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.

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.

Reversion analysis reveals the in vivo immunogenicity of a poorly MHC I-binding cancer neoepitope.

High-affinity MHC I-peptide interactions are considered essential for immunogenicity. However, some neo-epitopes with low affinity for MHC I have been reported to elicit CD8 T cell dependent tumor rejection in immunization-challenge studies. Here we show in a mouse model that a neo-epitope that poorly binds to MHC I is able to enhance the immunogenicity of a tumor in the absence of immunization. Fibrosarcoma cells with a naturally occurring mutation are edited to their wild type counterpart; the mutation is then re-introduced in order to obtain a cell line that is genetically identical to the wild type except for the neo-epitope-encoding mutation. Upon transplantation into syngeneic mice, all three cell lines form tumors that are infiltrated with activated T cells. However, lymphocytes from the two tumors that harbor the mutation show significantly stronger transcriptional signatures of cytotoxicity and TCR engagement, and induce greater breadth of TCR reactivity than those of the wild type tumors. Structural modeling of the neo-epitope peptide/MHC I pairs suggests increased hydrophobicity of the neo-epitope surface, consistent with higher TCR reactivity. These results confirm the in vivo immunogenicity of low affinity or 'non-binding' epitopes that do not follow the canonical concept of MHC I-peptide recognition.

An unbiased approach to defining bona fide cancer neoepitopes that elicit immune-mediated cancer rejection.

Identification of neoepitopes that are effective in cancer therapy is a major challenge in creating cancer vaccines. Here, using an entirely unbiased approach, we queried all possible neoepitopes in a mouse cancer model and asked which of those are effective in mediating tumor rejection and, independently, in eliciting a measurable CD8 response. This analysis uncovered a large trove of effective anticancer neoepitopes that have strikingly different properties from conventional epitopes and suggested an algorithm to predict them. It also revealed that our current methods of prediction discard the overwhelming majority of true anticancer neoepitopes. These results from a single mouse model were validated in another antigenically distinct mouse cancer model and are consistent with data reported in human studies. Structural modeling showed how the MHC I-presented neoepitopes had an altered conformation, higher stability, or increased exposure to T cell receptors as compared with the unmutated counterparts. T cells elicited by the active neoepitopes identified here demonstrated a stem-like early dysfunctional phenotype associated with effective responses against viruses and tumors of transgenic mice. These abundant anticancer neoepitopes, which have not been tested in human studies thus far, can be exploited for generation of personalized human cancer vaccines.

Mass spectrometry driven exploration reveals nuances of neoepitope-driven tumor rejection.

Neoepitopes are the only truly tumor-specific antigens. Although potential neoepitopes can be readily identified using genomics, the neoepitopes that mediate tumor rejection constitute a small minority, and there is little consensus on how to identify them. Here, for the first time, we use a combination of genomics, unbiased discovery MS immunopeptidomics and targeted MS to directly identify neoepitopes that elicit actual tumor rejection in mice. We report that MS-identified neoepitopes are an astonishingly rich source of tumor rejection mediating neoepitopes. MS has also demonstrated unambiguously the presentation by MHC I, of confirmed tumor rejection neoepitopes which bind weakly to MHC I; this was done using DCs exogenously loaded with long peptides containing the weakly binding neoepitopes. Such weakly MHC I-binding neoepitopes are routinely excluded from analysis, and our demonstration of their presentation, and their activity in tumor rejection, reveals a broader universe of tumor-rejection neoepitopes than presently imagined. Modeling studies show that a mutation in the active neoepitope alters its conformation such that its T cell receptor-facing surface is significantly altered, increasing its exposed hydrophobicity. No such changes are observed in the inactive neoepitope. These results broaden our understanding of antigen presentation and help prioritize neoepitopes for personalized cancer immunotherapy.

CD91 on dendritic cells governs immunosurveillance of nascent, emerging tumors.

The immune system detects aberrant, premalignant cells and eliminates them before the development of cancer. Immune cells, including T cells, have been shown to be critical components in eradicating these aberrant cells, and when absent in the host, incidence of cancer increases. Here, we show that CD91, a receptor expressed on antigen-presenting cells, is required for priming immune responses to nascent, emerging tumors. In the absence of CD91, effector immune responses are subdued, and tumor incidence and progression are amplified. We also show that, consequently, tumors that arise in the absence of CD91 express neo-epitopes with indices that are indicative of greater immunogenicity. Polymorphisms in human CD91 that are expected to affect ligand binding are shown to influence antitumor immune responses in cancer patients. This study presents a molecular mechanism for priming immune responses to nascent, emerging tumors that becomes a predictor of cancer susceptibility and progression.

