Stimulation of Subpopulations of Human Lymphocytes by a Vaccine Strain of Francisella tularensis

Abstract

When purified T lymphocytes from individuals vaccinated with a viable, attenuated strain of Francisella tularensis were incubated in vitro in the presence of heat-killed bacteria or a membrane preparation of the vaccine strain, they were stimulated to form blast cells and to synthesize deoxyribonucleic acid. The blast cells had the characteristics of T cells, being devoid of surface immunoglobulin and able to form rosettes with sheep erythrocytes. The stimulation occurred only when monocytes were present. A lymphocyte preparation enriched in B lymphocytes did not respond to the heat-killed bacteria or to the membrane preparation. In a stimulated mononuclear leukocyte preparation, about 70% of the blast cells formed rosettes with sheep erythrocytes, and 10 to 20% of them had surface immunoglobulin. The results show that there is an enlarged population of specifically committed T lymphocytes after tularemia vaccination. It is suggested that the lymphocyte stimulation test measures mainly T-lymphocyte reactivity when membranes or whole bacteria of F. tularensis LVS are used as antigen, and that the stimulation of human T lymphocytes by whole bacteria or bacterial membranes is completely monocyte or macrophage dependent. The present experimental procedure may provide a model for study of antigen-induced stimulation of human lymphocytes under controlled conditions. The technique used gave a reproducible, extremely purified preparation of T lymphocytes and a preparation of monocytes especially suitable for microcultures.

Immunospecific T-lymphocyte stimulation by membrane proteins from Francisella tularensis

“…They were then mixed with sheep erythrocytes to allow rosette formation, and the mixture was centrifuged on Lymphoprep as previously described (14). The cell suspension of the interphase between serum and Lymphoprep (non-T lymphocytes) was washed once in saline, distributed in microplates, collected onto glass-fiber filters, and assayed for radioactivity.…”

“…Cell-mediated immunity toward microbial antigens can be demonstrated by the lymphocyte stimulation test. This is valid for F. tularensisinduced stimulation, since most of the cells which respond in the test have the characteristics of T lymphocytes (14,15).…”

Immunogenicity and toxicity of lipopolysaccharide from Francisella tularensis LVS

“…Blood samples were obtained from six healthy adults previously vaccinated with F. tularensis LVS, and from four healthy adults denying knowledge of tularaemia or tularaemia vaccination. Lymphocytes were prepared from heparinized blood as previously described [29,30]. As antigen, LPS from F. tularensis was used at a concentration of 1.0 and 100 /~g ml -~.…”

“…Peripheral blood mononuclear adherent cells were prepared from heparinized blood of four human voluenteers as previously described [29,30]. The prepared cells (3 x 10 6 ml -~) were allowed to adhere to plastic culture dishes for 1-24 h in RPMI-1640 with 10% (v/v) inactivated normal human serum (obtained from healthy voluenteers) at 37°C.…”

Antigen from Francisella tularensis: nonidentity between determinants participating in cell-mediated and humoral reactions

“…These components were apparently removed by the preparatory procedure used. Lymphocytes responding specifically to F. tularensis have been reported to be the T lymphocytes mainly (28), and the antigen prepared should improve the test as a parameter of cell-mediated immunity. This is important, since the presence of cell-mediated but not humoral immunity correlates with protection against tularemia (17).…”