CD97 promotes spleen dendritic cell homeostasis through the mechanosensing of red blood cells

Abstract

Dendritic cells (DCs) are crucial for initiating adaptive immune responses. However, the factors that control DC positioning and homeostasis are incompletely understood. We found that type-2 conventional DCs (cDC2s) in the spleen depend on Gα₁₃ and adhesion G protein-coupled receptor family member-E5 (Adgre5, or CD97) for positioning in blood-exposed locations. CD97 function required its autoproteolytic cleavage. CD55 is a CD97 ligand, and cDC2 interaction with CD55-expressing red blood cells (RBCs) under shear stress conditions caused extraction of the regulatory CD97 N-terminal fragment. Deficiency in CD55-CD97 signaling led to loss of splenic cDC2s into the circulation and defective lymphocyte responses to blood-borne antigens. Thus, CD97 mechanosensing of RBCs establishes a migration and gene expression program that optimizes the antigen capture and presentation functions of splenic cDC2s.

Fig. 1.. Gα₁₃-ArhGEF1 signaling pathway is required in splenic cDC2s.

(A and B) Representative (A) flow cytometry profiles and (B) frequencies of DCIR2⁺CD8⁻ cDC2s in total B220⁻CD11c^hiI-Ab^hi DCs and (bottom) total splenocytes in Arhgef1^−/−, Cd11c-cre Gna13^fl/fl (labeled as Gna13^cKO), Arhgef1^−/− Cd11c-cre Gna13^fl/fl (labeled as Arhgef1^−/−Gna13^cKO or dKO), and control mice. Data are pooled from three independent experiments. (C) Representative distribution patterns of DCIR2⁺ cDC2s (red) relative to B cells [immunoglobulin D (IgD), blue] in spleens of mice of the indicated genotypes. Scale bar, 200 μm. Sections are representative of multiple cross sections from at least three mice of each type. (D to F) Mixed (50:50) BM chimeras were made with CD45.1 WT (Arhgef1^+/+) and CD45.2 Arhgef1^+/− or Arhgef1^−/− BM cells. (D) Representative flow cytometry profiles show gating strategies. (E) Equation for calculating the competitive competencies of CD45.2⁺ (gated as CD45.1⁻) population. (F) Plots showing CD45.2⁺ competency values in individual chimeras for the cDC2 compartment compared to B220⁺ follicular (FO) B cells. (G) Plots showing CD45.2⁺ competency values in WT:Gna13^WT and WT:Gna13^cKO chimeras for the cDC2 compartment compared to B220⁺ follicular (FO) B cells. (H) Flow cytometry profiles and (I) frequencies of in vivo anti-CD45-PE labeled cDC2s of indicated genotyped cells in (left) WT: Arhgef1^−/− or WT: (right) Gna13^cKO and their control mixed BM chimeras. Lines connect data from same animals. (J) Frequencies of cDC2s in blood of (left) Arhgef1^−/− or (right) Gna13^cKO and their control mice. In (D) to (J), data are pooled from two independent experiments. Each symbol represents one mouse and lines denote means. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.

Fig. 2.. CD97 functions upstream of Gα₁₃-ArhGEF1 in splenic cDC2s.

