Extended Data Figure 9. Mycl1 expression in CD8α⁺ DCs is necessary for normal T cell priming.

a, WT mice (black box) and BDCA2-DTR transgenic mice (open box) were treated with 125 ng diphtheria toxin (DT) per mouse 1 day before infection with 3×10³ LM-OVA. DT treatment was continued on days 1, 3, and 5 after infection, and splenocytes harvested on day 7 were stained for analysis. Shown is the quantitation of pDCs as a percent of all splenocytes to confirm efficiency of deletion (bar, SD; n=4 biological replicates per group, Student's t-test). b, Splenocytes described in (a) were re-stimulated with SIINFEKL peptide in vitro for 5 h. Shown is the quantitation of IFN-γ⁺ cells as a percent of CD8⁺ T cells (gated as Thy1.2⁺ CD8α⁺) (bar, SD; n=4 biological replicates, Student's t-test). c, Peripheral blood from Notch2^f/f mice (black box) and Cd11c-Cre Notch2^f/f mice (open box) was collected on day 7 after infection with 3×10³ LM-OVA. After red blood cell lysis, CD8⁺ T cells were stained with H-2K^b-SIINFEKL tetramer. Shown is the frequency of tetramer⁺ cells as a percent of CD8⁺ T cells (bar, SD; n=4 biological replicates, Student's t-test). d, BM from CD45.1⁺ WT mice and BM from CD45.2⁺ Zbtb46^dtr/dtr mice were mixed in a 50:50 ratio and injected into lethally irradiated WT recipient mice. Ten weeks after transplant, 400 ng DT was administered to each chimeric mouse and splenocytes harvested at 48 h, 72 h and 96 h after DT treatment were stained for analysis. Shown are two-color histograms of CD45.1 and CD45.2 expression for pre-gated DCs (CD11c⁺MHCII⁺, top panels) to determine relative donor chimerism and efficiency of deletion. e, Shown is an experimental outline for LM-OVA infection of BM chimeras following DC depletion and replenishment. f, BM chimeras were generated using a 50:50 ratio of Zbtb46^dtr/dtr BM and WT BM (DTR:WT), or of Zbtb46^dtr/dtr BM and Mycl1^gfp/gfp BM (DTR:Mycl1^gfp/gfp), or of Zbtb46^dtr/dtr BM and Batf3^−/− BM (DTR: Batf3^−/−). Twelve weeks after lethal irradiation and transplant, BM chimeras were infected according to the time course outlined in (e). Expansion of donor CD45.1⁺ OT-I CD8⁺ T cells was evaluated 7 days after infection with 300 LM-OVA. g, Shown is the total number of OT-I CD8⁺ T cells from infected DTR:WT, DTR:Mycl1^gfp/gfp , and DTR:Batf3^−/− chimeric mice described in (e, f). Data are from 2 independent experiments (bar, SD; n=8 infected biological replicates, one-way ANOVA Tukey's post hoc test). *, p<0.05; **, p<0.01; ns, p>0.05.