Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity.

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.

Inhibition of the dipeptidyl peptidase DPP4 (CD26) reveals IL-33-dependent eosinophil-mediated control of tumor growth.

Post-translational modification of chemokines mediated by the dipeptidyl peptidase DPP4 (CD26) has been shown to negatively regulate lymphocyte trafficking, and its inhibition enhances T cell migration and tumor immunity by preserving functional chemokine CXCL10. By extending those initial findings to pre-clinical models of hepatocellular carcinoma and breast cancer, we discovered a distinct mechanism by which inhibition of DPP4 improves anti-tumor responses. Administration of the DPP4 inhibitor sitagliptin resulted in higher concentrations of the chemokine CCL11 and increased migration of eosinophils into solid tumors. Enhanced tumor control was preserved in mice lacking lymphocytes and was ablated after depletion of eosinophils or treatment with degranulation inhibitors. We further demonstrated that tumor-cell expression of the alarmin IL-33 was necessary and sufficient for eosinophil-mediated anti-tumor responses and that this mechanism contributed to the efficacy of checkpoint-inhibitor therapy. These findings provide insight into IL-33- and eosinophil-mediated tumor control, revealed when endogenous mechanisms of DPP4 immunoregulation are inhibited.

Genetic Adaptation and Neandertal Admixture Shaped the Immune System of Human Populations.

Humans differ in the outcome that follows exposure to life-threatening pathogens, yet the extent of population differences in immune responses and their genetic and evolutionary determinants remain undefined. Here, we characterized, using RNA sequencing, the transcriptional response of primary monocytes from Africans and Europeans to bacterial and viral stimuli-ligands activating Toll-like receptor pathways (TLR1/2, TLR4, and TLR7/8) and influenza virus-and mapped expression quantitative trait loci (eQTLs). We identify numerous cis-eQTLs that contribute to the marked differences in immune responses detected within and between populations and a strong trans-eQTL hotspot at TLR1 that decreases expression of pro-inflammatory genes in Europeans only. We find that immune-responsive regulatory variants are enriched in population-specific signals of natural selection and show that admixture with Neandertals introduced regulatory variants into European genomes, affecting preferentially responses to viral challenges. Together, our study uncovers evolutionarily important determinants of differences in host immune responsiveness between human populations.

Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors.

The quantification and characterization of circulating immune cells provide key indicators of human health and disease. To identify the relative effects of environmental and genetic factors on variation in the parameters of innate and adaptive immune cells in homeostatic conditions, we combined standardized flow cytometry of blood leukocytes and genome-wide DNA genotyping of 1,000 healthy, unrelated people of Western European ancestry. We found that smoking, together with age, sex and latent infection with cytomegalovirus, were the main non-genetic factors that affected variation in parameters of human immune cells. Genome-wide association studies of 166 immunophenotypes identified 15 loci that showed enrichment for disease-associated variants. Finally, we demonstrated that the parameters of innate cells were more strongly controlled by genetic variation than were those of adaptive cells, which were driven by mainly environmental exposure. Our data establish a resource that will generate new hypotheses in immunology and highlight the role of innate immunity in susceptibility to common autoimmune diseases.

Publisher Correction: Natural variation in the parameters of innate immune cells is preferentially driven by genetic factors.

In the version of this article initially published, the name of one author was incorrect (James P. Santo). The correct name is James P. Di Santo. The error has been corrected in the HTML and PDF versions of the article.

Smoking changes adaptive immunity with persistent effects.

Individuals differ widely in their immune responses, with age, sex and genetic factors having major roles in this inherent variability1-6. However, the variables that drive such differences in cytokine secretion-a crucial component of the host response to immune challenges-remain poorly defined. Here we investigated 136 variables and identified smoking, cytomegalovirus latent infection and body mass index as major contributors to variability in cytokine response, with effects of comparable magnitudes with age, sex and genetics. We find that smoking influences both innate and adaptive immune responses. Notably, its effect on innate responses is quickly lost after smoking cessation and is specifically associated with plasma levels of CEACAM6, whereas its effect on adaptive responses persists long after individuals quit smoking and is associated with epigenetic memory. This is supported by the association of the past smoking effect on cytokine responses with DNA methylation at specific signal trans-activators and regulators of metabolism. Our findings identify three novel variables associated with cytokine secretion variability and reveal roles for smoking in the short- and long-term regulation of immune responses. These results have potential clinical implications for the risk of developing infections, cancers or autoimmune diseases.

