Accurate prediction of HLA class II antigen presentation across all loci using tailored data acquisition and refined machine learning.

Accurate prediction of antigen presentation by human leukocyte antigen (HLA) class II molecules is crucial for rational development of immunotherapies and vaccines targeting CD4+ T cell activation. So far, most prediction methods for HLA class II antigen presentation have focused on HLA-DR because of limited availability of immunopeptidomics data for HLA-DQ and HLA-DP while not taking into account alternative peptide binding modes. We present an update to the NetMHCIIpan prediction method, which closes the performance gap between all three HLA class II loci. We accomplish this by first integrating large immunopeptidomics datasets describing the HLA class II specificity space across all loci using a refined machine learning framework that accommodates inverted peptide binders. Next, we apply targeted immunopeptidomics assays to generate data that covers additional HLA-DP specificities. The final method, NetMHCIIpan-4.3, achieves high accuracy and molecular coverage across all HLA class II allotypes.

Machine learning reveals limited contribution of trans-only encoded variants to the HLA-DQ immunopeptidome.

Human leukocyte antigen (HLA) class II antigen presentation is key for controlling and triggering T cell immune responses. HLA-DQ molecules, which are believed to play a major role in autoimmune diseases, are heterodimers that can be formed as both cis and trans variants depending on whether the α- and ß-chains are encoded on the same (cis) or opposite (trans) chromosomes. So far, limited progress has been made for predicting HLA-DQ antigen presentation. In addition, the contribution of trans-only variants (i.e. variants not observed in the population as cis) in shaping the HLA-DQ immunopeptidome remains largely unresolved. Here, we seek to address these issues by integrating state-of-the-art immunoinformatics data mining models with large volumes of high-quality HLA-DQ specific mass spectrometry immunopeptidomics data. The analysis demonstrates highly improved predictive power and molecular coverage for models trained including these novel HLA-DQ data. More importantly, investigating the role of trans-only HLA-DQ variants reveals a limited to no contribution to the overall HLA-DQ immunopeptidome. In conclusion, this study furthers our understanding of HLA-DQ specificities and casts light on the relative role of cis versus trans-only HLA-DQ variants in the HLA class II antigen presentation space. The developed method, NetMHCIIpan-4.2, is available at https://services.healthtech.dtu.dk/services/NetMHCIIpan-4.2 .

Subcellular location of source proteins improves prediction of neoantigens for immunotherapy.

Antigen presentation via the major histocompatibility complex (MHC) is essential for anti-tumor immunity. However, the rules that determine which tumor-derived peptides will be immunogenic are still incompletely understood. Here, we investigated whether constraints on peptide accessibility to the MHC due to protein subcellular location are associated with peptide immunogenicity potential. Analyzing over 380,000 peptides from studies of MHC presentation and peptide immunogenicity, we find clear spatial biases in both eluted and immunogenic peptides. We find that including parent protein location improves the prediction of peptide immunogenicity in multiple datasets. In human immunotherapy cohorts, the location was associated with a neoantigen vaccination response, and immune checkpoint blockade responders generally had a higher burden of neopeptides from accessible locations. We conclude that protein subcellular location adds important information for optimizing cancer immunotherapies.

Localized ablative immunotherapy drives de novo CD8+ T-cell responses to poorly immunogenic tumors.

BACKGROUND: Localized ablative immunotherapies hold great promise in stimulating antitumor immunity to treat metastatic and poorly immunogenic tumors. Tumor ablation is well known to release tumor antigens and danger-associated molecular patterns to stimulate T-cell immunity, but its immune stimulating effect is limited, particularly against metastatic tumors. METHODS: In this study, we combined photothermal therapy with a potent immune stimulant, N-dihydrogalactochitosan, to create a local ablative immunotherapy which we refer to as laser immunotherapy (LIT). Mice bearing B16-F10 tumors were treated with LIT when the tumors reached 0.5 cm3 and were monitored for survival, T-cell activation, and the ability to resist tumor rechallenge. RESULTS: We found that LIT stimulated a stronger and more consistent antitumor T-cell response to the immunologically 'cold' B16-F10 melanoma tumors and conferred a long-term antitumor memory on tumor rechallenge. Furthermore, we discovered that LIT generated de novo CD8+ T-cell responses that strongly correlated with animal survival and tumor rejection. CONCLUSION: In summary, our findings demonstrate that LIT enhances the activation of T cells and drives de novo antitumor T-cell responses. The data presented herein suggests that localized ablative immunotherapies have great potential to synergize with immune checkpoint therapies to enhance its efficacy, resulting in improved antitumor immunity.

