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. 2020 Feb 19;21(4):e48925. doi: 10.15252/embr.201948925

Figure 1. Mature IS formation between DICER‐KO B cells and OT‐II T cells promotes class switching, proliferation, and survival.

Figure 1

DICER‐KO B cells were co‐cultured for 24 h with OTII‐derived T cells in the presence or absence of OVA.
  • A
    Representative confocal microscopy images of CMAC‐stained conjugates formed between B lymphocytes (isolated from DICER‐KO mouse splenocytes and pre‐activated with a mixture of LPS plus IL‐4) and CD4+ T cells (from OVA‐specific OT‐II transgenic mice); conjugates were formed either in the presence or in the absence of OVA. A representative experiment of at least three independent experiments is shown.
  • B–E
    Quantification of three representative experiments as in (A), showing the percentage of B cells forming conjugates (B), the accumulation at the IS of the TCR (C) and actin (D) at the IS, and the distance from the CD4+ T‐cell MTOC to the contact site with the B cell (E). Conjugates were assessed using the ImageJ plug‐in for IS analysis, and MTOC–B‐cell distance was measured with Imaris analysis software. Bar charts are representative of the mean of n ≥ 7 cells analyzed ± SEM from at least 3 independent experiments. Significance was assessed by unpaired Student's t‐test; *P < 0.05, **P < 0.01, ***P < 0.001.
  • F–J
    Flow cytometry analysis of B lymphocytes co‐cultured with OT‐II isolated T cells in the presence or absence of OVA, showing (F) IgG1 expression after 24 h of co‐culture, (G and H) B‐cell proliferation after 24, 48 and 72 h co‐culture (Ki67 expression in live B cells and cell violet tracer dilution, respectively), (I) survival (staining with the live marker DAPI), and (J) apoptosis (cleaved Caspase‐3 expression) 24, 48 and 72 h after IS formation.
Data information: Dot plots are representative of ≥ 4 independent experiments 48 h after IS formation, and bar charts show mean values ± SEM of at least three independent experiments at the indicated time points. Significance was assessed by paired Student's t‐test; *P < 0.05, **P < 0.01, ***P < 0.001.