Characterization of CD8+ T-cell response in acute and resolved hepatitis A virus infection.

BACKGROUND & AIMS: In contrast to the infection with other hepatotropic viruses, hepatitis A virus (HAV) always causes acute self-limited hepatitis, although the role for virus-specific CD8 T cells in viral containment is unclear. Herein, we analyzed the T cell response in patients with acute hepatitis by utilizing a set of overlapping peptides and predicted HLA-A2 binders from the polyprotein. METHODS: A set of 11 predicted peptides from the HAV polyprotein, identified as potential binders, were synthesized. Peripheral blood mononuclear cells (PBMCs) from patients were tested for IFNγ secretion after stimulation with these peptides and ex vivo with HLA-A2 tetramers. Phenotyping was carried out by staining with the activation marker CD38 and the memory marker CD127. RESULTS: Eight out of 11 predicted HLA-A2 binders showed a high binding affinity and five of them were recognized by CD8+ T cells from patients with hepatitis A. There were significant differences in the magnitude of the responses to these five peptides. One was reproducibly immunodominant and the only one detectable ex vivo by tetramer staining of CD8+ T cells. These cells have an activated phenotype (CD38hi CD127lo) during acute infection. Three additional epitopes were identified in HLA-A2 negative patients, most likely representing epitopes restricted by other HLA-class I-alleles (HLA-A11, B35, B40). CONCLUSIONS: Patients with acute hepatitis A have a strong multi-specific T cell response detected by ICS. With the tetramer carrying the dominant HLA-A2 epitope, HAV-specific and activated CD8+ T cells could be detected ex vivo. This first description of the HAV specific CTL-epitopes will allow future studies on strength, breadth, and kinetics of the T-cell response in hepatitis A.

Human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis show the imprints of an antigen-dependent process of somatic hypermutation and clonal selection.

The persistent presence of rheumatoid factors (RFs) in the circulation is a characteristic phenomenon in patients with rheumatoid arthritis (RA). Recent data indicate that RFs associated with seropositive RA are derived from terminally differentiated CD20-, CD38+ plasma cells (PCs) present in synovial fluids of the inflamed joints. These cells were shown to secrete RFs actively and are thought to originate from germinal centre (GC)-like structures present in the inflamed synovium. To obtain a representative image of the structural properties of IgM and IgG RFs associated with RA, phage antibody display libraries were constructed from CD38+ PCs isolated from the inflamed joints of RF-seropositive patients with RA. Subsequently, human IgG Fc-binding monoclonal phage antibodies were selected and analysed. The data suggest that RA-associated RFs are encoded by a diverse set of VL and a more restricted set of VH regions. VH gene family usage of PC-derived IgM- and IgG-RFs was found to be restricted to the VH1 and 3 gene families, with a preference for VH3, and many different VL genes were shown to contribute to RF specificity. Clonally related VH as well as VL sequences were identified, based on the presence of identical CDR3 regions and shared somatic mutations. In this B cell selection process base-pair substitutions as well as deletions of triplets in CDR regions, leaving the transcripts in frame, were involved. Together, these data provide further evidence for an Ag-driven immune response in the terminal differentiation into RF-producing PCs in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a GC reaction.

Polyreactivity of human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis.

Recent data indicate that rheumatoid factors (RFs) that occur in patients with rheumatoid arthritis (RA) are derived from Ig-producing terminally differentiated CD20-, CD38+ plasma cells present in synovial fluids (SFs). Phage antibody display libraries were constructed using CD38+ plasma cells isolated from SFs of two RF-seropositive RA patients. The libraries were enriched for phage antibodies (Phabs) binding to human IgG (HuIgG) Fc fragments and the sequences of their V genes were analysed. These data provided further evidence for an Ag-driven immune response in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a germinal center reaction. In the present study, the functional characteristics of these HuIgG Fc-binding monoclonal (mo) Phabs were further analysed in order to provide more insight into the specificity of HuIgG Fc-binding Phabs. Remarkably, all HuIgG Fc-binding moPhabs tested (n=48; derived from four different libraries) displayed polyreactivity. Structural analysis of the CDR3 regions revealed characteristic features of polyreactive Igs. Most H chain CDR3 regions harboured tryptophan/tyrosine-rich parts and approximately 60% of the L chain CDR3 regions of both RA patients displayed an identical stretch of amino acids (W/Y-D-S-S). Supportive for a dominant role of VH in specificity, exchange of VL regions with a single VH region yielded moPhabs with similar specificities. All together, the data suggest the presence of an Ag-driven process in the joints of patients with RA, including somatic mutation and clonal selection entailing isotype switching, resulting in the differentiation of B cells into polyreactive RF-secreting plasma cells.