Search

To target T-cells to the tumour area we created a recombinant protein of the bacterial superantigen (SAg) Staphylococcal enterotoxin A (SEA) and the Fab-fragment of a tumour-reactive antibody. This antibody-targeted SAg immunotherapy therapy has been shown to be highly efficient, eliminating > 95% of the pulmonary metastasis in mice carrying established melanoma micrometastases. Earlier studies demonstrated that elimination of the C215-expressing B16-melanoma lung metastasis was dependent on interferon (IFN)-γ release and expression of perforin. In the present study, therapeutic effector functions were analysed both locally at the tumour site and systemically in the spleen. In order to elucidate the role of each T-cell subset during Fab-SEA therapy, CD4 knock-out (KO) and CD8 KO mice were used. Tumour size reduction was statistically significant in Fab-SEA-based tumour therapy in both types of T-cell-deficient mice compared to wild-type mice. CD4 KO mice displayed a drastic reduction in the number of tumour-infiltrating macrophages and CD8[SUP+]T-cells. Therapy-induced accumulation of perforin-containing cells at the tumour site was significantly impaired in CD8 KO mice, and marginally in CD4 KO mice. Moreover, CD4 KO mice failed to produce substantial amounts of the tumour suppressive cytokine IFN-γ. This is in sharp contrast to normal mice where a massive local release was recorded. CD8 KO mice displayed a spontaneous production of interleukin (IL)-4 and IL-10 locally in the tumour. Neither normal nor CD4 KO mice produced detectable levels of these Th-2-associated cytokines. The high level of IL-10 was demonstrated to inhibit Fab-SEA tumour therapy, since the therapeutic efficacy was significantly higher in IL-10 KO mice. These results illustrate the importance of a finely tuned cellular collaboration to regulate the various phases of an efficient anti-tumour immune response. [ABSTRACT FROM AUTHOR]

Administration of staphylococcal enterotoxin A (SEA) to mice induces profound activation, cytokine production and cytotoxic activity of both CD4+ and CD8+ T cells, but subsequently activated cells are deleted or become anergic. This study demonstrates that administration of interleukin-2 (IL-2) can prevent sea-induced hyporesponsiveness in CD8+ cytotoxic T lymphocytes (CTL). Repeated injections with sea every fourth day resulted in severely reduced cytotoxic activity in the spleen, which correlated with a reduced number of sea-responsive T-cell receptor (TCR)-Vβ11+CD8+ cells. Studies of purified TCR-Vβ11+CD8+ cells showed that they possessed intact cytotoxic activity per cell compared with cells from mice given a single injection of SEA, indicating that deletion was the main mechanism for the reduced cytotoxic activity. Combined treatment with SEA and IL-2 increased the number of cytotoxic cells in the spleen after each SEA injection and prevented the down-regulation of cytotoxic activity. Increased cytotoxic activity could be related to increased number and proliferation of CDS+IL-2Rα+ cells, suggesting that administration of IL-2 maintained IL-2 responsiveness among CD8+ cells. Studies of sorted TCR-Vβ11+CD8+ cells demonstrated that combined treatment with SEA and IL-2 also increased cytotoxic activity per cell compared with treatment with SEA alone. Taken together, IL-2 administration in vivo augmented SEA-induced expansion of T cells as well as the cytotoxic activity per CTL, and prevented SEA-induced cell deletion. [ABSTRACT FROM AUTHOR]

We have investigated the effect of the superantigen staphylococcal enterotoxin A (SEA) on the balance between T-cell response and non-responsivess in T-cell receptor (TcR) Vβ3 transgenic mice. One injection of SEA resulted in a substantial activation of TcR Vβ3+ cells, whereas T cells from mice injected with repeated dosas 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 Vβ3+ 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 Vβ+ 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 turnout necrosis factor (TNF) were mainly produced by CD4+ T cells, while interferon-γ (IFN-+) 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-γ 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-γ production and cytotoxicity in the CD8+ T-cell compartment stay relatively intact. [ABSTRACT FROM AUTHOR]

