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Waldenström macroglobulinaemia (WM) is an indolent mature B cell lymphoma characterised by an infiltrate of heterogeneous B cells and hypersecretion of IgM. There are two distinct cellular populations that can be distinguished on morphology and immunophenotyping within the bone marrow. The predominant lymphoplasmacytic compartment arises at an earlier stage in ontogeny, and is responsible for the cytopenias noted during the symptomatic phase of the disease. This population is ably targeted by B cell immunodepletion. The smaller plasma cell compartment has been shown to be monotypic and to carry the MYD88L265P mutation noted in >90% of WM. Further, pathogenic IgM levels tend to correlate better with plasma cell burden as compared to lymphoplasmacytic cell burden within the bone marrow. B cell immunodepletion does not eradicate the plasma cell compartment resulting in persistent IgM levels and poor complete remission rates. In this review we discuss the different cellular compartments in WM and highlight evidence regarding the significance and impending function of the plasma cell compartment in WM. We suggest detection of plasma cells be incorporated into routine diagnostic algorithms, and highlight the need to trial incorporation of plasma cell depleting therapy into treatment algorithms to deepen responses and improve clinical outcomes., (Copyright © 2017 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Selectivity of autophagy is achieved by target recognition; however, the number of autophagy receptors identified so far is limited. In this study we demonstrate that a subset of tripartite motif (TRIM) proteins mediate selective autophagy of key regulators of inflammatory signaling. MEFV/TRIM20, and TRIM21 act as autophagic receptors recognizing their cognate targets and delivering them for autophagic degradation. MEFV recognizes the inflammasome components NLRP3, CASP1 and NLRP1, whereas TRIM21 specifically recognizes the activated, dimeric from of IRF3 inducing type I interferon gene expression. MEFV and TRIM21 have a second activity, whereby they act not only as receptors but also recruit and organize key components of autophagic machinery consisting of ULK1, BECN1, ATG16L1, and mammalian homologs of Atg8, with a preference for GABARAP. MEFV capacity to organize the autophagy apparatus is affected by common mutations causing familial Mediterranean fever. These findings reveal a general mode of action of TRIMs as autophagic receptor-regulators performing a highly-selective type of autophagy (precision autophagy), with MEFV specializing in the suppression of inflammasome and CASP1 activation engendering IL1B/interleukin-1β production and implicated in the form of cell death termed pyroptosis, whereas TRIM21 dampens type I interferon responses.

Harnessing the immune system to fight cancer is an exciting advancement in lung cancer therapy. Antitumor immunity can be augmented by checkpoint blockade therapy, which removes the inhibition/brakes imposed on the immune system by the tumor. Checkpoint blockade therapy with anti-programmed cell death protein 1 (anti-PD-1)/anti-programmed death ligand 1 (anti-PDL-1) antibodies causes tumor regression in about 25% of patients with lung cancer. In another approach, the immune system is forced or accelerated to attack the tumor through augmentation of the antitumor response against mutations carried by each lung tumor. This latter approach has become feasible since the advent of next-generation sequencing technology, which allows identification of the specific mutations that each individual lung tumor bears. Indeed lung cancers are now known to have high mutation rates, making them logical targets for mutation-directed immune therapies. We review how sequencing of lung cancer mutations leads to better understanding of how the immune system recognizes tumors, providing improved opportunities to track antitumor immunity and ultimately leading to the development of personalized vaccine strategies aimed at unleashing the host immune system to attack mutations in the tumor., (Copyright © 2016 University of Western Australia. Published by Elsevier Inc. All rights reserved.)

Vascular cell adhesion molecule 1 (VCAM-1) is an adhesion molecule assigned to the activated endothelium mediating immune cells adhesion and extravasation. However, its expression in renal carcinomas inversely correlates with tumor malignancy. Our experiments in clear cell renal cell carcinoma (ccRCC) cell lines demonstrated that von Hippel Lindau (VHL) loss, hypoxia, or PHD (for prolyl hydroxylase domain-containing proteins) inactivation decreased VCAM-1 levels through a transcriptional mechanism that was independent of the hypoxia-inducible factor and dependent on the nuclear factor κB signaling pathway. Conversely, VHL expression leads to high VCAM-1 levels in ccRCC, which in turn leads to better outcomes, possibly by favoring antitumor immunity through VCAM-1 interaction with the α4β1 integrin expressed in immune cells. Remarkably, in ccRCC human samples with VHL nonmissense mutations, we observed a negative correlation between VCAM-1 levels and ccRCC stage, microvascular invasion, and symptom presentation, pointing out the clinical value of VCAM-1 levels as a marker of ccRCC progression., (© 2017 Labrousse-Arias et al.)

Background: Mutations in PIK3CD and PIK3R1 cause activated PI3K-δ syndrome (APDS) by dysregulation of the PI3K-AKT pathway., Methods: We studied precursor and peripheral B-cell differentiation and apoptosis via flowcytometry. Furthermore, we performed AKT-phosphorylation assays and somatic hypermutations (SHM) and class switch recombination (CSR) analysis., Results: We identified 13 patients of whom 3 had new mutations in PIK3CD or PIK3R1. Patients had low total B-cell numbers with increased frequencies of transitional B cells and plasmablasts, while the precursor B-cell compartment in bone marrow was relatively normal. Basal AKT phosphorylation was increased in lymphocytes from APDS patients and natural effector B cells where most affected. PI3K mutations resulted in altered SHM and CSR and increased apoptosis., Conclusions: The B-cell compartment in APDS patients is affected by the mutations in PI3K. There is reduced differentiation beyond the transitional stage, increased AKT phosphorylation and increased apoptosis. This B-cell phenotype contributes to the clinical phenotype., (Copyright © 2017. Published by Elsevier Inc.)

