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Oncolytic virus (OV) is a replication competent virus that selectively invades cancer cells; as these cells die under the viral burden, the released virus particles proceed to infect other cancer cells. Oncolytic viruses are designed to also be able to stimulate the anticancer immune response. Thus, one may represent an OV by two parameters: its replication potential and its immunogenicity. In this paper we consider a combination therapy with OV and a checkpoint inhibitor, anti-PD-1. We evaluate the efficacy of the combination therapy in terms of the tumor volume at some later time, for example, 6 months from initial treatment. Since T cells kill not only virus-free cancer cells but also virus-infected cancer cells, the following question arises: Does increasing the amount of the checkpoint inhibitor always improve the efficacy? We address this question, by a mathematical model consisting of a system of partial differential equations. We use the model to construct, by simulations, an efficacy map in terms of the doses of the checkpoint inhibitor and the OV injection. We show that there are regions in the map where an increase in the checkpoint inhibitor actually decreases the efficacy of the treatment. We also construct efficacy maps with checkpoint inhibitor vs. the replication potential of the virus that show the same antagonism, namely, an increase in the checkpoint inhibitor may actually decrease the efficacy. These results have implications for clinical trials. [ABSTRACT FROM AUTHOR]

T-cells and antigen presenting cells are an essential part of the adaptive immune response system and how they interact is crucial in how the body effectively fights infection or responds to vaccines. Much of the experimental work studying interaction forces between cells has looked at the average properties of bulk samples of cells or applied microscopy to image the dynamic contact between these cells. In this paper we present a novel optical trapping technique for interrogating the force of this interaction and measuring relative interaction forces at the single-cell level. A triple-spot optical trap is used to directly manipulate the cells of interest without introducing foreign bodies such as beads to the system. The optical trap is used to directly control the initiation of cell-cell contact and, subsequently to terminate the interaction at a defined time point. The laser beam power required to separate immune cell pairs is determined and correlates with the force applied by the optical trap. As proof of concept, the antigen-specific increase in interaction force between a dendritic cell and a specific T-cell is demonstrated. Furthermore, it is demonstrated that this interaction force is completely abrogated when T-cell signalling is blocked. As a result the potential of using optical trapping to interrogate cellular interactions at the single cell level without the need to introduce foreign bodies such as beads is clearly demonstrated. [ABSTRACT FROM AUTHOR]

Experimental and mathematical studies in immunology have revealed that the dynamics of the programmed T cell response to vigorous infection can be conveniently modelled using a sigmoidal or a discontinuous immune response function. This paper hypothesizes strong synergies between this existing work and the dynamical behaviour of engineering systems with a variable structure control (VSC) law. These findings motivate the interpretation of the immune system as a variable structure control system. It is shown that dynamical properties as well as conditions to analytically assess the transition from health to disease can be developed for the specific T cell response from the theory of variable structure control. In particular, it is shown that the robustness properties of the specific T cell response as observed in experiments can be explained analytically using a VSC perspective. Further, the predictive capacity of the VSC framework to determine the T cell help required to overcome chronic Lymphocytic Choriomeningitis Virus (LCMV) infection is demonstrated. The findings demonstrate that studying the immune system using variable structure control theory provides a new framework for evaluating immunological dynamics and experimental observations. A modelling and simulation tool results with predictive capacity to determine how to modify the immune response to achieve healthy outcomes which may have application in drug development and vaccine design. [ABSTRACT FROM AUTHOR]

Enhancement or inhibition of cytokine signaling and corresponding immune cells responses are critical factors in various disease treatments. Cytokine signaling may be inhibited by cytokine-neutralizing antibodies (CNAs), which prevents further activation of cytokine receptors. However, CNAs may result in enhanced—instead of inhibitory—cytokine signaling (an “agonistic effect”) in various in vitro and in vivo experiments. This may lead to lack of efficacy or adverse events for cytokine-inhibiting based medicines. Alternatively, cytokine-antibody complexes may produce stronger signaling vs. cytokine alone, thereby increasing the efficacy of stimulating cytokine-based drugs, at equal or lower cytokine doses. In this paper, the effect of cytokine signaling enhancement by a CNA was studied in a generic mathematical model of interleukin-4 (IL-4) driven T-cell proliferation. The occurrence of the agonistic effect depends upon the antibody-to-cytokine binding affinity and initial concentrations of antibody and cytokine. Model predictions were in agreement with experimental studies. When the cytokine receptor consists of multiple subunits with substantially differing affinities (e.g., IL-4 case), the choice of the receptor chain to be blocked by the antibody is critical, for the agonistic effect to appear. We propose a generic mechanism for the effect: initially, binding of the CNA to the cytokine reduces free cytokine concentration; yet, cytokine molecules bound within the cytokine-CNA complex—and released later and over time—are “rescued” from earlier clearance via cellular internalization. Hence, although free cytokine-dependent signalling may be less potent initially, it will also be more sustained over time; and given non-linear dynamics, it will lead ultimately to larger cellular effector responses, vs. the same amount of free cytokine in the absence of CNA. We suggest that the proposed mechanism is a generic property of {cytokine, CNA, receptor} triads, both in vitro and in vivo, and can occur in a predictable fashion for a variety of cytokines of the immune system. [ABSTRACT FROM AUTHOR]

Lack of immunogenicity of cancer cells has been considered a major reason for their failure in induction of a tumor specific T cell response. In this paper, we present evidence that decitabine (DAC), a DNA methylation inhibitor that is currently used for the treatment of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and other malignant neoplasms, is capable of eliciting an anti-tumor cytotoxic T lymphocyte (CTL) response in mouse EL4 tumor model. C57BL/6 mice with established EL4 tumors were treated with DAC (1.0 mg/kg body weight) once daily for 5 days. We found that DAC treatment resulted in infiltration of IFN-γ producing T lymphocytes into tumors and caused tumor rejection. Depletion of CD8+, but not CD4+ T cells resumed tumor growth. DAC-induced CTL response appeared to be elicited by the induction of CD80 expression on tumor cells. Epigenetic evidence suggests that DAC induces CD80 expression in EL4 cells via demethylation of CpG dinucleotide sites in the promoter of CD80 gene. In addition, we also showed that a transient, low-dose DAC treatment can induce CD80 gene expression in a variety of human cancer cells. This study provides the first evidence that epigenetic modulation can induce the expression of a major T cell co-stimulatory molecule on cancer cells, which can overcome immune tolerance, and induce an efficient anti-tumor CTL response. The results have important implications in designing DAC-based cancer immunotherapy. [ABSTRACT FROM AUTHOR]

The spread of KPC-type carbapenemases is mainly attributed to the global dissemination of Klebsiella pneumoniae (KP) strains belonging to the clonal group (CG) 258, including sequence type (ST) 258 and other related STs. Two distinct clades of CG258-KP have evolved, which differ mainly for the composition of their capsular polysaccharides, and recent studies indicate that clade 1 evolved from an ancestor of clade 2 by recombination of a genomic fragment carrying the capsular polysaccharide (cps) locus. In this paper, we investigated the ability of two ST258-KP strains, KKBO-1 and KK207-1, selected as representatives of ST258-KP clade 2 and clade 1, respectively, to activate an adaptive immune response using ex vivo-stimulation of PBMC from normal donors as an experimental model. Our data showed that KKBO-1 (clade 2) induces a Th17 response more efficiently than KK207-1 (clade 1): the percentage of CD4+IL17+ cells and the production of IL-17A were significantly higher in cultures with KKBO-1 compared to cultures with KK207-1. While no differences in the rate of bacterial internalization or in the bacteria-induced expression of CD86 and HLA-DR by monocytes and myeloid dendritic cells were revealed, we found that the two strains significantly differ in inducing the production of cytokines involved in the adaptive immune response, as IL-1β, IL-23 and TNF-α, by antigen-presenting cells, with KKBO-1 being a more efficient inducer than KK207-1. The immune responses elicited by KK207-1 were comparable to those elicited by CIP 52.145, a highly virulent K. pneumoniae reference strain known to escape immune-inflammatory responses. Altogether, present results suggest that CG258-KP of the two clades are capable of inducing a different response of adaptive immunity in the human host. [ABSTRACT FROM AUTHOR]

Anaplasma marginale is the most prevalent tick-borne livestock pathogen and poses a significant threat to cattle industry. In contrast to currently available live blood-derived vaccines against A. marginale, alternative safer and better-defined subunit vaccines will be of great significance. Two proteins (VirB9-1 and VirB9-2) from the Type IV secretion system of A. marginale have been shown to induce humoral and cellular immunity. In this study, Escherichia coli were used to express VirB9-1 and VirB9-2 proteins. Silica vesicles having a thin wall of 6 nm and pore size of 5.8 nm were used as the carrier and adjuvant to deliver these two antigens both as individual or mixed nano-formulations. High loading capacity was achieved for both proteins, and the mouse immunisation trial with individual as well as mixed nano-formulations showed high levels of antibody titres over 107 and strong T-cell responses. The mixed nano-formulation also stimulated high-level recall responses in bovine T-cell proliferation assays. These results open a promising path towards the development of efficient A. marginale vaccines and provide better understanding on the role of silica vesicles to deliver multivalent vaccines as mixed nano-formulations able to activate both B-cell and T-cell immunity, for improved animal health. [ABSTRACT FROM AUTHOR]

