MHC-associated peptide proteomics enabling highly sensitive detection of immunogenic sequences for the development of therapeutic antibodies with low immunogenicity

ABSTRACT

Immunogenicity is a key factor capable of influencing the efficacy and safety of therapeutic antibodies. A recently developed method called MHC-associated peptide proteomics (MAPPs) uses liquid chromatography/mass spectrometry to identify the peptide sequences derived from a therapeutic protein that are presented by major histocompatibility complex class II (MHC II) on antigen-presenting cells, and therefore may induce immunogenicity. In this study, we developed a MAPPs technique (called Ab-MAPPs) that has high throughput and can efficiently identify the MHC II-presented peptides derived from therapeutic antibodies using magnetic nanoparticle beads coated with a hydrophilic polymer in the immunoprecipitation process. The magnetic beads could identify more peptides and sequence regions originating from infliximab and adalimumab in a shorter measurement time than Sepharose beads, which are commonly used for MAPPs. Several sequence regions identified by Ab-MAPPs from infliximab corresponded to immunogenic sequences reported by other methods, which suggests the method’s high potential for identifying significant sequences involved in immunogenicity. Furthermore, our study suggests that the Ab-MAPPs method can recognize the difference of a single amino acid residue between similar antibody sequences with different levels of T-cell proliferation activity and can identify potentially immunogenic peptides with high binding affinity to MHC II. In conclusion, Ab-MAPPs is useful for identifying the immunogenic sequences of therapeutic antibodies and will contribute to the design of therapeutic antibodies with low immunogenicity during the drug discovery stage.     

Properties of MHC Class I Presented Peptides That Enhance Immunogenicity

T-cells have to recognize peptides presented on MHC molecules to be activated and elicit their effector functions. Several studies demonstrate that some peptides are more immunogenic than others and therefore more likely to be T-cell epitopes. We set out to determine which properties cause such differences in immunogenicity. To this end, we collected and analyzed a large set of data describing the immunogenicity of peptides presented on various MHC-I molecules. Two main conclusions could be drawn from this analysis: First, in line with previous observations, we showed that positions P4–6 of a presented peptide are more important for immunogenicity. Second, some amino acids, especially those with large and aromatic side chains, are associated with immunogenicity. This information was combined into a simple model that was used to demonstrate that immunogenicity is, to a certain extent, predictable. This model (made available at http://tools.iedb.org/immunogenicity/) was validated with data from two independent epitope discovery studies. Interestingly, with this model we could show that T-cells are equipped to better recognize viral than human (self) peptides. After the past successful elucidation of different steps in the MHC-I presentation pathway, the identification of variables that influence immunogenicity will be an important next step in the investigation of T-cell epitopes and our understanding of cellular immune responses.     

Fast peptide exchange on major histocompatibility complex class I molecules by acidic stabilization of a peptide‐empty intermediate

The cell biology and biochemistry of peptide exchange on major histocompatibility complex class I (MHC‐I) proteins are of great interest in the study of immunodominance, which requires iterative optimization of peptide affinity, and cross‐presentation of pathogen and tumor antigens, in which endogenous peptides are exchanged for exogenous ones. Even though several methods exist to catalyze peptide exchange on recombinant MHC‐I proteins, the cellular conditions and mechanisms allowing for peptide exchange in vivo remain unclear. Here, we demonstrate that low pH, as present in endosomes, indeed triggers peptide exchange, and we dissect the individual steps of the exchange reaction. We find that low pH stabilizes the peptide‐empty forms of MHC‐I that occur as intermediates of the exchange reaction, and that is synergizes with dipeptides and with disulfide‐mediated stabilization of MHC‐I.     

Crystal structure of a non-canonical low-affinity peptide complexed with MHC class I: a new approach for vaccine design

Peptides bind with high affinity to MHC class I molecules by anchoring certain side-chains (anchors) into specificity pockets in the MHC peptide-binding groove. Peptides that do not contain these canonical anchor residues normally have low affinity, resulting in impaired pMHC stability and loss of immunogenicity. Here, we report the crystal structure at 1.6 A resolution of an immunogenic, low-affinity peptide from the tumor-associated antigen MUC1, bound to H-2Kb. Stable binding is still achieved despite small, non-canonical residues in the C and F anchor pockets. This structure reveals how low-affinity peptides can be utilized in the design of novel peptide-based tumor vaccines. The molecular interactions elucidated in this non-canonical low-affinity peptide MHC complex should help uncover additional immunogenic peptides from primary protein sequences and aid in the design of alternative approaches for T-cell vaccines.     

