As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2) has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR) signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell–specfic Rictor knockout (KO) mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons tyrosine kinase (pBtk) and phosphorylated SH2-containing inositol phosphatase (pSHIP), are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin) is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization.

Dock8 deficiency leads to immunodeficiency, and the role of Dock8 in B-cell development and function has been revealed; however, the role of DocK8 on B-cell receptor (BCR) signaling and function of memory B cells remains elusive. In this study, we generated a Dock8 knockout mouse model and collected peripheral blood mononuclear cells from Dock8 patients to study the effect of Dock8 deficiency on the BCR signaling and activation of memory B cells with confocal microscopy and total internal reflection fluorescence microscopy. The activation of key, positive upstream BCR signaling molecules, pCD19 and phosphorylated Brutons tyrosine kinase (pBtk), is reduced. Interestingly, the total protein and activated levels of Wiskott-Aldrich syndrome protein (WASP) are decreased in Dock8-deficient mouse B cells. Our previous research has shown that WASP positively regulates transcription; furthermore, we found that Dock8 regulates transcription. What we found in Dock8 patients can be a phenotype copied from Dock8 mice. The early activation of memory B cells from Dock8 patients is disrupted with reduced BCR clustering, B-cell spreading, and signalosome recruitment into the degree of naïve B cells, as well as the transition from naïve B cells to unswitched memory B cells. Overall, our study provides a novel mechanism for Dock8 regulation of BCR signaling by regulating transcription, as well as the underlying mechanism of noncompetence of memory B cells in Dock8 patients.

Key Points• Mst1 positively regulates CD19-mediated Btk signaling in B cells.• Mst1 regulates CD19 transcription through TEAD2 directly binding to the 39UTR of cd19.As a key regulator of hippo signaling pathway, Mst kinases are emerging as one of the key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. In

A pyramid strategy combining the crystal (Cry) 1A and 2A toxins in Bacillus thuringiensis (Bt) crops are active against many species of insects and nematode larvae. It has been widely used to delay pest adaption to genetically modified plants and broaden the insecticidal spectrum in many countries. Unfortunately, Cry2A can also bind with the specific receptor proteins of Cry1A. ATP-binding cassette (ABC) transporters can interact with Cry1A toxins as receptors in the insect midgut, and ABC transporter mutations result in resistance to Bt proteins. However, there is limited knowledge of the ABC transporters that specifically bind to Cry2Ab. Here, we cloned the ABCC1 gene in Helicoverpa armigera, which expressed at all larval stages and in nine different tissues. Expression levels were particularly high in fifth-instar larvae and Malpighian tubules. The two heterologously expressed HaABCC1 transmembrane domain peptides could specifically bind to Cry2Ab with high affinity levels. Moreover, transfecting HaABCC1 into the Spodoptera frugiperda nine insect cell significantly increased its mortality when exposed to Cry2Ab in vitro, and silencing HaABCC1 in H. armigera by RNA interference significantly reduced the mortality of larvae exposed to Cry2Ab in vivo. Altogether current results suggest that HaABCC1 serves as a functional receptor for Cry2Ab.

Wiskott-Aldrich syndrome (WAS) pediatric patients exhibit a deficiency in humoral immune memory. However, the mechanism by which Wiskott-Aldrich syndrome protein (WASP) regulates the differentiation and activation of memory B cells remains elusive. Here we examine the early activation events of memory B cells from the peripheral blood mononuclear cells of WAS patients and age-matched healthy controls (HCs) using total internal reflection fluorescence microscopy. In response to stimulation through the B-cell receptor (BCR), memory B cells from HCs showed significantly higher magnitudes of BCR clustering and cell spreading than naive B cells from the same individuals. This was associated with increases in CD19 recruitment to the BCR and the activation of its downstream signaling molecule Btk and decreases in FcγRIIB recruitment and the activation of its downstream molecule Src homology 2-containing inositol 5' phosphatase (SHIP). However, these enhanced signaling activities mediated by CD19 and Btk are blocked in memory B cells from WAS patients, whereas the activation of FcγRIIB and SHIP was increased. Although the expression levels of CD19, Btk, and FcγRIIB did not change between CD27(-) and CD27(+) B cells of HCs, the protein and mRNA levels of CD19 but not Btk and FcγRIIB were significantly reduced in both CD27(-) and CD27(+) B cells of WAS patients, compared with those of HCs. Overall, our study suggests that WASP is required for memory B-cell activation, promoting the activation by positive regulating CD19 transcription and CD19 recruitment to the BCR.

