Glomerulopathy induced by immunization with a peptide derived from the goodpasture antigen α3IV-NC1.

Mouse experimental autoimmune glomerulonephritis, a model of human antiglomerular basement membrane disease, depends on both Ab and T cell responses to the Goodpasture Ag noncollagenous domain 1 of the α3-chain of type IV collagen (α3IV-NC1). The aim of our study was to further characterize the T cell-mediated immune response. Repeated immunization with mouse α3IV-NC1 caused fatal glomerulonephritis in DBA/1 mice. Although two immunizations were sufficient to generate high α3IV-NC1-specific IgG titers, Ab and complement deposition along the glomerular basement membranes, and a nephrotic syndrome, two additional immunizations were needed to induce a necrotizing/crescentic glomerulonephritis. Ten days after the first immunization, α3IV-NC1-specific CD4(+) cells producing TNF-α, IFN-γ, or IL-17A were detected in the spleen. With the emergence of necrotizing/crescentic glomerulonephritis, ∼0.15% of renal CD4(+) cells were specific for α3IV-NC1. Using peptides spanning the whole α3IV-NC1 domain, three immunodominant T cell epitopes were identified. Immunization with these peptides did not lead to clinical signs of experimental autoimmune glomerulonephritis or necrotizing/crescentic glomerulonephritis. However, mice immunized with one of the peptides (STVKAGDLEKIISRC) developed circulating Abs against mouse α3IV-NC1 first detected at 8 wk, and 50% of the mice showed mild proteinuria at 18-24 wk due to membranous glomerulopathy. Taken together, our results suggest that autoreactive T cells are able to induce the formation of pathologic autoantibodies. The quality and quantity of α3IV-NC1-specific Ab and T cell responses are critical for the phenotype of the glomerulonephritis.

Synthesis and characterisation of self-assembled and self-adjuvanting asymmetric multi-epitope lipopeptides of ovalbumin.

Designing a lipopeptide (LP) vaccine with a specific asymmetric arrangement of epitopes may result in an improved display of antigens, increasing host-cell recognition and immunogenicity. This study aimed to synthesise and characterise the physicochemical properties of a library of asymmetric LP-based vaccine candidates that contained multiple CD4(+) and CD8(+) T-cell epitopes from the model protein antigen, ovalbumin. These fully synthetic vaccine candidates were prepared by microwave-assisted solid phase peptide synthesis. The C12 or C16 lipoamino acids were coupled to the N or C terminus of the OVA CD4 peptide epitope. The OVA CD4 LPs and OVA CD8 peptide constructs were then conjugated using azide-alkyne Huisgen cycloaddition to give multivalent synthetic vaccines. Physiochemical characterisation of these vaccines showed a tendency to self-assemble in aqueous media. Changes in lipid length and position induced self-assembly with significant changes to their morphology and secondary structure as shown by transmission electron microscopy and circular dichroism.

Identification of pathogenic T cell epitopes near cathepsin cleavage sites in thyroglobulin.

Experimental autoimmune thyroiditis, induced in mice after challenge with thyroglobulin (Tg), is known to be under the genetic control of the H2A(k) locus. Because cathepsins are known to influence proteolytic processing of Tg in vivo, we examined in this study whether putative H2A(k)-binding Tg epitopes, located near cathepsin cleavage sites within mouse Tg, have immunopathogenic properties. Cathepsin L, B, and D cleavage sites in mouse Tg were predicted based on homology with known cathepsin cleavage sites in rabbit Tg. We used an algorithm-based approach to identify H2A(k)-binding motifs within 20-aa residue segments adjacent to cathepsin cleavage sites, and five 12mer peptides encompassing these sequences were synthesized. Two of them, p2369 (aa 2369-2380) and p2439 (aa 2439-2450) were immunogenic, eliciting significant proliferative T cell responses using lymph node cells from peptide-primed mice and production of IL-2 and IFN-γ in recall assays in vitro. Both peptides induced experimental autoimmune thyroiditis upon direct challenge of CBA/J mice with peptide in CFA and by adoptive transfer of peptide-primed lymph node cells into naive recipient hosts, but neither peptide was characterized as dominant.

TCR-redirected human T cells inhibit hepatitis C virus replication: hepatotoxic potential is linked to antigen specificity and functional avidity.

Virus-specific CTL with high levels of functional avidity have been associated with viral clearance in hepatitis C virus (HCV) infection and with enhanced protective immunity. In chronic HCV infection, lack of antiviral CTL is frequently observed. In this study, we aim to investigate novel HCV TCRs that differ in Ag specificity. This involved isolating new HCV-specific murine TCRs that recognize a conserved HLA-A2-restricted CTL epitope within the nonstructural protein (NS) 5A viral protein and comparing them with TCRs recognizing another conserved CTL target in the NS3 viral protein. This was done by expressing the TCRs in human T cells and analyzing the function of the resulting TCR-transduced T cells. Our result indicates that these TCRs are efficiently assembled in transduced human T cells. They recognize peptide-loaded targets and demonstrate polyfunctional features such as IL-2, IFN-γ, and TNF-α secretion. However, in contrast to NS3-specific TCRs, the NS5A TCR-transduced T cells consist of a smaller proportion of polyfunctional T cells and require more peptide ligands to trigger the effector functions, including degranulation. Despite the differences, NS5A TCRs show effective inhibition of HCV replication in human hepatoma cells with persistent HCV RNA replication. Moreover, cellular injury demonstrated by aspartate aminotransferase release and cell death is less significant in the hepatoma cells following coincubation with NS5A TCR-transduced T cells, which is a property consistent with noncytotoxic antiviral CTLs. Our results suggest that HCV TCR-transduced T cells may be promising for the treatment of patients with chronic HCV infections.

