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Recent mathematical models have been developed to study the dynamics of chronic myelogenous leukemia (CML) under imatinib treatment. None of these models incorporates the anti-leukemia immune response. Recent experimental data show that imatinib treatment may promote the development of anti-leukemia immune responses as patients enter remission. Using these experimental data we develop a mathematical model to gain insights into the dynamics and potential impact of the resulting anti-leukemia immune response on CML. We model the immune response using a system of delay differential equations, where the delay term accounts for the duration of cell division. The mathematical model suggests that anti-leukemia T cell responses may play a critical role in maintaining CML patients in remission under imatinib therapy. Furthermore, it proposes a novel concept of an ''optimal load zone'' for leukemic cells in which the anti-leukemia immune response is most effective. Imatinib therapy may drive leukemic cell populations to enter and fall below this optimal load zone too rapidly to sustain the anti-leukemia T cell response. As a potential therapeutic strategy, the model shows that vaccination approaches in combination with imatinib therapy may optimally sustain the anti-leukemia T cell response to potentially eradicate residual leukemic cells for a durable cure of CML. The approach presented in this paper accounts for the role of the anti-leukemia specific immune response in the dynamics of CML. By combining experimental data and mathematical models, we demonstrate that persistence of anti-leukemia T cells even at low levels seems to prevent the leukemia from relapsing (for at least 50 months). As a consequence, we hypothesize that anti-leukemia T cell responses may help maintain remission under imatinib therapy. The mathematical model together with the new experimental data imply that there may be a feasible, low-risk, clinical approach to enhancing the effects of imatinib treatment. [ABSTRACT FROM AUTHOR]

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]

Head and neck cancer treatment often consists of surgical resection of the tumor followed by ionizing radiation (IR), which can damage surrounding tissues and cause adverse side effects. The underlying mechanisms of radiation-induced salivary gland dysfunction are not fully understood, and treatment options are scarce and ineffective. The wound healing process is a necessary response to tissue injury, and broadly consists of inflammatory, proliferative, and redifferentiation phases with immune cells playing key roles in all three phases. In this study, select immune cells were phenotyped and quantified, and certain cytokine and chemokine concentrations were measured in mouse parotid glands after IR. Further, we used a model where glandular function is restored to assess the immune phenotype in a regenerative response. These data suggest that irradiated parotid tissue does not progress through a typical inflammatory response observed in wounds that heal. Specifically, total immune cells (CD45+) decrease at days 2 and 5 following IR, macrophages (F4/80+CD11b+) decrease at day 2 and 5 and increase at day 30, while neutrophils (Ly6G+CD11b+) significantly increase at day 30 following IR. Additionally, radiation treatment reduces CD3- cells at all time points, significantly increases CD3+/CD4+CD8+ double positive cells, and significantly reduces CD3+/CD4-CD8- double negative cells at day 30 after IR. Previous data indicate that post-IR treatment with IGF-1 restores salivary gland function at day 30, and IGF-1 injections attenuate the increase in macrophages, neutrophils, and CD4+CD8+ T cells observed at day 30 following IR. Taken together, these data indicate that parotid salivary tissue exhibits a dysregulated immune response following radiation treatment which may contribute to chronic loss of function phenotype in head and neck cancer survivors. [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]

Fungal organisms contribute to significant human morbidity and mortality and Candida auris (C. auris) infections are of utmost concern due to multi-drug resistant strains and persistence in critical care and hospital settings. Pathogenesis and pathology of C. auris is still poorly understood and in this study, we demonstrate how the use of multiplex immunofluorescent imaging (MxIF) and single-cell analysis can contribute to a deeper understanding of fungal infections within organs. We used two different neutrophil depletion murine models (treated with either 1A8—an anti-Ly6G antibody, or RB6-8C5—an anti-Ly6G/Ly6C antibody; both 1A8 and RB6-8C5 antibodies have been shown to deplete neutrophils) and compared to wildtype, non-neutropenic mice. Following pathologist assessment, fixed samples underwent MxIF imaging using a C. albicans antibody (shown to be cross-reactive to C. auris) and immune cell biomarkers—CD3 (T cells), CD68 (macrophages), B220 (B cells), CD45 (monocytes), and Ly6G (neutrophils) to quantify organ specific immune niches. MxIF analysis highlighted the heterogenous distribution of C. auris infection within heart, kidney, and brain 7 days post-infection. Size and number of fungal abscesses was greatest in the heart and lowest in brain. Infected mice had an increased count of CD3+, CD68+, B220+, and CD45+ immune cells, concentrated around C. auris abscesses. CD68+ cells were predominant in wildtype (non-neutropenic mice) and CD3+/CD45+ cells were predominant in neutropenic mice, with B cells being the least abundant. These findings suggest a Th2 driven immune response in neutropenic C. auris infection mice models. This study demonstrates the value of MxIF to broaden understanding of C. auris pathobiology, and mechanistic understanding of fungal infections. [ABSTRACT FROM AUTHOR]

