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© 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...

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...

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.