MHC Class II-restricted Peptides Research Articles

Retraction.

Retraction: RajKishor Pandey, Manas Ranjan Dikhit, Avinash Kumar, Budheswar Dehury, Krishna Pandey, Roshan Kamal Topno, Pradeep Das, Sanjiva Bimal (2020) Evaluating the immunomodulatory responses of LdODC-derived MHC Class-II restricted peptides against VL. Parasite Immunology 42(4): e12699; https://doi.org/10.1111/pim.12699 The above article, published online on 24 January 2020, in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editors-in-Chief, Eleanor M. Riley and Richard K. Grencis, and John Wiley and Sons Ltd. Following publication, concerns were raised by a third party regarding similarities in Figures 3 and 5. The authors were unable to provide a satisfactory explanation and could not provide the original data. The retraction has been agreed because of concerns that the figures were duplicated, affecting the interpretation of the data and results presented.

Failure of thymic deletion and instability of autoreactive Tregs drive autoimmunity in immune-privileged liver.

The liver is an immune-privileged organ that can deactivate autoreactive T cells. Yet in autoimmune hepatitis (AIH), autoreactive T cells can defy hepatic control and attack the liver. To elucidate how tolerance to self-antigens is lost during AIH pathogenesis, we generated a spontaneous mouse model of AIH, based on recognition of an MHC class II–restricted model peptide in hepatocytes by autoreactive CD4+ T cells. We found that the hepatic peptide was not expressed in the thymus, leading to deficient thymic deletion and resulting in peripheral abundance of autoreactive CD4+ T cells. In the liver, autoreactive CD4+ effector T cells accumulated within portal ectopic lymphoid structures and maturated toward pathogenic IFN-γ and TNF coproducing cells. Expansion and pathogenic maturation of autoreactive effector T cells was enabled by a selective increase of plasticity and instability of autoantigen-specific Tregs but not of nonspecific Tregs. Indeed, antigen-specific Tregs were reduced in frequency and manifested increased IL-17 production, reduced epigenetic demethylation, and reduced expression of Foxp3. As a consequence, autoantigen-specific Tregs had a reduced suppressive capacity, as compared with that of nonspecific Tregs. In conclusion, loss of tolerance and the pathogenesis of AIH were enabled by combined failure of thymic deletion and peripheral regulation.

In Vitro Activation of Macrophages by an MHC Class II-restricted Trichomonas Vaginalis TvZIP8-derived Synthetic Peptide

ABSTRACT Background Macrophages play an important role in the inflammatory response towards pathogens and their effector functions depend on the mode of activation which is mediated by recognition of pathogen-associated molecular patterns, as peptides. Trichomonas vaginalis provokes an inflammatory response in the host in which macrophages are the first line of defense. This study aimed to analyze the effect of a specific peptide derived from the transporter TvZIP8 of T. vaginalis on the activation of macrophages. Methods An in silico approach based on computational prediction of epitopes was applied to detect potential murine MHC class II-restricted peptides from TvZIP8 that can activate macrophages and the immunomodulatory activity was evaluated by in vitro stimulation of murine macrophages. Results Based on binding scores, one peptide denominated TvZIP8-pep was selected for further analysis. In vitro stimulation with synthetic TvZIP8-pep triggered on murine macrophages the NO and H2O2 production and an overexpression of iNOS and NOX-2 genes. Also, a significant increase of pro-inflammatory cytokines: IL-1β, IL-6, and TNF-α, as well as, overexpression of the TLR4, MyD88, and NF-κB genes and NF-κB activation were observed on macrophages after stimulation with TvZIP8-pep in vitro. Moreover, higher levels of IFN-γ were detected in co-cultures using CD4 + T cells with TvZIP8-pep-stimulated macrophages Conclusion These results support the potential of TvZIP8 as a promising antigen to stimulates a specific macrophage response against T. vaginalis, but further analyses are required to evaluate their possible potential as a novel antigen for immunodiagnosis and/or vaccine against trichomoniasis.

Vaccinia Virus Vectors Targeting Peptides for MHC Class II Presentation to CD4+ T Cells.

