggested to be a negative regulatory compartment for T cell activation. The ratio of CD3dim
appears to vary depending on the strength of TCR stimulation. A strong TCR stimulus may result inmore CD3high
for endocytosis/degradation of TCR complexes, whereas a weak stimulus may result in more CD3dim
that are susceptible to CD28-PKCθ-mediated co-stimulation. This is consistent with previous data suggesting the function of c-SMAC in negative regulation by signal strength-dependent TCR degradation (Cemerski et al., 2007
). Our results suggest that c-SMACs may inherit ying and yang functions for the regulation of T cell activation: endocytosis/degradation of the TCR complex may mediate negative regulation through the CD3high
, whereas CD28-PKCθ accumulation could function for sustained T cell signaling through the spatially distinct region.
Our data depicted both spatial and temporal differences in the dynamics of PKCθ and Zap70/SLP-76 for MC formation. PKCθ may be recruited to the membrane toward diacylglycerol (DAG) or via phospholipase C-γ1 (PLCγ1)/DAG-independent manner (Villalba et al., 2002
), and functionally activated by membrane translocation (Bi et al., 2001
), while Zap70 and SLP-76 requires phosphorylation for the retention on cell surface by association with CD3 and LAT, respectively. Thus, PKCθ may associate with TCR-CD28 MCs via two steps—initial recruitment to the cell surface by DAG binding, followed by lateral movement to TCR-CD28 MCs through guidance by and/or association with CD28. Frequencies of cells with PKCθ translocation in CD28 Y189F, PI3K-bindingmutant, was lower than those in Δ16 mutant, in both T cell—APC and T cell—bilayer experiments (), whereas stimulation of the Y189F CD28-expressing cells with PMA plus anti-CD28 Abs augmented the proliferation, suggesting that involvement of PI3K for the membrane translocation of PKCθ and that T cell response was partially rescued by PMA-mediated PKCθ relocation ( and ).
Several mechanisms could explain the CD28-mediated membrane translocation and co-localization with PKCθ. Both molecules might be recruited through lipid raft. It has been reported that CD28 engagement re-distributes lipid raft, and CD28 and PKCθ associate with downstream molecules in lipid raft (Bi et al., 2001
; Viola et al., 1999
). However, we failed to observed cluster formation of a raft marker in our systems (data not shown). Alternatively, although PKCθ may be recruited through a DAG gradient generated at the T cell—APC interface (Spitaler et al., 2006
), we have not observed gradient of a DAG marker (unpublished observation). Moreover, cell adhesion by CD28 may contribute to the functional recruitment of PKCθ to CD28 clusters through cytoskeletal rearrangement (Kaga et al., 1998
). In this context, since filamin-A was reported to translocate into IS, associate with actin and PKCθ and enhanced IL-2 production in a CD28—CD80-dependent manner (Hayashi and Altman, 2006
; Tavano et al., 2006
), this mechanism might be involved in the co-localization of CD28 with PKCθ.
During T cell activation, the functional enhancement of T cell responses by CD28-mediated co-stimulation is prominent for T cell activation with weak TCR stimulus. Imaging studies have suggested that CD28 would support IL-2 production if no c-SMAC was generated (Purtic et al., 2005
) or if APC carried a low concentration of Ag or self-Ag (Wulfing et al., 2002
). In relation to these studies, we observed that CD28-PKCθ clusters could be generated even under the condition where c-SMAC is not clearly generated with lower affinity TCR, suggesting further differential regulation of two spatially distinct signaling clusters: peripheral MCs and annular clusters in c-SMAC may lead to the differential signaling pathways such as for Ca2+
/NFAT and NF-κB activation, respectively. The spatiotemporal regulation of TCR-CD28 cluster formation in c-SMAC may explain the complexity of intercommunicating molecules downstream of TCR and CD28.
At the outset of this study, we had strong evidence that TCR MCs generated initial and sustained signals and c-SMAC was primarily involved in signal termination. Whereas CD28 localization in IS had been extensively investigated, nano-scale imaging of co-stimulatory receptor CD28 and its signal transduction partners PI3K and PKCθ by TIRFM provided a new vision of two co-stimulatory mechanisms that are functionally critical: the early recruitment of PKCθ and PI3K to TCR-CD28 MCs and the late formation of a dynamic sub-region in c-SMAC enriched with PKCθ but depleted of TCR and PI3K.