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Antiplatelet-antibody-producing B cells play a key role in immune thrombocytopenia (ITP) pathogenesis; however, little is known about T-cell dysregulations that support B-cell differentiation. During the past decade, T follicular helper cells (TFHs) have been characterized as the main T-cell subset within secondary lymphoid organs that promotes B-cell differentiation leading to antibody class-switch recombination and secretion. Herein, we characterized TFHs within the spleen of 8 controls and 13 ITP patients. We show that human splenic TFHs are the main producers of interleukin (IL)-21, express CD40 ligand (CD154), and are located within the germinal center of secondary follicles. Compared with controls, splenic TFH frequency is higher in ITP patients and correlates with germinal center and plasma cell percentages that are also increased. In vitro, IL-21 stimulation combined with an anti-CD40 agonist antibody led to the differentiation of splenic B cells into plasma cells and to the secretion of antiplatelet antibodies in ITP patients. Overall, these results point out the involvement of TFH in ITP pathophysiology and the potential interest of IL-21 and CD40 as therapeutic targets in ITP., (© 2014 by The American Society of Hematology.)

Background: The forced expiratory volume at first second (FEV(1)) during spirometry reflects the severity of chronic obstructive pulmonary disease (COPD) and is known to be an important prognostic factor. It is uncertain whether the response to short-acting bronchodilators may predict long-term outcomes such as hospitalizations and mortality., Methods: We retrospectively studied a total of 1203 consecutive COPD patients without significant comorbidities during a mean (±SD) of 69 ± 39 months of follow-up. At baseline the subjects were classified as those with positive or negative bronchodilator test (BDT) and also in quartiles of absolute bronchodilator response to 400 μg of salbutamol. Hospital visits and mortality were the end points., Results: A positive bronchodilator test was observed in 332 (27.6%) of the patients. There were 73 (21.9%) deaths in patients with a positive BDT versus 253 (28.7%) in those with a negative BDT (p = 0.04). In adjusted Cox regression analysis a positive BDT was significantly associated with a prolonged time to first hospitalization. After stratifying the population by quartiles of response to BDT, a dose-response relationship was observed with the best outcomes in the quartile with highest level of airflow reversibility, even after controlling for age, sex, BMI, smoking status and baseline postbronchodilator FEV(1)., Conclusions: In a large population of well characterized COPD patients without significant comorbidities, those demonstrating higher levels of reversibility at baseline presented better long-term outcomes even after controlling for other known prognostic factors., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The pathogenic role of B cells in immune thrombocytopenia (ITP) has justified the therapeutic use of anti-CD20 antibodies such as rituximab (RTX). However, 60% of ITP patients do not respond to RTX. To decipher the mechanisms implicated in the failure of RTX, and because the spleen plays a well-recognized role in ITP pathogenesis, 12 spleens from ITP patients who had been nonresponders to RTX therapy were compared with 11 spleens from RTX-untreated ITP patients and 9 controls. We here demonstrate that in RTX-nonresponder ITP patients, preferential Th1 and Tc1 T lymphocyte polarizations occur, associated with an increase in splenic effector memory CD8(+) T-cell frequency. Moreover, in the RTX- nonresponder patient group, the CD8(+) T-cell repertoire displays a restricted pattern. In the blood, the phenotype of CD8(+) T cells before and after RTX treatment is not modified in responders or nonresponders. Altogether, these results demonstrate for the first time an activation of splenic CD8(+) T cells in ITP patients who did not respond to RTX and suggest their involvement in platelet destruction in these patients.

Immune thrombocytopenia (ITP) is an autoimmune disease with a complex pathogenesis. As in many B cell-related autoimmune diseases, rituximab (RTX) has been shown to increase platelet counts in some ITP patients. From an immunologic standpoint, the mode of action of RTX and the reasons underlying its limited efficacy have yet to be elucidated. Because splenectomy is a cornerstone treatment of ITP, the immune effect of RTX on this major secondary lymphoid organ was investigated in 18 spleens removed from ITP patients who were treated or not with RTX. Spleens from ITP individuals had follicular hyperplasia consistent with secondary follicles. RTX therapy resulted in complete B-cell depletion in the blood and a significant reduction in splenic B cells, but these patients did not achieve remission. Moreover, whereas the percentage of circulating regulatory T cells (Tregs) was similar to that in controls, splenic Tregs were reduced in ITP patients. Interestingly, the ratio of proinflammatory Th1 cells to suppressive Tregs was increased in the spleens of patients who failed RTX therapy. These results indicate that although B cells are involved in ITP pathogenesis, RTX-induced total B-cell depletion is not correlated with its therapeutic effects, which suggests additional immune-mediated mechanisms of action of this drug.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic disorder that occurs in elderly patients. One of the main diagnostic criteria is the accumulation of heterogeneous monocytes in the peripheral blood. We further explored this cellular heterogeneity and observed that part of the leukemic clone in the peripheral blood was made of immature dysplastic granulocytes with a CD14(-)/CD24(+) phenotype. The proteome profile of these cells is dramatically distinct from that of CD14(+)/CD24(-) monocytes from CMML patients or healthy donors. More specifically, CD14(-)/CD24(+) CMML cells synthesize and secrete large amounts of alpha-defensin 1-3 (HNP1-3). Recombinant HNPs inhibit macrophage colony-stimulating factor (M-CSF)-driven differentiation of human peripheral blood monocytes into macrophages. Using transwell, antibody-mediated depletion, suramin inhibition of purinergic receptors, and competitive experiments with uridine diphosphate (UDP)/uridine triphosphate (UTP), we demonstrate that HNP1-3 secreted by CD14(-)/CD24(+) cells inhibit M-CSF-induced differentiation of CD14(+)/CD24(-) cells at least in part through P2Y6, a receptor involved in macrophage differentiation. Altogether, these observations suggest that a population of immature dysplastic granulocytes contributes to the CMML phenotype through production of alpha-defensins HNP1-3 that suppress the differentiation capabilities of monocytes.

The differentiation of human peripheral blood monocytes into resident macrophages is driven by colony-stimulating factor-1 (CSF-1), which upon interaction with CSF-1 receptor (CSF-1R) induces within minutes the phosphorylation of its cytoplasmic tyrosine residues and the activation of multiple signaling complexes. Caspase-8 and -3 are activated at day 2 to 3 and contribute to macrophage differentiation, for example, through cleavage of nucleophosmin. Here, we show that the phosphatidylinositol-3 kinase and the downstream serine/threonine kinase AKT connect CSF-1R activation to caspase-8 cleavage. Most importantly, we demonstrate that successive waves of AKT activation with increasing amplitude and duration are required to provoke the formation of the caspase-8-activating molecular platform. CSF-1 and its receptor are both required for oscillations in AKT activation to occur, and expression of a constitutively active AKT mutant prevents the macrophage differentiation process. The extracellular receptor kinase 1/2 pathway is activated with a coordinated oscillatory kinetics in a CSF-1R-dependent manner but plays an accessory role in caspase activation and nucleophosmin cleavage. Altogether, CSF-1 stimulation activates a molecular clock that involves phosphatidylinositol-3 kinase and AKT to promote caspase activation. This oscillatory signaling pathway, which is coordinated with extracellular receptor kinase 1/2 oscillatory activation, involves CSF-1 and CSF-1R and controls the terminal differentiation of macrophages.