Leptospirosis is a global zoonosis caused by pathogenic Leptospira. The disease outcome is influenced by the interplay between innate and adaptive immune responses. Dendritic cells (DCs) play a crucial role in shaping the adaptive immune response. A recent study revealed that pathogenic Leptospira limited the activation of human monocyte-derived dendritic cells (MoDCs) compared to non-pathogenic Leptospira, but their impact on T-cell responses has not been investigated. Our study is the first to explore how viable pathogenic and non-pathogenic Leptospira affect the interaction between human MoDCs and T cells. We found that MoDCs infected with pathogenic leptospires (L. interrogans serovar Pomona and a clinical isolate, MoDCs-P) exhibited lower levels of CD80 and CD83 expression, suggesting partially impaired MoDC maturation, induced regulatory T cells (Tregs) while failing to induce CD4+ T cell proliferation, compared to MoDCs infected with non-pathogenic leptospires (L. biflexa serovar Patoc and L. meyeri serovar Ranarum, MoDCs-NP). In contrast, non-pathogenic leptospires enhanced MoDC maturation and induced higher T cell proliferation including IFN-γ-producing CD4+ T cells, indicative of a Th1-type response. Furthermore, pathogenic leptospires induced higher MoDC apoptosis through a cysteine aspartic acid-specific protease-3 (caspase-3)-dependent pathway and upregulated expression of the prostaglandin-endoperoxide synthase 2 (PTGS2) gene. Notably, prostaglandin E2 (PGE2), a product of the PTGS2 pathway, was found at higher levels in the sera of patients with acute leptospirosis and in the supernatant of MoDCs-P, possibly contributing to Treg induction, compared to those of healthy donors and MoDCs-NP, respectively. In conclusion, this study reveals a novel immunosuppressive strategy employed by pathogenic Leptospira to evade host immunity by partially impairing MoDC maturation and inducing Tregs. These findings deepen our understanding of leptospirosis pathogenesis in humans and may provide a novel strategy to modulate DCs for the prevention and treatment of the disease. Author summary: Dendritic cells (DCs) are critical antigen-presenting cells that link between innate and adaptive immune responses against pathogens. However, their role in the pathogenesis of human leptospirosis remains unclear. This study provides the first evidence of the impact of leptospires on MoDC-CD4+ T cell responses. We observed that pathogenic leptospires partially impaired MoDC maturation, potentially compromising their ability to induce CD4+ T cell proliferation while inducing regulatory T cells (Tregs). Notably, pathogenic leptospires upregulated the PTGS2 gene expression, resulting in elevated prostaglandin E2 (PGE2) levels, which may contribute to Treg activation. Furthermore, pathogenic leptospires trigger more MoDC apoptosis, partly through a caspase-3-dependent pathway, compared to non-pathogenic strains. These findings might reveal the mechanisms that pathogenic leptospires used to evade the host immune response, facilitating their dissemination within the host. Conversely, non-pathogenic leptospires promote MoDC maturation and stimulate the proliferation of IFN-γ-producing CD4+ T cells, potentially eliciting a Th1-type response. In conclusion, our study reveals a novel strategy employed by pathogenic leptospires for immune evasion while highlighting the immune-activating effects of non-pathogenic strains. [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]

The study of the initial phase of the adaptive immune response after first antigen encounter provides essential information on the magnitude and quality of the immune response. This phase is characterized by proliferation and dissemination of T cells in the lymphoid organs. Modeling and identifying the key features of this phenomenon may provide a useful tool for the analysis and prediction of the effects of immunization. This knowledge can be effectively exploited in vaccinology, where it is of interest to evaluate and compare the responses to different vaccine formulations. The objective of this paper is to construct a stochastic model based on branching process theory, for the dissemination network of antigen-specific CD4+ T cells. The devised model is validated on in vivo animal experimental data. The model presented has been applied to the vaccine immunization context making references to simple proliferation laws that take into account division, death and quiescence, but it can also be applied to any context where it is of interest to study the dynamic evolution of a population. [ABSTRACT FROM AUTHOR]

Background: T-cell epitopes play the important role in T-cell immune response, and they are critical components in the epitope-based vaccine design. Immunogenicity is the ability to trigger an immune response. The accurate prediction of immunogenic T-cell epitopes is significant for designing useful vaccines and understanding the immune system. Methods: In this paper, we attempt to differentiate immunogenic epitopes from non-immunogenic epitopes based on their primary structures. First of all, we explore a variety of sequence-derived features, and analyze their relationship with epitope immunogenicity. To effectively utilize various features, a genetic algorithm (GA)-based ensemble method is proposed to determine the optimal feature subset and develop the high-accuracy ensemble model. In the GA optimization, a chromosome is to represent a feature subset in the search space. For each feature subset, the selected features are utilized to construct the base predictors, and an ensemble model is developed by taking the average of outputs from base predictors. The objective of GA is to search for the optimal feature subset, which leads to the ensemble model with the best cross validation AUC (area under ROC curve) on the training set. Results: Two datasets named ‘IMMA2’ and ‘PAAQD’ are adopted as the benchmark datasets. Compared with the state-of-the-art methods POPI, POPISK, PAAQD and our previous method, the GA-based ensemble method produces much better performances, achieving the AUC score of 0.846 on IMMA2 dataset and the AUC score of 0.829 on PAAQD dataset. The statistical analysis demonstrates the performance improvements of GA-based ensemble method are statistically significant. Conclusions: The proposed method is a promising tool for predicting the immunogenic epitopes. The source codes and datasets are available in . [ABSTRACT FROM AUTHOR]