Mutations in the leucine-rich repeat kinase 2 ( LRRK2 ) gene are found in familial and idiopathic cases of Parkinson's disease (PD), but are also associated with immune-related disorders, notably Crohn's disease and leprosy. Although the physiological function of LRRK2 protein remains largely elusive, increasing evidence suggests that it plays a role in innate immunity, a process that also has been implicated in neurodegenerative diseases, including PD. Innate immunity involves macrophages and microglia, in which endogenous LRRK2 expression is precisely regulated and expression is strongly up-regulated upon cell activation. This brief report discusses the current understanding of the involvement of LRRK2 in innate immunity particularly in relation to PD, critically examining its role in myeloid cells, particularly macrophages and microglia., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Background: According to the World Health Organization, by the end of last year, about 37 million people throughout the world were diagnosed with AIDS and millions of people die each year from this disease., Objective: To develop an appropriate model which depicts the mechanism of the dynamics involved in the interactions between HIV and immune system in peripheral bloodstream of HIV infected individuals by considering the phenomena of virus mutation and taking into account the role of latently infected cells in speared of infection and considering the effects of antiretroviral drugs and occurrence of drug resistance in our model in order to assess the results obtained from applying different therapeutic methods., Methods: Two-dimensional CA model with Moor neighboring was developed. Various agents which they were referring to peripheral bloodstream particles of HIV infected individuals were defined. Then the biological rules were extracted from both expert knowledge and the authoritative articles. The extracted rules were applied for updating the states of these agents. The effects of using antiretroviral drug treatment were considered by applying drug's effectiveness of both of protease and reverse transcriptase inhibitors as two separate inputs of model., Results: Time evolution curves of concentrations of defined agents were shown as our results. In case of considering no treatment, our results showed that concentrations of healthy CD4+T cells reached the threshold of AIDS after a bout 250 weeks. By applying monotherapy method, the concentrations of these cells remained on the threshold of AIDS for a long time and applying combined antiretroviral therapy (cART) method leaded to increase the concentration of these cells 20% upper than threshold of AIDS. Also, by applying monotherapy and cART compared with no treatment, the concentrations of infected CD4+T cells 10% and 40% decreased further, respectively and for the level of viral load, leads to a reduction of almost 55% and 90%, respectively. Belated treatment, comparison with early treatment, caused almost 10% reduce (increase) in steady state concentrations of healthy (infected) cells.

Cancer accumulates 10s to 1000s of genomic mutations of which a fraction is immunogenic and may serve as an Achilles' heel of tumor cells. Mutation-specific T cells can recognize these antigens and destroy malignant cells. Strategies to immunotherapeutically address individual tumor mutations employing peptide or mRNA based vaccines are now actively investigated in mice and humans. An important step of determining the therapeutic potential of a mutanome vaccine is the detection of mutation reactive T-cell responses. In this chapter we provide protocols to identify and subtype mutation specific T cells in mice based on IFN-γ ELISpot and flow cytometry.

Candida albicans is among the most common human fungal pathogens, causing a broad range of infections, including life-threatening systemic infections. The cell wall of C. albicans is the interface between the fungus and the innate immune system. The cell wall is composed of an outer layer enriched in mannosylated glycoproteins (mannan) and an inner layer enriched in β-(1,3)-glucan and chitin. Detection of C. albicans by Dectin-1, a C-type signaling lectin specific for β-(1,3)-glucan, is important for the innate immune system to recognize systemic fungal infections. Increased exposure of β-(1,3)-glucan to the immune system occurs when the mannan layer is altered or removed in a process called unmasking. Nanoscale changes to the cell wall during unmasking were explored in live cells with atomic force microscopy (AFM). Two mutants, the cho1Δ/Δ and kre5Δ/Δ mutants, were selected as representatives that exhibit modest and strong unmasking, respectively. Comparisons of the cho1Δ/Δ and kre5Δ/Δ mutants to the wild type reveal morphological changes in their cell walls that correlate with decreases in cell wall elasticity. In addition, AFM tips functionalized with Dectin-1 revealed that the forces of binding of Dectin-1 to all of the strains were similar, but the frequency of binding was highest for the kre5Δ/Δ mutant, decreased for the cho1Δ/Δ mutant, and rare for the wild type. These data show that nanoscale changes in surface topology are correlated with increased Dectin-1 adhesion and decreased cell wall elasticity. AFM, using tips functionalized with immunologically relevant molecules, can map epitopes of the cell wall and increase our understanding of pathogen recognition by the immune system., (Copyright © 2016 American Society for Microbiology.)

Infectious bursal disease (also known as Gumboro disease) is an immunosuppressive viral disease specific to chickens. In spite of all the information amassed on the antigenic and immunological characteristics of the virus, the disease has not yet been brought fully under control. It is still prevalent in properly vaccinated flocks carrying specific antibodies at levels normally high enough to prevent the disease. Common causes apart, failure of vaccination against infectious bursal disease is associated mainly with early vaccination in flocks of unknown immune status and with the evolution of viruses circulating in the field, leading to antigenic drift and a sharp rise in pathogenicity. Various highly sensitive molecular techniques have clarified the viral determinants of antigenicity and pathogenicity of the infectious bursal disease virus. However, these markers are not universally recognised and tend to be considered as evolutionary markers. Antigenic variants of the infectious bursal disease virus possess modified neutralising epitopes that allow them to evade the action of maternally-derived or vaccine-induced antibodies. Autogenous or multivalent vaccines are required to control antigenic variants in areas where classical and variant virus strains coexist. Pathotypic variants (very virulent viruses) remain antigenically related to classical viruses. The difficulty in controlling pathotypic variants is linked to the difficulty of eliciting an early immune response, because of the risk of the vaccine virus being neutralised by maternal antibodies. Mathematical calculation of the optimal vaccination time and the use of vaccines resistant to maternally-derived antibodies have improved the control of very virulent viruses., (© OIE (World Organisation for Animal Health), 2016.)

