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Cooperation between monocytes and T lymphocytes is essential for several aspects of immunologic activation. We have utilized PHA and IL-2-activated human T cells to characterize the role of monocytes in the regulation of T cell-derived IFN-gamma production. The limited IFN-gamma production by isolated T cells in this culture system was increased more than 10-fold when monocytes were added. No influence of monocytes was observed on TNF production or T cell proliferation. Maximal level of IFN-gamma in the cell culture supernatants was obtained when monocytes were added within 12 h after activation of the T cells with IL-2 and PHA. Addition of monocytes 48 h after activation resulted in marginal production of IFN-gamma, suggesting that T cells are sensitive to the monocyte-related signal during a short time period after activation. Cell-to-cell contact between the T cells and accessory cells was found to be necessary for enhanced IFN-gamma production because separation of the cells with a semipermeable membrane abolished the effect. mAb blocking experiments suggested the involvement of the CD2/LFA-3 but not the LFA-1/ICAM-1 pathway in monocyte regulation of T cell synthesis of IFN-gamma. Chinese hamster ovary (CHO) cells transfected with LFA-3 (CHO-LFA-3) and HLA-DR4/LFA-3 (CHO-DR4/LFA-3) strongly enhanced T cell IFN-gamma production, whereas untransfected CHO cells, CHO cells transfected with ICAM-1 (CHO-DR4/ICAM-1), and HLA-DR4 (CHO-DR4) did not support IFN-gamma production. PCR analysis and in situ hybridization demonstrated enhanced IFN-gamma mRNA levels in T cells stimulated in the presence of CHO-DR4/LFA-3 compared with untransfected CHO cells, indicating that the CD2/LFA-3 pathway regulates IFN-gamma production at the mRNA level. CHO-LFA-3 and CHO-DR4/ICAM-1 cells mediated strong adhesion to T cells, whereas untransfected CHO cells and CHO-DR4 cells failed to mediate adhesion. This suggests that the ability of CHO-LFA-3 but not CHO-DR4/ICAM-1 cells to induce IFN-gamma production was attributed to signal transduction rather than cell adhesion only.

We have investigated the effect of the superantigen staphylococcal enterotoxin A (SEA) on the balance between T-cell response and non-responsiveness in T-cell receptor (TcR) V beta 3 transgenic mice. One injection of SEA resulted in a substantial activation of TcR V beta 3+ cells, whereas T cells from mice injected with repeated doses of SEA displayed a diminished response to a subsequent in vitro challenge. The reduced responsiveness became apparent when SEA was injected multiple times with short intervals. Proliferation and cytokine production in anergized T cells were severely reduced when stimulated with SEA in vitro, whereas cytotoxic T lymphocyte (CTL) activity remained unaffected. The dichotomy between these functions was examined in vitro with respect to different T-cell subsets. The total number of CD4+ T cells was reduced in the hyporesponsive spleens, compatible with cell deletion. The remaining CD4+ TcR V beta 3+ T cells showed anergy of all tested functions and did not respond to exogenous interleukin-2 (IL-2). In contrast, there was an expansion of CD8+ TcR V beta 3+ T cells with an intact cytotoxic activity. The in vitro proliferation and production of cytokines in the CD8+ compartment was impaired, but could be partially restored in the presence of exogenously added IL-2. Analysis of the cytokine response to SEA in vivo showed that IL-2 and tumour necrosis factor (TNF) were mainly produced by CD4+ T cells, while interferon-gamma (IFN-gamma) was predominantly released by CD8+ T cells. Induction of anergy resulted in a reduction of IL-2 and TNF mRNA levels, frequencies of producing cells as well as serum protein content. In contrast, there was only a moderate influence on the IFN-gamma level in vivo. The results suggest that SEA-induced hyporesponsiveness involves CD4+ cell deletion and a failure to produce cytokines in the remaining CD4+ T-cell compartment, while IFN-gamma production and cytotoxicity in the CD8+ T-cell compartment stay relatively intact.

Conjugates between monoclonal antibodies recognizing human cancer cells and the superantigen staphylococcal enterotoxin A (mAb-SEA) represent a potential novel approach to tumor therapy. Such mAb-SEA conjugates direct T-cells to lyse colon carcinoma cells in vitro. The synthesis of mAb-SEA conjugates which were prepared by introducing thiol groups on SEA and iodoacetyl or maleimide groups on mAb forming a stable thioether linkage between SEA and mAb is described. A hydrophilic spacer, composed of repeated ethylene oxide units, was constructed to increase the distance between SEA and mAb, preserving biological activity of both proteins. The degree of modification of mAb with SEA was determined with SDS-PAGE. Variables influencing the composition of the conjugates and their effect on the tumor-cell cytotoxicity were studied and optimal conditions for the synthesis were established. Functionally active mAb-SEA conjugates were prepared from a panel of different mAb and T-cell-dependent cytotoxicity against several human cancer types including colon, ovarial, breast, and renal cancer was obtained. This suggests that mAb-SEA conjugates may be of value in the treatment of human neoplastic disease.

The staphylococcal enterotoxins (SE) comprise a family of structurally related phage-encoded bacterial proteins, which are the most potent mitogens known for murine and human T lymphocytes. In this report we describe a novel cytotoxic mechanism, where SE directs human CD3+ T lymphocytes to mediate strong cytotoxicity against target cells of irrelevant nominal specificity. The SE-dependent cellular cytotoxicity (SDCC) occurred at picomolar concentrations of SE and involved the initial binding of the SE to the target cells and subsequent triggering of the cytotoxic T cells. SDCC was induced by SEA, SEB, SEC1 and SED, which indicates that this is a common property conserved among all SE. Certain antibodies to the HLA-DR molecule efficiently blocked SDCC. Major histocompatibility complex (MHC) class II+ RAJI cells and HLA-DR-transfected murine L cells were sensitive to SDCC, whereas the MHC class II- RJ.2.2.5 RAJI cell mutant and untransfected L cells were completely resistant to SDCC. These results demonstrate that the MHC class II antigen is the target molecule in SDCC. HLA-DR molecules acted as receptors for SE and the complex was recognized by T lymphocytes in a polyclonal fashion. SDCC was mediated by allospecific cytotoxic CD8+ T cells, by cloned CD8+ T cells and by fresh human peripheral blood mononuclear cells. The SDCC phenomenon provides a rapid, potent and specific mechanism for elimination of HLA-DR+ target cells. We suggest that SDCC is an important combat strategy, employed by the bacteria to avoid specific MHC class II antigen-dependent immune recognition, by inducing T-cell dependent autologous lysis of MHC class II-expressing cells.