(A and B) Cas9-based mutagenesis screen was performed by making chimeras reconstituted with Cas9-expressing BM cells transduced with sgRNA targeting specific genes, with Thy1.1 as a reporter. (A) Gating strategy for the screen. (B) Frequencies of cDC2s in sgRNA-Thy1.1⁺ DCs of chimeras with sgRNA targeting the indicated genes. Data are pooled from eight independent experiments. (C) Structural components of the CD97 short isoform. NTF, N-terminal fragment; CTF, C-terminal fragment; GAIN, GPCR autoproteolysis-inducing domain; GPS, GPCR proteolysis site; Stachel, putative tethered agonist peptide. Triangles indicate individual EGF domains. (D) Frequencies of cDC2s in (top) total DCs and (bottom) total splenocytes in WT mice treated with antibody to CD97 antibody or saline. (E) Frequencies of cDC2s in (top) total DCs and (bottom) total splenocytes in Adgre5^+/+ or Adgre5^−/− mice. Data are pooled from four independent experiments. (F) Representative distribution of DCIR2⁺ cDC2s (red) relative to B cells (IgD, blue) in spleens of Adgre5^+/+ or Adgre5^−/− mice. Scale bar, 200 μm. Sections are representative of multiple cross-sections from at least three mice of each type. (G and H) Mixed (50:50) BM chimeras were made with CD45.1 WT and CD45.2 Adgre5^+/+ or Adgre5^−/− BM cells. (G) The plots show CD45.2⁺ competency values in individual chimera for the cDC2 compartment compared with (left) total DCs or (right) B220⁺ follicular B (FO) cells. (H) Frequencies of in vivo anti-CD45-PE labeled cDC2s of indicated genotyped cells in mixed BM chimeras. (I) Frequencies of cDC2s in blood of Adgre5^−/− and control mice. Artery means blood was collected from celiac artery near splenic artery. Vein means blood was collected from portal vein after ligation of superior mesenteric vein. (J) BM chimeras were reconstituted with Adgre5^+/+ or Adgre5^−/− BM cells transduced with a retroviral construct encoding Adgre5 (1,2,4) or its mutants or empty vector, with Thy1.1 as a reporter. (Left) Representative histogram plots of surface CD97 on Thy1.1⁺ cDC2 cells in chimeras reconstituted as indicated. (Right) Color coding of histograms is as labeled in graph. Gray indicates isotype control. Frequencies of cDC2s in Thy1.1⁺ or Thy1.1⁻ DCs of chimeras reconstituted as indicated. In (G) to (J), data are pooled from two independent experiments. (K) Frequencies of cDC2s in sgRNA-Thy1.1⁺ or Thy1.1⁻ DCs of chimeras reconstituted with indicated genotyped BM cells transduced with sgRNA targeting Adgre5 or control. One of two independent experiments with similar results is shown. In (B), (D), (E), (G), and (I), each symbol represents one mouse and lines denote means. In (H), (J), and (K), lines connect data from same animals. * P<0.05; ** P<0.01; **** P<0.0001.

Fig. 3.. CD55 on RBCs is required for splenic cDC2 homeostasis.

(A) Frequencies of cDC2s in (left) total DCs and (right) total splenocytes in WT mice treated with antibody to CD55 or saline. (B) Frequencies of cDC2s in blood of WT mice 8 hours after treatment with antibody to CD55 or saline. (C) Frequencies of cDC2s in (left) total DCs and (right) total splenocytes in Cd55^+/+ or Cd55^−/− mice. (D) Frequencies of cDC2s in blood of Cd55^−/− and control mice. (E) Representative histogram plots of surface CD55 on cDC2s, cDC1s, and B cells in mice with indicated genotype. (F) Frequencies of cDC2s in total DCs in chimeras as indicated. (G) Frequencies of cDC2s in total DCs in chimeras reconstituted with 90% μMT (IgM null) and 10% Cd55^+/+ or Cd55^−/− BM cells. (H) Frequencies of cDC2s in total DCs in chimeras reconstituted with 90% Tcrb^−/− and 10% Cd55^+/+ or Cd55^−/− BM cells. (I) Frequencies of cDC2s in total DCs in chimeras reconstituted with 90% Rag1^−/− and 10% Cd55^+/+ or Cd55^−/− BM cells. In (A) to (I), data are pooled from two independent experiments. (J) Representative histogram plots of surface CD55 on RBCs in mice with indicated genotype. (K to M) RBC transfusions were performed from Cd55^−/− or Cd55^+/+ mice into the indicated recipient mice, and analysis was after 6 to 8 days. (K) Representative profile of purified Cd55^+/+ RBCs or Cd55^−/− RBCs transfused into Cd55^−/− or Cd55^+/+ recipients. (L) Frequencies of cDC2s in total DCs in mice with purified RBCs transfused as indicated. Data are pooled from three independent experiments. (M) Distribution of DCIR2⁺ cDC2s (red) and B cells (IgD, blue) in spleens as indicated. Scale bar, 200 μm. Representative of multiple cross sections from at least three mice of each type. (N) Frequencies of cDC2s in total DCs in Mpl^+/+ or Mpl^−/− mice. Data are pooled from two independent experiments. Each symbol indicates one mouse, and lines denote means. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.

