MARS an improved de novo peptide candidate selection method for non-canonical antigen target discovery in cancer.

Understanding the nature and extent of non-canonical human leukocyte antigen (HLA) presentation in tumour cells is a priority for target antigen discovery for the development of next generation immunotherapies in cancer. We here employ a de novo mass spectrometric sequencing approach with a refined, MHC-centric analysis strategy to detect non-canonical MHC-associated peptides specific to cancer without any prior knowledge of the target sequence from genomic or RNA sequencing data. Our strategy integrates MHC binding rank, Average local confidence scores, and peptide Retention time prediction for improved de novo candidate Selection; culminating in the machine learning model MARS. We benchmark our model on a large synthetic peptide library dataset and reanalysis of a published dataset of high-quality non-canonical MHC-associated peptide identifications in human cancer. We achieve almost 2-fold improvement for high quality spectral assignments in comparison to de novo sequencing alone with an estimated accuracy of above 85.7% when integrated with a stepwise peptide sequence mapping strategy. Finally, we utilize MARS to detect and validate lncRNA-derived peptides in human cervical tumour resections, demonstrating its suitability to discover novel, immunogenic, non-canonical peptide sequences in primary tumour tissue.

Targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient for generating responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins essential for tumorigenesis. We focused on targeting the unmutated peptide QYNPIRTTF discovered on HLA-A*24:02, which is derived from the neuroblastoma-dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (PC-CARs) through a counter panning strategy using predicted potentially cross-reactive peptides. We further proposed that PC-CARs can recognize peptides on additional HLA allotypes when presenting a similar overall molecular surface. Informed by our computational modelling results, we show that PHOX2B PC-CARs also recognize QYNPIRTTF presented by HLA-A*23:01, the most common non-A2 allele in people with African ancestry. Finally, we demonstrate potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that PC-CARs have the potential to expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and allow targeting through additional HLA allotypes in a clinical setting.

c-Myc uses Cul4b to preserve genome integrity and promote antiviral CD8+ T cell immunity.

During infection, virus-specific CD8+ T cells undergo rapid bursts of proliferation and differentiate into effector cells that kill virus-infected cells and reduce viral load. This rapid clonal expansion can put T cells at significant risk for replication-induced DNA damage. Here, we find that c-Myc links CD8+ T cell expansion to DNA damage response pathways though the E3 ubiquitin ligase, Cullin 4b (Cul4b). Following activation, c-Myc increases the levels of Cul4b and other members of the Cullin RING Ligase 4 (CRL4) complex. Despite expressing c-Myc at high levels, Cul4b-deficient CD8+ T cells do not expand and clear the Armstrong strain of lymphocytic choriomeningitis virus (LCMV) in vivo. Cul4b-deficient CD8+ T cells accrue DNA damage and succumb to proliferative catastrophe early after antigen encounter. Mechanistically, Cul4b knockout induces an accumulation of p21 and Cyclin E2, resulting in replication stress. Our data show that c-Myc supports cell proliferation by maintaining genome stability via Cul4b, thereby directly coupling these two interdependent pathways. These data clarify how CD8+ T cells use c-Myc and Cul4b to sustain their potential for extraordinary population expansion, longevity and antiviral responses.

Cryptic MHC-E epitope from influenza elicits a potent cytolytic T cell response.

