A Cancer Nanovaccine for Co-Delivery of Peptide Neoantigens and Optimized Combinations of STING and TLR4 Agonists.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment and led to complete and durable responses, but only for a minority of patients. Resistance to ICB can largely be attributed to insufficient number and/or function of antitumor CD8+ T cells in the tumor microenvironment. Neoantigen targeted cancer vaccines can activate and expand the antitumor T cell repertoire, but historically, clinical responses have been poor because immunity against peptide antigens is typically weak, resulting in insufficient activation of CD8+ cytotoxic T cells. Herein, we describe a nanoparticle vaccine platform that can overcome these barriers in several ways. First, the vaccine can be reproducibly formulated using a scalable confined impingement jet mixing method to coload a variety of physicochemically diverse peptide antigens and multiple vaccine adjuvants into pH-responsive, vesicular nanoparticles that are monodisperse and less than 100 nm in diameter. Using this approach, we encapsulated synergistically acting adjuvants, cGAMP and monophosphoryl lipid A (MPLA), into the nanocarrier to induce a robust and tailored innate immune response that increased peptide antigen immunogenicity. We found that incorporating both adjuvants into the nanovaccine synergistically enhanced expression of dendritic cell costimulatory markers, pro-inflammatory cytokine secretion, and peptide antigen cross-presentation. Additionally, the nanoparticle delivery increased lymph node accumulation and uptake of peptide antigen by dendritic cells in the draining lymph node. Consequently, nanoparticle codelivery of peptide antigen, cGAMP, and MPLA enhanced the antigen-specific CD8+ T cell response and delayed tumor growth in several mouse models. Finally, the nanoparticle platform improved the efficacy of ICB immunotherapy in a murine colon carcinoma model. This work establishes a versatile nanoparticle vaccine platform for codelivery of peptide neoantigens and synergistic adjuvants to enhance responses to cancer vaccines.

Die Kämpfe únd schláchten-the struggles and battles of innate-like effector T lymphocytes with microbes.

The large majority of lymphocytes belong to the adaptive immune system, which are made up of B2 B cells and the αß T cells; these are the effectors in an adaptive immune response. A multitudinous group of lymphoid lineage cells does not fit the conventional lymphocyte paradigm; it is the unconventional lymphocytes. Unconventional lymphocytes-here called innate/innate-like lymphocytes, include those that express rearranged antigen receptor genes and those that do not. Even though the innate/innate-like lymphocytes express rearranged, adaptive antigen-specific receptors, they behave like innate immune cells, which allows them to integrate sensory signals from the innate immune system and relay that umwelt to downstream innate and adaptive effector responses. Here, we review natural killer T cells and mucosal-associated invariant T cells-two prototypic innate-like T lymphocytes, which sense their local environment and relay that umwelt to downstream innate and adaptive effector cells to actuate an appropriate host response that confers immunity to infectious agents.

The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked to the induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for the regulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to the understanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

A molecular signature of lung-resident CD8+ T cells elicited by subunit vaccination.

Natural infection as well as vaccination with live or attenuated viruses elicit tissue resident, CD8+ memory T cell (Trm) response. Trm cells so elicited act quickly upon reencounter with the priming agent to protect the host. These Trm cells express a unique molecular signature driven by the master regulators-Runx3 and Hobit. We previously reported that intranasal instillation of a subunit vaccine in a prime boost vaccination regimen installed quick-acting, CD8+ Trm cells in the lungs that protected against lethal vaccinia virus challenge. It remains unexplored whether CD8+ Trm responses so elicited are driven by a similar molecular signature as those elicited by microbes in a real infection or by live, attenuated pathogens in conventional vaccination. We found that distinct molecular signatures distinguished subunit vaccine-elicited lung interstitial CD8+ Trm cells from subunit vaccine-elicited CD8+ effector memory and splenic memory T cells. Nonetheless, the transcriptome signature of subunit vaccine elicited CD8+ Trm resembled those elicited by virus infection or vaccination. Clues to the basis of tissue residence and function of vaccine specific CD8+ Trm cells were found in transcripts that code for chemokines and chemokine receptors, purinergic receptors, and adhesins when compared to CD8+ effector and splenic memory T cells. Our findings inform the utility of protein-based subunit vaccination for installing CD8+ Trm cells in the lungs to protect against respiratory infectious diseases that plague humankind.

