Bone Marrow Reconstitution Research Articles

From the Literature

From the Literature

Autoimmune responses are dependent on the MAVS innate pathway due to its natural co-stimulating effect on B cell activation and maturation

Abstract There is evidence that autoimmune disease might be induced by deficient regulation of innate immune pathways that normally induce type I interferon in response to pathogen-derived nucleic acids. To investigate a potential role for nucleic acid sensing pathways with cytosolic localization and more ubiquitous expression, we tested the requirement for MAVS (dsRNA-sensing) or STING (dsDNA-sensing) in the lupus autoimmune disease developed by FcγRIIB-KO (R2−/−) mice. These experiments revealed that STING was mildly protective, while MAVS was an essential factor for autoimmune disease in R2−/− mice. We found that MAVS deficiency, but not type I IFN receptor deficiency, completely eliminated autoantibodies and autoimmune pathology in R2−/− mice. MAVS deficiency also eliminated spontaneous activation of B and T cells, germinal center formation and follicular helper T cell development. Using mixed bone marrow reconstitution experiments, we determined that MAVS was required for autoreactivity in a B cell intrinsic manner, with a profound effect on B cell survival in the germinal center. MAVS deficiency did not alter BCR-proximal signaling, but resulted in diminished long term proliferative capacity of B cells, with lower cyclin D2 and Bcl-xL expression 24 hours after anti-IgM stimulation. MAVS deficiency also resulted in B cell developmental defects, fewer total B cells but a skewing toward marginal zone and against the follicular B cell program. Overall MAVS seems to provide its costimulatory effect independent of type I IFN, by enhancing the activation of NFκB in situations where T cell help or other modes of costimulation are not present and most importantly, by providing survival stimulus for autoreactive B cells.

Identification of a suppressor of autoimmune phenotype that affects deoxynucleotide synthesis in T cells

Abstract FcγRIIB-deficient mice represent a well-characterized animal model of systemic lupus erythematosus. They develop spontaneous anti-nuclear antibodies (ANA) and fatal glomerulonephritis when on the C57BL/6 (B6) background while the same mutation on the BALB/c background is phenotypically benign, indicating differences in lupus susceptibility between the BALB/c and B6 strains. We mapped the BALB/c derived protection to a 151KB genomic fragment on chromosome 12. Insertion of this fragment in transgenic form (A12Tg) results in protection from lupus disease. Mixed bone marrow reconstitution experiments pointed to a T cell intrinsic effect of the protective gene in the BALB/c A12 interval. This region contains a single gene, Rrm1, which encodes for a subunit of the ribonucleotide reductase responsible for the synthesis of deoxiribonucleotides. We hypothesize that reduced levels of this enzyme affect the ability of T cell to expand upon activation and that is why allelic differences in Rrm2 expression can alter tolerance in mouse models of lupus.

Bone marrow architecture reconstitution after hematopoietic grafting: A cross-sectional study

Abstract Objective Changes in bone marrow (BM) architecture and its remodeling after hematopoietic stem cell transplantation (HSCT) following a reduced-intensity conditioning regimen for treating malignant hematologic diseases are poorly documented. We assessed these changes and their correlation with the clinical course to investigate the dynamics of this process. Material and methods Hematology patients who received a reduced intensity conditioning (RIC) were followed after successful allogeneic or autologous HSCT. Patients had at least 100 days after hematopoietic grafting. Post-transplant BM architecture was assessed by expert examination of a trephine biopsy using age-adjusted scores and histopathology results were contrasted with clinical post-transplant evolution to determine meaningful associations. Results Twenty-seven HSCT recipients, 19 allogeneic and 8 autologous, were studied at a median 155 (100–721) days post-transplant. Eleven (40.7%) had hypocellular biopsies with around 50% of the expected cell composition for a normal individual at that age. Patients with increased fibrosis had lower peripheral white blood cell counts compared to those with normal reticulin distribution (p = 0.015). A decrease in overall survival (OS) was documented in the group with more severe myelofibrosis at a median 813 days of follow-up. Infectious complications were more frequent in patients receiving an allogeneic (n = 6) compared with the recipients of an autologous transplant (n = 0). Conclusions Despite normal peripheral blood count and composition, significant post-transplant hypocellularity, revealing incomplete bone marrow reconstitution, was documented after HSCT in patients conditioned with a RIC regimen. Severe BM fibrosis was associated to decrease OS.

The antiproliferative ELF2 isoform, ELF2B, induces apoptosis in vitro and perturbs early lymphocytic development in vivo

BackgroundELF2 (E74-like factor 2) also known as NERF (new Ets-related factor), a member of the Ets family of transcription factors, regulates genes important in B and T cell development, cell cycle progression, and angiogenesis. Conserved ELF2 isoforms, ELF2A, and ELF2B, arising from alternative promoter usage can exert opposing effects on target gene expression. ELF2A activates, whilst ELF2B represses, gene expression, and the balance of expression between these isoforms may be important in maintaining normal cellular function.MethodsWe compared the function of ELF2 isoforms ELF2A and ELF2B with other ELF subfamily proteins ELF1 and ELF4 in primary and cancer cell lines using proliferation, colony-forming, cell cycle, and apoptosis assays. We further examined the role of ELF2 isoforms in haemopoietic development using a Rag1-/-murine bone marrow reconstitution model.ResultsELF2B overexpression significantly reduced cell proliferation and clonogenic capacity, minimally disrupted cell cycle kinetics, and induced apoptosis. In contrast, ELF2A overexpression only marginally reduced clonogenic capacity with little effect on proliferation, cell cycle progression, or apoptosis. Deletion of the N-terminal 19 amino acids unique to ELF2B abrogated the antiproliferative and proapoptotic functions of ELF2B thereby confirming its crucial role. Mice expressing Elf2a or Elf2b in haemopoietic cells variously displayed perturbations in the pre-B cell stage and multiple stages of T cell development. Mature B cells, T cells, and myeloid cells in steady state were unaffected, suggesting that the main role of ELF2 is restricted to the early development of B and T cells and that compensatory mechanisms exist. No differences in B and T cell development were observed between ELF2 isoforms.ConclusionsWe conclude that ELF2 isoforms are important regulators of cellular proliferation, cell cycle progression, and apoptosis. In respect to this, ELF2B acts in a dominant negative fashion compared to ELF2A and as a putative tumour suppressor gene. Given that these cellular processes are critical during haemopoiesis, we propose that the regulatory interplay between ELF2 isoforms contributes substantially to early B and T cell development.

