Abstract
Haematopoietic stem cells (HSCs) home to the bone marrow via, in part, interactions with vascular cell adhesion molecule-1 (VCAM1)1,2,3. Once in the bone marrow, HSCs are vetted by perivascular phagocytes to ensure their self-integrity. Here we show that VCAM1 is also expressed on healthy HSCs and upregulated on leukaemic stem cells (LSCs), where it serves as a quality-control checkpoint for entry into bone marrow by providing ‘don’t-eat-me’ stamping in the context of major histocompatibility complex class-I (MHC-I) presentation. Although haplotype-mismatched HSCs can engraft, Vcam1 deletion, in the setting of haplotype mismatch, leads to impaired haematopoietic recovery due to HSC clearance by mononuclear phagocytes. Mechanistically, VCAM1 ‘don’t-eat-me’ activity is regulated by β2-microglobulin MHC presentation on HSCs and paired Ig-like receptor-B (PIR-B) on phagocytes. VCAM1 is also used by cancer cells to escape immune detection as its expression is upregulated in multiple cancers, including acute myeloid leukaemia (AML), where high expression associates with poor prognosis. In AML, VCAM1 promotes disease progression, whereas VCAM1 inhibition or deletion reduces leukaemia burden and extends survival. These results suggest that VCAM1 engagement regulates a critical immune-checkpoint gate in the bone marrow, and offers an alternative strategy to eliminate cancer cells via modulation of the innate immune tolerance.
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Data availability
Previously published data that were re-analysed here are available under accession codes GSE1035842 and GSE35008/GSE35010 (refs. 44,45). Human AML survival data were derived from the TCGA Research Network (http://cancergenome.nih.gov/) and re-analysed via the cBioPortal for Cancer Genomics from the Memorial Sloan Kettering Cancer Center (https://cbioportal.mskcc.org). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank J. W. Pollard for providing Csf1r-iCre mice, T. Papayannopoulou for providing Vcam1floxed mice, T. Takai and M. Rothenberg for providing Pirb−/− mice and S. A. Armstrong for providing the MLL-AF9 construct. We thank C. Prophete, C. Cruz, P. Ciero, G. Amatuni and A. Landeros for technical assistance, the University of Illinois and the Einstein Flow Cytometry Core Facility for cell sorting assistance, U. Steidl, A. Zahalka and K. Chronis for scientific discussions, and S. V. Buhl and M. D. Scharff of the Macromolecule Therapeutics Core at Einstein for technical assistance and guidance with VCAM1 mAb generation. S.P. and M.M. were supported by a New York Stem Cell Foundation-Druckenmiller Fellowship, J.C.B. by a Pew Latin America Fellowship and CONACYT (México), H.P. by a Training Program in Cellular and Molecular Biology and Genetics (T32 GM007491), D.K.B. by a NIH training grant (T32GM007288) and F.N. by the Japanese Society for the Promotion of Science. We thank the NIH (DK056638, HL069438, HL116340), the Leukemia and Lymphoma Society (LLS-TRP 6475-15) and the New York State Department of Health (NYSTEM IIRP C029570 and C029154) for support of the P.S.F. laboratory and the Cancer Center, University of Illinois Cancer Biology Targeted Grant for support of S.P. During the resubmission of this manuscript Dr Paul S. Frenette passed away on 26 July 2021.
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Authors and Affiliations
Contributions
S.P. designed the study, performed most of the experiments and analysed the data. Q.W. generated mice, provided assistance with experiments and interpreted data. M.M. provided assistance and expertise with AML experiments. D.Z. provided assistance with hVCAM1-MOLM-13 survival experiments. J.C.B. performed in vitro phagocytosis experiments. H.P. performed the parabiosis experiments. F.N. performed the in vitro colony forming assay and generated the hVCAM1-MOLM-13 line. A.D.S. performed FACS experiments for VCAM1 and PIR-B expression. B.A.B. performed the human GSE10358 data analysis. J.X. analysed the TCGA database for human AML. D.K.B. provided assistance with PIR-B and β2m−/− experiments. A.V. provided samples from patients with AML and human AML expertise. P.S.F. supervised and obtained funding for the study. S.P. and P.S.F. wrote the manuscript. All authors discussed the results and commented on the manuscript.
