Bone Marrow Transplantation, (1999) 23, 63–69 1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt Comparison of monocyte-dependent T cell inhibitory activity in GM- CSF vs G-CSF mobilized PSC products AG Ageitos1, ML Varney1, PJ Bierman2, JM Vose2, PI Warkentin1 and JE Talmadge1 1Department of Pathology and Microbiology, and 2Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA Summary: following transplant compared to normal individuals. PSCT patients, however, have a more rapid immune recovery than This study compares the immune properties of periph- do BMT patients.5–7,10 eral blood stem cell (PSC) products mobilized with dif- Stem cells are mobilized from the bone marrow (BM) ferent hematopoietic growth factors (HGFs) as well as into the peripheral blood (PB) after myelosuppressive apheresis products and peripheral blood leukocytes chemotherapy,12 chemotherapy combined with HGFs13–17 (PBL) from normal individuals. We found that mono- or during administration of HGFs.18–24 Accelerated hemato- cytes in mobilized PSC products appear to inhibit T cell poietic recovery is observed following PSCT as compared function independent of whether granulocyte colony- to transplantation with steady-state BM and is only stimulating factor (G-CSF) or granulocyte–macrophage observed when peripheral blood stem cell (PSC) products colony-stimulating factor (GM-CSF) was used for mobi- are collected after mobilization.21,24,25 The choice of HGF lization. In addition, the GF used to mobilize the stem used for mobilization may also be important as granulo- cell product may be less important to the CD4:CD8 cyte–macrophage colony-stimulating factor (GM-CSF) ratio than the extent of prior chemotherapy, as we interacts with earlier progenitors than does granulocyte found an inverse correlation between chemotherapy and colony-stimulating factor (G-CSF)26 and increases the fre- the CD4:CD8 ratio. In other observations, all apheresis quency of granulocytes, macrophages, eosinophils, mega- products, whether mobilized or unmobilized, contained karyocytes, erythroid precursors, and dendritic cells from significantly more monocytes compared to normal PBL. hematopoietic progenitors.27 In addition, the administration The mononuclear cells (MNC) from G-CSF or GM-CSF of GM-CSF28 or G-CSF29 after autologous BMT or PSCT mobilized PSC products had a similar T cell phytohem- also can enhance the endogenous cytotoxic and antimicro- agglutinin (PHA) mitogenic response that was signifi- bial30 activity of immune cells. cantly lower (P = 0.001 and P = 0.005, respectively) than G-CSF is used most frequently for mobilization and has non-mobilized apheresis products. We also examined been shown to enhance the production and maturation of neu- the T cell inhibitor (TI) activity of the MNC from the trophils in vitro and in vivo.27,31,32 G-CSF is unique among PSC products for allogeneic lymphocyte proliferation the regulators of granulopoiesis in that it not only stimulates and found that PSC products significantly reduced the the proliferation, but also induces the terminal maturation of proliferation of allogeneic PBL to PHA. A significant myeloid progenitor cells.33 Like other cytokines, G-CSF correlation (P = 0.001, r = 0.517) between the frequency exerts a pleiotropic effect on different cell populations. The of monocytes and TI activity also was observed. G-CSF receptor can be found on myeloid progenitor cells, Keywords: monocytes; stem cell mobilization; T cells; mature neutrophils, platelets, monocytes, and some T and B GM-CSF; G-CSF lymphoid cell lines.33 In addition, the PSC pool can be expanded by both G-CSF and GM-CSF.34–36 While it is well documented that the injection of HGFs mobilizes stem cells with different hematopoietic properties dependent on the The therapeutic efficacy of hematopoietic growth factor HGF, no studies to date have compared the immunologic (HGF) administration has been shown in a variety of dis- characteristics of PSC products mobilized with different 1,2 eases. One such utility includes rapid myelorestoration HGFs. following high-dose chemotherapy (HDT) and stem cell Analysis of G-CSF mobilized PSC products has shown transplantation. Although peripheral blood stem cell trans- that they contain at least three times more T cells than plantation (PSCT) has largely replaced bone marrow trans- steady-state BM products. However, the frequency of natu- plantation (BMT) due to a lower incidence of tumor-cell ral killer (NK) and T cells in these PSC products is in the 3 4–6 contamination and more rapid hematopoietic recovery, range expected for normal PB.37 We and others have 5–11 both BMT and PSCT patients are immunosuppressed reported9,38 that GM-CSF mobilized PSC products have a high frequency of monocytes. Furthermore, the phenotypic analysis of mobilized PSC products revealed normal levels Correspondence: Dr JE Talmadge, Department of Pathology and Micro- + + biology, University