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Objective. Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLA-B27 normally forms complexes with ␤ 2 -microglobulin (␤ 2 m) and peptide to form heterotrimers. However, an unusual characteristic of HLA-B27 is its ability to form ␤ 2 m-free heavy chain homodimers (HLA-B27 2 ), which, unlike classic HLA-B27, bind to killer cell immunoglobulinlike receptor 3DL2 (KIR-3DL2). Binding of HLA-B27 2 to KIR-3DL2-positive CD4؉ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLA-B27 2 in order to confirm its expression in SpA and to inhibit its proinflammatory properties.Methods. We generated monoclonal antibodies by screening a human phage display library positively against B27 2 and negatively against B27 heterotrimers. Conclusion. These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.Positivity for HLA-B27 is strongly associated with ankylosing spondylitis (AS) and other spondylarthritides (SpA) (1-3). However, despite extensive investigation, understanding of the pathogenic role of HLA-B27 is limited (4). The canonical HLA-B27 heterotrimer structure comprises an HLA heavy chain that is noncovalently associated with a monomorphic ␤ 2 -microglobulin (␤ 2 m) light chain and a short peptide derived from self proteins, viruses, or bacteria. These heterotrimeric complexes form in the endoplasmic reticulum and egress to the cell surface, where they are recognized by CD8ϩ cytotoxic T cells through their T cell receptors (5). HLA-B27 can also form ␤ 2 m-free

The cancer-testis antigen NY-ESO-1 is a potential target antigen for immune therapy expressed in a subset of patients with multiple myeloma. We generated chimeric antigen receptors (CARs) recognizing the immunodominant NY-ESO-1 peptide 157-165 in the context of HLA-A*02:01 to re-direct autologous CD8(+) T cells towards NY-ESO-1(+) myeloma cells. These re-directed T cells specifically lysed NY-ESO-1(157-165)/HLA-A*02:01-positive cells and secreted IFNγ. A total of 40% of CCR7(-) re-directed T cells had an effector memory phenotype and 5% a central memory phenotype. Based on CCR7 cell sorting, effector and memory CAR-positive T cells were separated and CCR7(+) memory cells demonstrated after antigen-specific re-stimulation downregulation of CCR7 as sign of differentiation towards effector cells accompanied by an increased secretion of memory signature cytokines such as IL-2. To evaluate NY-ESO-1 as potential target antigen, we screened 78 bone marrow biopsies of multiple myeloma patients where NY-ESO-1 protein was found to be expressed by immunohistochemistry in 9.7% of samples. Adoptively transferred NY-ESO-1-specific re-directed T cells protected mice against challenge with endogenously NY-ESO-1-positive myeloma cells in a xenograft model. In conclusion, re-directed effector- and central memory T cells specifically recognized NY-ESO-1(157-165)/ HLA-A*02:01-positive cells resulting in antigen-specific functionality in vitro and in vivo.

Key points• Stromal cell-interaction molecule (STIM) 2 senses Ca 2+ levels in the endoplasmic reticulum and activates Ca 2+ channels in the plasma membrane upon store depletion.• Here we report that STIM2 is preferentially activated by low agonist concentrations that cause mild reductions in endoplasmic reticulum Ca 2+ levels.• This shows that store-operated Ca 2+ entry is regulated through signal strength, with weak stimuli activating STIM2 and strong stimuli engaging STIM1.• The results help us to understand how receptor activation enables differential modulation of Ca 2+ entry over a range of agonist concentrations and levels of store depletion.Abstract Agonist-induced Ca 2+ oscillations in many cell types are triggered by Ca 2+ release from intracellular stores and driven by store-operated Ca 2+ entry. Stromal cell-interaction molecule (STIM) 1 and STIM2 serve as endoplasmic reticulum Ca 2+ sensors that, upon store depletion, activate Ca 2+ release-activated Ca 2+ channels (Orai1-3, CRACM1-3) in the plasma membrane. However, their relative roles in agonist-mediated Ca 2+ oscillations remain ambiguous. Here we report that while both STIM1 and STIM2 contribute to store-refilling during Ca 2+ oscillations in mast cells (RBL), T cells (Jurkat) and human embryonic kidney (HEK293) cells, they do so dependent on the level of store depletion. Molecular silencing of STIM2 by siRNA or inhibition by G418 suppresses store-operated Ca 2+ entry and agonist-mediated Ca 2+ oscillations at low levels of store depletion, without interfering with STIM1-mediated signals induced by full store depletion. Thus, STIM2 is preferentially activated by low-level physiological agonist concentrations that cause mild reductions in endoplasmic reticulum Ca 2+ levels. We conclude that with increasing agonist concentrations, store-operated Ca 2+ entry is mediated initially by endogenous STIM2 and incrementally by STIM1, enabling differential modulation of Ca 2+ entry over a range of agonist concentrations and levels of store depletion.