Dual roles for bone marrow-derived Sca-1 cells in cardiac function.

Recruitment of stem cells from the bone marrow (BM) is an important aspect of cardiac healing that becomes inefficient with age. We investigated the role of young stem cell antigen 1 (Sca-1)-positive BM cells on the aged heart by microarray analysis after BM reconstitution. Sca-1+ and Sca-1- BM cells from young green fluorescent protein (GFP)-positive mice were used to reconstitute the BM of aged mice. Myocardial infarction (MI) was induced 3 mo later. GFP+ cells were more abundant in the BM, blood, and heart of Sca-1+ mice, which corresponded to preserved cardiac function after MI. At baseline, Sca-1+ BM reconstitution increased cardiac expression of serum response factor, vascular endothelial growth factor A, and myogenic genes, but reduced the expression of Il-1β. After MI, inflammation was identified as a key difference between Sca-1- and Sca-1+ groups, as cytokine expression and cell surface markers associated with inflammatory cells were up-regulated with Sca-1+ reconstitution. Mac-3 and F4/80 staining showed that the postinfarction heart was composed of a mixture of GFP+ (donor) macrophages, GFP- (host) macrophages, and GFP+ cells that did not contribute to the macrophage population. This study demonstrates that Sca-1+ BM cells regulate cardiac healing though an acute inflammatory response and also before injury by stimulating formation of a beneficial cardiac niche.-Tobin, S. W., Li, S.-H., Li, J., Wu, J., Yeganeh, A., Yu, P., Weisel, R. D., Li, R.-K. Dual roles for bone marrow-derived Sca-1 cells in cardiac function.

Young Bone-Marrow Sca-1+ Stem Cells Rejuvenate the Aged Heart and Improve Function after Injury through PDGFR\u03b2-Akt pathway

Bone marrow (BM) reconstitution with young BM cells in aged recipients restores the functionality of cardiac resident BM-derived progenitors. This study investigated the cell type primarily responsible for this effect. We reconstituted old mice with BM cells from young or old mice and found that the number of stem cell antigen 1 (Sca-1) cells homing to the heart was significantly greater in young than old chimeras. We then reconstituted old mice with young BM Sca-1+ or Sca-1− cells. We found that Sca-1 cells repopulated the recipient BM and homed to the heart. The number of BM-derived cells in the aged myocardium co-expressing PDGFRβ was 3 times greater in Sca-1+ than Sca-1− chimeric mice. Sca-1+ chimeras had more active cell proliferation in the infarcted heart and improved ventricular function after MI. The improved regeneration involved activation of the PDGFRβ/Akt/p27Kip1 pathway. Sca-1+ stem cells rejuvenated cardiac tissue in aged mice. Restoration of the Sca-1+ subset of stem cells by BM reconstitution improved cardiac tissue regeneration after injury in aged mice.

β2-Adrenergic receptor-dependent chemokine receptor 2 expression regulates leukocyte recruitment to the heart following acute injury

Following cardiac injury, early immune cell responses are essential for initiating cardiac remodeling and tissue repair. We previously demonstrated the importance of β2-adrenergic receptors (β2ARs) in the regulation of immune cell localization following acute cardiac injury, with deficient leukocyte infiltration into the damaged heart. The purpose of this study was to investigate the mechanism by which immune cell-expressed β2ARs regulate leukocyte recruitment to the heart following acute cardiac injury. Chemokine receptor 2 (CCR2) expression and responsiveness to C-C motif chemokine ligand 2 (CCL2)-mediated migration were abolished in β2AR knockout (KO) bone marrow (BM), both of which were rescued by β2AR reexpression. Chimeric mice lacking immune cell-specific CCR2 expression, as well as wild-type mice administered a CCR2 antagonist, recapitulated the loss of monocyte/macrophage and neutrophil recruitment to the heart following myocardial infarction (MI) observed in mice with immune cell-specific β2AR deletion. Converse to β2AR ablation, β2AR stimulation increased CCR2 expression and migratory responsiveness to CCL2 in BM. Mechanistically, G protein-dependent β2AR signaling was dispensable for these effects, whereas β-arrestin2-biased β2AR signaling was required for the regulation of CCR2 expression. Additionally, activator protein 1 (AP-1) was shown to be essential in mediating CCR2 expression in response to β2AR stimulation in both murine BM and human monocytes. Finally, reconstitution of β2ARKO BM with rescued expression of a β-arrestin-biased β2AR in vivo restored BM CCR2 expression as well as cardiac leukocyte infiltration following MI. These results demonstrate the critical role of β-arrestin2/AP-1-dependent β2AR signaling in the regulation of CCR2 expression and recruitment of leukocytes to the heart following injury.

Introduction of Macrophage Tolerance By Human SIRPA Knock-in Enhances Peripheral Reconstitution of Human Hematopoiesis in Xenotransplantation Model

