Albert D. Donnenberg

ISOLATION OF CELL CYCLE FRACTIONS BY COUNTERFLOW CENTRIFUGAL ELUTRIATION

Counterflow centrifugal elutriation (CCE) has been used to fractionate cell populations on the basis of sedimentation properties, with minimal perturbation of metabolic function. Therefore, it is an ideal method for the isolation of cell cycle phase specific populations. We present modifications of the standard Beckman centrifugal elutriation system which permit standardization of the elutriation procedure and eliminate inter-run variability. We provide elutriation parameters for the cell cycle fractionation of a variety of cultured cell lines and suggest ways to improve the quality of the cell separations. In addition, we describe protocols for the fractionation of up to 3.50 X 10(8) cells in the small (JE-6B) Beckman elutriation system. This represents a four- to eight-fold increase in cell numbers over current cell fractionation procedures. Cell cycle populations containing greater than 95% G1, greater than 80% S, and greater than 70% G2/M were consistently obtained using these protocols. Finally, we analyzed phase-enriched fractions from several cultured cell lines for the cell cycle regulation of the enzyme thymidine kinase. The data confirm previous findings that CCE is an excellent means of obtaining physiologically unperturbed cell cycle phase specific fractions.

Development of a simplified counterflow centrifugation elutriation procedure for depletion of lymphocytes from human bone marrow

Graft-versus-host disease (GVHD) remains a major complication of allogeneic bone marrow (BM) transplantation. Techniques that effectively purge BM of mature T lymphocytes should reduce the incidence of GVHD and improve survival. We have developed a simplified, two-flow rate, fixed rotor speed counterflow centrifugation-elutriation (CCE) procedure that reproducibly depletes 99% of lymphocytes from Ficoll-Hypaque (F/H)-separated BM or BM buffy-coat. Two predetermined flow rates (24 and 28 ml/min) were used to purge small and intermediate-to-large lymphocytes, respectively, whereas faster sedimenting cells were recovered at the termination of the run. Lymphocyte depletion was substantiated by pan-T monoclonal antibody analysis as well as by complete loss of responsiveness of alloantigens and mitogens. Despite the lack of mature T cells, the depleted marrow fraction retained lymphoid colony-forming ability. Lymphocyte-purged marrow was obtained in high yield (72%), and retained high viability (>97%) and hematopoietic colony-forming ability (>99%). The ratio of total myeloid/erythroid colony-forming cells to T lymphocytes was 73-fold higher in the lymphocyte-depleted fraction than in unseparated BM. We concluded that a two-step CCE procedure can be used to rapidly deplete lymphocytes from both F/H-separated and buffy-coat BM inocula without altering hematopoietic capacity as measured by the in vitro clonogenic assays. It may be possible to adapt this procedure to the separation of the large number of marrow cells required for human BM transplantation.

Limiting dilution analysis of in vivo-activated (IL-2 responsive) peripheral blood lymphocytes in HIV-1-infected subjects

The progression of infection with human immunodeficiency virus, type 1 (HIV-1), is associated with a loss of helper T cell function, but the mechanism for this loss (e.g., decreased absolute number of helper cells, altered function of helper cells, or both) has not been delineated. Many studies have suggested that T-cell production of and/or responsiveness to the T cell growth factor interleukin-2 (IL-2) declines over the course of HIV-1 infection. Using a highly quantitative 6-day limiting dilution assay (LDA), we investigated whether the number and the proliferative capacity of circulating IL-2 responsive cells in patients with AIDS differ from those in patients in earlier stages of HIV-1 infection (asymptomatic or AIDS-related complex) and healthy seronegative individuals. The frequency of IL-2 responsive cells declined progressively in asymptomatic seropositive subjects, those with ARC, and those with AIDS. In contrast, the proliferative capacity of individual IL-2 responsive cells, as reflected by the magnitude of thymidine uptake per precursor, was reduced only in patients with frank AIDS and was normal in asymptomatic subjects and in those with ARC. These results suggest that the development of AIDS in the setting of HIV-1 infection may reflect a combination of qualitative as well as quantitative changes in lymphocyte function. They also suggest that analysis of lymphocyte responsiveness to IL-2 may provide a useful approach to prediction of the development of AIDS in individuals infected with HIV-1.