The skin is a very suitable organ for the induction of immune responses to vaccine antigens. Antigen delivery systems to the skin by needle and syringe directly deposit the antigen into the epidermal-dermal compartment, one of the most immunocompetent sites due to the presence of professional antigen-presenting cells aimed at the induction of antigen-specific T cells. In this study, we analyzed the amount of ovalbumin as an antigen delivered to the skin by a microneedle. When ovalbumin protein as an antigen was delivered to the skin of mice using a dissolving microneedle, it induced an immune response through the enhanced proliferation and cytokines production by the splenocytes and lymph nodes. Also, it effectively increased the ovalbumin-specific CD8+ T cell and CD4+ T cell population and induced an ovalbumin-specific CTL response against the graft of ovalbumin-expressing EG7 tumor cells in the immunized mice. Also, we identified the inhibition of tumor growth and prevention of tumor formation in the context of the therapeutic and prophylactic vaccine, respectively through EG-7 tumor mouse model. Finally, these data show the potential of patches as attractive antigen delivery vehicles. [ABSTRACT FROM AUTHOR]

We aimed to explore whether the combination of intradermal DNA vaccination, to boost immune response against melanoma antigens, and immune checkpoint blockade, to alleviate immunosuppression, improves antitumor effectiveness in a murine B16F10 melanoma tumor model. Compared to single treatments, a combination of intradermal DNA vaccination (ovalbumin or gp100 plasmid adjuvanted with IL12 plasmid) and immune checkpoint CTLA-4/PD-1 blockade resulted in a significant delay in tumor growth and prolonged survival of treated mice. Strong activation of the immune response induced by combined treatment resulted in a significant antigen-specific immune response, with elevated production of antigen-specific IgG antibodies and increased intratumoral CD8+ infiltration. These results indicate a potential application of the combined DNA vaccination and immune checkpoint blockade, specifically, to enhance the efficacy of DNA vaccines and to overcome the resistance to immune checkpoint inhibitors in certain cancer types. [ABSTRACT FROM AUTHOR]

Cancer cells harness immune checkpoints such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase 1 (IDO1) to evade immune control. Checkpoint inhibitors have demonstrated durable anti-tumor efficacy in human and preclinical models. Liver toxicity is one of the common immune-related adverse events associated with checkpoint inhibitors (CPIs) and its frequency and severity often increase significantly during CPI combination therapies. We aim to develop a mouse model to elucidate the immune mechanisms of CPI-associated liver toxicity. Co-administration of CTLA-4 blocking antibody, 9D9, and/or an IDO1 inhibitor, epacadostat in wild-type and PD-1-/- mice (to simulate the effect of PD1 blockade) synergistically induced liver injury and immune cell infiltration. Infiltrated cells were primarily composed of CD8+ T cells and positively associated with hepatocyte necrosis. Strikingly, sites of hepatocyte necrosis were frequently surrounded by clusters of mononuclear immune cells. CPI treatments resulted in increased expression of genes associated with hepatocyte cell death, leukocyte migration and T cell activation in the liver. In conclusion, blockade of immune checkpoints PD-1, CTLA-4, and IDO1 act synergistically to enhance T cell infiltration and activity in the liver, leading to hepatocyte death. [ABSTRACT FROM AUTHOR]

T cells are crucial players in obesity-mediated adipose tissue inflammation. We hypothesized that osteopontin (OPN), an inflammatory protein with enhanced activity when proteolytically cleaved, affects the number of viable T cells in adipose tissue and assessed inhibition of the interaction between T cells and thrombin and matrix metalloproteinases-cleaved OPN using antibodies and postimmune sera. Gene expression of T cell markers in adipose tissue from wild-type (wt) and Spp1-/- (OPN deficient) mice was analyzed after 16 weeks of high fat diet (HFD) or low fat diet (LFD) feeding. CD3, CD8 and OPN gene expression in omental adipose tissue from individuals with obesity was measured. OPN-T cell interactions were assessed with a fluorescence-based adhesion assay and blocked with antibodies targeting OPN. Comparison of T cell gene expression in adipose tissue from wt and Spp1-/- mice showed that OPN affected the number of T cells while in humans, levels of OPN correlated with T cell markers in omental adipose tissue. The interaction between T cells and cleaved OPN was blocked by postimmune sera following OPN peptide vaccinations and with monoclonal antibodies. In conclusion, levels of OPN affected the number of T cells in obesity and antibodies against cleaved OPN antagonize OPN-T cell interactions. [ABSTRACT FROM AUTHOR]

The genital tract of African women has been shown to differ from what is currently accepted as ‘normal’, defined by a pH≤4.5 and lactobacilli-dominated microbiota. Adolescent girls and young women (AGYW) from sub-Saharan Africa are at high risk for HIV, and we hypothesized that specific biological factors are likely to be influential. This study aimed to compare characteristics of vaginal health in HIV-negative AGYW (16-22-years-old), from two South African communities, to international norms. We measured plasma hormones, vaginal pH, presence of BV (Nugent scoring), sexually transmitted infections (multiplex PCR for Chlamydia trachomatis, Neisseria gonorrhoea, Trichomonas vaginalis, Mycoplasma genitalium) and candidiasis (Gram stain) in AGYW (n = 298) from Cape Town and Soweto. Cervicovaginal microbiota was determined by 16S pyrosequencing; 44 genital cytokines were measured by Luminex; and cervical T-cell activation/proliferation (CCR5, HLA-DR, CD38, Ki67) was measured by multiparametric flow cytometry. 90/298 (30.2%) AGYW were negative for BV, candidiasis and bacterial STIs. L. crispatus and L. iners were the dominant bacteria in cervicovaginal swabs, and the median vaginal pH was 4.7. AGYW with L. crispatus-dominant microbiota (42.4%) generally had the lowest cytokine concentrations compared to women with more diverse microbiota (34/44 significantly upregulated cytokines). Frequencies of CCR5+CD4+ T-cells co-expressing CD38 and HLA-DR correlated positively with interleukin (IL)-6, TNF-α, GRO-α, macrophage inflammatory protein (MIP)-1α, and IL-9. While endogenous oestrogen had an immune-dampening effect on IL-6, TNF-related apoptosis-inducing ligand (TRAIL) and IL-16, injectable hormone contraceptives (DMPA and Net-EN) were associated with significantly lower endogenous hormone concentrations (p<0.0001 for oestrogen and progesterone) and upregulation of 34/44 cytokines. Since genital inflammation and the presence of activated CD4+ T cells in the genital tract have been implicated in increased HIV risk in South African women, the observed high levels of genital cellular activation and cytokines from AGYW may point towards biological factors increasing HIV risk in this region. [ABSTRACT FROM AUTHOR]

Effective vaccine design relies on accurate knowledge of protection against a pathogen, so as to be able to induce relevant and effective protective responses against it. An ideal Human Immunodeficiency virus (HIV) vaccine should induce humoral as well as cellular immune responses to prevent initial infection of host cells or limit early events of viral dissemination. A Phase I HIV-1 prophylactic vaccine trial sponsored by the International AIDS Vaccine Initiative (IAVI) was conducted in India in 2009.The trial tested a HIV-1 subtype C vaccine in a prime-boost regimen, comprising of a DNA prime (ADVAX) and Modified Vaccine Ankara (MVA) (TBC-M4) boost. The trial reported that the vaccine regimen was safe, well tolerated, and resulted in enhancement of HIV-specific immune responses. However, preliminary immunological studies were limited to vaccine-induced IFN-γ responses against the Env and Gag peptides. The present study is a retrospective study to characterize in detail the nature of the vaccine-induced cell mediated immune responses among volunteers, using Peripheral Blood Mononuclear Cells (PBMC) that were archived during the trial. ELISpot was used to measure IFN-γ responses and polyfunctional T cells were analyzed by intracellular multicolor flow cytometry. It was observed that DNA priming and MVA boosting induced Env and Gag specific bi-functional and multi-functional CD4+ and CD8+ T cells expressing IFN-γ, TNF-α and IL-2. The heterologous prime-boost regimen appeared to be slightly superior to the homologous prime-boost regimen in inducing favorable cell mediated immune responses. These results suggest that an in-depth analysis of vaccine-induced cellular immune response can aid in the identification of correlates of an effective immunogenic response, and inform future design of HIV vaccines. [ABSTRACT FROM AUTHOR]

