TMs characterization using flow cytometry and immunofluorescent staining.

Frozen thymic sections were used for immunofluorescent staining to identify the localization of TMs. Thymuses were harvested from C57BL/6 mice aged six to ten months and enzymatically digested for single-cell preparation. Cells were stained with antibodies for flow cytometric analysis. A) Gating strategy for identifying TMs. B) Representative flow cytometry histogram showing MerTK expression on TMs (blue) compared to CD64-F4/80- lymphocytes (red). C) Identification of distinct TM subsets using TIMD4 and VCAM1 markers. D) Comparison of the percentages of TM subsets in six-month-old mice. E) Comparison of the percentages of MerTK+ cells between TM subsets. F) Representative flow cytometry plots showing TM reporter expressions in MafB-mCherry and Csf1r-EGFP mice. Data are presented as the mean ± SEM (n = 7 per group from 2 independent experiments). A Kruskal-Wallis test was performed for multiple comparisons between three groups (*p ≤ 0.05).

Distinguishing TMs from thymic and circulating monocytes.

Thymuses were harvested from six-month-old C57BL/6 mice and enzymatically digested for single-cell preparation. Cells were stained with antibodies for flow cytometric analysis. A) CD45 IV labeling of circulating leukocytes in the mouse thymus, distinguishing intravascular cells from thymic-resident populations. B) Comparison of the percentages of CD45 labeled cells between Ly6C+CD11b+ and CD64+F4/80+ cells in the thymus. C) Comparison of the percentages of CD45 labeled cells between TM subsets. D) Lack of expression of monocyte markers associated with TIMD4+VCAM1+ and TIMD4-VCAM1+ subsets. E) Comparative analysis of monocyte marker expression across TM subsets. Data are presented as mean ± SEM (n = 5-7 per group from 2 independent experiments). Statistical significance was assessed using a two-tailed Mann-Whitney test (**p ≤ 0.01) for comparisons between two groups. A Kruskal-Wallis test was performed for multiple comparisons between three groups (*p ≤ 0.05 and **p ≤ 0.01).

Single-cell transcriptomics reveals distinct subpopulations and gene expression profiles of thymic macrophages.

Thymuses were harvested from six-week-old C57BL/6 mice, and CD64⁺ cells were isolated by FACS for scRNA-seq using the 10x Genomics 3’ RNA-seq platform. For TM scRNA-seq, cells were pooled from six mice per sample, with each experiment performed independently in duplicate. The resulting dataset was integrated with published data from Dzhagalov’s lab where they sequenced from Cd11cYFP and MaFIA mice using Harmony for batch correction and alignment. A) UMAP clustering of TMs. B) Heatmap representation of DEGs across the five clusters. C) Comparison of macrophage numbers in the thymus and spleen of SpiC-/- mice. Data are presented as mean ± SEM (n = 4-5 per group from 2 independent experiments), analyzed using a two-tailed Mann-Whitney test (*p ≤ 0.05).

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

List of DEGs with an average log₂ fold change > 1 and an adjusted p-value < 0.01, ordered by adjusted log₂ fold change.

GO pathway analysis of TMs.

Thymuses were harvested from six-week-old C57BL/6 mice, and CD64⁺ cells were isolated by FACS for scRNA-seq using the 10x Genomics 3’ RNA-seq platform. For TM scRNA-seq, cells were pooled from six mice per sample, with each experiment performed independently in duplicate. The resulting dataset was integrated with published data from Dzhagalov’s laboratory where they sequenced from Cd11cYFP and MaFIA mice using Harmony for batch correction and alignment. A) Dotplot showing top 20 enriched GO pathways in TIMD4-VCAM1+CX3CR1+ TM subset based on highly expressed genes. B) Dotplot showing top 20 enriched GO pathways in TIMD4+VCAM1+CX3CR1- TM subset based on highly expressed genes.

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).

Ontology of TM subsets.

