Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer

Recently developed major histocompatibility complex (MHC) multimer technologies allow visualization and isolation of antigen-specific T cells. However, functional analysis and in vivo transfer of MHC multimer-stained cells is hampered by the persistence of T-cell receptor (TCR) MHC interactions and subsequently induced signaling events. As MHC monomers do not stably bind to TCRs, we postulated that targeted disassembly of multimers into MHC monomers would result in dissociation of surface-bound TCR ligands. We generated a new type of MHC multimers, which can be monomerized in the presence of a competitor, resulting in rapid loss of the staining reagent. Following dissociation, the T cells are phenotypically and functionally indistinguishable from untreated cells. This 'reversible' T-cell staining procedure, which maintains the specificity and sensitivity of MHC multimer staining while preserving the functional status of T lymphocytes, may be of broad benefit for ex vivo investigation of T-cell functions and clinical applications.     

Characterizing the impact of CD8 antibodies on class I MHC multimer binding

Many studies have suggested that CD8 Abs affect the binding of class I MHC tetramers/multimers to CD8(+) T cells, which has led to the interpretation that CD8 participates directly in multimer binding. In contrast, a recent publication has argued that CD8 Abs instead cause reorganization of TCR distribution and hence have an indirect effect on multimer binding to the TCR alone. We address these issues by testing the role of CD8 and the impact of CD8 Abs on the binding of normal and mutant multimers to Ag-specific mouse T cells. Our data suggest that, in this system, CD8 Abs act directly on CD8 and only mediate their effects on multimer binding when CD8 is capable of binding to the multimer. These data reinforce the paradigm that CD8 plays an active and direct role in binding of class I MHC multimers.     

Concurrent detection of circulating minor histocompatibility antigen-specific CD8+ T cells in SCT recipients by combinatorial encoding MHC multimers

Allogeneic stem cell transplantation (SCT) is a potentially curative treatment for patients with hematologic malignancies. Its therapeutic effect is largely dependent on recognition of minor histocompatibility antigens (MiHA) by donor-derived CD8⁺ T cells. Therefore, monitoring of multiple MiHA-specific CD8⁺ T cell responses may prove to be valuable for evaluating the efficacy of allogeneic SCT. In this study, we investigated the use of the combinatorial encoding MHC multimer technique to simultaneously detect MiHA-specific CD8⁺ T cells in peripheral blood of SCT recipients. Feasibility of this approach was demonstrated by applying dual-color encoding MHC multimers for a set of 10 known MiHA. Interestingly, single staining using a fluorochrome- and Qdot-based five-color combination showed comparable results to dual-color staining for most MiHA-specific CD8⁺ T cell responses. In addition, we determined the potential value of combinatorial encoding MHC multimers in MiHA identification. Therefore, a set of 75 candidate MiHA peptides was predicted from polymorphic genes with a hematopoietic expression profile and further selected for high and intermediate binding affinity for HLA-A2. Screening of a large cohort of SCT recipients resulted in the detection of dual-color encoded CD8⁺ T cells following MHC multimer-based T cell enrichment and short ex vivo expansion. Interestingly, candidate MiHA-specific CD8⁺ T cell responses for LAG3 and TLR10 derived polymorphic peptides could be confirmed by genotyping of the respective SNPs. These findings demonstrate the potency of the combinatorial MHC multimer approach in the monitoring of CD8⁺ T cell responses to known and potential MiHA in limited amounts of peripheral blood from allogeneic SCT recipients.     

Disulfide bond engineering to trap peptides in the MHC class I binding groove

Immunodominant peptides in CD8 T cell responses to pathogens and tumors are not always tight binders to MHC class I molecules. Furthermore, antigenic peptides that bind weakly to the MHC can be problematic when designing vaccines to elicit CD8 T cells in vivo or for the production of MHC multimers for enumerating pathogen-specific T cells in vitro. Thus, to enhance peptide binding to MHC class I, we have engineered a disulfide bond to trap antigenic peptides into the binding groove of murine MHC class I molecules expressed as single-chain trimers or SCTs. These SCTs with disulfide traps, termed dtSCTs, oxidized properly in the endoplasmic reticulum, transited to the cell surface, and were recognized by T cells. Introducing a disulfide trap created remarkably tenacious MHC/peptide complexes because the peptide moiety of the dtSCT was not displaced by high-affinity competitor peptides, even when relatively weak binding peptides were incorporated into the dtSCT. This technology promises to be useful for DNA vaccination to elicit CD8 T cells, in vivo study of CD8 T cell development, and construction of multivalent MHC/peptide reagents for the enumeration and tracking of T cells-particularly when the antigenic peptide has relatively weak affinity for the MHC.     

