Barbara J. Alter

Immune responses in vitro. I. Culture conditions for antibody synthesis.

Abstract The addition of mercaptoethanol to the culture medium and the optimization of other components has substantially improved the response of mouse spleen cells to in vitro immunization with heterologous RBC. As a result of these changes in the medium, the necessity of rocking and feeding cultures has been eliminated. The in vitro response of C 57 Bl spleen cells was as much as 30 times greater than that achieved in vivo . This suggests the loss of an in vivo control mechanism. Because the magnitude of responses was strain dependent, this culture system may facilitate the study of the genetic control of antibody synthesis.

Cell Mediated Immunity: Separation of Cells Involved in Recognitive and Destructive Phases

The mixed leukocyte culture (MLC) and the cell mediated lympholysis (CML) assays are used as in vitro models of the afferent, or recognitive, and efferent, or destructive, phases of the homograft reaction. Activity in both of these tests has been related to differences at the major histocompatibility complex, HL-A in man and H-2 in mouse. Recent evidence suggests that the presumed cell surface differences which lead to cell proliferation in MLC are different from those which act as a target for CML. Data are presented providing further support for this hypothesis; in addition separate cell populations may respond to the differences which activate cells in MLC and to the differences which serve as targets for CML. There thus appears to be a dichotomy both for genetic control of, and cell populations involved in, the recognitive and destructive phases of cell mediated immunity.

Lymphocyte reactivity in vitro: I. Cellular reconstitution of purified lymphocyte response

Abstract The in vitro mitotic response of human peripheral blood leukocytes to antigens to which the donor of the cells is sensitized or to allogeneic cells is not obtained if highly purified lymphocytes are used in culture. The addition to such nonresponding lymphocytes of a mitomycin C-treated adherent-cell preparation (enriched in macrophages) reconstitutes this response of the purified lymphocytes to either soluble antigen or purified allogeneic-stimulating cells. The data obtained are consistent with an essential role for the added cells, which can be either isogeneic or allogeneic to the responding lymphocytes. The degree of reconstitution is variable but depends to some extent on the number of added cells.

Enhancement of antibody synthesis in vitro by mercaptoethanol

Abstract The culture techniques described by Mishell and Dutton (1) and Marbrook (2, 3), in which a “primary” immune response can be initiated in a suspension of mouse spleen cells to heterologous erythrocyte antigens, offer great potential for analyzing cellular and molecular events involved in humoral responses. To further exploit these techniques, manipulations of culture conditions which either stimulate or inhibit the response would offer additional advantages for biochemical studies. The present report describes the enhancement of the number of antibody-producing cells formed by mouse spleen cells in culture by the reducing agent, mercaptoethanol (MET). This enhancement was found to be a result of the influence of MET on events that occur early in the response.

Lymphocyte reactivity in vitro: II. Soluble reconstituting factor permitting reponse of purified lymphocytes

Abstract Several reports have implicated an adherent cell population—enriched in macrophages—in the in vitro lymphocyte response to either soluble antigens to which the donor of the cells is sensitized or to allogeneic-stimulating cells. We have recently presented evidence which suggests that the role of the added cell population may be an essential one. A supernatant of the macrophage preparation, obtained by repeated centrifugations at 1650g to remove intact cells, will substitute for the macrophage preparation and allow purified cells to respond to allogeneic cells. Such purified cells would not respond otherwise. The existence of such a factor (conditioned medium reconstituting factor—CMRF) may cause us to reevaluate some previous roles suggested for the macrophage in immunological reactions.

TCGF Production for Cloning and Growth of Functional Human T Lymphocytes

In an effort to increase the potency of T cell growth factor (TCGF), several variables were examined for their effects on the production of TCGF. The following manipulations enhanced the potency of TCGF: first, the removal of adherent cells and addition of indomethacin to the producing cultures; second, irradiation with 1000 rads of the cells used to produce TCGF; and, third, the addition of Epstein‐Barr virus transformed lymphoblastoid (LCL) cells. It was also noted that the addition of irradiated feeder cells increased the efficiency of limiting dilution cloning.

