Structural Framework for Analysis of CD4+ T-Cell Epitope Dominance in Viral Fusion Proteins.

Antigen conformation shapes CD4+ T-cell specificity through mechanisms of antigen processing, and the consequences for immunity may rival those from conformational effects on antibody specificity. CD4+ T cells initiate and control immunity to pathogens and cancer and are at least partly responsible for immunopathology associated with infection, autoimmunity, and allergy. The primary trigger for CD4+ T-cell maturation is the presentation of an epitope peptide in the MHC class II antigen-presenting protein (MHCII), most commonly on an activated dendritic cell, and then the T-cell responses are recalled by subsequent presentations of the epitope peptide by the same or other antigen-presenting cells. Peptide presentation depends on the proteolytic fragmentation of the antigen in an endosomal/lysosomal compartment and concomitant loading of the fragments into the MHCII, a multistep mechanism called antigen processing and presentation. Although the role of peptide affinity for MHCII has been well studied, the role of proteolytic fragmentation has received less attention. In this Perspective, we will briefly summarize evidence that antigen resistance to unfolding and proteolytic fragmentation shapes the specificity of the CD4+ T-cell response to selected viral envelope proteins, identify several remarkable examples in which the immunodominant CD4+ epitopes most likely depend on the interaction of processing machinery with antigen conformation, and outline how knowledge of antigen conformation can inform future efforts to design vaccines.

Identification of an immunodominant region on a group A Streptococcus T-antigen reveals temperature-dependent motion in pili.

Group A Streptococcus (GAS) is a globally important pathogen causing a broad range of human diseases. GAS pili are elongated proteins with a backbone comprised repeating T-antigen subunits, which extend from the cell surface and have important roles in adhesion and establishing infection. No GAS vaccines are currently available, but T-antigen-based candidates are in pre-clinical development. This study investigated antibody-T-antigen interactions to gain molecular insight into functional antibody responses to GAS pili. Large, chimeric mouse/human Fab-phage libraries generated from mice vaccinated with the complete T18.1 pilus were screened against recombinant T18.1, a representative two-domain T-antigen. Of the two Fab identified for further characterization, one (designated E3) was cross-reactive and also recognized T3.2 and T13, while the other (H3) was type-specific reacting with only T18.1/T18.2 within a T-antigen panel representative of the major GAS T-types. The epitopes for the two Fab, determined by x-ray crystallography and peptide tiling, overlapped and mapped to the N-terminal region of the T18.1 N-domain. This region is predicted to be buried in the polymerized pilus by the C-domain of the next T-antigen subunit. However, flow cytometry and opsonophagocytic assays showed that these epitopes were accessible in the polymerized pilus at 37°C, though not at lower temperature. This suggests that there is motion within the pilus at physiological temperature, with structural analysis of a covalently linked T18.1 dimer indicating "knee-joint" like bending occurs between T-antigen subunits to expose this immunodominant region. This temperature dependent, mechanistic flexing provides new insight into how antibodies interact with T-antigens during infection.

Analysis of antibody binding specificities in twin and SNP-genotyped cohorts reveals that antiviral antibody epitope selection is a heritable trait.

Human immune responses to viral infections are highly variable, but the genetic factors that contribute to this variability are not well characterized. We used VirScan, a high-throughput epitope scanning technology, to analyze pan-viral antibody reactivity profiles of twins and SNP-genotyped individuals. Using these data, we determined the heritability and genomic loci associated with antibody epitope selection, response breadth, and control of Epstein-Barr virus (EBV) viral load. 107 EBV peptide reactivities were heritable and at least two Epstein-Barr nuclear antigen 2 (EBNA-2) reactivities were associated with variants in the MHC class II locus. We identified an EBV serosignature that predicted viral load in peripheral blood mononuclear cells and was associated with variants in the MHC class I locus. Our study illustrates the utility of epitope profiling to investigate the genetics of pathogen immunity, reports heritable features of the antibody response to viruses, and identifies specific HLA loci important for EBV epitope selection.

Structural assessment of HLA-A2-restricted SARS-CoV-2 spike epitopes recognized by public and private T-cell receptors.

T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T-cell receptors (TCRs) bound to their peptide-MHC targets is lacking. Here we determine the structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures reveal the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of the TCRs to recognize natural variants of RLQ but not YLQ. Neither TCR recognizes homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.

Variations of P40 and P63 Immunostains in Different Grades of Esophageal Squamous Cell Carcinoma: A Potential Diagnostic Pitfall.

