Heterogeneity and ontogeny of mouse thymic macrophages reveal a requirement for Csf1r-expressing myeloid cells during early T cell development
- Department of Immunology, University of Toronto, Canada
- Biological Sciences, Sunnybrook Research Institute, Canada
- Toronto General Hospital Research Institute, University Health Network, Canada
- Ted Rogers Centre for Heart Research, Translational Biology and Engineering Program, Canada
- Peter Munk Cardiac Centre, University Health Network, Canada
Figures
Figure 1
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) MafbmCherry and Csf1rEGFP 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).
Figure 2
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).
Figure 3
Single-cell transcriptomic profiles of thymic macrophages.
CD64 + cells from six-week-old C57BL/6 thymuses were analyzed by 10 X 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).
Figure 4
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.
Figure 5 with 1 supplement
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 two to four independent experiments).
Figure 5—figure supplement 1
Changes in TM numbers during thymic development.
Thymuses were harvested from mice 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 two to four independent experiments).
Figure 6
FLT3-Cre reporter history in thymic myeloid populations.
(A) In Flt3Cre × Rosa26mTmG 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).
Figure 7
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).
Figure 8 with 1 supplement
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/6 J (CD45.2) control FTOCs. CD45.2 denotes the congenic allele of the C57BL/6 J 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).
Figure 8—figure supplement 1
MaFIA transgene and reporter expression in thymic macrophages.
(A) The Csf1r-driven MaFIA transgene encodes EGFP together with a membrane-targeting low-affinity nerve growth factor receptor segment (dLNGFR) and an FKBP-Fas suicide module. dLNGFR is a structural component of the fusion construct and is not used as a lineage marker. (B) AP20187 is a synthetic homodimerizer that binds the engineered FKBP domains, bringing Fas intracellular domains together and inducing apoptosis in transgene-expressing cells. (C) Representative Csf1rEGFP signal in MaFIA TMs (green) and C57BL/6 J controls (gray). (D, E) Csf1rEGFP distributions and mean fluorescence intensity across TIMD4+VCAM1+, TIMD4- VCAM1+, and TIMD4- VCAM1- populations. Data are mean ± SEM (n=4 per group from two independent experiments), Kruskal-Wallis test (**p≤0.01).
Figure 9
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+CD8 SP 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).
Figure 10
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).
Tables
Table 1
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.
| Ly6C+ monocytes | TIMD4+ VCAM1+ CX3CR1- | TIMD4- VCAM1+ CX3CR1+ | |||
|---|---|---|---|---|---|
| Lsp1 | Ahnak | Cd163 | Ccl2 | Gpnmb | Lhfpl2 |
| Sirpb1a | Cytip | Mrc1 | Ccl4 | Tnfsf13b | Chst2 |
| Gm34084 | Ifitm2 | Mertk | Adgre1 | Lhfpl2 | Mmp2 |
| Alcam | S100a11 | Spic | Adgre2 | Chst2 | Pmepa1 |
| Cbfa2t3 | Ramp1 | Timd4 | Hmox1 | Mmp2 | Oas3 |
| Sirpb1b | Lyst | Vcam1 | Tlr7 | Pmepa1 | Cst7 |
| Cnn2 | Ccl6 | Clec1b | Axl | Oasl1 | Zmynd15 |
| Samsn1 | Itga4 | Stab2 | Fgr | Cst7 | H2-Q7 |
| Itgam | Il17ra | Fcgrt | Ccl8 | Zmynd15 | Ccr5 |
| Alox5ap | Ccr2 | Tgm2 | Id2 | H2-M2 | H2-T22 |
| Emb | Ly6c2 | P2ry12 | Sirpa | Sh3pxd2b | Bst2 |
| Traf1 | Il1b | Cd86 | Runx1 | Kctd14 | Stat2 |
| Dpp4 | Batf3 | Tlr4 | Stat5b | Ifit1 | Cebpa |
| S100a10 | Rbpj | Il18 | Cd84 | Fam20c | Il1rn |
