Evidence for a functional role of IgE anticitrullinated protein antibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease involving inflammation of the joints. Among the autoantibodies described in RA, anticitrullinated protein antibodies (ACPAs) are highly specific and predictive for RA. In addition, ACPAs have been implicated in the pathogenesis of RA. However, a direct functional response of immune cells from ACPA(+) RA patients toward citrullinated proteins has not been demonstrated. In this study, we show that exposure to citrullinated antigens leads to activation of basophils from ACPA(+) RA patients within 20 minutes. This was not observed after exposure of basophils to noncitrullinated control antigens or after stimulation of basophils from ACPA(-) RA patients and healthy controls. Basophil activation was correlated with the binding of citrullinated proteins to basophils. Furthermore, serum from ACPA(+) RA patients in contrast to that from ACPA(-) RA patients could specifically sensitize human FcepsilonRI expressing rat basophil cells (RBL), enabling activation by citrullinated proteins. Mast cell degranulation products such as histamine levels were enhanced in synovial fluid of ACPA(+) RA patients as compared with ACPA(-) RA and osteoarthritis patients. In addition, histamine levels in synovial fluid from ACPA(+) RA patients correlated with IgE levels, suggesting degranulation of mast cells by cross-linking IgE. Immunohistochemistry on synovial biopsies demonstrated an increased number of degranulated CD117(+) mast cells in ACPA(+) RA patients; IgE and FcepsilonRI expression in synovial mast cells from ACPA(+) RA patients was increased. In conclusion, our results show an immunological response of immune cells from ACPA(+) RA patients in a citrulline-specific manner. Moreover, these data indicate a role for IgE-ACPAs and FcepsilonRI-positive cells in the pathogenesis of RA.

Characterization of an immuno 'stealth' derivative of the herpes simplex virus thymidine-kinase gene.

The cellular immune response against transgene-encoded neoantigens is a potential hurdle in gene therapy applications where long-term expression of transgenes is desired. Here a new optimized derivative of the herpes simplex virus 1-thymidine-kinase (HSV1-TK) gene is described. The HSV-TK gene is frequently used in experimental studies on gene-directed enzyme prodrug therapy. In the optimized gene, the HSV-TK coding region is fused with the codons for the Gly-Ala repeat of the Epstein-Barr virus nuclear-antigen 1 to prevent proteasomal degradation of the HSV-TK. To measure the protective effect in vitro, a model cytotoxic T lymphocyte epitope derived from the ovalbumin was inserted in the TK. Cells expressing the GAr-modified TK do not present TK-derived peptides in the major histocompatibility complex. Furthermore, conservative nucleotide substitutions were introduced, which prevent splicing, as well as mutations that render the TK-expressing cells more sensitive to ganciclovir (GCV). The GAr HSV-TK fusion protein is fully functional in vitro. This HSV-TK gene may be especially useful in those gene therapy applications where an immune response against the transgene-encoded product would frustrate the treatment.

Creation of immune 'stealth' genes for gene therapy through fusion with the Gly-Ala repeat of EBNA-1.

A major obstacle in gene-therapy protocols is T-cell-mediated destruction of transgene-expressing cells. Therefore new approaches are needed to prevent rapid clearance of transduced cells. We exploited the Gly-Ala repeat (GAr) domain of the Epstein-Barr virus nuclear antigen-1, since the GAr prevents cytotoxic T-lymphocyte-epitope generation. Here we show that three different enzymes (viz. the E. coli LacZ gene encoded beta-galactosidase, firefly luciferase, and HSV1 thymidine kinase) fused with the GAr retained their function. Moreover, linking GAr with beta-galactosidase successfully prevented recognition of GAr-LacZ-expressing cells by beta-galactosidase-specific CTL. Nonetheless, vaccination with a GAr-LacZ adenovirus or with an allogeneic cell line expressing GAr-LacZ resulted in the induction of beta-gal-specific CTL. This demonstrates that the GAr domain does not inhibit cross presentation of antigens, but only affects breakdown of endogenously synthesized proteins. These data demonstrate how the GAr domain can be exploited to create immuno'stealth' genes by hiding transgene products from CTL-mediated immune attack.