The distinction of esophageal squamous cell carcinoma (SCC) from adenocarcinoma (adenoCA) using targeted therapies has become critical for small biopsies. In the United States, esophageal SCC is relatively uncommon compared with AdenoCA, with only few detailed immunohistochemical (IHC) studies on esophageal SCC. We characterized p40 and p63 IHC across various grades of squamous differentiation in esophageal SCC and compared their sensitivities between esophageal SCC and adenoCA. Twenty-eight esophageal SCC and 26 esophageal adenoCA (control group) samples were stained for p40, p63, and CK5/6. All hematoxylin-and-eosin-stained SCC slides were reviewed. Tumors were graded according to the World Health Organization classification: well, moderately, or poorly differentiated (WD, MD, and PD, respectively). Considering morphological heterogeneity, individual differentiation components within the same tumor were scored separately (0% to 100%) according to the proportion of immunoreactive cells and marked as positive (≥5%) or negative (<5%). Among 28 esophageal SCC, 15 had mixed intratumoral differentiation. There were 16 WD, 19 MD, and 14 PD components. P40 immunoreactivity was significantly lower in WD than in MD or PD components (P<0.001), P63 immunoreactivity patterns were similar (P<0.001), while CK5/6 showed no differences (P>0.05). The sensitivities for SCC components were 98% (P40), 100% (P63), and 100% (CK5/6), while those for esophageal AdenoCA were significantly lower: 4% (P40), 4% (P63), and 8% (CK5/6). P40 and P63 were sensitive and specific for routine esophageal SCC diagnosis. However, their immunostaining was significantly lower in WD SCC than in higher grade tumors. IHC results for small biopsy specimens should be interpreted carefully, particularly in WD components.

Antigen-Specific In Vivo Killing Assay.

The in vivo killing assay allows the quantification of the antigen-specific killing capacity of Cytotoxic CD8+ T Lymphocytes (CTLs) in mice. CTLs are indeed known for the lysis of cells expressing foreign or modified antigen peptides on their MHC class I molecules. Here we describe the detailed protocol used for the in vivo specific lysis of cells expressing the H-2 Kb immunodominant CD8+ T-cell epitope of the OVA protein: an 8 amino acid peptide corresponding to the 257-264 region of OVA (SIINFEKL).

The dominantly expressed class II molecule from a resistant MHC haplotype presents only a few Marek's disease virus peptides by using an unprecedented binding motif.

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.

Autoantibodies to DSC3 in Pemphigus Exclusively Recognize Calcium-Dependent Epitope in Extracellular Domain 2.

Pemphigus is a group of autoimmune bullous diseases characterized by the presence of autoantibodies against adhesion molecules, desmogleins, and desmocollins (DSCs). The pathogenicity of anti-DSC3 antibodies in pemphigus has been demonstrated; however, its characteristics have not yet been elucidated. We aimed to analyze the characteristics of anti-DSC3 antibodies using DSC3 domain‒swapped desmoglein 2 molecules in which the prosequence and five extracellular (EC) domains of desmoglein 2 were replaced with the corresponding domains of human DSC3. Using these proteins, we established an ELISA and analyzed sera from 56 patients with pemphigus. In 34 pemphigus sera positive for DSC3 full-EC domains, 15 sera (44.1%) were positive for EC2 domain, whereas other domains were rarely positive. We assessed the reactivity to a calcium-dependent epitope in DSC3 by ELISA with EDTA. The reactivity with the EC2 domain was mostly compromised in the presence of EDTA. In the in vitro assay, IgG from patients with paraneoplastic pemphigus preadsorbed with EC2 prevented both reduction of DSC3 and keratinocyte dissociation as compared with that with EDTA-treated EC2. This study revealed a predominant recognition of calcium-dependent epitopes in EC2 domain by anti-DSC3 antibodies and its pathogenicity on keratinocyte adhesion through DSC3 depletion.

Heterotypic immunity against vaccinia virus in an HLA-B*07:02 transgenic mousepox infection model.

