Cell isolation. PBMCs from adult blood were isolated by Ficoll-Histopaque (MilliporeSigma) gradient centrifugation and cryopreserved in freezing medium (90% FBS + 10% DMSO; ATCC) for batch analysis. Prenatal organs were processed within 2 hours of collection as previously described (14). Briefly, the MLNs and SI were dissected, washed, and cut into 1 cm fragments. Mucus from the SI was removed by 3 × 20-minute DTT washes, and the SI epithelial cells (IECs) were removed by 3 × 20-minute EDTA washes at 37°C. The MLNs and SI were digested in collagenase IV (Life Technologies) and DNase (Roche) for 45 minutes at 37°C, then filtered, and lymphocytes were enriched by 20/40/80 Percoll (GE Healthcare, now Cytiva) gradient centrifugation. SI and MLN CD4+ T cells were further enriched by negative selection using the EasySep Human CD4 T Cell Isolation Kit (STEMCELL Technologies). In select experiments (Supplemental Figure 2, A–C) CD4+ T cells were isolated as described above from cryopreserved MLN and SI tissue fragments. Thymocyte single-cell suspensions were obtained by pressing minced tissue through a 70 μm strainer (Corning). Primary naive CD4+ T cells were obtained from mature CD4–single-positive (CD4 SP) thymocytes. Mature CD1a–CD4+TCRαβ+ SP thymocytes were obtained by negative selection using the EasySep Biotin Positive Selection Kit (STEMCELL Technologies) with the modification of an added wash step after staining of whole thymocytes with mAbs for CD34, CD56, CD14, CD11c, CD19, CD1a, and CD8a as previously described (52). Viability was measured with trypan blue (MilliporeSigma).

Antibodies and flow cytometry. Cells were incubated in 2% FCS in PBS with 1 mM EDTA with human Fc block (STEMCELL Technologies) and stained with Aqua LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) and fluorochrome-conjugated antibodies against surface markers. Intracellular protein and cytokine staining was performed using the Foxp3/Transcription Factor Staining Buffer Set (Tonbo Biosciences). Mouse and rat anti-human mAbs used in this study included IFN-γ FITC (clone 25723.11, BD Biosciences, catalog 340449), TCRαβ Percp 710 (clone IP26, Invitrogen, catalog 46-9986-42), TCRγδ Pe-CF594 (clone B1, BD Biosciences, catalog 562511), CD45RA PE-Cy7 (clone HI100, BD Biosciences, catalog 560675), CD4 APC-H7 (clone L200, BD Biosciences, catalog 560837), PLZF APC (clone 6318100, R&D Systems, catalog IC2944A), TCR Va7.2 BV605 (clone 3C10, BioLegend, catalog 351720), CD45RO BV650 (clone UCHL1, BioLegend, catalog 304232), TNF-α BV711 (clone MAb11, BioLegend, catalog 502940), CD161 BV785 (clone HP-3G10, BioLegend, catalog 339930), CD45 BUV395 (clone HI30, BD Biosciences, catalog 563792), CD3 BUV737 (clone UCHT1, BD Biosciences, catalog 612750), CD8 APC-R700 (clone RPA-T8, BD Biosciences, catalog 565166), IL-22 FITC (clone 22URT1, Invitrogen, catalog 11-7229-42), IFN-γ BV421 (clone 4S.B3, BD Biosciences, catalog 564791), IL-2 BV711 (clone 5344.111, BD Biosciences, catalog 563946), IL-17A BV421 (clone BL168, BioLegend, catalog 512322), IFN-γ BV711 (clone 4S.B3, BioLegend, catalog 502539), IL-13 PE (clone JES10-5A2, BioLegend, catalog 501903), IL-4 BV421 (clone MP4-25D2, BioLegend, catalog 500826), Vα7.2 Biotin (clone 3C10, BioLegend, catalog 351724), Va2.4 Biotin (clone 6B11, Invitrogen, catalog 13-5806-82), Streptavidin APC-R700 (BD Biosciences, catalog 565144), CD154 PE (clone TRAP1, BD Biosciences, catalog 555700), TNF-α PECy7 (clone MAb11, BD Biosciences, catalog 557647), CTV BV421 (Thermo Fisher Scientific, catalog C34571), IFN-γ BV605 (clone 4S.B3, BioLegend, catalog 502536), CD8 BV711 (clone RPA-T8, BD Biosciences, catalog 563677), CD215 BV421 (clone JM7A4, BD Biosciences, catalog 747704), CD127 BV786 (clone HIL-7R-M21, BD Biosciences, catalog 563324), CD25 FITC (clone M-A251, BioLegend, catalog 356106), CD8 FITC (clone RPA-T8, BioLegend, catalog 301050), CD161 BV711 (clone DX12, BD Biosciences, catalog 563865), FoxP3 PE (clone PCH101, Invitrogen, catalog 12-4776-41), PD-1 BV605 (clone EH12.2H7, BioLegend, catalog 329924), CD8 PeCY7 (clone RPA-T8, BD Biosciences, catalog 557746), Streptavidin BV605 (BioLegend, catalog 405229), CCR7 BV421 (clone G043H7, BioLegend, catalog 353208), CD4 APC-Cy7 (clone RPA-T4, BioLegend, catalog 300517), and Aqua LD (Invitrogen, catalog L34957).