NOD background is one of the critical determinants for the efficient human cell engraftment in xenotransplantation models. We have reported that the NOD-specific polymorphism of Sirpa allows the mouse SIRPA to bind human CD47, preventing activation of recipient macrophages to engulf human hematopoietic cells (Nat Immunol. 2007). We then developed a C57BL/6.Rag2nullIl2rgnull (BRG) mouse line harboring the NOD-type Sirpa, named BRGS, in which the xenograft efficiency of human hematopoiesis was equal to that in NOD.Rag1nullIl2rgnull strain (Blood 2013). However, there are several unsolved problems in the current xenograft models including BRGS: The reconstituted human hematopoiesis is B-lymphoid dominant; the engraftment levels of human erythrocytes are quite low; and the distribution of human cells to peripheral tissues is not enough. We have reported that the binding affinity of recipient SIRPA with human CD47 is a decisive factor for the efficiency of xenotransplantation (Exp. Hematol.2014). Thus, we consider that introduction of human SIRPA confers much more efficient engraftment to recipients compared to that of NOD SIRPA.Here, we newly generated human SIRPA knock-in mice on C57BL/6 background and intercrossed these mice with BRG mice to obtain the BRGhS (C57BL/6.Rag2nullIl2rgnullHuman SIRPA) mice. The BRGhS mice were born healthy and displayed good fertility. In contrast to Sirpanullmice, the BRGhS mouse did not develop anemia and thrombocytopenia, probably because human SIRPA partially bound to mouse CD47.We found that the BRGhS macrophages have a better affinity for human CD47 than the BRGS macrophages by protein binding assay (Kd=7.2nM vs. 11.4nM, p=0.04). Human CD47 binding to the BRGhS macrophages activated the SHP-1 phosphatase, an anti-phagocytic signal. Consistent with this, when macrophages of each strain were cultured with human HSCs, the number of HSCs engulfed by the BRGhS macrophages was much less than those by the BRGS macrophages. To quantify the reconstitution activity of human hematopoiesis in vivo, we transplanted 5 × 103 CD34+CD38- human cord blood cells intrafemorally into irradiated BRGhS or BRGS mice. At 10-12 weeks after transplantation, both strains showed successful reconstitution, and the frequency of human CD45+ cells in the bone marrow of BRGhS was better than that of BRGS (80.2% vs. 72.3% p=0.09). However, the reconstitution of human erythrocytes/megakaryocytes lineages is limited in both strains. The frequency of human CD45+ cells was maintained at least until 24 weeks after transplantation. Surprisingly, the frequency of human CD45+cells in the spleen and peripheral blood (PB) of BRGhS was significantly higher than that of BRGS (Spleen: 37.8% vs. 11.2% p=0.01, PB: 11.4% vs. 5.2% p<0.01). To examine the phagocytic activity of macrophages against circulating human cells in vivo, human erythrocytes were injected into both strains and their clearance was monitored. Transfused erythrocytes were rapidly eliminated from the circulation of the BRGS recipients, but they remained detectable in the BRGhS recipients.The drastic improvement of the reconstitution in peripheral tissues and the much less elimination of infused human erythrocytes suggest that the introduction of human SIRPA strongly inhibits phagocytosis by macrophages, especially in peripheral tissues. Recently, we reported that robust erythroid reconstitution in bone marrow was obtained by introduction of Kit mutation into the BRGS strain (Stem Cell Reports, in press). Thus, combination Kit mutation with human SIRPA knock-in should be a powerful tool for establishing a more faithful humanized model with circulating mature human blood cells. DisclosuresAkashi:Celgene: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Sunitomo Dainippon Pharma: Consultancy; Bristol Meyers Squibb: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Astellas Pharma: Research Funding; Shionogi & Co., Ltd: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

The Preclinical Efficacy of a Novel Telomerase Inhibitor, Imetelstat, in AML - a Randomized Trial in Patient-Derived Xenografts

Replicative immortality depends on telomerase activation in the majority of cancers including acute myeloid leukemia (AML). Imetelstat is a covalently lipidated 13-mer oligonucleotide that competitively inhibits telomerase activity. Clinical efficacy of imetelstat has recently been reported in essential thrombocythemia and myelofibrosis. We investigated the efficacy of imetelstat in AML using a randomized trial in patient-derived xenografts (PDX).To establish an AML PDX cohort, primary bone marrow (BM) or peripheral blood (PB) samples from 31 AML patients were transplanted into NOD.Cg Prkdcscid Il2rgtm1Wjl Tg (CMV-IL3,CSF2,KITLG)1Eav/Mlo (NSGS). Engraftment was defined by reconstitution of BM and spleen with CD33+ donor cells, PB circulating blasts, anemia or thrombocytopenia. The success rate for engraftment was 70.4% and was independent of cytogenetics.Fifteen independent AML patient samples (n=12 recipients/sample) were tested for efficacy of imetelstat. After engraftment, mice were randomized and treated with imetelstat (15 mg/kg body weight) or control phosphate-buffered saline (PBS) intraperitoneally every 48-72h. Across the entire cohort, survival was improved for imetelstat- vs. PBS-treated PDX (Hazard ratio PBS: 5.299; 3.379 ± 8.312; p < 0.0001; Cox proportional hazards model). Imetelstat delayed the expansion of human AML cells in 14 PDX (93.3%). AML patient samples were divided into 2 groups based on survival outcomes, "Sustained responders" (imetelstat extended survival > 2-fold or > 4 weeks; 9 AML patient samples; 60%) or "Poor responders" (6 AML patient samples, 40%). European LeukemiaNet (ELN)-karyotypic subtypes differed trend-wise between groups: favourable (100% sustained, 0% poor response to imetelstat); intermediate-1, intermediate-2 and poor (45.5% sustained, 54.5% poor response to imetelstat; p = 0.1, Fisher’s exact test). Next generation sequencing revealed baseline AML PDX genetic profiles. FLT3, NRAS, TET2, RAD21 as well as 15 genes annotated in DNA damage, cell cycle regulation and apoptosis were mutated exclusively in the sustained responders group. Poor responders revealed mutations in ETV6, FANCM, MKI67, WT1 and 16 additional genes regulating growth factor independence and evasion of apoptosis. Imetelstat response was associated with reduced quiescence and induction of γ-H2AX levels in AML populations (n=4-6 / group / 3 individual AML patient samples): γ-H2AX (MFI normalized to PBS; PBS: 1.0 ± 0.02; imetelstat: 1.24 ± 0.02; p < 0.0001, Student’s t test), quiescence (%Ki67-diploid AML cells; PBS: 11.62% ± 0.87; imetelstat: 6.9% ± 0.68; p < 0.001, Student’s t test). The presence of spliceosome mutations (SRSF2, U2AF1) was not correlated with response (p = 0.5; Fisher’s exact test).To investigate the effects on normal human hematopoiesis, CD34-enriched cord blood was transplanted into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG, n=5-6/group/donor in 2 replicate experiments). Engraftment was confirmed at 4 weeks post-transplant by PB chimerism (>=1%). Mice were treated with imetelstat or vehicle control from 4 weeks. After 10 weeks treatment, BM cellularity and donor engraftment were significantly reduced whereas the frequency of the CD34+CD38low HSC-enriched population was unchanged (donor 1 PBS: 0.35% ± 0.03%, imetelstat: 0.23% ± 0.06%, p = 0.1; donor 2 PBS: 0.45% ± 0.05%, imetelstat: 0.92% ± 0.35%, p = 0.2, Student’s t test). HSC cell cycle and γ-H2AX levels were unchanged. PB and spleen B-cell engraftment were reduced (donor 1 spleen PBS: 77.74% ± 4.66%, imetelstat: 45.48% ± 9.03%, p < 0.05; donor 2 spleen PBS: 93.95% ± 0.40%, imetelstat: 89.1% ± 2.52%, p = 0.09, Student’s t test), whereas the myeloid lineage was elevated or relatively preserved (donor 1 spleen PBS: 3.29% ± 0.71%, imetelstat: 7.53% ± 1.6%, p < 0.05; donor 2 spleen PBS: 1.20% ± 0.12%, imetelstat: 4.49% ± 1.60%, p = 0.07, Student’s t test).In summary, imetelstat demonstrates efficacy in a significant proportion of AML PDX (60%, 9 out of 15 individual AML patient samples). Robust responses to imetelstat were associated with favourable cytogenetic risk groups and mutations in pathways controlling DNA damage. The effects on normal human hematopoiesis were modest and predominantly seen in the B-lymphocyte lineage with relative preservation of myeloid and stem cell populations. Further study is warranted to understand the preclinical and clinical efficacy of imetelstat in AML. DisclosuresLane:Janssen: Other: i have done consulting (once) for janssen..