Background: Cellular T-helper (Th)17 infiltrates dominate skin inflammation in filaggrin-deficient flaky tail (ft/ft) mice, and Th17 cells are found in both the skin and blood of patients with acute atopic dermatitis. However, the potential role of loss-of-function mutations in the filaggrin gene (FLG) for increased peripheral Th17 cells is unclear., Objectives: To study whether mutations in FLG influence the frequency of peripheral Th17 cells., Methods: We studied blood samples from six adults with mutations in FLG and five controls without mutations for frequencies of cytokine-producing CD4(+) T cells. We evaluated ft/ft mice and wild-type (WT) control mice for interleukin (IL)-17-producing CD4(+) T cells in naive mice and following 2,4-dinitrofluorobenzene (DNFB) challenge. In addition, the T-cell receptor (TCR) Vβ-chain repertoire was analysed by flow cytometry., Results: Human studies showed increased frequency of peripheral Th17 cells in FLG mutation carriers when compared with WT individuals. Mouse studies showed increased frequency of peripheral Th17 cells in adult ft/ft mice but not in 2-week-old ft/ft mice. Moreover, altered TCR Vβ-chain repertoire was found in ft/ft mice when compared with WT mice. An increased frequency of CD4(+) Vβ10(+) T cells producing IL-17 was found in the spleen of adult ft/ft mice when compared with WT mice. Finally, DNFB challenge induced an increased number of Th17 cells in ft/ft mice compared with WT mice., Conclusions: Deficiency of filaggrin appeared to be a driver of increased peripheral levels of Th17 cells. This increase must be acquired as peripheral Th17 cells were found in adult ft/ft mice but not in 2-week-old ft/ft mice indicating involvement of exogenous factors., (© 2016 British Association of Dermatologists.)

Broadly neutralizing antibodies (bnAbs) against the N332 supersite of the HIV envelope (Env) trimer are the most common bnAbs induced during infection, making them promising leads for vaccine design. Wild-type Env glycoproteins lack detectable affinity for supersite-bnAb germline precursors and are therefore unsuitable immunogens to prime supersite-bnAb responses. We employed mammalian cell surface display to design stabilized Env trimers with affinity for germline-reverted precursors of PGT121-class supersite bnAbs. The trimers maintained native-like antigenicity and structure, activated PGT121 inferred-germline B cells ex vivo when multimerized on liposomes, and primed PGT121-like responses in PGT121 inferred-germline knockin mice. Design intermediates have levels of epitope modification between wild-type and germline-targeting trimers; their mutation gradient suggests sequential immunization to induce bnAbs, in which the germline-targeting prime is followed by progressively less-mutated design intermediates and, lastly, with native trimers. The vaccine design strategies described could be utilized to target other epitopes on HIV or other pathogens., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Laminins are ubiquitous constituents of the basement membranes with major architectural and functional role as supported by the fact that absence or mutations of laminins lead to either lethal or severely impairing phenotypes. Besides genetic defects, laminins are involved in a wide range of human diseases including cancer, infections, and inflammatory diseases, as well as autoimmune disorders. A growing body of evidence implicates several laminin chains as autoantigens in blistering skin diseases, collagenoses, vasculitis, or post-infectious autoimmunity. The current paper reviews the existing knowledge on autoimmunity against laminins referring to both experimental and clinical data, and on therapeutic implications of anti-laminin antibodies. Further investigation of relevant laminin epitopes in pathogenic autoimmunity would facilitate the development of appropriate diagnostic tools for thorough characterization of patients' antibody specificities and should decisively contribute to designing more specific therapeutic interventions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

α1-Antitrypsin deficiency (A1ATD) is an inherited disorder caused by mutations in SERPINA1, leading to liver and lung disease. It is not a rare disorder but frequently goes underdiagnosed or misdiagnosed as asthma, chronic obstructive pulmonary disease (COPD) or cryptogenic liver disease. The most frequent disease-associated mutations include the S allele and the Z allele of SERPINA1, which lead to the accumulation of misfolded α1-antitrypsin in hepatocytes, endoplasmic reticulum stress, low circulating levels of α1-antitrypsin and liver disease. Currently, there is no cure for severe liver disease and the only management option is liver transplantation when liver failure is life-threatening. A1ATD-associated lung disease predominately occurs in adults and is caused principally by inadequate protease inhibition. Treatment of A1ATD-associated lung disease includes standard therapies that are also used for the treatment of COPD, in addition to the use of augmentation therapy (that is, infusions of human plasma-derived, purified α1-antitrypsin). New therapies that target the misfolded α1-antitrypsin or attempt to correct the underlying genetic mutation are currently under development.

Defective regulation of type I interferon response is associated with severe inflammatory phenotypes and autoimmunity. Type I interferonopathies are a clinically heterogenic group of Mendelian diseases with a constitutive activation of this pathway that might present as atypical, severe, early onset rheumatic diseases. Skin vasculopathy with chilblains and livedo reticularis, interstitial lung disease, and panniculitis are common. Recent studies have implicated abnormal responses to nucleic acid stimuli or defective regulation of downstream effector molecules in disease pathogenesis. As observed for IL1-β and autoinflammatory diseases, knowledge of the defects responsible for type I interferonopathies will likely promote the development of targeted therapy.

Invariant Natural Killer T (iNKT) cells are a unique subset of T lymphocytes that have been implicated in both promoting and suppressing a multitude of immune responses. In mice, iNKT cells express T cell antigen receptors (TCRs) comprising a unique TCRα rearrangement between the Trav11 and Traj18 gene segments. When paired with certain Trbv TCRβ chains, these TCRs recognize lipid antigens presented by the major histocompatibility complex (MHC) class I-like molecule, CD1d. Until recently, the sole model of iNKT deficiency targeted the Jα18, which is absolutely required to form the TCR with the appropriate antigenic specificity. However, these mice were demonstrated to have a large reduction in TCR repertoire diversity, which could confound results arising from studies using these mice. Here, we have created a new NKT-deficient mouse strain using transcription activator-like effector nuclease (TALEN) technology to only disrupt the expression of Jα18, leaving the remaining Jα repertoire unperturbed. We confirm that these mice lack iNKT cells and do not respond to lipid antigen stimulation while the development of conventional T cells, regulatory T cells, and type Ib NKT cells is normal. This new mouse strain will serve as a new model of iNKT cell deficiency to facilitate our understanding of iNKT biology.