Use of soluble MHC class II/peptide multimers to detect antigen-specific T cells in human disease

Most techniques that identify antigen-specific T cells are dependent on the response of these cells to the relevant antigen in culture. Soluble multimers of MHC molecules, when occupied with the same peptide, will bind selectively to T cells specific for that MHC/peptide complex. Techniques to produce fluorescent MHC class II/peptide multimers have recently been developed. These reagents provide a method to facilitate detection and isolation of antigen-specific CD4⁺ T cells and they represent a new research tool to study these cells in patients with immune-mediated diseases.     

Parallel detection of antigen-specific T cell responses by combinatorial encoding of MHC multimers

Fluorescently labeled multimeric complexes of peptide-MHC, the molecular entities recognized by the T cell receptor, have become essential reagents for detection of antigen-specific CD8(+) T cells by flow cytometry. Here we present a method for high-throughput parallel detection of antigen-specific T cells by combinatorial encoding of MHC multimers. Peptide-MHC complexes are produced by UV-mediated MHC peptide exchange and multimerized in the form of streptavidin-fluorochrome conjugates. Eight different fluorochromes are used for the generation of MHC multimers and, by a two-dimensional combinatorial matrix, these eight fluorochromes are combined to generate 28 unique two-color codes. By the use of combinatorial encoding, a large number of different T cell populations can be detected in a single sample. The method can be used for T cell epitope mapping, and also for the monitoring of CD8(+) immune responses during cancer and infectious disease or after immunotherapy. One panel of 28 combinatorially encoded MHC multimers can be prepared in 4 h. Staining and detection takes a further 3 h.     

Single chamber microbial fuel cell with spiral anode for dairy wastewater treatment

The circulating population of peripheral T lymphocytes obtained from a blood sample can provide a large amount of information about an individual's medical status and history. Recent evidence indicates that the detection and functional characterization of antigen-specific T cell subsets within the circulating population may provide a diagnostic indicator of disease and has the potential to predict an individual's response to therapy. In this report, a microarray detection platform that combines grating-coupled surface plasmon resonance imaging (GCSPRI) and grating-coupled surface plasmon coupled emission (SPCE) fluorescence detection modalities were used to detect and characterize CD4(+) T cells. The microspot regions of interest (ROIs) printed on the array consisted of immobilized antibodies or peptide loaded MHC monomers (p/MHC) as T cell capture ligands mixed with additional antibodies as cytokine capture ligands covalently bound to the surface of a corrugated gold sensor chip. Using optimized parameters, an unlabeled influenza peptide reactive T cell clone could be detected at a frequency of 0.1% in a mixed T cell sample using GCSPRI. Additionally, after cell binding was quantified, differential TH1 cytokine secretion patterns from a T cell clone cultured under TH1 or TH2 inducing conditions was detected using an SPCE fluorescence based assay. Differences in the secretion patterns of 3 cytokines, characteristic of the inducing conditions, indicated that differences were a consequence of the functional status of the captured cells. A dual mode GCSPRI/SPCE assay can provide a rapid, high content T cell screening/characterization tool that is useful for diagnosing disease, evaluating vaccination efficacy, or assessing responses to immunotherapeutics.     

Visualization of p53(264-272)/HLA-A*0201 complexes naturally presented on tumor cell surface by a multimeric soluble single-chain T cell receptor

Intracellular Ags are processed into small peptides that are presented on cell surfaces in the context of HLA class I molecules. These peptides are recognized by TCRs displayed by CD8+ T lymphocytes (T cells). To date, direct identification and quantitation of these peptides has relied primarily on mass spectrometry analysis, which is expensive and requires large quantities of diseased tissues to obtain useful results. Here we demonstrate that multimerization of a soluble single-chain TCR (scTCR), recognizing a peptide from p53 presented in the context of HLA-A2.1, could be used to directly visualize and quantitate peptide/MHC complexes on unmanipulated human tumor cells. Tumor cells displaying as few as 500 peptide/MHC complexes were readily detectable by flow cytometry. The scTCR/multimers exhibited exquisite recognition capability and could distinguish peptides differing in as little as a single amino acid. We also demonstrate that scTCR/multimers could specifically stain human tumors generated in mice, as well as tumors obtained from patient biopsies. Thus, scTCR/multimers represent a novel class of immunostaining reagents that could be used to validate, quantitate, or monitor epitope presentation by cancer cells.     