Epitope-based peptide vaccine can elicit T-cell immunity against SARS-CoV-2 to clear the infection. However, finding the best epitope from the whole antigen is challenging. A peptide screening using immunoinformatics usually starts from MHC-binding peptide, immunogenicity, cross-reactivity with the human proteome, to toxicity analysis. This pipeline classified the peptides into three categories, i.e., strong-, weak-, and non-binder, without incorporating the structural aspect. For this reason, the molecular detail that discriminates the binders from non-binder is interesting to be investigated. In this study, five CTL epitopes against HLA-A*02:01 were identified from the coarse-grained molecular dynamics-guided immunoinformatics screening. The strong binder showed distinctive activities from the non-binder in terms of structural and energetic properties. Furthermore, the second residue from the nonameric peptide was most important in the interaction with HLA-A*02:01. By understanding the nature of MHC-peptide interaction, we hoped to improve the chance of finding the best epitope for a peptide vaccine candidate. [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]

Agent-based simulation is a powerful method for investigating the complex interplay of the processes occurring in a lymph node during an adaptive immune response. We have previously established an agent-based modeling framework for the interactions between T cells and dendritic cells within the paracortex of lymph nodes. This model simulates in three dimensions the "random-walk" T cell motility observed in vivo, so that cells interact in space and time as they process signals and commit to action such as proliferation. On-lattice treatment of cell motility allows large numbers of densely packed cells to be simulated, so that the low frequency of T cells capable of responding to a single antigen can be dealt with realistically. In this paper we build on this model by incorporating new numerical methods to address the crucial processes of T cell ingress and egress, and chemotaxis, within the lymph node. These methods enable simulation of the dramatic expansion and contraction of the T cell population in the lymph node paracortex during an immune response. They also provide a novel probabilistic method to simulate chemotaxis that will be generally useful in simulating other biological processes in which chemotaxis is an important feature. [ABSTRACT FROM AUTHOR]

Background: Regulatory T cells are central actors in the maintenance of tolerance of self-antigens or allergens and in the regulation of the intensity of the immune response during infections by pathogens. An understanding of the network of the interaction between regulatory T cells, antigen presenting cells and effector T cells is starting to emerge. Dynamical systems analysis can help to understand the dynamical properties of an interaction network and can shed light on the different tasks that can be accomplished by a network. Methodology and Principal Findings: We used a mathematical model to describe a interaction network of adaptive regulatory T cells, in which mature precursor T cells may differentiate into either adaptive regulatory T cells or effector T cells, depending on the activation state of the cell by which the antigen was presented. Using an equilibrium analysis of the mathematical model we show that, for some parameters, the network has two stable equilibrium states: one in which effector T cells are strongly regulated by regulatory T cells and another in which effector T cells are not regulated because the regulatory T cell population is vanishingly small. We then simulate different types of perturbations, such as the introduction of an antigen into a virgin system, and look at the state into which the system falls. We find that whether or not the interaction network switches from the regulated (tolerant) state to the unregulated state depends on the strength of the antigenic stimulus and the state from which the network has been perturbed. Conclusion/Significance: Our findings suggest that the interaction network studied in this paper plays an essential part in generating and maintaining tolerance against allergens and self-antigens. [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 purpose of this study was to compare the effect of a gluten-free diet and/or antibiotics on tetanus vaccine induced immunoglobulin G titers and immune cell levels in BALB/c mice. The gluten-free diet was associated with a reduced anti-tetanus IgG response, and it increased the relative abundance of the anti-inflammatory Bifidobacterium significantly in some of the mice. Antibiotics also led to gut microbiota changes and lower initial vaccine titer. After a second vaccination, neither gluten-free diet nor antibiotics reduced the titers. In the spleen, the gluten-free diet significantly increased regulatory T cell (Treg) fractions, CD4+ T cell activation, and tolerogenic dendritic cell fractions and activation, which extend the downregulating effect of the Treg. Therefore, the systemic effect of the gluten-free diet seems mainly tolerogenic. Antibiotics reduced the fractions of CD4+ T and B cells in the mesenteric lymph nodes. These results suggest that vaccine response in mice is under influence of their diet, the gut microbiota and the interplay between them. However, a gluten-free diet seems to work through mechanisms different from those induced by antibiotics. Therefore, diet should be considered when testing vaccines in mice and developing vaccines for humans. [ABSTRACT FROM AUTHOR]

