An Interleukin 9-ZBTB18 axis promotes germinal center development of memory B cells [ATAC-seq]

Memory B cell (MBC) development from germinal centers (GCs) entail profound changes in cell cycling, localization and survival. Here we examined the mechanisms that induce the memory program, focusing on interleukin (IL)-9, given its importance for normal recall antibody responses. Using adoptive transfer and radiation chimera models, we found that T cell-derived IL-9 was required for MBC development and function. In contrast, B cells deficient in IL-9 generated functionally normal MBCs that support antibody recall normally. IL-9 induced expression of the transcriptional repressor ZBTB18 in GC memory precursor cells and MBCs. ZBTB18 was dispensable for naïve B cell activation and GC formation but required for development of GC-derived MBCs. ZBTB18 directly repressed expression of a suite genes encoding cyclin and cyclin-dependent kinases, pro-apoptotic genes Bid and Casp3, and the GC-retaining factor S1pr2. Lack of IL-9-mediated instruction or intrinsic programming by ZBTB18 impaired GC-derived MBC development and antibody recall. Thus, an IL-9-ZBTB18 axis instructs development of functional B cell memory from GCs. Overall design: Mice were immunized intraperitoneally with 100 µg NP-KLH (conjugate ratios20, Biosearch Technologies) mixed with 1 µg lipopolysaccharide (LPS, Sigma) in alum (Thermo Scientific). Single-cell suspension of splenocytes was stained in MACS buffer (PBS supplemented with 1% FBS and 5 mM EDTA) containing Fc blocker (supernatant from 2.4G2 cell culture) for 20 min on ice before surface staining with primary antibodies in appropriate concentrations. 5×104 naïve B cells or GCMP cells were sorted from Cd79acre/+ mice and Cd79acre/+Zbtb18fl/fl mice, respectively. Cells were collected by gentle centrifugation (500x g, 5 minutes) at 4 °C, and then resuspended in lysis buffer (10 mM Tris.Cl, 10 mM NaCl, 3 mM MgCl2, 0.1% (v/v) Igepal CA-630) and incubated on ice for 10 min. Nuclei were collected and resuspended into 50 µl tagmentation mix, which contains 10 µl 5×TTBL (Vazyme), 5 µl TTE Mix V50 and 35 µl ddH2O. Samples were incubated at 37 °C for 30 min. After tagmentation, fragmented DNA was isolated by using VAHTS DNA Clean Beads. ATAC libraries were amplified for 12 PCR cycles using TruePrepTM Index Kit V2 for Illumina (Vazyme #TD202). 0.55×VAHTSTM DNA Clean Beads were used to exclude the High–molecular weight DNA, followed by positive selection with 1×beads. High-quality DNA libraries were sequenced on the Illumina HiSeq2000.