MHC Multimer: A Molecular Toolbox for Immunologists

The advent of the major histocompatibility complex (MHC) multimer technology has led to a breakthrough in the quantification and analysis of antigen-specific T cells. In particular, this technology has dramatically advanced the measurement and analysis of CD8 T cells and is being applied more widely. In addition, the scope of application of MHC multimer technology is gradually expanding to other T cells such as CD4 T cells, natural killer T cells, and mucosal-associated invariant T cells. MHC multimer technology acts by complementing the T-cell receptor-MHC/peptide complex affinity, which is relatively low compared to antigen-antibody affinity, through a multivalent interaction. The application of MHC multimer technology has expanded to include various functions such as quantification and analysis of antigen-specific T cells, cell sorting, depletion, stimulation to replace antigen-presenting cells, and single-cell classification through DNA barcodes. This review aims to provide the latest knowledge of MHC multimer technology, which is constantly evolving, broaden understanding of this technology, and promote its widespread use.     

Human immunodeficiency virus type 1 Gag assembly through assembly intermediates

Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37 degrees C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles in the E. coli cells. In contrast, expression at 30 degrees C resulted in the production of only the 60 S form of Gag multimer, and crescent-shaped structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37 degrees C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30 degrees C reaction produced only the 60 S multimers. However, when the 60 S multimers were cross-linked so as not to allow conformational changes, in vitro assembly reactions at 37 degrees C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37 degrees C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag.     

Conformational and structural characteristics of peptides binding to HLA-DR molecules.

A fundamental characteristic of MHC class I and class II proteins is their unusual capacity to form stable complexes with a wide spectrum of peptide ligands. In this study, sets of peptide analogues containing long chain-biotinylated lysine individually substituted for each amino acid in the sequence have been used to explore the structural requirements for the formation of peptide-MHC class II protein complexes. Based on the ability of the analogs to bind both the MHC protein and fluorescent streptavidin, receptor contact residues were identified and from their spacing the conformation of the bound peptides could be inferred. Six separate peptides were studied; three defined by HLA-DR1Dw1-restricted T cells, and three identified by T cells restricted through alleles other than HLA-DR1Dw1. The similar patterns of fluorescent signals observed when the former three peptides were studied indicated that they shared conformational features when bound to HLA-DR1Dw1. In contrast when the latter three peptides were examined, the data indicated that they shared some but not all of the conformational features characteristic of the peptides known to elicit HLA-DR1Dw1-restricted T cells. When the peptide sequences were aligned based on the critical contact residues, two positions of structural homology were apparent. In each sequence, an amino acid with a bulky hydrophobic side chain could be identified separated by four residues from a small amino acid. These minimal structural requirements were consistent with recent experiments demonstrating that only a small number of side chains in the peptide were necessary for binding to the MHC protein.     

The noncollagenous domain 1 of type X collagen. A novel motif for trimer and higher order multimer formation without a triple helix.

In this study, we test the hypothesis that the carboxyl noncollagenous (NC1) domain of collagen X is sufficient to direct multimer formation without a triple helix. Two peptides containing the NC1 domain of avian collagen X have been synthesized using a bacterial expression system and their properties characterized. One peptide consists only of the NC1 domain, and the other is a chimeric molecule with a noncollagenous A domain of matrilin-1 fused to the N terminus of NC1. The NC1 peptide alone forms a 45-kDa trimer under native conditions, suggesting that NC1 contains all the information for trimerization without any triple helical residues. This trimeric association is highly thermostable without intermolecular disulfide bonds. This indicates that the NC1 domain contributes to the remarkable structural stability of collagen X. Chemical cross-linking of the NC1 trimer results in a series of varying sized multimers, the smallest of which is a trimer. Therefore the NC1 trimer is sufficient to form higher order multimers. The chimeric A-NC1 peptide forms a homotrimer by itself, and a series of heterotrimers with the NC1 peptide via the NC1 domain. Thus the NC1(X) domain directs multimer formation, even in a noncollagenous molecule.     

MHC-based detection of antigen-specific CD8+ T cell responses

The hallmark of adaptive immunity is its ability to recognise a wide range of antigens and technologies that capture this diversity are therefore of substantial interest. New methods have recently been developed that allow the parallel analysis of T cell reactivity against vast numbers of different epitopes in limited biological material. These technologies are based on the joint binding of differentially labelled MHC multimers on the T cell surface, thereby providing each antigen-specific T cell population with a unique multicolour code. This strategy of ‘combinatorial encoding’ enables detection of many (at least 25) different T cell populations per sample and should be of broad value for both T cell epitope identification and immunomonitoring.     