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Competing interests
S.P., Q.W. and P.S.F. are co-inventors on a patent application using anti-VCAM1 antibodies (patent W02017205560A1). P.S.F. serves as a consultant for Pfizer, has received research funding from Ironwood Pharmaceuticals and is a shareholder of Cygnal Therapeutics. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 VCAM1 is expressed on HSCs and progenitor cells.
(a, b) Gating strategies for the analyses of HSC and progenitor populations. (c) Representative histograms of VCAM1 expression levels in the populations represented. (d) Percentage of VCAM1 positive cells within progenitor cell populations from the bone marrow (BM) and spleen (n = 3 biological replicates). (e) FACS analysis of the BM and spleen of Csf1r-iCre;loxp-TdTomato transgenic mice showing the recombination efficiency of Csf1r-iCre in phagocytes, HSC and MPP (n = 3 biological replicates). Error bars, mean ± s.e.m. LMPP (lymphoid primed MPPs, LSK Flt3+); GMP (granulocyte macrophage progenitors, Lineage− c-Kit+ Sca1− CD34+ FcγRII/III+); CMP (common myeloid progenitors, Lineage− c-Kit+ Sca1− CD34+ FcγR-); MEP (megakaryocytic erythroid progenitors, Lineage−c-Kit+ Sca1− CD34− FcγR-) and CLP (common lymphoid progenitors, Lineage− c-Kitlow Sca1low Flt3+ IL7Rα+).
Extended Data Fig. 2 Vcam1-deficient HSCs exhibit normal viability, cell cycle and proliferation.
(a) Outline of experimental strategy. (b) Survival curve of recipient mice given lethal radiation and transplanted with 2 million BM nuclear cells (BMNCs) from Vcam1fl/fl (Control, n = 7) and Vcam1Csf1r-iCre (n = 8) mice, non-competitive transplantation. (c) Donor engraftment following secondary competitive reconstitution assay of BM from mice that survived the primary transplantation shown in (b) (Vcam1fl/fl n = 4; Vcam1Csf1r-iCre n = 5 mice). (d) Outline of experimental strategy. (e) Donor engraftment following competitive reconstitution assay from Vcam1fl/fl (n = 9) and Vcam1Csf1r-iCre (n = 10) mice. (f) Contribution of 200 sorted DAPI– LSK CD48– CD150+ VCAM1+ and VCAM1– HSCs to peripheral blood following competitive reconstitution. (n = 6 biological replicates) (g) Absolute number of BMNCs, and (h) HSCs and MPPs per femur in Vcam1fl/fl and Vcam1Csf1r-iCre mice (n = 5 biological replicates). (i) Colony output on day 7 of BM colony-forming unit in culture from Vcam1fl/fl and Vcam1Csf1r-iCre mice. GEMM: granulocyte, macrophage, erythroid and megakaryocyte; GM: granulocyte and macrophage; M: macrophage; G: granulocyte; BFU-E: erythroid (n = 3 biological replicates). (j) Concentration of white blood cells (WBC), erythrocytes (RBC) and platelets (PLTs) in the blood of Vcam1Csf1r-iCre mice as compared to littermate Vcam1fl/fl (n = 12 biological replicates). (k) Percentage of viable (Annexin V− DAPI−) HSC and MPP in the BM of Vcam1fl/fl and Vcam1Csf1r-iCre mice. (n = 3 biological replicates) (l) Cell cycle analysis, using anti-Ki67 and Hoechst 33342 staining, of HSCs from Vcam1fl/fl and Vcam1Csf1r-iCre mice (n = 4 biological replicates). Error bars, mean ± s.e.m. Box plots: media, whiskers: minimum and maximum. Log-rank analysis was used for the Kaplan-Meier survival curves in (b). Unpaired two-tailed student’s t test (c-l). Significant P values are indicated in the figure.
Extended Data Fig. 3 The distribution of HSCs in the mouse BM is not altered after Vcam1 deletion in Csf1r-iCre+ cells.
