BackgroundThe efficacy of B cell-depleting therapies for rheumatoid arthritis underscores antibody-independent functions of effector B cells such as cognate T–B interactions and production of pro-inflammatory cytokines. Receptor activator of nuclear factor κB ligand (RANKL) is a key cytokine involved in bone destruction and is highly expressed in synovial fluid B cells in patients with rheumatoid arthritis. In this study we sought to clarify the generation mechanism of RANKL+ effector B cells and their impacts on osteoclast differentiation.MethodsPeripheral blood and synovial fluid B cells from healthy controls and patients with rheumatoid arthritis were isolated using cell sorter. mRNA expression of RANKL, osteoprotegerin, tumor necrosis factor (TNF)-α, and Blimp-1 was analyzed by quantitative real-time polymerase chain reaction. Levels of RANKL, CD80, CD86, and CXCR3 were analyzed using flow cytometry. Functional analysis of osteoclastogenesis was carried out in the co-culture system using macrophage RAW264 reporter cells.ResultsRANKL expression was accentuated in CD80+CD86+ B cells, a highly activated B-cell subset more abundantly observed in patients with rheumatoid arthritis. Upon activation via B-cell receptor and CD40, switched-memory B cells predominantly expressed RANKL, which was further augmented by interferon-γ (IFN-γ) but suppressed by interleukin-21.Strikingly, IFN-γ also enhanced TNF-α expression, while it strongly suppressed osteoprotegerin expression in B cells. IFN-γ increased the generation of CXCR3+RANKL+ effector B cells, mimicking the synovial B cell phenotype in patients with rheumatoid arthritis. Finally, RANKL+ effector B cells in concert with TNF-α facilitated osteoclast differentiation in vitro.ConclusionsOur current findings have shed light on the generation mechanism of pathogenic RANKL+ effector B cells that would be an ideal therapeutic target for rheumatoid arthritis in the future.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0957-6) contains supplementary material, which is available to authorized users.

Human anti‐programmed death‐1 (PD‐1) antibody possesses the capability to revitalize host T cells and has been an effective therapy for metastatic malignant melanoma (MM). The precise subsets of T cells predominantly activated by anti‐PD‐1, however, have not yet been clarified. In this study, peripheral blood mononuclear cells obtained from MM patients scheduled to receive anti‐PD‐1 (nivolumab) therapy, and healthy subjects (HS), were systematically examined on flow cytometry to identify changes in the proportion of immune cell subsets. Compared with HS, MM patients prior to therapy had an increased proportion of activated CD8+ T cells with effector memory phenotypes (Tem), and PD‐1 positive subsets of CD4+ central memory T cells (Tcm) and T‐helper (Th)17 cells. After a single course of anti‐PD‐1 therapy, MM patients had an increase in activated Tem and Tcm subsets of CD4+ and CD8+ T cells, and activated Th1 plus T‐helper follicular 1 cells. There was no consistent change in the proportion of Tfh cells, B cells, natural killer cells, or dendritic cells. The observed activated phenotypes were attenuated during the course of therapy, but regulatory T cells belonging to the CD3+CD4+CD45RO+CD25high fraction increased at disease progression. Taken together, anti‐PD‐1 therapy modulates systemic immune reactions and exerts anti‐tumor effects, not only by revitalizing Tem and Tcm of CD4+ and CD8+ T cells, but also via a shift to a Th1 phenotype.

Background:Anti-programmed cell death 1 antibody nivolumab is a promising agent for various cancers. Immune-related adverse events are recognized; however, bi-cytopenia with nivolumab has not been reported.Case presentation:A 73-year-old man was diagnosed with advanced primary malignant melanoma of the esophagus with liver, lung, and lymph node metastases. Previous therapies including dacarbazine and radiation of 39 Gy to the esophageal region were performed, but the liver metastases deteriorated. The patient was then administered nivolumab (2 mg/kg, every 3 weeks). After 3 cycles, the esophageal tumor and lymph nodes showed marked reductions in size, the lung metastases disappeared, and the liver metastases shrank partially. The treatment continued with 7 cycles for 4 months. However, severe anemia and thrombocytopenia appeared in the 6th cycle, and intermittent blood transfusions were required. The patient received high-dose intravenous methylprednisolone therapy for bi-cytopenia, but it was ineffective. Seven months after the initiation of nivolumab, the patient died of tumor. Although the mechanisms of bi-cytopenia were unclear, it could have been induced by nivolumab.Conclusion:The present case shows a rare but serious life-threatening bi-cytopenia possibly associated with nivolumab and suggests the importance of awareness of hematological adverse events during nivolumab therapy.