Hematopoietic Stem Cells Respond Heterogeneously to Inflammatory Signals in an Age-Dependent Manner

Hematopoietic stem cells (HSCs) have the unique responsibility to produce balanced immune cell output throughout an organism's life. Importantly, they must do so robustly despite a plethora of external stress, including frequent inflammatory challenge. With age, the accumulation of these stresses leads to impaired HSC function and myeloid-biased output. Aged HSCs are also more prone to pathological hematopoiesis, such as myeloproliferative disorder, leukemia and autoimmune diseases. However, little is known about the subcellular mechanisms that govern the inflammatory response of HSCs with age, which in turn might contribute to pathologic transformation.We show that young hematopoietic stem and progenitor cells (HSPCs) demonstrate a robust transcriptional response to toll-like receptor (TLR) ligands. Interestingly, this response is similar to that of mature immune cell types such as dendritic cells. Using single-cell proteomic assays, we found that young HSPCs secrete a diverse array of myeloid and lymphoid cytokines. However, when challenged with TLR ligands in vivo, young mice acutely increase myeloid-biased output but return rapidly to baseline hematopoietic output of both lymphoid and myeloid cells. Moreover, inflammatory challenge of young long-term HSCs in vitro did not perturb the function and output of these cells in bone marrow reconstitution experiments.In contrast to their counterparts from young mice, we found HSPCs obtained from aged mice have a diminished ability to secrete cytokines in response to TLR ligands. Furthermore, they secrete a homogenous subset of myeloid-biased cytokines. When challenged with TLR ligands in vivo, aged mice acutely increased myeloid output and maintain elevated myeloid output for several months implying memory of the inflammatory challenge. Consistent with this, we also found that pre-stimulation of aged HSCs prior to bone marrow transplant results in a sustained increase in myeloid output compared to unstimulated aged HSCs.To elucidate the differential heterogeneity between young and aged HSPCs in response to TLR signaling, we next performed single-cell RNA sequencing (RNA-seq) experiments. We found that the sustained myeloid output in aged mice after TLR stimulation is largely due to expansion of a myeloid-biased HSC subset in the aged HSC pool. By characterizing the gene expression networks that define these myeloid-biased HSCs under stimulation conditions, we were then able to identify a myeloid-biased HSC subset in both the unperturbed young and aged HSC pools. Moreover, we found that these cells are more abundant in aged mice at steady-state, and that these HSCs demonstrate a unique response to inflammatory challenge. We further identify putative transcriptional regulators, including Klf4, Klf5, Ikzf1 and Stat3, among others, that define gene expression in these myeloid-biased HSCs. We further show that loss of function of these factors can differentially alter myeloid output in young and aged mice both in vitro and in vivo.Our results demonstrate that there is a differential response of young and aged HSCs to inflammatory signals. Using single-cell RNA-seq and protein secretion studies, we elucidate the molecular heterogeneity of the HSC pool at steady state and with TLR stimulation. By resolving heterogeneous subsets of cells in both the young and aged HSC pool, and by uncovering the transcriptional regulators that influence their function, we thus propose a new model of inflammatory hematopoiesis that may have implications to understanding age-related defects in immune development. DisclosuresNo relevant conflicts of interest to declare.

SETD2, a H3K36 Lysine Methyltransferase, Is Essential for Adult Normal Hematopoiesis and Leukemia Stem Cells

Hematopoietic stem cells (HSCs) are characterized by their capability for self-renewal and multi-potency. Hematopoiesis is dynamically controlled by the interplay between epigenetic and transcriptional networks. Dysregulation of these networks can lead to unfitness of hematopoiesis, cell transformation, and hematological diseases. The human SETD2 gene was originally isolated from HSCs and progenitors. SETD2 is a histone methyltransferase, which specifically catalyzes tri-methylation of histone 3 lysine 36 (H3K36me3). SETD2 functions as a tumor suppressor, as loss-of-function mutations have been identified in many cancers. However, the role of SETD2 in hematopoiesis has not been fully understood.To assess the function of Setd2 in hematopoiesis, we generated three Setd2 mouse alleles with Crispr/CAS9 technology; Setd2F2478/WT knock-in, Setd2Exon6-Δ/WT, and Setd2-Exon6flox/flox/Mx1-Cre conditional knockout alleles, as homozygous Setd2 mutation showed embryonic lethality. Setd2-F2478 point mutation, which is located in the SRI domain, can express SETD2 mutant protein but completely lose the interaction with RNA pol II. Setd2Exon6-Δ/WT allele results in a frame shift and nonsense mediated decay of Setd2 mRNA and protein.After induction of excision with pIpC injection, Setd2-exon6flox/flox/Mx1-Cre+ (Setd2Exon6-Δ/Δ) mice showed severe anemia, increased platelet count, and a reduction in bone marrow (BM) cellularity compared to wild-type (WT) mice, while Setd2F2478/WT and Setd2Exon6-Δ/WT mice did not show any obvious hematological changes. The Lin- Sca-1+ c-Kit+ (LSK) population in Setd2Exon6-Δ/Δ mice was 2.5-fold decreased compared to those in WT, while the LSK populations in Setd2F2478/WT and Setd2Exon6-Δ/WT mice were comparable with those in WT. Interestingly, all three of these Setd2 mutant alleles showed a higher frequency of Lin- Sca-1- c-Kit+ (LK) cells in the BM. In the LK populations, we found an increased CMP population in Setd2F2478/WT and Setd2Exon6-Δ/WT mice; of note, the CMP population in the Setd2Exon6-Δ/Δ mice had disappeared while the MEP population expanded with higher expression of CD16/32. Next, to assess the function of the HSPCs, we performed CFU assays and competitive bone marrow transplantations (CBMT). Consistent with our phenotypic findings, the number of colonies derived from Setd2F2478/WT and Setd2Exon6-Δ/WT BM cells was increased in the first two passages, while the number of colonies derived from Setd2Exon6-Δ/Δ mice was significantly decreased. In CBMT, we found that mice transplanted with Setd2Exon6-Δ/Δ BM cells showed anemia and an impaired BM reconstitution, compared to the control (p = 0.0002). On the other hand, the Setd2F2478/WT and Setd2Exon6-Δ/WT models showed comparable capabilities of BM reconstitution. Taken together, these results suggest that Setd2 has an essential role in the maintenance of adult hematopoiesis.SETD2 mutations (mainly one allele mutation) have been frequently identified in acute leukemia, especially in about 22% of MLL leukemia. To understand the role of SETD2 in leukemic stem cells, Setd2 mutant mice were bred with the Mll-AF9 knock-in mouse. The Mll-AF9/ Setd2F2478/WT and Mll-AF9/ Setd2Exon6-Δ/WT mice showed higher frequencies of LK and LSK populations compared to Mll-AF9 mice, indicating that Setd2 mutations may increase the stemness of leukemia stem cells (LSCs). The cells derived from Mll-AF9/ Setd2F2478/WT and Mll-AF9/ Setd2Exon6-Δ/WT mice resulted in a significantly higher yield of colonies and growth advantage in serial replating CFU assay compared to the cells derived from Mll-AF9 mice. After BMT of equal numbers of cells from Mll-Af9 or Mll-AF9/ Setd2F2478/WT mice into recipient mice, the Mll-AF9/ Setd2F2478/WTBMT mice developed leukemia with significantly shortened latencies compared with MLL-Af9 BMT mice.In conclusion, our data suggests that Setd2 plays an important role in maintaining normal HSPCs. Half the doses of Setd2 can still maintain the normal hematopoiesis while a total loss of Setd2 leads to a failure of hematopoiesis. In leukemia, heterozygous mutants of Setd2 can accelerate leukemogenesis by expanding LSCs. Whether the remaining WT allele is required for leukemia maintenance is unclear. Further reduction of Setd2 levels, or complete deletion of the other WT allele, may diminish SETD2-mutated leukemia. Such tumor vulnerability can be explored as a therapeutic strategy. DisclosuresNo relevant conflicts of interest to declare.

