Structural and physical features that distinguish tumor-controlling from inactive cancer neoepitopes.

Neoepitopes arising from amino acid substitutions due to single nucleotide polymorphisms are targets of T cell immune responses to cancer and are of significant interest in the development of cancer vaccines. However, understanding the characteristics of rare protective neoepitopes that truly control tumor growth has been a challenge, due to their scarcity as well as the challenge of verifying true, neoepitope-dependent tumor control in humans. Taking advantage of recent work in mouse models that circumvented these challenges, here, we compared the structural and physical properties of neoepitopes that range from fully protective to immunologically inactive. As neoepitopes are derived from self-peptides that can induce immune tolerance, we studied not only how the various neoepitopes differ from each other but also from their wild-type counterparts. We identified multiple features associated with protection, including features that describe how neoepitopes differ from self as well as features associated with recognition by diverse T cell receptor repertoires. We demonstrate both the promise and limitations of neoepitope structural analysis and predictive modeling and illustrate important aspects that can be incorporated into neoepitope prediction pipelines.

CD8+ T Cell-Dependent Antitumor Activity In Vivo of a Mass Spectrometry-Identified Neoepitope despite Undetectable CD8+ Immunogenicity In Vitro.

Identification of neoepitopes that can control tumor growth in vivo remains a challenge even 10 y after the first genomics-defined cancer neoepitopes were identified. In this study, we identify a neoepitope, resulting from a mutation in the junction plakoglobin (Jup) gene (chromosome 11), from the mouse colon cancer line MC38-FABF (C57BL/6). This neoepitope, Jup mutant (JupMUT), was detected during mass spectrometry of MHC class I-eluted peptides from the tumor. JupMUT has a predicted binding affinity of 564 nM for the Kb molecule and a higher predicted affinity of 82 nM for Db. However, whereas structural modeling of JupMUT and its unmutated counterpart Jup wild-type indicates that there are little conformational differences between the two epitopes bound to Db, large structural divergences are predicted between the two epitopes bound to Kb. Together with in vitro binding data with RMA-S cells, these data suggest that Kb rather than Db is the relevant MHC class I molecule of JupMUT. Immunization of naive C57BL/6 mice with JupMUT elicits CD8-dependent tumor control of a MC38-FABF challenge. Despite the CD8 dependence of JupMUT-mediated tumor control in vivo, CD8+ T cells from JupMUT-immunized mice do not produce higher levels of IFN-γ than do naive mice. The structural and immunological characteristics of JupMUT are substantially different from those of many other neoepitopes that have been shown to mediate tumor control.

Prediction of cancer neoepitopes needs new rules.

Tumors are immunogenic and the non-synonymous point mutations harbored by tumors are a source of their immunogenicity. Immunologists have long been enamored by the idea of synthetic peptides corresponding to mutated epitopes (neoepitopes) as specific "vaccines" against tumors presenting those neoepitopes in context of MHC I. Tumors may harbor hundreds of point mutations and it would require effective prediction algorithms to identify candidate neoepitopes capable of eliciting potent tumor-specific CD8+ T cell responses. Our current understanding of MHC I-restricted epitopes come from the observance of CD8+ T cell responses against viral (vaccinia, lymphocytic choriomeningitis etc.) and model (chicken ovalbumin, hen egg lysozyme etc.) antigens. Measurable CD8+ T cell responses elicited by model or viral antigens are always directed against epitopes possessing strong binding affinity for the restricting MHC I alleles. Immense collective effort to develop methodologies combining genomic sequencing, bioinformatics and traditional immunological techniques to identify neoepitopes with strong binding affinity to MHC I has only yielded inaccurate prediction algorithms. Additionally, new evidence has emerged suggesting that neoepitopes, which unlike the epitopes of viral or model antigens have closely resembling wild-type counterparts, may not necessarily demonstrate strong affinity to MHC I. Our bearing need recalibration.

Endocannabinoid system acts as a regulator of immune homeostasis in the gut.

