Donor pretreatment with progenipoietin-1 is superior to granulocyte colony-stimulating factor in preventing graft-versus-host disease after allogeneic stem cell transplantation

The granulocyte colony-stimulating factor (G-CSF) and Fit-3 receptor agonist progenipoietin-1 (ProGP-1) has potent effects on dendritic cell (DC) expansion and may be an alternative to G-CSF for the mobilization of stem cells for allogeneic stem cell transplantation (SCT). We studied the ability of stem cell grafts mobilized with this agent to induce graft-versus-host disease (GVHD) to minor and major histocompatibility antigens in the well-described B6 --> B6D2F1 SCT model. ProGP-1, G-CSIF, or control diluent was administered to donor B6 mice. ProGP-1 expanded all cell lineages in the spleen, and unseparated splenocytes from these animals produced large amounts of interleukin 10 (IL-10) and transforming growth factor beta (TGFbeta) whereas the expression of T-cell adhesion molecules was diminished. Transplantation survival was 0%, 50%, and 90% in recipients of control-, G-CSF-, and ProGP-1-treated allogeneic donor splenocytes, respectively (P < .0001). Donor pretreatment with ProGP-1 allowed a 4-fold escalation in T-cell dose over that possible with G-CSF. Donor CD4 T cells from allogeneic SCT recipients of ProGP-1 splenocytes demonstrated an anergic response to host antigen, and cytokine production (interferon gamma [IFNγ], IL-4, and IL-10) was also reduced while CD8 T-cell cytotoxicity to host antigens remained intact. Neither CD11c(hi) DCs nor CD11c(dim)/B220(hi) DCs from ProGP-1-treated animals conferred protection from GVHD when added to control spleen. Conversely, when equal numbers of purified T cells from control-, G-CSF-, or ProGP-1-treated allogeneic donors were added to allogeneic T-cell-depleted control spleen, survival at day 60 was 0%, 15%, and 90%, respectively (P < .0001). The improved survival in recipients of ProGP-1 T cells was associated with reductions in systemic tumor necrosis factor alpha generation and GVHD of the gastrointestinal tract. We conclude that donor pretreatment with ProGP-1 is superior to G-CSIF for the prevention of GVHD after allogeneic SCT, primarily due to incremental affects on T-cell phenotype and function

Granulocyte-colony stimulating factor increases CD123hi blood dendritic cells with altered CD62L and CCR7 expression

Changes in blood dendritic cell (BDC) counts (CD123(hi)BDC and CD11c(+)BDC) and expression of CD62L, CCR7, and CD49d were analyzed in healthy donors, multiple myeloma (MM), and non-Hodgkin lymphoma (NHL) patients, who received granulocyte-colony stimulating factor (G-CSF) containing peripheral blood stem cell (PBSC) mobilization protocols. Low-dose G-CSF in healthy donors (8-10 mug/ kg/d subcutaneously) and high-dose G-CSF in patients (30 mug/kg/d) increased CD123(hi)BDC (2- to 22-fold, mean 3.7 x 10(6)/ L-17.7 x 10(6)/L and 1.9 x 10(6)/L-12.0 x 10(6)/ L) in healthy donors and MM but decreased CD11c(+)BDC (2- to 10-fold, mean 5.7 x 10(6)/L-1.6 x 10(6)/L) in NHL patients, on the day of apheresis, compared with steady state. After apheresis, CD123(hi)BDC counts remained high, whereas low CD11c(+)BDC counts tended to recover in the following 2-5 days. Down-regulation of CD62L and up-regulation of CCR7 on CD123(hi)BDC were found in most healthy donors and MM patients. CD49d expression was unchanged. Thus, PBSC mobilization may change BDC counts by altering molecules necessary for BDC homing from blood into tissues.

Monitoring dendritic cells in clinical practice using a new whole blood single-platform TruCOUNT assay

