Figure 5. Flcn limits TGF-β signaling to control T_RM programming.

(A) sgNTC (Ametrine⁺)- and sgFlcn (GFP⁺)-transduced OT-I cells from siIEL of the same host were profiled by scRNA-seq at days 4.5 and 7.5 p.i. (n = 2 per group; pooled from 2 mice). UMAP plots depicting three clusters (cycling, Itgae⁻, and Itgae⁺) and Itgae expression.

(B) Violin plots showing activity scores of core T_RM signature,⁷ core T_CIRC signature,⁷ curated siIEL T_RM signature,⁷ and curated T_CM signature⁷ (see STAR Methods) among indicated 3 clusters.

(C) UMAP plot showing Slingshot trajectory analysis⁵² and pseudotime plot of indicated sgRNA-transduced siIEL OT-I cells at days 4.5 and 7.5 p.i.

(D) Plot depicting transcription factor motif enrichment analysis (including Smad3) comparing sgFlcn- versus sgNTC-transduced siIEL OT-I cells that were profiled by ATAC-seq at day 7.5 p.i.

(E) Violin plots showing activity scores of TGF-β-activated and TGF-β-suppressed signatures⁵³ (see STAR Methods) in indicated sgRNA-transduced siIEL OT-I cells from scRNA-seq profiling in (A).

(F) pSmad2-Smad3 levels (relative to spike; based on MFI) in indicated sgRNA-transduced OT-I cells from siIEL or spleen on day 7.5 p.i. (n = 3 per group).

(G) CD103 expression (based on MFI) on WT or Flcn-deficient (from Cd4^CreFlcn^fl/fl mice) CD8⁺ T cells cultured with rhTGF-β1 (or vehicle) for 5 days (see STAR Methods) (n = 3 technical replicates per group).

(H) Real-time PCR analysis of Tgfbr1 and Tgfbr2 expression in indicated sgRNA-transduced OT-I cells from siIEL or spleen at day 7.5 p.i. (n ≥ 3 per group).

(I and J) Frequencies (relative to spike) and numbers of total (I) or CD69⁺CD103⁺ siIEL OT-I cells (J) transduced with indicated sgRNAs at day 12 p.i. (from dual-color transfer system) (n ≥ 4 per group).

(K) pSmad2-Smad3 levels (relative to spike; based on MFI) in indicated sgRNA-transduced OT-I cells from siIEL at day 9 p.i. (from dual-color transfer system) (n ≥ 3 per group).

(L) WT (from Flcn^fl/flCas9⁺ mice) or Flcn-deficient (from Cd4^CreFlcn^fl/flCas9⁺ mice) OT-I cells that were transduced or co-transduced with indicated sgRNAs. Frequency of CD103⁺ siIEL OT-I cells at day 7.5 p.i. is shown. Fold-change relative to sgNTC is indicated (n = 6 per group).

(M) CD103 expression (based on MFI) on CD8⁺ T cells cultured with rhTGF-β1 (or vehicle) for 5 days (see STAR Methods) (n = 3 technical replicates per group).

(N) Pre-activated OT-I cells were transferred into Ctrl RPMI medium or Arg⁻ RPMI medium containing rhTGF-β1 (or vehicle) for 2.5 days (see STAR Methods). Frequency of CD69⁺CD103⁺ OT-I cells is shown (n = 4 technical replicates per group).

(O) GSEA enrichment plots showing the core T_RM signature⁷ in cells cultured in Arg⁻ versus Ctrl medium, cells cultured in Ctrl medium plus rhTGF-β1 versus Ctrl medium, or cells cultured in Arg⁻ medium plus rhTGF-β1 versus Ctrl medium plus rhTGF-β1 (as in N).

(P–R) Schematic for LM-OVA infection in mice fed control or low-protein diet (P). OT-I cell numbers in indicated tissues (Q) or frequency and number of CD69⁺CD103⁺ siIEL OT-I cells (R) at day 10 p.i. (n = 9–10 per group).

(S) Nuclear Tfeb MFI in siIEL OT-I cells from mice fed indicated diets (n > 30 cells per group). Wilcoxon rank sum test (B and E), two-tailed unpaired Student’s t test (F and Q–S), one-way ANOVA (H–L and N), two-way ANOVA (G and M). NS, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data (mean ± SEM) are compiled from two (K, L, Q, and R) or represent ≥2 (F), one (H–J and S), or three (G, M, and N) experiments.

See also Figure S5.