A lentiviral vector encoding fusion of light invariant chain and mycobacterial antigens induces protective CD4+ T cell immunity.

Lentiviral vectors (LVs) are highly efficient at inducing CD8+ T cell responses. However, LV-encoded antigens are processed inside the cytosol of antigen-presenting cells, which does not directly communicate with the endosomal major histocompatibility complex class II (MHC-II) presentation pathway. LVs are thus poor at inducing CD4+ T cell response. To overcome this limitation, we devised a strategy whereby LV-encoded antigens are extended at their N-terminal end with the MHC-II-associated light invariant chain (li), which contains an endosome-targeting signal sequence. When evaluated with an LV-encoded polyantigen composed of CD4+ T cell targets from Mycobacterium tuberculosis, intranasal vaccination in mice triggers pulmonary polyfunctional CD4+ and CD8+ T cell responses. Adjuvantation of these LVs extends the mucosal immunity to Th17 and Tc17 responses. A systemic prime and an intranasal boost with one of these LV induces protection against M. tuberculosis. This strategy improves the protective power of LVs against infections and cancers, where CD4+ T cell immunity plays an important role.

Monitoring Intracellular Routing of Internalized Antigens by Immunofluorescence Microscopy.

Antigen-presenting cells (APCs), especially macrophages and dendritic cells (DCs), are important for the induction of an adaptive immune response through their phagocytic capacity. APCs internalize extracellular antigens and, dependent on their intracellular localization, antigen-derived peptides are presented on MHC I or MHC II molecules. In context of antigen presentation and T cell activation tracking of internalized antigens is of high interest. In this article, we provide an immunofluorescence protocol and illustrate the analysis of intracellular routing of internalized antigens using the example of the model-antigen ovalbumin (OVA) in bone marrow-derived dendritic cells (BM-DCs). This protocol describes a procedure to stain such cells with an antibody against EEA-1, a marker for early endosomes, which can be easily adapted to other endosome markers, antigen-presenting cells, or antigens.