Suzanne L. Kirby

Differential Roles for CCR5 Expression on Donor T Cells during Graft-versus-Host Disease Based on Pretransplant Conditioning

The coordinated expression of chemokines and receptors may be important in the directed migration of alloreactive T cells during graft-vs-host disease (GVHD). Recent work demonstrated in a murine model that transfer of CCR5-deficient (CCR5−/−) donor cells to nonconditioned haploidentical recipients resulted in reduced donor cell infiltration in liver and lymphoid tissues compared with transfer of CCR5+/+ cells. To investigate the function of CCR5 during GVHD in conditioned transplant recipients, we transferred CCR5−/− or wild-type C57BL/6 (B6) T cells to lethally irradiated B6D2 recipients. Unexpectedly, we found an earlier time to onset and a worsening of GVHD using CCR5−/− T cells, which was associated with significant increases in the accumulation of alloreactive CD4+ and CD8+ T cells in liver and lung. Conversely, the transfer of CCR5−/− donor cells to nonirradiated recipients led to reduced infiltration of target organs, confirming previous studies and demonstrating that the role of CCR5 on donor T cells is dependent on conditioning of recipients. Expression of proinflammatory chemokines in target tissues was dependent on conditioning of recipients, such that CXCL10 and CXCL11 were most highly expressed in tissues of irradiated recipients during the first week post-transplant. CCR5−/− T cells were shown to have enhanced migration to CXCL10, and blocking this ligand in vivo improved survival in irradiated recipients receiving CCR5−/− T cells. Our data indicate that the effects of inhibiting CCR5/ligand interaction on donor T cells during GVHD differ depending on conditioning of recipients, a finding with potentially important clinical significance.

Comparison of Granulocyte Colony-Stimulating Factor (G-CSF)-Mobilized Peripheral Blood Progenitor Cells and G-CSF-Stimulated Bone Marrow as a Source of Stem Cells in HLA-Matched Sibling Transplantation

HLA-identical bone marrow or stem cell transplantation from a sibling is the preferred treatment for patients with chronic myelogenous leukemia, bone marrow failure syndromes, relapsed acute leukemia, and specific inborn errors of metabolism. Several groups have shown that granulocyte colony-stimulating factor (G-CSF)--mobilized peripheral blood progenitor cells (PBPCs) obtained from HLA-matched siblings are effective in reconstitution of marrow function after marrow ablative conditioning therapy. To evaluate whether G-CSF treatment before bone marrow harvest leads to enhanced recovery of PBPC counts and recovery from limited graft-versus-host disease (GVHD), we assessed the outcome of a sequential cohort of patients treated identically and then given either G-CSF--mobilized PBPCs or G-CSF--stimulated bone marrow from HLA-identical siblings. We show that the time to neutrophil engraftment is identical in the 2 cohorts, whereas platelet engraftment is earlier with the use of PBPCs. The incidence of acute GVHD was decreased, and that of chronic GVHD significantly decreased, in the group receiving bone marrow. Overall survival was not different between the 2 groups. Thus, G-CSF--stimulated bone marrow offers a source of stem cells that allows for early neutrophil engraftment with a decreased risk of GVHD.

Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies.

Our recent gene expression profiling analyses demonstrated that Wnt2 is highly expressed in Flk1+ cells, which serve as common progenitors of endothelial cells, blood cells, and mural cells. In this report, we characterize the role of Wnt2 in mesoderm development during embryonic stem (ES) cell differentiation by creating ES cell lines in which Wnt2 was deleted. Wnt2-/- embryoid bodies (EBs) generated increased numbers of Flk1+ cells and blast colony-forming cells compared with wild-type EBs, and had higher Flk1 expression at comparable stages of differentiation. Although Flk1+ cells were increased, we found that endothelial cell and terminal cardiomyocyte differentiation was impaired, but hematopoietic cell differentiation was enhanced and smooth muscle cell differentiation was unchanged in Wnt2-/- EBs. Later stage Wnt2-/- EBs had either lower or undetectable expression of endothelial and cardiac genes compared with wild-type EBs. Consistently, vascular plexi were poorly formed and neither beating cardiomyocytes nor α-actinin-staining cells were detectable in later stage Wnt2-/- EBs. In contrast, hematopoietic cell gene expression was upregulated, and the number of hematopoietic progenitor colonies was significantly enhanced in Wnt2-/- EBs. Our data indicate that Wnt2 functions at multiple stages of development during ES cell differentiation and during the commitment and diversification of mesoderm: as a negative regulator for hemangioblast differentiation and hematopoiesis but alternatively as a positive regulator for endothelial and terminal cardiomyocyte differentiation.