Sumoylation coordinates the repression of inflammatory and anti-viral gene-expression programs during innate sensing.

Innate sensing of pathogens initiates inflammatory cytokine responses that need to be tightly controlled. We found here that after engagement of Toll-like receptors (TLRs) in myeloid cells, deficient sumoylation caused increased secretion of transcription factor NF-κB-dependent inflammatory cytokines and a massive type I interferon signature. In mice, diminished sumoylation conferred susceptibility to endotoxin shock and resistance to viral infection. Overproduction of several NF-κB-dependent inflammatory cytokines required expression of the type I interferon receptor, which identified type I interferon as a central sumoylation-controlled hub for inflammation. Mechanistically, the small ubiquitin-like modifier SUMO operated from a distal enhancer of the gene encoding interferon-ß (Ifnb1) to silence both basal and stimulus-induced activity of the Ifnb1 promoter. Therefore, sumoylation restrained inflammation by silencing Ifnb1 expression and by strictly suppressing an unanticipated priming by type I interferons of the TLR-induced production of inflammatory cytokines.

Dipeptidylpeptidase 4 inhibition enhances lymphocyte trafficking, improving both naturally occurring tumor immunity and immunotherapy.

The success of antitumor immune responses depends on the infiltration of solid tumors by effector T cells, a process guided by chemokines. Here we show that in vivo post-translational processing of chemokines by dipeptidylpeptidase 4 (DPP4, also known as CD26) limits lymphocyte migration to sites of inflammation and tumors. Inhibition of DPP4 enzymatic activity enhanced tumor rejection by preserving biologically active CXCL10 and increasing trafficking into the tumor by lymphocytes expressing the counter-receptor CXCR3. Furthermore, DPP4 inhibition improved adjuvant-based immunotherapy, adoptive T cell transfer and checkpoint blockade. These findings provide direct in vivo evidence for control of lymphocyte trafficking via CXCL10 cleavage and support the use of DPP4 inhibitors for stabilizing biologically active forms of chemokines as a strategy to enhance tumor immunotherapy.

In silico tools for accurate HLA and KIR inference from clinical sequencing data empower immunogenetics on individual-patient and population scales.

Immunogenetic variation in humans is important in research, clinical diagnosis and increasingly a target for therapeutic intervention. Two highly polymorphic loci play critical roles, namely the human leukocyte antigen (HLA) system, which is the human version of the major histocompatibility complex (MHC), and the Killer-cell immunoglobulin-like receptors (KIR) that are relevant for responses of natural killer (NK) and some subsets of T cells. Their accurate classification has typically required the use of dedicated biological specimens and a combination of in vitro and in silico efforts. Increased availability of next generation sequencing data has led to the development of ancillary computational solutions. Here, we report an evaluation of recently published algorithms to computationally infer complex immunogenetic variation in the form of HLA alleles and KIR haplotypes from whole-genome or whole-exome sequencing data. For both HLA allele and KIR gene typing, we identified tools that yielded >97% overall accuracy for four-digit HLA types, and >99% overall accuracy for KIR gene presence, suggesting the readiness of in silico solutions for use in clinical and high-throughput research settings.

Functional analysis via standardized whole-blood stimulation systems defines the boundaries of a healthy immune response to complex stimuli.

Standardization of immunophenotyping procedures has become a high priority. We have developed a suite of whole-blood, syringe-based assay systems that can be used to reproducibly assess induced innate or adaptive immune responses. By eliminating preanalytical errors associated with immune monitoring, we have defined the protein signatures induced by (1) medically relevant bacteria, fungi, and viruses; (2) agonists specific for defined host sensors; (3) clinically employed cytokines; and (4) activators of T cell immunity. Our results provide an initial assessment of healthy donor reference values for induced cytokines and chemokines and we report the failure to release interleukin-1α as a common immunological phenotype. The observed naturally occurring variation of the immune response may help to explain differential susceptibility to disease or response to therapeutic intervention. The implementation of a general solution for assessment of functional immune responses will help support harmonization of clinical studies and data sharing.

Polygenic risk for skin autoimmunity impacts immune checkpoint blockade in bladder cancer.

