A reliable method for enrichment of neutrophils from peripheral blood in barramundi (Lates calcarifer)

Neutrophils are a short-lived, terminally differentiated, innate immune cell, that are critical first responders during infection. Research into neutrophil-pathogen interactions in fish has primarily employed cells derived from the pro-nephros and nephros. Since these sites are also the location of neutrophil and other immune cell development, there may be some ambiguity in maturation and functional ability of these cells, and difficulty in differentiating the effects of neutrophils from those of macrophages and monocytes. In contrast, peripheral blood circulating neutrophils are mature and ready to respond, thus it may be more physiologically relevant to use these cells for immune studies when evaluating interactions with blood-borne pathogens. The enrichment of tropical, euryhaline fish blood cells cannot follow classic mammalian enrichment methods for several reasons: Fish have nucleated red blood cells (RBC's), a high number of reticulocytes, a very low number of granulocytic leukocytes and an osmotic tolerance, rendering techniques such as water lysis ineffective. Enrichment of neutrophils, while minimizing RBC contamination, is imperative for studies where luminescence or fluorescence signals may be confounded by background from an overabundance of RBC's. We have optimized a method for enriching neutrophils from peripheral blood, with an initial settlement step employing 6% dextran (Mr 450,000–650,000), for 30–60 min at room temperature, followed by density separation on an 8-step Percoll density gradient. This method provides a cell suspension comprising 20–50% neutrophils, free of contamination from reticulocytes. These are then suitable for luminometric or fluorometric downstream analyses

Human CLEC9A antibodies deliver Wilms' tumor 1 (WT1) antigen to CD141+ dendritic cells to activate naïve and memory WT1‐specific CD8+ T cells

Objectives Vaccines that prime Wilms' tumor 1 (WT1)‐specific CD8+ T cells are attractive cancer immunotherapies. However, immunogenicity and clinical response rates may be enhanced by delivering WT1 to CD141+ dendritic cells (DCs). The C‐type lectin‐like receptor CLEC9A is expressed exclusively by CD141+ DCs and regulates CD8+ T‐cell responses. We developed a new vaccine comprising a human anti‐CLEC9A antibody fused to WT1 and investigated its capacity to target human CD141+ DCs and activate naïve and memory WT1‐specific CD8+ T cells. Methods WT1 was genetically fused to antibodies specific for human CLEC9A, DEC‐205 or β‐galactosidase (untargeted control). Activation of WT1‐specific CD8+ T‐cell lines following cross‐presentation by CD141+ DCs was quantified by IFNγ ELISPOT. Humanised mice reconstituted with human immune cell subsets, including a repertoire of naïve WT1‐specific CD8+ T cells, were used to investigate naïve WT1‐specific CD8+ T‐cell priming. Results The CLEC9A‐WT1 vaccine promoted cross‐presentation of WT1 epitopes to CD8+ T cells and mediated priming of naïve CD8+ T cells more effectively than the DEC‐205‐WT1 and untargeted control‐WT1 vaccines. Conclusions Delivery of WT1 to CD141+ DCs via CLEC9A stimulates CD8+ T cells more potently than either untargeted delivery or widespread delivery to all Ag‐presenting cells via DEC‐205, suggesting that cross‐presentation by CD141+ DCs is sufficient for effective CD8+ T‐cell priming in humans. The CLEC9A‐WT1 vaccine is a promising candidate immunotherapy for malignancies that express WT1

IL-1 beta enhances CD40 ligand-mediated cytokine secretion by human dendritic cells (DC): A mechanism for T cell-independent DC activation

CD40 ligand (CD40L) is a membrane-bound molecule expressed by activated T cells. CD40L potently induces dendritic cell (DC) maturation and IL-12p70 secretion and plays a critical role during T cell priming in the lymph nodes. IFN-γ and IL-4 are required for CD40L-mediated cytokine secretion, suggesting that T cells are required for optimal CD40L activity. Because CD40L is rapidly up-regulated by non-T cells during inflammation, CD40 stimulation may also be important at the primary infection site. However, a role for T cells at the earliest stages of infection is unclear. The present study demonstrates that the innate immune cell-derived cytokine, IL-1β, can increase CD40L-induced cytokine secretion by monocyte-derived DC, CD34-derived DC, and peripheral blood DC independently of T cell-derived cytokines. Furthermore, IL-1β is constitutively produced by monocyte-derived DC and monocytes, and is increased in response to intact Escherichia coli or CD40L, whereas neither CD34-derived DC nor peripheral blood DC produce IL-1β. Finally, DC activated with CD40L and IL-1β induce higher levels of IFN-γ secretion by T cells compared with DC activated with CD40L alone. Therefore, IL-1β is the first non-T cell-derived cytokine identified that enhances CD40L-mediated activation of DC. The synergy between CD40L and IL-1β highlights a potent, T cell-independent mechanism for DC activation during the earliest stages of inflammatory responses

Cross-priming of CD8+ T cells by viral and tumor antigens is a robust phenomenon

Cross-priming refers to the activation of naive CD8+ T cells by antigen-presenting cells that have acquired nominal antigens from another cell. The biological relevance of cross-priming of CD8+ T cells has recently been challenged (Zinkernagel, R. M., Eur. J. Immunol. 2002. 32: 2385-2392), on the basis that responses are weak or poorly quantitated, and the determinants recognized are undefined. Here we show that cross-priming is a robust process that elicits vigorous primary responses to multiple peptides in two well-defined systems. Our findings support the relevance of cross-priming in CD8+ T cell responses to viruses and tumor cells, and demonstrate that cross-priming elicits CD8+ T cells to determinants generated by the endogenous processing pathway