Thymuses were harvested from 11–14-week-old Flt3-Cre × Rosa-mTmG mice and enzymatically digested to obtain single-cell suspensions. Cells were stained with antibodies for flow cytometric analysis. A) Schematic representation of the Flt3-Cre × Rosa-mTmG reporter mouse model. B) Representative flow cytometry plots showing mTomato (Tomato) and mGFP (GFP) reporter expression across TIMD4+VCAM1+, TIMD4-VCAM1+, and TIMD4- VCAM1- subsets. C) Comparison of reporter expression percentages among TM subsets. Data are presented as mean ± SEM (n = 7 per group from 2 independent experiments). Statistical significance was assessed using the Mann-Whitney test for pairwise comparisons, followed by the Holm-Šídá method to correct for multiple comparisons (***p ≤ 0.001).

TIMD4-VCAM1+ TM subset is HSC derived.

Thymuses were harvested from 11–14-week-old Flt3-Cre × Rosa-mTmG mice (Control) and Flt3-Cre × Rosa-mTmG mice in the CCR2 knockout background (Ccr2-/-), followed by enzymatic digestion to prepare single-cell suspensions. Cells were stained with antibodies for flow cytometric analysis. A) Representative flow cytometry plots showing TM subsets from Ccr2-/- and control mice. B) Comparison of TIMD4+VCAM1+ TM subset percentage and absolute numbers between Ccr2-/- and control mice. C) Comparison of TIMD4-VCAM1+ TM subset percentage and absolute numbers between Ccr2-/- and control mice. D) Comparison of TIMD4-VCAM1- TM percentage and absolute numbers between Ccr2-/- and control mice. Data are presented as mean ± SEM (n = 6–7 per group from 2 independent experiments). Statistical significance was assessed using a two-tailed Mann-Whitney test (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001).

FTOC analysis of TM function during T cell development in MaFIA mice.

Thymuses from E15.5 MaFIA mice were used to establish FTOCs. Cultures were treated with 2.5 mM AP20187 to deplete Csf1r-expressing cells on the day of setup and again the following day. On day 2, FTOC films were transferred to fresh media plates, and samples were analyzed by flow cytometry on day 6 to assess T cell development kinetics. A) Schematic representation of the TMs depletion workflow in FTOC culture. B) Percentages and absolute numbers of EPCAM⁺ cells in FTOC samples on day 2, comparing control and AP20187-treated groups, analyzed by flow cytometry. C) Total thymocyte numbers on day 6, comparing control and AP20187-treated groups. D) Absolute numbers of DCs on day 6, comparing control and AP20187-treated groups. DCs were gated as CD45+CD11c+MHC-IIhi. Data are presented as mean ± SEM (n = 8–11 per group from 3 independent experiments). Statistical significance was assessed using a two-tailed Mann-Whitney test (*p ≤ 0.05 and ***p ≤ 0.001). E) Thymuses from embryonic day 15.5 (E15.5) MaFIA (Csf1r-EGFP) and congenic C57BL/6 (45.2) mice were used to setup FTOCs. Cultures were treated with 2.5 mM AP20187 to deplete Csf1r-expressing cells on the day of setup and again the following day. On day 2, FTOC films were transferred to fresh media plates, and samples were analyzed by flow cytometry on day 6 to assess T cell development kinetics. Comparison of TM percentages between control and AP20187-treated MaFIA mice. F) Comparison of TM absolute numbers in control and AP20187-treated MaFIA FTOCs, as well as in AP20187-treated C57BL/6 FTOCs. G) Comparison of TIMD4+VCAM1+CX3CR1- numbers between control and AP20187-treated MaFIA mice. H) Comparison of TIMD4-VCAM1+CX3CR1+ numbers between control and AP20187-treated MaFIA mice. Data are presented as the mean ± SEM (n = 8–11 per group from 3 independent experiments). Statistical significance was assessed using a two-tailed Mann-Whitney test (*p ≤ 0.05 and **p ≤ 0.01) for comparisons between two groups. A Kruskal-Wallis test was performed for multiple comparisons between three groups (*p ≤ 0.05).

