Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as 'non-self' neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, ∼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide-MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.

Neuropilin-1 is identified as a surface marker to distinguish different Foxp3+ T reg cell subsets under homeostatic conditions.

Pattern recognition receptors (PRRs) play an essential role in a macrophage's response to mycobacterial infections. However, how these receptors work in concert to promote this macrophage response remains unclear. In this study, we used bone marrow-derived macrophages isolated from mannose receptor (MR), complement receptor 3 (CR3), MyD88, Toll-like receptor 4 (TLR4), and TLR2 knockout mice to examine the significance of these receptors in mediating a macrophage's response to a mycobacterial infection. We determined that mitogenactivated protein kinase (MAPK) activation and tumor necrosis factor-␣ (TNF-␣) production in macrophage infected with Mycobacterium avium or M smegmatis is dependent on myeloid differentiation factor 88 (MyD88) and TLR2 but not TLR4, MR, or CR3. Interestingly, the TLR2-mediated production of TNF-␣ by macrophages infected with M smegmatis required the ␤-glucan receptor dectin-1. A similar requirement for dectin-1 in TNF-␣ production was observed for macrophages infected with M bovis Bacillus Calmette-Guerin (BCG), M phlei, M avium 2151-rough, and M tuberculosis H37Ra. The limited production of TNF-␣ by virulent M avium 724 and M tuberculosis H37Rv was not dependent on dectin-1. Furthermore, dectin-1 facilitated interleukin-6 (IL-6), RANTES (regulated on activation, normal T expressed and secreted), and granulocyte colony-stimulating factor (G-CSF) production by mycobacteriainfected macrophages. These are the first results to establish a significant role for dectin-1, in cooperation with TLR2, to activate a macrophage's proinflammatory response to a mycobacterial infection. IntroductionThe immune system has the complex task of separating friend from foe. To accomplish this mission, the immune system has evolved receptors that recognize molecules present on pathogenic organisms but which show limited interaction with host components. These receptors, referred to as pattern recognition receptors (PRRs), function to promote an innate immune response and include such members as the mannose receptor (MR), scavenger receptors, and Toll-like receptors (TLRs), among others. PRRs bind to conserved microbial structures called pathogen-associated molecular patterns (PAMPs). The expression of these receptors allows the immune system to recognize a wide variety of pathogens that express one or more of these PAMPs, and their engagement initiates the subsequent immune response. Not surprisingly, the PRRs are expressed on cells of the innate immune system, including macrophages, neutrophils, dendritic cells, and natural killer (NK) cells. 1 Binding of microbial products by PRRs elicits a signaling response within the leukocyte, resulting in the production of specific immune modulators. Which PRRs are engaged and in what combination, along with the specific ligands involved, will dictate the overall response by the immune cell. This complexity allows the immune system to tailor its response to a specific pathogen, yet remain flexible enough to recognize a large number of potential pathogens.One of the most...

Thymically derived Foxp3+ regulatory T cells (tTregs) constitute a unique T cell lineage that is essential for maintaining immune tolerance to self and immune homeostasis. However, Foxp3 can also be turned on in conventional T cells as a consequence of antigen exposure in the periphery, under both non-inflammatory and inflammatory conditions. These so-called peripheral Tregs (pTregs) participate in the control of immunity at sites of inflammation, especially at the mucosal surfaces. Although numerous studies have assessed in vitro generated Tregs (termed induced or iTregs), these cells most often do not recapitulate the functional or phenotypic characteristics of in vivo generated pTregs. Thus, there are still many unanswered questions regarding the T cell receptor (TCR) repertoire and function of pTregs as well as conditions under which they are generated in vivo, and the degree to which these characteristics identify specialized features of pTregs versus features that are shared with tTregs. In this review, we summarize the current state of our understanding of pTregs and their relationship to the tTreg subset. We describe the recent discovery of unique cell surface markers and transcription factors (including Neuropilin-1 and Helios) that can be used to distinguish tTreg and pTreg subsets in vivo. Additionally, we discuss how the improved ability to distinguish these subsets provided new insights into the biology of tTregs versus pTregs and suggested differences in their function and TCR repertoire, consistent with a unique role of pTregs in certain inflammatory settings. Finally, these recent advances will be used to speculate on the role of individual Treg subsets in both tolerance and autoimmunity.

Regulatory T cells (Treg) play a central role in counteracting inflammation and autoimmunity. A more complete understanding of cellular heterogeneity and the potential for lineage plasticity in human Treg subsets may identify markers of disease pathogenesis and facilitate the development of optimized cellular therapeutics. To better elucidate human Treg subsets, we conducted direct transcriptional profiling of CD4+FOXP3+Helios+ thymic-derived Treg (tTreg) and CD4+FOXP3+Helios− T cells, followed by comparison to CD4+FOXP3−Helios− T conventional (Tconv) cells. These analyses revealed that the coinhibitory receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) was highly expressed on tTreg. TIGIT and the costimulatory factor CD226 bind the common ligand CD155. Thus, we analyzed the cellular distribution and suppressive activity of isolated subsets of CD4+CD25+CD127lo/− T cells expressing CD226 and/or TIGIT. We observed TIGIT is highly expressed and upregulated on Treg following activation and in vitro expansion and is associated with lineage stability and suppressive capacity. Conversely, the CD226+TIGIT− population was associated with reduced Treg purity and suppressive capacity following expansion, along with a marked increase in IL-10 and effector cytokine production. These studies provide additional markers to delineate functionally distinct Treg subsets that may help direct cellular therapies and provide important phenotypic markers for assessing the role of Treg in health and disease.