IL-27 regulates monopoiesis during infection.

A) Schematic of infection and TAM dosing strategy for Procr-Ai6 mice. B) Procr-Ai6 mice were infected and representative flow plots of LTHSCs (Lin (CD3, NK1.1, B220, Ly6G)-, Sca-1+, CD117 (cKit)+, CD135-, CD48-, CD150+) from naïve (left) and infected (right) mice are shown. Proportions of zsGreen+ LTHSCs were then quantified in both groups. C) Proportions of zsGreen+ monocytes (CD3-, B220-, CD11b+, Ly6C+, Ly6G-) in the spleen and PECs of naïve and infected mice. D) Schematic of infection of WT and IL-27p28-/- mice for E-F. E) Numbers of LTHSCs and granulocyte progenitors (GPs) (CD3-, NK1.1-, B220-, CD117+, CD34+, CD16/32hi, Ly6C+, CD135-, CD115-) in the bone marrow of WT and IL-27p28-/- infected mice throughout infection. F) Monocyte progenitors (MPs) (CD3-, NK1.1-, B220-, CD117+, CD34+, CD16/32hi, Ly6C+, CD135-, CD115+; orange box) in the bone marrow of infected WT and IL-27p28-/- mice throughout infection. Representative flow plots are shown (left) and quantified (right). Statistical significance was tested by one-way ANOVA with Sidak’s correction. *, **, and *** correspond to p-values ≤ 0.05, 0.01, and 0.001, respectively. N=3-5 mice/group and data shown are representative of 2-3 experiments.

IL-27 regulates monopoiesis during infection in a cell intrinsic manner.

A) CD4-IL-27R mice were infected and MPs (orange box) measured at 5 dpi in the BM. Representative flow plots are shown (left) and quantified (right). B) Schematic of BM chimeras generated from WT (CD45.1) and IL-27R-/- (CD45.1.2) bulk bone marrow that were then infected and used in C-D. C) The proportion of LTHSCs (left) and MPs (right) from each respective donor lineage in the bone marrow of both the single (left two bars) and mixed (right two bars) chimeras at 5 dpi. D) Representative flow plots pre-gated on splenic monocytes (left plot) and then down-gated on CCR2hiCX3CR1lo (right plot) monocytes at 5 dpi. WT derived monocytes are shown in the solid-dark gray while IL-27R-/- derived cells are in the dashed-light gray. The proportion of each donor lineage that contribute to mature CCR2hiCX3CR1lo splenic monocytes at 5 dpi were then quantified (far right). Statistical significance was tested by either Welch’s T-test (A) or one-way ANOVA with Sidak’s correction (C-D). *, **, and *** correspond to p-values ≤ 0.05, 0.01, and 0.001, respectively. N=3-5 mice/group and data shown are representative of 2-3 experiments.

IL-27p28 is produced in the BM during infection.

A) WT mice were infected, and the bone marrow harvested throughout infection. BM was either washed with media or cultured for 24 hrs. and supernatant collected. Both the wash and supernatant were analyzed by ELISA for IL-27p28. B) IL-27p28-GFP reporter mice were infected and their bone marrow analyzed by flow cytometry throughout infection. A representative flow plot at 5 dpi of bulk marrow is shown (left) and proportion of immune cell contribution to the GFP+ population measured (right). C) Numbers of IL-27p28-GFP+ cells in the spleen, blood, and BM of infected mice were quantified throughout infection (left). Proportions of GFP+ CD45+ cells were analyzed by cell type in the blood, spleen, and PEC during infection (left). D) Femurs from infected Procr-tdTomato+p28-GFP+ mice were harvested, sectioned, and imaged at 40x magnification. The total imaged femur is shown (top), with zoomed in sections shown below for increased detail of cellular localization (bottom). Numbers of Procr-tdTomato+ and p28-GFP+ cells were counted in eight randomly selected regions throughout the marrow (see Supp. Fig. 4). GFP+ cells were normalized to tdTomato+ cells and the eight counted regions classified as areas of hyper-or hypo-vascularization according to proximity of the region to the labeled vasculature. This allowed quantification of the localization of cells (bottom right).

Expression of the IL-27R subunits gp130 and IL-27Rα during hematopoietic development.

