Bovine neonate natural killer cells are fully functional and highly responsive to interleukin-15 and to NKp46 receptor stimulation

Natural killer (NK) cells are key components of the innate immune system with their killing and cytokine producing abilities. Bovine NK cells have been characterized as NKp46+/CD3− lymphocytes, but little is known about these cells in neonatal calves. As the newborn calf, with an insufficiently developed acquired immunity, has to employ the innate immune system, we wanted to investigate whether neonate NK cells had the same characteristics as cells from older calves. Freshly isolated neonate and calf NK cells presented the same resting CD2+/CD25low/CD8−/low phenotype. Neonates less than 8 days old had one third of the circulating NKp46+ cells of older calves, but the NK cells proliferated more actively in vitro in the presence of interleukin (IL)-2 or IL-15. Moreover, neonate NK cells were more cytotoxic both in an NKp46 mediated redirected lysis assay and in direct killing of a bovine cell line MDBK when cultured in the presence of IL-15. Neonate and calf NK cells cultured in the presence of IL-2 and then stimulated with IL-12 produced similar dose-dependent interferon (IFN)-γ amounts, while IL-15 cultured NK cells did not give such a response whatever the age. However, neonatal NK cells cultured in IL-15 and stimulated by IL-12 concomitantly with cross-linking of NKp46, produced 4 to 5 times more IFN-γ than calf NK cells. These data suggest that although present in lower number at birth, neonate NK cells are fully functional and are more responsive to IL-15 and activation through the NKp46 receptor than NK cells from older calves

Ovine CD16+/CD14− blood lymphocytes present all the major characteristics of natural killer cells

Natural killer (NK) cells have a key role in the innate immune response against pathogens because of their cytotoxic properties and production of interferon-gamma (IFN-γ). Some insight into ruminant NK cell biology has been gained through the characterization of bovine NK cells as NKp46+/CD3− cells. However, ovine NK cells have been little studied because of the lack of specific antibodies. Most NK cells in humans and cattle express CD16. We found that an antibody against human CD16 that cross-reacts with bovine NK cells also recognizes cell populations in ovine peripheral blood mononuclear cells. Using double labelling with CD14 revealed the same profile as described in other species, and we identified a putative NK cell population. We therefore sorted this ovine CD16+/CD14− cell population and tested it for NK cell characteristics. More than 80% of sorted CD16+/CD14− cells expressed perforin. After a week of culture in the presence of IL-2 and IL-15, ovine CD16+/CD14− cells had become large cells with intra-cytoplasmic granules containing perforin, and the vast majority displayed an activated CD2−/low/CD25+/CD8+ phenotype, as observed for bovine NKp46+/CD3− cells. Moreover, these cells expressed transcripts for the NKp46 receptor, and were cytotoxic in a CD16-mediated redirected lysis assay against a murine cell line, P815, and in a direct lysis assay against the ovine cell line, IDO5. Finally, ovine CD16+/CD14− cells having expanded for 7 days in culture secreted IFN-γ in response to IL-12 in a dose-dependent manner. Taken together, these findings led us to conclude that the ovine CD16+/CD14− lymphocyte sub-population displays the phenotype and functional characteristics of NK cells

Evaluating the Efficiency of Isotope Transmission for Improved Panel Design and a Comparison of the Detection Sensitivities of Mass Cytometer Instruments

International audienceThe recent introduction of mass cytometry, a technique coupling a cell introduction system generating a stream of single cells with mass spectrometry, has greatly increased the number of parameters that can be measured per single cell. As with all new technology there is a need for dissemination of standardization and quality control procedures. Here, we characterize variations in sensitivity observed across the mass range of a mass cytometer, using different lanthanide tags. We observed a five-fold difference in lanthanide detection over the mass range and demonstrated that each instrument has its own sensitivity pattern. Therefore, the selection of lanthanide combinations is a key step in the establishment of a staining panel for mass cytometry-based experiments, particularly for multicenter studies. We propose the sensitivity pattern as the basis for panel design, instrument standardization and future implementation of normalization algorithms

Towards a universal definition of the dendritic cell subsets using comparative transcriptomics across warm blooded vertebrates

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

Abstract 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

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

International audienceAbstract 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

NK cell immune responses differ after prime and boost vaccination

International audienceA better understanding of innate responses induced by vaccination is critical for designing optimal vaccines. Here, we studied the diversity and dynamics of the NK cell compartment after prime-boost immunization with the modified vaccinia virus Ankara using cynomolgus macaques as a model. Mass cytometry was used to deeply characterize blood NK cells. The NK cell sub-phenotype composition was modified by the prime. Certain phenotypic changes induced by the prime were maintained over time and, as a result, the NK cell composition prior to boost differed from that before prime. The key phenotypic signature that distinguished NK cells responding to the boost from those responding to the prime included stronger expression of several cytotoxic, homing, and adhesion molecules, suggesting that NK cells at recall were functionally distinct. Our data reveal potential priming or imprinting of NK cells after the first vaccine injection. This study provides novel insights into prime-boost vaccination protocols that could be used to optimize future vaccines

Pig Skin Includes Dendritic Cell Subsets Transcriptomically Related to Human CD1a and CD14 Dendritic Cells Presenting Different Migrating Behaviors and T Cell Activation Capacities

Swine skin is one of the best structural models for human skin, widely used to probe drug transcutaneous passage and to test new skin vaccination devices. However, little is known about its composition in immune cells, and among them dendritic cells (DC), that are essential in the initiation of the immune response. After a first seminal work describing four different DC subpopulations in pig skin, we hereafter deepen the characterization of these cells, showing the similarities between swine DC subsets and their human counterparts. Using comparative transcriptomic study, classical phenotyping as well as in vivo and in vitro functional studies, we show that swine CD163(pos) dermal DC (DDC) are transcriptomically similar to the human CD14(pos) DDC. CD163(pos) DDC are recruited in inflamed skin, they migrate in inflamed lymph but they are not attracted toward CCL21, and they modestly activate allogeneic CD8 T cells. We also show that CD163(low) DDC are transcriptomically similar to the human CD1a(pos) DDC. CD163(low) DDC migrate toward CCL21, they activate allogeneic CD8 and CD4 T cells and, like their potential human lung counterpart, they skew CD4 T cells toward a Th17 profile. We thus conclude that swine skin is a relevant model for human skin vaccination