Extended Data Fig. 7. CRISPR/Cas9-mediated gene knockdown of SMARTA cells.

Related to Fig. 7

a, Schematic of the CRISPR/Cas9-mediated gene knockdown of SMARTA cell system. Details are in the Method section.

b, Representative flow cytometry of RNP transfection from more than four independent experiment; each dot represents one RNP+ group (n = 9). Data are mean ± s.d. Quantification of cell viability and transfection efficiency of RNP⁺ and RNP⁻ (electroporation without RNP) SMARTA cells.

c, Frequency of crRNA⁺ CD45.1⁺ SMARTA cells among total CD4 T cells from spleen of LCMV_Arm infected mice in Fig.7a. Two independent experiments were performed; each dot represents one crRNA⁺ group (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test.

d,e,f,g, WT or Bcl6^f/fPrdm1^f/f Cre^CD4 SMARTA CD4 T cells transfected with crCd8, crGata3, crRunx2, crRunx3, and crKlf2 were transferred to C57BL/6 host mice, followed by infection with LCMV_Arm (e,f,g) or immunization with KLH-gp₆₁ (d) of the host mice, and analyzed 6–7 days later. crRNA⁺ SMARTA cells were FACS sorted from spleens (LCMV_Arm infection) or dLN (KLH-gp₆₁ immunization) of host mice. Gene knockdown efficiencies were measured by flow cytometry (d), mRNA qPCR (e), or Western blot analysis (f,g). shRunx3-RV⁺ SMATRA cells and pMIG-Klf2-RV⁺ SMARTA cells were used as positive controls. Two independent experiments were performed; Each dot represents one mouse (n = 5). Data are mean ± s.d., unpaired two-tailed Student’s t-test. Details are in the Method section.

h, Schematic of the CRISPR/Cas9-mediated gene knockdown of SMARTA cell system used for testing Gata3 in LCMV_Arm infection. WT or Bcl6^f/fPrdm1^f/f Cre^CD4 SMARTA CD4 T cells transfected with crCd8 or crGata3 were transferred to C57BL/6 host mice, followed by infection of the host mice with LCMV_Arm, and analyzed 6–7 days later. Related to Fig.7c and Extended Data Fig.7i,l,m.

i,j,k, Frequency of crRNA⁺ CD45.1⁺ SMARTA cells among total CD4 T cells from spleen of LCMV_Arm infected mice in Fig.7d and Extended Data Fig.7h. Two (i) or three (j,k) independent experiments were performed; each dot represents one mouse (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test.

l, Representative flow cytometry of T_FH SMARTA cell subsets from spleen of LCMV_Arm infected mice in Extended Data Fig.7h. Two independent experiments were performed; Each dot represents one mouse (n = 4). Data are mean ± s.d., unpaired two-tailed Student’s t-test.

m, Quantification of expression of PSGL1 in Extended Data Fig.7h, gated on SMARTA cells. CD44^lo naive CD4 T cells were used as a negative control.