The cysteine residues in the cytoplasmic tail of CD8 α are required for its coreceptor function

109

CD8+ tumor-infiltrating lymphocytes (TIL) are severely deficient in cytolysis, a defect that may permit tumor escape from immune-mediated destruction. Because lytic function is dependent upon TCR signaling, we have tested the hypothesis that primary TIL have defective signaling by analysis of the localization and activation status of TIL proteins important in TCR-mediated signaling. Upon conjugate formation with cognate target cells in vitro, TIL do not recruit granzyme B+ granules, the microtubule-organizing center, F-actin, Wiskott-Aldrich syndrome protein, nor proline rich tyrosine kinase-2 to the target cell contact site. In addition, TIL do not flux calcium nor demonstrate proximal tyrosine kinase activity, deficiencies likely to underlie failure to fully activate the lytic machinery. Confocal microscopy and fluorescence resonance energy transfer analyses demonstrate that TIL are triggered by conjugate formation in that the TCR, p56lck, CD3ζ, LFA-1, lipid rafts, ZAP70, and linker for activation of T cells localize at the TIL:tumor cell contact site, and CD43 and CD45 are excluded. However, proximal TCR signaling is blocked upon conjugate formation because the inhibitory motif of p56lck is rapidly phosphorylated (Y505) and COOH-terminal Src kinase is recruited to the contact site, while Src homology 2 domain-containing protein phosphatase 2 is cytoplasmic. Our data support a novel mechanism explaining how tumor-induced inactivation of proximal TCR signaling regulates lytic function of antitumor T cells.

Section: Discussionmentioning

confidence: 89%

Defective Proximal TCR Signaling Inhibits CD8+ Tumor-Infiltrating Lymphocyte Lytic Function

Koneru

Schaer

Monu

et al. 2005

109

“…The reason for this difference is unknown, because in solution CD8␣␣ and CD8␣␤ bind to MHC class I molecules with similar affinities (15). Moreover, the binding site of CD8 for lck resides in the cytoplasmic tail of CD8␣ (3,4). We have previously observed that on cells, CD8␣␤ strengthens TCR-ligand binding more than CD8␣␣ (12).…”

Section: Discussionmentioning

confidence: 96%

“…In contrast, NK cells or intestinal T cells express homodimeric CD8, composed of disulfide-linked CD8␣ (1,2). The Ig domain of CD8 interacts with the constant domain of MHC class I molecules (1,2), and the cytoplasmic tail of CD8␣ can associate with the src kinase p56 lck (lck), 3 by means of a zinc cation, chelating vicinal cysteines on both molecules (3,4). Therefore, this association is disrupted by EDTA or iodoacetamide.…”

mentioning

confidence: 99%

Essential Role of CD8 Palmitoylation in CD8 Coreceptor Function

Arcaro

Grégoire

Boucheron

et al. 2000

Self Cite

159

164

To investigate the molecular basis that makes heterodimeric CD8αβ a more efficient coreceptor than homodimeric CD8αα, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252–260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8β and that this allows partitioning of CD8αβ, but not of CD8αα, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8β abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3ζ in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8β is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

“…Although the cytoplasmic domain of the CD8␣-chain contains the binding site for p56 lck required for the initiation of early T cell signaling (17)(18)(19), it is palmitoylation of the CD8␤-chain that facilitates the partitioning of CD8 into lipid rafts (20,21). Studies in mice indicated a role for the CD8␤ cytoplasmic tail in thymic development and activation of CD8 ϩ T cells although it contains no known protein binding motifs (22)(23)(24)(25).…”

Section: Ature Human Cytotoxic Mhc Class I (Mhc-i)mentioning

confidence: 99%

“…One microgram of total RNA was subjected to first strand cDNA synthesis. The oligo(dT) [12][13][14][15][16][17][18] -primed reverse transcriptase (RT) reaction was conducted in a total volume of 20 l either with SuperScript II RT or without the enzyme (ϪRT control) according to the manufacturer's protocol (Sprint Powerscript, Clontech). A total of 1 l of cDNA was used for each TaqMan measurement.…”

Section: Preparation Of Rna and Cdna And Procedures For Quantitative Pmentioning

confidence: 99%

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

Thakral

Dobbins

Devine

et al. 2008

The CD8αβ heterodimer functions as a coreceptor with the TCR, influencing the outcome of CD8+ T cell responses to pathogen-infected and tumor cells. In contrast to the murine CD8B gene, the human gene encodes alternatively spliced variants with different cytoplasmic tails (M-1, M-2, M-3, and M-4). At present, little is known about the expression patterns and functional significance of such variants. We used quantitative RT-PCR to demonstrate differential mRNA expression patterns of these splice variants in thymocytes and in resting, memory, and activated primary human CD8+ T cells. In total CD8+ T cells, mRNA levels of the M-1 variant were the most predominant and levels of M-3 were the least detected. The M-4 isoform was predominant in effector memory CD8+ T cells. Upon stimulation of CD8+ T cells, the M-2 variant mRNA levels were elevated 10–20-fold relative to resting cells in contrast to the other isoforms. Curiously, the M-2 isoform was not expressed on the cell surface in transfected cell lines. Using fluorescent chimeras of the extracellular domain of mouse CD8β fused to the cytoplasmic tails of each isoform, the M-2 isoform was localized in a lysosomal compartment regulated by ubiquitination of a lysine residue (K215) in its cytoplasmic tail. In contrast, upon short-term stimulation, the M-2 protein localized to the cell surface with the TCR complex. The relatively recent evolution of CD8B gene splice variants in the chimpanzee/human lineage is most likely important for fine-tuning the CD8+ T cell responses.