Immunotherapy has fundamentally changed the landscape of cancer treatment. Despite the encouraging results with the checkpoint modulators, response rates vary widely across tumor types, with a majority of patients exhibiting either primary resistance without a significant initial response to treatment or acquired resistance with subsequent disease progression. Hematopoietic progenitor kinase 1 (HPK1) is predominantly expressed in hematopoietic cell linages and serves as a negative regulator in T cells and dendritic cells (DC). While HPK1 gene knockout (KO) studies suggest its role in anti-tumor immune responses, the involvement of kinase activity and thereof its therapeutic potential remain unknown. To investigate the potential of pharmacological intervention using inhibitors of HPK1, we generated HPK1 kinase dead (KD) mice which carry a single loss-of—function point mutation in the kinase domain and interrogated the role of kinase activity in immune cells in the context of suppressive factors or the tumor microenvironment (TME). Our data provide novel findings that HKP1 kinase activity is critical in conferring suppressive functions of HPK1 in a wide range of immune cells including CD4+, CD8+, DC, NK to Tregs, and inactivation of kinase domain was sufficient to elicit robust anti-tumor immune responses. These data support the concept that an HPK1 small molecule kinase inhibitor could serve as a novel agent to provide additional benefit in combination with existing immunotherapies, particularly to overcome resistance to current treatment regimens. [ABSTRACT FROM AUTHOR]

This article describes the procedures used to isolate pure B-cell populations from whole blood using various Miltenyi magnetic-activated cell sorting (MACS) bead Isolation kits. Such populations are vital for studies investigating the functional capacity of B-cells, as the presence of other cell types may have indirect effects on B-cell function through cell-cell interactions or by secretion of several soluble molecules. B-cells can be isolated by two main approaches: 1) Negative selection—in which B-cells remain "untouched" in their native state; this is advantageous as it is likely that B-cells remain functionally unaltered by this process. 2) Positive selection–in which B-cells are labelled and actively removed from the sample. We used three Negative B-cell isolation kits as well as the Positive B-cell isolation kit from Miltenyi and compared the purity of each of the resulting B-cells fractions. Contamination of isolated B-cell fractions with platelets was the conclusive finding for all of the isolation techniques tested. These results illustrate the inefficiency of current available MACS B-cell isolation kits to produce pure B-cell populations, from which concrete findings can be made. As such we suggest cell sorting as the preferred method for isolating pure B-cells to be used for downstream functional assays. [ABSTRACT FROM AUTHOR]

Uveal melanoma (UM) is the most common primary intraocular cancer in adults. In the present study, we aimed to characterize the immunological features of primary UM cancer and to provide an association with prognostic markers and outcome. Also, we assessed the influence of the microenvironment on the expression of inhibitory immune checkpoints in UM. Genes of interest included MHC Class I and Class II molecules, as well as inhibitory immune-checkpoints, i.e. PDL1, PDL2, B7-H3, B7-H4, TBFRSF6B, CD47, CD155, GAL9, HVEM and CD200. We observed significant lower levels of MHC genes in UM cells as compared to normal uveal melanocytes. Unexpectedly however, the expression levels of most of the analyzed inhibitory immune-checkpoint genes were not different in cancer cells as compared to normal melanocytes, with the exception of CD200 and HVEM, that resulted significantly reduced. On the other hand, PDL1 inversely correlated with OS, PFS and thickness of the tumor. Also, PDL1, along with PDL2, expression significantly increased under inflammatory conditions. Finally, for the first time, we propose a possible role for CD47 in the immune evasive properties of UM. We show here that CD47 is significantly upregulated by UM cells following inflammatory stimuli and that it represents a good independent predictor of disease progression. The results from this study may propel advances in the development of immune-based therapies for UM patients. [ABSTRACT FROM AUTHOR]

Rationale: HSP90B1, also known as gp96, is a chaperone for multiple Toll-like receptors (TLRs) and is necessary for TLR-mediated inflammatory responses in murine myeloid cells. The molecule is also expressed in T-cells though its specific role is unknown. We hypothesized that human HSP90B1 regulates monocyte and T-cell responses to Mycobacterium tuberculosis (Mtb) and bacilli Calmette-Guerin (BCG) and that its variants are associated with susceptibility to TB disease. Methods: We screened 17 haplotype-tagging SNPs in the HSP90B1 gene region for association with BCG-induced T-cell cytokine responses using both an ex-vivo whole blood assay (N = 295) and an intracellular cytokine staining assay (N = 180) on samples collected 10 weeks after birth. Using a case-control study design, we evaluated the same SNPs for association with TB disease in a South African pediatric cohort (N = 217 cases, 604 controls). A subset of these SNPs was evaluated for association with HSP90B1 expression in human monocytes, monocyte-derived dendritic cells, and T-cells using RT-PCR. Lastly, we used CRISPR/Cas9 gene editing to knock down HSP90B1 expression in a human monocyte cell line (U937). Knockdown and control cell lines were tested for TLR surface expression and control of Mtb replication. Results: We identified three SNPs, rs10507172, rs10507173 and rs1920413, that were associated with BCG-induced IL-2 secretion (p = 0.017 for rs10507172 and p = 0.03 for rs10507173 and rs1920413, Mann-Whitney, dominant model). SNPs rs10507172 and rs10507173 were associated with TB disease in an unadjusted analysis (p = 0.036 and 0.025, respectively, dominant model) that strengthened with sensitivity analysis of the definite TB cases, which included only those patients with microbiologically confirmed Mtb (p = 0.007 and 0.012, respectively). Knockdowns of HSP90B1 in monocyte cell lines with CRISPR did not alter TLR2 surface expression nor influence Mtb replication relative to controls. Conclusion: Among infants, an HSP90B1 gene-region variant is associated with BCG-induced IL-2 production and may be associated with protection from TB disease. HSP90B1 knockdown in human monocyte-like cell lines did not influence TLR2 surface localization nor Mtb replication. Together, these data suggest that HSP90B1 regulates T-cell, but not monocyte, responses to mycobacteria in humans. [ABSTRACT FROM AUTHOR]

In the immune system, T cells can quickly discriminate between foreign and self ligands with high accuracy. There is evidence that T-cells achieve this remarkable performance utilizing a network architecture based on a generalization of kinetic proofreading (KPR). KPR-based mechanisms actively consume energy to increase the specificity beyond what is possible in equilibrium. An important theoretical question that arises is to understand the trade-offs and fundamental limits on accuracy, speed, and dissipation (energy consumption) in KPR and its generalization. Here, we revisit this question through numerical simulations where we simultaneously measure the speed, accuracy, and energy consumption of the KPR and adaptive sorting networks for different parameter choices. Our simulations highlight the existence of a “feasible operating regime” in the speed-energy-accuracy plane where T-cells can quickly differentiate between foreign and self ligands at reasonable energy expenditure. We give general arguments for why we expect this feasible operating regime to be a generic property of all KPR-based biochemical networks and discuss implications for our understanding of the T cell receptor circuit. [ABSTRACT FROM AUTHOR]

Antibody dependent enhancement (ADE) has most often been associated with dengue virus (DENV). Studies using leukemia cell lines suggest that DENV specific antibodies can enhance Zika virus (ZIKV) infectivity, and vice versa. To examine the mechanisms of ADE of ZIKV infection in primary human cells, we assessed 40 serum samples obtained from convalescent DENV-1 or DENV-3 infected subjects. All sera tested exhibited high binding potency, while modest or none neutralization activities against ZIKV. Primary CD14+ monocytes, rather than B and T cells in peripheral blood mononuclear cells (PBMCs), were found to be the mediators of the enhancement of ZIKV infectivity by DENV immune sera. Monocyte-derived immature dendritic cells (DCs), but not mature DCs were highly permissive to ZIKV infection, whereas neither immature nor mature DCs could mediate enhanced ZIKV infection in the presence of DENV immune sera. In addition, antibody blocking of either FcγRI (CD64), or FcγRII (CD32), or FcγRIII (CD16) resulted in diminished ADE of ZIKV infection. Our findings provide an improved understanding of the pathogenesis of ZIKV infection, and inform rational vaccine design. [ABSTRACT FROM AUTHOR]

Congenital Cytomegalovirus infection (cCMV) is the leading infection in determining permanent long-term impairments (LTI), and its pathogenesis is largely unknown due to the complex interplay between viral, maternal, placental, and child factors. The cellular activity, considered to be the result of the response to exogenous and endogenous factors, is captured by the determination of gene expression profiles. In this study, we determined whole blood transcriptomes in relation to cCMV, CMV viral load and LTI development at 6 years of age by using RNA isolated from neonatal dried blood spots (DBS) stored at room temperature for 8 years. As DBS were assumed to mainly reflect the neonatal immune system, particular attention was given to the immune pathways using the global test. Additionally, differential expression of individual genes was performed using the voom/limma function packages. We demonstrated feasibility of RNA sequencing from archived neonatal DBS of children with cCMV, and non-infected controls, in relation to LTI and CMV viral load. Despite the lack of statistical power to detect individual genes differences, pathway analysis suggested the involvement of innate immune response with higher CMV viral loads, and of anti-inflammatory markers in infected children that did not develop LTI. Finally, the T cell exhaustion observed in infected neonates, in particular with higher viral load, did not correlate with LTI, therefore other mechanisms are likely to be involved in the long-term immune dysfunction. Despite these data demonstrate limitation in determining prognostic markers for LTI by means of transcriptome analysis, this exploratory study represents a first step in unraveling the pathogenesis of cCMV, and the aforementioned pathways certainly merit further evaluation. [ABSTRACT FROM AUTHOR]

