Fig 2: Multicolor‐barcoded multiplex RNAi by miR‐AB retrovirus in CD8+ T cellsThis RNAi experiment was carried out as described in Fig 5 but used another SplashRNA‐designed shRNAmirs targeting CD127, CD90, CD44, or CD8. CD4‐specific shRNAmirs were used as controls. The data are representative of two biologically independent experiments.

Fig 3: Poly(I:C) affects the MLL-AF4 signature gene expression program in fetal liver Mll-AF4+ hematopoietic stem and progenitor cells in vitro. Expression of (A)Mll-AF4, (B)Mll, (C)Af4, (D)Cdk6, (E)Hmga2, (F)Hoxa9, (G)Lmo2, (H)Mcl1, (I)Meis1, (J)Bcl2, (K)Twist1, (L)E2a, (M)Ikaros, (N)Pax5, (O)Il7r, and (P)Flt3 in FL Mll-AF4+ LSK after a 48-hour in vitro stimulation with mock/PBS, poly(I:C), or LPS. Expression of (Q)Tlr3, (R)Tlr4, (S)Mll, (T)Af4, (U)Hmga2, (V)Hoxa9, and (W)Cdk6 in FL control (Mll-AF4–VEC-Cre+) LSK cells after a 48-hour in vitro stimulation with mock/PBS, poly(I:C), or LPS. Statistical differences were assessed using a nonparametric Wilcoxon test with a bilateral p value (*p < 0.05, **p < 0.01, p < 0.001; n = 3 or 4).

Fig 4: Comparison of RNAi efficiency mediated by miR‐E versus miR‐AB in vitro and in vivo Two top‐ranked SplashRNA‐designed shRNAmirs targeting human Nurd complex components and a control shRNAmir targeting human CD4 were cloned into both miR‐AB and miR‐E lentiviral vectors with Venus as reporter. The lentiviruses packaged by these constructs were used for transduction of multiple human cell lines. RNAi efficiency was determined by western blot (WB) analysis of target protein levels in the lysates of the transduced cells. β‐actin served as loading control.A shERWOOD‐designed shRNAmir targeting mouse transcriptional factor Tbx21 and a control shRNAmir targeting mouse CD4 were cloned into both miR‐AB and miR‐E retroviral vectors with Ametrine as reporter. The packaged viruses were used for transduction of primary mouse P14 TCR transgenic CD8+ T cells, which were used for evaluation of CD8+ T cells differentiation in vivo as described previously(Wang et al, 2018) (see details in the Materials and Methods section).Representative FACS plots of differentiation marker CD127/KLRG1 staining of the gated P14 CD8+ T cells and the statistical summary of SLEC subsets were shown to indicate CD8+ T cell differentiation. Data are shown as mean ± SD and n = 5 mice/group. * indicates significant difference (P < 0.01, two‐tailed unpaired Student's t‐test). Data are from one representative experiment of two biological replicates.The Tbx21 RNAi efficiency mediated by miR‐AB vs. miR‐E in the transduced in vitro‐expanded P14 CD8+ T cells was determined by western blot analysis of T‐bet protein level. β‐actin served as loading control.The RNAi efficiency of Pten.1524 and Pten.932 shRNAmir mediated by miR‐AB versus miR‐E was tested in NIH‐3T3 cells in the context of < 30% transduction efficiency (GFP as reporter). Representative western blots of Pten protein expression of the FACS‐sorted GFP+ cells from two biological replicates were shown. β‐actin served as loading control.Three shRNAmirs targeting the GFP reporter of a lentiviral vector in miR‐AB or miR‐E backbone were transduced into MC38 or 293T cells. The RNAi efficiency was determined by FACS analysis of GFP in the context of < 30% transduction efficiency. Data are shown as mean ± SD and n = 3 technical replicates. * indicates significant difference (P < 0.05). NS indicates no significant difference (P > 0.05, two‐tailed unpaired Student's t‐test). Source data are available online for this figure.

Fig 5: Inbred mouse strains exhibit different numbers of respiratory Treg cells. Analysis of Treg populations according to CD25, Foxp3, CD39 and CD127 expression indicates significantly increased CD4+ CD25high (A), CD4+ CD25high Foxp3+ (B), CD4+ CD25high Foxp3+ CD39+ (C), and CD4+ CD25+ CD127low/neg (D) Treg subsets in BALB/c mice in comparison to C3H and DBA mice. Results are representative for n≥ 5 independent animals per strain (mean ± SEM; *p<0.05; ***p<0.001).