The superantigen Staphylococcal enterotoxin A (SEA) conjugated to tumour-specific monoclonal antibodies (mAb) directs T cells to lyse tumour cells in the absence of major histocompatibility complex (MHC) class II. In contrast, the conjugate bound to MHC class II-negative tumour cells did not activate resting T cells to proliferate. The SEA-C215 mAb conjugate, when presented on the CA215 antigen-expressing Colo205 cells, required either signalling with CD28 mAb or CHO cells expressing the natural CD28 ligand, B7, to activate the T cells. The CD28/B7 co-stimulatory effect was further enhanced when the B7 and the tumour antigen were present on the same cell, decreasing the superantigen amount required for activation with a factor of 104. No influence of B7 was seen when the single CA215 or double CA215/B7 transfectants were used as targets for superantigen conjugate-dependent cytotoxicity. This suggests that the low affinity T-cell receptor (TcR) interaction of superanfigen in the absence of MHC class Il antigens is sufficient for induction of cytotoxicity but requires additional CD28/B7 signalling to result in proliferation of resting T cells. [ABSTRACT FROM AUTHOR]

The response of mouse T cells to the superantigen staphylococcal enterotoxin A (SEA) requires 1000fold higher concentrations compared to human T cells. In order to develop a sensitive model for SEA studies in mice, the immunopharmacology has been studied in T-cell receptor (TcR) Vβ3 transgenic (TGVβ3) and non-transgenic (non-TG) C57B1/6 mice. The frequency of SEA-responsive T cells in the TGVβ3 mice exceeded 90%, whereas a 10-fold lower frequency was seen in normal C57B1/6 mice. Nanograms of SEA injected intravenously into TGVβ3 mice indued strong cytolytic T lymphocyte (CTL) activity against SEA-coated major histocompatibility complex (MHC) class II+ B-lymphoma cells, whereas administration of 1000-fold higher amounts of SEA to non-TG littermates or normal C57B1/6 mice induced only a moderate response. Kinetic analysis demonstrated that the CTL activity was more rapidly detectable in TG mice, but substantial levels were seen 2 days after SEA injection in both TGVβ3 and non-TG mice. The cytotoxic T-cell response induced by SEA in TGVβ3 and nonTG mice was completely MHC class II dependent, as SEA-coated MHC class II-transfected syngeneic B16 melanoma cells but not untransfected B16 cells were sensitive to lysis. Large amounts of turnout necrosis factor-α (TNF-α) and interferon-y (IFN-γ) accumulated in serum of TGVβ3 mice after injection of 10 ng SEA, whereas only marginal amounts were recorded in non-TG even after injection of 100 µg SEA. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated that SEA-induced TNF-α and IFN-γ mRNA reached maximal levels 1 hr after SEA administration in TGVβ3 mice, whereas peak serum levels of TNF-α, and IFN-γ proteins were recorded after 2 hr. Comparison of the mRNA levels of a panel of cytokines in the TGVβ3 and nonTG mice revealed that almost similar amounts of interleukin-1 (IL-1) were induced in both strains, whereas IL-4 was only detected at significant levels in the TGVβ3 mouse. The results suggest that TGVβ3 mice are suitable for studying in vivo immune responses to superantigens at concentrations comparable to the potent effects elicited in humans. Moreover, this model is useful for detailed studies on the dynamic regulation of T-cell activation and anergy induced by superantigens. [ABSTRACT FROM AUTHOR]

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 Il antigen-dependent immune recognition, by inducing T-cell dependent autologous lysis of MHC class II-expressing cells. [ABSTRACT FROM AUTHOR]

