Delivery of loaded MR1 monomer results in efficient ligand exchange to host MR1 and subsequent MR1T cell activation.

MR1-restricted T cells have been implicated in microbial infections, sterile inflammation, wound healing and cancer. Similar to other antigen presentation molecules, evidence supports multiple, complementary MR1 antigen presentation pathways. To investigate ligand exchange pathways for MR1, we used MR1 monomers and tetramers loaded with 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) to deliver the antigen. Using MR1-deficient cells reconstituted with wild-type MR1 or MR1 molecules that cannot bind 5-OP-RU, we show that presentation of monomer-delivered 5-OP-RU is dependent on cellular MR1 and requires the transfer of ligand from the soluble molecule onto MR1 expressed by the antigen presenting cell. This mode of antigen delivery strengthens the evidence for post-ER ligand exchange pathways for MR1, which could represent an important avenue by which MR1 acquires antigens derived from endocytosed pathogens.

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.

A Kmer-based paired-end read de novo assembler and genotyper for canine MHC class I genotyping.

The major histocompatibility complex class I (MHC-I) genes are highly polymorphic. MHC-I genotyping is required for determining the peptide epitopes available to an individual's T-cell repertoire. Current genotyping software tools do not work for the dog, due to very limited known canine alleles. To address this, we developed a Kmer-based paired-end read (KPR) de novo assembler and genotyper, which assemble paired-end RNA-seq reads from MHC-I regions into contigs, and then genotype each contig and estimate its expression level. KPR tools outperform other popular software examined in typing new alleles. We used KPR tools to successfully genotype152 dogs from a published dataset. The study discovers 33 putative new alleles, finds dominant alleles in 4 dog breeds, and builds allele diversity and expression landscapes among the 152 dogs. Our software meets a significant need in biomedical research.

Deaza-modification of MR1 ligands modulates recognition by MR1-restricted T cells.

MR1-restricted T (MR1T) cells recognize microbial small molecule metabolites presented on the MHC Class I-like molecule MR1 and have been implicated in early effector responses to microbial infection. As a result, there is considerable interest in identifying chemical properties of metabolite ligands that permit recognition by MR1T cells, for consideration in therapeutic or vaccine applications. Here, we made chemical modifications to known MR1 ligands to evaluate the effect on MR1T cell activation. Specifically, we modified 6,7-dimethyl-8-D-ribityllumazine (DMRL) to generate 6,7-dimethyl-8-D-ribityldeazalumazine (DZ), and then further derivatized DZ to determine the requirements for retaining MR1 surface stabilization and agonistic properties. Interestingly, the IFN-γ response toward DZ varied widely across a panel of T cell receptor (TCR)-diverse MR1T cell clones; while one clone was agnostic toward the modification, most displayed either an enhancement or depletion of IFN-γ production when compared with its response to DMRL. To gain insight into a putative mechanism behind this phenomenon, we used in silico molecular docking techniques for DMRL and its derivatives and performed molecular dynamics simulations of the complexes. In assessing the dynamics of each ligand in the MR1 pocket, we found that DMRL and DZ exhibit differential dynamics of both the ribityl moiety and the aromatic backbone, which may contribute to ligand recognition. Together, our results support an emerging hypothesis for flexibility in MR1:ligand-MR1T TCR interactions and enable further exploration of the relationship between MR1:ligand structures and MR1T cell recognition for downstream applications targeting MR1T cells.

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.

Single-cell RNA sequencing reveals localized tumour ablation and intratumoural immunostimulant delivery potentiate T cell mediated tumour killing.