Wiskott-Aldrich syndrome protein (WASp) is a hematopoietic-specific regulator of actin nucleation. Wiskott-Aldrich syndrome (WAS) patients show immunodeficiencies, most of which have been attributed to defective T-cell functions. T follicular helper (Tfh) cells are the major CD4+ T-cell subset with specialized B-cell helper capabilities. Aberrant Tfh cells activities are involved in immunopathologies such as autoimmunity, immunodeficiencies, and lymphomas. We found that in WAS patients, the number of circulating Tfh cells was significantly reduced due to reduced proliferation and increased apoptosis, and Tfh cells were Th2 and Th17 polarized. The expression of inducible costimulator (ICOS) in circulating Tfh cells was higher in WAS patients than in controls. BCL6 expression was decreased in total CD4+ T and Tfh cells of WAS patients. Mirroring the results in patients, the frequency of Tfh cells in WAS knockout (KO) mice was decreased, as was the frequency of BCL6+ Tfh cells, but the frequency of ICOS+ Tfh cells was increased. Using WAS chimera mice, we found that the number of ICOS+ Tfh cells was decreased in WAS chimera mice, indicating that the increase in ICOS+ Tfh cells in WAS KO mice was cell extrinsic. The data from in vivo CD4+ naive T-cell adoptive transfer mice as well as in vitro coculture of naive B and Tfh cells showed that the defective function of WASp-deficient Tfh cells was T-cell intrinsic. Consistent findings in both WAS patients and WAS KO mice suggested an essential role for WASp in the development and memory response of Tfh cells and that WASp deficiency causes a deficient differentiation defect in Tfh cells by downregulating the transcription level of BCL6.

The purpose of this study was to assess the genetic characteristics of six breeds of Chinese local sheep using 19 microsatellite loci and to effectively validate statistical methods for individual assignment based on informative microsatellites. All the six breeds deviated from Hardy-Weinberg equilibrium expectations, while the majority of markers complied. The polymorphism information content (PIC) of overall loci for the six populations ranged from 0.283 (SRCRSP5) to 0.852 (OarVH72). Tibetan sheep were the most diverse population with the highest mean allelic richness (6.895), while Ujmuqin (UQ) harboured the lowest allelic richness (6.000). The F-statistics for the six populations were F(IS)  = -0.172, F(IT)  = -0.082 and F(ST)  = 0.077, respectively. Furthermore, the pair-wise F(IS) revealed a moderate genetic differentiation among populations (P < 0.01), indicating that all breeds can be considered genetically independent entities. The lowest genetic differentiation was between Tengchong (TC) and UQ (F(ST)  = 0.041), and the highest one was between TC and Fat-tailed Han (F(ST)  = 0.111). In comparing the three statistical models, we note that the seven microsatellite loci (MAF65, OarJMP58, SRCRSP9, MCM140, OarAE129, BM8125 and SRCRSP5) commonly used for individual assignment will ensure a powerful detection of individual origin, with accuracy up to 91.87%, when the likelihood-based method is used. Overall, these findings shed light onto the genetic characteristics of Chinese indigenous sheep and offer a set of microsatellite loci that is simple, economic and highly informative for individual assignment of Chinese sheep.

As a critical linker between mTORC1 and mTORC2, Akt is important for the cell metabolism. The role of Akt in the function and development of B and T cells is well characterized, however, the role of Akt for development and function of iNKT cells is unknown. iNKT cells bridge the adaptive and innate immunity, and in this study, we found that the differentiation of NKT17 cells and IL17 production of NKT17 cells were disrupted in Akt2 KO mice. ICOS has been demonstrated to be critical for the differentiation of NKT17 cells and we found that ICOS mRNA and protein expression was reduced in Akt2 KO iNKT cells. As a consequence, phosphorylation of FoxO-1 was downregulated in Akt2 KO thymocytes but the sequestration of FoxO-1 in the nucleus of Akt2 KO iNKT cells was increased. The negative feedback loop between ICOS and FoxO-1 has been demonstrated in CD4+T follicular helper cells. Therefore our study has revealed a new intracellular mechanism in which Akt2 regulates ICOS expression via FoxO-1 and this signaling axis regulates the differentiation and function of NKT17 cells. This study provides a new linker between cell metabolism and function of iNKT cells.