peptide-MHC Class Research Articles

Rab11a Regulates MHC Class II Recycling and Promotes Antigenic Peptide Exchange in Recycling Endosomes of Mature Dendritic Cells

Abstract Dendritic cells (DCs) specialize in antigen capture and maintain a highly dynamic pool of peptide-MHC class II complexes (MHC-II) that continuously internalized and degraded in immature DCs. By contrast, in mature DCs pMHC-II internalizes but is efficiently recycled back to the plasma membrane for recognition by CD4 T cells. Regulation of pMHC-II biosynthesis and degradation is essential for optimal DC function, however the molecular basis of pMHC-II recycling and the cellular machinery that orchestrates pMHC-II trafficking are incompletely understood. In the present study we found that internalized pMHC-II on mature DCs, but not immature DCs, colocalizes with the regulator of endosome recycling Rab11a. Using a lentivirus encoded shRNA, we show that Rab11a knock-down inhibits recycling of pMHC-II in mature DCs. Moreover, Rab11a knock-down results in the intracellular accumulation of internalized surface pMHC-II. We also show that Rab11a knock-down in mature DCs specifically inhibits presentation of a “recycling endosome” epitope of internalized influenza hemagglutinin only in mature DCs. Our study thus identifies Rab11a as an important regulator of pMHC-II recycling that can enhance presentation of certain antigenic peptides in mature DCs.

Antigen Processing in the Endoplasmic Reticulum Is Monitored by Semi-Invariant αβ TCRs Specific for a Conserved Peptide-Qa-1b MHC Class Ib Ligand.

Ag processing in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP) is central to presentation of a normal peptide-MHC class I (MHC I) repertoire. Alternations in ERAAP function cause dramatic changes in the MHC I-presented peptides, which elicit potent immune responses. An unusual subset of CD8+ T cells monitor normal Ag processing by responding to a highly conserved FL9 peptide that is presented by Qa-1b, a nonclassical MHC Ib molecule (QFL) in ERAAP-deficient cells. To understand the structural basis for recognition of the conserved ligand, we analyzed the αβ TCRs of QFL-specific T cells. Individual cells in normal wild-type and TCRβ-transgenic mice were assessed for QFL-specific TCR α- and β-chains. The QFL-specific cells expressed a predominant semi-invariant TCR generated by DNA rearrangement of TRAV9d-3-TRAJ21 α-chain and TRBV5-TRBD1-TRBJ2-7 β-chain gene segments. Furthermore, the CDR3 regions of the α- as well as β-chains were required for QFL ligand recognition. Thus, the αβ TCRs used to recognize the peptide-Qa-1 ligand presented by ERAAP-deficient cells are semi-invariant and likely reflect a conserved mechanism for monitoring the fidelity of Ag processing in the ER.

Trogocytosis of peptide–MHC class II complexes from dendritic cells confers antigen-presenting ability on basophils

Th2 immunity plays important roles in both protective and allergic responses. Nevertheless, the nature of antigen-presenting cells responsible for Th2 cell differentiation remains ill-defined compared with the nature of the cells responsible for Th1 and Th17 cell differentiation. Basophils have attracted attention as a producer of Th2-inducing cytokine IL-4, whereas their MHC class II (MHC-II) expression and function as antigen-presenting cells are matters of considerable controversy. Here we revisited the MHC-II expression on basophils and explored its functional relevance in Th2 cell differentiation. Basophils generated in vitro from bone marrow cells in culture with IL-3 plus GM-CSF displayed MHC-II on the cell surface, whereas those generated in culture with IL-3 alone did not. Of note, these MHC-II-expressing basophils showed little or no transcription of the corresponding MHC-II gene. The GM-CSF addition to culture expanded dendritic cells (DCs) other than basophils. Coculture of basophils and DCs revealed that basophils acquired peptide-MHC-II complexes from DCs via cell contact-dependent trogocytosis. The acquired complexes, together with CD86, enabled basophils to stimulate peptide-specific T cells, leading to their proliferation and IL-4 production, indicating that basophils can function as antigen-presenting cells for Th2 cell differentiation. Transfer of MHC-II from DCs to basophils was also detected in draining lymph nodes of mice with atopic dermatitis-like skin inflammation. Thus, the present study defined the mechanism by which basophils display MHC-II on the cell surface and appears to reconcile some discrepancies observed in previous studies.