Purpose: A fundamental challenge in the era of next-generation sequencing (NGS) is to design effective treatments tailored to the mutational profiles of tumors. Many newly discovered cancer mutations are difficult to target pharmacologically; however, T-cell-based therapies may provide a valuable alternative owing to the exquisite sensitivity and specificity of antigen recognition. To explore this concept, we assessed the immunogenicity of a panel of genes that are common sites of driver mutations in follicular lymphoma, an immunologically sensitive yet currently incurable disease., Experimental Design: Exon capture and NGS were used to interrogate tumor samples from 53 patients with follicular lymphoma for mutations in 10 frequently mutated genes. For 13 patients, predicted mutant peptides and proteins were evaluated for recognition by autologous peripheral blood T cells after in vitro priming., Results: Mutations were identified in 1-5 genes in 81% (43/53) of tumor samples. Autologous, mutation-specific CD8(+) T cells were identified in 23% (3/13) of evaluated cases. T-cell responses were directed toward putative driver mutations in CREBBP and MEF2B. Responding T cells showed exquisite specificity for mutant versus wild-type proteins and recognized lymphoma cells expressing the appropriate mutations. Responding T cells appeared to be from the naïve repertoire, as they were found at low frequencies and only at single time points in each patient., Conclusions: Patients with follicular lymphoma harbor rare yet functionally competent CD8(+) T cells specific for recurrent mutations. Our results support the concept of using NGS to design individualized immunotherapies targeting common driver mutations in follicular lymphoma and other malignancies. Clin Cancer Res; 22(9); 2226-36. ©2015 AACR., (©2015 American Association for Cancer Research.)

Tumor promotion is an early and critical stage during lung adenocarcinoma (ADC). We previously demonstrated that Tlr4 mutant mice were more susceptible to butylated hydroxytoluene (BHT)-induced pulmonary inflammation and tumor promotion in comparison to Tlr4-sufficient mice. Our study objective was to elucidate the underlying differences in Tlr4 mutant mice in innate immune cell populations, their functional responses, and the influence of these cellular differences on ADC progenitor (type II) cells following BHT-treatment. BALB (Tlr4-sufficient) and C.C3-Tlr4(Lps-d)/J (BALB(Lpsd); Tlr4 mutant) mice were treated with BHT (promoter) followed by bronchoalveolar lavage (BAL) and flow cytometry processing on the lungs. ELISAs, Club cell enrichment, macrophage function, and RNA isolation were also performed. Bone marrow-derived macrophages (BMDM) co-cultured with a type II cell line were used for wound healing assays. Innate immune cells significantly increased in whole lung in BHT-treated BALB(Lpsd) mice compared to BALB mice. BHT-treated BALB(Lpsd) mice demonstrated enhanced macrophage functionality, increased epithelial wound closure via BMDMs, and increased Club cell number in BALB(Lpsd) mice, all compared to BALB BHT-treated mice. Cytokine/chemokine (Kc, Mcp1) and growth factor (Igf1) levels also significantly differed among the strains and within macrophages, gene expression, and cell surface markers collectively demonstrated a more plastic phenotype in BALB(Lpsd) mice. Therefore, these correlative studies suggest that distinct innate immune cell populations are associated with the differences observed in the Tlr4-mutant model. Future studies will investigate the macrophage origins and the utility of the pathways identified herein as indicators of immune system deficiencies and lung tumorigenesis.

Somatic mutations are important drivers of cancer development. Accumulating evidence suggests that a significant subset of mutations result in neo-epitopes recognized by autologous T cells and thus may constitute the Achilles' heel of tumor cells. T cells directed against mutations have been shown to have a key role in clinical efficacy of potent cancer immunotherapy modalities, such as adoptive transfer of autologous tumor infiltrating lymphocytes and immune checkpoint inhibitors. Whereas these findings strengthen the idea of a prominent role of neo-epitopes in tumor rejection, the systematic therapeutic exploitation of mutations was hampered until recently by the uniqueness of the repertoire of mutations ('the mutanome') in every patient's tumor. This review highlights insights into immune recognition of neo-epitopes and novel concepts for comprehensive identification and immunotherapeutic exploitation of individual mutations., (Copyright © 2015. Published by Elsevier Ltd.)

Following transmission, HIV-1 adapts in the new host by acquiring mutations that allow it to escape from the host immune response at multiple epitopes. It also reverts mutations associated with epitopes targeted in the transmitting host but not in the new host. Moreover, escape mutations are often associated with additional compensatory mutations that partially recover fitness costs. It is unclear whether recombination expedites this process of multi-locus adaptation. To elucidate the role of recombination, we constructed a detailed population dynamics model that integrates viral dynamics, host immune response at multiple epitopes through cytotoxic T lymphocytes, and viral evolution driven by mutation, recombination, and selection. Using this model, we compute the expected waiting time until the emergence of the strain that has gained escape and compensatory mutations against the new host's immune response, and reverted these mutations at epitopes no longer targeted. We find that depending on the underlying fitness landscape, shaped by both costs and benefits of mutations, adaptation proceeds via distinct dominant pathways with different effects of recombination, in particular distinguishing escape and reversion. When adaptation at a single epitope is involved, recombination can substantially accelerate immune escape but minimally affects reversion. When multiple epitopes are involved, recombination can accelerate or inhibit adaptation depending on the fitness landscape. Specifically, recombination tends to delay adaptation when a purely uphill fitness landscape is accessible at each epitope, and accelerate it when a fitness valley is associated with each epitope. Our study points to the importance of recombination in shaping the adaptation of HIV-1 following its transmission to new hosts, a process central to T cell-based vaccine strategies., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