Fig. 4.. CD55-mediated CD97 NTF extraction is dependent on shear stress.

(A) Representative (left) histogram and (right) geometric mean fluorescence intensity (MFI) of surface CD97 expression on cDC2s in mice with blood transfusion as indicated. Data are pooled from three independent experiments. (B) MFI of surface CD97 on in vivo PE labeled (PE⁺) and PE non-labeled (PE⁻) cDC2s in WT mice that had been treated 8 hours earlier with antibody to CD55 or saline. Data are pooled from two independent experiments. (C) Chimeras were reconstituted with Adgre5^−/− BM transduced with Adgre5 (1,2,4)-T419G mutant, with Thy1.1 as a reporter. MFI of surface CD97 on in vivo PE labeled (PE⁺) and PE non-labeled (PE⁻) Thy1.1⁺ cDC2s in chimeras that had been treated 8 hours earlier with antibody to CD55 or saline. One of two independent experiments with similar results is shown. (D) MFI of surface CD97 expression on transferred Cd55^−/− cDC2s in blood collected from heart and distal ligated IVC of recipients as indicated (movie S1). Data are pooled from two independent experiments. (E and F) Splenocytes from (E) Cd55^−/− mice or (F) CD97 T419G expressing BM chimeras were cocultured for 1 hour at RT with Cd55^−/− or Cd55^+/+ RBCs, on a shaker or not. Representative (left) histogram and (right) MFI of surface CD97 expression on cDC2s are shown. Data are pooled from two independent experiments. In (A), (E), and (F), color coding of histograms is as for graphs, and gray lines indicate isotype control. In (A) to (C), (E), and (F), each symbol indicates one mouse, and lines denote means. In (D), lines connect data from the same animal. * P<0.05; *** P<0.001; **** P<0.0001.

Fig. 5.. CD97 pathway deficiency leads to increased F-actin and cDC2 motility.

(A and B) Representative (left) histogram and (right) MFI of F-actin in cDC2s from (A) Gna13^cKO or (B) Adgre5^−/− and control mice. Data are pooled from two independent experiments. (C and D) Representative (C) histogram and (D) MFI of F-actin expression on in vivo PE labeled (PE⁺) and PE non-labeled (PE⁻) cDC2s in WT mice treated for 8 hours with antibody to CD55 or saline. Data are pooled from two independent experiments. (E and F) GSEA of Mrtfa^−/− down-regulated genes compared with Adgre5^−/− or Arhgef1^−/− cDC2 dataset. (E) Enrichment profiles for genes that are down-regulated (P_adj< 0.001) in Mrtfa^−/− BMDCs (29) compared with (top) Adgre5^−/− and (bottom) Arhgef1^−/− cDC2 datasets. (F) Genes in the core enrichment, presented as a cluster-analyzed heatmap of expression levels of Adgre5^+/+ and Adgre5^−/− cDC2 cells. (G to I) The motility of splenic cDC2 was observed with intravital two-photon microscopy in Arhgef1^+/− Batf3^−/− Cd11c-YFP and Arhgef1^−/− Batf3^−/− Cd11c-YFP chimeras. Dextran-TRITC was used to label large blood vessels. (G) Example of migratory cDC2 and leaving cDC2 (white arrowhead and dashed track, respectively) in Arhgef1^+/− Batf3^−/− CD11c-YFP or Arhgef1^−/− Batf3^−/− CD11c-YFP chimera spleen (movies S2 and S3). Time indicated in min:sec. Scale bar, 20 μm. (H) Frequencies of migratory cells (cDC2 with a minimum track length of 30 μm) in chimeras as indicated. (I) Frequencies of “leaving cells” (cDC2 that enter large vessels) in chimeras as indicated (movies S5 to S8). Each symbol indicates one movie in the supplementary materials. Lines denote the means. Data are pooled from five independent experiments. (J) Frequencies of YFP⁺ DCs in blood of Arhgef1^−/− Batf3^−/− and control chimeras. Data are pooled from two independent experiments. (K to N) Spleens from CD45.1 WT:CD45.2 Gna13^cKO or CD45.1 WT:CD45.2 Gna13^WT mixed chimeric mice were transplanted into CD45.1/2 WT recipients. The percentages of donor derived B cells and cDC2s from the recipient blood and transplanted spleens were examined 8 to 9 hours after surgery. (K) Representative flow cytometry profiles show gating strategies. (L) Equation for calculating the migration competencies of CD45.2⁺ population into blood. (M) Plots showing CD45.2⁺ competency values in individual chimera for cDC2s leaving spleen into blood. (N) Plots showing CD45.2⁺ competency values in individual chimera for B cells leaving spleen into blood. Data are pooled from three independent experiments. Each symbol indicates one mouse. Lines denote the means. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.

