技术资料

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

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

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971,rs17615,rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls,P=0.016,OR=0.90 (0.82-0.98)). Two of these SNPs are in exon 10,directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC),and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact. View Publication

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge,no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach,we established the genetic relationship between the cell lines and the primary patient cells,and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patient's disease. Importantly,we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity,the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover,these cell lines will provide an invaluable tool to better understand AL,from the combined perspectives of amyloidogenic protein structure and amyloid formation,genetics,and cell biology. View Publication

Developing vaccine strategies to generate high numbers of Ag-specific CD8 T cells may be necessary for protection against recalcitrant pathogens. Heterologous prime-boost-boost immunization has been shown to result in large quantities of functional memory CD8 T cells with protective capacities and long-term stability. Completing the serial immunization steps for heterologous prime-boost-boost can be lengthy,leaving the host vulnerable for an extensive period of time during the vaccination process. We show in this study that shortening the intervals between boosting events to 2 wk results in high numbers of functional and protective Ag-specific CD8 T cells. This protection is comparable to that achieved with long-term boosting intervals. Short-boosted Ag-specific CD8 T cells display a canonical memory T cell signature associated with long-lived memory and have identical proliferative potential to long-boosted T cells Both populations robustly respond to antigenic re-exposure. Despite this,short-boosted Ag-specific CD8 T cells continue to contract gradually over time,which correlates to metabolic differences between short- and long-boosted CD8 T cells at early memory time points. Our studies indicate that shortening the interval between boosts can yield abundant,functional Ag-specific CD8 T cells that are poised for immediate protection; however,this is at the expense of forming stable long-term memory. View Publication