Thymic epithelial cells require lipid kinase Vps34 for CD4 but not CD8 T cell selection.

The generation of a functional, self-tolerant T cell receptor (TCR) repertoire depends on interactions between developing thymocytes and antigen-presenting thymic epithelial cells (TECs). Cortical TECs (cTECs) rely on unique antigen-processing machinery to generate self-peptides specialized for T cell positive selection. In our current study, we focus on the lipid kinase Vps34, which has been implicated in autophagy and endocytic vesicle trafficking. We show that loss of Vps34 in TECs causes profound defects in the positive selection of the CD4 T cell lineage but not the CD8 T cell lineage. Utilizing TCR sequencing, we show that T cell selection in conditional mutants causes altered repertoire properties including reduced clonal sharing. cTECs from mutant mice display an increased abundance of invariant chain intermediates bound to surface MHC class II molecules, indicating altered antigen processing. Collectively, these studies identify lipid kinase Vps34 as an important contributor to the repertoire of selecting ligands processed and presented by TECs to developing CD4 T cells.

A single-cell analysis of thymopoiesis and thymic iNKT cell development in pigs.

Many aspects of the porcine immune system remain poorly characterized, which poses a barrier to improving swine health and utilizing pigs as preclinical models. Here, we employ single-cell RNA sequencing (scRNA-seq) to create a cell atlas of the early-adolescent pig thymus. Our data show conserved features as well as species-specific differences in cell states and cell types compared with human thymocytes. We also describe several unconventional T cell types with gene expression profiles associated with innate effector functions. This includes a cell census of more than 11,000 differentiating invariant natural killer T (iNKT) cells, which reveals that the functional diversity of pig iNKT cells differs substantially from the iNKT0/1/2/17 subset differentiation paradigm established in mice. Our data characterize key differentiation events in porcine thymopoiesis and iNKT cell maturation and provide important insights into pig T cell development.

A nanovaccine for enhancing cellular immunity via cytosolic co-delivery of antigen and polyIC RNA.

Traditional approaches to cancer vaccines elicit weak CD8+ T cell responses and have largely failed to meet clinical expectations. This is in part due to inefficient antigen cross-presentation, inappropriate selection of adjuvant and its formulation, poor vaccine pharmacokinetics, and/or suboptimal coordination of antigen and adjuvant delivery. Here, we describe a nanoparticle vaccine platform for facile co-loading and dual-delivery of antigens and nucleic acid adjuvants that elicits robust antigen-specific cellular immune responses. The nanovaccine design is based on diblock copolymers comprising a poly(ethylene glycol)-rich first block that is functionalized with reactive moieties for covalent conjugation of antigen via disulfide linkages, and a pH-responsive second block for electrostatic packaging of nucleic acids that also facilitates endosomal escape of associated vaccine cargo to the cytosol. Using polyIC, a clinically-advanced nucleic acid adjuvant, we demonstrated that endosomolytic nanoparticles promoted the cytosolic co-delivery of polyIC and protein antigen, which acted synergistically to enhance antigen cross-presentation, co-stimulatory molecule expression, and cytokine production by dendritic cells. We also found that the vaccine platform increased the accumulation of antigen and polyIC in the local draining lymph nodes. Consequently, dual-delivery of antigen and polyIC with endsomolytic nanoparticles significantly enhanced the magnitude and functionality of CD8+ T cell responses relative to a mixture of antigen and polyIC, resulting in inhibition of tumor growth in a mouse tumor model. Collectively, this work provides a proof-of-principle for a new cancer vaccine platform that strongly augments anti-tumor cellular immunity via cytosolic co-delivery of antigen and nucleic acid adjuvant.

Nano-Particulate Platforms for Vaccine Delivery to Enhance Antigen-Specific CD8+ T-Cell Response.

