Supplemental Figure 1. Gating strategy for the identification ofPLZF Supplemental Figure1.Gatingstrategyfortheidentification fetal tissues,representativeintestinalsampleshown.
SSC-A 0 50K 100K 150K 200K 250K FSC-A 0 50K 100K 150K
CD4 APC H7 200K 0 103 104 105 1 Vα7.2 BV605 250K 0 4
10 FSC-A
3 0 50K 100K 150K 200K 250K FSC-H 0 10 50K 4
100K 10 5
150K 2
PLZF APC 200K 0 103 104 250K CD161 BV711 0 TCRβ PercPe710 10 0 103 104 105 3 Viability dye AQUA Viability 0 10 4
+ T cellsamong 5 10 10 3 3 5
10 4
10 5
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 10 0 -3 10 PercP e710 TCR αβ PercP 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- semi-invariant Vα7.2+ V α 7.2 BV605 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 - PLZF- PLZF T cells T cells CCR7 CD45RA CD45RA CD161 CD161 CD161 CD161 PLZF + PLZF+ T cells PLZF T cells - T cells SIIT cells IL-18R
PLZF SIIT cells PD-1 Vα7.2 Vα7.2 IL-18R semi-invariant innate T cells
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