Partial enzymatic deglycosylation preserves the structure of cleaved recombinant HIV-1 envelope glycoprotein trimers

The trimeric envelope glycoprotein complex (Env) is the focus of vaccine development programs aimed at generating protective humoral responses to human immunodeficiency virus type 1 (HIV-1). N-Linked glycans, which constitute almost half of the molecular mass of the external Env domains, produce considerable structural heterogeneity and are a major impediment to crystallization studies. Moreover, by shielding the peptide backbone, glycans can block attempts to generate neutralizing antibodies against a substantial subset of potential epitopes when Env proteins are used as immunogens. Here, we describe the partial deglycosylation of soluble, cleaved recombinant Env trimers by inhibition of the synthesis of complex N-glycans during Env production, followed by treatment with glycosidases under conditions that preserve Env trimer integrity. The partially deglycosylated trimers are stable, and neither abnormally sensitive to proteolytic digestion nor prone to aggregation. Moreover, the deglycosylated trimers retain or increase their ability to bind CD4 and antibodies that are directed to conformational epitopes, including the CD4-binding site and the V3 region. However, as expected, they do not react with glycan-dependent antibodies 2G12 and PGT123, or the C-type lectin receptor DC-SIGN. Electron microscopic analysis shows that partially deglycosylated trimers have a structure similar to fully glycosylated trimers, indicating that removal of glycans does not substantially perturb the structural integrity of the trimer. The glycan-depleted Env trimers should be useful for structural and immunogenicity studies.     

A structural difference limited to one residue of the antigenic peptide can profoundly alter the biological outcome of the TCR-peptide/MHC class I interaction

The vesicular stomatitis virus (VSV) octapeptide RGYVYQGL binds to H-2K(b) and triggers a cytotoxic T cell response in mice. A variant peptide, RGYVYEGL (E6) with a glutamic acid for glutamine replacement at position 6 of the VSV peptide, elicits a T cell response with features that are quite different from those elicited by the wild-type VSV peptide. The differences found in the nature of the T cells responding to the E6 peptide include changes in both the V beta elements and the sequences of the complementarity-determining region 3 loops of their TCRs. Further experiments found that the E6 peptide can act as an antagonist for VSV-specific T cell hybridomas. To determine whether these differences in V beta usage, complementarity-determining region 3 sequences, and the switch from agonism to antagonism are caused by a conformational change on the MHC, the peptide, or both, we determined the crystal structure of the variant E6 peptide bound to H-2K(b). This structure shows that the only significant structural difference between H-2K(b)/E6 and the previously determined H-2K(b)/VSV is limited to the side chain of position 6 of the peptide, with no differences in the MHC molecule. Thus, a minor conformational change in the peptide can profoundly alter the biological outcome of the TCR-peptide/MHC interaction.     

Peptide-MHC Cellular Microarray with Innovative Data Analysis System for Simultaneously Detecting Multiple CD4 T-Cell Responses

Background

Peptide:MHC cellular microarrays have been proposed to simultaneously characterize multiple Ag-specific populations of T cells. The practice of studying immune responses to complicated pathogens with this tool demands extensive knowledge of T cell epitopes and the availability of peptide:MHC complexes for array fabrication as well as a specialized data analysis approach for result interpretation.

Methodology/Principal Findings

We co-immobilized peptide:DR0401 complexes, anti-CD28, anti-CD11a and cytokine capture antibodies on the surface of chamber slides to generate a functional array that was able to detect rare Ag-specific T cell populations from previously primed in vitro T cell cultures. A novel statistical methodology was also developed to facilitate batch processing of raw array-like data into standardized endpoint scores, which linearly correlated with total Ag-specific T cell inputs. Applying these methods to analyze Influenza A viral antigen-specific T cell responses, we not only revealed the most prominent viral epitopes, but also demonstrated the heterogeneity of anti-viral cellular responses in healthy individuals. Applying these methods to examine the insulin producing beta-cell autoantigen specific T cell responses, we observed little difference between autoimmune diabetic patients and healthy individuals, suggesting a more subtle association between diabetes status and peripheral autoreactive T cells.

Conclusions/Significance

The data analysis system is reliable for T cell specificity and functional testing. Peptide:MHC cellular microarrays can be used to obtain multi-parametric results using limited blood samples in a variety of translational settings.     

Characterization of inhibitory T cells induced by an analog of type II collagen in an HLA-DR1 humanized mouse model of autoimmune arthritis

Introduction

We used DR1 transgenic mice and covalently linked DR1 multimers to characterize analog-specific inhibitory T cells in collagen-induced arthritis (CIA). Because of the low numbers of antigen-specific T cells in wild-type mice, functional T-cell studies in autoimmune arthritis have been challenging. The use of T-cell receptor (TCR) transgenic mice has provided useful information, but such T cells may not represent the heterogeneous T-cell response that occurs in natural settings. Our focus was to develop tools to identify and characterize the population of immunoregulatory T cells induced in wild-type mice by an analog peptide of CII_259-273, which contains amino acid substitutions at positions 263 (N) and 266 (D) (analog peptide A12).

