Cross-priming allows dendritic cells (DCs) to induce cytotoxic T cell (CTL) responses to extracellular antigens. DCs require cognate 'licensing' for cross-priming, classically by helper T cells. Here we demonstrate an alternative mechanism for cognate licensing by natural killer T (NKT) cells recognizing microbial or synthetic glycolipid antigens. Such licensing caused cross-priming CD8alpha(+) DCs to produce the chemokine CCL17, which attracted naive CTLs expressing the chemokine receptor CCR4. In contrast, DCs licensed by helper T cells recruited CTLs using CCR5 ligands. Thus, depending on the type of antigen they encounter, DCs can be licensed for cross-priming by NKT cells or helper T cells and use at least two independent chemokine pathways to attract naive CTLs. Because these chemokines acted synergistically, this can potentially be exploited to improve vaccinations.

CD81 (TAPA-1) is a member of the widely expressed and evolutionary conserved tetraspanin family that forms complexes with a variety of other cell surface receptors and facilitates hepatitis C virus entry. Here, we show that CD81 is specifically required for the formation of lamellipodia in migrating dendritic cells (DCs). Mouse CD81 ؊/؊ DCs, or murine and human CD81 RNA interference knockdown DCs lacked the ability to form actin protrusions, thereby impairing their motility dramatically. Moreover, we observed a selective loss of Rac1 activity in the absence of CD81, the latter of which is exclusively required for integrin-dependent migration on 2-dimensional substrates. Neither integrin affinity for substrate nor the size of basal integrin clusters was affected by CD81 deficiency in adherent DCs. However, the use of total internal reflection fluorescence microscopy revealed an accumulation of integrin clusters above the basal layer in CD81 knockdown cells. Furthermore, ␤1-or ␤2-integrins, actin, and Rac are strongly colocalized at the leading edge of DCs, but the very fronts of these cells protrude CD81-containing membranes that project outward from the actin-integrin area. Taken together, these data suggest a thus far unappreciated role for CD81 in the mobilization of preformed integrin clusters into the leading edge of migratory DCs on 2-dimensional

Dendritic cells (DCs) are the main inducers and regulators of cytotoxic T lymphocyte (CTL) responses against viruses and tumors. One checkpoint to avoid misguided CTL activation, which might damage healthy cells of the body, is the necessity for multiple activation signals, involving both antigenic as well as additional signals that reflect the presence of pathogens. DCs provide both signals when activated by ligands of pattern recognition receptors and “licensed” by helper lymphocytes. Recently, it has been established that such T cell licensing can be facilitated by CD4+ T helper cells (“classical licensing”) or by natural killer T cells (“alternative licensing”). Licensing regulates the DC/CTL cross-talk at multiple layers. Direct recruitment of CTLs through chemokines released by licensed DCs has recently emerged as a common theme and has a crucial impact on the efficiency of CTL responses. Here, we discuss recent advances in our understanding of DC licensing for cross-priming and implications for the temporal and spatial regulation underlying this process. Future vaccination strategies will benefit from a deeper insight into the mechanisms that govern CTL activation.

Cross-presentation allows dendritic cells (DCs) to load antigenic peptides on MHC-I molecules, which is important for activating cytotoxic T cells (CTL) that combat tumors and viruses. We recently showed that, under steady-state conditions, antigens internalized by DCs through the Mannose Receptor (MR) are efficiently cross-presented (1). The MR conveyed antigen into early endosomes, where peptide was loaded on MHC-I (2, 3). Recently, Segura et al. (4) reported that MR-mediated endocytosis is involved in cross-presentation only under inflammatory and not steady-state conditions (4), which, at first sight, contradicted our findings (1). They concluded that the MR operates only in bone marrow-or monocyte-derived DCs but not in CD8 + DCs, which are thought to be most relevant for in vivo cross-presentation in mice. Certainly, many immunologic phenomena may be explained by differential involvement of DC subsets, but in this case, two alternative explanations more likely explain the findings of Segura et al. (4). First, Segura et al. (4) reported that splenic CD8 + DCs do not express the MR and thus, cannot use it for ovalbumin (OVA) uptake. This statement was reached using splenic DCs isolated by collagenase digestion, which were subsequently exposed to OVA. However, collagen fragments are MR ligands (5). The massive amounts of such fragments, which are inevitably generated during collagenase digestion, bind the MR and cause its internalization. Segura et al. (4) stained for MR expression on the DC surface and thus, failed to detect intracellular MR. We had previously used intracellular staining and detected the MR within splenic DCs (2). Splenic DCs did endocytose OVA through the MR when we used a collagenasefree DC isolation technique (Fig. 1). Moreover, MR-mediated endocytosis by bone marrow-derived DCs was abolished when lysates from collagenase-digested spleens were added, confirming their ability to block the MR (Fig. 1). Second, antigen dose is a major determinant of immunity. Previously, we had injected mice with 0.1 mg OVA and showed that the absence of the MR reduced cross-presentation severely, albeit not completely (1). This confirmed its dominant

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