For phosphoflow staining, 1 million CD4+ SP thymocytes were stimulated with IL-7 at 5 ng/mL for 30 minutes at room temperature. The reaction was stopped by adding Fix/Perm and subsequently stained for p-STAT5B (pY694, BD Biosciences) in a 96-well plate using the Foxp3/Transcription Factor Staining Buffer Set. All data were acquired with an LSR/Fortessa Dual SORP flow cytometer (BD Biosciences) and analyzed with FlowJo V10.0.8 (TreeStar) software. Expansion index was calculated using FlowJo software according to the formula expansion index = (1 – PF)/(1 – Dil), where PF = fraction of the original population dividing at leads once during the culture period and Dil = percentage of cells in the final population that have divided.

Bulk RNA-Seq. T cells were isolated as described above from the intestine of 3 individual samples, and PLZF+CD4+TCRαβ+ T cells were sorted on proxy surface markers (Va7.2–, CD45RA–, CCR7–, CD161+, IL18R+, PD1+) using FACSAria2 SORP (BD Biosciences) as detailed in Supplemental Figure 1A and as previously described (14). Half the sorted cells were stimulated with 50 ng/mL PMA (Santa Cruz Biotechnology) and 5 μg/mL ionomycin (MilliporeSigma) for 3 hours at 37°C in 4% O2; the remaining cells were left unstimulated. RNA was extracted and purified with the Dynabeads mRNA DIRECT Purification Kit (Thermo Fisher Scientific). mRNA libraries were constructed using the Nugen/Nextera XT Library Prep Kit (Illumina), and 3 samples (3 donors) were sequenced on an Illumina HiSeq 4000 by the Functional Genomics Core Facility at UCSF. The reads from the Illumina HiSeq sequencer in fastq format were verified for quality control using the fastqc software package. Reads were aligned to the human genome (hg38) and read counts aggregated by gene using the Ensembl GRCh38.78 annotation using STAR (53). Transcriptional analysis of unstimulated prenatal PLZF+CD4+ T cells used for comparison was previously published (14). Differential gene expression analysis was performed on all genes with at least 10 reads with DESeq2, version 1.16.1 (54). Volcano plots were created with ggplot2 depicting DE genes (log2 fold-change > 0.5, FDR ≤ 0.05).

ScRNA-Seq. Single, live PLZF+CD4+TCRαβ+ T cells from the SI were sorted on proxy markers (Va7.2–, CD45RA–, CCR7–, CD161+, IL18R+, PD1+) using the same strategy as that for the purification of these cells for bulk RNA-Seq described above, as outlined in Supplemental Figure 1A, and as previously described (14). Postsort purity was more than 89% as determined by intracellular staining for PLZF. Single cells were captured by droplet-based microfluidics, then lysed, and sequencing libraries were prepared in a single bulk reaction following the 10x Genomics protocol. Transcripts were sequenced using a HiSeq4000 System (Illumina). FASTQ files from each multiplexed capture library were mapped to a custom reference containing GRCh19 using the Cell Ranger (v2.0.0) (10x Genomics) count function. After demultiplexing cells into samples, Seurat (v3.1.5) was used to perform quality control filtering of cells. Cells were retained if they a) contained ≥500 reads, b) contained ≤5% reads coming from mitochondrial genes, and c) were called a singlet by Demuxlet, leaving 23,676 cells for downstream analysis. Data were log-normalized, and then principal component analysis was run on variable features selected by the FindVariableFeatures function after scaling with regression of percentage mitochondrial and nReads per cell. UMAP and Louvain clustering were performed with Seurat defaults and resolution 0.3. Differential gene expression between subsets of PLZF+CD4+ T cells was performed using FindAllMarkers with Seurat’s MAST implementation (55). Genes were deemed significantly different if FDR < 0.05, if average fold-change > 1.2, and the gene was detected in > 5% of cells in either comparison group. Visualizations were created with dittoSeq (v1.6.0) (56). Gene set analyses were performed using Metascape (57) with visualizations created in R with ggplot2.