Impairment of T cell development in the absence of TMs.

Thymuses from embryonic day 15.5 (E15.5) MaFIA mice were used to establish FTOCs. Cultures were treated with 2.5 mM AP20187 to deplete Csf1r-expressing cells on the day of setup and again the following day. On day 2, FTOC films were transferred to fresh media plates, and samples were analyzed by flow cytometry on day 6 to assess T cell development kinetics. A) Representative flow cytometry plot showing T cell populations defined by CD4 and CD8 markers. B) Comparison of percentages and absolute numbers of developing T cells between control and AP20187-treated FTOC samples. C) Ratio of CD3⁺CD4⁺ single-positive (CD4SP) to CD3⁺CD8⁺ single-positive (CD8SP) thymocytes in control versus AP20187-treated FTOCs. Data are presented as the mean ± SEM (n = 8–11 per group from 3 independent experiments). D) Comparison of percentage and absolute numbers of γδ T cells between control and AP20187-treated FTOC samples. Data are presented as the mean ± SEM (n = 6–7 per group from 2 independent experiments). Statistical significance was assessed using the Mann-Whitney test, followed by the Holm-Šídák method to correct for multiple comparisons (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001).

TMs are critical for β-selection during T cell development.

Thymuses from embryonic day 15.5 (E15.5) MaFIA mice were used to establish FTOCs. Cultures were treated with 2.5 mM AP20187 to deplete Csf1r-expressing cells on the day of setup and again the following day. On day 2, FTOC films were transferred to fresh media plates, and samples were analyzed by flow cytometry on day 6 to assess T cell development kinetics. A) Representative flow cytometry plot showing DN cell populations defined by CD44 and CD25 markers B) Comparison of percentages and absolute numbers of DN cells between control and AP20187-treated FTOC samples. Data are presented as mean ± SEM (n = 8–11 per group from 3 independent experiments). C) Comparison of CD27+ DN3 cells percentage between control and AP20187-treated FTOC samples. D) Comparison of CD27+ DN4 cells percentage between control and AP20187-treated FTOC samples. Data are presented as mean ± SEM (n = 6–8 per group from 2 independent experiments). Statistical significance was assessed using the Mann-Whitney test for pairwise comparisons, followed by the Holm-Šídá method to correct for multiple comparisons (*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001).

Changes in TM numbers during thymic development.

Thymuses were harvested from mouse at various developmental time points and enzymatically digested to obtain single-cell suspensions. Cells were stained with antibodies for flow cytometry analysis. A) Total thymocyte numbers at different ages. B) Comparison of TM numbers and percentages across different ages of mice. Data are presented as mean ± SEM (n = 6-12 per group from 2-4 independent experiments).

TM analysis in MaFIA mice.

Thymuses were harvested from six-week-old MaFIA mice and enzymatically digested for single-cell preparation. Cells were stained with antibodies for flow cytometric analysis. A) Schematic representation of the transgene elements in the MaFIA mouse strain. B) Schematic representation of the mechanism by which AP20187 binds FKBP-expressing cells, leading to the induction of apoptosis. C) Representative images showing Csf1r-GFP reporter expression in TMs from MaFIA TM (green) and C75BL/6J (WT) TMs (grey) mice. D) Representative histogram showing Csf1r-EGFP reporter expression across TIMD4+VCAM1+CX3CR1-, TIMD4-VCAM1+CX3CR1+, and TIMD4-VCAM1- TM subsets. E) Comparison of Csf1r-EGFP means fluorescence intensity (MFI) across TIMD4+VCAM1+CX3CR1-, TIMD4-VCAM1+CX3CR1+, and TIMD4-VCAM1- TM subsets. Data are presented as mean ± SEM (n = 4 per group from 2 independent experiments). A Kruskal-Wallis test was performed for multiple comparisons between three groups (**p ≤ 0.01).