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IntroductionClassic differentiation of naive CD4 ϩ T cells into different T helper (Th) subsets, including Th1, Th2, and Th17, occurs in lymphoid tissues after contact with antigen-presenting cells that produce polarizing cytokines. These Th subsets in turn orchestrate diverse immune responses also mediated by production of distinct cytokines. Because aberrant Th1 or Th17 activities have the potential to trigger chronic inflammatory and autoimmune diseases, 1,2 effector Th responses in healthy persons are under tight regulation mediated in part by CD4 ϩ regulatory T cells (Tregs) that are thymic-derived or naive T cell-inducible. 3 Understanding how Th1/Th17/Treg differentiation and expansion are controlled is likely to provide an explanation of how inflammation may be sustained in pathologic environments.More recently, human monocytes were shown to trigger and polarize Th responses 4,5 as well as to both stimulate and suppress T-cell responses during infection and in autoimmune diseases. 5,6 Monocytes, which are generally regarded as precursors of tissue macrophages and dendritic cells, 7 can be phenotypically divided based on surface expression of CD14 (lipopolysaccharide receptor) and CD16 (low affinity Fc␥ receptor III) expression into subsets, each with distinct functional activities. The major monocyte subpopulation characterized by high CD14 but no CD16 expression (CD14 hi CD16 Ϫ ), also referred to as classic monocytes, have higher phagocytic activity. 8 The minor CD16 ϩ cells produce higher TNF after stimulation and expand under infectious or inflammatory conditions. 9,10 With regards to the control of Th differentiation and reactivation, the specific role of the monocyte subsets has not been fully characterized.Immune thrombocytopenia (ITP) is an autoimmune bleeding disease resulting from decreased platelet production as well as accelerated platelet destruction mediated in part by autoantibody-based destruction mechanisms. 11 ITP patients harbor activated platelet-autoreactive T cells with increasing cytokine imbalance toward IL-2 and IFN-␥ 12-14 as well as altered Treg numbers and function. [15][16][17][18][19][20] A shift toward stimulatory monocytes with enhanced Fc␥R-mediated phagocytic capacity further supports a generalized immune dysregulation in ITP. 21 More recently, studies reported increased Th17 cells or IL-17 cytokine in ITP patients, 22-24 implicating a possible role for Th17 cells in ITP immunopathology, although 2 reports did not detect any difference. 25,26 Among the treatment options available to ITP patients, the recently licensed thrombopoietic agents, by increasing platelet production, have yielded overall durable responses in patients with persistent, chronic, and/or refractory ITP while on treatment. 27 Interestingly, improved Treg function in ITP patients was associated with increased platelet counts after the use of these agents, 28 despite apparent lack of immunomodulatory activity associated with such agents. Similarly, improved Treg compartment was reported in ITP patients w...