Vaccines remain the most effective way to protect populations against deathly infectious diseases. Several disadvantages associated with the traditional vaccines that use whole pathogens have led to the development of alternative strategies including the use of recombinant subunit vaccines. Subunit vaccines are, in general, safer than whole pathogens but tend to be less immunogenic due to the lack of molecular cues that are typically found on whole pathogens. To enhance immunogenicity, the subunit antigen  can be administered with adjuvants that stimulate the innate immune system as a means to steer the quality and magnitude of the adaptive immune response. Novel classes of adjuvants are formulated using particle-based platforms such as virus-like particles, liposomes, and polymeric nanoparticles. These particle-based systems present antigens in ways reminiscent of whole pathogens. Such platforms offer several advantages that include co-delivery of antigen along with innate immune stimulators in a highly immunogenic format. Here we describe our recent efforts to synthesize, characterize, and validate two promising nanoparticle-based delivery systems and demonstrate their potential to induce antigen-specific CD8+ T cell responses, essential in clearing infection with intracellular pathogens, such as viruses and bacteria, and eradicating tumors.

Novel HLA-B7-restricted human metapneumovirus epitopes enhance viral clearance in mice and are recognized by human CD8+ T cells.

Human metapneumovirus (HMPV) is a leading cause of acute lower respiratory tract illness in children and adults. Repeated infections are common and can be severe in young, elderly, and immunocompromised persons due to short-lived protective humoral immunity. In turn, few protective T cell epitopes have been identified in humans. Thus, we infected transgenic mice expressing the common human HLA MHC-I allele B*07:02 (HLA-B7) with HMPV and screened a robust library of overlapping and computationally predicted HLA-B7 binding peptides. Six HLA-B7-restricted CD8+ T cell epitopes were identified using ELISPOT screening in the F, M, and N proteins, with M195-203 (M195) eliciting the strongest responses. MHC-tetramer flow cytometric staining confirmed HLA-B7 epitope-specific CD8+ T cells migrated to lungs and spleen of HMPV-immune mice. Immunization with pooled HLA-B7-restricted peptides reduced viral titer and protected mice from virulent infection. Finally, we confirmed that CD8+ T cells from HLA-B7 positive humans also recognize the identified epitopes. These results enable identification of HMPV-specific CD8+ T cells in humans and help to inform future HMPV vaccine design.

Know thy immune self and non-self: Proteomics informs on the expanse of self and non-self, and how and where they arise.

T cells play an important role in the adaptive immune response to a variety of infections and cancers. Initiation of a T cell mediated immune response requires antigen recognition in a process termed MHC (major histocompatibility complex) restri ction. A T cell antigen is a composite structure made up of a peptide fragment bound within the antigen-binding groove of an MHC-encoded class I or class II molecule. Insight into the precise composition and biology of self and non-self immunopeptidomes is essential to harness T cell mediated immunity to prevent, treat, or cure infectious diseases and cancers. T cell antigen discovery is an arduous task! The pioneering work in the early 1990s has made large-scale T cell antigen discovery possible. Thus, advancements in mass spectrometry coupled with proteomics and genomics technologies make possible T cell antigen discovery with ease, accuracy, and sensitivity. Yet we have only begun to understand the breadth and the depth of self and non-self immunopeptidomes because the molecular biology of the cell continues to surprise us with new secrets directly related to the source, and the processing and presentation of MHC ligands. Focused on MHC class I molecules, this review, therefore, provides a brief historic account of T cell antigen discovery and, against a backdrop of key advances in molecular cell biologic processes, elaborates on how proteogenomics approaches have revolutionised the field.

Natural Killer T Lymphocytes Integrate Innate Sensory Information and Relay Context to Effector Immune Responses.

It is now appreciated that a group of lymphoid lineage cells, collectively called innate-like effector lymphocytes, have evolved to integrate information relayed by the innate sensory immune system about the state of the local tissue environment and to pass on this context to downstream effector innate and adaptive immune responses. Thereby, innate functions engrained into such innate-like lymphoid lineage cells during development can control the quality and magnitude of an immune response to a tissue-altering pathogen and facilitate the formation of memory engrams within the immune system. These goals are accomplished by the innate lymphoid cells that lack antigen-specific receptors, γδ T cell receptor (TCR)-expressing T cells, and several αß TCR-expressing T cell subsets-such as natural killer T cells, mucosal-associated invariant T cells, et cetera. Whilst we briefly consider the commonalities in the origins and functions of these diverse lymphoid subsets to provide context, the primary topic of this review is to discuss how the semi-invariant natural killer T cells got this way in evolution through lineage commitment and onward ontogeny. What emerges from this discourse is the question: Has a "limbic immune system" emerged (screaming quietly in plain sight!) out of what has been dubbed "in-betweeners"?

