CD8 Raft Localization Is Induced by Its Assembly into CD8αβ Heterodimers, Not CD8αα Homodimers

Abstract

The coreceptor CD8 is expressed as a CD8␣␤ heterodimer on major histocompatibility complex class I-restricted TCR␣␤ T cells, and as a CD8␣␣ homodimer on subsets of memory T cells, intraepithelial lymphocytes, natural killer cells, and dendritic cells. Although the role of CD8␣␣ is not well understood, it is increasingly clear that this protein is not a functional homologue of CD8␣␤. On major histocompatibility complex class I-restricted T cells, CD8␣␤ is a more efficient TCR coreceptor than CD8␣␣. This property has for the mouse protein been attributed to the recruitment of CD8␣␤ into lipid rafts, which is dependent on CD8␤ palmitoylation. Here, these divergent distributions of CD8␣␤ and CD8␣␣ are demonstrated for the human CD8 proteins as well. However, although palmitoylation of both CD8␣ and CD8␤ chains was detected, this modification did not contribute to raft localization. In contrast, arginines in the cytoplasmic domain are crucial for raft localization of CD8␤␤. Most strikingly, the assembly of a non-raft localized CD8␤ chain with a non-raft localized CD8␣ chain resulted in raft-localized CD8␣␤ heterodimers. Using chimeric CD8 proteins, this property of the heterodimer was found to be determined by the assembly of CD8␣ and CD8␤ extracellular regions. The presence of two CD8␣ extracellular regions, on the other hand, appears to preclude raft localization. Thus, heterodimer formation and raft association are intimately linked for CD8␣␤. These results emphasize that lipid raft localization is a key feature of human CD8␣␤ that clearly distinguishes it from CD8␣␣.The cell surface glycoprotein CD8 plays an important role in the development and function of MHC 3 class I-restricted T cells. CD8 fulfils its function through interacting via its extracellular domain with MHC class I proteins and via its intracellular domain with the tyrosine kinase Lck (1, 2). An interaction of both the T cell receptor (TCR) and CD8 with MHC proteins enhances the adhesion between the T cell and the antigen-presenting cell. In addition, this dual interaction juxtaposes Lck to the TCR complex, resulting in phosphorylation of CD3 and TCR chains and, thus, the initiation of signaling (3, 4). CD4 performs a corresponding coreceptor role on MHC class IIrestricted T helper cells (5, 6). However, CD8 and CD4 are structurally distinct; CD4 is a monomer, whereas CD8 is a disulfide-linked heterodimer of a CD8␣ and CD8␤ chain. Yet, like CD8, CD4 binds to MHC proteins and interacts with Lck (2, 7). In addition, both proteins have been shown to localize to lipid rafts (8 -11), which are believed to act as key platforms for TCR signaling.The concept of lipid rafts is based on the segregation of lipids into liquid-ordered and liquid-disordered phases (12,13). Raft formation at the plasma membrane is dependent on cholesterol, which tightly packs sphingolipids that contain saturated fatty acids, into a liquid-ordered domain. The phospholipids on the other hand, with at least one unsaturated fatty acid, are in the liquid-disordered phase. Detergent-in...

CD4 and CD8 T cells require different membrane gangliosides for activation

“…WT G3 G2 WT G3 G2 WT G3 G2 WT G3 G2 WT G3 G2 WT G3 G2 extended-GM1b Although there are some commonalities between the mechanisms of TCR-mediated signaling in CD4 + T cells and CD8 + T cells, the two subsets do have different cellular and molecular modifications. CD4 and CD8 can localize to lipid rafts by palmitoylation in their juxtamembrane region (10,11), yet raft localization seems not to be determined by lipid modification alone (11,12). To ensure that CD4 and CD8 undergo proper intracellular trafficking and successful localization on the plasma membrane, it might be vital for CD4/CD8 to interact with rafts carrying a specific ganglioside.…”

Differential Expression of the Human CD8β Splice Variants and Regulation of the M-2 Isoform by Ubiquitination

“…Another variant described by DiSanto et al (29) that we have designated as M-4 (also known as transcript variant 2) possesses the transmembrane domain and lacks exons VII and VIII but carries exon IX, giving rise to a unique 36 amino acid sequence in the 3Ј region. All isoforms contain the membrane proximal cytoplasmic domain of 8 amino acids encoded by the C1 exon that has a cysteine required for palmitoylation (20,32), but the rest of the tails range in size from 3 to 39 amino acids (Fig. 1B).…”

Trogocytosis Is a Gateway to Characterize Functional Diversity in Melanoma-Specific CD8+ T Cell Clones

“…On day 14 of TIL initiation, the lymphocytes were washed with PBS, resuspended in PBS supplemented with 0.5% BSA, and stained with FITCconjugated HLA-A*0201/MART-1 [26][27][28][29][30][31][32][33][34][35] dextramer (Immudex, Copenhagen, Denmark) for 30 min at 4˚C. The lymphocytes were then incubated with allophycocyanin-conjugated mouse anti-human CD8 (eBioscience) for an additional 30 min at 4˚C and washed.…”