Endogenous cannabinoids (endocannabinoids) are small molecules biosynthesized from membrane glycerophospholipid. Anandamide (AEA) is an endogenous intestinal cannabinoid that controls appetite and energy balance by engagement of the enteric nervous system through cannabinoid receptors. Here, we uncover a role for AEA and its receptor, cannabinoid receptor 2 (CB2), in the regulation of immune tolerance in the gut and the pancreas. This work demonstrates a major immunological role for an endocannabinoid. The pungent molecule capsaicin (CP) has a similar effect as AEA; however, CP acts by engagement of the vanilloid receptor TRPV1, causing local production of AEA, which acts through CB2. We show that the engagement of the cannabinoid/vanilloid receptors augments the number and immune suppressive function of the regulatory CX3CR1hi macrophages (Mϕ), which express the highest levels of such receptors among the gut immune cells. Additionally, TRPV1-/- or CB2-/- mice have fewer CX3CR1hi Mϕ in the gut. Treatment of mice with CP also leads to differentiation of a regulatory subset of CD4+ cells, the Tr1 cells, in an IL-27-dependent manner in vitro and in vivo. In a functional demonstration, tolerance elicited by engagement of TRPV1 can be transferred to naïve nonobese diabetic (NOD) mice [model of type 1 diabetes (T1D)] by transfer of CD4+ T cells. Further, oral administration of AEA to NOD mice provides protection from T1D. Our study unveils a role for the endocannabinoid system in maintaining immune homeostasis in the gut/pancreas and reveals a conversation between the nervous and immune systems using distinct receptors.

Reagent-Free and Rapid Assessment of T Cell Activation State Using Diffraction Phase Microscopy and Deep Learning.

CD8+ T cells constitute an essential compartment of the adaptive immune system. During immune responses, naïve T cells become functional, as they are primed with their cognate determinants by the antigen presenting cells. Current methods of identifying activated CD8+ T cells are laborious, time-consuming and expensive due to the extensive list of required reagents. Here, we demonstrate an optical imaging approach featuring quantitative phase imaging to distinguish activated CD8+ T cells from naïve CD8+ T cells in a rapid and reagent-free manner. We measured the dry mass of live cells and employed transport-based morphometry to better understand their differential morphological attributes. Our results reveal that, upon activation, the dry cell mass of T cells increases significantly in comparison to that of unstimulated cells. By employing deep learning formalism, we are able to accurately predict the population ratios of unknown mixed population based on the acquired quantitative phase images. We envision that, with further refinement, this label-free method of T cell phenotyping will lead to a rapid and cost-effective platform for assaying T cell responses to candidate antigens in the near future.

CD11c+ MHCIIlo GM-CSF-bone marrow-derived dendritic cells act as antigen donor cells and as antigen presenting cells in neoepitope-elicited tumor immunity against a mouse fibrosarcoma.

Dendritic cells play a critical role in initiating T-cell responses. In spite of this recognition, they have not been used widely as adjuvants, nor is the mechanism of their adjuvanticity fully understood. Here, using a mutated neoepitope of a mouse fibrosarcoma as the antigen, and tumor rejection as the end point, we show that dendritic cells but not macrophages possess superior adjuvanticity. Several types of dendritic cells, such as bone marrow-derived dendritic cells (GM-CSF cultured or FLT3-ligand induced) or monocyte-derived ones, are powerful adjuvants, although GM-CSF-cultured cells show the highest activity. Among these, the CD11c+ MHCIIlo sub-set, distinguishable by a distinct transcriptional profile including a higher expression of heat shock protein receptors CD91 and LOX1, mannose receptors and TLRs, is significantly superior to the CD11c+ MHCIIhi sub-set. Finally, dendritic cells exert their adjuvanticity by acting as both antigen donor cells (i.e., antigen reservoirs) as well as antigen presenting cells.

Dendritic cells sequester antigenic epitopes for prolonged periods in the absence of antigen-encoding genetic information.

Studies with a number of viral systems have shown, on the basis of the ability of a host to prime naïve T cells, that viral antigens persist in the infected host well beyond complete clearance of the infection and even when viral antigen is undetectable by the most sensitive methods. This has led to a reasonable assumption that the antigen persists through persistence of antigen-encoding genetic information (DNA or RNA) that resides in the host at a subdetectable level. Here, we demonstrate that epitopes, or epitope precursors, of a model antigen (ovalbumin) persist in a host for prolonged periods (weeks), well beyond the time at which the intact antigen has disappeared, and in the complete absence of genetic information encoding it. Dendritic cells are shown to be the site of this epitope sequestration in vivo, as well as in cultures in vitro. For sequestration to occur, the uptaken antigen must be significantly large, that is, the epitope and its 18-mer precursor are not sequestered. Dendritic cells are shown to create an hsp90-dependent intracellular pool of epitopes or epitope precursors that continues to release epitopes for presentation on the major histocompatibility complex I molecules for prolonged periods. Demonstration of such long-term sequestration of antigenic epitopes inside dendritic cells presents new opportunities for stimulation of immune response against cancers and viruses.

