The outcome of cross-priming during virus infection is not directly linked to the ability of the antigen to be cross-presented.

The initiation of CD8(+) T cell (CTL) immune responses can occur via cross-priming. Recent data suggested a relationship between cross-presentation and immunodominance of epitope-specific T cells. To test this association, we evaluated the efficacy of cross-presentation for several virus epitopes in vitro and examined if this can be extrapolated in vivo. Employing lymphocytic choriomeningitis virus (LCMV), we demonstrate that the cross-presentation and cross-priming of LCMV antigens were dominated by NP396, but not NP205 when analyzing the LCMV-NP. Although with LCMV-GP, cross-presentation was dominated by GP276, and cross-priming was dominated by GP33. Importantly, although NP396 was significantly more efficient than GP33 in cross-presentation, cross-priming of their specific CTL was comparable. In a subsequent virus challenge after cross-priming, GP33-specific CTL dominated the response. Accordingly, based on our data, the ability of viral epitopes to be cross-presented in vitro does not entirely reflect what would occur in cross-priming. Thus, weak cross-presenting antigens may still cross-prime an efficient CTL response depending on other in vivo elements such as the naïve T-cell precursor frequencies.

Cross, but not direct, presentation of cell-associated virus antigens by spleen macrophages is influenced by their differentiation state.

The initiation of T-cell immune responses requires professional antigen-presenting cells. Emerging data point towards an important role for macrophages (Mphi) in the priming of naïve T cells. In this study we analyzed the efficiency and the mechanisms by which Mphi derived from spleen (Sp-Mphi) or bone marrow (BM-Mphi) present Lymphocytic choriomeningitis virus (LCMV) antigens to epitope-specific T cells. We demonstrate that because of phagosomal maturation, Sp-Mphi downregulate their ability to cross-present cell-associated, but not soluble, antigens, as they are further differentiated in culture without altering their capacity to directly present virus antigens after infection. We propose that Sp-Mphi are extremely efficient at direct and cross-presentation. However, if these cells undergo further M-CSF-dependent maturation, they will adapt to be more scavenger and phagocytic and concurrently reduce their cross-presenting capacity. Accordingly, Sp-Mphi can have an important role in regulating T-cell responses through cross-presentation depending on their differentiation state.

Delivery of Exogenous Antigens to Induce Cytotoxic CD8+ T Lymphocyte Responses.

Vaccines intended to induce a cytotoxic CD8+ T-cell response are highly sought after. However, some of these vaccines can be problematic if they replicate in the host. An alternative strategy is to exploit cross-presentation of exogenous antigens to express peptides on major histocompatibility complex (MHC) class I molecules. During cross-presentation, the delivered exogenous antigen can be taken up and processed through diverse mechanisms. Here, we will discuss the recent advances regarding the complex nature of the cross-priming process and the models that reflect its relevance in vivo. Moreover, we summarize current data that explore potential adjuvants and vaccine vectors that deliver antigens to activate CD8+ T cells relying on cross-presentation.

An efficient culture method for generating large quantities of mature mouse splenic macrophages.

In this study, we established an efficient in vitro culture method for generating mature splenic macrophages (Sp-Mphi). Splenocytes were cultured in the presence of conditioned medium containing macrophage colony-stimulating factor (M-CSF) for 7 days post post-isolation and the generated Sp-Mphi were characterized phenotypically and functionally. Through this method, 9 x 10(6)/mouse Sp-Mphi were obtained in comparison to 2 x 10(5)/mouse when Mphi were cultured in regular medium. In addition, the purity of these cells was as high as 80% by day 5 and >90% by day 7 of culturing, confirmed with Mphi-specific markers. The increased Sp-Mphi yields, in the presence of M-CSF, point towards the existence of a precursor population in the spleen that can be influenced to differentiate into Sp-Mphi. Moreover, we compared the maturation of generated Sp-Mphi to conventional bone marrow-derived Mphi (BM-Mphi) in vitro. Interestingly, Sp-Mphi exhibited lower capacity to phagocytose dead cells after 3 days of maturation, but showed similar internalizing capacity after 5 and 7 of maturation to BM-Mphi cultured for the same time period. Importantly, Sp-Mphi upregulated the expression of several surface markers such as MOMA-2 and CD68 while downregulating SIGN-R1 after 7 days, indicating that these Sp-Mphi undergo further maturation in vitro due to culturing in M-CSF. Taken together, we describe and validate a method for generating Sp-Mphi in large quantities and high purity. These data should prove valuable in future studies characterizing the functions and maturation of Sp-Mphi.

Cross-priming of a single viral protein from lymphocytic choriomeningitis virus alters immunodominance hierarchies of CD8+ T cells during subsequent viral infections.

Immunogenic epitopes that stimulate CD8+ T cells can be organized into an immunodominance hierarchy, based on their ability to induce T-cell priming and subsequent expansion. Cytotoxic CD8+ T cells can be primed through the cross-priming pathway, where exogenous viral proteins are acquired by professional antigen-presenting cells (pAPCs). We have previously reported that lymphocytic choriomeningitis nucleoprotein (LCMV-NP) expressed in HEK cells (HEK-NP) induces cross-priming of CD8+ T cells in vivo. In this study, we have used this HEK-NP model to study the effects of LCMV-NP cross-priming on the LCMV immunodominance hierarchy following viral challenge. Our results highlight the contribution of cross-priming to the immune response, since the T-cell hierarchy was significantly altered as a result of exogenous processing of a single virus protein, and this phenomenon was maintained throughout the memory response. Moreover, as a result of cross-priming, in vivo CD8+ T-cell killer activity was enhanced during subsequent virus assaults. These findings have significant implications for immunotherapy because they demonstrate that exogenous delivery of specific T-cell epitopes can be utilized to manipulate the host's CD8+ T-cell memory immunodominance responses.

TLR engagement prior to virus infection influences MHC-I antigen presentation in an epitope-dependent manner as a result of nitric oxide release.

Microorganisms contain PAMPs that can interact with different TLR-Ls. Cooperative signals from these receptors may modify innate and adaptive immune responses to invading pathogens. Therefore, a better understanding of the role TLRs play in initiating host defense during infections requires assessing the influence of multiple TLR engagement on pAPC activation and antigen presentation. In this study, we investigated the effects of combined TLR2, TLR3, or TLR4 engagement on DC activation and the presentation of LCMV antigens focusing on the major epitopes derived from NP and GP proteins encoded by the virus. Our results demonstrate that combined TLR ligation affected antigen presentation of NP(205-212), GP(33-41), and GP(276-286), but not NP(396-404). The altered antigen presentation was associated with changes in proteasomal activities and NO production as a result of TLR engagement. Taken together, the data demonstrate that combined TLR ligation could result in changes of innate effectors that may directly influence the adaptive immune response.