The extent to which unconventional forms of antigen presentation drive T cell immunity is unknown. By convention, CD8 T cells recognize viral peptides, or epitopes, in association with classical major histocompatibility complex (MHC) class I, or MHC-Ia, but immune surveillance can, in some cases, be directed against peptides presented by nonclassical MHC-Ib, in particular the MHC-E proteins (Qa-1 in mice and HLA-E in humans); however, the overall importance of nonclassical responses in antiviral immunity remains unclear. Similarly uncertain is the importance of 'cryptic' viral epitopes, defined as those undetectable by conventional mapping techniques. Here we used an immunopeptidomic approach to search for unconventional epitopes that drive T cell responses in mice infected with influenza virus A/Puerto Rico/8/1934. We identified a nine amino acid epitope, termed M-SL9, that drives a co-immunodominant, cytolytic CD8 T cell response that is unconventional in two major ways: first, it is presented by Qa-1, and second, it has a cryptic origin, mapping to an unannotated alternative reading frame product of the influenza matrix gene segment. Presentation and immunogenicity of M-SL9 are dependent on the second AUG codon of the positive sense matrix RNA segment, suggesting translation initiation by leaky ribosomal scanning. During influenza virus A/Puerto Rico/8/1934 infection, M-SL9-specific T cells exhibit a low level of egress from the lungs and strong differentiation into tissue-resident memory cells. Importantly, we show that M-SL9/Qa-1-specific T cells can be strongly induced by messenger RNA vaccination and that they can mediate antigen-specific cytolysis in vivo. Our results demonstrate that noncanonical translation products can account for an important fraction of the T cell repertoire and add to a growing body of evidence that MHC-E-restricted T cells could have substantial therapeutic value.

HIV-1-Infected CD4+ T Cells Present MHC Class II-Restricted Epitope via Endogenous Processing.

HIV-1-specific CD4+ T cells (TCD4+s) play a critical role in controlling HIV-1 infection. Canonically, TCD4+s are activated by peptides derived from extracellular ("exogenous") Ags displayed in complex with MHC class II (MHC II) molecules on the surfaces of "professional" APCs such as dendritic cells (DCs). In contrast, activated human TCD4+s, which express MHC II, are not typically considered for their APC potential because of their low endocytic capacity and the exogenous Ag systems historically used for assessment. Using primary TCD4+s and monocyte-derived DCs from healthy donors, we show that activated human TCD4+s are highly effective at MHC II-restricted presentation of an immunodominant HIV-1-derived epitope postinfection and subsequent noncanonical processing and presentation of endogenously produced Ag. Our results indicate that, in addition to marshalling HIV-1-specific immune responses during infection, TCD4+s also act as APCs, leading to the activation of HIV-1-specific TCD4+s.

Cross-HLA targeting of intracellular oncoproteins with peptide-centric CARs.

The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction.

STAT3-BDNF-TrkB signalling promotes alveolar epithelial regeneration after lung injury.

Alveolar epithelial regeneration is essential for recovery from devastating lung diseases. This process occurs when type II alveolar pneumocytes (AT2 cells) proliferate and transdifferentiate into type I alveolar pneumocytes (AT1 cells). We used genome-wide analysis of chromatin accessibility and gene expression following acute lung injury to elucidate repair mechanisms. AT2 chromatin accessibility changed substantially following injury to reveal STAT3 binding motifs adjacent to genes that regulate essential regenerative pathways. Single-cell transcriptome analysis identified brain-derived neurotrophic factor (Bdnf) as a STAT3 target gene with newly accessible chromatin in a unique population of regenerating AT2 cells. Furthermore, the BDNF receptor tropomyosin receptor kinase B (TrkB) was enriched on mesenchymal alveolar niche cells (MANCs). Loss or blockade of AT2-specific Stat3, Bdnf or mesenchyme-specific TrkB compromised repair and reduced Fgf7 expression by niche cells. A TrkB agonist improved outcomes in vivo following lung injury. These data highlight the biological and therapeutic importance of the STAT3-BDNF-TrkB axis in orchestrating alveolar epithelial regeneration.

Kinetically distinct processing pathways diversify the CD8+ T cell response to a single viral epitope.

The source proteins from which CD8+ T cell-activating peptides are derived remain enigmatic. Glycoproteins are particularly challenging in this regard owing to several potential trafficking routes within the cell. By engineering a glycoprotein-derived epitope to contain an N-linked glycosylation site, we determined that optimal CD8+ T cell expansion and function were induced by the peptides that are rapidly produced from the exceedingly minor fraction of protein mislocalized to the cytosol. In contrast, peptides derived from the much larger fraction that undergoes translocation and quality control are produced with delayed kinetics and induce suboptimal CD8+ T cell responses. This dual system of peptide generation enhances CD8+ T cell participation in diversifying both antigenicity and the kinetics of peptide display.

Standard screening methods underreport AAV-mediated transduction and gene editing.

