Identification of a naturally processed HLA-A*02:01-restricted CTL epitope from the human tumor-associated antigen Nectin-4.

Nectin-4 is a tumor antigen present on the surface of breast, ovarian and lung carcinoma cells. It is rarely present in normal adult tissues and is therefore a candidate target for cancer immunotherapy. Here, we identified a Nectin-4 antigenic peptide that is naturally presented to T cells by HLA-A2 molecules. We first screened the 502 nonamer peptides of Nectin-4 (510 amino acids) for binding to and off-rate from eight different HLA class I molecules. We then combined biochemical, cellular and algorithmic assays to select 5 Nectin-4 peptides that bound to HLA-A*02:01 molecules. Cytolytic T lymphocytes were obtained from healthy donors, that specifically lyzed HLA-A2(+) cells pulsed with 2 out of the 5 peptides, indicating the presence of anti-Nectin-4 CD8(+) T lymphocytes in the human T cell repertoire. Finally, an HLA-A2-restricted cytolytic T cell clone derived from a breast cancer patient recognized peptide Nectin-4145-153 (VLVPPLPSL) and lyzed HLA-A2(+) Nectin-4(+) breast carcinoma cells. These results indicate that peptide Nectin-4145-153 is naturally processed for recognition by T cells on HLA-A2 molecules. It could be used to monitor antitumor T cell responses or to immunize breast cancer patients.

Transmembrane BAFF from rheumatoid synoviocytes requires interleukin-6 to induce the expression of recombination-activating gene in B lymphocytes.

OBJECTIVE: B cells that accumulate in the synovial tissue of rheumatoid arthritis (RA) patients revise their receptors due to coordinate expression of recombination-activating gene 1 (RAG-1) and RAG-2 genes. The aim of this study was to determine the mechanisms that control this re-expression. METHODS: B cells from healthy control subjects were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA and osteoarthritis (OA). Re-expression of RAG messenger RNA (mRNA) and proteins was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescence. Activity of RAG enzymes was evaluated by flow cytometry to measure variations in immunoglobulin kappa and lambda light chain expression and by ligation-mediated-PCR to assess specific DNA breaks. Blocking antibodies, short hairpin RNA, and recombinant cytokine were used to identify the molecules involved in RAG re-expression. RESULTS: RA FLS, but not OA FLS, induced B cells to re-express RAG mRNA and proteins. Enzymes were functional, since the kappa-to-lambda ratios decreased and specific DNA breaks were detectable after coculture with RA FLS. Transmembrane BAFF provided the first signal of RAG re-expression, since its down-regulation in RA FLS prevented RAG gene transcription in B cells. The failure of transmembrane BAFF from OA FLS to induce RAG suggests that a second signal was provided by RA FLS. Interleukin-6 (IL-6) is a candidate, since blockade of its receptors precluded transcription of RAG genes by RA FLS. Unless supplemented with IL-6, OA FLS were unable to induce RAG gene expression in normal B cells. CONCLUSION: Two independent signals are required for the induction of RAG gene expression in B cells that infiltrate the synovium of patients with RA.

Signaling pathways regulating RAG expression in B lymphocytes.

Development of B-cell lymphopoiesis is dependent on the presence of recombination activating genes RAG1 and RAG2 enzymes. They control the rearrangements of immunoglobulin variable, diversity and joining region segments, and allow progression of the cellular maturation. RAG1 and RAG2 are successively up- and down-regulated at each B-cell stage to progressively generate a B-cell receptor for which unforeseeable antigenic specificity results from a stochastic process. Therefore, in autoreactive immature B cells, new round of RAG re-expression can be observed to eliminate self-reactivity. In some circumstances, RAG up-regulation can also be found in peripheral mature B lymphocytes, specifically in autoimmune diseases. It is therefore of utmost importance to unravel signaling pathways that trigger RAG induction in normal and pathological conditions. Therapeutic modulation of cytokines or intracellular contacts involved in RAG expression might restrict the development of inappropriate autoimmune repertoire.

RAG-mediated secondary rearrangements of B-cell antigen receptors in rheumatoid synovial tissue.

Rheumatoid arthritis (RA) induces major changes in synovial tissue (ST) and cartilage and bone destruction. Still, its pathogenesis is poorly understood. Accumulating evidence points to an important role for B lymphocytes. Rheumatoid-ST is characterized by activation of the synoviocytes and infiltrated by various inflammatory cells such as B and T lymphocytes. The infiltrate is diffuse or organized as germinal centers (GCs). These accommodate the immune response and favor self-tolerance breakdown. Receptor revision in B cells results from re-expression of the recombination activating genes (RAGs) which reinitiate immunoglobulin gene recombination, and modify the B-cell antigen receptor accordingly. In rheumatoid ST, secondary VDJ rearrangements occur and RAG proteins are detected. The mechanism that triggers and controls this revision remains elusive. We favor the hypothesis that such an uncontrolled process leads to autoimmunity.

Expression and reexpression of recombination activating genes: relevance to the development of autoimmune states.

Like all antibodies, autoreactive antibodies are generated in developing B cells in the bone marrow by variable (V), diversity (D), and joining (J) recombination under the regulation of recombination activating gene (RAG) 1 and RAG2 proteins. Deletion, anergy, and receptor edition prevent the emergence of autoreactive B cells. In the periphery, somatic hypermutation during the course of germinal center responses can lead to the emergence of autoreactive and low-affinity antibody-producing B cells. Deletion and receptor revision regulate autoreactive and inappropriate B cells. Defects in central or peripheral tolerance mechanisms associated with RAG expression could contribute to the appearance of autoreactive B cells. We demonstrate the presence of RAG(+) B cells in CD5-expressing cells outside germinal centers. Our data suggest that receptor revision in the periphery also may occur in unusual sites when B cells are induced to express CD5. This revision may correspond to a novel regulation checkpoint in which impaired control of RAG expression could generate autoreactive B cells and lead to autoimmune states.