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.

Fast bootstrapping-based estimation of confidence intervals of expression levels and differential expression from RNA-Seq data.

SUMMARY: This note presents IsoEM2 and IsoDE2, new versions with enhanced features and faster runtime of the IsoEM and IsoDE packages for expression level estimation and differential expression. IsoEM2 estimates fragments per kilobase million (FPKM) and transcript per million (TPM) levels for genes and isoforms with confidence intervals through bootstrapping, while IsoDE2 performs differential expression analysis using the bootstrap samples generated by IsoEM2. Both tools are available with a command line interface as well as a graphical user interface (GUI) through wrappers for the Galaxy platform. AVAILABILITY AND IMPLEMENTATION: The source code of this software suite is available at https://github.com/mandricigor/isoem2. The Galaxy wrappers are available at https://toolshed.g2.bx.psu.edu/view/saharlcc/isoem2_isode2/. CONTACT: imandric1@student.gsu.edu or ion@engr.uconn.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

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.

Aldehyde modification and alum coadjuvancy enhance anti-TNF-α autovaccination and mitigate arthritis in rat.

Experimental vaccination to induce antibodies (Abs) capable of cytokine antagonism shows promise as a novel immunotherapy for chronic inflammatory disease. We prepared a hybrid antigen consisting of residues 141-235 of rat TNF-α fused to the C-terminus of glutathione-S-transferase (GST), chemically modified to incorporate aldehyde residues, for development of an auto-vaccine eliciting anti-rTNF-α Abs. In rat immunization the soluble aldehyde-modified fusion protein did not generate observable Ab responses. By contrast, vaccination with the aldehyde-modified fusion protein adsorbed on alum induced anti-TNF-α autoAbs with high titer and neutralizing activity. Induction of adjuvant arthritis in rats pre-immunized with unmodified fusion protein or a control protein in alum resulted in severe inflammation and joint damage, whereas the disease induced in rats immunized with the aldehyde-bearing fusion protein in alum was markedly attenuated. Similar results were obtained in a collagen-induced rat arthritis model. Anti-collagen II IgG Ab titers did not deviate significantly in groups pre-immunized with modified fusion protein and control protein, suggesting that anti-TNF vaccination did not skew the immune response related to disease induction. This study demonstrates synergy between particulate alum and protein bound carbonyl residues for enhancement of protein immunogenicity. The antigen-specific co-adjuvant system could prove advantageous for breaking tolerance in emerging auto-vaccination therapies targeting inflammatory cytokines as well as for enhancing a broader category of subunit vaccines. Aldehyde adduction introduces a minimal modification which, together with the established use of alum as a safe adjuvant for human use, could be favorable for further vaccine development.

Reactive immunization suppresses advanced glycation and mitigates diabetic nephropathy.

Agents that inhibit glycation end products by reducing the carbonyl load from glycation and glycoxidation are an emerging pharmacologic approach to treat complications of diabetes. We previously demonstrated that antibodies generated to the glycoprotein keyhole limpet hemocyanin (KLH) can cross-link with reactive carbonyl residues on protein conjugates. Here, we immunized streptozotocin-induced diabetic rats with KLH to assess the capacity of the elicited antibodies to intercept carbonyl residues on glycated proteins and to mitigate glycation-related pathology. Compared with diabetic rats immunized with adjuvant alone, KLH-immunized diabetic rats had decreased levels of glycated peptides in sera and demonstrated a reduction in albuminuria, proteinuria, deposition of glycation end products in the kidney, and histologic damage. In vitro, low molecular weight glycated peptides from rat serum reacted with anti-KLH antibodies at a faster rate than normal IgG and selectively modified the lambda chains. The reaction products contained peptide sequences from type I collagen alpha chain, albumin, and LDL receptor-related protein. These adduction reactions were inhibited by free KLH and by reduction of glycated peptides with borohydride. In summary, these results suggest that inherent reactivity of Ig light chains provides a natural mechanism for the removal of cytotoxic glycation products. This reactivity can be augmented by glycoprotein-specific reactive immunization, a potential biopharmaceutical approach to glycation-related pathology.