Extended Data Fig. 6: The evaluation of T-B cell doublet’s stability.
(a) Splenocytes from CTV labeled (CTV + ) TdTom and unlabeled (CTV − ) ZsGreen K/BxN TH17 fate-mapping mice were mixed at 1:1 ratio in vitro (mixed group). For the control group, Splenocytes from ZsGreen K/BxN TH17 fate-mapping mice were used without mixing with CTV+ cells (unmixed group). Representative plots and quantitative data indicating (I) CD4+ T cells gated from singlet gate were further gated for CD4 vs. CTV. This control group showing a ~ 1:1 ratio of T cells from CTV+ and CTV − TH17 fate-mapping mice. (II) ZsGreen+ TFH cells were gated to include doublets (purposefully not exclude the doublets) were further gated for CD4 vs. B220. The T-B conjugates were then gated for CD4 vs. CTV (n = 2/unmixed group; n = 16/mixed group, from two experiments). The CTV + T-B conjugates of ZsGreen+ TFH cells indicates that the B cells were from CTV+ TdTom mouse. (b) CD4+ T cells were enriched from the spleens of TH17 fate-mapping mice using positive selection with CD4 microbeads (Miltenyi Biotec). The enriched CD4+ T cells (which included single cells and T-B doublets) were stained with antibodies against CD4, TCRβ, CD19, GITR, CXCR5, and PD-1. Doublets with B cells bound to non-TH17/TFH, TH17, ZsGreen− TFH, or ZsGreen+ TFH cells were sorted. Non-TH17/TFH and B cell conjugates were then combined with TH17/B cell conjugates as the non-TFH doublet group. The conjugates were examined under a microscope, and two representative images of conjugates from the (I) non-TFH group, (II) ZsGreen− TFH group, and (III) ZsGreen+ TFH group are shown. (c) Compiled data on the percentage of surviving conjugates from Extended Data Fig. 6b, I-III using 16 randomly selected fields in each group with 4 samples (each sample/symbol represented one mouse except one sample was a combination of two mice) is shown. To calculate the surviving doublet %, we counted each cell number in each field and divided that number by two; this is the “would-be” doublet number. We then divided the observed real doublet number by the would-be doublet number x100 to obtain the surviving doublet % in each field. In Extended Data Fig. 6, data are presented as mean values ± s.e.m. One-way ANOVA with Tukey’s multiple comparisons test in c.
