Adjuvant System 04 (AS04) combines the TLR4 agonist MPL (3-O-desacyl-4′-monophosphoryl lipid A) and aluminum salt. It is a new generation TLR-based adjuvant licensed for use in human vaccines. One of these vaccines, the human papillomavirus (HPV) vaccine Cervarix, is used in this study to elucidate the mechanism of action of AS04 in human cells and in mice. The adjuvant activity of AS04 was found to be strictly dependent on AS04 and the HPV Ags being injected at the same i.m. site within 24 h of each other. During this period, AS04 transiently induced local NF-κB activity and cytokine production. This led to an increased number of activated Ag-loaded dendritic cells and monocytes in the lymph node draining the injection site, which further increased the activation of Ag-specific T cells. AS04 was also found to directly stimulate those APCs in vitro but not directly stimulate CD4+ T or B lymphocytes. These AS04-induced innate responses were primarily due to MPL. Aluminum salt appeared not to synergize with or inhibit MPL, but rather it prolonged the cytokine responses to MPL at the injection site. Altogether these results support a model in which the addition of MPL to aluminum salt enhances the vaccine response by rapidly triggering a local cytokine response leading to an optimal activation of APCs. The transient and confined nature of these responses provides further supporting evidence for the favorable safety profile of AS04 adjuvanted vaccines.

A small percentage of human T lymphocytes, predominantly CD8+ T cells, express receptors for HLA class I molecules of natural killer type (NK-R) that are inhibitory for T-cell antigen receptor (TCR)-mediated functions. In the present study, it is demonstrated that the various NK-R molecules typically expressed by NK cells are also expressed on periheral blood T lymphocytes. These CD3+ NK-R+ cells have a cell surface phenotype typical of memory cells as indicated by the expression of CD45RO and CD29 and by the lack of CD28 and CD45RA. Furthermore, by the combined use of anti-TCR Vf3-specific antibodies and a semiquantitative polymerase chain reaction assay, the TCR repertoire in this CD3+ NK-R+ cell subset was found to be skewed; in fact, one or two V8 families were largely represented, and most of the other Vj8s were barely detected. In addition, analysis of recombinant clones of the largely represented V,B families demonstrated that these Vf3s were oligoclonally or monoclonally expanded.

and CD8؉ cells expressed CD94, the simultaneous expression of NKG2A (i.e., the other component of the CD94͞NKG2A inhibitory NKR) was confined to CD8 ؉ cells. Similar data were obtained in T cell populations activated in mixed lymphocyte cultures in the presence of IL-15. The expression of CD94͞NKG2A led to an impairment of allo-specific cytolytic activity by mixed lymphocyte culture-derived T cell populations or clones. Remarkably, cytolysis could be restored by the addition of anti-CD94 mAb, i.e., by masking the inhibitory NKRs.

After vaccination of melanoma patients with MAGE antigens, we observed that even in the few patients showing tumor regression, the frequency of anti-vaccine T cells in the blood was often either undetectable or <10−5 of CD8 T cells. This frequency being arguably too low for these cells to be sole effectors of rejection, we reexamined the contribution of T cells recognizing other tumor antigens. The presence of such antitumor T cells in melanoma patients has been widely reported. To begin assessing their contribution to vaccine-induced rejection, we evaluated their blood frequency in five vaccinated patients. The antitumor cytotoxic T lymphocyte (CTL) precursors ranged from 10−4 to 3 × 10−3, which is 10–10,000 times higher than the anti-vaccine CTL in the same patient. High frequencies were also observed before vaccination. In a patient showing nearly complete regression after vaccination with a MAGE-3 antigen, we observed a remarkably focused antitumoral response. A majority of CTL precursors (CTLp's) recognized antigens encoded by MAGE-C2, another cancer-germline gene. Others recognized gp100 antigens. CTLp's recognizing MAGE-C2 and gp100 antigens were already present before vaccination, but new clonotypes appeared afterwards. These results suggest that a spontaneous antitumor T cell response, which has become ineffective, can be reawakened by vaccination and contribute to tumor rejection. This notion is reinforced by the frequencies of anti-vaccine and antitumor CTLs observed inside metastases, as presented by Lurquin et al. (Lurquin, C., B. Lethé, V. Corbière, I. Théate, N. van Baren, P.G. Coulie, and T. Boon. 2004. J. Exp. Med. 201:249–257).

Natural killer (NK) cells express surface receptors for defined groups of HLA class I alleles. The specific interaction between these receptors and HLA class I molecules expressed on target cells results in inhibition of NK-mediated target cell lysis. In this report, we analyzed whether similar mechanisms were operating in cytolytic T lymphocytes (CTLs) capable of lysing NK-sensitive target cells. T cell clones were screened for their ability to lyse K562 target cells. The selected clones expressed either gamma delta or alpha beta TCR. The majority of these clones failed to lyse the HLA class I+ R8/15375 cell line; however, upon addition of the previously described A6-136 (IgM) or 6A4 F(ab')2 anti-HLA class I mAbs, target cells were efficiently lysed. Lysis of autologous phytohemagglutinin blasts in the presence of anti-HLA class I mAbs occurred primarily with TCR gamma delta+ CTLs. Recognition of HLA class I molecules on target cells implies the expression of NK-related specific receptors in CTL clones. Indeed, phenotypic analysis of > 300 CTL clones with NK-like activity revealed that 28% expressed p58 molecules (specific for HLA-C alleles) while 30% expressed CD94 molecules (specific for the Bw6 specificity). These receptor molecules were found to function as inhibitory receptors, as revealed by the effect of anti-p58 or anti-CD94 mAbs (of IgG isotype) on the lysis of the Fc gamma R+ K562 target cells.(ABSTRACT TRUNCATED AT 250 WORDS)