Terje Kalland

CHARACTERIZATION OF TWO DISTINCT MHC CLASS II BINDING SITES IN THE SUPERANTIGEN STAPHYLOCOCCAL ENTEROTOXIN A

Bacterial superantigens (SAgs) are potent activators of T lymphocytes and play a pathophysiological role in Gram‐positive septic shock and food poisoning. To characterize potential MHC class II binding sites of the bacterial SAg staphylococcal enterotoxin (SE) A, we performed alanine substitution mutagenesis throughout the C‐terminus and at selected sites in the N‐terminal domain. Four amino acids in the C‐terminus were shown to be involved in MHC class II binding. Three of these amino acids, H225, D227 and H187, had a major influence on MHC class II binding and appeared to be involved in coordination of a Zn2+ ion. Alanine substitution of H225 and D227 resulted in a 1000‐fold reduction in MHC class II affinity. Mutation at F47, which is equivalent to the F44 previously shown to be central in the MHC class II binding site of the SAg, SEB, resulted in a 10‐fold reduction in MHC class II affinity. The combination of these mutations in the N‐ and C‐terminal sites resulted in a profound loss of activity. The perturbation of MHC class II binding in the various mutants was accompanied by a corresponding loss of ability to induce MHC class II‐dependent T cell proliferation and cytotoxicity. All of the SEA mutants were expressed as Fab‐SEA fusion proteins and found to retain an intact T cell receptor (TCR) epitope, as determined in a mAb targeted MHC class II‐independent T cell cytotoxicity assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine Inhibits Interleukin 1 Production by Lipopolysaccharide-Stimulated Human Peripheral Blood Monocytes

Histamine inhibited the production of interleukin 1 (IL‐1) induced by lipopolysaccharide (LPS) in cultures of purified human peripheral blood monocytes. The effect of histamine on IL‐1 production was dose‐dependent and significant at histamine concentrations of 10−4‐10−5m. The histamine H2 receptor agonists dimaprit and 4‐methylhisiamine, hut not the H1 receptor agonists 2‐pyridylethylamine. aminoethylthiazole and 2‐methylhistamine, modulated the IL‐1 production in a similar manner to histamine. The inhibitory effects of histamine could be reversed by the H2 receptor antagonist cimetidine but not by the H1 receptor antagonist mepyramine. This indicates that the inhibitory effects of histamine on LPS‐induced IL‐1 production are mediated through H2 receptors on human peripheral blood monocytes.

Staphylococcal enterotoxins direct and trigger CTL killing of autologous HLA-DR+ mononuclear leukocytes and freshly prepared leukemia cells

Attempts have been made to induce cytolytic T cells to kill target cells that do not express the appropriate target molecules by crosslinking the T cells and the target cells in various ways. One successful strategy has been to use heteroconjugates or bispecific monoclonal antibodies reacting with T cell molecules with activating properties (e.g., mab directed to CD3/TCR) and target cell surface antigens. In this report we show that Staphylococcal enterotoxins (SE) direct human T lymphocytes to execute cytotoxicity toward MHC class II-expressing Raji cells, but not against MHC class II-deficient Raji mutant RJ 2.2.5. Both HLA-DR+ and HLA-DR- effector T lymphocytes are effective in the killing of Raji cells coated with SE. The Staphylococcal enterotoxin-dependent cell-mediated cytotoxicity (SDCC) is a rapid T lymphocyte-mediated cytolytic mechanism killing the targets within an hour of incubation. HLA-DR+ target cells are sensitized to be killed within minutes of incubation with picomolar concentrations of SE. SE-sensitized Raji cells remain targets for SDCC after overnight culture at 37 degrees C, demonstrating that the sensitive state is relatively stable. SEA- and SEB-selective cytolytic T cell lines were established to illustrate the clonal variability of SDCC effectors with respect to SE specificity. We also demonstrate that autologous monocytes and activated T lymphocytes as well as B lymphocytes and freshly prepared HLA-DR+ leukemic cells are excellent targets in SDCC.

Superantigen-based tumor therapy: in vivo activation of cytotoxic T cells.

We have recently demonstrated that the superantigen staphylococcal enterotoxin A (SEA) targets in vitro activated cytotoxic T lymphocytes against tumor cells expressing major histocompatibility complex (MHC) class II antigens. In this report we analyze the use of SEA as an immunoactivator in vivo. Treatment of mice with SEA activated a fraction of CD3+ T cells apparently as a function of their T cell receptor Vβ expression. SEA induced interleukin-2 receptor expression and proliferation in both CD4+ and CD8+ T cells. This proliferative response was dose-dependent (0.1 – 100 µg/mouse), peaked during day 1 after treatment and declined to background levels within 4 days. The cytotoxic response, measured as cytotoxicity to SEA-coated MHC class II+ target cells (staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity, SDCC), was maximal at a dosage of 1 µg SEA/mouse. The SDCC was confined to the CD8+ T cell compartment, peaked 2 days after treatment and declined to background levels within 4 days. A second injection of SEA on day 5 after the first SEA treatment resulted in SDCC function with kinetics and magnitude identical to that seen after one injection. These results pave the way for the use of SEA in the treatment of MHC class II+ tumors.

Effects of the novel immunomodulator LS 2616 on the delayed-type hypersensitivity reaction to Bordetella pertussis in the rat

Treatment of Sprague-Dawley rats with the compound LS 2616, a quinoline-3-carboxamide, enhanced the delayed-type hypersensitivity response (DTH) to recall antigens as judged by the specific accumulation of inflammatory cells after challenge with Bordetella pertussis in the pleural space. LS 2616 was effective when given both before sensitization and at the time of secondary antigen challenge. The effect of LS 2616 on DTH was dose-dependent. LS 2616 was highly effective in enhancing DTH in rats with suppressed cell-mediated immunity after treatment with anti-thymocyte globulin or prednisolone, indicating its possible value in the immunosuppressed state.