The monogenic autoinflammatory diseases are rare, genetic disorders resulting in constitutive innate immune defects leading to excessive response to danger signals, spontaneous activation of inflammatory mediators or loss of inhibitory regulators. During the past 15 years, a growing number of monogenic inflammatory diseases have been described and their respective responsible genes identified. The proteins encoded by these genes are involved in the regulatory pathways of inflammation and are mostly expressed in cells of the innate immune system. Although a group of patients exhibit episodic systemic inflammation (periodic fevers), these disorders are mediated by continuous overproduction and release of pro-inflammatory mediators, notably IL-1β, and are best considered as autoinflammatory diseases rather than periodic fevers. The most common autoinflammatory diseases are familial Mediterranean fever (FMF), TNF receptor-associated periodic syndrome (TRAPS), mevalonate kinase deficiency/hyperimmunoglobulin D syndrome (MKD/HIDS) and the cryopyrin-associated periodic syndromes (CAPS). Clinical features often include fever, cutaneous rash, serosal involvement and acute phase reactants. Autoantibodies are usually absent but may accompany certain syndromes. Diagnosis remains clinical and is based on the different phenotypic features. Genetic diagnosis is of utmost importance, but must be performed judiciously and interpreted cautiously. Treatment with biologic agents that block proinflammatory cytokines, particularly IL-1, has proved to be dramatically effective in many patients. Still, in many cases of autoinflammation no genetic abnormalities are detected and treatment remains suboptimal, raising the question of novel pathogenic mutations in unexplored genes and pathways.

We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Current treatments to restore neurological deficits caused by axonal disconnection following central nervous system (CNS) injury are extremely limited. Protein phosphatase 2A (PP2A), one of the main serine-threonine phosphatases in mammalian cells, dephosphorylates collapsin response mediator protein-2 (CRMP2) in the developing CNS. In our study, we found that the major CNS inhibiting substrates, including chondroitin sulfate proteoglycans (CSPGs) and myelin associated glycoproteins (MAG), activated epidermal growth factor receptor (EGFR), but inactivated PP2A and downstream CRMP2. Both EGFR inactivation and PP2A activation promoted axon elongation in vitro in the presence of inhibitory substrates. EGFR blockage by AG1478 selectively attenuated the inactive form of PP2A in pY307 phosphorylation, thus increasing PP2A activity. EGFR activation by EGF attenuated PP2A activity, whereas mutation of Y307 to phenylalanine abolished the effect. Furthermore, PP2A activity was down-regulated immediately after spinal cord injury (SCI) in rats. Chronic application of d-erythro-sphingosine (DES), the PP2A agonist, to spinal cord-lesioned rats enhanced the activity of this phosphatase and dephosphorylated CRMP2 around the lesion. PP2A activation induced significant axon sprouting in the lesioned spinal cord and promoted function recovery after SCI. These findings suggest that PP2A works downstream of EGFR and dephosphorylates CRMP2 after CNS injury. Therefore, therapies targeting PP2A may be effective following SCI., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose: Deficiencies in CD19 and CD81 (forming the CD19-complex with CD21 and CD225) cause a severe clinical phenotype. One CD21 deficient patient has been described. We present a second CD21 deficient patient, with a mild clinical phenotype and compared the immunobiological characteristics of CD21 and CD19 deficiency., Methods: CD21 deficiency was characterized by flowcytometric immunophenotyping and sequencing. Real-time PCR, in vitro stimulation and next generation sequencing were used to characterize B-cell responses and affinity maturation in CD21(-/-) and CD19(-/-) B cells., Results: A compound heterozygous mutation in CD21 caused CD21 deficiency. CD21(-/-) B cells responded normally to in vitro stimulation and AID was transcribed. Affinity maturation was less affected by CD21 than by CD19 deficiency., Conclusions: Both CD21 and CD19 deficiencies cause hypogammaglobulinemia and reduced memory B cells. CD19 deficiency causes a more severe clinical phenotype. B-cell characteristics reflect this, both after in vitro stimulation as in affinity maturation., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Autosomal recessive human ZAP70 deficiency is a rare cause of combined immunodeficiency (CID) characterized by defective CD4 T cells and profound CD8 T cell lymphopenia. Herein, we report two novel patients that extend the molecular genetics, the clinical and functional phenotypes associated with the ZAP70 deficiency. The patients presented as infant-onset CID with severe infections caused by varicella zoster virus and live vaccines. Retrospective TCR excision circle newborn screening was normal in both patients. One patient carried a novel non-sense mutation (p.A495fsX75); the other a previously described misense mutation (p.A507V). In contrast to CD4 T cells, the majority of the few CD8 T cells showed expression of the ZAP70-related tyrosine kinase SYK that correlated with residual TCR signaling including calcium flux and degranulation. Our findings highlight the differential requirements of ZAP70 and SYK during thymic development, peripheral homeostasis as well as effector functions of CD4 and CD8 T cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The clinical course of Hepatitis C Virus (HCV) infection is highly variable between infected individual hosts: up to 80% of acutely HCV infected patients develop a chronic infection while 20% clear infection spontaneously. Spontaneous clearance of HCV infection can be predicted by several factors, including symptomatic acute infection, favorable IFNL3 polymorphisms and gender. In our study, we explored the possibility that variants in HCV cell entry factors might be involved in resistance to HCV infection. In a same case patient highly exposed but not infected by HCV, we previously identified one mutation in claudin-6 (CLDN6) and a rare variant in occludin (OCLN), two tight junction proteins involved in HCV entry into hepatocytes. Here, we conducted an extensive functional study to characterize the ability of these two natural variants to prevent HCV entry. We used lentiviral vectors to express Wildtype or mutated CLDN6 and OCLN in different cell lines and primary human hepatocytes. HCV infection was then investigated using cell culture produced HCV particles (HCVcc) as well as HCV pseudoparticles (HCVpp) expressing envelope proteins from different genotypes. Our results show that variants of CLDN6 and OCLN expressed separately or in combination did not affect HCV infection nor cell-to-cell transmission. Hence, our study highlights the complexity of HCV resistance mechanisms supporting the fact that this process probably not primarily involves HCV entry factors and that other unknown host factors may be implicated.