(a) Representative whole−mount images of the sternal BM of Control and Vcam1Csf1r-iCre mice and magnified high power view. The dashed outline denotes bone-BM border. Arterioles (Art) are identified by CD31+ CD144+ Sca1+ expression. Phenotypic HSCs are identified by Lineage- CD41− CD48− CD150+ expression, and megakaryocytes (Mk) are distinguished by their size, morphology and CD41+ CD150+ expression. Representative images of n = 8 independent sternum segments (b, c) Localization of HSCs relative to (b) arterioles and (c) Mks in Vcam1fl/fl and Vcam1Csf1r-iCre mice. (n = 104 HSCs in Vcam1fl/fl, n = 90 HSCs in Vcam1Csf1r-iCre) (d) Number of BMNCs, MPP and HSC per femur in Vcam1fl/fl and Vcam1Csf1r-iCre mice after 5-FU injection (Vcam1fl/fl day 0 n = 5, day 5 n = 2, day 8-20 n = 3, day 25 n = 4; Vcam1Csf1r-iCre day 0 n = 5, day 5-15 n = 2, day 20-25 n = 4 biological replicates). (e) Spleen cellularity, and (f) number of HSCs and MPPs per spleen in Vcam1fl/fl and Vcam1Csf1r-iCre mice (n = 5 biological replicates). Error bars, mean ± s.e.m. Two-sample Kolmogorov–Smirnov tests were used for comparisons of distribution patterns in (b) and (c). Unpaired two-tailed student’s t test (d,e,f). Significant P values are indicated in the figure.
Extended Data Fig. 4 VCAM1 is a “don’t-eat-me” signal.
(a) Contribution of 300 sorted DAPI– LSK CD48– CD150+ Vcam1+ (n = 8 biological replicates) or Vcam1– (n = 7 biological replicates) HSCs to peripheral blood following competitive reconstitution. (b) Quantification of tri-lineage (myeloid, B lymphoid, and T lymphoid cells) engraftment 16 weeks post-transplantation. Vcam1+ (n = 8 biological replicates), Vcam1– (n = 7 biological replicates). (c) Representative BM FACS plots showing donor HSC contribution to recipient CD45.2+ LSK CD48– CD150+ Vcam1+ and Vcam1– HSCs compartment, at 16 weeks from the mice analysed in (a) and (b). (d) Absolute number of donor (CD45.2) Vcam1fl/fl and Vcam1Csf1r-iCre cells that homed to the BM of lethally irradiated CD45.1 recipients, 3 hours after injection (n = 5 biological replicates). (e) Representative FACS plots of the in vivo phagocytosis assay in Fig. 1e. (f) Outline of experiment strategy. Lethally irradiated CD45.1 recipients were transplanted competitively with 2 million of Vcam1Csf1r-iCre or Vcam1fl/fl BMNCs. At day 6 the BM of recipient mice were analysed by FACS and the number of host phagocytic Gr1high monocytes, Gr1low monocytes and macrophages quantified. (n = 4 mice). Error bars, mean ± s.e.m. Box plots: media, whiskers: minimum and maximum. Unpaired two-tailed student’s t test. Significant P values are indicated in the figure.
Extended Data Fig. 5 Csf1r-iCre transgene is genetically linked to the MHC locus.
(a) Representative FACS plots of DAPI− BMNCs cells from the same H-2b/q MHC-haplotype heterozygous mouse. Cells were stained with antibodies against MHC-I and II subclasses corresponding to the H-2b (C57BL/6 strain) and H-2q (FVB/N strain) haplotypes. (b) Calculation of the frequency of recombination between the Csf1r-iCre transgene and the MHC locus. (c) Quantification of tri-lineage (myeloid, B cell and T cell) engraftment in the blood of mice analyzed in Fig. 2b-d (Vcam1fl/fl;H-2b/b (n = 7); Vcam1Csf1r-iCre;H-2b/b (n = 6); Vcam1fl/fl;H-2b/q (n = 12); Vcam1Csf1r-iCre;H-2b/q (n = 10 biological replicates)). (d) Quantification of the in vitro phagocytosis of syngeneic and haplotype-mismatched control and Vcam1-null Lineage– cells incubated with freshly sorted phagocytes (Macrophages n = 6, Gr1high Monocytes n = 5 biological replicates). (e-f) Recipient mice were treated with a monoclonal anti-CD8 antibody or IgG control, lethally irradiated, and transplanted with 1 million BMNCs from haplotype-mismatched control Vcam1fl/fl or Vcam1Csf1r-iCre mice. (e) Representative FACS plots and percentage of T cell populations in the peripheral blood of mice treated with anti-CD8 antibody or IgG control before transplantation. (f) Survival curves of recipient mice depleted of CD8+ T cells and BM transplanted (n = 5 biological replicates). Error bars, mean ± s.e.m. Box plots: media, whiskers: minimum and maximum. Log-rank analysis was used for the Kaplan-Meier survival curves in (f) and one-way ANOVA analyses followed by Tukey’s multiple comparison tests (c, d). Significant P values are indicated in the figure.