Vaccination with vaccinia virus (VACV) elicits heterotypic immunity to smallpox, monkeypox, and mousepox, the mechanistic basis for which is poorly understood. It is generally assumed that heterotypic immunity arises from the presentation of a wide array of VACV-derived, CD8+ T cell epitopes that share homology with other poxviruses. Herein this assumption was tested using a large panel of VACV-derived peptides presented by HLA-B*07:02 (B7.2) molecules in a mousepox/ectromelia virus (ECTV)-infection, B7.2 transgenic mouse model. Most dominant epitopes recognized by ECTV- and VACV-reactive CD8+ T cells overlapped significantly without altering immunodominance hierarchy. Further, several epitopes recognized by ECTV-reactive CD8+ T cells were not recognized by VACV-reactive CD8+ T cells, and vice versa. In one instance, the lack of recognition owed to a N72K variation in the ECTV C4R70-78 variant of the dominant VACV B8R70-78 epitope. C4R70-78 does not bind to B7.2 and, hence, it was neither immunogenic nor antigenic. These findings provide a mechanistic basis for VACV vaccination-induced heterotypic immunity which can protect against Variola and Monkeypox disease. The understanding of how cross-reactive responses develop is essential for the rational design of a subunit-based vaccine that would be safe, and effectively protect against heterologous infection.

Serological tests using Sporothrix species antigens for the accurate diagnosis of sporotrichosis: a meta-analysis.

Some species of the fungus Sporothrix cause a chronic granulomatous infection in humans and animals called sporotrichosis. In the last decades, some research into serological tests has been carried out by different groups for the rapid detection of this infection. We performed a systematic review of the literature with meta-analysis to evaluate studies using Sporothrix spp. antigens and to evaluate their accuracy for sporotrichosis diagnostic. We searched Scopus, MEDLINE, Web of Science, GALE, Technology Research Database, DOA, Elsevier, SciELO, and Google Scholar Databases. The united results of sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio with their corresponding 95% confidence intervals (CI) were assessed. A total of 15 assays from 8 studies using 7 different serological methods and 8 different antigens were analyzed. The studies were performed in the USA, Brazil, and Venezuela from 1973 until 2015 and presented good quality. A high heterogeneity for sensitivity [I2 = 90.7%; 87% CI = (84-89), P < 0.001] and specificity [I2 = 89.2%; 93% CI = (92-95), P < 0.001] was observed. The performance of diagnostic tests was 0.93. Enzyme-linked immunosorbent assay was the main tool used, and the ConA-binding fraction antigen of the strain 1099-18 appears as a promising diagnostic biomarker candidate.

Functional Recognition by CD8+ T Cells of Epitopes with Amino Acid Variations Outside Known MHC Anchor or T Cell Receptor Recognition Residues.

Peptide-based vaccines can be safer and more cost effective than whole organism vaccines. Previous studies have shown that inorganic polystyrene nanoparticles (PSNPs) covalently conjugated to the minimal immunodominant peptide epitope from murine liver stage malaria (SYIPSAEKI) induced potent CD8+ T cell responses. Many pathogens, including malaria, have polymorphic T cell epitope regions. Amino acid changes in positions that are contact residues for the T cell receptor (TCR) often alter the specific cross-reactivity induced by the peptide antigen, and it is largely assumed that changes outside of these residues have little impact. Herein, each amino acid residue (except major histocompatibility complex (MHC) anchors) was systematically changed to an alanine. Peptide epitopes with altered amino acids outside T cell contact residues were still recognized by T cells induced by PSNPs-SYIPSAEKI (KI) vaccines, albeit at lower levels, except for the variant SYIPSAAKI (A7). PSNPs-SYIPSAAKI vaccines further elicited high responses to the index KI peptide. None of the epitopes displayed altered peptide ligand (APL) antagonism in vitro, and re-stimulating SYIPSAEKI and SYIPSAAKI together synergistically enhanced IFN-γ production by the T cells. These results show epitope variation in non-TCR recognition residues can have effects on T cell reactivity, suggesting that such natural variation may also be driven by immune pressure. Additionally, when re-modelling peptides to enhance the cross-reactivity of vaccines, both TCR recognition and non-recognition residues should be considered.

A Tolerogenic Role of Cathepsin G in a Primate Model of Multiple Sclerosis: Abrogation by Epstein-Barr Virus Infection.

Using a non-human primate model of the autoimmune neuroinflammatory disease multiple sclerosis (MS), we have unraveled the role of B cells in the making and breaking of immune tolerance against central nervous system myelin. It is discussed here that B cells prevent the activation of strongly pathogenic T cells present in the naïve repertoire, which are directed against the immunodominant myelin antigen MOG (myelin oligodendrocyte glycoprotein). Prevention occurs via destructive processing of a critical epitope (MOG34-56) through the lysosomal serine protease cathepsin G. This effective tolerance mechanism is abrogated when the B cells are infected with Epstein-Barr virus, a ubiquitous γ1-herpesvirus that entails the strongest non-genetic risk factor for MS.