Conventional methods to discern adeno-associated virus (AAV) vector transduction patterns are based on high, stable expression of a reporter gene. As a consequence, conventionally described tropisms omit cell types that undergo transient transduction, or have low but undetectable levels of reporter expression. This creates a blind spot for AAV-based genome editing applications because only minimal transgene expression is required for activity. Here, we use editing-reporter mice to fill this void. Our approach sensitively captures both high and low transgene expression from AAV vectors. Using AAV8 and other serotypes, we demonstrate the superiority of the approach in a side-by-side comparison with traditional methods, demonstrate numerous, previously unknown sites of AAV targeting, and better predict the gene editing footprint after AAV-CRISPR delivery. We anticipate that this system, which captures the full spectrum of transduction patterns from AAV vectors in vivo, will be foundational to current and emerging AAV technologies.

Recombinant Poxviruses: Versatile Tools for Immunological Assays.

The study of antigen processing and presentation is critical to our understanding of the mechanisms that govern immune surveillance. A typical requirement of assays designed to examine antigen processing and presentation is the de novo biosynthesis of a model antigen. Historically, Vaccinia virus, a poxvirus closely related to Cowpox virus, has enjoyed widespread use for this purpose. Recombinant poxvirus-based expression has a number of advantages over other systems. Poxviruses accommodate the insertion of large pieces of recombinant DNA into their genome, and recombination and selection are relatively efficient. Poxviruses readily infect a variety of cell types, and they drive rapid and high levels of antigen expression. Additionally, they can be utilized in a variety of assays to study both MHC class I restricted and MHC class II restricted antigen processing and presentation. Ultimately, the numerous advantages of poxvirus recombinants have made the Vaccinia expression system a mainstay in the study of processing and presentation over the past two decades. In an attempt to address one shortcoming of Vaccinia virus while simultaneously retaining the benefits inherent to poxviruses, our laboratory has begun to engineer recombinant Ectromelia viruses. Ectromelia virus, or mousepox, is a natural pathogen of murine cells and performing experiments in the context of a natural host-pathogen relationship may elucidate unknown factors that influence epitope generation and host response. This chapter will describe several recombinant poxvirus system protocols used to study both MHC class I and class II antigen processing and presentation, as well as provide insight and troubleshooting techniques to improve the reproducibility and fidelity of these experiments.

Impact of epitope density on CD8+ T cell development and function.

Effective immune responses against intracellular pathogens and tumors frequently rely upon CD8+ cytotoxic T lymphocytes (CTLs). In turn, CTL detection of foreign material from viruses and bacteria depends on antigen presentation by the MHC class I pathway. The underpinnings of antigen processing and presentation and, subsequent T cell activation and immunological memory development, have been extensively studied, leading to a better understanding of the balance between antigen dose, context, and, the T cell activation threshold. Still, the complexity of this process leads to apparent contradictions that hinder construction of rational strategies for generating optimal CD8 + T cell responses in a variety of settings. In this review we consolidate the current knowledge around the effects of peptide MHC I complex (pMHC) density and kinetics on CD8 + T cell responses and function during the acute phase of an infection.

CD8 + T-cell responses in vaccination: reconsidering targets and function in the context of chronic antigen stimulation.

Cytotoxic CD8 T cells play important roles in eliminating infected and transformed cells. Owing to their potential for therapeutic applications, significant efforts are dedicated toward developing CD8 T cell-based vaccines. Thus far, CD8 T-cell vaccination strategies have had limited success therapeutically in contrast to those targeting antibody-based immunity. However, if the current challenges and gaps in the understanding of T-cell biology are overcome, the full potential of rational CD8 T-cell vaccine design might be realized. Here, we review recent progress in this direction, focusing on target selection and maintenance of function in the settings of chronic infections and cancers.

Somatic HLA Mutations Expose the Role of Class I-Mediated Autoimmunity in Aplastic Anemia and its Clonal Complications.