| Plac8 | Ptgs1 | Mapk14 | Cp | Cxcl16 | |
| Stap1 | Pparg | Spsb1 | |||
| Sirpb1c | Adamdec1 | Gpnmb | |||
| Rnase6 | Cd38 | Tnfsf13b | |||
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6 J | The Jackson Laboratory | JAX:000664; RRID:IMSR_JAX:000664 | Wild-type controls and source strain for adult and timed-pregnancy experiments |
| Genetic reagent (M. musculus) | MaFIA; Csf1r-EGFP/Fas-FKBP | The Jackson Laboratory | JAX:005070; RRID:IMSR_JAX:005070 | Macrophage Fas-Induced Apoptosis model used for inducible depletion of Csf1r+myeloid cells |
| Genetic reagent (M. musculus) | MafB-mCherry-Cre | The Jackson Laboratory | JAX:029664; RRID:IMSR_JAX:029664 | Reporter/Cre line used for thymic macrophage subset characterization |
| Genetic reagent (M. musculus) | Csf1r-EGFP | The Jackson Laboratory | JAX:018549; RRID:IMSR_JAX:018549 | Reporter line used to detect Csf1r-expressing macrophages |
| Genetic reagent (M. musculus) | Spic−/− | The Jackson Laboratory | JAX: 013039; RRID:IMSR_JAX:013039 | SpiC knockout mice; exact stock identifier not reported in manuscript |
| Genetic reagent (M. musculus) | Flt3-Cre×Rosa-mTmG | The Jackson Laboratory | Rosa-mTmG: JAX:007676; RRID:IMSR_JAX:007676 | Fate-mapping mice; provided by Dr. Slava Epelman as stated in the manuscript |
| Genetic reagent (M. musculus) | Ccr2−/− | The Jackson Laboratory | JAX: 004999; RRID:IMSR_JAX:004999 | Ccr2 knockout line used to backcross into fate mapping Flt3-Cre×Rosa mTmG mice, as stated in the manuscript |
| Genetic reagent (M. musculus) | Flt3-Cre×Rosa mTmG; Ccr2−/− background | PMID:24439267 | Not commercially available | Fate-mapping mice in Ccr2 knockout background; provided by Dr. Slava Epelman as stated in the manuscript |
| Antibody | APC anti-mouse CD64/FcγRI (clone X54-5/7.1, mouse monoclonal) | BioLegend | Cat#:139305; RRID:AB_11219205; Cat#:139306; RRID:AB_11219391 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | BUV805 anti-mouse F4/80 (clone T45-2342, rat monoclonal) | BD Biosciences | Cat#:749282; RRID:AB_2873657 | Flow cytometry (1:200) |
| Antibody | FITC anti-mouse MERTK/Mer (clone 2B10C42, rat monoclonal) | BioLegend | Cat#:151503; RRID:AB_2617034; Cat#:151504; RRID:AB_2617035 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | PE anti-mouse Tim-4 (clone RMT4-54, rat monoclonal) | BioLegend | Cat#:130005; RRID:AB_1227807; Cat#:130006; RRID:AB_2201843 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | PE/Cyanine7 anti-mouse CD106/VCAM1 (clone 429/MVCAM.A, rat monoclonal) | BioLegend | Cat#:105719; RRID:AB_2214047; Cat#:105720; RRID:AB_2214046 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | Brilliant Violet 605 anti-mouse CX3CR1 (clone SA011F11, mouse monoclonal) | BioLegend | Cat#:149027; RRID:AB_2565937 | Flow cytometry (1:200) |
| Antibody | Brilliant Violet 785 anti-mouse CD192/CCR2 (clone SA203G11, rat monoclonal) | BioLegend | Cat#:150621; RRID:AB_2721565 | Flow cytometry (1:200) |
| Antibody | APC/Cyanine7 anti-mouse/human CD11b (clone M1/70, rat monoclonal) | BioLegend | Cat#:101225; RRID:AB_830641; Cat#:101226; RRID:AB_830642 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | Brilliant Violet 711 anti-mouse Ly-6C (clone HK1.4, rat monoclonal) | BioLegend | Cat#:128037; RRID:AB_2562630 | Flow cytometry (1:200) |
| Antibody | Alexa Fluor 700 anti-mouse I-A/I-E (clone M5/114.15.2, rat monoclonal) | BioLegend | Cat#:107621; RRID:AB_493726; Cat#:107622; RRID:AB_493727 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | BUV395 anti-mouse CD11c (clone N418, Armenian hamster monoclonal) | BD Biosciences | Cat#:744180; RRID:AB_2742045 | Flow cytometry (1:200) |