Roles of heat-shock proteins in innate and adaptive immunity.

Heat-shock proteins (HSPs) are the most abundant and ubiquitous soluble intracellular proteins. In single-cell organisms, invertebrates and vertebrates, they perform a multitude of housekeeping functions that are essential for cellular survival. In higher vertebrates, their ability to interact with a wide range of proteins and peptides--a property that is shared by major histocompatibility complex molecules--has made the HSPs uniquely suited to an important role in organismal survival by their participation in innate and adaptive immune responses. The immunological properties of HSPs enable them to be used in new immunotherapies of cancers and infections.

The inhibitory effect of metals and other ions on acid phosphatase activity from Vigna aconitifolia seeds.

Sensitivity of acid phosphatase from Vigna aconitifolia seeds to metal ions, fluoride, and phosphate was examined. All the effectors had different degree of inhibitory effect on the enzyme. Among metal ions, molybdate and ferric ion were observed to be most potent inhibitors and both exhibited mixed type of inhibition. Acid phosphatase activity was inhibited by Cu2+ in a noncompetitive manner. Zn and Mn showed mild inhibition on the enzyme activity. Inhibition kinetics analysis explored molybdate as a potent inhibitor for acid phosphatase in comparison with other effectors used in this study. Fluoride was the next most strong inhibitor for the enzyme activity, and caused a mixed type of inhibition. Phosphate inhibited the enzyme competitively, which demonstrates that inhibition due to phosphate is one of the regulatory factors for enzyme activity.

Isolation and enzymatic properties of a nonspecific acid phosphatase from Vigna aconitifolia seeds.

Acid phosphatase (EC 3.1.3.2) from Vigna aconitifolia seeds was purified to apparent homogeneity by using ammonium sulfate fractionation and cation-exchange chromatography [carboxymethyl (CM) cellulose]. The enzyme was 228-fold purified with 14.6% recovery. Analytical gel filtration chromatography on Sephadex G-200 column showed that Mr of native enzyme was 58 kDa and denaturing PAGE demonstrated that it was made up of two subunits of 24 and 27 kDa. The enzyme showed its optimum activity at pH 5.0 and 60°C. It exhibited broad substrate specificity and showed a higher specificity constant for para-nitrophenyl phosphate, Na ß-naphthyl phosphate, and adenosine monophosphate (AMP). Cu²âº, Mo6⁺, Fe³âº, phosphate, and fluoride ions were reported as strong inhibitors for the enzyme. Active site study for the enzyme demonstrated that tryptophan and aspartic acid may be important for the catalysis.

Cancer neoepitopes viewed through negative selection and peripheral tolerance: a new path to cancer vaccines.

A proportion of somatic mutations in tumors create neoepitopes that can prime T cell responses that target the MHC I-neoepitope complexes on tumor cells, mediating tumor control or rejection. Despite the compelling centrality of neoepitopes to cancer immunity, we know remarkably little about what constitutes a neoepitope that can mediate tumor control in vivo and what distinguishes such a neoepitope from the vast majority of similar candidate neoepitopes that are inefficacious in vivo. Studies in mice as well as clinical trials have begun to reveal the unexpected paradoxes in this area. Because cancer neoepitopes straddle that ambiguous ground between self and non-self, some rules that are fundamental to immunology of frankly non-self antigens, such as viral or model antigens, do not appear to apply to neoepitopes. Because neoepitopes are so similar to self-epitopes, with only small changes that render them non-self, immune response to them is regulated at least partially the way immune response to self is regulated. Therefore, neoepitopes are viewed and understood here through the clarifying lens of negative thymic selection. Here, the emergent questions in the biology and clinical applications of neoepitopes are discussed critically and a mechanistic and testable framework that explains the complexity and translational potential of these wonderful antigens is proposed.