Dendritic cells (DC) from distinct DC subsets are essential contributors to normal human immune responses. Despite this, reliable assays that enable DC to be counted precisely have been slow to evolve. We have now developed a new single-platform flow cytometric assay based on TruCOUN(TM) beads and the whole blood Lyse/No-Wash protocol that allows precise counting of the CD14(-) blood DC subsets: CD11c(+)CD16(-) DC, CD11c(+)CD16(+) DC, CD123(hi) DC, CD1c(+) DC and BDCA-3(+) DC. This assay requires 50 mul of whole blood; does not rely on a hematology blood analyser for the absolute DC counts; allows DC counting in EDTA samples 24 It after collection; and is suitable for cord blood and peripheral blood. The data is highly reproducible with intra-assay and inter-assay coefficients of variation less than 3% and 11%, respectively. This assay does not produce the DC-T lymphocyte conjugates that result in DC counting abnormalities in conventional gradient-density separation procedures. Using the TruCOUNT assay, we established that absolute blood DC counts reduce with age in healthy individuals. In preliminary studies, we found a significantly lower absolute blood CD11c(+)CD16(+) DC count in stage III/IV versus stage I/II breast carcinoma patients and a lower absolute blood CD123(hi) DC count in multiple myeloma patients, compared to age-matched controls. These data indicate that scientific progress in DC counting technology will lead to the global standardization of DC counting and allow clinically meaningful data to be obtained. (C) 2003 Elsevier B.V. All rights reserved.

Either interleukin-12 or interferon-gamma can correct the dendritic cell defect induced by transforming growth factor beta(1) in patients with myeloma

The poor response to immunotherapy in patients with multiple myeloma (MM) indicates that a better understanding of any defects in the immune response in these patients is required before effective therapeutic strategies can be developed. Recently we reported that high potency (CMRF44(+)) dendritic cells (DC) in the peripheral blood of patients with MM failed to significantly up-regulate the expression of the B7 co-stimulatory molecules, CD80 and CD86, in response to an appropriate signal from soluble trimeric human CD40 ligand. This defect was caused by transforming growth factor beta(1) (TGFbeta(1)) and interleukin (IL)-10, produced by malignant plasma cells, and the defect was neutralized in vitro with anti-TGFbeta(1). As this defect could impact on immunotherapeutic strategies and may be a major cause of the failure of recent trials, it was important to identify a more clinically useful agent that could correct the defect in vivo. In this study of 59 MM patients, the relative and absolute numbers of blood DC were only significantly decreased in patients with stage III disease and CD80 up-regulation was reduced in both stage I and stage III. It was demonstrated that both IL-12 and interferon-gamma neutralized the failure to stimulate CD80 up-regulation by huCD40LT in vitro. IL-12 did not cause a change in the distribution of DC subsets that were predominantly myeloid (CD11c+ and CDw123-) suggesting that there would be a predominantly T-helper cell type response. The addition of IL-12 or interferon-gamma to future immunotherapy trials involving these patients should be considered.

A population of HLA-DR+ immature cells accumulates in the blood dendritic cell compartment of patients with different types of cancer

Dendritic cell (DC) defects are an important component of immunosuppression in cancer. Here, we assessed whether cancer could affect circulating DC populations and its correlation with tumor progression. The blood DC compartment was evaluated in 136 patients with breast cancer, prostate cancer, and malignant glioma. Phenotypic, quantitative, and functional analyses were performed at various stages of disease. Patients had significantly fewer circulating myeloid (CD11c(+)) and plasmacytoid (CD123(+)) DC, and a concurrent accumulation of CD11c(-)CD123(-) immature cells that expressed high levels of HLA-DR+ immature cells (DR+IC). Although DR+IC exhibited a limited expression of markers ascribed to mature hematopoietic lineages, expression of HLA-DR, CD40, and CD86 suggested a role as antigen-presenting cells. Nevertheless, DR+IC had reduced capacity to capture antigens and elicited poor proliferation and interferon-gamma secretion by T-lymphocytes. Importantly, increased numbers of DR+IC correlated with disease status. Patients with metastatic breast cancer showed a larger number of DR+IC in the circulation than patients with local/nodal disease. Similarly, in patients with fully resected glioma, the proportion of DR+IC in the blood increased when evaluation indicated tumor recurrence. Reduction of blood DC correlating with accumulation of a population of immature cells with poor immunologic function may be associated with increased immunodeficiency observed in cancer.

The colony-stimulating factor 1 receptor is expressed on dendritic cells during differentiation and regulates their expansion

The lineage of dendritic cells (DC), and in particular their relationship to monocytes and macrophages, remains obscure. Furthermore, the requirement for the macrophage growth factor CSF-1 during DC homeostasis is unclear. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced GFP in cells of the myeloid lineage, we determined that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation. Furthermore, plasmacytoid DC and all CD11c(high) DC subsets are reduced by 50-70% in CSF-1-deficient osteopetrotic mice, confirming that CSF-1 signaling is required for the optimal differentiation of DC in vivo. These data provide additional evidence that the majority of tissue DC is of myeloid origin during steady state and supports a close relationship between DC and macrophage biology in vivo.