Extended Data Fig. 6 Vcam1 “don’t-eat-me” activity is partially regulated by PIR-B.
(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.
Extended Data Fig. 7 Loss of Vcam1 inhibits the establishment and progression of haplotype-mismatched and syngeneic MLL-AF9-induced AML.
(a) Outline of experimental strategy. (b) Representative FACS plots of BM leukaemic stem cells (LSCs) from control AML-Vcam1fl/fl (top) and AML-Vcam1Csf1r-iCre (bottom) primary recipients, 55 days after transplantation. (c) Histogram showing the presence of leukaemic VCAM1+ LSCs derived from Vcam1Csf1r-iCre mice in the BM of moribund secondary recipient mice, 103 days post-transplant. (d-e) Analysis of blood (left graph) and BM aspirates (right graph) from primary recipients transplanted with syngeneic Vcam1fl/fl or Vcam1Csf1r-iCre-transduced MLL-AF9-GFP LSKs, after 115 and 130 days. The red circle shows a leukemic mouse in which AML was derived from an escaped VCAM1+ clone. Left panel: P = 0.0153 (P = 0.003 if the VCAM1+ highlighted mouse is excluded); right panel: P = 0.1011 (P = 0.03 if the VCAM1+ highlighted mouse is excluded) (Vcam1fl/fl n = 10, Vcam1Csf1r-iCre n = 8 biological replicates) (e) Histogram showing the presence of leukaemic VCAM1+ LSCs in the BM of the sick primary recipient mouse of syngeneic Vcam1Csf1r-iCre AML, highlighted with red circle in (d). (f) MLL-AF9-GFP+ cells were incubated in the presence of anti-VCAM1 blocking antibody, isotype control or camptothecin-positive control. After 4.5 hours incubation, apoptotic cells were identified by Annexin V staining, as determined by FACS (n = 4 biological replicates). Error bars, mean ± s.e.m. Mann-Whitney tests (d) and one-way ANOVA analyses followed by Tukey’s multiple comparison tests (f). Significant P values are indicated in the figure.
Extended Data Fig. 8 Treatment of wild-type mice with a blocking anti-VCAM1 monoclonal antibody.
(a) Wild-type mice were daily injected with IgG control or blocking rat anti-mouse VCAM1 antibody for 5 days. Mice were analyzed 1 day after the last injection. BM cells from treated groups were incubated with an anti-rat antibody and after washing stained for phenotypic HSCs and probed for VCAM1 expression. (b) BM cellularity and (c) absolute number of HSC, MPP and LSK per femur and (d) per ml of blood in healthy C57BL/6 mice treated for 5 days with daily injections of either anti-VCAM1 or IgG control antibody (n = 5 biological replicates). (e) Peripheral blood was drawn post-treatment and hematology lab analysis was performed. White blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), platelets (PLT), neutrophils (Neut.), lymphocytes (Lymph.), reticulocytes (Retic.) (n = 5 biological replicates). (f) Colony-forming unit (CFU) in culture from the BM of wild-type mice injected with IgG1 or anti-VCAM1 antibody for 5 days. GEMM: granulocyte, macrophage, erythroid and megakaryocyte; GM: granulocyte and macrophage; BFU-E: erythroid (n = 3 biological replicates). (g) Contribution of 200 sorted DAPI– LSK CD48– CD150+ HSCs isolated from wild-type mice 1 day after IgG1 or anti-VCAM1 antibody treatment as in (a) to the peripheral blood following competitive reconstitution (n = 6 biological replicates). (h) Quantification of tri-lineage (myeloid, B lymphoid, and T lymphoid cells) engraftment from the mice in (g) (n = 6 biological replicates). (i) Survival curve of wild-type mice daily injected with IgG control or blocking anti-mouse VCAM1 antibody for 10 days. (n = 6 biological replicates). Error bars, mean ± s.e.m. Unpaired two-tailed student’s t test (b-h), and log-rank analysis was used for the Kaplan-Meier survival curves in (i). Significant P values are indicated in the figure.
Extended Data Fig. 9 Mechanisms of VCAM1 antibody inhibition in leukemogenesis.