ERAMER: A novel in silico tool for prediction of ERAP1 enzyme trimming.

MHC class I pathway consists of four main steps: proteasomal cleavage in the cytosol in which precursor proteins are cleaved into smaller peptides, which are then transported into the endoplasmic reticulum by the transporter associated with antigen processing, TAP, for further processing (trimming) from the N-terminal region by an ER resident aminopeptidases 1 (ERAP1) enzyme, to generate optimal peptides (8-10 amino acids in length) to produce a stable MHCI-peptide complex, that get transited via the Golgi apparatus to the cell surface for presentation to the cellular immune system. Several studies reported specificities related to the ERAP1 trimming process, yet there is no in silico tool for the prediction of the trimming process of the ERAP1 enzyme. In this paper, we provide and implement a prediction model for the trimming process of the ERAP1 enzyme.

Harnessing the antigenic fingerprint of each individual cancer for immunotherapy of human cancer: genomics shows a new way and its challenges.

The idea that individual tumors are antigenically unique has been around since the very dawn of our recognition of adaptive immune response to tumors. That idea has inspired a small number of attempts at individualized immunotherapy of human cancers. Such previous attempts for solid tumors have been hobbled by an inability to define the individually unique antigenic repertoire of tumors because of technological difficulties. The new availability of rapid and cheap high throughput DNA sequencing promises to overcome that hurdle. Using this new ability, coupled with bio-informatic tools, it is now possible to define the immunogenic repertoire of any tumor to a high degree of granularity within a practical time frame and an acceptable cost. The development of these ideas, and a small number of such studies that underscore this promise, is discussed. This new way--of characterizing the tumor immunome through characterization of the tumor genome--has distinct challenges, including selection of the appropriate peptides, choosing methods of immunizations that can incorporate tens of epitopes, and addressing issues of antigenic heterogeneity of tumors. However, tools for meeting these challenges exist and are emergent.

Mechanism of dichotomy between CD8+ responses elicited by apoptotic and necrotic cells.

Apoptotic cells are significantly more immunogenic than necrotic cells, even though both forms are identical in antigenic content. When a combination of apoptotic and necrotic cells are used to immunize, the phenotype conferred by apoptotic cells, i.e., high immunogenicity, is dominant. However, necrotic cells are not immunosuppressive or tolerogenic. Apoptotic and necrotic cells are taken up by antigen-presenting cells in an equivalent manner. The priming of naïve T cell response is also equivalent. However, the CD8+ T cells elicited by apoptotic cells expand, accumulate, and express effector function, while those primed by the necrotic cells do not. This dichotomy does not extend to CD4+ cells. Apoptotic and necrotic cells elicit equivalent CD4+ T cell priming, accumulation, and function. The deficit in CD8+ T cell function elicited by necrotic cells can be overcome to varying degrees by anti-CD40 antibody and ligands for TLR4 and TLR9; conversely, the immunogenicity of apoptotic cells can be abrogated by blocking anti-CD154 antibody. Our results indicate that immunization with apoptotic cells leads to engagement of CD40 on antigen-presenting cells; this is essential for their ability to elicit mature functional CD8+ cells. The necrotic cells fail to engage CD40, and this failure is the basis of their lack of immunogenicity. These differences have consequences for the understanding of mechanisms of cross-presentation and for efforts toward immunotherapy of cancers and autoimmune pathologies.

In vivo efficacy and synergistic interaction of 16α-hydroxycleroda-3, 13 (14) Z-dien-15, 16-olide, a clerodane diterpene from Polyalthia longifolia against methicillin-resistant Staphylococcus aureus.

The Staphylococcus aureus bacterium, a nosocomial pathogen often causing untreatable and lethal infection in patients, mutated to become resistant to all the first-line drugs. The present study details the potential of clerodane diterpene 16α-hydroxycleroda-3, 13 (14) Z-dien-15, 16-olide (CD) isolated from Polyalthia longifolia against methicillin-resistant S. aureus (MRSA) through in vitro and in vivo assays. Minimum inhibitory concentration (MIC) of CD exhibited significant anti-MRSA activity (15.625-31.25 mg/l) against reference strain and seven clinical isolates, while time kill assays at graded MICs indicated 2.78-9.59- and 2.9-6.18-fold reduction in growth of reference strain and clinical isolates of S. aureus, respectively. The combined effect of the CD and 7.5 % NaCl resulted in significant reduction in microbial count within 24 h, indicating the loss of the salt tolerance ability of S. aureus. Further, release of 260-nm absorbing material and flow cytometric analysis revealed an increased uptake of propidium iodide. These assays may indicate the membrane-damaging potential of CD. The molecule CD was found to interact synergistically with clinically used antibiotics (FICI ≤ 0.5) against all clinical isolates. In infected mice, CD significantly (P < 0.001) lowered the systemic microbial load in blood, liver, kidney, lung and spleen tissues and did not exhibit any significant toxicity at 100 mg/kg body weight.

