Fig. 5: Tfr cells restrain lymph node and intragraft germinal center B cell differentiation.
a Schematic of Tfr cell deletion using Tfr-DTR mice. Tfr-Con (Foxp3CreYFPCxcr5wt) or Tfr-DTR (Foxp3CreYFPCxcr5LoxSTOPLoxDTR) mice received diphtheria toxin (DT), CTLA4Ig (on day 2) and Balb/c kidneys. b Gating strategy and quantification of CXCR5+ follicular T, Tfh (gated as CD4+CD19-CXCR5+FoxP3-), Tfr (gated as CD4+CD19-CXCR5+FoxP3+), and CXCR5-FoxP3+ T cells in total CD4+ T cells from the dLNs. n = 4 mice for the Tfr-Con group and n = 5 for the Tfr-DTR group. c Gating strategy (left) and quantification (right) of CD19+GL7+FAS+ GC B cells. n = 4 mice for the Tfr-Con group and n = 5 for the Tfr-DTR group. d. Gating strategy (left) and quantification (right) of Naïve B cells (CD38+IgG1-CD19+), memory-like B cells (CD38+IgG1+CD19+), and IgG1+ B cells in the dLN (n = 4 for Tfr-Con and n = 5 for Tfr-DTR). e Gating strategy and quantification of Tfh and Tfr cells in kidney allografts (n = 4 for Tfr-Con and n = 5 for Tfr-DTR). f Gating strategy and quantification of GC-like B cells (GL7+CD19+) in kidney allografts. n = 4 mice for each group. g Schematic of GC B single cell culture assays. Single GC B cells from dLN or grafts were sorted and cultured with NB21 feeder cells for 6 days. Culture supernatants were pre-screened for IgG positivity and further assessed for DSA reactivity. h DSA signal of individual IgG+ clones from LN GC B and graft B cells from either control or Tfr-DTR mice 20 days after transplantation. The dotted line indicates the level of detection threshold assessed by signal using syngeneic cells. i Frequency of DSA clones (from all IgG+ clones). Numbers indicate the total number of IgG clones analyzed. Data is from one experiment and is representative of two independent experiments. Statistics: Error bars show mean ± SEM. Student’s two-tailed unpaired T test for 2-group comparisons. Source data are provided as a Source Data file. Portions created in BioRender. Zhang, H. (2025) https://BioRender.com/b29q775.
