1. Volume 47, Issue 2, Pages 349-362.e5 (August 2017)
T Cell Zone Resident Macrophages Silently Dispose of Apoptotic Cells in the Lymph Node 
Myriam Baratin, Léa Simon, Audrey Jorquera, Clément Ghigo, Doulaye Dembele, Jonathan Nowak, Rebecca Gentek, Stephan Wienert, Frederick Klauschen, Bernard Malissen, Marc Dalod, Marc Bajénoff 
Immunity 
Volume 47, Issue 2, Pages e5 (August 2017)
DOI: /j.immuni
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2. Immunity 2017 47, 349-362.e5DOI: (10.1016/j.immuni.2017.07.019)
Copyright © 2017 Elsevier Inc. Terms and Conditions

3. Figure 1 Lymph Node Paracortex Hosts CX3CR1hi MERTK+ Macrophages
(A) LN sections from Cx3cr1gfp/+ mice were stained for B220, CD3, and MERTK expression in order to locate B cell follicles, T cell zone, and macrophages. Scale bars represent 50 μm (left panels), 10 μm (right panels). n = 4 mice.
(B) LN sections from Cx3cr1gfp/+ mice were stained for CD3, CD169, or F4/80 expression to localize CX3CR1+ CD169+ SSM, CX3CR1+ CD169− F4/80+ MMM, and all other CD169− macrophages. Scale bars represent 100 μm. n = 4 mice.
(C) CD11b+ enriched LN cells from Cx3cr1gfp/+ mice were stained for MERTK, CD11b, and CD169 to identify MERTK+ CX3CR1+ CD169+ SSM and the T cell zone MERTK+ CX3CR1+ CD169− TZMC by flow cytometry (dot plots). Histograms represent the expression of additional markers (red line) and isotype controls (shaded gray) expressed by TZMC. n = 3 experiments, with 2 mice each.
(D) RFP and GFP expression in CD8α DC, CD11b DC, and TZM isolated from Cx3cr1gfp/+ Cd11cCre/+ Rosalsl-rfp/+ (upper panels, red line), Cx3cr1gfp/+ Clec9aCre/+Rosalsl-rfp/+ (middle panels, red line), and Zbtb46gfp/+ mice (lower panels, red line). The shaded gray line in each histogram represents Cre− or WT controls. n = 3 experiments, with 2 mice each.
(E) Heatmaps showing the expression patterns of core macrophages genes or core DC genes (brown font) in TZMC compared to SP and LN CD8α or CD11b DC and to brain macrophages. The expression pattern of Itgax and Csf1r is also shown (black font) as the corresponding proteins are often misused to identify DCs versus monocytes/macrophages. Three replicates are shown for each subset. LN or SP origin of each subset is indicated respectively by a pink or an orange bar. Cells from the ImmGen dataset are highlighted by gray bars and our own data by black bars on the top of the heatmap. See also Figures S1, S2, and S6.
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4. Figure 2
TZM Are Derived from BM Precursors That Seed the LN Paracortex after Birth and Differentiate into Long Lived Macrophages
(A) Cx3cr1gfp/CreER Rosalsl-rfp/+ mice were treated with tamoxifen in order to induce RFP expression in all CX3CR1+ cells including monocytes, brain macrophages, and TZM. As monocytes are short-lived cells continuously replenished by CX3CR1− HSC, they are rapidly replaced by RFP− monocytes. As a result, the fraction of RFP+ cells in a macrophages population that is continuously renewed by monocytes should progressively decrease over time (left panel). The percentage of RFP+ brain macrophages and TZM was assessed in newborn and 6-week-old Cx3cr1gfp/CreER Rosalsl-rfp/+ mice that received tamoxifen at E13. n = 2 experiments, with 2 to 3 mice each. Data are depicted as mean ± SEM (middle panel). Six-week-old Cx3cr1CreER Rosalsl-rfp/+ mice were fed with tamoxifen diet for 1 month. 1, 2, 5, and 6 months later, the percentage of RFP+ brain macrophages, TZM, blood monocytes, and total LN DC were determined by flow cytometry (right panel). n = 3 experiments, with 3 mice per time point. Data are depicted as mean ± SEM (right panel).
(B) Cx3cr1gfp/+ mice were shield irradiated with a lead cover protecting their entire body with the exception of the rear legs and reconstituted with monocyte depleted BM cells isolated from Ubctdt Cx3cr1gfp/+ mice. The percentage of tdT+ donor cells in shield protected organs is expected to increase only in macrophages subsets replaced by HSC derived progenitors (left panel). The percentage of donor brain macrophages, blood monocytes, and TZM located in protected LN were determined 1 month later (neonate, middle panel) or 1, 2, and 6 months (adult, right panel) Cx3cr1gfp/+ mice. n = 3 experiments, with 2 to 3 mice per time point. Data are depicted as mean ± SEM. See also Figure S3.
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5. Figure 3 TZM Proliferate upon LN Swelling
(A) Cx3cr1CreER/+ Ubow+/+ adult mice were treated with tamoxifen in order to trigger the stochastic acquisition of CFP and YFP by CX3CR1+ cells. One month later, when CFP and YFP expressing monocytes were no longer detected in their blood (not shown), mice were injected in their rear footpads with CFA. Seven days later, inflamed popliteal LN and non-draining LN were analyzed by confocal microscopy for the presence of monocolored clusters of TZM indicative of local proliferation. n = 4 mice for each condition. Scale bars represent 100 μm.