Immune response during tumor therapy with antibody-superantigen fusion proteins.

To engineer superantigens (SAg) to express tumor reactivity, we genetically fused the Fab‐part of the tumor‐reactive MAb C215 and the bacterial SAg staphylococcal enterotoxin A (SEA). Treatment of mice carrying established lung micrometastases of the C215‐transfected syngeneic B16 melanoma with 3‐4 daily injections of C215Fab‐SEA resulted in strong anti‐tumor effects, while only moderate effects were seen when treatment was given every 4th day (intermittent treatment). High serum levels of IL‐2, TNF‐α, IFN‐γ and strong induction of CTLs (cytotoxic T lymphocytes) were noted after priming with the fusion protein. T cells responded well to 3 daily injections of C215Fab‐SEA and then gradually entered a hyporesponsive state, characterized by a reduced ability to produce IL‐2, TNF‐α and IFN‐γ and failure to mediate cytotoxicity in vitro. Intermittent treatment was characterized by increased levels of IL‐10, concomitant with accentuated loss of IL‐2, TNF‐α and IFN‐γ production. A 10‐fold increase in SEA‐reactive TCR VB3+ CD4+ cells was observed in the spleen, while a loss of TCR VB3+ CD8+ and VB11+ CD8+ cells was noted. This is in striking contrast to injections of native SEA which induced a marked deletion of TCR VB3+ CD4+ T cells, but not of CD8+ cells. Recovery of the THI cytokine profile occurred within 1–2 weeks, while restoration of cytotoxicity required several months and correlated with recovery of TCR VB3+ CD8+ and TCR VB11+ CD8+ T cells. These results show that the temporal relationship of SAg stimulations dictates the cytokine profile. Moreover, different mechanisms appear to regulate hyporesponsiveness in CD4+ and CD8+ T cells.

Costimulation by B7-1 and LFA-3 targets distinct nuclear factors that bind to the interleukin-2 promoter: B7-1 negatively regulates LFA-3-induced NF-AT DNA binding.

We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-kappaB nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1- and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-kappaB complexes induced in B7-1- and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position -500 to +60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-kappaB, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancer-promoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA-3 was unaffected. Our data strongly suggest a selectivity in induction of nuclear factors by the CD2-LFA-3 and CD28-B7-1 pathways. This selectivity may contribute to regulation of the levels of IL-2 induced by LFA-3 and B7-1 costimulation and favor autocrine and paracrine T-cell responses, respectively.

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A.

SummaryActivation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5−CD56+) as well as T (CD5+) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.

Exposure of neonatal female mice to diethylstilbestrol persistently impairs NK activity through reduction of effector cells at the bone marrow level

Female NMRI mice neonatally treated with diethylstilbestrol (DES) show persistent impairment of various immune parameters, including NK activity. The present study demonstrated that NK activity was reduced in all lymphoid compartments and thus is not due to a simple redistribution of effector cells. Poly I:C was unable to augment NK activity in DES treated animals in vitro and in vivo. This defect was not attributable to inability to produce interferon in response to poly I:C since interferon-beta was also unsuccessful in augmenting NK activity in vitro. Moreover, the lack of response was not dependent on alterations in macrophage function, as evident from experiments showing that macrophages from DES-exposed animals resulted in a normal enhancement of NK activity in response to poly I:C when mixed with lymphocytes from control animals. Studies at the single cell level revealed that the reduced NK activity was the result of a reduced number of target-binding effector cells, and that the individual cells actually binding a target killed the target in an apparently normal fashion. Adoptive transfer of bone marrow cells between control and neonatally DES-treated animals showed that the reduced number of effector cells was determined at the bone marrow level.

Repeated treatment with antibody-targeted superantigens strongly inhibits tumor growth.

Superantigens (SAg) are microbial proteins with the capacity to activate a large proportion of T cells. We have developed a novel approach for cancer immunotherapy by genetically fusing the SAg staphylococcal enterotoxin A (SEA) to a Fab‐fragment of a tumor‐specific antibody. Repeated exposure to SEA induces a state of unresponsiveness including cell deletion and functional hyporesponsiveness, i.e., anergy. In this study we have developed improved therapeutic schedules to allow repeated injections of Fab‐SEA, limit development of immunological unresponsiveness and promote maximal anti‐tumor response. Four daily injections of Fab‐SEA to mice carrying B16‐C215 lung metastases resulted in 90–95% reduction in the number of metastases. However, the animals did retain a minimal residual tumor disease. The immune system was in a hyporesponsive state after 4 daily Fab‐SEA injections, and further injections did not improve therapy. Two repeated cycles, each comprising 4 daily injections of Fab‐SEA, significantly prolonged the survival and resulted in complete cure of a fraction of the animals. A rest period of 10 days between the cycles was required to mount an efficient secondary anti‐tumor response. This secondary immune response was characterized by partial recovery of cytokine production i.e., interleukin‐2, interferon‐γ and tumor necrosis factor‐α. Strong CTL activity was detected in animals that had rested for 8 weeks between the 2 cycles. Interestingly, irrespective of the resting period, the CD4+ SEA‐reactive T cells expanded in response to all 4 additional Fab‐SEA injections both locally and in spleen. In contrast, only marginal expansion of CD8+ T cells was seen if restimulation was given within 1 month. Our data show that potent anti‐tumor effector functions can be induced after repeated stimulation cycles with a SAg‐monoclonal antibody fusion protein resulting in a CD4+ T cell‐dependent cytokine release, prolonged survival and induction of complete cures. Int. J. Cancer 76:274‐283, 1998.© 1998 Wiley‐Liss, Inc.