(a) BM cellularity and (b) number of MOLM-13 cells that homed to the BM of recipient mice (n = 5 Mock, n = 4 hVCAM1 biological replicates). (c) Percentage of Annexin V+ apoptotic (n = 5 biological replicates) and (d) proliferating BrdU+ MOLM-13 cells in the BM of recipients (n = 5 biological replicates). (e) Survival analysis of mice with established MOLM-13 AML ( > 1% peripheral human CD45+ cells) and treated with daily injections (for 10 days) of IgG1 and anti-mouse Vcam1 monoclonal antibody which does not cross-react with human VCAM1. (f) Survival analysis of mice with established hVCAM1-MOLM-13 AML ( > 1% peripheral human CD45+) treated with daily injections (10 days) of anti-human VCAM1 monoclonal antibody or with PBS or clodronate liposomes. (g) Human Mock- and hVCAM1-MOLM-13 cells incubated with anti-human VCAM1 Fab fragments and with secondary antibodies specific against mouse IgG kappa light chain (left) or against mouse IgG Fc portion. (h) Survival analysis of mice with established hVCAM1-MOLM-13 AML and treated with anti-human VCAM1 monoclonal antibody, anti-human VCAM1 Fab fragments or with IgG1 for 5 days. (i) In vivo phagocytosis assay testing the effect of integrin alpha4 blocking antibody in the clearance of transplanted haplotype-mismatched cells (n = 4 biological replicates). (j) In vitro phagocytosis of Mock-MOLM-13 and hVCAM1-MOLM13 cells, in the presence of anti-alpha4, anti-beta1, anti-alpha4-beta7 blocking antibodies, compared with IgG. Values were normalized to the maximum number of events measured across technical replicates (n = 6). Error bars, mean ± s.e.m. Box plots: media, whiskers: minimum and maximum. Unpaired two-tailed student’s t test (a-d, i). Log-rank analysis was used for the Kaplan-Meier survival curves in (e, f, h). Two-way ANOVA with multiple comparisons correction (j). ns, non-significant. Significant P values are indicated in the figure.
Extended Data Fig. 10 VCAM1 is broadly expressed across human AML subgroups.
(a-b) Published gene expression microarray data from 183 patients (GSE10358) was analyzed for the expression of VCAM1 across French-American-British (FAB) subgroups of human AML. (a) VCAM1 is broadly expressed across all FAB AML subgroups with the highest expression on the M6 subgroup. (b) Association between VCAM1 expression and human AML mutations. The only statistically significant difference detected was a lower expression of VCAM1 in NPM1 mutated AML compared to the others. DNMT3A, mutation of the DNMT3A gene; FLT3-ITD, internal tandem duplication of the FLT3 gene; FLT3-TKD, tyrosine kinase domain mutation of the FLT3 gene; IDH1 and 2, mutation of the IDH1 and 2 genes; NPM1, mutation of the NPM1 gene. Box plots: media, whiskers: minimum and maximum. Mann-Whitney test (a, b). (c) VCAM1 gene expression data from sorted human AML BM samples and healthy controls (GSE35008 and GSE35010). Lineage− CD34+ CD38− CD90+ cells (referred to as LT-HSCs), Lineage− CD34+ CD38− CD90− cells (referred to as ST-HSCs), and Lineage− CD34+ CD38+ CD123+ CD45RA+ cells (referred to as GMPs). Cytogenetic abnormalities are depicted as: NK, normal karyotype; CK, complex karyotype; 7q, deletion of chromosome 7 (LT-HSCs healthy, NK, 7q n = 4, CK n = 5; ST-HSCs healthy,7q n = 6, NK, CK n = 4; GMP healthy, 7q n = 6, NK n = 4, CK n = 5 human samples). Box plots: media, whiskers: minimum and maximum. One-way ANOVA analyses followed by Tukey’s multiple comparison tests (c). (d) Representative flow-cytometry plots depicting the phagocytosis of human #V6 AML sample by human CD14+ monocytes when treated with anti-VCAM1, compared with the IgG1 control. CTFR, cell trace far red. (e) Percentage of live human CD45+ AML cells in the blood of leukaemic mice comparing pre- and post-treatment (IgG1 control (n = 10 biological replicates) or anti-human VCAM1 antibody (n = 9 biological replicates). Data from the mice in Figs. 5i and j. Significant P values are indicated in the figure.
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Pinho, S., Wei, Q., Maryanovich, M. et al. VCAM1 confers innate immune tolerance on haematopoietic and leukaemic stem cells. Nat Cell Biol 24, 290–298 (2022). https://doi.org/10.1038/s41556-022-00849-4
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DOI: https://doi.org/10.1038/s41556-022-00849-4