Polarized expression of cytokines in cell conjugates of helper T cells and splenic B cells.

Abstract

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 y 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 antigenspecific. 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.The introduction of proteinaceous foreign antigens (Ags) into an animal generally results in the proliferation and differentiation of B cells that then secrete antibodies (Abs), which bind specifically to the foreign Ags. The B-cell response is largely controlled by soluble growth and differentiation factors, collectively termed lymphokines or cytokines (1, 2), which are produced by Ag-specific helper T cells (Th cells).It is unclear yet how selective and regulated B-cell responses can be generated by the secretion of cytokines such as interferon y (IFN-y) and interleukins 2, 4, and 5 (IL-2, IL-4, and IL-5, respectively), since these cytokines are not Agspecific or even cell-type-specific (2). In addition, each of these cytokines can have multiple effects on the cells and when present together they can either antagonize or synergize each other (3). However, the addition of crude or purified cytokines to resting B cells in the absence of Th cells failed to cause B-cell proliferation and differentiation, implying that specific cell-cell interactions may be additionally required for effective B-cell responses (4-7). The molecular events that take place during such Th-cell-B-cell (Th-B) interactions are still largely unknown.We have described an in vitro model system to study at the single-cell level events that occur within the first minutes of the interaction of cloned Th cells and Ag-presenting B-cell (B-APC) lymphomas or hybridomas (8). By using immunofluorescence microscopy, we observed the clustering of the T-cell receptors (TCRs) for Ag, CD4, and lymphocyte function-associated antigen 1 (LFA-1) membrane re...

Requirement for Phosphoinositide 3-Kinase p110δ Signaling in B Cell Antigen Receptor-Mediated Antigen Presentation

“…The 2R.50 T cells express MHC class II (I-A d )-restricted TCR specific for rabbit IgG and were maintained and used as previously described (34,35). The LK35.2 B cell transfectant (HyHEL10), which expresses a hen egg lysozyme (HEL)-specific IgM BCR, and 2G7 T cells, which express a MHC class II (I-E k )-restricted TCR specific for HEL [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] , were gifts from F. Batista (Medical Research Laboratory of Molecular Biology, Cambridge, U.K.) and were maintained and used as previously described (36).…”

Imaging immune cell interactions and functions: SMACs and the Immunological Synapse

“…When the T-B conjugates were labeled intracellularly, after several hours of interaction, with antibodies specific for IL-2, IL-4. IL-5, and IFN-gamma the newly synthesized cytokines were heavily localized in the T cell at the contact site with the bound B cell [5]. Longer term (24-48 h) microscopic observations of Ag-specific conjugates between T cells and multiple bound small resting B cells demonstrated that only the B cells that were facing the site of cytokines production in the cells underwent proliferation [6] (Fig.…”

Cytotoxic immunological synapses do not restrict the action of interferon-γ to antigenic target cells

“…In conjugates of helper T cells and antigen-presenting B cells, IL-2, IFN-γ, IL-4, and IL-5 were all localized at the microtubule organizing center (MTOC), in the area of the T cell apposed to the B cell (3). The authors proposed that this polarized cytokine production was involved in the antigen-specific activation of B cells (4).…”