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Objectives: This study aimed to investigate the anti-PD-1 inhibitor pembrolizumab as a potential agent for use in non-muscle-invasive bladder cancer (NMIBC) by conducting a Phase 1 safety run-in study to assess the safety and tolerability of intravesical pembrolizumab after transurethral resection of the bladder tumour (TURBT). Patients and methods: Eligible patients had recurrent NMIBC for which adjuvant treatment post TURBT was a reasonable treatment option, Eastern Cooperative Oncology Group Performance Status (ECOG PS) 0–1 and adequate end-organ function. Pembrolizumab was administered by intravesical instillation once weekly for a total of six doses. Intra-patient dose escalation was performed in three paired patient cohorts with doses starting at 50 mg and increasing through 100 mg to a maximum of 200 mg. Adverse events (AEs) were assessed using Common Terminology Criteria for Adverse Events (CTCAE) v4.03 with dose limiting toxicity (DLT) defined as a clinically significant, drug-related, Grade 4 haematological or Grade 3 or higher non-haematological toxicity occurring within 7 days of administration of the first treatment at a given dose for that patient. Results: Six patients were treated with no DLTs seen during dose escalation. Drug-related AEs were of low grade and included dysuria and fatigue. All patients completed six doses of treatment as planned. Pharmacokinetic and pharmacodynamic assays did not detect any pembrolizumab in the serum following repeated intravesical administration, and no changes in peripheral immune cell populations were observed. Conclusions: Administration of intravesical pembrolizumab was well tolerated and did not raise any safety concerns in patients with NMIBC following TURBT. There was no evidence of systemic absorption or systemic immune effects following intravesical administration. Further studies are required to assess whether intravesical administration has anti-tumour activity.

Precise, analogue regulation of gene expression is critical for cellular function in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity. Here we report on a method for precise control of gene expression levels in mammalian cells using engineered microRNA response elements (MREs). First, we measure the efficacy of thousands of synthetic MRE variants under the control of an endogenous microRNA by high-throughput sequencing. Guided by this data, we establish a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to precisely control the expression of user-specified genes. We apply this technology to tune the T-cell co-inhibitory receptor PD-1 and to explore how antigen expression influences T-cell activation and tumour growth. Finally, we employ CRISPR/Cas9 mediated homology directed repair to introduce miSFITs into the BRCA1 3′UTR, demonstrating that this versatile tool can be used to tune endogenous genes., Analogue regulation of gene expression is important for normal function in mammals but existing genetic technologies are designed to achieve ON/OFF control. Here the authors develop synthetic microRNA silencing-mediated fine-tuners (miSFITs) to precisely control target gene expression levels.

Six Salmonella Typhi or Paratyphi human challenge studies were conducted, and daily stool cultures performed. Vi-containing vaccines reduced bacterial shedding, Ty21a or an experimental vaccine did not. Higher Vi immunoglobulin G titers were associated with reduced shedding., Background Shedding of Salmonella Typhi or Paratyphi in the stool or urine leads to contamination of food or water, which is a prerequisite for transmission of enteric fever. Currently, there are limited data on the effect of vaccination or prior exposure on stool shedding. Methods Six Salmonella Typhi or Paratyphi human challenge studies were conducted between 2011 and 2017. Participants were either unvaccinated or vaccinated with 1 of 4 vaccines: Vi-polysaccharide (Vi-PS), Vi-tetanus-toxoid conjugate vaccine (Vi-TT), live oral Ty21a vaccine, or an experimental vaccine (M01ZH09). Daily stool cultures were collected for 14 days after challenge. Results There were 4934 stool samples collected from 430 volunteers. Participants who received Vi-PS or Vi-TT shed less than unvaccinated participants (odds ratio [OR], 0.34; 95% confidence interval [CI], 0.15–0.77; P = .010 and OR, 0.41; 95% CI, 0.19–0.91, P = .029 for Vi-PS and Vi-TT, respectively). Higher anti-Vi immunoglobulin G titers were associated with less shedding of S. Typhi (P < .0001). A nonsignificant reduction in shedding was associated with Ty21a vaccine (OR, 0.57; 95% CI, 0.27–1.20; P = .140). Individuals previously exposed to S. Typhi shed less than previously unexposed individuals (OR, 0.30; 95% CI, 0.1–0.8; P = .016). Shedding of S. Typhi was more common than S. Paratyphi. Conclusions Prior vaccination with Vi vaccines, or natural infection, reduces onward transmission of S. Typhi. Field trials of Vi-TT should be designed to detect indirect protection, reflecting the consequence of reduced stool shedding observed in the human challenge model.