Article Recommendations from Our Deputy and Senior Editors

Image: Dendritic cells from Broz ML & Krummel MF. Cancer Immunol Res 2015; 3:313-19Cross-presentation of tumor antigens is key to the activation of T cells by dendritic cells. Siglec-G was found to interfere with peptide–MHC class I complex formation through inhibition of NOX2-mediated phagosome alkalinization, a condition conducive to optimal peptide loading.Ding Y,…, Cao X. Nature Immunol 2016 Oct;17:1167–75.Image: https://commons.wikimedia.org/wiki/File:Non-small_cell_lung_carcinoma_--_high_mag.jpgThe Keynote-024 trial was designed to compare the mAb pembrolizumab with chemotherapies as first-line treatments for NSCLC. Patients, preselected to have >50% PD-L1+ tumors, survived longer and with fewer serious adverse effects with pembrolizumab than chemotherapy.Reck M,…, Brahmer JR, M.D., for the KEYNOTE-024 Investigators. NEJM 2016 Oct 9; doi:10.1056/NEJMoa1606774.Image: Photo by David Dixon http://www.geograph.org.uk/photo/3492475Melanomas responsive to anti–CTLA-4 blockade are sensitive to IFNγ. IFNGR1-knockdown tumors in mice, and tumors from nonresponder patients, jump this hurdle by deleting or accumulating mutations in their IFNγ response pathways, which promotes tumor survival despite CTLA-4 blockade. This may represent a common mechanism for resistance to checkpoint blockade therapy.Gao J, …, Sharma P. Cell 2016 Oct 6;167:397–404.Image: https://ghr.nlm.nih.gov/art/large/squamous-cell-carcinoma.jpegPatients with HNSCC with tumors resistant to standard platinum-based treatment were randomized to receive anti-PD-1 (nivolumab) or another standard therapy. Nivolumab significantly prolonged survival, PD-L1 status notwithstanding, compared with standard therapies, and patients experienced fewer serious adverse effects.Ferris RL,…, Gillison ML. NEJM 2016 Oct 9; doi: 10.1056/NEJMoa1602252.Image: Türeci, Ö,…, Sahin U. Clin Can Res 2016; 22: 1885–96The proteasome cleaves proteins into peptides presented by class I on the cell surface. About a third of the HLA peptidome was found to consist of spliced-together peptides, creating new epitopes and a mechanism for presenting a broader spectrum of epitopes relevant to immunobiology and autoimmunity that are not accounted for in current prediction algorithms.Liepe J,…, Mishto M. Science 2016 Oct 21;354:354–8.Image: https://commons.wikimedia.org/wiki/File:Myeloblast_with_Auer_rod_smear_2010-01-27.JPGNK memory cells were tested as antitumor therapeutics in vitro and in vivo in a xenografted mouse model. These cells were effective cancer cell killers and also proved safe and promisingly effective in a small phase I clinical trial.Romee R,…, Fehniger TA. Science Transla Med 2016 Sept 21; 8:357ra123.

Highlights of This Issue

RNA interference (RNAi) technology has enabled the inhibition of previously undruggable targets at the mRNA level. Whereas clinical applications of RNAi have proven to be most successful for targeting mRNAs in the normal liver, drug delivery challenges have hindered its application in oncology. Ganesh and colleagues have identified and characterized potent tumor-selective RNAi nanoparticles, capable of silencing β-catenin mRNA and protein in both primary and metastatic tumors. Demonstration of strong efficacy against Wnt-dependent colorectal and hepatocellular tumors suggest a good opportunity for clinical development for this novel class of oligonucleotide-based therapeutics.Disrupting microtubule function represents a promising approach to treat glioblastoma multiforme (GBM), a devastating and intractable subtype of cancer, but antitubulin agents suffer from poor brain penetrance. Cherry and colleagues report the identification of a new brain-penetrant antitubulin agent, ST-11, that destabilizes microtubules, promotes chromosomal lingering that disrupts mitosis, and activates apoptosis in GBM cells. ST-11 reaches the brain after peripheral injection and dose-dependently reduces tumor burden in an orthotopic GBM mouse model without producing overt side effects. These findings indicate that ST-11 represents a promising scaffold to develop novel therapeutic modalities to treat GBM.Mimicking a viral infection can increase the antigenicity of tumor cells. Peptide–MHC class I complexes flag infected cells for their elimination by CD8 T effector cells. Technical flaws have hampered the idea of antibody-mediated delivery of peptide–MHC class I complexes to tumor cells for a long time. Schmittnaegel and colleagues overcame these technical issues with a new class of peptide MHC class I–fused antibodies named pMHC-IgGs. The new format can be expressed as a full immunoglobulin fusion and can be designed as mono- or bivalent antibodies and enable advancement of the concept towards clinical testing.Antiangiogenic therapy is a promising option for the treatment of glioma and other solid tumors, but resistance remains a formidable challenge. Park and colleagues discovered POSTN is responsible for acquired resistance to antiangiogenic therapy in glioma stem-like cells. Resistance to antiangiogenic therapy regulated by POSTN was associated with increased expression of mesenchymal markers. The combination of antiangiogenic therapy with downregulated expression of POSTN by shRNA dramatically reduced resistance-signaling and tumor invasion but extended overall animal survival significantly. These findings provide important basis for the development of the treatment for glioblastoma patients and identify POSTN as a novel therapeutic target in glioblastoma.