Hevylite and Freelite Normalisation Is a Surrogate Marker for MRD Negativity Post-ASCT

IntroductionEvaluation of minimal residual disease (MRD) in multiple myeloma (MM) by multi-parameter flow cytometry (MFC) is a biomarker for risk of relapse. Applicability of MFC in routine practice has been challenging with suboptimal marrow samples and sampling needed at multiple time points. Sensitive serological assays which can detect MRD and have prognostic utility are needed to optimize the management of MM patients. The HevyliteTM assay (The Binding Site, UK) evaluates serum heavy/light chain ratios for IgG, IgA and IgM. We hypothesised the normalisation of serological markers is a good surrogate for plasma cell reconstitution in a healthy bone marrow (BM) microenvironment. The aim of this study was to prospectively compare Hevyliteand FreeliteTM assays with MFC of BM plasma cells from patients receiving autologous stem cell transplants (ASCT). We wanted to investigate if a correlation exists between MFC MRD negativity and the serological normalisation of Hevylite plus Freelite assays.MethodsHevylite, Freelite, SPEP, IFE on serum and MFC on bone marrow were performed on 23 patients post induction therapy and at a median of 90 days post-ASCT. Serological tests were performed again in available patients at 275 days post-ASCT. The combination of Freelite and Hevylite, which measures involved (iHLCr) and uninvolved (uHLCr) heavy light chain (HLC)-pairs, allows investigation of plasma cell reconstitution and its prognostic value in transplant patients. Six-colour MFC was performed as previously published (Rawstron et al. JCO 2013). IMWG uniform response criteria were used to categorise patients. Kaplan Meier estimates of Progression Free Survival (PFS) from time of transplantation until disease progression or death (whichever came first) were calculated and log-rank test was used for comparisons across groups. Sensitivity, specificity and ROC area analysis were used to assess performance of serologic response tests against flow MRD negativity, which is considered gold standard.ResultsTotal of 23 patients were recruited to the study, 8 female (34.8%), 15 male (65.2%), with median age 61 (46-71). Isotype and ISS distribution were as follows; 11 IgG (47.8%), 4 IgA (17.4%), 1 IgM (4.35%), 7 LC only (30.4%) and 8 ISS1 (34.8%), 6 ISS2 (26.1%), 9 ISS3 (39.1%). Median marrow PC involvement at diagnosis was 45%, (20-90%) (N=18). At diagnosis, median LDH levels 192 IU/L, (153-475), median Hb 109.5 g/l, (72-152), with renal impairment noted in 6/23 (26.1%) and abnormal skeletal survey 15/23 (65.2%). High risk MM FISH was reported in 1/23 (4.35%), unknown 11/23 (47.8%) with rest determined to have standard risk. Median time from start of induction to HDM/ASCT 8 months (6.5-20) and median number of lines of treatment before HDM/ASCT was 1. Two deaths were noted on study. Median follow-up of surviving patients from diagnosis was 31 months (10-42). VGPR or better was achieved in 18/23 (88%) patients post-transplant by IMWG criteria.Hevylite ratio (iHLCr and uHLCr) normalised in 11/23 (48%) patients, with Freelite ratio (FLCr) normalisation in 14/23 (61%). MFC MRD negativity at 3 months post-ASCT was noted in 6/23 (26%) patients, in whom the post-ASCT Hevylite ratio and FLCr were also normalised. Comparing response assessments post-ASCT using standard IMWG criteria, MFC MRD, HLCr normalisation and FLCr normalisation, we found that MFC MRD is more sensitive to pick up residual disease over other tests. MFC MRD positivity correlates with early disease progression as MRD negativity significantly improves PFS outcomes (not reached vs 21 months) p=0.01 (Figure 1a). Patients with both HCLr and FLCr normalisation have significantly better PFS outcomes (not reached vs 21 months) p=0.04 (Figure 1b). ROC curve comparison of MFC MRD negativity and HLCr and FLCr normalisation tests, shows a 100% (95% CI 54.1% - 100%) sensitivity and 82% specificity (95% CI 56.6 - 96.2%) for serological normalisation. This demonstrates that HLCr and FLCr normalisation serves as a useful surrogate marker for MFC MRD negativity in our study.ConclusionsWe report the first prospective study demonstrating HLCr and FLCr normalisation post-transplant is a useful surrogate marker for flow MRD negativity, which is a widely accepted gold standard. This data requires further validation in a larger cohort of post-ASCT MM patients. [Display omitted] DisclosuresRamasamy:Celgene: Honoraria, Research Funding.