Heterotypic immunity against vaccinia virus in an HLA-B*07:02 transgenic mousepox infection model.

Vaccination with vaccinia virus (VACV) elicits heterotypic immunity to smallpox, monkeypox, and mousepox, the mechanistic basis for which is poorly understood. It is generally assumed that heterotypic immunity arises from the presentation of a wide array of VACV-derived, CD8+ T cell epitopes that share homology with other poxviruses. Herein this assumption was tested using a large panel of VACV-derived peptides presented by HLA-B*07:02 (B7.2) molecules in a mousepox/ectromelia virus (ECTV)-infection, B7.2 transgenic mouse model. Most dominant epitopes recognized by ECTV- and VACV-reactive CD8+ T cells overlapped significantly without altering immunodominance hierarchy. Further, several epitopes recognized by ECTV-reactive CD8+ T cells were not recognized by VACV-reactive CD8+ T cells, and vice versa. In one instance, the lack of recognition owed to a N72K variation in the ECTV C4R70-78 variant of the dominant VACV B8R70-78 epitope. C4R70-78 does not bind to B7.2 and, hence, it was neither immunogenic nor antigenic. These findings provide a mechanistic basis for VACV vaccination-induced heterotypic immunity which can protect against Variola and Monkeypox disease. The understanding of how cross-reactive responses develop is essential for the rational design of a subunit-based vaccine that would be safe, and effectively protect against heterologous infection.

Co-delivery of Peptide Neoantigens and Stimulator of Interferon Genes Agonists Enhances Response to Cancer Vaccines.

Cancer vaccines targeting patient-specific neoantigens have emerged as a promising strategy for improving responses to immune checkpoint blockade. However, neoantigenic peptides are poorly immunogenic and inept at stimulating CD8+ T cell responses, motivating a need for new vaccine technologies that enhance their immunogenicity. The stimulator of interferon genes (STING) pathway is an endogenous mechanism by which the innate immune system generates an immunological context for priming and mobilizing neoantigen-specific T cells. Owing to this critical role in tumor immune surveillance, a synthetic cancer nanovaccine platform (nanoSTING-vax) was developed that mimics immunogenic cancer cells in its capacity to efficiently promote co-delivery of peptide antigens and the STING agonist, cGAMP. The co-loading of cGAMP and peptides into pH-responsive, endosomolytic polymersomes promoted the coordinated delivery of both cGAMP and peptide antigens to the cytosol, thereby eliciting inflammatory cytokine production, co-stimulatory marker expression, and antigen cross-presentation. Consequently, nanoSTING-vax significantly enhanced CD8+ T cell responses to a range of peptide antigens. Therapeutic immunization with nanoSTING-vax, in combination with immune checkpoint blockade, inhibited tumor growth in multiple murine tumor models, even leading to complete tumor rejection and generation of durable antitumor immune memory. Collectively, this work establishes nanoSTING-vax as a versatile platform for enhancing immune responses to neoantigen-targeted cancer vaccines.

Nur77 controls tolerance induction, terminal differentiation, and effector functions in semi-invariant natural killer T cells.

Semi-invariant natural killer T (iNKT) cells are self-reactive lymphocytes, yet how this lineage attains self-tolerance remains unknown. iNKT cells constitutively express high levels of Nr4a1-encoded Nur77, a transcription factor that integrates signal strength downstream of the T cell receptor (TCR) within activated thymocytes and peripheral T cells. The function of Nur77 in iNKT cells is unknown. Here we report that sustained Nur77 overexpression (Nur77tg) in mouse thymocytes abrogates iNKT cell development. Introgression of a rearranged Vα14-Jα18 TCR-α chain gene into the Nur77tg (Nur77tg;Vα14tg) mouse rescued iNKT cell development up to the early precursor stage, stage 0. iNKT cells in bone marrow chimeras that reconstituted thymic cellularity developed beyond stage 0 precursors and yielded IL-4-producing NKT2 cell subset but not IFN-γ-producing NKT1 cell subset. Nonetheless, the developing thymic iNKT cells that emerged in these chimeras expressed the exhaustion marker PD1 and responded poorly to a strong glycolipid agonist. Thus, Nur77 integrates signals emanating from the TCR to control thymic iNKT cell tolerance induction, terminal differentiation, and effector functions.

