New approaches to prediction of immune responses to therapeutic proteins during preclinical development.

Clinical studies show that immunogenicity observed against therapeutic proteins can limit efficacy and reduce the safety of the treatment. It is therefore beneficial to be able to predict the immunogenicity of therapeutic proteins before they enter the clinic. Studies using deimmunized proteins have highlighted the importance of T-cell epitopes in the generation of undesirable immunogenicity. In silico, in vitro, ex vivo and in vivo methods have therefore been developed that focus on identification of CD4+ T-cell epitopes in the sequence of therapeutic proteins. A case study of existing therapeutic proteins is presented to review these different approaches in order to assess their utility in predicting immunogenic potential.

Exocytosis of CTLA-4 is dependent on phospholipase D and ADP ribosylation factor-1 and stimulated during activation of regulatory T cells.

CTLA-4 is an essential protein in the regulation of T cell responses that interacts with two ligands found on the surface of APCs (CD80 and CD86). CTLA-4 is itself poorly expressed on the T cell surface and is predominantly localized to intracellular compartments. We have studied the mechanisms involved in the delivery of CTLA-4 to the cell surface using a model Chinese hamster ovary cell system and compared this with activated and regulatory human T cells. We have shown that expression of CTLA-4 at the plasma membrane (PM) is controlled by exocytosis of CTLA-4-containing vesicles and followed by rapid endocytosis. Using selective inhibitors and dominant negative mutants, we have shown that exocytosis of CTLA-4 is dependent on the activity of the GTPase ADP ribosylation factor-1 and on phospholipase D activity. CTLA-4 was identified in a perinuclear compartment overlapping with the cis-Golgi marker GM-130 but did not colocalize strongly with lysosomal markers such as CD63 and lysosome-associated membrane protein. In regulatory T cells, activation of phospholipase D was sufficient to trigger release of CTLA-4 to the PM but did not inhibit endocytosis. Taken together, these data suggest that CTLA-4 may be stored in a specialized compartment in regulatory T cells that can be triggered rapidly for deployment to the PM in a phospholipase D- and ADP ribosylation factor-1-dependent manner.

Inhibition of human T cell proliferation by CTLA-4 utilizes CD80 and requires CD25+ regulatory T cells.

CD28 and CTLA-4 are opposing regulators of T cell activation, triggered by the two ligands CD80 and CD86. How these ligands promote either T cell activation via CD28 or inhibition via CTLA-4 is not understood. Using CD80 and CD86 molecules expressed on transfected cells, we have identified a major difference between these ligands in that CD80 transfectants have the ability to inhibit activation of resting human peripheral blood T cells via interaction with CTLA-4, whereas CD86 transfectants do not. Rather, CTLA-4-CD86 interactions appear to contribute towards T cell proliferation. We also observed that CTLA-4 function is strongly influenced by TCR stimulation, effects being observed only at relatively low levels of TCR stimulation. The kinetics of CD80-CTLA-4 interactions revealed that CTLA-4 inhibition took place within the first 8 h of T cell stimulation, despite there being little measurable CTLA-4 expression on the majority T cells. However, significant amounts of CTLA-4 were observed in the CD25(+) CD4(+) subset of T cells which, when removed from the cultures, accounted for the CTLA-4 inhibition observed. Overall, these data provide evidence that CD80 and CD86 differ in their interactions with CTLA-4 and that CD80 appears to be the preferential inhibitory ligand for CTLA-4 working via a population of CD4(+) CD25(+) CTLA-4(+) regulatory T cells.