T. Barlozzari

Natural killer activity in the rat: IV. Distribution of large granular lymphocytes (LGL) following intravenous and intraperitoneal transfer

Highly enriched populations of rat large granular lymphocytes (LGL) and T lymphocytes were prepared on discontinuous density gradients of Percoll, labeled with either 111In-oxine or 51Cr and injected either intravenously (iv) or intraperitoneally (ip) into normal syngeneic recipients. Following iv inoculation of labeled LGL or T cells into normal recipients, a large proportion of radioactivity (18 to 33%) was recovered within minutes in the lungs. By 2 to 4 hr following transfer, significantly more LGL (13.5%) than T cells (6.4%) remained in the lungs. This difference persisted through 48 hr (5.4 vs 0.8%). Decreasing levels of radioactivity in the lungs were accompanied by corresponding increases in counts in the spleen and liver. At early time points, a significantly higher proportion of T cells was found to distribute to the spleen, while labeled LGL persisted for longer periods in the blood as well as in the lungs. Following ip inoculation into normal recipients, there was a slow clearance of radiolabeled LGL and T cells from the peritoneal cavity, with less than 20% of the radiolabel found in peripheral organs by 24 hr. These results demonstrate a distribution pattern for LGL and T cells that resembles the previously reported proportions of these cells in various organs. In addition, these studies provide a firm basis for the formulation of further experiments to examine the usefulness of adoptive immunotherapy with LGL or immune T cells.

In vivo reactivity of mouse natural killer (NK) cells against normal bone marrow cells

Abstract An in vivo role for mouse natural killer (NK) cells in the rapid rejection of transplantable tumors has been previously demonstrated, using an assay of elimination of [125I]iododeoxyuridine-labeled tumor cells from the lungs and other organs. We have now used the same technique to examine the role of NK cells in in vivo clearance of syngeneic or allogeneic bone marrow cells from normal mice. The degree of clearance from the lungs or liver, at 4 hr after intravenous inoculation of radiolabeled bone marrow cells, correlated with the levels of NK activity in the recipients. Young CBA mice, with high NK activity, showed substantially more clearance of bone marrow cells than SJL mice, with low NK activity. Within the same strain, mice at 7 weeks of age had higher in vivo as well as in vitro reactivity than did 30-week-old mice. These differences were seen with syngeneic bone marrow cells, but there was stronger in vivo reactivity against parental cells by F1 hybrid recipients. Depression of NK activity by pretreatment of mice with cyclophosphamide, silica, or carrageenan also caused decreased clearance of syngeneic or parental bone marrow cells. Conversely, augmentation of NK activity by pretreatment of the mice with polyinosine:polycytidine resulted in increased in vivo clearance. These results indicate that NK cells may be involved in the in vivo control of growth of some normal cells as well as of some tumor cells.

NATURAL SUPPRESSOR CELLS FOR MURINE NK ACTIVITY

Publisher Summary This chapter discusses the presence of suppressor cells in some natural situations and their possible role in the physiologic regulation of natural killer (NK) activity. In mice, NK activity is absent at birth, begins to appear around 3 weeks of age, peaks at 6–8 weeks, and declines to low levels after 12 weeks of age. The low NK activity of spleen cells of aged mice was not associated with detectable suppressor cells. Normal mice of various inbred strains have been shown to vary considerably in their levels of spontaneous NK activity. Murine NK activity has a characteristic tissue distribution, with relatively high NK activity present in the spleen and peripheral blood and little or no activity in the thymus or peritoneal cavity. The frequent association between low NK activity and suppressor cells suggests that inhibition by macrophages or other cells at the effector phase may account for, or at least contribute to, the low cytolytic activity. The presence of suppressor cells in the peritoneal cavity, a site with low NK activity, but not in the spleen, an organ with high NK activity, suggests a possible regulatory role for these cells.

REGULATION OF IN VIVO REACTIVITY OF NATURAL KILLER (NK) CELLS

Publisher Summary This chapter focuses on regulation of in vivo reactivity of natural killer (NK) cells. The results with normal bone marrow cells are consistent with previous indications that NK cells mediate in vivo natural resistance against bone marrow transplants but differ in that some reactivity has been found against syngeneic cells and the stronger reactivity of allogeneic or parental cells, suggesting an autoreactive nature of NK cells. In view of the accumulating evidence for a possible in vivo role of NK cells in defenses against tumor cells, virus infections, and in homeostatic regulation of normal nonneoplastic tissues, it is particularly important to develop procedures to more definitively document their in vivo importance and to determine how to manipulate the levels of in vivo natural reactivity. The suppressor cells present in the spleen of normal SJL/J mice could account for the lower reconstitution of such mice and for the low NK activity of this strain. The data support the in vivo relevance of in vitro defined suppressor cells for NK activity.

CELL SURFACE ANTIGENIC CHARACTERISTICS OF RAT LARGE GRANULAR LYMPHOCYTES (LGL)

Publisher Summary This chapter examines cell-surface antigenic characteristics of rat large granular lymphocytes (LGL). LGL have distinct azurophilic granules in their cytoplasm and have been shown to be highly associated with in vitro natural killer (NK) activity. Studies were conducted with monoclonal antibodies and continuous flow microfluorometry to examine the cell surface antigenic characteristics of highly enriched rat LGL, relative to those of T cells, monocytes, and polymorphonuclear leukocytes (PMNs). All experiments were performed with peripheral blood leukocytes (PBL) from W/Fu or rnu/+ rats. Monocytes were isolated from PBL by adherence on FBS coated tissue culture flasks. LGL, T cells, and PMN were prepared from nylon wool (NW) nonadherent PBL by centrifugation on a discontinuous density gradient of Percoll. Viability was over 95% as judged by trypan blue exclusion. Morphological analyses of these fractions are fraction 2—LGL (75 to 90%), fraction 3—T cells (>80%), and fraction 4—PMN (60 to 90%). The results demonstrate that like human LGL and mouse NK cells, rat LGL are an antigenically distinct subpopulation of lymphocytes, which share some characteristics with both T cells and monocytes.