Goat and rabbit anti-human lymphotoxin sera, IgG and F(ab')2 reagents were investigated for their capacity to effect a specific alloimmune lymphocyte-mediated cytotoxic reaction. The cytotoxic reaction employed human peripheral blood or adenoid lymphocytes sensitized in MLC to allogeneic B lymphocyte cell lines and lysis was measured in a short-term 51Cr-release assay. A polyspecific anti-LT sera (anti-WS), made against unfractionated whole supernatants from lectin-activated lymphocytes and its IgG and F(ab')2 fragments, was found to be a potent inhibitor of this reaction when the anti-WS reagent was present throughout the assay period. Absorption studies indicated the anti-WS was inhibiting cytolysis at the level of effector cell or its products. Two broadly defined antibody specificities were involved in the cytolytic-inhibitory activity of the polyspecific anti-LT; i) antigens present on the normal lymphocyte cell surface; and ii) lymphocyte surface antigens associated with activated cells. These results correlate with the previously defined antigenic structure of the LT Cx and alpha H classes. Anti-LT sera reactive with the smaller m.w. alpha and beta classes and subclasses were not inhibitory, although the anti-beta sera showed a moderate enhancing activity. The results indicated that several anti-LT antibody specificities may be required to inhibit alloimmune cytolysis. The results suggest LT molecules may mediate lymphocyte-induced alloimmune cytolysis as a multi-component toxin system, rather than as an individual toxin.

The mechanism of inhibition of one human cytotoxic T lymphocyte-mediated (CTL) reaction in vitro by a polyspecific antiserum directed against soluble products of activated lymphocytes was investigated. This antiserum was made against serum-free culture supernatants from lectin-inactivated human lymphocytes (anti-WS). The anti-WS shows strong neutralizing activities for both soluble and membrane forms of human lymphotoxins. The anti-WS was investigated for its site(s) of inhibition of the alloimmune CTL reaction relative to the calcium-dependent phase. The anti-WS was found to neutralize the CTL reaction subsequent to the calcium-dependent phase, suggesting the anti-WS neutralized a lytic effector mechanism. A fluid-phase immunoadsorption assay was developed to analyze the biochemical characteristics of the antigenic determinant(s) recognized by the anti-WS. Unfractionated supernatants or defined m.w. regions corresponding to the m.w. classes of the human LT system were used to adsorb the anti-WS. The data indicated that unfractionated supernatants and LT-Cx fractions (>200,000 m.w.) significantly reduced the capacity of the anti-WS to inhibit the alloimmune CTL reaction. α-LT fractions (70 to 90,000 m.w.) partially adsorbed the cytolytic inhibitory antibodies of the anti-WS, whereas β and γ LT fractions (40 to 50,000 and <20,000 m.w., respectively) were ineffective. These results support the concept that the mechanism of inhibition mediated by the anti-WS was by its capacity to interact with multiple antigenic species of LT molecules, and thus implies that lymphotoxins are involved as a multicomponent system of cytotoxins in the lytic effector mechanism of human alloimmune cytotoxic T lymphocytes. The LT-Cx appears to represent the most likely molecular candidate for the lytic effector mechanism. A model is presented that attempts to integrate the biochemical properties of the LT system with the cellular mechanism(s) of the alloimmune cytotoxic reaction scheme.

The biochemical and immunological properties of a cell toxin(s) released into the fluid phase and expressed on the surface of lectin-activated alloimmune human cytotoxic lymphocytes has been investigated. The results indicate that the initial cytotoxin(s) released into the supernatant from alloimmune lymphocytes represents a precursor form of lymphotoxin (pre-LT), and in this precursor form, the α and β antigens exist as masked or cryptic determinants. The pre-LT form in unfractionated supernatants was defined immunologically by neutralization with a polyspecific anti-LT antisera (anti-WS), and its lack of reactivity with anti-α and anti-β LT antisera. However, gel filtration chromatographic profile of the pre-LT cytotoxic activity was heterogeneous and showed multiple m.w. classes that were characteristic of the chromatograms of traditionally defined LT. These fractionated components were now readily neutralized with anti-α LT antiserum. In addition, the pre-LT cytotoxic activity in unfractionated supernatants treated by various physical or chemical means rendered the cytotoxic activity neutralizable by an anti-α LT antiserum, indicating an immunologic relationship between the pre-LT form and components of the LT system. The antigens associated with pre-LT form were detectable, in part, on the LT-Cx and γ LT molecules by a fluid phase immunoadsorption assay. A functional immunoadsorption assay was used to detect the antigenic determinants of the pre-LT form on the cell surface of alloimmune lymphocytes before and after lectin activation. α LT-associated antigens were detectable on cytotoxic effectors only after lectin activation. These results imply LT may function as cell surface delivered cytotoxic molecules. LT activity was not detected in supernatants obtained from alloimmune cytotoxic reactions, however, the pre-LT and α-LT neutralizing activities of the anti-LT sera were significantly diminished after the incubation of these sera in the cytotoxic reaction and indicated that LT molecules were produced during the cytotoxic reaction, and LT remained closely associated with the lymphocyte:target cell conjugates. The relationship of the pre-LT form to the capacity of the various anti-LT antisera to affect the human alloimmune cytotoxic reaction is discussed. The results provide additional support to the concept that the LT system is involved in the lytic mechanism of cytotoxic T cells.

These studies demonstrate that specific and nonspecific cytolytic human T cells can release LT forms in vitro. Nonspecific cytolytic T cells were derived from IL 2-dependent cultures initiated by allogeneic mixed lymphocyte reaction (AMLR). Specific cytolytic T cells (CTL) were derived from IL 2-dependent T cell clones, initiated by mixed lymphocyte reaction and specific for class II antigens. alpha-LT was the major lytic component released by these cells in IL 2-dependent cultures. However, on Con A stimulation or contact with target cells, both AMLR and CTL effectors release a new LT form. The new LT form released by AMLR and CTL effectors appear similar, for they both elute from gel filtration at 60,000 to 70,000 m.w. and migrate as a single peak with an Rf of 0.4 on 7% native PAGE tube gels. Moreover, testing these materials on a panel of target cells in vitro indicates that they are both nonspecific, and lyses NK-resistant target cells in vitro. Additional studies revealed that in vitro lytic activity of this form(s) is not affected by either anti-alpha-LT serum or a monoclonal reagent which inactivates macrophage cell toxins (MCT) and tumor necrosis factor (TNF). However, when these two immunologic reagents are tested together, activity is totally neutralized. Thus, this LT form expresses antigens in common with alpha-LT, MCT, and TNF. Finally, studies with NK-CF and NK-LT forms revealed that they were also completely neutralized with a mixture of anti-LT and monoclonal anti-TNF antibody. These data suggest that certain macrophage- and lymphocyte-derived cell toxins are interrelated.