© F e r r a t a S t o r t i F o u n d a t i o nphoid T FH , cT FH cells can express significant levels of surface programmed cell death-1 (PD-1), 31 although the function of PD-1 on T FH cells remains controversial since it is associated with both promotion 16,17 and inhibition of B-cell responses. 18,[32][33][34] Taken together, these reports underscore the need for better characterization of markers for cT FH cells displaying defined functions not only in steady state but also in diseases. Remarkably, PD-1 has been described as a member of the growing family of inhibitory receptors also referred to as immune checkpoints, responsible for aborting T-cell responses. 35 Interestingly, another member of the immune checkpoint family, TIGIT (T-cell immunoreceptor with Ig and immunoreceptor tyrosinebased inhibitory domains), was reported to be overexpressed on both tonsillar and cT FH cells, 17 and was shown to be involved in interactions between T cells and follicular dendritic cells to regulate B-cell responses. 36,37 However, the functional activity of TIGIT on T FH cells, including cT FH cells, has not been studied to date.In this study, we took the approach of using TIGIT and PD-1 to characterize the phenotype and function of circulating T FH subsets and to investigate whether expression of these molecules on cT FH cells modulated their functions in healthy volunteer donors and in a group of chronically transfused SCD patients with or without alloantibodies. Methods Human samplesAll studies were approved by the Institutional Review Boards of the New York Blood Center (NYBC). De-identified fresh leukopaks were obtained from healthy donors at the NYBC. For SCD patients' samples, blood was obtained solely from discard apheresis waste bags obtained during erythrocytapheresis procedures at the Children's Hospital of Philadelphia (see Online Supplementary Material for details). T-cell studiesFreshly-sorted CD4 + T-cell subsets and autologous naïve or memory B cells were used (see Online Supplementary Material for details). Blocking antibodies for TIGIT 38 and PD-1 34,39 were preincubated with sorted T cells before being co-cultured with autologous B cells. Results PD- Expression of ICOS, CD40L and IL-21 by TIGIT + cT FH cellsWe next tested whether TIGIT + cT FH cells were functionally different from cT FH + cT FH populations from a small number of healthy donors (n=3 or 4) were sorted and their ability to express T FH -associated co-stimulatory markers and cytokines following stimulation was compared to those of sorted autologous PD-1 -/TIGIT -subsets (gating strategy shown in Online Supplementary Figure S1). As a control, sort-purified autologous CXCR5 -non-cT FH cells expressing TIGIT E. Godefroy et al. 1416haematologica | 2015; 100(11) A B C© F e r r a t a S t o r t i F o u n d a t i o n ("TIGIT+") or not ("TIGIT-") were also tested. We first monitored expression of co-stimulatory molecules CD40L and ICOS, both specialized in providing Bcell help, on T-cell subsets before or after stimulation by immunos...

A CBL-interacting protein kinase (CIPK) gene, BnCIPK6, was isolated in Brassica napus. Through yeast two-hybrid screening, 27 interaction partners (including BnCBL1) of BnCIPK6 were identified in Brassica napus. Interaction of BnCIPK6 and BnCBL1 was further confirmed by BiFC (bimolecular fluorescence complementation) in plant cells. Expressions of BnCIPK6 and BnCBL1 were significantly up-regulated by salt and osmotic stresses, phosphorous starvation, and abscisic acid (ABA). Furthermore, BnCIPK6 promoter activity was intensively induced in cotyledons and roots under NaCl, mannitol, and ABA treatments. Transgenic Arabidopsis plants with over-expressing BnCIPK6, its activated form BnCIPK6M, and BnCBL1 enhanced high salinity and low phosphate tolerance, suggesting that the functional interaction of BnCBL1 and BnCIPK6 may be important for the high salinity and phosphorous deficiency signalling pathways. In addition, activation of BnCIPK6 confers Arabidopsis plants hypersensitive to ABA. On the other hand, over-expression of BnCIPK6 in Arabidopsis cipk6 mutant completely rescued the low-phosphate-sensitive and ABA-insensitive phenotypes of this mutant, further suggesting that BnCIPK6 is involved in the plant response to high-salinity, phosphorous deficiency, and ABA signalling.

Red blood cell alloimmunization is a major complication of transfusion therapy. Host immune markers that can predict antibody responders remain poorly described. As regulatory T cells (Tregs) play a role in alloimmunization in mouse models, we analyzed the Treg compartment of a cohort of chronically transfused patients with sickle cell disease (SCD, n = 22) and β-thalassemia major (n = 8) with and without alloantibodies. We found reduced Treg activity in alloantibody responders compared with nonresponders as seen in mice. Higher circulating anti-inflammatory IL-10 levels and lower IFN-γ levels were detected in non-alloimmunized SCD patients. Stimulated sorted CD4+ cells from half of the alloimmunized patients had increased frequency of IL-4 expression compared with nonresponders, indicating a skewed T helper (Th) 2 humoral immune response in a subgroup of antibody responders. All patients had increased Th17 responses, suggesting an underlying inflammatory state. Although small, our study indicates an altered immunoregulatory state in alloantibody responders which may help future identification of potential molecular risk factors for alloimmunization.