Accurate MHC Motif Deconvolution of Immunopeptidomics Data Reveals a Significant Contribution of DRB3, 4 and 5 to the Total DR Immunopeptidome.

Mass spectrometry (MS) based immunopeptidomics is used in several biomedical applications including neo-epitope discovery in oncology, next-generation vaccine development and protein-drug immunogenicity assessment. Immunopeptidome data are highly complex given the expression of multiple HLA alleles on the cell membrane and presence of co-immunoprecipitated contaminants. The absence of tools that deal with these challenges effectively and guide the analysis and interpretation of this complex type of data is currently a major bottleneck for the large-scale application of this technique. To resolve this, we here present the MHCMotifDecon that benefits from state-of-the-art HLA class-I and class-II predictions to accurately deconvolute immunopeptidome datasets and assign individual ligands to the most likely HLA molecule, allowing to identify and characterize HLA binding motifs while discarding co-purified contaminants. We have benchmarked the tool against other state-of-the-art methods and illustrated its application on experimental datasets for HLA-DR demonstrating a previously underappreciated role for HLA-DRB3/4/5 molecules in defining HLA class II immune repertoires. With its ease of use, MHCMotifDecon can efficiently guide interpretation of immunopeptidome datasets, serving the discovery of novel T cell targets. MHCMotifDecon is available at https://services.healthtech.dtu.dk/service.php?MHCMotifDecon-1.0.

Improved Prediction of MHC II Antigen Presentation through Integration and Motif Deconvolution of Mass Spectrometry MHC Eluted Ligand Data.

Major histocompatibility complex II (MHC II) molecules play a vital role in the onset and control of cellular immunity. In a highly selective process, MHC II presents peptides derived from exogenous antigens on the surface of antigen-presenting cells for T cell scrutiny. Understanding the rules defining this presentation holds critical insights into the regulation and potential manipulation of the cellular immune system. Here, we apply the NNAlign_MA machine learning framework to analyze and integrate large-scale eluted MHC II ligand mass spectrometry (MS) data sets to advance prediction of CD4+ epitopes. NNAlign_MA allows integration of mixed data types, handling ligands with multiple potential allele annotations, encoding of ligand context, leveraging information between data sets, and has pan-specific power allowing accurate predictions outside the set of molecules included in the training data. Applying this framework, we identified accurate binding motifs of more than 50 MHC class II molecules described by MS data, particularly expanding coverage for DP and DQ beyond that obtained using current MS motif deconvolution techniques. Furthermore, in large-scale benchmarking, the final model termed NetMHCIIpan-4.0 demonstrated improved performance beyond current state-of-the-art predictors for ligand and CD4+ T cell epitope prediction. These results suggest that NNAlign_MA and NetMHCIIpan-4.0 are powerful tools for analysis of immunopeptidome MS data, prediction of T cell epitopes, and development of personalized immunotherapies.

Immunological ignorance is an enabling feature of the oligo-clonal T cell response to melanoma neoantigens.