Genomic instability, which is a hallmark of cancer, is generally thought to occur in the middle to late stages of tumourigenesis, following the acquisition of permissive molecular aberrations such as TP53 mutation or whole genome doubling. Tumours with somatic POLE exonuclease domain mutations are notable for their extreme genomic instability (their mutation burden is among the highest in human cancer), distinct mutational signature, lymphocytic infiltrate, and excellent prognosis. To what extent these characteristics are determined by the timing of POLE mutations in oncogenesis is unknown. Here, we have shown that pathogenic POLE mutations are detectable in non-malignant precursors of endometrial and colorectal cancer. Using genome and exome sequencing, we found that multiple driver mutations in POLE-mutant cancers show the characteristic POLE mutational signature, including those in genes conventionally regarded as initiators of tumourigenesis. In POLE-mutant cancers, the proportion of monoclonal predicted neoantigens was similar to that in other cancers, but the absolute number was much greater. We also found that the prominent CD8+ T-cell infiltrate present in POLE-mutant cancers was evident in their precursor lesions. Collectively, these data indicate that somatic POLE mutations are early, quite possibly initiating, events in the endometrial and colorectal cancers in which they occur. The resulting early onset of genomic instability may account for the striking immune response and excellent prognosis of these tumours, as well as their early presentation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A complex network of inflammatory genes is closely linked to somatic cell transformation and malignant disease. Immune cells and their associated molecules are responsible for detecting and eliminating cancer cells as they establish themselves as the precursors of a tumour. By the time a patient has a detectable solid tumour, cancer cells have escaped the initial immune response mechanisms. Here, we describe the development of a double binary zebrafish model that enables regulatory programming of the myeloid cells as they respond to oncogene-activated melanocytes to be explored, focussing on the initial phase when cells become the precursors of cancer. A hormone-inducible binary system allows for temporal control of expression of different Ras oncogenes (NRasQ61K, HRasG12V and KRasG12V) in melanocytes, leading to proliferation and changes in morphology of the melanocytes. This model was coupled to binary cell-specific biotagging models allowing in vivo biotinylation and subsequent isolation of macrophage or neutrophil nuclei for regulatory profiling of their active transcriptomes. Nuclear transcriptional profiling of neutrophils, performed as they respond to the earliest precursors of melanoma in vivo, revealed an intricate landscape of regulatory factors that may promote progression to melanoma, including Serpinb1l4, Fgf1, Fgf6, Cathepsin H, Galectin 1 and Galectin 3. The model presented here provides a powerful platform to study the myeloid response to the earliest precursors of melanoma., Summary: The authors present an innovative double binary zebrafish model for exploring regulatory mechanisms that govern the myeloid response at the onset of melanoma.

Invariant natural killer T (iNKT) cells are a distinct subset of innate-like lymphocytes bearing an invariant T-cell receptor, through which they recognize lipid antigens presented by monomorphic CD1d molecules. Upon activation, iNKT cells are capable of not only having a direct effector function but also transactivating NK cells, maturing dendritic cells, and activating B cells, through secretion of several cytokines and cognate TCR-CD1d interaction. Endowed with the ability to orchestrate an all-encompassing immune response, iNKT cells are critical in shaping immune responses against pathogens and cancer cells. In this review, we examine the critical role of iNKT cells in antitumor responses from two perspectives: (i) how iNKT cells potentiate antitumor immunity and (ii) how CD1d+ tumor cells may modulate their own expression of CD1d molecules. We further explore hypotheses to explain iNKT cell activation in the context of cancer and how the antitumor effects of iNKT cells can be exploited in different forms of cancer immunotherapy, including their role in the development of cancer vaccines.

In each influenza season, a distinct group of young, otherwise healthy individuals with no risk factors succumbs to life-threatening infection. To better understand the cause for this, we analyzed a broad range of immune responses in blood from a unique cohort of patients, comprising previously healthy individuals hospitalized with and without respiratory failure during one influenza season, and infected with one specific influenza A strain. This analysis was compared with similarly hospitalized influenza patients with known risk factors (total of n = 60 patients recruited). We found a sustained increase in a specific subset of proinflammatory monocytes, with high TNF-α expression and an M1-like phenotype (independent of viral titers), in these previously healthy patients with severe disease. The relationship between M1-like monocytes and immunopathology was strengthened using murine models of influenza, in which severe infection generated using different models (including the high-pathogenicity H5N1 strain) was also accompanied by high levels of circulating M1-like monocytes. Additionally, a raised M1/M2 macrophage ratio in the lungs was observed. These studies identify a specific subtype of monocytes as a modifiable immunological determinant of disease severity in this subgroup of severely ill, previously healthy patients, offering potential novel therapeutic avenues.

In each influenza season, a distinct group of young, otherwise healthy individuals with no risk factors succumbs to life-threatening infection. To better understand the cause for this, we analyzed a broad range of immune responses in blood from a unique cohort of patients, comprising previously healthy individuals hospitalized with and without respiratory failure during one influenza season, and infected with one specific influenza A strain. This analysis was compared with similarly hospitalized influenza patients with known risk factors (total of n = 60 patients recruited). We found a sustained increase in a specific subset of proinflammatory monocytes, with high TNF-α expression and an M1-like phenotype (independent of viral titers), in these previously healthy patients with severe disease. The relationship between M1-like monocytes and immunopathology was strengthened using murine models of influenza, in which severe infection generated using different models (including the high-pathogenicity H5N1 strain) was also accompanied by high levels of circulating M1-like monocytes. Additionally, a raised M1/M2 macrophage ratio in the lungs was observed. These studies identify a specific subtype of monocytes as a modifiable immunological determinant of disease severity in this subgroup of severely ill, previously healthy patients, offering potential novel therapeutic avenues., In a cohort of influenza patients, previously healthy and young patients who succumbed to life-threatening disease were defined by high levels of circulating M1-like, TNF-αhi monocytes.