BACKGROUND: Metastatic breast cancer poses great challenge in cancer treatment. N-dihydrogalactochitosan (GC) is a novel immunoadjuvant that stimulates systemic immune responses when administered intratumourally following local tumour ablation. A combination of photothermal therapy (PTT) and GC, referred to as localized ablative immunotherapy (LAIT), extended animal survival and generates an activated B cell phenotype in MMTV-PyMT mouse mammary tumour microenvironment (TME). However, how T cell populations respond to LAIT remains to be elucidated. METHODS: Using depletion antibodies, we studied the contributions of CD8+ and CD4+ T cells to the therapeutic effect of LAIT. Using single-cell RNA-sequencing (scRNAseq), we analysed tumour-infiltrating T cell heterogeneity and dissected their transcriptomes upon treatments of PTT, GC, and LAIT (PTT+GC). RESULTS: Loss of CD8+ T cells after LAIT abrogated the therapeutic benefits of LAIT. Ten days after treatment, proportions of CD8+ and CD4+ T cells in untreated TME were 19.2% and 23.0%, respectively. Upon LAIT, both proportions were increased to 25.5% and 36.2%, respectively. In particular, LAIT increased the proportions of naïve and memory cells from a resting state to an activated state. LAIT consistently induced the expression of co-stimulatory molecules, type I IFN responsive genes, and a series of antitumor cytokines, Ifng, Tnf, Il1, and Il17 in CD8+ and CD4+ T cells. LAIT also induced immune checkpoints Pdcd1, Ctla4, and Lag3 expression, consistent with T cell activation. Relevant to clinical translation, LAIT also upregulated genes in CD8+ and CD4+ T cells that positively correlated with extended survival of breast cancer patients. CONCLUSIONS: Overall, our results reveal that LAIT prompts immunological remodelling of T cells by inducing broad proinflammatory responses and inhibiting suppressive signalling to drive antitumour immunity.

Major histocompatibility complex class I molecule expression by pancreatic cancer cells is regulated by activation and inhibition of the epidermal growth factor receptor.

Pancreatic cancer is one of the deadliest neoplasms, with a dismal 5-year survival rate of only 10%. The ability of pancreatic cancer cells to evade the immune system hinders an anti-tumor response and contributes to the poor survival rate. Downregulation of major histocompatibility complex (MHC) class I cell-surface expression can aid in immune evasion by preventing endogenous tumor antigens from being presented to cytotoxic T cells. Earlier studies suggested that epidermal growth factor receptor (EGFR) signaling can decrease MHC class I expression on certain cancer cell types. However, even though erlotinib (a tyrosine kinase inhibitor that targets EGFR) is an approved drug for advanced pancreatic cancer treatment, the impact of EGFR inhibition or stimulation on pancreatic cancer cell MHC class I surface expression has not previously been analyzed. In this current study, we discovered that EGFR affects MHC class I mRNA and protein expression by human pancreatic cancer cell lines. We demonstrated that cell-surface MHC class I expression is downregulated upon EGFR activation, and the MHC class I level at the surface is elevated following EGFR inhibition. Furthermore, we found that EGFR associates with MHC class I molecules. By defining a role in pancreatic cancer cells for activated EGFR in reducing MHC class I expression and by revealing that EGFR inhibitors can boost MHC class I expression, our work supports further investigation of combined usage of EGFR inhibitors with immunotherapies against pancreatic cancer.

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.

Antigen presentation and interferon signatures in B cells driven by localized ablative cancer immunotherapy correlate with extended survival.

Rationale: B cells have emerged as key regulators in protective cancer immunity. However, the activation pathways induced in B cells during effective immunotherapy are not well understood. Methods: We used a novel localized ablative immunotherapy (LAIT), combining photothermal therapy (PTT) with intra-tumor delivery of the immunostimulant N-dihydrogalactochitosan (GC), to treat mice bearing mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT). We used single-cell RNA sequencing to compare the transcriptional changes induced by PTT, GC and PTT+GC in B cells within the tumor microenvironment (TME). Results: LAIT significantly increased survival in the tumor-bearing mice, compared to the treatment by PTT and GC alone. We found that PTT, GC and PTT+GC increased the proportion of tumor-infiltrating B cells and induced gene expression signatures associated with B cell activation. Both GC and PTT+GC elevated gene expression associated with antigen presentation, whereas GC elevated transcripts that regulate B cell activation and GTPase function and PTT+GC induced interferon response genes. Trajectory analysis, where B cells were organized according to pseudotime progression, revealed that both GC and PTT+GC induced the differentiation of B cells from a resting state towards an effector phenotype. The analyses confirmed upregulated interferon signatures in the differentiated tumor-infiltrating B cells following treatment by PTT+GC but not by GC. We also observed that breast cancer patients had significantly longer survival time if they had elevated expression of genes in B cells that were induced by PTT+GC therapy in the mouse tumors. Conclusion: Our findings show that the combination of local ablation and local application of immunostimulant initiates the activation of interferon signatures and antigen-presentation in B cells which is associated with positive clinical outcomes for breast cancer. These findings broaden our understanding of LAIT's regulatory roles in remodeling TME and shed light on the potentials of B cell activation in clinical applications.