The live-attenuated yellow fever vaccine 17D induces broad and potent T cell responses against several viral proteins in Indian rhesus macaques—implications for recombinant vaccine design

The yellow fever vaccine 17D (YF17D) is one of the most effective vaccines. Its wide use and favorable safety profile make it a prime candidate for recombinant vaccines. It is believed that neutralizing antibodies account for a large measure of the protection afforded to YF17D-vaccinated individuals, however cytotoxic T lymphocyte (CTL) responses have been described in the setting of YF17D vaccination. YF17D is an ssRNA flavivirus that is translated as a full-length polyprotein, several domains of which pass into the lumen of the endoplasmic reticulum (ER). The processing and presentation machinery for MHC class I-restricted CTL responses favor cytoplasmic peptides that are transported into the ER by the transporter associated with antigen presentation proteins. In order to inform recombinant vaccine design, we sought to determine if YF17D-induced CTL responses preferentially targeted viral domains that remain within the cytoplasm. We performed whole YF17D proteome mapping of CTL responses in six Indian rhesus macaques vaccinated with YF17D using overlapping YF17D peptides. We found that the ER luminal E protein was the most immunogenic viral protein followed closely by the cytoplasmic NS3 and NS5 proteins. These results suggest that antigen processing and presentation in this model system is not preferentially affected by the subcellular location of the viral proteins that are the source of CTL epitopes. The data also suggest potential immunogenic regions of YF17D that could serve as the focus of recombinant T cell vaccine development.     

Single-Molecule Motions of MHC Class II Rely on Bound Peptides

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells.     

Large Scale Characterization of the LC13 TCR and HLA-B8 Structural Landscape in Reaction to 172 Altered Peptide Ligands: A Molecular Dynamics Simulation Study

The interplay between T cell receptors (TCRs) and peptides bound by major histocompatibility complexes (MHCs) is one of the most important interactions in the adaptive immune system. Several previous studies have computationally investigated their structural dynamics. On the basis of these simulations several structural and dynamical properties have been proposed as effectors of the immunogenicity. Here we present the results of a large scale Molecular Dynamics simulation study consisting of 100 ns simulations of 172 different complexes. These complexes consisted of all possible point mutations of the Epstein Barr Virus peptide FLRGRAYGL bound by HLA-B*08:01 and presented to the LC13 TCR. We compare the results of these 172 structural simulations with experimental immunogenicity data. We found that simulations with more immunogenic peptides and those with less immunogenic peptides are in fact highly similar and on average only minor differences in the hydrogen binding footprints, interface distances, and the relative orientation between the TCR chains are present. Thus our large scale data analysis shows that many previously suggested dynamical and structural properties of the TCR/peptide/MHC interface are unlikely to be conserved causal factors for peptide immunogenicity.     

POPI: predicting immunogenicity of MHC class I binding peptides by mining informative physicochemical properties

MOTIVATION: Both modeling of antigen-processing pathway including major histocompatibility complex (MHC) binding and immunogenicity prediction of those MHC-binding peptides are essential to develop a computer-aided system of peptide-based vaccine design that is one goal of immunoinformatics. Numerous studies have dealt with modeling the immunogenic pathway but not the intractable problem of immunogenicity prediction due to complex effects of many intrinsic and extrinsic factors. Moderate affinity of the MHC-peptide complex is essential to induce immune responses, but the relationship between the affinity and peptide immunogenicity is too weak to use for predicting immunogenicity. This study focuses on mining informative physicochemical properties from known experimental immunogenicity data to understand immune responses and predict immunogenicity of MHC-binding peptides accurately. RESULTS: This study proposes a computational method to mine a feature set of informative physicochemical properties from MHC class I binding peptides to design a support vector machine (SVM) based system (named POPI) for the prediction of peptide immunogenicity. High performance of POPI arises mainly from an inheritable bi-objective genetic algorithm, which aims to automatically determine the best number m out of 531 physicochemical properties, identify these m properties and tune SVM parameters simultaneously. The dataset consisting of 428 human MHC class I binding peptides belonging to four classes of immunogenicity was established from MHCPEP, a database of MHC-binding peptides (Brusic et al., 1998). POPI, utilizing the m = 23 selected properties, performs well with the accuracy of 64.72% using leave-one-out cross-validation, compared with two sequence alignment-based prediction methods ALIGN (54.91%) and PSI-BLAST (53.23%). POPI is the first computational system for prediction of peptide immunogenicity based on physicochemical properties. AVAILABILITY: A web server for prediction of peptide immunogenicity (POPI) and the used dataset of MHC class I binding peptides (PEPMHCI) are available at http://iclab.life.nctu.edu.tw/POPI     

