Interplay of T-cell receptor and interleukin-2 signalling in Vγ2Vδ2 T-cell cytotoxicity.

Human peripheral blood Vγ2Vδ2 T cells are important for host defence and tumour immunity. Their unusual T-cell receptor (TCR) recognizes small molecule phosphoantigens; stimulated cells produce inflammatory cytokines and are potently cytotoxic for a variety of tumours. However, molecular mechanisms linking phosphoantigen stimulation and cytotoxicity are incompletely understood. We know that isopentenyl pyrophosphate (IPP) activates mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/Erk) and phosphoinositide 3-kinase (PI-3K)/Akt pathways; specific inhibition of Erk or Akt significantly impairs the functional response to IPP. We now show that interleukin-2 also activates MEK/Erk and PI-3K/Akt pathways but on its own, fails to induce cytokine expression or cytotoxicity. Hence, MEK/Erk and PI-3K/Akt activation are necessary but not sufficient to induce effector responses in Vγ2Vδ2 T cells and a TCR-dependent signal is still required for tumour cell killing. Cyclosporin A, an inhibitor of calcineurin, blocked calcium-dependent nuclear translocation of nuclear factor of activated T cell (NFAT) and significantly reduced IPP-induced cytokine production, degranulation and cytotoxicity. The IPP-induced calcium mobilization and NFAT translocation were necessary to activate Vγ2Vδ2 effector functions; interleukin-2, acting on the MEK/Erk pathway, regulated the strength of these responses. The TCR has a specific role in Vγ2Vδ2 T-cell killing of tumour cells, which is distinct from its role in triggering cellular proliferation in response to phosphoantigens.

Furin cleavage of the HIV-1 Tat protein.

Extracellular human immunodeficiency virus-1 (HIV-1) Tat protein and Tat-derived peptides are biologically active but mechanisms of Tat processing are not known. Within the highly conserved basic region of HIV-1 Tat protein (amino acids, a.a. 48-56), we identified two putative furin cleavage sites and showed that Tat protein was cleaved in vitro at the second site, RQRR\ (a.a. 53-56\). This in vitro cleavage was blocked by a monoclonal antibody that binds near the cleavage site or by the furin inhibitor alpha-1 PDX. Monocytoid cells rich in furin also degraded Tat and this process was slowed by the furin inhibitor or the specific monoclonal antibody. Furin processing did not affect the rates for Tat uptake and nuclear accumulation in HeLa or Jurkat cells, but the transactivation activity was greatly reduced. Furin processing is a likely mechanism for inactivating extracellular HIV-1 Tat protein.

CXCR4-dependent HIV-1 infection of differentiated epithelial cells.

Epithelial cells constitute a physical barrier to sexual transmission of HIV, but are also a source of cytokines that could alter infection efficiency. We studied HIV infection of the human colonic epithelial cell line HCT116, which is a model for differentiation of intestinal mucosal epithelium. Differentiated HCT116 cells had increased expression of cell surface C-X-C chemokine receptor type-4 (CXCR4) that mediated HIV entry, despite the apparent absence of cell surface CD4. HIV infection in differentiated HCT116 cells increased the levels of IL-1alpha, and IFN-alpha mRNA even though only 1% of cells had integrated provirus. The inefficient, CXCR4-mediated infection of differentiated HCT116 cells supports the view that epithelial cells are a barrier and not a portal for HIV transmission. However, low level infection of epithelial cells could trigger the release of cytokines that indirectly increase the transmission rate.