CD4+ helper T cells play important roles in providing help to B cells, macrophages, and cytotoxic CD8+ T cells, but also exhibit direct effector functions against a variety of different pathogens. In contrast to CD8+ T cells, CD4+ T cells typically exhibit broader specificities and undergo less clonal expansion during many types of viral infections, which often makes the identification of virus-specific CD4+ T cells technically challenging. In this study, we have generated recombinant vaccinia virus (VacV) vectors that target I-Ab-restricted peptides for MHC class II (MHC-II) presentation to activate CD4+ T cells in mice. Conjugating the lymphocytic choriomeningitis virus immunodominant epitope GP61-80 to either LAMP1 to facilitate lysosomal targeting or to the MHC-II invariant chain (Ii) significantly increased the activation of Ag-specific CD4+ T cells in vivo. Immunization with VacV-Ii-GP61-80 activated endogenous Ag-specific CD4+ T cells that formed memory and rapidly re-expanded following heterologous challenge. Notably, immunization of mice with VacV expressing an MHC-II-restricted peptide from Leishmania species (PEPCK335-351) conjugated to either LAMP1 or Ii also generated Ag-specific memory CD4+ T cells that underwent robust secondary expansion following a visceral leishmaniasis infection, suggesting this approach could be used to generate Ag-specific memory CD4+ T cells against a variety of different pathogens. Overall, our data show that VacV vectors targeting peptides for MHC-II presentation is an effective strategy to activate Ag-specific CD4+ T cells in vivo and could be used to study Ag-specific effector and memory CD4+ T cell responses against a variety of viral, bacterial, or parasitic infections.

Effects of Viral Peptide Presentation on CD4+ T Cell Responses to MHC Class II-Restricted Tumor Peptides

Background: Designing a vaccine against a defined tumor is a promising but complicated process that can be used as tumor immunotherapy. The presence of human telomerase reverse transcriptase (hTERT) has been proved in about 85% of tumors; therefore, this enzyme is a promising vaccine target. Most studies in tumor immunotherapy have focused on induction and reinforcement of CD8+ T cell responses against tumor antigens, but some evidence indicates that CD4+ T cell responses are important for induction of CD8+ T cell response and memory. Therefore, in this in vitro study, we combined an hTERT peptide with HIV peptides restricted to HLA-DRB1*11:03/04, the most common MHC class II molecule in Iran, to induce CD4+ T cell responses in healthy individuals and lung cancer patients. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation from nine healthy individuals and six lung cancer patients who were HLA-DRB1*11:03/04 positive. PBMCs were incubated with the tumoral and viral peptides for five days and then exposed to A549 cells to evaluate specific proliferative responses. The proliferation of PBMCs was examined by the MTT assay. Then, levels of secreted interferon-γ in culture media were analyzed by enzyme-linked immunosorbent assay (ELISA). Results: Proliferation of PBMCs was significantly greater in the presence of the tumoral peptide than in controls (P = 0.001). In addition, PBMC proliferation was greater when the viral peptides were added than with the tumoral peptide alone (P = 0.000). The IFN-γ secretion assay results supported the MTT assay (P = 0.000). Conclusions: In this study, we demonstrated that the hTERT-derived peptide induced CD4+ T cell proliferation in healthy individuals and patients with lung cancer. Both PBMC proliferation and IFN-γ secretion were further induced by the addition of tumor and viral peptides in combination.

Novel role for scavenger receptor B-I in neutrophilic asthma (IRC10P.419)