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.

A pipeline for identification and validation of tumor-specific antigens in a mouse model of metastatic breast cancer.

Cancer immunotherapy continues to make headway as a treatment for advanced stage tumors, revealing an urgent need to understand the fundamentals of anti-tumor immune responses. Noteworthy is a scarcity of data pertaining to the breadth and specificity of tumor-specific T cell responses in metastatic breast cancer. Autochthonous transgenic models of breast cancer display spontaneous metastasis in the FVB/NJ mouse strain, yet a lack of knowledge regarding tumor-bound MHC/peptide immune epitopes in this mouse model limits the characterization of tumor-specific T cell responses, and the mechanisms that regulate T cell responses in the metastatic setting. We recently generated the NetH2pan prediction tool for murine class I MHC ligands by building an FVB/NJ H-2q ligand database and combining it with public information from six other murine MHC alleles. Here, we deployed NetH2pan in combination with an advanced proteomics workflow to identify immunogenic T cell epitopes in the MMTV-PyMT transgenic model for metastatic breast cancer. Five unique MHC I/PyMT epitopes were identified. These tumor-specific epitopes were confirmed to be presented by the class I MHC of primary MMTV-PyMT tumors and their T cell immunogenicity was validated. Vaccination using a DNA construct encoding a truncated PyMT protein generated CD8 + T cell responses to these MHC class I/peptide complexes and prevented tumor development. In sum, we have established an MHC-ligand discovery pipeline in FVB/NJ mice, identified and tracked H-2Dq/PyMT neoantigen-specific T cells, and developed a vaccine that prevents tumor development in this metastatic model of breast cancer.

Alterations in the HLA-B*57:01 Immunopeptidome by Flucloxacillin and Immunogenicity of Drug-Haptenated Peptides.

Neoantigen formation due to the interaction of drug molecules with human leukocyte antigen (HLA)-peptide complexes can lead to severe hypersensitivity reactions. Flucloxacillin (FLX), a ß-lactam antibiotic for narrow-spectrum gram-positive bacterial infections, has been associated with severe immune-mediated drug-induced liver injury caused by an influx of T-lymphocytes targeting liver cells potentially recognizing drug-haptenated peptides in the context of HLA-B*57:01. To identify immunopeptidome changes that could lead to drug-driven immunogenicity, we used mass spectrometry to characterize the proteome and immunopeptidome of B-lymphoblastoid cells solely expressing HLA-B*57:01 as MHC-I molecules. Selected drug-conjugated peptides identified in these cells were synthesized and tested for their immunogenicity in HLA-B*57:01-transgenic mice. T cell responses were evaluated in vitro by immune assays. The immunopeptidome of FLX-treated cells was more diverse than that of untreated cells, enriched with peptides containing carboxy-terminal tryptophan and FLX-haptenated lysine residues on peptides. Selected FLX-modified peptides with drug on P4 and P6 induced drug-specific CD8+ T cells in vivo. FLX was also found directly linked to the HLA K146 that could interfere with KIR-3DL or peptide interactions. These studies identify a novel effect of antibiotics to alter anchor residue frequencies in HLA-presented peptides which may impact drug-induced inflammation. Covalent FLX-modified lysines on peptides mapped drug-specific immunogenicity primarily at P4 and P6 suggesting these peptide sites as drivers of off-target adverse reactions mediated by FLX. FLX modifications on HLA-B*57:01-exposed lysines may also impact interactions with KIR or TCR and subsequent NK and T cell function.

