Flow-cytometric characterization of thymic macrophage populations.

Thymuses from adult C57BL/6 mice were enzymatically digested and analyzed by flow cytometry. A) Gating strategy for CD64+ F4/80+ TMs. B) Representative MerTK histogram for TMs (blue) and CD64-F4/80-lymphocytes (red). C) TIMD4/VCAM1 definition of TIMD4+ VCAM1+, TIMD4-VCAM1+, and TIMD4-VCAM1-populations. D) Frequencies of the three TM populations. E) MerTK+ frequencies. F) MafB-mCherry and Csf1r-EGFP reporter expression evaluated within the same TIMD4/VCAM1 gates defined in panel C. Data are mean ± SEM (n = 7 per group from two independent experiments). Kruskal-Wallis test (*p ≤ 0.05).

Intravascular labeling and monocyte-associated marker expression distinguish thymic myeloid populations.

A) Short-term intravenous (i.v.) anti-CD45 labeling identifies blood-exposed cells. B) Frequencies of i.v. CD45-labeled cells in the broad Ly6C+ CD11b+ gate and the CD64+ F4/80+ macrophage gate. The Ly6C+ CD11b+ gate contains heterogeneous blood-exposed and parenchymal cells and is not equivalent to the TIMD4-VCAM1-gate. C) i.v. CD45 labeling within TIMD4/VCAM1-defined populations. D-E) Relative Ly6C, CCR2, and CX3CR1 signal across the three populations; descriptions are comparative rather than categorical. Data are mean ± SEM (n = 5-7 per group from two independent experiments). Mann-Whitney test for two-group comparisons and Kruskal-Wallis test for three-group comparisons (*p ≤ 0.05; **p ≤ 0.01).

Single-cell transcriptomic profiles of thymic macrophages.

CD64+ cells from six-week-old C57BL/6 thymuses were analyzed by 10X Genomics 3’ scRNA-seq in two independent experiments, each pooling six mice, and integrated with the Zhou et al. dataset using Harmony. A) UMAP showing five annotated myeloid clusters; panel colors denote cluster identity. B) Heatmap of row-scaled differentially expressed genes; yellow and purple represent higher and lower relative scaled expression, respectively, and are not equivalent to flow-cytometric fluorescence intensity. C) Total CD64+ F4/80+ macrophage numbers in thymus and spleen of Spic-/- and control mice; TM subpopulations were not separately quantified in this panel. Data are mean ± SEM (n = 4-5 per group from two independent experiments), Mann-Whitney test (*p ≤ 0.05).

Differentially expressed genes (DEGs) from scRNA-seq analysis.

List of DEGs with an average log fold change > 1 and an adjusted p-value < 0.01, ordered by adjusted log fold change.

Gene Ontology pathway analysis of TM populations.

A) Top enriched pathways in TIMD4-VCAM1+ CX3CR1+ TMs. B) Top enriched pathways in TIMD4+ VCAM1+ TMs. The panels display pathway-level enrichment; individual genes mentioned in the text are reported in Table 1 and are not plotted separately in this Figure.

Developmental progression of TM subsets.

Thymuses were harvested from mice at various developmental time points and enzymatically digested to prepare single-cell suspensions. Cells were stained with antibodies for flow cytometry analysis. A) Expression of TM markers across different age groups. B) Comparison of the numbers and percentages of TM in mice at different ages. Data are presented as mean ± SEM (n = 4-12 per group from 2-4 independent experiments).

Flt3-Cre reporter history in thymic myeloid populations.

A) In Flt3-Cre × Rosa-mTmG mice, Cre-mediated recombination switches membrane Tomato expression to membrane GFP in cells with Flt3 expression history. B) Representative Tomato/GFP profiles within TIMD4+ VCAM1+, TIMD4-VCAM1+, and TIMD4-VCAM1-gates. Tomato+ GFP+ events are interpreted as reporter-transition/persistence events and not as a distinct lineage. C) Reporter frequencies among the three populations. CX3CR1 phenotype is defined independently in Figure 2 and is not displayed in this Figure. Data are mean ± SEM (n = 7 per group from two independent experiments). Mann-Whitney tests with Holm-Šídák correction (***p ≤ 0.001).

CCR2 dependence of TIMD4/VCAM1-defined thymic myeloid populations.

A) Representative TIMD4/VCAM1 profiles from Ccr2-/- and control mice. B-D) Frequencies and absolute numbers of TIMD4+ VCAM1+, TIMD4-VCAM1+, and TIMD4-VCAM1-populations. CX3CR1 phenotype is defined independently in Figure 2 and is not displayed in this Figure. Data are mean ± SEM (n = 6-7 per group from two independent experiments). Two-tailed Mann-Whitney test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

Efficient depletion of Csf1r-expressing myeloid cells in MaFIA fetal thymic organ culture.

E15.5 MaFIA thymus lobes were treated with AP20187 at culture initiation and the following day, transferred to fresh FTOC media on day 2, and analyzed on day 6. A) Experimental workflow. B) EpCAM+ CD45-thymic epithelial-cell frequencies and numbers. C) Total thymocyte numbers. D) CD45+ CD11c+ MHC-IIhigh DC numbers. E-F) TM frequencies and absolute numbers in untreated and AP20187-treated MaFIA FTOCs and in AP20187-treated C57BL/6J (CD45.2) control FTOCs. CD45.2 denotes the congenic allele of the C57BL/6J control. G-H) Numbers of TIMD4+ VCAM1+ and TIMD4-VCAM1+ TMs. The preservation of epithelial-cell and control-FTOC TM numbers, together with depletion of both MaFIA TM populations, demonstrates transgene-dependent perturbation of the Csf1r-expressing thymic myeloid compartment. Data are mean +/− SEM (n = 8-11 per group from three independent experiments). Mann-Whitney or Kruskal-Wallis tests as indicated (*p <= 0.05; **p <= 0.01; ***p <= 0.001).

Csf1r-expressing myeloid cells support alpha-beta T cell development in FTOC.

A) Representative CD4/CD8 profiles. B) Frequencies and absolute numbers of thymocyte populations in untreated and AP20187-treated MaFIA FTOCs. C) CD3+ CD4SP-to-CD3+ CD8SP ratio. D) Frequencies and numbers of γδ T cells. Depletion reduced αβ-lineage DP thymocyte production while preserving γδ T cell output. Data are mean +/− SEM (n = 8-11 per group from three independent experiments; γδ T cells, n = 6-7 from two experiments). Mann-Whitney tests with Holm-Sidak correction (*p <= 0.05; **p <= 0.01; ***p <= 0.001).

Csf1r-expressing myeloid cells support progression across the DN3-to-DN4 checkpoint.

A) Representative CD44/CD25 profiles of DN populations. B) Frequencies and absolute numbers of DN subsets in untreated and AP20187-treated MaFIA FTOCs. C-D) Quantified frequencies of CD27+ DN3 and DN4 cells. The coordinated accumulation of DN3 cells, reduction of DN4 cells, and lower CD27 expression identify impaired progression at the β-selection transition. Data are mean +/− SEM (DN subsets, n = 8-11 from three independent experiments; CD27, n = 6-8 from two experiments). Mann-Whitney tests with Holm-Sidak correction (*p <= 0.05; **p <= 0.01; ***p <= 0.001).