PD-1 and PD-L1 act to restrict T cell responses in cancer and contribute to self-tolerance. Consistent with this role, PD-1 checkpoint inhibitors have been associated with immune-related adverse events (irAEs), immune toxicities thought to be autoimmune in origin. Analyses of dermatological irAEs have identified an association with improved overall survival (OS) following anti-PD-(L)1 therapy, but the factors that contribute to this relationship are poorly understood. We collected germline whole-genome sequencing data from IMvigor211, a recent phase 3 randomized controlled trial comparing atezolizumab (anti-PD-L1) monotherapy to chemotherapy in bladder cancer. We found that high vitiligo, high psoriasis, and low atopic dermatitis polygenic risk scores (PRSs) were associated with longer OS under anti-PD-L1 monotherapy as compared to chemotherapy, reflecting the Th17 polarization of these diseases. PRSs were not correlated with tumor mutation burden, PD-L1 immunohistochemistry, nor T-effector gene signatures. Shared genetic factors impact risk for dermatological autoimmunity and anti-PD-L1 monotherapy in bladder cancer.

Immune Profiling Enables Stratification of Patients With Active Tuberculosis Disease or Mycobacterium tuberculosis Infection.

BACKGROUND: Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb) infection and is a major public health problem. Clinical challenges include the lack of a blood-based test for active disease. Current blood-based tests, such as QuantiFERON (QFT) do not distinguish active TB disease from asymptomatic Mtb infection. METHODS: We hypothesized that TruCulture, an immunomonitoring method for whole-blood stimulation, could discriminate active disease from latent Mtb infection (LTBI). We stimulated whole blood from patients with active TB and compared with LTBI donors. Mtb-specific antigens and live bacillus Calmette-Guérin (BCG) were used as stimuli, with direct comparison to QFT. Protein analyses were performed using conventional and digital enzyme-linked immunosorbent assay (ELISA), as well as Luminex. RESULTS: TruCulture showed discrimination of active TB cases from LTBI (P < .0001, AUC = .81) compared with QFT (P = .45, AUC = .56), based on an interferon γ (IFNγ) readout after Mtb antigen (Ag) stimulation. This result was replicated in an independent cohort (AUC = .89). In exploratory analyses, TB stratification could be further improved by the Mtb antigen to BCG IFNγ ratio (P < .0001, AUC = .91). Finally, the combination of digital ELISA and transcriptional analysis showed that LTBI donors with high IFNγ clustered with patients with TB, suggesting the possibility to identify subclinical disease. CONCLUSIONS: TruCulture offers a next-generation solution for whole-blood stimulation and immunomonitoring with the possibility to discriminate active and latent infection.

Associations between untargeted plasma metabolomic signatures and gut microbiota composition in the Milieu Intérieur population of healthy adults.

Host-microbial co-metabolism products are being increasingly recognised to play important roles in physiological processes. However, studies undertaking a comprehensive approach to consider host-microbial metabolic relationships remain scarce. Metabolomic analysis yielding detailed information regarding metabolites found in a given biological compartment holds promise for such an approach. This work aimed to explore the associations between host plasma metabolomic signatures and gut microbiota composition in healthy adults of the Milieu Intérieur study. For 846 subjects, gut microbiota composition was profiled through sequencing of the 16S rRNA gene in stools. Metabolomic signatures were generated through proton NMR analysis of plasma. The associations between metabolomic variables and α- and ß-diversity indexes and relative taxa abundances were tested using multi-adjusted partial Spearman correlations, permutational ANOVA and multivariate associations with linear models, respectively. A multiple testing correction was applied (Benjamini-Hochberg, 10 % false discovery rate). Microbial richness was negatively associated with lipid-related signals and positively associated with amino acids, choline, creatinine, glucose and citrate (-0·133 ≤ Spearman's ρ ≤ 0·126). Specific associations between metabolomic signals and abundances of taxa were detected (twenty-five at the genus level and nineteen at the species level): notably, numerous associations were observed for creatinine (positively associated with eleven species and negatively associated with Faecalibacterium prausnitzii). This large-scale population-based study highlights metabolites associated with gut microbial features and provides new insights into the understanding of complex host-gut microbiota metabolic relationships. In particular, our results support the implication of a 'gut-kidney axis'. More studies providing a detailed exploration of these complex interactions and their implications for host health are needed.

A novel method to produce synthetic murine CXCL10 for efficient screening of functional variants.

