H. Sherwood Lawrence

The Cellular Transfer of Cutaneous Hypersensitivity to Tuberculin in Man

Summary and Conclusion 1. It has been possible to passively transfer in 12 consecutive instances cutaneous tuberculin hypersensitivity to tuberculin negative human recipients by means of an intradermal injection of viable leucocytes obtained from the blood of tuberculin positive human donors. 2. The effort at the passive transfer of cutaneous tuberculin hypersensitivity was unsuccessful when the leucocytes used, in the transfer were obtained from tuberculin negative donors. 3. The induced cutaneous hypersensitivity to tuberculin is a transient phenomenon of 4 days to 3 months duration, which can be produced again in the same individual. 4. The degree of induced hypersensitivity to tuberculin seems to bear a relationship to the dosage of leucocytes used and the degree of tuberculin hypersensitivity of the leucocyte donor.

Lymphocyte stimulation: transfer of cellular hypersensitivity to antigen in vitro.

Tuberculin-sensitive human lymphocytes cultured for 36 hours with tuberculin elaborate a soluble material which causes nonsensitive lymphocytes to respond to tuberculin in vitro by transformation and proliferation.

35 – THE PREPARATION AND PURIFICATION OF TRANSFER FACTOR*

Publisher Summary Transfer factor (TF) is the active moiety present in a particular population of circulating blood leukocytes of specifically sensitive human subjects that transfers cutaneous reactivity of the delayed type to nonsensitive individuals. TF preparations have equal potency when compared to the equivalent number of viable parent leukocytes from which transfer factor is derived. For the preparation of TF, the preliminary step involves the isolation and concentration of leukocytes from the plasma of heparinized venous blood— a procedure facilitated by the addition of a macromolecule to hasten erythrocyte sedimentation. The most important condition to be met if successful transfer is to occur is the selection of a donor with an intense degree of delayed cutaneous reactivity to the specific antigen under study. The use of leukocyte preparations obtained from donors who have negative skin reaction to the particular antigen will not result in a transfer of sensitivity no matter how often the recipient is skin tested with antigen.

The specificity of skin homograft rejection in man.

In the course of a study of the transfer of delayed hypersensitivity to skin homografts in man with leukocyte extracts,l it was noted that the specificity of homograft responses had not been evaluated as extensively in man2-5 as in animal species.6-10 This report describes an attempt to demonstrate the individual-specific character of skin homograft rejection in man on the basis of the experimental end point provided by the “white-graft” reaction. The application of a skin homograft to a human recipient results in its initial vascularization and proliferation, followed within 8 to 12 days by graft vessel thrombosis, hemorrhage, and escharification.” These changes are associated with development in the host of generalized altered reactivity to subsequent skin homografts from the same donor source. If such a second-set homograft is applied during the first 7 days following first-set rejection, the white-graft reaction is elicited.12 If the latent period after first-set rejection is lengthened to 10 to 15 days, the second-set graft exhibits the accelerated-rejection rea~t ion.’~ Accelerated rejection is associated with initial graft vascularization, followed by rapid vascular thrombosis and breakdown, with gross rejection on postoperative days 4 or 5 . In contrast, the white-graft reaction is characterized by a complete absence of vascularization on stereomicroscopic observation; the graft remains parchment white and opaque. This sharp end point, which denotes a prior specific immunological experience when contrasted with the behavior of control homografts, has been selected for the present study of homograft specificity in man.

Transfer factor--current status and future prospects.

We have detected new clues to the composition and function of “Transfer Factor” using the direct Leucocyte Migration Inhibition (LMI) test as an in vitro assay of Dialysates of Leucocyte Extracts (DLE). This approach has revealed two opposing antigen-specific activities to be present in the same >3500 <12,000 DA dialysis fraction — one activity is possessed of Inducer/Helper function (Inducer Factor). The opposing activity is possessed of Suppressor function (Suppressor Factor).When non-immune leucocyte populations are cultured with Inducer Factor they acquire the capacity to respond to specific antigen and inhibition of migration occurs. This conversion to reactivity is antigen- specific and dose-dependent. When immune leucocyte populations are cultured with Suppressor Factor their response to specific antigen is blocked and Inhibition of Migration is prevented.

A micromethod for evaluating the phagocytic activity of human macrophages by ingestion of radio-labelled polystyrene particles.