Functionally distinct subsets within the T-helper (CD4+) cell population have been described in man, rat and mouse. We have shown previously that the CEM+45R- human lymphocytes are producers of IL-2 within 24 hr of polyclonal stimulation and are the major interferon (IFN) producers. The mAbs 4B4 (CDw29) and Leu 8 (p80) are here used together with Leu-18 (CD45R) to characterize the subpopulations of the CD4+ human lymphocytes in more detail. The majority of the CD45R+ cells were CDw29- and the majority of the CDw29+ cells were CD45R-. Most of the CD45R+ cells (78%) were also p80+. A significant number of cells (12%) were CDw29-CD45R-. The predominant subpopulations were defined to be CDw29- CD45R+p80+ (31%), CDw29+ CE45R-p80-(24%) and CDW29+CD45R-p80+(18%). Within the CD4+CD45R- subpopulation different subsets vary in their capacities to produce IFN and to produce IL-2 within 24 hr after activation. CD4+CDw29-CD45R-p80- T lymphocytes produced the largest quantities of IFN and IL-2. [ABSTRACT FROM AUTHOR]

Superantigens activate T-cells by linking the T-cell receptor to MHC class II on antigen-presenting cells, and novel reactivity can be introduced by fusing the superantigen to a targeting molecule. Thus, an antibody-targeted superantigen, which activates T cells to destroy tumour cells, might be used as cancer therapy. A suitable target is the 5T4 oncofetal antigen, which is expressed on many carcinomas. We constructed a fusion protein from a Fab of a monoclonal antibody recognizing the 5T4 antigen, and an engineered superantigen. The recombinant product 5T4FabV13-SEA[SUBD227A] bound the 5T4 antigen expressed on the human non-small-cell lung cancer cell line Calu-1 with a K[SUBd] (of 1.2 nM while the substitution of Asp227 to Ala in the superantigen moiety reduced binding activity to MHC class II. 5T4FabV13-SEA[SUBD227A] tumour reactivity was demonstrated in 7/7 NSCLC samples by immunohistochemistry, while normal tissue reactivity was low to moderate. 5T4FabV13-SEA[SUBD227A] induced significant T-cell-dependent in vitro killing of sensitive 5T4 bearing Calu-1 cells, with maximum lysis at 10[SUP-10] M, while the capacity to lyse MHC class II expressing cells was approximately 1000 times less effective. Immunotherapy of 5T4FabV13-SEA[SUBD227A] against human NSCLC was investigated in SCID mice reconstituted with human peripheral blood mononuclear cells. Mice carrying intreperitoneally growing Calu-1 cells showed significant reduction in tumour mass and number after intravenous therapy with 5T4FabV13-SEA[SUBD227A]. Thus, 5T4FabV13-SEA[SUBD227A] has highly attractive properties for therapy of human NSCLC. [ABSTRACT FROM AUTHOR]

Previous work demonstrated that human cytotoxic T cells activated by superantigens can lyse major histocompatibility complex (MHC) class Il-positive target cells as well as MHC class II- negative tumour cells coated with conjugates of monoclonal antibodies and superantigens. In order to decrease MHC class II affinity, and therefore unwanted binding of the superantigen staphylococcal enterotoxin A (SEA) to MHC class H molecules, a point mutation was introduced into the SEA genes This mutation (SEAD227A) resulted in an approximately 3-log reduction of affinity to human leucocyte antigen (HLA)-DR, but cytotoxicity mediated by this mutant superantigen towards antibody-labelled tumour cells is as efficient as cytotoxicity mediated by the native superantigen. We therefore compared the T-cell activating potency of native and mutated SEA. Our data show that SEAD227A is 4- to 5-log less effective than native SEA when activation of resting T cells is assayed in terms of blast formation, expression of cell surface activation markers and cytokine release. Furthermore, presenting either SEA or SEAD227A to MHC Class II-neptive mononuclear cells by MHC class II-negative tumour cells did not result in significant blast formation of T cells, up-regulation of CD25 or cytokine release. This suggests that lysis of MHC class II-negative tumour cells is efficiently induced by monoclonal antibody targeted superantigen, while activation of resting T cells requires additional co-stimulatory signals. [ABSTRACT FROM AUTHOR]