Small Molecule Based Ex Vivo Expansion of CD34+CD90+CD49f+ Hematopoietic Stem & Progenitor Cells from Non-Enriched Umbilical Cord Blood Mononucleated Cells

Umbilical cord blood (UCB) transplantation in adults have slow hematopoietic recovery compared to bone marrow (BM) or peripheral blood grafts mainly due to low number of total nucleated cells (TNC) and hematopoietic stem & progenitor cells (HSPC). Current investigational clinical strategies focus on increasing HSPC dosage by expanding CD34 enriched grafts that have resulted in early neutrophil recovery followed by long term hematopoietic reconstitution.In an effort to expand HSPC, specifically those expressing primitive phenotype (CD34+CD90+CD49f+) from non-enriched UCB, we developed a proprietary library of 50 small molecules using structure-activity-relationship studies. Freshly-thawed UCB-mononucleated cells (MNC), were cultured in serum or animal component free expansion medium supplemented with optimal concentration of respective compound. The effects of the expansion protocol were measured based on phenotypic and functional assays. Cell cultures with basal cytokines served as control.Screening of the small molecule library showed negligible acute adverse effects on CD45+ leukocyte population and its viability within 72 hours compared to cytokine control. In long term expansion cultures lasting up to 11 days, one specific structural analog, C7, resulted in 1195.8±71.7-folds increase of absolute CD45+CD34+CD38-CD45RA- progenitors which was at least 9.2-folds higher than control cultures (P<0.01; n=4). Colony forming unit assay showed significant increase of granulocyte-macrophage colonies from C7 treated cells compared to cytokine control (P<0.01; n=6) although TNC expansion was comparable between the culture conditions. It was necessary for the cytokine cocktail to comprise of at least stem cell factor, thrombopoietin and Fms-related tyrosine kinase 3 ligand for mediating HSPC expansion in presence of C7, although further addition of insulin like growth factor binding protein 2 marginally boosted expansion (P<0.001; n=3). Majority of HSPC expansion occurred between the seventh and tenth day of the culture period. In MNC initiated cultures, addition of C7 boosted primitive HSPC (CD45+CD34+CD38-CD45RA-CD90+CD49f+) by 633.3±8.5-folds over 10 days which was at least 7.4-folds higher than control cultures (P<0.001; n=3). In cultures initiated with purified CD34+CD38- cells, there was at least 15.9-folds higher expansion of HSPC defined by CD45+CD34+CD38-CD45RA-CD90+ in presence of C7 compared to cytokine cultures (P<0.05; n=3). Expansion of HSPC by C7 was at least 2-folds higher in comparison to mesenchymal stromal co-culture system which is the only known clinical protocol that allows for UCB expansion without prior CD34/CD133 selection (P<0.001; n=6). Transplantation of C7 expanded UCB grafts (n=11) at equivalent dosage of 2.5x107 cells/kg to sub-lethally irradiated NOD SCID Gamma (NSG) mice resulted in 3.21- and 2.09-folds higher engraftment of human CD45+ cells in the peripheral blood by day 21 compared to non-expanded (P=0.0030; n=6) and cytokine expanded grafts (P=0.0005; n=12) respectively. The frequency of SCID repopulating cells contributing to early peripheral blood engraftment was 2.48-folds higher in C7 expanded graft compared to unmanipulated graft. C7 expanded graft sustained human cell engraftment over 19 weeks which were primarily myeloid cells (CD33+/CD15+) as opposed to non-expanded graft which consisted of CD3+ T cells. Analysis of NSG BM at week 19 post-transplantation, showed significantly better (P<0.0001) chimerism of human CD45 cells in female (n=15) recipient compared to male (n=14) irrespective of graft type (transplantation dosage: 2.5x107 cells/kg). C7 expanded graft gave comparable level of human CD45 and CD34 progenitor cell engraftment as that of the non-expanded grafts. Multi-lineage reconstitution of NSG BM comprising of both myeloid and lymphoid human cells could be achieved with the C7 expanded graft. At higher transplantation dosage of 5.0x107 cells/kg, the expanded grafts had a higher survival rate of 75% compared to 50% for non-expanded graft mainly due to lower incidence of graft-versus-host-disease.In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre-culture stem cell enrichment or stromal cell co-culture. The expanded UCB consists of phenotypically defined primitive HSPC that maintains in vitro and in vivo functionality. DisclosuresHwang:Celgene Corporation: Honoraria, Other: Travel Support; Roche Singapore: Honoraria, Other: Travel Support; Pfizer Singapore: Honoraria, Other: Travel Support; Novartis International AG: Honoraria, Other: Travel Support; Bristol-Myers Squibb Pte Ltd: Honoraria, Other: Travel Support; MSD Pharma (Singapore): Honoraria, Other: Travel Support; Sanofi Aventis Singapore: Honoraria, Other: Travel Support; Janssen-Cilag Singapore: Honoraria, Other: Travel Support.

Prostaglandin E2 Promotes Early Bone Marrow Reconstitution for Survival after Lethal Irradiation