Differentiating early mycosis fungoides (MF) from inflammatory dermatitis is a challenge. We compare the differential expression profile of early-stage MF samples and benign inflammatory dermatoses using microRNA (miRNA) arrays. 114 miRNAs were found to be dysregulated between these entities. The seven most differentially expressed miRNAs between these two conditions were further analyzed using RT-PCR in two series comprising 38 samples of early MFs and 18 samples of inflammatory dermatitis. A series of 51 paraffin-embedded samples belonging to paired stages of 16 MF patients was also analyzed. MiRNAs 26a, 222, 181a and 146a were differentially expressed between tumoral and inflammatory conditions. Two of these miRNAs (miRNA-181a and miRNA-146a) were significantly deregulated between early and advanced MF stages. Bioinformatic analysis showed FOXP3 expression to be regulated by these miRNAs. Immunohistochemistry revealed the level of FOXP3 expression to be lower in tumoral MFs than in plaque lesions in paraffin-embedded tissue. A functional study confirmed that both miRNAs diminished FOXP3 expression when overexpressed in CTCL cells. The data presented here suggest that the analysis of a restricted number of miRNAs (26a, 222, 181a and 146a) could be sufficient to differentiate tumoral from reactive conditions. Moreover, these miRNAs seem to be involved in MF progression. [ABSTRACT FROM AUTHOR]

Oxaliplatin is a platinum-based chemotherapeutic used for cancer treatment. Its use associates with peripheral neuropathies and chronic gastrointestinal side-effects. Oxaliplatin induces immunogenic cell death by provoking the presentation of damage associated molecular patterns. The damage associated molecular patterns high-mobility group box 1 (HMGB1) protein exerts pro-inflammatory cytokine-like activity and binds to toll-like receptors (namely TLR4). Gastrointestinal microbiota may influence chemotherapeutic efficacy and contribute to local and systemic inflammation. We studied effects of oxaliplatin treatment on 1) TLR4 and high-mobility group box 1 expression within the colon; 2) gastrointestinal microbiota composition; 3) inflammation within the colon; 4) changes in Peyer’s patches and mesenteric lymph nodes immune populations in mice. TLR4+ cells displayed pseudopodia-like extensions characteristic of antigen sampling co-localised with high-mobility group box 1 -overexpressing cells in the colonic lamina propria from oxaliplatin-treated animals. Oxaliplatin treatment caused significant reduction in Parabacteroides and Prevotella1, but increase in Prevotella2 and Odoribacter bacteria at the genus level. Downregulation of pro-inflammatory cytokines and chemokines in colon samples, a reduction in macrophages and dendritic cells in mesenteric lymph nodes were found after oxaliplatin treatment. In conclusion, oxaliplatin treatment caused morphological changes in TLR4+ cells, increase in gram-negative microbiota and enhanced HMGB1 expression associated with immunosuppression in the colon. [ABSTRACT FROM AUTHOR]

Background: In airway epithelium, thymus and activation-regulated chemokine (CCL17) and macrophage-derived chemokine (CCL22) are induced by defective epithelial barriers such as E-cadherin and attract the effector cells of Th2 immunity. However, the association between the epithelial barrier and CCL17 expression has not been studied in chronic rhinosinusitis with nasal polyp (CRSwNP). Thus, we aimed to evaluate the expression of CCL17 and its regulation by Th cytokines in nasal polyp (NP) epithelial cells. Methods: The expression and distribution of CCL17, CCL22, E-cadherin and/or epidermal growth factor receptor (EGFR) were measured using real-time PCR, western blot, and immunohistochemistry and compared between normal ethmoid sinus epithelium and NP epithelium. In addition, the expression level of CCL17 was determined in cultured epithelial cells treated with IL-4, IL-5, IL-13, TNF-α, and IFN-γ. Results: The expression of CCL17 was decreased in the NP epithelium compared to the epithelium of normal ethmoid sinus, whereas the expression of CCL22 was not decreased. E-cadherin was differentially distributed between the epithelium of normal ethmoid sinus and NP epithelium. EGFR was also decreased in NPs. Interestingly, the stimulation of cultured epithelial cells with Th2 cytokines, IL-4 and IL-5, resulted in an upregulation of CCL17 expression only in NP epithelial cells whereas the expression of CCL17 was increased in both normal epithelial cells and NP epithelial cells by Th1 cytokines. Conclusion: Our results suggest that the decreased expression of CCL17 in defective NP epithelium may be closely connected to NP pathogenesis and can be differentially regulated by cytokines in the NP epithelium of patients with CRSwNP. [ABSTRACT FROM AUTHOR]

Chemotherapy or chemoradiotherapy conditioning regimens required for bone marrow transplantation (BMT) cause significant morbidity and mortality as a result of insufficient immune surveillance mechanisms leading to increased risks of infection and tumor recurrence. Such conditioning causes host stromal cell injury, impairing restoration of the central (thymus) and peripheral (spleen and lymph node) T cell compartments and slow immune reconstitution. The chemokine, CCL21, produced by host stromal cells, recruits T- and B-cells that provide lymphotoxin mediated instructive signals to stromal cells for lymphoid organogenesis. Moreover, T- and B-cell recruitment into these sites is required for optimal adaptive immune responses to pathogens and tumor antigens. Previously, we reported that CCL21 was markedly reduced in secondary lymphoid organs of transplanted animals. Here, we utilized adenoviral CCL21 gene transduced dendritic cells (DC/CCL21) given by footpad injections as a novel approach to restore CCL21 expression in secondary lymphoid organs post-transplant. CCL21 expression in secondary lymphoid organs reached levels of naïve controls and resulted in increased T cell trafficking to draining lymph nodes (LNs). An increase in both lymphoid tissue inducer cells and the B cell chemokine CXCL13 known to be important in LN formation was observed. Strikingly, only mice vaccinated with DC/CCL21 loaded with bacterial, viral or tumor antigens and not recipients of DC/control adenovirus loaded cells or no DCs had a marked increase in the systemic clearance of pathogens (bacteria; virus) and leukemia cells. Because DC/CCL21 vaccines have been tested in clinical trials for patients with lung cancer and melanoma, our studies provide the foundation for future trials of DC/CCL21 vaccination in patients receiving pre-transplant conditioning regimens. [ABSTRACT FROM AUTHOR]

Leishmania (V.) braziliensis and Leishmania(L.) amazonensis are the most pathogenic agents of American Cutaneous Leishmaniasis in Brazil, causing a wide spectrum of clinical and immunopathological manifestations, including: localized cutaneous leishmaniasis (LCLDTH+/++), borderline disseminated cutaneous leishmaniasis (BDCLDTH±), anergic diffuse cutaneous leishmaniasis (ADCLDTH-), and mucosal leishmaniasis (MLDTH++++). It has recently been demonstrated, however, that while L. (V.) braziliensis shows a clear potential to advance the infection from central LCL (a moderate T-cell hypersensitivity form) towards ML (the highest T-cell hypersensitivity pole), L. (L.) amazonensis drives the infection in the opposite direction to ADCL (the lowest T-cell hypersensitivity pole). This study evaluated by immunohistochemistry the expression of Toll-like receptors (TLRs) 2, 4, and 9 and their relationships with CD4 and CD8 T-cells, and TNF-α, IL-10, and TGF-β cytokines in that disease spectrum. Biopsies of skin and mucosal lesions from 43 patients were examined: 6 cases of ADCL, 5 of BDCL, and 11 of LCL caused byL. (L.) amazonensis; as well as 10 cases of LCL, 4 of BDCL, and 6 of ML caused byL. (V.) braziliensis. CD4+ T-cells demonstrated their highest expression in ML and, in contrast, their lowest in ADCL. CD8+ T-cells also showed their lowest expression in ADCL as compared to the other forms of the disease. TNF-α+showed increased expression from ADCL to ML, while IL-10+and TGF-β+ showed increased expression in the opposite direction, from ML to ADCL. With regards to TLR2, 4, and 9 expressions, strong interactions of TLR2 and 4 with clinical forms associated with L. (V.) braziliensis were observed, while TLR9, in contrast, showed a strong interaction with clinical forms linked to L. (L.) amazonensis. These findings strongly suggest the ability of L. (V.) braziliensis and L. (L.) amazonensis to interact with those TLRs to promote a dichotomous T-cell immune response in ACL. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a granulomatous disease that has affected humanity for thousands of years. The production of cytokines, such as IFN-γ and TNF-α, is fundamental in the formation and maintenance of granulomas and in the control of the disease. Recently, the introduction of TNF-α-blocking monoclonal antibodies, such as Infliximab, has brought improvements in the treatment of patients with chronic inflammatory diseases, but this treatment also increases the risk of reactivation of latent tuberculosis. Our objective was to analyze, in an in vitro model, the influence of Infliximab on the granulomatous reactions and on the production of antigen-specific cytokines (TNF-α, IFN-γ, IL-12p40, IL-10 and IL-17) from beads sensitized with soluble Bacillus Calmette-Guérin (BCG) antigens cultured in the presence of peripheral blood mononuclear cells (PBMC) from TB patients. We evaluated 76 individuals, with tuberculosis active, treated and subjects with positive PPD. Granuloma formation was induced in the presence or absence of Infliximab for up to 10 days. The use of Infliximab in cultures significantly blocked TNF-α production (p <0.05), and led to significant changes in granuloma structure, in vitro, only in the treated TB group. On the other hand, there was a significant reduction in the levels of IFN-γ, IL-12p40, IL-10 and IL-17 after TNF-α blockade in the three experimental groups (p <0.05). Taken together, our results demonstrate that TNF-α blockade by Infliximab directly influenced the structure of granuloma only in the treated TB group, but negatively modulated the production of Th1, Th17 and regulatory T cytokines in the three groups analyzed. [ABSTRACT FROM AUTHOR]