Background & aims: HCV CD4+ and CD8+ specific T cells responses are functionally impaired during chronic hepatitis C infection. DAAs therapies eradicate HCV infection in more than 95% of treated patients. However, the impact of HCV elimination on immune responses remain controversial. Here, we aimed to investigate whether HCV cure by DAAs could reverse the impaired immune response to HCV. Methods: We analyzed 27 chronic HCV infected patients undergoing DAA treatment in tertiary care hospital, and we determined the phenotypical and functional changes in both HCV CD8+ and CD4+ specific T-cells before and after viral clearance. PD-1, TIM-3 and LAG-3 cell-surface expression was assessed by flow cytometry to determine CD4+ T cell exhaustion. Functional responses to HCV were analyzed by IFN-Ɣ ELISPOT, intracellular cytokine staining (IL-2 and IFN-Ɣ) and CFSE-based proliferation assays. Results: We observed a significant decrease in the expression of PD-1 in CD4+ T-cells after 12 weeks of viral clearance in non-cirrhotic patients (p = 0.033) and in treatment-naive patients (p = 0.010), indicating a partial CD4 phenotype restoration. IFN-Ɣ and IL-2 cytokines production by HCV-specific CD4+ and CD8+ T cells remained impaired upon HCV eradication. Finally, a significant increase of the proliferation capacity of both HCV CD4+ and CD8+ specific T-cells was observed after HCV elimination by DAAs therapies. Conclusions: Our results show that in chronically infected patients HCV elimination by DAA treatment lead to partial reversion of CD4+ T cell exhaustion. Moreover, proliferative capacity of HCV-specific CD4+ and CD8+ T cells is recovered after DAA's therapies. [ABSTRACT FROM AUTHOR]

More than 65 million people have been confirmed infection with SARS-CoV-2 and more than 1 million have died from COVID-19 and this pandemic remains critical worldwide. Effective vaccines are one of the most important strategies to limit the pandemic. Here, we report a construction strategy of DNA vaccine candidates expressing full length wild type SARS-CoV-2 spike (S) protein, S1 or S2 region and their immunogenicity in mice. All DNA vaccine constructs of pCMVkan-S, -S1 and -S2 induced high levels of specific binding IgG that showed a balance of IgG1/IgG2a response. However, only the sera from mice vaccinated with pCMKkan-S or -S1 DNA vaccines could inhibit viral RBD and ACE2 interaction. The highest neutralizing antibody (NAb) titer was found in pCMVkan-S group, followed by -S1, while -S2 showed the lowest PRNT50 titers. The geometric mean titers (GMTs) were 2,551, 1,005 and 291 for pCMVkan-S, -S1 and -S2, respectively. pCMVkan-S construct vaccine also induced the highest magnitude and breadth of T cells response. Analysis of IFN-γ positive cells after stimulation with SARS-CoV-2 spike peptide pools were 2,991, 1,376 and 1,885 SFC/106 splenocytes for pCMVkan-S, -S1 and -S2, respectively. Our findings highlighted that full-length S antigen is more potent than the truncated spike (S1 or S2) in inducing of neutralizing antibody and robust T cell responses. [ABSTRACT FROM AUTHOR]

In the present study, we evaluated adjuvant potential of Poria cocos polysaccharide (PCP) on the Th1-type immune responses of C57/BL6 mice against ovalbumin (OVA). We first determined the effect of PCP on maturation of murine bone marrow derived dendritic cells (BMDCs), PCP significantly upregulated surface expression of MHCII, CD40, CD80, CD86 and enhanced production of IL-6 and IL-12p40. In addition, PCP affected receptor-mediated endocytosis, but not pinocytosis in BMDCs. Furthermore, OVA + PCP immunization induced specific cytotoxic CD8+ T cell killing of OVA (257–264) peptide pulsed cell. When mice were immunized subcutaneously in a week interval with OVA + PCP. Serum were collected for measuring OVA-specific antibody and splenocytes were harvested for analyzing CD69, IFN-γ ELISpot and cytokines production. The result indicated that OVA-specific IgG, IgG2a and IgG1 antibody levels in serum were significantly elevated by PCP compared with control. PCP increased OVA-specific IFN-γ-secreting CD8+, CD4+ T cells, promoted CD8+ T cell proliferation and up-regulated Th-1 type (IFN-γ, IL-2) cytokine production. In conclusion, data suggest that PCP enhanced cellular immune response and possess potential as a vaccine adjuvant for Th1 immune response. [ABSTRACT FROM AUTHOR]