Studies in vitro of human macrophage function in health and disease have been impeded by the difficulty of obtaining such cells in sufficient number. Unlike animal species, the only readily available source of human macrophages are circulating monocytes. Herein, a method is described whereby the phagocytic rate of small numbers of glass-adherent mononuclear cells can be accurately measured. The method utilizes the ingestion by macrophages of technetium labelled polystyrene particles; both the radiolabel and ingestible substrate are readily available and the labelling process simple and efficient. The phagocytic rate can be expressed as radioactive counts per microgram of cell protein; data is also presented showing that the number of particles ingested per cell can be accurately derived.

A microculture system for the measurement of antigen-induced murine lymphocyte proliferation: Advantages of 5% horse serum and 5 × 10−5 M mercaptoethanol☆

Short term microculture systems which measure murine lymphocyte proliferative responses to mitogens are well established. We demonstrate here that these microculture methods are not suitable for antigen-induced responses because of the high levels of murine lymphocyte proliferation in control cultures associated with the use of fetal calf serum or human serum. We also show that this problem can be eliminated with the use of a combination of 5% horse serum and 5 X 10(-5) M mercaptoethanol. We describe an antigen-induced murine lymphocyte proliferation microculture system in which good stimulation indices are achieved and the lymphocyte proliferation in control cultures remain at a low level throughout the 7 day culture period.

Transfer Factor and Cellular Immunity

It has been shown that delayed hypersensitivity can be transferred both in vivo and in vitro by a cell-free substance elaborated by immune lymphocytes. This demonstration opens up the possibility that disease states associated with derangements in cellular immunity—perhaps including cancer—might be treated without the necessity of bypassing or surmounting the host defense mechanisms against whole cells.

SKIN HOMOGRAFT SENSITIVITY CROSS REACTIONS IN MAN

The genetic disparity encountered in the random populations of normal human subjects employed in previous studies concerned with the mechanisms of homograft rejection in man has introduced inescapable variables into interpretations of the observed results.lS2 Since there is no large series surveying the specificity of the homograft response in comparable to the array of data on this subject available in experimental animals,6-10 we have been prompted to study further this aspect of homograft sensitivity in man. The application of a skin homograft to a normal recipient results in its initial survival, followed within 8 to 12 days by a characteristic pattern of rejection which has been termed the homograft rejection phenomenonP These changes are associated with the development in the host of a state of generalized sensitivity to further skin homografts obtained from the same donor. If the host is challenged with a subsequent skin homograft from the same donor, he will exhibit either the accelerated rejection reaction, with ensuing hemorrhagic necrosis of the graft within the first 4 to 5 postoperative days, or the white graft reaclion, characterized by complete nonvascularization and a parchment white appearance of the graft.5 The duration of the latent period allowed between rejection of the first-set graft and application of the following graft from the same donor conditions the type of response observed. A short latent period of 1 to 5 days results in a white graft, while a longer period of 10 to 26 days elicits the accelerated rejection reaction. The development of objective criteria for the evaluation of skin homograft reactions in man has made possible an attempt to evaluate the influence of individual specificity on the response of the host to skin homografts. This paper presents data collected on the fate of 147 skin homografts performed in normal human subjects in the course of a comprehensive study of the immunology of skin homograft reactions in man, with particular reference to possible means of detecting individual or group specific factors concerned with homograft sensitization in man.

In vitro studies on transplantation immunity: II. The migration inhibition in homograft reactions in guinea pigs

Abstract The migration of peritoneal exudate cells from guinea pigs exhibiting transplantation immunity is inhibited in the presence of donor antigens. This inhibition of migration is demonstrable whether the donor transplantation antigens are presented in the form of viable cells (peritoneal exudate cells) or as particulate subcellular antigens (spleen microsomes). A greater degree of inhibition was observed when transplantation immunity was induced with lymphoid cells in Freuds adjuvant compared to sensitization with orthotopic skin grafts. There was no inhibition of migration in mixtures of normal allogeneic cells or when peritoneal cells from guinea pigs exhibiting tuberculin hypersensitivity were mixed with similar cells from normal animals. Finally, supernatants from cultures of sensitive lymphocytes plus donor antigens inhibited the migration of normal peritoneal cells indicating the presence of migration inhibitory factor (MIF) activity.