The efficiency of human cytomegalovirus pp65(495-503) CD8+ T cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases.

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8(+) CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65(495-503) is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65(495-503) generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65(495-503) epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65(495-503) epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65(495-503)-specific T cell activation, indicating the importance of ER aminopeptidases in pp65(495-503) generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65(495-503) epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.

Maintenance of T cell function in the face of chronic antigen stimulation and repeated reactivation for a latent virus infection.

Persisting infections are often associated with chronic T cell activation. For certain pathogens, this can lead to T cell exhaustion and survival of what is otherwise a cleared infection. In contrast, for herpesviruses, T cells never eliminate infection once it is established. Instead, effective immunity appears to maintain these pathogens in a state of latency. We used infection with HSV to examine whether effector-type T cells undergoing chronic stimulation retained functional and proliferative capacity during latency and subsequent reactivation. We found that latency-associated T cells exhibited a polyfunctional phenotype and could secrete a range of effector cytokines. These T cells were also capable of mounting a recall proliferative response on HSV reactivation and could do so repeatedly. Thus, for this latent infection, T cells subjected to chronic Ag stimulation and periodic reactivation retain the ability to respond to local virus challenge.

Antigen-specific cytotoxicity by invariant NKT cells in vivo is CD95/CD178-dependent and is correlated with antigenic potency.

Invariant NKT (iNKT) cells are a unique subset of T lymphocytes that rapidly carry out effector functions following activation with glycolipid Ags, such as the model Ag alpha-galactosylceramide. Numerous studies have investigated the mechanisms leading to Th1 and Th2 cytokine production by iNKT cells, as well as the effects of the copious amounts of cytokines these cells produce. Less is known, however, about the mechanisms of iNKT cell cytotoxicity. In this study, we investigated the effect of Ag availability and strength, as well as the molecules involved in iNKT cytotoxicity. We demonstrate that the iNKT cell cytotoxicity in vivo correlates directly with the amount of CD1d expressed by the targets as well as the TCR affinity for the target glycolipid Ag. iNKT cells from spleen, liver, and thymus were comparable in their cytotoxicity in vitro. Surprisingly, we show that the Ag-specific cytotoxicity of iNKT cells in vivo depended almost exclusively on the interaction of CD95 (Fas) with CD178 (FasL), and that this mechanism can be efficiently used for tumor protection. Therefore, unlike NK cells, which rely mostly on perforin/granzyme-mediated mechanisms, the Ag-specific cytotoxicity of iNKT cells in vivo is largely restricted to the CD95/CD178 pathway.

T cell retargeting with MHC class I-restricted antibodies: the CD28 costimulatory domain enhances antigen-specific cytotoxicity and cytokine production.

T cells require both primary and costimulatory signals for optimal activation. The primary Ag-specific signal is delivered by engagement of the TCR. The second Ag-independent costimulatory signal is mediated by engagement of the T cell surface costimulatory molecule CD28 with its target cell ligand B7. However, many tumor cells do not express these costimulatory molecules. We previously constructed phage display derived F(AB), G8, and Hyb3, Ab-based receptors with identical specificity but distinct affinities for HLA-A1/MAGE-A1, i.e., "TCR-like" specificity. These chimeric receptors comprised the FcepsilonRI-gamma signaling element. We analyzed whether linking the CD28 costimulation structure to it (gamma + CD28) could affect the levels of MHC-restricted cytolysis and/or cytokine production. Human scFv-G8(POS) T lymphocytes comprising the gamma + CD28 vs the gamma signaling element alone produced substantially more IL-2, TNF-alpha, and IFN-gamma in response to HLA-A1/MAGE-A1(POS) melanoma cells. Also a drastic increase in cytolytic capacity of scFv-G8(POS) T cells, equipped with gamma + CD28 vs the gamma-chain alone was observed.

Transfer of myelin-specific cells deviated in vitro towards IL-4 production ameliorates ongoing experimental allergic neuritis.

A causal role of IL-4 (Th2) production for recovery in experimental allergic neuritis (EAN) was indicated by experiments where Th1-like autoreactive cell populations, taken from the induction phase of the disease, were deviated to extensive secretion of IL-4 in a selective fashion, by ex vivo stimulation with autoantigen in the presence of IL-4. The deviated cells were adoptively transferred to EAN rats at a time just prior to the onset of clinical signs. This treatment ameliorated EAN compared with sham treatment. This therapeutic approach, with generation of autoreactive IL-4-secreting cells ex vivo followed by subsequent adoptive transfer, may become a new selective treatment of organ-specific autoimmune diseases since, in contrast to previous attempts, it is done in a physiological and technically easy way.