Healthcare workers concurrently may be at a higher risk of developing respiratory infections and allergic disease, such as asthma, than the general public. Increased incidence of allergic diseases is thought to be caused, in part, due to occupational exposure to chemicals that induce or augment Th2 immune responses. However, whether exposure to these chemical antimicrobials can influence immune responses to respiratory pathogens is unknown. Here, we use a BALB/c murine model to test if the Th2-promoting antimicrobial chemical triclosan influences immune responses to influenza A virus. Mice were dermally exposed to 2% triclosan for 7 days prior to infection with a sub-lethal dose of mouse adapted PR8 A(H1N1) virus (50 pfu); triclosan exposure continued until 10 days post infection (dpi). Infected mice exposed to triclosan did not show an increase in morbidity or mortality, and viral titers were unchanged. Assessment of T cell responses at 10 dpi showed a decrease in the number of total and activated (CD44hi) CD4+ and CD8+ T cells at the site of infection (BAL and lung) in triclosan exposed mice compared to controls. Influenza-specific CD4+ and CD8+ T cells were assessed using MHCI and MHCII tetramers, with reduced populations, although not reaching statistical significance at these sites following triclosan exposure. Reductions in the Th1 transcription factor T-bet were seen in both activated and tetramer+ CD4+ and CD8+ T cells in the lungs of triclosan exposed infected mice, indicating reduced Th1 polarization and providing a potential mechanism for numerical reduction in T cells. Overall, these results indicate that the immune environment induced by triclosan exposure has the potential to influence the developing immune response to a respiratory viral infection and may have implications for healthcare workers who may be at an increased risk for developing infectious diseases. [ABSTRACT FROM AUTHOR]

Chromoblastomycosis (CBM) is a chronic worldwide subcutaneous mycosis, caused by several dimorphic, pigmented dematiaceous fungi. It is difficult to treat patients with the disease, mainly because of its recalcitrant nature. The correct activation of host immune response is critical to avoid fungal persistence in the tissue and disease chronification. CD4+ T cells are crucial for the development of protective immunity to F. pedrosoi infection. Here, we investigated T helper cell response dynamics during experimental CBM. Following footpad injection with F. pedrosoi hyphae and conidia, T cells were skewed towards a Th17 and Th1 phenotype. The Th17 population was the main Th cell subset found in the infected area during the early stages of experimental murine CBM, followed by Th1 predominance in the later stages, coinciding with the remission phase of the disease in this experimental model. Depletion of CD25+ cells, which leads to a reduction of Treg cells in the draining lymph node, resulted in decline in fungal burden after 14 days of infection. However, fungal cells were not cleared in the later stages of the disease, prolonging CBM clinical features in those animals. IL-17A and IFN-γ neutralization hindered fungal cell elimination in the course of the disease. Similarly, in dectin-2 KO animals, Th17 contraction in the course of experimental CBM was accompanied by fungal burden decrease in the first 14 days of infection, although it did not affect disease resolution. In this study, we gained insight into T helper subsets' dynamics following footpad injections of F. pedrosoi propagules and uncovered their contribution to disease resolution. The Th17 population proved to be important in eliminating fungal cells in the early stages of infection. The Th1 population, in turn, closely assisted by Treg cells, proved to be relevant not only in the elimination of fungal cells at the beginning of infection but also essential for their complete elimination in later stages of the disease in a mouse experimental model of CBM. Author summary: Chromoblastomycosis is a chronic subcutaneous infection caused by several dimorphic, pigmented dematiaceous fungi. CD4+ T cells modulations are crucial for the proper immune response against this fungal infection and play a key role in CBM resolution in a self-healing mouse model. In this work we report Th17 cells as being the main CD4+ subpopulation in the infected area during the early stages of experimental murine CBM, followed by Th1 predominance in the later stages, coinciding with the remission phase of the disease in this experimental model. Depletion of CD25+ cells resulted in fungal burden reduction after 14 days of infection, but it compromised fungal clearing in later stages of the disease, prolonging CBM clinical features in those animals. In vivo analysis with IL-17A and IFN-γ neutralization hindered fungal cell elimination in the course of the disease. Dectin-2 deficiency was associated with impairment of Th17 response and fungal control in the early phase of CBM but did not affect disease resolution. In this study, we gained insight into T helper subsets' dynamics following footpad injections of F. pedrosoi fungal cells and uncovered their contribution to disease resolution. [ABSTRACT FROM AUTHOR]

