Prime and Boost Vaccination Elicit a Distinct Innate Myeloid Cell Immune Response.

Understanding the innate immune response to vaccination is critical in vaccine design. Here, we studied blood innate myeloid cells after first and second immunization of cynomolgus macaques with the modified vaccinia virus Ankara. The inflammation at the injection site was moderate and resolved faster after the boost. The blood concentration of inflammation markers increased after both injections but was lower after the boost. The numbers of neutrophils, monocytes, and dendritic cells were transiently affected by vaccination, but without any major difference between prime and boost. However, phenotyping deeper those cells with mass cytometry unveiled their high phenotypic diversity with subsets responding differently after each injection, some enriched only after the primary injection and others only after the boost. Actually, the composition in subphenotype already differed just before the boost as compared to just before the prime. Multivariate analysis identified the key features that contributed to these differences. Cell subpopulations best characterizing the post-boost response were more activated, with a stronger expression of markers involved in phagocytosis, antigen presentation, costimulation, chemotaxis, and inflammation. This study revisits innate immunity by demonstrating that, like adaptive immunity, innate myeloid responses differ after one or two immunizations.

In depth comparative phenotyping of blood innate myeloid leukocytes from healthy humans and macaques using mass cytometry.

Comparative immune-profiling of innate responses in humans and non-human primates is important to understand the pathogenesis of infectious and chronic inflammatory diseases as well as for the preclinical development of vaccines and immune therapies. However, direct comparisons of the two species are rare and were never performed using mass cytometry. Here, whole-blood-derived leukocytes from healthy humans and cynomolgus macaques were analyzed with mass cytometry. Two similar panels of around 30 monoclonal antibodies targeting human markers associated with innate myeloid cells to stain fixed human and macaque leukocytes were constructed. To compare the circulating innate cells from the two primate species, an analysis pipeline combining a clustering analysis by the Spanning-tree Progression Analysis of Density-normalized Events (SPADE) algorithm with a two-step hierarchical clustering of cells nodes and markers was used. Identical SPADE settings were applied to both datasets, except for the 20 clustering markers which slightly differed. A correlation analysis designed to compare the phenotypes of human and macaque cell nodes and based on 16 markers, including 15 shared clustering markers and CD19 for humans or CD20 for macaques, revealed similarities and differences between staining patterns. This study unique by the number of individuals (26 humans and 5 macaques) and the use of mass cytometry certainly contributes to better assess the advantages and limits of the use of non-human primates in preclinical research. © 2017 International Society for Advancement of Cytometry.

Redirecting soluble antigen for MHC class I cross-presentation during phagocytosis.

Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen-presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross-presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross-presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross-presentation pathway.