Abstract Asthma is a heterogeneous airway disease that has risen significantly in recent decades. While there has been a major research focus on eosinophilic asthma, the neutrophilic, Th17-predominant disease that characterizes severe asthmatics is less understood. Scavenger Receptor B-I (SR-BI) is a receptor mostly studied in the context of cellular uptake of cholesterol. Recently, we reported that SR-BI regulates the pulmonary innate immune response, but the role of SR-BI in asthma is unknown. Therefore, SR-BI+/+ and SR-BI-/- mice were sensitized on days 0 and 7 by oropharyngeal aspiration of ovalbumin (OVA) together with LPS and then challenged on day 14 with an aerosol OVA. Airway inflammation and cytokines were quantified 48hrs after challenge. Bone marrow-derived dendritic cells (BMDCs) from SR-BI+/+ and SR-BI-/- mice were co-cultured in the presence of OVA/LPS with CD4+ T cells from OT-II mice that carry a transgenic TCR specific for the MHC class II-restricted OVA peptide (OT-II Tg). Compared to SR-BI+/+ counterparts, SR-BI-/- mice had increased pulmonary neutrophils, Th17 cells, G-CSF and CXCL5 after OVA challenge, but had equivalent amounts of IL-4, IL-5, and eosinophils. BMDCs from SR-BI-/- mice have elevated levels of CD80 and CD86, and CD4+ OT-II Tg T cells produced more IL-17 after culture with OVA/LPS. SR-BI modulates the lung’s response to environmental adjuvants such as LPS by regulating Th17 polarization, suggesting a novel role for SR-BI in neutrophilic asthma.

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 (p53110-124/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 p53110-124/AcK120-reactive CD4(+) T cells. These findings prove that the epitope p53110-124/AcK120 is immunogenic for anti-tumor responses and is likely to be useful for cancer immunotherapy.

Epitope diversification driven by non-tumor epitope-specific Th1 and Th17 mediates potent antitumor reactivity

MHC class I-restricted peptide-based vaccination therapies have been conducted to treat cancer patients, because CD8+ CTL can efficiently induce apoptosis of tumor cells in an MHC class I-restricted epitope-specific manner. Interestingly, clinical responders are known to demonstrate reactivity to epitopes other than those used for vaccination; however, the mechanism underlying how antitumor T cells with diverse specificity are induced is unclear. In this study, we demonstrated that dendritic cells (DCs) that engulfed apoptotic tumor cells in the presence of non-tumor MHC class II-restricted epitope peptides, OVA323–339, efficiently presented tumor-associated antigens upon effector-dominant CD4+ T cell balance against regulatory T cells (Treg) for the OVA323–339 epitope. Th1 and Th17 induced tumor-associated antigens presentation of DC, while Th2 ameliorated tumor-antigen presentation for CD8+ T cells. Blocking experiments with anti-IL-23p19 antibody and anti-IL-23 receptor indicated that an autocrine mechanism of IL-23 likely mediated the diverted tumor-associated antigens presentation of DC. Tumor-associated antigens presentation of DC induced by OVA323–339 epitope-specific CD4+ T cells resulted in facilitated antitumor immunity in both priming and effector phase in vivo. Notably, this immunotherapy did not require pretreatment to reduce Treg induced by tumor. This strategy may have clinical implications for designing effective antitumor immunotherapies.

Generation of Multi-Antigen Specific T Cells for Adoptive Immunotherapy of Myeloid Leukemia and Identification of MHC Class I and II-Restricted Peptides for WT1, Proteinase 3 and Human Neutrophil Elastase