Recent studies have identified new melanoma antigens that are recognised by CD4+ T cells. Analysis of tumour-specific CD4+ T-cell responses may lead to the development of optimal anti-cancer vaccines that can induce an orchestrated effort of tumour-specific CD4+ and CD8+ T cells in the fight against cancer.

Systemic effects on T-cell-mediated antitumor immunity, on expression of T-cell adhesion/homing receptors, and on the promotion of T-cell infiltration of neoplastic tissue may represent key steps for the efficacy of immunological therapies of cancer. In this study, we investigated whether these processes can be promoted by s.c. administration of low-dose (0.5 microg/kg) recombinant human interleukin-12 (rHuIL-12) to metastatic melanoma patients. A striking burst of HLA-restricted CTL precursors (CTLp) directed to autologous tumor was documented in peripheral blood by a high-efficiency limiting dilution analysis technique within a few days after rHuIL-12 injection. A similar burst in peripheral CTLp frequency was observed even when looking at response to a single tumor-derived peptide, as documented by an increase in Melan-A/Mart-1(27-35)-specific CTLp in two HLA-A*0201+ patients by limiting dilution analysis and by staining peripheral blood lymphocytes (PBLs) with HLA-A*0201-melanoma antigen-A/melanoma antigen recognized by T cells (Melan-A/Mart)-1 tetrameric complexes. The CTLp burst was associated, in PBLs, with enhanced expression of T-cell adhesion/homing receptors CD11a/CD18, CD49d, CD44, and with increased proportion of cutaneous lymphocyte antigen (CLA)-positive T cells. This was matched by a marked increase, in serum, of soluble forms of the endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecules (VCAM)-1 and intercellular adhesion molecules (ICAM)-1. Infiltration of neoplastic tissue by CDS+ T cells with a memory and cytolytic phenotype was found by immunohistochemistry in eight of eight posttreatment metastatic lesions but not in five of five pretreatment metastatic lesions from three patients. Increased tumor necrosis and/or fibrosis were also found in several posttherapy lesions of two of three patients in comparison with pretherapy metastases. These results provide the first evidence that rHuIL-12 can boost the frequency of circulating antitumor CTLp in tumor patients, enhances expression of ligand receptor pairs contributing to the lymphocyte function-associated antigen-1/ICAM-1, very late antigen-4/VCAM-1, and CLA/E-selectin adhesion pathways, and promotes infiltration of neoplastic lesions by CD8+ memory T cells in a clinical setting.

Previous attempts to treat human malignancies by adoptive transfer of tumor-specific CTLs have been limited by the difficulty of isolating T cells of defined antigen specificity. The recent development of MHC class I/antigenic peptide tetrameric complexes that allow direct identification of antigen-specific T cells has opened new possibilities for the isolation and in vitro expansion of tumor-specific T cells. In the present study, we have derived polyclonal monospecific cell lines from circulating Melan-A-specific CTL precursors of HLA-A*0201+ melanoma patients by combining stimulation with recently identified peptide analogues of the immunodominant epitope from the melanoma-associated antigen Melan-A with staining with fluorescent HLA-A*0201/Melan-A peptide tetramers. In vitro expansion of antigen-specific CD8+ T cells was monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. This analysis revealed that Melan-A 26-35 peptide analogues were much more efficient than the parental peptides in stimulating a rapid in vitro expansion of antigen-specific CD8+ T cells. These cells were then isolated by tetramer-guided cell sorting and subsequently expanded in vitro by mitogen stimulation. The resulting polyclonal but monospecific CTLs fully cross-recognized the parental peptides and were able to efficiently lyse Melan-A-expressing tumor cells. Altogether, these results pave the way to a molecularly defined approach to antigen-specific adoptive transfer therapy of cancer.

Progress in human tumor immunology has recently been accelerated by new assays for antigen-specific cytotoxic T lymphocytes (CTLs). We have used tetrameric MHC class I complexes (tetramers) to study melanoma-specific CTLs both in vivo and in vitro, and have utilized the results to optimize vaccination strategies for patients. Tetramers have provided some of the best evidence to date that CTL responses against melanoma antigens arise spontaneously in patients. However, CTL responses to common (nonmutated) melanoma epitopes are generally weak or localized, and occur mostly in advanced metastatic disease, hence justifying early immunotherapeutic approaches. These observations led us to design a polyvalent vaccine construct for early administration to melanoma patients at high risk of progression. To compare possible vaccination protocols, we encoded this construct in several different vectors, and developed novel tetramers to track responses to the human melanoma epitopes in transgenic mice. Priming and boosting with the same poly-epitope construct encoded in heterologous vectors led to the expansion of CTLs with a single dominant specificity. Separating the antigens for independent presentation by antigen-presenting cells reversed the effect of immunodominance and induced a powerful polyvalent CTL response. These results provide important pointers for future vaccination trials, and tetramers will form an important tool in the immunomonitoring of these clinical studies.