CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Impaired fetal thymocyte development after efficient adenovirus-mediated inhibition of NF-kappa B activation.

We introduce a new experimental system combining adenovirus-mediated gene transfer and fetal thymic organ culture (FTOC). This system allowed us to efficiently express in developing thymocytes a mutant form of the NF-kappa B inhibitor I kappa B alpha (mut-I kappa B) and to study the maturation defects occurring when NF-kappa B activation is inhibited during fetal development. Fetal thymocytes infected with adenovirus containing mut-I kappa B were found to develop normally until the CD44-CD25+, CD4-CD8- double-negative stage, while production of more mature double-positive and single-positive populations was strongly decreased. Proliferation, as measured by the percentage of cells in cycle appeared normal, as did rearrangement and expression of the TCR beta-chain. However, apoptosis was much higher in FTOC infected with adenovirus containing mut-I kappa B than in FTOC infected with a control virus. Taken together, these results suggest that NF-kappa B plays a crucial role in ensuring the differentiation and survival of thymocytes in the early stages of their development.

Efficient adenoviral transfer of NF-kappaB inhibitor sensitizes melanoma to tumor necrosis factor-mediated apoptosis.

It has been suggested that the in vitro cytotoxicity of tumor necrosis factor (TNF) toward a number of transformed cell lines could make it a useful agent for anti-tumor therapy. However, many tumor cell lines are resistant to TNF-induced cell death. It has been shown that transcription factors of the NF-kappaB family, which are themselves activated by TNF, could protect cells against apoptotic cell death. To test whether melanoma cells, which are normally resistant to TNF-mediated killing, can be made susceptible by inhibiting the activation of NF-kappaB, we generated a recombinant adenovirus expressing a dominant mutant form of IkappaB alpha under the control of a CMV promoter. We show here that adenovirus-mediated inhibition of NF-kappaB function rendered melanoma cells susceptible to the cytotoxic effects of TNF, and thus that NF-kappaB-inhibiting adenoviruses could become useful adjuvants in TNF-based anti-tumor therapies.