The generation of high-affinity antibodies requires an efficient germinal center (GC) response. As differentiating B cells cycle between GC dark and light zones they encounter different oxygen pressures (pO2). However, it is essentially unknown if and how variations in pO2 affect B cell differentiation, in particular for humans. Using optimized in vitro cultures together with in-depth assessment of B cell phenotype and signaling pathways, we show that oxygen is a critical regulator of human naive B cell differentiation and class switch recombination. Normoxia promotes differentiation into functional antibody secreting cells, while a population of CD27++ B cells was uniquely generated under hypoxia. Moreover, time-dependent transitions between hypoxic and normoxic pO2 during culture - reminiscent of in vivo GC cyclic re-entry - steer different human B cell differentiation trajectories and IgG class switch recombination. Taken together, we identified multiple mechanisms trough which oxygen pressure governs human B cell differentiation.
Copyright © 2022 Koers, Marsman, Steuten, Tol, Derksen, ten Brinke, van Ham and Rispens.

h4>ABSTRACT/h4> Chimeric antigen receptor (CAR) costimulatory domains steer the phenotypic output of therapeutic T cells. In most cases these domains are derived from native immune receptors, composed of signaling motif combinations selected by evolution. To explore if non-natural combinations of signaling motifs could drive novel cell fates of interest, we constructed a library of CARs containing ∼2,300 synthetic costimulatory domains, built from combinations of 13 peptide signaling motifs. The library produced CARs driving diverse fate outputs, which were sensitive to motif combinations and configurations. Neural networks trained to decode the combinatorial grammar of CAR signaling motifs allowed extraction of key design rules. For example, the non-native combination of TRAF- and PLCγ1-binding motifs was found to simultaneously enhance cytotoxicity and stemness, a clinically desirable phenotype associated with effective and durable tumor killing. The neural network accurately predicts that addition of PLCγ1-binding motifs improves this phenotype when combined with TRAF-binding motifs, but not when combined with other immune signaling motifs (e.g. PI3K-or Grb2-binding motifs). This work shows how libraries built from the minimal building blocks of signaling, combined with machine learning, can efficiently guide engineering of receptors with desired phenotypes. h4>Graphical Abstract/h4>

OTULIN is a linear deubiquitinase that negatively regulates the nuclear factor κB (NF-κB) signaling pathway. Patients with OTULIN deficiency, termed as otulipenia or OTULIN-related autoinflammatory syndrome, present with early onset severe systemic inflammation due to increased NF-κB activation. We aimed to investigate additional disease mechanisms of OTULIN deficiency. Our study found a remarkable activation of type I interferon (IFN-I) signaling in whole blood, peripheral blood mononuclear cells, monocytes, and serum from patients with OTULIN deficiency. We observed similar immunologic findings in OTULIN-deficient cell lines generated by CRISPR. Mechanistically, we identified proteasome subunits as substrates of OTULIN deubiquitinase activity and demonstrated proteasome dysregulation in OTULIN-deficient cells as the cause of IFN-I activation. These results reveal an important role of linear ubiquitination in the regulation of proteasome function and suggest a link in the pathogenesis of proteasome-associated autoinflammatory syndromes and OTULIN deficiency.

Brain tumor–initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that about 8% of cells within the human glioblastoma microenvironment coexpress programmed cell death 1 (PD-1) and BTIC marker. Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation and self-renewal of BTICs. Phosphorylation of tyrosines within the cytoplasmic tail of PD-1 recruited Src homology 2–containing phosphatase 2 and activated the nuclear factor kB in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require programmed cell death ligand 1(PD-L1) ligation; thus, the therapeutic antibodies inhibiting PD-1/PD-L1 interaction could not overcome the growth advantage of PD-1 in BTICs. Last, BTIC-intrinsic PD-1 accelerated intracranial tumor growth, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a nonimmune resistance mechanism of patients with glioblastoma to PD-1– or PD-L1–blocking therapies.

Endothelial progenitor cells (EPCs) can enhance the recanalization of thrombosis during the progression of cerebral infarction. Prazosin plays a therapeutic role in expanding the peripheral vasculature and regulating infarction cardiosclerosis by inhibiting phosphoinositide signaling. However, the possible mechanisms underlying the therapeutic effects of prazosin have not been fully explored. The purpose of the present study was to analyze the anti-apoptotic effects of prazosin on EPCs in a rat cerebral infarction model. The results showed that prazosin treatment decreased apoptosis of EPCs. Prazosin treatment decreased the serum expression levels of the inflammatory factors, interleukin-1β and tumor necrosis factor-α in rats with cerebral infarctions as well as in EPCs in vitro. In addition, prazosin reduced the expression levels of Akt, NF-κB, phosphorylated (p)-Akt and p-NF-κB in EPCs and the middle cerebral artery of rats with cerebral infarction. These findings demonstrated that prazosin inhibited EPC apoptosis in the cerebral infarction rats through targeting the Akt/NF-κB signaling pathway. In conclusion, these results indicated that prazosin has a preventive effect on cerebral infarction by inhibiting EPC apoptosis and by inhibiting the inflammatory response in vitro and in vivo through regulating the Akt/NF-κB signaling pathway.
Copyright: © Liu et al.