Antigen processing by proteasomes: insights into the molecular basis of crypticity

Eight to eleven amino acid residues are the sizes of predominant peptides found to be associated with MHC class I molecules. Proteasomes have been implicated in antigen processing and generation of such peptides. Advanced methodologies in peptide elution together with sequence determination have led to the characterisation of MHC class I binding motifs. More recently, screening of random peptide phage display libraries and synthetic combinatorial peptide libraries have also been successfully used. This has led to the development and use of predictive algorithms to screen antigens for potential CTL epitopes. Not all predicted epitopes will be generated in vivo and the emerging picture suggests differential presentation of predicted CTL epitopes ranging from cryptic to immunodominant. The scope of this review is to discuss antigen processing by proteasomes, and to put forward a hypothesis that the molecular basis of immunogenicity can be a function of proteasomal processing. This may explain how pathogens and tumours are able to escape immunosurveillance by altering sequences required by proteasomes for epitope generation. Abbreviations: CTL – cytotoxic T lymphocytes; DRiPs – defective ribosomal products; ER – endoplasmic reticulum; Hsps – heat shock proteins; LMP – low molecular weight peptide; MHC – major histocompatibility complex; TAP – transporter associated with antigen processing.     

Cutting edge: receptor-mediated endocytosis of heat shock proteins by professional antigen-presenting cells

Immunization with heat shock proteins (HSPs) induces Ag-specific CTL responses. The specificity of the immune response is based on peptides associated with HSPs. To investigate how exogenous HSP/peptide complexes gain access to the MHC class I-restricted Ag presentation pathway, we incubated the monocytic cell line P388D1 and the dendritic cell line D2SC/1 with gold-labeled HSPs gp96 and HSC70. We show that HSPs bind specifically to the surface of these APCs and are internalized spontaneously by receptor-mediated endocytosis, demonstrating the existence of specific receptors for HSPs on these cells. In addition, we observe colocalization of internalized HSPs and surface MHC class I molecules in early and late endosomal structures. These findings provide possible explanations for the immunogenicity of HSP/peptide complexes and for the transfer of HSP-associated peptides onto MHC class I molecules.     

Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants

Defects in major histocompatibility complex (MHC) class I-restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.     

T cell recognition of transforming proteins encoded by mutated ras proto-oncogenes

Activated ras proto-oncogenes contribute to the pathogenesis of many animal and human malignancies. ras proto-oncogenes are generally activated by point mutations within codons 12 or 61, which result in the expression of ras protein (p21) bearing characteristic single amino acid substitutions at the corresponding residues. The purpose of the current study was to determine whether the presence of single transforming amino acid substitutions can render normal ras protein immunogenic and, thus, a possible target for T cell-mediated tumor therapy. In initial experiments, C57BL/6 mice were immunized with a synthetic peptide corresponding to residues 5 through 16 of p21 containing the transforming substitution of arginine for normal glycine at residue 12. The results demonstrated that class II MHC-restricted T cells which were specific for the peptide could be elicited, and that the peptide-induced T cells could specifically recognize the corresponding intact p21 ras protein. Recognition of p21 ras protein by peptide-specific T cells implies that C57BL/6 APC can process the activated ras protein in a fashion that allows presentation of digested protein by class II MHC molecules in a configuration similar to the configuration with synthetic peptide. Evaluation of the immunogenicity of peptides containing alternative transforming amino acid substitutions of ras protein demonstrated that some, but not all, were immunogenic in individual strains of mice. Therefore, although ras protein-specific T cells can be elicited by immunization with synthetic peptides, not all of the potential ras mutations commonly associated with malignancy may be recognizable by T cells from all individuals.     