We investigated immune responses to a lytic cytomegalovirus antigen (CMVpp52), and to a lytic human herpes virus (HHV) 6 antigen (HHV6p41), in systemic lupus erythematosus (SLE) patients and healthy controls (HCs), in order to clarify if the previously established impaired responses to Epstein-Barr virus (EBV) in SLE patients is a general defect in their responses against (all) HHVs. Multiplex Luminex technology results showed a normal induction of five quantified cytokines (interferon γ, interleukin(IL)12, IL17, IL10, and tumor necrosis factor α) in SLE patients compared to HCs upon stimulation with CMVpp52 and HHV6p41. However, flow cytometric results showed a reduced upregulation of the activation marker CD69 on T-cells from SLE patients (n = 17) compared to HCs (n = 17) upon stimulation with CMVpp52, indicating limited or defective CMVpp52-specific T-cells and/or poor antigen-presentation in SLE patients, and thereby possibly decreased control of the CMV infection. In conclusion, the dysfunctional immune response against EBV previously established in SLE patients does not seem to apply to the same degree regarding the immune responses against CMV or HHV6. Results designate that the main contributing HHV agent in development or exacerbation of SLE (in genetically predisposed individuals) is the previously determined uncontrolled EBV infection, and to a lesser extent CMV infection, and probably with no involvement of HHV6 infection. [ABSTRACT FROM AUTHOR]

Human bone marrow-derived mesenchymal stem cells (MSCs) have been observed to inhibit arthritis in experimental animal models such as collagen-induced arthritis. However, the exact anti-inflammatory mechanisms remain poorly understood. Interleukin-1 receptor antagonist (IL-1Ra) is an anti-inflammatory cytokine produced by immune and stromal cells. We postulated that MSCs could produce IL-1Ra and attenuate experimental arthritis. In this study, 5x106 MSCs were injected into the peritoneal cavity of IL-1Ra knockout (IL-1RaKO) mice. MSCs reduced the severity of the arthritis by histology and decreased pro-inflammatory cytokine levels in IL-1RaKO mice. The ratio of splenic T helper 17 (Th17) cells to regulatory T cells (Treg) was significantly decreased in MSC-injected IL-1RaKO mice. Purified splenic CD4+ T cells from mice in each of the treatment groups were cultured under Th17 polarizing conditions and analyzed by flow cytometry. Less expansion of the Th17 population was observed in the MSC-treated group. Interestingly, MSCs expressed inducible IL-1Ra against inflammatory environmental stimuli. Human recombinant IL-1Ra could suppress Th17 cells differentiation under Th17 polarizing conditions. These results indicate that IL-1Ra expressed by MSCs can inhibit Th17 polarization and decrease the immune response in IL-1RaKO mice. Therefore, MSC-derived IL-1Ra may inhibit inflammation in IL-1RaKO mice via effects on Th17 differentiation. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs are important mediators of post-transcriptional regulation of gene expression through RNA degradation and translational repression, and are emerging biomarkers of immune system activation/response after vaccination. Methods: We performed Next Generation Sequencing (mRNA-Seq) of intracellular miRNAs in measles virus-stimulated B and CD4+ T cells from high and low antibody responders to measles vaccine. Negative binomial generalized estimating equation (GEE) models were used for miRNA assessment and the DIANA tool was used for gene/target prediction and pathway enrichment analysis. Results: We identified a set of B cell-specific miRNAs (e.g., miR-151a-5p, miR-223, miR-29, miR-15a-5p, miR-199a-3p, miR-103a, and miR-15a/16 cluster) and biological processes/pathways, including regulation of adherens junction proteins, Fc-receptor signaling pathway, phosphatidylinositol-mediated signaling pathway, growth factor signaling pathway/pathways, transcriptional regulation, apoptosis and virus-related processes, significantly associated with neutralizing antibody titers after measles vaccination. No CD4+ T cell-specific miRNA expression differences between high and low antibody responders were found. Conclusion: Our study demonstrates that miRNA expression directly or indirectly influences humoral immunity to measles vaccination and suggests that B cell-specific miRNAs may serve as useful predictive biomarkers of vaccine humoral immune response. [ABSTRACT FROM AUTHOR]

We propose an agent-based model for the simulation of the autoimmune response in T1D. The model incorporates cell behavior from various rules derived from the current literature and is implemented on a high-performance computing system, which enables the simulation of a significant portion of the islets in the mouse pancreas. Simulation results indicate that the model is able to capture the trends that emerge during the progression of the autoimmunity. The multi-scale nature of the model enables definition of rules or equations that govern cellular or sub-cellular level phenomena and observation of the outcomes at the tissue scale. It is expected that such a model would facilitate in vivo clinical studies through rapid testing of hypotheses and planning of future experiments by providing insight into disease progression at different scales, some of which may not be obtained easily in clinical studies. Furthermore, the modular structure of the model simplifies tasks such as the addition of new cell types, and the definition or modification of different behaviors of the environment and the cells with ease. [ABSTRACT FROM AUTHOR]

Quantitative relationship between the magnitude of variation in minor histocompatibility antigens (mHA) and graft versus host disease (GVHD) pathophysiology in stem cell transplant (SCT) donor-recipient pairs (DRP) is not established. In order to elucidate this relationship, whole exome sequencing (WES) was performed on 27 HLA matched related (MRD), & 50 unrelated donors (URD), to identify nonsynonymous single nucleotide polymorphisms (SNPs). An average 2,463 SNPs were identified in MRD, and 4,287 in URD DRP (p<0.01); resulting peptide antigens that may be presented on HLA class I molecules in each DRP were derived in silico (NetMHCpan ver2.0) and the tissue expression of proteins these were derived from determined (GTex). MRD DRP had an average 3,670 HLA-binding-alloreactive peptides, putative mHA (pmHA) with an IC50 of <500 nM, and URD, had 5,386 (p<0.01). To simulate an alloreactive donor cytotoxic T cell response, the array of pmHA in each patient was considered as an operator matrix modifying a hypothetical cytotoxic T cell clonal vector matrix; each responding T cell clone’s proliferation was determined by the logistic equation of growth, accounting for HLA binding affinity and tissue expression of each alloreactive peptide. The resulting simulated organ-specific alloreactive T cell clonal growth revealed marked variability, with the T cell count differences spanning orders of magnitude between different DRP. Despite an estimated, uniform set of constants used in the model for all DRP, and a heterogeneously treated group of patients, higher total and organ-specific T cell counts were associated with cumulative incidence of moderate to severe GVHD in recipients. In conclusion, exome wide sequence differences and the variable alloreactive peptide binding to HLA in each DRP yields a large range of possible alloreactive donor T cell responses. Our findings also help understand the apparent randomness observed in the development of alloimmune responses. [ABSTRACT FROM AUTHOR]