Over the past few decades the extent to which cytotoxic T lymphocytes (CTLs) control human immunodeficiency virus (HIV) replication has been studied extensively, yet their role and mode of action remain controversial. In some studies, CTLs were found to kill a large fraction of the productively infected cells relative to the viral cytopathicity, whereas in others CTLs were suggested to kill only a small fraction of infected cells. In this review, we compile published estimates of CTL-mediated death rates, and examine whether these studies permit determining the rate at which CTLs kill HIV-1 infected cells. We highlight potential misinterpretations of the CTL-killing rates from the escape rates of mutants, and from perturbations of the steady state viral load during chronic infection. Our major conclusion is that CTL-mediated killing rates remain unknown. But contrary to current consensus, we argue that killing rates higher than one per day are perfectly consistent with the experimental data, which would imply that the majority of the productively infected cells could still die from CTL-mediated killing rather than from viral cytopathicity., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

When a T-cell Receptor (TcR) interacts with its cognate peptide-MHC (pMHC), it triggers activation of a signaling cascade that results in the elicitation of a T cell effector function. Different models have been proposed to understand which parameters are needed to obtain an optimal activation of the signaling. It was speculated that improving the binding of a TcR could bring a stronger pMHC recognition, hence a stronger stimulation of the T cell. However, it was recently shown that an increase in affinity does not seem to be sufficient to guarantee improved functionality. A combination of factors is necessary to place the modified TcR in an optimal functional window. We here compared the binding parameters of two mutants of the melanoma antigen peptide MART-127-35 specific TcR DMF5. The first mutant was previously isolated by others in a screen for improved TcR. It was reported to have an increased CD8-independent activity. We confirmed these data and showed that the enhancement was neither due to change in half life (t1/2) nor Kd of the pMHC-TcR complex. The second mutant was designed based on a previous report claiming that a particular polymorphic residue in the TRAV12-2 chain was stabilizing the TcR. We created a DMF5 mutant for this residue and showed that, unexpectedly, this TcR had acquired a reduced overall activity although the TcR-pMHC complex was more stable when compared to the TcR wild type complex (increased t1/2). In addition, the soluble TcR form of this mutant bound target cells less efficiently. From this we concluded that kinetic parameters do not always predict the superior functionality of mutant TcRs., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the development of one or more antiviral drugs will be necessary. A set of five single-domain antibody fragments (variable parts of the heavy chain of a heavy-chain antibody [VHHs]) with an in vitro neutralizing activity against poliovirus type 1 was developed previously (B. Thys, L. Schotte, S. Muyldermans, U. Wernery, G. Hassanzadeh-Ghassabeh, and B. Rombaut, Antiviral Res 87:257-264, 2010, http://dx.doi.org/10.1016/j.antiviral.2010.05.012), and their mechanisms of action have been studied (L. Schotte, M. Strauss, B. Thys, H. Halewyck, D. J. Filman, M. Bostina, J. M. Hogle, and B. Rombaut, J Virol 88:4403-4413, 2014, http://dx.doi.org/10.1128/JVI.03402-13). In this study, neutralization escape mutants were selected for each VHH. Sequencing of the P1 region of the genome showed that amino acid substitutions are found in the four viral proteins of the capsid and that they are located both in proximity to the binding sites of the VHHs and in regions further away from the canyon and hidden beneath the surface. Characterization of the mutants demonstrated that they have single-cycle replication kinetics that are similar to those of their parental strain and that they are all drug (VHH) independent. Their resistant phenotypes are stable, as they do not regain full susceptibility to the VHH after passage over HeLa cells in the absence of VHH. They are all at least as stable as the parental strain against heat inactivation at 44°C, and three of them are even significantly (P < 0.05) more resistant to heat inactivation. The resistant variants all still can be neutralized by at least two other VHHs and retain full susceptibility to pirodavir and 35-1F4., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Rapid advances in genetics are providing unprecedented insight into functions of the innate immune system with identification of the mutations that cause monogenic autoinflammatory disease. Cytokine antagonism is profoundly effective in a subset of these conditions, particularly those associated with increased interleukin-1 (IL-1) activity, the inflammasomopathies. These include syndromes where the production of IL-1 is increased by mutation of innate immune sensors such as NLRP3, upstream signalling molecules such as PSTPIP1 and receptors or downstream signalling molecules, such as IL-1Ra. Another example of this is interferon (IFN) and the interferonopathies, with mutations in the sensors STING and MDA5, the upstream signalling regulator AP1S3, and a downstream inhibitor of IFN signalling, ISG15. We propose that this can be extended to cytokines such as IL-36, with mutations in IL-36Ra, and IL-10, with mutations in IL-10RA and IL-10RB, however mutations in sensors or upstream signalling molecules are yet to be described in these instances. Additionally, autoinflammatory diseases can be caused by multiple cytokines, for example with the activation of NF-κB/Rel, for which we propose the term Relopathies. This nosology is limited in that some cytokine pathways may be degenerate in their generation or execution, however provides insight into likely autoinflammatory disease candidates and the cytokines with which newly identified mutations may be associated, and therefore targeted., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Inflammasomes are multi-protein complexes composed of a NOD-like receptor (NLR)/an AIM-like receptor (ALR), the adapter molecule apoptosis-associated speck-like protein that contains a CARD (ASC), and caspase-1. Active caspase-1 cleaves pro-IL-1β and pro-IL-18 to IL-1β and IL-18, resulting in inflammation. Genetic mutations in inflammasomes were first recognized to result in autoinflammatory diseases, which are characterized by the absence of both autoantibodies and autoreactive-T/B cells. However, there is increasing attention being placed on genetic polymorphisms that are involved in the components of inflammasomes, and these have implications for innate immunity and the natural history of autoimmune diseases. For example, while the NOD-like receptor family, pyrin domain containing 1 (NLRP1) haplotypes contributes to susceptibility to developing vitiligo; there are other single nucleotide polymorphisms (SNPs) that alters the susceptibility and severity of rheumatoid arthritis (RA) and juvenile idiopathic arthritis. Indeed, there are multiple factors that contribute to lowering the threshold of immunity and inflammasomes play a key role in this threshold. For example, IL-1β and IL-18 further perpetuate Th17 responses and endothelial cell damage, which potentiate a number of autoimmune diseases, including synovitis in RA, cardiovascular disease, and systemic lupus erythematosus (SLE). There is also increasing data on the role of innate immunity in experimental autoimmune encephalomyelitis (EAE), in lupus nephritis, and in a variety of autoimmune pathologies in which activation of the innate immune system is the driver for the adaptive system. Indeed, it is likely that the chronic pathology of autoimmunity is mediated in part by otherwise innocent bystander cells, augmented by inflammasomes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Currently, the concept of 'neuroinflammation' includes inflammation associated with neurodegenerative diseases, in which there is little or no infiltration of blood-derived immune cells into the brain. The roles of brain-resident and peripheral immune cells in these inflammatory settings are poorly understood, and it is unclear whether neuroinflammation results from immune reaction to neuronal dysfunction/degeneration, and/or represents cell-autonomous phenotypes of dysfunctional immune cells. Here, we review recent studies examining these questions in the context of Huntington's disease (HD), where mutant Huntingtin (HTT) is expressed in both neurons and glia. Insights into the cellular and molecular mechanisms underlying neuroinflammation in HD may provide a better understanding of inflammation in more complex neurodegenerative disorders, and of the contribution of the neuroinflammatory component to neurodegenerative disease pathogenesis., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Infection with hepatitis B virus (HBV) remains a worldwide health problem, and DNA-based vaccines against HBV have been tested for therapeutic applications. HBV possesses three envelope lipoproteins that are translated from a single reading-frame: large, middle, and small HBV surface antigens. Among these envelope proteins, the middle HBV surface antigen (MHBs) contains a constitutive N-linked glycosylation site at position 4 (Asn4) in the amino-terminal portion (MQWNSTTFHQ) of pre-S2 domain. Asn4 (shown in bold) is essential for secretion of viral particles and conserved among all serotypes of HBV, but its influence on the immunogenicity of MHBs remains unknown. Here, we constructed four MHBs genes carrying mutations, underlined, in the amino-terminal portion of pre-S2 domain. One mutant protein contains Q at position 4 (MQWQSTTFHQ). In addition, each of three mutant MHBs proteins contains a N-linked glycosylation site (N-X-S/T), relocated to position 5 (MQWQNTTFHQ), 6 (MQWQSNTSHQ) or 7 (MQWQSTNFTQ) in pre-S2 domain. The expression and immunogenic properties of mutant DNA vaccines were examined in 293T human renal epithelial cells and in BALB/c mice, respectively. We showed that Asn4 was critical for secretion and immunogenicity of MHBs. Moreover, the MHBs protein that carries a N-linked glycosylation site at position 5 or 7 retained the properties similar to wild-type MHBs. In contrast, the secretion-defective mutant protein carrying Asn at position 6 induced only marginal humoral and cellular immune responses in mice, despite the N-linked glycosylation. In conclusion, N-linked glycosylation at an appropriate position in pre-S2 domain is an essential requirement for DNA vaccine expressing MHBs.