A New Class of Bifunctional Major Histocompatibility Class I Antibody Fusion Molecules to Redirect CD8 T Cells.

Bifunctional antibody fusion proteins engaging effector T cells for targeted elimination of tumor cells via CD3 binding have shown efficacy in both preclinical and clinical studies. Different from such a polyclonal T-cell recruitment, an alternative concept is to engage only antigen-specific T-cell subsets. Recruitment of specific subsets of T cells may be as potent but potentially lead to fewer side effects. Tumor-targeted peptide-MHC class I complexes (pMHCI-IgGs) bearing known antigenic peptides complexed with MHC class I molecules mark tumor cells as antigenic and utilize the physiologic way to interact with and activate T-cell receptors. If, for example, virus-specific CD8(+) T cells are addressed, the associated strong antigenicity and tight immune surveillance of the effector cells could lead to efficacious antitumor treatment in various tissues. However, peptide-MHC class I fusions are difficult to express recombinantly, especially when fused to entire antibody molecules. Consequently, current formats are largely limited to small antibody fragment fusions expressed in bacteria followed by refolding or chemical conjugation. Here, we describe a new molecular format bearing a single pMHCI complex per IgG fusion molecule characterized by enhanced stability and expression yields. This molecular format can be expressed in a full immunoglobulin format and can be designed as mono- or bivalent antibody binders. Mol Cancer Ther; 15(9); 2130-42. ©2016 AACR.

Pan-Specific Prediction of Peptide-MHC Class I Complex Stability, a Correlate of T Cell Immunogenicity.

Binding of peptides to MHC class I (MHC-I) molecules is the most selective event in the processing and presentation of Ags to CTL, and insights into the mechanisms that govern peptide-MHC-I binding should facilitate our understanding of CTL biology. Peptide-MHC-I interactions have traditionally been quantified by the strength of the interaction, that is, the binding affinity, yet it has been shown that the stability of the peptide-MHC-I complex is a better correlate of immunogenicity compared with binding affinity. In this study, we have experimentally analyzed peptide-MHC-I complex stability of a large panel of human MHC-I allotypes and generated a body of data sufficient to develop a neural network-based pan-specific predictor of peptide-MHC-I complex stability. Integrating the neural network predictors of peptide-MHC-I complex stability with state-of-the-art predictors of peptide-MHC-I binding is shown to significantly improve the prediction of CTL epitopes. The method is publicly available at http://www.cbs.dtu.dk/services/NetMHCstabpan.

A triad of molecular regions contribute to the formation of two distinct MHC class II conformers.

MHC class II molecules present antigen-derived peptides to CD4T cells to drive the adaptive immune response. Previous work has established that class II αβ dimers can adopt two distinct conformations, driven by the differential pairing of transmembrane domain GxxxG dimerization motifs. These class II conformers differ in their ability to be loaded with antigen-derived peptide and to effectively engage CD4T cells. Motif 1 (M1) paired I-A(k) class II molecules are efficiently loaded with peptides derived from the processing of B cell receptor-bound antigen, have unique B cell signaling properties and high T cell stimulation activity. The 11-5.2mAb selectively binds M1 paired I-A(k) class II molecules. However, the molecular determinants of 11-5.2 binding are currently unclear. Here, we report the ability of a human class II transmembrane domain to drive both M1 and M2 class II conformer formation. Protease sensitivity analysis further strengthens the idea that there are conformational differences between the extracellular domains of M1 and M2 paired class II. Finally, MHC class II chain alignments and site directed mutagenesis reveals a triad of molecular regions that contributes to 11-5.2mAb binding. In addition to transmembrane GxxxG motif domain pairing, 11-5.2 binding is influenced directly by α chain residue Glu-71 and indirectly by the region around the inter-chain salt bridge formed by α chain Arg-52 and β chain Glu-86. These findings provide insight into the complexity of 11-5.2mAb recognition of the M1 paired I-A(k) class II conformer and further highlight the molecular heterogeneity of peptide-MHC class II complexes that drive T cell antigen recognition.

