Several recent studies have demonstrated that T-helper cell-dependent events during the initial priming period are required for the generation of CD8 ؉ T cell-mediated protective immunity. The underlying mechanisms of this phenomenon have not yet been determined, mostly because of difficulties in studying memory T cells or their precursor populations at early stages during immune responses. We identified IL-7 receptor (CD127) surface expression as a marker for long-living memory T cells, most importantly allowing the distinction between memory and effector T cells early after in vivo priming. The combination of surface staining for CD127 and CD62L further separates between two functionally distinct memory cell subsets, which are similar (if not identical) to cell subsets recently described as central memory T cells (CD127 high and CD62L high ) and peripheral effector memory T cells (CD127 high and CD62L low ). Using this new tool of memory T cell analysis, we demonstrate that CD8 ؉ T cell priming in the absence of T cell help or CD40L specifically alters the generation of the effector memory T cell subset, which appears to be crucial for immediate memory responses and long-term maintenance of effective protective immunity. Our data reveal a unique strategy to obtain information about the quality of long-term protective immunity early during an immune response, a finding that may be applied in a variety of clinical settings, including the rapid monitoring of vaccination success.

Upon first antigen encounter, naive CD8(+) T cells get activated, clonally expand, and can develop into very distinct subsets, such as short-living effector cells or different memory subpopulations. The origin of subset diversification is currently unknown, but qualitative and quantitative differences in early signals received by individual precursor cells have been suggested as a major determinant. We show that transfer of a single antigen-specific naive T cell into a normal recipient mouse allowed recovery of clonally expanded daughter cells upon immunization. With this experimental approach, we conclusively demonstrated that a wide range of diversity could develop out of a single precursor cell, including different types of effector and memory T cells. Interestingly, single-cell-derived subset diversification resembled that of polyclonal T cell responses in the same individual mouse, although differentiation patterns differed between immunization strategies. These data implicate that subset diversification is both shaped and synchronized during the expansion phase.

We analyzed whether the immunobiology of vaccinating plasmid DNA containing a transcription unit for OVA is influenced by immunostimulatory CpG motifs in the plasmid backbone. Indeed, plasmid DNA differentially activated in vitro myeloid and plasmacytoid dendritic cells (DCs) provided they expressed the CpG-DNA receptor, Toll-like receptor 9 (TLR9). Dependent on the DC subset, activation resulted in type 1 IFN production, while both DC subsets produced IL-6 and up-regulated expression of costimulatory molecules CD40 and CD86. In vivo, however, even upon repeated vaccination with plasmid DNA, priming of OVA-specific CTL and clonal expansion of SIINFEKL-specific CD8 T cells were equal in TLR9-positive and TLR9- or MyD88-negative mice. Overall, these results negate a dominant role of CpG-DNA/TLR9 interactions in long-term vaccination protocols.

CpG-rich oligonucleotides (CpG-ODN) bind to Toll-like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG-ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG-ODN resulted in antigen-specific T cell activation in local lymph nodes. In contrast, systemic application of CpG-ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3-dioxygenase (IDO) as indicated by the observation that CpG-ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1-methyl-tryptophan (1-MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG-ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG-ODN-induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG-ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down-modulation of immune responses by CpG-ODN may be possible in certain pathological conditions. See accompanying commentary: http://dx

Covalent linkage of immunostimulatory CpG-DNA to OVA (CpG-OVA complex) results in CpG-DNA-aided cross-presentation of OVA by dendritic cells (DCs). In this study, we analyzed the thesis that CpG-OVA complexes may be cross-presented by B cells to route internalized Ag into the class I MHC presentation pathway. First, we describe that conjugation of CpG-DNA to OVA enhances up to 40-fold internalization of OVA by B cells, which in turn generate the CD8 T cell epitope SIINFEKL complexed to MHC class I, albeit less efficiently than DCs. Furthermore, upon internalization, CpG-DNA conjugated to OVA stimulates B cells to up-regulate costimulatory molecules and cytokines including IL-12. Adoptive transfer of CpG-OVA complex-loaded wild-type B cells cross-primes naive CD8 T cells both in wild-type mice and in MyD88-deficient mice. Overall, these findings disclose attributes of B cells, including cross-presentation of exogenous Ag and cross-priming of naive CD8 T cells that hitherto have been considered as hallmarks restricted to DCs.

Covalent linkage of immunostimulatory CpG DNA to OVA results in CpG DNA-aided cross-presentation of OVA by dendritic cells (DCs). In vivo, cross-presentation is conditional for cross-priming of OVA-specific CD8 T cells. In this study, we investigated the involvement of the CpG DNA receptor Toll-like receptor (TLR)9 in CpG DNA-aided cross-presentation and cross-priming. Although CpG DNA-aided cross-presentation is not altered in TLR9-deficient cells, TLR9 is required for maturation of APC allowing cross-priming, as resulting in CTL function. These findings imply that TLR9 does not trigger endocytosis of CpG-OVA conjugates, but activates DCs downstream of endocytosis.

In contrast to infectious (live) vaccines are those based on subunit Ag that are notoriously poor in eliciting protective CD8 T cell responses, presumably because subunit Ags become insufficiently cross-presented by dendritic cells (DCs) and because the latter need to be activated to acquire competence for cross-priming. In this study, we show that CpG-Ag complexes overcome these limitations. OVA covalently linked to CpG-DNA (CpG-OVA complex), once it is efficiently internalized by DCs via DNA receptor-mediated endocytosis, is translocated to lysosomal-associated membrane protein 1 (LAMP-1)-positive endosomal-lysosomal compartments recently shown to display competence for cross-presentation. In parallel, CpG-OVA complex loaded DCs become activated and acquire characteristics of professional APCs. In vivo, a single s.c. dose of CpG-OVA complex (10 μg of protein) induces primary and secondary clonal expansion/contraction of Ag-specific CD8 T cells similar in kinetics to live vaccines; examples including Listeria monocytogenes genetically engineered to produce OVA (LM-OVA) and two viral vector-based OVA vaccines analyzed. Interestingly, CpG-OVA complex induced almost equal percentages of Ag-specific memory CD8 T cells as did infection with LM-OVA. A single dose vaccination with CpG-OVA complex protected mice against lethal doses of LM-OVA. These data underscore that the synergy imparted by CpG-OVA complex-mediated combined triggering of innate and specific immunity might be key to initiate CD8 T cell-based immunoprotection by synthetic vaccines based on subunit Ag.

The clinical association between viral infection and onset or exacerbation of autoimmune disorders remains poorly understood. Here, we examine the relative roles of molecular mimicry and nonspecific inflammatory stimuli in progression from infection to autoimmune disease. Murine herpes virus 1 (HSV-1 KOS) infection triggers T cell-dependent autoimmune reactions to corneal tissue. We generated an HSV-1 KOS point mutant containing a single amino acid exchange within the putative mimicry epitope as well as mice expressing a TCR transgene specific for the self-peptide mimic to allow dissection of two pathogenic mechanisms in disease induction. These experiments indicate that viral mimicry is essential for disease induction after low-level viral infection of animals containing limited numbers of autoreactive T cells, while innate immune mechanisms become sufficient to provoke disease in animals containing relatively high numbers of autoreactive T cells.