The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

The prognostic impact of mucinous carcinoma (MC) in colorectal cancer (CRC) has been subject to debate ever since the introduction of the classification of tumors according to their histological differentiation. MC is a distinct clinical and pathological entity within the spectrum of CRC and accounts for approximately 10-15 % of cases. Factors involved in MC development have not been completely understood, but clinical observations may lead to a better insight into the etiology of MC. In this article, we provide an in-depth review of the literature regarding etiological aspects of MC. We show that there are worldwide differences in the prevalence of MC, with low rates in Asian countries and higher rates in the western world. Moreover, MC is more commonly diagnosed in patients suffering from inflammatory bowel diseases or Lynch syndrome and an increased rate of MC is observed in patients with radiotherapy-induced CRCs. These findings are suggestive of a different oncogenic development. Identification of conditions that are associated with MC generates insight into the etiological pathways leading to the development of this special subtype.

Dendritic cells (DCs) are key in connecting innate and adaptive immunity. Their potential in inducing specific immune responses has made them interesting targets for immunotherapeutic approaches. Our research group was the first to exploit the naturally occurring myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in therapeutic vaccination trials against melanoma. To develop primary DC subsets as an optimal vaccine, the identification of a clinically applicable adjuvant activating both subsets is required. Although the expression of pathogen recognition receptors differs distinctly between the DC subsets, both pDCs and mDCs can respond to single-stranded RNA (ssRNA) via Toll-like receptors 7 and 8, respectively. Since ssRNA is easily degraded by RNases, we stabilized anionic RNA by complexing it with the positively charged protein protamine. This leads to the formation of protamine–RNA complexes with varying features depending on ionic content. We subsequently investigated the immunostimulatory effect of complexes that formed various salt concentrations on purified DC subsets. Both mDCs and pDCs upregulated maturation markers and produced pro-inflammatory cytokines in a dose-dependent way to the protamine–RNA complexes. This was dependent on endosomal acidification and correlated partly with the uptake of protamine–RNA complexes. Furthermore, both DC subsets induced T cell proliferation and IFN gamma secretion in a beneficial ratio to IL-10. These results indicate that protamine–RNA complexes can be used to stimulate human mDC and pDC ex vivo for use in immunotherapeutic settings.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-015-1746-9) contains supplementary material, which is available to authorized users.

Haploidentical hematopoietic stem cell transplantation (h-HSCT) represents an efficient curative approach for patients affected by hematologic malignancies in which the reduced intensity conditioning induces a state of immunologic tolerance between donor and recipient. However, opportunistic viral infections greatly affect h-HSCT clinical outcomes. Natural Killer (NK) cells are the first lymphocytes recovering after transplant and provide a prompt defense against human Cytomegalovirus (HCMV) infection/reactivation. By undertaking a longitudinal single cell computational profiling of multiparametric flow cytometry, we show that HCMV accelerates NK cell immune-reconstitution together with the expansion of CD158b1b2j pos /NKG2A neg /NKG2C pos /NKp30 low NK cells. The frequency of this subset correlates with HCMV viremia, further increases in recipients experiencing multiple episodes of viral reactivations and persists for months after the infection. The transcriptional profile of FACS-sorted CD158b1b2j pos NK cells confirmed the ability of HCMV to de-regulate NKG2C, NKG2A and NKp30 gene expression, thus inducing the expansion of NK cells with adaptive traits.These NK cells are characterized by the down-modulation of several gene pathways associated with cell migration, cell-cycle, effector-functions and by a state of metabolic/cellular exhaustion. This profile reflects the functional impairments of adaptive NK cells to produce IFN-, a phenomenon also due to the viral-induced expression of LAG-3 and PD-1 checkpoint-inhibitors.

Highlights d Human pDCs and cDC2s secrete a different set of chemokines upon activation d Activated human pDCs attract CD8 + T cells, CD56 + T cells, and gd T cells in vitro d cDC2-and pDC-based cancer vaccines attract lymphocytes in melanoma patient skin