Patients with sickle cell disease (SCD) often require transfusions to treat and prevent worsening anemia and other SCD complications. However, transfusions can trigger alloimmunization against transfused red blood cells (RBCs) with serious clinical sequelae. Risk factors for alloimmunization in SCD remain poorly understood. We recently reported altered regulatory T cell (Treg) and T helper (Th) responses with higher circulating Th1 (IFN-γ+) cytokines in chronically transfused SCD patients with alloantibodies as compared to those without alloantibodies. Since monocytes play a critical role in polarization of T cell subsets and participate in clearance of transfused RBCs, we tested the hypothesis that in response to RBC breakdown product, hemin, monocyte control of T cell polarization will differ between alloimmunized and non-alloimmunized SCD patients. Exogenous hemin induced Treg polarization in purified T-cell-monocyte cocultures from healthy volunteers through monocyte anti-inflammatory heme degrading enzyme HO-1. Importantly, hemin primarily through its effect on CD16+ monocytes induced an anti-inflammatory (higher Treg/lower Th1) polarization state in non-alloimmunized SCD group, whereas it had little effect in the alloimmunized group. Non-alloimmunized SCD CD16+ monocytes expressed higher basal levels of HO-1. Furthermore, IL-12, which contributed to a pro-inflammatory polarization state (low Treg/high Th1) in SCD, was dampened in hemin-treated stimulated monocytes from non-alloimmunized SCD patients, but not in alloimmunized group. These data suggest that unlike alloimmunized patients, non-alloimmunized SCD CD16+ monocytes in response to transfused RBC breakdown products promote an anti-inflammatory state that is less conductive to alloimmunization.

Regulation of gene expression by DNA-binding transcription factors is essential for proper control of growth and development in all organisms. In this study, we annotate and characterize growth and developmental phenotypes for transcription factor genes in the model filamentous fungus Neurospora crassa. We identified 312 transcription factor genes, corresponding to 3.2% of the protein coding genes in the genome. The largest class was the fungal-specific Zn2Cys6 (C6) binuclear cluster, with 135 members, followed by the highly conserved C2H2 zinc finger group, with 61 genes. Viable knockout mutants were produced for 273 genes, and complete growth and developmental phenotypic data are available for 242 strains, with 64% possessing at least one defect. The most prominent defect observed was in growth of basal hyphae (43% of mutants analyzed), followed by asexual sporulation (38%), and the various stages of sexual development (19%). Two growth or developmental defects were observed for 21% of the mutants, while 8% were defective in all three major phenotypes tested. Analysis of available mRNA expression data for a time course of sexual development revealed mutants with sexual phenotypes that correlate with transcription factor transcript abundance in wild type. Inspection of this data also implicated cryptic roles in sexual development for several cotranscribed transcription factor genes that do not produce a phenotype when mutated.

Phosphorus (P) is one of the essential nutrient elements for plant development. In this work, BnPHR1 encoding a MYB transcription activator was isolated from Brassica napus. The characterization of nuclear localization and transcription activation ability suggest BnPHR1 is a transcriptional activator. The tissue expression and histochemical assay showed that BnPHR1 was predominantly expressed in roots and modulated by exogenous Pi in transcriptional level in roots under Pi deficiency conditions. Furthermore, overexpression of BnPHR1 in both Arabidopsis and B. napus remarkably enhanced the expression of the Pi-starvation-induced genes including ATPT2 and BnPT2 encoding the high-affinity Pi transporter. Additionally, BnPHR1 can in vivo bind the promoter sequence of ATPT2 and BnPT2 in both Arabidopsis and B. napus. Possibly, due to the activation of ATPT2 and BnPT2, or even more high-affinity Pi transporters, the excessive Pi was accumulated in transgenic plants, resulting in the crucially Pi toxicity to cells and subsequently retarding plant growth. Given the data together, BnPHR1, as crucial regulator, is regulated by exogenous Pi and directly activates those genes, which promote the uptake and homeostasis of Pi for plant growth.