Abstract 2985Generating cytotoxic T lymphocytes (CTL) against malignant cells using defined leukemia associated antigen (LAA) vaccines, or ex vivo adoptively transferred T cells, is a promising approach to prevent or treat leukemia. Vaccine or T cell therapies have largely focused on CD8 responses to defined HLA-A2 restricted LAA peptides. Strategies targeting single peptides are limited to the recipient’s HLA type, lack of CD4 help, and risk of tumor immune escape. To overcome these constraints and develop T cell immunotherapy for patients with myeloid malignancies, we used entire peptide libraries of several LAA to generate multi-specific CD4+ and CD8+ leukemia–reactive CTL. We obtained 15mer peptide libraries overlapping by 11 aa for WT1, Proteinase 3 (Pr3), MAGE-A3, PRAME and human neutrophil elastase (HNE). PBMC of 10 healthy donors were stimulated with DC pulsed with peptide libraries of one antigen or the mix of 5 LAA (LAA mix). Cultures were re-stimulated weekly with peptide-pulsed DCs and peptide-recognition was tested by IFN γ-ELIspot. Positive cultures were further characterized by performing intracellular cytokine staining, measuring degranulation by CD107a release, and assessing in vitro cytolytic activity using peptide-pulsed autologous PHA blasts as targets in a flow cytometric killing assay.Nine out of 10 healthy donors responded to the LAA mix as assessed by IFN γ-ELIspot (mean: 471.2 SFU/2×105 cells, range: 296–968.5). The expanded CTL specifically recognized 3 antigens (mean, range 1–5), and PRAME was most frequently recognized (in 9 of 10 donors screened). Within donors we consistently saw recognition of the same profile of antigens irrespective of whether the CTL were induced with individual antigen or LAA mix, indicating that antigenic competition between LAA was not a limiting factor in our cultures. Correlating with ELIspot results, LAA CTL were cytotoxic to PHAB pulsed with the LAA mix (E:T=40:1; mean: 33.1%, range: 12.4–80.0%; negative control: PHAB: mean: 4.2%, range: 0–13.4%; n=7), as well as individual antigens (WT1: mean :12.2%, range: 0–26.1%; Pr3: mean: 9.7%, range: 5.6–13.7%; HNE: mean: 35.4%, range: 0–84.9%; MAGE-A3: mean: 19.9%, range: 15.2–28.2%; PRAME: mean: 16.7%, range: 3.8–37.5%). Cytotoxicity against the peptide library or the LAA mix was greater than against targets pulsed with the immunodominant peptide, demonstrating the benefit of a broad immune response. Cytolytic activity was also seen in donors with predominantly CD4+ response, suggesting a role for T helper cells in tumor lysis.The approach of stimulation with LAA mix was also feasible for epitope mapping. In addition to known peptides, we identified 16 new MHC class I and II-restricted peptides (WT1: 8, HNE: 6 and Pr3: 2). Thereby, up to 4 immunogenic peptides of two different antigens could be identified in one LAA mix culture. WT1 peptides showed class I and class II-restriction by blocking experiments with HLA antibodies. HNE peptides were mainly class I-restricted, with a dominant response against RRLACLFLACVLPAL in 5 of 7 donors.Thus, stimulation of healthy donor PBMC with LAA mix generates CTL without depending on known HLA-restrictions and may be a practical approach for adoptive immunotherapy of myeloid leukemia after stem cell transplantation. Moreover, the strategy enabled the identification of several new class I and II-restricted peptides. Importantly, in vitro cytolytic activity could also be shown for MHC class II-restricted peptides, indicating the importance of CD4+ T cells for anti-leukemia responses. Disclosures:No relevant conflicts of interest to declare.

Enhanced presentation of MHC class Ia, Ib and class II-restricted peptides encapsulated in biodegradable nanoparticles: a promising strategy for tumor immunotherapy

BackgroundMany peptide-based cancer vaccines have been tested in clinical trials with a limited success, mostly due to difficulties associated with peptide stability and delivery, resulting in inefficient antigen presentation. Therefore, the development of suitable and efficient vaccine carrier systems remains a major challenge.MethodsTo address this issue, we have engineered polylactic-co-glycolic acid (PLGA) nanoparticles incorporating: (i) two MHC class I-restricted clinically-relevant peptides, (ii) a MHC class II-binding peptide, and (iii) a non-classical MHC class I-binding peptide. We formulated the nanoparticles utilizing a double emulsion-solvent evaporation technique and characterized their surface morphology, size, zeta potential and peptide content. We also loaded human and murine dendritic cells (DC) with the peptide-containing nanoparticles and determined their ability to present the encapsulated peptide antigens and to induce tumor-specific cytotoxic T lymphocytes (CTL) in vitro.ResultsWe confirmed that the nanoparticles are not toxic to either mouse or human dendritic cells, and do not have any effect on the DC maturation. We also demonstrated a significantly enhanced presentation of the encapsulated peptides upon internalization of the nanoparticles by DC, and confirmed that the improved peptide presentation is actually associated with more efficient generation of peptide-specific CTL and T helper cell responses.ConclusionEncapsulating antigens in PLGA nanoparticles offers unique advantages such as higher efficiency of antigen loading, prolonged presentation of the antigens, prevention of peptide degradation, specific targeting of antigens to antigen presenting cells, improved shelf life of the antigens, and easy scale up for pharmaceutical production. Therefore, these findings are highly significant to the development of synthetic vaccines, and the induction of CTL for adoptive immunotherapy.