Understanding the B and T cell epitopes of spike protein of severe acute respiratory syndrome coronavirus-2: A computational way to predict the immunogens.

The 2019 novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) outbreak has caused a large number of deaths, with thousands of confirmed cases worldwide. The present study followed computational approaches to identify B- and T-cell epitopes for the spike (S) glycoprotein of SARS-CoV-2 by its interactions with the human leukocyte antigen alleles. We identified 24 peptide stretches on the SARS-CoV-2 S protein that are well conserved among the reported strains. The S protein structure further validated the presence of predicted peptides on the surface, of which 20 are surface exposed and predicted to have reasonable epitope binding efficiency. The work could be useful for understanding the immunodominant regions in the surface protein of SARS-CoV-2 and could potentially help in designing some peptide-based diagnostics. Also, identified T-cell epitopes might be considered for incorporation in vaccine designs.

Chromogenic immunohistochemical quadruplex provides accurate diagnostic differentiation of non-small cell lung cancer.

Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung malignancies, the vast majority of which are non-small cell lung carcinomas (NSCLC). Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or by immunohistochemistry, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung. Each marker is represented by a distinct color that can be read by a pathologist, using standard, bright field microscopy. We evaluated the ability of pathologists to differentiate NSCLCs using the multiplexed assay as compared to standard, single marker per slide diaminobenzidine (DAB)-based IHC. All cases in a cohort of 264 NSCLCs showed concordance of information (including positivity of stain, intensity of stain and coverage) between single IHC stains and the multiplex assay. This new multiplex IHC offers the capability to accurately diagnose and sub-classify primary lung NSCLCs, while conserving precious tissue for additional testing.

Utility of p63 and p40 in Distinguishing Polymorphous Adenocarcinoma and Adenoid Cystic Carcinoma.

OBJECTIVE: Adenoid cystic carcinoma and polymorphous adenocarcinoma are primarily the tumor of minor salivary glands. Both show certain morphological similarities, which limit their proper diagnosis in settings where there are obscuring factors and limited biopsy material. However, there is a considerable difference in treatment and prognosis, which raises the need to distinguish these two entities. In this study, we discuss the utility of two immunohistochemical stains, p63 and p40, in different combinations for distinguishing polymorphous adenocarcinoma from adenoid cystic carcinoma. MATERIALS AND METHODS: Two immunohistochemical stains, p63 and p40, were performed on 47 cases of adenoid cystic carcinoma and 23 cases of polymorphous adenocarcinoma. RESULTS: 36 out of 47 cases of adenoid cystic carcinoma showed p63+ve/p40+ve immunoprofile, followed by p63-ve/p40-ve immunoprofile, which is seen in10 cases of adenoid cystic carcinoma. However, 22 out of 23 cases of polymorphous adenocarcinoma displayed p63+ve/ p40-ve immunoprofile. p63-ve/p40+ve is the least frequent observed immunoprofile, which is seen in only one case of adenoid cystic carcinoma. CONCLUSION: On combining all possible immunoprofile combinations, p63+ve/p40-ve immunoprofile appears to be the most sensitive profile for distinguishing polymorphous adenocarcinoma from adenoid cystic carcinoma.

TCR-induced alteration of primary MHC peptide anchor residue.

The HLA-A*02:01-restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T-cells-1 (MART-1) protein, represents one of the best-studied tumor associated T-cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA-A*02:01 and TCRs from clinically relevant T-cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5-HLA-A*02:01-AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide-MHC anchoring. This "flexing" at the TCR-peptide-MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well-studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.

HLA-DO Modulates the Diversity of the MHC-II Self-peptidome.

Presentation of antigenic peptides on MHC-II molecules is essential for tolerance to self and for initiation of immune responses against foreign antigens. DO (HLA-DO in humans, H2-O in mice) is a nonclassical MHC-II protein that has been implicated in control of autoimmunity and regulation of neutralizing antibody responses to viruses. These effects likely are related to a role of DO in selecting MHC-II epitopes, but previous studies examining the effect of DO on presentation of selected CD4 T cell epitopes have been contradictory. To understand how DO modulates MHC-II antigen presentation, we characterized the full spectrum of peptides presented by MHC-II molecules expressed by DO-sufficient and DO-deficient antigen-presenting cells in vivo and in vitro using quantitative mass spectrometry approaches. We found that DO controlled the diversity of the presented peptide repertoire, with a subset of peptides presented only when DO was expressed. Antigen-presenting cells express another nonclassical MHC-II protein, DM, which acts as a peptide editor by preferentially catalyzing the exchange of less stable MHC-II peptide complexes, and which is inhibited when bound to DO. Peptides presented uniquely in the presence of DO were sensitive to DM-mediated exchange, suggesting that decreased DM editing was responsible for the increased diversity. DO-deficient mice mounted CD4 T cell responses against wild-type antigen-presenting cells, but not vice versa, indicating that DO-dependent alterations in the MHC-II peptidome could be recognized by circulating T cells. These data suggest that cell-specific and regulated expression of HLA-DO serves to fine-tune MHC-II peptidomes, in order to enhance self-tolerance to a wide spectrum of epitopes while allowing focused presentation of immunodominant epitopes during an immune response.