Toll-like receptor 9 signaling antagonizes the B cell receptor-dependent ability of B cells to process and present antigen to helper T cells.

Abstract B cells express both an adaptive antigen-specific B cell receptor (BCR) and innate Toll-like receptors (TLRs) allowing the functional outcome of the B cell’s engagement with antigen to be modulated in response to pathogen-derived TLR ligands. In T cell-dependent responses, the BCR both signals for B cell proliferation and differentiation and also internalizes bound antigen for processing and presentation to helper T cells. Here we show that although the presence of the TLR9 ligand, CpG, does not affect early BCR mediated events following antigen binding including phosphorylation of key components of the signaling cascade and the formation of BCR clusters on cell surface, CpG alters the outcome of BCR signaling, resulting in unique transcriptional profiles, enhanced proliferation and differentiation to antibody secreting cells. In contrast, CpG dramatically limits the ability of B cells to process and present antigen to helper T cells. In the presence of CpG, BCR-induced upregulation of CD86 and MHC class II expressions are antagonized and antigen internalized by the BCR is not properly trafficked to antigen processing compartments resulting in reduced numbers of peptide-MHC class II complexes on the B cell surface. Indeed, CpG treated antigen-specific B cells show a reduced ability to maintain contact with antigen-specific T cells and to activate antigen-specific helper T cell proliferation in vitro. These results suggest that TLR9 activation in T cell dependent responses would drive B cell toward proliferation and antibody secretion and away from events that are highly dependent on the ability of B cells to present antigen to helper T cells, such as entry into germinal centers and affinity selection.

Internalization of T-Cell Receptor Mimic Antibody RL6A by Brain Derived Endothelial Cells Analyzed by Flow Cytometry

Abstract T-cell receptor mimic monoclonal antibodies (TCRm) recognize peptide-MHC class I complexes. They combine the specificity of T-cell receptors with high binding affinities in the low nanomolar to picomolar range characteristic of monoclonal antibodies. The TCRm RL6A targets YLLPAIVHI peptide-HLA-A2 complexes, where the peptide is derived from the p68 RNA helicase protein. Previous work showed that this target is expressed by brain endothelial cells forming the blood-brain barrier (BBB). Upon binding, RL6A crosses the BBB via a receptor-mediated transcytosis mechanism. Confocal microscopy indicated that, after internalization, RL6A co-localized with the early endosomal marker (EEA1). Here we used conventional flow cytometry and imaging flow cytometry to quantitatively analyze the internalization process. Endothelial cell monolayers formed by hCMEC/D3 cells were incubated with unlabeled RL6A for time periods up to 120 min. Surface bound RL6A was detected by a secondary, PE-labeled antibody. RL6A disappeared in a biphasic pattern from the surface of hCMEC/D3 cells, with an initial half-life (t1/2) of internalization of about 3 min and a second, much slower phase (half life about 3 h) likely reflecting recycling to the membrane. In a complementary approach using imaging flow cytometry (Amnis ImagestreamX) and IDEAS analysis software, the internalization of AlexaFluor488-RL6A could be directly visualized and evaluated. The results confirmed that RL6A internalizes rapidly into hCMEC/D3 cells. In conclusion, flow cytometry is a suitable tool to analyze trafficking of specific peptide-HLA complexes in endothelial cells.