CD4+ T cell stimulation assays. Single-cell suspensions of primary CD4+ T cells isolated from the prenatal SI and MLNs were cultured in a 96-well U-bottom plate in complete RPMI (Gibco) supplemented with 10% FBS, 10 mM HEPES, 2 mM l-glutamine, and 1× nonessential amino acids (Gibco) and stimulated with plate-bound αCD3 mAb (clone HIT3a, BioLegend, catalog 300313) at 1 μg/mL and soluble αCD28 mAb (CD28.2, Invitrogen, catalog MA1-20792) at 2 μg/mL, and/or cells were activated with recombinant human IL-12 and IL-18 (R&D Systems), or IL-2 and IL-33, and/or IL-1β and IL-23 for 16–20 hours at 37°C in 4% O2, with Brefeldin A (eBioscience) added for the last 4 hours. All cytokines were used at 50 ng/mL and from PeproTech unless otherwise specified. After stimulation, cells were stained for intracellular cytokine production as described above. For allogeneic stimulation of prenatal CD4+ T cells, allogeneic CD14+ APCs were isolated from unrelated adult PBMCs with the CD14 Positive Selection Kit II (STEMCELL Technologies), plated at a density of 0.5 million CD14+ cells/well in a 96-well plate, and allowed to adhere. Nonadherent cells were removed after 30 minutes, and adherent cells were incubated with αMHC DP-DQ-DR mAb (BioLegend, clone Tü39) or isotype control (IgG2a, clone MOPC-173, BioLegend) at 10 μg/mL for 30 minutes at 37°C. Primary CD4+ T cells isolated from the prenatal SI were stained with CTV (Invitrogen) to differentiate these from residual adult T cells within CD14+ APCs and cocultured at a 4:1 (T cell/APC) ratio for 16 hours, with Brefeldin A added in the last 4 hours. Cells were stained for intracellular CD154 and cytokine production as described above.

Naive CD4+ T cell expansion, maturation, and differentiation assays. Primary naive CD4+ T cells obtained from mature CD4 SP thymocytes as described above were cultured with IL-7 (5 ng/mL), IL-2 (10 ng/mL), or IL-15 (10 ng/mL) for 7 days with media changes every 2–3 days. Cells were kept at 37°C and 4% O2 for the duration of the stimulation. In some cases, IL-7 doses were titrated as indicated, and/or TGF-β (10 ng/mL) was added for the duration of culture. TCR stimulation was provided by plate-bound αCD3 (clone HIT3a, BioLegend, catalog 300313) at 1 μg/mL unless otherwise specified and soluble αCD28 (clone CD28.2, BD, catalog 555725) at 2 μg/mL. For naive T cell maturation and differentiation assays, cells were washed and moved to a new 96-well plate at day 5–6, rested for 24 hours in the absence of cytokines, and matured further with IL-7 (5 ng/mL) and/or IL-2 (10 ng/mL) in the presence or absence of TCR stimulation as described above or under Th-skewing conditions for an additional 7 days. Cells were differentiated in the presence of TCR stimulation and IL-2 (10 ng/mL) for Th0, with the addition of IL-12 (2.5 ng/mL) for Th1, and the addition of IL-1b (10 ng/mL) and IL-23 (10 ng/mL) for Th17, in the presence or absence of IL-7 (5 ng/mL). For Th17 differentiation, cells were cultured in IMDM + 10% FBS with 1× nonessential amino acids, 2 mM l-glutamine, 10 mM HEPES, and penicillin/streptomycin. At day 14 of culture, cells were washed, replated, and rested for 24 hours as above, then restimulated with plate-bound αCD3 (1 μg/mL) and soluble αCD28 (2 μg/mL) or recombinant human IL-12 and recombinant human IL-18 (50 ng/mL) for 24 hours. Where indicated, cells were restimulated with PMA and ionomycin for 4 hours. Brefeldin A was added for the last 4 hours of each stimulation.

Chemical inhibition. Primary naive CD4+ T cells were labeled with CTV to track cell proliferation and cultured with IL-7 (10 ng/mL) in the presence or absence of chemical inhibitors at 37°C in 4% O2 and 5% CO2. Chemical inhibitors were used at the indicated concentrations and purchased from Selleck Chemicals unless otherwise indicated: 5 μM of SH-4-54 (catalog S7337), 500 nM PD0325901 (StemRD, catalog PD-010), 1 μM pictilisib/GDC-0941 (catalog S1065), 5 μM STAT3-IN-1 (catalog S0818), 50 μM JANEX-1 (catalog S5903), 10 μM solcitinib (catalog S5917), and 5 μM SIS3 (MilliporeSigma, catalog S0447-5MG). DMSO vehicle control was used at 1 μg/mL and 10 μg/mL. Expansion indices were defined as the fold-change in expansion of the overall culture and calculated in FlowJo. Expansion index is equivalent to the final count of proliferated cells divided by the starting cell count.