Fig. 6.. Functional defects of CD97 pathway deficient splenic cDC2s.

(A and B) Mixed BM chimeras (WT: Adgre5^+/+ or WT: Adgre5^−/−) were analyzed 3 hours after PKH26-labeled SRBC injection. (A) Representative (left) flow cytometry plot and (right) frequencies of PKH26 dye-positive cDC2s. (B) Representative (left) histogram and (right) MFI of surface CCR7 expression on cDC2s from chimeras. (C to E) CellTrace Violet (CTV)-labeled OT-II T cells were transferred into Gna13^cKO and Gna13^WT mice. Mice were analyzed 3 days after SRBC-OVA immunization. [(C and D)] Representative (C) flow cytometry plot of proliferation and (D) frequencies of OT-II cells in total splenocytes. (E) Frequencies of ICOS^hiPD-1^hi T cells among transferred OT-II T cells. (F) Frequencies of GC B cells in (left) Hy10⁺ B cells and (right) frequencies of Hy10⁺ GC B cells in splenocytes in Gna13^cKO and control mice. (G) Frequencies of GC B cells in splenic IgD⁻B220⁺ B cells in Adgre5^−/− and control mice after HKLM immunization. (H) Antibodies to RBC in the sera of Gna13^cKO and control mice 3 weeks after transfusion of HEL-OVA-conjugated mouse RBCs. In (D) to (H), each symbol indicates one mouse, and lines denote means. Data are pooled from [(A to E)] two, (F) three, or [(G and H)] four independent experiments. In (A) and (B), lines connect data from same animal. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.

Fig. 7.. CD97 action downstream of Irf4 in splenic cDC2s.

(A to E) Mixed (50:50) BM chimeras were made with CD45.1 WT (Irf4^WT) and CD45.2 Irf4^WT or Cd11c-cre Irf4^fl/fl (labeled as Irf4^cKO) BM cells. [(A and B)] Frequencies of (A) cDC2s in DCs and (B) in vivo PE-labeled cells in cDC2s in each compartment of the indicated chimeras. (C) Frequencies of cDC2s in blood of Irf4^cKO and control mice. [(D and E)] Representative (left) histogram and (right) MFI of (D) F-actin expression and (E) surface CD97 expression on cDC2s in each compartment of the mixed chimeras. (F and G) BM chimeras were reconstituted with Irf4^WT or Irf4^cKO BM cells transduced with Adgre5 (1,2,4) or empty vector, with Thy1.1 as a reporter. (F) Representative histogram plots of surface CD97 on Thy1.1⁺ and Thy1.1⁻ cDC2s and (G) frequencies of cDC2s in Thy1.1⁺ or Thy1.1⁻ DCs of chimeras reconstituted as indicated. In (A) to (E), each symbol indicates one mouse, and lines denote means. In each summary graph, data are pooled from two independent experiments. In (G), lines connect data from same animal. * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001.