The Length Distribution and Multiple Specificity of Naturally Presented HLA-I Ligands.

HLA-I molecules bind short peptides and present them for recognition by CD8+ T cells. The length of HLA-I ligands typically ranges from 8 to 12 aa, but variability is observed across different HLA-I alleles. In this study we collected recent in-depth HLA peptidomics data, including 12 newly generated HLA peptidomes (31,896 unique peptides) from human meningioma samples, to analyze the peptide length distribution and multiple specificity across 84 different HLA-I alleles. We observed a clear clustering of HLA-I alleles with distinct peptide length distributions, which enabled us to study the structural basis of peptide length distributions and predict peptide length distributions from HLA-I sequences. We further identified multiple specificity in several HLA-I molecules and validated these observations with binding assays. Explicitly modeling peptide length distribution and multiple specificity improved predictions of naturally presented HLA-I ligands, as demonstrated in an independent benchmarking based on the new human meningioma samples.

Toxoplasma gondii Histone 4 Affects Some Functions of Murine Ana-1 Macrophages In Vitro.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan that can infect almost all nucleated cells. Histone proteins and DNA form the nucleosomes, which are the fundamental building blocks of eukaryotic chromatin. Histone 4 is an essential component of a histone octamer. In the present study, T. gondii histone 4 (TgH4) was cloned and the regulatory effect of TgH4 on murine macrophages was characterized. Bioinformatics analysis revealed that TgH4 was highly conserved in structure. Recombinant TgH4 (rTgH4) protein was identified by sera from rats experimentally infected with T. gondii and native TgH4 in the total soluble protein of T. gondii tachyzoites was recognized by polyclonal antibodies against rTgH4, as indicated by immunoblotting analysis. Immunofluorescence assay showed that TgH4 binds to macrophages. Following incubation with rTgH4, the toll-like receptor 4 (TLR4) level of the macrophages was downregulated. Meanwhile, chemotaxis and the proliferation of macrophages were inhibited. However, rTgH4 can promote phagocytosis, apoptosis, and the secretion of nitric oxide, interleukin-6, and tumor necrosis factor-α from macrophages. Just 80 µg/ml rTgH4 can significantly elevate the secretion of interleukin-10 and interleukin-1ß (p < 0.05 and p < 0.01). Viewed together, these outcomes indicated that rTgH4 can affect the functions of murine macrophages in vitro.

Immature Dendritic Cell Therapy Confers Durable Immune Modulation in an Antigen-Dependent and Antigen-Independent Manner in Nonobese Diabetic Mice.

Dendritic cell (DC) immunotherapy has been effective for prevention of type 1 diabetes (T1D) in NOD mice but fails to protect if initiated after active autoimmunity. As autoreactivity expands inter- and intramolecularly during disease progression, we investigated whether DCs unpulsed or pulsed with ß cell antigenic dominant determinants (DD), subdominant determinants (SD), and ignored determinants (ID) could prevent T1D in mice with advanced insulitis. We found that diabetes was significantly delayed by DC therapy. Of interest, DCs pulsed with SD or ID appeared to provide better protection. T lymphocytes from DC-treated mice acquired spontaneous proliferating capability during in vitro culture, which could be largely eliminated by IL-2 neutralizing antibodies. This trend maintained even 29 weeks after discontinuing DC therapy and appeared antigen-independent. Furthermore, CD4+Foxp3+ T regulatory cells (Tregs) from DC-treated mice proliferated more actively in vitro compared to the controls, and Tregs from DC-treated mice showed significantly enhanced immunosuppressive activities in contrast to those from the controls. Our study demonstrates that DC therapy leads to long-lasting immunomodulatory effects in an antigen-dependent and antigen-independent manner and provides evidence for peptide-based intervention during a clinically relevant window to guide DC-based immunotherapy for autoimmune diabetes.