of Nebraska Medical Center, 600 South 42nd Street, of CD4 cells and an increased frequency of CD8 cells, Omaha, NE 68198-5660, USA which results in a decreased CD4:CD8 cell ratio as com- Received 29 May 1998; accepted 11 August 1998 pared to the PB of normal individuals. Monocytes in PSC products AG Ageitos et al 64 While the effects of GM-CSF and G-CSF on myeloid Isolated mononuclear cells (MNC) cells have been studied, the effect of HGF mobilization on lymphoid cells is not well understood. GM-CSF has been MNC were obtained from apheresis products and the hep- reported to amplify interleukin (IL)-2 supporting T cell pro- arinized venous blood of 49 normal donors by Ficoll– liferation,39 and in patients with non-Hodgkin’s lymphoma Hypaque gradient centrifugation. The interphase cells were (NHL), there is an increase in the absolute lymphocyte harvested, washed three times in Hanks’ balanced salt sol- count, particularly activated lymphocytes after GM-CSF ution (HBSS; Life Technologies; Gaithersburg, MD, USA), 40 41 and resuspended in RPMI-1640 medium supplemented with administration. Other reports have examined the ability ␮ of GM-CSF to induce T cell proliferation and found that 1mm HEPES, glutamine, 40 g/ml gentamycin (Life it upregulated the expression of IL-2 receptors on T lym- Technologies), and 10% fetal bovine serum (FBS). phocytes. We report here a comparison of the effect of G- CSF and GM-CSF mobilization on lymphocyte and mono- Proliferation assay cyte function and phenotype in PSC products. Phytohemagglutinin (PHA)-induced T cell proliferation was measured by determining 3H-thymidine incorporation into DNA. Cells (100 ␮l/well) were plated in quadruplicate Materials and methods in flat-bottomed wells of microtiter plates at 1 × 106 cells/ml. The cells were cultured in PHA (100 ␮l/well) at Patients and control group a final concentration of 0.5 ␮g/ml of media. On day 3, the ␮ 3 A total of 54 apheresis products, including HGF mobilized wells were pulsed for 18 h with 1 Ci of H-thymidine leukapheresis products from 36 patients and 18 non-mobil- (Amersham Life Sciences; Arlington Heights, IL, USA) ized apheresis products from healthy donors, were exam- and the cells harvested on day 4 using an automatic multi- ined. The samples were divided into three groups, accord- well harvester (Packard Instruments, Downers Grove, IL, ing to whether HGF was used for mobilization and whether USA). The filters were allowed to air dry, scintillation G-CSF or GM-CSF was used for mobilization. PSC apher- cocktail was added, and the samples were counted in a esis products were collected from 21 patients following Packard multi-well beta-plate counter. In all studies, a con- GM-CSF and from 15 patients following G-CSF mobiliz- trol of normal peripheral blood leukocytes (PBL) was ation. NHL patients who were eligible for PSCT at the Uni- included and the data are reported as the percent of the versity of Nebraska Medical Center (UNMC) were entered control sample. into these studies. The samples were obtained using proto- cols approved by the Institutional Review Board of UNMC Flow cytometry and following written informed consent. The clinical characteristics of both groups are shown in Table 1. Using Flow cytometric analyses were used for the phenotyping the same protocol as for the cancer patients, non-mobilized, studies. The cell populations were studied prior to Ficoll– apheresis products were obtained from normal donors and Hypaque separation and erythrocytes removed by lysis. Ali- served as a control group for the mobilized PSC apheresis. quots of 5 × 104 cells were stained with biotin-conjugated monoclonal antibodies (MoAb) to CD8 and CD14, a fluor- escein isothiocyanate (FITC)-labeled MoAb to CD3, and a phycoerythrin (PE)-labeled MoAb to CD4. After incubation Table 1 Characteristics of study patients and washing, streptavidin allophycocyanin (APC) was added as the third fluorochrome. The data were acquired Clinical features G-CSF GM-CSF on a FACStarPlus using a 40 mw, 488 nm argon laser and mobilized mobilized a 100 mW, 647 nm HeNe laser for excitation. Detailed data analysis was performed using Paint-A-Gate Plus software Number 15 21 Sex from Becton Dickinson (San Jose, CA, USA). Male 7 9 Female 8 12 Age (years) T cell inhibition assay Median 48 45 Range 27–64 14–63 The coculture assay used to measure the cellular inhibition Prior therapy of T cell function has been described previously.9,38,42,43 Chemotherapy 15 21 Briefly, Ficoll–Hypaque purified, normal PBL (1 × 105)as Radiation 6 responder cells were cocultured with varying numbers of Months of chemotherapy median 2 9a irradiated (500 cGy) putative inhibitor cells (MNC from PB range 1–5 2–25 or PSC product) starting at an inhibitor to responder ratio Tumor type (I:R) of 4:1. Four, two-fold dilutions of inhibitor cells (50 Non-Hodgkin’s lymphoma 15 16 ␮l) were made in 96-well flat bottom plates.