| Antibody | BUV563 anti-mouse CD45 (clone 30-F11, rat monoclonal) | BD Biosciences | Cat#:565710; RRID:AB_2722550; Cat#:612924; RRID:AB_2870209 | Flow cytometry (1:150); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | Purified anti-mouse CD16/32 Fc block (clone 93, rat monoclonal) | BioLegend | Cat#:101301; RRID:AB_312800; Cat#:101302; RRID:AB_312801 | Flow cytometry/Fc block (1:100); manuscript reports clone/vendor but not purchased size |
| Antibody | PE/Cyanine7 anti-mouse CD3ε (clone 145–2 C11, Armenian hamster monoclonal) | BioLegend | Cat#:100319; Cat#:100320; RRID:AB_312685 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | Brilliant Violet 711 anti-mouse CD4 (clone RM4-5, rat monoclonal) | BioLegend | Cat#:100549; RRID:AB_11219396; Cat#:100550; RRID:AB_2562099; Cat#:100557; RRID:AB_2562607 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | BUV395 anti-mouse CD8a (clone 53–6.7, rat monoclonal) | BD Biosciences | Cat#:563786; RRID:AB_2732919 | Flow cytometry (1:200) |
| Antibody | APC/Cyanine7 anti-mouse CD25 (clone PC61, rat monoclonal) | BioLegend | Cat#:102025; RRID:AB_830744; Cat#:102026; RRID:AB_830745 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | Brilliant Violet 785 anti-mouse/human CD44 (clone IM7, rat monoclonal) | BioLegend | Cat#:103041; RRID:AB_11218802; Cat#:103059; RRID:AB_2571953 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Antibody | Alexa Fluor 700 anti-mouse/human CD45R/B220 (clone RA3-6B2, rat monoclonal) | BioLegend | Cat#:103231; RRID:AB_493716; Cat#:103232 | Flow cytometry (1:200); manuscript reports clone/fluor/vendor but not purchased size |
| Commercial assay or kit | Spleen Dissociation Kit | Miltenyi Biotec | Cat#:130-095-926; RRID:SCR_024888 | Used for thymus dissociation prior to flow cytometry and sorting |
| Commercial assay or kit | Chromium Single Cell 3' Reagent Kit | 10 x Genomics | Cat#:PN-1000691 | Used for scRNA-seq library preparation; exact kit version not reported |
| Commercial assay or kit | anti-APC MicroBeads | Miltenyi Biotec | Cat#:130-090-855; RRID:AB_244367 | Used for CD64 + cell enrichment prior to sorting |
| Commercial assay or kit | LS Columns | Miltenyi Biotec | Cat#:130-042-401 | Used for positive selection of CD64 + cells |
| Chemical compound, drug | AP20187; B/B homodimerizer | Takara Bio | Cat#:635058 | Used for MaFIA-mediated depletion in FTOC; vendor/catalog not reported in manuscript |
| Software, algorithm | FlowJo | BD/Tree Star | RRID:SCR_008520 | Flow cytometry analysis |
| Software, algorithm | R (version 4.4.1) | R Project | RRID:SCR_001905 | Statistical computing and scRNA-seq analysis |
| Software, algorithm | Seurat | Satija Lab / CRAN | RRID:SCR_007322 | scRNA-seq processing, integration, clustering, SCTransform, FindMarkers |
| Software, algorithm | Harmony | Korsunsky et al., 2019 | RRID:SCR_022206 | Batch correction/data integration |
| Software, algorithm | Monocle3 | Trapnell Lab | RRID:SCR_018685 | Trajectory analysis / pseudotime inference |
| Software, algorithm | GraphPad Prism (version 10) | GraphPad | RRID:SCR_002798 | Statistical analysis and graphing |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/109219/elife-109219-mdarchecklist1-v1.docx
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Supplementary file 1
Supplemental tables.
- https://cdn.elifesciences.org/articles/109219/elife-109219-supp1-v1.docx
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Heterogeneity and ontogeny of mouse thymic macrophages reveal a requirement for Csf1r-expressing myeloid cells during early T cell development
eLife 15:RP109219.
https://doi.org/10.7554/eLife.109219.3
