The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck

Abstract

CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought.

Functional role of T-cell receptor nanoclusters in signal initiation and antigen discrimination

“…More CD3ζ molecules colocalized with Lck at 5 min than at later time points (Fig. 4D), supporting the notion that Lck may depart signaling clusters following early recruitment, resulting in an overall low level of colocalization as previously reported (39,40). Furthermore, although CD3ζ clusters contained pLat (Fig.…”

The CD4 and CD3δε Cytosolic Juxtamembrane Regions Are Proximal within a Compact TCR–CD3–pMHC–CD4 Macrocomplex

“…No difference in FRET E was observed between the PD-1T G ::PD-1T mCh negative control and our experimental CD3δT G ::CD4T mCh cells imaged on glass coverslips. This is consistent with data suggesting that CD4 and TCR do not pre-associate in an unengaged state (43). Adherence of CD3δT G ::CD4T mCh cells to glass coverslips functionalized with agonist pMHC resulted in a significant increase in CD3δT G ::CD4T mCh FRET E compared to cells on uncoated coverslips or the negative control cells, indicating that concurrent TCR and CD4 engagement of agonist pMHC positions CD3δ and CD4 in a close spatial relationship (Figure 1B).…”

“…In the case of CD3δ::CD4 FRET, coordinate binding of CD4 and TCR to an agonist pMHC creates a transient mEGFP and mCherry pairing for a subset of TCR and CD4 molecules that engage pMHC on an immobile surface. This is because CD4 and the TCR have been shown by super resolution imaging to inhabit distinct membrane domains (43). Consequently, the number of TCRs within a membrane domain that are available to engage the same pMHC as a CD4 molecules in a distinct membrane domain appears to be physically limited to those that are located at the boundary of each membrane domain (43).…”

Ionic CD3−Lck interaction regulates the initiation of T-cell receptor signaling

“…Genetic experiments show that Lck plays a more important role than Fyn in TCR signaling (10). Lck can either be loaded on coreceptor CD4/CD8 or remain free (11)(12)(13). It is suggested that Lck molecules are all loaded on coreceptors in thymocytes (12).…”

“…It is suggested that Lck molecules are all loaded on coreceptors in thymocytes (12). However, superresolution imaging shows that CD4 and Lck localize in different nanodomains in mature T cells (13). Functionally, the coreceptor contributes to antigen sensitivity of T cells but is not essential for T-cell activation in mature T cells (14).…”