M. Raffaella Zocchi

HIV-1 Tat: a polypeptide for all seasons

Abstract A plethora of mechanisms of action have been proposed for exogenous Tat to explain the pleiotropic, and sometimes controversial, extracellular effects reported for this molecule. Anna Rubartelli and colleagues discuss the molecular bases of these multiple functions.

NK Cell Activation by Dendritic Cells Is Dependent on LFA-1-Mediated Induction of Calcium-Calmodulin Kinase II: Inhibition by HIV-1 Tat C-Terminal Domain

In this study, we show that binding to autologous dendritic cells (DC) induces a calcium influx in NK cells, followed by activation of the calcium-calmodulin kinase II (CAMKII), release of perforin and granzymes, and IFN-γ secretion. CAMKII is induced via LFA-1: indeed, oligomerization of LFA-1 leads to CAMKII induction in NK cells. Moreover, release of lytic enzymes and cytotoxic activity is strongly reduced by masking LFA-1 or by adding CAMKII inhibitors such as KN62 and KN93, at variance with the inactive compound KN92. NK cell-mediated lysis of DC and IFN-γ release by NK cells upon NK/DC contact are inhibited by exogenous HIV-1 Tat: the protein blocks calcium influx and impairs CAMKII activation elicited via LFA-1 in NK cells, eventually inhibiting degranulation. Experiments performed with synthetic, overlapping Tat-derived peptides showed that the C-terminal domain of the protein is responsible for inhibition. Finally, both KN62 and Tat reduced the extension of NK/DC contacts, possibly affecting NK cell granule polarization toward the target. These data provide evidence that exogenous Tat inhibits NK cell activation occurring upon contact with DC: this mechanism might contribute to the impairment of natural immunity in HIV-1 infection.

Control of interleukin-18 secretion by dendritic cells: role of calcium influxes

Here we show that dendritic cells accumulate the precursor form of the leaderless secretory protein interleukin‐18 (pro‐interleukin‐18) in the cell cytosol and in organelles co‐fractionating with endolysosomes. Upon antigen specific contact with T lymphocytes, particulated pro‐interleukin‐18 decreases rapidly, and the cytokine appears extracellularly, suggesting that exocytosis of pro‐interleukin‐18‐containing organelles is induced. Exocytosis of secretory lysosomes is modulated by calcium: in agreement with this, calcium influx results in secretion of pro‐interleukin‐18. In turn, pro‐interleukin‐18 secretion induced by T cells is prevented by the calcium channel blocker nifedipine. Our results demonstrate a novel, calcium‐mediated mechanism of post‐translational regulation of secretion for interleukin‐18, that allows a fast release of the cytokine.

Leukocyte-associated Ig-like receptor-1 prevents granulocyte-monocyte colony stimulating factor-dependent proliferation and Akt1/PKB alpha activation in primary acute myeloid leukemia cells

The leukocyte‐associated Ig‐like receptor‐1 (LAIR‐1), a surface leukocyte receptor containing two immune receptor tyrosine‐based inhibitory motif (ITIM) is expressed on acute myeloid leukemia (AML) blasts isolated from peripheral blood or bone marrow of 17 patients (2 M0, 3 M1, 5 M2, 2 M4 and 5 M5 acording to French, American and British classification). Further, we provide evidence thatLAIR‐1 engagement inhibits granulocyte‐monocyte colony‐stimulating factor (GM‐CSF)‐induced proliferation of AML blasts. Indeed, leukemia cells stimulated with GM‐CSF were blocked in the G0/G1 phaseof the cell cycle and underwent apoptosis within 4 days after the engagement of LAIR‐1. Remarkably, LAIR‐1 was functional also in AML blasts which do not express CD33, mainly M4 and M5. Importantly, the LAIR‐1 ligation led to a strong inhibition of both GM‐CSF receptor‐mediated intracellular calcium increases, phosphorylation and activation of Akt1/protein kinase B alpha, a substrate of the phosphatidylinositol‐3 kinase. This last inhibitory effect was prevented by a synthetic peptide spanning the ITIM portion of LAIR‐1, suggesting the involvement of SHP‐1 phosphatase in LAIR‐1‐mediated inhibitory signal. Altogether, these findings indicate that the engagement of LAIR‐1 can down‐regulate GM‐CSF‐mediated survival and proliferation of AML blasts, suggesting an additional therapeutic approach to the treatment of AML patients.