The use of binding-prediction models to identify M. bovis-specific antigenic peptides for screening assays in bovine tuberculosis

The identification of MHC class II-restricted antigenic peptides for inclusion into vaccines and/or as diagnostic test reagents for mycobacterial infections remains a high research priority. To expedite discovery of such peptides, numerous bioinformatic tools have been developed to predict whether a given peptide is likely to form a stable binding interaction with MHC class II molecules. However, no prediction tool dedicated to the identification of bovine MHC (BoLA) class II-restricted peptides is currently available. Using experimental immunogenicity data derived from the stimulation of whole blood of Mycobacterium bovis-infected cattle with 105 individual M. bovis-derived peptides, we have compared the ability of a novel BoLA DRB3 structure-based prediction method (Hepitom) with the human MHC class II binding predictor model ProPred in predicting peptides that induce bovine T-cell activation. When a stringent cut off for considering peptide antigenicity was applied, the sensitivities of Hepitom and ProPred in detecting immunogenic peptides were 62% and 77%, respectively. In contrast, the Hepitom model showed greater specificity, with values of 66% and 34% for Hepitom and ProPred, respectively. Using all peptides, seven out of eleven M. bovis proteins were identified as being highly immunogenic. All but one of these antigens were also identified when just the Hepitom predicted peptides were used, while only four of the seven were identified using the ProPred predicted peptides. In conclusion, we demonstrate that the Hepitom model is a useful pre-screening tool to select peptides for further immunogenicity studies in cattle without major impact on the identification of antigenic M. bovis proteins.

Tumors Hamper the Immunogenic Competence of CD4+ T Cell-Directed Dendritic Cell Vaccination

Dendritic cells loaded with tumor-derived peptides induce protective CTL responses and are under evaluation in clinical trails. We report in this study that prophylactic administration of dendritic cells loaded with a MHC class II-restricted peptide derived from a model tumor Ag (Leishmania receptor for activated C kinase (LACK)) confers protection against LACK-expressing TS/A tumors, whereas therapeutic vaccination fails to cure tumor-bearing mice. Although CD4+ T cell-directed dendritic cell vaccination primed effector-like (CD44(high)CD62L(low), IL-2(+), IFN-gamma(+)) and central memory-like lymphocytes (CD44(high)CD62L(high), only IL-2(+)) in tumor-free mice, this was not the case in tumor-bearing animals in which both priming and persistence of CD4+ T cell memory were suppressed. Suppression was specific for the tumor-associated Ag LACK, and did not depend on CD25+ T cells. Because T cell help is needed for protective immunity, we speculate that the ability of tumors to limit vaccine-induced CD4+ T cell memory could provide a partial explanation for the limited efficacy of current strategies.

Characterization of the MUC1.Tg/MIN transgenic mouse as a model for studying antigen-specific immunotherapy of adenomas

A bigenic MUC1.Tg/MIN mouse model was developed by crossing Apc/(MIN/+) (MIN) mice with human MUC1 transgenic mice to evaluate MUC1 antigen-specific immunotherapy of intestinal adenomas. The MUC1.Tg/MIN mice developed adenomas at a rate comparable to that of MIN mice and had similar levels of serum MUC1 antigen. A MUC1-based vaccine consisting of MHC class I-restricted MUC1 peptides, a MHC class II-restricted pan-helper peptide, unmethylated CpG oligodeoxynucleotide and GM-CSF caused flattening of adenomas and significantly reduced the number of large adenomas. Immunization was successful in generating a MUC1-directed immune response evidenced by increased MUC1 peptide-specific anti-tumor cytotoxicity and IFN-gamma secretion by lymphocytes.