Oxidized lipid bodies block antigen cross-presentation in cancer by preventing trafficking of peptide-MHC class I complexes

Abstract Background Antigen cross presentation is a unique feature of dendritic cells (DCs) and plays an important role in the development of specific CD8+ T cell responses. It is established that the defect in cross-presentation by dendritic cells (DCs) in cancer results in ineffective antitumor immune responses and favors tumor escape. However, the mechanisms behind this phenomenon are unclear. Results In this study we found that tumor-derived factors impaired cross-presentation by differentiated conventional DCs without affecting expression of MHC class I, class II, or costimulatory molecules. The ability of DCs to stimulate antigen-specific T cells after loading with short peptide (directly bound to MHC class I) was fully preserved. We found that tumor-derived factors blocked trafficking of peptide-MHC class I (pMHC) complexes to the cell surface. These complexes were found to be entrapped into the lysosomes. This effect was caused by the accumulation of lipid bodies (LB) containing oxidized (ox) triglycerides (TAG) in DCs. Importantly LB containing non-oxidized TAG did not affect cross-presentation. Further experiments have identified the specific molecular mechanism responsible for oxTAG containing LB mediated block of cross-presentation in DCs. Conclusions Our results suggest a novel mechanism of regulation of cross-presentation in cancer

Fine-tuning of CD8(+) T-cell effector functions by targeting the 2B4-CD48 interaction.

Polyfunctionality and cytotoxic activity dictate CD8(+) T-cell efficacy in the eradication of infected and malignant cells. The induction of these effector functions depends on the specific interaction between the T-cell receptor (TCR) and its cognate peptide-MHC class I complex, in addition to signals provided by co-stimulatory or co-inhibitory receptors, which can further regulate these functions. Among these receptors, the role of 2B4 is contested, as it has been described as either co-stimulatory or co-inhibitory in modulating T-cell functions. We therefore combined functional, transcriptional and epigenetic approaches to further characterize the impact of disrupting the interaction of 2B4 with its ligand CD48, on the activity of human effector CD8(+) T-cell clones. In this setting, we show that the 2B4-CD48 axis is involved in the fine-tuning of CD8(+) T-cell effector function upon antigenic stimulation. Blocking this interaction resulted in reduced CD8(+) T-cell clone-mediated cytolytic activity, together with a subtle drop in the expression of genes involved in effector function regulation. Our results also imply a variable contribution of the 2B4-CD48 interaction to the modulation of CD8(+) T-cell functional properties, potentially linked to intrinsic levels of T-bet expression and TCR avidity. The present study thus provides further insights into the role of the 2B4-CD48 interaction in the fine regulation of CD8(+) T-cell effector function upon antigenic stimulation.

Standardization of cryopreserved peripheral blood mononuclear cells through a resting process for clinical immunomonitoring--Development of an algorithm.

Flow cytometry, as a powerful tool for immunomonitoring and quality control of peripheral blood mononuclear cells (PBMCs), is routinely used in clinical studies. However, flow cytometry based assays for cryopreserved peripheral blood mononuclear cells (cPBMCs) constitute a challenge. Down-regulation of surface and intracellular markers, as well as impairment of cell function might result from cryopreservation. Furthermore, protocols for resting cPBMCs are available but diverse. Therefore, we performed a standardization of the resting process concerning resting position, cell concentration, resting period and material of cell culture tubes as well as culture media. We further investigated the influence of resting on the phenotype and functionality of T cells comparing fresh PBMCs as gold standard to rested and non-rested cPBMCs. Polychromatic flow cytometry staining, peptide-MHC class I restricted tetramer staining and intracellular cytokine staining as major methods were used. Our results revealed that a horizontal position, a cell concentration of 2 to 5 × 10(6) cells/ml and an overnight resting phase is beneficial to eliminate dead or dying cells in cPBMCs with a mean cell loss of 14% overall cell populations. In addition, the quality and quantity of regulatory T cells and antigen specific T cells recovered upon resting. For multifunctional T cells a decrease of activation threshold in the way of a twofold mean fluorescence intensity (MFI) and increase of degranulation marker CD107a, co-stimulatory marker CD28, adhesion molecule CD62L as well as the ability to secrete antiviral cytokines like interferon gamma (IFN-γ), tumor necrosis factor (TNF), and interleukin 2 (IL-2) comparable to fresh PBMCs were observed. However, based upon our data resting is not helpful for the flow cytometric analyses of myeloid-derived suppressor cells (MDSCs) and large/intermediate size lymphocytes which rather decreased/vanished ex vivo. Therefore, we developed an algorithm to indicate for which cell population and for which type of analyses the resting process is useful or not.