Major Histocompatibility Complex Class II+ Invariant Chain Negative Breast Cancer Cells Present Unique Peptides that Activate Tumor-specific T Cells from Breast Cancer Patients

The major histocompatibility complex (MHC) class II-associated Invariant chain (Ii) is present in professional antigen presenting cells where it regulates peptide loading onto MHC class II molecules and the peptidome presented to CD4⁺ T lymphocytes. Because Ii prevents peptide loading in neutral subcellular compartments, we reasoned that Ii⁻ cells may present peptides not presented by Ii⁺ cells. Based on the hypothesis that patients are tolerant to MHC II-restricted tumor peptides presented by Ii⁺ cells, but will not be tolerant to novel peptides presented by Ii⁻ cells, we generated MHC II vaccines to activate cancer patients'' T cells. The vaccines are Ii⁻ tumor cells expressing syngeneic HLA-DR and the costimulatory molecule CD80. We used liquid chromatography coupled with mass spectrometry to sequence MHC II-restricted peptides from Ii⁺ and Ii⁻ MCF10 human breast cancer cells transfected with HLA-DR7 or the MHC Class II transactivator CIITA to determine if Ii⁻ cells present novel peptides. Ii expression was induced in the HLA-DR7 transfectants by transfection of Ii, and inhibited in the CIITA transfectants by RNA interference. Peptides were analyzed and binding affinity predicted by artificial neural net analysis. HLA-DR7-restricted peptides from Ii⁻ and Ii⁺ cells do not differ in size or in subcellular location of their source proteins; however, a subset of HLA-DR7-restricted peptides of Ii⁻ cells are not presented by Ii⁺ cells, and are derived from source proteins not used by Ii⁺ cells. Peptides from Ii⁻ cells with the highest predicted HLA-DR7 binding affinity were synthesized, and activated tumor-specific HLA-DR7⁺ human T cells from healthy donors and breast cancer patients, demonstrating that the MS-identified peptides are bonafide tumor antigens. These results demonstrate that Ii regulates the repertoire of tumor peptides presented by MHC class II⁺ breast cancer cells and identify novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.Cancer vaccines are a promising tool for cancer treatment and prevention because of their potential for inducing tumor-specific responses in conjunction with minimal toxicity for healthy cells. Cancer vaccines are based on the concept that tumor cells synthesize multiple peptides that are potential immunogens, and that with the appropriate vaccine protocol, these peptides will activate an efficacious antitumor response in the patient. Much effort has been invested in identifying and testing tumor-encoded peptides, particularly peptides presented by major histocompatibility complex (MHC)¹ class I, molecules capable of activating CD8⁺ T-cells that directly kill tumor cells (1, 2). Fewer studies have been devoted to identifying MHC class II-restricted peptides for the activation of tumor-reactive CD4⁺ T-cells despite compelling evidence that Type 1 CD4⁺ T helper cells facilitate the optimal activation of CD8⁺ T-cells and the generation of immune memory, which is likely to be essential for protection from metastatic disease.Activation of CD4⁺ T cells requires delivery of a costimulatory signal plus an antigen-specific signal consisting of peptide bound to an MHC II molecule. Most cells do not express MHC II or costimulatory molecules, so CD4⁺ T cells are typically activated by professional antigen presenting cells (APC), which endocytose exogenously synthesized antigen and process and present it in the context of their own MHC II molecules. This processing and presentation process requires Invariant chain (Ii), a molecule that is coordinately synthesized with MHC II molecules and prevents the binding and presentation of APC-encoded endogenous peptides (3, 4). As a result, tumor-reactive CD4⁺ T cells are activated to tumor peptides generated by the antigen processing machinery of professional APC, rather than peptides generated by the tumor cells. Because of the potential discrepancy in peptide generation between professional APC and tumor cells, and the critical role of Ii in preventing the presentation of endogenous peptides, we have generated “MHC II cancer vaccines” that consist of Ii⁻ tumor cells transfected with syngeneic MHC class II and CD80 genes. We reasoned that MHC II⁺Ii⁻CD80⁺ tumor cells may present a novel repertoire of MHC II-restricted tumor peptides that are not presented by professional APC, and therefore may be highly immunogenic. Once activated, CD4⁺ T cells produce IFNγ and provide help to CD8⁺ T cells and do not need to react with native tumor cells. Therefore, the MHC II vaccines have the potential to activate CD4⁺ Th1 cells that facilitate antitumor immunity. In vitro (5) and in vivo (5–7) studies with mice support this conclusion. In vitro studies with human MHC II vaccines further demonstrate that the absence of Ii facilitates the activation of MHC II-restricted tumor-specific CD4⁺ type 1 T cells of HLA-DR-syngeneic healthy donors and cancer patients, and that the vaccines activate CD4⁺ T cells with a distinct repertoire of T cell receptors (8–12). A critical negative role for Ii is also supported by studies of human acute myelogenous leukemia (AML). High levels of class II-associated invariant chain peptide (CLIP), a degradation product of Ii, by leukemic blasts is associated with poor patient prognosis (13, 14), whereas down-modulation of CLIP on AML cells increases the activation of tumor-reactive human CD4⁺ T cells (14, 15).We have now used mass spectrometry to identify MHC II-restricted peptides from MHC II⁺Ii⁻ and MHC II⁺Ii⁺ human breast cancer cells to test the concept that the absence of Ii facilitates the presentation of unique immunogenic MHC II-restricted peptides. We report here that a subset of MHC II-restricted peptides from HLA-DR7⁺ breast cancer cells are unique to Ii⁻ cells and are derived from source proteins not used by Ii⁺ cells. Ii⁻ peptides have high binding affinity for HLA-DR7 and activate tumor-specific T-cells from the peripheral blood of healthy donors and breast cancer patients. This is the first study to compare the human tumor cell MHC II peptidome in the absence or presence of Ii and to demonstrate that MHC II⁺Ii⁻ tumor cells present novel immunogenic MHC II-restricted peptides that are potential therapeutic reagents for cancer patients.     