Introduction: High dose total body irradiation (TBI) causes severe damage to the hematopoietic system, resulting in ablation of both red and white blood cells and a high probability of infection, hemorrhage, and death. This is known as the hematopoietic syndrome of the acute radiation syndrome (HS-ARS). TBI is used therapeutically, and incidental exposure through nuclear accidents or radical terrorism is a current threat for which preparation is critical. Safe, effective, and pragmatic agents against HS-ARS are needed. We have identified prostaglandin E2 (PGE2) as a promising medical countermeasure to promote hematopoietic recovery and increase survival from lethal irradiation. Here, using the PGE2 analog 16,16-dimethyl PGE2 (dmPGE2), pre-irradiation dosing strategies were assessed in survival studies with an established mouse model of HS-ARS, and time course analyses were performed to elucidate the early cellular events associated with PGE2 radioprotection.Methods: C57BL/6 mice (n = 20/group) were exposed to LD70/30 (872 cGy) or LD90/30 (904 cGy) and treated with one of the following four dmPGE2 dosing strategies: a single dose of 35 μg at time -30 min, -1 hr, or -3 hr, or a double-dose of 20 μg each at time -45 min and -15 min pre-irradiation. Thirty-day survival was evaluated. The double-dose regimen was further utilized to assess cellular effects over time following LD70/30. Peripheral blood (PB) and bone marrow (BM) cells were harvested on days 1, 2, 4, 7, and 10 post-irradiation (n = 3/group/day). BM was analyzed by flow cytometry with immunostaining for surface markers (Sca-1, c-Kit, CD150, CD48 and blood cell lineage markers), cell cycle (Hoechst/PyroninY), and apoptosis (AnnexinV/7-AAD). PB differentials were assessed by a veterinary hematology analyzer.Results: All dmPGE2 regimens conferred 100% survival to mice receiving LD70/30, and all regimens provided a distinct survival advantage to mice receiving LD90/30 with the double-dose strategy conferring 100% survival at LD90/30. The double-dose was then chosen to assess cellular effects of dmPGE2 throughout the critical 10-day time period following LD70/30. Within the BM, marker-defined HSC numbers were significantly preserved by day 1 post-irradiation compared to vehicle controls, with a drop to vehicle levels observed on day 2 only. Cell cycling frequency among primitive hematopoietic cells was also conserved on day 1, decreased on day 2, then restored to non-irradiated control levels by days 7-10, while that of the unprotected mice remained significantly lower. Differentiated BM cells (lineage marker-positive) were substantially less apoptotic by day 2 and, in significant contrast to the untreated mice, returned to control levels of basal apoptosis by days 7-10. Overall BM cell populations were found to be largely replenished within one week as compared to the vehicle-treated mice, which remained significantly depressed. Further, 73-88% of detectable BM cells from the untreated mice were non-viable by day 4 post-irradiation based on sub-diploid DNA content and did not recover through day 10; dmPGE2-treated mice maintained significantly greater BM cell viability at each time point, returning to only 17-26% sub-diploid by day 7 (non-irradiated controls demonstrated ~5% sub-diploid DNA content). A corresponding effect on PB resulted in significant rescue from neutropenia, anemia, and thrombocytopenia by or before day 10.Conclusions: A single treatment with PGE2 prior to lethal irradiation can facilitate virtually complete survival, though a double treatment regimen may be most effective; further work will test whether this may be primarily due to the combined higher dose or to the altered timing. This PGE2-mediated survival involves early protection of both primitive and differentiated hematopoietic BM cells, affecting cell number, cell cycling, and apoptosis. Early hematopoietic BM recovery subsequently rescues PB components, facilitating ultimate survival. With further study, PGE2 may serve to provide an effective radioprotectant with the potential to safeguard patients undergoing TBI therapy, as well as military personnel and first responders, from the risk of radiation-associated mortality. DisclosuresNo relevant conflicts of interest to declare.

Effect of Naoxintong capsule on endothelial progenitor cell mobilization and homing following bone marrow transplantation in a mouse hind limb ischemia model

Endothelial progenitor cells (EPCs) are precursor cells of endothelial cells. Signal molecules produced by ischemia and hypoxia can promote mobilization of bone marrow EPCs to peripheral circulation and formation of novel blood vessels in tissues that are damaged during heart attack. Naoxintong capsule (NXT) has the functions of promoting blood circulation, removing blood stasis, promoting the circulation of qi and relieving pain. The various components in NXT have protective effects on blood vessels and can effectively improve the symptoms of ischemia. However, its effect on EPCs is not clear. To study the intervention effect of NXT on mobilization and homing of peripheral blood EPCs, green fluorescent protein (GFP) transgenic mice were used for bone marrow transplantation (BMT) and then unilateral hind limb ischemia model (UHLI) were constructed. For BMT, wild-type ICR mice were irradiated by CS137 and then injected with 4×106 bone marrow cells isolated from GFP mice. The bone marrow reconstitution of recipients was assessed by quantification of GFP bone marrow-derived cells (BMDC) from transplanted mice 4 weeks after BMT. The UHLI model was duplicated by ligating femoral artery and divided into three groups: the model group, the NXT group (model+NXT) and the positive control group (model+simvastatin). Flow cytometry was used to detect the proportion of GFP positive cells and the peripheral blood EPCs levels at 1, 3, 7, 14 days before and after surgery. Ischemic tissue of gastrocnemius muscle was excised at 3 and 7 days after operation for immunofluorescence staining to detect the number of GFP+ cells. The bone marrow chimerism was achieved at day 28 after BMT. There was no significant difference in the percentage of GFP positive cells between BMT mice and GFP transgenic mice. NXT and simvastatin could significantly increase the number of peripheral blood EPCs 1,3 days after surgery. Three and seven days after operation, the number of homing EPCs was significantly higher in NXT group and positive control group than that in model group (P<0.001). In conclusion, NXT can obviously promote the mobilization and homing of EPCs.

Genetic and Pharmacologic Inhibition of the Chemokine Receptor CXCR2 Prevents Experimental Hypertension and Vascular Dysfunction.

The recruitment of leukocytes to the vascular wall is a key step in hypertension development. Chemokine receptor CXCR2 mediates inflammatory cell chemotaxis in several diseases. However, the role of CXCR2 in hypertension development and the underlying mechanisms remain unknown. Angiotensin II (490 ng·kg-1·min-1) or deoxycorticosterone acetate (DOCA) salt-induced mouse hypertensive models in genetic ablation, pharmacologic inhibition of CXCR2, and adoptive bone marrow transfer mice were used to determine the role of CXCR2 in hypertension (measured by radiotelemetry and tail-cuff system), inflammation (verified by flow cytometry and quantitative real-time polymerase chain reaction [PCR] analysis), vascular remodeling (studied by haematoxylin and eosin and Masson's trichrome staining), vascular dysfunction (assessed by aortic ring), and oxidative stress (indicated by nicotinamide adenine dinucleotide phosphate [NADPH] oxidase activity, dihydroethidium staining, and quantitative real-time PCR analysis). Moreover, the blood CXCR2+ cells in normotensive controls and hypertension patients were analyzed by flow cytometry. Angiotensin II significantly upregulated the expression of CXCR2 mRNA and protein and increased the number of CD45+ CXCR2+ cells in mouse aorta (n=8 per group). Selective CXCR2 knockout (CXCR2-/-) or pharmacological inhibition of CXCR2 markedly reduced angiotensin II- or DOCA-salt-induced blood pressure elevation, aortic thickness and collagen deposition, accumulation of proinflammatory cells into the vascular wall, and expression of cytokines (n=8 per group). CXCR2 inhibition also ameliorated angiotensin II-induced vascular dysfunction and reduced vascular superoxide formation, NADPH activity, and expression of NADPH oxidase subunits (n=6 per group). Bone marrow reconstitution of wild-type mice with CXCR2-/- bone marrow cells also significantly abolished angiotensin II-induced responses (n=6 per group). It is important to note that CXCR2 blockade reversed established hypertension induced by angiotensin II or DOCA-salt challenge (n=10 per group). Furthermore, we demonstrated that CXCR2+ proinflammatory cells were higher in hypertensive patients (n=30) compared with normotensive individuals (n=20). Infiltration of CXCR2+ cells plays a pathogenic role in arterial hypertension and vascular dysfunction. Inhibition of CXCR2 pathway may represent a novel therapeutic approach to treat hypertension.

