Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

IKKbeta-dependent NF-kappaB activation plays a key role in innate immunity and inflammation, and inhibition of IKKbeta has been considered as a likely anti-inflammatory therapy. Surprisingly, however, mice with a targeted IKKbeta deletion in myeloid cells are more susceptible to endotoxin-induced shock than control mice. Increased endotoxin susceptibility is associated with elevated plasma IL-1beta as a result of increased pro-IL-1beta processing, which was also seen upon bacterial infection. In macrophages enhanced pro-IL-1beta processing depends on caspase-1, whose activation is inhibited by NF-kappaB-dependent gene products. In neutrophils, however, IL-1beta secretion is caspase-1 independent and depends on serine proteases, whose activity is also inhibited by NF-kappaB gene products. Prolonged pharmacologic inhibition of IKKbeta also augments IL-1beta secretion upon endotoxin challenge. These results unravel an unanticipated role for IKKbeta-dependent NF-kappaB signaling in the negative control of IL-1beta production and highlight potential complications of long-term IKKbeta inhibition.

A core feature of protective T cell responses to infection is the robust expansion and diversification of naïve antigen-specific T cell populations into short-lived effector and long-lived memory subsets. By means of in vivo fate mapping, we found a striking variability of immune responses derived from individual CD8(+) T cells and show that robust acute and recall immunity requires the initial recruitment of multiple precursors. Unbiased mathematical modeling identifies the random integration of multiple differentiation and division events as the driving force behind this variability. Within this probabilistic framework, cell fate is specified along a linear developmental path that progresses from slowly proliferating long-lived to rapidly expanding short-lived subsets. These data provide insights into how complex biological systems implement stochastic processes to guarantee robust outcomes.

It has been proposed that two amino acid substitutions in the transcription factor FOXP2 have been positively selected during human evolution due to effects on aspects of speech and language. Here, we introduce these substitutions into the endogenous Foxp2 gene of mice. Although these mice are generally healthy, they have qualitatively different ultrasonic vocalizations, decreased exploratory behavior and decreased dopamine concentrations in the brain suggesting that the humanized Foxp2 allele affects basal ganglia. In the striatum, a part of the basal ganglia affected in humans with a speech deficit due to a nonfunctional FOXP2 allele, we find that medium spiny neurons have increased dendrite lengths and increased synaptic plasticity. Since mice carrying one nonfunctional Foxp2 allele show opposite effects, this suggests that alterations in cortico-basal ganglia circuits might have been important for the evolution of speech and language in humans.

Several recent studies have demonstrated that T-helper cell-dependent events during the initial priming period are required for the generation of CD8 ؉ T cell-mediated protective immunity. The underlying mechanisms of this phenomenon have not yet been determined, mostly because of difficulties in studying memory T cells or their precursor populations at early stages during immune responses. We identified IL-7 receptor (CD127) surface expression as a marker for long-living memory T cells, most importantly allowing the distinction between memory and effector T cells early after in vivo priming. The combination of surface staining for CD127 and CD62L further separates between two functionally distinct memory cell subsets, which are similar (if not identical) to cell subsets recently described as central memory T cells (CD127 high and CD62L high ) and peripheral effector memory T cells (CD127 high and CD62L low ). Using this new tool of memory T cell analysis, we demonstrate that CD8 ؉ T cell priming in the absence of T cell help or CD40L specifically alters the generation of the effector memory T cell subset, which appears to be crucial for immediate memory responses and long-term maintenance of effective protective immunity. Our data reveal a unique strategy to obtain information about the quality of long-term protective immunity early during an immune response, a finding that may be applied in a variety of clinical settings, including the rapid monitoring of vaccination success.

Aging is a major risk factor for a large number of disorders and functional impairments. Therapeutic targeting of the aging process may therefore represent an innovative strategy in the quest for novel and broadly effective treatments against age-related diseases. The recent report of lifespan extension in mice treated with the FDA-approved mTOR inhibitor rapamycin represented the first demonstration of pharmacological extension of maximal lifespan in mammals. Longevity effects of rapamycin may, however, be due to rapamycin's effects on specific life-limiting pathologies, such as cancers, and it remains unclear if this compound actually slows the rate of aging in mammals. Here, we present results from a comprehensive, large-scale assessment of a wide range of structural and functional aging phenotypes, which we performed to determine whether rapamycin slows the rate of aging in male C57BL/6J mice. While rapamycin did extend lifespan, it ameliorated few studied aging phenotypes. A subset of aging traits appeared to be rescued by rapamycin. Rapamycin, however, had similar effects on many of these traits in young animals, indicating that these effects were not due to a modulation of aging, but rather related to aging-independent drug effects. Therefore, our data largely dissociate rapamycin's longevity effects from effects on aging itself.