Quantitative Studies on the Mixed Lymphocyte Interaction in Rats

Antisera specific for the constant part Ctau of T cell receptor molecules in mice and rats have been produced in rabbits by immunization with idiotype-positive rat T cell molecules. Such antisera could be shown to react with the majority of normal T lymphocytes from both rats and mice. Mixed leukocyte culture-activated T blasts displayed a higher percentage of positive cells than concanovalin A- or phytohemaglutinin-induced T blasts. Lipopolysaccharide-induced B blasts were not reactive with the antiserum. Lyt-1-,2+,3+ blasts displayed a significantly brighter straining than the corresponding Lyt-1+,2-,3- blasts. Isolation of the reactive molecules from T cells by immunosorption yielded a 70,000-dalton single chain polypeptide as the dominant group of molecules. Plasmin caused the chain to split into 45,000- and 25,000-dalton polypeptides, with the smaller fragment displaying antigen-binding capacity. Molecules identical in size and plasma degradation patterns were isolated from Lyt-1+,2-3- and 1-,2+,3+ blasts. Preliminary functional data supported the view that the antiserum is directed against the constant region Ctau relevant receptor structures on immunocompetent T lymphocytes.

Section: Discussionmentioning

confidence: 99%

T cell receptors with allo-major histocompatibility complex specificity from rat and mouse. Similarity of size, plasmin susceptibility, and localization of antigen-binding region.

Binz¹,

Wigzell²

1981

“…Analytical MLI Cultures.--Culture conditions for analytical MLI were essentially as described previously (2). 106 parental strain peripheral blood lymphocytes (PBL) or thoracic duct lymphocytes (TDL) were incubated with 0.5 X 108 Ft hybrid PBL in sterile disposable glass culture tubes containing 1.0 ml Eagle's MEM with glutamine (2.5 mg/ml), penicillin (100 IU/ml), streptomycin (100 mg/ml), and fresh rat serum (5% vol/vol) from BN strain donors.…”

Section: Methodsmentioning

confidence: 99%

Specific Positive Selection of Lymphocytes Reactive to Strong Histocompatibility Antigens

Howard

Wilson

1974

Self Cite

The population of thymus-derlved (T) lymphocytes reactive to strong histocompatibility (H) 1 alloantigens (HARC) is peculiar in two respects in comparison to lymphocytes~responsive to other antigens: first, the T cells reactive to any given strong H antigen outnumber by two to three orders of magnitude the T cells responsive to more conventional antigens (1-3); and secondly, the frequency of HARC to a given H alloanfigen is not increased as a consequence of immunization in vivo (4-6). Just how fundamental these distinctions are between immune reactivity to H alloantigens and other antigen systems is not presently known. For example, the high frequency of HARC raises the possibility that these cells are multipotential, having the capacity to react to several different antigenic determinants. It is clear now that totipotentiality with respect to immune reactivity to H alloantigens can be excluded for HARC, since lymphocyte populations can be experimentally depleted of cells reactive to one H alloantigen, while retaining undiminished reactivity to other antigens (7-8). These "negative" selection experiments do not, however, allow conclusions concerning the ability of cells reactive to one H alloantigen to react to other H alloantigens or to conventional antigens. To examine this question it is essential to prepare pure populations of HARC with reactivity to a single H alloantigen so that the ability of these cells to react to other H alloantigens and to conventional antigens can be tested. This communication describes a procedure which appears to provide specific "positive" selection of cells reactive to chosen strong H alloantigens, and it also illustrates preliminary results obtained using this procedure.Previous studies have shown that parental strain T lymphocytes are induced to proliferate in the mixed lymphocyte interaction (MLI) when they are mixed with alloantigen-bearing cells derived from F1 animals (9), but disappear rapidly under the same culture conditions in the absence of antigen (2). This antigen-dependent survival of cells in the MLI could represent the progeny of lymphocytes specifically reactive to the priming antigens in the culture systems, and if so, these selected progeny should inherit the specific reactivity of their parents.

“…Relatively small fractions of B or T cells from immunized animals will respond to the sensitizing antigens (19,20), whereas a large fraction of the lymphocyte inoculum will be stimulated in vitro by phytomitogens (21,22). Therefore, the lymphocyte response to phytomitogens is generally considered to be nonspecific, 1 Abbreviations used in this paper: Con A, concanavalin A; HBSS, Hanks' balanced salt solution; MLC, mixed leukocyte culture; N-SRBC, neuraminidase-pretreated sheep erythrocytes; PHA, phytohemagglutinin; PWM, pokeweed mitogen.…”

mentioning

confidence: 99%

Cellular Interactions in the Proliferative Response of Human T and B Lymphocytes to Phytomitogens and Allogeneic Lymphocytes

Lohrmann

Novikovs

Graw

1974

163

The cellular events leading to proliferation of cultured human lymphocytes after in vitro stimulation with phytomitogens or allogeneic cells are poorly understood. Little is known on the nature of the proliferating lymphocytes, or whether interactions between different cell types are required for blastogenic transformation of the eventually responding lymphocytes. Investigating these questions, attention has to focus primarily on monocytes, and the different lymphocyte subpopulations.The role of the monocyte (or the monocyte-derived maerophage [1,2], respectively) in antigen-induced lymphocyte activation continues to be of great interest. Lymphocyte response to many antigens requires the presence of monocytes. Removal of glassadherent cells abolishes the in vitro antibody formation by mouse spleen cells against sheep erythrocytes (3) and antigen-induced transformation of human lymphocytes (4); addition of macrophages restores the lymphocyte reactivity (5, 6). Macrophagelymphocyte interaction is also required for lymphocyte activation by allogeneic histoincompatible lymphocytes in vitro, the mixed leukocyte culture (MLC) 1 (7-11). In contrast, lymphocyte activation by plant mitogens such as phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM) is widely attributed to direct interaction of the soluble mitogen with lymphocyte membrane receptors (12-15). Opinions about the role of monocytes in the mitogen-induced lymphocyte proliferation are controversial: reports that phagocytic cells inhibit the lymphocyte response to PHA (16) conflict with others that removal of phagocytic cells decreases lymphocyte activation by this mitogen (17, 18). Alter and Bach described potentiation of PHA-induced lymphocyte proliferation by monocytes (11), whereas Oppenheim and co-workers (4) observed this effect only at suboptimal doses of PHA.Lymphocytes can be separated into thymus-dependent (T) and thymus-independent (B) cells on the basis of ontogeny, function, and cell surface differentiation markers. Relatively small fractions of B or T cells from immunized animals will respond to the sensitizing antigens (19,20), whereas a large fraction of the lymphocyte inoculum will be stimulated in vitro by phytomitogens (21,22). Therefore, the lymphocyte response to phytomitogens is generally considered to be nonspecific, 1 Abbreviations used in this paper: Con A, concanavalin A; HBSS, Hanks' balanced salt solution; MLC, mixed leukocyte culture; N-SRBC, neuraminidase-pretreated sheep erythrocytes; PHA, phytohemagglutinin; PWM, pokeweed mitogen.