Immobilization and applications of glucose-6-phosphate dehydrogenase: a review.

Immobilized enzymes have been used extensively in the fields of food industry, materials processing, textiles, detergents, biochemical and chemical industries, biotechnology, and pharmaceuticals. Studies on immobilization of glucose-6-phosphate dehydrogenase have been less extensive than those for other industrially applicable enzymes. Immobilization of glucose-6-phosphate dehydrogenase has been carried out for the formation of biosensors for the estimation of glucose, ATP, phosphate, and so on. The present review deals with the attempts made for immobilization of glucose-6-phosphate dehydrogenase and its applications for various purposes.

Assays to Detect Cross-Dressing by Dendritic Cells In Vivo and In Vitro.

The presentation of peptides derived from exogenous antigens on major histocompatibility complex (MHC) class I molecules of antigen-presenting cells (APCs), termed cross-presentation, is crucial for the activation of cytotoxic T-lymphocytes during cell-mediated immune response. Typically, the APCs acquire exogenous antigens by (i) endocytosis of soluble antigens present in their external milieu, or (ii) through phagocytosis of dying/dead cancer cells or infected cells, followed by intracellular processing, before presentation by MHC I on the surface, or (iii) uptake of heat shock protein-peptide complexes generated in the antigen donor cells (3). In a fourth new mechanism, preformed peptide-MHC complexes can be directly transferred from the surface of antigen donor cells (i.e., cancer cells or infected cells) to that of APCs, without the need of further processing, in a process referred to as cross-dressing. Recently, the importance of cross-dressing in dendritic cell-mediated antitumor immunity and antiviral immunity has been demonstrated. Here, we describe a protocol to study cross-dressing of dendritic cells with tumor antigens.

Lung cDC1 and cDC2 dendritic cells priming naive CD8+ T cells in situ prior to migration to draining lymph nodes.

The current paradigm indicates that naive T cells are primed in secondary lymphoid organs. Here, we present evidence that intranasal administration of peptide antigens appended to nanofibers primes naive CD8+ T cells in the lung independently and prior to priming in the draining mediastinal lymph node (MLN). Notably, comparable accumulation and transcriptomic responses of CD8+ T cells in lung and MLN are observed in both Batf3KO and wild-type (WT) mice, indicating that, while cDC1 dendritic cells (DCs) are the major subset for cross-presentation, cDC2 DCs alone are capable of cross-priming CD8+ T cells both in the lung and draining MLN. Transcription analyses reveal distinct transcriptional responses in lung cDC1 and cDC2 to intranasal nanofiber immunization. However, both DC subsets acquire shared transcriptional responses upon migration into the lymph node, thus uncovering a stepwise activation process of cDC1 and cDC2 toward their ability to cross-prime effector and functional memory CD8+ T cell responses.

GeNeo: A Bioinformatics Toolbox for Genomics-Guided Neoepitope Prediction.

High-throughput DNA and RNA sequencing are revolutionizing precision oncology, enabling personalized therapies such as cancer vaccines designed to target tumor-specific neoepitopes generated by somatic mutations expressed in cancer cells. Identification of these neoepitopes from next-generation sequencing data of clinical samples remains challenging and requires the use of complex bioinformatics pipelines. In this paper, we present GeNeo, a bioinformatics toolbox for genomics-guided neoepitope prediction. GeNeo includes a comprehensive set of tools for somatic variant calling and filtering, variant validation, and neoepitope prediction and filtering. For ease of use, GeNeo tools can be accessed via web-based interfaces deployed on a Galaxy portal publicly accessible at https://neo.engr.uconn.edu/. A virtual machine image for running GeNeo locally is also available to academic users upon request.