Engagement of the leukocyte-associated Ig-like receptor-1 induces programmed cell death and prevents NF-κB nuclear translocation in human myeloid leukemias

Leukocyte‐associated Ig‐like receptor‐1 (LAIR‐1) is a surface molecule that functions as an inhibitory receptor on natural killer cells, T lymphocytes and monocytes. Here, we provide evidence that occupancy of LAIR‐1 on human myelomonocytic leukemic cell lines inhibits proliferation and leads to programmed cell death (PCD), evaluated by propidium iodide staining and transmission electron microscopy. Interestingly, PCD elicited via LAIR‐1 was not blocked by different caspase inhibitors, at variance with apoptosis induced via CD95 / Fas, which was prevented by the caspase‐1 and caspase‐8 specific inhibitors. In addition, we show that the p65 subunit of the nuclear factor κB (NF‐κB), constitutively expressed in the nucleus of these cell lines, was retained in the cytoplasm upon engagement of LAIR‐1. This was evident already 8 h after LAIR‐1 occupancy, when apoptosis was not yet detectable by fluorometric or ultrastructural analysis. Moreover, a reduction in inhibitor κBα phosphorylation was observed after LAIR‐1 engagement. As blocking of NF‐κB activation has been shown to rescue sensitivity to anti‐cancer drugs in solid tumors, we suggest that LAIR‐1 may represent a possible target for pharmacological approaches aimed to potentiate anti‐leukemic therapy.

Pertussis Toxin (PTX) B Subunit and the Nontoxic PTX Mutant PT9K/129G Inhibit Tat-Induced TGF-β Production by NK Cells and TGF-β-Mediated NK Cell Apoptosis

We show that the pertussis toxin B oligomer (PTX-B), and the PTX mutant PT9K/129G, which is safely administered in vivo, inhibit both transcription and secretion of TGF-β elicited by HIV-1 Tat in NK cells. Tat-induced TGF-β mRNA synthesis is also blocked by the ERK1 inhibitor PD98059, suggesting that ERK1 is needed for TGF-β production. Moreover, Tat strongly activates the c-Jun component of the multimolecular complex AP-1, whereas TGF-β triggers c-Fos and c-Jun. Of note, treatment of NK cells with PTX-B or PT9K/129G inhibits Tat- and TGF-β-induced activation of AP-1. TGF-β enhances starvation-induced NK cell apoptosis, significantly reduces transcription of the antiapoptotic protein Bcl-2, and inhibits Akt phosphorylation induced by oligomerization of the triggering NK cell receptor NKG2D. All these TGF-β-mediated effects are prevented by PTX-B or PT9K/129G through a PI3K-dependent mechanism, as demonstrated by use of the specific PI3K inhibitor, LY294002. Finally, PTX-B and PT9K/129G up-regulate Bcl-xL, the isoform of Bcl-x that protects cells from starvation-induced apoptosis. It is of note that in NK cells from patients with early HIV-1 infection, mRNA expression of Bcl-2 and Bcl-xL was consistently lower than that in healthy donors; interestingly, TGF-β and Tat were detected in the sera of these patients. Together, these data suggest that Tat-induced TGF-β production and the consequent NK cell failure, possibly occurring during early HIV-1 infection, may be regulated by PTX-B and PT9K/129G.

CD1+ thymocytes proliferate and give rise to functional cells after stimulation with monoclonal antibodies recognizing CD3, CD2 or CD28 surface molecules.

The signal requirements for activation and proliferation of CD1+ thymocytes have been studied in order to define whether this immature cell population could function as mature T cells do. We found that CD1+ cells expressed high levels of CD25 antigen upon triggering with specific monoclonal antibodies (mAbs) (anti-CD3, anti-CD2, anti-CD28) in association with low doses of Phorbol-13-myristate-12-acetate (PMA). More interestingly, we described that in the presence of PMA CD1+ thymocytes proliferate upon stimulation with anti-CD28 mAb as well as with a pair of anti-CD2 mAbs, without the need of exogenous interleukin-2 (IL2), whereas they respond to anti-CD3 mAb only if exogenous IL2 was provided. Furthermore, CD1+ cells stimulated under optimal proliferative conditions, gave rise to cell populations capable of lysing natural killer (NK)-sensitive (K562) and NK-resistant (MEL 10, Daudi, EPA1) tumor target cells. These data strongly support the idea that CD1+ thymocytes, under appropriate stimulations, display some of the functional capabilities of mature T cells.

Activation of CD3/TCR negative human thymocytes via CD28 molecule.

We have observed that the CD28 molecule was present on the cell surface of a large fraction of resting CD3- thymocytes (40 to 100%). Interestingly, the majority (greater than 90%) of surface CD3-CD28-cells reacted in the cytoplasm with anti-CD28 (CK248, 9.3) and anti-CD3 epsilon chain mAbs (Leu4, OKT3). Along this line, we found that CD28 surface expression could be induced within 18 hr on CD3-CD28- thymocytes using very low doses of phorbol-13-myristate-12-acetate (PMA). This event was accompanied by the appearance of CD25 and CD69 activation antigens but not of CD3/TCR complex. These results were further confirmed by immunoprecipitation studies. It is noteworthy that the T-cell activation pathway initiated via the CD28 molecule is functional in resting CD3- thymocytes in the presence of PMA and/or IL2. Finally, stimulation of CD3- immature thymocytes via CD28 gave rise to a large fraction (about one-third) of CD3-CD8+ cells.