Using allele-specific amplification method (ARMS), a highly sensitive one-stage allele-specific PCR, we have evaluated the incidence of NRAS and KRAS2 activating mutations (codons 12, 13, and 61) in 62 patients with either monoclonal gammopathy of undetermined significance (MGUS) or multiple myeloma (MM), primary plasma-cell leukemia (P-PCL), and also in human myeloma cell lines (HMCL). NRAS and/or KRAS2 mutations were found in 54.5% of MM at diagnosis (but in 81% at the time of relapse), in 50% of P-PCL, and in 50% of 16 HMCL. In contrast, the occurrence of such mutations was very low in MGUS and indolent MM (12.50%). Of note, KRAS2 mutations were always more frequent than NRAS. The validity of the technique was assessed by direct sequencing of cell lines and of some patients. Multiple mutations found in two patients were confirmed by subcloning exon PCR amplification products, testing clones with our method, and sequencing them. Thus, these early mutations could play a major role in the oncogenesis of MM and P-PCL.

Vγ9Vδ2 T cells, a major γδ PBL subset in human adults, have been previously implicated in dendritic cell (DC) licensing, owing to their high frequency in peripheral tissues and their ability to produce inflammatory cytokines upon recognition of a broad array of conserved Ags. Although these observations implied efficient recognition of Ag-expressing immature DC (iDC) by Vγ9Vδ2 T cells, the role played by DC subsets in activation of these lymphocytes has not been carefully studied so far. We show that iDC, and to a lesser extent mature DC, potentiated Th1 and Th2 cytokine, but not cytolytic or proliferative responses, of established Vγ9Vδ2 T cell clones and ex vivo memory Vγ9Vδ2 PBL stimulated by synthetic agonists. The ability of iDC to potentiate Vγ9Vδ2 production of inflammatory cytokines required for their own maturation suggested that Vγ9Vδ2 T cells, despite their strong lytic activity, could promote efficient iDC licensing without killing at suboptimal Ag doses. Accordingly Vγ9Vδ2 cells induced accelerated maturation of Ag-expressing iDC but not “bystander” DC, even within mixed cell populations comprising both Ag-expressing and nonexpressing iDC. Furthermore Vγ9Vδ2 cells induced full differentiation into IL-12-producing cells of iDC infected by Vγ9Vδ2-stimulating mycobacteria that were otherwise unable to induce complete DC maturation. In conclusion the ability of iDC to selectively potentiate cytokine response of memory Vγ9Vδ2 T cells could underlie the adjuvant effect of these lymphocytes, and possibly other natural memory T cells, on conventional T cell responses.

Although gammadelta T cells express clonally distributed T-cell receptors (TCRs), a hallmark of adaptive immunity, they are classically considered as innate-like effectors, owing to the high frequency of preactivated gammadelta T cells, with restricted antigen recognition repertoire in particular tissue locations. Actually, such features are shared only by a fraction of gammadelta T-cell subsets located in the skin and reproductive organ mucosa in rodents or in peripheral blood in humans. By contrast, other gammadelta subsets, e.g. those found in rodent and human spleen, show diverse antigenic reactivity patterns and mixed naive/memory phenotypes. Thus, gammadelta T cells are made of both 'primitive' subsets endowed with innate-like properties and 'evolved' subsets able to mount anamnestic responses like conventional major histocompatibility complex-restricted alphabeta T cells. In this article, we show that human gammadelta T cells, although heterogeneous, do share recurrent innate features that distinguish them from mainstream alphabeta T cells. In particular, most of them are activated on TCR- or natural killer receptor-mediated recognition of a restricted set of conserved yet poorly defined endogenous stress determinants. This rather simple recognition mechanism allows human gammadelta T cells to discriminate healthy cells from altered cells and to exert a variety of immunostimulatory or regulatory functions. The recent availability of synthetic gammadelta T-cell agonists mimicking these natural stress-induced ligands have fostered development of immunotherapeutic strategies, with broad indications against infectious and tumor diseases, which are briefly reviewed here.

γδ T cells, a major innate-like T cell subset, are thought to play in vivo an important role in innate and adaptive immune responses to various infection agents like parasites, bacteria, or viruses but the mechanisms contributing to this immune process remain ill defined. Owing to their ability to recognize a broad set of microbial molecular patterns, TLRs represent a major innate pathway through which pathogens induce dendritic cells (DC) maturation and acquisition of immunostimulatory functions. In this study, we studied the effects of various TLR ligands on the activation of human Vγ9Vδ2 T cells, a main human γδ PBL subset, which has been recently involved in the licensing of mycobacteria-infected DC. Both TLR3 and TLR4, but not TLR2 ligands, induced a rapid, strong, and exclusive IFN-γ production by Vγ9Vδ2 T cells. This γδ subset contributed to a large extent to the overall PBL IFN-γ response induced after short-term TLR stimulation of human PBMC. Importantly, this phenomenon primarily depended on type I IFN, but not IL-12, produced by monocytic DC upon TLR engagement. Vγ9Vδ2 T cells were similarly activated by plasmacytoid DC upon TLR8/9 activation or Yellow Fever virus infection. Moreover TLR-induced Vγ9Vδ2 IFN-γ noncytolytic response led to efficient DC polarization into IL-12p70-producing cells. Our results support an adjuvant role played by Vγ9Vδ2 T cells along microbial infections through a particular cross-talk with pathogen-associated molecular patterns-activated DC. Moreover they provide new insights into the mechanisms underlying functional activation of this unique peripheral innate-like T cell subset during viral infections.