The impact of intratumoral heterogeneity (ITH) and the resultant neoantigen landscape on T cell immunity are poorly understood. ITH is a widely recognized feature of solid tumors and poses distinct challenges related to the development of effective therapeutic strategies, including cancer neoantigen vaccines. Here, we performed deep targeted DNA sequencing of multiple metastases from melanoma patients and observed ubiquitous sharing of clonal and subclonal single nucleotide variants (SNVs) encoding putative HLA class I-restricted neoantigen epitopes. However, spontaneous antitumor CD8+ T cell immunity in peripheral blood and tumors was restricted to a few clonal neoantigens featuring an oligo-/monoclonal T cell-receptor (TCR) repertoire. Moreover, in various tumors of the 4 patients examined, no neoantigen-specific TCR clonotypes were identified despite clonal neoantigen expression. Mature dendritic cell (mDC) vaccination with tumor-encoded amino acid-substituted (AAS) peptides revealed diverse neoantigen-specific CD8+ T responses, each composed of multiple TCR clonotypes. Isolation of T cell clones by limiting dilution from tumor-infiltrating lymphocytes (TILs) permitted functional validation regarding neoantigen specificity. Gene transfer of TCRαß heterodimers specific for clonal neoantigens confirmed correct TCR clonotype assignments based on high-throughput TCRBV CDR3 sequencing. Our findings implicate immunological ignorance of clonal neoantigens as the basis for ineffective T cell immunity to melanoma and support the concept that therapeutic vaccination, as an adjunct to checkpoint inhibitor treatment, is required to increase the breadth and diversity of neoantigen-specific CD8+ T cells.

MHC-I Genotype Restricts the Oncogenic Mutational Landscape.

MHC-I molecules expose the intracellular protein content on the cell surface, allowing T cells to detect foreign or mutated peptides. The combination of six MHC-I alleles each individual carries defines the sub-peptidome that can be effectively presented. We applied this concept to human cancer, hypothesizing that oncogenic mutations could arise in gaps in personal MHC-I presentation. To validate this hypothesis, we developed and applied a residue-centric patient presentation score to 9,176 cancer patients across 1,018 recurrent oncogenic mutations. We found that patient MHC-I genotype-based scores could predict which mutations were more likely to emerge in their tumor. Accordingly, poor presentation of a mutation across patients was correlated with higher frequency among tumors. These results support that MHC-I genotype-restricted immunoediting during tumor formation shapes the landscape of oncogenic mutations observed in clinically diagnosed tumors and paves the way for predicting personal cancer susceptibilities from knowledge of MHC-I genotype.

Immunodominant West Nile Virus T Cell Epitopes Are Fewer in Number and Fashionably Late.

Class I HLA molecules mark infected cells for immune targeting by presenting pathogen-encoded peptides on the cell surface. Characterization of viral peptides unique to infected cells is important for understanding CD8(+) T cell responses and for the development of T cell-based immunotherapies. Having previously reported a series of West Nile virus (WNV) epitopes that are naturally presented by HLA-A*02:01, in this study we generated TCR mimic (TCRm) mAbs to three of these peptide/HLA complexes-the immunodominant SVG9 (E protein), the subdominant SLF9 (NS4B protein), and the immunorecessive YTM9 (NS3 protein)-and used these TCRm mAbs to stain WNV-infected cell lines and primary APCs. TCRm staining of WNV-infected cells demonstrated that the immunorecessive YTM9 appeared several hours earlier and at 5- to 10-fold greater density than the more immunogenic SLF9 and SVG9 ligands, respectively. Moreover, staining following inhibition of the TAP demonstrated that all three viral ligands were presented in a TAP-dependent manner despite originating from different cellular compartments. To our knowledge, this study represents the first use of TCRm mAbs to define the kinetics and magnitude of HLA presentation for a series of epitopes encoded by one virus, and the results depict a pattern whereby individual epitopes differ considerably in abundance and availability. The observations that immunodominant ligands can be found at lower levels and at later time points after infection suggest that a reevaluation of the factors that combine to shape T cell reactivity may be warranted.

Human Leukocyte Antigen-Presented Macrophage Migration Inhibitory Factor Is a Surface Biomarker and Potential Therapeutic Target for Ovarian Cancer.