Host Absence of CCR5 Potentiates Dendritic Cell Vaccination

Previous work has shown that dendritic cells (DCs) express specific chemokine receptors that allow for coordinated movement in vivo. To test the in vivo relevance of this, we used a murine melanoma system and knockout mice to investigate the function of the chemokine receptor CCR5 and its ligands, CCR ligand (CCL)3 and CCL5. We found that the lack of CCR5 in the host mouse resulted in delayed tumor growth, but this effect was overcome at a higher tumor load. With the administration of tumor charged DCs, CCR5−/− mice that had previously been injected with tumor were completely protected from tumor. This effect was dependent on the dose of tumor cells and the expression of CCR5 on the DC and its absence in the host. In contrast, the loss of the CCR5 ligand, CCL3, led to an early delay in tumor growth that did not persist, while the absence of the CCR5 ligand, CCL5, had no effect. Blocking the activity of CCR5 in the host may represent a new strategy for enhancing the activity of a therapeutic melanoma DC vaccine.

The Chemokine, CCL3, and Its Receptor, CCR1, Mediate Thoracic Radiation–Induced Pulmonary Fibrosis

Patients receiving thoracic radiation often develop pulmonary injury and fibrosis. Currently, there are no effective measures to prevent or treat these conditions. We tested whether blockade of the chemokine, CC chemokine ligand (CCL) 3, and its receptors, CC chemokine receptor (CCR) 1 and CCR5, can prevent radiation-induced lung inflammation and fibrosis. C57BL/6J mice received thoracic radiation, and the interaction of CCL3 with CCR1 or CCR5 was blocked using genetic techniques, or by pharmacologic intervention. Lung inflammation was assessed by histochemical staining of lung tissue and by flow cytometry. Fibrosis was measured by hydroxyproline assays and collagen staining, and lung function was studied by invasive procedures. Irradiated mice lacking CCL3 or its receptor, CCR1, did not develop the lung inflammation, fibrosis, and decline in lung function seen in irradiated wild-type mice. Pharmacologic treatment of wild-type mice with a small molecule inhibitor of CCR1 also prevented lung inflammation and fibrosis. By contrast, mice lacking CCR5 were not protected from radiation-induced injury and fibrosis. The selective interaction of CCL3 with its receptor, CCR1, is critical for radiation-induced lung inflammation and fibrosis, and these conditions can be largely prevented by a small molecule inhibitor of CCR1.

MyD88 provides a protective role in long-term radiation-induced lung injury

Purpose: The role of innate immune regulators is investigated in injury sustained from irradiation as in the clinic for cancer treatment or from a nuclear incident. The protective benefits of flagellin signaling through Toll-like receptors (TLR) in an irradiation setting warrant study of a key intracellular adaptor of TLR signaling, namely Myeloid differentiation primary response factor 88 (MyD88). The role of MyD88 in regulating innate immunity and Nuclear factor kappa-B (NF-κB)-activated responses targets this critical factor for influencing injury and recovery as well as maintaining immune homeostasis. Materials and methods: To examine the role of MyD88, we examined immune cells and factors during acute pneumonitic and fibrotic phases in Myd88-deficient animals receiving thoracic gamma (γ)-irradiation. Results: We found that MyD88 supports survival from radiation-induced injury through the regulation of inflammatory factors that aid in recovery from irradiation. The absence of MyD88 resulted in unresolved pulmonary infiltrate and enhanced collagen deposition plus elevated type 2 helper T cell (Th2) cytokines in long-term survivors of irradiation. Conclusions: These results based only on a gene deletion model suggest that alterations of MyD88-dependent inflammatory processes impact chronic lung injury. Therefore, MyD88 may contribute to attenuating long-term radiation-induced lung injury and protecting against fibrosis.