Determinants governing T cell receptor α/ß-chain pairing in repertoire formation of identical twins.

The T cell repertoire in each individual includes T cell receptors (TCRs) of enormous sequence diversity through the pairing of diverse TCR α- and ß-chains, each generated by somatic recombination of paralogous gene segments. Whether the TCR repertoire contributes to susceptibility to infectious or autoimmune diseases in concert with disease-associated major histocompatibility complex (MHC) polymorphisms is unknown. Due to a lack in high-throughput technologies to sequence TCR α-ß pairs, current studies on whether the TCR repertoire is shaped by host genetics have so far relied only on single-chain analysis. Using a high-throughput single T cell sequencing technology, we obtained the largest paired TCRαß dataset so far, comprising 965,523 clonotypes from 15 healthy individuals including 6 monozygotic twin pairs. Public TCR α- and, to a lesser extent, TCR ß-chain sequences were common in all individuals. In contrast, sharing of entirely identical TCRαß amino acid sequences was very infrequent in unrelated individuals, but highly increased in twins, in particular in CD4 memory T cells. Based on nucleotide sequence identity, a subset of these shared clonotypes appeared to be the progeny of T cells that had been generated during fetal development and had persisted for more than 50 y. Additional shared TCRαß in twins were encoded by different nucleotide sequences, implying that genetic determinants impose structural constraints on thymic selection that favor the selection of TCR α-ß pairs with entire sequence identities.

Defects in Antiviral T Cell Responses Inflicted by Aging-Associated miR-181a Deficiency.

Generation of protective immunity to infections and vaccinations declines with age. Studies in healthy individuals have implicated reduced miR-181a expression in T cells as contributing to this defect. To understand the impact of miR-181a expression on antiviral responses, we examined LCMV infection in mice with miR-181ab1-deficient T cells. We found that miR-181a deficiency delays viral clearance, thereby biasing the immune response in favor of CD4 over CD8 T cells. Antigen-specific CD4 T cells in mice with miR-181a-deficient T cells expand more and have a broader TCR repertoire with preferential expansion of high-affinity T cells than in wild-type mice. Importantly, generation of antigen-specific miR-181a-deficient CD8 effector T cells is particularly impaired, resulting in lower frequencies of CD8 T cells in the liver even at time points when the infection has been cleared. Consistent with the mouse model, CD4 memory T cells in individuals infected with West Nile virus at older ages tend to be more frequent and of higher affinity.

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.

Precision mouse models with expanded tropism for human pathogens.

A major limitation of current humanized mouse models is that they primarily enable the analysis of human-specific pathogens that infect hematopoietic cells. However, most human pathogens target other cell types, including epithelial, endothelial and mesenchymal cells. Here, we show that implantation of human lung tissue, which contains up to 40 cell types, including nonhematopoietic cells, into immunodeficient mice (lung-only mice) resulted in the development of a highly vascularized lung implant. We demonstrate that emerging and clinically relevant human pathogens such as Middle East respiratory syndrome coronavirus, Zika virus, respiratory syncytial virus and cytomegalovirus replicate in vivo in these lung implants. When incorporated into bone marrow/liver/thymus humanized mice, lung implants are repopulated with autologous human hematopoietic cells. We show robust antigen-specific humoral and T-cell responses following cytomegalovirus infection that control virus replication. Lung-only mice and bone marrow/liver/thymus-lung humanized mice substantially increase the number of human pathogens that can be studied in vivo, facilitating the in vivo testing of therapeutics.

Variability in German Cockroach Extract Composition Greatly Impacts T Cell Potency in Cockroach-Allergic Donors.