Protein extraction and cytokine level quantification from solid tissue. Fragments of tissue (10 mg) were taken from the SI lamina propria and MLNs and placed in chilled lysis buffer (1 μL/mL protease inhibitor, 3 μL/mL 500 mM PMSF in RIPA buffer). Tissue was disrupted by mixing 20 times with a 1 mL pipette tip cut to a 2 mm opening and then vortexed for 15 seconds. Tubes were shaken at 4°C for 20 minutes at 300 rpm, and then the entire process was repeated. Tissue was centrifuged at 400g for 10 minutes at 4°C and supernatant was collected. Tissue IL-7 and IL-15 concentrations were measured via cytokine bead array using the Human Hematopoietic Stem Cell Panel Kit (LEGENDplex). IL-12p70, IL-18, IL-1b, and IL-23 concentrations were measured using the Human Inflammation Panel 1 Kit (LEGENDplex).

IL-7 ELISA. Single-cell suspensions of SI IECs and SI lamina propria were obtained as described above, and live, lamina propria CD45+ and CD45– cell fractions were sort-purified. Whole protein content was extracted from each of these populations as described above. Samples were kept on ice at all times. Whole protein concentrations were measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, catalog 23227) and stored at –80°C until further analysis. Samples were thawed on ice, normalized in 1× PBS, and subsequently analyzed for the presence of human IL-7 using the precoated IL-7 human ELISA kit (Thermo Fisher Scientific, catalog EHIL7) according to the manufacturer’s instructions.

Immunofluorescence. Immunofluorescence staining was performed on prefixed, whole SI sections, embedded in coils in OCT of prenatal samples (22-week gestational age). Tissues were sectioned to 10 μM using Leica cryostat CM 1950 set to –20°C. Tissue sections were later fixed in acetone for 5 minutes at –20°C, rehydrated in PBS for 10 minutes, rinsed with 0.05% TBS-Tween, and then permeabilized in 0.1% PBS–Triton X-100 for 15 minutes, all at room temperature in a humid container. Slides were then blocked for 1 hour at room temperature in a humid container in blocking buffer (10% calf serum, 1.5% BSA, 130 mM glycine, 1× Triton X-100 in PBS). Primary anti–IL-7 (IL-7 rabbit anti-human, Thermo Fisher Scientific: catalog PA519844) was used at 1:50 in blocking buffer and incubated overnight at 4°C in humid container, which was not included for control slides. Slides were washed 3 times for 30 minutes in PBS. Secondary anti-rabbit donkey in C555 antibody (Thermo Fisher Scientific, catalog 20038-1) was used at 1:300 in 0.05% PBS/Tween 20 for 2 hours at room temperature. Slides were washed 3 times for 30 minutes in blocking buffer. Slides were blocked with new blocking buffer (supplemented with 0.1% fish skin gelatin, 5% rat serum, and 1% donkey serum) overnight at 4°C in humid container. Preconjugated anti-CD4-APC (Becton Dickinson, mouse, clone RPA-T4, catalog 561841) was then applied for 4 hours at room temperature in supplemented blocking buffer at 1:200. Slides were washed 3 times for 30 minutes in PBS. Slides were then mounted with Fluoromount G + DAPI (Thermo Fisher Scientific, catalog 00-4959-52) and coverslipped. Slides were imaged immediately with ECHO microscope, using channels DAPI, Cy5, and Texas red at various exposures.

Statistics. Data were analyzed using Wilcoxon’s test for paired nonparametric data and 1-way ANOVA with post hoc Tukey’s honestly significant differences test for comparison of 3 or more groups. A P value less than 0.05 was considered significant. Box plot upper and lower hinges represent the first and third quartiles, the center line indicates the median, and the whiskers extend from the hinge to the highest and lowest value no further than 1.5× interquartile range from the hinge unless otherwise stated.

Study approval. PBMCs were isolated from Trima residues from Trima Apheresis collection kits and were obtained from healthy donors after receipt of written informed consent at the Blood Centers of the Pacific. Human prenatal tissues (16 to 22 weeks gestational age) were obtained from terminations of pregnancy after maternal written informed consent with approval from and under the guidelines of the UCSF Research Protection program. Samples were excluded in the cases of known maternal infection, intrauterine prenatal demise, and/or known or suspected chromosomal abnormality.

Data availability. All raw RNA-Seq data analyzed in this study have been deposited on NCBI GEO (GSE213522). Values for all data points found in graphs are in the Supporting Data Values file.