Respiratory syncytial virus (RSV) is the single most important cause of serious lower respiratory tract disease in infants and young children worldwide and a high priority for vaccine development. Despite over 50 years of research, however, no vaccine is yet available. One block to vaccine development is an incomplete understanding of the aberrant memory response to the formalin-inactivated RSV vaccine (FI-RSV) given to children in the 1960s. This vaccine caused enhanced respiratory disease (ERD) with later natural RSV infection. Concern that any non-live virus vaccine may also cause ERD has blocked development of subunit vaccines for young children. A number of animal FI-RSV studies suggest various immune mechanisms behind ERD. However, other than limited data from the original FI-RSV trial, there is no information on the human ERD-associated responses. An in vitro model with human blood specimens may shed light on the immune memory responses likely responsible for ERD. Memory T cell responses to an antigen are guided by the innate responses, particularly dendritic cells that present an antigen in conjunction with co-stimulatory molecules and cytokine signaling. Our in vitro model involves human monocyte derived dendritic cells (moDC) and allogenic T cell cultures to assess innate responses that direct T cell responses. Using this model, we evaluated human responses to live RSV, FI-RSV, and subunit RSV G vaccines (G-containing virus-like particles, G-VLP). Similar to findings in animal studies, FI-RSV induced prominent Th2/Th17-biased responses with deficient type-1 responses compared to live virus. Responses to G-VLPs were similar to live virus, i.e. biased towards a Th1 and not a Th2/Th17. Also mutating CX3C motif in G gave a more pronounced moDC responses associated with type-1 T cell responses. This in vitro model identifies human immune responses likely associated with ERD and provides another pre-clinical tool to assess the safety of RSV vaccines. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is an intracellular parasite that persistently infects the CNS and that has genetically distinct strains which provoke different acute immune responses. How differences in the acute immune response affect the CNS immune response is unknown. To address this question, we used two persistent Toxoplasma strains (type II and type III) and examined the CNS immune response at 21 days post infection (dpi). Contrary to acute infection studies, type III-infected mice had higher numbers of total CNS T cells and macrophages/microglia but fewer alternatively activated macrophages (M2s) and regulatory T cells (Tregs) than type II-infected mice. By profiling splenocytes at 5, 10, and 21 dpi, we determined that at 5 dpi type III-infected mice had more M2s while type II-infected mice had more pro-inflammatory macrophages and that these responses flipped over time. To test how these early differences influence the CNS immune response, we engineered the type III strain to lack ROP16 (IIIΔrop16), the polymorphic effector protein that drives the early type III-associated M2 response. IIIΔrop16-infected mice showed a type II-like neuroinflammatory response with fewer infiltrating T cells and macrophages/microglia and more M2s and an unexpectedly low CNS parasite burden. At 5 dpi, IIIΔrop16-infected mice showed a mixed inflammatory response with more pro-inflammatory macrophages, M2s, T effector cells, and Tregs, and decreased rates of infection of peritoneal exudative cells (PECs). These data suggested that type III parasites need the early ROP16-associated M2 response to avoid clearance, possibly by the Immunity-Related GTPases (IRGs), which are IFN-γ- dependent proteins essential for murine defenses against Toxoplasma. To test this possibility, we infected IRG-deficient mice and found that IIIΔrop16 parasites now maintained parental levels of PECs infection. Collectively, these studies suggest that, for the type III strain, rop16III plays a key role in parasite persistence and influences the subacute CNS immune response. Toxoplasma is a ubiquitous intracellular parasite that establishes an asymptomatic brain infection in immunocompetent individuals. However, in the immunocompromised and the developing fetus, Toxoplasma can cause problems ranging from fever to chorioretinitis to severe toxoplasmic encephalitis. Emerging evidence suggests that the genotype of the infecting Toxoplasma strain may influence these outcomes, possibly through the secretion of Toxoplasma strain-specific polymorphic effector proteins that trigger different host cell signaling pathways. While such strain-specific modulation of host cell signaling has been shown to affect acute immune responses, it is unclear how these differences influence the subacute or chronic responses in the CNS, the major organ affected in symptomatic disease. This study shows that genetically distinct strains of Toxoplasma provoke strain-specific CNS immune responses and that, for one strain (type III), acute and subacute immune responses and parasite survival are heavily influenced by a polymorphic parasite gene (rop16III). [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]

The ability to appropriately mimic human disease is critical for using animal models as a tool for understanding virus pathogenesis. In the case of Nipah virus (NiV), infection of humans appears to occur either through inhalation, contact with or consumption of infected material. In two of these circumstances, respiratory or sinusoidal exposure represents a likely route of infection. In this study, intermediate-size aerosol particles (~7 μm) of NiV-Malaysia were used to mimic potential routes of exposure by focusing viral deposition in the upper respiratory tract. Our previous report showed this route of exposure extended the disease course and a single animal survived the infection. Here, analysis of the peripheral immune response found minimal evidence of systemic inflammation and depletion of B cells during acute disease. However, the animal that survived infection developed an early IgM response with rapid development of neutralizing antibodies that likely afforded protection. The increase in NiV-specific antibodies correlated with an expansion of the B cell population in the survivor. Cell-mediated immunity was not clearly apparent in animals that succumbed during the acute phase of disease. However, CD4+ and CD8+ effector memory cells increased in the survivor with correlating increases in cytokines and chemokines associated with cell-mediated immunity. Interestingly, kinetic changes of the CD4+ and CD8bright T cell populations over the course of acute disease were opposite from animals that succumbed to infection. In addition, increases in NK cells and basophils during convalescence of the surviving animal were also evident, with viral antigen found in NK cells. These data suggest that a systemic inflammatory response and “cytokine storm” are not major contributors to NiV-Malaysia pathogenesis in the AGM model using this exposure route. Further, these data demonstrate that regulation of cell-mediated immunity, in addition to rapid production of NiV specific antibodies, may be critical for surviving NiV infection. [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]