CD1d-deficiency results in a selective deletion of NKT cells in mice that is reported to prevent murine allergic airway disease (AAD). Because we find 2–3 fold lower basal IL-4 production in CD1d- mice than in wild-type (WT) mice, we hypothesized that the contribution made by NKT cells to AAD would depend on the strength of the stimulus used to induce the disease. Consequently, we compared CD1d-deficient mice to WT mice in the development of AAD, using several models of disease induction that differed in the type and dose of allergen, the site of sensitization and the duration of immunization. Surprisingly we found equivalent allergic inflammation and airway disease in WT and CD1d- mice in all models investigated. Consistent with this, NKT cells constituted only ~2% of CD4+ T cells in the lungs of mice with AAD, and IL-4-transcribing NKT cells did not expand with disease induction. Concerned that the congenital absence of NKT cells might have caused a compensatory shift within the immune response, we administered an anti-CD1d monoclonal Ab (mAb) to block NKT function before airway treatments, before or after systemic sensitization to antigen. Such Ab treatment did not affect disease severity. We suggest that the differences reported in the literature regarding the significance of NKT cells in the induction of allergic airway disease may have less to do with the methods used to study the disease and more to do with the animals themselves and/or the facilities used to house them. [ABSTRACT FROM AUTHOR]

Obesity promotes excessive inflammation, which is associated with senescence-like changes in visceral adipose tissue (VAT) and the development of type 2 diabetes (T2DM) and cardiovascular diseases. We have reported that a unique population of CD44hi CD62Llo CD4+ T cells that constitutively express PD-1 and CD153 exhibit cellular senescence and cause VAT inflammation by producing large amounts of osteopontin. Weight loss improves glycemic control and reduces cardiovascular disease risk factors, but its long-term effects on cardiovascular events and longevity in obese individuals with T2DM are somewhat disappointing and not well understood. High-fat diet (HFD)-fed obese mice were subjected to weight reduction through a switch to a control diet. They lost body weight and visceral fat mass, reaching the same levels as lean mice fed a control diet. However, the VAT of weight reduction mice exhibited denser infiltration of macrophages, which formed more crown-like structures compared to the VAT of obese mice kept on the HFD. Mechanistically, CD153+ PD-1+ CD4+ T cells are long-lived and not easily eliminated, even after weight reduction. Their continued presence maintains a self-sustaining chronic inflammatory loop via production of large amounts of osteopontin. Thus, we concluded that T-cell senescence is essentially a negative legacy effect of obesity. [ABSTRACT FROM AUTHOR]

Rationale: Patients with chronic rhinosinusitis with nasal polyps (CRSwNP) have been shown to be vitamin D3 (VD3) deficient, which is associated with more severe disease and increased polyp size. To gain mechanistic insights into these observational studies, we examined the impact of VD3 deficiency on inflammation and VD3 metabolism in an Aspergillus fumigatus (Af) mouse model of chronic rhinosinusitis (Af-CRS). Methods: Balb/c mice were fed control or VD3 deficient diet for 4 weeks. Mice were then sensitized with intraperitoneal Af, and one week later given Af intranasally every three days for four weeks while being maintained on control or VD3 deficient diet. Airway function, sinonasal immune cell infiltrate and sinonasal VD3 metabolism profiles were then examined. Results: Mice with VD3 deficiency had increased Penh and sRaw values as compared to controls as well as exacerbated changes in sRaw when coupled with Af-CRS. As compared to controls, VD3 deficient and Af-CRS mice had reduced sinonasal 1α-hydroxylase and the active VD3 metabolite, 1,25(OH)2D3. Differential analysis of nasal lavage samples showed that VD3 deficiency alone and in combination with Af-CRS profoundly upregulated eosinophil, neutrophil and lymphocyte numbers. VD3 deficiency exacerbated increases in monocyte-derived dendritic cell (DC) associated with Af-CRS. Conversely, T-regulatory cells were decreased in both Af-CRS mice and VD3 deficient mice, though coupling VD3 deficiency with Af-CRS did not exacerbate CD4 or T-regulatory cells numbers. Lastly, VD3 deficiency had a modifying or exacerbating impact on nasal lavage levels of IFN-γ, IL-6, IL-10 and TNF-α, but had no impact on IL-17A. Conclusions: VD3 deficiency causes changes in sinonasal immunity, which in many ways mirrors the changes observed in Af-CRS mice, while selectively exacerbating inflammation. Furthermore, both VD3 deficiency and Af-CRS were associated with altered sinonasal VD3 metabolism causing reductions in local levels of the active VD3 metabolite, 1,25(OH)2D3, even with adequate circulating levels. [ABSTRACT FROM AUTHOR]

The oral cavity is home to unique resident microbial communities whose interactions with host immunity are less frequently studied than those of the intestinal microbiome. We examined the stimulatory capacity and the interactions of two oral bacteria, Porphyromonas gingivalis (P. gingivalis) and Fusobacterium nucleatum (F. nucleatum), on Dendritic Cell (DC) activation, comparing them to the effects of the well-studied intestinal microbe Escherichia coli (E. coli). Unlike F. nucleatum and E. coli, P. gingivalis failed to activate DCs, and in fact silenced DC responses induced by F. nucleatum or E. coli. We identified a variant strain of P. gingivalis (W50) that lacked this immunomodulatory activity. Using biochemical approaches and whole genome sequencing to compare the two substrains, we found a point mutation in the hagA gene. This protein is though to be involved in the alteration of the PorSS/gingipain pathway, which regulates protein secretion into the extracellular environment. A proteomic comparison of the secreted products of the two substrains revealed enzymatic differences corresponding to this phenotype. We found that P. gingivalis secretes gingipain(s) that inactivate several key proinflammatory mediators made by DCs and/or T cells, but spare Interleukin-1 (IL-1) and GM-CSF, which can cause capillary leaks that serve as a source of the heme that P. gingivalis requires for its survival, and GM-CSF, which can cause epithelial-cell growth. Taken together, our results suggest that P. gingivalis has evolved potent mechanisms to modulate its virulence factors and dampen the innate immune response by selectively inactivating most proinflammatory cytokines. [ABSTRACT FROM AUTHOR]

Cytotoxic T-lymphocytes (CTLs) play a key role in immunity against cancer; however, the induction of CTL responses with currently available vaccines remains difficult. Because several reports have suggested that pigmentation and immunity might be functionally linked, we investigated whether melanin can act as an adjuvant in vaccines. Short synthetic peptides (8–35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides. Immunization with melanin-bound peptides efficiently triggered CTL responses in mice, even against self-antigens and at a very low dose of peptides (microgram range). Immunization against a tumor antigen inhibited the growth of established tumors in mice, an effect that was abrogated by the depletion of CD8+ lymphocytes. These results demonstrate the efficacy of melanin as a vaccine adjuvant. [ABSTRACT FROM AUTHOR]

Antigen exposure to oral mucosa is generally thought to lead to immune tolerance induction. However, very little is known about the subset composition and function of dendritic cells (DC) migrating from human oral mucosa. Here we show that migratory DC from healthy human gingival explants consist of the same phenotypic subsets in the same frequency distribution as DC migrating from human skin. The gingival CD1a+ Langerhans cell and interstitial DC subsets lacked CXCR4 expression in contrast to their cutaneous counterparts, pointing to different migration mechanisms, consistent with previous observations in constructed skin and gingival equivalents. Remarkably, without any exogenous conditioning, gingival explants released higher levels of inflammatory cytokines than human skin explants, resulting in higher DC migration rates and a superior ability of migrated DC to prime allogeneic T cells and to induce type-1 effector T cell differentiation. From these observations we conclude that rather than an intrinsic ability to induce T cell tolerance, DC migrating from oral mucosa may have a propensity to induce effector T cell immunity and maintain a high state of alert against possible pathogenic intruders in the steady state. These findings may have implications for oral immunization strategies. [ABSTRACT FROM AUTHOR]

