Hannah Kupfer

We describe the intracellular localization, by double immunofluorescence microscopy, of four cytokines that were produced during the prolonged interaction of cloned helper T cells with resting splenic B cells. When two rabbit immunoglobulin-specific helper-T-cell clones were mixed, either separately or together, with splenic B cells in the presence of the antigen rabbit anti-mouse immunoglobulin antibodies, stable T-cell-B-cell conjugates were seen up to 29 hr later. Microscopic observations of these cells revealed that interferon gamma and interleukin 2, inside one of the T-cell clones, and interleukins 4 and 5, inside the other T-cell clone, were concentrated very close to the T-cell-B-cell contact area. The cytokines were not seen in the T cells prior to their interaction with the B cells and their production was strictly antigen-specific. These studies show, at the single-cell level, that helper-T-cell clones can remain bound to splenic B cells long enough for the T cells to produce cytokines, which are synthesized near the bound B cells. We propose that the polarized synthesis of the cytokines may result in their directed secretion toward the bound B cells. By locally secreting the cytokines, which are not antigen-specific, at the contacting T-cell-B-cell membranes, where T- and B-cell surface receptors are engaged and clustered, the helper T cells can induce selective and specific B-cell responses.

Antigen (Ag)-specific T helper (Th) cells regulate the proliferation and differentiation of Ag-specific B cells by secreting cytokines and by expressing activating receptors like gp39. In vitro, the cytokines and the activating receptors function in an Ag-nonspecific manner. It is unclear, therefore, how Ag specificity is imposed on B cell responses in physiological Th-B cell interactions. Here we studied, at the single cell level, the interactions between cloned Th cells and small splenic B cells, which served as Ag-specific antigen-presenting cells (APCs) to the Th cells. Digital confocal immunofluorescence microscopy of Th-B cell conjugates revealed significant variability in the molecular and cellular properties of these interactions, in spite of the fact that all the interactions in this system were expected to be Ag specific. After 30 h of incubation B cells began to divide, and this process was entirely dependent on the presence of both Th cells and Ag. Immunofluorescence microscopic studies showed that essentially all the mitotic B cells were bound to Th cells and faced the microtubule organizing center (MTOC) in the Th cells where interleukin 4 was highly concentrated. Other B cells that were bound to the same Th cells but were not close to the Th-MTOC remained in interphase. These results provide the first direct structural and functional evidence that the site of interaction of B cells with Th cells affects their immune response. We propose that, during Ag-induced Th-B cell interactions, B cells that are bound facing the Th-MTOC proliferate preferentially because they are the recipients of locally secreted cytokines. In addition, these B cells may interact with newly expressed receptors, which may also be locally inserted into the Th membrane. The polarized delivery of activating molecules towards the Th-bound APCs may impose functional specificity on effector molecules that otherwise are not Ag specific.