German cockroach extract is used clinically to evaluate allergen-specific sensitization and for subcutaneous allergen-specific immunotherapy, though there are no guidelines for standardization in its manufacture. We performed an immunological evaluation of 12 different cockroach extracts prepared from different sources and their potency to induce allergen-specific T cell reactivity. PBMC from 13 cockroach allergic donors were expanded in vitro with 12 different German cockroach extracts. After culture expansion, cells were re-stimulated with the different extracts and T cell responses were assessed by FluoroSpot (IL-5, IFNγ and IL-10 production). In parallel to the extracts, single allergen peptide pools for allergens from groups 1, 2, 4, 5, and 11 were tested to determine allergen immunodominance. Furthermore, to assess allergy specificity, PBMC from 13 non-allergic donors were also tested with the most potent extract and T cell responses were compared to the allergic cohort. Dramatic variations in T cell reactivity were observed to the different cockroach extract batches. Response magnitudes varied over 3 logs within a single donor. IL-5 production in the allergic cohort was significantly higher compared to the non-allergic cohort (p=0.004). Allergen content determination by ELISA detected much lower concentrations of Bla g 5 compared to Bla g 1 and 2. Mass spectrometric analysis revealed that Bla g 5 was present in similar amounts to Bla g 1 and 2 in extracts made from whole body, whereas it was not detected in extracts made from fecal matter, suggesting that Bla g 5 is not excreted into feces. Different donors exhibit different response patterns to different extracts, potentially dependent on the donor-specific T cell allergen immunodominance pattern and the allergen content of the extract tested. These findings have dramatic implications for the selection of potent extracts used for diagnostic purposes or allergen-specific immunotherapy.

Role of MAIT cells in pulmonary bacterial infection.

Mucosal-associated invariant T (MAIT) cells represent a population of innate T cells that is highly abundant in humans. MAIT cells recognize metabolites of the microbial vitamin B pathway that are presented by the major histocompatibility complex (MHC) class I-related protein MR1. Upon bacterial infection, activated MAIT cells produce diverse cytokines and cytotoxic effector molecules and accumulate at the site of infection, thus, MAIT cells have been shown to be protective against various bacterial infections. Here, we summarize the current knowledge of the role of MAIT cells in bacterial pulmonary infection models.

Urinary Peptides As a Novel Source of T Cell Allergen Epitopes.

Mouse allergy in both laboratory workers and in inner-city children is associated with allergic rhinitis and asthma, posing a serious public health concern. Urine is a major source of mouse allergens, as mice spray urine onto their surroundings, where the proteins dry up and become airborne on dust particles. Here, we tested whether oligopeptides that are abundant in mouse urine may contribute to mouse allergic T cell response. Over 1,300 distinct oligopeptides were detected by mass spectrometry analysis of the low molecular weight filtrate fraction of mouse urine (LoMo). Posttranslationally modified peptides were common, accounting for almost half of total peptides. A pool consisting of 225 unique oligopeptides of 13 residues or more in size identified within was tested for its capacity to elicit T cell reactivity in mouse allergic donors. Following 14-day in vitro stimulation of PBMCs, we detected responses in about 95% of donors tested, directed against 116 distinct peptides, predominantly associated with Th2 cytokines (IL-5). Peptides from non-urine related proteins such as epidermal growth factor, collagen, and Beta-globin accounted for the highest response (15.9, 9.1, and 8.1% of the total response, respectively). Peptides derived from major urinary proteins (MUPs), kidney androgen-regulated protein (KAP), and uromodulin were the main T cell targets from kidney or urine related sources. Further ex vivo analysis of enrichment of 4-1BB expressing cells demonstrated that LoMo pool-specific T cell reactivity can be detected directly ex vivo in mouse allergic but not in non-allergic donors. Further cytometric analysis of responding cells revealed a bone fide memory T cell phenotype and confirmed their Th2 polarization. Overall, these data suggest that mouse urine-derived oligopeptides are a novel target for mouse allergy-associated T cell responses, which may contribute to immunopathological mechanisms in mouse allergy.