Mycetoma is a neglected tropical disease, endemic in many tropical and subtropical regions, characterised by massive deformity and disability and can be fatal if untreated early and appropriately. Interleukins (IL) -35 and IL-37 are newly discovered cytokines that play an important role in suppressing the immune system. However, the expression of these interleukins in patients with Madurella mycetomatis (M. mycetomatis) induced eumycetoma has not yet been explored. The aim of this study is to determine the levels of IL-1 family (IL-1β, IL-37) and IL-12 family (IL-12, IL-35) in a group of these patients and the association between these cytokines levels and the patients’ demographic characteristics. The present, case-control study was conducted at the Mycetoma Research Centre, Soba University Hospital, University of Khartoum, Sudan and it included 140 individuals. They were divided into two groups; group I: healthy controls [n = 70; median age 25 years (range 12 to 70 years)]. Group II: mycetoma patients [n = 70 patients; median age 25 (range 13 to 70 years)]. Cytokines levels were measured in sera using enzyme linked immunosorbent assay (ELISA). There was a significant negative correlation between IL-1β and IL-12 levels and lesion size and disease duration, while IL-37 and IL-35 levels were significantly positively correlated with both lesion size and disease duration. The analysis of the risk factors of higher circulatory levels of IL-37 in patients of mycetoma showed a negative significant association with IL-1β cytokine, where a unit increment in IL-1β will decrease the levels of IL-37 by 35.28 pg/ml. The levels of IL-37 among the patients with a duration of mycetoma infection ≤ 1 year were significantly low by an average of 18.45 pg/ml compared to patients with a mycetoma infection’s duration of ≥ 5years (reference group). Furthermore, the risk factors of higher levels of IL-35 in mycetoma patients revealed a negative significant association with IL-12, as a unit increment in IL-12 decreases the levels of IL-35 by 8.99 pg/ml (p < 0.001). Levels of IL-35 among the patients with duration of mycetoma infection ≤ one year were significantly low on average by 41.82 pg/ml (p value = 0.002) compared to patients with a duration of mycetoma infection ≥ 5 years (reference group). In conclusion, this study indicates that both IL-35 and IL-37 are negatively associated with the levels of IL-1β and IL-12 in eumycetoma mycetoma infection; and high levels of IL-37 and IL-35 may have a negative impact on disease progression. [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]

The T-cell (TCR) repertoire relies on the diversity of receptors composed of two chains, called α and β, to recognize pathogens. Using results of high throughput sequencing and computational chain-pairing experiments of human TCR repertoires, we quantitively characterize the αβ generation process. We estimate the probabilities of a rescue recombination of the β chain on the second chromosome upon failure or success on the first chromosome. Unlike β chains, α chains recombine simultaneously on both chromosomes, resulting in correlated statistics of the two genes which we predict using a mechanistic model. We find that ∼35% of cells express both α chains. Altogether, our statistical analysis gives a complete quantitative mechanistic picture that results in the observed correlations in the generative process. We learn that the probability to generate any TCRαβ is lower than 10−12 and estimate the generation diversity and sharing properties of the αβ TCR repertoire. [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]

CCR5 plays immune functions and is the coreceptor for R5 HIV-1 strains. It exists in diverse conformations and oligomerization states. We interrogated the significance of the CCR5 structural diversity on HIV-1 infection. We show that envelope glycoproteins (gp120s) from different HIV-1 strains exhibit divergent binding levels to CCR5 on cell lines and primary cells, but not to CD4 or the CD4i monoclonal antibody E51. This owed to differential binding of the gp120s to different CCR5 populations, which exist in varying quantities at the cell surface and are differentially expressed between different cell types. Some, but not all, of these populations are antigenically distinct conformations of the coreceptor. The different binding levels of gp120s also correspond to differences in their capacity to bind CCR5 dimers/oligomers. Mutating the CCR5 dimerization interface changed conformation of the CCR5 homodimers and modulated differentially the binding of distinct gp120s. Env-pseudotyped viruses also use particular CCR5 conformations for entry, which may differ between different viruses and represent a subset of those binding gp120s. In particular, even if gp120s can bind both CCR5 monomers and oligomers, impairment of CCR5 oligomerization improved viral entry, suggesting that HIV-1 prefers monomers for entry. From a functional standpoint, we illustrate that the nature of the CCR5 molecules to which gp120/HIV-1 binds shapes sensitivity to inhibition by CCR5 ligands and cellular tropism. Differences exist in the CCR5 populations between T-cells and macrophages, and this is associated with differential capacity to bind gp120s and to support viral entry. In macrophages, CCR5 structural plasticity is critical for entry of blood-derived R5 isolates, which, in contrast to prototypical M-tropic strains from brain tissues, cannot benefit from enhanced affinity for CD4. Collectively, our results support a role for CCR5 heterogeneity in diversifying the phenotypic properties of HIV-1 isolates and provide new clues for development of CCR5-targeting drugs. [ABSTRACT FROM AUTHOR]

Sequencing of the T cell receptor repertoire is a powerful tool for deeper study of immune response, but the unique structure of this type of data makes its meaningful quantification challenging. We introduce a new method, the Gamma-GPD spliced threshold model, to address this difficulty. This biologically interpretable model captures the distribution of the TCR repertoire, demonstrates stability across varying sequencing depths, and permits comparative analysis across any number of sampled individuals. We apply our method to several datasets and obtain insights regarding the differentiating features in the T cell receptor repertoire among sampled individuals across conditions. We have implemented our method in the open-source R package powerTCR. [ABSTRACT FROM AUTHOR]

