Fetal naïve CD4⁺ T cells display functional differences compared to adult naïve CD4⁺ T cells. (A) Flow cytometric analysis of the phenotype of naïve CD4⁺ T cells isolated from fetal mLNs (18–22 g.w.) and adult peripheral blood mononuclear cells (PBMC) (25–35 y.o.). Panels labeled (i) depict initial gating on CD3⁺CD4⁺ T cells showing CD45RA vs. CCR7 staining and those labeled (ii) depict CD45RA⁺CCR7⁺ cells (highlighted in black in panel i) subsequently gated on CD25 CD95⁻ cells that are considered “naïve” CD4⁺ T cells (highlighted in black in panel ii). Data are representative of at least 3 independent donors. (B) Sorted naïve CD4⁺ T cells were either left unstimulated, or cultured for 6 days in the presence of irradiated allogeneic PBMC. Proliferation of fetal and adult naïve CD4⁺ T cells was measured by CFSE dilution and analyzed by flow cytometry. Tregs were phenotypically identified by upregulation of CD25 and Foxp3.

Fetal and adult HSPC give rise to mature SP4 thymocytes that show similar functional differences to fetal and adult peripheral naïve CD4⁺ T cells. (A) Representative flow cytometry graphs showing thymocyte populations isolated from SCID-hu Thy/Liv implants following transplantation of donor (HLA-A2⁺) CD34⁺ HSPC isolated from fetal liver, fetal BM, or adult BM. Panels labeled (i) show initial gating on CD4 vs. CD8 to select for SP4 thymocytes and those labeled (ii) show subsequent gating on donor-derived (HLA-A2⁺) CD3⁺ mature SP4 thymocytes. (B) Flow cytometric analysis of the frequencies of CD25⁺Foxp3⁺ Tregs dervied from fetal liver, fetal BM or adult BM hematopoietic progenitors. (C) Quantification of CD25⁺Foxp3⁺ Treg frequencies observed in 3–5 different thymic implants from each group. (D) Flow cytometric analysis of proliferation and Foxp3 expression by SP4 thymocytes derived from fetal liver, fetal BM, or adult BM HSPC in response to stimulation with irradiated allogeneic PBMCs (7-day stimulation). (E) Quantification of the frequencies of CD25⁺Foxp3⁺ Tregs after stimulation with allogeneic PBMCs. Data show at least three separate implants for each group. Data in bar graphs represent mean +/− standard deviation and statistical significance was measured by Student’s t-test.

Fetal and adult naïve CD4⁺ T cells show significant differences in gene signature. (A) Unbiased cluster analysis on the basis of gene expression for fetal (mLN) and adult (PBMC) naïve CD4⁺ T cells and CD4⁺CD25⁺ Tregs. (B–D) Scatterplots of pairwise global gene expression comparisons comparing (B) fetal naïve CD4⁺ T cells and adult naïve CD4⁺ T cells, (C) fetal and adult CD4⁺CD25⁺ Tregs, and (D) CD4⁺CD25⁺ Tregs and naïve CD4⁺ T cells (in both fetal and adult). Gene expression values are plotted on a log scale. Genes that were differentially expressed between groups (determined using a 5% false-discovery rate and fold-change >= 2) are indicated in red and blue. Specific genes that were differentially expressed based on age or subset are highlighted in green.

Fetal and adult HSPC give rise to SP4 thymocytes with distinct gene signatures. (A) Unbiased cluster analysis based on gene expression for SP4 thymocytes from fetal BM, fetal liver, and adult BM hematopoietic progenitors.(B) Total number of genes found to be significantly different comparing SP4 thymocytes derived from fetal and adult progenitor populations. (C) Principle component analysis showing clear separations between SP4 thymocyte populations and peripheral T cells (PC1) as well as differences between fetal and adult SP4 and peripheral naïve CD4⁺ T cell populations (PC2). (D) Scatter plot depicting genes that are differentially expressed between fetal and adult SP4 thymocytes, and between fetal and adult peripheral naïve CD4⁺ T cells. (E) Scatter plot depicting genes that are differentially expressed between adult and fetal naïve T cells, and between adult naïve and adult Tregs. For both (D) and (E), differential expression was determined using a false discovery rate of ≤ 5% and fold-change of ≥ two-fold. The significance of overlap between groups of genes was determined using a chi-squared test for independence.