5 250K 250K 10 3 4 200K 200K 1 10 150K 150K 3 2 10 100K 100K PercPe710 β 0 50K 50K TCR SSC-A 0 0 FSC-A 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 103 104 105 FSC-A FSC-H Viability dye AQUA 5 10 4 4 10 4 10 5 3 3 10 10 0 0 CD4 APC H7 APC PLZF 0 103 104 105 0 103 104 105 Vα7.2 BV605 CD161 BV711 Supplemental Figure 1. Gating strategy for the identification of PLZF+ T cells among fetal tissues, representative intestinal sample shown. A B ** Naive Memory 100 5 10 80 MFI: 3403±1065 MFI: 4626±1347 4 10 60 3 10 MFI: 40 1417 0 20 PLZF APC PLZF ±930 0 103 104 105 MFI PLZF (% of Max) 0 CD161 BV711 PLZF- PLZF+ PLZF+ CD45RO+ Naive CD45RO+ C + + + - D PLZF CD161 PLZF CD161 30 **** *** 20±5 15±6 ** PLZF+ CD161+ PLZF+ CD161- 20 PLZF- TRM cells + 10 PLZF- adult PB % CD4 0 5 10 8±6 0±0 Small Large 4 Intestine Intestine 10 3 10 0 CD69 PE 0 103 104 105 CD103 FITC E MLN PLZF- SI PLZF- adult control 5 1±0 10 1±1 0±0 4 10 3 PercP e710 PercP 10 αβ 0 -3 10 TCR 10-3 0 103 104 105 Nur77 PE Supplemental Figure 2. Fetal PLZF+ CD4+ T cells display markers of resident memory T cells. (A) Representative flow plots of PLZF and CD161 staining among intestinal Naive (CD45RO-, CD45RA+, CCR7+) and memory (CD45RO+, CD45RA-) cells. The MFI (average ± SD) of PLZF is indicated for each population (n=7). (B) Normalized frequencies of PLZF MFI among indicated populations of intestinal Vα7.2- CD4+ TCRαβ+ cells. (C) Representative flow plots of CD69 and CD103 staining among intestinal TEM (CD45RA-, CCR7-) cells. Gating based on adult PBMCs. (D) Frequencies of resident memory T cells (TRM) (CD69+ CD103+) among fetal intestinal Vα7.2- CD4+ TCRαβ+ cells. (E) Representative flow plots of endogenous Nur77 expression among PLZF- CD4 T cells derived from fetal SI and MLN compared to adult PBMCs. Numbers in flow cytometry plots represent frequencies of gated populations ± SD. Circles represent individual donors. Box plot whiskers span minimum and maximum, line represents median. Kruskal Wallis paired ANOVA with Dunn’s multiple comparison test (B, D). **p < 0.01, ***p<0.001 ****p < 0.0001. Gated on Vα 7.2+ CD161hi TCRαβ+ A C PLZF+ T cells: Adult PB CD45RA- 61±11 0±0 CD161+ CD4+ IL-18R+ Vα7.2- PD-1+ CCR7- - PLZF T cells: Fetal small TCRαβ+ - intestine CD45RA 4±2 0±0 T cells CD161- IL-18R- - PD-1 7.2 BV605 semi-invariant α Vα7.2+ V innate T cells: MR1-5 OP RU PE MR1-5 6FP PE Vα7.2+ CD161+ B D post-sort purity: PLZF PLZF+ T cells SIIT cells + T cells T - PLZF- PLZF T cells T cells CCR7 CD45RA CD45RA CD161 CD161 CD161 CD161 PLZF + PLZF+ T cells PLZF T cells - T cells T SIIT cells IL-18R PLZF SIIT cells PD-1 Vα7.2 Vα7.2 IL-18R semi-invariant innate T cells T innate CD4 E ZBTB16 KLRB1 Vα7.2 5000 * 6000 ** 4000 * 3000 4000 IL-18R 2000 2000 CD161 1000 Norm. counts 0 Norm. counts 0 PLZF+ PLZF- SIIT PLZF+ PLZF- SIIT PDCD1 IL18R1 4000 ** 15000 ** 3000 10000 2000 5000 1000 Norm. counts 0 Norm. counts 0 PLZF+ PLZF- SIIT PLZF+ PLZF- SIIT Supplemental Figure 3. Characteristics of sorted fetal T cell populations. (A) Schematic representation of the strategy for the identification and isolation of sorted RNAseq T cell populations. (B) Sorting strategy for PLZF+ CD4+ T cells, PLZF- CD4+ T cells, and semi-invariant innate Vα7.2+ CD161+ T cells gated on live, TCRαβ+ intestinal T cells. (C) Representative flow plots of MAIT cell staining of adult PB (n=3) and fetal intestinal T cells (n=7). (D) Representative flow plots of post-sort purity after intra-nuclear staining for PLZF+ CD4+ T cells, PLZF- CD4+ T cells, and semi-invariant innate Vα7.2+ CD161+ T (SIIT) cells. (E) Boxplot quantification of RNAseq normalized read counts of indicated genes among sorted populations. *p<0.05, **p<0.01. Kruskal Wallis with Dunn’s multiple comparison test (E). A 1 1 1 1 1 1 1 1 1 1 1 1 AS AML F JUN IL32 IL7R SELL J CKLF ITGAE TGFB1 CXCR3 MAP3K8 TNFSF14 0 0 0 0 0 0 0 0 0 0 0 0 + CD4 T cells: 1 1 1 1 1 1 1 1 1 1 1 + PLZF - PLZF CD5 CD69 CD59 CD38 CCL5 CCR6 CCR4 CD226 ANXA1 BCL2A1 CD40LG 0 0 0 0 0 0 0 0 0 0 0 Cell type: Stem cells Lymphoid Correlation analysis: D Myeloid B Non-Immune Cluster 4 Cluste1 rs Myeloid-enriched Clusters: Cluste1 