A) Schematic ball-and-stick model of hierarchical hematopoietic development. Cell type colors are maintained for reference in the following panels. B) Progenitors and progeny shown in (A) were analyzed in WT and infected mice (at 5 dpi) by flow cytometry for the gp130 receptor. 10,000 live cells were concatenated from n=3 mice, and representative flow plots are shown. C) Cells in (B) were analyzed for expression of the IL-27Rα. D) Bulk bone marrow was isolated from IRGM1-dsRed reporter mice. 22 x 106 cells were plated per condition and stimulated with 20 ng/ml of IL-27 for 0, 6, or 48 hours and RFP expression measured by flow cytometry.

Developmental expression of hematopoietic cytokine receptors.

A) Expression of CD132 (IL-2Rγ) and B) CD131 (GM-CSF/IL-3/IL-5Rβ) was analyzed by flow cytometry on progenitors and progeny in the BM of naïve and 5 dpi mice as in Fig. 4. C) Expression of IL-6R⍺ and D) GM-CSFR⍺ were measured as in (A).

IL-27 limits infection induced HSPC polarization.

WT and IL-27R-/- mice were infected for 5 days, LTHSCs sorted, and cultured in MethoCult for 10-12 days before being analyzed by flow cytometry. A) 10,000 live cells from n=3-4 mice/ group (WT (naïve+5 dpi) and IL-27R-/- (5 dpi)) were concatenated and dimensionally reduced via UMAP. B) Numbers of cells in each cluster from (A) were measured. C) X-shift analysis was used to identify the expression level of each marker used in the clustering performed in (A). 5 of the dominant clusters from (B) are shown. D) Clusters 1, 10, and 11, all involved in monocyte development, are highlighted. E) Contribution of Clusters 1, 10, and 11 to each genotype are shown (left) and quantified (right). F) Bone marrow chimeras were generated from infected WT and IL-27R-/- mice (left). The contribution of donor cells to mature monocytes was then measured at 14 weeks post-transplant (right). Statistical significance was tested by one-way ANOVA with Sidak’s correction. *, ***, and **** correspond to p-values ≤ 0.05, 0.001, and 0.0001, respectively. N=3-5 mice/group and data shown are representative of 2-3 repeated experiments.

Testing the impact of IL-27 on functionality and fitness of HSPCs during infection.

A) WT and IL-27R-/- were infected, treated with 5 mg BRDU, and kept on 1mg/ml BRDU in the drinking water throughout infection. Incorporation of BRDU was then measured in Lin-Sca-1+cKit+ (LSK) progenitor cells in comparison to Ki67 at 5 dpi. Representative flow plots are shown (left) and the proportion of BRDU+Ki67+ cells quantified (right). B) Schematic for the generation of BM chimeras from infected mice used in (C). C) Numbers of LTHSCs from recipients receiving donor marrow from either uninfected or infected mice were measured based on genotype. D) Secondary transplant chimeras from mice that originally received naïve or infected marrow where generated as shown (left). Survival of recipients was then assessed (right).

IL-27 regulates monopoiesis during infection.

A) The proportion of zsGreen+ monocyte dendritic cell progenitors (MDPs) (CD3-, NK1.1-, B220-, CD117+, CD34+, CD16/32lo, CD115+), monocytes, and neutrophils (CD3-, B220-, CD11b+, Ly6C+, Ly6G+) in the BM of naïve and infected Procr-Ai6 mice. B) The proportion of zsGreen+ CCR2hi/lo CX3CR1hi/lo monocytes in the spleen and PECs in naïve and infected Procr-Ai6 mice. C) Number of MDPs in WT and IL-27p28-/- mice in the bone marrow during infection. D) Numbers of monocytes in the liver in WT and IL-27p28-/- throughout infection. E) WT and IL-27p28-/- mice were injected I.V. with fluorescent anti-CD45 to label immune cells in the vasculature. Numbers of I.V. label- and CCR2hiCX3CR1lo monocytes in the liver were then quantified throughout infection. Statistical significance was tested by one-way ANOVA with Sidak’s correction. *, **, and *** correspond to p-values ≤ 0.05, 0.01, and 0.001, respectively. N=3-5 mice/group and data shown are representative of 2-3 experiments.

Gating strategy for flow cytometric analysis of cells in the bone marrow and periphery.