Large scale separation of human bone marrow by counterflow centrifugation elutriation

Counterflow centrifugation elutriation (CCE) has been used to separate moderate quantities of bone marrow (BM) into distinct cell populations for further in vitro investigation. Recently, this technique was employed to reduce the incidence of graft-versus-host disease (GVHD) in human allogeneic bone marrow transplantation (BMT) by removing the majority of mature lymphocytes from the graft. Unfortunately, current methods employing the small (J6-B) elutriator rotor require time consuming preseparation steps and multiple runs. We report our experience with a fixed rotor speed, two-flow rate elutriation procedure using the new Beckman JE-10X rotor system which can separate more than 10(10) nucleated BM cells in 30 min. Three fractions were obtained which differed in size, morphology, and immunologic capacity. The large cell fraction is suitable for allogeneic BMT since it is radically depleted of mature T lymphocytes and retains high clonogeneic capacity.

Characterization of the natural suppressor cell population in adult rat bone marrow.

Natural suppressor cell activity (NSCA) has been ascribed to a subset of cells present in human and murine hematopoietic tissues which can suppress a variety of lymphocyte responses without MHC restriction. We investigated NSCA in lymphocyte‐depleted rat bone marrow (BM) which is used as a model for prevention of graft vs host disease (GVHD) following allogeneic BM transplantation (BMT). The T‐cell depleted fraction obtained after elutriation contained higher levels of NSCA than the unseparated BM. Further separation of this graft fraction by discontinuous Percoll gradient centrifugation revealed high levels of radiosensitive NSCA in the low density (< 1.070) fraction which represented 0.5% of the original BM population. These cells were of blast morphology, stained intensely with a dansylated derivative of cyclosporine A (dans CsA) and weakly expressed macrophage/granulocyte antigens and non‐specific esterase (NSE). These cells were initially non‐adherent but proliferated in culture to produce intensely NSE positive, adherent, phagocytic cells of macrophage morphology. We conclude that the highly suppresses, radiosensitive cell present in rat BM may be of early progenitor or monocyte lineage. The grafting of natural suppressor (NS) cells and progenitor cells may affect graft/host immunoregulation and their characterization may provide insight into GVH biology and graft rejection.

Immunity to herpes simplex virus type 2 (HSV-2): I. Development of virus-specific lymphoproliferative and leukocyte migration inhibition factor responses in HSV-2-infected guinea pigs

Abstract The development of virus-specific cell-mediated immune (CMI) memory and effector response was studied in strain 13/N guinea pigs infected with herpes simplex virus type 2 (HSV-2) (G). The indirect leukocyte migration inhibition factor (LIF) and the lymphocyte transformation (LT) assays, chosen as probable indicators of effector and memory responses, respectively, were performed simultaneously on spleen cells (SC) obtained at varying times after infection and cultured in the presence of uv-inactivated HSV-2 (G) antigen. Kinetic and dose-response analyses revealed: (i) a time-dependent increase in the magnitude and antigen sensitivity of the LT response as well as a time-dependent decrease in the in vitro “doubling time,” both suggestive of immune maturation, and (ii) a biphasic pattern of LIF production in vitro consisting of an “early” component generated within the first 24 hr in culture, and a “late” component detected between 3 and 6 days in culture. “Late” LIF production correlated well with the lymphoproliferative response and appeared to require the presence of glass-adherent cells and active cell division.

Determining Matrilines by Antibody Response to Exotic Antigens

The antibody responses of female Microtus pennsylvanicus inoculated with a series of antigens not normally encountered under field conditions were examined, and the kinetics of maternally acquired antibody loss in their offspring were determined. The initial antibody response in adults was rapid, peaking in 4–9 weeks, and long-lasting, with a half life of 4–5 months. Antibody levels in females were unaffected by parity, and more than one antigen could be given without affecting circulating-antibody titers. Antibody titers could be enhanced with additional inoculations. Maternal antibody in offspring increased until weaning at 3 weeks then declined exponentially. Minimal detectable titers were reached at 7–11 weeks. In many instances, maternal antibody remained detectable even after offspring reached adult (35 g) size. Examination of uninoculated wild-caught voles showed only one of 130 tests produced a “false” positive response. The exotic-antigen technique may be generally applicable for determining maternity in small, secretive mammals.