Prediction of MHC Binding Peptides and Antigenic Peptides from Ascaris lumbricoides

The parasitic disease Acariasis is the major concern because of its morbidity and mortality issue. In this investigation, we predicted the binding peptides of the MHC class I and MHC class II by Position Specific Scoring Matrices (PSSM) and Support Vector Machine (SVM) algorithms. We predicted the binding affinity of Cytochrome c oxidase subunit 2 (mitochondrion) having a 232 amino acids long residue sequence, which shows 224 nonamers. We predicted the peptide binding affinity to MHC I moles are as 8mer_H2_Db(The binding thresholds: 33.04,optimal score:52.494), 9mer_H2_Db(Optimal Score: 50.232, Binding Threshold: 17.96),10mer_H2_Db (The Optimal Score: 58.858, Binding Threshold: 41.32), 11mer_H2_Db(Optimal Score: 79.495, Binding Threshold: 56.96) and MHC- II molecules are as I_Ab.p, I_Ad.p, I_Ag7. We also study integrates prediction of peptide MHC class I binding; proteasomal C terminal cleavage and TAP transport efficiency by using sequence and properties of the amino acids. We also found the binding of peptides to different alleles by using Position Specific Scoring Matrix. PSSM based server will predict the peptide binders of Cytochrome c oxidase subunit 2 from Ascaris lumbricoides sequence to, which are found antigenic epitopes region in protein.

Regulatory T Cell Induction and Retention in the Lungs Drives Suppression of Detrimental Type 2 Th Cells During Pulmonary Cryptococcal Infection.

Lethal disease caused by the fungus Cryptococcus neoformans is a consequence of the combined failure to control pulmonary fungal replication and immunopathology caused by induced type 2 Th2 cell responses in animal models. In order to gain insights into immune regulatory networks, we examined the role of regulatory T (Treg) cells in suppression of Th2 cells using a mouse model of experimental cryptococcosis. Upon pulmonary infection with Cryptococcus, Treg cells accumulated in the lung parenchyma independently of priming in the draining lymph node. Using peptide-MHC class II molecules to identify Cryptococcus-specific Treg cells combined with genetic fate-mapping, we noted that a majority of the Treg cells found in the lungs were induced during the infection. Additionally, we found that Treg cells used the transcription factor, IFN regulatory factor 4, to dampen harmful Th2 cell responses, as well as mediate chemokine retention of Treg cells in the lungs. Taken together, induction and IFN regulatory factor 4-dependent localization of Treg cells in the lungs allow Treg cells to suppress the deleterious effects of Th2 cells during cryptococcal infection.

Enhanced Detection of Antigen-Specific CD4+ T Cells Using Altered Peptide Flanking Residue Peptide-MHC Class II Multimers.

Fluorochrome-conjugated peptide–MHC (pMHC) class I multimers are staple components of the immunologist’s toolbox, enabling reliable quantification and analysis of Ag-specific CD8+ T cells irrespective of functional outputs. In contrast, widespread use of the equivalent pMHC class II (pMHC-II) reagents has been hindered by intrinsically weaker TCR affinities for pMHC-II, a lack of cooperative binding between the TCR and CD4 coreceptor, and a low frequency of Ag-specific CD4+ T cell populations in the peripheral blood. In this study, we show that peptide flanking regions, extending beyond the central nonamer core of MHC-II–bound peptides, can enhance TCR–pMHC-II binding and T cell activation without loss of specificity. Consistent with these findings, pMHC-II multimers incorporating peptide flanking residue modifications proved superior for the ex vivo detection, characterization, and manipulation of Ag-specific CD4+ T cells, highlighting an unappreciated feature of TCR–pMHC-II interactions.

Gapped sequence alignment using artificial neural networks: application to the MHC class I system

Many biological processes are guided by receptor interactions with linear ligands of variable length. One such receptor is the MHC class I molecule. The length preferences vary depending on the MHC allele, but are generally limited to peptides of length 8-11 amino acids. On this relatively simple system, we developed a sequence alignment method based on artificial neural networks that allows insertions and deletions in the alignment. We show that prediction methods based on alignments that include insertions and deletions have significantly higher performance than methods trained on peptides of single lengths. Also, we illustrate how the location of deletions can aid the interpretation of the modes of binding of the peptide-MHC, as in the case of long peptides bulging out of the MHC groove or protruding at either terminus. Finally, we demonstrate that the method can learn the length profile of different MHC molecules, and quantified the reduction of the experimental effort required to identify potential epitopes using our prediction algorithm. The NetMHC-4.0 method for the prediction of peptide-MHC class I binding affinity using gapped sequence alignment is publicly available at: http://www.cbs.dtu.dk/services/NetMHC-4.0.

