Regulatory CD4+ T Cells Recognize Major Histocompatibility Complex Class II Molecule-Restricted Peptide Epitopes of Apolipoprotein B.

BACKGROUND: CD4+ T cells play an important role in atherosclerosis, but their antigen specificity is poorly understood. Immunization with apolipoprotein B (ApoB, core protein of low density lipoprotein) is known to be atheroprotective in animal models. Here, we report on a human APOB peptide, p18, that is sequence-identical in mouse ApoB and binds to both mouse and human major histocompatibility complex class II molecules. METHODS: We constructed p18 tetramers to detect human and mouse APOB-specific T cells and assayed their phenotype by flow cytometry including CD4 lineage transcription factors, intracellular cytokines, and T cell receptor activation. Apolipoprotein E-deficient ( Apoe-/-) mice were vaccinated with p18 peptide or adjuvants alone, and atherosclerotic burden in the aorta was determined. RESULTS: In human peripheral blood mononuclear cells from donors without cardiovascular disease, p18 specific CD4+ T cells detected by a new human leukocyte antigen-antigen D related-p18 tetramers were mostly Foxp3+ regulatory T cells (Tregs). Donors with subclinical cardiovascular disease as detected by carotid artery ultrasound had Tregs coexpressing retinoic acid-related orphan receptor gamma t or T-bet, which were both almost absent in donors without cardiovascular disease. In Apoe-/- mice, immunization with p18 induced Tregs and reduced atherosclerotic lesions. After peptide restimulation, responding CD4+ T cells identified by Nur77-GFP (green fluorescent protein) were highly enriched in Tregs. A new mouse I-Ab-p18 tetramer identified the expansion of p18-specific CD4+ T cells on vaccination, which were enriched for interleukin-10-producing Tregs. CONCLUSIONS: These findings show that APOB p18-specific CD4+ T cells are mainly Tregs in healthy donors, but coexpress other CD4 lineage transcription factors in donors with subclinical cardiovascular disease. This study identifies ApoB peptide 18 as the first Treg epitope in human and mouse atherosclerosis.

Novel and shared neoantigen derived from histone 3 variant H3.3K27M mutation for glioma T cell therapy.

The median overall survival for children with diffuse intrinsic pontine glioma (DIPG) is less than one year. The majority of diffuse midline gliomas, including more than 70% of DIPGs, harbor an amino acid substitution from lysine (K) to methionine (M) at position 27 of histone 3 variant 3 (H3.3). From a CD8+ T cell clone established by stimulation of HLA-A2+ CD8+ T cells with synthetic peptide encompassing the H3.3K27M mutation, complementary DNA for T cell receptor (TCR) α- and ß-chains were cloned into a retroviral vector. TCR-transduced HLA-A2+ T cells efficiently killed HLA-A2+H3.3K27M+ glioma cells in an antigen- and HLA-specific manner. Adoptive transfer of TCR-transduced T cells significantly suppressed the progression of glioma xenografts in mice. Alanine-scanning assays suggested the absence of known human proteins sharing the key amino acid residues required for recognition by the TCR, suggesting that the TCR could be safely used in patients. These data provide us with a strong basis for developing T cell-based therapy targeting this shared neoepitope.

Immunodominance in allergic T-cell reactivity to Japanese cedar in different geographic cohorts.

BACKGROUND: Japanese cedar (JC) pollen is a common trigger for allergic rhinitis in Japan. Pollen proteins targeted by IgE, including Cry j 1 and Cry j 2, and isoflavone reductase (IFR) have been identified. OBJECTIVE: To compare antigen-specific IgE titers and T-cell responses to JC pollen-derived extract and peptides in cohorts with high and low pollen exposure. METHODS: Peripheral blood mononuclear cells from JC pollen allergic or nonallergic patients who have lived in Japan for at least 1 year and JC pollen allergic patients who have never been to Japan were tested for T-cell responses against JC pollen extract and peptide pools derived from Cry j 1, Cry j 2, or IFR. T-cell reactivity was assessed by interleukin 5 and interferon γ production by ELISPOT. RESULTS: JC pollen-specific T-cell reactivity and IgE titers were significantly higher in the allergic compared with the nonallergic Japanese cohort, which was also associated with different patterns of polysensitization. Interestingly, a significant overlap was observed in the hierarchy of the T-cell epitopes in the allergic Japanese cohort compared with the allergic non-Japanese cohort. In all 3 cohorts, T-cell reactivity was dominantly directed against peptides from the major allergens Cry j 1 and 2, with few T-cell responses detected against IFR. CONCLUSION: Our studies identify common denominators of T-cell reactivity in patient populations with different sensitization patterns, suggesting that generally applicable immunotherapeutic approaches might be developed irrespective of exposure modality.

TepiTool: A Pipeline for Computational Prediction of T Cell Epitope Candidates.

Computational prediction of T cell epitope candidates is currently being used in several applications including vaccine discovery studies, development of diagnostics, and removal of unwanted immune responses against protein therapeutics. There have been continuous improvements in the performance of MHC binding prediction tools, but their general adoption by immunologists has been slow due to the lack of user-friendly interfaces and guidelines. Current tools only provide minimal advice on what alleles to include, what lengths to consider, how to deal with homologous peptides, and what cutoffs should be considered relevant. This protocol provides step-by-step instructions with necessary recommendations for prediction of the best T cell epitope candidates with the newly developed online tool called TepiTool. TepiTool, which is part of the Immune Epitope Database (IEDB), provides some of the top MHC binding prediction algorithms for number of species including humans, chimpanzees, bovines, gorillas, macaques, mice, and pigs. The TepiTool is freely accessible at http://tools.iedb.org/tepitool/. © 2016 by John Wiley & Sons, Inc.

Previously undescribed grass pollen antigens are the major inducers of T helper 2 cytokine-producing T cells in allergic individuals.

T cells play an important role in the pathogenesis of allergic diseases. However, the proteins considered as potential immunogens of allergenic T-cell responses have traditionally been limited to those that induce IgE responses. Timothy grass (TG) pollen is a well-studied inhaled allergen for which major IgE-reactive allergens have also been shown to trigger T helper 2 (Th2) responses. Here we examined whether other TG pollen proteins are recognized by Th2 responses independently of IgE reactivity. A TG pollen extract was analyzed by 2D gel electrophoresis and IgE/IgG immunoblots using pooled sera from allergic donors. Mass spectrometry of selected protein spots in combination with de novo sequencing of the whole TG pollen transcriptome identified 93 previously undescribed proteins for further study, 64 of which were not targeted by IgE. Predicted MHC binding peptides from the previoulsy undescribed TG proteins were screened for T-cell reactivity in peripheral blood mononuclear cells from allergic donors. Strong IL-5 production was detected in response to peptides from several of the previously undescribed proteins, most of which were not targeted by IgE. Responses against the dominant undescribed epitopes were associated with the memory T-cell subset and could even be detected directly ex vivo after Th2 cell enrichment. These findings demonstrate that a combined unbiased transcriptomic, proteomic, and immunomic approach identifies a greatly broadened repertoire of protein antigens targeted by T cells involved in allergy pathogenesis. The discovery of proteins that induce Th2 cells but are not IgE reactive may allow the development of safer immunotherapeutic strategies.