Extended Data Fig. 6: Vcam1 “don’t-eat-me” activity is partially regulated by PIR-B.
From: VCAM1 confers innate immune tolerance on haematopoietic and leukaemic stem cells
(a) Vcam1 is efficiently depleted in Vcam1Mx1-Cre bone marrow HSCs, as seen by FACS (n = 3 biological replicates), 3 weeks after the first poly I:C (pIpC) injection. PolyI:C was administered intraperitoneally (i.p.) every other day at 5 mg/kg to a total of 3 doses. (b) Outline of competitive bone marrow transplantation experimental strategy. (c) Quantification of long-term reconstituting HSCs from syngeneic Vcam1fl/fl and Vcam1Mx1-Cre mice after poly I:C treatment by competitive reconstitution assay in the blood (Vcam1fl/fl n = 5, Vcam1Mx1-Cre n = 3 biological replicates). (d) Phospho-flow analysis of tyrosine phosphorylation (P-TYR) levels in host CD45.1+ PIR-B+ phagocytic cells transplanted with syngeneic and haplotype-mismatched CD45.2 Vcam1fl/fl and Vcam1Csf1r-iCre cells, at day 6. P-TYR levels are represented as median fluorescent intensity (MFI) normalised to the basal P-TYR levels of phagocytic cells in Pirb-/- mice (n = 5 biological replicates). (e) Donor haematopoietic cell engraftment following competitive reconstitution from syngeneic and haplotype mismatch Vcam1fl/fl and Vcam1Csf1r-iCre cells into Pirb-/- recipients (n = 6 biological replicates). (f, g) Donor haematopoietic reconstitution of syngeneic Vcam1fl/fl and Vcam1Vav-iCre cells following competitive reconstitution assays into (f) Pirb-/- (Vcam1fl/fl n = 6, Vcam1Vav-iCre n = 8 biological replicates) or (g) wild-type mice (Vcam1fl/fl n = 7, Vcam1Vav-iCre n = 9 biological replicates), 1-week post-transplantation. Donor cell engraftment was evaluated by detecting the levels of donor male DNA in the female recipient blood, by real-time PCR (e-g). (h) Quantification of the in vitro phagocytosis of syngeneic and haplotype-mismatched control and Vcam1-null Lineage– cells incubated with freshly sorted Pirb-/- phagocytes (macrophages n = 4, Gr1high monocytes n = 5 biological replicates). Error bars, mean ± s.e.m. Box plots: media, whiskers: minimum and maximum. Unpaired two-tailed student’s t test (a, c, f, g) and one-way ANOVA analyses followed by Tukey’s multiple comparison tests (d, e, h). Significant P values are indicated in the figure.