Antitumor immune responses depend on the infiltration of solid tumors by effector T cells, a process guided by chemokines. In particular, the chemokine CXCL10 has been shown to play a critical role in mediating recruitment of CXCR3 + cytolytic T and NK cells in tumors, though its use as a therapeutic agent has not been widely explored. One of the limitations is due to the rapid inactivation of CXCL10 by dipeptidyl peptidase 4 (DPP4), a broadly expressed enzyme that is active in plasma and other bodily fluids. In the present study, we describe a novel method to produce synthetic CXCL10 that is resistant to DPP4 N-terminal truncation. Using a Fmoc solid-phase peptide synthesis approach, synthetic murine WT CXCL10 was produced, showing similar biochemical and biological properties to the recombinant protein. This synthesis method supported production of natural (amino acid substitution, insertion or deletion) and non-natural (chemical modifications) variants of CXCL10. In association with a functional screening cascade that assessed DPP4-mediated cleavage, CXCR3 signaling potency and chemotactic activity, we successfully generated 20 murine CXCL10 variants. Among those, two non-natural variants with N-methylated Leu3 (MeLeu3) and a reduced amide bond between Pro2 and Leu3 (rLeu3), respectively, showed resistance to DPP4 truncation but decreased CXCR3 signaling and chemotactic activity. Interestingly, MeLeu3 and rLeu3 CXCL10 behaved as DPP4 inhibitors, preventing the truncation of WT CXCL10. This study highlights the potential of using Fmoc solid-phase chemistry in association with biochemical and biological characterization to rapidly identify CXCL10 variants with desired properties. These novel methods unlock the opportunity to develop DPP4 resistant CXCL10 variants, as well as other chemokine substrates, while maintaining chemotactic properties.

Germline genetic polymorphisms influence tumor gene expression and immune cell infiltration.

Cancer immunotherapy has emerged as an effective therapy in a variety of cancers. However, a key challenge in the field is that only a subset of patients who receive immunotherapy exhibit durable response. It has been hypothesized that host genetics influences the inherent immune profiles of patients and may underlie their differential response to immunotherapy. Herein, we systematically determined the association of common germline genetic variants with gene expression and immune cell infiltration of the tumor. We identified 64,094 expression quantitative trait loci (eQTLs) that associated with 18,210 genes (eGenes) across 24 human cancers. Overall, eGenes were enriched for their being involved in immune processes, suggesting that expression of immune genes can be shaped by hereditary genetic variants. We identified the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene as a pan-cancer type eGene whose expression levels stratified overall survival in a subset of patients with bladder cancer receiving anti-PD-L1 (atezolizumab) therapy. Finally, we identified 103 gene signature QTLs (gsQTLs) that were associated with predicted immune cell abundance within the tumor microenvironment. Our findings highlight the impact of germline SNPs on cancer-immune phenotypes and response to therapy; and these analyses provide a resource for integration of germline genetics as a component of personalized cancer immunotherapy.

Distinctive roles of age, sex, and genetics in shaping transcriptional variation of human immune responses to microbial challenges.

The contribution of host genetic and nongenetic factors to immunological differences in humans remains largely undefined. Here, we generated bacterial-, fungal-, and viral-induced immune transcriptional profiles in an age- and sex-balanced cohort of 1,000 healthy individuals and searched for the determinants of immune response variation. We found that age and sex affected the transcriptional response of most immune-related genes, with age effects being more stimulus-specific relative to sex effects, which were largely shared across conditions. Although specific cell populations mediated the effects of age and sex on gene expression, including CD8+ T cells for age and CD4+ T cells and monocytes for sex, we detected a direct effect of these intrinsic factors for the majority of immune genes. The mapping of expression quantitative trait loci (eQTLs) revealed that genetic factors had a stronger effect on immune gene regulation than age and sex, yet they affected a smaller number of genes. Importantly, we identified numerous genetic variants that manifested their regulatory effects exclusively on immune stimulation, including a Candida albicans-specific master regulator at the CR1 locus. These response eQTLs were enriched in disease-associated variants, particularly for autoimmune and inflammatory disorders, indicating that differences in disease risk may result from regulatory variants exerting their effects only in the presence of immune stress. Together, this study quantifies the respective effects of age, sex, genetics, and cellular heterogeneity on the interindividual variability of immune responses and constitutes a valuable resource for further exploration in the context of different infection risks or disease outcomes.