IntroductionB-cell receptor (BCR) signaling guides critical cell fate decisions in B cells during ontogeny. 1,2 BCRs can generate tolerogenic signals to purge or silence B cells that bind to self-antigens, and immunogenic signals to expand B cells that are specific for foreign antigens. Thus, BCR signaling must be properly regulated at the various stages of B-cell development, as aberrant regulation of BCR signaling potentially leads to autoimmunity and B-cell malignancies.On BCR ligation by antigens, the Src-family protein tyrosine kinase (PTK) Lyn and Syk are initially activated. Syk propagates the signal by phosphorylating downstream signaling molecules, causing the activation of critical signaling intermediates phosphoinositol 3-kinase (PI3K) and phospholipase C (PLC)␥2. PI3K activates Akt kinase, which is important for B-cell survival. 3 PLC␥2 activation induces the release of intracellular Ca 2ϩ and the activation of protein kinase C (PKC), which cause the activation of mitogen-activated protein kinases (MAPKs; ERK, JNK, and p38 MAPK) and of transcription factors, including NF-B and NF-AT. These molecules regulate further downstream molecules that are responsible for determining B-cell fates such as survival, growth, and differentiation. 1,2 Casitas B-lineage lymphoma (Cbl) proteins are E3 ubiquitin ligases that regulate signals of various receptors by promoting the ubiquitination of signaling components. 4,5 Tyrosine phosphorylation of Cbl proteins is critical for their function. 6 Mammalian Cbl proteins consist of 3 members, c-Cbl, Cbl-b, and Cbl-3, among which c-Cbl and Cbl-b are expressed in hematopoietic cells. 7 In B cells, Cbl proteins associate with Syk and B-cell linker (BLNK), and negatively regulate BCR signaling. 8,9 B cell-specific ablation of c-Cbl/Cbl-b proteins in mice causes aberrant BCR signaling as well as impaired B-cell anergy, culminating in the development of systemic lupus erythematosus (SLE)-like disease. 10 In addition, c-Cbl is hypophosphorylated on tyrosine in advanced stages of chronic lymphocytic leukemia (CLL). 11 These findings suggest that Cbl-mediated regulation of BCR signaling is critical for the fate decisions of self-reactive and malignant B cells.Adaptors are noncatalytic molecules that integrate the spatial and temporal assembly of multiprotein complexes involved in the survival, growth, and differentiation of B cells. We previously showed that the B lymphocyte adaptor molecule of 32 kDa (Bam32)/DAPP1 regulates BCR signaling/internalization and B-cell survival. 12,13 The SH3KBP1 (SH3-domain kinase-binding protein 1) gene, which is also known as CIN85 (c-Cbl interacting protein of 85 kDa), encodes an adaptor that is independently identified by several groups and contains 3 SH3 domains, a proline-rich region, and a coiled-coil domain. [14][15][16][17] Early studies showed that in nonimmune cells, CIN85 regulates the clathrindependent internalization of receptor tyrosine kinases (RTKs) such as epidermal growth factor receptors (EGFRs). 18,19 The formation of the ternary...

SummaryInterleukin-33 (IL-33) induces T helper type 2 (Th2) cytokine production and eosinophilia independently of acquired immunity, leading to innate immunity-mediated allergic inflammation. Allergy-related innate myeloid cells such as eosinophils, basophils and mast cells express the IL-33 receptor (IL-33R), but it is still unknown how IL-33 regulates allergic inflammation involving these cells and their progenitors. Here, we revealed that the functional IL-33R was expressed on eosinophil progenitors (EoPs), basophil progenitors (BaPs) and mast cell progenitors (MCPs). In the presence of IL-33, these progenitors did not expand, but produced a high amount of Th2 and pro-inflammatory cytokines such as IL-9, IL-13, IL-1b and IL-6. The amount of cytokines produced by these progenitors was greater than that by mature cells. In vivo, IL-33 stimulated the expansion of EoPs, but it was dependent upon the elevated serum IL-5 that is presumably derived from type 2 innate lymphoid cells that express functional IL-33R. These data collectively suggest that EoPs, BaPs and MCPs are not only the sources of allergy-related granulocytes, but can also be sources of allergy-related cytokines in IL-33-induced inflammation. Because such progenitors can differentiate into mature granulocytes at the site of inflammation, they are potential therapeutic targets in IL-33-related allergic diseases.