70‐kDa heat shock proteins: Specific interactions with HLA‐DR molecules and their peptide fragments

Heat shock protein 70 (HSP70):peptide complexes are involved in MHC class I and class II-restricted antigen presentation enabling enhanced activation of antigen-specific T cells. Here, we investigated the potential of bacterial and mammalian HSP70 molecules to interact with peptide fragments from HLA-DR and the corresponding complete HLA-DR molecules. Peptide fragments were found to interact with DnaK, the HSP70 homologue from E. coli, but less with stress-inducible human Hsp70. Only a peptide sequence exclusively found in rheumatoid arthritis-protective HLA-DR molecules did not interact with DnaK. Subsequently, we investigated the interaction of complete HLA-DR molecules with HSP70 and detected a specific HSP70:HLA-DR interaction, with highest affinity for human stress-inducible Hsp70. In contrast to the peptide fragments, no allele-specific differences in Hsp70 affinity were detected with complete HLA-DR molecules. Interaction with HLA-DR molecules was increased at lowered pH values, whereas HSP70-chaperoned peptides were released at acidic pH, thus HSP70 could serve as scanner and carrier for antigenic peptides of self or foreign origin and transfer chaperoned peptides onto MHC class II molecules in acidic late endosomal compartments. Our findings indicate that direct interaction between mammalian HSP70 and HLA-DR molecules could be involved in the HSP70-mediated enhancement of MHC class II-restricted peptide presentation and CD4(+) T cell activation.

HBcAg-specific CD4+CD25+regulatory T cells modulate immune tolerance and acute exacerbation on the natural history of chronic hepatitis B virus infection

Acute exacerbations (AEs) of chronic hepatitis B (CH-B) are accompanied by increased T cell responses to hepatitis B core and e antigens (HBcAg/HBeAg). Why patients are immunotolerant (IT) to the virus and why AEs occur spontaneously on the immunoactive phase remain unclear. The role of HBcAg-specific CD4(+)CD25(+) regulatory T (T(reg)) cells in AE and IT phases was investigated in this study. The SYFPEITHI scoring system was employed to predict MHC class II-restricted epitope peptides on HBcAg overlapping with HBeAg that were used for T(reg)-cell cloning and for the construction of MHC class II tetramers to measure T(reg) cell frequencies (T(reg) f). The results showed that HBcAg-specific T(reg) f declined during AE accompanied by increased HBcAg peptide-specific cytotoxic T lymphocyte frequencies. Predominant Foxp3-expressing T(reg) cell clones were generated from patients on the immune tolerance phase, while the majority of Th1 clones were obtained from patients on the immunoactive phase. T(reg) cells from liver and peripheral blood of CH-B patients express CD152 and PD1 antigens that exhibit suppression on PBMCs proliferation to HBcAg. These data suggest that HBcAg peptide-specific T(reg) cells modulate the IT phase, and that their decline may account for the spontaneous AEs on the natural history of chronic hepatitis B virus infection.

Peptide-binding assays and HLA II transgenic Aβ° mice are consistent and complementary tools for identifying HLA II-restricted peptides

The identification of MHC class II-restricted peptides has become a priority for the development of peptide-based prophylactic and therapeutic vaccines. The aim of this study was to assess the correlations between peptide-binding assays on purified HLA II molecules and immunization of human HLA II transgenic mice deficient in murine class II molecules (Aβ°). We used as models two MHC class II-restricted peptides, one derived from the HIV Nef regulatory protein (Nef 56–68) and the other from the Schistosoma mansoni 28-kDa glutathione-S-transferase (Sm28GST 190–211). High correlations were found between the two approaches, which showed that the Nef 56–68 and Sm28GST 190–211 peptides may represent promiscuous ligands for HLA-DQ and for HLA-DR molecules, respectively . We suggest a rational method based on the combination of peptide-binding assays and HLA II transgenic mice experiments as consistent and complementary tools for selecting T helper epitopes.

Defining MHC class II T helper epitopes for WT1 tumor antigen.

