An Interleukin 9-ZBTB18 axis promotes germinal center development of memory B cells

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. 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. GC B cells were enriched from pooled splenocytes of 3 to 4 Cd79acre/+Zbtb18fl/fl or Cd79acre/+ mice with anti-mouse GL7 (clone GL7, eBioscience) 28 days after NP-KLH immunization. Memory precursor cells (GL7+FAS+CD38+IgD-CD19+) were then sorted into 4 replicates of ~200 cells per PCR tube containing the lysis buffer. Samples were reverse transcribed with SuperScript™ II Reverse Transcriptase (Catalog:18064022, Invitrogen) and the complement DNA library was constructed with TruePrep DNA Library Prep Kit for Illumina (Catalog:TD501, Vazyme), following the Smart-seq2 protocol. Amplified products were purified with VAHTS DNA Clean Beads (Vazyme) and quantified using Qubit and 2100 Bioanalyzer (Agilent). Quantitative RT-PCR was used for quality control. All libraries were sequenced on a HiSeq X Ten sequencer (Illumina).