Radio-resistant recipient bone marrow (BM) macrophages (MACS) are necessary for hematopoietic stem cell (HSC) engraftment post transplantation

For decades it has been assumed that lethal irradiation in bone marrow transplantation experiments ablates the entire host hematopoietic system but preserves the host non-hematopoietic bone marrow stroma. This postulate is at the basis of transplantation chimera experiments in mice to test involvement of hematopoietic cells versus non-hematopoietic stroma. As BM-Macs support HSC niche homeostasis, we examined whether host-derived BM-Macs persist lethal irradiation and play a role in HSC engraftment. Recipient MacGreen mice (expressing GFP in myeloid cells under the control of Csf1r promoter) were lethally irradiated (11.5Gy) and transplanted with sorted syngeneic B6.SJL CD45.1+ Lin-Kit+Sca1+ sorted cells. Flow cytometry analyses of BM 2-30 weeks (wk) post-transplant confirmed more than 99% donor chimerism of monocytes and granulocytes validating ablation of recipient HSC. In contrast, GFP+CD11b+F4/80+CD169+VCAM-1+ERHR3+Ly6Gneg recipient BM-Macs were detected throughout the time-course. A 5.9 fold expansion of these recipient BM-Macs occurred between wk 2 and 5 ( from 45,000 to 270,000 cells/femur) post-transplant which coincided with increased number of phenotypic donor HSC (GFP-Lin-Kit+Sca1+CD48-CD150+). Host BM-Mac proliferation was cell autonomous in the absence of host HSC and granulocytes. Recipient Macs in spleen displayed different frequency and longevity kinetics that correlated with transient post-Tx splenic extramedullary haematopoiesis. In situ, GFP+F480+ recipient BM-Macs were enriched in perivascular microenvironments within both central BM and endosteal regions. These GFP+ host-derived macrophages persisted long-term forming 8.4% of BM macrophages 7months post-transplant. To evaluate the importance of these host-derived radiation-resistant macrophages, we selectively depleted recipient BM-Macs using CD169-DTR mice transplanted with syngeneic ubiquitous GFP+ HSC. Depletion of recipient CD169+ Macs abated engraftment of donor phenotypic HSC by 70% at 5 wk post-transplant and reduced BM reconstitution potential in competitive secondary transplants. In conclusion BM contains a myeloablation-resistant self-repopulating Mac subset that is necessary for efficient and/or sustained HSC BM engraftment following transplantation. Therefore interpretation of transplantation chimera experiments must take into account the persistence of this subset of host macrophages that support donor HSC engraftment.

Human tonsil‑derived mesenchymal stromal cells enhanced myelopoiesis in a mouse model of allogeneic bone marrow transplantation.

Mesenchymal stromal cells (MSCs) have therapeutic potential for repairing tissue damage and are involved in immune regulation. MSCs are predominantly isolated from bone marrow (BM), adipose tissue or placental tissue. Further to these well-known sources, the isolation of MSCs from human tonsils was previously reported. The aim of the present study was to investigate a potential role for tonsil-derived MSCs (T-MSCs) in BM reconstitution and application towards supplementing hematopoiesis in a mouse model of BM transplantation (BMT). Eight-week-old BALB/c female mice received 80 mg/kg busulfan (Bu)/200 mg/kg cyclophosphamide (Cy) conditioning chemotherapy for BM ablation. Subsequently, human T-MSCs were injected into the Bu/Cy-treated mice with or without BM cells (BMCs) obtained from allogeneic C57BL/6 male mice. After 3 weeks, peripheral blood and BM was collected for analysis. The red blood cell count in the group that received BMCs had almost returned to normal, whereas mononuclear cell counts and BM cellularity were most improved in the T-MSCs + BMCs group. These results indicate that the T-MSCs enhanced myelopoiesis in the allogeneic BMT mouse model, as evidenced by the restoration of BM with hematopoietic cells, as well as increased myeloid colony formation in vitro. Therefore, T-MSCs may provide a source of MSCs to facilitate myelopoiesis and megakaryocytosis following BMT.

Mutual Interference between Cytomegalovirus and Reconstitution of Protective Immunity after Hematopoietic Cell Transplantation.

Hematopoietic cell transplantation (HCT) is a therapy option for aggressive forms of hematopoietic malignancies that are resistant to standard antitumoral therapies. Hematoablative treatment preceding HCT, however, opens a “window of opportunity” for latent Cytomegalovirus (CMV) by releasing it from immune control with the consequence of reactivation of productive viral gene expression and recurrence of infectious virus. A “window of opportunity” for the virus represents a “window of risk” for the patient. In the interim between HCT and reconstitution of antiviral immunity, primarily mediated by CD8+ T cells, initially low amounts of reactivated virus can expand exponentially, disseminate to essentially all organs, and cause multiple organ CMV disease, with interstitial pneumonia (CMV-IP) representing the most severe clinical manifestation. Here, I will review predictions originally made in the mouse model of experimental HCT and murine CMV infection, some of which have already paved the way to translational preclinical research and promising clinical trials of a preemptive cytoimmunotherapy of human CMV disease. Specifically, the mouse model has been pivotal in providing “proof of concept” for preventing CMV disease after HCT by adoptive transfer of preselected, virus epitope-specific effector and memory CD8+ T cells bridging the critical interim. However, CMV is not a “passive antigen” but is a pathogen that actively interferes with the reconstitution of protective immunity by infecting bone marrow (BM) stromal cells that otherwise form niches for hematopoiesis by providing the structural microenvironment and by producing hematopoietically active cytokines, the hemopoietins. Depending on the precise conditions of HCT, reduced homing of transplanted hematopoietic stem- and progenitor cells to infected BM stroma and impaired colony growth and lineage differentiation can lead to “graft failure.” In consequence, uncontrolled virus spread causes morbidity and mortality. In the race between viral BM pathology and reconstitution of antiviral immunity following HCT, exogenous reconstitution of virus-specific CD8+ T cells by adoptive cell transfer as an interventional strategy can turn the balance toward control of CMV.