Autologous Bone Marrow Transplantation - Blog Science Connections

698 T Lymphocyte CFU After ABMT
cers, 1 melanoma, and 1 acute myeloid leukemia. Nineteen patients received
intensive chemotherapy (Table 1 ) and one patient (with acute myeloid leukemia)
received chemotherapy followed by total body irradiation (80 Gy).
Cryopreserved autologous bone marrow was reinfused following intensive
therapy; no subsequent maintenance chemotherapy was administered.
Characteristics of the 20 patients are summarized in Table 1.
Analysis of T-Cell Subpopulations
Peripheral blood T lymphocyte subsets were studied at regular intervals
after ABMT. T lymphocytes and T cell subsets were determined by binding of
the following monoclonal antibodies: Tl 1 (pan T cell, cluster of differentiation
CD2), T4 (T helper/inducer CD4) and T8 (T suppressor/cytotoxic CD8), Bl
(B lineage), 12 (HLA D/DR-related la antigen), and IL2R1 (TAC antihuman IL2
receptor), obtained from Coulter Immunology (Hialeah, FL); OKT3 (CD3)
and OKT6 (Thymocytes CD1), purchased from Ortho Pharmaceutical
Laboratories (Raritan, NJ). T lymphocyte and T cell subsets were counted by
indirect fluorescence and flow cytometry (Epics®, Coulter). Absolute CD4-
and CD8-positive T cell counts from the leukocyte count, percentage of
lymphocytes in the differential count, and the percentages of CD 1, CD2, CD3,
CD4, CD8, l + , and TAC + cells were calculated. Control values were determined
in 25 healthy subjects.
T-Cell Colony Assay
Peripheral blood from patients or control subjects was collected in anticoagulant
solution. Mononuclear cells were isolated after Ficoll-Hypaque
gradient centrifugation. Cells from the interface were collected and washed in
modified Dulbecco's medium (Gibco, Paisley, Scotland). Cells (10 6 ) were
seeded for colony formation in the modified Dulbecco's medium in 35-mm
Petri dishes (Nunclon, Denmark) in 0.5 ml soft agar (0.33%) on a 2.5-ml harder
agar base (0.5%); 0.03 ml PHA (phytohemagglutinin) (Wellcome, HA15 Dartford,
England) per Petri dish was added to the lower agar layer. Autologous
plasma (20%) and mercaptoethanol 5 x 10 5
M (Merck, Schuchlardt, West
Germany) were added to both layers. Colonies of more than 50 cells were
counted 7 days afterseeding. Cultures were incubated at 37° C in a humidified
incubator with 10% C0 2
. Recombinant human interleukin 2 (rIL2), generously
provided by Professor Fiers (University of Ghent, Belgium), was added to the
underlayer at a total dose of either 25 or 50 U.
Cytomegalovirus
Cytomegalovirus (CMV) infection was investigated by assaying serum
antibodies IgM and IgG with an Elisa method, using viral urine cultures and
histologic samples when necessary.