Flu-HA-B (306-318) MHC II DRB1*01:01

Identification and in vitro expansion of functional antigen-specific CD25+ FoxP3+ regulatory T cells in hepatitis C virus infection

CD4(+)CD25(+) regulatory T cells (CD25(+) Tregs) play a key role in immune regulation. Since hepatitis C virus (HCV) persists with increased circulating CD4(+)CD25(+) T cells and virus-specific effector T-cell dysfunction,we asked if CD4(+)CD25(+) T cells in HCV-infected individuals are similar to natural Tregs in uninfected individuals and if they include HCV-specific Tregs using the specific Treg marker FoxP3 at the single-cell level. We report that HCV-infected patients display increased circulating FoxP3(+) Tregs that are phenotypically and functionally indistinguishable from FoxP3(+) Tregs in uninfected subjects. Furthermore,HCV-specific FoxP3(+) Tregs were detected in HCV-seropositive persons with antigen-specific expansion,major histocompatibility complex class II/peptide tetramer binding affinity,and preferential suppression of HCV-specific CD8 T cells. Transforming growth factor beta contributed to antigen-specific Treg expansion in vitro,suggesting that it may contribute to antigen-specific Treg expansion in vivo. Interestingly,FoxP3 expression was also detected in influenza virus-specific CD4 T cells. In conclusion,functionally active and virus-specific FoxP3(+) Tregs are induced in HCV infection,thus providing targeted immune regulation in vivo. Detection of FoxP3 expression in non-HCV-specific CD4 T cells suggests that immune regulation through antigen-specific Treg induction extends beyond HCV.

Association of HLA-DR1 with the allergic response to the major mugwort pollen allergen: molecular background

BACKGROUND: Mugwort pollen allergens represent the main cause of pollinosis in late summer. The major allergen,Art v 1,contains only one single immunodominant,solely HLA-DR-restricted T cell epitope (Art v 125-36). The frequency of HLA-DRB1*01 is highly increased in mugwort-allergic individuals and HLA-DR1 serves as restriction element for Art v 125-36. However,Art v 125-36 also binds to HLA-DR4 with high affinity and DR1-restricted Art v 125-36 -specific T cell receptors can be activated by HLA-DR4 molecules. To understand the predominance of HLA-DR1 in mugwort allergy in spite of the degeneracy in HLA/peptide-binding and TCR-recognition,we investigated the molecular background of Art v 125-36 /MHC/TCR interactions in the context of HLA-DR1 compared to -DR4.

RESULTS: The majority of Art v 125-36 -specific T cell lines and clones from HLA-DR1 carrying,mugwort pollen-allergic donors reacted to synthetic and naturally processed Art v 1-peptides when presented by HLA-DR1 or HLA-DR4 expressing antigen presenting cells. However,at limiting peptide concentrations DR1 was more effective in T cell stimulation. In addition,the minimal epitope for 50% of Art v 125-36 -specific T cells was shorter for DR1 than for DR4. In vitro binding assays of Art v 125-36 mutant peptides to isolated DR1- and DR4-molecules indicated similar binding capacities and use of the same register. In silico simulation of Art v 125-36 binding to HLA-DR1 and -DR4 suggested similar binding of the central part of the peptide to either molecule,but a higher flexibility of the N- and C-terminal amino acids and detachment at the C-terminus in HLA-DR1.

CONCLUSION: The predominance of HLA-DR1 in the response to Art v 125-36 may be explained by subtle conformation changes of the peptide bound to DR1 compared to DR4. Computer simulation supported our experimental data by demonstrating differences in peptide mobility within the HLA-DR complex that may influence TCR-binding. We suggest that the minor differences observed in vitro may be more relevant in the microenvironment in vivo,so that only presentation by HLA-DR1,but not -DR4 permits successful T cell activation.

Potential of an altered peptide ligand of lipocalin allergen Bos d 2 for peptide immunotherapy

BACKGROUND: Peptide immunotherapy is a promising alternative for treating allergic diseases. One way to enhance the efficacy of peptide immunotherapy is to use altered peptide ligands (APLs) that contain amino acid substitutions compared with the natural peptide.