Antibodies to stem cell marker antigens reduce engraftment of hematopoietic stem cells

Hematopoietic stem cells (HSCs) have enormous potential for use in transplantation and gene therapy. However, the frequency of repopulating HSCs is often very low; thus, highly effective techniques for cell enrichment and maintenance are required to obtain sufficient cell numbers for therapeutic use and for studies of HSC physiology. Common methods of HSC enrichment use antibodies recognizing HSC surface marker antigens. Because antibodies are known to alter the physiology of other cell types, we investigated the effect of such enrichment strategies on the physiology and lineage commitment of HSCs. We sorted HSCs using a method that does not require antibodies: exclusion of Hoechst 33342 to isolate side population (SP) cells. To elucidate the effect of antibody binding on this HSC population, we compared untreated SP cells with SP cells treated with the Sca‐1+c‐Kit+Lin− (SKL) antibody cocktail prior to SP sorting. Our findings revealed that HSCs incubated with the antibody cocktail had decreased expression of the stem cell‐associated genes c‐Kit, Cd34, Tal‐1, and Slamf1 relative to untreated SP cells or to cells treated with polyclonal isotype control antibodies. Moreover, SKL antibodies induced cycling in SP cells and diminished their ability to confer long‐term hematopoietic engraftment in lethally irradiated mice. Taken together, these data suggest that antibody‐based stem cell isolation procedures can have negative effects on HSC physiology.

csf1 is required for early embryonic macrophage development: Characterization of the csf1op/csf1op mutation in ES cell-derived macrophages

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Biosynthesis of Proteochondroitin Sulfate by HL-60 Human Promyelocytic Cells

Human promyelocytic cells (HL‐60) were labeled with 35S‐sulfate and either 3H‐glucosamine or 3H‐serine as precursors. Accumulation of 35S‐labeled macromolecules was approximately linear for up to 96 h, with a mean cell:medium ratio of 5.5:1, although activity/105 viable cells reached a plateau level after 24 h. Virtually none of the cell‐associated proteoglycan was removed by trypsinization, consistent with a predominantly intracellular localization. Proteoglycan heterogeneity was investigated by DEAE‐Sephacel chromatography, isopyknic CsCI gradient centrifugation, and gel filtration chromatography. HL‐60 cells appeared to synthesize a single proteoglycan species, Kav = 0.46 on Sepharose CL‐4B and Kav = 0.32 on Sepharose CL‐6B, recovered primarily from the high‐density fractions of a dissociative CsCI gradient (ρ > 1.40 g/l). Degradation products of lower charge density, lower buoyant density, and lower hydrodynamic size were also present, mainly in the cell pellets. The major proteoglycan was found to contain chondroitin sulfate chains of average Mr = 14.5 kD, yielding virtually 100% 4‐sulfated disaccharides on digestion with chondroitinase ABC. The proteoglycan was resistant to trypsin, chymotrypsin, plasmin, and papain, and the core protein Mr was approximately 20 kD by molecular sieve chromatography. Induction of HL‐60 cells with 0.15 dimethyl sulfoxide (DMSO) resulted in differentiation to a more mature granulocytic phenotype and was associated with a reduction in 35S‐sulfate incorporation to 45% of control values or 32%, expressed as activity/105 cells. Proteoglycans synthesized by DMSO‐treated cells were identical to those from untreated cells in terms of hydrodynamic size, glycosaminoglycan Mr, and sulfation.