A modern approach for epitope prediction: identification of foot-and-mouth disease virus peptides binding bovine leukocyte antigen (BoLA) class I molecules.

Major histocompatibility complex (MHC) class Imolecules regulate adaptive immune responses through the presentation of antigenic peptides to CD8+ T cells. Polymorphisms in the peptide binding region of class I molecules determine peptide binding affinity and stability during antigen presentation, and different antigen peptide motifs are associated with specific genetic sequences of class I molecules. Understanding bovine leukocyte antigen (BoLA), peptide-MHC class I binding specificities may facilitate development of vaccines or reagents for quantifying the adaptive immune response to intracellular pathogens, such as foot-and-mouth disease virus (FMDV). Six synthetic BoLA class I (BoLA-I) molecules were produced, and the peptide binding motif was generated for five of the six molecules using a combined approach of positional scanning combinatorial peptide libraries (PSCPLs) and neural network-based predictions (NetMHCpan). The updated NetMHCpan server was used to predict BoLA-I binding peptides within the P1 structural polyprotein sequence of FMDV (strain A24 Cruzeiro) for Bo-LA-1*01901, BoLA-2*00801, BoLA-2*01201, and BoLA-4*02401. Peptide binding affinity and stability were determined for these BoLA-I molecules using the luminescent oxygen channeling immunoassay (LOCI) and scintillation proximity assay (SPA). The functional diversity of known BoLA alleles was predicted using theMHCcluster tool, and functional predictions for peptide motifs were compared to observed data from this and prior studies. The results of these analyses showed that BoLA alleles cluster into three distinct groups with the potential to define BBoLA supertypes.^ This streamlined approach identifies potential T cell epitopes from pathogens, such as FMDV, and provides insight into T cell immunity following infection or vaccination.

Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification.

A key event in the generation of a cellular response against malicious organisms through the endocytic pathway is binding of peptidic antigens by major histocompatibility complex class II (MHC class II) molecules. The bound peptide is then presented on the cell surface where it can be recognized by T helper lymphocytes. NetMHCIIpan is a state-of-the-art method for the quantitative prediction of peptide binding to any human or mouse MHC class II molecule of known sequence. In this paper, we describe an updated version of the method with improved peptide binding register identification. Binding register prediction is concerned with determining the minimal core region of nine residues directly in contact with the MHC binding cleft, a crucial piece of information both for the identification and design of CD4(+) T cell antigens. When applied to a set of 51 crystal structures of peptide-MHC complexes with known binding registers, the new method NetMHCIIpan-3.1 significantly outperformed the earlier 3.0 version. We illustrate the impact of accurate binding core identification for the interpretation of T cell cross-reactivity using tetramer double staining with a CMV epitope and its variants mapped to the epitope binding core. NetMHCIIpan is publicly available at http://www.cbs.dtu.dk/services/NetMHCIIpan-3.1 .

Characterization of the Antigen-Specific CD4(+) T Cell Response Induced by Prime-Boost Strategies with CAF01 and CpG Adjuvants Administered by the Intranasal and Subcutaneous Routes.

The design of heterologous prime-boost vaccine combinations that optimally shape the immune response is of critical importance for the development of next generation vaccines. Here, we tested different prime-boost combinations using the tuberculosis vaccine antigen H56 with CAF01 or CpG ODN 1826 adjuvants, administered by the parenteral and nasal routes. Using peptide-MHC class II tetramers, antigen-specific CD4+ T cells were tracked following primary and booster immunizations. Both parenteral priming with H56 plus CAF01 and nasal priming with H56 plus CpG elicited significant expansion of CD4+ tetramer-positive T cells in the spleen; however, only parenterally primed cells responded to booster immunization. Subcutaneous (SC) priming with H56 and CAF01 followed by nasal boosting with H56 and CpG showed the greater expansion of CD4+ tetramer-positive T cells in the spleen and lungs compared to all the other homologous and heterologous prime-boost combinations. Nasal boosting exerted a recruitment of primed CD4+ T cells into lungs that was stronger in subcutaneously than nasally primed mice, in accordance with different chemokine receptor expression induced by primary immunization. These data demonstrate that SC priming is fundamental for eliciting CD4+ T cells that can be efficiently boosted by the nasal route and results in the recruitment of antigen-experienced cells into the lungs. Combination of different vaccine formulations and routes of delivery for priming and boosting is a strategic approach for improving and directing vaccine-induced immune responses.