Data from SIV-infected macaques indicate that virus-specific cytotoxic T lymphocytes (CTL) are mostly present in the extrafollicular (EF) compartment of the lymphoid tissue, with reduced homing to the follicular (F) site. This contributes to the majority of the virus being present in the follicle and represents a barrier to virus control. Using mathematical models, we investigate these dynamics. Two models are analyzed. The first assumes that CTL can only become stimulated and expand in the extrafollicular compartment, with migration accounting for the presence of CTL in the follicle. In the second model, follicular CTL can also undergo antigen-induced expansion. Consistent with experimental data, both models predict increased virus compartmentalization in the presence of stronger CTL responses and lower virus loads, and a more pronounced rise of extrafollicular compared to follicular virus during CD8 cell depletion experiments. The models, however, differ in other aspects. The follicular expansion model results in dynamics that promote the clearance of productive infection in the extrafollicular site, with any productively infected cells found being the result of immigration from the follicle. This is not observed in the model without follicular CTL expansion. The models further predict different consequences of introducing engineered, follicular-homing CTL, which has been proposed as a therapeutic means to improve virus control. Without follicular CTL expansion, this is predicted to result in a reduction of virus load in both compartments. The follicular CTL expansion model, however, makes the counter-intuitive prediction that addition of F-homing CTL not only results in a reduction of follicular virus load, but also in an increase in extrafollicular virus replication. These predictions remain to be experimentally tested, which will be relevant for distinguishing between models and for understanding how therapeutic introduction of F-homing CTL might impact the overall dynamics of the infection. [ABSTRACT FROM AUTHOR]

Human herpesviruses are antigenically rich agents that induce strong CD8+T cell responses in primary infection yet persist for life, continually challenging T cell memory through recurrent lytic replication and potentially influencing the spectrum of antigen-specific responses. Here we describe the first lytic proteome-wide analysis of CD8+ T cell responses to a gamma1-herpesvirus, Epstein-Barr virus (EBV), and the first such proteome-wide analysis of primary versus memory CD8+ T cell responses to any human herpesvirus. Primary effector preparations were generated directly from activated CD8+ T cells in the blood of infectious mononucleosis (IM) patients by in vitro mitogenic expansion. For memory preparations, EBV-specific cells in the blood of long-term virus carriers were first re-stimulated in vitro by autologous dendritic cells loaded with a lysate of lytically-infected cells, then expanded as for IM cells. Preparations from 7 donors of each type were screened against each of 70 EBV lytic cycle proteins in combination with the donor’s individual HLA class I alleles. Multiple reactivities against immediate early (IE), early (E) and late (L) lytic cycle proteins, including many hitherto unrecognised targets, were detected in both contexts. Interestingly however, the two donor cohorts showed a different balance between IE, E and L reactivities. Primary responses targeted IE and a small group of E proteins preferentially, seemingly in line with their better presentation on the infected cell surface before later-expressed viral evasins take full hold. By contrast, target choice equilibrates in virus carriage with responses to key IE and E antigens still present but with responses to a select subset of L proteins now often prominent. We infer that, for EBV at least, long-term virus carriage with its low level virus replication and lytic antigen release is associated with a re-shaping of the virus-specific response. [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]

Background: Africa bears the burden of approximately 70% of global HIV infections and 90% of global schistosome infections. We sought to investigate the impact of schistosome infection at the time of HIV-1 seroconversion on the speed of HIV-1 disease progression, as measured by the outcome CD4+ T-cell (CD4) counts <350 cells/μL and/or death. We hypothesized that people who had been infected with Schistosoma spp. at the time they acquired HIV-1 infection would have impaired antiviral immune response, thus leading them to progress twice as fast to a CD4 count less than 350 cells/μL or death than would people who had been free of schistosomes at time of HIV-1 seroconversion. Methods and principal findings: We conducted a longitudinal study in Tanzania from 2006 to 2017 using stored blood spot samples, demographic surveillance and sero-survey data from the community, and a review of clinical charts. A competing risk analysis was performed to look at the difference in time to reaching CD4 counts < 350 cells/μL and/or death in HIV-1-infected people who were infected versus not infected with Schistosoma spp. at time of HIV-1 seroconversion. We found an 82% reduction in risk of reaching the outcome in seroconverters who had been infected with Schistosoma (subHazard Ratio = 0.18[0.068,0.50], p = 0.001) after adjusting for age, occupation, clinic attendance and time-dependent covariates. Conclusions: Our study demonstrates that people with schistosome infection at the time of HIV-seroconversion develop adverse HIV outcomes more slowly than those without. The findings are contrary to our original hypothesis. Our current longitudinal findings suggest complex interactions between HIV-1 and schistosome co-infections that may be modulated over time. We urge new immunological studies to investigate the long-term impact of schistosome infection on HIV-1 viral load and CD4 counts as well as related immunologic pathways. [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]