(B) All confocal datasets were processed to generate Voronoi tessellated pictures (step 1). For each original dataset the positions of all TZM were computationally scrambled (step 2) using ClusterQuant software (see STAR Methods).
(C) The experimentally observed numbers of TZM per monocolored TZM cluster (circles) were compared to the computationally scrambled datasets (squares). p value: ∗∗∗∗<10−4; ∗<0.05. Bars represent median.
(D) Adult Cx3cr1gfp/+ mice were shield irradiated and reconstituted with Ubctdt BM cells. One month later, auricular LN inflammation was induced by CFA injection in their ears. The graph shows the percentage of TZM chimerism relative to monocyte chimerism in auricular draining (dLN) and non-draining (ndLN) protected LN. n = 2 experiments, with 3 mice each. Bars represent mean. ns = non significant.
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6. Figure 4
TZM but Not DC Act as Professional Efferocytes in the LN T Cell Zone
(A) LN sections from a Cx3cr1gfp/+ mice were stained for MERTK expression and DNA content. The percentage of TZM with no vacuole, DNA containing vacuoles, or DNA-free vacuoles was determined by imaging. n = 4 mice. Data are depicted as mean ± SEM.
(B and C) LN sections from Cx3cr1gfp/+ mice were stained for DNA, MERTK, and active caspase 3 (B) or apoptotic cells (TUNEL, C) and analyzed by confocal microscopy. Scale bars represent 10 μm. n = 4 mice.
(D) Heatmap showing the expression pattern of genes known to be involved in efferocytosis in TZM and SP CD8α DC. Three replicates are shown for each subset.
(E) Cx3cr1gfp/+ mice were co-injected with irradiated (pink) and non-irradiated (cyan) labeled T lymphocytes (T). Two, 8 hr and 12 hr later, the ratio of irradiated T versus non-irradiated T (left graph) as well as the percentage of irradiated and non-irradiated T engulfed by TZM (right graph) were determined by flow cytometry and imaging respectively. Bars represent mean. Right panels show representative pictures of LN sections collected 12 hr post transfer and stained for MERTK expression. Arrowheads point to apoptotic T engulfed by TZM while inserts show high magnification of some of these TZM. Scale bars represent 20 μm (left panel), 5 μm (right panel). n = 2 experiments, with 4 mice per time point.
(F) SP DC were isolated from Ubctdt mice and injected s.c. in the rear footpads of Cx3cr1gfp/+ mice. Thirty-six, 48, and 72 hr later, popliteal LN sections were stained for MERTK expression and used to determine the density of donor tdT+ DC in the LN. n = 2 experiments, with 3 mice per time point. Data are depicted as mean ± SEM. Arrowheads in confocal pictures point to donor DC engulfed by TZM. Scale bar represents 10 μm.
(G) LN sections from Cx3cr1gfp/+ LangerinCre/+ Rosalsl-rfp/+ mice were stained for MERTK expression. Arrowheads point to LANGERIN+ cells phagocytosed by TZM. Scale bar represents 10 μm. n = 3 mice.
(H) Cx3cr1gfp/+ Clec9a+/+ Rosalsl-rfp/+ mice, containing RFP+ GFP− CD8α DC and RFP− GFP+ TZM, were injected with labeled irradiated T. Twelve hours later, LNs were stained for MERTK expression and analyzed by confocal microscopy. The total number of phagocytes that engulfed apoptotic T was determined and used to calculate the percentage of TZM and DC among the T-phagocytosing cells. n = 4 mice. Data are depicted as mean ± SEM.
(I) C57BL/6 mice were fully irradiated and reconstituted with a mixture of BM cells isolated from Cx3cr1gfp/gfp (CX3CR1 deficient) and Ubctdt Cx3cr1gfp/+ (CX3CR1 proficient) mice. Two months later, chimeric mice were injected with labeled irradiated T and their LN analyzed 12 hr later by imaging. Confocal pictures were used to determine the percentage of CX3CR1 proficient and deficient cells among the phagocytosing TZM. n = 5 mice. Bars represent mean. See also Figure S4.
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7. Figure 5 TZM Do Not Prime nor Tolerize Naive CD4 T Cells
(A) Cx3cr1gfp/+ LN cells were stained for CD40, CD80, CD86 expression and analyzed by flow cytometry. Histograms show the expression of the indicated markers (red line) by TZM and CD64− MHCII+ CD11chi resident (res) DCs. Shaded gray: isotype control. n = 3 mice.
(B) OT-II T were stained with the Cell Trace Violet (CTV) proliferation tracer and co-cultured with either splenic DC or TZM both loaded with the OT-II T specific OVA peptide (OT-II peptide). Three days after, CTV dilution was assessed by flow cytometry. Data are representative of three independent experiments.
(C) CTV labeled OT-II T were co-cultured with TZM loaded or not with the OT-II peptide. The next day, OT-II peptide loaded DC were added to the wells. Three days later, CTV dilution as well as the expression of Foxp3, TNF-α, IL-4, IFN-γ and IL-10 expressed by OT-II T were assessed by flow cytometry. TGF-β was added to the co-culture as a positive control of Treg conversion. Histograms and dot plots are representative of two independent experiments. See also Figure S5.
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