OBJECTIVE: To evaluate the potential of an APL of the immunodominant epitope of lipocalin allergen Bos d 2 for peptide immunotherapy.

METHODS: Peripheral blood CD4(+) T-cell responses of 8 HLA-DR4-positive subjects to the natural ligand of Bos d 2 (p127-142) or to an APL (pN135D) were analyzed by MHC class II tetramer staining after in vitro expansion with the peptides. Long-term T-cell lines (TCLs) were induced with the peptides,and the cytokine production,cross-reactivity,and T-cell receptor Vbeta subtype expression of the TCLs were analyzed.

RESULTS: CD4(+) T cells specific for both p127-142 and pN135D were readily detected in peripheral blood after a single in vitro stimulation. Whereas the TCLs induced with p127-142 were T(H)2/T(H)0-deviated,those induced with pN135D were T(H)1/T(H)0-deviated and highly cross-reactive with p127-142. Moreover,the pN135D-induced TCLs appeared to use a broader repertoire of T-cell receptor Vbeta subtypes than those induced with p127-142.

CONCLUSION: An APL of an immunodominant allergen epitope was able to induce a novel T(H)1-deviated T-cell population cross-reactive with the natural epitope in vitro. This cell population could have a therapeutic immunomodulatory function in vivo through bystander suppression.

CLINICAL IMPLICATIONS: These results support the idea that altered peptide ligands may be used to enhance the efficacy of peptide immunotherapy.

Protective Allele for Multiple Sclerosis HLA-DRB1*01:01 Provides Kinetic Discrimination of Myelin and Exogenous Antigenic Peptides

Risk of the development of multiple sclerosis (MS) is known to be increased in individuals bearing distinct class II human leukocyte antigen (HLA) variants,whereas some of them may have a protective effect. Here we analyzed distribution of a highly polymorphous HLA-DRB1 locus in more than one thousand relapsing-remitting MS patients and healthy individuals of Russian ethnicity. Carriage of HLA-DRB1*15 and HLA-DRB1*03 alleles was associated with MS risk,whereas carriage of HLA-DRB1*01 and HLA-DRB1*11 was found to be protective. Analysis of genotypes revealed the compensatory effect of risk and resistance alleles in trans. We have identified previously unknown MBP153-161 peptide located at the C-terminus of MBP protein and MBP90-98 peptide that bound to recombinant HLA-DRB1*01:01 protein with affinity comparable to that of classical antigenic peptide 306-318 from the hemagglutinin (HA) of the influenza virus demonstrating the ability of HLA-DRB1*01:01 to present newly identified MBP153-161 and MBP90-98 peptides. Measurements of kinetic parameters of MBP and HA peptides binding to HLA-DRB1*01:01 catalyzed by HLA-DM revealed a significantly lower rate of CLIP exchange for MBP153-161 and MBP90-98 peptides as opposed to HA peptide. Analysis of the binding of chimeric MBP-HA peptides demonstrated that the observed difference between MBP153-161,MBP90-98,and HA peptide epitopes is caused by the lack of anchor residues in the C-terminal part of the MBP peptides resulting in a moderate occupation of P6/7 and P9 pockets of HLA-DRB1*01:01 by MBP153-161 and MBP90-98 peptides in contrast to HA308-316 peptide. This leads to the P1 and P4 docking failure and rapid peptide dissociation and release of empty HLA-DM-HLA-DR complex. We would like to propose that protective properties of the HLA-DRB1*01 allele could be directly linked to the ability of HLA-DRB1*01:01 to kinetically discriminate between antigenic exogenous peptides and endogenous MBP derived peptides.

A high throughput MHC II binding assay for quantitative analysis of peptide epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid,quantitative insights into immunogenic epitope identification,deletion,or design(1 ,2). Here,a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols(1 ,3-5). Specifically,the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot,this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular,the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.