Prolonged chronic stress has deleterious effects on immune function and is associated with numerous negative health outcomes. The spleen harbors one-fourth of the body’s lymphocytes and mediates both innate and adaptive immune responses. However, the subset of splenic lymphocytes that respond, either adaptively or maladaptively, to various stressors remains largely unknown. Here we investigated the effects of unpredictable chronic mild stress (CMS) exposure on spleen composition in male mice housed in two different caging conditions: standard caging (Cntl) and enriched environment (EE). EE-caged mice exhibited the greatest absolute number of splenocytes and CMS exposure significantly lowered splenocyte numbers in both caging conditions. Glucocorticoid production, measured by mean fecal corticosterone metabolites (FCM), was significantly lower in EE-caged mice vs. Cntl-caged mice. Surprisingly, CMS exposure resulted in an increase in mean FCM in EE-caged mice, but no significant change in Cntl-caged mice. CMS altered the splenic B:T lymphocyte ratio; it reduced the frequency of B cells, but increased the frequency of T cells in EE-caged mice. Splenocyte number and B:T lymphocyte ratio showed a negative relationship with mean FCM. EE-caged mice had a lower frequency of immature and germinal B cells than Cntl-caged mice. CMS markedly increased the frequency of immature and marginal zone B cells, but decreased the frequency of follicular B cells in both caging conditions. Mean FCM correlated positively with frequency of immature, marginal zone and germinal center B cells, but negatively with frequency of follicular B cells. To conclude, splenic immune cells, particularly B lymphocyte composition, are modulated by caging environment and stress and may prime mice differently to respond to immune challenges. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate differences in T helper cell sub-types and osteoclast (OCs) precursors in peripheral blood between patients affected by early rheumatoid arthritis (eRA) and healthy controls. The effect of administration of cholecalcipherol on clinical and laboratory parameters was subsequently evaluated, by a parallel, randomized double blind, placebo controlled trial. Thirty nine eRA patients and 31 age-matched controls were enrolled and compared for levels of 25OH vitamin D, T helper cell sub-types, OCs precursors including both classical and non-classical and pro-inflammatory cytokines at baseline. Eligible patients were female ≥18 years of age with a diagnosis of RA, as defined by the American College of Rheumatology 2010 criteria for <6 months prior to inclusion in the study. Patients with auto-immune or inflammatory diseases other than RA were excluded. Patients treated with glucocorticoids (GCs), disease modifying activity drugs and biologic agents within the past 6 months were also excluded. In the second phase of the study, eRA patients were randomly assigned to standard treatment with methotrexate (MTX) and GCs with (21) or without (18) cholecalcipherol (300,000 IU) and followed for 3 months; the randomization was done by computer generated tables to allocate treatments. Three patients didn’t come back to the follow up visit for personal reasons. None of the patients experienced adverse events. The main outcome measures were T cells phenotypes, OCs precursors and inflammatory cytokines. Secondary outcome measure were clinical parameters. In eRA, 25OH vitamin D levels were significantly lower. CD4+/IFNγ+,CD4+/IL4+, CD4+/IL17A+ and CD4+IL17A+IFNγ+, cells were increased in eRA as well as non-classical OCs precursors, whereas T regulatory cells were not altered. TNFα, TGFβ1, RANKL, IL-23 and IL-6 were increased in eRA. Non-classical OCs, IL-23 and IL-6 correlated with disease severity and activity. Standard treatment with MTX and GC ameliorated clinical symptoms and reduced IL-23, whereas it did not affect CD4+ cells sub-sets nor OCs precursors. After 3 months, the combined use of cholecalcipherol significantly ameliorated the effect of treatment on global health. In eRA, a significant imbalance in T CD4+ sub-types accompanied by increased levels of non-classical OCs precursors and pro-inflammatory cytokines was observed. A single dose of cholecalcipherol (300,000 IU) combined with standard treatment significantly ameliorates patients general health. [ABSTRACT FROM AUTHOR]

Many biopharmaceuticals (BPs) are known to be immunogenic in the clinic, which can result in modified pharmacokinetics, reduced efficacy, allergic reactions and anaphylaxis. During recent years, several technologies to predict immunogenicity have been introduced, but the predictive value is still considered low. Thus, there is an unmet medical need for optimization of such technologies. The generation of T cell dependent high affinity anti-drug antibodies plays a key role in clinical immunogenicity. This study aimed at developing and evaluating a novel in vitro T cell:PBMC assay for prediction of the immunogenicity potential of BPs. To this end, we assessed the ability of infliximab (anti-TNF-α), rituximab (anti-CD20), adalimumab (anti-TNF-α) and natalizumab (anti-α4-integrin), all showing immunogenicity in the clinic, to induce a CD4+ T cells response. Keyhole limpet hemocyanin (KLH) and cytomegalovirus pp65 protein (CMV) were included as neo-antigen and recall antigen positive controls, respectively. By analyzing 26 healthy donors having HLA-DRB1 alleles matching the European population, we calculated the frequency of responding donors, the magnitude of the response, and the frequency of BP-specific T cells, as measured by 3[H]-thymidine incorporation and ELISpot IL-2 secretion. KLH and CMV demonstrated a strong T cell response in all the donors analyzed. The frequency of responding donors to the BPs was 4% for infliximab, 8% for adalimumab, 19% for rituximab and 27% for natalizumab, which is compared to and discussed with their respective observed clinical immunogenicity. This study further complements predictive immunogenicity testing by quantifying the in vitro CD4+ T cell responses to different BPs. Even though the data generated using this modified method does not directly translate to the clinical situation, a high sensitivity and immunogenic potential of most BPs is demonstrated. [ABSTRACT FROM AUTHOR]

Introduction: HIV causes defects in memory B cells in children, but the mechanisms of those defects have not been fully elucidated. One possible mechanism is the lack of T-cell help to B cells during immune reactions. However, few studies have assessed the effect of HIV on follicular helper T cells (TFH cells) in children. Methods: In this study, follicular-homing CD4 T cells and memory B cells were assessed in HIV-infected children and compared with children from the community. CXCR5 and CD45RO were used as markers of follicular-homing T cells and memory T cells, respectively. Memory TFH cells were identified as CD3+CD8-CD4+CXCR5+CD45RO+PD1+. Central memory T cells were identified based on CCR7 expression. Relationship between the proportions of follicular-homing CD4 T cells and memory B cells were determined in multivariable regression models. Results: Highly viremic HIV-infected children had lower proportions of memory TFH cells when compared with community control children. In multivariable analyses, high proportions of memory TFH cells were associated with increased percentages of resting memory B cells after adjusting for other covariates. Conclusion: The impact of HIV on follicular helper T cells could influence the accumulation of memory B cells in HIV-infected children. [ABSTRACT FROM AUTHOR]

Research on human immunology has been hindered by the lack of optimal small animal models, given that the protective immune responses of human and non-human species show significant differences. However, due to ethical constraints[] and the high cost of clinical trials, it is urgent to improve the current animal models that can mimic faithfully human physiology, particularly the human immune system (HIS). HIS mice had been generated recently by engrafting human hematopoietic stem cells (hHSCs) or human peripheral mononuclear cells (hPBMCs) into highly immuno-deficient mice such as NSG, NOG or NRG mice. However, a major experimental drawback for studies using these models is the rapid onset of Graft-versus-Host Disease (GvHD). In the present study, we overcome this limitation by generating new immuno-deficient mice named "HUMAMICE" (HLA-A2+/+/DR1+/+/H-2-β2m-/-/IAβ-/-/Rag2-/-/IL2rγ-/-/Perf-/- mice), which expressed human HLA molecules instead of mouse MHC molecules (H-2), and whose immuno-deficient status was reversed by transferring functional HLA-matched PBMCs thus producing mice with an immuno-competent status with a functional human immune system. We showed that in this HLA-matched context, the hPBMC-transfer led to high lymphocytes engraftment rates without GvHD over three months in this novel mouse model. Furthermore, to evaluate the utility of the hPBMC-HUMAMICE, we immunized them with commercial vaccine of Hepatitis B virus (HBsAg, Hepvac@) which resulted in robust and reproducible production of high levels of HBsAg-specific antibodies, implying that both transferred T and B lymphocytes were functional in HUMAMICE. These responses are comparable to those observed in human clinical trials with this identical vaccine. In conclusion, these findings indicated that the HLA-matched-hPBMC-HUMAMICE represents a promising model for dissecting human immune responses in various human diseases, including infectious diseases, cancers and tumors, and to facilitate the development of novel vaccines and cellular therapies. [ABSTRACT FROM AUTHOR]

In an effort to make affordable vaccines suitable for the regions most affected by HIV-1, we have constructed stable vaccines that express an HIV-1 subtype C mosaic Gag immunogen (BCG-GagM, MVA-GagM and DNA-GagM). Mosaic immunogens have been designed to address the tremendous diversity of this virus. Here we have shown that GagM buds from cells infected and transfected with MVA-GagM and DNA-GagM respectively and forms virus-like particles. Previously we showed that a BCG-GagM prime MVA-GagM boost generated strong cellular immune responses in mice. In this study immune responses to the DNA-GagM and MVA-GagM vaccines were evaluated in homologous and heterologous prime-boost vaccinations. The DNA homologous prime boost vaccination elicited predominantly CD8+ T cells while the homologous MVA vaccination induced predominantly CD4+ T cells. A heterologous DNA-GagM prime MVA-GagM boost induced strong, more balanced Gag CD8+ and CD4+ T cell responses and that were predominantly of an effector memory phenotype. The immunogenicity of the mosaic Gag (GagM) was compared to a naturally occurring subtype C Gag (GagN) using a DNA homologous vaccination regimen. DNA-GagN expresses a natural Gag with a sequence that was closest to the consensus sequence of subtype C viruses sampled in South Africa. DNA-GagM homologous vaccination induced cumulative HIV-1 Gag-specific IFN-γ ELISPOT responses that were 6.5-fold higher than those induced by the DNA-GagN vaccination. Similarly, DNA-GagM vaccination generated 7-fold higher levels of cytokine-positive CD8+ T cells than DNA-GagN, indicating that this subtype C mosaic Gag elicits far more potent immune responses than a consensus-type Gag. Cells transfected and infected with DNA-GagM and MVA-GagM respectively, expressed high levels of GagM and produced budding virus-like particles. Our data indicates that a heterologous prime boost regimen using DNA and MVA vaccines expressing HIV-1 subtype C mosaic Gag is highly immunogenic in mice and warrants further investigation in non-human primates. [ABSTRACT FROM AUTHOR]

