A Lysosomal Protein Negatively Regulates Surface T Cell Antigen Receptor Expression by Promoting CD3ζ-Chain Degradation

Abstract

Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

“…4B). In agreement with a previous report (18), cell stimulation by pervanadate depleted CD3ζ-eGFP from the plasma membrane and increased the number of endocytic vesicles containing CD3ζ-eGFP (Movie S6), although a significant fraction of internalized CD3ζ was concentrated in vesicles clearly distinct from lysosomes. This demonstrates that although some of the activated CD3ζ is targeted for lysosomes, a subpopulation of internalized, tyrosine-phosphorylated CD3ζ is retained in the endosomal compartment.…”

“…Accumulation of CD3ζ in the perinuclear vesicles was previously observed for endogenous and GFP-tagged CD3ζ in several Jurkat clones, naïve T cells, and T-cell hybridomas (8,(15)(16)(17)(18) and is consistent with constitutive internalization and recycling of the TcR/CD3 described in various primary cells and cell lines. Indeed, time-lapse microscopy shows that the ZIP reportercontaining vesicles actively move within the cytoplasm and exchange with the plasma membrane (Movie S3).…”

“…Prior studies (2,3,18,19) indicated that internalized TcR/ CD3 in stimulated cells is targeted for degradation; therefore, we examined colocalization of the reporter or CD3ζ-eGFP with lysosomes using immunofluorescent staining with the lysosomal markers LAMP1 or LysoTracker Red. In unstimulated cells, only a fraction of internalized CD3ζ colocalized with fluorescent lysosomes (Fig.…”

Critical role of the IgM Fc receptor in IgM homeostasis, B-cell survival, and humoral immune responses

“…For analyzing FcμR expression in various B-cell subpopulations, cells were first incubated with a rat IgG 2b antimouse CD16/CD32 monoclonal antibody (clone 2.4G2; BD Biosciences) to block FcγR and then stained with either anti-FcμR (4B5) or an isotype control antibody (clone eBR2a; eBioscience), and further stained with phycoerythrin (PE)-conjugated anti-rat IgG 2a (clone RG7/1.30; BD Biosciences). After washing, the cells were further stained with FITC-, allophyocyanin (APC)-, or PE/Cy7-conjugated antibodies against various surface molecules expressed during Bcell development, maturation, activation, and differentiation as described (22,40). Memory and GC B cells were identified essentially as described (41).…”

“…The purity of the B cells was >95% as judged by staining with anti-B220. The cells were seeded and analyzed for proliferation by 3 H-thymidine incorporation as described previously (40). For the survival assay, the cells were stained with 7-aminoactinomycin D and analyzed by FACS.…”