The severe clinical symptoms of inherited CD59 deficiency confirm the importance of CD59 as essential complement regulatory protein for protection of cells against complement attack, in particular protection of hematopoietic cells and human neuronal tissue. Targeted complement inhibition might become a treatment option as suggested by a case report. The easy diagnostic approach by flow cytometry and the advent of a new treatment option should increase the awareness of this rare differential diagnosis and lead to further studies on their pathophysiology., (Copyright © 2015 Elsevier Inc. All rights reserved.)

T lymphocyte migration is crucial for adaptive immunity. Manipulation of signaling molecules controlling cell migration combined with in-vitro cell migration analysis provides a powerful research approach. Microfluidic devices, which can precisely configure chemoattractant gradients and allow quantitative single cell analysis, have been increasingly applied to cell migration and chemotaxis studies. However, there are a very limited number of published studies involving microfluidic migration analysis of genetically manipulated immune cells. In this study, we describe a simple microfluidic method for quantitative analysis of T cells expressing transfected chemokine receptors and other cell migration signaling probes. Using this method, we demonstrated chemotaxis of Jurkat transfectants expressing wild-type or C-terminus mutated CCR7 within a gradient of chemokine CCL19, and characterized the difference in transfectant migration mediated by wild-type and mutant CCR7. The EGFP-tagged CCR7 allows identification of CCR7-expressing transfectants in cell migration analysis and microscopy assessment of CCR7 dynamics. Collectively, our study demonstrated the effective use of the microfluidic method for studying CCR7 mediated T cell transfectant migration. We envision this developed method will provide a useful platform to functionally test various signaling mechanisms at the cell migration level., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Predicting the emergence of new pathogenic strains is a key goal of evolutionary epidemiology. However, the majority of existing studies have focussed on emergence at the population level, and not within a host. In particular, the coexistence of pre-existing and mutated strains triggers a heightened immune response due to the larger total pathogen population; this feedback can smother mutated strains before they reach an ample size and establish. Here, we extend previous work for measuring emergence probabilities in non-equilibrium populations, to within-host models of acute infections. We create a mathematical model to investigate the emergence probability of a fitter strain if it mutates from a self-limiting strain that is guaranteed to go extinct in the long-term. We show that ongoing immune cell proliferation during the initial stages of infection causes a drastic reduction in the probability of emergence of mutated strains; we further outline how this effect can be accurately measured. Further analysis of the model shows that, in the short-term, mutant strains that enlarge their replication rate due to evolving an increased growth rate are more favoured than strains that suffer a lower immune-mediated death rate ('immune tolerance'), as the latter does not completely evade ongoing immune proliferation due to inter-parasitic competition. We end by discussing the model in relation to within-host evolution of human pathogens (including HIV, hepatitis C virus, and cancer), and how ongoing immune growth can affect their evolutionary dynamics.