Identification of Four-Jointed Box 1 (FJX1)-Specific Peptides for Immunotherapy of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is highly prevalent in South East Asia and China. The poor outcome is due to late presentation, recurrence, distant metastasis and limited therapeutic options. For improved treatment outcome, immunotherapeutic approaches focusing on dendritic and autologous cytotoxic T-cell based therapies have been developed, but cost and infrastructure remain barriers for implementing these in low-resource settings. As our prior observations had found that four-jointed box 1 (FJX1), a tumor antigen, is overexpressed in NPCs, we investigated if short 9–20 amino acid sequence specific peptides matching to FJX1 requiring only intramuscular immunization to train host immune systems would be a better treatment option for this disease. Thus, we designed 8 FJX1-specific peptides and implemented an assay system to first, assess the binding of these peptides to HLA-A2 molecules on T2 cells. After, ELISPOT assays were used to determine the peptides immunogenicity and ability to induce potential cytotoxicity activity towards cancer cells. Also, T-cell proliferation assay was used to evaluate the potential of MHC class II peptides to stimulate the expansion of isolated T-cells. Our results demonstrate that these peptides are immunogenic and peptide stimulated T-cells were able to induce peptide-specific cytolytic activity specifically against FJX1-expressing cancer cells. In addition, we demonstrated that the MHC class II peptides were capable of inducing T-cell proliferation. Our results suggest that these peptides are capable of inducing specific cytotoxic cytokines secretion against FJX1-expressing cancer cells and serve as a potential vaccine-based therapy for NPC patients.     

Tertiary motifs as building blocks for the design of protein‐binding peptides

Despite advances in protein engineering, the de novo design of small proteins or peptides that bind to a desired target remains a difficult task. Most computational methods search for binder structures in a library of candidate scaffolds, which can lead to designs with poor target complementarity and low success rates. Instead of choosing from pre‐defined scaffolds, we propose that custom peptide structures can be constructed to complement a target surface. Our method mines tertiary motifs (TERMs) from known structures to identify surface‐complementing fragments or “seeds.” We combine seeds that satisfy geometric overlap criteria to generate peptide backbones and score the backbones to identify the most likely binding structures. We found that TERM‐based seeds can describe known binding structures with high resolution: the vast majority of peptide binders from 486 peptide‐protein complexes can be covered by seeds generated from single‐chain structures. Furthermore, we demonstrate that known peptide structures can be reconstructed with high accuracy from peptide‐covering seeds. As a proof of concept, we used our method to design 100 peptide binders of TRAF6, seven of which were predicted by Rosetta to form higher‐quality interfaces than a native binder. The designed peptides interact with distinct sites on TRAF6, including the native peptide‐binding site. These results demonstrate that known peptide‐binding structures can be constructed from TERMs in single‐chain structures and suggest that TERM information can be applied to efficiently design novel target‐complementing binders.     