Acquired aplastic anemia (aAA) is an acquired deficiency of early hematopoietic cells, characterized by inadequate blood production, and a predisposition to myelodysplastic syndrome (MDS) and leukemia. Although its exact pathogenesis is unknown, aAA is thought to be driven by Human Leukocyte Antigen (HLA)-restricted T cell immunity, with earlier studies favoring HLA class II-mediated pathways. Using whole exome sequencing (WES), we recently identified two aAA patients with somatic mutations in HLA class I genes. We hypothesized that HLA class I mutations are pathognomonic for autoimmunity in aAA, but were previously underappreciated because the Major Histocompatibility Complex (MHC) region is notoriously difficult to analyze by WES. Using a combination of targeted deep sequencing of HLA class I genes and single nucleotide polymorphism array (SNP-A) genotyping we screened 66 aAA patients for somatic HLA class I loss. We found somatic HLA loss in eleven patients (17%), with thirteen loss-of-function mutations in HLA-A*33:03, HLA-A*68:01, HLA-B*14:02 and HLA-B*40:02 alleles. Three patients had more than one mutation targeting the same HLA allele. Interestingly, HLA-B*14:02 and HLA-B*40:02 were significantly overrepresented in aAA patients, compared to ethnicity-matched controls. Patients who inherited the targeted HLA alleles, regardless of HLA mutation status, had a more severe disease course with more frequent clonal complications as assessed by WES, SNP-A, and metaphase cytogenetics, and more frequent secondary MDS. The finding of recurrent HLA class I mutations provides compelling evidence for a predominant HLA class I-driven autoimmunity in aAA, and establishes a novel link between aAA patients' immunogenetics and clonal evolution.

The elucidation of non-classical MHC class II antigen processing through the study of viral antigens.

By convention, CD4+ T cells are activated predominantly by Major Histocompatibility Complex class II-bound peptides derived from extracellular (exogenous) antigens. It has been known for decades that alternative sources of antigen, particularly those synthesized within the antigen-presenting cell, can also supply peptides but the impact on TCD4+ responses, sometimes considerable, has only recently become appreciated. This review focuses on the contributions that studies of viral antigen have made to this shift in perspective, concluding with discussions of relevance to rational vaccine design, autoimmunity and cancer immunotherapy.

Endogenous antigen processing drives the primary CD4+ T cell response to influenza.

By convention, CD4+ T lymphocytes recognize foreign and self peptides derived from internalized antigens in combination with major histocompatibility complex class II molecules. Alternative pathways of epitope production have been identified, but their contributions to host defense have not been established. We show here in a mouse infection model that the CD4+ T cell response to influenza, critical for durable protection from the virus, is driven principally by unconventional processing of antigen synthesized within the infected antigen-presenting cell, not by classical processing of endocytosed virions or material from infected cells. Investigation of the cellular components involved, including the H2-M molecular chaperone, the proteasome and γ-interferon-inducible lysosomal thiol reductase revealed considerable heterogeneity in the generation of individual epitopes, an arrangement that ensures peptide diversity and broad CD4+ T cell engagement. These results could fundamentally revise strategies for rational vaccine design and may lead to key insights into the induction of autoimmune and anti-tumor responses.

Virus-encoded ectopic CD74 enhances poxvirus vaccine efficacy.

Vaccinia virus (VV) has been used globally as a vaccine to eradicate smallpox. Widespread use of this viral vaccine has been tempered in recent years because of its immuno-evasive properties, with restrictions prohibiting VV inoculation of individuals with immune deficiencies or atopic skin diseases. VV infection is known to perturb several pathways for immune recognition including MHC class II (MHCII) and CD1d-restricted antigen presentation. MHCII and CD1d molecules associate with a conserved intracellular chaperone, CD74, also known as invariant chain. Upon VV infection, cellular CD74 levels are significantly reduced in antigen-presenting cells, consistent with the observed destabilization of MHCII molecules. In the current study, the ability of sustained CD74 expression to overcome VV-induced suppression of antigen presentation was investigated. Viral inhibition of MHCII antigen presentation could be partially ameliorated by ectopic expression of CD74 or by infection of cells with a recombinant VV encoding murine CD74 (mCD74-VV). In contrast, virus-induced disruptions in CD1d-mediated antigen presentation persisted even with sustained CD74 expression. Mice immunized with the recombinant mCD74-VV displayed greater protection during VV challenge and more robust anti-VV antibody responses. Together, these observations suggest that recombinant VV vaccines encoding CD74 may be useful tools to improve CD4⁺ T-cell responses to viral and tumour antigens.