Representative flow plots from the bone marrow (top) and spleen (bottom) are shown indicating the gating strategy used to identify various cell populations. The combination of surface markers used are listed in the tables (bottom) and are color coded to correspond to both the developmental schematic pictured as well as the corresponding flow gate on the representative plots.

IL-27 regulates monopoiesis during infection and post-irradiation in a cell intrinsic manner.

A) IL-27R⍺ expression on CD4+ (CD19-CD3+CD8⍺-) T cells, CD8+ (CD19-CD3+CD4-) T cells, and B cells (CD19+CD3-) in CD4-27R mice at 5 dpi. B) The proportion of LTHSCs (left) and MPs (right) from each respective donor lineage in the bone marrow post-reconstitution and pre-infection. C) The proportion of CCR2hiCX3CR1lo monocytes from each respective donor lineage in the spleen and liver post-reconstitution and pre-infection. Statistical significance was tested by one-way ANOVA with Sidak’s correction. *, **, and *** correspond to p-values ≤ 0.05, 0.01, and 0.001, respectively. N=3-5 mice/group and data shown are representative of 2 repeated experiments.

IL-27p28 is produced in the bone marrow during infection.

A) IL-27p28-GFP mice were infected, and the numbers of each CD45+, immune cell type that expressed GFP in the BM or periphery quantified throughout infection. B) Levels of IL-27p28-GFP expression were measured in bulk monocytes (red) and dendritic cells (blue) in the bone marrow (left) and spleen (right). C) Femurs from naïve IL-27p28-GFP mice were stained for Sca-1 and imaged at 40x magnification. Zoomed in images are shown (right) of the indicated areas, with the yellow arrows indicating positively stained, Sca-1+ cells. D) The merged image shown in Fig. 3D is separated by channel (GFP, tdTomato, and AF647, from top to bottom). Eight randomly selected regions that were used for quantification of either GFP+ or tdTomato+ cells are shown (top), and the quantification of cells in each region is shown next to the corresponding florescence channel (right).

IL-27Rα transcript expression during hematopoiesis and differential sensitivity of LTHSC and CMP to IL-27.

A) Expression levels of IL27RA are shown from the Haemosphere RNA-seq data set and online tool as well as the Blood Spot data set and visualizer (B). C) Representative flow plots of IRGM1-RFP expression in LTHSCs and CMPs 48 hrs. post-stimulation with IL-27.

Developmental expression of additional cytokine receptors.

Indicated cytokine receptors were analyzed in naive and infected mice as described in Fig. 4-5.

Gating strategy for sorting of LTHSCs used in monocyte differentiation.

LTHSCs were sorted from WT naïve, WT 5 dpi, and IL-27R-/- 5 dpi mice. The gating strategy is shown as well as the purity of sorted samples (last panel).

Development and validation of an unbiased flow cytometry approach for the analysis of MethoCult colonies.

WT and IL-27R-/- mice were infected, LTHSCs sorted and cultured in MethoCult for either 5 or 10 days. A) Colony phenotypes were analyzed by microscopy and counted by eye. B) Colonies were collected at either 5 or 10 days post-culture (dpc) and analyzed by flow cytometry. 500 live cells from n=3-4 mice/ group were concatenated and dimensionally reduced via UMAP. UMAP plots are shown, and Ki67 expression is overlayed. C) X-shift analysis was used to identify the expression level of each marker used in the clustering performed in (B). D) Numbers of cells in each cluster from (B) were measured. E) Contribution of each genotype to each cluster was quantified. Statistical significance was tested by one-way ANOVA with Sidak’s correction. *, ***, and **** correspond to p-values ≤ 0.05, 0.001, and 0.0001, respectively. N=3-4 mice/group and data shown are from 1 of 3 experiments.

Testing HSPC survival and fitness.

A) LTHSCs from WT and IL-27R-/- BM at 5 dpi was stained for Annexin V (AV) expression and free-amine staining viability dye (LD). This allowed the measurement of apoptotic (AV+LD-), late apoptotic (AV+LD+), and necrotic (AV-LD+) cells. The proportion of LTHSCs in each of these stages was quantified. B) Monocyte genotype was analyzed at 9 weeks post-transplant (wpt) from either naïve WT (CD45.1) or IL-27R-/- (CD45.1.2) donors (top) or from infected donors (bottom), including a 1:1 mix of infected WT and IL-27R-/- donors. Statistical significance was tested by Welch’s t-test.