Survivre et vivre: When iNKT cells met a Hippo.

Invariant natural killer T (iNKT) cells are innate-like lymphocytes with unique signaling requirements for their development and differentiation. In this issue of JEM, Raynor et al. (https://doi.org/10.1084/jem.20191157) report that the Hippo signaling pathway controls the maturation and effector differentiation of iNKT cells by modulating cellular metabolism.

NKT Cells Join the Two Step for Inflammasome-Independent IL-1ß Release.

The cytokine interleukin-1ß (IL-1ß) is critical for antimicrobial defenses; the inflammasome pathway typically controls IL-1ß release, but pathogens often evade this pathway. In this issue, Donado et al. (2020) describe an alternative, two-cell model, to instruct inflammasome-independent IL-1ß release.

Mucosal Immunization with a pH-Responsive Nanoparticle Vaccine Induces Protective CD8+ Lung-Resident Memory T Cells.

Tissue-resident memory T cells (TRM) patrol nonlymphoid organs and provide superior protection against pathogens that commonly infect mucosal and barrier tissues, such as the lungs, intestine, liver, and skin. Thus, there is a need for vaccine technologies that can induce a robust, protective TRM response in these tissues. Nanoparticle (NP) vaccines offer important advantages over conventional vaccines; however, there has been minimal investigation into the design of NP-based vaccines for eliciting TRM responses. Here, we describe a pH-responsive polymeric nanoparticle vaccine for generating antigen-specific CD8+ TRM cells in the lungs. With a single intranasal dose, the NP vaccine elicited airway- and lung-resident CD8+ TRM cells and protected against respiratory virus challenge in both sublethal (vaccinia) and lethal (influenza) infection models for up to 9 weeks after immunization. In elucidating the contribution of material properties to the resulting TRM response, we found that the pH-responsive activity of the carrier was important, as a structurally analogous non-pH-responsive control carrier elicited significantly fewer lung-resident CD8+ T cells. We also demonstrated that dual-delivery of protein antigen and nucleic acid adjuvant on the same NP substantially enhanced the magnitude, functionality, and longevity of the antigen-specific CD8+ TRM response in the lungs. Compared to administration of soluble antigen and adjuvant, the NP also mediated retention of vaccine cargo in pulmonary antigen-presenting cells (APCs), enhanced APC activation, and increased production of TRM-related cytokines. Overall, these data suggest a promising vaccine platform technology for rapid generation of protective CD8+ TRM cells in the lungs.

AS03-Adjuvanted H5N1 Avian Influenza Vaccine Modulates Early Innate Immune Signatures in Human Peripheral Blood Mononuclear Cells.

BACKGROUND: Adjuvant System 03 (AS03) markedly enhances responses to influenza A/H5N1 vaccines, but the mechanisms of this enhancement are incompletely understood. METHODS: Using ribonucleic acid sequencing on peripheral blood mononuclear cells (PBMCs) from AS03-adjuvanted and unadjuvanted inactivated H5N1 vaccine recipients, we identified differentially expressed genes, enriched pathways, and genes that correlated with serologic responses. We compared bulk PBMC findings with our previously published assessments of flow-sorted immune cell types. RESULTS: AS03-adjuvanted vaccine induced the strongest differential signals on day 1 postvaccination, activating multiple innate immune pathways including interferon and JAK-STAT signaling, Fcγ receptor (FcγR)-mediated phagocytosis, and antigen processing and presentation. Changes in signal transduction and immunoglobulin genes predicted peak hemagglutinin inhibition (HAI) titers. Compared with individual immune cell types, activated PBMC genes and pathways were most similar to innate immune cells. However, several pathways were unique to PBMCs, and several pathways identified in individual cell types were absent in PBMCs. CONCLUSIONS: Transcriptomic analysis of PBMCs after AS03-adjuvanted H5N1 vaccination revealed early activation of innate immune signaling, including a 5- to 8-fold upregulation of FcγR1A/1B/1C genes. Several early gene responses were correlated with HAI titer, indicating links with the adaptive immune response. Although PBMCs and cell-specific results shared key innate immune signals, unique signals were identified by both approaches.