The product of Wilms' tumor gene 1 (WT1) is overexpressed in diverse human tumors, including leukemia, lung and breast cancer, and is often recognized by antibodies in the sera of patients with leukemia. Since WT1 encodes MHC class I-restricted peptides recognized by cytotoxic T lymphocytes (CTL), WT1 has been considered as a promising tumor-associated antigen (TAA) for developing anticancer immunotherapy. In order to carry out an effective peptide-based cancer immunotherapy, MHC class II-restricted epitope peptides that elicit anti-tumor CD4(+) helper T lymphocytes (HTL) will be needed. In this study, we analyzed HTL responses against WT1 antigen using HTL lines elicited by in vitro immunization of human lymphocytes with synthetic peptides predicted to serve as HTL epitopes derived from the sequence of WT1. Two peptides, WT1(124-138) and WT1(247-261), were shown to induce peptide-specific HTL, which were restricted by frequently expressed HLA class II alleles. Here, we also demonstrate that both peptides-reactive HTL lines were capable of recognizing naturally processed antigens presented by dendritic cells pulsed with tumor lysates or directly by WT1+ tumor cells that express MHC class II molecules. Interestingly, the two WT1 HTL epitopes described here are closely situated to known MHC class I-restricted CTL epitopes, raising the possibility of stimulating CTL and HTL responses using a relatively small synthetic peptide vaccine. Because HTL responses to TAA are known to be important for promoting long-lasting anti-tumor CTL responses, the newly described WT1 T-helper epitopes could provide a useful tool for designing powerful vaccines against WT1-expressing tumors.

Simultaneous Induction of CD4 T Cell Tolerance and CD8 T Cell Immunity by Semimature Dendritic Cells

Previous studies suggested that depending on their maturation state, dendritic cells (DC) could either induce T cell tolerance (immature and semimature DC) or T cell activation (mature DC). Pretreatment of C57BL/6 mice with encephalitogenic myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide-loaded semimature DC protected from MOG-induced autoimmune encephalomyelitis. This protection was mediated by IL-10-producing CD4 T cells specific for the self Ag. Here we show that semimature DC loaded with the MHC class II-restricted nonself peptide Ag (OVA) induce an identical regulatory T cell cytokine pattern. However, semimature DC loaded simultaneously with MHC class II- and MHC class I-restricted peptides, could efficiently initiate CD8 T cell responses leading to autoimmune diabetes in a TCR-transgenic adoptive transfer model. Double-peptide-loaded semimature DC also induced simultaneously in the same animal partially activated CD8 T cells with cytolytic function as well as protection from MOG-induced autoimmune encephalomyelitis. Our study suggests that the decision between tolerance and immunity not only depends on the DC, but also on the type and activation requirements of the responding T cell.

Long-Term Stable Expanded Human CD4+ T Cell Clones Specific for Human Cytomegalovirus Are Distributed in Both CD45RAhigh and CD45ROhigh Populations

T cells play an important role in the control of human CMV (HCMV) infection. Peripheral blood CD4+ T cell proliferative responses to the HCMV lower tegument protein pp65 have been detected in most healthy HCMV carriers. To analyze the clonal composition of the CD4+ T cell response against HCMV pp65, we characterized three MHC class II-restricted peptide epitopes within pp65 in virus carriers. In limiting dilution analysis, we observed high frequencies of pp65 peptide-specific CD4+ T cells, many of which expressed peptide-specific cytotoxicity in addition to IFN-gamma secretion. We analyzed the clonal composition of CD4+ T cells specific for defined HCMV peptides by generating multiple independent peptide-specific CD4+ clones and sequencing the TCR beta-chain. In a given carrier, most of the CD4+ clones specific for a defined pp65 peptide had identical TCR nucleotide sequences. We used clonotype oligonucleotide probing to quantify the size of individual peptide-specific CD4+ clones in whole PBMC and in purified subpopulations of CD45RAhighCD45ROlow and CD45RAlowCD45ROhigh cells. Individual CD4+ T cell clones could be large (0.3-1.5% of all CD4+ T cells in PBMC) and were stable over time. Cells of a single clone were distributed in both the CD45RAhigh and CD45ROhigh subpopulations. In one carrier, the virus-specific clone was especially abundant in the small CD28-CD45RAhigh CD4+ T cell subpopulation. Our study demonstrates marked clonal expansion and phenotypic heterogeneity within daughter cells of a single virus-specific CD4+ T cell clone, which resembles that seen in the CD8+ T cell response against HCMV pp65.

Peptide immunisation of HLA-DR-transgenic mice permits the identification of a novel HLA-DRbeta1*0101- and HLA-DRbeta1*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-DRbeta1*0101 (HLA-DR1) and HLA-DRbeta1*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.