Recombinant poxviruses: versatile tools for immunological assays.

The study of antigen processing and presentation is critical to our understanding of the mechanisms that govern immune surveillance. A typical requirement of assays designed to examine antigen processing and presentation is the de novo biosynthesis of a model antigen. Historically, Vaccinia virus (VACV), a poxvirus closely related to Cowpox, has enjoyed widespread use for this purpose. Recombinant poxvirus-based expression has a number of advantages over other systems. Poxviruses accommodate the insertion of large pieces of recombinant DNA into their genome, and recombination and selection are relatively efficient. Poxviruses readily infect a variety of cell types, and they drive rapid and high levels of antigen expression. Additionally, they can be utilized in a variety of assays to study both MHC class I-restricted and MHC class II-restricted antigen processing and presentation. Ultimately, the numerous advantages of poxvirus recombinants have made the Vaccinia expression system a mainstay in the study of processing and presentation over the past two decades. In an attempt to address one shortcoming of VACV while simultaneously retaining the benefits inherent to poxviruses, our laboratory has begun to engineer recombinant Ectromelia viruses. Ectromelia virus, or mousepox, is a natural pathogen of murine cells and performing experiments in the context of a natural host-pathogen relationship may elucidate unknown factors that influence epitope generation and host response. This chapter describes several recombinant poxvirus system protocols used to study both MHC class I and class II antigen processing and presentation, as well as provides insight and troubleshooting techniques to improve the reproducibility and fidelity of these experiments.

Perforin-dependent CD4+ T-cell cytotoxicity contributes to control a murine poxvirus infection.

CD4(+) T cells are generally regarded as helpers and regulators of the immune response. Although cytolytic CD4(+) T cells have been described, whether those generated during the course of a viral infection play a role in virus control remains unknown. Here we show that during acute infection with ectromelia virus, the mouse homolog of the human virus of smallpox, large numbers of CD4(+) T cells in the draining lymph node and liver of resistant mice have a cytotoxic phenotype. We also show that these cells kill targets in vivo in a perforin-dependent manner and that mice with specific deficiency of perforin in CD4(+) T cells have impaired virus control. Thus, perforin-dependent CD4(+) T-cell killing of infected cells is an important mechanism of antiviral defense.

Comparable polyfunctionality of ectromelia virus- and vaccinia virus-specific murine T cells despite markedly different in vivo replication and pathogenicity.

Vaccinia virus (VACV) stimulates long-term immunity against highly pathogenic orthopoxvirus infection of humans (smallpox) and mice (mousepox [ectromelia virus {ECTV}]) despite the lack of a natural host-pathogen relationship with either of these species. Previous research revealed that VACV is able to induce polyfunctional CD8(+) T-cell responses after immunization of humans. However, the degree to which the functional profile of T cells induced by VACV is similar to that generated during natural poxvirus infection remains unknown. In this study, we monitored virus-specific T-cell responses following the dermal infection of C57BL/6 mice with ECTV or VACV. Using polychromatic flow cytometry, we measured levels of degranulation, cytokine expression (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]), and the cytolytic mediator granzyme B. We observed that the functional capacities of T cells induced by VACV and ECTV were of a similar quality in spite of the markedly different replication abilities and pathogenic outcomes of these viruses. In general, a significant fraction (≥50%) of all T-cell responses were positive for at least three functions both during acute infection and into the memory phase. In vivo killing assays revealed that CD8(+) T cells specific for both viruses were equally cytolytic (∼80% target cell lysis after 4 h), consistent with the similar levels of granzyme B and degranulation detected among these cells. Collectively, these data provide a mechanism to explain the ability of VACV to induce protective T-cell responses against pathogenic poxviruses in their natural hosts and provide further support for the use of VACV as a vaccine platform able to induce polyfunctional T cells.