Gene variants that disrupt TCR signaling can cause severe immune deficiency, yet less disruptive variants are sometimes associated with immune pathology. Null mutations of the gene encoding the scaffold protein Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76), for example, cause an arrest of T cell positive selection, whereas a synthetic membrane-targeted allele allows limited positive selection but is associated with proinflammatory cytokine production and autoantibodies. Whether these and other enigmatic outcomes are due to a biochemical uncoupling of tolerogenic signaling, or simply a quantitative reduction of protein activity, remains to be determined. In this study we describe a splice variant of Lcp2 that reduced the amount of wild-type SLP-76 protein by ~90%, disrupting immunogenic and tolerogenic pathways to different degrees. Mutant mice produced excessive amounts of proinflammatory cytokines, autoantibodies, and IgE, revealing that simple quantitative reductions of SLP-76 were sufficient to trigger immune dysregulation. This allele reveals a dose-sensitive threshold for SLP-76 in the balance of immunity and immune dysregulation, a common disturbance of atypical clinical immune deficiencies., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Tumor-associated macrophages are widely studied in solid tumors. The distribution of macrophages in lymph node samples was found to be associated with the prognosis of lymphoma patients. However, the role of macrophages in leukemia and their functional and phenotypic characteristics in hematopoietic malignancies have not been defined. In this study, we examined the distribution and functional and phenotypic characteristics of macrophages in a Notch1-induced mouse model of T cell acute lymphoblastic leukemia (T-ALL). The distribution of macrophages in bone marrow (BM) and spleen, which are proposed as BM and spleen leukemia-associated macrophages (LAMs), were different during the development of leukemia. LAMs stimulated the proliferation of T-ALL cells and had higher migration activity. RNA-sequencing analysis revealed that gene expression profiles of BM and spleen LAMs showed considerable differences. RT-PCR analysis showed that LAMs expressed both M1- and M2-associated phenotypic genes, but they expressed much lower levels of TGF-β1, VEGF-A, and CSF-1 than did tumor-associated macrophages from B16 melanoma. Furthermore, spleen LAMs more potently stimulated the proliferation of T-ALL cells compared with BM LAMs. Moreover, LAMs could be subdivided into M1-like (CD206(-)) and M2-like (CD206(+)) groups. Both CD206(+) and CD206(-) LAMs stimulated the proliferation of T-ALL cells, although CD206(+) LAMs expressed higher levels of most M1- and M2-associated genes. These results suggested the functional and phenotypic characteristics of LAMs, which were modified by organ specific microenvironments. Our results broaden our knowledge about macrophages in malignant microenvironments from solid tumors to leukemia., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Cellular immune responses that protect against tumors typically have been attributed to CD8 T cells. However, CD4 T cells also play a central role. It was shown recently that, in a patient with metastatic cholangiocarcinoma, CD4 T cells specific for a peptide from a mutated region of ERBB2IP could arrest tumor progression. This and other recent findings highlight new opportunities for CD4 T cells in cancer immunotherapy. In this article, I discuss the role and regulation of CD4 T cells in response to tumor Ags. Emphasis is placed on the types of Ags and mechanisms that elicit tumor-protective responses. I discuss the advantages and drawbacks of cancer immunotherapy through personalized genomics. These considerations should help to guide the design of next-generation therapeutic cancer vaccines., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The cytotoxic T-cell immune response plays an important role in the prevention of influenza infection and reducing of the illness severity. The knowledge about mechanisms of the virus-specific CD8+ T-cell induction in humans is necessary for better understanding of influenza epidemiology and vaccine development. Due to application of new immunological and genetic methods in last years, considerable amount of.data became available in the literature about CD8+ T-cell immune responses to different influenza A viruses. This review summarizes these data. The main attention is paid to (i) heterosubtypic CTL responses to conservative immunodominant sites; (ii) mechanisms of viral escape from the virus-specific CTLs by means of evolutional escape-mutations; (iii) influence of the HLA haplotype on CD8+ T-cell immune responses. The importance of these data for immunology and vaccinology is discussed.

Antibody-mediated immunotherapy is effective in humanized mice when combinations of broadly neutralizing antibodies (bNAbs) are used that target nonoverlapping sites on the human immunodeficiency virus type 1 (HIV-1) envelope. In contrast, single bNAbs can control simian-human immunodeficiency virus (SHIV) infection in immune-competent macaques, suggesting that the host immune response might also contribute to the control of viremia. Here, we investigate how the autologous antibody response in intact hosts can contribute to the success of immunotherapy. We find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively administered bNAbs by preventing the emergence of bNAb viral escape variants., (© 2014 Klein et al.)

This study was designed to determine whether treatment with erythropoietin (EPO) could protect cerebral microvasculature against the pathological consequences of endothelial nitric oxide (NO) synthase uncoupling. Wild-type and GTP cyclohydrolase I (GTPCH-I)-deficient hph1 mice were administered EPO (1000 U/kg/day, s.c., 3 days). Cerebral microvessels of hph1 mice demonstrated reduced tetrahydrobiopterin (BH4) bioavailability, increased production of superoxide anions and impaired endothelial NO signaling. Treatment of hph1 mice with EPO attenuated the levels of 7,8-dihydrobiopterin, the oxidized product of BH4, and significantly increased the ratio of BH4 to 7,8-dihydrobiopterin. Moreover, EPO decreased the levels of superoxide anions and increased NO bioavailability in cerebral microvessels of hph1 mice. Attenuated oxidation of BH4 and inhibition of endothelial NO synthase uncoupling were explained by the increased expression of antioxidant proteins, manganese superoxide dismutase, and catalase. The protective effects of EPO observed in cerebral microvessels of hph1 mice were also observed in GTPCH-I siRNA-treated human brain microvascular endothelial cells exposed to EPO (1 U/mL or 10 U/mL; 3 days). Our results suggest that EPO might protect the neurovascular unit against oxidative stress by restoring bioavailability of BH4 and endothelial NO in the cerebral microvascular endothelium. We demonstrate that treatment with erythropoietin (EPO) could protect cerebral microvasculature against the pathological consequences of endothelial nitric oxide (NO) synthase uncoupling. Our results suggest that EPO might protect the neurovascular unit against oxidative stress by restoring bioavailability of tetrahydrobiopterin (BH4) and endothelial nitric oxide., (© 2014 International Society for Neurochemistry.)