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.

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.

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.

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.

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.

Heat-Shock Proteins.

Heat-shock proteins (HSPs), or stress proteins, are abundant and highly conserved, present in all organisms and in all cells. Selected HSPs, also known as chaperones, play crucial roles in folding and unfolding of proteins, assembly of multiprotein complexes, transport and sorting of proteins into correct subcellular compartments, cell-cycle control and signaling, and protection of cells against stress and apoptosis. More recently, HSPs have been shown to be key players in immune responses: during antigen presentation as well as cross-priming, they chaperone and transfer antigenic peptides to class I and class II molecules of the major histocompatibility complexes. In addition, extracellular HSPs can stimulate and cause maturation of professional antigen-presenting cells of the immune system, such as macrophages and dendritic cells. They also chaperone several toll-like receptors, which play a central role in innate immune responses. HSPs constitute a large family of proteins that are often classified based on their molecular weight as Hsp10, Hsp40, Hsp60, Hsp70, Hsp90, etc. This unit contains a table that lists common HSPs and summarizes their characteristics including (a) name, (b) subcellular localization, (c) known function, (d) chromosome assignment, (e) brief comments, and (f) references. © 2022 Wiley Periodicals LLC.

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.

Sympathetic nervous tone limits the development of myeloid-derived suppressor cells.

Sympathetic nerves that innervate lymphoid organs regulate immune development and function by releasing norepinephrine that is sensed by immune cells via their expression of adrenergic receptors. Here, we demonstrate that ablation of sympathetic nervous system (SNS) signaling suppresses tumor immunity, and we dissect the mechanism of such immune suppression. We report that disruption of the SNS in mice removes a critical α-adrenergic signal required for maturation of myeloid cells in normal and tumor-bearing mice. In tumor-bearing mice, disruption of the α-adrenergic signal leads to the accumulation of immature myeloid-derived suppressor cells (MDSCs) that suppress tumor immunity and promote tumor growth. Furthermore, we show that these SNS-responsive MDSCs drive expansion of regulatory T cells via secretion of the alarmin heterodimer S100A8/A9, thereby compounding their immunosuppressive activity. Our results describe a regulatory framework in which sympathetic tone controls the development of innate and adaptive immune cells and influences their activity in health and disease.

Cross-dressing of CD8α+ Dendritic Cells with Antigens from Live Mouse Tumor Cells Is a Major Mechanism of Cross-priming.

Live cells are the most abundant sources of antigen in a tumor-bearing host. Here, we used live tumor cells as source of antigens to investigate the mechanism underlying their immunogenicity in murine tumor models. The live tumor cells were significantly more immunogenic than irradiated or apoptotic tumor cells. We examined the interaction of live and apoptotic tumor cells with major subsets of antigen-presenting cells, i.e., CD8α+ dendritic cells (DC), CD8α- DCs, plasmacytoid DCs, and CD169+ macrophages at skin draining lymph nodes. The CD8α+ DCs captured cell-associated antigens from both live and apoptotic tumor cells, whereas CD169+ macrophages picked up cell-associated antigens mostly from apoptotic tumor cells. Trogocytosis and cross-dressing of membrane-associated antigenic material from live tumor cells to CD8α+ DCs was the primary mechanism for cross-priming of tumor antigens upon immunization with live cells. Phagocytosis of apoptotic tumor cells was the primary mechanism for cross-priming of tumor antigens upon immunization with apoptotic or irradiated cells. These findings clarify the mechanism of cross-priming of cancer antigens by DCs, allowing for a greater understanding of antitumor immune responses.

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.

Low-Level Inhibition of Hsp90 Forces Cells to Tip Their (Antigenic) Hand.

Low-level inhibition of hsp90 enhances the antigenicity of cells whereas high-level inhibition diminishes antigenicity. The mechanism(s) by which hsp90 determines antigenicity are only partially clear. Regardless, these observations have profound implications in protein trafficking and antigen presentation and suggest a novel way to enhance the potency of existing anticancer agents.See related article by Jaeger et al., p. 6392.

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.

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.

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.

Tumor Control Index as a new tool to assess tumor growth in experimental animals.

Measurement of tumor diameters, tumor volumes, or area under the curve has been traditionally used to quantitate and compare tumor growth curves in immune competent as well as immune-compromised mice and rats. Here, using tumor growth data from a large number of mice challenged with live tumor cells, we describe the use of a new composite parameter, Tumor Control Index (TCI) as an alternative method to do the same. This index, comprised of three distinct values, the Tumor Inhibition Score, Tumor Rejection Score, and Tumor Stability Score, provides a complete picture of nearly every aspect of tumor growth in large numbers of animals, can be deduced automatically from tumor diameter or volume data, and can be used to compare several groups of animals in different experiments. This automatically derivable index also corresponds neatly to the use of complete and partial responses and tumor stability data generated in human tumors, and can be used to assess the efficacy of interventions to be used in clinical studies.

Neoepitopes as cancer immunotherapy targets: key challenges and opportunities.

Over the last half century, it has become well established that cancers can elicit a host immune response that can target them with high specificity. Only within the last decade, with the advances in high-throughput gene sequencing and bioinformatics approaches, are we now on the forefront of harnessing the host's immune system to treat cancer. Recently, some strides have been taken toward understanding effective tumor-specific MHC I restricted epitopes or neoepitopes. However, many fundamental questions still remain to be addressed before this therapy can live up to its full clinical potential. In this review, we discuss the major hurdles that lie ahead and the work being done to address them.

Neoepitopes of Cancers: Looking Back, Looking Ahead.

The search for specificity in cancers has been a holy grail in cancer immunology. Cancer geneticists have long known that cancers harbor transforming and other mutations. Immunologists have long known that inbred mice can be immunized against syngeneic cancers, indicating the existence of cancer-specific antigens. With the technological advances in high-throughput DNA sequencing and bioinformatics, the genetic and immunologic lines of inquiry are now converging to provide definitive evidence that human cancers are vastly different from normal tissues at the genetic level, and that some of these differences are recognized by the immune system. The very vastness of genetic changes in cancers now raises different question. Which of the many cancer-specific genetic (genomic) changes are actually recognized by the immune system, and why? New observations are now beginning to probe these vital issues with unprecedented resolution and are informing a new generation of studies in human cancer immunotherapy.