SUMMARY Humoral immunity relies on the efficient differentiation of memory B cells (MBCs) into antibody-secreting cells (ASCs). T helper (Th) signals upregulate B cell receptor (BCR) signaling by potentiating Src family kinases through increasing CD45 phosphatase activity (CD45 PA). In this study, we show that high CD45 PA in MBCs enhances BCR signaling and is essential for their effective ASC differentiation. Mechanistically, Th signals upregulate CD45 PA through intensifying the surface binding of a CD45 ligand, Galectin-1. CD45 PA works as a sensor of T cell help and defines high-affinity germinal center (GC) plasma cell (PC) precursors characterized by IRF4 expression in vivo . Increasing T cell help in vitro results in an incremental CD45 PA increase and enhances ASC differentiation by facilitating effective induction of the transcription factors IRF4 and BLIMP1. This study connects Th signals with BCR signaling through Galectin-1-dependent regulation of CD45 PA and provides a mechanism for efficient ASC differentiation of MBCs.

Background/Aims As autophagy is linked to several pathological conditions, like cancer and neurodegenerative diseases, it is crucial to understand its regulatory signaling network. In this study, we investigated the role of the serum- and glucocorticoid-induced protein kinase 1 (SGK1) in the control of autophagy. Methods To measure autophagic activity in vivo, we quantified the abundance of the autophagy conjugates LC3-PE (phosphatidylethanolamine) and ATG12-ATG5 in tissue extracts of SGK1 wild-type (Sgk1 +/+) and knockout (Sgk1 −/−) mice that were either fed or starved for 24 h prior sacrifice. In vitro, we targeted SGK1 by RNAi using GFP-WIPI1 expressing U-2 OS cells to quantify the numbers of cells displaying newly formed autophagosomes. In parallel, these cells were also assessed with regard to LC3 and ULK1 by quantitative Western blotting. Results The abundance of both LC3-PE (LC3-II) and ATG12-ATG5 was significantly increased in red muscle tissues of SGK1 knockout mice. This was found in particular in fed conditions, suggesting that SGK1 may keep basal autophagy under control in red muscle in vivo. Under starved conditions, significant differences were observed in SGK1-deficient white muscle tissue and, under fed conditions, also in the liver. In vitro, we found that SGK1 silencing provoked a significant increase of cells displaying WIPI1-positive autophagosomes and autophagosomal LC3 (LC3-II). Moreover, autophagic flux assessments revealed that autophagic degradation significantly increased in the absence of SGK1, strongly suggesting that SGK1 inhibits both autophagosome formation and autophagic degradation in vitro. In addition, more ULK1 protein lacking the inhibitory, TORC1-specific phosphorylation at serine 758 was detected in the absence of SGK1. Conclusions Combined, our data strongly support the idea that SGK1 inhibits the process of autophagy. Mechanistically, our data suggest that SGK1 should act upstream of ULK1 in regulating autophagy, and we hypothesize that SGK1 contributes to the regulation of ULK1 gene expression.

Chronic lymphocytic leukaemia (CLL) is characterised by malignant mature-like B cells. Supportive to CLL cell survival is chronic B-cell receptor (BCR) signalling; however, emerging evidence demonstrates CLL cells proliferate in response to Thelper (Th) cells in a CD40L-dependent manner. We showed provision of Th stimulation via CD40L upregulated CD45 phosphatase activity and BCR signalling in non-malignant B cells. Consequently, we hypothesised Th cell upregulation of CLL cell CD45 activity may be an important regulator of CLL BCR signalling and proliferation. Using patient-derived CLL cells in a culture system with activated autologous Th cells, results revealed increases in both Th and CLL cell CD45 activity, which correlated with enhanced downstream antigen receptor signalling and proliferation.Concomitantly increased was the surface expression of Galectin-1, a CD45 ligand, and CD43, a CLL immunophenotypic marker. Galectin-1/CD43 double expression defined a proliferative CLL cell population with enhanced CD45 activity. Targeting either Galectin-1 or CD43 using silencing, pharmacology, or monoclonal antibody strategies dampened CD45 activity and CLL cell proliferation. These results highlight a mechanism where activated Th cells drive CLL cell BCR signalling and proliferation via Galectin-1 and CD43-mediated regulation of CD45 activity, identifying modulation of CD45 phosphatase activity as a potential therapeutic target in CLL.