T cells recognize cancer cells via HLA/peptide complexes, and when disease overtakes these immune mechanisms, immunotherapy can exogenously target these same HLA/peptide surface markers. We previously identified an HLA-A2-presented peptide derived from macrophage migration inhibitory factor (MIF) and generated antibody RL21A against this HLA-A2/MIF complex. The objective of the current study was to assess the potential for targeting the HLA-A2/MIF complex in ovarian cancer. First, MIF peptide FLSELTQQL was eluted from the HLA-A2 of the human cancerous ovarian cell lines SKOV3, A2780, OV90, and FHIOSE118hi and detected by mass spectrometry. By flow cytometry, RL21A was shown to specifically stain these four cell lines in the context of HLA-A2. Next, partially matched HLA-A*02:01+ ovarian cancer (n = 27) and normal fallopian tube (n = 24) tissues were stained with RL21A by immunohistochemistry to assess differential HLA-A2/MIF complex expression. Ovarian tumor tissues revealed significantly increased RL21A staining compared with normal fallopian tube epithelium (P < 0.0001), with minimal staining of normal stroma and blood vessels (P < 0.0001 and P < 0.001 compared with tumor cells) suggesting a therapeutic window. We then demonstrated the anticancer activity of toxin-bound RL21A via the dose-dependent killing of ovarian cancer cells. In summary, MIF-derived peptide FLSELTQQL is HLA-A2-presented and recognized by RL21A on ovarian cancer cell lines and patient tumor tissues, and targeting of this HLA-A2/MIF complex with toxin-bound RL21A can induce ovarian cancer cell death. These results suggest that the HLA-A2/MIF complex should be further explored as a cell-surface target for ovarian cancer immunotherapy.

Cancer immunotherapy. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells.

T cell immunity directed against tumor-encoded amino acid substitutions occurs in some melanoma patients. This implicates missense mutations as a source of patient-specific neoantigens. However, a systematic evaluation of these putative neoantigens as targets of antitumor immunity is lacking. Moreover, it remains unknown whether vaccination can augment such responses. We found that a dendritic cell vaccine led to an increase in naturally occurring neoantigen-specific immunity and revealed previously undetected human leukocyte antigen (HLA) class I-restricted neoantigens in patients with advanced melanoma. The presentation of neoantigens by HLA-A*02:01 in human melanoma was confirmed by mass spectrometry. Vaccination promoted a diverse neoantigen-specific T cell receptor (TCR) repertoire in terms of both TCR-ß usage and clonal composition. Our results demonstrate that vaccination directed at tumor-encoded amino acid substitutions broadens the antigenic breadth and clonal diversity of antitumor immunity.

A novel T-cell receptor mimic defines dendritic cells that present an immunodominant West Nile virus epitope in mice.

We used a newly generated T-cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10-mer) or SSVWNATTA (9-mer) in complex with class I MHC antigen H-2D(b) , were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8(+) T cells can detect 10- and 9-mer length variants similarly. This result varies from the idea that a given T-cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10-mer was presented on the surface of uninfected and infected CD8α(+) CD11c(+) dendritic cells, which suggests the use of direct and cross-presentation pathways. In contrast, CD11b(+) CD11c(-) cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell-type specific mechanisms of antigen presentation in vivo.

Identification of class I HLA T cell control epitopes for West Nile virus.

The recent West Nile virus (WNV) outbreak in the United States underscores the importance of understanding human immune responses to this pathogen. Via the presentation of viral peptide ligands at the cell surface, class I HLA mediate the T cell recognition and killing of WNV infected cells. At this time, there are two key unknowns in regards to understanding protective T cell immunity: 1) the number of viral ligands presented by the HLA of infected cells, and 2) the distribution of T cell responses to these available HLA/viral complexes. Here, comparative mass spectroscopy was applied to determine the number of WNV peptides presented by the HLA-A*11:01 of infected cells after which T cell responses to these HLA/WNV complexes were assessed. Six viral peptides derived from capsid, NS3, NS4b, and NS5 were presented. When T cells from infected individuals were tested for reactivity to these six viral ligands, polyfunctional T cells were focused on the GTL9 WNV capsid peptide, ligands from NS3, NS4b, and NS5 were less immunogenic, and two ligands were largely inert, demonstrating that class I HLA reduce the WNV polyprotein to a handful of immune targets and that polyfunctional T cells recognize infections by zeroing in on particular HLA/WNV epitopes. Such dominant HLA/peptide epitopes are poised to drive the development of WNV vaccines that elicit protective T cells as well as providing key antigens for immunoassays that establish correlates of viral immunity.