Dying to replicate: the orchestration of the viral life cycle, cell death pathways, and immunity.

Manipulation of cell death pathways has been identified as a common feature of host-microbe interactions. We examine two examples: influenza A as a representative acute infection and cytomegalovirus as an example of chronic infection. From the perspective of viral entry, replication, and transmission, we identify points of interconnection with the host response to infection, namely the induction of host cell death, inflammation, and immunity. Following from this analysis, we argue that the evolution and fine-tuned regulation of death-associated genes may result from constant microbial pressure--past and present--that helped to support and coordinate cell death programs within the host. Interestingly, the delay in host cell death allows time for the virus to replicate while perturbations in cell death allow the host cell to initiate an immune response. This may represent a genetically encoded trade-off ensuring survival of both host and virus, or it may be a part of the complex agenda of infectious microbes.

New tools for pathology: a user's review of a highly multiplexed method for in situ analysis of protein and RNA expression in tissue.

Tumor cell heterogeneity and tumor cell-stromal interactions are being explored as determinants of disease progression and treatment resistance in solid tumor and hematological malignancies. As such, tools simultaneously capable of highly multiplexed profiling of tissues' protein and RNA content, as well as interrogation of rare or single cells, are required to precisely characterize constituent tumor cell populations, infiltrating lymphocytes and stromal elements. Access to spatial relationships will enable more precise characterization of tumors, support patient stratification and may help to identify novel drug targets. Multiple platforms are being developed to address these critical unmet needs. The NanoString digital spatial profiling (DSP) platform enables highly multiplexed, spatial assessment of protein and/or RNA targets in tissues by detecting oligonucleotide barcodes conjugated via a photocleavable linker to primary antibodies or nucleic acid probes. Although this platform enables high-dimensional spatial interrogation of tissue protein and RNA expression, a detailed understanding of its composition, function and chemistry is advisable to guide experimental design and data interpretation. The purpose of this review is to provide an independent, comprehensive description of the DSP technology, including an overview of NanoString's capture and antibody barcode conjugation chemistries, experimental workflow, data output and analysis methods. The DSP technology will be discussed in the context of other highly multiplexed immunohistochemistry methods, including imaging mass cytometry and multiplexed ion beam imaging, to inform potential users of the advantages and limitations of each. Additional issues such as preanalytical variability, sampling and specimen adequacy will be considered with respect to the platforms to inform potential experimental design. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CXCR3/CXCL10 Axis Shapes Tissue Distribution of Memory Phenotype CD8+ T Cells in Nonimmunized Mice.

The preimmune repertoire consists of mature T lymphocytes that have not yet been stimulated in the periphery. Memory phenotype (MP) cells have been reported as part of the preimmune repertoire (i.e., T cells bearing memory markers despite lack of engagement with cognate Ag); however, little is known about their trafficking and function. In this study, we hypothesized that MP cells, naive to TCR stimulation, constitute a transient population that traffics to tissues during development. Using mutant and transgenic animals with a monospecific TCR, we discovered increased numbers of MP CD8+ T cells circulating in nonimmunized Cxcr3-/- and Cxcl10-/- mice compared with wild-type animals. Phenotypic differences included decreased numbers of preimmune MP Ag-specific T cells in the skin and thymus and a distinct pattern of activation upon TCR engagement. Our results show for the first time, to our knowledge, an important role for CXCR3 and CXCL10 in the tissue distribution of preimmune MP cells.

Visualizing the functional diversification of CD8+ T cell responses in lymph nodes.

CD8(+) T cell responses generate effector cells endowed with distinct functional potentials but the contribution of early events in this process is unclear. Here, we have imaged T cells expressing a fluorescent reporter for the activation of the interferon-γ (IFN-γ) locus during priming in lymph nodes. We have demonstrated marked differences in the efficiency of gene activation during stable T cell-dentritic cell (DC) contacts, influenced in part by signal strength. Imaging the first cell division, we have demonstrated that heterogeneity in T cell functional potential was largely apparent as T cells initiated clonal expansion. Moreover, by analyzing the fate of single activated T cells ex vivo, we have provided evidence that these early differences resulted in clonal progenies with distinct functional properties. Thus, the early set of T cell-DC interactions in lymph nodes largely contribute to the heterogeneity of T cell responses through the generation of functionally divergent clonal progenies.