Antigenic requirements for triggering of cytotoxic t lymphocytes.

The cotnplexity of T lymphocyte subclasses that respond to alloantigens can be analyzed from at least two perspectives: first, by identification and separation of the different subclasses on the basis of their adherence properties, differentiation antigens present on their surface or other characteristics; and second, by analyzing the alloantigenic systems to which the cells respond. We have focused primarily on the second of these approaches utilizing genetically-defined antigens of the major histocompatibility complex (MHC) as the stimulating factors evoking T lymphocyte responses. It is from this perspective that we shall analyze the requirements for the triggering of T lymphocytes, discussing the cytotoxic T lymphocyte (CTL) in detail with attendant comments about helper and suppressor T lymphocytes. We have used a number of different test systems that appear to provide information in this regard; four of these will be discussed. First, the in vitro generation of helper, suppressor and cytotoxic T lymphocytes in a primary mixed lymphocyte culture (MLC) sensitization system; second, secondary

Ultrastructure of effector--target cell interaction in secondary cell-mediated lympholysis.

The ultrastructure of the secondary cell‐mediated lympholysis (CML) reaction and the effects on interacting lymphocytes of colchicine, cytochalasin B, and effector cell‐Specific antisera were examined using transmission and scanning electron microscopy. Surface labelling of cytotoxic secondary effector cells with cationized ferritin allowed them to be distinguished from unlabelled target lymphocytes. Effector target interactions were characterized by intercellular junctions involving extensive areas of membrane opposition and interdigitation and extension of pseudopod‐like processes by the effector cell. The abolition of such interactions when effector populations were pretreated with anti‐Ly2 sera plus complement demonstrated target cell destruction in secondary CML to be dependent on the activity of restimulated cytotoxic T lymphocytes. Cytochalasin B and colchicine dramatically decreased the numbers of Specific effector‐target cell interactions observed. Although the data presented do not allow the possible activity of soluble lytic factors associated with the effector cell surface to be ruled out, they suggest that target cell lysis in the secondary CML system examined results From immune‐specific binding of alloantigen‐sensitized effectors to targets and osmotic effects which follow localized disruption of the target cell membrane.

Typing an Unrelated Panel with PLT Cells: Association with DW Clusters

Primed LD typing (PLT) cells prepared in one laboratory (Madison) were shipped in the frozen state and tested in Tübingen on a separate panel that bad been typed with homozygous typing cells. Those PLT cells that had been grouped, on the basis of their reaction with rest cells of the Madison panel, as defining an HLA PI antigen showed identical or nearly identical patterns of reactivity with the Tübingen panel. Clear association between certain PL antigens and DW clusters as defined with homozygous typing cells could be demonstrated. Of particular interest may be combinations of certain PLT reactions with D‐locus‐typed cells, where the primed cells do not react as expected from the targets HLA‐D type.

T LYMPHOCYTE REACTIVITIES TO ALLO- AND ALTERED-SELF ANTIGENS

ABSTRACT The diversity of T lymphocytes that play a role in the response to alloantigens and altered-self antigens is great. In addition to the classical Ly 1, helper T lymphocytes and Ly 2, cytotoxic T lymphocytes, it is now clear that one must consider participation by suppressor T lymphocytes that are separate from the cytotoxic T lymphocytes, and Ly 1,2 T lymphocytes that are involved at the precursor level in the response to K/D alloantigenic stimuli as well as in the response to “altered-self” antigens. It appears that there are two alternative pathways that T lymphocytes can use in their response to foreign antigens: first, a pathway consisting of collaboration between Ly 1, helper T lymphocytes and Ly 2, cytotoxic T lymphocytes and second, a pathway dependent on an Ly 1,2 cell at the precursor level. The factors that may influence the balance between these two pathways as well as consideration of the nature of the antigenic stimuli needed to elicit a secondary response, perhaps dependent on which pathway(s) is used in the primary, are discussed.