技术资料

We investigated the contribution of host immune cells to bacterial killing in a whole-blood bactericidal activity (WBA) assay,an ex vivo model used to test efficacy of drugs against mycobacterium tuberculosis (Mtb). We performed WBA assays with immuno-magnetic depletion of specific cell types,in the presence or absence of rifampicin. Innate immune cells decreased Mtb growth in absence of drug,but appeared to diminish the cidal activity of rifampicin,possibly attributable to intracellular bacterial sequestration. Adaptive immune cells had no effect with or without drug. The WBA assay may have potential for testing adjunctive host-directed therapies acting on phagocytic cells. View Publication

Phospho flow is a powerful approach to detect cell signaling aberrations,identify biomarkers and assess pharmacodynamics,and can be performed using cryopreserved samples. The effects of cryopreservation on signaling responses and the reproducibility of phospho flow measurements are however unknown in many cell systems. Here,B lymphocytes were isolated from healthy donors and patients with the B cell malignancy chronic lymphocytic leukemia and analyzed by phospho flow using phospho-specific antibodies targeting 20 different protein epitopes. Cells were analyzed both at basal conditions and after activation of cluster of differentiation 40 (CD40) or the B cell receptor. Pharmacodynamics of the novel pathway inhibitor ibrutinib was also assessed. At all conditions,fresh cells were compared to cryopreserved cells. Minimal variation between fresh and frozen samples was detected. Reproducibility was tested by running samples from the same donors in different experiments. The results demonstrate reproducibility across different phospho flow runs and support the use of cryopreserved samples in future phospho flow studies of B lymphocytes. View Publication

TET enzymes are dioxygenases that promote DNA demethylation by oxidizing the methyl group of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Here,we report a close correspondence between 5hmC-marked regions,chromatin accessibility and enhancer activity in B cells,and a strong enrichment for consensus binding motifs for basic region-leucine zipper (bZIP) transcription factors at TET-responsive genomic regions. Functionally,Tet2 and Tet3 regulate class switch recombination (CSR) in murine B cells by enhancing expression of Aicda,which encodes the activation-induced cytidine deaminase (AID) enzyme essential for CSR. TET enzymes deposit 5hmC,facilitate DNA demethylation,and maintain chromatin accessibility at two TET-responsive enhancer elements,TetE1 and TetE2,located within a superenhancer in the Aicda locus. Our data identify the bZIP transcription factor,ATF-like (BATF) as a key transcription factor involved in TET-dependent Aicda expression. 5hmC is not deposited at TetE1 in activated Batf-deficient B cells,indicating that BATF facilitates TET recruitment to this Aicda enhancer. Our study emphasizes the importance of TET enzymes for bolstering AID expression and highlights 5hmC as an epigenetic mark that captures enhancer dynamics during cell activation. View Publication

Extracellular cold-inducible RNA-binding protein (CIRP) exaggerates inflammation and tissue injury in sepsis. Neutrophil extracellular traps (NETs) are released by activated neutrophils during sepsis. NETs contribute to pathogen clearance,but excessive NET formation (NETosis) causes inflammation and tissue damage. Peptidylarginine deiminase 4 (PAD4) is associated with NETosis by increasing histone citrullination and chromatin decondensation. We hypothesized that CIRP induces NETosis in the lungs during sepsis via upregulating PAD4 expression. Sepsis was induced in C57BL/6 wild-type (WT) and CIRP-/- mice by cecal ligation and puncture (CLP). After 20 h of CLP induction,NETs in the lungs of WT and CIRP-/- mice were quantified by flow cytometry by staining the single cell suspensions with MPO and CitH3 Abs. PAD4 expression in the lungs of WT and CIRP-/- mice after sepsis was assessed by Western blotting. In vitro effects of recombinant mouse (rm) CIRP for NETosis and PAD4 expression in the bone marrow-derived neutrophils (BMDN) were assessed by flow cytometry and Western blotting,respectively. After 20 h of CLP,NETosis in the lungs was significantly decreased in CIRP-/- mice compared to WT mice,which also correlated with the decreased PAD4 expression. Intratracheal administration of rmCIRP into WT mice significantly increased NETosis and PAD4 expression in the lungs compared to vehicle-injected mice. In vitro culture of BMDN with rmCIRP significantly increased NETosis and PAD4 expression compared to PBS-treated control. Fluorescence microscopy revealed typical web-like structures consistent with NETs in rmCIRP-treated BMDN. Thus,CIRP serves as a novel inducer of NETosis via PAD4 during sepsis. View Publication

RNA polymerase III (Pol III) is an essential 17-subunit complex responsible for the transcription of small housekeeping RNAs such as transfer RNAs and 5S ribosomal RNA. Biallelic variants in four genes (POLR3A,POLR3B,and POLR1C and POLR3K) encoding Pol III subunits have previously been found in individuals with (neuro-) developmental disorders. In this report,we describe three individuals with biallelic variants in POLR3GL,a gene encoding a Pol III subunit that has not been associated with disease before. Using whole exome sequencing in a monozygotic twin and an unrelated individual,we detected homozygous and compound heterozygous POLR3GL splice acceptor site variants. RNA sequencing confirmed the loss of full-length POLR3GL RNA transcripts in blood samples of the individuals. The phenotypes of the described individuals are mainly characterized by axial endosteal hyperostosis,oligodontia,short stature,and mild facial dysmorphisms. These features largely fit within the spectrum of phenotypes caused by previously described biallelic variants in POLR3A,POLR3B,POLR1C,and POLR3K. These findings further expand the spectrum of POLR3-related disorders and implicate that POLR3GL should be included in genetic testing if such disorders are suspected. View Publication

Broadly neutralizing HIV-1 antibodies (bNAbs) isolated from infected subjects display protective potential in animal models. Their elicitation by immunization is thus highly desirable. The HIV-1 envelope glycoprotein (Env) is the sole viral target of bnAbs,but is also targeted by binding,non-neutralizing antibodies. Env-based immunogens tested so far in various animal species and humans have elicited binding and autologous neutralizing antibodies but not bNAbs (with a few notable exceptions). The underlying reasons for this are not well understood despite intensive efforts to characterize the binding specificities of the elicited antibodies; mostly by employing serologic methodologies and monoclonal antibody isolation and characterization. These approaches provide limited information on the ontogenies and clonal B cell lineages that expand following Env-immunization. Thus,our current understanding on how the expansion of particular B cell lineages by Env may be linked to the development of non-neutralizing antibodies is limited. Here,in addition to serological analysis,we employed high-throughput BCR sequence analysis from the periphery,lymph nodes and bone marrow,as well as B cell- and antibody-isolation and characterization methods,to compare in great detail the B cell and antibody responses elicited in non-human primates by two forms of the clade C HIV Env 426c: one representing the full length extracellular portion of Env while the other lacking the variable domains 1,2 and 3 and three conserved N-linked glycosylation sites. The two forms were equally immunogenic,but only the latter elicited neutralizing antibodies by stimulating a more restricted expansion of B cells to a narrower set of IGH/IGK/IGL-V genes that represented a small fraction (0.003-0.02{\%}) of total B cells. Our study provides new information on how Env antigenic differences drastically affect the expansion of particular B cell lineages and supports immunogen-design efforts aiming at stimulating the expansion of cells expressing particular B cell receptors. View Publication