Various procedures were used to derive continuously growing cytotoxic T lymphocyte (CTL) clones from a primary culture containing responder cells from immunized mice and 3-(p-sulfophenyldiazo)-4-hydroxylphenyl acetic acid (SP)- or fluorescein isothiocyanate (FL)-coupled stimulator cells. It seems likely that CTL have to undergo some change, possibly genetic, to be able to grow continuously in T cell growth factor conditioned medium in the absence of any stimulator or filler cells. The most convenient and reliable procedure to generate CTL clones with different specificities was to establish from several aliquots of a primary culture cell populations continuously growing in medium conditioned with T cell growth factor(s). Clones with different specificities segregated in the different populations. SP- and FL-specific CTL clones restricted to H-2Kk and H-2Dd and two FL-specific CTL clones with no apparent H-2 restriction are described.

A new system to obtain large numbers of functionally competent hapten-specific effector helper T cells has been developed. Mice were primed in the tail with syngeneic spleen cells derivatized with one of three different haptens: 2,4,6-trinitrophenyl, fluorescein isothiocyanate and 3-(p-sulfophenyldiazo)-4-hydroxyphenyl. Draining lymph node cells were subsequently restimulated in culture with the homologous antigen for periods up to 6 weeks. More than 99% and 90% of the cells recovered from these preparative cultures, expressed Thy-1 and Lyt-1 antigens, respectively. These cells proliferated specifically in response to the homologous stimulation by haptenated syngeneic spleen cells, and they were found to display very high levels of hapten-specific helper activity, as assayed in a new, universal method for testing T -B cell cooperation, where every B cell can potentially respond to specific T cell help. Coculture of hapten-derivatized, functionally competent B cells with hapten-specific T cells obtained from the preparative cultures, resulted in the appearance, and exponential increase with time, of very large numbers of IgM and IgG plaque-forming cells. This helper activity could be shown to be: specific for the immunizing hapten and to increase with the time of in vitro helper cell enrichment, which also resulted in higher average avidities expressed by the helper cell populations. This methodology appears valuable for structural studies on hapten-specific helper cells, as well as for the functional analysis of effector helper -B cell interactions.

Cytotoxic T lymphocyte (CTL) responses were obtained in vitro to cells coupled with several different haptens. The degree of lysis of target cells was dependent on the amount of hapten coupled to stimulator and target cells. Spleen cells from normal mice responded to high-hapten density cells but not to low-hapten density cells. However, spleen cells from immunized CBA mice could be stimulated in vitro by low-hapten density cells to generate effector cells able to lyse low-hapten density cells. In vitro primed responder cells could be restimulated in the presence of the original hapten-coupled stimulator cells or in the presence of supernatant from concanavalin A-stimulated mouse or rat spleen cells. Large number of hapten-specific and H-2-restricted CTL could be generated by repeated exposure to fresh supernatant.

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XX cells from XX/XY hemopoietic chimeras do not express male determinants in a way to render them either stimulators or targets for male-specific cytotoxic lymphocytes. XX- but not XY-responder T cells from chimeras can be activated to lyse allogeneic male target cells; T cells from normal XX mice depleted of alloreactive T cells, however, cannot be sensitized to lyse allogeneic XY targets. The results imply that T cells recognize the Y-antigen and H-2 as distinct entities, and that in chimeras, they acquire the potential to react against allogeneic XY cells.