rs 32 0.54 TNF Cluster 1: 65 FURIN Lymphoid0 TGFB1 IFNGR1 Cluster 4: 0.5 CLEC7A Myeloid, EGR2 ID2 Lymphoid C3AR1 NLRP3 Cluster 6: JAK2 Stem cells GAB2 P2RY14 MAP3K8 Cluster 3: PTPN22 All cells BCL2A1 Cluster 5: REL ZC3H12A All cells TNFRSF9 NR4A3 Cluster 2: CST7 Myeloid CCL5 Spearman’s ρ 1 0.5 0 0.5 C E Cluster 2 C1: Immune response Myeloid- C2: Immune response enriched CSF1 C2: Lipid metabolism NFIL3 C3: Immune regulation DUSP5 ATF3 C4: Immune response NAMPT C5: Tissue development PTGS2 C6: Cell cycle MYO1E AHR 0 20 40 60 80 CAPG %genes in pathway PLIN2 LGALS3 ATP1B3 DAPK1 HIST1H2BK ISG15 Z-scale PPARG 3 2 1 0 -1 -2 SEC24D SERINC5 Supplemental Figure 4. Distinct transcriptional signature of fetal PLZF+ CD4+ T cells. (A) Normalized read counts of selected T cell memory- and activation-associated genes identified by RNAseq among sorted memory PLZF+ (red) and PLZF- (white) CD4+ T cells. (B) Correlation analysis using the Human Primary Cell Atlas as a reference of the differentially expressed genes (>2 fold, FDR<0.05) in PLZF+ CD4 T cells as compared to PLZF- CD4+ T cells revealing clusters of genes (C1-C6) that are co-expressed in the same cell types. (C) Pathway analysis of gene clusters identified by correlation analysis (B) was performed and gene pathways were organized into sub-clusters based on shared genes. Bar graphs show the % gene enrichment of each gene pathway within the indicated cluster. (D-E) Heatmap shows color-coded relative enrichment of differentially expressed genes in PLZF+ CD4+ T cells relative to PLZF- CD4+ T cells among indicated cell types identified from the Human Primary Cell Atlas for (D) Cluster 4 and (E) Cluster 2. Selected myeloid-en- riched genes involved in immune response and immune regulation are labeled. IFNγ TNFα A B ns SI PLZF+ CD161+ SI PLZF+ CD161- adult PB no PMA control 100 ns 5 10 TNFα IFNγ 80 4 57±28 43±26 20±11 0 10 60 3 10 40 PeCy7 α 0 20 TNF 28±10 20±8 9±6 0 % cytokine production 0 0 103 104 105 IFNγ FITC SI CD4+ PLZF+ PLZF+ T cells: CD161- CD161+ C D 40 15 Spearman ρ=0.35 Spearman ρ=0.34 cells p=0.15 + p=0.2 T T cells + 30 10 CD4 - CD4 - 20 PLZF + PLZF 5 α + γ 10 %TNF %IFN 0 0 17 18 19 20 21 22 23 17 18 19 20 21 22 23 GA (weeks) GA (weeks) E SI PLZF+ SI PLZF- adult PB 4 10 3 10 FITC γ 0 0±0 0±0 0.1±0 IFN 0 103 104 105 IL-10 Pe * F SI PLZF+ SI PLZF- no PMA control G 60 5 10 50 4 10 40 cells + 3 30 10 PeCy7 20 % IL-2 0 α 45±11 29±10 0±0 10 TNF 0 103 104 105 0 IL-2 FITC PLZF+ PLZF- T cells T cells H I SI PLZF+ SI PLZF- no PMA control 20 ns 5 10 15 4 10 cells + 3 10 10 % IL-8 FITC γ 5 0 8±3 8±6 0±0 IFN 0 103 104 105 0 IL-8 BV421 PLZF+ PLZF- T cells T cells Supplemental Figure 5. SI-PLZF+ CD4 T cells are poised for rapid production of Th1 cytokines. All samples gated on live Vα7.2- CD4+ TCRαβ+ cells. (A) Representative flow plots of intracellular TNFα and IFNγ staining of indicated populations (n=18) after PMA/Ionomycin stimulation of small intestine (SI) compared to adult PB CD4+ T cells. Gates drawn on unstimulated control. (B) Frequencies of IFNγ+ and TNFα+ cells within indicated populations of SI-PLZF+ CD4+ T cells after PMA/Ionomycin stimulation. (C, D) Association between gestational age (GA) and (C) IFNγ and (D) TNFα production by SI PLZF- CD4+ T cells in response to PMA/Ionomycin, Spearman’s rank correlation. Circles represent individual donors. (E) Representative flow plots of intracellular IL-10 expression after PMA/Ionomycin stimulation within indicated populations of SI CD4 T cells (n=5). (F, H) Representative flow plots of intracellular (F) IL-2 and (H) IL-8 after PMA/Ionomycin stimulation within indicated populations of SI CD4 T cells. Gates drawn on unstimulated control. (G, I) Frequencies of (G) IL-2+ and (I) IL-8+ cells within indicated populations of SI CD4 T cells after PMA/Ionomycin stimulation. Numbers in the flow cytometry plots correspond to the mean ± SD of gated frequencies. Circles represent individual donors. Box plot whiskers span minimum and maximum, line represents median. Kruskal Wallis paired ANOVA with Dunn’s multiple comparison test (B), Wilcoxon matched-pairs signed rank test (G, I).