Objectives: CD4+CD25+FOXP3+ regulatory T cells (Treg) inhibit the anti-tumour immune response and reduce the effect of cancer immunotherapy. Although studies have demonstrated that the number and suppressive activity of Treg increase with age, it is not clear whether these changes correlate with a higher incidence of tumours in the elderly. This study was designed to explore the relationship between increase in CD4+CD25+FOXP3+ Treg and the higher risk of lung cancer in the elderly. Methods: Seventy lung cancer patients and 60 sex- and age-matched controls were recruited. Both groups were divided into three subgroups based on their age (young, middle-aged, or elderly). The proportion of CD4+CD25+FOXP3+ /CD4+ T cells was detected using flow cytometry, and the level of FOXP3 mRNA in the peripheral blood was examined with real-time RT-PCR. Results: The levels of CD4+CD25+FOXP3+/CD4+ T cells and FOXP3 mRNA were significantly higher in lung cancer patients than in healthy controls (t = 7.16, P < 0.01 and t = 3.65, P < 0.01, respectively). Within the healthy groups, the elderly group had larger proportion of CD4+CD25+FOXP3+ Treg (F = 32.54, P < 0.01) and higher FOXP3 mRNA expression (F = 4.76, P < 0.01) than their younger counterparts. Among the six subgroups, the elderly lung cancer patients exhibited the highest levels of both CD4+CD25+FOXP3+ Treg (11.81 ± 2.40%) and FOXP3 mRNA (3.14 ± 1.30). Conclusions: The accumulation of CD4+CD25+FOXP3+ Treg with age correlates well with the increasing incidence of lung cancer in the elderly. [ABSTRACT FROM AUTHOR]

Background: Atrial fibrillation (AF) is the most common arrhythmia and despite obvious clinical importance remains its pathogenesis only partially explained. A relation between inflammation and AF has been suggested by findings of increased inflammatory markers in AF patients. Objective: The goal of this study was to characterize morphologically and functionally CD45-positive inflammatory cell populations in atrial myocardium of patients with AF as compared to sinus rhythm (SR). Methods: We examined 46 subjects (19 with AF, and 27 in SR) undergoing coronary bypass or valve surgery. Peroperative bioptic samples of the left and the right atrial tissue were examined using immunohistochemistry. Results: The number of CD3+ T-lymphocytes and CD68-KP1+ cells were elevated in the left atrial myocardium of patients with AF compared to those in SR. Immune cell infiltration of LA was related to the rhythm, but not to age, body size, LA size, mitral regurgitation grade, type of surgery, systemic markers of inflammation or presence of diabetes or hypertension. Most of CD68-KP1+ cells corresponded to dendritic cell population based on their morphology and immunoreactivity for DC-SIGN. The numbers of mast cells and CD20+ B-lymphocytes did not differ between AF and SR patients. No foci of inflammation were detected in any sample. Conclusions: An immunohistochemical analysis of samples from patients undergoing open heart surgery showed moderate and site-specific increase of inflammatory cells in the atrial myocardium of patients with AF compared to those in SR, with prevailing population of monocyte-macrophage lineage. These cells and their cytokine products may play a role in atrial remodeling and AF persistence. [ABSTRACT FROM AUTHOR]

Whole malaria sporozoite vaccine regimens are promising new strategies, and some candidates have demonstrated high rates of durable clinical protection associated with memory T cell responses. Little is known about the anatomical distribution of memory T cells following whole sporozoite vaccines, and immunization of nonhuman primates can be used as a relevant model for humans. We conducted a chemoprophylaxis with sporozoite (CPS) immunization in P. knowlesi rhesus monkeys and challenged via mosquito bites. Half of CPS immunized animals developed complete protection, with a marked delay in parasitemia demonstrated in the other half. Antibody responses to whole sporozoites, CSP, and AMA1, but not CelTOS were detected. Peripheral blood T cell responses to whole sporozoites, but not CSP and AMA1 peptides were observed. Unlike peripheral blood, there was a high frequency of sporozoite-specific memory T cells observed in the liver and bone marrow. Interestingly, sporozoite-specific CD4+ and CD8+ memory T cells in the liver highly expressed chemokine receptors CCR5 and CXCR6, both of which are known for liver sinusoid homing. The majority of liver sporozoite-specific memory T cells expressed CD69, a phenotypic marker of tissue-resident memory (TRM) cells, which are well positioned to rapidly control liver-stage infection. Vaccine strategies that aim to elicit large number of liver TRM cells may efficiently increase the efficacy and durability of response against pre-erythrocytic parasites. [ABSTRACT FROM AUTHOR]

Fibroblast activation protein alpha (FAP) is a unique dual peptidase of the S9B serine protease family, being capable of both dipeptidyl peptidase and endopeptidase activities. FAP is expressed at low level in healthy adult organs including the pancreas, cervix, uterus, submaxillary gland and the skin, and highly upregulated in embryogenesis, chronic inflammation and tissue remodelling. It is also expressed by cancer-associated stromal fibroblasts in more than 90% of epithelial tumours. FAP has enzymatic and non-enzymatic functions in the growth, immunosuppression, invasion and cell signalling of tumour cells. FAP deficient mice are fertile and viable with no gross abnormality, but little data exist on the role of FAP in the immune system. FAP is upregulated in association with microbial stimulation and chronic inflammation, but its function in infection remains unknown. We showed that major populations of immune cells including CD4+ and CD8+ T cells, B cells, dendritic cells and neutrophils are generated and maintained normally in FAP knockout mice. Upon intranasal challenge with influenza virus, FAP mRNA was increased in the lungs and lung-draining lymph nodes. Nonetheless, FAP deficient mice showed similar pathologic kinetics to wildtype controls, and were capable of supporting normal anti-influenza T and B cell responses. There was no evidence of compensatory upregulation of other DPP4 family members in influenza-infected FAP-deficient mice. FAP appears to be dispensable in anti-influenza adaptive immunity. [ABSTRACT FROM AUTHOR]

Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells. [ABSTRACT FROM AUTHOR]

Background: Burn injury initiates an acute inflammatory response that subsequently drives wound repair. However, acute disruption to the immune response is also common, leading to susceptibility to sepsis and increased morbidity and mortality. Despite increased understanding of the impact of burn injury on the immune system in the acute phase, little is known about long-term consequences of burn injury on immune function. This study was established to determine whether burn injury has long-term clinical impacts on patients’ immune responses. Methods: Using a population-based retrospective longitudinal study and linked hospital morbidity and death data from Western Australia, comparative rates of hospitalisation for respiratory infections in burn patients and a non-injured comparator cohort were assessed. In addition, a mouse model of non-severe burn injury was also used in which viral respiratory infection was induced at 4 weeks post-injury using a mouse modified version of the Influenza A virus (H3NN; A/mem/71-a). Results and conclusions: The burn injured cohort contained 14893 adult patients from 1980–2012 after removal of those patients with evidence of smoke inhalation or injury to the respiratory tract. During the study follow-up study a total of 2,884 and 2,625 respiratory infection hospital admissions for the burn and uninjured cohorts, respectively, were identified. After adjusting for covariates, the burn cohort experienced significantly elevated admission rates for influenza and viral pneumonia (IRR, 95%CI: 1.73, 1.27–2.36), bacterial pneumonia (IRR, 95%CI: 2.05, 1.85–2.27) and for other types of upper and lower respiratory infections (IRR, 95% CI: 2.38, 2.09–2.71). In the mouse study an increased viral titre was observed after burn injury, accompanied by a reduced CD8 response and increased NK and NKT cells in the draining lymph nodes. This data suggests burn patients are at long-term increased risk of infection due to sustained modulation of the immune response. [ABSTRACT FROM AUTHOR]

The self-nonself discrimination hypothesis remains a landmark concept in immunology. It proposes that tolerance breaks down in the presence of nonself antigens. In strike contrast, in statistics, occurrence of nonself elements in a sample (i.e., outliers) is not obligatory to violate the null hypothesis. Very often, what is crucial is the combination of (self) elements in a sample. The two views on how to detect a change seem challengingly different and it could seem difficult to conceive how immunological cellular interactions could trigger responses with a precision comparable to some statistical tests. Here it is shown that frustrated cellular interactions reconcile the two views within a plausible immunological setting. It is proposed that the adaptive immune system can be promptly activated either when nonself ligands are detected or self-ligands occur in abnormal combinations. In particular we show that cellular populations behaving in this way could perform location statistical tests, with performances comparable to t or KS tests, or even more general data mining tests such as support vector machines or random forests. In more general terms, this work claims that plausible immunological models should provide accurate detection mechanisms for host protection and, furthermore, that investigation on mechanisms leading to improved detection in “in silico” models can help unveil how the real immune system works. [ABSTRACT FROM AUTHOR]