VCAM1 Confers Innate Immune Tolerance on Hematopoietic and Leukemic Stem Cells

Hematopoietic stem cells (HSCs) home to the bone marrow (BM) via, in part, the interactions with Vascular Cell Adhesion Molecule-1 (VCAM1). Upon migrating into the BM, HSCs are vetted by perivascular phagocytes to ensure their self-integrity. Here, we show that VCAM1 is also expressed on healthy Lineage⁻ c-Kit⁺ Sca1⁺ CD48⁻ CD150⁺ HSCs and upregulated on acute myeloid leukemia (AML) cells where it serves as a quality-control checkpoint for entry into BM by providing 'don't-eat-me' stamping in the context of major histocompatibility complex (MHC) class-I presentation. Using a line in which HSCs are hemizygous for MHC (H-2^b/H-2^q) and Vcam1 can be conditionally deleted by breeding Vcam-1 floxed (Vcam1^fl/fl) mice with a Csf1r-iCre transgenic line (referred to as Vcam1^cKO), we find that Vcam1 deletion in HSCs leads to striking defects in hematopoietic recovery when donor and recipients are mismatched (donor H-2^b/q; recipient H-2^b). Remarkably, wild-type donor BM cells with haplotype-mismatched genotype exhibit engraftment and survival similar to their syngeneic (H-2^b) Vcam1^fl/fl (control) and Vcam1^cKO counterparts. In vivo phagocytic assays show that Vcam1^cKO;H-2^b/q HSCs are phagocytosed by host H-2^b immune phagocytes whereas Vcam1-sufficient H-2^b/q HSCs are not targeted. Clearance is not dependent on irradiation-induced damage since the same phenotype is observed in parabiotic mice where Vcam1^cKO;H-2^b/q HSCs are not able to engraft their wild-type (H-2^b) parabiont partner. Mechanistically, we show that MHC-mismatched HSCs are recognized at least in part by paired Ig-like receptor-B (PIR-B), a negative regulator of immune cells upon recognition of MHC-I molecules, expressed on α4β1⁺ murine phagocytic myeloid cells. Transplantation assays revealed that in absence of PIR-B inhibitory signals, Vcam1 deletion leads to significant reductions (~60%) in the early engraftment of syngeneic Vcam1^cKO;H-2^b/b cells compared to Vcam1^fl/fl;H-2^b/b cells, suggesting that the absence of VCAM1 also promotes cell clearance by syngeneic Pirb^-/- phagocytes. That VCAM1 expression can provide innate immune tolerance indicates that this pathway may be of use for cancer cells. Indeed, alterations in VCAM1 expression has been reported in various cancer cell types, including AML. To evaluate the effect of genetic Vcam1 deletion and MHC-mismatch on AML progression, we have transduced Vcam1^cKO;H-2^b/q and Vcam1^fl/fl;H-2^b/q cells with the pMSCV-MLL-AF9-GFP oncogene. Strikingly, analysis of primary AML recipient BM reveals a marked reduction (>99%) of phenotypic Vcam1^cKO leukemic stem cells compared to Vcam1^fl/fl control. Accordingly, the survival of secondary AML recipient mice is significantly prolonged in mice harboring Vcam1^cKO AML cells relative to Vcam1^fl/fl AML. Leukemia progression is markedly enhanced by phagocyte depletion (using clodronate liposomes), and the engraftment defect of Vcam1^cKO AML cells is completely rescued, indicating an important role for phagocytes in the establishment of Vcam1-deficient AML. Analysis of The Cancer Genome Atlas (TCGA) database reveals that high VCAM1 expression is associated with the poor prognosis of patients with AML (P=0.03). In addition, VCAM1 is significantly overexpressed in Lineage⁻ CD34⁺ CD38⁻ CD90⁻ short-term repopulating HSCs from sorted human AML BM samples. VCAM1 overexpression in human AML MOLM13 cancer cells promotes disease progression and significantly (P=0.005) shorter survival. Furthermore, VCAM1 blockade using novel monoclonal antibodies after implantation of primary human AML into immunodeficient mice blunts disease progression, and extends survival of mice. Altogether, these results suggest that VCAM1 engagement serves as a quality-control checkpoint for entry into BM by providing 'don't-eat-me' stamping, allowing HSCs to migrate and engraft, and offers a novel strategy to eliminate cancer cells via modulation of the innate immune tolerance.