Identification of naturally processed HLA-A2--restricted proinsulin epitopes by reverse immunology.

Type 1 diabetes is thought to result from the destruction of beta-cells by autoantigen-specific T-cells. Observations in the NOD mouse model suggest that CD8+ cytotoxic T-cells play an essential role in both the initial triggering of insulitis and its destructive phase. However, little is known about the epitopes derived from human beta-cell autoantigens and presented by HLA class I molecules. We used a novel reverse immunology approach to identify HLA-A2-restricted, naturally processed epitopes derived from proinsulin, an autoantigen likely to play an important role in the pathogenesis of type 1 diabetes. Recombinant human proinsulin was digested with purified proteasome complexes to establish an inventory of potential COOH-terminals of HLA class I-presented epitopes. Cleavage data were then combined with epitope predictions based on the SYFPEITHI and BIMAS algorithms to select 10 candidate epitopes; 7 of these, including 3 with a sequence identical to murine proinsulin, were immunogenic in HLA-A2 transgenic mice. Moreover, six of six tested peptides were processed and presented by proinsulin-expressing cells. These results demonstrate the power of reverse immunology approaches. Moreover, the novel epitopes may be of significant interest in monitoring autoreactive T-cells in type 1 diabetes.

HLA class I epitope discovery in type 1 diabetes.

Type 1 diabetes mellitus (T1DM) results from the destruction of beta cells by autoantigen-specific T cells. In the non-obese diabetic (NOD) mouse model, CD8+ T cells play an essential role in both the initial triggering of insulitis and its destructive phase, and proinsulin (PI) is one of the dominant target antigens (Ags). However, little is known about the beta cell epitopes presented by HLA class I molecules and recognized by human CD8+ T cells. We and other groups recently applied reverse immunology approaches to identify HLA class I-restricted PI epitopes. To establish an inventory of potential naturally processed epitopes, whole human PI or the transitional region between the B-chain and C-peptide were digested with purified proteasome complexes. By combining proteasome digestion data with epitope prediction algorithms, candidate epitopes restricted by HLA-A2.1 and other HLA class I molecules were identified. We validated immunogenicity and natural processing of the identified PI epitopes in HLA-A2.1-transgenic mice, while others demonstrated recognition of multiple PI epitopes by CD8+ T cells from T1DM and healthy subjects in the context of different HLA class I molecules. These results demonstrate the power of reverse immunology strategies for epitope discovery. DNA vaccination of HLA-transgenic mice may be another rapid and efficient reverse immunology approach to map additional epitopes derived from other T1DM Ags, such as IA-2 and glutamic acid decarboxylase 65 (GAD 65). Transfer of this information to Elispot- and MHC tetramer-based assay formats should allow to reliably detect and characterize autoreactive CD8+ T cell responses in T1DM, and may open new avenues for early T1DM diagnosis and immune intervention.

Complexity, contradictions, and conundrums: studying post-proteasomal proteolysis in HLA class I antigen presentation.

The vast majority of the peptides produced during protein degradation by the cytosolic proteasome-ubiquitin system are consecutively hydrolyzed to single amino acids by multiple cytosolic peptidases preferring intermediate length or short substrates. The small fraction of peptides surviving the aggressive cytosolic environment can be recruited for presentation by major histocompatibility complex (MHC) class I molecules. However, such peptides may frequently have to be adapted to the strict MHC class I-binding requirements by one or several N-terminal-trimming steps. A recent model proposes that an initial step, in which peptides of 15 or more residues are shortened by cytosolic tripeptidylpeptidase II, is followed by additional trimming by cytosolic or endoplasmic reticulum (ER) aminopeptidases. In humans, at least two ER resident aminopeptidases, ERAP1 and ERAP2, contribute to trimming of human leukocyte antigen class I ligands. These interferon-gamma-regulated metallopeptidases show distinct substrate preferences and may have to act in a concerted fashion to remove some complex or longer N-terminal extensions and to trim the full spectrum of precursor peptides. This task is likely facilitated by the formation of presumably heterodimeric ERAP1-2 complexes. RNA interference experiments suggest that both enzymes are important for normal antigen presentation, but precise determination of the extent and the cellular context of their requirement will be left to future experimentation.