Methods

DR1 multimers, developed by loading empty class II molecules with exogenous peptide, provide a method for visualizing antigen-specific T cells with flow cytometry. However, the low binding avidity of A12 for the major histocompatibility complex (MHC) made this strategy untenable. To overcome this problem, we generated DR1 multimers in which the analog peptide A12 was covalently linked, hoping that the low-avidity analog would occupy enough binding clefts to allow detection of the responsive T cells.

Results

Staining with the tetramer revealed that A12-specific T cells were readily detectable at 10 days after immunization. These CD4(+) T cells are a highly selective subset of the TCR repertoire and have a limited clonality. Analysis of cytokine expression showed that cells detected by tetramer (A12) expressed primarily suppressive cytokines (interleukin-4 (IL-4) and IL-10) in response to collagen, compared with control cells. Although they did not express Fox-p3, they were extremely effective in preventing and suppressing inflammatory arthritis.

Conclusions

In summary, our studies showed that the use of covalently linked multimers allows characterization of analog-specific T cells that are otherwise difficult to detect. The suppressive character of the analog-specific T-cell response suggests that these cells attenuate autoimmunity and differ significantly in phenotype from the inflammatory T cells predominantly found in arthritic joints. Such reagents will become powerful tools to study T-cell responses in RA patients in upcoming clinical trials.     

Branched short elastin-like peptides with temperature responsiveness obtained by EDTA-mediated multimerization

Elastin-like peptides (ELPs) exhibit a reversible phase transition, known as coacervation, triggered by temperature changes. This property makes them useful as stimuli-responsive molecular materials for various applications. Among ELPs, short peptide chain lengths have some advantages over long peptide chain lengths because short ELPs can be easily obtained by chemical synthesis, allowing the use of various amino acids, including D-type and unnatural amino acids, at any position in the sequence. Moreover, the incorporated amino acids readily affect the temperature-responsive behavior of ELPs. However, to be utilized in various applications, it is necessary to develop short ELPs and to investigate their temperature-responsive properties. To obtain further insights into the temperature-responsive behavior of the short ELPs, we investigated branched short ELP analogs composed of (FPGVG)_n chains (n = 1 or 2, abbreviated as F1 and F2, respectively). We synthesized multimers composed of four F1 chains or two to four F2 chains using ethylenediaminetetraacetic acid (EDTA) as a central component of multimerization. Our results show that the multimers obtained exhibited coacervation in aqueous solutions whereas linear F1 or F2 did not. Furthermore, the structural features of the obtained multimers were the same as those of linear (FPGVG)₄. In this study, we demonstrated that molecules capable of coacervation can be obtained by multimerization of F1 or F2. The temperature-responsive molecules obtained using short ELPs make it possible to use them as easy-to-synthesize peptide tags to confer temperature responsiveness to various molecules, which will aid the development of temperature-responsive biomaterials with a wide variety of functions.     

Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils

Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ_1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ_1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ_1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.     

Induction of tumor-reactive T helper responses by a posttranslational modified epitope from tumor protein p53

Posttranslational modifications regulate the function and stability of proteins, and the immune system is able to recognize some of these modifications. Therefore, the presence of posttranslational modifications increases the diversity of potential immune responses to a determinant antigen. The stimulation of tumor-specific CD4⁺ helper T lymphocytes (HTLs) is considered important for the production of anti-tumor antibodies by B cells and for the generation and persistence of CD8⁺ cytotoxic T lymphocytes, and in some instances, HTLs can directly reduce tumor cell growth. Identification of MHC class II-restricted peptide epitopes from tumor-associated antigens including those generated from posttranslational protein modifications should enable the improvement of peptide-based cancer immunotherapy. We describe here an MHC class II binding peptide from the tumor protein p53, which possesses an acetylated lysine at position 120 (p53_{110-124/AcK120}) that is effective in eliciting CD4⁺ T cell responses specific for the acetylated peptide. Most importantly, the acetylated peptide-reactive CD4 HTLs recognized the corresponding naturally processed posttranslational modified epitope presented by either dendritic cells loaded with tumor cell lysates or directly on tumors expressing p53 and the restricting MHC class II molecules. Treatment of tumor cells with a histone deacetylase inhibitor augmented their recognition by the p53_{110-124/AcK120}-reactive CD4⁺ T cells. These findings prove that the epitope p53_{110-124/AcK120} is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.