Viruses such as lactate dehydrogenase-elevating virus and adenovirus induce in vivo a polyclonal activation of murine B lymphocytes, followed by a marked increase in the production of immunoglobulin G2a (IgG2a). The role of T lymphocytes in this phenomenon was studied by injection of an anti-CD4 monoclonal antibody able to inhibit the T-helper function. This treatment profoundly depressed the production of IgG2a, whereas it had no effect on the proliferation of B cells. Activated B cells obtained from such infected and treated mice remained able to produce various immunoglobulin isotypes after exposure to an appropriate stimulus. In particular, gamma interferon, which is known to be secreted after viral infection, induced the production of IgG2a. These observations support the hypothesis that the influence of viruses on the switch of immunoglobulins is mediated by T-helper lymphocytes.
It was recently shown that T cells, macrophages and fibroblasts produce growth factors for B cell hybridomas and plasmacytomas. These factors were subsequently identified as members of a new family of cytokines on the basis of NH2-terminal amino acid sequence analyses. Using T cell-derived interleukin-HP1 (HP1), purified to homogeneity, as the prototype of this family, we examined the effects of these molecules in conventional polyclonal B cell activation assays with anti-immunoglobulin antibodies or dextran sulfate as co-stimulators. In the absence of other cytokines, the only significant effect of HP1 was to stimulate moderately the proliferation of anti-immunoglobulin-activated B cells. By contrast, in conjunction with interleukin 1, HP1 became a major growth and differentiation factor not only for B cells activated with anti-immunoglobulin antibodies but also for dextran sulfate-stimulated and even for unstimulated B cells. In fact, with respect to cell proliferation or IgM synthesis, the IL 1-HP1 combination proved to be equivalent to B cell stimulatory factors like IL 4 or IL 5. This B cell stimulatory activity was not due to the presence of a contaminant in the HP1 preparation because it was also observed with purified plasmacytoma growth factors derived from macrophages and fibroblasts, and could be inhibited by a monoclonal anti-HP1 antibody.
Interleukin (IL) 6 was compared to other macrophage-derived products for its capacity to support the proliferation of accessory cell-depleted T cells. Monoclonal anti-IL6 antibodies were found to inhibit completely the "accessory activity" of macrophage supernatants, thus demonstrating the central role played by IL6 in T cell activation. IL6 was apparently more critical for initiating than in maintaining T cell proliferation because anti-IL6 antibodies lost all inhibitory activity when added late to the culture. Moreover, IL6 was not the only accessory factor required for optimal T cell proliferation. Using low-density cultures to minimize the number of contaminating accessory cells, we found that significant proliferation of CD4 cells was obtained only in the presence of both IL6 and IL1. In contrast, with CD8 cells substantial proliferation was obtained with IL6 alone. This response could, however, be enhanced by IL1. Tumor necrosis factor (TNF) and granulocyte/macrophage colony-stimulating factor showed no activity in these assays. The concentrations of IL1 and of IL6 required to support optimal proliferation were 10 pg/ml and 1 ng/ml, respectively. Analysis of the mechanisms underlying T cell activation by IL1 and IL6 indicated that both cytokines were required for optimal production of IL2 but that IL6 alone was sufficient to confer IL2 responsiveness. For CD8 cells, this effect was observed with doses of IL6 about 100 times lower than those required for the induction of IL2 secretion (0.001 vs. 0.1 ng/ml). TNF, which was not capable of inducing IL2 secretion, was also found to induce IL2 responsiveness but only at a concentration approximately equal to 1000 times higher than that of IL6. In accordance with these observations, IL6 and to a lesser extent TNF were found to enhance IL2R expression by CD8 cells. Interestingly, this enhancing effect was totally dependent on the presence of IL2.
A murine monoclonal antibody (mAb; 7D6) that was mitogenic for T cells was derived from 129/Sv animals immunized with a T helper clone from C57BL/6 origin. Fluoresceinated 7D6 labeled T cells from most common mouse strains but not from 129/Sv and LP/J animals, and this labeling was inhibited by the anti-CD3 epsilon mAb 145-2C11. The mitogenicity of 7D6 for T cells had a similar strain specificity. The antibody immunoprecipitated the T cell receptor (TcR) complex from a T cell hybridoma. After dissociation of this immunoprecipitate with detergents, the CD3 gamma and epsilon chains were retained by the 7D6 antibody. Immunoprecipitation data were also obtained with COS cells transfected with the CD3 gamma, delta or epsilon chains alone, in pairs or together. They confirmed that 7D6 bound the CD3 gamma epsilon pair, suggesting that the antibody recognizes a conformational epitope formed by gamma epsilon pairing, whereas 145-2C11 bound both gamma epsilon and delta epsilon pairs. These results, therefore, add to current information about TcR structure and subunit stoichiometry. We have demonstrated that the 7D6 mAb specifically binds to a CD3 dimer comprised of gamma and epsilon chains. We thus provide additional evidence that indicates that two CD3 epsilon chains are found within the receptor, one linked to CD3 gamma and the other to CD3 delta.
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