Live‐cell super‐resolution imaging of actin using LifeAct‐14 with a PAINT‐based approach

We present direct‐LIVE‐PAINT, an easy‐to‐implement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of direct‐LIVE‐PAINT with an actin‐binding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that direct‐LIVE‐PAINT can be used to image actin structures below the diffraction‐limit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells.     

HLA-DR-presented peptide repertoires derived from human monocyte-derived dendritic cells pulsed with blood coagulation factor VIII

Activation of T-helper cells is dependent upon the appropriate presentation of antigen-derived peptides on MHC class II molecules expressed on antigen presenting cells. In the current study we explored the repertoire of peptides presented on MHC class II molecules on human monocyte derived dendritic cells (moDCs) from four HLA-typed healthy donors. MHC class II-bound peptides could be routinely recovered from small cultures containing 5 × 10(6) cells. A fraction of the identified peptides were derived from proteins localized in the plasma membrane, endosomes, and lysosomes, but the majority of peptides that were presented on MHC class II originate from other organelles. Subsequently, we studied the antigen-specific peptide repertoire after endocytosis of a soluble antigen. Blood coagulation factor VIII (FVIII) was chosen as the antigen since our current knowledge on MHC class II presented peptides derived from this immunogenic therapeutic protein is limited. Analysis of the total repertoire of MHC class II-associated peptides revealed that per individual sample 20-50 FVIII-derived peptides were presented on FVIII-pulsed moDCs. Repertoires of FVIII-derived peptides eluted from moDCs derived from a panel of four HLA typed donors revealed that some MHC class II-presented FVIII peptides were presented by multiple donors, whereas the presentation of other FVIII peptides was donor-specific. In total 32 different core peptides were presented on FVIII-pulsed moDCs from four HLA-typed donors. Together our findings provide an unbiased approach to identify peptides that are presented by MHC class II on antigen-loaded moDCs from individual donors.     

Peptide immunisation of HLA-DR–transgenic mice permits the identification of a novel HLA-DRβ1*0101– and HLA-DRβ1*0401–restricted epitope from p53

Because of the central role of CD4⁺ T cells in antitumour immunity, the identification of the MHC class II–restricted peptides to which CD4⁺ T cells respond has become a priority of tumour immunologists. Here, we describe a strategy permitting us to rapidly determine the immunogenicity of candidate HLA-DR–restricted peptides using peptide immunisation of HLA-DR–transgenic mice, followed by assessment of the response in vitro. This strategy was successfully applied to the reported haemaglutinin influenza peptide HA(307–319), and then extended to three candidate HLA-DR–restricted p53 peptides predicted by the evidence-based algorithm SYFPEITHI to bind to HLA-DR1*0101 (HLA-DR1) and HLA-DR1*0401 (HLA-DR4) molecules. One of these peptides, p53(108–122), consistently induced responses in HLA-DR1– and in HLA-DR4–transgenic mice. Moreover, this peptide was naturally processed by dendritic cells (DCs), and induced specific proliferation in the splenocytes of mice immunised with p53 cDNA, demonstrating that immune responses could be naturally mounted to the peptide. Furthermore, p53(108–122) peptide was also immunogenic in HLA-DR1 and HLA-DR4 healthy donors. Thus, the use of this transgenic model permitted the identification of a novel HLA-DR–restricted epitope from p53 and constitutes an attractive approach for the rapid identification of novel immunogenic MHC class II–restricted peptides from tumour antigens, which can ultimately be incorporated in immunotherapeutic protocols.     

Pseudomonas exotoxin-mediated delivery of exogenous antigens to MHC class I and class II processing pathways

Peptides associated with class II MHC molecules are normally derived from exogenous proteins, whereas class I MHC molecules normally associate with peptides from endogenous proteins. We have studied the ability of Pseudomonas exotoxin A (PE) fusion proteins to deliver exogenously added antigen for presentation by both MHC class I and class II molecules. A MHC class II-restricted antigen was fused to PE; this molecule was processed in a manner typical for class II-associated antigens. However, a MHC class I-restricted peptide fused to PE was processed by a mechanism independent of proteasomes. Furthermore, we also found that the PE fusion protein was much more stable in normal human plasma than the corresponding synthetic peptide. We believe that effective delivery of an antigen to both the MHC class I and class II pathways, in addition to the increased resistance to proteolysis in plasma, will be important for immunization.